Abstract 11758: Prevention of Radiation-Induced Coronary Microvascular Leakage by the Restoration of an Endothelial Tight-Junction Protein

Abstract

Introduction: In cancer survivors exposed to ionizing radiation (IR) exposure to the chest and heart, coronary endothelial injury and disruption of vascular barrier integrity is noted. The mechanisms driving the IR-induced leakiness of the coronary microvasculature are not known. There is no therapy to prevent such injury. Hypothesis: IR exposure downregulates a tight junction protein claudin-1 (cldn1), which can lead to altered endothelial barrier integrity. Ac-SDKP, an endogenous peptide, prevents cldn1 loss and preserves coronary vascular barrier function. Methods: We tested the constitutive and physiological effects of cldn1 both in vitro and in vivo . Human coronary vascular endothelial cells (HMVECs) lacking cldn1 by siRNA silencing were used for loss-of-function studies. Newly developed vascular-specific (VE-cadherin promotor-linked) cldn1 overexpressing transgenic (TG) mice were used for cldn1 gain-of-function studies. Mice were exposed to 45Gy chest radiation in fractionated doses (3Gy/day). A subgroup of irradiated mice was treated with Ac-SDKP (3.2mg/kg/day) for 3-weeks. Evans blue dye was injected via tail-vein 30 mins before euthanasia and dye extravasation through coronary microvasculature was quantified. Cldn1 expression was examined by Western blot and in vitro cell permeability was examined by FITC-dextran permeability assays. Results: Cldn1 silencing with siRNA showed increased permeability in HMVECs (control, 1.0±0.01, siRNA, 1.3±0.01; p=0.0002). Cardiac irradiation significantly decreased myocardial cldn1 (control 1.0±0.27, IR 0.45±0.31; p=0.001). Consequently, there was a significant increase in myocardial Evans blue dye extravasation compared to controls (control 1.0±0.22, IR 1.3±0.23, p=0.039). Cldn1 overexpression mice had decreased dye extravasation compared to WT controls (WT 1.0±0.23, TG 0.38±0.27 p=0.002). Ac-SDKP restored cldn1 protein expression, and inhibited dye leakage in the myocardium after IR exposure (0.57±0.21, p=0.048 vs IR). Conclusions: Our study shows that IR exposure induces endothelial barrier disruption through a reduced expression of endothelial tight-junction protein, cldn1. Ac-SDKP prevents these effects, at least in part, by preventing loss of claudin-1.