Estrogen attenuates the adventitial contribution to neointima formation in injured rat carotid arteries

OBJECTIVE: This study tested, in ovariectomized rats, whether (1) adventitial activation plays a role in the vascular injury response, and (2) inhibition of adventitial activation and the subsequent wave of cell proliferation moving from adventitia to neointima contributes to the estrogen-induced attenuation of neointima formation in balloon injured carotid arteries. METHODS: Ovariectomized Sprague-Dawley rats were treated with either 17 beta-estradiol or vehicle beginning 72 h prior to balloon injury of the right common carotid artery and were sacrificed at 0, 3, 7, 14 and 28 days after injury. BrdU was administered 18 h and 12 h prior to sacrifice in order to quantitate mitotic activity in adventitia, media and neointima of the damaged vessel at specified times post injury. RESULTS: Adventitial activation, evidenced by positive BrdU staining, was evident on the day of injury, peaked on day 3 and was resolved by day 7, thus preceding neointima formation. Numbers of BrdU labeled cells in adventitia on day 3 were significantly reduced in estrogen treated rats compared to controls. BrdU labeled cells were undetectable in media on the day of injury, appeared at day 3 and disappeared by day 14. Neointima appeared at day 7 and increased in area throughout the period of observation. Neointimal area and numbers of BrdU labeled cells in neointima were significantly reduced in estrogen treated rats compared to controls. These findings suggest that there is a wave of cell proliferation moving in an adventitia-to-lumen direction following endoluminal injury of the rat carotid artery and that estrogen modulates this proliferative response to injury. CONCLUSION: These results support the hypothesis that adventitial activation contributes to the vascular injury response and that estrogen reduces this contribution.     

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.     

Adventitial remodeling after angioplasty is associated with expression of tenascin mRNA by adventitial myofibroblasts

OBJECTIVES: The purpose of this study was to determine the temporospatial expression of tenascin-C (TnC) in balloon-injured rat and porcine arteries. BACKGROUND: Recent studies suggest that cell migration, in addition to cell proliferation, is a critical component of neointima formation after vascular injury. We have previously shown that adventitial myofibroblasts synthesize growth factors that contribute to the formation of neointima after arterial injury. We have also shown that the extracellular matrix protein, TnC, regulates cell migration. Consequently, we investigated the temporospatial expression of TnC by myofibroblasts after vascular injury. METHODS: In situ hybridization and immunohistochemistry were used to investigate the temporospatial expression of TnC in injured arteries. Northern and Western blots were used to determine the in vitro expression of TnC. RESULTS: In situ hybridization revealed that the major site of TnC expression early after vascular injury was the adventitial myofibroblasts. Immunohistochemical staining demonstrated that TnC expression began in adventitial myofibroblasts three days after injury. Tenascin-C expression, however, did not persist in this region. Rather, it moved progressively across the vascular wall toward the luminal surface. By one week, TnC expression reached the developing neointima. In vitro, myofibroblasts did not express TnC mRNA under basal conditions. In contrast, angiotensin II and PDGF-BB, factors that have been implicated in remodeling of balloon-injured arteries, markedly upregulated TnC mRNA. CONCLUSIONS: Tenascin-C is expressed in response to balloon injury. Tenascin-C expression begins with adventitial myofibroblasts. Over a period of 7 to 14 days, expression moves progressively across the vessel wall to the neointima. We hypothesize that adventitial myofibroblasts are actively involved in the formation of neointima and that TnC facilitates migration of these cells during adventitial remodeling.     

Angiotensin II type 1 receptor blockade attenuates in-stent restenosis by inhibiting inflammation and progenitor cells

The precise mechanism by which angiotensin II type 1 receptor blocker reduces in-stent restenosis in clinical trials is unclear. We, therefore, investigated the mechanism of in-stent neointima formation. Male cynomolgus monkeys and rabbits were fed a high-cholesterol diet and were allocated to untreated control and type 1 receptor blocker groups. Five days after grouping, multilink stents were implanted in the iliac artery. The type 1 receptor blocker reduced the development of in-stent neointima formation by approximately 30% in rabbits and monkeys. To investigate potential mechanisms, we examined the expression of renin-angiotensin system markers, all of which increased in monocytes and smooth muscle-like cells in the neointima and media within 7 days. The type 1 receptor blocker attenuated increased oxidative stress, the enhanced expression of markers of the rennin-angiotensin system and monocyte chemoattractant protein-1, and macrophage infiltration. The effects of type 1 receptor blocker on the differentiation of peripheral blood mononuclear cells into vascular progenitor cells were also examined. Treatment with type 1 receptor blocker suppressed the enhanced differentiation to smooth muscle progenitor cells induced by stenting. The type 1 receptor blocker attenuated in-stent neointima formation by inhibiting redox-sensitive inflammatory changes and by reducing recruitment of the progenitor cells. These potential actions of type 1 receptor blocker on inflammation and progenitor cells constitute a novel mechanism of suppression of in-stent restenosis by type 1 receptor blocker.     

Expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the pig coronary artery injury model: comparison of plain old balloon angioplasty and stent implantation

OBJECTIVES: Restenosis after coronary intervention usually occurs due to coronary remodeling or neointimal formation, but inflammation is also important especially after stent implantation. Adhesion molecules are important in the recruitment of inflammatory cells into the neointima and in the phenotypical changes of vascular smooth muscle cells. To examine the role of adhesion molecules in the pathogenesis of restenosis, immunohistochemical expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was investigated in the pig coronary injury model. METHODS: Left anterior descending coronary arteries of pigs were injured using a balloon. Two weeks after the injury, balloon injury was performed again in the balloon group and a Palmaz-Schatz stent was implanted in the stent group. Pigs were sacrificed at 1, 2 and 4 weeks. Immunohistochemical analysis was performed using ICAM-1, VCAM-1, macrophage and alpha-smooth muscle actin antibodies. RESULTS: In non-injured vessels, weak immunoreactivities of ICAM-1 and VCAM-1 were observed in the endothelium and media. In injured sites, ICAM-1 and VCAM-1 were found in the inflammatory cells and smooth muscle cells in the neointima from 1 week, and strong immunoreactivities were seen around the strut in the stent group. Although the immunoreactivities peaked at 2 weeks in the balloon group, strong immunoreactivities were still seen at 4 weeks in the stent group. Regenerated endothelial cells were positive for both antibodies from 2 weeks. CONCLUSIONS: The expression of ICAM-1 and VCAM-1 lasted longer in the stent group than in the balloon group, suggesting the occurrence of late restenosis after stent implantation. Control of the inflammatory response including adhesion molecules is essential for further reduction of restenosis after stent implantation.     

Local statin therapy differentially interferes with smooth muscle and endothelial cell proliferation and reduces neointima on a drug-eluting stent platform

OBJECTIVE: Therapeutic strategies to provide local inhibition of mitogen mediated proliferation and migration of human coronary artery smooth muscle cells (CASMC) by means of drug-eluting stents have been shown to enable effective limitation of neointimal hyperplasia. However, currently available drug-eluting stents utilize compounds that may also adversely affect endothelial regrowth, thus possibly precipitating subsequent cardiac events. Accordingly, identification of compounds that differentially inhibit smooth muscle and endothelial cell migration and proliferation could be of substantial clinical usefulness. METHODS AND RESULTS: In addition to lipid lowering, statins are known to display auxiliary pleiotropic activities. The purpose of this study was to evaluate the effect of local administration of cerivastatin on proliferation, migration and cytotoxicity of CASMC as well as coronary artery endothelial cells (CAEC) and to evaluate the effect of cerivastatin-coated stents on the inhibition of neointima formation as well as endothelial regrowth within the stented vessel. Cerivastatin displayed a differential effect on CASMC as compared to CAEC with regard to proliferation and migration; both were more profoundly inhibited in CASMC. Appreciable cytotoxicity and pro-apoptotic effects were low in both cell lines at therapeutic concentrations. Cerivastatin-elution led to significant inhibition of neointima formation in the rat carotid stent model, endothelial coverage of in-stent vascular tissue was similar with control and cerivastatin-eluting stents. CONCLUSIONS: As proof of principle, our study provides evidence that local application of a HMG-CoA reductase inhibitor on a drug-eluting stent platform can efficiently limit neointima formation. Consequently, these compounds warrant further clinical evaluation to confirm this finding. Our data further suggest that the anti-restenotic effect of local statin administration might be associated with a more protective interaction with the endothelium than that observed with compounds currently employed on drug-eluting stents.     

Time course of vascular remodeling, formation of neointima and formation of neoadventitia after angioplasty in a porcine model.

BACKGROUND: Vascular remodeling is the major cause of restenosis after coronary balloon angioplasty but the pathophysiology of this process is not known. OBJECTIVE: To examine the time courses of vascular remodeling, formation of neointima and adventitial changes after coronary angioplasty. DESIGN: An experimental study on pigs using coronary angiography, intravascular ultrasound (IVUS), and histology. METHODS: Deep vessel-wall injury was induced by conventional balloon angioplasty in the circumflex and right coronary arteries, and by retraction of a chain-encircled balloon in the left anterior descending artery. Angiography in all three arteries and IVUS measurements in circumflex and left anterior descending arteries were performed before and after angioplasty, and at follow-up on days 0, 1, 4, 7, 14, 28, and 56 (n = 5 in each group). Serial IVUS measurements were used to determine vascular remodeling. Formation of neointima and neoadventitia was measured by histomorphometry. RESULTS: Angiographically evident loss of lumen and ultrasonographically detectable constrictive remodeling occurred between day 7 and day 28. IVUS measurements showed that late loss of lumen (days 28 and 56) was correlated to vascular remodeling but not to the increase in wall area (neointima plus media). Histomorphometry revealed that neointima was present from day 7 and that amount of neointima increased up to day 28. Area of adventitia increased during the first 4 days and remained unchanged thereafter. Adventitial neovascularization by vasa vasorum was observed from day 4 onward. CONCLUSIONS: Formation of neoadventitia precedes late loss of lumen, constrictive remodeling, and formation of neointima. The time course of vascular remodeling coincides with growth of neointima rather than with changes in the adventitia.     

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.     

Roscovitine抑制大鼠颈总动脉球囊损伤后内膜增生的研究

目的:观察roscovitine对球囊损伤后大鼠颈总动脉血管平滑肌细胞及内膜增生的抑制作用,以期提供新型的支架涂层药物。方法:建立大鼠颈总动脉球囊损伤模型模拟经皮冠状动脉腔内介入术(PCI)术后再狭窄,干预组损伤局部给予roscovitine(200μmol/L)孵育10分钟。14天后取材,免疫荧光染色观察roscovitine对局部血管平滑肌细胞增殖的作用;HE染色观察roscovitine对内膜增生的作用。结果:本研究建立了大鼠颈总动脉球囊损伤模型,球囊损伤后14天,局部血管平滑肌细胞增殖活跃、内膜增生明显。Roscovitine干预后,血管平滑肌细胞增殖率明显降低,内膜增生被显著抑制,管腔狭窄率和内膜中膜面积比值显著降低。结论:Roscovitine显著抑制大鼠颈总动脉球囊损伤后局部血管平滑肌细胞增殖,进而有效抑制内膜增生,降低再狭窄发生率。     

Transforming growth factor-beta-dependent events in vascular remodeling following arterial injury

Constrictive remodeling has been identified as a major contributor to restenosis following angioplasty. Characterization of transforming growth factor-beta (TGF-beta)-mediated cellular events in the adventitia and their contribution to vascular remodeling, however, has not previously been studied in detail. The balloon catheter denudation model was performed on rat carotid artery, and groups of rats were treated with vehicle or a TGF-beta inhibitor, a soluble TGF-beta receptor type II (TGF-beta R:Fc). Adventitial cell proliferation, which peaked 4 days after injury, was characterized by the de novo formation of several cell layers surrounding the outer adventitia and this process was not dependent upon TGF-beta activity. These neoadventitial cells expressed an abundance of collagen type I and a fetal isoform of fibronectin containing the EIIIA domain, and the expression of both proteins was suppressed in the presence of TGF-beta R:Fc. Lumenal narrowing was apparent 14 days after injury. Inhibition of TGF-beta signaling promoted vessel enlargement. As a result, lumen size did not change despite neointima formation. In conclusion, adventitial fibrosis with abundant collagen matrix deposition but not adventitial cell proliferation is dependent upon endogenous TGF-beta activity. Furthermore, inhibition of TGF-beta signaling prevents injury-induced reduction in lumen area by promoting vessel enlargement.     

Kinetics of adventitial repair in the rat carotid model.

BACKGROUND: Discrepancies between success in experimental animals with a variety of pharmacologic strategies and failure with such agents in clinical trials have raised questions concerning the mechanism of restenosis. Recent observations suggest a potential implication for the adventitial (Adv) layer in neointimal formation. METHODS: The purpose of this study was to examine the Adv changes in the rat carotid artery subjected to balloon injury. These changes were characterized by morphometric, immunohistochemical, and electron microscopy analyses, with special attention devoted to early time-points post-injury. RESULTS: We report that the most important adventitial changes occurred in the first 48 h post-injury. Within 2 h there was extensive cell-loss by apoptosis and oncosis in the Adv and in the media; this was followed by the rapid onset of proliferation and a parallel slow increase in Adv thickening, reaching a maximum at 7 days. We further demonstrate an early migration of these Adv cells to the media and neointima. Moreover, we characterize the Adv cell phenotype with a panel of antibodies. Within 48 h after injury, a population of Adv cells expressed alpha-actin and vinculin with a maximum expression 7 days post-injury. At that time, these Adv cells started to express smooth muscle myosin heavy chain, a specific marker of smooth muscle cells. In parallel, we report an impaired production of elastic fibres in the Adv and medial layer. CONCLUSIONS: We reported a detailed time-course of adventitial changes after rat carotid injury (cell death, proliferation, migration and differentiation) that supports an important role of adventitia in neointima formation.     

茶多酚对兔颈总动脉血管成形术后再狭窄的影响

为探讨茶多酚对血管成形术后动脉中膜平滑肌增生及胶原增生的影响,以及与组织型纤溶酶原激活物和血管紧张素Ⅱ活性改变的关系,将雄性新西兰白兔30只随机分为对照组、低剂量茶多酚组和高剂量茶多酚组,用球囊导管剥脱右颈总动脉内皮,造成内皮及中膜损伤,分别在术前、术后3、7、11、14、22和28 d采动脉血应用酶联免疫法测血浆组织型纤溶酶原激活物活性及放射免疫法测血管紧张素Ⅱ血清水平,术后28 d处死动物并取右颈总动脉观察动脉中膜平滑肌和胶原增生程度.结果发现,高剂量茶多酚组组织型纤溶酶原激活物血浆活性为0.169±0.067 IU/L,低剂量茶多酚组为0.141±0.043 IU/L,对照组为0.126±0.043 IU/L,高剂量茶多酚组高于对照组和低剂量茶多酚组,差异具有显著性(P<0.05).高剂量茶多酚组血管紧张素Ⅱ血清水平为1 229±283 ng/L,低剂量茶多酚组为1 302±284 ng/L,对照组为1 309±263 ng/L,三组动物术后血管紧张素Ⅱ血清水平比较差异无显著性.高剂量茶多酚组动脉中膜胶原含量为50.1%+5.82%、低剂量茶多酚组为56.7%±2.3%,对照组为62.8%±2.1%,高剂量茶多酚组低于对照组及低剂量茶多酚组,差异具有显著性 (P<0.05);低剂量茶多酚组低于对照组,差异具有显著性(P<0.001).高剂量茶多酚组中膜平滑肌细胞计数为0.022±0.006/μm2,低剂量茶多酚组为0.034±0.008/μm2,对照组为0.033±0.007/μm2,高剂量茶多酚组低于对照组及低剂量茶多酚组,差异具有显著性(P<0.01).结果提示,高剂量茶多酚可提高血管成形术后血浆组织型纤溶酶原激活物活性,对血管紧张素Ⅱ血清水平无显著性影响,可抑制动脉中膜胶原及平滑肌细胞的增生,这可能有助于减轻或预防动脉血管成形术后再狭窄.     

外膜炎症诱发载脂蛋白E基因敲除鼠冠状动脉粥样硬化病灶

研究载脂蛋白E基因敲除(载脂蛋白E°)小鼠冠状动脉内粥样硬化病灶的分布、组成与动脉外膜炎症的关系.取载脂蛋白E°小鼠心脏作连续切片,Movat法染色,追踪冠状动脉主干及其心肌内的小分支;寻找病灶,观察病灶内组成,分析其分布规律.复制小鼠股动脉外膜无菌性炎症模型,用免疫组织化学方法检查内膜粘附分子的表达.结果发现,冠状动脉主干内有延伸病灶,在主干以下分支(包括心肌内小分支)内有在原位生成的病灶,在两类病灶相邻的外膜有炎性细胞浸润,外膜炎症面积大于动脉粥样硬化病灶累及的内膜面积,亦发现一些部位血管外有炎性细胞浸润,而尚无病灶形成.原位病灶均发生于心室壁,大的原位病灶多发生在左室壁心肌内、血管分支处和乳头肌附近的冠状动脉分支内.股动脉外膜炎症可诱发内膜表达细胞间粘附分子1和血管细胞粘附分子1,同时伴白细胞的附壁.以上提示血管外膜炎症是小鼠冠状动脉内病灶的一个始动环节.     

Neointimal growth can be influenced by local adventitial gene manipulation via a needle injection catheter.

Revascularization by percutaneous transluminal coronary angioplasty is limited in the long-term by restenosis, which is luminal renarrowing in the first 6 months after the procedure. Smooth muscle cell proliferation is thought to be an important factor in restenosis; this leads to neointima formation and arterial lumen narrowing. Local therapy delivered perivascularly may have an effect on events in the neointima and reduce restenosis. The effect of delivering expression vector plasmids for senescent cell-derived inhibitor SDI-1, which regulates cell proliferation, and its antisense, into the perivascular tissue of injured arteries was investigated in a porcine arterial injury model using a needle injection catheter. Transfection efficiency, biological effect and plasmid dissemination were evaluated in arterial and organ tissue sections between 2 days and 4 months. A limited number of adventitial, medial and neointimal cells were transfected up to 4 months. sdi gene transfer did not result in a change in neointima. Transfer of antisense sdi resulted in an increase in neointima after 3 weeks. No DNA plasmid was detected in control tissues. Liposomally-mediated adventitial local gene delivery is feasible and safe using the needle injection catheter in a porcine model. A limited number of cells was transfected, with expression of transfected genes up to 4 months after delivery. A transient biological effect with increased neointima was observed after delivery of the antisense sdi gene.     

Inhibition of neointima formation by tranilast in pig coronary arteries after balloon angioplasty and stent implantation

OBJECTIVES: We evaluated the effect of orally administered tranilast, N-(3,4-dimethoxycinnamoyl) anthranilic acid, on histologic and histomorphometric changes after angioplasty or stent implantation in pig coronary arteries. BACKGROUND: Tranilast, which has antikeloid and antiallergic properties and therefore may modulate the fibrotic and inflammatory tissue responses to angioplasty and stenting, has been shown to inhibit angiographic restenosis in small clinical trials. However, its effect on histomorphometric changes in coronary arteries after angioplasty and stenting is unknown. METHODS: Following initial pharmacokinetic studies in two pigs to determine desirable plasma levels of orally administered tranilast, 36 crossbred juvenile pigs were randomized to placebo or tranilast before undergoing balloon angioplasty in both the left anterior descending and left circumflex plus stent implantation in the right coronary artery. Oral tranilast was administered at 3 g/day starting 3 days before coronary injury and continued for 28 days until euthanasia. Injured vessels were harvested and sections analyzed by computer-assisted microscopic planimetry. RESULTS: In balloon-injured vessels, tranilast was associated with a 37% reduction in neointimal area normalized to fracture length (0.47 +/- 0.01 vs. 0.74 +/- 0.03 mm; p < 0.001) and a 23% reduction in adventitial area normalized to vessel size (0.43 +/- 0.02 vs. 0.56 +/- 0.03; p = 0.003). In stented arteries, neointimal area normalized to injury score was 32% lower in the tranilast-treated group compared to control (1.94 +/- 0.17 vs. 2.86 +/- 0.29; p = 0.01). CONCLUSIONS: In pig coronary arteries, tranilast was associated with a reduction in neointima formation and adventitial reaction after balloon injury. In stented vessels, tranilast was associated with a reduction in neointima formation normalized to injury score.     

兔颈动脉外膜损伤术后氧化应激中p38 MAPK的表达变化

目的: 探讨p38 MAPK在兔颈动脉外膜损伤致内膜病变氧化应激中的作用。方法: 采用高脂饮食喂养及胶原酶消化＋机械分离的方法,建立兔颈动脉外膜损伤致内膜病变的模型。测定术后不同时间点,对照组和阿托伐他汀组家兔血脂指标（TC,TG,LDL-C和HDL-C）、新生内膜面积（NIA）、活性氧物质（ROS）的产生情况,观察磷酸化p38 MAPK的表达及定位。结果: 从颈动脉外膜损伤术后1 d始,ROS的产生和磷酸化p38 MAPK的表达即增加并持续至少28 d。阿托伐他汀干预后,与对照组比较,各时间点ROS的产生及磷酸化p38的表达均下调（P<0.05）,TC及LDL-C的水平下降（P<0.05）,新生内膜形成减少（P<0.05）。结论: 氧化应激水平的升高和p38 MAPK的持续激活,是血管外膜损伤致新生内膜形成的可能机制之一。     

Altered endothelin-1 binding following balloon angioplasty of pig coronary arteries: effect of the ETA receptor antagonist, LU 135252.

OBJECTIVE: Since raised levels of endothelin-1 (ET-1) have been detected in the human coronary sinus following percutaneous transluminal angioplasty (PTCA) we investigated the role of ET-1 in the etiology of vascular restenosis. METHODS: Balloon angioplasty of coronary arteries was performed in pigs and the animals were treated with placebo or the endothelin (ETA) receptor antagonist LU 135252 (30 mg/kg/day). After 4 weeks vascular stenosis and the distribution of endothelin and its receptors was evaluated. RESULTS: The pronounced neointima formation in the control group (neointima:media ratio = 0.87 +/- 0.36) was significantly reduced by LU 135252 (0.43 +/- 0.30, P < 0.001). Angioplasty caused a significant increase in medial ETA (approximately 275%, P < 0.026) and ETB (approximately 250%, P < 0.001) binding to injured, compared with non-injured segments, an effect that was also reduced by LU 135252 (ETA = 11.5% increase; ETB = 14% increase). The neointima of control animals exhibited ET-1 like immunoreactivity as well as ETA and ETB binding sites. CONCLUSION: These data indicate that endothelin is locally-released from endothelial and vascular smooth muscle cells following angioplasty which binds to ETA and ETB receptor sites in the neointima and media. Since administration of the ETA antagonist LU 135252 markedly reduces neointima formation and medial ET binding, we conclude that vascular smooth muscle cell proliferation and subsequent neointima formation is mediated predominantly via ETA receptors. These data underscore the therapeutic potential of ETA antagonists in reducing the degree of restenosis following vascular injury.     

Myofibroblast involvement in glycosaminoglycan synthesis and lipid retention during coronary repair

Myofibroblasts of adventitial origin have been linked to neointimal formation and remodeling after coronary injury. Accordingly, the goal of this study was to examine whether myofibroblasts contribute to focal accumulation of glycosaminoglycans (GAGs) and lipids during coronary repair. GAG synthesis was assessed by ex vivo labeling of balloon-injured porcine coronary arteries with (14)C-glucosamine. The synthesis of total GAGs transiently increased at 8 days in the normolipemic model (a 2.2-fold increase over baseline, p < 0.05). The majority of newly synthesized GAGs were sensitive to chondroitin ABC lyase (chondroitin/dermatan sulfate GAGs). Versican was localized to myofibroblast-rich regions in the adventitia and neointima [positive for alpha-smooth muscle (SM) actin, negative for h-caldesmon and SM myosin heavy chain]. In contrast, the adjacent SM-rich media showed no increase in versican expression. The association between injury-induced GAG accumulation and lipid retention was examined at 2 weeks after coronary injury in the hyperlipemic model. Lipid (Oil Red O) accumulated in the neointima and adventitia, but not in the adjacent media. Coronary repair under hyperlipemic conditions was associated with macrophage infiltration (19 +/- 5 vs. 3 +/- 2% of neointimal cells in normolipemic animals, p < 0.001) and increased neointimal formation (1.8 +/- 0.5 vs. 1.0 +/- 0.3 mm(2) in normolipemic animals, p < 0.01). In conclusion, this study demonstrated a transient increase in GAG synthesis following coronary injury. Chondroitin sulfate proteoglycans (e.g., versican) were rapidly synthesized by activated adventitial and neointimal cells which could contribute to early lipid retention in injured vessels.     

Neointimal expression of rapamycin receptor FK506-binding protein FKBP12: postinjury animal and human in-stent restenosis tissue characteristics

Despite excellent clinical results for sirolimus (rapamycin)-eluting stents, the exact mechanisms of antirestenotic activity and affected cellular targets are incompletely understood. Therefore, we determined the presence and tem- porospatial expression pattern of FKBP12, the primary intracellular receptor of rapamycin, in rat carotid arteries after balloon injury, as well as in human in-stent restenosis and primary stable coronary atheroma. FKBP12 expression was assessed by immunohistochemistry. Rat carotid arteries revealed maximal expression in 57.7 +/- 4.0% of neointimal cells at day 7. A large proportion of these FKBP12+ cells showed luminally confined co-expression with dendritic cell markers. Despite a considerably thicker neointima at day 28, presence of FKBP12 decreased (8.5 +/- 1.9%, p = 0.02) with a scattered pattern in luminal and deep neointima. Likewise, human in-stent restenosis atherectomy specimens (time after stent implantation 2-12 months) revealed a comparable extent of cellular rapamycin receptor expression (9.3 +/- 1.0%) that significantly differed from that found in primary stable atheroma (1.3 +/- 0.4%, p < 0.001). In conclusion, the rapamycin receptor is predominantly present during early neointima formation, while mature neointimal atheromas show a relatively low expression without confinement to luminal areas. Co-expression of FKBP12 and dendritic cell markers suggests that dendritic cells may be another important target for early and long-term rapamycin effects.     

Hirulog-like peptide reduces balloon catheter injury induced neointima formation in rat carotid artery without increase in bleeding tendency

Vascular restenosis is one of the major concerns for the management of coronary artery disease using therapeutic vascular procedures. Treatments with thrombin-specific inhibitors, hirudin or hirulog-1, reduced ischemic events in coronary artery disease patients. Early started and prolonged infusions of these thrombin inhibitors partially prevented balloon catheter injury induced restenosis or neointima formation in experimental animal models, but increased the bleeding tendency. Hirulog-like peptide (HLP) was rationally designed to enhance the inhibition of the binding of thrombin to its receptor with less interruption of coagulation activity in comparison to hirulog-1. A single infusion of HLP for 4 h started 0.5 h before balloon catheter injury reduced neointima formation by 36% in rat carotid artery compared to vehicle controls. Tail bleeding time and activated partial thromboplastin time during HLP infusion were not significantly different from vehicle controls, but were significantly shorter than during heparin or hirulog-1 infusion. HLP treatment attenuated the expression of platelet-derived growth factor in the neointima of injured arteries. HLP also inhibited thrombin-induced thymidine incorporation in cultured baboon aortic smooth muscle cells. The findings suggest that HLP may substantially inhibit balloon catheter injury induced neointima formation without noticeable increase in bleeding tendency in rats. The inhibition by HLP of the expression of platelet-derived growth factor and of the smooth muscle cell proliferation in the vascular wall potentially contributes to the preventive effect of the new thrombin inhibitor on injury-induced neointima formation in the vascular wall.