Basic fibroblast growth factor: its role in the control of smooth muscle cell migration.

The formation of an intimal lesion in an injured artery is the consequence of the replication and migration of smooth muscle cells. Recent studies have implicated basic fibroblast growth factor (bFGF) as an important mediator of replication in the arterial media, and platelet-derived growth factor as an important mediator of migration. However, the degree of arterial trauma produced during injury has a significant influence on the time of onset of intimal thickening, suggesting that factors released from damaged smooth muscle cells may affect migration. We have investigated the role of one of these factors, bFGF, in smooth muscle cell migration in vivo. We found that 1) deendothelialization of the rat carotid artery results in significantly more migration when it is accompanied by traumatic injury to the underlying smooth muscle; 2) the rate of migration in arteries that have been gently deendothelialized is significantly stimulated by systemic injection of bFGF; and 3) inhibition of bFGF with a blocking antibody significantly reduces the amount of migration after traumatic deendothelializing injury with a balloon catheter. These findings suggest that bFGF plays an important role in the mediation of smooth muscle cell migration after arterial injury.     

抑制大鼠动脉损伤后早期中膜平滑肌细胞增殖可降低内膜肥厚

目的:探讨早期中膜平滑肌细胞增殖与内膜肥厚的关系。方法:球囊导管损伤大鼠动脉后,在不同时期投予血管紧张素转换酶抑制剂(ACEI)(temocapril-HCl,10mg·kg^-1·d^-1),观察平滑肌细胞BrdU(5-溴脱氧尿嘧啶尿苷)标记率和内膜面积。结果:损伤后动脉中膜平滑肌BrdU阳性细胞出现于术后24h,BrdU标记率呈双峰期变化,第1期在1-3d,峰值在2d为5.3%,第2期在3-7d,峰值在5d为2.7%。在第1个增殖期投予temocapril显著抑制第2个增殖期的BrdU标记率(0.05±0.02)%,对照组为(4.50±0.27)%(P＜0.01),同时也显著抑制了术后10d内膜面积的增加(10670.1μm²±7713.3μm²)(P＜0.01)。而仅在第2个增殖期投药,则不能抑制内膜面积的增加(83499.5μm²±31360.0μm²)(P＞0.05),其程度与未投药组相当。结论:球囊导管损伤后动脉中膜平滑肌细胞第1个增殖期是内膜形成所必需的。     

Wound healing in the media of the normolipemic rabbit carotid artery injured by air drying or by balloon catheter de-endothelialization.

The response to air-dry injury to the carotid artery of the normolipemic rabbit was compared with the response to de-endothelialization with a balloon catheter. Air drying induced an inflammatory response that resembled arteritis rather than atherosclerosis. There was medial damage, neutrophil but not macrophage infiltration, and fibrin formation, limited smooth muscle proliferation, which regressed after 3 months, and no lipid deposition. Within 1 week the smooth muscle cells were mainly of the secretory phenotype, and a neointima had formed. At 4 weeks the neointimal proliferation continued, but most cells showed a contractile phenotype. By 3 months, the lesion consisted of fibromuscular thickening with few small smooth muscle cells. Balloon injury induced minimal medial damage and continuing intimal proliferation with no evidence of regression by 3 months. It is concluded that air drying the carotid artery induces smooth muscle damage as well as endothelial cell loss, and this stimulates a wound-healing mechanism that is different from the response to selective intimal injury.     

Endothelial regeneration. II. Restitution of endothelial continuity

With a modified balloon catheter the endothelium of rat thoracic aortae was completely removed to study the interaction between two important responses of the vessel wall to intimal injury: endothelial regeneration and formation of an intimal fibrocellular thickening. Endothelial cells deriving from the uninjured intercostal arteries regenerated by migration followed by proliferation and proceeded as a continuous sheet at a rate of approximately 0.07 mm. per day in the circumferential direction and approximately 6 times faster in the axial direction. Smooth muscle cells appeared in the intima only in areas which were not covered by regenerating endothelium 7 days after injury. The smooth muscle cells formed a multilayered fibrocellular intimal lesion which reached the maximal thickness after 3 weeks. The continuous sheet of regenerating endothelium covered the intimal smooth muscle cells at a slower rate; 6 weeks after injury large areas located most distant from the source of regenerating endothelium still showed modified smooth muscle cells lining the lumen. However, platelets did not adhere to these smooth muscle cells, and the total amount of intimal thickening did not increase between 3 and 6 weeks after injury. We conclude that, in response to intimal injury, endothelial regeneration precedes the accumulation of intimal smooth muscle cells, and that injured intimal areas, which are rapidly covered by continuous endothelium, are protected from the development of a fibrocellular intimal lesion.     

Phenotypic features of smooth muscle cells during the evolution of experimental carotid artery intimal thickening. Biochemical and morphologic studies

Balloon catheter denudation of rat carotid artery that results in significant medial damage is followed by marked intimal smooth muscle cell (SMC) proliferation associated with limited endothelial regrowth. In this report we demonstrate that: (a) SMC of the carotid media, preceding their intimal proliferation, develop a cytoskeletal profile and morphology consistent with a de-differentiated SMC phenotype; and (b) both medial and intimal SMC subsequently revert to a cytoskeletal profile and morphology reflecting incomplete but significant re-differentiation toward normal SMC phenotype. Specifically, early after balloon injury, SMC of the media and those that have migrated into the intima contain decreased amounts of actin, desmin, and tropomyosin and increased amounts of vimentin; moreover, beta-actin becomes the dominant actin isoform, whereas alpha-actin decreases as compared with that found in normal medial SMC. Late after balloon injury, actin is still less abundant, however, desmin, tropomyosin, and vimentin return toward normal values and both medial and intimal SMC again show a predominance of alpha-actin, although the endothelium does not regenerate over the central surface of intimal thickening in this model. The SMC surface to volume ratio significantly decreases early after balloon injury, whereas it is not significantly different late after balloon injury as compared with that of SMC of the normal carotid media. We demonstrate, furthermore that: (c) adjacent luminal SMC are interconnected by gap junctions and develop focal tight junctions, a feature not reported previously to occur in smooth muscle; these cells however do not form any well defined membrane specialization with the leading edge of endothelium, supporting the view that presence of modified SMC on the luminal surface of chronically denuded vessels is not responsible for the cessation of endothelial regrowth.     

CD4+ mononuclear cells induce cytokine expression, vascular smooth muscle cell proliferation, and arterial occlusion after endothelial injury.

Studies of T cell-deficient or immunosuppressed animals undergoing arterial endothelial denudation have yielded conflicting results as to the contribution of the immune system to neointimal vascular smooth muscle cell accumulation and proliferation. We investigated the cell types and cytokine expression associated with intimal hyperplasia occurring 14 days after balloon angioplasty of the carotid artery in Sprague-Dawley rats. Immunohistological studies using monoclonal antibodies showed that the carotid luminal occlusion observed was associated with smooth muscle cell proliferation and neointimal accumulation of large numbers of CD4+, ED1+ mononuclear cells but no T cells. There was also wide-spread staining for the inflammatory cytokine interleukin-1B (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and IL-8, as well as dense expression of the potent smooth muscle mitogens platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), and protein S. The relationship of smooth muscle cell proliferation to monocyte/macrophage accumulation and cytokine expression was tested by daily intraperitoneal administration for 14 days of a rat CD4-specific monoclonal antibody, BWH-4 (500 micrograms/day). Morphometric analysis at day 14 showed that the intimal area of animals treated with CD4 monoclonal antibody comprised 7% +/- 4% of the arterial wall compared with 50% +/- 6% in control animals (n = 6/group, P < 0.001). In addition, immunohistological studies showed that CD4 monoclonal antibody treatment markedly reduced the intimal accumulation of mononuclear and smooth muscle cells and essentially abrogated expression of the cytokines PDGF-BB, TGF-beta, IL-1 beta, TNF-alpha, and IL-8, plus the anticoagulant molecule, protein S. Our results document the extensive expression in vivo of cytokines that in vitro promote vascular smooth muscle cell proliferation, and suggest that CD4+ mononuclear cells or their secreted products play a key role in the pathogenesis of intimal hyperplasia after endothelial injury. Furthermore, these observations may have clinical relevance in the development of novel strategies to prevent arteriosclerosis.     

Factors controlling smooth-muscle cell proliferation.

Injury to the arterial wall normally elicits a rapid and significant increase in smooth-muscle cell (SMC) replication with the subsequent development of intimal lesions. A variety of factors have been proposed to control SMC replication, but recent work has highlighted the role of basic fibroblast growth factor (bFGF) and platelet-derived growth factor in this process. In the carotid artery of the uninjured rat, we have shown that the SMCs express mRNA for bFGF and that bFGF can be readily extracted from these arteries. Following mechanical injury to the artery, ie, after balloon injury, we suggested that bFGF is released from damaged cells and then stimulates adjacent SMCs. In support of this concept, the infusion of a blocking antibody to bFGF was found to significantly inhibit the early SMC replication induced by use of a balloon catheter. The addition of the antibody at the time of injury, however, did not inhibit the development of intimal lesions. In contrast, studies by us and other investigators have shown that platelet-derived growth factor is not directly important for SMC replication after balloon injury, but that it plays a key role in stimulating the migration of SMCs into the intima. Intimal SMC replication was not inhibited with antibodies to either bFGF or platelet-derived growth factor. Therefore, while significant inroads have been made in understanding the initial events, we still do not fully understand all the processes involved in the proliferation of arterial intimal lesions.     

Kinetics of cellular proliferation after arterial injury. II. Inhibition of smooth muscle growth by heparin

Heparin inhibits intimal thickening after arterial injury. Whether this effect is due to inhibition of medial smooth muscle cell (SMC) migration, SMC proliferation in the intima, or synthesis and deposition of connective tissue has not been evident. In this study we have investigated these possibilities in a rat carotid balloon injury model. Heparin (0.3 mg/kg/hour) was administered intravenously by means of osmotic pumps to experimental animals, and controls received lactated Ringer's solution. Smooth muscle proliferation (thymidine index), intimal smooth muscle accumulation, and endothelial regeneration were measured at intervals between 0 and 28 days. Total smooth muscle growth as determined biochemically at 14 days was markedly inhibited by heparin if the pumps were placed 24 hours before or at the time of injury and less so if inserted 48 or 96 hours after injury. SMC thymidine indices were maximal in the media at 4 days and in the intima at 7 days for injured arteries of both heparin-treated and control rats; at each time point SMC proliferation and intimal thickening were less in heparin-treated rats. The volume of connective tissue in the intima was the same in both groups at 28 days. Medial SMC migration into the intima was diminished by heparin treatment, but endothelial regeneration was not affected. These results support the hypothesis that heparin is a specific inhibitor of SMC migration and proliferation and is most effective if started before SMC enter S-phase.     

Kinetics of cellular proliferation after arterial injury. III. Endothelial and smooth muscle growth in chronically denuded vessels

The healing of injured rat carotid chronically denuded of endothelium was studied at late times after balloon injury. One year after injury the endothelial layer was not fully regenerated and had ceased proliferating. The denuded areas of these vessels were covered with luminal smooth muscle cells which strongly resembled endothelium but did not stain with factor VIII.R.Ag antibody. These cells were actively proliferating and formed a surface which was weakly thrombogenic. Indium-111 platelet studies showed that over 24 hours there was a slight but significant adherence of platelets to the denuded surface. Luminal smooth muscle cell proliferation was matched by cell loss and did not produce further intimal thickening or an increase in total cell number. These results demonstrate that damaged conduit arteries can exist in a stable state without endothelium and do not develop thrombosis even after prolonged periods of time.     

Angiotensin-converting enzyme inhibitor versus angiotensin II, AT1 receptor antagonist. Effects on smooth muscle cell migration and proliferation after balloon catheter injury.

The angiotensin-converting enzyme (ACE) inhibitor, benazeprilat and the angiotensin II (Ang II), AT1-specific receptor antagonist, DuP753, were compared for their effects on intimal lesion formation as well as smooth muscle cell (SMC) proliferation and migration in Sprague Dawley rats after carotid balloon injury. Both the ACE inhibitor (benazeprilat, 3 mg/kg/day) and the AT1 antagonist (DuP 753, 10 mg/kg/day) significantly reduced intimal lesion formation after balloon injury (by 35% and 49%, respectively). Medial SMC proliferation after injury was reduced 53% by the AT1 antagonist; however, the ACE inhibitor had no effect on SMC proliferation. SMC migration was reduced 94% by the AT1 antagonist and 68% by the ACE inhibitor. These data demonstrate the importance of Ang II in SMC proliferation and migration after balloon injury. They also demonstrate that in the balloon injury model, the ACE inhibitor reduced intimal lesion size by inhibiting SMC migration alone without affecting SMC proliferation. A more pronounced reduction in lesion size was obtained after AT1 antagonism, however, when both SMC migration and proliferation were inhibited.     

Eliminating Arterial Pulsatile Strain by External Banding Induces Medial but Not Neointimal Atrophy and Apoptosis in the Rabbit

We have examined the role of vessel pulsation and wall tension on remodeling and intimal proliferation in the rabbit infrarenal abdominal aorta. A rigid perivascular polyethylene cuff was used to reduce vessel systolic diameter by 25%, producing a region of reduced circumferential strain. At 6 weeks postoperatively, reduced circumferential strain caused medial atrophy, with 45% reduction of medial area and 30% loss of medial smooth muscle cells. Apoptotic cell death was indicated by DNA fragmentation, propidium iodide staining, and cell morphology. Cuffing the aorta after balloon denudation produced medial atrophy but did not inhibit neointimal growth. At 1 week postoperatively, intimal thickness was slightly decreased in regions with reduced strain; however, intimal thickening in regions of reduced strain was not different from control segments at 3 weeks postoperatively (intimal area was 0.37 ± 0.05 mm² with reduced strain and 0.50 ± 0.08 for controls, mean ± SEM). We conclude that circumferential strain is a major factor controlling medial structure and cell number, whereas growth of the neointima after injury is not significantly affected by either reduced strain or extensive medial cell death. Vessel cuffing represents a new model of blood vessel remodeling in vivo that involves extensive smooth muscle cell apoptosis.     

大鼠血管球囊损伤促进血小板活化、凝血酶受体及血管紧张素Ⅱ1型受体的mRNA表达

目的该研究通过建立大鼠主动脉内皮球囊损伤模型，观察血小板活化、凝血酶受体及血管紧张素Ⅱ1型受体的变化。方法将大鼠随机分为对照组(n=24)和手术组(n=24)，并于术后3、7、14和28d取主动脉，通过组织学检查、放射免疫法和反转录聚合酶链反应(RT-PCR)技术检测血管球囊损伤后内膜增生的过程、血小板表面GMP-140的数目、血管AT1受体和凝血酶受体mRNA表达的变化。结果①凝血酶受体mRNA在正常血管组织表达极弱，球囊损伤术后3d已显著增加，术后14d达峰值，术后28d开始下降。②AT1受体mRNA于术后3d明显增高，并持续至术后14d，术后28d恢复至对照水平。③GMP-140于术后3d明显升高，术后7d开始下降。④内皮损伤术后3d已有增殖的血管平滑肌细胞(VSMC)移行至内膜层，术后7d内膜开始增生，术后14d VSMC的增殖及内膜增生更为明显，术后28d VSMC的增殖明显减弱，细胞外基质增加，内膜继续增生。结论血小板活化、凝血酶受体和AT。受体mRNA表达增加参与了血管内皮损伤后内膜增生的过程。     

The aortic intima. II. Repair of the aortic lining after mechanical denudation.

This article reports the ultrastructure of the aortic lining during the repair of mechanically denuded aortic intima in the rat. Three main features were observed: a) Although platelets form a pavement on the exposed components of the aortic intima, platelet thrombi do not form on the denuded surface. b) During the first weeks after injury, a temporary false endothelial lining is formed by modified intimal smooth muscle cells. While the modified smooth muscle cells do not constitute a continuous cell layer, they are like true endothelial cells in that platelets do not adhere to the cell membrane of either cell type. c) A continuous layer of true endothelial cells is formed within 2 months after the original injury. Even after reestablishment of a continuous endothelium, however, abnormalities persist in the form of incompletely formed intercellular junctions. This abnormal endothelium is associated with areas of intimal smooth muscle cell proliferation. These observations are compatible with two alternative interpretations of the role of endothelial injury in the intimal proliferation seen following injury to the vessel wall: a) persistent defects in the endothelium may result in proliferation of underlying arterial smooth muscle cells or b) the proliferation, in converse, may in some manner delay the healing process of the overlying endothelium.     

Thrombospondin deposition in rat carotid artery injury.

The balloon catheter injury model was used to determine the relative contributions of vascular smooth muscle cells (SMC) and platelets to thrombospondin (TSP) antigen deposition in the artery wall. Rat carotid arteries were denuded of endothelium, exposing the thrombogenic subendothelial extracellular matrix (ECM) to the circulation. Rats were killed after 1 hour, or 5, 10, or 20 days. Thrombospondin antigen deposition in the injured arteries was assessed using a specific polyclonal antiserum raised in rabbit against rat platelet TSP and a sensitive silver-enhanced immunogold staining method. Faint immunostaining for TSP antigen was detected, associated mostly with cells, in the media of the carotid artery of the nonoperated controls. One hour after balloon catheter injury, however, prominent cell-associated immunostaining was evident in the media; extracellular matrix staining was negligible. At this time, large foci of immunostaining were present on the lumenal surface of the vessel. Intimal proliferation was evident on most stained sections of tissue taken 5 days after balloon injury. Thrombospondin antigen immunostaining was markedly increased compared to nonoperated controls in all sections, regardless of the degree of intimal thickening. Thrombospondin immunostaining remained associated with cells in the neointima and media; extracellular matrix staining remained negligible. Ten days after endothelial injury, immunostaining for TSP antigen was detected in all layers of the artery, but was greater in the neointima and media. Reaction product was still associated only with cells. Thrombospondin antigen levels, as detected by this procedure, remained high in the injured tissue through 10 days of observation but appeared less prominent 20 days after injury. At this time extracellular matrix staining was obvious and cell-associated staining was reduced. These data support the hypotheses that thrombospondin (TSP) expression by vascular smooth muscle cells is an early response to injury and that the primary source of TSP antigen in injured artery is the vascular smooth muscle cells (SMC). These results support data derived from in vitro studies of TSP secretion.     

Model system to study interaction of platelets with damaged arterial wall. II. Inhibition of smooth muscle cell proliferation by dipyridamole and AH-P719

A new in vivo model for the initial events in atherogenesis was employed to investigate drugs which may inhibit intimal muscle cell proliferation following repeated limited endothelial cell injury. An artery forceps was placed over the central artery of the ear of an anesthetized rabbit for 30 min. The forceps were removed, blood flow resumed in the vessel, and platelets contacted the damaged vessel wall. When a vessel was injured two or more times the smooth muscle cells of the media migrated into the intima and proliferated there between 1 and 3 weeks after the last injury despite restoration of an apparently intact endothelium. The intima of control undamaged vessels sometimes contained a few individual smooth muscle cells while vessels injured two, four, or six times showed correspondingly increasing numbers of layers of intimal smooth muscle cells covering increasing amounts of the intima. Arteries from thrombocytopenic rabbits showed, at most, a single layer of smooth muscle cells covering a small area. In rabbits pretreated with dipyridamole (1.5 mg/kg) for 3 days before each injury, proliferation was also limited to a small area. Neither aspirin (8 mg/kg) nor ticlopidine (40 mg/kg, 5X over 3 days), which inhibit platelet aggregation ex vivo, nor the continuous presence of heparin (800 U/kg, bid), reported to inhibit smooth muscle cell growth in vitro and in vivo, prevented smooth muscle cell proliferation in response to two injuries. However, a potent inhibitor of platelet cyclic-adenosine monophosphate (cAMP) phosphodiesterase, AH-P719 (1.5 or 2.1 mg/kg), was able to inhibit intimal smooth muscle cell proliferation in doses that inhibited platelet aggregation ex vivo.     

Doxycycline modulates smooth muscle cell growth, migration, and matrix remodeling after arterial injury

The tetracyclines function as antibiotics by inhibiting bacterial protein synthesis, but recent work has shown that they are pluripotent drugs that affect many mammalian cell functions including proliferation, migration, apoptosis, and matrix remodeling. Because all of these processes have been implicated in arterial intimal lesion development, the objective of these studies was to examine the effect of doxycycline treatment using a well-characterized model of neointimal thickening, balloon catheter denudation of the rat carotid artery. Rats were treated with 30-mg/kg/day doxycycline. Doxycycline reduced the activity of matrix metalloproteinase (MMP)-2 and MMP-9 in the arterial wall, and inhibited smooth muscle cell migration from media to intima by 77% at 4 days after balloon injury. Replication of smooth muscle cells in the intima at 7 days was reduced from 28.3 plus minus 2.5% in controls to 17.0 +/- 2.8% in doxycycline-treated rats. The synthesis of elastin and collagen was not affected, but accumulation of elastin was blocked in the doxycycline-treated rats. By contrast, collagen accumulation was not affected, which led to the formation of a more collagen-rich intima. At 28 days after injury, the intimal:medial ratio was significantly reduced from 1.67 +/- 0.09 in control rats to 1.36 +/- 0.06 in the doxycycline-treated rats. This study shows that doxycycline is an effective inhibitor of cell proliferation, migration, and MMP activity in vivo. Further study in more complicated models of atherosclerosis and restenosis is warranted.     

Fas ligand gene therapy for vascular intimal hyperplasia

Fas, a member of the tumor necrosis factor receptor super-family, is expressed in all cell types examined, while physiologic expression of Fas ligand (FasL) is found predominantly in activated T-lymphocytes, vascular endothelial cells, and "immune-privileged" tissues. Activation of Fas following FasL binding activates caspases, which results in apoptosis. In the vasculature, there may be a delicate balance between cell proliferation and apoptosis in vascular smooth muscle cells. Shifts in this balance could account for the accumulation of vascular smooth muscle cells in response to arterial injury, a major feature of vascular intimal hyperplasia. Intimal hyperplasia occurs in more than a third of patients receiving percutaneous transluminal balloon angioplasty. Stenting with or without coating significantly reduces the incidence rate of angiographic restenosis and that of target vessel revascularization. However, "in-stent" intimal hyperplasia/restenosis remains a challenge for clinical cardiologists. Although both the cell types and mechanisms that contribute to intimal hyperplasia in response to vascular injury remain controversial, vascular smooth muscle cell migration and proliferation appear to play an important role in the process. In animal models, cytotoxic and cytostatic gene therapy strategies targeted at the vascular smooth muscle cells have shown therapeutic potential for the treatment of vascular intimal hyperplasia. However, Fas ligand-based gene therapy appears to offer several advantages. In this review article, we will discuss the mode of FasL/Fas signaling in vascular smooth muscle cells and its therapeutic implications. We will also compare the relative merits of FasL with other cytotoxic and cytostatic gene therapy approaches for the treatment of intimal hyperplasia.     

Decorin inhibition of PDGF-stimulated vascular smooth muscle cell function: potential mechanism for inhibition of intimal hyperplasia after balloon angioplasty

Decorin is a small proteoglycan that binds to transforming growth factor-beta (TGF-beta) and inhibits its activity. However, its interaction with platelet-derived growth factor (PDGF), involved in arterial repair after injury, is not well characterized. The objectives of this study were to assess decorin-PDGF and decorin-PDGF receptor (PDGFR) interactions, the in vitro effects of decorin on PDGF-stimulated smooth muscle cell (SMC) functions and the in vivo effects of decorin overexpression on arterial repair in a rabbit carotid balloon-injury model. Decorin binding to PDGF was demonstrated by solid-phase binding and affinity cross-linking assays. Decorin potently inhibited PDGF-stimulated PDGFR phosphorylation. Pretreatment of rabbit aortic SMC with decorin significantly inhibited PDGF-stimulated cell migration, proliferation, and collagen synthesis. Decorin overexpression by adenoviral-mediated gene transfection in balloon-injured carotid arteries significantly decreased intimal cross-sectional area and collagen content by approximately 50% at 10 weeks compared to beta-galactosidase-transfected or balloon-injured, non-transfected controls. This study shows that decorin binds to PDGF and inhibits its stimulatory activity on SMCs by preventing PDGFR phosphorylation. Decorin overexpression reduces intimal hyperplasia and collagen content after arterial injury. Decorin may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.     

Intimal healing. The pattern of reendothelialization and intimal thickening.

Studies were undertaken to investigate further the basis of intimal proliferation and the identity of the surface lining cell in rabbit aortas subjected to extensive deendothelialization. Endothelial cells were selectively removed by passage of an inflated balloon catheter through the arterial lumen. The healing response was evaluated at intervals up to 36 weeks by several techniques: 1) permeability to Evans blue, 2) reappearance of endothelial cells as indicated by the specific marker, adsorbed goat anti-rabbit tissue factor-horseradish peroxidase, 3) planimetric measurements of intimal thickness, and 4) electron microscopy. The results indicate that endothelial cell recovery progressed slowly and that it extended only from areas spared denudation. The regions not covered by endothelial cells were lined by cells of smooth muscle cell origin. Such areas were permeable to Evans blue-protein complex, and their luminal smooth muscle cells were associated with connective tissue-like material at their luminal surface; this material apparently acted as a base for platelet accumulation. The present findings indicate that the lumen of the extensively denuded vessel is lined by either endothelial or smooth muscle cells and that intimal healing is related to restoration of endothelial cell cover. In addition, intimal thickening reached a maximum well before reendothelialization was complete.     

L-精氨酸对大鼠血管损伤后内膜增生及相关细胞周期调控基因表达的影响

目的:研究L-精氨酸(L-Arg)对大鼠血管损伤后内膜增生及相关细胞周期调控基因表达的影响。方法:大鼠分为假手术组,球囊损伤组(又分为术后48h、7d和14d亚组)及球囊损伤+L-Arg组。取各组实验动脉段测新生内膜面积,并采用免疫组化及计算机图像分析法测细胞周期依赖性激酶2(CDK2)、细胞周期蛋白E(cyclinE)和增殖细胞核抗原(PCNA)的表达水平。结果:球囊损伤后14d组的血浆NO水平低于假手术组(P＜0.01),CDK2、cyclinE及PCNA均于球囊损伤术后48h在中膜表达,第7d、14d在内膜表达,中膜几无表达,随着内膜增厚表达水平上升。与球囊损伤后14d组相比,球囊损伤+L-Arg组的血浆NO水平增高(P＜0.01),新生内膜面积少59.1%(P＜0.01),CDK2、cyclinE及PCNA的阳性表达指数分别低36.1%,46.3%和76.2%(P均＜0.01)。结论:L-Arg可有效抑制血管损伤后内膜增生,其机制可能与逆转CDK2、cyclinE及PCNA的过表达从而抑制血管平滑肌细胞增殖有关。