Yajin Liu

Hyperhomocysteinemia activates the aryl hydrocarbon receptor/CD36 pathway to promote hepatic steatosis in mice

Hyperhomocysteinemia (HHcy) is associated with liver diseases such as fatty liver and hepatic fibrosis; however, the underlying mechanism is still largely unknown. The current study aimed to explore the signaling pathway involved in HHcy‐induced hepatic steatosis (HS). C57BL/6 mice were fed a high‐methionine diet (HMD) for 4 and 8 weeks to establish the HHcy mouse model. Compared to a chow diet, the HMD induced hepatic steatosis and elevated hepatic expression of CD36, a fatty acid transport protein. The increased CD36 expression was associated with activation of the aryl hydrocarbon receptor (AHR). In primary cultured hepatocytes, high levels of homocysteine (Hcy) treatment up‐regulated CD36 and increased subsequent lipid uptake; both were significantly attenuated by small interfering RNA (siRNA) knockdown of CD36 and AHR. Chromatin immunoprecipitation assay revealed that Hcy promoted binding of AHR to the CD36 promoter, and transient transfection assay demonstrated markedly increased activity of the AHR response element by Hcy, which was ligand dependent. Mass spectrometry revealed significantly increased hepatic content of lipoxin A4 (LXA4), a metabolite of arachidonic acid, in HMD‐fed mice. Furthermore, overexpression of 15‐oxoprostaglandin 13‐reductase 1, a LXA4 inactivation enzyme, inhibited Hcy‐induced AHR activation, lipid uptake, and lipid accumulation. Moreover, LXA4‐induced up‐regulation of CD36 and lipid uptake was inhibited by AHR siRNA in vitro in hepatocytes. Finally, treatment with an AHR antagonist reversed HHcy‐induced lipid accumulation by inhibiting the AHR‐CD36 pathway in mice. Conclusion: HHcy activates the AHR‐CD36 pathway by increasing hepatic LXA4 content, which results in hepatic steatosis. (Hepatology 2016;64:92‐105)

Inhibition of soluble epoxide hydrolase alleviated atherosclerosis by reducing monocyte infiltration in Ldlr(-/-) mice.

RATIONALE Circulating monocytes play pivotal roles in chronic inflammatory diseases. Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid, are known to have anti-inflammatory effects and are hydrolyzed by soluble epoxide hydrolase (sEH). OBJECTIVE We aimed to investigate the effect of sEH inhibition in atherogenesis. METHODS AND RESULTS Mice with low-density lipoprotein receptor deficiency (Ldlr(-/-)) with or without sEH inhibitor, and Ldlr/sEH double-knockout (DK) mice were fed a Western-type diet (WTD) for 6weeks to induce arteriosclerosis. Both sEH inhibition and gene depletion decreased the WTD-induced hyperlipidemia, plaque area and macrophage infiltration in mice arterial wall. Ly6C(hi) infiltration of monocytes remained similar in blood, spleen and bone marrow of DK mice, but was decreased in aortic lesions. To further assess the role of sEH or EETs in monocyte/macrophage infiltration in atherogenesis, we transplanted DK bone marrow into Ldlr(-/-) recipients, and then fed mice the WTD. Aortic lesions and Ly6C(hi) monocyte infiltration were reduced in mice with transplanted bone marrow of DK mice without diminishing the cholesterol level. Furthermore, sEH inhibition or gene depletion increased the ratio of EETs/DHETs and diminished the expression of P-selectin glycoprotein ligand 1 (PSGL-1) in mice peripheral-blood mononuclear cells. Monocyte adhesion to P-selectin and to tumor necrosis factor α-activated endothelial cells was also diminished by sEH inhibition. CONCLUSION sEH inhibition and gene depletion attenuated atherosclerosis in mice by decreasing the infiltration of monocytes into the artery wall. EET and PSGL-1 may play pivotal roles in monocyte/macrophage infiltration and atherogenesis.

Spectrum evaluation-assisted eicosanoid metabolomics for global eicosanoid profiling in human vascular endothelial cells

Eicosanoids are hundreds of metabolites derived from poly‐unsaturated fatty acids (PUFAs), which regulate biological processes from multiple angles via a complex metabolic network. Targeted eicosanoid metabolomics is used to study the eicosanoid profile in biological samples but only for eicosanoids with available standards. To expand the coverage of eicosanoids detected, we identified the eicosanoids without available standards by estimation of the retention time and comparison of the MS/MS spectra with the reference ones which was collected in a database from literature. Scheduled multiple reaction monitoring‐ information dependent acquisition‐ enhanced product ion (sMRM‐IDA‐EPI) scan mode was applied in this method, which was called Spectrum Evaluation‐assisted Eicosanoid Metabolomics (SEEM). By using this method, 243 eicosanoids (167 without standards) could be relatively quantified with precision over 90 percent. We applied the method to analyze the global profile of eicosanoids secreted by human umbilical vascular endothelial cells at the basal level and with n‐3 PUFA treatment. 26 putative eicosanoids showed altered levels, despite no available standards. In general, n‐3 PUFA treatment increased most of their own metabolites and decreased the epoxy‐, hydroxyl‐ and keto‐ linoleic acid metabolites. The application of the SEEM method proved its potency of identification and quantification of eicosanoids without standards.

Yes-Associated Protein Promotes Angiogenesis via Signal Transducer and Activator of Transcription 3 in Endothelial Cells

Rationale: Angiogenesis is a complex process regulating endothelial cell (EC) functions. Emerging lines of evidence support that YAP (Yes-associated protein) plays an important role in regulating the angiogenic activity of ECs. Objective: The objective of this study was to specify the effect of EC YAP on angiogenesis and its underlying mechanisms. Method and Results: In ECs, vascular endothelial growth factor reduced YAP phosphorylation time and dose dependently and increased its nuclear accumulation. Using Tie2Cre-mediated YAP transgenic mice, we found that YAP promoted angiogenesis in the postnatal retina and tumor tissues. Mass spectrometry revealed signal transducer and activator of transcription 3 (STAT3) as a potential binding partner of YAP in ECs. Western blot and immunoprecipitation assays indicated that binding with YAP prolonged interleukin 6–induced STAT3 nuclear accumulation by blocking chromosomal maintenance 1–mediated STAT3 nuclear export without affecting its phosphorylation. Moreover, angiopoietin-2 expression induced by STAT3 was enhanced by YAP overexpression in ECs. Finally, a selective STAT3 inhibitor or angiopoietin-2 blockage partly attenuated retinal angiogenesis in Tie2Cre-mediated YAP transgenic mice. Conclusions: YAP binding sustained STAT3 in the nucleus to enhance the latter’s transcriptional activity and promote angiogenesis via regulation of angiopoietin-2.

Metabolic profiling of murine plasma reveals eicosapentaenoic acid metabolites protecting against endothelial activation and atherosclerosis

Atherosclerosis results from a maladaptive inflammatory response initiated by the intramural retention of LDL in susceptible areas of the arterial vasculature. The ω‐3 polyunsaturated fatty acids (ω‐3) have protective effects in atherosclerosis; however, their molecular mechanism is still largely unknown. The present study used a metabolomic approach to reveal the atheroprotective metabolites of ω‐3 and investigate the underlying mechanisms.

Elevating ATP-binding cassette transporter G1 improves re-endothelialization function of endothelial progenitor cells via Lyn/Akt/eNOS in diabetic mice.

Endothelial progenitor cell (EPC) dysfunction contributes to diabetes-induced delay in endothelium repair after vessel injury, prominently associated with diabetic cardiovascular complications such as neointima formation. ATP-binding cassette transporter G1 (ABCG1) promotes cholesterol efflux to HDL, which may favorably affect EPC function. However, whether ABCG1 improves EPC function, especially in diabetes, remains unknown. Here we investigated the role of ABCG1 in EPCs by using Tie2-mediated ABCG1 transgenic (Tie2- ABCG1Tg) mice. Mice were injected with streptozotocin to induce diabetes mellitus. As compared with wild-type (WT) mice, in Tie2- ABCG1Tg mice, diabetes-impaired EPC migration and tube formation were reversed. In vitro gain-of-function and loss-of-function studies further revealed that ABCG1-overexpressing EPCs showed increased migration and tube formation and differentiation via the Lck/Yes-related novel protein tyrosine kinase /Akt/endothelial NO synthase pathway by enhancing cellular cholesterol efflux. Finally, type 1 and type 2 diabetic mouse models with arterial injury were intravenously injected with labeled EPCs from WT or Tie2- ABCG1Tg mice. Re-endothelialization in diabetic mice was improved to a greater extent by injection of ABCG1-overexpressing than WT EPCs. Our study demonstrated that ABCG1 in EPCs improved repair after vascular injury in diabetes by increasing EPC function such as migration, tube formation and differentiation, and subsequent re-endothelialization. ABCG1 might be a promising therapeutic target for diabetes-associated vascular diseases.-Shi, Y., Lv, X., Liu, Yan., Li, B., Liu, M., Yan, M., Liu, Yaj., Li, Q., Zhang, X., He, S., Zhu, M., He, J., Zhu, Yan, Zhu, Yi, Ai, D. Elevating ATP-binding cassette transporter G1 improves re-endothelialization function of endothelial progenitor cells via Lyn/Akt/eNOS in diabetic mice.

Improving the cytological diagnosis of high-grade serous carcinoma in ascites with a panel of complementary biomarkers in cell blocks

Precise cytological diagnosis of pelvic high‐grade serous carcinoma (HGSC) in ascites is important for tumour staging, therapeutic decision‐making and prognostic evaluation. However, it can often be difficult to distinguish metastatic HGSC cells from reactive mesothelial cells based on morphology alone. Immunocytochemical analysis of ascites cell blocks has been used to obtain accurate diagnosis and provide a reliable basis for treatment decisions in the clinic. This study was performed to determine whether a panel of antibodies is necessary to achieve high specificity and sensitivity for the identification of HGSC cells.

Macrophage Raptor Deficiency-Induced Lysosome Dysfunction Exacerbates Nonalcoholic Steatohepatitis

Background & Aims Nonalcoholic steatohepatitis (NASH) is an increasingly prevalent nonalcoholic fatty liver disease, characterized by inflammatory cell infiltration and hepatocellular damage. Mammalian target of rapamycin complex 1 (mTORC1) has been investigated extensively in the context of cancer, including hepatocellular carcinoma. However, the role of mTORC1 in NASH remains largely unknown. Methods mTORC1 activity in macrophages in human mild and severe NASH liver was compared. Mice with macrophage-specific deletion of the regulatory-associated protein of mTOR (Raptor) subunit and littermate controls were fed a high-fructose, palmitate, and cholesterol diet for 24 weeks or a methionine- and choline-deficient diet for 4 weeks to develop NASH. Results We report that in human beings bearing NASH, macrophage mTORC1 activity was lower in livers experiencing severe vs mild NASH liver. Moreover, macrophage mTORC1 disruption exacerbated the inflammatory response in 2 diet-induced NASH mouse models. Mechanistically, in response to apoptotic hepatocytes (AHs), macrophage polarization toward a M2 anti-inflammatory phenotype was inhibited in Raptor-deficient macrophages. During the digestion of AHs, macrophage mTORC1 was activated and coupled with dynamin-related protein 1 to facilitate the latter’s phosphorylation, leading to mitochondrial fission-mediated calcium release. Ionomycin or A23187, calcium ionophores, prevented Raptor deficiency–mediated failure of lysosome acidification and subsequent lipolysis. Blocking dynamin-related protein 1–dependent mitochondria fission impaired lysosome function, resulting in reduced production of anti-inflammatory factors such as interleukins 10 and 13. Conclusions Persistent mTORC1 deficiency in macrophages contributes to the progression of NASH by causing lysosome dysfunction and subsequently attenuating anti-inflammatory M2-like response in macrophages during clearance of AHs.