Linda Shapiro, Ph.D.
Center for Vascular Biology
University of Connecticut Health Center
263 Farmington Avenue
Farmington, CT 06030-3501
|Linda Shapiro, Ph.D.
Mallika Ghosh, Ph.D.
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Research in the Shapiro Laboratory concentrates on
understanding the regulation and function of cell surface
peptidases in the angiogenic endothelium of tumors and in
cardiovascular disease. Numerous cell surface peptidases are
strikingly upregulated on angiogenic endothelial cells
leading to the hypothesis that these may functionally
cooperate in enzymatic cascades to regulate angiogenesis and
endothelial cell function. While the angiogenic significance
of proteases that cleave large proteins (such as the matrix
metalloproteases) is well documented, increasing evidence
supports a role for peptidases (metAP2, CD13, APA, PSMA) as
angiogenic regulators as well. The fact that these enzymes
metabolize small peptide substrates suggests that small
molecule regulators of angiogenesis exist which have yet to
be identified and whose mechanisms are unknown. Indeed, we
have shown that individually, two peptidases, CD13 and PSMA,
are potent regulators of angiogenesis, and our investigation
of their regulatory mechanisms and their possible
interaction is the current focus of the laboratory.
We have shown that inhibition of CD13/APN blocks
endothelial morphogenesis and invasion and we continue to
examine the molecular mechanisms responsible for its
angiogenic regulatory capabilities, particularly its role in
invasion. Recent studies from our lab suggest that CD13
regulates signal transduction pathways leading to invasion
by participating in plasma membrane organization via its
interaction with membrane cholesterol. Further
investigations have indicated that CD13 also functions as an
adhesion molecule where it mediates inflammatory cell
interactions as well as endothelial/extracellular matrix
interactions in a signal-transduction-dependent manner, which
has strong implications for regulation of inflammatory
leukocyte trafficking and angiogenic cell invasion.
Furthermore, CD13 is expressed on adult pluripotent stem
cells and may play a role in stem cell trafficking as well.
Finally, high levels of CD13 are found in the serum of
patients with certain types of cancers and inflammatory
diseases and we are currently investigating the mechanisms
regulating its release from the cell surface and CD13’s
utility as a serum biomarker of chemoprevention in breast
cancer and myocardial infarction. We have recently produced
a conditional CD13 knockout mouse and are actively
characterizing CD13’s contribution to various physiologic
and pathologic processes by specifically inactivating the
gene in specific tissues.
Our investigation into the function of a second cell
surface peptidase, PSMA, has shown that this peptidase also
regulates cell signaling, albeit by an apparently different
mechanism. Investigation of PSMA’s regulation of endothelial
cell adhesion led to the very interesting discovery that
PSMA is a component of a complex regulatory loop that
controls integrin signaling and PAK1 activation. Invasion
studies with PSMA-null cells showed that PSMA regulates cell
invasion by controlling signaling from beta1 integrins to
focal adhesion kinase (FAK) and PAK1. We showed that PSMA
interacts with the actin-binding protein filamin A, and
disruption of this interaction decreases the peptidase
activity of PMSA and phosphorylation of PAK1 in cultured
cells. The interaction of PMSA with the cytoskeleton via
filamin A allows a feedback signal from integrin beta1 and
PAK that holds PMSA activity in check. Inhibition of PAK by
expression of a peptide corresponding to its autoinhibitory
domain enhanced the association of PMSA with filamin A, thus
increasing its peptidase activity. These studies suggest an
extracellular-matrix-derived, small molecule PSMA substrate
that superactivates beta1 integrins, thus regulating
angiogenesis and cell invasion. The manuscript describing
this work was recently featured as a ‘highlight of the
recent literature’ by the editors of Science.
Investigation into the regulation and function of these
molecules will increase our understanding of molecular
mechanisms controlling blood vessel formation in a variety
of diseases such as cancer, heart disease, inflammatory
disorders, diabetic retinopathy, and arthritis.
Linda Shapiro plans to extend her laboratory’s
observations on the roles of cell surface peptidases in
angiogenesis and cell invasion. We have conditional CD13
knockout animals and complete PSMA knockouts, so both in
vitro and in vivo studies are planned. For CD13, experiments
will be directed toward its role as an adhesion molecule on
endothelial cells and monocytes and its potential regulation
of inflammation, inflammatory diseases and cancer. Specific
areas of research will elucidate its interacting adhesion
partners, its place in the established paradigm of leukocyte
trafficking, characterization of the signal transduction
cascades induced by CD13 and investigation of its
internalization and re-expression on the membrane. In
addition, studies will continue on the mechanism of CD13
shedding and the function of soluble CD13 in inflammation
and stem cell trafficking. Studies regarding PSMA will focus
on its role as an angiogenic regulator and its control of
cell invasion via integrin signaling. Specifically we will
concentrate on identifying its angiogenic/integrin
activating peptide substrate, the concept of regulation of
angiogenesis by small extracellular matrix derived-peptide
fragments and the role of PSMA in prostate tumorigenesis and
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Current projects and potential rotation projects:
- Investigation of signal transduction pathways
induced by CD13 ligation in endothelial cells that lead
to increased cell adhesion and their roles in
inflammatory leukocyte trafficking.
- Structure/function analysis of CD13’s signaling and
adhesion functions using chimeric mouse/human molecules.
- Characterization of the role of CD13 in inflammation
in response to bacterial infection.
- Characterization of CD13’s participation in various
animal models of disease.
Investigation of CD13’s contribution to leukocyte
trafficking in inflammatory disease.
- Molecular dissection of the reorganization of the
monocyte cytoskeleton following CD13 ligation.
- Assess the role of CD13 as an adhesion molecule of
endothelial junctions. CD13 relocates to the cell-cell
junctions as cells become confluent, suggesting it
participates in junction formation and endothelial
- Characterization of the role of upregulated CD13
expression following myocardial ischemia in mouse models
of myocardial infarction (in collaboration with Bruce
Liang, Calhoun Cardiology Center).
- Investigation into serum CD13 as a biomarker for
inflammatory and cardiovascular diseases.
- Structure/function analysis of PSMA’s regulation of
beta 1 integrin signaling.
- Assessing PSMA as an endothelial adhesion molecule.
- Assessing endothelial PSMA in the angiogenesis
during wound healing.
- Investigating the role of PSMA in prostate cancer
metastasis. PSMA regulates prostate cancer cell invasion
suggesting it may regulate escape from the primary tumor
and access to metastatic sites.
- Investigation into how PSMA expression on tumor
blood vessels affects endothelial/basal lamina
interactions and vessel permeability.
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