Monocrotaline-induced Pulmonary Arterial Hypertension In Rats Research Articles

Autophagy and ubiquitin-dependent proteolysis processes in left ventricular mass loss in pulmonary arterial hypertension

The goal of this study was to evaluate the intensity of autophagy and ubiquitin-dependent proteolysis processes occurring in myocardium of left ventricle (LV) in subsequent stages of pulmonary arterial hypertension (PAH) to determine mechanisms responsible for LV mass loss in a monocrotaline-induced PAH rat model. LV myocardium samples collected from 32 Wistar rats were analyzed in an early PAH group (n = 8), controls time-paired (n = 8), an end-stage PAH group (n = 8), and their controls (n = 8). Samples were subjected to histological analyses with immunofluorescence staining, autophagy assessment by western blotting, and evaluation of ubiquitin-dependent proteolysis in the LV by immunoprecipitation of ubiquitinated proteins. Echocardiographic, hemodynamic, and heart morphometric parameters were assessed regularly throughout the experiment. Considerable morphological and hemodynamic remodeling of the LV was observed over the course of PAH. The end-stage PAH was associated with significantly impaired LV systolic function and a decrease in LV mass. The LC3B-II expression in the LV was significantly higher in the end-stage PAH group compared to the early PAH group (p = 0.040). The measured LC3B-II/LC3B-I ratios in the end-stage PAH group were significantly elevated compared to the controls (p = 0.039). Immunofluorescence staining showed a significant increase in the abundance of LC3 puncta in the end-stage PAH group compared to the matched controls. There were no statistically significant differences in the levels of expression of all ubiquitinated proteins when comparing both PAH groups and matched controls. Autophagy may be considered as the mechanism behind the LV mass loss at the end stage of PAH.

Effects of nitric oxide inhalation on pulmonary arterial impedance: differences between normal and pulmonary hypertension male rats.

Nitric oxide (NO) inhalation improves pulmonary hemodynamics in participants with pulmonary arterial hypertension (PAH). Although it can reduce pulmonary vascular resistance (PVR) in PAH, its impact on the dynamic mechanics of pulmonary arteries and its potential difference between control and participants with PAH remain unclear. PA impedance provides a comprehensive description of PA mechanics. With an arterial model, PA impedance can be parameterized into peripheral pulmonary resistance (Rp), arterial compliance (Cp), characteristic impedance of the proximal arteries (Zc), and transmission time from the main PA to the reflection site. This study investigated the effects of inhaled NO on PA impedance and its associated parameters in control and monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) male rats (6/group). Measurements were obtained at baseline and during NO inhalation at 40 and 80 ppm. In both groups, NO inhalation decreased PVR and increased the left atrial pressure. Notably, its impact on PA impedance was frequency dependent, as revealed by reduced PA impedance modulus in the low-frequency range below 10 Hz, with little effect on the high-frequency range. Furthermore, NO inhalation attenuated Rp, increased Cp, and prolonged transmission time without affecting Zc. It reduced Rp more pronouncedly in MCT-PAH rats, whereas it increased Cp and delayed transmission time more effectively in control rats. In conclusion, the therapeutic effects of inhaled NO on PA impedance were frequency dependent and may differ between the control and MCT-PAH groups, suggesting that the effect on the mechanics differs depending on the pathological state.NEW & NOTEWORTHY Nitric oxide inhalation decreased pulmonary arterial impedance in the low-frequency range (<10 Hz) with little impact on the high-frequency range. It reduced peripheral pulmonary resistance more pronouncedly in pulmonary hypertension rats, whereas it increased arterial compliance and transmission time in control rats. Its effect on the mechanics of the pulmonary arteries may differ depending on the pathological status.

Neutrophil extracellular traps promote proliferation of pulmonary smooth muscle cells mediated by CCDC25 in pulmonary arterial hypertension

BackgroundPrevious studies have indicated that neutrophil extracellular traps (NETs) play a pivotal role in pathogenesis of pulmonary arterial hypertension (PAH). However, the specific mechanism underlying the impact of NETs on pulmonary artery smooth muscle cells (PASMCs) has not been determined. The objective of this study was to elucidate underlying mechanisms through which NETs contribute to progression of PAH.MethodsBioinformatics analysis was employed in this study to screen for potential molecules and mechanisms associated with occurrence and development of PAH. These findings were subsequently validated in human samples, coiled-coil domain containing 25 (CCDC25) knockdown PASMCs, as well as monocrotaline-induced PAH rat model.ResultsNETs promoted proliferation of PASMCs, thereby facilitating pathogenesis of PAH. This phenomenon was mediated by the activation of transmembrane receptor CCDC25 on PASMCs, which subsequently activated ILK/β-parvin/RAC1 pathway. Consequently, cytoskeletal remodeling and phenotypic transformation occur in PASMCs. Furthermore, the level of NETs could serve as an indicator of PAH severity and as potential therapeutic target for alleviating PAH.ConclusionThis study elucidated the involvement of NETs in pathogenesis of PAH through their influence on the function of PASMCs, thereby highlighting their potential as promising targets for the evaluation and treatment of PAH.

Transcriptome-Wide N6-Methyladenosine Alternations in Pulmonary Arteries of Monocrotaline-Induced Pulmonary Arterial Hypertension in Rats and Novel Therapeutic Targets.

N6-methyladenosine (m6A) is a post-transcriptional epigenetic change with transcriptional stability and functionality regulated by specific m6A-modifying enzymes. However, the significance of genes modified by m6A and enzymes specific to m6A regulation in the context of pulmonary arterial hypertension (PAH) remains largely unexplored. MeRIP-seq and RNA-seq were applied to explore variances in m6A and RNA expression within the pulmonary artery tissues of control and monocrotaline-induced PAH rats. Functional enrichments were analyzed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. To screen candidate m6A-related genes, the STRING and Metascape databases were used to construct a protein-protein interaction network followed by a real-time PCR validation of their expression. The expression level of an m6A regulator was further investigated using immunohistochemical staining, immunofluorescence, and Western blot techniques. Additionally, proliferation assays were conducted on primary rat pulmonary artery smooth muscle cells (PASMCs). We identified forty-two differentially expressed genes that exhibited either hypermethylated or hypomethylated m6A. These genes are predominantly related to the extracellular matrix structure, MAPK, and PI3K/AKT pathways. A candidate gene, centromere protein F (CENPF), was detected with increased expression in the PAH group. Additionally, we first identified an m6A reader, leucine rich pentatricopeptide repeat containing (LRPPRC), which was downregulated in the PAH rat model. The in vitro downregulation of Lrpprc mediated by siRNA resulted in the enhanced proliferation and elevated expression of Cenpf mRNA in primary rat PASMCs. Our study revealed a modified transcriptome-wide m6A landscape and associated regulatory mechanisms in the pulmonary arteries of PAH rats, potentially offering a novel target for therapeutic strategies in the future.

Gut Fungal Microbiota Alterations in Pulmonary Arterial Hypertensive Rats.

The gut microbiome's imbalance has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), yet the contribution of the gut mycobiome remains largely unclear. This study delineates the gut mycobiome profile in PAH and examines its interplay with the bacterial microbiome alterations. Fecal samples from monocrotaline-induced PAH rats and matched controls were subjected to internal transcribed spacer 1 (ITS1) sequencing for fungal community assessment and 16S ribosomal RNA (rRNA) gene sequencing for bacterial community characterization. Comparative analysis revealed no significant disparities in the overall mycobiome diversity between the PAH and control groups. However, taxonomic profiling identified differential mycobiome compositions, with the PAH group exhibiting a significant enrichment of genera such as Wallemia, unidentified_Branch02, Postia, Malassezia, Epicoccum, Cercospora, and Alternaria. Conversely, genera Xeromyces, unidentified_Plectosphaerellaceae, and Monilia were more abundant in the controls. Correlations of Malassezia and Wallemia abundance with hemodynamic parameters were observed. Indications of bidirectional fungal-bacterial community interactions were also noted. This investigation reveals distinct gut mycobiome alterations in PAH, which are intricately associated with concurrent bacterial microbiome changes, suggesting a possible contributory role of gut fungi in PAH pathophysiology. These findings underscore the potential for novel gut mycobiome-targeted therapeutic interventions in PAH management.

In vivo pharmacokinetic study of vanillic acid in monocrotaline-induced pulmonary arterial hypertension rats and its tissue distribution.

Vanillic acid (VA) is a bioactive chemical present in many food plants and fruits. It has been shown to have a protective effect on pulmonary tissues in monocrotaline-induced pulmonary arterial hypertension, as well as an intervention effect on right ventricular remodeling. The purpose of this study was to develop and test a reliable method for assessing VA utilizing ultra-performance liquid chromatography-high resolution mass spectrometry using caffeic acid as the internal standard. Across diverse substrates, the correlation coefficient for VA ranged from 0.9992 to 0.9995. The method's intraday precision was <13.53% (RSD), and its accuracy (RE) ranged from -9.88 to 4.35%. The precision across days was <13.69% (RSD), while the accuracy ranged from 2.16 to 10.94% (RE). The extraction recoveries ranged from 80.30 to 118.81%, with a lower limit of quantification of 20 ng/mL. The approach was successfully applied to pharmacokinetic and tissue distribution studies of VA in rat plasma after gavage administration, and the pharmacokinetic parameters of VA in the plasma of the monocrotaline-induced pulmonary arterial hypertension were significantly different from those of the control group.

Therapeutic potential and protective role of GRK6 overexpression in pulmonary arterial hypertension

Abnormal proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key mechanism in the development of pulmonary arterial hypertension (PAH). Signal transducer and activator of transcription 3 (STAT3) signalling plays a critical role in modulating PASMC proliferation, and G-protein-coupled receptor kinase 6 (GRK6) regulates the STAT3 pathway. However, the mechanism underlying the relationship between GRK6 and PAH remains unclear. In this study, we aimed to investigate the role of GRK6 in PAH and determine its potential as a therapeutic target. We utilised hypoxia- and SU5416-induced PAH mouse models and a monocrotaline-induced PAH rat model to analyse the involvement of GRK6. We conducted gain- and loss-of-function experiments using mouse PASMCs. Modulation of GRK6 expression was achieved via a lentiviral vector in vitro and an adeno-associated virus serotype 1 encoding GRK6 in vivo. GRK6 was significantly downregulated in the lung tissues of PAH mice and rats, predominantly in PASMCs. Knockout of GRK6 exacerbated PAH, while both therapeutic and prophylactic overexpression of GRK6 alleviated PAH, as evidenced by a reduction in right ventricular systolic pressure, right ventricular wall to left ventricular wall plus ventricular septum ratio, pulmonary vascular media thickness, and pulmonary vascular muscularisation. Mechanistically, GRK6 overexpression attenuated hypoxia-induced PASMC proliferation and STAT3 phosphorylation. Conversely, knockdown of GRK6 promoted hypoxia-induced proliferation, which was mitigated by a STAT3 inhibitor. Our findings highlight the potential protective and beneficial roles of GRK6 in PAH; we propose a lung-targeted GRK6 gene therapy utilizing adeno-associated virus serotype 1 as a potential treatment approach for patients with PAH.

Ferrostatin-1 Blunts Right Ventricular Hypertrophy and Dysfunction in Pulmonary Arterial Hypertension by Suppressing the HMOX1/GSH Signaling.

Ferroptosis plays a critical role in pulmonary arterial hypertension (PAH)-induced right ventricular (RV) dysfunction, but key genes remain largely unclear. We here identified HMOX1 as an essential ferroptosis-related differentially expressed gene in PAH by bioinformatic analysis using FerrDb, GSE119754, and GSE3675 datasets, respectively. Notably, there were marked increases in HMOX1 and iron levels in RV of monocrotaline-induced PAH rats with reduced TAPSE levels. More importantly, treatment with ferrostatin-1 effectively attenuated RV hypertrophy, remodeling, myocardial fibrosis, and dysfunction in PAH rats. In cultured H9C2 cells and primary neonatal rat cardiomyocytes, pretreatment with ferrostatin-1 and knockdown HMOX1 by siRNA strikingly blunted hypoxia-induced promotion of lipid peroxidation, ferroptosis, and cardiomyocyte injury by potentiating glutathione (GSH) and nitric oxide signaling, respectively. In summary, ferrostatin-1 attenuates RV hypertrophy, fibrosis, and dysfunction in PAH by suppressing the HMOX1/GSH signaling. Targeting HMOX1 ferroptosis signaling functions as a potential therapeutic strategy for patients with PAH.

T18/S19 diphosphorylation of myosin regulatory light chain impairs pulmonary artery relaxation in monocrotaline-induced pulmonary hypertensive rats.

Phosphorylation of Ser19 (S19-p) on the myosin regulatory light chain (MLC2) is critical for arterial contraction. It has been shown that elevated RhoA-dependent kinase (ROCK) activity or decreased MLC phosphatase (MLCP) activity leads to further phosphorylation of Thr18 (T18/S19-pp), which has been linked to vasospastic diseases. However, this phenomenon has not yet been studied in the context of pulmonary arterial hypertension (PAH). In the monocrotaline-induced PAH (PAH-MCT) rat model, we observed a significant delay in pulmonary artery (PA) relaxation following high potassium-induced contraction, which persisted even with the use of an L-type calcium channel blocker or in a calcium-free solution. Immunoblot analysis showed increased levels of both S19-p and T18/S19-pp in unstimulated PAs from PAH-MCT rats. Proteomics analysis revealed a reduction in soluble guanylate cyclase (sGC) and protein kinase G (PKG) levels, and immunoblotting confirmed decreased levels of MYPT1 (a component of MLCP) and increased ROCK in PAH-MCT. In the control PAs, the pharmacological inhibition of sGC with ODQ resulted in a prominent delay of relaxation and increased T18/S19-pp as in PAH-MCT. The delayed relaxation and the T18/S19-pp in PAH-MCT were reversed by ROCK inhibitor, Y27632, while not by membrane permeable 8-Br-cGMP. The delayed relaxation and T18/S19-diP in the ODQ-treated control PA were also reversed by Y27632. Taken together, the decreased sGC and MLCP, and increased ROCK increased T18/S19-pp, which leads to the decreased ability of PA to relax in PAH-MCT rats. PA specific inhibition of ROCK or activation of MLCP are expected to serve as potential drugs in the treatment of PAH.

Abstract 11414: Anti-Mir-7110-3p as a Therapeutic Molecule in Monocrotaline Induced Pulmonary Arterial Hypertension Rat Model

Introduction: Pulmonary arterial hypertension (PAH) is a chronic condition marked by unusually increased blood pressure in the pulmonary artery which transports blood from the heart to lungs. Advances in PAH treatment have improved PAH patients outcomes and prognosis, but still continues to be a life-threatening condition and also the current treatment strategies have failed to address the underlying cellular and molecular problems. MicroRNAs are short noncoding RNAs that suppress gene expression by binding to the 3' UTR of their target mRNA. According to our prior findings, there is evidence that miR-7110 has a prominent role in PAH. Hypothesis: In this study, we assessed the hypothesis to determine whether anti-miR-7110-3p can help to prevent PAH in monocrotaline-induced PAH (MCT-PAH) rat model via PKCδ/STAT3 signaling pathway. Methods: Pulmonary arterial smooth muscle cells (PASMCs) were transfected with anti-miR-7110-3p to investigate the effect of miR-7110-3p on PASMCs proliferation and apoptosis in in vitro condition. To induce PAH, male Sprague Dawley rats were given an intraperitoneal injection of 50 mg/kg monocrotaline. The expression of miR-7110-3p/PKCδ/STAT3 were assessed using RT-qPCR and western blotting analysis, while the activity of miR-7110-3p and its target STAT3 was determined using luciferase reporter assay. Systolic pulmonary arterial pressure was recorded using PowerLab data acquisition system and after obtaining the readings the rats were immediately sacrificed. The lower lobe of right lung tissue was collected, fixed in 4 % paraformaldehyde, sectioned and processed for histological analyses. Results: Our results demonstrated that miR-7110-3p has a proliferative and anti-apoptotic effect on PASMCs. Additionally, in vitro studies revealed that miR-7110-3p/ PKCδ/STAT3 axis in involved in PAH. Our histological analyses suggested that anti-miR-7110-3p was able to reduce the pulmonary vascular remodeling. As a result, the relationship between miR-7110-3p, STAT3 and anti-miR-7110-3p is an important step in gene expression analyses. Conclusions: In conclusion, our findings suggest that miR-7110-3p may be used as a biomarker and anti-miR-7110-3p can be used as a therapeutic molecule for the treatment of PAH.

Saluisn-β contributes to endothelial dysfunction in monocrotaline-induced pulmonary arterial hypertensive rats

BackgroundThe endothelial dysfunction and the consequent attenuated pulmonary vasodilatation are the major causes of elevated pulmonary arterial resistance and pressure in pulmonary arterial hypertension (PAH). Current study aimed to explore the effects of a TOR2A gene product, salusin-β, on endothelium-dependent vascular relaxation and the progression of PAH in monocrotaline (MCT)-induced PAH rats as well as the relevant signaling pathway. MethodsAcetylcholine (ACh)-induced dose-dependent relaxation was used to evaluate the endothelium-dependent vasodilatation function. ResultsThe salusin-β level in plasma and pulmonary artery (PA) in MCT-PAH rats were significantly increased, while the ACh-induced endothelium-dependent vasodilatation was attenuated. After salusin-β incubation or overexpression of salusin-β gene, the endothelium-dependent relaxation was further deteriorated, while anti-salusin-β IgG incubation or knockdown of salusin-β improved it in PAH rats. The superoxide anions scavenger NAC or the antioxidant apocynin inhibited the effect of salusin-β, while the SOD inhibitor DETC further enhanced it. The nitric oxide (NO) synthase inhibitor L-NAME almost blocked the effect of anti-salusin-β IgG. Silencing of salusin-β in PAH rats decreased right ventricular (RV) systolic pressure, RV hypertrophy index, NAD(P)H oxidase activity and ROS level, and increased the eNOS activity and NO level of PA. Overexpression of salusin-β played opposite roles. ConclusionsThe elevated saluisn-β level in PAH rats plays important roles in the reduction of endothelium-dependent vasodilatation and participates in the progression of PAH through stimulating NAD(P)H oxidase-ROS production and inhibiting eNOS-NO release.

Comprehensive analysis of the expression of N6-methyladenosine RNA methylation regulators in pulmonary artery hypertension.

Background: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by pulmonary vascular remodeling. The development of PAH involves N6-methyladenosine (m6A) modification. However, the functional role of m6A regulators in PAH and the underlying regulatory mechanisms remain unknown so far. Methods: Microarray data (GSE149713) for monocrotaline induced PAH (MCT-PAH) rat models were downloaded and screened for differentially expressed genes (DEGs) and m6A regulators. Next, we screened for differentially expressed m6A regulators in endothelial cells (ECs), smooth muscle cells (SMCs), fibroblasts, interstitial macrophages, NK cells, B cells, T cells, regulatory T cells (Tregs) using scRNA sequencing data. The target DEGs of m6A regulators in ECs, SMCs, fibroblasts, and Tregs were functionally annotated using the Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In addition, the cellular interaction analysis was performed to reveal the receptor—ligand pairs regulated by m6A regulators. Pseudo-time trajectory analyses were performed and a ceRNA network of lncRNAs-miRNAs-mRNAs was constructed in SMCs. Furthermore, the RNA transcriptome sequencing data for the SMCs isolated from idiopathic PAH (IPAH) patients (GSE144274) were validated for differentially expressed m6A regulators. Moreover, the HNRNPA2B1 levels in the lung samples from PAH patients and MCT-PAH were determined using immunohistochemistry. Results: The m6A regulators were observed to be dysregulated in PAH. HNRNPA2B1expression level was increased in the PASMCs of scRNAs and IPAH patients. The target DEGs of HNRNPA2B1 were enriched in the regulation of muscle cell differentiation and vasculature development in PASMCs. The HNRNPA2B1 expression levels determined were consistent with the proliferation-related and collagen synthesis-related gene COL4A1. Moreover, the predicted transcription factors (TFs) foxd2/3 and NFκB could be involved in the regulation of HNRNPA2B1. HNRNPA2B1 might be regulating SMCs proliferation and phenotypic transition via rno-miR-330–3p/TGFβR3 and rno-miR-125a-3p/slc39a1. In addition, HNRNPA2B1 was observed to be highly expressed in the lung samples from MCT-PAH rat models and patients with PAH. Conclusion: In summary, the present study identified certain key functional m6A regulators that are involved in pulmonary vascular remodeling. The investigation of m6A patterns might be promising and provide biomarkers for diagnosis and treatment of PAH in the future.

18β-Glycyrrhetinic acid ameliorates endoplasmic reticulum stress-induced inflammation in pulmonary arterial hypertension through PERK/eIF2α/NF-κB signaling.

Endoplasmic reticulum stress (ERS)-induced inflammation participates in the occurrence of pulmonary arterial hypertension (PAH) by promoting pulmonary vascular remodeling, which involved in the activation of PERK/eIF2α/NF-κB signaling pathway. 18β-Glycyrrhetinic acid (18β-GA) has been found efficacious for attenuating PAH through its anti-remodeling effects in our previous research and it remains unclear whether 18β-GA has an effect on the remodeling caused by ERS-induced inflammation. In this study, we made observations in monocrotaline-induced PAH rats and found improvement of hemodynamic and histopathological parameters, decreases in the right ventricular hypertrophy index, and alleviation of pulmonary vascular remodeling after 18β-GA administration in vivo. Moreover, 18β-GA could significantly inhibit the proliferation and DNA synthesis of human pulmonary arterial smooth muscle cells (HPASMCs) induced by platelet-derived growth factor BB. At the cellular and molecular levels, we found that 18β-GA could significantly reduce the accumulation of misfolded protein in rat lung tissue, inhibit ERS activation, reduce the expression of GRP78, p-PERK, p-eIF2α, and p-NF-κB p65, and increase IκB protein expression. 18β-GA could inhibit the migration of NF-κB into the nucleus, reduce the contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatant of HPASMCs, and reduce GRP78, p-PERK, p-eIF2α, p-NF-κB p65, TNF-α, IL-6, and MCP-1 protein expression, increase IκB protein expression in HPASMCs. According to what we observed, this study indicated that 18β-GA could treat PAH, which is related to the inhibition of PERK/eIF2α/NF-κB signaling pathway.

Effect of selective dopamine D3 and D4 receptor agonists on trunk pulmonary artery vascular reactivity from control and monocrotaline-treated rats.

Dopamine D1-like and D2-like receptors are expressed in the pulmonary arteries, however there is a little information about their effect on vascular tone in pulmonary circulation, even the vascular effect of activation of the dopamine D3 and D4 subtypes in physiological and pathological conditions such as pulmonary hypertension is unknown. The objective of this study was to evaluate the vascular response of trunk pulmonary artery rings from saline and monocrotaline-treated rats in the presence of selective dopamine receptor agonists. In trunk pulmonary artery rings with intact and denuded endothelium, cumulative concentration-response curves were performed for phenylephrine, acetylcholine, and dopamine receptor agonists (apomorphine-D2-like, SKF38393-D1, quinpirole-D2/D3, 7-OH-DPATD3, and PD168077-D4) alone and in the presence of corresponding selective dopamine receptor antagonists (SCH23390-D1, raclopride-D2/D3, U99194 maleate-D3, and L-745,870-D4). Contractile and relaxant effects generated during the activation with phenylephrine and acetylcholine, respectively, were significantly reduced in intact and denuded endothelium trunk pulmonary artery rings from monocrotaline rats in comparison with control rats. All dopamine receptor agonists, except the 7-OH-DPAT, produced significant vascular relaxation in intact trunk pulmonary artery rings precontracted with phenylephrine in both experimental groups. Also, the vascular relaxation of SKF38393, and particularly apomorphine and PD168077 was significant in denuded endothelium trunk pulmonary artery rings from control and monocrotaline groups. Furthermore, the vasorelaxation induced by these dopamine agonists was significantly reduced in pulmonary preparations from monocrotaline-treated rats in comparison to that recorded in preparations from control rats. The effect of dopamine receptor agonists decreased significantly in the presence of the corresponding antagonist in both experimental groups. The results support that dopamine D4 receptor agonist induces significant vascular relaxation, whereas dopamine D3 receptor agonist induces vasoconstriction in intact and denuded endothelium trunk pulmonary artery rings in control and monocrotaline-induced pulmonary arterial hypertension rats.

Resveratrol Preserves Mitochondrial Function By Sirt3 Activation In A Right Ventricle Dysfunction Model

<h3>Introduction</h3> Pulmonary Arterial Hypertension (PAH) leads to right ventricular (RV) failure by increasing pulmonary vasculature pressure. Previously we showed that resveratrol (RES) has anti-hypertrophic properties and improved RV function in PAH. However, underlying mechanisms are not fully understood. Of note, sirtuins modulation has been identified as critical mediators that induce cardioprotection. <h3>Objective</h3> To elucidate the contribution of sirtuins in RV protection by RES in a monocrotaline-induced PAH rat model. <h3>Methodology</h3> The Internal Committee for Care and Handling of Laboratory Animals of the Tecnologico de Monterrey approved all animal procedures (protocols 2017-006, 2019-019). Myocyte function and mitochondrial membrane potential (ΔΨm) was characterized by confocal microscopy; mitochondrial respiration was assessed using high-resolution respirometry and calcium retention capacity (CRC) by fluorescence; Western Blot and immunoprecipitation evaluated post-translational modification. Data are presented as percentages, n>3. Statistical significance was set at <i>p</i>< 0.05. PAH is prone to mitochondrial permeability transition pore (mPTP) opening, thus decreasing the mitochondrial membrane potential. The compromised cellular energetics affect cardiomyocyte function by decreasing sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity and delaying myofilament unbinding, disrupting cell relaxation. RES partially protects mitochondrial integrity by deacetylating cyclophilin-D, a critical component of the mPTP, increasing SIRT3 expression and activity and preventing mPTP opening. <h3>Results</h3> PAH is prone to mitochondrial permeability transition pore (mPTP) opening by decrease 81% CRC, thus reducing the mitochondrial membrane potential by 47%. The compromised cellular energetics showed by 26% decrease in mitochondrial oxidative phosphorylation affect cardiomyocyte function by decreasing shortening and less efficient with slower time to peak shortening (TTPS, 81%) and half relaxation (TTHR, 41%). RES partially protects mitochondrial integrity by decreasing 51% cyclophilin-D hiperacetylation, a critical component of the mPTP, and increasing 3.5 fold SIRT3 expression thus preventing mPTP opening. <h3>Conclusion</h3> Preventing CypD hyperacetylation through SIRT3 partially contributes to preserving mitochondrial function, which holds an energetic cellular capability to maintain myocyte function. These findings provide new insight into the mechanisms underlying RES protection.

High magnesium mitigates the vasoconstriction mediated by different types of calcium influx from monocrotaline-induced pulmonary hypertensive rats.

What is the central question of this study? What is the involvement of Mg2+ in mitigating the vasoconstriction in pulmonary arteries and smaller pulmonary arteries in the monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model? What are the main finding and its importance? Both store-operated Ca2+ entry- and receptor-operated Ca2+ entry-mediated vasoconstriction were enhanced in the MCT-PAH model. High magnesium inhibited vasoconstriction by directly antagonizing Ca2+ and increasing NO release, and this was more notable in smaller pulmonary arteries. Increased extracellular magnesium concentration has been shown to attenuate the endothelin-1-induced contractile response via the release of nitric oxide (NO) from the endothelium in proximal pulmonary arteries (PAs) of chronic hypoxic mice. Here, we further examined the involvement of Mg2+ in the inhibition of vasoconstriction in PAs and distal smaller pulmonary arteries (sPAs) in a monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model. The data showed that in control rats vasoconstriction in sPAs is more intense than that in PAs. In MCT-PAH rats, store-operated Ca2+ entry (SOCE)- and receptor-operated Ca2+ entry (ROCE)-mediated contraction were significantly strengthened. However, there was no upregulation of the vasoconstriction mediated by voltage-dependent calcium entry (VDCE). Furthermore, high magnesium greatly inhibited VDCE-mediated contraction in PAs rather than sPAs, which was the opposite of the ROCE-mediated contraction. Moreover, monocrotaline pretreatment partly eliminated the endothelium-dependent vasodilatation in PAs, which in sPAs, however, was still promoted by magnesium due to the increased NO release in pulmonary microvascular endothelial cells (PMVECs). In conclusion, the findings suggest that both SOCE- and ROCE-mediated vasoconstriction in the MCT-PAH model are enhanced, especially in sPAs. The inhibitory effect of high magnesium on vasoconstriction can be achieved partly by its direct role as a Ca2+ antagonist and partly by increasing NO release in PMVECs.

Effects of Crocin on CCL2/CCR2 Inflammatory Pathway in Monocrotaline-Induced Pulmonary Arterial Hypertension Rats.

Pulmonary arterial hypertension (PAH) is a malignant cardiopulmonary disease, in which pulmonary arterial remodeling is regarded as the prominent pathological feature. So far, the mechanism of PAH is still unclear, so its treatment remains a challenge. However, inflammation plays an important part in the occurrence and progression of PAH. It is well known that crocin has anti-inflammatory properties, so we investigated whether crocin could be a potential drug for the treatment of PAH rat models. Rats injected subcutaneously with monocrotaline (MCT) were treated with crocin via a gastric tube daily for four weeks. The results showed that crocin treatment significantly reduced the right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) in the PAH rat models. Moreover, crocin treatment reduced the proliferation of pulmonary arteriole smooth muscle cells (PASMCs). In addition, crocin treatment not only relieved inflammatory cell infiltration and collagen fiber hyperplasia in the lung and right ventricle, but also decreased the expression of the CCL2/CCR2 inflammatory pathway in the lung of PAH rat models. Furthermore, crocin treatment reduced the inflammatory cytokines and oxidative stress responses. In summary, crocin may play a protective role in MCT-induced PAH rats by alleviating inflammatory response, improving pulmonary arterial remodeling, and preventing PAH. Therefore, crocin as a new treatment for PAH may be quite worthy of consideration.

Profiling and Molecular Mechanism Analysis of Long Non-Coding RNAs and mRNAs in Pulmonary Arterial Hypertension Rat Models.

Pulmonary arterial hypertension (PAH) is an immune-mediated disease with poor prognosis and associated with various inflammatory immune diseases. In fact, its pathogenesis is far from clear. Although long non-coding RNAs (lncRNAs) have been implicated in PAH, the molecular mechanisms remain largely unknown. For the first time, in lungs of monocrotaline-induced PAH rat models, we simultaneously detected the expression profiles of lncRNAs and mRNAs by high-throughput sequencing, and explored their roles with bioinformatics analysis and cell assay to discover more potential pathogenesis about PAH. Our data identified that a total of 559 lncRNAs and 691 mRNAs were differentially expressed in lungs during the pathogenesis of PAH. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that these dysregulated lncRNAs and mRNAs participated in important biological processes and pathways of PAH, among which inflammatory and immune responses represented the chief enriched pathway. The lncRNA-mRNA co-expression network was developed to uncover the hidden interactions between lncRNAs and mRNAs. Further, the expression levels of lncRNAs (NONRATT018084.2, NONRATT009275.2, NONRATT007865.2, and NONRATT026300.2) and mRNAs (LGALS3, PDGFC, SERPINA1, and NFIL3) were confirmed using quantitative real-time PCR. In the end, lncRNA NONRATT009275.2 could facilitate macrophage polarization to M2 type and be involved in inflammatory immune response. In conclusion, this study provided candidate drug targets and potential roles on lncRNAs in the pathogenesis of PAH, and several key regulatory genes were identified, which laid the initial foundation for further mechanism study in PAH.

Downregulation of Soluble Guanylate Cyclase and Protein Kinase G in the Pulmonary Artery Leads to the Sensitization to Thromboxane A2 in the Monocrotaline‐induced Pulmonary Hypertensive Rats

Thromboxane A2(TXA2) relates to physiological and pathophysiological conditions of pulmonary artery (PA). Here, we examined the role of TXA2 receptor (TP)-mediated signaling in the pathophysiology of pulmonary arterial hypertension (PAH). The responsiveness to contractile agonist in PA could be determined by signals related to vasodilation such as nitric oxide (NO), soluble guanylate cyclase (sGC) and cGMP-dependent kinase (PKG) pathway. In this study, the TP receptor agonist (U46619)-induced contraction and modulatory role of NO/cGMP signaling were investigated in a monocrotaline-induced PAH rat model (PAH-MCT). PA from PAH-MCT exhibited higher sensitivity to U46619, that is decreased EC50. Lower expression of eNOS, sGC, and PKG were observed while higher expression of RhoA-dependent kinase 2 (ROCK2) was revealed by immunoblot analysis in the PA from PAH-MCT and control. The higher sensitivity to U46619 in PAH-MCT was ameliorated by 8-Br-cGMP, a membrane-permeable cGMP analogue, but not by NO donor, sodium nitroprusside (SNP 30 µM). In contrast, in the control PA, sGC inhibition by 1H- [1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) 10 µM lowered the threshold of U46619-induced contraction. In the presence of ODQ, SNP treatment did not present its regulatory effect whereas the addition of 8-Br-cGMP lowered the sensitivity to U46619. The inhibition of ROCK by Y-27632 also lowered the sensitivity to U46619 in both control and PAH-MCT. The study suggests that the attenuation of NO/cGMP signaling and the upregulation of ROCK2 increase the sensitivity to TXA2 in the PAH animal, which might have pathophysiological implications in patients with PAH.

Dual-Functional MN-08 Attenuated Pulmonary Arterial Hypertension Through Vasodilation and Inhibition of Pulmonary Arterial Remodeling.

[Figure: see text].