Adenovirus-mediated gene transfer of TIMP-4 reduces neointimal hyperplasia in balloon-injured rat carotid artery

आऋऑऎऊࣽईࣜऋंࣜ उँऀअࣿࣽईࣜ ࣿऋईईँःँएࣜऋंࣜऌईࣽ Journal of Medical Colleges of PLA 26 (2011) 53–62

www.elsevier.com/locate/jmcpla

Adenovirus-mediated gene transfer of TIMP-4 reduces neointimal hyperplasia in balloon-injured rat carotid arteryƿ Zhong Li*, He Guoxiang, Song Zhiyuan, Ran Boli Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China Received 20 March 2011; Accepted 15 April 2011

Abstract Objective: To determine the effects of a recombinant replication-deficient adenovirus encoding human tissue inhibitor of metalloproteinase-4 (Ad.TIMP-4) on vascular smooth muscle cell (VSMC) function in vitro and neointimal development in the injured rat carotid artery. Methods: Western blotting, gelatin zymography and reverse zymography were used to characterize the expression and functional activity of the TIMP-4 secreted by Ad.TIMP-4-infected VSMCs. The migration and proliferation of VSMCs in vitro were separately detected by using Millicell-PCF invasion chambers and [ 3 H]-thymidine incorporation assay. Immunohistochemistry and morphometric analysis were used to determine the local expression of TIMP-4 and its effect on neointima development in a rat carotid artery balloon injury model. Results: VSMCs infected with Ad.TIMP-4 expressed functionally active human TIMP-4 which increased with the duration of infection. TIMP-4 expression inhibited VSMC migration, but not significantly affect VSMC proliferation. In a balloon-injured rat carotid artery model, a significant 62% reduction in neointimal area was found in Ad.TIMP-4–infected vessels at 14 days after injury. Ad.TIMP-4 infection had no effect on medial area. Conclusion: Our results indicated TIMP-4 over expression can significantly inhibit the migration of cultured VSMCs and prevent neointimal formation after vascular injury. Our findings provide additional evidence that TIMP-4 could play an important role in vascular pathophysiology, and may be an important therapeutic target for future drug development. Keywords: Tissue inhibitor of metalloproteinase-4; Vascular smooth muscle cells; Restenosis; Vascular injury;

1. Background ƿ

Supported by the National Natural Science Foundation of China (30630056)

* Corresponding author.

After arterial injury, vascular smooth muscle cells (VSMCs) proliferate and migrate, to form a

Tel.:  86-23-68765673

neointima that accumulates extracellular matrix in

E-mail address: [email protected] (Zhong L.)

the late stages of the restenotic process [1, 2]. It is

54

Zhong Li et al. / Journal of Medical Colleges of PLA 26 (2011) 53–62

believed that the extracellular matrix homeostasis is the basis of maintaining the integrity of the vessel

2. Materials and methods

wall. The process of migration of VSMCs requires degradation

of

basement

membrane

and

2.1. Generation of adenovirus vectors

extracellular matrix surrounding the cells [3, 4]. Matrix metalloproteinases (MMPs) were identified

Human TIMP-4 cDNA contained within the

to be the most important proteinases in maintaining

plasmid pCI-neo is a kind gift of Dr Yuenian E.Shi

the extracellular matrix homeostasis [5, 6]. A

(Long Island Jewish Medical Center, USA). The

number of evidence supports the notion that MMP

TIMP-4 cDNA was excised from pCI-neo TIMP-4

expression and activation play a critical role in the

and cloned into the PUC19 vector. Correct insertion

remodeling of the vessel wall that occurs after

and orientation were confirmed by restriction

experimental injury. Both MMP-2 and MMP-9

enzyme analysis and sequence analysis. TIMP-4

expression and activation increase after balloon

cDNA was then cloned into plasmid pDC315

catheter injury to vessels in several animal models

(Microbix Biosystems). The recombinant repli-

[7–9].

cation-deficient adenoviruses expressing TIMP-4

All of the active MMPs are inhibited by a class

were generated by liposome cotransfected of pDC315

of low-molecular-weight proteins known as tissue

TIMP-4

inhibitors of metalloproteinases (TIMPs). Four

pBHGloxƸE1, 3Cre (Microbix Biosystems) in 293

forms of TIMP, TIMP-1, -2, -3 and -4, have so far

cells. The recombinant adenovirus clones were

been described. It has been identified in different

screened for the TIMP-4 cDNA by PCR. The

animal models that over expression of TIMP-1, -2,

screened plaques were amplified to transfect 293

or -3 can respectively prevent VSMC migration

cells. The high-titer stocks of viral particles were

and/or proliferation and inhibit the neointimal

produced in 293 cells and concentrated on CsCl2

development after vascular injury [10–12]. TIMP-4

gradients. An adenovirus vector expressing the

is a new member of TIMPs, which shows a specific

bacterial

expression in cardiovascular tissues [13, 14]. A

amplified and used as a negative control.

previous

study

has

demonstrated

that

with

adenovirus

ȕ-galactosidase

gene

genomic

plasmid

(Ad.LacZ)

was

over

expression TIPM-4 could inhibit the migration of

2.2. VSMC culture and adenovirus infection

VSMCs through a matrix-coated membrane in vitro [ 15 ]. However, it is unclear whether TIMP-4

The primary VSMCs were isolated from the

can influence the biological behaviors of VSMC in

thoracic aortas of male Wistar rats, and used at

vivo. Therefore, we constructed a recombinant

passage 4 to 6. The immunocytochemistry using an

replication-deficient adenovirus expressing TIMP-4

anti-Į-smooth

(Ad.TIMP-4) to investigate its effects on VSMC

confirmed staining in 98% of cells. Eighty percent

function in cell culture and neointimal development

confluent

in the injured rat carotid artery.

serum-free RPMI 1640 in 24 h before infection. The

muscle

monolayers

actin were

antibody

(Sigma)

transferred

to

cells were incubated with Ad.TIMP-4 or Ad.LacZ

55

Zhong Li et al. / Journal of Medical Colleges of PLA 26 (2011) 53–62

for 1 h. The infection media were then replaced

gelatin zymography, recombinant human MMP-2

with serum-free RPMI 1640. The VSMCs were

(rhMMP-2, Sigma, 1, 0.5 and 0.25 ng) were run on

5

plated into six-well plates at 1×10 cells/well and

a 10% gel containing 1 mg/ml of gelatin (Sigma).

left in complete media for 24 h. Immediately before

Gels were washed in 2.5% Triton X-100 for 3 h and

infection, triplicate wells were trypsinized and

incubated for 15 h at 37 ć in a solution containing

counted for an accurate cell count. The cells in the

conditional media from Ad.TIMP-4- or Ad.LacZ-

remaining wells were infected at the required

infected cells before staining with Coomassie blue.

multiplicity of infection (MOI) in 2 ml fresh

For reverse zymography, the medium samples of

complete media and left for 18 h. The media were

cultured VSMCs infected with Ad.TIMP-4 or

removed, and the cells were washed and left in 2 ml

Ad.LacZ at MOI 5, MOI 50 and MOI 100 were run

fresh complete media until the required time point.

on a 10% gel containing 1 mg/ml of gelatin. Gels were washed and incubated for 15 h at 37 ć in

2.3. Western blot analysis

buffers plus rhMMP-2 before Coomassie blue staining.

VSMCs were analyzed by Western blotting at 1, 4, 6, 8, 10, 14, and 18 d after Ad.TIMP-4

2.5. Invasion and proliferation assay in vitro

infection. The infected cells were kept in culture until the required time point. The media were

Millicell-PCF invasion chambers (Millipore)

replaced with fresh serum-free RPMI 24 h before

coated with a barrier of reconstituted basement

collection.

TIMP-4

membrane proteins (Matritgel) were used for

(rhTIMP-4, Sigma) used as positive control. Protein

invasion studies. VSMCs were infected at an MOI

was loaded and run on a 10% SDS-polyacrylamide

of 100 as described above. At 1, 5 and 10 d after

gel

infection

and

Recombination

transferred

to

human

nitrocellulose.

Goat

the

VSMCs

were

trypsinized

and

anti-human TIMP-4 polyclonal antibody (Santa

suspended in serum-free RPMI 1640 at a density of

Cruz Biotechnology) was added at 1:1 000 for 2 h

3×10 5/ml. A volume of 250 ȝl cell suspension was

and probed with 1:1 000 horseradish peroxidase-

then placed in the upper chambers, and 50 ȝl RPMI

conjugated rabbit anti-goat monoclonal antibody

1640 with or without 10% platelet-derived growth

(Santa Cruz Biotechnology). A chemiluminescent

factor (PDGF, R&D Systems) was placed in the

(Pierce Biotechnology) detection method was used

lower chambers. The chambers were incubated at 37

to visualize the signal.

ć, 5% CO 2 for 4 h. The cells invading to the lower side of the filters were fixed and stained with

2.4. Gelatin zymography and reverse gelatin

hematoxylin/eosin. The average number of cells

zymography

from 4 randomly chosen high-power (×400) fields on the lower side of the filters was counted per

TIMP-4 activities were assayed by gelatin zymography

and

reverse

gelatin

chamber. Each sample was performed in triplicate.

zymography,

Data are presented as number of invaded cells/field.

respectively, as described by Webb et al [15]. For

DNA synthesis was assayed by incorporation

56

of

Zhong Li et al. / Journal of Medical Colleges of PLA 26 (2011) 53–62

[ 3H]-thymidine

VSMCs

(5×10

4

into

proliferating

cells/well)

were

VSMCs.

plated

into

individual wells of a 6-well tissue culture plate and

2.7. Immunohistochemistry and morphometric analysis of rat carotid arteries

then infected with either Ad.TIMP-4 or Ad.LacZ at 100 MOI. DNA synthesis per well was estimated at

Sections harvested at 2 and 14 d after gene

1, 5 and 10 d after infection. After pulsing with

transfer were incubated for 2 h with 1:200 goat

3

[ H]-thymidine (1 mCi/ml) for 18 h, the cells were

anti-human TIMP-4 polyclonal antibodies (Santa

harvested onto glass fiber filter paper, and the

Cruz

3

and

were

followed

by

was

30-minute incubation with biotinylated anti-goat

Three

IgG (Santa Cruz Biotechnology). Sections were

experiments were performed and each sample was

then stained with extravidin-peroxidase (Sigma) and

assayed in quadruplicate.

diaminobenzidine (Sigma).

amount

of

quantified

incorporated by

liquid

[ H]-thymidine

Biotechnology),

scintillation.

Rat vessels were fixed and immersed in 4%

2.6. Rat carotid injury and adenovirus infection

paraformaldehyde for 1 h at 2 and 14 d after balloon

in vivo

injury (6 rats in each groups). Thereafter, paraffin sections

The rat carotid artery balloon injury model was

of

Hematoxylin

vessel and

segments

Eosin

(HE).

were

stained

Morphometric

based on a model described by Webb et al [16].

analyses were performed on 3 cross sections for

Briefly, adult male Wistar rats weighing 400–600 g

each vessel, and the cross-sectional area of the

were used for animal models. The left common

intima and media was measured by computer image

carotid artery was injured with a 2F Fogarty balloon

analysis system (Image pro plus4.5), and calculated

catheter. Immediately after injury, 50 ȝl of a

the ratio of intima and media (I/M).

solution

containing

2.5×10

12

pfu

of

either

Ad.TIMP-4 or Ad.LacZ was introduced into the

3. Results

injured carotid artery with a 24-gauge intravenous catheter. Total dwell time for the solution in the

3.1. Adenovirus-mediated human TIMP-4 transfer

injured carotid artery was 30 min. Thereafter, the

to VSMCs

external carotid artery was ligated, and blood flow through the internal carotid artery was reestablished.

The time course of Ad-mediated human

At varying time intervals after gene transfer, rats

TIMP-4 expression in rat VSMCs was demonstrated

were killed, and the left carotid arteries were

by Western blot analysis of conditioned media.

removed for analysis. In a subset of animals, fresh

Results showed that TIMP-4 protein expression

tissues were frozen in liquid nitrogen, and protein

increased with the duration of infection. TIMP-4

was extracted for Western blot analysis of human

protein reached the highest level at 10 days, was

TIMP-4 as described above to conform the presence

constant up to 2 weeks after infection and markedly

of human TIMP-4 protein after in vivo gene

reduced at 18 days. The protein was not detected in

transfer.

Ad.LacZ-infected cells (negative control) (Fig. 1).

57

Zhong Li et al. / Journal of Medical Colleges of PLA 26 (2011) 53–62

1

Band

140

2

3

4

5

6

120

Ad.TIMP-4

100 %TIMP-4

80 60

Ad.LacZ

40

A

A

20 1 1

2

2

3 3

4 4

5 5

6 6

7 7

8 8

9

1

2

3

4

5

6

7

9

B

B

Fig. 1. TIMP-4 expression in cultured rat VSMCs infected with Ad.TIMP-4 by quatitative image analysis

Fig. 2. Activity of adenovirus mediated TIMP-4

(A) and Western blot analysis (B). 1: Ad .LacZ; 2:

expression assessed by gelatin zymography (A) and

recombination human TIMP-4; 3–9: conditioned media

reverse zymography (B). A: At 10 d after infected with

from Ad.TIMP-4-infected VSMCs analyzed by Western

Ad.LacZ

blotting at 18, 14, 10, 8, 6, 4, and 1 d respectively after

conditioned media of VSMCs were analyzed using

infection. rhTIMP-4 (23 kDa) was used as a positive

rhMMP-2 (1, 0.5 and 0.25 ng). Lane 1 and 2: 1 ng; Lane

control. Conditioned media from Ad.Lacz-infected

3 and 4: 0.5 ng; Lane 5 and 6: 0.25 ng. B. Conditioned

VSMCs was used as a negative control.

media of VSMCs were analyzed at 10 d after infected

.

with Ad.LacZ MOI 100 (lane 1), and Ad.TIMP-4 MOI

MOI

100

and

Ad.TIMP-4

MOI

100,

100 (lane 2 and 3), MOI 50 (lane 4 and 5) and MOI 5

3.2.

Biological

activity

of

human

TIMP-4

(lane 6 and 7) respectively.

transferred to VSMCs Both

gelatin

zymography

and

reverse

3.3. Migration and proliferation of VSMCs

zymography were used to determine whether the TIMP-4 secreted by Ad.TIMP-4-infected VSMCs

The effects of infection with Ad.TIMP-4 on

was functionally active. Fig. 2A shows the results

VSMC migration were examined (Fig. 3). Few cells

of a gelatin zymography in which conditional media

migrated in the absence of the chemoattractant; the

from

Ad.TIMP-4-infected

addition of PDGF caused almost 8-fold increase in

VSMCs were included in the buffer used to develop

the number of cells that migrated across the barrier.

MMP inhibition activity. Inhibition of MMP

The inhibitory effect of Ad.TIMP-4 significantly

activity was seen when the gel was incubated in

increased with the duration of post-infection time

Ad.TIMP-4 conditional media, whereas activity was

(P<0.01). At 10 d after infection, VSMCs infected

unaffected by Ad.LacZ. The biological activity of

with

the TIMP-4 was also demonstrated by reverse

(78.7±9.3)% compared with cells infected with

zymography (Fig. 2B).

Ad.LacZ (P<0.01).

either

Ad.LacZ-

or

Ad.TIMP-4

showed

an

inhibition

of

58

Zhong Li et al. / Journal of Medical Colleges of PLA 26 (2011) 53–62

1 d after Infection

Ad.LacZ

5 d after Infection 1.4 Mean intimal/medial ratio

Number of migratory VSMCs

10 d after Infection

40 35 30 25 20 15 10 5 0 Uninfected cells

Uninfected cells+PDGF

Ad.TIMP-4

Ad.LacZ+ PDGF

1.2 1 0.8 0.6 0.4 0.2 0

Ad.TIMP4+PDGF

Ad.LacZ

Ad.TIMP-4

Fig. 3. Amount of migratory VSMCs infected with

Fig. 5. Average results on effect of Ad.TIMP-4 on

Ad.TIMP-4 and Ad.LacZ respectively. The amount of

neointima development in the injured rat carotid artery.

migratory VSMCs was detected at 1, 5 and 10 d after

Average intimal/medial area in rat carotid arteries at 14

infection with Ad.TIMP-4 and Ad.LacZ respectively.

d after infected with either Ad.TIMP-4 or Ad.LacZ.

Uninfected cells with or without PDGF placed in the lower chamber were used as controls. Results are shown as mean number of cells form four random high-power microscopy per chamber and are representative of three experiments.

We

examined

the

effect

of

Ad.TIMP-4

infection on the proliferation of VSMCs. At 1, 5 and 10

d

after

infection,

the

incorporation

of

3

[ H]-thymidine was found no significant difference in VSMCs infected with Ad.TIMP-4 compared

[3H]-thymidine incorporation

Unifected 7000

Ad.LacZ

either Ad.LacZ or uninfected control cells (Fig. 4).

Ad.hTIMP-4

3.4. Effect of Ad.TIMP-4 on neointima development

6000 5000 4000

Successful transgenic expression in vivo was

3000

confirmed by Western blot analysis arterial extracts

2000

for

1000

Ad.TIMP-4-infected arteries was found, but was

0

absent in Ad.LacZ-infected arteries (data not 1

5 Days after infection

10

Fig. 4. Change of [ 3 H]-thymidine incorporation into VSMCs infected with Ad.TIMP-4, Ad.LacZ and uninfected cells. Data shown are the means±SD of quadruplicate values and are representative of three experiments.

human

TIMP-4.

A

23-kDa

band

in

shown). To examine the effect of Ad.TIMP-4 infection

on

neointimal

accumulation,

both

neointimal and medial areas were measured at 2 and 14 d after injury. At 2 d, Ad.TIMP-4 infection had no significant effect on the neointimal and medial areas (data not shown). At 14 d, the average results

59

Zhong Li et al. / Journal of Medical Colleges of PLA 26 (2011) 53–62

are

shown

in

Fig.

5

and

representative

we

investigated

the

potential

effect

of

photomicrographs are shown in Fig. 6. A significant

adenovirus-mediated gene transfer of TIPM-4 on

62% reduction in neointimal area was found in

the neointima development after endovascular

2

mm )

injury. Our in vitro experiments showed that the

compared with Ad.LacZ-infected vessels (0.68±

secretion of biologically active TIMP-4 inhibited

Ad.TIMP-4-infected

vessels

(0.26±0.09

2

0.05 mm ; P<0.01). However, there is no difference

VSMC migration, but not significantly affect

in

and

VSMC proliferation. In a balloon-injured rat carotid

Ad.LacZ-infected vessels (0.21±0.06 vs 0.19±0.04

artery model, our results showed that arterial gene

medial

area

for

Ad.TIMP-4

2

mm ; P>0.05). The average ratio of neointimal to

transfer

medial area was showed a significant difference

hyperplasia after vascular injury.

between Ad.TIMP-4- and Ad.LacZ-infected vessels 2

of

Previous

TIMP-4 studies

attenuates have

neointimal

demonstrated

that

(1.17±0.20 vs 0.60±0.11 mm ; P<0.01) (Fig. 5).

TIMP-1, -2, or -3 over expression inhibited SMC

These results showed that Ad.TIMP-4 infection to

invasion through reconstituted basement membrane,

the rat carotid artery after injury resulted in a

which is consistent with ours [10–12]. MMP-2 is

markedly decrease in early neointima development.

constitutively expressed, and its mRNA shows a slight decrease 1 day after balloon injury, whereas

4. Discussion

the relative activation of MMP-2 increases during the 5- to 14-day period after injury [5–7]. Our in

TIMP-4 is a 23-kDa protein that inhibits

vitro experiment showed that TIMP-4 protein

MMP-1, MMP-3, MMP-7, and MMP-9 and shows a

expression in VSMCs infected with Ad.TIMP-4

particular interaction with MMP-2 [17, 18]. We

increased with the duration of infection. TIMP-4

chose to study TIMP-4 because of its specific tissue

protein reached the highest level at 10 d, and was

expression: transcripts are abundant in the human

constant up to 2 weeks after infection. Therefore,

heart but occur at low levels in most other organs,

the consistent time frame of the changes with

which suggest a possible cardiovascular specificity

MMP-2 may contribute to the inhibitory invasion of

for this metalloproteinase inhibitor. In this study,

over-expression TIMP-4.

A

B

C

D

Fig. 6. Effect of Ad.TIMP-4 on neointima development in the injured rat carotid artery. Representative HE-stained sections of rat carotid artery (A and B) and immunohistochemistry sections (C and D) taken at 14 d after balloon injury and infection with either Ad.TIMP-4 (A and C) or Ad.LacZ (B and D).

60

Zhong Li et al. / Journal of Medical Colleges of PLA 26 (2011) 53–62

Previous studies have shown that MMPs also

increased VSMC apoptosis [21]. This may provide

play important roles in cell proliferation. Some

another explanation to the inhibitory effect of

MMP inhibitors also showed inhibitory effects on

TIMP-4 in vivo.

cell growth [19]. However, the effects of TIMPs on

TIMP-4, as well as TIMP-1, -2 and -3 have

cell growth cannot always be clearly reconciled

been demonstrated that their over expression

with their ability to abrogate MMP activity. TIMP-1

reduced neointimal hyperplasia. But their inhibitory

had no effect on cell proliferation [9, 20], while

mechanisms may be diverse. Further studies are

TIMP-2 caused a dose-dependent inhibition of cell

needed to compare their inhibitory effects and

number increase and BrdU incorporation without

levels in the same cellular or animal models. The

over

combined use of Ad vectors that code for different

expression also caused a dose-dependent inhibition

TIMPs genes may enhance the likelihood that

of the increase in cell number [12, 20]. We

Ad-mediated gene therapy may be successful in the

observed the effect of TIMP-4 on the proliferation

treatment of restenosis after endovascular injury.

affecting

cell

death

20]. TIMP-3

[11,

of VSMCs. Our results showed that over expressing TIMP-4

had

no

significant

effect

on

the

proliferation of cultured VSMCs. This result largely

5. Conclusion

parallels the findings of Gao et al who used Our

adenoviral vectors to express TIMP-4 that were

data

demonstrated

TIMP-4

over-

transferred to the arterial wall [21]. We also

expression can significantly inhibit the migration of

detected

cultured VSMCs and prevent neointimal formation

the

cell

cycle

parameters

by

flow

cytometry, and found the percentage of cells during

after

G 0-G 1, G 2-M and S periods in Ad.TIMP-4-infected

additional evidence that TIMP-4 could play an

VSMCs was similar with Ad.LacZ-infected VSMCs,

important role in vascular pathophysiology, and

which indicated that cell cycle progression of

may be an important therapeutic target for future

VSMCs were

drug development.

not

affected

by

TIMP-4

over

vascular

injury.

Our

findings

provide

expression (data not shown). It is known that extracellular matrix contact can mediate cell survival; in contrast, an imbalance

References

in the matrix composition of the arterial wall might mediate the cell loss. TIMP-3 has been shown to

1. Clowes AW, Reidy MA, Clowes MM. Mechanisms of

cause apoptosis of VSMCs, but TIMP-1 does not

stenosis after arterial injury. Lab Invest, 1983; 49 (2):

appear to share this property in vitro [20]. Another

208–215.

study also found that over expression of TIMP-4

2. Glagov S. Intimal hyperplasia, vascular modeling,

markedly increased apoptotic cells without chang-

and the restenosis problem. Circulation, 1994; 89 (6):

ing their proliferation in vitro experiments, and

2888–2891.

importantly, over expression of human TIMP-4 in

3. Bosman FT, Stamenkovic I. Functional structure and

the wall of balloon-injured rat carotid artery also

composition of the extracellular matrix. J Pathol,

61

Zhong Li et al. / Journal of Medical Colleges of PLA 26 (2011) 53–62

2003; 200 (4): 423–428.

12. George SJ, Lloyd CT, Angelini GD, et al. Inhibition

4. Pauly RR, Passaniti A, Bilato C, et al. Migration of

of late vein graft neointima formation in human and

cultured vascular smooth muscle cells through a

porcine

basement

expression of tissue inhibitor of metalloproteinase-3.

membrane

barrier

requires

type

IV

collagenase activity and is inhibited by cellular differentiation. Circ Res, 1994;75 (1): 41–54.

models

by

adenovirus-mediated

over-

Circulation, 2000;101 (3): 296–304. 13. Greene J, Wang M, Liu YE, et al. Molecular cloning

5. Bendeck MP, Zempo N, Clowes A W, et al. Smooth

and characterization of human tissue inhibitor of

muscle cell migration and matrix metalloproteinase

metalloproteinase 4. J Biol Chem, 1996; 271 (48):

expression after arterial injury in the rat. Circ Res,

30375–30380.

1994; 75 (3): 539–545.

14. Leco KJ, Apte SS, Taniguchi GT, et al. Murine

6. Dollery CM, McEwan JR, Henney AM. Matrix

tissue inhibitor of metalloproteinases-4 (Timp-4):

metalloproteinases and cardiovascular disease. Circ

cDNA isolation and expression in adult mouse

Res, 1995; 77 (5): 863–868.

tissues. FEBS Lett, 1997; 401 (2–3): 213–217.

7. Zempo N, Kenagy RD, Au YP, et al. Matrix metalloproteinases

of

vascular

wall

cells

are

increased in balloon-injured rat carotid artery. J Vasc Surg, 1994; 20 (2): 209–217.

of

basement

regulated by vascular injury in rats. Circ Res, 1999; 84 (5): 498–504. 16. Webb KE, Henney AM, Anglin S, et al. Expression

8. Southgate KM, Fisher M, Banning AP, et al. Upregulation

15. Dollery CM, McEwan JR, Wang M, et al. TIMP-4 is

of matrix metalloproteinases and their inhibitor

membrane-degrading

TIMP-1 in the rat carotid artery after balloon injury.

metalloproteinase secretion after balloon injury of pig

Arterioscler Thromb Vasc Biol, 1997; 17 (9):

carotid arteries. Circ Res, 1996; 79 (6): 1177–1187.

1837–1844.

9. D'Armiento J. Matrix metalloproteinase disruption of

17. Liu YE, Wang M, Greene J, et al. Preparation and

the extracellular matrix and cardiac dysfunction.

characterization of recombinant tissue inhibitor of

Trends Cardiovasc Med, 2002; 12 (3): 97–101.

metalloproteinase 4 (TIMP-4). J Biol Chem, 1997;

10. Dollery CM, Humphries SE, McClelland A, et al.

272 (33): 20479–20483.

Expression of tissue inhibitor of matrix metallo-

18. Bigg HF, Shi YE, Liu YE, et al. Specific, high

proteinases 1 by use of an adenoviral vector inhibits

affinity binding of tissue inhibitor of metallo-

smooth

reduces

proteinases-4 (TIMP-4) to the COOH-terminal

neointimal hyperplasia in the rat model of vascular

hemopexin-like domain of human gelatinase A.

balloon

TIMP-4 binds progelatinase A and the COOH-

muscle

injury.

cell

migration

Circulation,

and

1999;

99

(24):

3199–3205. 11. Cheng L, Mantile G, Pauly R, et al. Adenovirus-

terminal domain in a similar manner to TIMP-2. J Biol Chem, 1997; 272 (24): 15496 –15500.

mediated gene transfer of the human tissue inhibitor

19. Zempo N, Koyama N, Kenagy RD, et al. Regulation

of metalloproteinase-2 blocks vascular smooth

of vascular smooth muscle cell migration and

muscle cell invasiveness in vitro and modulates

proliferation in vitro and in injured rat arteries by a

neointimal development in vivo. Circulation, 1998;

synthetic

98 (20): 2195–2201.

Arterioscler Thromb Vasc Biol, 1996; 16 (1): 28–33.

matrix

metalloproteinase

inhibitor.

62

Zhong Li et al. / Journal of Medical Colleges of PLA 26 (2011) 53–62

20. Baker AH, Zaltsman AB, George SJ, et al. Divergent

21. Guo YH, Gao W, Li Q, et al. Tissue inhibitor of

effects of tissue inhibitor of metalloproteinase-1, -2,

metalloproteinases-4 suppresses vascular smooth

or -3 overexpression on rat vascular smooth muscle

muscle cell migration and induces cell apoptosis.

cell invasion, proliferation, and death in vitro.

Life Sci, 2004;75 (20): 2483–2493.

TIMP-3 promotes apoptosis. J Clin Invest, 1998; 101 (6): 1478–1487.

(Editor Guo Jianxiu)