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. 2011 Jun 24;286(25):22665-77.
doi: 10.1074/jbc.M110.202135. Epub 2011 May 2.

Mechanisms of urokinase plasminogen activator (uPA)-mediated atherosclerosis: role of the uPA receptor and S100A8/A9 proteins

Stephen D Farris  1 , Jie Hong Hu, Ranjini Krishnan, Isaac Emery, Talyn Chu, Liang Du, Michal Kremen, Helén L Dichek, Elizabeth Gold, Stephen A Ramsey, David A Dichek
Affiliations

Mechanisms of urokinase plasminogen activator (uPA)-mediated atherosclerosis: role of the uPA receptor and S100A8/A9 proteins

Stephen D Farris et al. J Biol Chem. .

Abstract

Data from clinical studies, cell culture, and animal models implicate the urokinase plasminogen activator (uPA)/uPA receptor (uPAR)/plasminogen system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/uPAR/plasminogen stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression and mice genetically deficient in uPAR to elucidate mechanisms of uPA/uPAR/plasminogen-accelerated atherosclerosis and aneurysm formation. We found that macrophage-specific uPA overexpression accelerates atherosclerosis and causes aortic root dilation in fat-fed Ldlr(-/-) mice (as we previously reported in Apoe(-/-) mice). Macrophage-expressed uPA accelerates atherosclerosis by stimulation of lesion progression rather than initiation and causes disproportionate lipid accumulation in early lesions. uPA-accelerated atherosclerosis and aortic dilation are largely, if not completely, independent of uPAR. In the absence of uPA overexpression, however, uPAR contributes modestly to both atherosclerosis and aortic dilation. Microarray studies identified S100A8 and S100A9 mRNA as the most highly up-regulated transcripts in uPA-overexpressing macrophages; up-regulation 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 up-regulated in the aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is up-regulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Macrophage 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 reveal uPAR independence of these actions and implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease.

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Figures

FIGURE 1.
FIGURE 1.
PA activity of medium conditioned by bone marrow-derived macrophages and explanted aortae. Medium conditioned by macrophages (A) or aortae (B) of Ldlr−/− mice (either nontransgenic (0/0) or hemizygous for the SR-uPA transgene (+/0)) was assayed for PA activity by the addition of plasminogen and the plasmin substrate S-2251. Data points are values from individual mice; bars are group means. p values are from the Mann-Whitney test.
FIGURE 2.
FIGURE 2.
Aortic root atherosclerosis in 15-week-old mice. Aortic roots from Ldlr−/− Plaur+/+ mice (either nontransgenic (0/0) or hemizygous for the SR-uPA transgene (+/0)) were sectioned and stained, and lesions were measured. A, shown is the intimal lesion area. Data points are values from individual mice; bars are group means. B and C, shown are representative sections from each of the genotypes with a hematoxylin and eosin stain; arrows = coronary ostia; size bars = 500 μm. p value is from a t test.
FIGURE 3.
FIGURE 3.
Plasma uPA antigen and PA activity in medium conditioned by bone marrow-derived macrophages and explanted aortae. A, plasma uPA was measured by ELISA. Medium conditioned by macrophages (B) or aortae (C) from Ldlr−/− mice (either nontransgenic (0/0) or hemizygous for the SR-uPA transgene (+/0) and either Plaur+/+ or Plaur−/−) was assayed for PA activity by the addition of plasminogen and the plasmin substrate S-2251. Data points are values from individual mice; bars are group means. p values comparing SR-uPA+/0 versus SR-uPA0/0 groups are from Mann-Whitney tests; p values comparing Plaur+/+ and Plaur−/− groups are from t tests.
FIGURE 4.
FIGURE 4.
Aortic root atherosclerosis in 25-week-old mice. Aortic roots from Ldlr−/− Plaur+/+ or Ldlr−/− Plaur−/− mice (either nontransgenic (0/0) or hemizygous for the SR-uPA transgene (+/0)) were sectioned and stained, and lesions were measured. A, the intimal lesion area is shown. Data points are values from individual mice; bars are group means. B–E, representative sections are from each of the genotypes and hematoxylin- and eosin-stained; size bars = 500 μm. p values are from t tests except for comparison of SR-uPA+/0 Plaur+/+ versus SR-uPA0/0 Plaur+/+ groups (Mann-Whitney test).
FIGURE 5.
FIGURE 5.
Aortic surface atherosclerosis in 25-week-old mice. Aortae from Ldlr−/− Plaur+/+ or Ldlr−/− Plaur−/− mice (either nontransgenic (0/0) or hemizygous for the SR-uPA transgene (+/0)) were pinned and stained with Sudan IV, and lesions were measured. A, the percentage of surface of each aorta staining positive with Sudan IV is shown. Data points are values from individual mice; bars are group means. B–E, shown are representative aortae from each of the genotypes. p values are from t tests.
FIGURE 6.
FIGURE 6.
S100A9 and S100A8 mRNA in macrophages and aortae of Apoe−/− mice. Total RNA was extracted from peritoneal macrophages or aortae; S100A9 and S100A8 mRNA were measured by qRT-PCR, with normalization to GAPDH mRNA. A and B, S100A9 and S100A8 mRNA in peritoneal macrophages from SR-uPA0/0 or SR-uPA+/0 mice are shown. C and D, S100A9 and S100A8 mRNA in aortae of SR-uPA0/0 recipients of either SR-uPA0/0 or SR-uPA+/0 bone marrow are shown. E and F, S100A9 and S100A8 mRNA in aortae of SR-uPA0/0 or SR-uPA+/0 mice are shown. Data points are individual mice; bars are group medians. p values are from Mann-Whitney tests. AU, arbitrary units.
FIGURE 7.
FIGURE 7.
S100A9 protein in extracts of peritoneal macrophages of nontransgenic versus SR-uPA+/0 mice (all Apoe−/−). Mice were fed a high fat diet from 5 until 10 weeks of age, peritoneal macrophages were harvested, and protein was extracted and analyzed. A, shown is a Western blot of macrophage extracts from individual nontransgenic and SR-uPA+/0 mice (upper panel). The blot was stripped and reprobed to detect GAPDH (lower panel). B, densities of the S100A9 bands in A and from three additional mice in each group, normalized to the corresponding GAPDH densities are shown. Data points are from individual mice from two independent experiments; bars are group means, with the mean of the nontransgenic group in each experiment set to 1.0. The p value is from t tests. AU, arbitrary units.
FIGURE 8.
FIGURE 8.
S100A9 mRNA, measured in nontransgenic macrophages. Peritoneal macrophages harvested from nontransgenic Apoe−/− mice were treated for 6 h with DMEM alone or with DMEM previously conditioned by SR-uPA+/0 or SR-uPA0/0 Apoe−/− macrophages. RNA was extracted, and S100A9 and GAPDH mRNA were measured by qRT-PCR. Data are the means ± S.E. of three independent experiments performed with a total of 4–8 wells of cultured macrophages for each group. The mean of the DMEM-only group in each experiment was set to 1.0. p values are from t tests. AU, arbitrary units.
FIGURE 9.
FIGURE 9.
Model of pathways through which macrophage uPA expression could accelerate atherosclerosis. The model is constructed based on data from the present study and others showing that macrophage overexpression of uPA promotes early lesion lipid accumulation as well as later lesion macrophage accumulation, increases macrophage migration, enhances vascular matrix metalloproteinase (MMP) activity, accelerates lesion progression, and stimulates macrophage S100A8/A9 expression (–16). 1, uPA-expressing macrophages begin to accumulate within an early lesion. 2, These macrophages secrete S100A8/A9 (SA8/9) themselves and can also up-regulate S100A8/A9 expression in other plaque cells (red arrows; see also Fig. 8) including macrophages and potentially in endothelial cells and smooth muscle cells, both of which can express S100A8/A9 (62, 63). 3, S100A8/A9 secreted by lesion cells increases monocyte CD11b expression and adhesion to endothelial ICAM-1 (57, 58). 4, as monocytes differentiate to macrophages, they up-regulate uPA expression, which enhances their migration into the lesion (14). 5, S100A8/A9 stimulation of endothelium also loosens tight junctions and promotes endothelial apoptosis (60, 64), effects that facilitate entry of both monocyte/macrophages and lipids to the artery wall. 6, lesion-derived S100A8/A9 binds to RAGE on endothelial cells and TLR4 on macrophages and smooth muscle cells, up-regulating expression of atherogenic cytokines MCP-1 and IL-6 in endothelium, and increasing lipid uptake by cells within the lesion (63, 65). 7, increased endothelial basement membrane proteolysis by uPA/Plg-activated matrix metalloproteinases (MMPs) (16) also increases artery wall permeability. 8, proteolysis of matrix protein within lesions exposes neoepitopes with apolipoprotein-binding activity, increasing lesion lipid retention.
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