The results suggested that PVAT increased several pro-angiogenic factor levels (MCP-1, IL-6, GM-CSF) and also up-regulated the expression of anti-angiogenic factor (PF-4) (Fig.?6). Open in a separate window Fig.?6 Mouse angiogenesis antibody array for supernatant of transplanted adipose tissue. 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-B 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-B. Electronic supplementary material The online version of this article (10.1186/s12967-018-1481-z) contains supplementary material, which is available to authorized users. test when comparisons were made between two groups. Values are expressed as mean??SEM, tube formation assay. c Ex vivo mouse aortic ring angiogenesis. d Immunostaining for CD31 of mouse aorta in c. e Statistical analysis for c (n?=?6). * em p? /em ?0.05 compared with vehicle group, HOKU-81 ** em p? /em ?0.01 compared with vehicle group, # em p? /em ?0.05 compared with PVAT group We next tested ex vivo angiogenesis via mouse aortic ring assay. The supernatant of transplanted PVAT markedly promoted the ex vivo mouse aortic ring angiogenesis which was confirmed by immunostaining of CD31 (Fig.?5c, d). When ER stress in PVAT was inhibited by 4-PBA, the angiogenesis effect would become weaker. Thus, Rabbit polyclonal to Synaptotagmin.SYT2 May have a regulatory role in the membrane interactions during trafficking of synaptic vesicles at the active zone of the synapse from the in vitro, ex vivo and in vivo evidences, we concluded that PVAT could promote angiogenesis, which could be attenuated HOKU-81 by ER stress inhibitor. Mouse angiogenesis antibody array for angiogenic factors produced by transplanted adipose tissue In spite of angiogenic effect of PVAT, it is still unkown about the related angiogenic factors playing an important role in the angiogenic process. Therefore, we determined to screen out these factors by mouse angiogenesis antibody array which could detect 24 antibodies directed to proteins involved in angiogenesis. The results suggested that PVAT increased several pro-angiogenic factor levels (MCP-1, IL-6, GM-CSF) and also up-regulated the expression of anti-angiogenic factor (PF-4) (Fig.?6). Open in a separate window Fig.?6 Mouse angiogenesis antibody array for supernatant of transplanted adipose tissue. HOKU-81 a Mouse angiogenesis antibody array detected 24 antibodies. b Statistical analysis for HOKU-81 a (n?=?3) ER stress upregulated GM-CSF expression of adipocytes by a transcriptional mechanism The results of angiogenesis antibody array revealed that 4-PBA reduced GM-CSF expression produced by PVAT. Then, we established the models of ER stress in adipocytes. We treated adipocytes with ER stress inducer tunicamycin (TM) (1?g/ml) or vehicle (DMSO) in the presence or absence of 5?mM 4-PBA. QRT-PCR results showed that TM induced GM-CSF gene expression in 3T3-L1 adipocytes and peaked at the 4th hour (Fig.?7a). Elisa results suggested the supernatant of adipocytes treated by TM had higher GM-CSF level than control, and 4-PBA attenuated GM-CSF expression (Fig.?7b). Open in a separate window Fig.?7 ER stress upregulated GM-CSF expression by a transcriptional mechanism. a GM-CSF mRNA levels of adipocytes treated with TM (1?g/ml) in different time..
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