Results of our studies suggest that resveratrol, an abundant red wine polyphenol, interacts with endothelial cells in vitro to result in morphologic and functional changes. The elongated shape, interspersed with long, tortuous projections displayed by resveratrol-treated BPAEC (Fig. 1,2,3) has apparent functional significance since these cells resisted detachment from the plastic coverslips under simulated arterial shear stress conditions (Fig. 4). The resveratrol-induced cellular changes could represent a mechanism of minimizing endothelial damage by shearing forces in vivo. Moreover, resistance to detachment could also make endothelial cells less likely to dislodge and become part of a growing thrombotic plug.
Modulation of endothelial cell shape by resveratrol
Size, shape, mutual orientation, and intercellular contacts in endothelium are not incidental and statistic events, but are precisely and dynamically regulated. Reversible change of endothelial cell shape, mutual orientation of cells in certain directions, changes in intercellular contacts, are controlled by a number of factors, such as, cooperative stimulation of the receptors or the systems of second messengers [32,33], dietary ingredients, e.g., retinoids , exercise [35,36], and fluid-imposed shear stress [37,38,39]. For example, endothelial cells are known to elongate and reorient their cytoskeletons in the direction of flow as a normal physiological response to prolonged shear stress . Results of the present studies show that treatment with resveratrol induces a morphologic change from a stellar to an elongated shape (Figure 1). 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.
The mechanism underlying this cytoskeletal rearrangement due to shear stress is ill-defined, but it has been found to be linked to tyrosine kinase activity, levels of intracellular calcium, intact actin microfilaments, and functional microtubule network, but is independent of protein kinase C, intermediate filaments, and stretch- and shear-activated mechanosensitive K+ channels . We therefore sought to determine whether a similar mechanism could account for the resveratrol-mediated endothelial shape change. Our approach involved using a number of selective inhibitors for various signaling pathway. These studies showed that quin2-AM ([Ca2+] inhibitor), added together with resveratrol, clearly abolished the endothelial cell shape change induced by the polyphenol. In contrast, chelerythrine, a PKC inhibitor, which added alone triggered a significant shape change in BPAEC, had no effect on resveratrol-elicited morphological change (Table 1). Analyzed as a whole, these experiments suggest that the change in cellular phenotype, as a consequence of resveratrol:EC interaction, is dependent on Ca2+, 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.
Prolonged shear stress leading to mechanotransduction signaling has been shown to activate the MAP kinase pathway in EC. Activation of ERK1 and ERK2, the major components of the MAPK pathway, presumably induces shear-specific transcription factors, such as c-fos/c-jun and NFκB, ultimately leading to changes in gene expression [30,31]. Western blot analysis of control and 25 μM and 100 μM resveratrol-treated BPAEC showed that there was no increase in active ERK 1/2 at the 6 h and 2 day time points. Rather, the decline in activated ERK 1/2 seen from days 2 to 4 of control cells, was effectively suppressed by resveratrol, suggesting that the polyphenol may act by affecting the stability of active ERK1/2 (Figure 6). This may involve modulation of MAPK phosphatase-1 (MKP-1), a member of the immediate-early response gene product functioning as a dual specificity phosphase to reverse MAPK [40,41,42,43,44], by resveratrol. Previously, it has been reported that MKP-1 is rapidly induced in rat carotid arterial wall following balloon catheter injury . 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
Another significant contribution of the present research is the demonstration that resveratrol promoted a greater adherence of BPAEC to the cultured vehicle in vitro. In vivo, 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
The endothelial cell lining of the blood vessel is extremely sensitive to damage from reactive oxygen species (ROS), the results of which are losses of both microvascular metabolic function and barrier properties . To minimize such oxidant damage, cells rely on the production of nitric oxide (NO) by the enzyme nitric oxide synthase (eNOS). Biological functions attributed to NO include vasodilation , inhibition of platelet adhesion and aggregation , reduction of expression of adhesion molecules and chemokines [49,50,51,52], and suppression of cell growth and migration [53,54]. Therefore, we investigated whether resveratrol treatment could lead to increased eNOS expression in endothelial cells. We determined that resveratrol treatment did induce eNOS expression at all time points tested (6 h, 2 days, and 4 days). These results agreed with a previous report from this laboratory . The peak expression of eNOS occurred at 2 days in 100 μM resveratrol-treated cells. These findings could mean that dietary resveratrol is capable of providing a gradual yet sustained increase in NO. The ability of resveratrol to induce S/G2 growth arrest [20,21] suggests an additional mechanism for cells to rapidly and efficiently repair any endothelial cell damage. Overall, the collective effect of resveratrol would be to decrease endothelial injury and exposure of the subendothelial matrix, which would lessen the probability of formation of atherosclerotic plaques and the development of CHD [53,54,55].