Effect of non-nucleoside reverse transcriptase inhibitors on inflammatory responses in endothelial cells

Abstract

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are one of the first-line treatments for patients with human immunodeficiency virus (HIV). However, epidemiological studies suggest an increased prevalence of cardiovascular diseases (CVD) in NNRTIs-treated HIV-infected patients. Since inflammation is a risk factor of CVD, the present study aimed to determine whether or not NNRTIs, efavirenz (EFV) and nevirapine (NPV), stimulate vascular inflammation.

Human umbilical vein endothelial cells (HUVECs) were incubated with EFV (5 or 25 μM), NPV (5 or 25 μM) or their vehicle (dimethyl sulfoxide, 0.1%) for 2 or 24 hours in the absence (the control conditon), or presence of hydrogen peroxide [H2O2 (50 μM), to mimic oxidative stress] or lipopolysaccharides [LPS (10 μg/ml), to mimic bacterial infection]. The releases of inflammatory mediators by HUVECs, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8) and C-reactive proteins (CRP), were determined by measuring their levels in the medium with ELISA. The mRNA levels of TNF-α, IL-6 and IL-8, and the protein presence of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-1 and -2 in HUVECs were measured with real time-polymerase chain reaction assay and Western immunoblotting, respectively.

After incubation with EFV (25 μM) for 2 or 24 hours, the number of viable cells, measured by the colorimetric assay with MTT, was significantly reduced. HUVECs treated with this concentration of EFV for 24 hours also showed reduced protein expression of eNOS. Despite these seemingly impairment of endothelial function, the release of CRP and TNF-α, mRNA level of IL-6 and IL-8, and the protein expressions of iNOS and COX-2, in the absence or presence of H2O2 or LPS, were not affected by EFV. As such, it is unlikely that EFV can induce significant inflammatory responses in endothelial cells. In view of the plasma level of EFV after an oral intake (12.89 μM) was lower than the high concentration (25 μM) used in the study, it is unlikely that the clinical use of EFV would cause endothelial cell death.

By contrast, NPV, at both concentrations tested, did not affect the protein expressions of iNOS, COX-1 and COX-2, and the mRNA level of TNF-α, IL-6 and IL-8 in HUVECs, but after 24 hours reduced CRP release in the presence of H2O2 (at high concentration) and IL-6 release in the presence of LPS (at low concentration). These findings suggest that NPV unlikely causes endothelial inflammation. NPV also did not affect the number of viable cells, thus indicating that it does not have cytotoxic effects on endothelial cells. Nevertheless, NPV down-regulated eNOS in the presence of H2O2 after 24 hours; this effect was observed at both concentrations tested, which are within the plasma concentrations in human, thus suggesting that it may have the potential to impair endothelial function during oxidative stress.

In summary, the present findings suggest that NNRTIs would not cause significant endothelial inflammation. However, they may impair endothelial function thereby increasing the risk of CVD after chronic treatment.