Regulatory Mechanisms Addressing Diabetic Vasculopathy

解决糖尿病血管病变的调节机制

• 批准号: 10718850
• 负责人: Kanhaiya Singh
• 金额: $ 38.25万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-07 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10718850
• 关键词: Accounting; Address; Adjuvant; Adult; Affect; Angioblast; Blood Vessels; CDH5 gene; Cadherins; Cardiovascular system; Cell Compartmentation; Cells; Clinical; Clinical Research; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; CpG Islands; Cyclic AMP-Dependent Protein Kinases; DNA; DNA Methylation; Dermal; Development; Diabetes Mellitus; Diglycerides; Dinoprostone; Down-Regulation; Eicosanoids; Elements; Endothelial Cells; Endothelium; Environment; Epigenetic Process; Exhibits; Gene Combinations; Gene Expression; Gene Targeting; Generations; Genes; Granulation Tissue; Guide RNA; Healthcare; Human; Hyperglycemia; Hypermethylation; Infection; Injury; Inositol; Ischemia; Knockout Mice; Literature; Macrophage; Mediating; Medicare; Methylation; Molecular; Mus; Myelogenous; Myeloid Cells; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Outcome; Outcome Study; PIK3CG gene; PLCgamma2; Pathway interactions; Patients; Perfusion; Phosphatidic Acid; Population; Prevalence; Process; Production; Protein Dephosphorylation; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Recovery of Function; Regimen; Regulation; Reporting; Research; Role; Signal Pathway; Signal Transduction; Skin; TREM2 gene; Testing; Therapeutic; Therapeutic Intervention; Tissues; Vascular Diseases; Vascular Endothelial Growth Factors; Vascularization; Work; aging population; angiogenesis; beneficiary; blood vessel development; cadherin 5; chronic wound; combination gene therapy; cost; demethylation; diabetic; diabetic ulcer; diabetic wound healing; epigenetic silencing; expectation; falls; functional outcomes; improved; in vivo; injured; lipid mediator; lymphatic vessel; non-diabetic; prevent; promoter; public health relevance; single-cell RNA sequencing; therapy outcome; tool; tripolyphosphate; vasculogenesis; wound; wound healing; wound vascularization

ABSTRACT The prevalence of diabetes, obesity, and vascular disease in an aging population is fueling a surge in chronic skin wounds, which affect >8 million Medicare beneficiaries at a cost of >$96 billion per year. An estimated 2.5% of US population is affected by chronic wounds. Diabetic ulcers (DU), complicated by infection/ischemia/high reactive oxygen species, often suffer from poor perfusion. Reliance on VEGF therapy to improve perfusion makes logical sense, yet clinical study outcomes fall far short of expectations. Possible factors limiting VEGF therapy outcomes include insufficient local concentration of VEGF signaling partners and regression of immature vessels. It is critically important to troubleshoot barriers and to establish a therapeutic regimen that delivers desirable functional outcomes. To circumvent the low-efficiency hurdle of VEGF single-gene “monotherapy”, development of “combined gene therapy” was initiated to deliver of two or more angiogenic molecules to substantially enhance efficacy. Current literature presents compelling evidence recognizing a critical role of active lipid mediators such as eicosanoids in enabling VEGF-dependent angiogenesis. However, the implementation of VEGF therapy has not tested the significance of these lipid mediators as necessary adjuvant. Recently we have reported that in diabetic ischemic skin endothelial cells, PLCγ2 levels remain low accounting for diminished efficiency of VEGF therapy. However, the underlying mechanisms of such downregulation remain unknown. Emerging evidence demonstrate that hyperglycemia leads to DNA methylation and epigenetic silencing in the complex interplay between genes and the environment in DU subjects. In addition to endothelium, PLCγ2 enables macrophage function via TREM2 signaling. TREM2+ macrophages participate in tissue angiogenesis post-injury. The central hypothesis is that hyperglycemia dependent loss of vascular PLCγ2– myeloid TREM2 crosstalk compromises VEGF-induced increase in diabetic wound-edge vascularization. Gene targeted DNA demethylation has potential to rescue PLCγ2 gene expression. The proposed line of work is the first to study CRISPR/dCas9-based in vivo targeted epigenetic editing of dermal endothelial cells as a therapeutic intervention for enabling VEGF therapy. Three aims are proposed: Aim 1. Determine the epigenetic mechanisms underlying PLCγ2 regulation of VEGF signaling under hyperglycemic conditions. Aim 2. Elucidate the molecular mechanisms by which PLCγ2 regulates wound tissue vascularization. Aim 3. Evaluate the functional significance of endothelial-targeted PLCγ2 augmentation in vascular-myeloid crosstalk as it relates to diabetic wound vascularization. The proposed research will unveil the critical role of PLCγ2 and the impact of this important element in VEGF signaling to help improve the efficiency and robustness of vasculogenic VEGF therapy in diabetic wound healing.

摘要 糖尿病、肥胖和血管疾病在老龄化人口中的流行正在推动慢性病的激增。 皮肤伤口，影响超过800万医疗保险受益人，每年花费超过960亿美元。估计2.5% 的美国人口受到慢性伤口的影响。糖尿病溃疡（DU），并发感染/缺血/高 活性氧物质常常遭受灌注不良。依赖VEGF治疗改善灌注 这在逻辑上是合理的，但临床研究结果远远低于预期。可能限制VEGF的因素 治疗结果包括VEGF信号传导伙伴的局部浓度不足和未成熟的 船舶.排除障碍并建立治疗方案至关重要， 理想的功能结果。为了规避VEGF单基因“单一疗法”的低效障碍， “联合基因疗法”的发展开始将两种或多种血管生成分子递送到 显著提高功效。目前的文献提供了令人信服的证据，承认 活性脂质介质，如类花生酸，使VEGF依赖性血管生成。但 VEGF治疗的实施还没有测试这些脂质介质作为必要辅助剂的意义。 最近，我们报道了在糖尿病缺血的皮肤内皮细胞中，PLCγ2水平保持低水平， 降低VEGF治疗的效率。然而，这种下调的潜在机制仍然存在， 未知新的证据表明，高血糖症导致DNA甲基化和表观遗传 在DU受试者中基因和环境之间复杂相互作用的沉默。除了内皮， PLCγ2通过TREM 2信号传导使巨噬细胞发挥功能。TREM 2+巨噬细胞参与组织 损伤后血管生成。中心假设是高血糖依赖性血管PLCγ2- 髓样TREM 2串扰损害VEGF诱导的糖尿病伤口边缘血管化的增加。基因 靶向DNA去甲基化有可能挽救PLCγ2基因的表达。拟议的工作路线是 首次研究基于CRISPR/dCas 9的真皮内皮细胞体内靶向表观遗传编辑作为治疗方法 干预以实现VEGF治疗。提出了三个目标：目标1。确定表观遗传机制 高血糖条件下PLCγ2对VEGF信号转导的潜在调节。目标二。阐明分子 PLCγ2调节创伤组织血管化的机制。目标3。评估功能意义 内皮靶向PLCγ2增强血管-骨髓串扰，因为它与糖尿病伤口有关 血管化拟议的研究将揭示PLCγ2的关键作用以及这一重要作用的影响。 在VEGF信号传导中的元件，以帮助提高血管生成VEGF治疗的效率和稳健性， 糖尿病伤口愈合