Navarro Angels, Perez Ricardo E, Rezaiekhaligh Mo, Mabry Sherry M, Ekekezie Ikechukwu I
Section of Neonatology, Children's Mercy Hospitals & Clinics, Kansas City, MO 64108, USA.
Am J Physiol Lung Cell Mol Physiol. 2008 Oct;295(4):L543-51. doi: 10.1152/ajplung.90262.2008. Epub 2008 Jul 25.
The lymphatic vasculature functions to maintain tissue perfusion homeostasis. Defects in its formation or disruption of the vessels result in lymphedema, the effective treatment of which is hampered by limited understanding of factors regulating lymph vessel formation. Mice lacking T1alpha/podoplanin, a lymphatic endothelial cell transmembrane protein, have malformed lymphatic vasculature with lymphedema at birth, but the molecular mechanism for this phenotype is unknown. Here, we show, using primary human lung microvascular lymphatic endothelial cells (HMVEC-LLy), that small interfering RNA-mediated silence of podoplanin gene expression has the dramatic effect of blocking capillary tube formation in Matrigel. In addition, localization of phosphorylated ezrin/radixin/moesin proteins to plasma membrane extensions, an early event in the capillary morphogenic program in lymphatic endothelial cells, is impaired. We find that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA early (by 30 min) after plating on Matrigel, and Rac1 shows a delay in its activation. Further indication that podoplanin action is linked to RhoA activation is that use of a cell-permeable inhibitor of Rho inhibited lymphatic endothelial capillary tube formation in the same manner as did podoplanin gene silencing, which was not mimicked by treatment with a Rac1 inhibitor. These data clearly demonstrate that early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression. To our knowledge, this is the first time that a mechanism has been suggested to explain the role of podoplanin in lymphangiogenesis.
淋巴管系统的功能是维持组织灌注稳态。其形成缺陷或血管破坏会导致淋巴水肿，由于对调节淋巴管形成的因素了解有限，其有效治疗受到阻碍。缺乏T1α/血小板反应蛋白（一种淋巴管内皮细胞跨膜蛋白）的小鼠，出生时淋巴管系统畸形并伴有淋巴水肿，但其这种表型的分子机制尚不清楚。在此，我们使用原代人肺微血管淋巴管内皮细胞（HMVEC-LLy）表明，小干扰RNA介导的血小板反应蛋白基因表达沉默对基质胶中毛细血管管形成具有显著的阻断作用。此外，磷酸化埃兹蛋白/根蛋白/膜突蛋白在质膜延伸部位的定位（这是淋巴管内皮细胞毛细血管形态发生程序中的早期事件）受到损害。我们发现，血小板反应蛋白表达降低的细胞在接种到基质胶上后早期（30分钟）不能正确激活小GTP酶RhoA，而Rac1的激活出现延迟。进一步表明血小板反应蛋白的作用与RhoA激活相关的是，使用一种细胞可渗透的Rho抑制剂抑制淋巴管内皮毛细血管管形成的方式与血小板反应蛋白基因沉默相同，而Rac1抑制剂处理则不能模拟这种效果。这些数据清楚地表明，淋巴管生成过程中RhoA的早期激活（这对于毛细血管网络的成功建立是必需的）依赖于血小板反应蛋白的表达。据我们所知，这是首次提出一种机制来解释血小板反应蛋白在淋巴管生成中的作用。
