The primary objective of the proposed research is to investigate the molecular mechanism/s involved in highly active anti-retroviral therapy (HAART)-induced dysfunctions in endothelial cells (ECs). We have shown that HIV-1 protease inhibitors (PIs) suppress adipogenic differentiation and that the inflammatory cytokine TNFalpha and HIV-1 Tat protein affect the clonogenic ability and differentiation potential of the endothelial precursors, mesenchymal progenitor cells (MPCs). We have also shown that at therapeutic concentrations, HAART (zidovudine, efavirenz, and indinavir or nelfinavir) exposure of human aortic ECs (HAECs) suppressed insulin induced NO production and eNOS gene expression, which was further decreased by TNFalpha HAART increased leukocyte/monocyte adhesion and induced cell adhesion molecule (CAM) gene expression, which was further enhanced in the presence of TNFalpha. We are therefore proposing to test the hypothesis that chronic exposure to HAART drugs alters the action of insulin in regulating NO production by the vascular endothelial cells which is further inhibited by TNFalpha and HIV-1 Tat protein. To test this hypothesis the following specific aims will be achieved: (1) To determine the effects of HAART and TNFalpha on endothelial-leukocyte adhesion and migration. HUVECs and HAECs will be exposed to varying concentrations of anti-retroviral drugs as a function of time in the presence or absence of TNFalpha and/or Tat protein. The changes in leukocyte/monocyte adhesion to ECs and trans-endothelial migration (TEM) will be determined. Expression of cell adhesion molecules (V-CAM, I-CAM and E-selectin), and activation of metalloproteases (MMPs) in ECs will be determined by RTPCR and ELISA, and zymography, respectively. The role of CAM specific neutralizing antibodies and/or MMP inhibitors will be determined. (2) To determine the effects of HAART on proliferation and differentiation of marrow derived MPCs. Cells will be exposed to single drugs and/or combinations of HAART, TNFalpha and HIV-1 Tat protein and differentiation towards endothelial lineage will be monitored. Effects on leukocyte adhesion and NO production in differentiating MPCs will be determined. (3) To delineate the molecular mechanisms of HAART induced endothelial dysfunction. We will test the hypothesis that HAART drugs, individually or in combination abrogate insulin signaling thus decreasing NO production and increasing oxidative stress in the ECs. Effect of HAART drugs on insulin signaling will be determined by immunolabeling of insulin receptor substrate (IRS-1) phosphorylation and by transient transfection and gel mobility shift assays for transcription factors, NF-kappaB and AP-1 and reversal of HAART induced leukocyte adhesion with insulin sensitizers will be determined. Effect of HAART drugs on NO and ROS production in ECs will be monitored in the presence or absence of NO-donors, NO synthase inhibitor, free radical quencher and insulin sensitizers in overcoming the HAART and TNFalpha induced EC dysfunctions will be determined. (4) To determine the in vivo effects of chronic HAART exposure on EC dysfunction in mice. To test the proposed hypotheses in vivo, three different groups of mice will be used (a) control (b) TNFalpha receptor knockout and (c) Tat transgenic. EC dysfunction, hyperinsulinemia and hyperglycemia in each group of mice will be determined with or without the administration of HAART and/or NO donor or NOS inhibitor. These studies should provide evidence in support of our hypotheses that disruption of NO production by HAART results in endothelial dysfunction, which is further exacerbated in the presence of TNFalpha and/or Tat protein.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL073691-04
Application #
7086954
Study Section
AIDS and Related Research 8 (AARR)
Program Officer
Mcdonald, Cheryl
Project Start
2003-07-01
Project End
2008-06-30
Budget Start
2006-07-14
Budget End
2008-06-30
Support Year
4
Fiscal Year
2006
Total Cost
$340,938
Indirect Cost
Name
Tulane University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
053785812
City
New Orleans
State
LA
Country
United States
Zip Code
70118
Chandra, Surabhi; Mondal, Debasis; Agrawal, Krishna C (2009) HIV-1 protease inhibitor induced oxidative stress suppresses glucose stimulated insulin release: protection with thymoquinone. Exp Biol Med (Maywood) 234:442-53
Chandra, Surabhi; Murthy, Subramanyam N; Mondal, Debasis et al. (2009) Therapeutic effects of Nigella sativa on chronic HAART-induced hyperinsulinemia in rats. Can J Physiol Pharmacol 87:300-9
Pradhan, Leena; Mondal, Debasis; Chandra, Surabhi et al. (2008) Molecular analysis of cocaine-induced endothelial dysfunction: role of endothelin-1 and nitric oxide. Cardiovasc Toxicol 8:161-71
Mondal, Debasis; Pradhan, Leena; Ali, Mussa et al. (2004) HAART drugs induce oxidative stress in human endothelial cells and increase endothelial recruitment of mononuclear cells: exacerbation by inflammatory cytokines and amelioration by antioxidants. Cardiovasc Toxicol 4:287-302