Cesario Bianchi

The Effects of Therapeutic Sulfide on Myocardial Apoptosis in Response to Ischemia – Reperfusion Injury

OBJECTIVE Ischemia-reperfusion (I/R) injury, often encountered clinically, results in myocardial apoptosis and necrosis. Hydrogen sulfide (H(2)S) is produced endogenously in response to ischemia and thought to be cardioprotective, although its mechanism of action is not fully known. This study investigates cardioprotection provided by exogenous H2S, generated as sodium sulfide on apoptosis following myocardial I/R injury. METHODS The mid-LAD coronary artery in Yorkshire swine (n=12) was occluded for 60 min, followed by reperfusion for 120 min. Controls (n=6) received placebo, and treatment animals (n=6) received sulfide 10 min prior to and throughout reperfusion. Hemodynamic, global, and regional functional measurements were obtained. Evans blue/TTC staining identified the area-at-risk (AAR) and infarction. Serum CK-MB, troponin I, and FABP were assayed. Tissue expression of bcl-2, bad, apoptosis-inducing-factor (AIF), total and cleaved caspase-3, and total and cleaved PARP were assessed. PAR and TUNEL staining were performed to assess apoptotic cell counts and poly-ADP ribosylation, respectively. RESULTS Pre-I/R hemodynamics were similar between groups. Post-I/R, mean arterial pressure (mmHg) was reduced by 30.2+/-4.3 in controls vs 8.2+/-6.9 in treatment animals (p=0.01). +LV dP/dt (mmHg/s) was reduced by 1308+/-435 in controls vs 403+/-283 in treatment animals (p=0.001). Infarct size (% of AAR) in controls was 47.4+/-6.2% vs 20.1+/-3.3% in the treated group (p=0.003). In treated animals, CK-MB and FABP were lower by 47.0% (p=0.10) and 45.1% (p=0.01), respectively. AIF, caspase-3, and PARP expression was similar between groups, whereas cleaved caspase-3 and cleaved PARP was lower in treated animals (p=0.04). PAR staining was significantly reduced in sulfide treated groups (p=0.04). TUNEL staining demonstrated significantly fewer apoptotic cells in sulfide treated animals (p=0.02). CONCLUSIONS Sodium sulfide is efficacious in reducing apoptosis in response to I/R injury. Along with its known effects on reducing necrosis, sulfides effects on apoptosis may partially contribute to providing myocardial protection. Exogenous sulfide may have therapeutic utility in clinical settings in which I/R injury is encountered.

Hydrogen Sulfide Therapy Attenuates the Inflammatory Response in a Porcine Model of Myocardial Ischemia – Reperfusion Injury

INTRODUCTION Hydrogen sulfide is produced endogenously in response to myocardial ischemia and thought to be cardioprotective. The mechanism underlying this protection has yet to be fully elucidated, but it may be related to sulfides ability to limit inflammation. This study investigates the cardioprotection provided by exogenous hydrogen sulfide and its potential anti-inflammatory mechanism of action. METHODS The mid left anterior descending coronary artery in 14 Yorkshire swine was acutely occluded for 60 minutes, followed by reperfusion for 120 minutes. Controls (n = 7) received placebo, and treatment animals (n = 7) received sulfide 10 minutes before and throughout reperfusion. Hemodynamic and functional measurements were obtained. Evans blue and triphenyl tetrazolium chloride staining identified the area at risk and infarction. Coronary microvascular reactivity was assessed. Tissue was assayed for myeloperoxidase activity and proinflammatory cytokines. RESULTS Pre-ischemia/reperfusion hemodynamics were similar between groups, whereas post-ischemia/reperfusion mean arterial pressure was reduced by 28.7 +/- 5.0 mm Hg in controls versus 6.7 +/- 6.2 mm Hg in treatment animals (P = .03). Positive first derivative of left ventricular pressure over time was reduced by 1325 +/- 455 mm Hg/s in controls versys 416 +/- 207 mm Hg/s in treatment animals (P = .002). Segmental shortening in the area at risk was better in treatment animals. Infarct size (percent of area at risk) in controls was 41.0% +/- 7.8% versus 21.2% +/- 2.5% in the treated group (P = .036). Tissue levels of interleukin 6, interleukin 8, tumor necrosis factor-alpha, and myeloperoxidase activity decreased in the treatment group. Treated animals demonstrated improved microvascular reactivity. CONCLUSIONS Therapeutic sulfide provides protection in response to ischemia/reperfusion injury, improving myocardial function, reducing infarct size, and improving coronary microvascular reactivity, potentially through its anti-inflammatory properties. Exogenous sulfide may have therapeutic utility in clinical settings in which ischemia/reperfusion injury is encountered.

Effect of hydrogen sulfide in a porcine model of myocardial ischemia-reperfusion: Comparison of different administration regimens and characterization of the cellular mechanisms of protection

Objective: We investigate the impact of different regimens of parenteral hydrogen sulfide (H2S) administration on myocardium during ischemia-reperfusion (IR) and the molecular pathways involved in its cytoprotective effects. Methods: Eighteen male Yorkshire pigs underwent 60 minutes of mid-left anterior descending coronary artery occlusion followed by 120 minutes of reperfusion. Pigs received either placebo (control, n = 6) or H2S as a bolus (bolus group, n = 6, 0.2 mg/kg over 10 seconds at the start of reperfusion) or as an infusion (infusion group, n = 6, 2 mg·kg−1·h−1 initiated at the onset of ischemia and continued into the reperfusion period). Myocardial function was monitored throughout the experiment. The area at risk and myocardial necrosis was determined by Monastral blue/triphenyl tetrazolium chloride staining. Apoptosis and the expression pattern of various intracellular effector pathways were investigated in the ischemic territory. Coronary microvascular reactivity to endothelium-dependent and endothelium-independent factors was measured. Results: H2S infusion but not bolus administration markedly reduce myocardial infarct size (P < 0.05) and improve regional left ventricular function, as well as endothelium-dependent and endothelium-independent microvascular reactivity (P < 0.05). The expression of B-cell lymphoma 2 (P = 0.059), heat shock protein 27 and αB-crystallin (P < 0.05) were lower in H2S-treated groups. Infusion of H2S caused higher expression of phospho-glycogen synthase kinase-3 β isoform(P < 0.05) and lower expression of mammalian target of rapamycin and apoptosis-inducing factor (P < 0.05). Bolus of H2S caused higher expression of phospho-p44/42 MAPK extracellular signal-regulated kinase and lower expression of Beclin-1 (P < 0.05). The expression of caspase 3 and cleaved caspase 3 were lower (P < 0.05), whereas the expression of phospho-Bad(Ser136) was higher in the bolus group versus control and infusion groups (P < 0.05). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cell count was lower in both H2S-treated groups compared with the control (P < 0.05). Conclusions: This study demonstrates that infusion of H2S is superior to a bolus alone in reducing myocardial necrosis after IR injury, even though some markers of apoptosis and autophagy were affected in both H2S-treated groups. Thus, the current results indicate that infusion of H2S throughout IR may offer better myocardial protection from IR injury.

Resveratrol Improves Myocardial Perfusion in a Swine Model of Hypercholesterolemia and Chronic Myocardial Ischemia

Background— Resveratrol may provide protection against coronary artery disease. We hypothesized that supplemental resveratrol will improve cardiac perfusion in the ischemic territory of swine with hypercholesterolemia and chronic myocardial ischemia. Methods and Results— Yorkshire swine were fed either a normal diet (control, n=7), a hypercholesterolemic diet (HCC, n=7), or a hypercholesterolemic diet with supplemental resveratrol (100 mg/kg/d orally, HCRV, n=7). Four weeks later, an ameroid constrictor was placed on the left circumflex artery. Animals underwent cardiac MRI and coronary angiography 7 weeks later before euthanasia and tissue harvest. Total cholesterol was lowered about 30% in HCRV animals (P<0.001). Regional wall motion analysis demonstrated a significant decrease in inferolateral function from baseline to 7 weeks in HCC swine (P=0.04). There was no significant change in regional function in HCRV swine from baseline to 7 weeks (P=0.32). Tissue blood flow during stress was 2.8-fold greater in HCRV swine when compared with HCC swine (P=0.04). Endothelium-dependent microvascular relaxation response to Substance P was diminished in HCC swine, which was rescued by resveratrol treatment (P=0.004). Capillary density (PECAM-1 staining) demonstrated fewer capillaries in both HCC and HCRV swine versus control swine (P=0.02). Immunoblot analysis demonstrated significantly greater expression in HCRV versus HCC swine of the following markers of angiogenesis: VEGF (P=0.002), peNOS (ser1177) (P=0.04), NFkB (P=0.004), and pAkt (thr308) (P=0.001). Conclusions— Supplemental resveratrol attenuates regional wall motion abnormalities, improves myocardial perfusion in the collateral dependent region, preserves endothelium-dependent coronary vessel function, and upregulates markers of angiogenesis associated with the VEGF signaling pathway.

Endostatin and angiostatin are increased in diabetic patients with coronary artery disease and associated with impaired coronary collateral formation

Coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Because of the diffuse nature of their disease, diabetic patients may be at risk for incomplete revascularization, highlighting a potential role for proangiogenic therapy in this group. This study investigates molecular mechanisms of angiogenesis in diabetic patients. Myocardial tissue was harvested from patients undergoing coronary artery bypass grafting [nondiabetic (ND) 11, type 2 diabetic (DM) 10]. Expression of angiostatin, endostatin, their precursors (plasminogen and collagen XVIII, respectively), enzymes leading to their production [matrix metalloprotease (MMP)-2 and -9, cathepsin L], and an inhibitor of MMPs (tissue inhibitor of metalloproteinase) was assessed with Western blotting. MMP activity was assessed. Coronary collateralization was graded by Rentrop scoring of angiograms. Plasminogen and collagen XVIII expression were similar between groups. Angiostatin expression trended to increase 1.24-fold (P = 0.07), and endostatin expression increased 2.02-fold in DM patients relative to ND (P = 0.02). MMP-9 expression was no different between groups, whereas MMP-2 expression decreased 1.8-fold in diabetics (P = 0.003). MMP-2 and -9 activity decreased 1.33-fold (P = 0.03) and 1.57-fold (P = 0.04), respectively, in diabetic patients. Cathepsin L expression was 1.38-fold higher in diabetic patients (P = 0.02). Coronary collateralization scores were ND 2.1 +/- 0.37 vs. DM 1.0 +/- 0.4 (P = 0.05). Myocardial endostatin expression correlated strongly with the percentage of hemoglobin A(1c) (r = 0.742, P = 0.0001). Myocardial expression of angiostatin and endostatin demonstrated significant negative linear correlations with coronary collateralization (angiostatin r = -0.531, P = 0.035, endostatin r = -0.794, P = 0.0002). Diabetic patients with CAD exhibit increased levels of the antiangiogenic proteins angiostatin and endostatin and differential regulation of the enzymes governing their production relative to ND patients. Myocardial levels of these proteins show significant correlation to coronary collateralization. These findings offer potential new therapeutic targets for enhancing proangiogenic therapy and insight into the angiogenic impairments seen in diabetes.

Hypoxia Inducible Factor-1α, Endothelial Progenitor Cells, Monocytes,Cardiovascular Risk, Wound Healing, Cobalt and Hydralazine:A Unifying Hypothesis

Bone marrow-derived mononuclear cells differentiate into endothelial cells in adult animals, including humans. These cells, endothelial progenitor cells (EPCs), play central roles in neovascularization in a variety of physiological and pathological processes. EPCs numbers are clinically relevant; in patients with vascular disease, EPC numbers are predictive of hard clinical endpoints and correlate with vascular health in patients without manifest atherosclerosis. EPCs express CXCR4 which allows homing to sites of neovascularization. The homing signal released by the target tissues is SDF-1 which is the ligand for CXCR4. With release of SDF-1 and reversal of the marrow/periphery gradient, EPCs are mobilized to the periphery where they are recruited to SDF-1 expressing tissues. The SDF-1/CXCR4 axis is the final common pathway for EPC mobilization by hypoxia, angiogenic peptides and G-CSF. Expression of SDF-1 in target tissues and CXCR4 in EPCs as well as angiogenic cytokines such as VEGF are regulated by hypoxia inducible factor-1 alpha (HIF-1 alpha). This paper discusses evidence suggesting that depressed HIF-1 alpha-mediated gene programming is the most fundamental of all cardiovascular risk factors and discusses the manipulation of this system with existing drugs such as cobalt or hydralazine. By stabilizing HIF-1 alpha protein, these compounds will enhance EPC mobilization and function, thereby improving cardiovascular health overall. This paper discusses why previous studies with EPC transplantation or mobilization with G-CSF have had negative results and proposes the use of Cobalt and Hydralazine to enhance EPC function to overcome the dysfunctional EPC phenotype that is seen in patients with vascular disease or cardiovascular risk factors.

Inhibition of the Cardiac Angiogenic Response to Surgical FGF-2 Therapy in a Swine Endothelial Dysfunction Model

Background—Discrepancy exists between the potent effects of therapeutic angiogenesis in laboratory animals and the marginal results observed in patients with advanced coronary artery disease. In vitro and small animal data suggest that angiogenesis may depend on locally available nitric oxide (NO), but the impact of endothelial dysfunction on therapeutic angiogenesis in the myocardium has been unclear. We compared the effects of clinically applicable angiogenesis methods in swine in which endothelial dysfunction was experimentally induced to that observed in normal swine. Methods and Results—Miniswine were fed either a regular (N=13) or hypercholesterolemic diet (N=13) for 20 weeks. Hypercholesterolemic swine showed coronary endothelial dysfunction on videomicroscopy. Animals from both groups received 100 &mgr;g of perivascular sustained-release fibroblast growth factor (FGF)-2 in the lateral myocardial territory, previously made ischemic by placement of an ameroid constrictor around the circumflex artery. After 4 weeks of FGF-2 therapy, lateral myocardial perfusion was significantly lower in hypercholesterolemic than in normocholesterolemic swine, both at rest and during pacing (0.44±0.04 versus 0.81±0.15 mL/min/g at rest, respectively; P =0.006; and 0.50±0.06 versus 0.71±0.10 mL/min/g during pacing; P =0.02). Hypercholesterolemic swine showed no net increase in perfusion from FGF-2 treatment. Endothelial cell density and FGF receptor-1 expression were significantly lower in the lateral territory of hypercholesterolemic versus normocholesterolemic animals. Conclusions—The cardiac angiogenic response to FGF-2 treatment using clinically applicable methods was markedly inhibited in hypercholesterolemic swine with coronary endothelial dysfunction. These findings suggest that coronary endothelial dysfunction is major obstacle to the efficacy of clinical angiogenesis protocols and constitutes a target toward making angiogenesis more effective in patients with advanced coronary disease.

Chymase Inhibition Reduces Infarction and Matrix Metalloproteinase-9 Activation and Attenuates Inflammation and Fibrosis after Acute Myocardial Ischemia/Reperfusion

Chymase is activated after acute myocardial ischemia/reperfusion (AMI-R) and is associated with an early activation of matrix metalloproteinase-9 (MMP-9), which increases infarct size after experimental AMI, and late fibrosis. We assessed the effect of chymase inhibition on myocardial protection and early signs of fibrosis after AMI-R. Fourteen pigs underwent AMI-R and received intravenously either vehicle (V; n = 7) or chymase inhibitor (CM; n = 7). Separately, rat myocardial fibroblast was incubated with vehicle (n = 4), low-dose chymase (n = 4), high-dose chymase (n = 4), or high-dose chymase plus chymase inhibitor (n = 4). Infarct size (V, 41 ± 5; CM, 24 ± 5; P < 0.01) and serum troponin T (P = 0.03) at the end of reperfusion were significantly reduced in CM. Chymase activity in both the area at risk (AAR) (P = 0.01) and nonischemic area (P = 0.02) was significantly lower in CM. Myocardial levels of pro, cleaved, and cleaved/pro-MMP-9 in the AAR were significantly lower in CM than V (P < 0.01, < 0.01, and = 0.02, respectively), whereas phospho-endothelial nitric-oxide synthase (eNOS) (P < 0.01) and total eNOS (P = 0.03) were significantly higher in CM. Apoptotic cells (P = 0.05), neutrophils (P < 0.05), and MMP-9-colocalizing mast cells (P < 0.05) in the AAR were significantly reduced in CM. Interleukin-18 (P < 0.05) and intercellular adhesion molecule-1 (P < 0.05) mRNA levels were significantly lower in CM. In cultured cardiac fibrosis, Ki-67-positive cells were significantly higher in the high-dose chymase groups (P < 0.03). This study demonstrates that chymase inhibition plays crucial roles in myocardial protection related to MMP-9, inflammatory markers, and the eNOS pathway. It may also attenuate fibrosis induced by activated chymase after AMI-R.

The cardiac microvasculature in hypertension, cardiac hypertrophy and diastolic heart failure.

Recent studies revealed an exceedingly high mortality with diastolic heart failure that was previously regarded as relatively benign compared to systolic heart failure. Prominent risk factors for diastolic heart failure are increasing age, hypertension and diabetes. These risk factors are associated with coronary microvascular rarefaction and resultant decreased coronary flow reserve, thereby rendering the myocardium vulnerable to ischemia. We discuss the importance of angiogenic gene programming in preserving the coronary microvasculature, preserving cardiac function and altering disease course. Further, we discuss the possible utility of therapies that activate hypoxia inducible factor-1 in preventing rarefaction of the coronary microvasculature and maintaining cardiac diastolic function.

Myocardial protection by PJ34, a novel potent poly (ADP-ribose) synthetase inhibitor.

BACKGROUND The activation of poly (ADP-ribose) synthetase plays an important role in the pathogenesis leading to myocardial ischemia-reperfusion injury. The aim of this study was to determine if a novel potent inhibitor of poly (ADP-ribose) synthetase, PJ34, provides myocardial protection. METHODS Pigs were subjected to 60 minutes of regional ischemia followed by 180 minutes of reperfusion. Ten mg/kg of PJ34 (PJ34; n = 6) was administrated intravenously (treated group) from 15 to 5 minutes before reperfusion followed by 3 mg/kg/hour of PJ34 from 5 minutes before reperfusion to the end of 180 minutes reperfusion. Control pigs (n = 7) received vehicle only. Arterial and left ventricular pressure and coronary flow were monitored. RESULTS The PJ34 showed significant reduction on infarct size (37.5%+/-4.5% and 50.5%+/-4.8% of the area at risk) for PJ34 and control pigs groups, respectively, (p < 0.05). Significant reduction in postsystolic shortening, as well as improvement on segment shortening, and positive first derivative of pressure over time (+dP/dt) maximum were also observed in PJ34 versus control pigs (p < 0.05). CONCLUSIONS Our results suggest that PJ34 provides cardioprotection by decreasing myocardial infarct size and enhancing postischemic regional and global functional recovery.