Apolipoprotein A-I mimetic peptide inhibits atherosclerosis by increasing tetrahydrobiopterin via regulation of GTP-cyclohydrolase 1 and reducing uncoupled endothelial nitric oxide synthase activity.

BACKGROUND AND AIMS: The apolipoprotein A-I mimetic peptide D-4F, among its anti-atherosclerotic effects, improves vasodilation through mechanisms not fully elucidated yet. METHODS: Low-density lipoprotein (LDL) receptor null (LDLr-/-) mice were fed Western diet with or without D-4F. We then measured atherosclerotic lesion formation, endothelial nitric oxide synthase (eNOS) phosphorylation and its association with heat shock protein 90 (HSP90), nitric oxide (NO) and superoxide anion (O2•-) production, and tetrahydrobiopterin (BH4) and GTP-cyclohydrolase 1 (GCH-1) concentration in the aorta. Human umbilical vein endothelial cells (HUVECs) and aortas were treated with oxidized LDL (oxLDL) with or without D-4F; subsequently, BH4 and GCH-1 concentration, NO and O2•- production, eNOS association with HSP90, and endothelium-dependent vasodilation were measured. RESULTS: Unexpectedly, eNOS phosphorylation, eNOS-HSP90 association, and O2•- production were increased, whereas BH4 and GCH-1 concentration and NO production were reduced in atherosclerosis. D-4F significantly inhibited atherosclerosis, eNOS phosphorylation, eNOS-HSP90 association, and O2•- generation but increased NO production and BH4 and GCH-1 concentration. OxLDL reduced NO production and BH4 and GCH-1 concentration but enhanced O2•- generation and eNOS association with HSP90, and impaired endothelium-dependent vasodilation. D-4F inhibited the overall effects of oxLDL. CONCLUSIONS: Hypercholesterolemia enhanced uncoupled eNOS activity by decreasing GCH-1 concentration, thereby reducing BH4 levels. D-4F reduced uncoupled eNOS activity by increasing BH4 levels through GCH-1 expression and decreasing eNOS phosphorylation and eNOS-HSP90 association. Our findings elucidate a novel mechanism by which hypercholesterolemia induces atherosclerosis and D-4F inhibits it, providing a potential therapeutic approach.

Simvastatin inhibits POVPC-mediated induction of endothelial-to-mesenchymal cell transition.

Endothelial-to-mesenchymal transition (EndMT), the process by which an endothelial cell (EC) undergoes a series of molecular events that result in a mesenchymal cell phenotype, plays an important role in atherosclerosis. 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine, is a proinflammatory lipid found in atherosclerotic lesions. Whether POVPC promotes EndMT and how simvastatin influences POVPC-mediated EndMT remains unclear. Here, we treated human umbilical vein ECs with POVPC, simvastatin, or both, and determined their effect on EC viability, morphology, tube formation, proliferation, and generation of NO and superoxide anion (O2•-). Expression of specific endothelial and mesenchymal markers was detected by immunofluorescence and immunoblotting. POVPC did not affect EC viability but altered cellular morphology from cobblestone-like ECs to a spindle-like mesenchymal cell morphology. POVPC increased O2- generation and expression of alpha-smooth muscle actin, vimentin, Snail-1, Twist-1, transforming growth factor-beta (TGF-ß), TGF-ß receptor II, p-Smad2/3, and Smad2/3. POVPC also decreased NO production and expression of CD31 and endothelial NO synthase. Simvastatin inhibited POVPC-mediated effects on cellular morphology, production of O2•- and NO, and expression of specific endothelial and mesenchymal markers. These data demonstrate that POVPC induces EndMT by increasing oxidative stress, which stimulates TGF-ß/Smad signaling, leading to Snail-1 and Twist-1 activation. Simvastatin inhibited POVPC-induced EndMT by decreasing oxidative stress, suppressing TGF-ß/Smad signaling, and inactivating Snail-1 and Twist-1. Our findings reveal a novel mechanism of atherosclerosis that can be inhibited by simvastatin.

Circulating extracellular vesicles from patients with valvular heart disease induce neutrophil chemotaxis via FOXO3a and the inhibiting role of dexmedetomidine.

We previously demonstrated that circulating extracellular vesicles (EVs) from patients with valvular heart disease (VHD; vEVs) contain inflammatory components and inhibit endothelium-dependent vasodilation. Neutrophil chemotaxis plays a key role in renal dysfunction, and dexmedetomidine (DEX) can reduce renal dysfunction in cardiac surgery. However, the roles of vEVs in neutrophil chemotaxis and effects of DEX on vEVs are unknown. Here, we investigated the impact of vEVs on neutrophil chemotaxis in kidneys and the influence of DEX on vEVs. Circulating EVs were isolated from healthy subjects and patients with VHD. The effects of EVs on chemokine generation, forkhead box protein O3a (FOXO3a) pathway activation and neutrophil chemotaxis on cultured human umbilical vein endothelial cells (HUVECs) and kidneys in mice and the influence of DEX on EVs were detected. vEVs increased FOXO3a expression, decreased phosphorylation of Akt and FOXO3a, promoted FOXO3a nuclear translocation, and activated the FOXO3a signaling pathway in vitro. DEX pretreatment reduced vEV-induced CXCL4 and CCL5 expression and neutrophil chemotaxis in cultured HUVECs via the FOXO3a signaling pathway. vEVs were also found to suppress Akt phosphorylation and activate FOXO3a signaling to increase plasma levels of CXCL4 and CCL5 and neutrophil accumulation in kidney. The overall mechanism was inhibited in vivo with DEX pretreatment. Our data demonstrated that vEVs induced CXCL4-CCL5 to stimulate neutrophil infiltration in kidney, which can be inhibited by DEX via the FOXO3a signaling. Our findings reveal a unique mechanism involving vEVs in inducing neutrophils chemotaxis and may provide a novel basis for using DEX in reducing renal dysfunction in valvular heart surgery.

Valsartan reduced the vulnerability to atrial fibrillation by preventing action potential prolongation and conduction slowing in castrated male mice.

INTRODUCTION: Deficiency of testosterone was associated with the susceptibility of atrial fibrillation (AF). Angiotensin-II (AngII) receptor antagonists were shown to reduce AF by improving atrial electrical remodeling. This study investigated the effects and mechanism of valsartan, an AngII receptor antagonist, on the susceptibility to AF with testosterone deficiency. METHODS AND RESULTS: Five-week-old male ICR mice were castrated and valsartan was administered orally (50 mg/kg/d). High-frequency electrical stimulation method was used to induce atrial arrhythmia. Patch-clamp technique was used for recording action potential duration (APD), transient outward potassium current ( I to ), sustained outward potassium current ( I ksus ), and late sodium current ( I Na-L ). Optical mapping technique was used to examine atrial conduction velocity (CV). The expression of connexin40 (Cx40) and Cx43 were detected by Western blot analysis. The occurrence rate of AF was significantly increased in castrated mice and APDs measured at 50% and 90% repolarization were markedly prolonged in castrated mice than controls, which were alleviated by the administration of valsartan. Valsartan suppressed the increase of INa-L and rescued the reduction of Ito and Iksus in castrated mice. The left atrial CV in castrated mice was decreased and the expression of Cx43 reduced than controls, which were restored after valsartan treatment. CONCLUSIONS: Valsartan reduced the susceptibility of AF in castrated mice, which may be related to the inhibition of action potential prolongation and improvement of atrial conduction impairment. This study indicates that valsartan may represent a useful agent for the prevention of AF pathogenesis in elderly male patients.

The oxidized phospholipid POVPC impairs endothelial function and vasodilation via uncoupling endothelial nitric oxide synthase.

Endothelial dysfunction is an early stage of atherosclerosis. We recently have shown that 25-hydroxycholesterol found in atherosclerotic lesions could impair endothelial function and vasodilation by uncoupling and inhibiting endothelial nitric oxide synthase (eNOS). 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), the oxidation product of oxidized low-density lipoprotein, is another proinflammatory lipid and has also been found in atherosclerotic lesions. However, whether POVPC promotes atherosclerosis like 25-hydroxycholesterol remains unclear. The purpose of this study was to explore the effects of POVPC on endothelial function and vasodilation. Human umbilical vein endothelial cells (HUVECs) were incubated with POVPC. Endothelial cell proliferation, migration and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation (O2-) were determined. The expression and phosphorylation of endothelial nitric oxide synthase (eNOS), AKT, PKC-ßII and P70S6K as well as the association of eNOS and heat shock protein 90 (HSP90) were detected by immunoblotting and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining. The expression of Bcl-2, Bax, and Cleaved Caspase 3 were detected by immunoblotting. Finally, aortic ring from C57BL6 mice were isolated and treated with POVPC and the endothelium-dependent vasodilation was evaluated. POVPC significantly inhibited HUVECs proliferation, migration, tube formation, decreased NO production but increased O2- generation. POVPC inhibited the phosphorylation of Akt and eNOS at Ser1177, increased activation of PKC-ßII, P70S6K and the phosphorylation of eNOS at Thr495, reduced the association of HSP90 with eNOS. Meanwhile, POVPC induced endothelial cell apoptosis by inhibiting Bcl-2 expression, increasing Bax and cleaved caspase-3 expressions as well as caspase-3 activity and impaired endothelium-dependent vasodilation. These data demonstrated that POVPC impaired endothelial function by uncoupling and inhibiting eNOS as well as by inducing endothelial cell apoptosis. Therefore, POVPC may play an important role in the development of atherosclerosis and may be considered as a potential therapeutic target for atherosclerosis.