High-dose atorvastatin improves hypercholesterolemic coronary endothelial dysfunction without improving the angiogenic response.

BACKGROUND: Although 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) can restore endothelial function in coronary disease, in vitro and murine studies have shown their effects on myocardial angiogenesis to be biphasic and dose dependent. We investigated the functional and molecular effects of high-dose atorvastatin on the endogenous angiogenic response to chronic myocardial ischemia in hypercholesterolemic swine. METHODS AND RESULTS: Yucatan pigs were fed either a normal (NORM group; n=7) or high-cholesterol diet, with (CHOL-ATR group; n=7) or without (CHOL group; n=6) atorvastatin (3 mg/kg per day) for 13 weeks. Chronic ischemia was induced by ameroid constrictor placement around the circumflex artery. Seven weeks later, microvessel relaxation responses, myocardial perfusion, and myocardial protein expression were assessed. The CHOL group demonstrated impaired microvessel relaxation to adenosine diphosphate (29+/-3% versus 61+/-6%, CHOL versus NORM; P<0.05), which was normalized in the CHOL-ATR group (67+/-2%; P=NS versus NORM). Collateral-dependent myocardial perfusion, adjusted for baseline, was significantly reduced in the CHOL group (-0.27+/-0.07 mL/min per gram versus NORM; P<0.001) as well as the CHOL-ATR group (-0.35+/-0.07 mL/min per gram versus NORM; P<0.001). Atorvastatin treatment was associated with increased phosphorylation of Akt (5.7-fold increase versus NORM; P=0.001), decreased vascular endothelial growth factor expression (-68+/-8%; P<0.001 versus NORM), and increased expression of the antiangiogenic protein endostatin (210+/-48%; P=0.004 versus NORM). CONCLUSIONS: Atorvastatin improves hypercholesterolemia-induced endothelial dysfunction without appreciable changes in collateral-dependent perfusion. Increased myocardial expression of endostatin, decreased expression of vascular endothelial growth factor, and chronic Akt activation associated with atorvastatin treatment may account for the diminished angiogenic response.

Hypercholesterolemia impairs the myocardial angiogenic response in a swine model of chronic ischemia: role of endostatin and oxidative stress.

BACKGROUND: Recent studies have shown that angiogenesis is regulated by a balance between activators and inhibitors. We investigated the effects of hypercholesterolemia on the functional angiogenic response and collateral formation induced by chronic myocardial ischemia and the expression of angiogenic mediators. METHODS: Twelve Yucatan miniswine, fed either a normal (NORM, n = 6) or high cholesterol (HCHO, n = 6) diet for 13 weeks, underwent ameroid constrictor placement around the circumflex artery. Three weeks later, myocardial perfusion was quantified using isotope-labeled microspheres. Seven weeks after ameroid placement, coronary microvascular responses and myocardial perfusion were assessed. Vascular density was evaluated by PECAM-1 (CD-31) staining, and vascular endothelial growth factor, endothelial nitric oxide synthase, endostatin, and angiostatin protein levels were determined. Myocardial protein oxidation was quantified. RESULTS: Coronary microvessels from HCHO pigs showed significant endothelial dysfunction. Baseline-adjusted myocardial flow at 7 weeks was significantly reduced in the HCHO animals (-0.002 +/- 0.06 versus +0.23 +/- 0.09 mL/min/g, HCHO versus NORM, p = 0.04). Endostatin expression was significantly increased in the HCHO pigs (2.2-fold, p = 0.001 versus NORM). There was a mild reduction in myocardial vascular endothelial growth factor expression (-29% +/- 14%, p = 0.09) in HCHO animals, but no difference in expression of endothelial nitric oxide synthase and angiostatin. The HCHO animals demonstrated increased myocardial protein oxidation compared with the NORM group (+155% +/- 21%, p = 0.03 versus NORM). CONCLUSIONS: Ischemia-induced angiogenesis is inhibited in hypercholesterolemic pigs with a concomitant increase in endostatin expression and oxidative stress. These findings suggest that under conditions of hypercholesterolemia, coronary collateral development may be regulated by endogenous angiogenesis inhibitors such as endostatin as well as reactive oxygen species.

Effects of L-arginine on the endogenous angiogenic response in a model of hypercholesterolemia.

BACKGROUND: The angiogenic properties of vascular endothelial growth factor and fibroblast growth factor-2 are mediated in part through nitric oxide release, whose availability is decreased in endothelial dysfunction associated with advanced coronary artery disease. We examined the influence of L-arginine supplementation on the endogenous angiogenic response to ischemia in a porcine model of hypercholesterolemia. METHODS: Eighteen Yucatan pigs were fed either a normal (NORM, n=6) or a high-cholesterol diet, with (CHOL-ARG, n=6) or without (CHOL, n=6) L-arginine (100 mg/kg/day), throughout the experiment. All pigs underwent ameroid constrictor placement on the circumflex artery (LCx). Seven weeks later, endothelium-dependent coronary microvascular responses to fibroblast growth factor-2 and vascular endothelial growth factor were assessed by videomicroscopy. Perfusion was assessed with radioactive microspheres; angiogenesis was evaluated by platelet-endothelial cell adhesion molecule-1 (CD-31) staining. Regional myocardial function was assessed by sonomicrometry. Expression of endothelial nitric oxide synthase and inducible nitric oxide synthase was measured by Western blot analyses. RESULTS: Pigs from the CHOL group showed significant endothelial dysfunction in the LCx territory. The dysfunction was normalized partially by L-arginine supplementation, which restored the response in the LCx territory to the level of the nonischemic anterior wall. L-arginine supplementation resulted in increases of perfusion, density of capillary endothelial, and level of endothelial nitric oxide synthase in the ischemic region. Despite these findings, no improvement in myocardial regional function was found. CONCLUSIONS: L-arginine supplementation can partially restore endothelium-dependent vasorelaxation and improve myocardial perfusion in a swine model of chronic myocardial ischemia with hypercholesterolemia-induced endothelial dysfunction. These findings suggest a putative role for L-arginine in combination with growth factor therapy for end-stage coronary artery disease.

Effects of L-arginine on fibroblast growth factor 2-induced angiogenesis in a model of endothelial dysfunction.

BACKGROUND: Nitric oxide availability, which is decreased in advanced coronary artery disease associated with endothelial dysfunction, is an important mediator of fibroblast growth factor-2 (FGF-2)-induced angiogenesis. This could explain the disappointing results of FGF-2 therapy in clinical trials despite promising preclinical studies. We examined the influence of L-arginine supplementation to FGF-2 therapy on myocardial microvascular reactivity and perfusion in a porcine model of endothelial dysfunction. METHODS AND RESULTS: Eighteen pigs were fed either a normal (NORM, n=6) or high cholesterol diet, with (HICHOL-ARG, n=6) or without (HICHOL, n=6) L-arginine. All pigs underwent ameroid placement on the circumflex artery and 3 weeks later received surgical FGF-2 treatment. Four weeks after treatment, endothelial-dependent coronary microvascular responses and lateral myocardial perfusion were assessed. Endothelial cell density was determined by immunohistochemistry. FGF-2, fibroblast growth receptor-1, endothelial-derived nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and syndecan-4 levels were determined by immunoblotting. Pigs from the HICHOL group showed endothelial dysfunction in the circumflex territory, which was normalized by L-arginine supplementation. FGF-2 treatment was ineffective in the HICHOL group (circumflex/left anterior descending blood flow ratios: 1.01 (rest) and 1.01 (pace), after and before treatment). Addition of L-arginine improved myocardial perfusion in response to FGF-2 at rest (ratio 1.13, P=0.02 versus HICHOL) but not during pacing (ratio 0.94, P=NS), and was associated with increased protein levels of iNOS and eNOS. CONCLUSIONS: L-arginine supplementation can partially restore the normal response to endothelium-dependent vasorelaxants and myocardial perfusion in response to FGF-2 treatment in a swine model of hypercholesterolemia-induced endothelial dysfunction. These findings suggest a role for L-arginine in combination with FGF-2 therapy for end-stage coronary artery disease.

Potent adenylate cyclase agonist forskolin restores myoprotective effects of ischemic preconditioning in rat hearts after myocardial infarction.

BACKGROUND: The purpose of this study was to determine whether ischemic preconditioning (IPC) provides myoprotective effects in post-myocardial infarction (MI) hearts, and whether beta adrenergic signaling is involved in IPC. METHODS: Rats were subjected to either ligation of the left anterior descending coronary artery (LAD) resulting in MI, or a sham operation. Two weeks later, hearts were isolated and perfused. Six groups (n = 7 each) were studied: group 1, control (sham operation); group 2, sham operation + IPC; group 3, post-MI; group 4, post-MI + IPC; group 5, post-MI + forskolin; group 6, post-MI + forskolin + IPC. IPC consisted of two cycles of 5 minutes of global ischemia. The adenylate cyclase agonist forskolin (1.0 x 10(-5) M) was administered in post-MI hearts either alone (group 5) or for 5 minutes before IPC (group 6). All hearts were then subjected to 20 minutes of global ischemia followed by 120 minutes of reperfusion, after which infarct size was measured. Concentrations of endogenous catecholamines and myocardial mRNA expression of beta 2 adrenergic receptor were measured in the post-MI model. RESULTS: (1) IPC reduced infarct size in shams, from 34.7 +/- 5.2% in group 1 to 21.4 +/- 3.8% in group 2, but did not affect infarct size in post-MI hearts (group 3 versus group 4). (2) Forskolin combined with IPC reduced infarct size in post-MI hearts to 29.3 +/- 3.4% (group 6), but not in group 5 where the value was 39.3 +/- 4.8%. (3) Beta 2 adrenergic receptor mRNA expression in post-MI hearts was significantly decreased as compared with sham-operated animals. CONCLUSIONS: The results indicate that downregulation of beta adrenergic receptors in post-MI hearts may be associated with ineffectiveness of IPC, and that beta adrenergic signaling, especially in relation to adenylate cyclase activation, may be required to generate the IPC response in post-MI hearts.

Oral hypoglycemic sulfonylurea glimepiride preserves the myoprotective effects of ischemic preconditioning.

BACKGROUND: To investigate whether the sulfonylurea glimepiride affects the myoprotective effects of ischemic preconditioning (IPC), isolated rabbit hearts were perfused with Krebs-Henseleit solution. METHODS: Eight hearts underwent IPC consisting of two cycles of 5 min global ischemia and reperfusion. Six hearts received a 5-min infusion of 10 microM glimepiride, six hearts received a 5-min infusion of 50 microM glimepiride, and seven hearts received a 5-min infusion of 10 microM glibenclamide before IPC. Seven hearts received a 5-min infusion of the selective mitochondrial K(ATP) channel opener diazoxide (50 microM). Other hearts received a 5-min infusion of 10 microM glimepiride (n = 6), 50 microM glimepiride (n = 6), or 10 microM glibenclamide (n = 7) before diazoxide. Seven hearts served as a control. All groups then were subjected to 1 h of regional ischemia, followed by 1 h of reperfusion. LV pressures, monophasic action potential duration (APD(50)), and infarct size were measured. RESULTS: Both IPC and diazoxide significantly prolonged APD(50) and preserved diastolic function at 60 min of reperfusion compared to control. In addition, both groups reduced infarct size compared to control. Glibenclamide, but not glimepiride reversed these effects. CONCLUSION: Glimepiride offers less cardiovascular effects than glibenclamide, possibly due to its lower affinity for the mitochondrial K(ATP) channels.

Na(+)/H(+) exchanger inhibitor HOE642 offers myoprotection in senescent myocardium independent of ischemic preconditioning mechanisms.

BACKGROUND: Inhibition of Na(+)/H(+) exchange has been shown to provide functional protection during ischemia and reperfusion in mature heart. This study was undertaken to elucidate the effect of Na(+)/H(+) exchange inhibitor HOE642 in the aged rabbit heart. METHODS: Isolated rabbit hearts were subjected to 1 h of left descending coronary artery (LAD) ischemia and 1 h of reperfusion. To determine the effects of HOE642 on ischemia/reperfusion injury, seven aged or mature hearts received the Na(+)/H(+) exchange inhibitor HOE642 (1 microM) for 15 min before the ischemia and for 30 min after reperfusion. Seven aged (more than 135 weeks) or mature (15-20 weeks) rabbit hearts served as a control (untreated) with no interventions. Left ventricular pressures, monophasic action potentials and coronary flows were measured throughout the experiment and infarct size was detected at the end of experiment. RESULTS: (1) In the mature hearts, HOE642 improved postischemic functional recovery (63.1 +/- 5.0% vs. 84.4 +/- 5.4%, mature untreated vs. mature HOE, p < 0.05) and reduced infarct size as compared to untreated hearts (42.0 +/- 2.5% vs. 24.8 +/- 2.3%, mature untreated vs. mature HOE, p < 0.05). (2) Although infarct size in aged untreated hearts was significantly decreased as compared to mature untreated hearts (42.0 +/- 2.5% vs. 19.3 +/- 1.6%, mature untreated vs. aged untreated, p < 0.05), there are no significant differences regarding postischemic functional recovery between mature and aged untreated hearts (63.1 +/- 5.0% vs. 59.5 +/- 5.9%, mature untreated vs. aged untreated, p = n.s.). (3) In the aged hearts, HOE642 improved postischemic functional recovery as compared to untreated hearts (59.5 +/- 5.9% vs. 85.9 +/- 8.1%, aged untreated vs. aged HOE, p < 0.05). CONCLUSION: Na(+)/H(+) exchange inhibitor HOE642 is effective against ischemia-reperfusion injury in senescent as well as mature hearts.

HMG-CoA reductase inhibitor cerivastatin prolonged rat cardiac allograft survival by blocking intercellular signals.

BACKGROUND: The development of atherosclerotic cardiovascular complications caused by hyperlipidemia is a common and serious problem for long-term survivors of organ transplantation. However, adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and lymphocyte function-associated antigen (LFA)-1 are involved in allograft rejection, possibly by providing costimulatory signals. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor cerivastatin has been shown to suppress ICAM-1 expression in acute inflammatory responses. METHODS: In this study, we evaluated the immunosuppressive effects of cerivastatin in rat cardiac allografts. The hearts of Fischer rats were transplanted heterotopically into Lewis rats. Cerivastatin (2 mg/kg) was administrated intraperitoneally to recipients for 7 consecutive days from the day before transplantation. RESULTS: Graft survival in the cerivastatin-treated group (n = 8) was significantly longer than in controls (n = 10) (24.6 +/- 2.2 days vs 10.2 +/- 1.3 days, p < 0.05). Mixed lymphocyte reaction (MLR) showed that on Day 8 after grafting, the proliferative response of alloreactive T cells against F344 alloantigen in cerivastatin-treated rats was significantly more suppressed than in Lewis rats. The Interleukin-2 concentration of supernatant in MLR cultures in the cerivastatin-treated group was lower than in the control group. Immunohistochemical analysis showed that the percentage of CD4-positive cells to infiltrating mononuclear cells was less prominent in the cerivastatin-treated group (9.8% +/- 2.2%) than in the control group (20.9% +/- 3.2%). CONCLUSIONS: The HMG-CoA reductase inhibitor cerivastatin effectively suppressed acute graft rejection, possibly by blocking intercellular signals via ICAM/LFA-1, and cerivastatin may be a candidate for treating patients with hyperlipidemia who undergo organ transplantation.

Long-term treatment with nipradilol, a nitric oxide-releasing beta-adrenergic blocker, enhances postischemic recovery and limits infarct size.

BACKGROUND: This study examines whether the chronic administration of nipradilol, a nitric oxide-releasing beta-adrenergic blocker, decreases ischemia-reperfusion injury. METHODS: Rats were treated with nipradilol (10 mg/kg per day orally) or a vehicle alone for 4 weeks. Isolated rat hearts were assigned to one of five groups (each n = 6): global ischemia groups treated with the vehicle or with nipradilol were subjected to 20 minutes of ischemia; ischemic preconditioning groups treated with the vehicle or with nipradilol were subjected to 3 minutes of ischemic preconditioning; and the L-arginine group treated with the vehicle received 1 mmol/L of L-arginine before global ischemia. Hemodynamic variables and coronary flow were recorded continuously. Nitrites and nitrates levels were measured 60 minutes after reperfusion, and the infarct size was determined. In another series (each n = 6), lipid peroxidation was investigated. RESULTS: In the nipradilol group, significant preservation of the left ventricular pressure and coronary flow, as well as the level of nitrates and nitrites, was observed, compared with the global ischemia group. The infarct size was also significantly reduced in the ischemic preconditioning (23.5%+/-5.47%), L-arginine (25.6%+/-5.59%), and especially the nipradilol (10.7%+/-1.65%) groups. However, in the nipradilol plus ischemic preconditioning group, the protective effects were eliminated. Lipid peroxidation after nipradilol treatment was significantly reduced before and after global ischemia, compared with the global ischemia group. CONCLUSIONS: The chronic administration of nipradilol improves postischemic functional recovery and infarct size, partly by preventing the formation of lipid peroxides. These cardioprotective effects were, however, abolished by ischemic preconditioning.