Abstract Body: Introduction: Polymerase delta–interacting protein 2 (POLDIP2) is a multifunctional adaptor protein that regulates mitochondrial function, redox signaling, and cytoskeletal dynamics and is critical for vascular biology. Endothelial cells maintain vascular homeostasis by integrating metabolic adaptation, barrier integrity, inflammatory signaling, and vessel remodeling. Under pathological conditions such as ischemia and metabolic stress, endothelial metabolic reprogramming and endothelial-to-mesenchymal transition (EndMT) promote endothelial dysfunction. However, the molecular mechanisms linking endothelial metabolism to phenotypic transition remain poorly defined. Here, we investigate POLDIP2 as a key regulator of endothelial metabolism and EndMT and define its role in driving endothelial dysfunction.
Methods: POLDIP2 was silenced (siRNA) or overexpressed (adenovirus) in primary Human Aortic Endothelial Cells (HAECs). Western blot, and beta-galactosidase staining (p7-8 HAECs were aged for 1.5 weeks) were employed to examine the effects of POLDIP2 on cellular metabolism and senescence.
Results: Overexpression of POLDIP2 led to increased expression of metabolic regulatory proteins PFKFB3 and Hexokinase 2 (HK2) by 100% (p<0.05) while silencing decreased expression by approximately 50% (p<0.05). Increased expression of the glycolytic enzymes PFKFB3 and HK2 has been implicated in metabolic reprogramming that supports EndMT progression. Next, pluripotency-associated transcription factors OCT4 and KLF4 which are associated with cellular de-differentiation and EndMT were examined. Overexpression of POLDIP2 induced expression of OCT4 and increased expression of KLF4. However, silencing of POLDIP2 did not significantly affect expression of KLF4 and OCT4. Senescence, measured by beta-galactosidase staining was decreased by about 20% (p<0.05) in cells lacking POLDIP2.
Conclusion: Collectively, these findings identify POLDIP2 as a central regulator linking endothelial metabolism and EndMT. Altered POLDIP2 expression reinforces a metabolic phenotype that sustains EndMT and endothelial dysfunction under conditions of overexpression, while constraining EndMT and preserving endothelial function upon silencing. POLDIP2 thus drives a cellular program associated with loss of endothelial identity and maladaptive metabolic activation. Together, these data position POLDIP2 as a promising mechanistic target for vascular–metabolic intervention.