Nkx2‐5 Is Expressed in Atherosclerotic Plaques and Attenuates Development of Atherosclerosis in Apolipoprotein E–Deficient Mice

Figures

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  Figure 1. NK2 homeobox 5 (Nkx2‐5) expression in human normal vessels and atherosclerotic plaques. A, Immunofluorescence assay of Nkx2‐5 in internal mammary arteries (normal, upper) and atherosclerotic lesions of carotid arteries (plaque, lower). For colocalization analysis, sections were costained for Nkx2‐5 (green) and CD68 (red, macrophage marker), α‐SMA (red, smooth muscle cell marker), or von Willebrand Factor (vWF; red, endothelial marker). 4′,6‐diamidino‐2‐phenylindole (DAPI) was used for nucleus staining (blue). Arrows indicate Nkx2‐5 and cell‐specific marker double‐positive cells. B, Quantitative analysis of Nkx2‐5‐positive cells in (A). n=5 for each group. C, Western blot for Nkx2‐5 expression in human control vessels (saphenous vein and internal mammary artery) and carotid atherosclerotic plaques. n=12 for atherosclerotic plaques, 10 for internal mammary artery, and 14 for saphenous vein. HE indicates hematoxylin and eosin; α‐SMA, alpha‐smooth muscle actin.

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  Figure 2. NK2 homeobox 5 (Nkx2‐5) exerts protective effect in the initiation and progression of atherosclerosis in ApoE−/− mice. A and B, ApoE−/− mice fed a chow diet were administrated adenovirus and sacrificed at 20 weeks. Cross‐sections of aortic sinus were stained with Oil Red O, and lesion areas were quantified. Data are expressed as mean±SEM (n=12 for Ad‐EV, 14 for Ad‐Nkx2‐5, 12 for Ad‐shNC, and 13 for the Ad‐shNkx2‐5–treated group). C and D, ApoE−/− mice fed a Western diet were administrated adenovirus and sacrificed at 22 weeks. Cross‐sections of aortic sinus were stained with Oil Red O, and lesion areas were quantified. Data are expressed as mean±SEM (n=14 for Ad‐EV, 13 for Ad‐Nkx2‐5, 15 for Ad‐shNC, and 13 for the Ad‐shNkx2‐5–treated group). E and F, ApoE−/− mice fed a Western diet were administrated adenovirus and sacrificed at 22 weeks. Lesion areas of thoracoabdominal aorta were evaluated by en face staining with Oil Red O. Data are expressed as mean±SEM (n=12 per group).

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  Figure 3. Histological analysis of aortic sinus lesions of ApoE−/− mice fed a Western diet. A, Images show representative sections of aortic sinus from each group of mice stained with hematoxylin and eosin (HE), Masson's trichrome (Masson), elastica van Gieson (EVG), anti‐α‐SMA antibody, and anti‐F4/80 antibody. B, Collagen content, smooth muscle cells, macrophages, fibrous cap area, and necrotic core area are quantified as a percent of total plaque area. Elastic lamina destruction of the tunica media was evaluated by quantifying the ruptures of elastic fibers. Arrows in α‐SMA staining panels indicate smooth muscle cells favoring fibrous caps formation, and arrows in EVG staining panels indicate ruptures of elastic fibers of tunica media. Data are expressed as mean±SEM (n=12 per group). α‐SMA indicates alpha‐smooth muscle actin.

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  Figure 4. Effects of NK2 homeobox 5 (Nkx2‐5) on MMP2/9 expression and activities in ApoE−/− mice fed a Western diet. A, Cross‐sections of aortic sinus were immunostained with antibodies against MMP2 and MMP9. Staining with rabbit IgG isotype was used as the negative control for MMP2 and MMP9. B, Quantification of histochemical staining of MMP2 and MMP9. Positive stained areas were quantified as a percentage of total plaque area. Data are expressed as mean±SEM (n=12 per group). C, In situ zymographic analysis of MMP2/9 activities in aortic sinus. Localization of green fluorescence indicates gelatinolytic activities of MMP2 and MMP9, and 4′,6‐diamidino‐2‐phenylindole (DAPI) was used for nucleus staining. All images were obtained under identical conditions of laser beam intensity and exposure time; representative images are displayed. D, Quantitative analysis of fluorescence intensity in (C). Data are expressed as mean±SEM (n=12 per group). DQ indicates dye‐quenched; IgG, immunoglobulin G; MMP, matrix metalloproteinase.

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  Figure 5. NK2 homeobox 5 (Nkx2‐5) regulates expression of MMPs and TIMPs in human monocyte‐derived macrophages and human aortic smooth muscle cells. A, Representative immunoblot for MMPs and TIMPs in human peripheral blood monocyte‐derived macrophages infected with Ad‐EV or Ad‐Nkx2‐5. B, Representative immunoblot for MMPs and TIMPs in human aortic smooth muscle cells infected with Ad‐EV or Ad‐Nkx2‐5. C, Quantification of band density in (A). Results are expressed as fold of control group. Data represent the mean±SEM of 3 independent experiments. *P<0.05 versus Ad‐EV group. D, Quantification of band density in (B). Results are expressed as fold of control group. Data represent the mean±SEM of 3 independent experiments. *P<0.05 versus Ad‐EV group. MMP indicates matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase.

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  Figure 6. NK2 homeobox 5 (Nkx2‐5) directly regulates MMPs/TIMPs transcription. Luciferase (Luc) reporter constructs containing promoters of MMPs or TIMPs were cotransfected with an internal control plasmid pRL‐TK into HEK293 cells, followed by infection with Ad‐Nkx2‐5 or Ad‐EV. Schematic representation in the left panel demonstrates the range of promoters, and the numbers indicate nucleotide positions relative to the transcription initiation site. The right panel demonstrates relative luciferase activities, which are expressed as a percent of values determined in the Ad‐EV‐treated group. Data represent the mean±SEM of 3 independent experiments. *P<0.05 versus the Ad‐EV‐treated group. MMP indicates matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase.

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  Figure 7. NK2 homeobox 5 (Nkx2‐5) regulates the expression of extra functional genes in macrophages, smooth muscle cells, and endothelial cells. Cells treated with Ad‐Nkx2‐5 or Ad‐EV for 24 hours were subjected to real‐time polymerase chain reaction analysis. A, Relative mRNA expression levels of genes involved in inflammation and cholesterol metabolism of monocyte‐derived macrophages. B, Relative mRNA expression levels of genes involved in apoptosis and phenotype transformation of aortic smooth muscle cells. C, Relative mRNA expression levels of endothelial factors involved in arterial vasomotoricity, chemokine production, adhesion, and angiogenesis. Data represent the mean±SEM of 3 independent experiments. *P<0.05 versus the Ad‐EV‐treated group. ICAM‐1 indicates intercellular adhesion molecule‐1; qPCR, quantitative polymerase chain reaction; VCAM‐1, vascular cell adhesion molecule‐1.

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  Figure 8. NK2 homeobox 5 (Nkx2‐5) inhibits monocyte‐endothelial adhesion and decreases expression of adhesion molecules in early atherosclerosis. A, Upper panel: representative images used for quantification of peripheral blood monocytes (PBMC; green, calcein AM) attached to aortic endothelial cells (HAECs, blue, DAPI). Middle panel: effects of Nkx2‐5 on expression of vascular cell adhesion molecule‐1 (VCAM‐1) in endothelial cells (representative immunofluorescence image: green for VCAM‐1 and blue for cell nuclei stained with DAPI). Lower panel: effects of Nkx2‐5 on expression of E‐selectin in endothelial cells (representative immunofluorescence image: red for E‐selectin and blue for cell nuclei stained with DAPI). B, Quantification of adhesion. The number of PBMC adhered to per 100 HAECs was calculated. Results are expressed as a percent of values determined in the Ad‐EV‐treated group. Data represent the mean±SEM of 3 independent experiments. C, Representative immunoblot for intercellular adhesion molecule‐1 (ICAM‐1), VCAM‐1, E‐selectin, and P‐selectin in endothelial cells infected with Ad‐EV or Ad‐Nkx2‐5. D, Cross‐sections of early atherosclerotic lesions in aortic sinus were…