Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-02-01
2002-03-19
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S023000, C435S024000, C435S962000, C435S968000, C435S975000
Reexamination Certificate
active
06358699
ABSTRACT:
BACKGROUND
1. EDNO regulates vascular tone.
Endothelium-derived nitric oxide (EDNO) is the most potent endogenous vasodilator known, and, by its effect upon vascular resistance and cardiac contractility, is a major regulator of blood pressure (Moncada and Higgs, 1993; Cooke and Dzau, 1997). NO exerts its effects as a vasodilator, in part, by stimulating soluble guanylate cyclase to produce cGMP. A deficiency of EDNO (as in the endothelial NOS knockout, or with administration of NOS antagonists), causes hypertension (Dananberg et al., 1993; Shesely et al., 1996). An overproduction of nitric oxide (NO) (as in sepsis), causes hypotension and cardiovascular collapse (Rees et al.,1990; Petros et al., 1991).
NO is released from the endothelium in response to a wide variety of physiologic stimuli. For over a century physiologists have recognized that as blood flow increases through a conduit vessel, the vessel dilates. This flow-mediated vasodilation is dependent upon the integrity of the endothelium, and is largely due to the release of EDNO in response to endothelial shear stress (Cooke et al., 1990; Cooke et al., 1991a). Endothelial cells also respond to pharmacological stimuli. Most vasoconstrictors, such as norepinepherine, 5-hydroxytryptamine, and angiotensin II, also stimulate NO release by the endothelium (Moncada and Higgs, 1993; Cooke and Dzau, 1997).
In this way the endothelium modulates vascular contractility. These responses have physiological consequences. For example, during exercise or with mental stress, myocardial oxygen demands increase. In normal individuals the epicardial coronary arteries dilate to accommodate the need for increased coronary blood flow. By contrast, individuals with coronary artery disease have a dysfunctional endothelium with reduced EDNO production and/or activity. In these individuals, a paradoxical coronary artery constriction is observed with exercise or mental stress that contributes to reduced coronary blood flow, resulting in myocardial ischeni ia (Cox et al., 1989; Zeiher et al., 1989).
In addition to its role as a vasodilator, EDNO is potent inhibitor of vascular smooth muscle (VSM) proliferation. The proliferation of cultured VSM cells is inhibited by exogenous NO donors and cGMP analogues (Garg and Hassid, 1989). Gene transfer of endothelial NOS into the balloon-injured rat carotid artery in vivo demonstrably increases NO release for days after the transfection, and significantly reduces myointimal hyperplasia due to proliferation of intimal vascular smooth muscle cells (von der Leyen, et al., 1995).
EDNO also affects vascular structure by inhibiting the interaction of circulating blood elements with the vessel wall. Platelet adherence and aggregation is inhibited by EDNO (Radomski et al., 1987; Stamler et al., 1989). The adherence and infiltration of leukocytes into the vessel wall during experimental inflammation is reduced by exogenous administration of NO donors, and is enhanced by administration of NOS antagonists (Lefer et al., 1993; Gáboury et al, 1993).
To summarize, in states of vascular injury or inflammation, a deficiency of NO contributes to thrombosis, leukocyte infiltration, and vascular smooth muscle proliferation.
2. The role of NO in atherosclerosis
Atherosclerosis is the major cause of disability in this country and is responsible for 500,000 deaths annually due to coronary artery disease and cerebral vascular attack. Atherosclerosis is accelerated by hyper-cholesterolemia, hypertension, diabetes mellitus, tobacco use, elevated levels of lipoprotein(a) (“Lp(a)”) and homocysteine. Intriguingly, all of these disorders are characterized in humans by an endothelial vasodilatory dysfunction well before there is any clinical evidence of atherosclerosis (Cooke and Dzau, 1997). In all of these conditions, the abnormality appears to be due in large part to a perturbation of the NOS pathway. In most of these conditions, the abnormality is reversed or ameliorated by the administration of the NO precursor, L-arginine (Cooke and Dzau, 1997). L-arginine is metabolized by NOS to citrulline and NO.
Dr. John Cooke and coworkers were the first to demonstrate that endothelial vasodilator dysfunction could be reversed by administration of the NO precursor. In hypercholesterolemic rabbits, administration of L-arginine normalizes the NO-dependent vasodilation to acetylcholine (Girerd et al., 1990; Cooke et al., 1991b). Subsequently, Dr. Cooke and others have demonstrated that acute administration of L-arginine can reverse endothelial vasodilator dysfunction that is observed in the coronary and peripheral circulation in patients with atherosclerosis, and in subjects at risk for atherosclerosis.
Because NO has inhibitory effects on many of the key processes that promote atherosclerosis (monocyte adherence, platelet aggregation, vascular smooth muscle proliferation), Cooke postulated that chronic enhancement of vascular NO production could inhibit atherogenesis. Indeed, his lab demonstrated that in hypercholesterolemic rabbits, chronic oral administration of L-arginine could enhance vascular NO activity (Cooke et al., 1992; Wang et al., 1994; Tsao et al., 1994). This effect was associated with a striking reduction in vascular lesions. By contrast, administration of NOS antagonists reduced vascular NO synthesis, increased endothelial adhesiveness for monocytes, and accelerated lesion formation (Tsao et al., 1994; Naruse et al, 1994; Cayatte et al, 1994). Cooke and others have shown that EDNO exerts its effects on atherogenesis by suppressing the expression and the signaling of endothelial adhesion molecules such as VCAM-1, and by reducing the expression of chemokines such as monocyte chemotactic protein-1 (Marui et al., 1993; Tsao et al., in press). The inhibition of adhesion signaling by NO appears to be mediated by cGMP, whereas the transcriptional effects of NO appear to be due, in part, to its abrogation of an oxidant-sensitive transcriptional pathway mediated by NF6B (Marui et al., 1993; Tsao et al., in press; Tsao et al., 1995).
Surprisingly, the administration of L-arginine in hypercholesterolemic rabbits with pre-existing lesions not only slows the progression of disease, but actually induces regression of atherosclerosis (Candipan et al., 1996).
Accordingly, enhancement of vascular NO may represent a novel therapeutic strategy for cardiovascular disease. The initial studies in humans are encouraging. Cooke and others have recently demonstrated that chronic oral administration of L-arginine in hypercholesterolemic humans or those with coronary artery disease can enhance vascular NO activity (as assessed by vascular reactivity studies and measurement of urinary nitrogen oxides), inhibit platelet aggregability, and reduce the adhesiveness of peripheral blood mononuclear cells (Bode-Böger et al., 1994; Wolfe et al., 1995; Theilmeier et al., in press; Lerman et al., 1997).
3. ADMA, a deter minant of endothelial dysfunction and novel risk factor for atherosclerosis
ADMA (asymmetric dimethylarginine) is an endogenous antagonist of nitric oxide synthase. Several years ago, Vallance and Moncada demonstrated that, in uremic rats and in patients with renal failure, plasma ADMA levels were elevated 5-10-fold from normal values of about 1 micromolar (Vallance et al., 1992a,b). Plasma from uremic animals and patients (but not controls) induced the constriction of isolated vascular rings. This vasoconstriction was reversed by L-arginine. Moreover, infusions of ADMA into the brachial artery of normal volunteers caused a significant increase in forearm vascular resistance at concentrations of ADMA that are found in patients with renal failure (Vallance et al., 1992b).
Recently, the enzyme that is responsible for degrading ADMA (dimethylarginine dimethylaminohydrolase, or DDAH), has been characterized. An antagonist to DDAH has been developed which blocks ADMA degradation (MacAllister et al., 1996). When the DDAH antagonist is added to vascular rings in vitro, a gradual increase in tone is observed. Again, this vasoconstriction is reversed by L-argi
Balint Robert F.
Cooke John P.
Mutz Mitchell Wayne
Cooke Pharma
Leary Louise N.
Rae-Venter Barbara
Rae-Venter Law Group P.C.
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