The Role of Endothelin in the Pathogenesis of Atherosclerosis

Mark C. Kowala Department of Biochemistry Bristol-Myers Squibb Pharmaceutical Research Institute Princeton, New Jersey 08543

The Role of Endothelin in the Pathogenesis of Atherosclerosis

1. Introduction Endothelin (ET) is a potent vasoactive peptide isolated from cultured endothelial cells (Yanigasawa et al., 1988). Three isopeptides of ET (ET-1, ET-2, ET-3) mediate their action by binding with two subtypes of endothelin receptors denoted as ETAand ETB(Arai et al., 1990; Sakurai et al., 1990). The amino acid sequence of the guanine nucleotide binding protein coupled receptors are 60% homologous (Arai et al., 1990). However, depending on their cellular location, they are functionally distinct. ET binding to ETAand ETB receptors on vascular smooth muscle cells produces contraction (Ihara et al., 1992; Sudjarwo et al., 1992; LaDouceur et al., 1993; Godfraind, 1993; White et al., 1993; Opgaard et al., 1994; Teerlink et al., 1994). ET activation of ETBreceptors on the endothelial cells produces smooth muscle cell relaxation via the release of nitric oxide and prostacyclin (de Nucci et al., 1988; Fukuroda et al., 1992; Ihara et al., 1992; Sudjarwo et al., 1992; Advances tn Pharmacology, Volume 37

Copyright 0 1997 h y Academic Press, Inc. All rights of reproduction in any form reserved.

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Hirata et al., 1993; Karaki et al., 1993; Tsukahara et al., 1994).The powerful vasoactive properties of ET influenced much of the early research toward its role on vascular tone, blood pressure, and hypertension. More recent evidence indicated that ET may participate in vascular diseases, such as atherosclerosis. Pertinent information supporting this concept is summarized and interpreted, but first the pathogenesis of atherosclerosis is described briefly to provide a context for the role of ET in this disease process.

II. Atherosclerosis: Lipoprotein Deposition to Plaque Rupture Hypercholesterolemia accelerates the transcytosis of plasma low density lipoprotein (LDL) across endothelial cells lining large arteries, and within the intima, these lipid particles undergo structural and biochemical modifications (Simionescu and Simionescu, 1993). Modified lipoproteins (called liposomes) rich in unesterified cholesterol accumulate in the extracellular matrix (Kruth, 1984; Simionescu et al., 1986). Arterial LDL becomes oxidized by free oxygen radicals, hydrogen peroxide, and enzymes such as lipoxygenases and phospholipases (Steinberg et al., 1989; Berliner et al., 1995). A lipid by-product of this reaction, lysophosphatidylcholine, is a chemoattractant for monocytes and lymphocytes (Quinn et a/., 1988). In vitro, lysophosphatidylcholine and oxidized LDL upregulate endothelial cell genes for platelet-derived growth factor (PDGF),monocyte chemoattractant protein-1 (MCP-1), and macrophage colony-stimulating factor (M-CSF), which are chemoattractants for monocytes (Berliner et al., 1995). Lysophosphatidylcholine stimulates the expression of intracellular adhesion molecule1(ICAM-1)and vascular cell adhesion molecule-1 (VCAM-1)on endothelial cells (Kume et al., 1992). Monocytes are recruited to the artery wall by tethering to the adhesion molecules, and after sensing a chemotactic gradient, they squeeze between adjacent endothelial cells toward the modified lipoproteins (Gerrity et al., 1979). The monocyte-derived macrophages bind and internalize the oxidized LDL via the scavenger receptor (Steinberg et al., 1989) and the Fc receptor (Stanton et al., 1992). Macrophages may also internalize LDL/proteoglycan complexes (Salisbury et al., 1985)or phagocytose aggregated masses of lipoproteins (Khoo et al., 1988). Through any of these pathways, macrophages perform their rudimentary housecleaning function during the early stages of the disease. The intracellular cholesterol becomes esterified and forms numerous cytoplasmic lipid droplets, gwing the macrophages their foamy appearance, hence the term foam cells. A massive accumulation of macrophage-foam cells leads to the first macroscopically visible lesion, the fatty streak. Macrophage-foam cells release a myriad of vasoactive peptides, lipid mediators, growth factors, cytokines, and chemokines that recruit additional

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blood-borne monocytes, lymphocytes, and medial smooth muscle cells into the intima and stimulate the proliferation of these cells. They include angiotensin 11, thromboxane A2 (TXA2),leukotriene B, (LTB4), interleukin-10 (IL-lo), interleukin-6 (IL-6), tumor necrosis factor (TNF), PDGF, heparinbinding epidermal growth factor (HB-EGF), basic fibroblast growth factor (bFGF), transforming growth factor-0 (TGF-P), macrophage-derived growth factor, M-CSF, MCP-1, and interleukin-8 (IL-8) (Ross, 1993; Berliner et al., 1995). Activated macrophages also produce free radicals, such as HzOzand Or, that probably further chemically modify arterial lipoproteins. The smooth muscle cells recruited from the media accumulate lipid, proliferate, and synthesize connective tissue, such as collagen. Smooth muscle cells generate numerous growth factors and inflammatory mediators, including PDGF, bFGF, HB-EGF, TGF-0, TNF, IL-1, IL-6, MCP-1, LTB4, TXA2, and angiotensin I1 (Ross, 1993). In the early atherosclerotic lesion, these factors act in a paracrine and autocrine fashion to amplify the recruitment of macrophages, lymphocytes, and smooth muscle cells and to increase the rate of cellular proliferation and connective tissue synthesis. Ultimately, the fatty streak transforms into a fibrofatty plaque that protrudes into the arterial lumen. Such lesions consist of a fibrous cap of smooth muscle cells and a connective tissue matrix surrounding a necrotic core of cholesterol crystals, with inflammatory foci occurring at the leading edges of the lesion. Thus, atherosclerosis is a chronic inflammatory response triggered by the continuous accumulation of arterial LDL. The developing fibrous cap of the atheroma is comparable to the growth of scar tissue following sublethal injury. The endothelial cells lining fatty lesions sometimes retract, allowing the formation of small mural thrombi (Faggiotto and Ross, 1984), and the subsequent exposure of the artery to thrombin, fibrinogen, fibrin, and PDGF may upregulate some inflammatory genes and accelerate the disease process. In some instances, the mural thrombi are permanently incorporated onto the lesion surface by encroaching fibrotic tissue, leading to an episodic enlargement of the plaque (Fuster et al., 1992). In the worst case, a plaque may rupture where the formation of a temporary or permanent occluding thrombus produces the clinical sequel of either unstable angina or myocardial infarction (Fuster et al., 1992).The cause of plaque rupture is unknown, but it may be related to arterial vasospasm at the site of the plaque (Kaski, 1991),increased blood shear forces (Karin0 etal., 1987; Glagov et al., 1988; Weinberger et al., 1988), changes in intraluminal coronary pressure or tone (Nobuyoshi et al., 1991; Lin et al., 1988), calcification of the atheroma (Demer et al., 1994), internal hemorrhage of intimal neovessels (Libby, 1995), or bending and twisting of the coronary arteries during the cardiac cycle (Fuster et al., 1992).Occlusive thrombi over plaques routinely originate at sites of intense focal inflammation (van der Wal et al., 1994) and where the fibrous cap is thin and flimsy (Richardson et al., 1989; Loree et al., 1992). It has been proposed that T lymphocyte-derived interferon-? (IFN-

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y ) inhibits smooth muscle cell proliferation (Hansson et a/., 1988) and

collagen synthesis, rendering the plaques susceptible to rupture (Libby, 1995). In situ zymographic techniques detected active matrix metalloproteinases in the vulnerable shoulder regions of atherosclerotic plaques, suggesting that matrix degradation contributes to lesion instability (Galis et al., 1994).Therefore, atherosclerotic plaques are benign until they fracture, and this event is less dependent on plaque size and degree of stenosis. A thin fibrous cap and focal inflammation are characteristics of plaques that are prone to rupture (Falk et al., 1995).

111. Elevated Plasma Endothelin with Hyperlipidemia, Atherosclerosis, Transplantation Arteriosclerosis, and Diabetes Several studies demonstrated that plasma ET concentrations are elevated in hypercholesterolemic patients (Bath and Martin, 1991; Haak et al., 1994) and in those with ischemic heart disease (Yasuda et al., 1990). In patients with symptomatic atherosclerosis, plasma ET levels increase compared with normal subjects, and there is a significant correlation between plasma ET concentration and the number of vascular sites with atherosclerosis (Lerman et al., 1991). Arendt et al. (1993) observed incremental increases in plasma ET levels in control patients vs those with hypercholesterolemia, coronary artery disease, and unstable angina. Plasma ET concentrations are elevated in hyperlipidemic pigs (Lerman et a/., 1993) and rats (Horio et al., 1991; Miyauchi et al., 1992). In patients undergoing cardiac transplantation, the plasma ET level becomes higher (Lerman et al., 1992; Haas et al., 1993; Dorent et al., 1994), and it is elevated in patients with non-insulin-dependent diabetes mellitis (Morise et al., 1995). Thus, plasma ET increases with cardiovascular risk factors, such as hypercholesterolemia and diabetes, and also when symptomatic atherosclerosis is present. Organ cultures of atherosclerotic rabbit aortas secrete more ET-1 than normal arteries (Patrignani et al., 1992). The release of arterial ET into the vascular compartment is unexpected, since in vitro endothelial cells secrete this peptide on the basolatera1 side (Wagner et al., 1992), suggesting that plasma ET levels may, in fact, underestimate the degree of ET synthesis in atherosclerotic arteries.

IV. Endothelin and I t s Receptors in Atherosclerotic Lesions Further evidence for ET involvement in atherogenesis was obtained from atherectomy specimens of human atherosclerotic plaques that had an increased expression of mRNA for preproendothelin (Winkles et al., 1993), and ET-1-like immunoreactivity was detected in vascular smooth muscle

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cells and endothelial cells (Lerman et al., 1991). In human coronary plaques, immunohistochemistry revealed that ET-1 was in macrophage-rich areas, in hypercellular regions with many microvessels, and in areas with evidence of previous hemorrhage (Zeiher et al., 1995). Both smooth muscle cells and macrophages were labeled for ET-1, and the highest expression occurred in active plaques of patients with crescendo angina or angina after myocardial infarction (Zeiher et al., 1995). Endothelial cells and subendothelial myointima1 cells or macrophages of the anterior descending coronary artery were positively stained for ET in hypercholesterolemic pigs (Lerman et al., 1993). In a rat model of chronic cardiac allograft rejection, ET-1 mRNA was elevated in rejected allografts at 7, 28, and 75 days (Watschinger et al., 1995). Immunohistochemistry indicated that ET-1 expression was localized predominantly in ED-1-positive macrophages infiltrating the myocardium and the arterial neointima (Watschinger et al., 1995). In the balloon-injured rabbit carotid artery, there was increased immunostaining for ET in the neointima containing smooth muscle cells at 24 and 72 h and at 4 weeks (Azuma et al., 1994). Since cultured endothelial cells (Stewart et al., 1990; Wagner et al., 1992; O’Reilly et al., 1993), smooth muscle cells (Kame et al., 1991; O’Reilly et al., 1993), monocytes, and macrophages (Ehrenreich et al., 1990) synthesize and secrete ET-1, it is likely that these cells may be actively producing ET in atherosclerotic and arteriosclerotic lesions. Endothelin receptors have also been identified in human atherosclerotic lesions. Messenger RNA for ETA and ETBreceptors was detected in human carotid artery atherectomy specimens, but their level of expression decreased compared with human aortas without lesions (Winkles et al., 1993). It is unknown whether these carotid plaques were active or stable. In vitro receptor autoradiography of human atherosclerotic coronary arteries demonstrated that ETA receptors were in the media, and non-ETAreceptors localized in areas of neovascularization (Dashwood et al., 1994). In sitti hybridization of atherosclerotic plaques from hyperlipidemic hamsters indicated that mRNA for ETA and ETBreceptors localized in endothelial cells and medial smooth muscle cells, as well as in intimal macrophage-foam cells and smooth muscle cells (Kowala et al., 1995). Neointimal smooth muscle cells in balloon-injured rabbit carotid arteries contained ETBreceptors, whereas the media had ETA receptors, as determined by displacement with biotinylated ET-1, BQ-123 (ETAreceptor antagonist), and Ro-462005 (ETA/ETB receptor antagonist) (Azuma et al., 1995). By in situ hybridization, mRNA for ETA receptors was detected in neointimal smooth muscle cells of the rat carotid artery following balloon catheter injury (Ferrer et al., 1995). ETA and ET, receptors localize on vascular smooth muscle cells (Ihara et al., 1992; Sudjarwo et al., 1992; LaDouceur et al., 1993; Godfraind, 1993; White et al., 1993; Opgaard et al., 1994; Teerlink et al., 1994). On the other hand, ETBreceptors are in human umbilical cord endothelial cells

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(HUVEC) (Winkles et al., 1993; Fujitani et al., 1992; O’Reilly et al., 1993) and in venous endothelium of various tissues (Ghoneim et al., 1993; Hagiwara et al., 1993; Durham et al., 1993). Endothelial ETA receptors were identified in rabbit aortic valves (Amano et al., 1994) and in human and rat brain microvessels (Stanimirovic et al., 1994a, b; Vigne and Frelin, 1994; Ladoux and Frelin, 1991). Peritoneal macrophages contain ETBreceptors (Kishino et al., 1991). In the hamster model, expression of mRNA for ETA receptors in endothelial cells and macrophages of the fibrofatty plaque possibly suggests that under atherogenic conditions, there is an alteration of ET receptor phenotype or an increased expression of one subtype of receptor, in this case the ETA receptor. Interestingly, IL-1p induced ETA receptor expression in human vascular smooth muscle cells (Newman et al., 1995). In vitro, rat aortic smooth muscle cells at the 10th-15th passage express the ETA receptor phenotype, whereas by the 30th-35th passage, ETB receptors predominate (Eguchi et al., 1994). These results further support the concept that the expression of ET receptor subtypes is changeable. In summary, ET and its receptors are localized in endothelial cells, macrophages, and smooth muscle cells of atherosclerotic and arteriosclerotic lesions and also in the same cells isolated in vitro.

V. Endothelin and Atherosclerosis: In Vivo Studies There is compelling in vivo evidence that endothelin promotes vascular disease. Infusion of exogenous ET accelerated smooth muscle cell accumulation in the neointima of the balloon-injured rabbit and rat carotid arteries (Trachtenberg et al., 1993; Douglas et al., 1994). Conversely, either a mixed ETA/ETB receptor antagonist (SB-209670) or a selective ETA receptor antagonist (BMS-182874)reduced the size of the smooth muscle cell-rich neointima in the rat model of intimal hyperplasia (Douglas et al., 1994; Ferrer et al., 1995). Based on in vitro data, it was presumed that the ET receptor antagonists inhibited smooth muscle cell proliferation. In the hamster model of atherosclerosis, the selective ETA receptor antagonist BMS- 182874 inhibited the formation of the fatty streak by reducing the number and the size of the macrophage-foam cells (Kowala et al., 1995). Increasing doses of BMS182874 (19, 38, 75, 150 pmol/kg) resulted in parallel decreases in fatty streak area and in the ET-l-induced pressor response (Fig. 1). The results suggested that ET receptors and, thus, ET promoted the early inflammatory phase of atherosclerosis. There is evidence that ET acts as an inflammatory mediator. For example, ET-1 activation of the ETA receptor causes the release of histamine from mast cells (Yamamura et al., 1994), which may explain how ET increases vascular permeability in the rat mesentary and in the hamster cheek pouch possibly through the contraction of postcapillary venule endothelial cells (Kurose et al., 1993; Mayhan and Rubinstein, 1994).

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In summary, ET receptor antagonists retarded the accumulation of intimal smooth muscle cells after balloon injury and decreased arterial macrophagefoam cell number and size during hyperlipidemia. Therefore, blocking the effects of ET with receptor antagonists diminishes the progression of vascular diseases in vivo.

VI. Stimuli for Endothelin Production In vitro, oxidized LDL and acetylated LDL increase preproendothelin mRNA in HUVEC and stimulate the release of ET, whereas native LDL and very low density lipoprotein (VLDL)were almost ineffective (Boulanger et al., 1992; Horio et al., 1993).Thrombin augmented the stimulatory effects of oxidized LDL (Boulanger et al., 1992). Oxidized and acetylated LDL activate human monocyte-derived macrophages to release ET into the culture medium (Martin-Nizard et al., 1991). Vasoactive peptides, growth factors, and inflammatory cytokines, such as angiotensin 11, vasopressin, thrombin, IL-10, TGF-P, and TNF-a, stimulate endothelial cells to increase mRNA expression for preproendothelin, which leads to ET secretion ( Yoshizumi et al., 1990; Boulanger et al., 1992; Maemura et al., 1992; Emori et al., 1992; Imai et al., 1992; Marsen et al., 1995; Hollenberg et al., 1994; Golden

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et al., 1995). Thrombin’s effect on ET production occurred through the breakdown of endothelial cell phosphoinositide, leading to the activation of protein kinase C and increases in intracellular Ca2+(Emori et al., 1992). Under conditions mimicking diabetes, both glucose and insulin stimulated endothelial cell gene transcription and synthesis of ET (Yamauchi et al., 1990; Hu et al., 1993). The effect of insulin on ET generation occurred through the insulin receptor coupled to the tyrosine kinase pathway (Hu et al., 1993). Shear stress also induces endothelial ET-1 gene expression through Ca2+ and protein kinase C-dependent pathways (Morita et al., 1994). Thus, a variety of atherogenic and inflammatory stimuli upregulate ET gene expression and protein synthesis in vitro.

VII. Effect of Endothelin on Prostaglandin, Cytokine, and Chemokine Production

ET-1 stimulates endothelial cell production of ET itself (Saijonmaa et al., 1992) and of TNF-a, IL-6, TXA2, prostacyclin, prostaglandin E2 (PGEZ), LTB,, and 5-hydroxyeicosatetraenoic acid (5-HETE) (Helset et al., 1993; Muck et al., 1993; Xin et al., 1995). In response to ET, human monocytes undergo Ca2+mobilization, which is accompanied by an increased release of 0 2 , IL-6, arachidonic acid, TXA2, and PGEz (Haller et al., 1991; Millul et al., 1991; McMillen et al., 1993, 1995). Enhanced IL-6 production by monocytes may augment hepatic synthesis of fibrinogen (Baumann et al., 1987; Otto et al., 1987), and elevated plasma fibrinogen is an independent risk factor for cardiovascular disease (Kannel et al., 1992; Ernst, 1993). ET activates monocyte production of IL-8 (Helset et al., 1994), which is a mitogen and chemoattractant for smooth muscle cells (Yue et al., 1994) and endothelial cells (Koch et al., 1992). Monocytes treated with ET also increase their production of MCP-1 (Helset et al., 1994), a powerful monocyte and lymphocyte chemoattractant. Therefore, ET is capable of promoting the synthesis of several characterized inflammatory mediators.

VIII. Endothelin Promotes Atherosclerosis: Potential Mechanisms Monocyte invasion of the arterial wall is an essential step toward the development of the fatty streak (Joris et al., 1983). ET was reported to be a chemoattractant for human peripheral blood monocytes (Achmad and Rao, 1992). However, this finding was not reproduced (Bath et al., 1990; Helset et al., 1994). ET-1 may indirectly recruit monocytes by stimulating arterial macrophages (and not endothelial cells) to synthesize MCP-1, a potent chemoattractant for monocytes (Helset et al., 1994). Human brain

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endothelial cells stimulated with ET-1, ET-2, or ET-3 increase the level of Eselectin, ICAM-l, and VCAM-1 expression (McCarron et al., 1993).Taken together, local synthesis of arterial ET-1 may stimulate endothelial cells and arterial macrophages to produce adhesion molecules and a chemotactic gradient, respectively, which promotes peripheral blood monocyte diapedesis into the arterial intima. In addition, elevated synthesis of TXAL by ET may contribute to atherogenesis, since this prostaglandin promotes the formation of the fatty streak (Skrinska et al., 1988). The recruitment of medial smooth muscle cells to the intima and their subsequent proliferation transform the fatty streak into a fibrous plaque. The first evidence linking ET with atherosclerosis was provided by Komuro et ul., (1988), where ET-1 induced the proto-oncogenes c-myc and c-fos and promoted the synthesis of DNA in rat aortic smooth muscle cells (RASMC). Increased DNA synthesis in RASMC treated with ET-1 was blocked by the ETAreceptor antagonists BQ-123 and BMS-182874 (Ohlstein et al., 1992; Ferrer et al., 1995) and by a nonselective ETA/ETBreceptor antagonist SB209670 (Douglas et al., 1994).The mitogenic effect of ET-1 was synergistically enhanced by epidermal growth factor and TGF-fl but not PDGF (Hirata et a/., 1989). Maximal RASMC DNA synthesis occurs 48 h after ET-1 stimulation, indicating that the delayed mitogenesis is due to the production of additional autocrine and paracrine growth factors (Weber et al., 1994). ET-1 induces PDGF-AA and TGF-P transcription after 3-10 h in RASMC (Hahn et al., 1990), suggesting that these growth factors drive the delayed mitogenic response. ET-1 treatment of RASMC also increased Ca2+mobilization, phosphoinositide metabolism, and phosphorylation of a substrate for protein kinase C (Weber et al., 1994). Unlike RASMC, only some human vascular smooth muscle cell lines are mitogenic for ET, and there is a correlation between mitogenicity and number of ET receptors (Kanse et al., 1995). It appears that the contractile smooth muscle cell phenotype had higher specific binding of [12sI]ET-1 (i.e., more ET receptors) and greater DNA synthesis than the synthetic phenotype (Serradeil-Le Gal et al., 1991). Complementary to ET stimulating smooth muscle cell growth factor production, PDGF-AA, TGF-fl, angiotensin 11, and ET-1 itself increase the expression of preproendothelin mRNA and subsequent ET-1 synthesis (Hahn et al., 1990). As mentioned previously, ET-1 activates monocyte production of IL-8 (Helset et ul., 1994), a chemokine that is mitogenic and chemotactic for smooth muscle cells (Yue et ul., 1994). IL-8 and ET-3 induce endothelial cell proliferation iM vitro (Wren et al., 1993; Koch et al., 1992), and IL-8 is capable of causing angiogenesis (Koch et al., 1992). Therefore, ET-1 controls smooth muscle cell proliferation in a paracrine and autocrine fashion, and the ability of ET to upregulate gene expression of a variety of factors, and vice versa, provides a powerful amplifying mechanism for smooth muscle cell chemotaxis and proliferation.

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Another component of plaque growth is the expansion of the extracellular matrix. ET-1 increases fibronectin transcription and synthesis in vascular smooth muscle cells (Hahn et al., 1993). In addition, rat cardiac fibroblasts and kidney mesangial cells produce more collagen when treated with ET-1 and ET-3 (Ruiz-Ortega et al., 1994; Guarda et al., 1993). Thus, ETpossibly initiates smooth muscle cell synthesis of connective tissue. Taken together, the in vitro studies indicate that arterial ET may facilitate monocyte diapedesis, stimulate smooth muscle cell migration and proliferation, and promote the synthesis of connective tissue-all obligatory steps in the formation of an atherosclerotic plaque.

IX. Endothelin and Arterial Vasospasm Coronary artery spasm appears to be a consequence of a local nonspecific hyperreactivity of medial smooth muscle cells. It may participate in acute coronary syndromes by interrupting blood flow and contribute to plaque fissuring and thrombosis (Kaski, 1991). ET has been suggested to play a role in coronary vasospasm (Igarashi et al., 1989; Toyo-oka et al., 1991). Patients infused with acetylcholine or ergonovine who developed coronary artery spasm had elevated plasma ET concentrations (Toyo-oka et al., 1991). Isolated rings of human mammary and coronary artery treated with threshold concentrations of ET-1 had amplified contractions to norepinephrine and serotonin (Yang et al., 1990). In hypercholesterolemic monkeys, the atherosclerotic illiac arteries had potentiated constrictor responses to ET-1 compared with normal vessels (Lopez et al., 1990). Monocytes incubated with strips of guinea pig carotid artery augmented the contraction induced by ET-1 (Magazine et al., 1994), suggesting that inflammatory cells enhance vasoconstriction in response to ET. ET-1 induced the contraction of distal preresistant human coronary arteries via ETAreceptors, and other ET receptors are responsible for contraction of the proximal segments (Godfraind, 1993). In a nutshell, there is no direct evidence that ET causes or promotes coronary vasospasm (due to the absence of appropriate models). Nevertheless, the data suggest that arterial reactivity increases with the combination of ET, atherosclerosis, and inflammation.

X. Synopsis The evidence supporting the participation of ET in atherogenesis is summarized in Tables I and 11. Based on current information, the following hypothesis is proposed. In the earliest stages of atherosclerosis, oxidized LDL stimulates arterial endothelial cells and macrophages to synthesize ET. Some of the peptide is secreted into the circulation, thereby raising plasma

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TABLE I in Vitro Evidence for ET Participation in Atherosclerosis ET receptors are on endothelial cells, macrophages, and smooth muscle cells Oxidized LDL induces endothelial cell and macrophage production of ET ET is a direct smooth muscle cell mitogen ET is an indirect smooth muscle cell mitogen via induction of PDGF, TGF-P, IL-8 ET, PDGF-AA, IL-I, TGF-P, and angiotensin I1 induce ET production ET induces IL-16, IL-6, TNF-a, and free oxygen radical production ET induces endothelial cell proliferation (and possibly angiogenesis via IL-8) ET induces inflammatory lipid mediator production, e.g., 5-HETE, TXAL, LTB, ET induces inflammatory chemokine production, e.g., MCP-1, IL-8 ET induces adhesion molecule expression, e.g., E-selectin, ICAM-1, VCAM-1 ET induces synthesis of connective tissue, such as fibronectin and collagen Insulin and glucose increase ET production

ET concentrations. Arterial ET activates endothelial cells in an autocrine and paracrine fashion to upregulate E-selectin, ICAM-1, and VCAM-1 expression. Similtaneously, ET stimulates resident intimal macrophages to produce MCP-1, which establishes a chemotactic gradient. Therefore, ET may indirectly facilitate monocyte adhesion and emigration into the artery wall. Subendothelial macrophages stimulated by ET increase the generation of free oxygen radicals that further oxidize arterial LDL and promote foam cell formation. Arterial ET activates resident intimal smooth muscle cell production of PDGF, TGF-P, and macrophage synthesis of IL-8, which promote the recruitment and proliferation of medial smooth muscle cells. Fibronectin generation by smooth muscle cells also becomes elevated. This scenario is illustrated in Fig. 2. There is in vitro evidence for the effects of ET on smooth muscle cell migration and proliferation, but in vivo data on these parameters are absent. Further gaps are apparent regarding the effects of ET on monocyte adhesion, migration, macrophage-foam cell formation, and connective tissue synthesis. Despite the missing pieces of mechanistic TABLE II In Vivo Evidence for ET Participation in Atherosclerosis Plasma ET increases with hyperlipidemia, atherosclerosis, transplantation arteriosclerosis, and diabetes ET is located in endothelial cells, smooth muscle cells, and macrophages of atherosclerotic and arteriosclerotic lesions ET receptors are o n endothelial cells, smooth muscle cells, and macrophages of atherosclerotic and arteriosclerotic lesions Infusion of exogenous ET accelerates neointimal smooth musle cell accumulation after balloon injury ETA/ETBor ETAreceptor antagonists decrease neointimal smooth muscle cell accumulation after balloon injury ET,, receptor antagonist decrease macrophage-foam cell accumulation during h ypercholesterolemia

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FIGURE 2 Diagram illustrating the hypothetical mechanisms whereby ET promotes atherosclerosis. Plasma LDL infiltrates into the artery wall and becomes minimally modified LDL (mmLDL) and, ultimately, oxidized LDL (oxLDL). Oxidized LDL stimulates endothelial cells and resident intimal macrophages to synthesize and secrete ET. Arterial ET stimulates endothelial cells to upregulate E-selectin, [CAM-1, and VCAM-1 expression and activates macrophages to produce MCP-1, which facilitate monocyte recruitment to the artery wall. Arterial ET stimulates intimal smooth muscle cell and macrophage production of ET, PDGF, TGF-P, and IL-8, which promote the recruitment and replication of medial smooth muscle cells. Fibronectin (FN)generation by smooth muscle cells is also increased. Macrophages stimulated by ET release free oxygen radicals that may further oxidize arterial LDL. ET also upregulates production of inflammatory mediators, such as TNF-a, TXAL,LTBs and 5-HETE.

data, the in uiuo studies with the ET receptor antagonists indicate that ET promotes atherosclerosis and arteriosclerosis. Obviously, ET is not the central player in the atherosclerotic process. Instead, it is part of a complex web of interacting vasoactive factors, prostaglandins, leukotrienes, growth factors, cytokines, and chemokines. ET upregulates gene transcription and protein synthesis of many inflammatory mediators and growth factors, and the reverse is also true, which forcefully amplifies the inflammatory and wound healing processes. With so many overlapping intercellular signalling pathways, there appears to be tremendous redundancy during the course of this disease; however, a selective ETAreceptor antagonist significantly inhibited early atherosclerosis in the hamster model. This suggests that blocking ET receptors interrupts the amplification process, or inhibits the generation of crucial autocrine and paracrine factors acting downstream of ET. The role of ET in clinical events, such as unstable angina and myocardial infarction, is less certain. ET is localized within inflammatory cells of active plaques that are prone to rupture, but the lack of models studying this phenomenon accounts for the absence of functional information. The potent vasoconstrictive properties of ET suggest a role in vasospasm and supportive

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TABLE Ill Evidence for ET Causing Vasospasm Plasma ET increases when acetylcholine or crgonvine induces coronary spasm Threshold concentrations of ET potentiate coronary artery vasoconstriction to serotonin and norepinephrine Atherosclerotic arteries had potentiated constrictor responses to ET-1 Monocytes potentiate ET-1-induced contraction of the carotid artery

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