Human lungs single-cell RNA sequencing data from healthy donors (elderly and young; GEO accession no. GSE122960) were analyzed to isolate and specifically study gene expression in alveolar type II cells. Colocalization of angiotensin-converting enzyme 2 (ACE2) and TMPRSS2 enables severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2) to enter the cells. Expression levels of these genes in the alveolar type II cells of elderly and young patients were comparable and, therefore, do not seem to be responsible for worse outcomes observed in coronavirus disease 2019 (COVID-19) affected elderly. In cells from the elderly, 263 genes were downregulated and 95 upregulated. Superoxide dismutase 3 (SOD3) was identified as the top-ranked gene that was most downregulated in the elderly. Other redox-active genes that were also downregulated in cells from the elderly included activating transcription factor 4 (ATF4) and metallothionein 2A (M2TA). ATF4 is an endoplasmic reticulum stress sensor that defends lungs via induction of heme oxygenase 1. The study of downstream factors known to be induced by ATF4, according to Ingenuity Pathway AnalysisÔ, identified 24 candidates. Twenty-one of these were significantly downregulated in the cells from the elderly. These downregulated candidates were subjected to enrichment using the Reactome Database identifying that in the elderly, the ability to respond to heme deficiency and the ATF4-dependent ability to respond to endoplasmic reticulum stress is significantly compromised. SOD3-based therapeutic strategies have provided beneficial results in treating lung disorders including fibrosis. The findings of this study propose the hypotheses that lung-specific delivery of SOD3/ATF4-related antioxidants will work in synergy with promising antiviral drugs such as remdesivir to further improve COVID-19 outcomes in the elderly. Antioxid. Redox Signal. 00, 000-000.
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.
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