Neointimal Smooth Muscle Cells Research Articles

Continuation of trastuzumab-based therapy beyond disease progression in metastatic breast cancer patients – a retrospective one center analysis

Despite decades of research, anastomotic intimal hyperplasia remains a major cause of delayed prosthetic arterial graft failure. Previously, we reported profound upregulation of thrombospondin-2 (TSP-2) mRNA in neointimal smooth muscle cells after prosthetic arterial bypass graft placement. TSP-2 is an antiangiogenic matricellular protein with specific functions yet unknown. In this study, we hypothesized that inhibition of TSP-2 in human aortic smooth muscle cells (HAoSMCs) would reduce cell proliferation and migration in vitro, providing a therapeutic target to mitigate intimal hyperplasia.HAoSMCs were transfected with TSP-2 small interfering ribonucleic acid (siRNA) using a commercial transfection reagent. Gene silencing was evaluated using semiquantitative real-time polymerase chain reaction. ELISA was used to measure TSP-2 protein levels in cell culture supernatants. Cell migration and proliferation were assessed using scratch wound assays and alamar blue assays, respectively. Attachment assays were performed to assess the effect of TSP-2 silencing on HAoSMC adhesion to fibronectin.TSP-2 siRNA achieved consistent target gene silencing at 48 hours post-transfection in HAoSMCs. This single transfection allowed suppression of TSP-2 protein expression for more than 30 days. TSP-2 gene silencing did not affect HAoSMC migration or proliferation. MMP-2 levels were also unaffected by changes in TSP-2 protein levels. However, HAoSMC attachment to fibronectin improved significantly in cells treated with TSP-2 siRNA.siRNA-mediated TSP-2 silencing of human aortic HAoSMCs improved cell attachment but had no effect on cell migration or proliferation. The effect on cell attachment was unrelated to changes in MMP activity.

The Source of Neointimal Cells in Vein Grafts: Does the Origin Matter?

The focus of this Commentary is the existing plasticity in the origin of neointimal vascular smooth muscle cells and endothelial cells after vein grafting.

Nicotine Inhibits Apoptosis and Stimulates Proliferation in Aortic Smooth Muscle Cells Through a Functional Nicotinic Acetylcholine Receptor

Atherosclerosis and neointimal hyperplasia formation are induced by alterations in the homeostatic balance between cell growth and cell death. Apoptosis is a physiological cell death process that, when deregulated, may be involved in many pathological conditions. Cigarette smoking is a primary risk factor for vascular disease and nicotine seems to exert its atherogenic effects in part through the increase of smooth muscle cell (SMC) proliferation. The aim of this study was to investigate the effect of nicotine on SMC apoptosis. Nicotine added for 24 and 72 h to serum deprived cell cultures resulted in a decrease of apoptotic SMCs. The inhibition was direct and not mediated by platelet-derived growth factor, basic fibroblast growth factor, and transforming growth factor beta(1), autocrinally released by nicotine-treated SMCs, because it was not influenced by addition of specific neutralizing antibodies. Apoptosis inhibition as well as the proliferation increase, and basic fibroblast growth factor expression on nicotine-treated SMCs were blocked by nicotinic acetylcholine receptor antagonists, including alpha-bungarotoxin, a competitive antagonist of alpha subunits of nicotinic receptor. In conclusion, we propose that nicotine could lead to the increase of neointimal SMCs in vascular lesions by inducing the inhibition of physiological SMC apoptosis and the increase of SMC proliferation. We also showed that nicotine signaling occurs as a result of activation of the classical nicotine receptor pathways.

Abstract 397: Perivascular Fat Tissue Plays An Atheroprotective Role In Neointimal Formation After Angioplasty

Background: Obesity is associated with chronic inflammation and increases risk for cardiovascular disease and type 2 diabetes. Recently visceral, as opposed to subcutaneous, fat tissue confers the most cardio-metabolic risk. The aim of the present study is to determine whether perivascular fat tissue contributes to neointimal formation after angioplasty. Methods and Results: An endovascular injury was induced in the femoral arteries of wild-type mice without or with removal of perivascular adipose tissue. Neointimal area and Neointima/Media ratio were markedly enhanced by removal of perivascular adipose mice (P=0.05). Visceral fat tissue (VAT) or subcutaneous fat tissue (SAT) were harvested from GFP-transgenic mice and transplanted around the injured femoral without endogenous perivascular fat. Neointimal formation was markedly enhanced in the VAT-transplantation group compared with the SAT-transplantation group (P=0.05). Real-time PCR analysis showed that the transplanted VAT had significantly higher proinflammatory cytokines, TNF alpha, IL-6, MCP-1, and PAI-1, mRNA levels than the SAT (P=0.05). Next, the effects of VAT and SAT on smooth muscle cell proliferation were investigated in vitro. Conditioned medium was prepared from extracts of VAT and SAT. Cell proliferation was measured by MTS assay. VAT stimulated smooth muscle cell proliferation compared with SAT (P=0.05). Conclusions: The present study demonstrates that perivascular adipose tissue protects neointimal formation after angioplasty. VAT stimulates neointimal formation and smooth muscle cell proliferation, probably by secreting proinflammatory cytokines, at least in part. Adipocytokines, particularly secreted from perivascular fat tissue, may play a role in the pathogenesis of neointimal formation after angioplasty.

Smooth Muscle Cells Healing Atherosclerotic Plaque Disruptions Are of Local, Not Blood, Origin in Apolipoprotein E Knockout Mice

Signs of preceding episodes of plaque rupture and smooth muscle cell (SMC)-mediated healing are common in atherosclerotic plaques, but the source of the healing SMCs is unknown. Recent studies suggest that activated platelets adhering to sites of injury recruit neointimal SMCs from circulating bone marrow-derived progenitor cells. Here, we analyzed the contribution of this mechanism to plaque healing after spontaneous and mechanical plaque disruption in apolipoprotein E knockout (apoE-/-) mice. To determine the origin of SMCs after spontaneous plaque disruption, irradiated 18-month-old apoE-/- mice were reconstituted with bone marrow cells from enhanced green fluorescent protein (eGFP) transgenic apoE-/- mice and examined when they died up to 9 months later. Plaque hemorrhage, indicating previous plaque disruption, was widely present, but no bone marrow-derived eGFP+ SMCs were detected. To examine the origin of healing SMCs in a model that recapitulates more features of human plaque rupture and healing, we developed a mechanical technique that produced consistent plaque disruption, superimposed thrombosis, and SMC-mediated plaque healing in apoE-/- mice. Mechanical plaque disruption was produced in irradiated apoE-/- mice reconstituted with eGFP+ apoE-/- bone marrow cells and in carotid bifurcations cross-grafted between apoE-/- and eGFP+ apoE-/- mice. Apart from few non-graft-derived SMCs near the anastomosis site in 1 transplanted carotid bifurcation, no SMCs originating from outside the local arterial segment were detected in healed plaques. Healing SMCs after atherosclerotic plaque disruption are derived entirely from the local arterial wall and not circulating progenitor cells in apoE-/- mice.

Expression of HIF-1α in Injured Arteries Controls SDF-1α–Mediated Neointima Formation in Apolipoprotein E–Deficient Mice

Hypoxia-inducible factor (HIF)-1alpha is the regulatory subunit of a transcriptional complex, which controls the recruitment of multipotent progenitor cells and tissue repair in ischemic tissue by inducing stromal cell-derived factor (SDF)-1alpha expression. Because HIF-1alpha can be activated under normoxic conditions in smooth muscle cells (SMCs) by platelet products, we investigated the role of HIF-1alpha in SDF-1alpha-mediated neointima formation after vascular injury. Wire-induced injury of the left carotid artery was performed in apolipoprotein E-deficient mice. HIF-1alpha expression was increased in the media as early as 1 day after injury, predominantly in SMCs. Nuclear translocation of HIF-1alpha and colocalization with SDF-1alpha was detected in neointimal cells after 2 weeks. HIF-1alpha mRNA expression was induced at 6 hours after injury as determined by real-time RT-PCR. Inhibition of HIF-1alpha expression by local application of HIF-1alpha-siRNA reduced the neointimal area by 49% and significantly decreased the neointimal SMCs content compared with control-siRNA. HIF-1alpha and SDF-1alpha expression were clearly diminished in neointimal cells of HIF-1alpha-siRNA treated arteries. HIF-1alpha expression is directly involved in neointimal formation after vascular injury and mediates the upregulation of SDF-1alpha, which may affect the stem cell-based repair of injured arteries.

Y-Box Binding Protein-1 Controls CC Chemokine Ligand-5 (CCL5) Expression in Smooth Muscle Cells and Contributes to Neointima Formation in Atherosclerosis-Prone Mice

The CC chemokine CCL5/Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) is upregulated in mononuclear cells or deposited by activated platelets during inflammation and has been implicated in atherosclerosis and neointimal hyperplasia. We investigated the influence of the transcriptional regulator Y-box binding protein (YB)-1 on CCL5 expression and wire-induced neointimal hyperplasia. Analysis of the CCL5 promoter revealed potential binding sites for YB-1, and interaction of YB-1 with a sequence at position -204/-173 was confirmed by DNA binding assays. Both YB-1 expression and CC chemokine ligand-5 (CCL5) mRNA expression were increased in neointimal versus medial smooth muscle cells, as analyzed by real-time polymerase chain reaction. Overexpression of YB-1 in smooth muscle cells (but not macrophages) enhanced CCL5 transcriptional activity in reporter assays, mRNA and protein expression, and CCL5-mediated monocyte arrest. Carotid arteries of hyperlipidemic apolipoprotein E-deficient mice were subjected to intraluminal transfection with a lentivirus encoding YB-1 short hairpin RNA or empty vector directly after wire injury. Double immunofluorescence revealed YB-1 expression in neointimal smooth muscle cells but not macrophages and colocalization with neointimal CCL5, which was downregulated by YB-1 short hairpin RNA. Neointima formation was decreased significantly after YB-1 knockdown compared with controls and was associated with a diminished content of lesional macrophages. A reduction of lesion formation by YB-1 knockdown was not observed in apolipoprotein E-deficient mice deficient in the CCL5 receptor CCR5 or after treatment with the CCL5 antagonist Met-RANTES, which indicates that YB-1 effects were dependent on CCL5. The transcriptional regulator YB-1 mediates CCL5 expression in smooth muscle cells and thereby contributes to neointimal hyperplasia, thus representing a novel target with which to limit vascular remodeling.

Rapamycin regulates synthesis of transforming growth factor-beta1 and extracellular matrix in arterial neointimal smooth muscle cells

Rapamycin regulates synthesis of transforming growth factor-beta1 and extracellular matrix in arterial neointimal smooth muscle cells

Angiotensin-converting enzyme inhibitor reduces proadhesive activity of neointimal smooth muscle cells through attenuating constitutive NF-kB activity

RESULTS: During the study period, 63 patients met inclusion criteria. Mean pre-procedural observation was 36( / 24) months and mean follow-up was 11.7( / 9) months. Renal impairment occurred in 30% of patients, whereas 65% experienced a worsening inverse creatinine slope. With multivariate analysis, the presence of intrinsic nephropathy and worsening creatinine slope were independent predictors of renal impairment during the follow-up period (P 0.05). Table. Independent Predictors of Developing Renal Insufficiency After TRF for EVAR

Thrombospondin-1 Activates Medial Smooth Muscle Cells and Triggers Neointima Formation Upon Mouse Carotid Artery Ligation

Thrombospondin-1 (TSP1) is described as a positive regulator of vascular smooth muscle growth in cell culture. However, insight into the in vivo effects of TSP1 on smooth muscle cell (SMC) function is lacking. We analyzed wild-type (WT) and TSP1-deficient (Tsp1-/-) mice in a carotid artery ligation model, in which neointimal lesions form without overt mechanical damage to the endothelium. On ligation, the expression of TSP1 increased strongly in the matrix of neointima and adventitia. In the early phase after ligation (day 3 to 7), activation, proliferation, and migration of medial SMCs were delayed and impaired in Tsp1-/- mice, in parallel with defective upregulation of metalloproteinase (MMP)-2 activity. As a result, Tsp1-/- arteries developed smaller neointimal lesions, a thicker media but comparably attenuated patency as in WT arteries, 28 days after ligation. Furthermore, medial and neointimal SMCs in Tsp1-/- mice produced more collagen, more osteopontin, and displayed weaker smooth muscle actin staining than WT SMCs, indicative of a modified SMC phenotype in Tsp1-/- mice. Arterial SMC activation in the absence of TSP1 is delayed and dysregulated, reducing neointima formation, on mild vascular injury.

Rosiglitazone Attenuates Transplant Arteriosclerosis After Allogeneic Aorta Transplantation in Rats

Transplant arteriosclerosis is a leading cause of chronic transplant dysfunction and is characterized by occlusive neointima formation in intragraft arteries. Development of transplant arteriosclerosis is refractory to conventional immunosuppressive drugs and adequate therapy is not available. In this study, we determined the efficacy of the synthetic peroxisome proliferator-activated receptor (PPAR)-gamma agonist rosiglitazone to attenuate the development of transplant arteriosclerosis in rat aortic allografts. Lewis aortic allografts were transplanted into Brown Norway recipient rats. Recipient rats received either approximately 5 mg rosiglitazone/day (starting 1 week before transplantation until the end of the experiment) or were left untreated. Transplant arteriosclerosis was quantified using morphometric analysis. Alloreactivity was measured in vitro using mixed lymphocyte reactions. Regulatory T cell frequency and function were analyzed using flow cytometry and in vitro suppression assays, respectively. Intragraft gene expression was analyzed using real-time polymerase chain reaction. Finally, medial and neointimal vascular smooth muscle cell proliferation was analyzed in vitro. Rosiglitazone significantly reduced transplant arteriosclerosis development 8 weeks after transplantation (P<0.01 vs. nontreated). Rosiglitazone reduced T cell alloreactivity which was not mediated through modulation of CD4+CD25+FoxP3+ regulatory T cells. Reduced development of transplant arteriosclerosis coincided with reduced intragraft expression of stromal-derived factor-1alpha and platelet-derived growth factor receptor-beta. Finally, rosiglitazone reduced growth-factor-driven proliferation of both medial and neointimal vascular smooth muscle cells in vitro, which was not mediated through PPARgamma. PPARgamma agonists may offer a new therapeutic strategy in clinical transplantation to attenuate the development of transplant arteriosclerosis and thereby chronic transplant dysfunction.

Cytokine and toll-like receptor dependent signalling in neointimal smooth muscle cells

Introduction: NF-κB is known to play a central role in the molecular and cellular events of intimal hyperplasia (IH). Since non-specific NF-κB inhibition risks deleterious effects on cell viability we sought to elucidate specific cytokine and TLR-dependent NF-κB pathways in control and neointimal SMCs with a view to targeted inhibition.

The origin of neointimal smooth muscle cells in transplant arteriosclerosis from recipient bone-marrow cells in rat aortic allograft

In order to investigate the origin of neointimal smooth muscle cells in transplant arteriosclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female Wistar rats. Four weeks after transplantation, the aortic transplant model was established by means of micro-surgery in rats. The recipients were divided into 4 groups: female Wistar-female Wistar aortic isografts, female SD female Wistar aortic allografts, male SD-male Wistar aortic allografts, female SD-chimera Wistar aortic allografts. Eight weeks after transplantation, aortic grafts were removed at autopsy and processed for histological evaluation and immunohistochemistry. The results indicated that excessive accumulation of alpha-SMA-positive smooth muscle cells resulted in significant neointima formation and vascular lumen stricture in rat aortic allografts. Neointima assay revealed that the neointimal area and NIA/MA ratio of transplanted artery were significantly increased in all of aortic allograft groups as compared with those in aortic isograft group (P<0.01). Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method. The Sry gene-specific PCR was performed, and the result showed that a distinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic allograft group respectively, but not in the female-female aortic allograft group. It was suggested that recipient bone-marrow cells, as the origin of neointimal smooth muscle cells, contributed to the pathological neointimal hyperplasia of aortic allograft and transplant arteriosclerosis.

Chemokines in vascular remodeling

The arterial vessel wall response to a variety of injuries consists in structural changes, which can result in luminal narrowing and aggravation of the underlying disease. This arterial remodeling is characterized by neointima formation and medial thickening, inflammatory cell recruitment and endothelial dysfunction. Chemokines and the corresponding receptors have been shown to participate at every step of the remodeling process. The monocyte chemotactic protein (MCP)-1/CC motif receptor 2 (CCR2) axis induces monocyte infiltration of the injured vessel wall and can stimulate proliferation of smooth muscle cells (SMCs) in models of restenosis, cardiac allograft vasculopathy (CAV), pulmonary hypertension, and systemic hypertension. In contrast, stromal cell-derived factor (SDF)-1 alpha and its receptor CXC motif receptor 4 (CXCR4) are centrally involved in the neointimal recruitment of SMC progenitor cells (SPCs), presumably in response to SMC apoptosis, in restenosis and CAV. The RANTES (Regulated upon activation, normally T-cell expressed, and presumably secreted) receptors CC motif receptor 1 (CCR1) and CC motif receptor 5 (CCR5) affect intimal monocyte infiltration and neointimal growth, which could be due to the deposition of platelet-derived RANTES on activated endothelial cells. Fractalkine is expressed on neointimal SMCs and thus mediates the arrest of monocytes. Interestingly, reendothelialization of injured vessels appears to primarily depend on CXC motif ligand 1 (CXCL1). These chemokine effects form a complex network, which operates in all mechanisms of vascular remodeling. The detailed understanding of the function of the chemokine network in the remodeling process may allow specific disease intervention.

The Universal NF-kappaB Inhibitor A20 Protects From Transplant Vasculopathy by Differentially Affecting Apoptosis in Endothelial and Smooth Muscle Cells

Transplant vasculopathy (TV) is an accelerated form of atherosclerosis resulting in chronic rejection of vascularized allografts. The causes of TV are multifactorial and integrate at the level of the vascular wall, leading to a phenotypic switch of endothelial cells (ECs) and smooth muscle cells (SMCs). A20 is a NF-κB-dependent stress response gene in ECs and SMCs with potent anti-inflammatory effect in both cell types through blockade of NF-κB. A20 expression in ECs and SMCs correlates with the absence of TV in rat kidney allografts and long-term functioning human kidney allografts. We demonstrate that A20 protects ECs from tumor necrosis factor, Fas, and natural killer cell-mediated apoptosis by inhibiting proteolytic cleavage of caspase 8. A20 also safeguards ECs from complement-mediated necrosis. Hence, effectively shutting down cell death pathways initiated by inflammatory and immune offenders associated with TV. In contrast, A20 sensitizes SMCs to cytokine and Fas-mediated apoptosis through a novel nitric oxide (NO)-dependent mechanism. The unexpected proapoptotic effect of A20 in SMCs translates in vivo by the regression of established neointimal carotid lesions following balloon angioplasty in rats. Antedating apoptosis of SMCs, expression of the inducible NO synthase increases in A20-expressing neointimal SMCs, corroborating the involvement of NO in causing the proapoptotic effect of A20 in SMCs. Combined anti-inflammatory and anti- or proapoptotic functions of A20 in ECs and SMCs respectively qualify the positive effect of A20 upon vascular remodeling and healing. We propose that A20-based therapies may be effective in prevention and treatment of TV.

Smooth muscle cell proliferation but not neointimal formation is dependent on alloantibody in a murine model of intimal hyperplasia.

Transplant coronary artery disease is the pre-eminent cause of late cardiac allograft failure. It is primarily characterized by a concentric intimal hyperplasia, which we designate transplant intimal hyperplasia (TIH). Although the pathogenesis of TIH is predominately immune driven, the specific role of alloantibodies in the disease process remains undefined. In this study we investigated the contribution of alloantibodies to the development of TIH in a murine model. Orthotopic, carotid artery transplantation was performed between B10A(2R) (H-2(h2)) donor mice and B-cell deficient muMT(-/-) knockout or wild-type C57BL/6 (H-2(b)) recipients in the absence of immunosuppression. Grafts were harvested at 35 days and subjected to planimetry and immunohistochemistry. Alloantibodies were detectable in wild-type recipients within 7 days of transplantation and recipients developed marked TIH at 35 days. Allografts harvested from B-cell deficient recipient mice also developed TIH, which was comparable in severity with wild-type recipients. However, whereas allografts from wild-type recipients showed marked intimal smooth muscle cell (SMC) proliferation, the neointima in B-cell deficient recipients lacked SMCs. Post-transplantation administration of anti-donor serum to muMT(-/-) recipients restored neointimal SMC population but did not influence the severity of TIH. Significant neointimal formation occurs in the absence of alloantibodies but lacks a SMC component. Therefore, SMC migration and proliferation is antibody dependent.

Inhibition of Carotis Venous Bypass Graft Disease by Intraoperative Nucleic Acid-Based Therapy in Rabbits

Bypass graft stenosis after venous revascularisation procedures is characterised by massive neointimal and vascular smooth muscle cell proliferation triggered via endothelin-1 synthesis in the vessel wall. Decoy oligodesoxynucleotides (ODN) against the transcription factor activator protein-1 (AP-1) inhibits pre-pro-endothelin-1 expression. In 20 rabbits, an end-to-side jugular vein bypass to the carotid artery was performed: (group A) 8 grafts were treated with consensus AP-1 decoy ODN, (group B) 8 with mutated control ODN and (group C) 4 received no treatment. Explantation, histomorphometric and immunohistochemical evaluation was performed after 28 days. Median intimal thickness of groups: (A) 28.3 microm, (B) 48.4 microm, (C) 71.1 microm. The decoy ODN-treated group showed a significant reduction of neointima formation ( P = 0.029) and a downregulation of the endothelin receptor. In this model, neointima formation was reduced by local transfection with consensus decoy ODN against AP-1. Endothelin A and B receptor expression is downregulated. Molecular target nucleic acid-based therapies seem to be a future means of overcoming neointima proliferation in pressure-induced venous graft failure. Intraoperative local application makes it easy to use in routine revascularisation procedures.

Neutralization of Interleukin-18 Inhibits Neointimal Formation in a Rat Model of Vascular Injury

Studies in humans and animal models suggest that interleukin-18 (IL-18) plays a crucial role in vascular pathologies. IL-18 is a predictor of cardiovascular death in angina and is involved in atherotic plaque destabilization. Higher IL-18 plasma levels also are associated with restenosis after coronary artery angioplasty performed in patients with acute myocardial infarction. We investigated the effective role of IL-18 in neointimal formation in a balloon-induced rat model of vascular injury. Endothelial denudation of the left carotid artery was performed by use of a balloon embolectomy catheter. Increased expression of IL-18 and IL-18Ralpha/beta mRNA was detectable in carotid arteries from days 2 to 14 after angioplasty. The active form of IL-18 was highly expressed in injured arteries. Strong immunoreactivity for IL-18 was detected in the medial smooth muscle cells at days 2 and 7 after balloon injury and in proliferating/migrating smooth muscle cells in neointima at day 14. Moreover, serum concentrations of IL-18 were significantly higher among rats subjected to vascular injury. Treatment with neutralizing rabbit anti-rat IL-18 immunoglobulin G significantly reduced neointimal formation (by 27%; P < 0.01), reduced the number of proliferating cells, and inhibited interferon-gamma, IL-6, and IL-8 mRNA expression and nuclear factor-kappaB activation in injured arteries. In addition, in vitro data show that IL-18 affects smooth muscle cell proliferation. These results identify a critical role for IL-18 in neointimal formation in a rat model of vascular injury and suggest a potential role for IL-18 neutralization in the reduction of neointimal development.

Protective Genes in the Vessel Wall: Modulators of Graft Survival and Function

Protecting a vascularized graft from transplant arteriosclerosis requires inhibition of host immune effectors, but protective responses emanating from the graft aimed at maintaining/restoring "homeostasis" might be equally important. Expression of the "protective" genes A20, heme-oxygenase-1 (HO-1), Bcl-xL, inducible nitric oxide synthase (iNOS), and others in the vessel wall of rodent allografts and xenografts correlates with absence of transplant arteriosclerosis. Given the antiapoptotic and anti-inflammatory functions of these genes in endothelial cells and their anti-inflammatory/antiproliferative and sometimes proapoptotic function in neointimal smooth muscle cells, we hypothesize that their expression survives to limit graft injury by maintaining vascular integrity, controlling inflammation and promoting healing. Beyond this beneficial effect, their expression in the vessel wall may also positively impact the alloimmune response.

Edge restenosis: impact of low dose irradiation on cell proliferation and ICAM-1 expression

BackgroundLow dose irradiation (LDI) of uninjured segments is the consequence of the suggestion of many authors to extend the irradiation area in vascular brachytherapy to minimize the edge effect. Atherosclerosis is a general disease and the uninjured segment close to the intervention area is often atherosclerotic as well, consisting of neointimal smooth muscle cells (SMC) and quiescent monocytes (MC). The current study imitates this complex situation in vitro and investigates the effect of LDI on proliferation of SMC and expression of intercellular adhesion molecule-1 (ICAM-1) in MC.MethodsPlaque tissue from advanced primary stenosing lesions of human coronary arteries (9 patients, age: 61 ± 7 years) was extracted by local or extensive thrombendarterectomy. SMC were isolated and identified by positive reaction with smooth muscle α-actin. MC were isolated from buffy coat leukocytes using the MACS cell isolation kit. For identification of MC flow-cytometry analysis of FITC-conjugated CD68 and CD14 (FACScan) was applied. SMC and MC were irradiated using megavoltage photon irradiation (CLINAC2300 C/D, VARIAN, USA) of 6 mV at a focus-surface distance of 100 cm and a dose rate of 6 Gy min-1 with single doses of 1 Gy, 4 Gy, and 10 Gy. The effect on proliferation of SMC was analysed at day 10, 15, and 20. Secondly, total RNA of MC was isolated 1 h, 2 h, 3 h, and 4 h after irradiation and 5 μg of RNA was used in standard Northern blot analysis with ICAM-1 cDNA-probes.ResultsBoth inhibitory and stimulatory effects were detected after irradiation of SMC with a dose of 1 Gy. At day 10 and 15 a significant antiproliferative effect was found; at day 20 after irradiation cell proliferation was significantly stimulated. Irradiation with 4 Gy and 10 Gy caused dose dependent inhibitory effects at day 10, 15, and 20. Expression of ICAM-1 in human MC was neihter inhibited nor stimulated by LDI.ConclusionThus, the stimulatory effect of LDI on SMC proliferation at day 20 days after irradiation may be the in vitro equivalent of a beginning edge effect. Extending the irradiation area in vascular brachytherapy in vivo may therefore merely postpone and not inhibit the edge effect. The data do not indicate that expression of ICAM-1 in quiescent MC is involved in the process.