Endothelial dysfunction after hypoxia-reoxygenation: do in vitro models work?

Hypoxia-reoxygenation (H/R) causes tissue injury, mainly due to free radical production and leukocyte activation. H/R-induced endothelial damage is widely described, however in pharmacological research, there are only sporadic functional studies investigating in vitro vascular H/R. This methodological study compares results of in vivo and in vitro functional experiments. In canine and porcine in vivo experiments hearts were subjected to regional or global ischemia and reperfusion. Blood flow was measured on the left anterior descending coronary artery with a perivascular ultrasonic probe. Endothelium-dependent and -independent vasodilation was assessed after single-bolus intracoronary administration of acetylcholine and sodium nitroprusside (SNP). In organ bath experiments, isolated porcine coronary and rat aortic rings were investigated. Hypoxia (30, 45, 60, 120 min) was induced in the chamber by gassing with 95% N2-5% CO2. (pO2 < 30 mm Hg) During the subsequent reoxygenation (30 min), gassing was changed to 95% O2-5% CO2. The dose-dependent vasoresponse to acetylcholine, bradykinin and SNP was investigated in precontracted rings under normoxic conditions and after H/R. Endothelial function assessed by coronary blood flow measurements was impaired after ischemia-reperfusion in vivo. Although the typical hypoxic vasomotor response could be observed in vitro, no impairment of endothelial function could be proven after H/R in any groups. We conclude that endothelial injury occurring in vessel rings during in vitro H/R is too slight (probably due to lack of activated leukocytes) and cannot be demonstrated in functional measurements. Therefore the experimental model of in vitro vascular H/R is not suited for reliable investigation of pharmacological attempts.

Improvement of aging-associated cardiovascular dysfunction by the orally administered copper(II)-aspirinate complex.

BACKGROUND: Aging-associated nitro-oxidative stress causes tissue injury and activates proinflammatory pathways that play an important role in the pathogenesis of aging-associated cardiovascular dysfunction. It has been recently reported, that the copper(II)-aspirinate complex (CuAsp) exerts not only the well-known anti-inflammatory and platelet antiaggregating effects of aspirin, but, due to its superoxide dismutase mimetic activity, it acts as a potent antioxidant as well. In this study we investigated the effects of CuAsp on aging-associated myocardial and endothelial dysfunction. METHODS AND RESULTS: Aging and young rats were treated for 3 weeks with vehicle, or with CuAsp (200 mg/kg per day per os). Left ventricular pressure-volume relations were measured by using a microtip pressure-volume conductance catheter, and indexes of contractility (e.g., slope of end-systolic pressure-volume relationships [ESPVR] [E(es)], and dP/dt(max) - end-diastolic volume [EDV]) were calculated. In organ bath experiments for isometric tension with isolated aortic rings, endothelium-dependent and -independent vasorelaxation were investigated by using acetylcholine and sodium nitroprusside. When compared to the young controls, aging rats showed impaired left ventricular contractility (E(es), 0.51 +/- 0.04 vs. 2.16 +/- 0.28 mmHg/microL; dP/dt(max) - EDV, 10.71 +/- 2.02 vs. 37.23 +/- 4.18 mmHg/sec per microL; p < 0.05) and a marked endothelial dysfunction (maximal relaxation to acetylcholine: 66.66 +/- 1.30 vs. 87.09 +/- 1.35%; p < 0.05). Treatment with CuAsp resulted in reduced nitro-oxidative stress, improved cardiac function (E(es), 1.21 +/- 0.17 vs. 0.51 +/- 0.04 mmHg/microL; dP/dt(max) - EDV, 23.40 +/- 3.34 vs. 10.71 +/- 2.02 mmHg/sec per microL; p < 0.05) and higher vasorelaxation to acetylcholine in aging animals (94.83 +/- 0.73 vs. 66.66 +/- 1.30%; p < 0.05). The treatment did not influence the cardiovascular functions of young rats. CONCLUSIONS: Our results demonstrate that oxidative stress and inflammatory pathways contribute to the pathogenesis of cardiovascular dysfunction in the aging organism, which can be reversed by CuAsp.

Endothelial dysfunction after long-term cold storage in HTK organ preservation solutions: effects of iron chelators and N-alpha-acetyl-L-histidine.

BACKGROUND: To improve the rate of successful heart transplantations, organ preservation should be optimized in cardiac transplantation surgery. Iron-dependent oxidative damage and iron-independent, chloride-dependent injury of the endothelium have been described after cold ischemic storage and reperfusion, leading to an enhanced rate of complications and unfavorable outcomes. This screening study tested the effects of iron chelator supplementation in different histidine-tryptophan-ketoglutarate (HTK) organ preservation solutions on endothelial function in a long-term storage model of the isolated rat aorta. METHODS: Isolated rat aortic rings underwent a 24-hour cold ischemic preservation in different HTK solutions supplemented with iron chelators of low (deferoxamine) and high (LK-614) membrane permeability. In vascular reactivity measurements we investigated the phenyleprine-induced contraction and both endothelium-dependent and -independent vasorelaxation by using cumulative concentrations of acetylcholine and sodium nitroprusside with and without an additional external oxidant injury during re-oxygenation. RESULTS: Traditional HTK solution, Custodiol, failed to prevent endothelial dysfunction in our experiments. Use of chloride-poor HTK solutions containing N-alpha-acetyl-l-histidine with and without supplementation with LK-614, but not with deferoxamine, resulted in significant improvement of impaired endothelial function; moreover, complete protection of the endothelium was feasible after 24-hour cold storage. Endothelium-independent functions of vascular smooth muscle were not affected in any of the groups. CONCLUSIONS: Our results demonstrate the important pathophysiologic role of iron-dependent oxidative injury in the development of endothelial dysfunction after cold storage, which can be prevented by cell-permeable iron chelators.

Poly(ADP-Ribose) polymerase inhibition improves endothelial dysfunction induced by hypochlorite.

Reactive oxygen species, such as myeloperoxidase-derived hypochlorite, induce oxidative stress and DNA injury. The subsequent activation of the DNA-damage-poly(ADP-ribose) polymerase (PARP) pathway has been implicated in the pathogenesis of various diseases, including ischemia-reperfusion injury, circulatory shock, diabetic complications, and atherosclerosis. We investigated the effect of PARP inhibition on the impaired endothelium-dependent vasorelaxation induced by hypochlorite. In organ bath experiments for isometric tension, we investigated the endothelium-dependent and endothelium-independent vasorelaxation of isolated rat aortic rings using cumulative concentrations of acetylcholine and sodium nitro-prusside. Endothelial dysfunction was induced by exposing rings to hypochlorite (100-400 microM). In the treatment group, rings were preincubated with the PARP inhibitor INO-1001. DNA strand breaks were assessed by the TUNEL method. Immunohistochemistry was performed for 4-hydroxynonenal (a marker of lipid peroxidation), nitrotyrosine (a marker of nitrosative stress), and poly(ADP-ribose) (an enzymatic product of PARP). Exposure to hypochlorite resulted in a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings, which was significantly improved by PARP inhibition, whereas the endothelium-independent vasorelaxation remained unaffected. In the hypochlorite groups we found increased DNA breakage, lipidperoxidation, and enhanced nitrotyrosine formation. The hypochloride-induced activation of PARP was prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of hypochlorite-induced endothelial dysfunction, which can be prevented by PARP inhibitors.

Effect of recombinant aprotinin on postoperative blood loss and coronary vascular function in a canine model of cardiopulmonary bypass.

OBJECTIVE: Aprotinin is a widely used serine protease inhibitor during cardiopulmonary bypass to reduce blood loss and preserve platelet function. However, the bovine-derived aprotinin can induce hypersensitivity reaction with fatal complications. Furthermore, vascular effects of aprotinin are not completely elucidated. The current study is designed to investigate the effects of recently developed recombinant aprotinin on blood loss and coronary vascular function in a clinically relevant canine model of cardiopulmonary bypass without aortic cross-clamping and cardioplegia. METHODS: Twenty-four dogs underwent cardiopulmonary bypass without aortic cross-clamping and cardioplegia. Dogs were divided into three groups in a blinded fashion: control animals (n=8) received placebo, aprotinin treatment groups received bovine (n=8) or recombinant aprotinin (n=8) according to the Hammersmith method. The doses of bovine and recombinant aprotinin were the same. Coagulation parameters and blood loss were measured regularly at different time points. Endothelium-dependent and -independent vasorelaxation were investigated in isolated left anterior descendent coronary arterial rings by using acetylcholine and bradykinin or sodium nitroprusside and adenosine, respectively. RESULTS: Postoperative blood loss was significantly reduced in the aprotinin-treated groups in comparison to control and there was no significant difference between the two aprotinin-treated groups. Endothelium-dependent relaxation of coronary arteries to acetylcholine and bradykinin was unaffected in the aprotinin treatment groups. Both types of aprotinin significantly increased vasorelaxation to adenosine when compared with controls, but did not affect that to sodium nitroprusside. CONCLUSIONS: The effectiveness of recombinant aprotinin on blood loss was equivalent to bovine-derived aprotinin. Neither types of aprotinin impaired endothelium-dependent relaxation in a canine model of cardiopulmonary bypass.

Poly(ADP-ribose) polymerase inhibition improves endothelial dysfunction induced by reactive oxidant hydrogen peroxide in vitro.

Reactive oxygen species, such as hydrogen peroxide (H(2)O(2)) induce oxidative stress and DNA-injury. The subsequent activation of poly(ADP-ribose) polymerase (PARP) has been implicated in the pathogenesis of various cardiovascular diseases including ischaemia-reperfusion injury, circulatory shock, diabetic complications and atherosclerosis. We investigated the effect of PARP-inhibition on endothelial dysfunction induced by H(2)O(2). In vascular reactivity measurements on isolated rat aortic rings we investigated the phenylephrine-induced contraction, and endothelium-dependent and -independent vasorelaxation by using cumulative concentrations of acetylcholine and sodium nitroprusside. Endothelial dysfunction was induced by exposing the rings to H(2)O(2) (200 and 400 muM) for 30 min. In the treatment group, rings were preincubated with the potent PARP-inhibitor INO-1001. DNA strand breaks were assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. Immunohistochemical analysis was performed for poly(ADP-ribose) (the enzymatic product of PARP) and for apoptosis inducing factor (a pro-apoptotic factor regulated by PARP). Exposure to H(2)O(2) resulted in reduced contraction forces and a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings (maximal relaxation to acetylcholine: 86.21+/-1.574% control vs. 72.55+/-1.984% H(2)O(2) 200 muM vs. 66.86+/-1.961% H(2)O(2) 400 muM; P<0.05). PARP-inhibition significantly improved the acetylcholine-induced vasorelaxation (77.75+/-3.019% vs. 66.86+/-1.961%; P<0.05), while the contractility remained unaffected. The dose-response curves of endothelium-independent vasorelaxation to sodium nitroprusside did not differ in any groups studied. In the H(2)O(2) groups immunohistochemical analysis showed enhanced PARP-activation and nuclear translocation of apoptosis inducing factor, which were prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of H(2)O(2)-induced endothelial dysfunction, which can be prevented by PARP inhibitors.

The peroxynitrite decomposition catalyst FP15 improves ageing-associated cardiac and vascular dysfunction.

Overproduction of oxidants and free radicals in ageing tissues induces nitro-oxidative stress, which has recently been implicated in the pathogenesis of cardiovascular dysfunction associated with ageing. Peroxynitrite, a strong cytotoxic oxidant damages proteins and DNA and activates several pathways causing tissue injury, including the peroxynitrite-poly(ADP-ribose) polymerase (PARP) pathway. In this study, we investigated the effectiveness of the peroxynitrite decomposition catalyst FP15 on ageing-associated cardiac and vascular dysfunction. Young and ageing rats were treated with vehicle or FP15 intraperitoneally. Using a microtip Millar pressure catheter we performed left ventricular blood pressure analysis to assess systolic and diastolic function. Endothelium-dependent and -independent vasorelaxation of isolated aortic rings were investigated by using acetylcholine and sodium nitroprusside. Ageing animals showed a marked reduction of systolic and diastolic cardiac function and loss of endothelium-dependent relaxant responsiveness of aortic rings. FP15-treatment significantly improved cardiac performance and endothelial function. Immunohistochemical staining confirmed that FP15 effectively reduced nitrosative stress and prevented the activation of PARP in the aortic wall of ageing rats. Our results demonstrate the importance of endogenous peroxynitrite-overproduction in the pathogenesis of ageing-associated cardiovascular dysfunction. Pharmacological decomposition of peroxynitrite by FP15 may represent a novel therapeutic utility to improve cardiac and vascular dysfunction associated with ageing.