Prolyl-Hydroxylase Inhibition Preserves Endothelial Cell Function in a Rat Model of Vascular Ischemia Reperfusion Injury

Abstract

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 prolylhyroxylase domain-containing enzyme (PHD). Reduction of PHD activity during hypoxia leads to stabilization and accumulation of hypoxia inducible factor (HIF) 1a. 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% 6 1% versus NaOCl 44% 6 4% versus DMOG 68% 6 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.

Addition of Vardenafil into Storage Solution Protects the Endothelium in a Hypoxia-Reoxygenation Model

“…5 Different research groups have aimed to develop individual methods to maintain vascular function during storage. 17,24 In our laboratory, we demonstrated a maintained vascular function with new iron chelators after 24 hours of ischaemia. 19 In accordance with the literature, in the present study we also found an impaired vasodilatative capacity in aortic rings after prolonged cold storage for 24 hours followed by warm HOCl-induced reperfusion injury by using vitro measurements.…”

“…To simulate the whole reperfusion process the well established method of reoxygenation and rewarming with additional HOCl incubation was applied. 16,17 After prolonged cold storage and high reperfusion levels of ROS, including other less toxic oxygen species, HOCl is released from activated leucocytes and severely impairs endothelial cells and NO production, and induces apoptosis in endothelial cells and in the vessel wall through oxidative stress. After prolonged cold storage and reperfusion, high levels of ROS severely impair endothelial cells and NO production.…”

“…Isometric contractions were recorded using isometric force transducers of a myograph (159901A; Radnoti Glass Technology), digitised, stored, and displayed with the IOX Software System (EMKA Technologies, Paris, France). The aortic rings (n ¼ 15e20 from N ¼ 8e12 animals in each group) were placed under a resting tension of 2 g (which was found to be optimal in preliminary experiments 7,17 ) and equilibrated for 60 minutes. During this period, tension was adjusted periodically to the desired level and the KrebseHenseleit solution was changed every 30 minutes.…”

“…Following the manufacturer's protocol (Chemicon International, Temecula, CA, USA), TUNEL staining was performed as described previously. 17 cGMP immunohistochemical staining. Rehydrated sections were blocked (3% goat serum) and rabbit polyclonal anti-cGMP primary antibody (Abcam, Cambridge, UK) incubation was performed (1:1,000) for 2 hours at room temperature.…”

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

“…17 cGMP immunohistochemical staining. Rehydrated sections were blocked (3% goat serum) and rabbit polyclonal anticGMP primary antibody (Abcam, Cambridge, UK) incubation was performed (1:1,000) for 2 hours at room temperature.…”