Novel Mechanism of Retinal Capillary Expansion

在美国，糖尿病视网膜病变（DR）是导致失明的主要原因。增生性视网膜病变（PDR）涉及严重的炎症和血管生成反应，导致视网膜变性。血管损伤之后是血管新生的爆发，迅速形成一个更复杂的毛细血管网络，其中有渗漏和脆弱的血管。我们在视网膜内皮细胞（REC）的基质培养中发现了类似的过程。众所周知，在基质上的内皮细胞1)停止增殖，2)在24-48小时内形成毛细血管样管，3)不侵入基质，4)塌陷成团块，5)死亡。因为这被认为是实验的终点，所以没有实验超出这一点。然而，我们报告的发现自发细胞存活后，管塌陷。这些存活的细胞1)增殖，2)迁移，3)形成球形菌落，存活数周，4)侵入基质，5)可以重新组装成更大的管状细胞。我们将这种新现象命名为“二次发芽”。由于存活菌落的血管生成表型与初始细胞明显不同，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继发发芽。因此，来自糖尿病和糖尿病视网膜病变REC的培养物应该比来自正常患者的培养物表现出更高的发芽能力。具体目标# 1：确定“崩溃前和崩溃后”的细胞外基质是否不同，以及这些改变是否会诱导“崩溃后”菌落的发芽表型。具体目标2：确定“崩溃前和崩溃后”的细胞是否不同，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；如果这些变化是永久性的，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模拟是否能增强REC的二次发芽能力。具体目标4：确定NL、DM和DR患者衍生的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。参与二次发芽的分子的发现和调控可以为PDR和其他与新生血管相关的疾病提供新的有效治疗方法。

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.