age-dependent changes in myocardial matrix metalloproteinasetissue inhibitor of metalloproteinase...

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dependent ECM remodeling.

D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

tissue function by providing a scaffold on which cells

migrate, grow, and differentiate [3]. A major constituent of

classes based on in vitro substrate specificity for various

ECM components. MMP activity is inhibited by tissue

inhibitors of metalloproteinases (TIMPs), a family currently

Ps are involved in

Cardiovascular Research 66 (2

by guest on April 4, 2014

http://cardiovascres.oxfordjournals.org/D

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the ECM is fibrillar collagen. Under basal conditions in theKeywords: Matrix metalloproteinase; Tissue inhibitor of metalloproteinase; Extracellular matrix; Matrix remodeling

1. Introduction

Cardiovascular disease is a leading cause of morbidity

and mortality that occurs with increasing incidence in the

elderly [1,2]. With aging, the myocardium undergoes

structural remodeling and hypertrophy. An important

component of structural remodeling is remodeling of the

extracellular matrix (ECM). The ECM integrates cell and

young adult, collagen turnover occurs at a rate that allows

for normal replacement and the maintenance of left

ventricular (LV) structure and function. The determinants

that regulate collagen turnover, particularly as a function of

aging, remain poorly understood.

Matrix metalloproteinases (MMPs) are key enzymes

involved in ECM turnover. The MMP family is comprised

of more than 25 individual members divided into specific114 Doughty Street, P.O. Box 250778, Charleston, SC 29425, United StatesbRalph A. Johnson Veterans Administration Medical Center, Charleston, SC 29425, United States

Received 30 August 2004; received in revised form 3 November 2004; accepted 24 November 2004

Avaialble online 13 December 2004

Time for primary review 28 days

Abstract

Objective: To evaluate the effects of aging on left ventricular (LV) geometry, collagen levels, matrix metalloproteinase (MMP) and tissue

inhibitor of metalloproteinase (TIMP) abundance, and myocardial fibroblast function.

Methods: Young (3-month-old; n=28), middle-aged (MA; 15-month-old; n=17), and old (23-month-old; n=16) CB6F1 mice of both sexes

were used in this study. Echocardiographic parameters were measured; collagen, MMP, and TIMP levels were determined for both the

soluble and insoluble protein fractions; and fibroblast function was evaluated.

Results: LV end-diastolic dimensions and wall thickness increased in both MA and old mice, accompanied by increased soluble protein and

decreased insoluble collagen. Immunoblotting revealed differential MMP/TIMP profiles. Compared to MA levels, MMP-3, MMP-8, MMP-

9, MMP-12, and MMP-14 increased, and TIMP-3 and TIMP-4 decreased in the insoluble fraction of old mice, suggesting increased

extracellular matrix (ECM) degradative capacity. Fibroblast proliferation was blunted with age.

Conclusion: This study, for the first time, identified specific differences in cellular and extracellular processes that likely contribute to age-Age-dependent changes in myoca

inhibitor of metalloproteinase

Merry L. Lindseya,*, Danielle K. Goshorna

Jennifer W. Hendricka, Joseph T. Mingoiaa

aDivision of Cardiothoracic Surgery Research, Room 629, Strom Thurmond0008-6363/$ - see front matter D 2004 European Society of Cardiology. Publish

doi:10.1016/j.cardiores.2004.11.029

* Corresponding author. Tel.: +1 843 876 5186; fax: +1 843 876 5187.

E-mail address: [email protected] (M.L. Lindsey).al matrix metalloproteinase/tissue

ofiles and fibroblast function

hristina E. Squiresa, G. Patricia Escobara,

rah E. Sweterlitscha, Francis G. Spinalea,b

arch Building, 770 MUSC Complex, Medical University of South Carolina,

005) 410419

www.elsevier.com/locate/cardiorescomposed of four members [4]. MMseveral cardiovascular disease processes, including plaque

rupture [5,6]; aneurysm formation and rupture [7]; LV

ed by Elsevier B.V. All rights reserved.

Medical University of South Carolina.

scular



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2.2. Echocardiography and tissue collection

For echocardiographic acquisition, the mice were anes-

thetized with 12% isoflurane (depending on the individual

mouse), with heart rates maintained at z400 beats perminute [17]. Echocardiographic acquisition and analysis

were performed using the Sonos 5500 (Agilent Technolo-

gies) equipped with a high-band linear 15.6 MHz transducer

to obtain m-mode images. LV dimensions and wall thick-

ness were measured as previously described [18]. Echocar-

diography was not performed on one MA and one old

mouse because the mice died before echocardiograms were

acquired.

For tissue collection, the mice were anesthetized with 5%

isoflurane, the coronary vasculature was flushed with saline,

and the heart was excised. The LV and RV were separatedremodeling following pressure and/or volume overload [8

12]; and during all stages of congestive heart failure

progression [1315]. Whether age-related changes in MMPs

and TIMPs provide a molecular mechanism for changes in

ECM structure has not been demonstrated.

The myocardial fibroblast is a predominant cell regulat-

ing ECM turnover by altering (1) ECM synthesis and

deposition, (2) ECM degradation and turnover through the

production and release of MMPs, and/or (3) mechanical

tension on the collagen network [16]. Based on previous

observations, age-related changes in fibroblast function

could provide a cellular mechanism for changes in ECM

structure. Therefore, the goal of this study was to evaluate

aging mice for changes in LV structure; altered levels of

collagen, MMPs, and TIMPs; and differences in fibroblast

function.

2. Methods

2.1. Mice

CB6F1 mice are the F1 hybrid of C57BL/6 and BALB/c

mice and were selected because they are genetically

heterogeneous and display hybrid vigor. Young (3 months

old; n=11 female and 17 male), middle-aged (MA; 15

months old; n=6 female and 12 male), and old (23 months

old; n=6 female and 11 male) CB6F1 mice were purchased

from the Aged Rodent Colonies maintained by the National

Institute of Aging. The age groups (3, 15, and 23 months

old) correspond to young adult, middle-aged adult, and old

but not senescent adult, respectively. All animal procedures

were conducted in accordance with the Guide for the Care

and Use of Laboratory Animals (NIH Publication No. 85-

23, revised 1996). All studies were approved by the

Institutional Animal Care and Use Committee at the

M.L. Lindsey et al. / Cardiovaand weighed individually. The LV was divided longitudi-

nally into two halves so that each half contained septum andfree wall. One half was snap frozen for immunoblotting and

the other half was used immediately for myocardial

fibroblast isolation.

2.3. Protein isolation and collagen content

A highly reproducible sample fractionation protocol

based on differential protein solubility was used to extract

proteins from LV samples. To extract easily soluble proteins

(e.g., cytoplasmic proteins), the LV half was homogenized

in soluble lysis buffer (0.25 M sucrose and 1 mM EDTA)

and centrifuged at 13,000 g for 10 min [19]. To extractinsoluble proteins (e.g., membrane proteins), the insoluble

pellet was resuspended in chaotropic membrane extraction

reagent (7 M urea, 2 M thiourea, and the detergent

amidosulfobetaine-14; Sigma). Fractionation purity was

confirmed by immunoblotting for thioredoxin (N90% inthe soluble fraction) and SERCA2 (N95% in the insolublefraction). Protein concentrations were determined using the

Bradford assay. Because of the high urea content, insoluble

protein extracts were diluted 1:40 to ensure compatibility

with the Bradford assay. All samples (5 Ag) were run on gelsto confirm the accuracy of protein concentrations.

Collagen volume fractions were determined in protein

extracts using the microplate picrosirius red assay [20,21].

Soluble collagen was measured in the soluble fraction;

insoluble collagen was measured in the insoluble fraction.

Equal amounts of myocardial extracts (10 Ag total protein)were added to triplicate wells of a 96-well microtiter plate.

The samples were dried in the incubator and stained for 1 h

with 100 Al of 0.1% picrosirius red in saturated picric acid(w/v). The dye was solubilized in 100 Al of 0.1 M NaOH,and the plates were read by spectrophotometry (Multiskan

MCC/340) at an absorbance of 540 nm. Vitrogen 100

purified collagen (Collagen Biomaterials) was used as a

positive control and to generate a standard curve. The

amount of collagen per 10 Ag total protein was obtainedfrom the standard curve and multiplied by the total protein

to give total collagen levels. Total collagen levels were

divided by the initial LV wet weight to obtain microgram

collagen per milligram LV wet weight.

2.4. Immunoblotting

Immunoblotting was performed as described previously

[22] using antibodies for MMP-2, MMP-3, MMP-9, MMP-

12, MMP-13, MMP-14, TIMP-1, and TIMP-4 (Chemicon),

MMP-7, MMP-8, and TIMP-2 (Oncogene), TIMP-3 and

Mac-3 (Cedarlane), and the discoidin domain receptor

(DDR2; Genex Bioscience). With the exception of MMP-

2, the MMP antibodies recognized both pro and active

forms (Table 1). A sample of breast cancer homogenate was

run on each gel as a positive control and to verify the correct

molecular weight size band. Recombinant proteins were

Research 66 (2005) 410419 411also used as positive controls when further confirmation was

required. All samples for each set were run on one of two

cular



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26-well 420% Criterion TrisHCl gels (Bio-Rad). The two

gels and two blots were handled identically and simulta-

neously to minimize intergel variability. Ponceau staining of

the blots confirmed the successful transfer, and the positive

control sample, which was run on both gels, served to

confirm consistency between the two blots.

2.5. Myocardial fibroblast isolation

Myocardial fibroblast isolation from mice is technically

challenging, and yields vary tremendously depending on

which strain of mice is used (unpublished observation).

Several mouse myocardial fibroblast isolation protocols have

been published, from which we developed the following

protocol [2325]. The LV half was placed into cold

Dulbeccos modified eagle media (DMEM; Gibco-BRL/

Invitrogen; Grand Island, NY) with 10% fetal bovine serum

(FBS) and 1% antibioticantimycotic solution (penicillin,

streptomycin, amphotericin B; Cellgro) and used immedi-

ately for fibroblast isolation using the outgrowth technique.

Each LV was used to establish separate primary cultures. The

cells displayed characteristic fibroblast morphology, and cell

Table 1

MMP and TIMP antibodies used for immunoblotting

Antibody Company Catalog # MMP forms

recognized

MMP-2 Chemicon AB19015 pro

MMP-3 Chemicon AB810 pro, active

MMP-7 Oncogene PC492 pro, active

MMP-8 Oncogene PC493 pro, active





TIMP-1 Chemicon AB8116

TIMP-2 Oncogene IM11L

TIMP-3 Cedarlane CL2T3

TIMP-4 Chemicon AB816

M.L. Lindsey et al. / Cardiovas412cultures were confirmed to be pure fibroblasts by immuno-

cytochemistry using a composite of antibodies. The anti-

bodies used were against vimentin (Sigma; positive), desmin

(Sigma; negative), factor VIII (Sigma; negative), the

discoidin domain receptor 2 (DDR2; Genex Bioscience;

positive), a smooth muscle actin (Sigma; positive), andproly-4-hydroxylase (Chemicon; positive) [26,27]. Passage

14 cells were used for the functional assays.

2.6. Fibroblast functional assays and protein levels

Proliferation, migration, and adhesion indexes were

evaluated using protocols published for mice, rat, and/or

dog and modified as described below. For proliferation

assays, myocardial fibroblasts were plated at 1104 per wellin quadruplicate on 0.2% gelatin-coated 24-well plates and

incubated for 24 h. The wells were washed with phosphate-

buffered saline, and the cells were fixed for 20 min withzincformalin (Anatech). The plates were stained with 1%

methylene blue. After eluting stain with acid alcohol (0.05

M HCl in 50% ethanol), the plates were read at an

absorbance of 620 nm [2830].

To compare migration properties, fibroblasts were main-

tained in serum-free media (DMEM supplemented with a

1 solution of insulin, transferrin, and selenium, 0.1%bovine serum albumin, 10 Ag/mL ascorbate, and 1antibiotic/antimycotic solution) for 24 h and then passaged.

Serum starved cells were seeded (3104 per well induplicate wells) into the upper compartment of a transwell

tissue-culture-treated insert (Costar Brand, Corning, Acton,

MA) of 24-well tissue culture plates. Preliminary studies

were performed to determine optimal cell densities and

incubation times. FBS (10%) served as a chemoattractant

and was placed in the lower chamber of each well.

Following 4 h incubation, fibroblasts that had migrated to

the underside of the membrane were fixed and stained with

1% methylene blue as described above. The migration index

was calculated as a percentage of the average optical density

for the young fibroblast group.

For adhesion rates, myocardial fibroblasts were plated at

3104 per well in duplicate on ECM-coated 24-well plates(BD BioCoat Cellware; Becton Dickinson, Franklin Lakes,

NJ). Substrates analyzed for fibroblast adhesion character-

istics were used at concentrations recommended [31] and

included plastic, gelatin (0.2 mg/mL), laminin (2 Ag/mL),fibronectin (2.5 Ag/mL), collagen I (5 Ag/mL), and collagenIV (5 Ag/mL). Plates were incubated for 10 min and thenwashed with PBS to remove nonadherent cells. Initial

experiments using 30- and 60-min incubations demonstrated

that saturation was already reached by 10 min. Fibroblasts

were fixed and stained with 1% methylene blue as described

above.

To analyze protein levels in fibroblast cell pellets,

confluent fibroblasts were incubated in serum-free media

for 48 h, and the cell pellets were homogenized in

chaotropic membrane extraction reagent to obtain total

protein. Immunoblotting was performed using antibodies for

a smooth muscle actin (Sigma) and MMP-9.

2.7. Data analysis

Data are presented as meanFstandard error of the mean(S.E.M.). Statistical analyses were performed using Inter-

cooled Stata 8.0 for Windows (Stata, College Station, TX).

Echocardiographic, proliferation, and adhesion data were

analyzed by repeated measures analysis of variance, with

comparisons between individual groups made using the

Bonferroni-corrected t-test. A two-tailed value of pb0.05was considered statistically significant. The migration data

were normalized as a percentage of the average value for the

young or MA group and analyzed using a one-sample t-test

to compare MA and old values to young or MA levels set at

Research 66 (2005) 410419100%. Immunoblotting data were analyzed as total MMP

data (which included all forms of the enzyme in the pooled

soluble and insoluble fractions) and for each MMP form

individually for each of the two fractions. A one-sample t-

test was used to compare MA and old values to the average

progressed in the same direction from the young to MA to

old groups; (2) changes that occurred from the young to MA

group and then remained stable in the old group; (3)

changes that occurred only in the old group; and (4) changes

that occurred in one direction in MA and then reversed

direction in the old group [32].

3. Results

3.1. Effect of aging on LV morphometrics

LV mass, end-diastolic dimensions, and posterior wall

thickness increased in the MA and old groups, while

fractional shortening was preserved (Table 2). The LV mass

calculated from echocardiographic measurements correlated

significantly with LV mass taken at necropsy (r=0.85;

pb0.001; n=61).

3.2. Effect of aging on total protein and collagen levels

Protein from young (n=24), MA (n=15), and old LV

samples (n=14) were differentially extracted into soluble and

insoluble fractions. The percentage of total protein obtained for

the combined fractions per initial wet weight was 14.7F0.5%,

Table 2

LV geometry, necropsy data, and total protein levels in aging mice

Young Middle-aged Old

Sample size (n) 28 17 16

Heart rate (bpm) 446F9 470F10 475F14End diastolic

dimensions (mm)

4.32F0.09 4.92F0.09* 4.87F0.12*

Fractional shortening (%) 33F1 29F1 30F1Posterior wall thickness

in diastole (mm)

0.75F0.01 0.82F0.02* 0.84F0.02*

Left ventricle mass (mg) 96F2 152F7* 155F8*Body weight (g) 26.0F0.4 39.5F1.3* 41.7F1.2*Left ventricle mass to

tibia length (mg/mm)

5.6F0.2 8.8F0.5* 8.3F0.5*

Soluble protein

(Ag/mg LV wet wt)28.2F1.4 35.7F2.2* 35.1F1.8*

Insoluble protein

(Ag/mg LV wet wt)109.4F6.2 103.0F6.3 98.9F5.3

Soluble collagen

(Ag/mg LV wet wt)4.3F0.4 4.9F0.4 4.8F0.4

Insoluble collagen

(Ag/mg LV wet wt)17.2F1.4 13.7F1.3 12.6F0.9*

* pb0.05 young vs. middle-aged or old mice.

M.L. Lindsey et al. / Cardiovascular Research 66 (2005) 410419 413

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nloaded from young value. Linear regressions were performed to deter-

mine relationships between groups, using age as the

independent variable. Changes that occurred with aging

were divided into four types: (1) changes that continuallyFig. 1. Aging differentially regulates MMP-8 and TIMP-4 expression. Soluble MM

the old age group (B)+breast cancer homogenate positive control sample. Soluble T

in the old age group (D). All immunoblots are representative. All samples were ru

shown for each age group.

uest on April 4, 2014P-8 levels were unchanged (A), while insoluble MMP-8 levels increased in

IMP-4 levels were unchanged (C), while insoluble TIMP-4 levels decreased14.2F0.7%, and 14.0F0.7% for young, MA, and old groups(p=0.68), indicating that equal yields were recovered.

Concordant with increases in LV mass, total protein and

collagen levels increased in the MA and old groups. Proteinn on one of two gels. For each set, two lanes from each of the two gels are

levels were increased in the soluble and insoluble fractions,

while collagen levels increased in the soluble fraction only.

When normalized to the initial LV wet weight, protein levels

remained elevated in the soluble fraction, indicating that the

increase in soluble protein was greater in proportion to the

increase in LV mass (Table 2). The increase in soluble

protein contributed to an increased soluble to insoluble

protein ratio. The ratios were 0.27F0.02, 0.36F0.03, and0.36F0.02 for young, MA, and old groups, respectively( pb0.05 for young vs. MA and young vs. old). In contrastcollagen levels decreased in the insoluble fraction, indicat-

ing that insoluble collagen levels were not maintained for

the given increase in LV mass (Table 2). The decrease in

insoluble collagen contributed to an increased soluble to

insoluble collagen ratio. The ratios were 0.28F0.030.38F0.03, and 0.41F0.04 for young, MA, and old groupsrespectively ( pb0.05 for young vs. old).

3.3. Effect of aging on MMP and TIMP profiles

MMP and TIMP levels were determined by immuno-

blotting. Representative immunoblots are shown in Fig. 1

Total MMP and TIMP levels were analyzed as a percent

change from young and MA levels (Table 3). MMP-2

MMP-7, MMP-8, and TIMP-2 levels were not changed

between the young and MA or old groups or between MA

and old groups. Compared to young levels, MMP-3, MMP-

9, MMP-12, MMP-14, and TIMP-1 were decreased and

TIMP-3 increased in the MA group. MMP-13, MMP-14

and TIMP-4 were decreased in the old group. Compared to

MA levels, MMP-3, MMP-9, and MMP-14 were increased

and TIMP-4 decreased in the old group.

Table 3

Total myocardial MMP and TIMP levels in middle-aged and old mice, as a

percentage of young or middle-aged levels

Middle-aged

(% young)

Old

(% young)

Old

(% middle-aged)

Sample size, n 712 1011 1011

MMP-2 169F40 154F34 91F20MMP-3 70F10* 113F12 162F17*MMP-7 82F18 93F10 114F12MMP-8 85F9 108F11 127F12MMP-9 68F10* 117F11 173F16*MMP-12 72F12* 84F10 117F14MMP-13 80F10 72F4* 89F5MMP-14 50F8* 77F7* 154F15*TIMP-1 24F3* 60F25 252F103TIMP-2 79F19 93F13 119F16TIMP-3 128F12* 97F17 76F13TIMP-4 102F11 81F7* 80F7*

The densitometric units of all immunoreactive bands in both fractions were

combined to obtain a total MMP or TIMP value. Data are presented as a

percentage (meanFS.E.M.%) of the average density for the young ormiddle-aged groups, which were set at 100%.

* pb0.05 young vs. middle-aged or old or middle-aged vs. old.

M.L. Lindsey et al. / Cardiovascular Research 66 (2005) 410419414

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Myocardial MMP and TIMP levels in young, middle-aged, and old LV

Sample size MW (kD) SolubleYoung Middle-aged

1821 1012

MMP-2 72 989F135 1644F402MMP-3 57 1188F139 514F73*

45 648F102 679F226MMP-7 28 1409+138 1153F260

19 ND ND

MMP-8 64 ND ND

58 603F51 486F81MMP-9 92 381F44 301F36*

88 ND ND

MMP-12 54 298F45 171F55*194F34 227F53

MMP-13 60 141F24 118F1848 173F24 120F22*

MMP-14 65 440F74 138F27*54 189F39 95F13*45 184F27 120F25*40 217F31 134F23*

TIMP-1 29 1073F203 258F35*TIMP-2 28 731+167 575F140TIMP-3 24 1786F350 2271F430TIMP-4 28 896F47 856F83

24 ND ND

Data are presented as densitometric (arbitrary) units (meanFSEM). NDnot dete* pb0.05 young vs. middle-aged or old.y pb0.05 middle-aged vs. old.

uest on April 4, 2014Insoluble

Old Young Middle-aged Old

1012 1422 912 1012

1368F373 70F11 141F40 265F92479F38* 166F22 690F135* 583F99*314F61*,y 143F20 267F58 693F167*,y

1315F139 ND ND NDND ND ND ND

ND ND ND ND

557F50 143F22 140F47 264F51*,y

461F79 833F70 504F124* 992F78y

ND ND ND ND

43F5*,y 230F34 170F45 262F5896F10*y 589F80 363F106 627F70y

90F6*,y ND ND ND125F13* ND ND ND314F73y 109F25 132F59 285F68*,y

100F14* ND ND ND150F14* ND ND ND68F4*,y ND ND ND649F266 ND ND ND683F93 ND ND ND2753F443 1331F352 1591F583 113F32*,y

725F84 202F35 269F57 157F29y

ND 23F4 15F3 20F2y

cted in that fraction.,

,

,

.

,

,

3.4. Effect of aging on cell numbers in vivo

Fig. 2. Summary of matrix metalloproteinase (MMP) and tissue inhibitor of matrix metalloproteinase (TIMP) changes with aging. Four types of changes

occurred: (1) progressive change from young to middle-aged to old groups; (2) change from young to middle-aged and old groups but not between middle-aged

etween young and middle-aged groups then a reverse in direction between middle-

t detected.




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MMP and TIMP levels from each fraction were compared to

young levels (Table 4). MMP-7, MMP-13, TIMP-1, and

TIMP-2 were detected in the soluble fraction only. MMP-2,

MMP-7, TIMP-2, and TIMP-4 levels were not changed

between the young and MA or old groups. In the soluble

fraction, MMP-3, MMP-9, MMP-12, MMP-13, MMP-14,

and TIMP-1 levels decreased in the MA group. Soluble

MMP-3, MMP-12, MMP-13, and MMP-14 levels remained

decreased in the old group. In the insoluble fraction, MMP-3

levels increased and MMP-9 levels decreased in the MA

group. MMP-3, MMP-8, and MMP-14 levels were

increased, while TIMP-3 levels were decreased in the

insoluble fraction of the old group.

MMP and TIMP levels in the old group were also

compared to MA levels (Table 4). MMP-2, MMP-7, TIMP-

1, and TIMP-2 were not different between the MA and old

groups. In the soluble fraction of the old group, MMP-3,

MMP-12, and MMP-13 levels decreased, and MMP-14

levels increased. In the insoluble fraction of the old group,

MMP-3, MMP-8, MMP-9, MMP-12, and MMP-14 levels

and old groups; (3) change only in old group; or (4) change in one direction b

aged and old groups. z increased; A decreased; NCno change; NDnoincreased, and the levels of TIMP-3 and TIMP-4 decreased.

Changes between the groups are summarized and charac-

terized in Fig. 2.

Fig. 3. Fibroblasts isolated from old (n=13) mice displayed lower

proliferative capacity compared to fibroblasts isolated from young (n=19)

and middle-aged (n=14) mice (*pb0.05 for young vs. old and ypb0.05 formiddle-aged vs. old).Levels of Mac-3, a macrophage marker, were not

different between the groups, suggesting that increased

MMP levels were not due to increased macrophage

numbers. Levels of DDR2, a fibroblast marker, decreased

in the MA and old groups to 64F13% and 74F8% of younglevels, respectively ( pb0.05 for both).

3.5. Effect of aging on fibroblast function and MMP-9

expression

Fibroblasts are the principal cell regulators of ECM

turnover. Given the changes in collagen, MMP, and TIMP

levels, we characterized the age-related fibroblast pheno-

type. We successfully established primary cultures from 58

of the 63 LV samples used for fibroblast isolation. Of the

five samples that did not establish, all were from young LVFig. 4. MMP-9 levels were decreased in fibroblasts isolated from the MA

(n=11) and old (n=10) groups compared with young (n=14). (Top)

Representative immunoblots for MMP-9 and a smooth muscle actin.(Bottom) The ratio of MMP-9 to a smooth muscle actin relative to younglevels (*pb0.05 for young vs. middle-aged and old).

14

samples (four female and one male). The cell counts at first

passage were 39F5104 cells for young (n=14),29F6104 cells for MA (n=11), and 33F8104 cells forold mice (n=10; p=0.48), indicating the equal and adequate

yields were obtained for all three age groups. For these

mice, the time from isolation to first passage was 19F1 daysfor the young, 20F3 days for MA, and 21F1 days for theold mice ( p=0.72), indicating that the rate of establishing

culture was similar between the three groups. All fibroblasts

were positive for a smooth muscle actin, indicating thatsome differentiation into a myofibroblast phenotype

occurred in culture.

Fibroblasts from old mice displayed lower proliferative

capacity than fibroblasts from young and MA mice (Fig. 3).

There was no correlation between proliferation and passage

M.L. Lindsey et al. / Cardiovascular416



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number(r=0.02; p=0.88), indicating that proliferation rates

did not change with passaging. The migration index,

normalized to the average of young values (n=13), was

61F6% for MA (n=12; pb0.001) and 81F10% for oldmyocardial fibroblasts (n=9; p=0.11). Adhesion to plastic

(n=812), gelatin (n=1319), fibronectin (n=1319), lam-

inin (n=814), collagen I (n=1319), and collagen IV

(n=1319) were similar for all age groups ( p=n.s.). Changes

in adhesive characteristics therefore did not explain the

decrease in migration in the MA group. Immunoblotting of

cell pellets demonstrated equal levels of a smooth muscleactin between the three groups ( p=0.48 and 0.41 for young

vs. MA and old, respectively). MMP-9 levels, normalized to

a smooth muscle actin levels, were lower in fibroblastsisolated from the MA and old groups (Figs. 3 and 4).

3.6. Regression analyses

To determine which parameters correlated with age,

regression analyses were performed using age as the

independent variable (Table 5). Insoluble MMP-3, insoluble

MMP-14, and total collagen correlated positively with age.

Soluble MMP-12, soluble MMP-14, insoluble collagen, and

fibroblast proliferation correlated negatively with age.

Table 5

Regression analyses using age as the independent variable

Dependent variable r p

LV mass 0.65 b0.001MMP-3 45 kD insoluble 0.57 0.001

Soluble protein 0.43 0.001

Soluble to insoluble protein ratio 0.42 0.002

Soluble to insoluble collagen ratio 0.41 0.003

Posterior wall thickness (diastole) 0.41 0.007

End-diastolic dimension 0.39 0.010

MMP-14 65 kD insoluble 0.39 0.022

Total collagen 0.28 0.041

MMP-12 54 kD soluble 0.53 b0.001MMP-14 40 kD soluble 0.51 b0.001Fibroblast Proliferation 0.48 b0.001Insoluble collagen 0.35 0.011

TIMP-1 soluble 0.31 0.0464. Discussion

4.1. Major findings

LV remodeling resulting in LV hypertrophy is a common

occurrence in the aging myocardium [2]. The present study

hypothesized that age-related LV hypertrophy was a result

of changes in ECM remodeling. Accordingly, the goal of

this study was to examine the effects of aging on LV

structure, collagen, MMP, and TIMP levels, and myocardial

fibroblast function in mice. The unique findings of this

study were as follows. First, the age-related increase in LV

mass was due to increases in both LV dimensions and wall

thickness. Second, soluble protein levels increased and

insoluble collagen levels decreased with age. Soluble MMPs

decreased, insoluble MMPs increased, and TIMPs decreased

in both fractions. Third, fibroblasts have decreased func-

tional capacity and produce less MMP-9 with age.

This study provides mechanistic insight into ECM

remodeling events that occur as a function of aging. The

shift in MMP localization from soluble to insoluble fractions

may indicate increased recruitment of the MMP to an

insoluble substrate. Increased MMP activity correlated with

decreased insoluble collagen levels, suggesting that ECM

degradation was stimulated in aging mice. The primary

difference between MA and old mice was a further shift in

MMPs from soluble to insoluble fractions, suggesting that

increased MMP degradative capacity serves an important

function in regulating collagen levels in old mice. The

continued increase in MMP levels in old mice suggests that

increased MMP activity is required to maintain increased

LV size. This is the first study to correlate age-dependent

changes in LV structure with changes in specific MMPs and

TIMPs.

4.2. Aging effects on LV size

Age-related LV hypertrophy occurred in CB6F1 mice.

Body weight and LV mass increased significantly with

aging, similar to previous studies in other mice strains, rats,

and humans [23,33]. None of these parameters were

different between the MA and old groups, indicating that

growth occurred primarily during the transition from young

to MA and was maintained thereafter (a type 2 change).

Increases in both LV dimension and wall thickness

contributed to the increase in LV mass.

4.3. Aging effects on collagen, MMP, and TIMP levels

With age, decreased soluble MMP and TIMP levels

accompanied the increase in soluble protein, and increased

insoluble MMP and decreased insoluble TIMP levels

accompanied the decrease in insoluble collagen. Together,

the results suggest two interpretations. First, the decrease in

Research 66 (2005) 410419insoluble collagen levels is likely due to increased MMP

and decreased TIMP levels in the insoluble fraction.

scular



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Changes in collagen solubility reflect changes in the

biochemical characteristics of the collagen. Coupled with

an overall increase in total protein, collagen synthesis may

be increased but matched with a greater increase in collagen

degradation. Alternatively, the increased soluble to insoluble

ratio may indicate a change in collagen type or degree of

cross-linking [34]. Second, changes in the soluble fraction

(decreased MMP-12 and MMP-14 and increased protein)

correlated with the increase in wall thickness and provided a

mechanism for increased LV size with aging.

A major finding of this study was that changes to specific

MMPs occur during aging rather than a global change in all

MMP levels. In the soluble fraction, the predominant types

of changes were type 1 (progressive changes with aging)

and type 4 changes (directional change in MA that reversed

in the old group), indicating that changes in soluble MMP

levels occurred throughout the aging process. In contrast,

type 3 changes (change only in the old group) characterized

the insoluble fraction. A primary difference between the

MA and old groups, therefore, was the increased levels of

MMPs and decreased levels of TIMPs in the insoluble

fraction. The changes in MMP levels between MA and old

groups indicate a change in myocardial ECM composition,

while LV mass is still maintained. The use of three time

points allowed nonlinear changes to be assessed and the rate

of change to be determined [32]. Interestingly, changes in

total MMP levels from the pooled fractions mirrored

changes in the soluble but not insoluble fraction. The

increases in total MMP-3, MMP-9, and MMP-14 suggest

increased synthesis of these particular MMP types. The

differential MMP-3 expression between soluble and insolu-

ble fractions highlights the need to use fractionated samples

to obtain information on age-related distribution patterns as

shifts between fractions cannot be discerned by monitoring

total myocardial levels. The decrease in MMP-3, MMP-9,

MMP-12, and MMP-14 in the soluble fraction was

accompanied by increases of the same MMPs in the

insoluble fraction, suggesting that increased binding to

insoluble substrates occurred with aging.

Proteins likely to be found in the soluble fraction include

cytoplasmic and easily soluble ECM proteins. Proteins likely

to be found in the insoluble fraction include membrane and

insoluble ECM proteins. The fact that MMP-7 and TIMP-1,

known soluble proteins, were only seen in the soluble fraction

confirms this idea. MMP-14, a membrane-bound MMP type,

is known to recycle intracellularly and may be found in both

fractions [35]. The age-related change in MMP-14 local-

ization suggests that degradation and signaling pathways

through this MMP were altered.

MMP-3 is a well-established activator of other MMPs

[36], and the increase of MMP-3 in the insoluble fraction

supports the idea of increased collagen degradation with

aging. The localization of MMP-12 around the vasculature

supports a role for MMP-12 in regulating changes in arterial

M.L. Lindsey et al. / Cardiovacompliance and stiffening that occur with aging [37]. MMP-

14 is a membrane-type MMP with putative roles inpericellular proteolysis and signaling [35,38]. The decreases

in soluble MMP-12 and MMP-14 would support the

accumulation of soluble ECM fragments. Matricryptins,

biologically active enzymatic ECM degradation products,

have been shown to stimulate signaling pathways and affect

cell growth [39]. The attenuation of MMPs in the soluble

fraction potentially promotes the accumulation of ECM

fragments and affects downstream signaling cascades. The

fact that fibroblast proliferation and migration were altered

with aging suggests a role for these ECM fragments in

regulating fibroblast function. The results from this study

provide the foundation for future studies that evaluate the

effects of ECM fragments on LV structure and ECM

remodeling, particularly during aging.

4.4. Aging effects on fibroblast function

Myocardial fibroblasts exhibited decreased proliferation

and migration in the MA group. Proliferation decreased

further in the old age group. In the old mice, the decrease in

fibroblast proliferation rates corresponded with the decrease

in myocardial DDR2 levels, which together suggest a

decreased number of fibroblasts in the aging myocardium.

In the MA mice, the fact that DDR2 levels were decreased

in vivo but fibroblast proliferation was unaltered in vitro

suggests that apoptotic rates may be increased. Apoptosis

was not evaluated in this study. Concordant with our results,

migration and proliferation are both attenuated in dermal

fibroblasts isolated from mice of increasing age [40]. The

decrease in fibroblast-derived MMP-9 was consistent with

the drop in myocardial MMP-9 levels in the MA group. The

rebound in myocardial MMP-9 in the old group is likely due

to other cell contributors. We did not observe a change in

Mac-3 levels, suggesting that macrophage levels were

unaltered with age. The amount of MMP-9 produced per

macrophage could be higher, however. Alternatively,

smooth muscle, endothelial, and mast cells also potentially

contribute MMP-9 [15]. The decreased proliferative and

migratory capacities in aging fibroblasts suggest that global

fibroblast function may be impaired, which would provide

an age-related cellular mechanism for altered ECM levels.

Studies examining the response of aging myocardial

fibroblasts to growth factor and cytokine stimulation would

support this concept.

4.5. Clinical relevance

MMPs have demonstrated roles in several components of

LV remodeling, including the inflammatory, angiogenic, and

hypertrophic responses. MMP inhibition has been used to

successfully attenuate LV remodeling following myocardial

infarction [41,42] and during the progression to heart failure

[43]. The data presented here establish age-related baseline

differences in LV echocardiographic parameters, ECM and

Research 66 (2005) 410419 417MMP levels, and fibroblast functions that may influence the

effects of MMP inhibition during disease pathologies such

dysfunction. J Clin Invest 2000;106:85766.

[15] Spinale FG. Matrix metalloproteinases: regulation and dysregulation

in the failing heart. Circ Res 2002;90:52030.

M.L. Lindsey et al. / Cardiovascular Research 66 (2005) 410419418



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form and function. Cardiovasc Pathol 1998;7:12733.

[17] Roth DM, Swaney JS, Dalton ND, Gilpin EA, Ross Jr J. Impact of

anesthesia on cardiac function during echocardiography in mice. Am J[12] Stroud JD, Baicu CF, Barnes MA, Spinale FG, Zile MR. Viscoelastic

properties of pressure overload hypertrophied myocardium: effect of

serine protease treatment. Am J Physiol Heart Circ Physiol 2002;282:

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[13] Li YY, Feldman AM. Matrix metalloproteinases in the progression of

heart failure: potential therapeutic implications. Drugs 2001;61:123952.

[14] Lindsey M, Lee RT. MMP inhibition as a potential therapeutic

strategy for CHF. Drug News Perspect 2000;13:3504.as pressure overload and myocardial infarction. These

results reveal unique targets for future interventions to

modify age-dependent matrix remodeling.

Acknowledgements

The authors acknowledge the following sources of

support: HL-10337 (MLL), HL-75360 (MLL), HL-45024

(FGS), HL-97012 (FGS), P01-48788 (FGS), and a VA

Career Development Award (FGS).

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Age-dependent changes in myocardial matrix metalloproteinase/tissue inhibitor of metalloproteinase profiles and fibroblast functionIntroductionMethodsMiceEchocardiography and tissue collectionProtein isolation and collagen contentImmunoblottingMyocardial fibroblast isolationFibroblast functional assays and protein levelsData analysis

ResultsEffect of aging on LV morphometricsEffect of aging on total protein and collagen levelsEffect of aging on MMP and TIMP profilesEffect of aging on cell numbers in vivoEffect of aging on fibroblast function and MMP-9 expressionRegression analyses

DiscussionMajor findingsAging effects on LV sizeAging effects on collagen, MMP, and TIMP levelsAging effects on fibroblast functionClinical relevance

AcknowledgementsReferences

age-dependent changes in myocardial matrix metalloproteinasetissue inhibitor of metalloproteinase...

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