Cytoskeletal and contractile proteins modulate microvascular cell function.
Endothelial cell migration and proliferation, central steps in both physiologic and
pathologic angiogenesis, require cytoskeletal-dependent remodeling, which is achieved by
the dynamic regulation of the β-actin network. Specifically, the β-actin
network has previously been shown to be enriched in regions of motile cytoplasm, and
modulated by its isoactin-specific barbed-end capping ... read moreprotein, βcap73. We
hypothesize that regulated over-expression of βcap73 could disrupt angiogenesis by
capping β-actin-filament assembly, thus inhibiting the incipient cellular
migration and microvascular morphogenesis. Indeed, upon infection of capillary
endothelial cells (cEC) with an adenovirus encoding the full-length βcap73
(Ad-βcap73), there is a robust cellular rounding response that occurs
concomitantly with cytoskeletal disruption. Further, we demonstrate that over-expression
of Ad-βcap73 inhibits cEC migration in wound healing studies. Quantitative in
vitro angiogenesis assays reveal that Ad-βcap73 prevents endothelial cells from
forming capillary-like networks, and also induces the collapse of preformed endothelial
tubes. In testing whether βcap73 impairs angiogenic events by inducing
anoikis/apoptosis, we demonstrate that Ad-βcap73 infection activates a
caspase-3-mediated cell death response as observed by quantitative Western blotting and
immunofluorescence analyses. To confirm and extend these findings in vivo, we have
engineered a 'two-mouse' transgenic approach, to specifically induce βcap73
over-expression within the post-natal vascular endothelium using a hypoxia-induced model
of retinopathy of prematurity. Indeed, βcap73 over-expression reduces the
pathologic angiogenic response by approximately 30%. This work demonstrates that
endothelial cell-specific targeting of actin-mediated assembly processes may be used as
a novel therapeutic approach to eradicate unwanted angiogenesis accompanying disease.
Vascular tone regulates blood pressure and tissue perfusion, and is modulated by
vascular smooth muscle cells and pericytes. Phosphorylation of the myosin light chain,
which is controlled by opposing actions of myosin light chain phosphatase (MLCP) and
myosin light chain kinase (MLCK), modulates mural cell contractile phenotype. While the
RhoA/Rho Kinase (ROCK) pathway has been shown to control pericyte contractile phenotype,
the mechanisms linking RhoGTPase activity and the cytoskeleton remain incompletely
understood. Recently, the myosin phosphatase-RhoA interacting protein (MRIP) has been
described to mediate the RhoA/ROCK-directed inactivation of MLCP, by localizing RhoA and
MLCP to stress fibers. Here, we report a novel interaction between MRIP and the
β-actin specific capping protein, βcap73. Dramatic alterations in
cytoskeletal architecture accompany increased pericyte contractility upon MRIP
knockdown. Ultimately, these MRIP-silenced, hyper-contractile pericytes are unable to
maintain contact-mediated endothelial cell growth arrest. This newly characterized
interaction between MRIP and βcap73 should offer insights into the actomyosin
dynamics controlling pericyte contractility, and inappropriate regulation of this
complex might have implications during microvascular pathologies, including essential
hypertension and proliferative diabetic
Thesis (Ph.D.)--Tufts University, 2011.
Submitted to the Dept. of Cellular & Molecular Physiology.
Advisors: Ira Herman, and Brent Cochran.
Committee: Laura Liscum, Howard Surks, and Michael Gimbrone.
Keywords: Physiology, and Biology.read less