Functional Studies of HIV-Tat Effects on Endothelium

描述(申请人提供)：最初的建议集中在HIV-1TAT与大血管、脐静脉和骨髓内皮细胞的相互作用。虽然在实现这些目标方面取得了进展，但很明显，HIV-1TAT的病理生理学是在微血管内皮细胞，特别是淋巴源的微血管内皮细胞。这是因为艾滋病的主要内皮细胞改变是在卡波西肉瘤(KS)的起源中发生的，KS的起源细胞是淋巴管。因此，我们将工作的重点转移到富含淋巴源性细胞的真皮微血管内皮细胞(DMEC)上。另一个焦点转移到了KSHV/HHV8。这种伽玛疱疹病毒似乎是KS的关键病原体。其包膜糖蛋白Gb与DMEC表面的α3beta1整合素结合。为了建立更接近于HIV-1和KSHV/HHV8混合感染体内致病机制的体外系统，我们开始研究Tat和GB对DMEC的影响。我们发现，淋巴管内皮细胞中的标志性血管内皮生长因子受体VEGFR3/Flt4与α3β1整合素连接相关，并且在缺乏同源的VEGF配体的情况下被Gb或TAT激活。此外，GB或TAT通过VEGFR3诱导DMEC迁移和增殖。这些关于α3beta1整合素与VEGFR3关系的数据表明，VEGFR3可能是KSHV的辅助受体，或者其信号转导促进了KSHV的进入。新的特异性目标解决了TAT和GB可能在改变内皮细胞功能和KSHV感染方面具有协同作用的假设：(1)进一步研究KSHV GB激活α3beta1整合素如何激活VEGFR3；(2)研究VEGFR3是否参与KSHV进入微血管内皮细胞；以及(3)表征HIV-1 Tat和KSHV GB之间功能相关的协同作用。这些研究旨在提供有关艾滋病合并感染宿主的新信息，从而为制定预防或治疗KS的战略提供基础。

DESCRIPTION (provided by applicant): The initial proposal focused on interactions of HIV-1 tat with large vessel umbilical vein and marrow endothelium. While progress was made in fulfilling the aims, it became clear that the pathophysiology of HIV-1 tat was in microvascular endothelium, particularly that of lymphatic origin. This was because the primary endothelial alteration in AIDS is in the genesis of Kaposi's sarcoma (KS), whose cell of origin is lymphatic. Thus, we shifted the focus of our work to dermal microvascular endothelium (DMEC) that are enriched with cells of lymphatic origin. The other shift in focus was to KSHV/HHV8. This gamma herpes virus appears to be the key etiological agent in KS. Its envelope glycoprotein, gB, binds to the alpha3beta1 integrin on the surface of DMEC. To develop in vitro systems that more closely model in vivo pathogenesis in coinfection with HIV-1 and KSHV/HHV8, we began to study the effects of tat and gB on DMEC. We found that VEGFR3/FLT4, the signature VEGF receptor in lymphatic endothelium, associated with, and was activated upon, alpha3beta1 integrin ligation by gB or tat in the absence of cognate VEGF ligands. Furthermore, gB or tat caused DMEC migration and proliferation via VEGFR3. These data on the association of the alpha3beta1 integrin with VEGFR3 suggested that VEGFR3 may either act as a coreceptor for KSHV or that its signaling facilitates KSHV entry. The new specific aims address the hypothesis that tat and gB may have cooperative effects on altering endothelial cell function and KSHV infection: (1) to further characterize how alpha3beta1 integrin activation by KSHV gB can activate VEGFR3; (2) to investigate whether VEGFR3 is involved in entry of KSHV into microvascular endothelial cells; and (3) to characterize functionally relevant cooperative interactions of HIV- 1 tat and KSHV gB. These studies are designed to provide new information on the coinfected host with AIDS and thereby provide a foundation for the design of preventive or therapeutic strategies against KS.