 by Ang II.
Inhibition of FPPS modulates the Ang II-induced MAPK activation in cultured VSMCs
Treatment with Ang II 0.1 μM for 15 min triggered the phosphorylation of p38, JNK and ERK1/2 in the VSMCs, which is in accordance with the findings of a previous study (7). To investigate whether the activation of all 3 MAPKs is involved in the molecular mechanisms underlying the ability of Iban to suppress the Ang II-induced increase in the expression of CTGF, the VSMCs were treated with specific inhibitors of MAPKs (SB203580 for p38, PD98059 for ERK1/2 and SP600125 for JNK) pior to stimulation with Ang II. Both the p38 inhibitor and JNK inhibitor diminished the Ang II-induced production of CTGF (Fig. 5). Furthermore, pre-incubation with Iban inhibited the Ang II-induced activation of p38 and JNK, but not that of ERK1/2 (Fig. 6), suggesting that the activation of p38 and JNK is involved in the downregulation of CTGF by Iban. These data indicate that the inhibition of FPPS modulates the Ang II-induced production of CTGF through the regulation of the p38 MAPK and JNK pathways in cultured VSMCs.
Discussion
In the present study, we demonstrated that FPPS expression is induced by Ang II in cultured VSMCs from SD rats. Iban, an FPPS inhibitor, inhibited the Ang II-induced cell proliferation and the production of CTGF in the cultured VSMCs. The underlying mechanisms involved the inhibition of RhoA signaling and the modulation of the p38 MAPK and JNK pathways.
Through the inhibition of osteoclastic activity and bone resorption, nitrogen-containing bisphosphonates (N-BPs) including alendronate, Iban and zoledronate are commonly used for the treatment of bone-related diseases in clinical practice (17,18). However, extensive research has focused on their potential effects on cardiovascular diseases. Due to their inhibitory effects on the mevalonate pathway and particularly FPPS, they now act as a tool used to investigate the role of FPPS in various diseases (19,20). FPPS has been implicated in the development of various cardiovascular diseases, including myocardial and vascular remodeling and endothelial dysfunction in SHRs (12–16). The inhibition of FPPS by alendronate or its knockdown by RNA interference has been shown to prevent cardiac hypertrophy and fibrosis both in vivo and in vitro (12–14). Moreover, the cardiac-specific overexpression of FPPS has been shown to induce cardiac hypertrophy and heart failure in mice (15). In addition, in a recent study of ours, we demonstrated that the inhibition of FPPS prevented NE-induced fibrotic responses, including cell proliferation, the hydroxyproline content and CTGF protein expression in SHR-VSMCs (16), indicating that FPPS may function as a potent regulator of vascular remodeling.
The exaggerated VSMC activities and excessive ECM protein accumulation induced by Ang II are thought to be key to the devolepment of vascular remodeling (3–9). A number of pharmacological agents, such as anti-thrombotics, antiplatelet agents, angiotensin-converting enzyme inhibitors, as well as mechanical and cellular approaches have been used in an attempt to attenuate this pathophysiological process (21,22). A growing numbers of studies in the literature have demonstrated that bisphosphonates may play an important role in inhibiting the development of atherosclerosis and neointimal hyperplasia in animal models. These effects are thought to be mediated by the transient systemic inactivation of monocytes and macrophages (23,24). However, in a balloon-injured rat carotid artery model, both the systemic and local delivery of zoledronate was shown to prevent intimal hyperplasia (25). Additionly, in a rabbit carotid anastomosis model, zoledronic acid (ZA), a third generation of N-BPs, was demonstrated to inhibit neointimal hyperplasia and decrease VSMC intensity, suggesting that it may have a direct inhibitory effect on VSMCs (26). Indeed, etidronate, another bisphosphonate, has been reported to exert an inhibitory effect on the growth of VSMCs from SHRs (27). Similarly, in cultured rat VSMCs, ZA has also been reported to inhibit the proliferation, adhesion and migration of VSMCs without the induction of necrosis or apoptosis (28). Recently, ZA was reported to inhibit the growth of stimulated human arotic smooth muscle cells, as well as their proliferation adhesion and migration, but had no effect on quiescent cells. The addition of GGOH significantly reversed the ZA-mediated alteration in cellular viability and RAP1A/B prenylation, suggesting that ZA modulates the mevalonate pathway and inhibits the prenylation of GTPase binding proteins (29). Taken together, the above data point out that BPs or N-BPs may play an important role in vascular remodeling by modulating cell signaling, including the prenylation of small signaling proteins (e.g., Ras, Rac, Rab and Rho) through the mevalonate pathway. However, the underlying mechanisms through which BPs or N-BPs inhibit VSMC activity and prevent intimal hyperplasia in animal models induced by FPPS were not elucidated in the above-mentioned studies.
In our previous study, we reported that Iban inhibited NE-induced fibrotic responses in SHR-VSMCs through the modulation of the Ras kinase and p38 pathways (16). In the present study, we further demonstrated that Iban also inhibits fibrotic responses in VSMCs induced by Ang II. As shown in Fig. 1 and in our previous study (16), Iban alone did not influence cell proliferation and CTGF expression in quiescent VSMCs, but markedly reduced the Ang II-induced increase in cell proliferation and CTGF expression (Figs. 2 and 3). Besides, Ang II upregulated FPPS in the cultured VSMCs. To determine whether the anti-fibrotic effects of Iban depend on the modulation of FPPS, analogues or antagonists of the mevalonate pathway were used in this study. The results revealed that addition of GGOH, but not that of FOH, partly reversed the anti-fibrotic effects of Iban (Fig. 3). In addition, GGTI-286, a specific inhibitor of GGTase I, but not FTI-276 (a selective inhibitor of farnesyltransferase), mimicked the anti-fibrotic effects of Iban (Fig. 3). Since RhoA is a geranylgeranylated protein (30), our results suggest that RhoA signaling is involved in this process.
In a previous study, it was clearly demonstrated that pretreatment with RhoA and ROCK inhibitors diminished the Ang II-induced overexpression of CTGF, indicating that Ang II increases the production of CTGF in VSMCs through the RhoA/ROCK pathway (7). In the present study, the inhibition of FPPS with Iban diminished the Ang II-induced RhoA activation in cultured VSMCs, suggesting that FPPS may be involved in the modulation of Ang II-induced CTGF production through the RhoA/ROCK pathway. This is consistent with the results of other studies on the effects of in vivo and in vitro alendronate treatment with hypertrophic response induced by Ang II in cultured neonatal ventricular myocytes and animal models (12,13). However, the levels of isoprenoid intermediates, such as FOH or GGOH were not measured after the suppression of FPPS. The knockdown of FPPS by RNA interference in VSMCs is another critical issue to be confirmed in view of the role of FPPS in Ang II-mediated vascular remodeling. Besides, the in vivo effect of the vascular-specific interference of FPPS in vascular remodeling needs to be validated in animal models. These limitations require further investigation.
It is well known that MAPKs are a widely distributed group of enzymes. The consisting of 3 isoforms (ERK, p38 and JNK) and play an important role in Ang II-mediated vascular remodeling (7,8,31). To determine whether Iban modulates the Ang II-induced overexpression of CTGF by altering the activity of the MAPK pathway, VSMCs were pre-treated with the specific inhibitor of the MAPK pathway prior to stimulation with Ang II. As shown in Fig. 5, both the JNK and p38 MAPK inhibitor diminished the Ang II-induced production of CTGF. This is consistent with the findings of a pregvious study on the effects of HMG-CoA reductase inhibitors (statins) on the modulation of Ang II-mediated vascular responses (7). Furthermore, as previously described (7) and shown in Fig. 6 in the present study, treatment with Ang II triggered the phosphorylation of all 3 MAPKs. However, only the phosphorylation of JNK and p38 was diminished by treatment with Iban. These data indicate that JNK and p38 MAPK may be involved in the Iban-mediated inhibition of CTGF overexpression induced by Ang II in VSMCs.
In conclusion, in this study, we provide evidence that FPPS expression is elevated in Ang II-stimulated VSMCs. The ihibibition of FPPS by Iban attenuates the Ang II-induced increase in cell proliferation and CTGF expression in VSMCs, and the underlying mechanisms, at least in part, involve the modulation of RhoA activity, and the p38 and JNK pathways.
Acknowledgments
This study was supported by the grants from the Natural Science Foundation of Zhejiang province (Project for Young Scientists, no. LQ13H020004)."Fast service, good food but parking sucks so call your order in ahead of time."
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