Ruby A. Fernandez, Jun Wan, Shanshan Song, Kimberly A. Smith, Yali Gu, Mohammad Tauseef, Haiyang Tang, Ayako Makino, Dolly Mehta, and Jason X.-J. Yuan Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois; Department of Medicine, University of Illinois at Chicago, Chicago, Ilinois; Division of Translational and Regenerative Medicine, Department of Medicine, The University of Arizona College of Medicine, Tucson, Arizona; and Department of Physiology, The University of Arizona College of Medicine, Tucson, Arizona

An increase in cytosolic free Ca2+ ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is not only a major trigger for pulmonary vasoconstriction but also an important stimulus for PASMC proliferation.We have previously shown both extracellular Ca2+‐sensing receptor (CaSR), a G protein‐coupled receptor, and TRPC6, a transient receptor potential cation channel, are upregulated in PASMC from idiopathic pulmonary arterial hypertension (IPAH) patients. Furthermore, extracellular Ca2+‐induced increase in [Ca2+]cyt was enhanced in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) compared to normal patients. However, the implication of increased CaSR and TRPC6 in pulmonary hypertension remains unknown. In this study, our data show the inhibition of CaSR by siRNA significantly inhibited hypoxia‐induced proliferation in both normal and IPAH PASMC. Furthermore, IPAH‐PASMC treated with siRNA targeting CaSR or TRPC6 inhibited Ca2+‐induced Ca2+ entry, suggesting functional coupling of CaSR and TRPC6. In addition, PDGF‐induced proliferation in IPAH‐PASMC was inhibited by blocking either CaSR or TRCP6 via inhibitors NPS2143 and 2‐APB, respectfully, while NPS2143 was unable to block PDGF‐induced proliferation in normal PASMC. The data from this study indicate that upregulation of CaSR is functionally coupled to TRPC6 channels to increase [Ca2+]cyt; the enhanced extracellular Ca2+‐induced increase in [Ca2+]cyt due to upregulation of CaSR and TRPC6 plays an important role in PASMC proliferation. This suggests that CaSR could be a novel therapeutic candidate for the treatment of PH.

Rationale: Sustained pulmonary vasoconstriction and excessive vascular remodeling are major causes of elevated pulmonary vascular resistance which leads to increased pulmonary arterial pressure in patients with pulmonary hypertension. Hypoxic-inducible factor (HIF) and its upstream regulators have been linked to the hypoxia response in vascular remodeling and the development of pulmonary hypertension. In this study, we aimed at defining whether increased HIF1α and/or HIF2α, due to endothelial cell specific deletion of prolyl hydroxylase domain protein 2 (PHD2) under normoxic condition are involved in or required for the initiation and progression of pulmonary hypertension. Methods: PHD2, HIF1α and HIF2α conditional knockout mice were created. Right ventricle systolic pressures (RVSP), right ventricular hypertrophy by RV/(LV+S) ratios, and small pulmonary artery smooth muscle layer thickness were measured. Pulmonary arterial smooth muscle cells (PASMCs) and pulmonary arterial endothelial cells (PAECs) were isolated from wild type (WT) or knockout (KO) mice, followed with cell-based assays. Results: We report here that mice with targeted deletion of PHD2 developed severe pulmonary hypertension under normoxic condition. Conditional and inducible deletion of HIF2α in endothelial cells, but not smooth muscle cells, dramatically protected mice from hypoxia-induced pulmonary hypertension. HIF2α KO mice had significantly lower RVSP, RV/(LV+S) ratios, and displayed less pulmonary vascular remodeling when exposed to hypoxia compared to their WT mice. Conclusion: This work shows that the endothelium is responsible for the development of pulmonary hypertension and it demonstrates a crucial role of PHD2/HIF signaling for hypoxic response in pulmonary hypertension. These findings unveil temporally and spatially distinct functions for HIFs in the development of pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease that, if left untreated, eventually leads to right heart failure and death. Elevated pulmonary arterial pressure (PAP) in patients with PAH is mainly caused by an increase in pulmonary vascular resistance (PVR). Sustained vasoconstriction and excessive pulmonary vascular remodeling are two major causes for elevated PVR in patients with PAH. Excessive pulmonary vascular remodeling is mediated by increased proliferation of pulmonary arterial smooth muscle cells (PASMC) due to PASMC dedifferentiation from a contractile or quiescent phenotype to a proliferative or synthetic phenotype. Increased cytosolic Ca(2+) concentration ([Ca(2+)]cyt) in PASMC is a key stimulus for cell proliferation and this phenotypic transition. Voltage-dependent Ca(2+) entry (VDCE) and store-operated Ca(2+) entry (SOCE) are important mechanisms for controlling [Ca(2+)]cyt. Stromal interacting molecule proteins (e.g., STIM2) and Orai2 both contribute to SOCE and we have previously shown that STIM2 and Orai2, specifically, are upregulated in PASMC from patients with idiopathic PAH and from animals with experimental pulmonary hypertension in comparison to normal controls. In this study, we show that STIM2 and Orai2 are upregulated in proliferating PASMC compared with contractile phenotype of PASMC. Additionally, a switch in Ca(2+) regulation is observed in correlation with a phenotypic transition from contractile PASMC to proliferative PASMC. PASMC in a contractile phenotype or state have increased VDCE, while in the proliferative phenotype or state PASMC have increased SOCE. The data from this study indicate that upregulation of STIM2 and Orai2 is involved in the phenotypic transition of PASMC from a contractile state to a proliferative state; the enhanced SOCE due to upregulation of STIM2 and Orai2 plays an important role in PASMC proliferation.

Pulmonary arterial hypertension (PAH) is a rare but progressive and deadly disease caused by functional and structural changes in the pulmonary vasculature, which lead to an increase in pulmonary vascular resistance. Regardless of the initial pathogenic trigger, the major causes of increased pulmonary vascular resistance in patients with PAH are sustained pulmonary vasoconstriction, pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular wall stiffness. Despite expanding research into the diagnosis and treatment of pulmonary hypertension, death rates from pulmonary hypertension have continued to increase 2.5% per year for women and 0.9% per year for men during the past decade.1 Patients with PAH, if untreated, die mainly because of progressive right heart failure, and the response of the right ventricle (RV) to the increased afterload is an important determinant of outcome in patients.2 During the development of pulmonary hypertension, an initial adaptive response of the RV to the increased afterload is to increase its wall thickness and contractility with varying degrees of RV hypertrophy.3 However, with disease progression, sustained long-term pressure overload of the RV can lead to progressive contractile dysfunction and eventually cause RV failure with further RV dilation. Little is known about the molecular and cellular mechanisms, which underlie the development of RV failure. The mechanism that determines the transition of RV function from compensated hypertrophy to decompensated failure is also uncertain. Article, see p 56 MicroRNAs (miRNAs), as crucial regulators of cardiovascular development and cardiac remodeling, have attracted increasing interest in recent years. Drake et al4 compared the gene expression patterns between RV hypertrophy in hypoxia-induced …

HomeCirculation ResearchVol. 114, No. 1Deacetylation of MicroRNA-124 in Fibroblasts Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBDeacetylation of MicroRNA-124 in FibroblastsRole in Pulmonary Hypertension Nicole M. Pohl, Ruby A. Fernandez, Kimberly A. Smith and Jason X.-J. Yuan Nicole M. PohlNicole M. Pohl From the Division of Pulmonary, Critical Care, Sleep, and Allergy Medicine, Department of Medicine; Department of Pharmacology; and Center for Cardiovascular Research, University of Illinois at Chicago, Chicago, IL. Search for more papers by this author , Ruby A. FernandezRuby A. Fernandez From the Division of Pulmonary, Critical Care, Sleep, and Allergy Medicine, Department of Medicine; Department of Pharmacology; and Center for Cardiovascular Research, University of Illinois at Chicago, Chicago, IL. Search for more papers by this author , Kimberly A. SmithKimberly A. Smith From the Division of Pulmonary, Critical Care, Sleep, and Allergy Medicine, Department of Medicine; Department of Pharmacology; and Center for Cardiovascular Research, University of Illinois at Chicago, Chicago, IL. Search for more papers by this author and Jason X.-J. YuanJason X.-J. Yuan From the Division of Pulmonary, Critical Care, Sleep, and Allergy Medicine, Department of Medicine; Department of Pharmacology; and Center for Cardiovascular Research, University of Illinois at Chicago, Chicago, IL. Search for more papers by this author Originally published3 Jan 2014https://doi.org/10.1161/CIRCRESAHA.113.302838Circulation Research. 2014;114:5–8The molecular mechanisms involved in the development of pulmonary hypertension (PH) remain unclear, although many investigators have demonstrated that abnormalities in gene expression in pulmonary vascular fibroblasts, smooth muscle cells, and endothelial cells are involved in the pathogenesis of PH. The control of gene expression is a complicated process, involving multiple layers of regulation. There are 3 distinct mechanisms of epigenetic regulation, DNA methylation, histone modifications, and gene silencing mediated by microRNAs (miRNAs). DNA methylation occurs on cytosine residues in CpG regions and is regulated by DNA methyltransferases (DNMTs). DNA methylation is essential for normal development, and 60% to 80% of the human genome CpGs are methylated. Methylation of most CpGs is constant, changing only in response to different cellular processes. In cancers and other diseases, hypermethylation of so-called CpG islands, which are CG-dense regions close to transcription start sites, found in tumor suppressor genes has been reported, leading to gene silencing. These data demonstrate that DNA methylation status is a frequently altered epigenetic modification in human diseases. In addition to DNA methylation, histone modifications represent another layer of regulation of gene expression. For the transcription machinery to be recruited to their target genes, the DNA needs to be accessible. The ability of the transcription machinery to reach the DNA is mainly controlled by histone acetyltransferases and histone deacetylases (HDACs). Histone acetyltransferases acetylate lysine residues and relax the chromatin structure, allowing for transcription factors to bind to the DNA and activate transcription. HDACs remove acetyl residues from histones, resulting in a condensed chromatin structure and transcriptional repression. The last layer of gene expression regulation is controlled by miRNAs, which are small noncoding RNAs that bind to their complementary sequence in the 3′ untranslated regions of their target mRNAs, resulting in gene silencing.Article, see p 67These pathways of gene regulation are often altered in many human diseases, such as cancer, leading to uncontrolled cell growth, migration, and invasion. In fact, changes in epigenetic modifications have recently been associated with PH, a disease that is associated with increased cell proliferation and decreased cell death of pulmonary arterial smooth muscle cells, pulmonary arterial endothelial cells, and adventitial fibroblasts. Recent studies of fawn-hooded rats, which spontaneously develop PH, have demonstrated that superoxide dismutase 2 (SOD2) expression is decreased in pulmonary arteries and plexiform lesions because of hypermethylation of CpG islands in the SOD2 gene.1 Reversal of the methylation status via DNMT1 inhibition rescued SOD2 expression and inhibited proliferation and increased cell apoptosis of fawn-hooded rat pulmonary arterial smooth muscle cells. Histone acetylation has also been shown to play an important role in the development of PH. Increased HDAC expression has been reported in lung tissues from patients with idiopathic pulmonary arterial hypertension as well as in lung tissues from hypoxia-induced pulmonary hypertensive (HPH) rats.2 The HDAC inhibitors valproic acid and suberoylanilide hydroxamic acid attenuated and reversed the development of HPH in rats and decreased proliferation in human pulmonary arterial smooth muscle cell and bovine fibroblasts. Additionally, a small-molecule HDAC inhibitor, which selectively inhibits class I HDACs, has been shown to suppress hypoxia-induced cardiopulmonary remodeling in rats.3 Recent studies have also implicated miRNAs in the development of PH. miR-204 expression has been shown to be decreased in animal models of PH and in human patient samples, and rescue of miR-204 reverses PH in rats.4 Additionally, miR-17 has been shown to be upregulated in HPH mice and monocrotaline-induced PH rats, and inhibition of miR-17 improved the PH phenotype of HPH mice and monocrotaline-induced PH rats.5 Several other miRNAs and miRNA targets have been identified to be involved in the development of PH. miRNAs have also been shown to regulate the expression of DNMTs and HDACs, adding on to the multitude of layers of regulation of gene expression.A study by Wang et al6 published in the current issue of Circulation Research is a good example of the complex relationship between gene regulation and gene expression. The authors used a large animal model of neonatal calves with HPH and focused their studies on adventitial fibroblasts, the most abundant cell type in the adventitia. In neonatal HPH calves, adventitial fibroblasts have undergone phenotypic changes, resulting in increased proliferation, migration, and inflammation activities. Wang et al6 identified that miR-124 expression is decreased in adventitial fibroblasts isolated from calves and humans with severe PH. Interestingly, miR-124 expression remained unchanged in fibroblasts isolated from control rats and mice compared with experimental HPH rodent models, indicating that the function of miR-124 in PH is species-specific. miR-124 expression has also been shown to be decreased in several cancers, resulting in increased cell proliferation and migration, 2 phenotypic changes that also occur in PH. Using miR-124 inhibitors and mimics in human and bovine fibroblasts, Wang et al6 set out to determine whether miR-124 also regulates cell growth and migration in PH fibroblasts. The authors found that overexpression of miR-124 decreased the proliferation and migration rate of PH fibroblasts. Similarly, inhibition of miR-124 in normal control fibroblasts increased cell proliferation and migration, demonstrating that miR-124 regulates cell proliferation and migration in fibroblasts. These data suggest that loss of miR-124 in PH fibroblasts is a major contributor to the constitutively activated phenotype of the adventitial fibroblasts.To elucidate the mechanism by which miR-124 regulates cell proliferation and migration, the authors screened transcript levels of cell cycle–related genes and found that miR-124 positively regulates Notch1, PTEN, FOXO3, p21/Cip1, and p27/Kip1, all of which were reduced in PH fibroblasts (Figure). Next, the authors sought to determine the target of miR-124 upstream of the cell-cycle regulator genes. Previous published data identified that miR-124 targets polypyrimidine tract–binding protein (PTBP)-1 and that PTBP1 affects Notch1 signaling.7,8 Therefore, Wang et al6 focused their studies on PTBP1, which is an abundantly expressed RNA-binding protein involved in several posttranscriptional regulation events, such as repressing RNA alternative splicing events, activation of translation driven by internal ribosomal entry sites, and RNA localization and stability. Wang et al6 determined that PTBP1 expression is increased in bovine and human PH fibroblasts as well as in pulmonary artery adventitia from humans and calves with severe PH compared with control fibroblasts and control tissue. Overexpression of miR-124 (using miR-124 mimics) in human and bovine PH fibroblasts inhibited PTPB1 expression, whereas inhibition of miR-124 (using anti-miR-124) in control cells increased PTBP1 expression. Additional luciferase assays were conducted to prove that PTBP1 is a direct target of miR-124. Furthermore, Wang et al6 showed that PTBP1 is upstream of Notch1 and negatively regulates the cell-cycle–related genes Notch1, PTEN, FOXO3, p21, and p27. The authors then elegantly designed an experiment to prove that PTBP1 expression is responsible for the increased proliferation rate of PH fibroblasts and that it is the action of miR-124 on PTBP1 that inhibits cell proliferation in PH fibroblasts. They designed a PTBP1 overexpression vector, which encodes the full-length mRNA without the 3′ untranslated region and hence the binding site for miR-124. Cotransfecting the miR-124–resistant PTBP1 vector with miR-124 mimics did not reduce PH fibroblast proliferation, demonstrating that miR-124 exerts its antiproliferative effects in PH fibroblasts by negatively regulating PTBP1. The novel findings in this study demonstrate that miR-124 is inhibited in PH fibroblasts, and this inhibition results in an increase of an RNA-binding protein, which posttranscriptionally regulates gene expression, adding to the complexity of gene regulation in PH.Download figureDownload PowerPointFigure. Proposed mechanism contributing to the constitutively activated phenotype in pulmonary hypertensive fibroblasts. In healthy subjects, microRNA (miR)-124 is acetylated (AC) and transcribed, inhibiting HIF-2α, polypyrimidine tract–binding protein (PTBP) 1, and MCP-1 gene expression, thereby maintaining a normal balance between cell growth, migration, and inflammation in the adventitia. In idiopathic pulmonary arterial hypertension patients and in hypoxia-induced pulmonary hypertensive calve fibroblasts, miR-124 expression is inhibited by class I histone deacetylases (HDACs). A decrease in miR-124 expression increases PTBP1, HIF-2α, and MCP-1. PTBP1 inhibits cell-cycle regulators Notch1 and PTEN. PTEN has been shown to be both a direct and indirect target of Notch1, resulting in different expression levels. The decrease in PTEN subsequently leads to an increase of AKT1/2 and decreased FOXO3/p27/p21, resulting in increased cell proliferation and migration. Additionally, PTEN inhibition leads to the activation of AKT/mTOR pathway, which increases cell growth by inhibiting 4EBP1/2 and activating p70S6K. The exact function of Notch1 inhibition in the diseased phenotype is not clear and requires further investigation. Upregulated expression of MCP-1 leads to β-integrin activation, causing migration and infiltration of monocytes and macrophages into the adventitia, thereby increasing inflammation and contributing to the activated fibroblast phenotype, which is characterized by enhanced cell proliferation, migration, and inflammation.Another interesting finding in this study is that Notch1 is downregulated in PH fibroblasts. The authors demonstrated that miR-124 inhibits PTBP1, resulting in increased Notch1 and PTEN expression, and suggested that in PH fibroblasts where miR-124 is inhibited, PTEN downregulation may be dependent on Notch1 inhibition (Figure). In the absence of Notch signaling, CBF-1 (also called RBP-Jκ) is bound to the promoter region of the target gene in a repressor complex. On activation of Notch signaling, the Notch intracellular domain binds to CBF-1, displacing the corepressors, and results in transcriptional activation of target genes. Based on previous studies that identified PTEN as a direct target of Notch1, the authors suggest that PTBP1-dependent inhibition of Notch1 results in decreased PTEN expression.9 However, several other studies suggest that PTEN is negatively regulated by HES1, a transcriptional repressor and major downstream target of Notch1.10,11 Notch signaling has been shown to be proproliferative in some cancers, and increased Notch3 expression has been demonstrated in PH patient samples and is required for the development of PH in murine models.12 The authors note that Notch activation is highly dependent on cellular and environmental context, and previous studies involving Notch3 have been performed in pulmonary arterial smooth muscle cells. It is, therefore, likely that Notch1 may have different functions in adventitial fibroblasts. The authors clearly demonstrated that a decrease in PTEN leads to the development of the activated phenotype of fibroblasts. This is consistent with recently established findings showing that the AKT/mTOR pathway, which is inhibited by PTEN, is activated in PH13 (Figure). It is then possible that PTBP1-mediated inhibition of Notch1 and PTEN are independent of each other, resulting in 2 different downstream pathways. Although the authors established the role of PTEN in PH fibroblasts, further studies need to be performed to determine how PTBP1 inhibits PTEN and Notch1 signaling and whether PTBP1 directly inhibits the expression of these proteins or whether PTBP1 regulates the expression of an intermediate protein, which in turn inhibits PTEN and Notch1.Wang et al6 also demonstrated in their study that HIF-2α levels are elevated in PH fibroblasts compared with control fibroblasts and that HIF-1α levels remained unchanged. Furthermore, they demonstrated that miR-124 controls HIF-2α expression in these fibroblasts, thereby additionally contributing to increased cell proliferation. Most studies in the pulmonary circulation have focused on the upregulation of HIF-1α, but recent studies have shown that both isoforms have tissue-specific and cell-specific expression patterns and both contribute independently to cell growth.8,14 It would be interesting to determine whether HIF-1α is important for the initial switch to the activated fibroblast phenotype and which mechanisms control HIF-2α activation in PH fibroblasts, because the authors do not provide any evidence showing that HIF-2α is a direct target of miR-124. It is possible that HIF-2α is an indirect target of miR-124 and that other effectors controlled by miR-124 increase HIF-2α.Increased inflammatory activity is another hallmark associated with the activated phenotype of PH fibroblasts, and Wang et al6 demonstrated that miR-124 is directly involved in this process. The authors show that PH fibroblasts from calves and humans have elevated levels of MCP-1, a chemoattractant for macrophages and monocytes. Transfection of miR-124 mimics in PH fibroblasts decreased MCP-1 transcript and protein levels, whereas transfection of anti-miR-124 in control fibroblasts increased MCP-1 expression. The application of luciferase assays further confirmed that MCP-1 is a direct target of miR-124, thereby directly contributing to the activated phenotype of PH fibroblasts.Finally, the authors discovered the mechanism behind the decreased expression of miR-124. Because it has previously been shown that miR-124 itself is subject to epigenetic modifications, the authors investigated whether miR-124 is epigenetically silenced in PH fibroblasts. Interestingly, they discovered that treatment of PH fibroblasts with the HDAC inhibitors suberoylanilide hydroxamic acid, Apidicin, and OSU42, led to a significant increase of miR-124 while decreasing the direct targets of miR-124, PTBP1, and MCP-1 (Figure). This suggests that miR-124 expression is decreased in PH fibroblasts through epigenetic modifications, specifically through the removal of acetylation marks on histones, resulting in a more condensed chromatin structure and inhibition of transcription. Such an epigenetic event would explain the constitutively activated phenotype of PH fibroblasts, which has been shown to be reversible through the application of HDAC inhibitors. The authors discuss that similar observations have been made in synoviocytes from patients with rheumatoid arthritis as well as in cancer cells. Further studies are needed to determine whether HDAC inhibitors are able to prevent and reverse PH in HPH calves and whether other miRNAs or mRNAs are epigenetically modified in activated PH fibroblasts.In summary, Wang et al6 provide compelling evidence that loss of miR-124 is directly involved in the development of activated PH fibroblasts, leading to PH. miR-124 expression is lost in PH fibroblasts from calves and idiopathic pulmonary arterial hypertension patients, causing increased expression of HIF-2α and the RNA-binding protein PTBP1, which in turn inhibits cell-cycle regulators Notch1/PTEN/FOXO3/p21 and p27, which ultimately leads to an increase in cell proliferation and migration (Figure). Additionally, inhibition of miR-124 increases MCP-1 expression, resulting in increased inflammation. The study by Wang et al6 reveals multiple targets for the development of therapeutic strategies (eg, HDAC inhibitors, PTBP1 inhibitors, or miR-124 mimics) for the treatment of patients with idiopathic pulmonary arterial hypertension.Sources of FundingThis work was supported, in part, by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL066012, HL115014, and HL098053).DisclosuresNone.FootnotesThe opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.Correspondence to Jason X.-J. Yuan, MD, PhD, Department of Medicine, University of Illinois at Chicago, COMRB 3131, MC 719, 909 South Wolcott Ave, Chicago, IL 60612. E-mail [email protected]References1. Archer SL, Marsboom G, Kim GH, Zhang HJ, Toth PT, Svensson EC, Dyck JR, Gomberg-Maitland M, Thébaud B, Husain AN, Cipriani N, Rehman J. Epigenetic attenuation of mitochondrial superoxide dismutase 2 in pulmonary arterial hypertension: a basis for excessive cell proliferation and a new therapeutic target.Circulation. 2010; 121:2661–2671.LinkGoogle Scholar2. Zhao L, Chen CN, Hajji N, Oliver E, Cotroneo E, Wharton J, Wang D, Li M, McKinsey TA, Stenmark KR, Wilkins MR. Histone deacetylation inhibition in pulmonary hypertension: therapeutic potential of valproic acid and suberoylanilide hydroxamic acid.Circulation. 2012; 126:455–467.LinkGoogle Scholar3. Cavasin MA, Demos-Davies K, Horn TR, et al. Selective class I histone deacetylase inhibition suppresses hypoxia-induced cardiopulmonary remodeling through an antiproliferative mechanism.Circ Res. 2012; 110:739–748.LinkGoogle Scholar4. Courboulin A, Paulin R, Giguère NJ, Saksouk N, Perreault T, Meloche J, Paquet ER, Biardel S, Provencher S, Côté J, Simard MJ, Bonnet S. Role for miR-204 in human pulmonary arterial hypertension.J Exp Med. 2011; 208:535–548.CrossrefMedlineGoogle Scholar5. Pullamsetti SS, Doebele C, Fischer A, Savai R, Kojonazarov B, Dahal BK, Ghofrani HA, Weissmann N, Grimminger F, Bonauer A, Seeger W, Zeiher AM, Dimmeler S, Schermuly RT. Inhibition of microRNA-17 improves lung and heart function in experimental pulmonary hypertension.Am J Respir Crit Care Med. 2012; 185:409–419.CrossrefMedlineGoogle Scholar6. Wang D, Zhang H, Li M, Frid MG, Flockton AR, McKeon BA, Yeager ME, Fini MA, Morrell NW, Pullamsetti SS, Velegala S, Seeger W, McKinsey TA, Sucharov CC, Stenmark KR. MicroRNA-124 controls the proliferative, migratory, and inflammatory phenotype of pulmonary vascular fibroblasts.Circ Res. 2014; 114:67–78.LinkGoogle Scholar7. Cheung HC, Corley LJ, Fuller GN, McCutcheon IE, Cote GJ. Polypyrimidine tract binding protein and Notch1 are independently re-expressed in glioma.Mod Pathol. 2006; 19:1034–1041.CrossrefMedlineGoogle Scholar8. Makeyev EV, Zhang J, Carrasco MA, Maniatis T. The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing.Mol Cell. 2007; 27:435–448.CrossrefMedlineGoogle Scholar9. Whelan JT, Forbes SL, Bertrand FE. CBF-1 (RBP-J kappa) binds to the PTEN promoter and regulates PTEN gene expression.Cell Cycle. 2007; 6:80–84.CrossrefMedlineGoogle Scholar10. Liu S, Ma X, Ai Q, Huang Q, Shi T, Zhu M, Wang B, Zhang X. NOTCH1 functions as an oncogene by regulating the PTEN/PI3K/AKT pathway in clear cell renal cell carcinoma.Urol Oncol. 2013; 31:938–948.CrossrefMedlineGoogle Scholar11. Palomero T, Sulis ML, Cortina M, et al. Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia.Nat Med. 2007; 13:1203–1210.CrossrefMedlineGoogle Scholar12. Li X, Zhang X, Leathers R, Makino A, Huang C, Parsa P, Macias J, Yuan JX, Jamieson SW, Thistlethwaite PA. Notch3 signaling promotes the development of pulmonary arterial hypertension.Nat Med. 2009; 15:1289–1297.CrossrefMedlineGoogle Scholar13. Houssaini A, Abid S, Mouraret N, Wan F, Rideau D, Saker M, Marcos E, Tissot CM, Dubois-Randé JL, Amsellem V, Adnot S. Rapamycin reverses pulmonary artery smooth muscle cell proliferation in pulmonary hypertension.Am J Respir Cell Mol Biol. 2013; 48:568–577.CrossrefMedlineGoogle Scholar14. Ahmad A, Ahmad S, Malcolm KC, Miller SM, Hendry-Hofer T, Schaack JB, White CW. Differential regulation of pulmonary vascular cell growth by hypoxia-inducible transcription factor-1α and hypoxia-inducible transcription factor-2α.Am J Respir Cell Mol Biol. 2013; 49:78–85.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails January 3, 2014Vol 114, Issue 1 Advertisement Article InformationMetrics © 2014 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.113.302838PMID: 24385500 Originally publishedJanuary 3, 2014 KeywordsPTBP1 protein, humanhypertension, pulmonaryhistone deacetylasesPDF download Advertisement SubjectsHypertension

Rationale: An increase in cytosolic free Ca2+ in pulmonary arterial smooth muscle cells (PASMC) is not only a major trigger for pulmonary vasoconstriction but also an important stimulus for PASMC proliferation. Our previous data showed that the extracellular calcium sensing receptor (CaSR) expression and function were both enhanced in PASMC from idiopathic PAH patients. However, the implication of CaSR in the development of PAH remains elusive.Methods: CaSR and parathyroid hormone double knockout (DK) mice were used to test the hypothesis that CaSR modulates pulmonary vascular tone and the response to hypoxia. Wild type (WT) and DK mice were exposed to 10% oxygen for up to three weeks. Pulmonary vascular remodeling was evaluated using tissue morphometrics. Right ventricle systolic pressures (RVSP) and right ventricular hypertrophy by, RV/(LV+S) ratios, were measured to evaluate pulmonary hypertensive development. We also isolated PASMC from WT or DK mice, followed with cell proliferation assay and cytosolic Ca2+ concentration ([Ca2+]cyt) measurement.Results: WT and DK mice displayed normal systemic and pulmonary arterial pressure under normoxic conditions. However, compared to WT mice, DK mice had significantly lower RVSP, RV/LV+S ratios, and displayed less pulmonary vascular remodeling when exposed to hypoxia. Extracellular Ca2+ caused an increase in [Ca2+]cyt in PASMC isolated from hypoxic WT mice. However, restoration of extracellular Ca2+ had little effect on [Ca2+]cyt in DK mice. Additionally, a decrease in store‐operated Ca2+ entry (SOCE) was observed in PASMC from DK mice, as compared to SOCE in PASMC from WT mice.Conclusion: These data indicate that CaSR plays an important role in the development and progression of pulmonary vascular remodeling in mice with PH. CaSR deletions prevent the development of hypoxia‐induced PAH in mice via its modulation of extracellular Ca2+ entry in PASMC, suggesting that CaSR may be a novel therapeutic candidate for the treatment of PAH.Grant Funding Source: Supported by NIH (HL066012; HL115014)

The molecular mechanisms involved in the development of pulmonary hypertension (PH) remain unclear, although many investigators have demonstrated that abnormalities in gene expression in pulmonary vascular fibroblasts, smooth muscle cells, and endothelial cells are involved in the pathogenesis of PH. The control of gene expression is a complicated process, involving multiple layers of regulation. There are 3 distinct mechanisms of epigenetic regulation, DNA methylation, histone modifications, and gene silencing mediated by microRNAs (miRNAs). DNA methylation occurs on cytosine residues in CpG regions and is regulated by DNA methyltransferases (DNMTs). DNA methylation is essential for normal development, and 60% to 80% of the human genome CpGs are methylated. Methylation of most CpGs is constant, changing only in response to different cellular processes. In cancers and other diseases, hypermethylation of so-called CpG islands, which are CG-dense regions close to transcription start sites, found in tumor suppressor genes has been reported, leading to gene silencing. These data demonstrate that DNA methylation status is a frequently altered epigenetic modification in human diseases. In addition to DNA methylation, histone modifications represent another layer of regulation of gene expression. For the transcription machinery to be recruited to their target genes, the DNA needs to be accessible. The ability of the transcription machinery to reach the DNA is mainly controlled by histone acetyltransferases and histone deacetylases (HDACs). Histone acetyltransferases acetylate lysine residues and relax the chromatin structure, allowing for transcription factors to bind to the DNA and activate transcription. HDACs remove acetyl residues from histones, resulting in a condensed chromatin structure and transcriptional repression. The last layer of gene expression regulation is controlled by miRNAs, which are small noncoding RNAs that bind to their complementary sequence in the 3′ untranslated regions of their target mRNAs, resulting in gene silencing. Article, see p 67 These pathways of gene …

Over the past decade a wealth of information has been divulged on stem cells present in the lung both in the pulmonary vasculature and the respiratory tract. Cells have been identified with the capability of repopulating the lung and others that contribute to the pathogenesis or, conversely, have therapeutic benefit in pulmonary vascular disease. The isolation of a single-resident lung stem cell capable of repopulating any lung epithelium still remains elusive. What is currently known about stem and progenitor cells in the lung suggests that a non-classical stem cell hierarchy exists with a novel array of cellular mechanisms controlling proliferation and differentiation of such cells. This chapter serves to provide an up-to-date review of what is currently known about stem and progenitor cells within the lung.

Disordered pulmonary arterial endothelial cell (PAEC) growth underlies the formation of angio‐proliferation and intimal lesions, obliterating the small pulmonary arteries (PAs), resulting in irreversible structural alterations, or pulmonary vascular remodeling in pulmonary arterial hypertension (PAH) patients.ObjectiveTo demonstrate that transient receptor potential channel (TRPC6) contributes to PAEC proliferation and in the development of PAH.ResultsPAEC isolated from Idiopathic PAH (IPAH) patients showed an enhanced VEGF‐induced [Ca2+]cyt and proliferation. In parallel, TRPC6 (mRNA and protein) expression was markedly up regulated in IPAH compared to normal subjects. Interestingly, knockdown of TRPC6 in IPAH‐PAEC using siRNA significantly attenuated VEGF‐induced rise in [Ca2+]cyt and proliferation. Deletion of either TRPC6 (TRPC6−/−) or TRPC4 (TRPC4−/−) abolished thapsigargin‐induced store Ca2+ release‐activated Ca2+ entry (SOCE) in mice PAEC. In addition, TRPC6−/− mice showed significant abrogation of hypoxia‐induced changes in the right ventricular systolic pressure and right ventricular hypertrophy suggesting its role in vascular remodeling associated with PAH.ConclusionOur studies provide a comprehensive picture of TRPC6‐mediated Ca2+ entry mechanisms in contributing to an increased PAEC proliferation and vascular remodeling associated with PAH.

Pulmonary hypertension (PH) is a progressive disease which, if left untreated, will eventually lead to right heart failure and death. Elevated pulmonary arterial pressure (PAP) in patients with PH is mainly caused by an increase in pulmonary vascular resistance (PVR). Sustained vasoconstriction and excessive pulmonary vascular remodeling are two of the major causes for elevated PVR in patients with PH. Excessive pulmonary vascular remodeling is mediated by increased proliferation of pulmonary arterial smooth muscle cells (PASMC) due to PASMC dedifferentiation from a contractile or quiescent to a proliferative or synthetic phenotype. Increased cytosolic Ca2+ concentration ([Ca2+]cyt) in PASMC is a key stimulus for cell proliferation and this phenotypic transition. Voltage dependent Ca2+ entry (VDCE) and store-operated Ca2+ entry (SOCE) are important mechanisms for controlling [Ca2+]cyt. Stromal interacting molecule proteins (e.g., STIM2) and Orai2 both contribute to SOCE and we have previously shown that STIM2 and Orai2, specifically, are upregulated in PASMC from idiopathic pulmonary arterial hypertension (IPAH) patients and from animals with experimental pulmonary hypertension in comparison to normal controls. Our data show that STIM2 and Orai2 are upregulated in proliferating PASMC compared with contractile phenotype of PASMC. Additionally, a switch in Ca2+ regulation is observed in correlation with a phenotype transition from contractile PASMC to proliferative PASMC. PASMC in a contractile state have increased VDCE, while in the proliferative state they have increased SOCE. The data from this study indicate that upregulation of STIM2 and Orai2 is involved in the phenotypic transition of PASMC from a contractile state to a proliferative state; the enhanced SOCE due to upregulation of STIM2 and Orai2 plays an important role in PASMC proliferation.

Pulmonary hypertension (PH) is a fatal disease attributed to increased pulmonary vascular resistance (PVR). Elevated PVR is partially due to sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling. Pulmonary arterial smooth muscle cells (PASMC) in their natural state are plastic and exist in two different phenotypes; a differentiated, contractile phenotype and a dedifferentiated, more proliferative phenotype. An increase in cytosolic free Ca 2+ concentration ([Ca 2+ ] cyt ) is a major stimulus for PASMC proliferation, contributing to pulmonary vascular remodeling. This study characterizes the phenotypic switch observed when freshly dissociated PASMC are cultured from normal male rats or in PASMC isolated from experimental PH rats. Western blot analysis showed freshly isolated pulmonary arteries (PA) express contractile markers that were decreased in cultured PASMC, demonstrating a switch to a proliferative phenotype. Cultured PASMC showed an increase in expression of store-operated and receptor-operated Ca 2+ channels Orai2, STIM2, and TRPC6 compared to freshly dissociated PASMC. Additionally, an increase in store-operated Ca 2+ entry (SOCE) and a decrease in voltage dependent Ca 2+ influx in cultured PASMC was observed when compared to freshly dissociated PASMC, suggesting SOCE plays a greater role in proliferating PASMC. Consistently, isometric force studies showed greater contraction induced by voltage-dependent Ca 2+ channel (VDCC) activation than SOCE induced contraction in freshly isolated pulmonary artery rings. Furthermore, Orai2, STIM2, and TRPC6 were upregulated in proliferative PASMC compared to quiescent PASMC. Knockdown of Orai2 was able to attenuate SOCE in proliferative PASMC demonstrating Orai2 is necessary for SOCE. These data suggest upregulation of STIM2 promotes the phenotypical transition of PASMC from a contractile to a proliferative phenotype by inhibiting VDCC and activating SOC, and that increased expression of Orai2 and TRPC6 channels mediate the increased [Ca 2+ ] cyt seen in proliferating cells. Understanding the molecular mechanisms which regulate [Ca 2+ ] cyt and PASMC proliferation is critical for the development of novel therapies for PH.

A recent study from our group demonstrated that the Ca(2+)-sensing receptor (CaSR) was upregulated, and the extracellular Ca(2+)-induced increase in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) was enhanced in pulmonary arterial smooth muscle cells from patients with idiopathic pulmonary arterial hypertension and animals with experimental pulmonary hypertension (PH). However, it is unclear whether CaSR antagonists (for example, NPS2143) rescue the development of experimental PH. We tested the rescue effects of NPS2143 in rats with monocrotaline (MCT)-induced PH and mice with chronic hypoxia-induced PH. For the NPS2143 treatment group, rats and mice were i.p. injected with NPS2143 once per day from days 14 to 24. Four weeks after MCT injection or exposure to normobaric hypoxia, the right ventricular (RV) systolic pressure, right heart hypertrophy (RV/LV+S ratio) and RV myocardial fibrosis were rescued or nearly restored to normal levels by NPS2143 treatment. The rescue effects of NPS2143 on experimental PH further support a critical role for the CaSR in the PH mechanism. Therefore, NPS2143 may be a promising potential treatment for pulmonary arterial hypertension.

Pulmonary circulation is an important circulatory system in which the body brings in oxygen. Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that predominantly affects women. Sustained pulmonary vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular stiffness are the major causes for the elevated pulmonary vascular resistance (PVR) in patients with PAH. The elevated PVR causes an increase in afterload in the right ventricle, leading to right ventricular hypertrophy, right heart failure, and eventually death. Understanding the pathogenic mechanisms of PAH is important for developing more effective therapeutic approach for the disease. An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC migration and proliferation which lead to pulmonary vascular wall thickening and remodeling. It is thus pertinent to define the pathogenic role of Ca(2+) signaling in pulmonary vasoconstriction and PASMC proliferation to develop new therapies for PAH. [Ca(2+)](cyt) in PASMC is increased by Ca(2+) influx through Ca(2+) channels in the plasma membrane and by Ca(2+) release or mobilization from the intracellular stores, such as sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER). There are two Ca(2+) entry pathways, voltage-dependent Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCC) and voltage-independent Ca(2+) influx through store-operated Ca(2+) channels (SOC) and receptor-operated Ca(2+) channels (ROC). This paper will focus on the potential role of VDCC, SOC, and ROC in the development and progression of sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in PAH.

RATIONALE:A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased resting [Ca(2+)](cyt) and enhanced Ca(2+) influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH).OBJECTIVE:We examined whether the extracellular Ca(2+)-sensing receptor (CaSR) is involved in the enhanced Ca(2+) influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension.METHODS AND RESULTS:In normal PASMC superfused with Ca(2+)-free solution, addition of 2.2 mmol/L Ca(2+) to the perfusate had little effect on [Ca(2+)](cyt). In IPAH-PASMC, however, restoration of extracellular Ca(2+) induced a significant increase in [Ca(2+)](cyt). Extracellular application of spermine also markedly raised [Ca(2+)](cyt) in IPAH-PASMC but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca(2+)-induced [Ca(2+)](cyt) rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca(2+)-mediated [Ca(2+)](cyt) increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia.CONCLUSIONS:The extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca(2+) influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension.

Rationale: A rise in cytosolic Ca concentration ([Ca ]cyt) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased resting [Ca ]cyt and enhanced Ca influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH). Objective: We examined whether the extracellular Ca -sensing receptor (CaSR) is involved in the enhanced Ca influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension. Methods and Results: In normal PASMC superfused with Ca -free solution, addition of 2.2 mmol/L Ca to the perfusate had little effect on [Ca ]cyt. In IPAH-PASMC, however, restoration of extracellular Ca 2 induced a significant increase in [Ca ]cyt. Extracellular application of spermine also markedly raised [Ca 2 ]cyt in IPAH-PASMC but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca -induced [Ca ]cyt rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca -mediated [Ca ]cyt increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia. Conclusions: The extracellular Ca -induced increase in [Ca ]cyt due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension. (Circ Res. 2012;111:469-481.)

influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension. Methods and Results: In normal PASMC superfused with Ca -free solution, addition of 2.2 mmol/L Ca to the perfusate had little effect on [Ca ]cyt. In IPAH-PASMC, however, restoration of extracellular Ca 2 induced a significant increase in [Ca ]cyt. Extracellular application of spermine also markedly raised [Ca 2 ]cyt in IPAH-PASMC but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca -induced [Ca ]cyt rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca -mediated [Ca ]cyt increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced