Aloperine protects pulmonary hypertension via triggering PPARγ signaling and inhibiting calcium regulatory pathway in pulmonary arterial smooth muscle cells.

Previous studies have reported the beneficial role of Aloperine (ALO), an active vasodilator purified from the seeds and leaves of the herbal plant Sophora alopecuroides L., on experimental pulmonary hypertension (PH); however, detailed mechanisms remain unclear. In this study, monocrotaline-induced PH (MCT-PH) rat model and primarily cultured rat distal pulmonary arterial smooth muscle cells (PASMCs) were used to investigate the mechanisms of ALO on experimental PH, pulmonary vascular remodeling, and excessive proliferation of PASMCs. Results showed that first, ALO significantly prevented the disease development of MCT-PH by inhibiting right ventricular systolic pressure (RVSP) and right ventricular hypertrophy indexed by the Fulton Index, normalizing the pulmonary arterials (PAs) remodeling and improving the right ventricular function indexed by transthoracic echocardiography. ALO inhibited the excessive proliferation of both PAs and PASMCs. Then, isometric tension measurements showed vasodilation of ALO on precontracted PAs isolated from both control and MCT-PH rats via activating the KCNQ channel, which was blocked by specific KCNQ potassium channel inhibitor linopirdine. Moreover, by using immunofluorescence staining and nuclear/cytosol fractionation, we further observed that ALO significantly enhanced the PPARγ nuclear translocation and activation in PASMCs. Transcriptome analyses also revealed activated PPARγ signaling and suppressed calcium regulatory pathway in lungs from MCT-PH rats treated with ALO. In summary, ALO could attenuate MCT-PH through both transient vasodilation of PAs and chronic activation of PPARγ signaling pathway, which exerted antiproliferative roles on PASMCs and remodeled PAs.NEW & NOTEWORTHY Aloperine attenuates monocrotaline-induced pulmonary hypertension (MCT-PH) in rats by inhibiting the pulmonary vascular remodeling and proliferation of pulmonary arterial smooth muscle cells (PASMCs). In mechanism, Aloperine not only exerts a transient KCNQ-dependent vasodilation in precontracted pulmonary arteries (PAs) from both control and MCT-PH rats but also activates PPARγ nuclear translocation and signaling transduction in PASMCs, which chronically inhibits the calcium regulatory pathway and proliferation of PASMCs.

Airway delivery of Streptococcus salivarius is sufficient to induce experimental pulmonary hypertension in rats.

BACKGROUND AND PURPOSE: The causal relationship between altered host microbiome composition, especially the respiratory tract microbiome, and the occurrence of pulmonary hypertension (PH) has not yet been studied. An increased abundance of airway streptococci is seen in patients with PH compared with healthy individuals. This study aimed to determine the causal link between elevated airway exposure to Streptococcus and PH. EXPERIMENTAL APPROACH: The dose-, time- and bacterium-specific effects of Streptococcus salivarius (S. salivarius), a selective streptococci, on PH pathogenesis were investigated in a rat model established by intratracheal instillation. KEY RESULTS: Exposure to S. salivarius successfully induced typical PH characteristics, such as elevated right ventricular systolic pressure (RVSP), right ventricular hypertrophy (Fulton's index) and pulmonary vascular remodelling, in a dose- and time-dependent manner. Moreover, the S. salivarius-induced characteristics were absent in either the inactivated S. salivarius (inactivated bacteria control) treatment group or the Bacillus subtilis (active bacteria control) treatment group. Notably, S. salivarius-induced PH is characterized by elevated inflammatory infiltration in the lungs, in a pattern different from the classic hypoxia-induced PH model. Moreover, in comparison with the SU5416/hypoxia-induced PH model (SuHx-PH), S. salivarius-induced PH causes similar histological changes (pulmonary vascular remodelling) but less severe haemodynamic changes (RVSP, Fulton's index). S. salivarius-induced PH is also associated with altered gut microbiome composition, suggesting potential communication of the lung-gut axis. CONCLUSION AND IMPLICATIONS: This study provides the first evidence that the delivery of S. salivarius in the respiratory tract could cause experimental PH in rats.

Microbiome and metabolome dysbiosis of the gut-lung axis in pulmonary hypertension.

Previous studies have suggested that dysbiosis of the gut microbiota is associated with the development of pulmonary hypertension (PH). In this study, we established a left pulmonary artery ligation (LPAL)-induced PH rat model due to high flow and hemodynamic stress and investigated the association between gut microbiota composition and host metabolome signatures (in both gut and lung tissues) by using multiomics and correlation analysis. The results showed that LPAL successfully induced PH, characterized by increased right ventricular systolic pressure, right ventricular hypertrophy and pulmonary vascular remodelling. Moreover, gut pathological abnormalities were observed in association with dramatic alterations in the gut microbiome and metabolome as well as the lung metabolome. The increased bacterial genus Sporobacter and decreased genera Eubacterium, Eubacteriaceae, Deltaproteobacteria and Desulfovibrio featured the altered gut microbiome in LPAL-PH versus SHAM rats. Moreover, imbalanced abundance of protective metabolites (e.g., butyrate, propionate) and pathogenic metabolites (e.g., proinflammatory mediators) were seen in the gut metabolome of LPAL-PH versus SHAM rats. In addition, the altered gut microbiome strongly correlated with the altered metabolome patterns in both the gut and lung of LPAL-PH rats. In conclusion, this study revealed significant gut dysbiosis in LPAL-PH rats, characterized by altered gut microbiota composition, in association with specific changes in gut and lung metabolome profiles. These findings enriched our understanding of the unique signature of the gut microbiome and the close association of the "gut-lung axis" in LPAL-PH induced by long-term high flow, leading to novel therapeutic, diagnostic or management paradigms for this subtype of PH.

Upregulation of mechanosensitive channel Piezo1 involved in high shear stress-induced pulmonary hypertension.

INTRODUCTION: Piezo1 is an important mechanosensitive channel implicated in vascular remodeling. However, the role of Piezo1 in different types of vascular cells during the development of pulmonary hypertension (PH) induced by high shear stress is largely unknown. MATERIALS AND METHODS: We used a rat PH model established by left pulmonary artery ligation (LPAL, for 2-5 weeks), which mimics the high flow and hemodynamic stress, to study Piezo1 contribution to pulmonary vascular remodeling. RESULTS: Right ventricular systolic pressure (RVSP), a surrogate measure for pulmonary arterial systolic pressure, and right ventricular wall thickness, a measure for right ventricular hypertrophy, were significantly increased in LPAL rats compared with Sham-control (SHAM) rats. Rats in LPAL-5w groups developed remarkable pulmonary vascular remodeling, while phenylephrine-induced contraction and acetylcholine-induced relaxation were both significantly inhibited in these rats. Upregulation of Piezo1, in association with increase in cytosolic Ca2+ concentration ([Ca2+]cyt), was observed in pulmonary arterial smooth muscle cells (PASMCs) from LPAL-2w and LPAL-5w rats in comparison to the SHAM controls. Piezo1 upregulation in PASMCs from LPAL rats was directly related to Yes-associated protein (YAP)/ TEA domain transcription factor 4 (TEAD4). Piezo1 expression was also upregulated in the whole-lung tissue of LPAL rats. The endothelial upregulation of Piezo1 was related to transcriptional regulation by RELA (p65) and lung inflammation. CONCLUSION: The upregulation of Piezo1 in both PASMCs and ECs coordinates with each other via different cell signaling pathways to cause pulmonary vascular remodeling in LPAL-PH rats, providing novel insights into the cell-type specific pathogenic roles of Piezo1 in shear stress-associated experimental PH.

Upregulation of Piezo1 (Piezo Type Mechanosensitive Ion Channel Component 1) Enhances the Intracellular Free Calcium in Pulmonary Arterial Smooth Muscle Cells From Idiopathic Pulmonary Arterial Hypertension Patients.

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Mitomycin C induces pulmonary vascular endothelial-to-mesenchymal transition and pulmonary veno-occlusive disease via Smad3-dependent pathway in rats.

BACKGROUND AND PURPOSE: Pulmonary veno-occlusive disease (PVOD) is a rare disease characterized by the obstruction of small pulmonary veins leading to pulmonary hypertension. However, the mechanisms underlying pulmonary vessel occlusion remain largely unclear. EXPERIMENTAL APPROACH: A mitomycin C (MMC)-induced PVOD rat model was used as in vivo animal model, and primarily cultured rat pulmonary microvascular endothelial cells (PMVECs) were used as in vitro cell model. KEY RESULTS: Our data suggested an endothelial-to-mesenchymal transition (EndoMT) may be present in the pulmonary microvessels isolated from either PVOD patients or MMC-induced PVOD rats. In comparison to the control vessels, vessels from both PVOD patients and PVOD rats had co-localized staining of specific endothelial marker von Willebrand factor (vWF) and mesenchymal marker α-smooth muscle actin (α-SMA), suggesting the presence of cells that co-express endothelial and mesenchymal markers. In both the lung tissues of MMC-induced PVOD rats and MMC-treated rat PMVECs there were decreased levels of endothelial markers (e.g. VE-cadherin and CD31) and increased mesenchymal markers (e.g. vimentin, fibronectin and α-SMA) were detected indicating EndoMT. Moreover, MMC-induced activation of the TGFß/Smad3/Snail axis, while blocking this pathway with either selective Smad3 inhibitor (SIS3) or small interfering RNA (siRNA) against Smad3, dramatically abolished the MMC-induced EndoMT. Notably, treatment with SIS3 remarkably prevented the pathogenesis of MMC-induced PVOD in rats. CONCLUSIONS AND IMPLICATIONS: Our data indicated that targeted inhibition of Smad3 leads to a potential, novel strategy for PVOD therapy, likely by inhibiting the EndoMT in pulmonary microvasculature.

Dysregulation of BMP9/BMPR2/SMAD signalling pathway contributes to pulmonary fibrosis and pulmonary hypertension induced by bleomycin in rats.

BACKGROUND AND PURPOSE: Pulmonary hypertension related to pulmonary fibrosis is classed as WHO Group III, one of the most common groups which lacks effective treatment options. In this study, we aimed to uncover the underlying mechanisms, particularly the involvement of the BMP9/BMPR2/SMAD signalling pathway, in this subtype of pulmonary hypertension. EXPERIMENTAL APPROACH: Male Sprague Dawley rats were used to establish a model of pulmonary hypertension with pulmonary fibrosis, induced by bleomycin. Haemodynamic and lung functions were measured, along with histological and immunohistochemical examinations. Primary cultures of rat pulmonary microvascular endothelial cells (PMVECs) were analysed with western blots, apoptosis assays and immunohistochemistry. KEY RESULTS: Early (7 days) after bleomycin treatment of rats, pulmonary arterial thickening and severe loss of pulmonary arterial endothelium were observed, followed (14 days) by increased right ventricular systolic pressure and right ventricular hypertrophy. Marked down-regulation of the BMP9/BMPR2/SMAD signalling pathway was markedly down-regulated in lung tissues from bleomycin-treated rats (throughout the 7- to 35-day treatment period) and bleomycin-treated rat PMVECs, along with excessive cell apoptosis and loss of pulmonary arterial endothelium. Treatment with recombinant human bone morphogenetic protein 9 (rhBMP9) attenuated these aspects of bleomycin-induced pulmonary hypertension, by restoring disrupted BMP9/BMPR2/SMAD signalling. CONCLUSION AND IMPLICATIONS: In bleomycin-treated rats, early and persisting suppression of the BMP9/BMPR2/SMAD signalling pathway triggered severe loss of pulmonary arterial endothelium and subsequent pulmonary arterial vascular remodelling, contributing to the development of pulmonary hypertension. Therapeutic approaches reinforcing BMP9/BMPR2/SMAD signalling might be ideal strategies for this subtype of pulmonary hypertension. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc.

MicroRNA­4500 suppresses tumor progression in non­small cell lung cancer by regulating STAT3.

Research has revealed that microRNA (miR)­4500 is downregulated in non­small cell lung cancer (NSCLC), and miR­4500 suppresses tumor growth by targeting lin­28 homolog B and NRAS proto­oncogene, GTPase. In the present study, it was reported that signal transducer and activator of transcription 3 (STAT3) may function as a novel target gene for miR­4500 in NSCLC. The experiments conducted in the present study confirmed that the miR­4500 expression was decreased in NSCLC tissues and cells compared with adjacent normal tissues and a normal lung cell line. miR­4500 suppressed the cell proliferation, migration, invasion and promoted apoptosis of the human NSCLC cell lines A549 and H1975. Expression of STAT3 was negatively correlated with miR­4500 expression in vivo. A luciferase reporter assay suggested that miR­4500 directly targeted the 3' untranslated region of STAT3. The tumor inhibition effect of small interfering RNA STAT3 in A549 and H1975 lines may be partially impaired by a miR­4500 inhibitor. The results of the present study suggests that miR­4500 may be a tumor suppressor and a potential therapeutic target in NSCLC.

Upregulated microRNA­671­3p promotes tumor progression by suppressing forkhead box P2 expression in non­small­cell lung cancer.

In the present study, the expression of microRNA (miR)­671­3p in non­small­cell lung cancer (NSCLC) was detected via reverse transcription­quantitative polymerase chain reaction analysis, and its role in cell proliferation, apoptosis, migration and invasion was investigated via Cell Counting Kit­8, colony formation, flow cytometry, Transwell and scratch assays, respectively. It was observed that the expression of miR­671­3p was upregulated in NSCLC tissues and cell lines (A549 and H1975). Treatment with miR­671­3p inhibitors suppressed cell proliferation, migration and invasion, and increased apoptosis in vitro, suggesting that miR­671­3p functions as an oncogene in NSCLC. In addition, forkhead box P2 (FOXP2) has been reported to be a tumor suppressor that is downregulated in several types of cancer, and its low expression was confirmed in NSCLC tissues and cell lines in the current study via western blotting. The results of the luciferase reporter assay also demonstrated that miR­671­3p targeted directly the 3'­untranslated region of FOXP2. Furthermore, overexpression of FOXP2 in A549 and H1975 cell lines suppressed the growth, migration and invasion, and promoted apoptosis, whereas these effects were reversed by transfection with miR­671­3p mimics, suggesting that miR­671­3p promoted tumor progression via regulating FOXP2. Taken together, the results reported in the present study implied that miR­671­3p may be a potential therapeutic target in NSCLC.