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Human sex redefined: The idea of two sexes is simplistic. Biologists now think there is a wider spectrum than that

Posted in Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Reproductive Biology & Bio Instrumentation on February 19, 2015| Leave a Comment »

Human sex redefined: The idea of two sexes is simplistic. Biologists now think there is a wider spectrum than that

Reporter: Aviva Lev-Ari, PhD, RN

As a clinical geneticist, Paul James is accustomed to discussing some of the most delicate issues with his patients. But in early 2010, he found himself having a particularly awkward conversation about sex.

A 46-year-old pregnant woman had visited his clinic at the Royal Melbourne Hospital in Australia to hear the results of an amniocentesis test to screen her baby’s chromosomes for abnormalities. The baby was fine — but follow-up tests had revealed something astonishing about the mother. Her body was built of cells from two individuals, probably from twin embryos that had merged in her own mother’s womb. And there was more. One set of cells carried two X chromosomes, the complement that typically makes a person female; the other had an X and a Y. Halfway through her fifth decade and pregnant with her third child, the woman learned for the first time that a large part of her body was chromosomally male1. “That’s kind of science-fiction material for someone who just came in for an amniocentesis,” says James.

Sex can be much more complicated than it at first seems. According to the simple scenario, the presence or absence of a Y chromosome is what counts: with it, you are male, and without it, you are female. But doctors have long known that some people straddle the boundary — their sex chromosomes say one thing, but their gonads (ovaries or testes) or sexual anatomy say another. Parents of children with these kinds of conditions — known as intersex conditions, or differences or disorders of sex development (DSDs) — often face difficult decisions about whether to bring up their child as a boy or a girl. Some researchers now say that as many as 1 person in 100 has some form of DSD2.

When genetics is taken into consideration, the boundary between the sexes becomes even blurrier. Scientists have identified many of the genes involved in the main forms of DSD, and have uncovered variations in these genes that have subtle effects on a person’s anatomical or physiological sex. What’s more, new technologies in DNA sequencing and cell biology are revealing that almost everyone is, to varying degrees, a patchwork of genetically distinct cells, some with a sex that might not match that of the rest of their body. Some studies even suggest that the sex of each cell drives its behaviour, through a complicated network of molecular interactions. “I think there’s much greater diversity within male or female, and there is certainly an area of overlap where some people can’t easily define themselves within the binary structure,” says John Achermann, who studies sex development and endocrinology at University College London’s Institute of Child Health.

These discoveries do not sit well in a world in which sex is still defined in binary terms. Few legal systems allow for any ambiguity in biological sex, and a person’s legal rights and social status can be heavily influenced by whether their birth certificate says male or female.

“The main problem with a strong dichotomy is that there are intermediate cases that push the limits and ask us to figure out exactly where the dividing line is between males and females,” says Arthur Arnold at the University of California, Los Angeles, who studies biological sex differences. “And that’s often a very difficult problem, because sex can be defined a number of ways.”

That the two sexes are physically different is obvious, but at the start of life, it is not. Five weeks into development, a human embryo has the potential to form both male and female anatomy. Next to the developing kidneys, two bulges known as the gonadal ridges emerge alongside two pairs of ducts, one of which can form the uterus and Fallopian tubes, and the other the male internal genital plumbing: the epididymes, vas deferentia and seminal vesicles. At six weeks, the gonad switches on the developmental pathway to become an ovary or a testis. If a testis develops, it secretes testosterone, which supports the development of the male ducts. It also makes other hormones that force the presumptive uterus and Fallopian tubes to shrink away. If the gonad becomes an ovary, it makes oestrogen, and the lack of testosterone causes the male plumbing to wither. The sex hormones also dictate the development of the external genitalia, and they come into play once more at puberty, triggering the development of secondary sexual characteristics such as breasts or facial hair.

Source: www.nature.com

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Allogeneic Transfusion Reactions

Posted in Coagulation Therapy and Internal Bleeding, Hematology, Hematopoiesis, Human Immune System in Health and in Disease, Immuno-Oncology & Genomics, Immunodiagnostics on February 18, 2015| Leave a Comment »

Allogeneic Transfusion Reactions

Writer and Curator: Larry H. Bernstein, MD, FCAP

Introduction

Transfusion Medicine owes much to the discovery of the ABO Blood Groups
by Landsteiner. This was a landmark in the history of immunology.  The next
important discovery was the RhD system in immune hemolytic anemia of the
newborn, which was important in the history of neonatology.  As blood banking
became critical in the preparation, storage, transport, and transfusion of blood
during the Second World War, there was the elucidation of the Kell, MNSs,
Kidd, Duffy, Lewis, and other red cell antigenic systems.  The type and screen
for antibodies, and for red cell compatibility in the crossmatch became routine,
and a formal classification of the antigenic components was created.
Incompatibilities in transfusion mediated reactions were expressed as
allogeneic transfusion reactions.  The red cell was not the only component,
as whole blood was ultimately broken into red cell units, plasma, and platelets,
and eventually a neutrophilic component.  With the development of solid organ
transplantation, the definition of the HLA antigen was a large focus for
compatibility.  Today, the traditional problems of transfusion immuno-compatibility have become displaced in large measure by transplantation
issues.  This piece and that which follows will address allogeneic
transplantation reactions and graft-versus-host disease.

Blood Groups

Although all blood is made of the same basic elements, not all blood is
alike. In fact, there are eight different common blood types, which are
determined by the presence or absence of certain antigens – substances that can trigger an immune response if they are foreign
to the body. Since some antigens can trigger a patient’s immune system
to attack the transfused blood, safe blood transfusions depend on careful
blood typing and cross-matching.

There are four major blood groups determined by the presence or absence
of two antigens – A and B – on the surface of red blood cells:

Group A – has only the A antigen on red cells (and B antibody in the plasma)
Group B – has only the B antigen on red cells (and A antibody in the plasma)
Group AB – has both A and B antigens on red cells (but neither A nor B
antibody in the plasma)
Group O – has neither A nor B antigens on red cells (but both A and B
antibody are in the plasma)

There are very specific ways in which blood types must be matched for a
safe transfusion. See the chart below:

http://www.redcrossblood.org/sites/arc/files/images/ab_top.gif

In addition to the A and B antigens, there is a third antigen called the
Rh factor, which can be either present (+) or absent ( – ). In general,
Rh negative blood is given to Rh-negative patients, and Rh positive blood
or Rh negative blood may be given to Rh positive patients.

O positive is the most common blood type. Not all ethnic groups have the
same mix of these blood types. Hispanic people, for example, have a relatively
high number of O’s, while Asian people have a relatively high number of B’s.
The mix of the different blood types in the U.S. population is:

	Caucasians	African American	Hispanic	Asian
O +	37%	47%	53%	39%
O –	8%	4%	4%	1%
A +	33%	24%	29%	27%
A –	7%	2%	2%	0.5%
B +	9%	18%	9%	25%
B –	2%	1%	1%	0.4%
AB +	3%	4%	2%	7%
AB –	1%	0.3%	0.2%	0.1%

Some patients require a closer blood match than that provided by the ABO
positive/negative blood typing. For example, sometimes if the donor and
recipient are from the same ethnic background the chance of a reaction
can be reduced. That’s why an African-American blood donation may be
the best hope for the needs of patients with sickle cell disease, 98 percent
of whom are of African-American descent.

http://www.redcrossblood.org/learn-about-blood/blood-types

Whether your blood group is type A, B, AB or O is based on the blood types
of your mother and father.

This chart shows the potential blood types you may inherit.

Parent 1

Parent 2

Possible
blood
type
of
child

A blood type (also called a blood group) is a classification of blood based on
the presence or absence of inherited antigenic substances on the surface of
red blood cells (RBCs). These antigens may be proteins, carbohydrates,
glycoproteins, or glycolipids, depending on the blood group system. Some of
these antigens are also present on the surface of other types of cells of
various tissues. Several of these red blood cell surface antigens can stem
from one allele (or very closely linked genes) and collectively form a blood
group system. Blood types are inherited and represent contributions from
both parents. A total of 33 human blood group systems are now recognized
by the International Society of Blood Transfusion (ISBT). The two most
important ones are ABO and the RhD antigen; they determine someone’s
blood type (A, B, AB and O, with +, − or Null denoting RhD status).

Many pregnant women carry a fetus with a blood type which is different from
their own, and the mother can form antibodies against fetal RBCs. Sometimes
these maternal antibodies are IgG, a small immunoglobulin, which can cross
the placenta and cause hemolysis of fetal RBCs, which in turn can lead to
hemolytic disease of the newborn called erythroblastosis fetalis, an illness
of low fetal blood counts that ranges from mild to severe. Sometimes this is
lethal for the fetus; in these cases it is called hydrops fetalis.

ABO_blood_type.svg

http://upload.wikimedia.org/wikipedia/commons/thumb/3/32/ABO_blood_type.svg/824px-ABO_blood_type.svg.png

Blood type (or blood group) is determined, in part, by the ABO blood group antigens

A complete blood type would describe a full set of 30 substances on the surface
of RBCs, and an individual’s blood type is one of many possible combinations
of blood-group antigens. Across the 33 blood groups, over 600 different blood-
group antigens have been found, but many of these are very rare, some being
found mainly in certain ethnic groups.

Almost always, an individual has the same blood group for life, but very rarely
an individual’s blood type changes through addition or suppression of an
antigen in infection, malignancy, or autoimmune disease. Another more
common cause in blood type change is a bone marrow transplant. Bone-marrow transplants are performed for many leukemias and lymphomas,
among other diseases. If a person receives bone marrow from someone
who is a different ABO type (e.g., a type A patient receives a type O bone
marrow), the patient’s blood type will eventually convert to the donor’s type.

Some blood types are associated with inheritance of other diseases; for example,
the Kell antigen is sometimes associated with McLeod syndrome. Certain
blood types may affect susceptibility to infections, an example being the
resistance to specific malaria species seen in individuals lacking the Duffy
antigen. The Duffy antigen, presumably as a result of natural selection, is
less common in ethnic groups from areas with a high incidence of malaria.

The Rh system (Rh meaning Rhesus) is the second most significant blood-
group system in human-blood transfusion with currently 50 antigens.
The most significant Rh antigen is the D antigen, because it is the most
likely to provoke an immune system response of the five main Rh antigens.
It is common for D-negative individuals not to have any anti-D IgG or
IgM antibodies, because anti-D antibodies are not usually produced
by sensitization against environmental substances. However, D-negative
individuals can produce IgG anti-D antibodies following a sensitizing event:
possibly a fetomaternal transfusion of blood from a fetus in pregnancy or
occasionally a blood transfusion with D positive RBCs. Rh disease can
develop in these cases. Rh negative blood types are much less common
in proportion of Asian populations (0.3%) than they are in White (15%).
The presence or absence of the Rh antigens is signified by the + or − sign,
so that for example the A− group does not have any of the Rh antigens.

http://en.wikipedia.org/wiki/Blood_type

Allogeneic blood transfusions: benefit, risks and clinical indications
in countries with a low or high human development index

Carlos Marcucci, Caveh Madjdpour and Donat R. Spahn
Br Med Bull (2004) 70 (1): 15-28. http://dx.doi.org:/10.1093/bmb/ldh023

The risks associated with allogeneic red blood cell (RBC) transfusions differ
significantly between countries with low and high human development indexes
(HDIs). In countries with a low HDI, the risk of infection (HIV, HBV, HCV and
malaria) is elevated. In contrast, in countries with a high HDI, immunological
reactions (hemolytic transfusion reactions, alloimmunization and immuno-suppression) are predominant. Therefore the overall risk associated with RBC
transfusions in low HDI countries is much more significant than that in high HDI
countries. In view of these risks, the limited efficacy of RBC transfusion and its
high costs, this procedure should be used sparingly and rationally.

Red blood cell (RBC) transfusions originating from an unrelated donor are known
as allogeneic RBC transfusions. In the West, i.e. in countries with a high human
development index (HDI), which is an index based on life expectancy, literacy,
enrolment in further education and per capita income, >50% of RBC transfusions
are used in trauma and surgery to compensate for major blood loss.

RBC transfusions are certainly beneficial in specific situations, but are accompanied
by many risks and side effects. Several recent studies have suggested that RBC
transfusions are associated with major adverse outcomes and high costs. In addition,
RBC transfusions are a limited resource and blood shortages can occur at times.

The risk of viral transmission via RBC transfusions has decreased considerably
in recent years in high HDI countries, although new transfusion-transmitted
viruses have been discovered.14 In contrast, in countries with medium or low
HDIs, the risk of transmission of infectious diseases may still be extremely high.

Most reviews consider risks on transfusion-transmissible infections and
immunological reactions, associated with RBC transfusions, that are only
applicable to Western countries, i.e. countries with a high HDI. Although 83%
of the global population live in countries with medium and low HDIs, they
have access to only 40% of the global blood supply. Most notably, all blood
donations in high HDI countries are screened for transfusion-transmissible
infections, whereas only 57% of blood donations in medium and low HDI
countries are tested. In addition, the tests used for blood screening are
not always comparable. For example, only four of the 19 countries
participating in the ‘Workshop of the Directors of National Blood Transfusion
Services’, held in Harare, Zimbabwe, in 2000, used p24 antigen testing for
HIV blood screening.18 Moreover, the blood donation rate per 1000 population
is almost 20 times higher in developed countries than in countries with a low HDI.
Regular non-remunerated volunteers, who are the safest donors, provide 98%
of donations in high HDI countries. In contrast, such donors are a minority in
low HDI countries where up to 60% of donated blood comes from relatives of
the anemic patient or from paid donors.

Acute extravascular hemolytic transfusion reaction due to
anti-Kpa antibody missed by electronic crossmatch

Ruth Padmore, Philip Berardi, …, Doris Neurath, Elianna Saidenberg
Transfusion and Apheresis Science 51 (2014) 168–171
http://dx.doi.org/10.1016/j.transci.2014.08.011

Background: Kpa antigen is a low incidence red blood cell antigen within the Kell
system. Anti-Kpa alloantibody may be associated with acute and delayed hemolytic
transfusion reactions.
Case Study: We report a case of a clinically significant acute extravascular
hemolytic transfusion reaction mediated by previously unrecognized (and
undetected) anti-Kpa alloantibody. This reaction occurred in a patient who
met all criteria for electronic crossmatch, resulting in the transfusion of an
incompatible red cell unit.
Results: Post-transfusion investigation showed the transfused red cell unit
was crossmatch compatible at the immediate spin phase but was 3 + incompatible
at the antiglobulin phase.No evidence of intravascular hemolysis was observed
upon visual comparison of the pre and post-transfusion peripheral blood plasma.
Further testing showed the presence of anti-Kpa antibody. The clinical course
of the patient included acute febrile and systemic reaction.
Conclusion: Acute extravascular hemolytic transfusion reaction may occur due
to undetected anti-Kpa alloantibody. Various strategies for crossmatching are
discussed in the context of antibodies to low incidence antigens.

Transfusion-related mortality: the ongoing risks of allogeneic blood
transfusion and the available strategies for their prevention

Eleftherios C. Vamvakas and Morris A. Blajchman
Blood. 2009; 113: 3406-3417
http://dx.doi.org:/10.1182/blood-2008-10-167643

As the risks of allogeneic blood transfusion (ABT)–transmitted viruses were
reduced to exceedingly low levels in the US, transfusion-related acute lung injury
(TRALI), hemolytic transfusion reactions (HTRs), and transfusion-associated
sepsis (TAS) emerged as the leading causes of ABT related deaths. Since 2004,
preventive measures for TRALI and TAS have been implemented, but their
implementation remains incomplete. Infectious causes of ABT-related deaths
currently account for less than 15% of all transfusion-related mortality, but the
possibility remains that a new transfusion-transmitted agent causing a fatal
infectious disease may emerge in the future. Aside from these established
complications of ABT, randomized controlled trials comparing recipients of
non–white blood cell (WBC)–reduced versus WBC-reduced blood components
in cardiac surgery have documented increased mortality in association with
the use of non-WBC–reduced ABT. ABT-related mortality can thus be further
reduced by universally applying the policies of avoiding prospective donors
alloimmunized to WBC antigens from donating plasma products, adopting
strategies to prevent HTRs, WBC-reducing components transfused to patients
undergoing cardiac surgery, reducing exposure to allogeneic donors through
conservative transfusion guidelines and avoidance of product pooling, and
implementing pathogen-reduction technologies to address the residual risk of TAS as well as the potential risk of a transfusion transmitted agent to emerge
in the foreseeable future.

Red blood cell-incompatible allogeneic hematopoietic progenitor cell
transplantation

S D Rowley, M L Donato and P Bhattacharyya
Bone Marrow Transplantation (2011) 46, 1167–1185; http://dx.doi.org:/10.1038/bmt.2011.135

Transplantation of hematopoietic progenitor cells from red cell-incompatible
donors occurs in 30–50% of patients. Immediate and delayed hemolytic
transfusion reactions are expected complications of red cell-disparate
transplantation and both ABO and other red cell systems such as Kidd
and rhesus can be involved. The immunohematological consequences of
red cell-incompatible transplantation include delayed red blood cell recovery,
pure red cell aplasia and delayed hemolysis from viable lymphocytes carried
in the graft (‘passenger lymphocytes’). The risks of these reactions, which
may be abrupt in onset and fatal, are ameliorated by graft processing and
proper blood component support. Red blood cell antigens are expressed on
endothelial and epithelial tissues in the body and could serve to increase
the risk of GvHD. Mouse models indicate that blood cell antigens may
function as minor histocompatibility antigens affecting engraftment. Similar
observations have been found in early studies of human transplantation
for transfused recipients, although current conditioning and immuno-suppressive regimens appear to overcome this affect. No deleterious effects
from the use of red cell-incompatible hematopoietic grafts on transplant
outcomes, such as granulocyte and platelet engraftments, the incidences
of acute or chronic GvHD, relapse risk or OS, have been consistently
demonstrated. Most studies, however, include limited number of patients,
varying diagnoses and differing treatment regimens, complicating the
detection of an effect of ABO-incompatible transplantation. Classification
of patients by ABO phenotype ignoring the allelic differences of these
antigens also may obscure the effect of red cell-incompatible transplantation
on transplant outcomes.

Severe hemolytic transfusion reaction due to anti-A1 following allogeneic
stem cell transplantation with minor ABO incompatibility

Çiğdem Akalın Akkok, Håkon Haugaa, Anders Galgerud, Lorentz Brinch
Transfusion and Apheresis Science 2013; 48(1), Pages 63–66
http://dx.doi.org/10.1016/j.transci.2012.07.006

Blood components should be compatible both with the recipient and the
donor in the ABO incompatible allogeneic stem cell transplantation setting.
A patient with blood type A2 received peripheral blood stem cells from a
blood type O donor. The patient was in critical condition due to treatment-
related toxicity. He had acquired anti-A1 that was unfortunately overlooked.
Following transfusion of A1 red blood cells in error, he developed a severe
hemolytic transfusion reaction. Anti-A1 is rarely clinically significant.
We discuss the role of passenger lymphocytes in development of the anti-A1, and stress the importance of investigating unusual/atypical reactions
in blood typing.

Transfusion Support of Allogeneic Stem Cell Transplant Recipients

Kate Chipperfield MD FRCPC 21 Feb 2012

After this session, the learner will be able to:

Provide an overview of transfusion issues in allogeneic stem cell transplant.
Discuss the potential consequences of ABO mismatch between recipient
and donor.
Understand the rationale for ABO/D group selection of blood product support
peri-stem cell transplant.
Appreciate the special impact of umbilical cord blood stem cell transplant.
Briefly outline variable practices in transfusion support of stem cell
transplantation.

Pre-transplant

Leukocyte Reduction

– reduction in HLA Alloimmunization (TRAP study)

– reduction in CMV infection in seronegative candidates*

Avoidance of directed donations
Irradiation of cellular blood products

– Only from start of SCT conditioning (to end of GVHD prophylaxis or
lymphs >1 x 109/L)

Issues with this

– as soon as identified as potential SCT recipient*

– prevention of microchimerism in intended recipient (donor lymphocytes)

ABO and SCT

Any allogeneic SCT will be one of:

– ABO-Identical

– Major ABO Incompatible

– Minor ABO Incompatible

– Major and Minor incompatible (Bidirectional)

(more not shown)

Major ABO Blood Group Mismatch Increases the Risk for Graft Failure
after Unrelated Donor Hematopoietic Stem Cell Transplantation

Mats Remberger, E Watz, O Ringdén, J Mattsson, A Shanwell, A Wikman
Biology of Blood and Marrow Transplantation 13:675-682 (2007)
http://dx.doi.org:/10.1016/j.bbmt.2007.01.084

Two hundred twenty-four patients with leukemia transplanted with an unrelated
donor between 1991 and 2003 at the Karolinska University Hospital were
analyzed according to association between graft failure and ABO, RhD, MNSs,
and Kidd blood group antigen compatibility. Median age was 29 years
(range: 0-55). Conditioning consisted of total-body irridiation or busulfan-based myeloablative conditioning. A bone marrow graft was given to 152
patients, and 72 patients received peripheral blood stem cells. Most patients
received graft-versus-host disease prophylaxis with cyclosporine and MTX.
Graft failure (GF) was seen in 6 (2.7%) patients. In the multivariate analysis
major ABO mismatch (odds ratio [OR] 14.9, 95% confidence interval
[CI] 2.01-110, P = .008) and HLA-allele mismatch (6.42, 1.19-34.8, P = .03)
was significantly associated to GF. In patients with and without major ABO
mismatch the incidence of GF was 7.5% and 0.6% (P = .02), respectively.
Using an ABO major mismatched graft increases the risk for GF after
unrelated donor hematopoietic stem cell transplantation.

Perioperative transfusion-related acute lung injury: The Canadian
Blood Services experience

Asim Alam, Mary Huang, Qi-Long Yi, Yulia Lin, Barbara Hannach
Transfusion and Apheresis Science 50 (2014) 392–398
http://dx.doi.org/10.1016/j.transci.2014.04.008

Purpose: Transfusion-related acute lung injury (TRALI) is a devastating transfusion-associated adverse event. There is a paucity of data on the incidence and
characteristics of TRALI cases that occur perioperatively. We classified
suspected perioperative TRALI cases reported to Canadian Blood Services
between 2001 and 2012, and compared them to non-perioperative cases
to elucidate factors that may be associated with an increased risk of developing
TRALI in the perioperative setting. Methods: All suspected TRALI cases
reported to Canadian Blood Services (CBS) since 2001 were reviewed by
two experts or, from 2006 to 2012, the CBS TRALI Medical Review Group
(TMRG). These cases were classified based on the Canadian Consensus
Conference (CCC) definitions and detailed in a database. Two additional
reviewers further categorized them as occurring within 72 h from the onset of
surgery (perioperative) or not in that period (non-perioperative). Various
demographic and characteristic variables of each case were collected and
compared between groups. Results: Between 2001 and 2012, a total of
469 suspected TRALI cases were reported to Canadian Blood Services;
303 were determined to be within the TRALI diagnosis spectrum. Of those,
112 (38%) were identified as occurring during the perioperative period.
Patients who underwent cardiac surgery requiring cardiopulmonary bypass
(25.0%), general surgery (18.0%) and orthopedics patients (12.5%) represented
the three largest surgical groups. Perioperative TRALI cases comprised more
men (53.6% vs. 41.4%, p = 0.04) than non-perioperative patients. Perioperative
TRALI patients more often required supplemental O2 (14.3% vs. 3.1%, p = 0.0003),
mechanical ventilation (18.8% vs. 3.1%), or were in the ICU (14.3% vs. 3.7%,
p = 0.0043) prior to the onset of TRALI compared to non-perioperative TRALI
patients. The surgical patients were transfused on average more components
than non-perioperative patients (6.0 [SD = 8.3] vs. 3.6 [5.2] products per patient,
p = 0.0002). Perioperative TRALI patients were transfused more plasma (152
vs. 105, p = 0.013) and cryoprecipitate (51 vs. 23, p < 0.01) than nonperioperative
TRALI patients. There was no difference between donor antibody test results
between the groups. Conclusion: CBS data has provided insight into the
nature of TRALI cases that occur perioperatively; this group represents a
large proportion of TRALI cases.

Platelet allo-antibodies identification strategies forpreventing and
managing platelet refractoriness

Basire, C.Picard
Transfusion Clinique et Biologique 21(2014)193–206
http://dx.doi.org/10.1016/j.tracli.2014.08.140

Platelet refractoriness is a serious complication for patients receiving recurrent
platelet transfusions ,which can be explained by non-immune and immune causes.
Human Leukocyte Antigens (HLA) allo-immunization, especially against HLA
class I, is the major cause for immune platelet refractoriness. To a lesser extent,
alloantibodies against specific Human Platelet Antigen (HPA) are also involved.
Pregnancy, transplantation and previous transfusions can lead to allo-immune reaction against platelet antigens. After transfusion, platelet count
is decreased by accelerated platelet destruction related to antibodies
fixation on incompatible platelet antigens. New laboratory tests for allo-antibodies identification were developed to improve sensibility and specificity,
especially with the LUMINEX® technology. The good use and interpretation
of these antibodies assays can improve strategies for platelet refractoriness
prevention and management with a patient adapted response. Compatible
platelets units can be selected according to their identity with recipient
typing or immune compatibility regarding HLA or HPA antibodies or HLA
epitope compatibility. Prospective studies are needed to further confirm the
clinical benefit of new allo-antibodies identification methods and consensus
strategies for immune platelet refractoriness management.

For Anti-HLA-Specific Donor Antibodies Detection By Flow Cytometry
Cytotoxic Crossmatches Comparison of Methods

Cervelli, F. Pisani, A. Aureli, R. Azzarone, .., A. Famulari, and F. Papola Transplantation Proceedings, 45, 2761e2764 (2013)
http://dx.doi.org/10.1016/j.transproceed.2013.07.023

Anti-HLA-specific donor antibodies induce rapid, irreversible destruction of
the transplant (hyperacute rejection) that today happens rarely due to
immunologic studies prospective crossmatch of patients awaiting the kidney
graft. The usual approach for pretransplant donor/recipient evaluation is
based on 2 methods: (1) the cytotoxic complement crossmatch (CDC) and
(2) the flow cytometric crossmatch  (FCX). The CDC crossmatch is positive
when complement-fixing antibodies are present, an absolute contra-
indication to kidney transplantation. The more sensitive FCX-positive
crossmatch detects low concentrations of unable to fix performed
antibodies complement. It is an  “index” of possible damage due to
accelerated rejection. The target of our study was to develop a cytotoxic
flow cytometry crossmatch (cFCX) that detected cytotoxic antibodies
move sensitively  than the traditional CDC method and also was less
subjective and more standardized for interpretation studying sera from
23 patients; the cFCX showed the requested efficiency characteristics even
in an emergency. In addition, the new method permitted one to calculate a
cutoff for positivity (average value of the negative control at + 2 standard
deviations), assuring an “objective” interpretation of the results that agreed
with the CDC but was more sensitive and accurate allowing solution of
ambiguous results for cases of “doubt”-positive CDC crossmatch.
Furthermore, our aim was to correlate the effect of the strength of the anti-HLA
antibodies determined by mean fluorescence intensity value of LabScreen
Single Antigen beads with results of CDC, cFCX, and FCX methods.

Allogeneic Stem Cell Transplants and Associated Incompatibities

Analysis of Donor and Recipient ABO Incompatibility and Antibody-
Associated Complications after Allogeneic Stem Cell Transplantation
with Reduced-Intensity Conditioning

Emma Watz, Mats Remberger, Olle Ringden, Joachim Lundahl, et al.
Biol Blood Marrow Transplant 20 (2014) 264e271
http://dx.doi.org/10.1016/j.bbmt.2013.11.011

Allogeneic hematopoietic stem cell transplantation (HSCT) can be performed
across the ABO blood group barrier. The impact of ABO incompatibility on
clinical outcome is controversial. A retrospective analysis of 310 patients who
underwent HSCT with reduced-intensity conditioning between 1998 and 2011
was performed to investigate the frequency and clinical implications of anti-RBC
antibodies in passenger lymphocyte syndrome (PLS) after minor ABO mismatch
(mm), persistent or recurring recipient type ABO antibodies (PRABO) after major
ABO mm HSCT, and autoimmune hemolytic anemia (AIHA). Transplantation
characteristics and clinical outcome were analyzed by univariate and multivariate
analysis for groups with or without anti-RBC antibodies. ABO blood group
incompatibility did not affect clinical outcome despite an increased requirement
of blood transfusion. Twelve patients with AIHA, 6 patients with PLS, and 12
patients with PRABO post-HSCT were identified. AIHA did not affect overall
survival (OS) or transplant-related mortality (TRM), but patients with AIHA had
a lower incidence of grades II to IV acute graft-versus-host disease (P < .05).
OS in the PLS group was 0% compared with 61% in the whole group receiving
minor ABO mm transplants (P < .001). Comparing PRABO patients with those
receiving a major ABO mm HSCT, the OS was 17% versus 73% (P <.002) and
TRM was 50% versus 21% (P < .03). At our center, PLS after minor ABO mm
and PRABO antibodies after major ABO mm HSCT are significant risk factors
for decreased OS and TRM. Our results suggest that occurrence of unexpected
ABO antibodies after HSCT warrant a wider investigation individual to find the
underlying cause.

Current Trends in Clinical Studies of Allogeneic Hematopoietic Stem Cell
Transplantation

Sophie Pilon, D Jedrysiak, D Sheppard, CN Bredeson, J Tay, DS Allan
Biol Blood Marrow Transplant 21 (2015) 364e381
http://dx.doi.org/10.1016/j.bbmt.2014.09.014

Allogeneic hematopoietic stem cell transplantation (HSCT) is a specialized
intervention performed at select centers worldwide. The extent to which
specific aspects of care in allogeneic HSCT have been studied and the
types of studies performed for different aspects of care remains incompletely
documented. Studies in allogeneic HSCT were systematically identified from
selected high-profile transplant journals between July 2010 and June 2011
and previously reported in a study addressing the definition of clinical outcomes
in HSCT. All articles were retrieved and assessed for study characteristics and
categorized by specific aspects of care related to allogeneic HSCT. One
hundred sixteen articles were retrieved and reviewed in detail by investigators.
The most studied aspect of care was conditioning regimens. Transfusion
practices were the most understudied aspect of care. Interestingly, most
studies included both adult and pediatric patients. Studies involving all
hematological malignancies were encountered more often than disease-
specific studies. Geographically, most patients described in the published reports
were treated only in North America or only in Europe. Most studies were
retrospective (78), and 25 reported on multicenter registry data. Of the 38
prospective studies, 8 were randomized controlled trials (RCTs) and
predominantly focused on prevention and treatment of graft-versus-host disease
(GVHD) and infections. Median follow-up was longer in retrospective registry
studies (54 months) and shortest in RCTs (32 months). The proportion of
positive outcomes in retrospective and prospective studies was remarkably
high (>80% for all categories) and not significantly different across all aspects
of care (P > .05). When comparing RCTs and registry data studies, this proportion
was similar and high (95% and 100%, respectively, P >.05). Our study highlights
the established and important role of retrospective registry studies for many
aspects of care and suggests RCTs may be most relevant for studies on
infectious complications and GVHD.

Efficacy and Long-Term Outcome of Treatment for Pure Red Cell Aplasia
after Allogeneic Stem Cell Transplantation from Major ABO-Incompatible
Donors

Makoto Hirokawa, T Fukuda, K Ohashi, …, H Sakamaki, for The PRCA
Collaborative Study Group
Biol Blood Marrow Transplant 19 (2013) 1026e1032
http://dx.doi.org/10.1016/j.bbmt.2013.04.004

No standard of care for pure red cell aplasia (PRCA) after major ABO-
incompatible hematopoietic stem cell transplantation (HSCT) has been
established. We conducted a retrospective cohort study to learn the
efficacy and outcome of treatment for PRCA. One hundred forty-five
recipients who showed delayed recovery of erythropoiesis and survived
>100 days after transplantation without early disease progression were
selected from 2846 records of major ABO-incompatible transplantation
in the registry database in Japan, and detailed data of 46 recipients
were collected. Treatment of PRCA, such as rapid tapering of calcineurin
inhibitors, corticosteroids, or additional immunosuppressants, was given
to 22 patients but not to the other 24 patients. The overall response rate
of the treatment group was 54.5%. The number of days from diagnosis of
PRCA to recovery of reticulocytes >1% and the cumulative number of red
blood cell transfusions were not significantly different between the 2 groups.
Infections accounted for the death of 7 of 11 patients in the treatment group.
Univariate analysis identified 5 variables influencing survival, including graft-
versus-host disease, disease progression, and treatment of PRCA; disease
progression remained as the only factor negatively affecting survival by
multivariate analysis. The present study could not provide supportive
evidence for the beneficial effects of treatment for PRCA after major
ABO-mismatched HSCT.

Current therapy of myelodysplastic syndromes

Amer M. Zeidan, Yuliya Linhares, Steven D. Gore
Blood Reviews 27 (2013) 243–259
http://dx.doi.org/10.1016/j.blre.2013.07.003

After being a neglected and poorly-understood disorder for many years, there
has been a recent explosion of data regarding the complex pathogenesis of
myelodysplastic syndromes (MDS). On the therapeutic front, the approval of
azacitidine, decitabine, and lenalidomide in the last decade was a major
breakthrough. Nonetheless, the responses to these agents are limited and
most patients progress within 2 years. Allogeneic stem cell transplantation
remains the only potentially curative therapy, but it is associated with significant
toxicity and limited efficacy. Lack or loss of response after standard therapies
is associated with dismal outcomes. Many unanswered questions remain
regarding the optimal use of current therapies including patient selection,
response prediction, therapy sequencing and combinations, and management
of resistance. It is hoped that the improved understanding of the underpinnings
of the complex mechanisms of pathogenesis will be translated into novel
therapeutic approaches and better prognostic/predictive tools that would
facilitate accurate risk-adaptive therapy.

Donor Selection for Killer Immunoglobulin-like Receptors B Haplotype
of the Centromeric Motifs Can Improve the Outcome after HLA-Identical
Sibling Hematopoietic Stem Cell Transplantation

Huifen Zhou, Xiaojing Bao, …, Miao Wang, Depei Wu, Jun He
Biol Blood Marrow Transplant 20 (2014) 98e105
http://dx.doi.org/10.1016/j.bbmt.2013.10.017

After hematopoietic stem cell transplantation (HSCT), natural killer (NK) cell
alloreactivity in HLA cells of recipients is regulated by killer immunoglobulin-like
receptors (KIRs) on donor NK cells. The effect of KIRs on HSCT outcomes
is controversial, particularly in those undergoing HLA-identical sibling HSCT.
In this study, effects of KIR and HLA genotypes on the HSCT outcome were
investigated in a 5-year retrospective study comprising 219 patient-donor pairs
undergoing HLA-identical sibling HSCT for myeloid and lymphoid malignancies.
We found that 39.7% (87 of 219) of these pairs, which were KIR mismatched,
had better overall survival (OS) and reduced grade III to IV acute graft-versus-
host disease (aGVHD), especially in acute myeloid leukemia (AML) patients.
Bx1 donor KIR genotype with haplotype B on a telomeric region was a risk
factor for the OS and relapse-free survival (RFS). Donor centromeric (c) and
telomeric (t) KIR haplotype analysis showed that donor KIR cB-tA/tB was
associated with improved OS and RFS compared with cA-tA or cA-tB.
Furthermore, donor KIR B haplotype of the centromeric motifs (Cen-B) was
an independent beneficial factor in improving OS and RFS and in protecting
from relapse after HSCT. In AML patients, the occurrence of a GVHD was
significantly lower in HLA-C1 group compared with that in HLA-C2 group,
although such effect was not observed in patients with acute lymphoblastic
leukemia or chronic myelogenous leukemia. Our results suggest that KIR
could impact outcome and donor KIR haplotype with Cen-B confer significant
survival benefits to HLA-identical sibling HSCT.

TEL-AML1 Corrupts Hematopoietic Stem Cells to Persist in the Bone
Marrow and Initiate Leukemia

Jeffrey W. Schindler, D Van Buren, A Foudi, O Krejci, J Qin, SH Orkin, and H Hock
Cell Stem Cell 5, 43–53, July 2, 2009
http://dx.doi.org:/10.1016/j.stem.2009.04.019

The initial steps in the pathogenesis of acute leukemia remain incompletely
understood. The TELAML1 gene fusion, the hallmark translocation in Childhood
Acute Lymphoblastic Leukemia and the first hit, occurs years before the clinical
disease, most often in utero. We have generated mice in which TEL-AML1
expression is driven from the endogenous promoter and can be targeted to
specific populations. TEL-AML1 renders mice prone to malignancy after
chemical mutagenesis when expressed in hematopoietic stem cells (HSCs),
but not in early lymphoid progenitors. We reveal that TEL-AML1 markedly
increases the number of HSCs and predominantly maintains them in the
quiescent (G0) stage of the cell cycle. TEL-AML1+ HSCs retain self renewal
properties and contribute to hematopoiesis, but fail to out-compete normal
HSCs. Our work shows that stem cells are susceptible to subversion by weak
oncogenes that can subtly alter their molecular program to provide a latent
reservoir for the accumulation of further mutations.

Factors Affecting the Outcome of Related Allogeneic Hematopoietic
Cell Transplantation in Patients with Fanconi Anemia

Mouhab Ayas, K Siddiqui, A Al-Jefri, , …, A Al-Musa, A Al-Seraihy
Biol Blood Marrow Transplant 20 (2014) 1599e1603
http://dx.doi.org/10.1016/j.bbmt.2014.06.016

Hematopoietic cell transplantation (HCT) can cure bone marrow failure in
patients with Fanconi Anemia (FA), and it is generally accepted that these
patients should receive low-intensity conditioning because of the underlying
DNA repair defect in their cells. Outcomes for recipients of matched related
HCT have generally been favorable, but only a few studies have scrutinized
the factors that may affect the eventual outcome of these patients. This
retrospective analysis of 94 pediatric patients with FA who underwent related
HCT at King Faisal Specialist Hospital & Research Center was carried out to
attempt to identify factors that may affect outcome. Results showed overall
survival (OS) probabilities of 92.5%, 89%, and 86% at 1, 5, and 10 years,
respectively. In univariate analysis, use of higher dose cyclophosphamide
(CY) (60 mg/kg) conditioning was associated with a better 10-year OS than
lower dose CY (20 mg/kg) conditioning (91% versus 82%, respectively;
P < .035), and use of radiation-containing regimens was associated with a
significantly lower 10-year OS than nonradiation regimens (76% versus 91%,
respectively; P < .005). Of the 4 regimens used in this study, the fludarabine-
based regimen was associated with the highest survival (95.2%; P < .034).
The use of the higher dose CY (60 mg/kg) was associated with a
significantly increased incidence of hemorrhagic cystitis (HC) (20% versus 5.6%
respectively; P < .049). Three patients (3%) developed squamous cell carcinoma
(2 oropharyngeal and 1 genitourinary), at 9.4, 5.4, and 13.3 years after HCT;
2 of them had radiation containing conditioning. In conclusion, our data suggest
that although using a higher dose CY (60 mg/kg)
conditioning regimen may be associated with better survival, it is also associated
with a significantly increased risk of HC. The addition of fludarabine to the low-dose
CY (20 mg/kg) is associated with the best survival. On the other hand, radiation-
containing regimens are associated with significantly lower survival.

Donor Cell Leukemia: A Review

Daniel H. Wiseman
Biol Blood Marrow Transplant 17: 771-789 (2011)
http://dx.doi.org:/10.1016/j.bbmt.2010.10.010

Relapse of acute leukemia following hematopoietic stem cell transplantation
(HSCT) usually represents return of an original disease clone, having evaded
eradication by pretransplant chemo-/radiotherapy, conditioning, or posttransplant
graft-versus-leukemia (GVL) effect. Rarely, acute leukemia can develop
de novo in engrafted cells of donor origin. Donor cell leukemia (DCL) was
first recognized in 1971, but for many years, the paucity of reported cases
suggested it to be a rare phenomenon. However, in recent years, an upsurge
in reported cases (in parallel with advances in molecular chimerism monitoring)
suggest that it may be significantly more common than previously appreciated;
emerging evidence suggests that DCL might represent up to 5% of all post-
transplant leukemia ‘‘relapses.’’ Recognition of DCL is important for several
reasons. Donor-derivation of the leukemic clone has implications when selecting
appropriate therapy, because seeking to enhance an allogeneic GVL effect
would intuitively not have the same role as in standard recipient-derived
relapses. There are also broader implications for donor selection and workup,
particularly given the growing popularity of nonmyeloblative HSCTand
corresponding rising age of the potential donor pool. Identification of DCL
raises potential concerns over future health of the donor, posing ethical
dilemmas regarding responsibilities toward donor notification (particularly
in the context of cord blood transplantation). The entity of DCL is also of
research interest, because it might provide a unique human model for studying
the mechanisms of leukemogenesis in vivo. This review presents and collates
all reported cases of DCL, and discusses the various strategies, controversies,
and pitfalls when investigating origin of posttransplant relapse. Putative etiologic
factors and mechanisms are proposed, and attempts made to address the
difficult ethical questions posed by discovery of donor-derived malignancy
within a HSCT recipient.

Feasible Outcomes of T Cell-Replete Haploidentical Stem Cell
Transplantation with Reduced-Intensity Conditioning in Patients
with Myelodysplastic Syndrome

Seung-Hwan Shin, Jung-Ho Kim, Young-Woo Jeon, …,, Woo-Sung Min,
Yoo-Jin Kim, Je-Hwan Lee
Biol Blood Marrow Transplant 21 (2015) 342e349
http://dx.doi.org/10.1016/j.bbmt.2014.10.031

Even with the recent optimization of haploidentical stem cell transplantation
(SCT), its role for patients with myelodysplastic syndrome (MDS) or acute
myeloid leukemia evolving from MDS (sAML) should be validated. We
analyzed the outcomes of consecutive 60 patients with MDS or sAML
who received T cell-replete haploidentical SCT after reduced-intensity
conditioning with fludarabine, busulfan, and rabbit antithymocyte globuline
800 cGy total body irradiation. Patients achieved a rapid neutrophil
engraftment after a median of 12 days (range, 8 to 23) and an early
immune reconstitution without high incidences of acute graft-versus-host
disease (GVHD) II to IV and chronic GVHD (36.7% and 48.3%, respectively).
After a median follow-up of 4 years, incidence of relapse and nonrelapse mortality
and rate of overall survival and disease-free survival was 34.8%, 23.3%, 46.8%,
and 41.9%, respectively. In multivariate analysis, the disease status at peak was
a significant predictor for relapse (lower-risk MDS versus higher-risk MDS or sAML;
hazard ratio [HR], 5.69; 95% confidence interval [CI], 1.45 to 22.29; P <.013)
and disease-free survival (HR, 4.44; 95% CI, 1.14 to 17.34; P <.032). Chronic
GVHD was an additional significant predictor for relapse (no versus yes; HR,
2.87; 95% CI, 1.03 to 7.51; P <.043). Our T cell-replete haploidentical SCT
may be a feasible option for patients with MDS and sAML without conventional
donors.

Extramedullary Relapse of Acute Leukemia after Allogeneic Hematopoietic
Stem Cell Transplantation: Different Characteristics between Acute
Myelogenous Leukemia and Acute Lymphoblastic Leukemia

Ling Ge, Fan Ye, X Mao, …, C Ruan, Depei Wu, Xiaowen Tang
Biol Blood Marrow Transplant 20 (2014) 1040e1047
http://dx.doi.org/10.1016/j.bbmt.2014.03.030

Extramedullary relapse (EMR) of acute leukemia (AL) after allogeneic
hematopoietic stem cell transplantation (allo-HSCT) is a contributor to post-
transplantation mortality and remains poorly understood, especially the
different characteristics of EMR in patients with acute myelogenous
leukemia (AML) and those with acute lymphoblastic leukemia (ALL).
To investigate the incidence, risk factors, and clinical outcomes of EMR
for AML and ALL, we performed a retrospective analysis of 362 patients
with AL who underwent allo-HSCT at the First affiliated Hospital of Soochow
University between January 2001 and March 2012. Compared with patients
with AML, those with ALL had a higher incidence of EMR (12.9% versus
4.6%; P < .009). The most common site of EMR was the central nervous
system, especially in the ALL group. Multivariate analyses identified the
leading risk factors for EMR in the patients with AML as advanced disease
status at HSCT, hyperleukocytosis at diagnosis, history of extramedullary
leukemia before HSCT, and a total body irradiationebased conditioning
regimen, and the top risk factors for EMR in the patients with ALL as
hyperleukocytosis at diagnosis, adverse cytogenetics, and transfusion
of peripheral blood stem cells. The prognosis for EMR of AL is poor,
and treatment options are very limited; however, the estimated 3-year
overall survival (OS) was significantly lower in patients with AML
compared with those with ALL (0 versus 18.5%; P < .000). The
characteristics of post-allo-HSCT EMR differed between the patients
with AML and those with ALL, possibly suggesting different pathogenetic
mechanisms for EMR of AML and EMR of ALL after allo-HSCT; further
investigation is needed.

French Multicenter 22-Year Experience in Stem Cell Transplantation
for Beta-Thalassemia Major: Lessons and Future Directions

Claire Galambrun, C Pondarré, Yves Bertrand, …,C Badens, I Thuret, for the
French Rare Disease Center for Thalassemia and the French Society of Bone
Marrow Transplantation
Biol Blood Marrow Transplant 19 (2013) 62e68
http://dx.doi.org/10.1016/j.bbmt.2012.08.005

Although hematopoietic stem cell transplantation (HSCT) offers curative
potential for beta-thalassemia major (beta-TM), it is associated with a
variable but significant incidence of graft rejection. We studied the French
national experience for improvement over time and the potential benefit
of antithymocyte globulin (ATG). Between December 1985 and December
2007, 108 patients with beta-TM underwent HSCT in 21 different French
transplantation centers. The majority of patients received a matched sibling
transplant (n = 96) and a busulfan- and cyclophosphamide-based conditioning
regimen (n = 95), also with ATG in 57 cases. Ninety five of the 108 patients
survived, with a median follow-up of 12 years. Probabilities of 15-year survival
and thalassemia-free survival after first HSCT were 86.8% and 69.4%, respectively.
Graft failure occurred in 24 patients, 11 of whom underwent a second HSCT.
The use of ATG was associated with a decrease in rejection rate from 35% to 10%.
Thalassemia-free survival improved significantly with time, reaching 83% in the
54 patients undergoing HSCT after 1994 (median time of HSCT). In view of the
increased risk of graft rejection after matched sibling HSCT, current French
national guidelines recommend, for all children at risk for beta-TM, the systematic
addition of ATG to the myeloablative conditioning regimen and special attention
to optimize transfusion and chelation therapy in the pretransplantation period.

Extramedullary Relapse of Acute Myelogenous Leukemia after
Allogeneic Hematopoietic Stem Cell Transplantation: Better
Prognosis Than Systemic Relapse

Melhem Solh, Todd E. DeFor, Daniel J. Weisdorf, Dan S. Kaufman
Biol Blood Marrow Transplant 18: 106-112 (2012)
http://dx.doi.org:/10.1016/j.bbmt.2011.05.023

Allogeneic hematopoietic cell transplantation (HSCT) is considered a curative
treatment for acute myelogenous leukemia (AML). Extramedullary relapse after
HSCT for AML is a rare event and is less well defined than systemic, hematologic
relapse. We retrospectively studied all patients with AML (n = 436) who underwent
HSCT at the University of Minnesota between 1996 and 2008 who developed
either a bone marrow (BM) or extramedullary (EM) relapse, and examined the
incidence and risk factors for BM and EM relapse. Of 128 patients who relapsed
post-HSCT, 25 had relapse in EM sites, either isolated (n = 13) or with concurrent
BM relapse (n = 12). Relapse sites included bone (n = 1), central nervous system
(n=5 6), gastrointestinal (n=5 4), lymphatic (n = 4), skin (n = 5), genitourinary
(n=5 1), pulmonary (n = 1), and soft tissue (n = 3). The time to relapse was longer
in the EM sites (median, 328 days vs 168 days). Patients with EM relapse were
more likely to have had preceding acute graft-versus-host disease (GVHD) (77%
vs 49%; P = .03) or chronic GVHD (46% vs 15%; P < .02) compared with those
with BM relapse. The 6-month survival post-relapse was significantly better in
patients with isolated EM relapse (69%) compared with those with combined
EM and BM relapse (8%) or those with BM relapse alone (27%) (P <.01).
Compared with local therapy alone, systemic therapy yielded better 6-month
survival in patients with EM relapse. This study suggests differing pathogenesis
of BM relapse versus EM relapse of AML after allogeneic HSCT. GVHD and its
accompanying graft-versus-leukemia effect may better protect BM sites, but
patients with EM relapse have better responses to combined therapy and
improved survival compared with those with BM relapse.

Hematopoietic Cell Transplantation for Thalassemia: A Global
Perspective BMT Tandem Meeting 2013

Parinda A. Mehta, Lawrence B. Faulkner
Biol Blood Marrow Transplant 19 (2013) S70eS73
http://dx.doi.org/10.1016/j.bbmt.2012.10.025

Hematopoietic cell transplantation (HCT) remains the sole available curative
option for patients with β-thalassemia major. Expanded and improved supportive
therapies for thalassemia now routinely extend the life span of affected individuals
well into adulthood. Consequently, in regions of the world where this care is
readily available, HCT has been pursued infrequently, in part owing to concerns
about an expected lack of balance between risks and benefits. More recently,
however, recognition of significant health problems in older patients with
thalassemia, along with recognition of increased risks of graft-versus-host
disease (GVHD), graft rejection, and impaired organ function leading to inferior
HCT outcomes in this particular group, seem to be turning the wheels and tipping
the balance again in the direction of consideration for earlier HCTs. In contrast,
in countries where thalassemia is most prevalent (>100,000 new children born
each year in Middle East and southeast Asia), lack of supportive care standards
together with often insufficient access to dedicated health care facilities, results
in the majority of these children not reaching adulthood, further supporting the
need for expanded access to HCT for these patients. The cost of HCT is equivalent
to that of a few years of noncurative supportive care, such that HCT in low-risk
young children with a compatible sibling is justified not only medically and ethically
but also financially. International cooperation can play a major role in increasing
access to safe and affordable HCT in countries where there is a considerable
shortage of transplantation centers. In this article, we review the current status
of bone marrow transplantation for thalassemia major, with particular emphasis
on a global prospective.

Hematopoietic Stem Cell Transplantation in Autoimmune Diseases: The
Ahmedabad Experience

AV Vanikar, PR Modi, RD Patel, KV Kanodia, VR Shah, VB Trivedi, HL Trivedi
Transplantation Proceedings, 39, 703–708 (2007)
http://dx.doi.org:/10.1016/j.transproceed.2007.01.070

Introduction. Autoimmune disease represents a (AD) breakdown of natural
tolerance against autoreactive antigens leading to a high mortality and morbidity.
The reaction is usually polyclonal; T- and B-cell components of the hematopoietic
system are responsible for disease progression. Allogeneic/ autologous
hematopoietic stem cell transplantation (HSCT) are the current modalities
for treating drug-resistant AD. Patients and Methods. We present a single-
center retrospective evaluation of allogeneic HSCT with nonmyelo-ablative,
low-intensity conditioning in nine patients (five males, four females) with
pemphigus vulgaris (PV) and 27 patients with systemic lupus erythematosus
(SLE; 3 males, 24 females). The mean follow-up period was 4.24 years for PV
and 4.9 years for SLE. Cytokine-mobilized HSC from unmatched related donors,
with mean dose of 21.3108 nucleated cells/kg body weight (BW; mean CD34⁺
count, 6 x 10⁶/kg BW) was administered in to the thymus as well as the portal and
peripheral circulations of recipients. Cyclosporine (4 + 1 mg/kg BW per day) and
prednisolone (10 mg/kg BW per day) were administered for 6 months to protect
mixed chimerism. A subset of patients with cross-gender donors were analyzed
for peripheral blood chimerism at 1 month post-HSCT and every 3 months
thereafter. Results. Sustained clinical remission with peripheral lymphohemato-
poietic chimerism of 0.7 + 0.3% was observed in PV, whereas SLE relapsed after
mean of 7.35 months of disease-free interval associated with fall in chimerism
from 5 + 3% to 0.08 + 0.03%. Conclusion. HSCT was effective to achieve early
clinical remission of PV; and in SLE relapsed after a 7.35-month disease-free
interval accompanied by a fall in mixed lymphohematopoietic chimerism.

Hematopoietic Stem Cell Transplantation in Children and Young Adults
with Secondary Myelodysplastic Syndrome and Acute Myelogenous
Leukemia after Aplastic Anemia

Ayami Yoshimi, B Strahm, I Baumann, I Furlan, S Schwarz, …, CM Niemeyer
Biol Blood Marrow Transplant 20 (2014) 421-434
http://dx.doi.org/10.1016/j.bbmt.2013.11.031

Secondary myelodysplastic syndrome and acute myelogenous leukemia
(sMDS/sAML) are the most serious secondary events occurring after immuno-
suppressive therapy in patients with aplastic anemia. Here we evaluate the
outcome of hematopoietic stem cell transplantation (HSCT) in 17 children
and young adults with sMDS/sAML after childhood aplastic anemia. The
median interval between the diagnosis of aplastic anemia and the development
of sMDS/sAML was 2.9 years (range, 1.2 to 13.0 years). At a median age of
13.1 years (range, 4.4 to 26.7 years), patients underwent HSCT with bone
marrow (n = 6) or peripheral blood stem cell (n = 11) grafts from HLA-
matched sibling donors (n = 2), mismatched family donors (n = 2), or
unrelated donors (n = 13). Monosomy 7 was detected in 13 patients. The
preparative regimen consisted of busulfan, cyclophosphamide, and melphalan
in 11 patients and other agents in 6 patients. All patients achieved neutrophil
engraftment. The cumulative incidence of grade II-IV acute graft-versus-host
disease (GVHD) was 47%, and that of chronic GVHD was 70%. Relapse
occurred in 1 patient. The major cause of death was transplant-related
complication (n = 9).Overall survival and event-free survival at 5 years after
HSCT were both 41%.In summary, this study indicates that HSCT is a curative
therapy for some patients with sMDS/sAML after aplastic anemia. Future
efforts should focus on reducing transplantation-related mortality.

Hematopoietic stem cells: An overview

Youssef Mohamed Mosaad
Transfusion and Apheresis Science 51 (2014) 68–82
http://dx.doi.org/10.1016/j.transci.2014.10.016

Considerable efforts have been made in recent years in understanding the
mechanisms that govern hematopoietic stem cell (HSC) origin, development,
differentiation, self-renewal, aging, trafficking, plasticity and trans-differentiation.
Hematopoiesis occurs in sequential waves in distinct anatomical locations during
development and these shifts in location are accompanied by changes in the
functional status of the stem cells and reflect the changing needs of the
developing organism. HSCs make a choice of either self-renewal or committing
to differentiation. The balance between self-renewal and differentiation is
considered to be critical to the maintenance of stem cell numbers. It is still
under debate if HSC can rejuvenate infinitely or if they do not possess ‘‘true”
self-renewal and undergo replicative senescence such as any other somatic
cell. Gene therapy applications that target HSCs offer a great potential for the
treatment of hematologic and immunologic diseases. However, the clinical
success has been limited by many factors. This review is intended to
summarize the recent advances made in the human HSC field, and will
review the hematopoietic stem cell from definition through development
to clinical applications.

HLA epitope based matching for transplantation

René J. Duquesnoy
Transplant Immunology 31 (2014) 1–6
http://dx.doi.org/10.1016/j.trim.2014.04.004

As important risk factors for transplant rejection and failure, HLA antibodies
are now recognized as being specific for epitopes which can be defined
structurally with amino acid differences between HLA alleles. Donor–recipient
compatibility should therefore be assessed at the epitope rather than the
antigen level. HLA Matchmaker is a computer algorithm that considers each
HLA antigen as a series of small configurations of polymorphic residues
referred to as eplets as essential components of HLA epitopes. It includes
epitopes on antigens encoded by all HLA-A, B, C, DR, DQ and DP loci as
well as MICA. HLA epitopes have two characteristics namely antigenicity, i.e.
the reactivity with antibody and immunogenicity, i.e. the ability of eliciting
an antibody response. This article addresses the relevance of determining
epitope-specificities of HLA antibodies, the effect of epitope structure on
technique-dependent antibody reactivity and the identification of acceptable
mismatches for sensitized patients considered for transplantation. Permissible
mismatching for non-sensitized patients aimed to prevent or reduce HLA
antibody responses could consider epitope loads of mismatched antigens and
the recently developed nonself-self paradigm of epitope immunogenicity.

Impact of HLA Mismatch Direction on the Outcome of Unrelated Bone
Marrow Transplantation: A Retrospective Analysis from the Japan
Society for Hematopoietic Cell Transplantation

Junya Kanda, T Ichinohe, S Fuji, Y Maeda, K Ohashi, …, Y Atsuta,
Y Kanda, on behalf of the HLA Working Group of the Japan Society
for Hematopoietic Cell Transplantation
Biol Blood Marrow Transplant 21 (2015) 305e311
http://dx.doi.org/10.1016/j.bbmt.2014.10.015

The relative desirability of an unrelated donor with a bidirectional 1-locus
mismatch (1MM-Bi), a 1-locus mismatch only in the graft-versus-host direction
(1MM-GVH), or a 1-locus mismatch only in the host versus-graft direction
(1MM-HVG) is not yet clear. We analyzed adult patients with leukemia or
myelodysplastic syndrome who received a first allogeneic stem cell transplant
from an HLA-A, -B, -C, and -DRB1 matched or 1-allele mismatched unrelated
donor in Japan. The effects of 1MM-Bi (n = 1020), 1MM-GVH (n = 83), and
1MM-HVG (n = 83) compared with a zero mismatch (0MM) (n = 2570)
were analyzed after adjusting for other significant variables. The risk of
grades III to IV acute graft-versus-host disease (GVHD) was higher with
marginal significance in the 1MM-GVH group than in the 0MM group
(hazard ratio, 1.85; P = .014). However, there was no significant difference
in overall or nonrelapse mortality between the 1MM-GVH and 0MM groups.
There was no significant difference in acute GVHD or overall or nonrelapse
mortality between the 1MM-HVG and 0MM groups. The risks of acute
GVHD and overall mortality were significantly higher in the 1MM-Bi group
than in the 0MM group. These findings indicate that unrelated donors with
1MM-GVH and 1MM-HVG are both good candidates for patients without an
HLA-matched unrelated donor in a Japanese cohort.

ABO incompatibility between donor and recipient and clinical
outcomes in allogeneic stem cell transplantation

James Goldman, Jane Liesveld, Diane Nichols, Joanna Heal, Neil Blumberg
Leukemia Research 27 (2003) 489–491 PII: S0145-2126(02)00259-X

We performed a retrospective, cohort study to evaluate the impact on recipient
survival of ABO incompatibility between recipient and donor after allogeneic
stem cell transplantation, primarily involving marrow-derived cells. No
statistically significant difference was noted in survival for 153 patients
with acute or chronic leukemia or myelodysplastic syndrome receiving
ABO identical or ABO mismatched allografts. Five patients who had
allografts that were bidirectionally incompatible (both donor cells and
plasma incompatible) did have significantly poorer survival than the other
recipients, similar to the experience reported in one other cohort study.
However, these patients had other risks for mortality, including being
older and receiving transplants from matched, unrelated donors. Our
data do not support a significant role for ABO donor–recipient matching in
allogeneic stem cell transplantation.

Allogeneic Hematopoietic Cell Transplantation Outcomes in
Acute Myeloid Leukemia: Similar Outcomes Regardless of
Donor Type

Erica D. Warlick, RP de Latour, R Shanley, …, Gerard Socie
Biol Blood Marrow Transplant 21 (2015) 357e363
http://dx.doi.org/10.1016/j.bbmt.2014.10.030

The use of alternative donor transplants is increasing as the transplantation-
eligible population ages and sibling donors are less available. We
evaluated the impact of donor source on transplantation outcomes for
adults with acute myeloid leukemia undergoing myeloablative (MA) or
reduced-intensity conditioning (RIC) transplantation. Between January 2000
and December 2010, 414 consecutive adult patients with acute myeloid
leukemia in remission received MA or RIC allogeneic transplantation from
either a matched related donor (n = 187), unrelated donor (n = 76), or
umbilical cord blood donor (n = 151) at the University of Minnesota or
Hôpital St. Louis in Paris. We noted similar 6-year overall survival
across donor types: matched related donor, 47% (95% confidence interval
[CI], 39% to 54%); umbilical cord blood, 36% (95% CI, 28% to 44%);
matched unrelated donor, 54% (95% CI, 40% to 66%); and mismatched
unrelated donor, 51% (95% CI, 28% to 70%) (P < .11). Survival differed
based on conditioning intensity and age, with 6-year survival of 57% (95% CI,
47% to 65%), 39% (95% CI, 28% to 49%), 23% (95% CI, 6% to 47%), 47%
(95% CI, 36% to 57%), and 28% (95% CI, 17% to 41%) for MA age 18 to 39,
MA age 40þ, or RIC ages 18 to 39, 40 to 56, and 57 to 74, respectively
(P < .01). Relapse was increased with RIC and lowest in younger patients
receiving MA conditioning (hazard ratio, 1.0 versus 2.5 or above for all RIC
age cohorts), P <.01. Transplantation-related mortality was similar across
donor types. In summary, our data support the use of alternative donors as
a graft source with MA or RIC for patients with acute myeloid leukemia
when a sibling donor is unavailable.

A Novel Reduced-Intensity Conditioning Regimen for Unrelated Umbilical
Cord Blood Transplantation in Children with Nonmalignant Diseases

Suhag H. Parikh, A Mendizabal, CL Benjamin, KV Komandur, J Antony, et al.
Biol Blood Marrow Transplant 20 (2014) 326e336
http://dx.doi.org/10.1016/j.bbmt.2013.11.021

Reduced-intensity conditioning (RIC) regimens have the potential to decrease
transplantation-related morbidity and mortality. However, engraftment
failure has been prohibitively high after RIC unrelated umbilical cord blood
transplantation (UCBT) in chemotherapy-naïve children with nonmalignant
diseases (NMD). Twenty-two children with a median age of 2.8 years, many
with severe comorbidities and prior viral infections, were enrolled in a novel RIC
protocol consisting of hydroxyurea, alemtuzumab, fludarabine, melphalan, and
thiotepa followed by single UCBT. Patients underwent transplantation for
inherited metabolic disorders (n = 8), primary immunodeficiencies (n = 9),
hemoglobinopathies (n = 4) and Diamond Blackfan anemia (n = 1). Most
umbilical cord blood (UCB) units were HLA-mismatched with median infused
total nucleated cell dose of 7.9 107/kg. No serious organ toxicities were
attributable to the regimen. The cumulative incidence of neutrophil engraftment
was 86.4% (95% confidence interval [CI], 65% to 100%) in a median of 20
days, with the majority sustaining > 95% donor chimerism at 1 year. Cumulative
incidence of acute graft-versus-host disease (GVHD) grades II to IV and III to IV
by day 180 was 27.3% (95% CI, 8.7% to 45.9%) and 13.6% (95 CI, 0% to 27.6%),
respectively. Cumulative incidence of extensive chronic GVHD was 9.1% (95%
CI, 0% to 20.8%). The primary causes of death were viral infections (n ¼ 3), acute
GVHD (n = 1) and transfusion reaction (n ¼ 1). One-year overall and event-free
survivals were 77.3% (95% CI, 53.7% to 89.8%) and 68.2% (95% CI, 44.6%
to 83.4%) with 31 months median follow-up. This is the first RIC protocol
demonstrating durable UCB engraftment in children with NMD. Future risk-
based modifications of this regimen could decrease the incidence of viral
infections. (www.clinicaltrials.gov/NCT00744692).

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Real-time perfusion imaging more beneficial to men without known CAD – Healio

Posted in Uncategorized on February 18, 2015| Leave a Comment »

In men without known CAD, the addition of perfusion imaging to real-time myocardial contrast echocardiography appears to improve prognostic capability compared with conventional stress echocardiography, according to recent findings.

Source: www.healio.com

See on Scoop.it – Cardiovascular and vascular imaging

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The Hospital Is No Place for a Heart Attack

Posted in Uncategorized on February 18, 2015| Leave a Comment »

Studies show having a heart attack in a hospital is deadlier than coming to the ER after suffering an attack at home or work. A group of 12 hospitals are trying to fix that.

Source: www.wsj.com

See on Scoop.it – Cardiovascular and vascular imaging

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German Database: Poor Prognosis of Critical Limb Ischemia Patients Linked in … – TCTMD

Posted in Uncategorized on February 18, 2015| Leave a Comment »

TCTMD
German Database: Poor Prognosis of Critical Limb Ischemia Patients Linked in …

Source: www.tctmd.com

See on Scoop.it – Cardiovascular and vascular imaging

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Four translational research winners in BiOS competition

Posted in Uncategorized on February 18, 2015| Leave a Comment »

Lunchtime forum at Photonics West rewards four research projects with high clinical potential.

Source: optics.org

See on Scoop.it – Cardiovascular Disease: PHARMACO-THERAPY

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Bacteria jump between species more easily than previously thought

Posted in Infectious Disease & New Antibiotic Targets, Infectious Disease Immunodiagnostics, Innovation in Immunology Diagnostics on February 18, 2015| Leave a Comment »

Bacteria jump between species more easily than previously thought

Reporter: Aviva Lev-Ari, PhD, RN

A new study suggests that bacteria may be able to jump between host species far easier than was previously thought. Researchers discovered that a single genetic mutation in a strain of bacteria infectious to humans enables it jump species to also become infectious to rabbits. The discovery has major implications for how we assess the risk of bacterial diseases that can pass between humans and animals. It is well known that relatively few mutations are required to support the transmission of viruses — such as influenza — from one species to another. Until now it was thought that the process was likely to be far more complicated for bacteria.

Scientists at the universities of CEU Cardenal Herrera (Spain) and Glasgow and Edinburgh (UK) studied a strain of bacteria called Staphylococcus aureus ST121, which is responsible for widespread epidemics of disease in the global rabbit farming industry. The team looked at the genetic make-up of ST121 to work out where the strain originated and the changes that occurred that enabled it to infect rabbits. They found that ST121 most likely evolved through a host jump from humans to rabbits around 40 years ago with a genetic mutation at a single site in the bacterial DNA code the cause for this.

The discovery transforms our understanding of the minimal genetic changes that are required for bacteria to infect different species. ST121 is found in the respiratory tract and on the skin of some people. While it is usually harmless, the bacteria can cause a variety of conditions from minor skin infections to meningitis and sepsis. In rabbits, the bacteria can cause serious skin infections.

Source: www.sciencedaily.com

See on Scoop.it – Cardiovascular Disease: PHARMACO-THERAPY

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Complex nerve-cell signaling traced back to common ancestor of humans and sea anemones

Posted in Cell Biology, Cell Biology, Signaling & Cell Circuits on February 18, 2015| Leave a Comment »

Complex nerve-cell signaling traced back to common ancestor of humans and sea anemones

Reporter: Aviva Lev-Ari, PhD, RN

New research shows that a burst of evolutionary innovation in the genes responsible for electrical communication among nerve cells in our brains occurred over 600 million years ago in a common ancestor of humans and the sea anemone. The research, led by Timothy Jegla, an assistant professor of biology at Penn State University, shows that many of these genes, which when mutated in humans can lead to neurological disease, first evolved in the common ancestor of people and a group of animals called cnidarians, which includes jellyfish, coral, and sea anemones.

“Our research group has been discovering evidence for a long time that most major signaling systems in our neurons are ancient, but we never really knew when they first appeared,” Jegla said. “We had always assumed that we would be able to trace most of these signaling systems to the earliest nervous systems, but in this paper we show that this is not the case. It looks like the majority of these signaling systems first appear in the common ancestor that humans share with jellyfish and sea anemones.”

Electrical impulses in nerve cells are generated by charged molecules known as ions moving into and out of the cell through highly specialized ion-channel proteins that form openings in the cell membrane. The new research focuses on the functional evolution of the genes that encode the proteins for potassium channels — ion channels that allow potassium to flow out of nerve cells, stopping the cell’s electrical impulses. “The channels are critical for determining how a nerve cell fires electrical signals,” said Jegla. “It appears that animals such as sea anemones and jellyfish are using the same channels that shape electrical signals in our brains in essentially the same way.”

“Humans and sea anemones went their separate ways evolutionarily speaking roughly 600 million years ago,” said Jegla, “so we know that the mechanisms we use to generate impulses in our neurons must be at least that old.”

Recent genome sequences from comb jellies, which also have nervous systems, show that they are a more ancient group of animals than sea anemones and might even be the oldest type of animals that are still living today. “When we looked at comb jellies, we found that the potassium channels looked very different — most of the channel types found in humans were missing,” said Jegla. “We could trace only one kind of the human potassium channels that we looked at all the way back to comb jellies, but we find almost all of them in sea anemones.”

Source: science.psu.edu

See on Scoop.it – Cardiovascular Disease: PHARMACO-THERAPY

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Action of Hormones on the Circulation

Posted in Ca2+ triggered activation, Calcium Signaling, Calmodulin, Calmodulin Kinase and Contraction, Cardiovascular Pharmacogenomics, Cardiovascular Research, Cell Biology, Curation, Cytoskeleton, Disease Biology, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Genetics & Pharmaceutical, Genomic Endocrinology, Innovations, Metabolism, Metabolomics, Molecular Genetics & Pharmaceutical, Myocardial Ischemia, Myocardial Metabolism, Nephrology, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Pharmaceutical Discovery, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged brochospasm, curculation, Endothelin Receptors, endothelins, Endothelium, GPR signaling, maocardium, receptor binding, vasoconstriction, vasospasm on February 17, 2015| Leave a Comment »

Action of Hormones on the Circulation

Writer and Curator: Larry H. Bernstein, MD, FCAP

Introduction

This is perhaps the most difficult piece to write, unexpectedly. I have done a careful search for related material using different search phrases. It is perhaps because of the great complexity of the topic, which is inextricably linked to sepsis, the Systemic Inflammatory Response Syndrome SIRS), and is poised differently than the neural innervation of the hormonal response and circulation, as in the previous piece. In the SIRS mechanism, we find a very large factor in glucocorticoids, the cytokine shower (IL-1, IL-6, TNF-α), and gluconeogenesis, with circulatory changes. In this sequence, it appears that we are focused on the arteriolar and bronchial smooth muscle architecture, the adrenal medulla, vasoconstriction and vasodilation, and another set of peptide interactions. This may be concurrent with the other effects described.

Related articles in Pharmaceutical Intelligence Journal:

Endothelial Function and Cardiovascular Disease

Pathologist and Author: Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2012/10/25/endothelial-function-and-cardiovascular-disease/

Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination?

Curator: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/10/19/clinical-trials-results-for-endothelin-system-pathophysiological-role-in-chronic-heart-failure-acute-coronary-syndromes-and-mi-marker-of-disease-severity-or-genetic-determination/

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author and Curator of an Investigator Initiated Study: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator of an Investigator Initiated Study: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

Cardiovascular Disease (CVD) and the Role of Agent Alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

Curator and Investigator Initiated Study: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Innervation of Heart and Heart Rate

Writer and Curator: Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/02/15/innervation-of-heart-and-heart-rate/

αllbβ3 Antagonists As An Example of Translational Medicine Therapeutics

Larry H Bernstein, MD, FCAP, Reporter and curator

http://pharmaceuticalintelligence.com/2013/10/12/%CE%B1llb%CE%B23-antagonists-as-an-example-of-translational-medicine-therapeutics/

Cardiac Contractility & Myocardium Performance: Therapeutic Implications for Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC

http://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differences/

Ca2+-Stimulated Exocytosis: The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-and-calcium-release-related-contractile-dysfunction-ryanopathy/

Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

Advanced Topics in Sepsis and the Cardiovascular System at its End Stage

Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-sepsis-and-the-cardiovascular-system-at-its-end-stage/

For most comprehensive Bibliography on the Ryanodine receptor calcium release channel complex and for FIGURES illustrating the phenomenon, see

Pharmacol Ther. 2009 August; 123(2): 151–177.

http://dx.doi.org:/10.1016/j.pharmthera.2009.03.006

PMCID: PMC2704947

Ryanodine receptor-mediated arrhythmias and sudden cardiac death

Lynda M. Blayney[low asterisk] and F. Anthony Lai

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2704947/

Oxidized Calcium Calmodulin Kinase and Atrial Fibrillation

Author: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/10/26/oxidized-calcium-calmodulin-kinase-and-atrial-fibrillation/

Contributions to cardiomyocyte interactions and signaling

Author and Curator: Larry H Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/10/21/contributions-to-cardiomyocyte-interactions-and-signaling/

Cardiac Contractility & Myocardium Performance: Therapeutic Implications for Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Editor: Justin Pearlman, MD, PhD, FACC, Author and Curator: Larry H Bernstein, MD, FCAP, and Article Curator: Aviva Lev-Ari, PhD, RN

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

Action of hormones on the circulation

Limbic system mechanisms of stress regulation: Hypothalamo-pituitary-adrenocortical axis

James P. Herman, Michelle M. Ostrander, Nancy K. Muelle, Helmer Figueiredo
Prog in Neuro-Psychopharmacol & Biol Psychiatry 29 (2005) 1201 – 1213
http://dx.doi.org:/10.1016/j.pnpbp.2005.08.006

Limbic dysfunction and hypothalamo-pituitary-adrenocortical (HPA) axis dysregulation are key features of affective disorders. The following review summarizes our current understanding of the relationship between limbic structures and control of ACTH and glucocorticoid release, focusing on the hippocampus, medial prefrontal cortex and amygdala. In general, the hippocampus and anterior cingulate/prelimbic cortex inhibit stress-induced HPA activation, whereas the amygdala and perhaps the infralimbic cortex may enhance glucocorticoid secretion. Several characteristics of limbic–HPA interaction are notable: first, in all cases, the role of given limbic structures is both region- and stimulus-specific. Second, limbic sites have minimal direct projections to HPA effector neurons of the paraventricular nucleus (PVN); hippocampal, cortical and amygdalar efferents apparently relay with neurons in the bed nucleus of the stria terminalis, hypothalamus and brainstem to access corticotropin releasing hormone neurons. Third, hippocampal, cortical and amygdalar projection pathways show extensive overlap in regions such as the bed nucleus of the stria terminalis, hypothalamus and perhaps brainstem, implying that limbic information may be integrated at subcortical relay sites prior to accessing the PVN. Fourth, these limbic sites also show divergent projections, with the various structures having distinct subcortical targets. Finally, all regions express both glucocorticoid and mineralocorticoid receptors, allowing for glucocorticoid modulation of limbic signaling patterns. Overall, the influence of the limbic system on the HPA axis is likely the end result of the overall patterning of responses to given stimuli and glucocorticoids, with the magnitude of the secretory response determined with respect to the relative contributions of the various structures.

representations of the HPA axis

Diagrammatic representations of the HPA axis of the rat. HPA responses are initiated by neurosecretory neurons of medial parvocellular paraventricular nucleus (mpPVN), which secretes ACTH secretagogues such as corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) in the hypophysial portal circulation at the level of the median eminence. These secretagogues promote release of ACTH into the systemic circulation, whereby it promotes synthesis and release of glucocorticoids at the adrenal cortex.

When exposed to chronic stress, the HPA axis can show both response Fhabituation_ and response Ffacilitation_. FHabituation_ occurs when the same (homotypic) stressor is delivered repeatedly, and is characterized by progressive diminution of glucocorticoid responses to the stimulus. Systemic administration of a mineralocorticoid receptor antagonist is sufficient to block habituation, implying a role for MR signaling in this process. It should be noted that HPA axis habituation is highly dependent on both the intensity and predictability of the stressful stimulus. FFacilitation_ is observed when animals repeatedly exposed to one stimulus are presented with a novel (heterotypic). In chronically stressed animals, exposure to a novel stimulus results in rise in glucocorticoids that is as large as or greater than that seen in a chronic stress naıve animal. Importantly, facilitation can occur in the context of chronic stress-induced elevations in resting glucocorticoids levels, suggesting that this process involves a bypass or override of negative feedback signals.

Hippocampal regulation of the HPA axis appears to be both region- and stressor-specific. Using a sequential lesion approach, our group has noted that the inhibitory effects of the hippocampus on stress-induced corticosterone release and CRH/AVP mRNA expression are likely subserved by neurons resident in the ventral subiculum-caudotemporal CA1. In addition to spatial specificity, hippocampal regulation of the HPA axis also appears to be specific to certain stress modalities; our studies indicate that ventral subiculum lesions cause elevated glucocorticoid secretion following restraint, open field or elevated plus maze exposure, but not to ether inhalation or hypoxia.

The research posits an intricate topographical organization of prefrontal cortex output to HPA regulatory circuits. The anatomy of medial prefrontal cortex efferents may illuminate this issue. The infralimbic cortex projects extensively to the anterior bed nucleus of the stria terminalis, medial and central amygdala and the nucleus of the solitary tract, all of which are implicated in stress excitation. In contrast, the prelimbic cortex has minimal input to these structures, but projects to the ventrolateral preoptic area, dorsomedial hypothalamus and peri-PVN region, areas implicated in stress inhibition. Thus, the infralimbic and prelimbic/anterior cingulate components of the prefrontal cortex may play very different roles in HPA axis regulation. Like other limbic regions, the influence of the amygdala on the HPA axis is stressor- and region-specific. The medial amygdala shows intense c-fos induction following stressors such as restraint, swimming, predator exposure and social interaction.

Despite the prominent involvement of the hippocampus, medial prefrontal cortex and amygdala in HPA axis regulation, there is limited evidence of direct innervation of the PVN by these structures. Rather, these regions appear to project to a number of basal forebrain, hypothalamic and brainstem cell populations that in turn innervate the medial parvocellular PVN. Thus, in order to access principle stress effector neurons, information from the limbic system requires an intermediary synapse. In the bed nucleus of the stria terminalis and hypothalamus, the majority of these intermediary neurons are GABAergic. For example, the bed nucleus of the stria terminalis, ventrolateral preoptic area, dorsomedial hypothalamic nucleus and peri-PVN region all contain rich populations of neurons expressing the GABA marker glutamatic acid decarboxylase (GAD) 65/67.

The organization of the peri-PVN cell groups is particularly interesting. In the case of the ventral subiculum and to a lesser extent, the medial prefrontal cortex, terminal fields can be observed in the immediate surround of the PVN, corresponding to areas containing substantial numbers of GABA neurons. Importantly, dendrites of PVN neurons are largely confined within the nucleus proper, indicating that limbic afferents are unlikely to interact directly with the PVN neurons themselves. The peri-PVN GABA neurons are activated by glutamate, and likely express glutamate receptor subunits. These neurons also up-regulate GAD65 mRNA following chronic stress, commensurate with involvement in long-term HPA regulation. Injections of a general ionotroptic glutamate receptor antagonist into the PVN surround potentiates glucocorticoid responses to restraint, consistent with blockade of glutamate excitation of these GABA neurons. The data are consistent with an interaction between the excitatory limbic structures and inhibitory PVN-regulatory cells at the level of the PVN surround.

Brainstem stress-modulatory pathways likely relay excitatory information to the PVN. For example, the nucleus of the solitary tract provides both catecholaminergic (norepinephrine) and non-catecholaminergic (e.g., glucagon-like peptide-1 (GLP-1) input to the medial parvocellular. Norepinephrine is released into the PVN following stress and is believed to activate CRH neurons via alpha-1 adrenergic receptors. The role of this pathway is thought to be associated with systemic stressors, as selective destruction of PVN norepinephrine input using anti-dopamine beta hydroxylase-saporin conjugate blocks responses to 2-deoxy-glucose but not restraint. In contrast, blockade of central GLP-1 receptors using exendin 9–36 markedly inhibits responsiveness to both lithium chloride and novelty, suggesting that this non-catecholaminergic cell population may play a more general role in stress integration.

The existence of these putative two-neuron circuits lends important insight into the nature of stress information processing. Anatomical data support the hypothesis that the vast majority of medial prefrontal cortex and ventral subicular inputs to subcortical stress relays are glutamate-containing. As can be appreciated, pyramidal cells of the medial prefrontal cortex and subiculum richly express mRNA encoding vesicular glutamate transporter-1 (VGlut1), a specific marker of glutamate neurons. Combined retrograde tracing/in situ hybridization studies performed in our lab indicate that the vast majority of cortical and hippocampal afferents to PVN-projecting regions (e.g., bed nucleus of the stria terminalis, dorsomedial hypothalamus, ventrolateral medial preoptic area) indeed contain VGlut1, verifying a glutamatergic input to these areas. In contrast, the majority of amygdalar areas implicated in stress regulation express glutamic acid decarboxylase (GAD) 65 or 67 mRNA, suggesting a GABAergic phenotype; indeed, the vast majority of medial and central amygdaloid projections to PVN relays are GABAergic.

representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus

Diagrammatic representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus. The medial prefrontal cortex (mPFC) subsumes neurons of the prelimbic (pl), anterior cingulate (ac) and infralimbic cortices (il), which appear to have different actions on the HPA axis stress response. The pl/ac send excitatory projections (designated as dark circles, filled line with arrows) to regions such as the peri-PVN zone and bed nucleus of the stria terminalis (BST), both of which send direct GABAergic projections to the medial parvocellular PVN (delineated as open circles, dotted lines ending in squares). This two-neuron chain is likely to be inhibitory in nature. In contrast, the infralimbic cortex projects to regions such as the nucleus of the solitary tract (NTS), which sends excitatory projections to the PVN, implying a means of PVN excitation from this cortical region. The ventral subiculum (vSUB) sends excitatory projections to numerous subcortical regions, including the posterior BST, peri-PVN region, ventrolateral region of the medial preoptic area (vlPOA) and ventrolateral region of the dorsomedial hypothalamic nucleus (vlDMH), all of which send GABAergic projections to the PVN and are likely to communicate transsynaptic inhibition. The medial amygdaloid nucleus (MeA) sends inhibitory projections to GABAergic PVN-projecting populations, such as the BST, vlPOA and peri-PVN, eliciting a transsynaptic disinhibition. A similar arrangement likely exists for the central amygdaloid nucleus (CeA), which sends GABAergic outflow to the ventrolateral BST and to a lesser extent, the vlDMH. The CeA also projects to GABAergic neurons in the NTS, which may disinhibit ascending projections to the PVN.

Inotropes and vasopressors: more than haemodynamics!

Hendrik Bracht, E Calzia, M Georgieff, J Singer, P Radermacher and JA Russell
British Journal of Pharmacology (2012) 165 2009–2011
http://dx.doi.org:/10.1111/j.1476-5381.2011.01776.x

Circulatory shock is characterized by arterial hypotension requiring fluid resuscitation combined with inotropes and/or vasopressors to correct the otherwise life-threatening impairment of oxygen supply to peripheral tissues. Catecholamines represent the current therapeutic choice, but this standard is only based on empirical clinical experience. Although there is evidence that some catecholamines may be better than others, it is a matter of debate which one may be the most effective and/or the safest for the different situations. In their review in this issue of the British Journal of Pharmacology, Bangash et al. provide an overview of the pharmacology as well as the available clinical data on the therapeutic use of endogenous catecholamines, their synthetic derivatives and a range of other agents (vasopressin and its analogues, PDE inhibitors and levosimendan). The authors point out that, despite well-established receptor pharmacology, the clinical effects of these treatments are poorly understood. Hence, further investigations are essential to determine which catecholamine, or, in a broader sense, which alternative vasopressor and/or inotrope is the most appropriate for a particular clinical condition.

LINKED ARTICLES This article is a commentary on Bangash et al., pp. 2015–2033 of this issue and is commented on by De Backer and Scolletta, pp. 2012–2014 of this issue. To view Bangash et al. visit http://dx.doi.org/10.1111/j.1476-5381.2011.01588.x and to view De Backer and Scolletta visit http://dx.doi.org/10.1111/j.1476-5381.2011.01746.x

In the present issue of the British Journal of Pharmacology, Bangash et al. (2012) review the pharmacology as well as the available clinical data on the therapeutic use of various inotropes and vasopressor agents used for the hemodynamic management of (septic) shock. By definition, circulatory shock is characterized by arterial hypotension that necessitates immediate intervention to maintain the balance of tissue oxygen supply and demand. In practice, the longer and the more frequent periods of hypotension are present in a patient, the less likely is survival, and early aggressive resuscitation is associated with improved outcome. Besides fluid administration to increase the circulating blood volume, in most cases, vasoactive drugs are required to restore an adequate perfusion pressure, and up to now, catecholamines represent the current therapeutic choice. According to their pharmacological profile, catecholamines are traditionally used for their predominant inotropic, vasodilating or constrictor effects.

Clinicians should not forget two fundamental aspects of catecholamine action. First, because of the ubiquitous presence of adrenoceptors, endogenous catecholamines. as well as their synthetic derivatives, have pronounced effects on virtually all tissues (many of which were described several years ago), in particular on the immune system (van der Poll et al., 1996; Flierl et al., 2008), on energy metabolism (Cori and Cori, 1928; Bearn et al., 1951) and on gastrointestinal motility (McDougal and West, 1954). Second, the adrenoceptor density and responsiveness to catecholamines are markedly altered by both the underlying disease and the ongoing catecholamine. Bangash et al. (2012) have to be commended that they not only describe the various endogenous catecholamines and their synthetic derivatives but also thoroughly discuss possible alternatives, such as vasopressin and its analogues, PDE inhibitors and levosimendan.

Inhibitory effects of cortisone and hydrocortisone on human Kv1.5 channel currents

Jing Yu, Mi-Hyeong Park, Su-Hyun Jo
Eur J Pharmacol 746 (2015) 158–166 http://dx.doi.org/10.1016/j.ejphar.2014.11.007

Glucocorticoids are the primary hormones that respond to stress and protect organisms from dangerous situations. The glucocorticoids hydrocortisone and its dormant form, cortisone, affect the cardiovascular system with changes such as increased blood pressure and cardioprotection. Kv1.5 channels play a critical role in the maintenance of cellular membrane potential and are widely expressed in pancreatic β-cells, neurons, myocytes, and smooth muscle cells of the pulmonary vasculature. We examined the electrophysiological effects of both cortisone and hydrocortisone on human Kv1.5 channels expressed in Xenopus oocytes using a two-microelectrode voltage clamp technique. Both cortisone and hydrocortisone rapidly and irreversibly suppressed the amplitude of Kv1.5 channel current with IC50 values of 50.2 + 74.2 μM and 33.4 + 73.2 μM, respectively, while sustained the current trace shape of Kv1.5 current. The inhibitory effect of cortisone on Kv1.5 decreased progressively from – 10mV to +30 mV, while hydrocortisone’s inhibition of the channel did not change across the same voltage range. Both cortisone and hydrocortisone blocked Kv1.5 channel currents in a non-use-dependent manner and neither altered the channel’s steady-state activation or inactivation curves. These results show that cortisone and hydrocortisone inhibited Kv1.5 channel currents differently. Kv1.5 channels were more sensitive to hydrocortisone than to cortisone.

In conclusion, cortisone and hydrocortisones rapidly and irreversibly blocked human Kv1.5 channels expressed in Xenopus oocytes in a closed state without altering activation and inactivation gating. These data provide a possible mechanism for GC effects on the cardiovascular system. The detailed mechanism of the interaction between GCs and human Kv1.5 channels merits further exploration.

Inflammasome and cytokine blocking strategies in autoinflammatory disorders

Monika Moll, Jasmin B. Kuemmerle-Deschner
Clinical Immunology (2013) 147, 242–275 http://dx.doi.org/10.1016/j.clim.2013.04.008

Autoinflammatory disorders are characterized by usually unprovoked recurrent episodes of features of inflammation caused by activation of the innate immune system. Many autoinflammatory disorders – the monogenetic defects in particular – are associated with alterations of inflammasomes. Inflammasomes are complex multimolecular structures, which respond to “danger” signals by activation of cytokines. Among these, IL-1 is the key player of the innate immune response and inflammation. Consequently, IL-1 blocking strategies are specific pathway targeting therapies in autoinflammatory diseases and applied in CAPS, colchicine-resistant FMF, TRAPS, HIDS and DIRA. A number of rare genetic disorders involve inflammasome malfunction resulting in enhanced inflammatory response. IL-1 inhibition to date is the most successful specific therapy in autoinflammatory disorders. Here, current treatment strategies in autoinflammatory disorders are reviewed with a focus on inflammasome and cytokine inhibition.

Autoinflammatory disorders have been defined as “clinical disorders marked by abnormally increased inflammation, mediated predominantly by the cells and molecules of the innate immune system.” This means that in autoinflammatory disorders autoantibodies or antigen related T-cells are usually absent. These are features of the adaptive immune system and found in autoimmune diseases.
In general, autoinflammatory disorders are characterized by a large spectrum of rather non-specific systemic and organ-specific signs and symptoms of inflammation. In some diseases specific symptoms are observed like hearing loss in Muckle–Wells syndrome or CNS-disease in NOMID/CINCA. Most autoinflammatory disorders are associated with high levels of serum amyloid A (SAA) during inflammatory attacks and high risk of life-threatening amyloidosis. In most cases the disease will start in infancy and childhood. Only rarely primary manifestations in adulthood are reported.
Because recurrent fevers have been the most prominent feature of this group of diseases, historically they have been summarized under the term “hereditary periodic fever syndromes”. With the deeper understanding of the underlying pathophysiologic mechanisms on the genetic and cellular level, the more comprehensive term “autoinflammatory syndromes”.
Along with the detection of the genetic origin of the autoinflammatory disorders, the cellular pathomechanism leading to the resulting inflammation has been described. A number of genes, which are affected by mutations in autoinflammatory disorders, encode proteins forming intracellular complexes called inflammasomes. External and endogenous “dangers” are recognized by these “danger sensors” and are able to induce an inflammatory reaction. Microbial components from infectious agents such as LPS, flagellin, lipoteichoic acid from bacteria, peptidoglycan or double-stranded DNA from viruses, or inorganic crystalline structures such as uric acid crystals, display pathogen-associated molecular patterns (PAMPs). These and endogenous damage-associated molecular patterns (DAMPs) like heat-shock proteins, the chromatin-associated protein high-mobility group box 1 (HMGB1), hyaluronan fragments, ATP, uric acid, and DNA which are released with cellular waste and injury stimulate the inflammasome. Also, the myeloid related proteins MRP8 and 14 (also known as S100A8 and S100A9) which are used as biomarkers, belong to the group of DAMPs. In addition to PAMPs and DAMPs, the inflammasome may interact with and be stimulated by proteins such as pyrin, proline–serine–threonine phosphatase interacting protein 1 (PSTPIP1), mevalonate kinase (MK) and NLRP7. All of these may also be altered in structure and function by monogenetic mutations.
As a consequence of inflammasome activation, a large variety of cytokines are produced and released by cells of the innate immune system (monocytes, macrophages, dendritic cells). They include the IL-1 family (IL-1, IL-18, IL-33), the TNF family (TNF-α, LT-α), the IL-6 family (IL-6, IL-11), the IL-17 family (IL-17A, IL-25), and type 1 IFNs (IFN-α, IFN-β). These cytokines play redundant roles depending on the cause and pathway of inflammation in the respective disease. Therefore, therapeutic strategies targeting only one cytokine should be expected to be inadequate to treat inflammatory disorders. However, improvement observed in diabetes mellitus Type 2 after blockade of IL-1 indicates that targeting one cytokine, even in a polygenic, complex inflammatory disorder, may cause beneficial effects. Regarding the inflammatory pathogenesis involved in the disease, Goldbach–Mansky and co-workers have classified the monogenetic autoinflammatory disorders as IL-1 mediated (CAPS and DIRA), partially IL-1 mediated (FMF, HIDS, PAPA) and mediated by other pathways (TRAPS, Blau-syndrome, Majeed’s syndrome, cherubism and IL-10 receptor deficiency).

Intracellular signaling pathways and therapeutic targets in autoinflammatory diseases. In autoinflammatory diseases, complex intracellular pathways lead to activation of the inflammatory response, particularly IL-1β activation and release, but also induction of NFκB and TNFα. Several mechanisms may activate the inflammasome, one crucial step in the IL-1 pathway. These include DAMPs (1), K+-efflux (2), activation of ROS (3) by ATP, anorganic crystals, membrane perturbation and proteases which are released from lysosomes damaged by β-amyloid, and heat shock proteins (4). NFκB may be induced by PAMPs via toll like receptors (5), IL-1β-signaling (6) or UPR (7). Activated NFκB eventually leads to the release of pro-inflammatory cytokines like IL-1, IL-6 and TNFα (8). Most of these steps to activation have been identified as targets for anti-inflammatory therapies, which are either already used in clinical practice or still experimental. IL-1- (a), TNF- (b), and IL-6 (c) inhibition are established safe and effective treatment strategies in many autoinflammatory diseases. Thalidomide (d) probably inhibits activation of IκB and is also part of routine treatment. Still experimental strategies include inhibition of PAMPs (e), DAMPs (f), potassium efflux (g), ROS by antioxidants (h), heat shock proteins (i), or caspase-1 (k). Caspase-inhibitors have entered clinical trials.

Colchicine has been used for the treatment of inflammatory disorders for centuries. Colchicine is effective in gout, but also in Behcet’s disease and FMF, where it is able to prevent amyloidosis. The drug affects many cell types and accumulates preferentially in neutrophils. Although its mode of action is still unclear it has microtubule destabilizing properties which may be part of its effects. Additional effects such as alteration of adhesion molecule expression, chemotaxis, and ROS generation also impact inflammation. Colchicine is generally tolerated well. However gastrointestinal, hematologic, and neuromuscular side-effects occur, when the administered dose is too high.

Inflammasome activation by heat shock proteins may be prevented by direct inhibition of HSP. HSP90 inhibition was effective in reducing gout-like arthritis in an animal model. Targeting caspase-1 (caspase-1-inhibitors) may be a strategy which has even greater potential in the treatment of autoimmune diseases and autoinflammatory disorders. IL-1 converting enzyme/caspase inhibitor VX-765 was able to inhibit IL-β-secretion in LPS-stimulated cells from FCAS and control subjects. A new IL-1 inhibitor, gevokizumab or Xoma 052 has entered clinical pilot trials. Therapeutic targets particularly for the protein-misfolding autoinflammatory diseases could be chemical chaperones and drugs that stimulate autophagy. Also inhibiting the signaling molecules that mediate the UPR activation which causes activation of the innate immune system and exacerbate inflammation could be a target.

To date IL-1 blockade is the most effective therapy in most monogenetic autoinflammatory diseases — in intrinsic and in extrinsic inflammasom-opathies. The most favorable effects are seen in the treatment of cryopyrin associated periodic syndromes like FACS, MWS and CINCA. But IL-1-blockade is also effective in other diseases like DIRA, TRAPS, PFAPA, colchicine-resistant FMF etc. IL-1 inhibition also has a role in multifactorial and common autoinflammatory diseases like diabetes, gout and artherosclerosis.

Endothelin—Biology and disease

Al-karim Khimji, Don C. Rockey
Cellular Signalling 22 (2010) 1615–1625
http://dx.doi.org:/10.1016/j.cellsig.2010.05.002

Endothelins are important mediators of physiological and pathophysiologic processes including cardiovascular disorders, pulmonary disease, renal diseases and many others. Additionally, endothelins are involved in many other important processes such as development, cancer biology, wound healing, and even neurotransmission. Here, we review the cell and molecular biology as well as the prominent pathophysiological aspects of the endothelin system.

Endothelin-1 (ET-1) was originally isolated from porcine aortic endothelial cells and is a 21 amino acid cyclic peptide, with two disulphide bridges joining the cysteine amino acids (positions 1–15 and 3–11) at the N-terminal end and hydrophobic amino acids at the c-terminal end of the peptide (Fig. 1). The C-terminal end contains the amino acids that bind to the receptor, the N-terminal end determines the peptide’s binding affinity to the receptor (see Fig. 1). There appear to be at least 2 other endothelin isoforms including endothelin-2 (ET-2) and endothelin-3 (ET-3), which differ from ET-1 in two and six amino acid residues, respectively.

Endothelin (ET) structure

Endothelin (ET) structure. Endothelin is a 21 amino acid cyclic peptide, with two disulphide bridges joining the cysteine residues at positions 1–15 and 3–11. The C-terminal end containsamino acids that appear tomediate receptor binding,while the N-terminal residues determine the peptide’s binding affinity to the receptor. The amino acids highlighted in black in panels (b) and (c) show differences in ET-2 and ET-3 compared to ET-1. As can be seen, the remainder of the primary sequence of the different family members is identical.

Endothelin-1 biosynthetic pathway

Endothelin-1 biosynthetic pathway. Preproendothelin mRNA is synthesized via transcriptional activation of the preproendothelin gene. The translational product is a 203-amino acid peptide known as preproendothelin, which is cleaved at dibasic sites by furin-like endopeptidases to form big endothelins. These biologically inactive, 37- to 41-amino acid intermediates, are cleaved at Trp21–Val 22 by a family of endothelin-converting enzymes (ECE) to produce mature ET-1. The pathway for endothelin-2 and -3 is presumed to be similar.

The endothelin peptides are produced through a set of complex molecular processes. Preproendothelins are synthesized via transcriptional activation of the preproendothelin gene, which is regulated by c-fos and c-jun, nuclear factor-1, AP-1 and GATA-2. The translational product is a 203-amino acid peptide known as preproendothelin which is cleaved at dibasic sites by furin-like endopeptidases to form big endothelins. These biologically inactive 37- to 41-amino acid intermediates are cleaved at Trp21–Val 22 by a family of endothelin-converting enzymes (ECE) to produce mature ET-1.

Three isoforms of ECE have been reported, namely ECE-1, ECE-2 and ECE-3; ECE-1 and ECE-2 are most prominent. (Endothelin receptors are widely distributed in many different tissues and cells, there is a marked difference in cell and tissue distribution patterns between the two receptor subtypes i.e. ETA and ETB. [ET Receptors: Endothelial cells -ETB Vascular tone, clearance of circulating ET-1]). ECEs belong to the M13 group of proteins—which is a family that includes neutral endopeptidases, kell blood group antigens (Kell), a peptide from phosphate regulating gene (PEX), X-converting enzyme (XCE), “secreted” endopeptidases, and the ECEs. M13 family members contain type II integral membrane proteins with zinc metalloprotease activity, and their function is inhibited by phosphoramidon. Four variants of ECE-1 have been reported in humans, namely ECE-1a, ECE-1b, ECE-1c and ECE-1d which are a result of alternate splicing of ECE-1mRNA. ECE-1 appears to be localized in the plasma cell membrane and its optimal activity is atpH7; it processes big ETs both intracellularly and on the cell surface. It is distributed predominantly in smooth muscle cells. ECE-1 can also hydrolyze other proteins including bradykinin, substance P, and insulin. ECE-2 is localized to the trans-Golgi network and is expressed abundantly in neural tissues and endothelial cells. Its optimal activity is at pH5; the acidic activity marks ECE-2 as an intracellular enzyme. Substrate selectivity experiments indicate that both ECE-1 and ECE-2 show preference for big ET-1 over big ET-2 or big ET-3.

Although there has been controversy about the precise repertoire of endothelin receptors, it appears that the endothelins exert their actions through two major receptor subtypes known as ETA and ETB receptors. ETA and ETB receptors belong to the superfamily of G-protein coupled receptors and contain seven transmembrane domains of 22–26 hydrophobic amino acids among approximately 400 total amino acids. The ETA receptor is found predominantly in smooth muscle cells and cardiac muscles, whereas the ETB receptor is abundantly expressed in endothelial cells.

ET-1 signaling is extremely complicated and ET receptor activation leads to diverse cellular responses through interaction in a chain of pathways that includes the G-protein-activated cell surface receptor, coupling G-proteins and phospholipase (PLC) pathway and other G protein-activated effectors. In one of the canonical signaling pathways, ETA induced activation of phospholipase C leads to the formation of inositol triphosphate and diacylglcerol from phosphatidylinositol. Inositol 1,4,5 triphosphate (IP3) then diffuses to specific receptors on the endoplasmic reticulum and releases stored Ca2+ into the cytosol. This causes a rapid elevation in intracellular Ca2+, which in turn causes cellular contraction and then vasoconstriction; the vasoconstrictive effects of ET persist despite dissociation of ET-1 from the receptor, perhaps because the levels of intracellular calcium remain elevated or because endothelin signaling pathways remain activated for prolonged time periods.

Endothelin signaling – smooth muscle cells

Endothelin signaling – smooth muscle cells. ET receptor stimulation leads to diverse cellular responses in a chain of pathways that include the G protein bg activation. This is followed by activation of a variety of different downstream cascades. For example, shown on the left, ETA induced activation of phosphatidyl inositol specific phospholipase C (PI-PLC) leads to the formation of inositol triphosphate (IP3) and diacylglcerol (DAG) from phosphoinositol 4,5 bisphosphate (PIP2). Inositol 1, 4, 5 triphosphate (IP3) then diffuses to specific receptors on the endoplasmic reticulum and releases stored Ca2+ into the cytosol. This causes a rapid elevation in intracellular Ca2+, which in turn causes cellular contraction

Endothelin signaling – endothelial cells.

Endothelin signaling – endothelial cells. ET-1 stimulates NO production in endothelial cells by activation of endothelial cell NO synthase (eNOS). This occurs via ET-1’s activation of the ET-B receptor and the PI3-K/Akt pathway, which in turn stimulates phosphorylation of eNOS, with subequent conversion of L-arginine to L-citrulline and at the same time, generating NO. In addition shear stress, G-protein coupled receptors (GPCR), transient receptor potential channel (TRPC) and receptor tyrosine kinase (RTK) are also activators of eNOS. As a result, NO diffuses to stellate cell, where it directly activates the heme moiety of soluble guanylate cyclase, leading to the production of cyclic GMP. Intracellular cyclic GMP leads to activation of protein kinase G (PKG) resulting in relaxation of stellate cells – offsetting ET’s contractile effect on stellate cells.

The plasma levels of endothelin do not correlate with either the presence of essential hypertension or its severity, presumably, due to the fact that endothelin appears to be biologically active in a paracrine or autocrine fashion (i.e., rather than in an endocrine fashion. Systemic administration of ET-1 in low doses produces a modest increase in blood pressure which is normalized by selective ETA receptor blockade. In experimental models, long-term infusion with ET-1 leads to stroke and renal injury, which can be prevented with long-term administration of selective ETA receptor antagonists. Apart from its direct vasoconstrictor effects, mediated by smooth muscle cell contraction in the arterial system, ET-1 also indirectly enhances the vasoconstrictor effects of other neurohumoral and endocrine factors and may potentiate essential hypertension via this mechanism. For example, ET-1 induces conversion of angiotensin I to angiotensin II in in vitro models and stimulates adrenal synthesis of epinephrine and aldosterone. Thus there is cross-talk between the endothelin and renin–angiotensin–aldosterone systems—to synergistically act to facilitate vasoconstriction. In aggregate, the data suggest that dysregulation of the endothelin system contributes to multisystem complications of hypertension such as progressive renal disease, cerebrovascular diseases, atherosclerosis, and cardiac disease.

ET-1 in the renal system is synthesized in vascular endothelial cells and epithelial cells of the collecting ducts. Both ET receptors are present in renal vasculature and epithelial cells where ETB is the predominant receptor type. Renal vasculature is relatively more sensitive to the vasoconstrictive effects of ET-1 than any other vasculature and it causes constriction of both afferent and efferent renal arterioles.

ET-1 administration in humans significantly reduces renal blood flow, glomerular filtration rate and urine volume. In addition to its hemodynamic effects, ET-1 system is also involved in salt and water reabsorption, acid-base balance, promotion of mesangial cell growth and activation of inflammatory cells. ET-1 has been implicated in the pathophysiology of acute renal injury, chronic renal failure as well as renal remodeling. Transgenic mice overexpressing ET-1 develop glomerulosclerosis, interstitial fibrosis and reduced renal function. Increased ET-1 and ET receptor upregulation has been described in various animal models of acute renal injury and also in patients with chronic renal failure. Additionally, plasma ET-1 levels have been shown to correlate with the severity of chronic renal failure.

ET-1 is produced and released by airway epithelial cells, macrophages, and pulmonary vascular endothelial cells. Endothelin receptors are similarly widely distributed in airway smooth muscle cells, the pulmonary vasculature, and in the autonomic neuronal network lining tracheal muscles. ET-1 has a potent bronchoconstrictor effect. In animal models, intravenous ET-1 injection led to a dose-dependent increase in airway resistance. The increase in airway resistance is in part due to enhanced production of thromboxanes with subsequent activation of thromboxane receptors and smooth muscle cell proliferation. The ET system has been emphasized in a number of pulmonary disorders, including asthma, cryptogenic fibrosing alveolitis, and pulmonary hypertension. Increased lung vasculature ET-1 immunoreactivity has been reported in both animals and patients with pulmonary hypertension and increases in ET-1 immunoreactivity correlate with the degree of pulmonary vascular resistance, disorders such as pulmonary hypertension, myocardial infarction, heart failure, neoplasia, vascular disorders, wound healing, and many others.

Endothelin and endothelin antagonism: Roles in cardiovascular health and disease

Praveen Tamirisa, William H. Frishman, and Anil Kumar
Am Heart J 1995;130:601-10

Endothelin is a naturally occurring polypeptide substance with potent vasoconstrictive actions. It was originally described as endotensin or endothelial contracting factor in 1985 by Hickey et al., who reported on the finding of a potent stable vasoconstricting substance produced by cultured endothelial cells. Subsequently, Yanagisawa et al. isolated and purified the substance from the supernatant of cultured porcine aortic and endothelial
cells and then went on to prepare its complementary deoxyribonucleic acid (cDNA). This substance was renamed endothelin.

Endothelin is the most potent vasoconstrictor known to date. Its chemical structure is closely related to certain neurotoxins (sarafotoxins) produced by scorpions and the burrowing asp (Atractaspis engaddensis). Endothelins have now been isolated in various cell lines from several organisms. They are now considered to be autocoids or cytokines 4 because of their wide distribution, their expression during ontogeny and adult life, their primary role as intracellular factors, and the complexity of their biologic effects.

The superfamily of endothelins and sarafotoxins have two main branches with four members each. Endothelin is a polypeptide consisting of 21 amino acids. There are three closely related isoforms endothelin-1, endothelin-2, and endothelin-3 (ET1, ET2, and ET3, respectively), which differ in a few of the amino acid constituents. The fourth member, called ET4 or vasoactive intestinal constrictor, is considered to be the murine form ofET2. The endothelin molecules have several conserved amino acids, including the last six carboxyl (C)-terminal amino acids and four cysteine residues, which form two intrachain disulfide bonds between residues 1 and 15 and 3 and 11. These residues may have biologic implications particularly in relation to three dimensional structure and function. The main differences in the endothelin isopeptides reside in their amino (N)-terminal segments. There is a very high degree of sequence similarity between the two branches (approximately 60%) and within the constituent members of a branch (71% to 95%).

Endothelin has been demonstrated to be produced from endothelial and nonendothelial cells. The synthesis of endothelins parallels that of the various peptide hormones in that a precursor polypeptide is sequentially cleaved to generate the active form. Recently, endothelin-converting enzyme (ECE) was cloned. ECE acts at an essential step in the production of active forms of endothelins. The fully formed molecule is then broken down into inactive peptides by as yet uncharacterized proteases. Some candidates are the lysosomal protective protein (deamidase) and enkephalinase (neutral endopeptidase EC 24.11). The regulation of endothelin production occurs predominantly at the levels of transcription and translation. No storage
vesicles containing endothelin have been identified. The genes for the various endothelin isoforms have been sequenced and are found to be scattered in different chromosomes. Current evidence suggests that they arose from a common ancestor by exon duplication.

Factors known to release endothelinThrombinTransforming growth factor-~Arginine vasopressinHypoxia

Phorbol ester

Glucose

Angiotensin II

Cyclosporin

Insulinlike growth factor

Bombesin

Cortisol

Low-density lipeprotein cholesterol

Hypercholesterolemia

Changes in shear stress on vascular wall

Receptor affinities
Receptor	Affinity
ETA	ET1 > ET2 > ET3
ETB	ET1 = ET2 = ET3
ETC	ET3 > ET1

Intracellular signal transduction pathways activated by endothelins (ETs)

Intracellular signal transduction pathways activated by endothelins (ETs). Activated ET receptor stimulates phospholipase C (PLC) and phospholipase A₂ (PLA2). Activated ET receptor also stimulates voltage-dependent calcium channels (VDC) and probably receptor-operated calcium channel (ROC). Inositol triphosphate (IP3) elicits release of calcium ion from caffeine-sensitive calcium store. Protein kinase C (PKC) activated by diacylglycerol (DG) sensitizes contractile apparatus. Increased concentration of intracellular free calcium ion ([Ca2+]_i induces contraction. Cyclooxygenase products (prostacyclin [PGI₂], prostaglandin E₂[PGE₂], and thromboxane A₂ [TXA₂]) modify contraction. G, G protein; IP₂, inositol biphosphate; IP₃, inositol triphosphate; PIP₂, phosphatidyl inositol biphosphate. (From Masaki T et al. Circulation 1991;84: 1460.)

Systemic hypertension. Endothelin is the most potent vasoconstrictor known to date and has an exceptionally long duration of physiologic action. The influence of endothelin in maintaining normal blood pressure and its role in the cause of systemic hypertension remain unclear. Intravenous injections of endothelin in animals cause a transient decrease in systolic blood pressure (ETB) followed by a prolonged pressor response (ETA). The vasoconstrictor action is mediated by ETA receptors in the vascular smooth muscle, whereas the predominant vasodilation effect is mediated by the ETB receptors on the endothelial cells that cause release of prostacyclin and nitric oxide. Therefore the overall predominant hemodynamic effect of endothelin in a given organ depends on the receptor type being stimulated, its location, and its relative abundance.

Angiotensin II has been found to increase endothelin concentrations in vitro from endo thelial cells, suggesting one mechanism by which angiotensin-converting-enzyme (ACE) inhibition could function in vivo. ACE inhibitors also can indirectly interfere with endothelin: increased concentrations of bradykinin decrease endothelin release (by acting through bradykinin 2 receptors, stimulation of which cause increased nitric oxide release). ACE inhibitors can cause regression of intimal hyperplasia, whereas other antihypertensive drugs are ineffective in this regard.

Myocardial ischemia. Myocardial ischemia can enhance the release of endothelin by cardiomyocytes and increase its vasoactive effects. Infusion of the ET1 isoform directly into the coronary circulation of animals results in the development of myocardial infarction, with impaired ventricular functioning and the development of arrhythmias. Endothelin has been shown to lower the threshold for ventricular fibrillation in dogs. An increase in ET1 has been observed in cardiac tissue after experimental myocardial infarction in rats, and pretreatment with an antiendothelin ϒ-globulin in this model can reduce infarct size by as much as 40%. Infusion of ETA receptor antagonist drugs before an ischemic insult can also reduce infarct size in animals.

Plasma endothelin concentrations can predict hemodynamic complications in patients with myocardial infarction. Patients with the highest plasma endothelin concentrations after myocardial infarction have the highest creatine phosphokinase (CPK) and CPK MB-isoenzyme concentrations and the lowest angiographically determined ejection fractions.

Left ventricular function and congestive heart failure. Endothelin exhibits potent inotropic activity in isolated hearts, cardiac muscle strips, isolated cells, and instrumented intact animals. High-affinity receptors for endothelin have been demonstrated in the atria and the ventricles. Intravenous administration of the ET1 isoform produces delayed prolonged augmentation of left ventricular performance in addition to its biphasic vasoactive effects of transient vasodilation followed by sustained vasocontraction.

Endothelin is a potent secretogogue of atrial natriuretic factor, which is a naturally occurring antagonist of endothelin. The ETA receptor appears to mediate endothelin’s actions of vasoconstriction and the stimulation of atrial natriuretic factor secretion, and the ETB receptor mediates endothelin-induced vasodilation and activation of the renin-angiotensin-aldosterone system. Urinary water excretion is mediated through both receptors, but sodium excretion is mediated through the ETA receptor.

Increased concentrations of endothelin described in patients with congestive heart failure are predictive of increased mortality risk. It also has been suggested that increased concentrations of endothelin may play an important role in the increased systemic vascular resistance observed in congestive heart failure.

There is early clinical evidence that treatment with ETA receptor antagonists and ECE inhibitors can influence favorably the course of human heart failure. ACE inhibitors may also benefit patients with heart failure because of their antiendothelin actions.

Pulmonary hypertension. Expression of ET1 in the lung has been studied by immunocytochemistry and hybridization in situ in specimens from patients with pulmonary hypertension of primary or secondary causes. In contrast to normal lung, specimens from patients with pulmonary hypertension exhibit abundant ET2 immunostaining, particularly over endothelium of markedly hypertrophied muscular pulmonary arteries and plexogenic lesions. Endothelin has been suggested as a potent vasoconstrictor and growth-promoting factor in the pathophysiologic pathophysiologic mechanisms of pulmonary hypertension.

Ventricular and vascular hypertrophy. Endothelin increases DNA synthesis in vascular smooth-muscle ceils, cardiomyocytes, fibroblasts, glial cells, mesangial cells, and other cells; causes expression of protooncogenes; causes cell proliferation; and causes hypertrophy. It acts in synergy with various factors such as transforming growth factor, epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor and insulin to potentiate cellular transformation and replication. This synergy suggests that all of these factors act through common pathways involving PKC and cyclic adenosine monophosphate. Endothelin per se may not be a direct mediator of angiogenesis but may function as a comitogenic factor.

Neointima formation after vascular wall trauma. The efficacy of coronary angioplasty is limited by the high incidence of restenosis. ET1 induces cultured vascular smooth-muscle cell proliferation by activation of the ETA-receptor subtype, a response that normally is attenuated by an intact, functional endothelium. In addition, ET1 also induces the expression and release of several protooncogenes and growth factors that modulate smooth-muscle cell migration, proliferation, and matrix formulation. In addition to inhibiting smooth-muscle cell proliferation in vitro, endothelin-receptor antagonism with SB 209670 ameliorates the degree of neointima formation observed after rat carotid artery angioplasty. The observations raise the possibility that ET1 antagonists will serve as novel therapeutic agents in the control of restenosis.

Nonspecific endothelin antagonists
ECE inhibitorsAngiotensin-converting-enzyme inhibitorsAngiotensin II receptor blocking agentsCalcium-entry blocking agentsPotassium-channel opening agentsAdenosineNitroglycerin

SUMMARY

Endothelin is the most potent mammalian vasoconstrictor yet discovered. Its three isoforms play leading roles in regulating vascular tone and causing mitogenesis. The isoforms bind to two major receptor subtypes (ETA and ETB), which mediate a wide variety of physiologic actions in several organ systems. Endothelin may also be a disease marker or an etiologic factor in ischemic heart disease, atherosclerosis, congestive heart failure, renal failure, myocardial and vascular wall hypertrophy, systemic hypertension, pulmonary hypertension, and subarachnoid hemorrhage. Specific and nonspecific receptor antagonists and ECE inhibitors that have been developed interfere with endothelin’s function. Many available cardiovascular therapeutic agents, such as angiotensin-converting-enzyme inhibitors, calcium-entry blocking drugs, and nitroglycerin, also may interfere with endothelin release or may modify its activity. The endothelin antagonists have great potential as agents for use in the treatment of a wide spectrum of disease entities and as biologic probes for understanding the actions of endothelin in human beings.

Endothelin receptor antagonists

Sophie Motte, Kathleen McEntee, Robert Naeije
Pharmacology & Therapeutics 110 (2006) 386 – 414
http://dx.doi.org:/10.1016/j.pharmthera.2005.08.012

Endothelin receptor antagonists (ERAs) have been developed to block the effects of endothelin-1 (ET-1) in a variety of cardiovascular conditions. ET-1 is a powerful vasoconstrictor with mitogenic or co-mitogenic properties, which acts through the stimulation of 2 subtypes of receptors [endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) receptors]. Endogenous ET-1 is involved in a variety of conditions including systemic and pulmonary hypertension (PH), congestive heart failure (CHF), vascular remodeling (restenosis, atherosclerosis), renal failure, cancer, and cerebrovascular disease. The first dual ETA/ETB receptor blocker, bosentan, has already been approved by the Food and Drug Administration for the treatment of pulmonary arterial hypertension (PAH). Trials of endothelin receptor antagonists in heart failure have been completed with mixed results so far. Studies are ongoing on the effects of selective ETA antagonists or dual ETA/ETB antagonists in lung fibrosis, cancer, and subarachnoid hemorrhage. While non-peptidic ET-1 receptor antagonists suitable for oral intake with excellent bioavailability have become available, proven efficacy is limited to pulmonary hypertension, but it is possible that these agents might find a place in the treatment of several cardiovascular and non-cardiovascular diseases in the coming future.

Proposed mechanism by which ET-1 triggers vasoconstriction and vascular remodeling. Activation of G-protein-coupled endothelin receptors leads to stimulation of phospholipase C (PLC) which hydrolyses phosphatidyl inositol biphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG). DAG opens receptor-operated Ca++ channels (ROC) while IP3 induces Ca++ mobilization from the sarcoplasmic reticulum (SR) and opens store-operated Ca++ channels (SOC) directly or indirectly by store depletion to further increase cytosolic Ca++. This Ca++ increase may also trigger Ca++ release from the SR through ryanodine receptors. Depolarization induced by the opening of non-selective cationic channels (NSCC) via ET-1 and Ca++-activated Cl[1] channels as well as by the inhibition of voltage-gated K+ channels (Kv), opens voltage-dependent Ca++ channels (VDCC) to further increase the Ca++ entry across the plasma membrane. The cytosolic Ca++ increase may also activate Na/H exchangers resulting in alkalinization of the cells and promoting Ca++ influx by activating the Na/Ca exchanger. In addition, the elevated cytosolic Ca++ concentrations and DAG activate the protein kinase C and thus promote cell cycle progression by the Ca++/calmodulin complex (Ca++/CaM) and induction of proto-oncogenes. The intracellular signaling cascade induced by activation of ETB receptor is similar to the ETA receptor one, in stimulating the activation of PLC, generating IP3 and DAG and mobilizing of calcium. However, the PLA2 is also activated via ETB receptors to release prostaglandins (PG) and thromboxane A2 (TXA2).

Endothelin-1 increases isoprenaline-enhanced cyclic AMP levels in cerebral cortex

Marıa J. Perez-Alvareza, MC Calcerrada, F Hernandez, RE Catalan, AM Martınez
Regulatory Peptides 88 (2000) 41–46 PII: S0167-0115(99)00118-4

We examined the effect of ET-1 on cyclic AMP levels in rat cerebral cortex. The peptide caused a concentration-dependent increase of [³ H] cyclic AMP accumulation after 10 min of treatment. This effect was due to adenosine accumulation since it was inhibited by the treatment with adenosine deaminase. ET-1, apart from being able to increase cyclic AMP, also potentiated the cyclic AMP generated by isoprenaline in the presence of adenosine deaminase. Experiments performed in the presence of BQ-123 or BQ-788, specific ET_A or ET_B receptor antagonists respectively indicated that ET was the receptor involved. This effect was dependent on extracellular and B intracellular calcium concentration. These findings suggest that ET-1 plays a modulatory role in cyclic AMP generation systems in cerebral cortex.

Endothelins And Asthma

Roy G. Goldie and Peter J. Henry
Life Sciences I999; 65(1), pp. I-15, PI1 SOO24-3205(98)00614-6

In the decade since endothelin-1 (ET-l) and related endogenous peptides were first identified as vascular endothelium-derived spasmogens, with potential pathophysiological roles in vascular diseases, there has been a significant accumulation of evidence pointing to mediator roles in obstructive respiratory diseases such as asthma. Critical pieces of evidence for this concept include the fact that ET-l is an extremely potent spasmogen in human and animal airway smooth muscle and that it is synthesised in and released from the bronchial epithelium. Importantly, symptomatic asthma involves a marked enhancement of these processes, whereas asthmatics treated with anti-inflammatory glucocorticoids exhibit reductions in these previously elevated indices. Despite this profile, a causal link between ET-l and asthma has not been definitively established. This review attempts to bring together some of the evidence suggesting the potential mediator roles for ET-l in this disease.

Endothelial Cell Peroxisome Proliferator–Activated Receptor ϒ Reduces Endotoxemic Pulmonary Inflammation and Injury

Aravind T. Reddy, SP Lakshmi, JM Kleinhenz, RL Sutliff, CM Hart, and R. Reddy
J Immunol 2012; 189:5411-5420
http://www.jimmunol.org/content/189/11/5411

Bacterial endotoxin (LPS)-mediated sepsis involves severe, dysregulated inflammation that injures the lungs and other organs Bacterial endotoxin (LPS)-mediated sepsis involves severe, dysregulated inflammation that injures the lungs and other organs, often fatally. Vascular endothelial cells are both key mediators and targets of LPS-induced inflammatory responses. The nuclear hormone receptor peroxisome proliferator–activated receptor ϒ (PPARϒ) exerts anti-inflammatory actions in various cells, but it is unknown whether it modulates inflammation through actions within endothelial cells. To determine whether PPARϒ acts within endothelial cells to diminish endotoxemic lung inflammation and injury, we measured inflammatory responses and mediators in mice with endothelial-targeted deletion of PPARϒ. Endothelial cell PPARϒ (ePPARϒ) knockout exacerbated LPS-induced pulmonary inflammation and injury as shown by several measures, including infiltration of inflammatory cells, edema, and production of reactive oxygen species and proinflammatory cytokines, along with upregulation of the LPS receptor TLR4 in lung tissue and increased activation of its downstream signaling pathways. In isolated LPS-stimulated endothelial cells in vitro, absence of PPARϒ enhanced the production of numerous inflammatory markers. We hypothesized that the observed in vivo activity of the ligand-activated ePPARϒ may arise, in part, from nitrated fatty acids (NFAs), a novel class of endogenous PPARϒ ligands.
Supporting this idea, we found that treating isolated endothelial cells with physiologically relevant concentrations of the endogenous NFA 10-nitro-oleate reduced LPS-induced expression of a wide range of inflammatory markers in the presence of PPARϒ, but not in its absence, and also inhibited neutrophil mobility in a PPARϒ-dependent manner. Our results demonstrate a key protective role of ePPARϒ against endotoxemic injury and a potential ePPARϒ-mediated anti-inflammatory role for NFAs.

Endothelins in health and disease

Rahman Shah
European Journal of Internal Medicine 18 (2007) 272–282
http://dx.doi.org:/10.1016/j.ejim.2007.04.002

Endothelins are powerful vasoconstrictor peptides that also play numerous other roles. The endothelin (ET) family consists of three peptides produced by a variety of tissues. Endothelin-1 (ET-1) is the principal isoform produced by the endothelium in the human cardiovascular system, and it exerts its actions through binding to specific receptors, the so-called type A (ETA) and type B (ETB) receptors. ET-1 is primarily a locally acting paracrine substance that appears to contribute to the maintenance of basal vascular tone. It is also activated in several diseases, including congestive heart failure, arterial hypertension, atherosclerosis, endothelial dysfunction, coronary artery diseases, renal failure, cerebrovascular disease, pulmonary arterial hypertension, and sepsis. Thus, ET-1 antagonists are promising new agents. They have been shown to be effective in the management of primary pulmonary hypertension, but disappointing in heart failure. Clinical trials are needed to determine whether manipulation of the ET system will be beneficial in other diseases.

The production of ET receptors is affected by several factors. Hypoxia, cyclosporine, epidermal growth factor, basic fibroblast growth factor, cyclic AMP, and estrogen upregulate ET_A receptors in some tissues, and C-type natriuretic hormone, angiotensin II, and perhaps basic fibroblast growth factor up-regulate ET_B receptors. In contrast, the endothelins, angiotensin II, platelet-derived growth factor, and transforming growth factor down-regulate ET_Areceptors, whereas cyclic AMP and catecholamines down-regulate ET_Breceptors.

The ET_A receptor contains 427 amino acids and binds with the following affinity: ET-1N>T-2>ET-3. It is predominantly expressed in vascular smooth muscle cells and cardiac myocytes. Its interaction with ET-1 results in vasoconstriction and cell proliferation. In contrast, the ET_B receptor contains 442 amino acids and binds all endothelins with equal affinity. It is predominantly expressed on vascular endothelial cells and is linked to an inhibitory G protein. Activation of ET_B receptors stimulates the release of NO and prostacyclin, prevents apoptosis, and inhibits ECE-1 expression in endothelial cells. ET_B receptors also mediate the pulmonary clearance of circulating ET-1 and the re-uptake of ET-1 by endothelial cells.

All three endothelins cause transient endothelium dependent vasodilatation before the development of constriction, though this is most apparent for ET-1. Endothelins induce vasodilatation via the endothelial cell ET_B receptors through generation of endothelium-derived dilator substances (Fig. 3), including nitric oxide (NO), which perhaps acts by physiologically antagonizing ETA receptor mediated vasoconstriction. The transient early vasodilator actions of the endothelins are attenuated by NO synthase inhibitors. Additionally, ET-1 increases generation of prostacyclin by cultured endothelial cells, whereas cyclo-oxygenase inhibitors potentiate ET-1-induced constriction, suggesting that vasodilator prostaglandins play a similar modulatory role.

It has been proposed that ET-1 can affect vascular tone indirectly through its effect on the sympathetic nervous system, and it has been shown that that ET-1 may increase peripheral sympathetic activity through postsynaptic potentiation of the effects of norepinephrine. While in vitro low concentrations of ET-1 potentiate the effects of other vasoconstrictor hormones, including norepinephrine and serotonin, these findings have not been confirmed in vivo in the forearm resistance bed of healthy subjects. In addition to its action on vascular vasomotion, ET-1 is thought to be a mediator in the vascular remodeling process. It seems that ET-1 interactions with the renin–angiotensin–aldosterone system play a significant role in this remodeling process.

Vascular actions of endothelin-1

Vascular actions of endothelin-1. Modified from – Galie N, Manes A, Branzi A; The endothelin system in pulmonary arterial hypertension. Cardiovasc Res 2004;61:227–37.

ET-1 appears to have a diverse role as a modulator of vascular tone and growth and as a mediator in many cardiovascular and non-cardiovascular diseases. To date, no disease entity, however, has been attributed solely to an abnormality in ET-1. Yet, ET-1 receptor antagonists have been studied in clinical trials involving a wide spectrum of cardiovascular diseases, though the only proven efficacy has been in patients with PAH.

Learning points

Endothelins are powerful vasoconstrictors and major regulators of vascular tone.
The endothelin (ET) family consists of three peptides (ET-1 ∼60%, ET-2 ∼30%, and ET-3 ∼10%) produced by a variety of tissues.
ET-1 is the principal isoform produced by the endothelium in the human cardiovascular system and appears to be foremost a locally acting paracrine substance rather than a circulating endocrine hormone.
Several human studies suggest that circulating ET-1 levels, which are elevated in heart failure and pulmonary hypertension, correlate with the prognosis of the disease.
ET-1 antagonists have been shown to be effective in the management of primary pulmonary hypertension, but disappointing in heart failure.
Clinical trials are needed to investigate the role of ET-1 receptor antagonists for other conditions, as ET-1 levels have been shown to be elevated in arterial hypertension, atherosclerosis, endothelial dysfunction, coronary artery disease, renal failure, cerebrovascular disease, and sepsis.

In Vitro Stability and Intestinal Absorption Characteristics of Hexapeptide Endothelin Receptor Antagonists

Hyo-kyung Han, BH Stewart, AM Doherty, WL Cody and GL Amidon
Life Sciences. I998; 63(18), pp. 1599-1609. PI1 SOO24-3205(98)00429-9

Endothelins are potent vasoconstrictor peptides which have a wide range of tissue distribution and three receptor subtypes (ET_A ET_B and ET_C). Among the linear hexapeptide ET_A / ET_B receptor antagonists, PD 145065 (Ac-D-Bhg-L-Leu-L-Asp-L-Ile-L-Ile-L-Trp, Bhg = (10,ll -dihydro-5H-dibenzo[a,d]cyclohepten-5-yl)-Gly) and PD 156252 (Ac-o-Bhg-L-Leu-L-Asp-L-Ile-(N-methyl)-L-Ile-L-Trp) were selected to evaluate the metabolic stability and intestinal absorption in the absence and/or in the presence of protease inhibitors. In vitro stability of both compounds was investigated in fresh plasma, lumenal perfusate, intestinal and liver homogenates. PD 156252 was more stable than PD 145065 in intestinal tissue homogenate (63.4% vs. 20.5% remaining) and liver homogenate (74.4% vs. 35.5 % remaining), while both compounds showed relatively good stability in the fresh plasma (94.5% vs. 86.7% remaining) and lumenal perfusate (85.8% vs. 72.3% remaining). The effect of protease inhibitors on the degradation of PD 145065 and PD 156252 was also investigated. Amastatin, thiorphan, chymostatin and the mixture of these three inhibitors were effective in reducing the degradation of both compounds. The pharmacokinetic parameters of PD 156252, calculated by using a non-compartmental model, were 6.95 min (terminal half-life), 191 mL (V_ss), and 25.5 mL/min (Cl_tot) after intravenous administration in rats. The intestinal absorption of PD 156252 in rats was evaluated in the absence and/or in the presence of protease inhibitors. The results indicate that the major elimination pathway of PD 156252 appears to be the biliary excretion and protease inhibitors increase the intestinal absorption of PD 156252 through increasing metabolic stability.

Inhibitory and facilitatory presynaptic effects of endothelin on sympathetic cotransmission in the rat isolated tail artery

Violeta N. Mutafova-Yambolieva & David P. Westfall
British Journal of Pharmacology (1998) 123, 136 – 142

1 The present study was undertaken to determine the modulatory effects of the endothelin peptides on the neurogenically-induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5′-triphosphate (ATP) from the sympathetic nerves of endothelium-free segments of the rat isolated tail artery. The electrical field stimulation (EFS, 8 Hz, 0.5 ms, 3 min) evoked over¯ow of NA and ATP, in the absence of endothelins, was 0.035+0.002 pmol mg71 tissue and 0.026+0.002 pmol mg71 tissue, respectively.

2 Endothelin-1 (ET-1; 1 ± 30 nM) significantly reduced the EFS evoked overflow of both NA and ATP. The maximum inhibitory effect was produced by a peptide concentration of 10 nM, the amount of NA overflow being 0.020+0.002 pmol mg71 and that of ATP overflow 0.015+0.001 pmol mg71. Higher peptide concentrations (100 and 300 nM) reversed the EFS-evoked overflow of NA to control levels and that of ATP to above control levels. The inhibitory effect of ET-1 (10 nM) was resistant to the selective ETA receptor antagonist cyclo-D-Trp-D-Asp(ONa)-Pro-D-Val-Leu (BQ-123) but was prevented by ETB receptor desensitization with sarafotoxin S6c (StxS6c) or by ETB receptor blockade with N, cis-2,6-dimethyl-piperidinocarbonyl-L-gmethylleucyl-D-1-methoxycarbonyl-tryptophanyl-D-norleucine (BQ-788).

3 StxS6c, upon acute application, exerted a dual effect on transmitter release. At concentrations of 0.001 ± 0.3 nM the peptide significantly reduced the EFS-evoked NA overflow, whereas at concentrations of 1 ± 10 nM it caused a significant increase in the evoked overflow of both ATP and NA. Both the maximum inhibitory effect of StxS6c at a concentration of 0.003 nM approximately 85% reduction of NA overflow and 40% of ATP overflow) and the maximum facilitatory effect of the peptide at a concentration of 3 nM (approximately 400% increase of ATP overflow and 200% of NA overflow) were completely antagonized by either BQ-788 or by StxS6c-induced ETB receptor desensitization.

4 ET-3 (10 ± 100 nM) did not aect the EFS evoked overflow of either ATP or NA, but at a concentration of 300 nM significantly potentiated the release of both transmitters (0.118+ 0.02 pmol mg71 tissue ATP overflow and .077+0.004 pmol mg71 NA overflow). This effect was prevented either by BQ-123 or by BQ-788.

5 In summary, the endothelin peptides exerted both facilitatory and inhibitory effects on the neurogenically-induced release of the sympathetic cotransmitters ATP and NA in the rat tail artery. Both transmitters were modulated in parallel indicating that the endothelins do not differentially modulate the release of NA and ATP in this tissue.

Involvement of the central adrenomedullin peptides in the baroreflex

Meghan M. Taylo, Cynthia A. Keown, Willis K. Samson
Regulatory Peptides 112 (2003) 87– 93
http://dx.doi.org:/10.1016/S0167-0115(03)00026-0

The peptides derived from post-translational processing of preproadreno-medullin are produced in and act on areas of the autonomic nervous system important for blood pressure regulation. We examined the role of endogenous, brain-derived adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) in the central nervous system arm of the baroreflex by using passive immunoneutralization to block the actions of the endogenous peptides. Our results indicate that the preproadrenomedullin-derived peptides do not play a role in sensing changes in blood pressure (baroreflex sensitivity), but the adrenomedullin peptides do regulate the speed with which an animal returns to a normal, stable blood pressure. These findings suggest that endogenous, brain-derived AM and PAMP participate in the regulation of autonomic activity in response to baroreceptor activation and inactivation.

Pharmacological characterization of cardiovascular responses induced by endothelin-1 in the perfused rat heart

Keiji Kusumoto, A Fujiwara, S Ikeda, T Watanabe, M Fujino
Eur J Pharmacology 296 (1996) 65-74 SSDI 0014-2999(95)00680-X

The effects of the endothelin receptor antagonist TAK-044 (cyclo[D-α-aspartyl-3-[(4-phenylpiperazin-l-yl)carbonyl]-L-alanyl-L-α-aspartyl-D-2-(2-thienyl)-glycyl-L-leucyl-D-tryptophyl] disodium salt) and BQ-123 (cyclo[D-Asp-Pro-D-VaI-Leu-D-Trp]) were studied in the rat heart to characterize the receptor subtypes responsible for the cardiovascular actions of endothelin-1. Endothelin-1 induced a transient decrease and subsequent increase in perfusion pressure in perfused rat hearts, and increased left ventricular developed pressure. TAK-044 diminished these endothelin-l-induced responses (100 pmol/heart) with IC50 values of 140, 57 and 1.3 nM, respectively. BQ-123 (1-30/µM) partially inhibited the endothelin-l-induced hypertension (30-40%) in the rat heart, and failed to inhibit the hypotension. The positive inotropic effect of endothelin-1 was abolished by BQ-123. Neither indomethacin (10/µM) nor N’°-nitro-L-arginine methyl ester (100/_pM) attenuated the endothelin-l-induced hypotension. TAK-044 and BQ-123 attenuated the positive inotropic effect of endothelin-1 in rat papillary muscles. In rat cardiac membrane fractions, TAK-044 and BQ-123 inhibited [¹²⁵I]endothelin-1 binding to endothelin ET A receptors with IC₅₀ values of 0.39 + 0.6 and 36 + 9 nM, respectively, whereas only TAK-044 potently blocked the endothelin ET B receptor subtype (IC50 value: 370 + 180 nM). These results suggest that endothelin-1 modulates cardiovascular functions in the rat heart by activating both endothelin ET A and endothelin ET B receptors, all of which are sensitive to TAK-044.

Molecular Pharmacology and Pathophysiological Significance of Endothelin

Katsutoshi Goto, Hiroshi Hama and Yoshitoshi Kasuya
Jp J Pharmacol 1996; 72: 261-290

Since the discovery of the most potent vasoconstrictor peptide, endothelin, in 1988, explosive investigations have rapidly clarified much of the basic pharmacological, biochemical and molecular biological features of endothelin, including the presence and structure of isopeptides and their genes (endothelin- 1, -2 and -3), regulation of gene expression, intracellular processing, specific endothelia converting enzyme (ECE), receptor subtypes (ET_A and ET_B), intracellular signal transduction following receptor activation, etc. ECE was recently cloned, and its structure was shown to be a single transmembrane protein with a short intracellular N-terminal and a long extracellular C-terminal that contains the catalytic domain and numerous N-glycosylation sites. In addition to acute contractile or secretory actions, endothelin has been shown to exert long-term proliferative actions on many cell types. In this case, intracellular signal transduction appears to converge to activation of mitogen-activated protein kinase. As a recent dramatic advance, a number of non-peptide and orally active receptor antagonists have been developed. They, as well as current peptide antagonists, markedly accelerated the pace of investigations into the true pathophysiological roles of endogenous endothelin-1 in mature animals.

The discovery of endothelin in 1988 soon triggered explosive investigations of a worldwide scale, presumably due to its unusual characteristics; i.e., marked potency and long-lasting pressor actions. As a result, most of the basic problems concerned with the science of endothelin have rapidly been solved; e.g., features and regulations of the expression of endothelin genes, biosynthetic pathways including characterization and cloning of endothelin converting enzyme, pharmacological, biochemical and molecular-biological identification of endothelin receptor subtypes, intracellular signal transduction following receptor activation, and discovery of various receptor agonists and antagonists. In addition to its potent cardiovascular actions, endothelin-1 shows a wide variety of biological effects, including contraction of nonvascular smooth muscle (intestinal, tracheal, broncheal, mesangial, bladder, uterine and prostatic smooth muscle), stimulation of neuropeptides, pituitary hormone and atrial natriuretic peptide release and aldosterone biosynthesis, modulation of neurotransmitter release, and increase of bone resorption. Furthermore, endothelin-1 has mitogenic properties and causes proliferation and hypertrophy of a number of cell types, including vascular smooth muscle cells, cardiac myocytes, mesangial cells, bronchial smooth muscle cells and fibroblasts. Endothelin-1 also induces the expression of several protooncogenes (c fos, C -Jun, c-myc, etc.).

These actions, whereby endothelin- 1 might influence the development of cellular hypertrophy/hyperplasia, are of potential significance in pathophysiological conditions associated with long-term changes in cardiovascular tissues, e.g., hypertension, myocardial infarction, chronic heart failure, vascular restenosis following balloon angioplasty, and atherosclerosis. These pathophysiological conditions are usually associated with increased plasma levels of endothelin-1, although the correlation is relatively poor. Nevertheless, a considerable increase in the tissue content of endothelin-1 has been gradually uncovered in many cases of these conditions. Even if the concentration of endothelin-1 at the cell surface is not high enough to induce contraction, it is well known that subthreshold concentrations of endothelin will enhance or potentiate the contraction produced by other vasoconstrictors (e.g., norepinephrine, serotonin, angiotensin II), indicating the existence of cross-talk among various vasoactive substances. Another important cross-talk among these substances may be mutual enhancement or inhibition of their expression in various tissues. In addition to these interactions, the true physiological and/or pathophysiological roles of each of the endothelin family peptide and receptor subtypes remain to be investigated.

Hydrogen Sulfide and Endothelium-Dependent Vasorelaxation

Jerzy Bełtowski, and Anna Jamroz-Wiśniewska
Molecules 2014, 19, 21183-21199; http://dx.doi.org:/10.3390/molecules191221183

In addition to nitric oxide and carbon monoxide, hydrogen sulfide (H₂S), synthesized enzymatically from L-cysteine or L-homocysteine, is the third gasotransmitter in mammals. Endogenous H₂S is involved in the regulation of many physiological processes, including vascular tone. Although initially it was suggested that in the vascular wall H₂S is synthesized only by smooth muscle cells and relaxes them by activating ATP-sensitive potassium channels, more recent studies indicate that H₂S is synthesized in endothelial cells as well. Endothelial H₂S production is stimulated by many factors, including acetylcholine, shear stress, adipose tissue hormone leptin, estrogens and plant flavonoids. In some vascular preparations H₂S plays a role of endothelium-derived hyperpolarizing factor by activating small and intermediate-conductance calcium-activated potassium channels. Endothelial H₂S signaling is up-regulated in some pathologies, such as obesity and cerebral ischemia-reperfusion. In addition, H₂S activates endothelial NO synthase and inhibits cGMP degradation by phosphodiesterase thus potentiating the effect of NO-cGMP pathway. Moreover, H₂S-derived polysulfides directly activate protein kinase G. Finally, H₂S interacts with NO to form nitroxyl (HNO)—a potent vasorelaxant. H₂S appears to play an important and multidimensional role in endothelium-dependent vasorelaxation.

GPCR modulation by RAMPs

Debbie L. Hay, David R. Poyner, Patrick M. Sexton
Pharmacology & Therapeutics 109 (2006) 173 – 197
http://dx.doi.org:/10.1016/j.pharmthera.2005.06.015

Our conceptual understanding of the molecular architecture of G-protein coupled receptors (GPCRs) has transformed over the last decade. Once considered as largely independent functional units (aside from their interaction with the G-protein itself), it is now clear that a single GPCR is but part of a multifaceted signaling complex, each component providing an additional layer of sophistication. Receptor activity modifying proteins (RAMPs) provide a notable example of proteins that interact with GPCRs to modify their function. They act as pharmacological switches, modifying GPCR pharmacology for a particular subset of receptors. However, there is accumulating evidence that these ubiquitous proteins have a broader role, regulating signaling and receptor trafficking. This article aims to provide the reader with a comprehensive appraisal of RAMP literature and perhaps some insight into
the impact that their discovery has had on those who study GPCRs.

RAMPs were first identified during attempts to expression clone a receptor for the neuropeptide calcitonin gene related peptide (CGRP; McLatchie et al., 1998). Historical evidence had suggested that CGRP acted through a GPCR, as its binding had proven sensitive to GTP analogues and stimulation of various tissues and cells led to the accumulation of cAMP, suggesting activation of a Gs-coupled GPCR. However, attempts to clone such a receptor proved difficult. A putative canine CGRP receptor, RDC-1, was identified in 1995, but the original findings have not been replicated and current IUPHAR guidelines do not consider this receptor a genuine CGRP receptor (Kapas & Clark, 1995; Poyner et al., 2002). Shortly afterward, a further orphan receptor (CL, a close homologue of the calcitonin receptor) was shown to be activated by CGRP when transfected into HEK293 cells (Aiyar et al., 1996). This finding posed something of a conundrum since earlier attempts to examine the function of this receptor (or its rat homologue) in Cos 7 cells had not given positive results with CGRP.
Given the apparent functionality of the human CL receptor in HEK293 cells, the rat homologue was also transfected into this cell type and now responded to CGRP (Han et al., 1997). The authors speculated that there was a factor present in HEK293 cells that conferred high affinity for CGRP on the receptor.

In 1998, McLatchie and colleagues confirmed this speculation and provided new insights into the way that GPCRs and their pharmacology can be regulated (McLatchie et al., 1998). It was discovered that a novel family of single transmembrane domain proteins, termed RAMPs, was required for functional expression of CL at the cell surface, explaining why it had been so difficult to observe CGRP binding or function when CL was transfected into cells lacking RAMP expression (Fluhmann et al., 1995; Han et al., 1997; McLatchie et al., 1998). RAMPs were first identified from a library derived from SK-N-MC cells, cells known to express CGRP receptors. An expression-cloning strategy was utilized, whereby an SK-N-MC cDNA library was transcribed and the corresponding cRNA was used for injection into Xenopus oocytes. Cystic
fibrosis transmembrane regulator chloride conductance, a reporter for cAMP formation, was strongly potentiated by a single cRNA pool (in the presence of CGRP). Subsequently, a single cDNA encoding a 148-amino-acid protein comprising RAMP1 was isolated. The structure of the protein was unexpected, as it was not a GPCR and it did not respond to CGRP in mammalian cells. Thus, it was postulated that RAMP1 might potentiate CGRP receptors. A CL/RAMP1 co-transfection experiment supported this hypothesis.

CGRP/AM on the outside of the cell and did not simply act as anchoring/chaperone proteins for CL. RAMPs therefore provide a novel mechanism for modulating receptor–ligand specificity. The unique pharmacological profiles supported by RAMPs are discussed in later sections.

Fig. (not shown). CGRP1 receptor-specific small molecule antagonists. The small molecule antagonist BIBN4096 BS (brown) is a specific antagonist of the CGRP1 receptor, acting at the interface between RAMP1 and the CL receptor to inhibit CGRP action. At least part of the binding affinity for BIBN4096 BS arises from interaction with Trp74 (red) of RAMP1. In contrast, antagonists that bind principally to the CL component of the complex will not discriminate between different CL/RAMP complexes.

The classic function attributed to RAMPs is their ability to switch the pharmacology of CL, thus providing a novel mechanism for modulating receptor specificity. Thus, the CL/RAMP1 complex is a high affinity CGRP receptor, but in the presence of RAMP2, CL specificity is radically altered, the related peptide AM being recognized with the highest affinity and the affinity for CGRP being reduced ¨100-fold. While AM is the highest affinity peptide, CGRP is recognized with moderate, rather than low affinity. Indeed, depending on the species and the form of CGRP (h vs. a), the separation between the 2 peptides can be as little as 10-fold (Hay et al., 2003a). This may particularly be true if receptor components of mixed species are used. The detailed pharmacology of the CGRP and AM receptors formed by RAMP interaction with CL has recently been reviewed (Born et al., 2002; Poyner et al., 2002; Hay et al., 2004; Kuwasako et al., 2004).

Fig. (not shown). The broadening spectrum of RAMP–receptor interactions. RAMPs can interact with multiple receptor partners. All RAMPs interact with the calcitonin receptor-like receptor (CL-R), the calcitonin receptor (CTR), and the VPAC1 receptor, while the glucagon and PTH1 receptors interact with RAMP2, the PTH2 receptor with RAMP3, and the calcium sensing receptor (CalS-R) with RAMP1 or RAMP3. The consequence of RAMP interaction varies. For the CL and CalS receptors, RAMPs play a chaperone role, allowing cell surface expression. For the CL and calcitonin receptors, RAMP interaction leads to novel receptor binding phenotypes . There is also evidence that RAMP interaction will modify signaling, and this has been seen for the VPAC1–RAMP2 heterodimer and for calcitonin receptor/RAMP complexes. In many instances, however, the consequence of RAMP interaction has yet to be defined.

Overall, the distribution data presented so far are supportive of the hypothesis that RAMP and CL or calcitonin receptor combinations are able to account for the observed CGRP, AM, and AMY pharmacology. A salient point for CGRP receptors relates to the cerebellum, where the lack of CL mRNA in some studies despite abundant CGRP binding has prompted speculation of alternative CGRP receptors (Oliver et al., 2001; Chauhan et al., 2003). Nevertheless, this apparent lack is study dependent and CL has been identified in cerebellum in other studies.

Some consideration has been given to the potential role that RAMPs may have in modifying receptor behaviors other than ligand binding pharmacology. An additional functional consequence might be that of alteration of receptor signaling characteristics.

While there is currently little evidence for signaling modifications of CL-based receptors in association with RAMPs, a completely different paradigm is evident for the VPAC1 receptor. This receptor has strong interactions with all 3 RAMPs, but its pharmacology, in terms of agonist binding, does not appear to be modified by their presence. On the other hand, there was a clear functional consequence of RAMP2 overexpression with the VPAC1 receptor where PI hydrolysis was specifically augmented relative to cAMP, which did not change. The potency of the response (EC50 of vasoactive intestinal peptide) was not altered, but the maximal PI hydrolysis response was elevated in the presence of RAMP2 . It has been suggested that this may reflect a change in compartmentalization of the receptor signaling complex. Such augmentation was not evident for the interaction of the VPAC1 receptor with RAMP1 or RAMP3; in these cases, the outcome of heterodimerization may be more subtle or involve the modification of different receptor parameters such as trafficking.

RAMPs transformed our understanding of how receptor pharmacology can be modulated and provided a novel mechanism for generating receptor subtypes within a subset of family B GPCRs. Their role has now broadened and they have been shown to interact with several other family B GPCRs, in 1 case modifying signaling parameters. There is now evidence to suggest that their interactions also reach into family C, and possibly family A, GPCRs, indicating that their function may not be restricted to modulation of a highly specific subset of receptors. Indeed, many aspects of RAMP function remain poorly understood, and the full extent of their action remains to be explored.

Receptor activity modifying proteins

Patrick M. Sexton, Anthony Albiston, Maria Morfis, Nanda Tilakaratne
Cellular Signalling 13 (2001) 73-83 PII: S0898-6568(00)00143-1

Our understanding of G protein-coupled receptor (GPCR) function has recently expanded to encompass novel protein interactions that underlie both cell-surface receptor expression and the exhibited phenotype. The most notable examples are those involving receptor activity modifying proteins (RAMPs). RAMP association with the calcitonin (CT) receptor-like receptor (CRLR) traffics this receptor to the cell surface where individual RAMPs dictate the expression of unique phenotypes. A similar function has been ascribed to RAMP interaction with the CT receptor (CTR) gene product. This review examines
our current state of knowledge of the mechanisms underlying RAMP function.

It is now evident that RAMPs can interact with receptors other than CRLR. Expression of amylin receptor phenotypes requires the coexpression of
RAMPs with the CTR gene product. However, as seen in CRLR, the phenotype engendered by individual RAMPs was distinct. In COS-7 or rabbit aortic endothelial cells (RAECs), RAMP1 and RAMP3 induced amylin receptors that differ in their affinity for CGRP, while RAMP2 was relatively ineffective in inducing amylin receptor phenotype. RAMP2 can also induce an amylin receptor phenotype, which is distinct from either the RAMP1- or RAMP3-induced receptors. However, the efficacy of RAMP2 was highly dependent upon the cellular background and the isoform of CTR used in the study.

In humans, the major CTR variants differ by the presence or absence of a 16 amino acid insert in the first intracellular domain, with the insert negative isoform (hCTRI1ÿ) being the most commonly expressed form and the variant used for initial studies with RAMPs. Unlike hCTRI1ÿ, cotransfection of the hCTRI1+ variant with any of the RAMPs into COS-7 cells caused strong induction of amylin receptor phenotype. The hCTR isoforms differ in their ability to activate signaling pathways (presumably due to an effect on G protein coupling) and to internalize in response to agonist treatment, which may suggest a role for G proteins in the ability of RAMPs to alter receptor phenotype.

There are at least three potential consequences of RAMP interaction with its associating receptors. The first is trafficking of receptor protein from an intracellular compartment to the cell surface. The second is an alteration in
the terminal glycosylation of the receptor, and the third is alteration of receptor phenotype, presumably through a direct or indirect effect on the ligand-binding site.

potential actions of RAMPs

Schematic diagram illustrating potential actions of RAMPs. (A) RAMPs facilitate the trafficking of CRLR from an intracellular compartment to the cell surface. (B) RAMP1 (but not RAMP2 or RAMP3) modifies the terminal glycosylation
of CRLR. (C) The cell surface RAMP1±CRLR complex is a Type 1 CGRP receptor, which displays a 1:1 stoichiometry. (D,E) Cell surface RAMP2±CRLR and RAMP3±CRLR complexes are adrenomedullin receptors. (F,G) For at least RAMP1 and RAMP3, RAMPs form stable homodimers, although the function
of these complexes is unknown. (H) Unlike CRLR, the CTR gene product is trafficked to the cell surface in the absence of RAMPs, where it displays classical CTR phenotype. (I,J) RAMP1± and RAMP3±CTR complexes form distinct amylin receptors. RAMP2 can also generate a separate amylin receptor phenotype (not illustrated). (C ±E,I,J) RAMPs are trafficked with either receptor to the plasma membrane. (K) For all three RAMP±CRLR complexes, agonist treatment causes clathrin-mediated internalization of both CRLR and RAMP.
(L) The majority of the internalized complex is targeted to the lysosomal-degradation pathway.

The data from Zumpe et al. suggest that RAMP2 interacts more weakly with the hCTRI1ÿ than RAMP1, and that the affinity of this interaction derives principally from the transmembrane domain/C-terminus (Ct) of the RAMPs. As RAMP3 induces an amylin receptor phenotype in COS-7 cells where RAMP2 is relatively weak, it is inferred that RAMP3 interaction with the hCTRI1ÿ is probably greater than that of RAMP2. Nonetheless, this has not been examined empirically. Given the recent data suggesting a potential role for G protein coupling in expression of RAMP-induced phenotype, it is also possible that the strength of RAMP interaction is, at least partially, dictated by receptor-G protein or RAMP-G protein interaction.

The discovery of RAMPs has led to a greater understanding of the nature of receptor diversity. However, although much progress has been made into elucidating the molecular mechanism of RAMP action, emerging data continue to open up new areas for investigation. These include identification of other RAMP-interacting receptors, understanding of the role of specific G proteins in RAMP-receptor function and the potential importance of RAMP regulation in disease progression. It also seems likely that the RAMP-receptor interface can provide a useful target for future drug development.

Cardiovascular endothelins: Essential regulators of cardiovascular homeostasis

Friedrich Brunner, C Bras-Silva, AS Cerdeira, AF Leite-Moreira
Pharmacology & Therapeutics 111 (2006) 508 – 531
http://dx.doi.org:/10.1016/j.pharmthera.2005.11.001

The endothelin (ET) system consists of 3 ET isopeptides, several isoforms of activating peptidases, and 2 G-protein-coupled receptors, ETA and ETB, that are linked to multiple signaling pathways. In the cardiovascular system, the components of the ET family are expressed in several tissues, notably the vascular endothelium, smooth muscle cells, and cardiomyocytes. There is general agreement that ETs play important physiological roles in the regulation of normal cardiovascular function, and excessive generation of ET isopeptides has been linked to major cardiovascular pathologies, including hypertension and heart failure. However, several recent clinical trials with ET receptor antagonists were disappointing.

In the present review, the authors take the stance that ETs are mainly and foremost essential regulators of cardiovascular function, hence that antagonizing normal ET actions, even in patients, will potentially do more harm than good. To support this notion, we describe the predominant roles of ETs in blood vessels, which are (indirect) vasodilatation and ET clearance from plasma and interstitial spaces, against the background of the subcellular mechanisms mediating these effects. Furthermore, important roles of ETs in regulating and adapting heart functions to different needs are addressed, including recent progress in understanding the effects of ETs on diastolic function, adaptations to changes in preload, and the interactions between endocardial-derived ET-1 and myocardial pump function. Finally, the potential dangers (and gains) resulting from the suppression of excessive generation or activity of ETs occurring in some cardiovascular pathological states, such as hypertension, myocardial ischemia, and heart failure, are discussed.

Figure (not shown): Synthesis of ET and its regulation. The release of active ET-1 is controlled via regulation of gene transcription and/or endothelin converting enzyme activity. ET-1 synthesis is stimulated by several factors, of which hypoxia seems to be the most potent in humans (see text). ET-1 formation is down-regulated by activators of the NO/cGMP pathway and other factors.

Figure (not shown): Vascular actions of ET. In healthy blood vessels, the main action of ET-1 is indirect vasodilatation mediated by ETB receptors located on endothelial cells. Their activation generates a Ca2+ signal via PLC that turns on the generation of NO, prostacyclin, adrenomedullin, and other mediators that are powerful relaxants of smooth muscle. On the other hand, binding of ET-1 to ETA receptors located on smooth muscle cells will lead to vascular contraction (physiological effect) and/or wall thickening, inflammation, and tissue remodeling (pathological effects). These latter effects may partly be mediated by vascular ETB2 receptors in certain disease states. Smooth muscle cell signaling involves DAG formation, PKC activation, and extracellular Ca2+ recruited via different cation channels. The specificity of the cellular response resides at the level of G proteins, that is, G-as or G-aq in the case of ETA, G-ai or G-aq for ETB.

signal transduction mechanisms involved in ET-1-mediated positive (left) and negative (right) inotropic effects

Summary of proposed signal transduction mechanisms involved in ET-1-mediated positive (left) and negative (right) inotropic effects. Left: Stimulation of ETA receptors causes Gq protein-directed activation of PLC, formation of IP3 and DAG, and activation of NHE-1. Increased contractile force is the result of (i) Ca2+ release from the sarco(endo)plasmic reticulum, (ii) sensitization of cardiac myofilaments to Ca2+ due to cellular alkalosis, and (iii) increased Ca2+ influx through the NCX operating in reverse mode. The contribution of voltage-gated L-type Ca2+ channels to the systolic Ca2+ transient is unknown, as is the role of myocyte ETB2 receptors. Right: The ET receptor subtypes mediating negative inotropic effects are poorly known. Two main signaling mechanisms involve (i) inhibition of adenylyl cyclase (AC), guided by a G protein, of unknown binding preference, which results in decreased levels of cAMP; (ii) cGMP-mediated activation of phosphatases that dephosphorylate putative targets resulting from cAMP/protein kinase A (PKA) activation. Other kinases like PKC and PKG have also been implicated in accentuated force antagonism.

Adrenomedullin (11–26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla

Kazuo Kitamuraa,*, Eizaburo Matsuia, Jhoji Katoa, Fumi Katoha
Peptides 22 (2001) 1713–1718 PII: S0196-9781(01)00529-0

Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from pheochromocytoma tissue. Both the ring structure and the C-terminal amide structure of AM are essential for its hypotensive activity. We have developed an RIA which recognizes the ring structure of human AM. Using this RIA, we have characterized the molecular form of AM in bovine adrenal medulla. Gel filtration chromatography revealed that three major peaks of immunoreactive AM existed in the adrenal medulla. The peptide corresponding to Mr 1500 Da was further purified to homogeneity. The peptide was determined to be AM (11–26) which has one intramolecular disulfide bond. Amino acid sequences of bovine AM and its precursor were deduced from the analyses of cDNA encoding bovine AM precursor. The synthetic AM (11–26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. The hypertensive activity lasted about one minute, and a dose dependent increase in heart rate was also observed. The present data indicate that AM (11–26) is a major component of immunoreactive AM in bovine adrenal medulla and shows pressor activity.

The pressor effect of AM(11–26) was examined by methods similar to those reported for Neuropeptide Y.

We have established a sensitive RIA system using a monoclonal antibody which recognizes the ring structure of human AM. Human AM antiserum recognized the peptide with high affinity at a final dilution of 1:2,800,000. The half maximal inhibition of radioiodinated ligand binding by human AM was observed at 10 fmol/tube. From 1 to 128 fmol/tube of AM was measurable by this RIA system. The intra- and inter-assay coefficients of variance were less than 6% and 9%, respectively. This RIA had 100% cross-reactivity with human AM(13–31), (1–25), (1–52)Gly and AM(1–52)CONH2, but less than 1% cross-reactivity with rat AM.

Sephadex G-50 gel-filtration of strongly basic peptide extract (SP-III) in bovine adrenal medulla identified three major peaks of immunoreactive AM. One emerged at the identical position of authentic AM, the other two unknown peaks were eluted later at molecular weights estimated to be 3000 and 1500 Da, respectively. The peptide corresponding to Mr 1500 Da was further purified.

The purified peptide (20 pmol) was subjected to a gas phase sequencer, and the amino acid sequence was determined up to the 16th residue, which was found to be C terminus . It was found that the purified peptide was AM (11–26). The structure of AM (11–26) was confirmed by chromatographic comparison with native AM (11–26) as well as a synthetic AM (11–26), which has one intramolecular disulfide bond.

3 clones were isolated, and the clone designated pBAM-2, which harbored the longest insert of 1,438 base, was used for sequencing. The bovine AM cDNA contained a single open reading frame encoding a putative 188 amino acid polypeptide. The first 21-residue peptide is thought to be a signal peptide. The bovine AM propeptide contains three signals of dibasic amino acid sequences, Lys-Arg or Arg-Arg. The first Lys-Arg followed proadrenomedullin N-terminal 20 peptide (PAMP) sequences. AM is located between the second signal of Lys-Arg and the third signal of Arg-Arg. Gly residues, which are donors of C-terminal amide structure of PAMP and AM, are found before the first and third signal of Lys-Arg and Arg-Arg. Bovine AM consists of 52 amino acids and is identical to human AM with exception of four amino acids. Bovine PAMP consists of 20 amino acids and is identical to human PAMP with exception of one amino acid. The present cDNA sequence encoding bovine AM precursor is almost identical to those of the reported AM cDNA sequences from bovine aortic endothelial cells. However, a difference in one amino acid was found in the sequences of signal peptide. In addition, three different residues of nucleotides were found in the noncoding region of cDNA encoding bovine preproadreno-medullin.

AM(11–26) elicited a potent hypertensive effects in unanesthetized rats.
When AM(11–26) at 20 nmol/kg was injected i.v., the maximum increase of mean blood pressure was 50 7.1 mmHg. Similarly, the synthetic AM(11–26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. (Blood pressure increase; F(3, 20 = 13.845, P < 0.0001). Injection of saline did not affects blood pressure and heart rate. The hypertensive activity lasted about 70 s, and a dose dependent increase of heart rate was also observed (Heart rate increase; F(3, 20) = 6.151, P = 0.0039).

We have isolated and characterized bovine AM(11–26) from bovine adrenal medulla as an endogenous peptide. The hallmark biological effects of AM are vasodilation and hypotensive effects in the vascular systems of most species. The mature form of AM has one ring structure formed by an intramolecular disulfide bond and a C terminal amide structure, both of which are essential for the hypotensive and other biological activities of AM. Watanabe et al. reported that the synthetic N-terminal fragment of human AM, AM (1–25)COOH and other related peptides, show vasopressor activity in anesthetized rats. The present purification and characterization of AM(11–26) indicate that the ring structure of AM may function as a biologically active endogenous peptide. The peptide corresponding to Mr 1,500 Da was further purified to homogeneity.

The purified peptide was found to be AM(11–26) which has one intramolecular disulfide bond. The structure of AM(11–26) was confirmed by chromatographic comparison with native AM(11–26) as well as a synthetic specimen which was prepared according to the determined sequence. The structure of bovine AM and related peptides were determined by cDNA analysis encoding bovine AM. Bovine AM consists of 52 amino acids whose sequence is identical to the human sequences with the exception of four amino acids. Furthermore, according to the cDNA analysis and chromatographic comparison of the synthetic AM(11–26) and purified AM, is now determined to be cystine. It should be noted that the structure of bovine AM(11–26) is identical to human AM(11–26).

It is well known that many peptide hormones and neuropeptides are processed from larger, biologically inactive precursors by the specific processing enzyme. It usually recognizes pairs of basic amino acids, processing signals, such as primarily Lys-Arg and Arg-Arg. AM (11–26) is not flanked by such a processing signal, but it was reproducibly observed in bovine adrenal medulla peptide extract. The molar ratio of AM(11–26)/AM was estimated to be 40%. The ratio varied from 5% to 50% according to the individual specimen, but the minor peak corresponding to 1,500 Da was reproducibly observed, suggesting that AM(11–26) is an endogenous peptide. It is likely that AM(11–26) is biosynthesized from AM or AM precursor by a specific enzyme.

In contrast to AM, synthetic bovine AM(11–26) caused potent hypertensive effects in unanesthetized rats. The hypertensive activity of AM(11–26) seems to be comparable to that of AM(1–25) as reported by Watanabe et al. It was unexpected that AM(11–26) would cause a dose dependent increase of heart rate in unanesthetized rats because vasopressor activity normally causes bradycardia through baroreceptor activation. The hypertensive mechanism is not fully understood, but it has been reported that the vasopressor effect of AM(1–25) might be caused by the release of endogenous catecholamine. We speculate that the released catecholamine counters the baroreceptor function resulting in an increased heart rate and blood pressure. It is possible that AM(11–26) participates in blood pressure control as an endogenous peptide.

A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides.

Tanenao Eto
Peptides 22 (2001) 1693–1711 PII: S0196-9781(01)00513-7

Adrenomedullin (AM), identified from pheochromocytoma and having 52 amino acids, elicits a long-lasting vasodilatation and diuresis. AM is mainly mediated by the intracellular adenylate cyclase coupled with cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) -cyclic guanosine monophosphate (cGMP) pathway through its specific receptor. The calcitonin receptor-like receptor (CLCR) and receptor-activity modifying protein (RAMP) 2 or RAMP3 models have been proposed as the candidate receptor. AM is produced mainly in cardiovascular tissues in response to stimuli such as shear stress and stretch, hormonal factors and cytokines. Recently established AM knockout mice lines revealed that AM is essential for development of vitelline vessels of embryo. Plasma AM levels elevate in cardiovascular diseases such as heart failure, hypertension and septic shock, where AM may play protective roles through its characteristic biological activities. Human AM gene delivery improves hypertension, renal function, cardiac hypertrophy and nephrosclerosis in the hypertensive rats. AM decreases cardiac preload and afterload and improves cardiac contractility and diuresis in patients with heart failure and hypertension. Advances in gene engineering and receptor studies may contribute to further understandings of biological implication and therapeutic availability of AM.

AM acts as a circulating hormone as well as elicits multiple biological activities in a paracrine or autocrine manner. Among them the most characteristic biological activity of AM is a very powerful hypotensive activity caused by dilatation of resistance vessels. A sensitive and specific radioimmunoassay demonstrated that AM circulates in blood and occurs in a variety of tissues. Plasma AM levels elevate in various diseases including cardiovascular and renal disorders or septic shock. Thus, AM may be involved in pathophysiological processes in these diseases, especially in disorders controlling circulation and body fluid. In this short review, the history of AM and proadrenomedullin N-terminal 20 peptide (PAMP) will be reviewed with special references to biological properties and function, receptors, gene engineering and clinical viewpoints. This review includes oral presentations from the aforementioned symposium; some of which have not yet been published. These unpublished oral presentations are quoted in this paper from the abstracts of this symposium.

Preproadrenomedullin, which consists of 185 amino acids and contains a 21-amino acid signal peptide, is processed to synthesize proadrenomedullin and finally AM. In the proadrenomedullin, a unique twenty amino acid sequence followed by a typical amidation signal known as Gly-Lys-Arg, is included in the N-terminal region. This novel 20 residues peptide with carboxyl terminus of Arg-CONH2 is also present in vivo and is termed “proadrenomedullin N-terminal 20 peptide (PAMP).” PAMP elicits a potent hypotensive activity in anesthetized rats.

Although widely distributed in the adenophypophysis and the neural lobe of pituitary glands, AM and PAMP occur in cell-specific, but not overlapping, patterns in the anterior pituitary. This cell-specific expression of each peptide may be explained by differences in posttranslational processing of AM gene. As such, potential pituitary specific transcription factor binding sites, gonadotropic-specific element (GSE) and a binding site for steroidogenic factor-l (SF-1) are found in the 5flanking region of human and mouse AM gene. SF-1 is a member of the steroid receptor superfamily that has been shown necessary for gonadotrope differentiation within the pituitary. In addition, one putative binding sequence of Pit-1 has been reported in mouse AM gene promoter position.

A specific AM binding protein (AMBP-1) in human plasma was isolated and the purified protein was identified as human complement factor H. AM and factor H interaction may interfere with the radioimmunoassay quantification of circulating AM. Factor H enhances AM-mediated induction of cAMP in fibroblast; augments the AM-mediated growth of a cancer cell line; and suppresses the bactericidal capability of AM on Escherichia coli. Conversely, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I. The augmentation of AM actions indicates that AMBP may facilitate the binding of AM to its receptor. In addition, the existence of AMBP suggests that large amounts of AM may circulate bound to this plasma protein.

In rat vascular smooth muscle cells, the CGRP, CGRP1 receptor antagonist, competitively inhibits the intracellular accumulation of cAMP induced by AM. Vasodilation of the rat mesenteric vascular bed elicited by AM and CGRP is also blocked by CGRP. Similar effects of CGRP are observed in the isolated rat heart and its microvasculature. Thus, CGRP1 receptor can mediate some effects of AM, but AM has a low affinity at CGRP2 receptor. Two distinct AM labeled bands with a molecular weight of 120 and 70 kDa was reported in the cultured rat vascular smooth muscle cell membrane. Therefore, the binding specificity and characteristics of the AM receptor may differ regionally by organ or tissue.

Two more RAMP proteins, RAMP2 and RAMP3, were discovered from database searches. These proteins share approximately 30% homology with RAMP1. Co-expression of RAMP2 or RAMP3 with CRLR appears to constitute AM receptor. RAMP2 and RAMP3 are indistinguishable in terms of AM binding. The RAMPs are required to transport CRLR to the plasma membrane. RAMP1 presents CRLR as a mature glycoprotein at the cell surface to form a CGRP receptor. However, receptors transported by RAMP2 or RAMP3 are core glycosylated and then become AM receptors. Three putative N-glycosylation sites Asn 60, Asn 112 and Asn 117 are present in the amino-terminal extracellular domain of the human CRLR. When the glycosylation of a myc-tagged CRLR was inhibited, specific ¹²⁵I-CGRP and -AM binding were blocked in parallel. Substitution of the Asn 117 by threonine abolished CGRP and AM binding in the face of intact N-glycosylation and cell surface expression. RAMPs are accessory proteins of CTR and CRLR at the cell surface where they define AM, amylin, calcitonin and CGRP specificity.

The receptor component protein (RCP) was cloned on the basis of its ability to potentiate the endogenous Xenopus oocyte CGRP receptor. RCP is a cytosolic protein with no similarity to RAMPs, consists of a hydrophobic 146 amino acids and is obtained from the Corti organ of guinea pig. RCF plays an essential role for signal-transduction of CGRP and AM, and interacts with CRLR directly within the cells. Thus, a functional AM or CGRP receptor seems to consist of at least three proteins: CRLR, RAMP and RCP, coupling the receptor to the intracellular signal-transduction pathway.

By using a chimera of the CRLR and green fluorescent protein (GFP), the study demonstrated that CRLR-GFP failed to generate responses to CGRP or AM without RAMP2 or RAMP3 in HEK 293 cells. When coexpressed with RAMP2 or RAMP3, CRLR-GFP appeared on the cell membrane and activated an intracellular cAMP production and calcium mobilization. Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs.

The discovery of RAMPs has promoted our understandingthat some of the biological activities of AM are blocked by CGRP receptor antagonist, whereas other biological activities are blocked only by AM receptor antagonist, which indicates the possible existence of AM receptor in dual nature. RAMP association with CRLR traffics this receptor to the cell surface where individual RAMPs dictate the expression of unique phenotypes such as CGRP receptor or AM receptors. Apart from receptor trafficking and glycosylation, the RAMPs may interact directly with the receptors in the cell surface modifying their affinities for the ligands.

Since AM was discovered by monitoring the elevating activity of cAMP in rat platelets, cAMP appears to be its major second messenger. Dose-dependent intracellular production of cAMP induced by AM has been confirmed in various tissues and cells. Moreover, information on the role of NO in alternative signal-transduction pathways for AM is available.

The vasodilating effect of AM is reduced by the blockade of NO synthetase activity with NG-nitro-L-arginine methylester (L-NAME), indicating that NO may at least partly contribute to the AM-induced vasodilation. However, the degree of NO contribution to vasodilation varies depending upon the organ or tissue and the species. NO synthetase inhibitor in the pulmonary vascular beds of rat significantly attenuates the AM-induced vasodilation, but it does not occur in cats. Thus, NO seems to be an important AM mediator despite regional and interspecies variation.

In bovine aortic endothelial cells, AM increases intracellular ionic calcium (Ca²⁺) and causes the accumulation of cAMP. This increase in intracellular Ca²⁺ may be involved in the activation of phospholipase C, thereby producing inducible NO synthetase and subsequently NO. NO transferred to medial smooth muscle cells may activate cGMP-mediating smooth muscle cells vasodilatation. In contrast, AM lowers both cytosolic Ca²⁺ and Ca²⁺ sensitivity in smooth muscle cells of pig coronary arteries and intracellular Ca²⁺ in rat renal arterial smooth muscle cells.

Among the multi-functional properties of AM, the most characteristic one is an intensive, long-lasting hypotension that is dose-dependent in humans, rats, rabbits, dogs, cats and sheep. AM dilates resistance vessels in the kidneys, brain, lung, hindlimbs in animals as well as in the mesentery. Moreover, AM elicits relaxation of ring preparations of the aorta and cerebral arteries. An i.v. injection of human AM to conscious sheep causes a dose dependent fall of blood pressure, an increase in heart rate and cardiac output with a small reduction in stroke volume, as well as a marked decrease in total peripheral resistance. Coronary blood flow increases in parallel with the increase in coronary conductance. These cardiovascular responses return to the control level by 40 min after the injection.

The low-dose infusion of AM administered to conscious sheep on a low-salt diet antagonizes the vasopressor actions of administered angiotensin II while stimulating cardiac output and heart rate. AM may control cardiovascular homeostasis in part through antagonism of the vasopressor action of angiotensin II. AM inhibits the secretion of endothelin-1 from the vascular endothelial cells and proliferation of vascular smooth muscle cells. In the cultured cardiomyocytes as well as cardiac fibroblasts, AM inhibits protein synthesis in these cells in an autocrine or a paracrine manner, which may result in modulating the cardiac growth. AM inhibits bronchial constriction induced by acetylcholine or histamine in a dose-dependent manner, indicating the important role of AM on airway function and its usefulness for the management of bronchial asthma. AM inhibits secretion of aldosterone from the adrenal cortex. When infused directly into the adrenal arterial supply of conscious sheep, AM directly inhibits the acute stimulation of aldosterone by angiotensin II, KCl and ACTH while not affecting basal or chronic aldosterone secretion or cortisol secretion stimulated by ACTH. AM co-exists in insulin-producing cells and it inhibits insulin secretion dose-dependently in isolated rat islets.

The N-terminal region of preproadrenomedullin, the precursor of AM, contains a unique 20-residue sequence followed by Gly-Lys-Arg, a typical amidation signal, which was termed as proadrenomedullin N-terminal 20 peptide (PAMP). PAMP was purified from porcine adrenal medulla and human pheochromo-cytoma by using radioimmunoassay for the peptide and its complete amino acid sequence was determined. In addition to the original form of PAMP [1–20], PAMP [9–20] has recently been purified from the bovine adrenal medulla. The amino acid sequences of both forms of PAMP are identical to amino acid sequences deduced by cDNA analysis and their carboxyl terminus of Arg is amidated. The distribution of PAMP is similar to that of human AM, due to the fact that PAMP as well as human AM is biosynthesized from an AM precursor.

AM is processed from its precursor, proadrenomedullin, as the intermediate or immature form, AM-glycine (AM[1–52]-COOH, immature AM). Subsequently, immature AM is converted to the biologically active mature form, AM [1–52]-CONH2 (mature AM) by enzymatic amidation. The AM circulating in the human blood stream (total AM), thus, consists of both mature AM and immature AM. In earlier studies, plasma AM levels were measured by using radioimmunoassay recognizing the entire AM molecule (AM [1–52]), which reflects plasma total AM levels, as previously described.

In healthy volunteers severe exercise elevates the plasma AM levels with an increase in plasma norepinephrine and exaggerated sympathetic nerve activity. In heart transplant recipients, maximal exercise induces an increase in plasma AM that is inversely related to mean blood pressure. AM, therefore, may participate in blood pressure regulation during exercise even after heart transplantation.

When compared with healthy controls, the plasma AM levels are increased in patients with a variety of diseases: congestive heart failure, myocardial infarction, renal diseases, hypertensive diseases, diabetes mellitus, acute phase of stroke, and septic shock.

Adrenomedullin and central cardiovascular regulation

Meghan M. Taylor, Willis K. Samson
Peptides 22 (2001) 1803–1807 PII: S0196-9781(01)00522-8

Adrenomedullin gene products have been localized to neurons in brain that innervate sites known to be important in the regulation of cardiovascular function. Those sites also have been demonstrated to possess receptors for the peptide and central administrations of adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) elevate blood pressure and heart rate in both conscious and anesthetized animals. The accumulated evidence points to a role of the sympathetic nervous system in these cardiovascular effects. These sympathostimulatory actions of AM and PAMP have been hypothesized to be cardioprotective in nature and to reflect the central nervous system (CNS) equivalent of the direct cardiostimulatory effects of the peptides in the periphery. This review summarizes the most recent data on the CNS actions of the adrenomedullin gene-derived peptides and suggests future strategies for the elucidation of the physiologic relevance of the already demonstrated, pharmacologic actions of these peptides.

Adrenomedullin and related peptides: receptors and accessory proteins

Roman Muff, Walter Born, Jan A. Fischer
Peptides 22 (2001) 1765–1772 PII: S0196-9781(01)00515-0
Adrenomedullin (AM), α- and β-calcitonin gene-related peptide (CGRP), amylin and calcitonin (CT) are structurally and functionally related peptides. The structure of a receptor for CT (CTR) was elucidated in 1991 through molecular cloning, but the structures of the receptors for the other three peptides had yet to be elucidated. The discovery of receptor-activity-modifying proteins (RAMP) 1 and -2 and their co-expression with an orphan receptor, calcitonin receptor-like receptor (CRLR) has led to the elucidation of functional CGRP and AM receptors, respectively. RAMP1 and -3 which are co-expressed with CTR revealed two amylin receptor isotypes. Molecular interactions between CRLR and RAMPs are involved in their transport to the cell surface. Heterodimeric complexes between CRLR or CTR and RAMPs are required for ligand recognition.

Pharmacological profiles of receptors of the adrenomedullin peptidefamily
AMR	AM>CGRP>>amylin=CT
CTR	CT>amylin>>CGRP=AM
CGRPR	CGRP>AM>>amylin=CT
AmylinR	AmylinsCTCGRP>>hCT>AM

Specific AM binding sites have been identified in many tissues including the heart, blood vessels, lung and spleen. Based on pharmacological evidence two receptor isotypes have been distinguished, for instance in rat astrocytes and NG108–15 cells. One AM receptor isotype recognizes CGRP and CGRP(8–37). The other receptor isotype specific for the AM ligand and antagonized by AM(22–52) does not recognize CGRP to any great extent. Both isotypes of the receptors have been shown to interact poorly with amylin and CT (Table). Biological actions of AM include vaso- and bronchodilation, and CNS transmitted inhibition of water intake.

CGRP receptors are widely distributed in the nervous and cardiovascular systems. To date, two isotypes have been described. On pharmacological evidence, CGRP1 receptors, such as those identified in human SK-N-MC neuroblastoma cells, recognize intact CGRP and CGRP(8–37) with similar potency, unlike a linear analog lacking the disulfide bridge. CGRP2 receptors,
on the other hand, interact with the linear analog but not with CGRP(8–37). These CGRP receptor isotypes cross-react with AM to some extent, but only minimally with amylin and CT. CGRP shares potent vasodilatory actions with AM, and has chronotropic and inotropic actions in the heart. The ionotropic actions are indirectly brought about via activation of the sympathetic nervous system. There is evidence to suggest the existence of α- or β-CGRP preferring receptor isotypes in both the central nervous system and peripheral tissues.

RAMP1, -2 and -3 are widely expressed, suggesting that RAMPs may have
important functions beyond those of the adrenomedullin family of receptors. To this end, RAMP1 and -3 are thought to reduce cell surface expression of angiotensin (AT) AT1 and AT2 receptors.

RAMP2 and CRLR are expressed in vascular smooth muscle cells, and RAMP1 expression was increased by dexamethasone. Moreover, increased levels of RAMP2 and CRLR were observed in the kidney and heart of rats with obstructive nephropathy and congestive heart failure, respectively. RAMP2
and CRLR levels were reduced, and RAMP3 levels were increased during lipopolysaccharide induced sepsis in rats.

The GABAB receptor 1 is retained as an immature glycoprotein in the cytosol unless co-expressed with GABAB receptor 2 isotype. Heterodimers of fully functional opioid receptors δ and κ result in a novel receptor displaying binding and functional properties distinct from those of the δ or κ receptors alone. Heterodimerization therefore facilitates receptor expression and defines ligand specificity also in G protein-coupled receptor families A and C. Moreover, heterodimers of metabotropic glutamate 1receptor (family C) and adenosine A1 receptors (family A) have been observed. As yet there is no evidence for homo or heterodimerization of family B receptors. Cysteines conserved in the extracellular N-terminal domain in all the receptors of family B and RAMPs suggest that RAMPs are truncated forms of receptors that interact as heterodimers with CRLR and CTR.

The discovery of RAMPs in combination with CRLR and CTR has led to the molecular identification of CGRP1, CGRP/amylin, AM and amylin receptor complexes. The physiological advantage of heterodimers between seven transmembrane domain receptors and the RAMPs required for the functional expression of the adrenomedullin, CGRP and amylin receptors remains to be demonstrated.

Angiotensin II, From Vasoconstrictor to Growth Factor: A Paradigm Shift

Sasa Vukelic, Kathy K. Griendling
Circ Res. 2014;114:754-757
http://dx.doi.org:/10.1161/CIRCRESAHA.114.303045

Angiotensin II (Ang II) is today considered as one of the essential factors in the pathophysiology of cardiovascular disease, producing acute hemodynamic and chronic pleiotropic effects. Although now it is widely accepted that these chronic effects are important, Ang II was initially considered only a short-acting, vasoactive hormone. This view was modified a quarter of a century ago when Dr Owens and his group published an article in Circulation Research with initial evidence that Ang II can act as a growth factor that regulates cell hypertrophy. They showed in a series of elegant experiments that Ang II promotes hypertrophy and hyperploidy of cultured rat aortic smooth muscle cells. However, Ang II had no effect on hyperplasia. These findings led to a paradigm shift in our understanding of the roles of growth factors and vasoactive substances in cardiovascular pathology and helped to redirect basic and clinical renin–angiotensin system research during the next 25 years. Ang II is now known to be a pleiotropic hormone that uses multiple signaling pathways to influence most processes that contribute to the development and progression of cardiovascular diseases, ranging from hypertrophy, endothelial dysfunction, cardiac remodeling, fibrosis, and inflammation to oxidative stress.

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Data Scientist on a Quest to Turn Computers Into Doctors

Posted in Artificial Intelligence Applications in Health Care on February 17, 2015| Leave a Comment »

Data Scientist on a Quest to Turn Computers Into Doctors

Reporter: Aviva Lev-Ari, PhD, RN

Some of the world’s most brilliant minds are working as data scientists at places like Google, Facebook, and Twitter—analyzing the enormous troves of online information generated by these tech giants—and for hacker and entrepreneur Jeremy Howard, that’s a bit depressing. Howard, a data scientist himself, spent a few years as the president of the Kaggle, a kind of online data scientist community that sought to feed the growing thirst for information analysis. He came to realize that while many of Kaggle’s online data analysis competitions helped scientists make new breakthroughs, the potential of these new techniques wasn’t being fully realized. “Data science is a very sexy job at the moment,” he says. “But when I look at what a lot of data scientists are actually doing, the vast majority of work out there is on product recommendations and advertising technology and so forth.”

So, after leaving Kaggle last year, Howard decided he would find a better use for data science. Eventually, he settled on medicine. And he even did a kind of end run around the data scientists, leveraging not so much the power of the human brain but the rapidly evolving talents of artificial brains. His new company is called Enlitic, and it wants to use state-of-the-art machine learning algorithms—what’s known as “deep learning”—to diagnosis illness and disease.

Publicly revealed for the first time today, the project is only just getting off the ground—“the big opportunities are going to take years to develop,” Howard says—but it’s yet another step forward for deep learning, a form of artificial intelligence that more closely mimics the way our brains work. Facebook is exploring deep learning as a way of recognizing faces in photos. Google uses it for image tagging and voice recognition. Microsoft does real-time translation in Skype. And the list goes on.

But Howard hopes to use deep learning for something more meaningful. His basic idea is to create a system akin to the Star Trek Tricorder, though perhaps not as portable. Enlitic will gather data about a particular patient—from medical images to lab test results to doctors’ notes—and its deep learning algorithms will analyze this data in an effort to reach a diagnosis and suggest treatments. The point, Howard says, isn’t to replace doctors, but to give them the tools they need to work more effectively. With this in mind, the company will share its algorithms with clinics, hospitals, and other medical outfits, hoping they can help refine its techniques. Howard says that the health care industry has been slow to pick-up on the deep-learning trend because it was rather expensive to build the computing clusters needed to run deep learning algorithms. But that’s changing.

The real challenge, Howard says, isn’t writing algorithms but getting enough data to train those algorithms. He says Enlitic is working with a number of organizations that specialize in gathering anonymized medical data for this type of research, but he declines to reveal the names of the organizations he’s working with. And while he’s tight-lipped about the company’s technique now, he says that much of the work the company does will eventually be published in research papers.

Source: www.wired.com

See on Scoop.it – Cardiovascular Disease: PHARMACO-THERAPY

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Archive for February, 2015

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