Neointimal Layer Research Articles

Reduced cGMP signaling associated with neointimal proliferation and vascular dysfunction in late-stage atherosclerosis.

Atherosclerosis is associated with alterations in nitric oxide (NO)/cGMP signaling. In early stages of the disease, inflammatory and possibly other cells produce reactive oxygen species that scavenge vasoprotective NO. In addition to the oxidative stress, expression and activity of enzymes downstream to NO formation may also be affected. Here, we show in the aortas of chronically hypercholesterolemic rabbits (a model of late-stage atherosclerosis), both subunits and specific activity of the NO receptor soluble guanylyl cyclase (sGC) were significantly reduced, whereas overall NO synthase activity was unaffected. These changes were most prominent in the neointimal layer, wherein cGMP-dependent protein kinase I (cGK) levels also were reduced. Additionally, a protein (p38(nt)) that was constitutively tyrosine-nitrated was detected, and its expression was significantly reduced in atherosclerotic aorta. Phosphorylation of the cGK substrate vasodilator-stimulated phosphoprotein (VASP) at Ser-239, an established biochemical endpoint of NO/cGMP signaling, also was reduced. Thus, late-stage atherosclerosis is associated not only with enhanced NO breakdown but also with altered NO reception and cGMP signaling. Preferential down-regulation in neointima suggests a direct connection of these changes to neointimal proliferation and vascular dysfunction and provides a rationale for future pharmacotherapy using classical and novel sGC activators.

Redox processes underlying the vascular repair reaction.

Accumulating evidence indicates that vascular dysfunction in atherosclerosis, hypertension, and diabetes is either caused by or accompanied by oxidative stress in the vessel wall. In particular, the role of redox processes as mediators of vascular repair and contributors to post-angioplasty restenosis is increasingly evident. Yet the pathophysiology of such complex phenomena is still unclear. After vascular injury, activation of enzymes such as NADPH oxidase leads to a marked increase in superoxide generation, proportional to the degree of injury, which rapidly subsides. Such early superoxide production is significantly greater after stent deployment, as compared to balloon injury. Recent data suggest the persistence of low levels of oxidant stress during the vascular repair reaction in neointimal and medial layers. Despite the compensatory increase in expression of iNOS and nNOS, nitric oxide bioavailability is reduced because of increased reaction rates with superoxide, yielding as by-products reactive nitrogen/oxygen species that induce protein nitration. Concurrently, the activity of vascular superoxide dismutases exhibits a sustained decrease following injury. This decreased activity appears to be a key contributor to vasoconstrictive remodeling and a major determinant of the occurrence of nitrative/oxidative stress. Replenishment of superoxide dismutase (SOD), as well as treatment with vitamins C and E or the lipid-lowering drug probucol and its analogs, led to decrease in constrictive remodeling and improved vessel caliber. Better understanding of the redox pathophysiology of vascular repair should help clarify the pathogenesis of many other vascular conditions and may provide novel therapeutic strategies to prevent vascular lumen loss.

Late neointimal tissue growth behind the stent after intravascular γ-radiation

Purpose To determine the nature of the changes of the vascular wall after intravascular brachytherapy in stented arteries leading to incomplete stent apposition. Methods and materials Stents were implanted in the infrarenal aortas of rabbits, and γ-intravascular brachytherapy (18 Gy) or a sham radiation procedure was immediately implemented. The arteries were harvested at 6 months for histologic analyses. Results The external elastic lamina area, as well as the vascular wall area behind the stent, were significantly greater in irradiated vs. control arteries (8.94 ± 0.68 mm 2 vs. 6.87 ± 0.40 mm 2 [ p <0.001] and 1.56 ± 0.13 mm 2 vs. 0.72 ± 0.07 mm 2 [ p <0.001], respectively). The ratio of the intimal area behind the stent related to the total intimal area was greater in the irradiated segments (control vs. irradiated: 9.0% ± 5.9% vs. 55.3% ± 15.5%, p <0.05). Neointimal growth of the irradiated vessels outside the stent was characterized by marked fibrin depositions and an inflammatory response around the stent struts. Conclusion Our study revealed the presence of a neointimal layer specifically located behind the stent, which represented the result of an unhealed fibrin-rich tissue growth process 6 months after intravascular brachytherapy.

Pathological mechanisms of fatal late coronary stent thrombosis in humans.

Coronary stent deployment is associated with a low incidence of acute thrombosis. However, late stent thrombosis (LST) has likely been underrecognized clinically, and pathological descriptions are lacking. LST was defined as an acute thrombus within a stent that had been in place > or =30 days. Cases of LST were selected from a registry of human coronary stents submitted for analysis. Thirteen cases of LST (9 men, 4 women) were identified. The mean duration from implantation to thrombosis was 3.6+/-3.5 months (range, 1 to 11.9 months). The causes of death were sudden cardiac death (n=10), acute myocardial infarction (n=2), and heart failure (n=1). The pathological mechanisms of LST were as follows: (1) stenting across ostia of major arterial branches (5 cases); (2) exposure to radiation therapy (3 cases); (3) plaque disruption in the nonstented arterial segment within 2 mm of the stent margin (2 cases); (4) stenting of markedly necrotic, lipid-rich plaques with extensive plaque prolapse (2 cases); and (5) diffuse in-stent restenosis (1 case). Twelve cases demonstrated a failure to form a completely healed neointimal layer overlying stent struts. Underlying in-stent restenosis was present in only 4 (31%) of 13 cases. LST is a potentially fatal complication of coronary stenting. Stenting across branch ostia, disruption of adjacent vulnerable plaques, radiation therapy, and extensive plaque prolapse can precipitate LST. Impaired intimal healing (ie, the failure to form a complete neointimal layer over stent struts) extends the window during which stents are prone to thrombosis.

Alternative method of rapid drying vascular specimens for scanning electron microscopy.

To report the use of hexamethyldisilazane (HMDS) as an alternative to critical point drying for preparing stented canine peripheral vessels for scanning electron microscopy (SEM). Vascular specimens were fixed in 4% formaldehyde overnight, dehydrated in a graded ethanol series, followed by immersion in 100% hexamethyldisilazane. After air drying, the specimens were mounted on stainless steel stubs, coated with gold, and examined in the SEM. The electron micrographs were of high quality, showing the layers of the vascular wall and the incorporated stent covered by a neointimal layer. The micrographs were comparable to corresponding histological sections, but detailed endothelial patterns were more visible. HMDS treatment and subsequent air drying provides good quality scanning electron micrographs that reveal both endothelial patterns and the layered architecture of stented vessels. The disadvantage of HMDS drying may be a shrinkage and distortion similar to other drying agents. Ease of handling, low cost, and a high rate of success are advantages that favor HMDS desiccation over other drying methods.

Transluminal stent graft repair with Wallgraft endoprosthesis in a porcine arteriovenous graft pseudoaneurysm model.

Pseudoaneurysm is a known complication of arteriovenous grafts in chronic hemodialysis and can result in graft disruption or thrombosis if left untreated. This study evaluated the safety and efficacy of endovascular repair with Wallgraft endoprosthesis (Boston Scientific, Inc, Watertown, Mass) in a porcine arteriovenous graft (AVG) pseudoaneurysm model. Bilateral groin AVG pseudoaneurysms (n = 18) were created with an oversized Dacron interposition graft within a polytetrafluoroethylene femoral AVG in nine domestic swine and allowed to mature 28 +/- 4 days (standard deviation). Transluminal placement of Wallgraft was performed to exclude the pseudoaneurysm from the AVG circulation. Hemodialysis was performed (400 mL/min x 1 hour, with intravenous heparin 30 units/kg) every 4 days for a total of 6 weeks via 15-gauge needles in the treated AVG pseudoaneurysm site. Arteriography and duplex ultrasound scan were performed to determine AVG patency and pseudoaneurysm flow. Histologic evaluation was performed to determine Wallgraft morphology. In vitro pulsatile flow chamber was used to determine maximal flow volume without peri-Wallgraft endoleak. All AVG pseudoaneurysms were successfully excluded with the Wallgrafts. Twelve AVG (67%) remained patent at the completion of the study. No Wallgraft migration occurred from hemodialysis. Transient peri-Wallgraft endoleak (<2 hours after hemodialysis) was present in 13 of 18 (72%) and four of 12 (33%) AVG pseudoaneurysms by weeks 1 and 6, respectively. With maintenance of an intraluminal pressure of 80, 100, 120, 140, and 160 mm Hg in the pulsatile flow chamber, the maximal flow rates without peri-Wallgraft endoleak were 625 +/- 120, 650 +/- 145, 620 +/- 95, 425 +/- 110, and 262 +/- 86 mL/min. Scanning electron microscopy showed a neointimal layer covered with thrombus on the Wallgraft surface. Endoluminal placement of Wallgraft endoprosthesis provides adequate structural support for continuous hemodialysis after AVG pseudoaneurysm exclusion. Transient blood flow in the pseudoaneurysm cavity may occur immediately after the hemodialysis, which may represent the effect of heparin used during hemodialysis. This study suggests Wallgraft is a safe and effective treatment for AVG pseudoaneurysm and permits continuous hemodialysis.

Flow-induced neointimal regression in baboon polytetrafluoroethylene grafts is associated with decreased cell proliferation and increased apoptosis.

We have previously shown that baboon grafts subjected to elevated shear stress exhibit an increase in luminal area through atrophy of the neointimal layer. This study was designed to investigate the smooth muscle cell (SMC) growth kinetics during early regression and evaluate the influence of nitric oxide (NO) in the regulation of this process. Sixteen baboons underwent bilateral polytetrafluorethylene aortoiliac graft placement. After development of a neointima over an 8-week period, blood flow through one graft was increased with placement of a downstream arteriovenous fistula. Grafts were harvested at 4 (n = 6), 7 (n = 5), and 14 (n = 5) days and assessed for neointimal cross-sectional area, SMC proliferation and apoptosis, and macrophage infiltration. High-flow grafts were compared with contralateral normal-flow controls. Eleven baboons underwent an identical experimental preparation to evaluate the effect of NO inhibition. Eight weeks after graft implantation, the animals were treated with an initial bolus (100 mg/kg) followed by continuous infusion (60 mg/kg/d) of either N(G)-nitro-L-arginine methyl ester (L-NAME; n = 6) or the inactive stereoisomer N(G)-nitro-D-arginine methyl ester (n = 5). Grafts were harvested at 7 days and evaluated with the experimental endpoints detailed previously. Distal fistula placement resulted in a 3.8-fold increase in mean centerline velocity and wall shear stress. Grafts harvested during the initial 14 days after flow manipulation showed a progressive reduction in neointimal cross-sectional area. This change was associated with a decrease in subendothelial SMC proliferation and an increase in neointimal SMC apoptosis, the latter being in the region adjacent to the graft. Animals treated with L-NAME showed a 20% reduction in platelet cyclic guanosine monophosphate and a 17% reduction in serum nitrate/nitrite concentrations. Despite this inhibition of NO production, no effect on the flow-dependent changes in neointimal area, cell proliferation, or apoptosis was observed in the L-NAME-treated baboons. The local hemodynamic environment within healing prosthetic grafts modulates neointimal SMC proliferation and apoptosis. An increase in graft flow leads to atrophy of the neointima.

ERK1/2 mediates PDGF-BB stimulated vascular smooth muscle cell proliferation and migration on laminin-5

During restenosis following arterial injury, vascular smooth muscle cells (VSMCs) form a neointimal layer in arteries by changing from a differentiated, contractile phenotype to a dedifferentiated, migratory, and proliferative phenotype. Several growth factors, cytokines, and extracellular matrix components released following injury have been implicated in these phenotypic changes. We have recently detected the expression of laminin-5, an ECM protein found predominantly in epithelial tissues, in the arterial vasculature. Here we report that ln-5 expression by VSMC is upregulated by platelet-derived growth factor (PDGF-BB), epidermal growth factor, basic fibroblast growth factor, and transforming growth factor-β1. Adhesion to ln-5 specifically enhances PDGF-BB-stimulated VSMC proliferation and migration. PD98059, a specific inhibitor of the ERK1/2 members of the Mitogen Activated Protein kinase family, increases both VSMC adhesion to ln-5 and blocks PDGF-BB-stimulated VSMC migration on ln-5. These results suggest that adhesion to ln-5 mediates a PDGF-BB-stimulated VSMC response to vascular injury via an ERK1/2 signaling pathway.

Contribution of the Helix-Loop-Helix Factor Id2 to Regulation of Vascular Smooth Muscle Cell Proliferation

Smooth muscle cell (SMC) proliferation plays a key role in vascular proliferative disorders. The molecular mechanisms that control cell cycle entry of SMCs in response to vascular injury are not well understood. Id2 (inhibitor of DNA binding) is a member of the helix-loop-helix (HLH) family of transcription regulators that are known to promote cell cycle progression. Thus, we investigated the role of Id2 in SMC growth and cell cycle regulation. The results demonstrated that overexpression of Id2 resulted in a significant enhancement of SMC growth via increased S-phase entry. A possible mechanism of Id2-enhanced SMC growth is via regulation of p21 expression, as overexpression of Id2-inhibited transcriptional activity of a 2.3-kb p21 promoter/luciferase reporter construct as well as p21 protein levels. Id2 enhancement of SMC growth and inhibition of p21 expression were dependent on phosphorylation of Id2 by cyclin E/cdk2, as an Id2 cDNA containing a mutation in the cdk2 phosphorylation site (serine 5) failed to regulate SMC cell cycle progression or p21 promoter activity. The mechanism of cyclin E/cdk2 control of the Id2 effect may in part involve regulation of nuclear transport; unlike wild-type Id2, the Id2 mutant was not transported to the nucleus. Finally, in a rat carotid model of arterial injury, Id2 was expressed in a temporal pattern that parallels the kinetics of cellular proliferation. In summary, these results provide evidence that the Id2 protein is integrated into the cell cycle regulatory cascade that results in SMC proliferation following vascular injury and suggest that this effect is at least in part via a cdk2-dependent inhibition of p21 gene expression.

Increased expression of TGF-β1 and IGF-I in inflammatory stenotic lesions of hemodialysis fistulas

Hemodialysis fistula dysfunction due to stenotic lesions remains a frequent cause of hospitalization for hemodialysis patients. Transforming growth factor-beta(TGF-beta) and insulin-like growth factor-I (IGF-I) are known to be involved in atherogenesis. The latent TGF-beta1 binding protein-1 (LTBP-1) targets extracellular matrix (ECM) interactions and is involved in the regulation of TGF-beta latency. We investigated the expression of TGF-beta1, LTBP-1 and IGF-I in 15 occluded or severely narrowed vein segments of primary arteriovenous fistulas, in 29 non-stenosed control veins from uremic, pre-dialysis patients, and in 15 non-stenosed control saphenous veins obtained from patients undergoing aortocoronary bypass grafting. Immunohistochemistry was performed on snap-frozen tissue specimens using antibodies recognizing either the latency-associated peptide of TGF-beta1 (96-1), LTBP-1 (Ab39) or IGF-I. Serum levels of TGF-beta1 and IGF-I were determined by commercially available IRMA. In stenosed hemodialysis fistulas, a pronounced intimal thickening with deposition of ECM was observed with light and electron microscopy. Infiltrating cells were seen in stenosed vessels, mostly in areas of intimal hyperplasia and in the media. TGF-beta1, LTBP-1 and IGF-I expression were mostly localized in the neointimal and medial layers, and were significantly higher than in the control groups. A positive correlation between the presence of inflammatory cells and the staining intensity for TGF-beta1, LTBP-1 and IGF-I was found in all vessels analyzed. Neointimal thickening of primary arteriovenous fistulas represents a local inflammatory process and appears to be associated with increased protein expression of TGF-beta1 and IGF-I. While local IGF-I is likely to stimulate smooth muscle cell proliferation in this setting, TGF-beta1 may be an important trigger of ECM production and deposition.

P160 Bcr mediates platelet-derived growth factor activation of extracellular signal-regulated kinase in vascular smooth muscle cells.

The human Bcr gene was originally identified by its presence in the chimeric Bcr/Abl oncogene, which is causative for chronic myeloblastic leukemia. Because Bcr encodes a serine/threonine protein kinase, we studied its kinase activity and determined the role of Bcr in the PDGF signaling pathway to ERK1/2 activation and DNA synthesis in rat aortic smooth muscle cells (RASMCs). In RASMCs, platelet-derived growth factor-BB (PDGF) stimulated Bcr kinase activity, with a maximum at 1 minute. Because phosphatidylinositol 3'-kinase (PI3-K) is essential for Bcr/Abl leukemogenesis, we evaluated the role of mouse PDGF-beta-receptor binding sites for PI3-K (Y708, Y719) and for phospholipase C-gamma (Y977, Y989) in PDGF-mediated Bcr kinase activation. The mutant PDGF receptor Y708F/Y719F but not Y977F/Y989F showed significantly reduced Bcr kinase activity. To determine the role of Bcr in PDGF-mediated signal transduction events leading to ERK1/2 and its downstream Elk1 transcription activation, wild-type (WT) and kinase-negative (KN) Bcr were transiently expressed in RASMCs. Bcr WT enhanced, whereas Bcr KN inhibited, PDGF-stimulated ERK1/2 and Elk1 transcriptional activity. Overexpression of Bcr also enhanced PDGF-induced Ras/Raf-1 activity and DNA synthesis, but this regulation is independent of the kinase activity of Bcr. Finally, we found that Bcr expression was increased in the neointimal layer after balloon injury of rat carotid artery. These results demonstrated the importance of Bcr in PDGF-mediated events, such as activation of Ras, Raf-1, ERK1/2, and Elk1, and stimulation of DNA synthesis.

Angiotensin converting enzyme inhibitor restrains inflammation-induced vascular injury in mice.

Recent advances in molecular genetics made the mouse model important for studying the genetic basis of hypertension and vascular diseases such as the components of the renin-angiotensin system. This study was undertaken to investigate the role of angiotensin converting enzyme (ACE) in the mouse vascular injury model. Inflammation-induced vascular injury was created by placing a polyethylene cuff around the femoral artery of 12-14-week-old male FVB/N mice. Cuffed arteries were harvested and applied to reverse transcriptase-polymerase chain reaction analysis and immunohistochemistry for ACE. Subsequently, the effects of an ACE inhibitor, perindopril (3 mg/kg per day), on neointimal thickening were examined 2 weeks after cuff placement The influence of a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg per day) on the effects of perindopril was also examined. ACE mRNA expression increased in a time-dependent manner up to 2 weeks after cuff placement. Immunoreactive ACE was localized in the endothelium in the intact artery, while positive staining was observed in the medial and neointimal layer as well as in the periadventitial region of the cuffed artery. The intimamedia area ratio was significantly decreased by perindopril treatment (vehicle, 0.75+/-0.10; perindopril, 0.32+/-0.04; P< 0.05). The effect of perindopril was abrogated by coadministration of L-NAME whereas L-NAME alone did not affect the intima-media ratio (L-NAME, 0.66+/-0.11; perindopril + L-NAME, 0.72+/-0.09). This study provides evidence that ACE plays a role in cuff-induced neointimal thickening in mice. Nitric oxide may contribute, at least in part, to the inhibitory effects of perindopril.

Vascular injury induces posttranscriptional regulation of the Id3 gene: cloning of a novel Id3 isoform expressed during vascular lesion formation in rat and human atherosclerosis.

The molecular mechanisms that regulate the proliferation of smooth muscle cells (SMCs) of the vasculature in response to injury are poorly understood. Members of the inhibitor of DNA binding (Id) class of helix-loop-helix transcription factors are known to regulate the growth of a variety of cell types; however, the expression of the various Id genes in SMCs and in vascular lesions has not been examined. In the present study, the yeast 2-hybrid system was used to clone Id genes from a cultured rat aortic SMC library. By use of ubiquitous E proteins as bait, Id3 and a novel isoform of Id3 (Id3a) were cloned. Id3a is the product of alternative splicing of the Id3 gene, resulting in inclusion of a 115-bp "coding intron," which encodes a unique 29-amino acid carboxyl terminus for the Id3a protein. Unlike Id3, Id3a mRNA was not detected in the normal rat carotid artery. However, after balloon injury, Id3a was abundantly expressed throughout the neointimal layer. In addition, mRNA of the human homologue of Id3a (Id3L) was detected in human carotid atherosclerotic plaques. Adenovirus-mediated overexpression of these Id3 isoforms in cultured rat aortic SMCs revealed that infection of SMCs with an adenovirus overexpressing Id3a (in contrast to Id3) resulted in a significant decrease in cell number versus AdLacZ-infected cells. DNA fragmentation analysis suggested that this decrease in SMC viability was due to increased apoptotic activity in cells infected with adenovirus overexpressing Id3a. These results provide evidence that alternative splicing of the Id3 gene may represent an important mechanism by which neointimal SMC growth is attenuated during vascular lesion formation.

Marked neointimal lipoprotein lipase increase in distinct models of proclivity to atherosclerosis: a feature independent of endothelial layer integrity

Lipoprotein lipase (LPL) in the arterial wall has been proposed to enhance the retention of apoB-containing lipoproteins, an early event in atherosclerosis. As the neointima is considered the primary site of lipid accumulation in atherogenesis, the arterial expression and location of LPL was investigated in distinct experimental models of neointimal formation in normolipidemic rabbits and rats. Neointima elicited by balloon aortic denudation or raised beneath an anatomically intact endothelial layer by placing a silastic collar around the common carotid artery, both showed a striking LPL immunostaining that mostly co-localized with neointimal smooth muscle cells. Besides, increased LPL protein and mRNA in deendothelialized aortas was demonstrated by Western and Northern blot analysis, respectively, suggesting an enhanced expression of LPL in injured arteries. It was concluded that LPL is increased in neointima developed in either denuded vessels or arteries with a preserved endothelium, a finding which suggests that LPL abundance may be an attribute of the neointima, whatever the stimulus that promotes its formation. On the basis of former evidence concerning the role of LPL in lipid retention, this study provides a possible explanation for the injury-induced vessel susceptibility to atherosclerosis, and the particular proneness of the neointimal layer to lipid accretion.

Nifedipine suppresses neointimal thickening by its inhibitory effect on vascular smooth muscle cell growth via a MEK-ERK pathway coupling with Pyk2.

The aim of this study was to determine whether nifedipine could suppress an atherogenic process such as balloon-injured intimal thickening in vivo and the proliferation of vascular smooth muscle cells (VSMC) in vitro. First, we examined the in vivo effect of nifedipine to determine whether it could suppress intimal thickening induced by balloon catheterization. Sprague-Dawley (SD) rats were divided into three groups (L, nifedipine 0.3 mg kg(-1) day(-1); H, nifedipine 3 mg kg(-1) day(-1); C, no nifedipine), and Alzet((R)) osmotic pumps were implanted in their backs for continuous administration. The neointimal layers were completely occupied by proliferated VSMC, and the area ratios of neointima/media treated with nifedipine significantly decreased dose-dependently compared to those of the control. Neither blood pressure nor lipid levels changed among the three groups. We next evaluated the in vitro effect of nifedipine on the proliferation of cultured rat VSMC. Nifedipine decreased the values of [(3)H]-thymidine incorporation and total cellular protein content as well as the levels of phosphorylated extracellular signal-regulated protein kinase (ERK) 1/2, mitogen-activated protein kinase kinase (MEK) 1/2, and even the phosphorylation of Pyk2, in dose-dependent fashions. In addition, nifedipine suppressed the levels of proliferative cell nuclear antigen (PCNA) dose-dependently in both VSMC and balloon-injured thoracic aortae. These results indicate that nifedipine has an inhibitory effect on intimal thickening by attenuating intimal VSMC proliferation, suggesting that nifedipine could be effective for preventing the progression of atherosclerotic plaque as in restenosis after angioplasty.

A New Stent Graft

The use of stents improves the result after balloon coronary angioplasty. Restenosis due to neointimal hyperplasia and proliferation of smooth muscle cells are, however, a concern. In the present report, we studied the prevention of restenosis to allow endothelial cell migration and growth to proceed through micropores using our developed stent graft with micropored segmented polyurethane (SPU) thin film in a normal beagle model. Our developed stent graft was made from Palmaz stent and micropored SPU thin film. The SPU film was arranged into four different micropore densities around the circumference: no micropores, arrangement 4; micropores of 30mum in diameter with an orderly distance of 250mum; (arrangement 1), 500mum; (arrangement 2) and 125mum (arrangement 3) between the neighboring two pores. Micropores were made using the Excimer laser ablation technique. The Palmaz stent was wrapped with micropored film, sutured, and glued with DMF (dimethyl formamide) under aid of a microscope. These stents were placed in the common carotid arteries of beagles (n = 5). They were sacrificed at 1 month, and a histological study and scanning electron microscopy study were performed for evaluation of endoluminal endothelialization. In 10 arteries applied with stent grafts, there was no severe stenosis although it did occur to some extent. All stented arteries were patent. Endothelial cell migration and growth through micropores were observed histologically on micropored SPU thin film in this model, which did not affect the intraluminal diameter. In most non-porous regions, significant thrombi were found between the SPU film and the neointimal layer. On the other hand, in the porous region, little thrombosis was observed except in the lowest density region. In 125mum of distance between two neighboring pores, the neointimal layer was the thinnest, which was suitable for wide intraluminal space after placement of a stent graft. Endothelial cell migration and growth through micropores were confirmed in the animal model using our developed micropored stent graft. The proceeding of their migration was controlled by micropore density under a constant micropore diameter. The stent graft with micropored SPU thin film is promising for the prevention of restenosis due to neointimal hyperplasia.

Autologous Vein Stent-Graft: Feasibility Study

To evaluate expandable stents healed into vein wall as autologous vein stent-grafts for endoluminal grafting. Balloon expandable stents were placed into external jugular veins of eight dogs. Stent and vein patency was followed by ultrasonography. Five weeks after stent placement, jugular veins with endothelialized stent were harvested. The autologous vein stent-grafts were divided into two groups. In group A, autologous vein stent-grafts (n = 3) were placed immediately into Baker solution for microscopic examination. In group B, autologous vein stent-grafts (n = 3) underwent mechanical manipulation; they were compressed, mounted on angioplasty balloon, pushed through a 9-F sheath and dilated. The autologous vein stent-graft endothelialization and changes after mechanical manipulation were evaluated by light and electron microscopy. Stent placement was successful in seven dogs. One stent migrated into the pulmonary artery. One well placed stent was damaged by compression dressing and thrombosed. At 5 weeks, gross and microscopic examinations revealed the autologous vein stent-grafts were fully covered by a 0.115- +/- 0.036-mm-thick neointimal layer. Small wall thrombus was observed in one autologous vein stent-graft. Repeated manipulations did not result in any intimal damage or stent loosening in the autologous vein stent-grafts. Expandable stents healed into a vein have potential to be used as autologous vein stent-grafts for endoluminal grafting without risk of disruption during percutaneous transcatheter introduction.

Endovascular Healing Is Inadequate for Fixation of Dacron Stent-grafts in Human Aortoiliac Vessels

Background migration and kinking of stent-grafts can occur late after endovascular aneurysm repair. It is unknown if endovascular grafts incorporate enough to be permanently anchored. In this report, healing of aortic stent-grafts was assessed in humans. Patients and methods we retrieved 23 Dacron stent-grafts from patients treated for an aortic aneurysm since 1993. Twelve stent-grafts were explanted at late conversion to open repair and 11 at autopsy. The deaths were unrelated to graft fixation. The median age of the patients was 74 years (IQR 55–84 years) and the grafts were explanted 9 months (1–31 months) after insertion. Microscopic slides were prepared by conventional techniques or by cutting and grinding arterial specimens embedded in plastic with the stent-grafts in situ. Results the stent-grafts detached readily from the native arteries at surgery or autopsy, except when the stents had hooks or barbs which engaged the vessel wall. A space filled with poorly organised blood components persisted between the graft and the aortic wall 2.5 years after implantation. No firm incorporation of the grafts was observed proximally in the aneurysm neck or distally in the iliac segment. A friable neo-intimal layer covered parts of the luminal aspect of the grafts. Conclusions endovascular healing provides poor fixation of Dacron stent-grafts in humans. At present, fixation relies on the mechanical properties of the stent-grafts.

Reduction in atherosclerotic lesion size in pigs by alphaVbeta3 inhibitors is associated with inhibition of insulin-like growth factor-I-mediated signaling.

Insulin-like growth factor-I (IGF-I) is a potent stimulant of smooth muscle cell (SMC) migration and proliferation and has been implicated in the development of experimental atherosclerotic lesions. Because optimal stimulation of SMC in vitro by IGF-I requires ligand occupancy of alphaVbeta3, these studies were conducted to determine whether alphaVbeta3 antagonists would result in a change in lesion size and whether they could alter IGF-I-mediated actions. Clamps were placed on the carotid and femoral arteries of normal pigs that had been fed a high-cholesterol diet for 4 weeks. alphaVbeta3 inhibitors (SC-69000, SC-65811) (10(-6) mol/L) or saline were infused for 2 weeks into the peristenotic area. Lesion area, the number of SMC layers, and proliferating cell nuclear antigen positive cells were determined in a 1.2-mm segment of each artery. Lesion areas were 304 788+/-113 453 micron(2) (saline), compared with 149 799+/-35 456 micron(2) (SC-69000) (P<0.01). Lesion areas in arteries treated with SC-64258, a compound that does not bind to alphaVbeta3, were 310 284+/-160 467 micron(2), P=not significant. In a second experiment, lesion areas were 110 391+/-17 347 micron(2) (saline) and 59 533+/-17 568 micron(2) (SC-65811, P<0.001). Neointimal SMC layers were reduced by SC-65811 from 7.4+/-4.5 to 3.0+/-0.4 (P<0.001). To determine whether IGF-I action was altered, IGF binding protein-5, which is synthesized in response to IGF-I, was analyzed. IGF-I binding protein-5 mRNA abundance was reduced by 67+/-8% in the 6 lesions treated with SRL-69000 compared with saline controls (P<0.001). We conclude that alphaVbeta3 antagonists block the development of lesions in pigs that have been induced by a high-cholesterol diet and stenosis, and the effect of these compounds is associated with their ability to inhibit IGF-I-mediated signaling.

Inhibition of rat vascular smooth muscle cell proliferation in vitro and in vivo by recombinant replication-competent herpes simplex virus.

The proliferation of vascular smooth muscle cells (VSMCs) is a common feature associated with vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. We examined the antiproliferative effects of recombinant replication-competent herpes simplex virus (HSV), hrR3, to proliferative VSMCs both in vitro and in vivo. Early passages of Sprague-Dawley rat VSMCs were infected with hrR3 at a low multiplicity of infection (0.01 to 1.0) to examine the in vitro cytotoxic activity of this recombinant HSV to VSMCs in a proliferative state. Sprague-Dawley rats underwent balloon dilatation injury of the left carotid artery to induce neointimal formation. The injured carotid arteries were infected with hrR3 five days after balloon injury. Two weeks after injury, the left carotid arteries were fixed, and the areas of the neointimal and medial layers were analyzed microscopically. Because the reporter Escherichia coli lacZ gene in hrR3 is expressed only in infected cells in which the virus is actively replicating, virus replication was confirmed by X-gal staining. A morphometric analysis revealed that there were significant differences in the intima/media ratio between the HSV-treated group and mock-infected group (0. 354+/-0.068 and 1.08+/-0.055, respectively). In the histological study (X-gal staining), positive X-gal staining was observed chiefly in the VSMCs in the medial layer just beneath the internal elastic lamina, indicating active viral replication. Virus-mediated cytocidal therapy using recombinant HSV vector is a promising modality for the treatment of the restenosis after balloon angioplasty.