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Mechanisms of urokinase plasminogen activator (uPA)-mediated atherosclerosis

ABSTRACT: Data from clinical studies, cell culture, and animal models implicate the urokinase (uPA)/Plasminogen (Plg) system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/Plg stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression to clarify mechanisms of uPA/Plg-accelerated atherosclerosis and aneurysm formation. Microarray studies were performed to identify potential mediators of uPA-accelerated atherosclerosis. These studies identified S100A8 and S100A9 mRNA as the most highly upregulated transcripts in uPA-overexpressing macrophages; upregulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also upregulated in aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is upregulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Bioinformatics analysis of the microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm—in a second animal model—that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease.

ORGANISM(S): Mus musculus

PROVIDER: GSE29028 | GEO | 2011/05/03

SECONDARY ACCESSION(S): PRJNA140485

REPOSITORIES: GEO

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Project description:Data from clinical studies, cell culture, and animal models implicate the urokinase (uPA)/Plasminogen (Plg) system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/Plg stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression to clarify mechanisms of uPA/Plg-accelerated atherosclerosis and aneurysm formation. Microarray studies were performed to identify potential mediators of uPA-accelerated atherosclerosis. These studies identified S100A8 and S100A9 mRNA as the most highly upregulated transcripts in uPA-overexpressing macrophages; upregulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also upregulated in aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is upregulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Bioinformatics analysis of the microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm—in a second animal model—that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease. Six independent biological replicate RNA samples were prepared from thioglycollate-elicited peritoneal macrophages from mice of two different genotypes: SR-uPA+/0 transgenic (overexpress uPA in macrophages) and nontransgenic, respectively), for a total of 12 independent RNA samples. Both transgenic and nontransgenic mice were Apoe-/- and were fed Western diet from 5-15 weeks before peritoneal macrophages were elicited. In addition, from the six samples of each genotype, a pooled sample was prepared by combining the six genotype-specific samples. Each of the two pooled samples was assayed on the BeadChip in two technical replicates, for a total of 16 hybridizations performed using two Illumina Mouse Ref-8 v1.1 chips.

Project description:Background: Cardiovascular diseases remain the leading cause of morbidity and mortality worldwide, most of which are caused by atherosclerosis. Discerning processes that participate in macrophage-to-foam cell formation are critical for understanding the basic mechanisms underlying atherosclerosis. To explore the molecular mechanisms of foam cell formation, the differentially expressed proteins were identified. Methods: In this paper, human monocytes, macrophage colony-stimulating factor induced macrophages, and oxidized low-density lipoprotein induced foam cells were cultured, and tandem mass tag (TMT) labeling combined with mass spectrometry (MS) were performed to find associations between foam cell transformation and proteome profiles. Results: Totally, 5146 quantifiable proteins were identified, among which 1515 and 182 differentially expressed proteins (DEPs) were found in macrophage/monocyte and foam cell/macrophage, respectively, using a cutoff of 1.5-fold change. Subcellular localization analysis revealed that downregulated DEPs of macrophages/monocytes were mostly located in the nucleus and upregulated DEPs of foam cells/macrophages mostly located in the plasma membrane and extracellular. Functional analysis of DEPs demonstrated that cholesterol metabolism related proteins were upregulated in foam cells, whereas the immune response-related proteins were downregulated in foam cells. The protein-interaction network showed that the DEPs with the highest interaction intensity between macrophages and foam cells were mainly concentrated in lysosomes and the endoplasmic reticulum. Conclusions: This study for the first time to perform quantitative proteomic investigation by TMT labeling and LC-MS/MS to identify differentially expressed proteins in human monocyte, macrophage, and foam cell. The results confirmed cholesterol metabolism was upregulated in foam cells, while immune response was suppressed, which suggested that foam cells were not the population that promote inflammation. In addition, KEGG enrichment analysis and protein-protein interaction indicated that the differentially expressed proteins locating in the endoplasmic reticulum and lysosomes may be key targets to regulate foam cell formation. These data provide a basis for identifying the potential proteins associated with the molecular mechanism involved in the transformation of macrophages to foam cells.

Project description:Background: Atherosclerosis leads high mortality, highlighting an urgent need for new therapeutic strategies. Protein kinases orchestrate multiple cellular events in atherosclerosis and may provide new therapeutic targets for atherosclerosis. Here, we identified a protein kinase, WEE1 G2 checkpoint kinase (WEE1), promoting inflammatory response in atherosclerosis. Methods: The ApoE-/- mice without macrophage-specific WEE1 deletion (ApoE-/-WEE1f/f) and ApoE-/- mice with macrophage-specific WEE1 deletion (ApoE-/-WEE1MCKO) were generated using bone marrow transplantation. These mice and WEE1 inhibitor MK1775 were used in a high-fat diet (HFD)-induced atherosclerotic model. Human atherosclerotic tissues, mouse primary peritoneal macrophages (MPMs), 293T cells, and recombinant proteins were utilized to investigate the pathogenic role and underlying mechanisms of WEE1. Results: We identified up-regulated p-WEE1 in macrophages in atherosclerotic lesions of HFD-fed ApoE-/- mice. Transcriptome sequencing analysis indicated that WEE1 promotes oxLDL-induced inflammation in macrophages. We then demonstrated that macrophage-specific deletion or pharmacological inhibition of WEE1 attenuates atherosclerosis by reducing inflammation both in vivo and in vitro. The overexpression of wild-type WEE1 or activating-mutant WEE1, but not inactivating-mutant WEE1, exacerbates inflammation in macrophages. Mechanistically, transcriptome sequencing analysis and co-immunoprecipitation followed by quantitative proteomics analysis identified p65 as a binding protein of WEE1. We confirmed that WEE1 directly interacts with the RHD domain of p65 via kinase domain and phosphorylates p65 at S536, thereby facilitating subsequent NF-κB activation and inflammatory response in macrophages. Conclusions: Our findings demonstrated that macrophage WEE1 promotes inflammatory atherosclerosis by directly binding to p65 and phosphorylate it at S536. This study provides WEE1 as a new p65 regulator in inflammation and a potential therapeutic target for atherosclerosis.

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Mechanisms of urokinase plasminogen activator (uPA)-mediated atherosclerosis

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