The long range goal of research in my laboratory is to understand the mechanisms that
control microvascular growth and permeability barrier function. Our current work is
focused on defining the mechanisms that regulate the expression and signal transduction
functions of VEGF (vascular endothelial growth factor) and PEDF (pigment epithelial
derived factor). VEGF is a potent angiogenic and permeability increasing growth factor
which is known to have primary role in pathological angiogenesis. PEDF is an angiostatic
factor that has recently been shown to block the effects of VEGF in increasing vascular
permeability in the retina.
Projects
Role of the uPA/uPAR System in Diabetes/high Glucose-induced Increases in Endothelial
Cell Permeability.
This project seeks to develop new therapies for diabetic retinopathy by targeting
the urokinase/urokinase receptor system (uPA/uPAR). Our previous work has shown that
diabetes/high glucose-induced injury of the retinal vasculature is mediated by oxidative
stress-induced increases in expression of VEGF, which causes breakdown of the blood-retinal
barrier due to activation of the uPA/uPAR system. Our preliminary data link these
events to diabetes' action in decreasing the expression of the anti-angiogenic, neuro-trophic
growth factor PEDF. PEDF is known to block the angiogenic and permeability-inducing
functions of VEGF. Studies of diabetic retinopathy and diseases characterized by breakdown
of the blood-retinal barrier and retinal neovascularization have shown that increases
in retinal VEGF are correlated with decreases in PEDF. Oxidative stress reduces PEDF
by increasing the formation of matrix metalloproteinases 2 and 9 (MMP2, MMP9), which
degrade and inactivate PEDF. We have evidence that diabetes-induced increases in uPAR
are associated with increases in MMP9 and decreases in PEDF. Moreover deletion of
the uPAR gene prevents MMP9 release, preserves PEDF and protects the blood-retinal
barrier. We also have data showing that diabetes-induced neuronal/glial cell death
is correlated with decreases in PEDF. Based on these data, we hypothesize that diabetes
and high glucose induce breakdown of the blood-retinal barrier and neuro-glial cell
death by causing activation of the uPA/uPAR system and decreasing PEDF. We are testing
this hypothesis with experiments using a combination of transgenic animal and tissue
culture models and specific inhibitors of the uPA/uPAR system. These studies will
set the stage for developing therapies for targeting both neural and vascular pathology
and preventing diabetic retinopathy, the leading cause of blindness in working age
adults in the US today.
Signaling Mechanisms by which Oxidative Stress Increases VEGF Expression in Retinal
Microvascular Endothelial Cells
This project seeks to test the general hypothesis that over-espression of VEGF and
retinal neovascularization during ischemic retinopathy critically involve activation
of NAD(P)H oxidase via angiotensin II. Recent clinical and experimental findings have
implicated angiotensin II in retinal VEGF over-expression, vascular hyperpermeability
and neovascularization in diabetes and other forms of ischemic retinopathy. Angiotensin
II is known to cause endothelial cell dysfunction in various forms of cardiovascular
disease due to its action inducing superoxide production by NAD(P)H oxidase. In macrovascular
endothelial cells, angiotensin II induces activation of NAD(P)H and "uncoupling" of
eNOS to generate additional superoxide, leading to formation of peroxynitrite and
decreased bioavailability of nitric oxide. Our preliminary data suggest that retinal
neovascularization during ischemic retinopathy is associated with increased vascular
expression and activity of NAD(P)H oxidase and peroxynitrite formation. Our studies
using cultured endothelial cells show that peroxynitrite induces activation of the
VEGF transcriptional regulator STAT3 and increased VEGF expression. Based on these
observations, we hypothesize that retinal neovascularization during ischemic retinopathy
critically involves activation of NAD(P)H oxidase via angiotensin II, leading to eNOS
"uncoupling", ONOO- formation and VEGF over-expression. We are testing this hypothesis
using a combination of transgenic animal and tissue culture models and well-established
cell biology approaches.
NAD(P)H Oxidase As a Therapeutic Target in Diabetic Retinopathy
The long term objective of this project is to determine the role of the superoxide generating
enzyme NAD(P)H oxidase in diabetic retinopathy and to evaluate the potential usefulness
of NAD(P)H oxidase inhibitors as a therapy. In diabetic retinopathy vision loss can
result from retinal swelling due to fluid leakage from the retinal blood vessels or
from vitreoretinal neovascularization. Previous research in diabetic patients and
experimental models indicates that overexpression of VEGF plays a major role in both
of these alterations. Diabetes-induced increases in the formation of reactive oxygen
species (ROS) including superoxide anion have been shown to play a key role in vascular
injury associated with increased VEGF expression. Our studies in a mouse model for
ischemic retinopathy indicate that superoxide formation by NAD(P)H oxidase has a key
role in hypoxia-induced increases in VEGF expression and retinal neovascularization
and inhibition of NAD(P)H oxidase inhibitor blocks these alterations. The NAD(P)H
oxidase enzyme is a major source of superoxide generation during hypoxia and it has
been suggested to serve as an oxygen sensor that responds to hypoxia by producing
superoxide. NAD(P)H oxidase in phagocytic cells and vascular endothelial cells consists
of two membranous subunits, gp91phox and p22phox as well as two cytosolic subunits,
p47phox and p67phox, and the low molecular weight G protein Rac-1. During diabetes
endothelial cells, leukocytes and microglial cells become activated and are potential
sources of NAD(P)H oxidase-derived superoxide formation. Knocking out the catalytic
subunit gp91phox has been shown to prevent neuronal injury after cerebral ischemia-reperfusion
injury. Thus, it is likely that NAD(P)H oxidase has a key role in diabetic retinopathy.
Our preliminary data show that increased expression of gp91phox is correlated with
diabetes-induced oxidative stress and that inhibiting NAD(P)H oxidase blocks the effects
of high glucose in stimulating increases in VEGF expression in vitro. Based on these
observations, we hypothesize that diabetes causes increases in VEGF expression and
break-down of the blood-retinal barrier via induction of gp91phox and activation of
NAD(P)H oxidase. To test this hypothesis, we are conducting experiments using specific
inhibitors for NAD(P)H oxidase, mice deficient in gp91phox.
Behzadian, M.A., Bartoli, M., El-Remessy, A.B., Al-Shabrawey, M., Platt, D.H., Liou,
G.I., Caldwell, R.W., Caldwell, R.B. Cellular and Molecular Mechanisms of Retinal Angiogenesis, What have we learned from
in vitro models? In "Retinal and Choroidal Angiogenesis", Ed. J.S. Penn, In Press,
2006.
Kaesemeyer, W.H., Jin, L. Caldwell, R.B., Caldwell, R.W. Drug-Induced Endothelial Cell Dysfunction. In "Nitric Oxide and its
Biomedical Significance", Ed: G. Stefano, 2003.
g term objective of this project is to determine the role of the superoxide generating
enzyme NAD(P)H oxidase in diabetic retinopathy and to evaluate the potential usefulness
of NAD(P)H oxidase inhibitors as a therapy. In diabetic retinopathy vision loss can
result from retinal swelling due to fluid leakage from the retinal blood vessels or
from vitreoretinal neovascularization. Previous research in diabetic patients and
experimental models indicates that overexpression of VEGF plays a major role in both
of these alterations. Diabetes-induced increases in the formation of reactive oxygen
species (ROS) including superoxide anion have been shown to play a key role in vascular
injury associated with increased VEGF expression. Our studies in a mouse model for
ischemic retinopathy indicate that superoxide formation by NAD(P)H oxidase has a key
role in hypoxia-induced increases in VEGF expression and retinal neovascularization
and inhibition of NAD(P)H oxidase inhibitor blocks these alterations. The NAD(P)H
oxidase enzyme is a major source of superoxide generation during hypoxia and it has
been suggested to serve as an oxygen sensor that responds to hypoxia by producing
superoxide. NAD(P)H oxidase in phagocytic cells and vascular endothelial cells consists
of two membranous subunits, gp91phox and p22phox as well as two cytosolic subunits,
p47phox and p67phox, and the low molecular weight G protein Rac-1. During diabetes
endothelial cells, leukocytes and microglial cells become activated and are potential
sources of NAD(P)H oxidase-derived superoxide formation. Knocking out the catalytic
subunit gp91phox has been shown to prevent neuronal injury after cerebral ischemia-reperfusion
injury. Thus, it is likely that NAD(P)H oxidase has a key role in diabetic retinopathy.
Our preliminary data show that increased expression of gp91phox is correlated with
diabetes-induced oxidative stress and that inhibiting NAD(P)H oxidase blocks the effects
of high glucose in stimulating increases in VEGF expression in vitro. Based on these
observations, we hypothesize that diabetes causes increases in VEGF expression and
break-down of the blood-retinal barrier via induction of gp91phox and activation of
NAD(P)H oxidase. To test this hypothesis, we are conducting experiments using specific
inhibitors for NAD(P)H oxidase, mice deficient in gp91phox.
