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 _,herpes virus appears to be the key etiological agent in KS. Its envelope glycoprotein, gB, binds to the _x3131integrin 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, _3_1 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 c_3131integrin 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 et3[31 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.
