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Richard
K. Assoian, Ph.D.
Professor of
Pharmacology
Member, Graduate Group in Pharmacological Sciences
Member, Cell and Molecular Biology Graduate Group
University of Pennsylvania School of Medicine
Ph.D. (Biochemistry), 1981, University of Chicago
Department of Pharmacology
University of Pennsylvania School of Medicine
Room 167, Johnson Pavilion
3620 Hamilton Walk
Philadelphia, PA 19104-6084
Phone: 215-898-7157; Fax: 215-573-5656
e-mail: rka@pharm.med.upenn.edu
Click for the laboratory website
Research Summary
Mammalian cells decide whether or not to proliferate by sensing growth regulatory
signals in their extracellular environment. Mitogenic growth factors and the
extracellular matrix (ECM) provide these extracellular signals for most cell
types. My laboratory is interested in understanding how growth factors and the
ECM cooperate to regulate cell proliferation. Our particular focus has been
in understanding the anchorage requirement for growth, i.e. why cells must be
attached to an ECM in order to proliferate. Experimentally, we ask how cell
adhesion affects the activity of the nuclear enzymes called cyclin-dependent
kinases (cdks), because these are the enzymes responsible for mediating progression
through the cell cycle.
Over the past
several years, we have shown that the anchorage requirement for proliferation
is restricted to the G1 phase of the cell cycle and that each of the G1 phase
cell cycle events typically attributed to growth factors (induction of cyclin
D1, activation of cyclin E-cdk2, downregulation of the cdk inhibitory proteins,
p21 and p27, phosphorylation of the retinoblastoma protein, and induction of
cyclin A) actually requires a co-regulation by growth factors and the ECM. These
results have allowed us to devise a model showing how growth factors and the
ECM cooperate to confer the phenotypes of mitogen- and anchorage-dependence
to cells.
We are now
asking how signaling by growth factor receptors and integrins (the receptors
for ECM proteins) regulates the cyclin-cdk events outlined above. We are identifying
cytosolic signal transduction pathways that are regulated coordinately by growth
factor receptors and integrins and asking if these pathways lead to regulation
of cyclin D1 and p21 in the nucleus. The ERK subfamily of MAP kinases is an
important aspect of this work since ERK activation leads to the induction of
both cyclin D1 and p21. Similarly, we are trying to identify cis-elements on
the cyclin D1 and p21 promoters that are regulated by growth factor receptor
and integrin signaling.
We have also
become interested in identifying signaling systems that regulate the timing
of cell cycle events. For example, we are asking if there are specific signals
that restrict the induction of cyclin D1 to mid-G1 phase. These studies have
led us to the rho family GTPases and have nucleated a strong interest in signaling
by the cytoskeleton and how cytoskeletal-dependent signaling events modulate
receptor-dependent signaling events.
Finally, we
are examining the regulation of cyclin-dependent kinases in atherosclerosis.
Aortic smooth muscle cells are thought to undergo a conversion from a quiescent
("contractile") to proliferating ("synthetic") state in
atherogenesis. Much attention has been paid to the roles of local mitogenic
and antimitogenic factors in inducing smooth muscle cell proliferation, but
synthetic SMCs characteristic of the atherosclerotic lesion also have an abnormal
ECM and undergo changes in the expression of their cell-surface integrins. We
are asking if the concepts we establish from studying signaling by the ECM and
integrins in fibroblasts are applicable to understanding the onset of arterial
smooth muscle cell proliferation in atherosclerosis.
Key References
Bottazzi, M.E., Zhu,
X., Bohmer, R.M., and Assoian, R.K. (1999) Sequential regulation of p21cip1
expression by growth factors and the extracellular matrix reveals a role for
transient ERK activity in G1 phase. J. Cell Biol. 146:1255-1264.
Roovers, K.R.,
and Assoian, R.K. (2000) Integrating the MAP kinase signal into the G1 phase
cell cycle machinery. Bioessays 22: 818-826.
Assoian, R.K.
and Schwartz, M.A. (2001) Coordinate signaling by integrins and receptor tyrosine
kinases in the regulation of G1 phase cell cycle progression. Curr. Op. Genetics
and Dev. 11: 48-53.
Aplin, A.E.,
Stewart, S.A., Assoian, R.K., and Juliano, R.L. (2001) Integrin-mediated adhesion
regulates ERK nuclear translocation and phosphorylation of Elk-1. J. Cell Biol.
153:273-282.
Bottazzi, M.E., Buzzai,
M., Brechot, C, and Assoian, R.K. (2001) Distinct effects of mitogens and the
cytoskeleton on CREB and pocket protein phosphorylation controls the extent
and timing of cyclin A promoter activity. Molecular and Cellular Biology 21:
7607-7616.
Welsh, C.F., Roovers, K.,
Villanueva, J., Zhao, X., Schwartz, M.A, and Assoian, R.K. (2001) Timing of
cyclin D1 expression within G1 phase is controlled by Rho. Nature Cell Biology
3: 950-957.
Honors, Awards and Professional
Activities
Member, CBY-2/CDF-3 study
section, NIH, 1997-2001
Chair, 2002 Signaling by Adhesion Receptors Gordon Research Conference
Editor, Journal of Cell Science
Associate Editor, Molecular Biology of the Cell
Editorial Board Member, Molecular and Cellular Biology
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Paul
H. Axelsen, M.D.
Associate Professor
of Pharmacology and Medicine
Member, Graduate Group in Pharmacological Sciences
Member, Biochemistry and Molecular Biophysics Graduate Group
University of Pennsylvania School of Medicine
Associate, Johnson Foundation for Molecular Biophysics
M.D., 1982, Mayo Medical School
Department
of Pharmacology
University of Pennsylvania School of Medicine
Room 105, Johnson Pavilion
Room 131, John Morgan Building
3610 Hamilton Walk
Philadelphia, PA 19104-6084
Phone: 215-898-9238; Fax: 215-573-2236
email: axe@pharm.med.upenn.edu
Click here for the laboratory website
Research
Summary
My laboratory is seeking to understand how pharmacologically active molecules
recognize their target sites of action, and how they accomplish their function
after binding to their target site. We use computer graphics modeling, molecular
dynamics simulation, various forms of optical spectroscopy (infrared and fluorescence),
NMR, and X-Ray crystallography, with a view towards the rational design of new
pharmacological agents for use against infections, cardiovascular, and neurological
diseases. Further information is available on the laboratory web site at http://axe2.med.upenn.edu.
Key References
Membrane-Induced Folding of Cecropin A. Silvestro, L., and P.H. Axelsen.
Biophysical Journal 79:1465-1477, 2000.
The Structural
Biology of Ligand Recognition by Vancomycin. Loll, P.J., and P.H. Axelsen. Annual
Reviews of Biophysics and Bioengineering 29:265-289, 2000.
Accelerated
Accumulation of Amyloid b Proteins on Oxidatively Damaged Lipid Membranes. Koppaka,
V., and P.H. Axelsen. Biochemistry 39:10011-10016, 2000.
Prothrombinase
Acceleration by Oxidatively Damaged Phospholipids. Weinstein, E.A., Li, H.,
Lawson, J.A., Rokach, J., FitzGerald G.A., and Axelsen, P.H. Journal of Biological
Chemistry 275:22925-22930, 2000.
The Structure
of Human Lipoprotein A-I: Evidence for the "Belt" Model. Koppaka,
V., Silvestro, L., Engler, J., Brouillette, C.G., and P. H. Axelsen. Journal
of Biological Chemistry, 274:14541-14544, 1999.
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Garret
A. FitzGerald, M.D.
Robinette Professor
of Cardiovascular Medicine
Elmer Bobst Professor of Pharmacology
Chair, Department of Pharmacology
Director, Center for Experimental Therapeutics
Director, Clinical Research Center
Member, Graduate Group in Pharmacological Sciences
Member, Genomics Graduate Group
M.D., B.Ch. (1974), University College, Dublin
Department
of Pharmacology
University of Pennsylvania School of Medicine
Room 153, Johnson Pavilion
3620 Hamilton Walk
Philadelphia, PA 19104-6084
Phone: 215-898-1184; Fax: 215-573-9135
e-mail: garret@spirit.gcrc.upenn.edu
Research
Interests
The Pharmacology
of COX Inhibition
A particular interest is to elucidate the cardiovascular biology of COXs and
its implication for the use of aspirin, traditional NSAIDs and COX-2 inhibitors.
Studies in mice and zebrafish complement studies of the genetic and environmental
factors which modulate response to this interesting class of compounds.
Key References
McAdam, B.F., Catella-Lawson, F., Mardini, I.A., Kapoor, S., Lawson, J.A.,
and FitzGerald, G.A. Systemic biosynthesis of prostacyclin by cyclooxygenase
(COX)-2: The human pharmacology of a selective inhibitor of COX-2. Proc. Natl.
Acad. Sci. (USA) 96: 272-277, 1999.
Rocca, B.,
Spain, L., Pure, E., Patrono, C., and FitzGerald, G.A. Distinct and coordinated
roles of prostaglandin H-synthesis 1 and 2 in T-cell development. J. Clin.
Invest. 103: 1469-1477, 1999.
Practico,
D., Cyrus, T., Li, H., Rokach, J., and FitzGerald, G.A. Acceleration of atherogenesis
by COX-1 dependent prostanoid formation in LDL receptor knockout mice. Proc.
Natl. Acad. Sci. USA 98: 3358-3363, 2001.
FitzGerald,
G.A. and Patrono, C. The coxibs, selective inhibitors of cyclooxygenase-2.
N. Engl. J. Med. 345:433-443, 2001
Grosser,
T., Cheskis, E., Pack, M.A., and FitzGerald, G.A. Developmental expression
of functional cyclooxygenases in zebrafish. Proc. Natl. Acad. Sci. USA 99:
8418-8423, 2002. [Article Commentary: Prescott, S. M. and Yost, H. J. The
COXss of Danio: From mechanistic model to experimental therapeutics. Proc.
Natl. Acad. Sci. USA 99: 9084-9086, 2002.]
Eicosanoid
Receptor Biology
Prostacyclin has potent vasodilator and platelet inhibitory properties. However,
its role in vivo is poorly understood. Mice deficient in the prostacyclin
receptor and mice overexpressing and lacking the thromboxane receptor are
being employed to investigate this phenomenon. Current research also aims
to investigate the molecular pharmacology of transporters, membrane and nuclear
receptors activated by prostanoids, their interaction with proteins and integration
into signaling pathways.
Key References
Vezza, R., Habib, A., and FitzGerald, G.A. Differential coupling of the thromboxane
receptor isoforms with Gh. J. Biol. Chem. 274: 12774-12779, 1999.
Smyth, E.M.,
Austin, S.C., Reilly, M.P., and FitzGerald, G.A. Internalization and sequestration
of the human prostacyclin receptor. J. Biol. Chem 275: 32037-32045, 2000.
Cheng, Y.,
Austin, S.C., Rocca, B., Koller, B.H., Coffman, T.M., Lawson, J.A. and FitzGerald,
G.A. Role of prostacyclin in the cardiovascular response to thromboxane A2.
Science 269: 539-541, 2002. [Commentary: Vane, J. R. Biomedicine. Back to an
aspirin a day? Science 296: 539-541, 2002.] Abstract
Full
Text
Isoeicosanoids
Isoeicosanoids are free radical catalyzed products of arachidonic acid with
potential utility as indices of oxidant stress. Methods for analyzing representatives
of distinct families of isomers continue to be developed, mass spectrometry.
The mechanisms of formation of these compounds and their potential activities
as incidental ligands at G protein coupled and nuclear receptors, are being
characterized. Alterations in isoeicosanoid generation are beging related
to indices of oxidant injury to DNA and proteins and functional outcome in
syndromes of oxidant injury in model systems and in humans.
Key References
Praticò, D., Tangirala, R.K., Rader, D.J., Rokach, J., and FitzGerald,
G.A. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis
in ApoE-deficient mice. Nature Medicine 4: 1189-1192, 1998.
Meagher,
E.A., Barry, O.P., Burke, A., Lucey, M., Rokach, J., and FitzGerald, G.A.
Alcohol-induced generation of lipid peroxidation products in humans. J. Clin.
Invest. 104: 805-813, 1999.
Audoly, L.P.,
Rocca, B., Fabre, J.-E., Koller, B.H., Thomas, D., Loeb, A., Coffman, T.M.,
and FitzGerald, G.A. Cardiovascular responses to the isoprostanes, iPF2ά-III
and iPE2-III
are mediated via the thromboxane A2 receptor in vivo. Circulation
101: 2833-2840, 2000.
Meagher,
E.A., Barry, O.P., Lawson, J.A., Rokach, R., and FitzGerald, G.A. Effects
of Vitamin E on lipid peroxidation in healthy volunteers. J. Am. Med. Assn.
285: 1178-1182, 2001.
Honors & Professional
Activities
Established Investigator,
American Heart Association (1985-1990)
Member, Association of American
Physicians
Fellow, American Association for the Advancement of Science
Member, American Society for Clinical Investigation
Past &
Present Study Section Memberships
NIH
AHA
Wellcome Trust
Editorial
Board Memberships
Journal of Clinical Investigation
Circulation
Trends in Cardiovascular Medicine
Journal of Biological Chemistry (past)
Journal of Pharmacology and Experimental Therapeutics (past)
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Colin
Funk, Ph.D.
Professor of
Pharmacology and Medicine
Member, Graduate Group in Pharmacological Sciences
Member, Center for Experimental Therapeutics
University of Pennsylvania School of Medicine
Ph.D. (Experimental Medicine) 1985, McGill University (Canada)
Department
of Pharmacology
University of Pennsylvania School of Medicine
Room 814, BRBII/III
421 Curie Boulevard
Philadelphia, PA 19104-6160
Phone: 215-898-0254; Fax: 215-573-9004
e-mail: colin@spirit.gcrc.upenn.edu
Research
Interests
Eicosanoids comprise a class of lipid mediators that include leukotrienes and
prostaglandins, which derive from the actions of lipoxygenase or cyclooxygenase
enzymes. Eicosanoids exert potent actions in airways, vascular tissue, inflammatory
cells and the reproductive system via specific membrane G protein-coupled receptors.
We have generated several lipoxygenase deficient mouse strains by gene targeting
and are in the midst of preparing additional transgenic mice to explore lipoxygenase,
cyclooxygenase and leukotriene receptor functions. Atherosclerosis, asthma and
inflammation models are studied in these mice. The cell and molecular biology
of intracellular lipoxygenase trafficking and the pharmacology of cysteinyl
leukotriene receptors are being investigated.
Key References
Chen, X-S., Sheller, J.R., Johnson, E.N., and Funk, C.D. Role of leukotrienes
revealed by targeted disruption of the 5-lipoxygenase gene. Nature 372: 179-182
(1994).
Johnson, E.N., Brass, L.,
and Funk, C.D. Increased platelet sensitivity to ADP in mice lacking platelet-type
12-lypoxygenase. Proc. Natl. Acad. Sci. USA, 95: 3100-3105 (1998).
Kennedy, C.R.J., Zhang,
Y., Brandon, S., Guan, Y., Coffee, K., Funk, C.D., Magnuson, M.A., Oates, J.A.,
Breyer, M.D., and Breyer, R.M. Salt-sensitive hypertension and reduced fertility
in mice lacking the prostaglandin EP2 receptor. Nature Medicine, 5: 217-220
(1999).
Martin, V., Ronde, P.J.,
Lerner, E., Wong, A., and Funk, C.D. Leukotriene binding, signaling and analysis
of HIV co-receptor function in mouse and human leukotriene B4 receptor transfected
cells. J. Biol. Chem. 274: 8597-8603 (1999).
Cyrus, T., Witztum, J.L.,
Rader, D.J., Tangirala, R.K., Fazio, S., Linton, M.F., and Funk, C.D. Disruption
of the 12/15-lipoxygenase gene diminishes atherosclerosis in apoE deficient
mice. J. Clin. Invest. 103: 1597-1604 (1999).
Huang, J.T., Welch, J.S.,
Ricote, M., Binder, C., Willson, T.M., Kelly, C., Witztum, J.L., Funk, C.D.,
Conrad, D., and Glass, C.K. Interleukin-4-dependent production of PPAR ligands
in macrophages by 12/15-lipoxygenase. Nature, 400: 378-382 (1999).
Chen, X-S. and Funk, C.D.
The N-terminal "beta-barrel" domain of 5-lipoxygenase is essential
for nuclear membrane translocation. J. Biol. Chem. 276: 811-818 (2001).
Cyrus, T., Pratico, D.,
Witztum, J.L., Rader, D.J., FitzGerald, G.A. and Funk, C.D. Absence of 12/15-lipoxygenase
expression decreases lipid peroxidation and atherogenesis in apolipoprotein
E deficient mice. Circulation, 103, 2277-2282 (2001).
Martin, V., Sawyer, N.,
Stocco, R., Unett, D., Lerner, M.R., Abramovitz, M., Funk, C.D. Molecular cloning
and functional characterization of murine cysteinyl leukotriene D4 receptors.
Biochem. Pharmacol., 62, 1193-1200 (2001).
Miller, Y.I., Chang, M.K.,
Funk, C.D., Feramisco, J.R., Witztum, J.L. 12/15-Lipoxygenase enhances site-specific
actin polymerization in macrophages phagocytosing apoptotic cells. J. Biol.
Chem., 276, 19431-19439 (2001).
Hui, Y., Yang, G., Galczenski,
H., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., and Funk, C.D. The murine
cysteinyl leukotriene 2 (cysLT2) receptor: cDNA and genomic cloning, alternative
splicing, and in vitro characterization. J. Biol. Chem., 276, 47489-47495 (2001).
Funk, C.D. and Cyrus, T.
12/15-lipoxygenase, oxidative modification of LDL and atherogenesis. Tr. Cardiov.
Med., 11, 116-124 (2001).
Funk, C.D. Prostaglandins
and leukotrienes: advances in eicosanoid biology. Science, 294, 1871-1875 (2001).
Kulkarni, S., Das, S., Funk,
C.D. Murray, D., and Cho, W. A molecular basis of specific subcellular localization
of the C2-like domain of 5-lipoxygenase. J. Biol. Chem. 277, 13167-13174 (2002).
Awards, Honors and Memberships
in Honorary Societies
Research Career Development Award - NIH-NHLBI (1991 - 1996)
Member, American Association for the Advancement of Science (1995 - present)
American Society of Biochemistry and Molecular Biology (1996 - present)
Editorial Board Member, Journal of Biological Chemistry (1996 - 2000)
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David
R. Manning, Ph.D.
Professor of
Pharmacology
Member, Graduate Group in Pharmacological Sciences
University of Pennsylvania School of Medicine
Ph.D. (Physiology/Pharmacology), 1980, University of California, San Diego
Department
of Pharmacology
University of Pennsylvania School of Medicine
Room 114, John Morgan Building
3620 Hamilton Walk
Philadelphia, PA 19104-6084
Phone: 215-898-1775; Fax: 215-573-2236
e-mail: manning@pharm.med.upenn.edu
Research
Summary
The major focus of the laboratory is the process by which the binding of hormones
to cell-surface receptors is translated into the regulation of target enzymes
and ion channels. The laboratory is specifically interested in pathways of transduction
defined by GTP-binding regulatory proteins (G proteins). Current studies include
mapping linkages among receptors and G proteins in intact cells, defining post-translational
modifications of G protein subunits that influence targeting and protein-protein
interactions, and exploring the roles of novel G proteins. Considerable effort
in the laboratory is devoted toward understanding the relevance of G proteins
to cell proliferation. Several growth factors, such as thrombin, bombesin, and
lysophosphatidic acid, for example, utilize G proteins as the sole means by
which they stimulate cell replication. Attempts to identify the G proteins activated
by these growth factors and the pathways engaged to achieve DNA synthesis, especially
those relevant to the regulation of transcription factors, are underway. Interests
of the laboratory also extend to the development of methods to evaluate the
communication of receptors with G proteins, with an emphasis placed on determinants
of efficacy. We have been able to monitor variations in the activation of individual
G proteins directly using GTP(gamma)S-binding coupled to immunoprecipitation.
Among the covalent modifications being investigated are N-myristoylation, palmitoylation,
and phosphorylation. These modifications occur at or near the amino terminus
of G protein alpha subunits and are relevant to membrane anchorage and interactions
with beta/gamma heterodimers. Finally, the laboratory maintains an active interest
in Gz, a G protein specific to platelets and neurons. Issues of function are
being pursued with a bias toward roles in differentiation and/or stimulus-secretion
coupling.
Key References
Chen, C.A., and D.R. Manning.
Regulation of G(alpha)i palmitoylation by activation of the 5-hydroxytryptamine-1A
receptor. J. Biol. Chem. 275:23516-23522, 2000.
Fan, X., L.F. Brass, M.
Poncz, F. Spitz, P. Maire, and D.R. Manning. The alpha subunits of Gz and Gi
interact with the eyes absent transcription cofactor Eya2, preventing its interaction
with the Six class of homeodomain-containing proteins. J. Biol. Chem. 275:32129-32134,
2000.
Chen, C.A., and D.R. Manning.
Regulation of G proteins by covalent modification. Oncogene 20:1643-1652, 2001.
Siehler, S., Y. Wang, X.
Fan, R.T. Windh, and D.R. Manning. Sphingosine 1-phosphate activates nuclear
factor-(kappa)B through Edg receptors: Activation through Edg-3 and Edg-5, but
not Edg-1, in human embryonic kidney 293 cells. J. Biol. Chem. 276:48733-48739,
2001.
Windh, R.T., and D.R. Manning.
Analysis of G protein activation in Sf9 and mammalian cells by agonist-promoted
[35S]GTP(gamma)S binding. Meth. Enzymol. 344:3-14, 2002.
Manning, D.R. Measures of
efficacy using G proteins as endpoints: Differential engagement of G proteins
through single receptors. Mol. Pharmacol. In press.
Honors and Professional
Activities
Established Investigator, American Heart Association (1989-1994)
Dean's Award for Excellence in Graduate Student Training (1994)
Cellular Biology and Physiology-2 Study Section (1994-1999; Chairman, 1997-1999)
Editorial Board, The Journal of Biological Chemistry (1995-2004)
Lindback Award for Distinguished Teaching (1999)
Editorial Board, Molecular Pharmacology (2002-2005)
Chair-elect, Division of Molecular Pharmacology (2002)
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Vladimir
R. Muzykantov, M.D., Ph.D.
Assistant Professor
of Pharmacology
University of Pennsylvania School of Medicine
M.D., 1980, Moscow Medical School
Ph.D., (Biochemistry) 1985, Russian Cardiology Research Center (Moscow)
Institute for Environmental Medicine
University of Pennsylvania School of Medicine
1st Floor, John Morgan Building
3620 Hamilton Walk
Philadelphia, PA 19104-6068
Phone: 215-898-9823; Fax: 215-898-0868
e-mail: muzykantov@pharm.med.upenn.edu
Research
Interests: Drug/gene targeting and vascular biology
Particular areas of interest/expertise include: recognition of the surface antigens
on the normal or pathologically altered endothelial cells; vascular inflammation
and leukocytes adhesion; mechanisms of oxidative stress and antioxidant protection
of the endothelium; evaluation of specific markers of endothelial injury; immunotargeting
of antioxidant enzymes, fibrinolytics and genes to the pulmonary endothelium;
pulmonary pathophysiology; lung ischemia/reperfusion; prolongation of enzymes
life-time in the bloodstream; controlled elimination of radiolabeled antibodies
or pathogens from the bloodstream; exploration of red blood cells as carriers
for prolonged circulation and site-specific delivery of drugs (fibrinolytics
and anticoagulants); regulation of fibrinolysis and complement; mechanisms and
regulation of intracellular targeting/trafficking of drugs.
Research
Summary
Laboratory is focused on several projects. First is the targeting of drugs (enzymes
either degrading or generating oxidants, fibrinolytics, interferon, antisense
oligos and genes) to the pulmonary vascular endothelium. The purpose is to develop
strategies for controlled site-specific delivery of a drug to the defined subcellular
compartments of the pulmonary endothelium. For example, genetic material must
be delivered into the nucleus, antioxidants must accumulate in the cytoplasm,
and fibrinolytics must avoid internalization. We therefore study how carrier
antibodies and their derivatives recognize endothelium, and characterize cellular
trafficking and local effects of the targeted agents in cell cultures, perfused
animal lungs and in intact animals. Our research includes identification of
the molecules localized on the surface of endothelium useful as targets for
drug delivery to either normal or pathologically challenged endothelium. Endothelium-specific
antigens may serve as such targets. Affinity carriers that are currently explored
in our laboratory include monoclonal antibodies (and their fragments) to: angiotensin-converting
enzyme (ACE), thrombomodulin and surface adhesion molecules, ICAM, PECAM, P-
and E-selectins. We have characterized carriers and their modifications providing:
i) a drug with an affinity to endothelium (recognition and targeting) and, ii)
drug delivery in a proper cellular compartment (sub-cellular addressing). Targeting
to either surface (by non-internalizable carriers) or intracellularly has been
documented in cell culture, perfused lungs and in rodents in vivo.
Second, we
explore red blood cells (RBC) as natural carriers for drugs. We have developed
an original methodology for effective conjugation of large amounts of a drug
(e.g., fibrinoytic enzymes or receptors for plasminogen activators) on RBC,
without loss of biocompatibility of the complex. Conjugation provides prolongation
of half-life of plasminogen activators in vivo by orders of magnitude and offers
specific transfer of the conjugated protein (tPA, uPA-receptor) to the pulmonary
endothelium. Both mechanism of the transfer (tentatively via exchange of GPI-anchored
membrane proteins between RBC and endothelium) and potential therapeutic applications
of RBC-conjugated fibrinolytics (treatment/prevention of pulmonary embolism/deep
vein thrombosis) are in the focus of the research. We also explore RBC as carriers
for intracellular drug delivery in phagocyte cells in the reticuloendothelial
tissue (liver and spleen) and endothelial cells.
Key References
V.Muzykantov, E.Atochina, H.Ischiropoulos, S.Danilov and A.Fisher (1996) Immunotargeting
of antioxidant enzymes to the pulmonary endothelium. Proc.Natl.Acad.Sci.USA,
93, 5213-5218.
V.Muzykantov, E.Barnathan, E.Atochina, S.Danilov and A.B.Fisher (1996) Targeting
of antibody-conjugated plasminogen activators to the pulmonary vasculature.
J.Pharmacol.Exp.Therap., 279:1026-1034.
E.Atochina, I.Balyasnikova, S.Danilov, D.Granger, A.Fisher and V.Muzykantov
(1998) Immunotargeting of catalase to ACE or ICAM-1 protects perfused rat lungs
against oxidative stress. Am.J.Physiol.(Lung)., 19:L806-L817.
V.Muzykantov, M.Christofidou, I.Balyasnikova, D.Harshaw, L.Schultz, A.Fisher
and S.Albelda (1999) Streptavidin facilitates internalization and pulmonary
targeting of an anti-endothelial cell antibody (PECAM): a strategy for intraendothelial
drug delivery. Proc.Natl.Acad.Sci.USA, 96:2379-2384.
A.Scherpereel, R.Wiewrodt,
M.Christofidou-Solomidou, R.Gervais, J-C.Murciano, S.M.Albelda and V.R.Muzykantov
(2001) Cell-selective intracellular delivery of a foreign enzyme to endothelium
in vivo using vascular immunotargeting FASEB.J., 15:416-426.
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Domenico
Praticò, M.D.
Research Assistant
Professor of Pharmacology
M.D., (1998) University of Rome "La Sapienza," School of Medicine,
Rome, Italy
Department
of Pharmacology
University of Pennsylvania School of Medicine
Room 812, BRBII/III
421 Curie Boulevard
Philadelphia, PA 19104-6160
Phone: 215-573-6641; Fax: 215-573-9004
e-mail: domenico@spirit.gcrc.upenn.edu
Research
Summary
Oxidant Stress and Atherogenesis
Atherosclerosis is the leading cause of death among persons with a Western
lifestyle. Although elevated plasma levels of low-density lipoprotein (LDL)
is clearly a major risk factor for cardiovascular disease, it appears that LDL
contributes to the atherosclerotic process only after having been oxidatively
modified (ox-LDL). Several lines of evidence implicate ox-LDL as an atherogenic
agent. However, our ability to obtain direct evidence in support of this hypothesis
has been limited by the paucity of methods which permit investigation of the
oxidant process in vivo. Isoprostanes (iPs) are a newly described class of lipids,
which are produced by free radical catalyzed peroxidation of polyunsaturated
fatty acids. We developed analytical methods to measure specific members of
this class. Because of their mechanism of formation and chemical stability,
they afford the unique opportunity to investigate non-invasively the role of
lipid peroxidation and oxidant stress as a mechanism of disease, and to select
rational doses of putative antioxidant drugs and vitamins for clinical evaluation.
We have examined the effects of a natural antioxidant, vitamin E, on a mouse
model of atherosclerosis, the apolipoprotein (apo) E knockout (KO) mouse. We
found that, by contrast with the wild type animals, apoE KO have age-dependent
increases of their urinary, plasma and vascular levels of iPs, which correlated
with the evolution of the aortic vascular lesions. When apoE KO mice received
vitamin E, a significant reduction in urinary and plasma iP levels with values
similar to wild type animals was observed as early as eight weeks after treatment
began. Vitamin E suppressed the increased vascular levels of iPs and retarded
the development of atherosclerosis by 50%, despite persistent hyper-cholesterolemia.
These studies offer the first demonstration that lipid peroxidation plays a
functional role in atherogenesis in vivo and provide "proof of principle"
that urinary iPs can be used as a non-invasive, biochemical basis for dose titration
and selection of patients in interventional trials with antioxidant. We have
now confirmed and extended these observations in another mouse model of atherosclerosis,
the LDL-receptor KO. Our current studies are designed to address the role of
lipid peroxidation in sustaining established atherosclerosis. Furthermore, we
are exploring the molecular and cellular mechanisms involved in advanced atherosclerosis.
Oxidant
Stress and Neurodegeneration
Several compelling lines of evidence have implicated oxidative stress and free
radical damage in the pathogenesis and possibly etiology of Alzheimer's disease
(AD). Oxidant stress seems to be involved in all of the known pathogenic mechanisms
governing the disease, including cytoskeletal phosphorylation, apoE genotype
and amyloid cascade. Oxidative damage in the central nervous system tissue will
prominently manifest as lipid peroxidation because of this tissue's enrichment
in polyunsaturated fatty acids. We have shown that iPs are increased specifically
in affected brain regions and in the ventricular cerebrospinal fluid (CSF) of
AD patients, as compared with carefully characterized control subjects. In order
to investigate whether this increase is an inevitable consequence of neurodegeneration
or if oxidative damage can occur early in the course of AD, we investigated
iP biosynthesis in patients with clinical diagnosis of AD. We found that AD
patients have increased iP levels in the urine, plasma and CSF compared to controls.
These levels were highly correlated with "classical biomnarker" of
AD pathology (CSF tau and amyloid), with measure of cognitive and functional
impairment, and with the number of apoE ,4 allele. This suggests that lipid
peroxidation is increased early in the course of AD, that iP might be a useful
biomarker of brain oxidative damage in AD. We are now using a mouse model of
AD amyloidosis (Tg 2576) in order to investigate mechanisms responsible for
the increase in lipid peroxidation and whether this is secondary to brain amyloid
deposition or they are two independent features of the disease.
Platelets
and Thromboxane Atherogenesis
Thromboxane (Tx) and Prostacyclin (PGI2) are biologically active lipid mediators
produced by the metabolism of arachidonic acid through the cyclooxygenase pathway.
Tx formation and action on platelets and vascular cells has been implicated
in cardiovascular disease and atherogenesis. By contrast, PGI2 inhibits platelet
activation and counteracts the vascular effects of Tx. The role of Tx in atherogenesis
has been established in randomized controlled clinical trials with platelet-inhibitors
drugs such as aspirin. Much less is known about the potential contribution of
platelet activation to atherogenesis. We wished to determine whether inhibitors
of platelet activation and Tx formation in vivo would reduce atherosclerosis
progression and whether inhibition of PGI2 would accelerate the disease in the
fat-fed LDL R KO. We used indomethacin, which suppressed platelet function and
Tx formation ex vivo and in vivo and partially inhibited PGI2 biosynthesis,
and nimesulide which specifically depressed PGI2 but had no effect on Tx and
platelet function. Neither drug altered serum lipids. Indomethacin reduced the
extent of atheroscelrosis by 55% whereas nimesulide failed to increase the rate
of atherogenesis. These studies suggest that platelet-derived Tx contributes
significantly to atherogenesis. A related area of research interest addresses
the hypothesis that expression level and activity of the thromboxane receptor
(TP) is also of functional relevance to atherogenesis, by using pharmacological
and genetic approaches. We are addressing the hypothesis that specific TP antagonism
retards the initiation and progression of atherogenesis and modulates platelet
and vascular function in the LDLR KO mouse. Furthermore, we shall cross the
TP knockout mouse with the LDLR KO mouse to address the hypothesis that TP deletion
retards the development and the extent of atherosclerosis in the LDLR KO mouse.
Finally, we shall cross mice that overexpress the vascular TP with the LDLR
KO mouse to address the hypothesis that increased TP expression and activity
accelerates the development and the extent of atherosclerosis in LDLR KO. Integration
of these studies using pharmacological tools and transgenic animals will allow
us to determine the functional relevance of the TP in atherogenesis and vascular
biology, issues of broad clinical importance.
Key References
Praticò, D., Tangirala, R.K., Hörkkö, S., Witztum, J.L.,
Palinski, W., FitzGerald, G.A. Circulating autoantibodies to oxidized cardiolipin
correlate with isoprostane F2"-VI levels and the extent of atherosclerosis
in ApoE-deficient mice: Modulation by Vitamin E. Blood 97:459-464, 2001.
Praticò,
D., Cyrus, T., Zhang, Z.B., Li, H., FitzGerald, G.A. Acceleration of Atherogenesis
by COX-1-dependent Prostanoid Formation in LDL Receptor Knockout mice. Proc.
Natl. Acad. Sci. USA 98: 3358-3363, 2001.
Praticò,
D., Clark, C.M., Lee, M-Y.V., Trojanowski, J.Q., Rokach, J., FitzGerald, G.A.
Increased 8,12-iso-iPF2"-VI in Alzheimer's Disease: Correlation of a non-invasive
index of lipid peroxidation with disease severity. Ann. Neurology 48: 809-812,2000.
Praticò,
D., Iuliano, L., Amerio, G., Tang, L.X., Rokach, J, Sabatino, G., Violi, F.
Down's syndrome is associated with increased 8,12-iso-iPF2"-VI levels:
Evidence for enhanced ipid peroxidation in vivo. Ann. Neurology 48: 795-798,2000.
Praticò,
D., Cyrus, T., Li, H., FitzGerald, G.A. Endogenous biosynthesis of Thromboxane
and Prostacyclin in 2 distinct murine models of atherosclerosis. Blood 96:3823-3826,
2000.
Praticò,
D., Rokach, J., Tangirala RK. Brains of aged Apolipoprotein E-deficient mice
have increased levels of F2-isoprostanes, in vivo markers of lipid peroxidation.
J. Neurochem. 73: 736-741, 1999.
Praticò,
D., Tangirala, R.K., Rader, D.J., Rokach, J. and FitzGerald, G.A. Vitamin E
suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient
mice. Nat. Med. 4:1189-92, 1998.
Praticò,
D., Barry, O.P., Lawson, J., Adiyaman, M., Huang, S-W., Khanapure, H., Iuliano,
L., Rokach, J. and FitzGerald, G.A. IPF2a-I: A novel index of lipid peroxidation
in humans. Proc. Natl. Acad. Sci. USA 95:3449-54, 1998.
Praticò,
D., Lee V.M-Y., Trojanowski, J.Q., Rokach, J. and FitzGerald, G.A. Increased
F2-isoprostanes in Alzheimer's disease: Evidence of enhanced lipid peroxidation
in vivo. FASEB J. 12:1777-83, 1998.
Praticò,
D., Iuliano, L., Mauriello, A., Spagnoli, L., Lawson, J.A., Maclouf, J., Violi,
F. and FitzGerald, G.A. Localization of distinct F2-isoprostanes in human atherosclerotic
lesions. J. Clin. Invest. 100:2028-34, 1997.
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Wenchao
Song, Ph.D.
Associate Professor
of Pharmacology
Member, Center For Experimental Therapeutics
Member, Graduate Group in Pharmacological Sciences
University of Pennsylvania School of Medicine
Ph.D. (Biochemistry), 1989, University of Wales (United Kingdom)
Department
of Pharmacology
University of Pennsylvania School of Medicine
Room 1351, BRBII/III
421 Curie Boulevard
Philadelphia, PA 19104-6160
Phone: 215-573-6641; Fax: 215-573-9004
e-mail: song@spirit.gcrc.upenn.edu
Research Summary
Vascular and autoimmune disease biology of membrane complement regulatory
proteins
Complement is a form of natural
immunity that plays an essential role in host defense. However, activated complement
must be carefully regulated to avoid autologous tissue damage. Prevention of
autologous complement attack is in part achieved by specific membrane regulatory
proteins on the cell surface. Decay-accelerating factor (DAF, CD55) and CD59
are two key membrane complement regulatory proteins on mammalian cells. The
roles of CD55 and CD59 in preventing complement-mediated vascular injury and
autoimmune end organ damage are being investigated in our laboratory using single
and double knockout mice that are deficient in CD55 or/and CD59.
Role of
estrogen metabolism in intracrine and paracrine estrogen regulation
Elicitation of biological responses by estrogen in target tissues requires
the presence of estrogen receptor as well as receptor-active ligand in the local
microenvironment. While much attention has been devoted to the study of the
receptors in estrogen target tissues, the concept is emerging that tissue estrogen
sensitivity may also be regulated by ligand availability through metabolic transformation
in situ. Estrogen sulfotransferase catalyzes the specific sulfoconjugation and
inactivation of estrogen and is expressed in a number of estrogen target tissues.
Using a knockout mouse that is deficient in the estrogen sulfotransferase gene,
we are studying the physiological role of estrogen sulfation in intracrine and
paracrine estrogen regulation.
Key References
Sun, X., Funk,
C.D., Deng, C., Sahu, A., Lambris, J.D., and Song, W.-C. Role of decay-accelerating
factor in regulating complement activation on the erythrocyte surface as revealed
by gene targeting. Proc. Natl. Acad. Sci. USA 96: 628-633. (1999).
Qian, Y. M.,
Qin, X., Miwa, T., Sun X., Halperin, J. A. and Song, W.-C. Identification and
functional characterization of a new gene encoding the mouse terminal complement
inhibitor CD59. J. Immunology. 165:2528-2534. (2000).
Song, W.-C.,
Sarrias, M. R. and Lambris, J. D. Complement and innate immunity. Immunopharmacology
49: 187-198. (2000).
Song, W.-C.
and Melner, M H. Editorial: Steroid transformation enzymes as critical regulators
of steroid action in vivo. Endocrinology 141: 1587-1589. (2000).
Qian, Y. M.,
Tong, M.H., Sun, X., Li, X., Richa, J. and Song, W.-C. Targeted disruption of
the mouse estrogen sulfotransferase gene reveals a role of estrogen metabolism
in intracrine and paracrine estrogen regulation. Endocrinology 142: 5342-5350
(2001).
Sogabe, H.,
Nangaku, M., Miwa, T., Ishibashi, Y., Wada, T., Fujita, T., Sun, X., Madaio,
M.P. and Song, W.-C. Increased susceptibility of decay-accelerating factor (DAF)
deficient mice to anti-GBM glomerulonephritis. J. Immunology 167: 2791-2797
(2001).
Miwa, T. and
Song, W.-C. Membrane complement regulatory proteins: insight from animal studies
and relevance to human diseases. Int. Immunopharmacology 1(3): 445-459 (2001).
Miwa, T., Zhou,
L., Hilliard, B., Molina, H. and Song, W.-C. Crry but not CD59 and DAF is indispensable
for murine erythrocyte protection in vivo from spontaneous complement attack.
Blood 99: 3707-3716. (2002).
Tong, M. H.
and Song, W.-C. Estrogen sulfotransferase: discrete and androgen-dependent expression
in the male reproductive tract and demonstration of an in vivo function in the
mouse epididymis. Endocrinology 143: 3144-3151 (2002).
Miwa, T., Maldonado,
M. A., Sun, X., Cai, D.W., Werth, V., Madaio, M. P., Eisenberg, R. A. and Song,
W.-C. Deletion of decay-accelerating factor (DAF, CD55) exacerbates autoimmune
disease development in MRL/lpr mice. Am. J. Pathol. (In press) (2002).
Molina, H., Miwa, T., Zhou,
L., Hilliard, B., Mastellos, D., Maldonado, M.A., Lambris, J.D. and Song, W.-C.
Complement-mediated clearance of erythrocytes: mechanism and delineation of
the regulatory roles of Crry and DAF. Blood (In press) (2002).
Honors and
Professional Activities
Established Investigator, American Heart Association (2000-2003)
Editorial Board, Endocrinology (2002-2005)
Organizing Committee, Complement Associated Diseases, Animal Models and Therapeutics
Workshop, Santorini, Greece, Oct 10-14, 2001
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Steven
A. Thomas, M.D., Ph.D.
Assistant Professor,
Pharmacology
Member, Graduate Group in Pharmacological Sciences
Member, Neuroscience Graduate Group
University of Pennsylvania School of Medicine
M.D., Ph.D. (Neuroscience), 1991, University of Michigan
Department
of Pharmacology
University of Pennsylvania School of Medicine
Room 103, John Morgan Building
3620 Hamilton Walk
Philadelphia, PA 19104-6084
Phone: 215-573-4950; Fax: 215-573-2236
e-mail: thomas@pharm.med.upenn.ed
Research
Summary
Broadly, the lab studies the development and physiology of the mammalian brain.
One goal is to define the systems that contribute to specific behaviors and
to understand the mechanisms that underlie these behaviors. Such knowledge will
ultimately permit the prevention and treatment of mental illness. Recent advances
in transgenic technology allow the analysis of specific genetic alterations
in the context of the whole organism. The ability to add, delete or modify genes
is particularly useful in the analysis of complex organ systems such as the
brain, where roughly half of all genes are thought to be uniquely expressed.
The lab focuses on the adrenergic nervous system in which norepinephrine (NE)
and epinephrine are the classic neurotransmitters. By genetically eliminating
the biosynthetic enzyme for NE, dopamine b-hydroxylase (DBH), mutant mice that
completely lack NE and epinephrine have been created. These mice are conditional
mutants in that NE can be restored to the adrenergic terminals by supplying
a synthetic amino acid precursor of NE, dihydroxyphenylserine. The lab is pursuing
several fundamental observations that resulted from the creation of these mutant
mice, including the essential role of NE in fetal development and the critical
role of NE in the expression of maternal behavior. In addition, the lab is investigating
the roles of NE in neuronal development; synaptic plasticity, learning and memory;
arousal, circadian activity and sleep/wake cycle; and mechanisms of antidepressant
action. A variety of techniques are being utilized, including molecular genetics,
gene expression assays, immunohistochemistry, autoradiography, electrophysiology
and behavioral tests. Because dopamine (DA) is the endogenous precursor of NE,
the adrenergic terminals release DA instead of NE in the Dbh-/- mice. As a weak
agonist at the adrenergic receptors, DA may ameliorate potential phenotypes
due to the absence of NE. Alternatively, ectopic release of DA may cause phenotypes
that are not due to the absence of NE. To address these questions, mice are
being created that have neither DA nor NE in their adrenergic terminals. This
will be achieved by knocking out the first enzyme in the pathway, tyrosine hydroxylase
(TH), and then restoring TH expression to the dopaminergic neurons specifically
by knocking in the TH gene to the dopamine transporter (DAT) locus. This model
will also be conditional in that NE will be restored by supplying the product
of TH, L-DOPA.
Key References
Thomas, S. A., Matsumoto, A. M., and Palmiter, R. D. Noradrenaline is essential
for mouse fetal development. Nature 374: 643-646, 1995.
Thomas, S.
A. and Palmiter, R. D. Mice lacking noradrenaline and adrenaline have reduced
cold tolerance, normal adiposity and increased metabolism and food intake. Nature
387: 94-97, 1997.
Thomas, S.
A. and Palmiter, R. D. Disruption of the dopamine b-hydroxylase gene suggests
a role for norepinephrine in motor function, learning, and memory. Behav. Neurosci.
111: 579-589, 1997.
Thomas, S.
A. and Palmiter, R. D. Impaired maternal behavior in mice lacking norepinephrine
and epinephrine. Cell 91: 583-592, 1997.
Thomas, S.
A., Marck, B. T., Palmiter, R. D., and Matsumoto, A. M. Restoration of norepinephrine
and reversal of phenotypes in mice lacking dopamine ß-hydroxylase. J.
Neurochem. 70: 2468-2476, 1998.
Cryan, J. F.,
Dalvi, A., Jin, S. -H., Hirsch, B. R., Lucki, I. and Thomas, S. A. (2001) Use
of dopamine ß-hydroxylase deficient mice to determine the role of norepinephrine
in the mechanism of action of antidepressant drugs. J. Pharmacol. Exp. Ther.:
298:947-953.
Honors and
Professional Activities
Young Investigator Award, National Alliance for Research on Schizophrenia and
Depression
I.J. Kopin Fellowship
for Excellence in Catecholamine Research, Foundation for Catecholamine Research
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Alexander
Steven Whitehead, D.Phil.
Professor of
Pharmacology
Member, Graduate Group in Pharmacological Sciences
Director, Center for Pharmacogenetics
University of Pennsylvania School of Medicine
D.Phil. (Biochemistry/Genetics), 1982, Oxford University
Center for Pharmacogenetics
University of Pennsylvania School of Medicine
Room 153, Johnson Pavilion
3610 Hamilton Walk
Philadelphia, PA 19104-6084
Phone: 215-898-2332; Fax: 215-573-9135
e-mail: aswhitehead@pharm.med.upenn.edu
Research
Summary
The control of expression of the major acute phase "Serum Amyloid A"
proteins (SAAs). This family of proteins has been extensively mapped and characterized
in a number of vertebrate species, and the members are differentially regulated
by cytokines during inflammation. Two SAAs are induced by up to a thousandfold
and are the focus of our interest. We are defining the precise manner in which
the principal upregulation, which is via increased transcription, is mediated.
In addition, we are investigating the second mechanism whereby SAA production
is regulated, namely translational modulation and mRNA stability, both of which
appear to be influenced by elements in the SAA mRNA 5' and 3' untranslated regions.
SAA is the precursor of the protein that is deposited in the inflammation-driven
condition secondary amyloidosis, and our ultimate aim is to devise strategies
to counteract its overproduction in inflammatory diseases. Recently, we have
initiated a study to establish whether the hyperinducible cytokine responsive
SAA promoter can be used to direct the production of therapeutic molecules (e.g.
cytokine antagonists), in gene therapy of inflammatory diseases such as rheumatoid
arthritis.
The role of
genetic variants of enzymes of the methionine metabolic pathway in human disease.
We and others have established that a common mildly dysfunctional "thermolabile"
variant of the folate-dependent enzyme methylenetetrahydrofolate reductase (MTHFR)
is significantly associated with a number of conditions ranging from neural
tube defects to cardiovascular disease. There are also intriguing hints that
the variant may be under-represented among the very elderly which suggests that
its pathogenic activity may be of a high enough order to constitute a risk factor
that predisposes to premature death. We are using developing cell culture and
animal models to better understand the mechanism whereby the MTHFR variant exerts
its pathogenic effects and are continuing to perform genetic studies of various
disease categories. As the dysfunction in MTHFR activity can be largely corrected
with folate supplementation, there are some interesting public health and health
policy issues that arise from this work.
Key References
Gallagher, P.M, Meleady, R., Shields, D.C., Tan, K.S., McMaster, D., Rozen,
R., Evans, A., Graham, I.M. and Whitehead, A.S.: Homocysteine and risk of coronary
heart disease: evidence for a common gene mutation. Circulation 94: 2154-2158,
1996.
McCormack, C.C., Hobson,
A.H., Doyle, S., Jackson, J., Kilty, C. and Whitehead, A.S. Generation of soluble
recombinant human acute phase serum amyloid A2 (A-SAA2) protein and its use
in development of an A-SAA specific ELISA. J. Immunol. Methods 198: 101-110,
1996.
Jensen, L.E., Hiney, M.,
Shields, D.C., Uhlar, C.M., Lindsay, A.J. and Whitehead, A.S. Acute phase proteins
in salmonids: evolutionary analysis and acute phase response. J. Immunol. 158:
384-392, 1997.
Uhlar, C.M., Grehan, S.,
Steel, D.M., Steinkasserer, A. and Whitehead, A.S. Use of the acute phase serum
amyloid A2 (SAA2) gene promoter in the analysis of pro- and anti-inflammatory
mediators: differential kinetics of SAA2 promoter induction by IL-1b and TNFa
compared to IL-6. J. Immunol. Methods 203: 123-130, 1997.
Grehan, S., Uhlar, C.M.,
Sim, R.B., Herbert, J., and Whitehead, A.S. Expression of a biologically active
recombinant mouse interleukin-1 receptor antagonist (IL-1ra) and its use in
vivo to modulate aspects of the acute phase response. J. Immunol. 159: 369-378,
1997.
Molloy, A.M., Daly, S.,
Mills, J.L., Kirke, P.N., Whitehead, A.S., Ramsbottom, D., Conley, M.R., Weir,
D.G. and Scott, J.M. Thermolabile variant of 5,10-methylenetetrahydrofolate
reductase associated with low red cell folates: implications for folate intake.
Lancet 349: 1591-1593, 1997.
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William
M. Armstead, Ph.D.
Research Associate
Professor
Department of Anesthesia
Department
of Anesthesia
University of Pennsylvania School of Medicine
34th and Spruce Streets
Philadelphia, PA 19104-4283
Phone: 215-573-3674; Fax: 215-349-5078
e-mail: armsteaw@mail.med.upenn.edu
Research
Interests
Control of cerebral hemodynamics during physiologic and pathologic conditions
such as traumatic brain injury and cerebral ischemia/reperfusion.
Research
Techniques
Closed cranial window for measurement of pial artery diameter and collection
of cortical periarachnoid CSF for vasoactive metabolite concentration determination
by RIA, fluid percussion brain injury, global cerebral ischemia, radiolabelled
microsphere regional cerebral blood flow determination.
Research
Summary
My laboratory is interested in characterizing the role of several vasoactive
systems (opioids, endothelin, prostaglandins) in impaired cerebral hemodynamic
control following fluid percussion brain injury, a mimic of shaken impact syndrome,
as a function of age in an in vivo model. My laboratory is also interested in
characterizing the contribution of these same vasoactive systems to altered
cerebral hemodynamic control following global cerebral hypoxia/ischemia in a
newborn animal model. The latter injury model mimics the hypoxia/ischemia that
may occur in the neonate with problems associated with delivery or respiratory
management post delivery.
Key Words
opioids, newborn,
brain injury, endothelin, nitric oxide, cerebral circulation
Key References
Armstead W. M. Role
of endothelin-1 in age dependent cerebrovascular hypotensive responses after
brain injury. Am. J. Physiol, 274:H1884-H1894, 1999.
Armstead W.
M. Relationship between nociceptin/orphanin FQ and cerebral hemodynamics following
hypoxia/ischemia in piglets. Am J Physiol, 278:H477-H483, 2000.
Kulkarni, M.
and W. M. Armstead. Superoxide generation links nociceptin/orphanin FQ (NOC/oFQ)
release to impaired N-methyl-D-aspartate cerebrovasodilation after brain injury.
Stroke, 31:1990-1996, 2000.
Armstead, W.
M. Role of nociceptin/orphanin FQ in age dependent cerebral hemodynamic effects
of brain injury. J Neurotrauma, 17:751-764, 2000.
Armstead, W.
M. Noc/oFQ and NMDA contribute to piglet hypoxic ischemic hypotensive cerebrovasodilation
impairment. Ped Res, 51:586-591, 2002.
Kulkarni M. and W. M. Armstead,
Relationship between NOC/oFQ, dynorphin, and COX-2 activation in impaired NMDA
cerebrovasodilation after brain injury. J Neurotrauma, 19:965-973, 2002.
Honors and Professional
Activities
Established Investigator,
American Heart Association (1994-1999)
Editorial Board, Microcirculation (1998-present)
Brain 2 Study Section, American Heart Association (2002-present)
Councilor, Division of Cardiovascular Pharmacology, American Soc of Pharm &
Exp Ther (2002-present)
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Lawrence
F. Brass, M.D., Ph.D.
Professor of
Medicine and Pharmacology
Associate Director, Center for Experimental Therapeutics
Associate Dean for the Combined Degree and Physician Scholars Programs
Member, Graduate Group in Pharmacological Sciences
Member, Cell and Molecular Biology Graduate Group
Member, Biochemistry and Molecular Biophysics Graduate Group
University of Pennsylvania School of Medicine
Ph.D. (Biochemistry), 1975, Case Western Reserve University
M.D., 1977, Case Western Reserve University
Hematology-Oncology
Division
University of Pennsylvania School of Medicine
Room 915, BRBII/III
421 Curie Boulevard
Philadelphia, PA 19104-6160
Phone: 215-573-3540; Fax: 215-573-7039
e-mail: brass@mail.med.upenn.edu
Research Summary
The studies in my laboratory focus on the molecular mechanisms
underlying vascular biology and pathology. Despite major medical advances over
the past 20 years, atherosclerotic cardiovascular disease and vascular thrombosis
remain among the major causes of death and chronic illness in the United States.
Our studies are directed toward understanding the intracellular events that
underlie these disorders in vascular cells. To do this, we study the signal
transduction pathways that allow platelets to respond to extracellular events.
Topics that we currently have under investigation include the role of Eph
tyrosine kinase receptors and their ligands in the late events of platelet
activation and Signaling events downstream of Gi family members in platelets.
We use a variety of methods and approaches and make extensive use of genetically-engineered
mice and in vivo models of thrombosis.
Keywords
vascular biology, thrombosis, atherosclerosis, signal transduction, platelets,
endothelial cells, G protein coupled receptors, integrins.
Key References
J. Yang, J. Wu, M.A. Kowalska, A. Dalvi, N. Prevost, P.J. O'Brien, D.R. Manning,
M. Poncz, I. Lucki, J.A. Blendy and L.F. Brass. Loss of signaling through the
novel G protein, Gz, results in abnormal platelet activation and altered responses
to psychoactive drugs. Proc. Nat. Acad. Sci. 97: 9984-89, 2000.
Brass, L.F.:
Small cells, big issues. Nature 409: 145-146, 2000.
Woulfe,D.,
Yang,J. and Brass,L.: ADP and platelets: the end of the beginning. J. Clin.
Invest. 107: 1503-1505, 2001.
P.J. O'Brien,
M. Molino, M. Kahn and L. Brass: Protease-activated receptors: theme and variations.
Oncogene 20: 1570-1581, 2001.
Brass,L.F.:
Platelets and proteases. Nature 413: 26-27, 2001.
D. Woulfe,
H. Jiang, R. Mortensen, J. Yang and L.F. Brass. Activation of Rap1B by Gi family
members in platelets. J. Biol. Chem. 277: 23382-23390, 2002.
N. Prevost,
D. Woulfe, T. Tanaka and L.F. Brass. Interactions between Eph kinases and ephrins
provide a novel mechanism to support platelet aggregation once cell-to-cell
contact has occurred. Proc. Nat. Acad. Sci 99: 9219-9224, 2002.
Awards,
Honors and Membership in Honorary Societies
Established Investigator,
American Heart Association, 1985-90
American Society for Clinical Investigation, 1987
Excellence in Teaching Award, Penn Medical School Class of 1999, 1998
Outstanding Teaching Award, Penn Medical School Class of 2001, 1998
IXth Biennial Award for Contributions to Hemostasis Research, International
Society of Thrombosis and Hemostasis, 1999
Excellence in Teaching Award, Penn Medical School Class of 2002, 2000
Outstanding Course Director Award, Penn Med Class of 2003, 2001
Christian R. and Mary F. Lindback Foundation Award for Distinguished Teaching,
University of Pennsylvania, 2001
Outstanding Course Director Award, Penn Med Class of 2004, 2001
Editorial Boards
Journal of Biological Chemistry,
1994-1999
Journal of Biological Chemistry, 2001-2006
Blood, 1996-2001
Arteriosclerosis, Thrombosis and Vascular Biology, 1999-2000
Membership in National
Scientific Review Panels
Merit Review Board in Hematology,
U.S. Veterans Administration, 1987-1990
Chair, Merit Review Board in Hematology, U.S. Veterans Admin., 1989-1990
Vascular Biology Review Board, American Heart Association, 1988-1990
Hematology II Study Section, NIH HLBI, 1997-2000
Conference organization
Co-Chair, FASEB Conference
on Thrombin and Vascular Medicine, 1997
Chair, Gordon Conference on Hemostasis, 2000
Chair, Committee on Platelets, American Society of Hematology, 2001
FASEB Conference on Proteases and Protease Receptors, 2005
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Sriram
Krishnaswamy, Ph.D.
Associate Professor
of Pediatrics
University of Pennsylvania School of Medicine
Stokes Investigator, Children's Hospital of Philadelphia
Ph.D. (Biochemistry), 1984, Syracuse University
Howard Hughes
Medical Institute
Children's Hospital of Philadelphia
Room 302F, Abramson Pediatric Research Center
34th and Civic Center Boulevard
Philadelphia, PA 19104-4318
Phone: 215-590-3346; Fax: 215-590-2320
e-mail: skrishna@mail.med.upenn.edu
Research
Summary
Blood coagulation proceeds through a series of discrete proteolytic activation
steps that convert a precursor zymogen to an active trypsin-like serine proteinase
that participates in the succeeding catalytic step. This cascade transduces
a small initiating stimulus into a highly amplified clotting response. Although
the serine proteinase possesses the necessary catalytic machinery, in several
of the coagulation reactions, proteolytic activity is greatly increased when
the serine proteinase binds to a protein cofactor on a membrane surface to form
a enzyme complex stabilized by reversible macromolecular interactions. This
process localizes the reactions to the site of injury and further amplifies
the clotting response.
Despite high
homology to trypsin, the serine proteinases of coagulation catalyze the cleavage
of their protein substrates with high and defined specificity. Our research
is directed towards investigating the molecular basis for the specificity of
these enzyme complexes and how the reversible interactions that stabilize the
enzyme complex modulate and enhance the catalytic function of the proteinase.
Our studies
with prothrombinase, the enzyme complex that catalyses thrombin formation from
prothrombin, are largely focused on the mechanisms of substrate specificity
of the enzyme complex and relating the physical biochemistry of the interactions
that stabilize the complex to specific contributions to enzymic function. Specificity
of the coagulation reactions has implicitly been assumed to arise from the specific
recognition of residues surrounding the site of cleavage with the active site
of the enzyme. Instead, we have shown that binding specificity for prothrombin
by prothrombinase is achieved by a docking of the substrate to extended macromolecular
recognition sites (exosites) on the enzyme that are removed from the catalytic
site. This initial docking interaction tethers the substrate to the enzyme and
facilitates otherwise unfavorable interactions between elements surrounding
the scissile bond and the active site of the proteinase leading to bond cleavage.
Our current efforts are directed towards using fluorescence spectroscopy, thermodynamics,
protein chemistry and recombinant derivatives of the substrate and enzyme to
resolve the determinants of binding specificity in this enzyme system and the
contributions of binding interactions to the enhanced catalytic efficiency of
the enzyme complex.Similar findings have also been made in the activation of
factor X by the VIIa-tissue factor complex.
This suggests
that exosite binding may be a common strategy by which narrow and distinctive
binding specificity for protein substrates is achieved by otherwise homologous
serine proteinases. In the case of VIIa-TF, our studies focus on the unique
and unexplained mechanism of enzyme inhibition by tissue factor pathway inhibitor
(TFPI). TFPI is a product dependent inhibitor of the enzyme complex that regulates
the initiation of coagulation. We are investigating the possibility that exosite
binding by factor Xa serves to deliver the inhibitor to the VIIa-TF complex.
We are also investigating the basis for the atypical ability of VIIa-TF to act
on two protein substrates (factors X and IX) with high catalytic efficiency.
Key References
Betz, A., Vlasuk, G.P., Bergum, P.W. and Krishnaswamy, S. (1997) Selective Inhibition
of the Prothrombinase Complex: Factor Va alters macromolecular recognition of
a tick anticoagulant peptide mutant by factor Xa. Biochemistry 36, 181-191
Krishnaswamy,
S. and Walker, R.K. (1997) Contribution of the Prothrombin Fragment 2 Domain
to the Function of Factor Va in the Prothrombinase Complex. Biochemistry 36,
3319-3330
Krishnaswamy,
S. and Betz, A. (1997) Exosites Determine Macromolecular Substrate Recognition
by Prothrombinase. Biochemistry, 36, 12080 -12086
Baugh, R.J.,
Broze, G.J. and Krishnaswamy, S. (1998) Regulation of Extrinsic Pathway Factor
Xa Formation by Tissue Factor Pathway Inhibitor. J. Biol. Chem., 273, 4378-4386
Betz, A. and
Krishnaswamy, S. (1998) Regions remote from the site of cleavage determine macromolecular
substrate recognition by the prothrombinase complex. J. Biol. Chem., 273, 10709-10718
Baugh, R.J.,
Dickinson, C.D., Ruf, W. and Krishnaswamy, S. (2000) Exosite Interactions Determine
the Affinity of Factor X for the Extrinsic Xase Complex. J. Biol. Chem., 275,
28826-28833
Boskovic, D.
and Krishnaswamy, S. (2000) Exosite Binding Tethers the Macromolecular Substrate
to the Prothrombinase Complex and Directs Cleavage at Two Spatially Distinct
Sites. J. Biol. Chem., 275, 38561-38570
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Robert
J. Levy, M.D.
William J.
Rashkind Professor of Pediatrics and Professor of Pharmacology
Member, Graduate Group in Pharmacological Sciences
University of Pennsylvania School of Medicine
M.D., 1970, Johns Hopkins University School of Medicine
Department
of Pediatrics
Children's Hospital of Philadelphia
Room 302F, Abramson Pediatric Research Center
34th and Civic Center Boulevard
Philadelphia, PA 19104-4318
Phone: 215-590-3346; Fax: 215-590-2320
e-mail: levyr@email.chop.edu
Research
Summary
Heart Valve Disease: Research programs underway concerning the developmental
basis for aortic valve disease, mechanistic studies of progression of calcific
aortic stenosis, serotonin mechanisms in heart valve disease, and novel biomaterials
for heat valve prostheses.
Gene delivery
stents: plasmid or viral vectors configured in sustained release preparations
onto vascular stents for site specific vascular gene therapy.
Myocardial
gene delivery systems for cardiac arrhythmias: Investigations of controlled
release plasmid and viral vector systems for sustained expression of mutant
ion channels in re-entrant pathways.
Key References
Webb, C.L., Nguyen, N.M., Schoen, F.J., Levy, R.J.: Calcification of allograft
aortic wall in a rat subdermal model. Pathophysiology and inhibition by Al3+
and aminodiphosphonate preincubations, American Journal of Pathology 141(2):487-496,
1992.
Labhasetwar, V., Underwood,
T., Schwendeman, S.P., Levy, R.J.: Iontophoresis for modulation of cardiac drug
delivery, Proceedings of the National Academy of Science USA 92:2612-2616, 1995.
Vyavahare, N., Hirsch, D.,
Lerner, E., Baskin, J.Z., Schoen, F.J., Bianco, R., Kruth, H.S., Zand, R., Levy,
R.J.: Prevention of bioprosthetic heart valve calcification by ethanol preincubation:
efficacy and mechanisms. Circulation 95:479-488, 1997.
Alferiev I, Vyavahare NR,
Song CX, Levy RJ. Elastomeric polyurethanes modified with geminal bisphosphonate
groups. Journal of Polymer Science. 39:105-116, 2000.
Klugherz BD, Jones PL, Cui
X, Chen W, Meneveau NF, DeFelice S, Connolly J, Wilensky R, Levy RJ. Gene delivery
from a DNA-controlled release stent in porcine coronary arteries. Nature Biotechnology,
18:1181-1184, 2000.
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Bruce
T. Liang, M.D.
Associate Professor
of Medicine and Pharmacology
Member, Graduate Group in Pharmacological Sciences
University of Pennsylvania School of Medicine
M.D., 1982, Harvard Medical School
e-mail: langb@mail.med.upenn.edu
Research
Summary
Regulation, function and signaling mechanism of adenosine receptors: Studies
are carried out to determine the cellular and molecular mechanisms underlying
the regulation and function of adenosine A1 and A3 receptors, which coexist
on the cardiac myocyte. The molecular events following receptor activation include
activation of phospholipase C and D as well as tyrosine kinase.
Function of
a novel cardiac P2 purinergic receptor: Studies are conducted to evaluate the
function of a novel cardiac P2 purinergic receptor mediating stimulation of
transsarcolemmal calcium entry and myocyte contractility. Molecular events following
activation of this receptor include a protein kinase C-mediated phosphorylation
of a ligand-gated ion channel. Other studies are carried out to determine the
function of and the mechanism underlying a P2Y receptor-mediated glucose transport.
Mechanisms
of ischemic preconditioning: Using a cardiac myocyte model of simulated ischemia,
studies are conducted to determine the cellular and molecular mechanisms by
which ischemic preconditioning is achieved.
Key References
Podrasky, E., Xu, D. and Liang, B.T.: A novel phospholipase C- and cyclic AMP-independent
positive inotropic mechanism via the P2 purinergic receptor in cardiac ventricular
myocyte. American J. Physiol. 273 (Heart Circ. Physiol. 42): H2380-H2387, 1997.
Dougherty, C, Barucha, J.,
Jacobson, K.A., and Liang, B.T.: Cardiac myocytes rendered ischemia-resistant
by expressing the human adenosine A1 or A3 receptor. FASEB J. 12: 1785-1792,
1998.
Liang, B.T. and Jacobson,
K.A.: A physiological role of adenosine A3 receptor: sustained cardioprotection.
Proc. Natl. Acad. Sci. U.S.A. 95: 6995-6999, 1998.
Stambaugh, K., Elliott,
G., Jacobson, K.A., and Liang, B.T.: Additive effects of late preconditioning
produced by monophosphoryl lipid A and the early preconditioning mediated by
adenosine receptors and KATP channel. Circ. 100:3300-3307, 1999.
Ruppelt, A., Liang, B.T.
and Soto, S.: Cloning, functional characterization and developmental distribution
of a ligand-gated P2 purinoceptor channel. Progress in Brain Research 120: 81-90,
1999.
Parsons, M., Young, L.,
Lee, J-E., Jacobson, K.A., and Liang, B.T.: Distinct cardioprotective effects
of adenosine mediated by differential coupling of receptor subtypes to phospholipase
C and D. FASEB J. 14: 1423-1431, 2000.
Jacobson, K.A., Xie, R.,
Young, L., Chang, L. and Liang, B.T.: A novel pharmacological approach to treating
cardiac ischemia: binary conjugates of A1 and A3 adenosine receptor agonists.
J. Biol. Chem. 275: 30272-30279, 2000.
Mei, Q. and Liang, B.T.:
Activation of P2 purinergic receptor enhances cardiac contractility in isolated
perfused rat and mouse hearts. Am. J. Physiol, in press, 2001.
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