Severin Semsroth

The Angiogenic Factor Secretoneurin Induces Coronary Angiogenesis in a Model of Myocardial Infarction by Stimulation of Vascular Endothelial Growth Factor Signaling in Endothelial Cells

Background—Secretoneurin is a neuropeptide located in nerve fibers along blood vessels, is upregulated by hypoxia, and induces angiogenesis. We tested the hypothesis that secretoneurin gene therapy exerts beneficial effects in a rat model of myocardial infarction and evaluated the mechanism of action on coronary endothelial cells. Methods and Results—In vivo secretoneurin improved left ventricular function, inhibited remodeling, and reduced scar formation. In the infarct border zone, secretoneurin induced coronary angiogenesis, as shown by increased density of capillaries and arteries. In vitro secretoneurin induced capillary tubes, stimulated proliferation, inhibited apoptosis, and activated Akt and extracellular signal-regulated kinase in coronary endothelial cells. Effects were abrogated by a vascular endothelial growth factor (VEGF) antibody, and secretoneurin stimulated VEGF receptors in these cells. Secretoneurin furthermore increased binding of VEGF to endothelial cells, and binding was blocked by heparinase, indicating that secretoneurin stimulates binding of VEGF to heparan sulfate proteoglycan binding sites. Additionally, secretoneurin increased binding of VEGF to its coreceptor neuropilin-1. In endothelial cells, secretoneurin also stimulated fibroblast growth factor receptor-3 and insulin-like growth factor-1 receptor, and in coronary vascular smooth muscle cells, we observed stimulation of VEGF receptor-1 and fibroblast growth factor receptor-3. Exposure of cardiac myocytes to hypoxia and ischemic heart after myocardial infarction revealed increased secretoneurin messenger RNA and protein. Conclusions—Our data show that secretoneurin acts as an endogenous stimulator of VEGF signaling in coronary endothelial cells by enhancing binding of VEGF to low-affinity binding sites and neuropilin-1 and stimulates further growth factor receptors like fibroblast growth factor receptor-3. Our in vivo findings indicate that secretoneurin may be a promising therapeutic tool in ischemic heart disease.

S-Nitroso Human Serum Albumin Improves Oxygen Metabolism during Reperfusion after Severe Myocardial Ischemia

Nitric oxide (NO) supplementation may modify myocardial oxygen consumption and vascular function after ischemia. We investigated the effects of the NO donor, S-nitroso human serum albumin (S-NO-HSA), on cardiac oxygen metabolism during controlled reperfusion on normothermic cardiopulmonary bypass after severe myocardial ischemia. Pigs randomly received either S-NO-HSA or human serum albumin prior to and throughout global myocardial ischemia. Myocardial oxygen utilization is impaired at the onset of reperfusion, which is not amenable to S-NO-HSA. However, NO supplementation during ongoing supply dependency of oxygen consumption eventually leads to greater myocardial oxygen delivery and consumption. In conjunction with a better washout of lactate, this indicates an improved capillary perfusion in the S-NO-HSA group during reperfusion, which results in a better contractile function post bypass.

Bilirubin rinse of the graft ameliorates ischemia reperfusion injury in heart transplantation

Ischemia and reperfusion contribute to substantial organ damage in transplantation. Clinically feasible measures for the prevention thereof are scarce. We tested whether rinsing rodent hearts with the antioxidant bilirubin ameliorates ischemia reperfusion injury (IRI). Left ventricular end‐diastolic pressure (LVEDP), left ventricular developed pressure (LVDevP), rate per pressure product (RPP), coronary flow, maximum (+dP/dt) and minimum (−dP/dt) rate of contraction were analyzed in Lewis rat hearts rinsed with bilirubin prior to reperfusion on a Langendorff apparatus after 12 h of cold ischemia. In vivo, isogenic C57Bl/6 mouse hearts rinsed with bilirubin were transplanted after 12 h of cold ischemia. Cardiac function and apoptosis were assessed 24 h after reperfusion. Heart lysates recovered 15 min after reperfusion were probed for the total and the phosphorylated forms of extracellular signal‐related protein kinases (ERK), JNK, p38‐MAPK, and Akt. In isolated perfused hearts, bilirubin rinse resulted in significantly lower LVEDP and improved LVDevP, RPP, coronary flow, +dP/dt and −dP/dt. In vivo, after reperfusion, all mitogen‐activated protein kinases (MAPKs) were suppressed significantly by bilirubin pretreatment. Bilirubin rinse improved cardiac scores (3.4 ± 0.5 vs. 2.0 ± 1.0 in controls, P < 0.05) and significantly suppressed apoptosis. Ex vivo administration of bilirubin to heart grafts prior reperfusion ameliorates IRI and provides a simple and effective tool to ameliorate outcome in heart transplantation.

872. Highly Efficient Virus-Mediated Gene Transfer to Human Vein Grafts Via Intraluminal Pressure Delivery

Top of pageAbstract Introduction Long term patency rates for coronary artery bypass grafting (CABG) using autologous saphenous vein are poor, showing 10 years post-CABG patency rates of 41%. To date, effective and proven pharmacologic interventions to prolong vein graft patency are lacking. Gene therapy seems particularly well suited for the prevention or postponement of vein graft failure since (1) the stimulation of smooth muscle cell proliferation appears to largely be an early and transient process, and (2) the target tissue is easily accessible ex vivo during the surgical procedure. However, a major obstacle in efficient gene transfer to human vein grafts is the heterogenity of vessel quality due to age, preexisting veinous disease, and surgical trauma. In this study we developed a clinically relevant viral delivery technique to achieve a reproducible and efficient gene transfer in human vein grafts. Methods 46 intact human saphenous veins (age 68+/|[minus]|7) from patients undergoing coronary bypass surgery were cut into rings 15 mm in length. A small piece of each vein was tested for endothelial viability with trypan blue. Individual vein rings were endoluminally incubated with adenoviral vectors expressing two reportergenes (Ad.CMV.lacZ/GFP, 1|[times]|10E11 pfu/mL) with different intraluminal pressures (50, 100, 150mmHg) for 30 or 60 minutes. Veinsegments were cultured for 6days after transfection and harvested for cryopreservation. Transfection efficiency was evaluated by X-gal staining and GFP- Westernblot; endothelial integrity and neointima-hyperplasia were evaluated by CD31-staining, Elastica Van Gieson-staining and H-E-staining. Results Veins which underwent endoluminal gene transfer without pressure showed only poor transfection efficiency (12%+/|[minus]|7). In contrast, veins wich were pressure-transfected with either 100 or 150 mmHg showed high transfection efficiency throughout the vessel wall (75%|[minus]|+/|[minus]|13 and 91+/|[minus]|7% respectively, p<0.02). Veins transfected for 60 minutes showed no significant difference in transfection efficiency versus 30 minute incubation. However, CD31 staining revealed a significant loss of endothelium in the high pressure group (150 mmHg), whereas the 100 mmHg pressure group showed only minor endothelial damage compared to control veins, irrespective of incubation time. Discussion This study presents an optimized transfection protocol for gene transfer in human vein grafts. We here show for the first time that viral transfection using carefully regulated supraphysiological endoluminal pressure greatly enhances transfection efficiency in human veins. Using continuous intraluminal pressure-monitoring and careful evaluation of endothelial integrity, we found optimal conditions for highly efficient and reproducible transfection using 100 mmHg intraluminal pressure for 30 minutes. Establishing a safe and efficient delivery method in a clinically relevant model for human cardiovascular gene therapy is an important aspect of translation from pre-clinical to clinical gene therapy.