Verma Sumit S, Sen Chandan K, Srivastava Rajneesh, Gnyawali Surya C, Katiyar Parul, Sahi Ajay K, Kumar Manishekhar, Rustagi Yashika, Liu Sheng, Pandey Diksha, Abouhashem Ahmed S, Fehme Leila N W, Kacar Sedat, Mohanty Sujit K, Faden-McCormack Julie, Murphy Michael P, Roy Sashwati, Wan Jun, Yoder Mervin C, Singh Kanhaiya
Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA; Department of Surgery, Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA.
Mol Ther. 2025 Mar 5;33(3):950-969. doi: 10.1016/j.ymthe.2025.01.034. Epub 2025 Jan 25.
Diabetic wounds are complicated by underlying peripheral vasculopathy. Reliance on vascular endothelial growth factor (VEGF) therapy to improve perfusion makes logical sense, yet clinical study outcomes on rescuing diabetic wound vascularization have yielded disappointing results. Our previous work has identified that low endothelial phospholipase Cγ2 (PLCγ2) expression hinders the therapeutic effect of VEGF on the diabetic ischemic limb. In this work, guided by single-cell RNA sequencing of human wound edge, we test the efficacy of gene-targeted therapeutic demethylation intending to improve VEGF-mediated neovascularization. PLCγ2 expression was diminished in all five identified diabetic wound-edge endothelial subclusters encompassing arterial, venous, and capillary cells. Such low expression was associated with hypermethylated PLCγ2 promoter. PLCγ2 promoter was also hypermethylated at murine diabetic ischemic wound edge. To specifically demethylate endothelial PLCγ2 promoter during VEGF therapy, a CRISPR-dCas9-based demethylation cocktail was delivered to the ischemic wound edge using tissue nanotransfection (TNT) technology. Demethylation-based upregulation of PLCγ2 during VEGF therapy improved wound tissue blood flow with an increased abundance of von Willebrand factor (vWF)/PLCγ2 vascular tissue elements by activating p44/p42-mitogen-activated protein kinase (MAPK) → hypoxia-inducible factor [HIF]-1α pathway. Taken together, TNT-based delivery of plasmids to demethylate the PLCγ2 gene promoter activity led to significant improvements in VEGF therapy for cutaneous diabetic wounds, resulting in better perfusion and accelerated wound closure.
糖尿病伤口因潜在的外周血管病变而变得复杂。依靠血管内皮生长因子（VEGF）疗法来改善灌注在逻辑上是合理的，然而，在挽救糖尿病伤口血管化方面的临床研究结果却令人失望。我们之前的工作已经确定，内皮型磷脂酶Cγ2（PLCγ2）低表达会阻碍VEGF对糖尿病缺血肢体的治疗效果。在这项工作中，以人伤口边缘的单细胞RNA测序为指导，我们测试了基因靶向治疗性去甲基化的疗效，旨在改善VEGF介导的新生血管形成。在所有五个已确定的糖尿病伤口边缘内皮亚群（包括动脉、静脉和毛细血管细胞）中，PLCγ2表达均降低。这种低表达与PLCγ2启动子的高甲基化有关。在小鼠糖尿病缺血伤口边缘，PLCγ2启动子也发生了高甲基化。为了在VEGF治疗期间特异性地使内皮PLCγ2启动子去甲基化，使用组织纳米转染（TNT）技术将基于CRISPR-dCas9的去甲基化混合物递送至缺血伤口边缘。在VEGF治疗期间，基于去甲基化的PLCγ2上调通过激活p44/p42-丝裂原活化蛋白激酶（MAPK）→缺氧诱导因子[HIF]-1α途径，改善了伤口组织血流，增加了血管性血友病因子（vWF）/PLCγ2血管组织成分的丰度。综上所述，基于TNT的质粒递送使PLCγ2基因启动子活性去甲基化，显著改善了VEGF对皮肤糖尿病伤口的治疗效果，导致更好的灌注和加速伤口愈合。
