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.

Current Projects

1. 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.

2. 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.

3. 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.

Grant Support as Principal Investigator

Principal Investigator: National Institutes of Health, National Eye Institute, R01 (EY04618), Blood-retinal Barrier Changes in Retinopathy.

Principal Investigator: National Institutes of Health, National Eye Institute, R01 (EY11766), Cellular Mechanisms of Retinal Angiogenesis.

Principal Investigator: Veterans Administration, Career Development Award, New Strategies for Treatment of Diabetic Retinopathy.

Principal Investigator: Alcon Laboratories, Research Contract, Inhibitory Effects of Pharmaceutical Reagents on Diabetes-induced Blood-retinal Barrier Breakdown.

Co-Investigator: National Institutes of Health, National Heart, Blood & Lung Institute, R01(HL070215), Endothelial Cell Dysfunction in Models of Oxidative Stress

Sponsor: Juvenile Diabetes Foundation International, Postdoctoral Fellowship, Dr. Mohamed Al-Shabrawey, Role of NAD(P)H Oxidase in Diabetic Retinopathy.

Lab

Honors and Awards

Invited Speaker

Selected Publications

Journal Articles

El-Remessy, A.B., Al-Shabrawey, M., Platt, D.H., Bartoli, M., Behzadian, M.A., Ghaly, N., Tsai, N., Motamed, K., and Caldwell, R.B. 2007. Peroxynitrite mediates VEGF's angiogenic signal and function via a nitration-independent mechanism in endothelial cells. Faseb J. (In Press).

Crandall, J., Matragoon, S., Khalifa, Y.M., Borlongan, C., Tsai, N.T., Caldwell, R.B., and Liou, G.I. Neuroprotective and Intraocular Pressure-Lowering Effects of (-)Delta-Tetrahydrocannabinol in a Rat Model of Glaucoma. Ophthalmic Res 39:69-75, 2007.

Romero, M.J., Platt, D.H., Caldwell, R.B., and Caldwell, R.W. Therapeutic use of citrulline in cardiovascular disease. Cardiovasc Drug Rev 24:275-290, 2006.

Tawfik, H.E., El-Remessy, A.B., Matragoon, S., Ma, G., Caldwell, R.B., Caldwell RW. Simvastatin Improves Diabetes-Induced Coronary Endothelial Dysfunction. J Pharmacol Exp Ther. Jul 18 2006.

Ambati, B.K., Patterson, E., Jani, P., Jenkins, C., Higgins, E., Singh, N., Suthar, T., Vira, N., Smith, K., and Caldwell, R. Soluble vascular endothelial growth factor receptor-1 contributes to the corneal anti-angiogenic barrier. Br J Ophthalmol, (In Press) 2006.

Ambati, B.K., Nozaki, M., Singh, N., Takeda, A., Jani, P.D., Suthar, T., Albuquerque, R.J., Richter, E., Sakurai, E., Newcomb, M.T., Kleinman, M.E., Caldwell, R.B., et al.. Corneal avascularity is due to soluble VEGF receptor-1. Nature 443, 993-997, 2006.

Ma, G., Al-Shabrawey, M., Johnson, J., Caldwell, R.B., Caldwell, R.W. Protection against myocardial ischemia/reperfusion injury by short term diabetes: role of VEGF-induced angiogenesis and activation of cell survival signaling. Naunyn-Schmiedeberg's Arch. Pharmacol. In press 2006.

Tawfik, A. Jin, L. Banes-Berceli, A.K. Caldwell, R.B., Ogbi, S., Shirley, A., Barber D., Catravas, J.D., Stern, D.M., Fulton, D., Caldwell, R.W., Marrero, M.B. Hyperglycemia and reactive Oxygen Species mediate apoptosis in aortic endothelial cells through Janus kinase 2. Vascul Pharmacol. 43(5):320-6. 2006.

DH Platt, M Bartoli, A El-Remessy, M AL-Shabrawey, D Fulton, RB Caldwell. Peroxynitrite-mediated Activation of VEGF Transcription in vascular cells via signal transducer and activator of transcription 3. Free Radic Biol Med. 15;39:1353-61. 2006.

A.B. El-Remessy, M. Al-Shabrawey, Y. Khalifa, R.B. Caldwell and G.I. Liou. Neuroprotective and blood-retina barrier preserving effects of cannabidiol on experimental diabetes. Am J Pathol. 168(1):235-44, 2006.

Caldwell, R.B., Bartoli, M., Behzadian, M.A., El-Remessy, A., Al-Shabrawey, M, Platt, D.H., Liou, G.I., Caldwell, R.W. Vascular endothelial growth factor and diabetic retinopathy: Role of oxidative stress. Curr. Drug Targets 6(4):511-24, 2005.

Al-Shabrawey, M., Bartoli, M., Platt, D.H., El-Remessy, A.B., Matragoon, S., Behzadian, M.A., Caldwell, R.W., Caldwell, R.B. Inhibition of NADPH oxidase activity blocks VEGF over-expression and angiogenesis in ischemic retinopathy. Am. J. Pathol., 167(2):599-607, 2005.

Caldwell, R.B., Bartoli, M., Behzadian, M.A., El-Remessy, A., Al-Shabrawey, M, Platt, D.H., Liou, G.I., Caldwell, R.W. Vascular endothelial growth factor and diabetic retinopathy: Role of oxidative stress. Curr. Drug Targets. 6:511-524, 2005.

El-Remessy, A.B., Bartoli, M. Platt, D.H., Fulton, D., Caldwell, R.B. Oxidative stress inactivates VEGF survival signaling in retinal endothelial cells via PI-3 kinase tyrosine nitration. J. Cell Sci., 118:243-252, 2005.

Liou, G.I., Samuel, S., Matragoon, S., Goss, K.H., Santoro, I., Groden, J., Hunt, R.C., Wang, F., Miller, S.S., Caldwell, R.B., Rustgi, A.K., Singh, H., Marcus, D.M. Alternative splicing of the APC gene in the neural retina and retinal pigment epithelium. Mol Vis. 10:383-391, 2004.

Salis, M.B., Graiani, G., Desortes, E., Caldwell, R B., Madeddu, P., Emanueli, C. Nerve growth factor supplementation reverses the impairment, induced by Type 1 diabetes, of hindlimb post-ischaemic recovery in mice. Diabetologia, 47:1055-1063, 2004.

Abou-Mohamed, G., Johnson, J.A., Jin, L., El-Remessy, A.B., Do, K., Kaesemeyer, W.H., Caldwell, R.B., Caldwell, R.W. Roles of superoxide, peroxynitrite and protein kinase C in the development of tolerance to nitroglycerin. J. Pharmacol. Exp. Therap., 308:289-299, 2004.

El-Remessy, A.B, Khalil, I.E., Matragoon S., Abou-Mohamed, G., Tsai, N.J., Roon, P., Caldwell, R.B., Caldwell, R.W., Green, K., Liou, G.I. Neuroprotective effect of (-)Delta9-tetrahydrocannabinol and cannabidiol in N-methyl-D-aspartate-induced retinal neurotoxicity: involvement of peroxynitrite. Am. J. Pathol., 163:1997-2008, 2003.

Al-Shabrawey, M. El-Remessy, A.B., Gu, X., Brooks, S.E., S. Hamed, M.S., Huang, P., Caldwell, R.B. Normal vascular development in mice deficient in endothelial NO synthase: Possible role of neuronal NO synthase, Molec. Vision, 9:549-558, 2003.

Caldwell, R.B., Bartoli, M., Behzadian, M.A., El-Remessy, A., Al-Shabrawey, M, Platt, D.H., Caldwell, R.W. Vascular endothelial growth factor and diabetic retinopathy: Pathophysiological mechanisms and treatment perspectives. Diabetes Metabolism Research & Reviews, 19:442-455, 2003.

Bartoli, M., Platt, D., Lemtalsi, T., Gu, X., Brooks, S.E., Marrero, M.B., Caldwell, R.B. VEGF differentially activates STAT3 in microvascular endothelial cells. Faseb J. 17:1562-1564, 2003.

Gu, X., El-Remessy, A., Brooks, S.E., Al-Shabrawey, M., Tsai, N.T., Caldwell, R.B. Hyperoxia induces retinal vascular endothelial cell apoptosis through formation of peroxynitrite. Am J Physiol Cell Physiol, 285:C546-54, 2003.

El-Remessy A., Abou-Mohamed, G., Caldwell, R.W., Caldwell, R.B., High glucose increases tyrosine nitration and superoxide anion formation in endothelial cells: Role of eNOS uncoupling and aldose reductase activation. Invest. Ophthalmol. Vis. Sci., 44:3135-3143, 2003.

El-Remessy A., M.A. Behzadian, G. Abou-Mohamed, T. Franklin, R.W. Caldwell, R.B. Caldwell, Peroxyntitrite increases vascular permeability in experimental diabetes by a mechanism involving increased expression of VEGF and urokinase plasminogen activator receptor (uPAR). Am. J. Pathol., 162:1995-2004, 2003.

Behzadian M.A., Windsor L.J., Ghaly N., Liou, G..I.., Tsai, N-T., Caldwell, R.B., VEGF-induced paracellular permeability in cultured endothelial cells involves urokinase and its receptor. Faseb J. 17:752-4. Epub 2003.

Book Chapters

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.

Education and Training

Research Experience & Academic Appointments

Institutional Faculty List