Most acute coronary events occur in the upstream region of stenotic atherosclerotic plaques that experience laminar shear stress (LSS) elevated above normal physiological levels. Many studies have described the atheroprotective effect on endothelial behavior of normal physiological LSS (approximately 15 dynes/cm2) compared to static or oscillatory shear stress (OSS), but it is unknown whether the levels of elevated shear stress imposed by a stenotic plaque would preserve, enhance or reverse this effect. Therefore we used transcriptomics and related functional analyses to compare human endothelial cells exposed to laminar shear stress of 15 (LSS15-normal) or 75 dynes/cm2 (LSS75-elevated). LSS75 upregulated expression of 145 and downregulated expression of 158 genes more than twofold relative to LSS15. Modulation of the metallothioneins (MT1-G, -M, -X) and NADPH oxidase subunits (NOX2, NOX4, NOX5, and p67phox) accompanied suppression of reactive oxygen species production at LSS75. Shear induced changes in dual specificity phosphatases (DUSPs 1, 5, 8, and 16 increasing and DUSPs 6 and 23 decreasing) were observed as well as reduced ERK1/2 but increased p38 MAP kinase phosphorylation. Amongst vasoactive substances, endothelin-1 expression decreased whereas vasoactive intestinal peptide (VIP) and prostacyclin expression increased, despite which intracellular cAMP levels were reduced. Promoter analysis by rVISTA identified a significant over representation of ATF and Nrf2 transcription factor binding sites in genes upregulated by LSS75 compared to LSS15. In summary, LSS75 induced a specific change in behavior, modifying gene expression, reducing ROS levels, altering MAP kinase signaling and reducing cAMP levels, opening multiple avenues for future study. J. Cell. Physiol. 226: 2841–2848, 2011. © 2011 Wiley-Liss, Inc.

Background: Rupture of advanced atherosclerotic plaques accounts for most life-threatening myocardial infarctions. Classical (M1) and alternative (M2) macrophage activation could promote atherosclerotic plaque progression and rupture by increasing production of proteases, including matrix metalloproteinases (MMPs). Lymphocyte-derived cytokines may be essential for generating M1 and M2 phenotypes in plaques, although this has not been rigorously tested until now.Methods and results: We validated the expression of M1 markers (iNOS and COX-2) and M2 markers (arginase-1, Ym-1, and CD206) and then measured MMP mRNA levels in mouse macrophages during classical and alternative activation in vitro. We then compared mRNA expression of these genes ex vivo in foam cells from subcutaneous granulomas in fat-fed immune-competent ApoE knockout (KO) and immune-compromised ApoE/Rag-1 double-KO mice, which lack all T and B cells. Furthermore, we performed immunohistochemistry in subcutaneous granulomas and in aortic root and brachiocephalic artery atherosclerotic plaques to measure the extent of M1/M2 marker and MMP protein expression in vivo. Classical activation of mouse macrophages with bacterial lipopolysaccharide in vitro increased MMPs-13, -14, and -25 but decreased MMP-19 and TIMP-2 mRNA expressions. Alternative activation with IL-4 increased MMP-19 expression. Foam cells in subcutaneous granulomas expressed all M1/M2 markers and MMPs at ex vivo mRNA and in vivo protein levels, irrespective of Rag-1 genotype. There were also similar percentages of foam cell macrophages (FCMs) carrying M1/M2 markers and MMPs in atherosclerotic plaques from ApoE KO and ApoE/Rag-1 double-KO mice.Conclusion: Classical and alternative activation leads to distinct MMP expression patterns in mouse macrophages in vitro. M1 and M2 polarization in vivo occurs in the absence of T and B lymphocytes in either granuloma or plaque FCMs.

BackgroundThelper1 (Th1) lymphocytes have been previously implicated in atherosclerotic plaque growth but their role in plaque vulnerability to rupture is less clear. We investigated whether T-bet knockout that prevents Th1 lymphocyte differentiation modulates classical (M1) macrophage activation or production of matrix degrading metalloproteinases (MMPs) and their tissue inhibitors, TIMPs.Methods & ResultsWe studied the effect of T-bet deletion in apolipoproteinE (ApoE) knockout mice fed a high fat diet (HFD) or normal chow diet (ND). Transcript levels of M1/M2 macrophage polarization markers, selected MMPs and TIMPs were measured by RT-qPCR in macrophages isolated from subcutaneous granulomas or in whole aortae. Immunohistochemistry of aortic sinus (AS) and brachiocephalic artery (BCA) plaques was conducted to quantify protein expression of the same factors.Deletion of T-bet decreased mRNA for the M1 marker NOS-2 in granuloma macrophages but levels of M2 markers (CD206, arginase-1 and Ym-1), MMPs-2, -9, -12, -13, -14 and -19 or TIMPs-1 to -3 were unchanged. No mRNA differences were observed in aortic extracts from mice fed a HFD for 12 weeks. Moreover, AS and BCA plaques were similarly sized between genotypes, and had similar areas stained for NOS-2, COX-2, MMP-12 and MMP-14 proteins. T-bet deletion increased MMP-13, MMP-14 and arginase-1 in AS plaques. After 35 weeks of ND, T-bet deletion reduced the size of AS and BCA plaques but there were no differences in the percentage areas stained for M1 or M2 markers, MMPs-12, -13, -14, or TIMP-3.ConclusionsAbsence of Th1 lymphocytes is associated with reduced plaque size in ApoE knockout mice fed a normal but not high fat diet. In either case, M1 macrophage polarization and expression of several MMPs related to plaque instability are either maintained or increased.

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Introduction Atherosclerosis is a chronic inflammatory disease, in which foam cell macrophage (FCM) accumulation is initiated by hypercholesterolemia. Classical (M1) and alternative (M2) activation of FCMs could play important roles in atherosclerotic plaque progression and rupture, in part by increasing production of matrix metalloproteinases (MMPs), which directly destabilise plaques. Thelper1(Th1) lymphocytes are believed essential for classical macrophage activation in plaque by secreting cytokines including interferon gamma, although this hypothesis has not been rigorously tested. Methods and Results We compared mRNA levels of putative markers for M1/M2 macrophage activation, MMPs and tissue inhibitors of MMPs (TIMPs) in FCMs from subcutaneous sponges implanted into high fat fed ApoE knockout (EO) and ApoE/Tbet double knockout (DKO) mice; the later lack Th1 lymphocytes. Furthermore, we performed immunohistochemistry(IHC) on aortic sinuses (AS) and brachiocephalic arteries (BCA) to compare markers of classical/M1(iNOS) or alternative/M2(Arg1) activation and MMP/TIMP protein expression. Only male mice were used. Data for mRNA (amplicon copies/ng RNA) and the proportion of staining in the plaque (% of total plaque area) are reported as mean ± SEM. FCMs in subcutaneous sponges from DKO mice had significantly less iNOS mRNA than those from EO mice (22 ± 3 vs 90 ± 13) and the same was true for MMP13 (7000 ± 1600 vs 15000 ± 2300). Unexpectedly, however, Arg-1 was also significantly less in DKO FCMs (32400 ± 4100 vs 55400 ± 5400). There were no differences in MMP12 (~60000), MMP14 (~6) or TIMP3 (~1500) transcript levels. DKO mice had significantly smaller plaques than EO mice in the AS but not the BCA. Although ASplaques were more macrophage rich than BCAplaques, IHC showed no difference in the percentage of plaque area occupied by macrophages in DKO and EO mice, either in the AS (70.9 ± 6.4% vs 69.1 ± 4.6%) or BCA (48.9 ± 7.2% vs 48.4 ± 4.9%). ASplaques of DKO mice were significantly enriched in smooth muscle cells vs EO mice (29.01 ± 0.17% vs 14.87 ± 0.06%) but the opposite was observed in BCAplaques (9.15 ± 0.05% vs 16.13 ± 0.08%). There were no significant differences between DKO and EO mice in the proportion of plaque area positive for iNOS (~10–20%) in either site. However, MMP13 protein expression (<20% in either site) was significantly increased in the AS in DKOvsEO mice (37.1 ± 6.8% vs 17.2 ± 2.6%) and the same was true for Arg-1 (<10% in either site) (5.58 ± 2.31% vs 0.91 ± 0.54%, respectively); both opposite of the sponge results. MMP12 protein expression was overall high (~50% of plaque area) and significantly less in DKOvsEO mice (22.6 ± 3.0% vs 56.1 ± 13.5%) in the BCA. Although there was more MMP14 protein in AS than BCA plaques (~50% vs ~30% respectively), there were no differences between the two genotypes. Finally, TIMP3 protein expression was significantly increased in AS plaques in DKOvsEO mice (41.1 ± 6.3% vs 27.2 ± 4.0%). Conclusion Deletion of Th1 lymphocytes altered several properties of...

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