Pathogenesis Of Pulmonary Hypertension Research Articles

Single cell transcriptomic analyses reveal diverse and dynamic changes of distinct populations of lung interstitial macrophages in hypoxia-induced pulmonary hypertension.

Hypoxia is a common pathological driver contributing to various forms of pulmonary vascular diseases leading to pulmonary hypertension (PH). Pulmonary interstitial macrophages (IMs) play pivotal roles in immune and vascular dysfunction, leading to inflammation, abnormal remodeling, and fibrosis in PH. However, IMs' response to hypoxia and their role in PH progression remain largely unknown. We utilized a murine model of hypoxia-induced PH to investigate the repertoire and functional profiles of IMs in response to acute and prolonged hypoxia, aiming to elucidate their contributions to PH development. We conducted single-cell transcriptomic analyses to characterize the repertoire and functional profiles of murine pulmonary IMs following exposure to hypobaric hypoxia for varying durations (0, 1, 3, 7, and 21 days). Hallmark pathways from the mouse Molecular Signatures Database were utilized to characterize the molecular function of the IM subpopulation in response to hypoxia. Our analysis revealed an early acute inflammatory phase during acute hypoxia exposure (Days 1-3), which was resolved by Day 7, followed by a pro-remodeling phase during prolonged hypoxia (Days 7-21). These phases were marked by distinct subpopulations of IMs: MHCIIhiCCR2+EAR2+ cells characterized the acute inflammatory phase, while TLF+VCAM1hi cells dominated the pro-remodeling phase. The acute inflammatory phase exhibited enrichment in interferon-gamma, IL-2, and IL-6 pathways, while the pro-remodeling phase showed dysregulated chemokine production, hemoglobin clearance, and tissue repair profiles, along with activation of distinct complement pathways. Our findings demonstrate the existence of distinct populations of pulmonary interstitial macrophages corresponding to acute and prolonged hypoxia exposure, pivotal in regulating the inflammatory and remodeling phases of PH pathogenesis. This understanding offers potential avenues for targeted interventions, tailored to specific populations and distinct phases of the disease. Moreover, further identification of triggers for pro-remodeling IMs holds promise in unveiling novel therapeutic strategies for pulmonary hypertension.

IL-6/gp130 signaling in CD4+ T cells drives the pathogenesis of pulmonary hypertension

Pulmonary arterial hypertension (PAH) is characterized by stenosis and occlusions of small pulmonary arteries, leading to elevated pulmonary arterial pressure and right heart failure. Although accumulating evidence shows the importance of interleukin (IL)-6 in the pathogenesis of PAH, the target cells of IL-6 are poorly understood. Using mice harboring the floxed allele of gp130, a subunit of the IL-6 receptor, we found substantial Cre recombination in all hematopoietic cell lineages from the primitive hematopoietic stem cell level in SM22α-Cre mice. We also revealed that a CD4+ cell-specific gp130 deletion ameliorated the phenotype of hypoxia-induced pulmonary hypertension in mice. Disruption of IL-6 signaling via deletion of gp130 in CD4+ T cells inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and suppressed the hypoxia-induced increase in T helper 17 cells. To further examine the role of IL-6/gp130 signaling in more severe PH models, we developed Il6 knockout (KO) rats using the CRISPR/Cas9 system and showed that IL-6 deficiency could improve the pathophysiology in hypoxia-, monocrotaline-, and Sugen5416/hypoxia (SuHx)-induced rat PH models. Phosphorylation of STAT3 in CD4+ cells was also observed around the vascular lesions in the lungs of the SuHx rat model, but not in Il6 KO rats. Blockade of IL-6 signaling had an additive effect on conventional PAH therapeutics, such as endothelin receptor antagonist (macitentan) and soluble guanylyl cyclase stimulator (BAY41-2272). These findings suggest that IL-6/gp130 signaling in CD4+ cells plays a critical role in the pathogenesis of PAH.

Right main pulmonary artery distensibility on dynamic ventilation CT and its association with respiratory function

BackgroundHeartbeat-based cross-sectional area (CSA) changes in the right main pulmonary artery (MPA), which reflects its distensibility associated with pulmonary hypertension, can be measured using dynamic ventilation computed tomography (DVCT) in patients with and without chronic obstructive pulmonary disease (COPD) during respiratory dynamics. We investigated the relationship between MPA distensibility (MPAD) and respiratory function and how heartbeat-based CSA is related to spirometry, mean lung density (MLD), and patient characteristics.MethodsWe retrospectively analyzed DVCT performed preoperatively in 37 patients (20 female and 17 males) with lung cancer aged 70.6 ± 7.9 years (mean ± standard deviation), 18 with COPD and 19 without. MPA-CSA was separated into respiratory and heartbeat waves by discrete Fourier transformation. For the cardiac pulse-derived waves, CSA change (CSAC) and CSA change ratio (CSACR) were calculated separately during inhalation and exhalation. Spearman rank correlation was computed.ResultIn the group without COPD as well as all cases, CSACR exhalation was inversely correlated with percent residual lung volume (%RV) and RV/total lung capacity (r = -0.68, p = 0.003 and r = -0.58, p = 0.014). In contrast, in the group with COPD, CSAC inhalation was correlated with MLDmax and MLD change rate (MLDmax/MLDmin) (r = 0.54, p = 0.020 and r = 0.64, p = 0.004) as well as CSAC exhalation and CSACR exhalation.ConclusionIn patients with insufficient exhalation, right MPAD during exhalation was decreased. Also, in COPD patients with insufficient exhalation, right MPAD was reduced during inhalation as well as exhalation, which implied that exhalation impairment is a contributing factor to pulmonary hypertension complicated with COPD.Relevance statementAssessment of MPAD in different respiratory phases on DVCT has the potential to be utilized as a non-invasive assessment for pulmonary hypertension due to lung disease and/or hypoxia and elucidation of its pathogenesis.Key points• There are no previous studies analyzing all respiratory phases of right main pulmonary artery distensibility (MPAD).• Patients with exhalation impairment decreased their right MPAD.• Analysis of MPAD on dynamic ventilation computed tomography contributes to understanding the pathogenesis of pulmonary hypertension due to lung disease and/or hypoxia in patients with expiratory impairment.Graphical

Identification of Serum Interleukin-22 as Novel Biomarker in Pulmonary Hypertension: A Translational Study.

Growing evidence suggests the crucial involvement of inflammation in the pathogenesis of pulmonary hypertension (PH). The current study analyzed the expression of interleukin (IL)-17a and IL-22 as potential biomarkers for PH in a preclinical rat model of PH as well as the serum levels in a PH patient collective. PH was induced by monocrotalin (60 mg/kg body weight s.c.) in 10 Sprague Dawley rats (PH) and compared to 6 sham-treated controls (CON) as well as 10 monocrotalin-induced, macitentan-treated rats (PH_MAC). Lung and cardiac tissues were subjected to histological and immunohistochemical analysis for the ILs, and their serum levels were quantified using ELISA. Serum IL levels were also measured in a PH patient cohort. IL-22 expression was significantly increased in the lungs of the PH and PH_MAC groups (p = 0.002), whereas increased IL17a expression was demonstrated only in the lungs and RV of the PH (p < 0.05) but not the PH_MAC group (p = n.s.). The PH group showed elevated serum concentrations for IL-22 (p = 0.04) and IL-17a (p = 0.008). Compared to the PH group, the PH_MAC group demonstrated a decrease in IL-22 (p = 0.021) but not IL17a (p = n.s.). In the PH patient collective (n = 92), increased serum levels of IL-22 but not IL-17a could be shown (p < 0.0001). This elevation remained significant across the different etiological groups (p < 0.05). Correlation analysis revealed multiple significant relations between IL-22 and various clinical, laboratory, functional and hemodynamic parameters. IL-22 could serve as a promising inflammatory biomarker of PH with potential value for initial diagnosis, functional classification or even prognosis estimation. Its validation in larger patients' cohorts regarding outcome and survival data, as well as the probability of promising therapeutic target structures, remains the object of further studies.

Noncoding RNA Lipotherapeutics: A Promising Breakthrough in Pulmonary Hypertension Treatment.

Pulmonary Hypertension (PH) is a complex cardiovascular disorder characterized by elevated blood pressure in the pulmonary arteries. Current therapeutic approaches for PH have limitations in addressing the underlying molecular mechanisms. This article explores the potential of noncoding RNAs (ncRNAs), including microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), delivered through Lipid-Based Nanoparticles (LNPs) as a novel treatment strategy. These ncRNAs play critical roles in regulating vascular function and are implicated in PH pathogenesis. LNPs provide a promising method for the efficient and targeted delivery of ncRNAs. Advances in LNP technology, including the incorporation of R8 peptide modification, have shown promise in enhancing the delivery and efficacy of ncRNAs in PH models. Challenges such as biocompatibility, toxicity, and precise targeting must be addressed as these therapies move toward clinical application. The potential of personalized medicine and the integration of artificial intelligence in LNP design are discussed as prospects. In conclusion, ncRNA lipotherapeutics delivered via LNPs offer a transformative approach to treating PH, potentially leading to more effective management and improved patient outcomes in the future. However, continued research and clinical trials are necessary to fully realize their therapeutic potential in the field of PH treatment.

Impact of hyperoxia on the phenotype of pulmonary artery smooth muscle cells

Objective: To investigate the influence of varied oxygen (O2) concentration environments on the phenotypic transformation of pulmonary artery smooth muscle cells (PASMC) and the mechanism of pulmonary hypertension. Methods: Primary rat PASMC were isolated and cultured through the process of enzymatic digestion. Following identification, the stable passaged PASMC were subjected to a 6-hour incubation in sealed containers with normal O2 content (group C) and relative O2 content comprising 55% (group H55), 75% (group H75), and 95% (group H95). mRNA and protein expression of α-Actin (α-SMA), smooth muscle 22α (SM22α), osteopontin (OPN), and matrix metalloproteinase-2 (MMP-2) were measured using real-time quantitative PCR and western blot analysis. Results: The H55 group displayed no significant difference from the C group in terms of mRNA and relative protein expression levels for α-SMA, SM22α, OPN, and MMP-2 (all P>0.05). On the other hand, groups H75 and H95 exhibited a reduction in mRNA and relative protein expression of α-SMA and SM22α, along with an increase in mRNA and relative protein expression of OPN and MMP-2 when compared with both the C and H55 groups (all P<0.05). The H95 group showed a higher relative mRNA expression of MMP-2 as compared to the H75 group (P<0.05). Conclusions: Oxygen concentration environments of 75% or higher can serve as the foundation for the pathogenesis of pulmonary hypertension, essentially by inducing a phenotypic transformation in PASMC towards adopting a robust secretory function. This induction is contingent upon the concentration of oxygen present.

Inactivation of Malic Enzyme 1 in Endothelial Cells Alleviates Pulmonary Hypertension.

Pulmonary hypertension (PH) is a progressive cardiopulmonary disease with a high mortality rate. Although growing evidence has revealed the importance of dysregulated energetic metabolism in the pathogenesis of PH, the underlying cellular and molecular mechanisms are not fully understood. In this study, we focused on ME1 (malic enzyme 1), a key enzyme linking glycolysis to the tricarboxylic acid cycle. We aimed to determine the role and mechanistic action of ME1 in PH. Global and endothelial-specific ME1 knockout mice were used to investigate the role of ME1 in hypoxia- and SU5416/hypoxia (SuHx)-induced PH. Small hairpin RNA and ME1 enzymatic inhibitor (ME1*) were used to study the mechanism of ME1 in pulmonary artery endothelial cells. Downstream key metabolic pathways and mediators of ME1 were identified by metabolomics analysis in vivo and ME1-mediated energetic alterations were examined by Seahorse metabolic analysis in vitro. The pharmacological effect of ME1* on PH treatment was evaluated in PH animal models induced by SuHx. We found that ME1 protein level and enzymatic activity were highly elevated in lung tissues of patients and mice with PH, primarily in vascular endothelial cells. Global knockout of ME1 protected mice from developing hypoxia- or SuHx-induced PH. Endothelial-specific ME1 deletion similarly attenuated pulmonary vascular remodeling and PH development in mice, suggesting a critical role of endothelial ME1 in PH. Mechanistic studies revealed that ME1 inhibition promoted downstream adenosine production and activated A2AR-mediated adenosine signaling, which leads to an increase in nitric oxide generation and a decrease in proinflammatory molecule expression in endothelial cells. ME1 inhibition activated adenosine production in an ATP-dependent manner through regulating malate-aspartate NADH (nicotinamide adenine dinucleotide plus hydrogen) shuttle and thereby balancing oxidative phosphorylation and glycolysis. Pharmacological inactivation of ME1 attenuated the progression of PH in both preventive and therapeutic settings by promoting adenosine production in vivo. Our findings indicate that ME1 upregulation in endothelial cells plays a causative role in PH development by negatively regulating adenosine production and subsequently dysregulating endothelial functions. Our findings also suggest that ME1 may represent as a novel pharmacological target for upregulating protective adenosine signaling in PH therapy.

The nitric oxide-soluble guanylate cyclase-cGMP pathway in pulmonary hypertension: from PDE5 to soluble guanylate cyclase.

The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway plays a key role in the pathogenesis of pulmonary hypertension (PH). Targeted treatments include phosphodiesterase type 5 inhibitors (PDE5i) and sGC stimulators. The sGC stimulator riociguat is approved for the treatment of pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH). sGC stimulators have a dual mechanism of action, enhancing the sGC response to endogenous NO and directly stimulating sGC, independent of NO. This increase in cGMP production via a dual mechanism differs from PDE5i, which protects cGMP from degradation by PDE5, rather than increasing its production. sGC stimulators may therefore have the potential to increase cGMP levels under conditions of NO depletion that could limit the effectiveness of PDE5i. Such differences in mode of action between sGC stimulators and PDE5i could lead to differences in treatment efficacy between the classes. In addition to vascular effects, sGC stimulators have the potential to reduce inflammation, angiogenesis, fibrosis and right ventricular hypertrophy and remodelling. In this review we describe the evolution of treatments targeting the NO-sGC-cGMP pathway, with a focus on PH.

Expression of Inflammatory Genes in Murine Lungs in a Model of Experimental Pulmonary Hypertension: Effects of an Antibody-Based Targeted Delivery of Interleukin-9.

Pathogenesis of pulmonary hypertension (PH) is a multifactorial process driven by inflammation and pulmonary vascular remodeling. To target these two aspects of PH, we recently tested a novel treatment: Interleukin-9 (IL9) fused to F8, an antibody that binds to the extra-domain A of fibronectin (EDA+ Fn). As EDA+ Fn is not found in healthy adult tissue but is expressed during PH, IL9 is delivered specifically to the tissue affected by PH. We found that F8IL9 reduced pulmonary vascular remodeling and attenuated PH compared with sham-treated mice. To evaluate possible F8IL9 effects on PH-associated inflammatory processes, we analysed the expression of genes involved in pulmonary immune responses. We applied the monocrotaline (MCT) model of PH in mice (n = 44). Animals were divided into five experimental groups: sham-induced animals without PH (control, n = 4), MCT-induced PH without treatment (PH, n = 8), dual endothelin receptor antagonist treatment (dual ERA, n = 8), F8IL9 treatment (n = 12, 2 formats with n = 6 each), or with KSFIL9 treatment (KSFIL9, n = 12, 2 formats with n = 6 each, KSF: control antibody with irrelevant antigen specificity). After 28 days, a RT-PCR gene expression analysis of inflammatory response (84 genes) was performed in the lung. Compared with the controls, 19 genes exhibited relevant (+2.5-fold) upregulation in the PH group without treatment. Gene expression levels in F8IL9-treated lung tissue were reduced compared to the PH group without treatment. This was the case especially for CCL20, CXCL5, C-reactive protein, pentraxin related (CRPPR), and Kininogen-1 (KNG1). In accordance with the hypothesis stated above, F8IL9 treatment diminished the upregulation of some genes associated with inflammation in a PH animal model. Therefore, we hypothesize that IL9-based immunocytokine treatment will likely modulate various inflammatory pathways.

FUNDC1/USP15/Drp1 ameliorated TNF-α-induced pulmonary artery endothelial cell proliferation by regulating mitochondrial dynamics.

Mitochondrial dysfunction in pulmonary artery endothelial cells (PAECs) is related to the pathogenesis of pulmonary hypertension (PH). The mitochondrial receptor protein FUN14 domain containing 1 (FUNDC1) was found to be involved in pulmonary artery smooth muscle cell proliferation in PH. However, its role in PAECs remains unclear. We investigated FUNDC1 expression in the pulmonary artery endothelium in both monocrotaline-induced animal models and TNF-α-stimulated cell models. Additionally, the effect of FUNDC1 on PAECs proliferation and its possible mechanism were also investigated. We observed decreased FUNDC1 protein levels in animals and in vitro in PAECs. FUNDC1 deficiency in PAECs upregulated the expression of the deubiquitination enzyme ubiquitin-specific peptidase 15 (USP15), enhanced dynamin-related protein1 (Drp1)-mediated mitochondrial division, and increased mitochondrial ROS levels via the deubiquitination of Drp1. Additionally, FUNDC1 deficiency increased aerobic glycolysis, the production of ATP and lactic acid, and glucose uptake. FUNDC1 overexpression inhibited PAECs proliferation. Moreover, FUNDC1 overexpression in combination with a mitochondrial division or aerobic glycolysis inhibitor enhanced its inhibitory effect on cell proliferation. Our study findings suggest that FUNDC1 deficiency induced by inflammation can promote PAECs proliferation by regulating mitochondrial dynamics and cell energy metabolism via the USP15/Drp1 pathway.

Contribution of Mitochondrial Reactive Oxygen Species to Chronic Hypoxia-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) resulting from chronic hypoxia (CH) occurs in patients with chronic obstructive pulmonary diseases, sleep apnea, and restrictive lung diseases, as well as in residents at high altitude. Previous studies from our group and others demonstrate a detrimental role of reactive oxygen species (ROS) in the pathogenesis of CH-induced PH, although the subcellular sources of ROS are not fully understood. We hypothesized that mitochondria-derived ROS (mtROS) contribute to enhanced vasoconstrictor reactivity and PH following CH. To test the hypothesis, we exposed rats to 4 weeks of hypobaric hypoxia (PB ≈ 380 mmHg), with control rats housed in ambient air (PB ≈ 630 mmHg). Chronic oral administration of the mitochondria-targeted antioxidant MitoQ attenuated CH-induced decreases in pulmonary artery (PA) acceleration time, increases in right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary arterial remodeling. In addition, endothelium-intact PAs from CH rats exhibited a significantly greater basal tone compared to those from control animals, as was eliminated via MitoQ. CH also augmented the basal tone in endothelium-disrupted PAs, a response associated with increased mtROS production in primary PA smooth muscle cells (PASMCs) from CH rats. However, we further uncovered an effect of NO synthase inhibition with Nω-nitro-L-arginine (L-NNA) to unmask a potent endothelial vasoconstrictor influence that accentuates mtROS-dependent vasoconstriction following CH. This basal tone augmentation in the presence of L-NNA disappeared following combined endothelin A and B receptor blockade with BQ123 and BQ788. The effects of using CH to augment vasoconstriction and PASMC mtROS production in exogenous endothelin 1 (ET-1) were similarly prevented by MitoQ. We conclude that mtROS participate in the development of CH-induced PH. Furthermore, mtROS signaling in PASMCs is centrally involved in enhanced pulmonary arterial constriction following CH, a response potentiated by endogenous ET-1.

Patent Ductus Arteriosus and Bronchopulmonary Dysplasia–Associated Pulmonary Hypertension

Bronchopulmonary dysplasia (BPD) is often associated with pulmonary vascular disease and secondary pulmonary hypertension (PH). The pathogenesis of BPD-associated PH (BPD-PH) is complex and involves prenatal and postnatal factors that disrupt pulmonary vascular development, and patent ductus arteriosus (PDA) is a factor potentially associated with risk of BPD-PH that has been identified in very recent studies. To explore the association of PDA with BPD-PH using a bayesian model-averaged (BMA) meta-analysis of studies. PubMed and Embase were searched up to April 2023. Key search terms included BPD and PH. Studies examining infants with gestational age 32 weeks or less and reporting data on PDA and risk of BPD-PH. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and the Meta-Analysis of Observational Studies in Epidemiology reporting guidelines. Two independent reviewers extracted data, with a third reviewer checking for accuracy and completeness. Data pooling and effect size calculations were performed by BMA. The primary outcome was BPD-PH. BMA was used to calculate Bayes factors (BFs). The BF10 is the ratio of the probability of the data under the alternative hypothesis (H1, association of PDA with BPD-HP) over the probability of the data under the null hypothesis (H0). A total of 32 studies (8513 infants) were included. BMA showed that the evidence in favor of H1 was weak for any PDA (BF10 = 2.90; 10 studies), moderate for hemodynamically significant PDA (BF10 = 3.77; 3 studies), and extreme for surgically ligated or catheter-occluded PDA (BF10 = 294.9; 16 studies). In contrast, the evidence in favor of H0 was weak for medically treated PDA (BF10 = 0.55; 6 studies). In addition, BMA found strong evidence in favor of H1 when prolonged exposure to PDA was analyzed as a dichotomous variable (BF10 = 11.80; 6 studies) and extreme evidence (BF10 = 113.60; 3 studies) when PDA exposure time was analyzed as a continuous variable. In this bayesian meta-analysis, the data suggest that prolonged exposure to PDA might be associated with increased risk of pulmonary vascular disease in extremely preterm infants. This highlights the need to monitor for PH in high-risk preterm infants with prolonged exposure to PDA and to incorporate PH risk into clinical decisions regarding PDA management.

The Emerging Therapeutic Role of Prostaglandin E2 Signaling in Pulmonary Hypertension.

Mild-to-moderate pulmonary hypertension (PH) is a common complication of chronic obstructive pulmonary disease (COPD). It is characterized by narrowing and thickening of the pulmonary arteries, resulting in increased pulmonary vascular resistance (PVR) and ultimately leading to right ventricular dysfunction. Pulmonary vascular remodeling in COPD is the main reason for the increase of pulmonary artery pressure (PAP). The pathogenesis of PH in COPD is complex and multifactorial, involving chronic inflammation, hypoxia, and oxidative stress. To date, prostacyclin and its analogues are widely used to prevent PH progression in clinical. These drugs have potent anti-proliferative, anti-inflammatory, and stimulating endothelial regeneration properties, bringing therapeutic benefits to the slowing, stabilization, and even some reversal of vascular remodeling. As another well-known and extensively researched prostaglandins, prostaglandin E2 (PGE2) and its downstream signaling have been found to play an important role in various biological processes. Emerging evidence has revealed that PGE2 and its receptors (i.e., EP1-4) are involved in the regulation of pulmonary vascular homeostasis and remodeling. This review focuses on the research progress of the PGE2 signaling pathway in PH and discusses the possibility of treating PH based on the PGE2 signaling pathway.

A Review of Serotonin in the Developing Lung and Neonatal Pulmonary Hypertension.

Serotonin (5-HT) is a bioamine that has been implicated in the pathogenesis of pulmonary hypertension (PH). The lung serves as an important site of 5-HT synthesis, uptake, and metabolism with signaling primarily regulated by tryptophan hydroxylase (TPH), the 5-HT transporter (SERT), and numerous unique 5-HT receptors. The 5-HT hypothesis of PH was first proposed in the 1960s and, since that time, preclinical and clinical studies have worked to elucidate the role of 5-HT in adult PH. Over the past several decades, accumulating evidence from both clinical and preclinical studies has suggested that the 5-HT signaling pathway may play an important role in neonatal cardiopulmonary transition and the development of PH in newborns. The expression of TPH, SERT, and the 5-HT receptors is developmentally regulated, with alterations resulting in pulmonary vasoconstriction and pulmonary vascular remodeling. However, much remains unknown about the role of 5-HT in the developing and newborn lung. The purpose of this review is to discuss the implications of 5-HT on fetal and neonatal pulmonary circulation and summarize the existing preclinical and clinical literature on 5-HT in neonatal PH.

Inactivation of catalytic and non-catalytic PI3-kinase gamma function enhances hypoxia-induced pulmonary hypertension in mice

Abstract Rationale: Pulmonary hypertension (PH) is a pulmonary vascular disease characterised by chronically elevated pulmonary arterial mean pressure, increased pulmonary vascular resistance and right ventricular (RV) dysfunction and hypertrophy. The pathogenesis is characterised by vasoconstriction of pulmonary vessels and pulmonary vascular remodelling. PI 3-kinase γ (PI3Kγ) is activated via G protein-coupled receptors and abundantly expressed in all cell types involved in the pathogenesis of PH including leukocytes, cardiomyocytes, and endothelial cells (EC). The catalytic function of PI3Kγ is involved in numerous processes that are important for both vascular remodelling and maladaptive cardiac hypertrophy, including leukocyte recruitment and EC proliferation and survival. On the other hand, its non-catalytic function affects cyclic adenosine monophosphate (cAMP) production and thus cardiac contractility. Furthermore, inactivation of PI3Kγ impairs nitric oxide (NO) production by endothelial NO synthase (eNOS), which may lead to increased vascular resistance. Therefore, the aim of our study was to investigate the role of both catalytic and non-catalytic functions of PI3Kγ in the pathogenesis of PH. Methods PI3Kγ knockout mice (PI3Kγ-/-), as well as mice with a catalytically inactive form of PI3Kγ (PI3KγKD/KD) were analysed in vivo using the hypoxia-induced mouse model of PH (21 days at 10% O2 hypoxia (HOX)). To specifically inhibit the non-catalytic function of PI3Kγ, C57BL/6N mice were treated with a mimetic peptide (MP) via intratracheal instillation and studied in the same model. Systolic right ventricular pressure (RVSP) was measured using a Millar pressure catheter inserted via the jugular vein. RV hypertrophy was determined by Fulton's index (RV/LV+S). Results PI3Kγ-/- mice showed significantly increased RVSP after three weeks of hypoxia compared to WT littermate controls (HOX WT 35.64±3.21mmHg vs HOX PI3Kγ-/- 37.04±2.31mmHg;p=0.0049). A significant increase in RVSP was also detected in PI3KγKD/KD and MP-treated mice compared to respective WT controls (HOX WT 34.67±3.83mmHg vs. HOX PI3KγKD/KD 37.9±2.19mmHg;p=0.0404 and HOX vehicle 34.19±2.74mmHg vs. HOX MP 37.61±2.46mmHg;p=0.0111). Under normoxic conditions (NOX), an increased RVSP was already measured in PI3Kγ-/- mice (NOX WT 26.13±1.2mmHg vs. NOX PI3Kγ-/- 28.07±0.88;p=0.0157). Heart rate and systemic blood pressure remained unchanged. A significant increase in RV hypertrophy was only evident for PI3KγKD/KD compared to WT controls (HOX WT 0.38±0.06 vs. HOX PI3KγKD/KD 0.46±0.09mmHg;p=0.0037). Conclusion The results show that both catalytic and non-catalytic inactivation of PI3Kγ in vivo do not counteract the pathogenesis of PH, but conversely enhance it. In this context, reduced phosphorylation of eNOS may play a crucial role, leading to increased vasoconstriction, whereas increased cAMP levels may help the RV to adapt to the increased pressure. The exact mechanisms will be analysed in the future.

Unleashing the Potential of Nrf2: A Novel Therapeutic Target for Pulmonary Vascular Remodeling.

Pulmonary vascular remodeling, characterized by the thickening of all three layers of the blood vessel wall, plays a central role in the pathogenesis of pulmonary hypertension (PH). Despite the approval of several drugs for PH treatment, their long-term therapeutic effect remains unsatisfactory, as they mainly focus on vasodilation rather than addressing vascular remodeling. Therefore, there is an urgent need for novel therapeutic targets in the treatment of PH. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a vital transcription factor that regulates endogenous antioxidant defense and emerges as a novel regulator of pulmonary vascular remodeling. Growing evidence has suggested an involvement of Nrf2 and its downstream transcriptional target in the process of pulmonary vascular remodeling. Pharmacologically targeting Nrf2 has demonstrated beneficial effects in various diseases, and several Nrf2 inducers are currently undergoing clinical trials. However, the exact potential and mechanism of Nrf2 as a therapeutic target in PH remain unknown. Thus, this review article aims to comprehensively explore the role and mechanism of Nrf2 in pulmonary vascular remodeling associated with PH. Additionally, we provide a summary of Nrf2 inducers that have shown therapeutic potential in addressing the underlying vascular remodeling processes in PH. Although Nrf2-related therapies hold great promise, further research is necessary before their clinical implementation can be fully realized.

Abstract 16402: Pitx2c-Conditional Knockout Mice May Become a Useful Model to Elucidate the Pathogenesis of Post-Capillary Pulmonary Hypertension

Introduction: Pulmonary hypertension (PH) is an intractable disease that causes cardiopulmonary dysfunction due to elevated pulmonary arterial pressure. The pathogenesis of PH varies greatly depending on whether the primary lesion is upstream or downstream of the pulmonary capillaries, and the latter, post-capillary PH (pc-PH) caused by pulmonary vein stenosis/obstruction or left heart disease, currently has not an appropriate animal models to study its pathophysiology and pharmacological effects. Hypothesis: We hypothesized that a tissue-specific deletion of a gene involved in the formation of pulmonary veins could create a pathological mouse model of pc-PH. Methods: Focusing on the homeobox transcription factor Pitx2c, which has been reported to be indispensable for the formation of pulmonary veins, we generated Pitx2c-conditional knockout (cKO) mice specific for pulmonary vein and atrial myocardium by crossing Pitx2c-floxed mouse lines with Cre mouse lines for sarcolipin, a sarcoplasmic reticulum membrane protein. Then, we examined the cardiac morphology and hemodynamics of the Pitx2c-cKO mice. Results: Tissue weights of 8-week-old Pitx2c-cKO mice showed that the relative weights of both atria (especially the right atrium), right ventricle and lungs were significantly increased in Pitx2c-cKO mice, while the relative weight of the left ventricle was decreased. Furthermore, echocardiography performed on 3-month-old Pitx2c-cKO mice revealed that the right ventricle was markedly enlarged in Pitx2c-cKO mice, with enlarged pulmonary artery annulus diameter and increased velocity time integral (VTI) of blood flow through the right ventricular outflow tract. Pitx2c-cKO mice also had significantly decreased left ventricular end-diastolic diameter (LVDd), end-systolic diameter (LVDs), and mass (LVM). On the other hand, there was no difference in left ventricular fractional shortening (%FS) or tricuspid annular plane systolic excursion (TAPSE), indicating no cardiac dysfunction in Pitx2c-cKO mice. Cardiac catheterization also revealed elevated right ventricular pressure in some Pitx2c-cKO mice. Conclusion: These primary findings suggest that the Pitx2c-cKO mice may be a model for understanding the pathogenesis of pc-PH.

Abstract 18479: Lack of Catalytic and Non-Catalytic Function of PI3 Kinase Gamma in Mice Increases Hypoxia-Induced Pulmonary Hypertension

Rationale: Pulmonary hypertension (PH) is characterised by vasoconstriction and remodelling of pulmonary vessels, leading to elevated pulmonary arterial pressure and right ventricular (RV) hypertrophy. PI 3-kinase γ (PI3Kγ) is expressed in leukocytes, cardiomyocytes, and endothelial cells (EC) which are all involved in the pathogenesis of PH. The catalytic function of PI3Kγ is involved in leukocyte recruitment and EC proliferation which are important for vascular remodelling and maladaptive cardiac hypertrophy whereas its non-catalytic function affects cardiac contractility via cAMP signaling. Furthermore, inactivation of PI3Kγ impairs nitric oxide production by eNOS, which may lead to increased vascular resistance. Therefore, we investigated the role of both catalytic and non-catalytic functions of PI3Kγ in the pathogenesis of PH. Methods: PI3Kγ knockout mice (PI3Kγ -/- ), as well as mice with a catalytically inactive form of PI3Kγ (PI3Kγ KD/KD ) were exposed to hypoxia-induced PH (21 days at 10% O 2 hypoxia (HOX)). Systolic right ventricular pressure (RVSP) was measured using a Millar pressure catheter inserted via the jugular vein. RV hypertrophy was determined by Fulton's index. Results: PI3Kγ -/- mice showed significantly increased RVSP after hypoxia compared to WT controls (HOX WT 34.16±3.21 mmHg vs HOX PI3Kγ -/- 37.04±2.31 mmHg; p=0.0049). Already under normoxia (NOX), an increased RVSP was measured in PI3Kγ -/- mice (NOX WT 26.13±1.2mmHg vs. NOX PI3Kγ -/- 28.07±0.88 mmHg, p=0.0157). A significant increase in RVSP was also detected in PI3Kγ KD/KD (HOX WT 34.67±2.02 mmHg vs. HOX PI3Kγ KD/KD 37.95±1.39 mmHg; p=0.0228). Here, a significant increase in RV hypertrophy was evident for PI3Kγ KD/KD mice (HOX WT 0.38±0.06 vs. HOX PI3Kγ KD/KD 0.46±0.09; p=0.0037) which was not detected in PI3Kγ -/- compared to WT controls after hypoxia. Conclusion: The results show that both catalytic and non-catalytic inactivation of PI3Kγ in vivo do not counteract the pathogenesis of PH, but conversely enhance it. In this context, reduced phosphorylation of eNOS may play a crucial role, leading to increased vasoconstriction, whereas increased cAMP levels in PI3Kγ -/- may help the RV to adapt to the increased pressure. The exact mechanisms will be analysed in the future.

Impact of inhalation exposure to cigarette smoke on the pathogenesis of pulmonary hypertension primed by monocrotaline in rats.

Extensive, long-term exposure to cigarette smoke (CS) was recently suggested to be a risk factor for pulmonary hypertension, although further validation is required. The vascular effects of CS share similarities with the etiology of pulmonary hypertension, including vascular inflammation and remodeling. Thus, we examined the influence of CS exposure on the pathogenesis of monocrotaline (MCT)-induced pulmonary hypertension, hypothesizing that smoking might accelerate the development of primed pulmonary hypertension. CS was generated from 3R4F reference cigarettes, and rats were exposed to CS by inhalation at total particulate matter concentrations of 100-300 μg/L for 4h/day, 7days/week for 4weeks. Following 1 week of initial exposure, rats received 60 mg/kg MCT and were sacrificed and analyzed after an additional 3weeks of exposure. MCT induced hypertrophy in pulmonary arterioles and increased the Fulton index, a measure of right ventricular hypertrophy. Additional CS exposure exacerbated arteriolar hypertrophy but did not further elevate the Fulton index. No significant alterations were observed in levels of endothelin-1 and vascular endothelial growth factor, or in hematological and serum biochemical parameters. Short-term inhalation exposure to CS exacerbated arteriolar hypertrophy in the lung, although this effect did not directly aggravate the overworked heart under the current experimental conditions.

The association of eicosanoids and eicosanoid-related metabolites with pulmonary hypertension.

Eicosanoids are bioactive lipids that regulate systemic inflammation and exert vasoactive effects. Specific eicosanoid metabolites have previously been associated with pulmonary hypertension (PH), yet their role remains incompletely understood. We studied 482 participants with chronic dyspnoea who underwent clinically indicated cardiopulmonary exercise testing (CPET) with invasive haemodynamic monitoring. We performed comprehensive profiling of 888 eicosanoids and eicosanoid-related metabolites using directed non-targeted mass spectrometry, and examined associations with PH (mean pulmonary arterial pressure (mPAP) >20 mmHg), PH subtypes and physiological correlates, including transpulmonary metabolite gradients. Among 482 participants (mean±sd age 56±16 years, 62% women), 200 had rest PH. We found 48 eicosanoids and eicosanoid-related metabolites that were associated with PH. Specifically, prostaglandin (11β-dhk-PGF2α), linoleic acid (12,13-EpOME) and arachidonic acid derivatives (11,12-DiHETrE) were associated with higher odds of PH (false discovery rate q<0.05 for all). By contrast, epoxide (8(9)-EpETE), α-linolenic acid (13(S)-HOTrE(γ)) and lipokine derivatives (12,13-DiHOME) were associated with lower odds. Among PH-related eicosanoids, 14 showed differential transpulmonary metabolite gradients, with directionality suggesting that metabolites associated with lower odds of PH also displayed pulmonary artery uptake. In individuals with exercise PH, eicosanoid profiles were intermediate between no PH and rest PH, with six metabolites that differed between rest and exercise PH. Our findings highlight the role of specific eicosanoids, including linoleic acid and epoxide derivatives, as potential regulators of inflammation in PH. Of note, physiological correlates, including transpulmonary metabolite gradients, may prioritise future studies focused on eicosanoid-related pathways as important contributors to PH pathogenesis.