© 1995 American Heart Association, Inc.
Articles |
In Vivo Effect of TGF-ß1
Enhanced Intimal Thickening by Administration of TGF-ß1 in Rabbit Arteries Injured With a Balloon Catheter
From the Second Department of Internal Medicine (T.K., K. Tamura, K. Takahashi, Y.S., N.M.) and the Second Department of Pathology (B.A.), School of Medicine, Chiba University, Chiba; the Kirin Brewery Co, Ltd (H.O.), Maebashi City; and the Department of Pathology, Osaka City University Medical School (N.K., M.U.), Osaka, Japan.
Correspondence to Tetsuto Kanzaki, MD, Second Department of Internal Medicine, School of Medicine, Chiba University, 1-8-1, Inohana, Chiba City 260, Japan.
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Abstract |
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Abstract The in vivo effect of transforming growth factor–ß1 (TGF-ß1) was studied in a model system in which arterial intimal thickening was induced by injury of rabbit arteries with a balloon catheter (BCI). Intimal area and its ratio to medial area in carotid arteries after BCI were significantly higher in rabbits treated with 10 µg/kg TGF-ß1 and 10 mg/kg aspirin IV QD (TGF-ß1 group) than in those treated with 10 mg/kg aspirin IV QD only (control group). Intimal cell numbers in the TGF-ß1 and control groups were not significantly different from each other, but matrix volume in the intimal layer was significantly higher in the TGF-ß1 group. By immunohistochemical and Northern blot analyses, the fibronectin content in carotid intimal and medial layers was greater in the TGF-ß1 group compared with that in the control group. Thus, in intimal thickenings induced by BCI, TGF-ß1 mainly enhanced the formation of matrix containing fibronectin. Moreover, the mRNAs of TGF-ß type I and type II receptors were detected in carotid arteries 7 and 14 days after, but not before, BCI. Thus, TGF-ß1 influences the process of intimal thickening induced by BCI through a receptor-mediated mechanism in vivo. The significance of this fact is discussed in relation to the development of atherosclerosis.
Key Words: TGF-ß1 • intimal thickening • balloon catheter injury • extracellular matrix • fibronectin
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Introduction |
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TGF-ß1 belongs to the TGF-ß superfamily, which includes several types of TGF-ß (namely, TGF-ß1, -ß2, -ß3, -ß4, and -ß5), inhibins, activins, Mullerian-inhibiting substance, bone morphogenic proteins, and products of the Vg-l gene and decapentaplegic gene complex.1 2 TGF-ß1 is a multifunctional protein that has been implicated in the regulation of a variety of physiological and developmental processes.1 2 TGF-ßs exert their effects by binding to cell-surface receptors,1 and three distinct receptors (types I, II, and III) have been identified in most cells, including arterial SMCs. However, type III receptors may not be directly involved in signal transduction.3
Migration of SMCs from the media to the intima and SMC proliferation are important in arterial intimal thickening.4 Related to this migration and proliferation are various growth factors,5 including TGF-ß1, a bifunctional regulator that is dependent on species, cell phenotype, growth conditions, and interactions with other growth factors.6 7 These characteristics suggest that TGF-ß1 can both accelerate and retard SMC migration and proliferation in vivo. Moreover, TGF-ß1 has been found to control extracellular matrix protein production and degradation by SMCs in vitro.8 Furthermore, studies of transgenic mice and of the targeted expression of TGF-ß1 have shown that it affects tumorigenesis and immune and inflammatory systems function in vivo.9 10 However, little is known about the roles of TGF-ß1 in the initiation, progression, and regression of atherosclerosis in vivo.
BCI is one of several forms of arterial injury that have
been used to investigate specific aspects of arterial
intimal thickening.11 Jawien et al12 found
that by increasing SMC migration, PDGF-BB infusion enhanced the intimal
thickening induced by BCI in rats, and Ferns et al13
reported a 40% reduction in intimal thickening after administration of
an anti-PDGF antibody, with no change in the thymidine labeling index 8
days after performance of BCI. Lindner and Reidy14
observed that basic fibroblast growth factor stimulated SMC
proliferation in arteries after BCI, and Majesky et al15
showed that within 6 hours of BCI, expression of TGF-ß1 mRNA and
protein in arteries increased, continued to increase for at least 14
days after injury, and was associated with the overexpression of
fibronectin, collagen-
2(I), and
collagen-1(III). Taken together, these reports indicate
that a single factor cannot explain the mechanism of intimal thickening
induced by BCI. In this article, we report the in vivo effects of
TGF-ß1 on intimal thickening in arteries after BCI as a model of
atherosclerosis.
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Methods |
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Chemicals
TGF-ß1 was purified from Escherichia coli transfected with its cDNA to >95% homogeneity as estimated by SDS-PAGE. Before it was injected, TGF-ß1 was dissolved in 1 mmol/L HCl/PBS. Acetylsalicylic acid (aspirin) was purchased from Sigma Chemical Co, [
-32P]dCTP was from
New England Nuclear, and ß-actin cDNA was from Oncogene Science
Inc. Human TGF-ß type I and type II receptor cDNAs were kindly
provided by Dr Kohei Miyazono (Ludwig Institute for Cancer Research,
Uppsala Branch, Uppsala, Sweden). Fibronectin cDNAs
(pFH 1, pFH 111, and pFH 154) were donated by the Japanese Cancer
Research Resources Gene Bank,16 and collagen type III cDNA
was a gift from Dr Akira Ooshima (Wakayama Medical College).
BCI and Injection of TGF-ß1
Two groups of male Japanese White rabbits (control, n=5;
TGF-ß, n=7) weighing 2 kg were fed 100 g/d of standard chow (Oriental
Kobo Co) for 14 days. BCI of the right carotid artery was performed on
day 14 as described.17 18 This procedure was done under
sterile conditions while the animals were under pentobarbital
anesthesia and met the ethical standards regarding
experiments with animals. The rabbits then received injections of 10
µg/kg TGF-ß1 and 10 mg/kg aspirin IV QD for 14 days (TGF-ß1
group) or 10 mg/kg aspirin IV QD only (control group) and were killed
on day 28 by injection of sodium pentobarbital (100 mg/kg).
Measurement of Thickening of Intimal and Medial
Layers
To determine the optimum time for evaluation of intimal
thickening, in preliminary experiments cross sections of injured right
carotid arteries were examined 0, 7, 14, 21, and 28 days after BCI. The
first sign of intimal thickening was recognized on day 7 and was
observed to increase until day 21. In this study, the intimal
thickening value 14 days after injury was used for data collection.
Fourteen days after BCI, the right carotid artery was removed, cut into four segments of equal length, and prepared for light and electron microscopy. The sections were prepared for light microscopy by staining with hematoxylin-eosin and van Gieson's elastic stain. The areas of intimal and medial layers in arterial cross sections were calculated by using an image scanner as described.17 18 The intimal thickening in one right carotid artery was designated as the mean intimal-medial area ratio in the four segments. Data on intimal and medial areas and on intimal thickening in right carotid arteries in the control (n=5) and TGF-ß1 (n=7) groups were expressed as medians. The rank sum test was used for comparison of the two groups.
Measurement of Cell Numbers, Cell Volumes, and Matrix Volumes in
the Arterial Intimal Layer
Cell numbers in the intimal layer of the four
cross-sectional segments were calculated as the mean number of
nuclei. To determine the volume densities of cells and of extracellular
matrix in control and TGF-ß1 groups, at least 12 electron
photomicrographs of the cross-sectional intimal layers were taken
at the midpoint of the injured carotid artery. Volume densities were
estimated by the point-count method, using a transparent grid with
169 test points as described.19 20 The rank sum test was
used for comparison of the two groups.
Immunohistochemical Study of BCI Arteries
The artery segments were snap-frozen, stored at
-80°C, serially sectioned at a thickness of 4 µm, and fixed
in acetone. Every first section was stained with hematoxylin and eosin;
the three remaining sections were used for immunohistochemical
staining.
For identification of cellular fibronectin, we used a mouse monoclonal antibody (DH1, Biohit) that is specific to the extradomain of cellular fibronectin and thus, recognizes only cellular fibronectin. For identification of collagen type III, we used a mouse monoclonal antibody (III-53, Fuji Chemical Industries Ltd) that cross-reacts with rabbit antibody, the specificity of which has been reported.21 22 An immunoperoxidase avidin-biotin complex system with NiCl2 color modification was used in all instances.23 24 Sections were counterstained with methyl green.
RT-PCR of the TGF-ß Receptors
Total RNA was purified by the LiCl-urea
method25 from medial and intimal layers of carotid
arteries 7 and 14 days after BCI and from those of uninjured rabbits.
RT-PCR was performed as described by Tsuchida et al.26 In
brief, total RNA (2 µg) was incubated at 37°C for 1 hour in 33 µL
of reverse transcriptase buffer (45 mmol/L Tris-HCl [pH 8.3], 68
mmol/L KCl, 15 mmol/LDTT, 9 mmol/L MgCl2, 0.08 mg/mL
bovine serum albumin, and 1.8 mmol/L each dNTP) containing
RNase inhibitor, 0.2 µg random-hexamer primers, and
cloned murine reverse transcriptase (Pharmacia LKB Biotechnology).
After a 1-hour incubation at 37°C, 10 µL of the reaction mixture
was removed for PCR. Reactions were performed in 10 mmol/L Tris-HCl, pH
8.3, containing 1.5 mmol/L MgCl2, 50 mmol/L KCl, 0.2
mmol/L of each dNTP, 20 pmol each of sense and antisense primers, and
2.5 U Taq polymerase (Perkin-Elmer Cetus). PCR primers were
prepared at nucleotide sequences 511 through 531 (sense),
1329 through 1350 (antisense), 94 through 116 (sense), and 591 through
610 (antisense) of the human TGF-ß type I receptor27 and
at nucleotide sequences 251 through 271 (sense), 931
through 949 (antisense), 868 through 893 (sense), and 1348 through 1371
(antisense) of rat TGF-ß type II receptor.26 PCR
conditions were as follows: 30 cycles at 94°C (1 minute), 60°C (2
minutes), and 72°C (3 minutes), followed by 10 minutes at 72°C.
After PCR amplification, an aliquot of each amplification mixture was
separated by 1.5% agarose gel electrophoresis and visualized by
ethidium bromide staining.
Southern Blot Analysis
Agarose gels were soaked sequentially in 0.5 mol/L
NaOH–1.5 mol/L NaCl for 1 hour, neutralized in 1 mol/L Tris-HCl [pH
8.0]–1.5 mol/L NaCl for 1 hour, and then transferred by blotting to a
Hybond N+ membrane (Amersham). Southern hybridization was
performed in 50% formamide, 5x SSC (1x SSC contains 15 mmol/L sodium
citrate and 150 mmol/L NaCl, pH 7.4), 5x Denhardt's solution, 0.1%
SDS, 50 mmol/L sodium phosphate buffer (pH 6.5), and 0.1 mg/mL salmon
sperm DNA at 42°C overnight with a human cDNA of TGF-ß type I or
type II receptor. These TGF-ß cDNAs contained the amplified region
without the primer sequences and were labeled with the Megaprime DNA
labeling system (Amersham) as a probe. The membrane was washed three
times for 20 minutes each in 0.1x SSC and 0.1% SDS at 42°C, dried,
and exposed to Amersham Hyperfilm MP.
Northern Blot Analysis
Northern blot analysis was performed as described
by Claesson-Welsh et al.28 In brief, 20 µg total
RNAs from BCI arteries of control and experimental rabbits were
electrophoresed on a 1% agarose gel in the presence of formaldehyde
and blotted to a Hybond N+ membrane. The membrane was
hybridized at 42°C under conditions identical to those described for
Southern blot analysis, using 32P-labeled cDNA as a
probe.
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Results |
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Effects of TGF-ß1 Injection on Various Organs of Rabbits
First, 10 µg/kg IV QD of TGF-ß1 alone was injected into rabbits for 14 days, but no remarkable histological changes were observed in the liver, kidney, spleen, lung, heart, or various arteries in rabbits without BCI (data not shown). These findings suggested that TGF-ß1 alone had no morphological effects on intact arteries.
Next, we injected TGF-ß1 into rabbits after BCI, which induces intimal thickening via migration and proliferation of SMCs. Injections of 10 µg/kg IV of TGF-ß1 into rabbits after BCI resulted in remarkable thrombus formation: in the TGF-ß1–treated group (n=9, 36 segments), 67% of all segments from injured carotid arteries had a thrombus, and some showed complete occlusion of the arterial lumen. In contrast, no thrombus formation was observed in the control group after BCI (n=7, 28 segments). This preliminary experiment was performed without aspirin. Afterward, final experiments were conducted in control (n=5) and TGF-ß (n=7) groups.
In instances of marked thrombus formation or complete occlusion, it is difficult to evaluate the direct effect of TGF-ß1 on intimal thickening because blood platelets contain many biochemical factors that influence arterial intimal thickening and/or blood flow is blocked by obstruction of the lumen. To prevent thrombus formation after BCI, aspirin was used.29 After BCI, rabbits were treated with 10 µg/kg IV TGF-ß1 and 10 mg/kg aspirin QD (TGF-ß1 group) or 10 mg/kg aspirin QD alone (control group) for 14 days. The rate of thrombus formation was only 6% in injured carotid arterial segments in the TGF-ß1 group and 0% in the control group.
Blood Parameters of TGF-ß1–Treated
Rabbits
No significant differences were observed in body weights or
blood cell counts (data not shown). Values for serum total protein,
albumin, aspartate aminotransferase, alanine aminotransferase,
alkaline phosphatase, blood urea nitrogen, creatinine,
electrolytes, total cholesterol, triglyceride,
HDL cholesterol, and phospholipid were also not
significantly different between the control and TGF-ß1 groups (data
not shown).
Effects of TGF-ß1 Injection on Arterial Intimal and
Medial Areas After BCI
As shown in Fig 1
and Table 1,
intimal area measurements in both groups of rabbits after BCI were
0.418 mm2 (range, 0.263 to 0.471; n=7) and 0.251
mm2 (range, 0.03 to 0.322; n=5), respectively, which were
significantly different (P<.05). Medial area measurements
were 0.782 mm2 (range, 0.600 to 1.137; n=7) and 0.731
mm2 (range, 0.667 to 0.890; n=5), respectively, which were
not significantly different. Thus, the intimal-medial
ratio of thickening in the TGF-ß1 group (46.0%; range, 33.2% to
67.9%; n=7) was significantly different from that in the control group
(29.7%; range, 4.5% to 39.7%; n=5).
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Cell Numbers, Cell Volumes, and Matrix Volumes in the
Arterial Intimal Layer After BCI
Electron microscopy showed that 
95% of cells in the intima
were SMCs. The numbers of cells in the intima after BCI were higher in
the TGF-ß1 group than in the control group, but this increase was not
significant (Table 2). As shown in Table 2, cell volumes in the arterial intimal layers in the
TGF-ß1 group were significantly different from those in the control
group (28.0%; range, 22.3% to 36.1%; n=7 versus 38.3%; range,
34.5% to 51.9%; n=5; P<.05). Similarly, matrix volumes of
the arterial intimal layer in the TGF-ß1 group were also
significantly higher than those of the control group (72.1%; range,
63.9% to 77.7%; n=7 versus 62.0%; range, 48.1% to 65.5%; n=5;
P<.05). In contrast, the calculated sizes of intimal SMCs
were not significantly different between the TGF-ß1 and control
groups. The finding that cell volume in the intima was lower despite no
decrease in cell number can be explained by the fact that cell density
was lower in the TGF-ß1 group (data not shown).
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Immunohistochemical Study of Arteries After BCI
In the control group, mildly thickened intimas stained
positively with anti-human cellular fibronectin antibody, whereas
the media stained only weakly (Fig 2A).
In the TGF-ß1 group, both the media and the markedly thickened intima
stained strongly and positively with this antibody (Fig 2B). Type III collagen was diffusely stained in intimal
and medial layers in both groups (data not shown). By
immunohistochemistry, intimal thickening in the control group was found
to be less than that of carotid arteries after BCI, as shown in Fig 1A
. This was probably due to use of different sections
of carotid artery after BCI.
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mRNA Expression of Fibronectin and Collagen Type III in Arteries
After BCI
Fibronectin mRNA was expressed in carotid arteries 14 days
after BCI but not in arteries without BCI. The size of this mRNA was
7.8 kb, somewhat similar to that of human and rat fibronectin
mRNA.30 31 The intensity ratio of fibronectin to
ß-actin mRNA expression was about five times stronger after BCI
in the TGF-ß1 group than in the control group (Fig 3), suggesting that fibronectin synthesis
was increased by injection of TGF-ß1. However, no difference in
collagen type III mRNA was detected by Northern blot analysis
between control and TGF-ß1 groups (data not shown).
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Expression of TGF-ß Receptor mRNA After BCI
To determine whether intimal thickening after BCI was induced
directly by TGF-ß1 injection, we examined the expression of TGF-ß
type I and type II receptors in both the media and intima. mRNAs of
TGF-ß type I and type II receptors were detected by
RT-PCR26 7 and 14 days after BCI but not before BCI (Figs 4A and 5A). The sizes of the bands
acquired from PCR analysis were the same as those predicted by
the TGF-ß receptors. Moreover, the same results were obtained by
RT-PCR using different pairs of primers (data not shown). These results
were also confirmed on Southern blots by using cDNA that contained the
amplified region of the TGF-ß type I and type II receptors without
the primer as a probe (Figs 4B and 5B). The only difference was that a
very thin band for the TGF-ß type I receptor was observed in control
arterial media (without BCI) by Southern blot
analysis. This finding suggests that TGF-ß1 exerts its
function by binding to SMCs in BCI-treated arteries and that no intimal
thickening in uninjured carotid arteries from TGF-ß1–injected
rabbits can be correlated with the absence of detectable TGF-ß type
II receptors.
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Discussion |
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The present results have shown that TGF-ß1 stimulates intimal thickening in arteries after in vivo BCI and is associated with the expression of TGF-ß type I and type II receptor mRNAs. The intimal thickening in carotid arteries after BCI was almost the same with and without 1 µg/kg TGF-ß1 (data not shown). However, injection of 10 µg/kg TGF-ß1 and 10 mg/kg aspirin IV QD was effective on intimal thickening after BCI. We could not use higher concentrations of TGF-ß1 because its availability was limited. The plasma half-life of active TGF-ß1 has been reported to be
3
minutes in rats,32 suggesting that the plasma
concentration of TGF-ß1 is <1 pg/mL 1 hour after injection of 10
µg/kg if the injected TGF-ß1 is not biochemically
"recruited." It is unclear whether this relatively short duration
of the effective level of TGF-ß1 can exert its biologic effects in
vivo. It is possible that the injected TGF-ß1 is conserved on the
cell surface with ß-glycan or in the extracellular
matrix33 34 and can then be supplied for a long time.
TGF-ß1 is secreted by many cell types, including platelets and
macrophages.6 35 Majesky et al15 have
reported that TGF-ß1 is produced during arterial repair
after injury and is associated with increases in fibronectin,
collagen-2(I), and collagen-1(III) gene
expressions. The present study shows that TGF-ß1 enhances intimal
thickening of rabbit arteries after BCI, suggesting the significance of
TGF-ß1 in the formation of intimal thickenings or
atheromatous lesions.
Many investigators have reported the in vitro effects of TGF-ß1 on the migration and proliferation of SMCs and SMC matrix synthesis. Koyama et al7 observed the bifunctional effects of TGF-ß1 on the migration of rat SMCs; ie, TGF-ß1 at concentrations of 10 to 50 pg/mL enhanced the migration of rat SMCs but dose-dependently inhibited PDGF-induced migration at concentrations of 1 to 1000 pg/mL. Morisaki et al36 reported that the growth of rabbit SMCs was inhibited by TGF-ß1 at concentrations of 1 to 10 000 pg/mL. Goodman and Majack37 found that TGF-ß1 inhibited proliferation of SMCs in sparse cultures but stimulated their proliferation in confluent cultures. Davidson et al38 showed that TGF-ß1 acted as a mitogen in confluent porcine SMCs. In the present study, increases in the number of intimal SMCs in the TGF-ß1 group were not significant. Our data indicate that after BCI, the intimal thickening induced by TGF-ß1 is mainly due to an increased amount of intimal extracellular matrix produced by SMCs. However, it is difficult to clearly determine whether the increases in the amount of matrix are due to a direct effect of TGF-ß1 or not.
Immunohistochemical study showed that in TGF-ß1–treated rabbits
compared with controls, fibronectin content was increased in intimal
and medial layers and mRNA fibronectin expression was also enhanced
after BCI. These results suggest that some of the increase in intimal
extracellular matrix content in the TGF-ß1 group is due to an
increase in fibronectin synthesis. Davidson et al38 and
Majack et al39 reported that TGF-ß1 stimulated in vitro
synthesis of several components of the extracellular matrix, ie,
thrombospondin, elastin, and 1-collagen. These reports
are consistent with our in vivo finding that TGF-ß1 increases
the synthesis of intimal extracellular matrix.
Nabel et al40 reported that in normal pig arteries, TGF-ß1 expression by direct gene transfer resulted in substantial extracellular matrix production accompanied by intimal and medial hyperplasia. The in vivo effect of TGF-ß1 on intimal thickening in their model was similar to that in ours, but different, in that we did not detect intimal thickening in noninjured arteries of TGF-ß1–treated rabbits. Our data show that the mRNAs of TGF-ß receptors are expressed in carotid arteries after BCI but not in uninjured arteries. These differences may be due to local concentrations of TGF-ß1. It is probably difficult to maintain a high arterial concentration of TGF-ß1 by a single injection, because the plasma half-life of active TGF-ß1 is short. However, it is also possible that arterial lumen was injured by catheter insertion at the time of direct gene transfer.
The effects of exogenous TGF-ß1 can be expressed through TGF-ß type I and type II receptors after BCI in arteries. Platelets, macrophages, and endothelial cells are known to express TGF-ß1,6 35 41 and intimal SMCs in the BCI model also express TGF-ß1 6 hours to 14 days after injury.15 Indeed, human arterial atherosclerotic lesions express more TGF-ß1 than do arteries without such lesions.42 However, TGF-ß1 is usually synthesized and secreted in its latent form43 44 45 and is activated by removal of an LAP from the latent TGF-ß1.43 Several reports have proposed mechanisms for the activation of latent TGF-ß1 in vitro: cleavage within the N-terminal region of LAP by protease,43 enzymatic removal of carbohydrate structures from LAP,44 and coculture of endothelial cells with SMCs45 cause partial, but not complete, activation of TGF-ß1. Further studies on the activation of latent TGF-ß1 in vivo are necessary to determine the significance of endogenous TGF-ß1 in intimal thickening.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We thank Dr Kohei Miyazono (Ludwig Institute for Cancer Research, Uppsala Branch, Uppsala, Sweden) and Dr Akira Ooshima (Wakayama Medical Collage) for gifts of human TGF-ß type I and II receptor cDNAs and collagen type III cDNA, respectively.
Received May 10, 1994; accepted June 28, 1995.
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