Abstract 13872: CD70 Modulates Vascular Smooth Muscle Phenotype and Metabolism by Regulating Cellular Redox State

Abstract

Introduction: The tumor necrosis factor superfamily (TNFSF) displays a wide array of vascular effects; however, the role of the TNFSF member CD70 is still being elucidated in the vasculature. We have recently described how CD70 modulates endothelial cell function and redox status by altering nitric oxide bioavailability and intracellular reactive oxygen species (ROS). However, the role of CD70 in vascular smooth muscle cells (VSMCs) is not known. We hypothesized that CD70 can alter VSMC phenotype and mitochondrial function through regulation of intracellular ROS. Methods: Human aortic VSMCs (AoVSMCs) were transfected with control or CD70-directed siRNA (n = 5-6). Cellular phenotype was assessed by scratch assay and collagen matrix gel contraction assay. Cell lysates were analyzed by Western blot and quantitative RT-PCR. Intracellular cytosolic hydrogen peroxide levels were measured using the fluorescent biosensor Hyper7 following stimulation by auranofin (50-70 cells per group averaged over 4 experiments). Metabolic profiling was performed with Seahorse assay, and mitochondrial membrane potential was assessed using TMRM dye (120 cells per group averaged over 3 experiments). Results: Following CD70 knockdown, AoVSMCs demonstrated augmented wound closure and decreased collagen matrix contraction. Expression of synthetic/proliferative markers, including vimentin, alpha-1 type I collagen, osteopontin, caldesmon-1, and c-Myc, was increased by up to 50%, and contractile markers including smoothelin, synemin, and synaptopodin-2 were decreased by up to 50%. Cytosolic hydrogen peroxide production was increased, with expression of NADPH oxidase (NOX) 4 and gp91phox (the catalytic subunit of NOX2) both increased by over 2-fold. CD70 knockdown led to a pronounced alteration in VSMC metabolic profile, with significantly decreased oxygen consumption rate (25%), extracellular acidification rate (45%), and maximal respiratory capacity (46%), as well as a decrease in mitochondrial membrane potential (37%). Conclusions: CD70 exerts a novel regulatory effect on VSMC phenotype; loss of CD70 promotes transition to a synthetic phenotype characterized by increased ROS with consequent metabolic reprogramming and mitochondrial dysfunction.