Polychlorinated biphenyl-induced VCAM-1 expression is attenuated in aortic endothelial cells isolated from caveolin-1 deficient mice

Abstract

Exposure to environmental contaminants, such as polychlorinated biphenyls (PCBs), is a risk factor for the development of cardiovascular diseases such as atherosclerosis. Vascular cell adhesion molecule-1 (VCAM-1) is a critical mediator for adhesion and uptake of monocytes across the endothelium in the early stages of atherosclerosis development. The upregulation of VCAM-1 by PCBs may be dependent on functional membrane domains called caveolae. Caveolae are particularly abundant in endothelial cell membranes and involved in trafficking and signal transduction. The objective of this study was to investigate the role of caveolae in PCB-induced endothelial cell dysfunction. Primary mouse aortic endothelial cells (MAECs) isolated from caveolin-1-deficient mice and background C57BL/6 mice were treated with coplanar PCBs, such as PCB77 and PCB126. In addition, siRNA gene silencing technique was used to knockdown caveolin-1 in porcine vascular endothelial cells. In MAECs with functional caveolae, VCAM-1 protein levels were increased after exposure to both coplanar PCBs, whereas expression levels of VCAM-1 were not significantly altered in cells deficient of caveolin-1. Furthermore, PCB-induced monocyte adhesion was attenuated in caveolin-1-deficient MAECs. Similarly, siRNA silencing of caveolin-1 in porcine endothelial cells confirmed the caveolin-1-dependent VCAM-1 expression. Treatment of cells with PCB77 and PCB126 resulted in phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2), and pharmacological inhibition of ERK1/2 diminished the observed PCB-induced increase in monocyte adhesion. These findings suggest that coplanar PCBs induce adhesion molecule expression, such as VCAM-1, in endothelial cells, and that this response is regulated by caveolin-1 and functional caveolae. Our data demonstrate a critical role of functional caveolae in the activation and dysfunction of endothelial cells by coplanar PCBs.

Figure 1

Characterization of endothelial cells. (A) Fluorescent microscopy of mouse and porcine endothelial cells demonstrating uptake of Dil-Ac-LDL labeling. Pictures were taken at 400× magnification. (B) Fluorescent microscopy of mouse aortic endothelial cells (MAECs) and porcine endothelial cells (PECs) that express PECAM-1 (FITC, green) and caveolin-1 (Cav-1) (Texas Red, red). (C) Caveolin-1 protein expression in mouse and porcine endothelial cells determined by SDS-PAGE and Western blot analysis. β-actin was used as loading control.

Figure 2

Expression of VCAM-1 in porcine endothelial cells exposed to coplanar PCBs. (A) Expression of mRNA in porcine endothelial cells exposed to DMSO or coplanar PCBs (PCB77 and PCB126) at a concentration of 2.5 µM for 16 h was measured using real-time PCR. Results represent the mean ± SEM, with n=3. Experiments were repeated a minimum of three times. (B) Expression of VCAM-1 protein after PCB treatment for 16 h. Densitometry results were normalized to β-actin. Results represent the mean ± SEM, with n=3. Experiments were repeated a minimum of three times. The Western blot picture shown is a representative of three independent blots. *Significantly different compared to DMSO control.

Figure 3

Monocyte adhesion to PECs exposed to coplanar PCBs. PECs were exposed to 2.5 µM PCB77 or PCB126 for 16 h. Human THP-1 monocytes were activated with TNF-α and loaded with the fluorescent probe calcein. Activated and calcein loaded monocytes were added to endothelial monolayers for adhesion. After washing, adhered monocytes were counted using a fluorescent microscope. Results represent the mean ± SEM, with n=3. Experiments were repeated a minimum of three times. Representative microphotographs showing adhered monocytes on endothelial cell monolayers. *Significantly different compared to DMSO control.

Figure 4

Expression of VCAM-1 in PECs lacking caveolin-1 (Cav-1). PECs treated with Cav-1 siRNA or control siRNA were exposed to PCB77 or PCB126 at 2.5 µM for 16 h. VCAM-1 protein levels in control siRNA (A) and Cav-1 siRNA (B) were observed using Western blot analysis. Densitometry results were normalized to β-actin, and represent the mean ± SEM, with n=3. Experiments were repeated a minimum of three times. The Western blot picture shown is a representative of three independent blots. (C) Monocyte adhesion to control and Cav-1 siRNA tansfected PECs were exposed to PCB126 (2.5 µM, 16 h) and TNF-α (4 ng/ml, 16 h). *Significantly different compared to DMSO control. #Significantly different compared to corresponding control siRNA group.

Figure 5

Expression of VCAM-1 in MAECs exposed to coplanar PCBs. Cells were exposed to DMSO or 2.5 µM PCB77 or PCB126 for 16 h, and protein levels were measured using Western blot analysis. Protein expression levels were detected in PCB treated MAECs containing caveolin-1 (A) and in caveolin-1-deficient cells (B). Densitometry results were normalized to β-actin, and represent the mean ± SEM (n=3) of three independent experiments. The Western blot picture shown is a representative of three independent blots. *Significantly different compared to DMSO control.

Figure 6

Monocyte adhesion to MAECs exposed to coplanar PCBs. MAECs were exposed to PCB77 or PCB126 at 2.5 µM for 16 h. Human THP-1 monocytes were activated with TNF-α and loaded with the fluorescent probe calcein. Activated and calcein loaded monocytes were added to endothelial monolayers for adhesion. After washing, adhered monocytes were counted using a fluorescent microscope. Results represent the mean ± SEM, with n=3. Experiments were repeated a minimum of three times. Representative microphotographs show adhered monocytes on endothelial cell monolayers. *Significantly different compared to DMSO control. #Significantly different compared to corresponding control C57BL/6 mouse endothelial cells.

Figure 7

Dose- and time-dependent phosphorylation of ERK1/2 MAPK by PCB126 in PECs (A). PECs were treated with 0–7.5 µM PCB126 for 16 h to observe a dose-dependent phosphorylation of ERK1/2. PECs were treated with 2.5 µM PCB126 for 0.5–16 h to determine time-dependent phosphorylation of ERK1/2. Western blot analysis was performed to observe phosphorylation of ERK1/2 using a monoclonal antibody. Experiments were repeated a minimum of three times. The Western blot picture shown is a representative of three independent blots. Phosphorylation of ERK1/2 MAPK by PCB77 or PCB126 in PECs (B). PECs were pretreated with 10 µM of PD98059 for 1 h followed by treatment with PCB77 or PCB126 at 2.5 µM for 16 h. Western blot analysis was performed to observe phosphorylation of ERK1/2 using a monoclonal antibody. Densitometry results were normalized to total ERK1/2, and represent the mean ± SEM, with n=3. Experiments were repeated a minimum of three times. The Western blot picture shown is a representative of three independent blots. (C) Expression of VCAM-1 mRNA in porcine endothelial cells exposed to DMSO or PCB126 at a concentration of 2.5 µM for 16 h after pretreatment with PD98059. mRNA was measured using real-time PCR. Results represent the mean ± SEM, with n=3. Experiments were repeated a minimum of three times. *Significantly different compared to DMSO control. #Significantly different compared to corresponding DMSO control.

Figure 8

Monocyte adhesion to PECs after pretreatment with PD98059 followed by exposure to PCB77 or PCB126 (2.5 µM for 16 h). Human THP-1 monocytes were activated with TNF-α and loaded with the fluorescent probe calcein. Activated and calcein loaded monocytes were added to endothelial monolayers for adhesion. After washing, adherend monocytes were counted using a fluorescent microscope. Representative microphotographs show adherend monocytes on endothelial cell monolayers. *Significantly different compared to DMSO control. #Significantly different compared to corresponding control C57BL/6 mouse endothelial cells.

Figure 9

Phosphorylation of ERK1/2 MAPK by PCB77 and PCB126 in MAECs. MAECs were treated with PCB77 or PCB126 at 2.5 µM for 16 h. Western blot analysis was performed to observe phosphorylation of ERK1/2 using a monoclonal antibody. Densitometry results were normalized to total ERK1/2, and represent the mean ± SEM, with n=3. Experiments were repeated a minimum of three times. The Western blot picture shown is a representative of three independent blots. *Significantly different compared to DMSO control. #Significantly different compared to corresponding Cav-1 +/+ C57BL/6 MAECs.