Endothelial Phospholipase Cγ2 Improves Outcomes of Diabetic Ischemic Limb Rescue Following VEGF Therapy

Therapeutic VEGF replenishment has met with limited success for the management of critical limb threatening ischemia (CLTI). To improve outcomes of VEGF therapy we applied single-cell RNA sequencing technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using Chromium Next GEM Single-cell 3' Kit v3.1. Using appropriate quality control measures, 36,487 cells were chosen for downstream analysis. scRNA-seq studies identified that although VEGF signaling was not significantly altered in diabetic vs non-diabetic skin, phospholipase-C-Gamma-2 (PLCg2) was down-regulated. The significance of PLCg2 in VEGF mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In these cells, VEGF enhanced mitochondrial function as indicated by elevation in oxygen consumption rate and extracellular acidification rate. VEGF-dependent increase in cell metabolism was blunted in response to PLCg2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCg2 in type-1 (streptozotocin-injected) and type-2 (db/db) diabetic ischemic tissue. Non-viral topical tissue nanotransfection (TNT) delivery of CDH5 promoter driven PLCg2-ORF promoted the rescue of hind-limb ischemia in diabetic mice. Improvement of blood flow was also associated with higher abundance of VWF⁺/CD31⁺ and VWF⁺/SMA⁺ immunohistochemical staining. TNT-based gene delivery was not associated with tissue edema, a commonly noted complication associated with pro-angiogenic gene therapies. Taken together our study demonstrates that TNT mediated delivery of endothelial PLCg2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.