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.

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.

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.

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.

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.

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.

Effect of endothelial and adventitial injury on somatostatin receptor expression.

BACKGROUND: The somatostatin analog, angiopeptin, inhibits intimal hyperplasia formation; although the specific somatostatin receptor (SSTR) subtypes transducing this effect are unknown. The purpose of this study was to determine the expression of SSTR subtypes in rat iliac arteries after balloon catheter endothelial injury and perivascular dissection. METHODS: Male rats received balloon endothelial injury to their left common and external iliac arteries with or without circumferential arterial dissection. The right arteries served as controls. At 1 and 2 months after intimal injury, animals were killed and their iliac arteries harvested and studied for SSTR expression by using immunocytochemical and molecular techniques. Quantitative polymerase chain reaction was used to determine the level of SSTR expression. RESULTS: Normal rat iliac arteries expressed only SSTR2 and 3. After balloon endothelial injury, there was significant upregulation of SSTR2 messenger RNA at 1 and 2 months after injury as compared with controls (1 month, 1.8 +/- 0.3 vs 0.4 +/- 0.1 zmol, P < .001; 2 months, 2.7 +/- 0.5 vs 1.1 +/- 0.2 zmol, P < .001). The addition of adventitial dissection to endothelial injury also showed a significant increase in SSTR2 expression (1 month, 2.4 +/- 0.4 vs 0.8 +/- 0.2, P < .05; 2 months, 1.3 +/- 0.3 vs 0.7 +/- 0.3, P < .05), but not significantly greater than that seen after balloon endothelial injury alone. Immunocyto-chemical studies also demonstrated an increase in SSTR2 immunoreactivity on the luminal surface of the endothelial cells in the balloon catheter-injured arteries. CONCLUSIONS: These findings show that SSTR2 is the primary SSTR that is upregulated after injury and likely mediates the effects of somatostatin analogs on intimal hyperplasia.

Assessing Photofrin uptake in atherosclerosis with a fluorescent probe: comparison with photography and tissue measurements.

The purpose of this study was to assess Photofrin porfimer sodium (P*) concentration in atherosclerotic plaque (ASP) using a fluorescence detector (Fluoroprobe) compared with fluorescent photography and chemical extraction of P*. ASP was created in the aortoiliac segments of Yucatan miniswine by a combination of balloon endothelial injury and 2% cholesterol and 15% lard diet for 7 weeks. At that time, swine were given P* I.V. in one of the following single dosages: Group I, 2.5; Group II, 1.0; or Group III, 0.5 mg/kg. Swine were sacrificed 24 hours later and aortoiliac and control carotid artery segments removed. Fluorescence was determined from these segments using photographic techniques, the Fluoroprobe, and a spectrofluorometer after chemical extraction. ASP were identified in all swine using photography and the Fluoroprobe. The intensity of fluorescence measured with the Fluoroprobe for Groups I to III was 1,098 +/- 524, 471 +/- 337, and 295 +/- 173 units, respectively (P < 0.01). The tissue concentration of P* in ASP from each group was 130.4 +/- 82.7, 10.0 +/- 1.2, and 9.1 +/- 0.6 ng/g, respectively (P < 0.01). There was a linear correlation between the fluorescence intensity measured with the Fluoroprobe and the extracted tissue concentration (r = 0.88, P < 0.0001). This study showed that a fluorescent detector such as the Fluoroprobe accurately detects the uptake of P* into atherosclerotic plaque.

[Morphological and biochemical study of the lipid components of the fetal aorta]. / Studio morfologico e biochimico della componente lipidica della aorta fetale.

We made a biochemical and histochemical study of the lipidic component of intima of fetal aortas on 8 autopsy cases (7 +/- 2 months aged) arrived at our observation in the Pathology's Institute of II Faculty of Naples. We made a study with freeze-sections stained with Oil-Red 0 and after dissociation of the intima by the adventitia, it is valued biochemically the lipidic peroxidation studying the levels of malonyldialdehyde (MDA) like indirect marker of peroxidation. It is known that is present a lipidic component in the intima of fetal aorta whether intracellular or extracellular (Fig. 1, 2). Sometimes this component can accumulate until to determinate true lipidic striae. The aim of this study is a detection of MDA in lipids extracted from human fetal aortas. MDA levels was measured by Thiobarbituric method (TBA): lipids were extracted both intima and adventitia by Chloroform/methanol method, after surgery immediately. The results are expressed in nMoles/mg of lipids +/- Standard Deviation. Controls of spontaneous lipid peroxidation was take at a different times. It is known that in vitro incubation of LDL with cultured endothelial cells, smooth muscle cells or macrophages leads to peroxidation of LDL phospholipids and oxidatively modified LDL become atherogenic via foam cells production. In addition lipid peroxidation was formed by the direct peroxidation of unsaturated fatty acids and their esters are capable of further lipoperoxide production by oxygen free radical; chain reactions. In this context lipid peroxidation could be an important factor in the first stage of human pathophysiological development and this phenomenon may be related by an early free radical production.