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Introduction: Balloon angioplasty is a commonly applied procedure for treating atherosclerotic vascular diseases. However, the maintenance of long-term lumen patency is relatively difficult due to the occurrence of restenosis. Previous research has shown that the occurrence of vascular wall inflammation is associated with higher rates of restenosis. Sophocarpine (SPC) can exert various therapeutic effects such as anti-oxidation, anti-inflammation, anti-tumor, antivirus and immune regulation. This study aimed to investigate whether SPC can alleviate intimal hyperplasia following balloon injury in a rat carotid artery model. Methods: Twenty Sprague–Dawley rats were randomly assigned to four groups: (i) control, (ii) balloon injury, (iii) balloon injury followed by saline injection, and (iv) balloon injury followed by SPC administration. Each group contained five rats. A high-pressure balloon of 3 mm × 20 mm was placed in the carotid artery. The balloon was inflated to a pressure of 8 atmospheres to carry out rat carotid artery balloon injury model. The areas of neointimal and media were determined by Verhoeff_Van Gieson staining, and the intima-to-media (I:M) ratios were subsequently evaluated. After that, the protein levels of IL-6, IL-1β, MCP-1, NF-κB, TNF-α, VCAM-1, ICAM-1 and eNOS were measured. Results: The ratio of I:M was remarkably higher in the balloon injury group than in the control group (p < 0.01). SPC could significantly decrease the ratio of I:M compared with the balloon injury group (p < 0.01). Besides, the protein levels of IL-6, IL-1β, MCP-1, NF-κB, TNF-α, ICAM-1 and VCAM-1 were increased in rat carotid arteries exposed to balloon injury (p < 0.01), and treatment with SPC could attenuate these effects (p < 0.05). Furthermore, balloon injury inhibited the protein expression of eNOS (p < 0.01), and SPC could elevate its level (p < 0.05). Conclusions: SPC could alleviate an intimal hyperplasia in balloon-injured carotid artery, and the mechanisms underlying this protective effect might be due to its inhibitory potency against inflammation signals. Our study also implies the potential applicability of SPC in treating restenosis after balloon angioplasty.

Introduction Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been shown to reduce the risk of cardiovascular events independently of glycemic control. The possibility that SGLT2 inhibitors improve endothelial regeneration and vascular restenosis is unknown. Purpose To examine whether dapagliflozin, a selective SGLT2 inhibitor, could prevent neointima thickening induced by balloon injury and, if so, to determine the underlying mechanisms. The effect of dapagliflozin was compared to that of losartan, an angiotensin type 1 receptor (AT1R) antagonist. Methods Saline, dapagliflozin (1.5 mg/kg/day), or losartan (30 mg/kg/day) were administered orally for 5 weeks to male Wistar rats. Balloon injury of the left carotid artery was performed 1 week after starting the treatment and sacrificed 4 weeks later. Vascular reactivity was assessed on left (injured) and right (healthy) carotid artery rings. The extent of neointima was assessed by histomorphometric analysis, changes of target factors by immunofluorescence, RT-qPCR and histochemistry. Results Dapagliflozin and losartan treatments reduced neointima thickening by 32% and 27%, respectively. Blunted contractile responses to phenylephrine and relaxations to acetylcholine and down-regulation of eNOS were observed in the injured artery. These effects were not modified by the dapagliflozin or the losartan treatments. RT-qPCR investigations indicated an increased in gene expression of inflammatory (IL-1beta, ITGAM, VCAM-1), oxidative (p47phox, p22phox) and fibrotic (TGF-beta1) markers and a decreased of eNOS in the injured carotid. However, these changes were not affected by the pharmacological treatments. By contrast, significant increased levels of AT1R angiotensin receptor and NTPDase1 (CD39) ectonucleotidase were observed in the restenotic carotid artery of the dapagliflozin group. Histochemical analysis evidenced important NTPDase1 activity in the neointima. Conclusions Dapagliflozin effectively reduced neointimal thickening. As the contribution of AT1R and P2Y2 ATP receptor in smooth muscle cell proliferation and neointima formation has been reported in the literature, the present data suggest that dapagliflozin prevents restenosis through interfering with angiotensin and/or extracellular nucleotides signaling. SGLT2 transporter represent potential new target for limiting vascular restenosis. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): This work was supported by AstraZeneca

Objectives This study aimed to preliminarily explore the effects of the soft tissue mobilization of pushing on Qiao-Gong (MPQ) on biomechanical properties of the carotid artery using an animal model of carotid atherosclerosis (CAS). Methods Fifty rabbits were randomly divided into 4 groups: animals with CAS treated with MPQ (CAS-MPQ [n = 15]); animals with CAS treated without MPQ (CAS [n = 15]); normal animals treated with MPQ (normal-MPQ [n = 10]); and a blank control group (n = 10). The MPQ procedure consisted of soft tissue mobilization of the Qiao-Gong acupoint on the front edge of the sternocleidomastoid muscle applied from top to bottom, by flat pushing with the thumb repeatedly for 20 times. Disease in the CAS models was induced by carotid artery balloon injury combined with a high-fat diet for 12 weeks. At the end of modeling, carotid color Doppler ultrasonography examination was performed to confirm which animal models were successfully induced with CAS, excluding model rabbits without typical CAS at the same time. Then, MPQ was applied on rabbits in the CAS-MPQ and the normal-MPQ groups for 3 weeks. By contrast, rabbits in the other 2 groups were fed normally without MPQ. Uniaxial failure tests were later performed on carotid arteries in all 4 groups, and at the end of the study, a 2-way factorial analysis of variance of the results was conducted. Results (1) At the end of modeling, 10 rabbits in the CAS-MPQ group and 9 in the CAS group were included with typical carotid atherosclerotic characteristics. (2) Young’s elastic modulus of the rabbit carotid artery increased more significantly in the CAS-MPQ group than the CAS group. (3) Compared with normal rabbit carotid arteries, atherosclerotic carotid arteries had lower levels of ultimate stress and ultimate strain but higher levels of ultimate load. Conclusions The uniaxial tensile mechanical properties of the rabbit atherosclerotic carotid artery were impaired after MPQ.

Introduction Detection of vulnerable plaques is critically important for the selection of appropriate treatment and/or the prevention of atherosclerosis and ensuing cardiovascular diseases. In order to clarify the utility of [ 11 C]acetate for atherosclerosis imaging, we determined the uptake and metabolism of acetate by in vitro studies using rabbit atherosclerotic arteries and [ 14 C]acetate. Methods Rabbits were fed with a conventional (n=5) or a 0.5% cholesterol diet (n=6). One side of the iliac–femoral arteries was injured by a balloon catheter. Radioactivity levels in the iliac–femoral arteries were measured after incubation in DMEM containing [1- 14 C]acetate for 60 min (% dpm/mg tissue). Radioactive components in the homogenized arteries were partitioned into aqueous, organic, and residue fractions by the Folch method, and analyzed by thin-layer chromatography (TLC). Results The radioactivity level in the injured arteries of rabbits fed with the 0.5% cholesterol diet (atherosclerotic arteries) was significantly higher than that in either the non-injured or injured arteries of rabbits fed with the conventional diet ( p Conclusions The level of [ 14 C]acetate-derived radioactivity into the arteries was increased by balloon injury and the burden of a cholesterol diet. Water-soluble metabolites were the dominant components with radioactivity in the atherosclerotic lesions. These results provide a biological basis for imaging atherosclerotic lesions by PET using [ 11 C]acetate.

Objective Chronic stress may lead to augmented incidence rates of coronary and cerebrovascular diseases associated with atherosclerosis. However, few studies have focused on the effect of chronic stress on atherosclerosis plaque formation. Therefore, this study was designed to directly evaluate how chronic stress affects atherosclerosis. Methods Thirty rabbits were divided into three groups: the control group, balloon-injury operation + high-fat diet model group, and chronic stress + balloon-injury operation + high-fat diet model group. Physical and social stress were induced, and proteomic methods were applied to identify specific markers. Results After protein determination, the chronic stress + balloon-injury operation + high-fat diet model group exhibited significant upregulation of the following apoptosis-related proteins: UBE2K, caspase 3, caspase 9, BAX, P53, and FAS. In particular, real-time polymerase chain reaction showed that the protein expression of caspase 9 was significantly downregulated in the stress group compared with the non-stress groups. However, the other proteins showed significantly increased expression in the stress group. Conclusion Chronic stress may promote cell apoptosis in the physiopathologic process of atherosclerosis.

Background. The safety of cervical rotatory manipulation (CRM) is still controversial, especially in patients with carotid artery atherosclerosis (CAS). The study aimed to investigate the effects of CRM on carotid plaques in vulnerability. Methods. 50 rabbits were randomly divided into four groups: model rabbits with CRM [CAS-CRM (n=15)]; model rabbits without CRM [CAS (n=15)]; normal rabbits with CRM [Normal-CRM (n=10)]; and Blank-control group (n=10). CAS disease models were induced by carotid artery balloon injury combined with a high-fat diet for 12 weeks. Then, CRM technique was performed in CAS-CRM and Normal-CRM groups for 3 weeks. In the end, determination of serum level of hs-CRP and Lp-PLA2, histological analysis under HE and Masson trichromic staining, and immunohistochemical analysis with CD34 and CD68 antibody were completed in order. Results. Carotid stenosis rates on successful model rabbits ranged from 70% to 98%. The CAS-CRM group had an increased level of hs-CRP (PP Conclusion. CRM therapy may increase the vulnerability of carotid plaque in rabbits with severe CAS.

Background Restenosis after percutaneous coronary intervention in coronary heart disease remains an unsolved problem. Clusterin (CLU) (or Apolipoprotein [Apo] J) levels have been reported to be elevated during the progression of postangioplasty restenosis and atherosclerosis. However, its role in neointimal hyperplasia is still controversial. Objective To elucidate the role Apo J in neointimal hyperplasia in a rat carotid artery model in vivo with or without rosuvastatin administration. Methods Male Wistar rats were randomly divided into three groups: the control group (n = 20), the model group (n = 20) and the statin intervention group (n = 32). The rats in the intervention group were given 10mg /kg dose of rosuvastatin. A 2F Fogarty catheter was introduced to induce vascular injury. Neointima formation was analyzed 1, 2, 3 and 4 weeks after balloon injury. The level of Apo J was measured by real-time PCR, immunohistochemistry and western blotting. Results Intimal/medial area ratio (intimal/medial, I/M) was increased after balloon-injury and reached the maximum value at 4weeks in the model group; I/M was slightly increased at 2 weeks and stopped increasing after rosuvastatin administration. The mRNA and protein levels of Apo J in carotid arteries were significantly upregulated after rosuvastatin administration as compared with the model group, and reached maximum values at 2 weeks, which was earlier than in the model group (3 weeks). Conclusion Apo J served as an acute phase reactant after balloon injury in rat carotid arteries. Rosuvastatin may reduce the neointima formation through up-regulation of Apo J. Our results suggest that Apo J exerts a protective role in the restenosis after balloon-injury in rats.

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

Recently, the evidence showed that interleukin-37 (IL-37) was expressed in the foam-like cells of atherosclerotic coronary and carotid artery plaques in IL-37-transgenic mice, suggesting that interleukin-37 is involved in atherosclerosis-related diseases. The purpose of this study was to determine the change of IL-37 in atherosclerotic plaque, the effect of atorvastatin on IL-37 and the association between IL-37 and Smad3 in atherosclerotic disease. Rabbits were subjected to atherosclerosis by the immunologic injury composite with balloon injury (BI). Some rabbits received atorvastatin treatment from 6 weeks to 12 weeks. Serum levels of IL-37 were assessed at baseline, 6 weeks and 12 weeks in normal, atherosclerotic and atorvastatin groups. Protein and RNA levels of IL-37 atherosclerotic plaque from abdominal aorta were processed at 12 weeks. Abdominal aorta including atherosclerotic plaque was immunostained with IL-37 and Smad3. Serum IL-37 significantly increased in atherosclerotic disease, and this increase could be reduced by the atorvastatin treatment. IL-37 and Smad3 were accumulated in the macrophage-derived foam cells in the plaque and significantly increased in protein and RNA levels. Atorvastatin treatment could significantly suppress the increase of both IL-37 and Smad3. Plasma level of IL-37 and the IL-37 expression of the plaque were significantly increased in atherosclerotic disease. This increase could be suppressed by the atorvastatin treatment. In addition, Smad3 might be required for IL-37 activity during the atherosclerotic physiologic process.

Aims The effects of hormone replacement therapy with estrogen on cardiovascular disease in postmenopausal women are still controversial. In the present study, we examined the effects of an endothelin (ET) receptor antagonist (ERA) and/or angiotensin receptor blocker (ARB) on neointimal formation following vascular injury in ovariectomized (OVX) female rats. Main methods Female rats were divided into intact female and OVX groups. The right carotid artery was subjected to balloon injury, and harvested 2 weeks later. Key findings In the intact female groups, treatment with ARB (L-158809; 1 mg/kg/day) for two weeks after the injury significantly decreased neointimal formation, whereas treatment with the ERA (J-104132; 10 mg/kg/day) did not affect neointimal formation. On the other hand, the ERA markedly decreased neointimal formation after the injury in the OVX groups; however, neointimal formation was not significantly improved by the ARB treatment. In addition, the combined treatment with 17β-estradiol (20 μg/kg/day) or the ERA and ARB markedly suppressed neointimal formation after the balloon injury in the OVX groups, whereas no combinational effects were observed due to the combined treatment with 17β-estradiol and the ERA. Significance These results suggest that ERAs have estrogen-like vasoprotective effects on neointimal formation following balloon injury in OVX rats. ERAs may be useful as an alternative therapy to prevent vascular disease in postmenopausal women.

Background Clinical studies have suggested a benefit for hyperbaric oxygen (HBO) treatment in decreasing symptomatic restenosis after coronary angioplasty. We hypothesize that HBO treatment will decrease hyperplastic intimal area after arterial balloon injury in a rat. Methods Fifty-four male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were randomly assigned either to room air (n = 27) or a treatment group (n = 27) receiving HBO treatment (2 hyperbaric treatments of 100% oxygen at 2 bars for 90 min). A 2F balloon-tipped catheter was used to injure the right common carotid arteries, which were harvested at 7, 14, and 28 days. Postinjury intimal thickness and area were measured from hematoxylin–eosin-stained specimens at each time point. Computer-assisted histomorphometry was used to calculate maximal intimal thickness, relative intimal thickness (ratio of intimal to intimal plus medial thickness), intimal area, and the intimal to medial area ratio. Results There was a 42.3% reduction in maximal intimal thickness (P = 0.0012) and a 36.5% reduction in intimal area (P = 0.0337) at day 28 in the HBO-treated group (mean ± standard error [SE], 0.0425 ± 0.0054 mm for maximal thickness and 0.065 ± 0.0056 mm2 for area) when compared to the normoxic group (0.0737 ± 0.004 mm for maximal thickness and 0.0413 ± 0.0074 mm2 for area). The relative intimal thickness also showed a 28.3% reduction at day 28 in the HBO-treated group (ratio of 0.38 ± 0.0329) compared to the normoxic group (ratio of 0.53 ± 0.0141; P = 0.0065). Conclusions Our results indicate that HBO treatment decreases maximal intimal thickness and intimal area of the carotid artery after balloon injury. This could have significant clinical implications on the increasing number of endovascular interventions in vascular surgery and cardiology.

Temporal consequences of neurocompensation to balloon injury on endothelinergic functionality in rat contralateral carotid were evaluated. Rats underwent balloon injury in left carotid and were treated with CP-96345 (NK1 antagonist). Concentration-response curves for endothelin-1 were obtained in contralateral (right) carotid at 2, 8, 16, 30, or 45 days after surgery in the absence or presence of BQ-123 (ETA antagonist), BQ-788 (ETB antagonist), or Tempol (superoxide-dismutase mimic). Endothelin-1-induced calcium mobilization was evaluated in functional assays carried out with BQ-123, BQ-788, or Tempol. Endothelin-1-induced NADPH oxidase-driven superoxide generation was measured by lucigenin chemiluminescence assays performed with BQ-123 or BQ-788. Endothelin-1-induced contraction was increased in contralateral carotid from the sixteenth day after surgery. This response was restored in CP-96345-treated rats. Endothelium removal or BQ-123 did not change endothelin-1-induced contraction in contralateral carotid. This response was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced calcium mobilization, which was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced lucigenin chemiluminescence, which was restored by BQ-788. We conclude that the NK1-mediated neurocompensatory response to balloon injury elicits a contractile hyperreactivity to endothelin-1 in rat contralateral carotid by enhancing the muscular ETB-mediated NADPH oxidase-driven generation of superoxide, which activates calcium channels.

Percutaneous transluminal angioplasty is frequently used in patients with severe arterial narrowing due to atherosclerosis. However, it induces severe arterial injury and an inflammatory response leading to restenosis. Here, we studied a potential activation of the endocannabinoid system and the effect of FA amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in arterial injury. We performed carotid balloon injury in atherosclerosis-prone apoE knockout (apoE(-/-)) and apoE(-/-)FAAH(-/-) mice. Anandamide levels were systemically elevated in apoE(-/-) mice after balloon injury. ApoE(-/-)FAAH(-/-) mice had significantly higher baseline anandamide levels and enhanced neointima formation compared with apoE(-/-) controls. The latter effect was inhibited by treatment with CB1 antagonist AM281. Similarly, apoE(-/-) mice treated with AM281 had reduced neointimal areas, reduced lesional vascular smooth-muscle cell (SMC) content, and proliferating cell counts. The lesional macrophage content was unchanged. In vitro proliferation rates were significantly reduced in CB1(-/-) SMCs or when treating apoE(-/-) or apoE(-/-)FAAH(-/-) SMCs with AM281. Macrophage in vitro adhesion and migration were marginally affected by CB1 deficiency. Reendothelialization was not inhibited by treatment with AM281. In conclusion, endogenous CB1 activation contributes to vascular SMC proliferation and neointima formation in response to arterial injury.

In animal models, increased tissue receptor for advanced glycation end products and its ligands, including N-epsilon-(carboxymethyl)lysine (CML), are critically implicated in postprocedural intimal hyperplasia after balloon injury. In patients undergoing percutaneous coronary interventions with stenting, we investigated whether plasma levels of CML and the soluble form of receptor for advanced glycation end products (sRAGE) changed during poststenting follow-up.We studied 81 patients with coronary artery disease who underwent successful percutaneous coronary interventions. Plasma levels of CML and sRAGE were measured before intervention, and at 1 day and 180 days of follow-up.CML levels increased significantly at day 1 after stenting and persisted at an elevated level at 180 days (P=0.013), whereas sRAGE levels increased significantly at 180 days (P=0.011). CML levels were significantly higher in multivessel-treated patients than in single-vessel-treated patients both at 1 day and 180 days of follow-up. In addition, CML values were positively associated with the extent of stent area at 1 day and 180 days of follow-up (r=0.278, P=0.022 and r=0.315, P=0.012, respectively). In logistic regression analysis, only the extent of stent area predicted adverse clinical events at 180-day follow-up (P=0.03, odds ratio=14.25, confidence interval=1.25-162.2).This study supports the hypothesis that increased circulating levels of CML occurred in the presence of vascular injury. This persistent rise of CML could amplify an inflammatory phenomenon triggered by stent placement and thus contributes to coronary artery disease progression.

Background Several animal models have facilitated the evaluation and pathological understanding of atherosclerosis, but a definitive animal model of coronary atherosclerosis is not available. We therefore developed low density lipoprotein receptor knockout (LDLR-KO) pigs with hypercholesterolemia, a model which rapidly developed coronary atherosclerosis following balloon injury. Methods and Results We deleted LDLR exon regions from cultured porcine fetal fibroblasts and cloned LDLR knockout (LDLR-KO) embryos microinjecting fetal fibroblast nuclei into enucleated oocytes. Twelve LDLR-KO pigs were fed a 2.0% cholesterol and 20% fat diet. Baseline serum LDL cholesterol level was 510.0±86.1 mg/dL. Balloon injury was created in 46 coronary segments and necropsy were obtained 2, 4, 8 and 12 weeks later. Coronary artery sections were reviewed to evaluate lesion progression. We found lipid accumulation with foam cells and inflammatory cells beginning four weeks after balloon injury. The mean ratio of macrophages to plaque area was significantly higher in the four- weeks and eight-week animals compared with those at 2-weeks (8.79% ± 5.98% and 17.00% ± 10.38% vs. 1.14% ± 1.88%, P < 0.0001). At 12 weeks the ratio decreased toward the level at 2 week level (4.00% ± 4.56%, P = 0.66 vs. baseline). Advanced coronary atherosclerotic lesions contained lipid pools at eight-weeks with fibrous components beginning at 12 weeks. Conclusions We developed a model of rapid coronary atherosclerosis using LDLR KO pigs with balloon injury. This model may be useful for preclinical evaluation of medication or devices, and may also help investigate mechanisms of plaque progression.

Background. Transplant arteriosclerosis is a leading cause of chronic transplant dysfunction and is characterized by occlusive neointima formation in intragraft arteries. Development of transplant arteriosclerosis is refractory to conventional immunosuppressive drugs and adequate therapy is not available. In this study, we determined the efficacy of the synthetic peroxisome proliferator-activated receptor (PPAR)-gamma agonist rosiglitazone to attenuate the development of transplant arteriosclerosis in rat aortic allografts.Methods. Lewis aortic allografts were transplanted into Brown Norway recipient rats. Recipient rats received either similar to 5 mg rosiglitazone/day (starting 1 week before transplantation until the end of the experiment) or were left untreated. Transplant arteriosclerosis was quantified using morphometric analysis. Alloreactivity was measured in vitro using mixed lymphocyte reactions. Regulatory T cell frequency and function were analyzed using flow cytometry and in vitro suppression assays, respectively. Intragraft gene expression was analyzed using real-time polymerase chain reaction. Finally, medial and neointimal vascular smooth muscle cell proliferation was analyzed in vitro.Results. Rosiglitazone significantly reduced transplant arteriosclerosis development 8 weeks after transplantation (P Conclusion. PPAR gamma agonists may offer a new therapeutic strategy in clinical transplantation to attenuate the development of transplant arteriosclerosis and thereby chronic transplant dysfunction.

BACKGROUND Restenosis after revascularization procedures is accelerated in persons with type 2 diabetes. AIM The current study tested the hypothesis that the neointimal response to endovascular injury is enhanced in female obese Zucker (OZ) rats, a model of type 2 diabetes. METHODS Animals were randomized to receive either a standard diet (SD) or a diabetogenic diet (DD) for 6 weeks. Four weeks later, balloon injury of the right common carotid artery was induced. All rats were euthanized 2 weeks after injury. Lean Zucker (LZ) rats served as controls. RESULTS At the time of death, plasma glucose was elevated in OZ rats fed a SD (208 +/- 13 mg/dl) and a DD (288 +/- 21 mg/dl) compared to corresponding LZ rats (SD: 153 +/- 8; DD: 132 +/- 7 mg/dl). The ratio of high-density lipoprotein cholesterol (HDLc) to total cholesterol (Totc), an index of atherogenicity, was reduced in OZ rats on both diets (SD: 0.77 +/- 0.06; DD: 0.80 +/- 0.09) compared to LZ controls (SD: 1.11 +/- 0.02; DD: 1.20 +/- 0.05). Histomorphometric analysis of injured arteries showed that the intima to media (I : M) ratio was significantly increased in OZ (1.37 +/- 0.07) compared to LZ (0.79 +/- 0.08) rats. Elevations in plasma glucose and triglycerides (Tg) correlated positively and decreases in HDLc negatively with an increased I : M ratio. Administration of the DD did not further enhance the I : M ratio in LZ (0.87 +/- 0.06) or OZ (1.29 +/- 0.09) rats. CONCLUSIONS These results suggest that neointima formation following endoluminal injury of the carotid artery is enhanced at an early stage in the development of diabetes mellitus.

The aim of this study was to validate in vivo measurement of intravascular ultrasound (IVUS) for the analysis of structural and functional vessel wall alterations in a chronic animal model. Furthermore, the relation between functional and structural alteration of the vessel wall should be investigated. Fifteen cholesterol-fed rabbits (1%) and 15 control rabbits underwent balloon injury of the abdominal aorta. Immediately before and after balloon traumatization as well as 2 and 6 weeks later IVUS depiction of 10 aortal vessel segments was performed (n = 1100 measurements). In vivo IVUS measurements and morphometric analysis of the neointimal area of same aortal segments showed a high correlation (n = 148, r = 0.844, p < 0.001). Plaque area determined by morphometry revealed larger areas than the evaluation by IVUS (0.162 ± 0.138 vs. 0.130 ± 0.126 mm2, p < 0.001). Before balloon traumatization, pulsatility of the aortal vessel segments was less in cholesterol-fed rabbits (0.067 vs. 0.090, p < 0.01) and neointimal index higher (0.003 vs. 0). Investigation using IVUS 2 and 6 weeks after balloon traumatization demonstrated a continuous loss of arterial distensibility and an increase of neointimal index, being more pronounced in the cholesterol-fed group. As demonstrated by IVUS the loss of distensibility preceded the atherosclerotic alterations. Our investigation suggests using IVUS in this animal model is a reliable setting for long-term investigation of characteristics of the vessel wall. We could demonstrate that altered function of the vessel wall precedes the structural atherosclerotic vessel wall alterations.

Percutaneous coronary intervention [PCI or percutaneous transluminal coronary angioplasty (PTCA)] has been developed into a mature interventional treatment for atherosclerotic cardiovascular disease. However, the long-term therapeutic effect is compromised by the high incidence of vascular restenosis following angioplasty, and the underlying mechanisms of vascular restenosis have not yet been fully elucidated. In the present study, we investigated the role of the gap junction (GJ) protein, connexin 43 (Cx43), in the development of vascular restenosis. To establish vascular restenosis, rat carotid arteries were subjected to balloon angioplasty injury. At 0, 7, 14 and 2 days following balloon injury, the arteries were removed, and the intimal/medial area of the vessels was measured to evaluate the degree of restenosis. We found that the intimal area gradually increased following balloon injury. Intimal hyperplasia and restenosis were particularly evident at 14 and 28 days after injury. In addition, the mRNA and protein expression of Cx43 was temporarily decreased at 7 days, and subsequently increased at 14 and 28 days following balloon injury, as shown by RT-PCR and western blot analysis. To determine the involvement of Cx43 in vascular restenosis, the lentivirus vector expressing shRNA targeting Cx43, Cx43-RNAi-LV, was used to silence Cx43 in the rat carotid arteries. The knockdown of Cx43 effectively attenuated the development of intimal hyperplasia and vascular restenosis following balloon injury. Thus, our data indicate the vital role of the GJ protein, Cx43, in the development of vascular restenosis, and provide new insight into the pathogenesis of vascular reste-nosis. Cx43 may prove to be a novel potential pharmacological target for the prevention of vascular restenosis following PCI.

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models – both resulting in artery stenosis but each underlying a different pathway of development – are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.

Here we aimed to investigate the effects of atorvastatin on accelerated reendothelialization after carotid balloon injury. A mouse model of carotid arterial injury was established, followed by intragastric administration of atorvastatin at a dose of 0.6 mg·(kg body mass)–1·d–1. Pathological sections of carotid artery stained with hematoxylin and eosin were observed under light microscopy. Expression levels of eNOS mRNA and protein were detected with real-time quantitative PCR and Western blot analysis, respectively. Proliferation and differentiation of endothelial progenitor cells (EPCs) were observed after treatment, in vitro. Reendothelialization appeared on the neovascular surface, while intimal hyperplasia was inhibited after treatment with atorvastatin. Numbers of CD31-positive cells increased after atorvastatin treatment, as did the number of leucocyte antigen positive cells. The expression of cell markers, such as CD34, eNOS, and VEGF-R, were higher in the atorvastatin-treated group of mononuclear cells. EPC numbers increased with the concentration of atorvastatin. The expression of eNOS mRNA was reduced in the mice with carotid artery injury that were treated with normal saline. The expression levels of eNOS protein were increased in atorvastatin treatment group. In conclusion, atorvastatin stimulates EPCs to differentiate into endothelial cells and promotes the repair of carotid arterial injury.

After percutaneous transluminal coronary angioplasty (PTCA), 30–50% of the patients may present with restenosis within 6 months. The aim of this study was to search for a preventive remedy against the balloon injury-induced neointima formation. Ginseng, with its wide indications on immune and cardiovascular functions, has prompted us to explore its role in neointima formation. In the present study, we aimed to explore if a standardized Panax Ginseng extract G115 was able to inhibit neointimal formation. With BrdU luminencence assay, maximal proliferation of rat smooth muscle cells was reduced to 24% of control values by G115. Norepinephrine-induced vasocontraction was antagonized in 21% and 44% by 1.44mg/ml and 2.88mg/ml of G115, respectively. Neointima-to-lumen area ratio of balloon-injured rat carotid arteries was reduced 77.3% by G115 as compared to the sham control. These results demonstrate the preventive effects of ginsenosides on angioplasty-mediated neointima formation.

BACKGROUND Intracoronary radiation has shown the potential to reduce formation of neointima in porcine models of restenosis. OBJECTIVE To investigate the feasibility, safety, and efficacy of a new 'deployable-balloon' device with radioisotope 32P integrated into the balloon material. METHODS Ten swine were subjected to balloon-overstretching injury in 20 coronary arteries and were randomly allocated to receive a radiation dose of 0, 15, or 20Gy prescribed to 1 mm from the surface of the radioactive balloon material. The animals were killed 4 weeks after the procedure. Their coronaries were perfusion fixed and stained. Vessel parameters (area of intima and length of fracture) and area of thrombus area were analyzed by computer-aided histomorphometry. RESULTS Radiation treatment with the new 32P balloon device was performed without complications. The lengts of fracture for the three groups were similar (NS). Formation of neointima after balloon injury was less in members of the radiated groups than it was in controls (area of intima/length of fracture was 0.70 +/-0.12 mm for controls, 0.08+/-0.13mm with 15 Gy, and 0.07+/-0.17 mm with 20 Gy; P < 0.001). Vessels treated with 20 Gy had a greater total area of thrombus (0.00+/-0.00 versus 0.51+/-0.98 mm2, P< 0.01). CONCLUSIONS Intracoronary radiation therapy using a new 32P deployable-balloon system is feasible and safe. A radiation dose of 15 Gy was sufficient to inhibit neointimal response in the porcine coronary-balloon-injury model.

Intimal thickening and formation of extracellular matrix are parts of the repair process after vascular injury. Similar processes occur after coronary angioplasty. Prior studies have shown that losartan inhibits intimal thickening in rat carotid arteries following balloon injury. However, the effects of losartan in reducing the collagen content of arteries after balloon injury have not been examined. The objectives of this study were to determine the change in collagen content after balloon injury and to analyse the mechanisms of reduction of collagen content and intimal thickening. Losartan (15 mg/kg/d) was administered orally from six days before to eight weeks after balloon injury in rabbits. Collagen content was measured histologically by the use of circularly polarised images of picrosirius red-stained sections. Collagen content in arterial intima was found to be significantly lower in the losartan-treated group (n=12) than in the control group (n=12) (21.6%±5.2% vs. 43.8%±7.6%, p 2 vs. 0.52±0.29 mm2, 0.55±0.21 vs. 0.97±0.25, respectively, p

Neointimal formation in response to arterial injury is a major contributing element in restenosis after coronary balloon angioplasty and stenting. Endovascular irradiation has been reported to be effective in reducing restenosis. The purpose of this study was to investigate the effect of beta-emitting holmium-166 for the inhibition of neointimal formation in porcine coronary artery. A total of 34 pigs weighing 25 to 30 kg underwent oversized balloon injury (balloon/artery ratio, 1.3:1.4) at the proximal portion of the left anterior descending and circumflex arteries. One artery was randomly assigned to receive radiation after injury. Ho-166 was left in the balloon within the delivery catheter for a period sufficient to deliver 9 Gy and 18 Gy to a depth of 1 mm from the surface of the balloon. Four weeks later, pigs were sacrificed and hearts were perfusion-fixed, followed by histopathologic analysis and planimetry for measurement of maximal intimal thickness, intimal area, and fracture length. The coronary segment of the pigs in the control group had neointimal area of 1.18 ± 0.55 mm2; the pigs in the 9-Gy group had neointimal area of 0.68 ± 0.40 mm2 (P >.05 vs control); and the pigs in the 18-Gy group had neointimal area of 0.29 ± 0.12 mm2 (P >.01 vs control). The maximal intimal thickness in the 18-Gy group (0.14 ± 0.11 mm) was significantly reduced compared with the maximal intimal thickness in the control group (0.48 ± 0.13 mm) (P > .01). Intracoronary radiation with liquid Ho-166 contained in a perfusion balloon catheter is feasible and effective in reducing neointimal formation after coronary overstretch injury in pigs. Therefore intracoronary irradiation on the injured segment may further reduce restenosis after balloon injury.

The aim of this study was to determine whether a single local probucol administration could suppress neointimal formation in balloon-injured rat carotid artery. Rats were divided into four groups; (i) rats receiving no probucol (control group); (ii) rats receiving topical application of 50 mg probucol at the time of ballooning (p-top group); (iii) rats fed chow containing 1% probucol (p-fed group; (iv) rats receiving both topical application of 50 mg probucol and chow containing 1% probucol (p-top/fed group). Although serum total cholesterol levels did not significantly differ between the control and the p-top groups at two weeks after balloon injury, the p-top group had a smaller intimal/medial ratio or intimal area than the control group (0.97 +/- 0.11 vs. 0.53 +/- 0.08 or 0.15 +/- 0.02 mm2 vs. 0.08 +/- 0.02 mm2 respectively, P0.01). Neointimal formation was suppressed to a similar extent in the p-fed group, in which serum total cholesterol level was approximately 40% lower than that in the control group. Thus, a single local delivery of probucol can suppress neointimal formation in balloon-injured rat carotid artery without altering serum lipid level.

Summary. Background: IGF-1 is a potent mitogen for vascular smooth muscle cells, but exerts protective effects on endothelial cells that may trigger antiatherogenic mechanisms. Objectives: This study was designed to test the hypothesis that an IGF-1 excess following arterial injury prevents neointima formation and vascular stenosis. Methods: Rats were subjected to carotid balloon injury and treated with IGF-1 (1.2 mg kg−1 per die) or saline for 10 days. Results: In IGF-1 treated animals, high tissue levels of eNOS, Akt and its phosphorylated form were found, confirming activation of IGF-1-dependent signaling pathways. IGF-1 markedly reduced neointima formation and post-injury arterial stenosis. IGF-1 exerted proliferative and anti-apoptotic effects in the media of injured carotids, but inhibited mitotic activity and induced apoptosis in the neointima. Furthermore, IGF-1 stimulated mobilization of progenitor endothelial cells and re-endothelialization of the injured arteries. L-NAME administration inhibited IGF-1 vasculoprotective effects. Conclusions: IGF-1 attenuates post-injury carotid stenosis by exerting differential effects in the neointima and tunica media with regard to the key components of the response to injury. The data point to a novel role of IGF-1 as a potent vasculoprotective factor.

Present experiments were designed to investigate the effects of ovariectomy (OVX) and estrogen replacement (ER) on neointimal formation after balloon injury of the rat carotid artery. Young adult female rats were divided into 3 groups of sham operation (control), ovariectomy, and ovariectomy plus estrogen replacement. Estrogen replacement was initiated by implanting a sustained release pellet containing water-soluble 17beta-estradiol 1 week after the ovariectomy. Carotid arteries were harvested 2 weeks after the balloon injury for determinations. The balloon injury caused intimal hyperplasia, which was accompanied by the impaired endothelium-dependent relaxation and cyclic GMP production, and accumulation of asymmetric dimethylarginine (ADMA) as an endogenous NOS inhibitor. Bilateral ovariectomy accelerated the intimal hyperplasia. The acceleration was accompanied by the enhanced impairment of NO production, attenuated reendothelialization, and enhanced accumulation of ADMA. The estrogen replacement improved the accelerated intimal hyperplasia with concomitant improvement of the impaired NO production and accumulated asymmetric dimethylarginine, and facilitated reendothelialization. These results suggests that the enhanced impairment of NO production, which possibly results from the accumulated asymmetric dimethylarginine and lack of reendothelialization, may contribute to the acceleration of intimal hyperplasia by ovariectomy and that estrogen replacement effectively improves the intimal hyperplasia by restoring the impaired NO production through reducing endogenous NOS inhibitor and facilitating reendothelialization.

Background: Arterial intimal hyperplasia and following restenosis may be inhibited by estrogens. We investigated the effect of a synthetic steroid hormone, Tibolon: (a) on intima hyperplasia and restenosis in vivo, and (b) on production of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), endothelial cell proliferation and apoptosis in vitro. Methods: Influence of Tibolon treatment (0.1 mg/kg body weight, during 3 days before and 3 weeks after the operation as a drinking solution once daily) on neointimal formation (measured by morphometry) and arterial wall damage (by qualitative histology) were investigated in vivo using an animal model of balloon injury of carotid artery. In human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1), the effect of Tibolon (0.1 µg/ml) on eNOS and VEGF was assessed by ELISA. Cell proliferation was induced by VEGF165 and measured by BrdU incorporation assay, cell apoptosis was detected colorimetrically measuring DNA fragmentation. Results: Balloon injury resulted in neointima formation and prominent damage of the carotid artery wall. Treatment with Tibolon increased luminal area, decreased intimal area and intima to media ratio, and promoted better reparation of damaged vessel wall. In vitro, Tibolon treatment did not influence the expression of eNOS protein in HUVEC as well as cell proliferation rate but reduced apoptosis of endothelial cells by about 40%. Additionally, this treatment suppressed basal and IL-1β-stimulated synthesis of VEGF in HMEC-1. Conclusions: Tibolon treatment suppressed neointimal formation and promoted better reparation of damaged vessel wall in carotid artery after balloon injury. This positive effect seems to be associated with improved endothelial cell survival resulting possibly in increased NO production. It might be also related to the decrease of VEGF generation.

We investigated the effects of SK-1080, a novel angiotensin AT1 receptor antagonist, on neointimal proliferation in the rat carotid artery after balloon injury, together with its effects on the impaired endothelium-dependent vascular relaxation. SK-1080 (0.3 and 1.0 mg/kg/day) was orally administered in balloon-injured rats for 21 days (from 6 days before to 14 days after balloon injury). SK-1080 (1 mg/kg) exerted significant effects on three important parameters associated with the intimal thickening induced by balloon injury (50.0% reduction in neointimal area, 42.7% reduction in stenosis ratio and 69.1% increase in lumen/total area ratio). Acetylcholine-induced relaxation was significantly reduced in the balloon-injured carotid arteries (64.0 ± 9.1%), and this impairment of acetylcholine-induced relaxation was significantly restored by SK-1080 (maximal relaxation: 87.1 ± 6.5 and 88.6 ± 1.9% at 0.3 and 1.0 mg/kg, respectively, p < 0.05). However, the endothelial-independent, sodium nitroprusside-induced relaxation was clearly demonstrated and did not differ in carotid arteries from all treatment groups. Furthermore, acetylcholine-induced relaxation was completely inhibited by L-NAME but not by indomethacin. SK-1080 caused a slight hypotension 1 day before balloon injury (8.7%), which gradually returned to the baseline 6 and 13 days after balloon injury. These results suggest that SK-1080 may have therapeutic potential for the treatment of vascular diseases such as restenosis and atherosclerosis.

To evaluate the effect of hypercholesterolemia on apoptosis and proliferation after vascular injury, iliac arteries of hypercholesterolemic (HC) and normocholesterolemic (NC) rabbits were examined after balloon injury using TUNEL, immunohistochemical staining of PCNA, macrophages, smooth muscle actin and p53. In media, apoptosis occurred massively early after injury and then decreased. HC did not affect this early post-injury apoptosis but significantly increased apoptosis 14 days later (D14). Immediate apoptosis in media was followed by active proliferation. HC sustained a high activity of proliferation until D14. The changes of immunoreactivity to p53 over the same 14 day period parallel that of apoptosis. In intima, where cells were scarce initially, proliferative activity reached a peak at D7 and then decreased. HC significantly enhanced proliferation at D14. In intima proliferation was accompanied by a later low-level apoptosis. HC significantly enhanced this low-level apoptosis at D14. These effects of HC resulted in significantly increased areas of intima and media. The fundamental difference between HC and NC was the infiltration of macrophages in HC. In conclusion, balloon injury induces early massive p53-associated apoptosis followed by proliferation in media, whereas in intima, it induces active proliferation followed by a low-level apoptosis. Hypercholesterolemia does not affect the early post-injury apoptosis but enhances proliferation and low-level apoptosis at a later stage, which in turn results in intimal and medial hyperplasia.

Experimental models have brought an impressive wealth of data demonstrating the protective effects of various antioxidants. Comparison among models is difficult, since there are major variations regarding types of model, dose and timing of antioxidant, and primary objectives to assess response to injury.

The phenomenon of restenosis following vascular interventional procedures continues to be a major obstacle to the long-term success of these procedures. Worldwide, thousands of coronary interventions are performed yearly, including percutaneous transluminal coronary angioplasty (PTCA), coronary stenting, as well as directional and rotational atherectomy. Although these procedures are generally equivalent in their acute success rate, variable amounts of clinical restenosis are noted, ranging from a low of 20% for intracoronary stenting to as high as 60% for atherectomy (1–2, 15). The same phenomenon in different form plagues the long-term success of whole organ transplantation.