Lymph vs. blood angiogenesis: functional differences

描述(由申请人提供)： 尽管淋巴管生成在肿瘤转移等重要的病理生理过程中很重要，但对淋巴管再生的生理学了解远远少于血管生成的生理学。事实上，人们普遍认为淋巴管生成的许多方面与血管生成的那些方面类似。这项生物工程研究将解决这两个过程之间的差异，使用体外模型以及我们实验室开发的一种新的再生皮肤淋巴管生成的动物模型。我们利用该模型对淋巴管发育的初步观察表明：1)液体通道的形成先于淋巴管内皮细胞(LEC)的组织，2)LEC在组织成血管之前以单个细胞的形式在间质内迁移，3)蛋白酶活性、液体通道、VEGF-C蛋白表达和LEC的迁移都主要发生在上游端，与淋巴流动的方向一致，4)间质流量的减少中断了毛细淋巴管的组织，尽管上皮细胞迁移和血管生成相同。在该模型中获得的这些观察结果表明，淋巴管生成和血管生成之间存在明显和根本的差异，在萌发的血管表面，基质金属蛋白酶的活性允许毛细血管进展，而BEC总是与其邻近的细胞连接并暴露在管腔中，即使在增殖和推进的时候也是如此。这也是淋巴发育受间质血流影响的第一个证据。根据提供的初步数据，我们的工作假设如下：间质液体流动是淋巴管生成的启动事件，通过引起基质金属蛋白酶上调和沿流动方向的运输(“流动”)，导致液体通道的形成和LEC沿这些通道的定向迁移。然后，LEC组织起来，将原油流体通道重塑为功能最优的毛细淋巴管网络。本研究将探索这一假说，并将淋巴管的功能与其形成过程从几个方面联系起来，将淋巴管生成中的生化和生物物理/运输介质联系起来，关注液体通道、细胞外基质(ECM)的重塑、LEC迁移、VEGF-C表达和淋巴管毛细血管网络的形成。将阐明蛋白酶在通道形成中的作用，以及血管内皮生长因子-C的有丝分裂和形态发生功能。在体外，我们将把LEC对液体和生长因子的反应与在体内观察到的淋巴管生成过程中的LEC行为相关联，并将这些结果与体外BEC的反应进行比较。

DESCRIPTION (provided by applicant): The physiology of lymphatic regeneration is much less understood than that of angiogenesis, in spite of the importance of lymphangiogenesis in important pathophysiological processes such as tumor metastasis. Indeed, it is generally assumed that many aspects of lymphangiogenesis parallel those in angiohgenesis. This bioengineering study will address the differences between these two processes, using in vitro models as well as a novel animal model of lymphangiogenesis in regenerating skin, developed in our laboratory. Our preliminary observations of lymphatic development using this model indicate that: 1) fluid channel formation occurs prior to lymphatic endothelial cell (LEC) organization, 2) LECs migrate as single cells within the interstitial space before organizing into vessels, 3) protease activity, fluid channeling, VEGF-C protein expression, and LEC migration all occur predominantly from the upstream end, consistent with the direction of lymph flow, and 4) reduction of interstitial flow interrupted lymphatic capillary organization, despite identical epithelial cell migration and angiogenesis. These observations obtained in this model suggest clear and fundamental differences between lymphangiogenesis and blood angiogenesis, where MMP activity at the surface of the sprouting vessel allow capillary progression and where BECs are always connected to their neighboring cells and exposed to a lumen, even while proliferating and advancing. They also represent the first evidence that lymphatic development is influenced by interstitial flow. Based upon preliminary data presented, our working hypothesis is as follows: Interstitial fluid flow is an initiating event in lymphangiogenesis by causing MMP upregulation and transport ("streaming") in the direction of flow, leading to fluid channel formation and directed LEC migration along those channels. The LECs then organize to remodel the crude fluid channels into a functionally optimal network of lymphatic capillaries. This investigation will explore this hypothesis and connect the function of the lymphatics to the process of their formation in several regards, correlating biochemical and biophysical/transport mediators in lymphangiogenesis, with attention to fluid channeling, remodeling of the extracellular matrix (ECM), LEC migration, VEGF-C expression, and lymphatic capillary network formation. The role of protease streaming in channel formation will be clarified, as will the mitogenic vs. morphogenetic function of VEGF-C. In vitro, we will correlate LEC responses to fluid and growth factors with LEC behavior observed in vivo during lymphangiogenesis, and we will compare those results to BEC responses in vitro.