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Foam Cell Formation In Vivo Converts Macrophages to a Pro-Fibrotic Phenotype.

Thomas AC, Eijgelaar WJ, Daemen MJ, Newby AC - PLoS ONE (2015)

Bottom Line: Unexpectedly, the hepatic fibrosis pathway, associated with platelet derived growth factor and transforming growth factor-β action, was also over-represented.Several collagen polypeptides and proteoglycan core proteins as well as connective tissue growth factor and fibrosis-related FOS and JUN transcription factors were up-regulated in foam cell macrophages.We conclude that foam cell formation in vivo leads to a pro-fibrotic macrophage phenotype, which could contribute to plaque stability, especially in early lesions that have few vascular smooth muscle cells.

View Article: PubMed Central - PubMed

Affiliation: Bristol Heart Institute, University of Bristol, Bristol, United Kingdom.

ABSTRACT
Formation of foam cell macrophages, which sequester extracellular modified lipids, is a key event in atherosclerosis. How lipid loading affects macrophage phenotype is controversial, with evidence suggesting either pro- or anti-inflammatory consequences. To investigate this further, we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in the subcutaneous granulomas of fed-fat ApoE mice and normal chow fed wild-type mice in vivo. Consistent with previous studies, LXR/RXR pathway genes were significantly over-represented among the genes up-regulated in foam cell macrophages. Unexpectedly, the hepatic fibrosis pathway, associated with platelet derived growth factor and transforming growth factor-β action, was also over-represented. Several collagen polypeptides and proteoglycan core proteins as well as connective tissue growth factor and fibrosis-related FOS and JUN transcription factors were up-regulated in foam cell macrophages. Increased expression of several of these genes was confirmed at the protein level in foam cell macrophages from subcutaneous granulomas and in atherosclerotic plaques. Moreover, phosphorylation and nuclear translocation of SMAD2, which is downstream of several transforming growth factor-β family members, was also detected in foam cell macrophages. We conclude that foam cell formation in vivo leads to a pro-fibrotic macrophage phenotype, which could contribute to plaque stability, especially in early lesions that have few vascular smooth muscle cells.

No MeSH data available.


Related in: MedlinePlus

Phosphorylated SMAD proteins in RAW 264.7 cells and sponge or artery macrophages from mice.RAW cells were exposed to 10 ng/ml TGFβ1 for 45 minutes. A) phospho-SMAD2 and B) phospho-SMAD3 were present in the cytoplasm and nucleus of many cells. pSMAD2 was also found in C) the cytoplasm and nucleus (arrows) from isolated FCMs, but only in the D) cytoplasm of NFMs. E) pSMAD is present in the cytoplasm and nuclei (arrows) of plaques in a brachiocephalic artery a fat-fed ApoE  mouse. E’) higher magnification of plaque in E. F) negative control in a section from the same plaque. G) RAW negative control staining (rabbit IgG). Red/orange = cytoplasmic staining, pink = nuclear staining, blue = nuclei (DAPI), green = autofluorescence. A, B, E’, G magnification x 1000, C, D, E, F magnification x 400.
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pone.0128163.g008: Phosphorylated SMAD proteins in RAW 264.7 cells and sponge or artery macrophages from mice.RAW cells were exposed to 10 ng/ml TGFβ1 for 45 minutes. A) phospho-SMAD2 and B) phospho-SMAD3 were present in the cytoplasm and nucleus of many cells. pSMAD2 was also found in C) the cytoplasm and nucleus (arrows) from isolated FCMs, but only in the D) cytoplasm of NFMs. E) pSMAD is present in the cytoplasm and nuclei (arrows) of plaques in a brachiocephalic artery a fat-fed ApoE mouse. E’) higher magnification of plaque in E. F) negative control in a section from the same plaque. G) RAW negative control staining (rabbit IgG). Red/orange = cytoplasmic staining, pink = nuclear staining, blue = nuclei (DAPI), green = autofluorescence. A, B, E’, G magnification x 1000, C, D, E, F magnification x 400.

Mentions: Members of the TGF family signal through phosphorylation and nuclear translocation of SMADs, especially SMAD2. Hence we hypothesised that FCMs in sponges and plaques might contain increased levels of nuclear pSMAD2 detectable by immunohisto/cytochemistry. As a positive control, we first showed that TFGβ1 quickly stimulated SMAD2 and SMAD3 phosphorylation and translocation to the nucleus by 45 minutes in mouse RAW cells (Fig 8A and 8B and Table B in S1 File). Staining for nuclear pSMAD2 (pink) was found in 50 ± 12% of isolated FCMs; many also had cytoplasmic staining (red). By contrast, nuclear pSMAD2 staining was detected in only 10 ± 10% of NFM (n = 3, P = 0.0110, Fig 8C and 8D). Furthermore, 74–76% of the FCMs in brachiocephalic artery plaques had pSMAD2 present in their nuclei (pink), with many also having pSMAD2 in their cytoplasm (orange) (Fig 8E and 8E’). No staining was observed in an IgG control section from the same plaque or in IgG control RAW cells (Fig 8F and 8G). This provides strong evidence for SMAD2 signalling in FCMs in sponges and plaques.


Foam Cell Formation In Vivo Converts Macrophages to a Pro-Fibrotic Phenotype.

Thomas AC, Eijgelaar WJ, Daemen MJ, Newby AC - PLoS ONE (2015)

Phosphorylated SMAD proteins in RAW 264.7 cells and sponge or artery macrophages from mice.RAW cells were exposed to 10 ng/ml TGFβ1 for 45 minutes. A) phospho-SMAD2 and B) phospho-SMAD3 were present in the cytoplasm and nucleus of many cells. pSMAD2 was also found in C) the cytoplasm and nucleus (arrows) from isolated FCMs, but only in the D) cytoplasm of NFMs. E) pSMAD is present in the cytoplasm and nuclei (arrows) of plaques in a brachiocephalic artery a fat-fed ApoE  mouse. E’) higher magnification of plaque in E. F) negative control in a section from the same plaque. G) RAW negative control staining (rabbit IgG). Red/orange = cytoplasmic staining, pink = nuclear staining, blue = nuclei (DAPI), green = autofluorescence. A, B, E’, G magnification x 1000, C, D, E, F magnification x 400.
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pone.0128163.g008: Phosphorylated SMAD proteins in RAW 264.7 cells and sponge or artery macrophages from mice.RAW cells were exposed to 10 ng/ml TGFβ1 for 45 minutes. A) phospho-SMAD2 and B) phospho-SMAD3 were present in the cytoplasm and nucleus of many cells. pSMAD2 was also found in C) the cytoplasm and nucleus (arrows) from isolated FCMs, but only in the D) cytoplasm of NFMs. E) pSMAD is present in the cytoplasm and nuclei (arrows) of plaques in a brachiocephalic artery a fat-fed ApoE mouse. E’) higher magnification of plaque in E. F) negative control in a section from the same plaque. G) RAW negative control staining (rabbit IgG). Red/orange = cytoplasmic staining, pink = nuclear staining, blue = nuclei (DAPI), green = autofluorescence. A, B, E’, G magnification x 1000, C, D, E, F magnification x 400.
Mentions: Members of the TGF family signal through phosphorylation and nuclear translocation of SMADs, especially SMAD2. Hence we hypothesised that FCMs in sponges and plaques might contain increased levels of nuclear pSMAD2 detectable by immunohisto/cytochemistry. As a positive control, we first showed that TFGβ1 quickly stimulated SMAD2 and SMAD3 phosphorylation and translocation to the nucleus by 45 minutes in mouse RAW cells (Fig 8A and 8B and Table B in S1 File). Staining for nuclear pSMAD2 (pink) was found in 50 ± 12% of isolated FCMs; many also had cytoplasmic staining (red). By contrast, nuclear pSMAD2 staining was detected in only 10 ± 10% of NFM (n = 3, P = 0.0110, Fig 8C and 8D). Furthermore, 74–76% of the FCMs in brachiocephalic artery plaques had pSMAD2 present in their nuclei (pink), with many also having pSMAD2 in their cytoplasm (orange) (Fig 8E and 8E’). No staining was observed in an IgG control section from the same plaque or in IgG control RAW cells (Fig 8F and 8G). This provides strong evidence for SMAD2 signalling in FCMs in sponges and plaques.

Bottom Line: Unexpectedly, the hepatic fibrosis pathway, associated with platelet derived growth factor and transforming growth factor-β action, was also over-represented.Several collagen polypeptides and proteoglycan core proteins as well as connective tissue growth factor and fibrosis-related FOS and JUN transcription factors were up-regulated in foam cell macrophages.We conclude that foam cell formation in vivo leads to a pro-fibrotic macrophage phenotype, which could contribute to plaque stability, especially in early lesions that have few vascular smooth muscle cells.

View Article: PubMed Central - PubMed

Affiliation: Bristol Heart Institute, University of Bristol, Bristol, United Kingdom.

ABSTRACT
Formation of foam cell macrophages, which sequester extracellular modified lipids, is a key event in atherosclerosis. How lipid loading affects macrophage phenotype is controversial, with evidence suggesting either pro- or anti-inflammatory consequences. To investigate this further, we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in the subcutaneous granulomas of fed-fat ApoE mice and normal chow fed wild-type mice in vivo. Consistent with previous studies, LXR/RXR pathway genes were significantly over-represented among the genes up-regulated in foam cell macrophages. Unexpectedly, the hepatic fibrosis pathway, associated with platelet derived growth factor and transforming growth factor-β action, was also over-represented. Several collagen polypeptides and proteoglycan core proteins as well as connective tissue growth factor and fibrosis-related FOS and JUN transcription factors were up-regulated in foam cell macrophages. Increased expression of several of these genes was confirmed at the protein level in foam cell macrophages from subcutaneous granulomas and in atherosclerotic plaques. Moreover, phosphorylation and nuclear translocation of SMAD2, which is downstream of several transforming growth factor-β family members, was also detected in foam cell macrophages. We conclude that foam cell formation in vivo leads to a pro-fibrotic macrophage phenotype, which could contribute to plaque stability, especially in early lesions that have few vascular smooth muscle cells.

No MeSH data available.


Related in: MedlinePlus