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.

Gasdermin D-Mediated Pyroptosis Promotes the Development of Atherosclerosis.

Atherosclerosis is a chronic inflammatory cardiovascular disease with a high-morbidity and mortality rate. An increasing number of studies have addressed the crucial contribution of gasdermin D (GSDMD)-mediated pyroptosis, which is triggered by the inflammasomes to the development of atherosclerosis. However, the underlying mechanism is still unclear. This study aimed to uncover the detailed role of GSDMD in the development of atherosclerosis. An atherosclerotic model was established in Gsdmd-/-/Ldlr-/- mice and Gsdmd+/+/Ldlr-/- mice fed with a high-fat diet. The atherosclerotic lesions, the activation of GSDMD, and the expression level of inflammatory cytokines and chemokines were evaluated. Gsdmd deletion ameliorated the atherosclerotic lesion sizes and the infiltration of immune cells and inflammatory cells in the aortas of mice. Additionally, Gsdmd deletion suppressed the pyroptosis of macrophages and endothelial cells induced by the serum of Ldlr-/- mice fed with a high-fat diet. Furthermore, the formation of neutrophil extracellular traps was also attenuated by knockout of Gsdmd. Bone marrow chimeras confirmed that the genetic deficiency of Gsdmd in both immune cells and intrinsic cells played a role in the promotion of arteriosclerosis. Collectively, our study demonstrated that Gsdmd deletion hindered the pathogenesis of atherosclerosis by inhibiting endothelial cell and macrophage cell death, and the formation of neutrophil extracellular traps.

CPT1 regulates the proliferation of pulmonary artery smooth muscle cells through the AMPK-p53-p21 pathway in pulmonary arterial hypertension.

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a dominant role in the development of pulmonary arterial hypertension (PAH). Some studies and our previous work found that disturbance of fatty acid metabolism existed in PAH. However, the mechanistic link between fatty acid catabolism and cell proliferation remains elusive. Here, we identified an essential role and signal pathway for the key rate-limiting enzyme of mitochondrial fatty acid ß-oxidation, carnitine palmitoyltransferase (CPT) 1, in regulating PASMC proliferation in PAH. We found that CPT1 was highly expressed in rat lungs and pulmonary arteries in monocrotaline-induced PAH, accompanied by decreased adenosine triphosphate (ATP) production and downregulation of the AMPK-p53-p21 pathway. Platelet-derived growth factor (PDGF)-BB upregulated the expression of CPT1 in a dose- and time-dependent manner. PASMC proliferation and ATP production induced by PDGF-BB were partly reversed by the CPT1 inhibitor etomoxir (ETO). The overexpression of CPT1 in PASMCs also promoted proliferation and ATP production and subsequently inhibited the phosphorylation of AMPK, p53, as well as p21 in PASMCs. Furthermore, AMPK was activated by ETO, which increased the expression of p53 and p21, and the proportion of cells in the cell cycle G2/M phase in response to PDGF-BB stimulation in PASMCs. Our work reveals a novel mechanism of CPT1 regulating PASMC proliferation in PAH, and regulation of CPT1 may be a potential target for therapeutic intervention in PAH.

Pulmonary arterial hypertension induced by a novel method: Twice-intraperitoneal injection of monocrotaline.

Pulmonary arterial hypertension (PAH) in humans manifests as a chronic process. However, PAH induced by high-dose monocrotaline (MCT) in animals occurs as a subacute process. To establish a chronic PAH model, rats were randomly divided into three groups, control (ctrl), single injection (SI), and twice injection (TI) groups. Rats in the SI group received a single intraperitoneal injection of 40 mg/kg MCT on day 0. Rats in the TI group received twice injections of 20 mg/kg MCT on days 0 and 7. Survival percentage, characteristic changes of pulmonary arterial variables, and right ventricular features were evaluated. Thirty-five days after the first MCT injection, survival percentage in TI group was higher than that in the SI group. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVHI), pulmonary vascular remodeling, serum tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) were higher either in SI or in TI 28 and 35 days after the first MCT injection. The rats in the SI and TI groups exhibited higher right ventricle end diastolic diameter (RVEDD) and lower adjusted pulmonary artery acceleration time (PAAT/HR), tricuspid annular plane systolic excursion (TAPSE), cardiac output (CO) and right ventricle fractional shortening (RVFS) when compared with controls. However, mPAP, RVHI, TAPSE, PAAT/HR, CO, TNFα, and IL-6 were lower and RVEDD were higher in the TI group than in the SI group. Pulmonary macrophage infiltration and right ventricle (RV) fibrosis were lower in TI than SI groups. The cardiomyocyte cross-sectional area and the beta myosin heavy chain (MYH7) mRNA level of RV were lower in TI than SI, whereas alpha myosin heavy chain (MYH6) was increased. These results show that two intraperitoneal injections of 20 mg/kg MCT with seven days interval could induce a model similar to chronic PAH with increased survival percentage in rats. Impact statement We demonstrated previously that a single intraperitoneal injection of 40 mg/kg MCT produced a subacute, not chronic, PAH model in rats, and the short survival periods of these rats did not represent adequately the chronic PAH seen in humans. To overcome this limitation, in this study, the single dose of 40 mg/kg MCT was divided into twice injections of 20 mg/kg with an interval of seven days. This modified administration of MCT produced an animal model much more similar to chronic PAH with prolonged survival and characteristic changes of structures and function in pulmonary arteries and right ventricles.