The role of the adventitia in the arterial response to angioplasty: the effect of intravascular radiation

PURPOSE: In the current series of experiments we have characterized cell proliferation leading to vascular lesion formation in a porcine model for post-angioplasty restenosis and examined the mechanism of action of intravascular beta irradiation in the prevention of lesion formation in this model. METHODS AND MATERIALS: Juvenile male pigs were subjected to balloon overstretch injury of the left anterior descending and circumflex coronary arteries using clinical angioplasty catheters. Proliferating cells were labelled by injections of 50 mg/kg of bromo-deoxyuridine (BrDU) 24, 16 and 8 hrs prior to sacrifice and were detected by immunohistochemistry using a specific antibody to BrDU. In some cases, BrDU was given as a pulse 3 days after angioplasty and the animals sacrificed on day 14 to follow the migration of the cells which had proliferated earlier. Characterization of the proliferating cells was performed by immunohistochemistry using antibodies to specific cytoskeletal proteins specific for smooth muscle cells and myofibroblasts. Some vessels were treated at the time of angioplasty with 14 or 28 Gy (to a depth of 2 mm) intravascular irradiation using a flexible catheter with a pure beta emitter 90 SR/Y and the effect on cell proliferation and terminal transferase-mediated UTP nick-end labelling (TUNEL) examined 3 or 7 days later. RESULTS: The first major site of cell proliferation between 2-3 days after angioplasty is the adventitia and not the medial wall. Seven days after angioplasty cell proliferation is predominant in the neointima and is reduced in the media and adventitia. Differential staining with antibodies directed against smooth muscle alpha actin and other cytoskeletal proteins indicates that the proliferating adventitial cells are myofibroblasts. Pulse label studies with BrDU indicates that the proliferating adventitial myofibroblasts migrate into the neointima and contribute to the mass of the restenosis lesion. Fourteen days after angioplasty the myofibroblasts in the neointima and the adventitia express alpha smooth muscle actin and form a fibrotic scar in the adventitia surrounding the injury site. Endovascular irradiation appears to inhibit development of the restenosis lesion by significantly reducing cell proliferation in the media and adventitia at early time points after injury. There were no significant differences in the percent of TUNEL labelled cells in the irradiated vessels compared to controls. Alpha actin staining of myofibroblasts in the adventitia was reduced in the irradiated vessels suggesting a positive effect of intravascular irradiation on vascular remodeling. CONCLUSIONS: These studies have shown that adventitial myofibroblasts contribute to the problem of post-angioplasty restenosis by proliferating, forming a fibrotic scar surrounding the injury site, and migrating into the neointima. We hypothesize that the adventitial fibrosis which develops at the injury site contributes to negative vascular remodeling associated with clinical restenosis. Experiments in which vessels were exposed to intravascular irradiation at the time of angioplasty indicate that this treatment reduces post-angioplasty restenosis by inhibiting early cell proliferation in the media and adventitia and by preventing the fibrotic changes in the adventitia without a corresponding increase in cellular death or apoptosis in these tissues.     

Activation of mitogen-activated protein kinases (ERK/JNK) and AP-1 transcription factor in rat carotid arteries after balloon injury

Smooth muscle cell proliferation is a key event in neointimal formation after balloon angioplasty. The molecular signals that mediate this process have yet to be identified. Mitogen-activated protein (MAP) kinases are thought to play a pivotal role in transmitting transmembrane signals required for cell proliferation in vitro. The present studies were designed to investigate whether the signal transduction pathways of MAP kinases were involved in the development of restenosis in the injured arteries. Rat carotid arteries were isolated at various time points after balloon injury, and activities of MAP kinases, including extracellular signal-regulated kinases (ERK), and stress activated protein kinases (SAPK)/c-Jun N-terminal protein kinases (JNK), were determined in protein extracts of the vasculature using protein kinase assay and Western blot analysis. After balloon angioplasty, ERK2 and JNK1 activities in the vessel wall increased rapidly, reached a high level in 5 minutes and maintained for 1 hour. A sustained increase in ERK2 kinase activity was observed over the next 7 days in the arterial wall and 14 days in neointima after injury. In contrast, opposite and uninjured arteries did not show significant changes in these kinase activities. Concomitantly, Western blot analysis confirmed that the ERK2 kinase in the injured vessels was indeed activated or phosphorylated, showing a slowly migrating species of a 42-kDa protein containing phosphorylated tyrosine. Kinase activation is followed by an increase in c-fos and c-jun gene expression and enhanced activator protein 1 (AP-1) DNA-binding activity. Thus, balloon injury rapidly activates the MAP kinases in rat carotid arteries. These kinase activations may be crucial in mediating smooth muscle cell proliferation in response to vascular angioplasty.     

Temporal expression of c-fos mRNA following balloon injury in the rat common carotid artery

OBJECTIVE: Restenosis is a common problem which limits the effectiveness of percutaneous transluminal coronary angioplasty (PTCA). The cellular mechanisms of restenosis appear to involve smooth muscle cell (SMC) migration to the neointima in response to mitogens and growth factors, resulting in proliferation and deposition of cells in the lumen of the vessel. An antibody directed against PDGF attenuates this response in the rat. Thus, signaling cascades induced by growth factors including PDGF may be important targets for therapeutic intervention. METHODS: Since a number of growth factors activate c-fos via the p21-ras signaling pathway, we examined c-fos expression in a time course experiment involving restenotic lesions in rat carotid arteries. Sections of arteries collected at 1, 3, 7, 14 and 28 days following balloon injury were hybridized using a fluorescein-labeled RNA probe to c-fos. Immunohistochemistry was performed with antibodies to proliferating cell nuclear antigen (PCNA) and alpha-smc actin to characterize cellular constituents of the neointima, and detect any correlation between fos expression and PCNA localization. RESULTS: Expression of c-fos was low at day 1. By day 3, the media and adventitia were positively stained. At days 7 and 14, most cells in the neointima were labeled. By day 28, c-fos was expressed mainly in scattered cells along the luminal surface. Control sections revealed little labeling and confirmed specific staining by the antisense strand, PCNA localization and c-fos expression were similar at days 1, 3, 7 and 28, but at day 14 c-fos was expressed throughout the lesion, with PCNA localized mainly along the luminal edge. The majority of the cells making up the neointima stained rather intensely for alpha-smc actin, identifying them as SMCs. CONCLUSIONS: Results of these experiments indicate that, while c-fos expression correlates with lesion formation, it may be associated with a cellular process distinct from proliferation in this model.     

Dynamic imaging of the pancreas using real-time endoscopic ultrasonography with secretin stimulation

BACKGROUND: Tissue factor (TF) is a transmembrane glycoprotein that, after binding to factor VII/VIIa, initiates the extrinsic coagulation pathway, resulting in thrombin generation and its sequelae. Thrombin has been shown to induce TF mRNA in endothelium, monocytes, and smooth muscle cells, further perpetuating the thrombogenic cycle. This study was designed to determine the effect of specific inhibition of thrombin by recombinant hirudin (r-hirudin) on TF distribution after balloon angioplasty in the cholesterol-fed rabbit femoral artery and porcine coronary artery models. METHODS AND RESULTS: Thirty-five femoral arteries from 32 cholesterol-fed New Zealand White rabbits and 84 coronary arteries from 55 Yorkshire-Albino swine were studied by use of a recently developed in situ method of TF localization based on digoxigenin labeling of recombinant factor VIIa (Dig-VIIa), with correlative studies of TF immunoreactivity by use of anti-rabbit (AP-1) or anti-human (sTF) antibodies. At sites of balloon angioplasty in rabbit femoral or pig coronary arteries (double or single injury), TF-antibody and Dig-VIIa staining were noted in association with endothelial cells, smooth muscle cells, and foam cells and within the fibrous tissue matrix primarily of the adventitia and neointima. Staining was significantly greater after balloon angioplasty than in vessels that had not undergone angioplasty but was similar after single and double balloon injury. Animals treated with r-hirudin (rabbits, 1 mg/kg bolus plus 2-hour infusion; pigs, 1 mg/kg bolus plus 0.7 mg x kg(-1) x d(-1) infusion for 14 days with implantable pump) had diminished TF-antibody and Dig-VIIa staining 28 days after balloon angioplasty compared with controls (bolus heparin only). This effect was more prominent on the neointima and was more striking in the porcine than the rabbit model. CONCLUSIONS: TF expression, persistent 1 month after balloon angioplasty in rabbit femoral arteries and porcine coronary arteries, is attenuated by specific thrombin inhibition with hirudin. These results suggest that thrombin inhibition, in addition to its effect on acute thrombus formation and its effect on luminal narrowing by plaque in experimental animals, may result in a prolonged reduction in thrombogenicity of the restenotic plaque through this effect on TF expression.     

Endovascular beta-radiation to reduce restenosis after coronary balloon angioplasty: results of the beta energy restenosis trial (BERT)

BACKGROUND: In the porcine overstretch injury model of restenosis, endovascular beta-radiation reduces neointima formation. To determine whether this therapy could be applied to patients with coronary artery disease, a special device was developed to allow delivery of 12 encapsulated 90Sr/Y sources, measuring a total of 30 mm, to various sites within the coronary arterial tree. This study was designed to evaluate the feasibility of the delivery of 12, 14, or 16 Gy at 2 mm after balloon angioplasty of stenoses of native coronary vessels. METHODS AND RESULTS: Delivery of beta-radiation was attempted in 23 patients after successful balloon angioplasty. Source delivery was successful in 21 of the 23 patients (91%). There was no in-hospital or 30-day morbidity or mortality. Follow-up quantitative coronary arteriography in 20 patients demonstrated a late loss of 0.05 mm, a late loss index of 4%, and a restenosis rate of 15%. The use of the beta-emitter 90Sr/Y significantly reduced treatment time and operator exposure compared with previous trials with the gamma-emitter 192Ir. CONCLUSIONS: In this study, the administration of endovascular beta-radiation after angioplasty was safe and feasible and substantially altered the postangioplasty late lumen loss, resulting in a lower-than-expected rate of restenosis. On the basis of these encouraging results, a multicenter, randomized trial with operators and patients blinded to treatment assignment is planned.     

Apoptosis and cell proliferation after porcine coronary angioplasty

BACKGROUND: Angioplasty initiates a number of responses in the vessel wall including cellular migration, proliferation, and matrix accumulation, all of which contribute to neointima formation and restenosis. Cellular homeostasis within a tissue depends on the balance between cell proliferation and apoptosis. METHODS AND RESULTS: Profiles of apoptosis and proliferation were therefore examined in a porcine PTCA injury model over a 28-day period. Forty-two arteries from 21 pigs, harvested at the site of maximal injury at 1, 6, and 18 hours, and 3, 7, 14, and 28 days after PTCA, were examined (n=3 animals per time point). Uninjured arteries were used as controls. Apoptosis was demonstrated by the terminal uridine nick-end labeling (TUNEL) method, transmission electron microscopy (TEM), and DNA fragmentation. Cells traversing the cell cycle were identified by immunostaining for proliferating cell nuclear antigen (PCNA). Apoptosis was not detected in control vessels at all time points nor at 28 days after PTCA. Apoptotic cells were identified at all early time points with a peak at 6 hours (5.1+/-0.26%; compared to uninjured artery, P<0.001) and confirmed by characteristic DNA ladders and TEM findings. Regional analysis showed apoptosis within the media, adventitia, and neointima peaked at 18 hours, 6 hours, and 7 days after PTCA, respectively. In comparison, PCNA staining peaked at 3 days after PTCA (7.16+/-0.29%; compared to 1.78+/-0.08% PCNA-positive cells in the uninjured artery, P<0.001). Profiles of apoptosis and cell proliferation after PTCA were discordant in all layers of the artery except the neointima. These profiles also differed between traumatized and nontraumatized regions of the arterial wall. Immunostaining with cell-type specific markers and TEM analysis revealed that apoptotic cells included vascular smooth muscle cells (VSMCs), inflammatory cells, and adventitial fibroblasts. CONCLUSIONS: These results suggest that the profile of apoptosis and proliferation after PTCA is regional and cell specific, and attempts to modulate either of these events for therapeutic benefit requires recognition of these differences.     

High-dose chemotherapy and peripheral blood stem cell infusion in patients with non-Hodgkin''s lymphoma: results of outpatient treatment in community cancer centers

There is interest in the role of growth factors in the genesis of arterial remodeling. We studied local administration of basic fibroblast growth factor (bFGF) to coronary lesions to determine whether there is a difference in remodeling and whether neovascularization could be induced in such stenoses and distal myocardium. Pigs were randomized to balloon infusion of either saline or bFGF at each thermally injured arterial site. After the animals were killed, their internal elastic lamina, neointima, and lumen areas were measured. Capillaries were counted in the arteries and myocardium. There was a greater loss of lumen and internal elastic in the bFGF group. The neointima, media, and myocardium in the bFGF treated arteries had statistically more capillaries. This study showed that local intracoronary bFGF, at a dose that results in arterial luminal revascularization in injured segments, adversely affects arterial remodeling. Thus, the angiogenic response to exogenous bFGF may be offset by concomitant shrinkage of injured arterial segments.     

Gene transfer of human prostacyclin synthase prevents neointimal formation after carotid balloon injury in rats

BACKGROUND AND PURPOSE: A disordered proliferative process in the vascular wall is thought to underlie the pathogenesis of restenosis after percutaneous transluminal angioplasty and carotid endarterectomy. A growth inhibitory property of overexpressed prostacyclin (PGI2) synthase (PGIS) was recently implicated in the pathological proliferation of vascular smooth muscle cells (VSMC) in vitro. Here, we investigated the effects of increased PGI2 synthesis on the pathological proliferation of VSMCs. METHODS: The cDNA encoding human PGIS was transfected into endothelium-denuded rat carotid arteries after arterial balloon injury with the use of hemagglutinating virus Japan (HVJ). HVJ liposome vector complex without PGIS cDNA was used for vehicle control. The level of 6-keto PGF1alpha, a stable hydrolyzed metabolite of PGI2, the histological distribution of the immunoreactivity for human PGIS and the ratio of neointimal/medial area were analyzed. RESULTS: In the analyses of 6-keto PGF1alpha, the level in the carotid arteries was significantly elevated 3 days after PGIS expression-vector transfection compared with that in the arteries after vehicle transfection. Seven days after human PGIS expression-vector transfection, the PGIS cDNA-transfected neointimal cells were strongly positive for human PGIS immunoreactivity in 81% sections examined. Fourteen days after the injury, the ratio of neointimal/medial area was 1.2+/-0.4 in the PGIS expression-vector transfected group, which was significantly smaller than that of the vehicle control group, 1.7+/-0.5; P<0.01. CONCLUSIONS: It was thus demonstrated that the gene transfer of human PGIS expression-vector into rat carotid arteries resulted in the increased production of human PGI2 in the vascular wall, the expression of human PGIS in the developing neointima and significantly inhibited the neointimal formation generated after balloon injury.     

Effects of locally administered argatroban on restenosis after balloon angioplasty: experimental and clinical study

1. This study was undertaken to evaluate the preventive effects of locally administered argatroban, a competitive inhibitor of thrombin-induced platelet activation, on restenosis after balloon angioplasty. 2. A hydrogel-coated balloon catheter was immersed three times in argatroban/saline solution (1 mg/mL) for 60 s, inflated to a pressure of 606 kPa and left in the rabbit common carotid artery for 1 min. The same procedure was performed, without drug, as a control. The pharmacokinetics of delivered argatroban in the arterial wall were assessed using [14C]-argatroban. Platelet deposition 2 h after balloon injury was quantified by fluorescence studies using antiplatelet antibody. Vascular smooth muscle cell (VSMC) proliferation 3 days after balloon injury was assessed by immunohistochemical staining for proliferative cell nuclear antigen (PCNA). In a clinical study, we divided 50 elective patients into two groups: argatroban and control. 3. In the experimental study, the mean quantities of argatroban at 0, 2 and 6 h after deflation were 24.63, 0.49 and 0.11 nmol/g wet weight of artery, respectively. Argatroban was undetected 24 h after deflation. Two hours after deflation, argatroban-treated arteries showed less platelet adhesion than saline-treated controls. The mean number of PCNA-positive cells was 16.9 and 43.8% in the argatroban and control groups, respectively (P < 0.01). In the clinical study, the mean late gain loss was 8.2 and 27.3% in the argatroban and control groups, respectively (P < 0.05). The mean late restenosis rate was 11.1 and 41.4% in the argatroban and control groups, respectively (P < 0.05). 4. These data suggest that blood coagulation plays a significant role in VSMC proliferation after balloon injury and that locally administered argatroban using hydrogel-coated balloon catheter may prevent post-percutaneous transluminal coronary angioplasty restenosis.     

Probucol decreases neointimal formation in a swine model of coronary artery balloon injury. A possible role for antioxidants in restenosis

BACKGROUND: Restenosis after percutaneous transluminal coronary angioplasty is the major limitation of the long-term success of this procedure. The process of restenosis is similar to an accelerated form of atherosclerosis. Thus, therapeutic interventions that limit the progression and initiation of atherosclerosis may be beneficial in the treatment of restenosis. One such intervention is the antioxidant drug probucol, which has demonstrated benefit in animal models of atherosclerosis. METHODS AND RESULTS: Twenty-six female domestic swine were divided into three study groups (control, n = 9; low-dose probucol, n = 9; high-dose probucol, n = 8) before oversized balloon injury of the left anterior descending and left circumflex coronary arteries. Probucol (1 g/d, low-dose group; 2 g/d, high-dose group) was administered 2 days before balloon injury and was continued until the swine were killed 2 weeks after balloon injury. Morphometric analysis of the injured arteries included the intimal area (square millimeters), maximal intimal thickness (millimeters), and residual lumen (ratio of luminal to intimal plus luminal area). Treatment with high-dose probucol significantly reduced neointimal formation compared with control animals (decreases of 36% in intimal area, P = .007; 20% in maximal intimal thickness, P = NS; and an increase of 15% in residual lumen, P = .02). CONCLUSIONS: The major finding of this study is that the antioxidant drug probucol reduces neointimal formation after oversized balloon injury in a swine model of restenosis. This suggests that active oxygen species may play a role in restenosis.     

The relation between de novo atherosclerosis remodeling and angioplasty-induced remodeling in an atherosclerotic Yucatan micropig model

Geometric remodeling in de novo atherosclerosis and in restenosis after balloon angioplasty constitutes a change in total arterial circumference that, together with plaque growth or neointima formation, determines the lumen of the artery. The heterogeneous nature of arterial obstructions raises the question of whether early and late outcomes (restenosis) of angioplasty are affected by the degree and direction of de novo atherosclerotic remodeling. This study was designed to assess the relationship between atherosclerotic remodeling and the degree and mechanism of restenosis after balloon angioplasty. Atherosclerosis was induced in 27 peripheral arteries of 18 Yucatan micropigs by a combination of denudation and atherogenic diet. Balloon angioplasty was performed, with serial intravascular ultrasound and quantitative angiography before and after intervention and at 42 days' follow-up. We used the relative media-bounded area (MBA), defined as the MBA of the treated site divided by the MBA of the reference, before angioplasty as a measure of remodeling in de novo atherosclerosis and late MBA loss as a measure of remodeling after balloon angioplasty. Relative MBA before angioplasty was not correlated with angiographic and echographic acute gain after balloon angioplasty (r=.22, P=.28 and r=.14, P=.48) or with late lumen loss (r=-.05, P=.81 and r=.19, P=.33). No correlation was found between relative MBA and late MBA loss (r=.14 and P=.48). In the atherosclerotic Yucatan micropig, remodeling during de novo atherosclerosis has no relevance for acute gain and late lumen loss after balloon angioplasty. Both the direction and the extent of remodeling after balloon angioplasty are not related to the direction and extent of remodeling during de novo atherosclerosis.     

The atherosclerotic Yucatan animal model to study the arterial response after balloon angioplasty: the natural history of remodeling

OBJECTIVE: Remodeling in de novo atherosclerosis and in restenosis after balloon angioplasty constitutes a change in total arterial circumference which, together with plaque growth or neointimal formation, determines the lumen of the artery. To better understand the fundamental biology of neointimal formation, remodeling and their interaction, animal studies are needed. In this study, we described in detail the methodology used and the natural history of neointimal formation and remodeling after balloon angioplasty in atherosclerotic Yucatan micropigs. METHODS AND RESULTS: Atherosclerosis was induced in 60 peripheral arteries of sixteen Yucatan micropigs by a combination of denudation and atherogenic diet. Balloon angioplasty was performed in 38 arteries, with serial intravascular ultrasound (IVUS) and quantitative angiography before and after intervention and at 2, 4, 7, 14 or 42 days follow-up. Remodeling, expressed as late media-bounded area (MBA) loss, increased progressively over time. At 42 days, late MBA loss after balloon angioplasty was significantly different compared to late MBA loss in control arteries, 2.2 +/- 1.0 versus -0.3 +/- 1.1 mm2 and p = 0.02. Late lumen loss increased over time and was highest at 42 days after balloon angioplasty (2.8 +/- 0.7 mm2). The contribution of neointimal formation to late lumen loss decreased over time and the contribution of late MBA loss to late lumen increased over time and was highest at 42 days (78%). Medial necrosis was 48% at two days after balloon angioplasty and the repopulation of the media was almost completed at seven days. CONCLUSION: Remodeling following balloon angioplasty has an early onset and progresses with neointimal formation to cause restenosis over the standard 42-day time course for Yucatan micropigs. This correlates to six months renarrowing in humans. In this model, atherosclerosis and the natural history of restenosis, both with respect to neointimal formation and remodeling, resemble the human disease quite closely.     

Conjugated equine estrogens inhibit progression of atherosclerosis but have no effect on intimal hyperplasia or arterial remodeling induced by balloon catheter injury in monkeys

OBJECTIVES: This study sought to determine the effects of estrogen treatment on atherosclerosis progression and the proliferative and structural responses of the atherosclerotic arteries to injury. BACKGROUND: Estrogen treatment suppresses the intimal response to arterial injury in nonatherosclerotic rodents and rabbits and inhibits the in vitro proliferation of smooth muscle cells. However, the effect of estrogen on the response of atherosclerotic arteries to transmural injury, as occurs in balloon catheter angioplasty in humans, is unknown. METHODS: Forty-six ovariectomized cynomolgus monkeys were fed an atherogenic diet for 30 months; 25 received 175 microg/day of conjugated equine estrogens, and 21 served as untreated control animals. All animals underwent balloon catheter injury of the left iliac artery. Subsets of animals underwent a necropsy study at 4, 7, 14 and 28 days after injury; injured and contralateral (uninjured) arteries were pressure-fixed and evaluated morphometrically. RESULTS: Estrogen treatment resulted in a 37% decrease (p < 0.05) in atherosclerosis (plaque area) in the uninjured artery. In response to injury, arterial cell proliferation increased at days 4 and 7, and intimal area was increased two- to threefold at day 28 (p < 0.05). Although estrogen treatment resulted in a trend toward decreased arterial cell proliferation at day 4, there was evidence of increased cell proliferation in both media and intima at day 7 (p < 0.05). However, there was no effect of estrogen treatment on intimal area or indexes of arterial remodeling in the injured artery at day 28 (p > 0.4). CONCLUSIONS. In contrast to previous studies of nonatherosclerotic animals, the results indicate that in the circumstance of transmural injury to arteries of primates with preexisting atherosclerosis, estrogen does not suppress arterial neointimal or structural responses to injury.     

Wound healing: a paradigm for lumen narrowing after arterial reconstruction

PURPOSE: The intimal hyperplasia hypothesis that equates lumen narrowing after arterial injury with intimal mass has recently been challenged. Evidence has emerged to suggest that lumen narrowing is caused in large part by changes in artery wall geometry rather than intimal mass per se. We have begun to explore this hypothesis in a unique nonhuman primate model of atherosclerosis. METHODS: Monkeys who were fed an atherogenic diet for 3 to 5 years underwent experimental angioplasty of the left iliac artery. The contralateral iliac artery served as an intraanimal control. Arteries were removed 2, 4, 7, 14, 28, or 112 days later for analysis (6 or 13 per time point). Angioplasty dilated arteries by fracturing atheroma and stretching or tearing the media. Cross-sections of injured arteries were analyzed for expression of extracellular matrix components and cell surface integrins that are important in wound healing. Antibodies, riboprobes, or histochemical stains specific for fibrin, hyaluronan, versican (chondroitin sulfate-containing proteoglycan), procollagen-I, elastin, and the alpha 2 beta 1 and alpha V beta 3 integrins were used. RESULTS: A thin mural thrombus was seen at sites of denudation and plaque fracture (days 2 to 7). This provisional matrix was invaded by leukocytes (days 2 to 4) and alpha-actin-positive smooth muscle cells (SMCs; days 4 to 7). Thrombus was replaced by SMCs expressing hyaluronan and the associated versican proteoglycans (day 14). Versican was expressed throughout the neointima as it enlarged (day 28), but expression later subsided (day 112). Procollagen-I expression initially increased in the adventitia (day 4) and then in the forming neointima (day 14). Procollagen-I expression was found to persist within the adventitia and in the neointima in SMCs nearest the lumen (days 28 to 112). Elastin staining was prominent within the mature neointima (day 112) but not at earlier time points. Integrin expression also increased within the injured artery wall. alpha v beta 3 staining (fibrin[ogen] receptor) increased in the injured media (days 2 to 7) and was then seen throughout the early neointima (day 7). Low level expression of alpha V beta 3 subsequently persisted within the forming neointima (day 28). alpha 2 beta 1 (collagen receptor) expression increased in the neointima in SMCs nearest the lumen (day 28). CONCLUSIONS: Lumen narrowing after angioplasty in this model of atherosclerosis is caused largely by decreased artery wall diameter. The pattern of matrix and integrin expression within the injured artery wall is in many ways analogous to that of healing wounds. These observations suggest that tissue contraction may play a role in lumen narrowing at sites of arterial reconstruction. Strategies to inhibit wound contraction may prove effective in preventing restenosis.     

Propofol preserves the viability of isolated rat hepatocyte suspensions under an oxidant stress

OBJECTIVES: This study evaluated the long-term impact of endoluminal low power red laser light (LPRLL) on restenosis in an atherosclerotic rabbit model. BACKGROUND: Despite widespread application of balloon angioplasty for treatment of coronary artery disease, restenosis limits its clinical benefits. Restenosis is a complex process and may be partly attributed to the inability of the vascular endothelium to regenerate and cover the denuded area at the site of arterial injury. We previously demonstrated that LPRLL stimulates endothelial cell proliferation in vitro and contributes to rapid endothelial regeneration after balloon injury in nonatherosclerotic rabbits. METHODS: Rabbit abdominal aortas (n = 12) were treated in separate zones with balloon dilation and balloon dilation plus laser illumination. Endoluminal laser therapy was performed using a laser-balloon catheter delivering a single dose of 10 mW for 3 min from a helium-neon laser (632 nm). Angiography was performed before and after treatment and was repeated 8 weeks before harvesting the aortas. RESULTS: Quantitative angiographic analysis demonstrated no differences in the minimal lumen diameter (MLD) between the two zones before treatment; an increase in the MLD in both zones after balloon angioplasty and a significant versus slight reduction of the MLD in the balloon treatment versus balloon plus laser zones at 8 weeks. Histologic examination showed a very high level of myointimal hyperplasia in the balloon treatment zones but a minimal level in the LPRLL-treated zones. Morphometric analysis revealed a statistically significant difference in the lumen area, intimal area and intima/media ratio between the balloon versus balloon plus laser treatment sites. CONCLUSIONS: Our experimental data indicate that endoluminal irradiation with LPRLL prevents restenosis after balloon angioplasty in an atherosclerotic rabbit model.     

Reduction of restenosis after angioplasty in an atheromatous rabbit model by suicide gene therapy

BACKGROUND: Gene delivery of the thymidine kinase (tk) gene combined with ganciclovir (GCV) limits intimal hyperplasia after abrasion of normal arteries. However, the low efficiency of adenoviral-mediated gene transfer to atherosclerotic arteries has raised concerns about the applicability of this strategy to the prevention of restenosis. METHODS AND RESULTS: A replication-defective adenoviral vector expressing tk (Ad-RSVtk) demonstrated selective toxicity toward GCV-treated arterial smooth muscle cells, with oligonucleolytic cleavage suggesting apoptosis. In vivo, after demonstration of tk expression after Ad-RSVtk delivery, the combination of Ad-RSVtk followed by GCV was tested in a rabbit model of angioplasty of atheromatous iliac arteries. Angioplasty (8 atm, 20 minutes) was performed by use of a hydrogel balloon coated with Ad-RSVtk (4x10(9) plaque forming units). GCV was infused (25 mg.kg(-1) I.V. BID) from days 2 through 7 after angioplasty in 8 of 12 rabbits. Four weeks later, morphometric analysis demonstrated a reduced intima-to-media ratio in the group receiving combination therapy compared with Ad-RSVtk alone (3.0+/-1.2 versus 5.2+/-0.5, P<.018). GCV per se had no effect on intimal hyperplasia after arterial injury. CONCLUSIONS: In vitro, Ad-RSVtk demonstrates selective toxicity toward GCV-treated arterial smooth muscle cells involving apoptosis. In vivo, GCV conditions reduction of neointimal formation after percutaneous delivery of Ad-RSVtk during angioplasty of atheromatous arteries.     

Inhibition of smooth muscle cell proliferation and neointimal growth by low-anticoagulant heparin

Low-anticoagulant heparin (LA-heparin) obtained by affinity chromatography on antithrombin III Sepharose inhibits the proliferation of cultured arterial smooth muscle cells in an in vitro bioassay system as effectively as standard heparin. A growth inhibition of smooth muscle cells of about 60% is achieved when LA-heparin or heparin is added to the culture medium to a concentration of 50 micrograms/ml. In normolipemic rats LA-heparin suppresses the formation of neointimal thickenings and stenosis after balloon catheter-induced deendothelialization of the carotid artery. In terms of mass a dose of 5 mg/kg body weight/d given subcutaneously twice daily one week before and 2 weeks after balloon injury the cross sectional area of the neointima is reduced to 36% as compared with the nontreated control group (100%). This 64% reduction is statistically highly significant (p < 0.001). After treatment with 0.5 mg LA-heparin/kg/d the reduction of the neointima was 11% (p < 0.05). At a dose of 5 mg/kg body weight single or repeated administrations of LA-heparin caused only a small and transient increase in activated partial thromboplastin time values. The results show that subcutaneous administration of LA-heparin very effectively prevents smooth muscle cell proliferation and balloon catheter-induced neointimal growth. The well tolerated systemic application of this chemically non-modified LA-heparin might justify clinical trials for prevention of restenosis after percutaneous transluminal coronary angioplasty or other invasive cardiovascular interventions without complications of bleeding.     

Evidence that implicates the parathyroid hormone-related peptide in vascular stenosis. Increased gene expression in the intima of injured carotid arteries and human restenotic coronary lesions

Proliferation of vascular smooth muscle cells (VSMCs) is considered to be one key event underlying the pathophysiology of restenosis after angioplasty. The parathyroid hormone-related peptide (PTHrP) and its receptor, a local autocrine and paracrine regulator of cellular growth in a variety of normal cell types, have been reported in the vicinity of VSMCs. To investigate how PTHrP might be involved in the process of neointimal formation after balloon angioplasty, we examined PTHrP expression in balloon-denuded rat carotid arteries and human coronary arteries that had been retrieved by directional atherectomy. In rat carotid arteries, the RNase protection assay and in situ hybridization demonstrated that PTHrP mRNA expression increased fourfold to sixfold 1 to 7 days after denudation and continued for 28 days, coincident with downregulation of PTH/PTHrP receptor mRNA expression. In situ hybridization and immunohistochemistry revealed that PTHrP expression in balloon-denuded carotid arteries was mainly localized to the neointima. To confirm the involvement of the PTHrP in human coronary artery restenotic lesions, immunohistochemical analysis of human coronary atherectomy specimens (23 primary and 10 restenotic lesions) was then performed. The number of intimal cells that expressed PTHrP protein was significantly higher in restenotic (407 +/- 53 cells/mm2; range, 143 to 739) than in stable angina (50 +/- 12 cells/mm2; range, 18 to 132; P<.05) or unstable angina (129 +/- 16 cells/mm2; range, 21 to 232; P<.05) specimens. These data demonstrate that PTHrP gene expression in VSMCs markedly increases during neointimal formation, supporting the hypothesis that PTHrP may play an important role in vascular stenosis as a regulator of VSMC proliferation.     

Inhibition of leucocyte and platelet adhesion reduces neointimal hyperplasia after arterial injury

Restenosis following coronary angioplasty is though to result from migration and proliferation of medial smooth muscle cells. However, the factors that initiate this proliferation are still unknown. In a rabbit model of carotid artery injury, we tested the hypothesis that activated platelets and leucocytes might contribute to the development of neointimal hyperplasia. Following arterial injury, rabbits received either no treatment, R15.7, a monoclonal antibody against the leucocyte CD11/CD18 adhesion complex, aurintricarboxylic acid (ATA), a substance that inhibits platelet glycoprotein Ib-von Willebrand factor interaction, or the combination of R15.7 and ATA. After 21 days, the extent of neointimal hyperplasia was evaluated by planimetry on histological arterial sections. The area of neointima averaged 0.51 +/- 0.07 mm2 in control animals and it was significantly reduced by administration of either R15.7 or ATA alone to 0.12 +/- 0.05 and 0.20 +/- 0.01 mm2, respectively (p < 0.05 vs controls for both groups). The animals that received the combination of R15.7 and ATA showed a further reduction in neointimal hyperplasia, as compared to animals that received ATA alone (p < 0.05 vs ATA alone). These data indicate that platelets and leucocytes play an important role in the pathophysiology of neointimal hyperplasia in this experimental model. Interventions that reduce platelet and leucocyte adhesion to vessel wall might have beneficial effects in reducing restenosis following coronary angioplasty.