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. 2021 Jan;41(1):220-233.
doi: 10.1161/ATVBAHA.120.315255. Epub 2020 Oct 22.

Local M-CSF (Macrophage Colony-Stimulating Factor) Expression Regulates Macrophage Proliferation and Apoptosis in Atherosclerosis

Satyesh K Sinha  1   2   3   4 , Aika Miikeda  1   2   3 , Zachary Fouladian  1   2   3 , Margarete Mehrabian  1   2   3 , Chantle Edillor  1   2   3 , Diana Shih  1   2   3 , Zhiqiang Zhou  1   2   3 , Manash K Paul  3 , Sarada Charugundla  1   2   3 , Richard C Davis  1   2   3 , Tripathi B Rajavashisth  1   2   3   4   5 , Aldons J Lusis  1   2   3
Affiliations

Local M-CSF (Macrophage Colony-Stimulating Factor) Expression Regulates Macrophage Proliferation and Apoptosis in Atherosclerosis

Satyesh K Sinha et al. Arterioscler Thromb Vasc Biol. 2021 Jan.

Abstract

Objective: Previous studies have shown that deficiency of M-CSF (macrophage colony-stimulating factor; or CSF1 [colony stimulating factor 1]) dramatically reduces atherosclerosis in hyperlipidemic mice. We characterize the underlying mechanism and investigate the relevant sources of CSF1 in lesions. Approach and Results: We quantitatively assessed the effects of CSF1 deficiency on macrophage proliferation and apoptosis in atherosclerotic lesions. Staining of aortic lesions with markers of proliferation, Ki-67 and bromodeoxyuridine, revealed around 40% reduction in CSF1 heterozygous (Csf1+/-) as compared with WT (wild type; Csf1+/+) mice. Similarly, staining with a marker of apoptosis, activated caspase-3, revealed a 3-fold increase in apoptotic cells in Csf1+/- mice. Next, we determined the cellular sources of CSF1 contributing to lesion development. Cell-specific deletions of Csf1 in smooth muscle cells using SM22α-Cre (smooth muscle protein 22-alpha-Cre) reduced lesions by about 40%, and in endothelial cells, deletions with Cdh5-Cre (VE-cadherin-Cre) reduced lesions by about 30%. Macrophage-specific deletion with LysM-Cre (lysozyme M-Cre), on the other hand, did not significantly reduce lesions size. Transplantation of Csf1 null (Csf1-/-) mice bone marrow into Csf1+/+ mice reduced lesions by about 35%, suggesting that CSF1 from hematopoietic cells other than macrophages contributes to atherosclerosis. None of the cell-specific knockouts affected circulating CSF1 levels, and only the smooth muscle cell deletions had any effect on the percentage monocytes in the circulation. Also, Csf1+/- mice did not exhibit significant differences in Ly6Chigh/Ly6Clow monocytes as compared with Csf1+/+.

Conclusions: CSF1 contributes to both macrophage proliferation and survival in lesions. Local CSF1 production by smooth muscle cell and endothelial cell rather than circulating CSF1 is the primary driver of macrophage expansion in atherosclerosis.

Keywords: apoptosis; atherosclerosis; colony stimulating factors; macrophages; monocytes.

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Figures

Figure 1.
Figure 1.. CSF1 deficiency reduces macrophage proliferation and promotes macrophage apoptosis.
(A-F) Macrophages in lesions from hypercholesterolemic CSF1 wild type (Csf1+/+, top) and heterozygous deficient (Csf1+/−, bottom) mice were examined for presence of Ki-67 (A, B), BrdU incorporation (C, D), or activated Caspase-3 (E, F). Representative images are shown at magnification X10 (A, E) and X20 (C) along with the percent positive macrophages (B, D, F). (G-M) Hypercholesterolemic Csf1+/+ and Csf1+/− mice were examined for lesion morphology at magnification X4 (G) and lesion area (H). total plasma cholesterol (I) HDL cholesterol (J) non-HDL cholesterol (K) plasma CSF1 (L) and circulating monocyte percent (M).
Figure 1.
Figure 1.. CSF1 deficiency reduces macrophage proliferation and promotes macrophage apoptosis.
(A-F) Macrophages in lesions from hypercholesterolemic CSF1 wild type (Csf1+/+, top) and heterozygous deficient (Csf1+/−, bottom) mice were examined for presence of Ki-67 (A, B), BrdU incorporation (C, D), or activated Caspase-3 (E, F). Representative images are shown at magnification X10 (A, E) and X20 (C) along with the percent positive macrophages (B, D, F). (G-M) Hypercholesterolemic Csf1+/+ and Csf1+/− mice were examined for lesion morphology at magnification X4 (G) and lesion area (H). total plasma cholesterol (I) HDL cholesterol (J) non-HDL cholesterol (K) plasma CSF1 (L) and circulating monocyte percent (M).
Figure 1.
Figure 1.. CSF1 deficiency reduces macrophage proliferation and promotes macrophage apoptosis.
(A-F) Macrophages in lesions from hypercholesterolemic CSF1 wild type (Csf1+/+, top) and heterozygous deficient (Csf1+/−, bottom) mice were examined for presence of Ki-67 (A, B), BrdU incorporation (C, D), or activated Caspase-3 (E, F). Representative images are shown at magnification X10 (A, E) and X20 (C) along with the percent positive macrophages (B, D, F). (G-M) Hypercholesterolemic Csf1+/+ and Csf1+/− mice were examined for lesion morphology at magnification X4 (G) and lesion area (H). total plasma cholesterol (I) HDL cholesterol (J) non-HDL cholesterol (K) plasma CSF1 (L) and circulating monocyte percent (M).
Figure 1.
Figure 1.. CSF1 deficiency reduces macrophage proliferation and promotes macrophage apoptosis.
(A-F) Macrophages in lesions from hypercholesterolemic CSF1 wild type (Csf1+/+, top) and heterozygous deficient (Csf1+/−, bottom) mice were examined for presence of Ki-67 (A, B), BrdU incorporation (C, D), or activated Caspase-3 (E, F). Representative images are shown at magnification X10 (A, E) and X20 (C) along with the percent positive macrophages (B, D, F). (G-M) Hypercholesterolemic Csf1+/+ and Csf1+/− mice were examined for lesion morphology at magnification X4 (G) and lesion area (H). total plasma cholesterol (I) HDL cholesterol (J) non-HDL cholesterol (K) plasma CSF1 (L) and circulating monocyte percent (M).
Figure 1.
Figure 1.. CSF1 deficiency reduces macrophage proliferation and promotes macrophage apoptosis.
(A-F) Macrophages in lesions from hypercholesterolemic CSF1 wild type (Csf1+/+, top) and heterozygous deficient (Csf1+/−, bottom) mice were examined for presence of Ki-67 (A, B), BrdU incorporation (C, D), or activated Caspase-3 (E, F). Representative images are shown at magnification X10 (A, E) and X20 (C) along with the percent positive macrophages (B, D, F). (G-M) Hypercholesterolemic Csf1+/+ and Csf1+/− mice were examined for lesion morphology at magnification X4 (G) and lesion area (H). total plasma cholesterol (I) HDL cholesterol (J) non-HDL cholesterol (K) plasma CSF1 (L) and circulating monocyte percent (M).
Figure 2.
Figure 2.. Absence of SMC-derived CSF1 reduces lesion area.
(A-G) Female mice carrying floxed alleles of Csf1 (Csf1fl/+ or Csf1fl/fl) and with (+) or without (−) SM22α-Cre (the latter labeled ”controls”) were bred and examined for lesion morphology (A), lesion sizes (B), total cholesterol (C), HDL cholesterol (D), non-HDL cholesterol (E), circulating CSF1 levels (F) and monocytes % (G). (H-K) Ki-67 positive macrophages (H, I) and EdU positive macrophages in control and Csf1fl/fl;SM22α-Cre mice (J, K) were quantitated. Control- Csf1fl/fl and Csf1fl/+ mice on a WT background (no Cre). Merged- CD68 (red), Ki-67 or EdU (green), and DAPI (blue).
Figure 2.
Figure 2.. Absence of SMC-derived CSF1 reduces lesion area.
(A-G) Female mice carrying floxed alleles of Csf1 (Csf1fl/+ or Csf1fl/fl) and with (+) or without (−) SM22α-Cre (the latter labeled ”controls”) were bred and examined for lesion morphology (A), lesion sizes (B), total cholesterol (C), HDL cholesterol (D), non-HDL cholesterol (E), circulating CSF1 levels (F) and monocytes % (G). (H-K) Ki-67 positive macrophages (H, I) and EdU positive macrophages in control and Csf1fl/fl;SM22α-Cre mice (J, K) were quantitated. Control- Csf1fl/fl and Csf1fl/+ mice on a WT background (no Cre). Merged- CD68 (red), Ki-67 or EdU (green), and DAPI (blue).
Figure 2.
Figure 2.. Absence of SMC-derived CSF1 reduces lesion area.
(A-G) Female mice carrying floxed alleles of Csf1 (Csf1fl/+ or Csf1fl/fl) and with (+) or without (−) SM22α-Cre (the latter labeled ”controls”) were bred and examined for lesion morphology (A), lesion sizes (B), total cholesterol (C), HDL cholesterol (D), non-HDL cholesterol (E), circulating CSF1 levels (F) and monocytes % (G). (H-K) Ki-67 positive macrophages (H, I) and EdU positive macrophages in control and Csf1fl/fl;SM22α-Cre mice (J, K) were quantitated. Control- Csf1fl/fl and Csf1fl/+ mice on a WT background (no Cre). Merged- CD68 (red), Ki-67 or EdU (green), and DAPI (blue).
Figure 3.
Figure 3.. Absence of EC-derived CSF1 reduces lesion area.
(A-G) Female mice carrying floxed alleles of Csf1 (Csf1fl/+ or Csf1fl/fl) and with (+) or without (−) Cdh5-Cre (the latter labeled ”controls”) were bred and examined for lesion morphology (A), lesion sizes (B), total cholesterol (C), HDL cholesterol (D), non-HDL cholesterol (E), circulating CSF1 levels (F), and monocytes % (G). (H-I) Ki-67 positive macrophages in control and Csf1fl/fl;Cdh5-Cre mice were quantitated. Control- Csf1fl/fl and Csf1fl/+ mice on a WT background (no Cre). Merged- CD68 (red), Ki-67 (green), and DAPI (blue).
Figure 3.
Figure 3.. Absence of EC-derived CSF1 reduces lesion area.
(A-G) Female mice carrying floxed alleles of Csf1 (Csf1fl/+ or Csf1fl/fl) and with (+) or without (−) Cdh5-Cre (the latter labeled ”controls”) were bred and examined for lesion morphology (A), lesion sizes (B), total cholesterol (C), HDL cholesterol (D), non-HDL cholesterol (E), circulating CSF1 levels (F), and monocytes % (G). (H-I) Ki-67 positive macrophages in control and Csf1fl/fl;Cdh5-Cre mice were quantitated. Control- Csf1fl/fl and Csf1fl/+ mice on a WT background (no Cre). Merged- CD68 (red), Ki-67 (green), and DAPI (blue).
Figure 3.
Figure 3.. Absence of EC-derived CSF1 reduces lesion area.
(A-G) Female mice carrying floxed alleles of Csf1 (Csf1fl/+ or Csf1fl/fl) and with (+) or without (−) Cdh5-Cre (the latter labeled ”controls”) were bred and examined for lesion morphology (A), lesion sizes (B), total cholesterol (C), HDL cholesterol (D), non-HDL cholesterol (E), circulating CSF1 levels (F), and monocytes % (G). (H-I) Ki-67 positive macrophages in control and Csf1fl/fl;Cdh5-Cre mice were quantitated. Control- Csf1fl/fl and Csf1fl/+ mice on a WT background (no Cre). Merged- CD68 (red), Ki-67 (green), and DAPI (blue).
Figure 4.
Figure 4.. Absence of macrophage-derived CSF1 does not reduce lesion area.
(A-G) Female mice carrying floxed alleles of Csf1 (Csf1fl/+ or Csf1fl/fl) and with (+) or without (−) LysM-Cre (the latter labeled ”controls”) were bred and examined for lesion morphology (A), lesion sizes (B), total cholesterol (C), HDL cholesterol (D), non-HDL cholesterol (E), circulating CSF1 levels (F), and monocytes % (G). Control- Csf1fl/fl and Csf1fl/+ mice on a WT background (no Cre).
Figure 4.
Figure 4.. Absence of macrophage-derived CSF1 does not reduce lesion area.
(A-G) Female mice carrying floxed alleles of Csf1 (Csf1fl/+ or Csf1fl/fl) and with (+) or without (−) LysM-Cre (the latter labeled ”controls”) were bred and examined for lesion morphology (A), lesion sizes (B), total cholesterol (C), HDL cholesterol (D), non-HDL cholesterol (E), circulating CSF1 levels (F), and monocytes % (G). Control- Csf1fl/fl and Csf1fl/+ mice on a WT background (no Cre).
Figure 5:
Figure 5:. Transplantation of bone marrow (BM) from Csf1−/− mice into wild type mice reduces atherosclerosis.
BM cells from male Csf1−/− or Csf1+/+ donor mice were transplanted into lethally irradiated Ldlr−/− female recipients and after 6 weeks placed on an atherogenic diet for 11-12 weeks. The mice were then examined for lesion morphology (A), lesion area (B), plasma total cholesterol (C), circulating CSF1 levels (D), and lymphocyte (Lym), monocyte (Mono) and granulocyte (Gran) percents (N=15 for Csf1+/+ and N=10 for Csf1−/− BM) (E). The percentage of Ki-67 positive macrophages in mice transplanted with Csf1+/+ BM and Csf1−/− BM was quantitated (F, G). Merged- CD68 (red), Ki-67 (green), and DAPI (blue).
Figure 5:
Figure 5:. Transplantation of bone marrow (BM) from Csf1−/− mice into wild type mice reduces atherosclerosis.
BM cells from male Csf1−/− or Csf1+/+ donor mice were transplanted into lethally irradiated Ldlr−/− female recipients and after 6 weeks placed on an atherogenic diet for 11-12 weeks. The mice were then examined for lesion morphology (A), lesion area (B), plasma total cholesterol (C), circulating CSF1 levels (D), and lymphocyte (Lym), monocyte (Mono) and granulocyte (Gran) percents (N=15 for Csf1+/+ and N=10 for Csf1−/− BM) (E). The percentage of Ki-67 positive macrophages in mice transplanted with Csf1+/+ BM and Csf1−/− BM was quantitated (F, G). Merged- CD68 (red), Ki-67 (green), and DAPI (blue).
Figure 5:
Figure 5:. Transplantation of bone marrow (BM) from Csf1−/− mice into wild type mice reduces atherosclerosis.
BM cells from male Csf1−/− or Csf1+/+ donor mice were transplanted into lethally irradiated Ldlr−/− female recipients and after 6 weeks placed on an atherogenic diet for 11-12 weeks. The mice were then examined for lesion morphology (A), lesion area (B), plasma total cholesterol (C), circulating CSF1 levels (D), and lymphocyte (Lym), monocyte (Mono) and granulocyte (Gran) percents (N=15 for Csf1+/+ and N=10 for Csf1−/− BM) (E). The percentage of Ki-67 positive macrophages in mice transplanted with Csf1+/+ BM and Csf1−/− BM was quantitated (F, G). Merged- CD68 (red), Ki-67 (green), and DAPI (blue).
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