The role of PKCepsilon in diabetic retinopathy

描述(申请人提供)：项目总结。申请者是一名内科科学家，已经在乔治·L·金博士的实验室完成了博士后研究，在申请者扩大他在新领域的培训期间，金博士将继续担任导师，包括视网膜内皮细胞生物学和糖尿病视网膜并发症的动物模型。这项培训将由乔斯林糖尿病中心毕坦眼科研究所的劳埃德·保罗·艾洛博士共同指导。职业发展奖将使申请者成为视网膜疾病领域的独立研究人员，并有助于实现了解生长因子在糖尿病眼并发症中的作用的长期目标。在糖尿病黄斑水肿和增殖性糖尿病视网膜病变中，血管内皮生长因子(VEGF)表达上调起核心作用。蛋白激酶C(PKC)亚型a、(3和e)在糖尿病视网膜中被激活，但到目前为止，缺乏对PKC功能进行亚型特异性操作的动物模型的数据。在初步结果中，在内皮细胞培养中，用小干扰RNA(SiRNA)下调PKCE对血管内皮生长因子刺激的信号转导和细胞功能的几个途径有显著影响，包括一氧化氮的产生和细胞增殖，而在下调PKCA(3或8)后，影响很小或相反。此外，玻璃体内注射PKCE siRNA抑制了血管内皮生长因子刺激的视网膜血管通透性。因此，这一应用的中心假设是，PKCE亚型在介导血管内皮生长因子刺激的Akt、eNOS和AMP激活的蛋白激酶(AMPK)信号转导中具有定量上的主要作用，并且在糖尿病黄斑水肿和增殖性视网膜病变的发生中起关键作用。这些研究的具体目的是：1.研究PKCE在视网膜内皮细胞中激活的机制及其调控的细胞功能；2.确定PKCE基因敲除或过表达对血管通透性的影响；3.基因敲除或过表达PKCE功能对视网膜缺血新生血管的影响。关联性。这项拟议的研究有望对糖尿病患者黄斑水肿和增殖性视网膜病变的预防产生积极影响，因为它将确立信号分子PKCE作为这些过程的关键介质和治疗这些疾病的潜在药物靶点的有用性。

DESCRIPTION (provided by applicant): Project summary. The applicant is a physician-scientist who has completed his post-doctoral fellowship in the laboratory of Dr. George L. King, who will continue to function as a mentor while the applicant expands his training in new fields, including retinal endothelial cell biology and animal models of retinal complications of diabetes. This training will be co-mentored by Dr. Lloyd Paul Aiello at the Beetham Eye Institute at Joslin Diabetes Center. The Career Development Award would allow the applicant to become an independent researcher in the field of retinal disease and help achieve the long-term objective of understanding growth factor actions in diabetic eye complications. In diabetic macular edema and proliferative diabetic retinopathy, upregulation of vascular endothelial growth factor (VEGF) plays a central role. Protein kinase C (PKC) isoforms a, (3, and e are activated in the diabetic retina, but so far there has been a lack of data from animal models with isoform-specific manipulation of PKC function. In preliminary results, downregulation of PKCe with small interfering RNA (siRNA) in endothelial cell culture had a dramatic effect on several pathways of VEGF-stimulated signaling and cell function, including nitric oxide production and cell proliferation, with minor or opposite effects seen after downregulation of PKCa, (3, or 8. Further, intravitreal injection of PKCe siRNA inhibited VEGF-stimulated retinal vascular permeability in rats. Therefore, the central hypothesis of this application is that PKCe isoform has a quantitatively major role in mediating VEGF-stimulated Akt, eNOS, and AMP-activated protein kinase (AMPK) signaling, and is critical for development of macular edema and proliferative retinopathy in diabetes. The specific aims are: i. To characterize mechanisms of activation of PKCe in retinal endothelial cells and cell function regulated by PKCe; 2. To determine the effect of PKCe knockout or overexpression on vascular permeability during VEGF stimulation and in diabetes; 3. the effect of knockout or overexpression of PKCe function on ischemic neovascularization in the retina. Relevance. The proposed research is expected to have a positive impact on the prevention of macular edema and proliferative retinopathy in patients with diabetes because it will establish the usefulness of the signaling molecule PKCe as a key mediator of these processes and a potential drug target for these conditions.