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

Abstract

BackgroundAlthough, 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 ResultsAortic 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.ConclusionEven 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.

Reperfusion injury and reactive oxygen species: The evolution of a concept

“…While the conditions used to simulate I/R in vitro are arguably artificial due to the absence of other relevant cell populations and non-physiological environmental conditions (e.g., pO2, pH), the in vitro models have shown a remarkable level of consistency in reproducing the phenotypic responses of tissues to I/R [32] . Similarities in the i n vitro and in vivo responses to I/R (H/R) have been demonstrated using cardiac myocytes [35] , intestinal enterocytes [36] , alveolar epithelium [34] , neurons [37] , hepatocytes [38] , adipocytes [39] , and arterial grafts [40] . Endothelial cell (EC) monolayers subjected to H/R have proven to be extraordinarily accurate in simulating the diverse microvascular alterations that are elicited by H/R, including (1) an enhanced production of ROS [41] , (2) increased expression of adhesion molecules with a consequent increase in the adhesivity of EC to leukocytes (neutrophils and T-lymphocytes) [42] , [43] , (3) diminished EC barrier function [44] , and (4) the development of a procoagulant/prothrombotic phenotype [45] ( Fig.…”

Exosomes Derived from Mesenchymal Stem Cells Ameliorate Hypoxia/Reoxygenation-Injured ECs via Transferring MicroRNA-126

“…Vascular endothelial cells (ECs) are critical in maintaining vascular homeostasis [1], and endothelial dysfunction is involved in various ischemic diseases such as limb ischemia, ischemic stroke, and myocardial ischemia [2, 3]. Under ischemia, ECs suffered from hypoxia and reoxygenation (H/R) injury and contribute to the pathogenesis of ischemic diseases [3]. Therefore, understanding EC regulation and protection under H/R injury is pivotal in developing novel preventive and therapeutic strategies for ischemic diseases.…”

Extracellular histones and xenotransplantation

“…Graft survival is significantly affected by IRI, which leads to graft failure by vascular injury, 53 IRI is an important factor that may contribute to graft dysfunction, acute rejection, and graft survival. Moreover, IRI can activate the complement system in xenotransplantation.…”