Differential Effects of Human Immunodeficiency Virus Nef Variants on Pulmonary Vascular Endothelial Cell Dysfunction

<b>Background:</b> Human Immunodeficiency Virus (HIV) infections remain a source of cardiopulmonary complications among people receiving antiretroviral therapy. Still to this day, pulmonary hypertension (PH) severely affects the prognosis in this patient population. The persistent expression of HIV proteins, even during viral suppression, has been implicated in vascular dysfunction; however, little is known about the specific effects of these proteins on the pulmonary vasculature. This study investigates the impact of Nef variants derived from HIV-positive pulmonary hypertensive and normotensive donors on pulmonary vascular cells in vitro. <b>Methods:</b> We utilized well-characterized Nef molecular constructs to examine their effects on cell adhesion molecule gene expression (<i>ICAM1</i>, <i>VCAM1</i>, and <i>SELE</i>), pro-apoptotic gene expression (<i>BAX</i>, <i>BAK</i>), and vasoconstrictive endothelin-1 (<i>EDN1</i>) gene expression in endothelial nitric oxide synthase (eNOS) nitric oxide and the production and secretion of pro-inflammatory cytokines over 24, 48, and 72 h post-transfections with Nef variants. <b>Results:</b> HIV Nef variants SF2, NA7, and PH-associated Fr17 and 3236 induced a significant increase in adhesion molecule gene expression of <i>ICAM1</i>, <i>VCAM1</i>, and <i>SELE</i>. Pulmonary normotensive Nef 1138 decreased <i>ICAM1</i> gene expression, but had increased <i>VCAM1</i>. PH Nef ItVR showed a consistent decrease in <i>ICAM1</i> and no changes in <i>SELE</i> and <i>VCAM1</i> expression. Further gene expression analyses of pro-apoptotic genes <i>BAX</i> and <i>BAK</i> demonstrated that Nef NA7, SF2, normotensive Nef 1138, and PH Nef Fr8, Fr9, Fr17, and 3236 variants significantly increased gene expression for apoptosis. Normotensive Nef 1138, as well as PH Nef Fr9 and ItVR, all displayed a statistically significant decrease in <i>BAX</i> expression. The expression of <i>EDN1</i> had a statistically significant increase in samples treated with Nef NA7, SF2, normotensive Nef 2044 and PH Nef 3236, Fr17, and Fr8. Notably, PH-associated Nef variants sustained pro-inflammatory cytokine production, including IL-2, IL-4, and TNFα, while anti-inflammatory cytokine levels remained insufficient. Furthermore, eNOS was transiently upregulated by all Nef variants except for normotensive Nef 2044. <b>Conclusions:</b> The distinct effects of Nef variants on pulmonary vascular cell biology highlight the complex interplay between Nef, host factors, and vascular pathogenesis according to the variants.