171 Deleting TH1-lymphocytes Influences Macrophage Activation and Metalloproteinase Expression in apoe Null Mice

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Abstract

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.

Methods and Results

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 FCMs in subcutaneous sponges and atherosclerotic plaques. However, the effects were the opposite in sponges and in atherosclerotic plaques and do not fit the M1/M2 paradigm. Smaller size, increased smooth muscle cells, decreased expression of MMP12 and increased TIMP3 in AS plaques of DKO mice could imply a deleterious effect of Th1 cells on plaque stability.

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