|
The cause of decreased nitric oxide (NO) that could potentially lead to HIV related Pulmonary Arterial Hypertension can be attributed to the presence of proteins made by the HIV-1 virus. The presence of HIV-1 proteins may upregulate the production of harmful reactive oxygen species (ROS) and depletes nitric oxide (NO), which can have deleterious consequences in various cell types. However, it has been shown that glutathione restoration reverses nitric oxide depletion and the detrimental effects of ROS. Nef, a protein produced and secreted by the HIV-1 virus, is thought to contribute to a decrease in NO. However, the mechanism is unknown. We tested the possible role of the HIV-1 protein Nef in decreased NO levels by examining its effects on eNOS expression and oxidative stress in human pulmonary artery endothelial cells (HPAEC) by using Toxilight and Amplex Red Assays, and Real Time Polymerase Chain Reaction. (RT-PCR). Our results show no statistically significant differences in cell death, hydrogen peroxide and glutathione production between cells treated with varying concentrations of Nef. However, our data demonstrates a decrease in eNOS mRNA levels as the concentration of Nef protein increased.
Approximately 39 million individuals worldwide are infected with Human Immunodeficiency Virus (HIV-1). With the advent of improved HIV therapies, HIV-1-infected individuals are showing increased survival. However, with the improved survival, HIV-1 patients are now developing non-infectious complications such as HIV-1 related Pulmonary Arterial Hypertension (HIV-PAH). Pulmonary Arterial Hypertension (PAH) is a disease characterized by increased pulmonary artery constriction and blockage, as well as endothelial dysfunction and vasculature remodeling. Pulmonary artery endothelial cells from PAH patients are shown to produce decreased amounts of NO. This suggest that NO plays a major protective role against PAH. Nitric oxide (NO) is an important vasodilator of the pulmonary vasculature. NO is generated by eNOS (endothelial nitric oxide synthase). NO levels can be affected by altering eNOS expression. In addition, reactive oxygen species (ROS) deplete NO. Both mechanisms lead to vasoconstriction within the pulmonary arteries which is characteristic of PAH. NO is thought to be decreased by HIV-1 however, the mechanism is not well characterized. In this study we tested the possible role of the HIV-1 protein Nef in decreased NO by examining its effects on eNOS expression and oxidative stress in human pulmonary artery endothelial cells (HPAEC).
Human Pulmonary Artery Endothelial Cells (HPAEC) were grown in EGM-2 medium. Cells were maintained in at 37°C with 5% CO2. Confluent HPAEC were then acclimated twenty-four hours prior to treatment in FBS and Heparin-free EGM-2 medium. HPAEC were then separated into three groups according to Nef treatment. Group one was treated with 0 ng of Nef and served as the control group. Group two was treated with 10 ng of Nef, and Group three was treated with 20 ng of Nef. The cells were then incubated for twenty-four hours at 37°C. Following twenty-four hour incubation at 37°C, supernatant, protein collected using Tissue Protein Extraction Reagent (T-PER) and RNA collected using RNA Bee were seperated and stored for analysis. Gene expression analyses were performed by real-time PCR using the LightCycler system (Roche). cDNA templates were amplified with gene-specific primer sets (SuperArray, Inc). All transcripts were detected using SYBR Green I (Molecular Probes, Inc). Transcripts were normalized to the housekeeping gene, β-Globin. Toxilight Assays were performed to assess cell death. Adenylate Kinase (AK) is released following the death of a cell and the assay detects the amount of AK in the medium of the cells using Adenylate Kinase Detection Reagent (AKDR).Amplex Red detects the amount of hydrogen peroxide in the surrounding medium of cells. Using Amplex Red reagent and horseradish peroxidase, the assay is able to detect hydrogen peroxide released from cells as a result of oxidative damage.
Our results show that eNOS mRNA expression is decreased in HPAEC treated with 20 ng/mL of Nef as analyzed by RT-PCR. However, our results show no statistically significant differences in cell death, hydrogen peroxide release and glutathione production between cells treated with varying concentrations of Nef. It is noteworthy to mention that though hydrogen peroxide release between the cells treated with Nef and those that weren’t were not statistically significant, there still was an increase in hydrogen peroxide release as the concentration of Nef increased.
Our results suggest that the HIV-1 protein Nef may potentially contribute to HIV-related Pulmonary Arterial Hypertension (HIV-PAH) by decreasing eNOS mRNA expression. This decrease in expression may be responsible for the decrease in nitric oxide (NO). Since nitric oxide is the primary vasodilator for the pulmonary vasculature, decreases in NO can potentially lead to vasoconstriction and increase susceptibility to HIV-PAH. In the future, we would want to determine whether the HIV-1 protein Nef has a similar effect on iNOS mRNA expression. Another future direction would be to determine how Nef is directly affecting eNOS expression and this could be possible by perhaps analyzing the eNOS promoter. There is also the possibility of analyzing NO levels in HPAEC corresponding to decreases in mRNA expression as a result of Nef treatment and to directly treat pulmonary arteries with Nef protein to see if there are alterations in vasoreactivity.
This material is based upon work supported by the Howard Hughes Medical Institute under Grant No. 52005873 and NIH HL 070892
Tables and Figures
Figure 1. Amplex Red Assay show no significant differences in H2O2 release among three treatment groups (A). Toxilight Assay demonstrates that Nef exposure does not increase cell death as the amount of Nef protein increases (B).
Figure 2. HPAEC treated with 20 ng/mL of Nef showed significant decreases in eNOS mRNA expression (A). Nef exposure produced no significant differences in glutathione levels in HPAEC (B).
|
