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Important but Unseen Human Embryo Developmental Stages Mimicked in Lab
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
Scientists have created embryo-like structures that mimic a crucial yet not much known stage of human development. The structures, created from stem cells and called gastruloids, are the first to form a 3D assembly that lays out how the body will take shape. The gastruloids developed rudimentary components of a heart and nervous system, but lacked the components to form a brain and other cell types that would make them capable of becoming a viable fetus.
Human embryos take a momentous leap in their third week, when the largely homogeneous ball of cells starts to differentiate and develop specific characteristics of the body parts they will become, a process known as gastrulation. During this process, the embryo elongates and lays down a body plan with a head and tail, often called the head-to-tail axis. But scientists have never seen this process live in action. That is partly because many countries have regulations that stop embryos from being grown in the laboratory for research beyond 14 days.
Over the past years, several research groups have cultured embryonic stem-cell structures that model when cells start to differentiate. The latest model developed at the University of Cambridge, UK and their collaborators in the Netherlands, Showed for the first time what happens when the blueprint for the body’s development is laid out, around 18–21 days after conception. Genetic analysis showed that the cells formed were those that would eventually go on to form muscles in the trunk, vertebrae, heart and other organs.
If everything is done properly, the cells develop into 3D structures on their own — and then spontaneously mimic the gastrulation process. Although they display certain key features of a 21-day-old embryo, the gastruloids reach that stage after just 72 hours and survive for maximum 4 days before collapsing. Scientists will probably use the model to make structures that are even more realistic representations of early development.
The model could help scientists to understand the role of genetics and environmental factors in different disorders. The artificial structures make it possible to avoid ethical concerns about doing research on human embryos. But as the structures become more advanced and life-like, there may be ethical restrictions.
Michael Rudnicki, who has done pioneering work in muscle stem cell biology and muscle regeneration, and whose work has been featured several times on this blog, has struck again. Rudnicki, who serves as director of the Regenerative Medicine Program at The Ottawa Hospital and a professor at the University of Ottawa and holds the prestigious Canada Research Chair in Molecular Genetics, teamed up with workers from the Sprott Centre for Stem Cell Research and the Sinclair Centre for Regenerative Medicine to investigate the role of muscle-specific stem cells in patients who suffer from Duchenne muscular dystrophy. This new earth-shaking study, which was published in the journal Nature Medicine (November 16, 2015), has changed the way we think about muscular dystrophy and will almost certainly force people to rethink the treatments and cures for this dreadful disease.
According to this new study, Duchenne muscular dystrophy directly affects muscle stem cells, and is, largely a disease of muscle stem cells.
Rudicki said: “For nearly 20 years, we’ve thought that the muscle weakness observed in patients with Duchenne muscular dystrophy is primarily due to problems in their muscle fibers, but our research shows that it is also due to intrinsic defects in the function of their muscle stem cells. This completely changes our understanding of Duchenne muscular dystrophy and could eventually lead to far more effective treatments.”
Muscular dystrophy comes in several different forms, but the predominant sign of muscular dystrophy is progressive muscle weakness. Altogether, muscular dystrophy refers to a group of more than 30 genetic diseases, all of which cause progressive weakness and degeneration of skeletal muscles used during voluntary movement. Approximately half of all who suffer from muscular dystrophy have Duchenne muscular dystrophy (DMD). Because muscular dystrophy results from mutations in the dystrophin gene, which is on the X chromosome, the vast majority of muscular dystrophy patients are male. Girls can be carriers of muscular dystrophy and can be mildly affected.
Interestingly, somewhere around one-third of boys who suffer from DMD have no family history of the disease. Because the dystrophin gene is so large, spontaneous mutations in it are probably relatively common.
The signs and symptoms typically appear between the ages of 2 and 3, and may include frequent falls, difficulty getting up from a lying or sitting position, trouble running and jumping, a strange, shuffling way of walking or having a tendency to walk on their toes, calf muscles that are abnormally large, muscle pain and stiffness, and some learning disabilities.
Muscular dystrophy affects all ethnic groups and occurs globally. It affects around 1 in every 3,500 to 6,000 male births each year in the United States. DMD affects approximately one in 3,600 boys.
Because DMD results from mutations in the dystrophin gene, the vast majority of muscular dystrophy research was based on a simple model in which the Dystrophin protein played a structural role in the structural integrity of muscle fibers. Abnormal versions of the Dystrophin protein caused the muscle fibers to become damaged and die as a result of contraction. Dystrophin anchors the cytoskeleton of the muscle fibers, which are essential for muscle contraction, to the muscle cell membrane, and then to the extracellular matrix outside the cell that serves as a foundation upon which the muscle cells are built.
However in this current study, Rudnicki and his team discovered that muscle stem cells also express the dystrophin protein. This is a revelation because Dystrophin was thought to be protein that ONLY appeared in mature muscle. However, in this study, it became exceedingly clear that in the absence of Dystrophin, muscle stem cells generated ten-fold fewer muscle precursor cells, and, consequently, far fewer functional muscle fibers. Dystrophin is also a component of a signal transduction pathway that allows muscle stem cells to properly ascertain if they need to replace dead or dying muscle. Muscle stem cells repair the muscle in response to injury or exercise by dividing to generate precursor cells that differentiate into muscle fibers.
Even though Rudnicki used mice as a model system in these experiments, the Dystrophin protein is highly conserved in most vertebrate animals. Therefore, it is highly likely that these results will also apply to human muscle stem cells.
Gene therapy experiments and trials are in progress and even show some promise, but Rudnicki’s work tells us that gene therapy approaches must target muscle stem cells as well as muscle fibers if they are to work properly.
“We’re already looking at approaches to correct this problem in muscle stem cells,” said Dr. Rudnicki.
This paper has received high praise from the likes of Ronald Worton, who was one of the co-discovers of the dystrophin gene with Louis Kunkel in 1987.
Early pathogenesis of Duchenne muscular dystrophy modelled in patient-derived human induced pluripotent stem cells
Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by a dystrophin deficiency. Effective suppression of the primary pathology observed in DMD is critical for treatment. Patient-derived human induced pluripotent stem cells (hiPSCs) are a promising tool for drug discovery. Here, we report an in vitro evaluation system for a DMD therapy using hiPSCs that recapitulate the primary pathology and can be used for DMD drug screening. Skeletal myotubes generated from hiPSCs are intact, which allows them to be used to model the initial pathology of DMD in vitro. Induced control and DMD myotubes were morphologically and physiologically comparable. However, electric stimulation of these myotubes for in vitro contraction caused pronounced calcium ion (Ca2+) influx only in DMD myocytes. Restoration of dystrophin by the exon-skipping technique suppressed this Ca2+ overflow and reduced the secretion of creatine kinase (CK) in DMD myotubes. These results suggest that the early pathogenesis of DMD can be effectively modelled in skeletal myotubes induced from patient-derived iPSCs, thereby enabling the development and evaluation of novel drugs.
Duchenne muscular dystrophy (DMD) is characterised by progressive muscle atrophy and weakness that eventually leads to ambulatory and respiratory deficiency from early childhood1. It is an X-linked recessive inherited disease with a relatively high frequency of 1 in 3500 males1,2.DMD, which is responsible for DMD, encodes 79 exons and produces dystrophin, which is one of the largest known cytoskeletal structural proteins3. Most DMD patients have various types of deletions or mutations in DMD that create premature terminations, resulting in a loss of protein expression4. Several promising approaches could be used to treat this devastating disease, such as mutation-specific drug exon-skipping5,6, cell therapy7, and gene therapy1,2.
Myoblasts from patients are the most common cell sources for assessing the disease phenotypes of DMD11,12. …Previous reports have shown that muscle cell differentiation from DMD patient myoblasts is delayed and that these cells have poor proliferation capacity compared to those of healthy individuals11,12. Our study revealed that control and DMD myoblasts obtained by activating tetracycline-dependent MyoD transfected into iPS cells (iPStet-MyoD cells) have comparable growth and differentiation potential and can produce a large number of intact and homogeneous myotubes repeatedly.
The pathogenesis of DMD is initiated and progresses with muscle contraction. The degree of muscle cell damage at the early stage of DMD can be evaluated by measuring the leakage of creatine kinase (CK) into the extracellular space15. Excess calcium ion (Ca2+) influx into skeletal muscle cells, together with increased susceptibility to plasma membrane injury, is regarded as the initial trigger of muscle damage in DMD19,20,21,22,23,24. Targeting these early pathogenic events is considered essential for developing therapeutics for DMD.
In this study, we established a novel evaluation system to analyse the cellular basis of early DMD pathogenesis by comparing DMD myotubes with the same clone but with truncated dystrophin-expressing DMD myotubes, using the exon-skipping technique. We demonstrated through in vitro contraction that excessive Ca2+ influx is one of the earliest events to occur in intact dystrophin-deficient muscle leading to extracellular leakage of CK in DMD myotubes.
Generation of tetracycline-inducible MyoD-transfected DMD patient-derived iPSCs (iPStet-MyoD cells)
Figure 1: Generation and characterization of control and DMD patient-derived Tet-MyoD-transfected hiPS cells. Full size image
Morphologically and physiologically comparable intact myotubes differentiated from control and DMD-derived hiPSCs
Figure 2: Morphologically and physiologically comparable skeletal muscle cells differentiated from Control-iPStet-MyoD and DMD-iPStet-MyoD. Full size image
Exon-skipping with AO88 restored expression of Dystrophin in DMD myotubes differentiated from DMD-iPStet-MyoD cells
Figure 3: Restoration of dystrophin protein expression by AO88. Full size image
Restored dystrophin expression attenuates Ca2+ overflow in DMD-Myocytes
Figure 4: Restored expression of dystrophin diminishes Ca2+ influx in DMD muscle in response to electric stimulation. Full size image
Ca2+ influx provokes skeletal muscle cellular damage in DMD muscle
Figure 5: Ca2+ influx induces prominent skeletal muscle cellular damage in DMD-Myocytes. Full size image
Skeletal muscle differentiation in myoblasts from DMD patients is generally delayed compared to that in healthy individuals11,36,37. Our differentiation system successfully induced the formation of myotubes from DMD patients, and the myotubes displayed analogous morphology and maturity compared with control myotubes (Fig. 2a–c). Comparing myotubes generated from patient-derived iPS cells with those derived from the same DMD clones but expressing dystrophin by application of the exon-skipping technique enabled us to demonstrate the primary cellular phenotypes in skeletal muscle solely resulting from the loss of the dystrophin protein (Fig. 4b). Our results demonstrate that truncated but functional dystrophin protein expression improved the cellular phenotype of DMD myotubes.
In DMD, the lack of dystrophin induces an excess influx of Ca2+ , leading to pathological dystrophic changes22. We consistently observed excess Ca2+ influx in DMD-Myocytes compared to Control-Myocytes (Supplementary Figure S3a and S3b) in response to electric stimulation. TRP channels, which are mechanical stimuli-activated Ca2+ channels40that are expressed in skeletal muscle cells41, can account for this pathogenic Ca2+ influx…
In conclusion, our study revealed that the absence of dystrophin protein induces skeletal muscle damage by allowing excess Ca2+ influx in DMD myotubes. Our experimental system recapitulated the early phase of DMD pathology as demonstrated by visualisation and quantification of Ca2+ influx using intact myotubes differentiated from hiPS cells. This evaluation system significantly expands prospective applications with regard to assessing the effectiveness of exon-skipping drugs and also enables the discovery of drugs that regulate the initial events in DMD.
Duchenne muscular dystrophy affects stem cells, University of Ottawa study finds
New treatments could one day be available for the most common form of muscular dystrophy after a study suggests the debilitating genetic disease affects the stem cells that produce healthy muscle fibres.
The findings are based on research from the University of Ottawa and The Ottawa Hospital, published Monday in the journal Nature Medicine.
For nearly two decades, doctors had thought the muscular weakness that is the hallmark of the disease was due to problems with human muscle fibers, said Dr. Michael Rudnicki, the study’s senior author.
The new research shows the specific protein characterized by its absence in Duchenne muscular dystrophy normally exists in stem cells.
Dystrophin protein found in stem cells
“The prevailing notion was that the protein that’s missing in Duchenne muscular dystrophy — a protein called dystrophin — was not involved at all in the function of the stem cells.”
When the genetic mutations caused by Duchenne muscular dystrophy inhibit the production of dystrophin in stem cells, those stem cells produce significantly fewer precursor cells — and thus fewer properly functioning muscle fibres. Further, stem cells need dystrophin to sense their environment to figure out if they need to divide to produce more stem cells or perform muscle repair work.
July 25, 2011|By Thomas H. Maugh II, Los Angeles Times
A genetic technique that allows the body to work around a crucial mutation that causes Duchenne muscular dystrophy increased the mass and function of muscles in a small group of patients with the devastating disease, paving the way for larger clinical trials of the drug. The study in a handful of boys age 5 to 15 showed that patients receiving the highest level of the drug, called AVI-4658 or eteplirsen, had a significant increase in production of a missing protein and increases in muscle fibers. The study demonstrated that the drug is safe in the short term. Results were reported Sunday in the journal Lancet.
Duchenne muscular dystrophy affects about one in every 3,500 males worldwide. It is caused by any one of several different mutations that affect production of a protein called dystrophin, which is important for the production and maintenance of muscle fibers. Affected patients become unable to walk and must use a wheelchair by age 8 to 12. Deterioration continues through their teens and 20s, and the condition typically proves fatal as muscle failure impairs their ability to breathe.
This study is designed to assess the efficacy, safety, tolerability, and pharmacokinetics (PK) of AVI-4658 (eteplirsen) in both 50.0 mg/kg and 30.0 mg/kg doses administered over 24 weeks in subjects diagnosed with Duchenne muscular dystrophy (DMD).
A Randomized, Double-Blind, Placebo-Controlled, Multiple Dose Efficacy, Safety, Tolerability and Pharmacokinetics Study of AVI-4658(Eteplirsen),in the Treatment of Ambulant Subjects With Duchenne Muscular Dystrophy
Dystrophin is expressed in differentiated myofibers, in which it is required for sarcolemmal integrity, and loss-of-function mutations in the gene that encodes it result in Duchenne muscular dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells), in which it associates with the serine-threonine kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to localize the cell polarity regulator Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, which also display a loss of polarity, abnormal division patterns (including centrosome amplification), impaired mitotic spindle orientation and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors that are needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD not only is caused by myofiber fragility, but also is exacerbated by impaired regeneration owing to intrinsic satellite cell dysfunction.
This e-Book is a comprehensive review of recent Original Research on METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases. It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences. Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation.The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012. All new articles on this subject, will continue to be incorporated, as published with periodical updates.
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Table of Contents forMetabolic Genomics & Pharmaceutics, Vol. I
Chapter 1: Metabolic Pathways
Chapter 2: Lipid Metabolism
Chapter 3: Cell Signaling
Chapter 4: Protein Synthesis and Degradation
Chapter 5: Sub-cellular Structure
Chapter 6: Proteomics
Chapter 7: Metabolomics
Chapter 8: Impairments in Pathological States: Endocrine Disorders; Stress
Hypermetabolism and Cancer
Chapter 9: Genomic Expression in Health and Disease
In the imtroduction to this series of discussions I pointed out JEDS Rosalino’s observation about the construction of a complex molecule of acetyl coenzyme A, and the amount of genetic coding that had to go into it. Furthermore, he observes – Millions of years later, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.
acetylCoA
In the tutorial that follows we find support for the view that mechanisms and examples from the current literature, which give insight into the developments in cell metabolism, are achieving a separation from inconsistent views introduced by the classical model of molecular biology and genomics, toward a more functional cellular dynamics that is not dependent on the classic view. The classical view fits a rigid framework that is to genomics and metabolomics as Mendelian genetics if to multidimentional, multifactorial genetics. The inherent difficulty lies in two places:
Interactions between differently weighted determinants
A large part of the genome is concerned with regulatory function, not expression of the code
The goal of the tutorial was to achieve an understanding of how cell signaling occurs in a cell. Completion of the tutorial would provide
a basic understanding signal transduction and
the role of phosphorylation in signal transduction.
Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin
In addition – detailed knowledge of –
the role of Tyrosine kinases and
G protein-coupled receptors in cell signaling.
serine
threonine
protein kinase
We are constantly receiving and interpreting signals from our environment, which can come
in the form of light, heat, odors, touch or sound.
The cells of our bodies are also
constantly receiving signals from other cells.
These signals are important to
keep cells alive and functioning as well as
to stimulate important events such as
cell division and differentiation.
Signals are most often chemicals that can be found
in the extracellular fluid around cells.
These chemicals can come
from distant locations in the body (endocrine signaling by hormones), from
nearby cells (paracrine signaling) or can even
be secreted by the same cell (autocrine signaling).
Signaling molecules may trigger any number of cellular responses, including
changing the metabolism of the cell receiving the signal or
result in a change in gene expression (transcription) within the nucleus of the cell or both.
controlling the output of ribosomes.
To which I would now add..
result in either an inhibitory or a stimulatory effect
The three stages of cell signaling are:
Cell signaling can be divided into 3 stages:
Reception: A cell detects a signaling molecule from the outside of the cell.
Transduction: When the signaling molecule binds the receptor it changes the receptor protein in some way. This change initiates the process of transduction. Signal transduction is usually a pathway of several steps. Each relay molecule in the signal transduction pathway changes the next molecule in the pathway.
Response: Finally, the signal triggers a specific cellular response.
Signal Transduction – ligand binds to surface receptor
Membrane receptors function by binding the signal molecule (ligand) and causing the production of a second signal (also known as a second messenger) that then causes a cellular response. These types of receptors transmit information from the extracellular environment to the inside of the cell.
by changing shape or
by joining with another protein
once a specific ligand binds to it.
Examples of membrane receptors include
G Protein-Coupled Receptors and
Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance.
Intracellular receptors are found inside the cell, either in the cytopolasm or in the nucleus of the target cell (the cell receiving the signal).
Note that though change in gene expression is stated, the change in gene expression does not here imply a change in the genetic information – such as – mutation. That does not have to be the case in the normal homeostatic case.
This point is the differentiating case between what JEDS Roselino has referred as
a fast, adaptive reaction, that is the feature of protein molecules, and distinguishes this interaction from
a one-to-one transcription of the genetic code.
The rate of transcription can be controlled, or it can be blocked. This is in large part in response to the metabolites in the immediate interstitium.
This might only be
a change in the rate of a transcription or a suppression of expression through RNA.
Or through a conformational change in an enzyme
Swinging domains in HECT E3 enzymes
Since signaling systems need to be
responsive to small concentrations of chemical signals and act quickly,
cells often use a multi-step pathway that transmits the signal quickly,
while amplifying the signal to numerous molecules at each step.
Signal transduction pathways are shown (simplified):
Signal Transduction
Signal transduction occurs when an
extracellular signaling molecule activates a specific receptor located on the cell surface or inside the cell.
In turn, this receptor triggers a biochemical chain of events inside the cell, creating a response.
Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.
The signal can be amplified at any step. Thus, one signaling molecule can cause many responses.
In 1970, Martin Rodbell examined the effects of glucagon on a rat’s liver cell membrane receptor. He noted that guanosine triphosphate disassociated glucagon from this receptor and stimulated the G-protein, which strongly influenced the cell’s metabolism. Thus, he deduced that the G-protein is a transducer that accepts glucagon molecules and affects the cell. For this, he shared the 1994 Nobel Prize in Physiology or Medicine with Alfred G. Gilman.
Guanosine monophosphate structure
In 2007, a total of 48,377 scientific papers—including 11,211 e-review papers—were published on the subject. The term first appeared in a paper’s title in 1979. Widespread use of the term has been traced to a 1980 review article by Rodbell: Research papers focusing on signal transduction first appeared in large numbers in the late 1980s and early 1990s.
Signal transduction involves the binding of extracellular signaling molecules and ligands to cell-surface receptors that trigger events inside the cell. The combination of messenger with receptor causes a change in the conformation of the receptor, known as receptor activation.
This activation is always the initial step (the cause) leading to the cell’s ultimate responses (effect) to the messenger. Despite the myriad of these ultimate responses, they are all directly due to changes in particular cell proteins. Intracellular signaling cascades can be started through cell-substratum interactions; examples are the integrin that binds ligands in the extracellular matrix and steroids.
Integrin
Most steroid hormones have receptors within the cytoplasm and act by stimulating the binding of their receptors to the promoter region of steroid-responsive genes.
steroid hormone receptor
Various environmental stimuli exist that initiate signal transmission processes in multicellular organisms; examples include photons hitting cells in the retina of the eye, and odorants binding to odorant receptors in the nasal epithelium. Certain microbial molecules, such as viral nucleotides and protein antigens, can elicit an immune system response against invading pathogens mediated by signal transduction processes. This may occur independent of signal transduction stimulation by other molecules, as is the case for the toll-like receptor. It may occur with help from stimulatory molecules located at the cell surface of other cells, as with T-cell receptor signaling. Receptors can be roughly divided into two major classes: intracellular receptors and extracellular receptors.
Signal transduction cascades amplify the signal output
Signal transduction cascades amplify the signal output
G protein-coupled receptors (GPCRs) are a family of integral transmembrane proteins that possess seven transmembrane domains and are linked to a heterotrimeric G protein. Many receptors are in this family, including adrenergic receptors and chemokine receptors.
Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling
signal transduction pathways
Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling
Signal transduction by a GPCR begins with an inactive G protein coupled to the receptor; it exists as a heterotrimer consisting of Gα, Gβ, and Gγ. Once the GPCR recognizes a ligand, the conformation of the receptor changes to activate the G protein, causing Gα to bind a molecule of GTP and dissociate from the other two G-protein subunits.
The dissociation exposes sites on the subunits that can interact with other molecules. The activated G protein subunits detach from the receptor and initiate signaling from many downstream effector proteins such as phospholipases and ion channels, the latter permitting the release of second messenger molecules.
Receptor tyrosine kinases (RTKs) are transmembrane proteins with an intracellular kinase domain and an extracellular domain that binds ligands; examples include growth factor receptors such as the insulin receptor.
insulin receptor and and insulin receptor signaling pathway (IRS)
To perform signal transduction, RTKs need to form dimers in the plasma membrane; the dimer is stabilized by ligands binding to the receptor.
RTKs
The interaction between the cytoplasmic domains stimulates the autophosphorylation of tyrosines within the domains of the RTKs, causing conformational changes.
Subsequent to this, the receptors’ kinase domains are activated, initiating phosphorylation signaling cascades of downstream cytoplasmic molecules that facilitate various cellular processes such as cell differentiation and metabolism.
Signal-Transduction-Pathway
As is the case with GPCRs, proteins that bind GTP play a major role in signal transduction from the activated RTK into the cell. In this case, the G proteins are
members of the Ras, Rho, and Raf families, referred to collectively as small G proteins.
They act as molecular switches usually
tethered to membranes by isoprenyl groups linked to their carboxyl ends.
Upon activation, they assign proteins to specific membrane subdomains where they participate in signaling. Activated RTKs in turn activate
small G proteins that activate guanine nucleotide exchange factors such as SOS1.
Once activated, these exchange factors can activate more small G proteins, thus
amplifying the receptor’s initial signal.
The mutation of certain RTK genes, as with that of GPCRs, can result in the expression of receptors that exist in a constitutively activate state; such mutated genes may act as oncogenes.
Integrin
Integrin
Integrin-mediated signal transduction
An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).
Integrins are produced by a wide variety of cells; they play a role in
cell attachment to other cells and the extracellular matrix and
in the transduction of signals from extracellular matrix components such as fibronectin and collagen.
Ligand binding to the extracellular domain of integrins
changes the protein’s conformation,
clustering it at the cell membrane to
initiate signal transduction.
Integrins lack kinase activity; hence, integrin-mediated signal transduction is achieved through a variety of intracellular protein kinases and adaptor molecules, the main coordinator being integrin-linked kinase.
As shown in the picture, cooperative integrin-RTK signaling determines the
timing of cellular survival,
apoptosis,
proliferation, and
differentiation.
integrin-mediated signal transduction
Integrin signaling
ion channel
A ligand-gated ion channel, upon binding with a ligand, changes conformation
to open a channel in the cell membrane
through which ions relaying signals can pass.
An example of this mechanism is found in the receiving cell of a neural synapse. The influx of ions that occurs in response to the opening of these channels
induces action potentials, such as those that travel along nerves,
by depolarizing the membrane of post-synaptic cells,
resulting in the opening of voltage-gated ion channels.
RyR and Ca+ release from SR
An example of an ion allowed into the cell during a ligand-gated ion channel opening is Ca2+;
it acts as a second messenger
initiating signal transduction cascades and
altering the physiology of the responding cell.
This results in amplification of the synapse response between synaptic cells
by remodelling the dendritic spines involved in the synapse.
In eukaryotic cells, most intracellular proteins activated by a ligand/receptor interaction possess an enzymatic activity; examples include tyrosine kinase and phosphatases. Some of them create second messengers such as cyclic AMP and IP3,
cAMP
Inositol_1,4,5-trisphosphate.svg
the latter controlling the release of intracellular calcium stores into the cytoplasm.
Many adaptor proteins and enzymes activated as part of signal transduction possess specialized protein domains that bind to specific secondary messenger molecules. For example,
calcium ions bind to the EF hand domains of calmodulin,
allowing it to bind and activate calmodulin-dependent kinase.
calcium movement and RyR2 receptor
PIP3 and other phosphoinositides do the same thing to the Pleckstrin homology domains of proteins such as the kinase protein AKT.
Signals can be generated within organelles, such as chloroplasts and mitochondria, modulating the nuclear
gene expression in a process called retrograde signaling.
Recently, integrative genomics approaches, in which correlation analysis has been applied on transcript and metabolite profiling data of Arabidopsis thaliana, revealed the identification of metabolites which are putatively acting as mediators of nuclear gene expression.
Omega-3 (ω-3) fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA). The main omega-3 fatty acids in the mammalian body are
α-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).
Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain,
DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total.
DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on.
On the other hand,
zinc is the most abundant trace metal in the human brain.
There are many scientific studies linking zinc, especially
excess amounts of free zinc, to cellular death.
Neurodegenerative diseases, such as Alzheimer’s disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between
omega-3 fatty acids, zinc transporter levels and
free zinc availability at cellular levels.
Many other studies have also suggested a possible
omega-3 and zinc effect on neurodegeneration and cellular death.
Therefore, in this review, we will examine
the effect of omega-3 fatty acids on zinc transporters and
the importance of free zinc for human neuronal cells.
Moreover, we will evaluate the collective understanding of
mechanism(s) for the interaction of these elements in neuronal research and their
significance for the diagnosis and treatment of neurodegeneration.
Epidemiological studies have linked high intake of fish and shellfish as part of the daily diet to
reduction of the incidence and/or severity of Alzheimer’s disease (AD) and senile mental decline in
Omega-3 fatty acids are one of the two main families of a broader group of fatty acids referred to as polyunsaturated fatty acids (PUFAs). The other main family of PUFAs encompasses the omega-6 fatty acids. In general, PUFAs are essential in many biochemical events, especially in early post-natal development processes such as
cellular differentiation,
photoreceptor membrane biogenesis and
active synaptogenesis.
Despite the significance of these
two families, mammals cannot synthesize PUFA de novo, so they must be ingested from dietary sources. Though belonging to the same family, both
omega-3 and omega-6 fatty acids are metabolically and functionally distinct and have
opposing physiological effects. In the human body,
high concentrations of omega-6 fatty acids are known to increase the formation of prostaglandins and
thereby increase inflammatory processes [10].
the reverse process can be seen with increased omega-3 fatty acids in the body.
Many other factors, such as
thromboxane A2 (TXA2),
leukotriene
B4 (LTB4),
IL-1,
IL-6,
tumor necrosis factor (TNF) and
C-reactive protein,
which are implicated in various health conditions, have been shown to be increased with high omega-6 fatty acids but decreased with omega-3 fatty acids in the human body.
Dietary fatty acids have been identified as protective factors in coronary heart disease, and PUFA levels are known to play a critical role in
immune responses,
gene expression and
intercellular communications.
omega-3 fatty acids are known to be vital in
the prevention of fatal ventricular arrhythmias, and
are also known to reduce thrombus formation propensity by decreasing platelet aggregation, blood viscosity and fibrinogen levels
.Since omega-3 fatty acids are prevalent in the nervous system, it seems logical that a deficiency may result in neuronal problems, and this is indeed what has been identified and reported.
The main
In another study conducted with individuals of 65 years of age or older (n = 6158), it was found that
only high fish consumption, but
not dietary omega-3 acid intake,
had a protective effect on cognitive decline
In 2005, based on a meta-analysis of the available epidemiology and preclinical studies, clinical trials were conducted to assess the effects of omega-3 fatty acids on cognitive protection. Four of the trials completed have shown
a protective effect of omega-3 fatty acids only among those with mild cognitive impairment conditions.
A trial of subjects with mild memory complaints demonstrated
an improvement with 900 mg of DHA.
We review key findings on
the effect of the omega-3 fatty acid DHA on zinc transporters and the
importance of free zinc to human neuronal cells.
DHA is the most abundant fatty acid in neural membranes, imparting appropriate
fluidity and other properties,
and is thus considered as the most important fatty acid in neuronal studies. DHA is well conserved throughout the mammalian species despite their dietary differences. It is mainly concentrated
in membrane phospholipids at synapses and
in retinal photoreceptors and
also in the testis and sperm.
In adult rats’ brain, DHA comprises approximately
17% of the total fatty acid weight, and
in the retina it is as high as 33%.
DHA is believed to have played a major role in the evolution of the modern human –
in particular the well-developed brain.
Premature babies fed on DHA-rich formula show improvements in vocabulary and motor performance.
Analysis of human cadaver brains have shown that
people with AD have less DHA in their frontal lobe
and hippocampus compared with unaffected individuals
Furthermore, studies in mice have increased support for the
protective role of omega-3 fatty acids.
Mice administrated with a dietary intake of DHA showed
an increase in DHA levels in the hippocampus.
Errors in memory were decreased in these mice and they demonstrated
reduced peroxide and free radical levels,
suggesting a role in antioxidant defense.
Another study conducted with a Tg2576 mouse model of AD demonstrated that dietary
DHA supplementation had a protective effect against reduction in
drebrin (actin associated protein), elevated oxidation, and to some extent, apoptosis via
decreased caspase activity.
Zinc
Zinc is a trace element, which is indispensable for life, and it is the second most abundant trace element in the body. It is known to be related to
growth,
development,
differentiation,
immune response,
receptor activity,
DNA synthesis,
gene expression,
neuro-transmission,
enzymatic catalysis,
hormonal storage and release,
tissue repair,
memory,
the visual process
and many other cellular functions. Moreover, the indispensability of zinc to the body can be discussed in many other aspects, as
a component of over 300 different enzymes
an integral component of a metallothioneins
a gene regulatory protein.
Approximately 3% of all proteins contain
zinc binding motifs .
The broad biological functionality of zinc is thought to be due to its stable chemical and physical properties. Zinc is considered to have three different functions in enzymes;
catalytic,
coactive and
Indeed, it is the only metal found in all six different subclasses
of enzymes. The essential nature of zinc to the human body can be clearly displayed by studying the wide range of pathological effects of zinc deficiency. Anorexia, embryonic and post-natal growth retardation, alopecia, skin lesions, difficulties in wound healing, increased hemorrhage tendency and severe reproductive abnormalities, emotional instability, irritability and depression are just some of the detrimental effects of zinc deficiency.
Proper development and function of the central nervous system (CNS) is highly dependent on zinc levels. In the mammalian organs, zinc is mainly concentrated in the brain at around 150 μm. However, free zinc in the mammalian brain is calculated to be around 10 to 20 nm and the rest exists in either protein-, enzyme- or nucleotide bound form. The brain and zinc relationship is thought to be mediated
through glutamate receptors, and
it inhibits excitatory and inhibitory receptors.
Vesicular localization of zinc in pre-synaptic terminals is a characteristic feature of brain-localized zinc, and
its release is dependent on neural activity.
Retardation of the growth and development of CNS tissues have been linked to low zinc levels. Peripheral neuropathy, spina bifida, hydrocephalus, anencephalus, epilepsy and Pick’s disease have been linked to zinc deficiency. However, the body cannot tolerate excessive amounts of zinc.
The relationship between zinc and neurodegeneration, specifically AD, has been interpreted in several ways. One study has proposed that β-amyloid has a greater propensity to
form insoluble amyloid in the presence of
high physiological levels of zinc.
Insoluble amyloid is thought to
aggregate to form plaques,
which is a main pathological feature of AD. Further studies have shown that
chelation of zinc ions can deform and disaggregate plaques.
In AD, the most prominent injuries are found in
hippocampal pyramidal neurons, acetylcholine-containing neurons in the basal forebrain, and in
somatostatin-containing neurons in the forebrain.
All of these neurons are known to favor
rapid and direct entry of zinc in high concentration
leaving neurons frequently exposed to high dosages of zinc.
This is thought to promote neuronal cell damage through oxidative stress and mitochondrial dysfunction. Excessive levels of zinc are also capable of
inhibiting Ca2+ and Na+ voltage gated channels
and up-regulating the cellular levels of reactive oxygen species (ROS).
High levels of zinc are found in Alzheimer’s brains indicating a possible zinc related neurodegeneration. A study conducted with mouse neuronal cells has shown that even a 24-h exposure to high levels of zinc (40 μm) is sufficient to degenerate cells.
If the human diet is deficient in zinc, the body
efficiently conserves zinc at the tissue level by compensating other cellular mechanisms
to delay the dietary deficiency effects of zinc. These include reduction of cellular growth rate and zinc excretion levels, and
redistribution of available zinc to more zinc dependent cells or organs.
A novel method of measuring metallothionein (MT) levels was introduced as a biomarker for the
assessment of the zinc status of individuals and populations.
In humans, erythrocyte metallothionein (E-MT) levels may be considered as an indicator of zinc depletion and repletion, as E-MT levels are sensitive to dietary zinc intake. It should be noted here that MT plays an important role in zinc homeostasis by acting
as a target for zinc ion binding and thus
assisting in the trafficking of zinc ions through the cell,
which may be similar to that of zinc transporters
Zinc Transporters
Deficient or excess amounts of zinc in the body can be catastrophic to the integrity of cellular biochemical and biological systems. The gastrointestinal system controls the absorption, excretion and the distribution of zinc, although the hydrophilic and high-charge molecular characteristics of zinc are not favorable for passive diffusion across the cell membranes. Zinc movement is known to occur
via intermembrane proteins and zinc transporter (ZnT) proteins
These transporters are mainly categorized under two metal transporter families; Zip (ZRT, IRT like proteins) and CDF/ZnT (Cation Diffusion Facilitator), also known as SLC (Solute Linked Carrier) gene families: Zip (SLC-39) and ZnT (SLC-30). More than 20 zinc transporters have been identified and characterized over the last two decades (14 Zips and 8 ZnTs).
Members of the SLC39 family have been identified as the putative facilitators of zinc influx into the cytosol, either from the extracellular environment or from intracellular compartments (Figure 1).
The identification of this transporter family was a result of gene sequencing of known Zip1 protein transporters in plants, yeast and human cells. In contrast to the SLC39 family, the SLC30 family facilitates the opposite process, namely zinc efflux from the cytosol to the extracellular environment or into luminal compartments such as secretory granules, endosomes and synaptic vesicles; thus decreasing intracellular zinc availability (Figure 1). ZnT3 is the most important in the brain where
it is responsible for the transport of zinc into the synaptic vesicles of
glutamatergic neurons in the hippocampus and neocortex,
Figure 1: Subcellular localization and direction of transport of the zinc transporter families, ZnT and ZIP. Arrows show the direction of zinc mobilization for the ZnT (green) and ZIP (red) proteins. A net gain in cytosolic zinc is achieved by the transportation of zinc from the extracellular region and organelles such as the endoplasmic reticulum (ER) and Golgi apparatus by the ZIP transporters. Cytosolic zinc is mobilized into early secretory compartments such as the ER and Golgi apparatus by the ZnT transporters. Figures were produced using Servier Medical Art, http://www.servier.com/. http://www.hindawi.com/journals/jnme/2012/173712.fig.001.jpg
Figure 2: Early zinc signaling (EZS) and late zinc signaling (LZS). EZS involves transcription-independent mechanisms where an extracellular stimulus directly induces an increase in zinc levels within several minutes by releasing zinc from intracellular stores (e.g., endoplasmic reticulum). LSZ is induced several hours after an external stimulus and is dependent on transcriptional changes in zinc transporter expression. Components of this figure were produced using Servier Medical Art, http://www.servier.com/ and adapted from Fukada et al. [30].
omega-3 fatty acids in the mammalian body are
α-linolenic acid (ALA),
docosahexenoic acid (DHA) and
eicosapentaenoic acid (EPA).
In general, seafood is rich in omega-3 fatty acids, more specifically DHA and EPA (Table 1). Thus far, there are nine separate epidemiological studies that suggest a possible link between
increased fish consumption and reduced risk of AD
and eight out of ten studies have reported a link between higher blood omega-3 levels
DHA and Zinc Homeostasis
Many studies have identified possible associations between DHA levels, zinc homeostasis, neuroprotection and neurodegeneration. Dietary DHA deficiency resulted in
increased zinc levels in the hippocampus and
elevated expression of the putative zinc transporter, ZnT3, in the rat brain.
Altered zinc metabolism in neuronal cells has been linked to neurodegenerative conditions such as AD. A study conducted with transgenic mice has shown a significant link between ZnT3 transporter levels and cerebral amyloid plaque pathology. When the ZnT3 transporter was silenced in transgenic mice expressing cerebral amyloid plaque pathology,
a significant reduction in plaque load
and the presence of insoluble amyloid were observed.
In addition to the decrease in plaque load, ZnT3 silenced mice also exhibited a significant
reduction in free zinc availability in the hippocampus
and cerebral cortex.
Collectively, the findings from this study are very interesting and indicate a clear connection between
zinc availability and amyloid plaque formation,
thus indicating a possible link to AD.
DHA supplementation has also been reported to limit the following:
amyloid presence,
synaptic marker loss,
hyper-phosphorylation of Tau,
oxidative damage and
cognitive deficits in transgenic mouse model of AD.
In addition, studies by Stoltenberg, Flinn and colleagues report on the modulation of zinc and the effect in transgenic mouse models of AD. Given that all of these are classic pathological features of AD, and considering the limiting nature of DHA in these processes, it can be argued that DHA is a key candidate in preventing or even curing this debilitating disease.
In order to better understand the possible links and pathways of zinc and DHA with neurodegeneration, we designed a study that incorporates all three of these aspects, to study their effects at the cellular level. In this study, we were able to demonstrate a possible link between omega-3 fatty acid (DHA) concentration, zinc availability and zinc transporter expression levels in cultured human neuronal cells.
When treated with DHA over 48 h, ZnT3 levels were markedly reduced in the human neuroblastoma M17 cell line. Moreover, in the same study, we were able to propose a possible
neuroprotective mechanism of DHA,
which we believe is exerted through
a reduction in cellular zinc levels (through altering zinc transporter expression levels)
that in turn inhibits apoptosis.
DHA supplemented M17 cells also showed a marked depletion of zinc uptake (up to 30%), and
free zinc levels in the cytosol were significantly low compared to the control
This reduction in free zinc availability was specific to DHA; cells treated with EPA had no significant change in free zinc levels (unpublished data). Moreover, DHA-repleted cells had
low levels of active caspase-3 and
high Bcl-2 levels compared to the control treatment.
These findings are consistent with previous published data and further strengthen the possible
correlation between zinc, DHA and neurodegeneration.
On the other hand, recent studies using ZnT3 knockout (ZnT3KO) mice have shown the importance of
ZnT3 in memory and AD pathology.
For example, Sindreu and colleagues have used ZnT3KO mice to establish the important role of
ZnT3 in zinc homeostasis that modulates presynaptic MAPK signaling
required for hippocampus-dependent memory
Results from these studies indicate a possible zinc-transporter-expression-level-dependent mechanism for DHA neuroprotection.
Integrins, Cadherins, Signaling and the Cytoskeleton
Curator: Larry H. Bernstein, MD, FCAP
We have reviewed the cytoskeleton, cytoskeleton pores and ionic translocation under lipids. We shall now look at this again, with specific attention to proteins, transporters and signaling.
Integrins and extracellular matrix in mechanotransduction
Lindsay Ramage
Queen’s Medical Research Institute, University of Edinburgh,
Edinburgh, UK
Cell Health and Cytoskeleton 2012; 4: 1–9
Integrins are a family of cell surface receptors which
mediate cell–matrix and cell–cell adhesions.
Among other functions they provide an important
mechanical link between the cells external and intracellular environments while
the adhesions that they form also have critical roles in cellular signal-transduction.
Cell–matrix contacts occur at zones in the cell surface where
adhesion receptors cluster and when activated
the receptors bind to ligands in the extracellular matrix.
The extracellular matrix surrounds the cells of tissues and forms the
structural support of tissue which is particularly important in connective tissues.
Cells attach to the extracellular matrix through
specific cell-surface receptors and molecules
including integrins and transmembrane proteoglycans.
Integrins work alongside other proteins such as
cadherins,
immunoglobulin superfamily
cell adhesion molecules,
selectins, and
syndecans
to mediate
cell–cell and
cell–matrix interactions and communication.
Activation of adhesion receptors triggers the formation of matrix contacts in which
bound matrix components,
adhesion receptors,
and associated intracellular cytoskeletal and signaling molecules
form large functional, localized multiprotein complexes.
Cell–matrix contacts are important in a variety of different cell and
tissue properties including
embryonic development,
inflammatory responses,
wound healing,
and adult tissue homeostasis.
This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.
Integrins are a family of αβ heterodimeric receptors which act as
cell adhesion molecules
connecting the ECM to the actin cytoskeleton.
The actin cytoskeleton is involved in the regulation of
cell motility,
cell polarity,
cell growth, and
cell survival.
The integrin family consists of around 25 members which are composed of differing
combinations of α and β subunits.
The combination of αβ subunits determines
binding specificity and
signaling properties.
In mammals around 19 α and eight β subunits have been characterized.
Both α and β integrin subunits contain two separate tails, which
penetrate the plasma membrane and possess small cytoplasmic domains which facilitate
the signaling functions of the receptor.
There is some evidence that the β subunit is the principal
site for
binding of cytoskeletal and signaling molecules,
whereas the α subunit has a regulatory role. The integrin
tails
link the ECM to the actin cytoskeleton within the cell and with cytoplasmic proteins,
such as talin, tensin, and filamin. The extracellular domains of integrin receptors bind the ECM ligands.
The ECM is a complex mixture of matrix molecules, including -glycoproteins, collagens, laminins, glycosaminoglycans, proteoglycans,
and nonmatrix proteins, – including growth factors.
These can be categorized as insoluble molecules within the ECM, soluble molecules, and/or matrix-associated biochemicals, such as systemic hormones or growth factors and cytokines that act locally.
The integrin receptor formed from the binding of α and β subunits is shaped like a globular head supported by two rod-like legs (Figure 1). Most of the contact between the two subunits occurs in the head region, with the intracellular tails of the subunits forming the legs of the receptor.6 Integrin recognition of ligands is not constitutive but is regulated by alteration of integrin affinity for ligand binding. For integrin binding to ligands to occur the integrin must be primed and activated, both of which involve conformational changes to the receptor.
The integrins are composed of well-defined domains used for protein–protein interactions. The α-I domains of α integrin subunits comprise the ligand binding sites. X-ray crystallography has identified an α-I domain within the β subunit and a β propeller domain within the α subunit which complex to form the ligand-binding head of the integrin.
The use of activating and conformation-specific antibodies also suggests that the β chain is extended in the active integrin. It has since been identified that the hybrid domain in the β chain is critical for integrin activation, and a swing-out movement of this leg activates integrins.
Figure Integrin binding to extracellular matrix (ECM). Conformational changes to integrin structure and clustering of subunits which allow enhanced function of the receptor.
Integrin extracellular binding activity is regulated from inside the cell and binding to the ECM induces signals that are transmitted into the cell.15 This bidirectional signaling requires
dynamic,
spatially, and
temporally regulated formation and
disassembly of multiprotein complexes that
form around the short cytoplasmic tails of integrins.
Ligand binding to integrin family members leads to clustering of integrin molecules in the plasma membrane and recruitment of actin filaments and intracellular signaling molecules to the cytoplasmic domain of the integrins. This forms focal adhesion complexes which are able to maintain
not only adhesion to the ECM
but are involved in complex signaling pathways
which include establishing
cell polarity,
directed cell migration, and
maintaining cell growth and survival.
Initial activation through integrin adhesion to matrix recruits up to around 50 diverse signaling molecules
to assemble the focal adhesion complex
which is capable of responding to environmental stimuli efficiently.
Mapping of the integrin
adhesome binding and signaling interactions
identified a network of 156 components linked together which can be modified by 690 interactions.
The binding of the adaptor protein talin to the β subunit cytoplasmic tail is known to have a key role in integrin activation. This is thought to occur through the disruption of
inhibitory interactions between α and β subunit cytoplasmic tails.
Talin also binds
to actin and to cytoskeletal and signaling proteins.
This allows talin to directly link activated integrins
to signaling events and the cytoskeleton.
Genetic programming occurs with the binding of integrins to the ECM
Signal transduction pathway activation arising from integrin-
ECM binding results in changes in gene expression of cells
and leads to alterations in cell and tissue function. Various
different effects can arise depending on the
cell type,
matrix composition, and
integrins activated.
One way in which integrin expression is important in genetic programming is in the fate and differentiation of stem cells.
Osteoblast differentiation occurs through ECM interactions
with specific integrins
to initiate intracellular signaling pathways leading to osteoblast-specific gene expression
disruption of interactions between integrins and collagen;
fibronectin blocks osteoblast differentiation and
Disruption of α2 integrin prevents osteoblast differentiation, and activation of the transcription factor
It has been suggested that integrin-type I collagen interaction is necessary for the phosphorylation and activation of osteoblast-specific transcription factors present in committed osteoprogenitor cells.
A variety of growth factors and cytokines have been shown to be important in the regulation of integrin expression and function in chondrocytes. Mechanotransduction in chondrocytes occurs through several different receptors and ion channels including integrins. During osteoarthritis the expression of integrins by chondrocytes is altered, resulting in different cellular transduction pathways which contribute to tissue pathology.
In normal adult cartilage, chondrocytes express α1β1, α10β1 (collagen receptors), α5β1, and αvβ5 (fibronectin) receptors. During mechanical loading/stimulation of chondrocytes there is an influx of ions across the cell membrane resulting from activation of mechanosensitive ion channels which can be inhibited by subunit-specific anti-integrin blocking antibodies or RGD peptides. Using these strategies it was identified that α5β1 integrin is a major mechanoreceptor in articular chondrocyte responses to mechanical loading/stimulation.
Osteoarthritic chondrocytes show a depolarization response to 0.33 Hz stimulation in contrast to the hyperpolarization response of normal chondrocytes. The mechanotransduction pathway in chondrocytes derived from normal and osteoarthritic cartilage both involve recognition of the mechanical stimulus by integrin receptors resulting in the activation of integrin signaling pathways leading to the generation of a cytokine loop. Normal and osteoarthritic chondrocytes show differences at multiple stages of the mechanotransduction cascade (Figure 3). Early events are similar; α5β1 integrin and stretch activated ion channels are activated and result in rapid tyrosine phosphorylation events. The actin cytoskeleton is required for the integrin-dependent Mechanotransduction leading to changes in membrane potential in normal but not osteoarthritic chondrocytes.
Cell–matrix interactions are essential for maintaining the integrity of tissues. An intact matrix is essential for cell survival and proliferation and to allow efficient mechanotransduction and tissue homeostasis. Cell–matrix interactions have been extensively studied in many tissues and this knowledge is being used to develop strategies to treat pathology. This is particularly important in tissues subject to abnormal mechanical loading, such as musculoskeletal tissues. Integrin-ECM interactions are being used to enhance tissue repair mechanisms in these tissues through differentiation of progenitor cells for in vitro and in vivo use. Knowledge of how signaling cascades are differentially regulated in response to physiological and pathological external stimuli (including ECM availability and mechanical loading/stimulation) will enable future strategies to be developed to prevent and treat the progression of pathology associated with integrin-ECM interactions.
Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels
Matthews, DR. Overby, R Mannix and DE. Ingber
1Vascular Biology Program, Departments of Pathology and Surgery, Children’s Hospital, and 2Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, MA J Cell Sci 2006; 119: 508-518. http://dx.doi.org:/10.1242/jcs.02760
To understand how cells sense and adapt to mechanical stress, we applied tensional forces to magnetic microbeads bound to cell-surface integrin receptors and measured changes in bead isplacement with sub-micrometer resolution using optical microscopy. Cells exhibited four types of mechanical responses: (1) an immediate viscoelastic response;
(2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces;
(3) later adaptive cell stiffening with sustained (>15 second) static stresses; and
(4) a large-scale repositioning response with prolonged (>1 minute) stress.
Importantly, these adaptation responses differed biochemically. The immediate and early responses were affected by
inhibiting tension through interference with Rho signaling,
similar to the case of the immediate and early responses, but it was also prevented by
blocking mechanosensitive ion channels or
by inhibiting Src tyrosine kinases.
All adaptive responses were suppressed by cooling cells to 4°C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.
Microtubule-Stimulated ADP Release, ATP Binding, and Force Generation In Transport Kinesins
J Atherton, I Farabella, I-Mei Yu, SS Rosenfeld, A Houdusse, M Topf, CA Moores
1Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, University of London, London, United Kingdom; 2Structural Motility, Institut Curie, Centre National de la Recherche Scientifique, Paris, France; 3Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States
eLife 2014;3:e03680. http://dx.doi.org:/10.7554/eLife.03680
Kinesins are a large family of microtubule (MT)-based motors that play important roles in many cellular activities including
mitosis,
motility, and
intracellular transport
Their involvement in a range of pathological processes also highlights their significance as therapeutic targets and the importance of understanding the molecular basis of their function They are defined by their motor domains that contain both the microtubule (MT) and ATP binding sites. Three ATP binding motifs—the P-loop, switch I, switch II–are highly conserved among kinesins, myosin motors, and small GTPases. They share a conserved mode of MT binding such that MT binding, ATP binding, and hydrolysis are functionally coupled for efficient MT-based work.
The interior of a cell is a hive of activity, filled with proteins and other items moving from one location to another. A network of filaments called microtubules forms tracks along which so-called motor proteins carry these items. Kinesins are one group of motor proteins, and a typical kinesin protein has one end (called the ‘motor domain’) that can attach itself to the microtubules.
The other end links to the cargo being carried, and a ‘neck’ connects the two. When two of these proteins work together, flexible regions of the neck allow the two motor domains to move past one another, which enable the kinesin to essentially walk along a microtubule in a stepwise manner.
Atherton et al. use a technique called cryo-electron microscopy to study—in more detail than previously seen—the structure of the motor domains of two types of kinesin called kinesin-1 and kinesin-3. Images were taken at different stages of the cycle used by the motor domains to extract the energy from ATP molecules. Although the two kinesins have been thought to move along the microtubule tracks in different ways, Atherton et al. find that the core mechanism used by their motor domains is the same.
When a motor domain binds to the microtubule, its shape changes, first stimulating release of the breakdown products of ATP from the previous cycle. This release makes room for a new ATP molecule to bind. The structural changes caused by ATP binding are relatively small but produce larger changes in the flexible neck region that enable individual motor domains within a kinesin pair to co-ordinate their movement and move in a consistent direction. This mechanism involves tight coupling between track binding and fuel usage and makes kinesins highly efficient motors.
A number of kinesins drive long distance transport of cellular cargo with dimerisation allowing them to take multiple 8 nm ATP-driven steps toward MT plus ends. Their processivity depends on communication between the two motor domains, which is achieved via the neck linker that connects each motor domain to the dimer-forming coiled-coil
Kinesins are a superfamily of microtubule-based
ATP-powered motors, important for multiple, essential cellular functions.
How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy.
All our reconstructions have, as their asymmetric unit, a triangle-shaped motor domain bound to an αβ-tubulin dimer within the MT lattice (Figure 1). The structural comparisons below are made with respect to the MT surface, which, at the resolution of our structures (∼7 Å, Table 1), is the same (CCC > 0.98 for all). As is well established across the superfamily, the major and largely invariant point of contact between kinesin motor domains and the MT is helix-α4, which lies at the tubulin intradimer interface (Figure 1C, Kikkawa et al., 2001).
However, multiple conformational changes are seen throughout the rest of each domain in response to bound nucleotide (Figure 1D). Below, we describe the conformational changes in functionally important regions of each motor domain starting with the nucleotide-binding site, from which all other conformational changes emanate.
The nucleotide-binding site (Figure 2) has three major elements: (1) the P-loop (brown) is visible in all our reconstructions;
(2) loop9 (yellow, contains switch I) undergoes major conformational changes through the ATPase cycle; and
(3) loop11 (red, contains switch II) that connects strand-β7 to helix-α4,
the conformation and flexibility of which is determined by MT binding and motor nucleotide state.
Movement and extension of helix-α6 controls neck linker docking
the N-terminus of helix-α6 is closely associated with elements of the nucleotide binding site suggesting that its conformation alters in response to different nucleotide states. In addition, because the orientation of helix-α6 with respect to helix-α4 controls neck linker docking and because helix-α4 is held against the MT during the ATPase cycle,
conformational changes in helix-α6 control movement of the neck linker.
Mechanical amplification and force generation involves conformational changes across the motor domain
A key conformational change in the motor domain following Mg-ATP binding is peeling of the central β-sheet from the C-terminus of helix-α4 increasing their separation (Figure 3—figure supplement 2); this is required to accommodate rotation of helix-α6 and consequent neck linker docking (Figure 3B–E).
Peeling of the central β-sheet has previously been proposed to arise from tilting of the entire motor domain relative to static MT contacts, pivoting around helix-α4 (the so-called ‘seesaw’ model; Sindelar, 2011). Specifically, this model predicts that the major difference in the motor before and after Mg-ATP binding would be the orientation of the motor domain with respect to helix-α4.
Kinesin mechanochemistry and the extent of mechanistic conservation within the motor superfamily are open questions, critical to explain how MT binding, and ATP binding and hydrolysis drive motor activity. Our structural characterisation of two transport motors now allows us to propose a model that describes the roles of mechanochemical elements that together drive conserved MT-based motor function.
Model of conserved MT-bound kinesin mechanochemistry. Loop11/N-terminus of helix-α4 is flexible in ADP-bound kinesin in solution, the neck linker is also flexible while loop9 chelates ADP. MT binding is sensed by loop11/helix-α4 N-terminus, biasing them towards more ordered conformations.
We propose that this favours crosstalk between loop11 and loop9, stimulating ADP release. In the NN conformation, both loop11 and loop9 are well ordered and primed to favour ATP binding, while helix-α6—which is required for mechanical amplification–is closely associated with the MT on the other side of the motor domain. ATP binding draws loop11 and loop9 closer together; causing
(1) tilting of most of the motor domain not contacting the MT towards the nucleotide-binding site,
(2) rotation, translation, and extension of helix-α6 which we propose contributes to force generation, and
(3) allows neck linker docking and biases movement of the 2nd head towards the MT plus end.
In both motors, microtubule binding promotes
ordered conformations of conserved loops that
stimulate ADP release,
enhance microtubule affinity and
prime the catalytic site for ATP binding.
ATP binding causes only small shifts of these nucleotide-coordinating loops but induces
large conformational changes elsewhere that
allow force generation and
neck linker docking towards the microtubule plus end.
Family-specific differences across the kinesin–microtubule interface account for the
distinctive properties of each motor.
Our data thus provide evidence for a
conserved ATP-driven
mechanism for kinesins and
reveal the critical mechanistic contribution of the microtubule interface.
Phosphorylation at endothelial cell–cell junctions: Implications for VE-cadherin function
I Timmerman, PL Hordijk, JD van Buul
Cell Health and Cytoskeleton 2010; 2: 23–31
Endothelial cell–cell junctions are strictly regulated in order to
control the barrier function of endothelium.
Vascular endothelial (VE)-cadherin is one of the proteins that is crucial in this process. It has been reported that
phosphorylation events control the function of VE-cadherin.
This review summarizes the role of VE-cadherin phosphorylation in the regulation of endothelial cell–cell junctions and highlights how this affects vascular permeability and leukocyte extravasation.
The vascular endothelium is the inner lining of blood vessels and
forms a physical barrier between the vessel lumen and surrounding tissue;
controlling the extravasation of fluids,
plasma proteins and leukocytes.
Changes in the permeability of the endothelium are tightly regulated. Under basal physiological conditions, there is a continuous transfer of substances across the capillary beds. In addition the endothelium can mediate inducible,
transient hyperpermeability
in response to stimulation with inflammatory mediators,
which takes place primarily in postcapillary venules.
However, when severe, inflammation may result in dysfunction of the endothelial barrier in various parts of the vascular tree, including large veins, arterioles and capillaries. Dysregulated permeability is observed in various pathological conditions, such as tumor-induced angiogenesis, cerebrovascular accident and atherosclerosis.
Two fundamentally different pathways regulate endothelial permeability,
the transcellular and paracellular pathways.
Solutes and cells can pass through the body of endothelial cells via the transcellular pathway, which includes
vesicular transport systems, fenestrae, and biochemical transporters.
The paracellular route is controlled by
the coordinated opening and closing of endothelial junctions and
thereby regulates traffic across the intercellular spaces between endothelial cells.
Endothelial cells are connected by
tight, gap and
adherens junctions,
of which the latter, and particularly the adherens junction component,
vascular endothelial (VE)-cadherin,
are of central importance for the initiation and stabilization of cell–cell contacts.
Although multiple adhesion molecules are localized at endothelial junctions, blocking the adhesive function of VE-cadherin using antibodies is sufficient to disrupt endothelial junctions and to increase endothelial monolayer permeability both in vitro and in vivo. Like other cadherins, VE-cadherin mediates adhesion via homophilic, calcium-dependent interactions.
This cell–cell adhesion
is strengthened by binding of cytoplasmic proteins, the catenins,
to the C-terminus of VE-cadherin.
VE-cadherin can directly bind β-catenin and plakoglobin, which
both associate with the actin binding protein α-catenin.
Initially, α-catenin was thought to directly anchor cadherins to the actin cytoskeleton, but recently it became clear that
α-catenin cannot bind to both β-catenin and actin simultaneously.
Data using purified proteins show that
monomeric α-catenin binds strongly to cadherin-bound β-catenin;
in contrast to the dimer which has a higher affinity for actin filaments,
indicating that α-catenin might function as a molecular switch regulating cadherin-mediated cell–cell adhesion and actin assembly.
Thus, interactions between the cadherin complex and the actin cytoskeleton are more complex than previously thought. Recently, Takeichi and colleagues reported that
the actin binding protein EPLIN (epithelial protein lost in neoplasm)
can associate with α-catenin and thereby
link the E-cadherin–catenin complex to the actin cytoskeleton.
Although this study was performed in epithelial cells,
an EPLIN-like molecule might serve as
a bridge between the cadherin–catenin complex and
the actin cytoskeleton in endothelial cells.
Next to β-catenin and plakoglobin, p120-catenin also binds directly to the intracellular tail of VE-cadherin.
Numerous lines of evidence indicate that
p120-catenin promotes VE-cadherin surface expression and stability at the plasma membrane.
Different models are proposed that describe how p120-catenin regulates cadherin membrane dynamics, including the hypothesis
that p120-catenin functions as a ‘cap’ that prevents the interaction of VE-cadherin
with the endocytic membrane trafficking machinery.
In addition, p120-catenin might regulate VE-cadherin internalization through interactions with small GTPases. Cytoplasmic p120-catenin, which is not bound to VE-cadherin, has been shown to
decrease RhoA activity,
elevate active Rac1 and Cdc42, and thereby is thought
to regulate actin cytoskeleton organization and membrane trafficking.
The intact cadherin-catenin complex is required for proper functioning of the adherens junction. Mutant forms of VE-cadherin which
lack either the β-catenin, plakoglobin or p120 binding regions reduce the strength of cell–cell adhesion.
Moreover, our own results showed that
interfering with the interaction between α-catenin and β-catenin,
using a cell-permeable peptide which encodes the binding site in α-catenin for β-catenin,
resulted in an increased permeability of the endothelial monolayer.
Several mechanisms may be involved in the regulation of the organization and function of the cadherin–catenin complex, including endocytosis of the complex, VE-cadherin cleavage and actin cytoskeleton reorganization. The remainder of this review primarily focuses on the
role of tyrosine phosphorylation in the control of VE-cadherin-mediated cell–cell adhesion.
Regulation of the adhesive function of VE-cadherin by tyrosine phosphorylation
It is a widely accepted concept that tyrosine phosphorylation of components of the VE–cadherin-catenin complex
Correlates with the weakening of cell–cell adhesion.
One of the first reports that supported this idea showed that the level of phosphorylation of VE-cadherin was
high in loosely confluent endothelial cells, but
low in tightly confluent monolayers,
when intercellular junctions are stabilized.
In addition, several conditions that induce tyrosine phosphorylation
of adherens junction components, like
v-Src transformation
and inhibition of phosphatase activity by pervanadate,
have been shown to shift cell–cell adhesion from a strong to a weak state. More physiologically relevant;
permeability-increasing agents such as
histamine,
tumor necrosis factor-α (TNF-α),
thrombin,
platelet-activating factor (PAF) and
vascular endothelial growth factor (VEGF)
increase tyrosine phosphorylation of various components of the cadherin–catenin complex.
A general idea has emerged that
tyrosine phosphorylation of the VE-cadherin complex
leads to the uncoupling of VE-cadherin from the actin cytoskeleton
through dissociation of catenins from the cadherin.
However, tyrosine phosphorylation of VE-cadherin is required for efficient transmigration of leukocytes.
This suggests that VE-cadherin-mediated cell–cell contacts
are not just pushed open by the migrating leukocytes, but play
a more active role in the transmigration process.
A schematic overview of leukocyte adhesion-induced signals leading to VE-cadherin phosphorylation
Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin
Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin.
Notes: A) Permeability-inducing agents such as thrombin, histamine and VEGF, induce tyrosine phosphorylation (pY) of VE-cadherin and the associated catenins. Although the specific consequences of catenin tyrosine phosphorylation in endothelial cells are still unknown, VE-cadherin tyrosine phosphorylation results in opening of the cell–cell junctions (indicated by arrows) and enhanced vascular permeability. How tyrosine phosphorylation affects VE-cadherin adhesiveness is not yet well understood; disrupted binding of catenins, which link the cadherin to the actin cytoskeleton, may be involved. VEGF induces phosphorylation of VE-cadherin at specific residues, Y658 and Y731, which have been reported to regulate p120-catenin and β-catenin binding, respectively. Moreover, VEGF stimulation results in serine phosphorylation (pSer) of VE-cadherin, specifically at residue S665, which leads to its endocytosis. B) Adhesion of leukocytes to endothelial cells via ICAM-1 increases endothelial permeability by inducing phosphorylation of VE-cadherin on tyrosine residues. Essential mediators, such as the kinases Pyk2 and Src, and signaling routes involving reactive oxygen species (ROS) and Rho, have been shown to act downstream of ICAM-1. Different tyrosine residues within the cytoplasmic domain of VE-cadherin are involved in the extravasation of neutrophils and lymphocytes, including Y658 and Y731. (β: β-catenin, α: α-catenin, γ: γ-catenin/plakoglobin).
N-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs
BT Jamal, MN Nita-Lazar, Z Gao, B Amin, J Walker, MA Kukuruzinska
Cell Health and Cytoskeleton 2009; 1: 67–80
N-glycosylation of E-cadherin has been shown to inhibit cell–cell adhesion. Specifically, our recent studies have provided evidence that the reduction of E-cadherin N-glycosylation promoted the recruitment of stabilizing components, vinculin and serine/ threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs) and enhanced the association of AJs with the actin cytoskeleton. Here, we examined the details of how
N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions.
Using the hypoglycosylated E-cadherin variant, V13, we show that
V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein.
This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that
increased association of PP2A with V13-containing AJs promoted their tethering to microtubules.
On the other hand, V13/γ-catenin complexes associated more with vinculin, suggesting that they
mediated the interaction of AJs with the actin cytoskeleton.
N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin.
These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through
the assembly of distinct β-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components.
Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle
Matteo Vatta, and Georgine Faulkner,
1 Departments of Pediatrics (Cardiology), Baylor College of Medicine, Houston, TX 2 Department of Reproductive and Developmental Sciences, University of Trieste, Trieste, Italy
3 Muscular Molecular Biology Unit, International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy
The heart is a force-generating organ that responds to
self-generated electrical stimuli from specialized cardiomyocytes.
This function is modulated
by sympathetic and parasympathetic activity.
In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle, cardiomyocytes
depend on their highly evolved and specialized cytoskeletal apparatus.
Defects in components of the cytoskeleton, in the long term,
affect the ability of the cell to compensate at both functional and structural levels.
In addition to the structural remodeling,
the myocardium becomes increasingly susceptible to altered electrical activity leading to arrhythmogenesis.
The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive. Here I will review
the current knowledge of the molecular and functional relationships between the cytoskeleton and ion channels
and, I will discuss the future impact of new data on molecular cardiology research and clinical practice.
Myocardial dysfunction in the end-stage failing heart is very often associated with increasing
susceptibility to ventricular tachycardia (VT) and ventricular fibrillation (VF),
both of which are common causes of sudden cardiac death (SCD).
Among the various forms of HF,
myocardial remodeling due to ischemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM)
is characterized by alterations in baseline ECG,
which includes the
prolongation of the QT interval,
as well as QT dispersion,
ST-segment elevation, and
T-wave abnormalities,
especially during exercise. In particular, subjects with
severe left ventricular chamber dilation such as in DCM can have left bundle branch block (LBBB), while right bundle branch block (RBBB) is more characteristic of right ventricular failure. LBBB and RBBB have both been repeatedly associated with AV block in heart failure.
The impact of volume overload on structural and electro-cardiographic alterations has been noted in cardiomyopathy patients treated with left ventricular assist device (LVAD) therapy, which puts the heart at mechanical rest. In LVAD-treated subjects,
QRS- and both QT- and QTc duration decreased,
suggesting that QRS- and QT-duration are significantly influenced by mechanical load and
that the shortening of the action potential duration contributes to the improved contractile performance after LVAD support.
Despite the increasing use of LVAD supporting either continuous or pulsatile blood flow in patients with severe HF, the benefit of this treatment in dealing with the risk of arrhythmias is still controversial.
Large epidemiological studies, such as the REMATCH study, demonstrated that the
employment of LVAD significantly improved survival rate and the quality of life, in comparison to optimal medical management.
An early postoperative period study after cardiac unloading therapy in 17 HF patients showed that in the first two weeks after LVAD implantation,
HF was associated with a relatively high incidence of ventricular arrhythmias associated with QTc interval prolongation.
In addition, a recent retrospective study of 100 adult patients with advanced HF, treated with an axial-flow HeartMate LVAD suggested that
the rate of new-onset monomorphic ventricular tachycardia (MVT) was increased in LVAD treated patients compared to patients given only medical treatment,
while no effect was observed on the development of polymorphic ventricular tachycardia (PVT)/ventricular fibrillation (VF).
The sarcomere
The myocardium is exposed to severe and continuous biomechanical stress during each contraction-relaxation cycle. When fiber tension remains uncompensated or simply unbalanced,
it may represent a trigger for arrhythmogenesis caused by cytoskeletal stretching,
which ultimately leads to altered ion channel localization, and subsequent action potential and conduction alterations.
Cytoskeletal proteins not only provide the backbone of the cellular structure, but they also
maintain the shape and flexibility of the different sub-cellular compartments, including the
plasma membrane,
the double lipid layer, which defines the boundaries of the cell and where
ion channels are mainly localized.
The interaction between the sarcomere, which is the basic for the passive force during diastole and for the restoring force during systole. Titin connects
the Z-line to the M-line of the sarcomeric structure
(Figure 1).
In addition to the strategic
localization and mechanical spring function,
titin is a length-dependent sensor during
stretch and promotes actin-myosin interaction
Titin is stabilized by the cross-linking protein
telethonin (T-Cap), which localizes at the Z-line and is also part of titin sensor machinery (Figure 1).
The complex protein interactions in the sarcomere entwine telethonin to other
Z-line components through the family of the telethonin-binding proteins of the Z-disc, FATZ, also known as calsarcin and myozenin.
FATZ binds to
calcineurin,
γ-filamin as well as the
spectrin-like repeats (R3–R4) of α-actinin-2,
the major component of the Z-line and a pivotal
F-actin cross-linker (Figure 1).contractile unit of striated muscles, and
the sarcolemma,
the plasma membrane surrendering the muscle fibers in skeletal muscle and the muscle cell of the cardiomyocyte,
determines the mechanical plasticity of the cell, enabling it to complete and re-initiate each contraction-relaxation cycle.
At the level of the sarcomere,
actin (thin) and myosin (thick) filaments generate the contractile force,
while other components such as titin, the largest protein known to date, are responsible for
the passive force during diastole and for the restoring force during systole, and (titin).
the Z-line to the M-line of the sarcomeric structure
(Figure 1).
In addition to the strategic
localization and mechanical spring function,
it acts as a length-dependent sensor during stretch and
promotes actin-myosin interaction.
Stabilized by the cross-linking protein telethonin (T-Cap),
titin localizes at the Z-line and is
part of titin sensor machinery
Another cross-linker of α-actinin-2 in the complex Z-line scaffold is
the Z-band alternatively spliced PDZ motif protein (ZASP),
which has an important role in maintaining Z-disc stability
in skeletal and cardiac muscle (Figure 1).
ZASP contains a PDZ motif at its N-terminus,
which interacts with C-terminus of α-actinin-2,
and a conserved sequence called the ZASP like motif (ZM)
found in the alternatively spliced exons 4 and 6.
It has also been reported
to bind to the FATZ (calsarcin) family of Z-disc proteins (Figure 1).
The complex protein interactions in the sarcomere entwine telethonin to other Z-line components through the family of the telethonin-binding proteins of the
Z-disc,
FATZ, also known as calsarcin and
myozenin
FATZ binds to calcineurin,
γ-filamin as well as the
spectrin-like repeats (R3–R4) of α-actinin-2, the major component of the Z-line and a pivotal F-actin cross-linker (Figure 1).
sarcomere structure
Figure 1. Sarcomere structure
The diagram illustrates the sarcomeric structure. The Z-line determines the boundaries of the contractile unit, while Titin connects the Z-line to the M-line and acts as a functional spring during contraction/relaxation cycles.
Sarcomeric Proteins and Ion Channels
In addition to systolic dysfunction characteristic of dilated cardiomyopathy (DCM) and diastolic dysfunction featuring hypertrophic cardiomyopathy (HCM), the clinical phenotype of patients with severe cardiomyopathy is very often associated with a high incidence of cardiac arrhythmias. Therefore, besides fiber stretch associated with mechanical and hemodynamic impairment, cytoskeletal alterations due to primary genetic defects or indirectly to alterations in response to cellular injury can potentially
affect ion channel anchoring, and trafficking, as well as
functional regulation by second messenger pathways,
causing an imbalance in cardiac ionic homeostasis that will trigger arrhythmogenesis.
Intense investigation of
the sarcomeric actin network,
the Z-line structure, and
chaperone molecules docking in the plasma membrane,
has shed new light on the molecular basis of
cytoskeletal interactions in regulating ion channels.
In 1991, Cantiello et al., demonstrated that
although the epithelial sodium channel and F-actin are in close proximity,
they do not co-localize.
Actin disruption using cytochalasin D, an agent that interferes with actin polymerization, increased Na+ channel activity in 90% of excised patches tested within 2 min, which indicated that
the integrity of the filamentous actin (F-actin) network was essential
for the maintenance of normal Na+ channel function.
Later, the group of Dr. Jonathan Makielski demonstrated that
actin disruption induced a dramatic reduction in Na+ peak current and
slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation.
These data were the first to support a role for the cytoskeleton in cardiac arrhythmias.
F-actin is intertwined in a multi-protein complex that includes
the composite Z-line structure.
Further, there is a direct binding between
the major protein of the Z-line, α-actinin-2 and
the voltage-gated K+ channel 1.5 (Kv1.5), (Figure 2).
The latter is expressed in human cardiomyocytes and localizes to
the intercalated disk of the cardiomyocyte
in association with connexin and N-cadherin.
Maruoka et al. treated HEK293 cells stably expressing Kv1.5 with cytochalasin D, which led to
a massive increase in ionic and gating IK+ currents.
This was prevented by pre-incubation with phalloidin, an F-actin stabilizing agent. In addition, the Z-line protein telethonin binds to the cytoplasmic domain of minK, the beta subunit of the potassium channel KCNQ1 (Figure 2).
Molecular interactions between the cytoskeleton and ion channels
Figure 2. Molecular interactions between the cytoskeleton and ion channels
The figure illustrates the interactions between the ion channels on the sarcolemma, and the sarcomere in cardiac myocytes. Note that the Z-line is connected to the cardiac T-tubules. The diagram illustrates the complex protein-protein interactions that occur between structural components of the cytoskeleton and ion channels. The cytoskeleton is involved in regulating the metabolism of ion channels, modifying their expression, localization, and electrical properties. The cardiac sodium channel Nav1.5 associates with the DGC, while potassium channels such as Kv1.5, associate with the Z-line.
Ion Channel Subunits and Trafficking
Correct localization is essential for ion channel function and this is dependent upon the ability of auxiliary proteins to
shuttle ion channels from the cytoplasm to their final destination such as
the plasma membrane or other sub-cellular compartments.
In this regard, Kvβ-subunits are
cytoplasmic components known to assemble with the α-subunits of voltage-dependent K+ (Kv) channels
at their N-terminus to form stable Kvα/β hetero-oligomeric channels.
When Kvβ is co-expressed with Kv1.4 or Kv1.5, it enhances Kv1.x channel trafficking to the cell membrane without changing the overall protein channel content. The regulatory Kvβ subunits, which are also expressed in cardiomyocytes, directly decrease K+ current by
accelerating Kv1.x channel inactivation.
Therefore, altered expression or mutations in Kvβ subunits could cause abnormal ion channel transport to the cell surface, thereby increasing the risk of cardiac arrhythmias.
Ion Channel Protein Motifs and Trafficking
Cell membrane trafficking in the Kv1.x family may occur in a Kvβ subunit-independent manner through specific motifs in their C-terminus. Mutagenesis of the final asparagine (N) in the Kv1.2 motif restores the leucine (L) of the Kv1.4 motif
re-establishing high expression levels at the plasma membrane in a Kvβ-independent manner
Cytoskeletal Proteins and Ion Channel Trafficking
Until recently, primary arrhythmias such as LQTS have been almost exclusively regarded as ion channelopathies. Other mutations have been identified with regard to channelopathies. However, the conviction that primary mutations in ion channels were solely responsible for
the electrical defects associated with arrhythmias
has been shaken by the identification of mutations in the
ANK2 gene encoding the cytoskeletal protein ankyrin-B
that is associated with LQTS in animal models and humans.
Ankyrin-B acts as a chaperone protein, which shuttles the cardiac sodium channel from the cytoplasm to the membrane. Immunohistochemical analysis has localized ankyrin-B to the Zlines/T-tubules on the plasma membrane in the myocardium. Mutations in ankyrin-B associated with LQTS
alter sodium channel trafficking due to loss of ankyrin-B localization at the Z-line/transverse (T)-tubules.
Reduced levels of ankyrin-B at cardiac Z-lines/T-tubules were associated with the deficiency of ankyrin-B-associated proteins such as Na/K-ATPase, Na/Ca exchanger (NCX) and inositol-1, 4, 5-trisphosphate receptors (InsP3R).
Dystrophin component of the Dystrophin Glycoprotien Complex (DGC)
Synchronized contraction is essential for cardiomyocytes, which are connected to each other via the extracellular matrix (ECM) through the DGC. The N-terminus domain of dystrophin
binds F-actin, and connects it to the sarcomere, while
the cysteine-rich (CR) C-terminus domain ensures its connection to the sarcolemma (Figure 2).
The central portion of dystrophin, the rod domain, is composed of
rigid spectrin-like repeats and four hinge portions (H1–H4) that determine the flexibility of the protein.
Dystrophin possesses another F-actin binding domain in the Rod domain region, between the basic repeats 11- 17 (DysN-R17).
Dystrophin, originally identified as the gene responsible for Duchenne and Becker muscular dystrophies (DMD/BMD), and later for the X-linked form of dilated cardiomyopathy (XLCM), exerts a major function in physical force transmission in striated muscle. In addition to its structural significance, dystrophin and other DGC proteins such as syntrophins are required for the
correct localization,
clustering and
regulation of ion channel function.
Syntrophins have been implicated in ion channel regulation. Syntrophins contain two pleckstrin homology (PH) domains, a PDZ domain, and a syntrophin-unique (SU) C-terminal region. The interaction between syntrophins and dystrophin occurs at the PH domain distal to the syntrophin N-terminus and through the highly conserved SU domain. Conversely, the PH domain proximal to the N-terminal portion of the protein and the PDZ domain interact with other membrane components such as
phosphatidyl inositol-4, 5-bisphosphate,
neuronal NOS (nNOS),
aquaporin-4,
stress-activated protein kinase-3, and
5,
thereby linking all these molecules to the dystrophin complex (Figure 2).
Among the five known isoforms of syntrophin, the 59 KDa α1-syntrophin isoform is the most highly represented in human heart, whereas in skeletal muscle it is only present on the
sarcolemma of fast type II fibers.
In addition, the skeletal muscle γ2-syntrophin was found at high levels only at the
postsynaptic membrane of the neuromuscular junctions.
In addition to syntrophin, other scaffolding proteins such as caveolin-3 (CAV3), which is present in the caveolae, flask-shaped plasma membrane microdomains, are involved
in signal transduction and vesicle trafficking in myocytes,
modulating cardiac remodeling during heart failure.
CAV3 and α1-syntrophin, localizes at the T-tubule and are part of the DGC. In addition, α1-syntrophin binds Nav1.5, while
caveolin-3 binds the Na+/Ca2+ exchanger, Nav1.5 and the L-type Ca2+ channel as well as nNOS and the DGC (Figure 2).
Although ankyrin-B is the only protein found mutated in patients with primary arrhythmias, other proteins such as caveolin-3 and the syntrophins if mutated may alter ion channel function.
Conclusions
It is important to be aware of the enormous variety of clinical presentations that derive from distinct variants in the same pool of genetic factors. Knowledge of these variants could facilitate tailoring the therapy of choice for each patient. In particular, the recent findings of structural and functional links between
the cytoskeleton and ion channels
could expand the therapeutic interventions in
arrhythmia management in structurally abnormal myocardium, where aberrant binding
between cytoskeletal proteins can directly or indirectly alter ion channel function.
Executive Summary
Arrhythmogenesis and myocardial structure
Rhythm alterations can develop as a secondary consequence of myocardial structural abnormalities or as a result of a primary defect in the cardiac electric machinery.
Until recently, no molecular mechanism has been able to fully explain the occurrence of arrhythmogenesis in heart failure, however genetic defects that are found almost exclusively in ion channel genes account for the majority of primary arrhythmias such as long QT syndromes and Brugada syndrome. The contractile apparatus is linked to ion channels
The sarcomere, which represents the contractile unit of the myocardium not only generates the mechanical force necessary to exert the pump function, but also provides localization and anchorage to ion channels.
Alpha-actinin-2, and telethonin, two members of the Z-line scaffolding protein complex in the striated muscle associate with the potassium voltage-gated channel alpha subunit Kv1.5 and the beta subunit KCNE1 respectively.
Mutations in KCNE1 have previously been associated with the development of arrhythmias in LQTS subjects.
Mutations in both alpha-actinin-2, and telethonin were identified in individuals with cardiomyopathy. The primary defect is structural leading to ventricular dysfunction, but the secondary consequence is arrhythmia.
Ion channel trafficking and sub-cellular compartments
Ion channel trafficking from the endoplasmic reticulum (ER) to the Golgi complex is an important check-point for regulating the functional channel molecules on the plasma membrane. Several molecules acting as chaperones bind to and shuttle the channel proteins to their final localization on the cell surface
Ion channel subunits such as Kvβ enhance Kv1.x ion channel presentation on the sarcolemma. The α subunits of the Kv1.x potassium channels can be shuttled in a Kvβ-independent manner through specific sequence motif at Kv1.x protein level.
In addition, cytoskeletal proteins such as ankyrin-G bind Nav1.5 and are involved in the sodium channel trafficking. Another member of the ankyrin family, ankyrin-B was found mutated in patients with LQTS but the pathological mechanism of ankyrin-B mutations is still obscure, although the sodium current intensity is dramatically reduced.
The sarcolemma and ion channels
The sarcolemma contains a wide range of ion channels, which are responsible for the electrical propagating force in the myocardium.
The DGC is a protein complex, which forms a scaffold for cytoskeletal components and ion channels.
Dystrophin is the major component of the DGC and mutations in dystrophin and DGC cause muscular dystrophies and X-linked cardiomyopathies (XLCM) in humans. Cardiomyopathies are associated with arrhythmias
Caveolin-3 and syntrophins associate with Nav1.5, and are part of the DGC. Syntrophins can directly modulate Nav1.5 channel function.
Conclusions
The role of the cytoskeleton in ion channel function has been hypothesized in the past, but only recently the mechanism underlying the development of arrhythmias in structurally impaired myocardium has become clearer.
The recently acknowledged role of the cytoskeleton in ion channel function suggests that genes encoding cytoskeletal proteins should be regarded as potential candidates for variants involved in the susceptibility to arrhythmias, as well as the primary target of genetic mutations in patients with arrhythmogenic syndromes such as LQTS and Brugada syndrome.
Studies of genotype-phenotype correlation and and patient risk stratification for mutations in cytoskeletal proteins will help to tailor the therapy and management of patients with arrhythmias.
Summary – Volume 4, Part 2: Translational Medicine in Cardiovascular Diseases
Author and Curator: Larry H Bernstein, MD, FCAP
We have covered a large amount of material that involves
the development,
application, and
validation of outcomes of medical and surgical procedures
that are based on translation of science from the laboratory to the bedside, improving the standards of medical practice at an accelerated pace in the last quarter century, and in the last decade. Encouraging enabling developments have been:
1. The establishment of national and international outcomes databases for procedures by specialist medical societies
2. The identification of problem areas, particularly in activation of the prothrombotic pathways, infection control to an extent, and targeting of pathways leading to progression or to arrythmogenic complications.
5. This has become possible because of the advances in our knowledge of key related pathogenetic mechanisms involving gene expression and cellular regulation of complex mechanisms.
This completes what has been presented in Part 2, Vol 4 , and supporting references for the main points that are found in the Leaders in Pharmaceutical Intelligence Cardiovascular book. Part 1 was concerned with Posttranslational Modification of Proteins, vital for understanding cellular regulation and dysregulation. Part 2 was concerned with Translational Medical Therapeutics, the efficacy of medical and surgical decisions based on bringing the knowledge gained from the laboratory, and from clinical trials into the realm opf best practice. The time for this to occur in practice in the past has been through roughly a generation of physicians. That was in part related to the busy workload of physicians, and inability to easily access specialty literature as the volume and complexity increased. This had an effect of making access of a family to a primary care provider through a lifetime less likely than the period post WWII into the 1980s.
However, the growth of knowledge has accelerated in the specialties since the 1980’s so that the use of physician referral in time became a concern about the cost of medical care. This is not the place for or a matter for discussion here. It is also true that the scientific advances and improvements in available technology have had a great impact on medical outcomes. The only unrelated issue is that of healthcare delivery, which is not up to the standard set by serial advances in therapeutics, accompanied by high cost due to development costs, marketing costs, and development of drug resistance.
I shall identify continuing developments in cardiovascular diagnostics, therapeutics, and bioengineering that is and has been emerging.
Abstract:Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.
In order to identify how chemical compounds target genes and affect the physiology of the cell, tests of the perturbations that occur when treated with a range of pharmacological chemicals are required. By examining the haploinsufficiency profiling (HIP) and homozygous profiling (HOP) chemogenomic platforms, Lee et al.(p. 208) analyzed the response of yeast to thousands of different small molecules, with genetic, proteomic, and bioinformatic analyses. Over 300 compounds were identified that targeted 121 genes within 45 cellular response signature networks. These networks were used to extrapolate the likely effects of related chemicals, their impact upon genetic pathways, and to identify putative gene functions
A team of cardiovascular researchers from the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai, Sanford-Burnham Medical Research Institute, and University of California, San Diego have identified a small, but powerful, new player in thIe onset and progression of heart failure. Their findings, published in the journal Nature on March 12, also show how they successfully blocked the newly discovered culprit.
Investigators identified a tiny piece of RNA called miR-25 that blocks a gene known as SERCA2a, which regulates the flow of calcium within heart muscle cells. Decreased SERCA2a activity is one of the main causes of poor contraction of the heart and enlargement of heart muscle cells leading to heart failure.
Using a functional screening system developed by researchers at Sanford-Burnham, the research team discovered miR-25 acts pathologically in patients suffering from heart failure, delaying proper calcium uptake in heart muscle cells. According to co-lead study authors Christine Wahlquist and Dr. Agustin Rojas Muñoz, developers of the approach and researchers in Mercola’s lab at Sanford-Burnham, they used high-throughput robotics to sift through the entire genome for microRNAs involved in heart muscle dysfunction.
Subsequently, the researchers at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai found that injecting a small piece of RNA to inhibit the effects of miR-25 dramatically halted heart failure progression in mice. In addition, it also improved their cardiac function and survival.
“In this study, we have not only identified one of the key cellular processes leading to heart failure, but have also demonstrated the therapeutic potential of blocking this process,” says co-lead study author Dr. Dongtak Jeong, a post-doctoral fellow at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai in the laboratory of the study’s co-senior author Dr. Roger J. Hajjar.
Publication: Inhibition of miR-25 improves cardiac contractility in the failing heart.Christine Wahlquist, Dongtak Jeong, Agustin Rojas-Muñoz, Changwon Kho, Ahyoung Lee, Shinichi Mitsuyama, Alain Van Mil, Woo Jin Park, Joost P. G. Sluijter, Pieter A. F. Doevendans, Roger J. : Hajjar & Mark Mercola. Nature (March 2014) http://www.nature.com/nature/journal/vaop/ncurrent/full/nature13073.html
“Junk” DNA Tied to Heart Failure
Deep RNA Sequencing Reveals Dynamic Regulation of Myocardial Noncoding RNAs in Failing Human Heart and Remodeling With Mechanical Circulatory Support
The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.
Junk DNA was long thought to have no important role in heredity or disease because it doesn’t code for proteins. But emerging research in recent years has revealed that many of these sections of the genome produce noncoding RNA molecules that still have important functions in the body. They come in a variety of forms, some more widely studied than others. Of these, about 90% are called long noncoding RNAs (lncRNAs), and exploration of their roles in health and disease is just beginning.
The Washington University group performed a comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.
In their study, the researchers found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.
“The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support,” wrote the researchers. “These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.”
‘Junk’ Genome Regions Linked to Heart Failure
In a recent issue of the journal Circulation, Washington University investigators report results from the first comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.
“We took an unbiased approach to investigating which types of RNA might be linked to heart failure,” said senior author Jeanne Nerbonne, the Alumni Endowed Professor of Molecular Biology and Pharmacology. “We were surprised to find that long noncoding RNAs stood out.
In the new study, the investigators found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.
“We don’t know whether these changes in long noncoding RNAs are a cause or an effect of heart failure,” Nerbonne said. “But it seems likely they play some role in coordinating the regulation of multiple genes involved in heart function.”
Nerbonne pointed out that all types of RNA molecules they examined could make the obvious distinction: telling the difference between failing and nonfailing hearts. But only expression of the long noncoding RNAs was measurably different between heart failure associated with a heart attack (ischemic) and heart failure without the obvious trigger of blocked arteries (nonischemic). Similarly, only long noncoding RNAs significantly changed expression patterns after implantation of left ventricular assist devices.
Heart failure is a complex disease with a broad spectrum of pathological features. Despite significant advancement in clinical diagnosis through improved imaging modalities and hemodynamic approaches, reliable molecular signatures for better differential diagnosis and better monitoring of heart failure progression remain elusive. The few known clinical biomarkers for heart failure, such as plasma brain natriuretic peptide and troponin, have been shown to have limited use in defining the cause or prognosis of the disease.1,2 Consequently, current clinical identification and classification of heart failure remain descriptive, mostly based on functional and morphological parameters. Therefore, defining the pathogenic mechanisms for hypertrophic versus dilated or ischemic versus nonischemic cardiomyopathies in the failing heart remain a major challenge to both basic science and clinic researchers. In recent years, mechanical circulatory support using left ventricular assist devices (LVADs) has assumed a growing role in the care of patients with end-stage heart failure.3 During the earlier years of LVAD application as a bridge to transplant, it became evident that some patients exhibit substantial recovery of ventricular function, structure, and electric properties.4 This led to the recognition that reverse remodeling is potentially an achievable therapeutic goal using LVADs. However, the underlying mechanism for the reverse remodeling in the LVAD-treated hearts is unclear, and its discovery would likely hold great promise to halt or even reverse the progression of heart failure.
Efficacy and Safety of Dabigatran Compared With Warfarin in Relation to Baseline Renal Function in Patients With Atrial Fibrillation: A RE-LY (Randomized Evaluation of Long-term Anticoagulation Therapy) Trial Analysis
In patients with atrial fibrillation, impaired renal function is associated with a higher risk of thromboembolic events and major bleeding. Oral anticoagulation with vitamin K antagonists reduces thromboembolic events but raises the risk of bleeding. The new oral anticoagulant dabigatran has 80% renal elimination, and its efficacy and safety might, therefore, be related to renal function. In this prespecified analysis from the Randomized Evaluation of Long-Term Anticoagulant Therapy (RELY) trial, outcomes with dabigatran versus warfarin were evaluated in relation to 4 estimates of renal function, that is, equations based on creatinine levels (Cockcroft-Gault, Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI]) and cystatin C. The rates of stroke or systemic embolism were lower with dabigatran 150 mg and similar with 110 mg twice daily irrespective of renal function. Rates of major bleeding were lower with dabigatran 110 mg and similar with 150 mg twice daily across the entire range of renal function. However, when the CKD-EPI or MDRD equations were used, there was a significantly greater relative reduction in major bleeding with both doses of dabigatran than with warfarin in patients with estimated glomerular filtration rate ≥80 mL/min. These findings show that dabigatran can be used with the same efficacy and adequate safety in patients with a wide range of renal function and that a more accurate estimate of renal function might be useful for improved tailoring of anticoagulant treatment in patients with atrial fibrillation and an increased risk of stroke.
Aldosterone Regulates MicroRNAs in the Cortical Collecting Duct to Alter Sodium Transport.
ABSTRACT A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells.
Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport.
Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein.
A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3′ untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.
2. Diagnostic Biomarker Status
A prospective study of the impact of serial troponin measurements on the diagnosis of myocardial infarction and hospital and 6-month mortality in patients admitted to ICU with non-cardiac diagnoses.
ABSTRACT Troponin T (cTnT) elevation is common in patients in the Intensive Care Unit (ICU) and associated with morbidity and mortality. Our aim was to determine the epidemiology of raised cTnT levels and contemporaneous electrocardiogram (ECG) changes suggesting myocardial infarction (MI) in ICU patients admitted for non-cardiac reasons.
cTnT and ECGs were recorded daily during week 1 and on alternate days during week 2 until discharge from ICU or death. ECGs were interpreted independently for the presence of ischaemic changes. Patients were classified into 4 groups: (i) definite MI (cTnT >=15 ng/L and contemporaneous changes of MI on ECG), (ii) possible MI (cTnT >=15 ng/L and contemporaneous ischaemic changes on ECG), (iii) troponin rise alone (cTnT >=15 ng/L), or (iv) normal. Medical notes were screened independently by two ICU clinicians for evidence that the clinical teams had considered a cardiac event.
Data from 144 patients were analysed [42% female; mean age 61.9 (SD 16.9)]. 121 patients (84%) had at least one cTnT level >=15 ng/L. A total of 20 patients (14%) had a definite MI, 27% had a possible MI, 43% had a cTNT rise without contemporaneous ECG changes, and 16% had no cTNT rise. ICU, hospital and 180 day mortality were significantly higher in patients with a definite or possible MI.Only 20% of definite MIs were recognised by the clinical team. There was no significant difference in mortality between recognised and non-recognised events.At time of cTNT rise, 100 patients (70%) were septic and 58% were on vasopressors. Patients who were septic when cTNT was elevated had an ICU mortality of 28% compared to 9% in patients without sepsis. ICU mortality of patients who were on vasopressors at time of cTNT elevation was 37% compared to 1.7% in patients not on vasopressors.
The majority of critically ill patients (84%) had a cTnT rise and 41% met criteria for a possible or definite MI of whom only 20% were recognised clinically. Mortality up to 180 days was higher in patients with a cTnT rise.
Prognostic performance of high-sensitivity cardiac troponin T kinetic changes adjusted for elevated admission values and the GRACE score in an unselected emergency department population.
ABSTRACT To test the prognostic performance of rising and falling kinetic changes of high-sensitivity cardiac troponin T (hs-cTnT) and the GRACE score.
Rising and falling hs-cTnT changes in an unselected emergency department population were compared.
635 patients with a hs-cTnT >99th percentile admission value were enrolled. Of these, 572 patients qualified for evaluation with rising patterns (n=254, 44.4%), falling patterns (n=224, 39.2%), or falling patterns following an initial rise (n=94, 16.4%). During 407days of follow-up, we observed 74 deaths, 17 recurrent AMI, and 79 subjects with a composite of death/AMI. Admission values >14ng/L were associated with a higher rate of adverse outcomes (OR, 95%CI:death:12.6, 1.8-92.1, p=0.01, death/AMI:6.7, 1.6-27.9, p=0.01). Neither rising nor falling changes increased the AUC of baseline values (AUC: rising 0.562 vs 0.561, p=ns, falling: 0.533 vs 0.575, p=ns). A GRACE score ≥140 points indicated a higher risk of death (OR, 95%CI: 3.14, 1.84-5.36), AMI (OR,95%CI: 1.56, 0.59-4.17), or death/AMI (OR, 95%CI: 2.49, 1.51-4.11). Hs-cTnT changes did not improve prognostic performance of a GRACE score ≥140 points (AUC, 95%CI: death: 0.635, 0.570-0.701 vs. 0.560, 0.470-0.649 p=ns, AMI: 0.555, 0.418-0.693 vs. 0.603, 0.424-0.782, p=ns, death/AMI: 0.610, 0.545-0.676 vs. 0.538, 0.454-0.622, p=ns). Coronary angiography was performed earlier in patients with rising than with falling kinetics (median, IQR [hours]:13.7, 5.5-28.0 vs. 20.8, 6.3-59.0, p=0.01).
Neither rising nor falling hs-cTnT changes improve prognostic performance of elevated hs-cTnT admission values or the GRACE score. However, rising values are more likely associated with the decision for earlier invasive strategy.
ABSTRACT: Under normal circumstances, most intracellular troponin is part of the muscle contractile apparatus, and only a small percentage (< 2-8%) is free in the cytoplasm. The presence of a cardiac-specific troponin in the circulation at levels above normal is good evidence of damage to cardiac muscle cells, such as myocardial infarction, myocarditis, trauma, unstable angina, cardiac surgery or other cardiac procedures. Troponins are released as complexes leading to various cut-off values depending on the assay used. This makes them very sensitive and specific indicators of cardiac injury. As with other cardiac markers, observation of a rise and fall in troponin levels in the appropriate time-frame increases the diagnostic specificity for acute myocardial infarction. They start to rise approximately 4-6 h after the onset of acute myocardial infarction and peak at approximately 24 h, as is the case with creatine kinase-MB. They remain elevated for 7-10 days giving a longer diagnostic window than creatine kinase. Although the diagnosis of various types of acute coronary syndrome remains a clinical-based diagnosis, the use of troponin levels contributes to their classification. This Editorial elaborates on the nature of troponin, its classification, clinical use and importance, as well as comparing it with other currently available cardiac markers.
ABSTRACT: Although redefinition for acute myocardial infarction (AMI) has been proposed few years ago, to date it has not been universally adopted by many institutions. The purpose of this study is to evaluate the diagnostic, prognostic and economical impact of the new diagnostic criteria for AMI. Patients consecutively admitted to the emergency department with suspected acute coronary syndromes were enrolled in this study. Troponin T (cTnT) was measured in samples collected for routine CK-MB analyses and results were not available to physicians. Patients without AMI by traditional criteria and cTnT > or = 0.035 ng/mL were coded as redefined AMI. Clinical outcomes were hospital death, major cardiac events and revascularization procedures. In-hospital management and reimbursement rates were also analyzed. Among 363 patients, 59 (16%) patients had AMI by conventional criteria, whereas additional 75 (21%) had redefined AMI, an increase of 127% in the incidence. Patients with redefined AMI were significantly older, more frequently male, with atypical chest pain and more risk factors. In multivariate analysis, redefined AMI was associated with 3.1 fold higher hospital death (95% CI: 0.6-14) and a 5.6 fold more cardiac events (95% CI: 2.1-15) compared to those without AMI. From hospital perspective, based on DRGs payment system, adoption of AMI redefinition would increase 12% the reimbursement rate [3552 Int dollars per 100 patients evaluated]. The redefined criteria result in a substantial increase in AMI cases, and allow identification of high-risk patients. Efforts should be made to reinforce the adoption of AMI redefinition, which may result in more qualified and efficient management of ACS.
Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of application with substantial intrinsic hurdles, but where human translation is now occurring.
Acellular Biomaterials: An Evolving Alternative to Cell-Based Therapies
Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of applications with substantial intrinsic hurdles, but where human translation is now occurring.
Instructive Nanofiber Scaffolds with VEGF Create a Microenvironment for Arteriogenesis and Cardiac Repair
Angiogenic therapy is a promising approach for tissue repair and regeneration. However, recent clinical trials with protein delivery or gene therapy to promote angiogenesis have failed to provide therapeutic effects. A key factor for achieving effective revascularization is the durability of the microvasculature and the formation of new arterial vessels. Accordingly, we carried out experiments to test whether intramyocardial injection of self-assembling peptide nanofibers (NFs) combined with vascular endothelial growth factor (VEGF) could create an intramyocardial microenvironment with prolonged VEGF release to improve post-infarct neovascularization in rats. Our data showed that when injected with NF, VEGF delivery was sustained within the myocardium for up to 14 days, and the side effects of systemic edema and proteinuria were significantly reduced to the same level as that of control. NF/VEGF injection significantly improved angiogenesis, arteriogenesis, and cardiac performance 28 days after myocardial infarction. NF/VEGF injection not only allowed controlled local delivery but also transformed the injected site into a favorable microenvironment that recruited endogenous myofibroblasts and helped achieve effective revascularization. The engineered vascular niche further attracted a new population of cardiomyocyte-like cells to home to the injected sites, suggesting cardiomyocyte regeneration. Follow-up studies in pigs also revealed healing benefits consistent with observations in rats. In summary, this study demonstrates a new strategy for cardiovascular repair with potential for future clinical translation.
Along with scientific and regulatory issues, the translation of cell and tissue therapies in the routine clinical practice needs to address standardization and cost-effectiveness through the definition of suitable manufacturing paradigms.
Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1
Author and Curator: Larry H Bernstein, MD, FCAP
and
Curator: Aviva Lev-Ari, PhD, RN
Part 1 of Volume 4 in the e-series A: Cardiovascular Diseases and Translational Medicine, provides a foundation for grasping a rapidly developing surging scientific endeavor that is transcending laboratory hypothesis testing and providing guidelines to:
Target genomes and multiple nucleotide sequences involved in either coding or in regulation that might have an impact on complex diseases, not necessarily genetic in nature.
Target signaling pathways that are demonstrably maladjusted, activated or suppressed in many common and complex diseases, or in their progression.
Enable a reduction in failure due to toxicities in the later stages of clinical drug trials as a result of this science-based understanding.
Enable a reduction in complications from the improvement of machanical devices that have already had an impact on the practice of interventional procedures in cardiology, cardiac surgery, and radiological imaging, as well as improving laboratory diagnostics at the molecular level.
Enable the discovery of new drugs in the continuing emergence of drug resistance.
Enable the construction of critical pathways and better guidelines for patient management based on population outcomes data, that will be critically dependent on computational methods and large data-bases.
What has been presented can be essentially viewed in the following Table:
Summary Table for TM – Part 1
There are some developments that deserve additional development:
1. The importance of mitochondrial function in the activity state of the mitochondria in cellular work (combustion) is understood, and impairments of function are identified in diseases of muscle, cardiac contraction, nerve conduction, ion transport, water balance, and the cytoskeleton – beyond the disordered metabolism in cancer. A more detailed explanation of the energetics that was elucidated based on the electron transport chain might also be in order.
2. The processes that are enabling a more full application of technology to a host of problems in the environment we live in and in disease modification is growing rapidly, and will change the face of medicine and its allied health sciences.
Electron Transport and Bioenergetics
Deferred for metabolomics topic
Synthetic Biology
Introduction to Synthetic Biology and Metabolic Engineering
http://www.ibiology.org Lecturers generously donate their time to prepare these lectures. The project is funded by NSF and NIGMS, and is supported by the ASCB and HHMI.
Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”.
Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching. Learn more about how Kris became a scientist at
Prather 1: Synthetic Biology and Metabolic Engineering 2/6/14IntroductionLecture Overview In the first part of her lecture, Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”. The key material in building these machines is synthetic DNA. Synthetic DNA can be added in different combinations to biological hosts, such as bacteria, turning them into chemical factories that can produce small molecules of choice. In Part 2, Prather describes how her lab used design principles to engineer E. coli that produce glucaric acid from glucose. Glucaric acid is not naturally produced in bacteria, so Prather and her colleagues “bioprospected” enzymes from other organisms and expressed them in E. coli to build the needed enzymatic pathway. Prather walks us through the many steps of optimizing the timing, localization and levels of enzyme expression to produce the greatest yield. Speaker Bio: Kristala Jones Prather received her S.B. degree from the Massachusetts Institute of Technology and her PhD at the University of California, Berkeley both in chemical engineering. Upon graduation, Prather joined the Merck Research Labs for 4 years before returning to academia. Prather is now an Associate Professor of Chemical Engineering at MIT and an investigator with the multi-university Synthetic Biology Engineering Reseach Center (SynBERC). Her lab designs and constructs novel synthetic pathways in microorganisms converting them into tiny factories for the production of small molecules. Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching.
Calcium Cycling in Synthetic and Contractile Phasic or Tonic Vascular Smooth Muscle Cells
in INTECH
Current Basic and Pathological Approaches to
the Function of Muscle Cells and Tissues – From Molecules to HumansLarissa Lipskaia, Isabelle Limon, Regis Bobe and Roger Hajjar
Additional information is available at the end of the chapter http://dx.doi.org/10.5772/48240
1. Introduction
Calcium ions (Ca ) are present in low concentrations in the cytosol (~100 nM) and in high concentrations (in mM range) in both the extracellular medium and intracellular stores (mainly sarco/endo/plasmic reticulum, SR). This differential allows the calcium ion messenger that carries information
as diverse as contraction, metabolism, apoptosis, proliferation and/or hypertrophic growth. The mechanisms responsible for generating a Ca signal greatly differ from one cell type to another.
In the different types of vascular smooth muscle cells (VSMC), enormous variations do exist with regard to the mechanisms responsible for generating Ca signal. In each VSMC phenotype (synthetic/proliferating and contractile [1], tonic or phasic), the Ca signaling system is adapted to its particular function and is due to the specific patterns of expression and regulation of Ca.
For instance, in contractile VSMCs, the initiation of contractile events is driven by mem- brane depolarization; and the principal entry-point for extracellular Ca is the voltage-operated L-type calcium channel (LTCC). In contrast, in synthetic/proliferating VSMCs, the principal way-in for extracellular Ca is the store-operated calcium (SOC) channel.
Whatever the cell type, the calcium signal consists of limited elevations of cytosolic free calcium ions in time and space. The calcium pump, sarco/endoplasmic reticulum Ca ATPase (SERCA), has a critical role in determining the frequency of SR Ca release by upload into the sarcoplasmic
sensitivity of SR calcium channels, Ryanodin Receptor, RyR and Inositol tri-Phosphate Receptor, IP3R.
Synthetic VSMCs have a fibroblast appearance, proliferate readily, and synthesize increased levels of various extracellular matrix components, particularly fibronectin, collagen types I and III, and tropoelastin [1].
Contractile VSMCs have a muscle-like or spindle-shaped appearance and well-developed contractile apparatus resulting from the expression and intracellular accumulation of thick and thin muscle filaments [1].
Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs
Figure 1. Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs.
Left panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Contractile re-sponse is initiated by extracellular Ca influx due to activation of Receptor Operated Ca (through phosphoinositol-coupled receptor) or to activation of L-Type Calcium channels (through an increase in luminal pressure). Small increase of cytosolic due IP3 binding to IP3R (puff) or RyR activation by LTCC or ROC-dependent Ca influx leads to large SR Ca IP3R or RyR clusters (“Ca -induced Ca SR calcium pumps (both SERCA2a and SERCA2b are expressed in quiescent VSMCs), maintaining high concentration of cytosolic Ca and setting the sensitivity of RyR or IP3R for the next spike.
Contraction of VSMCs occurs during oscillatory Ca transient.
Middle panel: schematic representa tion of atherosclerotic vessel wall. Contractile VSMC are located in the media layer, synthetic VSMC are located in sub-endothelial intima.
Right panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Agonist binding to phosphoinositol-coupled receptor leads to the activation of IP3R resulting in large increase in cytosolic Ca calcium pumps (only SERCA2b, having low turnover and low affinity to Ca depletion leads to translocation of SR Ca sensor STIM1 towards PM, resulting in extracellular Ca influx though opening of Store Operated Channel (CRAC). Resulted steady state Ca transient is critical for activation of proliferation-related transcription factors ‘NFAT).
Abbreviations: PLC – phospholipase C; PM – plasma membrane; PP2B – Ca /calmodulin-activated protein phosphatase 2B (calcineurin); ROC- receptor activated channel; IP3 – inositol-1,4,5-trisphosphate, IP3R – inositol-1,4,5- trisphosphate receptor; RyR – ryanodine receptor; NFAT – nuclear factor of activated T-lymphocytes; VSMC – vascular smooth muscle cells; SERCA – sarco(endo)plasmic reticulum Ca sarcoplasmic reticulum.
Time for New DNA Synthesis and Sequencing Cost Curves
By Rob Carlson
I’ll start with the productivity plot, as this one isn’t new. For a discussion of the substantial performance increase in sequencing compared to Moore’s Law, as well as the difficulty of finding this data, please see this post. If nothing else, keep two features of the plot in mind: 1) the consistency of the pace of Moore’s Law and 2) the inconsistency and pace of sequencing productivity. Illumina appears to be the primary driver, and beneficiary, of improvements in productivity at the moment, especially if you are looking at share prices. It looks like the recently announced NextSeq and Hiseq instruments will provide substantially higher productivities (hand waving, I would say the next datum will come in another order of magnitude higher), but I think I need a bit more data before officially putting another point on the plot.
cost-of-oligo-and-gene-synthesis
Illumina’s instruments are now responsible for such a high percentage of sequencing output that the company is effectively setting prices for the entire industry. Illumina is being pushed by competition to increase performance, but this does not necessarily translate into lower prices. It doesn’t behoove Illumina to drop prices at this point, and we won’t see any substantial decrease until a serious competitor shows up and starts threatening Illumina’s market share. The absence of real competition is the primary reason sequencing prices have flattened out over the last couple of data points.
Note that the oligo prices above are for column-based synthesis, and that oligos synthesized on arrays are much less expensive. However, array synthesis comes with the usual caveat that the quality is generally lower, unless you are getting your DNA from Agilent, which probably means you are getting your dsDNA from Gen9.
Note also that the distinction between the price of oligos and the price of double-stranded sDNA is becoming less useful. Whether you are ordering from Life/Thermo or from your local academic facility, the cost of producing oligos is now, in most cases, independent of their length. That’s because the cost of capital (including rent, insurance, labor, etc) is now more significant than the cost of goods. Consequently, the price reflects the cost of capital rather than the cost of goods. Moreover, the cost of the columns, reagents, and shipping tubes is certainly more than the cost of the atoms in the sDNA you are ostensibly paying for. Once you get into longer oligos (substantially larger than 50-mers) this relationship breaks down and the sDNA is more expensive. But, at this point in time, most people aren’t going to use longer oligos to assemble genes unless they have a tricky job that doesn’t work using short oligos.
Looking forward, I suspect oligos aren’t going to get much cheaper unless someone sorts out how to either 1) replace the requisite human labor and thereby reduce the cost of capital, or 2) finally replace the phosphoramidite chemistry that the industry relies upon.
IDT’s gBlocks come at prices that are constant across quite substantial ranges in length. Moreover, part of the decrease in price for these products is embedded in the fact that you are buying smaller chunks of DNA that you then must assemble and integrate into your organism of choice.
Someone who has purchased and assembled an absolutely enormous amount of sDNA over the last decade, suggested that if prices fell by another order of magnitude, he could switch completely to outsourced assembly. This is a potentially interesting “tipping point”. However, what this person really needs is sDNA integrated in a particular way into a particular genome operating in a particular host. The integration and testing of the new genome in the host organism is where most of the cost is. Given the wide variety of emerging applications, and the growing array of hosts/chassis, it isn’t clear that any given technology or firm will be able to provide arbitrary synthetic sequences incorporated into arbitrary hosts.
Dr. Jon Rowley and Dr. Uplaksh Kumar, Co-Founders of RoosterBio, Inc., a newly formed biotech startup located in Frederick, are paving the way for even more innovation in the rapidly growing fields of Synthetic Biology and Regenerative Medicine. Synthetic Biology combines engineering principles with basic science to build biological products, including regenerative medicines and cellular therapies. Regenerative medicine is a broad definition for innovative medical therapies that will enable the body to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. Regenerative therapies that are in clinical trials today may enable repair of damaged heart muscle following heart attack, replacement of skin for burn victims, restoration of movement after spinal cord injury, regeneration of pancreatic tissue for insulin production in diabetics and provide new treatments for Parkinson’s and Alzheimer’s diseases, to name just a few applications.
While the potential of the field is promising, the pace of development has been slow. One main reason for this is that the living cells required for these therapies are cost-prohibitive and not supplied at volumes that support many research and product development efforts. RoosterBio will manufacture large quantities of standardized primary cells at high quality and low cost, which will quicken the pace of scientific discovery and translation to the clinic. “Our goal is to accelerate the development of products that incorporate living cells by providing abundant, affordable and high quality materials to researchers that are developing and commercializing these regenerative technologies” says Dr. Rowley
NHMU Lecture featuring – J. Craig Venter, Ph.D. Founder, Chairman, and CEO – J. Craig Venter Institute; Co-Founder and CEO, Synthetic Genomics Inc.
J. Craig Venter, Ph.D., is Founder, Chairman, and CEO of the J. Craig Venter Institute (JVCI), a not-for-profit, research organization dedicated to human, microbial, plant, synthetic and environmental research. He is also Co-Founder and CEO of Synthetic Genomics Inc. (SGI), a privately-held company dedicated to commercializing genomic-driven solutions to address global needs.
In 1998, Dr. Venter founded Celera Genomics to sequence the human genome using new tools and techniques he and his team developed. This research culminated with the February 2001 publication of the human genome in the journal, Science. Dr. Venter and his team at JVCI continue to blaze new trails in genomics. They have sequenced and a created a bacterial cell constructed with synthetic DNA, putting humankind at the threshold of a new phase of biological research. Whereas, we could previously read the genetic code (sequencing genomes), we can now write the genetic code for designing new species.
The science of synthetic genomics will have a profound impact on society, including new methods for chemical and energy production, human health and medical advances, clean water, and new food and nutritional products. One of the most prolific scientists of the 21st century for his numerous pioneering advances in genomics, he guides us through this emerging field, detailing its origins, current challenges, and the potential positive advances.
His work on synthetic biology truly embodies the theme of “pushing the boundaries of life.” Essentially, Venter is seeking to “write the software of life” to create microbes designed by humans rather than only through evolution. The potential benefits and risks of this new technology are enormous. It also requires us to examine, both scientifically and philosophically, the question of “What is life?”
J Craig Venter wants to digitize DNA and transmit the signal to teleport organisms
A technology trend analyst offers an overview of synthetic biology, its potential applications, obstacles to its development, and prospects for public approval.
In addition to boosting the economy, synthetic biology projects currently in development could have profound implications for the future of manufacturing, sustainability, and medicine.
Before society can fully reap the benefits of synthetic biology, however, the field requires development and faces a series of hurdles in the process. Do researchers have the scientific know-how and technical capabilities to develop the field?
Biology + Engineering = Synthetic Biology
Bioengineers aim to build synthetic biological systems using compatible standardized parts that behave predictably. Bioengineers synthesize DNA parts—oligonucleotides composed of 50–100 base pairs—which make specialized components that ultimately make a biological system. As biology becomes a true engineering discipline, bioengineers will create genomes using mass-produced modular units similar to the microelectronics and computer industries.
Currently, bioengineering projects cost millions of dollars and take years to develop products. For synthetic biology to become a Schumpeterian revolution, smaller companies will need to be able to afford to use bioengineering concepts for industrial applications. This will require standardized and automated processes.
A major challenge to developing synthetic biology is the complexity of biological systems. When bioengineers assemble synthetic parts, they must prevent cross talk between signals in other biological pathways. Until researchers better understand these undesired interactions that nature has already worked out, applications such as gene therapy will have unwanted side effects. Scientists do not fully understand the effects of environmental and developmental interaction on gene expression. Currently, bioengineers must repeatedly use trial and error to create predictable systems.
Similar to physics, synthetic biology requires the ability to model systems and quantify relationships between variables in biological systems at the molecular level.
The second major challenge to ensuring the success of synthetic biology is the development of enabling technologies. With genomes having billions of nucleotides, this requires fast, powerful, and cost-efficient computers. Moore’s law, named for Intel co-founder Gordon Moore, posits that computing power progresses at a predictable rate and that the number of components in integrated circuits doubles each year until its limits are reached. Since Moore’s prediction, computer power has increased at an exponential rate while pricing has declined.
DNA sequencers and synthesizers are necessary to identify genes and make synthetic DNA sequences. Bioengineer Robert Carlson calculated that the capabilities of DNA sequencers and synthesizers have followed a pattern similar to computing. This pattern, referred to as the Carlson Curve, projects that scientists are approaching the ability to sequence a human genome for $1,000, perhaps in 2020. Carlson calculated that the costs of reading and writing new genes and genomes are falling by a factor of two every 18–24 months. (see recent Carlson comment on requirement to read and write for a variety of limiting conditions).
Startup to Strengthen Synthetic Biology and Regenerative Medicine Industries with Cutting Edge Cell Products
Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging
Futurists have touted the twenty-first century as the century of biology based primarily on the promise of genomics. Medical researchers aim to use variations within genes as biomarkers for diseases, personalized treatments, and drug responses. Currently, we are experiencing a genomics bubble, but with advances in understanding biological complexity and the development of enabling technologies, synthetic biology is reviving optimism in many fields, particularly medicine.
Michael Brooks holds a PhD in quantum physics. He writes a weekly science column for the New Statesman,and his most recent book is The Secret Anarchy of Science.
The basic idea is that we take an organism – a bacterium, say – and re-engineer its genome so that it does something different. You might, for instance, make it ingest carbon dioxide from the atmosphere, process it and excrete crude oil.
That project is still under construction, but others, such as using synthesised DNA for data storage, have already been achieved. As evolution has proved, DNA is an extraordinarily stable medium that can preserve information for millions of years. In 2012, the Harvard geneticist George Church proved its potential by taking a book he had written, encoding it in a synthesised strand of DNA, and then making DNA sequencing machines read it back to him.
When we first started achieving such things it was costly and time-consuming and demanded extraordinary resources, such as those available to the millionaire biologist Craig Venter. Venter’s team spent most of the past two decades and tens of millions of dollars creating the first artificial organism, nicknamed “Synthia”. Using computer programs and robots that process the necessary chemicals, the team rebuilt the genome of the bacterium Mycoplasma mycoides from scratch. They also inserted a few watermarks and puzzles into the DNA sequence, partly as an identifying measure for safety’s sake, but mostly as a publicity stunt.
What they didn’t do was redesign the genome to do anything interesting. When the synthetic genome was inserted into an eviscerated bacterial cell, the new organism behaved exactly the same as its natural counterpart. Nevertheless, that Synthia, as Venter put it at the press conference to announce the research in 2010, was “the first self-replicating species we’ve had on the planet whose parent is a computer” made it a standout achievement.
Today, however, we have entered another era in synthetic biology and Venter faces stiff competition. The Steve Jobs to Venter’s Bill Gates is Jef Boeke, who researches yeast genetics at New York University.
Boeke wanted to redesign the yeast genome so that he could strip out various parts to see what they did. Because it took a private company a year to complete just a small part of the task, at a cost of $50,000, he realised he should go open-source. By teaching an undergraduate course on how to build a genome and teaming up with institutions all over the world, he has assembled a skilled workforce that, tinkering together, has made a synthetic chromosome for baker’s yeast.
Stepping into DIYbio and Synthetic Biology at ScienceHack
We got a crash course on genetics and protein pathways, and then set out to design and build our own pathways using both the “Genomikon: Violacein Factory” kit and Synbiota platform. With Synbiota’s software, we dragged and dropped the enzymes to create the sequence that we were then going to build out. After a process of sketching ideas, mocking up pathways, and writing hypotheses, we were ready to start building!
The night stretched long, and at midnight we were forced to vacate the school. Not quite finished, we loaded our delicate bacteria, incubator, and boxes of gloves onto the bus and headed back to complete our bacterial transformation in one of our hotel rooms. Jammed in between the beds and the mini-fridge, we heat-shocked our bacteria in the hotel ice bucket. It was a surreal moment.
While waiting for our bacteria, we held an “unconference” where we explored bioethics, security and risk related to synthetic biology, 3D printing on Mars, patterns in juggling (with live demonstration!), and even did a Google Hangout with Rob Carlson. Every few hours, we would excitedly check in on our bacteria, looking for bacterial colonies and the purple hue characteristic of violacein.
Most impressive was the wildly successful and seamless integration of a diverse set of people: in a matter of hours, we were transformed from individual experts and practitioners in assorted fields into cohesive and passionate teams of DIY biologists and science hackers. The ability of everyone to connect and learn was a powerful experience, and over the course of just one weekend we were able to challenge each other and grow.
Returning to work on Monday, we were hungry for more. We wanted to find a way to bring the excitement and energy from the weekend into the studio and into the projects we’re working on. It struck us that there are strong parallels between design and DIYbio, and we knew there was an opportunity to bring some of the scientific approaches and curiosity into our studio.
There are two bloggers who have brought a clear vision to the growing importance of Pleuripotential stem cell research, applications, and noted risks. They are M Buratov and David O’Connell.
I repost some work that needs more attention. The technology has improved, and there are a number of successful applications. The treatment of the cells, and the ability to put them on a stable and nontoxic resorbable matrix is a bioengineering advance.
Skeletal muscle – that type of voluntary muscle that allows movement – has proven difficult to grow in the laboratory. While particular cells can be differentiated into skeletal muscle cells, forming a coherent, structurally sound skeletal muscle is a tough nut to crack from a research perspective. Another problem dogging muscle research is the difficulty growing new muscle in patients with muscle diseases such as muscular dystrophy or other types of disorders that weaken and degrade skeletal muscle. Now research groups at the Boston Children’s Hospital Stem Cell Program have reported that they can boost the muscle mass and even reverse the disease of mice that suffer from a type of murine muscular dystrophy. To do this, this group use a combination of three different compounds that were identified in a rapid culture system.
This ingenious rapid culture system uses
the cells of zebrafish (Danio rerio) embryos to screen for these muscle-inducing compounds.
These single cells are placed into the well of a 96-well plate, and then treated with various compounds to determine if those chemical induce the muscle formation. To facilitate this process,
the zebrafish embryo cells express a very special marker that consists of the myosin light polypeptide 2 gene fused to a red-colored protein called “cherry.”
When cells become muscle, they express the myosin light polypeptide 2 gene at high levels. Therefore, any embryo cell that differentiates into muscle should glow a red color.
Zebrafish embryos myosin light polypeptide 2 gene fused to a red-colored protein called “cherry.”
(A) myf5-GFP;mylz2-mCherry double-transgenic expression recapitulates expression of the endogenous genes. myf5-GFP is first detected at the 11-somite stage. mylz2-mCherry expression is not observed until 32 hpf. Scale bars represent 200 mm.
(B) myf5-GFP;mylz2-mCherry embryos were dissociated at the oblong stage and cultured in zESC medium. Images were taken 48 hr after plating. Scale bars represent 250 mm.
Once a cocktail of muscle-inducing chemicals were identified in this assay, those same chemicals were used to treat induced pluripotent stem cells made from cells taken from patients with muscular dystrophy. Those iPSCs were treated with the combination of chemicals identified in the zebrafish embryo screen as muscle inducing agents.
The results were outstanding. Leonard Zon from the Division of Hematology/Oncology, Children’s Hospital Boston and Dana-Farber Cancer Institute and his colleagues showed that
a combination of basic Fibroblast Growth Factor, an adenylyl cyclase activator called forskolin, and the GSK3β inhibitor BIO
induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs).
Furthermore, these muscle cells produced
engraftable myogenic progenitors that contributed to muscle repair
when implanted into mice with a rodent form of muscular dystrophy.
Representative hematoxylin and eosin staining (H&E) images and immunostaining on TA sections of preinjured NSG mice injected with 1 3 105 iPSCs at day 14 of differentiation. Muscles injected with BJ, 00409, or 05400 iPSC-derived cells stain positively for human d-Sarcoglycan protein (red). Fibers were counterstained with Laminin (green). No staining is observed in PBS-injected mice or when 00409 fibroblast cells were transplanted. Because the area of human cell engraftment could not be specifically distinguished on H&E stained sections, which must be processed differently from sections for immunostaining, the H&E images shown do not represent the same muscle region as that shown in immunofluorescence images. Scale bars represent 100 mm, n = 3 per sample.
Zon hopes that clinical trials can being soon in order to translate these remarkable results into patients with muscle loss within the next several years. Zon and his co-workers are also screening compounds to address other types of disorders beyond muscular dystrophy.
This paper represents the application of shear and utter genius. However, there is one caveat. The mice into which the muscles were injected were immunodeficient mice who immune systems are unable to reject transplanted tissues. In human patients with muscular dystrophy,
an immune response against dystrophin, the defective protein, has been an enduring problem (for a review of this, see T. Okada and S. Takeda, Pharmaceuticals (Basel). 2013 Jun 27;6(7):813-836).
While there have been some technological developments that might circumvent this problem,
transplanting large quantities of muscle cells might be beyond the pale.
Muscular dystrophy results from disruption of an important junction between the muscle and substratum to which the muscle is secured. This connection is mediated by
the “dystrophin-glycoprotein complex.”
Structural disruptions of this complex (shown below) lead to
unanchored muscle that cannot contract properly, and
Dystrophin-glycoprotein complex. Molecular structure of the dystrophin-glycoprotein complex and related proteins superimposed on the sarcolemma and subsarcolemmal actin network (redrawn from Yoshida et al. [5], with modifications). cc, coiled-coil motif on dystrophin (Dys) and dystrobrevin (DB); SGC, sarcoglycan complex;SSPN, sarcospan; Syn, syntrophin; Cav3, caveolin-3; N and C, the N and C termini, respectively; G, G-domain of laminin; asterisk indicates the actin-binding site on the dystrophin rod domain; WW, WW domain.
This is a remarkable advance, but until the host immune response issue is satisfactorily addressed, it will remain a problem.
Whole Bone Marrow Transplantations into the Heart: Hope or Hype?
Bone marrow, that squishy material that resides inside your bones, especially your long bones, is a treasure-trove of stem cells. Bone marrow has blood-making stem cells called
“hematopoietic stem cells” or HSCs, and
a small subset of bone marrow stem cells can make blood vessels. These blood vessel-making stem cells are called
“endothelial progenitor cells,” or EPCs.
HSCs are the main stem cells in bone marrow that allows bone marrow transplants to reconstitute the blood cell formation system. People who have cancers of the blood system and have had their own bone marrow
completely destroyed by ionizing radiation or drugs like busulphan or cyclophosphamide
require bone marrow transplants to refurbish their own decimated bone marrow.
When a leukemia or lymphoma patient receives a bone marrow transplant, the stem cells in the bone marrow proliferate and reconstitute the patient’s blood-making and immune capacity (See R. Haas, et al. High-dose therapy and autologous peripheral blood stem cell transplantation in patients with multiple myeloma. Recent Results in Cancer Research 2011;183:207-38; and Ronjon Chakraverty and Stephen Mackinnon, Allogeneic Transplantation for Lymphoma. Journal of Clinical Oncology2011;29(14):1855-63). Bone marrow also has a supportive tissue called “stroma.”
Bone marrow stroma growing on plates coated with spider silk protein.
Stromal cells do not make blood, but it plays an essential supportive role in blood making. The main component of the stroma are the mesenchymal stem cells,: or MSCs. MSCs can readily differentiate into fat, bone, or muscle,but a wide variety of experiments have shown that MSCs can also become heart muscle, blood vessels, glial cells, neurons, and several other cell types.There are other types of stem cells as well that include
Given the ability of bone marrow to reconstruct another patient’s bone marrow, could it heal another tissue? This question was given a very strange answer when women who had bone marrow transplants from male donors were found to have heart cells that contained a Y chromosome. Since human females have cells with two X chromosomes,
the only source of these cells was the bone marrow transplant (see Arjun Deb, et al. Bone marrow-derived cardiomyocytes are present in adult human heart: A study of gender-mismatched bone marrow transplantation patients. Circulation 2003;107(9):1247-9). This finding suggested that bone marrow could be used to heal the hearts of patients who had suffered a heart attack.
Such notions were tested in mice. The experimental strategy was rather simple in principle; experimentally induce a heart attack in laboratory mice and then transplant human bone marrow stem cells into the hearts to see if these cells could help heal these hearts. The initial experiments in mice were astounding. Not only did the implanted bone marrow cells regenerate over half of the heart,
the implanted bone marrow cells expressed a bevy of heart-specific genes and
the hearts of the bone marrow recipient mice worked extremely well (Donald Orlic, et al. Transplanted adult bone marrow cells repair myocardial infarcts in mice. Annals of the New York Academy of Sciences2001;938:221-9; discussion 229-3).
Unfortunately, no one else could recapitulate Orlic’s remarkable studies, and when bone marrow cells were transplanted into mouse hearts in other labs, they helped heart function, but
they did not become anything like heart muscle cells (Leora Balsam, et al. Haematopoietic stem cells adopt mature haematopoietic fates in ischemic myocardium. Nature. 2004;428(6983):668-73).
In all cases the transplanted bone marrow cells helped improve the function of the hearts of mice that had recently experienced a heart attack, but there were hanging questions as to how they helped the heart.
Despite these uncertainties, several clinical trials examined the ability of a patient’s own bone marrow to heal their damaged heart. These trials took patients who had suffered a heart attack and
extracted their own bone marrow and
then transplanted into the heart of the heart attack patient.
A very noninvasive way to transplant the bone marrow that use catheter technologies that are used to perform angioplasty and apply stents (for an EXCELLENT video on this technology, see this link). The catheter
was used to introduce bone marrow stem cells into the heart by means of a catheter.
This precluded the need to crack the patient’s chest, and was quite safe, since it has already been used in angioplasty. Early Phase I studies just examined the safety of applying stem cells from bone marrow to the heart. While these early Phase I studies were small and nonrandomized, they universally found that procedure was safe. See the following references:
Birgit Assmus, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE -AMI). Circulation 2002;106:3009-17. 59 patients were treated with intracoronary bone marrow cells, the percent of the blood in the ventricle that was pumped per heartbeat (ejection fraction or EF; it is a major indicator of how well the heart is performing) increased; the tendency for the heart to enlarge decreased, the size of the heart scar decreased and the amount of blood flowing to the heart increased. One patient died during the course of the experiment, but no further cardiovascular events, including ventricular arrhythmias or syncope, occurred during one-year follow-up.
Bodo E. Strauer, et al. Repair of myocardium by autologous intracoronary mononuclear bone marrow transplantation in humans. Circulation 2002;106:1913-18. Results – Ten patients, were injected with intracoronary bone marrow cells 6-10 days after experiencing a heart attack. All in all, the amount of blood pumped per beat (stroke volume), increased, the myocardial scar shrunk, and blood supply to the rest of the heart increased.
Francisco Fernández=Avilés, et al. Experimental and clinical capability of human bone marrow cells after myocardial infarction. Circulation Research 2004;95:742-8. 20 recent heart attack patients who had suffered a heart attack ~13 days earlier received intracoronary bone marrow cells and, on the average, the EF increased, the volume that remains in the chambers after pumping (end-systolic volume or ESV) decreased (means the heart is beat more effectively), and the motion of the surfaces of the heart increased as well. There were no major adverse events.
Volker Schächinger, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: Final one-year results of the TOPCARE-AMI Trial. Journal of the American College of Cardiology 2004;44(8): 1690-1699. See the other TOPCARE-AMI summary above.
J. Bartunek, et al. Intracoronary injection of CD133-positive enriched bone marrow progenitor cells promotes cardiac recovery after recent myocardial infarction: feasibility and safety. Circulation. 2005;112(9 Suppl):I178-83. 19 recent heart attack patients received intracoronary bone marrow cells 10-13 days after suffering a heart attack and on the average, patients showed an increase in ejection fraction, increase in circulation throughout the heart and fewer dead cells in the heart. No major adverse effects.
These studies established the safety of the procedure, but they were small, and they were not tested against a placebo. Therefore, randomized studies were conducted to test the efficacy of bone marrow transplants in the heartto treat heart attack patients. Remember, drug treatments slow the heart down and delay further cardiac deterioration, but they do not address the problem of dead heart tissue.
Only regenerative treatments can potentially replace the dead heat tissue with new, living tissue.
Phase II studies and other studies that were combined Phase I/II studies examined just over 900 patients in almost 20 clinical trials and
the result overwhelmingly show that bone marrow transplants
significantly improve the function of the hearts of heart attack patients.
A few studies are negative, that is there are no statistically significant differences between the placebo and the experimental patients. However, the vast majority of the studies are positive, and those studies that are negative seem to have a viable explanation as to why they are so. These studies are listed below:
Shao-liang Chen, et al. Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. American Journal of Cardiology 2004;94(1): 92-95. In this study, 69 patients participated, but only 34 received the intracoronary bone marrow-derived mesenchymal stem cells approximately 18 days after experiencing a heart attack. Patients who had received the stem cells showed a significant increase in ejection fraction versus those patients that had received the placebo. There were no adverse reactions.
Junbo Ge, et al. Efficacy of emergent transcatheter transplantation of stem cells for treatment of acute myocardial infarction (TCT-Stami). Heart 2006;92(12):1764-7. 20 patients were treated, the moment they received angioplasty less than a day after they has experience a heart attack. 1o received the placebo and 10 received the bone marrow cells. Those who received the bone marrow cells showed enhanced ejection fraction, better heart circulation, and showed no signs of enlargement of the heart relative to the placebo group, which showed a decrease in EF, signs of heart enlargement and decreased heart circulation. There were no adverse reactions.
Wen Ruan, et al. Assessment of left ventricular segmental function after autologous bone marrow stem cells transplantation in patients with acute myocardial infarction by tissue tracking and strain imaging. Chinese Medical Journal 2005;118(14):1175-81. Less than one day after a heart attack, twenty patients were randomly treated with intracoronary injections of bone-marrow cells (N= 9) or diluted serum (n = 11). Echocardiograms at 1 week, 3 weeks and 3 and 6 months after treatment were used to assess the status of the patient’s hearts, and various means were used to assess left ventricular ejection fraction (LVEF), end-diastolic volume (EDV) and end-systolic volume (ESV). They found that bone marrow stem cells helped improve global and regional contractility and attenuate post-infarction left ventricular remodeling. There were clear increases in EF, and clear decreases in EDV and ESV. There were no adverse reactions.
Huang RC, et al. Long term follow-up on emergent intracoronary autologous bone marrow mononuclear cell transplantation for acute inferior-wall myocardial infarction. Long term follow-up on emergent intracoronary autologous bone marrow mononuclear cell transplantation for acute inferior-wall myocardial infarction. Zhonghua Yi Xue Za Zhi 2006; 86(16):1107-10. This article is only in Chinese, which I do not read. Therefore this is a summary of the abstract, which is in English. Forty patients who had just experience a heart attack were treated with angioplasty and intracoronary transplantation of autologous bone marrow cells (n = 20) or normal saline and heparin (n = 20) less than one day after the heart attack. After six months, the treated group had higher EFs and greater decrease in the size of the heart scar.
Kang Yao, et al. Administration of intracoronary bone marrow mononuclear cells on chronic myocardial infarction improves diastolic function. Heart 2008;94:1147-53. 47 patients who had just experienced a heart attack received either intracoronary infusion of bone marrow cells (24 of them), or a saline infusion (23 of them) 5-21 days after experiencing the heart attack. Bone marrow treatments did not lead to significant improvement of cardiac systolic function, infarct size or myocardial perfusion, but did lead to improvement in diastolic function.
Martin Penicka, et al. Intracoronary injection of autologous bone marrow-derived mononuclear cells in patients with large anterior acute myocardial infarction. Journal of the American College of Cardiology. 2007 49(24):2373-4. This study was a bit of a mess. It was prematurely terminated, and four patients died or had severely worsened heart failure during the study. The authors do not provide details on how they isolated and prepared their bone marrow stem cells, which turns out to be quite important. 27 patients were treated nine days after a heart attack with either intracoronary bone marrow cells (n = 17) or just angioplasty (n = 10). There were no significant differences between the two groups. Given the problems with this paper, the results do not inspire much confidence.
The BOOST study. Three papers – (1) Arnd Schaefer, et al. Impact of intracoronary bone marrow cell transfer on diastolic function in patients after acute myocardial infarction: results from the BOOST trial. European Heart Journal 2006;27(8):929-35. (2) Kai C. Wollert, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. The Lancet 2004;364(9429):141-8. (3) Gerd P. Meyer, et al. Intracoronary Bone Marrow Cell Transfer After Myocardial Infarction: Eighteen Months’ Follow-Up Data From the Randomized, Controlled BOOST (BOne marrOw transfer to enhance ST-elevation infarct regeneration) Trial. Circulation 2006;113:1287-94. This study examined 60 heart attack patients and treated 30 of them with intracoronary bone marrow stem cells and other 30 with just angioplasty 4-8 days after the heart attack. At six-months there was a significant increase in ejection fraction in the bone marrow-recipient group, but those differences between the bone marrow group and the control disappeared after six months and during the 18 month follow-up, no differences could be detected. At the five-year follow-up, no differences could be detected between the two groups. Therefore these authors suggested that early recovery is accelerated by bone marrow stem cells, but that these effects are not long-term. See Arnd Scharfer, et al. Long-term effects of intracoronary bone marrow cell transfer on diastolic function in patients after acute myocardial infarction: 5-year results from the randomized-controlled BOOST trial—an echocardiographic study. European Journal of Echocardiology 2010;11(2):165-71. No adverse effects were seen in this study.
Stefan Janssens, et al. Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled trial. The Lancet 2006;267(9505):113-121. This study treated 67 patients less than one day after experiencing a heart attack, and broke the patients into two groups, half of whom were treated with intracoronary bone marrow stem cells (n = 33), and the other half were treated just with angioplasty (n = 34). While there was no significant increase in ejection fraction in the treated group in comparison to the control group after four months, the bone marrow-treated patients showed increased shrinkage of the heart scar and increased regional heart contraction abilities. A follow-up study published in 2009 confirmed these improvements. See Lieven Herbots, et al. Improved regional function after autologous bone marrow-derived stem cell transfer in patients with acute myocardial infarction: a randomized, double-blind strain rate imaging study. European Heart Journal 2009;30(6):662-70.
REPAIR-AMI – Several papers: (1) Sandra Erbs, et al. Restoration of Microvascular Function in the Infarct-Related Artery by Intracoronary Transplantation of Bone Marrow Progenitor Cells in Patients With Acute Myocardial Infarction: The Doppler Substudy of the Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) Trial. Circulation 2007;116:366-74. (2) Throsten Dill, et al. Intracoronary administration of bone marrow-derived progenitor cells improves left ventricular function in patients at risk for adverse remodeling after acute ST-segment elevation myocardial infarction: Results of the Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction study (REPAIR-AMI) cardiac Magnetic Resonance Imaging substudy. American Heart Journal 2009;157(3):541-7. (3) Volker Schächinger, et al. Intracoronary infusion of bone marrow-derived mononuclear cells abrogates adverse left ventricular remodelling post-acute myocardial infarction: insights from the reinfusion of enriched progenitor cells and infarct remodelling in acute myocardial infarction (REPAIR-AMI) trial. European Journal of Heart Failure 2009;11(10):973-9. (4) Birgit Assmus, et al. Clinical outcome 2 years after intracoronary administration of bone marrow-derived progenitor cells in acute myocardial infarction. Circulation Heart Failure 2010;3(1):89-96. This large study used 204 patients and treated 102 of them with bone marrow cells and the others with just angioplasty and the infusion of a placebo 3-7 days after suffering a heart attack. This study definitively showed a significant increase in the ejection fraction in comparison to the placebo group. Likewise, the combined end point death and recurrence of heart attacks and rehospitalization for heart failure was significantly reduced in the bone marrow-treated group. A two-year follow-up also showed that these improvements still presisted after two years. No major adverse side effects were observed.
Jaroslav Meluzin, et al. Autologous transplantation of mononuclear bone marrow cells in patients with acute myocardial infarction: The effect of the dose of transplanted cells on myocardial function. American Heart Journal 2006;152(5):975(e9-15). Also see Roman Panovsky, et al. Cell Therapy in Patients with Left Ventricular Dysfunction Due to Myocardial Infarction. Echocardiography 2008;25(8): 888–897. This study is one of the few to address the dosage of bone marrow cells. These workers randomized 66 patients, and placed them into three groups: 22 of them received the placebo, 22 received a low dose of bone marrow cells (10,000,000 cells), and 22 received a high dose of bone marrow cells (100,000,000 cells). These treatments were given seven days after experiencing a heart attack. At 3 months after the treatment, the ejection fraction was significantly higher in the patients who had received the high dose of bone marrow cells and not the low dose patients. Again, these treatments were by means of intracoronary delivery, and no major adverse effects were observed.
The ASTAMI Study – Another fairly large study. (1) Ketil Lunde, et al. Exercise capacity and quality of life after intracoronary injection of autologous mononuclear bone marrow cells in acute myocardial infarction: Results from the Autologous Stem cell Transplantation in Acute Myocardial Infarction (ASTAMI) randomized controlled trial. American Heart Journal 2007;154(4):710.e1-8. (2) Jan Otto Beitnes, et al. Left ventricular systolic and diastolic function improve after acute myocardial infarction treated with acute percutaneous coronary intervention, but are not influenced by intracoronary injection of autologous mononuclear bone marrow cells: a 3 year serial echocardiographic sub-study of the randomized-controlled ASTAMI study. European Journal of Echocardiology 2011;12(2):98-106. (3) Ketil Lunde, et al. Autologous stem cell transplantation in acute myocardial infarction: The ASTAMI randomized controlled trial. Intracoronary transplantation of autologous mononuclear bone marrow cells, study design and safety aspects. Scandinavian Cardiovascular Journal 2005;39(3):150-8. (4) Jan Otto Beitnes, et al. Long-term results after intracoronary injection of autologous mononuclear bone marrow cells in acute myocardial infarction: the ASTAMI randomised, controlled study. Heart 2009;95:1983-9. (5) Einar Hopp, et al. Regional myocardial function after intracoronary bone marrow cell injection in reperfused anterior wall infarction – a cardiovascular magnetic resonance tagging study. Journal of Cardiovascular Magnetic Resonance 2011, 13:22This study examined 100 recent heart attack patients and treated 50 of them with intracoronary bone marrow cells and the remaining patients with just angioplasty, 5-7 days after a heart attack. Measurements of heart function at 3, 6, and 12 months, and 3 years after the procedure found no significant differences between the two groups, with the exception of a slightly increased exercise tolerance in the group that received the bone marrow cells. Both the control and the treated group showed the same low numbers of adverse reactions; none of which could be attributed directly to the treatment protocol. This study was negative and it is often brought up by proponents of embryonic stem cells as an example of the failure of bone marrow cells to heal a heart. However, the protocol that was used by the ASTAMI study to isolate and store the bone marrow cells was different from that used by the successful REPAIR-AMI group. Florian Seeger at the University of Frankfurt evaluated the two protocols and found that the ASTAMI bone marrow isolation protocol produced cells that showed poor viability and poor response to chemical factors that are made in the heart after a heart attack that summons stem cells to it and holds them there (See FH Seeger, et al. Cell isolation procedures matter: a comparison of different isolation protocols of bone marrow mononuclear cells used for cell therapy in patients with acute myocardial infarction. 2007;28(6):766-72). The ASTAMI research group has refused to accept that their bone marrow isolation protocol affected the efficacy of their bone marrow stem cells, but Seeger’s work was corroborated by the work of van Beem (see R.T. van Beem, et al. Recovery and functional activity of mononuclear bone marrow and peripheral blood cells after different cell isolation protocols used in clinical trials for cell therapy after acute myocardial infarction. Eurointervention 2008;4(1):133-8). Therefore, the ASTAMI clinical trial used poor quality bone marrow preparations that were destined to fail, and this clinical trial is no indication of the efficacy or lack of efficacy of bone marrow stem cells to treat failing hearts.
José Suárez de Lezo, et al. Regenerative Therapy in Patients With a Revascularized Acute Anterior Myocardial Infarction and Depressed Ventricular Function. Revista Espaňola de Cardiologia 2007;60(4):357-65. A small study treated 30 patients with either angioplasty (n = 10), a drug called G-CSF, which tends to bring bone marrow stem cells from the bone marrow and into the circulating blood (n = 10), or intracoronary bone marrow cell treatments (n = 10). The bone marrow=treat group showed a 20% increase in ejection fraction whereas the control and G-CSF-treated group only saw 6% and 4% increases, respectively. Patients received their treatments 5-9 days after their heart attacks.
The FINCELL Trial – Heikki V. Huikuri, et al. Effects of intracoronary injection of mononuclear bone marrow cells on left ventricular function, arrhythmia risk profile, and restenosis after thrombolytic therapy of acute myocardial infarction. European Heart Journal 2008;29(22):2723-2732. 2-6 days after experiencing a heart attack, 80 patients were randomly assigned to receive intracoronary either bone marrow cells (n = 40) or placebo (n = 40) during angioplasty. After 6 months, the bone marrow-treated group showed clear increases in ejection fraction in comparison to the control group. Also, several safety issues, such as “restenosis” or the narrowing of coronary arteries that surround the heart as a result of bone marrow treatments were addressed by this study, since some researchers suspected that bone marrow treatments increased the risk of restenosis. In this study, no increased incidence of restenosis was observed in the bone marrow-treated group.
REGENT Study – Michał Tendera, et al. Intracoronary infusion of bone marrow-derived selected CD34+CXCR4+ cells and non-selected mononuclear cells in patients with acute STEMI and reduced left ventricular ejection fraction: results of randomized, multicentre Myocardial Regeneration by Intracoronary Infusion of Selected Population of Stem Cells in Acute Myocardial Infarction (REGENT) Trial. European Heart Journal 2009;30(11):1313-21. This study examined 200 patients who had experienced a heart attack, and seven days after the heart attack, they treated these patients with either unselected bone marrow cells (n = 80), selected bone marrow cells (n = 80), or a placebo (n = 40). This large study did not find statistically significant differences between the three groups, but the control group did not show an increase in the ejection fraction, but the unselected and selected bone marrow-treated patients did.
The figure shown below is from the Tendera et al., paper that shows the compiled changes in ejection fraction between the three groups:
As you can see, the control group patients experienced a decrease in their ejection fractions, but the two bone marrow-treated groups experienced an increase, even if it was slight. The figure below shows the data for the sickest patients.
As can be seen, for those patients with the sickest hearts there was a significant difference in the increase in the injection fraction and other heart-associated factors. For this reason, this study does not seem definitive. There were three deaths (one in each group), no strokes, four heart attacks (two in the controls and one in each experimental group), and a low rate of re-narrowing of the heart blood vessels. Since this is from 200 total patients, this is a very low rate of adverse events.
15. Jay H. Tendera, et al. Results of a phase 1, randomized, double-blind, placebo-controlled trial of bone marrow mononuclear stem cell administration in patients following ST-elevation myocardial infarction. American Heart Journal 2010;160:428-34. In this study forty patients were treated with either intracoronary bone marrow cells or a placebo. The two groups showed no significant differences in ejection fraction after six months, but the bone marrow-treated group showed no enlargement of the heart in response to the heart attack, whereas the control group did. No adverse heart events occurred.
This summarizes the clinical trials that used bone marrow to treat patients who had experienced recent heart attacks (acute myocardial infarctions). The preponderance of the data clearly shows that this procedure is safe, and effective to treat heart attacks. Secondly, several analyses that take the data from these trials and group them together into one gigantic study (meta-analysis) have been published, and these studies also show that bone marrow treatments for recent heart attacks are safe and effective (for example, see Meng Jiang, et al. Randomized controlled trials on the therapeutic effects of adult progenitor cells for myocardial infarction: meta-analysis. Expert Opinion on Biological Therapy 2010;10(5):667-80).
A new take on efficient delivery in regenerative medicine
StemCells, Inc. Launches Alzheimer’s Disease Program Supported by California Institute for Regenerative Medicine (pharmaceuticalintelligence.com)
Introduction to Regenerative Therapy: How It Works (pharmaceuticalintelligence.com)
How nanotechnology can advance regenerative medicine (transbiotex.wordpress.com)
The Global Revolution in Biotechnology and the Impact of Regenerative Medicine (transbiotex.wordpress.com)
New Company Applies Regenerative Medicine to Corneal Transplants (transbiotex.wordpress.com)
Institute for Regenerative Medicine to Lead National Effort to Aid Wounded Warriors (transbiotex.wordpress.com)
The Alliance for Regenerative Medicine Gains Bipartisan Support for Gao Assessment of Federal Regenerative Medicine Activities (transbiotex.wordpress.com)
Joint development of the world’s first regenerative medicine/cell therapy business based on iPS cell technology
Dr. Jon Rowley and Dr. Uplaksh Kumar, Co-Founders of RoosterBio, Inc., a newly formed biotech startup located in Frederick, are paving the way for even more innovation in the rapidly growing fields of Synthetic Biology and Regenerative Medicine
Dr. Jon Rowley and Dr. Uplaksh Kumar, Co-Founders of RoosterBio, Inc., a newly formed biotech startup located in Frederick, are paving the way for even more innovation in the rapidly growing fields of Synthetic Biology and Regenerative Medicine. Synthetic Biology combines engineering principles with basic science to build biological products, including regenerative medicines and cellular therapies. Regenerative medicine is a broad definition for innovative medical therapies that will enable the body to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. Regenerative therapies that are in clinical trials today may enable repair of damaged heart muscle following heart attack, replacement of skin for burn victims, restoration of movement after spinal cord injury, regeneration of pancreatic tissue for insulin production in diabetics and provide new treatments for Parkinson’s and Alzheimer’s diseases, to name just a few applications.
While the potential of the field is promising, the pace of development has been slow. One main reason for this is that the living cells required for these therapies are cost-prohibitive and not supplied at volumes that support many research and product development efforts. RoosterBio will manufacture large quantities of standardized primary cells at high quality and low cost, which will quicken the pace of scientific discovery and translation to the clinic. “Our goal is to accelerate the development of products that incorporate living cells by providing abundant, affordable and high quality materials to researchers that are developing and commercializing these regenerative technologies” says Dr. Rowley.
RoosterBio’s current focus is to supply high volume research-grade cells manufactured with processes consistent with current Good Manufacturing Practices (cGMP). These cells will be used for tissue engineering research and cell-based product development. This will position RoosterBio to quickly move on to producing clinical-grade cells to be used in translational R&D and clinical studies.
“We have spent almost 20 years as cell and tissue technologists and have lived with the pain of needing to generate large amounts of cells for experiments this whole time. RoosterBio was founded to address this problem for cell and tissue engineers, saving them time and money, and accelerating their path to the clinic,” says Dr. Rowley. RoosterBio will supply cells, starting with adult human bone marrow-derived stem cells, at volumes that will allow for a more rapid pace of experimentation in the lab.
“We will also offer paired media that has been engineered to quickly and efficiently expand the supplied cells to hundreds of millions or billions of cells within 1-2 weeks, something that would take 4-8 weeks using cell and media systems currently on the market,” adds Dr. Kumar. “We aim to usher in a new era of productivity to the field, and we believe that our products will at least triple the efficiency of the average laboratory”.
RoosterBio, Inc. is located in the Frederick Innovative Technology Center on Metropolitan Court in Frederick. Dr. Rowley entered into the incubation program in October of this year, and already gained four full time employees, and has several academic and industrial collaborators lined up. This team has made remarkable progress and are already poised for their official product launch for their human bone marrow-derived Mesenchymal Stem Cells (hBM-MSC), anticipated in March 2014.
RoosterBio’s product formats have been extraordinarily well received by the market, and RoosterBio has already secured customers who are anxiously awaiting their product launch. “I am excited to see that someone is taking on the challenge of providing a sufficient number of MSCs to immediately start experiments upon their receipt. This saves us several weeks of time upfront waiting for cells to expand to volumes that allow us to begin experiments,” says Todd McDevitt, Director of the Stem Cell Engineering Center at the Georgia Institute of Technology. “For tissue engineering folks like myself, this means we can focus our time on high priority research questions and not spend the majority of our time performing routine cell culture.”
The Tissue Engineering and Regenerative Medicine industry is one of the fastest growing in the life science sector with the total expenditure in 2011 at $17.1 billion. This number is expected to increase in 2020 to $40.5 billion. The sales of stem cell products accounted for $1.38 billion in 2010 and is expected to reach $3.9 billion by the year 2014 and $8 billion in annual revenues by 2020.
About RoosterBio
RoosterBio is focused on building a robust and sustainable Regenerative Medicine industry. Our products are affordable and standardized primary cells and media, manufactured and delivered with highest quality and in formats that simplify product development efforts. RoosterBio products will accelerate the translation of cell therapy and tissue engineering technologies into the clinic.
Investigational Bioengineered Blood Vessel: Humacyte Presents Interim First-in-Human Data at the American Heart Association (AHA) Scientific Sessions 2013
Humacyte, Inc., presented the Results of Foundational U.S. Preclinical Studies of its Investigational Bioengineered blood vessel at the American Society of Nephrology’s ‘Kidney Week 2013’ Annual Meeting in Atlanta, GA.