Abstract Body (Do not enter title and authors here): Introduction: We recently identified the tumor necrosis superfamily member CD70 as a novel regulator of redox homeostasis and mitochondrial function in endothelial cells. Whether CD70 exerts a regulatory role on metabolism and cellular function in vascular smooth muscle cells (VSMCs) is unknown.
Methods: Human aortic VSMCs (AoVSMCs) were transfected with control or CD70-directed siRNA (n = 5-6). Metabolic function was assessed by Seahorse assay. Hydrogen peroxide, calcium, ATP, and NADH/NAD⁺ ratio were assayed in live cells with fluorescent biosensors. Semi-quantitative proteomics were performed using liquid chromatography-tandem mass spectrometry. VSMC phenotype was evaluated by scratch assay and collagen gel contraction assay. VSMC function was assessed in control versus global CD70 knockout mice using wire myography.
Results: CD70 knockdown significantly altered cellular metabolic profile, decreasing oxidative phosphorylation and glycolysis (Fig. 1A). This was accompanied by mitochondrial network dispersion, reduced mitochondrial membrane potential and impaired glucose-dependent ATP production (Fig. 1B-C). Markers for mitochondrial fusion and biogenesis, including the key regulator PGC1-alpha, were reduced (Fig. 1D-E). Cellular redox balance shifted towards an oxidized state with decreased NADH/NAD⁺ ratio (Fig. 1F) and enhanced intracellular hydrogen peroxide generation. Proteomic analysis using intensity-based absolute quantification and a false discovery rate of 5% revealed that CD70 knockdown led to activation of pro-oxidative proteins, alteration in proteins involved in mitochondrial dynamics, and decreased expression of contractile proteins, all with significant p values. CD70 knockdown reduced VSMC contractile function (Fig. 1G) and intracellular calcium flux; migratory capacity increased. CD70 knockout mice had reduced aortic contractile reserve (Fig. 1H).
Conclusions: CD70 has a previously unknown homeostatic role in maintaining VSMC metabolic and contractile function. Loss of CD70 disrupts mitochondrial networks and alters VSMC metabolism to promote phenotypic switching from a contractile to a migratory state. These results identify CD70 as a potential new target for regulating VSMC pathobiology.