Blockade of ZFX Alleviates Hypoxia-Induced Pulmonary Vascular Remodeling by Regulating the YAP Signaling.

High expression of the zinc finger X-chromosomal protein (ZFX) correlates with proliferation, aggressiveness, and development in many types of cancers. In the current report, we investigated the efficacy of ZFX in mouse pulmonary artery smooth muscle cells (PASMCs) proliferation during pulmonary arterial hypertension (PAH). PASMCs were cultured in hypoxic conditions. Real-time PCR and western blotting were conducted to detect the expression of ZFX. Cell proliferation, apoptosis, migration, and invasion were, respectively, measured by CCK-8, flow cytometry, wound scratchy, and transwell assays. Glycolytic ability was validated by the extracellular acidification rate and oxygen consumption rate. Transcriptome sequencing technology was used to explore the genes affected by ZFX knockdown. Luciferase and chromatin immunoprecipitation assays were utilized to verify the possible binding site of ZFX and YAP1. Mice were subjected to hypoxia for 21 days to induce PAH. The right ventricular systolic pressure (RVSP) was measured and ratio of RV/LV + S was calculated. The results show that ZFX was increased in hypoxia-induced PASMCs and mice. ZFX knockdown inhibited the proliferation, migration, and invasion of PASMC. Using RNA sequencing, we identify glycolysis and YAP as a key signaling of ZFX. ZFX knockdown inhibited Glycolytic ability. ZFX strengthened the transcription activity of YAP1, thereby regulating the YAP signaling. YAP1 overexpression reversed the effect of ZFX knockdown on hypoxia-treated PASMCs. In conclusion, ZFX knockdown protected mice from hypoxia-induced PAH injury. ZFX knockdown dramatically reduced RVSP and RV/(LV + S) in hypoxia-treated mice.

A Novel Protective Role for Matrix Metalloproteinase-8 in the Pulmonary Vasculature.

Rationale: Mechanical signaling through cell-matrix interactions plays a major role in progressive vascular remodeling in pulmonary arterial hypertension (PAH). MMP-8 (matrix metalloproteinase-8) is an interstitial collagenase involved in regulating inflammation and fibrosis of the lung and systemic vasculature, but its role in PAH pathogenesis remains unexplored. Objectives: To evaluate MMP-8 as a modulator of pathogenic mechanical signaling in PAH. Methods: MMP-8 levels were measured in plasma from patients with pulmonary hypertension (PH) and controls by ELISA. MMP-8 vascular expression was examined in lung tissue from patients with PAH and rodent models of PH. MMP-8-/- and MMP-8+/+ mice were exposed to normobaric hypoxia or normoxia for 4-8 weeks. PH severity was evaluated by right ventricular systolic pressure, echocardiography, pulmonary artery morphometry, and immunostaining. Proliferation, migration, matrix component expression, and mechanical signaling were assessed in MMP-8-/- and MMP-8+/+ pulmonary artery smooth muscle cells (PASMCs). Measurements and Main Results: MMP-8 expression was significantly increased in plasma and pulmonary arteries of patients with PH compared with controls and induced in the pulmonary vasculature in rodent PH models. Hypoxia-exposed MMP-8-/- mice had significant mortality, increased right ventricular systolic pressure, severe right ventricular dysfunction, and exaggerated vascular remodeling compared with MMP-8+/+ mice. MMP-8-/- PASMCs demonstrated exaggerated proliferation and migration mediated by altered matrix protein expression, elevated integrin-ß3 levels, and induction of FAK (focal adhesion kinase) and downstream YAP (Yes-associated protein)/TAZ (transcriptional coactivator with PDZ-binding motif) activity. Conclusions: MMP-8 is a novel protective factor upregulated in the pulmonary vasculature during PAH pathogenesis. MMP-8 opposes pathologic mechanobiological feedback by altering matrix composition and disrupting integrin-ß3/FAK and YAP/TAZ-dependent mechanical signaling in PASMCs.

Bioactive Compounds From Coptidis Rhizoma Alleviate Pulmonary Arterial Hypertension by Inhibiting Pulmonary Artery Smooth Muscle Cells' Proliferation and Migration.

ABSTRACT: Pulmonary arterial hypertension (PAH) is a devastating disorder characterized by excessive proliferation and vasoconstriction of small pulmonary artery vascular smooth muscle cells (PASMCs). Coptidis rhizoma (CR) because of the complexity of the components, the underlying pharmacological role and mechanism of it on PAH remains unknown. In this article, the network pharmacological analysis was used to screen the main active constituents of CR and the molecular targets that these constituents act on. Then, we evaluated the importance of berberine and quercetin (biologically active components of CR) on the proliferation and migration of PASMCs and vascular remodeling in experimental models of PAH. Our results showed that berberine and quercetin effectively inhibited the proliferation and migration of hypoxia-induced PASMCs in a manner likely to be mediated by the suppression of MAPK1, NADPH oxidase 4 (NOX4), and cytochrome P450 1B1 (CYP1B1) expression. Furthermore, berberine and quercetin treatment attenuates pulmonary hypertension, reduces right ventricular hypertrophy, and improves pulmonary artery remodeling in monocrotaline-induced pulmonary hypertension in rat models. In conclusion, this research demonstrates CR might be a promising treatment option for PAH, and the network pharmacology approach can be an effective tool to reveal the potential mechanisms of Chinese herbal medicine.

The Role of Regulatory T Cells in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a chronic, incurable condition characterized by pulmonary vascular remodeling, perivascular inflammation, and right heart failure. Regulatory T cells (Tregs) stave off autoimmunity, and there is increasing evidence for their compromised activity in the inflammatory milieu of PAH. Abnormal Treg function is strongly correlated with a predisposition to PAH in animals and patients. Athymic Treg-depleted rats treated with SU5416, an agent causing pulmonary vascular injury, develop PAH, which is prevented by infusing missing CD4+CD25highFOXP3+ Tregs. Abnormal Treg activity may also explain why PAH disproportionately affects women more than men. This mini review focuses on the role of Tregs in PAH with a special view to sexual dimorphism and the future promise of Treg therapy.

Fetal Gene Reactivation in Pulmonary Arterial Hypertension: GOOD, BAD, or BOTH?

Pulmonary arterial hypertension is a debilitating chronic disorder marked by the progressive obliteration of the pre-capillary arterioles. This imposes a pressure overload on the right ventricle (RV) pushing the latter to undergo structural and mechanical adaptations that inexorably culminate in RV failure and death. Thanks to the advances in molecular biology, it has been proposed that some aspects of the RV and pulmonary vascular remodeling processes are orchestrated by a subversion of developmental regulatory mechanisms with an upregulation of a suite of genes responsible for the embryo's early growth and normally repressed in adults. In this review, we present relevant background regarding the close relationship between overactivation of fetal genes and cardiopulmonary remodeling, exploring whether the reawakening of developmental factors plays a causative role or constitutes a protective mechanism in the setting of PAH.

Penetrance of Severe Pulmonary Arterial Hypertension in Response to Vascular Endothelial Growth Factor Receptor 2 Blockade in a Genetically Prone Rat Model Is Reduced by Female Sex.

Background We have previously reported important strain differences in response to SU5416 (SU, a vascular endothelial growth factor receptor 2 inhibitor) in rats and have identified a specific colony of Sprague-Dawley rats that are hyperresponsive (SDHR) to SU alone and develop severe pulmonary arterial hypertension (PAH) with a single injection of SU, even in the absence of hypoxia. Interestingly, SDHR rats exhibit incomplete penetrance of the severe PAH phenotype with an "all-or-none" response to SU alone, which provides a unique opportunity to assess the influence of female sex and sex hormones on susceptibility to PAH after endothelial injury in a genetically prone model. Methods and Results SDHR rats were injected with SU (20 mg/kg SC) and, in the absence of hypoxia, 72% of male but only 27% of female rats developed severe PAH at 7 weeks, which was associated with persistent endothelial cell apoptosis. This sex difference in susceptibility for severe PAH was abolished by ovariectomy. Estradiol replacement, beginning 2 days before SU (prevention), inhibited lung endothelial cell apoptosis and completely abrogated severe PAH phenotype in both male and ovariectomized female rats, while progesterone was only protective in ovariectomized female rats. In contrast, delayed treatment of SDHR rats with established PAH with estradiol or progesterone (initiated at 4 weeks post-SU) failed to reduce lung endothelial cell apoptosis or improve PAH phenotype. Conclusions Female sex hormones markedly reduced susceptibility for the severe PAH phenotype in response to SU alone in a hyperresponsive rat strain by abolishing SU-induced endothelial cell apoptosis, but did not reverse severe PAH in established disease.

The incidence rate of pulmonary arterial hypertension and scleroderma renal crisis in systemic sclerosis patients with digital ulcers on endothelin antagonist receptors (ERAs) and phosphodiesterase-5 inhibitors (PDE5i).

INTRODUCTION: Endothelin antagonist receptors (ERAs) and phosphodiesterase-5 inhibitors (PDE5i) are beneficial in pulmonary arterial hypertension (PAH) and digital ulcers (DU) and prevent from DU recurrences. Our study aimed to determine the difference in the incidence rate of PAH and scleroderma renal crisis (SRC) in patients with SSc and DU (SSc-DU) under ERAs/PDE5i or without treatment. METHODS: We conducted a retrospective cohort study including SSc-DU patients from the Spanish Scleroderma Registry (RESCLE). The primary outcome was the incidence rate of PAH and SRC in patients under ERAs/PDE5i or not. RESULTS: Some 544 patients out of 1817 (29.9%) in the RESCLE database had DU, 221 (40.6%) under ERAs/PDE5i and 323 (59.4%) not. The incidence rate (95% CI) difference between patients under treatment or not under did not reach statistical significance in PAH [-0.1 (-4.8, 4.69), P = 0.988] or in SRC [0.7 (-2.2, 3.7), P = 0.620]. However, the time from the first DU to the diagnosis of SRC was delayed in treated patients [mean (s.d.) 7.6 (5.8) years vs 2.9 (5.3); P = 0.021]. The dcSSc subset was more prevalent in the treatment group (36 vs 26%; P = 0.018), along with anti-topoisomerase I antibodies (34 vs 18%; P < 0.001) and tendon friction rubs (12 vs 6%; P = 0.038), whereas the lcSSc subset was more prevalent in the no-treatment group (57 vs 66%; P = 0.031) along with ACA (37 vs 46%; P = 0.031). CONCLUSION: There was no difference in the incidence rate of PAH and SRC between groups. However, treatment with ERAs and/or PDE5i appeared to delay the occurrence of SRC.

Preventive treatment with ginsenoside Rb1 ameliorates monocrotaline-induced pulmonary arterial hypertension in rats and involves store-operated calcium entry inhibition.

CONTEXT: Ginsenoside Rb1, the main active ingredient of ginseng, exhibits ex vivo depression of store-operated calcium entry (SOCE) and related vasoconstriction in pulmonary arteries derived from pulmonary hypertension (PH) rats. However, the in vivo effects of ginsenoside Rb1 on PH remain unclear. OBJECTIVE: This study explored the possibility of using ginsenoside Rb1 as an in vivo preventive medication for type I PH, i.e., pulmonary arterial hypertension (PAH), and potential mechanisms involving SOCE. MATERIALS AND METHODS: Male Sprague-Dawley rats (170-180 g) were randomly divided into Control, MCT, and MCT + Rb1 groups (n = 20). Control rats received only saline injection. Rats in the MCT + Rb1 and MCT groups were intraperitoneally administered single doses of 50 mg/kg monocrotaline (MCT) combined with 30 mg/kg/day ginsenoside Rb1 or equivalent volumes of saline for 21 consecutive days. Subsequently, comprehensive parameters related to SOCE, vascular tone, histological changes and hemodynamics were measured. RESULTS: Ginsenoside Rb1 reduced MCT-induced STIM1, TRPC1, and TRPC4 expression by 35.00, 31.96, and 32.24%, respectively, at the protein level. SOCE-related calcium entry and pulmonary artery contraction decreased by 162.6 nM and 71.72%. The mean pulmonary artery pressure, right ventricle systolic pressure, and right ventricular mass index decreased by 19.5 mmHg, 21.6 mmHg, and 39.50%. The wall thickness/radius ratios decreased by 14.67 and 17.65%, and the lumen area/total area ratios increased by 18.55 and 15.60% in intrapulmonary vessels with 51-100 and 101-150 µm o.d. CONCLUSION: Ginsenoside Rb1, a promising candidate for PH prevention, inhibited SOCE and related pulmonary vasoconstriction, and relieved MCT-induced PAH in rats.

Niclosamide attenuates lung vascular remodeling in experimental pulmonary arterial hypertension.

Despite advances in medical therapy, pulmonary arterial hypertension (PAH) remains an inexorably progressive and highly lethal disease. Signal transducer and activator of transcription (STAT)-3 is one of the main intracellular transcription factors implicated in PAH vascular remodeling. We hypothesized that niclosamide, a STAT3 inhibitor, would reduce vascular remodeling in an established pulmonary arterial hypertension model, thus enhancing cardiac function. Male Wistar rats were treated either with monocrotaline (60 mg/kg), to induce PAH, or saline (C group) by intraperitoneal injection. On day 14, PAH animals were randomly assigned to receive oral (1) saline (PAH-SAL); (2) niclosamide (75 mg/kg/day) (PAH-NICLO); (3) sildenafil (20 mg/kg/day) (PAH-SIL); or (4) niclosamide + sildenafil (PAH-NICLO + SIL), once daily for 14 days. On day 28, right ventricular systolic pressure was lower in all treated groups compared to PAH-SAL. Pulmonary vascular collagen content was lower in PAH-NICLO (37 ± 3%) and PAH-NICLO + SIL (37 ± 6%) compared to PAH-SAL (68 ± 4%), but not in PAH-SIL (52 ± 1%). CD-34, an endothelial cell marker, was higher, while vimentin, a mesenchymal cell marker, was lower in PAH-NICLO and PAH-NICLO + SIL compared to PAH-SAL, suggesting attenuation of endothelial-mesenchymal transition. Expression of STAT3 downstream targets such as transforming growth factor (TGF)-ß, hypoxia-inducible factor (HIF)-1, and provirus integration site for Moloney murine leukemia virus (PIM-1) in lung tissue was reduced in PAH-NICLO and PAH-NICLO + SIL compared to PAH-SAL. In conclusion, niclosamide, with or without sildenafil, mitigated vascular remodeling and improved right ventricle systolic pressure. This new role for a well-established drug may represent a promising therapy for PAH.

The newborn sheep translational model for pulmonary arterial hypertension of the neonate at high altitude.

Chronic hypoxia during gestation induces greater occurrence of perinatal complications such as intrauterine growth restriction, fetal hypoxia, newborn asphyxia, and respiratory distress, among others. This condition may also cause a failure in the transition of the fetal to neonatal circulation, inducing pulmonary arterial hypertension of the neonate (PAHN), a syndrome that involves pulmonary vascular dysfunction, increased vasoconstrictor tone and pathological remodeling. As this syndrome has a relatively low prevalence in lowlands (~7 per 1000 live births) and very little is known about its prevalence and clinical evolution in highlands (above 2500 meters), our understanding is very limited. Therefore, studies on appropriate animal models have been crucial to comprehend the mechanisms underlying this pathology. Considering the strengths and weaknesses of any animal model of human disease is fundamental to achieve an effective and meaningful translation to clinical practice. The sheep model has been used to study the normal and abnormal cardiovascular development of the fetus and the neonate for almost a century. The aim of this review is to highlight the advances in our knowledge on the programming of cardiopulmonary function with the use of high-altitude newborn sheep as a translational model of PAHN.

In vivo miR-138-5p inhibition alleviates monocrotaline-induced pulmonary hypertension and normalizes pulmonary KCNK3 and SLC45A3 expression.

BACKGROUND: The pathogenesis of pulmonary arterial hypertension (PAH) involves many signalling pathways. MicroRNAs are potential candidates involved in simultaneously coordinating multiple genes under such multifactorial conditions. METHODS AND RESULTS: MiR-138-5p is overexpressed in pulmonary arterial smooth muscle cells (PASMCs) from PAH patients and in lungs from rats with monocrotaline-induced pulmonary hypertension (MCT-PH). MiR-138-5p is predicted to regulate the expression of the potassium channel KCNK3, whose loss is associated with the development and progression of PAH. We hypothesized that, in vivo, miR-138-5p inhibition would restore KCNK3 lung expression and subsequently alleviate PAH. Nebulization-based delivery of anti-miR-138-5p to rats with established MCT-PH significantly reduced the right ventricular systolic pressure and significantly improved the pulmonary arterial acceleration time (PAAT). These haemodynamic improvements were related to decrease pulmonary vascular remodelling, lung inflammation and pulmonary vascular cell proliferation in situ. In vivo inhibition of miR-138-5p restored KCNK3 mRNA expression and SLC45A3 protein expression in the lungs. CONCLUSIONS: We confirmed that in vivo inhibition of miR-138-5p reduces the development of PH in experimental MCT-PH. The possible curative mechanisms involve at least the normalization of lung KCNK3 as well as SLC45A3 expression.

Dihydroartemisinin Attenuates Pulmonary Hypertension Through Inhibition of Pulmonary Vascular Remodeling in Rats.

Pulmonary arterial hypertension (PAH) is a malignant disease characterized by pulmonary arterial remodeling because of the abnormal proliferation and migration of pulmonary arterial smooth muscle cells. Dihydroartemisinin (DHA), an artemisinin derivative used to treat malaria, is able to inhibit fibrosis, neovascularization, and tumor proliferation. In this study, we hypothesized that DHA can be beneficial in treating PAH. To test this hypothesis, a rat model of pulmonary hypertension induced with monocrotaline (MCT) was used. Compared with MCT treatment alone, treatment with 50 or 100 mg/kg DHA significantly reduced the mean pulmonary arterial pressure (30.11 ± 2.48 mm Hg vs. 21.35 ± 3.04 mm Hg and 19.18 ± 1.98 mm Hg, respectively, both P < 0.01), right ventricular transverse diameter (4.36 ± 0.41 mm vs. 3.72 ± 0.24 mm and 3.67 ± 0.27 mm, respectively, both P < 0.01), pulmonary artery medial wall thickness (57.93 ± 11.14% vs. 34.45 ± 4.39% and 25.01 ± 6.66%, respectively, both P < 0.01), and increased tricuspid annular plane systolic excursion (1.34 ± 0.17 mm vs. 1.62 ± 0.3 mm and 1.62 ± 0.16 mm, respectively, both P < 0.05). We also found that DHA inhibited platelet-derived growth factor-BB-mediated pulmonary arterial smooth muscle cells proliferation and migration in a dose-dependent manner. Moreover, DHA downregulated ß-catenin levels while upregulating the levels of axis inhibition protein 2 (Axin2) and glycogen synthase kinase 3ß (GSK-3ß). Our findings suggest that DHA, which may be a potential candidate for PAH therapy, attenuates experimental pulmonary hypertension possibly by inhibiting pulmonary vascular remodeling.

Contribution of reactive oxygen species via the OXR1 signaling pathway in the pathogenesis of monocrotaline-induced pulmonary arterial hypertension: The protective role of Crocin.

AIM: Pulmonary arterial hypertension (PAH) identified by progressive increase in pulmonary vascular resistance and pressure, ultimately leading to right ventricular failure and sudden death. Oxidation resistance 1 (OXR1) and its downstream target genes has a pivotal role for defense against oxidative stress. But its molecular function is unknown in respiratory system disorders. This study designed to determine whether PAH associated with oxidative stress and OXR1 signaling pathway modulation. Also, Crocin co-treatment evaluated to determine the possible role and mechanism in pulmonary arterial hypertension. MAIN METHOD: The PAH model was induced by a single dose of MCT. It was given intraperitoneal administration of Crocin or saline for 21 consecutive days the other groups in this study. In the last day of experiment, hemodynamic parameter and right ventricular hypertrophy was evaluated as PAH index. The expression levels of OXR1, P21 and Nrf2 genes were detected through RT-PCR. Moreover, oxidative stress index and antioxidant capacity were measured and histological examination were used to determine the lung tissue injuries. KEY FINDINGS: Results of the current study demonstrated that the OXR1 and P21 gene expression significantly decrease in PAH which is associated with increase of lipid peroxidation and decrease antioxidant capacity in lung tissue. Crocin co-treatment significantly improved the hemodynamic, oxidative stress biomarkers and histological data of the PAH rats, which associated with increase of OXR1 and its downstream target genes. SIGNIFICANCE: This report reveals the critical role of OXR1 in pathogenesis of oxidative stress-related pulmonary disease. Current experiment also provides evidence that Crocin has a protective effect against MCT-induced pulmonary arterial hypertension by modulation of OXR1 signaling pathway in rats.

Growth Factor Midkine Aggravates Pulmonary Arterial Hypertension via Surface Nucleolin.

Pulmonary arterial hypertension (PAH) is a progressive fatal disease caused by pulmonary arterial remodeling. Midkine regulates cell proliferation and migration, and it is induced by hypoxia, but its roles in pulmonary arterial remodeling remain unclear. Serum midkine levels were significantly increased in PAH patients compared with control patients. Midkine expression was increased in lungs and sera of hypoxia-induced PAH mice. Hypoxia-induced pulmonary arterial remodeling and right ventricular hypertrophy were attenuated in midkine-knockout mice. Midkine-induced proliferation and migration of pulmonary arterial smooth muscle cells (PASMC) and epidermal growth factor receptor (EGFR) signaling were significantly increased under hypoxia, which also induced cell-surface translocation of nucleolin. Nucleolin siRNA treatment suppressed midkine-induced EGFR activation in vitro, and nucleolin inhibitor AS1411 suppressed proliferation and migration of PASMC induced by midkine. Furthermore, AS1411 significantly prevented the development of PAH in Sugen hypoxia rat model. Midkine plays a crucial role in PAH development through interaction with surface nucleolin. These data define a role for midkine in PAH development and suggest midkine-nucleolin-EGFR axis as a novel therapeutic target for PAH.

Resveratrol Prevents Right Ventricle Remodeling and Dysfunction in Monocrotaline-Induced Pulmonary Arterial Hypertension with a Limited Improvement in the Lung Vasculature.

Pulmonary arterial hypertension (PAH) is a life-threatening disease that is characterized by an increase in pulmonary vascular pressure, leading to ventricular failure and high morbidity and mortality. Resveratrol, a phenolic compound and a sirtuin 1 pathway activator, has known dietary benefits and is used as a treatment for anti-inflammatory and cardiovascular diseases. Its therapeutic effects have been published in the scientific literature; however, its benefits in PAH are yet to be precisely elucidated. Using a murine model of PAH induced by monocrotaline, the macroscopic and microscopic effects of a daily oral dose of resveratrol in rats with PAH were evaluated by determining its impact on the lungs and the right and left ventricular function. While most literature has focused on smooth muscle cell mechanisms and lung pathology, our results highlight the relevance of therapy-mediated improvement of right ventricle and isolated cardiomyocyte physiology in both ventricles. Although significant differences in the pulmonary architecture were not identified either micro- or macroscopically, the effects of resveratrol on right ventricular function and remodeling were observed to be beneficial. The values for the volume, diameter, and contractility of the right ventricular cardiomyocytes returned to those of the control group, suggesting that resveratrol has a protective effect against ventricular dysfunction and pathological remodeling changes in PAH. The effect of resveratrol in the right ventricle delayed the progression of findings associated with right heart failure and had a limited positive effect on the architecture of the lungs. The use of resveratrol could be considered a future potential adjunct therapy, especially when the challenges to making a diagnosis and the current therapy limitations for PAH are taken into consideration.

Impact of Nutrition on Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by sustained vasoconstriction, vascular remodeling, inflammation, and in situ thrombosis. Although there have been important advances in the knowledge of the pathophysiology of PAH, it remains a debilitating, limiting, and rapidly progressive disease. Vitamin D and iron deficiency are worldwide health problems of pandemic proportions. Notably, these nutritional alterations are largely more prevalent in PAH patients than in the general population and there are several pieces of evidence suggesting that they may trigger or aggravate disease progression. There are also several case reports associating scurvy, due to severe vitamin C deficiency, with PAH. Flavonoids such as quercetin, isoflavonoids such as genistein, and other dietary polyphenols including resveratrol slow the progression of the disease in animal models of PAH. Finally, the role of the gut microbiota and its interplay with the diet, host immune system, and energy metabolism is emerging in multiple cardiovascular diseases. The alteration of the gut microbiota has also been reported in animal models of PAH. It is thus possible that in the near future interventions targeting the nutritional status and the gut dysbiosis will improve the outcome of these patients.

Hypoxia induces an increase in intestinal permeability and pulmonary arterial pressures in neonatal Holstein calves despite feeding the flavonoid rutin.

The purposes of this study were to determine whether the naturally occurring flavonoid quercetin, as its glucorhamnoside rutin, reduces intestinal permeability and susceptibility to hypoxia-induced pulmonary hypertension in neonatal Holstein calves. A 2 × 2 between-subjects factorial design was conducted using Holstein steers (n = 16). Factors included oxygen level (simulated altitude of 4,572 m vs. 975 m) and quercetin supplementation as its glucorhamnoside rutin (4 g of quercetin per day vs. 0 g per day). Two days after arrival (d 0 of study) the calves were blocked by body mass into treatment groups, and treatments were initiated. Pulmonary arterial pressure, echocardiography, and serum concentrations of orally administered lactulose (0.45 g/kg) and mannitol (0.15 g/kg) were measured on d 12, 13, and 14, respectively. Calves were euthanized on d 15 and pulmonary tissues collected for semiquantitative scoring of histological lesions. Data were analyzed using linear regression, generalized estimating equations, and 2-sample proportion tests. Hypoxia, but not rutin, was found to be associated with intestinal permeability. The lactulose-mannitol ratio was 0.54 ± 0.13 (standard error) in hypoxic calves and 0.02 ± 0.13 in normoxic controls. Hypoxia increased mean pulmonary arterial pressure. Calves fed rutin under hypoxic conditions tended to have a lower mean pulmonary arterial pressure (59 ± 7 mmHg) than control calves (80 ± 7 mmHg) but similar pressures under normoxic conditions. Paradoxically, however, a greater proportion of calves fed rutin had histological evidence of pulmonary arteriolar medial hypertrophy and adventitial hyperplasia than did controls. In conclusion, the findings of this study indicate that hypoxia increased intestinal permeability in neonatal calves. The flavonoid quercetin, as its glucorhamnoside rutin, had no protective effect on intestinal permeability, and, although it tended to reduce the severity of hypoxia-induced pulmonary hypertension, a greater proportion of calves fed rutin had histological lesions consistent with pulmonary arteriolar remodeling.

Preventive Effect and Mechanism of Ethyl Acetate Extract of Sceptridium ternatum in Monocrotaline-Induced Pulmonary Arterial Hypertension.

OBJECTIVE: To observe the effect and molecular mechanism of ethyl acetate extract of Sceptridium ternatum (STE) on the monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). METHODS: The main chemical components of Sceptridium ternatum were determined, and the effects in PAH rats were observed. A total of 140 Sprague Dawley rats were randomly and equally divided into the normal group, the model group, the Bosentan group, and the STE groups (2.5, 5, 10 g/kg) by the random number table method. The characteristic indicators of PAH were measured, and immunohistochemistry was used to observe the lung tissue of rats. Morphological changes of the lung tissue were observed under the light microscope. RESULTS: Compared with the normal group, rats in the model group showed a significant increase in right ventricular free wall thickness (RVFWT), mean pulmonary arterial pressure (mPAP), mean right ventricular pressure (mRVP), max right ventricular pressure (max RVP), weight of right ventricle (RV), and lung index (LI), while a significant decrease in pulmonary artery acceleration time (PAAT, P<0.01). Compared with the model group, rats treated with STE had a significant decrease of RVFWT, mPAP, mRVP, max RVP, and RV, while a significant increase of PAAT (P<0.01). After injection of MCT, nuclear factor- κB (NF- κB) p65 and α -smooth muscle actin (α -SMA) expression levels were up-regulated, and on the contrary, the treatment groups showed a significant down-regulation without dose-dependent trend. CONCLUSIONS: STE can relieve the PAH in rats. STE may relieve pulmonary vascular disease and pulmonary injury by down-regulating the expression of NF- κB p65 and α -SMA.

Statin treatment prevents the development of pulmonary arterial hypertension in a nonhuman primate model of HIV-associated PAH.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by pulmonary vascular remodeling, elevated pulmonary arterial pressure, and right heart failure. Human immunodeficiency virus (HIV)-infected individuals have a higher incidence of PAH than the non-HIV infected population and evidence suggests a role for systemic and pulmonary inflammation in the pathogenesis of HIV-associated PAH. Due to their pleiotropic effects, including immune-modulatory and anti-inflammatory effects, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been considered for the treatment of PAH, with conflicting results. The effects of statins on HIV-associated PAH have not been specifically evaluated. We have developed a non-human primate (NHP) model of HIV-associated PAH that closely mimics HIV-PAH using simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta). We determined that treatment of healthy macaques with atorvastatin prior to and throughout SIV infection prevented the development of SIV-associated PAH. Additionally, SIV-infected macaques that initiated atorvastatin treatment during the early chronic disease stage had reduced incidence of PAH compared to untreated animals. Statin treatment reduced inflammatory mediators TGF-ß, MIP-1α, and TNF-α and the numbers of CD14dimCD16+ non-classical monocytes, and CD14+CCR7-CD163-CD206+ alveolar macrophages previously shown to be associated with SIV-PAH. These results support the concept that statins reduce inflammatory processes that contribute to PAH and may provide a safe and effective prophylactic strategy for the prevention of PAH in HIV-infected individuals.

Sevoflurane improves circulatory function and pulmonary fibrosis in rats with pulmonary arterial hypertension through inhibiting NF-κB signaling pathway.

OBJECTIVE: The aim of this study was to explore the influences of sevoflurane inhalation therapy on circulation function and pulmonary fibrosis in rats with pulmonary arterial hypertension (PAH) and the nuclear factor-κB (NF-κB) signaling pathway. MATERIALS AND METHODS: A total of 30 adult male Sprague-Dawley rats were randomly divided into three groups, including control group (CTL group, n=10), PAH group (n=10), and PAH + sevoflurane group (n=10) using a random number table. Subsequently, the pulmonary artery right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were measured. Rats in PAH group were subcutaneously injected with 60 mg/kg monocrotaline once to establish the model of PAH. 28 d later, the differences in the morphology of pulmonary tissues and the protein expression levels of phosphorylated inhibitory κB (p-IκB), p-P65, P65, cyclin D1, proliferating cell nuclear antigen (PCNA) and tubulin among the three groups were analyzed via hematoxylin-eosin (HE) staining and Western blotting, respectively. Meanwhile, the messenger ribonucleic acid (mRNA) expression level of P65 was determined via Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR). Additionally, changes in the expression levels of Ki-67 and α-smooth muscle actin (α-SMA) in rat pulmonary tissues of the three groups were evaluated through immunohistochemistry. RESULTS: According to HE staining results, compared with CTL group, rats in PAH group exhibited significant thickening of the pulmonary artery wall, reduction of the vascular lumen, inflammatory cell infiltration, and thrombosis in some small arteries. This indicated that the PAH model was successfully established in rats. Compared with PAH group, PAH + sevoflurane group showed a significantly improved morphology of rat pulmonary tissues. Western blotting demonstrated that the protein expression levels of p-IκB, p-P65, and P65 in rat pulmonary tissues of PAH group were remarkably higher than CTL group (p<0.01). However, they were notably down-regulated in PAH + sevoflurane group when compared with those in PAH group (p<0.05). The above experimental results suggested that the NF-κB signaling pathway in pulmonary tissues of rats in PAH group was activated and was inhibited by sevoflurane. Subsequent RT-qPCR results indicated that no significant (N.S.) differences were observed in the mRNA level of P65 among the three groups. Compared with CTL group, PAH group showed significantly up-regulated levels of Ki-67 and α-SMA in rat pulmonary tissues (p<0.01). However, their expression levels were markedly reduced in PAH + sevoflurane group when compared with PAH group (p<0.05). Finally, the detection of pulmonary circulatory function-related indicators illustrated that RVSP and RVHI increased significantly in PAH group in comparison with CTL group. However, they declined remarkably in PAH + sevoflurane group when compared with those in PAH group (p<0.05). CONCLUSIONS: Sevoflurane down-regulates the levels of p-IκB, p-P65, and P65 to repress the activation of the NF-κB signaling pathway. This may reduce pulmonary fibrosis and ultimately prevent PAH.