Metformin Attenuates Hypoxia-induced Endothelial Cell Injury by Activating the AMP-Activated Protein Kinase Pathway.

ABSTRACT: Metformin reduces the incidence of cardiovascular diseases, and potential underlying mechanisms of action have been suggested. Here, we investigated the role of metformin in endothelial cell injury and endothelial-mesenchymal transition (EndMT) induced by hypoxia. All experiments were performed in human cardiac microvascular endothelial cells (HCMECs). HCMECs were exposed to hypoxic conditions for 24, 48, 72, and 96 hours, and we assessed the cell viability by cell counting kit 8; metformin (2, 5, 10, and 20 mmol/L) was added to the cells after exposure to the hypoxic conditions for 48 hours. The cells were randomly divided into the control group, hypoxia group, hypoxia + metformin group, hypoxia + control small interfering RNA group, hypoxia + small interfering Prkaa1 (siPrkaa1) group, and hypoxia + siPrkaa1 + metformin group. Flow cytometry and cell counting kit 8 were used to monitor apoptosis and assess cell viability. Immunofluorescence staining was used to identify the CD31+/alpha smooth muscle actin+ double-positive cells. Quantitative real-time-PCR and Western blot were used for mRNA and protein expression analyses, respectively. Hypoxia contributed to endothelial injuries and EndMT of HCMECs in a time-dependent manner, which was mainly manifested as decreases in cell viability, increases in apoptotic rate, and changes in expression of apoptosis-related and EndMT-related mRNAs and proteins. Furthermore, metformin could attenuate the injuries and EndMT caused by hypoxia. After metformin treatment, phosphorylated-AMPK (pAMPK) and p-endothelial nitric oxide synthase expression increased, whereas p-mammalian target of rapamycin expression decreased. However, results obtained after transfection with siPrkaa1 were in contrast to the results of metformin treatment. In conclusion, metformin can attenuate endothelial injuries and suppress EndMT of HCMECs under hypoxic conditions because of its ability to activate the AMPK pathway, increase p-AMPK/AMP-activated protein kinase, and inhibit mammalian target of rapamycin.

Relationship between Plasma Trimethylamine N-Oxide Levels and Renal Dysfunction in Patients with Hypertension.

INTRODUCTION: Trimethylamine N-oxide (TMAO) is a metabolite produced by gut bacteria. Although increased TMAO levels have been linked to hypertension (HTN) and chronic kidney disease (CKD) with poor prognosis, no clinical studies have directly addressed the relationship between them. In this study, we investigated the relationship between TMAO and renal dysfunction in hypertensive patients. METHODS: We included healthy controls (n = 50), hypertensive patients (n = 46), and hypertensive patients with renal dysfunction (n = 143). Their blood pressure values were taken as the highest measured blood pressure. Renal function was evaluated using the estimated glomerular filtration rate. Plasma TMAO levels were measured using high-performance liquid chromatography tandem mass spectrometry. RESULTS: We found significant differences in plasma TMAO levels among the 3 groups (p < 0.01). The plasma TMAO of patients with HTN was significantly higher than that of healthy people, and the plasma TMAO of patients with HTN complicated by renal dysfunction was significantly higher than either of the other groups. Patients in the highest TMAO quartile were at a higher risk of developing CKD stage 5 than those in the lowest quartile. In the receiver operating characteristic curve, the area under the curve of TMAO combined with ß 2-macroglobulin for predicting renal dysfunction in patients with HTN was 0.85 (95% confidence interval 0.80-0.90). CONCLUSION: An elevated TMAO level reflects higher levels of HTN and more severe renal dysfunction. TMAO, combined with ß 2-macroglobulin levels, may assist in diagnosing CKD in hypertensive patients. Plasma TMAO has predictive value for early kidney disease in hypertensive patients.

Klotho improves diabetic cardiomyopathy by suppressing the NLRP3 inflammasome pathway.

AIMS: NLRP3 inflammasome activation is essential for the development and prognosis of diabetic cardiomyopathy (DCM). The anti-aging protein Klotho is suggested to modulate tissue inflammatory responses. The aim of the present study was to examine the protective effects of Klotho on DCM. MAIN METHODS: A streptozotocin-induced diabetes mouse model was established to assess the effects of Klotho in vivo, which was administered for 12 weeks. The characteristics of type 1 DCM were evaluated by general status, echocardiography, and histopathology. The expression of associated factors was determined by RT-qPCR and western blotting. Parallel experiments to determine the molecular mechanism through which Klotho prevents DCM were performed using H9C2 cells exposed to high glucose (35 mM). KEY FINDINGS: Diabetes-induced increases in serum creatine kinase-muscle/brain and lactate dehydrogenase levels, cardiac fibrosis, cardiomyocyte apoptosis, and cardiac dysfunction were ameliorated by Klotho. Additionally, Klotho suppressed TXNIP expression, NLRP3 inflammasome activation, and expression of the inflammatory cytokines tumor necrosis factor ɑ, interleukin-1ß, and interleukin-18 in vivo. In high glucose-cultured cardiomyocytes, Klotho and N-acetylcysteine significantly downregulated intracellular reactive oxygen species generation and TXNIP/NLRP3 inflammasome activation. Pretreatment of H9C2 cells with NLRP3 siRNA or Klotho prevented high glucose-induced inflammation and apoptosis in H9C2 cells. SIGNIFICANCE: Our results demonstrate that the protective effect of Klotho on diabetes-induced cardiac injury is associated with inhibition of the NLRP3 inflammasome pathway, suggesting its therapeutic potential for DCM.