FIGURE 1.

Gremlin-1 is expressed in atherosclerotic lesions in ApoE^−/− mice. Aortic tissue was obtained from ApoE^−/− (n = 10) and wild type (n = 7) mice at the age of 8 and 16 weeks, respectively. ApoE^−/− mice were fed with a cholesterol-rich diet for 4 or 12 weeks beginning at the age of 4 weeks. a, mRNA expression of Gremlin-1 or aldolase was determined by RT-PCR, and mRNA expression was quantified by densitometry and calculated in the same manner as Gremlin-1/aldolase. Data represent mean ± S.E. (**, indicates statistical significance of p < 0.01). b, Gremlin-1 expression was analyzed in aortic tissue derived from 16-week-old ApoE^−/− and wild type mice by in situ hybridization using specific probes. In situ hybridization revealed enhanced Gremlin-1 mRNA expression in atherosclerotic lesions. Arrows indicate Gremlin-1-positive cells. Representative images of three in situ hybridization analyses are shown (50). c, protein expression of Gremlin-1 in aortic tissue from 16-week-old ApoE^−/− and wild type mice was assessed by immunostaining using a mAb directed against human Gremlin-1 (upper left panels). Parallel sections were stained with anti-CD68 to detect monocytes/macrophages (upper right panels). Immunostaining with anti-Gremlin-1 showed that Gremlin-1 protein expression in aortic tissue of ApoE^−/− mice was substantially enhanced compared with the aortic specimen derived from wild type mice. Anti-CD68 staining revealed that Gremlin-1 expression in aortic lesions from ApoE^−/− mice was found primarily in monocytes/macrophages as shown by confocal colocalization studies (lower panel). Representative images of seven immunostainings are shown altogether. d, Gremlin-1 is expressed in isolated monocytes/macrophages and is released upon activation. RT-PCR analysis and immunoblotting with anti-Gremlin-1 showed that Gremlin-1 is expressed on the protein level in monocytes, macrophages/foam cells, and human aortic endothelial cells. The two immunoreactive bands represent the 23- and 28-kDa form of Gremlin-1 as also shown in the recombinant Gremlin-1 control. Representative results of five independently performed experiments are shown. e, Gremlin-1 protein expression was further analyzed by confocal microscopy after staining of permeabilized monocytes or macrophages/foam cells with fluorochrome-conjugated mAb directed against Gremlin-1 (red, phycoerythrin) or CD14 or CD68 (green, fluorescein isothiocyanate), respectively. f, isolated monocytes were stimulated with 1 μg/ml LPS for the indicated times. Intracellular protein expression of Gremlin-1 was determined in cell lysates by immunoblotting using an anti-human Gremlin-1 mAb. g, secreted Gremlin-1 was quantified in the cell supernatant by ELISA. Data represent the mean ± S.E. of five independent experiments. h, isolated monocytes were stimulated with vehicle control, LPS (1 μg/ml), oxidized LDL (oxLDL) (50 μg/ml), and TNF-α (100 ng/ml). Gremlin-1 was determined in the supernatant by ELISA after 6 h.