Development Of Pulmonary Vascular Remodeling Research Articles

The role and mechanism of FTO in pulmonary vessels

ABSTRACT Pulmonary vascular remodeling (PVR) is the main factor of pulmonary hypertension (PH). The pathological characteristics of PVR are vascular smooth muscle hyperplasia, hypertrophy, and extensive damage. In vivo experiments, the expression of FTO in PH rat lung tissues of different rat models of hypoxia PH was observed by immunohistochemical method. mRNA microarray analysis was used to analyze the differential expressed genes in rat lung tissues. In vitro experiments, we developed models of overexpression and knockdown of FTO to study the effect of FTO protein expression on cell apoptotic, cell cycle, and the abundance of m6A. The expression of FTO was increased in PH rats. FTO knockdown can inhibit the proliferation of PASMCs, thereby regulating the cell cycle and reducing the expression of Cyclin D1 and the abundance of m6A, while overexpression of FTO leads to increased expression of Cyclin D1 and the abundance of m6A. FTO destroys the stability of Cyclin D1 by regulating the abundance of Cyclin D1 m6A, causing cell cycle arrest and inducing cell proliferation, thus inducing the occurrence and development of PVR in PH.

Ythdf2 promotes pulmonary hypertension by suppressing Hmox1-dependent anti-inflammatory and antioxidant function in alveolar macrophages

Pulmonary hypertension (PH) is a devastating disease characterized by irreversible pulmonary vascular remodeling (PVR) that causes right ventricular failure and death. The early alternative activation of macrophages is a critical event in the development of PVR and PH, but the underlying mechanisms remain elusive. Previously we have shown that N6-methyladenosine (m6A) modifications of RNA contribute to phenotypic switching of pulmonary artery smooth muscle cells and PH. In the current study, we identify Ythdf2, an m6A reader, as an important regulator of pulmonary inflammation and redox regulation in PH. In a mouse model of PH, the protein expression of Ythdf2 was increased in alveolar macrophages (AMs) during the early stages of hypoxia. Mice with a myeloid specific knockout of Ythdf2 (Ythdf2Lyz2 Cre) were protected from PH with attenuated right ventricular hypertrophy and PVR compared to control mice and this was accompanied by decreased macrophage polarization and oxidative stress. In the absence of Ythdf2, heme oxygenase 1 (Hmox1) mRNA and protein expression were significantly elevated in hypoxic AMs. Mechanistically, Ythdf2 promoted the degradation of Hmox1 mRNA in a m6A dependent manner. Furthermore, an inhibitor of Hmox1 promoted macrophage alternative activation, and reversed the protection from PH seen in Ythdf2Lyz2 Cre mice under hypoxic exposure. Together, our data reveal a novel mechanism linking m6A RNA modification with changes in macrophage phenotype, inflammation and oxidative stress in PH, and identify Hmox1 as a downstream target of Ythdf2, suggesting that Ythdf2 may be a therapeutic target in PH.

Chitinase 3 like 1 contributes to the development of pulmonary vascular remodeling in pulmonary hypertension.

Chitinase 3 like 1 (CHI3L1) is the prototypic chitinase-like protein mediating inflammation, cell proliferation, and tissue remodeling. Limited data suggest CHI3L1 is elevated in human pulmonary arterial hypertension (PAH) and is associated with disease severity. Despite its importance as a regulator of injury/repair responses, the relationship between CHI3L1 and pulmonary vascular remodeling is not well understood. We hypothesize that CHI3L1 and its signaling pathways contribute to the vascular remodeling responses that occur in pulmonary hypertension (PH). We examined the relationship of plasma CHI3L1 levels and severity of PH in patients with various forms of PH, including group 1 PAH and group 3 PH, and found that circulating levels of serum CHI3L1 were associated with worse hemodynamics and correlated directly with mean pulmonary artery pressure and pulmonary vascular resistance. We also used transgenic mice with constitutive knockout and inducible overexpression of CHI3L1 to examine its role in hypoxia-, monocrotaline-, and bleomycin-induced models of pulmonary vascular disease. In all 3 mouse models of pulmonary vascular disease, pulmonary hypertensive responses were mitigated in CHI3L1-null mice and accentuated in transgenic mice that overexpress CHI3L1. Finally, CHI3L1 alone was sufficient to induce pulmonary arterial smooth muscle cell proliferation, inhibit pulmonary vascular endothelial cell apoptosis, induce the loss of endothelial barrier function, and induce endothelial-mesenchymal transition. These findings demonstrate that CHI3L1 and its receptors play an integral role in pulmonary vascular disease pathobiology and may offer a target for the treatment of PAH and PH associated with fibrotic lung disease.

Upregulation of endothelial hypoxia‐inducible factor‐2 alpha in animals with pulmonary hypertension

IntroductionIdiopathic pulmonary arterial hypertension (IPAH) is a life‐threatening disease with no cure. Pulmonary vascular remodeling due to increased pulmonary artery smooth muscle (PASMC) and lung vascular endothelial cell (LVEC) proliferation is a major cause for the elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in patients with IPAH. We have previously demonstrated that endothelial hypoxia‐inducible factor‐2α (HIF‐2α) contributes to the development of pulmonary vascular remodeling in LVEC isolated from IPAH patients. Moreover, HIF‐2α is involved in the initiation of endothelial‐to‐mesenchymal transition (EndMT) by upregulating Snai1 and Snai2 (EndMT drivers). However, the exact role of HIF‐2α in the development and progression of pulmonary hypertension (PH) remains elusive. We aimed to investigate whether endothelial HIF‐2α is involved in pulmonary vascular remodeling in animals with hypoxia/sugen‐induced PH (SuHx‐PH) and monocrotaline‐induced PH (MCT‐PH).MethodsHypoxia/sugen‐PH (SuHx‐PH) model in mice was induced by exposing animals to the combination of normobaric hypoxia (10% O2) and sugen 5416 (20 mg/kg, i.p., weekly) for 6 weeks. Monocrotaline‐MCT‐PH model in rats was induced by single administration of MCT (60 mg/kg, s.c.) followed by experimental measurements after 28 days. Body weight (BW) was collected from all animals weekly. Hemodynamic parameters including right ventricular systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP), the Fulton index (FI), right ventricular (RV) contractility, heart rate (HR) were measured to establish PH. The pulmonary artery (PA) wall thickness was estimated by H&E staining. The protein expression of Snai1 and HIF‐2α was determined by Western blot and immunocytochemistry. Normal human LVEC was treated with TGF‐β1 for 7 days to induce EndMT. LVEC isolated with patients with IPAH was treated with HIF‐2α inhibitor Compound 2 for 8‐48 hrs.ResultsExposure of mice to the SuHx (6 weeks) or rats to MCT (4 weeks) resulted in the significant increase of RVSP, mPAP, RV contractility, and FI compared to control animals. We did not detect any changes in HR between corresponded groups. BW was decreased by 7% after the first week of the SuHx treatment in mice. By the end of 6 weeks BW was significantly lower by 12% in SuHx mice compared with normoxic animals. BW in MCT‐treated rats was 5% lower after the first week and at the end of experiment compared to vehicle‐treated animals. Pulmonary vascular remodeling estimated by PA wall thickness was enhanced in both SuHx‐PH mice and MCT‐PH rats. The protein expression of HIF‐2α and Snai1 was higher in lung tissues isolated from SuHx‐PH mice and MCT‐PH rats compared to controls. Moreover, HIF‐2α and Snai1 was activated in normal human LVEC treated with TGF‐β1 (10ng/ml) for 7 days. Additionally, HIF‐2α inhibitor, Compound 2 (30µM) was able to downregulate Snai1 protein expression in LVEC isolated from IPAH patients after 16 hours.ConclusionsOur results indicate that HIF‐2α is involved in pulmonary vascular remodeling in animals with PH. Our studies support the premise that targeting HIF‐2α would be a novel therapeutic approach for the treatment of IPAH and other forms of PH.

Mechanosensitive channel Piezo1 is required for pulmonary artery smooth muscle cell proliferation.

Concentric pulmonary vascular wall thickening due partially to increased pulmonary artery (PA) smooth muscle cell (PASMC) proliferation contributes to elevating pulmonary vascular resistance (PVR) in patients with pulmonary hypertension (PH). Although pulmonary vasoconstriction may be an early contributor to increasing PVR, the transition of contractile PASMCs to proliferative PASMCs may play an important role in the development and progression of pulmonary vascular remodeling in PH. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) is a trigger for PASMC contraction and proliferation. Here, we report that upregulation of Piezo1, a mechanosensitive cation channel, is involved in the contractile-to-proliferative phenotypic transition of PASMCs and potential development of pulmonary vascular remodeling. By comparing freshly isolated PA (contractile PASMCs) and primary cultured PASMCs (from the same rat) in a growth medium (proliferative PASMCs), we found that Piezo1, Notch2/3, and CaSR protein levels were significantly higher in proliferative PASMCs than in contractile PASMCs. Upregulated Piezo1 was associated with an increase in expression of PCNA, a marker for cell proliferation, whereas downregulation (with siRNA) or inhibition (with GsMTx4) of Piezo1 attenuated PASMC proliferation. Furthermore, Piezo1 in the remodeled PA from rats with experimental PH was upregulated compared with PA from control rats. These data indicate that PASMC contractile-to-proliferative phenotypic transition is associated with the transition or adaptation of membrane channels and receptors. Upregulated Piezo1 may play a critical role in PASMC phenotypic transition and PASMC proliferation. Upregulation of Piezo1 in proliferative PASMCs may likely be required to provide sufficient Ca2+ to assure nuclear/cell division and PASMC proliferation, contributing to the development and progression of pulmonary vascular remodeling in PH.

The substitution of SERCA2 redox cysteine 674 promotes pulmonary vascular remodeling by activating IRE1α/XBP1s pathway

Pulmonary hypertension (PH) is a life-threatening disease characterized by pulmonary vascular remodeling, in which hyperproliferation of pulmonary artery smooth muscle cells (PASMCs) plays an important role. The cysteine 674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is the critical redox regulatory cysteine to regulate SERCA2 activity. Heterozygous SERCA2 C674S knock-in mice (SKI), where one copy of C674 was substituted by serine to represent partial C674 oxidative inactivation, developed significant pulmonary vascular remodeling resembling human PH, and their right ventricular systolic pressure modestly increased with age. In PASMCs, substitution of C674 activated inositol requiring enzyme 1 alpha (IRE1α) and spliced X-box binding protein 1 (XBP1s) pathway, accelerated cell cycle and cell proliferation, which reversed by IRE1α/XBP1s pathway inhibitor 4μ8C. In addition, suppressing the IRE1α/XBP1s pathway prevented pulmonary vascular remodeling caused by substitution of C674. Similar to SERCA2a, SERCA2b is also important to restrict the proliferation of PASMCs. Our study articulates the causal effect of C674 oxidative inactivation on the development of pulmonary vascular remodeling and PH, emphasizing the importance of C674 in restricting PASMC proliferation to maintain pulmonary vascular homeostasis. Moreover, the IRE1α/XBP1s pathway and SERCA2 might be potential targets for PH therapy.

C81. Pulse Dose Corticosteroid Improves Clinical Outcome in Pulmonary Hypertension Patient: A Potential of Immense Affordability and Availability Medication in Suburban Area

Abstract Background Pulmonary hypertension (PH) in adolescence is a devastating diagnosis with significant morbidity and mortality. Despite limited literature exists on treating PH with anti-inflammatory medications in humans, hereby we present the case of prednisolone induced clinical outcomes improvement in PH patient. Case Summary A 20-year-old woman presented with exertional dyspnea. Her past-two-years medical history was remarkable for tricuspid regurgitation with multiple readmission. Upon arrival, her saturation-75% and BP-110/60. She was in respiratory distress with JVP increased and high-pitched-holosystolic murmur. Laboratory studies was unremarkable. The x-ray revealed cardiomegaly and ECG showing RVH-RAE. Initial echocardiography demonstrated TR-Vmax 4.55m/s and PR-Vmax 3.62m/s. The RVSP estimated around 107mmHg, which was highly suggestive for PH. Due to the aggressive nature of the disease, her condition deteriorated despite management with IV-furosemide-(40mg/d), beta-blocker-(2.5mg/d), and sodium beraprost-(60µg/d). Following days, a 5-days pulse-dose-steroid (methylprednisolone) at 2mg/kg/day was administered. Her condition improved with saturation from 50% to 90% and significant reduced in dyspnea. The steroid was tapering down for another 7 days and she was discharged for further referral. Discussion Inflammation plays unequivocal role in the pathogenesis of PH, which was involved in the development of pulmonary vascular remodeling. The possible mechanism of prednisolone were related with the inhibition of platelet-derived growth factor (PDGF)-induced accelerated proliferation and migration of pulmonary artery smooth muscle cell, and prevented the activation of NF-κB, key transcription factor that controls inflammation, cell-proliferation, and apoptosis. Thus, given the ready availability and features of this medication compared with other PH-medication, we decided to utilize steroid in this patient.

C81. Pulse Dose Corticosteroid Improves Clinical Outcome in Pulmonary Hypertension Patient: A Potential of Immense Affordability and Availability Medication in Suburban Area

Abstract Background Pulmonary hypertension (PH) in adolescence is a devastating diagnosis with significant morbidity and mortality. Despite limited literature exists on treating PH with anti-inflammatory medications in humans, hereby we present the case of prednisolone induced clinical outcomes improvement in PH patient. Case Summary A 20-year-old woman presented with exertional dyspnea. Her past-two-years medical history was remarkable for tricuspid regurgitation with multiple readmission. Upon arrival, her saturation-75% and BP-110/60. She was in respiratory distress with JVP increased and high-pitched-holosystolic murmur. Laboratory studies was unremarkable. The x-ray revealed cardiomegaly and ECG showing RVH-RAE. Initial echocardiography demonstrated TR-Vmax 4.55m/s and PR-Vmax 3.62m/s. The RVSP estimated around 107mmHg, which was highly suggestive for PH. Due to the aggressive nature of the disease, her condition deteriorated despite management with IV-furosemide-(40mg/d), beta-blocker-(2.5mg/d), and sodium beraprost-(60µg/d). Following days, a 5-days pulse-dose-steroid (methylprednisolone) at 2mg/kg/day was administered. Her condition improved with saturation from 50% to 90% and significant reduced in dyspnea. The steroid was tapering down for another 7 days and she was discharged for further referral. Discussion Inflammation plays unequivocal role in the pathogenesis of PH, which was involved in the development of pulmonary vascular remodeling. The possible mechanism of prednisolone were related with the inhibition of platelet-derived growth factor (PDGF)-induced accelerated proliferation and migration of pulmonary artery smooth muscle cell, and prevented the activation of NF-κB, key transcription factor that controls inflammation, cell-proliferation, and apoptosis. Thus, given the ready availability and features of this medication compared with other PH-medication, we decided to utilize steroid in this patient.

Endothelial upregulation of mechanosensitive channel Piezo1 in pulmonary hypertension.

Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca2+ and Na+ influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here, we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared with normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca2+-dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag-1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, whereas downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag-1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Intraperitoneal injection of a Piezo1 channel blocker, GsMTx4, ameliorated experimental PH in mice. Taken together, our study suggests that membrane stretch-mediated Ca2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.

MicroRNA-663 prevents monocrotaline-induced pulmonary arterial hypertension by targeting TGF-β1/smad2/3 signaling

ObjectivePulmonary vascular remodeling due to excessive growth factor production and pulmonary artery smooth muscle cells (PASMCs) proliferation is the hallmark feature of pulmonary arterial hypertension (PAH). Recent studies suggest that miR-663 is a potent modulator for tumorigenesis and atherosclerosis. However, whether miR-663 involves in pulmonary vascular remodeling is still unclear. Methods and resultsBy using quantitative RT-PCR, we found that miR-663 was highly expressed in normal human PASMCs. In contrast, circulating level of miR-663 dramatically reduced in PAH patients. In addition, in situ hybridization showed that expression of miR-663 was decreased in pulmonary vasculature of PAH patients. Furthermore, MTT and cell scratch-wound assay showed that transfection of miR-663 mimics significantly inhibited platelet derived growth factor (PDGF)-induced PASMCs proliferation and migration, while knockdown of miR-663 expression enhanced these effects. Mechanistically, dual-luciferase reporter assay revealed that miR-663 directly targets the 3’UTR of TGF-β1. Moreover, western blots and ELISA results showed that miR-663 decreased PDGF-induced TGF-β1 expression and secretion, which in turn suppressed the downstream smad2/3 phosphorylation and collagen I expression. Finally, intratracheal instillation of adeno-miR-663 efficiently inhibited the development of pulmonary vascular remodeling and right ventricular hypertrophy in monocrotaline (MCT)-induced PAH rat models. ConclusionThese results indicate that miR-663 is a potential biomarker for PAH. MiR-663 decreases PDGF-BB-induced PASMCs proliferation and prevents pulmonary vascular remodeling and right ventricular hypertrophy in MCT-PAH by targeting TGF-β1/smad2/3 signaling. These findings suggest that miR-663 may represent as an attractive approach for the diagnosis and treatment for PAH.

CENPE contributes to pulmonary vascular remodeling in pulmonary hypertension

Hypoxic pulmonary vascular remodeling is a pathological feature of pulmonary hypertension (PH). Our results showed that centromere-associated protein E (CENPE) expression in PH patients and hypoxia-induced PH rats was significantly higher than that in normal controls. In addition, CENPE deficiency significantly inhibited the development of pulmonary vascular remodeling and right ventricular hypertrophy. Moreover, knocking out CENPE effectively inhibited the proliferation and induced the apoptosis of primary pulmonary artery smooth muscle cells (PASMCs) in vivo. Furthermore, CENPE silencing by small interference significantly inhibited abnormal proliferation, apoptosis resistance, migration, and cell cycle arrest in hypoxia-induced PASMCs. Interestingly, we found that CENPE might exert its biological effect by targeting the transcription of CDK1 proteins.

Cytokines, Chemokines, and Inflammation in Pulmonary Arterial Hypertension.

According to the World Symposium Pulmonary Hypertension (WSPH) classification, pulmonary hypertension (PH) is classified into five categories based on etiology. Among them, Group 1 pulmonary arterial hypertension (PAH) disorders are rare but progressive and often, fatal despite multiple approved treatments. Elevated pulmonary arterial pressure in patients with WSPH Group 1 PAH is mainly caused by increased pulmonary vascular resistance (PVR), due primarily to sustained pulmonary vasoconstriction and excessive obliterative pulmonary vascular remodeling. Growing evidence indicates that inflammation plays a critical role in the development of pulmonary vascular remodeling associated with PAH. While the role of auto-immunity is unclear, infiltration of inflammatory cells in and around vascular lesions, including T- and B-cells, dendritic cells, macrophages, and mast cells have been observed in PAH patients. Serum and plasma levels of chemokines, cytokines, and autoantibodies are also increased in PAH patients; some of these circulating molecules are correlated with disease severity and survival. Preclinical experiments have reported a key role of the inflammation in PAH pathophysiology in vivo. Importantly, anti-inflammatory and immunosuppressive agents have further exhibited therapeutic effects. The present chapter reviews published experimental and clinical evidence highlighting the canonical role of inflammation in the pathogenesis of PAH and as a major target for the development of anti-inflammatory therapies in patients with PAH.

Decreased MicroRNA‐153 Promotes Endothelial‐to‐Mesenchymal Transition in Idiopathic Pulmonary Arterial Hypertension

IntroductionIdiopathic pulmonary arterial hypertension (IPAH) is a fatal and progressive disease. Pulmonary vascular remodeling and vasoconstriction are the major factors for the increased pulmonary vascular resistance (PVR) and pulmonary arterial pressure in IPAH patients. Endothelial‐to‐mesenchymal transition (EndMT) has been recently implicated in the development and progression of pulmonary vascular remodeling in IPAH. EndMT is a biological process which converts fully differentiated and slowly growing endothelial cells (EC) into the highly‐proliferative myofibroblasts (myoFB). We previously reported that elevated endothelial hypoxia‐inducible factor‐2α (HIF‐2α or EPAS1) due to decreased prolyl hydroxylase domain protein 2 (PHD2 or EGLN1) contributes to EndMT in pulmonary hypertension (PH). However, the underlying molecular mechanisms of EndMT are still unknown. In this study, we aimed to investigate the role of microRNA (miR) in the regulation of EndMT. MiR is a small non‐coding RNA which is able to regulate gene expression post‐transcriptionally by inhibiting mRNA translation and stability. We hypothesize that decreased miR‐153 resultes in upregulating of SNAI1 and SNAI2 and the enhanced EndMT in lung vascular EC (LVEC) and contributes to pulmonary vascular remodeling in PH.MethodsLVEC from healthy subjects and patients with IPAH were used for this study. In silico analysis was perfomed by using standard online software (Targetscan.org). miR‐153 level was measured by real‐time RT‐PCR. Normal LVEC were transfected with miR‐153 inhibitor while IPAH‐LVEC were overexpressed with miR‐153 mimic. mRNA and protein expression SNAI1/2, PHD2, and HIF‐2α were measured by RT‐PCR, Western blot and ICC.ResultsIn silico analysis revealed that miR‐153 directly targets human SNAI1/2. Downregulated miR‐153 is correlated with increased SNAI1/2 expression and EndMT in LVEC from IPAH patients compared to normal LVEC. Transfroming growth factor‐β1 (TGF‐β1), a well‐known stimulus for EndMT, decreases miR‐153 and induces SNAI1/2 in normal human LVEC. PHD2 was downregulated while HIF‐2α was activated in TGF‐β1‐treated normal LVEC. Inhibition of miR‐153 in normal LVEC significantly upregulates SNAI1/2 expression while overexpression of miR‐153 in IPAH‐LVEC decreases SNAI1/2 level. Moreover, miR‐153 level was lower in lung tissues isolated from mice with endothelial specific deletion of egln1, which spontaneously develop severe PH due to increased HIF‐2α and enhanced EndMT.ConclusionThe decreased miR‐153 promotes phenotypical transition of LVEC to highly proliferative myoFB via enhancing HIF‐2α‐associated EndMT, and contributes to neointimal formation, intimal thickening and distal PA obliteration. The concentric PA wall thickening and intraluminal obliteration in arterioles directly increase PVR and right ventricular afterload in IPAH patients. We conclude that PHD2/HIF‐2α/miR‐153/SNAI axis drives the initiation and development of pulmonary vascular remodeling and obliterative lesions contributing to the development of PH.Support or Funding InformationThis work is supported in part by the grants from the National Heart, Lung and Blood Institute of the National Institutes of Health (HL135807).

Expression of Kv11.1 Channels in Lungs and Role in Pulmonary Hypertension

BackgroundWhile Kv11.1 potassium selective channels are well known for their contribution to the heart repolarization little is known about their importance for the lung function. Here we show that Kv11.1 channels are abundantly expressed in the human and rat lung tissues.Methods & ResultsWe found that, in the lungs of healthy humans and rats, Kv11.1 channel expression was limited to the large pulmonary arteries (PAs). In the lungs of human patients with chronic obstructive pulmonary disease (COPD) and of rats with pulmonary arterial hypertension (PAH), Kv11.1 channels were expressed not only in the large PAs, but also in the small PAs with the diameter <100 micrometers. The increase in the Kv11.1 channel expression followed the time course of the development of pulmonary vascular remodeling in PAH rats. Administration of dofetilide, a Kv11.1 blocker, to rats with PAH inhibited PAH‐associated pulmonary vascular remodeling.ConclusionsOur study indicates that Kv11.11 channels are expressed in the lung and that these channels mediate the development of pulmonary vascular remodeling. Dofetilide is an FDA approved drug used for treatment of atrial fibrillation. Thus, our findings raise a possibility of repurposing this antiarrhythmic drug for the treatment of pulmonary hypertension.Support or Funding InformationSupported by NIH

Effects of 2‐APB, a Stim‐Activated TRPC‐ORAI Channels Blocker, on the Vascular Remodeling and Pulmonary Hypertension Induced by Chronic Intermittent Hypoxia

Obstructive sleep apnea (OSA), a breathing disorder characterized by episodes of chronic intermittent hypoxia (CIH), is an independent risk for systemic hypertension. Furthermore, 20–60% of OSA patients develop moderate pulmonary hypertension (PH). Rats exposed to CIH developed pulmonary vascular remodeling and PH, but the mechanisms underlaying these responses are not well known. Stim‐activated TRPC‐ORAI channel (STOC), cationic calciumpermeable channels, are overexpressed in the lung and plays a key role in the development of pulmonary vascular remodeling and PH in animals exposed to sustained hypoxia. We previously demonstrated that STOC are overexpressed in lung of rats exposed to CIH. However, whether these channels are involved in the pulmonary vascular remodeling and PH induced by CIH have not been addressed. Thus, we studied the effects of treatment of 2‐aminoethyldiphenylborinate (2‐APB), a STOC blocker, in rats exposed to CIH. Male Sprague‐Dawley rats (~200g) were exposed for 28 days to CIH (5% O2, 12 times/h, for 8h). At 14 days of CIH, rats under anesthesia were implanted with osmotic pumps containing 2‐APB (10 mg/kg/day) or its vehicle, and then rats were exposed to CIH for 14 more days. At the end of CIH exposure, the right ventricular systolic pressure (RVSP) was measure under anesthesia with urethane/α‐cloralose (800 mg/kg, i.p). Animals were then euthanized and lungs dissected to determine vascular remodeling in arteries of 200–300 μm, and the mRNA levels of the STOC forming subunits TRPC1, TRPC4, TRPC6, ORAI 1 and ORAI 2 by qPCR. 2‐APB infusion prevented the increase of the RVSP (36.5 ± 1.1 mmHg vs. 24.7 ± 1.6 mmHg, 28d‐CIH vs. 2‐APB respectively), and the pulmonary vascular remodeling (66.3 ± 9.4 % vs 48.2 ± 3.5 %, CIH, 28d‐CIH vs. 2‐APB, respectively). In addition, 2‐APB treatment reverted the increase in the mRNA levels of TRPC1, TRPC4 and TRPC6, but induced an overexpression of ORAI 1, while ORAI 2 remained unchanged. Present results suggest that STOC plays a key role in the developing of the vascular remodeling and pulmonary hypertension induced by CIH.Support or Funding InformationSUPPORTED BY FONDECYT 1150040 and 1180341

Increased Smooth Muscle Kv11.1 Channel Expression in Pulmonary Hypertension and Protective Role of Kv11.1 Channel Blocker Dofetilide

Kv11.1 potassium channels are essential for heart repolarization. Prescription medication that blocks Kv11.1 channels lengthens the ventricular action potential and causes cardiac arrhythmias. Surprisingly little is known about the Kv11.1 channel expression and function in the lung tissue. Here we report that Kv11.1 channels were abundantly expressed in the large pulmonary arteries (PAs) of healthy lung tissues from humans and rats. Kv11.1 channel expression was increased in the lungs of humans affected by chronic obstructive pulmonary disease-associated pulmonary hypertension and in the lungs of rats with pulmonary arterial hypertension (PAH). In healthy lung tissues from humans and rats, Kv11.1 channels were confined to the large PAs. In humans with chronic obstructive pulmonary disease-associated pulmonary hypertension and in rats with PAH, Kv11.1 channels were expressed in both the large and small PAs. The increase in Kv11.1 channel expression closely followed the time-course of the development of pulmonary vascular remodeling in PAH rats. Treatment of PAH rats with dofetilide, an Kv11.1 channel blocker approved by the US Food and Drug Administration for use in the treatment of arrythmia, inhibited PAH-associated pulmonary vascular remodeling. Taken together, the findings from this study uncovered a novel role of Kv11.1 channels in lung function and their potential as new drug targets in the treatment of pulmonary hypertension. The protective effect of dofetilide raises the possibility of repurposing this antiarrhythmic drug for the treatment of patients with pulmonary hypertension.

Protective effects of 18β-glycyrrhetinic acid on pulmonary arterial hypertension via regulation of Rho A/Rho kinsase pathway

PurposeExcessive proliferation, migration and anti-apoptosis of pulmonary artery smooth muscle cells (PASMCs) are the basis for the development of pulmonary vascular remodeling, and it is the driving force for pulmonary arterial hypertension (PAH). 18β-glycyrrhetinic acid (18β-GA) is the main active substance extracted from Chinese herbal medicine licorice, with outstanding anti-inflammatory, anti-oxidation and anti-proliferative effects. Our team found in previous studies that 18β-GA has protective effects on monocrotaline-induced PAH in rats. However, the anti-angiogenic effect of 18β-GA on PAH remains unclear. Therefore, in order to further investigate whether the beneficial effects of 18β-GA on PAH are related to its antiproliferative effect, we conducted experiments in vivo and in vitro. Methods and resultsIn vivo, 18β-GA relieved mean pulmonary arterial pressure, right ventricular systolic pressure, and right ventricular hypertrophy index, improving pulmonary remodeling. In vitro, 18β-GA significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of HPASMCs, blocking the progression of G0/G1 to S phase of the cell cycle. Furthermore, after treatment with 18β-GA, the expression of Rho A, ROCK1, ROCK2 was decreased and ROCK activity was inhibited in HPASMC. In addition, 18β-GA also attenuated PDGF-induced changes in p27kip1, Bax and Bcl-2. ConclusionsIn summary, these results indicate that 18β-GA regulates the activity of RhoA-ROCK signaling pathway, inhibits the proliferation of HPASMCs, and has potential value in the treatment of PAH.

Metformin Prevents Progression of Experimental Pulmonary Hypertension via Inhibition of Autophagy and Activation of Adenosine Monophosphate-Activated Protein Kinase

Previous studies have shown that metformin (MET) prevents experimental pulmonary arterial hypertension (PAH) and that activation of autophagy is involved in the development of pulmonary vascular remodeling. However, the mechanism of how MET inhibits autophagy and reverses pulmonary vascular remodeling is still unclear. The objective of the present study was to investigate the role of autophagy in MET-induced hypoxia PAH protection and the underlying mechanisms. To examine the effects of MET on hypoxia, we treated rats with MET (100 mg/kg/day) after 3 weeks of hypoxia. Hemodynamic changes, weight of the right ventricle/left ventricle plus septum (RV/LV+S) ratio, and lung morphological features were examined after 3 weeks. In addition, alpha smooth muscle actin (α-SMA), p62, and PCNA were assessed by immunofluorescence and immunohistochemistry staining. BECN-1, LC3B, p62, and activation of adenosine monophosphate-activated protein kinase (AMPK) were analyzed by Western blotting. Cell proliferation was detected using the Cell Counting Kit-8 (CCK-8) and the 5-ethynyl-2′-deoxyuridine staining kit assay. Hypoxia induced increases in right ventricular systolic pressure and the RV/LV+S ratio, which were attenuated by MET treatment. MET also inhibited hypoxia-induced pulmonary vascular remodeling, collagen deposition, proliferation of pulmonary arterial smooth muscle cells, elevation of BECN-1 and the LC3B-II/-I ratio, and downregulation of p62. Further studies found that this process was mediated by inhibition of autophagy and activation of the AMPK signaling pathway.

Intermittent Hypoxia Induces Pulmonary Vascular Remodeling and Increases the Expression of STIM‐activated TRPC‐ORAI Channels in the Lung

Obstructive sleep apnea (OSA), a breathing disorder characterized by episodes of chronic intermittent hypoxia (CIH) and re‐oxygenation during sleep, is an independent risk for systemic hypertension. Furtheremore, 20–60% of OSA patients develop moderate pulmonary hypertension. Rats exposed to CIH developed pulmonary vascular remodeling, but the mechanisms underlaying the vascular remodeling are not well known. Stim‐activated TRP‐CORAI channel (STOC), cationic calcium‐permeable channels, are overexpressed in the lung and plays a key role in the development of pulmonary vascular remodeling in animals exposed to sustained hypoxia. However, whether these channels are involved in the pulmonary vascular remodeling induced by CIH have not been addressed. We studied the time‐course of the vascular pulmonary remodeling and the changes in STOC expression, in a rodent model of OSA. Male Sprague‐Dawley rats (~200g) were exposed for 14 to 28 days to CIH (5% O2, 12 times/h, for 8h). At 14, 21 and 28 days of CIH animals were euthanized and lungs dissected to determine the vascular remodeling index by histology analysis, and the pulmonary mRNA and protein levels of the STOC forming subunits TRPC1, TRPC4, TRPC6, ORAI 1, ORAI 2 and STIM1 by qPCR and western blot, respectively. Results were compared with age‐matched controls. CIH increased the pulmonary expression of TRPC 6, ORAI 1, ORAI 2 and STIM1 at 21 and 28 days compared with controls. In addition, CIH was associated to an increased in pulmonary arteries medial thickness at 21 and 28 days (66.6% ± 2.4, 66.3% ± 4.2 vs 45.4 ± 1.6, 21 and 28 days of CIH vs control respectively, p < 0.05). Present results shown that CIH increased the pulmonary STOC expression, and these changes paralleled the vascular remodeling.Support or Funding InformationFondecyt grants 1150040 and 1180341This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Berberine attenuates pulmonary arterial hypertension via protein phosphatase 2A signaling pathway both in vivo and in vitro.

Excessive proliferation, migration, and antiapoptosis of pulmonary artery (PA) smooth muscle cells (PASMCs) underlies the development of pulmonary vascular remodeling. The innervation of the PA is predominantly sympathetic, and increased levels of circulating catecholamines have been detected in pulmonary arterial hypertension (PAH), suggesting that neurotransmitters released by sympathetic overactivation may play an essential role in PAH. However, the responsible mechanism remains unclear. Here, to investigate the effects of norepinephrine (NE) on PASMCs and the related mechanism, we used 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, the proliferating cell nuclear antigen and the cell counting kit-8 assay to evaluate the proliferation of PASMCs, Boyden chamber migration, and wound-healing assays to assess migration and western blot analysis to investigate protein expression. We demonstrated that the phosphorylation level of the protein phosphatase 2A (PP2A) catalytic subunit (Y307) was higher in PAH patients and PAH models than in controls, both in vivo and in vitro. In addition, NE induced the proliferation and migration of PASMCs, which was attenuated by berberine (BBR), a Chinese herbal medicine, and/or PP2A overexpression. PP2A inhibition worsened NE-induced PAH and could not be reversed by BBR. Thus, PP2A is critical in driving PAH, and BBR may alleviate PAH via PP2A signaling pathways, thereby offering a potential therapeutic option for PAH.