11 In fact, flow cessation per se results in a significant JAK inhibitor reduction in endothelial vasoprotective pathways leading to cell activation and apoptosis. These negative effects of cold storage conditions, observed in cultured endothelial cells, are partly due to the loss of expression of the vasoprotective transcription factor Kruppel-like Factor 2 (KLF2) and can be prevented by adding a KLF2-inducer, such as simvastatin,12 to the cold preservation solution.11 Considering that endothelial protection during cold
storage represents a key factor for a successful transplantation, and that induction of KLF2-derived transcriptional programs confers endothelial protection, the main purpose of the present study was to evaluate the effects of cold storage on the hepatic endothelial vasoprotective phenotype and if supplementing a cold preservation solution with the KLF2-inducer simvastatin ameliorates
the hepatic I/R injury observed upon reperfusion. DHE: dihydroethidium; eNOS: endothelial nitric oxide synthase; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HEC: hepatic endothelial cells; HO-1: hemeoxygenase-1; ICAM-1: intercellular adhesion molecule 1; I/R: ischemia/reperfusion; KLF2: Kruppel-like Factor 2; LDH: lactate dehydrogenase; NO: nitric oxide; TM: thrombomodulin; UWS: University of Wisconsin solution. Male Wistar rats from Charles River Laboratories SA (Barcelona, Spain) weighing 275-300 g were used. The animals were
kept Z-VAD-FMK manufacturer in environmentally controlled animal facilities at the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS). All experiments were approved by the Laboratory Animal Care and Use Committee of the University of Barcelona and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, NIH Publication 86-23, revised 1996). Rat hepatic endothelial cells (HEC) were Montelukast Sodium isolated as described.13 Briefly, after perfusion of the livers with 0,015% collagenase A and isopycnic sedimentation of the resulting dispersed cells through a two-step density gradient of Percoll (25%-50%), monolayer cultures of HEC were established by selective attachment on a collagen I substrate. Cells were cultured (37°C, 5% CO2) in Roswell Park Memorial Institute (RPMI) 1640 as described.13 Highly pure and viable cells were used. After 2 hours of isolation, HEC were washed twice with phosphate-buffered saline (PBS) and lysed (no cold storage group) or incubated 16 hours at 4°C in University of Wisconsin solution (UWS) supplemented with simvastatin 1 μM (Calbiochem, Darmstadt, Germany) or its vehicle (dimethyl sulfoxide 0.1% vol/vol) (n = 4 per group). The dose of simvastatin used has been validated.11, 12 siRNA transfection was performed as described with minor modifications.
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