IPAH Patients Research Articles

Dephosphorylated uncarboxylated Matrix-Gla-Protein as candidate biomarker for immune-mediated vascular remodeling and prognosis in pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a disease characterized by pulmonary vascular remodeling. Since dephosphorylated-uncarboxylated Matrix Gla-Protein (dp-ucMGP) is associated with cardiovascular mortality in systemic sclerosis, a disease associated with PAH, and immune-system involvement in PAH is increasingly recognized, we investigated the relationship between dp-ucMGP, vascular remodeling and soluble immune-checkpoint proteins in PAH. This prospective cohort study included patients with idiopathic (I)PAH, connective tissue disease (CTD)-PAH, chronic thrombo-embolic PH (CTEPH) and CTD patients without PAH. Patients with IPAH and CTD-PAH were stratified by clinical signs of immune-mediated inflammatory disease (IMID). We measured dp-ucMGP plasma levels, soluble immune-checkpoint proteins (sICPs), and vascular smooth muscle cell (iVSMC) calcification. We found elevated dp-ucMGP levels in all PAH subtypes and CTD patients compared to healthy controls. PAH patients showed increased iVSMC calcification, but no direct correlation was found with dp-ucMGP. IMID-PAH patients had higher dp-ucMGP levels than non-IMID PAH patients. dp-ucMGP correlated with several sICPs in both IPAH and CTD patients; multiple sICPs were elevated in IMID PAH patients. High dp-ucMGP levels in IPAH patients were associated with worse survival. Our findings suggest dp-ucMGP as a potential biomarker of immune-mediated vascular remodeling in PAH. Hence, dp-ucMGP, could help identify PAH patients who might benefit from immunosuppressive therapies.

Mitochondria as a primary determinant of angiogenic modality in pulmonary arterial hypertension.

Impaired pulmonary angiogenesis plays a pivotal role in the progression of pulmonary arterial hypertension (PAH) and patient mortality, yet the molecular mechanisms driving this process remain enigmatic. Our study uncovered a striking connection between mitochondrial dysfunction (MD), caused by a humanized mutation in the NFU1 gene, and severely disrupted pulmonary angiogenesis in adult lungs. Restoring the bioavailability of the NFU1 downstream target, lipoic acid (LA), alleviated MD and angiogenic deficiency and rescued the progressive PAH phenotype in the NFU1G206C model. Notably, significant NFU1 expression and signaling insufficiencies were also identified in idiopathic PAH (iPAH) patients' lungs, emphasizing this study's relevance beyond NFU1 mutation cases. The remarkable improvement in mitochondrial function of PAH patient-derived pulmonary artery endothelial cells (PAECs) following LA supplementation introduces LA as a potential therapeutic approach. In conclusion, this study unveils a novel role for MD in dysregulated pulmonary angiogenesis and PAH manifestation, emphasizing the need to correct MD in PAH patients with unrecognized NFU1/LA deficiency.

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 remodeling 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, whereas 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 decorin (DCN). Expression of lumican, versican, and biglycan also positively correlated with collagen 1α1/1α2 expression in PASMCs in patients with IPAH and PH-PF. We demonstrated that transforming growth factor-beta (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.NEW & NOTEWORTHY Idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries (PAs) displayed the greatest proteoglycan (PG) changes, with PH associated with pulmonary fibrosis (PH-PF) and PH associated with chronic obstructive pulmonary disease (PH-COPD) following. Agrin, an endothelial cell-specific PG, was solely upregulated in IPAH. Among all cells, neo-intima-forming smooth muscle cells (SMCs) displayed the most significant PG increase. Increased levels of circulating decorin, lumican, and versican, mainly derived from SMCs, and adventitial fibroblasts, may serve as systemic indicators of pulmonary remodeling, reflecting perivascular fibrosis and neointima formation.

Abstract 159: Endothelial Fatty Acid-binding Proteins Contribute To The Pathogenesis Of Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Objectives: To determine whether upregulation of fatty-acid binding protein 4 and 5 (FABP4/5) is critical in pathogenesis of PAH. Methods: FABP4/5 expression was examined in PA endothelial cells (PAECs) and lung tissues from patients with PAH. Plasma proteome analysis was performed in human PAH samples. Echocardiography, hemodynamics, histology, and immunostaining were performed to evaluate the lung and heart PH phenotypes in Egln1 Tie2Cre (CKO) mice and Egln1 Tie2Cre /Fabp4-5 -/- (TKO) mice. Results: Both FABP4 and FABP5 were highly induced in ECs of CKO mice and PAECs from IPAH patients. Plasma levels of FABP4/5 were upregulated in IPAH patients and directly correlated with severity of hemodynamics and biochemical parameters. Genetic deletion of Fabp4-5 in CKO mice caused a reduction of right ventricular systolic pressure (RVSP) and RV hypertrophy, attenuated pulmonary vascular remodeling and prevented the right heart failure. Fabp4/5 deletion also normalized EC glycolysis, reduced ROS and HIF-2α expression, and decreased aberrant EC proliferation in CKO lungs. Conclusions: PH causes aberrant expression of FABP4/5 in pulmonary ECs which leads to enhanced EC glycolysis and hyperproliferation, contributing to the accumulation of arterial ECs and vascular remodeling and exacerbating the disease.

Abstract 2109: Nox1-mediated Endothelial Ros Propagates Hypoxia-induced Pulmonary Hypertension Via The Unfolded Protein Response.

Pulmonary arterial hypertension (PAH) is a devastating disease in which reactive oxygen species (ROS) play an important role. The disease is characterized by lung arterial cell proliferation (inward remodeling) leading to right ventricle (RV) failure. We postulated that NADPH oxidase 1 (NOX1)-derived ROS mediate endothelial cell (EC) proliferation and hemodynamic changes in PAH. Indeed, we found increased NOX1 (1.6±0.10-fold; n=7-9, p<0.01) in human pulmonary artery ECs (HPAECs) exposed to 24 hr hypoxia (Hx) vs. normoxia (Nx) (1% v. 21% O 2 , in vitro PAH phenotype). To test an in vivo NOX1 role, C57BL/6J (WT), mice were implanted with pumps delivering a NOX1-selective peptidic inhibitor (NoxA1ds) or scrambled control (Scr) and exposed to Nx (21% O 2 ) or Hx (10% O 2 ) for 3 wks plus vehicle or VEGF receptor antagonist Sugen 5416. Sugen/Hx (Su/Hx) increased RV max pressure (RVMP) 2-fold ± 0.20-fold (n=7-9, p<0.001) along with elevated mean pulmonary artery pressure (mPAP, 2-fold 0.25-fold, n=7-9, p<0.001), pulmonary vascular resistance (PVR, 2-fold±0.2-fold, n=7-9, p<0.001), and right ventricular max dP/dt (RVM dP/dt, 2-fold±0.2-fold, n=7-9, p<0.001) vs. Nx. NoxA1ds infusion significantly attenuated these rises. In parallel, RNA-Seq of HPAECs treated with NOX1 siRNA and exposed to Nx or Hx revealed the protein kinase R-like ER kinase (PERK) branch of the unfolded protein response (UPR) as NOX1-mediated. UPR promotes tumor growth in cancer due to positive effects on cell survival and proliferation. Indeed, phospho-PERK expression measured via WB increased by 1.5-fold ± 0.3-fold (n=9, p<0.01) in HPAECs exposed to Hx, which was completely reversed by siNOX1. Further, downstream members of the PERK-UPR pathway followed similar trends including phospho-eukaryotic translation initiation factor 2A (eIF2α, 3-fold ± 0.6-fold, n=9, p<0.05), activating transcription factor 4 (ATF4, 2.1-fold ± 0.4-fold, n=9, p<0.01), and protein disulfide-isomerase (PDI, 2.3-fold ± 0.65-fold, n=9, p<0.01). We corroborated these findings in cross-sections of Su/Hx mouse lungs and in autopsied iPAH patient lungs. The findings are consistent with NOX1 mediating activation of the UPR through PERK, which, in turn, induces EC hyperproliferation in PAH.

Differential expression and analysis of extrachromosomal circular DNAs as serum biomarkers in pulmonary arterial hypertension

BackgroundExtrachromosomal circular DNAs (eccDNAs) have been reported to play a key role in the occurrence and development of various diseases. However, the characterization and role of eccDNAs in pulmonary arterial hypertension (PAH) remain unclear.MethodsIn the discovery cohort, we first explored eccDNA expression profiles by Circle-sequencing analysis. The candidate eccDNAs were validated by routine polymerase chain reaction (PCR), TOPO-TA cloning and Sanger sequencing. In the validation cohort, 30 patients with PAH and 10 healthy controls were recruited for qPCR amplification to detect the candidate eccDNAs. Datas at the baseline were collected, including clinical background, biochemical variables, echocardiography and hemodynamic factors. Receiver operating characteristic curve was used to investigate the diagnostic effect of the eccDNA.ResultsWe identified a total of 21,741 eccDNAs in plasma samples of 3 IPAH patients and 3 individuals in good health, and the expression frequency, GC content, length distribution, and genome distribution of the eccDNAs were thoroughly characterized and analyzed. In the validation cohort, 687 eccDNAs were differentially expressed in patients with IPAH compared with healthy controls (screening threshold: |FC|≥2 and P < 0.05). Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the specific eccDNAs in IPAH were significantly enriched in calcium channel activity, the mitogen-activated protein kinase pathway, and the wnt signaling pathway. Verification queue found that the expression of eccDNA-chr2:131208878–131,424,362 in PAH was considerably higher than that in healthy controls and exhibited a high level of accuracy in predicting PAH with a sensitivity of 86.67% and a specificity of 90%. Furthermore, correlation analysis disclosed a significant association between serum eccDNA-chr2:131208878–131,424,362 and mean pulmonary artery pressure (mPAP) (r = 0.396, P = 0.03), 6 min walking distance (6MWD) (r = -0.399, P = 0.029), N-terminal pro-B-type natriuretic peptide (NT-proBNP) (r = 0.685, P < 0.001) and cardiac index (CI) (r = − 0.419, P = 0.021).ConclusionsThis is the first study to identify and characterize eccDNAs in patients with PAH. We revealed that serum eccDNA-chr2:131208878–131,424,362 is significantly overexpressed and can be used in the diagnosis of PAH, indicating its potential as a novel non-invasive biomarker.

Elderly Patients with Idiopathic Pulmonary Hypertension: Clinical Characteristics, Survival, and Risk Stratification in a Single-Center Prospective Registry.

The predictive value of the risk stratification scales in elderly patients with IPAH might differ from that in younger patients. It is unknown whether young and older IPAH patients have the same survival dependence on PAH-specific therapy numbers. The aim of this study was to evaluate the prognostic relevance of risk stratification scales and PAH medication numbers in elderly IPAH patients in comparison with young IPAH patients. A total of 119 patients from a prospective single-center PAH registry were divided into group I < 60 years old (n = 89) and group II ≥ 60 years old (n = 30). ESC/ERS, REVEAL, and REVEAL 2.0 risk stratification scores were assessed at baseline, as well as H2FpEF score and survival at follow-up. During a mean follow-up period of 2.9 years (1.63; 6.0), 42 (35.3%) patients died; at 1, 2, 3, 5, 7, and 10 years, survival was 95%, 88.6%, 78.5%, 61.7%, 48.5%, and 33.7%, respectively. No survival differences were observed between the two groups, despite the use of monotherapy in the elderly patients. The best predictive REVEAL value in elderly patients (IPAH patients ≥ 60 years) was AUC 0.73 (0.56-0.91), p = 0.03; and in patients with LHD comorbidities in the entire cohort, it was AUC 0.73 (0.59-0.87), p < 0.009. Factors independently associated with death in the entire cohort were CKD (p = 0.01, HR 0.2), the right-to-left ventricle dimension ratio (p = 0.0047, HR 5.97), and NT-proBNP > 1400 pg/mL (p = 0.008, HR 3.18). Risk stratification in the elderly IPAH patients requires a fundamentally different approach than that of younger patients, taking into account the initial limitations in physical performance and comorbidities that interfere with current assessment scores. The REVEAL score reliably stratifies patients at any age and LHD comorbidities. The initial monotherapy seems to be reasonable in patients over 60 years. Selection tools for initial combination PAH therapy in older IPAH patients with comorbidities need to be validated in prospective observational studies.

Disruption of DLL4/NOTCH1 Causes Dysregulated PPARγ/AKT Signaling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, activates NOTCH1 signaling and plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in PAECs activated AKT and decreased DLL4 expression. DLL4 loss was also seen in lungs of patients with IPAH and HPAH. Over-expression of DLL4 in PAECs induced BMPR2 promoter activity and exogenous DLL4 increased BMPR2 mRNA through NOTCH1 activation. Furthermore, DLL4/NOTCH1 signaling blocked AKT activation, decreased proliferation and reversed EndoMT in BMPR2-silenced PAECs and ECs from IPAH patients. PPARγ, suppressed by BMPR2 loss, was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH PAECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Finally, leniolisib, a well-tolerated oral PI3Kδ/AKT inhibitor, decreased cell proliferation, induced apoptosis and reversed markers of EndoMT in BMPR2-silenced PAECs. Restoring DLL4/NOTCH1/PPARγ signaling and/or suppressing AKT activation may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

Abstract 18413: Therapeutic Genome Editing of Endothelial FABP4 by Endothelium-Targeted Nanoparticle Delivery of the CRISPR/Cas9 System Inhibits Pulmonary Arterial Hypertension in Mice and Rats

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive vasoconstriction and obliterative vascular remodeling. The molecular mechanisms of obliterative pulmonary vascular remodeling remain elusive. Hypothesis: Therapeutic genome editing of endothelial Fabp4 will knockout FABP4 expression selectively in endothelial cells and inhibit PAH in mice and rats. Methods: Employing the newly developed endothelial cell (EC)-targeted nanoparticle delivery technology, plasmid DNA expressing CRISPR/Cas9 controlled by Cdh5 promoter and Fabp4 -specific guide RNAs driven by U6 promoter was administered i.v. to adult SD rats and Egln1 Tie2Cre mice which exhibit severe PAH and obliterative pulmonary vascular remodeling. One week after nanoparticle administration, the rats were injected with a single dose of monocrotaline (MCT) to induce PAH. At 4 weeks post-MCT, right ventricular systolic pressure (RVSP), ratio of right ventricular versus left ventricular plus septum (RV/LV+S) and pulmonary vascular remodeling were determined. Pharmacological FABP4 inhibitor was also employed to treat MCT rats as a comparison. Similarly, the pulmonary hypertensive phenotype in genome-edited Egln1 Tie2Cre mice was also characterized. Results: Nanoparticle delivery of the all-in-one CRISPR rCdh5 plasmid DNA knockout Fabp4 selectively in endothelial cells in adult rats and Egln1 Tie2Cre mice leading to reduced RVSP, RV hypertrophy and pulmonary vascular remodeling in MCT rats and also in Egln1 Tie2Cre mice. Obliterative pulmonary vascular remodeling seen in Egln1 Tie2Cre mice was inhibited and the mortality rate in endothelial Fabp4-deficient Egln1 Tie2Cre mice was reduced. Treatment of FABP4 inhibitor also inhibits PAH in MCT rats. We also observed a marked increase of FABP4 expression in endothelial cells of lung samples of IPAH patients. Overexpression of FABP4 in primary cultures of human lung microvascular endothelial cells resulted in a 70% increase of cell proliferation. Conclusions: These findings demonstrate endothelial FABP4 is a promising target for PAH therapy. EC-targeted nanoparticle delivery of the genome editing system may represent an effective and safer gene therapy approach for PAH treatment.

Abstract 16968: Hypoxia-Lactate Dehydrogenase A-Lactate Axis Mediated Metabolic Adaptation of Macrophage Promotes Vascular Remodeling of Pulmonary Hypertension

Introduction: Early recruitment of inflammatory macrophages with subsequent adaptation into pro-fibrotic phenotype have been reported essential for the development of PH. However, the underlying mechanism remains undetermined. Hypothesis: With enhanced glycolysis under hypoxic conditions, lactate dehydrogenase A(LDHA) mediated accumulation of lactate facilitates metabolic adaptation of macrophages and promotes the development of PH. Methods: Circulating monocytes, lactate, LDHA expression level were analyzed from patients with PH. The expression of LDHA, iNOS, and Arg1 in macrophages within lung tissue were examined on histopathology from patients with IPAH. Mice with myeloid specific knockout of Ldha were generated, and hemodynamic measurement, echocardiogram, histopathological examinations were conducted to evaluate pulmonary vascular remodeling using Su/Hx PH model. Bone marrow derived macrophage (BMDM) and vascular smooth muscle cells (SMCs) from mice were harvested for the mechanism study. Results: Lactate, monocytes and LDHA expression in PBMC were elevated in PH patients. On histopathology, perivascular macrophages infiltration with overexpression of LDHA, iNOS and Arg1 was noticed from lung tissue of IPAH patients, similar seen in both MCT-PH rats and Su/Hx mice. Mice with myeloid knockout of Ldha ( Ldha lyz2-/- ) shown decreased RVSP and improved right heart function on Echocardiogram. In vitro, inflammatory BMDM shown significantly elevated expression of LDHA and lactate under hypoxia, along with oversecreted growth factors including PDGF-β and promoting SMCs proliferation, which can be reversed by Ldha depletion. Conclusions: The hypoxia-LDHA-lactate axis plays a crucial role in metabolic adaption of macrophages under hypoxia condition, promoting SMCs proliferation and pulmonary vascular remodeling process. These findings suggest that targeting LDHA-lactate axis might exert therapeutic potential for PH.

Targeting IL-11 system as a treatment of pulmonary arterial hypertension

IL-11 is linked to fibrotic diseases, but its role in pulmonary hypertension is unclear. We examined IL-11's involvement in idiopathic pulmonary arterial hypertension (iPAH). Using samples from control (n = 20) and iPAH (n = 6) subjects, we assessed IL-11 and IL-11Rα expression and localization through RT-qPCR, ELISA, immunohistochemistry, and immunofluorescence. A monocrotaline-induced PAH model helped evaluate the impact of siRNA-IL-11 on pulmonary artery remodeling and PH. The effects of recombinant human IL-11 and IL-11Rα on human pulmonary artery smooth muscle cell (HPASMC) proliferation, pulmonary artery endothelial cell (HPAEC) mesenchymal transition, monocyte interactions, endothelial tube formation, and precision cut lung slice (PCLS) pulmonary artery remodeling and contraction were evaluated. IL-11 and IL-11Rα were over-expressed in pulmonary arteries (3.2-fold and 75-fold respectively) and serum (1.5-fold and 2-fold respectively) of patients with iPAH. Therapeutic transient transfection with siRNA targeting IL-11 resulted in a significant reduction in pulmonary artery remodeling (by 98%), right heart hypertrophy (by 66%), and pulmonary hypertension (by 58%) in rats exposed to monocrotaline treatment. rhIL-11 and soluble rhIL-11Rα induce HPASMC proliferation and HPAEC to monocyte interactions, mesenchymal transition, and tube formation. Neutralizing monoclonal IL-11 and IL-11Rα antibodies inhibited TGFβ1 and EDN-1 induced HPAEC to mesenchymal transition and HPASMC proliferation. In 3D PCLS, rhIL-11 and soluble rhIL-11Rα do not promote pulmonary artery contraction but sensitize PCLS pulmonary artery contraction induced by EDN-1.In summary, IL-11 and IL-11Rα are more highly expressed in the pulmonary arteries of iPAH patients and contribute to pulmonary artery remodeling and the development of PH.

SIRT3 Is a Critical Regulator of Mitochondrial Function of Fibroblasts in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a heterogeneous and life-threatening cardiopulmonary disorder in which mitochondrial dysfunction is believed to drive pathogenesis, although the underlying mechanisms remain unclear. To determine if abnormal SIRT3 (sirtuin 3) activity is related to mitochondrial dysfunction in adventitial fibroblasts from patients with idiopathic pulmonary arterial hypertension (IPAH) and hypoxic PH calves (PH-Fibs) and whether SIRT3 could be a potential therapeutic target to improve mitochondrial function, SIRT3 concentrations in control fibroblasts, PH-Fibs, and lung tissues were determined using quantitative real-time PCR and western blot. SIRT3 deacetylase activity in cells and lung tissues was determined using western blot, immunohistochemistry staining, and immunoprecipitation. Glycolysis and mitochondrial function in fibroblasts were measured using respiratory analysis and fluorescence-lifetime imaging microscopy. The effects of restoring SIRT3 activity (by overexpression of SIRT3 with plasmid, activation SIRT3 with honokiol, and supplementation with the SIRT3 cofactor nicotinamide adenine dinucleotide [NAD+]) on mitochondrial protein acetylation, mitochondrial function, cell proliferation, and gene expression in PH-Fibs were also investigated. We found that SIRT3 concentrations were decreased in PH-Fibs and PH lung tissues, and its cofactor, NAD+, was also decreased in PH-Fibs. Increased acetylation in overall mitochondrial proteins and SIRT3-specific targets (MPC1 [mitochondrial pyruvate carrier 1] and MnSOD2 [mitochondrial superoxide dismutase]), as well as decreased MnSOD2 activity, was identified in PH-Fibs and PH lung tissues. Normalization of SIRT3 activity, by increasing its expression with plasmid or with honokiol and supplementation with its cofactor NAD+, reduced mitochondrial protein acetylation, improved mitochondrial function, inhibited proliferation, and induced apoptosis in PH-Fibs. Thus, our study demonstrated that restoration of SIRT3 activity in PH-Fibs can reduce mitochondrial protein acetylation and restore mitochondrial function and PH-Fib phenotype in PH.

Pregnancy outcomes in women with pulmonary hypertension: a retrospective study in China

BackgroundIn recent years, with the development of monitoring conditions and the application of pulmonary vascular-targeted drugs, pregnancy outcomes in women with pulmonary hypertension (PH) have improved, but the maternal mortality rate is still high. The purpose of this study was to describe the maternal-foetal outcomes in pregnant women with PH.MethodsThe clinical data of 154 pregnant women with PH who were admitted to the Third Affiliated Hospital of Guangzhou Medical University from January 2011 to December 2020 were collected and descriptively analysed.ResultsAmong the 154 pregnant women with PH, 6 (3.9%) had idiopathic pulmonary arterial hypertension (iPAH), 41 (26.6%) had pulmonary arterial hypertension (PAH) associated with congenital heart disease (CHD-PAH), 45 (29.2%) had PAH related to other diseases (oPAH), and 62 (40.3%) had PH related to left heart disease (LHD-PH). The systolic pulmonary artery pressure (sPAP) was 36–49 mmHg in 53.2% of the patients, 50–69 mmHg in 22.1% of the patients and ≥ 70 mmHg in 24.7% of the patients. Five (3.2%) pregnant women died within 1 week after delivery; iPAH patients had the highest mortality rate (3/6, 50%). Fifty-four patients (35.1%) were admitted to the intensive care unit (ICU), and the incidence of heart failure during pregnancy was 14.9%. A total of 70.1% of the patients underwent caesarean section; 42.9% had premature infants; 28.6% had low-birth-weight (LBW) infants; 13.0% had very-low-birth-weight (VLBW) infants; 3.2% had extremely-low-birth-weight (ELBW) infants; 61% had small for gestational age (SGA) infants; and 1.9% experienced neonatal mortality.ConclusionThere were significant differences in the maternal-foetal outcomes in the iPAH, CHD-PAH, oPAH and LHD-PH groups. Maternal mortality was highest in the iPAH group; therefore, iPAH patients should be advised to prevent pregnancy. Standardized and multidiscipline-assisted maternal management is the key to improving maternal-foetal outcomes.

CTLA-4 Expression Is a Promising Biomarker of Idiopathic Pulmonary Arterial Hypertension and Allows Differentiation of the Type of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is an increasingly frequently diagnosed disease, the molecular mechanisms of which have not been thoroughly investigated. The aim of our study was to investigate subpopulations of lymphocytes to better understand their role in the molecular pathomechanisms of various types of PAH and to find a suitable biomarker that could be useful in the differential diagnosis of PAH. Using flow cytometry, we measured the frequencies of lymphocyte subpopulations CD4+CTLA-4+, CD8+ CTLA-4+ and CD19+ CTLA-4+ in patients with different types of PAH, namely pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH), pulmonary arterial hypertension associated with connective tissue disorders (CTD-PAH), chronic thromboembolic pulmonary hypertension (CTEPH) and idiopathic pulmonary arterial hypertension (iPAH), and in an age- and sex-matched control group in relation to selected clinical parameters. Patients in the iPAH group had the significantly highest percentage of CD4+CTLA-4+ T lymphocytes among all PAH groups, as compared to those in the control group (p < 0.001), patients with CTEPH (p < 0.001), CTD-PAH (p < 0.001) and CHD-PAH (p < 0.01). In iPAH patients, the percentages of CD4+CTLA-4+ T cells correlated strongly positively with the severity of heart failure New York Heart Association (NYHA) Functional Classification (r = 0.7077, p < 0.001). Moreover, the percentage of B CD19+CTLA-4+ cells strongly positively correlated with the concentration of NT-proBNP (r = 0.8498, p < 0.001). We have shown that statistically significantly higher percentages of CD4+CTLA-4+ (p ≤ 0.01) and CD8+ CTLA-4+ (p ≤ 0.001) T cells, measured at the time of iPAH diagnosis, were found in patients who died within 5 years of the diagnosis, which allows us to consider both of the above lymphocyte subpopulations as a negative prognostic/predictive factor in iPAH. CTLA-4 may be a promising biomarker of noninvasive detection of iPAH, but its role in planning the treatment strategy of PAH remains unclear. Further studies on T and B lymphocyte subsets are needed in different types of PAH to ascertain the relationships that exist between them and the disease.

Abstract 15464: Mitochondrial Function of Adventitial Fibroblasts in Pulmonary Hypertension is Regulated Through Sirt3 Deacetylase Activity

Introduction: Pulmonary hypertension (PH) is a heterogeneous and life-threatening cardiopulmonary disorder in which mitochondrial dysfunction drive pathogenesis. Although metabolic changes in PH draw attention of extensive investigation, the underlying mechanisms remain unclear. Hypothesis: Decreased Sirt3 mitochondrial deacetylase activity leads to increased mitochondrial enzyme acetylation level and mitochondrial dysfunction in pulmonary artery adventitial fibroblast cells isolated from IPAH patients (PH-Fibs). Methods: 1) qPCR and western blot to examine Sirt3 gene and protein levels in PH-Fibs and CO-Fibs, 2) western blotting of isolated mitochondrial protein acetylation level to examine the Sirt3 deacetylase activity, 3) immunostaining of mitochondrial SOD2 (direct target of Sirt3) acetylation level in lung tissues from PAH patients to examine Sirt3 deacetylase activity in vivo, 4) respiratory analysis and fluorescent lifetime imaging microscopy (FLIM) to analyze the glycolytic and mitochondrial function in cells, 5) overexpressing Sirt3 and supplement NAD + to investigate the therapeutic potential of regulating mitochondrial enzyme acetylation level and mitochondrial function in PH-Fibs. Results: PH-Fibs have decreased Sirt3 mRNA and protein levels compared to CO-Fibs. NAD + , a Sirt3 co-factor, is also decreased in PH-Fibs. Increased mitochondrial protein pan-acetylation levels and mitochondrial specific Sirt3 target (MnSOD2) were identified in cultured PH-Fibs, as well as lung vasculatures of IPAH patients, indicating the decreased mitochondrial deacetylase activity of Sirt3 in vitro and in vivo. We demonstrated increased glycolytic capacity and impaired mitochondrial function in PH-Fibs compared to CO-Fibs. Finally, by overexpressing Sirt3 level and at the same time increase NAD + availability, we proved that Sirt3 and NAD + can serve as therapeutic methods to synergistically improve mitochondrial function by regulating mitochondrial enzyme acetylation level in PH-Fibs. Conclusions: This study demonstrated that the mitochondrial deacetylase activity of Sirt3 as a critical mechanism regulating mitochondrial protein acetylation and mitochondrial function of fibroblast cells in IPAH patients.

Abstract 12025: Fatty Acid-Binding Proteins Promote Pulmonary Arterial Hypertension via Upregulation of Endothelial Glycolysis

Introduction: Pulmonary arterial hypertension (PAH) is a disaster disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Hypothesis: We hypothesis that endothelial fatty acid metabolism is critical for obstructive vascular remodeling in the pathogenesis of PAH. Methods: A severe mouse model of PH Egln1 Tie2Cre mice were bred with Fabp45 -/- mice to generate Egln1 Tie2Cre / Fabp45 -/- mice. Single-cell RNA sequencing (scRNA-seq) analysis and metabolomic analysis were used to profile the pulmonary cells in Egln1 Tie2Cre mice and Egln1 Tie2Cre / Fabp45 -/- mice. Human hPAEC from idiopathic PAH patients and healthy donors, monocrotaline (MCT)-induced and Sugen5416/hypoxia (SuHx)-induced PH rats were used to measure fatty acid-binding protein 4 and 5 (FABP4 and FABP5) expression. siRNA mediated knockdown of FABP4 and FABP5 and lentivirus mediated FABP4 and 5 overexpression were performed to study cell proliferation, apoptosis, glycolysis, fatty acid oxidation. Echocardiography, hemodynamics, histological and immunostaining assay were performed to evaluate the PH phenotypes. Results: Both FABP4 and 5 were highly induced in the ECs of Egln1 Tie2Cre mice and PAECs from IPAH patients, as well as the whole lungs of MCT and SuHx-induced PH rats. Knockdown or overexpression of FABP4-5 reduced or enhanced EC proliferation, starvation-induced Caspase 3/7 activity, glycolysis and fatty acid oxidation. Genetic deletion of Fabp4 and 5 in Egln1 Tie2Cre mice exhibited a reduction of right ventricular systolic pressure (RVSP), RV hypertrophy, and attenuation of pulmonary vascular remodeling, prevention of right heart failure. Fabp4-5 deletion also normalized EC glycolysis gene programming, reduced HIF-2a expression and endothelial proliferation in Egln1 Tie2Cre mice. Conclusions: FABP4 and 5 control EC glycolysis and contribute to the development of severe PH.

Abstract 10848: Adar1 Mediated Rna Editing Contributes to Vascular Remodeling

Intro: Adenosine deaminases acting on RNA 1 (ADAR1) catalyzes the conversion of adenosine (A) to inosine (I) on RNA transcripts. Due to the host immune response to non-edited double-stranded RNAs, ADAR1 deficiency has been linked to spontaneous type I interferon (IFN) signaling. Few studies have examined the role of RNA editing in vascular remodeling-associated diseases, including pulmonary arterial hypertension (PAH), a devastating disease that can lead to right heart failure and premature death. Hypothesis: ADAR1 mediated RNA editing plays a vital role in vascular diseases, particularly the pathogenesis of PAH. Methods and Results: Deeply sequenced RNA-seq data analysis using the SOAP2 program showed that IPAH patient smooth muscle cells (SMCs, N=3) have shown significantly fewer editing sites in transcripts than healthy SMCs (N=3) &amp; decreased levels of ADAR1 mRNA and protein. ADAR1 knockdown in healthy donor SMCs resulted in increased proliferation, migration and contractility, along with upregulated expression of IFN-b signaling-related molecules, such as MDA5 and ISG15. Furthermore, overexpression of MDA5, a dsRNA sensor of ADAR1-mediated immune response, in healthy SMCs induced activation of IFN-b signaling. A novel transgenic mouse model (ADAR1 SMC-KO ) was generated by knocking down ADAR1 (ADAR1 fl/fl ) selectively in smooth muscle actin-positive SMCs (SMA-CreER). Compared to controls, ADAR1 SMC-KO mice that underwent 3wk hypoxia developed severe pulmonary hypertension as evidenced by high RVSP (37mmHg, Fig1A) &amp; RVH(FI: 42%, Fig1B) with the excessive vascular remodeling in distal vessels. They also expressed higher expression of IFN-b in serum and increased recruitment of inflammatory cells, including macrophages and monocytes(Fig1C). Conclusions: This study reveals a novel molecular mechanism of ADAR1 contributing to abnormal vascular remodeling mediated by upregulation of Type 1 IFN-b, which could be central to the pathobiology of PAH.

Abstract P3009: Fatty Acid-binding Proteins Promote Pulmonary Arterial Hypertension Via Upregulation Of Endothelial Glycolysis

Introduction: Pulmonary arterial hypertension (PAH) is a disaster disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Hypothesis: We hypothesize that endothelial fatty acid metabolism is critical for obstructive vascular remodeling in the pathogenesis of PAH. Methods: A severe mouse model of PH Egln1 Tie2Cre mice were bred with Fabp45 -/- mice to generate Egln1 Tie2Cre / Fabp45 -/- mice. We applied single-cell RNA sequencing (scRNA-seq) to profile the pulmonary cells in Egln1 Tie2Cre mice and Egln1 Tie2Cre / Fabp45 -/- mice. Human hPAEC from idiopathic PAH patients and healthy donors were used to measure fatty acid-binding protein 4 and 5 (FABP4 and FABP5) expression. siRNA-mediated knockdown of FABP4 and FABP5 and lentivirus-mediated FABP4 and 5 overexpression were performed to study cell proliferation, apoptosis, and glycolysis. Results: scRNA-seq analysis demonstrated that both FABP4 and 5 were highly induced in the ECs of Egln1 Tie2Cre mice. PAECs from IPAH patients also showed higher expression of FABP4 and 5. Knockdown of FABP4-5 reduced EC proliferation, starvation-induced Caspase 3/7 activity, and glycolysis. Overexpression of FABP4-5 promoted EC glycolysis and proliferation. Genetic deletion of Fabp4 and 5 in Egln1 Tie2Cre mice exhibited a reduction of RVSP, RV hypertrophy, and reduction of EC glycolysis gene programming compared to Egln1 Tie2Cre mice. Conclusions: FABP4 and 5 control EC glycolysis and contribute to the development of PAH.

Idiopathic pulmonary arterial hypertension patients with a high H2FPEF-score: Insights from the Amsterdam UMC PAH-cohort.

The idiopathic pulmonary arterial hypertension (iPAH) phenotype is changing from a predominantly young female patient to an older, frequently obese patient of either sex. Many newly diagnosed iPAH-patients have risk factors for left ventricular diastolic dysfunction (LVDD), possibly affecting management and treatment. To determine whether the H2FPEF-score identifies a subgroup of iPAH-patients with blunted response to PAH-targeted treatment. We performed a retrospective analysis of 253 treatment-naïve iPAH-patients (1989-2019) with a confirmed diagnosis after right heart catheterization by a multidisciplinary team. Follow-up RHC measurements were available in 150 iPAH-patients. iPAH-patients were stratified by the H2FPEF-score; a score ≥5 identified a higher possibility of (concealed) LVDD. The presence of a high H2FPEF-score in incident iPAH-patients rose 30% in thirty years. Patients with a H2FPEF-score ≥5 were older, more often male and/or obese, and had more comorbidities than patients with a H2FPEF-score ≤1. A high H2FPEF-score was associated with worse survival and poor functional capacity. Right ventricular function was equally depressed among iPAH-groups. Imaging and invasive hemodynamic measurements suggested concealed LVDD in iPAH patients with a high H2FPEF-score. At follow-up, hemodynamic and functional responses were similar in iPAH-patients with a high or low H2FPEF-score. While a high H2FPEF-score in iPAH is associated with a worse prognosis and signs of LVDD, hemodynamic and functional responses to PAH treatment are not predicted by the H2FPEF-score.

Variation of PetCO2 during incremental exercise and severity of IPAH and CTEPH

Background and objectiveEnd-tidal PCO2 (PetCO2) patterns during exercise testing as well as ventilatory equivalents for CO2 have been reported for different pulmonary vascular diseases but seldomly for the significant differences in exercise response depending on the etiology of pulmonary hypertension. We aimed to compare PetCO2 change pattern in IPAH and CTEPH with varying severity during incremental cardiopulmonary exercise testing (CPET).Methods164 IPAH patients and 135 CTEPH patients referred to Shanghai Pulmonary Hospital between 2012 and 2019 were retrospectively recruited into the study. All patients performed CPET and also underwent right-heart catheterization (RHC). Forty-four healthy subjects also performed CPET and were included as controls.ResultsPetCO2 was significantly lower in IPAH and CTEPH patients as compared to normal subjects. Moreover, the PetCO2 did not rise, in fact fell from rest to anaerobic threshold (AT), then further decreased until peak in both IPAH and CTEPH. PetCO2 value at rest, unloaded, AT and peak were proportionately reduced as the World Health Organization functional class (WHO-Fc) increased in both IPAH and CTEPH patients. The PETCO2 in IPAH patients had significant differences during all phases of exercise between WHO-Fc I-II and III-IV subgroup. CTEPH also demonstrated significant difference except for PetCO2 at peak. PetCO2 values were significantly higher in IPAH during all phases of exercise as compared to CTEPH patients (all P < 0.001).PeakVO2%pred correlated significantly with PetCO2 at rest (r = 0.477, P < 0.001), AT (r = 0.609, P < 0.001) and peak exercise (r = 0.576, P < 0.001) in IPAH. N-terminal natriuretic peptide type-B (NT-proBNP) also correlated markedly with PetCO2, with a correlation coefficient of − 0.326 to − 0.427 (all P < 0.001). Additionally, PetCO2 at rest, at AT and at peak correlated positively with peakVO2%pred and showed an inverse correlation with NT-proBNP in CTEPH patients (all P < 0.05).ConclusionsPetCO2 during exercise in IPAH and CTEPH patients was significantly different from normal subjects. Moreover, PetCO2 values were significantly higher in IPAH during all phases of exercise as compared to CTEPH patients (all P < 0.001). PetCO2 was progressively more abnormal with increasing disease severity according to peakVO2%pred and WHO-Fc.