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Background and Purpose: Neointima is considered a critical event in the development of vascular occlusive disease. Nectandrin B from nutmeg functions as a potent AMP-activated protein kinase (AMPK) activators. The present study addressed whether nectandrin B inhibits intimal hyperplasia in guide wire-injured arteries and examined its molecular mechanism., Experimental Approach: Neointima was induced by guide wire injury in mouse femoral arteries. Cell proliferation and mechanism studies were performed in rat vascular smooth muscle cells (VSMC) culture model., Key Results: Nectandrin B increased AMPK activity in VSMC. Nectandrin B inhibited the cell proliferation induced by PDGF and DNA synthesis. Moreover, treatment of nectandrin B suppressed neointima formation in femoral artery after guide wire injury. We have recently shown that Pin1 plays a critical role in VSMC proliferation and neointima formation. Nectandrin B potently blocked PDGF-induced Pin1 and cyclin D1 expression and nectandrin B's anti-proliferation effect was diminished in Pin1 overexpressed VSMC. PDGF-induced phosphorylation of ERK and Akt was marginally affected by nectandrin B. However, nectandrin B increased the levels of p53 and its downstream target p21 and, also reversibly decreased the expression of E2F1 and phosphorylated Rb in PDGF-treated VSMC. AMPK inhibition by dominant mutant form of adenovirus rescued nectandrin B-mediated down-regulation of Pin1 and E2F1., Conclusions and Implications: Nectandrin B inhibited VSMC proliferation and neointima formation via inhibition of E2F1-dependent Pin1 gene transcription, which is mediated through the activation of an AMPK/p53-triggered pathway., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Introduction: The role of hypoxia-inducible factor-1α (HIF-1α) in pulmonary vascular remodeling is still undetermined. The objective of this study is to investigate the expression of HIF-1α and its role in proliferating neointimal lesions in a rat model of pulmonary arterial hypertension induced by monocrotaline (MCT) administration after left pneumonectomy., Methods: The rats were subjected to MCT (60 mg/kg, subcutaneously) 7 days after left pneumonectomy or sham surgery; controls with vehicle treatment after left pneumonectomy or sham surgery were also studied. On day 35, hemodynamic parameters of the rats were measured. The right lower lobes of the lungs were fixed for morphometric analysis. The expression of proliferating cell nuclear antigen and survivin was detected with Western blot. The expressions of HIF-1α and hexokinase-2 (HK-2) were detected with Western blot and immunohistochemistry assay., Results: The rats treated with MCT after pneumonectomy developed severe pulmonary arterial hypertension and marked medial thickening on day 35. The neointimal lesions in pulmonary arterioles were observed only in MCT-treated pneumonectomized rats. The severely injured pulmonary arterioles (intimal proliferation causing greater than 50% luminal occlusion) accounted for 40% of all the measured arterioles in rats treated by MCT after pneumonectomy. The intriguing finding showed that HIF-1α was predominantly expressed in neointimal lesion areas, paralleled with the increased expression of HK-2 in MCT-treated pneumonectomized rats, which was not observed in rats undergoing MCT treatment alone., Conclusions: The activation of HIF-1α/HK-2 axis is probably the key mediator responsible for the neointimal lesion formation in MCT-treated pneumonectomized rats.

Objectives: The aims of this study were to evaluate the effect of urinary trypsin inhibitor (UTI) on the regulation of inflammatory cytokines induced by lipopolysaccharide (LPS) and the reduction of neointimal formation in rabbits., Methods and Results: Rabbits subjected to iliac artery balloon injury were randomly divided into three groups: control group (balloon injury), LPS group (LPS + balloon injury) and UTI group (UTI + LPS + balloon injury). Systemic markers of inflammation (serum IL-1β and TNF-α levels measured by ELISA) were increased after LPS administration. Arterial nuclear factor-κB (NF-κB/p65) at 28 days after injury was 31.50 ± 7.08 % of total cells in controls and 73.50 ± 6.90 % in LPS group (P < 0.05). Morphometric analysis of the injured arteries at 28 days revealed significantly increased luminal stenosis (45.81 ± 5.31 vs 27.93 ± 2.85 %, P < 0.05) and neointima-to-media ratio (1.40 ± 0.15 vs 0.68 ± 0.12, P < 0.05) in LPS-treated animals compared with controls. This effect was reduced by UTI administration. Serum IL-1β and TNF-α levels and NF-κB/p65 expression were significantly increased in correlation with the severity of intimal hyperplasia and inhibited by UTI., Conclusions: Systemic inflammatory response concurrently with arterial vascular injury facilitated neointimal formation. UTI reduced neointimal hyperplasia by regulating inflammatory response and could be considered as a potential anti-restenosis supplement.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays a crucial role in stress-induced apoptosis. Recently, we have reported that suppressed macrophage apoptosis in ASK1 and apolipoprotein E double-knockout mice accelerates atheromatous plaques in the hyperlipidemia-induced atherosclerotic model. However, the pathogenic role of smooth muscle cell (SMC) apoptosis in atherosclerosis still remains unclear. We investigated neointimal remodeling in ligated carotid arteries of ASK1-deficient mice (ASK1(-/-)) for 3 weeks. ASK1(-/-) mice had significantly more suppressed intimal formation, inversely manifesting as potential anti-atherogenic aspects of ASK1 deficiency, characterized by fewer SMCs and less collagen synthesis; and fewer apoptotic SMCs, infiltrating T lymphocytes, and microvessels, associated with decreased apoptosis of luminal endothelial cells, compared with those of wild-type mice. Injured arteries of ASK1(-/-) mice also showed significantly down-regulated expression of pro-apoptotic markers, adhesion molecules, and pro-inflammatory signaling factors. Moreover, tumor necrosis factor-α-induced apoptosis was markedly suppressed in cultured aortic SMCs from ASK1(-/-) mice. These findings suggest that ASK1 accelerates mechanical injury-induced vascular remodeling with activated SMC migration via increased neovascularization and/or enhanced SMC and endothelial cell apoptosis. ASK1 expression, especially in the SMCs, might be crucial, and reciprocally responsible for various pro-atherogenic functions, and SMC apoptosis seems to be detrimental in this model., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Despite advances in contemporary stent technology, in-stent restenosis (ISR) remains the major limitation following revascularization procedures. We developed a porcine model of ISR to specifically investigate the preclinical outcomes of a novel drug-eluting balloon (DEB) in this particular setting. Fifteen pigs received bare metal stents in each of the major coronary arteries for 28 days to induce neointimal growth. Following repeat angiography, animals were allocated to fourdifferent treatment groups. The control group consisted of a bare angioplasty catheter, while the Pantera Lux™ (3.0 µg/mm(2) paclitaxel) (30 s inflation) was compared to two consecutive deployments of the Pantera Lux™ (60 s inflation each) and the commercial SeQuent(®) Please balloon (60 s inflation). Twenty-eight days following balloon deployment, the animals underwent repeat angiography and were subsequently sacrificed for histopathologic assessment. There was a trend in reduction of percent diameter stenosis in the DEB group versus control (13.9% vs. 20.4%), while longer inflation duration or consecutive DEB deployment had no additional growth-limiting effect. Neointimal thickness was reduced from 0.38 ± 0.13 to 0.30 ± 0.09 mm in the control versus DEB group. All DEB groups showed delayed vascular healing characterized by dose-dependent increases in fibrin deposition and neointimal cell vacuity. Investigation of DEB in a porcine model of ISR is feasible and more accurately represents human disease conditions. The magnitude of neointima suppression is lower than that observed in non-diseased animal models and is accompanied by delayed vascular healing.

Background: Due to intimal hyperplasia instent reobstruction in the femoropopliteal arterial segment is still an unsolved problem. Different techniques have been discussed in case of reintervention to guarantee longlasting patency rate., Methods: We conducted a randomized, controlled, pilot trial comparing Silverhawk atherectomy with percutaneous transluminal angioplasty (PTA) in patients with a first instent reobstruction in the femoropopliteal arterial segment, to evaluate intima media thickness (IMT) within the treated segment, as a parameter of recurrence of intimal hyperplasia., Results: In a total 19 patients were included: 9 patients in the atherectomy device and 10 patients in the PTA arm. IMT within the treated segment was statistically significantly elevated in all patients treated with the Silverhawk device versus the patients treated with PTA. The obvious differentiation in elevation of IMT in nonfavor for patients treated with the Silverhawk device started at month 2 (max IMT SH 0.178 mm vs. IMT PTA 0.1 mm, p = 0.001) with a spike at month 5 (max IMT SH 0.206 mm vs. IMT PTA 0.145 mm, p = 0.003) and a decline once again at month 6 (max IMT SH 0.177 mm vs. IMT PTA 0.121 mm, p = 0.02). The values for mean IMT performed the same way., Conclusions: Although Silverhawk atherectomy provides good results at first sight, in the midterm follow-up of treatment of first instent restenosis it did not perform better than PTA as it showed elevated reoccurrence of intimal media hyperplasia.

Vascular smooth muscle cell (VSMC) activation promotes a synthetic phenotype that underlies many vessel growth disorders. In this regard it has been suggested that the metabolic sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) has significant antigrowth and antimetastatic properties and may serve as a viable therapeutic target. In the current study we hypothesized that AMPK reduces neointima formation following balloon injury and that this occurs through reduction in VSMC proliferation and migration. Data reveal that local or systemic dosing with the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) significantly increased AMPK activity in vivo and inhibited neointima formation in rat carotid arteries 2 wk after injury. In primary VSMCs, AICAR inhibited migration and induced cytostatic growth arrest through increased protein phosphatase 2A-mediated inhibition of mitosis-promoting cyclin B. AICAR also significantly enhanced AMPK-specific T278 phosphorylation of the actin anticapping vasodilator-activated serum phosphoprotein, increased G- to F-actin ratios and stress fiber formation, and abrogated PDGF-stimulated S397 autophosphorylation of focal adhesion kinase, promigratory cytoplasmic accumulation of paxillin, and extracellular matrix proteolysis by matrix metalloproteinase-9. Together, these results provide compelling evidence that AMPK serves to inhibit vascular smooth muscle migration and proliferation through regulation of cytoskeletal/focal adhesion/ECM stability, increasing our knowledge of this important metabolic regulator and providing support for its continued investigation in the treatment of vascular growth disorders.

Purpose: To report the use of intravascular frequency-domain optical coherence tomography (FD-OCT) to detect and characterize in-stent neointimal tissue following infrapopliteal drug-eluting stent (DES) placement in patients suffering from critical limb ischemia., Methods: This prospective study included 12 patients (7 men; mean age 72.8±7.2 years) who had previously received 21 infrapopliteal sirolimus- or everolimus-eluting stents. The patients returned for regular angiographic follow-up or presented with clinical relapse of symptoms over a mean follow-up of 13.5±7.3 months (range 6-33), at which time FD-OCT imaging was performed. Study endpoints were technical imaging success, defined as successful FD-OCT acquisition and visualization of the arterial lumen and complete vessel wall, and the detection and characterization of in-stent neointimal hyperplasia according to widely accepted OCT criteria., Results: OCT imaging was successfully completed in 19 of the 21 stents. Binary in-stent restenosis (ISR>50%) was detected in 10/19 stents. Percent restenosis was higher after longer follow-up (60.6%±19.8% ≥1 year vs. 35.3%±20.6% <1 year, p=0.03) and in symptomatic vs. asymptomatic patients (61.5%±20.4% vs. 37.2%±19.3%, p=0.04). Neoatherosclerosis findings included lipid-laden neointima (16/19), neointimal neovascularization (13/19), neointimal calcifications (6/19), and thrombus (5/19); no cases of thin-cap fibroatheroma were identified. Neointimal calcifications were more frequent after ≥12 months of follow-up compared to <12 months (46.6% vs. 0%, p=0.02)., Conclusion: FD-OCT of the infrapopliteal arteries following DES placement is safe and feasible and may demonstrate features of developing neointimal neoatherosclerosis. The latter might play a key role as a mechanism of below-the-knee ISR and warrants further investigation.

Aims: To report the four-month and nine-month angiographic results as well as one-year clinical follow-up from the first-in-man study with the silicon carbide and sirolimus-eluting bioabsorbable polymer (poly-L-lactic acid (PLLA) polymer) -coated cobalt-chromium Orsiro stent., Methods and Results: A group of 30 patients with documented myocardial ischaemia related to a single de novo coronary stenosis up to 22 mm in length, in vessels with a 2.5 to 3.5 mm reference diameter, and between >50% and <90% diameter stenosis were enrolled at two sites. The primary endpoint of the study was in-stent late lumen loss at nine months. The secondary endpoints included major adverse cardiac events (MACE) at one year defined as the composite of cardiac death, ischaemia-driven target lesion revascularisation (TLR) and target vessel myocardial infarction (MI). Procedural success was 100%. Angiographic late lumen loss was 0.12±0.19 mm and 0.05±0.22 mm at four and nine months respectively. At one-year clinical follow-up, the composite MACE was 10% with one patient who died from cardiac death and two patients who had ischaemia-driven target lesion revascularisation. There was no report of MI or stent thrombosis., Conclusions: The Orsiro drug-eluting stent demonstrated potency with low rates of in-stent neointimal hyperplasia and cardiovascular events but warrants further evaluation in a larger population cohort with longer follow-up time points.

There have been no optical coherence tomographic (OCT) data directly comparing the pattern of strut coverage between the 2 second-generation drug-eluting stents in the early period. The aim of this prospective study was to evaluate early strut coverage using optical coherence tomography 3 months after Resolute zotarolimus-eluting stent (ZES-R) or everolimus-eluting stent (EES) implantation in de novo coronary artery lesions. A total of 40 patients who were suitable for the OCT procedure and consented to the study protocol were randomized 1:1 to receive either ZES-R or EES. Among these patients, 35 stented lesions (18 ZES-R, 17 EES) in 34 patients were evaluated by optical coherence tomography immediately and 3 months after stent implantation. Neointimal hyperplasia thickness, percentage of uncovered struts, and the proportion of malapposed struts were measured at 1-mm intervals. An uncovered strut was defined as having a neointimal hyperplasia thickness of 0 μm. At the 3-month OCT evaluation, mean neointimal hyperplasia thickness (ZES-R vs EES 74 ± 41 vs 75 ± 35 μm, p = 0.89) and mean percentage of uncovered struts (ZES-R vs EES 6.2 ± 6.9 vs 4.7 ± 5.1%, p = 0.62) were not significantly different between the groups. The percentage of malapposed struts was also similar between the groups (0.7 ± 2.2% for ZES-R and 0.7 ± 1.7% for EES, p = 0.64). Thrombi were documented in 3 stents (1 [5.6%] in a ZES-R vs 2 [11.8%] in EES, p = 0.60). In conclusion, early stent strut coverage on the basis of serial OCT evaluation was comparable between ZES-R and EES 3 months after stent implantation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: We hypothesized that the tissue components of in-stent restenosis (ISR) might differ between drug-eluting stents (DES) and bare-metal stents (BMS) and that these differences could be distinguished by qualitative and quantitative optical coherence tomography (OCT) analyses., Methods and Results: One-hundred and twenty-two initial ISR lesions (sirolimus-eluting stents: n=28; paclitaxel-eluting stents: n=51; BMS: n=43) were evaluated with OCT. Based on their OCT appearance, the lesions were classified as homogeneous, layered or heterogeneous. The optical properties of backscatter, attenuation and signal intensity of the neointimal tissue (NIT) were quantified. To evaluate the vascular response after balloon angioplasty (BA), the rate of reduction of the NIT area (NITA) was calculated (NITA before - after BA/NITA before BA at the minimum lumen cross-sectional area). Among the morphologic OCT patterns, the layered type was predominant with DES, whereas lesions were homogeneous with BMS (P<0.001). Backscatter and signal intensity were significantly higher with BMS (P<0.05 and P<0.001 respectively). The NITA reduction rate was significantly greater in the layered and heterogeneous groups than in the homogeneous group (P<0.01)., Conclusions: The morphologic OCT patterns of the NIT in ISR differed significantly between DES and BMS, probably reflecting pathologic differences. Layered and heterogeneous tissues might respond better than homogeneous tissue to simple balloon dilatation, suggesting a possible direction for OCT-based ISR treatment strategies.

The proliferation and migration of vascular smooth muscle cells (VSMCs) contributes importantly to the development of in-stent restenosis. Lithium has recently been shown to have beneficial effects on the cardiovascular system, but its actions in VSMCs and the direct molecular target responsible for its action remains unknown. On the other hand, PGC-1α is a transcriptional coactivator which negatively regulates the pathological activation of VSMCs. Therefore, the purpose of the present study is to determine if lithium chloride (LiCl) retards VSMC proliferation and migration and if PGC-1α mediates the effects of lithium on VSMCs. We found that pretreatment of LiCl increased PGC-1α protein expression and nuclear translocation in a dose-dependent manner. MTT and EdU incorporation assays indicated that LiCl inhibited serum-induced VSMC proliferation. Similarly, deceleration of VSMC migration was confirmed by wound healing and transwell assays. LiCl also suppressed ROS generation and cell cycle progression. At the molecular level, LiCl reduced the protein expression levels or phosphorylation of key regulators involved in the cell cycle re-entry, adhesion, inflammation and motility. In addition, in vivo administration of LiCl alleviated the pathophysiological changes in balloon injury-induced neointima hyperplasia. More importantly, knockdown of PGC-1α by siRNA significantly attenuated the beneficial effects of LiCl on VSMCs both in vitro and in vivo. Taken together, our results suggest that LiCl has great potentials in the prevention and treatment of cardiovascular diseases related to VSMC abnormal proliferation and migration. In addition, PGC-1α may serve as a promising drug target to regulate cardiovascular physiological homeostasis.

Purpose: Intimal thickening, which results from the response to arterial damage caused by therapeutic interventions or other reasons, is usually called as neointima. Neointimal hyperplasia is a main step in the pathogenesis of late-term restenosis, which is developed after vascular interventions. Reduction in nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling plays a substantial role in the pathogenesis of neointima formation. Phosphodiesterase V is detected in the peripheral coronary and pulmonary vascular smooth muscle cells and in the cardiac tissue. Based on the effects of phosphodiesterase V inhibitors on vascular smooth muscle cells, in the present study, the effect of tadalafil, a new member of phosphodiesterase V inhibitors, on neointimal hyperplasia was investigated in the rabbit carotid artery anastomosis model., Material and Method: Fourteen male New Zealand white rabbits weighing between 2.5-3 kg, were used. The rabbits were randomly divided into two equal groups; tadalafil group received oral tadalafil (2 mg/kg/day), and PBS group received sterile PBS solution (normal saline; 2 mg/kg/day) for 28 days after the surgery. The right carotid arteries of all rabbits were anastomosed in an end-to-end fashion using 8/0 polypropylene suture. The rabbits were sacrificed at the end of the postoperative period of 28 days. After sacrificing, firstly anastomosis segment on the right carotid artery and secondly a part of the left carotid artery (as control) of each rabbit were removed. Morphometric examination of tissue sections was performed under a light microscope connected to an image capture system., Results: There was a significant difference between the right and left carotid arteries in terms of intimal area and intima/media ratio both in tadalafil and PBS groups (p <0.001 for each). Intimal area and intima/media ratio were increased in the right carotid arteries compared to the left carotid arteries (p <0.001 for each). Besides, when the right carotid arteries of both groups were compared using covariance analysis, it was observed that intimal area and intima/media ratio in the anastomosis site were significantly reduced with tadalafil treatment (p <0.001)., Conclusion: The present study was promising in terms of tadalafil use as a new agent for the prevention of neointimal hyperplasia, which is the leading cause of late-term graft failure in vascular surgery.

Background: Cell transplantation has great potential for promoting endothelial repair and reducing the complications of percutaneous coronary intervention (PCI). The aim of this study was to investigate the effect of transplantation of human umbilical cord blood endothelial progenitor cells (EPCs) on injured arteries., Methods: Umbilical cord blood mononuclear cells were obtained from post-partum lying-in women, and EPCs were isolated, cultured, expanded and identified by immunofluorescence. The carotid arterial endothelium of New Zealand white rabbits was injured by dilatation with a 3F balloon, and the EPCs were injected into the lumen of the injured artery in the transplanted group (n = 16), while an equal volume of phosphated buffered saline (PBS) was injected into the control group after balloon injury (n = 16). The animals were sacrificed after either 2 or 4 weeks, and the grafted cells were identified by double immunofluorescence staining with human nuclear antigen (HNA) and CD31 antibodies. Arterial cross sections were analyzed by pathology, immunohistochemistry and morphometry to evaluate the reparative effects of EPCs. Proliferating cell nuclear antigen (PCNA) and transforming growth factor (TGF)-β1 mRNA expression were detected by reverse transcription-polymerase chain reaction (RT-PCR)., Results: Fluorescence-labeled EPCs were found in the neointima. The neointimal area and the neointimal/medial area ratio were significantly lower in the transplanted group than in the control group (P < 0.05). von Willebrand factor (vWF) immunohistostaining showed more VWF-positive cells in the transplanted animals than in the controls (8.75 ± 2.92 vs. 4.50 ± 1.77, P < 0.05). Compared with the control group, the transplanted group had lower expression of PCNA mRNA (0.67 ± 0.11 vs. 1.25 ± 0.40, P < 0.01) and higher expression of TGF-β1 mRNA (1.10 ± 0.21 vs. 0.82 ± 0.07, P < 0.05)., Conclusions: EPCs derived from human umbilical cord blood were successfully transplanted into injured vessels. The transplanted EPCs inhibited neointimal hyperplasia and promoted vascular re-endothelialization.

The timing and incidence of neointimal calcification after stenting (NIC) is largely unknown. The purpose of our study was to elucidate the characteristics of NIC. The presence of NIC in patients who underwent intravascular ultrasound between June 30, 2009 and June 30, 2012 was analyzed. The patients were divided into two groups based on the follow-up period: < 365 days or ≥ 365 days. A total of 181 images were analyzed. Those with NIC had a lower estimated glomerular filtration rate [51 (6-60) versus 61 (52-72) mL/minute/1.73 m²; P < 0.01] and longer time after stenting [3198 (1710-3684) versus 211 (180-516) days; P < 0.01] compared to those without NIC. NIC during short-term follow-up was observed only in patients who were on hemodialysis. On the other hand, NIC in the long-term follow-up was observed only in patients with bare metal stents. The development of NIC was related to renal function and time after stenting. NIC in the short-term and the long-term follow-up was observed only in patients who were on hemodialysis and who were implanted with a bare metal stent, respectively.

Objectives: Depletion of early growth response factor-1 (Egr-1) by a DNA enzyme, ED5, inhibits neointimal hyperplasia (NH) following vascular injury by an unknown mechanism. The aim of this study was to characterize the effects of ED5 in a rat carotid injury model in order to elucidate the mechanism by which ED5 inhibits NH., Methods: ED5 was transfected into the arterial wall of Wistar rats using FuGENE6 transfection reagent following artery balloon injury. Hematoxylin and eosin staining, immunohistochemistry, real-time reverse transcription polymerase chain reaction and Western blotting analysis were used to characterize the response to ED5., Results: NH decreased significantly in the ED5- plus FuGENE6-treated rats (p < 0.05) compared with the control groups, and this was accompanied by a reduced inflammatory response. Egr-1 mRNA and protein levels were significantly decreased in the ED5-treated group, as expected. The decrease in Egr-1 was accompanied by decreases in the mRNA and protein levels of PDGF-BB, Cyclin D1, CDK4, MCP-1, and ICAM-1 (p < 0.05)., Conclusions: Transfection of the Egr-1-specific synthetic DNA enzyme ED5 significantly reduced NH after injury in rats, at least in part, as a result of decreased expression of downstream proliferative genes such as PDGF-BB, Cyclin D1, CDK4, and the inflammatory factors MCP-1 and ICAM-1., (Copyright © 2013 S. Karger AG, Basel.)

TRPC-mediated Ca(2+) entry has been implicated in the control of smooth muscle proliferation and might represent a pivotal mechanism underlying in-stent restenosis. As we have observed significant expression of TRPC3 in human smooth muscle from the coronary artery as well as the aorta, we tested the efficiency of a recently discovered TRPC3 selective Ca(2+) entry blocker Pyr3 to prevent vascular smooth muscle proliferation and stent implantation-induced hyperplasia of human aorta. The effect of Pyr3 on proliferation was measured by detection of BrdU incorporation and PCNA expression in human coronary smooth muscle and microvascular endothelium, which displays significantly smaller expression levels of TRPC3 as compared with smooth muscle. Pyr3 inhibited smooth muscle proliferation but lacked detectable effects on endothelial proliferation. Measurements of ATP-induced Ca(2+) signals revealed that Pyr3 suppressed agonist-induced Ca(2+) entry more effectively in vascular smooth muscle than in endothelial cells. Inhibitory effects of Pyr3 on stent implantation-induced arterial injury was tested using a novel in vitro model of in-stent hyperplasia in human arteries based on organ typical culture of human aortic constructs. Pyr3 effectively prevented increases in tissue levels of PCNA and Ki-67 at 2 weeks after stent implantation into human aortae. Similarly, proliferation markers were significantly suppressed when implanting a Pyr3-releasing stent prototype as compared with a bare metal stent (BMS) control. Our results suggest TRPC3 as a potential target for pharmacological control of smooth muscle proliferation. Selectively inhibition of TRPC Ca(2+) entry channels in vascular smooth muscle is suggested as a promising strategy for in-stent restenosis prevention.

Objective: Endovascular injury induced by balloon withdrawal leads to the increased activation of matrix metalloproteinases (MMPs) in the vascular wall allowing the proliferated smooth muscle cells (SMCs) to digest the surrounding extracellular matrix and migrate from the media into the intima leading to the intimal thickening. The objective of this study was to examine the effect of Nelumbo nucifera leaf extract (NL) on intimal thickening of rat carotid artery injured by balloon catheter and on the proliferation and migration of cultured vascular smooth muscle cells (VSMCs) induced by tumor necrosis factor-α., Methods: NL was administered orally using gastric sonde at three different doses, 100 mg kg(-1) (NL100), 400 mg kg(-1) (NL400), and 800 mg kg(-1) (NL800) for 4 wk from the day of balloon injury in the rats. VSMC proliferation and migration were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Boyden chamber methods, whereas enzymatic action of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) was carried out by gelatin zymography, and MMP-9 protein expression, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase phosphorylations were assessed by Western blot analyses., Results: NL reduced the intimal thickening by suppressing VSMC's proliferation through inhibition of extracellular signal-regulated kinase 1/2 phosphorylation and their migration by reducing the expression of MMP-2 and -9 through inhibition of JNK1/2 phosphorylation., Conclusion: Thus, the results suggest that NL can be considered of therapeutic value in the prevention of atherosclerosis because restenosis after percutaneous transluminal coronary angioplasty can be considered a model of "accelerated atherosclerosis.", (Copyright © 2013 Elsevier Inc. All rights reserved.)

Previous intravascular ultrasound studies have shown that echolucent neointimal hyperplasia occasionally appears after bare-metal stent (BMS) or sirolimus-eluting stent (SES) implantation. Optical coherence tomography (OCT) studies have also demonstrated that paclitaxel-eluting stent (PES) restenosis exhibited similar images showing low signal intensity areas (LSIA) surrounding stent struts and three-layer appearance (TLA). The aim of the present study was to investigate the clinical significance of LSIA on OCT images in various types of stents. Fifty nine consecutive patients who underwent scheduled follow-up coronary angiography and OCT were enrolled. There was no significant difference in the prevalence of LSIA among the 3 stent groups (BMS 30%, SES 19%, PES 28%, P = 0.70). LSIA thickness was larger in the PES group than in the other stent groups (BMS 0.51 ± 0.21 mm, SES 0.35 ± 0.06 mm, PES 0.87 ± 0.19 mm, P < 0.01). The ratio of LSIA thickness to the neointimal thickness was also larger in PES compared with other stents (BMS 53 ± 9 %, SES 57 ± 8 %, PES 77 ± 5 %, P < 0.01). Also, LSIA thickness in patients with in-stent restenosis (ISR) was significantly larger than in those without ISR (0.37 ± 0.37 mm versus 0.12 ± 0.26 mm, P = 0.048). Our results suggest that LSIA might be involved in excessive neointimal formation, and that the healing response after PES implantation might be different from BMS or SES.

Background:  Adipose tissue-derived stem cells (ASC) produce a variety of cytokines that potentially mediate the proangiogenic and antiapoptotic effects of the ASC. We examined whether ASC produced angiopoietin-1 (Ang1) and whether Ang1 functionally mediated ASC-induced suppression of neointimal formation., Methods and Results:  Ang1 production was measured by enzyme-linked immunosorbent assay. Production of endogenous Ang1 by ASC was inhibited with small interfering RNA (siRNA) for Ang1. Overproduction of Ang1 was achieved with an adenovirus that expresses Ang1 (AdAng1). ASC expressing Ang1 siRNA, or AdAng1 were administered around the femoral artery after wire injury, and immunohistochemical analysis was performed to examine their effects on neointimal formation. ASC produced Ang1 in a time-dependent manner, especially when cultured in medium containing growth factors for vascular endothelial cells. When ASC were treated with Ang1 siRNA, the inhibitory effect of ASC on neointimal formation was significantly reduced. Knockdown of Ang1 significantly increased macrophage infiltration in the neointima, and significantly decreased endothelial regeneration. In contrast, forced expression of Ang1 using AdAng1 significantly suppressed neointimal formation and macrophage infiltration, and stimulated reendothelialization., Conclusions:  Ang1 was implicated in ASC-induced suppression of neointimal formation. The results also suggested that Ang1 inhibited neointimal formation via stimulation of reendothelialization and suppression of macrophage infiltration in the neointima.

Maintenance of normal endothelial function is critical to various aspects of blood vessel function, but its regulation is poorly understood. In this study, we show that disruption of baseline fibroblast growth factor (FGF) signaling to the endothelium leads to a dramatic reduction in let-7 miRNA levels that, in turn, increases expression of transforming growth factor (TGF)-β ligands and receptors and activation of TGF-β signaling, leading to endothelial-to-mesenchymal transition (Endo-MT). We also find that Endo-MT is an important driver of neointima formation in a murine transplant arteriopathy model and in rejection of human transplant lesions. The decline in endothelial FGF signaling input is due to the appearance of an FGF resistance state that is characterized by inflammation-dependent reduction in expression and activation of key components of the FGF signaling cascade. These results establish FGF signaling as a critical factor in maintenance of endothelial homeostasis and point to an unexpected role of Endo-MT in vascular pathology., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Autologous venous grafts, used to circumvent occluded coronary arteries during coronary artery bypass, often develop thrombosis and neointimal hyperplasia. During neointimal hyperplasia, vascular smooth muscle cells (VSMCs), exposed to substantially higher pressure and hemodynamic forces, proliferate and extracellular matrix accumulate causing narrowing of the vessel lumen. Activation of insulin-like growth factor-1 receptor (IGF-1R) has been confirmed to be critically involved in mechanical stretch-stimulated VSMC proliferation. However, the comprehensive mechanisms responsible for activation of IGF-1R in VSMCs by mechanical stretch remain unclear. This study found that miR-223 and miR-153, targeted to IGF-1R, were down-regulated in VSMCs under stretch stress by miRNA microarray analysis in conjunction with Target Scan analysis. Overexpression of miR-223 or miR-153 down-regulated IGF-1R expression and activity in VSMCs under stretch stress. Specifically, overexpression of miR-223 and miR-153 inhibited stretch stress-enhanced VSMC proliferation and the activity of PI3K-AKT signaling. In conclusion, our study indicates that miR-153 and miR-223 are reduced in VSMCs by stretch stress, contributing to IGF-1R activation and resultant VSMC proliferation. Thus, miR-153 and miR-223 may be viable therapeutic targets for mechanical stretch-induced neointimal hyperplasia in vein grafts., (Copyright © 2012. Published by Elsevier Inc.)

Osteopontin (OPN) is known to be one of the cytokines that is involved in the vascular inflammation caused by aldosterone (Ald). Previous reports have shown that Ald increases OPN expression, and the mechanisms for this remain to be clarified. In this study, we investigated how Ald increases OPN expression in the vascular smooth muscle cells (VSMCs) of rats. Ald increased OPN expression time dependently as well as dose dependently. This increase was diminished by spironolactone, a mineralocorticoid receptor (MR) antagonist. PD98059, an inhibitor of p42/44 MAPK pathway, and SB203580, an inhibitor of p38 MAPK pathway, suppressed Ald-induced OPN expression and secretion in VSMCs. VSMCs migration stimulated by aldosterone required OPN expression. In conclusion, these data suggest that Ald-induced OPN expression in VSMC is mediated by MR and signaling cascades involving ERK and p38 MAPK. These molecules may represent therapeutic targets for the prevention of pathological vascular remodeling.

Endovascular stent grafts used for treatment of arterial aneurysms require preclinical testing for investigation of biological responses following implantation. The preclinical evaluation process related to the safety and efficacy of these devices is limited by the absence of an in vitro aneurysmal blood vessel equivalent capable of providing high-throughput, cost-effective assessments. With this in mind, the focus of this work was to develop an aneurysm model consisting of human blood vessel cells. To create aneurysmal scaffolds, expanded polytetrafluoroethylene vascular grafts were dilated utilizing an angioplasty balloon. Stromal vascular fraction cells isolated from human adipose tissue were integrated with the scaffolds, and luminal flow of nutrient medium was executed for 14 days in a vascular bioreactor. Following bioreactor perfusion, histology verified that a neointimal lining of human tissue had formed. Immunohistochemistry and scanning electron microscopy revealed a flow-contacting layer of smooth muscle cells, characterizing the model as a representation of neointimal formation in an injured or diseased vessel. This study has demonstrated the engineering of a vascular construct containing an aneurysmal dilation. A tissue-engineered aneurysm model could provide an alternative to current nonbiological in vitro aneurysm models and serve as a practical tool in the progression of new devices toward in vivo studies., (Copyright © 2012 Wiley Periodicals, Inc.)

Introduction: A novel self-expanding, drug-eluting stent (DES) was designed to slowly release everolimus in order to prevent restenosis after percutaneous peripheral intervention. The purpose of this experimental animal study was to test the hypothesis that long-term local, stent-mediated delivery of everolimus would reduce neointimal hyperplasia in porcine iliac arteries., Methods: The iliac arteries of 24 Yucatan mini-swine were percutaneously treated with overlapping 8- × 28-mm self-expanding nitinol stents loaded with everolimus (225 μg/cm2 stent surface area) formulated in a poly(ethylene-co-vinyl alcohol) copolymer intended to deliver the drug in a sustained fashion over about 6 months (DES). Bare nitinol self-expanding stents (bare metal stent [BMS]) were implanted in an identical fashion on the contralateral side to serve as controls. After 3, 6, or 12 months, the animals were sacrificed and the stented arteries perfusion-fixed for histomorphometric analysis., Results: The chronic presence of everolimus in arterial tissue reduced stent-induced inflammation after 3 months (inflammation score: BMS 2.29±0.44 vs DES 0.17±0.17; P=.001) and 6 months (BMS 2.06±0.43 vs DES 0.50±0.5; P=.007), although some late inflammation was observed after drug exhaustion (BMS 1.00±0.25 vs DES 2.56±0.62 after 12 months; P=not significant [NS]). Treatment with locally delivered everolimus significantly reduced neointimal hyperplasia after 3 months (neointimal thickness: BMS 0.79±0.20 vs DES 0.37±0.04 mm; P=.03) and 6 months (BMS 0.73±0.14 vs DES 0.41±0.08 mm; P=.05), although the effect had dissipated after 12 months (BMS 0.68±0.11 vs DES 0.67±0.11 mm; P=NS). Remarkably, stent-induced neoatherosclerosis, characterized by the histologic presence of foamy macrophages and cholesterol clefts, was significantly attenuated by treatment with everolimus (atherogenic change scores at 3 months: BMS 0.56±0.15 vs DES 0.04±0.04; P=.003; 6 months: BMS 0.84±0.23 vs DES 0.00±0.00; P=.004; and 12 months: BMS 0.09±0.10 vs DES 0.19±0.19; P=NS)., Conclusions: In this experimental animal model, local arterial stent-mediated delivery of everolimus inhibited the formation of neointimal hyperplasia and neoatherosclerosis during the first 6 months. The effect was transient, however, as arterial morphology and histology appeared similar to control stented arteries after 12 months., (Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Although short-term outcomes in kidney transplantation have improved dramatically, long-term survival remains a major challenge. A key component of long-term, chronic allograft injury in solid organ transplants is arteriosclerosis characterized by vascular neointimal hyperplasia and inflammation. Establishing a model of this disorder would provide a unique tool not only to identify mechanisms of disease but also to test potential therapeutics for late graft injury. To this end, we utilized a mouse orthotopic renal transplant model in which C57BL/6J (H-2b) recipients were given either a kidney allograft from a completely mismatched Balb/cJ mouse (H-2d) or an isograft from a littermate. A unilateral nephrectomy was performed at the time of transplant followed by a contralateral nephrectomy on post-transplant day 7. Recipients were treated with daily cyclosporine subcutaneously for 14 days and then studied 8 and 12 weeks post transplantation. Renal function was significantly worse in allograft compared with isograft recipients. Moreover, the allografts had significantly more advanced tubulointerstitial fibrosis and profound vascular disease characterized by perivascular leukocytic infiltration and neointimal hyperplasia affecting the intrarenal blood vessels. Thus, we describe a feasible and reproducible murine model of intrarenal transplant arteriosclerosis that is useful to study allograft vasculopathy.

Sonic hedgehog (Shh) is a morphogen critically involved in development that is reexpressed in atherosclerotic lesions. It also stimulates proliferation of vascular smooth muscle cells (SMCs). Autophagy in vascular SMCs is known to promote SMC survival and increase plaque stability. The aim of this study was to investigate whether Shh induces autophagy of vascular SMCs. Our study showed that both Shh protein and microtubule-associated protein 1 light chain 3 (LC3)-II were increased in SMCs within neointimal lesions of mouse common carotid arteries. In cultured mouse aortic SMCs, recombinant mouse Shh stimulated LC3-II levels. Overexpression of wild-type mouse Shh through the tetracycline-regulated expression-inducible system in human aortic SMCs time-dependently increased the levels of LC3-II and also stimulated protein kinase B (AKT) phosphorylation. Pretreatment with AKT inhibitor IV (AKTI IV) inhibited AKT phosphorylation and the increase in LC3-II. Shh-induced autophagy was further confirmed by the formation of autophagosomes as detected by immunostaining and transmission electron microscopy, which was inhibited by AKTI IV. Shh further increased SMC LC3-II in the presence of bafilomycin A1, (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, and pepstatin A or siRNA for the autophagy-related gene 7 (ATG7). In addition, Shh induced SMC proliferation, which was inhibited not only by AKTI IV but also by cyclopamine, an inhibitor of Shh receptor. Inhibition of autophagy with 3-methyladenine (3-MA), bafilomycin A1, or ATG7 siRNA resulted in inhibition of cell proliferation. Treatment with 3-MA, AKTI IV, or cyclopamine inhibited neointima formation in mouse common carotid arteries. Taken together, our results have shown that Shh induces autophagy of vascular SMCs involving AKT activation, suggesting a role of autophagy in Shh-induced cellular responses.

Myocardin-related transcription factor (MRTF)-A is a Rho signalling-responsive co-activator of serum response factor (SRF). Here, we show that induction of MRTF-A expression is key to pathological vascular remodelling. MRTF-A expression was significantly higher in the wire-injured femoral arteries of wild-type mice and in the atherosclerotic aortic tissues of ApoE(-/-) mice than in healthy control tissues, whereas myocardin expression was significantly lower. Both neointima formation in wire-injured femoral arteries in MRTF-A knockout (Mkl1(-/-)) mice and atherosclerotic lesions in Mkl1(-/-); ApoE(-/-) mice were significantly attenuated. Expression of vinculin, matrix metallopeptidase 9 (MMP-9) and integrin β1, three SRF targets and key regulators of cell migration, in injured arteries was significantly weaker in Mkl1(-/-) mice than in wild-type mice. In cultured vascular smooth muscle cells (VSMCs), knocking down MRTF-A reduced expression of these genes and significantly impaired cell migration. Underlying the increased MRTF-A expression in dedifferentiated VSMCs was the downregulation of microRNA-1. Moreover, the MRTF-A inhibitor CCG1423 significantly reduced neointima formation following wire injury in mice. MRTF-A could thus be a novel therapeutic target for the treatment of vascular diseases.

Additional beneficial effects of angiotensin II type 1 (AT(1)) receptor blockers beyond AT(1) receptor blockade have been highlighted. Irbesartan is reported to act as an antagonist of the monocyte chemoattractant protein-1 (MCP-1) receptor, C-C chemokine receptor 2 (CCR2). We examined the possible synergistic effects of the combination of irbesartan with rosuvastatin on preventing vascular remodeling focusing on the MCP-1/CCR2 pathway. We observed that administration of irbesartan and CCR2 antagonist, propagermanium, at noneffective doses, decreased the neointima with a decrease in PCNA labeling index in the injured mouse femoral artery induced by cuff placement. We also observed that administration of a noneffective dose of rosuvastatin with propagermanium decreased the neointima area, suggesting that the inhibitory effect of rosuvastatin on neointima formation is at least partly attributable to blockade of the MCP-1/CCR2 pathway. Moreover, we demonstrated that the combination of irbesartan with rosuvastatin decreased neointima formation. MCP-1 mRNA level was significantly increased in injured femoral arteries, and administration of irbesartan with rosuvastatin decreased the mRNA levels of MCP-1, TNFα, and IL-1β, and increased PPARγ mRNA expression. These results suggest that the synergistic inhibitory effects of irbesartan with rosuvastatin on neointima formation may involve attenuation of MCP-1/CCR2 signaling., (Copyright © 2012 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.)

Background: Novel vascular scaffolds aim at equipoise between safety and efficacy. Intravascular optical coherence tomography (OCT) allows in-vivo serial assessment of stent-vessel interactions with high resolution and frequent sampling and may complement histology assessment. We investigated the vascular response to a novel absorbable coating sirolimus-eluting stent (AC-SES) by means of serial OCT and histology evaluation in a porcine model., Methods: One AC-SES and one bare-metal stent (BMS) were implanted in separate coronary arteries of three Yucatan mini-swine. Serial OCT was performed post procedure and at 3-, 28-, 90-, and 180-day follow-up. Normalized optical density (NOD) was used for the assessment of tissue response over time. Histological evaluation was performed at day 180., Results: A total of 6408 stent struts were analyzed. OCT revealed 100% of struts covered at 28 days, and a significant difference in NOD from 3 to 28 days (0.64 ± 0.07 vs 0.71 ± 0.05, respectively; P<.001) in the AC-SES group. Neointimal thickness was 0.14 ± 0.08 mm, 0.17 ± 0.11 mm, and 0.16 ± 0.09 mm in the AC-SES group and 0.18 ± 0.10 mm, 0.14 ± 0.09 mm, and 0.10 ± 0.08 mm in the BMS group, while rates of uncovered struts were 0%, 0%, and 3.1% and 1.4%, 7.8%, and 21.5%, respectively, at 28, 90, and 180 days. Minimal inflammation and a mature endothelialization were demonstrated in both groups by histology., Conclusion: OCT serial assessment of vascular response suggested NIH maturation 28 days following AC-SES implantation in pigs. These findings, coupled with histological demonstration of low inflammation scores and complete endothelial coverage as measured at 180 days, suggest a satisfactory healing response to AC-SES.

Aims: In the previous COREA-TAXUS trial, a 6-month adjunctive use of celecoxib reduced target-lesion revascularization (TLR) without increased thrombotic risk. We aimed to confirm the effects of 3-month celecoxib in patients receiving drug-eluting stent (DES) implantation in the larger prospective, randomized trial., Methods and Results: Patients (n = 909) treated for native coronary lesions were randomized into four groups: the control or the celecoxib group with stratification by stents: paclitaxel-eluting stent (PES) or zotarolimus-eluting stent (ZES). In the celecoxib group, 200 mg of celecoxib was given twice daily for 3 months after the procedure. The primary endpoint was in-stent late loss (LL) at 6 months. In-stent LL was significantly lower in the celecoxib group than the control group (0.64 ± 0.54 vs. 0.55 ± 0.47 mm, P = 0.02). The trend of LL reduction in the celecoxib group was maintained in the ZES and PES subgroups, although it did not reach statistical significance. There was a trend towards the reduced clinically driven TLR in the celecoxib group (5.7 vs. 3.2%, log-rank P = 0.09), but adverse cardiac events rate did not differ between the two groups (composite of cardiac death, non-fatal myocardial infarction, and TLR; 8.6 vs. 7.7%, log-rank P = 0.84). Non-fatal myocardial infarction and cardiac death occurred in 1.6% of the patients in the celecoxib group when compared with 0.2% in the control group (log-rank P = 0.03)., Conclusion: Three-month adjunctive celecoxib would be useful to reduce LL of DES. However, this study may raise the concern about increased thrombotic risk with celecoxib even in patients receiving dual anti-platelet therapy.

Bare metal and drug-eluting coronary stents suffer an inherent lack of vascular cell and blood compatibility resulting in adverse patient responses. We have developed a plasma-activated coating (PAC) for metallic coronary stents that is durable, withstands crimping and expansion, has low thrombogenicity and can covalently bind proteins, linker-free. This has been shown to enhance endothelial cell interactions in vitro and has the potential to promote biointegration of stents. Using the rabbit denuded iliac artery model, we show for the first time that PAC is a feasible coating for coronary stents in vivo. The coating integrity of PAC was maintained following implantation and expansion. The rate of endothelialization, strut coverage, neointimal response and the initial immune response were equivalent to bare metal stents. Furthermore, the initial thrombogenicity caused by the PAC stents showed a reduced trend compared to bare metal stents. This work demonstrates a robust, durable, non-cytotoxic plasma-based coating technology that has the ability to covalently immobilize bioactive molecules for surface modification of coronary stents. Improvements in the clinical performance of implantable cardiovascular devices could be achieved by the immobilization of proteins or peptides that trigger desirable cellular responses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Serial changes in strut coverage of drug-eluting stents (DESs), which are placed across side-branch vessels, remain unclear., Methods: The changes in strut coverage of DESs crossing side-branch vessels (size ≥2.0 mm) were serially evaluated by optical coherence tomography (OCT) in 30 patients at 9 months and 2 years after the index DES implantation. DESs were paclitaxel-eluting stents (PESs), sirolimus-eluting stents (SESs), and zotarolimus-eluting stents (ZESs), each in 10 patients. Measured neointimal hyperplasia (NIH) thickness of 0 μm on OCT was defined as an uncovered strut., Results: The percentage of uncovered side-branch struts significantly decreased from 55.7 ± 39.9% to 36.6 ± 32.0% (P<.0001) on serial follow-up: PES, 93.4 ± 10.5% to 67.6 ± 24.2%, P=.018; SES, 47.5 ± 34.4% to 29.6 ± 24.1%, P=.036; and ZES, 26.2 ± 34.8% to 12.4 ± 19.0%, P=.028. Among covered side-branch struts, the overall percentage of struts with NIH thickness more than 30 μm significantly increased from 36.3 ± 37.4% to 51.0 ± 36.0% (P<.0001). However, compared to other DES types, a significant increase in relatively thin NIH (0 to 30 μm) was observed in PESs (1.6 ± 3.4% to 17.4 ± 16.0%; P=.018)., Conclusion: Serial follow-up OCT examination showed a significant decrease in the percentage of uncovered side-branch struts, and the coverage pattern differed with DES type.

Objective: Because vein graft neointimal hyperplasia engenders vein graft failure, and because most vein graft neointimal cells derive from outside the vein graft, we sought to determine whether vein graft neointimal hyperplasia is affected by activity of the CXC chemokine receptor-4 (CXCR4), which is important for bone marrow-derived cell migration., Methods: In congenic Cxcr4(-/+) and wild-type (WT) recipient mice, we performed interposition grafting of the common carotid artery with the inferior vena cava (IVC) of either Cxcr4(-/+) or WT mice to create four surgically chimeric groups of mice (n ≥ 5 each), characterized by vein graft donor/recipient: WT/WT; Cxcr4(-/+)/WT; WT/Cxcr4(-/+); and Cxcr4(-/+)/Cxcr4(-/+); vein grafts were harvested 6 weeks postoperatively., Results: The agonist for CXCR4 is expressed by cells in the arterializing vein graft. Vein graft neointimal hyperplasia was reduced by reducing CXCR4 activity in vein graft-extrinsic cells, but not in vein graft-intrinsic cells: the rank order of neointimal hyperplasia was WT/WT ≈ Cxcr4(-/+)/WT > WT/Cxcr4(-/+) ≈ Cxcr4(-/+)/Cxcr4(-/+); CXCR4 deficiency in graft-extrinsic cells reduced neointimal hyperplasia by 39% to 47% (P < .05). Vein graft medial area was equivalent in all grafts except Cxcr4(-/+)/Cxcr4(-/+), in which the medial area was 60% ± 20% greater (P < .05). Vein graft re-endothelialization was indistinguishable among all three vein graft groups. However, the prevalence of medial leukocytes was 40% ± 10% lower in Cxcr4(-/+)/Cxcr4(-/+) than in WT/WT vein grafts (P < .05), and the prevalence of smooth muscle actin-positive cells was 45% ± 20% higher (P < .05)., Conclusions: We conclude that CXCR4 contributes to vein graft neointimal hyperplasia through mechanisms that alter homing to the vein graft of graft-extrinsic cells, particularly leukocytes., Clinical Relevance: The utility of autologous vein grafts is severely reduced by neointimal hyperplasia, which accelerates subsequent graft atherosclerosis. Our study demonstrates that vein graft neointimal hyperplasia is aggravated by activity of the cell-surface “CXC” chemokine receptor-4 (CXCR4), which is critical for recruitment of bone marrow-derived cells to sites of inflammation. Our model for CXCR4 deficiency used mice with heterozygous deficiency of Cxcr4. Consequently, our results suggest the possibility that a CXCR4 antagonist--like plerixafor, currently in clinical use--could be applied to vein grafts periadventitially, and perhaps achieve beneficial effects on vein graft neointimal hyperplasia., (Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Upon secretion, transforming growth factor β (TGFβ) is maintained in a sequestered state in extracellular matrix as a latent form. The latent TGFβ is considered as a molecular sensor that releases active TGFβ in response to the perturbations of the extracellular matrix at the situations of mechanical stress, wound repair, tissue injury, and inflammation. The biological implication of the temporal discontinuity of TGFβ storage in the matrix and its activation is obscure. Here, using several animal models in which latent TGFβ is activated in vascular matrix in response to injury of arteries, we show that active TGFβ controls the mobilization and recruitment of mesenchymal stem cells (MSCs) to participate in tissue repair and remodeling. MSCs were mobilized into the peripheral blood in response to vascular injury and recruited to the injured sites where they gave rise to both endothelial cells for re-endothelialization and myofibroblastic cells to form thick neointima. TGFβs were activated in the vascular matrix in both rat and mouse models of mechanical injury of arteries. Importantly, the active TGFβ released from the injured vessels is essential to induce the migration of MSCs, and cascade expression of monocyte chemotactic protein-1 stimulated by TGFβ amplifies the signal for migration. Moreover, sustained high levels of active TGFβ were observed in peripheral blood, and at the same time points following injury, Sca1+ CD29+ CD11b- CD45- MSCs, in which 91% are nestin+ cells, were mobilized to peripheral blood and recruited to the remodeling arteries. Intravenously injection of recombinant active TGFβ1 in uninjured mice rapidly mobilized MSCs into circulation. Furthermore, inhibitor of TGFβ type I receptor blocked the mobilization and recruitment of MSCs to the injured arteries. Thus, TGFβ is an injury-activated messenger essential for the mobilization and recruitment of MSCs to participate in tissue repair/remodeling., (Copyright © 2012 AlphaMed Press.)

Toward understanding the mechanisms of vascular wall remodeling, here we have studied the role of NFATc1 in MCP-1-induced human aortic smooth muscle cell (HASMC) growth and migration and injury-induced rat aortic wall remodeling. We have identified PKN1 as a novel downstream target of NFATc1-cyclin D1/CDK6 activity in mediating vascular wall remodeling following injury. MCP-1, a potent chemoattractant protein, besides enhancing HASMC motility, also induced its growth, and these effects require NFATc1-dependent cyclin D1 expression and CDK4/6 activity. In addition, MCP-1 induced PKN1 activation in a sustained and NFATc1-cyclin D1/CDK6-dependent manner. Furthermore, PKN1 activation is required for MCP-1-induced HASMC growth and migration. Balloon injury induced PKN1 activation in NFAT-dependent manner and pharmacological or dominant negative mutant-mediated blockade of PKN1 function or siRNA-mediated down-regulation of its levels substantially suppressed balloon injury-induced smooth muscle cell migration and proliferation resulting in reduced neointima formation. These novel findings suggest that PKN1 plays a critical role in vascular wall remodeling, and therefore, it could be a promising new target for the next generation of drugs for vascular diseases, particularly restenosis following angioplasty, stent implantation, or vein grafting.

Objectives: We aimed to determine the extent of acute endothelial cell loss and neointimal proliferation in the long-term in saphenous vein grafts (SVGs) exposed to defined distension pressures., Methods: During routine competence testing of SVGs for coronary artery bypass grafting (CABG), blinded peak pressure measurements were performed in 10 patients. In an experimental set-up, distension pressure-related endothelial damage was studied in the SVGs of 20 patients. In a subgroup (n = 10), each patient's SVG was divided into segments and subjected to four constant pressures (50, 100, 150 and 300 mmHg) for 30 min each. In another subgroup (n = 10), SVGs were exposed to a short phase of high pressure (low pressure followed by 300 mmHg for 5 min). Acute endothelial cell loss was quantified by CD31-immunostaining. After 2 weeks of organ culture, the neointimal proliferation was evaluated using histomorphometry. Pressure-related damage was compared with damage at baseline (0 mmHg)., Results: During routine competence testing for CABG, we revealed a median peak pressure of 355 mmHg (range: 240-639 mmHg). In the experimental set-up, significant acute endothelial cell loss occurred at all tested distension pressures: at 50 mmHg, the median endothelial cell loss was 29% (range: 20-51%, P = 0.015), at 100 mmHg 54% (range: 37-69%, P < 0.001), at 150 mmHg 75% (range: 41-88%, P < 0.001), at 300 mmHg 91% (range: 63-100%, P < 0.001) and at short high-pressure exposure 65% (range: 49-82%, P < 0.001) in comparison with 20% (range: 0-44%) at baseline. Significant neointimal proliferation occurred when a distension pressure of 50 mmHg was exceeded: at 50 mmHg, median neointimal proliferation was 97 µm (range: 60-380 µm, P = 0.176), at 100 mmHg 168 µm (range: 100-600 µm, P = 0.001), at 150 mmHg 183 µm (range: 160-440 µm, P < 0.001) at 300 mmHg 347 µm (range: 190-590 µm, P < 0.001) and at short high-pressure exposure 130 µm (range: 60-410 µm, P = 0.02) in comparison with 90 µm (range: 60-170 µm) at baseline., Conclusions: In vitro exposure of SVGs to low distension pressure ranges causes significant acute endothelial cell loss and crucial long-term damage, namely neointimal proliferation.

Epidemiological studies revealed an association between IUGR (intrauterine growth restriction) and an increased risk of developing CVDs (cardiovascular diseases), such as atherosclerosis or hypertension, in later life. Whether or not IUGR contributes to the development of atherosclerotic lesions, however, is unclear. We tested the hypothesis that IUGR aggravates experimentally induced vascular remodelling. IUGR was induced in rats by maternal protein restriction during pregnancy (8% protein diet). To detect possible differences in the development of vascular injury, a model of carotid artery ligation to induce vascular remodelling was applied in 8-week-old intrauterine-growth-restricted and control rat offspring. Histological and immunohistochemical analyses were performed in the ligated and non-ligated carotid arteries 8 weeks after ligation. IUGR alone neither caused overt histological changes nor significant dedifferentiation of VSMCs (vascular smooth muscle cells). After carotid artery ligation, however, neointima formation, media thickness and media/lumen ratio were significantly increased in rats after IUGR compared with controls. Moreover, dedifferentiation of VSMCs and collagen deposition in the media were more prominent in ligated carotids from rats after IUGR compared with ligated carotids from control rats. We conclude that IUGR aggravates atherosclerotic vascular remodelling induced by a second injury later in life.

Recent studies have reported the development of neoatherosclerosis inside stents and subsequent acute coronary syndrome secondary to disruption of neointimal hyperplasia. The aim of the study was to compare the characteristics of neointimal hyperplasia and its time course between bare metal stents (BMSs) and drug-eluting stents (DESs) using optical coherence tomography. A total of 138 stents were divided into 3 groups according to the follow-up period: early phase, <9 months (25 BMSs and 27 DESs); intermediate phase, ≥9 and <48 months (18 BMSs and 43 DESs); and delayed phase, ≥48 months (13 BMSs and 12 DESs). Optical coherence tomographic analysis included the presence of lipid-laden intima, percentage of lipid-rich plaque, and signal attenuation. The optical coherence tomographic findings were compared between the BMSs and DESs in each period, and the difference between the periods was also determined. In the early phase, a greater incidence of lipid-laden plaque (37% vs 8%, p = 0.02) and a greater percentage of lipid-rich plaque (12.9 ± 25.1% vs 1.2 ± 4.3%, p = 0.01) were found in the DESs than in the BMSs. In the intermediate phase, the DES group continuously showed a significantly greater incidence of lipid-laden plaque (63% vs 28%, p = 0.03) and greater percentage of lipid-rich plaque (24.8 ± 28.1% vs 4.1 ± 7.3%, p <0.01). In addition, signal attenuation was greater in the DES group, suggesting early changes in neointimal hyperplasia properties. In the delayed phase, lipid-laden plaque was the predominant type in both groups. In conclusion, lipid-rich neoatherosclerosis develops inside stents earlier in DESs than in BMSs. After 48 months, most restenotic stents will have developed lipid-laden neointima in both groups., (Copyright © 2012. Published by Elsevier Inc.)

An 80-year-old patient presented with severe in-stent restenosis of an everolimus-eluting stent implanted in the left anterior descending coronary artery 3 years previously. We obtained good angiographic result after paclitaxel-eluting balloon dilation. However, on optical coherence tomography (OCT), multiple, angiographically silent, in-stent, and edge-related dissections were readily recognized. Intravascular ultrasound (IVUS) revealed residual neointima with minor disruptions. At 9-month follow-up, an excellent angiographic result was demonstrated with complete resolution of the stent-related dissections on OCT. IVUS and OCT confirmed complete neointimal healing with a larger lumen. This case illustrates the value of OCT and IVUS to provide unique insights on the pathophysiological mechanisms and healing patterns of paclitaxel-eluting balloon treatment of in-stent restenosis.

Background: The Sparrow stent system (Biosensors International) consists of a self-expanding, ultra-thin nitinol stent mounted within a 0.014″ guidewire designed for small or tortuous coronary lesions. We compared the intravascular ultrasound (IVUS) findings between the novel self-expanding sirolimus-eluting stent (Sparrow-SES) and a conventional balloon-expandable sirolimus-eluting stent (Cypher-SES) in patients with small coronary disease., Methods: We examined 14 lesions treated with the Sparrow-SES from CARE II, compared with 22 small vessel lesions treated with Cypher-SES. IVUS examination was performed post-procedure and 8 months later. Volumetric data were standardized by length as volume index (VI; mm³/mm)., Results: While baseline stent VI trended smaller in Sparrow-SES, follow-up stent VI became similar between the 2 groups due to a significant increase of stent VI in self-expanding Sparrow-SES during the follow-up period. At 8 months, Sparrow-SES showed greater neointima than Cypher-SES (0.8 ± 0.6 mm³/mm vs 0.2 ± 0.2 mm³/mm; P<.001). However, the decrease in lumen VI was only 0.3 ± 0.7 mm³/mm in Sparrow-SES, as compared to 0.1 ± 0.3 mm³/mm in Cypher-SES (P=.259), since the late loss due to neointimal hyperplasia was partly counterbalanced by the chronic stent expansion in Sparrow-SES., Conclusion: While 8-month follow-up of Sparrow-SES revealed greater amounts of neointimal hyperplasia compared with conventional Cypher-SES, chronic stent expansion preserved the lumen by increasing stent volume. This novel, guidewire-based, self-expanding stent system designed to be delivered through complex anatomies may be useful to treat patients with small-caliber coronary lesions by offering sufficient lumen preservation at follow-up.

Objective: To evaluate direct versus indirect monocyte chemoattractant protein (MCP)-1/CCR2 signaling and to identify the cellular producers and effectors for MCP-1 during neointimal hyperplasia (NIH) development in vein grafts., Methods and Results: Genomic analysis revealed an overrepresentation of 13 inflammatory pathways in wild-type vein grafts compared with CCR2 knockout vein grafts. Further investigation with various vein graft-host combinations of MCP-1- and CCR2-deficient mice was used to modify the genotype of cells both inside (graft-intrinsic group) and outside (graft-extrinsic group) the vein wall. CCR2 deficiency inhibited NIH only when present in cells extrinsic to the graft wall, and MCP-1 deficiency required its effectiveness in cells both intrinsic and extrinsic to the graft wall to suppress NIH. Deletion of either MCP-1 or CCR2 was equally effective in inhibiting NIH. CCR2 deficiency in the predominant neointimal cell population had no impact on NIH. Direct MCP-1 stimulation of primary neointimal smooth muscle cells had minimal influence on cell proliferation and matrix turnover, confirming an indirect mechanism of action., Conclusions: MCP-1/CCR2 axis accelerates NIH via its signaling in graft-extrinsic cells, particularly circulating inflammatory cells, with cells both intrinsic and extrinsic to the graft wall being critical MCP-1 producers. These findings underscore the importance of systemic treatment for anti-MCP-1/CCR2 therapies.

Coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI), including stent insertion, are established therapies in both acute coronary syndromes (ACS) and symptomatic chronic coronary artery disease refractory to pharmacological therapy. These continually advancing treatments remain limited by failure of conduit grafts in CABG and by restenosis or thrombosis of stented vessel segments in PCI caused by neointimal hyperplasia, impaired endothelialisation and accelerated atherosclerosis. While pharmacological and technological advancements have improved patient outcomes following both procedures, when grafts or stents fail these result in significant health burdens. In this review we discuss the pathophysiology of vein graft disease and in-stent restenosis, gene therapy vector development and design, and translation from pre-clinical animal models through human clinical trials. We identify the key issues that are currently preventing vascular gene therapy from interfacing with clinical use and introduce the areas of research attempting to overcome these., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aim: In-stent restenosis (ISR), especially focal ISR, after percutaneous coronary intervention (PCI) remains one of the major clinical problems in the drug-eluting stent (DES) era. Several reports have revealed that excimer laser coronary angioplasty (ELCA) is useful for ISR; however, detailed findings after ELCA are unknown. Therefore, we investigated the condition of the neointima after ELCA for ISR with optical coherence tomography (OCT) and compared the OCT findings and clinical outcome between ELCA and cutting-balloon angioplasty (CBA)., Methods: Twenty-one consecutive patients with focal ISR who underwent ELCA or CBA were enrolled. All patients underwent 12- to 15-month follow-up coronary angiography. OCT was performed immediately after successful PCI to evaluate the neointimal condition in the ISR lesion. We compared the following OCT parameters between ELCA and CBA groups: maximal thickness of remaining in-stent neointima (MTN), number of tears, minimum lumen dimension (MLD), and minimum lumen area (MLA). We also evaluated clinical outcomes, including target vessel revascularization, acute myocardial infarction, death, and stent thrombosis., Results: MLA in the ELCA group (n = 10) was significantly larger than in the CBA group, and number of tears in the ELCA group was significantly lower than in the CBA group. A trend was shown toward lower TLR with ELCA versus CBA (10.0% vs 45.5%)., Conclusions: OCT immediately after ELCA for ISR lesions revealed larger lumen area and smaller number of tears compared with CBA, which may support favorable effects of ELCA for focal ISR.

Current drug eluting stent (DES) technology is not optimized with regard to the pharmacokinetics of drug delivery. A novel, absorbable-coating sirolimus-eluting stent (AC-SES) was evaluated for its capacity to deliver drug more evenly within the intimal area rather than concentrating drug around the stent struts and for its ability to match coating erosion with drug release. The coating consisted of absorbable poly-lactide-co-glycolic acid (PLGA) and crystalline sirolimus deposited by a dry-powder electrostatic process. The AC-SES demonstrated enhanced drug stability under simulated use conditions and consistent drug delivery balanced with coating erosion in a porcine coronary implant model. The initial drug burst was eliminated and drug release was sustained after implantation. The coating was absorbed within 90 days. Following implantation into porcine coronary arteries the AC-SES coating is distributed in the surrounding intimal tissue over the course of several weeks. Computational modeling of drug delivery characteristics demonstrates how distributed coating optimizes the load of drug immediately around each stent strut and extends drug delivery between stent struts. The result was a highly efficient arterial uptake of drug with superior performance to a clinical bare metal stent (BMS). Neointimal thickness (0.17±0.07 mm vs. 0.28±0.11 mm) and area percent stenosis (22±9% vs. 35±12%) were significantly reduced (p<0.05) by the AC-SES compared to the BMS 30 days after stent implantation in an overlap configuration in porcine coronary arteries. Inflammation was significantly reduced in the AC-SES compared to the BMS at both 30 and 90 days after implantation. Biocompatible, rapidly absorbable stent coatings enable the matching of drug release with coating erosion and provide for the controlled migration of coating material into tissue to reduce vicissitudes in drug tissue levels, optimizing efficacy and reducing potential toxicity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Stromal interaction molecules (STIM1 and STIM2) are single pass transmembrane proteins located mainly in the endoplasmic reticulum (ER). STIM proteins contain an EF-hand in their N-termini that faces the lumen side of the ER allowing them to act as ER calcium (Ca(2+)) sensors. STIM1 has been recognized as central to the activation of the highly Ca(2+) selective store-operated Ca(2+) (SOC) entry current mediated by the Ca(2+) release-activated Ca(2+) (CRAC) channel; CRAC channels are formed by tetramers of the plasma membrane (PM) protein Orai1. Physiologically, the production of inositol 1,4,5-trisphosphate (IP(3)) upon stimulation of phospholipase C-coupled receptors and the subsequent emptying of IP(3)-sensitive ER Ca(2+) stores are sensed by STIM1 molecules which aggregate and move closer to the PM to interact physically with Orai1 channels and activate Ca(2+) entry. Orai1 has two homologous proteins encoded by separate genes, Orai2 and Orai3. Other modes of receptor-regulated Ca(2+) entry into cells are store-independent; for example, arachidonic acid activates a highly Ca(2+) selective store-independent channel formed by heteropentamers of Orai1 and Orai3 and regulated by the PM pool of STIM1. Here, I will discuss results pertaining to the roles of STIM and Orai proteins in smooth muscle Ca(2+) entry pathways and their role in vascular remodelling.

Background: Stent luminal surface characteristics influence surface endothelialization. We hypothesize that luminal stent microgrooves created in the direction of coronary flow accelerate endothelial cell migration, resulting in lower levels of neointimal formation., Methods and Results: Surface coverage efficiency was evaluated in vitro by allowing human aortic endothelial cells (HAEC) to migrate onto microgrooved (G) or smooth (NG) surfaces. HAEC functionality was assessed by proliferation rate, apoptosis rate, nitric oxide production, and inflammatory markers TNF-α and VCAM-1 expression. Early endothelialization and restenosis studies were performed using the porcine coronary injury model. Stainless steel stents of identical design with (GS) and without (NGS) luminal microgrooves were used. The commercially available Multi-Link Vision (MLVS) stent of identical design was used as a control. The degree of GS and NGS surface endothelialization was compared at 3 days. Biocompatibility and tissue response outcomes were evaluated at 28 days. The in vitro study demonstrated that at 7 days the presence of surface microgrooves increased HAEC migration distance >2-fold. Cell proliferation rate and nitric oxide production were increased and apoptosis rate was decreased. There was no difference in inflammatory marker expression. At 3 days, coronary artery stent endothelialization was significantly increased in GS compared with NGS (81.3% versus 67.5%, P=0.0002). At 28 days, GS exhibited lower neointimal thickness compared with either NGS (21.1%, P=0.011) or MLVS (40.8%, P=0.014)., Conclusion: Parallel microgrooves on coronary stent luminal surfaces promote endothelial cell migration and positively influence endothelial cell function, resulting in decreased neointimal formation in the porcine coronary injury model.