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Abstract Background Perivascular adipose tissue (PVAT) accelerates plaque progression and increases cardiovascular risk. We tested the hypothesis that PVAT contributed to plaque vulnerability and investigated whether endoplasmic reticulum stress (ER stress) in PVAT played an important role in vulnerable plaque. Methods We transplanted thoracic aortic PVAT or subcutaneous adipose tissue as a control, from donor mice to carotid arteries of recipient apolipoprotein E deficient (apoE−/−) mice after removing carotid artery collar placed for 6 weeks. Two weeks after transplantation, ER stress inhibitor 4-phenyl butyric acid (4-PBA) was locally administrated to the transplanted PVAT and then animals were euthanized after 4 weeks. Immunohistochemistry was performed to quantify plaque composition and neovascularization. Mouse angiogenesis antibody array kit was used to test the angiogenic factors produced by transplanted adipose tissue. In vitro tube formation assay, scratch wound migration assay and mouse aortic ring assay were used to assess the angiogenic capacity of supernatant of transplanted PVAT. Results Ultrastructural detection by transmission electron microscopy showed transplanted PVAT was a mixed population of white and brown adipocytes with abundant mitochondria. Transplanted PVAT increased the intraplaque macrophage infiltration, lipid core, intimal and vasa vasorum neovascularization and MMP2/9 expression in plaque while decreased smooth muscle cells and collagen in atherosclerotic plaque, which were restored by local 4-PBA-treatment. Antibody array analysis showed that 4-PBA reduced several angiogenic factors [Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), MCP-1, IL-6] secreted by PVAT. Besides, conditioned medium from 4-PBA treated-PVAT inhibited tube formation and migration capacity of endothelial cells and ex vivo mouse aortic ring angiogenesis compared to conditioned medium from transplanted PVAT. mRNA expression and protein levels of GM-CSF were markedly elevated in adipocytes under ER stress which would be suppressed by 4-PBA. In addition, ER stress enhanced NF-κB binding to the promoter of the mouse GM-CSF gene in adipocytes confirmed by Chromatin immunoprecipitation analyses. Conclusions Our findings demonstrate that ER stress in PVAT destabilizes atherosclerotic plaque, in part through increasing GM-CSF paracrine via transcription factor NF-κB.

Abstract Background Colorectal cancer (CRC) is a worldwide cancer with rising annual incidence. New medications for patients with CRC are still needed. Recently, fluorescent chemical probes have been developed for cancer imaging and therapy. Signal transducer and activator of transcription 1 (STAT1) has complex functions in tumorigenesis and its role in CRC still needs further investigation. Methods RNA sequencing datasets in the NCBI GEO repository were analyzed to investigate the expression of STAT1 in patients with CRC. Xenograft mouse models, tail vein injection mouse models, and azoxymethane/dextran sodium sulfate (AOM/DSS) mouse models were generated to study the roles of STAT1 in CRC. A ligand-based high-throughput virtual screening approach combined with SWEETLEAD chemical database analysis was used to discover new STAT1 inhibitors. A newly designed and synthesized fluorescently labeled 4’,5,7-trihydroxyisoflavone (THIF) probe (BODIPY-THIF) elucidated the mechanistic actions of STAT1 and THIF in vitro and in vivo. Colonosphere formation assay and chick chorioallantoic membrane assay were used to evaluate stemness and angiogenesis, respectively. Results Upregulation of STAT1 was observed in patients with CRC and in mouse models of AOM/DSS-induced CRC and metastatic CRC. Knockout of STAT1 in CRC cells reduced tumor growth in vivo. We then combined a high-throughput virtual screening approach and analysis of the SWEETLEAD chemical database and found that THIF, a flavonoid abundant in soybeans, was a novel STAT1 inhibitor. THIF inhibited STAT1 phosphorylation and might bind to the STAT1 SH2 domain, leading to blockade of STAT1-STAT1 dimerization. The results of in vitro and in vivo binding studies of THIF and STAT1 were validated. The pharmacological treatment with BODIPY-THIF or ablation of STAT1 via a CRISPR/Cas9-based strategy abolished stemness and angiogenesis in CRC. Oral administration of BODIPY-THIF attenuated colitis symptoms and tumor growth in the mouse model of AOM/DSS-induced CRC. Conclusions This study demonstrates that STAT1 plays an oncogenic role in CRC. BODIPY-THIF is a new chemical probe inhibitor of STAT1 that reduces stemness and angiogenesis in CRC. BODIPY-THIF can be a potential tool for CRC therapy as well as cancer cell imaging.

Abstract Background Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells, and its mRNA destabilization activity is regulated by protein phosphorylation. Methods We generated an antibody against phospho-Serine316 located at the C-terminal NOT1-binding site and examined TTP phosphorylation in LPS-stimulated RAW264.7 cells. Knockout of TTP was created in RAW264.7 cells using CRISPR/Cas9 gene editing to explore TTP functions. Results We demonstrated that Ser316 was phosphorylated by p90 ribosomal S6 kinase 1 (RSK1) and p38-activated protein kinase (MK2) and dephosphorylated by Protein Phosphatase 2A (PP2A). A phosphorylation-mimic mutant of S316D resulted in dissociation with the CCR4-NOT deadenylase complex through weakening interaction with CNOT1. Furthermore, Ser316 and serines 52 and 178 were independently contributed to the CCR4-NOT complex recruitment in the immunoprecipitation assay using phosphor-mimic mutants. In RAW264.7 macrophages, TTP was induced, and Ser316 was phosphorylated through RSK1 and MK2 by LPS stimulation. Knockout of TTP resulted in TNFα mRNA increased due to mRNA stabilization. Overexpression of non-phosphorylated S316A TTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316 phosphorylation decreased the interaction with CNOT1. Conclusions Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316 phosphorylation via regulating the TTP interaction with the CCR4-NOT deadenylase complex.