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Sirolimus-eluting stent reduces restenosis after percutaneous coronary intervention. However, accumulating evidence suggests that sirolimus potentially affects re-endothelialization, leading to late thrombosis. Statins have protective effects on endothelium. Recently, statins are reported to increase the number of circulating endothelial progenitor cells (EPCs) and accelerate re-endothelialization after vascular injury. Here, we tested the hypothesis that fluvastatin has beneficial effect on re-endothelialization after local sirolimus treatment. We performed wire-mediated vascular injury to both sides of femoral arteries of wild-type mice and bone marrow chimeric mice. Either sirolimus (100 microg) or DMSO was administered locally to the perivascular area of the injured arteries. All mice received either fluvastatin (5 mg/kg/day) or vehicle by gavage starting at one week before the surgery until sacrifice. At 4 weeks after the surgery, re-endothelialization of the sirolimus-treated artery was significantly less than that of DMSO-treated one in the vehicle-treated mice as determined by the percentage of CD31-positive area (P<0.05). Systemic administration of fluvastatin accelerated the re-endothelialization in the sirolimus-treated artery to the similar degree of that in the DMSO-treated artery (P=NS). Contribution of bone marrow-derived cells to re-endothelialization was seldom observed in bone marrow chimeric mice regardless of fluvastatin administration. Fluvastatin significantly ameliorated proliferation (2.5-folds) and migration activities (2.3-folds) of mature endothelial cells impaired by sirolimus treatment (P<0.05, respectively). Fluvastatin increased endothelial nitric oxide synthase expression and decreased plasminogen activator inhibitor-1 expression in mature endothelial cell in the presence of sirolimus (P<0.05, respectively). Our findings suggest that fluvastatin has protective effects against impaired re-endothelialization after sirolimus treatment.

Because macrophages play a major role in atherosclerotic plaque destabilization, selective removal of macrophages represents a promising approach to stabilize plaques. We showed recently that the protein synthesis inhibitor cycloheximide, in contrast to puromycin, selectively depleted macrophages in rabbit atherosclerotic plaques without affecting smooth muscle cells (SMCs). The mechanism of action of these two translation inhibitors is dissimilar and could account for the differential effects on SMC viability. It is not known whether selective depletion of macrophages is confined to cycloheximide or whether it can also be achieved with translation inhibitors that have a similar mechanism of action. Therefore, in the present study, we investigated the effect of anisomycin, a translation inhibitor with a mechanism of action similar to cycloheximide, on macrophage and SMC viability. In vitro, anisomycin induced apoptosis of macrophages in a concentration-dependent manner, whereas SMCs were only affected at higher concentrations. In vivo, anisomycin selectively decreased the macrophage content of rabbit atherosclerotic plaques through apoptosis. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole] prevented anisomycin-induced macrophage death, without affecting SMC viability. SB202190 decreased anisomycin-induced p38 MAPK phosphorylation, did not alter c-Jun NH(2)-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. The latter effect was abolished by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene ethanolate], although the prevention of anisomycin-induced macrophage death by SB202190 remained unchanged. The JNK phosphorylation inhibitor SP600125 did not affect anisomycin-induced macrophage or SMC death. In conclusion, anisomycin selectively decreased the macrophage content in rabbit atherosclerotic plaques, indicating that this effect is not confined to cycloheximide. p38 MAPK, but not ERK1/2 or JNK, plays a major role in anisomycin-induced macrophage death.

Endothelin receptors (ET(A) and ET(B)) mediate responses to ET-1. ET(B) receptor function seems to differ between a similarly sized arterial and venous pair, the rat vena cava (RVC) and rat thoracic aorta (RA). ET(B) receptors mediate RVC contraction directly, but it is unclear whether ET(B) receptors mediate contraction in RA. Because of these apparent differences in ET(B) receptor-mediated vascular contraction, we hypothesize that relaxant ET(B)-receptor mechanisms in RVC would be different from those in RA. RA and RVC rings were isolated from rats for measurement of isometric contraction. When contracted with prostaglandin F-2alpha (PGF-2alpha) (20 microM), the ET(B) receptor agonist sarafotoxin-6c (S6c) (100 nM) significantly relaxed RA and RVC. N(omega)-Nitro-L-arginine (LNNA) (100 microM) or endothelial denudation abolished relaxation to S6c in RA. By contrast, S6c-induced relaxation of RVC was attenuated but not abolished by LNNA and endothelial denudation. RVC (PGF-2alpha-contracted) relaxed to low concentrations of ET-1, whereas under the same conditions RA responded with contraction. ET-1-induced relaxation in RA was observed only with ET(A) receptor blockade. Vessels from dopamine-beta-hydroxylase-ET(B) transgenic rats, which lack functional ET(B) receptors in the vasculature, were also used. RVC (PGF-2alpha-contracted) from these rats did not relax to ET-1. Thus, although both RA and RVC possess endothelial relaxant ET(B) receptors, RA and RVC differ in that relaxant ET(B) receptors may also be present in smooth muscle of RVC. Moreover, the mechanisms of endothelial cell ET(B) receptor-mediated relaxation in RA and RVC are not the same.

Advanced glycation endproducts (AGEs) are present in the vasculature and are associated with vascular disease. We determined levels of AGEs in eight distinct adult vascular tissues using tissue microarray (TMA) technology and associated these levels with clinical characteristics. Medium-to-large caliber blood vessels were harvested from 100 adult autopsies to create 17 TMAs. AGE levels were evaluated by IHC using a polyclonal anti-AGE antibody on over 700 unique blood vessels. Slides were digitally scanned, and quantitative analysis was performed using a color deconvolution image analysis technique. Medial AGE staining was strongly correlated between all eight blood vessels. In the media, AGE staining levels were significantly higher at older ages (p=0.009), in white subjects (p<0.001) and with longer postmortem interval (PMI; p<0.0001). These associations remained significant after simultaneous adjustment for age, race/ethnicity, PMI, and diabetes status. Diabetes was associated with elevated AGE levels but only after adjustment for confounding by clinical variables including race/ethnicity, hypertension, and kidney function. This extensive vascular study shows that AGE accumulation in the macrovasculature is a global process affecting atherosclerosis-prone and -resistant vessels. It also suggests ethnicity has a previously undescribed role in vascular tissue AGE levels. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

This study was designed to test the hypothesis that a medium-term simulated microgravity by tail-suspension (SUS) induces hypertrophic and atrophic changes in the common carotid artery and abdominal aorta with their innermost smooth muscle (SM) layers being most profoundly affected. The second purpose was to elucidate whether vascular local renin-angiotensin system (L-RAS) plays an important role in the differential remodeling of the two kinds of large arteries by examining the gene and protein expression of angiotensinogen (AO) and angiotensin II receptor type 1 (AT1R) and their localization in the vessel wall. The results showed that SUS induced an increase in the media thickness of the common carotid artery due to hypertrophy of the four SM layers and a decrease in the total cross-sectional area of the nine SM layers of the abdominal aorta without significant change in its media thickness. Irrespective of the nature of remodeling, the most prominent changes were in the innermost layers. Immunohistochemistry, in situ hybridization, Western blot, and real time quantitative PCR analysis revealed that SUS induced an up- and down-regulation in AO and AT1R expression in the common carotid artery and abdominal aorta, respectively. In conclusion, our findings have demonstrated some special features in the structural adaptation of large elastic arteries due to a medium-term simulated microgravity.

Background: Clinical studies have demonstrated that 50% of individuals with chronic renal disease (CRD) die of cardiovascular causes, including advanced calcific arterial and valvular disease; however, the mechanisms of accelerated calcification in CRD remain obscure, and no therapies can prevent disease progression. We recently demonstrated in vivo that inflammation triggers cardiovascular calcification. In vitro evidence also indicates that elastin degradation products may promote osteogenesis. Here, we used genetically modified mice and molecular imaging to test the hypothesis in vivo that cathepsin S (catS), a potent elastolytic proteinase, accelerates calcification in atherosclerotic mice with CRD induced by 5/6 nephrectomy., Methods and Results: Apolipoprotein-deficient (apoE(-/-))/catS(+/+) (n=24) and apoE(-/-)/catS(-/-) (n=24) mice were assigned to CRD and control groups. CRD mice had significantly higher serum phosphate, creatinine, and cystatin C levels than those without CRD. To visualize catS activity and osteogenesis in vivo, we coadministered catS-activatable and calcification-targeted molecular imaging agents 10 weeks after nephrectomy. Imaging coregistered increased catS and osteogenic activities in the CRD apoE(-/-)/catS(+/+) cohort, whereas CRD apoE(-/-)/catS(-/-) mice exhibited less calcification. Quantitative histology demonstrated greater catS-associated elastin fragmentation and calcification in CRD apoE(-/-)/catS(+/+) than CRD apoE(-/-)/catS(-/-) aortas and aortic valves. Notably, catS deletion did not cause compensatory increases in RNA levels of other elastolytic cathepsins or matrix metalloproteinases. Elastin peptide and recombinant catS significantly increased calcification in smooth muscle cells in vitro, a process further amplified in phosphate-enriched culture medium., Conclusions: The present study provides direct in vivo evidence that catS-induced elastolysis accelerates arterial and aortic valve calcification in CRD, providing new insight into the pathophysiology of cardiovascular calcification.

Although aortic or cardiac complications are common in patients with Marfan syndrome, the presence of an intracranial aneurysm is comparatively rare. In this study, the authors report on their experience with resection of a ruptured fusiform aneurysm of the posterior cerebral artery in a 30-year-old woman with Marfan syndrome. Microscopic examination of the resected tissue showed many Alcian blue-staining deposits, consistent with the presence of mucopolysaccharide in the tunica media and focal fragmentation of the internal elastic lamina.

Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE(-/-) mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin beta receptor (LTbetaR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE(-/-) mice with LTbetaR-Ig to interrupt LTbetaR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTbetaR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

Background: To investigate the relationship between the intima-media thickness of the carotid artery (CA-IMT) and its major risk factors in maintenance hemodialysis (MHD) patients., Methods: Seventy-five MHD patients and 30 healthy volunteers were enrolled. The MHD patients were divided into 3 subgroups according to their CA-IMT value., Results: CA-IMT values in the MHD group were significantly higher than those in the control group. The differences in age, systolic blood pressure (SBP), and levels of serum albumin, prealbumin, cholesterol and serum phosphate between the increased IMT group and the normal IMT group were significant. SBP and serum phosphate levels were also greater in the abnormal IMT group than those in the normal IMT group. Significant relationships were found between CA-IMT and age, SBP, and serum levels of phosphate, albumin and prealbumin. In multiple regression analysis, a high serum phosphate level, low serum prealbumin level and high SBP were significant independent risk factors of increased CA-IMT., Conclusions: CA-IMT was increased dramatically in MHD patients. A high serum phosphate level, low serum prealbumin level and high SBP may be associated with advanced arteriosclerosis., (Copyright 2009 S. Karger AG, Basel.)

We studied men with coronary atherosclerosis without acute coronary syndrome and determined typical valuable parameters of inflammatory (tumor necrotic factor, antagonist of receptor to interleukin [IL] 1, IL 6, IL 8, monocytes chemotactic protein 1, endothelial monocytes activating protein II), and destructive (matrix metalloproteinase [MMP] 3, MMP 7, MMP 9, tissue inhibitor of metalloproteinase) processes at consecutive stages of formation of coronary atherosclerotic plaque: "normal intimal tissue --> lipid stain --> early stable plaque --> unstable vulnerable plaque <--> stable plaque with fibrosis", and in 3 types of unstable plaques (lipid type, inflammatory erosive type, necrotic type).

Microparticles (MPs) are membrane vesicles harboring cell surface proteins and containing cytoplasmic components of the original cell. High levels of circulating MPs have been detected in pathological states associated with vascular dysfunction. We took advantage of the self-assembly method of tissue engineering to produce in vitro three vascular constructs from human vascular smooth muscle cells and fibroblasts to investigate the role of the adventitia in the modulation of vascular tone by MPs, comparing the contractile response of each of these constructs to histamine. The first two were composed of an adventitia (tissue-engineered vascular adventitia (TEVA)) or a media (tissue-engineered vascular media (TEVM)) solely, and the third one contained a media and an adventitia (tissue-engineered vascular media and adventitia (TEVMA)). In the three constructs, the results show that histamine induces contraction insensitive to blockade of inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) and not affected by MP treatment. MPs decreased NO production and nuclear factor (NF)-kappaB expression but did not affect superoxide anion (O(2)(-)) release in TEVA. MPs enhanced NF-kappaB expression but did not affect iNOS and COX-2 expression or NO or O(2)(-) release in TEVM. In TEVMA, MPs did not enhance NF-kappaB expression, but COX-2 expression was higher, and O(2)(-) release was lower. Thus, MPs affected NO, O(2)(-), NF-kappaB, and COX-2 in a subtle fashion to maintain the contractile response to histamine. The use of tissue-engineered vascular constructs results in a better understanding of the effect of MPs on human adventitia and media.

Cerebral aneurysm is a common disease with a high prevalence and can cause a catastrophic subarachnoid hemorrhage. To elucidate the molecular mechanism of the formation and progression of cerebral aneurysms, gene expression profiling was performed in experimentally induced rat cerebral aneurysms. The intima and media of cerebral arterial walls in rats with or without aneurysm induction were dissected respectively by a laser-microdissection technique. Changes in gene expression in the intima and media of aneurysmal walls were analyzed using Agilent Rat Oligo Microarrays, followed by a specific pathway analysis using GeneSpring software. Of the 41,012 genes examined, 633 were differentially expressed between a normal cerebral artery and a cerebral aneurysm in the intima, with 395 showing increased expression and 238 showing decreased expression. In the media, 1344 were differentially expressed, with 928 showing increased expression and 416 showing decreased expression. Specific pathway analysis revealed that increased gene expression was associated with proteinase, reactive oxygen species, growth factor, chemokine, complement, adhesion molecule and apoptosis in both the intima and the media of aneurysmal walls. Some genes showed an opposite expression pattern between the intima and the media indicating a different role between endothelial cells and vascular smooth muscle cells in cerebral aneurysm formation and progression. These data suggest that cerebral aneurysmal formation and progression are closely related to vascular inflammation, degeneration of extracellular matrix and apoptosis.

Nogo-B was recently identified as a novel vascular marker; the normally high vascular expression of Nogo-B is rapidly lost following vascular injury. Here we assess the potential therapeutic effects of Ad-Nogo-B delivery to injured vessels in vivo. Nogo-B overexpression following Ad-Ng-B infection of vascular smooth muscle cells (VSMCs) was shown to block proliferation and migration in a dose-dependent manner in vitro. We next assessed the effects of Ad-Ng-B treatment on neointima formation in two in vivo models of acute vascular injury. Adventitial delivery of Ad-Ng-B to wire-injured murine femoral arteries led to a significant decrease in the intimal area [0.014 mm(2) versus 0.030 mm(2) (P = 0.049)] and the intima:media ratio [0.78 versus 1.67 (P = 0.038)] as compared to the effects of Ad-beta-Gal control virus at 21 days after injury. Similarly, lumenal delivery of Ad-Ng-B to porcine saphenous veins prior to carotid artery grafting significantly reduced the intimal area [2.87 mm(2) versus 7.44 mm(2) (P = 0.0007)] and the intima:media ratio [0.32 versus 0.55 (P = 0.0044)] as compared to the effects following the delivery of Ad- beta-Gal, at 28 days after grafting. Intimal VSMC proliferation was significantly reduced in both the murine and porcine disease models. Gene delivery of Nogo-B exerts a positive effect on vascular injury-induced remodeling and reduces neointimal development in two arterial and venous models of vascular injury.

Cigarette smoke is associated with increased carotid intimal thickening or stroke. Preliminary work showed that exposure to smoke resulted in a 4.5-fold reduction of heat shock protein-70 (HSP70) expression in spleens of mice using gene microarray analysis. In the current study, we investigated the role of extracellular HSP70 in carotid intimal thickening of mice exposed to cigarette smoke. Intimal thickening was induced by placement of a cuff around the right carotid artery of mice. Cuff injury resulted in increased HSP70 mRNA expression in carotid arteries that persisted for 21 days. Cigarette smoke exposure decreased arterial HSP70 expression and significantly increased intimal thickening compared with mice exposed to air. Treatment of mice exposed to cigarette smoke with intravenous recombinant HSP70 attenuated intimal thickening through reduced phosphorylated extracellular signal-regulated kinase (pERK) expression in the arterial wall. In vitro experiments with rat aortic smooth muscle cells confirmed that recombinant HSP70 decreases pERK and proliferating cell nuclear antigen (PCNA) expression in cells exposed to cigarette smoke extract and H(2)O(2). Our study suggests that decreased expression of arterial HSP70 is an important mechanism by which exposure to cigarette smoke augments intimal thickening. The effects of recombinant HSP70 suggest a role for extracellular HSP70.

Objective: To determine whether systemic inflammation induced by chronic antigen-induced arthritis (AIA) accelerates vascular lesions in rabbits with atherosclerosis., Methods: Two models of atherosclerosis and chronic AIA were combined. Atherosclerosis was induced by coupling a hyperlipemic diet with an endothelial lesion at the femoral arteries, while chronic AIA was induced by ovalbumin injection. Markers in sera and peripheral blood mononuclear cells (PBMCs) as well as vessels and synovial membranes from the rabbits with the double phenotype (both chronic AIA and atherosclerosis) were compared with those from rabbits with each disease alone., Results: Serum levels of interleukin-6, C-reactive protein, and prostaglandin E(2) increased in rabbits with both chronic AIA and atherosclerosis as compared with healthy animals or animals with either chronic AIA alone or atherosclerosis alone. NF-kappaB binding and CCL2 and cyclooxygenase 2 (COX-2) expression were higher in PBMCs from rabbits with both chronic AIA and atherosclerosis than in PBMCs from healthy rabbits. The intima-media thickness ratio of femoral arteries was equally increased in rabbits with atherosclerosis alone and in rabbits with both chronic AIA and atherosclerosis, but the latter group showed a higher level of macrophage infiltration. Femoral CCL2 and COX-2 expression was increased in rabbits with both chronic AIA and atherosclerosis as compared with rabbits with atherosclerosis alone. In the aortas, vascular lesions were found in 27% of rabbits with atherosclerosis alone and in 60% of rabbits with both chronic AIA and atherosclerosis. Rabbits with both chronic AIA and atherosclerosis exhibited more severe synovitis and higher synovial expression of CCL2 than did rabbits with chronic AIA alone., Conclusion: The onset of chronic AIA in animals with atherosclerosis resulted in the local and systemic up-regulation of mediators of tissue inflammation and plaque instability associated with a higher incidence of aortic lesions. This model could represent a novel approach to the study of inflammation-associated atherosclerosis.

Aims: Cardiac allograft vasculopathy (CAV) continues to be an unsolved clinical problem requiring the development of new therapeutic strategies. We have previously demonstrated that ex vivo donor allograft treatment with decoy oligodeoxynucleotides (ODN) targeting the transcription factors, activator protein-1 (AP-1) or signal transducer and activator of transcription-1 (STAT-1), delays acute rejection and prolongs cardiac allograft survival. Here, we investigated whether this treatment regime also prevents the occurrence of CAV in a fully allogeneic rat heart transplantation model., Methods and Results: Wistar-Furth rat cardiac allografts were perfused ex vivo with AP-1 decoy ODN, STAT-1 decoy ODN, or buffer solution and transplanted into the abdomen of Lewis rats immunosuppressed with cyclosporine. Treatment with both decoy ODNs but not vehicle significantly attenuated the incidence and severity of CAV. Laser-assisted microdissection/real-time polymerase chain reaction as well as immunohistochemistry analyses revealed a significant increase in CD40 abundance in the coronary endothelial cells and medial smooth muscle cells on day 1 post transplantation which was virtually abolished upon AP-1 or STAT-1 decoy ODN treatment. While the AP-1 decoy ODN primarily attenuated basal CD40 expression, the STAT-1 decoy ODN suppressed tumour necrosis factor-alpha-/interferon-gamma-stimulated expression of CD40 in rat native endothelial cells., Conclusion: Treating donor hearts with decoy ODNs neutralizing AP-1 or STAT-1 at the time of transplantation prevents upregulation of CD40 expression in the graft coronary arteries and effectively inhibits CAV.

Elastin is not only one of fundamental components of arterial wall but also an albuminoid substance related to diseases such as aortic dissecting aneurysm. Distributions of elastic fiber in arterial wall were measured using image processing in animal experiment. Statistical results show that the elastic fiber content in arterial wall decreases along the ascending aorta, aortic arch and descending aorta, but the axial distribution of elastic fiber in aortic arch is uniform. Furthermore, the axial distributions in four representative circumferential positions nearly exhibit no notable differences. Hence, the circumferential distribution of elastic fiber in arterial wall is uniform. On the contrary, the radial distribution of elastic fiber in arterial wall is non-uniform, and the elastic fiber content in the internal layer decreased distinctly, compared with that in the middle layer and the external layer. Therefore, the blood vessel can be reasonably simplified as 3-dimensional symmetrical composite material with segments in axial direction and layers in radial direction to a certain extent.

Two well-recognized patterns of calcification occur in large- and medium-sized arteries, intimal calcification associated with atherosclerosis and medial calcification described by Mönckeberg. Calcification limited to the internal elastic lamina is a third pattern of calcification not previously reported in coronary arteries. Here we describe 19 cases of coronary artery internal elastic lamina calcification. We serially sectioned and examined the coronary arteries of 66 patients with advanced AIDS and 27 HIV- controls with other chronic illnesses. We observed calcification of the internal elastic lamina in 10 HIV+ patients and 9 controls. HIV- patients with internal elastic lamina calcification were significantly older than HIV- patients without it (P=0.008) and HIV+ patients with it (P=0.006). Occasionally, calcification encroached on adjacent intimal or medial tissue with mild fibrosis. There was frequent disruption of the internal elastic lamina but no evidence of inflammation. Calcification was the dominant histologic feature in all cases. Von Kossa, Alizarin red, and trichrome/elastic stains confirmed these findings. Patients with internal elastic lamina calcification often had extensive medical histories but did not suffer from chronic renal failure or other conditions known to cause calcium dysregulation. We describe coronary internal elastic lamina calcification in HIV+ patients and older HIV- adults. The clinical significance of this finding is unknown. It could lead to arterial stiffening and increased pulse pressure and could be mistaken for intimal calcification on coronary imaging. Internal elastic lamina calcification may result from premature aging due to HIV disease and chronic illness or from metabolic disorders in HIV+ patients.

Objective: The aortic media lesion is a key pathologic feature in thoracic aortic dissection. To identify key proteins in aortic media lesions that may contribute to its pathogenesis, we performed proteomic studies to find differentially expressed proteins in the media from diseased and normal thoracic aorta., Methods: Ascending aortic segments were obtained from patients with thoracic aortic dissection (n = 8) and age-matched normal donors (n = 6). The differentially expressed proteins of their media tissues were analyzed by 2-dimensional electrophoresis and mass spectrometry, and verified by Western blotting. Oxidative stress was measured by functional assays in a larger sample size (15 patients and 10 controls)., Results: Image analysis of the protein profiles from 2-dimensional gels revealed 126 differentially expressed proteins, of which 26 were identified by mass spectrometry. Among them, extracellular superoxide dismutase, an enzyme involved in oxidative stress, was selected for further studies. Western blotting showed that extracellular superoxide dismutase expression was more than 50% lower in patient samples than in controls (P < .001). Superoxide dismutase activity was consistently decreased (P < .001) and lipid peroxidation was increased (P = .019) in patient media homogenates compared with that in controls., Conclusion: Our results indicate that protein expression profiles in the aortic media from thoracic aortic dissection differ significantly from that of controls, which may provide important insights into the disease mechanisms. This study also suggests that increased oxidative stress may play an important role in the disease.

Aims: This retrospective study aimed to document and illustrate the histomorphological changes underlying peripheral vascular disease (PVD). More specifically, it aimed to analyse and quantify those changes that lead to lower limb amputations. Histological changes were assessed in relation to various clinical pathologies, and significant correlations were sought thereafter., Methods: A total of 1305 arterial segments were examined from 58 consecutive patients undergoing a lower limb amputation from January 2002 to December 2003. Serial arterial segments were taken from the femoral, popliteal, anterior tibial, posterior tibial, peroneal, and dorsalis pedis arteries, and the degrees of atherosclerotic stenosis and medial calcification were histologically quantified., Results: Atherosclerosis was associated with severe arterial stenosis. An increased occurrence of severe atherosclerotic narrowing coincided with increasing patient age (p = 0.0166), hypertension (p = 0.0019), and diabetes mellitus (p = 0.0036). The presence of medial calcification was an important pathological feature in patients under 70 years of age (p = 0.0308) and significantly more severe in those with diabetes mellitus (p<0.001)., Conclusion: Atherosclerosis and medial calcification are significant underlying lesions in diabetic patients undergoing lower limb amputation. Medial calcification can cause significant stiffening of the arterial wall and a reduction in its ability to respond to vasodilator stimuli.

We have recently demonstrated that stem cell antigen 1-positive (Sca-1(+)) progenitors exist in the vascular adventitia of apolipoprotein E-deficient (apoE(-/-)) mice and contribute to smooth muscle cell (SMC) accumulation in vein graft atherosclerosis. Using a combined proteomic and metabolomic approach, we now characterize these local progenitors, which participate in the formation of native atherosclerotic lesions in chow-fed apoE(-/-) mice. Unlike Sca-1(+) progenitors from embryonic stem cells, the resident Sca-1(+) stem cell population from the vasculature acquired a mature aortic SMC phenotype after platelet-derived growth factor-BB stimulation. It shared proteomic and metabolomic characteristics of apoE(-/-) SMCs, which were clearly distinct from wild-type SMCs under normoxic and hypoxic conditions. Among the differentially expressed proteins were key enzymes in glucose metabolism, resulting in faster glucose consumption and a compensatory reduction in baseline interleukin-6 secretion. The latter was associated with a marked upregulation of insulin-like growth factor binding proteins (IGFBPs) 3 and 6. Notably, reconstitution of interleukin-6 to levels measured in the conditioned medium of wild-type SMCs attenuated the elevated IGFBP expression in apoE(-/-) SMCs and their vascular progenitors. This coregulation of apoE, interleukin-6, and IGFBPs was replicated in wild-type SMCs from hypercholesterolemic mice and confirmed by silencing apoE expression in SMCs from normocholesterolemic mice. In summary, we provide evidence that Sca-1(+) progenitors contribute to native atherosclerosis in apoE(-/-) mice, that apoE deficiency and hypercholesterolemia alter progenitor cell behavior, and that inflammatory cytokines such as interleukin-6 act as metabolic regulators in SMCs of hyperlipidemic mice.

Aims: To study how the balance between tacrolimus elution and polymer degradation from drug-eluting stents (DES) affects neointimal thickening (NIT) in swine coronary arteries., Methods and Results: We assessed a fast-degrading high dose (2 microg/mm2), a slow degrading low dose (1 microg/mm2) or polymer-only coated DES (Pol) versus bare metal stent (BMS). Coronary segments were pre-injured with a balloon/artery ratio of 1.1 to 1.3. Then stents were implanted at that site with a stent/artery ratio of 1.1, with a follow-up period of 5 to 180 days. Histology showed a well endothelialised neointima (82 +/- 1% in high dose DES vs. 93 +/- 8% in BMS) already at five days, without differences in eNOS expression. Morphometry indicated that neointimal thickness in DES was significantly reduced as compared to BMS and Pol at 28 and 90 days. Polymer degradation products induced a distinct inflammatory response which was effectively suppressed in DES. Between 90 and 180 days, however, the slow degrading low-dose stent showed catch-up of NIT., Conclusions: Tacrolimus eluted from a biodegradable stent coating can suppress the inflammatory effect of the coating degradation products if the balance between the drug levels and the degradation products is favorable.

Aims: To determine and quantify calcitonin receptor (CTR) immunoreactivity associated with specific cell types within, and associated with, the endothelial layers, neo-intima, media and vasa vasorum of diseased radial and internal mammary arteries., Methods and Results: Immunohistochemistry and anti-CTR antibodies were used to identify positive cells within remnants of diseased human radial (n = 3) and internal mammary arteries (n = 4) that remained after bypass surgery. Three cell types expressed CTR, including endothelial cells, fibroblast-like cells within the neo-intima, and cellular structures aligned with the smooth muscle cells of the media. Other smaller cells within the surrounding parenchyma of the vasa vasorum of diseased vessels and blood-borne cells were also immunoreactive. Immunoquantification of CTR expression (Intensity x Proportional Area) in the endothelium (P < 0.05), neo-intima (P < 0.02) and media (P < 0.03) established a significant statistical correlation (Students' two-tailed t-test) with the ratio of intimal/media thickness., Conclusions: Increased immunoreactivity developed using anti-CTR antibodies was associated with specific cell types in the endothelial layers, neo-intima, media and vasa vasorum of diseased regions of radial and internal mammary arteries, in which there was an increased intimal/media ratio. Furthermore, CTR+, blood-borne cells present in the vessels of diseased regions suggest recruitment into these surrounding tissues.

PTHrP is produced in vessels and acts as a local modulator of tone. We recently reported that PTHrP(1-34) is able to induce vasorelaxation in rat uterine arteries, but in pregnancy, this response is blunted and becomes strictly endothelium dependent. The present study aimed to get insights into the mechanisms involved in these changes because the adaptation of uterine blood flow is essential for fetal development. On d 20 of gestation, RT-PCR analysis of uterine arteries showed that PTH/PTHrP receptor (PTH1R) mRNA expression was decreased, whereas that of PTHrP mRNA was increased. This was associated with a redistribution of the PTHrP/PTH1R system, with both PTH1R protein and PTHrP peptide becoming concentrated in the intimal layer of arteries from pregnant rats. On the other hand, the blunted vasorelaxation induced by PTHrP(1-34) in uterine arteries from pregnant rats was specifically restored by indomethacin and a specific cyclooxygenase-2 inhibitor, NS 398. This was associated with an increase in cyclooxygenase-2 expression and in 8-iso-prostaglandin F(2alpha) release when uterine arteries from pregnant rats were exposed to high levels of PTHrP(1-34). Most interestingly, 8-iso-prostaglandin F(2alpha) itself was able to increase PTHrP expression and reduce PTH1R expression in cultured rat aortic smooth muscle cells. These results suggest a local regulation of uterine artery functions by PTHrP during pregnancy resulting from PTH1R redistribution. Moreover, they shed light on a potential role of 8-iso-prostaglandin F(2alpha).

Context: Medial calcification of muscular arteries is known as Mönckeberg sclerosis (MS). Although this was first described in 1903, disagreement persists over its precise histologic appearance. Some, including Mönckeberg, have written that the media alone are calcified, whereas others maintain that both the media and internal elastic lamina (IEL) are involved. Since vascular calcification is of great interest to investigators and clinicians, defined criteria for classifying calcified arterial lesions are important., Objective: To clarify the histologic definition of MS with regard to calcification of the IEL., Design: We reviewed slides from 14 incisional and excisional surgical biopsies and autopsy specimens containing arteries with previously diagnosed MS. We looked specifically for medial and IEL calcification and used von Kossa, alizarin red, and trichrome/elastic stains to confirm our findings. We also performed a literature search on the histologic appearance of MS., Results: Both medial and IEL calcification were present in all specimens. Medial calcification extended alongside calcified IEL. In focal regions, calcification appeared limited to the IEL, with minimal medial calcification. Occasionally, calcified nodules in the media appeared separated from the IEL yet were connected to it in other planes of section. Despite these variations in appearance, IEL involvement was universal. Of 25 journal articles and texts, 10 state that MS involves the IEL with calcification, whereas 15 state or suggest that it does not., Conclusions: Our findings indicate MS involves both the IEL and media with calcification in spite of inconsistencies on this point in the medical literature.

Arterial calcification was previously viewed as an inevitable, passive, and degenerative process that occurred at the end stages of atherosclerosis. Recent studies, however, have demonstrated that calcification of arteries is a complex and regulated process. It may occur in conjunction with atherosclerosis or in an isolated form that is commonly associated with diabetes and renal failure. Higher artery calcium scores are associated with increased cardiovascular events, and some aspects of arterial calcification are similar to the biology of forming bone. Arterial calcification can thus be viewed as a distinct inflammatory arteriopathy, much like atherosclerosis and aneurysms, with its own contribution to cardiovascular morbidity and mortality. Current research involves efforts to define the complex interactions between cellular and molecular mediators of arterial calcification and, in particular, the role of endogenous calcification inhibitors. This review discusses the clinical relevance, cellular events, and suspected molecular pathways that control arterial calcification.

Objective: Hypoxia-inducible factor (HIF)-1alpha is the regulatory subunit of a transcriptional complex, which controls the recruitment of multipotent progenitor cells and tissue repair in ischemic tissue by inducing stromal cell-derived factor (SDF)-1alpha expression. Because HIF-1alpha can be activated under normoxic conditions in smooth muscle cells (SMCs) by platelet products, we investigated the role of HIF-1alpha in SDF-1alpha-mediated neointima formation after vascular injury., Methods and Results: Wire-induced injury of the left carotid artery was performed in apolipoprotein E-deficient mice. HIF-1alpha expression was increased in the media as early as 1 day after injury, predominantly in SMCs. Nuclear translocation of HIF-1alpha and colocalization with SDF-1alpha was detected in neointimal cells after 2 weeks. HIF-1alpha mRNA expression was induced at 6 hours after injury as determined by real-time RT-PCR. Inhibition of HIF-1alpha expression by local application of HIF-1alpha-siRNA reduced the neointimal area by 49% and significantly decreased the neointimal SMCs content compared with control-siRNA. HIF-1alpha and SDF-1alpha expression were clearly diminished in neointimal cells of HIF-1alpha-siRNA treated arteries., Conclusions: HIF-1alpha expression is directly involved in neointimal formation after vascular injury and mediates the upregulation of SDF-1alpha, which may affect the stem cell-based repair of injured arteries.

Recently, the role of bone marrow (BM)-derived endothelial cells and smooth muscle cells (SMCs) has been extensively studied in the pathogenesis of atherosclerosis. In this study we examined the effect of hypercholesterolemia on cuff-induced intimal thickening in LDL-receptor knockout (LDLR-/-) mice fed with a high-fat diet. We transplanted BM of green fluorescence protein (GFP)-transgenic mice to LDLR-/- mice to identify the cell lineage in the lesion. After BM transplantation mice were fed with a high-fat diet for 4 weeks and were then planted a polyethylene cuff on the right femoral artery. Two weeks after cuff placement, atherosclerotic lesions developed in the intima predominantly consisting of a massive accumulation of foam cells with a number of alpha smooth muscle actin (alphaSMA)- and GFP-positive cells. Adventitial small vessels were positive both for CD31 and GFP. Our data indicate that BM-derived cells can contribute to the development of atherosclerosis in the presence of hypercholesterolemia.

Hyperhomocysteinemia is considered an independent risk factor for atherosclerosis. The present study was designed to assess the effect of high level of serum homocysteine on other cardiovascular risk factors and markers in rats and to study its mode of action in initiating atherosclerosis. To address this issue, four different doses of methionine (0.1 g/kg, 0.25 g/kg, 0.5 g/kg, 1 g/kg) were orally administered to four groups (Group II, III, IV, V respectively) of rats (6 rats in each group) for a period of 8 weeks to get different level of homocysteine in serum. Group I was administered with saline and served as control. Our results revealed that the level of Total cholesterol, Triglyceride, and Oxidized low-density lipoproteins increased significantly with the increase in the level of serum homocysteine. The levels of Resistin, C-reactive protein and cysteinyl-leukotrienes were found to be significantly high in Group IV (P<0.001 vs Group I) and Group V (P<0.001 vs Group I) at 8 weeks. Total antioxidant capacity and nitrite/nitrate level in serum showed negative correlation with the increased dose of methionine. The mRNA expression and the enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase significantly increased only in livers of rats of Group V. Furthermore, high mRNA expression of P2 receptors and caveolin were found in aorta of rats administered with high dose of methionine (Group IV and V at 8 weeks). Data obtained from in-vitro effect of homocysteine on isolated aortic arch also showed induction in P2 receptors and caveolin with the increase in the concentration of homocysteine. These findings collectively suggest that hyperhomocysteinemia initiates atherosclerosis by modulating the cholesterol biosynthesis and by significantly inducing the level of other cardiovascular risk factors and markers, which play important role in initiating atherosclerosis.

Background: The formation of an occluding thrombus on a ruptured or eroded atherosclerotic plaque is the hallmark event leading to acute coronary syndromes, myocardial infarction, and sudden death in humans. However, other species are highly resistant to plaque complications, and the specific processes predisposing to plaque destabilization and thrombosis are poorly understood., Methods and Results: Mice carrying a null mutation of a gene regulating intracellular cholesterol transport (the Niemann-Pick C1 [Npc1] gene) were crossed with apolipoprotein E (Apoe) knockout mice to examine the effect of Npc1 on atherosclerotic lesion formation. Double-mutant mice showed greater lesion area compared with Apoe-/- littermates. Remarkably, the double mutants also developed large, protruding thrombi associated with the plaques and prominent medial degradation with inflammatory cell infiltration into the adventitia. Genetic studies suggested that the BALB background was permissive for plaque complications compared with C57BL/6J, and a BALB susceptibility locus was mapped by linkage analysis to chromosome 6. Examination of clotting parameters in double-knockout mice revealed that native clotting times were shortened and thrombin-antithrombin complex and soluble CD40 ligand levels were elevated compared with wild-type controls. In addition, cathepsin K was induced in Npc1-/- macrophages, and cathepsin K immunostaining and elastase activity were increased in proximal aortas of double-mutant mice compared with controls., Conclusions: A defect in intracellular cholesterol trafficking caused by the Npc1 null mutation predisposes to increased lesion formation, atherothrombosis, and medial degradation. Plaque complications may require a procoagulant state and an increased protease activity, leading to plaque destabilization.

It has been argued that smoking or overweight might contribute to hearing disorder by atherogenic narrowing of the nutrient arteries to the cochlea. The carotid intima-media thickness (CIMT) is a surrogate marker for generalized atherosclerosis. We analyzed a subgroup (n=2619) from a general population sample in north-eastern Germany aged 45-81 years (Study of Health in Pomerania, SHIP). Assessments included self-statements about hearing disorder and medical examinations of CIMT. Using ordinal logistic regression for data analysis and after adjustment for cigarettes per day, waist circumference, diabetes, exposure to noise, age and sex, we found CIMT remained a predictor of hearing disorder (odds ratio 1.8, 95% confidence interval 1.0-3.2). Cigarettes per day and waist circumference were related to CIMT but not to hearing disorder. The findings suggest a positive association between CIMT and hearing disorder.

In this study, the shape and the configuration of smooth muscle cells (SMCs) within the arterial wall are altered to investigate their influence on molecular transport across the media layer of the thoracic aorta wall. In a 2D geometry of the media layer containing SMCs, the finite-element method has been employed to simulate the diffusion of solutes through the media layer. The media is modeled as a heterogeneous system composed of SMCs having elliptic or circular cross sections embedded in a homogeneous porous medium made of proteoglycan and collagen fibers with an interstitial fluid filling the void. The arrangement of SMCs is in either ordered or disordered fashion for different volume fractions of SMCs. The interstitial fluid enters the media through fenestral pores, which are assumed to be distributed uniformly over the internal elastic lamina (IEL). Results revealed that in an ordered arrangement of SMCs, the concentration of adenosine 5'-triphosphate (ATP) over the surface of SMCs with an elliptic cross section is 5-8% more than those of circular SMCs in volume fractions of 0.4-0.7. The ATP concentration at the SMC surface decreases with volume fraction in the ordered configuration of SMCs. In a disordered configuration, the local ATP concentration at the SMC surface and in the bulk are strongly dependent on the distance between neighboring SMCs, as well as the minimum distance between SMCs and fenestral pores. Moreover, the SMCs in farther distances from the IEL are as important as those just beneath the IEL in disordered configurations. The results of this study lead us to better understanding of the role of SMCs in controlling the diffusion of important species such as ATP within the arterial wall.

Objective: Thrombospondin-1 (TSP1) is described as a positive regulator of vascular smooth muscle growth in cell culture. However, insight into the in vivo effects of TSP1 on smooth muscle cell (SMC) function is lacking., Methods and Results: We analyzed wild-type (WT) and TSP1-deficient (Tsp1-/-) mice in a carotid artery ligation model, in which neointimal lesions form without overt mechanical damage to the endothelium. On ligation, the expression of TSP1 increased strongly in the matrix of neointima and adventitia. In the early phase after ligation (day 3 to 7), activation, proliferation, and migration of medial SMCs were delayed and impaired in Tsp1-/- mice, in parallel with defective upregulation of metalloproteinase (MMP)-2 activity. As a result, Tsp1-/- arteries developed smaller neointimal lesions, a thicker media but comparably attenuated patency as in WT arteries, 28 days after ligation. Furthermore, medial and neointimal SMCs in Tsp1-/- mice produced more collagen, more osteopontin, and displayed weaker smooth muscle actin staining than WT SMCs, indicative of a modified SMC phenotype in Tsp1-/- mice., Conclusions: Arterial SMC activation in the absence of TSP1 is delayed and dysregulated, reducing neointima formation, on mild vascular injury.

Background: In the present study, an arterial tissue-engineered vascular media (TEVM) was produced from cultured human smooth muscle cells of the umbilical artery and we took advantage of this model to evaluate the regulation of contraction and the signalling pathways of polyphenols in arteries., Methods: Cultured human smooth muscle cells of the umbilical artery were used to produce arterial TEVMs. Contraction experiments were performed to determine intracellular targets involved in the modulation of contraction by polyphenols extract from red wine, Provinols (SEPPIC Groupe Air Liquide, Paris, France)., Results: Smooth muscle cells in arterial TEVM displayed a differentiated phenotype as demonstrated by the expression of alpha-smooth muscle actin, a vascular smooth muscle-specific marker, and tissue contraction in response to vasoconstrictor and vasodilator agents. Contractions caused by histamine were associated with an increase in [Ca(2+)](i) and a Ca(2+)-independent signalling pathway. The latter pathway involved mechanisms sensitive to protein kinase C, myosin light chain kinase, and Rho-associated protein kinase inhibitors. The regulation of contraction induced by Provinols shows that treatment of arterial TEVM with this compound significantly decreased histamine-induced contraction. This effect was associated with the inhibition of the Rho-associated protein kinase pathway and the decrease in alpha-smooth muscle actin expression., Conclusion: The use of arterial TEVM, brings new insights into the mechanisms by which polyphenols regulate vascular contraction in the human artery.

Objective: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a multi-system disorder characterized by progressive cyst formation in the kidneys. Serious complications of ADPKD are intracranial and aortic aneurysms. The condition is mainly caused by mutations in the PKD1 or PKD2 gene. We have carefully analyzed vascular remodeling in hypomorphic Pkd1(nl/nL) mouse model with dissecting aneurysms in the aorta., Methods and Results: Quantitative real-time polymerase chain reaction revealed that in the aorta the expression of normal Pkd1 is reduced to approximately 26%. Using (immuno)histochemistry we have characterized the pathogenetic sequence for dissecting aneurysm formation. The aorta shows regions with accumulation of matrix components between the elastin lamellae. This is followed by increased numbers of smooth muscle cells and locally weakening of the media. In the intima, accumulation of matrix components and detachment of endothelial cells from the elastin lamellae results in a tear. The combination of weak media and a tear in the intima leads to rupture of the vessel wall resulting in intramural bleeding., Conclusions: The Pkd1(nl/nl) mouse reveals that polycystin1 is implicated in maintenance of the vessel wall structural integrity, and it is a useful model for dissecting aneurysm formation studies.

Increase in the expression of leukocyte antigen-related (LAR) protein causes insulin resistance, an important contributor to atherosclerosis. However, the function of LAR in atherosclerosis is not known. To address whether LAR is important in the response of vascular cells to atherogenic stimuli, we investigated cell proliferation, migration, and insulin-like growth factor-1 receptor (IGF-1R) signaling in wild-type and LAR(-/-) mouse vascular smooth muscle cells (VSMC) treated with IGF-1. Absence of LAR significantly enhanced proliferation and migration of VSMC compared with wild-type cells after IGF-1 treatment. U0126 and LY249002, specific inhibitors of MAPK/ERK kinase (MEK) and phosphoinositide 3-kinase, respectively, inhibited IGF-1-induced DNA synthesis and migration in both wild-type and LAR(-/-) VSMC. IGF-1 markedly enhanced IGF-1R phosphorylation in both wild-type and LAR(-/-) VSMC, but the phosphorylation was 90% higher in knock-out cells compared with wild-type cells. Absence of LAR enhanced phosphorylation of insulin receptor substrate-1 and insulin receptor substrate-1-associated phosphoinositide 3-kinase activity in VSMC treated with IGF-1. IGF-1-induced phosphorylation of ERK1/2 also increased significantly in LAR(-/-) VSMC compared with wild-type cells. Furthermore, LAR directly binds to IGF-1R in glutathione S-transferase-LAR pull-down and IGF-1R immunoprecipitation experiments and recombinant LAR dephosphorylates IGF-1R in vitro. Neointima formation in response to arterial injury and IGF-1R phosphorylation in neointima increased significantly in LAR(-/-) mice compared with wild-type mice. A significant decrease in body weight, fasting insulin, and IGF-1 levels were observed in LAR(-/-) mice compared with wild-type mice. Together, these data indicate that LAR regulates IGF-1R signaling in VSMC and dysregulation of this phosphatase may lead to VSMC hyperplasia.

Objective: Intimal smooth muscle cells (SMCs) are dedifferentiated SMCs that have a powerful ability to proliferate and migrate. This cell-type is responsible for the development of intimal hyperplasia after vascular angioplasty. Retinoids, especially all-trans retinoid acid, are known to regulate many processes activated at sites of vascular injury, including modulation of SMC phenotype and inhibition of SMC proliferation. Intracellular levels of active retinoids are under firm control. A key enzyme is the all-trans retinoic acid-degrading enzyme cytochrome p450 isoform 26 (CYP26). Thus, an alternative approach to exogenous retinoid administration could be to increase the intracellular level of all-trans retinoic acid by blocking CYP26-mediated degradation of retinoids., Methods and Results: Vascular intimal and medial SMCs expressed CYP26A1 and B1 mRNA. Although medial cells remained unaffected, treatment with the CYP26-inhibitor R115866 significantly increased cellular levels of all-trans retinoic acid in intimal SMCs. The increased levels of all-trans retinoic acid induced retinoid-regulated genes and decreased mitogenesis., Conclusions: Blocking of the CYP26-mediated catabolism mimics the effects of exogenously administrated active retinoids on intimal SMCs. Therefore, CYP26-inhibitors offer a potential new therapeutic approach to vascular proliferative disorders.

Aldosterone plays a key role in cardiovascular and renal injury. The underlying mechanisms are not completely understood. Because the epidermal growth factor receptor (EGFR) is involved in the development of fibrosis and vascular dysfunction, upregulation of EGFR expression by aldosterone-bound mineralocorticoid receptor (MR) is an attractive hypothesis. We investigated the effect of aldosterone on EGFR expression in the aorta of adrenalectomized rats and in human aorta smooth muscle cells (HAoSMC) in primary culture. Aldosterone, but not dexamethasone, stimulated EGFR expression in vivo in the aorta as well as in HAoSMC. EGFR degradation was not affected. Aldosterone-induced EGFR expression in HAoSMC was dose dependent and prevented by spironolactone. Furthermore, incubation of HAoSMC with aldosterone led to enhanced EGF-induced ERK1/2 phosphorylation and an EGFR-dependent increase in media fibronectin. EGFR promoter reporter gene assay as well as chromatin immunoprecipitation data indicate that MR interacts with the EGFR promoter. With deletion constructs we gained evidence that this interaction takes place between the hMR and the EGFR promoter regions 316-163 (stronger activation site, EC50 approximately 1.0 nM) and 163-1 (weaker activation site, EC50 approximately 0.7 nM), which do not comprise canonical glucocorticoid response elements and are not activated by the human glucocorticoid receptor. The interactions require in part the NH2-terminal domains of MR. ELISA-based transcription factor DNA binding assay with in vitro synthesized hMR suggest direct binding to region 163-1. Our results indicate that aldosterone leads to enhanced EGFR expression via an interaction with the EGFR promoter, which is MR specific and could contribute to the aldosterone-induced increase in fibronectin abundance.

Atherosclerosis localizes at a bend andor bifurcation of an artery, and low density lipoproteins (LDL) accumulate in the intima. Hemodynamic factors are known to affect this localization and LDL accumulation, but the details of the process remain unknown. It is thought that the LDL concentration will be affected by the filtration flow, and that the velocity of this flow will be affected by deformation of the arterial wall. Thus, a coupled model of a blood flow and a deformable arterial wall with filtration flow would be invaluable for simulation of the flow field and concentration field in sequence. However, this type of highly coupled interaction analysis has not yet been attempted. Therefore, we performed a coupled analysis of an artery with multiple bends in sequence. First, based on the theory of porous media, we modeled a deformable arterial wall using a porohyperelastic model (PHEM) that was able to express both the filtration flow and the viscoelastic behavior of the living tissue, and simulated a blood flow field in the arterial lumen, a filtration flow field and a displacement field in the arterial wall using a fluid-structure interaction (FSI) program code by the finite element method (FEM). Next, based on the obtained results, we further simulated LDL transport using a mass transfer analysis code by the FEM. We analyzed the PHEM in comparison with a rigid model. For the blood flow, stagnation was observed downward of the bends. The direction of the filtration flow was only from the lumen to the wall for the rigid model, while filtration flows from both the wall to the lumen and the lumen to the wall were observed for the PHEM. The LDL concentration was high at the lumenwall interface for both the PHEM and rigid model, and reached its maximum value at the stagnation area. For the PHEM, the maximum LDL concentration in the wall in the radial direction was observed at the position of 3% wall thickness from the lumenwall interface, while for the rigid model, it was observed just at the lumenwall interface. In addition, the peak LDL accumulation area of the PHEM moved about according to the pulsatile flow. These results demonstrate that the blood flow, arterial wall deformation, and filtration flow all affect the LDL concentration, and that LDL accumulation is due to stagnation and the presence of filtration flow. Thus, FSI analysis is indispensable.

Inflammatory markers, particularly C-reactive protein (CRP), predict incident cardiovascular disease and are associated with the presence of subclinical atherosclerosis. The relations between multiple inflammatory markers and direct measures of atherosclerosis are less well established. Participants in the Offspring Cohort of the Framingham Heart Study (n = 2,885, 53% women, mean age 59 years) received routine assessments of common carotid artery intima-media thickness (CCA-IMT), internal carotid artery intima-media thickness (ICA-IMT), and the presence or absence of > or =25% carotid stenosis by ultrasonography. Circulating inflammatory markers assessed from an examination 4 years later included CRP, interleukin-6 (IL-6), intercellular adhesion molecule-1, monocyte chemoattractant protein-1, P-selectin, and CD40 ligand. Assessed as a group, inflammatory markers were significantly associated with ICA-IMT (p = 0.01), marginally with carotid stenosis (p = 0.08), but not with CCA-IMT. Individually, with an increase from the 25th to 75th percentile in IL-6, there were significant increases in ICA-IMT and carotid stenosis (for ICA-IMT, estimated fold increase 1.04, 95% confidence interval 1.03 to 1.06, p = 0.0004; for carotid stenosis, odds ratio 1.25, 95% confidence interval 1.06 to 1.47, p = 0.007) after adjustment for age, gender, and established risk factors for atherosclerosis. There was a similar significant multivariate-adjusted association of CRP with ICA-IMT but not with carotid stenosis. Smoking appeared to modify the associations of ICA-IMT with CRP (p = 0.009) and with IL-6 (p = 0.006); the association was more pronounced in current (vs former or never) smokers. In conclusion, there were modest associations of inflammatory markers, particularly IL-6, with carotid atherosclerosis. This association appears more pronounced in current smokers than in former smokers and nonsmokers.

Inflammation plays a central role in vascular repair, and spreads into perivascular tissue (PVT) following angioplasty. Chemokines (CK) and chemokine receptors (CKR) are key determinants of inflammatory chemotaxis. We sought to assess the arterial and perivascular expression of the CK CCL2 and CXCL2, and the CKR CCR2, CCR5, and CXCR4 in balloon-injured porcine coronary arteries. Vascular cells that express specific CK and CKR mRNA during post-angioplasty time course were detected by in situ hybridization (ISH), and expression was quantified by real time RT-PCR in PVT. CCL2 was maximal in PVT from 2 to 24h post injury, coincident with local macrophage-activation. Expression was upregulated in media and adventitia from 24h to 3 days, and in neointima at 7 days. CXCL2 was detected in media at 2 and 4h, and also in some neointimal cells. CCR2 and CCR5 were maximal in PVT at 24h and 3 days, respectively. Expression shifted to media and adventitia at 2 and 3 days, and to neointima and adventitia at 7 days, and was low at 14 days. CXCR4 was low in PVT, but was upregulated in media and adventitia at 2 and 3 days, as well as in neointima and adventitia at 7 days. In conclusion, PVT is the primary source of inflammatory CK and CKR early post-angioplasty. Specific sequential patterns of CK- and CKR-synthesis are identified that may regulate phase-specific chemotaxis by spatio-temporally differential expression during coronary response to injury.

Background and Purpose: Inflammation is a key mechanism in atherosclerosis. Variation in genes encoding inflammatory responses may therefore influence atherosclerosis risk possibly through interaction with chronic infections and proinflammatory environmental risk factors such as smoking, diabetes, and obesity. The Toll-like receptor family (TLRs) genes TLR2 and TLR4, both involved in the inflammatory process, are potential candidates and TLR-4 has been previously associated with cardiovascular disease, although other studies have failed to confirm this., Methods: A total of 3000 individuals from the prospective community-based Carotid Atherosclerosis Progression Study (CAPS) were genotyped for single nucleotide polymorphisms: TLR2 (Arg753Gln, -16934 A/T) and TLR4 (D299G, T399I). Associations were determined with common carotid artery intima-media thickness (IMT) at baseline and also progression of IMT over the 3-year follow-up period. Gene-environment interactions with high sensitive C-reactive protein, smoking, body mass index, and diabetes were determined., Results: There was no association between single nucleotide polymorphisms or haplotypes in either TLR4 or TLR2 and either baseline IMT or progression of IMT over the 3-year follow up. There were no interactions among the three proinflammatory risk factors. No genotype or haplotype was associated with high sensitive C-reactive protein., Conclusions: In this large community population, we found no evidence for genetic variation in these two TLRs being risk factors for increased IMT either directly or through interaction with proinflammatory risk factors. We were unable to confirm associations with the TLR4 polymorphisms reported in previous smaller studies.

Objective: Relationship between adult growth hormone deficiency (AGHD) and increased cardiovascular disease risk is very well known in hypopituitary patients treated with conventional hormone replacement therapy other than growth hormone (GH) administration. Endothelial dysfunction, an early and reversible event in pathogenesis of atherosclerosis, is associated with increased vascular smooth muscle tone, arterial stiffening and intima-media thickness (IMT). Coronary flow reserve (CFR) measurement by transthoracic Doppler echocardiography (TTDE) reflects coronary microvascular and endothelial functions, as a cheaper and an easy screening test. We have used TTDE to evaluate endothelial function and coronary microvascular function in AGHD., Design: Cross-sectional observational study., Patients: A total of 10 GH-deficient adults on conventional replacement therapy other than GH (4 males, 6 females; mean age 37 +/- 11 years) and 15 healthy subjects (7 males, 8 females; mean age 41 +/- 11 years) were studied. Patients and controls were all nonsmokers, normotensive and nondiabetic., Measurements: IGF-1, free T4, lipid profile, insulin, glucose, insulin resistance (IR), anthropometrical and physical parameters were recorded. CFR recordings and IMT measurements were performed using the Vivid 7 echocardiography device., Results: IMT were significantly higher in patients than controls (0.70 + 0.19 mm and 0.53 + 0.13 mm, respectively; P = 0.02). CFR was significantly lower in patients than in controls (1.96 +/- 0.35 and 2.62 +/- 0.45, respectively; P < 0.001). CFR was positively correlated with IGF-1 levels (r = 0.54, P = 0.005)., Conclusion: CFR is significantly lower in adults with GH deficiency than in controls. Direct correlation between CFR and IGF-1 concentrations suggests GH replacement could improve microvascular function and thereby could decrease cardiovascular morbidity and mortality in AGHD.

The authors review 20 cases of segmental arterial mediolysis (SAM) including 3 newly reported cases. SAM developed in areas of vascular distention in 2 of the latter cases: 1 in utero in the heart of a recipient of a twin transfusion syndrome and the other in the jejunum secondary to partial venous obstruction. In the third case, it occurred in a patient with Raynaud disease. Characterizing SAM are injurious and reparative lesions that occur in the media and/or at the adventitial medial junction. Four distinctive alterations are recognized: (1) mediolysis, (2) a tearing separation of the outer media from adventitia, (3) arterial gaps, and (4) a florid reparative response that replaces zones of mediolysis and fills areas of medial adventitial separation. The repair can transform SAM into lesions indistinguishable from common types of fibromuscular dysplasia (FMD.) A venous angiopathy involving large and medium-sized veins accompanies SAM. It features medial muscle vacuolar change with lysis leading to apparent separation of residual muscle bundles. Immunostaining shows endothelin-1 (ET-1) decorating adventitial capillaries in SAM and neighboring arteries, in capillaries of adjoining tissues, and outlining smooth muscle cell membranes in adjacent veins including those of the venous angiopathy. The significance of these changes is uncertain. Vasospasm is believed to cause SAM, but ET-1 is not the direct pressor agent responsible for this condition. The reason(s) for synthesis and release of ET-1 in SAM are still hypothetical, but local perturbations in vascular tone may be an important factor. ET-1 may be indirectly play a role in SAM by cross-talking and potentiating the activities of other vasoconstrictors such as norepinephrine and by orchestrating its reparative phase.

Objective: Ageing, plasma circulating C-reactive protein (CRP), oxidized low-density lipoprotein (OxLDL) and homocysteine (Hcy) are associated with atherosclerosis. The aim of this study was to evaluate the relationship between age, inflammatory and oxidative stress-related markers with functional and structural changes of the arteries in asymptomatic persons., Methods: CRP, OxLDL and Hcy were measured in 175 clinically healthy subjects, aged 40-70 years. Ultrasonography and pulse wave analysis were used to measure carotid intima-media thickness (IMT) and augmentation index (AIx)., Results: OxLDL was correlated with IMT (r = 0.24, P = 0.003), whereas CRP was correlated with AIx (r = 0.21, P = 0.005). No correlation was detected between Hcy and AIx or age-adjusted IMT. There was a significant association between AIx and age 50 years (r = 0.40; P = 0.001). In stepwise regression analysis age, weight, white blood cell count, OxLDL, heart rate and timing of the reflected waveform adjusted for height were significantly and independently associated with IMT (R = 0.41; P < 0.001). At the same time, AIx as the dependent variable correlated positively with age, gender, CRP and mean arterial pressure, and negatively with heart rate, weight and height, in stepwise regression analysis (R = 0.63; P < 0.001)., Conclusion: The results of the present study showed that CRP, OxLDL, Hcy and age are not similarly related to AIx and IMT in asymptomatic persons. The results suggest that CRP and younger age are related to arterial stiffness, whereas OxLDL and older age become more important determinants of structural changes of the arteries in asymptomatic persons.

Background: Inflammatory processes may play a pivotal role in the pathogenesis of cerebrovascular injury in salt-loaded, stroke-prone, spontaneously hypertensive rats (SHRSP). Thromboxane A2 (TP) receptor stimulation by 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha) is involved in the process of vascular inflammation., Objective: In the present study, we examined the involvement of TP receptor in the development of cerebrovascular damage in salt-loaded SHRSP., Methods: Nine-week-old SHRSP were fed a 0.4% NaCl or a 4% NaCl diet with or without ONO-8809 treatment (a TP receptor antagonist) for 5 weeks. Blood pressure, mortality, and the parameters of cerebrovascular inflammation and damage were compared between the groups. Moreover, we examined the effect of 8-iso-PGF2alpha infusion on cerebrovascular injury of SHRSP., Results: High salt intake in SHRSP significantly increased blood-brain barrier impairment and early mortality, which were suppressed by ONO-8809 treatment independent of changes in blood pressure. Salt loading also significantly increased superoxide production in basilar arteries of SHRSP, which was suppressed by ONO-8809 treatment. Macrophage accumulation and matrix metalloproteinase-9 (MMP-9) activity in the stroke-negative area in the contralateral cerebral cortex to the stroke lesion of salt-loaded SHRSP and 8-iso-PGF2alpha-treated SHRSP were significantly reduced by ONO-8809 treatment. The ONO-8809 treatment prevented thinning of the vessel layer in cerebral arterioles of salt-loaded SHRSP and 8-iso-PGF2alpha-treated SHRSP., Conclusions: These results suggest that TP receptor stimulation by 8-iso-PGF2alpha may involve salt loading-induced stroke through activation of cerebrovascular inflammation and damage.

Vascular calcification is an active, cell-mediated process that can involve either the intima or media of the blood vessels. The current methods used to clinically measure vascular calcification cannot distinguish between intimal (invariably atherosclerotic) and medial calcification. The high calcification scores seen in patients with end-stage renal disease likely represent a composite of high calcification burden in both sites. The severity of vascular calcification has been associated with a variety of findings including left ventricular hypertrophy and angiographic vascular stenosis as well as with all-cause and cardiovascular mortality. There is increasing evidence that disordered mineral metabolism participates in the process of vascular calcification and is one of the mechanisms whereby hyperphosphatemia, hypercalcemia, and hyperparathyroidism enhance cardiovascular and all-cause mortality of end-stage renal disease patients. There are no studies showing improved outcomes of ESRD patients with aggressive control of disordered mineral metabolism. However, the preponderance of evidence argues strongly in favor of aggressive management of these abnormalities from starting early in the course of chronic kidney disease in the hope of improving patient outcomes.

Coronary calcification is a potent predictor of cardiac events. In patients with chronic renal disease, both prevalence and intensity of coronary calcification are increased. It has remained uncertain whether it is the intima of the coronaries or the media that is calcified and whether the morphologic details of calcified plaques differ between renal and nonrenal patients. Autopsy samples of coronaries were obtained from standard sites in 23 renal and 23 age- and gender-matched nonuremic patients. Specimens were examined using light and electron microscopy, immunohistochemistry, backscatter imaging, and x-ray analysis. In coronaries, calcified plaques occupied a similar proportion of the intima area in renal versus nonrenal patients (17.3 +/- 11.9 versus 18.1 +/- 11.9%) but occupied a significantly higher proportion of the media (16.6 +/- 10.6 versus 3.8 +/- 2.31%). Expression of the proteins osteocalcin, C-reactive protein, TGF-beta, and collagen IV was significantly more intensive around coronary plaques of renal compared with nonrenal patients. The non-plaque-bearing intima of renal patients showed minimal staining for fetuin, but fetuin staining was seen surrounding calcified plaques. In addition, more pronounced deposition of C5b-9 was found around coronary plaques of renal patients, and glycophorin deposition pointed to more past intraplaque hemorrhage in renal patients. Calcification by electron backscatter analysis is more intense in the coronary media, but not if the intima is more intense in renal compared with nonrenal patients. A more marked inflammatory response in renal patients is suggested by more frequent presence and greater intensity of markers of inflammation.