Macrophages in human atheroma contain PPARgamma: differentiation-dependent peroxisomal proliferator-activated receptor gamma(PPARgamma) expression and reduction of MMP-9 activity through PPARgamma activation in mononuclear phagocytes in vitro

Abstract

Mononuclear phagocytes play an important role in atherosclerosis and its sequela plaque rupture in part by their secretion of matrix metalloproteinases (MMPs), including MMP-9. Peroxisomal proliferator-activated receptor gamma (PPARgamma), a transcription factor in the nuclear receptor superfamily, regulates gene expression in response to various activators, including 15-deoxy-delta12,14-prostaglandin J2 and the antidiabetic agent troglitazone. The role of PPARgamma in human atherosclerosis is unexplored. We report here that monocytes/macrophages in human atherosclerotic lesions (n = 12) express immunostainable PPARgamma. Normal artery specimens (n = 6) reveal minimal immunoreactive PPARgamma. Human monocytes and monocyte-derived macrophages cultured for 6 days in 5% human serum expressed PPARgamma mRNA and protein by reverse transcription-polymerase chain reaction and Western blotting, respectively. In addition, PPARgamma mRNA expression in U937 cells increased during phorbol 12-myristate 13 acetate-induced differentiation. Stimulation of PPARgamma with troglitazone or 15-deoxy-delta12,14-prostaglandin J2 in human monocyte-derived macrophages inhibited MMP-9 gelatinolytic activity in a concentration-dependent fashion as revealed by zymography. This inhibition correlates with decreased MMP-9 secretion as determined by Western blotting. Thus, PPARgamma is present in macrophages in human atherosclerotic lesions and may regulate expression and activity of MMP-9, an enzyme implicated in plaque rupture. PPARgamma is likely to be an important regulator of monocyte/macrophage function with relevance for human atherosclerotic disease.

Figure 1.

Expression of PPARγ in human atherosclerotic lesions. A: Low-power view of frozen section of human carotid lesions shows immunoreactive PPARγ next to the lipid core in the shoulder region with abundant macrophages present. Similar results were seen in other atherosclerotic specimens (n = 12). B: No immunoreactive PPARγ is detectable in parallel sections stained with PPARγ antibodies preabsorbed with the immunizing peptide, indicating that staining for PPARγ in (A) and (C) is specific. Similar results were seen with immunoglobulin G controls (not shown). C: High-power view of the area indicated by the rectangle in (A) shows PPARγ staining restricted to macrophage nuclei (see quantification in Results). D: In nonatherosclerotic arteries (n = 6), little PPARγ could be detected, with scant staining in occasional vascular smooth muscle cells.

Figure 2.

A: PPARγ mRNA and protein are expressed in cells of the monocyte/macrophage lineage. Upper panel: reverse transcription-polymerase chain reaction of PPARγ mRNA in freshly prepared monocytes (Mo), monocyte-derived macrophages (MØ), and PMA-differentiated U937 cells (U937) reveals a cDNA fragment of the expected size. Also shown are a 100-bp DNA ladder (MW) and negative control without cDNA (Co). Lower panel: Western blot analysis of PPARγ protein expression in nuclear extracts of freshly prepared monocytes (Mo), monocyte-derived macrophages (MØ), and U937 cells (U937) reveals a band of the appropriate size. The identity of this band is confirmed by co-migration with a band seen in PPARγ-transfected human skin fibroblasts (+) but not in similar but untransfected fibroblasts (−). All results shown were reproduced in three independent experiments. B (right): PPARγ mRNA expression in undifferentiated (Undiff.) and PMA-differentiated U937 cells (Diff.), as shown by Northern blot analysis. Differentiated U937 cells show increased PPARγ mRNA expression compared with undifferentiated cells. B (left): Ethidium bromide staining demonstrates equal loading of intact RNA. Results shown were reproduced in three independent experiments.

Figure 3.

A: PPARγ activators decrease MMP-9 gelatinolytic activity in supernatants from monocyte-derived macrophages (MØ) in a concentration-dependent fashion, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. Monocytes were cultured in 5% human serum in the presence or absence of troglitazone or 15 d-PGJ₂ as indicated. Supernatants of PMA-treated fibroblasts served as a control (Co). Gelatinolytic activity of freshly prepared monocytes is also shown (Mo). Equal amounts of lysates were loaded. Results shown were reproduced in three independent experiments. B: PPARγ activators decrease MMP-9 protein levels in supernatants from monocyte-derived macrophages (MØ). Vascular smooth muscle cells treated with PMA at 50 μg/L served as a control (Co). Supernatants from freshly prepared monocyte supernatant (Mo) are also shown. Similar results were reproduced in three independent experiments.