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.

抽象的 老龄化人群中糖尿病，肥胖和血管疾病的患病率正在加剧慢性 皮肤伤口每年影响800万Medicare受益人，费用> 960亿美元。估计2.5％ 美国人口中受到慢性伤口的影响。糖尿病性溃疡（DU），因感染/缺血/高而复杂 活性氧，通常灌注不良。依赖VEGF治疗以改善灌注 具有逻辑意义，但临床研究结果远远没有期望。限制VEGF的可能因素 治疗结果包括不足的局部VEGF信号伴侣和未成熟的回归 船只。对障碍进行故障排除并建立提供的治疗方案至关重要 理想的功能结果。为了避免VEGF单基因“单一疗法”的低效率障碍， 启动了“联合基因疗法”的发展，以递送两个或多个血管生成分子 大大提高效率。当前的文献提出了令人信服的证据，认识到 活跃的脂质介质（例如类黄花素）在启用VEGF依赖性血管生成中。但是， VEGF疗法的实施尚未在必要时测试这些脂质介质的重要性。 最近，我们报道说，在糖尿病性缺血性皮肤内皮细胞中，PLCγ2水平保持较低。 为了降低VEGF疗法的效率。但是，这种下调的基本机制仍然存在 未知。新兴证据表明，高血糖导致DNA甲基化和表观遗传 在DU受试者中的基因与环境之间的复杂相互作用中沉默。除了内皮外， PLCγ2通过TREM2信号传导启用巨噬细胞函数。 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性血管PLCγ2– 髓样Trem2串扰损害了VEGF引起的糖尿病伤口 - 边缘血管形成的增加。基因 靶向DNA脱甲基具有挽救PLCγ2基因表达的潜力。建议的工作是 首先研究基于CRISPR/DCAS9的体内靶向表观遗传学编辑真皮内皮细胞作为一种疗法 启用VEGF治疗的干预措施。提出了三个目标：目标1。确定表观遗传机制 在高血糖条件下，VEGF信号传导的基础PLCγ2调节。目标2。阐明分子 PLCγ2调节伤口组织血管形成的机制。目标3。评估功能意义 与糖尿病伤口有关的血管乳突串扰中的内皮靶向PLCγ2的增强 血管化。拟议的研究将揭示PLCγ2的关键作用以及这一重要的影响 VEGF信号的元素，以帮助提高血管生成VEGF治疗的效率和鲁棒性 糖尿病伤口愈合。