Transgenic overexpression of macrophage matrix metalloproteinase-9 exacerbates age-related cardiac hypertrophy, vessel rarefaction, inflammation, and fibrosis

The present study was the first to use mice with transgenic overexpression of matrix metalloproteinase-9 (MMP-9) in macrophages to examine the effects of macrophage-derived MMP-9 on cardiac aging. We found that an elevation in macrophage-derived MMP-9 induced a greater age-dependent cardiac hypertrophy and vessel rarefaction phenotype, which enhanced cardiac inflammation and fibrosis.

Abstract

Advancing age is an independent risk factor for cardiovascular disease. Matrix metalloproteinase-9 (MMP-9) is secreted by macrophages and robustly increases in the left ventricle (LV) with age. The present study investigated the effect of MMP-9 overexpression in macrophages on cardiac aging. We compared 16- to 21-mo-old C57BL/6J wild-type (WT) and transgenic (TG) male and female mice (n = 15–20/group). MMP-9 overexpression amplified the hypertrophic response to aging, as evidenced by increased LV wall thickness and myocyte cross-sectional areas (P < 0.05 for both). MMP-9 overexpression reduced LV expression of the angiogenesis-related factors ICAM-1, integrins α3 and β3, platelet/endothelial cell adhesion molecule-1, thrombospondin-1, tenascin-c, and versican (all P < 0.05). Concomitantly, the number of vessels in the TG was lower than WT LV (P < 0.05). This led to a mismatch in the muscle-to-vessel ratio and resulted in increased cardiac inflammation. Out of 84 inflammatory genes analyzed, 16 genes increased in the TG compared with WT (all P < 0.05). Of the elevated genes, 14 were proinflammatory genes. The increase in cardiac inflammation resulted in greater accumulation of interstitial collagen in TG (P < 0.05). Fractional shortening was similar between groups, indicating that global cardiac function was still preserved at this age. In conclusion, overexpression of MMP-9 in macrophages resulted in exacerbated cardiac hypertrophy in the setting of vessel rarefaction, which resulted in enhanced inflammation and fibrosis to augment the cardiac-aging phenotype. Our results provide evidence that macrophage-derived MMP-9 may be a therapeutic target in elderly subjects.

NEW & NOTEWORTHY The present study was the first to use mice with transgenic overexpression of matrix metalloproteinase-9 (MMP-9) in macrophages to examine the effects of macrophage-derived MMP-9 on cardiac aging. We found that an elevation in macrophage-derived MMP-9 induced a greater age-dependent cardiac hypertrophy and vessel rarefaction phenotype, which enhanced cardiac inflammation and fibrosis.

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aging is an independent risk factor for cardiovascular disease (CVD), and CVD remains a leading cause of death (8). Aging results in diastolic dysfunction and concentric remodeling in the left ventricle (LV), whereas systolic function is relatively preserved until senescence (26, 51). The cardiac ECM provides structural support for the myocardium, in addition to serving as a reservoir for cytokines, growth factors, and modulatory proteins (22). With advancing age, fibrillar collagens accumulate in the LV, resulting in the increase in myocardial stiffness (15, 26, 34). Matrix metalloproteinases (MMPs) are zinc-dependent enzymes that regulate LV function by degrading ECM to stimulate turnover (5). In addition, MMPs activate and inactivate cytokines and growth factors (49).

MMP-9 processes a wide variety of ECM substrates (collagens, fibronectin, and laminin) and non-ECM substrates (IL-1β, IL-6, and latent transforming growth factor-β) (45). This wide range of substrates implicates MMP-9 in the pathogenesis of many diseases, including CVD (45). MMP-9 is robustly produced by macrophages and promotes inflammatory cell infiltration by degrading myocardial ECM (18). In aging mice, MMP-9 levels increase in plasma and LV (9, 10, 52). Our group has demonstrated that age-related LV diastolic dysfunction and collagen accumulation are attenuated in MMP-9–null mice compared with wild-type (WT) mice (10). Because MMP-9 deletion improves the cardiac-aging phenotype, the hypothesis of this study was that overexpression of macrophage MMP-9 may enhance aging-induced LV inflammation and fibrosis.

MATERIALS AND METHODS

Animals.

All procedures were performed in accordance with the 2011 Guide for the Care and Use of Laboratory Animals (National Research Council, The National Academies Press, Washington, DC) and were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center. We used 16- to 21-mo-old male and female C57BL/6J WT and transgenic (TG) mice overexpressing human MMP-9 in macrophages to study genotype differences in the aged LV. TG mice carried the human MMP-9 cDNA (2.4 kb) transgene driven by the scavenger receptor A enhancer/promoter (4.5 kb), as described previously (7, 55). The old WT mice were a randomly selected subgroup from our previous studies (52, 53). For this study, all echocardiograms were analyzed by a single observer, and all downstream tissue analysis was performed at the same time for all samples and by individuals blinded to groups.

Echocardiography.

Echocardiographic measurements were performed using the Vevo 2100 system (VisualSonics, Toronto, ON, Canada) to measure LV structure and function. Mice were anesthetized using isoflurane (0.5–2% in an oxygen mix) and stationed on an isothermal pad. Images were taken at a minimum heart rate of 400 beats/min to maintain physiological relevance. LV dimensions and wall thickness were measured from the LV parasternal short axis M-mode view, and fractional shortening was calculated. Images from three cardiac cycles were used and averaged to measure each variable.

Tissue collection.

Mice were euthanized under anesthesia with isoflurane (2% in an oxygen mix). The hearts were flushed with saline (for cell isolation) or cardioplegic solution (for tissue collection) and excised from the chest cavity. The LV was separated from the right ventricle (RV) and sectioned transversely. For tissue collection, the middle section from old WT (n = 15) and TG (n = 20) mice was fixed in zinc formalin and used for histological analysis. The base was snap frozen and used for RNA extraction.

Cardiac macrophage isolation and real-time RT-PCR.

For macrophage isolation, LV tissue from old WT (n = 6) and TG (n = 7) mice was minced into 1- to 2-mm pieces under a sterile laminar flow hood and dissociated into single-cell suspension using collagenase II (600 U/ml; Worthington Biochemical, Lakewood, NJ) and DNase I (60 U/ml; AppliChem, Darmstadt, Germany) in HBSS (Thermo Fisher Scientific, Waltham, MA). After 1 h incubation at 37°C with mechanical dissociation applied every 15 min, the cell suspension was centrifuged and resuspended in HBSS and applied over preseparation filters (130-041-407; Miltenyi Biotec, Bergisch Gladbach, Germany) to remove nondissociated clumps. After lysing red blood cells (Red Blood Cell Lysis Solution; Miltenyi Biotec), cell suspension was purified using the anti-Ly-6G Microbeads kit (130-092-332; Miltenyi Biotec) and the magnetic MS column (130-042-201; Miltenyi Biotec) to remove neutrophils. The macrophages were separated from the flowthrough using an anti-CD11b Microbeads kit (130-049-601; Miltenyi Biotec) and a magnetic MS column. Macrophages were cultured in RPMI-1640 media (Thermo Fisher Scientific), supplemented with 10% FBS overnight, and RNA was extracted from the adherent cells with TRIzol (Thermo Fisher Scientific). cDNA was synthesized using the High-Capacity RNA-to-cDNA kit (4387406; Thermo Fisher Scientific), and TaqMan gene-expression assays were performed using specific primer for human MMP-9 (Hs00234579_m1; Thermo Fisher Scientific). Gene levels were normalized to hypoxanthine-guanine phosphoribosyltransferase 1 (Hprt1; Mm01545399_m1; Thermo Fisher Scientific). Real-time RT-PCR was performed according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, with the exception that only Hprt1 was used as the reference gene.

Histology.

Paraffin-embedded LV sections were de-paraffinized and rehydrated. Sections (5 μm) were stained with hematoxylin-eosin, biotinylated anti-Griffonia (Bandeiraea) simplicifolia lectin I (GSL-I), or Picrosirius Red (PSR). Sections stained with hematoxylin-eosin were used for myocyte cross-sectional analysis. The dimensions of five myocytes with central nuclei from five random regions were measured from each region for a total of 25 myocytes/LV. A GSL-I (B-1105, 1:100; Vector Laboratories, Burlingame, CA) was used to detect endothelial cells with 3,3′ diaminobenzidine chromogen, which was used to label positive staining, and eosin was used as a counterstain. All GSL-I-positive vessels were counted in each randomly selected region. For PSR staining, after the incubation in 0.2% phosphomolydbic acid, the LV sections were stained with 0.1% Sirius Red in saturated picric acid and washed in 0.01 N hydrochloric acid. Angiogenesis and collagen deposition were determined by GSL-I-positive and PSR-positive area/total tissue area for each image region. For image analysis, five random regions from each slide were scanned and analyzed (Image-Pro Analyzer 7.0).

RNA extraction from LV tissue and quantitative real-time RT-PCR.

RNA was extracted from the LV using the TRIzol reagent, and cDNA was synthesized using the RT² First Strand Kit (330401; Qiagen, Germantown, MD). To measure gene expression of 84 ECM and adhesion molecules or inflammatory cytokines and cytokine receptors, quantitative real-time RT-PCR was performed using RT² SYBR Green Rox qPCR Mastermix (330523; Qiagen) and 96-well array plates (PAMM-013E and PAMM-011E; Qiagen). The relative gene expression of individual target molecules was calculated by normalization of the cycle threshold (Ct) values of the target genes to the Ct values of Hprt1. The experiments were performed in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, with the exception that only Hprt1 was used as the reference gene (31).

Statistics.

Data are expressed as means ± SE. Two group comparisons between old WT and old TG mice for body weight, echocardiographic, gene array, myocyte cross-sectional area, GSL-I staining, and collagen area fraction data and between WT and TG for human MMP-9 expression were performed using unpaired t-test. Genotype comparisons of WT and TG across ages for echocardiographic data were performed using two-way ANOVA, followed by the Bonferroni multiple comparisons correction test. A P < 0.05 was considered significant.

RESULTS

Human MMP-9 gene was expressed in macrophages isolated from the LV of TG.

To verify the transgene expression of the human MMP-9 in this study, we quantified the expression of the human MMP-9 gene in cardiac macrophages. As expected, human MMP-9 was observed in TG macrophages but not in WT macrophages (Fig. 1). Human and mouse MMP-9 share 90% homology (81% identity) with a bit score of 724, and mice recognize human MMP-9 (39). The TG mouse has previously been shown to have 15-fold overexpression of macrophage MMP-9 (55).

Fig. 1.

Human matrix metalloproteinase-9 (MMP-9) was expressed in macrophages isolated from the left ventricles of macrophage MMP-9 transgenic (TG; n = 7) but not wild-type (WT; n = 6) mice. Macrophages were isolated from 16- to 21-mo-old mice. Gene expression was normalized to Hprt1 and is shown as 2^−ΔCt. Data are expressed as means ± SE.

Macrophage MMP-9 overexpression enhanced age-dependent global LV hypertrophy.

We previously reported that mice exhibit increased LV wall thickness with age (48, 52). Hypertrophy begins in middle age and can be observed in mice at ~15 mo of age. In the present study, echocardiographic analysis demonstrated that 16- to 21-mo-old WT and TG showed increased wall thickness compared with the respective young mice (Table 1). Interestingly, LV wall thickness in the TG was greater than the age-matched WT LV, indicating that overexpression of MMP-9 accentuated the hypertrophic response. Old WT and TG showed similar fractional shortening compared with young mice. Previously, we reported that the older WT mice (26–34 mo old) and not mice younger than 24 mo old exhibit decreased fractional shortening (10). Our data support the concept that LV dysfunction generally occurs after 26 mo of age for healthy WT mice, which is consistent with our present results (30). Of note, fractional shortening was similar between old WT and TG, indicating that overexpression of macrophage MMP-9 had no effects on myocyte contractility at this age (Table 1). The WT and TG mice of young and old groups had similar LV mass and LV mass/tibia ratio, indicating that the age observed in this study might be a key transition point where there was no increase in LV mass but significant LV wall thickening. The RV mass and RV mass/tibia ratio of the old TG mice were higher than young TG and age-matched WT mice, indicating that RV hypertrophy occurred in the old TG (Table 1). There were no significant sex differences in fractional shortening, wall thickness, or collagen content in the aged LV. Old male TG weighed more than age-matched female TG (P < 0.05).

Table 1.

Echocardiographic and necropsy analyses of young (3–6 mo old) and old (16–21 mo old) wild-type (WT) and macrophage matrix metalloproteinase-9 (MMP-9) transgenic (TG) mice

	Young WT	Old WT	Young TG	Old TG
n (Male)	11 (6)	15 (10)	20 (10)	20 (10)
Age, mo	3.7 ± 0.1	18.3 ± 0.6	4.7 ± 0.2	18.9 ± 0.4
Body weight, g	26.5 ± 1.0	29.8 ± 1.0*	24.7 ± 0.9	32.8 ± 1.3*
End systolic dimension, mm	2.1 ± 0.2	2.2 ± 0.1	1.9 ± 0.1	2.1 ± 0.1
End diastolic dimension, mm	3.4 ± 0.1	3.6 ± 0.1	3.2 ± 0.1	3.6 ± 0.1*
Fractional shortening, %	41 ± 3	39 ± 3	42 ± 2	42 ± 2
Interventricular septal wall thickness, diastole, mm	0.62 ± 0.02	0.74 ± 0.03*	0.69 ± 0.02	0.86 ± 0.03*†
Posterior wall thickness, diastole, mm	0.61 ± 0.04	0.65 ± 0.03	0.64 ± 0.02	0.71 ± 0.03
Mean wall thickness, diastole, mm	0.61 ± 0.02	0.70 ± 0.02*	0.66 ± 0.02	0.79 ± 0.02*†
Left ventricular mass, mg	87.6 ± 3.7	88.2 ± 3.8	80.0 ± 2.7	88.7 ± 2.9*
Left ventricular mass/tibia length	5.1 ± 0.2	5.0 ± 0.2	4.7 ± 0.1	5.0 ± 0.2
Right ventricular mass, mg	21.6 ± 1.7	21.8 ± 1.7	22.7 ± 1.0	26.5 ± 1.0*†
Right ventricular mass/tibia length	1.3 ± 0.1	1.3 ± 0.1	1.3 ± 0.1	1.5 ± 0.1*†

Data are expressed as means ± SE. Mean wall thickness, average of the left ventricle interventricular septum wall thickness and posterior wall thickness. WT, wild-type; MMP-9, macrophage matrix metalloproteinase-9; TG, transgenic mice.

^*P < 0.05 vs. respective young.

^†P < 0.05 vs. age-matched WT.

Macrophage MMP-9 overexpression enhanced age-dependent cardiomyocyte hypertrophy.

We previously demonstrated that myocyte size in old (15–18 mo old) WT is greater than young (6–9 mo old) WT (48, 52). Myocyte size was greater in the 16- to 21-mo-old TG compared with the WT control (Fig. 2). This result indicated that overexpression of MMP-9 only in macrophages promoted age-related myocyte hypertrophy—a finding consistent with the increase in LV wall thickness.

Fig. 2.

Macrophage matrix metalloproteinase-9 (MMP-9) overexpression increased myocyte hypertrophy in 16- to 21-mo-old mice. A: representative images of hematoxylin-eosin-stained sections from the left ventricles (LVs) of wild-type (WT; n = 9; left) and macrophage MMP-9 transgenic (TG; n = 6; right) mice. Original scale bars, 50 μm. B: myocyte cross-sectional areas were quantified by measuring the area of 5 random cells/section for a total of 25 myocytes/LV. Graph shows greater myocyte size in old TG compared with WT. Data are expressed as means ± SE.

Macrophage MMP-9 overexpression decreased angiogenic-relevant ECM and adhesion molecule expression.

To examine the effects of overexpression of macrophage MMP-9 on cardiac ECM, we measured 84 ECM and adhesion molecule genes (Table 2). Of the 84 genes evaluated, 10 increased, and 25 decreased in TG compared with WT (Table 3). Out of the 25 decreased genes, 7 genes have been reported to be associated with angiogenesis, suggesting that overexpression of macrophage MMP-9 altered the angiogenic landscape in the aged heart. With the increase in human MMP-9, mouse MMP-9 gene expression reduced by one-half.

Table 2.

ECM and adhesion molecules analyzed by gene array

Gene List
Adamts1	Contactin1	Emilin1	ItgaL	Lamb3	Mmp8	Spp1
Adamts2	Col1a1	Entpd1	ItgaM	Lamc1	Mmp9	Syt1
Adamts5	Col2a1	Fibulin1	ItgaV	Mmp10	Ncam1	Tgfbi
Adamts8	Col3a1	Fibronectin1	ItgaX	Mmp11	Ncam2	Thbs1
Ctnna1	Col4a1	Hapln1	Itgb1	Mmp12	Pecam1	Thbs2
Ctnna2	Col4a2	Hc	Itgb2	Mmp13	Periostin	Thbs3
Ctnnb1	Col4a3	Icam1	Itgb3	Mmp14	E-selectin	Timp1
Cd44	Col5a1	Itga2	Itgb4	Mmp15	L-selectin	Timp2
Cdh1	Col6a1	Itga3	Lama1	Mmp1a	P-selectin	Timp3
Cdh2	Vcan	Itga4	Lama2	Mmp2	Sgce	Tnc
Cdh3	Ctgf	Itga5	Lama3	Mmp3	Sparc	Vcam1
Cdh4	Ecm1	ItgaE	Lamb2	Mmp7	Spock1	Vitronectin

Adamts, a disintegrin and metalloproteinase with thrombospondin motif; Ctnn, catenin; Cd44, cluster of differentiation 44; Cdh, cadherin; Col, collagen; Vcan, versican; Ctgf, connective tissue growth factor; Entpd1, ectonucleoside triphosphate diphosphohydrolase 1; Hapln1, hyaluronan and proteoglycan link protein 1; Hc, hemolytic complement; Itg, integrin; Lam, laminin; Mmp, matrix metalloproteinase; Ncam, neural cell adhesion molecule; Pecam1, platelet/endothelial cell adhesion molecule 1; Sgce, sarcoglycan epsilon; Sparc, secreted protein acidic and rich in cysteine; Spock1, sparc/osteonectin; Spp1, secreted phosphoprotein 1; Syt1, synaptotagmin-1; Tgfbi, transforming growth factor β-induced protein; Thbs, thrombospondin; Timp, tissue inhibitor of metalloproteinase; Tnc, tenascin C; Vcam, vascular cell adhesion molecule.

Table 3.

By ECM and adhesion molecule gene array analysis, 10 genes were increased and 24 genes decreased in TG LV

	WT	TG	P
↑ in TG
Ctnna1	95.67 ± 4.73	176.21 ± 11.83	0.0006
Cdh2	52.23 ± 10.89	103.52 ± 11.09	0.0049
Emilin1	12.03 ± 1.04	19.65 ± 2.11	0.0033
Itga4	0.63 ± 0.11	0.77 ± 0.05	0.0434
Itgb2	2.81 ± 0.29	4.88 ± 0.55	0.0030
Lama2	7.19 ± 0.49	11.07 ± 1.25	0.0085
Lamb3	3.43 ± 0.21	4.29 ± 0.30	0.0270
Mmp8	0.09 ± 0.02	0.22 ± 0.04	0.0031
Mmp11	0.59 ± 0.09	0.71 ± 0.04	0.0434
Sparc	97.44 ± 7.41	123.72 ± 3.66	0.0117
↓ in TG
Ctnnb1	176.48 ± 6.17	144.32 ± 3.89	0.0008
Cdh3	0.043 ± 0.004	0.022 ± 0.002	0.0016
Col1a1	49.44 ± 4.97	28.00 ± 2.47	0.0009
Col3a1	82.11 ± 10.22	45.05 ± 5.40	0.0090
Col4a3	1.50 ± 0.14	0.63 ± 0.11	0.0003
Col5a1	21.92 ± 2.10	14.43 ± 1.36	0.0121
Ctgf	81.04 ± 15.23	45.73 ± 5.24	0.0434
Hapln1	0.557 ± 0.183	0.033 ± 0.005	0.0117
Hc	0.14 ± 0.03	0.05 ± 0.02	0.0434
Icam1	19.33 ± 1.91	9.17 ± 0.66	<0.0001
Itga3	5.58 ± 0.69	3.27 ± 0.13	0.0005
Itgae	0.75 ± 0.15	0.37 ± 0.08	0.0495
Itgb3	4.60 ± 0.54	1.67 ± 0.13	<0.0001
Itgb4	0.33 ± 0.04	0.15 ± 0.01	<0.0001
Lama3	3.00 ± 0.37	1.54 ± 0.16	0.0021
Lamc1	66.97 ± 4.79	51.86 ± 1.92	0.0266
Mmp9	0.54 ± 0.10	0.25 ± 0.06	0.0316
Mmp14	11.05 ± 0.88	5.45 ± 0.49	<0.0001
Ncam1	3.63 ± 0.70	1.28 ± 0.14	0.0434
Ncam2	0.053 ± 0.009	0.026 ± 0.004	0.0067
Pecam1	126.8 ± 8.88	88.31 ± 6.19	0.0038
Thbs1	65.47 ± 13.87	8.60 ± 1.34	0.0003
Thbs3	6.82 ± 0.80	3.90 ± 0.30	0.0031
Tnc	2.27 ± 0.30	1.18 ± 0.14	0.0085
Vcam	10.67 ± 1.45	5.87 ± 0.34	0.0266

Data are expressed as means ± SE. Gene expression was normalized to hypoxanthine-guanine phosphoribosyltransferase 1 and shown as 2^−ΔCt × 100 units. All genes listed were P < 0.05 vs. WT (WT, n = 10; TG, n = 8). Underlines, angiogenic genes.

Macrophage MMP-9 overexpression reduced vessel numbers in aged LV.

Because the ECM gene array result suggested that angiogenic stimuli were lower in the aged TG LV, we quantified blood vessel numbers in the myocardium (Fig. 3). TG LV had lower vessel density compared with WT. There was no age-dependent increase in vessel numbers in the WT LV despite the increase in myocyte hypertrophy and circulating angiogenic stimuli, which may lead to insufficient oxygen supply to hypertrophied myocytes in aged myocardium (52). This imbalance was exacerbated in the TG LV. Of note, MMP-9 deletion increases vessel numbers in the aged LV (52). These results indicate a strong relationship between MMP-9 and downstream angiogenic responses.

Fig. 3.

Transgenic (TG) overexpression of macrophage matrix metalloproteinase-9 (MMP-9) increased age-related vessel rarefaction. Blood vessels in the left ventricles (LVs) of 16- to 21-mo-old mice were stained with Griffonia (Bandeiraea) simplicifolia lectin I (GSL-I) and quantified. A: representative images showing GSL-I-positive cells (black stain) in the LVs of wild-type (WT; n = 6; left) and macrophage MMP-9 TG (n = 7; right) mice. Original scale bars, 100 μm. B: percentage of GSL-I-positive area/total area, showing that vessel area was smaller in old TG compared with WT. Data are expressed as means ± SE.

Macrophage MMP-9 overexpression induced LV inflamm-aging.

Inflammation plays an important role in cardiac aging, and the concept of inflamm-aging has been proposed to define a chronic proinflammatory status of aging occurring in the absence of overt infection (11, 17, 43). To assess LV inflammatory status, we measured the expression of inflammatory cytokines and cytokine receptors by gene array (Table 4). Of the 84 genes measured, 16 increased, and 10 decreased in TG compared with WT (Table 5). Out of the 16 increased genes, 14 genes were proinflammatory mediators, indicating an elevation in inflamm-aging induced by the overexpression of macrophage MMP-9. Both the present results and our previous data with the MMP-9–null mice showing a suppressed, age-dependent increase in LV inflammatory gene expression support the hypothesis that MMP-9 is a promoter of inflamm-aging in the myocardium (52).

Table 4.

Inflammatory cytokines and cytokine receptors analyzed by gene array

Gene List
Abcf1	Ccl22	Ccr3	Cxcl12	Il11	Il1r2	Ltb
Bcl6	Ccl24	Ccr4	Cxcl13	Il13	Il20	Mif
Cxcr5	Ccl25	Ccr5	Cxcl15	Il13ra1	Il2rb	Scye1
C3	Ccl3	Ccr6	Pf4	Il15	Il2rg	Spp1
Caspase1	Ccl4	Ccr7	Cxcl5	Il16	Il3	Tgfb1
Ccl1	Ccl5	Ccr8	Cxcl9	Il17b	Il4	Tnf
Ccl11	Ccl6	Ccr9	Cxcr3	Il18	Il5ra	Tnfrsf1a
Ccl12	Ccl7	Crp	Ccr10	Il1a	Il6ra	Tnfrsf1b
Ccl17	Ccl8	Cx3cl1	Ifng	Il1b	Il6st	Cd40lg
Ccl19	Ccl9	Cxcl1	Il10	Il1f6	Il8rb	Tollip
Ccl2	Ccr1	Cxcl10	Il10ra	Il1f8	Itgam	Xcr1
Ccl20	Ccr2	Cxcl11	Il10rb	Il1r1	Itgb2	Ltb

Abcf1, ATP-binding cassette subfamily F member 1; Bcl6, B cell leukemia/lymphoma 6; Cxcr, C-X-C chemokine receptor; C3, complement component 3; Ccl, chemokine (C-C motif) ligand; Ccr, CC- chemokine receptor; Crp, C-reactive protein; Cx3cl, chemokine (C-X3-C motif) ligand; Cxcl, chemokine (C-X-C motif) ligand; Pf, platelet factor; Ifng, IFN-γ; r, IL receptor; Itg, integrin; Lt, lymphotoxin; Mif, macrophage migration inhibitory factor; Scye1, small inducible cytokine subfamily E member 1; Spp1, secreted phosphoprotein 1; Tgfb1, transforming growth factor-β 1; Tnfrsf1, TNF receptor superfamily member 1; Cd40lg, CD40 ligand; Tollip, Toll-interacting protein; Xcr1, chemokine (C motif) receptor 1.

Table 5.

By inflammatory cytokine and cytokine receptor gene array analysis, 16 genes were increased and 10 decreased in TG LV

	WT	TG	P
↑ in TG
Abcf1	56.86 ± 2.89	84.56 ± 4.62	<0.0001
Ccl11	0.67 ± 0.22	1.51 ± 0.25	0.0225
Ccl12	1.22 ± 0.43	4.95 ± 0.73	0.0003
Ccl4	0.32 ± 0.07	0.62 ± 0.07	0.0081
Ccr2	0.88 ± 0.19	1.78 ± 0.15	0.0020
Ccr3	0.35 ± 0.06	1.13 ± 0.13	<0.0001
Ccr5	0.51 ± 0.20	2.72 ± 0.40	<0.0001
Cxcr3	0.17 ± 0.04	0.38 ± 0.07	0.0091
Il10rb	65.96 ± 7.93	124.36 ± 8.38	<0.0001
Il13ra1	13.62 ± 1.13	18.93 ± 0.78	0.0021
Il15	22.15 ± 2.02	32.81 ± 1.82	0.0015
Il2rg	8.06 ± 1.03	11.67 ± 0.80	0.0173
Itgb2	3.24 ± 0.28	5.05 ± 0.44	0.0024
Mif	47.43 ± 6.08	145.7 ± 8.48	<0.0001
Scye1	41.68 ± 4.04	76.53 ± 5.54	<0.0001
Tnfrsf1a	9.33 ± 0.62	14.72 ± 0.67	<0.0001
↓ in TG
C3	133.21 ± 11.06	90.97 ± 7.94	0.0093
Ccl19	24.52 ± 4.03	11.61 ± 0.87	0.0205
Ccl20	0.048 ± 0.005	0.030 ± 0.004	0.0193
Ccr9	1.44 ± 0.19	0.72 ± 0.13	0.0097
Il18	1.38 ± 0.21	0.63 ± 0.07	0.0044
Il1r1	13.59 ± 0.99	8.10 ± 0.43	0.0002
Il10	1.15 ± 0.27	0.37 ± 0.08	0.0155
Il13	0.21 ± 0.04	0.09 ± 0.02	0.0269
Il4	0.23 ± 0.05	0.08 ± 0.03	0.0352
Tgfb1	41.14 ± 3.56	27.54 ± 1.58	0.0051

Data are expressed as means ± SE. Gene expression was normalized to hypoxanthine-guanine phosphoribosyltransferase 1 and shown as 2^−ΔCt × 100 units. All genes listed were P < 0.05 vs. WT (WT, n = 10; TG, n = 8). Underlines, proinflammatory genes.

Macrophage MMP-9 overexpression stimulated collagen accumulation.

The accumulation of interstitial fibrillar collagen is a key component of cardiac aging and can occur through increased synthesis or decreased breakdown by MMPs (15, 26, 34). We have shown that collagen content increases in WT with age, and MMP-9 deletion attenuates the age-associated increase in collagen deposition, suggesting a fibrogenic role of MMP-9 in aged LV (10, 28). By PSR staining, collagen content was significantly elevated in TG compared with WT (Fig. 4). Our results indicated that TG overexpression of macrophage MMP-9 facilitated the accumulation of interstitial collagen in the myocardium of old mice.

Fig. 4.

Overexpression of macrophage matrix metalloproteinase-9 (MMP-9) increased collagen content in the left ventricles (LVs). LV sections were stained with Picrosirius Red (PSR) and quantified. A: representative images of PSR-stained LV sections from wild-type (WT; n = 10; left) and macrophage MMP-9 transgenic (TG; n = 8; right) mice. Original scale bars, 50 μm. B: percentage of the collagen area/total area, showing an increased collagen content in the old TG compared with WT. Data are expressed as means ± SE.

The mRNA levels of collagen I and III were reduced in TG compared with WT, suggesting that transgene overexpression of macrophage MMP-9 suppressed transcription of these collagens (Table 2). MMP-8 is a collagenase, which has proteolytic activity on collagen I and III (20, 50). MMP-8 mRNA levels were significantly higher in the LV of old TG mice compared with that of age-matched WT mice (Table 2). The accumulation of fibrillar collagens in the TG LV (Fig. 4) might cause a compensatory increase in MMP-8. Secreted protein acidic and rich in cysteine (SPARC), which promotes postsynthetic procollagen processing, increases in aged myocardium and facilitates age-related cardiac fibrosis and stiffness (3, 48). SPARC was higher in TG vs. WT (Table 2).

DISCUSSION

With the use of an MMP-9 deletion strategy, we have previously reported that MMP-9 is robustly produced by macrophages and plays a key role in cardiac aging (9, 13, 18, 52). In the present study, we extend our past findings by examining the effects of overexpression of human MMP-9 in mouse macrophages on cardiac aging. The significant findings of our study were that aging in the setting of macrophage MMP-9 overexpression resulted in the following: 1) exacerbated age-dependent LV hypertrophy, indicated by increases in both LV wall thickness and individual myocyte cross-sectional areas, 2) decreased angiogenic stimuli with concomitant enhanced vessel rarefaction, and 3) enhanced LV inflamm-aging and fibrosis. These results reveal that macrophage-derived MMP-9 is a prime modulator of cardiac aging.

With advancing age, a variety of changes occur in LV structure and function to induce the cardiac aging phenotype. Whereas these changes do not translate to impaired LV function until the very latest stages, they prime the LV to respond poorly to an extra-stress stimuli by reducing the cardiac reserve potential. These changes include increases to mechanical load and LV wall stress (26, 48). To compensate, LV wall thickening occurs to result in hypertrophy. The present study demonstrated that an age-dependent increase in LV wall thickness was greater in the TG, which provided evidence that overexpression of macrophage MMP-9 during cardiac aging accentuated the cardiac hypertrophic response. Myocyte hypertrophy, concomitant with myocyte cell loss, are major features of the aging heart (38). Our previous study showed that global MMP-9 deletion had no effect on age-related myocyte hypertrophy, suggesting that MMP-9 overexpression effects may be due to indirect effects on myocyte physiology (52). Both LV wall thickness and individual myocyte size were greater in TG compared with WT, indicating that the myocyte cell change translated to a global tissue-level change. We have previously seen that whereas an individual myocyte cross-sectional area increased with age, LV wall thickness did not, due to a simultaneous increase in cell loss (52).

Hypertrophied myocytes would need additional blood vessels to supply the increase in oxygen demand. Indeed, we previously observed an increase in proangiogenic stimuli in the plasma of old WT mice (52). Despite an age-related increase in angiogenic stimuli, the WT mice do not show an increase in the number of vessels, indicating unmatched angiogenic potential in old WT LV. Our group showed that MMP-9 deletion increases vessel numbers in LV of old mice, indicating that overexpression of MMP-9 may inhibit angiogenesis (52). Gene array results in the present study revealed that seven genes, which relate to angiogenesis, decreased in old TG compared with WT. Out of seven genes, four genes (integrin β3, tenascin C, platelet/endothelial cell adhesion molecule 1, and versican) are reported to promote angiogenesis, and one gene (ICAM-1) is expressed in vascular endothelial cells (1, 21, 24, 42, 54), whereas two genes (integrin α3 and thrombospondin 1) exhibit anti-angiogenic properties (27, 44). Integrins are essential components for angiogenesis; in particular, antagonists of integrin αvβ3 inhibit angiogenesis in many experimental models and are clinically examined for their therapeutic possibility against angiogenesis-dependent diseases (42). Integrin αvβ3 also mediates coronary vessel formation promoted by tenascin C (21). An age-associated increase in integrin β3 in the LV indicates its important role in the maintenance of vessel number in aging LV (52). Platelet/endothelial cell adhesion molecule 1 (Pecam1) is expressed on the endothelium and also on macrophages (12, 16, 19). The downregulation of Pecam1 in the TG LV is consistent with a lower vessel density. Although we did not measure VEGF or FGF2 expression in the TG LV, we previously showed that VEGF expression increases in the old WT LV compared with the respective young, and MMP-9 deletion enhanced an age-related increase in VEGF (52). These findings suggest that VEGF expression may be suppressed in the aged TG LV. Concomitant with decreases in expression of these angiogenic factors, blood vessel numbers were smaller in TG than WT. The present result indicates that vessel rarefaction was enhanced by MMP-9. With age, there is vessel rarefaction, which yields changes in blood vessels that are qualitative but may not be quantitative in the absence of pathology (25, 52). With MMP-9 overexpression in macrophages, there would be not only qualitative but also quantitative changes. This indicates that the myocyte-to-endothelial cell number has greater mismatch, starting a cascade of events that stimulate the cardiac aging phenotype.

Insufficient vascularization caused by MMP-9 overexpression might lead to a hypoxic environment due to an imbalance of oxygen supply to trigger an inflammatory response in the myocardium (35). Inflammation plays an important role in cardiac aging. Chronic low-grade systemic inflammation is observed in elderly individuals, even in the absence of overt disease (6). In WT, LV levels of inflammatory genes and numbers of macrophages increase with age (32, 52). The present study demonstrated that overexpression of macrophage MMP-9 enhanced the age-related cardiac inflammatory response. MMP-9 mediates age-related cardiac inflammation by decreasing anti-inflammatory alternatively activated (M2) macrophage polarization and increasing proinflammatory classically activated macrophage polarization (32). Cardiac tissue-resident macrophages in young mice exhibit an M2 macrophage-like phenotype, which may be important for tissue homeostasis (41). Alterations in composition, gene expression, and function of cardiac tissue macrophages precede overt cardiac aging, such as fibrosis and impairment in the inflammatory response (40). Overexpression of macrophage MMP-9 would disrupt homeostasis of cardiac tissue macrophages to a more proinflammatory status by decreasing M2 macrophages, resulting in stimulation and acceleration of cardiac aging.

Cardiac inflammation causes the release of fibrogenic cytokines and growth factors, all of which trigger the accumulation of ECM fibrillar collagens (22). Fibroblasts play principal roles in regulating ECM turnover. In aging fibroblasts, proliferative and migratory capacities decrease, suggesting impaired global fibroblasts function, which would contribute to an age-associated, altered ECM composition (30). Collagen solubility in the LV changes with age, indicating a change in collagen type and/or degree of crosslinking (30). Excessive, insoluble, crosslinked collagen fibril accumulation decreases compliance of the heart, which leads to myocardial stiffness, LV diastolic dysfunction, and adverse remodeling. Collagen content in the myocardium was increased by overexpression of macrophage MMP-9, suggesting that MMP-9-facilitated cardiac fibrosis. This result supports the idea that MMP-9 facilitates cardiac remodeling through, at least in part, the regulation of collagen levels. Despite an increase in collagen content in TG, the TG had lower mRNA levels of collagen I and III, suggesting negative feedback to transcription of these collagens. It also suggests that LV collagen accumulation in TG was not due to the changes in transcription of collagen genes. Collagens are synthesized as procollagens and released into the extracellular space, where their propeptide domains are cleaved, assembled, and crosslinked to form mature collagen fibers (14). One potential explanation for the mismatch between collagen gene expression and deposition is that post-translational cleavage and crosslinking processes might be accelerated in the aged TG LV. SPARC binds to newly secreted procollagen within the extracellular space and promotes the formation of mature crosslinked collagen fibrils (2, 4). SPARC increases in aged myocardium and facilitates age-related cardiac fibrosis and stiffness (3, 47, 48). Increased SPARC levels in the TG LV would accelerate post-translational processing, such that increased collagen processing would prevail over decreased transcription for a net increase in collagen accumulation. Although further study is needed, the accumulation of fibrillar collagens in the TG LV might relate to the induction of a compensatory increase in the expression of collagenases, particularly MMP-8. The balance between MMPs and tissue inhibitor of metalloproteinases (TIMPs) is critical in ECM remodeling, which contributes to cardiac function (36). Cardiac ECM remodeling is generally associated with increased MMPs and TIMPs (23). We previously found that MMP-3, -8, -9, -12, and -14 increase, whereas TIMP-3 and -4 decrease in the insoluble protein fraction of old mice compared with young and/or middle-aged mice, suggesting increased ECM degradative capacity (30). The present study found increased MMP-8 and -11, decreased MMP-14, and unchanged TIMP gene levels in TG compared with WT. The functional imbalance of MMPs and TIMPs might play roles in enhanced age-related collagen deposition in TG.

In the absence of pathology, normal cardiac aging is associated with LV concentric remodeling, diastolic dysfunction with relatively preserved systolic function, and chronic low-grade inflammation (6). With pathology, such as myocardial infarction (MI), there is an amplified cardiac response characterized by irreversible cell death, infiltration of excessive numbers of inflammatory cells, and robust release of inflammatory mediators and MMPs. At the later phase, a collagen-rich scar replaces extensive loss of cardiomyocytes, eventually resulting in adverse LV remodeling and systolic dysfunction (37, 46). We have shown that overexpression of macrophage MMP-9 attenuated the post-MI inflammatory response and the extent of LV dysfunction, whereas MMP-9 deletion suppressed LV dysfunction and inflammation in the mouse model of MI (13, 29, 55). Combined, both MMP-9 deletion and overexpression of macrophage MMP-9 have beneficial effects on post-MI remodeling. One interpretation is that the diverse effects of MMP-9 depend on the origin of MMP-9 and the physiological or pathological context. How overexpression of macrophage MMP-9 may regulate the inflammatory challenge post-MI in the setting of the aging LV remains to be determined.

MMP-9 robustly increases during several CVDs, including hypertension, atherosclerosis, and MI, and in these pathological states, macrophages are a primary source of MMP-9 (18). MMP-9 is secreted, not only by macrophages but also by myocytes, fibroblasts, vascular smooth muscle cells, endothelial cells, neutrophils, and mast cells (33). With age, fibroblast-derived MMP-9 decreases, and macrophage-derived MMP-9 increases in the myocardium (30).

In summary, this study demonstrated for the first time that human MMP-9 overexpression in mouse macrophages increased age-dependent myocyte hypertrophy and vessel rarefaction. Insufficient vessel formation to hypertrophied myocytes may, in part, exacerbate the cardiac inflammatory response, which leads to consequent ECM collagen deposition. Overexpression of macrophage MMP-9, therefore, exerts an adverse effect in the setting of cardiac aging. Figure 5 summarizes one hypothesized mechanism for how macrophage MMP-9 overexpression might exacerbate the cardiac aging phenotype. In the present study, we cannot exclude another hypothesis, namely that overexpression of macrophage MMP-9 might alter systemic vascular remodeling and resistance and increase blood pressure, leading to the observed cardiac hypertrophy and fibrosis. We previously measured systolic and diastolic blood pressure in young, middle-aged, and old WT and MMP-9 null mice (10). There were no age- or genotype- dependent changes in systolic or diastolic blood pressure in these mice, whereas the old WT but not MMP-9–null mice showed diastolic dysfunction and elevated collagen deposition. Further investigation into blood pressure and systemic arterial remodeling in the TG will provide the precise contribution of macrophage MMP-9 in arterial remodeling during aging.

Fig. 5.

Mechanistic diagram showing the hypothesized influence of macrophage matrix metalloproteinase-9 (MMP-9) in cardiac aging. In wild-type mice, myocyte hypertrophy occurs with advancing age, which increases oxygen demand. MMP-9 suppresses the increase in angiogenic stimuli in an aged heart, leading to insufficient angiogenesis and microcirculation. Imbalance of oxygen supply triggers an inflammatory response. Cardiac inflammation induces the production of fibrogenic cytokines, leading to accumulation of collagens and adverse remodeling. Transgenic overexpression of MMP-9 in macrophages exacerbates these effects of MMP-9 in cardiac aging, leading to greater inflammation and fibrosis, which may cause feedback to myocyte hypertrophy.

From our results, we conclude that macrophage-derived MMP-9 serves as an early upstream modifier of cardiac aging and that therapies targeted at its effects may prolong cardiac health.

GRANTS

This work was supported by National Institutes of Health Grants HL-075360, HL-129823, HL-051971, GM-114833, and GM-104357 and Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research & Development Award 5I01BX000505.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

H.T., P.L.C., A.Y., and R.P.I. performed experiments; H.T., P.L.C., A.Y., R.P.I., and M.L.L. analyzed data; H.T., P.L.C., and M.L.L. interpreted results of experiments; H.T. and P.L.C. prepared figures; H.T. drafted manuscript; H.T., P.L.C., A.Y., R.P.I., J.D., and M.L.L. edited and revised manuscript; H.T., P.L.C., A.Y., R.P.I., J.D., and M.L.L. approved final version of manuscript.