Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHE1-dependent endothelial cell apoptosis

Abstract

Aim: To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide (LPS)-accelerated atherosclerosis. Methods: Human umbilical vein endothelial cells (HUVECs) were exposed to LPS (100 ng/mL) in the presence of drugs tested. The activity of Na + /H + exchanger 1 (NHE1) and calpain, intracellular free Ca 2+ level ([Ca 2+ ] i ), as well as the expression of apoptosis-related proteins in the cells were measured. For in vivo study, ApoE-deficient (ApoE -/-) mice were fed high-fat diets with 0.5% (w/w) amiloride for 4 weeks and LPS (10 µg/mouse) infusion into caudal veins. Afterwards, atherosclerotic lesions, NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated. Results: LPS treatment increased NHE1 activity and [Ca 2+ ] i in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca 2+ -dependent protease calpain. Amiloride (1−10 µmol/L) significantly suppressed LPS-induced increases in NHE1 activity, [Ca 2+ ] i . and calpain activity. In the presence of the Ca 2+ chelator BAPTA (0.5 mmol/L), LPS-induced increase of calpain activity was also abolished. In LPS-treated HUVECs, the expression of Bcl-2 protein was significantly decreased without altering its mRNA level. In the presence of amiloride (10 µmol/L) or the calpain inhibitor ZLLal (50 µmol/L), the down-regulation of Bcl-2 protein by LPS was blocked. LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs. In the presence of amiloride, BAPTA or ZLLal, LPS-induced HUVEC apoptosis was significantly attenuated. In ApoE -/-mice, administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity, and reversed LPS-induced down-regulation of Bcl-2 expression. Conclusion: LPS stimulates NHE1 activity, increases [Ca 2+ ] i , and activates calpain, which leads to endothelial cell apoptosis related to decreased Bcl-2 expression. Amiloride inhibits NHE1 activity, thus attenuates LPS-accelerated atherosclerosis in mice.

Oral administration of Pantoea agglomerans-derived lipopolysaccharide prevents development of atherosclerosis in high-fat diet-fed apoE-deficient mice via ameliorating hyperlipidemia, pro-inflammatory mediators and oxidative responses

“…It must be pointed out that LPS injection is known to aggravate atherosclerosis. However, in in vivo studies, the route of LPS injection is limited to intravenous or intraperitoneal, and the doses of LPS usually range between 0.5 mg/kg and 2.5 mg/kg BW, which is sufficient to cause atherosclerosis in apoE-deficient mice [ 49 – 51 ]. On the other hand, oral administration of LPSp at a high dose (300 mg/kg BW, 4 weeks) caused no significant hepatotoxicity or nephrotoxicity in rat [ 52 ].…”

Direct Cardiac Actions of Sodium Glucose Cotransporter 2 Inhibitors Target Pathogenic Mechanisms Underlying Heart Failure in Diabetic Patients

“…Elevations in [Na + ] c and [Ca 2+ ] c are coupled to reduced mitochondrial [Ca 2+ ] ([Ca 2+ ] m ) in cardiomyocytes, which will decrease the energetic and redox function of mitochondria ( Murphy and Eisner, 2009 ; Bay et al, 2013 ). Elevated [Ca 2+ ] c is perceived in hyperglycemia- and LPS-induced endothelial dysfunction ( Wang et al, 2008 ; Cui et al, 2013 ), atrial fibrillation ( Neef et al, 2010 ) and in myocytes during IR injury ( Garcia-Dorado et al, 2012 ). Increases in [Na + ] c and [Ca 2+ ] c are caused by perturbed ion fluxes due to altered activity of ion channels and/or transporters, including the sodium-calcium exchanger (NCX), the sodium-hydrogen exchanger 1 (NHE-1), the ryanodine receptor regulating SR-calcium release and the sodium-potassium ATPase (Na + /K + ATPase), and targeting these ion regulating transporters has been proposed to improve cardiovascular function ( Baartscheer et al, 2003a ; Despa et al, 2012 ; Karmazyn, 2013 ; Luo et al, 2013 ; Sasahara et al, 2013 ).…”

Brucella infection inhibits macrophages apoptosis via Nedd4-dependent degradation of calpain2

“…The Ca 2+ i concentration was measured by using a Fluo-4 NW kit (Invitrogen) according to our previous described procedure (Cui et al, 2013;Zhao et al, 2012). Briefly, the cell culture medium was aspirated after infection or treatment, washed with HEPES buffer (pH 7.4) once, and 1 ml of HEPES buffer containing fluorescent dye was added to cultured cells.…”

“…Our previous studies have shown that increases of Ca 2+ i induced by lipopolysaccharide (LPS) enhances the activity of calpain in human umbilical vein endothelial cells (HUVECs) (Cui et al, 2013;Zhao et al, 2012). To address whether a similar mechanism was operative in Brucellainfected macrophages, the activity of calpain in macrophages was measured by using the fluorogenic peptide Suc-Leu-Leu-Val-Tyr-AMC as a substrate (Fig.…”