© 1996 American Heart Association, Inc.
Articles |
Transforming Growth Factor-ß1 Expression and Myofibroblast Formation During Arterial Repair
the Department of Medicine (Cardiology), Thomas Jefferson University, Philadelphia, Pa.
Correspondence to Yi Shi, MD, PhD, Thomas Jefferson University, Cardiovascular Research Center, Division of Cardiology, Suite 410N, 1025 Walnut St, Philadelphia, PA 19107.
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Abstract |
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Transforming growth factor-ß1 (TGF-ß1) plays a central role in tissue repair owing to its modulating effects on cell growth and the synthesis of extracellular matrix. We have previously shown that adventitial fibroblasts differentiate to myofibroblasts after endoluminal injury, thereby contributing to arterial remodeling. Since TGF-ß1 exerts several biologic actions attributed to myofibroblasts, we examined its role in myofibroblast formation in a porcine model of balloon overstretch coronary artery injury. TGF-ß1 transcripts were induced in numerous adventitial cells 2 days after injury (47±10%, P<.001 versus control). These cells displayed no smooth muscle (SM) markers, ie,
-SM actin or desmin, which suggested their fibroblastic origin. This was further corroborated by the rare presence of macrophages in the injured adventitia (3±1%). At 7 to 8 days, most TGF-ß1–expressing cells demonstrated -SM actin immunoreactivity. Their myofibroblast phenotype was confirmed by electron microscopy, which revealed microfilaments (stress fibers) and a well-developed rough endoplasmic reticulum. The distribution of TGF-ß1 transcripts by in situ hybridization was paralleled by the immunolocalization of intracellular and extracellular TGF-ß1 epitopes. At later times (>14 days after injury), the decrease in TGF-ß1 coincided with the disappearance of adventitial myofibroblasts, whereas the neointima exhibited longer TGF-ß1 expression. In conclusion temporal and spatial relationships between TGF-ß1 and myofibroblast formation suggest an important role for autocrine TGF-ß1 in the phenotypic modulation of vascular fibroblasts. Induction of TGF-ß1 expression may provide a differentiation signal for adventitial fibroblasts to become myofibroblasts, which affect arterial remodeling via their mechanical and synthetic properties.
Key Words: transforming growth factor-ß1 • myofibroblasts • adventitia • remodeling • restenosis
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Introduction |
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TGF-ß1 is involved in tissue repair by modulating the growth of mesenchymal cells, augmenting the synthesis of several ECM proteins, and facilitating migration of fibroblasts and macrophages.1 2 3 4 5 Increased expression of TGF-ß1 has been demonstrated in human restenotic lesions.6 In experimental settings arterial transfer of a TGF-ß1 construct or systemic administration of TGF-ß1 resulted in the formation of a neointima rich in ECM proteins.7 8 Conversely, anti–TGF-ß1 neutralizing antibodies reduced neointimal formation, further pointing to the important role of TGF-ß1 in vascular repair.9 Colocalization of TGF-ß1 and
-SM actin, followed by deposition of ECM proteins in the injured vessel, has been interpreted as evidence that SMCs that migrate from the media to the neointima are primarily involved in the repair process.
Myofibroblasts represent highly specialized mesenchymal cells that play a central role in tissue repair.10 It is believed that myofibroblasts are derived from fibroblasts and are linked to the process of wound healing.11 12 Their formation is marked by the development of bundles of microfilaments (stress fibers) and abundant connections with the surrounding ECM.13 These characteristics are consistent with the primary role of newly formed myofibroblasts to close an open wound by means of ECM protein synthesis (eg, collagens) and contraction. Subsequent studies have confirmed the presence of myofibroblasts in a wide range of other pathological conditions that are associated with fibrogenesis and organ remodeling.14 15 16 17 Although myofibroblasts from such diverse sources are heterogeneous, a common feature of their phenotype is expression of -SM actin. Our recent findings have indicated that adventitial fibroblasts differentiate to myofibroblasts (by acquiring -SM actin) in a porcine model of coronary artery injury.18 This process is associated with the deposition of collagen, resulting in adventitial remodeling. Furthermore, adventitial myofibroblasts, rather than medial SMCs, migrate toward the vessel lumen and constitute a major cellular component of the neointima following severe balloon-induced injury associated with medial dissection.19 Interestingly, other studies have demonstrated unfavorable geometric remodeling of injured vessels,20 21 22 not unlike tissue contraction in fibrocontractive disorders that are mediated by myofibroblasts.
Although exogenous TGF-ß1 has been shown to induce myofibroblast phenotype and matrix retraction in cultured nonvascular fibroblasts,23 24 25 26 the mechanisms underlying the formation of vascular myofibroblasts in vivo have not been elucidated. In this study we demonstrate the induction of TGF-ß1 expression in adventitial fibroblasts in a porcine model of coronary artery injury. The spatial and temporal relationships between TGF-ß1 expression and the formation of vascular myofibroblasts are illustrated. These findings suggest that TGF-ß1 provides the "signal" for fibroblasts to acquire a differentiated phenotype that imparts synthetic and mechanical properties.
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Methods |
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Animal Model
Domestic crossbred pigs (Sus scrofa, n=26) weighing 23 to 31 kg were premedicated with aspirin (650 mg PO), atropine (1 mg IM), and nifedipine (10 mg sublingual). The animals were sedated with ketamine (20 mg/kg IM) and xylazine (4 mg/kg IM). After endotracheal intubation the pigs were ventilated with isoflurane (1.75%) and O2 to maintain anesthesia throughout the experiment. The right common carotid artery was surgically exposed and heparin (10 000 U) was administered intra-arterially. The coronary ostia were cannulated under fluoroscopic guidance with an 8F SAL 1 guiding catheter, and intracoronary nitroglycerin was administered (100 µg). The coronary arteries were injured with an oversized (4.0 mm) balloon inflated three times (6 to 10 atm) for 30 seconds, whereas noninstrumented coronary arteries served as controls. Postsurgical therapy included aspirin 325 mg PO and ampicillin 250 mg IM for the next 2 days. The animals were euthanatized with an intravenous overdose of pentobarbital sodium (1950 mg) and phenytoin sodium (250 mg) (Euthasol, Delmarva Laboratory) at times indicated in the text. All experiments conformed with the position of the American Heart Association on research animal use and were in accordance with institutional guidelines.
Tissue Preparation and Preservation
Porcine coronary arteries and surrounding tissue were carefully removed, rinsed with PBS, and then immersed in tissue fixative (HistoChoice, Amresco) within 5 minutes of extraction. The specimens were sectioned into 
3-mm blocks and fixed for at least 5 hours. The tissues were then processed in a Tissue-Tek vacuum infiltration processor (Miles), embedded in paraffin, and cut into 5-µm-thick sections. Representative sections from each block of injured coronary artery were stained with Verhoeff's stain for elastic tissue to determine the location and extent of medial injury.27 The arteries with severe injury, defined as disruption of the IEL and media, were selected for this study. The vessels with mild injury, defined as punctate breaks in the IEL and incomplete medial damage, were excluded from further analysis. For immunohistochemistry the sections were adhered to gelatin-coated glass slides. For in situ hybridization specimens were adhered to charged and precleaned ProbeOn Plus slides (Fisher Scientific). At least three sections from each artery were examined by in situ hybridization and immunohistochemistry. The n value in the "Results" section represents the number of vessels.
For electron microscopy the expected site of coronary artery injury was sectioned into 3-mm blocks separated by an 1-mm block. The former were used for histology and immunohistochemistry to confirm the degree of injury and the localization of myofibroblasts by -SM actin immunostaining, respectively. The latter were rapidly subdivided into smaller transmural specimens and fixed in 2% glutaraldehyde in 0.1 mol/L Soerensen's phosphate buffer (pH 7.2 to 7.4). Tissue fragments were then postfixed in 1% OsO4. After dehydration in a graded series of ethanol, the specimens were embedded in an infiltration medium (Spurr). Ultrathin (100 nm) sections were cut on a Reichert Ultracut S microtome and sequentially stained in uranyl acetate and lead citrate. Sections that contained the adventitia adjacent to medial injury were examined at 80 kV in an electron microscope (JEOL-100 CX II).
In Situ Hybridization
The sections were deparaffinized twice in xylene for 5 minutes each, rehydrated in a graded series of alcohol, and rinsed in PBS for 5 minutes. The sections were then immersed in 2x SSC for 10 minutes and prehybridyzed for 2 hours at 42°C. The prehybridization solution contained 5x SSC, 0.1% N-lauroylsarcosine, 0.02% SDS, 50% formamide, and 2% blocking reagent (Boehringer Mannheim). The sections were hybridized with digoxigenin-labeled TGF-ß1 riboprobe (100 ng/mL) for 16 hours at 42°C in a MicroProbe humid chamber (Fisher Scientific) using capillary gap technology (Fisher Scientific). The labeled riboprobe probe was generated by in vitro transcription. In brief a 974-bp cDNA coding for murine TGF-ß1 in pGEM7Z plasmid (kindly provided by Dr H.L. Moses, Vanderbilt University, Nashville, Tenn) was linearized by HindIII for antisense cRNA or EcoRI for sense cRNA.28 The linearized DNA was purified by phenol-chloroform extraction and transcribed with SP6 (for sense probe) or T7 (for antisense probe) RNA polymerases with digoxigenin-labeled UTP as the substrate (Boehringer Mannheim Biochemica). Transcript yield was estimated by electrophoresis and ethidium bromide staining, and labeling efficiency was examined by dot blot analysis. Digoxigenin-labeled nucleic acids were detected by enzyme immunoassay and an enzyme-catalyzed color reaction (digoxigenin nucleic acid detection kit, Boehringer Mannheim), following the procedures provided by the supplier. In brief the hybridized sections were washed twice in 2x SSC for 30 minutes in washing buffer and then treated with DNase-free RNase (100 µg/mL, Sigma Chemical Co) in 2x SSC for another 30 minutes to eliminate the unbound probe. Then the sections were washed three times in 0.1x SSC in 0.1% SDS for 20 minutes each and incubated in blocking buffer for 30 minutes followed by incubation with anti–digoxigenin/alkaline phosphatase conjugate (1:500) for 5 hours in a humid chamber. After being washed twice, the sections were incubated with freshly prepared substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt for 48 hours. This results in the formation of insoluble blue precipitates, which identify hybrid molecules. Finally, the slides were counterstained for 1 minute with Nuclear Fast Red (Vector Laboratories). After dehydration in a graded series of ethanol, the specimens were placed in xylene for 3 minutes and covered with mounting medium (Permount) and coverslips. Two types of negative control were used, including one with a sense instead of an antisense probe and RNase digestion of tissue for 30 minutes before hybridization.
Immunohistochemistry
The Vectastain Elite ABC system (Vector Laboratories) was used for immunohistochemistry. Sections were deparaffinized, incubated with 0.6% H2O2 in methanol for 30 minutes, and blocked with 5% horse or goat serum. After being washed in PBS, the sections were incubated with primary antibodies for either 1 hour at room temperature or overnight at 4°C in a moisture chamber. The following primary antibodies were used: monoclonal mouse 1A4 antibody recognizing -SM actin (1:100, Sigma Diagnostics); monoclonal mouse DE-R-11 antibody recognizing intermediate filament desmin (1:50, Novocastra); polyclonal rabbit antibody LC-30 recognizing intracellular TGF-ß1 (1:100, kindly provided by Dr K.C. Flanders, National Institutes of Health, Bethesda, Md); polyclonal rabbit CC-30 antibody directed against extracellular TGF-ß1 (1:250, kindly provided by Drs K.C. Flanders and A. Roberts, National Institutes of Health)29 ; and monoclonal mouse anti-porcine macrophage IgG2b antibody (1:100, ATCC HB 142.1, American Type Culture Collection). Then the slides were washed, incubated with biotinylated secondary horse anti-mouse antibodies (1:2000, Vector Laboratories) or goat anti-rabbit antibodies (1:3000, Vector Laboratories) for 1 hour, visualized with diaminobenzidine substrate (Vector Laboratories), and counterstained with Gill's hematoxylin (Sigma Diagnostics). Positive controls for TGF-ß1 immunostaining included myocardium with discrete staining of cardiac myocytes with anti–LC-30 antibody or interstitial connective tissue with anti–CC-30 antibody.30 For the anti-macrophage antibody, positive controls consisted of lung specimens with alveolar macrophages. Negative control experiments were performed with nonimmune serum instead of the primary antibody.
For combined in situ hybridization and immunohistochemistry, in situ hybridization was carried out first with TGF-ß1 cRNA probe as described above. After incubation with the 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium salt substrate for 48 hours, the sections were immersed in 0.6% H2O2, immunostained with the -SM actin antibody as described above, and counterstained with Nuclear Fast Red.
Quantitative Measurements
All quantitative analyses were carried out in arteries that had a similar degree of medial injury. The adventitia was defined as the region between the inner border of the EEL outward to the edge of adipose tissue or myocardium. The neointima was delineated by the inner edge of the EEL, the border of dissected media, and the luminal edge of the neointima. To detect TGF-ß1 mRNA, sections were reviewed at high magnification. The cells were considered to have expressed TGF-ß1 mRNA when at least five grains were associated with each nucleus. To determine the percentage of TGF-ß1–positive cells by in situ hybridization, three separate fields, each with 
100 nuclei, were screened for each artery. To assess the percentage of macrophages in the injured arterial wall, 200 cells were counted in each arterial compartment. All quantitative measurements were carried out by two independent observers, with interobserver and intraobserver variabilities not >10%. Values were obtained for separate arteries and the results reported as mean±SEM. One-way ANOVA was used to compare repeated measurements. If the F test results were significant, Bonferroni analysis was carried out to determine differences among subgroups. A value of P<.05 was required to reject the null hypothesis.
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Results |
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TGF-ß1 Expression by In Situ Hybridization
Control coronary arteries (ie, uninjured vessels, n=4) displayed low-level expression of TGF-ß1 transcripts in medial SMCs and endothelium. Adventitial fibroblasts also showed a paucity of TGF-ß1 transcripts, with only infrequent TGF-ß1–expressing cells present adjacent to the vasa vasorum (2±1%, Fig 1A
).
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At 2 to 3 days after coronary artery injury (n=5), there was a marked increase in the number of cells that demonstrated the presence of TGF-ß1 transcripts within the adventitia (47±10%, P<.001 versus control vessels). These cells were predominantly localized in the vicinity of medial injury. As shown by combined in situ hybridization for TGF-ß1 mRNA and immunohistochemistry with -SM actin or desmin antibodies, TGF-ß1–expressing cells in the adventitia were negative for both -SM actin and desmin (Fig 1B
). Since TGF-ß1 can be expressed by either resident fibroblasts or recruited macrophages, immunostaining for porcine macrophages was carried out (n=4). To this end the injured adventitia contained rare macrophages (3±1%). Medial SMCs showed low TGF-ß1 expression and positive immunostaining with -SM actin and desmin antibodies (not shown).
At 7 to 8 days after arterial injury (n=6), adventitial cells continued to express TGF-ß1 (54±4%, P<.001 versus control vessels; NS versus 2 to 3 days). Macrophages remained infrequent in the adventitia (9±1%). Most TGF-ß1–expressing cells began to demonstrate positive immunoreactivity with -SM actin antibody (67±8%), which indicated myofibroblast formation (Fig 1C). Likewise, a thin layer of neointima contained a similar percentage of cells that coexpressed TGF-ß1 and -SM actin protein (70±7%, NS versus adventitia). At 28 to 35 days (n=4), the number of TGF-ß1–expressing cells was markedly reduced in the adventitia (12±6%, P<.005 versus 2 to 3 and 7 to 8 days; NS versus control vessels). In contrast, neointimal cells continuously expressed TGF-ß1 by in situ hybridization (73±4%).
TGF-ß1 by Immunostaining
The time course and immunolocalization of TGF-ß1 LC-30, -SM actin, and desmin after coronary artery injury are summarized in the Table. In control coronary arteries (n=4), medial SMCs demonstrated weak immunoreactivity with TGF-ß1 LC-30 antibody (Fig 2A). Most adventitial cells were negative for TGF-ß1, consistent with in situ hybridization findings. This feature contrasted with the appearance of focal areas of strong TGF-ß1 anti–LC-30 immunostaining in the adventitia 2 days after injury (n=5, Fig 2B).
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At 7 to 14 days after injury (n=6), TGF-ß1 anti–LC-30 staining was present in most of the adventitial cells adjacent to areas of medial dissection (Fig 2C). These cells were elongated and often localized in the vicinity of the thrombus on the luminal surface. The neointima demonstrated strong and uniform immunoreactivity with TGF-ß1 LC-30 and -SM actin antibodies (Fig 2D). At 28 to 35 days after injury (n=5), TGF-ß1 anti–LC-30 immunoreactivity was largely absent in the adventitia (Fig 2E), whereas neointimal cells remained uniformly positive (not shown). However, at 3 months after injury (n=5), TGF-ß1 anti–LC-30 immunostaining became patchy in the neointima adjacent to the vessel lumen (Fig 2F).
To examine whether TGF-ß1 anti–LC-30 immunostaining (predominantly intracellular TGF-ß1) was associated with the secretion of the extracellular form, we stained adjacent sections with CC-30 antibody against the extracellular TGF-ß1 epitope. At various time points, TGF-ß1 anti–LC-30 immunoreactivity in both the adventitia and neointima was associated with concordant localization of TGF-ß1 anti–CC-30 immunostaining (Fig 3).
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Ultrastructural Characteristics of Adventitial Myofibroblasts
To examine the ultrastructural changes associated with TGF-ß1 induction, the adventitia was examined by electron microscopy. Normal adventitial cells (devoid of TGF-ß1 expression) demonstrated typical fibroblast features, including a rough endoplasmic reticulum and the absence of microfilaments. At 1 to 2 weeks after injury, ie, when both TGF-ß1 transcripts and protein were abundant, adventitial cells displayed numerous actin microfilaments (stress fibers), which are unique characteristics of myofibroblasts (Fig 4A). Furthermore, these cells demonstrated a markedly dilated rough endoplasmic reticulum and appeared embedded in the ECM, reflecting their active synthetic phenotype. Likewise, neointimal cells exhibited typical features of myofibroblasts, suggesting a common origin for adventitial and neointimal cells after severe arterial injury (Fig 4B).
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Discussion |
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TGF-ß1 Expression in Adventitial Fibroblasts
Coronary arterial injury resulted in dynamic changes in TGF-ß1 expression, with the initial changes involving the adventitia, as reflected by in situ hybridization and immunohistochemistry findings. The increase in autocrine TGF-ß1 transcripts/protein in adventitial cells was apparent as early as 2 days after injury (Figs 1B and 2B
), a finding consistent with previously reported augmentation of TGF-ß1 transcripts in whole arterial extracts within 6 hours of balloon injury that lasted for 2 weeks.31 The question can be raised as to the mechanism(s) of TGF-ß1 induction after vascular injury, inasmuch as normal adventitial fibroblasts are devoid of this cytokine. The ability of TGF-ß1 to induce its own expression suggests that its release from degranulated platelets and activated macrophages may initiate TGF-ß1 upregulation in adventitial fibroblasts.32 33 Furthermore, platelet-derived growth factor released from platelets early after vascular insult could contribute to the induction of TGF-ß1.34 Although macrophages recruited to the site of injury can express TGF-ß1, they constitute only a small fraction of cells in the adventitia. Likewise, induction of TGF-ß1 expression in the adventitia was not due to migration of SMCs from the media or proliferation of pericytes, since TGF-ß1–expressing cells initially showed a paucity of -SM actin and desmin immunostaining. Importantly, medial expression of TGF-ß1 remained at basal levels, demonstrating no significant increase at any time after severe vascular injury.
The mere expression of a cytokine does not provide sufficient evidence of its function. In particular, this is relevant for studies regarding TGF-ß1, which can exist in a latent form.35 There are several lines of evidence, however, suggesting the functional role of autocrine TGF-ß1 expression in the process of myofibroblast formation. First, despite the release of TGF-ß1 immediately after vascular injury from exogenous sources (eg, platelets), myofibroblast formation was delayed (7 to 8 days). Second, a high rate of colocalization of TGF-ß1 and -SM actin at an early stage of myofibroblast formation was noted (7 to 8 days). Third, extracellular TGF-ß1, which likely includes the active cytokine, exhibited a distribution pattern similar to that of TGF-ß1 transcripts/intracellular TGF-ß1 protein expressed by adventitial fibroblasts/myofibroblasts. It should be emphasized that the present study demonstrates the association between TGF-ß1 expression and adventitial myofibroblast formation after medial dissection that exposes the adventitia to the vessel lumen. This exposure could facilitate contact between adventitial cells and activators of latent TGF-ß1 (eg, plasmin).36 37 In contrast, mild injury less likely involves the adventitia during arterial repair, inasmuch as no adventitial activation has been observed when medial continuity was preserved.19
TGF-ß1 and Myofibroblast Formation
Prior studies have shown that continuous subcutaneous delivery of exogenous TGF-ß1 results in the appearance of wound myofibroblasts.23 Granulocyte macrophage colony stimulating factor, the only other cytokine that exerts similar effects, caused macrophage accumulation, which suggested a contribution of their products (eg, TGF-ß1) in this response.38 39 The origin of myofibroblasts in various tissues has been a subject of controversy.40 41 42 The present study is the first to provide evidence that arterial injury elicits autocrine induction of TGF-ß1 in adventitial fibroblasts followed by the appearance of -SM actin, a differentiation marker that identifies them as myofibroblasts, 7 to 8 days after injury (Fig 1C). The mechanism of TGF-ß1–induced changes in -SM actin expression remains to be defined, and it could include regulation of transcription, translation, and protein turnover.
The sequential appearance of TGF-ß1 in adventitial and then neointimal cells (Fig 2) was consistent with our prior findings that adventitial myofibroblasts translocate into the gaps in the dissected media and contribute to the development of neointima.19 Thus, unlike healing wounds, wherein myofibroblasts are eliminated mainly by apoptosis, a significant fraction of myofibroblasts appeared to migrate to the luminal surface and form the neointima.19 Different patterns of desmin immunostaining in the adventitia and neointima (Table) are consistent with the plasticity of the myofibroblast phenotype.10 This finding may also suggest that expression of cytoskeletal protein markers differs in various layers of the arterial wall due to distinct myofibroblast functions in situ and diverse signaling mechanisms. Interestingly, SMCs appeared to play a limited role in the repair of severely injured coronary arteries, inasmuch as the paucity of SM myosin in both the adventitia and neointima at 7 to 14 days contrasted with sustained SM myosin immunoreactivity in the media (Y.S., unpublished data, 1996). The mechanism of neointimal formation in milder forms of coronary artery injury is likely different. It may involve either medial SMCs undergoing phenotypic modulation, or it may depend on differentiation of "nonmuscle" cells derived from the underlying media.43
Although TGF-ß1 is critical for the induction of differentiation of fibroblasts to myofibroblasts,23 24 sustained expression of TGF-ß1 is not required to maintain this highly specialized cell phenotype. As shown in this study, the decrease in TGF-ß1 expression in the neointima was associated with a persistent -SM actin immunostaining 3 months after severe injury (Table). The mechanisms that lead to the decrease in TGF-ß1 are poorly understood. The ECM itself can provide a negative-feedback loop that terminates TGF-ß1 synthesis.44 Decorin, a member of the proteoglycan family, neutralizes extracellular TGF-ß1 and presumably interrupts its autoinduction.45 In normal vessels decorin is preferentially expressed in the adventitia, which is associated with the absence of TGF-ß1.46
The augmented synthetic function of myofibroblasts derived from various tissues, with the accompanying deposition of collagens and fibronectin, is consistent with the profibrotic effects of TGF-ß1.15 47 48 Furthermore, myofibroblasts are capable of mediating contraction of their surrounding environment.12 13 26 Thus, TGF-ß1 expression that results in myofibroblast formation may affect remodeling of various tissues by means of synthetic and mechanical events after injury. Undoubtedly, vascular repair after catheter-based revascularization is much more complex in atherosclerotic, human coronary arteries than in animal models. However, a high incidence of adventitial exposure to the lumen that is often unrecognized by angiography occurs after coronary angioplasty and after newer revascularization procedures (eg, atherectomy or stenting). This may provide a basis for adventitial involvement in the arterial response to injury. Recent interest in geometric remodeling has been stimulated by clinical observations that point to late luminal loss after angioplasty.49 50 Neointimal myofibroblasts with prolonged expression of TGF-ß1 and sustained expression of -SM actin may render the cellular mechanism for these chronic, constrictive changes.
Conclusions
In this study we have demonstrated rapid induction of TGF-ß1 expression in adventitial fibroblasts by in situ hybridization and immunohistochemistry in porcine balloon-injured coronary arteries. TGF-ß1 expression was associated with differentiation of adventitial fibroblasts to myofibroblasts, as reflected by colocalization of TGF-ß1 transcripts and -SM actin. Myofibroblast phenotype in the adventitia was further confirmed by electron microscopy, which revealed abundant microfilaments and extensive rough endoplasmic reticulum. Although TGF-ß1–expressing myofibroblasts transiently appeared in the adventitia, the neointima exhibited sustained TGF-ß1 expression. In the context of the multifunctional properties of TGF-ß1, it is postulated that its role in vascular repair is mediated through the synthetic and mechanical properties of myofibroblasts.
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Acknowledgments |
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This study was supported in part by the National Institutes of Health, Bethesda, Md (grant HL55410 to Dr Zalewski); a Grant-in-Aid from the American Heart Association, Delaware, Inc (Dr Shi) and the Florida and Delaware Affiliates, Inc (Dr Zalewski). Dr Ali Fard was supported by a fellowship grant from the American Heart Association, Delaware Affiliate, Inc. The authors gratefully acknowledge the technical assistance of Dian Wang and Felicia Hayes. We express our gratitude to Drs Kathleen C. Flanders and Anita B. Roberts (National Cancer Institute, National Institutes of Health, Bethesda, Md) for providing the anti–TGF-ß1 antibodies (LC-1-30-1 and CC-1-30-1) and Dr H.L. Moses (Department of Cell Biology, Vanderbilt University, Nashville, Tenn) for supplying the murine TGF-ß1 cDNA probe. We are also indebted to Dr Elizabeth G. Nabel (Department of Medicine, University of Michigan Medical Center, Ann Arbor) for advice regarding antibodies that recognize porcine macrophages. The expert assistance of Bodil T. Tuma and Dr Renato V. Iozzo (Department of Pathology, Thomas Jefferson University, Philadelphia, Pa) with the electron microscopy is also acknowledged.
Received February 15, 1996;
revision received April 12, 1996;
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