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

Abstract

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. 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 μM and 100 μM 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. to elicit morphological and structural changes; the observed changes support the interpretation that resveratrol acts as a cardioprotective agent.

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 μM and 100 μM 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

to elicit morphological and structural changes; the observed changes support the interpretation that resveratrol acts as a cardioprotective agent.

Background

].

]. Thus resveratrol may act by a mechanism(s) closely resembling that triggered by shear stress.

Resveratrol induces morphologic change in endothelial cells

].

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.

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.

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.

Resveratrol treatment leads to increased adherence of BPAEC under simulated arterial flow conditions

, compared to the 0 min sample, a significant percentage of resveratrol-treated BPAEC remained attached to the plastic coverslips after 2 min and 5 min flow challenge. In comparison, under identical experimental conditions, cells adhering to the coverslips decreased dramatically in controls, with virtually no cells remaining after a 5 min simulated flow. These results suggest that a functional alteration, measured by greater adherence of cells under simulated arterial flow conditions, accompanied the change in cellular phenotype, following treatment with resveratrol.

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.

Effect of cell signaling inhibitors on cell elongation induced by resveratrol

further showed that cytoskeletal changes elicited by resveratrol also appeared to be linked to integrity of actin microfilaments and microtubule network as the morphologic changes were also substantially lowered in cells treated with cytochalasin D and nocodazole, which inhibit formation of actin microfilaments and microtubule network, respectively.

These experiments used passages 4-5 BPAEC cells. The cells on cover slips were incubated with inhibitors for 24 h, before 100 μM resveratrol was added and incubation was continued for an additional 40 h. An exception was quin2-AM, which was applied 1 h prior to resveratrol treatment.

Results in this table were averaged from 4 separate experiments, each performed with a different preparation of primary BPAEC's. Since growth of control cells as well as their response to resveratrol and addition of inhibitors differed between BPAEC preparations, cells showing the elongated shape in each experiment were calculated as a percent of total cells counted (ranging from 60-700). The numbers obtained in this manner were used to calculate the mean (%)± SD, as shown.

Effect of resveratrol on ERK1/2 activation and eIF4E, eNOS expression

.

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.

, 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.

Modulation of endothelial cell shape by resveratrol

). It is tempting to speculate that appearance of such cellular phenotype involves formation of stress fibers, which would rearrange the cytoskeleton in ways that facilitate better anchoring of the BPAEC to the culture substratum and contribute to the ability of BPAEC to resist simulated arterial flow challenge. Only studies in the future would validate such a possibility. It is also interesting to note that the observed changes in resveratrol-treated BPAEC are similar to that described for EC subjected to arterial shear. We propose that this could represent a physiologically-relevant mechanism by which resveratrol, as a polyphenolic constituent of red wine, contributes to cardioprotection by inducing resistance to potential damage by shearing forces.

, tyrosine kinases, and intact actin microfilament and microtubules. These results also raise the possibility that there is cellular heterogeneity within endothelial cells used in the studies, based on the fact that a subset displayed exquisite sensitivity to chelerythrine. Overall, these results support the notion that resveratrol and shear stress induce elongation of the EC cytoskeleton via an overlapping outside-in signaling mechanism.

]. Since no noticeable change in active ERK1/2 occurred at the 6 h or 2 day time points, where morphological changes clearly became visible, following treatment with resveratrol, it seems unlikely that the activation of MAPK is directly linked to the observed morphologic changes.

Responses of cultured endothelial cells to arterial shear stress

, such a cellular property could make endothelial cells less likely to dislodge to become part of a growing thrombotic plug. Restricted detachment could also imply that there is less degeneration of the endothelial cell monolayer, which, in turn, would reduce the exposure of the underlying subendothelial matrix components, thereby making platelet adhesion and aggregation less likely. The mechanism(s) responsible for the resveratrol-induced cellular properties remain to be further investigated. One possibility centers on modulation of the number of focal contact adhesion sites, and/or the effective redistribution of the focal contacts, as well as the increased production of the EC-specific integrin complex, by resveratrol, all of which could contribute to the cardioprotective mechanism of this polyphenol. However, it should be emphasized that these experiments involved growing EC on plastic, not on layers of subendothelial matrix components such as collagen or fibrinogen. The latter experimental format simulating a more physiologically relevant subendothelial matrices may yield vastly different results. These possibilities warrant further investigation.

Induction of eNOS and modulation of EC growth by resveratrol

].

Conclusions

to elicit morphological and functional changes. Specifically, resveratrol induced an elongated shape, interspersed with long, tortuous projections, in cultured BPAEC. Treated cells resisted detachment from the plastic coverslips under simulated arterial shear stress conditions. These results, combined with data from our previous studies, provide additional support for the notion that resveratrol acts as an anti-atherosclerotic agent. Whether these findings can be extrapolated to humans require further study.

Cell culture and treatment with resveratrol

cells/ml, were added to each of the wells. Twenty-four h after seeding, the coverslips were treated with 0,10, 25, 50, and 100 μM resveratrol and maintained for 2 days at 37°C until they were fixed and stained for confocal microscopy.

Preparation and staining of slides

Coverslips containing adhered BPAEC were washed for 5 min in TBS, fixed with 5% glutaraldehyde in 0.1 M cacodylate (pH 7.4) for 15 min, washed again for 5 min in TBS, then stored in 0.1 M cacodylate/7% sucrose at 4°C until staining. Cells were permeabilized with 0.1% Triton X-100/TBS for 3 min, washed twice with TBS, blocked twice for 3 min with 0.1% bovine serum albumin in TBS, and incubated with rhodamine-conjugated phalloidin for 20 min. Routinely, 5 μl of rhodamine-phalloidin, dissolved in 200 μl TBS, was used for each coverslip stained. The coverslips were wash with 0.1% BSA/TBS for 3 min, then mounted in an inverted position onto TBS-containing beveled glass slides and sealed with clear nail polish.

Perfusion

was achieved based on the dimensions of the flow chamber slit and a constant flow rate of 10 ml/min. After preincubation at 37°C, minimal medium was drawn directly by an eight roller peristaltic pump through the perfusion chamber containing the BPAEC-covered coverslip positioned 70 mm from the inlet valve at a rate of 10 ml/min for either 2 min or 5 min. A recirculating system was employed to recycle the medium after passing through the chamber.

Confocal microscopy

Prepared slides containing adhered BPAEC were examined using a BioRad confocal microscope. The cells were imaged following excitation of rhodamine-phalloidin with an argon laser beam at a wavelength of 554 nm using either an inverted- or an uprighted-staged microscope equipped with either a 10X or a 20X oil-free objective and an epifluorescent illumination attachment. Cells viewed in representative field screen captures were counted manually for cell morphology and classified as either normal or elongated.

Inhibitor study

]. Stock solutions were prepared by dissolving the inhibitors in DMSO. The inhibitors used and their stock solutions were as follows: quin2-AM (Sigma, 2 mM), herbimycin A (GibcoBRL, 0.175 mM), chelerythrine (Sigma, 0.4 mM), cytochalasin D (Sigma, 8 μM), nocodazole (Sigma, 0.66 mM). Quin2-AM, herbimycin A, chelerythrine, and cytochalasin D were stored at -20°C, and nocodazole was stored at 4°C. The stock solutions were diluted 1:200 in MEM to give the following final concentrations: quin2-AM (10 μM), herbimycin A (875 nM), chelerythrine (2 μM), cytochalasin D (40 nM), nocodazole (3.3 μM). Inhibitors were applied 24 h prior to the addition of resveratrol, except for quin2-AM, which was applied 1 h prior to resveratrol treatment. Since cells treated with various inhibitors grew less well, compared to controls, treatment with 100 μM resveratrol was reduced to approximately 40 h. Fixing and staining of cells for confocal microscopy was as described above.

Western blot analysis

, 1 mM dithiothreitol (DTT), 0.5% NP40, 5% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 10 μg/ml each of the protease inhibitors aprotinin, pepstatin, and leupeptin. Cell-free extracts were obtained by centrifugation in a microcentrifuge. Lysates (7-10 μg) from control and treated cells were separated on 10% SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes, and the membranes were incubated with the respective primary and secondary antibodies. The monoclonal primary antibodies used and their dilutions were as follows: Phosphorylated, active ERK1/2-P (RBI/Sigma, 1:1000), total ERK1/2 (RBI/Sigma, 1:1000), eIF4E (Santa Cruz, 1:250), eNOS (Sigma 1:1000). Membranes were probed with alkaline phosphatase-conjugated IgG (Santa Cruz, 1:1500) or horseradish peroxidase-conjugated IgG (Santa Cruz, 1:2000). Specific immunoreactive bands were identified by color reaction or enhanced chemiluminescence, respectively.

Acknowledgements

Supported in part by the Vivien Wu-Au Memorial Cancer Research Fund and an unrestricted grant from the Philip Morris Company to J.M.W., and by grant 9816832 from the National Science Foundation to K.M.L.