Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration ([Ca(2+)](i)) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca(2+) dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, [Ca(2+)](i), and migration in PASMCs. Blockade of Ca(2+) entry through voltage-dependent Ca(2+) or nonselective cation channels prevented the hypoxia-induced increase in PASMC [Ca(2+)](i), AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in [Ca(2+)](i) that results in increased AQP1 protein levels and cell migration.

Chronic hypoxia is an inciting factor for the development of pulmonary arterial hypertension. The mechanisms involved in the development of hypoxic pulmonary hypertension (HPH) include hypoxia-inducible factor 1 (HIF-1)-dependent transactivation of genes controlling pulmonary arterial smooth muscle cell (PASMC) intracellular calcium concentration ([Ca 2+ ] i ) and pH. Recently, digoxin was shown to inhibit HIF-1 transcriptional activity.In this study, we tested the hypothesis that digoxin could prevent and reverse the development of HPH. Mice were injected daily with saline or digoxin and exposed to room air or ambient hypoxia for 3 wk. Treatment with digoxin attenuated the development of right ventricle (RV) hypertrophy and prevented the pulmonary vascular remodeling and increases in PASMC [Ca 2+ ] i , pH, and RV pressure that occur in mice exposed to chronic hypoxia. When started after pulmonary hypertension was established, digoxin attenuated the hypoxia-induced increases in RV pressure and PASMC pH and [Ca 2+ ] i . These preclinical data support a role for HIF-1 inhibitors in the treatment of HPH.acriflavine | cardiac glycosides | pulmonary circulation P rolonged exposure to hypoxia occurs in physiological and pathological contexts, such as during a high-altitude sojourn or as a result of chronic obstructive pulmonary disease, respectively. Chronic hypoxia (CH) triggers maladaptive responses in the lung vasculature, leading to the development of hypoxic pulmonary hypertension (HPH) (1). Typically, HPH results from arteriolar constriction followed by vascular wall remodeling, which includes both thickening of the wall due to smooth muscle cell (SMC) and fibroblast proliferation, as well as extension of SMCs into previously nonmuscular precapillary arterioles. Over time, HPH causes right ventricle hypertrophy (RVH), which can lead to right ventricular (RV) failure and death.Although incompletely understood, some of the alterations in pulmonary arterial SMCs (PASMCs) that underlie the development of HPH have been delineated (2, 3). For example, in HPH, alterations in Ca 2+ and pH homeostasis contribute to growth and contraction of PASMCs. Our previous work demonstrated that HPH is characterized by increased PASMC intracellular pH (pH i ) due to increased activity and expression of Na + /H + exchanger isoform 1 (NHE1) (4, 5), and elevated intracellular Ca 2+ concentration ([Ca 2+ ] i ) (6) due to increased expression of canonical transient receptor potential (TRPC) proteins and enhanced Ca 2+ entry through nonselective cation channels (7). Critical aspects of the pathogenesis of HPH, including both the increase in basal [Ca 2+ ] i and alkalinization of PASMCs, are mediated by hypoxia-inducible factor 1 (HIF-1) (5, 7-9).HIF-1 is a heterodimeric transcription factor composed of HIF-1α and HIF-1β subunits that regulates the expression of hundreds of genes in response to hypoxia, including many genes associated with HPH (10). HIF-1β is ubiquitously expressed, whereas HIF-1α expression is O 2 -r...

Pisarcik S, Maylor J, Lu W, Yun X, Undem C, Sylvester JT, Semenza GL, Shimoda LA. Activation of hypoxia-inducible factor-1 in pulmonary arterial smooth muscle cells by endothelin-1. Am J Physiol Lung Cell Mol Physiol 304: L549 -L561, 2013. First published February 15, 2013 doi:10.1152/ajplung.00081.2012.-Numerous cellular responses to hypoxia are mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 plays a central role in the pathogenesis of hypoxic pulmonary hypertension. Under certain conditions, HIF-1 may utilize feedforward mechanisms to amplify its activity. Since hypoxia increases endothelin-1 (ET-1) levels in the lung, we hypothesized that during moderate, prolonged hypoxia ET-1 might contribute to HIF-1 signaling in pulmonary arterial smooth muscle cells (PASMCs). Primary cultures of rat PASMCs were treated with ET-1 or exposed to moderate, prolonged hypoxia (4% O2 for 60 h). Levels of the oxygen-sensitive HIF-1␣ subunit and expression of HIF target genes were increased in both hypoxic cells and cells treated with ET-1. Both hypoxia and ET-1 also increased HIF-1␣ mRNA expression and decreased mRNA and protein expression of prolyl hydroxylase 2 (PHD2), which is the protein responsible for targeting HIF-1␣ for O2-dependent degradation. The induction of HIF-1␣ by moderate, prolonged hypoxia was blocked by BQ-123, an antagonist of ET-1 receptor subtype A. The effects of ET-1 were mediated by increased intracellular calcium, generation of reactive oxygen species, and ERK1/2 activation. Neither ET-1 nor moderate hypoxia induced the expression of HIF-1␣ or HIF target genes in aortic smooth muscle cells. These results suggest that ET-1 induces a PASMC-specific increase in HIF-1␣ levels by upregulation of HIF-1␣ synthesis and downregulation of PHD2-mediated degradation, thereby amplifying the induction of HIF-1␣ in PASMCs during moderate, prolonged hypoxia. calcium; prolyl hydroxylase; pulmonary hypertension IN A VARIETY OF CHRONIC lung diseases, the pulmonary circulation is exposed to prolonged periods of hypoxia, often resulting in the development of pulmonary hypertension. Numerous studies have described the structural and functional changes that occur in the pulmonary circulation in response to chronic hypoxia (60). Structural remodeling, characterized by pulmonary arterial smooth muscle cell (PASMC) proliferation, intimal thickening, and extension of muscle into previously nonmuscular arterioles, is commonly observed with pulmonary hypertension (23,40). Changes in the vascular wall are accompanied by active contraction of vessels, evidenced by acute reduction in pulmonary arterial pressure in response to vasodilatory agents (42, 44). These pulmonary vascular changes result, in large part, from altered expression of genes encoding ion channels and transporters that control PASMC ion homeostasis, including increased expression of Na ϩ /H ϩ exchanger isoform 1 (NHE1) and the canonical transient receptor potential (TRPC) family members TRPC1 and TRPC6, as well as reduced levels of mRNA...

Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pHi), typically due to activation of Na+/H+ exchange (NHE). In the systemic vasculature, ET-1 increases pHi, Na+/H+ exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pHi and smooth muscle growth in the lung; however, the effect of ET-1 on basal pHi and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3–5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pHi and NHE activity induced by increasing concentrations of ET-1 (10−10 to 10−8 M). We found that application of exogenous ET-1 increased pHi and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pHi or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pHi.

Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca(2+)](i)) occurring through activation of voltage-dependent Ca(2+) channels (VDCC) even though ET-1-induced depolarization via inhibition of K(+) channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca(2+)](i) in PASMCs from rats exposed to CH (10% O(2), 3 wk) using the Ca(2+)-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca(2+)](i) by >70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca(2+)](i) to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca(2+)](i) in PASMCs occurs via Ca(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.

Increased PASMC contraction and growth during chronic hypoxia (CH) may be due to cytoskeletal rearrangement involving transmembrane and cytosolic proteins. We found that the ion transporter, NHE1, is upregulated in chronically hypoxic rats (10% O2; 3 wk) and is required for development of hypoxic pulmonary hypertension. In addition to regulating pH, NHE1 was recently found to bind phosphorylated ezrin (p‐ezrin), an actin filament binding protein. NHERF1 also binds p‐ezrin, at the same site as NHE1. Since the transmembrane spanning NHE1 can act as an anchor, but cytosolic NHERF1 cannot, we hypothesized that changes in NHE1/ezrin interactions during hypoxia enables actin filaments to be tethered to the cell membrane, promoting PASMC contraction and growth. Confocal images of immunofluorescent stained chronically hypoxic mouse lung sections confirmed co‐localization of p‐ezrin and smooth muscle cell‐specific α‐actin (SM‐actin). Immunoblots showed increased NHE1, increased p‐ezrin and decreased NHERF1 levels in response to hypoxia. Co‐immunoprecipitation studies showed increased NHE1 binding to p‐ezrin and SM‐actin with hypoxia (4% O2; 24 hr), and decreased NHERF1 binding. These results suggest reciprocal regulation of NHERF1 and NHE1 expression in PASMC during hypoxia, with both proteins competing to bind p‐ezrin. Increased NHE1/ezrin interactions may contribute to hypoxic pulmonary hypertension.

Prolonged exposure to hypoxia causes pulmonary hypertension due to contraction, proliferation and migration of PASMCs, which result from hypoxia‐induced elevations in basal intracellular calcium concentration ([Ca2+]i). Calpain, a Ca2+‐dependent cysteine protease, has two major isoforms, μ‐ and m‐calpain, that require μM and mM [Ca2+]i, respectively, for activation. Calpain is known to participate in fibroblast and endothelial cell migration and proliferation, and PASMCs exhibit increased calpain levels during hypoxia (Ma et al, JCI 2011). In this study, we hypothesized that reducing calpain activity through in vivo overexpression of calpastatin (CAST), an endogenous calpain inhibitor, or in vitro inhibition with the pharmacological agent, calpeptin, would impair migration of PASMCs. For in vivo experiments, wild type (WT) and CAST overexpressing mice were exposed to normoxia or chronic hypoxia (10% O2; 21 d). For in vitro experiments, PASMCs were isolated from WT or CAST mice before exposure to normoxia or hypoxia (4% O2; 24 h). PASMC migration was measured using a modified Boyden chamber transwell assay. For both in vitro and in vivo experiments, WT, but not CAST, PASMCs exhibited increased migration in response to hypoxia. In WT PASMCs, calpeptin prevented hypoxia‐induced migration. These results suggest that calpain activity is necessary to facilitate the migration of hypoxic PASMCs.

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