**2.7. Animal models in HIV-1 endothelial dysfunction**

**2.6. HIV-1 regulatory proteins**

354 Endothelial Dysfunction - Old Concepts and New Challenges

MAPK/ERK signaling pathway and caspase-3 [58].

with EC growth, migration, and angiogenesis [59, 60].

HIV-1 Tat protein is a trans-activating regulatory protein, which is essential for efficient transcription of the viral genome. Tat is a proto-cytokine promoting several disease conditions by

The HIV-1 viral protein Nef is a 27-kD myristoylated protein. It is not secreted by infected cells, but its interaction with membrane and host cell proteins is crucial to sustain its biological activity. Nef protein is involved in different intracellular functions including alteration of protein trafficking, cell signaling cascades, and inhibition of antibody maturation in B cells [49]. Nef is able to enhance HIV-1 infectivity by promoting the formation of nanotubes connecting HIV-1-infected cells to bystander cells [50]. In particular, transfer of Nef from a HIV-1-infected target cell to ECs through nanotubes supports EC activation, dysfunction, and death [51].

Similarly to many potent angiogenic growth factors such as vascular endothelial growth factor (VEGF) A, Tat has a basic domain rich in arginine and lysine residues that endows the viral protein of a potent and direct angiogenic activity [52, 53]. On the contrary, Nef contains multiple domains capable of interacting with the endocytic cellular machinery [54]. Tat and Nef are both capable of inducing apoptosis in ECs. Many studies demonstrate that Nef is able to induce and activate NADPH oxidase that drives ECs to go for apoptosis. Indeed, by significantly decreasing NO production and increasing superoxide anion production, Nef contributes to reactive oxygen species (ROS) production, cell oxidative stress, and cell death [55, 56]. Moreover, Nef was also found to potently induce EC apoptosis by activation of caspases [57]. Tat causes apoptotic death of ECs via either TNF-α secretion or through activation of the Fasdependent pathway. Additionally, Tat is able to promote apoptosis in ECs by activating the

In contrast to its proapoptotic effect, Tat may also exert an angiogenic activity through a multi-signaling-dependent pathway. Angiogenic activity promoted by Tat depends on binding and activation of the Flk-1/kinase insert domain receptor (Flk-1/KDR), a VEGF-A tyrosine kinase receptor, and on binding to integrin αvβ5 receptor and heparan sulfate proteoglycans. Tat interaction with cellular receptors leads to the activation of signaling pathways associated

Similarly to the HIV-1 structural protein p17, both Tat and Nef proteins trigger immune cells activation and inflammation. In fact, Tat promotes transmigration of monocytes through the endothelial barrier and inflammation by inducing ECs to express adhesion molecules as E-selectin, ICAM-1, VCAM-1, and ELAM-1 and to release IL-6 [61, 62]. Tat-induced EC activation is likely aimed to facilitate interaction of inflammatory cells with ECs and promote MCP-1 secretion by activation of PKC signaling pathway [63]. At the same time, Nef protein contributes to inflammation increasing the endothelial MCP-1 production through activation of the NF-kB signaling pathway [50]. It is worth noting that this activity is also promoted by the HIV-1 structural protein p17, following activation of the AP-1 signaling pathway [32] highlighting a remarkable redundancy in the biological activity of structural and regulatory proteins. Interestingly, it has been recently shown that Nef is also involved in the alteration of

modulating the function of immune cells, mesenchymal cells, and ECs [47, 48].

Although many improvements have been made in the development of animal models to study HIV-1-associated endothelial dysfunction, these models do not completely reproduce the pathophysiological features of endothelial dysfunction in humans.

A model of transgenic mice partially reproduces, but below expectations, the features of endothelial dysfunction observed during HIV-1 infection in humans [66]. Indeed, HIV-1-infected mice develop an adventitial mixed inflammatory cell migration, medial hypocellularity, and intimal hyperplasia following smooth muscle infiltration with sparing of the ECs. Furthermore, viral components are observed in smooth muscle cells, which in some instances proliferate in the absence of inflammation, remarking the conceptual principles of viral invasion [66]. The model of macaque species infected with the simian immunodeficiency virus (SIV) shares many more similarities than the transgenic mouse model, in term of disease, with HIV-1 infection and vascular diseases in humans. In an animal model based on macaques infected with a chimeric viral construct containing the HIV-1 Nef gene in a SIV backbone (SHIV-1-nef), the presence of complex vascular lesions has been demonstrated that are not evident in SIV-infected animals [67]. These findings seem to highlight a possible role of HIV-1 Nef in endothelial dysfunction leading to severe arterial disease. Interestingly, vascular alterations, subendothelial infiltration of immune cells, and significantly reduced levels of NO have been found in a model of Rhesus macaques infected by SIV and SHIV-1 [68].

Vasculogenic activity of p17 has been recently demonstrated using ex vivo and in vivo model [40–42]. The ex vivo rat aortic ring assay showed that p17 was able to promote vasculogenesis as potent as that observed using VEGF-A [40]. Similar results were obtained in the in vivo chick chorioallantoic membrane (CAM) assay, which highlighted the capability of p17 to generate allantoic neovessels as compared to control CAMs [40]. Matrigel plug assay has been used to test the lymphangiogenic activity of p17 in mice. Matrigel plugs containing the viral matrix protein were implanted into the dorsal subcutaneous tissue of C57BL/6 mice and after 10 days from the injection; matrigel plugs were immunostained with polyclonal antibody to lymphatic vessel endothelial receptor-1 (LYVE-1) identifying pronounced lymphatic vessel formation in p17-treated mice, compared to controls [42]. Interestingly, matrigel plugs containing a p17 variant derived from an Ugandan clade A1, named S75X and endowed with B cell growth-promoting activity, showed the presence of adipocyte infiltration observed at the histological level, thus suggesting that at least some p17 variants may trigger a possible interplay between angiogenesis, lymphangiogenesis, and adipogenesis [41].
