HDAC6: A Novel Histone Deacetylase Implicated in Pulmonary Arterial Hypertension.
ABSTRACT: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with limited therapeutic options. Although exposed to stressful conditions, pulmonary artery (PA) smooth muscle cells (PASMCs) exhibit a "cancer-like" pro-proliferative and anti-apoptotic phenotype. HDAC6 is a cytoplasmic histone deacetylase regulating multiple pro-survival mechanisms and overexpressed in response to stress in cancer cells. Due to the similarities between cancer and PAH, we hypothesized that HDAC6 expression is increased in PAH-PASMCs to face stress allowing them to survive and proliferate, thus contributing to vascular remodeling in PAH. We found that HDAC6 is significantly up-regulated in lungs, distal PAs, and isolated PASMCs from PAH patients and animal models. Inhibition of HDAC6 reduced PAH-PASMC proliferation and resistance to apoptosis in vitro sparing control cells. Mechanistically, we demonstrated that HDAC6 maintains Ku70 in a hypoacetylated state, blocking the translocation of Bax to mitochondria and preventing apoptosis. In vivo, pharmacological inhibition of HDAC6 improved established PAH in two experimental models and can be safely given in combination with currently approved PAH therapies. Moreover, Hdac6 deficient mice were partially protected against chronic hypoxia-induced pulmonary hypertension. Our study shows for the first time that HDAC6 is implicated in PAH development and represents a new promising target to improve PAH.
Project description:An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation leading to vascular wall thickening. Here, we report that STIM2 (stromal interaction molecule 2), a Ca2+ sensor in the sarcoplasmic reticulum membrane, is required for raising the resting [Ca2+]cyt in PASMCs from patients with pulmonary arterial hypertension (PAH) and activating signaling cascades that stimulate PASMC proliferation and inhibit PASMC apoptosis. Downregulation of STIM2 in PAH-PASMCs reduces the resting [Ca2+]cyt, whereas overexpression of STIM2 in normal PASMCs increases the resting [Ca2+]cyt The increased resting [Ca2+]cyt in PAH-PASMCs is associated with enhanced phosphorylation (p) of CREB (cAMP response element-binding protein), STAT3 (signal transducer and activator of transcription 3), and AKT, increased NFAT (nuclear factor of activated T-cell) nuclear translocation, and elevated level of Ki67 (a marker of cell proliferation). Furthermore, the STIM2-associated increase in the resting [Ca2+]cyt also upregulates the antiapoptotic protein Bcl-2 in PAH-PASMCs. Downregulation of STIM2 in PAH-PASMCs with siRNA (1) decreases the level of pCREB, pSTAT3, and pAKT and inhibits NFAT nuclear translocation, thereby attenuating proliferation, and (2) decreases Bcl-2, which leads to an increase of apoptosis. In summary, these data indicate that upregulated STIM2 in PAH-PASMCs, by raising the resting [Ca2+]cyt, contributes to enhancing PASMC proliferation by activating the CREB, STAT3, AKT, and NFAT signaling pathways and stimulating PASMC proliferation. The STIM2-associated increase in the resting [Ca2+]cyt is also involved in upregulating Bcl-2 that makes PAH-PASMCs resistant to apoptosis, and thus plays an important role in sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in patients with PAH.
Project description:Abstract Pulmonary vascular remodeling is the most important pathological characteristic of pulmonary arterial hypertension (PAH). No effective treatment for PAH is currently available because the mechanism underlying vascular remodeling is not completely clear. CD248, also known as endosialin, is a transmembrane protein that is highly expressed in pericytes and fibroblasts. Here, we evaluated the role of CD248 in pulmonary vascular remodeling and the processes of PAH pathogenesis. Activation of CD248 in pulmonary artery smooth muscle cells (PASMCs) was found to be proportional to the severity of PAH. CD248 contributed to platelet?derived growth factor?BB (PDGF?BB)?induced PASMC proliferation and migration along with the shift to more synthetic phenotypes. In contrast, treatment with Cd248 siRNA or the anti?CD248 therapeutic antibody (ontuxizumab) significantly inhibited the PDGF signaling pathway, obstructed NF??B p65?mediated transcription of Nox4, and decreased reactive oxygen species production induced by PDGF?BB in PAMSCs. In addition, knockdown of CD248 alleviated pulmonary vascular remodeling in rat PAH models. This study provides novel insights into the dysfunction of PASMCs leading to pulmonary vascular remodeling, and provides evidence for anti?remodeling treatment for PAH via the immediate targeting of CD248. CD248 as a novel therapeutic target in pulmonary arterial hypertension. Expression of CD248 contributed to platelet?derived growth factor?BB (PDGF?BB)?induced PASMC proliferation and migration along with the shift to more synthetic phenotypes. Besides, treatment of human PASMCs with PDGF?BB, to explore the PAH signaling, exhibited ERK and NF??B activation, enhanced NOX4 activity, and reactive oxygen species production.
Project description:Increased muscularity of small pulmonary vessels, involving enhanced proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a key component of the vascular remodeling underlying the development of pulmonary hypertension (PH). Stimuli such as growth factors and hypoxia induce PASMC alkalinization, proliferation, and migration through upregulation of the Na(+)/H(+) exchanger (NHE), inhibition of which prevents the development of hypoxia-induced vascular remodeling and PH. We wanted to explore whether NHE was also necessary for pathologic PASMC proliferation and migration in a model of pulmonary arterial hypertension (PAH), a severe form of PH not associated with persistent hypoxia. PASMCs were isolated from rats exposed to SU5416-hypoxia (SuHx) followed by return to normoxia and from vehicle controls. We measured resting intracellular pH (pHi) and NHE activity using the pH-sensitive fluorescent dye BCECF-AM. PASMC proliferation and migration were assessed using BrdU incorporation and transwell filters, respectively. NHE activity was increased in SuHx PASMCs, although resting pHi was unchanged. SuHx PASMCs also exhibited increased proliferation and migration relative to controls, which was attenuated in the setting of pharmacologic inhibition of NHE. Our findings suggest that increased NHE activity contributes to pathologic PASMC function in the SuHx model of PAH, although this effect does not appear to be mediated by global changes in pHi homeostasis.
Project description:Pulmonary arterial hypertension (PAH) is characterized by excessive pulmonary arterial smooth muscle cells (PASMCs) growth, partially in response to PDGF-BB but whether this is dependent on ?-catenin (?C) activation is unclear. Compared to healthy cells, PAH PASMCs demonstrate higher levels of proliferation both at baseline and with PDGF-BB that correlate with GSK3? dependent ?C activation. We show that ?C knockdown but not Wnt5a stimulation reduces PDGF-BB dependent growth and normalizes PAH PASMCs proliferation. These findings support that cross-talk between PDGF and Wnt signaling modulates PASMC proliferation and suggest that ?C targeted therapies could treat abnormal vascular remodeling in PAH.
Project description:Pulmonary arterial hypertension (PAH) is commonly associated with chronic hypoxemia in disorders such as chronic obstructive pulmonary disease (COPD). Prostacyclin analogs are widely used in the management of PAH patients; however, clinical efficacy and long-term tolerability of some prostacyclin analogs may be compromised by concomitant activation of the E-prostanoid 3 (EP3) receptor. Here, we found that EP3 expression is upregulated in pulmonary arterial smooth muscle cells (PASMCs) and human distal pulmonary arteries (PAs) in response to hypoxia. Either pharmacological inhibition of EP3 or Ep3 deletion attenuated both hypoxia and monocrotaline-induced pulmonary hypertension and restrained extracellular matrix accumulation in PAs in rodent models. In a murine PAH model, Ep3 deletion in SMCs, but not endothelial cells, retarded PA medial thickness. Knockdown of EP3? and EP3?, but not EP3?, isoforms diminished hypoxia-induced TGF-?1 activation. Expression of either EP3? or EP3? in EP3-deficient PASMCs restored TGF-?1 activation in response to hypoxia. EP3?/? activation in PASMCs increased RhoA-dependent membrane type 1 extracellular matrix metalloproteinase (MMP) translocation to the cell surface, subsequently activating pro-MMP-2 and promoting TGF-?1 signaling. Activation or disruption of EP3 did not influence PASMC proliferation. Together, our results indicate that EP3 activation facilitates hypoxia-induced vascular remodeling and pulmonary hypertension in mice and suggest EP3 inhibition as a potential therapeutic strategy for pulmonary hypertension.
Project description:Activation of the mammalian target of rapamycin complex 1 (mTORC1) subunit Raptor induces cell growth and is a downstream target of Akt. Elevated levels of aldosterone activate Akt, and, in pulmonary arterial hypertension (PAH), correlate with pulmonary arteriole thickening, which suggests that mTORC1 regulation by aldosterone may mediate adverse pulmonary vascular remodeling. We hypothesized that aldosterone-Raptor signaling induces abnormal pulmonary artery smooth muscle cell (PASMC) survival patterns to promote PAH. Remodeled pulmonary arterioles from SU-5416/hypoxia-PAH rats and monocrotaline-PAH rats with hyperaldosteronism expressed increased levels of the Raptor target, p70S6K, which provided a basis for investigating aldosterone-Raptor signaling in human PASMCs. Aldosterone (10(-9) to 10(-7) M) increased Akt/mTOR/Raptor to activate p70S6K and increase proliferation, viability, and apoptosis resistance in PASMCs. In PASMCs transfected with Raptor-small interfering RNA or treated with spironolactone/eplerenone, aldosterone or pulmonary arterial plasma from patients with PAH failed to increase p70S6K activation or to induce cell survival in vitro Optimal inhibition of pulmonary arteriole Raptor was achieved by treatment with Staramine-monomethoxy polyethylene glycol that was formulated with Raptor-small interfering RNA plus spironolactone in vivo, which decreased arteriole muscularization and pulmonary hypertension in 2 experimental animal models of PAH in vivo Up-regulation of mTORC1 by aldosterone is a critical pathobiologic mechanism that controls PASMC survival to promote hypertrophic vascular remodeling and PAH.-Aghamohammadzadeh, R., Zhang, Y.-Y., Stephens, T. E., Arons, E., Zaman, P., Polach, K. J., Matar, M., Yung, L.-M., Yu, P. B., Bowman, F. P., Opotowsky, A. R., Waxman, A. B., Loscalzo, J., Leopold, J. A., Maron, B. A. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension.
Project description:Background:Pulmonary arterial hypertension (PAH) is often characterized by cell proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). LncRNA cancer susceptibility candidate 2 (CASC2) has been revealed to be involved in PASMC injury in hypoxia-induced pulmonary hypertension. However, the exact molecular mechanisms whereby CASC2 regulates PASMC proliferation and migration are still incompletely understood. Methods:The expression levels of CASC2, miR-222 and inhibitor of growth 5 (ING5) were measured using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot, respectively. Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) assay. Wound healing assay was used to analyze cell migration ability. The relationship between miR-222 and CASC2 or ING5 was confirmed using bioinformatics analysis, luciferase reporter assay and RNA immunoprecipitation assay. Results:CASC2 was down-regulated in hypoxia-induced PASMCs in a dose- and time-dependent manner. Functional experiments showed that CASC2 overexpression could reverse hypoxia-induced proliferation and migration of PASMCs. Bioinformatics analysis indicated that CASC2 acted as a competing endogenous RNA of miR-222, thereby regulating the expression of ING5, the downstream target of miR-222, in PASMCs. In addition, rescue assay suggested that the inhibition mediated by CASC2 of hypoxia-induced PASMC proliferation and migration could be attenuated by miR-222 inhibition or ING5 overexpression. Conclusion:CASC2 attenuated hypoxia-induced PASMC proliferation and migration by regulating the miR-222/ING5 axis to prevent vascular remodeling and the development of PAH, providing a novel insight and therapeutic strategy for hypoxia-induced PAH.
Project description:Pulmonary arterial hypertension (PAH) is a rare progressive pulmonary vascular disorder associated with vascular remodeling and right heart failure. Vascular remodeling involves numerous signaling cascades governing pulmonary arterial smooth muscle cell (PASMC) proliferation, migration and differentiation. Glycogen synthase kinase 3beta (GSK3ß) is a serine/threonine kinase and can act as a downstream regulatory switch for numerous signaling pathways. Hence, we hypothesized that GSK3ß plays a crucial role in pulmonary vascular remodeling.All experiments were done with lung tissue or isolated PASMCs in a well-established monocrotaline (MCT)-induced PAH rat model. The mRNA expression of Wnt ligands (Wnt1, Wnt3a, Wnt5a), upstream Wnt signaling regulator genes (Frizzled Receptors 1, 2 and secreted Frizzled related protein sFRP-1) and canonical Wnt intracellular effectors (GSK3ß, Axin1) were assessed by real-time polymerase chain reaction and protein levels of GSK3ß, phospho-GSK3ß (ser 9) by western blotting and localization by immunohistochemistry. The role of GSK3ß in PASMCs proliferation was assessed by overexpression of wild-type GSK3ß (WT) and constitutively active GSK3ß S9A by [(3)H]-thymidine incorporation assay.Increased levels of total and phosphorylated GSK3ß (inhibitory phosphorylation) were observed in lungs and PASMCs isolated from MCT-induced PAH rats compared to controls. Further, stimulation of MCT-PASMCs with growth factors induced GSK3ß inactivation. Most importantly, treatment with the PDGFR inhibitor, Imatinib, attenuated PDGF-BB and FCS induced GSK3ß phosphorylation. Increased expression of GSK3ß observed in lungs and PASMC isolated from MCT-induced PAH rats was confirmed to be clinically relevant as the same observation was identified in human iPAH lung explants. Overexpression of GSK3ß significantly increased MCT-PASMCs proliferation by regulating ERK phosphorylation. Constitutive activation of GSK3ß (GSK3ß S9A, 9th serine replaced to alanine) inhibited MCT-PASMCs proliferation by decreasing ERK phosphorylation.This study supports a central role for GSK3ß in vascular remodeling processes and suggests a novel therapeutic opportunity for the treatment of PAH.
Project description:Pulmonary arterial hypertension (PAH) is an often fatal disease with limited treatment options. Whereas current data support the notion that, in pulmonary artery endothelial cells (PAECs), expression of transcription factor hypoxia inducible factor-1? (HIF-1?) is increased, the role of HIF-1? in pulmonary artery smooth muscle cells (PASMCs) remains controversial. This study investigates the hypothesis that, in PASMCs from patients with PAH, decreases in HIF-1? expression and activity underlie augmented pulmonary vascular contractility. PASMCs and tissues were isolated from nonhypertensive control patients and patients with PAH. Compared with controls, HIF-1? and Kv1.5 protein expression were decreased in PAH smooth muscle cells (primary culture). Myosin light chain (MLC) phosphorylation and MLC kinase (MLCK) activity-major determinants of vascular tone-were increased in patients with PAH. Cofactors involved in prolyl hydroxylase domain activity were increased in PAH smooth muscle cells. Functionally, PASMC contractility was inversely correlated with HIF-1? activity. In PASMCs derived from patients with PAH, HIF-1? expression is decreased, and MLCK activity, MLC phosphorylation, and cell contraction are increased. We conclude that compromised PASMC HIF-1? expression may contribute to the increased tone that characterizes pulmonary hypertension.-Barnes, E. A., Chen, C.-H., Sedan, O., Cornfield, D. N. Loss of smooth muscle cell hypoxia inducible factor-1? underlies increased vascular contractility in pulmonary hypertension.
Project description:In pulmonary arterial hypertension (PAH), excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) causes vascular medial thickening. Medial thickening is a histopathological hallmark of pulmonary vascular remodeling, the central disease process driving PAH progression. Pulmonary vascular remodeling causes stenosis and/or obstruction of small pulmonary arteries. This leads to increased pulmonary vascular resistance, elevated pulmonary arterial pressure, and ultimately right heart failure. To improve the survival of PAH patients, which remains at approximately 60% at 3 years after diagnosis, the development of novel PAH-targeted drugs is desired. To this end, a detailed understanding of the mechanisms underlying excessive PASMC proliferation and the medial thickening that ensues is necessary. However, a lack of in vitro models that recapitulate medial thickening impedes our deeper understanding of the pathogenetic mechanisms involved. In the present study, we applied 3-dimensional (3D) cell culture technology to develop a novel in vitro model of the pulmonary artery medial layer using human PAH patient-derived PASMCs. The addition of platelet-derived growth factor (PDGF)-BB, a mitogen known to promote excessive PASMC proliferation in PAH, resulted in increased thickness of the 3D-PAH media tissues. Conversely, administration of the PDGF receptor inhibitor imatinib or other clinical PAH drugs inhibited this medial thickening-inducing effect of PDGF-BB. Altogether, by using 3D cell culture technology, we report the generation of an in vitro model of medial thickening in PAH, which had hitherto not been successfully modeled in vitro. This model is potentially useful for assessing the ability of candidate PAH drugs to suppress medial thickening.