Projects List

Role of tetrahydrobiopterin in control of vascular endothelial function

Tetrahydrobiopterin (BH4) is an essential co-factor required for enzymatic activity of nitric oxide synthase (NOS). All three isoforms endothelial (eNOS), inducible (iNOS) and neuronal (nNOS) require optimal concentration of BH4 to produce nitric oxide (NO). Accumulating evidence suggests that reduced availability of BH4 causes “uncoupling” of eNOS leading to increased production of superoxide anion and formation of a potent oxidant, peroxynitrite, thereby imposing oxidative stress in vascular wall. BH4 is a molecular target for oxidation whereas peroxynitrite is a potent oxidant of BH4. Loss of NO caused by reduced availability of BH4 appears to be an important mechanism of endothelial dysfunction, phenomenon responsible for initiation and progression of atherosclerosis. Thus, understanding of the mechanisms responsible for biosynthesis and degradation of BH4 is critical for development of strategies in prevention and treatment of atherosclerosis. Our efforts have been directed towards identification of circulating substances and mechanical forces that may affect biosynthesis and/or degradation of BH4 in vascular endothelium. We employ endothelial cell culture as well as in vivo approaches in genetically modified mice to study BH4 metabolism and its effect on vascular disease.

Pathogenesis and therapy of endothelial dysfunction

In human body, endothelial cells occupy surface that is approximately equal to the surface of two tennis courts. Constant replacement of injured or detached endothelial cells is essential for preservation of normal cardiovascular function. Maintenance of endothelial integrity in vascular wall could be achieved by proliferation of mature endothelial cells or by incorporation of endothelial progenitor cells (EPCs). Regenerative function of EPCs is an important homeostatic mechanism in cardiovascular system. Methods for isolation and propagation of EPCs from circulating blood have been well established. Most importantly, ability of EPCs to stimulate endothelial repair have been demonstrated in experimental models of vascular injury suggesting that EPCs have high therapeutic potential. However, biology of EPCs is in not completely understood, and mechanisms underlying their therapeutic effects are unclear.

Aging is a major risk factor for the development of endothelial dysfunction and cardiovascular disease. The exact mechanisms of vascular aging are poorly understood, and consequently, we have no therapies aimed at modulating cardiovascular risk inherent to aging. Existing evidence suggests that in mammals, aging-induced deterioration of tissue functions occurs in part because of decline in the restorative capacity of stem/progenitor cells. This project is designed to determine molecular mechanisms responsible for deterioration of EPCs function induced by aging. Established model of balloon-induced injury of common carotid artery is employed to study the effect of aging on endothelial repair and vascular architecture. Transplantation of autologous EPCs is used to determine the effect of aging on regenerative capacity of EPCs. Cultured EPCs are used to analyze the role of nitric oxide, superoxide anion, and antioxidant defense system in regenerative functions of young and aged EPCs. Genetic modification of EPCs is tested to improve therapeutic potential of EPCs.

Development of novel therapeutic approaches to cerebral vasospasm

Cerebral vasospasm is the most common cause of death in patients with subarachnoid hemorrhage resulting in 30% mortality. Pathogenesis of cerebral vasospasm is complex and incompletely understood. Existing therapeutic approaches suffer from a number of limitations and improvements in prevention and treatment of vasospasm are needed. Our efforts have been directed towards genetic modification of cerebral arterial wall designed to enhance endogenous vasodilator systems and prevent or reduce vasoconstriction induced by subarachnoid hemorrhage. In our previous studies we used adenovirus-mediated gene transfer to deliver recombinant endothelial nitric oxide synthase (eNOS) into adventitial fibroblasts or endothelium. This resulted in increased local production of a potent vasodilator, nitric oxide (NO). Over-expression of eNOS protein in adventitia of canine cerebral arteries had beneficial effect on cerebral vasospasm providing support for genetic enhancement of vasodilator capacity as potential therapeutic approach to vasospasm. Furthermore, our in vitro studies on isolated human cerebral arteries demonstrated that eNOS gene transfer increased production of NO and enhanced relaxations in response to endothelium-dependent vasodilators. Thus, genetic modification of cerebral arteries from both experimental animals and humans can result in reduced vasoconstriction and enhanced vasodilatation. Introduction of these techniques in clinical arena requires further optimization of vectors safety, development of more effective gene delivery techniques, and identification of genes with the best therapeutic profile. These problems are currently being addressed by ongoing studies in our laboratory.