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.

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.

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.

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.

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.

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.

Caveolin-1 influences vascular protease activity and is a potential stabilizing factor in human atherosclerotic disease.

Caveolin-1 (Cav-1) is a regulatory protein of the arterial wall, but its role in human atherosclerosis remains unknown. We have studied the relationships between Cav-1 abundance, atherosclerotic plaque characteristics and clinical manisfestations of atherosclerotic disease.We determined Cav-1 expression by western blotting in atherosclerotic plaques harvested from 378 subjects that underwent carotid endarterectomy. Cav-1 levels were significantly lower in carotid plaques than non-atherosclerotic vascular specimens. Low Cav-1 expression was associated with features of plaque instability such as large lipid core, thrombus formation, macrophage infiltration, high IL-6, IL-8 levels and elevated MMP-9 activity. Clinically, a down-regulation of Cav-1 was observed in plaques obtained from men, patients with a history of myocardial infarction and restenotic lesions. Cav-1 levels above the median were associated with absence of new vascular events within 30 days after surgery [0% vs. 4%] and a trend towards lower incidence of new cardiovascular events during longer follow-up. Consistent with these clinical data, Cav-1 null mice revealed elevated intimal hyperplasia response following arterial injury that was significantly attenuated after MMP inhibition. Recombinant peptides mimicking Cav-1 scaffolding domain (Cavtratin) reduced gelatinase activity in cultured porcine arteries and impaired MMP-9 activity and COX-2 in LPS-challenged macrophages. Administration of Cavtratin strongly impaired flow-induced expansive remodeling in mice. This is the first study that identifies Cav-1 as a novel potential stabilizing factor in human atherosclerosis. Our findings support the hypothesis that local down-regulation of Cav-1 in atherosclerotic lesions contributes to plaque formation and/or instability accelerating the occurrence of adverse clinical outcomes. Therefore, given the large number of patients studied, we believe that Cav-1 may be considered as a novel target in the prevention of human atherosclerotic disease and the loss of Cav-1 may be a novel biomarker of vulnerable plaque with prognostic value.

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.

Intraobserver and interobserver variability and spatial differences in histologic examination of carotid endarterectomy specimens.

INTRODUCTION: Studies using histologic examination and protein analysis of atherosclerotic plaques are increasingly being performed, but reproducibility of plaque histology and variation of plaque composition among different parts of the plaque, which are key to reliability of these studies, are relatively unexplored. Therefore, this study investigated the intraobserver and interobserver variability of plaque histology and spatial variability in plaque composition. METHODS: Atherosclerotic plaques (n = 100) obtained during carotid endarterectomy were divided into 0.5-cm segments. Paraffin sections were stained and semiquantitatively analyzed (four categories: no, minor, moderate, and heavy) for fat, macrophages, smooth muscle cells, collagen, calcification, thrombus, and overall phenotype. First, to determine the intraobserver and interobserver reproducibility, two independent observers independently analyzed the plaques. Second, to investigate spatial variability in plaque composition, histologic appearances of the culprit lesions (0-segment) were compared with the histologic appearances of adjacent (+5 mm) and more distant (+10 mm) plaque segments of 30 specimens. RESULTS: The kappa values for intraobserver variability of fat, macrophages, smooth muscle cells, collagen, calcifications, thrombus, and overall phenotype were 0.83, 0.85, 0.71, 0.63, 0.81, 0.80, and 0.86, respectively, and kappa values for interobserver variability were 0.68, 0.74, 0.54, 0.59, 0.82, 0.75, and 0.71, respectively. Comparison of the histologic scorings of adjacent segments revealed a mean kappa of 0.40 (range, 0.33 to 0.60). When the culprit segment was compared with the more distant segment, the mean kappa was 0.24; however, in 91% of cases, the difference between the culprit segment and the distal segment was one category or less. CONCLUSION: Semiquantitative analysis of carotid atherosclerotic plaque histology was well reproducible, both intraobserver and interobserver. Although variation between different plaque segments in histologic appearance was observed, differences were small in almost all cases. Variability in histologic examination needs to be taken into account in studies comparing plaque imaging with histopathology and plaque research studies.

Levels of extra domain A containing fibronectin in human atherosclerotic plaques are associated with a stable plaque phenotype.

BACKGROUND: Extra domain A (EDA), splice-variant of fibronectin, is a Toll-like receptor 4 (Tlr4) ligand. Recently, EDA has been demonstrated to enhance atherogenesis in mice but human data on the role of EDA in atherosclerotic disease are lacking. We hypothesized that EDA is associated with unstable plaque phenotypes and that plasma EDA could serve as biomarker for atherosclerosis. METHODS: EDA levels were assessed in carotid endarterectomy specimen (206 patients) and related with plaque phenotype. In a second patient cohort, systemic EDA levels in atherosclerotic patients (73 patients) were compared to risk-factor matched controls (68 patients). RESULTS: EDA plaque levels were associated with characteristics of stable plaques; more smooth muscle cells (P=0.003), more collagen (P=0.071) and less fat (P=0.023). Concomitantly, asymptomatic patients showed higher EDA values in the plaque compared to symptomatic patients (P=0.004). EDA plasma levels did not differ between atherosclerotic patients versus controls (P=0.134). CONCLUSION: EDA plaque levels are higher in asymptomatic patients and are associated with a stable plaque phenotype. EDA is not a plasma marker for atherosclerotic disease. These results suggest that local presence of endogenous Tlr4 ligand EDA is not associated with in an unstable plaque phenotype in humans.

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.

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.

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.

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.

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.

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.

Increase in collagen turnover but not in collagen fiber content is associated with flow-induced arterial remodeling.

BACKGROUND: Degradation and synthesis of collagen are common features in arterial geometrical remodeling. Previous studies described an association between arterial remodeling and an increase in collagen fiber content after balloon injury. However, this does not exclude that the association between collagen content and remodeling depends on arterial injury since the association of collagen fiber content and arterial remodeling, without arterial injury, has not been investigated. The aim of the present study was to study the relation between flow-induced arterial geometrical remodeling, without arterial injury, and collagen synthesis and degradation, collagen fiber content and cell-migration-associated moesin levels. METHODS AND RESULTS: In 23 New Zealand White rabbits an arteriovenous shunt (AV shunt) was created in the carotid and femoral artery to induce a structural diameter increase or a partial ligation (n = 27 rabbits) to induce a diameter decrease. In both models, arterial remodeling was accompanied by increased procollagen synthesis, reflected by increased procollagen mRNA or Hsp47 protein levels. In both models, however, no changes were detected in collagen fiber content. Active MMP-2 and moesin levels were increased after AV shunting. CONCLUSIONS: Collagen synthesis and MMP-2 activation were associated with arterial remodeling. However, a change in collagen fiber content was not observed. These results suggest that, during flow-induced geometrical arterial remodeling, increases in collagen synthesis are used for matrix collagen turnover and cell migration but not to augment collagen fiber content.

Local overexpression of C-type natriuretic peptide ameliorates vascular adaptation of porcine hemodialysis grafts.

BACKGROUND: Outflow obstruction at the outflow tract of arteriovenous grafts contributes significantly to the poor patency rates of dialysis grafts in vivo. We addressed the potential of local periadventitial gene therapy at the outflow tract for improving access patency in a validated porcine model of arteriovenous grafts using an adenoviral vector encoding murine C-type natriuretic peptide (Ad.CNP). METHODS: Gene transfer efficiency and optimal virus concentration were determined using Ad.LacZ on porcine jugular veins in vivo (N= 2). Next, in 14 pigs, arteriovenous grafts were implanted bilaterally between the carotid artery and the jugular vein, followed local venous transduction with Ad.CNP (right) and Ad.mock (left). Transduction efficiency of Ad.CNP was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and cyclic guanosine monophosphate (cGMP) measurements (N= 2). Fourteen days after gene transfer, arteriovenous grafts were excised for histologic analysis (N= 12). RESULTS: Ad.LacZ transduction (1 x 10E10 IU) of porcine veins resulted in evident expression of beta-galactosidase, mainly in the adventitia. At termination, intima/media ratio was decreased by 37% in CNP-treated veins, predominantly due to medial thickening (Ad.CNP 3.1 +/- 0.6 mm(2) vs. Ad.mock 1.70 +/- 0.3 mm(2); P < 0.01) rather than decreased intimal hyperplasia (NS). Adventitial delivery of CNP resulted in increased external elastic lamina (EEL) (Ad.CNP 11.8 +/- 1.4 mm vs. Ad.mock 9.4 +/- 1.0 mm; P= 0.04) and luminal area (Ad.CNP 10.7 +/- 1.4 mm(2) vs. Ad.mock 8.8 +/- 1.7 mm(2); P= 0.05) at the venous anastomosis. CONCLUSION: Overexpression of CNP enhances venous medial thickening and increases outward remodeling in the outflow tract of porcine arteriovenous grafts. These findings underscore the potential of local gene-therapeutic interventions in preventing luminal narrowing at the outflow tract of hemodialysis grafts.

Matrix metalloproteinase inhibition reduces intimal hyperplasia in a porcine arteriovenous-graft model.

BACKGROUND: The patency of arteriovenous (AV) polytetrafluoroethylene grafts for hemodialysis is impaired by intimal hyperplasia (IH) at the venous outflow tract. IH mainly consists of vascular smooth muscle cells, fibroblasts, and extracellular matrix proteins. Because matrix metalloproteinases (MMPs) are enzymes able to degrade extracellular matrix proteins such as elastin and collagen and also stimulate migration of vascular smooth muscle cells, we hypothesized that BB2983 (a broad-spectrum MMP inhibitor) could reduce IH in AV grafts. METHODS: In 12 pigs, AV grafts were created bilaterally between the carotid artery and the jugular vein. Six pigs received the oral MMP inhibitor (MMPi), and six pigs served as a control. Four weeks after AV shunting, the grafts and adjacent vessels were excised and underwent histologic analysis. Quantification of elastin content was performed on Elastin von Gieson-stained sections. RESULTS: At the venous outflow tract, IH was strongly inhibited after MMPi when compared with the control group (1.02 +/- 0.26 mm(2) vs 2.14 +/- 0.38 mm(2); P =.027). The medial area did not differ significantly. In the control group elastin density decreased compared with nonoperated veins. This decrease was not observed in the MMPi group (nonoperated, 6.3% +/- 0.4%; MMPi, 7.2% +/- 0.7% vs untreated, 3.6% +/- 0.5%; P =.0004). Outward remodeling of the vein was not influenced by MMP inhibition. CONCLUSION: MMPi reduces IH formation at the venous outflow tract of AV grafts in pigs, probably by inhibiting elastin degradation. These data suggest that MMP inhibitors might be useful for minimizing IH in AV grafts, thus prolonging patency rates of AV grafts in patients on hemodialysis.

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.