HIV antiretroviral drug combination induces endothelial mitochondrial dysfunction and reactive oxygen species production, but not apoptosis.
Numerous reports now indicate that HIV patients administered long-term antiretroviral therapy (ART) are at a greater risk for developing cardiovascular diseases. Endothelial dysfunction is an initiating event in atherogenesis and may contribute to HIV-associated atherosclerosis. We previously reported that ART induces direct endothelial dysfunction in rodents. In vitro treatment of human umbilical vein endothelial cells (HUVEC) with ART indicated endothelial mitochondrial dysfunction and a significant increase in the production of reactive oxygen species (ROS). In this study, we determined whether ART-induced endothelial dysfunction is mediated via mitochondria-derived ROS and whether this mitochondrial injury culminates in endothelial cell apoptosis. Two major components of ART combination therapy, a nucleoside reverse transcriptase inhibitor and a protease inhibitor, were tested, using AZT and indinavir as representatives for each. Microscopy utilizing fluorescent indicators of ROS and mitochondria demonstrated the mitochondrial localization of ART-induced ROS. MnTBAP, a cell-permeable metalloporphyrin antioxidant, abolished ART-induced ROS production. As a final step in confirming the mitochondrial origin of the ART-induced ROS, HUVEC were transduced with a cytosolic- compared to a mitochondria-targeted catalase. Transduction with the mitochondria-targeted catalase was more effective than cytoplasmic catalase in inhibiting the ROS and 8-isoprostane (8-iso-PGF2alpha) produced after treatment with either AZT or indinavir. However, both mitochondrial and cytoplasmic catalase attenuated ROS and 8-iso-PGF2alpha production induced by the combination treatment, suggesting that in this case, the formation of cytoplasmic ROS may also occur, and thus, that the mechanism of toxicity in the combination treatment group may be different compared to treatment with AZT or indinavir alone. Finally, to determine whether ART-induced mitochondrial dysfunction and ROS production culminate in apoptosis, we performed the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), annexin V and 4',6-diamidino-2-phenylindole (DAPI) staining, and caspase-3 activity assays. However, none of these assays showed appreciable levels of ART-induced apoptosis. Our studies thus suggest that in endothelial cells, ART induces mitochondrial dysfunction with a concomitant increase in mitochondria-derived ROS. This compromised mitochondrial function may be one important factor culminating in endothelial dysfunction, without inducing an increase in apoptosis.
Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71103, USA.
Bo
B
Jiang
Valeria Y
VY
Hebert
Yuchi
Y
Li
J Michael
JM
Mathis
J Steven
JS
Alexander
Tammy R
TR
Dugas
D000256:Adenoviridae / Q000187:drug effects / Q000201:enzymology; D017304:Annexin A5 / Q000378:metabolism; D019380:Anti-HIV Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002374:Catalase / Q000096:biosynthesis; D002478:Cells, Cultured; D053938:DNA Fragmentation / Q000187:drug effects; D004338:Drug Combinations; D042783:Endothelial Cells / Q000187:drug effects*; D005456:Fluorescent Dyes; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors; D006801:Humans; D019469:Indinavir / Q000494:pharmacology; D007211:Indoles; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects*; D010101:Oxygen Consumption / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D015215:Zidovudine / Q000494:pharmacology
D017304:Annexin A5; D019380:Anti-HIV Agents; D004338:Drug Combinations; D005456:Fluorescent Dyes; D017320:HIV Protease Inhibitors; D007211:Indoles; D017382:Reactive Oxygen Species; D018894:Reverse Transcriptase Inhibitors; C007293:DAPI; D015215:Zidovudine; D019469:Indinavir; D002374:Catalase; D054303:HIV Reverse Transcriptase; D053148:Caspase 3