Macrophage overexpression of matrix metalloproteinase-9 in aged mice improves diastolic physiology and cardiac wound healing after myocardial infarction - PubMed (original) (raw)

Macrophage overexpression of matrix metalloproteinase-9 in aged mice improves diastolic physiology and cardiac wound healing after myocardial infarction

Cesar A Meschiari et al. Am J Physiol Heart Circ Physiol. 2018.

Abstract

Matrix metalloproteinase (MMP)-9 increases in the myocardium with advanced age and after myocardial infarction (MI). Because young transgenic (TG) mice overexpressing human MMP-9 only in macrophages show better outcomes post-MI, whereas aged TG mice show a worse aging phenotype, we wanted to evaluate the effect of aging superimposed on MI to see if the detrimental effect of aging counteracted the benefits of macrophage MMP-9 overexpression. We used 17- to 28-mo-old male and female C57BL/6J wild-type (WT) and TG mice ( n = 10-21 mice/group) to evaluate the effects of aging superimposed on MI. Despite similar infarct areas and mortality rates at day 7 post-MI, aging TG mice showed improved diastolic properties and remodeling index compared with WT mice (both P < 0.05). Macrophage numbers were higher in TG than WT mice at days 0 and 7 post-MI, and the post-MI increase was due to elevated cluster of differentiation 18 protein levels (all P < 0.05). RNA sequencing analysis of cardiac macrophages isolated from day 7 post-MI infarcts identified 1,276 statistically different (all P < 0.05) genes (994 increased and 282 decreased in TG mice). Reduced expression of vascular endothelial growth factor A, platelet-derived growth factor subunit A, and transforming growth factor-β3, along with elevated expression of tissue inhibitor of MMP-4, in macrophages revealed mechanisms of indirect downstream effects on fibroblasts and neovascularization. While collagen accumulation was enhanced in TG mice compared with WT mice at days 0 and 7 post-MI ( P < 0.05 for both), the post-MI collagen cross-linking ratio was higher in WT mice ( P < 0.05), consistent with increased diastolic volumes. Vessel numbers [by Griffonia ( Bandeiraea) simplicifolia lectin I staining] were decreased in TG mice compared with WT mice at days 0 and 7 post-MI ( P < 0.05 for both). In conclusion, macrophage-derived MMP-9 improved post-MI cardiac wound healing through direct and indirect mechanisms to improve diastolic physiology and remodeling. NEW & NOTEWORTHY Aging mice with macrophage overexpression of matrix metalloproteinase-9 have increased macrophage numbers 7 days after myocardial infarction, resulting in improved diastolic physiology and left ventricular remodeling through effects on cardiac wound healing.

Keywords: RNA sequencing; angiogenesis; cardiac wound healing; collagen; left ventricular physiology.

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Figures

Fig. 1.

Macrophage matrix metalloproteinase-9 overexpression in transgenic (TG) mice (n = 16) did not affect the day 7 following myocardial infarction (post-MI) survival rate compared with wild-type (WT) mice (n = 22).

Fig. 2.

Matrix metalloproteinase-9 overexpression increased macrophage cell numbers at day 0 (D0) and day 7 (D7) following myocardial infarction (post-MI). A: left ventricular sections were stained with anti-Mac-3 antibody; arrows depict positive staining. At D7 post-MI, macrophage numbers were increased in wild-type (WT) infarcts compared with D0 controls. The increase in macrophage numbers was greater in post-MI transgenic (TG) than WT control infarcts. B: CD18 was increased in WT and TG infarcts at D7 post-MI compared with D0. CD18 was increased in TG compared with WT infarcts. The spleen was used as a positive control (+). Values are means ± SE and graphed as individual value plots; n = 10–21 mice/group for immunohistochemistry and 7–10 mice/group for immunoblot analysis. *P < 0.05 vs. respective D0 values; #P < 0.05 vs. WT D7; †P < 0.05 vs. WT D0.

Fig. 3.

RNA sequencing (RNA-seq) of isolated cardiac macrophages from wild-type (WT) and transgenic (TG) mice at day 7 (D7) following myocardial infarction (post-MI). A: of the 23,843 genes examined, 1,276 genes were different between WT and TG groups (all P < 0.05 by _t_-test). Ingenuity pathway analysis showed the top 5 most important pathways. B: heat map of the top 50 genes ranked by P value and fold change (FC). Red, upregulation; green, downregulation. Top 3 ranked up- and downregulated genes, by P value and FC, are shown. C: heat map showing proinflammatory (red) and anti-inflammatory (blue) genes differentially expressed in WT and TG mice (all P < 0.05).

Fig. 4.

Macrophage matrix metalloproteinase-9 overexpression increased collagen (Col) content at baseline and following myocardial infarction (post-MI). A: left ventricular sections stained with picrosirius red demonstrated increased collagen content in transgenic (TG) mice compared with wild-type (WT) mice at day 0 (D0). The collagen-stained area was increased in WT and TG mice at day 7 (D7) post-MI compared with D0. Collagen content was increased in TG mice compared with WT mice at D7 post-MI. B: procollagen type I levels were decreased in WT mice at D7 post-MI compared with D0. Full-length collagen type I levels were increased in WT and TG mice at D7 post-MI compared with D0. Collagen type I fragment levels were increased in WT mice at D7 post-MI compared with D0 and decreased in TG mice compared with WT mice at D7 post-MI. C: procollagen type III and full-length collagen III levels were increased in WT and TG mice at D7 post-MI compared with D0. Collagen type III fragment levels were decreased in TG mice at D7 post-MI compared with D0. Purified full-length collagen type I and collagen type III were used as positive controls (+). Values are means ± SE and graphed as individual value plots; n = 10–21 mice/group for picrosirius red staining and 7–10 mice/group for immunoblot analysis. *P < 0.05 vs. respective D0 values; #P < 0.05 vs. WT D7; †P < 0.05 vs. WT D0.

Fig. 5.

Macrophage matrix metalloproteinase-9 overexpression reduced following myocardial infarction (post-MI) collagen cross-linking. A: lysyl oxidase (LOX) protein was increased in wild-type (WT) and transgenic (TG) infarcts at day 7 (D7) post-MI compared with day 0 (D0). No differences were observed between WT and TG infarcts at D7 post-MI. B: at D7 post-MI, the procollagen (Pro-Col) type I cross-linking ratio was reduced in TG mice compared with WT mice. Values are means ± SE and graphed as individual value plots; n = 7–10 mice/group. *P < 0.05 vs. respective D0 values; #P < 0.05 vs. WT D7.

Fig. 6.

Macrophage matrix metalloproteinase-9 overexpression reduced vessel numbers and angiogenesis signaling at baseline and after myocardial infarction (MI). A: left ventricular sections were stained with biotinylated Griffonia (Bandeiraea) simplicifolia lectin I (GSL-I) and quantified. GSL-I was measured in 5 random sections taken from the infarct region across the left ventricular free wall. The percentage of GSL-I-stained area per total area showing reduced vessel numbers in transgenic (TG) mice at day 0 (D0) was compared with that in wild-type (WT) mice at D0. The GSL-I-stained area was reduced in TG mice compared with WT mice at day 7 (D7) post-MI. B: gene expression levels of proangiogenic factors [VEGFA, platelet-derived growth factor subunit A (Pdgfa), VCAM-1, integrin-α5 (Itga5), and tissue inhibitor of metalloproteinase 4 (Timp4)] assessed by RNA sequencing (RNA-seq) in isolated cardiac macrophages. FPKM, fragments per kilobase of transcript per 1 × 106 mapped reads. C: VEGF188 (25 kDa) was increased in WT mice, but not in TG mice, at D7 post-MI compared with D0, and VEGF164 (22 kDa) was decreased in WT and TG mice at D7 post-MI compared with D0 controls. D: VCAM-1 was decreased in WT and TG mice at D7 post-MI compared with D0. Mouse hepatic tumor was used as a positive control (+). Values are means ± SE and graphed as individual value plots; n = 10–21 mice/group for GSL-I staining, 4–5 mice/group for RNA-seq, and 7–10 mice/group for immunoblot analysis. *P < 0.05 vs. respective D0 values; #P < 0.05 vs. WT D7; †P < 0.05 vs. WT D0.

Fig. 7.

Mechanistic diagram showing hypothesized effects of transgenic macrophage matrix metalloproteinase (MMP)-9 overexpression in the aging and following myocardial infarction (post-MI) left ventricle (LV). In the wild-type LV, MMP-9 is increased by aging and MI, which trigger CD18 expression and macrophage infiltration to further increase MMP-9. Increased inflammation leads to extracellular matrix (ECM) accumulation, collagen cross-linking, and impaired angiogenesis. Together, these changes result in enhanced LV diastolic volume and remodeling. In the transgenic LV, macrophage infiltration and ECM accumulation are increased further, and cross-linking is decreased to improve LV diastolic volume and LV remodeling. +, increase; −, decrease.

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Macrophage overexpression of matrix metalloproteinase-9 in aged mice improves diastolic physiology and cardiac wound healing after myocardial infarction - PubMed (original) (raw)