Time-dependent changes in atherosclerotic plaque composition in patients undergoing carotid surgery.

BACKGROUND: Time-dependent trends in the incidence of cardiovascular disease have been reported in high-income countries. Because atherosclerosis underlies the majority of cardiovascular diseases, we investigated temporal changes in the composition of atherosclerotic plaques removed from patients undergoing carotid endarterectomy. METHODS AND RESULTS: The Athero-Express study is an ongoing, longitudinal, vascular biobank study that includes the collection of atherosclerotic plaques of patients undergoing primary carotid endarterectomy in the province of Utrecht from 2002 to 2011. Histopathologic features of plaques of 1583 patients were analyzed in intervals of 2 years. The analysis included quantification of collagen, calcifications, lipid cores, plaque thrombosis, macrophages, smooth muscle cells, and microvessels. Large atheroma, plaque thrombosis, macrophages, and calcifications were less frequently observed over time, with adjusted odds ratios of 0.72 (95% confidence interval, 0.650-0.789), 0.62 (95% confidence interval, 0.569-0.679), 0.87 (95% confidence interval, 0.800-0.940), and 0.75 (95% confidence interval, 0.692-0.816) per 2-year increase in time, respectively. These changes in plaque characteristics were consistently observed in patient subgroups presenting with stroke, transient ischemic attack, ocular symptoms, and asymptomatic patients. Concomitantly, risk factor management and secondary prevention strategies among vascular patients scheduled for carotid endarterectomy significantly improved over the past decade. CONCLUSIONS: In conclusion, over the past decade, atherosclerotic plaques harvested during carotid endarterectomy show a time-dependent change in plaque composition characterized by a decrease in features currently believed to be causal for plaque instability. This appears to go hand in hand with improvements in risk factor management.

Composition of carotid atherosclerotic plaque is associated with cardiovascular outcome: a prognostic study.

BACKGROUND: Identification of patients at risk for primary and secondary manifestations of atherosclerotic disease progression is based mainly on established risk factors. The atherosclerotic plaque composition is thought to be an important determinant of acute cardiovascular events, but no prospective studies have been performed. The objective of the present study was to investigate whether atherosclerotic plaque composition is associated with the occurrence of future vascular events. METHODS AND RESULTS: Atherosclerotic carotid lesions were collected from patients who underwent carotid endarterectomy and were subjected to histological examination. Patients underwent clinical follow-up yearly, up to 3 years after carotid endarterectomy. The primary outcome was defined as the composite of a vascular event (vascular death, nonfatal stroke, nonfatal myocardial infarction) and vascular intervention. The cumulative event rate at 1-, 2-, and 3-year follow-up was expressed by Kaplan-Meier estimates, and Cox proportional hazards regression analyses were performed to assess the independence of histological characteristics from general cardiovascular risk factors. During a mean follow-up of 2.3 years, 196 of 818 patients (24%) reached the primary outcome. Patients whose excised carotid plaque revealed plaque hemorrhage or marked intraplaque vessel formation demonstrated an increased risk of primary outcome (risk difference=30.6% versus 17.2%; hazard ratio [HR] with [95% confidence interval]=1.7 [1.2 to 2.5]; and risk difference=30.0% versus 23.8%; HR=1.4 [1.1 to 1.9], respectively). Macrophage infiltration (HR=1.1 [0.8 to 1.5]), large lipid core (HR=1.1 [0.7 to 1.6]), calcifications (HR=1.1 [0.8 to 1.5]), collagen (HR=0.9 [0.7 to 1.3]), and smooth muscle cell infiltration (HR=1.3 [0.9 to 1.8]) were not associated with clinical outcome. Local plaque hemorrhage and increased intraplaque vessel formation were independently related to clinical outcome and were independent of clinical risk factors and medication use. CONCLUSIONS: The local atherosclerotic plaque composition in patients undergoing carotid endarterectomy is an independent predictor of future cardiovascular events.

Neutral Effects of Combined Treatment With GLP-1R Agonist Exenatide and MR Antagonist Potassium Canrenoate on Cardiac Function in Porcine and Murine Chronic Heart Failure Models.

Background: Ischemia-reperfusion and cardiac remodeling is associated with cardiomyocyte death, excessive fibrosis formation, and functional decline, eventually resulting in heart failure (HF). Glucagon-like peptide (GLP)-1 agonists are reported to reduce apoptosis and myocardial infarct size after ischemia-reperfusion. Moreover, mineralocorticoid receptor antagonists (MRAs) have been described to reduce reactive fibrosis and improve cardiac function. Here, we investigated whether combined treatment with GLP-1R agonist exenatide and MRA potassium canrenoate could minimize cardiac injury and limit HF progression in animal models of chronic HF. Methods and Results: Forty female Topigs Norsvin pigs were subjected to 150 min balloon occlusion of the left anterior descending artery (LAD). Prior to reperfusion, pigs were randomly assigned to placebo or combination therapy (either low dose or high dose). Treatment was applied for two consecutive days or for 8 weeks with a continued high dose via a tunneled intravenous catheter. Using 2,3,5-Triphenyltetrazolium chloride (TTC) staining we observed that combination therapy did not affect the scar size after 8 weeks. In line, left ventricular volume and function assessed by three-dimensional (3D) echocardiography (baseline, 7 days and 8 weeks), and cardiac magnetic resonance imaging (CMR, 8 weeks) did not differ between experimental groups. In addition, 36 C57Bl/6JRj mice underwent permanent LAD-occlusion and were treated with either placebo or combination therapy prior to reperfusion, for two consecutive days via intravenous injection, followed by continued treatment via placement of osmotic mini-pumps for 28 days. Global cardiac function, assessed by 3D echocardiography performed at baseline, 7, 14, and 28 days, did not differ between treatment groups. Also, no differences were observed in cardiac hypertrophy, assessed by heart weight/bodyweight and heart weight/tibia length ratio. Conclusion: In the current study, combined treatment with GLP-1R agonist exenatide and MR antagonist potassium canrenoate did not show beneficial effects on cardiac remodeling nor resulted in functional improvement in a small and large animal chronic HF model.

Histologic atherosclerotic plaque characteristics are associated with restenosis rates after endarterectomy of the common and superficial femoral arteries.

OBJECTIVES: This study assessed the predictive value of histologic plaque characteristics for the occurrence of restenosis after femoral artery endarterectomy. BACKGROUND: It would be advantageous if patients at increased risk for restenosis after arterial endarterectomy could be identified by histologic characteristics of the dissected plaque. Differences in atherosclerotic plaque composition of the carotid artery have been associated with restenosis rates after surgical endarterectomy. However, whether atherosclerotic plaque characteristics are also predictive for restenosis in other vascular territories is unknown. METHODS: Atherosclerotic plaques of 217 patients who underwent a common femoral artery endarterectomy (CFAE; n = 124) or remote superficial femoral artery endarterectomy (RSFAE; n = 93) were examined and scored microscopically for the presence of collagen, macrophages, smooth muscle cells, lipid core, intraplaque hemorrhage, and calcifications. The 12-month restenosis rate was assessed using duplex ultrasound imaging (peak systolic velocity [PSV] ratio >or=2.5). RESULTS: The 1-year restenosis rate was 66% (61 of 93) after RSFAE compared to 21% (26 of 124) after CFAE. Plaque with characteristics of high collagen and smooth muscle cell content were positively associated with the occurrence of restenosis, with odds ratios (ORs) of 2.90 (95% confidence interval [CI], 1.82-4.68) and 2.20 (1.50-3.20) for superficial femoral artery (SFA) and common femoral artery (CFA), respectively. SFA plaques showed significantly heavier staining for collagen (69% vs 31% for CFA; P < .001) and smooth muscle cells (64% vs 36% for CFA; P < .001). After multivariate analysis, the operation type (CFAE or RSFAE), gender, and the presence of collagen were independent predictive variables for restenosis after endarterectomy of the CFA and SFA. CONCLUSION: Plaque composition of the CFA and SFA differs. Furthermore, the dissection of a fibrous collagen-rich plaque is an independent predictive variable for restenosis after endarterectomy of the CFA and SFA.

Atherosclerotic plaque composition and occurrence of restenosis after carotid endarterectomy.

CONTEXT: Previous studies have assessed the predictive value of clinical and angiographic parameters for development of restenosis after vascular interventions. The composition of the atherosclerotic plaque at the intervention site has had limited evaluated as a marker for restenosis [corrected]. OBJECTIVE: To investigate the relationship between atherosclerotic plaque histology and the occurrence of restenosis after carotid endarterectomy. DESIGN, SETTING, AND PATIENTS: The Athero-Express study is a longitudinal vascular biobank study that includes the collection of atherosclerotic plaques of patients undergoing primary carotid endarterectomy. Five hundred patients were prospectively followed up between April 1, 2002, and March 14, 2006, to assess carotid artery restenosis measured by duplex ultrasound 1 year after the intervention. MAIN OUTCOME MEASURES: Risk of carotid restenosis in relation to predefined histological characteristics (macrophage and smooth muscle cell infiltration, collagen, calcifications, intraplaque bleeding, luminal thrombus, and lipid core size), adjusted for clinical characteristics (multivariate logistic regression analysis). RESULTS: At 1 year, 85 patients (17%) developed 50% or greater restenosis, including 40 patients (8%) who developed 70% or greater restenosis of the target vessel. Patients whose histological examination of the plaque revealed marked macrophage infiltration (n = 286) had a lower risk than those with none or minor macrophage infiltration (n = 214) of developing 50% or greater restenosis (risk difference, 11.5% vs 24.3%; adjusted odds ratio [OR], 0.43; 95% confidence interval [CI], 0.26-0.72) and a lower risk of developing 70% or greater restenosis (risk difference, 4.5% vs 12.6%; adjusted OR, 0.36; 95% CI, 0.17-0.74). Patients (n = 177) with a plaque having a large lipid core size (>40%) had a lower risk than those (n = 94) with a plaque having a lipid core size of less than 10% of developing 50% or greater restenosis (risk difference, 11.3% vs 25.5%; adjusted OR, 0.40; 95% CI, 0.19-0.81) and a lower risk of developing 70% or greater restenosis (risk difference, 5.6% vs 14.9%; adjusted OR, 0.42; 95% CI, 0.17-1.04), independent of clinical characteristics. CONCLUSIONS: Plaque composition is an independent predictor of restenosis after carotid endarterectomy. The dissection of a lipid-rich, inflammatory plaque is associated with reduced risk of restenosis.

Sirolimus-eluting stents to abolish intimal hyperplasia and improve flow in porcine arteriovenous grafts: a 4-week follow-up study.

BACKGROUND: The patency of arteriovenous (AV) expanded polytetrafluoroethylene (ePTFE) hemodialysis grafts is severely compromised by intimal hyperplasia (IH) at the venous anastomosis and in the venous outflow tract. We addressed the potential of primary placement of a sirolimus-eluting stent (SES) in a validated porcine model. METHODS AND RESULTS: In 25 pigs, ePTFE AV grafts were created bilaterally between the carotid artery and the jugular vein, whereupon a self-expandable nitinol stent (14 SESs and 11 bare-metal stents) was implanted over the venous anastomosis in 1 of the 2 grafts. After exclusion of technical failures and 1 unilateral occlusion, 16 pigs (9 SESs and 7 bare-metal stents) were included for further analysis. After 28 days, we measured graft flow and performed quantitative angiography. The pigs were then euthanized, and grafts with adjacent vessels were excised for histological analysis. Minimal luminal diameter was substantially larger in the SES group compared with unstented controls (5.9+/-0.2 versus 3.8+/-0.4 mm, respectively, P=0.01), which was accompanied by more prominent graft flow (SES, 1360+/-89 mL/min versus unstented, 861+/-83 mL/min, P=0.05). IH at the venous anastomosis was 77% less in the SES group compared with unstented controls (0.44+/-0.05 versus 1.92+/-0.5 mm2, respectively, P=0.01), whereas IH increased markedly when bare-metal stents were used (5.7+/-1.4 mm2, P=0.05). CONCLUSIONS: SESs in the venous outflow of AV grafts significantly reduce IH and increase vessel diameter and graft flow compared with unstented grafts. These findings suggest that SESs have the potential to improve primary patency of AV grafts in hemodialysis patients.

In vivo cell seeding with anti-CD34 antibodies successfully accelerates endothelialization but stimulates intimal hyperplasia in porcine arteriovenous expanded polytetrafluoroethylene grafts.

BACKGROUND: The patency of AV expanded polytetrafluoroethylene (ePTFE) grafts for hemodialysis is impaired by intimal hyperplasia (IH) at the venous outflow tract. The absence of a functional endothelial monolayer on the prosthetic grafts is an important stimulus for IH. In the present study, we evaluated the feasibility of capturing endothelial progenitor cells in vivo using anti-CD34 antibodies on ePTFE grafts to inhibit IH in porcine AV ePTFE grafts. METHODS AND RESULTS: In 11 pigs, anti-CD34-coated ePTFE grafts were implanted between the carotid artery and internal jugular vein. Bare ePTFE grafts were implanted at the contralateral side. After 3 (n=2) or 28 (n=9) days, the pigs were terminated, and the AV grafts were excised for histological analysis and SEM. At 3 and 28 days after implantation, 95% and 85% of the coated graft surface was covered by endothelial cells. In contrast, no cell coverage was observed in the bare graft at 3 days, whereas at 28 days, bare grafts were partly covered with endothelial cells (32%; P=0.04). Twenty-eight days after implantation, IH at the venous anastomosis was strongly increased in anti-CD34-coated grafts (5.96+/-1.9 mm2) compared with bare grafts (1.70+/-0.4 mm2; P=0.03). This increase in IH coincided with enhanced cellular proliferation at the venous anastomosis. CONCLUSIONS: Autoseeding with anti-CD34 antibodies results in rapid endothelialization within 72 hours. Despite persistent endothelial graft coverage, IH at the outflow tract is increased profoundly at 4 weeks after implantation. Further modifications are required to stimulate the protective effects of trapped endothelial cells.

Matrix metalloproteinase 2 is associated with stable and matrix metalloproteinases 8 and 9 with vulnerable carotid atherosclerotic lesions: a study in human endarterectomy specimen pointing to a role for different extracellular matrix metalloproteinase inducer glycosylation forms.

BACKGROUND AND PURPOSE: We studied matrix metalloproteinases (MMP) 2, 8, and 9 and extracellular matrix metalloproteinase inducer (EMMPRIN) levels in relation to carotid atherosclerotic plaque characteristics. METHODS: Carotid atherosclerotic plaques (n=150) were stained and analyzed for the presence of collagen, smooth muscle cell (SMC), and macrophages. Adjacent segments were used to isolate total protein to assess MMP-2 and MMP-9 activities and gelatin breakdown, MMP-8 activity, and EMMPRIN levels. RESULTS: Macrophage-rich lesions revealed higher MMP-8 and MMP-9 activities, whereas SMC-rich lesions showed higher MMP-2 activity. The levels of less glycosylated EMMPRIN-45kD were higher in SMC-rich lesions and lower in macrophage-rich plaques. EMMPRIN-45kD was associated with MMP-2 levels, whereas EMMPRIN-58kD was related to MMP-9 levels. CONCLUSIONS: MMP-2, MMP-8, and MMP-9 activities differed among carotid plaque phenotypes. Different EMMPRIN glycosylation forms are associated with either MMP-2 or MMP-9 activity, which suggests that EMMPRIN glycosylation may play a role in MMP regulation and plaque destabilization.

Statin treatment is not associated with consistent alterations in inflammatory status of carotid atherosclerotic plaques: a retrospective study in 378 patients undergoing carotid endarterectomy.

BACKGROUND AND PURPOSE: Anti-inflammatory qualities are held partially responsible for the reduction of cardiovascular events after statin treatment. We examined the phenotype of carotid atherosclerotic plaques harvested during carotid endarterectomy in relation to the previous use of different statins prescribed in clinical practice. METHODS: Three hundred and seventy-eight patients were included. Atherosclerotic plaques were harvested, immunohistochemically stained and semiquantitively examined for the presence of macrophages (CD68), smooth muscle cells, collagen and fat. Adjacent atherosclerotic plaques were used to study protease activity and interleukin levels. Patients' demographics were recorded and blood samples were stored. RESULTS: Serum cholesterol, low-density lipoprotein, apolipoprotein B, and C-reactive protein levels were lower in patients treated with statins compared with patients without statin treatment. Atheromatous plaques were less prevalent in patients receiving statins compared with patients without statin therapy (29% versus 42%, P=0.04). An increase of CD68 positive cells was observed in patients receiving statins compared with nonstatin treatment (P=0.05). This effect was specifically related to atorvastatin treatment. In patients treated with atorvastatin, the increased amount of CD68 positive cells were not associated with increased protease activity. In contrast, a dose-dependent decrease in protease activity was shown in the atorvastatin group. Interleukin 6 expression was lower in plaques obtained from patients treated with statins (P=0.04). CONCLUSIONS: Statin use may exert pleiotropic effects on plaque phenotype. However, not the presence of macrophages but activation with subsequent protease and cytokine release may be attenuated by statin use.

Involvement of furin-like proprotein convertases in the arterial response to injury.

BACKGROUND: Furin-like proprotein convertases (PCs) are proteolytic activators of proproteins, like membrane type 1-matrix metalloproteinase (MT1-MMP) and transforming growth factor beta (TGF-beta), that are described in the arterial response to injury. However, the involvement of furin-like PCs in the arterial response to injury has not been studied yet. We studied furin, MT1-MMP, MMP levels and TGF-beta signaling after arterial injury. We also investigated the effect of an inhibitor of furin-like PCs, alpha1-antitrypsin Portland (alpha1-PDX), on arterial injury following balloon dilation. METHODS AND RESULTS: NZW rabbit femoral and iliac arteries (N=42) were balloon dilated unilaterally and harvested after 2, 7, 14, 28 or 42 days. Furin mRNA levels were increased after 2 and 7 days. MMP-2 and MT1-MMP levels were increased after day 7 and TGF-beta signaling, by phosphorylating Smad 1/5 and 2/3, was increased at all time points. Inhibition of furin-like PCs, by adenoviral over-expression of alpha1-PDX, blocked proTGF-beta activation and Smad phosphorylation, and reduced MT1-MMP and MMP-2 activation (N=5). In vivo adventitial inhibition of furin-like PCs (N=9) resulted in a reduction of 13.1+/-5.2% in advential and 23.6+/-7.9% in intimal areas (P<0.05), but had no effect on lumen size due to decreased vessel areas. CONCLUSIONS: This study demonstrates that furin-like PCs are involved in the arterial response to injury possibly through activation of the TGF-beta-Smad signaling pathway and identifies furin-like PCs as a possible target to inhibit intimal hyperplasia.

Identification of atherosclerotic plaque components with intravascular ultrasound elastography in vivo: a Yucatan pig study.

BACKGROUND: Intravascular ultrasound elastography assesses the local strain of the atherosclerotic vessel wall. In the present study, the potential to identify different plaque components in vivo was investigated. METHODS AND RESULTS: Atherosclerotic external iliac and femoral arteries (n=24) of 6 Yucatan pigs were investigated. Before termination, elastographic data were acquired with a 20-MHz Visions catheter. Two frames acquired at end-diastole with a pressure differential of approximately 4 mm Hg were acquired to obtain the elastograms. Before dissection, x-ray was used to identify the arterial segments that had been investigated by ultrasound. Specimens were stained for collagen, fat, and macrophages. Plaques were classified as absent, early fibrous lesion, early fatty lesion, or advanced fibrous plaque. The average strains in the plaque-free arterial wall (0.21%) and the early (0.24%) and advanced fibrous plaques (0.22%) were similar. Higher average strain values were observed in fatty lesions (0.46%) compared with fibrous plaques (P=0.007). After correction for confounding by lipid content, no additional differences in average strain were found between plaques with and without macrophages (P=0.966). Receiver operating characteristic analysis revealed a sensitivity and a specificity of 100% and 80%, respectively, to identify fatty plaques. The presence of a high-strain spot (strain >1%) has 92% sensitivity and 92% specificity to identify macrophages. CONCLUSIONS: To the best of our knowledge, this is the first time that intravascular ultrasound elastography has been validated in vivo. Fatty plaques have an increased mean strain value. High-strain spots are associated with the presence of macrophages.

Age-related changes in plaque composition: a study in patients suffering from carotid artery stenosis.

OBJECTIVE: The extent of atherosclerotic plaque burden and the incidence of atherosclerosis-related cardiovascular events accelerate with increasing age. The composition of the plaque is associated with plaque thrombosis and acute coronary occlusion. Surprisingly, however, the relation between advancing age and atherosclerotic plaque composition is still unclear. In the present study, we investigated the association between plaque characteristics and advancing age in a population of patients with haemodynamically significant carotid artery stenosis. METHODS: Patients (N=383), ages 39-89 years, underwent carotid endarterectomy (CEA). Morphometric analysis was performed on the dissected atherosclerotic plaques to study the prevalence of fibrous and atheromatous plaques. Picro sirius red, haematoxylin eosin, alfa actin and CD68 stainings were performed to investigate the extent of collagen, calcification, smooth muscle cells and macrophages in carotid plaques, respectively. The presence of metalloproteinases-2 and -9 was assessed by ELISA. RESULTS: With aging, a decrease in fibrous plaques and an increase in atheromatous plaques were observed. This was accompanied by an age-associated decrease in smooth muscle cell content in carotid plaques. Macrophage content slightly increased with age. In addition, total matrix metalloprotease (MMP)-2 was negatively and MMP-9 positively related with age. Differences in plaque phenotype were most prominent for the youngest age quartile compared with older age quartiles. CONCLUSIONS: With increasing age, the morphology of atherosclerotic plaques from patients with carotid artery stenosis changes. Plaques become more atheromatous and contain less smooth muscle cells with increasing age. Local inflammation and MMP-9 levels slightly increased with age in plaques obtained from patients suffering from haemodynamically significant advanced atherosclerotic lesions.

Acute-phase protein haptoglobin is a cell migration factor involved in arterial restructuring.

Collagen turnover and cell migration are fundamental aspects of arterial restructuring. To identify mRNAs involved in blood flow-induced arterial restructuring, we performed subtraction polymerase chain reaction and found expression of haptoglobin mRNA in adventitial fibroblasts of rabbit arteries. Haptoglobin is highly expressed in liver, but its arterial expression and function are unknown. In vitro studies revealed that stimulation of haptoglobin expression by lipopolysaccharides in mice fibroblasts stimulated migration of wild-type fibroblasts but had no effect on migration of haptoglobin knockout fibroblasts. In vivo studies showed that flow-induced arterial restructuring was delayed in haptoglobin knockout mice. This new function of haptoglobin might be explained by facilitating cell migration through accumulation of a temporary gelatin matrix because cell culture showed that haptoglobin is involved in the breakdown of gelatin. We conclude that haptoglobin is highly expressed in arterial tissue and is involved in arterial restructuring. This new haptoglobin function may also apply to other functional and pathological restructuring processes such as angiogenesis, tissue repair, and tumor cell invasion.

Increased collagen turnover is only partly associated with collagen fiber deposition in the arterial response to injury.

OBJECTIVE: In the arterial response to injury, collagen breakdown has been studied extensively, but little is known on collagen synthesis and fiber formation. Here, we studied in vivo collagen synthesis and collagen fiber content in relation to collagen breakdown following arterial balloon injury. METHODS AND RESULTS: Twenty-five New Zealand White rabbits were balloon dilated in femoral and iliac arteries and terminated at 2, 7, 14 and 28 days. From day 7, both constrictive arterial remodeling and intimal hyperplasia were observed. Collagen degradation, synthesis and fiber content were studied using zymography, quantitative Polymerase Chain Reaction, Western blotting and picrosirius red staining. Collagen synthesis, reflected by procollagen I and heat shock protein 47 (Hsp47) expression, increased at day 2 with a maximum at day 14 and was accompanied by increased collagen breakdown as reflected by matrix metalloproteinase-1 and -2 levels. Collagen content in media and adventitia only increased between days 2 and 7 after balloon injury. CONCLUSIONS: In the first week after arterial injury, increased collagen content is associated with increased collagen synthesis and degradation. However, after 1 week, collagen turnover remains high in contrast to increased collagen fiber content. This suggests that after 1 week, collagen turnover is used for other processes like cell migration and arterial remodeling.

Increased arterial expression of a glycosylated haptoglobin isoform after balloon dilation.

OBJECTIVE: Haptoglobin is a novel cell migration factor that is expressed in arteries after sustained flow changes and involved in arterial restructuring. Arterial restructuring is the major determinant of arterial shrinkage after balloon dilation. Although the function of extrahepatic haptoglobin expression is not yet understood, local haptoglobin expression may provide the tissue with functionally different haptoglobin due to post-translational modifications. We hypothesized that haptoglobin expression is increased during arterial restructuring after balloon dilation and compared glycosylation patterns between arterial and liver haptoglobin. METHODS: Arterial haptoglobin expression was studied in rabbits at 0, 2, 7, 14 and 28 days after balloon dilation (n=36) using real-time polymerase chain reaction, Western blotting and in situ hybridization. Two-dimensional gel electrophoresis and lectin affinity blotting were used to identify liver and arterial haptoglobin glycoforms. RESULTS: Arterial haptoglobin mRNA (5.7-fold, P=0.01) and protein levels (1.4-fold, P=0.01) were increased after balloon dilation whereas liver haptoglobin expression remained constant. Haptoglobin was expressed in the adventitia of balloon dilated rabbit arteries, which was confirmed in human atherosclerotic arteries. Comparison between liver and arterial haptoglobin demonstrated the expression of artery-specific haptoglobin glycoforms. CONCLUSIONS: This study demonstrates that arterial haptoglobin expression is increased early after balloon dilation whereas liver haptoglobin expression does not change. Furthermore, arterial haptoglobin consists of an unique set of glycoforms compared to haptoglobin produced in the liver.

Matrix metalloproteinase inhibition reduces adventitial thickening and collagen accumulation following balloon dilation.

OBJECTIVE: Constrictive arterial remodeling following balloon angioplasty has been related to adventitial collagen accumulation and subsequent thickening and can be prevented by matrix metalloproteinase (MMP) inhibition. Following balloon dilation, we examined the effect of MMP inhibition on collagen turnover and the relationship between adventitial area and degree of constrictive remodeling. METHODS: In 12 non-atherosclerotic landrace pigs, balloon dilation was performed in 39 peripheral arteries with and without MMP inhibition. Follow up with intravascular ultrasound was performed at 42 days. Collagen content was quantified using polarized light and digital image microscopy. Procollagen expression was determined using immunochemistry and Western blotting. RESULTS: In the MMP inhibitor group, constrictive remodeling was inhibited at 42 days follow up. In control and MMP inhibitor groups, a positive relation was observed between adventitial thickness and degree of constrictive remodeling (P<0.001). Adventitial thickening and adventitial collagen content were reduced in the MMP inhibitor group (P=0.002 and P=0.001, respectively). Procollagen immunostaining, but not protein analysis on Western blotting, was decreased in the MMP inhibitor group. CONCLUSION: MMP inhibition impaired adventitial thickening by reduction of collagen content 42 days after balloon dilation. This might explain its inhibitory effect on constrictive remodeling.

Nitric oxide synthesis is involved in arterial haptoglobin expression after sustained flow changes.

The acute phase protein haptoglobin is highly expressed in arteries after sustained flow changes and involved in cell migration and arterial restructuring. In the liver, haptoglobin expression is mainly regulated by interleukin-6 (IL-6). In the artery, shear stress and NO influence IL-6 expression. In the present study, we demonstrate that NO synthesis is involved in the regulation of arterial haptoglobin expression after sustained flow changes. Decreased haptoglobin expression after NO inhibition coincided with decreased IL-6 levels. However, IL-6 knockout mice had normal arterial haptoglobin expression levels after sustained flow changes suggesting that other mediators may provide compensatory mechanisms for the regulation of arterial haptoglobin expression.

UVB-activated psoralen reduces luminal narrowing after balloon dilation because of inhibition of constrictive remodeling.

In this study we have explored the potential of PUVB (8-MOP + UVB) therapy for the reduction of luminal narrowing after arterial injury. In 15 rabbits, balloon dilation of iliac arteries was performed. In 20 arteries, dilation was combined with the delivery of pulsed ultraviolet light B (UVB) irradiation with 10 arteries being previously subjected to sensitizer infusion. Changes in vessel diameter, proliferation and extracellular matrix protein content at 6 weeks were evaluated by means of angiography and histomorphometry-immunohistochemistry. We found that PUVB, applied at the time of dilation, induced reduction in late loss (LL) at 6 weeks (percutaneous transluminal angioplasty vs UVB vs PUVB: 0.64 +/- 0.15 mm vs 0.61 +/- 0.05 mm vs 0.29 +/- 0.05 mm; p = 0.018). The same holds true for constrictive remodeling (0.53 +/- 0.15 mm vs 0.45 +/- 0.06 mm vs 0.15 +/- 0.05 mm; p = 0.016). In the irradiation groups, LL was independent of acute gain (AG), as opposed to the control. Collagen content increased significantly after PUVB in media and adventitia, without increased cellular proliferation in all vessel layers. Thus, PUVB at the time of dilation reduced luminal narrowing at follow-up without effecting proliferation. This effect was independent of AG and was associated with increased collagen content in media and adventitia.

High reproducibility of histological characterization by whole virtual slide quantification; an example using carotid plaque specimens.

OBJECTIVE: Tissue biobanks are an important source for discovery and validation studies aiming for new proteins that are causally related with disease development. There is an increasing demand for accurate and reproducible histological characterization, especially for subsequent analysis and interpretation of data in association studies. We assessed reproducibility of one semiquantative and two quantitative methods for histological tissue characterization. We introduce a new automated method for whole digital slide quantification. Carotid atherosclerotic plaques were used to test reproducibility. METHODS: 50 atherosclerotic plaques that were obtained during carotid endarterectomy were analysed. For the semiquantitative analysis, 6 different plaque characteristics were scored in categories by two independent observers, and Cohen's κ was used to test intra- and interobserver reproducibility. The computer-aided method (assessed by two independent observers) and automated method were tested on CD68 (for macrophages) and α smooth muscle actin (for smooth muscle cells) stainings. Agreement for these two methods (done on a continuous scale) was assessed by intraclass correlation coefficients (ICCs). RESULTS: For the semiquantitative analysis, κ values ranged from 0.55 to 0.69 for interobserver variability, and were slightly higher for intraobserver reproducibility in both observers. The computer-aided method yielded intra- and interobserver ICCs between 0.6 and 0.9. The new automated method performed most optimal regarding reproducibility, with ICCs ranging from 0.92 to 0.97. CONCLUSIONS: The analysis of performance of three methods for histological slide characterization on carotid atherosclerotic plaques showed high precision and agreement in repeated measurements for the automated method for whole digital slide quantification. We suggest that this method can fulfill the need for reproducible histological quantification.

Leukotriene B4 levels in human atherosclerotic plaques and abdominal aortic aneurysms.

BACKGROUND: Leukotriene B4 (LTB4) has been associated with the initiation and progression of atherosclerosis and abdominal aortic aneurysm (AAA) formation. However, associations of LTB4 levels with tissue characteristics and adverse clinical outcome of advanced atherosclerosis and AAA are scarcely studied. We hypothesized that LTB4 levels are associated with a vulnerable plaque phenotype and adverse clinical outcome. Furthermore, that LTB4 levels are associated with inflammatory AAA and adverse clinical outcome. METHODS: Atherosclerotic plaques and AAA specimens were selected from two independent databases for LTB4 measurements. Plaques were isolated during carotid endarterectomy from asymptomatic (n = 58) or symptomatic (n = 317) patients, classified prior to surgery. LTB4 levels were measured without prior lipid extraction and levels were corrected for protein content. LTB4 levels were related to plaque phenotype, baseline patient characteristics and clinical outcome within three years following surgery. Seven non-diseased mammary artery specimens served as controls. AAA specimens were isolated during open repair, classified as elective (n = 189), symptomatic (n = 29) or ruptured (n = 23). LTB4 levels were measured similar to the plaque measurements and were related to tissue characteristics, baseline patient characteristics and clinical outcome. Twenty-six non-diseased aortic specimens served as controls. RESULTS: LTB4 levels corrected for protein content were not significantly associated with histological characteristics specific for vulnerable plaques or inflammatory AAA as well as clinical presentation. Moreover, it could not predict secondary manifestations independently investigated in both databases. However, LTB4 levels were significantly lower in controls compared to plaque (p = 0.025) or AAA (p = 0.017). CONCLUSIONS: LTB4 levels were not associated with a vulnerable plaque phenotype or inflammatory AAA or clinical presentation. This study does not provide supportive evidence for a role of LTB4 in atherosclerotic plaque destabilization or AAA expansion. However, these data should be interpreted with care, since LTB4 measurements were performed without prior lipid extractions.