Abstract 4146447: Deficiency of Mitochondrial Serine Hydroxymethyltransferase Impairs Endothelial Cell Phenotype and Function

Introduction: Our previous study showed that endothelial cells (ECs), differentiated from human induced pluripotent stem cells (hiPSCs) derived from patients with type 2 diabetes (dia-hiPSCs), displayed impaired phenotypes and function, including lower intracellular glycine content, increased senescence and inflammation, and impaired mitochondrial function. Furthermore, of the four molecules that regulate glycine homeostasis (glycine transporter 1 and 2 and the cytoplasmic and mitochondrial serine hydroxymethyltransferases [cSHMT and mSHMT, respectively]), only mSHMT was downregulated in dia-hiPSCs derived ECs (dia-hiPSC-ECs). Thus, we aim to determine whether deficiency of mSHMT protein expression will impair EC phenotype and function due to increased oxidative stress. Methods: hiPSCs reprogrammed from the cells of healthy human subjects (nor-hiPSCs) were differentiated into ECs (nor-hiPSC-ECs), and transduced with adenoviral vectors carrying shRNA targeting mSHMT (Ad-shRNA/mSHMT) or Scramble (Ad-Scramble) for 48 hours, and cultured in endothelial growth medium for 14 days. Intracellular glycine content and ROS were measured by liquid chromatography and MitoSOX, respectively, cell senescence was assessed by β-galactosidase staining, mitochondrial membrane potential was visualized using JC-1 dye, and expression of intercellular adhesion molecule-1 (ICAM-1, an indicator of inflammation) protein was evaluated via Western blot. Results: Ad-shRNA/mSHMT starting 25 infectious unit/EC dramatically inhibited mSHMT protein expression, which was accompanied with significantly reduced glycine content in nor-hiPSC-ECs. Compared to Ad-scramble transduced nor-hiPSC-ECs, down-regulation of mSHMT, caused by Ad-shRNA-mSHMT, resulted in significant increase of senescence (increased abundance of β-gal and P53 expression) and inflammation (increased ICAM-1 protein expression), and significant decline in mitochondrial membrane potential (reduced red fluorescence intensity) in nor-hiPSC-ECs. Although low intracellular cellular glycine level can lead to oxidative stress as it is required for glutathione synthesis, increased oxidative stress, by MitoSOX labeling, was not found in nor-hiPSC-ECs with mSHMT knockdown. Conclusion: Deficiency of mSHMT impairs nor-hiPSC-EC phenotype and function, which is independent of oxidative stress.