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436 Nandini Nair, Hannah Valantine, and John P. Cooke

observed in patients with transplant arteriopathy. This impairment reflects abnormalities in the production or activity of several endothelial vasoactive substances. Most notably, there is a significant deficit in the nitric oxide synthase (NOS) pathway. The impairment of the NOS pathway contributes to alterations in vascular reactivity, structure, and interaction with circulating blood elements. Since endothelium-derived NO suppresses vascular cell proliferation and vascular inflammation, a deficit in vascular NO facilitates the initiation and progression of transplant arteriopathy. This chapter focuses on identification of factors adversely influencing endothelial dysfunction in transplant arteriopathy. Understanding the mechanisms of endothelial dysfunction may lead to endothelial-targeted therapies for prevention of transplant arteriopathy.

Vascular Alterations in Transplant Arteriopathy

Transplant arteriopathy is characterized by vascular inflammation and intimal proliferation, and it ultimately results in luminal stenosis of epicardial branches, occlusion of smaller vessels, and myocardial infarction. Histopathological analysis has revealed that morphologic manifestation of transplant arteriopathy may range from concentric, diffuse intimal hyperplasia to fibrofatty plaques indistinguishable from spontaneously occurring atherosclerosis [2].

Immunologic and nonimmunologic factors likely influence the evolution and progression of transplant arteriopathy. Allograft coronary endothelial cells can serve as potent stimulators (antigen-presenting cells) as well as targets of allogeneic lymphocyte reactivity. T-cell-interaction with graft endothelial cells, initiates and sustains the chronic immune response injury [3–5]. Furthermore, a number of conditions occurring in the context of transplantation may cause alterations in endothelial function, and the expression of adhesion molecules and chemokines, which participate in the inflammatory process leading to a prothrombogenic state. These predisposing conditions include preservation injury, ischemia–reperfusion, acute rejection, T cell activation, antibody deposition and complement fixation, and viral infection. The ongoing inflammation is thought to accelerate the development of transplant arteriopathy.

A major contributor to the inflammation and vascular cell proliferation that characterizes transplant arteriopathy is endothelial dysfunction. The allograft endothelium is made dysfunctional by a host of factors including ischemia–reperfusion during transplantation; metabolic abnormalities posttransplantation including dyslipidemia, insulin resistance, and hypertension due to the use of immunosuppressive agents; the direct effects of some immunosuppressive agents on endothelial function; and infectious agents, most notably cytomegalovirus (CMV).

Evaluation of Endothelial Vasodilator Function

of the Coronary Arteries

One of the important endothelial functions is its ability to modulate vessel tone. The healthy endothelium exerts a vasodilator influence by releasing a panoply of paracrine factors that relax vascular smooth muscle cell (VSMC), such as prostacyclin, endothelium-derived hyperpolarizing factor, and nitric oxide (NO). Endothelial vasodilator function of the epicardial coronary arteries is assessed by coronary angiography after intra-arterial infusions of acetylcholine or substance P. In addition, the endothelium can be stimulated to release vasodilator factors by increases in flow. The effect of increased flow can be assessed by infusing an endothelium-independent vasodilator (such as adenosine) downstream of the proximal epicardial coronary artery. During these interventions, coronary angiography permits measurement of the diameter of the epicardial coronary arteries. An increase in diameter of these conduit vessels is expected in response to stimulation of the endothelium.

The endothelial vasodilator function of the resistance vessels in the heart can be assessed by measuring coronary blood flow before and after intracoronary artery infusion of endothelium-dependent vasodilators such as substance P or acetylcholine. During these infusions, a Doppler flow wire is used to measure flow velocity, and thereby calculate coronary blood flow.

These approaches have been used to assess the endothelial function of the allograft coronary arteries. Endothelial dysfunction is commonly observed in the transplanted heart [6–8]. The etiology of endothelial dysfunction is multifactorial (Fig. 20.1) and time dependent. In the early months after transplant, epicardial vasodilatation is relatively preserved in response to tachycardia. During follow-up, exercise-induced flow-mediated endothelium-dependent vasodilation becomes impaired [9]. Some have estimated the prevalence of endothelial vasodilator dysfunction in the epicardial coronary arteries to be 20% to 30% of the patients during the first year and 30% to 40% at long-term follow-up [10]. This is almost certainly an underestimate, as endothelial dysfunction of the epicardial arteries was defined as a paradoxical vasoconstriction of >10% in response to acetylcholine [10]. The Stanford experience in recent years is that about 90% of cardiac transplant patients show endothelial vasodilator dysfunction, as defined by the absence of vasodilation or active vasoconstriction [11]. Epicardial endothelial dysfunction may be segmental in nature [12], which could cause a sampling error, and thereby account for some of the variability in reports of endothelial dysfunction. Furthermore, endotheliumdependent flow responses declined significantly (approximately 50%) in a 3-year follow-up period [13].

The severity of microvascular endothelial dysfunction does not correlate with vasodilator responses of the epicardial arteries suggesting independent determinants of the two processes [14, 15].

Endothelial Dysfunction and Loss of Vascular

Homeostasis

This endothelial dysfunction observed in the cardiac catheterization laboratory may contribute to alterations in vascular reactivity and coronary blood flow that adversely affect the allograft heart. The endothelium is a major determinant of vascular tone and blood flow. It synthesizes a variety of vasodilator substances, such as NO, prostacyclin, atrial natriuretic peptide, endothelium-derived hyperpolarizing factor, and adrenomedullin. The shear stress of coronary blood flow is a primary stimulus for the endothelial release of these substances. Endothelial vasodilator dysfunction may be associated with reduced release of vasodilators and increased endothelial production of vasoconstrictors such as endothelin and angiotensin II. Notably, a loss of the homeostatic balance between endothelial vasodilators (which predominate in health) and endothelial vasoconstrictors (which are increased in a number of vascular disorders, most notably arteriosclerosis) may contribute to adverse changes in vascular structure as well as vascular reactivity. Endotheliumderived NO is paradigmatic of an endothelial factor that is vasoprotective as well as vasodilatory. We and others have provided evidence that a reduction in the synthesis or bioactivity of endothelium-derived NO promotes processes favoring coronary vascular disease [16–19].

The Vasoprotective Effects of NO

Our studies have focused on endothelium-derived NO as a paradigm of an endothelial factor that regulates coronary vessel tone, vessel structure, and interaction of the vessel with circulating blood elements. Endotheliumderived NO is the most potent endogenous vasodilator known NO induces vasodilation by stimulating soluble guanylate cyclase to produce cyclic GMP (cGMP). NO has a short half-life, and avidly interacts with sulfhydrylcontaining proteins, heme proteins, and oxygen-derived free radicals. By virtue of its ability to nitrosylate proteins, it may change their activity or behavior [20]. The physiological importance of this endothelium-derived vasodilator is reflected by the significant increase in vascular resistance induced in animals and humans exposed to pharmacological antagonists of NOS [21].

Endothelium-derived NO also inhibits platelet adherence to the vessel wall. NO released into the lumen affects the behavior of circulating platelets. As platelets traverse the healthy myocardial microvasculature, they exhibit an elevation of cGMP, and a suppression of their aggregability. This effect of the healthy cardiac microvasculature can be suppressed by pharmacological antagonists of the NOS pathway. Furthermore, endothelium-derived NO inhibits leukocyte adherence to the vessel wall. An acute effect of NO to inhibit leukocyte adhesion is likely mediated by effects of NO on intracellular signaling of adhesion molecules [22]. A more chronic effect of NO is mediated by its suppression of specific adhesion molecules and chemokines. Finally,

440 Nandini Nair, Hannah Valantine, and John P. Cooke

endothelium-derived NO also inhibits VSMC proliferation [23–25]. This is partly mediated by an effect of NO to increase VSMC apoptosis [26]. These observations indicate that NO is an endogenous antiatherogenic molecule.

Impairment of the NOS Pathway in Atherosclerosis

Atherosclerosis and transplant arteriopathy share some common pathophysiological processes, hence it is instructive to review the evidence for a role of NOS impairment in atherosclerosis. In animal models and in patients, endothelium-mediated vasodilation is impaired [27, 28]. The mechanism of impairment may include endothelial generation of superoxide anion and increased degradation of NO; elaboration of vasoconstrictor prostanoids and endothelin; reduced elaboration of prostacyclin; and/or impaired biosynthesis of NO [29–31]. With respect to the latter defect, impaired biosynthesis of NO may be due to alterations in NOS affinity for L-arginine; lipid-induced impairment of the high-affinity cationic amino acid transporter (CAT); reduced availability of the cofactor tetrahydrobiopterin; or increased levels of asymmetric dimethylarginine (ADMA), the competitive inhibitor of NOS. Our group and others have accumulated extensive data to indicate that O2 − and ADMA are major determinants of endothelial vasodilator dysfunction induced by cardiovascular risk factors [32–35]. Whereas O2− degrades NO to reduce its bioactivity, ADMA inhibits NO synthesis. Moreover, recent data from our laboratory indicates that the elevations in O2− and ADMA are inextricably linked (Fig. 20.2).

Multiple lines of evidence point to a pathophysiological role for ADMA. We were the first to demonstrate that endothelial vasodilator dysfunction in hypercholesterolemic animals or humans could be reversed by administration of the NO precursor L-arginine [35, 36]. In patients with atherosclerotic or transplant coronary artery disease, the impairment of acetylcholine-induced

ARG

FIGURE 20.2. The effect of oxidative stress on intracellular signaling: the inextricable link between ADMA and oxidative stress.

vasodilation of the epicardial and coronary resistance vessels is reversed by an intravenous infusion of L-arginine. Several groups of investigators have observed an arginine-induced improvement in endothelium-dependent NOmediated vasodilation in patients with native or transplant coronary artery disease [37–40]. Since the Km of NOS for L-arginine is in a micromolar range [40], with plasma L-arginine levels at 50–100 m, L-arginine should not be rate limiting. There are several possible explanations for this “arginine paradox,” including effects of hypercholesterolemia and/or atherosclerosis upon NOS affinity; the effect of glutamine to inhibit activation of NOS; a reduction in the availability of the cofactor tetrahydrobiopterin; the colocalization to the caveolar membrane of NOS and CAT (the cationic amino acid transporter that mediates L-arginine influx) and reduced arginine transport due to lipid-induced impairment of the high-affinity CAT. Localization of NOS and CAT to the caveolar membrane may explain the sensitivity of the NOS pathway to reductions in extracellular L-arginine levels [41, 42]. However, there is accumulating data in preclinical and clinical studies, that ADMA is the major determinant of the “arginine paradox.”

ADMA: An Endogenous Inhibitor of NO synthesis

ADMA is a competitive inhibitor of NOS. This modified amino acid is derived from the methylation of internal arginine residues in proteins and the subsequent hydrolysis of these proteins [34]. ADMA is not produced by methylation of free arginine, and is not affected by L-arginine intake [43, 44]. ADMA is excreted by the kidney or metabolized by dimethylarginine dimethylaminohydrolase (DDAH) to citrulline and dimethylamine [45]. Normal plasma levels are 0.5 to 1 m [34]. Plasma levels of ADMA are elevated in a number of conditions associated with endothelial vasodilator dysfunction including renal failure, hypercholesterolemia, hyperhomocysteinemia, hypertension, diabetes mellitus, and heart failure [46–48]. In young hypercholesterolemic subjects, we have shown that plasma ADMA levels are doubled in association with an arginine-reversible impairment of flowmediated vasodilation (FMVD) of the brachial artery.

It appears that the levels of ADMA observed in these conditions are sufficient to explain the impairment of endothelial function, and to explain the observations made repeatedly by multiple investigators that the endothelial dysfunction is reversible by administration of exogenous L-arginine [35–39, 49, 50]. Faraci et al. [51] found that the IC50 for ADMA was 2 M. Added to crude purified preparations of eNOS, ADMA in a physiological/pathophysiological range (1 to 10 m) induces a dose-dependent inhibition of NO synthesis. At the concentrations observed in hypercholesterolemic patients, ADMA inhibits NO biosynthesis. It should also be noted that the plasma level of ADMA is only an indirect reflection of tissue levels. In endothelial cells regenerating after vascular injury the intracellular levels