Background: An emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments. Methods: Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry–based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry. Results: We detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1β expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results. Conclusions: In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.

Stiffening of large arteries is increasingly used as an independent predictor of risk and therapeutic outcome for small artery dysfunction in many diseases including pulmonary hypertension. The molecular mechanisms mediating downstream vascular cell responses to large artery stiffening remain unclear. We hypothesize that high pulsatility flow, induced by large artery stiffening, causes inflammatory responses in downstream pulmonary artery endothelial cells (PAECs) through toll-like receptor (TLR) pathways. To recapitulate the stiffening effect of large pulmonary arteries that occurs in pulmonary hypertension, ultrathin silicone tubes of variable mechanical stiffness were formulated and were placed in a flow circulatory system. These tubes modulated the simulated cardiac output into pulsatile flows with different pulsatility indices, 0.5 (normal) or 1.5 (high). PAECs placed downstream of the tubes were evaluated for their expression of proinflammatory molecules (ICAM-1, VCAM-1, E-selectin and MCP-1), TLR receptors and intracellular NF-κB following flow exposure. Results showed that compared to flow with normal pulsatility, high pulsatility flow induced proinflammatory responses in PAECs, enhanced TLR2 expression but not TLR4, and caused NF-κB activation. Pharmacologic (OxPAPC) and siRNA inhibition of TLR2 attenuated high pulsatility flow-induced pro-inflammatory responses and NF-κB activation in PAECs. We also observed that PAECs isolated from small pulmonary arteries of hypertensive animals exhibiting proximal vascular stiffening demonstrated a durable ex-vivo proinflammatory phenotype (increased TLR2, TLR4 and MCP-1 expression). Intralobar PAECs isolated from vessels of IPAH patients also showed increased TLR2. In conclusion, this study demonstrates for the first time that TLR2/NF-κB signaling mediates endothelial inflammation under high pulsatility flow caused by upstream stiffening, but the role of TLR4 in flow pulsatility-mediated endothelial mechanotransduction remains unclear.

Rationale : Pulmonary hypertensive remodeling is characterized by excessive proliferation, migration, and proinflammatory activation of adventitial fibroblasts. In culture, fibroblasts maintain a similar activated phenotype. The mechanisms responsible for generation/maintenance of this phenotype remain unknown. Objective : We hypothesized that aberrant expression of microRNA-124 (miR-124) regulates this activated fibroblast phenotype and sought to determine the signaling pathways through which miR-124 exerts effects. Methods and Results : We detected significant decreases in miR-124 expression in fibroblasts isolated from calves and humans with severe pulmonary hypertension. Overexpression of miR-124 by mimic transfection significantly attenuated proliferation, migration, and monocyte chemotactic protein-1 expression of hypertensive fibroblasts, whereas anti–miR-124 treatment of control fibroblasts resulted in their increased proliferation, migration, and monocyte chemotactic protein-1 expression. Furthermore, the alternative splicing factor, polypyrimidine tract–binding protein 1, was shown to be a direct target of miR-124 and to be upregulated both in vivo and in vitro in bovine and human pulmonary hypertensive fibroblasts. The effects of miR-124 on fibroblast proliferation were mediated via direct binding to the 3′ untranslated region of polypyrimidine tract–binding protein 1 and subsequent regulation of Notch1/phosphatase and tensin homolog/FOXO3/p21Cip1 and p27Kip1 signaling. We showed that miR-124 directly regulates monocyte chemotactic protein-1 expression in pulmonary hypertension/idiopathic pulmonary arterial hypertension fibroblasts. Furthermore, we demonstrated that miR-124 expression is suppressed by histone deacetylases and that treatment of hypertensive fibroblasts with histone deacetylase inhibitors increased miR-124 expression and decreased proliferation and monocyte chemotactic protein-1 production. Conclusions : Stable decreases in miR-124 expression contribute to an epigenetically reprogrammed, highly proliferative, migratory, and inflammatory phenotype of hypertensive pulmonary adventitial fibroblasts. Thus, therapies directed at restoring miR-124 function, including histone deacetylase inhibitors, should be investigated.

HomeCirculationVol. 127, No. 14Letter by Bogaard et al Regarding Article, "Histone Deacetylation Inhibition in Pulmonary Hypertension: Therapeutic Potential of Valproic Acid and Suberoylanilide Hydroxamic Acid" Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBLetter by Bogaard et al Regarding Article, "Histone Deacetylation Inhibition in Pulmonary Hypertension: Therapeutic Potential of Valproic Acid and Suberoylanilide Hydroxamic Acid" Harm J. Bogaard, MD, PhD Shiro Mizuno, MD, PhD Norbert F. Voelkel, MD Harm J. BogaardHarm J. Bogaard VU University Medical CenterDepartment of Pulmonary MedicineAmsterdam, Netherlands Shiro MizunoShiro Mizuno Kanazawa Medical UniversityDivision of Respiratory DiseaseUchinada, Ishikawa, Japan Norbert F. VoelkelNorbert F. Voelkel Virginia Commonwealth UniversityDepartment of MedicineRichmond, VA Originally published9 Apr 2013https://doi.org/10.1161/CIRCULATIONAHA.112.127092Circulation. 2013;127:e539To the Editor:Patients who have pulmonary arterial hypertension (PAH) are facing a battle on two fields: Whereas progressive vascular remodeling increases pulmonary vascular resistance, maladaptive myocardial remodeling eventually culminates in right-sided heart failure and death. PAH was initially understood as a condition of excessive pulmonary vasoconstriction, but today elements of exuberant cellular proliferation, unguided angiogenesis, and apoptosis-resistance are seen as central to the development of the disease.1 Although the transition from right-sided heart adaptation to failure has only recently become a focus of research, it has already become clear that the failing right ventricle is characterized by the morphological triad of apoptotic loss of cardiomyocytes, fibrosis, and loss of capillaries.2 As such, there is an obvious therapeutic dilemma in PAH that can be stated as follows: What is good for the lung (inhibition of angiogenesis and induction of apoptosis) may be bad for the heart. This concern also pertains to the possible therapeutic effects of histone deacetylase (HDAC) inhibitors in PAH. As elegantly shown by Zhao and coworkers,3 there are a number of reasons to postulate that the HDAC inhibitors valproic acid and suberoylanilide hydroxamic acid may mitigate pulmonary vascular remodeling in PAH through effects on lung endothelial cells or vascular smooth muscle cells. Apparently, HDAC inhibition partially reverses hypoxic pulmonary hypertension, as had already been shown by Cavasin et al.4 These results may seem encouraging; however, there is the real and serious concern that the same antiangiogenic and proapoptotic effects of HDAC inhibitors threaten the heart's adaptive response to pressure overload. Treatment with valproic acid or trichostatin A (another broad-spectrum HDAC inhibitor) of rats subjected to pulmonary artery banding, which triggers a complex adaptive response of the right ventricle, led to a severe impairment of right heart function, in association with apoptotic loss of cardiomyocytes, vascular rarefaction, and excessive fibrosis.5 That HDAC inhibition affected capillaries was shown because trichostatin A dose-dependently suppressed vascular endothelial growth factor, endothelial nitric oxide synthase, and angiopoietin-1 expression in cultured myocardial endothelial cells. This suggests that inhibition of adaptive angiogenesis was central to the detrimental effects of HDAC inhibition in the pressure-overloaded right ventricle. HDAC inhibition had little effect on the unstressed heart, and it is possible that HDAC inhibition doesn't interfere with normal right ventricular adaptation to a mild increase in afterload, such as in hypoxia, as shown by both Zhao et al3 and Cavasin et al.4 Unfortunately, it is very unlikely that HDAC inhibitors would completely reverse pulmonary vascular remodeling in all PAH patients in whom the vascular remodeling is not driven by hypoxia. Before we conduct very careful clinical studies, we are left with the possibility that HDAC inhibition may perhaps show some benefit in the lungs of some PAH patients but may be a disaster for the heart of others.Harm J. Bogaard, MD, PhDVU University Medical CenterDepartment of Pulmonary MedicineAmsterdam, NetherlandsShiro Mizuno, MD, PhDKanazawa Medical UniversityDivision of Respiratory DiseaseUchinada, Ishikawa, JapanNorbert F. Voelkel, MDVirginia Commonwealth UniversityDepartment of MedicineRichmond, VADisclosuresNone.References1. Voelkel NF, Gomez-Arroyo J, Abbate A, Bogaard HJ, Nicolls MR. Pathobiology of pulmonary arterial hypertension and right ventricular failure.Eur Respir J. 2012; 40:1555–1565.CrossrefMedlineGoogle Scholar2. Bogaard HJ, Natarajan R, Henderson SC, Long CS, Kraskauskas D, Smithson L, Ockaili R, McCord JM, Voelkel NF. Chronic pulmonary artery pressure elevation is insufficient to explain right heart failure.Circulation. 2009; 120:1951–1960.LinkGoogle Scholar3. 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 Scholar4. Cavasin MA, Demos-Davies K, Horn TR, Walker LA, Lemon DD, Birdsey N, Weiser-Evans MC, Harral J, Irwin DC, Anwar A, Yeager ME, Li M, Watson PA, Nemenoff RA, Buttrick PM, Stenmark KR, McKinsey TA. Selective class I histone deacetylase inhibition suppresses hypoxia-induced cardiopulmonary remodeling through an antiproliferative mechanism.Circ Res. 2012; 110:739–748.LinkGoogle Scholar5. Bogaard HJ, Mizuno S, Hussaini AA, Toldo S, Abbate A, Kraskauskas D, Kasper M, Natarajan R, Voelkel NF. Suppression of histone deacetylases worsens right ventricular dysfunction after pulmonary artery banding in rats.Am J Respir Crit Care Med. 2011; 183:1402–1410.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Cavasin M, Stenmark K and McKinsey T (2015) Emerging Roles for Histone Deacetylases in Pulmonary Hypertension and Right Ventricular Remodeling (2013 Grover Conference series), Pulmonary Circulation, 10.1086/679700, 5:1, (63-72), Online publication date: 1-Mar-2015. Kim J, Hwangbo C, Hu X, Kang Y, Papangeli I, Mehrotra D, Park H, Ju H, McLean D, Comhair S, Erzurum S and Chun H (2014) Restoration of Impaired Endothelial Myocyte Enhancer Factor 2 Function Rescues Pulmonary Arterial Hypertension, Circulation, 131:2, (190-199), Online publication date: 13-Jan-2015. April 9, 2013Vol 127, Issue 14 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.112.127092PMID: 23569123 Originally publishedApril 9, 2013 PDF download Advertisement SubjectsHeart FailurePulmonary Hypertension

Background— Epigenetic programming, dynamically regulated by histone acetylation, is a key mechanism regulating cell proliferation and survival. Little is known about the contribution of histone deacetylase (HDAC) activity to the development of pulmonary arterial hypertension, a condition characterized by profound structural remodeling of pulmonary arteries and arterioles. Methods and Results— HDAC1 and HDAC5 protein levels were elevated in lungs from human idiopathic pulmonary arterial hypertension and in lungs and right ventricles from rats exposed to hypoxia. Immunohistochemistry localized increased expression to remodeled vessels in the lung. Both valproic acid, a class I HDAC inhibitor, and suberoylanilide hydroxamic acid (vorinostat), an inhibitor of class I, II, and IV HDACs, mitigated the development of and reduced established hypoxia-induced pulmonary hypertension in the rat. Both valproic acid and suberoylanilide hydroxamic acid inhibited the imprinted highly proliferative phenotype of fibroblasts and R-cells from pulmonary hypertensive bovine vessels and platelet-derived growth factor–stimulated growth of human vascular smooth muscle cells in culture. Exposure to valproic acid and suberoylanilide hydroxamic acid was associated with increased levels of p21 and FOXO3 and reduced expression of survivin. The significantly higher levels of expression of cKIT, monocyte chemoattractant protein-1, interleukin-6, stromal-derived factor-1, platelet-derived growth factor-b, and S100A4 in R-cells were downregulated by valproic acid and suberoylanilide hydroxamic acid treatment. Conclusions— Increased HDAC activity contributes to the vascular pathology of pulmonary hypertension. The effectiveness of HDAC inhibitors, valproic acid, and suberoylanilide hydroxamic acid, in models of pulmonary arterial hypertension supports a therapeutic strategy based on HDAC inhibition in pulmonary arterial hypertension.

Detection of DNA methylation has produced promising results as biomarkers for head and neck squamous cell carcinoma (HNSCC). However, current panels are limited by an insufficient number of sensitive and specific tumor markers. MicroRNAs (miR) play an important role in tumorigenesis, and may represent a novel panel of molecules for the development of cancer biomarkers. We investigated methylation of three miRNA promoter sites of miR-9 (miR-9-1, miR-9-2, miR-9-3) in 107 human head and neck tissue samples and controls. We found methylations of miR-9-1 and miR-9-3 were higher in oral and oropharyngeal carcinomas than that in laryngeal carcinoma, achieving a combined sensitivity of 63% and 56%, respectively, for these two tumor types, compared to 21% for the laryngeal carcinoma. Quantitative PCR of miR-9 showed reduced expression associated with methylation of miR-9 in tumor tissues. To investigate the functional consequences of miR-9 methylation, we found that miR-9 methylation is correlated with miR-9 expression level in human HNSCC cell lines. Demethylation treatment using 5-aza-deoxycytidine restored its expression in a miR-9 methylated human HNSCC cell line UM-SCC22A. Furthermore, cell proliferation and viability was significantly inhibited, while PTEN expression was elevated after transfection of miR-9 into the UM-SCC22A cell line. In summary, our results suggest that methylations of miR-9-1 and miR-9-3 are sensitive and specific biomarkers for HNSCC, particularly for oral and oropharyngeal squamous cell carcinomas. In addition, miR-9 may function as a tumor suppressor in HNSCC through inhibition of cell proliferation and elevation of tumor suppressor PTEN.