Pentoxifylline reduces tumor necrosis factor-α and HIV-induced vascular endothelial activation

Abstract

Untreated HIV infection is associated with endothelial dysfunction and subsequent cardiovascular disease, likely due to both direct effects of the virus and to indirect effects of systemic inflammation on the vasculature. We have recently shown that treatment with the antiinflammatory agent pentoxifylline (PTX) improved in vivo endothelial function and reduced circulating levels of the inflammatory markers vascular cell adhesion molecule-1 (VCAM-1) and interferon-gamma-induced protein (IP-10) in HIV-infected patients. To delineate the mechanisms underlying this therapeutic effect, we tested whether clinically relevant concentrations of PTX suppress VCAM-1 or IP-10 release in cultivated human lung microvascular endothelial cells. Indeed, we found that tumor necrosis factor (TNF)-α-induced VCAM-1 was reduced with concentrations of PTX in the low nanomolar range, comparable to plasma levels in PTX-treated groups. We also investigated the effect of HIV proteins and found that HIV transactivator of transcription (HIV-Tat) and HIV-envelope-derived recombinant gp120 enhanced TNF-α-induced VCAM-1 gene expression in lung microvascular and coronary macrovascular endothelial cells, respectively. In addition, PTX and a NF-κB-specific inhibitor reduced this enhanced VCAM-1 gene induction in microvascular and macrovascular endothelial cells. These results provide novel insights in how the antiinflammatory agent PTX can directly reduce HIV-associated proinflammatory endothelial activation, which may underlie vascular dysfunction and coronary vascular diseases.

FIG. 1.

Pentoxifylline reduces tumor necrosis factor (TNF)-α-induced vascular cell adhesion molecule-1 (VCAM-1) at low concentrations. Human lung microvascular endothelial cells (HLMVECs) were incubated for 6 h with either (A) TNF-α (20 ng/ml) or (B) interferon (IFN)-γ (40 ng/ml) in the absence or presence of increasing concentrations of pentoxifylline (PTX). Total RNA was isolated and qRT-PCR was performed using primers specific for (A) VCAM-1 or (B) interferon-gamma-induced protein (IP-10). Relative gene expression levels were normalized to the endogenous control (EF1alpha) compared to target genes. All experiments were performed at least three times.

FIG. 2.

HIV transactivator of transcription (HIV-Tat) enhances TNF-α-induced VCAM-1 expression in HLMVECs. HLMVECs were treated with low TNF-α concentrations (1 ng/ml) alone or in combination with HIV-Tat (100 ng/ml) for 6 h. (A) qRT-PCR analysis of relative gene expression levels of VCAM-1. (B) Flow cytometry analysis of VCAM-1 surface expression. Inset depicts a typical example of VCAM expression (black dotted line HIV-Tat + TNF-α vs. gray dotted line TNF-α only). (C) Treatment with a high TNF-α concentration (20 ng/ml) alone or in combination with HIV-Tat (100 ng/ml) with or without treatment with PTX (200 nM) for 6 h. Immunofluorescence pictures captured for VCAM-1 staining in (i) vehicle-treated, (ii) TNF-α only-treated, (iii) HIV-Tat and TNF-α-treated, and (iv) HIV-Tat and TNF-α and PTX-treated endothelial cells. (D) Quantification of VCAM-1 staining was determined using MetaMorph image analysis software. All experiments were performed three times.

FIG. 3.

gp120 enhances TNF-α-induced VCAM-1 expression in HCAECs. HCAECs were treated with TNF-α (1 ng/ml) alone or in combination with gp120 (100 ng/ml) for 6 h and then analyzed for (A) relative VCAM-1 gene expression levels and (B) surface expression of VCAM-1 as determined by flow cytometry. Inset depicts a typical example of VCAM expression (black dotted line HIV-Tat + TNF-α vs. gray dotted line TNF-α only). Values were expressed as mean fluorescence intensity (FI) and normalized to the untreated control group.

FIG. 4.

Tat- and gp120-enhanced TNF-α-induced upregulation of VCAM-1 displays differential sensitivity to PTX and IKK inhibitors. HLMVECs were treated with TNF-α (1 ng/ml) in combination with HIV-Tat (100 ng/ml) with/without PTX (200 nM) or IKKI (100 nM) for 6 h and then analyzed for (A) relative VCAM-1 gene expression levels and (B) surface expression of VCAM-1 as determined by flow cytometry. HCAECs were also treated with TNF-α (1 ng/ml) and gp120 (100 ng/ml) with/without PTX (200 nM) or IKKI (100 nM) for 6 h and then analyzed for (C) relative VCAM-1 gene expression levels and (D) surface expression of VCAM-1 as determined by flow cytometry. Values were expressed as % inhibition of VCAM-1 as compared to the HIV-Tat + TNF or gp120 + TNF samples, respectively.