Adenovirus-mediated gene delivery in animal models of vascular injury has provided insights into the mechanisms underlying vessel wall pathologies. We have previously demonstrated that overexpression of the Gax transcription factor inhibits neointimal formation in rat and rabbit models of arterial injury. Here, we evaluate potential mechanisms for the reduction in stenotic lesion size due to Gax overexpression. At 3, 7 and 14 days after injury the Ad-Gax-infected arteries displayed a marked decrease in medial vascular smooth muscle cell number (3 days, 54% reduction P < 0.01; 7 days, 41% reduction p < 0.003; 14 days, 49% reduction p < 0.02). at 3 days after injury, pcna expression was attenuated in the ad-gax-treated vessels compared with control vessels (65% reduction p < 0.02), indicating a reduction in cellular proliferation. at 7 days and 14 days after injury ad-gax-infected arteries exhibited elevated number of tunel-positive medial vsmcs compared with control-treated arteries (7 days, 9.2-fold increase p < 0.03; 14 days, 17.2-fold increase p < 0.03), indicating an induction of apoptotic cell death. these data suggest that deregulated gax expression induces first cell cycle arrest and then apoptosis in the vascular smooth muscle cells that contribute to the neointimal layer. therefore, the efficacy of this therapeutic strategy appears to result from the ability of the gax transcriptional regulator to modulate multiple cellular responses.
The vascular smooth muscle cells (VSMCs) that line the normal vessel wall are quiescent, contractile, non-migratory and display a differentiated phenotype. Upon injury these VSMCs become activated, migrate and proliferate to form an intimal lesion.1 These lesions also exhibit high frequencies of VSMC apoptosis.2,3,4
In VSMCs, the decision to proliferate, differentiate, or undergo apoptosis may be linked through the actions of common nuclear factors that function as nodal point regulators. Recent investigations have begun to identify transcription factors that regulate VSMC phenotype including Gax,5 Ets-1,6 GATA-6,7,8 and MEF2A.9,10 These transcription factors are regulated, either positively or negatively, by mitogens suggesting a function in coordinating the expression of cell cycle and apoptosis regulatory genes during VSMC phenotypic modulation.11
Homeobox genes are transcription factors that function as pleiotropic regulators of cell fate, functioning in either a positive or negative fashion to regulate cell growth12,13 and viability.14,15,16,17 The muscle-specific homeobox gene gax, first identified in rat aorta smooth muscle, is highly expressed in quiescent VSMCs where it is down-regulated upon mitogen stimulation5 or vascular injury.18 Adenovirus-mediated expression of Gax (Ad-Gax) in culture inhibits cell cycle activity by arresting cells in the G1 phase of the cell cycle.19 In addition, Gax-expressing cells undergo apoptotic cell death under conditions of prolonged mitogen-activation while quiescent cells are refractive to Gax-induced apoptosis.20
When the effects of constitutive Gax expression were analyzed in vivo, neointimal hyperplasia was markedly attenuated in both rat carotid19 and rabbit iliac arteries21 following injury. Other investigators have reported similar results with adenovirus-mediated expression of cell cycle inhibitors or cytotoxic genes in the vessel wall.22,23,24,25 In most cases, the effect of adenovirus-mediated transgene overexpression at the cellular level was not analyzed. Here, we have elucidated a potential mechanism for the reduction in stenotic lesion size that occurs in the presence of constitutive Gax expression. At 3, 7 and 14 days after injury the Ad-Gax-infected arteries displayed a decrease in medial cell number which can be attributed to a reduction in cell proliferation and/or cell viability. At 3 days after injury PCNA expression was markedly attenuated in the Ad-Gax-treated vessels compared with the control vessels, suggesting that Gax overexpression inhibits cell cycle activity in vivo. At 7 and 14 days Ad-Gax-infected arteries displayed elevated numbers of TUNEL-positive medial VSMCs compared with saline- or Ad-β-gal-treated arteries, suggesting that Gax also induces apoptosis in vivo. Thus, Ad-Gax promotes vessel patency by inducing both cell cycle arrest and apoptosis in the VSMCs that contribute to the hyperproliferative lesion.
Gax mediated inhibition of neointimal hyperplasia
Balloon injury to the rat carotid artery results in the formation of a neointimal layer composed of proliferating VSMCs.26,27 Within hours of injury, Gax mRNA18 and protein (HP, unpublished data) are down-regulated in the medial VSMCs of the vessel wall. Saline-treated vessels or vessels infected with a control virus that expresses β-galactosidase (Ad-β-gal), develop considerable neointimal mass by 2 weeks following injury. In contrast, the proliferative response following balloon injury in arteries infected with a replication-deficient adenovirus expressing Gax (Ad-Gax) were markedly attenuated.19
We have expanded on this earlier study by examining neointimal formation in response to various doses of Ad-Gax. Rat carotid arteries were denuded with a balloon catheter and immediately exposed to 1 × 107 p.f.u. to 1 × 1010 p.f.u. of Ad-Gax or 1 × 1010 p.f.u. Ad-β-gal virus. Following a 20-min infection period, the viral solution was withdrawn and the ligatures were removed to restore circulation. Rats were killed 2 weeks later and quantitative morphometric analyses were performed on cross sections of the treated vessels. The Ad-β-gal-transduced carotid arteries had developed considerable neointimal thickening with a intima:media ratio of 1.31 ± 0.13. In contrast, Ad-Gax-treatment markedly reduced the hyperproliferative response to balloon injury at doses of 1 × 108 p.f.u. or greater. All vessels treated with the high doses of Ad-Gax displayed a reduced intima:media ratios of 0.53 ± 0.12 (1 × 108 p.f.u.), 0.68 ± 0.15 (1 × 109 p.f.u.), and 0.62 ± 0.15 (1 × 1010 p.f.u.). Statistical analysis confirmed that Ad-Gax treatment significantly inhibited the development of intimal thickening relative to the Ad-β-gal controls (Figure 1). In contrast, balloon-injured rat carotid arteries infected with a low dose of Ad-Gax (1 × 107) displayed neointimal formation (intima:media ratio 1.25 ± 0.14) similar to Ad-β-gal-treated or saline-treated vessels (intima:media ratio 1.4 ± 0.14).
Ad-Gax reduces medial cell number
Previous studies in rat carotid arteries have shown that balloon injury results in a 65% loss of medial VSMCs within 4 h.28 Under normal conditions the repopulation of the media is complete by 3 days.25 Thus, we assessed the inhibitory action of Ad-Gax on hyperplasia by examining medial VSMC number at 3, 7 and 14 days after injury in the Ad-Gax- and Ad-β-gal-treated arteries. At 3 days after injury the Ad-Gax-treated arteries displayed a 50% decrease in media cell number compared with the saline- and Ad-β-gal-treated vessels (Figure 2). At 7 days after injury, the Ad-Gax infected vessels displayed a 41% decrease in medial cell number compared with the saline-treated vessels. A decrease was also evident at 14 days after injury as arteries transduced with Ad-Gax contained only 37% and 59% of the medial cells found in Ad-β-gal- or saline-treated vessels, respectively.
Ad-Gax reduces medial PCNA expression
Since Ad-Gax infection inhibits cell cycle progression in vitro,19 PCNA expression was analyzed by immunohistochemistry to determine if Ad-Gax inhibits VSMC proliferation in vivo. Previous investigations have demonstrated that peak medial cell proliferation occurs between 48 and 72 h after balloon injury.27,29 At 3 days after injury arteries treated with Ad-Gax (Figure 3a) displayed decreases of 67% and 65% in PCNA expression compared with saline- and Ad-β-gal-treated vessels, respectively (Figure 3b). At later time-points (7 and 14 days) little or no medial PCNA expression was detected under any condition analyzed (not shown), consistent with previous reports.27,29 Thus, Gax overexpression appears to decrease VSMC populations at early time-points through cell cycle inhibition.
Gax induces VSMC apoptosis in vivo
To test whether enhanced VSMC apoptosis also contributed to a decrease in lesion size in Ad-Gax-treated vessels, DNA fragmentation analysis by TdT-mediated dUTP-fluorescein (TUNEL) labeling was performed on arterial sections that were harvested at 3, 7 and 14 days after injury. At 3 days after injury no significant difference in TUNEL-positive medial VSMCs was observed in the Ad-Gax transduced arteries compared with the saline- or Ad-β-gal-treated arteries. However, at 7 and 14 days after injury the Ad-Gax-infected vessels displayed a marked increase in TUNEL-positive medial VSMCs. Pyknotic nuclei, indicated by condensed chromatin, (Figure 4a) co-localized with TUNEL-positive cells in the Ad-Gax-treated arteries, providing further evidence that the TUNEL-positive cells are apoptotic. Quantitative analysis revealed that Ad-Gax infected arteries harvested at 7 and 14 days after injury displayed 22-fold (P < 0.05) and 44.3-fold (P < 0.01) increases, respectively, in the number of TUNEL-positive medial VSMCs compared with saline-treated or Ad-β-gal-infected arteries (Figure 4b). Thus, deregulated Gax expression induces apoptosis in vivo and may be responsible for a reduction in the hyperproliferative response of VSMCs to balloon injury at later time-points.
Though numerous cytostatic and cytotoxic agents have been delivered to the vessel wall to inhibit intimal hyperplasia, few studies have examined the mechanisms of transgene action at a cellular level in vivo. In carotid arteries, the expression of the transcription factor Gax is down-regulated upon injury.18 This down-regulation may be required for successful VSMC cell cycle progression or to ensure cell survival during the initial proliferative response. This appears to be the case in vivo since forced Gax expression inhibits injury-induced intimal hyperplasia.19,21 In this paper we addressed the cellular mechanisms that may be responsible for this inhibition.
We have evaluated the effects of Gax overexpression on intimal hyperplasia in balloon-injured rat carotid arteries at the cellular level. At 3, 7 and 14 days after injury, arteries infected with Ad-Gax displayed reduced medial cell populations compared with either saline- or Ad-β-gal-treated vessels. The medial cell decrease at 3 days after injury may be attributed to the growth arrest by Gax overexpression and the inhibition of VSMC repopulation of the injured vessels, as PCNA expression was decreased by 67% and 65% in the Ad-Gax-treated arteries compared with saline- or Ad-β-gal-transduced vessels, respectively. No significant difference in the apoptosis rate in the 3-day injured vessels was observed between the Ad-Gax and saline- or Ad-β-gal-treated arteries. However, by 7 or 14 days following injury, arteries treated with Ad-Gax exhibited a significant increase in medial-VSMC apoptosis compared with control arteries. Apoptosis induced by Gax overexpression was largely observed in the medial, but not the neointimal layer. Consistent with these observations, immunohistochemical analyses revealed positive transgene expression using anti-HA antibody in medial, but not neointimal VSMCs (not shown).
Since homeodomain proteins generally function as pleiotropic regulators through their abilities to activate and suppress batteries of downstream genes, it is possible that Gax overexpression following acute vascular injury might have multiple effects on medial VSMCs. Here, we have shown that Gax exhibits dual actions in VSMCs of balloon-injured rat carotid arteries, promoting both cell cycle arrest and apoptosis, consistent with previous in vitro observations.19,20 It is noteworthy that Gax is unable to induce apoptosis in quiescent VSMCs, apoptosis is observed only in proliferating cells,20 the subpopulation that forms the stenotic lesion in injured vessels.29 Such a lack of toxicity to quiescent VSMCs indicates that Ad-Gax should prove an advantageous therapeutic strategy for treating conditions characterized by VSMC hyperproliferation.
Materials and methods
Rat model of smooth muscle hyperplasia
The model of balloon injury was based on that described by Clowes et al.29 Male Sprague–Dawley rats weighing 400–500 g were anesthetized with an intraperitoneal injection of sodium pentobarbital (45 mg/kg, Abbot Laboratories, North Chicago, IL, USA). The bifurcation of the left common carotid artery was exposed through a midline incision and the left common, internal and external carotid arteries were temporarily ligated. A 2F embolectomy catheter (Baxter Edwards Healthcare, Irvine, CA, USA) was introduced into the external carotid and advanced to the distal ligation of the common carotid. The balloon was inflated with saline and drawn towards the arteriotomy site three times to produce a distending, de-endothelializing injury. A temporarily isolated segment of the artery was treated with saline or 1 × 107 p.f.u. to 1 × 1010 p.f.u. in a volume of 100 μl of either Ad-Gax19,20 or Ad-β-gal.19,20,30 The solution was injected via a cannula inserted just proximal to the carotid bifurcation and the vessel was incubated for 20 min, after which the solution was withdrawn and the cannula removed. All transgenes are expressed under control of the cytomegalovirus promoter. The proximal external carotid artery was ligated and blood flow was restored to the common carotid artery by release of the ligatures. The experimental protocol was approved by the Institutional Animal Care and Use Committee and complied with the ‘Guide for the Care and Use of Laboratory Animals’ (NIH Publication No. 86–23, revised 1985).
Rats were killed at 3, 7 and 14 days following treatment with an intraperitoneal injection of pentobarbital (100 mg/kg). The balloon injured segment of the left common carotid artery, from the proximal edge of the omohyoid muscle to the carotid bifurcation, was perfused with saline and dissected from the surrounding tissue. Tissue was fixed in 4% paraformaldehyde or 100% methanol until embedded in paraffin. Several 5-μm sections were cut from each tissue specimen. Separate sections from each specimen were stained with hematoxylin and eosin or with Richardson’s combination elastic-trichrome stain for conventional light microscopic analysis.
Histological images of cross sections of hematoxylin and eosin or elastic-trichrome-stained arterial sections were projected on to a digitizing board (Summagraphics, Fairfield, CT, USA) and the intimal, medial and luminal areas were measured by quantitative morphometric analysis using a computerized sketching program (MACMEASURE, version 1.9, National Institute of Mental Health, Bethesda, MD, USA).
Results were expressed as the mean ± s.e.m. Differences between groups were analyzed using an unpaired two-tailed Student’s t test.
TUNEL labeling and nuclear condensation
Paraformaldehyde fixed sections (5 μm) were deparaffinized and rehydrated. The tissue was permeabilized with 20 mg/ml of proteinase K for 30 min after which fluorescein-conjugated dUTP and TdT enzyme were added according to the manufacturer’s specifications (Boehringer Mannheim, Indianapolis, IN, USA; in situ death detection kit). Nuclei were counterstained with Hoechst 33258 (Sigma, St Louis, MO, USA), and mounted for examination using mounting media for fluorescence (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). Specimens were examined and photographed on a Nikon (Tokyo, Japan) Diaphot microscope equipped with a phase-contrast and epifluorescence optics.
Five-micron sections from uninjured and injured rat arterial tissue fixed in methanol were deparaffinized and blocked in 10% horse serum. Sections were incubated with mouse anti-PCNA antibody (Signet, Dedham, MA, USA) or mouse IgG (Sigma). Prostates from rats 3 days after castration were used as a positive control.31 A biotinylated horse-anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA, USA) followed by alkaline phosphatase (BioGenex, San Ramon, CA, USA) conjugated to streptavidin were used to detect primary antibody complexes. Visualization was accomplished using the Vector Blue alkaline phosphatase substrate kit III (Vector Laboratories).
This research was supported by National Institutes of Health grants HL50692 and AR40197 awarded to KW. KW was an established investigator of the American Heart Association during the course of this study. We thank Linda Whittaker for assistance in preparation of this manuscript.
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Perlman, H., Luo, Z., Krasinski, K. et al. Adenovirus-mediated delivery of the Gax transcription factor to rat carotid arteries inhibits smooth muscle proliferation and induces apoptosis. Gene Ther 6, 758–763 (1999) doi:10.1038/sj.gt.3300893
- smooth muscle
- growth arrest
- gene therapy
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