Remodeling In Pulmonary Hypertension Research Articles

Skeletal Muscle SIRT3 Deficiency Contributes to Pulmonary Vascular Remodeling in Pulmonary Hypertension Due to Heart Failure With Preserved Ejection Fraction.

Pulmonary hypertension (PH) is a major complication linked to adverse outcomes in heart failure with preserved ejection fraction (HFpEF), yet no specific therapies exist for PH associated with HFpEF (PH-HFpEF). We have recently reported on the role of skeletal muscle SIRT3 (sirtuin-3) in modulation of PH-HFpEF, suggesting a novel endocrine signaling pathway for skeletal muscle modulation of pulmonary vascular remodeling. In this study, we attempted to define the processes by which skeletal muscle SIRT3 defects affect pulmonary vascular health in PH-HFpEF. Skeletal muscle-specific Sirt3 knockout mice (Sirt3skm-/-) exhibited reduced pulmonary vascular density accompanied by pulmonary vascular proliferative remodeling and elevated pulmonary pressures. Using mass spectrometry-based comparative secretome analysis, we demonstrated elevated secretion of LOXL2 (lysyl oxidase homolog 2) in SIRT3-deficient skeletal muscle cells. Elevated circulation and protein expression levels of LOXL2 were also observed in plasma and skeletal muscle of Sirt3skm-/- mice, a rat model of PH-HFpEF, and humans with PH-HFpEF. In addition, expression levels of CNPY2 (canopy fibroblast growth factor signaling regulator 2), a known proliferative and angiogenic factor, were increased in pulmonary artery endothelial cells and pulmonary artery smooth muscle cells of Sirt3skm-/- mice and animal models of PH-HFpEF. CNPY2 levels were also higher in pulmonary artery smooth muscle cells of subjects with obesity compared with nonobese subjects. Moreover, treatment with recombinant LOXL2 protein promoted pulmonary artery endothelial cell migration/proliferation and pulmonary artery smooth muscle cell proliferation through regulation of CNPY2-p53 signaling. Last, skeletal muscle-specific Loxl2 deletion decreased pulmonary artery endothelial cell and pulmonary artery smooth muscle cell expression of CNPY2 and improved pulmonary pressures in mice with high-fat diet-induced PH-HFpEF. This study demonstrates a systemic pathogenic impact of skeletal muscle SIRT3 deficiency in remote pulmonary vascular remodeling and PH-HFpEF. This study suggests a new endocrine signaling axis that links skeletal muscle health and SIRT3 deficiency to remote CNPY2 regulation in the pulmonary vasculature through myokine LOXL2. Our data also identify skeletal muscle SIRT3, myokine LOXL2, and CNPY2 as potential targets for the treatment of PH-HFpEF.

A comprehensive map of proteoglycan expression and deposition in the pulmonary arterial wall in health and pulmonary hypertension.

Changes in the extracellular matrix of pulmonary arteries (PAs) are a key aspect of vascular remodelling in pulmonary hypertension (PH). Yet, our understanding of the alterations affecting the proteoglycan (PG) family remains limited. We sought to investigate the expression and spatial distribution of major vascular PGs in PAs from healthy individuals and various PH groups (chronic obstructive pulmonary disease: PH-COPD, pulmonary fibrosis: PH-PF, idiopathic: IPAH). PG regulation, deposition, and synthesis were notably heightened in IPAH, followed by PH-PF, with minor alterations in PH-COPD. Single-cell analysis unveiled cell-type and disease-specific PG regulation. Agrin expression, a basement membrane PG, was increased in IPAH, with PA endothelial cells (PAECs) identified as a major source. PA smooth muscle cells (PASMCs) mainly produced large-PGs, aggrecan and versican, and small-leucine-like proteoglycan (SLRP) biglycan, while the major PGs produced by adventitial fibroblasts were SLRP decorin and lumican. In IPAH and PF-PH, the neointima-forming PASMC population increased the expression of all investigated large-PGs and SLRPs, except fibroblast-predominant DCN. Expression of lumican, versican, and biglycan also positively correlated with collagen 1α1/1α2 expression in PASMCs of IPAH and PH-PF patients. We demonstrated that TGF-β regulates versican and biglycan expression, indicating their contribution to vessel fibrosis in IPAH and PF-PH. We furthermore show that certain circulating PG levels display a disease-dependent pattern, with increased decorin and lumican across all patient groups, while versican was elevated in PH-COPD and IPAH and biglycan reduced in IPAH. These findings suggest unique compartment-specific PG regulation in different forms of PH, indicating distinct pathological processes.

LOXL2 inhibition ameliorates pulmonary artery remodeling in pulmonary hypertension

Background: Conduit pulmonary arterial stiffening and the resultant increase in pulmonary vascular impedance has emerged as an important underlying driver of pulmonary arterial hypertension (PAH). Given that matrix deposition is central to vascular remodeling, we evaluated the role of the collagen crosslinking enzyme lysyl oxidase like 2 (LOXL2) in this study. Methods and Results: Human pulmonary artery smooth muscle cells (PASMCs) subjected to hypoxia showed increased LOXL2 secretion. LOXL2 activity and expression were markedly higher in primary PASMCs isolated from pulmonary arteries of the rat Sugen 5416 + hypoxia (SuHx) model of severe PH. Similarly, LOXL2 protein and mRNA levels were increased in pulmonary arteries (PA) and lungs of rats with PH (SuHx and monocrotaline (MCT) models). Pulmonary arteries (PAs) isolated from rats with PH exhibited hypercontractility to phenylephrine and attenuated vasorelaxation elicited by acetylcholine, indicating severe endothelial dysfunction. Tensile testing revealed a a significant increase in PA stiffness in PH. Treatment with PAT-1251, a novel small-molecule LOXL2 inhibitor, improved active and passive properties of the PA ex vivo. There was an improvement in right heart function as measured by right ventricular pressure volume loops in-vivo with PAT-1251. Importantly PAT-1251 treatment ameliorated PH, resulting in improved pulmonary artery pressures, right ventricular remodeling, and survival. Conclusion: Hypoxia induced LOXL2 activation is a causal mechanism in pulmonary artery stiffening in PH, as well as pulmonary artery mechanical and functional decline. LOXL2 inhibition with PAT-1251 is a promising approach to improve pulmonary artery pressures, right ventricular elastance, cardiac relaxation, and survival in PAH. This work was supported by a NHLBI grant R01HL105296 (to D.E.B.), a NHLBI grant R01HL148112 01 (to L.S.), two Stimulating and Advancing ACCM Research (StAAR) grants from the Department of Anesthesiology and Critical Care Medicine, JHU (to L.S. and J.S.), NHLBI grants R01HL073859 and R01HL126514 (to L.A.S) and a NHLBI K08 grant K08HL145132 (to J.S.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A New Cell Type in Pulmonary Veins

Pulmonary veins (PVs) carry oxygenated blood from lungs back to the heart and may play an important role in preventing blood backflow from the left atrium. Previous studies show that large PVs exhibit spontaneously pulsatile contractions in cardiomyocyte-like manner. However, the contractions of small PVs have not been investigated. We hypothesized that Ca2+ signaling mechanisms in the myocytes mediate small PV contractions. Small, 4th-order PVs from C57BL6 mice were obtained and imaged. Pressurized PVs showed ryanodine receptor-dependent Ca2+ signals and spontaneous contractions. Light sheet fluorescence microscopy and immunostaining showed the expression of troponin C, a cardiomyocyte marker, in the myocyte layer of small PVs, which was not observed in the pulmonary arteries, vena cava, or mesenteric veins. Based on these results, we termed these cells cardiomyocyte-like (CML) cells. In the study of mice with Myh11 Cre-driven tdTomato expression, Myh11 expression was observed in CML cells in small PVs, which was not seen in the smooth muscle cells (SMC) of small PAs. Endothelial protrusions crossing the connective tissue layer into the CML layer were also observed in PVs. Previous studies showed PV remodeling in pulmonary hypertension (PH), and impaired Ca2+ signaling in PAs contribute to higher pulmonary arterial pressure in PH. We further hypothesized that ryanodine receptor (RyR) Ca2+ signals in CML cells are impaired in PH. Results show that RyR Ca2+ signaling was reduced in CML cells of PH mice (injected with Su5416, subjected to chronic hypoxic conditions). Overall, our findings identify a new cell type in small PVs. Moreover, our data show that RyR Ca2+ signals in CML cells underlie spontaneous contractions in small PV, and are impaired in PH. We propose that RyR signaling mechanisms in CML cells may be possible targets for therapy in pulmonary vascular disorders. This work was supported by funding from the National Institute of Health to Swapnil Sonkusare (R01:HL146914, R01:HL157407). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

GLI1+ Cells Contribute to Vascular Remodeling in Pulmonary Hypertension.

The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs). Gli1Cre-ERT2; tdTomatoflox mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature. The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an Acta2high fraction of cells with pathways in cancer and MAPK signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation. Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies.

(10) - Physiological Mechanisms of Reverse Pulmonary Vascular Remodeling in Pulmonary Hypertension Due to Left Heart Disease

(10) - Physiological Mechanisms of Reverse Pulmonary Vascular Remodeling in Pulmonary Hypertension Due to Left Heart Disease

Unravling the Impact: Pulmonary Side Effects of Anti-seizure Medications

Background: Epilepsy is a chronic brain condition affecting over 50 million people worldwide. Several new anti-seizure medications (ASMs) have been introduced to treat epilepsy in recent decades. Objective: Nearby the specific therapeutic action, ASMs, like other types of pharmacotherapy, can produce various side effects. In this review, we shall analyze the different pharmaceutical classes of ASMs, their mechanism of action, and their interaction with the respiratory system. Methods: This manuscript is based on a retrospective review of English publications indexed by Pubmed, UpToDate and datasheets published by the European Medicines Agency and the Food and Drug Administration (FDA), using various terms reminiscent of ASMs and pulmonary function. Results: ASMs act on organism homeostasis in different ways, acting on lung function directly and indirectly and playing a protective or damaging role. A damaging direct lung involvement ranged from infections, hypersensitivity reactions, and respiratory depression to other structured pulmonary diseases. Meanwhile, a damaging indirect effect, might be constituted by pulmonary artery hypertension. On the other hand, a protective effect might be the expression of developmental processing, decreasing airway remodelling in asthma patients, vascular remodelling in pulmonary hypertension and, nonetheless, anti-inflammatory and immunomodulatory actions. Conclusion: An adequate awareness of ASMs effects on the respiratory system seems essential for better managing frail individuals or/and those predisposed to respiratory disorders to improve our patients' clinical outcomes.

Emerging Epigenetic Targets and Their Molecular Impact on Vascular Remodeling in Pulmonary Hypertension.

Pulmonary Hypertension (PH) is a terminal disease characterized by severe pulmonary vascular remodeling. Unfortunately, targeted therapy to prevent disease progression is limited. Here, the vascular cell populations that contribute to the molecular and morphological changes of PH in conjunction with current animal models for studying vascular remodeling in PH will be examined. The status quo of epigenetic targeting for treating vascular remodeling in different PH subtypes will be dissected, while parallel epigenetic threads between pulmonary hypertension and pathogenic cancer provide insight into future therapeutic PH opportunities.

Kiosk 10R-FB-09 - Right Heart Remodeling in Pulmonary Hypertension Associated with Left Heart Disease and Chronic Thromboembolic Pulmonary Hypertension: Insights FBom Cardiac Magnetic Resonance

Kiosk 10R-FB-09 - Right Heart Remodeling in Pulmonary Hypertension Associated with Left Heart Disease and Chronic Thromboembolic Pulmonary Hypertension: Insights FBom Cardiac Magnetic Resonance

The EPOR/CD131 heteroreceptoragonist has an endothelioprotective effect against the background of pulmonary hypertension caused by monocrotalin

Introduction: The abnormal increase in pulmonary pressure observed in pulmonary arterial hypertension (PAH) is a consequence of increased pulmonary vascular resistance due to progressive loss and obliteration of pulmonary arteries. The initial trigger is a combination of factors that lead to endothelial damage and impaired vascular regeneration. Aim: research the possibilities of pharmacological correction of pulmonary arterial hypertension induced by monocrotalin using the EPOR/CD131 heteroreceptor agonist with the laboratory code EP-11-3.
 Materials and Methods: The study of pharmacological activity on a model of monocrotaline induced PAH was carried out on male Sprague-Dawley rats weighing 180-220 grams. Monocrotaline (MCT) pulmonary hypertension was simulated in 30 animals using subcutaneous injection of MCT at a dose of 60 mg/kg. Seven days after the injection of MCT, the administration of the studied compounds began. The erythropoietin derivative with the laboratory code EP-11-3 and pHBSP administered subcutaneously at a dose of 25 mcg/kg once every 3 days for 21 days.
 Results: On the model of monocrotalin-induced PAH, it was shown that the erythropoietin derivative with the laboratory code EP-11-3 has a pronounced endothelioprotective effect, reducing the coefficient of endothelial dysfunction, statistically significantly increasing the expression of VEGF-R2 mRNA and reducing the expression of SDF-1 mRNA, reducing the concentrations of CT-1 and PNP, and reducing the signs of remodeling of the heart and pulmonary vessels.
 Conclusion: Erythropoietin derivative with laboratory code EP-11-3 has an endothelioprotective effect and reduces the manifestations of vascular remodeling in pulmonary hypertension caused by monocrotalin.

Abstract 14407: Right Ventricular Myocardial Stiffening is Associated With Pulmonary Arterial Stiffening in Pulmonary Hypertension

Introduction: Pulmonary hypertension (PH) increases vascular resistance in the lung leading to remodeling of the proximal pulmonary artery (PA). PH also leads to the right ventricular (RV) remodeling following increased RV afterload. Studies of tissue remodeling in PH have focused on either the RV or PA, leaving the understanding of the RV-PA remodeling relationship elusive. We hypothesize that PA proximal tissue stiffening is associated with RV free wall (RVFW) stiffening. Methods: The mild model of PH was developed by placing SD rats (n=6) in a hypoxia chamber (10% O2) for 3 weeks, followed by one week of normoxia. The severe model of PH was developed by injecting one dose of SU5416 (20 mg/kg sc) into CDF rats (n=6) followed by the hypoxia protocol. Control rats (n=6 each for SD and CDF) were kept in normoxia at all times. Each group was half male and half female. RVFW and main PA tissues were harvested and characterized through mechanical tests. Tissue stiffness was calculated as the slope of the ensemble stress-strain curve (sum of circumferential and longitudinal stress) at a large strain (>30%) for the RVFW and PA specimens. Results: RVSP increased, and PAT decreased in both models. These changes were significant only in the severe model (Figs. 1A, B). Similarly, RVFW and PA stiffnesses increased significantly only in the severe model (Figs. 1C, D). A significant positive correlation between RVFW and PA stiffness was observed (R2 = 0.59, p=0.0001, Fig. 1E). Conclusions. Our results demonstrate, for the first time, a significant correlation between RVFW and PA stiffening in PH. Proximal PA stiffening changes the pulsatile component of the RV afterload, contributing to the RVFW stiffening, which is known to be a key precursor of RV failure. Our results suggest that the proximal PA stiffening may worsen RV stiffening independent of increases in PA distal resistance. Further studies are needed to determine the role of resistive vs. pulsatile RV afterload in the outcome of RV remodeling.

Transcriptional profiling unveils molecular subgroups of adaptive and maladaptive right ventricular remodeling in pulmonary hypertension

Right ventricular (RV) function is critical to prognosis in all forms of pulmonary hypertension. Here we perform molecular phenotyping of RV remodeling by transcriptome analysis of RV tissue obtained from 40 individuals, and two animal models of RV dysfunction of both sexes. Our unsupervised clustering analysis identified ‘early’ and ‘late’ subgroups within compensated and decompensated states, characterized by the expression of distinct signaling pathways, while fatty acid metabolism and estrogen response appeared to underlie sex-specific differences in RV adaptation. The circulating levels of several extracellular matrix proteins deregulated in decompensated RV subgroups were assessed in two independent cohorts of individuals with pulmonary arterial hypertension, revealing that NID1, C1QTNF1 and CRTAC1 predicted the development of a maladaptive RV state, as defined by magnetic resonance imaging parameters, and were associated with worse clinical outcomes. Our study provides a resource for subphenotyping RV states, identifying state-specific biomarkers, and potential therapeutic targets for RV dysfunction.

Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension.

Epigenetic regulation of vascular remodeling in pulmonary hypertension (PH) is poorly understood. Transcription regulating, histone acetylation code alters chromatin accessibility to promote transcriptional activation. Our goal was to identify upstream mechanisms that disrupt epigenetic equilibrium in PH. Human pulmonary artery smooth muscle cells (PASMCs), human idiopathic pulmonary arterial hypertension (iPAH):human PASMCs, iPAH lung tissue, failed donor lung tissue, human pulmonary microvascular endothelial cells, iPAH:PASMC and non-iPAH:PASMC RNA-seq databases, NanoString nCounter, and cleavage under targets and release using nuclease were utilized to investigate histone acetylation, hyperacetylation targets, protein and gene expression, sphingolipid activation, cell proliferation, and gene target identification. SPHK2 (sphingosine kinase 2) knockout was compared with control C57BL/6NJ mice after 3 weeks of hypoxia and assessed for indices of PH. We identified that Human PASMCs are vulnerable to the transcription-promoting epigenetic mediator histone acetylation resulting in alterations in transcription machinery and confirmed its pathological existence in PH:PASMC cells. We report that SPHK2 is elevated as much as 20-fold in iPAH lung tissue and is elevated in iPAH:PASMC cells. During PH pathogenesis, nuclear SPHK2 activates nuclear bioactive lipid S1P (sphingosine 1-phosphate) catalyzing enzyme and mediates transcription regulating histone H3K9 acetylation (acetyl histone H3 lysine 9 [Ac-H3K9]) through EMAP (endothelial monocyte activating polypeptide) II. In iPAH lungs, we identified a 4-fold elevation of the reversible epigenetic transcription modulator Ac-H3K9:H3 ratio. Loss of SPHK2 inhibited hypoxic-induced PH and Ac-H3K9 in mice. We discovered that pulmonary vascular endothelial cells are a priming factor of the EMAP II/SPHK2/S1P axis that alters the acetylome with a specificity for PASMC, through hyperacetylation of histone H3K9. Using cleavage under targets and release using nuclease, we further show that EMAP II-mediated SPHK2 has the potential to modify the local transcription machinery of pluripotency factor KLF4 (Krüppel-like factor 4) by hyperacetylating KLF4 Cis-regulatory elements while deletion and targeted inhibition of SPHK2 rescues transcription altering Ac-H3K9. SPHK2 expression and its activation of the reversible histone H3K9 acetylation in human pulmonary artery smooth muscle cell represent new therapeutic targets that could mitigate PH vascular remodeling.

Downregulation of PDCD4 through STAT3/ATF6/autophagy mediates MIF-induced PASMCs proliferation/migration and vascular remodeling

To address the molecular mechanisms underlying macrophage migration inhibitory factor (MIF) induced pulmonary artery smooth muscle cells (PASMCs) proliferation, migration and vascular remodeling in pulmonary hypertension (PH), primary cultured rat PASMCs and monocrotaline (MCT)-induced rats with PH were applied in the present study. The results showed that MIF increased signal transducer and activator of transcription 3 (STAT3) phosphorylation, and then stimulated activating transcription factor 6 (ATF6) activation, subsequently triggered autophagy activation, which further led to programmed cell death factor 4 (PDCD4) lysosomal degradation, and eventually promoted PASMCs proliferation/migration. In lung tissues of MCT rats, MIF protein expression was elevated, phosphorylation of STAT3 and activation of ATF6 were increased, activation of autophagy was evident, and reduction of PDCD4 was observed. Intervention with MIF inhibitor 4-Iodo-6-phenylpyrimidine (4-IPP), ATF6 blocker melatonin or autophagy inhibitor chloroquine, confirmed the in vitro interaction among MIF, STAT3, ATF6, autophagy and PDCD4 in MCT induced rats with PH. Targeting MIF/STAT3/ATF6/autophagy/PDCD4 axis effectively prevented the development of PH by suppressing PASMCs proliferation and vascular remodeling. In conclusions, we demonstrate that MIF activates the STAT3/ATF6/autophagy cascade and then degrades PDCD4 leading to PASMCs proliferation/migration and pulmonary vascular remodeling, suggesting that intervention this axis might have potential value in management of PH.

Cerebellin-2 promotes endothelial-mesenchymal transition in hypoxic pulmonary hypertension rats by activating NF-κB/HIF-1α/Twist1 pathway

AimsEndothelial-mesenchymal transition (EndMT) is one of the critical factors leading to vascular remodeling in pulmonary hypertension (PH). Recent studies found that the expression of Cerebellin-2 (CBLN2) is significantly increased in the lung tissue of patients with PH, suggesting that CBLN2 may be closely related to the development of PH. This study aims to investigate the role and potential mechanism of CBLN2 in the hypoxia-induced EndMT of PH rats. Material and methodsHypoxia-induced PH rat model or EndMT cell model was constructed to investigate the role of CBLN2 in the process of endothelial mesenchymal transition during PH. The effects of CBLN2 siRNA, KC7F2 (HIF-1α inhibitor), and PDTC (NF-κB inhibitor) on hypoxia-induced EndMT were observed to evaluate the potential mechanism of CBLN2 in promoting EndMT. Key findingsThe right ventricular systolic pressure and pulmonary vascular remodeling index in hypoxia-treated rats were significantly increased. The transformation of endothelial cells (marked by CD31) to mesenchymal cells (marked by α-SMA) can be observed in the pulmonary vessels of PH rats, and the expression of CBLN2 in the intima was also significantly up-regulated. In the hypoxia-induced HPAECs, endothelial cell markers such as VE-cadherin and CD31 expression were significantly down-regulated, while mesenchymal-like cell markers such as α-SMA and vimentin were increased considerably, along with the increased expressions of CBLN2, p-p65, HIF-1α, and Twist1; CBLN2 siRNA, PDTC, and KC7F2 could inhibit those phenomena. SignificanceCBLN2 can promote EndMT by activating NF-κB/HIF-1α/Twist1 pathway. Therefore, CBLN2 may be a new therapeutic target for PH.

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.

Characterizing the role of pulmonary veins in pulmonary hypertension

Pulmonary veins (PVs) transport oxygenated blood from lungs back to the heart. Small PVs can play a crucial role in the pathogenesis of pulmonary hypertension. Studies have also shown PV remodeling in pulmonary hypertension and heart failure. However, unlike pulmonary arteries (PAs), which have been a major focus of pulmonary vascular studies, the signaling mechanisms in PVs have not been investigated under normal conditions and in pulmonary hypertension. We hypothesized that the vascular wall composition and cellular signaling mechanisms in PVs are distinct from PAs. We imaged 4th order PVs (~ 50 mm) from C57BL6 mice using spinning disk confocal imaging along the z-axis. In direct contrast to PAs, where a single endothelial cell (EC) layer is surrounded by alpha actin-positive smooth muscle cells (SMCs), PV walls did not show SMCs. Cardiac troponin C staining of PVs identified cardiac myocyte-like (CML) cells with striations surrounding the EC layer. The CML layer was not observed in small PAs, vena cava, and mesenteric veins. Confocal calcium imaging showed spontaneous calcium signals in CML cells from PVs that were inhibited by ryanodine (1 mM), a ryanodine receptor inhibitor. Cannulated PVs in pressure myography experiments showed spontaneous pulsatile constrictions, which were also inhibited by ryanodine. Collectively, our results suggest that ryanodine receptor calcium signals in CML cells underlie spontaneous contractions in small PVs. Our findings also indicate that signaling mechanisms in CML cells could be potential targets for therapy in pulmonary hypertension. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

SOX17 is a Critical Factor in Maintaining Endothelial Function in Pulmonary Hypertension by an Exosome-Mediated Autocrine Manner.

Endothelial dysfunction is considered a predominant driver for pulmonary vascular remodeling in pulmonary hypertension (PH). SOX17, a key regulator of vascular homoeostasis, has been found to harbor mutations in PH patients, which are associated with PH susceptibility. Here, this study explores whether SOX17 mediates the autocrine activity of pulmonary artery ECs to maintain endothelial function and vascular homeostasis in PH and its underlying mechanism. It is found that SOX17 expression is downregulated in the endothelium of remodeled pulmonary arteries in IPH patients and SU5416/hypoxia (Su/hypo)-induced PH mice as well as dysfunctional HPAECs. Endothelial knockdown of SOX17 accelerates the progression of Su/hypo-induced PH in mice. SOX17 overexpression in the pulmonary endothelium of mice attenuates Su/hypo-induced PH. SOX17-associated exosomes block the proliferation, apoptosis, and inflammation of HPAECs, preventing pulmonary arterial remodeling and Su/hypo-induced PH. Mechanistic analyses demonstrates that overexpressing SOX17 promotes the exosome-mediated release of miR-224-5p and miR-361-3p, which are internalized by injured HPAECs in an autocrine manner, ultimately repressing the upregulation of NR4A3 and PCSK9 genes and improving endothelial function. These results suggest that SOX17 is a key gene in maintaining endothelial function and vascular homeostasis in PH through regulating exosomal miRNAs in an autocrine manner.

Histone deacetylase inhibitors synergize with sildenafil to suppress purine metabolism and proliferation in pulmonary hypertension

RationaleSildenafil, a well-known vasodilator known to interfere with purinergic signaling through effects on cGMP, is a mainstay in the treatment of pulmonary hypertension (PH). However, little is known regarding its effects on the metabolic reprogramming of vascular cells, which is a hallmark of PH. Purine metabolism, especially intracellular de novo purine biosynthesis is essential for vascular cell proliferation. Since adventitial fibroblasts are critical contributors to proliferative vascular remodeling in PH, in this study we aimed to investigate if sildenafil, beyond its well-known vasodilator role in smooth muscle cells, impacts intracellular purine metabolism and proliferation of fibroblasts derived from human PH patients. MethodsIntegrated omics approaches (plasma and cell metabolomics) and pharmacological inhibitor approaches were employed in plasma samples and cultured pulmonary artery fibroblasts from PH patients. Measurements and main resultsPlasma metabolome analysis of 27 PH patients before and after treatment with sildenafil, demonstrated a partial, but specific effect of sildenafil on purine metabolites, especially adenosine, adenine, and xanthine. However, circulating markers of cell stress, including lactate, succinate, and hypoxanthine were only decreased in a small subset of sildenafil-treated patients. To better understand potential effects of sildenafil on pathological changes in purine metabolism (especially purine synthesis) in PH, we performed studies on pulmonary fibroblasts from PAH patients (PH-Fibs) and corresponding controls (CO-Fibs), since these cells have previously been shown to demonstrate stable and marked PH associated phenotypic and metabolic changes. We found that PH-Fibs exhibited significantly increased purine synthesis. Treatment of PH-Fibs with sildenafil was insufficient to normalize cellular metabolic phenotype and only modestly attenuated the proliferation. However, we observed that treatments which have been shown to normalize glycolysis and mitochondrial abnormalities including a PKM2 activator (TEPP-46), and the histone deacetylase inhibitors (HDACi), SAHA and Apicidin, had significant inhibitory effects on purine synthesis. Importantly, combined treatment with HDACi and sildenafil exhibited synergistic inhibitory effects on proliferation and metabolic reprogramming in PH-Fibs. ConclusionsWhile sildenafil alone partially rescues metabolic alterations associated with PH, treatment with HDACi, in combination with sildenafil, represent a promising and potentially more effective strategy for targeting vasoconstriction, metabolic derangement and pathological vascular remodeling in PH.

Pulmonary Vascular Remodeling in Pulmonary Hypertension.

Pulmonary vascular remodeling is the critical structural alteration and pathological feature in pulmonary hypertension (PH) and involves changes in the intima, media and adventitia. Pulmonary vascular remodeling consists of the proliferation and phenotypic transformation of pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs) of the middle membranous pulmonary artery, as well as complex interactions involving external layer pulmonary artery fibroblasts (PAFs) and extracellular matrix (ECM). Inflammatory mechanisms, apoptosis and other factors in the vascular wall are influenced by different mechanisms that likely act in concert to drive disease progression. This article reviews these pathological changes and highlights some pathogenetic mechanisms involved in the remodeling process.