BMP2/ALKs signaling system in diabetic retinopathy

摘要 血-视网膜屏障(BRB)破裂和内皮细胞基底膜增厚 细胞外基质(ECM)沉积是糖尿病视网膜病变(DR)发病机制的早期事件。 目前的治疗方法受到严重副作用的限制，包括ECM沉积。骨形态发生蛋白- 骨形态发生蛋白2(BMP2)是转化生长因子-β超家族的一种分泌型细胞因子，通过激活素受体启动信号传导 与Alk2和Alk3有高亲和力的蛋白激酶(Alk1、Alk2、Alk3和Alk6)。这个项目的目标是检验这一假说 在糖尿病中，BMP2损害BRB并通过依赖内皮细胞Alk2/3诱导ECM形成 机制。我们的假设得到了令人信服的初步发现的支持，即视网膜和循环中的BMP2 在糖尿病人和小鼠以及在人视网膜内皮细胞(HRECs)中表达上调 至高糖(HG)。重要的是，BMP2抑制剂减弱了HG的通透性和ECM沉积效应 在HREC中。此外，BMP2激活了规范和非规范通路(Smad/Runx2和p38 MAPK/NFB)。我们的假设预测，在糖尿病中，BMP2激活Smad和p38 MAPK通路整合在Wnt/-catenin处，诱导高通透性和细胞外基质沉积。我们将测试 1.激活的BMP2/ALKS信号系统对视网膜内皮细胞的作用 DR中的功能障碍：我们将使用以下方法将循环中BMP水平的变化与DR的发生联系起来 从糖尿病表型和基因组中登记的一大批糖尿病患者的血清样本 自身免疫(POODA)研究，并已被前瞻性监测糖尿病的发展 并发症包括最近15年的糖尿病视网膜病变。使用链脲佐菌素诱导的糖尿病小鼠模型，以及 我们将测定BMP2信号系统(BMP2，BMP4， BMP受体、Smad1/5/9、Runx2和BMP2的负调控因子，如noggin和BMP结合的内皮细胞 监管机构(BMPER)。随后，我们将研究药物抑制或基因操作的效果 BMP2/ALKS系统对BRB功能和ECM的影响。我们将使用内皮Alk2/3条件性基因敲除小鼠 在我们实验室和商业上可买到的BMPER缺陷小鼠(BMPER/−)和；2.两者都是典型的 非正则途径导致BMP2介导的视网膜内皮细胞功能障碍：WE 将测试a)在正常情况下，骨形态发生蛋白2/碱性磷酸酶信号对Smad1/5/9和p38/nFB信号通路的影响 或糖尿病情况，b)抑制Smad1/5/9或p38/NFB信号在糖尿病或骨形态发生蛋白2诱导下的作用 通透性和细胞外基质沉积，以及c)Wnt/连环蛋白通路作为潜在下游的作用 P38/NFB和Smad1/5/9通路靶点介导骨形态发生蛋白2诱导的视网膜微血管损伤 这一建议的翻译意义在于抑制BMP2/ALKS信号转导的治疗潜力 改善DR患者的视力结果，最终目标是克服当前的限制因素 预防ECM沉积的治疗方法。

Summary Breakdown of the blood-retinal barrier (BRB) and thickening of the endothelial cell basement membrane due to deposition of extracellular matrix (ECM) are early events in the pathogenesis of diabetic retinopathy (DR). Current treatments are limited by significant side effects, including ECM deposition. Bone morphogenetic protein- 2 (BMP2), a secreted cytokine belonging to the TGF-β superfamily initiates signaling through activin receptors like kinases (Alk1, 2, 3, and 6) with high affinity to Alk2 and 3. The goal of this project is to test the hypothesis that in diabetes, BMP2 compromises BRB and induces ECM formation through the endothelial Alk2/3-dependent mechanism. Our hypothesis is supported by compelling preliminary findings that retinal and circulating BMP2 are upregulated in diabetic humans and mice, as well as in human retinal endothelial cells (HRECs) subjected to high glucose (HG). Importantly, BMP2 inhibitors attenuate the permeability and ECM deposition effect of HG in HRECs. Moreover, BMP2 activates the canonical and non-canonical pathways (smad/Runx2 and p38 MAPK/NFB, respectively) in HRECs. Our hypothesis predicts that in diabetes BMP2 activates smad and p38 MAPK pathways integrate at wnt/-catenin to induce hyperpermeability and ECM deposition. We will test the following specific aims: 1. Activated BMP2/Alks signaling system contributes to retinal endothelial cell dysfunction in DR: We will correlate changes in circulating levels of BMPs to the development of DR using serum samples from a large cohort of diabetic patients enrolled in the Phenome and Genome of Diabetes Autoimmunity (PAGODA) study and have been prospectively monitored for the development of diabetic complications including DR in the last 15 years. Using a streptozotocin-induced diabetic mouse model, and HRECs subjected to HG, we will determine the levels and distribution of BMP2 signaling system (BMP2, BMP4, BMP receptors, smad1/5/9, Runx2, and negative regulators of BMP2 such as noggin and BMP-binding endothelial regulator (BMPER). Subsequently, we will study the effects of pharmacological inhibition or genetic manipulation of the BMP2/Alks system on BRB function, and ECM. We will use endothelial Alk2/3 conditional knockout mice developed within our lab and commercially available BMPER-deficient mice (BMPER+/−) and; 2. Both canonical and non-canonical pathways contribute to BMP2-mediated retinal endothelial cell dysfunction in DR: We will test a) the effect of modulation of BMP2/Alks signaling on the smad1/5/9 and p38/NFB pathways under normal or diabetic conditions, b) the effect of inhibition of smad1/5/9 or p38/NFB signaling on diabetes or BMP2-induced permeability and ECM deposition, and c) the role of the wnt/catenin pathway as a potential downstream target from both p38/NFB and smad1/5/9 pathways to mediate retinal microvascular damage induced by BMP2. The translational significance of this proposal is the therapeutic potential of inhibition of BMP2/Alks signaling to improve the visual outcomes in DR with the ultimate goal of overcoming the limiting factors of current therapies in the prevention of ECM deposition.