Induced cytoskeletal changes in bovine pulmonary artery endothelial cells by resveratrol and the accompanying modified responses to arterial shear stress

Abstract

Background: Atherosclerosis and coronary heart disease (CHD) are significant contributors to morbidity and mortality in developed countries. A noted exception is the low mortality of CHD in France, particularly the southwest region. This phenomenon, commonly referred to as the French paradox, may be associated with high consumption of red wine. We investigate whether the cardioprotective activity of red wine may involve the grape skin-derived polyphenol, resveratrol. We further test the possibility that resveratrol acts by modulating structural and functional changes in endothelial cells lining the blood vessel wall.

Results: Bovine pulmonary artery endothelial cells (BPAEC) were incubated with resveratrol, with and without concurrent exposure to simulated arterial shear stress. Resveratrol significantly affected proliferation and shape of BPAEC; growth was suppressed and cells became elongated, based on morphologic analysis of rhodamine-conjugated phalloidin stained F-actin by confocal microscopy. Using selective signaling inhibitors, we showed that the resveratrol-induced cellular phenotype was dependent on intracellular calcium and tyrosine kinase activities, and assembly of actin microfilaments and microtubules, but was unrelated to PKC activity. Exposure to simulated arterial flow revealed that, whereas controls cells easily detached from the culture support in a time-dependent manner, resulting in total cell loss after a 5 min challenge with simulated arterial flow conditions, a significant percentage of the treated cells remained attached to the cultured plastic coverslips under identical experimental conditions, suggesting that they adhered more strongly to the surface. Western blot analysis shows that whereas cells treated with 25 microM and 100 microM resveratrol had no change in total ERK1/2, treatment did result in an increase in phosphorylated ERK1/2, which probably involved stabilization of the active enzyme. An increase in nitric oxide synthase expression was detected as early as 6 h and persisted for up to 4 days of treatment.

Conclusions: Results of our studies show that resveratrol interacts with endothelial cells in vitro to elicit morphological and structural changes; the observed changes support the interpretation that resveratrol acts as a cardioprotective agent.

Figure 1

Resveratrol treatment produces elongation of BPAEC. Panel A. Example of stellar, cobblestone-like morphology characteristic of normal BPAECs grown in culture. Panel B. Example of elongated, spindle-shaped morphology characteristic of resveratrol-treated cells. Here cells were treated with 100 μM resveratrol and were viewed with 20X objective. Six experiments, each involving a different preparation of BPAEC, were performed with similar results.

Figure 2

Morphology of passage 6 BPAEC treated with the following concentrations of resveratrol: A) 0 μM; B) 10 μM; C) 25 μM; D) 50 μM. Cells were stained with rhodamine-phalloidin and viewed under 10X confocal microscopy. The same experiment was repeated three times, using passages 6-7 cells from two different preparation of BPAEC.

Figure 3

Resveratrol treatment causes a dose-dependent increase in BPAEC elongation. Cells from representative confocal microscopy fields from passages 5 and 6 were visually evaluated for overall change in cell morphology. A minimum of 300 cells were scored for each treatment condition.

Figure 4

Passage 7 BPAEC were treated with the following concentration of resveratrol as described in Materials and Methods and then subjected to simulated arterial flow durations, respectively. A) 0 μM, 0 min (control); B) 100 μM; C) 0 μM, 2 min; D) 100 μM, 2 min; E) 0 μM, 5 min; F) 100 μM, 5 min. Similar results were obtained using passage 6 BPAEC (data not shown). Four experiments, using passages 6-7 cells from two different preparation of BPAEC, were performed.

Figure 5

Biochemical changes in resveratrol-treated BPAEC. BPAEC were treated with the indicated concentrations of resveratrol and lysed after 6, 48, and 96 h, respectively. Lysates were run under 10% SDS-PAGE, and probed with antibodies for the various target proteins. Panel A. Changes in activated ERK1/2-P (representative of 3 experiments). Panel B. Changes in ERK1/2 (representative of 2 experiments). Panel C. Changes in eIF-4E. Panel D. Changes in eNOS. Panel E. Changes in actin. Lanes 1,4,7 correspond to control at 6, 48, and 96 h; lanes 2,5,8 correspond to BPAEC treated with 25 μM resveratrol for 6, 48, and 96 h; lanes 3,6,9 correspond to BPAEC treated with 100 μM resveratrol for 6, 48, and 96 h. The results represent the average of two experiments, each analyzed in duplicate or triplicate.

Figure 6

Time-dependent relative changes in expression of total ERK1/2, active ERK1/2, and eNOS. Intensity of signals corresponding to total ERK1/2 (panel A), active ERK1/2 (panel B), and eNOS (panel C) obtained by western blot analysis, similar to those shown in Figure 5, was quantified by actin-adjusted image analysis and plotted as a function of time of treatment with 25 μM and 100 μM resveratrol. The control value at 6 h was set at 100. The results represent the average of two experiments, each analyzed in duplicate or triplicate.