INTRODUCTION:

Vascular Endothelium

Vascular endothelium “the maestro of blood circulation” ¹, is the innermost single layer lining of polygonal flat epithelial cells of thickness 15-40 nm² which spreads continuously over the entire vasculature of the organism. It is located between the blood and VSMC layer of the blood vessels³ and is the largest endocrine gland⁴.

Functions of Vascular Endothelium

Vascular endothelium has biosynthetic, secretory, metabolic and immunologic functions⁵. It provides free flow of blood in arteries due to its anticoagulant and antithrombotic activities⁶. Vascular endothelium controls vascular tone, hemostasis, endothelial permeability, vascular growth and interaction between endothelium and leukocytes⁷.

Vascular Tone: Regulation of vasomotor tone is through release of EDRF (NO), EDCF, EDHF, PGI₂ and endothelins^8-12.

Hemostasis: Vascular endothelium secretes factor VIII antigen, von Willebrand’s factor (vWF), thrombomodulin and tissue plasminogen activator to regulate flow of blood^13-15

Regulation of Vascular Growth: Structural components of extracellular matrix such as collagen, elastin, glycosaminoglycans, matrix metalloproteinases and fibronectin are released from vascular endothelium^16-18. Growth of vascular smooth muscle cells is regulated by vascular endothelium by release of various growth promoting factors like fibroblast growth factor (FGF), vascular endothelial derived growth factor (VEGF), TGF α and β, insulin like growth factor-1 (IGF-1) and growth inhibiting factors such as activators of tyrosine kinases and angiopeptin^19-21.

Cellular Interaction: The vascular endothelium secretes leukocyte chemoattractant protein (interleukin-8), monocyte chemotactic protein 1 (MCP- 1), intercellular adhesion molecules 1 and 2 (ICAM-1 and ICAM-2), endothelial leukocyte adhesion molecule 1 (E-selectin), vascular cell adhesion molecule (VCAM-l) and GMP-140 to regulate interaction between leukocytes and vascular endotheliurn²².

Transport and Metabolism: The transport of lipids or water soluble macromolecules, glucose and amino acids across the vascular endothelium is regulated by intercellular tight junctions, trans-endothelial channels, caveolae, GLUT-1, GLUT-4 and cationic amino acid transporter system²³. Further, vascular endothelium metabolise plasma lipids, lipoproteins, adenine nucleotides, nucleosides, serotonin, catecholamines, bradykinin and angiotensin II²⁴.

Repair: Injury to vascular endothelium induces apoptotic cell death and release viable endothelial cells in circulation²⁵. The mechanisms to repair damaged vascular endothelium include inhibition of apoptotic cell death and replacement of damaged endothelial cells with its embryonic precursor cells.

Vascular Endothelial Dysfunction (VED):

Vascular endothelial dysfunction has been characterized as partial or complete loss of balance between vasorelaxation and vasoconstriction^26-27, thrombosis and thrombolysis²⁸, growth promotion and growth inhibition²⁹.

Implications of VED:

Vascular endothelial dysfunction is implicated in essential hypertension³⁰, congestive heart failure³¹, diabetes mellitus³², hyperhomocysteinemia³³, erectile dysfunction³⁴ and secondary complications of atherosclerosis³⁵.

Nitric Oxide and VED:

NO is responsible for various physiologic and pathophysiologic changes in vascular endothelium. Endothelial nitric oxide synthase (eNOS), an enzyme responsible for synthesis of nitric oxide, oxidizes guanidium nitrogen of L-arginine in presence of tetrahdrobiopeterin (BH₄), NADPH, FAD, O₂ and Ca⁺² to produce nitric oxide (NO) and L-citrulline. Uncoupling of eNOS stimulates generation of reactive oxygen species which binds to NO to produce inactive peroxynitrite³⁶. The reduced activity of DDAH and CAT-1 transporter of L-arginine, accumulation of ADMA and increase in activity of arginase, lowers the bioavailability of L-arginine and consequently inhibits biosynthesis of NO³⁷. The activation of eNOS due to decrease in caveolin-1 and increase in calmodulin expression as well as acylation and phosphorylation of eNOS enhances NO production³⁸. Moreover, decrease in expression or destabilization of mRNA for eNOS and gene polymorphism such as Glut28-Asp reduces formation of NO³⁹. The reduced bioavailability of BH₄and increase in BH₄ oxidation enhance uncoupling of eNOS and thereby, produces oxidative stress⁴⁰.

Thus, the decrease in biosynthesis and release of NO and increase in metabolism of NO produce vascular endothelial dysfunction. Moreover, NO has been reported to inhibit platelet adhesion, release of inflammatory cytokines and proliferation of smooth muscle cells and produce vasorelaxation⁴¹.

Oxidative Stress:

Diabetes mellitus, hyperhomocysteinemia, hypertension and hypercholesterolemia produce oxidative stress and uncoupling of eNOS⁴². The reduced supply of substrate or cofactors uncouples eNOS to inhibit formation of NO and consequently stimulates generation of superoxide⁴³. The increased production of superoxide anions quenches NO and consequently reduces its bioavailability⁴⁴. Moreover, the increased generation of reactive oxygen species oxidizes LDL to produce ox-LDL^41,45 which is engulfed by LOX-1 to reduce expression of eNOS⁴⁶.

Further, the reduced NO bioavailability stimulates the release of adhesion molecules like VCAM-1 and 2, ELAM and ICAM-1 and consequently stimulate adhesion of monocytes and formation of plaque⁴⁷. Macrophages engulfs lipids to form foam cells which ultimately produces fatty streaks⁴⁸. The foam cells in the fatty streak and overlying endothelium stimulates expression of MCP-1 and further potetiate chemo-attraction of monocyte⁴⁹. The decrease in NO, increase in oxidative stress and ox-LDL stimulate matrix metalloproteinases such as MMP-2 and MMP-9 to stimulate rupture of fibrous plaque and consequently activate proliferation of vascular smooth muscle cells⁵⁰. Thus, dysfunction of vascular endothelium occurs with reduced NO bioavailability, increased oxidative stress and increased expression of adhesion molecules which stimulate the formation of atherosclerotic plaque and precipitates in cardiovascular disorders.

L-arginine:

L-arginine is a substrate for NOS to biosynthesize NO⁵¹. Asymmetric dimethyl arginine (ADMA) is endogenous competitive inhibitor of L-arginine and inversely regulates biosynthesis of NO⁵². L-arginine level is modulated by arginase which converts arginine to ornithine and urea³⁷. The stimulation of arginase reduces bioavailability of L-arginine and modulates cellular NO production.

Caveolin:

The binding of caveolin-1 to eNOS inhibits activity of eNOS⁵³. The Ca²⁺-calmodulin complex inhibits the binding of caveolin-1 with eNOS to activate eNOS⁵⁴. The increase in expression of caveolin-1 and decrease in expression of calmodulin have been demonstrated to produce vascular endothelial dysfunction⁵⁵.

BH₄:

The cofactor tetrahydrobiopterin (BH₄) is required for substrate binding and stabilization of dimeric structure of eNOS to facilitate biosynthesis of NO⁵⁶. The GTP cyclohydrolase I (GTPCH), 6-pyruvoyl tetrahydrobiopterin synthase (PTPS) and sepiapterin reductase (SR) stimulate formation of BH₄⁵⁷. The decrease in expression or activity of GTPCH, PTPS, SR and DHFR reduce biosynthesis of BH₄⁵⁸. Deficiency of BH₄ stimulates the transfer of electrons to molecular oxygen and generates superoxide anion to produce oxidative stress⁵⁹. Uncoupling of eNOS has been reported to generate oxidative stress which consequently stimulates oxidation of BH₄ to produce vascular endothelial dysfunction⁶⁰.

Molecular Mechanism of VED:

Diabetes Mellitus:

The main cause of mortality and morbidity in diabetic patients is the association of microvascular (retinopathy, nephropathy and neuropathy) and macrovascular (coronary artery disease, atherosclerosis and peripheral vascular disease) complications⁶¹. Streptozotocin-induced diabetes mellitus has been shown to attenuate ACh-induced endothelium-dependent vasorelaxation but has not altered sodium nitroprusside evoked endothelium-indepenedent vasorelaxation⁶². Elevated glucose (hyperglycemia) is responsible for impaired endothelium-dependent vasorelaxation⁶³ and promotes generation of endothelium-derived vasoconstrictor prostanoids in rabbit aorta⁶⁴. Hyperglycemia triggers increased polyol pathway flux, altered cellular redox state, increased formation of diacylglycerol and subsequent activation of specific protein kinase C isoforms, and accelerated non-enzymatic formation of advanced glycation end products^65-67.

Plasma or tissue concentrations of superoxide dismutase, catalase, glutathione and ascorbic acid are reduced in both clinical and experimental diabetes⁶⁸. Antioxidants such as probucol⁶⁹, N-acetylcysteine⁷⁰, Vitamin E^71-72 and vitamin C⁷³ have prevented the development of endothelial dysfunction in clinical and experimental diabetes mellitus.

Hyperhomocysteinemia:

Hyperhomocysteinemia (Hhcy) is characterized by increase in plasma concentration of total homocysteine, associated with vascular disease and an independent risk factor for atherosclerosis and atherothrombosis^74-76. Homocysteine, a sulphur-containing amino acid⁷⁷, is formed during conversion of methionine to cysteine and it is either metabolised by trans-sulfuration pathway to cysteine or by remethylation pathway to methionine by methionine synthase⁷⁸. Methionine synthase uses vitamin B₁₂as co-factor and 5-Methyl-tetrahydrofolate as methyl donor⁷⁹. Normal endothelial cells detoxify homocysteine by releasing nitric oxide, forming S-nitroso-homocysteine in presence of oxygen⁸⁰, a potent platelet inhibitor and vasodilator⁸¹.

When excess of methionine is present, homocysteine enters trans-sulfuration pathway in which homocysteine condenses with serine to form cystathione in a reaction catalyzed by the vit. B₆-dependent enzyme cystathione β-synthase⁸². Cystathione is further hydrolysed to form cysteine, which in turn be incorporated into glutathione or further metabolized to sulfate and excreted in the urine⁸³.

Diet-induced hyperhomocysteinemia leads to impaired vasomotor regulation in vivo, endothelial antithrombotic function ex vivo⁸⁴ and impaired endothelium-dependent vasodialtion⁸⁵. Long term hyperhomocysteinemia damages endothelium to limit NO production and its bioavailability^86-87, induction of oxidative injury to endothelium⁸⁰, promote lipid peroxidation which decreases expression of eNOS⁸⁸ and decrease in cellular glutathione peroxidase (GPx-1) both in vitro and in vivo⁸⁹. Homocysteine-induced oxidative stress induces intimal injury, activate elastase and increase deposition of calcium and sulfated glycosaminoglycan. Hhcy induces oxidative stress by ADMA accumulation; an eNOS and iNOS inhibitor⁹⁰ which attenuates endothelial NO signalling⁹¹. The inhibitory effect of ADMA on NO synthesis is removed by dimethylarginine dimethylaminohydrolase (DDAH), which catalyzes the conversion of ADMA to L-arginine, citrulline and dimethylarginine⁹². Hhcy induces homocysteine auto-oxidation⁹³ and activate nicotinamide adenine dinucleotide phosphate NAD(P)H oxidase activity⁹⁴ to produce oxidative stress.

Hhcy stimulates proinflammatory responses⁹⁵ induced by expression of monocyte chemoattractant protein-1 and IL-8 through activation of NF-κB which stimulate production of cytokines, chemokines, leukocyte adhesion molecules and hemopoetic growth factors⁹⁶. It increases endoplasmic reticulum (ER) stress and activate unfolded protein response (UPR)⁹⁷ to induce apoptotic cell death⁹⁸.

Approaches to Ameliorate VED:

The delivery of eNOS and its gene⁹⁹, eNOS substrate¹⁰⁰ and its cofactors¹⁰¹, activators of eNOS¹⁰², scavengers of peroxynitrite¹⁰³, inhibitors of endothelial cell senescence¹⁰⁴ and NO donors¹⁰⁵ may be employed to improve vascular endothelial dysfunction.

The reduced expression or activity of eNOS can be modulated by ex vivo or in vivo delivery of eNOS gene. The transduction of recombinant eNOS in human saphenous vein has been reported to augment NO production¹⁰⁶. The in vivo eNOS gene transfer has been demonstrated to inhibit neointimal vascular lesions¹⁰⁷, reduce hypertension¹⁰⁶ and enhance vasorelaxation in carotid arteries obtained from hypercholesterolemic rabbits¹⁰⁸, and improve bioavailability of NO in stroke-prone spontaneously hypertensive rats¹⁰⁹. However, the overexpression of eNOS has been shown to accelerate development of atherosclerotic lesions in apolipoprotein E-deficient mice¹¹⁰.

The infusion of L-arginine, an eNOS substrate, has been noted to restore acetylcholine-induced coronary vasorelaxation in hypercholesterolemic patients and cholesterol-fed rabbits¹¹¹. Moreover, L-arginine has been demonstrated to protect heart from ischaemia-reperfusion injury, inhibit intimal hyperplasia after balloon induced endothelial injury, and decrease endothelial adhesiveness due to hypercholesterolemia^112-113.The systemic infusion of L-arginine to healthy subjects has been noted to induce vasodilation and inhibit platelet aggregation⁶⁸. Moreover administration of L-arginine in hypercholesterolemic rabbits has been reported to regress preexisting lesions due to the induction of apoptosis of macrophages¹¹⁴. However, L-Arginine overload may increase homocysteine formation, methylation stress and consequent attenuation of its beneficial effects^115,87.

BH₄, molecular oxygen, NADPH and FAD are cofactors for activation of eNOS¹¹⁶. BH₄ has been documented to improve endothelial function in familial hypercholesterolemia, nitroglycerin-tolerant and insulin-resistant rats^117-118. However, the excessive amount of tetrahydrobiopterin has been found to be deleterious due to excessive generation of superoxide anion¹¹⁹.

The supplementation of folic acid or its active circulating metabolite such as 5- methyltetrahydrofolate is noted to restore impaired endothelium dependent vasodilatation in patients with HHcy or hypercholesterolemia^120-121. Vitamin E and probucol which are potent antioxidants have been demonstrated to preserve endothelial function in hypercholesterolemic rabbits¹²². Vitamin C and E normalize genetic endothelial dysfunction through regulation of eNOS and NAD(P)H oxidase activity¹²³. PKC inhibition reversed hyperhomocysteinemia induced eNOS inactivation and threonine 495 phosphorylation in HAECs to improve endothelial dysfunction¹²⁴.

3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) may exert beneficial effects through decrease in cholesterol and LDL and/or increase in HDL. Oxidized LDL decreases transcription of eNOS and destabilizes its mRNA¹⁰². Moreover, oxidized LDL may suppress L-arginine uptake in platelets and endothelial cells¹¹¹. HDL improves vascular endothelial dysfunction due to its antioxidant action¹²⁵. It has been demonstrated that statins prevent suppression of mRNA of eNOS by oxidized LDL in endothelial cells. Moreover, statins stimulate eNOS through activation of protein kinase B/Akt and consequent phosphorylation of eNOS¹²⁶. Akt activation activate eNOS and reduces oxidative stress to improve diabetes and hyperhomocysteinemia induced vascular endothelial dysfunction⁶². Statins generate and regenerate tetrahydrobiopterin ¹²⁷ and consequently restore coupled state of eNOS to improve vascular endothelial dysfunction¹²⁸. Protein tyrosine phosphatase (PTPase) inhibitors has shown improvement in peripheral endothelial dysfunction in heart failure patients¹²⁹. Moreover, PTPase inhibitors improve hypercholesterolemia and hypertension induced vascular endothelial dysfunction¹³⁰. Angiotensin receptor blocker has improved endothelial dysfunction in patients with essential hypertension¹³¹. Moreover, Mas, a receptor for Angiotensin (ANG)-(1-7) preserve normal vasorelaxation and Mas agonists has improved endothelial dysfunction¹³².

ACE inhibitors improve vascular endothelial dysfunction in patients with CAD or congestive heart failure due to the inhibition of angiotensin II production, concomitant reduction in oxidative stress and stimulation of bradykinin- dependent activation of eNOS to produce NO^133,35. Chronic uses of angiotensin-converting enzyme inhibitors are noted to enhance expression of eNOS¹¹² and suppression of PAI-1¹³³.

17-β estradiol treatment to hypercholesterolemic rabbits with severe endothelial dysfunction has been found to improve endothelium dependent vasorelaxation¹³⁴ and decrease size of atheromas without altering serum cholesterol level due to activation of Akt and phosphorylation of eNOS¹³⁵. Moreover, PPAR-α agonists (fibrates) have been shown to increase half-life of eNOS mRNA ¹³⁶. The anti-inflammatory action of fibrates and their ability to suppress eNOS has been attributed to their effect due to PPAR-α¹³⁷.

Rho kinase inhibition has improved vascular endothelial dysfunction in coronary artery disease patients, diabetic and hyperhomocysteinemic rats^138-139. Protein Kinase A (PKA) inhibits Rho A activation¹⁴⁰. Moreover, PKA activator ameliorate diabetes and hyperhomocysteinemia induced vascular endothelial dysfunction¹⁴¹Taurine protects HUVECs from hyperglycemia induced endothelial dysfunction by downregulation of apoptosis and adhesion molecules¹⁴². Recently GLP-1 analogue, exendin-4 has shown its role in improvement of diabetes mellitus and hyperhomocysteinemia-induced vascular endothelial dysfunction¹⁴³.

Potential Target Sites for Vascular Endothelial Dysfunction
Protein Kinase A	Glucagon-Like Peptide-1
Peroxisome Proliferator Activated Receptors	Caveolin
Cholesteryl Ester Transfer Protein	Rho Kinase
Lipoprotein lipase	Poly (ADP ribose) polymerase
Sphingosine-1-phosphate	Protein Tyrosine Phosphatase
AGEs and Transketolase	Akt
Geranylgeranyltransferase	Angiotensin Converting Enzyme II
Dipeptidyl Peptidase –IV	Janus Kinase

Thus, it may be concluded that various new target sites are being explored and their modulators have shown role in amelioration of VED. New drugs which improve diabetes mellitus, hyperhomocysteinemia and other implications of VED, will be the novel future approach for management of VED and its complications. Therefore, various pathological targets are still needs to be explored employing other animal models and pharmacological interventions.

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Received on 09.01.2010 Modified on 05.02.2010

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