title: HIV Nef protein causes endothelial dysfunction in porcine pulmonary arteries and human pulmonary artery endothelial cells.
abstract: BACKGROUND
  Infection of human immunodeficiency virus (HIV) has been associated with several chronic diseases, including pulmonary artery hypertension and atherosclerosis. However, the underlying mechanisms of these vascular complications are largely unknown. The objective of this study was to test a novel hypothesis that HIV Nef, an accessory HIV protein, may directly affect endothelial functions and gene expression in pulmonary arteries.
  
  
  METHODS
  Fresh porcine pulmonary artery rings and human pulmonary artery endothelial cells (HPAECs) were treated with HIV Nef for 24 h. With a myograph device, vasomotor function was determined with thromboxane A2 analog, U46619, for contraction, bradykinin, and sodium nitroprusside for relaxation. The expression of endothelial nitric oxide synthase (eNOS) was determined with real-time PCR and immunohistochemistry. Nitric oxide (NO) production was determined by Calorimetric Nitric Oxide Assay kit. Superoxide anion levels were detected with lucigenin-enhanced chemiluminescence assay and dihydroethidium (DHE) staining.
  
  
  RESULTS
  The endothelium-dependent vasorelaxation in response to bradykinin was significantly reduced in HIV Nef-treated porcine pulmonary artery rings in a concentration-dependent manner. In response to bradykinin (10(-8) mol/L), HIV Nef (10 ng/mL) significantly reduced vasorelaxation by 32% compared with untreated controls (P < 0.05). In addition, HIV Nef significantly decreased eNOS expression in the vessels and HPAECs. HIV Nef at 10 ng/mL significantly decreased NO production in HPAECs by 21% compared with controls (P < 0.05). Furthermore, HIV Nef significantly increased superoxide anion production in porcine pulmonary arteries and HPAECs compared with controls (P < 0.05). Consequently, Mn (III) tetrakis porphyrin, a superoxide dismutase mimic, effectively blocked HIV Nef-induced vasomotor dysfunction and superoxide anion production. The specificity of HIV Nef action was confirmed by anti-Nef antibody blocking and Nef heat inactivation.
  
  
  CONCLUSIONS
  HIV Nef protein significantly decreases endothelium-dependent vasorelaxation in porcine pulmonary arteries. It also reduces eNOS expression and induces oxidative stress in both porcine pulmonary arteries and HPAECs. This study demonstrates a new mechanism of HIV Nef, which causes endothelial dysfunction and may contribute to the human pulmonary artery disease in HIV-infected patients.
authors.affiliation: Molecular Surgeon Research Center, Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Michael E. DeBakey VA Medical Center, Houston, Texas 77030, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Duffy
authors.forename: Xinwen
authors.initials: X
authors.lastname: Wang
authors.forename: Peter H
authors.initials: PH
authors.lastname: Lin
authors.forename: Qizhi
authors.initials: Q
authors.lastname: Yao
authors.forename: Changyi
authors.initials: C
authors.lastname: Chen
mesh_terms: D000818:Animals; D042783:Endothelial Cells / Q000187:drug effects*; D004730:Endothelium, Vascular / Q000187:drug effects*; D015870:Gene Expression; D006801:Humans; D023421:Models, Animal; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D018384:Oxidative Stress / Q000187:drug effects; D011651:Pulmonary Artery / Q000187:drug effects*; D013481:Superoxides / Q000378:metabolism; D013552:Swine; D014652:Vascular Diseases / Q000503:physiopathology*; D014664:Vasodilation / Q000187:drug effects; D014666:Vasomotor System / Q000187:drug effects; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000494:pharmacology*
chemical_list: D054311:nef Gene Products, Human Immunodeficiency Virus; D013481:Superoxides; D019001:Nitric Oxide Synthase