Novel Mechanism of Retinal Capillary Expansion

视网膜毛细血管扩张的新机制

• 批准号: 6651487
• 负责人: RAQUEL CASTELLON
• 金额: $ 15.15万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6651487
• 关键词: SDS polyacrylamide gel electrophoresis; angiogenesis; capillary bed; cell growth regulation; cell population study; cellular pathology; clinical research; diabetic retinopathy; extracellular matrix; extracellular matrix proteins; growth factor; human tissue; matrigel; medical complication; metalloendopeptidases; organ culture; phenotype; protein structure function; retina; western blottings

Diabetic retinopathy (DR) is a leading cause of blindness in the U.S. The proliferative type (PDR) involves severe inflammatory and angiogenic responses resulting in retinal degeneration. Vascular damage is followed by a burst of angiogenesis that rapidly creates an even more complex capillary network of leaky and fragile vessels. We have detected a similar process in Matrigel cultures of retinal endothelial cells (REC). It is well known that endothelial cells plated on this matrix 1) stop proliferating, 2) form capillary-like tubes for 24-48 hr, 3) do not invade the matrix, 4) collapse into clumps, and 5) die. Because this was thought to be the endpoint of the assay, no experiments extended beyond this point. However, we report the discovery of spontaneous cell survival following tube collapse. These surviving cells 1) proliferate, 2) migrate, 3) form spherical colonies that remain alive for weeks, 4) invade the matrix, and 5) can reassemble into larger tubes. We have named this novel phenomenon "secondary sprouting". Since the angiogenic phenotype of surviving colonies is markedly different from the initial cells, we hypothesize that these behaviors are caused by changes in the cells themselves and/or in the surrounding matrix. If diabetes induced cell injury promotes the sprouting phenotype, simulating such injuries in vitro should increase secondary sprouting. Consequently, cultures from diabetic and diabetic retinopathy REC should exhibit a higher sprouting capacity than those from normal patients. Specific aim #l: Determine if the "pre- and post-collapse" extracellular matrices are different and whether these alterations induce the sprouting phenotype of "post-collapse" colonies. Specific aim #2: To determine if the "pre- and post-collapse" cells are different and whether these differences are temporary or permanent. If changes are temporary, we will examine whether growth factor and metalloproteinase production results in the sprouting phenotype; if the changes are permanent, we will determine if they are due to terminal differentiation of the whole cell population or to selection of a specific subpopulation. Specific aim #3: Investigate whether simulation of diabetic cellular injuries enhances the secondary sprouting ability of REC. Specific aim #4: Determine if REC derived from NL, DM and DR patients have differential abilities to form secondary sprouts. These experiments could explain how cellular injury induces blood vessel regression as well as the subsequent expansion of the capillary bed. The discovery and modulation of molecules involved in secondary sprouting could provide novel and effective therapeutics for PDR and other conditions associated with neovascularization.

糖尿病视网膜病变（DR）是美国失明的主要原因。增殖型（PDR）涉及导致视网膜变性的严重炎症和血管生成反应。血管损伤之后是血管生成的爆发，其迅速产生甚至更复杂的渗漏和脆弱血管的毛细血管网络。我们已经在视网膜内皮细胞（REC）的基质胶培养物中检测到类似的过程。众所周知，铺在该基质上的内皮细胞1）停止增殖，2）形成毛细血管样管24-48小时，3）不侵入基质，4）塌陷成团块，和5）死亡。因为这被认为是试验的终点，所以没有实验延伸超过该点。然而，我们报告的发现自发细胞存活管崩溃。这些存活的细胞1）增殖，2）迁移，3）形成球形集落，保持存活数周，4）侵入基质，5）可以重新组装成更大的管。我们将这种新现象命名为“二次发芽”。由于存活集落的血管生成表型与初始细胞显着不同，我们假设这些行为是由细胞本身和/或周围基质的变化引起的。如果糖尿病诱导的细胞损伤促进了发芽表型，那么在体外模拟这种损伤应该会增加二次发芽。因此，糖尿病和糖尿病视网膜病变REC的培养物应表现出比正常患者更高的发芽能力。具体目标1：确定“塌陷前”和“塌陷后”细胞外基质是否不同，以及这些改变是否诱导“塌陷后”菌落的发芽表型。具体目标#2：确定“塌陷前”和“塌陷后”细胞是否不同，以及这些差异是暂时的还是永久的。如果变化是暂时的，我们将检查生长因子和金属蛋白酶的产生是否导致发芽表型;如果变化是永久性的，我们将确定它们是由于整个细胞群的终末分化还是由于特定亚群的选择。具体目标3：研究糖尿病细胞损伤的模拟是否增强REC的二次发芽能力。具体目标#4：确定来自NL、DM和DR患者的REC是否具有形成二次发芽的不同能力。这些实验可以解释细胞损伤如何诱导血管退化以及随后的毛细血管床扩张。发现和调节参与二次发芽的分子可以为PDR和其他与新血管形成相关的疾病提供新的有效治疗方法。