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。增殖型(PDR)包括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生成的爆发，迅速形成了一个由泄漏和脆弱的血管组成的更复杂的毛细血管网络。我们在视网膜内皮细胞(REC)的Matrigel培养中检测到了类似的过程。众所周知，种植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)坍塌成团，5)死亡。因为这被认为是化验的终点，所以没有实验超出这一点。然而，我们报告了在输卵管坍塌后自发细胞存活的发现。这些存活的细胞1)增殖，2)迁移，3)形成可存活数周的球形菌落，4)侵入基质，5)重新组装成更大的管。我们将这种新现象命名为“二次发芽”。由于存活的克隆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。如果糖尿病引起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，来自糖尿病和糖尿病视网膜病变REC的培养应该比来自正常患者的培养具有更高的发芽能力。具体目标#L：确定崩溃前和崩溃后的细胞外基质是否不同，以及这些变化是否会导致“崩溃后”菌落的萌发表型。具体目标2：确定“崩溃前和崩溃后”的细胞是否不同，以及这些差异是暂时的还是永久性的。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体的终末分化还是由于对特定亚群的选择。具体目的#3：研究模拟糖尿病细胞损伤是否能增强REC的二次发芽能力。具体目标#4：确定NL、DM和DR患者来源的REC是否具有不同的形成继发芽的能力。这些实验可以解释细胞损伤如何导致血管退化以及随后毛细血管床的扩张。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的治疗方法。

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.