Xin-Liang Ma

Diminished basal nitric oxide release after myocardial ischemia and reperfusion promotes neutrophil adherence to coronary endothelium.

We measured changes in basal release of nitric oxide and its effect on polymorphonuclear leukocyte (PMN) adherence to endothelial cells (ECs) in a feline model of myocardial ischemia (90 minutes) and reperfusion. Basal release of nitric oxide from the left anterior descending coronary artery (LAD) after myocardial ischemia/reperfusion and from the control left circumflex coronary artery (LCX) was assessed by NG-nitro L-arginine methyl ester (L-NAME)-induced vasocontraction. L-NAME induced a significant EC-dependent vasocontraction in control LCX rings (0.28 +/- 0.04 g), which was fully reversed by L-arginine but not D-arginine. L-NAME-induced vasocontraction of LAD rings was not significantly changed after 90 minutes of myocardial ischemia without reperfusion. However, 10 minutes of reperfusion reduced the L-NAME-induced vasocontraction to 0.13 +/- 0.04 g (p < 0.05), and this was restored by addition of 3 mM L-arginine but not D-arginine. Longer periods of reperfusion progressively decreased L-NAME-induced vasocontraction. After 270 minutes of reperfusion, L-NAME-induced vasocontraction was virtually abolished. Myocardial ischemia without reperfusion did not increase PMN adherence to ECs. However, PMN adherence to LAD ECs was significantly increased after 20 minutes of reperfusion (39 +/- 6 to 105 +/- 9 PMNs/mm2, p < 0.01), and incubation of LAD segments with L-arginine significantly attenuated this increase in PMN adherence. After 270 minutes of reperfusion, PMN adherence to LAD ECs was further increased to 224 +/- 10 PMNs/mm2 (p < 0.001). This increase in PMN adherence was almost completely blocked by MAb R15.7, a monoclonal antibody against CD18 of PMNs, and was significantly attenuated by MAb RR1/1, a monoclonal antibody against intercellular adhesion molecule-1 of ECs (p < 0.01). These results indicate that decreased basal release of endothelium-derived relaxing factor after myocardial ischemia/reperfusion precedes enhanced PMN adherence to the coronary endothelium, which may lead to PMN-induced myocardial injury.

Nitric Oxide Mediates the Antiapoptotic Effect of Insulin in Myocardial Ischemia-Reperfusion: The Roles of PI3-Kinase, Akt, and Endothelial Nitric Oxide Synthase Phosphorylation

Background—Recent evidence from cultured endothelial cell studies suggests that phosphorylation of endothelial nitric oxide synthase (eNOS) through the PI3-kinase–Akt pathway increases NO production. This study was designed to elucidate the signaling pathway involved in the antiapoptotic effect of insulin in vivo and to test the hypothesis that phosphorylation of eNOS by insulin may participate in the cardioprotective effect of insulin after myocardial ischemia and reperfusion. Methods and Results—Male Sprague-Dawley rats were subjected to 30 minutes of myocardial ischemia and 4 hours of reperfusion. Rats were randomized to receive vehicle, insulin, insulin plus wortmannin, or insulin plus L-NAME. Treatment with insulin resulted in 2.6-fold and 4.3-fold increases in Akt and eNOS phosphorylation and a significant increase in NO production in ischemic/reperfused myocardial tissue. Phosphorylation of Akt and eNOS and increase of NO production by insulin were completely blocked by wortmannin, a PI3-kinase inhibitor. Pretreatment with L-NAME, a nonselective NOS inhibitor, had no effect on Akt and eNOS phosphorylation but significantly reduced NO production. Moreover, treatment with insulin markedly reduced myocardial apoptotic death (P <0.01 versus vehicle). Pretreatment with wortmannin abolished the antiapoptotic effect of insulin. Most importantly, pretreatment with L-NAME also significantly reduced the antiapoptotic effect of insulin (P <0.01 versus insulin). Conclusions—These results demonstrated that in vivo administration of insulin activated Akt through the PI3-kinase–dependent mechanism and reduced postischemic myocardial apoptotic death. Phosphorylation of eNOS and the concurrent increase of NO production contribute significantly to the antiapoptotic effect of insulin.

In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury.

The cardioprotective effects of an mAb to P-selectin designated mAb PB1.3 was examined in a feline model of myocardial ischemia (MI) and reperfusion. PB1.3 (1 mg/kg), administered after 80 min of ischemia (i.e., 10 min before reperfusion), significantly attenuated myocardial necrosis compared to a non-blocking mAb (NBP1.6) for P-selectin (15 +/- 3 vs 35 +/- 3% of area at risk, P < 0.01). Moreover, endothelial release of endothelium derived relaxing factor, as assessed by relaxation to acetylcholine, was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with mAb PB1.3 compared to mAb NBP1.6 (67 +/- 6 vs 11 +/- 3, P < 0.01). This endothelial preservation was directly related to reduced endothelial adherence of PMNs in ischemic-reperfused coronary arteries. Immunohistochemical localization of P-selectin was significantly upregulated in the cytoplasm of endothelial cells that lined coronary arteries and veins after 90 min of ischemia and 20 min of reperfusion. The principal site of intracytoplasmic expression was in venous vessels. mAb PB1.3 significantly decreased (P < 0.01) adherence of unstimulated PMNs to thrombin and histamine stimulated endothelial cells in a concentration-dependent manner in vitro. These results demonstrate that PMN adherence to endothelium by P-selectin is an important early consequence of reperfusion injury, and a specific monoclonal antibody to P-selectin exerts significant endothelial preservation and cardioprotection in myocardial ischemia and reperfusion.

The role of L-arginine in ameliorating reperfusion injury after myocardial ischemia in the cat.

BackgroundMyocardial ischemia followed by reperfusion results in endothelial dysfunction characterized by a reduced release of endothelium-derived relaxing factor (EDRF). Because EDRF has been characterized as nitric oxide, we examined the ability of L-arginine, the substrate for nitric oxide synthesis, to protect in a feline model of myocardial ischemia plus reperfusion. Methods and ResultsThe effects of L-arginine were investigated in a 6-hour model of myocardial ischemia and reperfusion in pentobarbital-anesthetized cats. A bolus administration (30 mg/kg) of L-arginine, or its enantiomer D-arginine, was given followed by a continuous infusion of 10 mg/kg/min for 1 hour starting 10 minutes before reperfusion. Myocardial ischemia plus reperfusion in cats receiving D-arginine resulted in severe myocardial injury and endothelial dysfunction characterized by marked myocardial necrosis, high cardiac myeloperoxidase activity in ischemic cardiac tissue, and loss of acetylcholine and A-23187–induced endothelium-dependent relaxation in coronary artery rings. In contrast, myocardial ischemia plus reperfusion cats treated with L-arginine exhibited a reduced area of cardiac necrosis (16±2% versus 41±5% of area at risk, p<0.01), lower myeloperoxidase activity in the ischemic region (03±0.08 versus 0.8±0.10 units/100 mg tissue, p<0.05), and significant preservation of acetylcholine- (p<0.01) and A-23187-(p<0.01) induced endothelial-dependent relaxation. ConclusionsThese results demonstrate the ability of L-arginine to reduce necrotic injury in a cat model of myocardial ischemia plus reperfusion, and this reduction in infarct size is associated with the preservation of endothelial function and attenuation of neutrophil accumulation in ischemic cardiac tissue.

Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia.

Endothelial dysfunction occurs after myocardial ischemia and reperfusion characterized by a marked reduction in endothelium‐dependent relaxation (EDR) due to reduced release or action of endothelium‐derived relaxing factor (EDRF). This reduced EDR occurs in coronary rings isolated from cats 2.5 min after reperfusion and in isolated perfused cat hearts 2.5 min after reperfusion. No decrease in EDR occurs before reperfusion in either preparation, suggesting that this impairment in EDR occurs during reperfusion. The decrease in EDR occurs soon after the generation of superoxide radicals by the reperfused coronary endothelium. Accumulation of neutrophils and myocardial cell injury does not occur until 3–4.5 h after reperfusion. Thus, endothelial generation of superoxide radicals acts as a trigger mechanism for endothelial dysfunction which is then amplified by neutrophil adherence and diapedesis into the ischemic region enhancing post‐reperfusion ischemic injury. Agents that preserve endothelial function or inhibit neutrophil activation (e.g., superoxide dismutase, prostacyclin analogs, TGF‐β, antibodies to adhesive proteins) can protect against endothelial dysfunction and myocardial injury, if administered before reperfusion.—Lefer, A. M.; Tsao, P. S.; Lefer, D. J.; Ma, X.‐1. Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia. FASEB J. 5: 2029–2034; 1991.

Coronary endothelial and cardiac protective effects of a monoclonal antibody to intercellular adhesion molecule-1 in myocardial ischemia and reperfusion.

BackgroundIntercellular adhesion molecule-1 (ICAM-1) is a major ligand on endothelial cells for adherence of activated polymorphonuclear leukocytes (PMNs). The major purpose of this study was to study the effects of RR1/1, a monoclonal antibody against ICAM-1 (i.e., MAb RR1/1), on myocardial injury and endothelial dysfunction associated with myocardial ischemia and reperfusion. Methods and ResultsEither MAb RR1/1 (2 mg/kg, n=7), an antibody that was found to bind selectively to endothelial cells in the cat, or MAb R3.1 (2 mg/kg, n=7), a nonbinding control antibody, was given as an intravenous bolus 10 minutes before reperfusion. Two hundred eighty minutes later, hearts were excised. The left ventricle area-at-risk (AAR) was similar in MAb RR1/1 (297±2%) and MAb R3.1 (30±3%) groups. In MAb R3.1-treated cats, 90 minutes of myocardial ischemia plus 4.5 hours of reperfusion induced a significant myocardial injury (necrotic tissue/AAR, 28±2%), high myeloperoxidase activity (0.65±0.16 units/100 mg ischemic tissue), and a marked decrease in endothelium-dependent vasorelaxation in isolated left anterior descending coronary arteries (vasorelaxation to acetylcholine, 29±3%) with no change in endothelium-independent vasorelaxation (relaxation to NaNO2, 91±3%). However, cats treated with MAb RR1/1 developed significantly less myocardial necrosis (10±2% of the AAR, p<0.0l), lower myeloperoxidase activity in ischemic myocardial tissue (0.2±0.03 units/100 mg ischemic tissue, p<0.01), and enhanced vasorelaxant responses to endothelial-dependent relaxation to acetylcholine (53±5%) compared with ischemic/reperfused cats treated with MAb R3.1. Furthermore, addition of MAb RR1/1 in vitro significantly inhibited unstimulated PMN adherence to ischemicreperfused coronary artery endothelium. ConclusionsThese results suggest that ICAM-1-dependent PMN adherence plays an important role in reperfusion injury, and that PMN adherence and infiltration contribute significantly to coronary endothelial dysfunction.

Antibody to CD-18 exerts endothelial and cardiac protective effects in myocardial ischemia and reperfusion.

We studied the effects of MAbR15.7, an antibody directed against the common beta-chain (CD-18) of a family of neutrophil adherence glycoproteins, on endothelial dysfunction and myocardial injury in a model of myocardial ischemia and reperfusion in cats. Pentobarbital-anesthetized cats were subjected to 1.5 h occlusion of the left anterior descending coronary artery (LAD) and 4.5 h of reperfusion. MI + R resulted in severe myocardial injury and endothelial dysfunction, including significant elevation of plasma creatine kinase (CK) activity, marked myocardial necrosis, high cardiac myeloperoxidase (MPO) activity in ischemic cardiac tissue, and loss of response of LAD coronary rings to the endothelium-dependent vasodilators, acetylcholine (ACh) and A-23187. In contrast, MAbR15.7-treated cats exhibited a lower plasma CK activity at every time point observed after 2 h, a reduced area of cardiac necrosis (2 +/- 1 vs. 30.8 +/- 2.5% of area-at-risk, P less than 0.001), lower MPO activity in the ischemic region (P less than 0.01), and significantly preserved vasorelaxant responses of LAD coronary rings to endothelium-dependent vasodilators, ACh (P less than 0.001), and A-23187 (P less than 0.001). These results indicate that myocardial ischemia and reperfusion induces significant myocardial injury and endothelial dysfunction in the cat involving a CD18-dependent neutrophil adherence mechanism. Inhibition of neutrophil adherence to the endothelium exerts significant protective effects in this model of reperfusion injury.

Monoclonal antibody to L-selectin attenuates neutrophil accumulation and protects ischemic reperfused cat myocardium.

BackgroundInteraction of CDii/CD18 located on neutrophil membranes with its endothelial counterreceptor, intercellular adhesion molecule-1, plays a major role in polymorphonuclear leukocyte (PMN)- mediated endothelial dysfunction and myocardial injury associated with ischemia and reperfusion. However, PMN-derived L-selectin, which is thought to play an early role in PMN rolling along the vascular endothelium, has not been studied in a setting of myocardial ischemia and reperfusion. Methods and ResultsIn this study, we evaluated the effects of a monoclonal antibody against L-selectin, DREG-200, in a feline model of myocardial ischemia (1.5 hours) and reperfusion (4.5 hours). DREG-200 (1 mg/kg) or an isotype-matched IgGl antibody, MAb R3.1, which does not cross-react in cats, was administered as a bolus 10 minutes before reperfusion. In MAb R3.1-treated cats, myocardial ischemia followed by reperfusion resulted in significant coronary vascular endothelial dysfunction, elevated cardiac myeloperoxidase activity indicative of neutrophil accumulation in the ischemic myocardium, and severe myocardial injury. In contrast, administration of DREG-200 at 1 mg/kg significantly attenuated myocardial necrosis (14 ± 4 versus 32 ± 3 expressed as percentage of area at risk, p<.001) and attenuated coronary endothelial dysfunction (P<.01) associated with ischemia/reperfusion. Moreover, myeloperoxidase activity in the ischemic myocardium was significantly lower than MAb R3.1-treated cats (0.4 ± 0.1 versus 0.9 ± 0.2 U/100 mg tissue, p<.05). ConclusionThese results demonstrate that blocking L-selectin with DREG-200 exerts a significant cardioprotective effect in a feline model of myocardial ischemia and reperfusion, indicating that L-selectin plays a significant role in mediating PMN accumulation and PMN-induced endothelial and myocardial injury after ischemia and reperfusion.

Possible Involvement of Stress-Activated Protein Kinase Signaling Pathway and Fas Receptor Expression in Prevention of Ischemia/Reperfusion-Induced Cardiomyocyte Apoptosis by Carvedilol

Carvedilol, a new vasodilating beta-adrenoceptor antagonist and a potent antioxidant, produces a high degree of cardioprotection in a variety of experimental models of ischemic cardiac injury. Recent clinical studies in patients with heart failure have demonstrated that carvedilol reduces morbidity and mortality and inhibits cardiac remodeling. The present study was designed to explore whether the protective effects of carvedilol on the ischemic myocardium include inhibition of apoptosis of cardiomyocytes and, if so, to determine its mechanism of action. Anesthetized rabbits were subjected to 30 minutes of coronary artery occlusion followed by 4 hours of reperfusion. Detection of apoptosis of cardiomyocytes was based on the presence of nucleosomal DNA fragments on agarose gels (DNA ladder) and in situ nick end labeling. Carvedilol (1 mg/kg IV), administered 5 minutes before reperfusion, reduced the number of apoptotic myocytes in the ischemic area from 14.7 +/- 0.4% to 3.4 +/- 1.8% (77% reduction, P<.001). Propranolol, administered at equipotent beta-blocking dosage, reduced the number of apoptotic myocytes to 8.9 +/- 2.1% (39% reduction, P<.05). DNA ladders were observed in the hearts of all six vehicle-treated rabbits but only one of six carvedilol-treated rabbits (P<.01). Immunocytochemical analysis of rabbit hearts demonstrated an upregulation of Fas protein in ischemic cardiomyocytes, and treatment with carvedilol reduced both the intensity of staining as well as the area stained. Myocardial ischemia/reperfusion led to a rapid activation of stress-activated protein kinase (SAPK) in the ischemic area but not in nonischemic regions. SAPK activity was increased from 2.1 +/- 0.3 mU/mg (basal) to 8.9 +/- 0.8 mU/mg after 30 minutes of ischemia followed by 20 minutes of reperfusion. Carvedilol inhibited the activation of SAPK by 53.4 +/- 6.5% (P<.05). Under the same conditions, propranolol (1 mg/kg) had no effect on SAPK activation. Taken together, these results suggest that carvedilol prevents myocardial ischemia/reperfusion-induced apoptosis in cardiomyocytes possibly by downregulation of the SAPK signaling pathway, by inhibition of Fas receptor expression, and by beta-adrenergic blockade. The former two actions represent novel and important mechanisms that may contribute to the cardioprotective effects of carvedilol.

Signature microRNA Expression Profile of Essential Hypertension and Its Novel Link to Human Cytomegalovirus Infection

Background— Essential hypertension has been recognized as a disease resulting from a combination of environmental and genetic factors. Recent studies demonstrated that microRNAs (miRNAs) are involved in cardiac hypertrophy and heart failure. However, little is known about the roles of miRNAs in essential hypertension. Methods and Results— Using microarray-based miRNA expression profiling, we compared the miRNA expressions in plasma samples from 13 hypertensive patients and 5 healthy control subjects. Twenty-seven miRNAs were found to be differentially expressed. The expressions of selected miRNAs (miR-296–5p, let-7e, and a human cytomegalovirus [HCMV]–encoded miRNA, hcmv-miR-UL112) were validated independently in plasma samples from 24 hypertensive patients and 22 control subjects. The absolute expression levels of hcmv-miR-UL112, miR-296–5p, and let-7e were further determined in 127 patients and 67 control subjects (fold changes are 2.5, 0.5, and 1.7 respectively; all P<0.0001). Additionally, we demonstrated that interferon regulatory factor 1 is a direct target of hcmv-miR-UL112. Increased HCMV seropositivity and quantitative titers were found in the hypertension group compared with the control group (52.7% versus 30.9%, P=0.0005; 1870 versus 54 copies per 1 mL plasma, P<0.0001). Seropositivity, log-transformed copies of HCMV, and hcmv-miR-UL112 were independently associated with an increased risk of hypertension (odds ratio, 2.48; 95% confidence interval, 1.48 to 4.15; P=0.0005; odds ratio, 1.97; 95% confidence interval, 1.58 to 2.46; P<0.0001; and odds ratio, 2.55; 95% confidence interval, 1.98 to 3.27; P<0.0001, respectively). Conclusions— We report for the first time a circulating miRNA profile for hypertensive patients and demonstrate a novel link between HCMV infection and essential hypertension. These findings may reveal important insights into the pathogenesis of essential hypertension. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00420784.