Atrial natriuretic peptide preserves endothelial function during intimal hyperplasia

BACKGROUND: Atrial and C-type natriuretic peptides (ANP and CNP), acting through different receptors, have antiproliferative effects in vitro. Beneficial effects of CNP in vivo on early atherosclerosis have been described, but it is not known if ANP is antiproliferative in vivo. In the present study, the effects of chronic in vivo ANP were tested and compared with CNP on endothelial dysfunction and intimal thickening caused by peri-arterial collars. METHODS: Non-occlusive collars were placed bilaterally around the common carotid arteries of rabbits. One collar was filled with saline vehicle. The contralateral collar was filled with ANP or CNP (1 or 10 microM, n = 5-7) with slow replacement of peptide via mini-pump (1 or 10 fmol/h). RESULTS: After 7 days, endothelium-dependent vasorelaxation in saline-collared arteries was 33 +/- 3% of maximum [averaged over 0.03-1 muM acetylcholine (ACh)] compared to 64 +/- 2% in normal (uncollared) arteries (p < 0.05, n = 23). In vivo ANP restored the ACh relaxation to normal (e.g., 57 +/- 6%, 1 microM ANP), similar to effects seen with CNP in vivo. Endothelium-independent vasorelaxation of collared-vessels was not altered by either peptide. Intimal hyperplasia induced by the collars was not prevented by peri-arterial natriuretic peptides. In additional rabbits (n = 6), CNP (100 pmol/h) given directly into the lumen of collared carotid arteries for 7 days reduced neointima formation by 16 +/- 5% (p < 0.05), whereas ANP given intraluminally (100 pmol/h; n = 6) did not. CONCLUSIONS: The more potent actions of CNP on vascular smooth muscle cell migration and proliferation (established in vitro) may explain differences between the peptides on intimal hyperplasia in vivo. The major hallmark of atherosclerosis and restenosis, endothelial dysfunction, was prevented by chronic, peri-arterial administration of ANP or CNP.     

Adventitial application of the NADPH oxidase inhibitor apocynin in vivo reduces neointima formation and endothelial dysfunction in rabbits

OBJECTIVE: Reactive oxygen species including superoxide have been shown to promote atherogenesis. We previously showed that a major source of superoxide, the NADPH oxidase system, is upregulated in the intima and adventitia during remodelling induced by periarterial collars in rabbits. We have now examined the action of the NADPH oxidase inhibitor apocynin, given via the adventitia, on the neointima formation and endothelial function in this model. METHODS: Perivascular collars were implanted around the common carotid arteries of male NZW rabbits for 14 days to induce intimal thickening. The periarterial space of one collar was filled with apocynin (1 mM) while the contralateral collar with the vehicle (0.1% DMSO). RESULTS: After 14 days, local treatment with apocynin via the adventitia, reduced superoxide generation. In addition, apocynin significantly reduced neointima formation and proliferation of cells in both the neointima and adventitia. Moreover, NO-dependent vasorelaxation to acetylcholine, which is normally impaired in collared arteries, was improved, and apocynin suppressed the endothelial expression of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1. CONCLUSIONS: NADPH oxidase is implicated in vascular remodelling and superoxide-stimulated cell proliferation in the neointima contributes to intimal hyperplasia in this collar model. Targeting NADPH oxidase via adventitial drug delivery not only reduces superoxide generation, but also normalises endothelial cell function. Targeting the primary source of NADPH oxidase-derived superoxide is an effective approach to prevent deleterious arterial remodelling, providing a rationale for designing more efficacious and selective inhibitors of vascular NADPH oxidase as potential therapeutics for human vascular disease.     

Vascular neutral endopeptidase inhibition improves endothelial function and reduces intimal hyperplasia

OBJECTIVE: Neutral endopeptidase (NEP, EC 3.4.24.11) metabolises endogenous vasoactive peptides that may protect against atherogenesis. Since NEP is found in the adventitia of arteries, we investigated the anti-atherogenic effects of chronic adventitial NEP inhibition. METHODS: Intimal hyperplasia of rabbit carotid arteries was induced by placement of soft, non-occlusive, peri-arterial silastic collars. NEP localisation was studied with autoradiography 7 and 14 days after collar placement. Vascular NEP was inhibited in vivo by local superfusion of one collared carotid artery with Candoxatrilat (50 pmol/h), for 7 days (n = 7). The contralateral collar was filled with saline vehicle. After 7 days, ring segments of collared and normal (proximal to the collar) arteries were obtained and in vitro functional measurements, immunohistochemical determination of the pro-atherogenic factor plasminogen activator inhibitor-1 (PAI-1), localization of macrophages and morphometric analyses were carried out. RESULTS: Vascular NEP radiolabelled substrate binding, mainly in the media, was increased by approximately 50% after 7 days (n = 5; p < 0.05) and by approximately 300% after 14 days of collar placement (n = 5; p < 0.05). Compared with normal artery segments from the same animal, vehicle-filled collared sections displayed significantly impaired vasorelaxation to acetylcholine (endothelium-independent vasodilatation was preserved), increased PAI-1 immunostaining, macrophage accumulation and intimal thickening. In Candoxatrilat-treated collared arteries, vasorelaxation to acetylcholine was improved, along with reductions in PAI-1 levels, macrophage numbers and intimal area (all p < 0.05). CONCLUSION: Enhancing the activity of local, endogenous peptides by adventitial inhibition of vascular NEP may protect against early atherogenesis. This is of particular relevance to using adventitial therapies to prevent intimal hyperplasia leading to restenosis.     

Increased NADPH oxidase activity,gp91phox expression,and endothelium-dependent vasorelaxation during neointima formation in rabbits

Reactive oxygen species including superoxide and hydrogen peroxide are important mediators in atherogenesis. We investigated the enzymatic source of vascular superoxide and its role in endothelium-dependent vasorelaxation during neointima formation. Silastic collars positioned around carotid arteries of rabbits for 14 days induced neointimal thickening. Using lucigenin-enhanced chemiluminescence, superoxide production was detectable in collared artery sections, but not in controls, only after inactivation of endogenous Cu2+/Zn2+-superoxide dismutase (Cu2+/Zn2+-SOD) with diethyldithiocarbamate (DETCA). Dihydroethidium staining indicated that endothelium and adventitia were the major sites of superoxide generation. Superoxide production in DETCA-treated collared arteries was enhanced further by NADPH and was inhibited by diphenyleneiodonium, suggesting NADPH oxidase was the source of the radical in collared arteries. Moreover, real-time PCR demonstrated 11-fold higher expression of the gp91phox subunit of NADPH oxidase in collared arteries than in controls. In vascular reactivity studies, endothelium-dependent vasorelaxation to acetylcholine did not differ between collared and control sections. However, treatment with DETCA reduced relaxations to acetylcholine in collared rings, but not in controls. NADPH further reduced relaxations to acetylcholine in DETCA-treated collared sections, but not in controls. In DETCA/NADPH-treated collared rings, sensitivity to nitroprusside, in contrast to acetylcholine, exceeded that of controls. Moreover, further treatment of such rings with exogenous Cu2+/Zn2+-SOD restored acetylcholine relaxations without altering nitroprusside responses. Thus, early neointimal lesions induced by periarterial collars are associated with elevated gp91phox expression and increased NAPDH-oxidase-dependent superoxide production in endothelium and adventitia. However, endothelium-dependent vasorelaxation is largely preserved due to the actions of Cu2+/Zn2+-SOD and increased smooth muscle sensitivity to nitric oxide.     

Protection by shear stress from collar-induced intimal thickening: role of nitric oxide

Nitric oxide (NO) has potent relaxant and antiproliferative effects on vascular smooth muscle cells, which may represent an important antiatherosclerotic mechanism. Since one of the major stimuli for NO release is flow-related shear stress, we have investigated (1) the effect of increased shear stress on neointimal formation induced in the rabbit carotid artery by enclosing the vessel in a nonconstrictive silicone soft collar and (2) the role of NO in the antiproliferative effect of increased shear stress. Forty-three New Zealand White rabbits were used. High shear stress in the left common carotid artery (CCA) was induced by ligature of the contralateral right internal carotid artery; intimal thickening was produced by the positioning a nonconstrictive silicone soft collar around the left CCA. To evaluate the role of NO, N(G)-nitro-L-arginine methyl ester (L-NAME) was orally administered at a subpressor dose. In all rabbits, arterial blood pressure, heart rate, arterial diameters, and blood flow velocities of both CCAs were determined at days 0, 3, 7, and 14. At the end of the study, all rabbits were euthanized, and histological analyses were performed on both CCAs of each animal. The presence of the collar was associated with a marked degree of intimal hyperplasia (intimal/medial area ratio 29+/-3.0% in collared arteries compared with 3+/-0.7% in sham control [noncollared] arteries, P<0.001). The increase in blood flow almost completely inhibited neointimal formation and induced an increase in arterial diameter of approximately 30%. The effects of increased blood flow were reversed by the administration of L-NAME. In conclusion, we demonstrate that in collar-induced intimal thickening, a chronic increase in shear stress (1) almost completely inhibits intimal thickening, and (2) this protective effect is mediated by NO production.     

Increased intramural expression of plasminogen activator inhibitor type 1 after balloon injury: A potential progenitor of restenosis

Objectives. This study was performed to determine whether altered gene expression of plasminogen activator inhibitor type I (PAI-1) occurs within the arterial wall after experimentally induced balloon injury.Background. PAI-1, known to inhibit fibrinolysis in the circulation and to be present within atherosclerotic vessels, may influence proteolysis in the arterial wall and neointimal formation after angioplasty.Methods. In rabbit carotid arteries subjected to balloon injury, both PAI-1 gene and protein expression were assayed sequentially with the use of Northern blotting, in situ hybridization and immunohistochemical studies.Results. In uninjured, normal vessels PAI-1 messenger ribonucleic acid (mRNA) was not detectable by Northern blotting or in situ hybridization. However, injury was followed within 3 h by increases in PAI-1 mRNA (3.2 kb) of 5.9-fold compared with that in contralateral control carotid arteries (Northern blots). PAI-1 mRNA was detectable by in situ hybridization early after injury first in adventitia; after 24 h it was particularly prominent in the media. From 1 to 4 weeks after injury it was consistently detectable and was localized in neointimal vascular smooth muscle and endothelial cells at a time when neointimal thickening was marked. Cells of both types exhibited PAI-1 protein detected immunohistochemically. In vessels maintained in organ culture after balloon injury in vivo, sustained increases in PAI-1 activity appeared in conditioned media as well.Conclusions. Our results indicate that balloon injury simulating angioplasty in patients induces intramural expression of PAI-1 in vascular smooth muscle and endothelial cells. The decreased cell surface fibrinolytic activity likely to result from the increased PAI-1 expression may initiate or exacerbate mural thrombosis. Accordingly, excessive stimulation with clot-associated mitogens may stimulate vascular smooth muscle cell proliferation, which, coupled with increased accumulation of extracellular matrix attributable to decreased plasmin-mediated degradation, may contribute to restenosis.     

External collar inhibits balloon-induced intimal hyperplasia in rabbits

Intimal hyperplasia is a common complication following vascular interventions. To understand the underlying pathophysiology, the focus has mainly been on the intima and media. The adventitia has been less investigated, although adventitial hyperplasia is seen together with intimal hyperplasia. If the adventitial response is an important part of the process, the adventitia might be a target to inhibit intimal hyperplasia. In the present study we investigated whether an external collar attenuating the adventitial thickness could inhibit a balloon-induced intimal hyperplasia. The common carotid artery was injured in rabbits (n = 6) with a 3-french balloon catheter. The mid portion of the injured artery was encircled with a silicone collar (diameter = 2.0 mm). After 14 days the balloon-induced neointima was reduced by 54 +/- 6.3% underneath the collar. The adventitial and medial thickenings were also attenuated (36 +/- 8.7 and 44 +/- 4.3%, respectively). This study demonstrates that intimal hyperplasia following balloon injury can be inhibited with an external collar. This supports the idea of the adventitia as a potential target to inhibit intimal hyperplasia.     

Diverse effects of calcium channel blockers in the collar model

OBJECTIVE: Calcium channel blockers (CCBs) are among the most frequently prescribed cardiovascular drugs. It has been shown that these drugs have antiatherosclerotic effects in both experimental and clinical settings. However, calcium channel blockers have markedly different chemical structures and different effects on the cardiovascular system. We investigated the effect of CD-832, a Ca(+2) channel antagonist, on collar-induced intimal thickening, as well as accompanied reactivity changes in rabbit carotid artery. METHODS AND RESULTS: Rabbits received 5 mg/kg/day CD-832 or vehicle (polyethylene glycol, 0.5 ml/kg/day) intramuscularly for a week before and 2 weeks after the collar application. Histological and isometric force measurements were performed in segments from sham and collared carotid arteries. A three-week treatment with CD-832 did not inhibit the intimal thickening caused by perivascular application of a silicone collar. Potassium chloride (KCl), phenylephrine, 5-hydroxytryptamine (5-HT, serotonin) and histamine induced concentration-dependent contractions in both sham-operated (sham) and collared arteries. Collar-induced attenuations in maximum KCl, histamine, phenylephrine and 5-HT contractions were not affected by CD-832. Collaring caused an increase in pD2 values of 5-HT and a decrease in those of phenylephrine, histamine and acetylcholine. CD-832 did not affect the altered sensitivity to these agonists. CONCLUSIONS: These results demonstrate that, in rabbit carotid artery, CD-832 did not inhibit the collar-induced intimal thickening and did not affect the accompanying changes in vascular reactivity.     

Extracellular superoxide dismutase overexpression reduces cuff-induced arterial neointimal formation

The mechanisms of neointimal formation in cuff-injury models are still uncertain. To examine whether extracellular superoxide dismutase (EC-SOD) can reduce neointimal formation in a cuff-injury model, adenoviruses expressing EC-SOD (AxCAEC-SOD) or Escherichia coli beta-galactosidase (AxCALacZ) was injected between the cuff and the adventitia of rat femoral arteries. As a result, EC-SOD protein was effectively produced in the adventitia, as assessed by immunohistochemical staining. In comparison with cuff-treated control arteries and AxCALacZ-transfected arteries, neointimal formation was significantly reduced in AxCAEC-SOD-transfected arteries. Furthermore, proliferating smooth muscle cells in neointima and media were reduced by EC-SOD treatment. Similarly, augmented iNOS expression, apoptosis and collagen content in the vascular wall were also reduced by EC-SOD treatment. Reactive oxygen species (ROS) generation in tissue was reduced by EC-SOD expression, as assessed by dihydroethidium staining and coelenterazine chemiluminescence. These results suggest that ROS, especially superoxide anions at an adventitia, are responsible for neointimal formation in a cuff-injury model.     

Perivascular carotid collar placement induces neointima formation and outward arterial remodeling in mice independent of apolipoprotein E deficiency or Western-type diet feeding

We investigated the influence of apolipoprotein E deficiency and Western-type diet feeding on the development and composition of neointimal lesions induced by periadventitial carotid placement of a non-occlusive collar in mice. ApoE-/- and wild-type mice were fed a Western-type diet or chow diet for 4 weeks before collar surgery. Diets were continued after collar placement for 6 or 12 weeks. Compared to sham-operated arteries, collared carotids showed significant neointima formation, lumen loss, and outward remodeling in both apoE-/- and wild-type mice. These changes were not affected by either the genotype or the diet. Conversely, significant differences in neointima composition were detected between the two genotypes, with apoE-/- mice showing greater lipid deposition and lower SMC accumulation compared to wild-type mice, independent of the dietetic regimen. Altogether, the results of the present study indicate that although lesion composition may be influenced by genotype, neointima formation and arterial remodeling in the murine perivascular carotid collar model occur independent of the exposure to atherogenic diet or the presence of a sensitized genotype such as apoE-/-. The murine perivascular carotid collar model would thus be suitable for investigating neointima formation, arterial remodeling, and their potential pharmacological modulation in the setting of different genetic and dietary conditions.     

A disintegrin and metalloprotease 17 mediates neointimal hyperplasia in vasculature

The requirement of a metalloprotease, a disintegrin and metalloprotease 17 (ADAM17) for the growth of cultured vascular smooth muscle cells has been demonstrated in vitro. However, whether this metalloprotease is responsible for vascular remodeling in vivo remains unanswered. Rat carotid arteries were analyzed 2 weeks after a balloon angioplasty. The neointimal cells were strongly positive for ADAM17 immunostaining. Marked inhibition of intimal hyperplasia was observed in a dominant-negative ADAM17 adenovirus-treated carotid artery. Proliferating cell nuclear antigen-positive cells and phospho-epidermal growth factor receptor-positive cells in the neointima were reduced by dominant-negative ADAM17 as well. In contrast, the neointima formation, proliferating cell nuclear antigen-positive cells, and phospho-epidermal growth factor receptor-positive cells were markedly enhanced by wild-type ADAM17 adenovirus. In conclusion, ADAM17 activation is involved in epidermal growth factor receptor activation and subsequent neointimal hyperplasia after vascular injury. ADAM17 could be a novel therapeutic target for pathophysiological vascular remodeling.     

Local plasminogen activator inhibitor type 1 overexpression in rat carotid artery enhances thrombosis and endothelial regeneration while inhibiting intimal thickening

Elevated levels of plasminogen activator inhibitor type 1 (PAI-1) are found in advanced atherosclerotic plaque compared with normal vessel and may contribute to plaque progression and complications associated with plaque rupture. Increased expression of PAI-1 probably contributes to the thrombotic properties of advanced atherosclerotic plaque by impeding plasmin generation and degradation of fibrin. To test this hypothesis, we have deliberately created synthetic neointimas by seeding onto the denuded luminal surface of rat carotid arteries smooth muscle cells transduced with replication-defective retrovirus encoding rat PAI-1. This cell-based gene transfer method results in stable, long-term, and localized gene expression. PAI-1 overexpression increases mural thrombus accumulation at 4 days but decreases neointimal area by 30% and 25% at 1 week and 2 weeks, respectively. PAI-1 overexpression accelerates reendothelialization of injured arteries compared with control arteries at 1 week, 2 weeks, and 1 month. PAI-1 overexpression does not alter matrix accumulation at 1 week. Increased PAI-1 expression in the rat carotid artery enhances thrombosis and endothelial regeneration while inhibiting intimal thickening. These results suggest that PAI-1 could play a direct role in the development of advanced atherosclerotic plaque and in the repair of the diseased vessel after fibrous cap disruption.     

Effect of TGF-beta(1) antisense S-oligonucleotide on synthesis and accumulation of matrix proteoglycans in balloon catheter-injured neointima of rabbit carotid arteries

Arterial matrix proteoglycans (PG) are necessary for the maintenance of viscoelastic properties of the vessel wall, but excess levels, particularly of versican and biglycan in primary and restenotic intimal thickenings, are correlated with increased tissue volume and with atherogenicity. There is good evidence that the primary stimulus to increased PG synthesis, including versican and biglycan, is transforming growth factor-beta(1) (TGF-beta(1)). The aim of this study was to determine the effects of reducing endogenous TGF-beta(1) on rates and patterns of PG synthesis and on versican, biglycan and decorin accumulation in vivo. Rabbit common carotid arteries subjected to balloon catheter injury were treated with a TGF-beta(1) antisense phosphorothioate oligonucleotide applied in a pluronic gel to the adventitia. Control animals received a nonsense oligonucleotide or gel alone. TGF-beta(1) antisense (1) significantly (p < 0.005) inhibited, at day 2, the balloon catheter-induced increase in TGF-beta(1) mRNA relative to beta-actin mRNA; (2) inhibited intimal thickening at 23 days by approximately 40% (p < 0.05); (3) inhibited (p < 0.05) PG synthesis, measured by autoradiographic detection of [(3)H]glucosamine, in the media of day 2 ballooned carotids and in the subendothelial zone of day 23 neointima, and (4) decreased immunostaining intensity for versican (p < 0.03) and TGF-beta(1) (p < 0.001) in the neointima. Biglycan was reduced to a lesser extent but not significantly and decorin was not affected. Proliferating cell nuclear antigen indices were variable and not significantly changed. These findings confirm a role for TGF-beta(1) in developing neointima and demonstrate a specific effect on the synthesis, distribution, and accumulation of matrix PG, particularly versican.     

Characterization of an inhibitory effect of pioglitazone on balloon-injured vascular smooth muscle cell growth

This study investigates whether pioglitazone could suppress an atherogenic process such as balloon-injured carotid intimal thickening and the proliferation of vascular smooth muscle cells (VSMC). We first examined the effect of pioglitazone to determine whether it could suppress intimal thickening induced by balloon catheterization in Sprague-Dawley rats. After 14 days postcatheterization in the left common carotid artery, the neointimal layers were completely occupied by proliferated VSMC, and the area ratio of neointima to media treated with 10 mg/kg/d of pioglitazone was significantly decreased to 57%. Next, we evaluated the effect of pioglitazone on the proliferation of rat cultured VSMC. Piogliotazone dose-dependently decreased the values of DNA synthesis, total cellular protein content, phosphorylations of extracellular signal-regulated protein kinase 1/2 and mitogen-activated protein kinase kinase 1/2, and proliferative cell nuclear antigen in VSMC. Pioglitazone also inhibited the phosphorylation of Pyk2. We conclude that pioglitazone itself could be effective for suppressing the growth of VSMC and consequent carotid intimal thickening.     

Local expression of C-type natriuretic peptide suppresses inflammation,eliminates shear stress-induced thrombosis,and prevents neointima formation through enhanced nitric oxide production in rabbit injured carotid arteries

We previously observed that adenovirus-mediated expression of C-type natriuretic peptide (CNP) markedly inhibits neointima formation after balloon injury in rat carotid arteries, suggesting that CNP has multiple effects over its modest inhibitory effect on cellular proliferation. We hypothesized that local expression of CNP might have antithrombotic and antiinflammatory effects. Balloon-injured rabbit carotid arteries were infected with an adenovirus expressing human CNP (AdCNP), human tissue factor pathway inhibitor (AdTFPI), or bacterial beta-galactosidase (AdLacZ) or infused with saline. Seven days later, shear stress-induced thrombosis was evaluated by cyclic flow variation (CFV), reflecting recurrent cycles of thrombus formation and dislodgment. CFV was observed in all AdLacZ-infected and saline-infused arteries but not in arteries infected with AdCNP or AdTFPI even in the presence of epinephrine. Injury increased the expressions of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and infiltration of macrophages. However, these effects were markedly reduced in AdCNP-treated arteries but not in AdTFPI-infected ones. In AdCNP-infected arteries, injury-induced expression of inducible NO synthase (iNOS) was enhanced, leading to increased NO generation. Interestingly, when the enhanced NO production was inhibited, neither inhibitory effect was observed, and suppression of neointima formation by CNP was canceled. Our study demonstrates that overexpression of CNP shows antithrombotic and antiinflammatory effects and reduces neointima formation mainly through enhanced NO production.     

Decreased neointimal thickening after arterial wall injury in inducible nitric oxide synthase knockout mice

Mechanical injury in vivo results in the expression of the inducible form of nitric oxide synthase (iNOS) in vascular smooth muscle cells. However, the role of iNOS in modulating neointima formation after arterial wall injury is not clear. To determine whether the induction of iNOS gene expression promotes or attenuates the neointimal response to injury, we used a murine model of perivascular injury induced by placing a periadventitial collar around the carotid arteries in both wild-type and iNOS knockout mice (iNOS-KO mice). Periadventitial injury induced iNOS expression in the wild-type but not the iNOS-KO mice. Neointimal area and the intima/media ratio were significantly less in the iNOS-KO mice compared with the wild-type mice at 21 days. Injury-induced proliferation of medial cells and vascular cell adhesion molecule-1 expression were also attenuated in iNOS-KO mice compared with wild-type mice. The induction of iNOS and the activation of the nuclear factor-kappaB-mediated pathway were also demonstrated in an in vitro injury model. We conclude that mechanical injury in vivo and in vitro induces iNOS expression and that lack of iNOS expression attenuates neointima formation after perivascular arterial injury. Taken together, these findings suggest that iNOS expression after vascular injury may promote neointima formation.     

p27-p16 fusion gene inhibits angioplasty-induced neointimal hyperplasia and coronary artery occlusion

Inhibition of proliferative neointima formed by vascular smooth muscle cells is a potential target in preventing angioplasty-induced restenosis. We have created a potent antiproliferative by fusing the active regions of the p27 and p16 cell cycle inhibitors. Intravascular delivery of a replication-deficient adenoviral vector (AV) encoding this p27-p16 fusion protein, named W9, inhibited balloon injury-induced neointimal hyperplasia in rabbit carotid arteries. In a therapeutically more relevant model, AV-W9 was delivered to balloon-injured porcine coronary arteries in vivo using an infusion catheter. Of the three coronary arteries, two were injured with a 15-mm balloon catheter and either were left untreated or were treated with 10(12) viral particles of either AV-W9 or a control null virus. AV-W9 treatment significantly inhibited neointimal hyperplasia in this porcine arterial balloon injury model compared with untreated or control virus-treated vessels. The average intimal area of the AV-W9-treated group 10 days after balloon injury and treatment was 0.42+/-0.36 mm(2), whereas the AV-null group demonstrated an intimal area of 0.70+/-0.52 mm(2). At day 10 the average intimal thickness of the AV-W9-treated vessels was 9.1 microm (n=5, x 20 magnification) compared with 21.2 microm (n=5, x 20 magnification) in control virus-treated vessels. This trend was also observed at 28 days after balloon injury and gene transfer during which AV-W9-treated vessels demonstrated an average intimal thickness of 4.7 microm (n=8, x 20 magnification) compared with 13.3 microm (n=3, x 20 magnification) in control virus-treated vessels and 7.3 microm (n=5, x 20 magnification) in the sham-treated vessels. The AV-W9 treatment was safe and well tolerated. These data suggest that AV-W9 gene therapy may be useful in preventing angioplasty-induced intimal hyperplasia in the coronary artery.     

血管外膜剥离致内膜增生病变的动物模型

目的建立颈动脉外膜剥离引起内膜增生的动物模型,为研究血管外膜在动脉粥样硬化发生中的作用奠定基础。方法2g/L的Ⅱ型胶原酶消化颈动脉外膜后,眼科镊钝性剥离外膜,HE染色明确外膜剥离效果;免疫组织化学染色观察外膜剥离对内膜的影响。结果酶化学消化 眼科镊钝性剥离可有效剥离血管外膜;外膜剥离2周后可见相应内膜处出现增生性病变,病变成分为分泌型平滑肌细胞;而外膜保留侧正常。结论胶原酶消化 眼科镊钝性剥离的方法可有效剥离血管外膜,促进血管内膜增生性病变形成。     

Migration of adventitial myofibroblasts following vascular balloon injury: insights from in vivo gene transfer to rat carotid arteries

OBJECTIVES: Migration of adventitial fibroblasts, in addition to smooth muscle cell proliferation, plays a role in neointima formation following vascular injury. Previous studies have not directly addressed whether endogenous adventitial cells migrate towards the intima following balloon injury in the absence of medial dissection. We have employed an in vivo gene transfer technique to the rat carotid artery to directly label adventitial fibroblasts prior to balloon injury. METHODS: An adenoviral vector coordinating expression of nuclear targeted beta-galactosidase (AdLacZ) suspended in pluronic gel was applied to the perivascular surface of left carotid arteries of male Sprague-Dawley rats. Balloon catheter mediated vascular injury was performed on these arteries 4 days later and animals killed at 3, 7 and 14 days after injury. RESULTS: Expression of LacZ up to 14 days after application of the adenovirus was restricted only to the adventitia of uninjured arteries and absent from untransfected right carotid arteries. However, following balloon catheter injury, LacZ positive cells were observed within the medial layer of vessels by 3 days, and contributed to the population of cells within the neointima at 7-14 days. Adventitial cells in uninjured arteries did not express smooth muscle alpha-actin but after injury, LacZ positive cells migrating towards the lumen exhibited alpha-actin immunostaining, suggesting their change to a myofibroblastic phenotype. CONCLUSIONS: These findings provide direct evidence that adventitial fibroblasts migrate and contribute to neointima formation after balloon injury and show that in vivo gene transfer to the adventitia results in sustained transgene expression capable of labelling migrating adventitial cells within the media and neointima of injured vessels.