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

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

Writer and Curator: Larry H Bernstein, MD, FCAP
and
Curator and Content Editor: Aviva Lev-Ari, PhD, RN

This article is Part V in a series of TWELVE articles, listed at the end of this article,  on the

1. cytoskeleton,
2. calcium calmodulin kinase signaling,
3. muscle and nerve transduction, and
4. calcium,
5. Na+-K+-ATPase,
6. neurohumoral activity and vesicles vital and essential for all functions related to

• cell movement,
• migration, and
• contraction.

Calmodulin and Protein Kinase C Increase Ca–stimulated Secretion by Modulating
Membrane-attached Exocytic Machinery

YA Chen, V Duvvuri, H Schulmani, and RH.Scheller‡

From the ‡Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology,
and the Department of Neurobiology, Stanford University School of Medicine, Stanford, CA

J Biol Chem 1999; 274(37): 25469-26476.

The molecular mechanisms underlying the Ca2+ regulation of hormone and neurotransmitter release
are largely unknown.

Using a reconstituted [3H]norepinephrine release assay in permeabilized PC12 cells, we found

• essential proteins that support the triggering stage of Ca2+-stimulated exocytosis
• are enriched in an EGTA extract of brain membranes.

Fractionation of this extract allowed purification of two factors that stimulate secretion

• in the absence of any other cytosolic proteins.

These are calmodulin and protein kinase Ca (PKCa). Their effects on secretion were

• confirmed using commercial and recombinant proteins.

Calmodulin enhances secretion

• in the absence of ATP, whereas
• PKC requires ATP to increase secretion, suggesting that
• phosphorylation is involved in PKC-mediated stimulation
• but not calmodulin mediated stimulation.
• Both proteins modulate
  • The half-maximal increase was elicited by
    3 nM PKC and 75 nM calmodulin.

These results suggest that calmodulin and PKC increase Ca2+-activated exocytosis by

• directly modulating the membrane- or cytoskeleton-attached exocytic machinery
  downstream of Ca2+ elevation.

The abbreviations used are:

NE, norepinephrine; PKC, protein kinase C; CaM, calmodulin; SNAP-25, synaptosome-associated protein of 25 kDa; CAPS, calcium-dependent activator protein for secretion; SNARE, SNAP (soluble N-ethylmaleimide-sensitive factor attachment proteins) receptor; CaMK, Ca2+/calmodulin-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; AMP-PNP, adenosine 59-(b,g- imido) triphosphate;  HA, hydroxyapatite

*This work was supported in part by Conte Center Grant MH48108. The costs of publication of
this article were defrayed in part by the payment of page charges. This article has been marked
“advertisement” in accordance with 18 U.S.C. Section 1734.

The molecular mechanisms of presynaptic vesicle release have been extensively examined
by a combination of

• biochemical,
• genetic, and
• electrophysiological techniques.

A series of protein-protein interaction cascades have been proposed to lead to vesicle
docking and fusion (1–3). The SNARE protein family, including

• syntaxin, SNAP-25, and vesicle-associated membrane protein
  (VAMP, also called synaptobrevin),
• plays an essential role in promoting membrane fusion, and
• is thought to comprise the basic fusion machinery (4, 5).

In Ca2+-stimulated exocytosis, many additional proteins are important in the Ca2+ regulation
of the basic membrane trafficking apparatus. Calcium

• not only triggers rapid fusion of release-competent vesicles, but is also involved in
• earlier processes which replenish the pool of readily releasable vesicles (6).

Furthermore, it appears to be critical in initiating several forms of synaptic plasticity including

• post-tetanic potentiation (7).

The molecular mechanisms by which Ca2+ regulates these processes is not well understood.

PC12 cells have often been utilized to study Ca2+-activated exocytosis, as

• they offer a homogeneous cell population that possesses the same basic exocytic machinery as neurons (8).

In this study, we used an established cracked cell assay, in which

• [3H]norepinephrine (NE)1 labeled PC12 cells are
• permeabilized by mechanical “cracking” and
• then reconstituted for secretion of NE in the presence of test proteins (9).

Transmitter-filled vesicles and intracellular cytoskeletal structures

• remain intact in these cells,
• while cytosolic proteins leak out (10).

These cracked cells readily release NE upon addition of

• ATP,
• brain cytosol, and
• 1 mM free Ca2+
  • at an elevated temperature.

We term this a “composite assay,” as

• all essential components are added into one reaction mixture.

Alternatively, cracked cells can be

• first primed with cytosol and ATP, washed, then
• reconstituted for NE release with cytosol and Ca2+ (11).

This sequential priming-triggering protocol is useful

• for determining whether a protein acts early or late in the exocytic pathway, and
• whether its effect is dependent on Ca2+ or ATP.

This semi-intact cell system serves as

• a bridge between an in vitro system comprised of purified components, and
• electro-physiological systems that monitor release in vivo.
• It provides information on protein functions in a cell with an intact membrane infrastructure while being easily manipulatable.

Ca2+ regulation by membrane depolarization is no longer a concern, as
intra-cellular Ca2+ concentration can be controlled by a buffered solution.

• Indirect readout of neurotransmitter release using a postsynaptic cell is replaced by
• direct readout of [3H]NE released into the buffer.

Complications associated with interpreting overlapping

• exo- and endocytotic signals are also eliminated as only one round of exocytosis is measured.

Finally, concentration estimates are likely to be accurate, since

• added compounds do not need to diffuse long distances along axons and dendrites to their sites of action.

Using this assay, several proteins required for NE release have been purified from rat brain cytosol, including

• phosphatidyl-inositol transfer protein (12),
• phosphatidylinositol-4-phosphate 5-kinase (13), and
• calcium-dependent activator protein for secretion (CAPS) (9).

The validity of the cracked cell system is confirmed by the finding that

• phosphatidylinositol transfer protein and CAPS are mammalian homologues of
  • yeast SEC14p (12) and
  • nematode UNC31p, respectively (14),
• both proteins involved in membrane trafficking (15, 16).

Calmodulin is the most ubiquitous calcium mediator in eukaryotic cells, yet its involvement in membrane trafficking has not been well established. Some early studies showed

• that calmodulin inhibitors (17–19), anti-calmodulin antibodies (20,21),

or

• calmodulin binding inhibitory peptides (22) inhibited Ca2+-activated exocytosis.

However, in other studies, calmodulin-binding peptides and an anti-calmodulin antibody led to the conclusion that

• calmodulin is only involved in endocytosis,
• not exocytosis (23).

More recently, it was reported that

1. Ca2+/ calmodulin signals the completion of docking and
2. triggers a late step of homotypic vacuole fusion in yeast,

• thus suggesting an essential role for Ca2+/calmodulin in constitutive intracellular membrane fusion (24).

If calmodulin indeed plays an important role in exocytosis,

• a likely target of calmodulin is
• Ca2+/calmodulin-dependent protein kinase II (CaMKII),
  • a multifunctional kinase that is found on synaptic vesicles (25) and
  • has been shown to potentiate neurotransmitter release (26, 27).

Another Ca2+ signaling molecule, PKC, has also been implicated in regulated exocytosis.
In various cell systems, it has been shown that

• the phorbol esters stimulate secretion (28, 29).

It is usually assumed that phorbol esters effect on exocytosis is

• through activation of PKC,
• but Munc13-1 was recently shown to be a presynaptic phorbol ester receptor that enhances neurotransmitter release (30, 31),

which complicates the interpretation of some earlier reports. The mode of action of PKC remains controversial. There is evidence

• that PKC increases the intracellular Ca2+ levels by modulating plasma membrane Ca2+ channels (32, 33),
• that it increases the size of the release competent vesicle pool (34, 35), or
• that it increases the Ca2+ sensitivity of the membrane trafficking apparatus (36).

no consensus on these issues has been reached.

PKC substrates that have been implicated in exocytosis include

1. SNAP-25 (37),
2. synaptotagmin (28),
3. CAPS (38), and
4. nsec1 (39).

It is believed that upon phosphorylation, these PKC substrates might

• interact differently with their binding partners, which, in turn,
• leads to the enhancement of exocytosis.

In addition, evidence is accumulating that PKC and calmodulin interfere with each others actions, as

• PKC phosphorylation sites are embedded in the calmodulin-binding domains of substrates such as
• neuromodulin and
• neurogranin (40).

It is therefore possible that PKC could modulate exocytosis via

• a calmodulin-dependent pathway by synchronously releasing calmodulin from storage proteins.

In this study, we fractionated an EGTA extract of brain membranes in order to identify active components that could reconstitute release in the cracked cell assay system. We identified calmodulin and PKC as two active factors. Thus, we demonstrate that

• calmodulin and PKC play a role in the Ca2+ regulation of exocytosis, and provide further insight into the mechanisms of their action.

DISCUSSION

 In this study, we first identified an EGTA extract of brain membranes as a protein source

•  capable of reconstituting Ca2+- activated exocytosis in cracked PC12 cells.

EGTA only extracts a small pool of Ca2+-dependent membrane-associating proteins,

• it served as an efficient initial purification step.

Further protein chromatography led to the identification of two active factors in the starting extract,

• calmodulin and PKC,
• which together accounted for about half of the starting activity.

Upon confirmation with commercially obtained proteins, this result unambiguously demonstrated

• that calmodulin and PKC mediate aspects of Ca2+-dependent processes in exocytosis.

The finding that brain membrane EGTA extract alone is able

• to replace cytosol in supporting Ca2+-triggered NE secretion

 in PC12 cells is somewhat surprising. We suggest that the likely explanation is 2-fold.

1. some cytosolic proteins essential for exocytosis have a membrane-bound pool
   within permeabilized cells, whose activity might be sufficient for a normal level of exocytosis.
2. although the 100,000 3 g membrane pellet was washed to remove as many cytosolic proteins as possible,

• some cytosolic proteins that associate with membranes in a
  • Ca2+-independent manner are probably present in the membrane EGTA extract.
• these proteins likely constitute only a small percentage of the proteins in the extract, as
  • the characteristics of the activity triggered by the membrane extract
  • are quite different to that of cytosol (Fig. 2).

 Using an unbiased biochemical purification method, we demonstrated that

•  calmodulin and PKC directly modulate the exocytotic machinery downstream of Ca2+ entry
• they signal through membrane-attached molecules to increase exocytosis.

 These targets include integral and peripheral membrane proteins, and cytosolic proteins that have a significant
membrane-bound pool.  The modest stimulation by calmodulin and PKC on secretion might suggest a regulatory

role. However, it is also possible that some intermediates in their signaling pathways are in limiting amounts in the
cell ghosts, so that their full effects were not observed. Half-maximal stimulation was obtained at

• about 3 nM for PKC and
• at about 75 nM for calmodulin.

This is consistent with an enzymatic role for PKC, and predicts a high-affinity interaction between

• calmodulin and its substrate protein.

 Ca2+ regulates exocytosis at many different levels. Prior studies indicated that Ca2+ signaling occurs in

• the priming steps as well
• as in triggering steps (49, 50).

Our priming triggering protocol 

1. does not allow Ca2+-dependent priming events to be assayed, as EGTA is present in the priming reaction.
2. a different approach revealed the existence of both high and low Ca2+-dependent processes (Fig. 2).
3. this analysis indicated that late triggering events require high [Ca2+], whereas
4. early priming events require low [Ca2+]. If, as proposed, there is

a pronounced intracellular spatial and temporal [Ca2+] gradient from

• the point of Ca2+ entry during depolarization (51),
• perhaps triggered events occur closer to the point of Ca2+ entry,
• while Ca2+-dependent priming events occur further away from the point of Ca2+ entry.

Distinct Ca2+ sensors at these stages might be appropriately tuned to different [Ca2+] to handle different tasks.

By analyzing the Ca2+ sensitivity of calmodulin-and PKC-stimulated release, we addressed the question of

• whether calmodulin and PKC plays an early or a late role in vesicle release.
•  they both require relatively high [Ca2+] (Fig. 8B),
• implying that calmodulin and PKC both mediate late triggering events, consistent with some earlier reports
  (34, 52, 53).

In addition, it is interesting to note that PKC does not alter the calcium sensitivity of release in cracked cells, in contrast

to observations from the chick ciliary ganglion (36). Therefore, in contrast to previous electrophysiological studies (28),
we are able to limit the possible modes of PKC action in our system to an increase in the readily releasable vesicle pool or
release sites, or an enhancement of the probability of release of individual vesicles upon Ca2+ influx.

The experiments assaying the calcium sensitivity of release (Figs. 2, 5, and 8) demonstrated

• a drop in release at very high [Ca2+].

This decline in release at high [Ca2+] has been previously reported (49, 51), and may represent

• the true Ca2+ sensitivity of the Ca2+-sensing mechanism inside cells.

However, in our system, it could also be due to the activation of a variety of Ca2+ -activated proteases, as experiments are usually performed in the presence of crude extracts, which include unsequestered proteases.

What might the molecular targets of PKC and calmodulin be? An obvious calmodulin target molecule is CaMKII.

• but calmodulin’s effect on exocytosis is ATP-independent, rendering the involvement of a kinase unlikely.

 Calmodulin has also been shown to associate with

• synaptic vesicles in a Ca2+-dependent fashion through synaptotagmin (54),
• probably by binding to its C-terminal tail (55), and to promote Rab3A dissociation from synaptic vesicles (56).
• However, there was little calcium-dependent binding of calmodulin to synaptotagmin
  • either on synaptic vesicles, in a bead binding assay with recombinant proteins,
  • or in a calmodulin overlay (data not shown).

In addition, using immobilized calmodulin, we did not see

• significant Ca2+-dependent pull-down of synaptotagmin or Rab3A from rat brain extract (data not shown).

Recent work has suggested three other candidate targets for calmodulin, Munc13, Pollux, and CRAG (57).

• Pollux has similarity to a portion of a yeast Rab GTPase-activating protein, while
• CRAG is related to Rab3 GTPase exchange proteins.

Further work is required to investigate the role of their interactions with calmodulin in vivo.

The recent report that calmodulin mediates yeast vacuole fusion (24) is intriguing, as it raises the possibility that

• calmodulin, a highly conserved ubiquitous molecule,
  • may mediate many membrane trafficking events.

It is not yet known if

• the effector molecule of calmodulin is conserved or variable across species and different trafficking steps.

It is enticing to propose a model for Ca2+ sensing whereby

• calmodulin is a high affinity Ca2+ sensor for both constitutive and regulated membrane fusion.

1. In the case of constitutive fusion, calmodulin may be the predominant Ca2+ sensor.
2. In the case of slow, non-local exocytosis of large dense core granules, an additional requirement for
3. the concerted actions of other molecule(s) that are better tuned to intermediate rises in [Ca2+] might exist.

At the highly localized sites of fast exocytosis of small clear vesicles where high [Ca2+] is reached,

• specialized low affinity sensor(s) are likely required
• in addition to calmodulin to achieve membrane fusion.

Therefore, although calmodulin participates in multiple types of vesicle fusion,

• the impact of Ca2+ sensing by calmodulin on vesicle release likely varies.

Due to the fact that calmodulin binding to some proteins can be modulated by PKC phosphorylation, one might suspect

• PKC action on exocytosis proceeds through a calmodulin-dependent pathway.
• but the effects of calmodulin and PKC are additive within our system,
  • suggesting that PKC does not act by releasing calmodulin from a substrate
    • that functions as a calmodulin storage protein.

How Ca2+ regulates presynaptic vesicle release has been an open question for many years. By

• identifying calmodulin and PKC as modulators of Ca21-regulated exocytosis and clarifying their functions,
• we have extended our knowledge of the release process.

While the basic machinery of membrane fusion is becoming better understood,

• the multiple effects of Ca2+ on exocytosis remain to be elucidated at the molecular level.

In addition, the ways that Ca2+ regulation may be important to

• the mechanisms of synaptic plasticity in the central nervous system

EXPERIMENTAL PROCEDURES

Materials
Rat Brain Cytosol Preparation
Membrane EGTA Extract Preparation

Cracked Cell Assay

PC12 cells were maintained and [3H]NE labeled as described previously (11). Labeled cells were harvested by pipetting with ice-cold potassium glutamate buffer (50 mM Hepes, pH 7.2, 105 mM potassium glutamate, 20 mM potassium acetate, 2 mM EGTA) containing 0.1% bovine serum albumin. Subsequent manipulations were carried out at 0–4 °C. Labeled cells (1–1.5 ml/dish) were mechanically permeabilized passage through a stainless steel homogenizer. The cracked cells were adjusted to 11 mM EGTA and

• incubated on ice for 0.5–3 h, followed by three washes in which
• the cells were centrifuged at 800 3 g for 5 min and
• resuspended in potassium glutamate buffer containing 0.1% bovine serum albumin.

Composite Assay 

Each release reaction contains 0.5–1 million cracked cells, 1.5 mM free Ca2+, 2 mM MgATP,
and the protein solution to be tested in potassium glutamate buffer. Release reactions were initiated
by incubation at 30 °C and terminated by returning to ice. The supernatant of each reaction was
isolated by centrifugation at 2,500 3 g for 30 min at 4 °C, and the

• released [3H]NE was quantified by scintillation counting (Beckman LS6000IC).

Cell pellets were dissolved in 1% Triton X-100, 0.02% azide and similarly counted. NE release

• was calculated as a percentage of total [3H] in the supernatant.

Priming Assay

A priming reaction contains about

• 1–2 million cracked cells,
• 2 mM MgATP, and
• the protein solution to be tested.
• Ca2+ is omitted.

The primed cells were spun down, washed once with fresh potassium glutamate buffer, and

• distributed into two triggering reactions, each containing
• rat brain cytosol and free Ca2+
• The triggering reaction was performed at 30 °C for 3 min, and
• the NE release was measured
  • as in a composite assay.

Triggering Assay

Cracked cells were primed …, centrifuged, washed …, and

distributed into triggering reactions containing

• 1.5 mM free Ca2+ and the protein solution 

To inhibit any ATP dependent activity in the triggering reaction,  an

• ATP depletion system of

1. 1. hexokinase
   2. MgCl2,
   3. glucose or

• a non-hydrolyzable ATP analogue AMPPNP

was added into the triggering reaction. NE release was measured as above.

Free Ca2+ Concentration Determination

The range of Ca2+free in the release reaction (Fig. 2B) was achieved

• by adding Ca2+ into potassium glutamate buffer to reach final [Ca2+] total values of
  • 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 1.9, and 2.0 mM.
• The pH of the reaction was 7.24 when no Ca2+ was added and
• 7.04 when 2.0 mM Ca2+ was added, 
  • in the absence of protein extracts or cracked cells.

Fig. 2B.   The range of [Ca21]free in the release reaction (Fig. 2B)

Free Ca2+ concentrations were determined using video microscopic
measurements of fura-2 fluorescence (41). [Ca2+]free was calculated from the equation

• [Ca2+]free 5 Kd*3 (R 2 Rmin)/(Rmax 2 R) (42).

The values of Rmin, Rmax, and Kd* were determined in the following solutions: 

potassium glutamate buffer (PGB) containing

• 8 x 3 10^6 cracked cells/ml, 2 mM MgATP (PGB+CC)

1) Rmin:  PGB+CC and 10 mM additional EGTA;

2) Rmax: PBG+CC, and 10 mM total Ca2+;

3) Kd*: PGB+CC, 28 mM additional EGTA, and 18 mM total Ca2+, pH 7.2
([Ca2+]free 5 = 169 nM, determined in the absence of cells and MgATP

• based on fura-2 calibration in cell-free solutions).

These solutions were

1. incubated at 37 °C ,
2. mixed with fura-2 pentapotassium salt
   (100 mM; Molecular Probes, Eugene, OR), and
3. imaged.

This procedure allowed us to take into account

• changes in fura-2 properties
• caused by the presence of
  • permeabilized cells.

Duplicate measurements of the above range of [Ca2+] total gave

• the following average [Ca2+] free values:
• 106, 146, 277, 462, 971, 1468, 1847, and 2484 nM.

Purification of Active Proteins

All procedures were carried out at 4 °C or on ice. Membrane
EGTA extract of one or two bovine brain(s) was

1. filtered through cheesecloth and
2. loaded overnight onto a column packed with DEAE-Sepharose
   CL-6B beads (Amersham Pharmacia Biotech).

The column was then

1. washed with
   (20 mM Hepes, pH 7.5, 0.25 mM sucrose, 2 mM EGTA, 1 mM dithiothreitol) and 
2. step eluted with 10 column volumes of elution buffer
   (20 mM Hepes, pH 7.5, 2 mM EGTA, 400 mM KCl, 1 mM dithiothreitol).
   100 ml of every other fraction was
3. dialyzed overnight into PGB, and
4. tested in a composite release assay for activity.

• The active fractions were pooled and dialyzed into zero salt buffer
  (20 mM Hepes, pH 7.5, 2 mM EGTA) and
• batch bound to 10 ml of Affi-Gel Blue beads (Bio-Rad) or DyeMatrex-Green A beads (Amicon)

Blue beads were used in earlier experiments, and Green beads were used later to

• specifically deplete CAPS, which was known to bind to Green beads (9).

The unbound material was

1. collected,
2. concentrated to about 2 ml using a Centriprep-10 (Amicon), and
3. loaded onto a 120-ml HiPrep Sephacryl S-200 gel filtration column
   (Amersham Pharmacia Biotech).

Samples were run on the S-200 column in PGB at a flow rate of 7 ml/h.

• 10–50 ml of every other fraction was tested for
  • activity in the cracked cell composite assay, and
• two peaks of activity were observed (Fig. 3).

The first peak of activity had a predicted molecular mass of 85 kDa.
The corresponding material was

• adjusted to 10 mM potassium phosphate concentration (pH 7.2) and
• loaded onto a 1-ml column packed with hydroxyapatite Bio-Gel HT
  (Bio-Rad).

The bound material was

• eluted with a linear K-PO4 gradient from 10 to 500 mM (pH 7.2)
•  at a flow rate of about 0.1 ml/min, and
• 0.4–0.5-ml fractions were collected.
•  each fraction was dialyzed into PGB and
• tested for activity.

The fractions were also analyzed by

• SDS-PAGE and silver staining (Sigma silver stain kit).

The active material was concentrated and resolved

• on an 8% poly-acrylamide gel.

Two Coomassie-stained protein bands that matched the activity profile (Fig. 6)

• were excised from the gel,
• sequenced by the Stanford PAN facility.

The two polypeptide sequences obtained from the upper band were:

1. LLNQEEGEYYNVPIXEGD
2. IRSTLNPRWDESFT.

The only bovine protein that contains both polypeptides is PKCa.
The four polypeptide sequences obtained from the lower band were:

1. YELTGKFERLIVGLMRPPAY,
2. LIEILASRTNEQIHQLVAA,
3. MLVVLLQGTREEDDVVSEDL, and
4. EMSGDVRDVFVAIVQSVK.

Based on these sequences, the protein band was

• unambiguously identified to be bovine annexin VI.

The second S-200 peak has a predicted molecular mass of 25 kDa.
The corresponding material was

• dialyzed into zero salt buffer
  (20 mM Tris, pH 7.5, 1 mM EGTA) and
• injected onto a Mono-Q HR 5/5 FPLC column
  (Pharmacia).

The FPLC run was performed at 18 °C at 1 ml/min and

• 1-ml fractions were collected
• with a linear salt gradient from 0 to 1 M KCl over 71 ml.

The fractions containing proteins (determined by A280) were

• dialyzed into PGB and
• tested in the cracked cell assay.

Western Blot

Anti-calmodulin antibody and anti-PKC antibody were used, and

• ECL (Amersham) was used for detection.

RESULTS

A Membrane EGTA Extract Supports NE Release 

Brain cytosol, prepared as the supernatant of the brain homogenate,

• effectively stimulates NE release
• in the cracked cell assay (Fig. 1)
  as previously shown (9). 

We wondered whether crude extracts other than cytosol

• could support NE release, and we focused on
• extractable peripheral membrane proteins.

We found that a salt or EGTA extract of brain membranes,
membranes defined as the

• 100,000 3 g pellet of the crude homogenate,
• reconstituted secretion in the absence of cytosol.
• the salt extract only slightly enhanced NE release
  above background (data not shown), the 

EGTA extract not only stimulated NE release to a high level,

• similar to that supported by cytosol, but also
• had a higher specific activity than cytosol (Fig. 1). 

FIG. 1. The EGTA extract of brain membranes can support NE release in the absence of cytosol. Rat brain membrane EGTA extract (closed triangles) and rat brain cytosol (closed squares) were prepared as described under “Experimental Procedures.” NE release was measured in a composite reaction mixture of cracked cells, MgATP, Ca2+, and the indicated amount of crude extracts.

The ability of the membrane EGTA extract to support secretion is consistent with the fact that

• following cracking, the cells are immediately extracted with EGTA, and are presumably
• devoid of most membrane EGTA-extractable factors.

This also suggests that these factors, some of which are probably

• Ca2+-dependent membrane-associating proteins,
• participate in Ca2+- triggered exocytosis.

The Membrane EGTA Extract Is Enriched in Triggering Fators

NE release in cracked cells can be resolved into two sequential stages,

• an ATP-dependent priming stage and
• an ATP-independent Ca21-dependent triggering stage (11), and
• proteins can be tested for activity in either stage.

An effect in priming indicates

1. an early role for the protein, and
2. an effect in triggering a late ATP-independent role.

Since the protein composition of the

• membrane EGTA extract and cytosol are different,

we tested whether they had different activities

• in the priming stage versus the triggering stage.

We found that the membrane EGTA extract is enriched in factors that

• act during triggering stage of NE release, as
• the same amount of protein from the membrane EGTA extract as cytosol
• gave a higher stimulation in the triggering assay, but
• not in the priming assay (Fig. 2A). 

Regular cytosol is prepared in a buffer containing 2 mM EGTA, and thus

• presumably contains some of the proteins present in the membrane EGTA extract.

Cytosol prepared in the absence of EGTA showed an even lower specific activity

• in the triggering assay compared with regular cytosol (Fig. 2A).

Identification of Calmodulin as an Active Triggering Factor in the EGTA Extract

Biochemical fractionation of the bovine brain membrane EGTA extract was carried out

• to identify the active components capable of reconstituting NE release.

Activity was assayed in a composite reaction mixture containing

• cracked cells,
• ATP,
• Ca2+, and
• the test protein(s).

Except for the presence of bovine serum albumin in the basal buffer,

• no other proteins were added to the cell ghosts except for the test protein(s).

Initial tests indicated that at least

1. part of the activity in the membrane EGTA extract binds to and
2. can be efficiently eluted from an anion exchanger and hydroxyapatite resin,
3. but does not bind to Amicon color resins.

The starting material was, therefore, sequentially purified using

• DEAE, Affi-Gel Blue (or Matrex Green-A), and gel filtration chromotography.

Gel filtration fractionation indicated the presence of two peaks of activity with

• predicted molecular masses of 25 and 85 kDa, respectively (Fig. 3).

FIG. 3. Gel filtration chromatography reveals two stimulatory factors in the membrane EGTA extract.

In order to purify the active component(s) in the membrane EGTA extract, the crude extract from one bovine brain was fractionated chromatographically (see Experimental Procedures” for details). Fractions from a Sephacryl S-200 gel filtration column were tested for their activity in stimulating NE release in the composite assay. The two activity peaks have predicted molecular masses of 85 and 25 kDa, respectively. The arrows indicate the retention volume of standard proteins run on the same column.

The low molecular weight active factor was purified to homogeneity, as judged by a

• Coomassie-stained SDS-PAGE gel, after a subsequent Mono-Q fractionation (Fig. 4).

FIG. 4. The low molecular wen.ight active factor is calmodulin

A, the  membrane EGTA extract from one bovine brain (Start) was subjected to sequential fractionation on DEAE, Blue A, and
Sephacryl S-200 columns. The pooled material containing the activity after each chromotographic step was analyzed by SDS-
PAGE and Coomassie staining. The arrowheads indicate the presence of calmodulin in all the lanes. Calmodulin shows a
mobility shift depending on whether or not Ca2+ is present during electrophoresis (see panel C).
B, the active material  pooled from Sephacryl S-200 was fractionated on a Mono-Q FPLC column and the fractions
(5 ml/fraction) were tested for activity in a composite assay. The activity peak is shown.
C, the active Mono-Q fractions (5 ml/fraction) were subjected to SDS-PAGE in the presence of 1 mM EGTA or 0.1 mM Ca2+,
and the gels stained with Coomassie Blue.
D, fraction 47 (1 ml) was probed by Western blotting with a monoclonal anti-calmodulin antibody. No Ca2+ or EGTA was
added during SDS-PAGE.

We reasoned that the protein might be calmodulin (43) based on the following:

1) It is a relatively small protein (14–18 kDa) that is abundant in the
starting extract (Fig. 4A).
2) It elutes at a very high salt concentration (0.41 M KCl) on the
Mono-Q column.
3) It stains negatively in silver stain (data not shown).
4) Its electrophoretic mobility shifts depending on the presence or
absence of Ca21 (Fig. 4C).

A Western blot with an anti-calmodulin monoclonal antibody gave a
positive signal (Fig. 4D), confirming our prediction.

Properties of Calmodulin-stimulated Exocytosis

We used commercial calmodulin or bacterially expressed recombinant calmodulin to confirm our purification result; both sources of authentic calmodulin stimulated NE release as expected. Moreover, we found that calmodulin stimulates secretion in a triggering assay as well as in a composite assay (Fig. 5A).

The half-maximal increase was at 75 nM (250 ng/200 ml) final calmodulin concentration. This is within the broad
range of affinities between calmodulin and its various targets and suggests that the interaction between
calmodulin and its target molecule in exocytosis is in the physiological range. When the triggering reaction was
performed at different Ca2+ concentrations, calmodulin increased NE release only at high [Ca2+] (0.4 – 2 mM)
similar to the crude EGTA extract (Fig. 5B),

suggesting that calmodulin contributes to the triggering activity of the membrane EGTA extract.  Calmodulin’s affinity for Ca2+ has
been  reported to be around 1 mM (25),

• consistent with the Ca2+ requirement for
• calmodulin-stimulated secretion that we observed.

FIG. 5. Calmodulin stimulates NE release in the triggering stage.
A, calmodulin (obtained from Sigma) increased NE release in the
triggering assay in a dose-dependent fashion, in the absence of ATP
or any other cytosolic proteins. In this particular experiment, the
maximal release achieved by addition of rat brain cytosol was 46.5%.

B, the triggering assay was performed with different concentrations
of free Ca2+. Calmodulin (3 mg bacterially expressed recombinant
protein; closed squares) increased NE release with a similar Ca2+
sensitivity to rat brain membrane EGTA extract (10 mg; closed
triangles), as compared with conditions in which no protein was
added (open squares).

Western analysis with commercial protein as standards indicated that calmodulin 

•  constitutes about 5% of total proteins in the rat brain membrane EGTA extract
• and about 2% of total proteins in the rat brain cytosol (data not shown).

In addition, a significant amount of calmodulin appears to be left

• in the washed cell ghosts (data not shown).

Based on the activity of saturating levels of

• pure calmodulin (releasing 6–10% of total [3H]NE)
• and crude EGTA extract (releasing ;45% of total [3H]NE),

we estimated that

• calmodulin accounts for 13–22% of total activity of the extract.

Consistent with this,

• a high affinity calmodulin-binding peptide
  (CaMKIIa(291–312) (44), used at 5 mM) and
• an anti-calmodulin antibody (2 mg/200 ml)
• inhibited about 20% of the membrane EGTA extract-stimulated release
  (6.7 mg of extract added; data not shown).

We showed that calmodulin increased NE release

• in the triggering stage.

Since regular triggering reactions were performed

• in the absence of any added ATP,

this suggests that

• calmodulin enhanced secretion in an ATP-independent fashion.

Furthermore, residual ATP in the cell ghosts did not play a role, since

•  addition of a hexokinase ATP depletion system that
• can deplete millimolar concentrations of ATP
  • within a few minutes (11) had little effect, as did
  • addition of 5 mM AMPPNP,
• which blocks ATP-dependent enzymatic activity (Fig.8A).

Therefore, we ruled out the possibility that a kinase mediates calmodulin’s effect.

FIG. 8. PKC and calmodulin stimulate the late triggering reaction in
an ATP-dependent and ATP-independent manner respectively.
A, triggering assays were performed to test the activity of calmodulin
(recombinant; black bars) and PKC (purified rat brain PKC from
Calbiochem; shaded bars) in the absence of ATP. A regular triggering
assay is done in the absence of ATP (2ATP). To deplete residual ATP
in the cells, hexokinase-based ATP depletion was employed (1Hexo).
Alternatively, 5 mM AMP-PNP (1AMP-PNP) was added in the triggering
reaction. Under all three conditions, calmodulin increased release
as compared with the background (buffer only; white bars), whereas
PKC did not.
B, NE release in a composite assay was measured with varying
concentrations of free Ca2+ in the presence of 10 mg of calmodulin
(recombinant; closed triangles), 70 ng of PKC  (purified rat brain PKC
from Calbiochem; closed squares), or buffer only (open squares).

A series of calmodulin mutants from Paramecium and chicken were tested

• for their ability to enhance Ca2+-stimulated secretion, and
• none of the mutations abolished the calmodulin effect (data not shown).

These mutations include

• S101F, M145V, E54K, G40E/D50N, V35I/D50N within Paramecium
• calmodulin (45), and M124Q, M51A/V55A, and M51A/V55A/L32A
  within chicken calmodulin (46, 47).

The Paramecium calmodulin mutants are the result of

• naturally occurring mutations that result in aberrations in their behavior.

These mutants can be grouped into two categories according to their
behavior, reflecting their loss of either

1. a Ca2+-dependent Na1 current
    (calmodulin N-terminal lobe mutants: E54K, G40E/D50N, and
    V35I/D50N) or
2. a Ca21-dependent K1 current
   (calmodulin C-terminal lobe mutants: S101F and M145V) (45).

The chicken calmodulin mutants have been shown to

• differentially activate myosin light chain kinase
  (M124Q, M51A/V55A, and M51A/V55A/L32A),
  CaMKII (M124Q),  and CaMKIV (M124Q),

and the mutated residues are thought to be important in

• defining calmodulin’s binding specificity (46, 47).

Our finding that these mutant calmodulins can stimulate exocytosis suggests that

• calmodulin-binding domains similar to those of Paramecium Ca2+/calmodulin-dependent
  ion channels, myosin light chain kinase, CaMKII, and CaMKIV,
• are unlikely to mediate release utilizing the conserved SNARE fusion machinery, as they
• could be completely abolished by addition of exogenous syntaxin H3 domains (data not shown).
• the same molecular pathway was not activated, since their effects were additive (data not shown).

 

Acknowledgments—

We thank Diana Bautista and Dr. Richard S.Lewis for generous help

with [Ca21]free determination; Dr. Ching Kung for providing the Paramecium calmodulin

mutants, and Dr. Anthony R. Means for providing the chicken calmodulin mutants. We also

thank Dr. Jesse C. Hay for the initial setup of the cracked cell assay, and Dr. Suzie J.

Scales for helpful comments on the manuscript.

REFERENCES

1. Calakos, N., and Scheller, R. H. (1996) Physiol. Rev. 76, 1–29

2. Su¨ dhof, T. C. (1995) Nature 375, 645–653

3. Zucker, R. S. (1996) Neuron 17, 1049–1055

4. Hanson, P. I., Heuser, J. E., and Jahn, R. (1997) Curr. Opin. Neurobiol. 7, 310–315

5. Chen, Y. A., Scales, S. J., Patel, S. M., Doung, Y.-C., and Scheller, R. H. (1999) Cell 97, 165–174

6. Neher, E., and Zucker, R. S. (1993) Neuron 10, 21–30

7. Kamiya, H., and Zucker, R. S. (1994) Nature 371, 603–606

SOURCE

J Biol Chem 1999; 274(37): 25469-26476.

Other related articles published in this Open Access Online Scientific Journal include the following:

The role of ion channels in Na(+)-K(+)-ATPase: regulation of ion transport across the plasma membrane has been studies by our Team in 2012 and 2013. Chiefly, our sources of inspiration were the following:

1. 2013 Nobel work on vesicles and calcium flux at the neuromuscular junction Machinery Regulating Vesicle Traffic, A Major Transport System in our Cells The 2013 Nobel Prize in Physiology or Medicine is awarded to Dr. James E. Rothman, Dr. Randy W. Schekman and Dr. Thomas C. Südhof

• for their discoveries of machinery regulating vesicle traffic,
• a major transport system in our cells.

This represents a paradigm shift in our understanding of how the eukaryotic cell, with its complex internal compartmentalization, organizes

• the routing of molecules packaged in vesicles
• to various intracellular destinations,
• as well as to the outside of the cell

Specificity in the delivery of molecular cargo is essential for cell function and survival.

http://www.nobelprize.org/nobel_prizes/medicine/laureates/2013/advanced-medicineprize2013.pdf

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

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

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

2. Perspectives on Nitric Oxide in Disease Mechanisms

available on Kindle Store @ Amazon.com

http://www.amazon.com/dp/B00DINFFYC

http://pharmaceuticalintelligence.com/biomed-e-books/series-a-e-books-on-cardiovascular-diseases/
perspectives-on-nitric-oxide-in-disease-mechanisms-v2/

3. Professor David Lichtstein, Hebrew University of Jerusalem, Dean, School of Medicine

Lichtstein’s main research focus is the regulation of ion transport across the plasma membrane of eukaryotic cells.

His work led to the discovery that specific steroids that have crucial roles, as

• the regulation of cell viability,
• heart contractility,
• blood pressure and
• brain function.

His research has implications for the fundamental understanding of body functions,

• as well as for several pathological states such as
  • heart failure, hypertension
  • and neurological and psychiatric diseases.

Physiologist, Professor Lichtstein, Chair in Heart Studies at The Hebrew University elected
Dean of the Faculty of Medicine at The Hebrew University of Jerusalem

Reporter: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/12/18/physiologist-professor-lichtstein-chair-in-heart-studies-
at-the-hebrew-university-elected-dean-of-the-faculty-of-medicine-at-the-hebrew-university-of-jerusalem/

4. Professor Roger J. Hajjar, MD at Mount Sinai School of Medicine

Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension
and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-for-
pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

5.            Seminal Curations by Dr. Aviva Lev-Ari on Genetics and Genomics of Cardiovascular Diseases with a focus on Conduction and Cardiac Contractility

• Atrioventricular (AV) Conduction Disease (block): Human Mutations affecting the Voltage Clock

Aviva Lev-Ari, PhD, RN

• Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis

Aviva Lev-Ari, PhD, RN

•  Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics, 3/2010 – 3/2013

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

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

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

6. Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

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

http://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

This study presents  the possible correlation between Myocardial Ischemia (Coronary Artery Disease (CAD)) aka Ischemic Heart Disease (IHD) and single-nucleotide polymorphisms (SNPs) genes encoding several regulators involved in Coronary Blood Flow Regulation (CBFR), including

• ion channels acting in vascular smooth muscle and/or
• endothelial cells of coronary arteries.

They completely analyzed exon 3 of both KCNJ8 and KCNJ11 genes (Kir6.1 and Kir6.2 subunit, respectively) as well as

• the whole coding region of KCN5A gene (Kv1.5 channel).

The work suggests certain genetic polymorphisms may represent a non-modifiable protective factor that could be

• used to identify individuals at relatively low-risk for cardiovascular disease
  • an independent protective role of the
  • rs5215_GG against developing CAD and
  • a trend for rs5219_AA to be associated with protection against coronary microvascular dysfunction

Other related articles published on this Open Access Online Scientific Journal include the following:

ION CHANNEL and Cardiovascular Diseases

http://pharmaceuticalintelligence.com/?s=Ion+Channel

Calcium Role in Cardiovascular Diseases

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-
that-are-related-to-the-actin-cytoskeleton/

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-
skeleton-and-lipid-structures-in-signaling-and-cell-motility/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease
Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-
exchange-mechanism-in-health-and-disease/

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and
Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia,
Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-
involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-
post-ischemic-arrhythmia-similarities-and-differen/

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

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

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

Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary

Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-
for-pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure –
Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-
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vesicles with cell membranes during Neurotransmission
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http://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

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Mitochondria and its Role in Cardiovascular Diseases

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Mitochondrial Metabolism and Cardiac Function
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MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix Identified
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Mitochondrial Dynamics and Cardiovascular Diseases
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Mitochondrial Damage and Repair under Oxidative Stress
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Gene Expression: Algorithms for Protein Dynamics

Posted in Biological Networks, Gene Regulation and Evolution, Computational Biology/Systems and Bioinformatics, Genome Biology, Genomic Testing: Methodology for Diagnosis, Personalized and Precision Medicine & Genomic Research, Population Health Management, Genetics & Pharmaceutical, Scientist: Career considerations, Statistical Methods for Research Evaluation, tagged Alan Boyle, DNA, ENCODE, gene expression, National Human Genome Research Institute, Nucleic acid sequence, Stanford University School of Medicine on October 26, 2013| 1 Comment »

Gene Expression: Algorithms for Protein Dynamics

Reporter:  Aviva Lev-Ari, PhD, RN

Stanford-developed algorithm reveals complex protein dynamics behind gene expression

BY KRISTA CONGER

Michael Snyder

In yet another coup for a research concept known as “big data,” researchers at the Stanford University School of Medicine have developed a computerized algorithm to understand the complex and rapid choreography of hundreds of proteins that interact in mindboggling combinations to govern how genes are flipped on and off within a cell.

To do so, they coupled findings from 238 DNA-protein-binding experiments performed by the ENCODE project — a massive, multiyear international effort to identify the functional elements of the human genome — with a laboratory-based technique to identify binding patterns among the proteins themselves.

The analysis is sensitive enough to have identified many previously unsuspected, multipartner trysts. It can also be performed quickly and repeatedly to track how a cell responds to environmental changes or crucial developmental signals.

“At a very basic level, we are learning who likes to work with whom to regulate around 20,000 human genes,” said Michael Snyder, PhD, professor and chair of genetics at Stanford. “If you had to look through all possible interactions pair-wise, it would be ridiculously impossible. Here we can look at thousands of combinations in an unbiased manner and pull out important and powerful information. It gives us an unprecedented level of understanding.”

Snyder is the senior author of a paper describing the research published Oct. 24 in Cell. The lead authors are postdoctoral scholars Dan Xie, PhD, Alan Boyle, PhD, and Linfeng Wu, PhD.

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• » Snyder, Cherry receive National Human Genome Research Institute grants to advance work on ENCODE project

Proteins control gene expression by either binding to specific regions of DNA, or by interacting with other DNA-bound proteins to modulate their function. Previously, researchers could only analyze two to three proteins and DNA sequences at a time, and were unable to see the true complexities of the interactions among proteins and DNA that occur in living cells.

The challenge resembled trying to figure out interactions in a crowded mosh pit by studying a few waltzing couples in an otherwise empty ballroom, and it has severely limited what could be learned about the dynamics of gene expression.

The ENCODE, for the Encyclopedia of DNA Elements, project was a five-year collaboration of more than 440 scientists in 32 labs around the world to reveal the complex interplay among regulatory regions, proteins and RNA molecules that governs when and how genes are expressed. The project has been generating a treasure trove of data for researchers to analyze for the last eight years.

In this study, the researchers combined data from genomics (a field devoted to the study of genes) and proteomics (which focuses on proteins and their interactions). They studied 128 proteins, called trans-acting factors, which are known to regulate gene expression by binding to regulatory regions within the genome. Some of the regions control the expression of nearby genes; others affect the expression of genes great distances away.

The researchers used 238 data sets generated by the ENCODE project to study the specific DNA sequences bound by each of the 128 trans-acting factors. But these factors aren’t monogamous; they bind many different sequences in a variety of protein-DNA combinations. Xie, Boyle and Snyder designed a machine-learning algorithm to analyze all the data and identify which trans-acting factors tend to be seen together and which DNA sequences they prefer.

Wu then performed immunoprecipitation experiments, which use antibodies to identify protein interactions in the cell nucleus. In this way, they were able to tell which proteins interacted directly with one another, and which were seen together because their preferred DNA binding sites were adjoining.

“Before our work, only the combination of two or three regulatory proteins were studied, which oversimplified how gene regulators collaborate to find their targets,” Xie said. “With our method we are able to study the combination of more than 100 regulators and see a much more complex structure of collaboration. For example, it had been believed that a key regulator of cell proliferation called FOS typically only works with JUN protein family members. We show, in addition to JUN, FOS has different partners under different circumstances. In fact, we found almost all the canonical combinations of two or three trans-acting factors have many more partners than we previously thought.”

To broaden their analysis, the researchers included data from other sources that explored protein-binding patterns in five cell types. They found that patterns of co-localization among proteins, in which several proteins are found clustered closely on the DNA to govern gene expression, vary according to cell type and the conditions under which the cells are grown. They also found that many of these clusters can be explained through interactions among proteins, and that not every protein bound to DNA directly.

“We’d like to understand how these interactions work together to make different cell types and how they gain their unique identities in development,” Snyder said. “Furthermore, diseased cells will have a very different type of wiring diagram. We hope to understand how these cells go astray.”

Other Stanford co-authors include life science research assistant Jie Zhai and life science research associate Trupti Kawli, PhD.

The research was supported by the National Human Genome Research Institute (grants U54HG004558 and U54HG006996).

Information about Stanford’s Department of Genetics, which also supported the work, is available at http://genetics.stanford.edu.

PRINT MEDIA CONTACT

Krista Conger | Tel (650) 725-5371

kristac@stanford.edu

BROADCAST MEDIA CONTACT

M.A. Malone | Tel (650) 723-6912

mamalone@stanford.edu

Stanford Medicine integrates research, medical education and patient care at its three institutions – Stanford University School of Medicine, Stanford Hospital & Clinics and Lucile Packard Children’s Hospital. For more information, please visit the Office of Communication & Public Affairs site at

http://mednews.stanford.edu/.http://med.stanford.edu/ism/2013/october/snyder.html?goback=%2Egde_5180384_member_5799368448383397888#sthash%2EhU03LKIX%2Edpuf

 

Dynamic trans-Acting Factor Colocalization in Human Cells

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Cell, Volume 155, Issue 3, 713-724, 24 October 2013
Copyright © 2013 Elsevier Inc. All rights reserved.
10.1016/j.cell.2013.09.043

Authors

Dan Xie,Alan P. Boyle,Linfeng Wu,Jie Zhai,Trupti Kawli,Michael SnyderSee Affiliations

• Highlights
• Colocalization patterns of 128 TFs in human cells
• An application of SOMs to study high-dimensional TF colocalization patterns
• Colocalization patterns are dynamic through stimulation and across cell types
• Many TF colocalizations can be explained by protein-protein interaction

Summary

Different trans-acting factors (TFs) collaborate and act in concert at distinct loci to perform accurate regulation of their target genes. To date, the cobinding of TF pairs has been investigated in a limited context both in terms of the number of factors within a cell type and across cell types and the extent of combinatorial colocalizations. Here, we use an approach to analyze TF colocalization within a cell type and across multiple cell lines at an unprecedented level. We extend this approach with large-scale mass spectrometry analysis of immunoprecipitations of 50 TFs. Our combined approach reveals large numbers of interesting TF-TF associations. We observe extensive change in TF colocalizations both within a cell type exposed to different conditions and across multiple cell types. We show distinct functional annotations and properties of different TF cobinding patterns and provide insights into the complex regulatory landscape of the cell.

http://www.cell.com/abstract/S0092-8674%2813%2901217-8#!

Personal Omics Profiling Reveals Dynamic Molecular and Medical Phenotypes

Cell, Volume 148, Issue 6, 1293-1307, 16 March 2012
Copyright © 2012 Elsevier Inc. All rights reserved.
10.1016/j.cell.2012.02.009

Authors

Rui Chen,George I. Mias,Jennifer Li-Pook-Than,Lihua Jiang,Hugo Y.K. Lam,Rong Chen,Elana Miriami,Konrad J. Karczewski,Manoj Hariharan,Frederick E. Dewey,Yong Cheng,Michael J. Clark,Hogune Im,Lukas Habegger,Suganthi Balasubramanian,Maeve O’Huallachain,Joel T. Dudley,Sara Hillenmeyer,Rajini Haraksingh,Donald Sharon,Ghia Euskirchen,Phil Lacroute,Keith Bettinger,Alan P. Boyle,Maya Kasowski,Fabian Grubert,Scott Seki,Marco Garcia,Michelle Whirl-Carrillo,Mercedes Gallardo,Maria A. Blasco,Peter L. Greenberg,Phyllis Snyder,Teri E. Klein,Russ B. Altman,Atul J. Butte,Euan A. Ashley,Mark Gerstein,Kari C. Nadeau,Hua Tang,Michael Snyder

Department of Genetics, Stanford University School of Medicine, Stanford University, Stanford, CA 94305, USADivision of Systems Medicine and Division of Immunology and Allergy, Department of Pediatrics, Stanford University, Stanford, CA 94305, USACenter for Inherited Cardiovascular Disease, Division of Cardiovascular Medicine, Stanford University, Stanford, CA 94305, USADivision of Hematology, Department of Medicine, Stanford University, Stanford, CA 94305, USA

Personalized medicine aims to assess medical risks, monitor, diagnose and treat patients according to their specific genetic composition and molecular phenotype. The advent of genome sequencing and the analysis of physiological states has proven to be powerful (Cancer Genome Atlas Research Network, 2011). However, its implementation for the analysis of otherwise healthy individuals for estimation of disease risk and medical interpretation is less clear. Much of the genome is difficult to interpret and many complex diseases, such as diabetes, neurological disorders and cancer, likely involve a large number of different genes and biological pathways (Ashley et al., 2010,Grayson et al., 2011,Li et al., 2011), as well as environmental contributors that can be difficult to assess. As such, the combination of genomic information along with a detailed molecular analysis of samples will be important for predicting, diagnosing and treating diseases as well as for understanding the onset, progression, and prevalence of disease states (Snyder et al., 2009).

Presently, healthy and diseased states are typically followed using a limited number of assays that analyze a small number of markers of distinct types. With the advancement of many new technologies, it is now possible to analyze upward of 10⁵ molecular constituents. For example, DNA microarrays have allowed the subcategorization of lymphomas and gliomas (Mischel et al., 2003), and RNA sequencing (RNA-Seq) has identified breast cancer transcript isoforms (Li et al., 2011,van der Werf et al., 2007,Wu et al., 2010,Lapuk et al., 2010). Although transcriptome and RNA splicing profiling are powerful and convenient, they provide a partial portrait of an organism’s physiological state. Transcriptomic data, when combined with genomic, proteomic, and metabolomic data are expected to provide a much deeper understanding of normal and diseased states (Snyder et al., 2010). To date, comprehensive integrative omics profiles have been limited and have not been applied to the analysis of generally healthy individuals.

To obtain a better understanding of: (1) how to generate an integrative personal omics profile (iPOP) and examine as many biological components as possible, (2) how these components change during healthy and diseased states, and (3) how this information can be combined with genomic information to estimate disease risk and gain new insights into diseased states, we performed extensive omics profiling of blood components from a generally healthy individual over a 14 month period (24 months total when including time points with other molecular analyses). We determined the whole-genome sequence (WGS) of the subject, and together with transcriptomic, proteomic, metabolomic, and autoantibody profiles, used this information to generate an iPOP. We analyzed the iPOP of the individual over the course of healthy states and two viral infections (Figure 1A). Our results indicate that disease risk can be estimated by a whole-genome sequence and by regularly monitoring health states with iPOP disease onset may also be observed. The wealth of information provided by detailed longitudinal iPOP revealed unexpected molecular complexity, which exhibited dynamic changes during healthy and diseased states, and provided insight into multiple biological processes. Detailed omics profiling coupled with genome sequencing can provide molecular and physiological information of medical significance. This approach can be generalized for personalized health monitoring and medicine.

• Figure 1
• Figure 2
• Figure 3
• Figure 4
• Figure 5
• Figure 6

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Figure 1. Summary of Study

http://www.cell.com/abstract/S0092-8674%2812%2900166-3#Introduction

 

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

Posted in Biological Networks, Gene Regulation and Evolution, Biomedical Measurement Science, Calcium Signaling, Cell Biology, Signaling & Cell Circuits, tagged Aviva Lev-Ari, Bernard Katz, Heart Failure, Howard Hughes Medical Institute, James Rothman, Justin Pearlman, Larry H. Bernstein, Richard Scheller, SNARE, Stanford University School of Medicine, Thomas C. Südhof, Vascular smooth muscle on September 10, 2013| 11 Comments »

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

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

Image created by Adina Hazan 06/30/2021

This article is the Part X in a series of articles on Activation and Dysfunction of the Calcium Release Mechanisms in Cardiomyocytes and Vascular Smooth Muscle Cells.

The Series consists of the following articles:

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton

Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

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

http://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
 and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-exchange-mechanism-in-health-and-disease/

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

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

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

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

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

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

Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-for-pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

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

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

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

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

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

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

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

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

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

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

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

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

Part XI: Sensors and Signaling in Oxidative Stress

Larry H. Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

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

http://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

Introduction

Author: Larry H Bernstein, MD, FCAP 

This introduction is based on two sources:

#1:

Michael J. Berridge, Smooth muscle cell calcium activation mechanisms

The Babraham Institute, Babraham, Cambridge CB22 4AT, UK

J Physiol 586.21 (2008) pp 5047–5061

http://jp.physoc.org/content/586/21/5047.full.pdf

and

#2

Thomas C Südhof, A molecular machine for neurotransmitter release: synaptotagmin and beyond

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

Part IX of this series of articles discussed the mechanism of the signaling of smooth muscle cells by the interacting parasympathetic neural innervation that occurs by calcium triggering neurotransmitter release by initiating synaptic vesicle fusion.   It involves the interaction of soluble N-acetylmaleimide-sensitive factor (SNARE) and SM proteins, and in addition, the discovery of a calcium-dependsent Syt1 (C) domain of protein- kinase C isoenzyme, which binds to phospholipids.  It is reasonable to consider that it differs from motor neuron activation of skeletal muscles, mainly because the innervation is in the involuntary domain.   The cranial nerve rooted innervation has evolved comes from the spinal ganglia at the corresponding level of the spinal cord.  It is in this specific neural function that we find a mechanistic interaction with adrenergic hormonal function, a concept intimated by the late Richard Bing.  Only recently has there been a plausible concept that brings this into serious consideration.  Moreover, the review of therapeutic drugs that are used in blocking adrenergic receptors are closely related to the calcium-channels.  Interesting too is the participation of a phospholipid bound protein-kinase isoenzyme C calcium-dependent domain Syt1.  The neurohormonal connection lies in the observation by Katz in the 1950’s that the vesicles of the neurons hold and eject fixed amounts of neurotransmitters.

In Sudhof’s Lasker Award presentation he refers to the biochemical properties of synaptotagmin were found to precisely correspond to the extraordinary calcium-triggering properties of release, and to account for a regulatory pathway that also applies to other types of calcium-triggered fusion, for example fusion observed in hormone secretion and fertilization. At the synapse, finally, these interdependent machines — the fusion apparatus and its synaptotagmin-dependent control mechanism — are embedded in a proteinaceous active zone that links them to calcium channels, and regulates the docking and priming of synaptic vesicles for subsequent calcium-triggered fusion. Thus, work on neurotransmitter release revealed a hierarchy of molecular machines that mediate the fusion of synaptic vesicles, the calcium-control of this fusion, and the embedding of calcium-controlled fusion in the context of the presynaptic terminal at the synapse.  The neural transmission is described as a biological relay system. Neurotransmission kicks off with an electrical pulse that runs down a nerve cell, or neuron. When that signal reaches the tip, calcium enters the cell. In response, the neuron liberates chemical messengers—neurotransmitters—which travel to the next neuron and thus pass the baton.

He further stipulates that synaptic vesicle exocytosis operates by a general mechanism of membrane fusion that revealed itself to be a model for all membrane fusion, but that is uniquely regulated by a calcium-sensor protein called synaptotagmin.  Neurotransmission is thus a combination of electrical signal and chemical transport.

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

Several SMC types illustrate how signaling mechanisms have been adapted to control different contractile functions with particular emphasis on how Ca2+ signals are activated.

[1] Neural activation of vas deferens smooth muscle cells

Noradrenaline (NA) acts by stimulating α1-adrenoreceptors to produce InsP3, which then releases Ca2+ that may induce an intracellular Ca2+ wave similar to that triggered by the ATP-dependent entry of external Ca2+. In addition, the α1-adrenoreceptors also activate the smooth muscle Rho/Rho kinase signalling pathway that serves to increase the Ca2+ sensitivity of the contractile machinery.

[2] Detrusor smooth muscle cells

The bladder, which functions to store and expel urine, is surrounded by layers of detrusor SMCs. The latter have two operational modes: during bladder filling they remain relaxed but contract vigorously to expel urine during micturition. The switch from relaxation to contraction, which is triggered by neurotransmitters released from parasympathetic nerves, depends on the acceleration of an endogenous membrane oscillator that produces the repetitive trains of action potentials that drive contraction.

This mechanism of activation is also shared by [1], and uterine contraction.  SMCs are activated by membrane depolarization (ΔV) that opens L-type voltage-operated channels (VOCs) allowing external Ca2+ to flood into the cell to trigger contraction. This depolarization is induced either by ionotropic receptors (vas deferens) or a membrane oscillator (bladder and uterus). The membrane oscillator, which resides in the plasma membrane, generates the periodic pacemaker depolarizations responsible for the action potentials that drive contraction.

The main components of the membrane oscillator are the Ca2+ and K+ channels that sequentially depolarize and hyperpolarize the membrane, respectively. This oscillator generates the periodic pacemaker depolarizations that trigger each action potential. The resulting Ca2+ signal lags behind the action potential because it spreads into the cell as a slower Ca2+ wave mediated by the type 2 RYRs.

Neurotransmitters such as ATP and acetylcholine (ACh), which are released from parasympathetic axonal varicosities that innervate the bladder, activate or accelerate the oscillator by inducing membrane depolarization (ΔV).

[3]  The depolarizing signal that activates gastrointestinal, urethral and ureter SMCs is as follows:

A number of SMCs are activated by pacemaker cells such as the interstitial cells of Cajal (ICCs) (gastrointestinal and urethral SMCs) or atypical SMCs (ureter). These pacemaker cells have a cytosolic oscillator that generates the repetitive Ca2+ transients that activate inward currents that spread through the gap junctions to provide the depolarizing signal (ΔV) that triggers contraction.

[4]  Our greatest interest has been in this mechanism.  The rhythmical contractions of vascular, lymphatic, airway and corpus cavernosum SMCs depend on an endogenous pacemaker driven by a cytosolic Ca2+ oscillator that is responsible for the periodic release of Ca2+ from the endoplasmic reticulum. The periodic pulses of Ca2+ often cause membrane depolarization, but this is not part of the primary activation mechanism but has a secondary role to synchronize and amplify the oscillatory mechanism. Neurotransmitters and hormones act by modulating the frequency of the cytosolic oscillator.

Vascular or airway SMCs are driven by a cytosolic oscillator that generates a periodic release of Ca2+ from the endoplasmic reticulum that usually appears as a propagating Ca2+ wave.

Step 1. The initiation and/or modulation of this oscillator depends upon the action of transmitters and hormones such as ACh, 5-HT, NA and endothelin-1 (ET-1) that increase the formation of InsP3 and diacylglycerol (DAG), both of which promote oscillatory activity.

Step 2. The oscillator is very dependent on Ca2+ entry to provide the Ca2+ necessary to charge up the stores for each oscillatory cycle. The nature of these entry mechanisms vary between cell types.

Step 3. The entry of external Ca2+ charges up the ER to sensitize the RYRs and InsP3 receptors prior to the next phase of release. An important determinant of this sensitivity is the luminal concentration of Ca2+ and as this builds up the release channels become sensitive to Ca2+ and can participate in the process of Ca2+-induced Ca2+ release (CICR), which is responsible for orchestrating the regenerative release of Ca2+ from the ER. The proposed role of cyclic ADP-ribose (cADPR) in airway SMCs is consistent with this aspect of the model on the basis of its proposed action of stimulating the SERCA pump to enhance store loading and such a mechanism has been described in colonic SMCs.

Step 4. The mechanism responsible for initiating Ca2+ release may depend either on the RYRs or the InsP3 receptors (I). RYR channels are sensitive to store loading and the InsP3 receptors will be sensitized by the agonist-dependent formation of InsP3.

Step 5. This initial release of Ca2+ is then amplified by regenerative Ca2+ release by either the RYRs or InsP3 receptors, depending on the cell type.

Step 6. The global Ca2+ signal then activates contraction.

Step 7. The recovery phase depends on the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), that pumps some of the Ca2+ back into the ER, and the plasma membrane Ca2+-ATPase (PMCA), that pumps Ca2+ out of the cell.

Step 8. One of the effects of the released Ca2+ is to stimulate Ca2+-sensitive K+ channels such as the BK and SK channels that will lead to membrane hyperpolarization. The BK channels are activated by Ca2+ sparks resulting from the opening of RYRs.

Step 9.  Another action of Ca2+ is to stimulate Ca2+-sensitive chloride channels (CLCA) (Liu & Farley, 1996; Haddock & Hill, 2002), which result in membrane depolarization to activate the CaV1.2 channels that introduce Ca2+ into the cell resulting in further membrane depolarization (ΔV).

Step 10. This depolarization can spread to neighbouring cells by current flow through the gap junctions to provide a synchronization mechanism in those cases where the oscillators are coupled together to provide vasomotion.

SOURCE

Smooth muscle cell calcium activation mechanisms. Berridge MJ.
J Physiol. 2008; 586(Pt 21):5047-61.   http://dx.doi.org/10.1113/jphysiol.2008.160440

Synaptotagmin functions as a Calcium Sensor

Thomas C. Südhof is at the Department of Molecular and Cellular Physiology and the Howard Hughes Medical Institute, Stanford University School of Medicine, Palo Alto, California, USA

Prof.  Thomas C. Südhof explains:

Fifty years ago, Bernard Katz’s seminal work revealed that calcium triggers neurotransmitter release by stimulating ultrafast synaptic vesicle fusion. But how a presynaptic terminal achieves the speed and precision of calcium-triggered fusion remained unknown. My colleagues and I set out to study this fundamental problem more than two decades ago.

How do the synaptic vesicle and the plasma membrane fuse during transmitter release? How does calcium trigger synaptic vesicle fusion? How is calcium influx localized to release sites in order to enable the fast coupling of an action potential to transmitter release? Together with contributions made by other scientists, most prominently James Rothman, Reinhard Jahn and Richard Scheller, and assisted by luck and good fortune, we have addressed these questions over the last decades.

As he described below, we now know of a general mechanism of membrane fusion that operates by the interaction of SNAREs (for soluble N-ethylmaleimide–sensitive factor (NSF)-attachment protein receptors) and SM proteins (for Sec1/Munc18-like proteins). We also have now a general mechanism of calcium-triggered fusion that operates by calcium binding to synaptotagmins, plus a general mechanism of vesicle positioning adjacent to calcium channels, which involves the interaction of the so-called RIM proteins with these channels and synaptic vesicles. Thus, a molecular framework that accounts for the astounding speed and precision of neurotransmitter release has emerged. In describing this framework, I have been asked to describe primarily my own work. I apologize for the many omissions of citations to work of others; please consult a recent review for additional references1.

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

Outlook

Our work, together with that of other researchers, uncovered a plausible mechanism explaining how membranes undergo rapid fusion during transmitter release, how such fusion is regulated by calcium and how the calcium-controlled fusion of synaptic vesicles is spatially organized in the presynaptic terminal. Nevertheless, many new questions now arise that are not just details but of great importance. For example, what are the precise physicochemical mechanisms underlying fusion, and what is the role of the fusion mechanism we outlined in brain diseases? Much remains to be done in this field.

How calcium controls membrane fusion

The above discussion describes the major progress that was made in determining the mechanism of membrane fusion. At the same time, my laboratory was focusing on a question crucial for neuronal function: how is this process triggered in microseconds when calcium enters the presynaptic terminal?

While examining the fusion machinery, we wondered how it could possibly be controlled so tightly by calcium. Starting with the description of synaptotagmin-1 (Syt1)5, we worked over two decades to show that calcium-dependent exocytosis is mediated by synaptotagmins as calcium sensors.

Synaptotagmins are evolutionarily conserved transmembrane proteins with two cytoplasmic C2 domains (Fig. 3a)5,6. When we cloned Syt1, nothing was known about C2 domains except that they represented the ‘second constant sequence’ in protein-kinase C isozymes. Because protein kinase C had been shown to interact with phospholipids by an unknown mechanism, we speculated that Syt1 C2 domains may bind phospholipids, which we indeed found to be the case5. We also found that this interaction is calcium dependent6,7 and that a single C2 domain mediates calcium-dependent phospholipid binding (Fig. 3b)8. In addition, the Syt1 C2 domains also bind syntaxin-1 and the SNARE complex6,9. All of these observations were first made for Syt1 C2 domains, but they have since been generalized to other C2 domains.

As calcium-binding modules, C2 domains were unlike any other calcium-binding protein known at the time. Beginning in 1995, we obtained atomic structures of calcium-free and calcium-bound Syt1 C2 domains10 in collaboration with structural biologists, primarily Jose Rizo (Fig. 3c). These structures provided the first insights into how C2 domains bind calcium and allowed us to test the role of Syt1 calcium binding in transmitter release11.

The biochemical properties of Syt1 suggested that it constituted Katz’s long-sought calcium sensor for neurotransmitter release. Initial experiments in C. elegans and Drosophila, however, disappointingly indicated otherwise. The ‘synaptotagmin calcium-sensor hypothesis’ seemed unlikely until our electrophysiological analyses of Syt1 knockout mice revealed that Syt1 is required for all fast synchronous synaptic fusion in forebrain neurons but is dispensable for other types of fusion (Fig. 4)12. These experiments established that Syt1 is essential for fast calcium-triggered release, but not for fusion as such.

Although the Syt1 knockout analysis supported the synaptotagmin calcium-sensor hypothesis, it did not exclude the possibility that Syt1 positions vesicles next to voltage-gated calcium channels (a function now known to be mediated by RIMs and RIM-BPs; see below),

with calcium binding to Syt1 performing a role unrelated to calcium sensing and transmitter release. To directly test whether calcium binding to Syt1 triggers release, we introduced a point mutation into the endogenous mouse Syt1 gene locus. This mutation decreased the Syt1 calcium-binding affinity by about twofold11. Electrophysiological recordings revealed that this mutation also decreased the calcium affinity of neurotransmitter release approximately twofold, formally proving that Syt1 is the calcium sensor for release (Fig. 5). In addition to mediating calcium triggering of release, Syt1 controls (‘clamps’) the rate of spontaneous release occurring in the absence of action potentials, thus serving as an essential mediator of the speed and precision of release by association with SNARE complexes and phospholipids (Fig. 6a,b).

It was initially surprising that the Syt1 knockout produced a marked phenotype because the brain expresses multiple synaptotagmins6. However, we found that only three synaptotagmins—Syt1, Syt2 and Syt9—mediate fast synaptic vesicle exocytosis13. Syt2 triggers release faster, and Syt9 slower, than Syt1. Most forebrain neurons express only Syt1, but not Syt2 or Syt9, accounting for the profound Syt1 knockout phenotype. Syt2 is the predominant calcium sensor of very fast synapses in the brainstem14, whereas Syt9 is primarily present in the limbic system13. Thus, the kinetic properties of Syt1, Syt2 and Syt9 correspond to the functional needs of the synapses that contain them.

Parallel experiments in neuroendocrine cells revealed that, in addition to Syt1, Syt7 functions as a calcium sensor for hormone exocytosis. Moreover, experiments in olfactory neurons uncovered a role for Syt10 as a calcium sensor for insulin-like growth factor-1 exocytosis15, showing that, even in a single neuron, different synaptotagmins act as calcium sensors for distinct fusion reactions. Viewed together with results by other groups, these observations indicated that calcium-triggered exocytosis generally depends on synaptotagmin calcium sensors and that different synaptotagmins confer specificity onto exocytosis pathways.

We had originally identified complexin as a small protein bound to SNARE complexes (Fig. 6b)16. Analysis of complexin-deficient neurons showed that complexin represents a cofactor for synaptotagmin that functions both as a clamp and as an activator of calcium-triggered fusion17. Complexin-deficient neurons exhibit a phenotype milder than that of Syt1-deficient neurons, with a selective suppression of fast synchronous exocytosis and an increase in spontaneous exocytosis, which suggests that complexin and synaptotagmins are functionally interdependent.

How does a small molecule like complexin, composed of only ~130 amino acid residues, act to activate and clamp synaptic vesicles for synaptotagmin action? Atomic structures revealed that, when bound to assembled SNARE complexes, complexin contains two short a-helices flanked by flexible sequences (Fig. 6c). One of the a-helices is bound to the SNARE complex and is essential for all complexin function18. The second a-helix is required only for the clamping, and not for the activating function of complexin17. The flexible N-terminal sequence of complexin, conversely, mediates only the activating, but not the clamping, function of the protein. Our current model is that complexin binding to SNAREs activates the SNARE–SM protein complex and that at least part of complexin competes with synaptotagmin for SNARE complex binding. Calcium-activated synaptotagmin displaces this part of complexin, thereby triggering fusion-pore opening (Fig. 6a)1,18.

REFERENCES

1. Südhof, T.C. & Rothman, J.E. Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474–477 (2009).

2. Hata, Y., Slaughter, C.A. & Südhof, T.C. Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin. Nature 366, 347–351 (1993).

3. Burré, J. et al. a-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science 329, 1663–1667 (2010).

4. Khvotchev, M. et al. Dual modes of Munc18–1/SNARE interactions are coupled by functionally critical binding to syntaxin-1 N-terminus. J. Neurosci. 27, 12147–12155 (2007).

5. Perin, M.S., Fried, V.A., Mignery, G.A., Jahn, R. & Südhof, T.C. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature 345, 260–263 (1990).

6. Li, C. et al. Ca2+-dependent and Ca2+-independent activities of neural and nonneural synaptotagmins. Nature 375, 594–599 (1995).

7. Brose, N., Petrenko, A.G., Südhof, T.C. & Jahn, R. Synaptotagmin: a Ca2+ sensor on the synaptic vesicle surface. Science 256, 1021–1025 (1992).

8. Davletov, B.A. & Südhof, T.C. A single C2-domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid-binding. J. Biol. Chem. 268, 26386–26390 (1993).

9. Pang, Z.P., Shin, O.-H., Meyer, A.C., Rosenmund, C. & Südhof, T.C. A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent SNARE-complex binding in synaptic exocytosis. J. Neurosci. 26, 12556–12565 (2006).

10. Sutton, R.B., Davletov, B.A., Berghuis, A.M., Südhof, T.C. & Sprang, S.R. Structure of the first C2-domain of synaptotagmin I: a novel Ca2+/phospholipid binding fold. Cell 80, 929–938 (1995).

11. Fernández-Chacón, R. et al. Synaptotagmin I functions as a Ca2+-regulator of release probability. Nature 410, 41–49 (2001).

12. Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994).

13. Xu, J., Mashimo, T. & Südhof, T.C. Synaptotagmin-1, -2, and -9: Ca2+-sensors for fast release that specify distinct presynaptic properties in subsets of neurons. Neuron 54, 567–581 (2007).

14. Sun, J. et al. A dual Ca2+-sensor model for neuro-transmitter release in a central synapse. Nature 450, 676–682 (2007).

15. Cao, P., Maximov, A. & Südhof, T.C. Activity-dependent IGF-1 exocytosis is controlled by the Ca2+-sensor synaptotagmin-10. Cell 145, 300–311 (2011).

16. McMahon, H.T., Missler, M., Li, C. & Südhof, T.C. Complexins: cytosolic proteins that regulate SNAP-receptor function. Cell 83, 111–119 (1995).

17. Maximov, A., Tang, J., Yang, X., Pang, Z. & Südhof, T.C. Complexin controls the force transfer from SNARE complexes to membranes in fusion. Science 323, 516–521 (2009).

18. Tang, J. et al. Complexin/synaptotagmin-1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175–1187 (2006).

19. Wang, Y., Okamoto, M., Schmitz, F., Hofman, K. & Südhof, T.C. RIM: a putative Rab3-effector in regulating synaptic vesicle fusion. Nature 388, 593–598 (1997).

20. Kaeser, P.S. et al. RIM proteins tether Ca2+-channels to presynaptic active zones via a direct PDZ-domain interaction. Cell 144, 282–295 (2011).

21. Schoch, S. et al. RIM1a forms a protein scaffold for regulating neurotransmitter release at the active zone. Nature 415, 321–326 (2002).

22. Verhage, M. et al. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287, 864–869 (2000).

 

SOURCE

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

NATURE MEDICINE | SPOONFUL OF MEDICINE

Lasker Awards go to rapid neurotransmitter release and modern cochlear implant

09 Sep 2013 | 13:38 EDT | Posted by Roxanne Khamsi | Category: Neuroscience/mental health, Odds and ends

Posted on behalf of Arielle Duhaime-RossA very brainy area of research has scooped up one of this year’s $250,000 Lasker prizes, announced today: The Albert Lasker Basic Medical Research Award has gone to two researchers who shed light on the molecular mechanisms behind the rapid release of neurotransmitters—findings that have implications for understanding the biology of mental illnesses such as schizophrenia, as well the cellular functions underlying learning and memory formation.By systematically analyzing proteins capable of quickly releasing chemicals in the brain, Genentech’s Richard Scheller and Stanford University’s Thomas Südhofadvanced our understanding of how calcium ions regulate the fusion of vesicles with cell membranes during neurotransmission. Among Scheller’s achievements is the identification of three proteins—SNAP-25, syntaxin and VAMP/synaptobrevin—that have a vital role in neurotransmission and molecular machinery recycling. Moreover, Südhof’s observations elucidated how a protein called synaptotagmin functions as a calcium sensor, allowing these ions to enter the cell. Thanks to these discoveries, scientists were later able to understand how abnormalities in the function of these proteins contribute to some of the world’s most destructive neurological illnesses. (For an essay by Südhof on synaptotagmin, click here.)The Lasker-DeBakey Clinical Medical Research Award went to three researchers whose work led to the development of the modern cochlear implant, which allows the profoundly deaf to perceive sound. During the 1960s and 1970s Greame Clark of the University of Melbourne and Ingeborg Hochmair, CEO of cochlear implant manufacturer MED-EL, independently designed implant components that, when combined, transformed acoustical information into electrical signals capable of exciting the auditory nerve. Duke University’s Blake Wilson later contributed his “continuous interleaved sampling” system, which gave the majority of cochlear implant wearers the ability to understand speech clearly without visual cues. (For a viewpoint by Graeme addressing the evolving science of cochlear implants, click here.)Bill and Melinda Gates were also honored this year with the Lasker-Bloomberg Public Service Award. Through their foundation, the couple has made large investments in helping people living in developing countries gain access to vaccines and drugs. The Seattle-based Bill & Melinda Gates Foundation also runs programs to educate women about proper nutrition for their families and themselves. The organization has a broad mandate in public health; one of its most well known projects is the development of a low-cost toilet that will have the ability to operate without water.The full collection of Lasker essays, as well as a Q&A between Lasker president Claire Pomeroy and the Gateses, can be found here.

 http://blogs.nature.com/spoonful/2013/09/lasker-awards-go-to-rapid-neurotransmitter-release-and-modern-cochlear-implant.html

Summary

Author: Larry H Bernstein, MD, FCAP

Chapter IX focused on VSM of the artery and related the action of calcium-channel blockers (CCMs) to the presynaptic interruption of synaptic-vesicle fusion necessary for CA+ release that leads to neurotransmitter secretion.  Under the circumstance neurotransmitter activation, the is VSM contraction (associated with tone).  The effect of CCB action on neurotransmitter action, there is a resultant vascular dilation facilitating flow.    In this section, we extend the mechanism to other smooth muscle related action in various organs.

[1] Neural activation of vas deferens smooth muscle cells

Noradrenaline (NA) acts by stimulating α1-adrenoreceptors to produce InsP3, which then releases Ca2+ that may induce an intracellular Ca2+ wave similar to that triggered by the ATP-dependent entry of external Ca2+. In addition, the α1-adrenoreceptors also activate the smooth muscle Rho/Rho kinase signaling pathway that serves to increase the Ca2+ sensitivity of the contractile machinery.

[2]  Urinary bladder and micturition

The bladder, which functions to store and expel urine, is surrounded by layers of detrusor SMCs. The latter have two operational modes: during bladder filling they remain relaxed but contract vigorously to expel urine during micturition. The switch from relaxation to contraction, which is triggered by neurotransmitters released from parasympathetic nerves, depends on the acceleration of an endogenous membrane oscillator that produces the repetitive trains of action potentials that drive contraction.

SMCs are activated by membrane depolarization (ΔV) that opens L-type voltage-operated channels

This mechanism of activation is also shared by [1], and uterine contraction. SMCs are activated by membrane depolarization (ΔV) that opens L-type voltage-operated channels (VOCs) allowing external Ca2+ to flood into the cell to trigger contraction. This depolarization is induced either by ionotropic receptors (vas deferens) or a membrane oscillator (bladder and uterus). The membrane oscillator, which resides in the plasma membrane,  generates the periodic pacemaker depolarizations responsible for the action potentials that drive contraction.

The main components of the membrane oscillator are the Ca2+ and K+ channels that sequentially depolarize and hyperpolarize the membrane, respectively. This oscillator generates the periodic pacemaker   depolarizations that trigger each action potential. The resulting Ca2+ signal lags behind the action potential because it spreads into the cell as a slower Ca2+ wave mediated by the type 2 RYRs.   Neurotransmitters such as ATP and acetylcholine (ACh), which are released from parasympathetic axonal varicosities that innervate the bladder, activate or accelerate the oscillator by inducing membrane depolarization (ΔV).

[3] The depolarizing signal that activates gastrointestinal, urethral and ureter SMCs is as follows:

A number of SMCs are activated by pacemaker cells such as the interstitial cells of Cajal (ICCs) (gastrointestinal and urethral SMCs) or atypical SMCs (ureter). These pacemaker cells have a cytosolic oscillator that generates the repetitive Ca2+ transients that activate inward currents that spread through the gap junctions to provide the depolarizing signal (ΔV) that triggers contraction. Our greatest interest has been in this mechanism. The rhythmical contractions of vascular, lymphatic, airway and corpus cavernosum SMCs depend on an endogenous pacemaker driven by a cytosolic Ca2+ oscillator that is responsible for the periodic release of Ca2+ from the endoplasmic reticulum. The periodic pulses of Ca2+ often cause membrane depolarization, but this is not part of the primary activation mechanism but has a secondary role to synchronize and amplify the oscillatory mechanism. Neurotransmitters and hormones act by modulating the frequency of the cytosolic oscillator.

Vascular or airway SMCs are driven by a cytosolic oscillator that generates a periodic release of Ca2+ from the endoplasmic reticulum that usually appears as a propagating Ca2+ wave.

The following points are repeated:

Step 1. The initiation and/or modulation of this oscillator depends upon the action of transmitters and hormones such as ACh, 5-HT, NA and endothelin-1 (ET-1) that increase the formation of InsP3 and diacylglycerol (DAG), both of which promote oscillatory activity.

Step 2. The oscillator is very dependent on Ca2+ entry to provide the Ca2+ necessary to charge up the stores for each oscillatory cycle. The nature of these entry mechanisms vary between cell types.

Step 3. The entry of external Ca2+ charges up the ER to sensitize the RYRs and InsP3 receptors prior to the next phase of release.

The proposed role of cyclic ADP-ribose (cADPR) in airway SMCs is consistent with this aspect of the model on the basis of its proposed action of stimulating the SERCA pump to enhance store loading and such a mechanism has been described in colonic SMCs.

Step 4. The mechanism responsible for initiating Ca2+ release may depend either on the RYRs or the InsP3 receptors (I). RYR channels are sensitive to store loading and the InsP3 receptors will be sensitized by the agonist-dependent formation of InsP3.

The global Ca2+ signal then activates contraction

Smooth muscle cell calcium activation mechanisms. Berridge MJ.
J Physiol. 2008; 586(Pt 21):5047-61. http://dx.doi.org/10.1113/jphysiol.2008.160440

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Survivals Comparison of Coronary Artery Bypass Graft (CABG) and Percutaneous Coronary Intervention (PCI) / Coronary Angioplasty

Posted in Aortic Valve: TAVR, TAVI vs Open Heart Surgery, CABG, Cardiac & Vascular Repair Tools Subsegment, Cardiac and Cardiovascular Surgical Procedures, Ecosystems & Industrial Concentration in the Medical Device Sector, Frontiers in Cardiology and Cardiovascular Disorders, ISO 10993 for Product Registration: FDA & CE Mark for Development of Medical Devices and Diagnostics, Medical Devices R&D Investment, Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, Mitral Valve: Repair and Replacement, PCI, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Stents & Tools, Valves & Tools, tagged CABG, Coronary artery bypass surgery, Drug-eluting stent, Heart disease, Houston, Percutaneous coronary intervention, Stanford University School of Medicine, Stent, Texas Heart Institute, University of Michigan on June 23, 2013| 15 Comments »

Survivals Comparison of Coronary Artery Bypass Graft (CABG) and Percutaneous Coronary Intervention (PCI) / Coronary Angioplasty

Larry H. Bernstein, MD, Writer
And
Aviva Lev-Ari, PhD, RN, Curator

 

This is a summary of several studies, mostly reviewing one decade of work at Texas Heart Institute, Houston, TX.

Seminal treatments of the evolving methods, leading to a recent review of options for

• Survival comparison of CABD vs PCI
• Mitral valve repair or mitral valve replacement for the treatment of ischemic mitral regurgitation. This might further consolidate a series of articles in these chapters.

SOURCES

1. Bypass, Angioplasty Similar in Survival 10 Years After Heart Procedures, Survival Rates Differ Little. K Doheny. WebMD Health News   Oct. 15, 2007

http://www.webmd.com/heart-disease/news/20071015/bypass-angioplasty-similar-survival

2. Will Drug-Eluting Stents Replace Coronary Artery Bypass Surgery?

RM Reul,  Tex Heart Inst J. 2005; 32(3): 323–330

3. Will Stent Revascularization Replace Coronary Artery Bypass Grafting? JM Wilson Tex Heart Inst J. 2012; 39(6): 856–859

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528239/

4. Coronary Artery Bypass Surgery versus Coronary Stenting. Risk-Adjusted Survival Rates in 5,619 Patients. RP Villlareal,V-V Lee, MA Elayda, JM Wilson.  Tex Heart Inst J. 2002; 29(1): 3–9.

http://www.ncbi.nlm.nih.gov/pubmed/11995845

5. Should all ischemic mitral regurgitation be repaired? When should we replace?  DJ LaPar, IL Kron. Curr Opin Cardiol. 2011 March; 26(2): 113–117

6. Hybrid Cath Lab Combines Nonsurgical, Surgical Treatments

http://www.dicardiology.com/article/hybrid-cath-lab-combines-nonsurgical-surgical-treatments

Bypass, Angioplasty Similar in Survival 10 Years After Heart Procedures

The survival rates 10 years after coronary artery bypass surgery and angioplasty are similar, according to a new analysis of nearly 10,000 heart patients. Five years after the procedures, 90.7% of the bypass patients and 89.7% of the angioplasty patients were still alive, says  Mark A. Hlatky, MD, senior author of the analysis and a professor of health research and policy and professor of medicine at Stanford University School of Medicine in Palo Alto.

Hlatky and colleagues stress that their analysis only applies to a select group of heart patients: those for whom either procedure would be considered a reasonable choice. For patients who are eligible for either heart intervention, “either is feasible,” Hlatky tells WebMD. The report is released early online and will be published in the Nov. 20 issue of the Annals of Internal Medicine.

CABG vs. Angioplasty

The researchers evaluated the results of 23 clinical trials in which 5,019 patients (average age 61 years; 73% men) were randomly assigned to get angioplasty with or without stents (PCI), and 4,944 were assigned to get coronary artery bypass graft surgery (CABG) In angioplasty, interventional cardiologists push a balloon-like device into the coronary arteries and inflate the balloon to widen the vessel. An expandable wire mesh tube called a stent may be inserted to keep the vessel open. Some stents are coated with drugs meant to help prevent the artery from clogging up. In 2005, about 645,000 angioplasty procedures were done in the U.S. In bypass surgery, cardiac surgeons harvest a segment of a healthy blood vessel from another part of the body and use it to bypass the clogged artery or arteries, rerouting the blood to improve blood flow to the heart. About 261,000 bypass procedures were done in the U.S. in 2005.

Findings

Besides similar survival rates overall, the researchers found no significant survival differences between the two procedures for patients with diabetes, although earlier research had seemed to favor bypass surgery. Similar numbers of patients suffered heart attacks within five years of the procedures. While 11.9 of those who got angioplasty had a heart attack within five years, 10.9% of those who got bypass did. Repeat procedures were more common in angioplasty patients. While 46.1% of angioplasty patients who didn’t get a stent needed repeat procedures, 40.1% of those who got a stent did. But just 9.8% of surgery patients needed another procedure.  The study didn’t include information on drug-coated stents.

Second Opinions

The new analysis is “very complete,” says Kim A. Eagle, MD, director of the Cardiovascular Center and Albion Walter Hewlett Professor of Internal Medicine at the University of Michigan, Ann Arbor. The study shows, he says, that if either procedure is considered appropriate for an individual patient, the decision can rest on patient attitudes and preferences. Patients preferences might be based on lower need to repeat in favor of surgery, or on avoidance of surgery in favor of angioplasty. But it is important to note, acoording to Curtis Hunter at Santa-Monica-UCLA, that the studies cover the least sick with heart disease, so the two procedures are shown to be equal in a very small subset of the patients.

Coronary Artery Bypass Surgery versus Coronary Stenting – Risk-Adjusted Survival Rates in 5,619 Patients  THIJ. 2002

We used the Texas Heart Institute Cardiovascular Research Database to retrospectively identify patients who had undergone their 1st revascularization procedure with coronary artery bypass surgery (CABG; n=2,826) or coronary stenting (n=2,793) between January 1995 and December 1999. Patients were classified into 8 anatomic groups according to the number of diseased vessels and presence or absence of proximal left anterior descending coronary artery disease. Mortality rates were adjusted with proportional hazards methods to correct for baseline differences in severity of disease and comorbidity.

We found that in-hospital mortality was significantly greater in patients undergoing CABG than in those undergoing stenting (3.6% vs 0.75%; adjusted OR 8.4; P <0.0001). At a mean 2.5-year follow-up, risk-adjusted survival was equivalent (CABG 91%, stenting 95%; adjusted OR 1.26; P = 0.06). When subgroups matched for severity of disease were compared, no differences in risk-adjusted survival were seen. A survival advantage of stenting was noted in 3 categories of patients: those >65 years of age (OR 1.33, P = 0.049), those with non-insulin-requiring diabetes (OR 2.06, P = 0.002), and those with any noncoronary vascular disease (OR 1.59, P = 0.009).

In this nonrandomized observational study, CABG had a higher periprocedural mortality rate than did percutaneous stenting. At 2.5 years, however, the survival advantage of stenting was no longer evident. These data suggest that there is no intermediate-term survival advantage of CABG over stenting in patients who have multivessel disease with lesions that can be treated percutaneously. (Tex Heart Inst J 2002;29:3–9)

Fig. 1 Adjusted and unadjusted survival rates in all patients treated with CABG or PCI-stenting
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC101260/table/t3-2/?report=previmg

TABLE III. Multivariate Correlates of Intermediate-Term (2.5-Year) Mortality
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC101260/table/t3-2/?report=previmg

Fig. 2 Adjusted odds ratios comparing the results of CABG and PCI-stenting in the 8 anatomic subgroups.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC101260/bin/2FF2.jpg

TABLE IV. Intermediate-Term (2.5-Year) Survival According to Treatment in Each of the 8 Anatomic Groups
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC101260/bin/2TT4.jpg

Fig. 3 Adjusted odds ratios comparing the results of CABG and PCI-stenting in the various prespecified subsets.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC101260/bin/2FF3.gif

Will Drug-Eluting Stents Replace Coronary Artery Bypass Surgery?

Abstract

Introduction

The growth of the PCI industry and the consequent decline in the number of patients referred for CABG has produced much speculation about the future role of each type of intervention. Because the new drug-eluting stents allow PCI to be performed with lower rates of early restenosis than do bare-metal stents or percutaneous transluminal coronary angioplasty (PTCA) alone, 2–8 some have predicted that surgical revascularization will soon be obsolete.

CABG vs Pharmaco-Therapy

Randomized clinical trials performed during the 1970s and early 1980s clearly established the advantages of CABG over medical therapy in patients with triple-vessel CAD, left main coronary artery stenosis, double-vessel CAD with proximal left anterior descending (LAD) coronary artery stenosis, or left ventricular dysfunction. Problems arose subsequently because of the limitations built into the trial so that the results were biased in favor of medical therapy.  These were:

• stringent exclusion criteria that eliminated a large percentage of potential participants
• left main CAD and an ejection fraction of less than 0.40, eliminated patients for whom CABG would have been beneficial
• the high rate of crossover from the medical to the surgical groups

The numerous technical and technological advances made since these trials were completed limit the degree to which their results resemble those of the CAD treatments used today. The maximal medical therapy used during the trials did not routinely include lipid-lowering agents, β-blockers, angiotensin-converting enzyme (ACE) inhibitors, clopidogrel, or some of the other drugs currently used for CAD. Nor did the CABG groups benefit from advances that were subsequently made in preoperative imaging, perfusion and myocardial protection, anesthesia, and perioperative and intensive care practices. CABG did not then include the use of left internal mammary artery (LIMA) grafts, much less other arterial conduits. Finally, PCIs, including balloon angioplasty and stenting, were not included in these trials.

CABG vs PTCA

Randomized trials comparing PTCA with CABG revealed dramatically higher re-intervention rates in the PTCA groups and better angina relief in the CABG groups, although there were no significant differences in death or myocardial infarction rates. The Duke database study. 9 showed better survival rates with PTCA than with CABG in patients with single-vessel CAD, whereas CABG produced better survival than did PTCA in patients with severe, triple-vessel CAD.

These results are not necessarily representative of the results obtainable today with PTCA and CABG, for several reasons.

1.  stents were not used in the PTCA patients in these trials

2.  operative mortality rates for the CABG groups were higher than the rates currently found in the Society of Thoracic Surgeons (STS) database

3.  the inclusion/exclusion criteria of these studies eliminated a high percentage of those patients who might have benefited more from CABG than from PTCA

CABG vs Stents

The introduction of coronary artery stenting resulted in better outcomes than those produced by balloon angioplasty or by other adjuncts, including rotational atherectomy, brachytherapy, and laser angioplasty.  Since then, stent designs and delivery techniques have advanced considerably. The use of coronary stents has greatly decreased the necessity of emergent CABG for technical failure of PCI and for dissection or rupture of coronary arteries during PCI. Another major advance in the application of PCI is the use of the antiplatelet agent clopidogrel in addition to aspirin after PCI, as well as the use of glycoprotein (GP) IIb/IIIa receptor inhibitors during the procedure. These adjuncts have significantly reduced the incidence of acute and subacute thrombosis after PTCA with stenting.

Randomized trials comparing PTCA plus stenting with PTCA alone have shown that stenting significantly reduces rates of restenosis and re-intervention, as well as the frequency of emergent CABG.  On the other hand, randomized trials of stenting versus surgery have produced less conclusive results regarding the mid-term survival and freedom from adverse events.  For example, the Stent or Surgery (SOS) trial reported a greater need for repeat revascularization in the stent group (21%) than in the CABG group (6%) and a survival advantage in the CABG group (hazard ratio, 2.91; 95% CI, 1.29–6.53; P = 0.01) during the 3-year follow-up period. Additionally, angina and the use of anti-angina medications were less common in the CABG group at 1-year follow-up.

The ARTS and ERACI trials also reported an increased need for revascularization in the stent groups but did not show a survival advantage in the CABG groups. This was due in part to a higher operative mortality rate in the CABG group than reported in the STS database. Like the PCI versus CABG trials mentioned previously, these randomized trials involved a select group of patients with relatively low expected mortality rates and relatively high expected technical success with PCI.

Observational data in retrospective analyses of large patient databases comparing CABG with PCI plus stenting does indicate that, because of the greater invasiveness of surgical revascularization, CABG produces greater operative mortality than does PCI. However, in patients with multivessel CAD, the risk-adjusted survival rates at 2.5 years of follow-up are no better for PCI than for CABG, and 3 recent risk-adjusted observational studies showed that the CABG patients had a significant survival advantage at 3- to 8-year follow-up.   The CABG patients had significantly more preoperative risk factors than did the PCI patients in each study, so that unadjusted, the CABG groups in each study included significantly more patients with triple-vessel disease and fewer patients with double-vessel disease than did the PCI groups. Again, we have a moving target with recent advances in both surgery and PCI technology.

Disadvantages of Stenting

The Achilles’ heel of PCI is restenosis and the need for repeat revascularization. Stents have decreased the rate of acute and subacute  periprocedural thrombosis. The newer, drug-eluting stents (DESs) have improved in-stent restenosis rates, especially in the carefully selected patient populations studied in the early DES trials. In the RAVEL trial, the early reports of zero in-stent restenosis compared favorably with the 27% in-stent restenosis rates in the bare-metal stent control group at 6-month follow-up. However, the RAVEL trial excluded patients with lesions longer than 18 mm, ostial targets, calcified or thrombosed targets, or target arteries less than 2.5 mm in diameter.

The media frenzy that followed the release of these findings created a public demand for these new “miracle” stents that apparently did not re-occlude. Stories of CAD patients refusing conventional PCI and CABG —instead, adding their names to the list of patients waiting for U.S. Food and Drug Administration (FDA) approval of DESs—appeared to change the practice patterns of cardiologists and cardiac surgeons overnight.  And then there were the calls for class-action lawsuits and recall of various DES models. After the FDA approved the Cordis Cypher™ DES (Cordis Corporation, a Johnson & Johnson company; Miami Lakes, Fla), a few reports of subacute thrombosis and hypersensitivity reactions prompted the FDA to release a public health notification on 29 October 2003.

The SIRIUS trial had slightly less strict exclusion criteria than did the RAVEL trial, admitting patients with target lesions 2.5 to 3.5 mm in diameter and 15 to 30 mm long, as well as patients with diabetes mellitus (who constituted 26% of the total group).  The SIRIUS trial also differed from the RAVEL trial in that the reported end-point was in-segment restenosis, rather than in-stent restenosis. The results showed a significant advantage of DESs over bare-metal stents for preventing in-segment restenosis (9.2% vs 32.3%) and target failures (10.5% vs 19.5%), but major adverse cardiac events were more frequent in the DES group than in the bare-metal stent group (3.7% vs 1.0%). Interestingly, the 6-month restenosis rates of the bare-metal stents in the RAVEL and SIRIUS control groups were much higher than the 19% 12-month restenosis rate associated with bare-metal stents in an earlier study comparing bare-metal stents with PTCA. In fact, the restenosis rates in the RAVEL and SIRIUS control groups more closely resembled the 40% restenosis rate reported for the PTCA control group in the earlier study.

The practical advantages of DESs over bare-metal stents are evident; nonetheless, we still do not have sufficient mid-term or long-term clinical data to argue that PTCA with DESs is preferable to CABG in “real-world” patients who require revascularization. Although DESs will likely provide better outcomes than bare-metal stents for many patients for whom stenting is indicated, a general extrapolation of existing data to justify the use of DESs in patients for whom CABG is currently indicated is unknown, perhaps undeterminable because the lesion and patient characteristics that lead to the failure of PCI are multifactorial, and the size of the population with lesions having unfavorable characteristics , such as,

• longer
• total occlusion
• branch
• small-diameter
• calcified
• multiple
• left main
• ostial, and
• diffuse lesions

are being treated with PCI more often, as well as diabetics, multiple lesions, and patients with multiple comorbidities.

Advantages of CABG

Over the last 4 decades, surgical coronary artery revascularization techniques and technology have advanced significantly. As a result, despite an increasingly older and sicker patient population, CABG outcomes continue to improve. Observed operative mortality rates have decreased because advances in preoperative evaluation, including more precise coronary artery and myocardial imaging and diagnostic techniques, have allowed more appropriate patient selection and surgical planning. In addition, preoperative, intraoperative, and postoperative monitoring and therapeutic interventions have made CABG safer, even for critically ill and high-risk patients. Improvements in cardiopulmonary perfusion and careful myocardial protection, as well as the use of off-pump and on-pump beating- heart techniques in selected patients, have also decreased perioperative morbidity and mortality rates.

LIMA-to-LAD Long-Term Patency

The long-term benefits of CABG with regard to survival and quality of life are dependent on prolonged graft patency. The LIMA-to-LAD bypass, which is now performed in more than 90% of CABG procedures, shows excellent patency in 10- to 20-year angiographic follow-up studies, setting the gold standard with which other revascularization strategies should be compared. Tatoulis et al. reported that LIMA-to-LAD grafts had a 97.1% patency rate in patients who underwent angiography for cardiac symptoms. Those authors also found high patency rates at 5-year (98%), 10-year (95%), and 15-year (88%) follow-up. However, there are not yet long-term data on bare-metal stents or DESs, and by the time 10- or 20-year data are available, DESs probably will have been replaced by a newer, more advanced technology.

Because of the reported success of the LIMA-to-LAD bypass, other types of arterial conduits are also being used much more frequently. Conduit selection has become an area of great interest to cardiac surgeons, and conduit studies are expanding our understanding of the mechanisms of graft failure and ways to improve bypass graft patency. For example, studies have shown that patients who undergo CABG with both LIMA and right internal mammary artery (RIMA) conduits have better results than those who undergo CABG with one IMA and one or more saphenous vein grafts.

Techniques to Improve Conduit Patency

To maximize the odds of long-term graft patency, surgeons carefully harvest the graft as a pedicled or skeletonized conduit using “no touch” techniques. Using careful anastomotic technique to avoid excessive turbulence at the anastomosis site will prolong graft patency, and the quality of the conduit is crucial. Long-term graft patency depends not only on the conduit chosen but also on the target artery and the degree of stenosis proximal to the anastomosis. Maintaining flow patterns in the native artery, including residual flow (that is, competitive flow) and outflow, is important to avoid stasis in the graft, turbulence at the anastomosis, and vasospasm, especially in arterial conduits. Studies have shown an inverse relationship between the degree of proximal stenosis and graft patency. Targeting the LAD produces the highest patency rates. The characteristics of the target artery also determine graft patency, including –

1. the diameter of the target artery,

2. the presence or absence of diffuse disease within the artery,

3. whether or not the artery requires endarterectomy

Surgeons can avoid atheroembolic events by handling the aorta carefully or not at all. They can also improve safety by

1. using aggressive myocardial protection techniques;

2. avoiding the induction of inflammatory mediators; and

3. carefully controlling

• blood pressure,
• body temperature, and
• electrolyte and glucose levels.

Although there have been major innovations that have enabled surgeons to perform cardiac surgery (including CABG) less invasively, minimally invasive surgical procedures are useful only if they are at least as efficacious as conventional surgery. New technology is being developed to enhance the evolving field of minimally invasive coronary bypass surgery.

Hybrid Coronary Revascularization

As PCI technology improves and techniques of LIMA-to-LAD grafting become less invasive, hybrid coronary revascularization is becoming a distinct possibility. For example, a minimally invasive, off-pump, direct LIMA-to-LAD anastomosis can be combined with DES placement in a focal mid-right-coronary-artery lesion in a patient with complex proximal LAD lesions. Hybrid coronary revascularization procedures are currently being performed, with promising early results. A few centers now have hybrid operating rooms with cardiac surgical and coronary angiographic capabilities that make it possible to perform simultaneous hybrid coronary revascularizations.

Although coronary artery bypass grafting (CABG) remains the treatment of choice for certain types of coronary artery disease (CAD), percutaneous coronary intervention (PCI)—particularly coronary angioplasty with stenting—has become the most popular nonmedical treatment approach to CAD. Some have speculated that, with the advent of drug-eluting stents (DESs), PCI will replace CABG entirely. However, the complete disappearance of CABG is both unlikely and unwarranted, for several reasons. Published randomized trials of CABG, PCI, and medical approaches to CAD compared only highly selected subgroups of patients because of strict exclusion criteria that often favored the PCI cohorts. Therefore, their results do not constitute sufficient evidence for the superiority of PCI over CABG in all CAD patients requiring revascularization. As PCI indications broaden to include more complex lesions and more high-risk patients, outcomes will not remain as favorable. In addition, although PCI is less invasive than surgery, CABG offers more complete revascularization and better freedom from repeat revascularization. Furthermore, no long-term patency data on DESs yet exist, whereas excellent 10- and 20-year patency rates have been reported for the left internal mammary artery-to-left anterior descending artery graft used in most CABG procedures. While PCI has been changing, CABG has not been stagnant; recently, advances in many aspects of the CABG procedure have improved short- and long-term outcomes in CABG patients. Both CABG and PCI technologies will continue to advance, not necessarily exclusive of one another, but no data yet exist to suggest that DESs will render CABG obsolete any time soon. 

Will Stent Revascularization Replace Coronary Artery Bypass Grafting?

When we discuss revascularization outcomes, we are talking about 3 major endpoints: death, myocardial infarction, and symptom control. With respect to death, we know that revascularization benefits patients who have severe multivessel disease and left ventricular dysfunction or other physiologic indicators of high risk. 2-vessel disease with proximal left anterior descending coronary artery (LAD) stenosis has been accepted as an indication for revascularization, even though the supporting data come from a small subgroup in a single trial. There has been no success in proving that endovascular treatment has a positive impact on stable CAD, but it is relevant because we leave the native arteries relatively intact. Attempts to improve graft performance beyond the relatively spectacular performance of the pedicled internal mammary artery (IMA) graft to the LAD have been disappointing.

Fig. 1 Graph of graft patency shows deterioration rates over 10 years and the comparative superiority of using the internal mammary artery (IMA) instead of the saphenous vein (SVG).http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528239/bin/25FF1.gif

Percutaneous Transluminal Coronary Angioplasty

When angioplasty was introduced, the hope was for a method of revascularization that would rival coronary artery bypass grafting. However, the results were mixed. Angioplasty worked well in patients with no major risk factors, such as diabetes mellitus, but failed miserably in diabetic patients. In fact, the Bypass Angioplasty Revascularization Investigation (BARI)  taught us this: if revascularization is needed, regardless of physiologic markers of high risk, the use of percutaneous coronary intervention (PCI) is potentially harmful in comparison with an IMA bypass for the LAD.

Stents and Short-Term Outcomes

The use of stents drastically reduced the probability of emergent surgery after attempted; however, the probability of new lesion formation or restenosis after intervention did not decrease.

Fig. 2 Diagrams  show the calculated success (after percutaneous revascularization) of A) percutaneous transluminal coronary angioplasty (PTCA), and B) bare-metal and C) drug-eluting stenting in patients with 3-vessel coronary artery disease (CAD).
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528239/bin/25FF2.gif

At the same time, surgeons got better. Myocardial preservation techniques improved, and the use of the pedicled IMA graft changed the game. As a result, successful revascularization, meaning long-term success, became the domain of the surgeon. We at the Texas Heart Institute/St. Luke’s Episcopal Hospital (THI/SLEH) examined our long-term outcomes after stenting or surgery, and we initially reported that stenting was just as beneficial as surgery. This was in accord with the results of several trials: whenever placing a stent was feasible, stent therapy and surgery had the same outcome.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528239/bin/25TT1.jpg

Stents and Long-Term Outcomes

Later, when we looked at longer-term follow-up data and the effects of multiple procedures, this picture began to change. Stented patients underwent more procedures. When the risk of one surgical procedure was compared with that of multiple endovascular procedures, the outcomes became more similar, especially in patients with bifurcation lesions or lesions with severe calcification. Drug-eluting stents, with their promise of no restenosis, substantially increased interventional cardiologists’ reach, but not their grasp. In patients with multivessel disease and high-risk lesions, DES placement was almost as risky as surgery and did not yield the same long-term benefit.

Nevertheless, we found locally that the introduction of the DES, with its lower risk of restenosis, was treated as a blessing to proceed with stenting (Table I). This did not follow the data, but cardiologists continued anyway, given the promise of less restenosis. Early risk was discounted, glycoprotein IIb/IIIa inhibitor use declined overnight, and the rate of endovascular procedural complications rose to meet that of surgery without the promise of an IMA graft in our future.

Table I. Independent Predictors of 30-Day Major Adverse Cardiac Events and 3-Year Survival after Drug-Eluting Stent Placement
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528239/bin/25TT1.jpg

Comparing Stenting and Surgery

For decades, methods have been sought to quantify lesion complexity in order to compare the early and late risks associated with stenting versus surgery. Although no perfect system has been devised, the SYNTAX was an important step forward. The SYNTAX score is a simple, computer-based tool for evaluating the risk of complications or failure after PCI. And there are other tools for estimating the same complications after surgery. These estimates enable cardiologists to give patients objective advice regarding the revascularization method that has the best short- and long-term probability of success.

In the patient with non-life-threatening disease (that is, not left main or severe multivessel CAD with left ventricular dysfunction or severely impaired function), stent revascularization has become a reasonable, although not ideal, alternative to surgical revascularization. However, this is true only if stenting is confined to patients whose anatomy and physiology are suited to it—considerations that are well quantified in the SYNTAX score. Whenever questions arise as to the most appropriate therapy, the SYNTAX score should be weighed against clinical characteristics that affect surgical risk. This will guide discussions between the cardiologist, cardiovascular surgeon, patient, and treating physician.

I think that our THI risk is more useful than the other available scores. It uses simple clinical data and can be easily calibrated to the geographic location of its use. Other scores require data that might not be available at the time of clinical decision-making or at all—making such predictions hazardous, at best.

Conclusion

With regard to the chosen mode of revascularization, it is perhaps safe to say that the decision goes beyond the individual physician and must become collective. When a patient has multivessel disease, a reasoned approach must be taken, using these predictive tools and considering the patient’s wishes. Treatment decisions should include all interested parties: the patient, cardiologist, cardiovascular surgeon, and anesthesiologist. The time of ad hoc angioplasty for the patient with multivessel CAD has passed.

Should all ischemic mitral regurgitation be repaired? When should we replace?   Curr Opin Cardiol. 2011

Abstract

Purpose of review

Ischemic mitral regurgitation (IMR) is a major source of morbidity and mortality. Although mitral valve repair has become recently popularized for the treatment of IMR, select patients may derive benefits from replacement. The purpose of this review is to describe current surgical options for IMR and to discuss when mitral valve replacement (MVR) may be favored over mitral valve repair.

Recent findings

Current surgical options for the treatment of IMR include surgical revascularization alone, mitral valve repair, or MVR. Although surgical revascularization alone may benefit patients with mild–moderate IMR, most surgeons advocate the performance of revascularization in combination with either mitral valve repair or replacement. In the current era, mitral valve repair has proven to offer improved short-term and long-term survival, decreased valve-related morbidity, and improved left ventricular function compared with MVR. However, MVR should be considered for high-risk patients and those with specific underlying mechanisms of IMR.

Summary

In the absence of level one evidence, mitral valve repair offers an effective and durable surgical approach to the treatment of mitral insufficiency and remains the operation of choice for IMR. MVR, however, is preferred for select patients. Future randomized, prospective clinical trials are needed to directly compare these surgical techniques.

Introduction

Ischemic mitral regurgitation (IMR) describes insufficiency of the mitral valve in the setting of myocardial ischemia, resulting from coronary artery disease. Although IMR may present in the acute setting, usually as a papillary rupture (Carpentier type II), it is usually a consequence of chronic myocardial ischemia that typically presents weeks following a complete infarction. IMR describes mitral insufficiency in the absence of degenerative (structural) mitral valve disease. The underlying pathophysiologic mechanisms of IMR are often complex, resulting from several different structural changes involving left ventricular geometry, the mitral annulus, and the valvular/subvalvular apparatus. Although changes to any one component may result in detectable mitral valve insufficiency, moderate-to-severe IMR requiring surgical correction often involves the complex interplay of several co-existent anatomic changes. These underlying mechanisms result in clinically significant valve incompetence due to the combined effects of decreased ventricular function and restricted motion of the valve itself due to tethering.

IMR is a major source of patient morbidity and mortality. Although the frequency of IMR differs based upon imaging modality, estimates have suggested that nearly 20–30% of patients experience mitral insufficiency following myocardial infarction. Furthermore, its intimate association with heart failure and poor outcomes for suboptimal medical management further complicates the management of clinically significant IMR. Recent evidence suggests that moderate or severe mitral regurgitation may be associated with a three-fold increase in the adjusted risk of heart failure and a 1.6-fold increase in risk-adjusted mortality at 5-year follow-up. In addition, unfavorable patient profiles and co-existing comorbid disease, including renal failure, chronic obstructive pulmonary disease, diabetes, and impaired left ventricular function, further complicate the clinical picture for those with IMR. Consequently, surgical correction of this condition is often required.

The purpose of this review is to analyze published results for the surgical correction of IMR and to provide current opinion regarding the selection of mitral valve procedure in the setting of myocardial ischemia. Herein, we review current surgical options for IMR and discuss when MVR may be favored over mitral valve repair.

Surgical options for ischemic mitral regurgitation: surgical revascularization alone

Surgical revascularization alone with CABG may be beneficial for some patients. Although CABG alone may be performed in cases of mild-to-moderate IMR, for the treatment of severe IMR, evidence supports performance of CABG with a mitral valve. In fact, a lack of evidence exists to support the performance of CABG alone for severe IMR. In one retrospective review of propensity-matched cohorts, Diodato et al. suggested that addition of a mitral valve procedure to patients undergoing CABG for moderately severe to severe IMR did not increase mortality or improve survival over the performance of CABG alone. This study, however, was limited by small sample sizes (51 CABG + mitral valve repair vs. 51 CABG alone) and 3-year follow-up. To the contrary, substantial evidence exists to support the performance of surgical revascularization alone in cases of mild-to-moderate IMR.

A study by Aklog et al. investigated the role of CABG alone in the correction of moderate IMR. In their series of 136 patients with moderate IMR, they demonstrated that performance of revascularization alone conferred improvement of mitral regurgitation in 51% of patients with complete resolution in an additional 9%. Despite these results, 40% of patients remained with 3–4+ mitral regurgitation, leading the authors to conclude that CABG alone may not be the optimal therapy for most patients and suggest that concomitant mitral annuloplasty may improve results. Other series similarly suggest that complete resolution of functional IMR is uncommon following revascularization alone. Despite the presence of residual mitral regurgitation following revascularization, the impact of performance of CABG without a valve procedure on long-term survival remains ill defined. Currently, on-going prospective evaluation may help to define the potential role of revascularization alone for patients with moderate IMR. Until the completion of these trials, however, evidence supports the performance of surgical revascularization combined with a mitral valve procedure for moderate-to-severe mitral regurgitation.

Surgical revascularization with a mitral valve procedure

The majority of patients with moderate-to-severe IMR require surgical revascularization with a concomitant mitral valve procedure (MVR or mitral valve repair). Historically, these procedures have been associated with high morbidity and mortality as well as poor long-term. However, improved surgical techniques and postoperative management have improved contemporary outcomes. Those favoring mitral valve repair promote its beneficial effects on survival, preserved ventricular function, and the avoidance of long-term anticoagulation, whereas those favoring MVR argue that it ensures long-term freedom from recurrent mitral insufficiency.

Mitral valve replacement vs. mitral valve repair

The use of MVR for IMR eliminates the possibility of recurrent IMR. In addition, previous literature suggests improvements in surgical technique for MVR 29–32. For patients with IMR, MVR with preservation of the subvalvular apparatus using a chordal sparing technique has been shown to be beneficial 33. David and Ho 33 demonstrated a significant survival benefit for patients undergoing MVR with preservation of chordae tendineae (89%) compared with complete excision of the mitral valves (59%) in a cohort of 51 patients with IMR. In addition, Cohn et al. suggested disproportionate survival benefits favoring MVR in a cohort of 150 patients with both functional and structural IMR, concluding that survival following performance of mitral valve procedures for IMR was more dependent on underlying pathophysiology rather than surgical technique. More recently, series have suggested equivalent results for the MVR and mitral valve repair. Mantovani et al. report that prosthetic MVR and mitral valve repair offer very similar results for chronic IMR, demonstrating similar operative mortality and 5-year actuarial survival for both techniques. In a similar report, Magne et al.•• compared short-term and long-term outcomes for 370 patients undergoing mitral valve repair (n = 186) and MVR (n = 184) for IMR. Although operative mortality was lower for mitral valve repair compared with MVR (9.7 vs. 17.4%, P = 0.03), 6-year survival was similar for both operations (73 ± 4 vs. 67 ± 4%, P = 0.17). Type of procedure was also not an independent predictor of mortality following risk adjustment. As a result, the authors suggest that mitral valve repair is not superior to MVR for patients with IMR.

In contrast, other series favor the performance of mitral valve repair for functional IMR. Although several repair techniques exist, restrictive annuloplasty remains the most commonly performed operation 37• and has been shown to be beneficial in both functional and chronic IMR 38•. The purported benefits of improved survival, decreased valve-related morbidity, and improved left ventricular function have been previously established, and several series have reported lower hospital mortality with mitral valve repair compared with MVR.

The Cleveland Clinic published a landmark review of 482 patients undergoing mitral valve procedures for IMR to study the influence of mitral valve procedure type on survival 1. In this series, propensity-matched cohorts were compared: mitral valve repair (n = 397) vs. MVR (n = 85). Concomitant CABG was performed in 95% of operations, and annuloplasty for repair occurred in 98% of cases. After matching, patients were risk stratified into five quintiles. Group 1 represented the highest-risk patients with higher degrees of heart failure and emergent operations, and group 5 represented the lowest-risk patients. Subsequent survival analysis revealed that overall 5-year survival was poor for patients with IMR (58% mitral valve repair vs. 36% MVR, P = 0.08). Moreover, within matched quintiles, the highest-risk patients (quintile 1) had the worst survival, but survival was similar (P = 0.4) despite mitral valve procedure type. In contrast, survival favored mitral valve repair over replacement for quintiles III–V (P = 0.003).

In the absence of published randomized trials, two recently published meta-analyses provide more robust comparisons of the influence of surgical mitral valve repair or replacement. Shuhaiber and Anderson  compared outcomes of 29 studies, including over 10 000 patients. Study groups were stratified based upon mitral valve etiology into ischemic, degenerative/myxomatous, rheumatic, and mixed groups. Summary analyses indicated worse overall survival for MVR (early mortality odds ratio = 2.24 and total survival hazard ratio = 1.58) compared with repair. Mitral valve repair was also associated with lower rates of thromboembolism. Moreover, a nonsignificant trend toward lower 30-day mortality favored mitral valve repair for those with IMR. The most recent meta-analysis to date compared short-term and long-term survival of mitral valve repair vs. replacement specifically for IMR ••. In this analysis, nine studies were included based upon stringent exclusion criteria to ensure direct comparisons of survival for mitral valve procedures exclusively performed for IMR. Interestingly, in this series, although patients undergoing MVR were older, those undergoing repair often had higher rates of hypertension and diabetes with lower ejection fractions. Further, the proportion of patients with severe ventricular dysfunction was similar between procedure groups. These findings conflict with a common assumption that an inherent selection bias exists within published studies for the performance of mitral valve repair in healthier patients. Nevertheless, MVR was associated with worse short-term mortality (odds ratio = 2.667) and long-term mortality (hazard ratio = 1.35) compared with mitral valve repair, and the authors advocate that choice in mitral procedure should be based upon individual patient profile.

When not to repair ischemic mitral regurgitation?

Within the context of published literature and current dogma among practicing surgeons, the fundamental question of when not to repair an ischemic mitral valve remains. For several years, accumulated evidence supports the performance of mitral valve repair over replacement for the surgical treatment of functional IMR. The aforementioned benefits of repair include improved long-term survival, durability and efficacy, improved ventricular function, and avoidance of chronic anticoagulation therapy. Nevertheless, MVR still plays a select role in the treatment of IMR.

With respect to the performance of MVR, the use of bioprosthetic valves and the avoidance of mechanical valve replacement are preferred. This choice is largely driven by the avoidance of complications due to long-term anticoagulation use as well as by the belief that it is unlikely that the majority of patients requiring MVR are likely to encounter bioprosthetic deterioration in their lifetime. In addition, MVR with techniques to preserve the subvalvular apparatus should be performed when possible.

Summary

Undoubtedly, the debate regarding when to perform repair or replacement for IMR remains unsettled. In the recent era, mitral valve repair has proven efficacious and remains the preferred surgical strategy for most cases of IMR. MVR should be considered for severe tethering, complex or uncertain mechanisms of mitral insufficiency, regurgitation due to papillary muscle rupture, and perhaps for the sickest and highest-risk patients.

The present review was supported by Award Number 2T32HL007849-11A1 (D.J.L.) from the National Heart, Lung, and Blood Institute. The content is solely the responsibility of the authors.

Hybrid Cath Lab Combines Nonsurgical, Surgical Treatments  2008

A new cardiac treatment facility that couples the benefits of interventional cardiology with cardiothoracic surgery for critically ill newborns, children and adults has opened at Rush University Medical Center, Chicago.  Toshiba’s new biplane hybrid cardiac suite, which is one of only three facilities of its kind in the U.S., is equipped with the latest in continuous, real-time imaging technology and radio frequency identification (RFID) technology which allows “all-in-one-room” care. The suite allows collaboration between the surgeon and interventional cardiologist on complex heart problems. For example, fixing a very large hole in the heart can be done by inserting a catheter through a small incision in the chest rather than relying on major surgery to open the chest to reach the heart. “Now, interventional cardiologists and cardiothoracic surgeons working together in this suite will reduce the amount of time required to correct complex heart problems and reduce the emotional and physical stress placed on a patient and their family – which translates into less pain, less scarring and a faster recovery time,” Ziyad Hijazi, M.D., director of the new Rush Center for Congenital and Structural Heart Disease. The hybrid suite is equipped with the latest technology for minimally invasive interventional cardiology that involves the use of a catheter and an image-guidance system to thread tiny instruments through blood vessels to repair the heart. Through these special catheters, physicians at Rush can implant stents, artificial heart valves and insert patches for holes in the heart. In many complex cardiac cases, patients who would otherwise have no other option but to undergo open-heart bypass surgery can now have minimally invasive procedures that would otherwise not be available to them. “We can now communicate with colleagues and obtain their expertise in real time for very complex situations,” said Dr. Hijazi. “If physicians decide another procedure is needed, even surgery, the suite can be converted into an operating room and the surgical team can be assembled in the new suite ”Patients at Rush will stay in one place in the new hybrid cardiac suite where all the imaging technology and implantable devices that might be needed are stored and located. The additional ability it gives us to provide surgical treatments allows us to provide the most comprehensive care in the most sensitive manner for patients with often extremely fragile conditions.”  The new hybrid cardiac catheterization suite has the most advanced imaging technologies and can still get a precise, optimal image of any region of the heart regardless of the size or complexity of congenital heart disease. The imaging system also features eight-inch cardiac flat panel detectors designed to deliver distortion-free images. The suite also includes intravascular ultrasound machines, which takes real-time images to allow physicians to see the progress of the procedure taking place inside the patient’s body. A high-tech, automated clinical resource management system located in the suite stores and tracks the medication, surgical tools, medical devices, and implantable devices and supplies using the latest RFID enabled technology.

Hybrid Cath Lab/ORs Are the Way of the Future

Recent developments in cardiac surgery and interventional cardiology with new percutaneous alternatives for aneurysm repair, valve replacements, shunt closure devices and aortic arch reconstruction have led to the creation of integrated, hybrid cath lab/operating rooms (OR) that allow both surgical and intravascular procedures. These rooms offer both surgical equipment and high-end angiographic equipment. Creating such rooms requires special planning and design from both surgical and interventional cardiologists working closely together. Cath labs have high-quality fluoroscopy equipment, but generally are smaller rooms and lack the sterile requirements and equipment needed for surgical procedures. ORs tend to use lower quality mobile C-arms, which are not ideal for interventional procedures. The hybrids aim to provide the best of both worlds. The trend toward hybrid labs has been reinforced by digital angiography manufacturers partnering with surgical equipment companies to create easy-to-integrate hybrid room solutions with coordinated installation. Philips partners with both Skytron and Steris. Toshiba partners with MAQUET. GE Healthcare, Siemens and Toshiba also offer hybrid installations. Philips said while some hospitals want to combine interventional procedures with minimally invasive surgeries, they also want a properly equipped room in case emergency surgery is needed.

Philips said hybrids also allow hospitals with lower PCI numbers to get a bigger bang for their buck by allowing the same room to serve the needs of surgeons. Penn Presbyterian Medical Center in Philadelphia, PA, created a hybrid lab with help from Siemens, which opened in November. Wilson Szeto, M.D., cardio-thoracic surgeon, and William Matthai, M.D., interventionalist, both from Penn Presbyterian said hybrid labs are ideally suited for procedures that require both percutaneous and surgical interventions, percutaneous valve replacements, deploying percutaneous septal occluders or installing aortic stent grafts. Interventionalists can also be called in after cardiac surgery to perform a completion angiography.

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37. Banning AP, Westaby S, Morice MC, Kappetein AP, Mohr FW, Berti S, et al. Diabetic and nondiabetic patients with left main and/or 3-vessel coronary artery disease: comparison of outcomes with cardiac surgery and paclitaxel-eluting stents. J Am Coll Cardiol 2010;55(11):1067–75. [PubMed]

38. Laham RJ, Carrozza JP, Berger C, Cohen DJ, Kuntz RE, Baim DS. Long-term (4- to 6-year) outcome of Palmaz-Schatz stenting: paucity of late clinical stent-related problems. J Am Coll Cardiol 1996;28(4):820–6. [PubMed]

39. Rodriguez A, Bernardi V, Navia J, Baldi J, Grinfeld L, Martinez J, et al. Argentine Randomized Study: Coronary Angioplasty with Stenting versus Coronary Bypass Surgery in patients with Multiple-Vessel Disease (ERACI II): 30-day and one-year follow-up results. ERACI II Investigators [published erratum appears in J Am Coll Cardiol 2001;37(3):973–4]. J Am Coll Cardiol 2001;37(1):51–8. [PubMed]

40. Serruys PW, Unger F, Sousa JE, Jatene A, Bonnier HJ, Schonberger JP, et al. Comparison of coronary-artery bypass surgery and stenting for the treatment of multivessel disease. N Engl J Med 2001;344(15):1117–24. [PubMed]

41. Goy JJ, Kaufmann U, Goy-Eggenberger D, Garachemani A, Hurni M, Carrel T, et al. A prospective randomized trial comparing stenting to internal mammary artery grafting for proximal, isolated de novo left anterior coronary artery stenosis: the SIMA trial. Stenting vs Internal Mammary Artery. Mayo Clin Proc 2000;75(11):1116–23. [PubMed]

42. SoS Investigators. Coronary artery bypass surgery versus percutaneous coronary intervention with stent implantation in patients with multivessel coronary artery disease (the Stent or Surgery trial): a randomised controlled trial. Lancet 2002;360 (9338):965–70. [PubMed]

43. Reul RM. Will drug-eluting stents replace coronary artery bypass surgery? Tex Heart Inst J 2005;32(3):323–30. [PMC free article] [PubMed]

44. Sianos G, Morel MA, Kappetein AP, Morice MC, Colombo A, Dawkins K, et al. The SYNTAX Score: an angiographic tool grading the complexity of coronary artery disease. EuroIntervention 2005;1(2):219–27. [PubMed]

45. Madan P, Elayda MA, Lee VV, Wilson JM. Predicting major adverse cardiac events after percutaneous coronary intervention: the Texas Heart Institute risk score. Am Heart J 2008; 155(6):1068–74. [PubMed]

46. Gillinov AM, Wierup PN, Blackstone EH, et al. Is repair preferable to replacement for ischemic mitral regurgitation? J Thorac Cardiovasc Surg. 2001;122:1125–1141. [PubMed]

47. Grigioni F, Enriquez-Sarano M, Zehr KJ, et al. Ischemic mitral regurgitation: long-term outcome and prognostic implications with quantitative Doppler assessment. Circulation. 2001;103:1759–1764. [PubMed]

48. Lamas GA, Mitchell GF, Flaker GC, et al. Clinical significance of mitral regurgitation after acute myocardial infarction. Survival and Ventricular Enlargement Investigators. Circulation. 1997;96:827–833. [PubMed]

49. Bursi F, Enriquez-Sarano M, Nkomo VT, et al. Heart failure and death after myocardial infarction in the community: the emerging role of mitral regurgitation. Circulation. 2005;111:295–301. [PubMed]

50. Adams DH, Filsoufi F, Aklog L. Surgical treatment of the ischemic mitral valve. J Heart Valve Dis. 2002;11 (Suppl 1):S21–S25. [PubMed]

51. Filsoufi F, Salzberg SP, Adams DH. Current management of ischemic mitral regurgitation. Mt Sinai J Med. 2005;72:105–115. [PubMed]

52. Micovic S, Milacic P, Otasevic P, et al. Comparison of valve annuloplasty and replacement for ischemic mitral valve incompetence. Heart Surg Forum. 2008;11:E340–E345. [PubMed]

53. Aklog L, Filsoufi F, Flores KQ, et al. Does coronary artery bypass grafting alone correct moderate ischemic mitral regurgitation? Circulation. 2001;104 (12 Suppl 1):I68–I75. [PubMed]

54. Lam BK, Gillinov AM, Blackstone EH, et al. Importance of moderate ischemic mitral regurgitation. Ann Thorac Surg. 2005;79:462–470. discussion 462–470. [PubMed]

55. Ryden T, Bech-Hanssen O, Brandrup-Wognsen G, et al. The importance of grade 2 ischemic mitral regurgitation in coronary artery bypass grafting. Eur J Cardiothorac Surg. 2001;20:276–281. [PubMed]

56•. Goland S, Czer LS, Siegel RJ, et al. Coronary revascularization alone or with mitral valve repair: outcomes in patients with moderate ischemic mitral regurgitation. Tex Heart Inst J. 2009;36:416–424. This series documents current outcomes for the performance of CABG alone with/without concomitant mitral valve repair for ischemic mitral regurgitation. The authors report similar 5-year survival rates for both techniques; however, revascularization with repair resulted in significantly reduced mitral regurgitation grade, improved left ventricular function, and functional class compared with revascularization alone. This study provides an important comparison of these two techniques in the current surgical era. [PMC free article] [PubMed]

57••. Magne J, Girerd N, Senechal M, et al. Mitral repair versus replacement for ischemic mitral regurgitation: comparison of short-term and long-term survival. Circulation. 2009;120(11 Suppl):S104–S111. In this study, the authors compare postoperative outcomes for mitral valve repair and replacement for ischemic mitral regurgitation. Despite lower operative mortality following mitral valve repair, long-term survival was equivalent between surgical groups. This study adds important long-term comparisons of mitral valve procedures to accumulating data examining surgical treatments for ischemic mitral regurgitation. [PubMed]

58. Silberman S, Klutstein MW, Sabag T, et al. Repair of ischemic mitral regurgitation: comparison between flexible and rigid annuloplasty rings. Ann Thorac Surg. 2009;87:1721–1726. discussion 1726–1727. This study provides a contemporary comparison between the use of flexible and rigid annuloplasty rings for the surgical treatment of IMR. The authors report significantly improved clinical and hemodynamic results for rigid mitral annuloplasty rings compared with flexible rings. [PubMed]

59•. Tekumit H, Cenal AR, Uzun K, et al. Ring annuloplasty in chronic ischemic mitral regurgitation: encouraging early and midterm results. Tex Heart Inst J. 2009;36:287–292. This study reports early and midterm results for the use of flexible annuloplasty rings for the surgical treatment of chronic IMR. The authors demonstrate that use of flexible mitral valve annuloplasty conferred a reduction in left ventricular diameter with improved New York Heart Association functional class. This study reports current, encouraging results and provides a context for future investigations comparing flexible and rigid annuloplasty rings for chronic IMR. [PMC free article] [PubMed]

60. Shuhaiber J, Anderson RJ. Meta-analysis of clinical outcomes following surgical mitral valve repair or replacement. Eur J Cardiothorac Surg. 2007;31:267–275. [PubMed]

61••. Vassileva CM, Boley T, Markwell S, Hazelrigg S. Meta-analysis of short-term and long-term survival following repair versus replacement for ischemic mitral regurgitation. Eur J Cardiothorac Surg. 2010 [Epub ahead of print] This meta-analysis provides a comparison of nine published series specifically addressing the performance of mitral valve repair vs. replacement for IMR. The authors demonstrate worse short-term and long-term mortality for MVR. Their analysis offers an up-to-date and robust comparison of these two surgical techniques. [PubMed]

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Diagram of coronary angioplasty and stent placement (Photo credit: Wikipedia)

Denton A. Cooley, MD (Photo credit: Wikipedia)

English: A thoracic surgeon performs a mitral valve replacement at the Fitzsimons Army Medical Center. Slovenščina: Kirurgi med operacijo. (Photo credit: Wikipedia)

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A Blood Test to Identify Aggressive Prostate Cancer: a Discovery @ SRI International, Menlo Park, CA

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Interviews with Scientific Leaders, tagged Benign prostatic hyperplasia, BPH, Prostate, Prostate cancer, Prostate-specific antigen, PSA, Stanford University School of Medicine on May 16, 2013| 5 Comments »

A Blood Test to Identify Aggressive Prostate Cancer: a Discovery @ SRI International, Menlo Park, CA

Reporter: Aviva Lev-Ari, PhD, RN

• 

Dr. Lev-Ari was Director @ SRI International in the mid 1980s.

Denong Wang

Distinguished Scientist and Senior Program Director, Tumor Glycomics Laboratory, Center for Cancer and Metabolism

 

Denong Wang, Ph.D., is an SRI distinguished scientist and senior program director of the Tumor Glycome Laboratoryin the Center for Cancer and Metabolism in SRI Biosciences. Wang’s long-term research interest is in the carbohydrate moieties that are critical for self/non-self recognition and induction of antibody responses.

Wang’s team has established multiple platforms of carbohydrate microarrays and introduced these glycomics tools to explore the structural and antigenic diversities of the glycome. The main research focus of his lab is in the immunogenic sugar moieties. In the past few years, his group has contributed to the identification of immunologically potent glycan markers of SARS-CoV, Bacillus anthracis exosporium, and a number of human cancers.

Wang received his Ph.D. in immunology and glycobiology with the late Professor Elvin A. Kabat at Columbia University in 1993. After that, he entered the developing field of post-genomics research. Before joining SRI in 2010, he served as head of the Functional Genomics Division at Columbia University’s Genome Center from 1998 to 2003 and was director of Stanford University’s Tumor Glycome Laboratory from 2007 to 2010.

 http://www.sri.com/about/people/denong-wang

 

SRI International

A Blood Test to Identify Aggressive Prostate Cancer

SRI Blog

 

 

A Blood Test to Identify Aggressive Prostate Cancer

By Denong Wang at 9:15 AM PDT, Wed May 8, 2013

Comments

0

Prostate cancer is the second most common cancer in American men, killing nearly 30,000 per year. In 2004, I attended a conference where one of the nation’s leading researchers in the field declared that the gold-standard test for this disease was not successful at identifying dangerous invasive tumors. That triggered my interest in how to address the challenge of developing a blood test to detect the deadly form of prostate cancer.

After nearly a decade, my collaborators and I have found the first marker that specifically identifies the approximately six to eight percent of prostate cancers that are considered “aggressive,” meaning they will migrate to other parts of the body, at which point they are very difficult to treat. Although we have confirmed this marker, there is much to be done before a clinical application can be developed.

If further study confirms that the test is clinically reliable, it can provide a much-needed tool to differentiate between aggressive cancer and the majority of cases, which are slow-growing tumors with a low probability of migrating to other parts of the body (and thus don’t require special treatment, such as radical prostatectomy).

The current standard test looks at elevated blood prostate-specific antigen (PSA) levels, known as the PSA test. Dr. Thomas Stamey, an emeritus faculty member and urologist at the Stanford University School of Medicine, published his original findings in 1987 linking elevated blood PSA levels to prostate cancer. In 2004, Dr. Stamey declared that the PSA test was no longer useful for the diagnosis of prostate cancer. Rather, an elevated PSA level is now known to reflect the volume increase of a prostate, which could either be associated with a harmless increase in prostate size called benign prostatic hyperplasia (BPH), or be caused by cancer.

I began collaborating with Dr. Stamey and his Stanford colleague Dr. Donna Peehl to look for a new prostate cancer marker, hopefully one that would indicate the presence of aggressive prostate cancer through a blood test.  This is a very active area of research, with scientists exploring the idea from (1) a genomics perspective, (2) a proteomics perspective, and (3) a glycomics perspective, the latter of which entails using carbohydrate-based markers to identify cancer. My focus is the third area, where we are concentrating on how the immune system recognizes changes in the carbohydrates found on the surface of cancer cells compared with those on the surface of normal cells.  

SRI’s Tumor Glycome Laboratory has discovered a marker that appears to be associated with aggressive prostate cancer. The marker is an antibody that is produced against a carbohydrate molecule on the surface of aggressive prostate cancer cells, and is expressed in increasing levels that correlate with cancer severity. We call it a “cryptic” biomarker, since it only becomes an immunological target if something goes awry in the cell, such as a viral infection or the malignant transformation of normal cells to cancer.

This biomarker has the potential, with further development, to be used as a test to help diagnose aggressive prostate cancer. It is rewarding to have reached this point in our understanding of prostate cancer and toward a diagnostic test that ultimately could save lives.

Our research findings were published last year in the Journal of Proteomics & Bioinformatics (5:090-095, DOI:10.4172/jpb.1000218). Our latest study, published in Drug Development Research, lays the foundation for predicting which prostate cancer patients may develop more aggressive forms of the disease and directs the future design of more effective treatments [14(2):65-80, DOI: 10.1002/ddr.21063].

Anti‐Oligomannose Antibodies as Potential Serum Biomarkers of Aggressive Prostate Cancer

Abstract

This study bridges a carbohydrate microarray discovery and a large‐scale serological validation of anti‐oligomannose antibodies as novel serum biomarkers of aggressive prostate cancer (PCa). Experimentally, a Man9‐cluster‐specific enzyme‐linked immunosorbent assay was established to enable sensitive detection of anti‐Man9 antibodies in human sera. A large‐cohort of men with PCa or benign prostatic hyperplasia (BPH) whose sera were banked at Stanford University was characterized using this assay. Subjects included patients with 100% Gleason grade 3 cancer (n = 84), with Gleason grades 4 and/or 5 cancer (n = 204), and BPH controls (n = 135). Radical prostatectomy Gleason grades and biochemical (PSA) recurrence served as key parameters for serum biomarker evaluation. It was found that IgG^Man9 and IgM^Man9 were widely present in the sera of men with BPH, as well as those with cancer. However, these antibody reactivities were significantly increased in the subjects with the largest volumes of high grade cancer. Detection of serum IgG^Man9 and IgM^Man9 significantly predicted the clinical outcome of PCa post‐radical prostatectomy. Given these results, we suggest that IgG^Man9 and IgM^Man9 are novel serum biomarkers for monitoring aggressive progression of PCa. The potential of oligomannosyl antigens as targets for PCa subtyping and targeted immunotherapy is yet to be explored.

Authors:		Denong Wang, Laila Dafik, Rosalie Nolley, Wei Huang, Russell D. Wolfinger, Lai‐Xi Wang, Donna M. Peehl
Journal:		Drug Development Research
Year:		2013
Pages:		n/a
DOI:		10.1002/ddr.21063
Publication date:		11-02-2013

http://www.bionity.com/en/publications/522994/anti-oligomannose-antibodies-as-potential-serum-biomarkers-of-aggressive-prostate-cancer.html

 

Proteomics & Bioinformatics

N-glycan Cryptic Antigens as Active Immunological Targets in Prostate

Cancer Patients

Denong Wang*

Tumor Glycomics Laboratory, Center for Cancer Research, Biosciences Division, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025, USA

*Corresponding author: Dr. Denong Wang, Tumor Glycomics Laboratory,

Biosciences Division, SRI International, 333 Ravenswood Avenue, Menlo

Park, CA 94025, USA, Tel: +1 650 859-2789; Fax: +1 650 859-3153; E-mail:

denong.wang@sri.com

Received March 07, 2012; Accepted April 13, 2012; Published April 30, 2012

Citation: Wang D (2012) N-glycan Cryptic Antigens as Active Immunological

Targets in Prostate Cancer Patients. J Proteomics Bioinform 5: 090-095.

doi:10.4172/jpb.1000218

Copyright: © 2012 Wang D.

Abstract

Although tumor-associated abnormal glycosylation has been recognized for decades, information regarding host recognition of the evolving tumor glycome remains elusive. We report here a carbohydrate microarray analysis of a number of tumor-associated carbohydrates for their serum antibody reactivities and potential immunogenicity in humans. These are the precursors, cores and internal sequences of N-glycans. They are usually masked by other sugar moieties and belong to a class of glyco-antigens that are normally “cryptic”. However, viral expression of these carbohydrates may trigger host immune responses. For examples, HIV-1 and SARS-CoV display Man9 clusters and tri- or multi-antennary type II (Galβ1→4GlcNAc) chains (Tri/m-II), respectively; viral neutralizing antibodies often target these sugar moieties. We asked, therefore, whether prostate tumor expression of corresponding carbohydrates triggers antibody responses in vivo. Using carbohydrate microarrays, we analyzed a panel of human sera, including 17 samples from prostate cancer patients and 12 from men with Benign Prostatic Hyperplasia (BPH).

We observed that IgG antibodies targeting the Man9- or Tri-/m-II-autoantigens are readily detectable in the sera of men with BPH, as well as those with cancer. Importantly, these antibody activities were selectively increased in prostate cancer patients. Thus, human immune systems actively recognize these N-glycan cryptic carbohydrates and produce targeting antibodies. This finding shads a light on a class of previously less studied immunological targets of human cancers. Identifying the diagnostic, prognostic and therapeutic values of these targets will require further investigation.

http://www.omicsonline.org/0974-276X/JPB-05-090.pdf

 

 

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CD47: Target Therapy for Cancer

Posted in Advanced Drug Manufacturing Technology, Biomarkers & Medical Diagnostics, BioSimilars, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Prevention: Research & Programs, Cell Biology, Signaling & Cell Circuits, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Drug Delivery Platform Technology, Pharmaceutical R&D Investment, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Technology Transfer: Biotech and Pharmaceutical, tagged Antibody, Cancer - General, CD47, CD47-SIRPα pathway, Chemotherapy, Integrins, macrophages, monoclonal antibody, Phagocytic cells, PI3K/AKT pathway, radiotherapy, Signal-regulatory protein alpha, Signal-regulatory protein-alpha (SIRPα), Stanford University, Stanford University School of Medicine, Thrombospondin 1, Thrombospondin-1 (TSP-1), Tilda Barliya, Trastuzumab on May 7, 2013| 7 Comments »

CD47: Target Therapy for Cancer

Author/Curator: Tilda Barliya

• 

“A research team from Stanford University’s School of Medicine is now one step closer to uncovering a cancer treatment that could be applicable across the board in killing every kind of cancer tumor” (1). It appeared that their antibody-drug against the CD47 protein, enabled the shrinking of all tumor cells. After completing their animal studies the researchers now move into a human phase clinical trials. CD47 has been previously studied and evaluated for its role in multiple cells, some of this data however, is somewhat controversy. So where do we stand?

CD47

CD47 (originally named integrin-associated protein (IAP)) is a cell surface protein of the immunoglobulin (Ig) superfamily, which is heavily glycosylated and expressed by virtually all cells in the body and overexpressed in many types of cancer  including breast, ovarian, colon, prostate and others (3). CD47 was first recognized as a 50 kDa protein associated and copurified with the  Alpha-v-Beta-3 integrin in placenta and neutrophil granulocytes and later shown to have the capacity to regulate integrin function and the responsiveness of leukocytes to RGD-containing extracellular matrix proteins. CD47 has also been shown to be identical to the OA-3/OVTL3 antigen highly expressed on most ovarian carcinomas (4,5).

CD47 consists of an extracellular IgV domain, a five times transmembrane-spanning domain, and a short alternatively spliced cytoplasmic tail. In both humans and mice, the cytoplasmic tail can be found as four different splice isoforms ranging from 4 to 36 amino acids, showing different tissue expression patterns (3).

CD47 interactions (3, 6):

• Thrombospondin-1 (TSP-1) – a secreted glycoprotein that plays a role in vascular development and angiogenesis. Binding of TSP-1 to CD47 influences several fundamental cellular functions including cell migration and adhesion, cell proliferation or apoptosis, and plays a role in the regulation of angiogenesis and inflammation.
• Signal-regulatory protein-alpha (SIRPα) – an inhibitory transmembrane receptor present on myeloid cells. The CD47/SIRPα interaction leads to bidirectional signaling, resulting in different cell-to-cell responses including inhibition of phagocytosis, stimulation of cell-cell fusion, and T-cell activation.
• Integrins – several membrane integrins, most commonly integrin avb3. These interactions result in CD47/integrin complexes that effect a range of cell functions including adhesion, spreading and migration

These interactions with multiple proteins and cells types create several important functions, which include:

• Cell proliferation – cell proliferation is heavily dependent on cell type as both activation and loss of CD47 can result in enhanced proliferation. For example, activation of CD47 with TSP-1 in wild-type cells inhibits proliferation and reduces expression of stem cell transcription factors. In cancer cells however, activation of CD47 with TSP-1 increases proliferation of human U87 and U373 astrocytoma. it is likely that CD47 promotes proliferation via the PI3K/Akt pathway in cancerous cells but not normal cells (7).  Loss of CD47 allows sustained proliferation of primary murine endothelial cells and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters (8).
  
• Apoptosis – Ligation of CD47 by anti-CD47 mAbs was found to induce apoptosis in a number of different cell types (3). For example: Of the two SIRP-family members known to bind the CD47 IgV domain (SIRPα and SIRPγ), SIRPα as a soluble Fc-fusion protein does not induce CD47-dependent apoptosis, hile SIRPα or SIRPγ bound onto the surface of beads induces apoptosis through CD47 in Jurkat T cells and the myelomonocytic cell line U937.
• Migration – CD47  role on cell migration was first demonstrated in neutrophils, these effects were shown to be dependent on avb3 integrins, which interact with and are activated by CD47 at the plasma membrane. In cancer, Blocking CD47 function has been shown to inhibit migration and metastasis in a variety of tumor models. Blockade of CD47 by neutralizing antibodies reduced migration and chemotaxis in response to collagen IV in melanoma, prostate cancer and ovarian cancer-derived cells (9).
• Angiogenesis – The mechanism of the anti-angiogenic activity of CD47 is not fully understood, but introduction of CD47 antibodies and TSP-1 have been shown to inhibit nitric oxide (NO)-stimulated responses in both endothelial and vascular smooth muscle cells (10). More so, CD47 signaling influences the SDF-1 chemokine pathway, which plays a role in angiogenesis (11). (12)
• Inflammatory response – Interactions between endothelial cell CD47 and leukocyte SIRPγ regulate T cell transendothelial migration (TEM) at sites of inflammation. CD47 also functions as a marker of self on murine red blood cells which allows RBC to avoid phagocytosis. Tumor cells can also evade macrophage phagocytosis through the expression of CD47 (2, 13).

It appears that CD47 ligation induce different responses, depending on cell type and partner for ligation.

Therapeutic and clinical aspect of CD47 in human cancer:

CD47 is overexpressed in many types of human cancers  and its known function as a “don’t eat me” signal, suggests the potential for targeting the CD47-SIRPα pathway as a common therapy for human malignancies (2,13). Upregulation of CD47 expression in human cancers also appears to influence tumor growth and dissemination. First, increased expression of CD47 in several hematologic malignancies was found to be associated with a worse clinical prognosis, and in ALL to predict refractoriness to standard chemotherapies (13, 14-16). Second, CD47 was demonstrated to regulate tumor metastasis and dissemination in both MM and NHL (13, 17).

Efforts have been made to develop therapies inhibiting the CD47-SIRPα pathway, principally through blocking monoclonal antibodies directed against CD47, but also possibly with a recombinant SIRPα protein that can also bind and block CD47.

Chao MP et al. 2012 Combination strategies targeting CD47 in cancer

While monotherapies targeting CD47 were efficacious in several pre-clinical tumor models, combination strategies involving inhibition of the CD47-SIRPα pathway offer even greater therapeutic potential. Specifically, antibodies targeting CD47-SIRPα can be included in combination therapies with other therapeutic antibodies, macrophage-enhancing agents, chemo-radiation therapy, or as an adjuvant therapy to inhibit metastasis (13).

For example, anti-SIRPα antibody was found to potentiate  antibody-dependent cellular cytotoxicity (ADCC) mediated by the anti-Her2/Neu antibody trastuzumab against breast cancer cells (18).  CD47–SIRPα interactions and SIRPα signaling negatively regulate trastuzumab-mediated ADCC in vitro and antibody-dependent elimination of tumor cells in vivo

More so, chemo-radiation therapy-mediated upregulation of cell surface calreticulin may potentially augment the activity of anti-CD47 antibody. However, this approach may also lead to increased toxicity as cell surface calreticulin is expressed on non-cancerous cells undergoing apoptosis, a principle effect of chemo-radiation therapy (19).

Highlights:

• Phagocytic cells, macrophages, regulate tumor growth through phagocytic clearance
• CD47 binds SIRPα on phagocytes which delivers an inhibitory signal for phagocytosis
• A blocking anti-CD47 antibody enabled phagocytic clearance of many human cancers
• Phagocytosis depends on a balance of anti-(CD47) and pro-(calreticulin) signals
• Anti-CD47 antibody synergized with an FcR-engaging antibody, such as rituximab

Summary

Evasion of immune recognition is a major mechanism by which cancers establish and propagate disease. Recent data has demonstrated that the innate immune system plays a key role in modulating tumor phagocytosis through the CD47-SIRPα pathway. Careful development of reagents that can block the CD47/SIRPα interaction may indeed be useful to treat many forms of cancer without having too much of a negative side effect in terms of inducing clearance of host cells. Therapeutic approaches inhibiting this pathway have demonstrated significant efficacy, leading to the reduction and elimination of multiple tumor types.

Dr. Weissman says: “We are now hopeful that the first human clinical trials of anti-CD47 antibody will take place at Stanford in mid-2014, if all goes well. Clinical trials may also be done in the United Kingdom”. These clinical trials must be designed so that the data they generate will produce a valid scientific result!!!

REFERENCES

1. By Sara Gates:  Cancer Drug That Shrinks All Tumors Set To Begin Human Clinical Trials. http://www.huffingtonpost.com/2013/03/28/cancer-drug-shrinks-tumors_n_2972708.html

2. Willingham SB, Volkmer JP, Gentles AJ, Sahoo D, Dalerba P, Mitra SS, Wang J, Contreras-Trujillo H, Martin R, Cohen JD, Lovelace P, Scheeren FA, Chao MP, Weiskopf K, Tang C, Volkmer AK, Naik TJ, Storm TA, Mosley AR, Edris B, Schmid SM, Sun CK, Chua MS, Murillo O, Rajendran P, Cha AC, Chin RK, Kim D, Adorno M, Raveh T, Tseng D, Jaiswal S, Enger PØ, Steinberg GK, Li G, So SK, Majeti R, Harsh GR, van de Rijn M, Teng NN, Sunwoo JB, Alizadeh AA, Clarke MF, Weissman IL. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):6662-6667. http://www.pnas.org/content/early/2012/03/20/1121623109

3. Oldenborg PL. CD47: A Cell Surface Glycoprotein Which Regulates Multiple Functions of Hematopoietic Cells in Health and Disease. ISRN Hematology Volume 2013 (2013), Article ID 614619, 19 pages.  http://www.hindawi.com/isrn/hematology/2013/614619/

4. G. Campbell, P. S. Freemont, W. Foulkes, and J. Trowsdale, “An ovarian tumor marker with homology to vaccinia virus contains an IgV- like region and multiple transmembrane domains,”Cancer Research, vol. 52, no. 19, pp. 5416–5420, 1992. http://cancerres.aacrjournals.org/content/52/19/5416.long

5. L. G. Poels, D. Peters, Y. van Megen et al., “Monoclonal antibody against human ovarian tumor-associated antigens,” Journal of the National Cancer Institute, vol. 76, no. 5, pp. 781–791, 1986. http://www.ncbi.nlm.nih.gov/pubmed/3517452

6. CD47. Wikipedia. http://en.wikipedia.org/wiki/CD47

7. Sick E, Boukhari A, Deramaudt T, Rondé P, Bucher B, André P, Gies JP, Takeda K (February 2011). “Activation of CD47 receptors causes proliferation of human astrocytoma but not normal astrocytes via an Akt-dependent pathway”. Glia 59 (2): 308–319. http://www.ncbi.nlm.nih.gov/pubmed/21125662

8. Kaur S, Soto-Pantoja DR, Stein EV, Liu C, Elkahloun AG, Pendrak ML, Nicolae A, Singh SP, Nie Z, Levens D, Isenberg JS, Roberts DD.  “Thrombospondin-1 Signaling through CD47 Inhibits Self-renewal by Regulating c-Myc and Other Stem Cell Transcription Factors”. Sci Rep 2013: 3: 1673. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3628113/

9. Shahan TA, Fawzi A, Bellon G, Monboisse JC, Kefalides NA. “Regulation of tumor cell chemotaxis by type IV collagen is mediated by a Ca(2+)-dependent mechanism requiring CD47 and the integrin alpha(V)beta(3)”. J. Biol. Chem 2000. 275 (7): 4796–4802. http://www.jbc.org/content/275/7/4796

10. Isenberg JS, Ridnour LA, Dimitry J, Frazier WA, Wink DA, Roberts DD. “CD47 is necessary for inhibition of nitric oxide-stimulated vascular cell responses by thrombospondin-1”. J. Biol. Chem  2006. 281 (36): 26069–26080.  http://www.jbc.org/content/281/36/26069

11. Smadja DM, d’Audigier C, Bièche I, Evrard S, Mauge L, Dias JV, Labreuche J, Laurendeau I, Marsac B, Dizier B, Wagner-Ballon O, Boisson-Vidal C, Morandi V, Duong-Van-Huyen JP, Bruneval P, Dignat-George F, Emmerich J, Gaussem P. “Thrombospondin-1 is a plasmatic marker of peripheral arterial disease that modulates endothelial progenitor cell angiogenic properties”. Arterioscler. Thromb. Vasc. Biol  2011. 31 (3): 551–559. http://atvb.ahajournals.org/content/31/3/551

12. G. D. Grossfeld, D. A. Ginsberg, J. P. Stein et al., “Thrombospondin-1 expression in bladder cancer: association with p53 alterations, tumor angiogenesis, and tumor progression,” Journal of the National Cancer Institute 1997 vol. 89, no. 3, pp. 219–227. http://www.scopus.com/record/display.url?eid=2-s2.0-18744423089&origin=inward&txGid=9C86356DDB0B6816ACCBF90F9CA44E92.WlW7NKKC52nnQNxjqAQrlA%3a2

13. Chao MP, Weissman IL, Majeti R. “The CD47-SIRPα pathway in cancer immune evasion and potential therapeutic implications”. Curr. Opin. Immunol 2012. 24 (2): 225–32. http://www.sciencedirect.com/science/article/pii/S095279151200012X. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3319521/

14. Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, Gibbs KD, Jr, van Rooijen N, Weissman IL. Cd47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell. 2009;138(2):286–299. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726837/

15. Chao MP, Alizadeh AA, Tang C, Jan M, Weissman-Tsukamoto R, Zhao F, Park CY, Weissman IL, Majeti R. Therapeutic antibody targeting of cd47 eliminates human acute lymphoblastic leukemia.Cancer Res. 2011;71 (4):1374–1384. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3041855/

16. Chao MP, Alizadeh AA, Tang C, Myklebust JH, Varghese B, Gill S, Jan M, Cha AC, Chan CK, Tan BT, Park CY, et al. Anti-cd47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-hodgkin lymphoma. Cell. 2010;142(5):699–713. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943345/

17. Chao MP, Tang C, Pachynski RK, Chin R, Majeti R, Weissman IL. Extranodal dissemination of non-hodgkin lymphoma requires cd47 and is inhibited by anti-cd47 antibody therapy. Blood.2011;118(18):4890–4901. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208297/

18. Zhao XW, van Beek EM, Schornagel K, Van der Maaden H, Van Houdt M, Otten MA, Finetti P, Van Egmond M, Matozaki T, Kraal G, Birnbaum D, et al. Cd47-signal regulatory protein-alpha (sirpalpha) interactions form a barrier for antibody-mediated tumor cell destruction. Proc Natl Acad Sci U S A.2011;108(45):18342–18347. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215076/

19. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, Castedo M, Mignot G, Panaretakis T, Casares N, Metivier D, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med. 2007;13(1):54–61. http://www.ncbi.nlm.nih.gov/pubmed/17187072

Other related articles on this Open Access Online Scientific Journal include the following:

I. By: Larry Bernstein MD. Treatment for Metastatic HER2 Breast Cancer http://pharmaceuticalintelligence.com/2013/03/03/treatment-for-metastatic-her2-breast-cancer/

II. By: Tilda Barliya PhD. Colon Cancer.  http://pharmaceuticalintelligence.com/2013/04/30/colon-cancer/

III. By: Ritu Saxena PhD. In focus: Triple Negative Breast Cancer. http://pharmaceuticalintelligence.com/2013/01/29/in-focus-triple-negative-breast-cancer/

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Systems Pharmacology – Pathways to Patient Response @ BioIT World, April 9-11, 2013, World Trade Center, Boston, MA

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, BioSimilars, Computational Biology/Systems and Bioinformatics, Drug Delivery Platform Technology, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmaceutical Industry Competitive Intelligence, Pharmaceutical R&D Investment, Population Health Management, Genetics & Pharmaceutical, Technology Transfer: Biotech and Pharmaceutical, tagged Harvard Medical School, national cancer institute, National Institutes of Health, Stanford University School of Medicine, systems biology, Thomson Reuters on March 21, 2013| 12 Comments »

Reporter: Aviva Lev-Ari, PhD, RN

Track 6 focuses on how compounds (drugs) work in the body. How are they influenced by various ‘omics’? How do they vary by tissue? The practical implications of such a compound-centric approach are exciting: new targets, new screens, new markers, new understanding of drug failure mechanisms. The systems computational tool sets including multi-scale modeling, simulation, web-based platforms, etc. will be emphasized.

Final Agenda

Download Brochure | Pre-Conference Workshops

TUESDAY, APRIL 9

7:00 am Workshop Registration and Morning Coffee

8:00 Pre-Conference Workshops*

*Separate Registration Required

2:00 – 7:00 pm Main Conference Registration

4:00 Event Chairperson’s Opening Remarks

Cindy Crowninshield, RD, LDN, Conference Director, Cambridge Healthtech Institute

4:05 Keynote Introduction

Kevin Brode, Senior Director, Health & Life Sciences, Americas Hitachi Data Systems

» 4:15 PLENARY KEYNOTE

Do Network Pharmacologists Need Robot Chemists?

Andrew L. Hopkins, DPhil, FRSC, FSB, Division of Biological Chemistry and Drug Design, College of Life Sciences, University of Dundee

5:00 Welcome Reception in the Exhibit Hall with Poster Viewing
Welcome Reception Introduction, Sponsored by Okta

Drop off a business card at the CHI Sales booth for a chance to win 1 of 2 iPads® or 1 of 2 Kindle Fires®!*

*Apple ® and Amazon are not sponsors or participants in this program

WEDNESDAY, APRIL 10

7:00 am Registration and Morning Coffee

8:00 Chairperson’s Opening Remarks

Phillips Kuhl, Co-Founder and President, Cambridge Healthtech Institute

8:05 Keynote Introduction

Sanjay Joshi, CTO, Life Sciences, EMC Isilon

» 8:15 PLENARY KEYNOTE

Atul Butte, M.D., Ph.D., Division Chief and Associate Professor, Stanford University School of Medicine; Director, Center for Pediatric Bioinformatics, Lucile Packard Children’s Hospital; Co-founder, Personalis and Numedii

8:55 Benjamin Franklin Award & Laureate Presentation

9:15 Best Practices Award Program

9:45 Coffee Break in the Exhibit Hall with Poster Viewing

PHARMACODYNAMIC MODELS

10:50 Chairperson’s Remarks

Hugo Geerts, Ph.D., CSO, Computational Neuropharmacology, In Silico Biosciences

» Featured Speaker

11:00 Systems Pharmacology in a Post-Genomic Era

Peter Sorger, Ph.D., Professor, Systems Biology, Harvard Medical School; Co-Chair, Harvard Initiative in Systems Pharmacology

I will describe the emergence of “systems pharmacology” as a means to guide the creation of new molecular matter, study cellular networks and their perturbation by drugs, understand pharmaco-kinetics and pharmaco-dynamics in mouse and man and design and analyze clinical trial data. The approach combines mathematical modeling with empirical measurement as a means to tackle basic and clinical problems in pharmacology. Ultimately we aim for models that describe drug responses at multiple temporal and physical scales from molecular mechanism to whole-organism physiology.

11:30 Using Quantitative Systems Pharmacology for De-Risking Projects in CNS R&D

Hugo Geerts, Ph.D., CSO, Computational Neuropharmacology, In Silico Biosciences

Quantitative Systems Pharmacology is a computer based mechanistic modeling approach combining physiology, the functional imaging of genetics with the pharmacology of drug-receptor interaction and parameterized with clinical data and is a possible powerful tool for improving the success rate of CNS R&D projects. The presentation will include failure analyses of unsuccessful clinical trials, correct prospective identification of clinical problems that halted clinical development and estimation of genotype effects on the pharmacodynamics of candidate drugs.

12:00 pm Systems Pharmacology Approaches to Drug Repositioning

Svetlana Bureeva, Ph.D., Director, Professional Services, Thomson Reuters, IP & Science

Drug repositioning requires advanced computational approaches and comprehensive knowledgebase information to reach success. Thomson Reuters will present on recent advances in drug repositioning approaches, their validation and performance, best practices in using systems biology content, and successful case studies.

12:30 Luncheon Presentation (Sponsorship Opportunity Available) or Lunch on Your Own

HIGH CONTENT ANALYSIS: CANCER CELL LINES

1:40 Chairperson’s Remarks

William Reinhold, Manager, Genomics and Bioinformatics Group, Laboratory of Molecular Pharmacology (LMP), National Cancer Institute (NCI)

1:45 Systems Pharmacology Using CellMiner and the NCI-60 Cancerous Cell Lines

William Reinhold, Manager, Genomics and Bioinformatics Group, Laboratory of Molecular Pharmacology (LMP), National Cancer Institute (NCI)

CellMiner is a web-based application that allows rapid access to and comparison between 20,503 compound activities and the expression levels of 26,065 genes and 360 microRNAs. Included are 102 FDA-approved drugs as well as 53 in clinical trials. The tool is designed for the non-informatisist, and allows the user wide latitude in defining the question of interest. This opens the door to systems pharmacological studies for physicians, molecular biologists and others without bioinformatics expertise.

2:15 Oncology Drug Combinations at Novartis

Joseph Lehár, Ph.D., Associate Director, Bioinformatics, Oncology Translational Research, Novartis; Adjunct Assistant Professor, Bioinformatics, Boston University

Novartis is undertaking a large-scale effort to comprehensively describe cancer through the lens of cell cultures and tissue samples.  In collaboration with academic and industrial partners, we have generated mutation status, gene copy number, and gene expression data for a library of 1,000 cancer cell lines, representing most cancer lineages and common genetic backgrounds.  Most of these cell lines have been tested for chemosensitivity against ~1,200 cancer-relevant compounds, and we are systematically exploring drug combinations for synergy against ~100 prioritized CCLE lines.  We expect this large-scale campaign to enable efficient patient selection for clinical trials on existing cancer drugs, reveal many therapeutically promising drug synergies or anti-resistance combinations, and provide unprecedented detail on functional interactions between cancer signaling pathways.   I will discuss early highlights of this work and describe our plans to make use of this resource.

2:45 Sponsored Presentations (Opportunities Available)

3:15 Refreshment Break in the Exhibit Hall with Poster Viewing

PHARMACODYNAMIC MODELS FOR ONCOLOGY

3:45 Systems Biology in Cancer Immunotherapy: Applications in the Understanding of Mechanism of Action and Therapeutic Response

Debraj Guha Thakurta, Ph.D., Senior Scientist II & Group Leader, Systems Biology, Dendreon Corporation

We are using high-content platforms (DNA and protein microarrays, RNA-seq) in various stages of the development of cellular immunotherapies for cancer. We will provide examples of genomic applications that can aid in the mechanistic understanding and the discovery of molecular markers associated with the efficacy of a cancer immunotherapy..

4:15 Use of Systems Pharmacology to Aid Cancer Clinical Development

Anna Georgieva Kondic, Ph.D., MBA, Senior Principal Scientist, Modeling and Simulation, Merck Research Labs

The last few years have seen an increased use of physiologically-based pharmacokinetics and pharmacodynamics models in Oncology drug development. This is partially due to an improved mechanistic understanding of disease drivers and the collection of better patient-level quantitative data that lends itself to modeling. In this talk, a suite of studies where systems modeling was successfully used to inform either preclinical to clinical transition or clinical study design will be presented. The talk will complete with a potential systems pharmacology framework that can be used systematically in drug development.

4:45 Two-Edged Sword Role of the Mammalian DNA Methyltransferases: New Implication to Cancer Therapy Targeting the Epigenetic Pathway

Che-Kun James Shen, Ph.D., Distinguished Research Fellow, Institute of Molecular Biology, Academia Sinica

Methylation at the 5-position of cytosine (C) to generate 5-methylcytosine (5-mC) on the vertebrate genomes is an essential epigenetic modification that regulates different biological processes including carcinogenesis. This modification has been known to be accomplished by the combined catalytic actions of three DNA methyltransferases (DNMTs), the de novo enzymes DNMT3A/ DNMT3B and the maintenance enzyme DNMT1. This property of DNMTs and the imbalance of CpG methylation in cancer cells have led to the development of cancer therapeutic drugs/ chemicals targeting the DNA methylation activities of DNMTs. However, we have recently discovered that the mammalian DNMTs could also act as active DNA 5-mC demethylases in a Ca++ion-and redox state-dependent manner. This suggests new directions for re-investigation of the structures of DNMTs and their functions in the genome wide and/or local DNA methylation in the mammalian cells. In particular, the concept and strategies for drug therapy targeting the DNMTs may need to be re-evaluated.

5:15 Best of Show Awards Reception in the Exhibit Hall

6:15 Exhibit Hall Closes

Thursday, April 11

7:00 am Breakfast Presentation (Sponsorship Opportunity Available) or Morning Coffee

MODELING AND MINING TARGETS

8:45 Chairperson’s Opening Remarks

I-Ming Wang, Ph.D., Associate Scientific Director, Research Solutions and Bioinformatics, Informatics and Analysis, Merck Research Laboratory

8:50 Systems Biology Approach for Identification of New Targets and Biomarkers

I-Ming Wang, Ph.D., Associate Scientific Director, Research Solutions and Bioinformatics, Informatics and Analysis, Merck Research Laboratory

A representative gene signature was identified by an integrated analysis of expression data in twelve rodent inflammatory models/tissues. This “inflammatome” signature is highly enriched in known drug target genes and is significantly overlapped with macrophage-enriched metabolic networks (MEMN) reported previously. A large proportion of genes in this signature are tightly connected in several tissue-specific Bayesian networks built from multiple mouse F2 crosses and human tissue cohorts; furthermore, these tissue networks are very significantly overlapped. This indicates that variable expression in this set of co-regulated genes is the main driver of many disease states. Disease-specific gene sets with the potential of being utilized as biomarkers were also identified with the approach we applied. The identification of this “inflammatome” gene signature extends the coverage of MEMN beyond adipose and liver in the metabolic disease to multiple diseases involving various affected tissues.

9:20 Optimizing Therapeutic Index (TI) by Exploring Co-Dependencies of Target and Therapeutic Properties

Madhu Natarajan, Ph.D., Associate Director, Computational Biology, Discovery Research, Shire HGT

Conventional drug-discovery informatics workflows employ combinations of mechanistic/probabilistic in-silico methods to rank lists of targets; therapeutics are then developed for “optimal” targets. I describe a systems pharmacology approach that instead integrates systematic in-silico therapeutic perturbation with models of target/disease biology to identify conditions for optimal TI; non-intuitively optimal TI is sometimes achieved by pairing sub-optimal targets with therapeutics having appropriate properties.

9:50 Leveraging Mathematical Models to Understand Population Variability in Response to Cardiac Drugs

Eric Sobie, Ph.D., Associate Professor, Pharmacology & Systems Therapeutics, Icahn School of Medicine, Mount Sinai School of Medicine

Mathematical models of heart cells and tissues are sufficiently advanced that the models can predict mechanisms underlying pro-arrhythmic or anti-arrhythmic effects of drugs. At present, however, these models are not adequate for understanding variability across a population, i.e., why a drug may be effective in one patient but ineffective in another patient. I will describe novel computational approaches my laboratory has developed to quantify and predict differences between individuals in response to cardiac drugs.

10:20 Coffee Break in the Exhibit Hall and Poster Competition Winners Announced

10:45 Plenary Keynote Panel Chairperson’s Remarks

Kevin Davies, Ph.D., Editor-in-Chief, Bio-IT World

10:50 Plenary Keynote Panel Introduction

Yury Rozenman, Head of BT for Life Sciences, BT Global Services

» PLENARY KEYNOTE PANEL

11:05 The Life Sciences CIO Panel

Panelists:
Remy Evard, CIO, Novartis Institutes for BioMedical Research
Martin Leach, Ph.D., Vice President, R&D IT, Biogen Idec
Andrea T. Norris, Director, Center for Information Technology (CIT) and Chief Information Officer, NIH
Gunaretnam (Guna) Rajagopal, Ph.D., VP & CIO – R&D IT, Research, Bioinformatics & External Innovation, Janssen Pharmaceuticals
Cris Ross, Chief Information Officer, Mayo Clinic

12:15 pm Luncheon in the Exhibit Hall with Poster Viewing

MODELING MOLECULAR AND PATHOPHYSIOLOGICAL DATA

1:55 Chairperson’s Remarks

Jake Chen, Ph.D., Associate Professor, Indiana University School of Informatics & Purdue University Department of Computer Science; Director, Indiana Center for Systems Biology and Personalized Medicine

2:00 Predicting Adverse Side Effects of Drugs Using Systems Pharmacology

Jake Chen, Ph.D., Associate Professor, Indiana University School of Informatics & Purdue University Department of Computer Science; Director, Indiana Center for Systems Biology and Personalized Medicine

A new way of studying drug toxicity is to incorporate biomolecular annotation and network data with clinical observations of drug targets upon drug perturbations. I will describe the development of a novel computational modeling framework, with which we demonstrated the highest drug toxicity prediction accuracies ever reported by far. Adoption of this framework may have profound practical drug discovery implications.

2:30 Holistic Integration of Molecular and Physiological Data and Its Application in Personalized Healthcare

David de Graaf, Ph.D. President and CEO, Selventa

There are multiple industry-wide challenges in aggregating molecular and pathophysiological data for systems pharmacology to transform the process of drug discovery and development. One of the ways to address these challenges is to utilize a common computable biological expression language (BEL) that can provide a comprehensive knowledge network for new discoveries. An application of BEL and its use in identifying clinically relevant predictive biomarkers for patient stratification will be presented.

3:00 The Role of Informatics in ADME Pharmacogenetics

Boyd Steere, Ph.D., Senior Research Scientist, Lilly Research Laboraories, IT Research Informatics, Eli Lilly

The leveraging of pharmacogenetics to support decisions in early-phase clinical trial design requires informatics methods to integrate, visualize, and analyze heterogeneous data sets from many different discovery platforms.  This presentation describes challenges and solutions in making sense of diverse sets of genetic, protein, and metabolic data in support of ADME pharmacology projects.

3:30 A Systems Pharmacology Approach to Understand and Optimize Functional Selectivity for Non-Selective Drugs

Joshua Apgar, Principal Scientist, Systems Biology, Dept. of Immunology & Inflammation, Boehringer Ingelheim Pharmaceuticals, Inc.

Most commonly the selectivity of a compound is defined in an in vitro or cellular assay, and it is thought of as principally a function of the binding energy of the drug to its on-target and off-target proteins; however, in vivo functional selectivity is much more complicated, and is affected by systems level effects such as multiple feedback processes within and between the various on- and off-target pathways. These systems level processes are often impossible to reconstruct in vitro as they involve many cell types, tissues, and organs systems throughout the body. We show here that through mathematical modeling we were able to identify, in silico, molecular properties that are critical to driving functional selectivity. The models, although simple, capture the key systems pharmacology needed to understand the on- an off- target effects. Surprisingly, in this case, the key driver of functional selectivity is not the affinity of the drugs but rather the pharmacokinetics, with drugs having a short half-life predicted to be the most functionally selective.

4:00 Conference Adjourns

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CONCURRENT TRACKS
	Track 1: IT Infrastructure – Hardware Big Data Solutions and End-User Perspectives		Track 7: eClinical Trials Solutions Innovative Management in Clinical Trials
	Track 2: Software Development Technologies and Applications for Managing and Sharing Data		NEW THIS YEAR! Track 8: Data Visualization and Exploration Tools From Discovery to the Clinic
	Track 3: Cloud Computing Riding the Cloud to Next-Generation Computing		Track 9: Drug Discovery Informatics Thinking of Drugs Outside of the Box
	Track 4: Bioinformatics Understanding Massive Quantities of -omic Information across Research Initiatives		NEW THIS YEAR! Track 10: Clinical OmicsTools for Integrating and Interrogating Multiple ‘Omic Data Sets
	Track 5: Next-Generation Sequencing Informatics NGS, Genome-Scale Screening and HTP Proteomics		Track 11: Collaborations and Open Access Innovations Collaborative and Open Access Models for Advancing Research, Discovery and Personalized Medicine
	Track 6: Systems Pharmacology Pathways to Patient Response		Track 12: Cancer Informatics Applying Computational Biology to Cancer Research/Care

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WORKSHOPS – VIEW DETAILS

Morning Workshops Integrated Research Data Management Quality Practices for R&D Informatics Beyond the Cloud IT & Informatics in Support of Collaboration and Externalization The Repurposing Challenge

Afternoon Workshops Avoiding Intellectual Property Problems in Research Collaborations Using Information Technology Avoiding Cloud Gotchas Advancing the Use of EHR/EMR for Clinical Research and Drug Development Cloud Computing in Hospital Data Management and Integration Data Visualization in Biology: From the Basics to Big Data Software for Clinical Genomics IT Project Planning and Implementation

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SPONSOR & EXHIBITOR INFORMATION

2013 Prospectus is now available.

Reserve your space early and SAVE!

EVENT FEATURES

• Access All 12 Tracks for One Price
• Network with 2,500+ Global Attendees
• Hear 150+ Technology and Scientific Presentations
• Attend Bio-IT World’s Best Practices Awards
• Connect with Attendees Using CHI’s Intro-Net
• Participate in the Poster Competition
• Choose from 16 Pre-Conference Workshops
• See the Winners of the following 2013 Awards:
  Benjamin Franklin
  Best of Show
  Best Practices
• View Novel Technologies and Solutions in the Expansive Exhibit Hall
• And Much More!

KEYNOTE PRESENTERS:

Atul Butte, M.D., Ph.D., Division Chief and Associate Professor, Stanford University School of Medicine; Director, Center for Pediatric Bioinformatics, Lucile Packard Children’s Hospital; Co-founder, Personalis and Numedii

Andrew L. Hopkins, D.Phil, FRSC, FSB, Division of Biological Chemistry and Drug Design, College of Life Sciences, University of Dundee

PLENARY SESSION:

The Life Sciences CIO Panel

From managing big data and cloud computing capabilities to building virtual communities and optimizing drug development, the life sciences CIO has to be a firefighter, evangelist, visionary. In this special plenary roundtable, Bio-IT World invites a select group of CIOs from big pharma, academia and government to discuss the major issues facing today’s biosciences organization and the prospects for future growth and organizational success.

Special guests:
Remy Evard – CIO, Novartis Institutes for BioMedical Research
Martin Leach, Ph.D., Vice President, R&D IT, Biogen Idec
Andrea T. Norris – Director, Center for Information Technology (CIT) and CIO, NIH
Gunaretnam (Guna) Rajagopal, Ph.D., VP & CIO – R&D IT, Research, Bioinformatics & External Innovation, Janssen Pharmaceuticals
Cris Ross – CIO, Mayo Clinic

FEATURED SESSIONS:

Managing Big Data: The Genome Center Perspective

Panelists Include: Matthew Trunnell (Broad Institute)
Alexander (Sasha) Wait Zaranek, (Harvard Medical School/Clinical Future, Inc.)
Guy Coates (The Wellcome Trust Sanger Institute)

Building the IT Architecture of the New York Genome Center

Chris Dwan, Acting Senior Vice President, Information Technology and Research Computing, New York Genome Center
Kevin Shianna, Senior Vice President, Sequencing Operations, New York Genome Center
Jim Harding, CTO, Sabey Corporation
Sanjay Joshi, CTO, Life Sciences, EMC Isilon Storage Division

Robert B. Darnell, M.D., Ph.D., President & Scientific Director, New York Genome Center

Additional Speakers to be Announced

  

3/4

VIDEO CHANNEL

Cancer Trends Plenary Session (part II) – Bio-IT World Expo 2012

Bio-IT World Expo 2012 – 10th Anniversary Celebration

ClearTrial wins Best of Show 2012

CERF wins Best of Show 2012

OpsCode wins Best of Show 2012

Cambridge Semantics wins Best of Show 2012

Stephen Wolfram, Ph.D., part 1 – Keynote Presentation

BlueArc at 2011 Bio-IT World Conference & Expo

Roche innovative multi-touch environment for scientific decision

Praxeon DocumentLens

Yury Rozenman – Bio-IT World Expo 2011 Keynote Panel

Mark Boguski – Bio-IT World Expo 2011 Keynote Panel

Ken Buetow – Bio-IT World Expo 2011 Keynote Panel

Benjamin Heywood – Bio-IT World Expo 2011 Keynote Panel

Debora Goldfarb – Bio-IT World Expo 2011 Keynote Panel

Martin Leach – Bio-IT World Expo 2011 Keynote Panel

Jonathan Eisen – Ben Franklin Award 2011

Bryn Roberts part 1- Keynote Presentation

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