CARD24: Protective Effect of HemoTech - Blood Substitute Against Contrast Media-Induced Endothelial Toxicity: An In Vitro Study

Abstract

Background: Although the most common complication after intravascular administration of iodinated contrast media (CM) is contrast-induced nephropathy (CIN) there is some evidence suggesting CM toxicity to endothelial cells (EC). CIN mechanism is well defined and includes medullary ischemia and direct toxicity to renal tubular cells. The mechanism of CM-associated EC damage is still unclear. The purpose of this study was twofold: (1) to determine the response of normal and ischemic coronary artery EC to low-osmolar nonionic CM commonly used in percutaneous coronary interventions (PCI) and (2) to evaluate the protective effect of HemoTech used concomitantly with CM. HemoTech is Hb-based blood substitute pharmacologically modified with vasodilators, anti-inflammatory and antioxidant agents: ATP, adenosine and reduced glutathione (GSH), and is in the preclinical development phase of its intended use as perfusion fluid for PCI. Methods: In this study human coronary artery EC (Clonetics, Bio-Whittaker, San Diego, CA) were subcultured in EGM-MV medium to confluence on cover slips and 6-well cell cultured plates in a humidified atmosphere of 5% CO2 and 37 deg C. To mimic ischemia, EC were depleted from GSH by 24 hr treatment with 200 uM of BSO (L-buthionine-[S,R]-sulphoximine).Then, normal and ischemic EC were exposed for 1, 2, 4 and 18 hrs to 50 uL/mL of low-osmolar nonionic iodinated CM Optiray 350 (Ioversol Injection 74%, Mallinckrodt Pharmaceuticals) alone or with 0.4 mM of HemoTech and assessed for cellular redox state and oxidative stress (GSH, TBARS), inflammatory response (VCAM-1) and early and late apoptosis. Results: The results demonstrate a different response of normal and ischemic EC to CM. While normal EC showed delayed mild reaction, a response of ischemic EC was immediate and intensifies with time. In normal EC there was a depletion of GSH from 24.1±1.5 to 18.5±1.1 nM/mg protein (p<0.05) at 18 hr; however without changes in TBARS and only insignificant expression of VACAM-1 in the absence of apoptosis. On the contrary, CM showed to strongly affect ischemic EC causing further redox imbalance. At 2 hr GSH dropped from 13.6±0.9 to 10.3±0.6 (p<0.05) and progressively decreased to 9.1±0.5 (p<0.01) at 4 hr and 7.5±0.4 (p<0.001) at 18 hr. TBARS progressively increased from 0.84±0.03 to 0.98±0.04 nM/mg protein (p<0.05) at 2 hr, 1.1±0.07 (p<0.01) at 4 hr and 1.44±0.12 (p<0.001) at 18 hr. A slight expression of VACAM-1 was noted at 4 hr that reached the peak at 18 hr (p<0.01). Early apoptosis was detected at 18 hr. HemoTech treatment effectively restored the redox balance in ischemic EC and alleviated inflammatory and apoptotic responses to CM. All tested parameters were in normal range. Conclusions: Low-osmolar nonionic iodinated CM is well tolerated by normal but not ischemic coronary artery EC. HemoTech blood substitute with pharmacological properties of ATP, adenosine and GSH protects ischemic EC against CM-induced toxicity.