Furchgott and Zawadzki initially demonstrated that relaxation of vascular rings in response to acetylcholine was dependent on an intact endothelium and was abolished by removal of the endothelium (7). This relaxation is due to the release of an endogenous substance called nitric oxide (8,9,10). Nitric oxide elicits vascular smooth muscle dilation by activating soluble guanylyl cyclase, thereby increasing intracellular levels of cyclic guanosine monophosphate (cGMP).

Nitric oxide is the most potent endogenous vasodilator yet identified and contributes to the regulation of basal vascular tone. Nitric oxide is synthesized by nitric oxide synthase from the amino acid, L-arginine (11,12) in response to a wide variety of stimuli (Fig. 14-1). A number of vasoconstrictors, such as norepinephrine, vasopressin, thrombin, and endothelin, interact with specific receptors on the endothelial surface to induce the release of nitric oxide, thus modulating the direct vasoconstrictor effect of these substances. Aggregating platelets also release vasoactive substances (e.g., adenosine diphosphate and serotonin) that stimulate nitric oxide production and thereby prevent further platelet aggregation. Thus, in the presence of an intact endothelium, nitric oxide counterbalances the deleterious effects

of vasoconstrictive and thrombogenic stimuli. However, after endothelial cell injury, the predominant effects of these stimuli are mediated by their receptors on vascular smooth muscle, activation of which causes vasoconstriction (13,14,15). Thus, alterations in the bioavailability of nitric oxide in disease states might lead to increased vascular resistance and may be associated with changes in vascular structure and increased thrombosis.

Endothelial cells synthesize the vasodilator prostaglandins E₂ and I₂ (PGE₂ and PGI₂) from arachidonic acid. Increased systemic levels of vasodilator prostaglandins are observed in patients with heart failure. In patients with severe left ventricular dysfunction, the prostaglandin metabolites of PGE₂ and PGI₂ are three- to tenfold higher than levels measured in normal subjects (40). The mechanisms underlying increased prostaglandin levels in heart failure are not known. Local synthesis of vasoactive prostanoids is increased by hypoperfusion of regional vascular beds (e.g., the renal and coronary circulations [41,42] and production of prostaglandins may directly be stimulated by vasoconstrictor hormones, such as angiotensin, vasopressin, and norepinephrine [43–45]). The concentration of prostanoids is not increased in all patients, but levels of these vasodilator prostaglandins correlate directly with the concentrations of angiotensin II and plasma renin activity. Thus, it appears that synthesis of prostaglandins by blood vessels counterbalances vasoconstrictor mechanisms in heart failure.

The contribution of local vasodilator prostaglandins to vascular resistance has important clinical implications. Prostaglandin-synthetase inhibition in patients with heart failure causes a decline in cardiac output and an increase in pulmonary capillary wedge pressure, particularly in those subjects with the greatest activation of circulating vasoconstrictor hormones (40). Additionally, some pharmacological agents produce their beneficial effects in part by increasing the production of endogenous vasodilator prostaglandins. The vasodilation induced by captopril, nitroglycerin, nitroprusside, and hydralazine is attenuated by pretreatment with indomethacin (46,47,48). In addition to its effects on angiotensin, captopril inhibits the breakdown of bradykinin by kininase II. Bradykinin may be an important contributor to the short-term vasodilatory effect of captopril (49), potentially by stimulating release of prostacyclin (50). Acetylsalicylic acid, another inhibitor of cyclooxygenase, has been found to blunt the augmentation in endothelium-dependent vasodilation induced by enalaprilat in patients with mild heart failure, highlighting the contribution of prostaglandins to the peripheral vascular effects of angiotensin-converting enzyme (ACE) inhibitors in this population (51,52).

The endothelium also produces potent vasoconstrictor substances. Yanagisawa et al. identified the endothelium-derived vasoconstrictor, endothelin, in 1988 (53). Endothelin, a 21-amino acid peptide hormone, is the most potent endogenous vasoconstrictor substance identified to date. Endothelin is synthesized from its messenger RNA (mRNA) in endothelial cells as a pre-propeptide and is cleaved to big endothelin by an endogenous endopeptidase. Mature endothelin is then produced by an endothelin-specific converting enzyme, which is present in the lung and in other endothelial cells (54). Many of the same stimuli that increase release of nitric oxide (including shear stress, epinephrine, thrombin, angiotensin II, arginine vasopressin, and calcium ionophore) also increase transcription of the pre-proendothelin mRNA and increase release of endothelin (53,55,56,57,58).

Three isoforms of endothelin have been identified and sequenced: endothelin-1 (ET-1), ET-2, and ET-3. The ET-1 isoform is most widely associated with cardiovascular regulation. ET-1 acts through the endothelin A (ET_A) and endothelin B (ET_B) receptor subtypes. The ET_A receptor, found on vascular smooth muscle cells, mediates vasoconstriction as well as smooth muscle cell proliferation (53,59) (Fig. 14-3). ET_B is present on both vascular smooth muscle cells and endothelial cells, where it mediates ET-1-induced vasoconstriction and vasodilation, respectively (60,61) (Fig. 14-3).

Endothelin binds to these specific receptors and activates several intracellular signal transduction systems via guanosine triphosphate (GTP)-binding proteins. Endothelin stimulates phospholipase C (PLC) and opens voltage-dependent calcium channels. Activation of PLC degrades phosphoinositide to form inositol triphosphate (IP₃) and diacylglycerol. IP₃ releases calcium from intracellular stores, and diacylglycerol sensitizes contractile proteins via protein kinase C (62,63,64,65). Endothelin may also promote vasoconstriction by upregulation of the endogenous nitric oxide synthase inhibitor, asymmetric dimethyl-arginine, thus reducing nitric oxide synthesis (66). Endothelin also stimulates phospholipase A₂ to produce several vasoconstrictor prostanoids (62,64). The effects of endothelin are most prominent in adjacent vascular regions and, thus, endothelin serves as an autocoid modulator of arteriolar resistance.

The vascular effects of endothelin are complex and potentially clinically important. During exogenous infusion of endothelin there is an initial, brief vasodilation followed by prolonged vasoconstriction (60,61,67,68,69). Infusion of exogenous endothelin into normal animals increases systemic vascular resistance and consequently reduces cardiac output via cardiac and associated peripheral vascular effects (67,68,70). Chronic exposure of vascular smooth muscle cells to endothelin has been shown to cause proliferation in vitro (71,72,73). Like peptide growth factors, endothelin mediates cellular growth via tyrosine kinases and mitogen-activated protein kinase pathways (74).

A two- to threefold increase in plasma concentrations of endothelin has been observed in experimental heart failure induced by rapid ventricular pacing in the dog (75,76). Elevations in plasma endothelin concentration also occur in humans with stable or decompensated heart failure (76,77,78,79,80).

Plasma endothelin levels may be increased in heart failure by several mechanisms. Catecholamines, angiotensin II, and arginine vasopressin stimulate endothelin synthesis (53,56,81) and endothelin clearance also is decreased in heart failure. The pathophysiological significance of increased plasma endothelin levels has become more apparent with the advent of endothelin receptor antagonists. In a rat model of post-myocardial infarction heart failure, both ET_A and combined ET_A/B receptor antagonism improved acetylcholine-mediated vasodilation and reduced superoxide anion formation (82) (Fig. 14-4). Similarly, treatment with the ET_A receptor blocker, LU135252, improved flow-mediated vasodilation in the brachial artery of humans with heart failure (83). Blockade of endogenous endothelin with a short-term infusion of the nonselective antagonist bosentan, lowered ventricular filling pressures and systemic vascular resistance in patients with heart failure (84). However, the promise of both specific and combined endothelin receptor antagonists has not been realized in large clinical trials, suggesting that the effects and importance of the two endothelin receptors are poorly understood (85,86,87) (Table 14-2).

The presence of renin, angiotensinogen, and angiotensin II in blood vessels has been reported by a number of laboratories (93,94). The vessel wall distribution of renin has been examined by use of antirenin-specific antibody, and intense staining has been noted throughout the thickness of the aorta, large and smaller arteries, as well as arterioles, particularly in endothelial and smooth muscle cells (95). Additionally, kinetic analysis of arterial-venous angiotensin I and angiotensin II differences in humans indicates that both angiotensin I and angiotensin II are synthesized in vascular tissues (96).

The local synthesis of angiotensin II in the blood vessel wall has important physiological implications. Angiotensin II has multiple effects on the cardiovascular system, which include not only vasoconstriction, but also smooth muscle cell proliferation, myocardial hypertrophy, and altered ventricular remodeling (97). Conceptually, local increases in vascular angiotensin II can cause constriction of large arteries and resistance vessels via the angiotensin type I receptor, resulting in increased systemic vascular resistance and reduced arterial compliance. Local angiotensin II also may contribute directly to regional blood flow regulation by activating vascular receptors in specific circulations (e.g., the kidney). Angiotensin II receptors are also found on endothelial cells, and activation can result in release of both nitric oxide (98) and vasoconstrictor prostanoids (99). Angiotensin II also may alter vascular function by facilitating norepinephrine release from local noradrenergic nerve terminals (100). In addition to these mechanisms, there is data suggesting that angiotensin II activates membrane-bound NADH/NADPH-driven oxidases that promote superoxide anion generation in cultured vascular smooth muscle cells (101). Cells exposed to angiotensin II for 4 hours produce two- to sevenfold more superoxide anion than control cells. This increase can be reversed by co-incubation with an angiotensin II receptor antagonist. Angiotensin II-induced, but not norepinephrine-induced, hypertension is associated with impaired vascular response to acetylcholine (102). Treatment with the angiotensin II receptor antagonist, losartan, improves the response to acetylcholine and normalizes vascular superoxide production, implicating the angiotensin type-1 receptor as a mediator in this process. These autocoid mechanisms may affect regional vascular tone, even when circulating levels of angiotensin II are not increased.

Evidence to support the importance of local angiotensin II to arteriolar and conduit artery function is derived from experiments involving ACE inhibition or angiotensin receptor blockade. Local infusion of ACE inhibitors to humans causes limb and coronary vasodilation, indicating that local generation of angiotensin II may contribute to vascular resistance (103). Intra-arterial infusion of quinapril augments the forearm blood flow response to bradykinin. The increase in arterial flow caused by quinapril can be inhibited by L-NMMA, suggesting that this ACE inhibitor may increase vascular nitric oxide, perhaps through a bradykinin-mediated pathway (104). The compliance and diameter of large (brachial and carotid) arteries are increased by ACE inhibition, even at doses that do not reduce blood pressure (105,106). This latter observation suggests that the local vascular renin-angiotensin system activity influences distensibility of these conduit vessels.

The juxtaglomerular apparatus is capable of releasing large amounts of renin into the circulation. In patients with heart failure, low renal perfusion pressure, decreased distal tubular sodium load, increased sympathetic activity, and diuretic administration all increase systemic levels of renin and angiotensin II. The presence of a locally active renin-angiotensin system in the kidney, in sites other than the juxtaglomerular cells, has been documented using molecular biological, immunocytochemical, and biochemical techniques. Renin has been demonstrated in afferent and efferent arterioles and in the proximal tubule by antirenin antiserum staining (107,108). Cultured glomerular mesangial cells synthesize renin (93). Angiotensinogen mRNA expression in the renal cortex has been demonstrated and in situ hybridization studies have
shown that angiotensinogen mRNA is expressed principally in the proximal tubule (109,110), while renin mRNA is primarily localized to the juxtaglomerular cells (110). Intrarenal ACE also has been demonstrated by the presence of ACE mRNA (111). In addition to the vasculature, ACE has been localized to the proximal tubule brush border using immunohistochemical and radioligand binding techniques (112). Because all the components are found in the proximal tubule, local synthesis of angiotensin II has been hypothesized (110). Indeed, a micropuncture study demonstrated that proximal tubular fluid angiotensin II concentration is 1,000-fold greater than in the plasma (113). Local angiotensin II production might be a major factor in regulation of basal renal hemodynamics and sodium reabsorption.