FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.05-5435fje.
This Article
Right arrow Abstract Freely available
Right arrow Summary
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
fj.05-5435fjev1
20/10/1686    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mallawaarachchi, C. M.
Right arrow Articles by Siow, R. C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mallawaarachchi, C. M.
Right arrow Articles by Siow, R. C. M.
(The FASEB Journal. 2006;20:1686-1688.)
© 2006 FASEB

Antagonism of platelet-derived growth factor by perivascular gene transfer attenuates adventitial cell migration after vascular injury: new tricks for old dogs?

Chandike M. Mallawaarachchi*, Peter L. Weissberg* and Richard C. M. Siow*,{dagger},1

* Division of Cardiovascular Medicine, School of Clinical Medicine, University of Cambridge, Addenbrookes Hospital, Cambridge, UK; and

{dagger} Cardiovascular Division, School of Biomedical and Health Sciences, King’s College London, Guy’s Hospital Campus, London, UK

1Correspondence: Cardiovascular Division, School of Biomedical and Health Sciences, Kings College London, Guys Hospital Campus, London SE1 1UL, UK. E-mail: richard.siow{at}kcl.ac.uk

ABSTRACT

Migration of adventitial fibroblasts contributes to vascular remodeling after angioplasty. This study has used perivascular gene transfer of a truncated platelet-derived growth factor PDGF receptor (PDGFXR) to investigate whether antagonism of PDGF signaling alters adventitial cell migration after balloon injury in rat carotid arteries. Adenoviruses coordinating expression of ß-galactosidase (LacZ) and PDGFXR or LacZ and green fluorescent protein (GFP) were applied to the perivascular surface of arteries and balloon injury performed 4 days later. Vessels were excised at 3, 7, and 14 days to determine morphology and gene expression. Uninjured arteries only expressed LacZ positive cells in the adventitial compartment; however, after injury in LacZ and GFP transfected arteries, LacZ positive cells contributed to the population of cells within the media and neointima at 7–14 days. Overexpression of PDGFXR and LacZ resulted in a significant reduction in the number of LacZ labeled cells in the neointima after vascular injury, concomitant with reduced remodeling, collagen content, expression of matrix metalloproteinase-2, and increased levels of tissue inhibitors of metalloproteinase-1 and -2. We provide evidence that perivascular antagonism of PDGF attenuates remodeling and contribution of adventitial fibroblasts to neointima formation after balloon angioplasty. Perivascular gene transfer may represent a therapeutic strategy to reduce the incidence of restenosis.—Mallawaarachchi, C. M., Weissberg, P. L., and Siow, R. C. M. Antagonism of platelet-derived growth factor by perivascular gene transfer attenuates adventitial cell migration after vascular injury: New tricks for old dogs?


Key Words: restenosis • vascular remodeling • balloon angioplasty • PDGF

PERCUTANEOUS TRANSLUMINAL CORONARY angioplasty (PCTA) in humans for the treatment coronary artery occlusion often results in restenosis, affecting >30% of patients who undergo balloon angioplasty without stenting. The cellular events leading to postangioplasty restenosis are similar to those that cause accelerated atherosclerosis and vein graft failure after coronary artery bypass surgery (1 , 2) . Experimental balloon catheter mediated vascular injury in animal models results in migratory and proliferative responses of cells within the vessel wall in addition to matrix remodeling, characteristic events observed in restenosis and atherosclerosis (3) . Over the past decade the role of PDGF-B-chain (PDGF-B) in smooth muscle cell (SMC) migration and proliferation has been well characterized in vascular remodeling after angioplasty in humans and models of experimental balloon injury in rodents (3 , 4) . Antagonism of vascular PDGF-B signaling in the vessel wall through its sequestration by specific antibodies or inhibition of PDGF-ß-receptor kinase activity can attenuate these events and thereby limit vascular remodeling after angioplasty (4 5 6 7 8) . The contribution of the adventitia compartment of the vessel wall in the remodeling response to balloon angioplasty has received much less attention compared with the media and neointima and has been generally regarded as an "inert" layer providing extracellular matrix (ECM) support for the blood vessel and a scaffold for sympathetic nerve endings and the vasa vasorum. However, we and others (9 , 10) have established that functional changes in the adventitia layer do contribute to vascular remodeling after balloon injury, through the activation and migration of adventitial fibroblasts leading to neointima formation after vascular injury, partly under the influence of transforming growth factor-ß1 signaling (11) .

At present, a paucity of information exists on the roles that PDGF-B play in the adventitial response to vascular injury, particularly in the characterization of adventitial cell migration and their contribution to angioplasty-induced neointima formation. Therefore, the present study has used a localized in vivo perivascular adenoviral gene transfer approach to track migratory adventitial cells and assess neointima formation after experimental balloon catheter mediated injury in the rat carotid artery (10) and to determine whether antagonism of PDGF-B signaling in the adventitia modulates the remodeling process. Antagonism of PDGF-B signaling in the adventitia was achieved by adenoviral gene transfer of the truncated soluble extracellular region of the human PDGF-ß-receptor (PDGFXR). Loss of vessel lumen area, the contribution of adventitial cells to neointima formation, collagen deposition, and the expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinase-1 and -2 (TIMP-1, 2) were assessed after carotid artery balloon angioplasty. The soluble PDGFXR protein has been previously shown to bind PDGF-B with high affinity, but not PDGF-A, and thereby acts as a selective antagonist of the PDGF-B signaling pathway (12) . Overexpression of PDGFXR in rat aortic SMC has been reported to inhibit PDGF-B induced PDGF-ß-receptor tyrosine phosphorylation and DNA synthesis, while direct competition assays have demonstrated that PDGFXR inhibits PDGF-B binding to the full-length native PDGF-ß-receptor (7 , 12) . Our findings provide novel evidence that antagonism of PDGF-B in the adventitial compartment through perivascular PDGFXR gene transfer attenuates adventitial cell migration and vascular remodeling processes. Studies performed in both experimental animals and in human subjects have established the contribution of PDGF-B in "constrictive remodeling" after angioplasty; however, its "new tricks" in the adventitia provide additional insights in the mechanisms of action by which these "old dogs" contribute to restenosis.

MATERIALS AND METHODS

Adventitial gene transfer and carotid artery balloon injury
Recombinant replication-defective type 5 adenoviral vectors with the cytomegalovirus (cytomeglovirus) promoter coordinating expression of nuclear-targeted ß-galactosidase (ß-gal; AdLacZ), green fluorescent protein (AdGFP), or the soluble extracellular region (XR) of the human PDGF-ß-receptor (corresponding to amino acids 1–531, AdPDGFXR) were propagated and purified as described previously (7 , 10 , 11) . For adventitial gene transfer, AdLacZ and AdGFP (inactive controls) or AdLacZ and AdPDGFXR adenoviruses (1010 pfu/ml) were suspended together in pluronic F127 gel (BASF, 25% w/v) and maintained at 4°C. Animal studies were carried out under the approval of the Home Office, UK. Male Sprague-Dawley rats (3 months old, Charles River, wt range 380–450 g, n=12 per time point for each treatment group) were anesthetized with ketamine and xylazine, allowed to recover after surgical procedures, and killed by exsanguination and fixation in situ by retrograde aortic perfusion with saline containing formalin (2%) and glutaraldehyde (0.2%), as described previously before careful excision of the common carotid arteries to avoid damage to the adventitial layer (10 , 11) . The left common carotid artery was exposed under aseptic conditions and 200 µl of pluronic gel containing the adenovirus pairs applied to the adventitial surface of the artery, with minimal manipulation of the perivascular layer. Four days after adventitial gene transfer, the left carotid artery was injured by passing an inflated 2F Fogarty balloon catheter (Baxter) through the arterial lumen three times to elicit endothelial denudation and arterial distension. Rats were killed at 3, 7, or 14 days after adenoviral gene transfer only or at 3, 7, and 14 days after balloon mediated injury, and common carotid arteries were excised for histological analyses.

Assessment of PDGFXR vascular gene transfer
Arterial sections from common carotid arteries (200 µg) were excised at 7 or 14 days after adenoviral gene transfer and incubated in 1 ml of Dulbecco’s modified Eagles culture medium containing 5% fetal calf serum (FCS) at 37°C in a 5% CO2, 95% air atmosphere for 24 h. After incubation of arteries ex vivo, the conditioned culture media were collected and the levels of secreted soluble PDGFXR were determined by Western blot analyses using a specific primary antibody (Ab) against the extracellular region of the PDGF-ß-receptor (Santa Cruz Biotechnology). The total protein content in samples was determined using the bicinchoninic acid (BCA) assay (Pierce), and equal amounts were separated on SDS-PAGE gels. Resulting Western blot bands were quantified by scanning and computer densitometry.

In vitro cell migration assay to assess efficacy of PDGFXR
A transwell migration assay was used to determine the chemotactic activity of PDGF-B on cultured rat aortic smooth muscle cells (RASMC) to assess the inhibitory effect of PDGFXR overexpression on cell migration in vitro. RASMC were cultured from rat aortic explants in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS, and confluent cell cultures transfected with AdPDGFXR (50 MOI) for 24 h. After gene transfer (24–96 h), the conditioned media were collected and levels of secreted soluble PDGFXR determined by Western blot analyses using a specific primary Ab against the extracellular region of the PDGF-ß-receptor (Santa Cruz Biotechnology). The total protein content in samples was determined using the BCA assay (Pierce) and equal amounts were separated on SDS-PAGE gels. Resulting Western blots bands were quantified by scanning and computer densitometry. Cell migration was assessed as described previously using a modified Boyden chamber technique (13) . Briefly, RASMC, overexpressing PDGFXR 48 h after tansfection or control untransfected cells, were labeled with 5 µM calcein-acetoxymethyl ester (Molecular Probes) for 2 h at 37°C in DMEM containing 2% FCS, washed with PBS, and detached with trypsin. Cell suspensions were loaded on a 3 µm Fluorblok transwell chamber (Falcon) at a density of 50,000 cells/well, and the chamber was placed in a 24-well plate containing PDGF-B (10 ng/ml) in DMEM with 0.1% BSA for 24 h at 37°C. Migration of RASMC to the lower well chamber compartment was determined using a fluorescence microplate reader with excitation and emission wavelengths of 485 and 530 nM, respectively.

Immunohistochemical analyses
Localization of nuclear ß-gal (LacZ) transgene expression was performed on arterial segments by X-gal staining as described previously to minimize loss of blue chromogen labeling (10 , 11) . Expression of MMP-2, TIMP-1, and TIMP-2 was determined in common carotid artery sections using specific monoclonal mouse primary antibodies (Santa Cruz Biotechnology) and secondary biotinylated anti-mouse antibodies (Dako). Mouse IgG was used as a negative control instead of primary Ab. Immunostaining was visualized using the Vectastain Elite avidin-biotin complex system (Vector Laboratories). Collagen content in arterial sections was assessed by picrosirius red staining. All images were captured using an Olympus BX51 microscope fitted with a CCD camera and attached to a PC running Colorview 12 (Soft Imaging System) image analysis software.

Adventitial cell migration and morphometric analyses
After computer image acquisition of histological sections from 12 separate animals per time point, software analysis (Photoshop 5.02, Adobe) was used to quantify morphological changes in vessel lumen area, wall thickness, and proportion of blue stained ß-gal positive nuclei of adventitial cells contributing to the media and neointima and collagen deposition after balloon injury in six representative areas for each compartment of the vessel wall as described previously (10 , 11) . Results are reported as mean ± SE and data from 6 to 12 individual rats per treatment group evaluated using the unpaired Student’s t test with P < 0.05 considered statistically significant.

RESULTS

In the present study, initial experiments using cultured RASMC established that overexpression of PDGFXR in vitro resulted in the secretion of the soluble extracellular region of the PDGF-ß-receptor protein and attenuated RASMC migration in response to PDGF-B. We demonstrated by Western blot analyses that the 110 kDa soluble PDGF-ß-receptor protein fragment was present only in conditioned media of RASMC at 48 to 96 h after AdPDGFXR gene transfer but not in control or AdLacZ transfected cells (Fig. 1 A). The secretion of PDGFXR at 48 h was associated with a concomitant significant (P<0.05; n=3) attenuation (32±4%) of RASMC migration elicited by exogenous PDGF-B (10 ng/ml, 24 h) compared with the concentration of cell migration observed in control and AdLacZ transfected RASMC populations using a modified Boyden chamber technique, thereby confirming efficacy of PDGFXR gene transfer.


Figure 1
View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. PDGFXR gene transfer in rat aortic SMC and carotid arteries. Conditioned media were collected from confluent cultures of control (CTL) or AdPDGFXR transfected rat aortic SMC and levels (24–96 h) of secreted soluble PDGFXR determined by Western blot analyses (A) and quantified by densitometric analyses (B). Sections of AdGFP or AdPDFGXR transfected or control untranfected (CTL) common carotid arteries (200 µg) were excised at 7 or 14 days after gene transfer, placed in culture medium (37°C, 5% CO2) for 24 h and conditioned media collected. Levels of secreted soluble PDGFXR were determined by Western blot analyses (C) and quantified by densitometric analyses. Representative blots from 3 independent cell cultures or tissue sections from 6 different animals in each group are shown with coomassie blue staining of gels to indicate equal protein loading (*P<0.05).

To establish that in vivo perivascular gene transfer with AdPDGFXR resulted in enhanced secretion of the soluble extracellular region of the PDGF-ß-receptor, we demonstrated the presence of the 110 kDa protein only in conditioned media of carotid artery sections that had been transfected with AdPDGFXR and AdLacZ, and not in the conditioned media of arteries transfected with AdGFP and AdLacZ or control untransfected arteries, after ex vivo incubations for 24 h (Fig. 1B ). Levels of PDGFXR secreted were similar in arterial sections excised at 7 and 14 days after gene transfer, suggesting that perivascular PDGFXR transgene expression and secretion of the PDGF-ß-receptor fragment were maintained in vivo throughout this experimental time period.

After in vivo gene transfer with either of the adenoviral pairs AdLacZ and AdPDGFXR or AdLacZ and AdGFP, the adventitial compartment of transfected left carotid arteries exhibited an equal proportion of cells with blue stained ß-gal (LacZ) positive nuclei restricted solely to the adventitial compartment after adenoviral gene transfer regimens alone, with negligible changes in morphology of the vessel wall up to 14 days after delivery of adenoviral vectors (data not shown). Expression of ß-gal was not observed at any time in control right untransfected carotid arteries. These findings are consistent with our previous studies in which adenoviral gene delivery alone to the rat carotid artery perivascular surface resulted in a transfection efficiency of 34 ± 4% determined by X-gal and nuclear fast red staining, while the lack of ß-gal transgene expression in the medial layer of uninjured vessels establishes that only adventitial cells are initially transfected and subsequently observed migrating toward the lumen after balloon catheter mediated vascular injury (10 , 11) .

Cells exhibiting blue nuclear ß-gal staining were observed in the adventitial and medial layer of the vessel wall at 7 to 14 days after injury of left carotid arteries overexpressing perivascular ß-gal and GFP (Fig. 2 A and C). However, after experimental angioplasty, vessels transfected with AdPDGFXR and Adß-gal exhibited a significantly lower number of adventitial cells that had migrated in to the neointima (Fig. 2B and D ), as shown by the significant reduction in blue stained nuclei in the neointimal compartment compared with the number of ß-gal positive cells in the control Adß-gal and AdGFP transfected arteries (Fig. 3 ). Overexpression of PDGFXR significantly attenuated the loss in luminal area at 14 days after balloon injury, from 0.33 ± 0.03 mm2 in control injured arteries transfected with Adß-gal and AdGFP to 0.40 ± 0.04 mm2 in Adß-gal and AdPDGFXR transfected arteries (P<0.01; n=12). The increase in lumen area was associated with a significant decrease in thickness of the neointimal compartment after balloon injury in Adß-gal and AdPDGFXR transfected arteries at 7 and 14 days after balloon injury compared with control Adß-gal and AdGFP transfected vessels (Fig. 4 A). At 14 days after balloon injury, the neointimal to medial area ratios were significantly attenuated in carotid arteries overexpressing PDGFXR compared with control AdGFP and Adß-gal transfected vessels (Fig. 4B ). Taken together, these data indicate that antagonism of PDGF-B signaling through perivascular PDGFXR overexpression leads to an attenuation of constrictive vascular remodeling through diminished advential cell migration to the neointima.


Figure 2
View larger version (93K):
[in this window]
[in a new window]
  		Figure 2. Adventitial cell migration after carotid artery balloon injury. To assess adventitial cell migration, Adß-gal and AdGFP (inactive controls, A and C) or Adß-gal and AdPDGFXR (B and D) adenoviruses were suspended in pluronic gel and applied to perivascular adventitial surface of carotid arteries. Four days after gene transfer, left carotid arteries underwent balloon injury to elicit endothelial denudation and arterial distension. Localization of blue ß-gal positive nuclei of adventitial cells contributing to the medial and neointimal compartments were visualized after X-gal staining in sections excised at 7 (A and B) or 14 d (C and D) after balloon injury. a.: adventitia; m: media; n: neointima. Scale bar = 100 µm. Representative micrographs shown from 6–12 animals per treatment condition at each time point.


Figure 3
View larger version (9K):
[in this window]
[in a new window]
  		Figure 3. Quantification of adventitial cell migration. Localization of ß-gal transgene expression was determined by X-gal staining as described in Materials and Methods and the proportion of ß-gal positive nuclei in the neointimal compartment at 3, 7, and 14 days after balloon injury were quantified using chromagen separation PC software in micrographs of AdGFP and Adß-gal (open bars) or AdPDGFXR and Adß-gal (solid bars) transfected arterial sections, captured using a microscope fitted with a CCD camera. Data are mean ± SE; n = 6–12 animals per treatment time point, *P < 0.05.


Figure 4
View larger version (13K):
[in this window]
[in a new window]
  		Figure 4. Morphometric analyses of arterial sections. After computer image acquisition of hematoxilin and eosin stained paraffin embedded sections from AdGFP and Adß-gal (open bars) or AdPDGFXR and Adß-gal (solid bars) transfected and injured carotid arteries, software analysis was used to quantify morphological changes in neointimal thickness (A) and neointimal to medial area ratios (B) in six representative areas for each compartment of vessel wall in each arterial section as described previously (10 , 11) . Values are mean ± SE; n = 6–12 animals per treatment time point, *P < 0.05.

Immunohistological studies showed that perivascular overexpression of PDGFXR also significantly attenuated adventitial and medial MMP-2 expression at 7 days after balloon injury (Fig. 5 B) compared with levels in injured control carotid arteries transfected with Adß-gal and AdGFP (Fig. 5A ). However, adventitial immunoreactivity for TIMP-1 (Fig. 5C and D ) and TIMP-2 (Fig. 5E and F ) were significantly increasedin balloon injured arteries overexpressing adventitial PDGFXR compared withvessels transfected with Adß-gal and AdGFP. Moreover, picrosirius red staining revealed a significant decrease in vascular collagen content at 14 days after balloon injury in carotid arteries overexpressing adventitial PDGFXR compared with control Adß-gal and AdGFP transfected vessels (Fig. 6 A and C).


Figure 5
View larger version (78K):
[in this window]
[in a new window]
  		Figure 5. Expression of MMP-2, TIMP-1 and TIMP-2 after balloon injury. Immunostaining of MMP-2 (A and B), TIMP-1 (C and D), or TIMP-2 (E and F) in arteries excised 7 days after balloon injury were visualized. Left panels) Micrographs from control AdGFP and Adß-gal transfected vessels. Right panels) Sections from AdPDGFXR and Adß-gal transfected arteries. Positive immunoreactivity indicated by brown staining within the vessel wall; n = 6–12 animals per treatment condition. Scale bar = 100 µm.


Figure 6
View larger version (57K):
[in this window]
[in a new window]
  		Figure 6. Collagen content in arterial sections after balloon injury. Arterial sections were excised at 3, 7, and 14 days after vascular injury and collagen content was assessed by picrosirius red staining. A) AdGFP and Adß-gal transfected vessels. B) depicts a section from an AdPDGFXR and Adß-gal transfected artery. Representative micrographs shown from n = 6–12 animals per treatment condition at 14 d after balloon injury. Scale bar = 100 µm. C) Levels of collagen staining were quantified in six representative areas in each arterial section. Values are mean ± SE, n = 6–12 animals per treatment time point, * P < 0.05.

DISCUSSION

Our previous studies together with other recent reports have provided increasing evidence that adventitial cells contribute to constrictive remodeling after vascular injury by migrating to the neointima and synthesizing ECM (9 10 11) . PDGF-B plays a key role in the remodeling processes leading to restenosis after vascular angioplasty through its contribution to phenotypic modulation and proliferation of medial SMC (4 5 6 7 8) . Our present study represents the first demonstration that in vivo perivascular gene transfer of the extracellular region of the PDGF-ß-receptor antagonizes PDGF-B mediated migration of adventitial cells in balloon injured carotid arteries. Our findings clearly show that perivascular PDGFXR overexpression results in secretion of the soluble PDGF-ß-receptor for up to 14 days after gene transfer (Fig 1B ). Moreover, migration of cultured aortic SMC secreting the PDGF-ß-receptor was significantly attenuated in response to exogenous PDGF-B after in vitro transfection with AdPDGFXR. Taken together, it is likely that antagonism of perivascular PDGF-B signaling in the present study resulted in the attenuation of adventitial cell migration to the neointima, diminished loss of luminal area, and decreased vascular collagen content after carotid artery balloon injury.

Previous reports have shown that pharmacological inhibition of the PDGF-ß-receptor tyrosine kinase activity (5 , 6) or systemic administration of a PDGF-B Ab (8) inhibited vascular remodeling after angioplasty. However, our present study provides novel mechanistic evidence that localized perivascular gene transfer to antagonize PDGF-B mediated signaling before angioplasty attenuates the contribution of adventitial cell migration elicited by balloon injury (9 , 10) . It has been shown that levels of PDGF are elevated in the vessel wall after angioplasty (2 ,4) ; therefore, localized sequestration of PDGF-B, specifically in the adventitial compartment by perivascular PDGFXR gene delivery before balloon angioplasty contributes to the attenuation of luminal area loss and reduced neointima thickness after injury through reduced adventitial cell migration. However, it is possible that by restricting PDGFXR gene transfer to the adventitia in this study the contribution of medial SMC proliferation to neointimal hyperplasia was unaffected. Nevertheless, our study provides further direct evidence that adventitial cells play a significant role in the remodeling process after angioplasty (9 10 11) . We have recently reported that antagonism of perivascular transforming growth factor-ß1 signaling attenuates vascular remodeling (9) ; however, there remains a paucity of data regarding the effects of localized antagonism of adventitial PDGF-B through perivascular PDGFXR gene transfer. It is likely that antagonism of PDGF-B signaling by PDGFXR overexpression attenuates the phenotypic modulation of adventitial cells to myofibroblasts that express markers of SMC differentiation, such as smooth muscle {alpha}-actin (unpublished observation), which subsequently migrate toward the neointima and synthesize ECM after balloon angioplasty (10 , 11) . It is also possible that PDGFXR overexpression may have had an additional effect on medial VSMC proliferation, perhaps through its diffusion to the outer layers of the media. Moreover, migratory adventitial cells carrying the PDGFXR transgene are also likely to secrete PDGFXR within the media and neointima and therefore also contribute to reduced SMC migration and proliferation and attenuation of restenosis.

In the present study, overexpression of PDGFXR in the carotid artery adventitia resulted in a 38% decrease in both intimal thickness and intimal:medial (I:M) area ratio at 14 days after balloon injury. In comparison, Rutherford et al. (14) reported that systemic delivery of a specific PDGF-B Ab resulted in a 47% reduction in rat carotid artery intimal thickness and 59% reduction in I:M area ratio at 8 days after balloon injury. In a similar rat carotid artery injury model, Yamasaki et al. (15) showed that subcutaneous administration of SU9518, a PDGF receptor tyrosine kinase inhibitor, reduced intimal thickness by 33% and I:M area ratio by 39% at 14 days after balloon injury. These findings in studies using different strategies to antagonize PDGF signaling are entirely consistent with our present data that demonstrate a similar degree in attenuation of vascular remodeling. However, our novel approach through adopting perivascular delivery of PDGFXR differs from previous reports in the literature as we have sought to antagonize PDGF signaling locally in the adventitia at the site of vascular injury. Adenoviral delivery may have led to a diminution of transgene expression over time and hence the more modest effects observed are consistent with a likely overall lower concentration of effective temporal and spatial PDGF antagonism and therefore entirely comparable with findings in these other reports. The novel mechanistic emphasis of our study is that antagonism of perivascular PDGF attenuates the migration of specifically labeled adventitial cells and their contribution to neointima formation.

An imbalance between MMPs and TIMPs contributes to vessel remodeling in restenosis (16 17 18) . This may be mediated in part by facilitation of adventitial cell migration through degradation ECM proteins by enhanced MMP activity and decreased TIMP expression (16) . Antagonism of PDGF-B by AdPDGFXR gene transfer in the present study diminished MMP-2 and enhanced TIMP-1 and TIMP-2 expression in the adventitial and medial compartments of balloon injured arteries (Fig. 5) and decreased collagen deposition (Fig. 6) . MMP-2, TIMP1, and TIMP-2 are key mediators of cell migration through matrix degradation and vascular remodeling in atherosclerosis and restenosis (16 , 18) . Modulation of their expression through perivascular PDGFXR gene transfer may partially account for the attenuation of adventitial cell invasion toward the lumen after balloon injury since PDGF-B has also been shown to alter MMP and TIMP expression in cultured vascular SMC (16 , 19) .

Our present findings provide novel mechanistic insights in demonstrating a "new trick" for PDGF-B in playing a key role in adventitial cell migration and neointimal remodeling after vascular injury. It is possible that PDGF-B activates mitogen activated protein kinase (MAPK) pathways (20) that contribute to phenotypic modulation of fibroblasts and SMC leading to their migration, proliferation, and synthesis of matrix components such as collagen in the vascular remodeling processes (1 2 3 , 21) ; however, these interactions between PDGF-B and MAPK vascular signaling pathways remain to be elucidated in vivo. It is likely that PDGFXR overexpression was likely to have attenuated adventitial cell migration to a greater extent than their subsequent proliferation since PDGF-B has been shown to mediate smooth muscle chemotaxis to a greater extent after arterial injury (22) .

Another potentially exciting "new trick" for PDGF-B within the vessel wall may be in mediating the maturation of progenitor cells after vascular injury since PDGF-B has been recently shown to differentiate vascular progenitor cells into a myofibroblast phenotype (23 , 24) . It is exciting to speculate that such perivascular progenitor cells may reside within the adventitial compartment of vessels and become activated to differentiate into smooth muscle-like cells during the pathogenesis of atherosclerosis and restenosis after angioplasty under the influence of PDGF-B (25) . Although we have not directly examined this in the present study, it is conceivable that a subset of the ß-gal-labeled adventitial cells observed migrating to the neointima may have originated as adventitial "progenitor" cells. In addition, far from being an "inert" compartment of the vessel wall, adventitial fibroblasts have been recently shown to be a source of reactive oxygen species (ROS), under the influence of PDGF-B, thereby contributing to myofibroblast proliferation, migration, and vascular remodeling after balloon injury (26 , 27) . PDGF-B has also been recently shown not only to enhance ROS generation in cultured vascular SMCs via NAD(P)H oxidase activity (28) , but also to modulate peroxiredoxin-II expression, an endogenous hydrogen peroxide scavenging antioxidant gene, after angioplasty in a murine model of vascular remodeling (29) , thus providing additional evidence to support the "prooxidant" potential of PDGF-B signaling in restenosis. Our present study and other recent reports have established that both of these "old dogs," adventitial cells and PDGF-B, do play "new tricks" in their contribution to vascular remodeling. We have provided further mechanistic insights for gene therapy approaches to attenuate adventitial cell migration and ECM deposition through localized antagonism of PDGF-B by in vivo perivascular gene transfer of PDGFXR. It may be possible that gene delivery to the adventitia after PCTA procedures through coated drug-eluting perivascular stent cuffs (30) may represent a novel therapeutic strategy for the reduction in the incidence of restenosis.

ACKNOWLEDGMENTS

We thank Nichola Figg, University of Cambridge for expert technical assistance; Dr. Y. Takuwa, Kanazawa University, Japan for generously providing the AdPDGFXR adenovirus; Dr. James Uney, University of Bristol, UK for providing the green fluorescent protein adenovirus; and Dr. Salman Rahman, King’s College, University of London, for assistance with the in vitro cell migration assay. This study was supported by the British Heart Foundation.

Received for publication December 8, 2005. Accepted for publication March 31, 2006.

REFERENCES

  1. Ross, R. (1999) Atherosclerosis: an inflammatory disease. N. Engl. J. Med. 340,115-126[Free Full Text]
  2. Schwartz, S. M., Reidy, M. A., O’Brien, E. R. M. (1995) Assessment of factors important in atherosclerotic occlusion and restenosis. Thromb. Haemost. 74,541-551[Medline]
  3. Schwartz, S. M. (1997) Smooth muscle migration in atherosclerosis and restenosis. J. Clin. Invest. 100,S87-S89[Medline]
  4. Lindner, V., Giachelli, C. M., Schwartz, S. M., Reidy, M. A. (1995) A subpopulation of smooth muscle cells in injured rat arteries expresses platelet-derived growth factor–B chain mRNA. Circ. Res. 76,951-957[Abstract/Free Full Text]
  5. Levitzki, A. (2005) PDGF receptor kinase inhibitors for the treatment of restenosis. Cardiovasc. Res. 65,581-586[Abstract/Free Full Text]
  6. Myllarniemi, M., Calderon, L., Lemstrom, K., Buchdunger, E., Hayry, P. (1997) Inhibition of platelet-derived growth factor receptor tyrosine kinase inhibits vascular smooth muscle cell migration and proliferation. FASEB J. 11,1119-1126[Abstract]
  7. Deguchi, J., Namba, T., Hamada, H., Nakaoka, T., Abe, J., Sato, O., Miyata, T., Makuuchi, M., Kurokawa, K., Takuwa, Y. (1999) Targeting endogenous platelet-derived growth factor B-chain by adenovirus-mediated gene transfer potently inhibits in vivo smooth muscle proliferation after arterial injury. Gene Ther. 6,956-965[CrossRef][Medline]
  8. Ferns, G. A., Raines, E. W., Sprugel, K. H., Motani, A. S., Reidy, M. A., Ross, R. (1991) Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science 253,1129-1132[Abstract/Free Full Text]
  9. Sartore, S., Chiavegato, A., Faggin, E., Franch, R., Puato, M., Ausoni, S., Pauletto, P. (2001) Contribution of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent bystander to active participant. Circ. Res. 89,1111-1121[Abstract/Free Full Text]
  10. Siow, R. C., Mallawaarachchi, C. M., Weissberg, P. L. (2003) Migration of adventitial myofibroblasts after vascular balloon injury: insights from in vivo gene transfer to rat carotid arteries. Cardiovasc. Res. 59,212-221[Abstract/Free Full Text]
  11. Mallawaarachchi, C. M., Weissberg, P. L., Siow, R. C. (2005) Smad7 gene transfer attenuates adventitial cell migration and vascular remodeling after balloon injury. Arterioscler. Thromb. Vasc. Biol. 25,1383-1387[Abstract/Free Full Text]
  12. Duan, D. S., Pazin, M. J., Fretto, L. J., Williams, L. T. (1991) A functional soluble extracellular region of the platelet-derived growth factor (PDGF) ß-receptor antagonizes PDGF-stimulated responses. J. Biol. Chem. 266,413-418[Abstract/Free Full Text]
  13. Lambiase, P., Edwards, R., Anthopoulos, P., Rahman, S., Meng, Y., Bucknall, C., Redwood, S., Pearson, J., Marber, M. (2004) Circulating humoral factors and endothelial progenitor cells in patients with differing coronary collateral support. Circulation 109,2986-2992[Abstract/Free Full Text]
  14. Rutherford, C., Martin, W., Salame, M., Carrier, M., Anggard, E., Ferns, G. (1997) Substantial inhibition of neo-intimal response to balloon injury in the rat carotid artery using a combination of antibodies to platelet-derived growth factor-BB and basic fibroblast growth factor. Atherosclerosis. 130,45-51[CrossRef][Medline]
  15. Yamasaki, Y., Miyoshi, K., Oda, N., Watanabe, M., Miyake, H., Chan, J., Wang, X., Sun, L., Tang, C., McMahon, G., Lipson, K. E. (2001) Weekly dosing with the platelet-derived growth factor receptor tyrosine kinase inhibitor SU9518 significantly inhibits arterial stenosis. Circ. Res. 88,630-636[Abstract/Free Full Text]
  16. Galis, Z. S., Khatri, J. J. (2002) Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ. Res. 90,251-262[Abstract/Free Full Text]
  17. Shi, Y., Patel, S., Niculescu, R., Chung, W., Desrochers, P., Zalewski, A. (1999) Role of matrix metalloproteinases and their tissue inhibitors in the regulation of coronary cell migration. Arterioscler. Thromb. Vasc. Biol. 19,1150-1155[Abstract/Free Full Text]
  18. Newby, A. C. (2005) Dual role of matrix metalloproteinases (matrixins) in intimal thickening and atherosclerotic plaque rupture. Physiol. Rev. 85,1-31[Abstract/Free Full Text]
  19. Lau, H. K. (1999) Regulation of proteolytic enzymes and inhibitors in two smooth muscle cell phenotypes. Cardiovasc. Res. 43,1049-1059[Abstract/Free Full Text]
  20. Peppel, K., Zhang, L., Orman, E. S., Hagen, P. O., Amalfitano, A., Brian, L., Freedman, N. J. (2005) Activation of vascular smooth muscle cells by TNF and PDGF: overlapping and complementary signal transduction mechanisms. Cardiovasc. Res. 65,674-682[Abstract/Free Full Text]
  21. Ohashi, N., Matsumori, A., Furukawa, Y., Ono, K., Okada, M., Iwasaki, A., Miyamoto, T., Nakano, A., Sasayama, S. (2000) Role of p38 mitogen-activated protein kinase in neointimal hyperplasia after vascular injury. Arterioscler. Thromb. Vasc. Biol. 20,2521-2526[Abstract/Free Full Text]
  22. Buetow, B. S., Tappan, K. A., Crosby, J. R., Seifert, R. A., Bowen-Pope, D. F. (2003) Chimera analysis supports a predominant role of PDGFR-ß in promoting smooth-muscle cell chemotaxis after arterial injury. Am. J. Pathol. 163,979-984[Abstract/Free Full Text]
  23. Hu, Y., Zhang, Z., Torsney, E., Afzal, A. R., Davison, F., Metzler, B., Xu, Q. (2004) Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice. J. Clin. Invest. 113,1258-1265[CrossRef][Medline]
  24. Miyata, T., Iizasa, H., Sai, Y., Fujii, J., Terasaki, T., Nakashima, E. (2005) Platelet-derived growth factor-BB induces differentiation of bone marrow endothelial progenitor cell-derived cell line TR-BME2 into mural cells, and changes the phenotype. J. Cell. Physiol. 204,948-955[CrossRef][Medline]
  25. Torsney, E., Hu, Y., Xu, Q. (2005) Adventitial progenitor cells contribute to arteriosclerosis. Trends Cardiovasc. Med. 15,64-68[CrossRef][Medline]
  26. Rey, F. E., Pagano, P. J. (2002) The reactive adventitia: fibroblast oxidase in vascular function. Arterioscler. Thromb. Vasc. Biol. 22,1962-1971[Abstract/Free Full Text]
  27. Dourron, H. M., Jacobson, G. M., Park, J. L., Liu, J., Reddy, D. J., Scheel, M. L., Pagano, P. J. (2005) Perivascular gene transfer of NADPH oxidase inhibitor suppresses angioplasty-induced neointimal proliferation of rat carotid artery. Am. J. Physiol. 288,H946-H953
  28. Kreuzer, J., Viedt, C., Brandes, R. P., Seeger, F., Rosenkranz, A. S., Sauer, H., Babich, A., Nurnberg, B., Kather, H., Krieger-Brauer, H. I. (2003) Platelet-derived growth factor activates production of reactive oxygen species by NAD(P)H oxidase in smooth muscle cells through Gi1,2. FASEB J. 17,38-40[Abstract/Free Full Text]
  29. Choi, M. H., Lee, I. K., Kim, G. W., Kim, B. U., Han, Y. H., Yu, D. Y., Park, H. S., Kim, K. Y., Lee, J. S., Choi, C., et al (2005) Regulation of PDGF signaling and vascular remodeling by peroxiredoxin II. Nature 435,347-353[CrossRef][Medline]
  30. Pires, N. M., van der Hoeven, B. L., de Vries, M. R., Havekes, L. M., van Vlijmen, B. J., Hennink, W. E., Quax, P. H., Jukema, J. W. (2005) Local perivascular delivery of anti-restenotic agents from a drug-eluting poly(epsilon-caprolactone) stent cuff. Biomaterials. 26,5386-5394[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
P. J. Pagano and D. D. Gutterman
The adventitia: The outs and ins of vascular disease
Cardiovasc Res, September 1, 2007; 75(4): 636 - 639.
[Full Text] [PDF]

Home page
 Cardiovasc ResHome page
R. C.M. Siow and A. T. Churchman
Adventitial growth factor signalling and vascular remodelling: Potential of perivascular gene transfer from the outside-in
Cardiovasc Res, September 1, 2007; 75(4): 659 - 668.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Summary
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
fj.05-5435fjev1
20/10/1686    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mallawaarachchi, C. M.
Right arrow Articles by Siow, R. C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mallawaarachchi, C. M.
Right arrow Articles by Siow, R. C. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS