Indiana Center for Regenerative Medicine and Engineering, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN.
Sharkia Clinical Research Department, Ministry of Health and Population, Cairo, Egypt.
Diabetes. 2022 May 1;71(5):1149-1165. doi: 10.2337/db21-0830.
Therapeutic vascular endothelial growth factor (VEGF) replenishment has met with limited success for the management of critical limb-threatening ischemia. To improve outcomes of VEGF therapy, we applied single-cell RNA sequencing (scRNA-seq) 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 the 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 versus nondiabetic skin, phospholipase Cγ2 (PLCγ2) was downregulated. The significance of PLCγ2 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. The 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. Nonviral topical tissue nanotransfection technology (TNT) delivery of CDH5 promoter-driven PLCγ2 open reading frame promoted the rescue of hindlimb 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 proangiogenic 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.
治疗性血管内皮生长因子 (VEGF) 补充治疗在治疗严重肢体缺血方面的效果有限。为了提高 VEGF 治疗的效果，我们应用单细胞 RNA 测序 (scRNA-seq) 技术研究了人类糖尿病皮肤的内皮细胞。从人类皮肤中生成单细胞悬液，然后使用 Chromium Next GEM Single-cell 3' Kit v3.1 制备 cDNA。通过适当的质量控制措施，选择了 36487 个细胞进行下游分析。scRNA-seq 研究表明，尽管 VEGF 信号在糖尿病皮肤与非糖尿病皮肤之间没有明显改变，但磷脂酶 Cγ2 (PLCγ2) 下调。在培养的人微血管内皮细胞中评估了 PLCγ2 在 VEGF 介导的内皮细胞代谢和功能增加中的作用。在这些细胞中，VEGF 通过提高耗氧量和细胞外酸化率来增强线粒体功能。PLCγ2 抑制减弱了 VEGF 依赖性细胞代谢增加。因此，后续的挽救研究集中于了解在 1 型（链脲佐菌素注射）和 2 型（db/db）糖尿病缺血组织中存在或不存在内皮 PLCγ2 的情况下，VEGF 治疗的意义。非病毒局部组织纳米转染技术 (TNT) 递送 CDH5 启动子驱动的 PLCγ2 开放阅读框促进了糖尿病小鼠后肢缺血的挽救。血流改善也与更高丰度的 VWF+/CD31+和 VWF+/SMA+免疫组织化学染色相关。TNT 为基础的基因传递与组织水肿无关，组织水肿是与促血管生成基因治疗相关的常见并发症。总之，我们的研究表明，作为联合基因治疗的一部分，TNT 介导的内皮 PLCγ2 传递在糖尿病缺血肢体挽救中是有效的。
