Monocrotaline Model Research Articles

Endothelial characteristics of cardiac stem cell antigen-1 expressing cells and their relevance to right ventricular adaptation.

A growing body of evidence suggest that the stem cell antigen-1 expressing (Sca-1+) cells in the heart may be the cardiac endothelial stem/progenitor cells. Their endothelial cell (EC) functions, and their role in RV physiology and pathophysiology of right heart failure (RHF) remains poorly defined. This study investigated EC characteristics of rat cardiac Sca-1+ cells, assessed spatial distribution and studied changes in Sca1+ cells during RV remodelling in monocrotaline (MCT) model of pulmonary hypertension and RV remodeling. First, flow cytometry analysis of adult male and female Sprague Dawley (SD) and Fischer CDF rat heart cells was performed, and we observed that the majority of Sca-1+ cells also expressed CD31, an EC marker. Furthermore, Sca-1+ cells showed acetylated low-density lipoprotein (ac-LDL) uptake and lectin binding similar to CD31+ cells from the same heart. The Sca-1+ cells also demonstrated network formation when plated on Matrigel. In the MCT treated rats, we observed increase in RV hypertrophy that correlated with the reduction in the abundance of Sca-1+CD31+ cells in the RV. Together, the cardiac Sca-1+ cells in the heart are endothelial stem/progenitor-like cells. These cells have higher abundance in the RV and may play a role in RV adaptation.

Rapid adaptive regulation of systemic circulation is suppressed in pulmonary

Pulmonary arterial hypertension (PAH) is characterized by an increase of a pressure in the pulmonary circulation; PAH is accompanied by activation of the sympathetic (SNS) and the renin-angiotensin-aldosterone system (RAAS). However, PAH-associated changes in baroreceptor regulation of systemic circulation, which is tightly interwoven with SNS and RAAS, have not been studied. The baroreceptor response (BRR) was studied in a chronic monocrotaline (MCT) model of PAH in rats (Wistar, 290 ± 30 g, 2–4 months). Phenylephrine as an agonist of α1-adrenergic receptor and sodium nitroprusside as NO donor were gradually administered to chronically catheterized, non-anesthetized control animals and animals with PAH (4 weeks after MCT administration) to induce vasomotor responses. Mean arterial pressure and heart rate (HR) were recorded under the action of vasoactive compounds alone or under the action of vasoactive compounds in presence of angiotensin-II (ATII), atropine. The parameters characterizing baroreceptor change in HR including maximal and minimal heart rate (HRmax, HRmin), reflex tachycardia (TBRR) and bradycardia (BBRR), range (ABBR) and the baroreceptor response sensitivity index (SIBRR) were calculated. A significant decrease in HRmax, TBRR, ABBR (but not BBRR), as well as the sensitivity index of BRR was observed in rats with PAH. ATII induces significant and different changes in the BRR parameters in control rats and in rats with PAH if administered 4 weeks after the start of the experiment. In rats with PAH, ATII causes less pronounced changes in HRmax, TBRR, and BBRR than in control animals. ATII insignificantly affects parasympathetic component of the baroreceptor reflex in rats with PAH. Thus, at least in the MCT-mediated model in rats, PAH significantly deteriorates the baroreceptor regulation of HR. This effect manifests in a decrease in the range and sensitivity of the baroreceptor response. Also, PAH unequally affects the sympathetic and parasympathetic control of the baroreceptor regulation of HR. On the other hand, ATII exhibits weak ability to alter BRR in rats with HAP. In conclusion, PAH leads to a disfunction of immediate, reflex mechanisms HR and systemic circulation control.

Magnetic nanoradiotracers for targeted neutrophil detection in pulmonary arterial hypertension

BackgroundPulmonary arterial hypertension (PAH) is a severe disease characterized by elevated blood pressure in the pulmonary artery that can ultimately damage the right ventricle of the heart. PAH is pathophysiologically heterogeneous, which makes early diagnosis and treatment difficult. Inflammation is thought to be an important factor in the development and progression of this disease and may explain some of the observed interindividual differences. In the context of both acute and chronic inflammation, neutrophil recruitment to the lung has been suggested as a potential biomarker for studying PAH progression. However, there are currently no specific probes for its non-invasive in vivo detection. The imaging-based gold standard for assessing inflammation is [18F] fluorodeoxyglucose (18F-FDG), which is not cell specific. This highlights the urgent need for more specific molecular probes to support personalized medicine.MethodsThis study investigated the potential of magnetic nanoradiotracers based on ultrasmall iron oxide nanoparticles, functionalized with N-cinnamoyl-F-(D)L-F-(D)L-F peptide, to detect increased neutrophil infiltration in vivo in different PAH animal models via positron emission tomography. These nanoprobes target formyl peptide receptor 1, which is abundantly expressed in the cell membrane of neutrophils. To assess the benefit of these nanoprobes, their biodistribution was first assessed via magnetic resonance imaging and histology. Then, their lung uptake was compared by positron emission tomography with that of 18F-FDG in two types of PAH animal models with different profiles of inflammation and neutrophil infiltration: monocrotaline and double-hit Sugen-chronic hypoxia PAH rat models.ResultsOur targeted magnetic nanoradiotracer detected an increase in pulmonary neutrophil infiltration in both PAH models and distinguished between them, which was not possible with 18F-FDG PET.ConclusionsThis study underscores the importance of targeted imaging in providing an individualized and longitudinal evaluation of heterogeneous and multifactorial diseases such as PAH. The use of targeted multimodal nanoprobes, for magnetic resonance/positron emission tomography imaging has the potential to facilitate the diagnosis and monitoring of diseases, as well as the development of novel therapies.Graphical abstract

The role and mechanism of thrombospondin-4 in pulmonary arterial hypertension associated with congenital heart disease

BackgroundDue to a special hemodynamic feature, pulmonary vascular disease in pulmonary arterial hypertension associated with congenital heart disease (PAH-CHD) has two stages: reversible and irreversible. So far, the mechanism involved in the transition from reversible to irreversible stage is elusive. Moreover, no recognized and reliable assessments to distinguish these two stages are available. Furthermore, we found that compared with control and reversible PAH, thrombospondin-4 (THBS4) was significantly upregulated in irreversible group by bioinformatic analysis. Hence, we further verify and investigate the expression and role of THBS4 in PAH-CHD.MethodsWe established the monocrotaline plus aorto-cava shunt-induced (MCT-AV) rat model. We measured the expression of THBS4 in lung tissues from MCT-AV rats. Double immunofluorescence staining of lung tissue for THBS4 and α-SMA (biomarker of smooth muscle cells) or vWF (biomarker of endothelial cells) to identify the location of THBS4 in the pulmonary artery. Primary pulmonary artery smooth muscle cells (PASMCs) were cultivated, identified, and used in this study. THBS4 was inhibited and overexpressed by siRNA and plasmid, respectively, to explore the effect of THBS4 on phenotype transformation, proliferation, apoptosis, and migration of PASMCs. The effect of THBS4 on pulmonary vascular remodeling was evaluated in vivo by adeno-associated virus which suppressed THBS4 expression. Circulating level of THBS4 in patients with PAH-CHD was measured by ELISA.ResultsTHBS4 was upregulated in the lung tissues of MCT-AV rats, and was further upregulated in severe pulmonary vascular lesions. And THBS4 was expressed mainly in PASMCs. When THBS4 was inhibited, contractile markers α-SMA and MYH11 were upregulated, while the proliferative marker PCNA was decreased, the endothelial-mensenchymal transition marker N-cad was downregulated, proapototic marker BAX was increased. Additionally, proliferation and migration of PASMCs was inhibited and apoptosis was increased. Conversely, THBS4 overexpression resulted in opposite effects. And the impact of THBS4 on PASMCs was probably achieved through the regulation of the PI3K/AKT pathway. THBS4 suppression attenuated pulmonary vascular remodeling. Furthermore, compared with patients with simple congenital heart disease and mild PAH-CHD, the circulating level of THBS4 was higher in patients with severe PAH-CHD.ConclusionsTHBS4 is a promising biomarker to distinguish reversible from irreversible PAH-CHD before repairing the shunt. THBS4 is a potential treatment target in PAH-CHD, especially in irreversible stage.

LOXL2 inhibition ameliorates pulmonary artery remodeling in pulmonary hypertension.

Conduit pulmonary arterial stiffening and the resultant increase in pulmonary vascular impedance have 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 cross-linking enzyme lysyl oxidase like 2 (LOXL2) in this study. 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 the pulmonary arteries of the rat Sugen 5416 + hypoxia (SuHx) model of severe pulmonary hypertension (PH). Similarly, LOXL2 protein and mRNA levels were increased in the pulmonary arteries (PA) and lungs of rats with PH (SuHx and monocrotaline (MCT) models). Pulmonary arteries (PAs) isolated from the rats with PH exhibited hypercontractility to phenylephrine and attenuated vasorelaxation elicited by acetylcholine, indicating severe endothelial dysfunction. Tensile testing revealed 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. Hypoxia-induced LOXL2 activation is a causal mechanism in pulmonary artery stiffening in PH and pulmonary artery mechanical and functional decline. LOXL2 inhibition with PAT-1251 could be a promising approach to improve pulmonary artery pressures, right ventricular elastance, cardiac relaxation, and survival in PAH.NEW & NOTEWORTHY Pulmonary arterial stiffening contributes to the progression of PAH and the deterioration of right heart function. This study shows that LOXL2 is upregulated in rat models of PH. LOXL2 inhibition halts pulmonary vascular remodeling and improves PA contractility, endothelial function, and PA pressure, resulting in prolonged survival. Thus, LOXL2 is an important mediator of PA remodeling and stiffening in PH and a promising target to improve PA pressures and survival in PH.

Ultrasound Neuromodulation of an Anti-Inflammatory Pathway at the Spleen Improves Experimental Pulmonary Hypertension.

Inflammation is pathogenically implicated in pulmonary arterial hypertension; however, it has not been adequately targeted therapeutically. We investigated whether neuromodulation of an anti-inflammatory neuroimmune pathway involving the splenic nerve using noninvasive, focused ultrasound stimulation of the spleen (sFUS) can improve experimental pulmonary hypertension. Pulmonary hypertension was induced in rats either by Sugen 5416 (20 mg/kg SQ) injection, followed by 21 (or 35) days of hypoxia (sugen/hypoxia model), or by monocrotaline (60 mg/kg IP) injection (monocrotaline model). Animals were randomized to receive either 12-minute-long sessions of sFUS daily or sham stimulation for 14 days. Catheterizations, echocardiography, indices of autonomic function, lung and heart histology and immunohistochemistry, spleen flow cytometry, and lung single-cell RNA sequencing were performed after treatment to assess the effects of sFUS. Splenic denervation right before induction of pulmonary hypertension results in a more severe disease phenotype. In both sugen/hypoxia and monocrotaline models, sFUS treatment reduces right ventricular systolic pressure by 25% to 30% compared with sham treatment, without affecting systemic pressure, and improves right ventricular function and autonomic indices. sFUS reduces wall thickness, apoptosis, and proliferation in small pulmonary arterioles, suppresses CD3+ and CD68+ cell infiltration in lungs and right ventricular fibrosis and hypertrophy and lowers BNP (brain natriuretic peptide). Beneficial effects persist for weeks after sFUS discontinuation and are more robust with early and longer treatment. Splenic denervation abolishes sFUS therapeutic benefits. sFUS partially normalizes CD68+ and CD8+ T-cell counts in the spleen and downregulates several inflammatory genes and pathways in nonclassical and classical monocytes and macrophages in the lung. Differentially expressed genes in those cell types are significantly enriched for human pulmonary arterial hypertension-associated genes. sFUS causes dose-dependent, sustained improvement of hemodynamic, autonomic, laboratory, and pathological manifestations in 2 models of experimental pulmonary hypertension. Mechanistically, sFUS normalizes immune cell populations in the spleen and downregulates inflammatory genes and pathways in the lung, many of which are relevant in human disease.

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.

SiRNA Targeting ECE-1 Partially Reverses Pulmonary Arterial Hypertensionassociated Damage in a Monocrotaline Model.

The aim of this study was to develop a possible treatment for pulmonary arterial hypertension. Pulmonary arterial hypertension (PAH) is a rare disease characterised by a pulmonary arterial pressure greater than 20 mmHg. One of the factors that contribute to PAH is an increase in the production of endothelin-1, a polypeptide that increases vascular resistance in the pulmonary arteries, leading to increased pulmonary arterial pressure and right ventricular hypertrophy. The objective of this study was to design, synthesize, and evaluate two siRNAs directed against endothelin-1 in a rat model of PAH induced with monocrotaline. Wistar rats were administered monocrotaline (60 mg/kg) to induce a PAH model. Following two weeks of PAH evolution, the siRNAs were administered, and after two weeks, right ventricular hypertrophy was evaluated using the RV/LV+S ratio, blood pressure, weight, and relative expression of ECE-1 (Endothelin-converting enzyme-1) mRNA (messenger RNA) by RT-PCR (real-time PCR). The monocrotaline group showed an increase in the hypertrophy index and in ECE-1 mRNA, as well as a significant decrease in weight compared to the control group, while in the monocrotaline + siRNA group, a significant decrease was observed in the relative expression of ECE-1 mRNA, as well as in right ventricular hypertrophy. Based on the above information, we conclude that the administration of siRNAs directed to ECE-1 decreases the damage associated with PAH.

Exercise improves systemic metabolism in a monocrotaline model of pulmonary hypertension

Exercise training in pulmonary arterial hypertension (PAH) has been gaining popularity with guidelines now recommending it as an important adjunct to medical therapy. Despite improvements in function and quality of life, an understanding of metabolic changes and their mechanisms remain unexplored. The objective of this study was therefore to understand the metabolic basis of exercise in a monocrotaline model of PAH.24 male Wistar rats (age: 8–12 weeks and mean body weight: [262.16 ​± ​24.49] gms) were assigned to one of the four groups (i.e., Control, PAH, Exercise and PAH ​+ ​Exercise). The exercise groups participated in treadmill running at 13.3 ​m/min, five days a week for five weeks. Demographic and clinical characteristics were monitored regularly. Following the intervention, LC-MS based metabolomics were performed on blood samples from all groups at the end of five weeks. Metabolite profiling, peak identification, alignment and isotope annotation were also performed. Statistical inference was carried out using dimensionality reducing techniques and analysis of variance.Partial-least-squares discrimination analysis and variable importance in the projection scores showed that the model was reliable, and not over lifting. The analysis demonstrated significant perturbations to lipid and amino acid metabolism, arginine and homocysteine pathways, sphingolipid (p ​< ​0.05), glycerophospholipid (p ​< ​0.05) and nucleotide metabolism in PAH. Exercise, however, was seen to restore arginine (p ​< ​0.05) and homocysteine(p ​< ​0.000 1) levels which were independent effects, irrespective of PAH.Dysregulated arginine and homocysteine pathways are seen in PAH. Exercise restores these dysregulated pathways and could potentially impact severity and outcome in PAH.

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.

HS135, a novel activin and GDF trap, is highly efficacious in preclinical models of pulmonary hypertension and obesity-associated heart failure with preserved ejection fraction

Abstract Introduction Activins and growth differentiation factors (GDFs) signal via the Activin Type II receptor (ActRII) and are genetically and clinically validated drivers of heart failure (HF), pulmonary hypertension (PH) and obesity. Despite initial clinical success in PH, current inhibitors of Activin mediated signalling (eg, sotatercept, an ActRIIA receptor ectodomain Fc-fusion protein) cannot be dosed to full biological activity. HS135 is a rationally designed ActRII-based fusion protein that acts as a ligand trap for pathological Activins and GDFs while sparing BMP-9 to treat PH and obesity-associated HF. We previously demonstrated that HS135 achieves best-in-class in vitro potency against Activin and GDF targets, which translated into full in vivo target engagement (not observed with ActRIIA-Fc). Here, the differentiated efficacy profile of HS135 was explored in preclinical models of PH and HF with preserved Ejection Fraction (HFpEF). Methods HS135 PH efficacy was assessed using the rat monocrotaline (MCT) and the mouse transaortic constriction (TAC) models, while the murine High Fat Diet (HFD) and L-NAME model was utilized to explore the potential of HS135 in obesity-associated HFpEF. In the MCT model, rats were injected twice weekly for four weeks with HS135 or ActRIIA-Fc starting the day following MCT induction. In the TAC model, mice were injected twice weekly for four weeks with HS135 or ActRIIA-Fc two weeks following surgical intervention to establish TAC. HFpEF was established by feeding mice HFD in combination with L-NAME supplied in drinking water ad libitum for 5 weeks before commencing twice weekly treatment with HS135 or empagliflozin for three weeks. In each case, tissue remodelling in the heart and lungs was assessed by IHC and RNA-seq. In addition, changes in body composition as well as markers of adiposity were evaluated. Results Right ventricles (RV) of MCT treated animals showed a distinct gene expression profile by RNAseq particularly related to pathways of inflammation and energy balance. This profile was only modestly improved by ActRIIA-Fc whereas RVs of HS135-treated animals were nearly indistinguishable by RNAseq from naïve mice. Similarly, in the lung, HS135 was more efficacious than ActRIIA-Fc at improving inflammation markers and rebalancing Activin and BMP signalling. In the TAC and HFD/L-NAME models, HS135 was efficacious at modulating the left ventricle phenotype. Across all models of PH and HF, HS135 led to profound increases in lean mass and a more metabolically favourable muscle gene expression profile. Furthermore, in the HFD / L-NAME model, HS135 was able to improve fat mass and markers of adiposity. Conclusion HS135 best-in-class target engagement profile uniquely improves PH, HF, and metabolism across in vivo models. Collectively, these data support the development of HS135 as a novel agent in cardiopulmonary and cardiometabolic disease, including PH and obesity-associated HF.

Abstract 15534: Identification of Narciclasine, a Potential Drug to Treat PAH by Unbiased Pharmaco-Transcriptomic Study

Introduction: Pulmonary arterial hypertension (PAH) is a life-threatening vascular disorder characterized by persistent vasoconstriction and detrimental remodeling of the pulmonary blood vessels. This incurable disease is marked by a complex gene expression reprogramming that triggers, among other, metabolic disorders, heightened by inflammation, and the development of a pro-survival phenotype in pulmonary arterial smooth muscle cells (PASMC).We conducted an unbiased transcriptomic study using lung samples from 16 PAH patients and 12 non-PAH controls and identified 4013 differentially expressed genes. Leveraging in silico pharmaco-transcriptomic analysis (utilizing the Lincs and Sigcom databases), we identified narciclasine as a potential drug. Hypothesis: Consequently, we hypothesized that narciclasine could offer improvements in PAH. Methods and Results: In vivo , we administered narciclasine treatment (1 mg/kg, 2 weeks, gavage) in a murine model of PH (monocrotaline model, 60 mg/kg, sc.) and observed improved PH hemodynamics (RVSP, PAAT, TPR), reduced inflammation (CD68 immunofluorescence (IF)), decreased PASMC survival (PCNA and cleaved caspase 3 IF), and alleviated adverse vascular remodeling (Elastic van Gieson Stain Kit). In vitro , narciclasine exhibited a dose-dependent reduction of cell proliferation (IF: ki67; Western blot Survivin, PCNA and PLK1) and increased apoptosis PAH-PASMC (IF: cleaved caspase 3; Wb: Bax and Bcl2). Transcriptomic study (conducted in treated PASMCs) reveals that the therapeutic effects of narciclasine in PAH may be attributed to decreased glycosylation (Gene Ontology analysis). We further observed increased glycosylation in remodeled arteries from PAH lungs, PH preclinical models, and cultured PASMC (Periodic Acid Schiff Kit). Subsequently, we validated that narciclasine reduced glycosylation and downregulated the expression of glycosylation enzymes (by wb: GFAT, NGT, OGT) both in vitro and in vivo. Statistical tests used: unpaired t (2 groups), ANOVA (&gt; 2 groups). Conclusions: In conclusion, our unbiased pharmaco-transcriptomic study identifies narciclasine as a potential therapeutic drug for PAH. Notably, its efficacy appears to be mediated by the reduction of glycosylation.

Abstract 18286: Histone Acetyltransferase P300/CBP in Pulmonary Arterial Hypertension and Associated Right Ventricular Failure

INTRODUCTION Pulmonary arterial hypertension (PAH) is defined by increased pulmonary artery (PA) pressure and vascular remodeling, partly due to excessive proliferation and survival of PA smooth muscle cells (PASMCs). Initially, RV hypertrophy allows adaptation to increased resistance, but as the disease progresses, maladaptive remodeling takes place, leading to RV failure and death. Current therapies aim to promote vasodilatation, but none of them directly targets pathological lungs or RV remodeling. The histone acetyltransferases P300/CBP have been identified as central players driving gene expression in various cellular processes such as proliferation/apoptosis and hypertrophy/fibrosis, all of which are critical features of pathological lung and RV remodeling in PAH. Given their role in controlling gene transcription programs, we hypothesized that P300/CBP contributes to maladaptive remodeling. METHODS &amp; RESULTS We show by western blot (WB) and immunofluorescence (IF) increased P300 expression in isolated PASMCs and distal PAs from PAH patients compared to controls (p&lt;0.01) as well as in monocrotaline (MCT) and sugen-hypoxia rat models (p&lt;0.05). Similar results are observed in remodeled RV from PAH patients, MCT- and pulmonary artery banding-subjected rats (WB). In vitro, P300/CBP inhibition (CCS-1477 or siRNA) reduces H3K27 acetylation (WB, p&lt;0.05) in PAH-PASMCs and RV fibroblasts (RVFbs). This effect is accompanied by a decrease in PAH-PASMC proliferation and resistance to apoptosis [WB (PCNA, PLK1, Survivin), IF (Ki67, AnexinV), p&lt;0.01]. Similarly, P300/CBP inhibition reduces proliferation (WB, PCNA, Survivin) and activation/extracellular matrix production (WB, pSMAD2/3, aSMA, Fn, Col1, MMP2) in RVFbs. In addition, P300/CBP inhibition prevents phenylephrine-induced hypertrophy in H9C2 cells and adult rat cardiomyocytes (IF, p&lt;0.05). In vivo, administration of CCS-1477 reduces pulmonary vascular remodeling (Elastica Van Gieson, p&lt;0.05), improves pulmonary hemodynamics (p&lt;0.01) and attenuates RV fibrosis (Masson’s Trichrome, p&lt;0.05) in MCT rats with established PAH. Conclusion: We provide evidence that targeting P300/CBP may represent a promising avenue to tackle both lung and RV maladaptive remodeling in PAH.

Monocrotaline model of Pulmonary hypertension in immature rats from the perspective of Serotonergic regulation

Pulmonary hypertension (PH) is a multifactorial disease characterized by an average pulmonary artery pressure of 25mmHg or higher. Although PH is generally classified into five distinct groups, pulmonary arterial hypertension (PAH), especially the idiopathic subgroup (IPAH) or congenital heart disease (CHD) subgroup, has been the focus of most of our research from a serotonergic regulatory perspective. There are many models of pulmonary hypertension in experimental practice. Each model aim at certain goals and is based on various mechanisms. The monocrotaline model of pulmonary hypertension is one of the best models for studying pulmonary hypertension due to endothelial dysfunction. However, in experimental practice, this model is used only for adulthood. At present, the influence of the serotonergic system is not taken into account in the treatment of children with pulmonary hypertension. We have modified the monocrotaline model of pulmonary hypertension for immature rats. A positive correlation was found between the concentration of serotonin metabolites in the urine and the degree of pulmonary hypertension, which can become a potential marker of pulmonary hypertension.

Copaiba oil improves pulmonary nitric oxide bioavailability in monocrotaline-treated rats.

Oxidative stress is involved in increased pulmonary vascular resistance (PVR) and right ventricular (RV) hypertrophy, characteristics of pulmonary arterial hypertension (PAH). Copaiba oil, an antioxidant compound, could attenuate PAH damage. This study's aim was to determine the effects of copaiba oil on lung oxidative stress, PVR, and mean pulmonary arterial pressure (mPAP) in the monocrotaline (MCT) model of PAH. Male Wistar rats (170g, n=7/group) were divided into four groups: control, MCT, copaiba oil, and MCT+copaiba oil (MCT-O). PAH was induced by MCT (60mg/kg i.p.) and, after 1 week, the treatment with copaiba oil (400mg/kg/day gavage) was started for 14 days. Echocardiographic and hemodynamic measurements were performed. RV was collected for morphometric evaluations and lungs and the pulmonary artery were used for biochemical analysis. Copaiba oil significantly reduced RV hypertrophy, PVR, mPAP, and antioxidant enzyme activities in the MCT-O group. Moreover, increased nitric oxide synthase and decreased NADPH oxidase activities were observed in the MCT-O group. In conclusion, copaiba oil was able to improve the balance between nitric oxide and reactive oxygen species in lungs and the pulmonary artery and to reduce PVR, which could explain a decrease in RV hypertrophy in this PAH model.

5-HT2-Receptors and 5-HIAA – Therapeutic Targets for Evaluation of Severity, Progression and Effectiveness of Treatment in Immature Male Rats in a Monocrotalin Model of Pulmonary Hypertension

Suppression of the proliferation of vascular endothelial cells and the interaction of endothelial with smooth muscle cells in pulmonary hypertension (PH) are impaired. Participation of the 5-HT2a-receptor in the mitogenic effect on endothelial, and 5-HT2b-receptor – on vascular smooth muscle cells was revealed. The main organ that metabolizes serotonin is the lung. In the endothelial cells of the vessels of the lungs under the action of the enzyme monoamine oxidase And serotonin is converted to 5-hydroxyindoleacetic acid (5-HIAA), which is subsequently excreted in the urine. Currently, the role of 5-HT2-receptors is not taken into account in the treatment of children with pulmonary hypertension. We have modified the monocrotaline model of pulmonary hypertension for immature rats. A scheme for the administration of a 5-HT2-receptor blocker for the prevention and treatment of pulmonary hypertension in immature rats was developed and tested. A positive correlation was found between the concentration of 5-HIAA in urine and the degree of pulmonary hypertension, which can become a potential marker of pulmonary hypertension. The data obtained indicate the development of pulmonary hypertension in immature rats after a single injection of monocrotaline in the form of replacement of lung tissue with fibrous tissue, the development of pneumosclerosis and bronchiectasis. Also, in animals in this model, changes in the structure of the heart muscle and vascular wall are formed with the development of fibrous tissue, which may indicate the involvement of 5HT2-receptors in the activation of fibroblasts and, accordingly, in the pathogenesis of pulmonary hypertension.

Thoracic Spinal Cord Neuroinflammation as a Novel Therapeutic Target in Pulmonary Hypertension.

Pulmonary hypertension (PH) is associated with aberrant sympathoexcitation leading to right ventricular failure (RVF), arrhythmias, and death. Microglial activation and neuroinflammation have been implicated in sympathoexcitation in experimental PH. We recently reported the first evidence of thoracic spinal cord (TSC) neuroinflammation in PH rats. Here, we hypothesize that PH is associated with increased cardiopulmonary afferent signaling leading to TSC-specific neuroinflammation and sympathoexcitation. Furthermore, inhibition of TSC neuroinflammation rescues experimental PH and RVF. We performed transcriptomic analysis and its validation on the TSC of monocrotaline (n=8) and Sugen hypoxia (n=8) rat models of severe PH-RVF. A group of monocrotaline rats received either daily intrathecal microglial activation inhibitor minocycline (200 μg/kg per day, n=5) or PBS (n=5) from day 14 through 28. Echocardiography and right ventricle-catheterization were performed terminally. Real-time quantitative reverse transcription PCR, immunolocalization, microglia+astrocyte quantification, and terminal deoxynucleotidyl transferase dUTP nick end labeling were assessed. Plasma catecholamines were measured by ELISA. Human spinal cord autopsy samples (Control n=3; pulmonary arterial hypertension n=3) were assessed to validate preclinical findings. Increased cardiopulmonary afferent signaling was demonstrated in preclinical and clinical PH. Our findings delineated common dysregulated genes and pathways highlighting neuroinflammation and apoptosis in the remodeled TSC and highlighted increased sympathoexcitation in both rat models. Moreover, we validated significantly increased microglial and astrocytic activation and CX3CL1 expression in TSC of human pulmonary arterial hypertension. Finally, amelioration of TSC neuroinflammation by minocycline in monocrotaline rats inhibited microglial activation, decreased proinflammatory cytokines, sympathetic nervous system activation and significantly attenuated PH and RVF. Targeting neuroinflammation and associated molecular pathways and genes in the TSC may yield novel therapeutic strategies for PH and RVF.

Targeting Wnt-ß-Catenin-FOSL Signaling Ameliorates Right Ventricular Remodeling.

The ability of the right ventricle (RV) to adapt to an increased pressure afterload determines survival in patients with pulmonary arterial hypertension. At present, there are no specific treatments available to prevent RV failure, except for heart/lung transplantation. The wingless/int-1 (Wnt) signaling pathway plays an important role in the development of the RV and may also be implicated in adult cardiac remodeling. Molecular, biochemical, and pharmacological approaches were used both in vitro and in vivo to investigate the role of Wnt signaling in RV remodeling. Wnt/β-catenin signaling molecules are upregulated in RV of patients with pulmonary arterial hypertension and animal models of RV overload (pulmonary artery banding-induced and monocrotaline rat models). Activation of Wnt/β-catenin signaling leads to RV remodeling via transcriptional activation of FOSL1 and FOSL2 (FOS proto-oncogene [FOS] like 1/2, AP-1 [activator protein 1] transcription factor subunit). Immunohistochemical analysis of pulmonary artery banding -exposed BAT-Gal (β-catenin-activated transgene driving expression of nuclear β-galactosidase) reporter mice RVs exhibited an increase in β-catenin expression compared with their respective controls. Genetic inhibition of β-catenin, FOSL1/2, or WNT3A stimulation of RV fibroblasts significantly reduced collagen synthesis and other remodeling genes. Importantly, pharmacological inhibition of Wnt signaling using inhibitor of PORCN (porcupine O-acyltransferase), LGKK-974 attenuated fibrosis and cardiac hypertrophy leading to improvement in RV function in both, pulmonary artery banding - and monocrotaline-induced RV overload. Wnt- β-Catenin-FOSL signaling is centrally involved in the hypertrophic RV response to increased afterload, offering novel targets for therapeutic interference with RV failure in pulmonary hypertension.

The thromboxane receptor antagonist NTP42 promotes beneficial adaptation and preserves cardiac function in experimental models of right heart overload.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary artery pressure leading to right ventricular (RV) failure. While current PAH therapies improve patient outlook, they show limited benefit in attenuating RV dysfunction. Recent investigations demonstrated that the thromboxane (TX) A2 receptor (TP) antagonist NTP42 attenuates experimental PAH across key hemodynamic parameters in the lungs and heart. This study aimed to validate the efficacy of NTP42:KVA4, a novel oral formulation of NTP42 in clinical development, in preclinical models of PAH while also, critically, investigating its direct effects on RV dysfunction. The effects of NTP42:KVA4 were evaluated in the monocrotaline (MCT) and pulmonary artery banding (PAB) models of PAH and RV dysfunction, respectively, and when compared with leading standard-of-care (SOC) PAH drugs. In addition, the expression of the TP, the target for NTP42, was investigated in cardiac tissue from several other related disease models, and from subjects with PAH and dilated cardiomyopathy (DCM). In the MCT-PAH model, NTP42:KVA4 alleviated disease-induced changes in cardiopulmonary hemodynamics, pulmonary vascular remodeling, inflammation, and fibrosis, to a similar or greater extent than the PAH SOCs tested. In the PAB model, NTP42:KVA4 improved RV geometries and contractility, normalized RV stiffness, and significantly increased RV ejection fraction. In both models, NTP42:KVA4 promoted beneficial RV adaptation, decreasing cellular hypertrophy, and increasing vascularization. Notably, elevated expression of the TP target was observed both in RV tissue from these and related disease models, and in clinical RV specimens of PAH and DCM. This study shows that, through antagonism of TP signaling, NTP42:KVA4 attenuates experimental PAH pathophysiology, not only alleviating pulmonary pathologies but also reducing RV remodeling, promoting beneficial hypertrophy, and improving cardiac function. The findings suggest a direct cardioprotective effect for NTP42:KVA4, and its potential to be a disease-modifying therapy in PAH and other cardiac conditions.

Abstract 10487: In vivo Efficacy of HS135, a Novel Activin and GDF Trap for the Treatment of Pulmonary Hypertension (PH) and Metabolic Syndrome (MetS)

Introduction: PH is a disorder characterized by remodeling of the pulmonary vasculature which causes increased pulmonary vascular resistance and ultimately right ventricular (RV) failure leading to death. MetS is a recognized risk factor for PH, particularly in patients suffering from PH associated with left-sided heart failure (WHO Group 2 PH) for which there are no approved therapies. Activins and growth differentiation factors (GDFs) are implicated in both PH pathology as well as body composition and metabolism by genetic disease association, preclinical, translational, and clinical evidence. HS135 is a rationally designed first-in-class activin-receptor (ActR)-based fusion protein that addresses both vascular remodeling and MetS in PH without affecting hematological parameters; hematological effects limit the dosing of other ActR-based agents. Methods: HS135’s ability to counteract vascular remodeling and heart failure in PH was assessed using a rat monocrotaline (MCT) model: a single dose of 60 mg/kg MCT was administered on Day 0 followed by 4 weeks of twice weekly treatment with either HS135 (1, 4, or 16 mg/kg) or vehicle. PH efficacy was assessed at end of study by echocardiography and histological readouts. HS135’s ability to improve body composition and metabolism was assessed in wild type mice by administering HS135 (1, 3, 10, 20, or 50 mg/kg) or vehicle twice weekly for 3 weeks. The effect of HS135 on hematological parameters was assessed by whole blood cell count in non-human primates (NHPs) 2 weeks following a single administration of HS135 at 3 or 30 mg/kg. Results: HS135 at 3 or 30 mg/kg did not lead to changes in hematological parameters in NHPs. Compared to vehicle, HS135 was effective at counteracting the MCT-induced PH phenotype in rats as evidenced by significant reductions in Fulton index and improved echocardiography parameters. In wild type mice, HS135 led to significant improvements in body composition. Conclusions: Contrary to other ActR-based therapies, HS135 did not lead to increases in hematocrit which would limit its dosing. HS135 was efficacious at treating PH while simultaneously improving body composition in preclinical models. Clinical trials with HS135 are expected to commence within 2023.