The Amiloride Derivative Phenamil Attenuates Pulmonary Vascular Remodeling by Activating NFAT and the Bone Morphogenetic Protein Signaling Pathway

In response to vascular injury, vascular smooth muscle cells (vSMCs) undergo a unique process known as “phenotype modulation,” the transition from a quiescent, “contractile” phenotype to a proliferative, “synthetic” state (31, 32). Phenotypic plasticity is essential for vascular development and vascular repair after injury. However, aberrant switching from the contractile to synthetic phenotype, characterized by increased vSMC proliferation, increased migration, and decreased ability to contract, underlies the formation of various proliferative vascular disorders, including atherosclerosis, postangioplasty restenosis, lymphangioleiomyomatosis (LAM), and pulmonary artery hypertension (PAH) (40, 45). The transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) pathways play essential roles in cardiac myogenesis (24, 39), vasculogenesis (25), and angiogenesis. In vSMCs, TGF-β and BMP have been shown to promote the contractile phenotype and inhibit switching to the synthetic phenotype (20). Both TGF-βs and BMPs inhibit vSMC proliferation and migration and increase contractile vSMC gene expression (16, 20, 22). Loss-of-function mutations of the genes encoding receptors of TGF-βs and BMPs have been linked to vascular disorders, such as idiopathic PAH (IPAH) and hereditary hemorrhagic telangiectasia (41). Furthermore, BMP and TGF-β signaling is reduced when phenotype switching is induced both in cell culture by platelet-derived growth factor (PDGF) stimulation (5) or in vivo by using the monocrotaline (MCT) model (37) and chronic hypoxia (5). Together, these results suggest that inhibition of TGF-β or BMP signaling plays an important role in the pathogenesis of proliferative and obliterative vascular diseases. Phenamil, along with benzamil, dimethyl amiloride (DMA), and ethyl isopropyl amiloride (EIPA), are derivatives of the diuretic amiloride, and inhibitors of ion transporters. It has been demonstrated that dimethyl amiloride, a potent and specific inhibitor of the Na+/H+ transporter, can inhibit pulmonary artery smooth muscle cell (PASMC) proliferation in vitro and chronic-hypoxia-induced pulmonary hypertension in rats (35, 36). Unlike DMA, phenamil, benzamil, and amiloride are potent blockers of Na+ channels, including epithelial sodium channels (ENaCs) and acid-sensing ion channels (ASICs). vSMCs express low levels of ENaCs, but ASIC (acid-sensing ion channel protein 1 [ASIC1] to ASIC3) expression and function has been demonstrated (14). ASICs are essential for vSMC migration both at basal and PDGF-stimulated conditions (14). ASIC1 protein level is also elevated in pulmonary arteries under chronic-hypoxia treatment (17). The nuclear factor of activated T cell (NFAT) family of transcription factors, which includes five NFAT proteins (NFAT1 to NFAT5) and their splice variants, are known to regulate a range of genes in response to increases in the intracellular free calcium levels. NFATs were originally identified as transcription regulators in lymphoid cells, but it has since been shown that they play a critical role in a variety of cells, including vSMCs (21). At steady state, NFATs are phosphorylated, excluded from the nucleus, and thus inactive. Upon increase of the intracellular calcium concentration, calcineurin, a calmodulin-dependent phosphatase, dephosphorylates NFAT1 to NFAT4 proteins, which promotes nuclear translocation and association with DNA (21). A recent study suggested that phenamil facilitates BMP2-induced osteoblastic differentiation and mineralization through increased expression of Tribbles 3 (Trb3) protein in the mesenchymal stem cell line M2-10B4 cells (33). We previously identified Trb3 as a potent and crucial positive modulator of phenotype switch in vSMCs. Increased Trb3 was shown to promote the degradation of Smad ubiquitin regulatory factor 1 (Smurf1), a negative regulator of BMP Smad-dependent signaling (6). In comparison, the prosynthetic PDGF cytokine promotes downregulation of Trb3, which results in decreased Smad protein levels and vSMC contractile gene expression (5, 6). The pro-BMP signaling and thus procontractile function of Trb3 in vSMCs is inhibited when BMP type II receptor (BMPRII) is mutated in the same manner as in some IPAH patients. Furthermore, PASMCs from rats subjected to chronic hypoxia show decreased Trb3 protein expression and corresponding decreased BMP signaling. These results suggest that control of Trb3 expression and activity plays an important role in the careful control of the switch between the synthetic and contractile phenotypes. In this study, we are interested in determining whether a drug-induced increase in Trb3 expression can be used as a therapeutic strategy in models of PAH. We observed that phenamil induces Trb3 expression and attenuates hypoxia-induced PAH and vascular remodeling in rats. We demonstrate that in cultured pulmonary vSMCs, phenamil promotes transcriptional activation of Trb3 by activating the calcineurin-NFAT pathway through elevation of intracellular calcium concentration. Phenamil facilitates the procontractile effect of BMP signaling and the maintenance of a contractile phenotype. These data establish an ameliorating effect of elevated BMP signal and maintenance of the contractile phenotype in vSMCs during vascular remodeling via induction of Trb3.