Lymphangiogenesis or formation of new lymphatic vessels, is a tightly regulated process that is controlled by growth factor signaling and biomechanical cues. Lymphatic endothelial cells (LECs) undergo remodeling, migration and proliferation to invade the surrounding extracellular matrix (ECM) during both physiological and pathological lymphangiogenesis. This study optimized conditions for an in vitro 3D collagen-based model that induced LEC invasion and recapitulated physiological formation of lymphatic capillaries with lumens. Invasion of LECs was enhanced in the presence of Sphingosine 1-phosphate (S1P). Effects of various known lymphangiogenic factors, vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (bFGF), interleukin 8 (IL-8), and hepatocyte growth factor (HGF) were tested on LEC sprout formation synergistically with VEGF-C. Several of these growth factors significantly enhanced LEC invasion while synergistic effects of some of these further enhanced the sprouting density and lumen volume. To determine the contribution of specific ECM components, we analyzed the expression of different integrin subunits. Basal expression of the integrin α5 and integrin β1 subunits were high in LECs. The addition of fibronectin, which mediates cellular responses through these integrins, enhanced LEC sprouting density and sprout length dose-dependently. siRNA mediated knockdown of the integrin β1 subunit suppressed LEC invasion. Further, exposing LECs to the inflammatory mediator, LPS inhibited sprouting. This optimized model for LEC invasion, includes S1P, VEGF-C and fibronectin within a 3D collagen matrix, along with VEGF-C, VEGF-A, bFGF and HGF in the culture medium, providing a useful tool to investigate the effect of various lymphangiogenic factors and inhibitors.

中文翻译：

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在优化的3D胶原蛋白基质模型中，β1整合素在淋巴管内皮细胞侵袭中起关键作用

淋巴管生成或新的淋巴管的形成是受生长因子信号传导和生物力学提示控制的严格调控的过程。淋巴管内皮细胞（LECs）在生理和病理性淋巴管生成过程中都会发生重塑，迁移和增殖，从而侵入周围的细胞外基质（ECM）。这项研究优化了体外基于3D胶原蛋白的模型的条件，该模型可诱导LEC侵袭并概述具有内腔的淋巴毛细血管的生理形成。在1-磷酸鞘氨醇（S1P）的存在下，LEC的入侵得以增强。各种已知的淋巴管生成因子，血管内皮生长因子A（VEGF-A），碱性成纤维细胞生长因子（bFGF），白介素8（IL-8）的作用，肝细胞生长因子（HGF）和肝细胞生长因子（HGF）与VEGF-C协同作用对LEC发芽形成进行测试。这些生长因子中的几个显着增强了LEC侵袭，而其中一些的协同作用进一步增强了发芽密度和管腔体积。为了确定特定ECM组件的贡献，我们分析了不同整合素亚基的表达。LECs中整合素α5和整合素β1亚基的基础表达较高。纤连蛋白的添加通过这些整合素介导细胞反应，从而增加了LEC的发芽密度和发芽长度呈剂量依赖性。siRNA介导的整合素β1亚基的抑制抑制了LEC的侵袭。此外，将LECs暴露于炎症介质中，LPS抑制了发芽。这种针对LEC入侵的优化模型，包括S1P，