Arterial response to mild balloon injury in the normal rabbit: evidence for low proliferation rate in the adventitia.

BACKGROUND: The role of constrictive remodeling, spasm and proliferation (particularly in the adventitia) in the genesis of chronic lumen narrowing after balloon injury remains under debate. This study analyzed the time course of these components following mild injury in normal arteries. METHODS: Iliac injury was induced by balloon overstretch in 32 rabbits, sacrificed at timed intervals from day 3 to 28. Angiographic response to nitrates, morphometric, immunohistochemical and biochemical analysis were performed at each time point. RESULTS: Quantitative angiography showed a decrease in lumen diameter and no change in response to nitrates over time. On morphometric analysis, remodeling was usually constrictive, appeared as early as day 3 and was responsible for 69+/-14% of the histologic lumen area stenosis at day 28. Constrictive remodeling was correlated negatively to intimal hyperplasia (r= 0.51, P< 0.002) and positively to the lumen area stenosis (r= 0.92, P< 0.0001). Macrophages (labeled by anti-RAM 11 antibodies) were very rare at all time points. Immunohistochemistry identified a high rate of proliferating smooth muscle cells in the media (13+/-7%) and intima (49+/-8%) at day 7, which decreased rapidly. Proliferating cells in the adventitia were rare (3+/-2% at day 7). The number of proliferating cells was time-dependent (r= 0.82, P< 0.0001) and related to cyclin A mRNA measured by reverse transcription-polymerase chain reaction (r= 0.84, P< 0.0001). CONCLUSIONS: In this model, luminal loss was mainly caused by constrictive remodeling rather than intimal hyperplasia. Constrictive remodeling appeared early and was not time-dependent. Macrophages, spasm and adventitial proliferation did not contribute to this constrictive remodeling.

Pre-treatment with elastase improves the efficiency of percutaneous adenovirus-mediated gene transfer to the arterial media.

The endothelium and internal elastic lamina (IEL) appear to be the main barriers to adenovirus-mediated gene transfer to medial smooth muscle cells (SMC). The present randomized study tested whether controlled incubation with elastase enhanced the efficiency of catheter-based gene transfer to medial SMC by adenoviral vectors. After an initial safety dose ranging study, rabbits underwent balloon abrasion of the iliac endothelium followed by local incubation of either elastase (2 x 10(-7) IU over 5 min) or saline using a double balloon catheter (DBC). Then, adenoviral vectors (5 x 10(9) p.f.u.) carrying Cmv-Luc or RSV-beta gal reporter genes were instilled for 30 min. Three days later, the number of medial SMC expressing lacZ was increased in the elastase-treated arteries compared with saline-treated arteries (7.2 +/- 2.5 versus 2.3 +/- 0.9 cells per section, P = 0.003). Likewise, the amount of luciferase protein product was increased (70 +/- 32 versus 36 +/- 15 pg luciferase/mg tissue, P = 0.03). No vessel enlargement, light or electron microscopic evidence of injury or inflammation was seen in elastase-treated arteries up to 7 weeks. Preincubation with elastase increased transduction efficiency of catheter-based gene delivery of replication-defective adenoviral vectors to rabbit iliac arteries without detectable arterial damage.

Reduction of restenosis after angioplasty in an atheromatous rabbit model by suicide gene therapy.

BACKGROUND: Gene delivery of the thymidine kinase (tk) gene combined with ganciclovir (GCV) limits intimal hyperplasia after abrasion of normal arteries. However, the low efficiency of adenoviral-mediated gene transfer to atherosclerotic arteries has raised concerns about the applicability of this strategy to the prevention of restenosis. METHODS AND RESULTS: A replication-defective adenoviral vector expressing tk (Ad-RSVtk) demonstrated selective toxicity toward GCV-treated arterial smooth muscle cells, with oligonucleolytic cleavage suggesting apoptosis. In vivo, after demonstration of tk expression after Ad-RSVtk delivery, the combination of Ad-RSVtk followed by GCV was tested in a rabbit model of angioplasty of atheromatous iliac arteries. Angioplasty (8 atm, 20 minutes) was performed by use of a hydrogel balloon coated with Ad-RSVtk (4x10(9) plaque forming units). GCV was infused (25 mg.kg(-1) I.V. BID) from days 2 through 7 after angioplasty in 8 of 12 rabbits. Four weeks later, morphometric analysis demonstrated a reduced intima-to-media ratio in the group receiving combination therapy compared with Ad-RSVtk alone (3.0+/-1.2 versus 5.2+/-0.5, P<.018). GCV per se had no effect on intimal hyperplasia after arterial injury. CONCLUSIONS: In vitro, Ad-RSVtk demonstrates selective toxicity toward GCV-treated arterial smooth muscle cells involving apoptosis. In vivo, GCV conditions reduction of neointimal formation after percutaneous delivery of Ad-RSVtk during angioplasty of atheromatous arteries.

The Dispatch catheter as a delivery tool for arterial gene transfer.

OBJECTIVE: Most currently available percutaneous delivery methods for arterial gene therapy are limited by the need for a long incubation period, which may lead to unacceptable tissue ischemia, especially in the coronary vasculature. Conversely, shorter incubation times may result in inefficient gene transfer, especially in atheromatous arteries. A new local delivery autoperfusion multichamber catheter is now available which permits local delivery in the coronary arterial system without inducing myocardial ischemia. The present study aimed at evaluating the performance of this catheter for achieving arterial gene transfer using replication-defective adenoviral vectors in normal and atheromatous arteries. METHODS: A replication-defective adenoviral vector carrying a nuclear-targeted beta-galactosidase reporter gene (Ad-RSV beta gal, 5.10(9) plaque-forming units [pfu]) was delivered to the iliac arteries of normal (n = 7) and atheromatous (1% cholesterol diet + arterial abrasion) (n = 6) rabbits, via a multichamber autoperfusion balloon catheter (Dispatch, SciMed). Duration of gene delivery was 60 min. RESULTS: Three days later, marked expression of the reporter gene was detected by histochemistry in the endothelium at the delivery site (percentage of transfected cells: 16 +/- 8% / artery (range 11-25%). There was a low transduction rate in medial smooth muscle cells 0.7 +/- 0.4%/artery (range 0.3-1.1%). In atheromatous arteries, transduction was consistently achieved in the superficial layers of the neointima but was lower (1.1 +/- 0.5%/artery, range 0.3-1.7%). Transgene expression was detected by histochemistry in the liver of 3/13 animals, suggesting that there is a substantial risk of systemic dissemination of the viral vectors. CONCLUSION: Efficient arterial gene delivery to endothelial and superficial smooth muscle cells is feasible using local delivery of adenoviral vectors via the Dispatch autoperfusion catheter, in both normal and atheromatous arteries. This perfusion catheter may be a useful tool for coronary artery gene transfer.

Adenovirus-mediated arterial gene therapy for restenosis: problems and perspectives.

Restenosis remains the main limitation of interventional cardiology. Restenosis occurs when angioplasty-induced intimal hyperplasia as well as arterial remodelling result in flow-limiting renarrowing of the arterial lumen at the angioplasty site. Intimal hyperplasia is an important candidate for gene therapy since it is related to smooth muscle cell proliferation, which is an inviting target for molecular antiproliferative strategies. To date, adenoviral vectors are, by far, the most efficient vectors to perform in vivo arterial gene transfer. These vectors, as well as others, have been recently used to demonstrate that therapeutic genes encoding cytotoxic (herpes virus thymidine kinase) or cytostatic (hypophosphorylatable Rb, Gax, endothelial nitric oxide synthase) products successfully inhibit smooth muscle cell proliferation and related intimal hyperplasia. Despite substantial progress, major technical issues, including the toxicity of first-generation adenoviral vectors, inefficient transduction of atherosclerotic arteries, and the risk of extra-arterial transfection remain to be addressed before gene therapy is applied to clinical restenosis.

Perspectives of arterial gene therapy for the prevention of restenosis.

Restenosis remains the main limitation of interventional cardiology. Restenosis is an important target for gene therapy since it is frequent (30% of patients), costly (estimated $2 billion annually), refractory to all pharmacological therapies, and related, at least in part, to smooth muscle cell proliferation which is an inviting target for antiproliferative molecular strategies. Because cell division is ultimately controlled by intranuclear events, the protein product of genes selected for their antiproliferative effects usually remains inside the cells. Consequently, the transfer of growth-inhibitory genes needs to be efficient-i.e., involve a large proportion of smooth muscle cells populating the angioplasty site. To date, adenoviral vectors are, by far, the most efficient vectors to perform in vivo arterial gene delivery. These vectors, as well as others, have been recently used to demonstrate that therapeutic genes encoding cytolytic (thymidine kinase) or cytostatic (hypophosphorylatable retinoblastoma protein, endothelial nitric oxide synthase, gax, etc.) products successfully inhibit smooth muscle cell proliferation and related intimal hyperplasia. Despite substantial progress, major technical issues remain to be addressed before gene therapy is applied to clinical restenosis. First-generation recombinant adenoviruses evoke both cellular and humoral immune responses leading to local toxicity and transient gene expression. Moreover, the low efficiency of gene transfer to atherosclerotic arteries may further impair the biological effect of antiproliferative genes. Finally, restenosis is a multifactorial phenomenon in which intimal hyperplasia plays an important but not exclusive role. Prevention of constrictive remodeling should also be taken into account in an integrated genetic strategy to prevent restenosis.

[Prospects of prevention of restenosis after coronary angioplasty]. / Perspectives de prévention de la resténose après angioplastie coronaire.

The prevention of restenosis after coronary angioplasty has been marked over recent years by the failure of trials of drug treatment based on inhibition of arterial smooth muscle cell proliferation. This failure could be due to an insufficient concentration of the orally or intravenously administered drug in the lesion to be treated. Another reason for this failure of drug treatment could be the nonexclusive role of intimal hyperplasia in the pathophysiology of restenosis, which also appears to be related to a education of the overall calibre of the artery at the site of dilatation. The pathogenesis of this phenomenon, called "remodelling", remains obecure and is only partly prevented by insertion of stents, which is currently the only treatment able to decrease the number of new revascularization procedures for restenosis. This benefit is related to optimization of the initial result (stents avoiding early "recoil", secondary to the elastic recoil forces of the arterial wall), and possibly to prevention of late remodelling of the vessel at the site of dilatation, either tonic (vasomotor) or trophic. On the other hand, the benefit related to the absence of "remodelling" of the stended lesions is partly limited by intimal hyperplasia, exacerbated by the presence of the stent. The future therapeutic strategy could combine insertion of stents and prevention of smooth muscle cell proliferation by new treatment strategies acting at the molecular level. Encouraging preliminary results have already been obtained in animals with chimeric toxins, antisense strategies and especially gene therapy using defective adenoviral vectors for replication.

Reduction of intimal hyperplasia by naroparcil, a 4-methylumbelliferyl beta-D-xyloside analogue, after arterial injury in the hypercholesterolemic rabbit.

4-Methylumbelliferyl beta-D-xylosides (beta-D-xylosides) inhibit proteoglycan synthesis, and this is associated with reduced proliferation and extracellular matrix production by vascular smooth muscle cells. This study evaluated whether treatment with naroparcil, a beta-D-xyloside analogue, reduced intimal hyperplasia after arterial injury in the hypercholesterolemic rabbit. Forty-two rabbits were assigned to three groups that received either a 1% cholesterol-enriched diet (group 1, n = 15) or the same diet with added 100 mg.kg-1 naroparcil (group 2, n = 15) or 300 mg.kg-1 naroparcil (group 3, n = 12). All animals underwent iliac artery endothelial abrasion at day 14 and were killed at day 56. At the time of death, the angiographic minimal luminal diameter was significantly larger in both treated groups. Morphometric analysis showed a larger luminal area in treated rabbits (groups 2 and 3) compared with control rabbits (group 1) (0.75 +/- 0.54 and 0.85 +/- 0.61 mm2 versus 0.32 +/- 0.25 mm2, respectively; P < .05), with a decreased intimal thickness in groups 2 and 3 (average reduction of 37% and 39%, respectively, compared with group 1; P < .05) but without changes in medial area. Total vessel area was comparable among all groups. In the media, immunohistochemistry suggested reduced infiltration by macrophages and a larger fractional area of smooth muscle cells. There were no differences in plasma or arterial wall cholesterol content between groups. Plasma levels of glycosaminoglycans and dermatan sulfate content were increased only in group 3. In this model, oral treatment with naroparcil appears to preserve the arterial lumen and reduce intimal thickness after arterial injury.

Arterial gene transfer to rabbit endothelial and smooth muscle cells using percutaneous delivery of an adenoviral vector.

BACKGROUND: Previous investigations in live animals convincingly established that arterial gene transfer, while feasible, was compromised by a low transfection efficiency. More recent studies have shown that transfection efficiency may be substantially augmented by the use of recombinant adenoviral vectors. Most in vivo transfections reported to date, however, have used direct (operative) administration of the adenoviral vector. Clinical applications of arterial gene transfer (such as prevention of restenosis), however, would require local percutaneous delivery of the transgene. The present study was designed to extend in vivo intraoperative findings to percutaneous delivery system and to assess whether gene transfer remains site specific. METHODS AND RESULTS: A recombinant, replication-defective adenovirus modified to include an expression cassette for nucleus-targeted beta-galactosidase was introduced into rabbit iliac arteries in vivo using either a double-balloon catheter (DBC, n = 27) or a hydrogel-coated balloon catheter (HBC, n = 27). Contralateral arteries-normal, endothelium-denuded, or sham-transfected with a control adenoviral vector-served as controls. beta-Galactosidase expression was assessed by X-Gal staining. Cell-transduction efficiency was measured by morphometric analysis. Polymerase chain reaction (PCR) and histochemistry were used to detect the presence and/or expression of viral DNA in remote organs. Transgene expression was detected in all cases (46 of 46) between 3 and 14 days after transfection but was in no case detectable 28 days after transfection. In the DBC group, transgene expression was limited to endothelial cells when the endothelium was left intact and to rare medial cells (< 2.2%) when it had been removed. In contrast, HBC delivery resulted in transduction of up to 9.6% of medial smooth muscle cells (P = .0001). Optimized PCR and histochemistry failed to detect evidence of extra-arterial transfection except in a small number of cells (between 1 in 3 x 10(2) and 1 in 3 x 10(5) cells) in the livers of 2 animals in the DBC group. CONCLUSIONS: (1) Efficient, adenovirus-mediated, arterial gene transfer to endothelial and/or smooth muscle cells is feasible by percutaneous, clinically applicable techniques. (2) Consistent transfection of medial smooth muscle cells may be achieved when the endothelial layer is abraded. (3) Medial transfection is more efficient when an HBC, rather than a DBC, is used. (4) Percutaneous delivery of the adenoviral vector via HBC results in site-specific arterial gene transfer. Very-low-level extra-arterial transfection may occur, however, when the DBC is used.