Protease-activated receptors are potential regulators in the development of arterial endofibrosis in high-performance athletes.

OBJECTIVE: High-performance athletes can develop symptomatic arterial flow restriction during exercise caused by endofibrosis. The pathogenesis is poorly understood; however, coagulation enzymes, such as tissue factor (TF) and coagulation factor Xa, might contribute to the fibrotic process, which is mainly regulated through activation of protease-activated receptors (PARs). Therefore, the aim of this explorative study was to evaluate the presence of coagulation factors and PARs in endofibrotic tissue, which might be indicative of their potential role in the natural development of endofibrosis. METHODS: External iliac arterial specimens with endofibrosis (n = 19) were collected during surgical interventions. As control, arterial segments of the external iliac artery (n = 20) were collected post mortem from individuals with no medical history of cardiovascular disease who donated their body to medical science. Arteries were paraffinized and cut in tissue sections for immunohistochemical analysis. Positive staining within lesions was determined with ImageJ software (National Institutes of Health, Bethesda, Md). RESULTS: Endofibrotic segments contained a neointima, causing intraluminal stenosis, which was highly positive for collagen (+150%; P < .01) and elastin (+148%; P < .01) in comparison with controls. Intriguingly, endofibrosis was not limited to the intima because collagen (+213%) and elastin (+215%) were also significantly elevated in the media layer of endofibrotic segments. These findings were accompanied by significantly increased α-smooth muscle actin-positive cells, morphologically compatible with the presence of myofibroblasts. In addition, PAR1 and PAR4 and the membrane receptor TF were increased as well as coagulation factor X. CONCLUSIONS: We showed that myofibroblasts and the accompanying collagen and elastin synthesis might be key factors in the development of endofibrosis. The special association with increased presence of PARs, factor X, and TF suggests that protease-mediated cell signaling could be a contributing component in the mechanisms leading to endofibrosis.

Mechanical response of human subclavian and iliac arteries to extension, inflation and torsion.

Peripheral vascular trauma due to injuries of the upper and lower limbs are life-threatening, and their treatment require rapid diagnosis and highly-qualified surgical procedures. Experienced surgeons have recognized that subclavian arteries, affected by injuries of the upper limbs, require a more careful handling due to fragility than common iliac arteries, which are may be affected by injures of the lower limbs. We investigated these two artery types with comparable diameter to evaluate the differences in the biomechanical properties between subclavian and iliac arteries. Human subclavian and common iliac arteries of 14 donors either from the right or the left side (age: 63 yrs, SD: 19,9 female and 5 male) were investigated. Extension-inflation-torsion experiments at different axial strains (0-20%), transmural pressures (0-200 mmHg) and torsion (±25°) on preconditioned arterial tubes were performed. Residual stresses in both circumferential and axial direction were determined. Additionally, the microstructure of the tissues was determined via second-harmonic generation imaging and by histological investigations. At physiological conditions (pi=13.3 kPa, λz=1.1) common iliac arteries revealed higher Cauchy stresses in circumferential and axial directions but a more compliant response in the circumferential direction than subclavian arteries. Both arteries showed distinct stiffer behavior in circumferential than in axial direction. Circumferential stiffness of common iliac arteries at physiological conditions increased significantly with aging (r=-0.67,p=0.02). The median inversion stretches, where the axial force is basically independent of the transmural pressure, were determined to be 1.05 for subclavian arteries and 1.11 for common iliac arteries. Both arteries exhibited increased torsional stiffness, when either axial prestretch or inflation pressure was increased. Residual stresses in the circumferential direction were significantly lower for subclavian arteries than for common iliac arteries at measurements after 30 min (p=0.05) and 16hrs (p=0.01). Investigations of the collagen microstructure revealed different collagen fiber orientations and dispersions in subclavian and iliac arteries. The difference in the collagen microstructure revealed further that the adventitia seems to contribute significantly to the passive mechanical response of the tested arteries at physiological loadings. Histological investigations indicated pronounced thickened intimal layers in subclavian and common iliac arteries, with a thickness comparable to the adventitial layer. In conclusion, we obtained biomechanical differences between subclavian and common iliac arteries, which possibly resulted from their different mechanical loadings/environments and respective in vivo movements caused by their anatomical locations. The biomechanical differences explored in this study are well reflected by the microstructure of the collagen and the histology of the investigated arteries, and the results can improve trauma patient care and endovascular implant design. STATEMENT OF SIGNIFICANCE: During surgical interventions surgeons experienced that subclavian arteries (SAs) supplying the upper extremities, appear more fragile and prone to damage during surgical repair than common iliac arteries (CIAs), supplying the lower extremities. To investigate this difference in a systematic way the aim of this study was to compare the biomechanical properties of these two arteries from the same donors in terms of geometry, extension-inflation-torsion behavior, residual stresses, microstructure, and histology. In regard to cardiovascular medicine the material behavior of aged human arteries is of crucial interest. Moreover, the investigation of SA is important as it can help to improve surgical procedures at this challenging location. Over the long-term it might well be of value in the construction of artificial arteries for substituting native arteries. In addition, the analysis of mechanical stresses can improve design and material choice for endovascular implants to optimize long-term implant function.

Identification of differential gene expression patterns in human arteries from patients with chronic kidney disease.

Uremia accelerates atherosclerosis, but little is known about affected pathways in human vasculature. This study aimed to identify differentially expressed arterial transcripts in patients with chronic kidney disease (CKD). Global mRNA expression was estimated by microarray hybridization in iliac arteries ( n = 14) from renal transplant recipients and compared with renal arteries from healthy living kidney donors ( n = 19) in study 1. Study 2 compared nonatherosclerotic internal mammary arteries (IMA) from five patients with elevated plasma creatinine levels and age- and sex-matched controls with normal creatinine levels. Western blotting and immunohistochemistry for selected proteins were performed on a subset of study 1 samples. Fifteen gene transcripts were significantly different between the two groups in study 1 [fold changes (FC) > 1.05 and false discovery rates (FDR) < 0.005]. Most upregulated mRNAs associated with cellular signaling, apoptosis, TNFα/NF-κB signaling, smooth muscle contraction, and 10 other pathways were significantly affected. To focus attention on genes from genuine vascular cells, which dominate in IMA, concordant deregulated genes in studies 1 and 2 were examined and included 23 downregulated and eight upregulated transcripts (settings in study 1: FC > 1.05 and FDR < 0.05; study 2: FC > 1.2 and P < 0.2). Selected deregulated gene products were investigated at the protein level, and whereas HIF3α confirmed mRNA upregulation, vimentin showed upregulation in contrast to the mRNA results. We conclude that arteries from CKD patients display change in relatively few sets of genes. Many were related to differentiated vascular smooth muscle cell phenotype. These identified genes may contribute to understanding the development of arterial injury among patients with CKD.

Time-dependent differences in femoral artery plaque characteristics of peripheral arterial disease patients.

BACKGROUND AND AIMS: Peripheral arterial disease (PAD) is a common manifestation of atherosclerosis with an increasing incidence worldwide. The disease is still associated with high morbidity and mortality risks. Previous research in carotid arteries indicates that atherosclerotic plaque characteristics have stabilized over time in patients considered for surgery. It is currently unknown whether this time-dependent stabilization occurs in ilio-femoral arteries as well. Our objective was to analyze whether local ilio-femoral atherosclerotic plaque characteristics have changed over time. METHODS: 497 patients within the Athero-Express biobank who underwent primary endarterectomy of the iliac or femoral artery between 2002 and 2013 were analyzed. We investigated six histological plaque characteristics: calcification, collagen, fat content, intraplaque haemorrhage, macrophages and smooth muscle cells. RESULTS: Over the course of 10 years, we observed a lower percentage of all plaque characteristics that are considered indicators of a vulnerable plaque, such as: plaques with a large lipid core from 37.9% to 14.9% and plaques with intraplaque haemorrhage from 69.0% to 34.8% when the two-year cohorts 2003-2004 and 2011-2012 were compared, respectively. Multivariable analyses showed that time-dependent changes occurred independently of changing procedural and patient characteristics. CONCLUSIONS: In this cohort of peripheral arterial disease patients undergoing primary endarterectomy, we observed a time dependent shift of plaque characteristics towards a less lipid rich lesion with less intraplaque haemorrhage. These findings indicate research in cardiovascular disease would benefit from contemporary patient characteristics and plaque specimens to optimize translational potential.

Intimal sarcoma of the abdominal aorta and common iliac arteries presenting as epithelioid angiosarcoma of the skin: a case report.

Intimal sarcoma (IS) is the most common type of sarcoma of the aorta. IS tumor emboli can involve various organs, including the skin. However, a limited number of IS cases with an initial presentation of skin metastasis has been reported. Cutaneous metastasis as a form of epithelioid angiosarcoma (EAS) has not been well described. Herein, we present a 61-year-old Japanese man with an initial presentation of EAS of the skin, followed by multiple metastases to the skin as a form of EAS prior to detection of IS of the infrarenal aorta and common iliac arteries. In our case, the IS was CD31 and cytokeratin positive but did not express CD34 and factor VIII-related antigen. The EASs in our case exhibited diffuse CD31 expression, and focal factor VIII-related antigen and cytokeratin expression were observed throughout the tumor, including the neoplastic vascular structure; CD34 expression was not identifiable. IS metastasis to the skin has been documented as a form of angiosarcoma. However, IS metastasis has not been well described as a form of EAS. Our case could prove a morphological change from IS to EAS. Given the rarity of primary cutaneous EAS, it is recommended that primary sites other than the skin should be thoroughly investigated when EAS of the skin is encountered.

Investigation of the optimal collagen fibre orientation in human iliac arteries.

The distribution of collagen fibres plays a significant role in the mechanical behaviour of artery walls. Experimental data show that in most artery wall layers there are two (or more) in-plane symmetrically disposed families of fibres. However, a recent investigation revealed that some artery wall layers have only one preferred fibre direction, notably in the medial layer of human common iliac arteries. This paper aims to provide a possible explanation for this intriguing phenomenon. An invariant-based constitutive model is utilized to characterize the mechanical behaviour of tissues. We then use three different hypotheses to determine the 'optimal fibre angle' in an iliac artery model. All three hypotheses lead to the same result that the optimal fibre angle in the medial layer of the iliac artery is close to the circumferential direction. The axial pre-stretch, in particular, is found to play an essential role in determining the optimal fibre angle.

Clinical significance of preexisting microcalcification in the iliac artery in renal transplant recipients.

BACKGROUND: The clinical significance of preexisting microcalcification in the iliac artery is undetermined in renal transplant recipients. METHODS: We obtained iliac artery segments from 90 transplant recipients at the time of renal transplantation and performed von Kossa staining for microcalcification. The clinical significance of intimal microcalcification was evaluated with allograft survival rate, rate of graft function decline, and composite of any cardiovascular event or patient death. Expression of fetuin-A and C-reactive protein, key regulators of calcification, was also investigated in the iliac artery. RESULTS: Intimal microcalcification was positive in 48 (53.3%) patients, and its intensity was correlated positively with intimal C-reactive protein intensity (P = 0.019). Allograft survival in patients positive for intimal microcalcification was lower than patients who were negative (P = 0.017). The patients with positivity for both intimal microcalcification and fetuin-A showed lower allograft survival rate than patients with intimal microcalcification positivity alone (P = 0.012). The rate of renal graft function decline was significantly steeper in patients positive for intimal microcalcification than in patients who were negative (P = 0.036). In multivariate analysis, positivity for both intimal microcalcification and fetuin-A was an independent predictor for renal graft function decline (ß = -10.21; P = 0.011). The intimal microcalcification was not associated with composite-event free survival. CONCLUSION: Preexisting intimal microcalcification in the iliac artery predicts a lower allograft survival rate and rapid decline of allograft function. Positivity of fetuin-A with intimal microcalcification further reduces allograft survival rate and an independent predictor for renal graft function decline.

Effects of estradiol on transcriptional profiles in atherosclerotic iliac arteries in ovariectomized cynomolgus macaques.

OBJECTIVE: This study aimed to assess the in vivo effects of estradiol treatment on arterial gene expression in atherosclerotic postmenopausal female monkeys. METHODS: Eight ovariectomized cynomolgus monkeys were fed atherogenic diets for 6.5 years. The left iliac artery was biopsied before randomization to the estradiol group (human equivalent dose of 1 mg/d, n = 4) or the vehicle group (n = 4) for 8 months. The right iliac artery was obtained at necropsy. Transcriptional profiles in pretreatment versus posttreatment iliac arteries were compared to assess the responses of atherosclerotic arteries to estradiol. RESULTS: Iliac artery plaque size did not differ between the estradiol group and the placebo group at baseline or during the treatment period. Nevertheless, estradiol treatment was associated with increased expression of 106 genes and decreased expression of 26 genes in the iliac arteries. Estradiol treatment increased the expression of extracellular matrix genes, including the α1 chain of type I collagen, the α2 chain of type VI collagen, and fibulin 2, suggestive of an increase in the proportion or phenotype of smooth muscles or fibroblasts in lesions. Also increased were components of the insulin-like growth factor pathway (insulin-like growth factor 1, insulin-like growth factor binding protein 4, and insulin-like growth factor binding protein 5) and the Wnt signaling pathway (secreted frizzled-related protein 2, secreted frizzled-related protein 4, low-density lipoprotein receptor-related protein 6, and Wnt1-inducible signaling pathway protein 2). CONCLUSIONS: Estradiol treatment of monkeys with established atherosclerosis affected iliac artery gene expression, suggesting changes in the cellular composition of lesions. Moreover, it is probable that the presence of atherosclerotic plaque affected the gene expression responses of arteries to estrogen.

Adventitial lymphatics and atherosclerosis.

Lymphatic vessels are important in reverse cholesterol transport and play a crucial role in regression of atherosclerotic plaque in experimental animal models. Therefore, we attempted to analyze adventitial microcirculation including lymphatic vessels and adventitial macrophages in large human arteries in various stages of atherosclerosis. Eighty-one arterial segments of large arteries (iliac arteries and abdominal aortas) were obtained from deceased organ donors. Lymphatic vessels were identified using anti-LYVE-1 and anti-D2-40/podoplanin immunohistochemical staining. Adventitial blood vessels and macrophages were visualized using anti-CD-31 and anti-CD-68. Intimal thickness was measured under 100x magnification with an Olympus BX 41 light microscope using the visual mode analySIS 3.2 software. Lymphatic vessels were counted in each cross section of the examined arteries, and adventitial blood vessels (CD31+) were counted using the "hot spot" method. Statistical analysis was performed with Statistica 9.1 PL software (StatSoft, Cracow, Poland). Mann-Whitney, F-Cox, Chi-square, and Spearman's correlation tests were performed and the differences were considered significant at p < 0.05. Lymphatic and blood vessels in the adventitia of examined arteries were identified and quantified. Significant positive correlations were found between the number of adventitial lymphatics (LYVE-L +) and intimal thickness (r = 0.37; p < 0.05) as well as with age of the subjects (r = 0.3; p < 0.05). Thus, lymphatic vessels are present in the adventitia of large arteries in humans and the number of adventitial lymphatic vessels increases with progression of atherosclerosis as assessed by intimal thickness.

Determination of the layer-specific distributed collagen fibre orientations in human thoracic and abdominal aortas and common iliac arteries.

The established method of polarized microscopy in combination with a universal stage is used to determine the layer-specific distributed collagen fibre orientations in 11 human non-atherosclerotic thoracic and abdominal aortas and common iliac arteries (63 ± 15.3 years, mean ± s.d.). A dispersion model is used to quantify over 37 000 recorded fibre angles from tissue samples. The study resulted in distinct fibre families, fibre directions, dispersion and thickness data for each layer and all vessels investigated. Two fibre families were present for the intima, media and adventitia in the aortas, with often a third and sometimes a fourth family in the intima in the respective axial and circumferential directions. In all aortas, the two families were almost symmetrically arranged with respect to the cylinder axis, closer to the axial direction in the adventitia, closer to the circumferential direction in the media and in between in the intima. The same trend was found for the intima and adventitia of the common iliac arteries; however, there was only one preferred fibre alignment present in the media. In all locations and layers, the observed fibre orientations were always in the tangential plane of the walls, with no radial components and very small dispersion through the wall thickness. A wider range of in-plane fibre orientations was present in the intima than in the media and adventitia. The mean total wall thickness for the aortas and the common iliac artery was 1.39 and 1.05 mm, respectively. For the aortas, a slight thickening of the intima and a thinning of the media in increasingly distal regions were observed. A clear intimal thickening was present distal to the branching of the celiac arteries. All data, except for the media of the common iliac arteries, showed two prominent collagen fibre families for all layers so that two-fibre family models seem most appropriate.

Calcium scoring in unenhanced and enhanced CT data of the aorta-iliacal arteries: impact of image acquisition, reconstruction, and analysis parameter settings.

BACKGROUND: Several studies have been published on the matter of abdominal aortic and iliac calcifications and the association to clinical entities such as diabetes mellitus and renal failure. However, comparing of these studies is questionable since quantification methods for atherosclerosis differ. PURPOSE: To evaluate the effect of image acquisition settings, reconstruction parameters, and analysis methods on calcium quantification in the abdominal aorta. MATERIAL AND METHODS: Calcium scores were retrospectively determined on standardized abdominal CT scans of 15 patients. Two researchers obtained calcium scores with 10 different lower thresholds (LT) (130, 145, 160, 175, 200, 300, 400, 500, 600, 1000) in CT scans with and without contrast enhancement, with slice thicknesses (ST) varying between 2.0-5.0 mm for the non-contrast-enhanced series and between 1.0-5.0 mm for the contrast-enhanced series. In addition calcium scores obtained with two convolution kernels (B10f, B20f) were compared. Inter-observer variability was calculated. RESULTS: Calcium scoring at higher STs is overestimated compared to smaller STs and this effect was more pronounced with increasing calcium loads. Concerning the convolution kernel, scores obtained with kernel B10f were overestimated compared to kernel B20f. Increase of LT resulted in a decrease of the calcium score and scoring in contrast-enhanced series resulted in higher scores compared to non-contrast-enhanced series. These effects are more apparent in patients with higher calcium loads. Calcium scoring reproducibility with the reference standard is limited for the aorta-iliac trajectory, whereas scoring with the remaining settings is reproducible. CONCLUSION: Scores obtained with different settings cannot be compared. The inter-observer reproducibility was limited using the reference standard and practical difficulties were substantial. Scoring with higher LT, ST, and contrast enhancement is faster and has less practical difficulties.

Endothelial gene expression in regions of defined shear exposure in the porcine iliac arteries.

The effect of hemodynamic shear stress on endothelial gene expression was investigated in the porcine iliac arteries. A novel statistical approach was applied to computational fluid dynamics simulations of the iliac artery flow field to identify three anatomical regions likely to experience high, medium, and low levels of time average shear stress magnitude. Subsequently, endothelial cell mRNA was collected from these regions in the iliac arteries of six swine and analyzed by DNA microarray. Gene set enrichment analysis demonstrated a strong tendency for genes upregulated or downregulated in one of the extreme shear environments (low or high, relative to medium) to be regulated in the same direction in the other extreme shear environment. This tendency was confirmed for specific genes by real-time quantitative PCR. Specifically, beta-catenin, c-jun, VCAM-1, and MCP-1 were all upregulated in low and high shear stress regions relative to the medium shear stress region. eNOS expression was not significantly different in any of the regions. These results are consistent with the notion that endothelial cells chronically exposed to abnormally low or high shear levels in vivo exhibit similar genetic responses. Alternative explanations of this outcome are proposed, and its implications for the role of shear stress in atherogenesis are examined.

Imaging and quantitative analysis of atherosclerotic lesions by CARS-based multimodal nonlinear optical microscopy.

OBJECTIVE: The purpose of this study was to assess the ability of label-free multimodal nonlinear optical (NLO) microscopy to characterize, and thus enable quantitative in situ analyses of, different atherosclerotic lesion types, according to the original scheme suggested by the AHA Committee. METHODS AND RESULTS: Iliac arteries were taken from 24 male Ossabaw pigs divided into lean control and metabolic syndrome groups and were imaged by multimodal NLO microscopy where sum-frequency generation (SFG) and 2-photon excitation fluorescence (TPEF) were integrated on a coherent anti-Stokes Raman scattering (CARS) microscope platform. Foam cells, lipid deposits, matrices, and fibrous caps were visualized with submicron 3D resolution. Starting from the adaptive intimal thickening in the initial stage to the fibrous atheroma or mineralization in the advanced stages, lesions were visualized without labels. Histological staining of each lesion confirmed the lesion stages. Lipid and collagen contents were quantitatively analyzed based on the CARS and SFG signals. Lipid accumulation in thickened intima culminated in type IV whereas the highest collagen deposition was found in Type V lesions. Luminal CARS imaging showed the capability of viewing the location of superficial foam cells that indicate relatively active locus in a lesion artery. CONCLUSIONS: We have demonstrated the capability of CARS-based multimodal NLO microscopy to interrogate different stages of lesion development with subcellular detail to permit quantitative analysis of lipid and collagen contents.

In vivo differences between endothelial transcriptional profiles of coronary and iliac arteries revealed by microarray analysis.

Endothelial cells (ECs) from different vascular beds display a remarkable heterogeneity in both structure and function. Phenotypic heterogeneity among arterial ECs is particularly relevant to atherosclerosis since the disease occurs predominantly in major arteries, which vary in their atherosusceptibility. To explore EC heterogeneity between typical atheroprone and atheroresistant arteries, we used DNA microarrays to compare gene expression profiles of freshly harvested porcine coronary (CECs) and iliac artery (IECs) ECs. Statistical analysis revealed 51 genes that were differentially expressed in CECs relative to IECs at a false discovery rate of 5%. Seventeen of these genes are known to be involved in atherogenesis. Consistent with coronary arteries being more atherosusceptible, almost all putative atherogenic genes were overexpressed in CECs, whereas all atheroprotective genes were downregulated, relative to IECs. A subset of the identified genes was validated by quantitative polymerase chain reaction (PCR). PCR results suggest that the differences in expression levels between CECs and IECs for the HOXA10 and HOXA9 genes were >100-fold. Gene ontology (GO) and biological pathway analysis revealed a global expression difference between CECs and IECs. Genes in twelve GO categories, including complement immune activation, immunoglobulin-mediated response, and system development, were significantly upregulated in CECs. CECs also overexpressed genes involved in several inflammatory pathways, including the classical pathway of complement activation and the IGF-1-mediated pathway. The in vivo transcriptional differences between CECs and IECs found in this study may provide new insights into the factors responsible for coronary artery atherosusceptibility.

The epidemiology of abdominal aortic diameter.

BACKGROUND: The diameter of the abdominal aorta is central to the diagnosis of abdominal aortic aneurysm. This study aimed to determine the associations between the diameter of the abdominal aorta at three distinct locations and the traditional cardiovascular disease risk factors as well as calcified atherosclerosis. METHODS: A total of 504 patients (41% women) underwent whole body scanning by electron beam computed tomography (EBCT) and a standardized assessment for cardiovascular disease risk factors. The resulting EBCT images were retrospectively interrogated for the diameter of the abdominal aorta just inferior to the superior mesenteric artery (SMA), just superior to the aortic bifurcation, and at the midpoint between the SMA and bifurcation. RESULTS: Mean patient age was 57.8 years. The mean (SD) diameter was 21.3 (2.9) mm at the SMA, 19.3 (2.5) mm at the midpoint, and 18.6 (2.2) mm at the bifurcation. In a model containing the traditional cardiovascular disease risk factors, age (standardized beta = 0.96), male sex (beta = 3.06), and body mass index (standardized beta = 0.68) were significantly associated with increasing aortic diameter at the SMA (P < .01 for all). The significance of the associations for these variables was the same for aortic diameter at the midpoint and bifurcation. Furthermore, a 1-unit increment in the calcium score in the abdominal aorta and iliac arteries was associated with 0.13-mm (P < .01) and 0.09-mm (P = .02) increases, respectively, in aortic diameter at the SMA. The results were similar for the midpoint (beta = 0.19, P < .01; beta = 0.12, P = .01, respectively) and bifurcation (beta = 0.09, P < .04; beta = 0.09, P = .03, respectively). CONCLUSIONS: Age, sex, body mass index, and the presence and extent of calcified atherosclerosis in both the abdominal aorta and iliac arteries are significantly associated with increasing aortic diameter independent of the other cardiovascular disease risk factors.

Atherosclerosis is associated with plasminogen activator inhibitor type-1 in chronic haemodialysis patients.

AIM: The aim of the present report was to investigate the probable association of circulating levels of PAI-1 and expression of PAI-1 in internal iliac artery walls with atherosclerotic disease in chronic haemodialysis (HD) patients. METHODS: Sixty-eight non-diabetic HD patients and 50 age- and sex-matched healthy normotensive controls participated in the study. Atherosclerotic disease in both groups was assessed by measuring intima-media thickness (IMT) and plaque score of the common carotid arteries using an ultrasound scanner. Levels of serum PAI-1, C-reactive protein (CRP), interleukin (IL)-6 and lipids profile were measured. Internal iliac artery samples were obtained at the time of renal transplantation. Quantitative expression of PAI-1 in internal iliac artery walls was assessed by positive unit (pu) value using an immunohistochemical method. In addition, the IMT and carotid plaque score were analyzed in relation to circulating levels of PAI-1 and expression of PAI-1 in internal iliac artery walls. RESULTS: Compared with control subjects, HD patients had significantly increased common carotid artery (CCA)-IMT (P = 0.002). Atherosclerotic plaques were detected in 42 (61.76%) of HD patients and in two (4%) controls. The above ultrasonographic indices were correlated with age in HD patients (P < 0.001). A significant relationship was observed between IMT and systolic blood pressure (BP), low-density lipoprotein in HD patients (P < 0.001 and P < 0.001, respectively). In HD patients, IMT was significantly correlated with CRP and IL-6 (P < 0.001 and P < 0.001, respectively). In HD patients, a close correlation was found between serum PAI-1 level, CRP and IL-6 (P < 0.01 and P < 0.01, respectively). A close correlation was also found between PAI-1 pu value, CRP and IL-6 (P < 0.01 and P < 0.01 respectively). Serum PAI-1 level is highly correlated to PAI-1 pu value (P < 0.01). In HD patients, CCA-IMT and plaque score were correlated significantly with circulating levels of PAI-1(P < 0.01 and P < 0.05, respectively) and expression of PAI-1 in internal iliac artery walls (P < 0.01 and P < 0.05, respectively). Multivariate analysis showed that log CRP values were a strong independent contributor to CCA-IMT and plaque score (P = 0.03 and P = 0.04, respectively). Multivariate analysis showed that serum PAI-1 concentration was a strong independent correlate of CCA-IMT and carotid plaque score (P = 0.004 and P = 0.009, respectively). Multivariate analysis also showed that expression of PAI-1 in internal iliac artery walls was a strong independent correlate of CCA-IMT and carotid plaque score (P = 0.008 and P = 0.005, respectively). CONCLUSION: The circulating levels of PAI-1 and expression of PAI-1 in internal iliac artery walls were statistically associated with CRP, IL-6 and low-density lipoprotein cholesterol. Moreover, in HD patients, CCA-IMT and plaque score were correlated significantly with circulating levels of PAI-1 and expression of PAI-1 in internal iliac artery walls and the circulating levels of PAI-1 and expression of PAI-1 in internal iliac artery walls were independent predictors of carotid atherosclerosis including CCA-IMT and carotid plaque score. The correlations may suggest that increased circulating PAI-1 level and upregulated expression of PAI-1 in the vasculature could indicate a chronic endothelium activated state and PAI-1 may more precisely identify the risk of atherothrombosis and be useful as a target for anti-inflammatory treatment strategies.

ARFI imaging for noninvasive material characterization of atherosclerosis.

Cardiovascular disease (CVD) is the leading cause of death in the United States, with 70% of CVD mortalities the result of sequelae of atherosclerosis. An urgent need for enhanced delineation of vulnerable plaques has catalyzed the development of novel atherosclerosis imaging strategies that use X-ray computed tomography, magnetic resonance and ultrasound modalities. As suggested by the pathophysiology of plaque development and progression to vulnerability, insight to the focal material, i.e., mechanical, properties of arterial walls and plaques may enhance atherosclerosis characterization. We present acoustic radiation force impulse (ARFI) ultrasound in application to mechanically characterizing a raised focal atherosclerotic plaque in an iliac artery extracted from a relevant pig model. ARFI results are correlated to matched immunohistochemistry, indicating elastin and collagen composition. In regions of degraded elastin, slower recovery rates from peak ARFI-induced displacements were observed. In regions of collagen deposition, lower ARFI-induced displacements were achieved. This work demonstrates ARFI for characterizing the material nature of an atherosclerotic plaque.

Molecular imaging of atherosclerotic plaques using a human antibody against the extra-domain B of fibronectin.

Current imaging modalities of human atherosclerosis, such as angiography, ultrasound, and computed tomography, visualize plaque morphology. However, methods that provide insight into plaque biology using molecular tools are still insufficient. The extra-domain B (ED-B) is inserted into the fibronectin molecule by alternative splicing during angiogenesis and tissue remodeling but is virtually undetectable in normal adult tissues. Angiogenesis and tissue repair are also hallmarks of advanced plaques. For imaging atherosclerotic plaques, the human antibody L19 (specific against ED-B) and a negative control antibody were labeled with radioiodine or infrared fluorophores and injected intravenously into atherosclerotic apolipoprotein E-null (ApoE-/-) or normal wild-type mice. Aortas isolated 4 hours, 24 hours, and 3 days after injection exhibited a selective and stable uptake of L19 when using radiographic or fluorescent imaging. L19 binding was confined to the plaques as assessed by fat staining. Comparisons between fat staining and autoradiographies 24 hours after 125I-labeled L19 revealed a significant correlation (r=0.89; P<0.0001). Minimal antibody uptake was observed in normal vessels from wild-type mice receiving the L19 antibody and in atherosclerotic vessels from ApoE-/- mice receiving the negative control antibody. Immunohistochemical studies revealed increased expression of ED-B not only in murine but also in human plaques, in which it was found predominantly around vasa vasorum and plaque matrix. In summary, we demonstrate selective targeting of atheromas in mice using the human antibody to the ED-B domain of fibronectin. Thus, our findings may set the stage for antibody-based molecular imaging of atherosclerotic plaques in the intact organism.

Caveolin-1 can regulate vascular smooth muscle cell fate by switching platelet-derived growth factor signaling from a proliferative to an apoptotic pathway.

BACKGROUND: Caveolin-1 is a regulator of signaling events originating from plasma membrane microdomains termed caveolae. This study was performed to determine the regulatory role of caveolin-1 on the proliferative events induced by platelet-derived growth factor (PDGF) in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Treatment of VSMCs with PDGF for 24 hours resulted in a loss of caveolin-1 protein expression and plasma membrane-associated caveolae, despite a 3-fold increase in caveolin-1 mRNA. Pretreatment of VSMCs with chloroquine, an inhibitor of lysosomal function, inhibited the PDGF-induced loss of caveolin-1. These studies demonstrated that caveolin-1 was a target of PDGF signaling events. Adenoviral overexpression of caveolin-1 was associated with a switch in PDGF-induced signaling events from a proliferative response to an apoptotic response. This overexpression inhibited PDGF-induced expression of cyclin D1 in the presence of unaffected mitogen-activated protein kinase activation. CONCLUSIONS: Taken together, these studies suggest that caveolin-1 is an inhibitor of PDGF proliferative responses and might be capable of transforming PDGF-induced proliferative signals into death signals.

Cardiac ankyrin repeat protein (CARP) expression in human and murine atherosclerotic lesions: activin induces CARP in smooth muscle cells.

OBJECTIVE: Cardiac ankyrin repeat protein (CARP) is a transcription factor-related protein that has been studied most extensively in the heart. In the present study, we investigated the expression and the potential function of CARP in human and murine atherosclerosis. METHODS AND RESULTS: CARP expression was observed by in situ hybridization in endothelial cells lining human atherosclerotic plaques, whereas lesion macrophages were devoid of CARP. Furthermore, we established that CARP mRNA and smooth muscle (SM) alpha-actin antigen both colocalized in a subset of intimal smooth muscle cells (SMCs), whereas no CARP mRNA was encountered in quiescent SMCs in the media. The CARP mRNA-expressing intimal SMCs were distinct from intimal SMCs that synthesized the activation marker osteopontin or proliferating cell nuclear antigen. In addition, we showed that activin A, a member of the TGFbeta superfamily that prevents SMC-rich lesion formation, induced CARP mRNA expression in cultured SMCs. CONCLUSIONS: Based on our data and the knowledge that CARP reduces the proliferation of cultured SMCs, we propose that CARP is involved in inhibition of vascular lesion formation.