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CVP FELLOWS RESEARCH
PREDOCS
POSTDOCS
Courter, Don
Askari, Bardia
Nhan, Thomas
POSTDOCTORAL
Askari,
Bardia
Mentor: Karin E. Bornfeldt, PhD
It has been established that the majority of
adults with diabetes suffer from vascular complications. The progression of
atherosclerotic plaques is accelerated by diabetes, a phenomenon observed in
humans and in animal models of the diabetes. We had previously established that
elevated glucose concentration did not appear to directly increase the
proliferative response in cultured porcine and human vascular smooth muscle cell1.
Elevated glucose levels (25 mM) also did not appear to influence vascular smooth
muscle cell death.
In a porcine model of diabetes-accelerated
atherosclerosis2, fat-fed, diabetic animals had elevated plasma
triglyceride levels compared to normal-diet, diabetic and normal non-diabetic
animals. Previous studies have demonstrated that cis unsaturated, non-esterified
fatty acids such as oleic acid (OA, 18:1) and linoleic acid (LA, 18:2) have a
proliferative effect in cultured vascular smooth muscle4,5. LA and
OA also act synergistically with other growth factors5,6. I have
demonstrated that LA and OA potentiate the mitogenic effects of insulin-like
growth factor I (IGF-I) in cultured porcine smooth muscle cells7,
while positional and stereo-isomers of OA and LA, such as elaidic (trans
18:1) and conjugated-linoleic acid (c-LA) did not affect IGF-I-induced SMC
proliferation.
The mitogenic effect of IGF-I is mediated by
the PI-3 kinase/PK-B pathway and to lesser degree, the p42/p44 MAPK. OA and LA
did not effect the phosphorylation of Akt/PK-B, P70S6 or
MAP kinase pathways. Inhibition of the cyclooxygenase or the 5-and
12-lipoxygenase pathway also were without effect, indicating that oxidation
products such as 9- and 13-hydroxyoctadecaidenoic acid (HODE) do not play a role
in the potentiation of IGF-I. These results were confirmed by HPLC analysis of
[14C]-labeled OA and LA
SMC's, as ethyl acetate extraction of the supernatant demonstrated that no
autocrine factors were released in IGF-I-treated SMC's.
It has been demonstrated in other tissues that
ther is a differential distribution of fatty acids into distinct phospholipid
pools8. To determine whether OA and LA are incorporated into the
plasma, we preincubated the SMC’s with OA and LA prior to stimulation with IGF-I.
Pretreatment of the SMC’s with the fatty acids increased the potentiation of IGF-I-induced
proliferation, indicating that OA and LA are incorporated into the membrane.
Inhibition of phospholipase C and phospholipase A2
were without effect while inhibition of phospholipase D activity abolished the
potentiation of OA and LA. Inhibition of diacyl glycerol (DAG) kinase activity,
the enzyme responsible for removal of DAG, also increased the effects of IGF-I
on SMC proliferation, indicating that DAG may play an important role in IGF-I-induced
effects in SMC’s9.
Future experiments will attempt to a)
determine the membrane localization of OA and LA b) which products are
formed by the activity of PLD and c) how IGF-I modulated PLD activity.
References
1.
Suzuki LA, Poot M,
Gerrity RG, Bornfeldt KE: Diabetes accelerates smooth muscle accumulation in
atherosclerosis: Lack of direct growth-promoting effects of hyperglycemia. (Diabetes.
50:851-860, 2001)
2.
Gerrity RG, Natarajan
R, Nadler JL, Kimsey T: Diabetes-induced accelerated atherosclerosis in swine.
(Diabetes. 50:1654-1665,2001)
3.
Lu G, Morinelli TA,
Meier KE, Rosenzweig SA, Egan BM: Oleic acid-induced mitogenic signaling in
vascular smooth muscle cells: Role for protein kinase C. (Circ Res.
79:611-618, 1996)
4.
Rao GN, Alexander RW,
Runge MS: Linoleic acid and its metabolites, hydroperoxyoctadecadienoic acids,
stimulate c-fos, c-jun and c-myc mRNA expression, MAP kinase
activation and growth in rat aortic smooth muscle cells. (J. Clin. Invest.
96:842-847, 1995)
5.
Lu G, Meier KE, Jaffa
AA, Rosenzweig SA, Egan BM: Oleic acid and angiotensin II induce a synergistic
mitogenic response in vascular smooth muscle cells. (Hypertension
31:978-985, 1998)
6.
Kwok CF, Shih KC, Hwu
CM, Ho LT: Linoleic and oleic acid increase the endothelin-1 binding and action
in cultured rat aortic smooth muscle cells. (Metabolism,
40(11):1386-1389, 2000)
7.
Askari B, Gerrity RG,
Kramer F, Bornfeldt KE: Free fatty acids potentiate the mitogenic effects of
insulin-like growth factor-I. (61st Scientific Sessions of the
American Diabetes Association, June 2001)
8.
Zheng Z, Barkai AI,
Hungund BL: Effects of ethanol on the incorporation of free fatty acids into
cerebral membrane phospholipids. (Neurochem. Int. 28(5-6):551-556, 1996)
9.
Askari B, Carroll M,
Gerrity RG, Capparelli M, Kramer F: Insulin-like Growth Factor I-induced
Proliferation of Arterial Smooth Muscle cells: Role of Phospholipase D (in
preparation)
Mentor: Daniel F. Bowen-Pope, PhD
My work in the Bowen-Pope lab involves two
separate projects: a primary focus on rPTP-GMC1 "Kidphos" and a secondary focus
on PDGFα.
Kidphos is a receptor-like PTPase that was
discovered in a rat renal injury model. Kidphos protein is expressed by rat
renal mesangial cells, human renal podocytes, and mouse inner ear hair cells.
Kiphos has multiple transcripts. The largest transcript (7.5 kb) encodes for a
the receptor-like PTPase form of Kidphos. The smallest transcript (1.8 kb)
encodes for the "truncation-deletion" intracellular variant form of Kidphos that
has the catalytic domain but does not contain the transmembrane nor
extracellular domains and cannot encode a receptor PTPase. A "knock-out" mouse
was made where the transmembrane region was replaced such that a receptor-like
Kidphos cannot be expressed. Subsequently it was determined that the 1.8 kb
transcript is derived from an alternate promoter downstream of the transmembrane
domain; so a second Kidphos "knock-out" (KO) mouse is being developed where both
the receptor-like and intracellular variants of Kidphos with be eliminated.
We have not yet identified any
histolopathologic or physiolotic phenotype in the transmembrane homozygote KO
mice that cannot make the receptor-like form of Kidphos. This summer we had
germline transmission in the second line of catylic KO mice. Recently the F1
heterozygotes were crossed and genotyping and characterization of their pups are
imminent.
The majority of my work has centered on
determining the histologic and/or physiologic phenotype in the first line of KO
Kidphose mice. In addition, immunohistochemical (IHC) studies are in process to
determine the cellular expression of Kidphos protein in human, nonhuman primate,
and mouse tissues. As a component of this, portions of my work involve
characterization of a new peptide antibody to the C-terminal end of Kidphos.
The antibody studies have shown that we can detect the receptor-like form of
Kidphos protein in the sensory hair cells of the inner ear of mice and in the
renal glomerular podocytes. Tissues from the catalytic KO mice will be
characterized with our antibodies once they are available.
The secondary project is centered on stellate
(Ito) cells in the liver and the role of PDGFα
in carbon tetrachloride (CCl4) induced hepatic injury. Mice lacking PDGFâ die
during embryogenesis. In order to study the role of PDGFα,
a chimeric model is utilized where 8 cell embryos from mice carrying the human
globin gene (cellular marker) and wild type, heterozygous, or null for PRGFα
are fused with an 8 cell embryo from a different line of mice. These "chimeric"
mice are then injected with CCl4 and euthanized and necropsied at different time
points. Stellate cells stain positive by desmin IHC. Cells from the PDGFα
mice can be identified by in situ hybridization (ISH) to the human betaglobin
gene. Currently all the animal studies are completed and the tissues collected,
processed and embedded. The first phase verifying stellate cell proliferation
after hepatic injury by CCl4 is completed. The individual protocols for the DNA
ISH and desmin staining by IHC both work individually. Dual staining has been
accomplished but is in the process of being refined for optimum cell counting to
complete the quantitative analysis of the response of stellate cells containing
or lacking PDGFα
in CCl4 induce hepatic injury. This project is anticipated being completed
before this spring.
Mentor: Deborah A. Nickerson, PhD
Until quite recently, the challenge in
constructing SNP association studies has been simply finding a reasonable number
of SNPs in each candidate gene. Recent advances in sequencing throughput have
solved this problem, but now there are too many SNPs available within each gene
and investigators need to select a subset of SNPs for genotyping. We have been
working on algorithms for SNP selection which can identify a minimal subset of
SNPs which provide maximal information about any given region, starting from
complete sequence data in a modest sized SNP discovery population. The
resulting minimal set should allow investigators to determine whether any common
variant within a region is associated with significant disease risk. We are
working with several groups to extend these principles successfully into large
scale case control studies.
Mentor: Michael E. Rosenfeld, PhD
Since joining the laboratories of Dr. Steve
Schwartz and Dr. Michael Rosenfeld in July 2001, I have focused mainly on two
projects. In general, this work aims to evaluate the effects of prohibition of
macrophage cell death on the progression of late stage atherosclerosis lesions.
Our work is in mice with the hopeful applicability to human disease.
The first project is to determine the effect of
the loss of the MCP-1 receptor (CCR2) on macrophage infiltration into late stage
atherosclerotic lesions, in mice. To do so, we will be performing bone marrow
transplantation of macrophage progenitor cells from CCR2 knockout mice into
irradiated ApoE deficient mice after lesions have already formed.
The second project I am currently working on is
to develop a retroviral vector construct to be used for the overexpression of
intracellular glutathione in mouse macrophages. To do so, I am creating a
construct that contains a macrophage-specific promoter sequence along with
sequences encoding for the two glutathione-synthesis enzymes.
Mentor:
Åke
Lernmark, PhD
Type 1 diabetes mellitus (T1DM) is a major risk
factor for coronary heart disease and other clinical complications. In order to
better study both the direct causes of T1DM and its effects on other diseases,
we are genetically dissecting T1DM in the BioBreeding (BB) rat animal model of
T1DM. Using inbred rat strains that are genetically identical within strains,
but differ significantly between strains, we have genetically isolated a number
of genes, some of which are required for the development of T1DM in BB rats, and
others which protect against the development of T1DM. In particular, we have
isolated and sequenced the ~100kb genomic regions in rat (chromosome 4) and
mouse (chromosome 6) containing the Iddm1 lymphopenia (Lyp) gene,
a gene affecting late T-cell maturation in the thymus, and are now determining
which of the small number of candidates in the region is Lyp by using
resequencing and genomic rescue in transgenic rats. In addition, we are using
further genetic crosses to narrow the regions containing Iddm3 and
Iddm4, rat genes whose Fischer strain alleles show protective effects
against the development of T1DM. We hope to use the identified functions of
these genes, which have been shown genetically to cause or prevent T1DM in BB
rats, to identify the mechanisms involved in the development of T1DM, and later,
its complications.
Mentor: Stephen M. Schwartz, MD, PhD
In recent years, our laboratory has used cDNA
micro-array technology to identify novel molecules that have a potential role in
the regulation of smooth muscle cell migration, proliferation, and vascular
remodeling. Among the genes identified using this technology, our laboratory is
currently focusing on the characterization of the Regulators of G-protein
Signaling (RGS) family during pathological processes such as atherosclerosis,
restenosis and hypertension. However, since little information is known
concerning the role of RGS proteins in vascular biology, multiple approaches
will be required to completely understand the cellular mechanisms that are
regulated by this novel family. Extensive research in the last decade has
demonstrated the conservation of molecular pathways that regulate embryonic
development and cardiovascular disease. For this reason, we will be initiating
developmental studies to gain further insight into the molecular mechanisms that
can regulate vessel growth and smooth muscle cell differentiation. Our studies
will involve the characterization of RGS expression and function during
developmental stages that have been previously established to be critical time
points for vascular growth and remodeling.
Mentor: Peter H. Byers, MD
The fibrillar collagens are the predominant
proteins in the human body and are major components of several organ systems
including the cardiovascular system. Assembly of the triple helical procollagen
molecular is directed by the C-terminal propeptides, yet little is known about
the structure of these domains and the mechanisms that govern these processes.
The major objective of my work is to determine the three dimensional structure
of the trimeric C-propeptide of type III procollagen and then use informatics
strategies to make substitutions for all the different fibrillar collagens. I
will then use this structural data to determine the folding mechanisms and to
better understand how mutations affect these processes and lead to disease.
This overall objective has been broken down into several steps:
1. Clone the C-propeptide of type III
procollagen into expression vectors.
2. Transform yeast with expression vector.
3. Express the C-propeptide of type III
procollagen in yeast.
4. Isolate and purify the expressed trimeric
C-propeptide.
5. Crystallize the trimeric C-propeptide.
6. Determine the structure of the C-propeptide
by x-ray crystallography.
To this point, I have successfully cloned the
C-propeptide into appropriate vectors in frame with an initiation codon,
secretion signal and cleavage site. I have transformed the yeast, Pichia
Pastoris, with the plasmid and confirmed the presence of the procollagen
gene in the yeast genome by PCR. I am preparing to express the recombinant
strains and isolate procollagen C-propeptide.
Publications
during the funding period
1. Pace JM, Kuslich CD, Willing MC, Byers PH.
Disruption of one intra-chain disulphide bond in the carboxyl-terminal
propeptide of the proα1(I) chain of type I procollagen permits slow assembly and
secretion of overmodified, but stable procollagen trimers and results in mild
osteogenesis imperfecta. J Med Genet
2001 Jul;38(7):443-9.
2. Pace JM, Atkinson M, Willing MC, Wallis G,
Byers PH. Deletions and duplications of Gly-Xaa-Yaa triplet repeats in the triple
helical domains of type I collagen chains disrupt helix formation and result in
several types of osteogenesis imperfecta. Hum Mutat 2001
Oct;18(4):319-26.
3. Pace JM, Chitayat D, Atkinson M, Wilcox WR,
Schwarze U, Byers PH. A single amino acid
substitution (D1441Y) in the carboxyl-terminal propeptide of the proalpha1(I)
chain of type I collagen results in a lethal variant of osteogenesis imperfecta
with features of dense bone diseases. J Med Genet. 2002 Jan;39(1):23-9.
4. Byers PH, Schwarze U, Pace JM, Kuslich C,
Pepin M, Valiente E, Atkinson M. Osteogenesis imperfecta: classification,
molecular abnormalities, phenotype-genotype relationships, and implications for
treatment. In: Proceedings of Consensus Conference on Dentinogenesis
Imperfecta (In press).
5. Pace JM, Byers PH. Abnormal type I
procollagen chains bind molecular chaperones in a defect-specific manner and are
degraded by the cytosolic proteasome (in preparation).
Mentor: Daniel E. Sabath, MD, PhD
Current classification systems for B cell
lymphomas categorize these disorders based on morphology and a limited number of
molecular markers. However, each category of lymphoma likely encompasses
multiple biologically distinct processes with different natural histories and
responses to therapy. Theoretically, analyzing the expression of a large number
of markers will result in a lymphoma classification system that is more
predictive of prognosis and therapeutic response. To this purpose, we are
developing a lymphoma diagnostic microarray. Using microarrays of spotted cDNAs,
we examined the expression of ~15,000 human genes in pooled mRNA isolated from
15 benign tonsils, 17 benign lymph nodes, 12 mantle cell lymphomas (MCL), 14
follicular lymphomas (FL), or 16 small lymphocytic lymphomas (SLL). Cy3- and
Cy5-labeled cDNA was prepared from each mRNA pool and hybridized to different
microarrays. Since each microarray contained duplicate cDNA spots, four
fluorescence intensity values (two Cy3 and two Cy5) were obtained per cDNA for
each tissue type. To combine the Cy3 and Cy5 intensity values, we developed a
strategy to normalize Cy3 and Cy5 data based on second-order polynomial
equations. This method provided quadruplicate intensity measurements. We then
used the Student t test to determine the statistical significance of differences
in gene expression. We identified 140 genes that were 8-fold differentially
expressed (p=0.02) in at least one tissue relative to other tissues. Of 14 of
these genes, 11 (79%) were verified to be differentially expressed using real
time quantitative RT-PCR. Several distinct expression patterns were found. Genes
uniquely over-expressed in benign tissue (benign lymph node and tonsil) relative
to all types of malignancy included zinc finger protein 162 and ATP-binding
cassette protein ABCG2. Some genes were expressed preferentially in one lymphoma
type; e.g., crystallin mu and cyclin D1 were over-expressed only in MCL. Some
genes were over-expressed in some lymphomas but not others; e.g. apoliprotein D
was overexpressed in MCL and SLL but not FL. Finally, genes over-expressed in
all three lymphoma subtypes but not in benign tissue (benign lymph node and
tonsil) included MIG (monokine induced by gamma interferon). These genes have
the potential to be new diagnostic markers for lymphoma and will be incorporated
into custom microarrays to evaluate their diagnostic utility. In addition,
understanding the function of these genes will lead to new insights into the
pathophysiology of lymphoma and normal lymphoid tissue.
Mentor: David W. Raible
Zebrafish Cardiac Crest.
The neural crest makes a
critical contribution to the normal development of the heart. Crest derivatives
generate the ectomesenchyme that forms the septa and the outflow tracts. The
neural crest also forms the sensory, sympathetic and parasympathetic ganglia
that regulate heart function. We have been investigating factors that influence
cell fate choices within the vagal neural crest of zebrafish. We have focused on
the role of GDNF family ligands in developing zebrafish. Previous studies in
other vertebrates have shown that GDNF family ligands help specify different
vagal crest derivatives and the ligand binding subunit of the GDNF receptor, GFRa1,
and another GDNF family member receptor binding subunit, GFRa2,
are expressed in rat cardiac tissue (Hiltunen et al. 2000). In the past year we
have cloned the complete open reading frame of two GFRa1
orthologues and one GFRa2
orthologue in zebrafish. We have undertaken in vivo experiments to determine the
function of the zebrafish GFRa1
and GFRa2
orthologues in the developing embryo. We have shown both zebrafish GFRa1
orthologues are together necessary for the normal development of the enteric
nervous system precursors that are derived from the vagal neural crest but they
do not appear to be necessary for normal cardiac crest development. These
results are consistent with our previous results investigating the function of
zebrafish GDNF in vivo (Shepherd et al 2001). We are currently preparing a
manuscript reporting these results. In a second series of studies we have
continued characterize two novel zebrafish mutants that have defects in vagal
neural crest derived structures. Both mutants have abnormal heart development
that may result from defects in the cardiac crest. We are currently preparing a
manuscript detailing the initial characterization of these mutants. In addition
we have recently identified 3 new mutants that effect vagal neural crest derived
structures that also have cardiac defects.
References:
·
Hiltunen JO,
Laurikainen A, Airaksinen MS, Saarma M.
(2000) GDNF family receptors in the embryonic and postnatal rat heart and
reduced cholinergic innervation in mice hearts lacking ret or GFRalpha2. Dev
Dynamics 219:28-39
·
Iain T.
Shepherd, Christine Beattie and David W. Raible.
(2001) Functional analysis of zebrafish GDNF. Dev Biol 231:
420-435
PUBLICATIONS & MANUSCRIPTS IN
PREPARATION 2001
2001 Papers:
·
Iain T.
Shepherd, Christine Beattie and David W. Raible.
(2001) Functional analysis of zebrafish GDNF. Dev Biol 231: 420-435
2001 Abstracts:
·
Shepherd, T.
Linbo, and D. Raible (2001) A
genetic screen to identify zebrafish enteric nervous system mutants.
Abstracts for the Society for Developmental Biology Meeting 2001.
·
Iain T.
Shepherd and David W. Raible
(2001) Characterization and functional analysis of zebrafish GFRa1,
GFRa2
and RET. Abstracts for the Society for Neuroscience Meeting 2001.
2001 Manuscripts in Preparation:
·
Iain T.
Shepherd, David W. Raible
(2001) GFRa1
and RET orthologues are necessary for enteric nervous system development in
zebrafish.
·
Tor Linbo,
Iain T. Shepherd and David W. Raible
(2001) Zebrafish Mutations that affect the differentiation of post otic neural
crest.
Mentor: Cecilia M. Giachelli, PhD
Antagonists of the integrin αvβ3
inhibit angiogenesis by promoting endothelial cell apoptosis. We have
previously shown that the transcription factor NF-κB is specifically activated
when rat aortic endothelial cells were plated on αvβ3
ligands. NF-B activation was necessary for αvβ3
ligand-mediated cell survival. Here we show that rat smooth muscle cells (SMC)
also up-regulate NF-κB activity when adherent to the αvβ3
ligand osteopontin. The aim of this study is to determine structures in the β3
cytoplasmic tail that are involved in the signaling pathway leading to NF-κB
activation. To this end, we created a bicistronic retroviral vector encoding the
human integrin β3,
an internal ribosome entry site, and green fluorescent protein. Rat smooth
muscle cells were then engineered to stably over-express integrin β3
subunit. As detected by flow
cytometry, these cells had increased αvβ3
surface expression compared to cells infected only with the vector. When plated
on osteopontin, cells over-expressing β3
showed enhanced NF-kB activation, which was inhibited with a soluble blocking
antibody to αvβ3.
Currently, we are developing SMCs that over-express human β3
subunits with mutations in the
cytoplasmic tail to determine sequences required for NF-κB activation.
Mentor: Stephen M. Schwartz, MD, PhD
In the past year, Tom Nhan has developed an in
vitro system for characterizing macrophage death; specifically, in the context
of oxidized-LDL mediated toxicity. This current project has two primary goals:
1. Develop a model of macrophage death to
address its role in the progression of necrotic core in atherosclerotic plaques.
2. Identify key inhibitors and promoters of
macrophage death pathways as clinical targets.
The current work has identified that Fas-FasL
pathway, in addition to its primary role in the inflammatory response of
macrophages, also prime macrophages to undergo apoptotic death in response to
oxidized-LDL. The preliminary data from this work has led to one successful
grant for Dr. Mike Rosenfeld, two submitted grants for Dr. Karin Bornfeldt, and
the basis for the recently submitted grant in the lab of Dr. Steve Schwartz.
There are currently two manuscripts in preparation to address the role of
macrophage death and survival.
Mentor: Cecilia M. Giachelli, PhD
The avb3
integrin plays an essential role in angiogenesis by inhibiting endothelial cell
apoptosis. The mechanisms involved in conferring endothelial survival have not
been well defined. Recently, we have shown that
avb3
ligation on rat aortic endothelial cells (RAECs) specifically activates the
transcription factor NF-kB
(Scatena et al, 1998). The protective effect of
avb3
ligands was abolished by inhibiting NF-kB
nuclear translocation. To define the signaling mediators involved in
avb3–mediated
NF-kB
activation we transiently transfected RAECs with an NF-kB
response reporter construct in combination with wild type (WT) and dominant
negative (DN) constructs encoding known upstream mediators of NF-kB
signaling: inhibitor of kappa B kinase alpha (IKKb,
inhibitor of kappa B kinase beta (IKKb,
and NF-kB-inducing
kinase (NIK). Cells plated on the
avb3
ligand, osteopontin (OPN), showed
approximately a 4-fold induction of NF-kB
activity when compared to control cells plated poly-D-lysine (PDL). OPN-mediated
NF-kB
activity was increased upon NIK WT transfection and blocked following NIK DN
transfection. IKKb
appeared to be the main IkB-kinase,
since OPN-induced NF-kB
activity was enhanced in response to IKKb
WT transfection, and blocked in response to IKKb
DN. Finally, IB-a
phosphorylation, was observed in cells plated on OPN but not in cells plated on
PDL as measured by Western blot. These studies suggest that
avb3
ligation stimulates IKKb
activity via NIK, and leads to phosphorylation of IkBa
and subsequent NF-kB
activation. Future studies are aimed at gathering biochemical evidence for NIK/IKK
complex phosphorylation and functional activation, as well as defining the
upstream mediators of NIK activation in response to OPN-avb3
ligation.
Mentor: Daniel Storm, PhD
Cyclic adenosine 3’,5’-monophosphate (cAMP) is generated from ATP by adenylyl
cyclases (AC) in essentially all tissues. This molecule serves as secondary
messenger in the intracellular signal transduction of a wide variety of
extracellular stimuli. Currently, ten different isoforms (1-10) of have been
cloned and multiple isoforms may be expressed in each cell type.
To determine which AC isoform is activated in arterial smooth muscle cells
(SMCs) by prostaglandin E2 (PGE2). Cultured human SMCs when stimulated with
PGE2 (2uM) induced an increased in cAMP accumulation and this can be inhibited
by increase in intracellular calcium induced by A23187 (a calcium ionophore).
This suggests that the cyclase that is activated by PGE2 is type III AC.
Although the AC3 was originally isolated in olfactory neuroepithelium, this
isoform has been identified in other tissues as well and AC3 is the only calcium
inhibited cyclase. This inhibition is through the activation of the calcium-calmodulin
kinase II (CaMKII). This kinase phosphorylated AC3 at ser-1076 leading to the
down regulation of AC3. The enzymatic activity suggests that AC3 is expressed
in SMCs. This was confirmed by immunohistochemistry and western analysis of
human and murine arterial tissues.
This led us to examine the PGE2 stimulation of SMCs in the AC3 knockout mouse.
Due to the extreme low survival rate of the homozygote knockout animals, most of
our experiments were done using SMCs derived from heterozygote animals. Again
PGE2 stimulates cAMP accumulation in wild-type murine SMCs and this level of
stimulation is reduced by ~50% in the AC3 heterozygote cell lines. In addition,
we examined the role of AC3 in PGE2 mediated growth inhibition of SMCs. SMCs
were stimulated to proliferate with PDGF and this proliferation is habited by
PGE2. In wild-type SMCs, PGE2 induces a 65% reduction in DNA synthesis. In the
heterozygote and homozygote AC3 cell lines, PGE2 inhibits DNA synthesis by 35%.
This suggests that AC3 because of its unique regulatory property functions as an
integrator of growth inhibitory signal in SMCs.
These results just been published in the September issue of Journal of
Biological Chemistry.
-Wong, S. T. et al, 2001 JBC, Vol.
276(36):34206-34212
Mentor: Charles E. Murry, M. D., Ph. D.
Skeletal myoblasts transplanted into the heart
can form stable muscle grafts to replace scar tissue formed following myocardial
infarction. Despite its promise in experimental models and preliminary clinical
trials, the success of myoblast transplantation depends on the ability to
generate grafts of predictable and physiologically sufficient size. This has
proved difficult to accomplish even when the number of cells implanted and the
method of injection are kept constant. A method of selectively and reversibly
inducing proliferation in grafted myoblasts post-transplantation may allow more
precise control over graft size and improve cardiac function.
To induce proliferation specifically in
skeletal myoblasts, we created a chimeric receptor composed of a modified FK506
binding protein (FKBP) domain, F36V, fused with the intracellular domain of
fibroblast growth factor receptor-1 (FGFR-1), which we stably expressed in MM14
myoblasts. Treatment with a bivalent F36V ligand (AP20187; ARIAD
Pharmaceuticals) induced proliferation, prevented differentiation, and activated
MAP kinase signaling in transfected cells, similar to treatment with the
endogenous FGFR-1 ligand, basic fibroblast growth factor (bFGF). The dimerizer
had no effect on non-transfected control myoblasts. To determine whether these
effects are reversible, transfected myoblasts were cultured for 30 days in the
presence of AP20187, followed by dimerizer withdrawal. Upon removal of
dimerizer treatment, transfected myoblasts differentiated normally,
downregulated MAPK phosphorylation, and demonstrated reduced BrdU
incorporation. In summary, treatment of transfected myoblasts with a synthetic
dimerizer induces proliferation selectively and reversibly by signaling through
a chimeric growth factor receptor. Future studies will focus on the application
of this technology to stimulate proliferation of transplanted myoblasts in
vivo for cardiac repair.
Publications:
Whitney, M. L., Otto, K., Blau, C. A., Reinecke,
H. and C. E. Murry. Control of myoblast proliferation with a synthetic ligand.
J Biol Chem, in press, 2001.
Podium presentation:
Whitney, M. L. and C. E. Murry. Control of
skeletal muscle proliferation with a synthetic ligand: implications for cardiac
repair. Biomedical Engineering Society, October 4 - 7, 2001, Durham, North
Carolina.
Poster presentation:
Whitney, M. L., Nourse,
M.B. and Murry, C. E. Control of myoblast and endothelial cell proliferation for
cardiac repair. AHA Conference on Molecular, Integrative and Clinical
Approaches to Myocardial Ischemia, August 9 – 11, 2001, Seattle, Washington.
|
UW
Cardiovascular Pathology Training Program