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,1
* Division of Cardiovascular Medicine, School of Clinical Medicine, University of Cambridge, Addenbrookes Hospital, Cambridge, UK; and
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, Guy’s Hospital Campus, London SE1 1UL, UK. E-mail: richard.siow{at}kcl.ac.uk
SPECIFIC AIMS
Migration of adventitial fibroblasts contributes to vascular remodeling after percutaneous transluminal coronary angioplasty. This study has used in vivo perivascular adenoviral gene transfer of a soluble truncated platelet-derived growth factor (PDGF)-ßbeta;-receptor (PDGFXR) to investigate whether antagonism of PDGF signaling in the adventitia attenuates adventitial cell migration, collagen deposition, and expression of matrix metalloproteinases (MMPs) after balloon angioplasty in rat carotid arteries.
PRINCIPAL FINDINGS
1. Antagonism of PDGF-B by overexpression of the soluble PDGF-ßbeta;-receptor
Adenoviral pairs coordinating expression of ßbeta;-galactosidase (ßbeta;-gal; LacZ) and PDGFXR or LacZ and green fluorescent protein (GFP) were applied to the perivascular surface of arteries and balloon catheter mediated vascular injury performed 4 days later in some animals. Arterial sections from common carotid arteries were excised at 7 or 14 days after gene transfer and incubated in culture medium for 24 h. The conditioned media were collected, and levels of secreted soluble PDGFXR were elevated only in conditioned media of carotid artery sections that had been transfected with AdPDGFXR at both time points. Overexpression of PDGFXR in cultured rat aortic smooth muscle cells significantly (32±4%, P<0.05, n=3) attenuated their migration in response to exogenous PDFG-B, thereby confirming efficacy of PDGFXR gene transfer.
2. Attenuation of adventitial cell migration and vascular remodeling after vascular injury by PDGFXR gene transfer
Uninjured carotid arteries only expressed LacZ positive cells in the adventitia; however, after injury in control arteries transfected with LacZ and GFP, ßbeta;-gal positive adventitial cells contributed to the population of cells within the media and neointima at 7–14 days. Overexpression of PDGFXR and LacZ in the adventitia resulted in a significant reduction in the number of LacZ labeled cells in the neointima at 7 and 14 days after vascular injury compared with control LacZ and GFP transfected arteries (Fig. 1
) and was associated with a significant reduction in vascular remodeling as shown by an attenuation of luminal area loss, neointimal thickening and neointimal to medial area ratios.
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3. Attenuation of collagen deposition and MMP expression after vascular injury by PDGFXR gene transfer
Perivascular overexpression of PDGFXR significantly attenuated adventitial and medial MMP-2 expression at 7 days after balloon injury compared with levels in injured control carotid arteries transfected with Adßbeta;-gal and AdGFP. However, adventitial immunoreactivity for the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 was significantly elevated in balloon injured arteries transfected with AdPDGFXR and Adßbeta;-gal compared with vessels transfected with Adßbeta;-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 Adßbeta;-gal and AdGFP transfected vessels.
CONCLUSIONS AND SIGNIFICANCE
Percutaneous transluminal coronary angioplasty in humans for the treatment coronary artery occlusion often results in restenosis, affecting >30% of patients who undergo balloon angioplasty without stenting. 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 extracellular matrix (ECM). 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 smooth muscle cells. Our present study represents the first demonstration that in vivo perivascular gene transfer of the extracellular region of the PDGF-ßbeta;-receptor antagonizes PDGF-B mediated migration of adventitial cells in balloon injured carotid arteries. Our findings show that perivascular PDGFXR overexpression results in secretion of the soluble PDGF-ßbeta;-receptor for up to 14 days after gene transfer. Moreover, migration of cultured aortic smooth muscle cells secreting the PDGF ßbeta;-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-ßbeta;-receptor tyrosine kinase activity or systemic administration of a PDGF-B antibody 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. It has been shown that levels of PDGF are elevated in the vessel wall after angioplasty; 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 smooth muscle cell 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. We have recently reported that antagonism of perivascular transforming growth factor-ßbeta;1 signaling attenuates vascular remodeling; 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 smooth muscle cells (SMC) differentiation, such as smooth muscle 
-actin (unpublished observation), which subsequently migrate toward the neointima and synthesize ECM after balloon angioplasty.
An imbalance between MMPs and TIMPs contributes to vessel remodeling in restenosis. This may be mediated in part by facilitation of adventitial cell migration through degradation ECM proteins by enhanced MMP activity and decreased TIMP expression. 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 and decreased collagen deposition. MMP-2, TIMP1, and TIMP-2 are key mediators of cell migration through matrix degradation and vascular remodeling in atherosclerosis and restenosis. 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.
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 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; 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.
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. 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. Although we have not directly examined this in the present study, it is conceivable that a subset of the ßbeta;-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. PDGF-B has been recently shown not only to enhance ROS generation in cultured vascular smooth muscle cells via NAD(P)H oxidase activity but also to modulate peroxiredoxin-II expression, an endogenous hydrogen peroxide scavenging antioxidant gene, after angioplasty in a murine model of vascular remodeling, 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 the "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. It may be possible that gene delivery to the adventitia after percutaneous transluminal coronary angioplasty through coated drug-eluting perivascular stent cuffs may represent a novel therapeutic strategy to reduce the incidence of restenosis.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5435fje
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  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]
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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]
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