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疾病的激增 皮肤创伤，影响到800万医疗保险受益人，每年花费960亿美元。估计为2.5% 的美国人口受到慢性创伤的影响。糖尿病溃疡(DU)，合并感染/缺血/高 活性氧物种，经常遭受血流灌注不良的困扰。依赖血管内皮细胞生长因子治疗改善血流灌注 这在逻辑上是有道理的，但临床研究结果远远达不到预期。限制血管内皮生长因子的可能因素 治疗结果包括局部血管内皮生长因子信号转导伙伴浓度不足和幼稚的消退。 船只。解决障碍和建立一种治疗方案是至关重要的，它可以提供 理想的功能结果。为了绕过血管内皮生长因子单基因“单一疗法”的低效障碍， “联合基因疗法”的发展是为了将两个或更多的血管生成分子输送到 大幅提升药效。目前的文献提供了令人信服的证据，认识到 活性脂质介体，如二十烷基类，使血管内皮生长因子依赖的血管生成。然而， 血管内皮生长因子治疗的实施还没有检验这些脂质介质作为必要佐剂的意义。 最近我们报道了在糖尿病缺血的皮肤内皮细胞中，PLCγ2的水平仍然很低 对于血管内皮细胞生长因子治疗效率的降低。然而，这种下调监管的潜在机制仍然存在 未知。新的证据表明，高血糖会导致DNA甲基化和表观遗传学 DU受试者在基因和环境之间复杂相互作用中的沉默。除了内皮， PLCγ2通过TREM2信号实现巨噬细胞功能。TREM2+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的血管PLCγ2-丢失。 髓系TREM2串扰抑制了血管内皮生长因子诱导的糖尿病创缘血管生成的增加。基因 靶向去甲基化有可能挽救γ-2基因的表达。建议的工作范围是 首次研究基于CRISPR/dCas9的真皮内皮细胞体内靶向性表观遗传编辑作为治疗 能够进行血管内皮细胞生长因子治疗的干预。提出了三个目标：目标1.确定表观遗传机制 基础PLCγ2在高血糖条件下对血管内皮生长因子信号的调节.目的2.阐明分子 PLCγ2调节伤口组织血管形成的机制。目的3.评估功能意义 内皮靶向PLCγ2在血管-髓系串扰中的增强作用与糖尿病伤口的关系 血管形成。拟议的研究将揭示PLCγ2的关键作用以及这一重要的影响 血管内皮生长因子信号转导元件有助于提高血管源性血管内皮生长因子治疗的有效性和稳健性 糖尿病伤口愈合。