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

Abstract

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 (PLCγ2) was down-regulated. The significance of PLCγ2 in VEGF mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In HMECs, 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 PLCγ2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCγ2 in type-1 (streptozotocin-injected) and type-2 (db/db) diabetic ischemic tissue. Non-viral topical tissue nanotransfection (TNT) delivery of CDH5 promoter driven PLCγ2-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 PLCγ2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.

Genome-wide DNA hypermethylation opposes healing in patients with chronic wounds by impairing epithelial-mesenchymal transition

“…Genes such as KRT7 (60), KRT19 (61), KRT8 (61) and KRT18 (62), highly abundant in Kera2, are directly implicated in EMT. Human keratinocyte populations, KRT14 + KRT1 + and KRT19 + KRT7 + , referred to as Kera1 and Kera2 in this work, have been evident in data reported by us as well as other laboratories (63)(64)(65)(66)(67)(68). In those studies, Kera2 has been evident in human skin samples.…”

“…In those studies, Kera2 has been evident in human skin samples. Kera2 have been localized in the bottom/side of the epidermal rete ridges, hair follicles, glandular and ductal cells (63)(64)(65)(66)(67)(68). In this work, Kera2 pool of keratinocytes have been found to be abundant in the UW skin.…”

Inducible miR-1224 silences cerebrovascular Serpine1 and restores blood flow to the stroke-affected site of the brain

“…The biological functions of differentially expressed TCT-sensitive upregulated (contralateral versus peri-infract) microRNAs were analyzed using IPA (Ingenuity Systems) as described previously. 84 , 85 , 86 Briefly, the Excel file containing the list of differentially expressed microRNAs was loaded as a dataset in the IPA software. Core analysis was then performed to obtain enriched pathways represented by microRNAs using the Ingenuity Knowledge Base with default settings in IPA.…”

The NIDDK Diabetic Foot Consortium

“…An impaired skin barrier can lead to wound recurrence and abnormalities of the repaired skin of DFU patients may underlie the high rates of recidivism in diabetic wounds (30% recur within one year). 2,33 The hypothesis is that restoration of skin barrier function, a marker of functional wound closure, [34][35][36]…”