Expressional changes of the vascular antioxidant system in atherosclerotic coronary arteries

Oxidative stress induced by reactive oxygen species (ROS) plays an important role in atherogenesis, and the redox state is determined by the balance between antioxidants and the ROS generating system. To defend against enhanced ROS, mammalian cells have a complex network of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase. To clarify the role of the vascular antioxidant system, we investigated by immunohistochemistry the expressional changes of antioxidative enzymes in coronary arteries obtained from autopsied cases. In nonatherosclerotic coronary arteries, Cu/Zn SOD and Mn SOD were expressed in medial smooth muscle cells (SMC), whereas cytosolic GPx (GPx-1) was expressed mainly in endothelium and weakly in medial SMC. Catalase was expressed in medial SMC and endothelium. Progression of atherosclerosis did not result in an additional increase in the expression of antioxidative enzymes in SMC in the media or endothelium. However, migrating SMC and macrophages in atheromatous plaques expressed these four antioxidative enzymes intensively. Double staining with cell markers confirmed the cell-specific expression of the antioxidative enzymes. Thus, the expressional pattern showed regional heterogeneity. In response to oxidative stress, the vascular antioxidant system was upregulated in atherosclerotic lesions. The imbalance between vascular antioxidant and oxidant systems might play an important role in coronary atherogenesis.     

Increased tissue transglutaminase expression in human atherosclerotic coronary arteries

BACKGROUND AND OBJECTIVE: Transglutaminase 2 (TGase 2) is a calcium-dependent cross-linking enzyme that catalyzes a covalent iso-peptide bond between two proteins. Interestingly, this catalysis can activate the nuclear factor-kappaB (NF-kappaB) through the polymerization of the inhibitory protein of NF-kappaB (I-kappaB). The objective of the present study was to investigate the expression of TGase 2 in the human atherosclerotic human coronary artery, and the possible roles of TGase 2 in NF-kappaB activation. METHODS AND RESULTS: We explored whether expressions of TGase 2 and NF-kappaB are associated in atherosclerosis. Using human samples, we found that TGase 2 was markedly higher than normal in the neointimal tissue of atherosclerotic coronary arteries with atherosclerosis progression. TGase 2 activity was also increased approximately two-fold in the atherosclerotic vascular wall. In immunofluorescence analysis, NF-kappaB, COX-2, and TNF-alpha were co-localized at TGase 2-positive neointimal smooth muscle cells. A promoter assay test showed that NF-kappaB activity increased in both the human monocyte and human breast carcinoma cell by TGase 2, and that TGase 2-mediated NF-kappaB activation was reversed by TGase 2 siRNA. CONCLUSION: According to these results, we suggest that TGase 2 may function as an activator in the NF-kappaB pathway; this effect may occur in the atherosclerotic vessel wall.     

Selective attenuation of endothelium-mediated vasodilation in atherosclerotic human coronary arteries

This study was undertaken to determine whether atherosclerosis impairs relaxations mediated by endothelium-derived relaxing factor (EDRF) in human coronary arteries. Epicardial coronary arteries were obtained from the hearts of cardiac transplantation patients with or without histologically documented coronary atherosclerosis (atherosclerotic arteries were from patients aged 42-55 years, nonatherosclerotic arteries were from patients aged 14-24 years). Transverse strip preparations were mounted in organ baths for isometric tension recording. Tension was induced with prostaglandins F2 alpha. Indomethacin (10(-5) M) was present to prevent possible interference from endogenously formed prostaglandins. The EDRF-mediated relaxations in response to substance P (10(-10) to 10(-8) M), bradykinin (10(-9) to 10(-7) M), and Ca2+-ionophore A23187 (10(-9) to 10(-7) M) were significantly attenuated in atherosclerotic arteries. In deendothelialized tissues these compounds had no effect. In contrast, endothelium-independent relaxations induced by isoprenaline (10(-7) to 10(-5) M) were not affected by atherosclerosis. Atherosclerotic arteries showed also normal relaxations with high concentrations of glyceryl trinitrate (10(-8) to 10(-7) M), but reduced relaxations with a lower concentration of the compound (10(-9) M). Acetylcholine (10(-7) to 10(-6) M) only produced endothelium-dependent relaxations in 8 of 60 arterial preparations (with or without atherosclerosis). In most of the arteries, it was a direct vasoconstrictor (which may have masked EDRF release in many cases). Omission of indomethacin from the bath solution increased the incidence of moderate acetylcholine-induced relaxations (9 of 16 preparations). It is concluded that atherosclerosis attenuates EDRF-mediated vasospasm and myocardial ischemia.     

Connective tissue components in normal and atherosclerotic human coronary arteries

Human coronary arteries with various degrees of atherosclerosis were analyzed for the concentration of different types of glycosaminoglycans (GAGs). The changes in GAGs were considered against the background of macroscopic atherosclerosis, and the concentration of glycoprotein-bound hexosamines, collagen, calcium and cholesterol. The concentration of calcium was increased and that of hyaluronic acid decreased even in mildly atherosclerotic coronary arteries. The additional changes in advanced atherosclerosis included the increase of collagen and dermatan sulphate and the decrease of heparan sulphate. Cholesterol was increased in mild, and even further in advanced, atheroslcerosis. The concentrations of chondroitin sulphates and glycoprotein-bound hexosamines were not significantly affected by atherosclerosis.     

Short-term administration of estrogen and vascular responses of atherosclerotic coronary arteries.

OBJECTIVES. This experiment sought to determine the effect of short-term administration of estrogen on endothelium-dependent dilation in the coronary arteries of 13 surgically postmenopausal female cynomolgus monkeys. BACKGROUND. Long-term estrogen replacement therapy prevents impaired endothelium-dependent dilation of atherosclerotic coronary arteries in postmenopausal female monkeys. However, it remains unclear whether this action of estrogen is due to long-term effects on plasma lipids and atherogenesis or to direct short-term effects on the endothelium. METHODS. The monkeys consumed an atherogenic diet for 18 months after bilateral ovariectomy. Vascular responses were measured just before euthanasia and necropsy. Dextrose in water (control), acetylcholine, 10(-6)M, and nitroglycerin were infused for 2.5 min each both before and 20 min after intravenous injection of 54 ng ethinyl estradiol. RESULTS. Quantitative coronary angiography revealed that the arteries constricted (-17 +/- 3%) in response to intracoronary infusion of acetylcholine before estrogen treatment but dilated (+5 +/- 3%) 20 min after intravenous injection of ethinyl estradiol (p less than 0.05). Coronary arteries dilated in response to nitroglycerin both before and after administration of estrogen (p greater than 0.05). Vascular responses of coronary arteries, both before and after administration of estrogen, were not associated with variation in plasma lipid concentrations, blood pressure, heart rate or plaque size. CONCLUSIONS. Estrogen affects endothelium-dependent coronary dilation within 20 min of administration and may have rapid direct effects on the vascular endothelium.     

Morphology of the endothelium over atherosclerotic plaques in human coronary arteries

The right coronary arteries of six hearts removed from patients with atherosclerosis, who were undergoing cardiac transplantation, were perfused with 2% buffered glutaraldehyde for 20 minutes before preparation for scanning electron microscopy. Perfusion was started within five minutes of explanation. In two patients the artery was angiographically normal, in one it was irregular in outline, and three had focal segments with significant stenosis. None of the patients had concentrations of plasma lipids above 5.5 mmol/l. The endothelial surface showed widespread focal abnormalities ranging from adhesion and migration of monocytes to loss of individual endothelial cells. Larger areas of endothelial denudation with exposure of underlying collagen were also seen consistently. Loss of endothelial cells was associated with accumulation of monocytes, on and deep to the surface, as well as adhesion of platelets to the exposed subendothelial tissue. These results accord with the endothelial damage and platelet adhesion seen in hyperlipidaemic animals fed a high lipid diet.     

Overexpression of tissue-type plasminogen activator in atherosclerotic human coronary arteries.

The plasminogen activator (PA)/plasmin system is involved in various pathological processes that are considered important features of atherogenesis and atherothrombosis. These include the proteolysis of fibrin deposits and extracellular matrix components as well as the induction of cell migration and mitogenesis. Tissue-type PA (TPA) is a key enzyme mediating plasminogen to plasmin conversion. TPA plasma concentrations are elevated in patients with advanced atherosclerosis and correlate with an increased risk for myocardial infarction and stroke. In this study, we have analysed the content and expression of TPA in human coronary arteries and their relation to the presence and severity of atherosclerotic lesions. Methods: Segments of coronary arteries obtained from heart explants (n = 15) were classified by the presence and types of atherosclerotic lesions. TPA was quantitatively determined in protein extracts of intimal and medial layers. In situ hybridization and immunohistochemical analyses were performed on serial sections of representative tissue specimens. Results: PA activity entirely attributable to the presence of active TPA was consistently detected in the protein extracts. Extractable TPA antigen and activity showed a significant graded increase in relation to the presence and severity of atherosclerotic lesions. The ratios of active over total TPA were increased several-fold in extracts of advanced lesions despite a concomitant threefold increase in TPA complexed to its inhibitor PA-1. In macroscopically normal arterial segments and in early lesions, TPA was expressed in the endothelium and in colocalization with vascular smooth muscle cells (VSMCs). In advanced plaques, TPA mRNA was mainly detected in the lateral regions of the fibrous caps in association with migrating VSMCs and in the vicinity of the core areas infiltrated by CD68-positive macrophages. Conclusions: TPA content and expression is consistently increased in relation to the severity of the lesions in atherosclerotic coronary arteries. This may contribute to plaque destabilization and disruption. Conversely, the increased intramural TPA activity may counteract mural fibrin deposition.     

Immunohistochemical localization of endothelial cell-derived lipase in atherosclerotic human coronary arteries

OBJECTIVE: A novel lipoprotein lipase (LPL)-like gene, endothelial cell-derived lipase (EDL), was recently cloned from vascular endothelial cells. The presence of LPL in the vascular wall has been implicated in the progression of atherosclerosis through the bridging function between lipoprotein particles and matrix proteoglycans to enhance lipoprotein uptake into the vascular wall. The aim of this study was to investigate the local expression of EDL in human coronary arteries. METHODS AND RESULTS: Human coronary arterial specimens from 10 autopsied cases were examined by immunohistochemistry with polyclonal antibodies against specific synthetic EDL peptides. Immunohistochemical analysis revealed that EDL was expressed in endothelial cells and medial smooth muscle cells in non-atherosclerotic coronary arteries. In addition, EDL was expressed in infiltrating cells within atheromatous plaques as well as endothelial and smooth muscle cells. Double labeling immunofluorescence confirmed EDL positive-cells were endothelial cells, smooth muscle cells and macrophages. EDL immunoreactivity was also detected in neovasculature within atheromatous plaques in atherosclerotic coronary arteries. CONCLUSIONS: These results suggest that EDL may have unique functional roles in the pathogenesis of coronary artery diseases such as atherosclerosis as well as in lipid metabolism in the vessel wall.     

Effects of dietary L-arginine on the reactivity of canine coronary arteries

Experiments were designed to determine the effects of supplemental dietary L-arginine on the endothelial and smooth muscle function of canine coronary arteries. One group of dogs was fed the standard laboratory chow while another group was supplemented with 250 mg/kg per day L-arginine. All dogs had undergone bilateral reversed interposition saphenous vein grafting and received 325 mg/day oral aspirin. After 5 weeks of arginine feeding, left circumflex coronary arteries were removed, cut into rings, and suspended for the measurement of isometric force in organ chambers. Concentration-response curves were obtained to L-arginine, UK-14,304 (alpha2-adrenergic agonist) and A23187 (calcium ionophore) in the absence and presence of N(G)-monomethyl-L-arginine (L-NMMA) and tetraethylammonium (TEA) alone or in combination. Serum concentrations of L-arginine increased by about 20% following 2 weeks of arginine feeding and remained elevated throughout the study. In rings with and without endothelium contracted with prostaglandin F2alpha, L-arginine caused concentration-dependent contractions in rings from control animals but no significant change in tension in rings from arginine-fed animals. Contractions to L-arginine in control animals were reduced by either L-NMMA or TEA. Endothelium-dependent relaxations to the alpha2-adrenergic agonist were decreased with arginine feeding while relaxations to the calcium ionophore and the endothelium-derived factor nitric oxide were similar among groups. Relaxations to UK-14,304 were reduced by L-NMMA in both groups but by TEA only in rings from control animals. These results suggest that dietary supplementation with L-arginine modifies reactivity of endothelium and smooth muscle by at least two mechanisms: one associated with activation of potassium channels and the other with receptor-coupled release of nitric oxide.     

Morphology of the endothelium over atherosclerotic plaques in human coronary arteries.

The right coronary arteries of six hearts removed from patients with atherosclerosis, who were undergoing cardiac transplantation, were perfused with 2% buffered glutaraldehyde for 20 minutes before preparation for scanning electron microscopy. Perfusion was started within five minutes of explanation. In two patients the artery was angiographically normal, in one it was irregular in outline, and three had focal segments with significant stenosis. None of the patients had concentrations of plasma lipids above 5.5 mmol/l. The endothelial surface showed widespread focal abnormalities ranging from adhesion and migration of monocytes to loss of individual endothelial cells. Larger areas of endothelial denudation with exposure of underlying collagen were also seen consistently. Loss of endothelial cells was associated with accumulation of monocytes, on and deep to the surface, as well as adhesion of platelets to the exposed subendothelial tissue. These results accord with the endothelial damage and platelet adhesion seen in hyperlipidaemic animals fed a high lipid diet.     

Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries

To investigate the potential role of fluid mechanical factors in the localized genesis and development of atherosclerotic lesions in humans, the exact anatomic locations of atherosclerotic lesions and the flow patterns at such sites in left and right human coronary arteries were studied in detail by flow visualization and high-speed cinemicrographic techniques using five isolated, transparent human coronary arterial trees prepared postmortem. It was found that atherosclerotic plaques and wall thickenings in left and right coronary arteries were localized almost exclusively on the outer wall of one or both daughter vessels at major bifurcations and T-junctions, which left the flow-divider free of lesions, and along the inner wall of curved segments. When flow patterns in such vessels were studied in detail, it was discovered that these sites were where flow was either slow or disturbed with the formation of slow recirculation and secondary flows and where wall shear stress was low. The results indicate that the major hemodynamic factors directly related to the localization of atherosclerotic plaques and wall thickenings in the human arterial system are the low fluid velocity and the resultant low shear stress that acts on the vessel wall.     

Estrogen modulates responses of atherosclerotic coronary arteries

Although evidence indicates that estrogen replacement therapy reduces risk of coronary heart disease, the mechanism remains unknown. Among the possibilities are that estrogen replacement therapy may 1) inhibit growth of atherosclerotic plaque and 2) decrease the prevalence of transient myocardial ischemia and myocardial infarction by modulating vasomotion in atherosclerotic coronary arteries. Using quantitative coronary angiography, we determined vasomotor responses of atherosclerotic coronary arteries in ovariectomized cynomolgus monkeys; six were given physiological estrogen "replacement" by subcutaneous implants, and six were not. Intracoronary infusion of the endothelium-dependent dilator acetylcholine (1 X 10(-6) M) caused paradoxical constriction of coronary arteries (from 1.2 +/- 0.2 to 0.6 +/- 0.1 mm, p less than 0.05) in the estrogen-deficient monkeys. However, acetylcholine tended to minimally dilate the left circumflex coronary artery in estrogen-treated monkeys (from 1.2 +/- 0.2 to 1.5 +/- 0.2 mm, p greater than 0.2). Although estrogen replacement therapy reduced plaque extent in coronary arteries, altered vasomotion was not related to plaque extent. We conclude that estrogen modulates vasomotion of atherosclerotic coronary arteries of monkeys and speculate that estrogen-modulated constrictor responses of atherosclerotic coronary arteries may reduce the incidence of coronary heart disease in postmenopausal women.     

Enhanced expression of TLR4 in smooth muscle cells in human atherosclerotic coronary arteries

Toll-like receptors (TLRs) play an essential role in innate immunity as components of the primary defense system against microbial infections. It has become evident that TLRs are also involved in the pathogenesis of various cardiovascular diseases. However, the expression patterns of TLRs in the human coronary arteries of coronary artery disease (CAD) patients and the regulatory mechanisms of their expression remain unknown. The TLR4 expression patterns were invstigated by immunohistochemical analysis of coronary specimens obtained from autopsy cases or CAD patients by using directional coronary atherectomy. In atherosclerotic coronary arteries (n = 8), TLR4 immunoreactivity was colocalized with infiltrating inflammatory cells. Interestingly, vascular smooth muscle cells of atherosclerotic coronary arteries intensely expressed TLR4 even in the regions that had few inflammatory cells. In contrast, TLR4 expression was barely detected in the vascular smooth muscle cells of nonatherosclerotic coronary arteries (n = 4). Furthermore, intense expression of smooth muscle TLR4 was observed in the coronary arteries of CAD patients (n = 52). Stimulation with tumor necrosis factor α and angiotensin II increased the expression of TLR4 mRNA in cultured human vascular smooth muscle cells. Candesartan, an antagonist of the angiotensin II type 1 receptor (AT1), and N-acetylcystine inhibited angiotensin II-induced TLR4 mRNA expression in these cells. These findings suggest that the vascular smooth muscle cells of atherosclerotic coronary arteries may be activated to express TLR4. Furthermore, proinflammatory cytokines and oxidative stress in the inflammatory lesions might contribute to the enhanced expression of TLR4 in vascular smooth muscle cells of atherosclerotic arteries.