Endothelial Phospholipase Cγ2 Improves Outcomes of Diabetic Ischemic Limb Rescue Following VEGF Therapy
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posted on 2022-02-22, 20:44 authored by Yashika Rustagi, Ahmed S Abouhashem, Priyanka Verma, Sumit S Verma, Edward Hernandez, Sheng Liu, Manishekhar Kumar, Poornachander R Guda, Rajneesh Srivastava, Sujit K Mohanty, Sedat Kacar, Sanskruti Mahajan, Kristen E Wanczyk, Savita Khanna, Michael P Murphy, Gayle M Gordillo, Sashwati Roy, Jun Wan, Chandan K Sen, Kanhaiya SinghTherapeutic
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.