Both neointimal hyperplasia and inward remodeling contribute to restenosis and lumen loss. Nogo-B has been recently described as an inhibitor of vascular injury and neointimal hyperplasia. To determine whether Nogo-B expression may be a mediator of inward remodeling, we examine the localization of expression of Nogo-B in an in vivo model that examines both neointimal hyperplasia and inward remodeling. The rabbit carotid artery was subjected to balloon injury, outflow branch ligation to reduce flow, or both balloon injury and reduction in flow. In balloon injury-induced neointimal hyperplasia Nogo-B expression was reduced in the intima and media but stimulated in the adventitia. In low flow-induced inward remodeling medial Nogo-B expression was not reduced and adventitial Nogo-B expression was not stimulated. Low flow significantly augmented balloon injury-induced neointimal hyperplasia and was accompanied by reduced intimal and medial Nogo-B expression, and increased adventitial Nogo-B expression in both smooth muscle cells and macrophages. Low flow-induced inward remodeling is not associated with changes in medial Nogo-B expression and is distinct from injury-induced neointimal hyperplasia. Pharmacological strategies to inhibit neointimal hyperplasia and restenosis using normal flow models may only partially account for lumen loss and therefore may not accurately predict responses in patients with extensive outflow disease.
neointimal hyperplasia; flow-induced remodeling; Nogo-B; rabbit
Mural inflammation has been shown to contribute to the development of plaque, with the αVβ3 integrin highly expressed in atherosclerotic plaques. We herein examined αVβ3 integrin expression as a function of carotid atherosclerosis formation in the apolipoprotein E-deficient (apoE−/−) mouse.
Methods and results
Constrictive collars were placed around the left common carotid arteries of apo E−/− mice maintained on a high-fat diet (n = 14). Before and 21 days following collar placement, in vivo serial magnetic resonance imaging (MRI) measurements of the carotid aortic diameter were performed using a 7T magnetic resonance (MR) scanner. Near- infrared fluorescence (NIRF) imaging was performed (n = 6) using an in vivo imaging system 0–24 hours following administration of 1.0 nmol c(RGDyK)-Cy5.5 via the tail vein. A competition experiment was performed by the co-injection of a saturating dose of bicyclic RGD peptide H-Glu[cyclo(Arg-Gly-Asp-D-Tyr-Lys)]2 (n = 3). Following image acquisition and sacrifice at 24 hours after injection, carotid arteries were harvested for histological analyses. Neointima formation and arterial remodeling in the carotid arteries of apoE−/− mice were induced by the placement of a constrictive collar. Significantly greater fluorescent signals were obtained from constrictive collar left common carotid arteries as compared to uninvolved aortic segments in constrictive collar mice. Binding to stenotic lesions was efficiently blocked in competition experiments. Immunostaining confirmed the presence of mural αVβ3 integrin expression in macrophages in the neointima. Signal intensity increased in a macrophage density-dependent fashion in the stenotic segments.
Mural αVβ3 integrin expression, as determined using RGD-Cy5.5 near-infrared optical imaging, was increased in carotid arteries with constrictive collars in experimental mice. This expression can estimate the macrophage-bound inflammatory activity of atherosclerotic lesions.
near-infrared fluorescence (NIRF); macrophage; αVβ3 integrin; carotid atherogenesis
Wall shear stress is thought to play a critical role in the local development of atherosclerotic plaque and to affect plaque vulnerability. However, current models and hypotheses do not fully explain the link between wall shear stress and local plaque development. We aimed to investigate the relation between wall shear stress and local plaque development in surgically induced common carotid artery stenoses of hypercholesterolemic minipigs.
Materials, Methods and Results:
We created a surgically induced stenosis of the common carotid artery in 10 minipigs using a perivascular collar. We documented the flow and shear stress changes by ultrasound, magnetic resonance imaging, and computational fluid dynamics. Carotid plaques were documented by microscopy. Atherosclerotic lesions, in both pre-stenotic and post-stenotic segments, were associated with thrombus in the stenosed segment. In patent carotid arteries, atherosclerotic lesions were found in the post-stenotic segments only. Atherosclerotic lesions developed where low and oscillatory shear stress were present simultaneously, whereas low or oscillatory shear stress alone did not lead to lesion formation.
Low and oscillatory shear stress in combination promoted plaque development, including plaques with necrotic cores that are the key and dangerous characteristic of vulnerable plaques.
Atherosclerosis; carotid artery; magnetic resonance imaging; vulnerable plaque; wall shear stress
Stem/progenitor cell-based therapy has successfully been used as a novel therapeutic strategy for vascular diseases triggered by endothelial dysfunction. The aim of this study was to investigate the effects of mononuclear cell (MNC) therapy in situ on carotid cuff-induced occlusive thrombus in the apolipoprotein E knockout (apoE-/-) mouse.
Spleen-derived MNCs were isolated from green fluorescent protein (GFP)-transgenic mice for cell treatment. A cuff-induced thrombus model was produced by placing a nonconstrictive silastic collar around the left common carotid artery in 20-week-old female apoE-/- mice. After 10 days, the cuff was removed, and the animals received in situ MNCs (Cuff-MNC) or vehicle (Cuff-Vehicle) and were compared with sham-operated animals (Sham).
The histological analysis showed that the MNC treatment reverted occlusive thrombus formation compared to the vehicle and the vessel lumen area to that observed in the Sham group (MNC, 50 ± 4; Vehicle, 20 ± 4; Sham, 55 ± 2 x103 μm2; p < 0.01). The animals that underwent the carotid cuff placement developed compensatory vessel enlargement, which was reduced by the MNC therapy. In addition, the treatment was able to reduce superoxide anion production, which likely contributed to the reduced apoptosis that was observed. Lastly, the immunofluorescence analysis revealed the presence of endothelial progenitor cells (EPCs) in the carotid endothelia of the apoE-/- mice.
In situ short-term MNC therapy was able to revert cuff-induced occlusive thrombi in the carotid arteries of apoE-/- mice, possibly through the homing of EPCs, reduction of oxidative stress and decreased apoptosis.
ApoE-/-; Mononuclear cells; Thrombus; Cuff model
Peroxisome proliferator-activated receptor α is a member of the nuclear receptor superfamily. It modulates smooth muscle cell proliferation and inflammatory cytokines in vitro. In this study, we tested the hypothesis that PPARα would decrease the expression of monocyte chemoattractant protein-1 and tissue factor, and inhibit neointimal formation in a murine double carotid artery injury model. Carotid artery injury was performed in the PPARα knockout and wild type (WT) mice, treated and untreated with Wy14643, a PPARα activator. Up-regulated MCP-1 and TF expression and more neointimal formation were observed in the PPARα−/− mice compared with WT mice. The activation of PPARα resulted in further decreased neointimal formation. Our data further suggest that the decrease in neointimal formation is due to down-regulation of MCP-1 by PPARα resulting in decreased leukocyte infiltration and TF expression.
Introduction. The purpose of this study was to investigate the changes of chitinase 3-like 1 (CHI3L1) in the aorta of patients with coronary atherosclerosis and to determine whether inhibition of CHI3L1 by lentivirus-mediated RNA interference could stabilize atherosclerotic plaques in apolipoprotein E-knockout (ApoE−/−) mice. Methods. We collected discarded aortic specimens from patients undergoing coronary artery bypass graft surgery and renal arterial tissues from kidney donors. A lentivirus carrying small interfering RNA targeting the expression of CHI3L1 was constructed. Fifty ApoE−/− mice were divided into control group and CHI3L1 gene silenced group. A constrictive collar was placed around carotid artery to induce plaques formation. Then lentivirus was transfected into carotid plaques. Results. We found that CHI3L1 was overexpressed in aorta of patients with atherosclerosis and its expression was correlated with the atherosclerotic risk factors. After lentivirus transduction, mRNA and protein expression of CHI3L1 were attenuated in carotid plaques, leading to reduced plaque content of lipids and macrophages, and increased plaque content of collagen and smooth muscle cells. Moreover, CHI3L1 gene silencing downregulated the expression of local proinflammatory mediators. Conclusions. CHI3L1 is overexpressed in aorta from patients with atherosclerosis and the lentivirus-mediated CHI3L1 gene silencing could represent a new strategy to inhibit plaques progression.
Inflammation plays an essential role in the initiation and progression of atherosclerosis, but its role in vascular repair after mechanical arterial injury (i.e., percutaneous transluminal coronary angioplasty, PTCA) is unknown. In animal models of vascular injury, leukocytes are recruited as a precursor to intimal thickening. Furthermore, markers of leukocyte activation — in particular, increased expression of the β2-integrin Mac-1 (αMβ2, or CD11b/CD18), which is responsible for firm leukocyte adhesion to platelets and fibrinogen on denuded vessels — predict restenosis after PTCA. To determine whether Mac-1–mediated leukocyte recruitment is causally related to neointimal formation, we subjected mice lacking Mac-1 to a novel form of mechanical carotid artery dilation and complete endothelial denudation. We now report that the selective absence of Mac-1 impairs transplatelet leukocyte migration into the vessel wall, reducing leukocyte accumulation over time. Diminished medial leukocyte accumulation was accompanied by markedly reduced neointimal thickening after vascular injury. These data establish a role for inflammation in neointimal thickening and suggest that leukocyte recruitment to mechanically injured arteries may prevent restenosis.
The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), encodes a large cytoskeletal protein present in muscle fibers. While dystrophin in skeletal muscle has been extensively studied, the function of dystrophin in vascular smooth muscle is less clear. Here, we have analyzed the role of dystrophin in injury-induced arterial neointima formation.
We detected a down-regulation of dystrophin, dystroglycan and β-sarcoglycan mRNA expression when vascular smooth muscle cells de-differentiate in vitro. To further mimic development of intimal lesions, we performed a collar-induced injury of the carotid artery in the mdx mouse, a model for DMD. As compared with control mice, mdx mice develop larger lesions with increased numbers of proliferating cells. In vitro experiments demonstrate increased migration of vascular smooth muscle cells from mdx mice whereas the rate of proliferation was similar in cells isolated from wild-type and mdx mice.
These results show that dystrophin deficiency stimulates neointima formation and suggest that expression of dystrophin in vascular smooth muscle cells may protect the artery wall against injury-induced intimal thickening.
A precise understanding of the mechanism of human neointimal stenoses and atherosclerotic fibrous plaques, which give rise to thromboses in vital arteries, requires a suitable animal model that would mimic the same characteristics well. We developed a rabbit model of neointimal stenosis and fibrotic plaque rupture in the carotid artery to visualize the lesion progress and to characterize the lesion types according to the American Heart Association classification.
Twenty-eight healthy male New Zealand white rabbits were randomly divided into two groups: The rabbits in group A (n = 14) consumed a standard chow diet, and those in group B (n = 14) were injured via perivascular cold injury using liquid nitrogen at the right common carotid artery before being fed a high cholesterol diet (1.5%) for eight weeks. Plasma lipid evaluation was performed before the sacrificing of the rabbits. At the end of every week, at least 1 rabbit from group B was sacrificed for an analysis of lesion histopathology and calculation of the area ratios of the intima to media.
The plasma lipid level in group B was significantly higher than that in group A (p value < 0.05). The histopathological results revealed atherosclerosis characteristics such as endothelial layer destruction, fatty streaks and lipid-containing macrophages (foam cells) formation in the intima and media layers, extracellular lipid collections, smooth muscle cells proliferation and migration, neointima formation, intima thickening and deformation, fibrotic plaque formation, and finally plaque rupture. Statistical analysis revealed a significant increase in the intima-to-media ratio at the end of the eighth week (6.41 ± 0.27, p value < 0.05).
We successfully developed a rabbit model of neointimal stenosis and atherosclerotic fibrous connective tissue plaque rupture, which is not only quickly and easily reproducible and inexpensive but also without mortality. The merits of our model render the evaluation of neointimal stenoses and fibrotic plaques and their treatment strategies more feasible in humans.
Atherosclerosis; Carotid arteries; Plaque; atherosclerotic; Rabbits; Animals
Our previous studies showed that increased levels of cyclophilin A (CyPA) may be a valuable marker for predicting the severity of acute coronary syndromes and that interruption of CD137-CD137L interactions diminished the formation and progression of atherosclerosis in apolipoprotein E-deficient (ApoE−/−) mice. Here, we sought to determine whether the proinflammatory factor CyPA is involved in atherosclerosis regulated by CD137-CD137L interactions.
Methods and Results
A constrictive collar was placed around the right carotid arteries of ApoE−/− mice that were fed a high-fat diet to induce atherosclerotic plaque formation. After that, the mice were intraperitoneally injected with anti-CD137 or anti-CD137L in the presence or absence of the recombinant lentiviral vectors LVTHM-CyPA or pGC-FU-CyPA, respectively. Interestingly, activation of CD137-CD137L was negatively correlated with CyPA expression in vivo and in vitro. Stimulating CD137-CD137L interaction significantly increased CyPA, which was concurrent with the upregulation of proinflammatory cytokines, chemokines and matrix metalloproteinases and resulted in the promotion of atherosclerosis in ApoE-/- mice. Silencing CyPA could eliminate these effects, and restoration of CyPA effectively and consistently attenuated the atherosclerotic suppression phenotypes elicited by the blockade of CD137-CD137L.
These observations suggest that CD137-CD137L interactions mediated via regulation of CyPA contribute to the progression of atherosclerosis.
Neointimal hyperplasia involving smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) degradation is an important component of atherosclerosis. It develops as a response to vascular injury after balloon angioplasty and vascular graft placement. Matrix metalloproteinases (MMPs) induce SMC proliferation, migration and contribute to intimal hyperplasia by degrading ECM. PPARγ agonists inhibit SMC proliferation, migration and lesion formation. In this study, we aimed to investigate the effects of PPARγ agonist rosiglitazone on neointimal hyperplasia and gelatinase (MMP-2 and MMP-9) expressions in rabbit carotid anastomosis model.
New Zealand white rabbits (n = 13, 2.7–3.2 kg) were divided into placebo and treatment groups. Right carotid artery (CA) was transected and both ends were anastomosed. Treatment group (n = 6) received rosiglitazone (3 mg/kg/day/p.o.) and placebo group (n = 7) received PBS (phosphate buffered saline, 2.5 ml/kg/day/p.o.) for 4 weeks postoperatively. After the sacrification, right and left CAs were isolated. Morphometric analyses and immunohistochemical examinations for gelatinases were performed.
Intimal area (0.055 ± 0.005 control vs 0.291 ± 0.020 μm2 anastomosed, p < 0,05) and index (0.117 ± 0.002 control vs 0.574 ± 0.013 anastomosed, p < 0,01) significantly increased in anastomosed arteries compared to control arteries from placebo group. However, in rosiglitazone-treated group, intimal area (0.291 ± 0.020 PBS vs 0.143 ± 0.027 rosiglitazone, p < 0,05) and index (0.574 ± 0.013 PBS vs 0.263 ± 0.0078 rosiglitazone, p < 0,01) significantly decreased. Furthermore, gelatinase immunopositivity was found to have significantly increased in anastomosed arteries from placebo group and decreased with rosiglitazone treatment.
These results suggest that rosiglitazone may prevent neointimal hyperplasia, which is the most important factor involved in late graft failure, by inhibiting gelatinase enzyme expression.
Neointima; Rosiglitazone; Matrix metalloproteinases (MMPs); Rabbit
We have used antisense phosphorothioate oligonucleotides to define the role played by proliferating cell nuclear antigen (PCNA) in neointimal accumulation of smooth muscle cells in a rat carotid artery injury model. The short-term extraluminal delivery of 250 nmol of antisense oligonucleotides, but not control oligonucleotides, immediately after arterial injury produces a 77% suppression of PCNA mRNA after 24 h and a 52% decrease in the frequency of medial smooth muscle cells expressing PCNA after 72 h. This reduction in PCNA expression is accompanied by a 59% decrease in the frequency of proliferating medial smooth muscle cells at 3 d as measured by BudR staining and an 80% decrease in neointimal accumulation assessed morphometrically at 2 wk. Thus, the expression of PCNA is required for medial smooth muscle cell growth in vivo and for neointimal formation after arterial injury.
FOR THE FIRST 30 YEARS AFTER CAROTID ENDARTERECTOMY WAS FIRST DEVELOPED, anecdotal evidence was used to identify patients with internal carotid artery disease for whom this procedure would be appropriate. More recently, the appropriateness of carotid endarterectomy for symptomatic patients and asymptomatic subjects has emerged from 7 randomized trials. Risk of stroke and benefit from the procedure are greatest for symptomatic patients with at least 70% stenosis of the internal carotid artery. Within this group, carotid endarterectomy is most beneficial for the following patients: otherwise healthy elderly patients, those with hemispheric transient ischemic attack, those with tandem extracranial and intracranial lesions and those without evidence of collateral vessels. Risk of perioperative stroke and death is higher in the following groups, although they still benefit: patients with widespread leukoaraiosis, those with occlusion of the contralateral internal carotid artery and those with intraluminal thrombus. Patients with 50% to 69% stenosis experience lesser benefit, and some other groups may even be harmed by carotid endarterectomy, including women and patients with transient monocular blindness only. The procedure is indicated for patients presenting with lacunar stroke and for those with a nearly occluded internal carotid artery, but the benefit is muted. Patients with less than 50% stenosis do not benefit. In the largest randomized trial of asymptomatic subjects, the perioperative risk of stroke and death was very low (1.5%), but the results indicated that a prohibitively high number of subjects (83) must be treated to prevent one stroke in 2 years. The subsequent literature reported higher perioperative risks (2.8% to 5.6%). In asymptomatic individuals nearly half of the strokes that occur may be due to heart and small-vessel disease. These limitations counter any potential benefit. Another trial is in progress and may identify subgroups of asymptomatic subjects who would benefit. Meanwhile, most individuals without symptoms fare better with medical care.
The most commonly used procedures to induce arterial injury in mice are carotid artery ligation with cessation of blood flow and mechanically-induced denudation of endothelium in the carotid or the femoral arteries. Both procedures result in neointimal hyperplasia after two to three weeks. A survey of various inbred strain of mice shows that strain-specific differences in susceptibility to injury-induced neointimal hyperplasia are different than those for susceptibility to diet-induced atherosclerosis, with strains identified as susceptible to both neointimal hyperplasia and atherosclerosis, resistant to both, susceptible to atherosclerosis but resistant to neointimal hyperplasia, or resistant to atherosclerosis but susceptible to neointimal hyperplasia. Inflammatory cells such as T and B lymphocytes, which are contributory to atherosclerosis, are protective against injury-induced neointimal hyperplasia. In contrast, the infiltration of monocytes into the site of injury and their differentiation to macrophages favor neointimal hyperplasia similar to their pathogenic role in atherosclerosis. The regulatory role of lymphocytes and macrophages in neointimal hyperplasia is related to the production of cytokines such as interferon-γ and tumor necrosis factor-α, respectively. Interestingly, inducible nitric oxide synthase (iNOS) activity appears to inhibit neointimal hyperplasia in the endothelial denudation model but contributes to neointimal hyperplasia when arterial injury is induced by periadventitial cuff placement. The difference appears to be due to the time required for endothelial recovery and the participation of inflammatory cells. Thus, although arterial injury-induced neointimal hyperplasia results in similar vascular occlusion as progressive atherosclerosis, the pathology and mechanism of the two disease processes are quite different.
Neointimal Hyperplasia; Mouse Genetics; Nitric Oxide; Lymphocytes; Smooth Muscle Cells; Macrophages; Bone Marrow Progenitor Cells
Atherosclerosis and arterial injury-provoked neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins β-arrestin1 and -2 might regulate this pathologic process. Deficiency of β-arrestin2 in ldlr-/- mice reduced aortic atherosclerosis by 40%, and decreased the prevalence of atheroma SMCs by 35%—suggesting that β-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic WT, β-arrestin1-/-, and β-arrestin2-/- mice. Neointimal hyperplasia was enhanced in β-arrestin1-/- mice, and diminished in β-arrestin2-/- mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with GFP-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in β-arrestin2-/- mice was not altered by transplantation with either WT or β-arrestin2-/- bone marrow cells. After carotid injury, medial SMC ERK activation and proliferation were increased in β-arrestin1-/- and decreased in β-arrestin2-/- mice. Concordantly, thymidine incorporation, ERK activation and migration evoked by 7-transmembrane receptors were greater than WT in β-arrestin1-/- SMCs, and less in β-arrestin2-/- SMCs. Proliferation was less than WT in β-arrestin2-/- SMCs, but not in β-arrestin2-/- endothelial cells. We conclude that β-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration, and that these SMC activities are regulated reciprocally by β-arrestin2 and β-arrestin1. These findings identify inhibition of β-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.
arteriosclerosis; muscle; smooth; signal transduction; receptors; endothelium
Objective: To use intravascular ultrasound to investigate the effects of antiplatelet agents and other factors on neointimal proliferation after stent implantation for iliac artery stenosis.
Patients and methods: The subjects were 109 patients with peripheral arterial disease who underwent stent implantation in the iliac artery. Intravascular ultrasound was performed to evaluate lesion area, stent dilatation and neointimal proliferation before, just after, and six months after stenting. Multiple regression analysis was performed to examine the relationship of the neointimal proliferation rate with antiplatelet agents and other factors.
Results: At the time of stent implantation, a Palmaz stent resulted in a significant increase in lumen area compared with a Wallstent (p < 0.05). Six months later, self-expanding Wallstent and Luminexx stents showed a significant increase in the stent-lumen area (p < 0.05). The neointimal proliferation rate showed a significant negative correlation with beraprost and cilostazol (p < 0.05) and a significant positive correlation with serum creatinine (p < 0.01). There was no significant difference in the vessel lumen area including the proliferated intima among the three stents.
Conclusion: Suppression of neointimal proliferation can be achieved with beraprost or cilostazol whereas renal dysfunction may increase neointimal proliferation following stent implantation in iliac artery lesions.
beraprost; neointimal proliferation; stent implantation; iliac artery; peripheral arterial disease
Sulodexide, a glycosaminoglycan-containing compound, is known to have an antiproliferative effect on vascular smooth-muscle cells, in vitro, as well as antithrombotic and fibrinolytic effects. But there are few reports about the effect of neointimal proliferation in vivo. In this study, we examined whether Sulodexide was effective in the inhibition of neointimal proliferation after vascular injury. Ten-week-old Sprague-Dawley rats were subjected to vascular injury by endothelial denudation of the common carotid artery by using a balloon catheter. They were then allocated randomly into a control group (saline 2 ml for 3 days, and then 1 ml for 18 days, IM) and a treated group (Sulodexide 10 mg/kg/day for 3 days, and then 4 mg/kg/day for 18 days, IM). Three weeks after vascular injury, we analyzed the neointimal proliferation using morphometry. The neointimal proliferation was significantly reduced in the treated group compared to the control group (Ratio of neointimal area to medial area; 118.39 +/- 6.80% in the treated group, 177.25 +/- 17.25% in the control group). This result showed that Sulodexide might be effective in reducing the rate of restenosis after balloon angioplasty.
The aim of our study was to investigate whether citronella-spray collars offer a humane alternative to electric-shock collars to reduce the barking of domestic dogs. The Aboistop collar was applied to seven dogs with problematic barking behaviour by the dogs' owners in a series of case studies concurrently run. Vocalisation of the dogs was recorded in the problem context under baseline conditions, inactive collar conditions, and active collar conditions. The Aboistop collar was effective at reducing problem vocalization for only three of seven dogs and appeared to be most effective for dogs whose problem barking had developed more recently. The collar may be more humane than other punishment methods, but it did produce stress reactions which varied in severity across the dogs. Clinical Relevance. In our study, the collar was applied by the dogs' owners in order to test whether the collar would be effective when used by members of the public. While the results here are preliminary, they suggest that the collar may be effective for some dogs, but not for others, when applied by dog owners for the treatment of problem vocalisation. Further research is required to determine whether the collar could be effective when administered by a trained professional.
Purpose. We hypothesized that adventitial transplantation of blood outgrowth endothelial cells (BOEC) to the vein-to-graft anastomosis of polytetrafluoroethylene grafts will reduce neointimal hyperplasia by reducing hypoxia inducible factor-1α (HIF-1α), by increasing angiogenesis in a porcine model of chronic renal insufficiency with haemodialysis polytetrafluoroethylene grafts. Because matrix metalloproteinases (MMPs) have been shown to be involved with angiogenesis, the expression of MMPs and their inhibitors was determined.
Methods. Chronic renal insufficiency was created by subtotal renal infarction and 28 days later, arteriovenous PTFE grafts were placed bilaterally from the carotid artery to the jugular vein. Autologous blood outgrowth endothelial cells labeled with Lac Z were transplanted to the adventitia of the vein-to-graft anastomosis using polyglycolic acid scaffolding and scaffolding only to other side (control). Animals were killed 14 days later and vessels were explanted from the vein-to-graft anastomosis of both sides and underwent immunohistochemical analysis, western blotting and zymography for HIF-1α, MMP-2, MMP-9, TIMP-1 and TIMP-2. BOEC were also made hypoxic and normoxic for 12, 24 and 48 h to determine protein expression for MMPs and TIMPs.
Results. Under hypoxia, BOEC significantly increased the expression of pro MMP-2 by 12 h and TIMP-2 by 24 h when compared to normoxic cells (P < 0.05). Transplantation of BOEC resulted in a significant decrease in both HIF-1α and intima-to-media ratio with a significant increase in both pro and active MMP-9 when compared to control vessels (P < 0.05). MMP-9 activity was localized to the neointima of the transplanted vessels by immunohistochemistry. There was increased CD31 density with engraftment of BOEC cells into the neointima of both the transplanted vessels compared to controls (P = NS).
Conclusion. Transplantation of BOEC resulted in a significant decrease in intimal hyperplasia and HIF-1α with a significant increase in both pro and active MMP-9 that was localized to the neointima of transplanted vessels. The increase in MMP-9 offers a possible mechanism for angiogenesis and the reduced intima-to-media ratio. Furthermore, we observed that BOEC had homed to the neointima of the contralateral vessels that had increased levels of HIF-1α, suggesting that hypoxia may be an important stimulus for BOEC migration.
blood outgrowth endothelial cells; haemodialysis graft failure; hypoxia; restenosis; vascular biology
We hypothesized that 1) neointimal formation in a rat carotid balloon injury model could be reduced in vivo following targeted ultrasound delivery of rapamycin-loaded microbubbles (RMBs), and 2) the addition of dual mode ultrasound decreases the total amount of drug needed to reduce neointima formation.
Methods and Results
Balloon injury was performed in rat carotids to induce neointima formation. High or low doses of RMBs were injected I.V. and ruptured at the site of injury with ultrasound. Compared to non-treated injured arteries, neointima formation was reduced by 0% and 35.9% with 108 RMBs, and by 28.7% and 34.9% in arteries treated with 109 RMBs with and without ultrasound respectively.
Without ultrasound, 10-fold higher concentrations of RMBs were needed to reduce neointima formation by at least 28%, whereas 108 RMBs combined with ultrasound were sufficient to achieve the same therapeutic effect demonstrating that this technology may have promise for localized potent drug therapy.
Balloon Injury; Ultrasound; Microbubbles; Drug Delivery; Rats
Atherosclerotic disease is a leading cause of morbidity and mortality in developed
countries, and oxidized LDL (OxLDL) plays a key role in the formation, rupture, and
subsequent thrombus formation in atherosclerotic plaques. In the current study,
anti-mouse OxLDL polyclonal antibody and nonspecific IgG antibody were conjugated to
polyethylene glycol-coated ultrasmall superparamagnetic iron oxide (USPIO)
nanoparticles, and a carotid perivascular collar model in apolipoprotein E-deficient
mice was imaged at 7.0 Tesla MRI before contrast administration and at 8 h and 24 h
after injection of 30 mg Fe/kg. The results showed MRI signal loss in the carotid
atherosclerotic lesions after administration of targeted anti-OxLDL-USPIO at 8 h and
24 h, which is consistent with the presence of the nanoparticles in the lesions.
Immunohistochemistry confirmed the colocalization of the OxLDL/macrophages and iron
oxide nanoparticles. The nonspecific IgG-USPIO, unconjugated USPIO nanoparticles, and
competitive inhibition groups had limited signal changes (p <
0.05). This report shows that anti-OxLDL-USPIO nanoparticles can be used to directly
detect OxLDL and image atherosclerotic lesions within 24 h of nanoparticle
administration and suggests a strategy for the therapeutic evaluation of
atherosclerotic plaques in vivo.
atherosclerosis; molecular imaging; magnetic resonance imaging; low density lipoprotein
Vascular smooth muscle cell migration, proliferation, and differentiation are central to blood vessel development. Since neointimal formation after vascular injury may require the reexpression of a smooth muscle developmental sequence, we examined the expression of H19, a developmentally regulated gene, in rat blood vessels. Expression of the H19 gene is associated with the differentiation process that takes place during development of many tissues. Consistent with this, H19 was highly expressed in the 1-d-old rat aorta but was undetectable in the adult. H19 transcripts were only minimally detected in uninjured carotid artery but were abundant at 7 and 14 d after injury and were localized by in situ hybridization, primarily to the neointima. H19 transcript were undetectable in proliferating neointimal cells in culture but became highly abundant in postconfluent, differentiated neointimal cells. H19 transcripts were only minimally expressed in adult medial smooth muscle cells grown under the identical conditions. Thus, H19 may play an important role in the normal development and differentiation of the blood vessel and in the phenotypic changes of the smooth muscle cells, which are associated with neointimal lesion formation. The vascular injury model may be a useful system to use in examining the function of H19.
Inflammation is important to vascular repair following injury, modulating neointimal proliferation and remodeling. Previously, we have shown that a low-intensity inflammatory response aggravates neointimal formation following balloon and stent injury. The present study examined whether modulation of the extent and timing of nonspecific inflammation mediates the local vascular response in an additive unidirectional or rather a bidirectional fashion.
Methods and results
Rabbits subjected to denudation and balloon injury of the iliac artery were treated with low(1 µg/kg) or high (100µg/kg) doses of bacterial endotoxin (LPS) immediately after injury, or with early high-dose LPS administered 3 days prior to injury (preconditioning). Neointimal formation at 28 days was significantly increased in the low-dose group (0.537 ± 0.059mm2) as compared with controls (0.3 ± 0.03mm2). High-dose LPS did not significantly affect neointimal formation while early high dose significantly reduced neointima (0.296 ± 0.033 and 0.194 ± 0.025mm2, respectively, n = 12–14/group). Arterial wall and systemically circulating interleukin-1β levels, and monocyte CD14 activation correlated with neointimal formation. Vascular remodeling was accelerated in animals treated with low- or high-dose LPS while not affected in the preconditioned group. Remodeling index inversely correlated with arterial matrix metalloproteinase-2 levels 6 days after injury.
The extent and timing of nonspecific inflammation that is concurrent with vascular injury can determine different and opposite vascular repair patterns.
Inflammation; Vascular injury; Neointima; Restenosis
Occupation has been linked to cardiovascular disease (CVD) incidence and mortality, but few studies have investigated occupation in relation to early atherosclerotic disease. This study examined associations between various occupational characteristics and carotid artery intima-media thickness (IMT) in a multi-ethnic sample.
The Multi-Ethnic Study of Atherosclerosis (MESA) recruited 6814 adults aged 45e84 years and free of clinical CVD (response rate 60%, 51% female). Questionnaire data were used to determine occupational group (managerial/professional, sales/office, service, blue-collar), psychosocial job characteristics (ie, job demands, job control) and other sociodemographic information.
Common carotid artery (CCA)-IMT was greater for blue-collar jobs than for management/professional jobs (mean difference=0.012 mm, p=0.049) after adjustment for age, sex, race, place of birth (US or foreign born) and CVD risk factors. Compared to management/professional jobs, internal carotid artery (ICA)-IMT was greater for sales/office, service and blue-collar jobs (mean difference=0.071 mm, p<0.001; 0.057 mm, p=0.009; and 0.110 mm, p<0.001, respectively) after adjustment for age, sex, race and place of birth. The difference between blue-collar jobs and management/professional jobs remained significant after additional adjustment for CVD risk factors, income and education (mean difference=0.048 mm, p=0.045). Higher levels of control at work were associated with thinner CCA-IMT (mean difference=‒0.009 mm, p=0.016, adjusted for age, sex, race and place of birth) but not with ICA-IMT. Job demands had no significant association with IMT.
Blue-collar jobs and low levels of job control were associated with the development of subclinical atherosclerosis.
A periadventitial polymer system is an alternative local drug delivery technique to obtain and maintain high tissue levels of the drug at the site of vascular injury. To determine if local periadventitial delivery of dexamethasone decreases neointimal proliferation after balloon vascular injury, in three groups of Sprague-Dawley rats, 5% dexamethasone, 0.5% dexamethasone, and placebo silicone polymers were implanted around the left common carotid artery after balloon injury. In a fourth group, placebo polymers were implanted without balloon injury. Dexamethasone serum and tissue levels after polymer implantation were significantly higher in the 5% dexamethasone group compared with the 0.5% dexamethasone group. There was no neointima formation in any of the arterial segments covered with placebo polymers for 3 wk, but without balloon injury. In the arterial segments covered by the 5 and 0.5% dexamethasone polymers, there was a 76 and 75% reduction in intima/media ratios, respectively, compared with the placebo group (5% dexamethasone, 0.26 +/- 0.04; 0.5% dexamethasone, 0.27 +/- 0.03; placebo, 1.09 +/- 0.16, respectively; P < 0.0001). These results suggest that: (a) silicone polymers wrapped around the common carotid arteries for 3 wk did not, without balloon injury, stimulate neointimal proliferation in the rat model; (b) the activity of the drug-eluting polymer for suppressing intimal proliferation was chiefly, but not exclusively, site specific; and (c) transadventitial local delivery of dexamethasone at two different doses markedly inhibits neointimal proliferation after balloon vascular injury.