Gaseous hydrogen sulfide protects against myocardial ischemia-reperfusion injury in mice partially independent from hypometabolism.

BACKGROUND: Ischemia-reperfusion injury (IRI) is a major cause of cardiac damage following various pathological processes. Gaseous hydrogen sulfide (H2S) is protective during IRI by inducing a hypometabolic state in mice which is associated with anti-apoptotic, anti-inflammatory and antioxidant properties. We investigated whether gaseous H2S administration is protective in cardiac IRI and whether non-hypometabolic concentrations of H2S have similar protective properties. METHODS: Male C57BL/6 mice received a 0, 10, or 100 ppm H2S-N2 mixture starting 30 minutes prior to ischemia until 5 minutes pre-reperfusion. IRI was inflicted by temporary ligation of the left coronary artery for 30 minutes. High-resolution respirometry equipment was used to assess CO2-production and blood pressure was measured using internal transmitters. The effects of H2S were assessed by histological and molecular analysis. RESULTS: Treatment with 100 ppm H2S decreased CO2-production by 72%, blood pressure by 14% and heart rate by 25%, while treatment with 10 ppm H2S had no effects. At day 1 of reperfusion 10 ppm H2S showed no effect on necrosis, while treatment with 100 ppm H2S reduced necrosis by 62% (p<0.05). Seven days post-reperfusion, both 10 ppm (p<0.01) and 100 ppm (p<0.05) H2S showed a reduction in fibrosis compared to IRI animals. Both 10 ppm and 100 ppm H2S reduced granulocyte-influx by 43% (p<0.05) and 60% (p<0.001), respectively. At 7 days post-reperfusion both 10 and 100 ppm H2S reduced expression of fibronectin by 63% (p<0.05) and 67% (p<0.01) and ANP by 84% and 63% (p<0.05), respectively. CONCLUSIONS: Gaseous administration of H2S is protective when administered during a cardiac ischemic insult. Although hypometabolism is restricted to small animals, we now showed that low non-hypometabolic concentrations of H2S also have protective properties in IRI. Since IRI is a frequent cause of myocardial damage during percutaneous coronary intervention and cardiac transplantation, H2S treatment might lead to novel therapeutical modalities.

Renal effects of long-term darbepoetin alpha treatment in hypertensive TGR(mRen2)27 rats.

INTRODUCTION: Erytropoietin (EPO) has cytoprotective and angiogenic properties and has a beneficial effect in ischaemic conditions. Since the development of renal interstitial abnormalities are often associated with ischaemia, we studied the effects of the long-acting EPO analogue darbepoetin alpha (DA) on kidney damage in TGR(mRen2)27 (Ren2) rats. MATERIALS AND METHODS: Ren2 rats were randomised to DA or vehicle (VEH) or to DA + angiotensin converting enzyme inhibitor (ACEi) or VEH + ACEi. Sprague Dawley (SD) rats served as controls. Blood pressure was measured weekly and 24-h urine was collected to measure proteinuria. Blood samples were collected for creatinine and haematocrit. Kidneys were studied for inflammation and pre-fibrosis. Renal mRNA expression was studied for EPO, EPO-receptor, collagen-3α1 and kidney injury molecule-1 (KIM-1). RESULTS: DA had no effect on SBP, serum creatinine and proteinuria. Interstitial and glomerular α-SMA expression was significantly increased in Ren2. ACEi but not DA improved the increased renal inflammatory and pro-fibrotic profile in Ren2 rats. DA on top of ACEi further reduced glomerular α-SMA and KIM-1 expression. CONCLUSION: Long-term DA treatment has no beneficial effects on renal structural and functional changes in TGR(mRen2)27 rats in the time frame studied and the dose provided.

Heart failure-associated anemia: bone marrow dysfunction and response to erythropoietin.

Heart failure (HF)-associated anemia is common and has a poor outcome. Because bone marrow (BM) dysfunction may contribute to HF-associated anemia, we first investigated mechanisms of BM dysfunction in an established model of HF, the transgenic REN2 rat, which is characterized by severe hypertrophy and ventricular dilatation and SD rats as controls. Secondly, we investigated whether stimulation of hematopoiesis with erythropoietin (EPO) could restore anemia and BM dysfunction. After sacrifice, erythropoietic precursors (BFU-E) were isolated from the BM and cultured for 10 days. BFU-E were quantified and transcript abundance of genes involved in erythropoiesis were assayed. Number of BFU-E were severely decreased in BM of REN2 rats compared to SD rats (50 ± 6.2 vs. 6.4 ± 1.7, p < 0.01). EPO treatment increased hematocrit in the SD-EPO group (after 6 weeks, 49 ± 1 vs. 58 ± 1%, p < 0.01); however, in the mildly anemic REN2 rats, there was no effect (43 ± 1 vs. 44 ± 1%). This was paralleled by a 67% decrease in BFU-E in BM of REN2 rats compared to SD (p < 0.01). EPO significantly improved BFU-E in both SD and REN2 but could not restore this to control levels in the REN2 rats. Expression of several genes involved in differentiation (LMO2), mobilization (SDF-1), and iron incorporation (transferrin receptor) of the BM were differentially expressed in REN2 rats compared to SD rats, and EPO did not normalize this. Altogether, these results suggest that BM dysfunction is an important contributor to HF-associated anemia and that EPO is not an effective agent to treat HF-associated anemia.

Erythropoiesis stimulation in acute ischemic syndromes.

Erythropoietin (EPO) is a hematopoietic hormone with extensive nonhematopoietic properties. The discovery of an EPO receptor outside the hematopoietic system has fuelled research into the beneficial effects of EPO for various conditions, predominantly in cardiovascular disease. Experimental evidence has revealed the cytoprotective properties of EPO, and it seems that the EPO-EPO receptor system provides a powerful backbone against acute myocardial ischemia, gaining from the different properties of EPO. There is an ongoing discussion about possible discrepancy between preclinical and clinical effects of EPO on the cardiovascular system. Large, randomized, placebo-controlled clinical trials are underway to give a final verdict on EPO treatment for acute coronary syndromes.

Vascular endothelial growth factor is crucial for erythropoietin-induced improvement of cardiac function in heart failure.

AIMS: We intended to delineate the mechanisms of erythropoietin (EPO)-induced cardiac vascular endothelial growth factor (VEGF) production and to establish if VEGF is crucial for EPO-induced improvement of cardiac performance. METHODS AND RESULTS: The effects of EPO on VEGF expression were studied in cultured cardiac cells and EPO-treated hearts. The role of VEGF in EPO-induced neovascularization was studied with two distinct VEGF-neutralizing antibodies or irrelevant control IgG in an aortic sprouting assay and in rats with heart failure (HF) after myocardial infarction (MI) treated with EPO. EPO-alfa (10 IU/mL) was used in vitro and darbepoetin alfa (40 microg/kg/3 weeks, starting 3 weeks after MI) in vivo. EPO stimulated VEGF mRNA expression through the signal transducers and activators of transcription-3 (STAT-3) pathway in neonatal rat cardiomyocytes, but not in endothelial cells or fibroblasts. Similarly, the direct effects of EPO on endothelial sprouting were modest and VEGF independent. In rats with HF, EPO increased VEGF protein expression predominantly in cardiomyocytes, associated with a 37% increase in capillary density and improved cardiac performance. Administration of VEGF-neutralizing antibodies abrogated the salutary effects of EPO on cardiac microvascularization and function. VEGF neutralization attenuated EPO-induced proliferation of myocardial endothelial cells and reduced myocardial incorporation of endothelial progenitor cells (EPCs) in rats with alkaline phosphatase-labelled bone marrow cells. CONCLUSION: VEGF is crucial for EPO-induced improvement of cardiac function in HF. EPO fosters VEGF expression predominantly in cardiomyocytes, which in turn stimulates myocardial endothelial proliferation and incorporation of EPCs.

Estradiol-induced, endothelial progenitor cell-mediated neovascularization in male mice with hind-limb ischemia.

We investigated whether administration of estradiol to male mice augments mobilization of bone marrow-derived endothelial progenitor cells (EPC) and incorporation into foci of neovascularization after hind-limb ischemia, thereby contributing to blood flow restoration. Mice were randomized and implanted with placebo pellets or pellets containing low-dose estradiol (0.39 mg) or high-dose estradiol (1.7 mg). Hind-limb ischemia was induced by unilateral resection of the left femoral artery 1 week after pellet implantation, then EPC mobilization and functional recovery was evaluated. EPC recruitment was assessed in mice transplanted with bone marrow from transgenic donors expressing beta-galactosidase driven by the Tie-2 promoter. EPC culture assay performed 2 weeks after pellet implantation revealed a significantly greater (p<0.05) number of circulating EPCs in the high-dose estradiol group than in the low-dose estradiol and placebo groups. At 3 and 4 weeks after induction of hind-limb ischemia, perfusion was significantly greater (p<0.05) in high-dose estradiol mice than in mice implanted with the low-dose estradiol or placebo pellets. At 1 and 4 weeks after hind-limb ischemia surgery, more bone marrow-derived EPCs, identified as beta-galactosidase-positive cells, were observed in ischemic regions from high-dose estradiol animals than in low-dose (p<0.05) or placebo groups (p<0.05). These results indicate that estradiol dose-dependently increases the levels of EPCs in peripheral blood in male animals, improves the recovery of blood flow, and decreases limb necrosis after hind-limb ischemia, and that this enhancement occurs, in part, through augmentation of EPC mobilization and greater incorporation of bone marrow-derived EPCs into foci of neovascularization.

Therapeutic potential of erythropoietin in cardiovascular disease: erythropoiesis and beyond.

Erythropoietin (EPO) is a glycoprotein hormone implicated in the regulation of red blood cell production. Anemia is common in chronic heart failure (CHF) patients and associated with an inappropriately low EPO-production, suggesting a role for its recombinant human form (rhEPO) in treatment. Although safety concerns have been raised regarding treatment with rhEPO in patients with chronic kidney disease, treatment with rhEPO in patients with CHF has so far been safe and well tolerated. The effect of rhEPO on outcome in anemic CHF patients is under investigation in a phase III clinical trial. In addition to its erythropoietic effects, EPO has been detected in the cardiovascular system, fueling intense research into possible non-hematopoietic effects. EPO has been shown to exert protective effects on the heart during acute myocardial ischemia and improve cardiac function in experimental CHF. Acute protection is mediated through reduction of apoptotic cell death. Improvement of cardiac function in CHF is related to myocardial neovascularization. EPO exhibits a vast array of beneficial effects in cardiovascular disease. In addition to the correction of anemia in CHF, rhEPO might benefit patients with cardiovascular disease.

Left main coronary angioplasty in surgical and nonsurgical candidates: putative role for a preconditioned expert culture on clinical outcome.

The impact of patient- and operator-related clinical variables of success and evaluation of subsequent midterm effects of percutaneous treatment of left main coronary stenosis were assessed at a tertiary-referral high-volume angioplasty center in a retrospective observational study. A total of 118 consecutive surgical and nonsurgical patients with protected and unprotected left main (LM) lesions were treated by operators within a preconditioned expert culture. There were 57 protected and 61 unprotected patients, including 13 patients with an acute myocardial infarction (AMI). Mean age was 67 years (range 33-90). The length of the stenotic segment was 4.8 +/- 2.3 mm, mean lumen diameter was 1.1 +/- 0.6 mm, and percentage diameter stenosis was 63.6 +/- 14.6%. There were 7 (5.9%) in-hospital cardiac deaths that presented with AMI and cardiogenic shock. All 7 patients presented with unprotected LM lesions. Average follow-up was 8 months (range 1-36 months). Major adverse cardiac events (MACE) during follow-up comprised 8 (6.8%) cardiac deaths, 3 (2.5%) myocardial infarctions, 8 (6.8%) subjects with coronary bypass surgery, and 16 (13.6%) repeated angioplasties. The total event rate (MACE, n = 43) at the end of the follow-up period was 36.4%. There were more MACE in the unprotected group than in the protected group (41% vs. 31.6%, P<0.05). This study supports prior data on LM angioplasty. LM stenting in AMI showed less favorable in-hospital and late outcome.