The SphK1/S1P axis is a novel pathway in PAH that promotes PASMC proliferation, a major contributor to pulmonary vascular remodeling. Our results suggest that this pathway is a potential therapeutic target in PAH.

Rationale: Recent studies suggest that microRNAs (miRNAs) play important roles in regulation of pulmonary artery smooth muscle cell (PASMC) phenotype and are implicated in pulmonary arterial hypertension (PAH). However, the underlying molecular mechanisms remain elusive.Objectives: This study aims to understand the mechanisms regulating PASMC proliferation and differentiation by microRNA-17z92 (miR-17z92) and to elucidate its implication in PAH. Methods:We generated smooth muscle cell (SMC)-specific miR17z92 and PDZ and LIM domain 5 (PDLIM5) knockout mice and overexpressed miR-17z92 and PDLIM5 by injection of miR-17z92 mimics or PDLIM5-V5-His plasmids and measured their responses to hypoxia. We used miR-17z92 mimics, inhibitors, overexpression vectors, small interfering RNAs against PDLIM5, Smad, and transforming growth factor (TGF)-b to determine the role of miR-17z92 and its downstream targets in PASMC proliferation and differentiation.Measurements and Main Results: We found that human PASMC (HPASMC) from patients with PAH expressed decreased levels of the miR-17z92 cluster, TGF-b, and SMC markers. Overexpression of miR-17z92 increased and restored the expression of TGF-b 3 , Smad3, and SMC markers in HPASMC of normal subjects and patients with idiopathic PAH, respectively. Knockdown of Smad3 but not Smad2 prevented miR-17z92-induced expression of SMC markers. SMC-specific knockout of miR-17z92 attenuated hypoxiainduced pulmonary hypertension (PH) in mice, whereas reconstitution of miR-17z92 restored hypoxia-induced PH in these mice. We also found that PDLIM5 is a direct target of miR-17/20a, and hypertensive HPASMC and mouse PASMC expressed elevated PDLIM5 levels. Suppression of PDLIM5 increased expression of SMC markers and enhanced TGF-b/Smad2/3 activity in vitro and enhanced hypoxia-induced PH in vivo, whereas overexpression of PDLIM5 attenuated hypoxia-induced PH. Conclusions:We provided the first evidence that miR-17z92 inhibits PDLIM5 to induce the TGF-b 3 /SMAD3 pathway, contributing to the pathogenesis of PAH.

Pulmonary arterial hypertension (PAH) is a devastating disease without effective treatment. Despite decades of research and the development of novel treatments, PAH remains a fatal disease, suggesting an urgent need for better understanding of the pathogenesis of PAH. Recent studies suggest that microRNAs (miRNAs) are dysregulated in patients with PAH and in experimental pulmonary hypertension. Furthermore, normalization of a few miRNAs is reported to inhibit experimental pulmonary hypertension. We have reviewed the current knowledge about miRNA biogenesis, miRNA expression pattern, and their roles in regulation of pulmonary artery smooth muscle cells, endothelial cells, and fibroblasts. We have also identified emerging trends in our understanding of the role of miRNAs in the pathogenesis of PAH and propose future studies that might lead to novel therapeutic strategies for the treatment of PAH.

Human pulmonary artery smooth muscle cells (HPASMCs) express both adenosine monophosphate-activated protein kinase (AMPK) α1 and α2. We investigated the distinct roles of AMPK α1 and α2 in the survival of HPASMCs during hypoxia and hypoxia-induced pulmonary hypertension (PH). The exposure of HPASMCs to hypoxia (3% O2) increased AMPK activation and phosphorylation, and the inhibition of AMPK with Compound C during hypoxia decreased their viability and increased lactate dehydrogenase activity and apoptosis. Although the suppression of either AMPK α1 or α2 expression led to increased cell death, the suppression of AMPK α2 alone increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. It also resulted in the decreased expression of myeloid cell leukemia sequence 1 (MCL-1). The knockdown of MCL-1 or MCL-1 inhibitors increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. On the other hand, the suppression of AMPK α1 expression alone prevented hypoxia-mediated autophagy. The inhibition of autophagy induced cell death in HPASMCs. Our results suggest that AMPK α1 and AMPK α2 play differential roles in the survival of HPASMCs during hypoxia. The activation of AMPK α2 maintains the expression of MCL-1 and prevents apoptosis, whereas the activation of AMPK α1 stimulates autophagy, promoting HPASMC survival. Moreover, treatment with Compound C, which inhibits both isoforms of AMPK, prevented and partly reversed hypoxia-induced PH in mice. Taking these results together, our study suggests that AMPK plays a key role in the pathogenesis of pulmonary arterial hypertension, and AMPK may represent a novel therapeutic target for the treatment of pulmonary arterial hypertension.

In the pulmonary vasculature, the endothelial and smooth muscle cells are two key cell types that play a major role in the pathobiology of pulmonary vascular disease and pulmonary hypertension. The normal interactions between these two cell types are important for the homeostasis of the pulmonary circulation, and any aberrant interaction between them may lead to various disease states including pulmonary vascular remodeling and pulmonary hypertension. It is well recognized that the endothelial cell can regulate the function of the underlying smooth muscle cell by releasing various bioactive agents such as nitric oxide and endothelin-1. In addition to such paracrine regulation, other mechanisms exist by which there is cross-talk between these two cell types, including communication via the myoendothelial injunctions and information transfer via extracellular vesicles. Emerging evidence suggests that these nonparacrine mechanisms play an important role in the regulation of pulmonary vascular tone and the determination of cell phenotype and that they are critically involved in the pathobiology of pulmonary hypertension.Keywords: paracrine; myoendothelial injunction; microvesicles; vasoconstriction; vascular remodelingIn the pulmonary vasculature, endothelial cells (ECs) and smooth muscle cells (SMCs) are the key cell types involved in the regulation of vessel diameter in most of the pulmonary vascular tree and thereby they regulate pulmonary arterial pressure and total vascular resistance. ECs, which are strategically located as the innermost layer of the blood vessel and are in contact with the circulating blood, exert a delicate and constant influence on the underlying vascular smooth muscle cells (VSMCs). It is well recognized that the regulation of pulmonary vasoreactivity by the endothelium is predominantly accomplished by paracrine signaling through the release of various vasoactive agents such as nitric oxide (NO), endothelin-1 (ET-1) (1-3), and others (4-8). However, there is increasing evidence that the relationship between the EC and the SMC is not a simple one-way interaction from the endothelium to the SMC; rather, there are complicated interactions between them (9-11). Moreover, the communication patterns are not limited to paracrine signaling; cross-talk through myoendothelial gap junctions (MEJs), extracellular vesicles (EVs), and other mechanisms is critically involved (12)(13)(14)(15)(16)(17)(18)(19)(20). These mechanisms operate in a complicated but co-ordinated manner to maintain the homeostasis of the pulmonary circulation. Under pathological conditions, the interaction between ECs and VSMCs may be chronically altered so that a sustained increase in vasocontractility and abnormal vascular proliferation develops, which leads to high pulmonary artery pressure, vascular remodeling, right ventricular hypertrophy, and the clinical condition, pulmonary hypertension (PH) (1,(21)(22)(23)(24). This article discusses the recent progress in our knowledge of the interactions between ECs and SMCs thr...

Lung cancer is the leading cause of cancer-related death worldwide. Hypoxia is known to increase cancer cell migration and invasion. We have previously reported that hypoxia induces epithelial-mesenchymal transition (EMT) in lung cancer cells. However, it is unknown whether hypoxia promotes lung cancer cell migration and invasion via EMT and whether cyclic AMP (cAMP) dependent protein kinase (PKA) plays a role in this process. We found that hypoxia increased PKA activity and induced mRNA and protein expression of PKA catalytic subunit α (PKACA), and regulatory subunits R1A and R1B. Knockdown of HIF-1/2α prevented hypoxia-mediated induction of PKACA mRNA expression and PKA activity. Inhibition of PKA activity with chemical inhibitors prevented EMT induced by hypoxia and tumor growth factor β1. However, activation of PKA by forskolin and 8-Br-cAMP did not induce EMT. Furthermore, treatment with H89 and knockdown of PKACA prevented hypoxia-mediated, EMT, cell migration, and invasion, whereas overexpression of mouse PKACA rescued hypoxia-mediated migration and invasion in PKACA deficient cancer cells. Our results suggest that hypoxia enhances PKA activity by upregulating PKA gene expression in a HIF dependent mechanism and that PKA plays a key role in hypoxia-mediated EMT, migration, and invasion in lung cancer cells.

Human embryonic stem cells (hESCs) during long-term culture acquire chromosomal changes similar to those occurring in tumorigenesis. This was raised concerns about the progression from hESCs to malignant cells. This study aimed to investigate the changes in chromosomes, cell phenotype, and genes in culture-adapted hESCs to ascertain whether tumorigenic transformation occurred. By cytogenetic analysis we found progressive karyotypic changes from simple to complex in chHES-3, one of the hESC lines established in our laboratory, during a long-term suboptimal culture. We further compared chHES-3 cells at different karyotypic stages in cell surface markers, in vivo differentiation, cell cycle, apoptosis, and gene expression profiles. We found that the karyotypically aberrant chHES-3 had higher S-phase fraction in cell cycle distributions and antiapoptosis ability. In vivo differentiation of karyotypically normal chHES-3 resulted in relatively mature teratoma, whereas karyotypically aberrant chHES-3 formed immature teratoma (grade III), in which more primary neural epithelium was revealed by pathological analysis. The microarray analysis and real-time PCR results showed that some oncogenes were upregulated in karyotypically aberrant chHES-3 cells, whereas the genes related to differentiation were downregulated, and that Wnt signal pathway was activated. In conclusion, chHES-3 cells underwent deregulation of self-renewal and dysfunction of related genes in long-term culture adaptation, leading to malignant transformation.

BackgroundPreviously we found that smooth muscle cell (SMC)‐specific knockout of miR‐17~92 attenuates hypoxia‐induced pulmonary hypertension. However, the mechanism underlying miR‐17~92‐mediated pulmonary artery SMC (PASMC) proliferation remains unclear. We sought to investigate whether miR‐17~92 regulates hypoxia‐inducible factor (HIF) activity and PASMC proliferation via prolyl hydroxylases (PHDs).Methods and ResultsWe show that hypoxic sm‐17~92−/− mice have decreased hematocrit, red blood cell counts, and hemoglobin contents. The sm‐17~92−/− mouse lungs express decreased mRNA levels of HIF targets and increased levels of PHD2. miR‐17~92 inhibitors suppress hypoxia‐induced levels of HIF1α, VEGF, Glut1, HK2, and PDK1 but not HIF2α in vitro in PASMC. Overexpression of miR‐17 in PASMC represses PHD2 expression, whereas miR‐17/20a inhibitors induce PHD2 expression. The 3′‐UTR of PHD2 contains a functional miR‐17/20a seed sequence. Silencing of PHD2 induces HIF1α and PCNA protein levels, whereas overexpression of PHD2 decreases HIF1α and cell proliferation. SMC‐specific knockout of PHD2 enhances hypoxia‐induced vascular remodeling and exacerbates established pulmonary hypertension in mice. PHD2 activator R59949 reverses vessel remodeling in existing hypertensive mice. PHDs are dysregulated in PASMC isolated from pulmonary arterial hypertension patients.ConclusionsOur results suggest that PHD2 is a direct target of miR‐17/20a and that miR‐17~92 contributes to PASMC proliferation and polycythemia by suppression of PHD2 and induction of HIF1α.