Simvastatin protects against amyloid beta and HIV-1 Tat-induced promoter activities of inflammatory genes in brain endothelial cells

Abstract

Increased deposition of amyloid beta (Abeta) is characteristic for normal aging and human immunodeficiency virus-1 (HIV-1)-associated alterations of the central nervous system. In addition, both Abeta and HIV-1 are known to induce cellular oxidative stress and disruption of the blood-brain barrier (BBB). Therefore, we hypothesize that Abeta and HIV-1 protein Tat can potentiate their proinflammatory effects at the brain endothelium level. To address this hypothesis, we studied promoter activity of three proinflammatory genes in an in vitro BBB model of human brain microvascular endothelial cells (HBMEC) cocultured with a human astrocyte cell line producing Tat (SVGA-Tat cells) and exposed to Abeta. Treatment of HBMEC with Abeta(1-40) in the presence of SVGA-Tat cells resulted in a significant up-regulation of E-selectin, CC chemokine ligand-2, and interleukin-6 promoter activities and protein levels compared with the individual effects of Abeta or Tat. In addition, Abeta markedly amplified E-selectin promoter activity in HBMEC cocultured with HIV-1-infected Jurkat T cells. Simvastatin, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, effectively blocked proinflammatory reactions induced by Abeta in cocultures with SVGA-Tat cells or with HIV-1-infected Jurkat cells. The present study indicates that a combined exposure to Abeta and Tat or HIV-1 can synergistically potentiate the expression of inflammatory genes in brain endothelial cells. In addition, simvastatin may provide a beneficial influence by reducing these effects at the BBB level.

Fig. 1

Morphological characterization of immortalized HBMECs, control astrocytic cell line (SVGA), and SVGA overexpressing Tat. Top, phase-contrast micrographs of a confluent HB-MEC monolayer with the typical morphology of elongated, fusiform shape of endothelial cells cultured on a cell culture dish (A) and on a filter insert (B). Endothelial cells cultured on filter inserts have no morphological changes compared with those grown on cell culture dishes. Bottom, phase-contrast micrograph of confluent control SVGA astrocytes (C) and SVGA-Tat astrocytes (D). Control SVGA and SVGA-Tat cells exhibit similar morphology characteristic for astrocyte cultures, such as multipolar shape and overlapping morphology.

Fig. 2

Tat expression by SVGA-Tat astrocytes. A, control SVGA and SVGA-Tat astrocytes were transfected with the pEGFP LTR (bottom) or promoterless pEGFP (negative control, top) constructs. To confirm the specificity of Tat detection, normal SVGA astrocytes were transiently transfected with the pcDNA3 Tat86 expression vector and cotransfected with the pEGFP LTR or the promoterless pEGFP constructs. EGPF expression was detected by immunofluorescence microscopy. B, to quantify Tat transactivation, control SVGA and SVGA-Tat astrocytes were transfected with pGL3 LTR or pGL3 basic vector (negative control). As a positive control, SVGA astrocytes were transiently transfected with pcDNA3 Tat86 expression vector (or pcDNA3 empty vector) and pGL3 LTR. To normalize transfection rates, all cells were cotransfected with the R. reniformis luciferase reporter plasmid pRL-TK. Luciferase activity was analyzed by dual luciferase assay 20 h after transfections and normalized according to R. reniformis luciferase activity. Values are mean ± S.E.M. (n = 4–6).

Fig. 3

Aβ amplifies Tat-induced promoter activities of proinflammatory genes in brain endothelial cells. HBMECs cocultured with control or Tat-expressing astrocytes (SVGA and SVGA-Tat cells, respectively) were transfected with the firefly luciferase reporter constructs containing the human E-selectin (A), CCL-2 (B), and IL-6 (C) promoter sequences. Cells in the coculture system were treated with the indicated concentrations of Aβ(1-40) 24 h after transfections. Aβ(40-1) at the concentration of 1 µM was used as a negative control. Luciferase activity was analyzed in HBMECs after a 20-h exposure to Aβ. Values are mean ± S.E.M., n = 4. *, statistically different compared with the corresponding controls within the HBMECs plus SVGA or HBMECs plus SVGA-Tat cocultures. †, data in cocultures of HBMECs with SVGA-Tat are significantly different compared with cocultures of HBMEC with control SVGA and exposed to the corresponding concentration of Aβ.

Fig. 4

Aβ and Tat synergistically amplify protein expression of proinflammatory mediators in brain endothelial cells. A, HBMECs cultured on chambered slides were exposed to 1 µM Aβ(1-40) and/or conditioned media from SVGA or SVGA-Tat cells for 6 h. E-selectin immunoreactivity was determined by immunofluorescence microscopy. Red color reflects E-selectin-positive immunoreactivity, and blue color represents 4′,6-diamidino-2-phenylindole staining for DNA that visualizes the nuclei. Arrows indicate intensification of E-selectin immunoreactivity by Aβ and/or SVGA-Tat conditioned media. B and C, HBMECs were cocultured with SVGA-Tat or control SVGA cells and treated with Aβ(1-40) at 0.5 µM (B) or 1 µM (C). CCL-2 and IL-6 protein levels were determined in the culture media by enzyme-linked immunosorbent assay. Values are mean ± S.E.M. (n = 3–8). *, statistically different compared with the corresponding controls within the HBMECs plus SVGA or HBMECs plus SVGA-Tat cocultures. †, data in cocultures of HBMECs with SVGA-Tat are significantly different compared with cocultures of HBMECs with control SVGA and exposed to the corresponding concentration of Aβ.

Fig. 5

Simvastatin protects against Aβ and Tat-induced promoter activities of proinflammatory genes in brain endothelial cells. Transfections and luciferase activity assays were performed as described in the legend to Fig. 3. In addition, selected cultures were pretreated for 15 min with 10 µM (A and B) or 5 µM (C) simvastatin before coexposure to 1 µM (A and C) or 0.5 µM (B) Aβ(1-40) for 20 h. Values are mean ± S.E.M. (n = 4–6). *, statistically different compared with the corresponding controls within the HBMECs plus SVGA or HBMECs plus SVGA-Tat cocultures. †, Data in cocultures of HBMEC with SVGA-Tat are significantly different compared with cocultures of HBMECs with control SVGA and exposed to the corresponding concentration of Aβ. #, data in cocultures exposed to simvastatin are significantly different compared with the corresponding cocultures without added simvastatin.

Fig. 6

Simvastatin protects against Aβ and HIV-1-induced promoter activity of E-selectin in brain endothelial cells. Transfections with the pGL3 E-selectin promoter construct were performed as described in the legend to Fig. 3. The next day, the transfected HBMECs were placed in cocultures with normal or HIV-1-infected Jurkat cells, followed by a treatment with 1 µM Aβ(1-40) for 20 h. Selected cultures were pretreated for 15 min with simvastatin (10 µM) before Aβ(1-40) coexposure. E-selectin promoter activity was analyzed by luciferase assay and normalized to cellular protein levels. Values are mean ± S.E.M. (n = 6–10). *, statistically different compared with the corresponding controls within the cocultures of HBMECs with normal or HIV-1-infected Jurkat cells. †, data in cocultures of HBMECs with HIV-1-infected Jurkat cells are significantly different compared with cocultures of HBMEC with normal Jurkat cells and exposed to the corresponding concentration of Aβ. #, data in cocultures exposed to simvastatin are significantly different compared with the corresponding controls without added simvastatin.