DEVELOPING VISION--THE MECHANISM OF TISSUE REMODELING

了解发育程序化毛细管回归的机制是我们的长期目标。作为一个模型系统，我们使用位于眼睛前房的临时毛细血管网络--瞳孔膜(PM)。它的优点是可以通过角膜注射进行操作，从眼睛解剖时很容易进行整体检查，并在出生后退化。我们以前已经证明，(1)毛细血管退缩需要巨噬细胞，(2)巨噬细胞诱导内皮细胞和周细胞程序性死亡以驱动毛细血管退缩，(3)内皮细胞和周细胞死亡是细胞周期G1期接收到的巨噬细胞信号的结果。在目前的应用中，我们将寻求一个广泛的问题，即巨噬细胞如何调节毛细血管回归，目的如下。目的8：确定周细胞是否在调节PM消退中起作用。周细胞被认为具有维持毛细血管的作用，可能是通过营养支持。因此，我们将测试巨噬细胞诱导的周细胞死亡是否是PM回归的一个独特和必要的步骤。目的9：确定巨噬细胞是否独立于诱导细胞凋亡而影响细胞周期。巨噬细胞是内皮细胞有丝分裂原和细胞周期抑制因子的来源。这增加了一种可能性，即它们可能独立于它们在诱导细胞死亡中的作用来调节内皮细胞周期。目的10：确定巨噬细胞诱导的细胞周期依赖性程序性死亡是否需要转化生长因子β信号。转化生长因子β对血管内皮细胞具有促凋亡作用，是公认的细胞周期调节因子。我们已经证明，在沐浴PM的水中有活性的TGFbeta，并将使用多种策略来确定这是否是PCD或细胞周期调节所必需的。目的11：确定巨噬细胞诱导的PCD是否依赖于基质蛋白酶的作用。依赖于细胞周期状态的细胞死亡表明，在细胞周期限制点抑制基本信号可能是原因。我们将研究巨噬细胞产生的基质蛋白酶可能通过降解必要的基质配体而导致PCD的可能性。目前能详细研究毛细管回归机理的系统很少。有了我们开发的方法和本提案中概述的策略，我们有一个极好的机会来了解这一临床上重要的过程。我们所了解到的关于发育程序性毛细血管回归的知识很有可能适用于支持肿瘤生长或某些血管疾病的中心特征的毛细血管。

It is our long-term goal to understand the mechanism of developmentally programmed capillary regression. As a model system we use the pupillary membrane (PM), a transient capillary network situated in the anterior chamber of the eye. It has the advantage that it is accessible to manipulation through trans- corneal injection, is easily examined in whole-mount when dissected from the eye and regresses postnatally. We have previously shown, (1) that macrophages are required for capillary regression, (2) that macrophages induce programmed cell death in endothelial cells and pericytes to drive capillary regression, and (3) that endothelial cells and pericytes die as a consequence of a macrophage signal received in G1-phase of cell cycle. In the current application, we will pursue the broad question of how macrophages mediate capillary regression with the following aims. Aim 8: To determine whether pericytes have a role in regulating PM regression. Pericytes are thought to have a role in maintaining capillaries, perhaps through trophic support. Thus, we will test whether macrophage-induced pericyte death is a distinct and essential step in PM regression. Aim 9: To determine whether macrophages influence cell-cycling independent of the induction of apoptosis. Macrophages are a source of endothelial cell mitogens and suppressors of cell-cycle. This raises the possibility that they may regulate endothelial cell- cycle independent of their role in inducing cell death. Aim 10: To determine whether TGFbeta signaling is required for macrophage-induced cell-cycle dependent programmed cell death. TGFbeta can have a pro-apoptotic activity on endothelial cells and is a recognized regulator of cell-cycle. We have shown that there is active TGFbeta in the aqueous that bathes the PM and will determine, using a multiple strategies, whether this is required for PCD or regulation of cell-cycle. Aim 11: To determine whether macrophage-induced PCD is dependent upon the action of matrix proteases. A cell-cycle state dependent cell death suggests that inhibition of essential signaling at the restriction point of cell-cycle may be the cause. We will investigate the possibility that matrix proteases produced by macrophages may cause PCD by degrading essential matrix ligands. There are few systems where the mechanism of capillary regression can be studied in detail. With the methods we have developed and the strategies outlined in this proposal, we have an excellent opportunity to understand this clinically important process. There is every possibility that what we learn about developmentally programmed capillary regression will be applicable to the capillaries that support the growth of tumors or those that are the central feature of some vascular diseases.