Mouse Model Of Pulmonary Arterial Hypertension Research Articles

Ginsenoside Rg1 improves hypoxia-induced pulmonary vascular endothelial dysfunction through TXNIP/NLRP3 pathway-modulated mitophagy

BackgroundVascular endothelial dysfunction (VED) is one of the main pathogenic events in pulmonary arterial hypertension (PAH). Previous studies have demonstrated that the ginsenoside Rg1 (Rg1) can ameliorate PAH, but the mechanism by which Rg1 affects pulmonary VED in hypoxia-induced PAH remains unclear. MethodsNetwork pharmacology, molecular docking and other experiments were used to explore the mechanisms by which Rg1 affects PAH. A PAH mouse model was established via hypoxia combined with the vascular endothelial growth factor (VEGFR) inhibitor su5416 (SuHx), and a cell model was established via hypoxia. The functions of Rg1 in VED, oxidative stress, inflammation, mitophagy, and TXNIP and NLRP3 expression were examined. ResultsIn hypoxia-induced VED, progressive exacerbation of oxidative stress, inflammation, and mitophagy were observed, and were associated with elevated TXNIP and NLRP3 expression in vivo and in vitro. Rg1 improved hypoxia-induced impaired endothelium-dependent vasodilation and increased nitric oxide (NO) and endothelial NO synthase (eNOS) expression. Rg1, SRI37330 (a TXNIP inhibitor), MCC950 (an NLRP3 inhibitor), and Liensinine (a mitophagy inhibitor) attenuated oxidative stress, inflammation, and mitophagy by reducing the expression of TXNIP and NLRP3 in mice and cells. Furthermore, the combination of SB203580 (a mitophagy agonist) with Rg1 disrupted the protective effect of Rg1 on hypoxia-induced pulmonary artery and human pulmonary artery endothelial cells (HPAECs). ConclusionRg1 improves hypoxia-induced pulmonary vascular endothelial dysfunction through TXNIP/NLRP3 pathway-modulated oxidative stress, inflammation and mitophagy.

Empagliflozin Attenuates Pulmonary Arterial Remodeling Through Peroxisome Proliferator-Activated Receptor Gamma Activation.

The loss of peroxisome proliferator-activated receptor gamma (PPARγ) exacerbates pulmonary arterial hypertension (PAH), while its upregulation reduces cell proliferation and vascular remodeling, thereby decreasing PAH severity. SGLT2 inhibitors, developed for type 2 diabetes, might also affect signal transduction in addition to modulating sodium-glucose cotransporters. Pulmonary arterial smooth muscle cells (PASMCs) isolated from patients with idiopathic pulmonary arterial hypertension (IPAH) were treated with three SGLT2 inhibitors, canagliflozin (Cana), dapagliflozin (Dapa), and empagliflozin (Empa), to investigate their antiproliferative effects. To assess the impact of Empa on PPARγ, luciferase reporter assays and siRNA-mediated PPARγ knockdown were employed to examine regulation of the γ-secretase complex and its downstream target Notch3. Therapy involving daily administration of Empa was initiated 21 days after inducing hypoxia-induced PAH in mice. Empa exhibited significant antiproliferative effects on fast-growing IPAH PASMCs. Empa activated PPARγ to prevent formation of the γ-secretase complex, with specific impacts on presenilin enhancer 2 (PEN2), which plays a crucial role in maintaining γ-secretase complex stability, thereby inhibiting Notch3. Similar results were obtained in lung tissue of chronically hypoxic mice. Empa attenuated pulmonary arterial remodeling and right ventricle hypertrophy in a hypoxic PAH mouse model. Moreover, PPARγ expression was significantly decreased and PEN2, and Notch3 levels were increased in lung tissue from PAH patients compared with non-PAH lung tissue. Empa reverses vascular remodeling by activating PPARγ to suppress the γ-secretase-Notch3 axis. We propose Empa as a PPARγ activator and potential therapeutic for PAH.

High relative humidity environment alleviates hypoxia-induced pulmonary arterial hypertension in mice

The environment has long been considered a crucial factor influencing the onset and progression of pulmonary diseases. Environmental therapy is also a practical treatment approach for many conditions. While research has explored the effects of factors like air pressure and oxygen concentration on pulmonary arterial hypertension (PAH), the impact of air humidity on PAH has not been investigated. In this study, we examined the role of different air humidity levels in a mouse model of PAH by controlling relative humidity. We induced PAH in mice using 10 % hypoxia, which led to significant thickening of the pulmonary vasculature, elevated right ventricular systolic pressure, and an increased right ventricular hypertrophy index (RVHI). However, when exposed to an environment with 80–95 % relative humidity, there was a marked reduction in the extent of pulmonary vascular remodeling, decreased vascular thickening, and lower RVHI, effectively preserving right heart function. Notably, changes in the Bmpr2/Tgf-β signaling pathway were significant and may play a pivotal role in this protective effect. In summary, our findings indicate that high relative humidity confers a protective effect on hypoxia-induced PAH in mice, providing novel insights into potential treatments for PAH.

Erythrophagocytosis-induced ferroptosis contributes to pulmonary microvascular thrombosis and thrombotic vascular remodeling in pulmonary arterial hypertension

BackgroundWhether primary or just as a complication from the progression of pulmonary arterial hypertension (PAH), thrombosis seems to be an important player in this condition. The crosstalk between red blood cells (RBCs) and pulmonary microvascular endothelial cells (PMVECs) and their role in PAH remain undefined. ObjectivesThe goals of this study were to assess the role of RBC–PMVEC interaction in microvascular thrombosis and thrombotic vascular remodeling under hypoxic conditions. MethodsWe established an in vitro hypoxic coincubation model of RBC and PMVEC as well as a hypoxic mouse model. We investigated erythrophagocytosis (EP), ferroptosis, thrombosis tendency, and pulmonary hemodynamics in experimental PAH. ResultsIncreased EP in PMVEC triggered ferroptosis, enhanced procoagulant activity, and exacerbated vessel remodeling under hypoxic conditions. In the PAH mouse model induced by chronic hypoxia, EP-induced ferroptosis followed by upregulated TMEM16F led to a high tendency of thrombus formation and thrombotic vascular remodeling. Inhibition of ferroptosis or silencing of TMEM16F could alleviate hypercoagulable phenotype, reverse right ventricular systolic pressure, right ventricular hypertrophy index, and remodeling of pulmonary vessels. ConclusionThese results illustrate the pathogenic RBC–PMVEC interactions in PAH. Inhibition EP, ferroptosis, or TMEM16F could be a novel therapeutic target to prevent PAH development and thrombotic complications.

5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase promotes pulmonary arterial smooth muscle cell proliferation via the Ras signaling pathway.

Pulmonary arterial hypertension (PAH) is a debilitating vascular disorder characterized by abnormal pulmonary artery smooth muscle cell (PASMC) proliferation and collagen synthesis, contributing to vascular remodeling and elevated pulmonary vascular resistance. This study investigated the critical role of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) in cell proliferation and collagen synthesis in PASMCs in PAH. Here we show that ATIC levels are significantly increased in the lungs of monocrotaline (MCT)-induced PAH rat model, hypoxia-induced PAH mouse model, and platelet-derived growth factor (PDGF)-stimulated PASMCs. Inhibition of ATIC attenuated PDGF-induced cell proliferation and collagen I synthesis in PASMCs. Conversely, overexpression or knockdown of ATIC causes a significant promotion or inhibition of Ras and ERK activation, cell proliferation, and collagen synthesis in PASMCs. Moreover, ATIC deficiency attenuated Ras activation in the lungs of hypoxia-induced PAH mice. Furthermore, Ras inhibition attenuates ATIC overexpression- and PDGF-induced collagen synthesis and PASMC proliferation. Notably, we identified that transcription factors MYC, early growth response protein 1 (EGR1), and specificity protein 1 (SP1) directly binds to promoters of Atic gene and regulate ATIC expression. These results provide the first evidence that ATIC promotes PASMC proliferation in pulmonary vascular remodeling through the Ras signaling pathway.NEW & NOTEWORTHY Our findings highlight the important role of ATIC in the PASMC proliferation of pulmonary vascular remodeling through its modulation of the Ras signaling pathway and its regulation by transcription factors MYC, EGR1, and SP1. ATIC's modulation of Ras signal pathway represents a novel mechanism contributing to PAH development.

Exosomes enriched by miR-429-3p derived from ITGB1 modified Telocytes alleviates hypoxia-induced pulmonary arterial hypertension through regulating Rac1 expression

BackgroundRecent studies have emphasized the critical role of Telocytes (TCs)-derived exosomes in organ tissue injury and repair. Our previous research showed a significant increase in ITGB1 within TCs. Pulmonary Arterial Hypertension (PAH) is marked by a loss of microvessel regeneration and progressive vascular remodeling. This study aims to investigate whether exosomes derived from ITGB1-modified TCs (ITGB1-Exo) could mitigate PAH.MethodsWe analyzed differentially expressed microRNAs (DEmiRs) in TCs using Affymetrix Genechip miRNA 4.0 arrays. Exosomes isolated from TC culture supernatants were verified through transmission electron microscopy and Nanoparticle Tracking Analysis. The impact of miR-429-3p-enriched exosomes (Exo-ITGB1) on hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs) was evaluated using CCK-8, transwell assay, and inflammatory factor analysis. A four-week hypoxia-induced mouse model of PAH was constructed, and H&E staining, along with Immunofluorescence staining, were employed to assess PAH progression.ResultsForty-five miRNAs exhibited significant differential expression in TCs following ITGB1 knockdown. Mus-miR-429-3p, significantly upregulated in ITGB1-overexpressing TCs and in ITGB1-modified TC-derived exosomes, was selected for further investigation. Exo-ITGB1 notably inhibited the migration, proliferation, and inflammation of PASMCs by targeting Rac1. Overexpressing Rac1 partly counteracted Exo-ITGB1’s effects. In vivo administration of Exo-ITGB1 effectively reduced pulmonary vascular remodeling and inflammation.ConclusionsOur findings reveal that ITGB1-modified TC-derived exosomes exert anti-inflammatory effects and reverse vascular remodeling through the miR-429-3p/Rac1 axis. This provides potential therapeutic strategies for PAH treatment.Graphical 1. Identification of Differentially Expressed microRNAs (DEmiRs) in ITGB1 overexpressed TCs.2. Effects of Exo-ITGB1 or miR-429-3p on Hypoxia-Induced PASMCs in vitro.3. Exo-ITGB1 inhibits the hyper-proliferation and migration of PASMCs through regulating miR-429-3p/Rac1 axis in vitro.4. The therapeutic potential of Exo-ITGB1 in hypoxia-induced PAH model in vivo.

Dissecting molecular mechanisms underlying ferroptosis in human umbilical cord mesenchymal stem cells: Role of cystathionine γ-lyase/hydrogen sulfide pathway.

Ferroptosis can induce low retention and engraftment after mesenchymal stem cell (MSC) delivery, which is considered a major challenge to the effectiveness of MSC-based pulmonary arterial hypertension (PAH) therapy. Interestingly, the cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S) pathway may contribute to mediating ferroptosis. However, the influence of the CSE/H2S pathway on ferroptosis in human umbilical cord MSCs (HUCMSCs) remains unclear. To clarify whether the effect of HUCMSCs on vascular remodelling in PAH mice is affected by CSE/H2S pathway-mediated ferroptosis, and to investigate the functions of the CSE/H2S pathway in ferroptosis in HUCMSCs and the underlying mechanisms. Erastin and ferrostatin-1 (Fer-1) were used to induce and inhibit ferroptosis, respectively. HUCMSCs were transfected with a vector to overexpress or inhibit expression of CSE. A PAH mouse model was established using 4-wk-old male BALB/c nude mice under hypoxic conditions, and pulmonary pressure and vascular remodelling were measured. The survival of HUCMSCs after delivery was observed by in vivo bioluminescence imaging. Cell viability, iron accumulation, reactive oxygen species production, cystine uptake, and lipid peroxidation in HUCMSCs were tested. Ferroptosis-related proteins and S-sulfhydrated Kelch-like ECH-associating protein 1 (Keap1) were detected by western blot analysis. In vivo, CSE overexpression improved cell survival after erastin-treated HUCMSC delivery in mice with hypoxia-induced PAH. In vitro, CSE overexpression improved H2S production and ferroptosis-related indexes, such as cell viability, iron level, reactive oxygen species production, cystine uptake, lipid peroxidation, mitochondrial membrane density, and ferroptosis-related protein expression, in erastin-treated HUCMSCs. In contrast, in vivo, CSE inhibition decreased cell survival after Fer-1-treated HUCMSC delivery and aggravated vascular remodelling in PAH mice. In vitro, CSE inhibition decreased H2S levels and restored ferroptosis in Fer-1-treated HUCMSCs. Interestingly, upregulation of the CSE/H2S pathway induced Keap1 S-sulfhydration, which contributed to the inhibition of ferroptosis. Regulation of the CSE/H2S pathway in HUCMSCs contributes to the inhibition of ferroptosis and improves the suppressive effect on vascular remodelling in mice with hypoxia-induced PAH. Moreover, the protective effect of the CSE/H2S pathway against ferroptosis in HUCMSCs is mediated via S-sulfhydrated Keap1/nuclear factor erythroid 2-related factor 2 signalling. The present study may provide a novel therapeutic avenue for improving the protective capacity of transplanted MSCs in PAH.

Pericytes: The lung-forgotten cell type.

Pericytes are a heterogeneous population of mesenchymal cells located on the abluminal surface of microvessels, where they provide structural and biochemical support. Pericytes have been implicated in numerous lung diseases including pulmonary arterial hypertension (PAH) and allergic asthma due to their ability to differentiate into scar-forming myofibroblasts, leading to collagen deposition and matrix remodelling and thus driving tissue fibrosis. Pericyte-extracellular matrix interactions as well as other biochemical cues play crucial roles in these processes. In this review, we give an overview of lung pericytes, the key pro-fibrotic mediators they interact with, and detail recent advances in preclinical studies on how pericytes are disrupted and contribute to lung diseases including PAH, allergic asthma, and chronic obstructive pulmonary disease (COPD). Several recent studies using mouse models of PAH have demonstrated that pericytes contribute to these pathological events; efforts are currently underway to mitigate pericyte dysfunction in PAH by targeting the TGF-β, CXCR7, and CXCR4 signalling pathways. In allergic asthma, the dissociation of pericytes from the endothelium of blood vessels and their migration towards inflamed areas of the airway contribute to the characteristic airway remodelling observed in allergic asthma. Although several factors have been suggested to influence this migration such as TGF-β, IL-4, IL-13, and periostin, recent evidence points to the CXCL12/CXCR4 pathway as a potential therapeutic target. Pericytes might also play an essential role in lung dysfunction in response to ageing, as they are responsive to environmental risk factors such as cigarette smoke and air pollutants, which are the main drivers of COPD. However, there is currently no direct evidence delineating the contribution of pericytes to COPD pathology. Although there is a lack of human clinical data, the recent available evidence derived from in vitro and animal-based models shows that pericytes play important roles in the initiation and maintenance of chronic lung diseases and are amenable to pharmacological interventions. Therefore, further studies in this field are required to elucidate if targeting pericytes can treat lung diseases.

Circ-Ntrk2 acts as a miR-296-5p sponge to activate the TGF-β1/p38 MAPK pathway and promote pulmonary hypertension and vascular remodelling

BackgroundCircular RNAs (circRNAs), a novel class of non-coding RNAs, play an important regulatory role in pulmonary arterial hypertension (PAH); however, the specific mechanism is rarely studied. In this study, we aimed to discover functional circRNAs and investigate their effects and mechanisms in hypoxia-induced pulmonary vascular remodelling, a core pathological change in PAH.MethodsRNA sequencing was used to illustrate the expression profile of circRNAs in hypoxic PAH. Bioinformatics, Sanger sequencing, and quantitative real-time PCR were used to identify the ring-forming characteristics of RNA and analyse its expression. Then, we established a hypoxia-induced PAH mouse model to evaluate circRNA function in PAH by echocardiography and hemodynamic measurements. Moreover, microRNA target gene database screening, fluorescence in situ hybridisation, luciferase reporter gene detection, and western blotting were used to explore the mechanism of circRNAs.ResultsRNA sequencing identified 432 differentially expressed circRNAs in mouse hypoxic lung tissues. Our results indicated that circ-Ntrk2 is a stable cytoplasmic circRNA derived from Ntrk2 mRNA and frequently upregulated in hypoxic lung tissue. We further found that circ-Ntrk2 sponges miR-296-5p and miR-296-5p can bind to the 3′-untranslated region of transforming growth factor-β1 (TGF-β1) mRNA, thereby attenuating TGF-β1 translation. Through gene knockout or exogenous expression, we demonstrated that circ-Ntrk2 could promote PAH and vascular remodelling. Moreover, we verified that miR-296-5p overexpression alleviated pulmonary vascular remodelling and improved PAH through the TGF-β1/p38 MAPK pathway.ConclusionsWe identified a new circRNA (circ-Ntrk2) and explored its function and mechanism in PAH, thereby establishing potential targets for the diagnosis and treatment of PAH. Furthermore, our study contributes to the understanding of circRNA in relation to PAH.

Deficiency of filamin A in smooth muscle cells protects against hypoxia‑mediated pulmonary hypertension in mice.

Filamin A (FLNA) is a high molecular weight cytoskeleton protein important for cell locomotion. A relationship between FLNA mutations and pulmonary arterial hypertension (PAH) has previously been reported; however, the detailed mechanism remains unclear. The present study aimed to explore the role of FLNA in vascular smooth muscle cells during the development of PAH. Smooth muscle cell (SMC)‑specific FLNA‑deficient mice were generated and the mice were then exposed to hypoxia for 28days to build the mouse model of PAH. Human pulmonary arterial smooth muscle cells (PASMCs) were also cultured and transfected with FLNA small interfering RNA or overexpression plasmids to investigate the effects of FLNA on PASMC proliferation and migration. Notably, compared with control individuals, the expression levels of FLNA were increased in lung tissues from patients with PAH, and it was obviously expressed in the PASMCs of pulmonary arterioles. FLNA deficiency in SMCs attenuated hypoxia‑induced pulmonary hypertension and pulmonary vascular remodeling. Invitro studies suggested that absence of FLNA impaired PASMC proliferation and migration, and produced lower levels of phosphorylated (p)‑PAK‑1 and RAC1 activity. However, FLNA overexpression promoted PASMC proliferation and migration, and increased the expression levels of p‑PAK‑1 and RAC1 activity. The present study highlights the role of FLNA in pulmonary vascular remodeling; therefore, it could serve as a potential target for the treatment of PAH.

Identification of JPX-RABEP1 Pair as an Immune-Related Biomarker and Therapeutic Target in Pulmonary Arterial Hypertension by Bioinformatics and Experimental Analyses

Pulmonary arterial hypertension (PAH) is a pulmonary vascular disease characterized by pulmonary vascular remodeling and right heart enlargement the pathogenesis of PAH is complicated; no biologic-based therapy is available for the treatment of PAH, but recent studies suggest that inflammatory response and abnormal proliferation of pulmonary artery smooth muscle cells are the main pathogenic mechanism, while the role of immune-related long non-coding RNAs (lncRNAs) remains unclear. The aim of this study was to systematically analyze immune-related lncRNAs in PAH. Here, we downloaded a publicly available microarray data from PAH and control patients (GSE113439). A total of 243 up-regulated and 203 down-regulated differentially expressed genes (DEGs) were screened, and immune-related DEGs were further obtained from ImmPort. The immune-related lncRNAs were obtained by co-expression analysis of immune-related mRNAs. Then, immune-related lncRNAs-mRNAs network including 2 lncRNAs and 6 mRNAs was constructed which share regulatory miRNAs and have significant correlation. Among the lncRNA-mRNA pairs, one pair (JPX-RABEP1) was verified in the validating dataset GSE53408 and PAH mouse model. Furthermore, the immune cell infiltration analysis of the GSE113439 dataset revealed that the JPX-RABEP1 pair may participate in the occurrence and development of PAH through immune cell infiltration. Together, our findings reveal that the lncRNA-mRNA pair JPX-RABEP1 may be a novel biomarker and therapeutic target for PAH.

Salt-inducible kinase 1 deficiency promotes vascular remodeling in pulmonary arterial hypertension via enhancement of yes-associated protein-mediated proliferation

Pulmonary arterial remodeling at an early stage, including excessive proliferation and migration of smooth muscle cells, is a hallmark of pulmonary arterial hypertension (PAH). Salt-inducible kinases (SIKs) have been increasingly reported to play a key role in smooth muscle cell proliferation and phenotype switching, which may be associated with arterial remodeling. However, the potential effects of SIK1 in PAH and the underlying mechanisms have not been explored. The aim of this study was to determine whether reduced expression or inactivation of SIK1 is associated with pulmonary arterial remodeling in PAH and to elucidate whether it is related to the Hippo/Yes-associated protein (YAP) pathway. Using mouse models of PAH and hypoxia-stimulated hPASMCs, we observed that SIK1 expression was robustly reduced in lung tissues of PAH mice and hPASMCs cultured under hypoxia. In hypoxia-induced PAH mice, pharmacological SIK inhibition or AAV9-mediated specific smooth muscle SIK1 knockdown strongly aggravated pathological changes caused by hypoxia, including right ventricular hypertrophy and small pulmonary arterial remodeling. Meanwhile, in hypoxia-stimulated hPASMCs, SIK1 knockdown or inhibition promoted proliferation and migration under hypoxia, accompanied by decreased phosphorylation and increased nuclear accumulation of YAP, while SIK1 overexpression inhibited hypoxia-induced proliferation, migration and nuclear translocation of YAP in hPASMCs. YAP knockdown attenuated the increase in cell proliferation induced by HG-9-91-01 treatment or SIK1 siRNA transfection under hypoxia in hPASMCs. Here, we identified SIK1 as an antiproliferative factor in hypoxia-induced pulmonary arterial remodeling via YAP-mediated mechanisms. These results show that targeting SIK1 may be a promising therapeutic strategy for the treatment of PAH.

Pulmonary Vascular Remodeling Characteristics of Pulmonary Arterial Hypertension Mouse Model Induced by Left Pneumonectomy and Jugular Vein Injection of Monocrotaline Pyrrole

To compare and investigate the differences and characteristics of pulmonary vascular remodeling in three mouse models of pulmonary arterial hypertension (PAH) constructed by left pneumonectomy, jugular vein injection of monocrotaline pyrrole, and left pneumonectomy combined with jugular vein injection of monocrotaline pyrrole, to explore for a PAH animal model that approximates the clinical pathogenesis of PAH, and to create a model that will provide sound basis for thorough investigation into the pathogenesis of severe PAH. 59 male C57/BL mice (10-12 weeks, 24-30 g) were randomized into four groups, a control group ( n=9), a group that had left pneumonectomy (PE, n=15), a group that had jugular vein injection of monocrotaline pyrrole (MCTP, n=15), and the last group that had left pneumonectomy combined with jugular injection of monocrotaline pyrrole (P+M, n=20). To evaluate the effect of modeling and the characteristics of pulmonary vascular remodeling, hemodynamic and morphological parameters, including right ventricular systolic pressure (RVSP), right ventricle/(left ventricle plus septum) (RV/LV+S), percent of wall thickness in the pulmonary artery (WT%), muscularization of non-muscular arteries, neointima formation, and vascular obstruction score (VOS), were measured in each group. 1) Compared with those of the control group, the RVSP, RV/LV+S, WT%, and the degree of small pulmonary arteries muscularization in the P+M group were significantly increased ( P<0.01). The MCTP group had just slightly higher findings for these indicators ( P<0.05), while no significant change in these indicators was observed in the PE group ( P>0.05). 2) Neointima formation in the acinus pulmonary arteries, which caused obvious stenosis of the lumen, was observed in the P+M group, the VOS being 1.25±0.80 points ( P<0.001). In contrast, neointima formation was not observed in the MCTP group or the PE groups, the VOS being 0 point ( P>0.05). Left pneumonectomy combined with jugular intravenous injection of MCTP could induce severe PAH formation in mouse. The model provides a good simulation of neointima formation, the characteristic pathological change of clinical severe PAH.

Maresin 1 intervention reverses experimental pulmonary arterial hypertension in mice.

Pulmonary arterial hypertension (PAH) is a pulmonary vasculature obstructive disease that leads to right heart failure and death. Maresin 1 is an endogenous lipid mediator known to promote inflammation resolution. However, the effect of Maresin 1 on PAH remains unclear. The serum Maresin 1 concentration was assessed using UPLC. A mouse model of PAH was established by combining the Sugen 5416 injection and hypoxia exposure. After treatment with Maresin 1, the right ventricular systolic pressure (RVSP) and right ventricular function were measured by haemodynamic measurement and echocardiography, respectively. Vascular remodelling was evaluated by histological staining. Confocal microscopy and western blot were used to test related protein expression. In vitro cell migration, proliferation and apoptosis assays were performed in primary rat pulmonary artery smooth muscle cells (PASMCs). Western blotting and siRNA transfection were used to clarify the mechanism of Maresin 1. Endogenous serum Maresin 1 was decreased in PAH patients and mice. Maresin 1 treatment decreased RVSP and attenuated right ventricular dysfunction (RVD) in the murine PAH model. Maresin 1 reversed abnormal changes in pulmonary vascular remodelling, attenuating endothelial to mesenchymal transformation and enhancing apoptosis of α-SMA positive cells. Furthermore, Maresin 1 inhibited PASMC proliferation and promoted apoptosis by inhibiting STAT, AKT, ERK, and FoxO1 phosphorylation via LGR6. Maresin 1 improved abnormal pulmonary vascular remodelling and right ventricular dysfunction in PAH mice, targeting aberrant PASMC proliferation. This suggests Maresin 1 may have a potent therapeutic effect in vascular disease.

Caspase-8 Promotes Pulmonary Hypertension by Activating Macrophage-Associated Inflammation and IL-1β (Interleukin 1β) Production.

Macrophages are involved in the pathogenesis of pulmonary arterial hypertension (PAH). Caspase-8, an apical component of cell death pathways, is significantly upregulated in macrophages of PAH animal models. However, its role in PAH remains unclear. Caspase-8 plays a critical role in regulating inflammatory responses via inflammasome activation, cell death, and cytokine induction. This study investigated the mechanism of regulation of IL-1β (interleukin 1β) activation in macrophages by caspase-8. A hypoxia + SU5416-induced PAH mouse model and monocrotaline-induced rat model of PAH were constructed and the role of caspase-8 was analyzed. Caspase-8 and cleaved-caspase-8 were significantly upregulated in the lung tissues of SU5416 and hypoxia-treated PAH mice and monocrotaline-treated rats. Pharmacological inhibition of caspase-8 alleviated PAH compared with wild-type mice, observed as a significant reduction in right ventricular systolic pressure, ratio of right ventricular wall to left ventricular wall plus ventricular septum, pulmonary vascular media thickness, and pulmonary vascular muscularization; caspase-8 ablated mice also showed significant remission. Mechanistically, increased proliferation of pulmonary arterial smooth muscle cellss is closely associated with activation of the NLRP3 (NOD [nucleotide oligomerization domain]-, LRR [leucine-rich repeat]-, and PYD [pyrin domain]-containing protein 3) inflammasome and the IL-1β signaling pathway. Although caspase-8 did not affect extracellular matrix synthesis, it promoted inflammatory cell infiltration and pulmonary arterial smooth muscle cell proliferation via NLRP3/IL-1β activation during the development stage of PAH. Taken together, our study suggests that macrophage-derived IL-1β via caspase-8-dependent canonical inflammasome is required for macrophages to play a pathogenic role in pulmonary perivascular inflammation.

Decrease in LINC00963 attenuates the progression of pulmonary arterial hypertension via microRNA-328-3p/profilin 1 axis.

BackgroundPulmonary arterial hypertension (PAH) is a severe cardiopulmonary disease characterized by vascular hyperplasia and remodeling. Long noncoding RNA LINC00963 can regulate cell proliferation and metastasis in nonsmall cell lung cancer. However, the function of LINC00963 on PAH progression is rarely reported.MethodsQuantitative real‐time PCR was used to determine the expression levels of LINC00963, microRNA (miRNA)‐328‐3p, and profilin 1 (PFN1), as well as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF‐2), and hypoxia‐inducible factor (HIF)‐α. The protein level of PFN1 was measured by western blotting. The viability and migration of hypoxia‐induced pulmonary arterial smooth muscle cells (PASMCs) were assessed by 3‐(4, 5‐dimethyl‐2‐thiazolyl)‐2, 5‐diphenyl‐2‐h‐tetrazolium bromide, and transwell assays, respectively. The target relationships between miR‐328‐3p and LINC00963/PFN1 were confirmed by dual‐luciferase reporter assay. A PAH mouse model was conducted to explore the effects of hypoxia on cardiopulmonary functions.ResultsIn hypoxia‐induced PASMCs and PAH mouse model, high expression levels of LINC00963 and PFN1, and low expression of miR‐328‐3p, were determined. The viability, migration of hypoxia‐induced PASMCs, the expression of VEGF, FGF‐2, and HIF‐α were significantly repressed by transfection of si‐LINC00963 or miR‐328‐3p mimics. The inhibitory effects of LINC00963 silencing on cell viability, migration, and the levels of VEGF, FGF‐2, and HIF‐α were partly eliminated by miR‐328‐3p inhibitor or increasing the expression of PFN1. Hypoxia treatment increased the levels of RVSP, mPAP, and RV/(LV+S), as well as the thickness of pulmonary artery wall.ConclusionsSilencing of LINC00963 ameliorates PAH via modulating miR‐328‐3p/PFN1.

SNPs for Genes Encoding the Mitochondrial Proteins Sirtuin3 and Uncoupling Protein 2 Are Associated With Disease Severity, Type 2 Diabetes, and Outcomes in Patients With Pulmonary Arterial Hypertension and This Is Recapitulated in a New Mouse Model Lacking Both Genes.

BackgroundIsolated loss‐of‐function single nucleotide polymorphisms (SNPs) for SIRT3 (a mitochondrial deacetylase) and UCP2 (an atypical uncoupling protein enabling mitochondrial calcium entry) have been associated with both pulmonary arterial hypertension (PAH) and insulin resistance, but their collective role in animal models and patients is unknown.Methods and ResultsIn a prospective cohort of patients with PAH (n=60), we measured SNPs for both SIRT3 and UCP2, along with several clinical features (including invasive hemodynamic data) and outcomes. We found SIRT3 and UCP2 SNPs often both in the same patient in a homozygous or heterozygous manner, correlating positively with PAH severity and associated with the presence of type 2 diabetes and 10‐year outcomes (death and transplantation). To explore this mechanistically, we generated double knockout mice for Sirt3 and Ucp2 and found increasing severity of PAH (mean pulmonary artery pressure, right ventricular hypertrophy/dilatation and extensive vascular remodeling, including inflammatory plexogenic lesions, in a gene dose‐dependent manner), along with insulin resistance, compared with wild‐type mice. The suppressed mitochondrial function (decreased respiration, increased mitochondrial membrane potential) in the double knockout pulmonary artery smooth muscle cells was associated with apoptosis resistance and increased proliferation, compared with wild‐type mice.ConclusionsOur work supports the metabolic theory of PAH and shows that these mice exhibit spontaneous severe PAH (without environmental or chemical triggers) that mimics human PAH and may explain the findings in our patient cohort. Our study offers a new mouse model of PAH, with several features of human disease that are typically absent in other PAH mouse models.

Not a PERK in lungs under pressure

Physiology The vascular remodeling that occurs in pulmonary arterial hypertension (PAH) is eventually fatal because it leads to right ventricular heart failure. PAH induces the unfolded protein response (UPR) in vascular smooth muscle cells, which led Shimizu et al. to investigate the role of the UPR kinase PERK in disease pathology. Genetic ablation or pharmacological inhibition of PERK limited vascular remodeling in two different mouse models of PAH, preventing mice from developing right ventricular heart failure and improving survival. Sci. Signal. 14 , eabb3616 (2021).

Abstract 13575: Lack of Both Mitochondrial Proteins Sirt3 and Ucp2 in Mice Recapitulates Many Critical Features of Human Pulmonary Arterial Hypertension (PAH), Including Inflammatory Plexogenic Lesions, Supporting the Metabolic Theory of PAH

Loss-of function SNPs for Sirt3 (a mitochondrial deacetylase) and Ucp2 (an atypical uncoupling protein enabling mitochondrial calcium entry) have been associated with insulin resistance and obesity in humans; and patients with PAH have insulin resistance without being obese. In a cohort of PAH patients (n=60) we found these SNPs often both in the same patient in a homozygous or heterozygous manner and their presence correlated positively with the degree of PAH upon referral, the presence of type II diabetes and outcomes (death, transplantation). We generated and studied double KO mice for Sirt3 and Ucp2 using closed-chest right heart catheterization and echocardiography and found increasing severity of pulmonary hypertension (PHT) in Sirt3 +/- -Ucp2 +/ - , Sirt3 -/- -Ucp2 +/ - , Sirt3 +/- -Ucp2 -/- and Sirt3 -/- -Ucp2 -/- , associated with decreasing cardiac output and increasing right ventricular hypertrophy, dilatation and dysfunction (TAPSE), compared to wild-type (WT) mice. The LVEDP in all mice was normal. There was increasing severity of vascular remodeling with increasing levels of CD4 + cell infiltration, while Sirt3 -/- -Ucp2 +/- , Sirt3 +/- -Ucp2 -/- and Sirt3 -/- -Ucp2 -/- mice also developed frequent plexogenic lesions. In vivo and in vitro pulmonary artery smooth muscle cells (PASMC) expressed higher levels of Ki67, compared to WT mice. In vitro, the Sirt3 -/- -Ucp2 +/- , Sirt3 +/- -Ucp2 -/- and Sirt3 -/- -Ucp2 -/- PASMC exhibited more apoptosis-resistance, expressed higher nuclear levels of proliferative transcription factors (HIF1, NFATc2), exhibited decreased respiration and higher levels of glycolysis, similarly to previous reports of animal and human PAH PASMC. The Sirt3 -/- -Ucp2 -/- , but not the WT mice, also developed in vivo evidence of insulin resistance. Our work supports the metabolic theory of PAH and shows that these mice exhibit spontaneous severe PHT (without environmental or chemical triggers) in a gene dose-response dependent manner that mimics human PAH. No other mouse model of PHT has shown frequent and predictable plexogenic lesions. Our study is relevant to the PAH patients that carry loss-of-function SNPs of mitochondrial proteins and offers a new mouse model of PAH, with more features of human PAH than previous mice models.

Abstract 14532: Computational Drug Repurposing Identifies a Piperlongumine Analog That Controls a Gstp1-iscu Pathogenic Axis in Pulmonary Hypertension

Background and Hypothesis: Pulmonary arterial hypertension (PAH) is an incurable vascular disease for which chemotherapies are being considered for therapeutic development. There is no method reported to date for effective computational screening of these drugs for this disease. Big data analyses that leverage the molecular parallels between cancer and PH may define novel pathogenic mechanisms and facilitate repurposing of chemotherapies for PAH. More specifically, while functional deficiency of the iron-sulfur (Fe-S) biogenesis gene ISCU and oxidative metabolism in human pulmonary arterial endothelial cells (PAECs) is known to drive PAH, the pathogenic regulation of ISCU is not fully defined, and no tailored drugs have been identified to bolster ISCU activity. Methods and Results: We applied a computational algorithm EDDY (Evaluating Differential DependencY), which analyzes RNA sequencing data from 810 cancer cell lines exposed to 368 small molecules, in order to identify chemotherapeutics that depended upon rewired PH-related gene clusters. The top ranked drug was a piperlongumine (PL) analog (BRD2889) that was predicted to extensively rewire dependencies across PH gene clusters, mediated by ISCU. In vitro, coupling gain- and loss-of-function analyses of GSTP1 with BRD2889 exposure in PAECs, we found that BRD2889 inhibits glutathione S-transferase P1 (GSTP1), an enzyme which in turn catalyzes ISCU glutathionylation and increases its stability in hypoxia. Consequently, BRD2889 and GSTP1 knockdown phenocopy one another by increasing Fe-S-dependent Complex I activity and mitochondrial oxygen consumption while ameliorating pathogenic apoptosis. Consistent with these computational and in vitro results, in a mouse model of PAH (IL-6 transgenic mice in hypoxia), BRD2889 improved hemodynamic and molecular disease manifestations in vivo. Conclusions: Using a novel computational platform, we identified a coordinated connection between BRD342289 and GSTP1-ISCU axis, crucial to PAEC metabolism. This study offers insight to fundamental PH pathobiology and sets the stage for accelerated repurposing of chemotherapies such as BRD342289 in PH.