Canagliflozin inhibits PASMCs proliferation via regulating SGLT1/AMPK signaling and attenuates artery remodeling in MCT-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) was a devastating disease characterized by artery remodeling, ultimately resulting in right heart failure. The aim of this study was to investigate the effects of canagliflozin (CANA), a sodium-glucose cotransporter 2 inhibitor (SGLT2i) with mild SGLT1 inhibitory effects, on rats with PAH, as well as its direct impact on pulmonary arterial smooth muscle cells (PASMCs). PAH rats were induced by injection of monocrotaline (MCT) (40 mg/kg), followed by four weeks of treatment with CANA (30 mg/kg/day) or saline alone. Pulmonary artery and right ventricular (RV) remodeling and dysfunction in PAH were alleviated with CANA, as assessed by echocardiography. Hemodynamic parameters and structural of pulmonary arteriole, including vascular wall thickness and wall area, were reduced by CANA. RV hypertrophy index, cardiomyocyte hypertrophy, and fibrosis were decreased with CANA treatment. PASMCs proliferation was inhibited by CANA under stimulation by platelet-derived growth factor (PDGF)-BB or hypoxia. Activation of AMP kinase (AMPK) was induced by CANA treatment in cultured PASMCs in a time- and concentration-dependent manner. These effects of CANA were attenuated when treatment with compound C, an AMPK inhibitor. Abundant expression of SGLT1 was observed in PASMCs and pulmonary arteries, while SGLT2 expression was undetectable. SGLT1 increased in response to PDGF-BB or hypoxia stimulation, while PASMCs proliferation was inhibited and beneficial effects of CANA were counteracted by knockdown of SGLT1. Our research demonstrated for the first time that CANA inhibited the proliferation of PASMCs by regulating SGLT1/AMPK signaling and thus exerted an anti-proliferative effect on MCT-induced PAH.

Forsythoside B Mitigates Monocrotaline-Induced Pulmonary Arterial Hypertension via Blocking the NF-κB Signaling Pathway to Attenuate Vascular Remodeling.

Purpose: Pulmonary arterial hypertension (PAH) is a devastating disease with little effective treatment. The proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by the nuclear factor-κB (NF-κB) signaling activation plays a pivotal role in the pathogenesis of PAH. Forsythoside B (FTS•B) possesses inhibitory effect on NF-κB signaling pathway. The present study aims to explore the effects and mechanisms of FTS•B in PAH. Methods: Sprague-Dawley rats received monocrotaline (MCT) intraperitoneal injection to establish PAH model, and FTS•B was co-treated after MCT injection. Right ventricular hypertrophy and pulmonary artery pressure were measured by echocardiography and right heart catheterization, respectively. Histological alterations were detected by H&E staining and immunohistochemistry. FTS•B's role in PASMC proliferation and migration were evaluated by CCK-8 and wound healing assay. To investigate the underlying mechanisms, Western blotting, immunofluorescence staining and ELISA were conducted. The NF-κB activator PMA was used to investigate the role of NF-κB in FTS•B's protective effects against PAH. Results: FTS•B markedly alleviated MCT-induced vascular remodeling and pulmonary artery pressure, and improved right ventricular hypertrophy and survival. FTS•B also reversed PDGF-BB-induced PASMC proliferation and migration, decreased PCNA and CyclinD1 expression in vitro. The elevated levels of IL-1ß and IL-6 caused by MCT were decreased by FTS•B. Mechanistically, MCT-triggered phosphorylation of p65, IκBα, IKKα and IKKß was blunted by FTS•B. FTS•B also reversed MCT-induced nuclear translocation of p65. However, all these protective effects were blocked by PMA-mediated NF-κB activation. Conclusion: FTS•B effectively attenuates PAH by suppressing the NF-κB signaling pathway to attenuate vascular remodeling. FTS•B might be a promising drug candidate with clinical translational potential for the treatment of PAH.

Activation of CaMKII/HDAC4 by SDF1 contributes to pulmonary arterial hypertension via stabilization Runx2.

Stromal derived factor 1 (SDF1) has been shown to be involved in the pathogenesis of pulmonary artery hypertension (PAH). However, the detailed molecular mechanisms remain unclear. To address this, we utilized primary cultured rat pulmonary artery smooth muscle cells (PASMCs) and monocrotaline (MCT)-induced PAH rat models to investigate the mechanisms of SDF1 driving PASMCs proliferation and pulmonary arterial remodeling. SDF1 increased runt-related transcription factor 2 (Runx2) acetylation by Calmodulin (CaM)-dependent protein kinase II (CaMKII)-dependent HDAC4 cytoplasmic translocation, elevation of Runx2 acetylation conferred its resistance to proteasome-mediated degradation. The accumulation of Runx2 further upregulated osteopontin (OPN) expression, finally leading to PASMCs proliferation. Blocking SDF1, suppression of CaMKII, inhibition the nuclear export of HDAC4 or silencing Runx2 attenuated pulmonary arterial remodeling and prevented PAH development in MCT-induced PAH rat models. Our study provides novel sights for SDF1 induction of PASMCs proliferation and suggests that targeting SDF1/CaMKII/HDAC4/Runx2 axis has potential value in the management of PAH.

Hydroxycitric Acid Tripotassium Hydrate Attenuates Monocrotaline and Hypoxia-Induced Pulmonary Hypertension in Rats.

This study investigated the effects of hydroxycitric acid tripotassium hydrate on right ventricular function, myocardial and pulmonary vascular remodeling in rats with pulmonary hypertension, and possible mechanisms. METHODS: Pulmonary hypertension was induced in male Sprague-Dawley rats by a single subcutaneous injection of monocrotaline or hypoxic chamber. In vivo, inflammatory cytokine (including TNF-α, IL-1ß, IL-6, and TGF-ß, the level of SOD) expression, superoxide dismutase and hydrogen peroxide levels, and p-IκBα and p65 expressions were detected. In vitro, pulmonary artery smooth muscle cell proliferation and migration, ROS production, and hypoxia-inducible factor-1 expression were also studied. RESULTS: Hydroxycitric acid tripotassium hydrate decreased right ventricular systolic pressure and reduced right ventricular fibrosis and pulmonary vascular remodeling in rats with two kinds of pulmonary hypertension. Moreover, the expression of both inflammatory and oxidative stress factors was effectively reduced, and the p65 signaling pathway was found to be inhibited in this study. Additionally, hydroxycitric acid tripotassium hydrate inhibited human pulmonary artery smooth cell proliferation and migration in vitro. CONCLUSIONS: This study shows that hydroxycitric acid tripotassium hydrate can alleviate pulmonary hypertension caused by hypoxia and monocycloline in rats, improve remodeling of the right ventricle and pulmonary artery, and inhibit pulmonary artery smooth muscle cell proliferation and migration. The protective effects may be achieved by regulating inflammation and oxidative stress through the p65 signaling pathway.

Induced pluripotent stem cell-derived exosomes attenuate vascular remodelling in pulmonary arterial hypertension by targeting HIF-1α and Runx2.

AIMS: Pulmonary arterial hypertension (PAH) is characterized by extensive pulmonary arterial remodelling. Although mesenchymal stem cell (MSC)-derived exosomes provide protective effects in PAH, MSCs exhibit limited senescence during in vitro expansion compared with the induced pluripotent stem cells (iPSCs). Moreover, the exact mechanism is not known. METHODS AND RESULTS: In this study, we used murine iPSCs generated from mouse embryonic fibroblasts with triple factor (Oct4, Klf4, and Sox2) transduction to determine the efficacy and action mechanism of iPSC-derived exosomes (iPSC-Exo) in attenuating PAH in rats with monocrotaline (MCT)-induced pulmonary hypertension. Both early and late iPSC-Exo treatment effectively prevented the wall thickening and muscularization of pulmonary arterioles, improved the right ventricular systolic pressure, and alleviated the right ventricular hypertrophy in MCT-induced PAH rats. Pulmonary artery smooth muscle cells (PASMC) derived from MCT-treated rats (MCT-PASMC) developed more proliferative and pro-migratory phenotypes, which were attenuated by the iPSC-Exo treatment. Moreover, the proliferation and migration of MCT-PASMC were reduced by iPSC-Exo with suppression of PCNA, cyclin D1, MMP-1, and MMP-10, which are mediated via the HIF-1α and P21-activated kinase 1/AKT/Runx2 pathways. CONCLUSION: IPSC-Exo are effective at reversing pulmonary hypertension by reducing pulmonary vascular remodelling and may provide an iPSC-free therapy for the treatment of PAH.

Panax notoginseng saponins improve monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting ADAM10/Notch3 signaling pathway / 生理学报

In this study, we investigated the effects of Panax notoginseng saponins (PNS) on pulmonary vascular remodeling and ADAM10/Notch3 pathway in pulmonary arterial hypertension (PAH). PAH rat model was established, and male Sprague Dawley (SD) rats were randomly divided into control group, monocrotaline (MCT) group and MCT+PNS group, with 10 rats in each group. Rats in the control group were intraperitoneally injected with equal volume of normal saline. Rats in the MCT group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with the same volume of normal saline every day. Rats in the MCT+PNS group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with 50 mg/kg PNS every day. The modeling time of each group lasted for 21 days. After the model was established, the mean pulmonary artery pressure (mPAP) was measured by right heart catheterization technique, the right ventricular hypertrophy index (RVHI) was calculated, the microscopic morphology and changes of pulmonary vascular wall were observed by HE and Masson staining, and the expressions of ADAM10, Notch3, Hes-1, P27, PCNA, Caspase-3 proteins and mRNA in pulmonary vascular tissue of rats were detected by Western blot and qPCR. The expression and localization of Notch3 and α-SMA were detected by immunofluorescence staining. The protein expression of ADAM10 was detected by immunohistochemical staining. The results showed that compared with the control group, mPAP, RVHI, pulmonary vessels and collagen fibers in the MCT group were significantly increased, the expressions of ADAM10, Notch3, Hes-1, and PCNA protein and mRNA were significantly increased, while the expressions of P27 and Caspase-3 protein and mRNA were decreased significantly. Compared with the MCT group, mPAP and RVHI were significantly decreased, pulmonary vessels were significantly improved and collagen fibers were significantly reduced, the expressions of protein and mRNA of ADAM10, Notch3, Hes-1, and PCNA were decreased in MCT+PNS group, but the expressions of protein and mRNA of P27 and Caspase-3 were increased slightly. The results of immunofluorescence showed that Notch3 and α-SMA staining could overlap, which proved that Notch3 was expressed in smooth muscle cells. The expression of Notch3 in the MCT group was increased significantly compared with that in the control group, while PNS intervention decreased the expression of Notch3. Immunohistochemical staining showed that compared with the control group, the amount of ADAM10 in the MCT group was increased significantly, and the expression of ADAM10 in the MCT+PNS group was decreased compared with the MCT group. These results indicate that PNS can improve the PAH induced by MCT in rats by inhibiting ADAM10/Notch3 signaling pathway.