Background and Aims

Atherosclerosis is the inflammation of vascular wall triggered by initial dysfunction of the luminal endothelial cells potentiated by dyslipidemia. Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD+) -dependent mitochondrial deacetylase. The present study tested whether endothelial-selective Sirt3 deletion accelerates vascular inflammation and atherosclerosis and the potential effect of L-arginine to alleviate vascular inflammation associated with SIRT3 deletion.

Methods

Cdh5-Cre driven deletion of Sirt3-floxed allele was used to generate endothelium-specific Sirt3 knockout mice. Mice were injected with adeno-associated virus to overexpress PCSK9, and fed with high cholesterol diet to induce atherosclerosis. Human endothelial cell HUVECs and mouse brain microvascular endothelial cells were used for in vitro experiments.

Results

SIRT3 knockout further reduced endothelium-dependent vasodilatation impaired IL-1β in aorta, accompanied by increased production of mitochondrial superoxide. Such effect could be attenuated by treatment with NAD or L-arginine. Deletion of SIRT3 exacerbated atherosclerotic plaque formation in mice, accompanied by higher expression of E-selectin and more macrophage infiltration, with reduced vasodilatation. In human and mouse endothelial cells, SIRT3 knockdown enhanced upregulation of vascular inflammation markers including VCAM1, ICAM1, and CCL2 induced by IL1β or TNFα. Pre-treatment with NAD or L-arginine attenuated the upregulation of these adhesion molecules and chemokines. L-arginine also alleviated impaired mitochondrial respiration induced by TNFα in wild type and SIRT3 knockdown endothelial cells.

Conclusions

Our results support the role of mitochondrial SIRT3 against atherosclerotic inflammation and mitochondrial dysfunction. Further study is needed to identify the enzymes in L-arginine metabolism regulated by SIRT3 in endothelial cells (supported by RGC/GRF 14109519).

Background and Aims

The research into the pathophysiology of atherosclerosis has considerably increased our understanding of the complexity of the disease, but still many questions remain unanswered, and the burden of recurrent cardiovascular events remains unacceptable, despite optimal treatment with contemporary intervention and pharmacologic agents.

Our previous findings that prenylcysteine oxidase 1 (PCYOX1) represents a novel lipoprotein-associated protein, and that lipoproteins can themselves generate the reactive oxygen species H₂O₂ through the action of PCYOX1, prompted us to investigate the biological role of this unique enzyme in the development of atherosclerosis.

Methods

Gene silencing approaches with proteomic and biochemical analysis have been introduced to study PCYOX1 biological functions. In vivo localization of PCYOX1 has been performed in human atherosclerotic lesions and aortic arch of ApoE^-/- mice. A double knockout mouse model (PCYOX1^−/−/apoE^−/− mice) was treated with Western-type diet in order to study atherosclerotic lesion progression and the inflammatory status.

Results

Here, we provided evidence that the pro-oxidant enzyme Prenylcysteine Oxidase 1 (PCYOX1), in the human atherosclerotic lesions, is both synthesized locally and transported within the subintimal space by proatherogenic lipoproteins accumulating in the arterial wall during atherogenesis. Further, PCYOX1 deficiency in apoE^-/- mice retards atheroprogression, is associated with decreased features of lesion vulnerability and lower levels of lipid peroxidation, improves plasma lipid profile, and reduces inflammation.

Conclusions

Collectively, these findings identify the pro-oxidant enzyme PCYOX1 as a novel player in atherogenesis and, therefore, understanding the biology and mechanisms of all functions of this unique enzyme is likely to provide significant therapeutic opportunities in addressing atherosclerosis

Background and Aims

The aim of this study is to investigate the role of Grx1 in the onset of nutrient-stress induced obesity and atherosclerosis.

Methods

Age and sex-matched male and female Grx1^-/- and WT mice were a fed chow diet for 18 months. Body weights and fasting blood glucose levels were measured weekly, and atherosclerosis was accessed after 18 months. Myeloid Grx1 deficient mice (Grx1_Leuko^-/-) were generated by transplanting either WT or Grx1^-/- bone marrow (BM) cells into atherosclerosis-prone male and female LDLR^-/- mice and were fed a high-calorie diet (HCD) for 20 weeks. Monocyte priming was determined by in vivo Matrigel plug assay. MKP-1 activity was measured to examine monocyte function. Macrophage reprogramming was determined by targeted RT-qPCR using custom-designed 384 TaqMan® Gene Expression Array Cards.

Results

At 18 months of age, female Grx1^-/- mice were 30% heavier than males. Monocytes in 18-month old female Grx1^-/- mice were primed, and promoted the recruitment of monocyte-derived macrophages into adipose tissues, and developed early atherosclerotic lesions. Grx1 deficiency promotes the reprogramming of macrophages, notable induction of genes encoding ROS and RNS generating enzymes. This reprogramming is sexually dimorphic and dramatically exacerbated in macrophages from old female mice. Female but not male Grx1_Leuko^-/- mice recapitulated the obesogenic and atherogenic phenotype of female Grx1^-/- mice, showing accelerated weight gain (+14.6%), hyperglycemia, and accelerated atherosclerosis (+21%) compared to WT BM recipient mice.

Conclusions

We identified a critical and sexually dimorphic role for monocytic Grx1 activity in protecting monocytes and macrophages from HFD-induced reprogramming and dysfunction and preventing obesity and atherosclerosis.

Background and Aims

PLIN2 is the most prominent lipid droplet (LD)-associated protein in foam cells. PLIN2 stabilizes LDs and LDs protect PLIN2 from rapid degradation. LD-bound PLIN2 degradation is dependent on AMPK-phosphorylation via chaperone-mediated autophagy (CMA). PLIN2 is vital to maintain certain LD size and number. We have recently shown that the Ser251Pro SNP in PLIN2 influences macroautophagy, cholesterol efflux, cholesterol accumulation and is associated with subclinical atherosclerosis development in humans. Since a direct crosstalk between macroautophagy and CMA has been proposed, we aim to investigate whether the Ser251Pro SNP affects CMA and the degradation of PLIN2, thereby influencing lipophagy.

Methods

We utilized stably transfected HEK293 cells carrying either variant of PLIN2 loaded with oleate, followed by treatment with AMPK modulators: we measured PLIN2 and L2A levels by WB and colocalization of Hsc70/L2A by IHC. We used WT, ATG5^-/- and L2A^-/- MEF cells transiently transfected with PLIN2 variants: we measured LD number by IHC, PLIN2 levels by WB.

Results

Ser251Pro-dependent differences in lipid accumulation observed in WT MEF cells are abolished in both ATG5^-/- and L2A^-/- MEF cells. Cells carrying the Pro251 allele show lower L2A expression and less Hsc70/L2A colocalization, indicating fewer CMA-active lysosomes. Further, AMPK inhibition in HEK cells induces PLIN2 expression more in Ser251- than in Pro251-carrying cells.

Conclusions

The effect of the Ser251Pro SNP on lipid accumulation is dependent on both macroautophagy and CMA. The presence of the Pro251 allele reduces CMA-activity and modifies PLIN2 degradation via AMPK. Thus, PLIN2 influences lipophagy via crosstalk between macroautophagy and CMA.

Background and Aims

Oxidative stress-induced endothelial dysfunction is considered to exert a vital role in the development of atherosclerosis. In the present work, we appraise the cytoprotective property and anti-atherosclerosis effect of Z-Ligustilide (Z-Lig), which is derived from the Ligusticum species.

Methods

Potential NRF2 activators were screened and verified by luciferase reporter gene assay. The protein and mRNA levels of NRF2 and ARE-mediated genes, and GSH/GSSG level in EA.hy926 cells treated with Z-Lig were detected. The cytoprotective property of Z-Lig was assessed in the tert-butyl hydroperoxide (t-BHP)-evoked oxidative stress model. Cell viability and reactive oxygen species (ROS) levels in EA.hy926 cells were determined. An atherosclerosis model induced by HFD was used to determine the anti-atherosclerosis effect of Z-Lig in HFD-fed Ldlr-deficient mice.

Results

In vitro, 100 μM Z-Lig upregulated expressions of NRF2 and ARE-driven genes, promoted accumulation of nuclear NRF2 and unbound NRF2- KEAP1 complex in EA.hy926 cells. Furthermore, Z-Lig alleviated oxidative stress and cell injury caused by t-BHP via stimulation of the NRF2/ARE pathway. In vivo, intervention with 20 mg/kg Z-Lig markedly restrained atherosclerosis progression, including attenuation of HFD-induced atherosclerotic plaque formation, alleviation of lipid peroxidation and increase in antioxidant enzyme activity in aortas of HFD-fed Ldlr-/- mice. The chemopreventive effects of Z-Lig might be associated with the activation of NRF2 and ARE-driven genes.

Conclusions

The present study suggested that Z-Lig is an effective NRF2 activator, which can protect vascular endothelial cells from oxidative stress and rescue HFD-induced atherosclerosis.