Plumbagin reverses proliferation and resistance to apoptosis in experimental PAH.

Like cancer, pulmonary arterial hypertension (PAH) is characterised by a pro-proliferative and anti-apoptotic phenotype. In PAH, pulmonary artery smooth muscle cell (PASMC) proliferation is enhanced and apoptosis suppressed. The sustainability of this phenotype requires the activation of pro-survival transcription factors, such as signal transducer and activator of transcription (STAT)3 and nuclear factor of activated T-cells (NFAT). There are no drugs currently available that are able to efficiently and safely inhibit this axis. We hypothesised that plumbagin (PLB), a natural organic compound known to block STAT3 in cancer cells, would reverse experimental pulmonary hypertension. Using human PAH-PASMC, we demonstrated in vitro that PLB inhibits the activation of the STAT3/NFAT axis, increasing the voltage-gated K(+) current bone morphogenetic protein receptor type II (BMPR2), and decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)), rho-associated coiled-coil containing protein kinase (ROCK)1 and interleukin (IL)-6, contributing to the inhibition of PAH-PASMC proliferation and resistance to apoptosis (proliferating cell nuclear antigen (PCNA), TUNEL, Ki67 and anexine V). In vivo, PLB oral administration decreases distal pulmonary artery remodelling, mean pulmonary artery pressure and right ventricular hypertrophy without affecting systemic circulation in both monocrotaline- and suden/chronic hypoxia-induced PAH in rats. This study demonstrates that the STAT3/NFAT axis can be therapeutically targeted by PLB in human PAH-PASMC and experimental PAH rat models. Thus, PLB could be considered a specific and attractive future therapeutic strategy for PAH.

The nuclear factor of activated T cells in pulmonary arterial hypertension can be therapeutically targeted.

In pulmonary arterial hypertension (PAH), antiapoptotic, proliferative, and inflammatory diatheses converge to create an obstructive vasculopathy. A selective down-regulation of the Kv channel Kv1.5 has been described in human and animal PAH. The resultant increase in intracellular free Ca(2+) ([Ca(2+)](i)) and K(+) ([K(+)](i)) concentrations explains the pulmonary artery smooth muscle cell (PASMC) contraction, proliferation and resistance to apoptosis. The recently described PASMC hyperpolarized mitochondria and increased bcl-2 levels also contribute to apoptosis resistance in PAH. The cause of the Kv1.5, mitochondrial, and inflammatory abnormalities remains unknown. We hypothesized that these abnormalities can be explained in part by an activation of NFAT (nuclear factor of activated T cells), a Ca(2+)/calcineurin-sensitive transcription factor. We studied PASMC and lungs from six patients with and four without PAH and blood from 23 PAH patients and 10 healthy volunteers. Compared with normal, PAH PASMC had decreased Kv current and Kv1.5 expression and increased [Ca(2+)](i), [K(+)](i), mitochondrial potential (Delta Psi m), and bcl-2 levels. PAH but not normal PASMC and lungs showed activation of NFATc2. Inhibition of NFATc2 by VIVIT or cyclosporine restored Kv1.5 expression and current, decreased [Ca(2+)](i), [K(+)](i), bcl-2, and Delta Psi m, leading to decreased proliferation and increased apoptosis in vitro. In vivo, cyclosporine decreased established rat monocrotaline-PAH. NFATc2 levels were increased in circulating leukocytes in PAH versus healthy volunteers. CD3-positive lymphocytes with activated NFATc2 were seen in the arterial wall in PAH but not normal lungs. The generalized activation of NFAT in human and experimental PAH might regulate the ionic, mitochondrial, and inflammatory remodeling and be a therapeutic target and biomarker.

Oxygen activates the Rho/Rho-kinase pathway and induces RhoB and ROCK-1 expression in human and rabbit ductus arteriosus by increasing mitochondria-derived reactive oxygen species: a newly recognized mechanism for sustaining ductal constriction.

BACKGROUND: Constriction of the ductus arteriosus (DA) is initiated at birth by inhibition of O2-sensitive K+ channels in DA smooth muscle cells. Subsequent membrane depolarization and calcium influx through L-type calcium channels initiates functional closure. We hypothesize that Rho-kinase activation is an additional mechanism that sustains DA constriction. METHODS AND RESULTS: The effect of increased PO2 on the activity and expression of Rho-kinase was assessed in DAs from neonates with hypoplastic left-heart syndrome (n=15) and rabbits (339 term and 99 preterm rabbits). Rho-kinase inhibitors (Y-27632 and fasudil) prevent and reverse O2 constriction. Heterogeneity exists in the sensitivity of constrictors (PO2=endothelin=phenylephrine>KCl) and of fetal vessels (DA=pulmonary artery>aorta) to Rho-kinase inhibition. Inhibition of L-type calcium channels (nifedipine) or removal of extracellular calcium inhibits approximately two thirds of O2 constriction. Residual DA constriction reflects calcium sensitization, which persists after removal of extracellular calcium and blocking of sarcoplasmic reticulum Ca2+-ATPase. In term DA, an increase in PO2 activates Rho-kinase and thereby increases RhoB and ROCK-1 expression. Activation of Rho-kinase in DA smooth muscle cells is initiated by a PO2-dependent, rotenone-sensitive increase in mitochondrion-derived reactive O2 species. O2 effects on Rho-kinase are mimicked by exogenous H2O2. In preterm DAs, immaturity of mitochondrial reactive oxygen species generation is associated with reduced and delayed O2 constriction and lack of PO2-dependent upregulation of Rho-kinase expression. CONCLUSIONS: O2 activates Rho-kinase and increases Rho-kinase expression in term DA smooth muscle cells by a redox-regulated, positive-feedback mechanism that promotes sustained vasoconstriction. Conversely, Rho-kinase inhibitors may be useful in maintaining DA patency, as a bridge to congenital heart surgery.