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

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.


Related in: MedlinePlus

BrdU pulse labeling detects macrophage proliferation in situ and tracks monocyte incorporation into lesions. Mac‐3‐positive macrophages in lesions incorporated BrdU within 2 hours p.i. (A, arrows). At 24 hours p.i., BrdU‐positive monocytes were observed in the lumen, attached to the endothelium or extravasating into the intima (B, arrows in insets), in addition to macrophages within the lesion that had proliferated in situ (B and C, arrows). Aortic root sections from Apoe−/− mice on a chow diet at 12  (early, n=11) and 24 weeks (intermediate, n=9) were immunostained for BrdU both 2 and 24 hours after BrdU injection. Early lesion, 2 hours p.i. (n=6) (D) and 24 hours p.i. (n=5) (E). Intermediate lesion 2 hours p.i. (n=4) (G) and 24 hours p.i. (n=5) (H). Arrows indicate BrdU‐positive cells. Positive cells were quantified in 3 sections from each mouse and averaged. BrdU‐positive macrophages were classified based on their location within the lesion as peripheral (close to the endothelium), internal (deeper in the lesion), or attached (in the lumen attached to the endothelium). Quantification of early (F) and intermediate lesions (I). Bar=50 μm. *P<0.05 as measured by Mann–Whitney U test. BrdU indicates bromodeoxyuridine; p.i., postinjection.
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jah31705-fig-0004: BrdU pulse labeling detects macrophage proliferation in situ and tracks monocyte incorporation into lesions. Mac‐3‐positive macrophages in lesions incorporated BrdU within 2 hours p.i. (A, arrows). At 24 hours p.i., BrdU‐positive monocytes were observed in the lumen, attached to the endothelium or extravasating into the intima (B, arrows in insets), in addition to macrophages within the lesion that had proliferated in situ (B and C, arrows). Aortic root sections from Apoe−/− mice on a chow diet at 12  (early, n=11) and 24 weeks (intermediate, n=9) were immunostained for BrdU both 2 and 24 hours after BrdU injection. Early lesion, 2 hours p.i. (n=6) (D) and 24 hours p.i. (n=5) (E). Intermediate lesion 2 hours p.i. (n=4) (G) and 24 hours p.i. (n=5) (H). Arrows indicate BrdU‐positive cells. Positive cells were quantified in 3 sections from each mouse and averaged. BrdU‐positive macrophages were classified based on their location within the lesion as peripheral (close to the endothelium), internal (deeper in the lesion), or attached (in the lumen attached to the endothelium). Quantification of early (F) and intermediate lesions (I). Bar=50 μm. *P<0.05 as measured by Mann–Whitney U test. BrdU indicates bromodeoxyuridine; p.i., postinjection.

Mentions: Aortic sections were immunostained for BrdU and Mac‐3 and visualized by immunofluorescence. In the early lesion group sacrificed at 2 hours p.i. (Figure 4A), some of the Mac‐3‐positive macrophages were observed to express BrdU, a finding consistent with the positive Ki67 immunostaining (Figure 1B), and confirming that intimal aortic macrophages are proliferating locally, or in situ. No BrdU‐positive cells were observed in the lumen of the aorta at this time point. In the early lesion group sacrificed at 24 hours p.i., BrdU‐positive monocytes were identified in the lumen, attached to the endothelium, and extravasating through the endothelial layer (Figure 4B, arrows). BrdU‐positive cells were also observed in intermediate lesions, some located in the basal portion similar to the 2‐hour time point, but mostly located toward the luminal aspect of the lesion (Figure 4C, arrows).


Characterization of Proliferating Lesion ‐ Resident Cells During All Stages of Atherosclerotic Growth
BrdU pulse labeling detects macrophage proliferation in situ and tracks monocyte incorporation into lesions. Mac‐3‐positive macrophages in lesions incorporated BrdU within 2 hours p.i. (A, arrows). At 24 hours p.i., BrdU‐positive monocytes were observed in the lumen, attached to the endothelium or extravasating into the intima (B, arrows in insets), in addition to macrophages within the lesion that had proliferated in situ (B and C, arrows). Aortic root sections from Apoe−/− mice on a chow diet at 12  (early, n=11) and 24 weeks (intermediate, n=9) were immunostained for BrdU both 2 and 24 hours after BrdU injection. Early lesion, 2 hours p.i. (n=6) (D) and 24 hours p.i. (n=5) (E). Intermediate lesion 2 hours p.i. (n=4) (G) and 24 hours p.i. (n=5) (H). Arrows indicate BrdU‐positive cells. Positive cells were quantified in 3 sections from each mouse and averaged. BrdU‐positive macrophages were classified based on their location within the lesion as peripheral (close to the endothelium), internal (deeper in the lesion), or attached (in the lumen attached to the endothelium). Quantification of early (F) and intermediate lesions (I). Bar=50 μm. *P<0.05 as measured by Mann–Whitney U test. BrdU indicates bromodeoxyuridine; p.i., postinjection.
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jah31705-fig-0004: BrdU pulse labeling detects macrophage proliferation in situ and tracks monocyte incorporation into lesions. Mac‐3‐positive macrophages in lesions incorporated BrdU within 2 hours p.i. (A, arrows). At 24 hours p.i., BrdU‐positive monocytes were observed in the lumen, attached to the endothelium or extravasating into the intima (B, arrows in insets), in addition to macrophages within the lesion that had proliferated in situ (B and C, arrows). Aortic root sections from Apoe−/− mice on a chow diet at 12  (early, n=11) and 24 weeks (intermediate, n=9) were immunostained for BrdU both 2 and 24 hours after BrdU injection. Early lesion, 2 hours p.i. (n=6) (D) and 24 hours p.i. (n=5) (E). Intermediate lesion 2 hours p.i. (n=4) (G) and 24 hours p.i. (n=5) (H). Arrows indicate BrdU‐positive cells. Positive cells were quantified in 3 sections from each mouse and averaged. BrdU‐positive macrophages were classified based on their location within the lesion as peripheral (close to the endothelium), internal (deeper in the lesion), or attached (in the lumen attached to the endothelium). Quantification of early (F) and intermediate lesions (I). Bar=50 μm. *P<0.05 as measured by Mann–Whitney U test. BrdU indicates bromodeoxyuridine; p.i., postinjection.
Mentions: Aortic sections were immunostained for BrdU and Mac‐3 and visualized by immunofluorescence. In the early lesion group sacrificed at 2 hours p.i. (Figure 4A), some of the Mac‐3‐positive macrophages were observed to express BrdU, a finding consistent with the positive Ki67 immunostaining (Figure 1B), and confirming that intimal aortic macrophages are proliferating locally, or in situ. No BrdU‐positive cells were observed in the lumen of the aorta at this time point. In the early lesion group sacrificed at 24 hours p.i., BrdU‐positive monocytes were identified in the lumen, attached to the endothelium, and extravasating through the endothelial layer (Figure 4B, arrows). BrdU‐positive cells were also observed in intermediate lesions, some located in the basal portion similar to the 2‐hour time point, but mostly located toward the luminal aspect of the lesion (Figure 4C, arrows).

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&#8208;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&#8208;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&minus;/&minus; and LDLr&minus;/&minus; mice) and human coronary artery lesions, in&nbsp;situ proliferation of lesion&#8208;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&nbsp;situ. BrdU pulse labeling allowed for accurate identification of in&nbsp;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&nbsp;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&#8208;lymphocyte&#8208;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.


Related in: MedlinePlus