Péter Hegedűs

Prolyl-hydroxylase inhibition preserves endothelial cell function in a rat model of vascular ischemia reperfusion injury

Storage protocols of vascular grafts need further improvement against ischemia-reperfusion (IR) injury. Hypoxia elicits a variety of complex cellular responses by altering the activity of many signaling pathways, such as the oxygen-dependent prolyl-hyroxylase domain–containing enzyme (PHD). Reduction of PHD activity during hypoxia leads to stabilization and accumulation of hypoxia inducible factor (HIF) 1α. We examined the effects of PHD inhibiton by dimethyloxalylglycine on the vasomotor responses of isolated rat aorta and aortic vascular smooth muscle cells (VSMCs) in a model of cold ischemia/warm reperfusion. Aortic segments underwent 24 hours of cold ischemic preservation in saline or DMOG (dimethyloxalylglycine)-supplemented saline solution. We investigated endothelium-dependent and -independent vasorelaxations. To simulate IR injury, hypochlorite (NaOCl) was added during warm reperfusion. VSMCs were incubated in NaCl or DMOG solution at 4°C for 24 hours after the medium was changed for a supplied standard medium at 37°C for 6 hours. Apoptosis was assessed using the TUNEL method. Gene expression analysis was performed using quantitative real-time polymerase chain reaction. Cold ischemic preservation and NaOCl induced severe endothelial dysfunction, which was significantly improved by DMOG supplementation (maximal relaxation of aortic segments to acetylcholine: control 95% ± 1% versus NaOCl 44% ± 4% versus DMOG 68% ± 5%). Number of TUNEL-positive cell nuclei was significantly higher in the NaOCl group, and DMOG treatment significantly decreased apoptosis. Inducible heme-oxygenase 1 mRNA expressions were significantly higher in the DMOG group. Pharmacological modulation of oxygen sensing system by DMOG in an in vitro model of vascular IR effectively preserved endothelial function. Inhibition of PHDs could therefore be a new therapeutic avenue for protecting endothelium and vascular muscle cells against IR injury.

Myocardial reverse remodeling after pressure unloading is associated with maintained cardiac mechanoenergetics in a rat model of left ventricular hypertrophy

Pressure unloading represents the only effective therapy in increased afterload-induced left ventricular hypertrophy (LVH) as it leads to myocardial reverse remodeling (reduction of increased left ventricular mass, attenuated myocardial fibrosis) and preserved cardiac function. However, the effect of myocardial reverse remodeling on cardiac mechanoenergetics has not been elucidated. Therefore, we aimed to provide a detailed hemodynamic characterization in a rat model of LVH undergoing pressure unloading. Pressure overload was induced in Sprague-Dawley rats by abdominal aortic banding for 6 (AB 6th wk) or 12 wk (AB 12th wk). Sham-operated animals served as controls. Aortic debanding procedure was performed after the 6th experimental week (debanded 12th wk) to investigate the regression of LVH. Pressure unloading resulted in significant reduction of LVH (heart weight-to-tibial length ratio: 0.38 ± 0.01 vs. 0.58 ± 0.02 g/mm, cardiomyocyte diameter: 18.3 ± 0.1 vs. 24.1 ± 0.8 μm debanded 12th wk vs. AB 12th wk, P < 0.05), attenuated the extracellular matrix remodeling (Massons score: 1.37 ± 0.13 vs. 1.73 ± 0.10, debanded 12th wk vs. AB 12th wk, P < 0.05), provided protection against the diastolic dysfunction, and reversed the maladaptive contractility augmentation (slope of end-systolic pressure-volume relationship: 1.39 ± 0.24 vs. 2.04 ± 0.09 mmHg/μl, P < 0.05 debanded 12th wk vs. AB 6th wk, P < 0.05). In addition, myocardial reverse remodeling was also associated with preserved ventriculoarterial coupling and increased mechanical efficiency (50.6 ± 2.8 vs. 38.9 ± 2.5%, debanded 12th wk vs. AB 12th wk, P < 0.05), indicating a complete functional and mechanoenergetic recovery. According to our best knowledge, this is the first study demonstrating that the regression of LVH is accompanied by maintained cardiac mechanoenergetics.

Mild Type 2 Diabetes Mellitus Reduces the Susceptibility of the Heart to Ischemia/Reperfusion Injury: Identification of Underlying Gene Expression Changes

Despite clinical studies indicating that diabetic hearts are more sensitive to ischemia/reperfusion injury, experimental data is contradictory. Although mild diabetes prior to ischemia/reperfusion may induce a myocardial adaptation, further research is still needed. Nondiabetic Wistar (W) and type 2 diabetic Goto-Kakizaki (GK) rats (16-week-old) underwent 45 min occlusion of the left anterior descending coronary artery and 24 h reperfusion. The plasma glucose level was significantly higher in diabetic rats compared to the nondiabetics. Diabetes mellitus was associated with ventricular hypertrophy and increased interstitial fibrosis. Inducing myocardial infarction increased the glucose levels in diabetic compared to nondiabetic rats. Furthermore, the infarct size was smaller in GK rats than in the control group. Systolic and diastolic functions were impaired in W + MI and did not reach statistical significance in GK + MI animals compared to the corresponding controls. Among the 125 genes surveyed, 35 genes showed a significant change in expression in GK + MI compared to W + MI rats. Short-term diabetes promotes compensatory mechanisms that may provide cardioprotection against ischemia/reperfusion injury, at least in part, by increased antioxidants and the upregulation of the prosurvival PI3K/Akt pathway, by the downregulation of apoptotic genes, proinflammatory cytokine TNF-α, profibrogenic TGF-β, and hypertrophic marker α-actin-1.

Endothelial Dysfunction of Bypass Graft: Direct Comparison of In Vitro and In Vivo Models of Ischemia-Reperfusion Injury

BACKGROUND Although, ischemia/reperfusion induced vascular dysfunction has been widely described, no comparative study of in vivo- and in vitro-models exist. In this study, we provide a direct comparison between models (A) ischemic storage and in-vitro reoxygenation (B) ischemic storage and in vitro reperfusion (C) ischemic storage and in-vivo reperfusion. METHODS AND RESULTS Aortic arches from rats were stored for 2 hours in saline. Arches were then (A) in vitro reoxygenated (B) in vitro incubated in hypochlorite for 30 minutes (C) in vivo reperfused after heterotransplantation (2, 24 hours and 7 days reperfusion). Endothelium-dependent and independent vasorelaxations were assessed in organ bath. DNA strand breaks were assessed by TUNEL-method, mRNA expressions (caspase-3, bax, bcl-2, eNOS) by quantitative real-time PCR, proteins by Western blot analysis and the expression of CD-31 by immunochemistry. Endothelium-dependent maximal relaxation was drastically reduced in the in-vivo models compared to ischemic storage and in-vitro reperfusion group, and no difference showed between ischemic storage and control group. CD31-staining showed significantly lower endothelium surface ratio in-vivo, which correlated with TUNEL-positive ratio. Increased mRNA and protein levels of pro- and anti-apoptotic gens indicated a significantly higher damage in the in-vivo models. CONCLUSION Even short-period of ischemia induces severe endothelial damage (in-vivo reperfusion model). In-vitro models of ischemia-reperfusion injury can be limitedly suited for reliable investigations. Time course of endothelial stunning is also described.

Addition of vardenafil into storage solution protects the endothelium in a hypoxia-reoxygenation model

OBJECTIVE Based upon the well known protective effect of intracellular cyclic guanosine monophosphate (cGMP) accumulation, we tested the hypothesis that storage solution enriched with optimal concentration of the phosphodiestherase-5 inhibitor vardenafil could provide better protection of vascular grafts against reperfusion injury after long-term cold ischaemic storage. METHODS Isolated thoracic aorta obtained from rats underwent 24-h cold ischaemic preservation in physiological saline or vardenafil (10(-11) M)-supplemented saline solution. Reperfusion injury was simulated by hypochlorite (200 μM) exposure for 30 minutes. Endothelium-dependent vasorelaxation was assessed, and histopathological and molecular-biological examination of the aortic tissue were performed. RESULTS Compared with the control group, the saline group showed significantly attenuated endothelium-dependent maximal relaxation (Rmax) to acetylcholine after hypoxia-reoxygenation, which was significantly improved by vardenafil supplementation (Rmax control: 98 ± 1%; saline: 48 ± 6%; vardenafil: 75 ± 4%; p < .05). Vardenafil treatment significantly reduced DNA strand breaks (control: 10.6 ± 6.2%; saline: 72.5 ± 4.0%; vardenafil: 14.2 ± 5.2%; p < .05) and increased cGMP score in the aortic wall (control: 8.2 ± 0.6; saline: 4.5 ± 0.3; vardenafil: 6.7 ± 0.6; p < .05). CONCLUSIONS Our results support the view that impairment of intracellular cGMP signalling plays a role in the pathogenesis of the endothelial dysfunction induced by cold storage warm reperfusion, which can be effectively reversed by pharmacological phosphodiesterase-5 inhibition.

Short- and long-term effects of brain death on post-transplant graft function in a rodent model

OBJECTIVES Heart transplantation has become the most effective treatment for end-stage heart failure. Donors after brain death (BD) are currently the only reliable source for cardiac transplants. However, haemodynamic instability and cardiac dysfunction have been demonstrated in brain-dead donors and this could therefore also affect post-transplant graft function. We studied the effects of BD on cardiac function and its short-term (1 h) or long-term (5 h) impacts on graft function. METHODS In Lewis rats, BD was induced by inflation of a subdurally placed balloon catheter (n = 7). Sham-operated rats served as controls (n = 9). We continuously assessed cardiac function by left ventricular (LV) pressure-volume analysis. Then, 1 or 5 h after BD or sham operation, hearts were perfused with a cold preservation solution (Custodiol), then explanted, stored at 4°C in Custodiol and heterotopically transplanted. We evaluated graft function 1.5 h after transplantation. RESULTS BD was associated with decreased left ventricular contractility (ejection fraction: 37 ± 6 vs 57 ± 5%; maximum rate of rise of LV pressure dP/dtmax: 4770 ± 197 vs 7604 ± 348 mmHg/s; dP/dtmax-end-diastolic volume: 60 ± 7 vs 74 ± 2 mmHg/s; slope Emax of the end-systolic pressure-volume relationship: 2.4 ± 0.1 vs 4.4 ± 0.3 mmHg/µl; preload recruitable stroke work: 47 ± 9 vs 78 ± 3 mmHg; P <0.05) and relaxation (maximum rate of fall of left ventricular pressure dP/dtmin: -6638 ± 722 vs -11 285 ± 539 mmHg/s; time constant of left ventricular pressure decay Tau: 12.6 ± 0.7 vs 10.5 ± 0.4 ms; end-diastolic pressure-volume relationship: 0.22 ± 0.05 vs 0.09 ± 0.03 mmHg/µl, P <0.05) 45 min after its initiation and for the rest of 5 h compared with controls. Moreover, after transplantation, graft systolic and diastolic functions were impaired in the 5-h brain-dead group, while they were identical in the 1-h brain-dead group compared with the corresponding controls. CONCLUSIONS We established a well-characterized in vivo rat model to examine the influence of BD on cardiac function using a miniaturized technology for pressure-volume analysis. These results demonstrate that impaired donor cardiac function after short-term BD is reversible after transplantation and long-term BD renders hearts more susceptible to ischaemia/reperfusion injury.

Superiority of zinc complex of acetylsalicylic acid to acetylsalicylic acid in preventing postischemic myocardial dysfunction

The pathophysiology of ischemic myocardial injury involves cellular events, reactive oxygen species, and an inflammatory reaction cascade. The zinc complex of acetylsalicylic acid (Zn(ASA)2) has been found to possess higher anti-inflammatory and lower ulcerogenic activities than acetylsalicylic acid (ASA). Herein, we studied the effects of both ASA and Zn(ASA)2 against acute myocardial ischemia. Rats were pretreated with ASA (75 mg/kg) or Zn(ASA)2 (100 mg/kg) orally for five consecutive days. Isoproterenol (85 mg/kg, subcutaneously [s.c.]) was applied to produce myocardial infarction. After 17–22 h, animals were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally [i.p.]) and both electrical and mechanical parameters of cardiac function were evaluated in vivo. Myocardial histological and gene expression analyses were performed. In isoproterenol-treated rats, Zn(ASA)2 treatment normalized significantly impaired left-ventricular contractility index (Emax 2.6 ± 0.7 mmHg/µL vs. 4.6 ± 0.5 mmHg/µL, P < 0.05), increased stroke volume (30 ± 3 µL vs. 50 ± 6 µL, P < 0.05), decreased systemic vascular resistance (7.2 ± 0.7 mmHg/min/mL vs. 4.2 ± 0.5 mmHg/min/mL, P < 0.05) and reduced inflammatory infiltrate into the myocardial tissues. ECG revealed a restoration of elevated ST-segment (0.21 ± 0.03 mV vs. 0.09 ± 0.02 mV, P < 0.05) and prolonged QT-interval (79.2 ± 3.2 ms vs. 69.5 ± 2.5 ms, P < 0.05) by Zn(ASA)2. ASA treatment did not result in an improvement of these parameters. Additionally, Zn(ASA)2 significantly increased the mRNA-expression of superoxide dismutase 1 (+73 ± 15%), glutathione peroxidase 4 (+44 ± 12%), and transforming growth factor (TGF)-β1 (+102 ± 22%). In conclusion, our data demonstrate that oral administration of zinc and ASA in the form of bis(aspirinato)zinc(II) complex is superior to ASA in preventing electrical, mechanical, and histological changes after acute myocardial ischemia. The induction of antioxidant enzymes and the anti-inflammatory cytokine TGF-β1 may play a pivotal role in the mechanism of action of Zn(ASA)2.

Graft preservation with heparinized blood/saline solution induces severe graft dysfunction.

OBJECTIVES Vascular grafts are often stored in cold physiological saline/heparinized blood preservation solution. Until now, only in vitro studies investigated the effect of the aforementioned preservation solutions on endothelial function. The main goal of our study was to compare the storage effect of physiological saline and heparinized blood after short-time cold storage and warm reperfusion in a rat model of aortic transplantation. METHODS Aortic abdominal transplantations (n = 6-8/group) were performed in Lewis rats. The donor aortic arches were placed in cold physiological saline and heparinized blood solutions and stored for 2 h. After the 2 h ischaemia, the aortic arches were transplanted into the abdominal aorta of the recipient. Two, 24 h or 1 week after transplantation, implanted grafts were harvested. Endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside) vasorelaxation were investigated in organ bath experiments. DNA strand breaks were assessed by transferase-mediated dUTP nick-end labelling-method and mRNA expression by quantitative real-time polymerase chain reaction. In addition, the expression of CD-31 was also investigated by immunochemistry. RESULTS Severely impaired endothelial function and integrity of grafts were shown after 2 and 24 h reperfusion in both groups (maximal vasorelaxation control: 94 ± 1%, heparinized blood: 27 ± 4 and 17 ± 3%, saline 34 ± 5% and 28 ± 5%; CD-31 positive area control: 96 ± 1% blood: 38 ± 8% and 41 ± 6%, saline: 35 ± 12% and 41 ± 7%, respectively P < 0.05). After 1 week, endothelial function and integrity were partially recovered (maximal vasorelaxation: heparinized blood: 46 ± 4%, saline: 46 ± 2%, CD-31 positive area blood: 35 ± 4%; saline: 56 ± 5%, P < 0.05). In addition, mRNA levels of Bax, Bcl-2 and caspase-3 were significantly altered and DNA stand breaks were observed. CONCLUSIONS Storage with the generally used physiological saline and heparinized blood solutions is unable to protect the endothelium against cold ischaemia and warm reperfusion injury. A similar weak preservation effect was observed.

Is internal thoracic artery resistant to reperfusion injury? Evaluation of the storage of free internal thoracic artery grafts

Objectives The in situ internal thoracic artery (ITA) is recognized as the best conduit for coronary artery bypass surgery. The ITA—if it is used as an in situ graft—has a much higher late patency rate than any other arterial graft, including a free ITA graft. We sought to determine if the use of the ITA as an in situ/free graft and its storage in preservation solutions, have an effect on endothelial function. Methods The ITA was harvested as either a free or in situ graft in a porcine model. Free grafts were stored in different preservation solutions (saline, Custodiol and Tiprotec [both Köhler Chemie GmbH, Bensheim, Germany]). The ITA was anastomosed off pump to the left anterior descending artery (as in situ/free graft). Freshly harvested ITA served as a control. After 2 hours of reperfusion, the implanted grafts were harvested. The assessment of endothelial function, histopathological analysis, and gene expression were performed. Results Endothelial function and integrity were severely impaired after reperfusion in the free ITA groups, however, it was partially preserved in the Tiprotec group. Reperfusion injury resulted in increased nitro‐oxidative stress, DNA breakage, vascular cell adhesion protein 1, intercellular adhesion molecule‐1, and caspase‐3 scores, and a decreased endothelial nitric oxide synthase score in the free ITA groups. The in situ ITA graft showed no signs of injury. mRNA levels were significantly altered among the groups. Conclusions An early, severe endothelial dysfunction of the stored, free ITA as described, could be completely prevented by the use of an in situ ITA graft. Tiprotec might be a feasible option for storage of free arterial grafts during coronary artery bypass grafting.

Donor Preconditioning After the Onset of Brain Death With Dopamine Derivate n-Octanoyl Dopamine Improves Early Posttransplant Graft Function in the Rat

Heart transplantation is the therapy of choice for end‐stage heart failure. However, hemodynamic instability, which has been demonstrated in brain‐dead donors (BDD), could also affect the posttransplant graft function. We tested the hypothesis that treatment of the BDD with the dopamine derivate n‐octanoyl‐dopamine (NOD) improves donor cardiac and graft function after transplantation. Donor rats were given a continuous intravenous infusion of either NOD (0.882 mg/kg/h, BDD+NOD, n = 6) or a physiological saline vehicle (BDD, n = 9) for 5 h after the induction of brain death by inflation of a subdural balloon catheter. Controls were sham‐operated (n = 9). In BDD, decreased left‐ventricular contractility (ejection fraction; maximum rate of rise of left‐ventricular pressure; preload recruitable stroke work), relaxation (maximum rate of fall of left‐ventricular pressure; Tau), and increased end‐diastolic stiffness were significantly improved after the NOD treatment. Following the transplantation, the NOD‐treatment of BDD improved impaired systolic function and ventricular relaxation. Additionally, after transplantation increased interleukin‐6, tumor necrosis factor TNF‐α, NF‐kappaB‐p65, and nuclear factor (NF)‐kappaB‐p105 gene expression, and increased caspase‐3, TNF‐α and NF‐kappaB protein expression could be significantly downregulated by the NOD treatment compared to BDD. BDD postconditioning with NOD through downregulation of the pro‐apoptotic factor caspase‐3, pro‐inflammatory cytokines, and NF‐kappaB may protect the heart against the myocardial injuries associated with brain death and ischemia/reperfusion.