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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2461-2470.)
© 1997 American Heart Association, Inc.

Articles

Inhibition of Protein Tyrosine Kinases Attenuates Increases in Expression of Transforming Growth Factor-ß Isoforms and Their Receptors Following Arterial Injury

Michael R. Ward; Alex Agrotis; Peter Kanellakis; Rodney Dilley; Garry Jennings; ; Alex Bobik

From the Cell Biology Laboratory, Baker Medical Research Institute, and Alfred Baker Medical Unit, Alfred Hospital, Prahran, Australia.

Correspondence to Dr M. Ward, Cell Biology Laboratory, Baker Medical Research Institute, Commercial Rd, Prahran, VIC 3181, Australia. E-mail mward{at}Baker.edu.au

	Abstract

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Abstract Transforming growth factor-ß₁ (TGF-ß₁) has been implicated in neointima formation in mechanically injured vessels and in restenosis after angioplasty. To further understand the significance of TGF-ßs in neointima formation, we examined the temporal expression of three TGF-ß isoforms (-ß₁, -ß₂, and -ß₃), their receptors (ALK-2, ALK-5, and TßRII), and two putative TGF-ß responses (elevations in _v and ß₃ integrin mRNAs) in balloon catheter–injured rat carotid arteries and their dependency on tyrosine kinase activity. Using a standardized reverse transcriptase–polymerase chain reaction assay optimized to estimate mRNA levels, we observed distinct patterns of mRNA regulation for TGF-ß₁, -ß₂, and -ß₃ during the 48 hours immediately after injury, which were localized to the vessel's media. TGF-ß₁ mRNA increased 10-fold during this time while TGF-ß₃ mRNA also increased almost 2-fold. There were also increases in mRNAs encoding the TGF-ß type I receptors ALK-5 and ALK-2, as well as the type II receptor (TßRII). Eight hours after the injury, mRNA levels for ALK-2 and ALK-5 were on average 2-fold higher; mRNA encoding the type II receptor increased approximately 3-fold by 24 hours. There were also associated increases in TGF-ß₁, TGF-ß₃, ALK-5, and TßRII immunoreactive peptide levels. Peak increases in mRNAs for integrins _v and ß₃ averaged approximately 2-fold and 2.5-fold, respectively. Perivascular administration of the tyrosine kinase inhibitor genistein at the time of vessel injury markedly (>85%) inhibited elevations in mRNAs encoding TGF-ß₁, TGF-ß₃, TßRII, and the two integrins _v and ß₃, while application of its inactive chemically similar homologue daidzein did not prevent the injury-induced elevations in mRNA levels. Since the increases in integrins _v and ß₃ mRNA could be theoretically attributed to TGF-ß actions despite being dependent on tyrosine kinase activity, we examined whether the observed elevations in integrins _v and ß₃ were due to TGF-ß₁ secretion, using cultured rat carotid artery smooth muscle cells. TGF-ß₁ neutralizing antibodies specifically inhibited elevations in integrins _v and ß₃ mRNAs due to platelet-derived growth factor-BB and fibroblast growth factor-2. We conclude that multiple components of the TGF-ß system in vessels are activated following injury and influence expression of integrin receptors important for smooth muscle cell migration. Activation of the TGF-ß system appears to be highly dependent on tyrosine kinases.

Key Words: transforming growth factor-ß • receptors • integrins _v and ß₃ • protein tyrosine kinase inhibitors • balloon catheter injury

	Introduction

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Extensive remodeling of vessels is known to occur following mechanical injury such as that induced with an inflated balloon catheter. Depending on the vessel type, remodeling frequently involves the development of a functionally significant neointimal fibrocellular lesion, which in humans may limit the long-term success of balloon angioplasty in revascularizing tissues.¹ In the rat carotid artery injured with an inflated balloon catheter, neointima development and its ultimate size is in part dependent on early events in the injured media of the vessel associated with SMC proliferation and migration.² The growth factors initiating these early effects in the injured media are yet to be fully defined, although PDGF, FGF-2, and other receptor tyrosine kinase growth factors have been implicated.^{3 4 5 6 7 8 9 10} There is also evidence to suggest that growth factors activating serine-threonine kinase mechanisms such as TGF-ß are also likely to be important.^{11 12 13 14 15 16}

TGF-ß₁ has been reported to be elevated early after balloon catheter injury of the rat carotid artery,¹² but the mechanism by which TGF-ß₁ is elevated following vessel injury is not understood, despite its potential importance in SMC proliferation,¹⁷ migration,¹⁸ and neointima formation.^{13 14 15} Currently there is also no information regarding involvement of other TGF-ß isoforms or their receptor types in the healing processes associated with mechanically injured vessels. In earlier studies, neither TGF-ß₂ nor TGF-ß₃ mRNAs could be detected in either injured or uninjured rat carotid arteries.¹² Here we report on our experiments designed to determine which TGF-ß isoforms in addition to TGF-ß₁ are involved in the early responses to vessel injury, the receptor types expressed, and the extent to which they are regulated by tyrosine kinase–dependent mechanisms. We demonstrate a highly coordinated temporal expression of TGF-ß₁, TGF-ß₃, and their receptors early after injury and provide evidence that their induction after injury is dependent on tyrosine kinases. The results of our study also indicate that early elevations in integrins _v and ß₃ mRNAs, proteins important for cell migration,¹⁹ are most likely a consequence of activation of these components of the TGF-ß system.

	Methods

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Animals, Study Design, and Drug Administration
Male Sprague-Dawley rats weighing 400 to 500 g were obtained from a colony maintained at the Baker Medical Research Institute (BMRI), Melbourne, Australia. The left common carotid artery of rats was subjected to balloon catheter injury by surgical procedures approved by the Baker Medical Research Institute and Alfred Hospital Animal Experimentation Committee. Twenty-two of the rats were used to examine the time course of changes in mRNA levels encoding TGF-ß isoforms, their receptors, and _v and ß₃ integrins over a 48-hour period following balloon catheter injury and results were compared with those from uninjured vessels. An assessment of the contribution of adventitial mRNAs to the observed changes in vessel mRNAs was carried out using six rats 24 hours after injury. To determine whether the changes in mRNA levels of the growth factors and their receptors also influenced the levels of their respective peptides, immunohistochemistry using antibodies to specific peptide fragments of the growth factors/receptors was carried out on frozen sections of four carotid arteries 48 hours after balloon catheter injury and compared with results from uninjured arteries.

To assess the contribution of tyrosine kinases to the changes in TGF-ß mRNAs, their receptors, and responses, we applied 200 nmol of either genistein, a known tyrosine kinase inhibitor, or its inactive chemically related homologue daidzein (RBI) in saline containing 40% gel F-127 (Pluronic gel, BASF) to the adventitial side of the vessel immediately after balloon catheter injury (see discussion following). This solution forms a gel at 37°C and has previously been shown to allow penetration of drugs into the surrounding tissues for up to 24 hours.²⁰ The concentration of genistein chosen has previously been shown to specifically inhibit tyrosine kinase activity.²¹ Genistein and daidzein were dissolved in the F-127-containing solution at 4°C on the morning of surgery and kept on ice until just before their application around the balloon-injured carotid artery. Their effects on vessel levels of the different mRNAs (see above) were compared 24 hours after application and injury.

Operation and Tissue Collection
Injury to the rat carotid artery by using a balloon catheter was carried out as described previously.² Briefly, after anesthetizing the rats with a mixture of pentobarbital (30 mg/kg), methohexitone (40 mg/kg), and atropine sulfate (3 mg/kg), administered by IP injection, a midline neck incision was made and blunt dissection to the carotid bifurcation was performed. Through an arteriotomy in the external carotid, a 2F Fogarty arterial embolectomy catheter (Baxter) was passed to the aortic arch. The balloon was then inflated with 25 µL of saline and withdrawn with a rotating action to the bifurcation. This procedure was performed three times before the balloon catheter was removed and the external carotid artery ligated. In those animals receiving periadventitial genistein or daidzein, the vessel was dissected free of its surrounding connective tissue and 1 mL of saline containing Pluronic gel together with one or the other agent was placed around the vessel and allowed to gel. The incision was closed and the animals were allowed to recover in a humidified warmed chamber for 1 to 2 hours. Animals were killed at 8, 24, or 48 hours after the operations by administering pentobarbital (60 mg/kg IP). In addition, all animals received Evans blue dye (60 mg/kg IV), the resultant blue coloration confirming uniform removal of endothelium in the balloon catheter–damaged vessels.

Both left and right carotid arteries were rapidly dissected free of connective tissue and placed into cold saline. The right (uninjured) carotid artery was opened longitudinally and its endothelium removed using a moist cotton bud; removal was confirmed by using light microscopy. When required (see "Results"), the adventitia was stripped from the vessels with the aid of a dissecting microscope and snap-frozen in liquid nitrogen, then stored at -70°C until analyzed for the different mRNAs. Vessel segments reserved for immunohistochemistry were embedded in OCT ("Tissue Tek," Miles), frozen using isopentane (Unilab) in liquid nitrogen, and then stored at -70°C.

Cell Culture
Cultured vascular SMCs were obtained by enzyme digestion of six rat carotid arteries, injured 24 hours earlier with a balloon catheter. After removal of the adventitia with fine watchmaker's forceps under a dissecting microscope, the media was subjected to digestion with collagenase and elastase as previously described.¹⁷ Cells from the digest were harvested by centrifugation, resuspended in DMEM containing 10% fetal calf serum, and cultured in 30-mm tissue-culture plates in two independent cultures prepared from three vessels each. Cells exhibited a characteristic hill-and-valleys pattern typical of many vascular SMCs grown in cell culture.²² They were deprived of serum for 24 hours before being cultured for 24 hours in either DMEM or DMEM containing PDGF-BB (200 ng/mL), FGF-2 (50 ng/mL), or TGF-ß₁ (2 ng/mL), both with and without a TGF-ß₁ neutralizing antibody (2.5µg/mL, Becton Dickinson). The experiment was repeated on three separate occasions using cultured SMCs from both independent cultures.

Immunohistochemistry
Immunohistochemical detection of TGF-ß₁, TGF-ß₃, the type I TGF-ß receptor (ALK-5), and TßRII peptides was carried out using 4-µm frozen sections of vessels fixed in acetone (30 minutes at -20°C) and treated with 1% H₂O₂ for 10 minutes. Sections were washed in 0.1 mol/L PBS and after incubation in 10% horse serum for 30 minutes followed by further washings were incubated for 1 hour at room temperature in PBS containing a primary (anti-TGF-ß/receptor) antibody (1:1000) or control IgG. Sections were then washed in PBS and incubated with the appropriate biotinylated secondary antibody (1:200) for 1 hour. After further washes, staining was achieved using the avidin-biotin-peroxidase complex system (Vector Laboratories) and 3,3'-diaminobenzidine tetrahydrochloride as the chromogenic substrate; sections were then counterstained with hematoxylin. Sections were then examined visually, in a qualitative manner, for differences in coloration intensity between balloon-injured vessels and uninjured vessels by three independent investigators.

RT-PCR and Estimation of mRNAs
RNA Isolation
Total RNA was extracted from tissues using the method of Chomczynski and Sacchi²³ and resuspended in sterile water; any contaminating DNA was removed by incubating these RNA extracts with 2 U DNase (Stratagene), for 15 minutes at 37°C. Then 2 µL 2 mol/L sodium acetate followed by an equal volume of isopropanol was added and the precipitated RNA sedimented by centrifugation. The RNA pellet was washed by resuspension in 70% aqueous ethanol followed by centrifugation, and then dried at 37°C for 30 minutes. This purified RNA was dissolved in sterile water and quantitated by spectrophotometry at 260 nm.

RT-PCR
This reaction was optimized so that the PCR product reflected the levels of mRNA in the original tissues. The product amplification-RNA relationship was always kept in the log-linear phase (see "Results"). The number of cycles chosen to achieve this linearity are summarized in Table 1. The optimum number of cycles required for each target sequence was determined by amplifying reverse-transcribed mRNA of uninjured carotid arteries through a range of cycles and determining when the amounts of products plateaued; the number of cycles considered optimum occurred before the plateau of each PCR product-cycle number relationship (see Table 1). Different amounts of total RNA from a single sample were then amplified for the chosen cycle number to demonstrate that the amount of PCR product was proportional to the RNA. This procedure was performed for all mRNA species (see "Results"). The amount of PCR product for each target mRNA is expressed relative to the amount of PCR product for L7, a ribosomal protein that is encoded by a noninducible cell cycle–independent gene.²⁴ The identity of each PCR-amplified DNA fragment was confirmed by using specific restriction enzymes (see Table 1). Conditions for RT-PCR were as follows: Each RT incubation mixture contained 1 µL 25 mmol/L MgCl₂, 0.5 µL 10x PCR buffer (containing 500 mmol/L KCl, 100 mmol/L Tris-HCl, pH 8.3), 2 µL dNTP mix (containing 2.5 mmol/L of each of dATP, dCTP, dGTP, and dTTP), 0.25 µL 50 µmol/L random hexamers, 0.25 µL 20 U/µL RNase inhibitor, 0.25 µL 50 U/µL MuLV reverse transcriptase, and 0.75 µL of 0.27µg/µL total RNA (200 ng). After equilibration for 10 minutes at room temperature, reverse transcription was performed using a Hybaid Omnigene thermal cycler at 42°C for 15 minutes, followed by 5 minutes at 95°C. Samples were then placed on ice and PCR was performed with each reaction mixture containing 5 µL of RT product, 1 µL primers (containing 10 µmol/L sense and antisense primers), 0.5 µL 25 mmol/L MgCl₂, 2.0 µL 10x PCR buffer, 0.125 µL 5 U/µL Amplitaq DNA polymerase, and 0.125 µL 1µg/µL anti-Taq DNA polymerase antibody (MAb 8C1C Technogene), and 16.25 µL sterile distilled water. All components except for the antibody were from a GeneAmp RNA PCR core kit (Perkin-Elmer). Each PCR cycle consisted of the following stages: 94°C for 30 seconds, 60°C for 1 minute, and 72°C for 2 minutes, with a prolonged extension stage after the final cycle of 72°C for 8 minutes. PCR products were electrophoresed on 2% agarose gels at 120 mV (Progen), together with *X174 DNA digested with Hae III size markers (Promega). Gels were photographed under ultraviolet light with positive/negative film (Polaroid 665) and intensities on the negatives quantitated using laser densitometry (LKB 2222-010 Ultrascan XL, LKB).

View this table: [in this window] [in a new window]   Table 1. PCR Product Characteristics and the Number of PCR Cycles Chosen to Estimate the Different mRNAs in Vessel and Cell Extracts by the Standardized RT-PCR Procedure

Oligonucleotide Primer Pair Selection for RT-PCR
Oligonucleotide primer pairs for RT-PCR were either selected from the literature or designed using "Primer Detective" (TMJ Lowe, Clontech Labs), according to the following criteria: GC content 45% to 55%, melting point 76°C to 83.5°C, filtering hairpins and 3' homologies. They were for TGF-ß₁²⁵ sense: bp 784-803, antisense: bp 1205-1224 according to the published rat sequence²⁶ ; TGFß₂ sense: bp 604-633, antisense: bp 890-914 according to the published mouse sequence²⁷ ; TGF-ß₃ sense: bp 908-937, antisense: bp 1241-1266 according to the published rat sequence²⁸ ; ALK-5 sense: bp 79-102, antisense: bp 490-513 according to the published rat sequence²⁹ ; ALK-2 sense: bp 128-152, antisense: bp 435-459 according to the published rat sequence³⁰ ; TßRII sense: bp 252-272, antisense: bp 931-949 according to the published rat sequence,³¹ the ribosomal protein L7, sense: bp 143-162, antisense: bp 405-428 according to the published rat sequence³² ; integrin subunit _v sense: bp 42-66, antisense: bp 510-531 according to the published rat sequence³³ ; and integrin subunit ß₃ sense: 168-193, antisense: bp 422-447 according to the published rat sequence.³⁴

Materials
A TGF-ß₁ polyclonal purified chicken IgG raised against human TGF-ß₁ and purchased from Becton Dickinson was used for immunohistochemistry. It is also capable of neutralizing TGF-ß₁ (1 µg neutralizes 0.72 ng TGF-ß₁) and was used in the cell-culture experiments. A specific TGF-ß₃ rabbit polyclonal IgG antibody was purchased from Santa Cruz Biotechnologies, as were specific ALK-5 and TßRII rabbit polyclonal IgG antibodies. The biotinylated goat anti-turkey/chicken IgG was from Zymed, and biotinylated goat anti-rabbit IgG was from Vector Laboratories. PDGF-BB, FGF-2, and TGF-ß₁ were obtained from Sigma, Bachem, and Celltrix, respectively.

Statistical Methods
The effects of vessel injury or the different treatments were assessed using analysis of variance (nonparametric) and, where differences were detected between the groups, the Mann-Whitney rank sum test (balloon-injured vessels versus uninjured vessels) was used to detect differences between the uninjured vessels and the individual time point groups or paired t test (balloon-injured vessels treated with periadventitial daidzein versus uninjured vessels and balloon-injured vessels treated with genistein versus uninjured vessels), depending on whether the results were normally distributed, as indicated by the Kolmogorov-Smirnov test (Sigmastat, Jandel Scientific). Differences were considered statistically significant if P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Estimation of Vessel-Derived TGF-ßs, Their Receptors, and Integrin mRNAs
Before undertaking any measurements of mRNA levels using the standardized RT-PCR procedure, we determined the characteristics of the PCR products and the overall accuracy of our procedure. The conditions and oligonucleotide PCR primers used (see "Methods") generated single DNA fragments of the predicted sizes and characteristics with respect to restriction enzyme digestion (Table 1). In addition, the relationships between the number of cycles performed with each set of primers and the log of amount of PCR product generated, measured as product intensity by laser densitometry, were curvilinear in nature (not shown), but their linear ranges extended for up to 36 cycles for some products. The number of cycles chosen in this linear range for each mRNA species ranged from 23 for L7 to 36 cycles for TGF-ß₁, TGF-ß₂, and TßRII mRNAs (Table 1). Estimating the nine different mRNAs using the conditions summarized in Table 1 resulted in linear relationships between the amounts (100 to 400 ng) of RNA and the PCR product (Fig 1 and Table 2). When 200 ng of RNA was used to estimate mRNA, the coefficient of variation for the procedure averaged 10% over the nine different reactions. This amount of RNA was used for all estimations of mRNA in the different tissues.

View larger version (20K): [in this window] [in a new window]   Figure 1. Representative agarose gels and graphs showing the relationships between total RNA and amplified PCR products when oligonucleotide primers for TGF-ß1 and ALK-5 were used in the standardized RT-PCR procedure (see "Methods" for details).

View this table: [in this window] [in a new window]   Table 2. Regression Equations of the Relationships Between the Amounts of PCR Products Generated, Measured as Optical Density [P(DNA)], and the Amounts (ng) of RNA Used (RNA) in the Standardized RT-PCR Procedure

Arterial Injury and Expression of TGF-ß Isoforms
Since expression of multiple TGF-ß isoforms can theoretically be affected by injury to the vessel wall,^{34 35} we initially compared mRNA levels encoding TGF-ß₁, -ß₂, and -ß₃ in uninjured and injured vessels. Using RT-PCR, we detected mRNAs encoding all three isoforms in uninjured vessels. Their amplified cDNA fragments possessed the sizes and restriction enzyme digestion patterns expected from their cDNA sequences (Table 1). The pattern of mRNA expression for the three isoforms changed markedly after balloon catheter injury (Fig 2). Twenty-four hours after the injury, mRNA encoding TGF-ß₁ was significantly elevated, and by 48 hours, this elevation was approximately 10-fold (P<.05). In contrast, mRNA encoding TGF-ß₂ was not significantly affected early after injury but after 24 hours was reduced by approximately 90% (P<.05; Fig 2). Small elevations in TGF-ß₃ mRNA levels became apparent up to 24 hours after injury (approximately 70%, P<.05); 48 hours after injury, this elevation was still apparent although no longer statistically significant (P>.05; Fig 2).

View larger version (12K): [in this window] [in a new window]   Figure 2. Time course of changes in the expression of mRNAs encoding TGF-ß1, -ß2, and -ß3 relative to L7 in balloon catheter–injured arteries. Total RNA was extracted from uninjured and injured carotid arteries 8, 24, and 48 hours after performing the balloon catheter injury, and relative mRNA levels were estimated as described in "Methods." C indicates uninjured (right) carotid arteries and B8h, B24h, and B48h, injured (left) carotid arteries 8, 24, and 48 hours after inflicting the injury with an inflated balloon catheter. Results are the mean±SEM of 22, 7, 8, and 7 vessels from the different groups of animals, respectively. *P<.05 vs C (uninjured vessels; Mann-Whitney rank sum test after ANOVA by ranks).

To determine whether the increases in mRNAs encoding TGF-ß₁ and TGF-ß₃ were associated with increased levels of TGF-ß peptides within the injured vessel media, we compared by immunohistochemistry TGF-ß₁ and TGF-ß₃ peptide levels in vessels 48 hours after injury with levels in uninjured vessels. At this time, the vast majority of SMCs throughout the injured media stained positive for TGF-ß₁ peptides; in the uninjured vessel only occasional SMCs possessed TGF-ß₁ immunoreactive peptides, and staining in the media was relatively weak (Fig 3), consistent with the differences in TGF-ß₁ mRNA levels between injured and uninjured arteries (Figs 2 and 3). At this time, there were also more TGF-ß₃ immunopositive SMCs in the injured carotid artery, and staining was greater than in the uninjured carotid (Fig 3). No TGF-ß₁ or TGF-ß₃ immunopositive cells were apparent in the adventitia of the injured vessels.

View larger version (69K): [in this window] [in a new window]   Figure 3. Immunohistochemical localization of TGF-ß1 and -ß3 peptides in uninjured and injured carotid arteries 48 hours after balloon injury. Top, TGF-ß1/-ß3 immunoreactive peptides (indicated by the brown coloration) in uninjured carotid arteries (C); bottom, TGF-ß1/-ß3 immunoreactivity in the vessels 48 hours after balloon catheter injury (I). L indicates lumen. The examples shown reflect qualitatively the changes observed in vessels from each of the four animals in each group.

Arterial Injury and Expression of mRNAs Encoding TGF-ß Receptors
Because the effects of TGF-ß₁ and TGF-ß₃ following vessel injury are likely to be dependent on SMCs expressing specific receptor types, we examined the time course of changes in mRNA levels encoding the TßRII and two TGF-ß type I receptors, ALK-5 and ALK-2.³⁶ In uninjured vessels, mRNAs encoding both ALK-5 and ALK-2 were readily detectable. However, PCR product from mRNA encoding TßRII was present only in very low amounts and in some vessels (11 of the 22 examined) was undetectable (Fig 4). In the injured vessels, mRNA levels encoding all three receptors increased significantly (Fig 4); mRNA levels encoding ALK-5 increased twofold 8 hours after the injury (P<.05), and these levels were maintained at 48 hours. The elevations in mRNA encoding ALK-2 were also time dependent, with peak increases (twofold, P<.05) apparent 8 hours after injury; subsequently, mRNA levels declined and by 48 hours the increase was no longer statistically significant (P>.05; Fig 4); mRNA levels encoding the TßRII receptor increased more slowly, and 24 hours after injury the increase was threefold (P<.05) compared with the uninjured arteries. These levels were maintained at 48 hours (Fig 4).

View larger version (13K): [in this window] [in a new window]   Figure 4. Time course of changes in the expression of mRNAs encoding the TGF-ß receptors ALK-5, ALK-2, and TßRII relative to L7. Total RNA was extracted from uninjured and injured carotid arteries 8, 24, and 48 hours after performing the balloon catheter injury, and relative mRNA levels were estimated as described in "Methods." C indicates the uninjured (right) carotid artery, and B8h, B24h, and B48h, the injured (left) carotid artery 8, 24, and 48 hours after balloon catheter injury. Results are the mean±SEM of 22, 7, 8, and 7 vessels from the different groups of animals, respectively. *P<.05 vs C (uninjured vessels; Mann-Whitney rank sum test after ANOVA by ranks).

These increases in ALK-5 and TßRII mRNA levels in the injured vessels were also associated with increased levels of receptor peptides determined immunohistochemically. Forty-eight hours after injury, a greater number of SMCs possessed ALK-5 immunoreactive peptides compared with the uninjured vessels, and staining was also more intense in the injured media (Fig 5). A somewhat similar pattern of TßRII immunoreactive peptide expression was also seen in the injured vessel media at this time (Fig 5). No cells containing immunoreactive ALK-5 or TßRII peptides were apparent in the adventitia of the injured vessels.

View larger version (68K): [in this window] [in a new window]   Figure 5. Immunohistochemical localization of TGF-ß receptor peptides ALK-5 and TßRII in uninjured and injured carotid arteries after balloon injury. Top, ALK-5 and TßRII immunoreactive peptides (brown coloration) in uninjured vessels (C); bottom, ALK-5 and TßRII peptides in the vessels 48 hours after balloon catheter injury (I). L indicates lumen. The examples shown reflect qualitatively the changes observed in vessels from each of the four animals in each group.

Vessel Integrin mRNA Expression Increases Following Injury
Since TGF-ß₁ and activators of receptor tyrosine kinases, such as PDGF-BB and FGF-2 have the potential to influence SMC migration,^{9 18} we also examined the temporal expression of mRNAs encoding two integrin receptors implicated in cell migration, _v and ß₃.¹⁹ mRNA encoding integrin _v increased approximately 2-fold after vessel injury (P<.05; Fig 6), and peak increases occurred after 24 to 48 hours. Peak increases in integrin ß₃ mRNA occurred earlier, nearer 8 hours (Fig 6); at this time the increase was approximately 2.5-fold compared with levels in uninjured vessels (P<.05) and by 48 hours had returned to levels present in uninjured vessels (P>.05; Fig 6).

View larger version (15K): [in this window] [in a new window]   Figure 6. Time course of changes in expression of mRNAs encoding integrins v and ß3 relative to L7 in injured arteries. Total RNA was extracted from uninjured and injured carotid arteries 8, 24, and 48 hours after inflicting the balloon catheter injury, and mRNAs were estimated as described in "Methods." C indicates uninjured (right) carotid arteries and B8h, B24h, and B48h, injured (left) carotid arteries 8, 24, and 48 hours after the balloon catheter injury. Results are the mean±SEM of 22, 7, 8, and 7 vessels from the different groups, respectively. *P<.05 vs C (uninjured vessels; Mann-Whitney rank sum test after ANOVA by ranks).

Adventitial Genistein Attenuates Increases in mRNAs Encoding TGF-ßs and Receptors
We have previously suggested that activation of receptor tyrosine kinases most likely accounts for elevations in TGF-ß₁ mRNA levels.¹⁷ Since FGF-2 and PDGF are known to exert their effects on vascular smooth muscle early after injury, we examined how an inhibitor of tyrosine kinases, genistein, affected mRNA levels encoding the TGF-ß isoforms and their receptors in the injured vessel wall. Periadventitial administration of genistein and its inactive homologue daidzein differentially affected mRNAs encoding the TGF-ß isoforms and their receptors (Fig 7). Daidzein did not affect the significant elevation in TGF-ß₁ mRNA induced by the injury (P>.05), while in the genistein-treated injured vessels TGF-ß₁ mRNA levels were similar to those in uninjured carotid arteries (P>.05; Table 3). Similarly, genistein prevented the 24-hour increase in TGF-ß₃ mRNA in the injured vessels while daidzein was without effect (Fig 7, Table 3).

View larger version (51K): [in this window] [in a new window]   Figure 7. Typical agarose gels demonstrating the effects of balloon catheter injury plus genistein or daidzein treatment on mRNAs encoding the TGF-ß isoforms and receptors. Top, TGF-ß1, -ß2, -ß3, and L7 RT-PCR transcripts obtained from RNA extracts of uninjured carotid arteries or vessels injured 24 hours earlier (see "Results"). C indicates uninjured arteries and D and G, injured vessels treated with periadventitial daidzein or genistein, respectively. Bottom, ALK-5, ALK-2, TßRII, and L7 RT-PCR transcripts obtained from RNA extracts of carotid arteries described above. C indicates uninjured arteries and D and G, injured vessels treated with daidzein and genistein, respectively. All results are typical of five animals in each group.

View this table: [in this window] [in a new window]   Table 3. Effects of Periadventitial Daidzein and Genistein on mRNA Levels in Vessels 24 Hours After Injury

The increases in mRNAs encoding TGF-ß receptors in the injured vessels were also differentially affected by genistein and daidzein. Daidzein had no apparent effects on the elevations in ALK-5, ALK-2, and TßRII mRNA levels 24 hours after injury. In contrast, genistein application to the injured vessels greatly attenuated these increases in ALK-5, ALK-2, and TßRII (Fig 7 and Table 3).

Because balloon injury with or without periadventitial administration of genistein and daidzein involved surgical manipulation of the adventitia (see "Methods"), we also examined the extent to which balloon injury and adventitial manipulation induce changes in adventitial mRNAs encoding the TGF-ß isoforms and their receptors. Using RNA isolated from pooled adventitia taken from balloon-injured (24 hours) and uninjured vessels, the same RT-PCR procedures, and identical amounts of RNA (200 ng), we could detect only trace amounts of mRNA encoding TGF-ß₁, ALK-2, and ALK-5, while TGF-ß₂, TGF-ß₃, and TßRII were not detectable in extracts from surgically injured adventitial specimens. There was also no apparent difference in expression between uninjured and surgically injured adventitia (not shown), consistent with the changes in mRNAs' being restricted to the media of the vessels.

Relationships Between Tyrosine Kinases, TGF-ß₁, and Integrin mRNA Expression
Since integrin expression in SMCs has been reported to be elevated by TGF-ß₁ and PDGF-BB,²¹ we also investigated the extent to which tyrosine kinases and TGF-ß₁ might be responsible for the injury-induced elevations in _v and ß₃ integrin mRNAs.

In vivo integrin _v mRNA levels observed in injured vessels treated with periadventitial daidzein were significantly elevated compared with those in uninjured vessels (P<.05), while treatment with genistein attenuated the 24-hour postinjury increases and mRNA levels were not significantly different from those in control vessels (P>.05; Fig 8, Table 3); in these vessels, daidzein did not affect the elevation in mRNA due to injury. Similarly, daidzein was associated with significant elevations in integrin ß₃ mRNA in the injured vessels (P<.05), and levels were comparable to those due to injury alone, while genistein attenuated these increases to levels seen in uninjured vessels. These effects were attributed to medial SMCs, since neither _v or ß₃ mRNAs could be detected in RNA from surgically injured adventitia (not shown).

View larger version (22K): [in this window] [in a new window]   Figure 8. Typical agarose gels demonstrating the effects of genistein and daidzein on relative v and ß3 mRNA levels in injured carotid arteries. Vessels from the different groups were collected and their RNA was subjected to the standardized RT-PCR procedure as described in Fig 7 and "Methods." C indicates uninjured arteries and D and G, injured arteries treated with daidzein and genistein, respectively. Results are typical of five animals in each group.

Cultured SMCs were used to assess the extent to which active, secreted TGF-ß₁ regulated by tyrosine kinases might be responsible for the elevations in _v and ß₃ mRNAs in the injured vessels. Incubation of SMCs for 24 hours with PDGF-BB, FGF-2, or TGF-ß₁ elevated, in all instances, integrin _v mRNA levels, by approximately 300%, 90%, and 800%, respectively. Simultaneous incubation with the TGF-ß₁ neutralizing antibody completely abrogated these increases in mRNA (Fig 9); it did not, however, affect other non-TGF-ß–mediated responses induced by either PDGF-BB or FGF-2, such as increases in mRNAs encoding the disintegrin/metalloprotease MDC9 or the variably spliced hyaluronic acid receptor CD44v6 (not shown). Similarly, PDGF-BB and TGF-ß₁ increased integrin ß₃ mRNA during the 24-hour incubation period; the TGF-ß₁ neutralizing antibody completely prevented the TGF-ß₁ elevation in integrin ß₃ mRNA and the PDGF-BB–induced rise was attenuated by approximately 60% (Fig 9); mRNA encoding the ribosomal protein L7 was unaffected by the growth factors or neutralizing antibodies.

View larger version (51K): [in this window] [in a new window]   Figure 9. Typical agarose gels demonstrating the ability of a TGF-ß1 neutralizing antibody to modulate the expression of integrins v and ß3 mRNA when cultured carotid SMCs are exposed to growth factors. C indicates RT-PCR transcripts obtained from SMCs cultured in DMEM; P, SMCs cultured for 24 hours in DMEM/PDGF (200 ng/mL); F, SMCs cultured for 24 hours in DMEM/FGF-2 (50 ng/mL), and T, SMCs cultured for 24 hours in DMEM/TGF-ß1 (2 ng/mL). PA, FA, and TA represent RT-PCR transcripts obtained from SMCs treated as in P, F, and T but also containing the TGF-ß1-specific neutralizing antibody. L7 represents RT-PCR transcripts encoding part of the ribosomal protein L7. The results are typical of three experiments in two independent cultures.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Vessel healing is a highly ordered and well-coordinated process controlled by growth factors regulating the spatial and temporal expression of peptides directly participating in healing and vessel remodeling. TGF-ß₁ is thought to be one of the growth factors involved in these processes.^{13 15} In this study we have demonstrated that two TGF-ß isoforms are most likely involved, since TGF-ß₁ and -ß₃, as well as their type I and II receptors, are upregulated early after vessel injury. Their upregulation is dependent on protein tyrosine kinases and appears to involve the secretion by SMCs of at least one biologically active TGF-ß isoform (-ß₁), which in turn contributes to the increases in _v and ß₃ integrin gene activities, systems essential for early SMC migration.¹⁹

Previous studies on balloon catheter–injured carotid arteries described increases in TGF-ß₁ mRNA and peptide levels, but attempts to detect TGF-ß₂ and TGF-ß₃ were unsuccessful.¹² Using our standardized RT-PCR procedure optimized for relative measurements of vessel mRNA levels, we demonstrate differential regulation of these isoforms. During the 48-hour interval immediately after injury, both TGF-ß₁ and TGF-ß₃ mRNA and peptide levels increased in the media of the injured vessels, while mRNA encoding TGF-ß₂ fell to virtually undetectable levels, suggesting specific roles for both TGF-ß₁ and -ß₃ in the early phases of vessel healing. However, their precise functions at this time are somewhat unclear. In vitro the two isoforms exert apparently similar effects on SMCs,³⁷ but in vivo there are indications that TGF-ß isoforms may have some unique functions. For example, in healing skin, TGF-ß₃ has been shown to inhibit the fibrogenic properties of TGF-ß₁.³⁸ Also, TGF-ß₂ and TGF-ß₃, on the basis of their differential temporal expression, have been suggested to play distinct roles in growth and development during embryogenesis,³⁹ but following dermal injury, all three isoforms are simultaneously upregulated.⁴⁰ Clearly, further experimentation is required to determine the significance of these patterns of differential regulation in vivo. In the injured vessel, the increases in TGF-ß₁ and -ß₃ isoforms occur when the SMCs are either preparing for or in the process of migrating and/or proliferating.²

The effects of the TGF-ß isoforms are dependent on both the nature of their receptor types and the manner in which they interact with these receptors.^{36 41 42 43 44 45 46 47} In SMCs, as in other cell types, TGF-ß₁, -ß₂, and -ß₃ interact with the type I and II receptors to induce their effects.⁴⁸ TGF-ß is thought to initially bind to the type II receptor, and this binding is followed by interaction with and phosphorylation of the type I receptor, resulting in signal generation through serine-threonine kinase–dependent mechanisms.⁴⁸ In our studies, the levels of mRNA encoding both receptor types and their immunoreactive peptide levels increased rapidly after injury, in the media of the injured carotid artery. Since TGF-ß receptors are similarly increased following dermal wound healing,⁴⁰ it is possible that this pattern of increases in TGF-ß receptors is an essential component of any TGF-ß–initiated response to injury.

At present, very little is known about the mechanisms responsible for this coordinate upregulation of TGF-ß₁ and -ß₃ isoforms and their receptors in the injured vessel. In vitro increases in TGF-ß₁ gene transcription can be initiated through a variety of transcription factors, including the promoter-specific (SP-1) family of transcription factors,⁴⁹ shear stress–dependent factors,⁵⁰ or activating protein-1 (AP-1) complexes, while cAMP and AP-2 complexes have been reported to increase TGF-ß₃ gene transcription.^{51 52} Our studies with the tyrosine kinase inhibitor genistein indicate that after vessel injury, the increases in TGF-ß₁ and -ß₃ mRNA levels are primarily dependent on protein tyrosine kinase activity. This inhibitor does not affect other kinases, such as protein kinase A, protein kinase C, or the calmodulin-dependent kinases,²¹ and its closely related homologue daidzein, which does not affect protein tyrosine kinases,²⁴ did not affect the increases in mRNA levels in the injured vessels. It is likely that multiple protein tyrosine kinases contribute to the elevations in TGF-ß₁ and -ß₃ mRNA levels, since a number of receptor tyrosine kinases are known to be activated at this time. Together, these observations indicate an important primary role for tyrosine kinases in the regulation of TGF-ß₁ and -ß₃ activities in injured vessels. We have also demonstrated a dependency of the type I and II receptor mRNA increases on protein tyrosine kinase activities. However, it is unclear whether the increases in the receptors in the injured vessels are due to direct or indirect actions of tyrosine kinases. Recently, TGF-ß₁ has been reported to be a potent inducer of type I and II receptors, increasing their expression in U-937 cells between 9-fold and 14-fold.⁵³

It is well known that TGF-ßs are secreted as biologically inactive forms.⁵⁴ However, there are also reports which indicate that some cells, including cultured human SMCs, can secrete biologically active forms of TGF-ß, dependent in part on their ability to simultaneously produce the latent TGF-ß binding protein.^{55 56} Our studies indicate that cultured carotid SMCs in the presence of PDGF-BB or FGF-2 also produce biologically active TGF-ß₁, which in turn initiates additional cellular responses. SMCs isolated and cultured from injured arteries responded to PDGF-BB, FGF-2, and TGF-ß₁ by increasing their expression of _v and ß₃ integrin mRNA. These responses were greatly attenuated or abolished by the TGF-ß₁-specific neutralizing antibody. Together, these observations suggest that the early elevations in the integrin mRNAs in the injured vessels are dependent at least in part on the secretion of active TGF-ß₁. Since these integrin receptors are frequently involved in SMC migration,⁵⁷ it would appear that one of the functions of TGF-ß₁ early after injury is to facilitate early SMC migratory events in the injured vessels.

In these studies we used a standardized RT-PCR procedure to estimate the relative amounts of the different mRNAs rather than the more complex but potentially more accurate competitive RT-PCR assay procedure.^{58 59} After optimizing and carefully standardizing the conditions to obtain linearity between the amount of PCR product and the amount of RNA, using established methods,⁵⁸ we found the procedure to be easily capable of detecting small (50%) changes in relative mRNA levels. However, despite the fact that RT-PCR procedures are highly sensitive in detecting and estimating mRNA levels, all can suffer from the limitation that they provide little or no information about the possible presence of alternately spliced mRNA species that can encode variant protein products of the same gene. In this study we did not investigate whether injury affected the characteristics of the mRNAs encoding the various TGF-ß isoforms or their receptors. In addition, we were unable to detect significant levels of mRNA or protein of the TGF-ß isoforms or their receptors in the adventitia of the injured vessels. Potential interpretations of these data are that TGF-ß does not participate in healing an injured adventitia or that the adventitia does not play a major role in the response to injury in this animal model.

Taken together, our findings suggest that early after balloon catheter injury of the carotid artery, there is a rapid, tyrosine kinase–dependent upregulation of the TGF-ß system in medial SMCs resulting in large increases in TGF-ß₁, TGF-ß₃, and their type I and II receptors. Active TGF-ß₁ produced by the SMCs is most likely responsible for the early induction of integrins _v and ß₃ mRNA, key surface membrane proteins that participate in SMC migration. Recently, in humans, a monoclonal antibody to integrin ß₃ administered immediately after angioplasty has been shown to attenuate the frequency of restenosis, implicating integrin ß₃ as one early response of diseased atherosclerotic vessels to injury.⁶⁰ Whether an activated TGF-ß system similar to that seen in the injured rat carotid artery is responsible for integrin ß₃ expression in the diseased human vessels after angioplasty remains to be determined.

	Selected Abbreviations and Acronyms

 

DMEM = Dulbecco's modified Eagle's medium FGF = fibroblast growth factor PDGF = platelet-derived growth factor RT-PCR = reverse transcriptase–polymerase chain reaction SMC = smooth muscle cell TßRII = TGF-ß type II receptor TGF = transforming growth factor

	Acknowledgments

 
Dr Michael Ward is a recipient of a National Health and Medical Research Council postgraduate medical research scholarship. These studies have in part been funded by the NH&MRC and the National Heart Foundation of Australia.

Received March 19, 1997; accepted May 22, 1997.

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