Endothelial healing following defined injury to rabbit aorta. Depth of injury and mode of repair

A study has been made of the healing of a narrow deep injury to rabbit aortic endothelium which also involves damage to the media of the vessel. The injury was produced using a nylon catheter containing a wire filament; the injury was approximately 150 micron in width and damaged up to 3 elastic lamellae. Immediately after injury platelet aggregates were observed over the injured areas, several hours later large numbers of leukocytes were also seen to adhere. Two days after injury a non-thrombogenic neointimal surface was observed over deeply injured areas; endothelial cells could be identified covering the injured area at 6 days. The healing process following the injury has been directly compared with the healing of rabbit aortic endothelium following a superficial injury of similar width, where endothelial cells are removed without significant damage to the media of the vessel [1]. The results show that (a) following a narrow injury to the aorta which causes damage to the media platelet aggregation and proliferation of smooth muscle cells occurs, (b) despite the disruption of subendothelial components, endothelium rapidly regenerates over the narrow injured area, although not as quickly as for a superficial injury.     

Effect of regenerated endothelium on lipid accumulation in the arterial wall.

We tested the hypothesis that loss of endothelium results in increased transport of lipoprotein into the arterial wall, favors accumulation of lipid, and thus predisposes to atherosclerosis. In rabbits initially fed a diet low in lipid, the aortas were de-endothlialized with an intraarterial balloon catheter; 28 days later, the animals were divided into two groups. Group I animals were continued on a diet low in lipid and sacrificed at 8, 11, 13, and 15 weeks after de-endothelialization. Group II animals were fed the same diet supplemented with 0.5% cholesterol and sacrificed at comparable intervals. Aortas of group I animals revealed proliferative fibromuscular intimal thickening in both de-endothelialized and re-endothelialized areas, with little or no fatty change in the intima. In contrast, aortas of group II animals revealed slight to marked fatty change in the intima, characterized by accumulation of oil red O-positive material with anisotropic lipid inclusions. The greatest quantity of lipid was present in intimal thickening beneath regenerated endothelium, and not in adjacent intimal thickening lacking an endothelial lining. These results do not support the hypothesis that the absence of endothelium favors accumulation of lipid and predisposes to atherosclerosis. The experiments indicate that lipid accumulates preferentially in areas of intimal thickening covered by regenerated endothelium.     

Persistent dysfunction of regenerated endothelium after balloon angioplasty of rabbit iliac artery

This study investigated the vasodilator function of endothelium that regenerated after balloon angioplasty and the relation of this function to the extent of vascular injury and to subsequent intimal proliferation. Balloon angioplasty was performed in the left iliac artery of 47 New Zealand White rabbits. Vascular responses were examined in vitro 2 and 4 weeks after a "severe" injury (3.0-mm balloon) or a "moderate" injury (2.5-mm balloon). Both degrees of balloon injury caused complete endothelial denudation. Endothelial regrowth 2 weeks after either injury was confirmed histologically. Although the regenerated cells had irregular sizes and polygonal shapes and lacked the typical alignment in the direction of blood flow, immunocytochemical staining for factor VIII-related antigen identified these cells as endothelium. To study the vasodilator function of regenerated endothelium, rings of balloon-injured and control (contralateral) iliac arteries were suspended in organ chambers for recording of isometric force. Endothelium-dependent relaxation of balloon-injured vessels to acetylcholine and to the calcium ionophore A23187 were reduced at 2 and at 4 weeks after severe injury. After moderate injury, endothelium-dependent relaxations to these agents were reduced at 2 weeks but had normalized by 4 weeks. Endothelium-independent relaxation to sodium nitroprusside, however, was preserved in all study groups. Morphometric analysis revealed an inverse correlation between the degree of intimal thickening and maximal relaxation to acetylcholine (r = 0.45, p less than 0.01). Thus, there is a persistent attenuation of receptor- and nonreceptor-mediated endothelium-dependent relaxations after arterial injury. The regenerated cells have an altered morphological appearance, but staining for factor VIII-related antigen confirms their endothelial origin. The degree and duration of endothelial dysfunction depends on the severity of the initial injury and is related to the extent of intimal thickness.     

The arterial barrier to lipoprotein influx in the hypercholesterolemic rabbit. 1. Studies during the first two days after mild aortic injury

These studies examine the hypothesis that removal of aortic endothelium eliminates a barrier to lipoprotein cholesterol influx. The aortas of rabbits fed a cholesterol-rich diet from 7 to 16 days before balloon injury were studied 1 or 2 days after deendothelialization of specific areas of the aorta. By this design the aortic sterol content was near normal on the day of injury, but areas of injured and noninjured aorta were exposed to identical levels of elevated plasma cholesterol. Measuring the arterial [3H]- and [14C] cholesterol fractions accumulated during 2 different intervals after dosage in the same animals permitted calculation of total influx and fractional loss of aortic cholesterol. During the first 2 days after deendothelialization, total (unidirectional) cholesteryl ester influx in deendothelialized aorta was similar to that in adjacent uninjured aorta, but total influx of nonesterified cholesterol was increased. The calculated increase in influx of nonesterified cholesterol was not a result of an increase in hydrolysis of entered cholesteryl ester but probably represents increased exchange of labeled cholesterol between artery and plasma. These results suggest that subendothelial layers of the aorta of short-term cholesterol-fed rabbits function as significant barriers to lipoprotein influx and that processes other than increased permeation by lipoproteins initiate injury-induced lesions.     

Development of a large fibromuscular intimal thickening does not impair endothelium-dependent relaxation in the rabbit carotid artery

The release of endothelium-derived relaxing factor (EDRF) was examined in the rabbit carotid artery 6 weeks after denudation with an inflated balloon catheter in vivo. A concentric, fibromuscular intimal thickening of variable width developed in all areas lined with either regenerated endothelium or modified luminal smooth muscle cells. In vitro studies showed that in vessels precontracted with serotonin, only the re-endothelialized areas could relax to the endothelium-dependent dilators methacholine, substance P and the Ca2+ ionophore A23187. Re-endothelialized areas with large concentric, fibromuscular intimal thickening (between 10 and 20 cells thick) relaxed with a similar sensitivity and maximum to methacholine compared with control areas. It is concluded that newly generated endothelial cells release EDRF whilst the specialized lining smooth muscle cells present 6 weeks after injury do not, and that the presence of a large fibromuscular intima does not prevent EDRF from reaching the media to cause relaxation.     

球囊损伤大鼠主动脉内皮后血小板活化及凝血酶受体表达的变化

目的 探讨经皮冠状动脉腔内成形术后再狭窄的发生机制。方法建立大鼠主动脉内皮球囊损伤模型，分别于术后3天、7天、14天和28天，通过组织学检查、放射免疫法和逆转录一聚合酶链反应技术检测主动脉球囊损伤后内膜增生的情况、血小板表面GMP-140数目和凝血酶受体mRNA表达的变化。结果凝血酶受体mRNA在正常血管组织的表达较弱，球囊损伤术后第3天已显著增加，术后第14天达峰值，术后第28天开始下降。GMP-140于术后第3天明显升高，术后第7天开始下降。内皮损伤术后第3天已有增殖的血管平滑肌细胞移行至内膜层；术后第7天内膜开始增生；术后第14天血管平滑肌细胞的增殖及内膜增生更为明显；术后第28天血管平滑肌细胞的增殖明显减弱，细胞外基质增加，内膜继续增生。结论血管内皮损伤内膜增生的过程中血小板活化．凝血酶受体mRNA表达增加。     

Phenotypic and functional changes in regenerated porcine coronary endothelial cells : increased uptake of modified LDL and reduced production of NO

Porcine coronary arteries with regenerated endothelium exhibit impaired endothelium-dependent relaxations. Experiments were designed to analyze the structural and functional changes occurring in regenerated endothelial cells. Primary cultures from regenerated endothelium contained giant endothelial cells, with an increased number of cells with diameter >14.5 microm, a reduced ability to proliferate, and signs of apoptosis. The uptake of fluorescent acetylated LDL was increased 2-fold in cultures from regenerated endothelium. The increased uptake of acetylated LDL was confirmed ex vivo in injured coronary arteries. In cultures from regenerated endothelium, cGMP production was decreased under basal conditions and during stimulation with serotonin, bradykinin, and A23187. Thus, during regeneration, there is accelerated senescence of endothelial cells accompanied by increased incorporation of modified LDL and reduction of NO production without decrease in endothelial NO synthase expression. These alterations help to explain the altered endothelium-dependent responses 28 days after balloon injury.     

Arterial injury-induced smooth muscle cell proliferation in rats is accompanied by increase in polyamine synthesis and level

Proliferation of smooth muscle cells (SMC), enhancement of polyamine biosynthesis and increase in polyamine level in response to deendothelialization in the rat aorta were studied. [3H]Thymidine incorporation into SMC in aortas denuded with a balloon catheter began 25 h after injury, and maximal incorporation occurred 33-37 h after injury. Afterwards, [3H]thymidine incorporation declined, approaching the baseline level, but was slightly higher than that of sham-operated controls until 14 days after injury. Intimal thickening started 7 days after injury, and peaked at 21 days. Prior to these proliferative changes in aortic SMC, a rapid and transient increase in ornithine decarboxylase (ODC) activity was observed within 8 h after injury. There was no significant difference in ODC activity between injured and intact aortas after 4 days. The levels of polyamines, putrescine, spermidine, and spermine increased and were maximal at 48 h after injury, 8.1, 3.4 and 1.4 times the control levels, respectively. Increased levels of polyamines, in particular spermidine, continued until 7 days after injury. These results suggest that the enhancement of polyamine synthesis and the increased polyamine content of the aorta play important roles in the proliferation of SMC and in the development of intimal thickening, particularly in the initial proliferative response of medial SMC after deendothelialization.     

Catheter-induced intimal injury during routine coronary catheterization in dogs

The location and progression of changes in arterial permeability and structure were studied in nine dogs over a 13-week period following left coronary catheterization with standard coronary catheters. Changes in arterial permeability were analyzed by quantitating Evans blue dye (EBD) uptake over the aortic luminal surface (blue areas). Structural changes were assessed by light and electron microscopy. In the catheterized animals, compared to uncatheterized controls, we observed a significant increase in aortic luminal EBD uptake that was maximal 4 hours after the procedure but still present up to 13 weeks later. Microscopic analysis of "blue areas" shortly after the procedure revealed widespread endothelial denudation, platelet and leukocyte adherence, with occasional intimal avulsions, disruption of the internal elastica, and thrombi. The EBD uptake patterns in association with the electron microscopic findings in these animals suggested that 67-89% of the aortic endothelium was removed by the catheter during the procedure. The proliferative response that occurred following this catheter-induced injury produced fibrocellular intimal thickening in the aortas and left main coronary arteries of animals studied 10 to 92 days after the procedure. In the aortic root, such thickening was associated with incomplete re-endothelialization, thrombogenicity, and grossly abnormal permeability patterns. We conclude that significant catheter-induced intimal injury can occur during coronary angiography. In the canine model, such injury is associated with widespread fibrocellular intimal thickening and abnormal permeability that persists for at least 13 weeks after the procedure.     

Oxidized LDL receptor LOX-1 is involved in neointimal hyperplasia after balloon arterial injury in a rat model

OBJECTIVE: LOX-1 is a multi-ligand receptor originally identified as the endothelial oxidized LDL receptor. LOX-1 expression is also induced in smooth muscle cells in response to proinflammatory and oxidative stimuli. Here, we report on the role of LOX-1 in intimal hyperplasia, in which proinflammatory and oxidative stimuli are increased. METHODS AND RESULTS: Left common carotid artery of rat was injured by a balloon catheter. The expression of LOX-1 was significantly increased within 24 h after the balloon injury and peaked at day 7. LOX-1 expression was observed predominantly in medial smooth muscle cells until day 3, and then shifted to predominantly intimal smooth muscle cells. At day 14, the expression was concentrated in the regenerated endothelial cells. To examine the contributory role of LOX-1 in the growth of intimal smooth muscle cells, rats were administered anti-LOX1 antibody intravenously every 3 days after balloon injury. Anti-LOX-1 antibody administration effectively suppressed intimal hyperplasia, oxidative stress, and leukocyte infiltration compared with control IgG. These findings suggest the importance of LOX-1 expression in the pathogenesis of neointimal formation in conjunction with oxidative stress and leukocyte infiltration. CONCLUSION: The LOX-1 expressed in smooth muscle cells is involved in intimal hyperplasia in a rat model of balloon injury. Manipulation of LOX-1 activity is a novel potential therapeutic target to prevent restenosis after angioplasty.     

Targeted vascular delivery of antisense molecules using intravenous microbubbles.

OBJECTIVE: Perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microbubbles bind the antisense to the c-myc protooncogene (anti-c-myc) which prevents neointimal hyperplasia following vascular endothelial injury. The microbubbles also adhere to sites of damaged vascular endothelium and thus may be a method of systemically targeting delivery of anti-c-myc. METHODS: Laser scanning microscopy was performed on the aorta of 10 mice (five which were complement depleted) that received intravenous FITC-PESDA following aortic endothelial injury. C-myc expression was quantified following selective intracoronary injury in nine pigs that received intravenous (IV) anti-c-myc bound to PESDA. Finally, neointimal formation was measured following intracoronary stent deployment in 30 pigs that received either IV anti-c-myc alone or the same dose bound to PESDA. RESULTS: Fluorescent microscopy confirmed selective PESDA microbubble adherence to aortic endothelium in all mice with aortic injury. This binding was nearly abolished when serum complement was depleted prior to injury. C-myc expression at the site of coronary endothelial injury was significantly lower in pigs treated with systemic anti-c-myc bound to PESDA. There was a 33% reduction in % stenosis and a 28% reduction in intimal area at 45 days post-stent deployment in pigs that received IV antisense plus PESDA. The stent margins also had reduced neointimal formation. CONCLUSION: Systemic administration of anti-c-myc bound to PESDA microbubbles may be a good method for preventing coronary neointimal formation within and around implanted stents.     

Regions of constantly increased plasminogen activator activity along the intima of the normal aorta.

Histochemical studies of the aorta in newborn, young, and adult rats showed that the intima in the branching regions and at the beginning of the branches has a constantly increased plasminogen activator activity in comparison with the nonbranching regions. The aortic valve also shows constantly increased activator activity. The intimal activator activity is variable in the nonbranching regions, depending on the age and the anatomical area of the aorta. In young rats (20--30 days), the overall plasminogen activator activity in the aortic intima is higher than in adult rats (2--4 months), while the activity is variable in newborn rats. The intimal activator activity is generally higher in the thoracic aorta than in the abdominal aorta (in the nonbranching regions). The aortic wall has a low plasmin inhibitor capacity. The pattern of the endothelial plasminogen activator activity at different ages and anatomical areas of the aorta, in combination with the corresponding pattern of the aortic endothelial cell turnover, gives valuable information about the mechanism(s) of the production and normal (nonstressed) release of the endothelial plasminogen activator.     

L-精氨酸对糖尿病兔动脉损伤后内膜增生和内皮功能的影响

目的 :探讨糖尿病动脉损伤后内膜增生的机制和L 精氨酸对内膜增生及内皮功能的影响。方法 :2 8只糖尿病兔和 14只正常兔髂动脉行球囊损伤术后分为对照 (Con)组、糖尿病 (DM)组、L 精氨酸(L arg)组 ( 2 2 5 %L arg溶液饮水 )。术后 4周取髂动脉行图象分析或血管反应性测定 ,同时测定血浆中MDA、SOD和NO含量。结果 :DM组血浆中MDA水平明显低于Con组和L arg组 ,而SOD和NO水平明显降低。DM组内膜面积明显大于对照组 ( 0 2 5 5± 0 0 2 4vs.0 176± 0 0 2 9mm2 ,P <0 0 1) ,而L arg组内膜面积明显小于DM组 ( 0 15 3± 0 0 16,P <0 0 1)。L arg组髂动脉环对乙酰胆碱的舒张反应明显高于DM组。结论 :L arg能改善糖尿病兔的内皮功能 ,抑制动脉损伤后内膜增生     

Hypercholesterolemia enhances macrophage recruitment and dysfunction of regenerated endothelium after balloon injury of the rabbit iliac artery.

BACKGROUND. We studied the effects on and possible interaction of balloon denudation and hypercholesterolemia on large arteries in the rabbit with special regard to structure and vascular reactivity. METHODS AND RESULTS. New Zealand White rabbits fed a 1% cholesterol diet or a standard diet for 14 weeks underwent balloon denudation of the left iliac artery 4 weeks before death. Both the balloon-injured and the control iliac arteries were harvested for in vitro studies of vascular reactivity, for immunohistochemical staining with monoclonal antibodies directed at smooth muscle cells and macrophages, and for scanning electron microscopy. Balloon injury caused intimal smooth muscle proliferation with little macrophage infiltration and was followed by recovery of endothelium-dependent vasodilator function within 4 weeks. Hypercholesterolemia caused macrophage-rich lesions confined to the intima with moderate impairment of endothelial vasodilator function. Balloon injury in the setting of hypercholesterolemia caused intimal smooth muscle cell proliferation and intense macrophage infiltration throughout the arterial wall and severe impairment of endothelial vasodilator function. Scanning electron microscopy confirmed regrowth of the endothelium in all balloon-injured vessels. In the balloon-injured arteries of hypercholesterolemic animals, the regenerated endothelium exhibited areas of atypical morphology not seen after balloon injury or hypercholesterolemia alone. CONCLUSIONS. The present study shows that balloon injury, hypercholesterolemia, and their combination cause distinct lesions and functional disturbances. An arterial balloon injury in the setting of hypercholesterolemia produces a diffuse inflammatory response that is accompanied by a sustained impairment of endothelial function and a marked proliferative response.     

睾酮对雄性家兔腹主动脉损伤后内膜增生的作用

探讨雄激素睾酮对雄性家兔腹主动脉球囊损伤后内膜增生的作用,选择健康雄性新西兰白兔24只,随机分成对照组（假去势）,低睾酮血症组（单纯去势）和睾酮替代组（去势一周后加用长效十一酸睾酮单次肌注,14mg/kg),去势10天后对所有动物行腹主动脉球囊损伤术,损伤后两周检测血浆睾酮,一氧化氮,超氧化物歧化酶及丙二醛水平,并截取腹主动脉进行图像和增殖细胞核抗原分析。结果发现,假去势对照组和睾酮替代组新生内膜面积和增殖细胞核抗原增殖指数明显低于低睾酮血症组（P＜0．01）,且血浆一氧化氮水平明显上升,丙二醛水平显著下降（P＜0．01或0．05）。结果提示,生理水平的睾酮（内源或外源补充）有抑制动脉损伤介导的内膜增生作用,其机制可能与睾酮增加一氧化氮的产生和抗氧化作用有关。     

罗格列酮对大鼠胸主动脉球囊损伤后内皮再生和内膜增生的影响

目的探讨罗格列酮(rosiglitazone,RSG)对血管损伤后内皮再生和内膜增生的影响。方法制备大鼠胸主动脉球囊损伤模型,将SD大鼠随机分为RSG组、对照组和假手术组,于术后第7天和第14天处死动物。分别进行伊文思蓝染色观察内皮覆盖情况,细胞核增殖抗原(PCNA)免疫组化染色和组织形态学定量分析,并测量损伤后14 d时各组血清一氧化氮(NO)含量。结果RSG 7 d组和14 d组的再生内皮覆盖率分别为38.2%和75.2%,均较对照组(32.4%和60.4%)显著增加(P<0.05和P<0.01),且RSG 14 d组血清中的NO含量较对照组升高。球囊损伤后7 d内膜开始有少量增生,14 d时形成明显的新生内膜。RSG使损伤后14 d形成的新生内膜显著减少,内膜面积与中膜面积的比值(IA/MA)较对照组降低60.9%。与对照组比较,RSG 7 d组和14 d组新生内膜内的PCNA阳性表达指数均显著减少。结论RSG可以促进大鼠胸主动脉球囊损伤处的内皮再生,并减少新生内膜的形成。     

Intimal injury and thrombus formation

One of the most important factor of thrombosis is intimal injury. We examined the morphology of thrombogenesis in the large artery using the rabbit as an experimental animal. When the intima was injured by a balloon catheter, we observed the adhesion of platelets onto the denuded area of the intima with various degrees of aggregation. Fibrinogen, fibronectin and vWF were observed on the surface of the aggregated platelets. In the raised thrombi induced by a polyethylene tubing fibrin thread formation was observed among the aggregated platelets. In the injured site showing interruption of internal elastic lamina by polyethylene tubing or wire tubing prominent fibrin formation was observed. The intima showing fibrous thickening which was induced by polyethylene tubing 3 weeks before the second injury also revealed prominent fibrin formation after the second injury. These thickened intima is considered to have an availability of a large amount of tissue factor for the activation of coagulation system than the normal intima.     

球囊扩张术后大鼠腹主动脉钙调神经磷酸酶、血浆MCP-1的变化

目的探讨大鼠腹主动脉球囊扩张术后内膜增生、钙调神经磷酸酶(calcineurin,CaN)信号通路以及炎症水平的变化。方法雄性SD大鼠48只,随机分为假手术组24只和球囊组24只。球囊损伤术后30天取材,血管组织作HE染色,光学显微镜观察病理学改变,免疫组织化学检测钙调神经磷酸酶在血管壁中的表达;ELISA法测定血清单核细胞趋化蛋白1(monocyte chemoattractant protein-1,MCP-1)水平。结果球囊损伤后血管壁出现新生内膜,形成的新生内膜厚度不均。球囊组与假手术组相比较内膜增生、内膜/中膜厚度明显增加(P<0.05)。免疫组织化学检测血管壁上钙调神经磷酸酶表达,球囊组与假手术组相比表达明显增加(P<0.05)。血浆炎症因子血清单核细胞趋化蛋白1水平球囊组与假手术组相比差异有统计学意义(P<0.05)。结论球囊扩张术导致大鼠腹主动脉内膜增生,同时伴随钙调神经磷酸酶蛋白表达明显增强以及血浆的炎症因子血清单核细胞趋化蛋白1水平升高,提示钙调神经磷酸酶信号通路和炎症因子血清单核细胞趋化蛋白1可能在球囊损伤后内膜增生过程中起重要作用。     

Cells derived from regenerated endothelium of the porcine coronary artery contain more oxidized forms of apolipoprotein-B-100 without a modification in the uptake of oxidized LDL

Increased accumulation of lipoproteins and cholesterol within cells from regenerated endothelium may be responsible for their reported dysfunction. This study compared the presence and uptake of oxidized forms of low-density lipoprotein (LDL) in cells derived from native and regenerated endothelium. Four weeks after balloon denudation, primary cultures of native and regenerated endothelial cells were prepared from porcine coronary arteries. Regenerated endothelium stained more strongly using an antibody against oxidized lipoproteins. The increase in oxidized forms of apolipoprotein-B-100 exhibited by cells from regenerated endothelium was not due to an increase in extracellular-induced oxidation of native LDL, measured as the production of thiobarbituric-acid-reactive substances, being identical in both cell types. Intracellular cholesterol and cholesterol ester content were unchanged in regenerated cells. Using flow cytometry, accumulation of oxidized LDL was investigated further by quantifying the uptake of a mildly oxidized preparation of 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate-labelled LDL. The parameters of uptake, EC(50) and E(max), were not different between cells from native and regenerated endothelium suggesting that the number of LOX-1 receptors was identical in the two cell types. Moreover, a negative correlation between the increased uptake of acetylated LDL and decreased cGMP production in response to bradykinin was observed in cells from regenerated endothelium. Thus, the increased incorporation of modified LDL and their intracellular oxidation could be responsible for the alteration in NO production. The presence of oxidized forms of LDL may be a marker of endothelium regeneration and could be involved in the endothelial dysfunction of pig coronary arteries 4 weeks after balloon denudation.