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Proteome profiling in IL-1? and VEGF-activated human umbilical vein endothelial cells delineates the interlink between inflammation and angiogenesis.

ABSTRACT: Endothelial cells represent major effectors in inflammation and angiogenesis, processes that drive a multitude of pathological states such as atherosclerosis and cancer. Both inflammation and angiogenesis are interconnected with each other in the sense that many pro-inflammatory proteins possess proangiogenic properties and vice versa. To elucidate this interplay further, we present a comparative proteome study of inflammatory and angiogenic activated endothelial cells. HUVEC were stimulated with interleukin 1-? and VEGF, respectively. Cultured primary cells were fractionated into secreted, cytoplasmic and nuclear protein fractions and processed for subsequent LC-MS/MS analysis. Obtained protein profiles were filtered for fraction-specific proteins to address potential cross fractional contamination, subjected to comparative computational biology analysis (GO-Term enrichment analysis, weighted gene co-expression analysis) and compared to published mRNA profiles of IL-1? respectively VEGF stimulated HUVEC. GO Term enrichment analysis and comparative pathway analysis revealed features such as NOD and NfkB signaling for inflammatory activated HUVEC and VEGF and ErB signaling for VEGF-activated HUVEC with potential crosstalk via map kinases MAP2K2. Weighted protein co-expression network analysis revealed several potential hub genes so far not associated with driver function in inflammation or angiogenesis such as HSPG2, ANXA3, and GPI. "Classical" inflammation or angiogenesis markers such as IL6, CXCL8 or CST1 were found in a less central position within the co-expression networks. In conclusion, this study reports a framework for the computational biology based analysis of proteomics data applied to cytoplasmic, nucleic and extracellular fractions of quiescent, inflammatory and angiogenic activated HUVEC. Novel potential hub genes relevant for these processes were successfully identified.

SUBMITTER: Mohr T

PROVIDER: S-EPMC5472280 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Proteome profiling in IL-1β and VEGF-activated human umbilical vein endothelial cells delineates the interlink between inflammation and angiogenesis.

Mohr Thomas T Haudek-Prinz Verena V Slany Astrid A Grillari Johannes J Micksche Michael M Gerner Christopher C

PloS one 20170615 6

Endothelial cells represent major effectors in inflammation and angiogenesis, processes that drive a multitude of pathological states such as atherosclerosis and cancer. Both inflammation and angiogenesis are interconnected with each other in the sense that many pro-inflammatory proteins possess proangiogenic properties and vice versa. To elucidate this interplay further, we present a comparative proteome study of inflammatory and angiogenic activated endothelial cells. HUVEC were stimulated wit ...[more]

PMID: 28617818

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Project description:BackgroundMicroparticles (MPs) are small extracellular plasma membrane particles shed by activated and apoptotic cells, which are involved in the development of atherosclerosis. Our previous study found that microRNA (miR)-19b encapsulated within endothelial MPs (EMPs) may contribute to the upregulation of circulating miR-19b in unstable angina patients. Hypoxia is involved in atherosclerosis as a critical pathological stimulus. However, it still remains unclear whether the increase of miR-19b levels in EMPs is related to hypoxia and if the effect of miR-19b - wrapped within EMPs - stimulates hypoxia on vascular endothelial cells. This study aimed to explore the changes of miR-19b in EMPs induced by hypoxia as well as their effects on endothelial cells.MethodsHuman umbilical vein endothelial cells (HUVECs) were cultured in vitro and arranged to harvest EMPs in two parts: the first part consisted of EMPcontrol and EMPhypoxia and the second part included EMPvehicle, EMPNC mimic, and EMPmiR-19b mimic. Cell migration was detected by scratch migration and transwell chamber migration. Angiogenesis was assessed by tube formation assays. Furthermore, we predicted the target gene of miR-19b by bioinformatics analysis, and luciferase assay was used to verify the targeted gene of miR-19b. Data were analyzed by one-way analysis of variance. Student's t-test was used when two groups were compared.ResultsCompared with EMPcontrol- and EMPhypoxia-inhibited migration of cells by scratch migration assay (80.77 ± 1.10 vs. 28.37 ± 1.40, P < 0. 001) and transwell chamber migration assay (83.00 ± 3.46 vs. 235.00 ± 16.52, P < 0.01), the number of tube formations was markedly reduced by 70% in the EMPhypoxia group (P < 0.001) in vitro analysis of HUVECs. Meanwhile, a strong inhibition of migration and tube formation of HUVECs in the presence of miR-19b-enriched EMPmiR-19b mimic was observed. This effect might be due to the delivery of miR-19b in EMPs. Transforming growth factor-?2 (TGF?2) was predicted to be one of the target genes of miR-19b, and we further confirmed that TGF?2 was a direct target gene of miR-19b using the luciferase assay. The expression of TGF?2 in HUVECs was inhibited by treatment with EMPhypoxia and EMPmiR-19b mimic.ConclusionsMiR-19b in EMPs induced by hypoxia could reduce endothelial cell migration and angiogenesis by downregulating TGF?2 expression, which may have inhibited the progression of atherosclerosis.

Dataset Information

Proteome profiling in IL-1? and VEGF-activated human umbilical vein endothelial cells delineates the interlink between inflammation and angiogenesis.

Publications

Proteome profiling in IL-1β and VEGF-activated human umbilical vein endothelial cells delineates the interlink between inflammation and angiogenesis.

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