Proteomic and functional analysis on endothelial cell heterogeneity identifies key regulators in hyperglycemia-induced dysfunction

Endothelial cells (ECs) play a critical role in managing vascular homeostasis and neovascularization. EC functions vary significantly depending on their anatomic locations, especially for ECs forming macrovascular versus microvascular vessels. ECs possess heterogeneous signaling pathways, energy metabolism, and cellular behaviors that enable them to handle both physiological and hyperglycemic conditions. These variations can impact the efficacy of pharmacotherapy and influence the likelihood of unexpected side effects. In this study, we compared human aortic ECs (HAECs) and human dermal microvascular ECs (HDMVECs) to observe the functional and proteomic differences potentially contributing to EC heterogeneity. Compared with HAECs, HDMVECs exhibited faster proliferation, but lower migration and permeability. Under high glucose (HG), migration was worsened for both cell types, whereas proliferation was unaffected, and permeability increased for HDMVECs. Using proteomic analysis, we identified 126 proteins whose abundance was significantly different between HAECs and HDMVECs. Database for Annotation, Visualization, and Integrated Discovery (DAVID) analysis revealed their biological processes, cellular compartments, molecular functions, and pathways. Under high glucose, WARS1 increased whereas SOD2 decreased. Reversing WARS1 or SOD2 expression levels improved HDMVEC migration and permeability functions. The combined treatment of WARS1 knockdown and SOD2 overexpression fully restored EC migration and reduced permeability to levels comparable with those of their counterparts under normal glucose (NG) conditions. Furthermore, in the cutaneous wound in type 2 diabetic mice, the combination therapy of Wars1 knockdown and SOD2 overexpression accelerated the wound closure and augmented wound angiogenesis. Our studies provide novel molecular insights into EC heterogeneity and identified WARS1 and SOD2 as potential targets for dermal angiogenesis during tissue repair.<b>NEW & NOTEWORTHY</b> Our research highlights the functional and proteomic differences between human aortic endothelial cells (HAECs) and human dermal microvascular endothelial cells (HDMVECs). Importantly, we identified WARS1 as a novel target in HDMVECs. Together with SOD2, correcting the abnormalities of these two molecules, HDMVECs' migration and permeability can be fine-tuned under high glucose (HG) conditions. Furthermore, WARS1 knockdown and SOD2 overexpression accelerated wound healing in type 2 diabetic mice, highlighting the therapeutic potential of targeting WARS1 and SOD2 to address delayed wound healing in diabetes.