Conditional knockout of membrane‐type I matrix metalloproteinase in smooth muscle cells of adult mice alleviates atherosclerosis without affecting basic cardiovascular function

Figures

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  FIGURE 1. Expression of MMP14 and inducible SMC‐specific Mmp14 knockout. (A) H&E staining of human coronary artery sections, pathological intimal thickening (PIT) and fibroatheroma (FIT) (n = 7/group, scale bar = 400 µm). (B) Immunofluorescence of the regions indicated with a square in (A). DAPI: blue; MMP14: purple; and MYH11: green (scale bar = 50 µm). (C) Quantification of the mean fluorescence intensity of MMP14 and MYH11 co‐localization. (D) Relative Mmp14 mRNA level to Gapdh in different tissues of control and Mmp14 SMCiKO mice (n = 3/group). (E) Relative Mmp14 mRNA level to Gapdh in primary SMC isolated from the thoracic aortas of control and Mmp14 SMC‐CKO mice (n = 3 /group). (F) MMP14 protein levels of primary SMCs and its quantification. The relative levels were determined by normalizing the densitometry of MMP14 to that of VINCULIN on the same membrane (n = 4/group). (G and H) Body weight (G) and blood glucose levels (H) of control and Mmp14 SMC‐CKO mice (n = 7/group). (I and J) Fasting plasma cholesterol (I) and TG (J) profiles of control and Mmp14 SMC‐CKO mice (n = 7/group). n refers to biological replicates. Data are represented as mean ± S.D. p‐value was calculated by…

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  FIGURE 2. Effects of SMC‐specific Mmp14 knockout in adult mice on vascular function. (A and B) Maximum vasoconstrictor capacity to high K+ physiological saline solution (KPPS) in thoracic (A) and abdominal aorta of control and Mmp14 SMC‐CKO mice (n = 5/group). C and D, Vasoconstrictor response curve of the thoracic (C) and abdominal (D) aorta of control and Mmp14 SMC‐CKO mice to phenylephrine (PE) and area under the curve (AUC) (n = 5/group). (E and F) Mesenteric artery circumferential strain (E) and stress (F) in response to increasing pressure and area under the curve (AUC) in control and Mmp14 SMC‐CKO mice (n = 4/group). (G) Circumferential stress and strain in control and Mmp14 SMC‐CKO mice (n = 4/group). n refers to biological replicates. Data are represented as mean ± S.D. p‐value was calculated by unpaired two‐tailed Student's t‐test in panels (A)to (F). p‐value < .05 is considered significant.

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  FIGURE 3. Effects of SMC‐specific Mmp14 knockout in adult Ldlr−/− mice on atherosclerosis. (A) Relative Mmp14 mRNA level to Gapdh in different tissues of Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO mice at the endpoint (n = 3/group). (B and C) FPLC of fasting plasma cholesterol of Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO mice at baseline (B) and the endpoint (C) (n = 6/group). (D) Representative images of the aortas of Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO mice (scale bar = 3 mm). (E) Representative images and quantification of lesions of the aortic arch of Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO mice (scale bar = 1 mm, n = 6/group). (F) Representative images and quantification of oil red O‐stained aortic sinus of Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO mice at the endpoint (n = 8/group, scale bar = 0.5 mm). (G) Immunofluorescence staining of DAPI (blue) and α‐SMA (green) in the aortic sinus of Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO mice (n = 4/group) at the endpoint (scale bar = 200 µm, inset picture scale bar = 100 µm). (H) Representative pictures and quantification of picrosirius red‐stained aortic sinus of Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO mice (n =…

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  FIGURE 4. Single cell RNA seq analysis. (A) Dot blot showing the expression of the top two genes expressed in each cluster in the combined library of Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO groups. (B) UMAP analysis showing cells from the combined library of Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO groups coloured by cell cluster. (C) Heat map showing the expression of contraction and differentiation related genes, and dedifferentiation and lipid metabolism genes in the contractile and foam cell‐like SMC clusters.

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  FIGURE 5. Single cell RNA seq analysis. (A) Dot blot showing the expression of the top two genes expressed in each cluster in the SMC clusters. (B) Table shows the expression of the top 10 genes in each cluster in the SMC clusters. (C) UMAP analysis showing cells from the SMC clusters coloured by cell cluster. (D) Stacking blot showing the proportion of cells from Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO group within each SMC cluster. (E) Go‐pathway enrichment analysis for the upregulated genes in the fibroblast‐like SMC cluster. (F) Go‐pathway enrichment analysis for the upregulated genes in the foam cell‐like SMC cluster.

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  FIGURE 6. Effects of MMP14 deficiency on SMC proliferation and migration. (A) Relative mRNA levels of Acta2, Cnn1, Tagln, Col1a1, Col1a2, Col3a1, Fn1 and Mmp14 to Gapdh in primary SMCs isolated from control and Mmp14 SMC‐CKO mice (n = 3). B, Proliferation of primary SMCs isolated from control and Mmp14 SMC‐CKO mice (n = 10). (C) Representative pictures and quantification for primary SMCs migrated through collagen type I‐coated transwell (n = 3, scale bar = 0.5 mm). (D) Relative Mmp14 mRNA expression over Gapdh in HASMCs transfected with control or MMP14 siRNA (n = 3). (E) MMP14 levels and its relative levels, which was normalized to CALNEXIN on the same membrane, in HASMCs (n = 3). (F) Proliferation of HASMCs transfected with control or siMMP14 (n = 3). (G) Representative pictures and quantification of control and siMMP14 HASMCs migrated through collagen type I‐coated transwell (n = 3, scale bar = 100 µm). (H and I) Electron microscopy picture and quantification of the clear area of control explant (H) and Mmp14 SMC‐CKO explant (I) cultured in type I collagen gel (scale bar = 1 µm). (J) PDGFβ‐R protein expression and its quantification, which is normalized to Vinculin on the same blot in…

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  FIGURE 7. Effects of SMC‐specific Mmp14 conditional knockout on atherosclerosis regression. (A) Graphical illustration of the study design. 10–12‐week Ldlr−/ −/Mmp14 flox/Myh11‐Cre male mice were fed a Western Diet (WD) for 12 weeks, followed by administration of tamoxifen or olive oil daily for 5 consecutive days. Mice were then fed a regular chow Diet for 6 weeks before euthanasia and tissue collection. (B) FPLC of fasting plasma cholesterol of Ldlr−/ −/Mmp14 flox/Myh11‐Cre mice at baseline and after a 12‐week WD feeding (n = 6/group). (C) FPLC of fasting plasma cholesterol of Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO mice at endpoint (n = 6/group). (D) Body weight of mice at baseline throughout the study period (n = 6 mice per group). (E) Blood glucose of baseline, Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO mice at endpoint (n = 5–6/group). F, Representative images and quantification of oil red O‐stained aortic sinus of baseline, Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO mice at the endpoint (n = 5‐6/group, scale bar = 0.5 mm). (G) Representative pictures and quantification of oil red O‐stained aortas of baseline, Ldlr −/− control and Ldlr −/−/Mmp14 SMC‐CKO mice at the endpoint (n…

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  FIGURE 8. Inducible conditional SMC‐specific MMP14 knockout in mice. (A) Mechanism of inducible conditional SMC‐specific MMP14 knockout in mice (Mmp14 SMC‐CKO). (B) Mmp14 SMC‐CKO does not affect basic cardiac or vascular function in mice. (C) Mmp14 SMC‐CKO in Ldlr−/− mice reduces atherosclerosis progression after 16‐week WD. (D) Mmp14 SMC‐CKO promotes atherosclerosis regression in Ldlr−/− mice.