High‐Fat Diet‐Induced Degradation of the Protein Phosphatase 4 Regulatory Subunit 2 Protein Increases Protein Phosphatase 4 Activity in the Aorta and Mediates Endothelial Dysfunction and Vascular Damage

Background High fat diet (HFD)‐induced increases in free fatty acids in the blood increase the risk of cardiovascular diseases by compromising vascular function. We reported that palmitic acid decreases eNOS (endothelial nitric oxide synthase) phosphorylation at serine 633 by reducing the expression of the PP4R2 (protein phosphatase 4 regulatory subunit 2) protein and activating PP4, leading to endothelial dysfunction. This study aimed to elucidate the underlying mechanisms. Methods Studies were performed using C57BL/6J male mice fed an HFD or a normal fat diet for 12 weeks. PP4R2 protein expression was upregulated or PP4C protein expression was downregulated in the aorta by injecting Ppp4r2 overexpression (OE‐ Ppp4r2 ) or Ppp4c knockdown (sh Ppp4c ) lentiviral vectors into the tail vein, respectively. Mouse aortic endothelial cells were transfected with LV‐sh Ppp4c or LV‐OE‐ Ppp4r2 and then treated with palmitic acid. Results Decreases in PP4R2 protein levels increased PP4 activity and decreased phosphorylated‐eNOS Ser633 and NO levels in the aortas of HFD‐fed mice and palmitic acid‐treated mouse aortic endothelial cells. Either Ppp4r2 overexpression or Ppp4c knockdown inhibited PP4 activity, improved eNOS/NO function in the aorta and endothelial cells, and ameliorated HFD‐induced pathological changes in the aorta. Palmitic acid activated PP4 in mouse aortic endothelial cells by increasing degradation of the PP4R2 protein and enhancing the interactions between PP4R2 and PP4C. Ginkgolide B, a potential ligand of PP4R2 identified with an in silico virtual screen, promoted PP4R2 inhibition of PP4 and partially reversed eNOS/NO dysfunction. Conclusions The endothelial PP4R2 protein is essential for maintaining eNOS/NO function and vascular homeostasis. Boosting PP4R2 inhibitory effect on PP4 activity, like using ginkgolide B, offers a potential strategy for combating HFD‐induced vascular damage.