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Characterization of Proliferating Lesion ‐ Resident Cells During All Stages of Atherosclerotic Growth

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ABSTRACT

Background: Monocyte recruitment leads to accumulation of macrophage foam cells and contributes to atherosclerotic lesion growth. Recent studies have reported that lesion‐resident macrophages can proliferate and represent a major cellular component during lesion development. This study was designed to assess whether the rate of macrophage proliferation changes during well‐established stages of lesion growth and to characterize other populations of proliferating cells within these lesions.

Methods and results: Using murine models of atherosclerosis (Apoe−/− and LDLr−/− mice) and human coronary artery lesions, in situ proliferation of lesion‐resident cells at different stages of growth was assessed by staining for Ki67 and bromodeoxyuridine (BrdU). In early lesions, close to half of all actively growing macrophages were proliferating in situ. BrdU pulse labeling allowed for accurate identification of in situ proliferating macrophages compared to those derived from monocyte recruitment. Local macrophage proliferation declined as lesions advanced. Interestingly, intimal inflammatory cell infiltrates containing proliferating T lymphocytes were identified during the active phase of lesion growth and correlated with apoptotic cell death. Inflammatory cell infiltrates were completely resolved in advanced lesions and replaced with the necrotic core.

Conclusions: Our findings indicate that atherosclerotic lesions contain locally proliferating macrophages primarily during early and intermediate stages of lesion growth. Furthermore, T‐lymphocyte‐enriched inflammatory cell infiltrates represent a novel subset of proliferating cells within the atherosclerotic lesion that correlate with apoptosis and precede the necrotic core. These findings have novel implications in understanding the pathogenesis of atherosclerosis and may implicate proliferating T lymphocytes as a contributing factor to lesion progression and stability.

No MeSH data available.


Cell‐type characterization of inflammatory cell infiltrates (ICIs). Low‐magnification hematoxylin and eosin staining delineates the intimal ICI in an intermediate lesion. (A, adventitial infiltrate is marked with arrowheads). Higher magnification consecutive sections of the ICI immunostained for Ki67 (B), T‐lymphocyte marker CD3 (C), macrophage marker Mac‐3, (D), smooth muscle cell actin (SMA) (E), neutrophil marker myeloperoxidase (MPO) (F), M2 macrophage marker chitinase 3‐like 3 (YM1) (G) mannose receptor (MR) (H), and arginase I (Arg I) (I) and M1 macrophage marker inducible nitric oxide synthase (iNOS) (L). YM1‐ and MR‐positive areas are limited to ICIs, both in the intima and adventitia, whereas lesions without the presence of acute inflammation do not express these markers (J and K, arrows). Bar=100 μm.
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jah31705-fig-0005: Cell‐type characterization of inflammatory cell infiltrates (ICIs). Low‐magnification hematoxylin and eosin staining delineates the intimal ICI in an intermediate lesion. (A, adventitial infiltrate is marked with arrowheads). Higher magnification consecutive sections of the ICI immunostained for Ki67 (B), T‐lymphocyte marker CD3 (C), macrophage marker Mac‐3, (D), smooth muscle cell actin (SMA) (E), neutrophil marker myeloperoxidase (MPO) (F), M2 macrophage marker chitinase 3‐like 3 (YM1) (G) mannose receptor (MR) (H), and arginase I (Arg I) (I) and M1 macrophage marker inducible nitric oxide synthase (iNOS) (L). YM1‐ and MR‐positive areas are limited to ICIs, both in the intima and adventitia, whereas lesions without the presence of acute inflammation do not express these markers (J and K, arrows). Bar=100 μm.

Mentions: In addition to the identification of proliferating macrophages, prominent intimal ICIs containing proliferating cells (Figure 5A and 5B) were observed in some sections in the intermediate stage of lesion growth, typically associated with large apoptotic foci. These were not observed in early and advanced lesions. Large ICIs were also present in the adventitia underlying the intimal ICI (Figure 5A, arrowheads), and partially resemble previously described artery tertiary lymphoid organs (ATLOs).17, 26 Ki67 immunostaining showed that both the intimal and the adventitial ICIs were rich in proliferating cells (Figure 5B), most of which were CD3‐positive T lymphocytes (Figure 5C). They also contained macrophages (Mac‐3; Figure 5D), but very few smooth muscle cells (SMA; Figure 5E) or neutrophils (MPO; Figure 5F). Interestingly, the ICIs displayed high expression of the M2 markers, YM1 (Figure 5G) and MR (Figure 5H), while having low expression of the M2 marker, Arg 1 (Figure 5I), and the M1 marker, iNOS (Figure 5L). YM1‐ and MR‐positive cells were found solely in the ICIs, as indicated by lack of staining in other parts of the lesions (Figure 5J and 5K, arrows). Taken together, these findings suggest that T‐lymphocyte‐ and macrophage‐rich proliferating inflammatory infiltrates occur in the intima of intermediate lesions.


Characterization of Proliferating Lesion ‐ Resident Cells During All Stages of Atherosclerotic Growth
Cell‐type characterization of inflammatory cell infiltrates (ICIs). Low‐magnification hematoxylin and eosin staining delineates the intimal ICI in an intermediate lesion. (A, adventitial infiltrate is marked with arrowheads). Higher magnification consecutive sections of the ICI immunostained for Ki67 (B), T‐lymphocyte marker CD3 (C), macrophage marker Mac‐3, (D), smooth muscle cell actin (SMA) (E), neutrophil marker myeloperoxidase (MPO) (F), M2 macrophage marker chitinase 3‐like 3 (YM1) (G) mannose receptor (MR) (H), and arginase I (Arg I) (I) and M1 macrophage marker inducible nitric oxide synthase (iNOS) (L). YM1‐ and MR‐positive areas are limited to ICIs, both in the intima and adventitia, whereas lesions without the presence of acute inflammation do not express these markers (J and K, arrows). Bar=100 μm.
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jah31705-fig-0005: Cell‐type characterization of inflammatory cell infiltrates (ICIs). Low‐magnification hematoxylin and eosin staining delineates the intimal ICI in an intermediate lesion. (A, adventitial infiltrate is marked with arrowheads). Higher magnification consecutive sections of the ICI immunostained for Ki67 (B), T‐lymphocyte marker CD3 (C), macrophage marker Mac‐3, (D), smooth muscle cell actin (SMA) (E), neutrophil marker myeloperoxidase (MPO) (F), M2 macrophage marker chitinase 3‐like 3 (YM1) (G) mannose receptor (MR) (H), and arginase I (Arg I) (I) and M1 macrophage marker inducible nitric oxide synthase (iNOS) (L). YM1‐ and MR‐positive areas are limited to ICIs, both in the intima and adventitia, whereas lesions without the presence of acute inflammation do not express these markers (J and K, arrows). Bar=100 μm.
Mentions: In addition to the identification of proliferating macrophages, prominent intimal ICIs containing proliferating cells (Figure 5A and 5B) were observed in some sections in the intermediate stage of lesion growth, typically associated with large apoptotic foci. These were not observed in early and advanced lesions. Large ICIs were also present in the adventitia underlying the intimal ICI (Figure 5A, arrowheads), and partially resemble previously described artery tertiary lymphoid organs (ATLOs).17, 26 Ki67 immunostaining showed that both the intimal and the adventitial ICIs were rich in proliferating cells (Figure 5B), most of which were CD3‐positive T lymphocytes (Figure 5C). They also contained macrophages (Mac‐3; Figure 5D), but very few smooth muscle cells (SMA; Figure 5E) or neutrophils (MPO; Figure 5F). Interestingly, the ICIs displayed high expression of the M2 markers, YM1 (Figure 5G) and MR (Figure 5H), while having low expression of the M2 marker, Arg 1 (Figure 5I), and the M1 marker, iNOS (Figure 5L). YM1‐ and MR‐positive cells were found solely in the ICIs, as indicated by lack of staining in other parts of the lesions (Figure 5J and 5K, arrows). Taken together, these findings suggest that T‐lymphocyte‐ and macrophage‐rich proliferating inflammatory infiltrates occur in the intima of intermediate lesions.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Monocyte recruitment leads to accumulation of macrophage foam cells and contributes to atherosclerotic lesion growth. Recent studies have reported that lesion‐resident macrophages can proliferate and represent a major cellular component during lesion development. This study was designed to assess whether the rate of macrophage proliferation changes during well‐established stages of lesion growth and to characterize other populations of proliferating cells within these lesions.

Methods and results: Using murine models of atherosclerosis (Apoe−/− and LDLr−/− mice) and human coronary artery lesions, in situ proliferation of lesion‐resident cells at different stages of growth was assessed by staining for Ki67 and bromodeoxyuridine (BrdU). In early lesions, close to half of all actively growing macrophages were proliferating in situ. BrdU pulse labeling allowed for accurate identification of in situ proliferating macrophages compared to those derived from monocyte recruitment. Local macrophage proliferation declined as lesions advanced. Interestingly, intimal inflammatory cell infiltrates containing proliferating T lymphocytes were identified during the active phase of lesion growth and correlated with apoptotic cell death. Inflammatory cell infiltrates were completely resolved in advanced lesions and replaced with the necrotic core.

Conclusions: Our findings indicate that atherosclerotic lesions contain locally proliferating macrophages primarily during early and intermediate stages of lesion growth. Furthermore, T‐lymphocyte‐enriched inflammatory cell infiltrates represent a novel subset of proliferating cells within the atherosclerotic lesion that correlate with apoptosis and precede the necrotic core. These findings have novel implications in understanding the pathogenesis of atherosclerosis and may implicate proliferating T lymphocytes as a contributing factor to lesion progression and stability.

No MeSH data available.