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TGF-β signaling is critical for differentiation and survival of SMCs from NCSCs and for maintaining the contractile phenotype late in life (Humphrey et al., 2014; Mao et al., 2012; Xie et al., 2013). In NCSCs, p-Smad2 interacts with the myocardin-related transcription factor to regulate SMC marker gene expression. Deletion of Smad2 results in defective differentiation of NC cells into vascular SMCs in aortic arch MM-102 (Xie et al., 2013). TGF-β upregulates MYOCD expression, which is a master regulator of the expression of smooth muscle marker genes, including MYH11 (Davis-Dusenbery et al., 2011; Wang et al., 2003). In the SMCs derived from BAV-NCSCs, the signaling of the TGF-β canonical pathway significantly decreased as manifested by decreased pSMAD2 and CTGF. MYOCD expression levels were also downregulated. The decreased TGF-β signaling could impair the expression of MYOCD and thus down regulate SMC marker gene expression, including MYH11. Interestingly, two groups have reported separately that impaired canonical TGF-β signaling was also found in the ascending aortic tissue in BAV patients, and might potentiate the development of aneurysms in BAV (Grewal et al., 2014a; Paloschi et al., 2015). Our in vitro model provided another piece of evidence that defective TGF-β signaling might be the cause of the defective differentiation of NCSCs and the observed aortopathy in the BAV patients with ascending aortic aneurysm. On the other hand, impairment of the TGF-β pathway in SMCs resulted in hyperactive mTOR pathway and caused aneurysms in a SMCs conditional Tgfbr2 knockout mouse model, and rapamycin administration prevented aortic dilation (Li et al., 2014). In a Tsc2 knockout mouse model, elevated mTOR signaling decreased expression of SMCs contractile genes, and rapamycin promoted SMC contractile gene expression and prevented aneurysms (Cao et al., 2010). SMC differentiation is promoted by activating phosphatidylinositol 3-kinase (PI3K) and Akt which are negatively regulated by mTOR and S6 kinase 1 (S6K1). Rapamycin promotes SMC differentiation by inhibiting mTOR, S6K1 and activating Akt2 (Martin et al., 2007). In the BAV/TAA NCSC SMCs, we also found upregulation of phosphorylated S6, indicating upregulation of the mTOR pathway, which may also contribute to the downregulation of the contractile gene SMMHC expression. Also, the finding that rapamycin promotes MYH11 expression and contractile function in the BAV/TAA patient derived NCSC-derived SMCs raises the possibility that rapamycin might be a potent therapeutic drug for BAV associated aneurysms. The following are the supplementary data related to this article.
Conflict of Interest Statement
Funding Sources This study was supported by AATS Graham Foundation, Thoracic Surgery Foundation for Research and Education, and McKay Award, University of Michigan.
Author Contributions
Acknowledgements
Introduction Clevidipine is a 3rd generation dihydropyridine (DHP) that lowers blood pressure via selective antagonism of peripheral vascular smooth muscle (VSM) voltage-gated l-type calcium channels (CaV1.2) (Fig. 1A). Recently, clinical observations suggest that the mechanisms of clevidipine action are more complex than simple antagonism of peripheral VSM CaV1.2. In a clinical trial examining the safety and efficacy of clevidipine for blood pressure (BP) reduction in hypertensive acute heart failure (AHF), clevidipine showed a superior dyspnea-relieving benefit versus standard of care intravenous (IV) antihypertensives, including another DHP (Peacock et al., 2014). When compared to standard of care, dyspnea relief in patients receiving clevidipine was more robust and faster, and this benefit was only partially related to the blood pressure lowering actions of clevidipine. Importantly, this benefit was not conferred by nicardipine, another l-type blocking DHP agent. Clevidipine is also a potent pulmonary vasodilator with most of the PVR reduction occurring at doses that are lower than those needed to cause the majority of the reduction of systemic blood pressure (Cleviprex package insert and (Kieler-Jensen et al., 2000)) (Fig. 1A). Clevidipine effects on PVR as measured by overall response rate and magnitude of reduction are equivalent to the nitrovasodilators and clevidipine is also effective in nitrate non-responders (Nordlander et al., 2004). Lastly, similar to the observed potency increase for clevidipine effects on pulmonary vascular resistance (PVR), clevidipine has a spasmolytic effect in peripheral arteries that occurs at doses that are minimally effective for BP reduction (Huraux et al., 1997; Patel et al., 2012). This dose-response relationship between spasmolysis and BP reduction is not shared by other DHPs (Schmidt et al., 2010). In this study, we applied a reverse translational approach to understand these non-BP lowering effects of clevidipine by conducting further basic science studies on an already approved clinical drug.