Neointimal Cell Proliferation Research Articles

Neointimal hyperplasia after endoluminal injury in mice is dependent on tissue factor- and angiopoietin-2 dependent interferon gamma production by fibrocytes and macrophages.

The intimal hyperplasia (IH) and vascular remodelling that follows endovascular injury, for instance after post-angioplasty re-stenosis, results in downstream ischaemia and progressive end organ damage. Interferon gamma (IFNγ) is known to play a critical role in this process. In mouse models we have previously shown that fibrocytes expressing tissue factor (TF) are recruited early to the site of injury. Through thrombin generation and protease activated receptor-1 (PAR-1) activation, fibrocytes secrete angiopoietin-2, stimulate neointimal cell proliferation, inhibit apoptosis and induce CXCL-12 production, all of which contribute to the progressive IH that then develops. In this study we investigated the relationship between TF, angiopoietin-2 and IFNγ. IH developing in carotid arteries of wild-type mice 4 weeks after endoluminal injury contained a significant proportion of IFNγ+ fibrocytes and macrophages, which we show, using a previously defined adoptive transfer model, were derived from circulating CD34+ cells. IH did not develop after injury in IFNγ-deficient mice, except after transplantation of WT bone marrow or adoptive transfer of WT CD34+ cells. In vitro, CD34+ cells isolated from post-injury mice did not express IFNγ, but this was induced when provided with FVIIa and FX, and enhanced when prothrombin was also provided: In both cases IFNγ secretion was TF-dependent and mediated mainly through protease activated PAR-1. IFNγ was predominantly expressed by fibrocytes. In vivo, all IFNγ+ neointimal cells in WT mice co-expressed angiopoietin-2, as did the small numbers of neointimal cells recruited in IFNγ-/- mice. Adoptively transferred WT CD34+ cells treated with either an anti-TIE-2 antibody, or with siRNA against angiopoetin-2 inhibited the expression of IFNγ and the development of IH. TF-dependent angiopoietin-2 production by newly recruited fibrocytes, and to a lesser extent macrophages, switches on IFNγ expression, and this is necessary for the IH to develop. These novel findings enhance our understanding of the pathophysiology of IH and expose potential targets for therapeutic intervention.

A Novel Method of End-to-Side Microvascular Anastomosis Using T-Shaped Metal Stents: A Porcine Study.

End-to-side anastomosis requires highly specialized techniques. An easy end-to-side anastomosis technique enables anastomosis of vessels with different diameters and under various situations. We invented T-shaped metal stents and evaluated novel methods of end-to-side sutureless anastomosis, confirming their safety, effectiveness, and operability. We performed 8 end-to-side sutureless anastomoses in 4 7- to 11-month-old, male Mexican hairless piglets. After induction of anesthesia, the left femoral artery was resected by approximately 8 cm, and the superior and posterior stumps of the resected femoral artery underwent an end-to-side anastomosis with the right femoral artery by the placement of the metal stents with subsequent use of adhesive for the circumferential area. The patency of blood vessels and the presence of thrombosis were evaluated by ultrasonography or contrast-enhanced computed tomography and histology 4 weeks postoperatively. All the animals survived the procedure; no thrombosis was identified in any of the 8 anastomosis sites according to imaging studies performed 4 weeks postoperatively. Histological examination confirmed the probe patency of blood vessels and neointimal cell proliferation around stent branches. End-to-side anastomosis is possible with T-shaped metal stents. In the future, we aim for the practical application of these stents by improving their operability.

Periadventitial Delivery of Simvastatin-Loaded Microparticles Attenuate Venous Neointimal Hyperplasia Associated With Arteriovenous Fistula.

BackgroundVenous neointimal hyperplasia and venous stenosis (VS) formation can result in a decrease in arteriovenous fistula (AVF) patency in patients with end‐stage renal disease. There are limited therapies that prevent VNH/VS. Systemic delivery of simvastatin has been shown to reduce VNH/VS but local delivery may help decrease the side effects associated with statin use. We determined if microparticles (MP) composed of cyclodextrins loaded with simvastatin (MP‐SV) could reduce VS/VNH using a murine arteriovenous fistula model with chronic kidney disease.Methods and ResultsMale C57BL/6J mice underwent nephrectomy to induce chronic kidney disease. Four weeks later, an arteriovenous fistula was placed and animals were randomized to 3 groups: 20 μL of PBS or 20 μL of PBS with 16.6 mg/mL of either MP or MP‐SV. Animals were euthanized 3 days later and the outflow veins were harvested for quantitative reverse transcriptase–polymerase chain reaction analysis and 28 days later for immunohistochemistical staining with morphometric analysis. Doppler ultrasound was performed weekly. Gene expression of vascular endothelial growth factor‐A (Vegf‐A), matrix metalloproteinase‐9 (Mmp‐9), transforming growth factor beta 1 (Tgf‐β1), and monocyte chemoattractant protein‐1 (Mcp‐1) were significantly decreased in MP‐SV treated vessels compared with controls. There was a significant decrease in the neointimal area, cell proliferation, inflammation, and fibrosis, with an increase in apoptosis and peak velocity in MP‐SV treated outflow veins. MP‐SV treated fibroblasts when exposed to hypoxic injury had decreased gene expression of Vegf‐A and Mmp‐9.ConclusionsIn experimental arteriovenous fistulas, periadventitial delivery of MP‐SV decreased gene expression of Vegf‐A, Mmp‐9, Tgf‐β1 and Mcp‐1, VNH/VS, inflammation, and fibrosis.

Effect of bivalirudin on neointimal hyperplasia and endothelial proliferation in rabbit.

The aim of this study was to investigate the effects of bivalirudin on endothelial cell proliferation and neointimal hyperplasia in a rabbit carotid artery model. "New Zealand rabbits (n = 12)" weighing 2-3kg were randomly divided into two groups. Arteriotomy was performed to the rabbit carotid artery and closed with continuous suture technique. Group B (n = 6) as a control group received 150U/kg heparin sodium; however, group A (n = 6) was given 0.75mg/kg bivalirudin i.v. bolus and infusion 1.75mg/kg/hour (B01AE06-Bivalirudin 250mg) during perioperation period. At the end of the 28th day, the carotid artery segment was excised and evaluated histologically. All histological and immune staining analyzes were performed by two blind researchers in the treatment of rabbits. In the control group rabbit carotid artery sections, tunica intima was observed to thicken. In the bivalirudin group, intimal hyperplasia was less observed compared to the control group. No significant difference was observed between groups in tunica media thickness. Lumen diameter and lumen area were found to be wider in the experimental group. P value was found to be less than 0.05. Our study demonstrates that bivalirudin significantly affects and prevents neointimal hyperplasia and endothelial cell proliferation.

Medial artery calcification increases neointimal hyperplasia after balloon injury

Arterial calcification predicts accelerated restenosis after angioplasty and stenting. We studied the effects of calcification on neointimal hyperplasia after balloon injury in the rat carotid. Arterial calcification was induced by subcutaneous injection of vitamin D3 or by adventitial application of calcium chloride. After balloon catheter injury, neointimal hyperplasia was significantly increased in rats with medial calcification compared with controls. Neointimal cell proliferation in calcified arteries as assessed by proliferating cell nuclear antigen (PCNA) staining was also higher. In calcified arteries, bone morphogenetic protein 2 (BMP-2)levels were increased at the time of injury suggesting a possible explanation for the altered responses. In vascular smooth muscle cells (SMCs) grown under calcifying conditions , stimulation with BMP-2 significantly increased cell proliferation, however, this did not occur in those grown under non-calcifying conditions. These data suggest that neointimal hyperplasia is accelerated in calcified arteries and that this may be due in part to increased BMP-2 expression in medial SMCs. Treatments aimed at inhibiting restenosis in calcified arteries may differ from those that work in uncalcified vessels.

Sphigosine-1-phosphate receptor 1 promotes neointimal hyperplasia in a mouse model of carotid artery injury

Vascular remodeling, resulting from proliferation and migration of vascular smooth muscle cells (VSMCs), is a major cause of atherosclerosis and restenosis. The lysophospholipid mediator sphingosine-1-phosphate (S1P) regulates proliferation and migration of VSMCs via S1P-specific G protein-coupled receptors, including S1P receptor 1 (S1PR1) to S1PR3. However, the role of S1PR1 in vascular remodeling is not well understood. Therefore, in this study, we aimed to investigate the effect of S1PR1 on neointimal hyperplasia in a carotid artery ligation mouse model using transgenic C57Bl/6 mice that overexpressed S1PR1 (Tg-S1PR1) under the control of α-smooth muscle actin promoter. We found that S1PR1 expression in carotid artery was upregulated after carotid artery ligation in non-transgenic (nTg) mice. Tg-S1PR1 mice showed enhanced ligation-induced neointimal hyperplasia with increased neointimal cell proliferation, compared with control nTg mice. VSMCs isolated from Tg-S1PR1 mice showed enhanced proliferation and migration in response to S1P stimulation. VSMCs from Tg-S1PR1 mice showed greater expression of interleukin-6 (IL-6) compared with nTg mouse-derived VSMCs, and administration of IL-6-neutralizing antibody into Tg-S1PR1 mice suppressed neointimal hyperplasia. These results suggest that S1P-S1PR1 signaling plays an important role in neointimal hyperplasia after vascular injury via IL-6 production.

Vasoprotective Effects of Vildagliptin in Mice Is Masked by Metformin—Overlapping Mechanisms Implied

Preclinical and small clinical studies have demonstrated the direct cardiovascular protective effects of dipeptidyl peptidase 4 inhibitors (DPP-4Is). However, cardiovascular benefits have not been proven in large clinical trials that utilized multiple antidiabetic agents. We hypothesized that the cardiovascular protective effects of the first-line antidiabetic agent metformin (Met) affects those of DPP-4Is, so we evaluated the vasoprotective effects of vildagliptin (Vil) in combination with Met in mice. Methods: We randomly assigned C57BL6 and diabetic db/db mice (8 weeks old) to treatment with vehicle, Vil (3 mg/kg/d), Met (400 mg/kg/d), or the combination of Vil and Met. The mice were subjected to femoral artery wire injury to induce arterial remodeling. Four weeks after the injury, the femoral arteries were collected. Human umbilical vein endothelial cells (HUVECs) were used in the in vitro experiments. Results: In wild type mice, the biological parameters were similar among the groups. Both Vil and Met treatment reduced neointimal area and vascular cell proliferation to a similar extent (40% reduction) compared to that of the vehicle, and combining the two drugs did not further reduce those values. Similarly, both Vil and Met treatment reduced neointimal area in db/db mice (50% reduction) without affecting biological parameters, but no additive effect was found. In wild type mice, cotreatment with the nitric oxide (NO) synthase inhibitor completely abolished the vascular effects of both Vil and Met, suggesting that Vil and Met had common mechanisms that are dependent on endothelial NO. In HUVECs, Vil did not directly affect NO production. In contrast, glucagon like peptide (GLP)-1 did stimulate NO production. Met also stimulated NO production. However, the combination of GLP-1 and Met did not increase NO production. Conclusions: Metformin masks the vasoprotective effects of vildagliptin, possibly because of overlapping mechanisms that are dependent on endothelial NO. Disclosure H. Kushima: None. Y. Mori: None. M. Koshibu: None. M. Hiromura: None. K. Kohashi: None. M. Terasaki: None. T. Hirano: Speaker's Bureau; Self; Novo Nordisk Inc., AstraZeneca.

Sustained Thromboresistant Bioactivity with Reduced Intimal Hyperplasia of Heparin-Bonded Polytetrafluoroethylene Propaten Graft in a Chronic Canine Femoral Artery Bypass Model

Bypass graft thrombosis remains a significant mode of failure in prosthetic graft revascularization. The purpose of this investigation was to evaluate the long-term thromboresistant effect of heparin-bonded expanded polytetrafluoroethylene (ePTFE) graft using Carmeda BioActive Surface technology in a canine model. Bilateral femorofemoral artery bypass grafts with ePTFE grafts were performed in 25 adult grayhound dogs. In each animal, a heparin-bonded ePTFE graft (Propaten, WL Gore) was placed on one side, whereas a control nonheparin graft was placed on the contralateral side. The graft patency was assessed at 1, 6, 12, 18, and 24months (n=5 per group) following the bypass. Heparin bioactivity of the graft material was analyzed. The effect of intimal hyperplasia was also assessed. All bypass grafts were patent at 1 month. Significantly greater patency rates were noted in the Propaten group compared to the control group at 12, 18, and 24months, which were 84%, 80%, and 80% vs. 55%, 35%, and 20%, respectively (P<0.02). There was a significant reduction in the anastomotic neointimal area and neointimal cell proliferation in Propaten grafts compared with control grafts at all groups between 6 and 24months (P<0.05). Heparin bioactivity as measured by antithrombin binding assay was demonstrated in the Propaten graft between 1 and 24months. Mean heparin activities on Propaten grafts ranged from 26.3±6.4 pmol/cm2 to 18.4±8.7 pmol/cm2 between 1 and 24months, which were significantly greater than the control group (P<0.001). Differences between mean heparin activities of explanted Propaten graft samples at the various time points were nonsignificant (P>0.05). Heparin-bonded ePTFE graft provides a thromboresistant surface and reduced anastomotic intimal hyperplasia at 2 years. The stable heparin bioactivity of the Propaten graft confers an advantage in long-term graft patency.

Carotid artery ligation induced intimal thickening and proliferation is unaffected by ageing

Following interventions to treat atherosclerosis, such as coronary artery bypass graft surgery, restenosis occurs in approximately 40% of patients. Identification of proteins regulating intimal thickening could represent targets to prevent restenosis. Our group previously demonstrated that in a murine model of vascular occlusion, Wnt4 protein expression and β-catenin signalling was upregulated which promoted vascular smooth muscle cell (VSMC) proliferation and intimal thickening. In this study, the effect of age on VSMC proliferation, intimal hyperplasia and Wnt4 expression was investigated. In vitro proliferation of VSMCs isolated from young (2 month) or old (18–20 month) C57BL6/J mice was assessed by immunocytochemistry for EdU incorporation. As previously reported, 400 ng/mL recombinant Wnt4 protein increased proliferation of VSMCs from young mice. However, this response was absent in VSMCs from old mice. As our group previously reported reduced intimal hyperplasia in Wnt4+/− mice compared to wildtype controls, we hypothesised that impaired Wnt4 signalling with age may result in reduced neointimal formation. To investigate this, carotid artery ligation was performed in young and old mice and neointimal area was assessed 21 days later. Surprisingly, neointimal area and percentage lumen occlusion were not significantly affected by age. Furthermore, neointimal cell density and proliferation were also unchanged. These data suggest that although Wnt4-mediated proliferation was impaired with age in primary VSMCs, carotid artery ligation induced neointimal formation and proliferation were unchanged in old mice. These results imply that Wnt4-mediated proliferation is unaffected by age in vivo, suggesting that therapeutic Wnt4 inhibition could inhibit restenosis in patients of all ages.

Inhibition of Smooth Muscle β-Catenin Hinders Neointima Formation After Vascular Injury.

Smooth muscle cells (SMCs) contribute to neointima formation after vascular injury. Although β-catenin expression is induced after injury, whether its function is essential in SMCs for neointimal growth is unknown. Moreover, although inhibitors of β-catenin have been developed, their effects on SMC growth have not been tested. We assessed the requirement for SMC β-catenin in short-term vascular homeostasis and in response to arterial injury and investigated the effects of β-catenin inhibitors on vascular SMC growth. We used an inducible, conditional genetic deletion of β-catenin in SMCs of adult mice. Uninjured arteries from adult mice lacking SMC β-catenin were indistinguishable from controls in terms of structure and SMC marker gene expression. After carotid artery ligation, however, vessels from mice lacking SMC β-catenin developed smaller neointimas, with lower neointimal cell proliferation and increased apoptosis. SMCs lacking β-catenin showed decreased mRNA expression of Mmp2, Mmp9, Sphk1, and S1pr1 (genes that promote neointima formation), higher levels of Jag1 and Gja1 (genes that inhibit neointima formation), decreased Mmp2 protein expression and secretion, and reduced cell invasion in vitro. Moreover, β-catenin inhibitors PKF118-310 and ICG-001 limited growth of mouse and human vascular SMCs in a dose-dependent manner. SMC β-catenin is dispensable for maintenance of the structure and state of differentiation of uninjured adult arteries, but is required for neointima formation after vascular injury. Pharmacological β-catenin inhibitors hinder growth of human vascular SMCs. Thus, inhibiting β-catenin has potential as a therapy to limit SMC accumulation and vascular obstruction.

A retrospective analysis of catheter-based sources in intravascular brachytherapy

PurposeCoronary artery disease involves the deposition of plaque along the walls of a coronary artery leading to narrowed or blocked vessels (stenosis) and is one of the main causes of death in developed countries. Percutaneous transluminal coronary angioplasty (PTCA) is used to reverse stenosis. Restenosis (renarrowing) of the treated vessel is a major complication of PTCA. A metal mesh tube (stent) can be placed inside the vessel to prevent restenosis. Tissue stress incurred during PTCA and stenting can provoke neointimal cell proliferation leading to in-stent restenosis (ISR). Intravascular brachytherapy (IVBT), a form of internal radiotherapy, is used to treat ISR. Renewed interest in IVBT is being expressed as a treatment for patients with ISR in drug-eluting stents. Current treatment planning (TP) of IVBT is extremely limited and assumes human tissue can be approximated by water. The interactions of arterial plaque, guidewires, and the stent have been shown to attenuate radiation significantly but are ignored in TP. Other models have determined the degree of attenuation by each factor in isolation. For the first time, we create a model with several inhomogenities present to determine whether attenuation by multiple inhomogenities combines linearly or if a larger dose reduction than anticipated is realized. We are also able to evaluate a spatial distribution of dose around the source and in arterial walls. Methods and MaterialsA dosimetric analysis of two commercially available IVBT systems was performed in a Monte Carlo–based particle simulation (Geant4). Absorbed dose was calculated using a model of a human coronary artery with a calcified plaque and stent. Dose delivered in water was also calculated to evaluate the accuracy of a water approximation. ResultsDose as a function of θ shows significant variation around IVBT sources. For the Guidant Galileo, dose is reduced by 20% behind stent struts and as much as 66% in a region occluded by the guidewire, plaque, and stent. For the Novoste Beta Cath device, delivered dose is reduced by 19% and 58%, respectively, in the same regions. ConclusionsOur findings show that the water approximation used in clinical practice to calculate dose is inaccurate when inhomogeneities are present. Methods proposed for calculating dose perturbations in IVBT may underestimate the magnitude of dose reduction. Increasing source dwell time seems unlikely to resolve dosimetric issues in IVBT. The effectiveness of currently existing β-emitting devices may be reduced in patients with complex lesions at the treatment site. Investigation of new radioisotopes and off-centering devices should be considered to improve dose outcomes.

MicroRNA-33 protects against neointimal hyperplasia induced by arterial mechanical stretch in the grafted vein.

Mechanical factors play significant roles in neointimal hyperplasia after vein grafting, but the mechanisms are not fully understood. Here, we investigated the roles of microRNA-33 (miR-33) in neointimal hyperplasia induced by arterial mechanical stretch after vein grafting. Grafted veins were generated by the 'cuff' technique. Neointimal hyperplasia and cell proliferation was significantly increased, and miR-33 expression was decreased after 1-, 2-, and 4-week grafts. In contrast, the expression of bone morphogenetic protein 3 (BMP3), which is a putative target of miR-33, and the phosphorylation of smad2 and smad5, which are potential downstream targets of BMP3, were increased in the grafted veins. miR-33 mimics/inhibitor and dual luciferase reporter assay confirmed the interaction of miR-33 and BMP3. miR-33 mimics attenuated, while miR-33 inhibitor accelerated, proliferation of venous smooth muscle cells (SMCs). Moreover, recombinant BMP3 increased SMC proliferation and P-smad2 and P-smad5 levels, whereas BMP3-directed siRNAs had the opposite effect. Then, venous SMCs were exposed to a 10%-1.25 Hz cyclic stretch (arterial stretch) by using the FX4000 cyclic stretch loading system in vitro to mimic arterial mechanical conditions. The arterial stretch increased venous SMC proliferation and repressed miR-33 expression, but enhanced BMP3 expression and smad2 and smad5 phosphorylation. Furthermore, perivascular multi-point injection in vivo demonstrated that agomiR-33 not only attenuates BMP3 expression and smad2 and smad5 phosphorylation, but also slows neointimal formation and cell proliferation in grafted veins. These effects of agomiR-33 on grafted veins could be reversed by local injection of BMP3 lentivirus. The miR-33-BMP3-smad signalling pathway protects against venous SMC proliferation in response to the arterial stretch. miR-33 is a target that attenuates neointimal hyperplasia in grafted vessels and may have potential clinical applications.

Smooth muscle cell-specific Tgfbr1 deficiency attenuates neointimal hyperplasia but promotes an undesired vascular phenotype for injured arteries.

Neointimal hyperplasia (NIH) and inward wall remodeling cause arterial restenosis and failure of bypass vein grafts. Previous studies from our group suggest that transforming growth factor (TGF) β promotes these pathologies via regulating cell kinetics at the early stage and matrix metabolism at the late stage. Although these temporal TGF β effects may result from its signaling in different cell groups, the responsible cell type has not been identified. In the current study, we evaluated the effect of smooth muscle cell (SMC)‐specific TGF β signaling through its type I receptor TGFBR1 on NIH and wall remodeling of the injured femoral arteries (FAs). An inducible Cre/loxP system was employed to delete SMC Tgfbr1 (Tgfbr1 iko). Mice not carrying the Cre allele (Tgfbr1 f/f) served as controls. The injured FAs were evaluated on d3, d7, and d28 postoperatively. Tgfbr1 iko attenuated NIH by 92%, but had insignificant influence on arterial caliber when compared with Tgfbr1 f/f controls on d28. This attenuation correlated with greater cellularity and reduced collagen content. Compared with Tgfbr1 f/f FAs, however, Tgfbr1 iko FAs exhibited persistent neointimal cell proliferation and cell apoptosis, with both events at a greater rate on d28. Tgfbr1 iko FAs additionally contained fewer SMCs and more inflammatory infiltrates in the neointima and displayed a thicker adventitia than did Tgfbr1 f/f FAs. More MMP9 proteins were detected in the adventitia of Tgfbr1 iko FAs than in that of Tgfbr1 f/f controls. Our results suggest that disruption of SMC Tgfbr1 inhibits arterial NIH in the short term, but the overall vascular phenotype may not favor long‐term performance of the injured arteries.

Coronary Injury Score Correlates with Proliferating Cells and Alpha-Smooth Muscle Actin Expression in Stented Porcine Coronary Arteries.

Neointimal formation and cell proliferation resulting into in-stent restenosis is a major pathophysiological event following the deployment of stents in the coronary arteries. In this study, we assessed the degree of injury, based on damage to internal elastic lamina, media, external elastic lamina, and adventitia following the intravascular stenting, and its relationship with the degree of smooth muscle cell proliferation. We examined the smooth muscle cell proliferation and their phenotype at different levels of stent injury in the coronary arteries of domestic swine fed a normal swine diet. Five weeks after stent implantation, swine with and without stents were euthanized and coronaries were excised. Arteries were embedded in methyl methacrylate and sections were stained with H&E, trichrome, and Movat’s pentachrome. The expression of Ki67, α-smooth muscle actin (SMA), vimentin, and HMGB1 was evaluated by immunofluorescence. There was a positive correlation between percent area stenosis and injury score. The distribution of SMA and vimentin was correlated with the degree of arterial injury such that arteries that had an injury score >2 did not have immunoreactivity to SMA in the neointimal cells near the stent struts, but these neointimal cells were positive for vimentin, suggesting a change in the smooth muscle cell phenotype. The Ki67 and HMGB1 immunoreactivity was highly correlated with the fragmentation of the IEL and injury in the tunica media. Thus, the extent of coronary arterial injury during interventional procedure will dictate the degree of neointimal hyperplasia, in-stent restenosis, and smooth muscle cell phenotype.

Abstract 432: The Atypical Cadherin Fat1 Suppresses Mitochondrial Function to Control Vascular Smooth Muscle Cell Growth After Vascular Injury

In response to vascular injury, vascular smooth muscle cells (SMCs) undergo phenotypic switching, with enhanced cell cycle entry and migration, and loss of contractile protein expression. Previously, we found that the atypical cadherin Fat1 is upregulated in SMCs after vascular injury. In cultured SMCs, Fat1 is induced by growth factor stimulation, but limits SMC proliferation. Recently, we found that Fat1 species accumulate in SMC mitochondria and interact with critical proteins, including Complex I subunits. The factors governing and significance of mitochondrial activity during SMC response to vascular injury are unclear. We hypothesized that Fat1 controls mitochondrial activity to regulate SMC growth. In cultured SMCs, loss of Fat1 increased mitochondrial oxygen consumption rate (24.8±2.2 vs. 48.1±5.4 pMoles/min, WT vs. Fat1 knockout (KO), p&lt;0.001, n=10), maximal respiratory capacity (35.8±3.4 vs. 55.4±5.4 pMoles/min, WT vs. KO, p&lt;0.01, n=10), oxygen consumed for ATP production (13.1±1 vs. 27.3±3.3 pMoles/min, WT vs. KO, p=0.0005, n=10), and Complex I activity (1.0±0.04 vs.1.3±0.09 RU, WT vs. KO, p&lt;0.034, n=10). Reactive oxygen species (ROS) also increased in Fat1 KO SMCs (1426±313 vs.1848±329 dihydroethidium fluorescence intensity/cell, WT vs. KO, p&lt;0.002, n=3). SMCs lacking Fat1 exhibited enhanced growth (p&lt;0.0001, n=12) and dedifferentiation; adding Rotenone to inhibit Complex I function opposed this growth advantage (p&lt;0.0001, n=6). In a mouse model of vascular injury, SMC Fat1 deletion caused early and exuberant medial hyperplasia, (0.23±0.23 vs.7.16±1.62%, WT n=5 vs. KO n=8, p&lt;0.003, day 3 post-injury) and neointimal expansion (0.18±0.05 vs.0.55±0.1 I/M ratio, WT n=5 vs. KO n=8, p&lt;0.02, day 14 post-injury). We also found higher neointimal cell proliferation (7.7±2 vs. 26±7.1% of pHH3 positive nuclei, WT vs. KO, p=0.041, n=6) and ROS production in Fat1-deleted vessels after injury. In conclusion, we show that the atypical cadherin Fat1 communicates with mitochondrial Complex I to repress energy and ROS production, acting as a molecular “brake” on mitochondrial activity to suppress SMC growth after vascular injury. This novel mechanism could serve as an effective target in the treatment of hyperproliferative vascular diseases.

Effect of imatinib mesylate and rapamycin on the preformed intimal hyperplasia in rat carotid injury model.

PurposeIntimal hyperplasia (IH) is the main cause of restenosis or occlusion after vascular procedures. Imatinib mesylate and rapamycin are known to prevent IH. The purpose of this study was to evaluate the effect of these drugs on the regression of preformed IH in rat carotid injury model.MethodsIH was established in rat carotid arteries using a balloon catheter. The drug effects were assessed in vitro on proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMC) in the neointima. And in vivo studies were carried out in 4 groups: imatinib, rapamycin, combined, and no medication. After 2-week oral medication, morphometric analysis evaluated the number and density of neointimal cells, intima-to-media (I/M) ratio and cross-sectional area. Cell proliferation, apoptosis, and collagen changes were also investigated by immunohistochemical staining (IHCS).ResultsImatinib and rapamycin significantly inhibited VSMC proliferation and migration, and promoted apoptosis in vitro. In morphometric analysis, the number and density of neointimal cells decreased significantly in all medication groups compared with control group (P < 0.01). However, there was no significant difference in neointimal cross-sectional area and I/M ratio among groups. In IHCS, imatinib and rapamycin inhibited neointimal cell proliferation significantly. However, there was no significant change in cell apoptosis and collagen composition.ConclusionCombined treatment of with imatinib and rapamycin induced reduction of cell mass in preformed intimal hyperplasia, but failed to induce regression of intimal mass in this short-term medication study. Further studies will be needed with additional strategies of inducing lysis of the extracellular matrix.

Periadventitial atRA citrate-based polyester membranes reduce neointimal hyperplasia and restenosis after carotid injury in rats.

Oral all-trans retinoic acid (atRA) has been shown to reduce the formation of neointimal hyperplasia; however, the dose required was 30 times the chemotherapeutic dose, which already has reported side effects. As neointimal formation is a localized process, new approaches to localized delivery are required. This study assessed whether atRA within a citrate-based polyester, poly(1,8 octanediolcitrate) (POC), perivascular membrane would prevent neointimal hyperplasia following arterial injury. atRA-POC membranes were prepared and characterized for atRA release via high-performance liquid chromatography with mass spectrometry detection. Rat adventitial fibroblasts (AF) and vascular smooth muscle cells (VSMC) were exposed to various concentrations of atRA; proliferation, apoptosis, and necrosis were assessed in vitro. The rat carotid artery balloon injury model was used to evaluate the impact of the atRA-POC membranes on neointimal formation, cell proliferation, apoptosis, macrophage infiltration, and vascular cell adhesion molecule 1 (VCAM-1) expression in vivo. atRA-POC membranes released 12 μg of atRA over 2 wk, with 92% of the release occurring in the first week. At 24 h, atRA (200 μmol/l) inhibited [(3)H]-thymidine incorporation into AF and VSMC by 78% and 72%, respectively (*P = 0.001), with negligible apoptosis or necrosis. Histomorphometry analysis showed that atRA-POC membranes inhibited neointimal formation after balloon injury, with a 56%, 57%, and 50% decrease in the intimal area, intima-to-media area ratio, and percent stenosis, respectively (P = 0.001). atRA-POC membranes had no appreciable effect on apoptosis or proliferation at 2 wk. Regarding biocompatibility, we found a 76% decrease in macrophage infiltration in the intima layer (P < 0.003) in animals treated with atRA-POC membranes, with a coinciding 53% reduction in VCAM-1 staining (P < 0.001). In conclusion, perivascular delivery of atRA inhibited neointimal formation and restenosis. These data suggest that atRA-POC membranes may be suitable as localized therapy to inhibit neointimal hyperplasia following open cardiovascular procedures.

Abstract 502: Arterial Calcification Increases Neointimal Proliferation Following Balloon Injury in Rat Carotid Arteries

Introduction Arterial calcification predicts accelerated restenosis after angioplasty and stenting. We sought to determine how calcification affected neointimal accumulation after balloon injury in the rat carotid. Hypothesis We propose that transformed SMCs in calcified arteries respond to balloon injury in an accelerated manner through a mechanism involving bone-related factors. Methods Arterial calcification was induced in male Sprague-Dawley rats weighing 350-400 grams by subcutaneous administration of vitamin D3. Control rats received vehicle. Both groups underwent left carotid balloon injury one week following injection, a time at which significant medial calcification has developed. Arteries were harvested twenty-one days following balloon injury. The degree of neointimal cell proliferation was next assessed by immunohistochemical staining for PCNA. Neointimal hyperplasia was measured by morphometric analysis using standard imaging analysis software. Results At 3 weeks after carotid balloon injury, mean cellular proliferation as measured by PCNA staining was higher in the intima of calcified than control arteries (63.8 ± 6.0 versus 40.5 ± 9.5 cells/hpf, P=ns). Calcified arteries from Vitamin D3-treated rats, however, had significantly more intimal hyperplasia than control uncalcified arteries (intima-to-media ratio = 1.374 ± 0.071 versus 0.758 ± 0.080, P=0.0021). Intimal and medial areas were also individually larger in calcified than control arteries. Conclusions Neointimal hyperplasia is accelerated in Vitamin D3-treated rats through a mechanism that partially involves increased SMC proliferation. Calcified vessels may respond differently to balloon angioplasty and this may explain the poorer results of endovascular interventions in patients with arterial calcification. Other factors including accelerated migration and increased matrix synthesis may also be involved. Treatments to inhibit restenosis in calcified arteries may differ from those that work in uncalcified vessels.

Resveratrol inhibits neointimal formation after arterial injury through an endothelial nitric oxide synthase-dependent mechanism

Revascularization procedures used for treatment of atherosclerosis often result in restenosis. Resveratrol (RSV), an antioxidant with cardiovascular benefits, decreases neointimal formation after arterial injury by a mechanism that is still not fully clarified. Our main objective was to address the role of nitric oxide synthases (NOSes) and more specifically the endothelial-NOS (eNOS) isoform as a mediator of this effect.RSV (4mg/kg/day, s.c.) alone or in combination with the NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) (2mg/kg/day, s.c.) was given to Sprague-Dawley rats beginning at 3 days before arterial (carotid or aortic) injury. RSV reduced neointimal formation by 50% (P<0.01), decreased intimal cell proliferation by 37% (P<0.01) and reduced inflammatory markers such as PECAM and MMP-9 mRNA. These effects of RSV were all abolished by coadministration of l-NAME. Oral RSV (beginning at 5 days before arterial injury) reduced neointimal thickness after femoral wire injury in mice, however this effect was not observed in eNOS knockout mice. This is the first report of RSV decreasing neointimal cell proliferation and neointimal growth through an eNOS-dependent mechanism.

Pigment epithelium-derived factor reduces the PDGF-induced migration and proliferation of human aortic smooth muscle cells through PPARγ activation

Our previous study demonstrated that pigment epithelium-derived factor (PEDF) plays an important role in the proliferation and migration of human aortic smooth muscle cells (HASMCs). In the present study, we examined whether PEDF inhibited platelet-derived growth factor (PDGF)-stimulated HASMC migration and proliferation. PEDF dose-dependently reduced PDGF-induced HASMC migration and proliferation in vitro and also arrested cell cycle progression in the G0/G1 phase, and this was associated with decreased expression of cyclin D1, cyclin E, CDK2, CDK4, and p21 Cip1 and increased expression of the cyclin-dependent kinase inhibitor p27 Kip1. The antiproliferative and antimigratory effects of PEDF were partially blocked by the PPARγ antagonist GW9662, but not by the PPARα antagonist MK886. In in vivo studies, the femoral artery of C57BL/6 mice was endothelial-denuded and the mice injected intravenously with PEDF or vehicle. After 2 weeks, both the neointima/media area ratio and cell proliferation (proliferating cell nuclear antigen-positive cells) in the neointima were significantly reduced and again these effects were partially reversed by GW9662 pretreatment. Our data show that PEDF increases PPARγ activation, preventing entry of HASMCs into the cell cycle in vitro and reducing the neointimal area and cell proliferation in the neointima in vivo. Thus, PEDF may represent a safe and effective novel target for the prevention and treatment of vascular proliferative diseases.