Pulmonary Vascular Smooth Muscle Cells Research Articles

Plasma Proteomics Identifies B2M as a Regulator of Pulmonary Hypertension in Heart Failure With Preserved Ejection Fraction.

Pulmonary hypertension (PH) represents an important phenotype in heart failure with preserved ejection fraction (HFpEF). However, management of PH-HFpEF is challenging because mechanisms involved in the regulation of PH-HFpEF remain unclear. We used a mass spectrometry-based comparative plasma proteomics approach as a sensitive and comprehensive hypothesis-generating discovery technique to profile proteins in patients with PH-HFpEF and control subjects. We then validated and investigated the role of one of the identified proteins using in vitro cell cultures, in vivo animal models, and independent cohort of human samples. Plasma proteomics identified high protein abundance levels of B2M (β2-microglobulin) in patients with PH-HFpEF. Interestingly, both circulating and skeletal muscle levels of B2M were increased in mice with skeletal muscle SIRT3 (sirtuin-3) deficiency or high-fat diet-induced PH-HFpEF. Plasma and muscle biopsies from a validation cohort of PH-HFpEF patients were found to have increased B2M levels, which positively correlated with disease severity, especially pulmonary capillary wedge pressure and right atrial pressure at rest. Not only did the administration of exogenous B2M promote migration/proliferation in pulmonary arterial vascular endothelial cells but it also increased PCNA (proliferating cell nuclear antigen) expression and cell proliferation in pulmonary arterial vascular smooth muscle cells. Finally, B2m deletion improved glucose intolerance, reduced pulmonary vascular remodeling, lowered PH, and attenuated RV hypertrophy in mice with high-fat diet-induced PH-HFpEF. Patients with PH-HFpEF display higher circulating and skeletal muscle expression levels of B2M, the magnitude of which correlates with disease severity. Our findings also reveal a previously unknown pathogenic role of B2M in the regulation of pulmonary vascular proliferative remodeling and PH-HFpEF. These data suggest that circulating and skeletal muscle B2M can be promising targets for the management of PH-HFpEF.

GRK2-YAP signaling is implicated in pulmonary arterial hypertension development.

Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation of small pulmonary arterial vascular smooth muscle cells (PASMCs), endothelial dysfunction, and extracellular matrix remodeling. G protein-coupled receptor kinase 2 (GRK2) plays an important role in the maintenance of vascular tone and blood flow. However, the role of GRK2 in the pathogenesis of PAH is unknown. GRK2 levels were detected in lung tissues from healthy people and PAH patients. C57BL/6 mice, vascular smooth muscle cell-specific Grk2 -knockout mice ( Grk2ΔSM22 ), and littermate controls ( Grk2flox/flox ) were grouped into control and hypoxia mice ( n =8). Pulmonary hypertension (PH) was induced by exposure to chronic hypoxia (10%) combined with injection of the SU5416 (cHx/SU). The expression levels of GRK2 and Yes-associated protein (YAP) in pulmonary arteries and PASMCs were detected by Western blotting and immunofluorescence staining. The mRNA expression levels of Grk2 and Yes-associated protein ( YAP ) in PASMCs were quantified with real-time polymerase chain reaction (RT-PCR). Wound-healing assay, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, and 5-Ethynyl-2'-deoxyuridine (EdU) staining were performed to evaluate the proliferation and migration of PASMCs. Meanwhile, the interaction among proteins was detected by immunoprecipitation assays. The expression levels of GRK2 were upregulated in the pulmonary arteries of patients with PAH and the lungs of PH mice. Moreover, cHx/SU-induced PH was attenuated in Grk2ΔSM22 mice compared with littermate controls. The amelioration of PH in Grk2ΔSM22 mice was accompanied by reduced pulmonary vascular remodeling. In vitro study further confirmed that GRK2 knock-down significantly altered hypoxia-induced PASMCs proliferation and migration, whereas this effect was severely intensified by overexpression of GRK2 . We also identified that GRK2 promoted YAP expression and nuclear translocation in PASMCs, resulting in excessive PASMCs proliferation and migration. Furthermore, GRK2 is stabilized by inhibiting phosphorylating GRK2 on Tyr86 and subsequently activating ubiquitylation under hypoxic conditions. Our findings suggest that GRK2 plays a critical role in the pathogenesis of PAH, via regulating YAP expression and nuclear translocation. Therefore, GRK2 serves as a novel therapeutic target for PAH treatment.

Hypoxia Inducible Factor-1α in SM22α Expressing Cells Modulates Alveolarization.

Worldwide, the incidence of both preterm births and chronic lung disease of infancy, or bronchopulmonary dysplasia (BPD), remains high. Infants with BPD have larger and fewer alveoli, pathology that can persist into adulthood. Though hypoxia inducible factor-1α (HIF-1α) plays a significant role in mediating pulmonary angiogenesis and alveolarization, the cell specific role of HIF-1α remains incompletely understood. To determine whether HIF-1α, in a mesenchymal cells (MC) subset, mediates postnatal alveolarization. We generated mice with cell-specific deletion of HIF-1α by crossing SM22α-promoter-driven Cre mice with HIF-1αflox/flox mice (SM22α- HIF-1α-/-), determined SM22α-expressing cell identity using single cell RNA-seq, and interrogated clinical samples from preterm infants. Measurement and Main Outcomes: Deletion of HIF-1α in SM22α expressing cells had no effect on lung structure at day (d) 3 of life. However, at 8d there were fewer and larger alveoli, a difference that persisted into adulthood. Microvascular density, elastin organization, and peripheral branching of the lung vasculature were decreased in SM22α-HIF-1α-/-, compared to control, mice. Single cell RNA-seq demonstrated that 3 MC subtypes (myofibroblasts, airway and vascular smooth muscle cells (VSMC)) express SM22α. Pulmonary VSMC from SM22α- HIF-1α-/- had decreased angiopoetin 2 expression and, in co-culture experiments, a diminished capacity to promote angiogenesis that was rescued by angiopoetin-2. Angiopoetin-2 expression in the tracheal aspirates of preterm infants was inversely correlated with overall time on mechanical ventilation, a marker of disease severity. SM22α-specific HIF-1α expression drives peripheral angiogenesis and alveolarization in the lung, perhaps by promoting angiopoetin-2 expression.

Calcineurin Is a Universal Regulator of Vessel Function-Focus on Vascular Smooth Muscle Cells.

Calcineurin, a serine/threonine phosphatase regulating transcription factors like NFaT and CREB, is well known for its immune modulatory effects and role in cardiac hypertrophy. Results from experiments with calcineurin knockout animals and calcineurin inhibitors indicate that calcineurin also plays a crucial role in vascular function, especially in vascular smooth muscle cells (VSMCs). In the aorta, calcineurin stimulates the proliferation and migration of VSMCs in response to vascular injury or angiotensin II administration, leading to pathological vessel wall thickening. In the heart, calcineurin mediates coronary artery formation and VSMC differentiation, which are crucial for proper heart development. In pulmonary VSMCs, calcineurin/NFaT signaling regulates the release of Ca2+, resulting in increased vascular tone followed by pulmonary arterial hypertension. In renal VSMCs, calcineurin regulates extracellular matrix secretion promoting fibrosis development. In the mesenteric and cerebral arteries, calcineurin mediates a phenotypic switch of VSMCs leading to altered cell function. Gaining deeper insights into the underlying mechanisms of calcineurin signaling will help researchers to understand developmental and pathogenetical aspects of the vasculature. In this review, we provide an overview of the physiological function and pathophysiology of calcineurin in the vascular system with a focus on vascular smooth muscle cells in different organs. Overall, there are indications that under certain pathological settings reduced calcineurin activity seems to be beneficial for cardiovascular health.

Peroxisome proliferator-activated receptor gamma (PPARγ) activation: a potential treatment for ascites syndrome in broiler chickens

Ascites (serous fluid accumulation in the abdominal cavity) has been observed worldwide in fast growing broilers. Pulmonary vascular remodeling is an important pathological feature of broiler ascites syndrome. Peroxisome proliferators-activated receptor gamma (PPARγ) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) are expressed in pulmonary vascular endothelial cells and vascular smooth muscle cells (VSMC) where they participate in the regulation of normal pulmonary vascular function. The objective of the present study was to investigate the effects of omega-3 fatty acids (found in fish oil) and pioglitazone (PIO) as natural and synthetic PPARγ ligands supplementation on PPARγ and PGC-1α expression in the prevention of pulmonary arterial hypertension (PAH) syndrome in broiler chickens. The experiment was conducted with 4 treatment groups: 1) negative control, normal temperature conditions with basal diet; 2) positive control, low-temperature conditions with basal diet; 3) positive control+10 mg PIO/kg of weight/d and 4) positive control+1% FO. Each treatment had 5 replicates. Ascites heart index (RV/TV) was significantly (P < 0.05) reduced in chickens receiving FO (0.20) and PIO (0.21) compared to the positive control group (0.26). The addition of PIO in broilers under cold-induced ascites significantly increased the expression of PPARγ (9.44) and PGC-1α (5.81) genes in lung tissue compared to the negative control group (1.03, P < 0.05). Proliferative indexes of VSMC in pulmonary arteries such as PMT, PIT, and percentage wall thickness were significantly elevated in positive control group, indicating that pulmonary vascular remodeling occurred following VSMC proliferation in ascites. The vessel internal diameter was increased in FO and PIO groups. Based on these results, activation and expression of PPARγ and PGC-1α genes as a critical regulator of pulmonary artery smooth muscle cell using ligands, especially PIO, can be effective in reducing the incidence of PAH in broiler chickens.

Exosomal miR-663b from "M1" macrophages promotes pulmonary artery vascular smooth muscle cell dysfunction through inhibiting the AMPK/Sirt1 axis.

Inflammatory mediators from macrophages are proven to be involved in pulmonary vascular remodeling in pulmonary hypertension (PH). Here, this study intends to explore the mechanism of "M1" macrophage-derived exosomal miR-663b in pulmonary artery smooth muscle cells (PASMCs) dysfunctions and pulmonary hypertension. Hypoxia-treated PASMCs were utilized for constructing an in-vitro pulmonary hypertension model. THP-1 cells were treated with PMA (320 nM) and LPS (10 μg/mL) + IFN-γ (20 ng/ml) for eliciting macrophage "M1" polarization. Exosomes derived from "M1" macrophages were isolated and added into PASMCs. The proliferation, inflammation, oxidative stress, and migration of PASMCs were evaluated. RT-PCR or Western blot examined the levels of miR-663b and the AMPK/Sirt1 pathway. Dual luciferase activity assay and RNA pull-down assay were carried out for confirming the targeted association between miR-663b and AMPK. An in-vivo PH model was built. Macrophage-derived exosomes with miR-663b inhibition were used for treating the rats, and alterations of pulmonary histopathology were monitored. miR-663b was obviously up-regulated in hypoxia-elicited PASMCs and M1 macrophages. miR-663b overexpression boosted hypoxia-induced proliferation, inflammation, oxidative stress, and migration in PASMCs, whereas miR-663b low expression resulted in the opposite situation. AMPK was identified as a target of miR-663b, and miR-663b overexpression curbed the AMPK/Sirt1 pathway. AMPK activation ameliorated the damaging impact of miR-663b overexpression and "M1" macrophage exosomes on PASMCs. In vivo, "M1" macrophage exosomes with miR-663b low expression alleviated pulmonary vascular remodeling in pulmonary hypertension rats. Exosomal miR-663b from "M1" macrophage facilitates PASMC dysfunctions and PH development by dampening the AMPK/Sirt1 axis.

Crosstalk between BMP signaling and KCNK3 in phenotypic switching of pulmonary vascular smooth muscle cells

Pulmonary arterial hypertension (PAH) is a progressive and devastating disease whose pathogenesis is associated with a phenotypic switch of pulmonary arterial vascular smooth muscle cells (PASMCs). Bone morphogenetic protein (BMP) signaling and potassium two pore domain channel subfamily K member 3 (KCNK3) play crucial roles in PAH pathogenesis. However, the relationship between BMP signaling and KCNK3 expression in the PASMC phenotypic switching process has not been studied. In this study, we explored the effect of BMPs on KCNK3 expression and the role of KCNK3 in the BMP-mediated PASMC phenotypic switch. Expression levels of BMP receptor 2 (BMPR2) and KCNK3 were downregulated in PASMCs of rats with PAH compared to those in normal controls, implying a possible association between BMP/BMPR2 signaling and KCNK3 expression in the pulmonary vasculature. Treatment with BMP2, BMP4, and BMP7 significantly increased KCNK3 expression in primary human PASMCs (HPASMCs). BMPR2 knockdown and treatment with Smad1/5 signaling inhibitor substantially abrogated the BMP-induced increase in KCNK3 expression, suggesting that KCNK3 expression in HPASMCs is regulated by the canonical BMP-BMPR2-Smad1/5 signaling pathway. Furthermore, KCNK3 knockdown and treatment with a KCNK3 channel blocker completely blocked BMP-mediated anti-proliferation and expression of contractile marker genes in HPAMSCs, suggesting that the expression and functional activity of KCNK3 are required for BMP-mediated acquisition of the quiescent PASMC phenotype. Overall, our findings show a crosstalk between BMP signaling and KCNK3 in regulating the PASMC phenotype, wherein BMPs upregulate KCNK3 expression and KCNK3 then mediates BMP-induced phenotypic switching of PASMCs. Our results indicate that the dysfunction and/or downregulation of BMPR2 and KCNK3 observed in PAH work together to induce aberrant changes in the PASMC phenotype, providing insights into the complex molecular pathogenesis of PAH. [BMB Reports 2022; 55(11): 565-570].

Pathophysiology and pathogenic mechanisms of pulmonary hypertension: role of membrane receptors, ion channels, and Ca2+ signaling.

The pulmonary circulation is a low-resistance, low-pressure, and high-compliance system that allows the lungs to receive the entire cardiac output. Pulmonary arterial pressure is a function of cardiac output and pulmonary vascular resistance, and pulmonary vascular resistance is inversely proportional to the fourth power of the intraluminal radius of the pulmonary artery. Therefore, a very small decrease of the pulmonary vascular lumen diameter results in a significant increase in pulmonary vascular resistance and pulmonary arterial pressure. Pulmonary arterial hypertension is a fatal and progressive disease with poor prognosis. Regardless of the initial pathogenic triggers, sustained pulmonary vasoconstriction, concentric vascular remodeling, occlusive intimal lesions, in situ thrombosis, and vascular wall stiffening are the major and direct causes for elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension and other forms of precapillary pulmonary hypertension. In this review, we aim to discuss the basic principles and physiological mechanisms involved in the regulation of lung vascular hemodynamics and pulmonary vascular function, the changes in the pulmonary vasculature that contribute to the increased vascular resistance and arterial pressure, and the pathogenic mechanisms involved in the development and progression of pulmonary hypertension. We focus on reviewing the pathogenic roles of membrane receptors, ion channels, and intracellular Ca2+ signaling in pulmonary vascular smooth muscle cells in the development and progression of pulmonary hypertension.

Congenital diaphragmatic hernia: phosphodiesterase-5 and Arginase inhibitors prevent pulmonary vascular hypoplasia in rat lungs.

Severe pulmonary hypoplasia related to congenital diaphragmatic hernia (CDH) continues to be a potentially fatal condition despite advanced postnatal management strategies. To evaluate the effect of the antenatal sildenafil and 2(S)-amino-6-boronohexanoic acid (ABH-Arginase inhibitor) on lung volume, pulmonary vascular development, and nitric oxide (NO) synthesis in a Nitrofen-induced CDH rat model. Nitrofen-induced CDH rat model was used. Nitrofen was administrated on embryonic day(E) 9,5. At E14, five intervention groups were treated separately: Nitrofen, Nitrofen+Sildenafil, Nitrofen+ABH, Nitrofen+Sildenafil+ABH and Control. At term, offspring's lungs were weighed, some paraffin-embedded for histology, others snap-frozen to analyze eNOS, Arginase I-II expression, and activity. In CDH-bearing offsprings, ABH or Sildenafil+ABH preserved the total lung/body-weight index (p < 0.001), preventing pulmonary vascular smooth muscle cell hyperproliferation and improving lung morphometry. Sildenafil+ABH increased 1.7-fold the lung nitrite levels (p < 0.01) without changes in eNOS expression. Sildenafil and ABH improved the number of pulmonary vessels. These results suggest that in this CDH rat model, the basal activity of Arginase participates in the lung volume and, together with phosphodiesterase-5, regulates NOS activity in the term fetal lung. The combined treatment (Sildenafil+ABH) could revert some of the pulmonary features in CDH by improving the local NO synthesis and preventing smooth muscle cell hyperproliferation. This study presents Arginase inhibition as a new therapeutic target and the importance of the combined antenatal treatment to improve pulmonary vascular development in a congenital diaphragmatic hernia (CDH) rat model. This study shows that the action of an Arginase inhibitor (ABH) enhances the effects already described for sildenafil in this model. These results reinforce the importance of prenatal treatments' synergy in recovering the hypoplastic lung in the Nitrofen-induced CDH rat model.

Cysteine and glycine rich protein 2 exacerbates vascular fibrosis in pulmonary hypertension through the nuclear translocation of yes-associated protein and transcriptional coactivator with PDZ-binding motif

Pulmonary hypertension (PH) is a serious cardiovascular disease with a poor prognosis and high mortality. The pathogenesis of PH is complex, and the main pathological changes in PH are abnormal hypertrophy and vessel stiffness. Cysteine and glycine rich protein 2 (Csrp2), a member of the LIM-only family plays a key role in the response to vascular injury. However, its roles in vascular fibrosis and PH have not been clarified. Therefore, this study aimed to investigate whether Csrp2 can promote vascular fibrosis and to further explore the possible mechanisms. Csrp2 expression was increased in both the pulmonary vasculature of rats with PH and hypoxic pulmonary vascular smooth muscle cells (PASMCs). Hypoxia activated TGF-β1 and its downstream effector, SP1. Additionally, hypoxia activated the ROCK pathway and inhibited KLF4 expression. Silencing SP1 and overexpressing KLF4 reversed the hypoxia-induced increase in Csrp2 expression. Csrp2 knockdown decreased the expression of extracellular matrix (ECM) proteins and inhibited the nuclear translocation and expression of YAP/TAZ in hypoxic PASMCs. These results indicate that hypoxia induces Csrp2 expression through the TGF-β1/SP1 and ROCK/KLF4 pathways. Elevated Csrp2 promoted the nuclear translocation and expression of YAP/TAZ, leading to vascular fibrosis and the development of PH.

Akt-Dependent Glycolysis-Driven Lipogenesis Supports Proliferation and Survival of Human Pulmonary Arterial Smooth Muscle Cells in Pulmonary Hypertension.

Hyper-proliferation of pulmonary arterial vascular smooth muscle cells (PAVSMC) is an important pathological component of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Lipogenesis is linked to numerous proliferative diseases, but its role in PAVSMC proliferation in PAH remains to be elucidated. We found that early-passage human PAH PAVSMC had significant up-regulation of key fatty acids synthesis enzymes ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN), and increased unstimulated proliferation compared to control human PAVSMC. Treatment with an allosteric ACC inhibitor 5-tetradecyloxy-2-furoic acid (TOFA) significantly decreased proliferation and induced apoptosis of human PAH PAVSMC. Intracellular lipid content and proliferation of PAH PAVSMC were not reduced by incubation in lipid-depleted media but suppressed by a non-metabolizable analog of glucose 2-Deoxy-D-glucose (2-DG) and partially restored by addition of pyruvate. Protein kinase Akt was upregulated in human PAH PAVSMC in a sirtuin 7 (SIRT7)- and c-Jun N-terminal kinase (JNK)-dependent manner. Pharmacological inhibition of Akt down-regulated ACLY and ACC, significantly reduced intracellular lipid content, inhibited proliferation and induced apoptosis of human PAH PAVSMC. Taken together, these data demonstrate that human PAH PAVSMC have up-regulated lipogenesis, which is supported in an Akt- and glycolysis-dependent manner and is required for increased proliferation and survival. Our data suggest that there is a mechanistic link between glycolysis, lipogenesis, and the proliferation of human PAH PAVSMC and call for further studies to determine the potential attractiveness of a SIRT7/JNK-Akt-lipogenesis axis as a target pathway to inhibit PAVSMC hyper-proliferation in PAH.

Cysteine and glycine-rich protein 2 promotes hypoxic pulmonary vascular smooth muscle cell proliferation through the Wnt3α-β-catenin/lymphoid enhancer-binding factor 1pathway.

Pulmonary hypertension (PH) is mainly characterized by abnormal pulmonary vascular hyperplasia and vascular remodeling, but its mechanism is complicated and currently unclear. Cysteine and glycine-rich protein 2 (Csrp2) has been reported to promote cell proliferation and migration, and affect cell cycle progression. As a new invasive actin-binding factor, Csrp2 increased the invasion and even metastasis of some cancer cells. It was associated with tumor recurrence and chemotherapy resistance. However, the role of Csrp2 in PH remains unknown. We found that Csrp2 expression was increased both in pulmonary arteries (PAs) and smooth muscle cells (PASMCs) in PH. Csrp2 enhanced PASMC proliferation and phenotypic transition. The Wnt3α-β-catenin/lymphoid enhancer-binding factor 1 (LEF1) pathway is involved in cell proliferation and phenotypic transition regulated by Csrp2 expression. These results suggest that hypoxia downregulates YinYang-1 (YY1) and then increases Csrp2 expression. Increased Csrp2 promotes PASMC proliferation and phenotypic transition by activating the Wnt3α-β-catenin/LEF1 pathways, which leads to pulmonary vascular remodeling and even provides a new theoretical basis for studying the pathogenesis and therapeutic targets of PH.

Oxidative Stress and Antioxidative Therapy in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is clinically characterized by a progressive increase in pulmonary artery pressure, followed by right ventricular hypertrophy and subsequently right heart failure. The underlying mechanism of PAH includes endothelial dysfunction and intimal smooth muscle proliferation. Numerous studies have shown that oxidative stress is critical in the pathophysiology of PAH and involves changes in reactive oxygen species (ROS), reactive nitrogen (RNS), and nitric oxide (NO) signaling pathways. Disrupted ROS and NO signaling pathways cause the proliferation of pulmonary arterial endothelial cells (PAECs) and pulmonary vascular smooth muscle cells (PASMCs), resulting in DNA damage, metabolic abnormalities, and vascular remodeling. Antioxidant treatment has become a main area of research for the treatment of PAH. This review mainly introduces oxidative stress in the pathogenesis of PAH and antioxidative therapies and explains why targeting oxidative stress is a valid strategy for PAH treatment.

Recovery of hyperoxia‐induced DDAH1 suppression with FXR agonist GW4064 in human pulmonary microvascular endothelial cells

ObjectivePulmonary hypertension (PH) predicts mortality in patients with bronchopulmonary dysplasia (BPD), and chronic hyperoxia exposure contributes to BPD‐PH disease progression. BPD‐PH is characterized by aberrant pulmonary vascular wall proliferation/remodeling, which occludes the microvascular lumen, increasing pulmonary vascular resistance. Endogenous nitric oxide (NO) production by pulmonary vascular endothelial cells promotes apoptosis of pulmonary vascular endothelial and smooth muscle cells. In vitro studies of human pulmonary microvascular endothelial cells (hPMVEC) have shown that dimethylarginine dimethylaminohydrolase‐1 (DDAH1) promotes NO‐mediated apoptosis. DDAH1 metabolizes the majority of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial NO synthase. Therefore, our hypothesis is that pharmacologically enhancing DDAH1 expression in hPMVEC will restore endogenous endothelial NO production.MethodshPMVEC were grown at 37 ̊C to ~70% confluence. Farnesoid X receptor (FXR) agonist, GW4064 (1.25 µM) was used to bind FXR element (FXRE) of the DDAH1 promoter to enhance transcription. hPMVEC were pre‐treated with FXR agonist for 24 hours and then exposed to 85%O2 or 21%O2 for an additional 24 hours. DDAH1 protein expression was analyzed by Western blot. Groups were compared by 1‐way ANOVA.ResultsIn hPMVEC, hyperoxia exposure resulted in lower DDAH1 protein levels compared to hPMVEC grown in normoxia (N=6 in each group, p=0.003). Treatment with GW4064 prior to exposing to hyperoxia resulted in 4.3‐fold greater DDAH1 protein levels than in vehicle/85%O2 hPMVEC (N=6, p=0.044), and restored DDAH1 protein levels to 72% of that seen in the vehicle/21%O2exposed hPMVEC.ConclusionsChronic hyperoxia suppresses DDAH1 levels, and FXR agonist promotes DDAH1 recovery in hPMVEC. We speculate that promoting endogenous DDAH1 expression may prevent aberrant proliferation in the pulmonary vascular wall and thereby attenuate or prevent the vascular remodeling characteristic of pulmonary hypertension in BPD.

Noncanonical HIPPO/MST Signaling via BUB3 and FOXO Drives Pulmonary Vascular Cell Growth and Survival.

The MSTs (mammalian Ste20-like kinases) 1/2 are members of the HIPPO pathway that act as growth suppressors in adult proliferative diseases. Pulmonary arterial hypertension (PAH) manifests by increased proliferation and survival of pulmonary vascular cells in small PAs, pulmonary vascular remodeling, and the rise of pulmonary arterial pressure. The role of MST1/2 in PAH is currently unknown. To investigate the roles and mechanisms of the action of MST1 and MST2 in PAH. Using early-passage pulmonary vascular cells from PAH and nondiseased lungs and mice with smooth muscle-specific tamoxifen-inducible Mst1/2 knockdown, we found that, in contrast to canonical antiproliferative/proapoptotic roles, MST1/2 act as proproliferative/prosurvival molecules in human PAH pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts and support established pulmonary vascular remodeling and pulmonary hypertension in mice with SU5416/hypoxia-induced pulmonary hypertension. By using unbiased proteomic analysis, gain- and loss-of function approaches, and pharmacological inhibition of MST1/2 kinase activity by XMU-MP-1, we next evaluated mechanisms of regulation and function of MST1/2 in PAH pulmonary vascular cells. We found that, in PAH pulmonary arterial adventitial fibroblasts, the proproliferative function of MST1/2 is caused by IL-6-dependent MST1/2 overexpression, which induces PSMC6-dependent downregulation of forkhead homeobox type O 3 and hyperproliferation. In PAH pulmonary arterial vascular smooth muscle cells, MST1/2 acted via forming a disease-specific interaction with BUB3 and supported ECM (extracellular matrix)- and USP10-dependent BUB3 accumulation, upregulation of Akt-mTORC1, cell proliferation, and survival. Supporting our in vitro observations, smooth muscle-specific Mst1/2 knockdown halted upregulation of Akt-mTORC1 in small muscular PAs of mice with SU5416/hypoxia-induced pulmonary hypertension. Together, this study describes a novel proproliferative/prosurvival role of MST1/2 in PAH pulmonary vasculature, provides a novel mechanistic link from MST1/2 via BUB3 and forkhead homeobox type O to the abnormal proliferation and survival of pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts, remodeling and pulmonary hypertension, and suggests new target pathways for therapeutic intervention.

Expression of the microRNA‑30 family in pulmonary arterial hypertension and the role of microRNA‑30d‑5p in the regulation of pulmonary arterial smooth muscle cell toxicity and apoptosis

The biological processes of pulmonary artery vascular smooth muscle cells (PA-SMCs) and pulmonary artery endothelial cells in pulmonary arterial hypertension (PAH) are generally abnormal, with increased levels of proliferation and reduced levels of apoptosis. Although microRNAs (miRNAs/miRs) participate in a number of biological processes in a variety of diseases, such as tumors and infections, studies on the association between miRNAs and PAH are limited. In the present study, blood samples were collected from 6 patients with patent ductus arteriosus. The experimental group included 3 patients with severe PAH, while the control group included 3 patients without PAH. Microarray technology was used to detect the presence of any associated miRNAs. Moreover, a rat PAH model was established via left lung resection followed by monocrotaline injection, involving a total of 8 rats in the PAH group and 8 untreated rat in the control group. Reverse transcription-quantitative PCR was performed to verify the expression levels of the miR-30 family in the animal model. miR-30d-5p mimics and anti-miR-30d-5p were transfected into primary cultured PA-SMCs. Levels of cytotoxicity and cell apoptosis were examined, and Notch-3 expression levels were studied using western blotting. The results of the present study demonstrated that miR-30d-5p expression was downregulated in both patient blood and animal models of the PAH group compared with control groups. In primary cultured PA-SMCs, overexpression of miR-30d-5p attenuated the platelet-derived growth factor-induced toxicity of PA-SMCs, while knockdown of miR-30d-5p resulted in the increased toxicity of PA-SMCs compared with control group. The apoptosis rate of PA-SMCs increased with the overexpression of miR-30d-5p compared with control group. Moreover, the expression levels of Notch-3 in the miR-30d-5p group were significantly reduced compared with the anti-miR-30d-5p and miR-NC groups. In total, 10 circulating miRNAs that may be associated with PAH were discovered in the present study. Moreover, the expression of the miR-30 family was verified in animal models in vivo, and seven miRNAs in this family were discovered that may be associated with PAH. Additionally, miR-30d-5p was downregulated in both patients with PAH and animal models compared with control groups. Thus, the results of the present study demonstrated that the regulatory mechanism underlying PA-SMCs may be via the Notch-3 signaling pathway.

Abstract 12968: mitoTEMPO Improves Expression of Pulmonary Artery Soluble Guanylate Cyclase β1 and Lowers Exercise Associated Elevations in Pulmonary Artery Pressures in a Rat Model of Combined Pre- and Post-Capillary Pulmonary Hypertension

Introduction: Exercise induced pulmonary hypertension (EIPH) portends worse prognosis compared to isolated resting PH. Exercise increases mitochondrial reactive oxygen species (mROS) production in peripheral blood mononuclear cells of patients with heart failure. Metabolic syndrome related mROS contribute to EIPH by decreasing the expression of the vasodilatory enzyme soluble guanylate cyclase (sGC) via downregulating an sGC transcription factor, namely nuclear factor Y alpha (NFYA; Satoh et al in press). Hypothesis: mROS scavenging with mitoTEMPO will restore sGC expression and ameliorate EIPH in a rat model of combined pre-and post-capillary pulmonary hypertension (CpcPH). Methods: Obese ZSF1 rats treated with sugen were used to model CpcPH. Half received mitoTEMPO (2mM) for 2 weeks. CpcPH rats treated with vehicle and lean ZSF1 rats served as controls (n=6/group). Hemodynamics were evaluated at rest and during treadmill exercise. Cardiac function was assessed using Doppler-echocardiography. We cultured pulmonary artery vascular smooth muscle cells (PAVSMCs) from lean rats in palmitate, glucose and insulin (PGI) containing media to simulate the metabolic syndrome milieu. Cells were treated with vehicle or mitoTEMPO. Expression of sGCβ1 and NFYA in pulmonary arteries of animals and in cultured cells was quantified by Western Blot. Data were analyzed with repeated measures two-way ANOVA. Results: MitoTEMPO significantly improved EIPH by reducing pulmonary vascular resistance (fig1A), significantly improved right ventricular function (fig1B) and significantly restored NFYA/sGCβ1 expression in pulmonary arteries (fig1C) and PAVSMCs of rats with CpcPH (fig1D). MitoTEMPO significantly blunted PGI-induced downregulation of NFYA and sGCβ1 in PAVSMCs from lean rats (fig1E). Conclusions: Mitochondria targeted antioxidants can improve EIPH in CpcPH by restoring NFYA/sGCβ1 expression, identifying a potential therapeutic target for CpcPH.

Role of Long Non-Coding RNAs in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a debilitating condition of the pulmonary circulatory system that occurs in patients of all ages and if untreated, eventually leads to right heart failure and death. Despite existing medical treatment options that improve survival and quality of life, the disease remains incurable. Thus, there is an urgent need to develop novel therapies to treat this disease. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play critical roles in pulmonary vascular remodeling and PAH. LncRNAs are implicated in pulmonary arterial endothelial dysfunction by modulating endothelial cell proliferation, angiogenesis, endothelial mesenchymal transition, and metabolism. LncRNAs are also involved in inducing different pulmonary arterial vascular smooth muscle cell phenotypes, such as cell proliferation, apoptosis, migration, regulation of the phenotypic switching, and cell cycle. LncRNAs are essential regulators of gene expression that affect various diseases at the chromatin, transcriptional, post-translational, and even post-translational levels. Here, we focus on the role of LncRNAs and their molecular mechanisms in the pathogenesis of PAH. We also discuss the current research challenge and potential biomarker and therapeutic potentials of lncRNAs in PAH.

DUSP5-mediated inhibition of smooth muscle cell proliferation suppresses pulmonary hypertension and right ventricular hypertrophy.

Pulmonary hypertension (PH) is associated with structural remodeling of pulmonary arteries (PAs) because of excessive proliferation of fibroblasts, endothelial cells, and smooth muscle cells (SMCs). The peptide hormone angiotensin II (ANG II) contributes to pulmonary vascular remodeling, in part, through its ability to trigger extracellular signal-regulated kinase (ERK1/2) activation. Here, we demonstrate that the ERK1/2 phosphatase, dual-specificity phosphatase 5 (DUSP5), functions as a negative regulator of ANG II-mediated SMC proliferation and PH. In contrast to wild-type controls, Dusp5 null mice infused with ANG II developed PH and right ventricular (RV) hypertrophy. PH in Dusp5 null mice was associated with thickening of the medial layer of small PAs, suggesting an in vivo role for DUSP5 as a negative regulator of ANG II-dependent SMC proliferation. Consistent with this, overexpression of DUSP5 blocked ANG II-mediated proliferation of cultured human pulmonary artery SMCs (hPASMCs) derived from patients with idiopathic PH or from failed donor controls. Collectively, the data support a role for DUSP5 as a feedback inhibitor of ANG II-mediated ERK signaling and PASMC proliferation and suggest that disruption of this circuit leads to adverse cardiopulmonary remodeling.NEW & NOTEWORTHY Dual-specificity phosphatases (DUSPs) serve critical roles in the regulation of mitogen-activated protein kinases, but their functions in the cardiovascular system remain poorly defined. Here, we provide evidence that DUSP5, which resides in the nucleus and specifically dephosphorylates extracellular signal-regulated kinase (ERK1/2), blocks pulmonary vascular smooth muscle cell proliferation. In response to angiotensin II infusion, mice lacking DUSP5 develop pulmonary hypertension and right ventricular cardiac hypertrophy. These findings illustrate DUSP5-mediated suppression of ERK signaling in the lungs as a protective mechanism.

Vascular cell-specific roles of mineralocorticoid receptors in pulmonary hypertension.

Abnormalities that characterize pulmonary arterial hypertension include impairment in the structure and function of pulmonary vascular endothelial and smooth muscle cells. Aldosterone levels are elevated in human pulmonary arterial hypertension and in experimental pulmonary hypertension, while inhibition of the aldosterone-binding mineralocorticoid receptor attenuates pulmonary hypertension in multiple animal models. We explored the role of mineralocorticoid receptor in endothelial and smooth muscle cells in using cell-specific mineralocorticoid receptor knockout mice exposed to sugen/hypoxia-induced pulmonary hypertension. Treatment with the mineralocorticoid receptor inhibitor spironolactone significantly reduced right ventricular systolic pressure. However, this is not reproduced by selective mineralocorticoid receptor deletion in smooth muscle cells or endothelial cells. Similarly, spironolactone attenuated the increase in right ventricular cardiomyocyte area independent of vascular mineralocorticoid receptor with no effect on right ventricular weight or interstitial fibrosis. Right ventricular perivascular fibrosis was significantly decreased by spironolactone and this was reproduced by specific deletion of mineralocorticoid receptor from endothelial cells. Endothelial cell-mineralocorticoid receptor deletion attenuated the sugen/hypoxia-induced increase in the leukocyte-adhesion molecule, E-selectin, and collagen IIIA1 in the right ventricle. Spironolactone also significantly reduced pulmonary arteriolar muscularization, independent of endothelial cell-mineralocorticoid receptor or smooth muscle cell-mineralocorticoid receptor. Finally, the degree of pulmonary perivascular inflammation was attenuated by mineralocorticoid receptor antagonism and was fully reproduced by smooth muscle cell-specific mineralocorticoid receptor deletion. These studies demonstrate that in the sugen/hypoxia pulmonary hypertension model, systemic-mineralocorticoid receptor blockade significantly attenuates the disease and that mineralocorticoid receptor has cell-specific effects, with endothelial cell-mineralocorticoid receptor contributing to right ventricular perivascular fibrosis and smooth muscle cell-mineralocorticoid receptor participating in pulmonary vascular inflammation. As mineralocorticoid receptor antagonists are being investigated to treat pulmonary arterial hypertension, these findings support novel mechanisms and potential mineralocorticoid receptor targets that mediate therapeutic benefits in patients.