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Aims: Atherosclerosis is a chronic inflammatory disease that is initiated by the retention and accumulation of low-density lipoprotein in the artery, leading to maladaptive response of cells from the immune system and vessel wall. Strong evidence implicates indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of the kynurenine pathway of tryptophan (Trp) degradation, with immune regulation and anti-inflammatory mechanisms in different diseases. However, the role of IDO and the endogenous degradation of Trp have never been directly examined in atherosclerosis development. We used the IDO inhibitor 1-methyl-Trp (1-MT) to determine the role of IDO-mediated Trp metabolism in vascular inflammation and atherosclerosis., Methods and Results: Apoe(-/-) mice were treated with 1-MT in drinking water for 8 weeks. Systemic IDO inhibition led to a significant increase in atherosclerotic lesions that were ∼58 and 54% larger in the aortic arch and root, respectively. 1-MT treatment enhanced vascular inflammation, up-regulated VCAM-1 and CCL2, and increased CD68 macrophage accumulation into the plaque. Notably, the rise in VCAM-1 expression was not limited to the plaque but also found in smooth muscle cells (SMCs) of the tunica media. Furthermore, we found that IDO-dependent Trp metabolism by SMCs regulates VCAM-1 expression, and that 1-MT-induced acceleration of atherosclerosis and vascular inflammation can be reversed by exogenous administration of the Trp metabolite 3-hydroxyanthranilic acid (3-HAA)., Conclusion: IDO-mediated Trp metabolism regulates vascular inflammation and plaque formation in hypercholesterolaemic Apoe(-/-) mice. Our data establish that this pathway plays a major role in the pathological process of atherogenesis., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Objective: In this study the role of microRNA-145 expression in ascending aortic aneurysm wall media remodeling was examined., Methods: Aortic wall samples were obtained from 10 patients who had undergone surgery for ascending aortic aneurysm. Control aortic tissue samples were obtained from 10 patients who had undergone coronary artery bypass graft. The levels of microRNA-145 were analyzed using real time quantitative reverse transcriptase polymerase chain reaction, and western blots were used to determine the expressions of osteopontin (OPN) and collagen. In vitro cultures of vascular smooth muscle cells (VSMCs) were established from both patient groups. The VSMCs were transfected with either microRNA-145 mimics or microRNA-145 inhibitors to determine the effect of microRNA-145 on the expression of OPN and collagen. Furthermore, cells were co-transfected with microRNA-145 mimics and TGF-b1 siRNA to investigate whether TGF-b1 is involved in the process that microRNA-145 increases the expression of OPN and collagen., Results: Aortic microRNA-145, OPN and collagen III was increased in ascending aortic aneurysm patients compared with controls (p < .05). VSMCs transfected with microRNA-145 mimics increased the expression of both OPN (average of 1.59-fold, p < .05) and collagen III (mean of 1.71-fold, p < .05). Cells transfected with microRNA-145 inhibitors decreased expression of both OPN and collagen III compared with negative controls. In addition, the inhibition of TGF-b1 decreased the positive effect of microRNA-145 on the expression of OPN and collagen III., Conclusion: The increased expression of microRNA-145 promotes media remodeling through TGF-b1 in the aortic aneurysm wall.

Hypertension, the major cause of cardiovascular disease, is bidirectionally linked to arterial stiffness. Evidence shows that vascular calcification, either medial or intimal, induces arterial stiffening further worsening hypertension parallel to substantially increasing cardiovascular risk. The disturbance in the bone-vascular axis that leads to the increase of calcium deposition in the arterial wall may be the result of a shift in the functionality of bone marrow-derived circulating stem cells with a calcifying potential, namely osteoprogenitor cells. These cells deposit bone matrix proteins in the vascular wall that can subsequently become mineralized. The current notion is that these cells derive from diverse cell lines. The present review summarizes the current knowledge on the role of progenitor cells with a calcifying potential on arterial calcification, stiffness and hypertension.

Objective: Diabetes mellitus is one of the chronic metabolic diseases which is characterized by microvascular and macrovascular complications. This study was designed to investigate the improving the effects of amnioguanidine on aortic damage in a streptozotocin (STZ) induced diabetic rat model., Methods: Thirty-two male Sprague-Dawley rats divided into four groups as follows: Control, Aminoguanidine, Diabetes, and Diabetes+Aminoguanidine. Experimental diabetes was induced by single dose STZ (45 mg/kg) intraperitoneally. After administration of STZ, the DM+AMG group began to receive AMG (1 g/L) was prepared by dissolving in tap water during 10 weeks. At the end of the study, blood glucose levels were determined and rats were sacrified by ketamine anesthesia. Following routine tissue process, aortas were embedded in paraffin. Histochemical (H-E and Orcein) and immunohistochemical α-smooth muscle actin (α-SMA) stains were applied and the sections examined by light microscope. Statistical analysis was carried out using the SPSS 13.0 statistical program., Results: The rats in diabetes group had significantly higher blood glucose levels than the rats of control. The main histological alterations were detected in tunica media such as extensive thickening (414.32±9.62 μm), irregular of elastic fibers and intensive α-SMA staining in diabetic rats. The thickness of tunica media was statistically increased in DM group, when compared with the control group (p<0.001). On the other hand, AMG prevented disorganization of elastic fibers and overexpression of α-SMA. The mean thickness of tunica media was decreased significantly in DM+AMG (319.16±6.53 μm) compared with the DM group (p<0.001)., Conclusion: Our results demonstrate that AMG treatment may protect the impairment of aort structure at histological level.

Multiple clinical studies show that arterial stiffness, measured as pulse wave velocity (PWV), precedes hypertension and is an independent predictor of hypertension end organ diseases including stroke, cardiovascular disease and chronic kidney disease. Risk factor studies for arterial stiffness implicate age, hypertension and sodium. However, causal mechanisms linking risk factor to arterial stiffness remain to be elucidated. Here, we studied the causal relationship of arterial stiffness and hypertension in the Na-induced, stroke-prone Dahl salt-sensitive (S) hypertensive rat model, and analyzed putative molecular mechanisms. Stroke-prone and non-stroke-prone male and female rats were studied at 3- and 6-weeks of age for arterial stiffness (PWV, strain), blood pressure, vessel wall histology, and gene expression changes. Studies showed that increased left carotid and aortic arterial stiffness preceded hypertension, pulse pressure widening, and structural wall changes at the 6-week time-point. Instead, differential gene induction was detected implicating molecular-functional changes in extracellular matrix (ECM) structural constituents, modifiers, cell adhesion, and matricellular proteins, as well as in endothelial function, apoptosis balance, and epigenetic regulators. Immunostaining testing histone modifiers Ep300, HDAC3, and PRMT5 levels confirmed carotid artery-upregulation in all three layers: endothelial, smooth muscle and adventitial cells. Our study recapitulates observations in humans that given salt-sensitivity, increased Na-intake induced arterial stiffness before hypertension, increased pulse pressure, and structural vessel wall changes. Differential gene expression changes associated with arterial stiffness suggest a molecular mechanism linking sodium to full-vessel wall response affecting gene-networks involved in vascular ECM structure-function, apoptosis balance, and epigenetic regulation.

Objective: Abnormally low-flow conditions, sensed by the arterial endothelium, promote aneurysm rupture. Fibronectin (FN) is among the most abundant extracellular matrix proteins and is strongly upregulated in human aneurysms, suggesting a possible role in disease progression. Altered FN splicing can result in the inclusion of EIIIA and EIIIB exons, generally not expressed in adult tissues. We sought to explore the regulation of FN and its splicing and their possible roles in the vascular response to disturbed flow., Approach and Results: We induced low and reversing flow in mice by partial carotid ligation and assayed FN splicing in an endothelium-enriched intimal preparation. Inclusion of EIIIA and EIIIB was increased as early as 48 hours, with negligible increases in total FN expression. To test the function of EIIIA and EIIIB inclusion, we induced disturbed flow in EIIIAB(-/-) mice unable to include these exons and found that they developed focal lesions with hemorrhage and hypertrophy of the vessel wall. Acute deletion of floxed FN caused similar defects in response to disturbed flow, consistent with a requirement for the upregulation of the spliced isoforms, rather than a developmental defect. Recruited macrophages promote FN splicing because their depletion by clodronate liposomes blocked the increase in endothelial EIIIA and EIIIB inclusion in the carotid model., Conclusions: These results uncover a protective mechanism in the inflamed intima that develops under disturbed flow, by showing that splicing of FN mRNA in the endothelium, induced by macrophages, inhibits hemorrhage of the vessel wall., (© 2014 American Heart Association, Inc.)

Tissue preparation is the key to a successful MALDI mass spectrometry imaging experiment. A number of different tissue preparations methods have recently been reported for increased sensitivity and/or high spatial resolution analysis. In order to better benchmark these methods in terms of the information content and their suitability for analyzing small tissues containing small but distinct regions, we have performed an extensive comparison using technical and biological repeats as well as a fully randomized measuring sequence. We then demonstrate how the optimized tissue preparation method enables 30μm spatial resolution analysis of proteins from atherosclerotic arterial tissues, revealing proteins specific to the intima and media layers., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Introduction: Infusion of angiotensin-II (Ang-II) in apolipoprotein-E-deficient mice (Apo-E(-/-)) results in suprarenal abdominal aortic aneurysm (AAA) in 30-85% of cases. This study identifies the apparent mechanism by which some animals do, but others do not, develop AAA in this model., Methods: Male Apo-E(-/-) mice were infused with Ang-II (n=21) or saline (n=6) and sacrificed at 4 weeks. Aortas were excised, embedded in paraffin, sectioned (250 μm intervals), and stained. Sites of transmedial disruption (TMD) were identified and characterized, and their relationship to the 4 major aortic side branches (celiac, superior mesenteric, and renals) were determined., Results: The frequency of TMDs in Ang-II-infused mice that formed AAA (n=9) was similar to those that did not (n=12) (AAA vs. no-AAA: 25 of 36[69%] vs. 28 of 48[58%] branches, P=.3 by chi-square). All TMDs were at branch points. However, in animals with AAA, the mean maximum length of the TMDs was significantly larger (1.94±1.6 vs. 0.65±0.5mm, P=.007 by Mann Whitney U test), the #mac-2(+) macrophages per 0.01mm(2) of defect area was greater (32±10 vs. 19±11, P<.02 by Kruskal-Wallis with Conover-Inman post hoc), the % area of attempted repair occupied by collagen was less (17±13% vs. 44±15%, P=.0009 by Mann Whitney U test), and the density of collagen per unit length of media missing was also markedly less (0.13±0.2 vs. 1.14±1.0, P=.0001 by Mann Whitney U test)., Conclusions: Reinforcement of transmedial defects at branch points by wall matrix is a key intrinsic player in limiting AAA formation in the Ang-II-infused, Apo E(-/-) mouse and a potentially important mechanism-based therapeutic target for management of small, slowly progressing aneurysms., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Vascular calcification can be categorized into two different types. Intimal calcification related to atherosclerosis and elastin-specific medial arterial calcification (MAC). Osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) has been shown in both types; however, how this relates to initiation of vascular calcification is unclear. We hypothesize that the initial deposition of hydroxyapatite-like mineral in MAC occurs on degraded elastin first and that causes osteogenic transformation of VSMCs. To test this, rat aortic smooth muscle cells (RASMCs) were cultured on hydroxyapatite crystals and calcified aortic elastin. Using RT-PCR and specific protein assays, we demonstrate that RASMCs lose their smooth muscle lineage markers like alpha smooth muscle actin (SMA) and myosin heavy chain (MHC) and undergo chondrogenic/osteogenic transformation. This is indicated by an increase in the expression of typical chondrogenic proteins such as aggrecan, collagen type II alpha 1(Col2a1) and bone proteins such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, when calcified conditions are removed, cells return to their original phenotype. Our data supports the hypothesis that elastin degradation and calcification precedes VSMCs' osteoblast-like differentiation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

C3d deposition in peritubular capillaries has been demonstrated to indicate antibody-mediated alloresponse during renal transplantation. C3d deposition in renal arterioles in IgA nephropathy (IgAN), however, is poorly documented. Especially, its significance to the pathology of primary glomerulonephritis remains unclear. This retrospective study included 340 patients with IgAN who underwent renal biopsy at our center. C3d strongly positive deposition in arterioles was observed in 123 (36.2%) of the 340 cases, and weakly positive deposition of C3d was observed in 217 cases (63.8%). In the weakly positive group, C3d mainly deposited in the intima of arterioles. In the strongly positive group, C3d deposited in the intima and the media of arterioles, presenting as the medial thickening and sclerosis of varying severities. The prognosis was worse in the C3d strongly positive group than in the weakly positive group during a 2-year follow-up (P = .027). The predictive value of C3d deposition in the media of arterioles in patients with IgAN may be a useful marker for arteriolosclerosis indicating unfavorable clinical outcomes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Vascular calcification (VC), commonly encountered in renal failure, diabetes, and aging, is associated with a large increase in the risk for cardiovascular events and mortality. Calcification of the arterial media and of heart valves clearly plays a mediating role in this regard, whereas it is less clear how calcification of plaque influences atherogenesis and risk for plaque rupture. Vascular calcification is an active process in which vascular smooth muscle cells (VSMCs) adopt an osteoblastic phenotype and deposit hydroxyapatite crystals; apoptosis of VSMCs also promotes this deposition. Drivers of this phenotypic transition, which include elevated serum phosphate, advanced glycation end-products, bone morphogenetic proteins, inflammatory cytokines, and leptin, invariably induce oxidative stress in VSMCs, which appears to be a necessary and sufficient condition for induction of the runt-related transcription factor 2 gene (RUNX2) and the shift to osteoblastic behavior. Magnesium antagonizes the impact of phosphate on VSMC osteoblastic transition, both by a direct effect within VSMCs and by suppressing absorption of dietary phosphate. Antioxidants that suppress reduced nicotinamide adenine dinucleotide phosphate oxidase activity may have the potential to block the osteoblastic transition of VSMCs. Minimizing the absorption of dietary phosphate may also be helpful in this regard, particularly in renal failure, and it can be achieved with plant-based dietary choices, avoidance of phosphate additives, and administration of pharmaceutical phosphate binders, supplemental magnesium, and niacin. Good vitamin K status opposes VC by optimizing the γ-carboxylation of matrix Gla protein, a physiological antagonist of VC. Adequate but not excessive vitamin D status also appears to discourage VC. Etidronate, a structural analogue of pyrophosphate, has shown potential for blocking VC.

An excessive consumption of a diet rich in saturated fatty acids is a key factor in pathogenesis of cardiovascular diseases which are strictly connected with leptin imbalance in the vessels. However, whether vitamin E supplementation would influence leptin expression in aortic layers is still unknown. For 3 or 6 weeks male Wistar rats were fed a high-fat (20% w/w) diet with lard as dietary fat source with or without vitamin E supplementation (50 mg/100 g of diet). The 6-week intake of an atherogenic diet increased total cholesterol (TC) and high density lipoprotein cholesterol (HDL) plasma levels simultaneously lowering TC/HDL ratio (ANOVA p≤0.0001 for all three parameters). After longer period of feeding it was stated that leptin expression in all three aortic layers was enhanced (ANOVA p≤0.0001 for endothelium, tunica media and adventitia, respectively) with decreased leptin plasma concentration (ANOVA p≤0.0001). After both periods of feeding vitamin E supplementation caused an increase in plasma HDL content and a decrease of TC/HDL ratio. In the 3-week experiment vitamin E addition caused a decrease in leptin plasma levels (Fisher's test, 3L versus 3LE: p≤0.002) and an increase in leptin expression in all three aortic layers (Fisher's test, 3L versus 3LE p≤0.005, p≤0.01 and p≤0.05 respectively for endothelium, tunica media and adventitia). The contradictory results were observed in the 6-week experiment in which vitamin supplementation decreased leptin expression in the aortic endothelium (Fisher's test, 6L versus 6LE: p≤0.001) with lack of changes in the other two layers of the aorta and plasma. The study showed that vitamin E supplementation influenced leptin expression in aortic layers in rats fed atherogenic diet differently depending on the length of feeding period. It may suggest that vitamin E consumption plays an important role in the control of leptin status in the endothelium.

Objectives: The aim of the study was to determine whether the release by macrophages of matrix metalloproteinase (MMP)-12 and vascular endothelial growth factor (VEGF) - leading to inflammation, matrix degradation and neoangiogenesis - represents an effective pathway that underlies aortic wall remodeling in Stanford type A acute aortic dissection (AAD)., Methods: Twenty-one consecutive patients with no genetic predisposition, with Stanford type A AAD were selected. In each patient, the levels of serum VEGF, MMP-12, serum interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1 were evaluated using enzyme-linked immunosorbent assay. Ascending aortic specimens were collected for immunohistochemical identification of any presence of inflammatory infiltrate, VEGF and CD31 expression., Results: A significant increase in serum VEGF (p = 0.044), MMP-12 (p = 0.007), IL-6 (p = 0.0001), IL-8 (p = 0.0001) and MCP-1 (p = 0.0001) levels was observed in the AAD group compared to the control group. Furthermore, all AAD samples were positive for VEGF in the tunica media and showed vessel growth and immune-inflammatory infiltrate. A large number of cases (62.79%) showed inflammation at the edge of the dissection and approximately half (51.42%) showed neovessels growing at the edge of the dissection., Conclusions: The results suggest that VEGF-mediated angiogenesis and matrix degradation play a role in AAD. Finally, we believe that MMP-12 should be considered a marker of AAD., (© 2013 S. Karger AG, Basel.)

The process of β-amyloid accumulation in cerebral vessels is presented. Cerebral amyloid angiopathy (CAA) was confirmed during an autopsy. It was diagnosed according to the Boston criteria. Cerebral amyloid angiopathy can involve all kinds of cerebral vessels (cortical and leptomeningeal arterioles, capillaries and veins). The development of CAA is a progressive process. β-amyloid appears first in the tunica media, surrounding smooth muscle cells, and in the adventitia. β-amyloid is progressively accumulated, causing a gradual loss of smooth muscle cells in the vessel wall and finally replacing them. Then, the detachment and delamination of the outer part of the tunica media results in the "double barrel" appearance, fibrinoid necrosis, and microaneurysm formation. Microbleeding with perivascular deposition of erythrocytes and blood breakdown products can also occur. β-amyloid can also be deposited in the surrounding of the affected vessels of the brain parenchyma, known as "dysphoric CAA". Ultrastructurally, when deposits of amyloid fibers were localized in or outside the arteriolar wall, the degenerating vascular smooth muscle cells were observed. In the Institute of Psychiatry and Neurology the study was carried out in a group of 48 patients who died due to intracerebral hemorrhage caused by sporadic CAA.

Background: Continuous-flow left ventricular assist devices (LVADs) provide durable, reliable, energy-efficient long-term support. However, the biologic effects of continuous flow are not completely known. Therefore, we examined aortic wall morphology in patients with heart failure before and after prolonged circulatory support with a continuous-flow LVAD., Methods: After applying a partial aortic occlusion vascular clamp in the lower half of the ascending aorta, we removed samples of aortic wall tissue and then attached the outflow graft of the pump. Samples were obtained from 11 patients (9 men and 2 women, mean age 65 ± 7 years) with severe heart failure at the time of LVAD implantation. We obtained matched specimens at explantation after heart transplantation (n = 5) or autopsy (n = 6). These specimens were removed from the distal ascending aorta, remote from the aortic anastomotic site. Tissue sections were stained with hematoxylin and eosin, Movat's pentachrome and Masson's trichrome. Smooth muscle actin immunohistochemistry was performed on all sections. To evaluate the morphology of the aortic wall media, we quantitatively graded tissue sections for medial thickness, medial degenerative changes, smooth muscle cell (SMC) disorientation and depletion, elastic fiber fragmentation and depletion, medial fibrosis and atherosclerotic changes., Results: The mean duration of support was 140 ± 136 days (range 87 to 580 days). The histologic evaluation and comparison of specimens obtained before and after LVAD support showed significantly increased foci of medial degeneration, SMC depletion, elastic fiber fragmentation, medial fibrosis and atherosclerotic changes after LVAD support. Mean medial thickness was not significantly different after LVAD support. We observed similar changes between samples obtained at transplantation and those obtained at autopsy., Conclusions: After continuous-flow LVAD support, the morphology of the aortic wall media was altered in all of our patients. The clinical relevance of these findings is unknown., (© 2013 International Society for Heart and Lung Transplantation. All rights reserved.)

Background: Data about the structure and immunohistochemistry of the lenticulostriate arteries (LSAs), although very important for medical research and clinical practice, have been rarely reported in literature., Materials and Methods: Fourty serially sectioned LSAs were stained with hematoxilin and eosin, and prepared for immunohistochemistry., Results: Our examination revealed a typical endothelial lining and a narrow subendothelial space with subintimal smooth muscle cells occasionally. The internal elastic lamina was fragmented or absent in the smallest LSAs branches. The mediacoat, with a mean diameter of 148.5 μm, contained typical smooth muscle cells which formed 14.2 layers on average and showed a positive immune reactions for alfa-actin, desmine, laminin and collagen IV. The thin adventitial coat contained fibroblasts, collagen fibers, and nerve bundles, with the strongest immunopositivity to thyrosin hydroxilase. The immune reactions against CD31 and CD34 proteins,endothelial nitric oxide synthase, S 100 protein, neurofilament protein and synaptophysin,seem to be performed in the LSAs wall for the first time. Similarly,the thickness of the LSAs wall and its coats have never been reported, nor the number of the smooth muscle cell layers., Conclusions: Our results related to the structure and immunohistochemistry of the LSAs could be important in cerebrovascular pathology, neurology and neurosurgery.

Objectives: To evaluate the effect of the novel angiotensin receptor blocker Fimasartan on the development of atherosclerosis and plaque stabilization in an animal model., Methods: Twenty-four rabbits received an aortic balloon injury from 30 cm to a level just above the aortic valve to the iliac bifurcation using 3 Fr Fogarty catheters on third day of the experiment, followed by a 1% cholesterol diet for 8 weeks. The rabbits were randomized to receive placebo or 3 or 6 mg · kg⁻¹ · d⁻¹ Fimasartan. The study was double blinded. The rabbits started receiving their medications 2 days before the aortic balloon injury and treatment continued. Atherosclerosis burden was determined by calculating the intima-media ratio of the infrarenal portion of the aorta because the bulk of the atherosclerotic burden was limited to the infrarenal region. The frequency of plaque disruption with thrombosis and the proportions of the plaques that were occupied by macrophages, smooth muscle cells, and collagen were determined., Results: Relative to the placebo group, the Fimasartan-treated rabbits had less atherosclerosis [intima-media ratio (mean ± SEM) of 1.14 ± 0.21 vs. 1.51 ± 0.26, P = 0.005], fewer disrupted plaques with thrombi (3 of 16 vs. 5 of 8, P = 0.047), lower proportion of macrophages (17.5% ± 2.5% vs. 26% ± 3.5%, P = 0.03), higher proportion of smooth muscle cells (43.5% ± 8.3% vs. 11.9% ± 2.1%, P = 0.001), and higher proportion of collagen (34.3% ± 6.4% vs. 19.7% ± 2.1%, P = 0.02)., Conclusions: These results show that the newly developed angiotensin receptor blocker, Fimasartan, attenuated atherosclerosis progression and reduced macrophage accumulation in the rabbit aortic plaques.

This experiment was performed to explore the relationship between 5-hydroxytryptamine (5-HT) levels in pulmonary arterioles and in pulmonary vascular remodelling in broilers. Pulmonary arterial hypertension was induced by injecting cellulose microparticles intravenously. Pulmonary hypertension syndrome (PHS) morbidity, right ventricle/total ventricle weight ratio (RV/TV), packed cell volume (PCV), haemoglobin concentration (HB), vessel wall area to vessel total area ratio (WA/TA) and mean tunica media thickness in pulmonary arterioles (mMTPA) were measured. Proliferating cell nuclear antigen (PCNA), argyrophilic nucleolar organizer region proteins (Ag-NORs) and 5-HT content in pulmonary arterioles were determined. The results showed that injecting cellulose microparticles intravenously in broilers could successfully increase the PHS morbidity, significantly elevate RV/TV, PCV and HB, significantly increase mMTPA and WA/TA, and significantly increase the argyrophilic particles in smooth muscle cell nucleoli, PCNA-positive cells in the medial layer, and the 5-HT content in pulmonary arterioles. Correlation analysis showed that the level of 5-HT was strongly positively correlated with PCNA and Ag-NORs. The results indicated that the increase of 5-HT in the tunica media could possibly promote the proliferation of smooth muscle cells in pulmonary arterioles and thus the occurrence of pulmonary vascular remodelling.

Aim: Smooth muscle cell (SMC) phenotypic switching in the aortic media may play a critical role in the pathogenesis of thoracic aortic dissection (TAD). However, few investigations are available and most of the observations are based on histological examinations without in vitro evidence. This study, which was performed both in vivo and in vitro, was designed to investigate SMC phenotypic diversity between dissected and unaffected aortic media., Methods: Using optimized explant technique, aortic medial SMCs were obtained from patients with TAD and controls. In vivo and in vitro expression of α-smooth muscle actin (α-SMA), smooth muscle-myosin heavy chain 2 (SM-MHC-2), smooth muscle-calponin (SM-Calponin), Vimentin, osteopontin (OPN) and non-muscle myosin heavy chain B (SMemb) were evaluated by immunostaining and immunoblotting. SMC proliferation was also analyzed., Results: Although the majority of SMCs from the dissected media displayed an elongated, spindle- or triangle-like shape as control SMCs, there were some oval or flat, quadrate cells in the dissection cultures. In contrast with controls, SMCs derived from the dissected media uniformly showed the negative staining for the contractile proteins and the intense staining for the synthetic markers. Similarly, in vitro protein levels of α-SMA, SM-MHC-2, SM-Calponin and Vimentin were significantly decreased to 60.1% (P<0.05), 12.0% (P<0.01), 23.1% (P<0.01) and 32.5% (P<0.01) respectively, whereas those of OPN and SMemb were markedly elevated by5.7- and 10.3-fold respectively (P<0.01 for both). In vivo expression of the phenotypic markers showed the parallel results. Furthermore, SMCs derived from the dissected media exhibited the enhanced proliferation (P<0.05)., Conclusion: We have established a simple and potent method to acquire SMCs from the dissected and unaffected aortic media. Compared to the contractile SMCs in the unaffected media, those in the dissected media manifest phenotypic switching from the contractile to the synthetic type. The primary cultures can be subsequently used as in vitro models and contribute to further elucidating the etiopathogenesis of TAD.

Objective: Inflammatory cell activation plays a key role in atherosclerotic plaque growth and acute complications. While secretion of proteases and inflammatory cytokines are likely involved in the development of plaque instability, the precise mechanistic pathways are not well understood., Methods and Results: Based on our previous study, we crossed Toll-like receptor 3 (Tlr3)(-/-) mice with a unique BALB-Apoe(-/-)Npc1(-/-) plaque complication-susceptible mouse model, as well as the widely-used B6-Ldlr(-/-) atherosclerosis model, to test the role of TLR3 signaling in the development of plaque instability. TLR3-deficient mice showed no change in aortic root lesion area, but displayed a marked increase in collagen and smooth muscle cell (SMC) content of lesions. Notably, Apoe(-/-)Npc1(-/-)Tlr3(-/-) mice exhibited a 50% reduction in the incidence of medial destruction, a precursor to aortic aneurysm formation. MMP-2 activity was markedly reduced in aortic extracts from Apoe(-/-)Npc1(-/-)Tlr3(-/-) compared to controls, while both MMP-2 and -9 activities were reduced in Ldlr(-/-)Tlr3(-/-) extracts. Consistent with the in vivo data, TLR3 deficiency suppressed MMP-2 activity induced by TNF-α or polyinosine-polycytidylic acid in macrophages from Apoe(-/-)Npc1(-/-) mice., Conclusions: TLR3 plays a critical role in regulating the degradation of extracellular matrix in lesions, in part by modulation of macrophage MMP-2 and -9 activities., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

In normal development and pathology, the vascular system depends on complex interactions between cellular elements, biochemical molecules, and physical forces. The electrokinetic vascular streaming potential (EVSP) is an endogenous extremely low frequency (ELF) electrical field resulting from blood flowing past the vessel wall. While generally unrecognized, it is a ubiquitous electrical biophysical force to which the vascular tree is exposed. Extracellular matrix elastin plays a central role in normal blood vessel function and in the development of atherosclerosis. It was hypothesized that ELF fields of low amplitude would alter elastin accumulation, supporting a link between the EVSP and the biology of vascular smooth muscle cells. Neonatal rat aortic smooth muscle cell cultures were exposed chronically to electrical fields characteristic of the EVSP. Extracellular protein accumulation, DNA content, and electron microscopic (EM) evaluation were performed after 2 weeks of exposure. Stimulated cultures showed no significant change in cellular proliferation as measured by the DNA concentration. The per-DNA normalized protein in the extracellular matrix was unchanged while extracellular elastin accumulation decreased 38% on average. EM analysis showed that the stimulated cells had a 2.85-fold increase in mitochondrial number. These results support the formulation that ELF fields are a potential factor in both normal vessel biology and in the pathogenesis of atherosclerotic diseases including heart disease, stroke, and peripheral vascular disease., (Copyright © 2013 Wiley Periodicals, Inc.)

Objectives: Increasing knowledge is required for a better comprehension of the etiology of thoracic aortic aneurysm (TAA). The aim of this study was to highlight the modulations in vascular cell phenotypes, including myofibroblasts (MFs), in human TAA specimens compared to healthy aortas., Methods: histology, RT-PCR and immunohistochemical analysis of a panel of molecules, including ED-A Fibronectin (Fn), smoothelin, CD34 and alpha-smooth muscle actin (alpha-SMA), selected on the basis of their informative potential as markers of smooth muscle cells (SMCs) and MF phenotypic modulation, were performed on all samples., Results: The media of TAAs was characterized by the absence of smoothelin, the unaltered expression of alpha-SMA accompanied by an alteration of its distribution pattern, and by the activated expression of the ED-A isoform of Fn. We found a concentration of round-shaped cells exclusively in the adventitia and in the perivascular tissue of TAAs, also rich in vasa vasorum, largely expressing alpha-SMA, while a sub-population also expressed ED-A Fn and CD34. CD34 was expressed by several cells in the intima of TAAs, together with cells expressing cytoplasmatic ED-A Fn and alpha-SMA in comparison to healthy aortas., Conclusion: TAA specimens show an altered expression and localization of SMC and MF differentiation markers in comparison to healthy aortas, with possible implications on remodeling.

Background: Inflammation is a common feature in the majority of cardiovascular disease, including Diabetes Mellitus (DM). Levels of pro-inflammatory markers have been found in increasing levels in serum from diabetic patients (DP). Moreover, levels of Cyclooxygenase-2 (COX-2) are increased in coronary arteries from DP., Methods: Through a cross-sectional design, patients who underwent CABG were recruited. Vascular smooth muscle cells (VSMC) were cultured and COX-2 was measured by western blot. Biochemical and clinical data were collected from the medical record and by blood testing. COX-2 expression was analyzed in internal mammary artery cross-sections by confocal microscopy. Eventually, PGI2 and PGE2 were assessed from VSMC conditioned media by ELISA., Results: Only a high glucose concentration, but a physiological concentration of triglycerides exposure of cultured human VSMC derived from non-diabetic patients increased COX-2 expression .Diabetic patients showed increasing serum levels of glucose, Hb1ac and triglycerides. The bivariate analysis of the variables showed that triglycerides was positively correlated with the expression of COX-2 in internal mammary arteries from patients (r(2) = 0.214, P < 0.04)., Conclusions: We conclude that is not the glucose blood levels but the triglycerides levels what increases the expression of COX-2 in arteries from DP.

Studies indicate that risk for type 2 diabetes mellitus (T2D) or cardiovascular disease is detectable in childhood, though these disorders may not emerge until adulthood. This study was aimed to assess the markers of endothelial dysfunction in patients with the family history of T2D from South Indian population. A total of 450 subjects were included in the study comprising Group I (n = 200) of T2D, Group II (n = 200) of age- and sex-matched healthy controls, Group III (n = 25) of children of T2D patients and Group IV (n = 25) of children of healthy controls. Results showed that intimal medial thickening (IMT) was significantly higher in T2D patients, compared with control subjects with no family history of diabetes. The fasting plasma glucose, glycated hemoglobin, serum total cholesterol, triglyceride, LDL-cholesterol, apolipoprotein B (ApoB) and high-sensitive C-reactive protein (hsCRP) levels were significantly increased, whereas HDL-cholesterol and serum nitrite levels were significantly decreased in T2D patients. However, children of T2D patients who were not diabetic did not show significant increase in the IMT, as compared to those of healthy controls. In conclusion, the present study demonstrate that IMT was significantly higher in the T2D patients and increased with age and family history. The increased levels of lipids, hsCRP, IMT and decreased nitrite levels might contribute to the risk of endothelial dysfunction in patients with T2D. However, further studies are warranted with other biomarkers of endothelial dysfunction in T2D patients with increased sample size.

The rapid proliferation of smooth muscle cells (SMCs) contributes to atherosclerotic plaque formation and neointimal thickening in other occlusive vascular diseases. In cancer cells, rapid cell proliferation is often accompanied by DNA damage, division aberrations, elevated cell apoptosis, or accumulation of abnormal cells. However, little is known about division fidelity in vascular disorders. We have analyzed the cell division fidelity during the rapid SMC proliferation that occurs after balloon injury of the rat carotid artery using en face confocal microscopy of the full thickness of the vessel wall. SMCs newly migrated to the neointima had increased division defects and increased apoptosis compared with SMCs in the subjacent media, despite comparable mitosis rates. Protein kinase Cα and the receptor for hyaluronic acid-mediated motility (RHAMM) regulate division fidelity in cultured neointimal SMCs. The centrosomal targeting sequence of RHAMM was required for localization to the mitotic spindle and spindle organization. Dynein and RHAMM colocalized in the spindle area and were part of a complex. Dynein inhibition caused spindle defects similar to RHAMM or protein kinase C inhibition. Our study uncovered abnormalities in rapidly proliferating SMCs after arterial injury that could contribute to the growth of atherosclerotic plaques and reduce plaque stability by triggering apoptosis, and it described a mechanism by which RHAMM and dynein coordinate division fidelity in neointimal SMCs., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Laser microdissection (LMD) is a novel methodology for noncontact isolation of tissue regions or cells for subsequent molecular analysis. Although it is an upcoming field, its combination with proteomics for differential analysis remains not very well explored, since amount of protein obtained after LMD is scarce. We have combined LMD arterial layer isolation with saturation labeling DIGE, successfully achieving differential analysis of healthy and pathological intima and media layers. Identification of differential spots could be performed in whole tissue extract as reference proteome, since studied regions are subproteomes of the aforementioned.

Introduction: Migration of the smooth muscle cells (SMCs) to the tunica media in the saphenous vein (SV) transplants is facilitated by matrix metalloproteinases (MMPs). The aim of this study was to identify any associations between expression of MMP-2 or endogenous tissue inhibitors (TIMP-2 and TIMP-3) in the SV segments and late failure of the SV grafts., Methods: Two hundred consecutive patients with a mean age of 63.1 ± 8.9 years who underwent primary isolated venous CABG were examined. Patients were retrospectively split into two subgroups, with the SV graft disease (SVGD (+); n = 47) or without it (SVGD (-); n = 153). In the SV segments, immunohistochemical analysis of the expression of the MMP-2, TIMP-2, and -3 was performed., Results: In the SVGD (+) patients, tissue expression of MMP-2 was stronger, whereas that of both TIMPs was weaker than in the SVGD (-) patients. In majority of the SV segments obtained from the SVGD (-) individuals, a balance in MMP and TIMP expressions was found, whereas an upregulation of MMP-2 expression was usually noted in the SVGD (+) subjects., Conclusion: The strong expression of MMP-2 accompanied by reduced immunostaining of both TIMPs is associated with the development of the SV graft disease and unfavorable CABG outcomes.

High phosphate-induced phenotypic switching of smooth muscle cells (SMCs) into osteogenic cells is critical for the formation of arterial medial calcification in chronic kidney disease. Because vascular calcification is also prevalent in type 2 diabetes, we examined whether glucose concentration affects high phosphate-induced SMC phenotypic switching and calcification. First, the formation of arterial medial calcification was compared among 4 groups: adenine-fed uremic rats, streptozotocin-injected hyperglycemic rats, adenine-fed and streptozotocin-injected uremic/hyperglycemic rats, and control rats. Calcification was obvious in uremic and uremic/hyperglycemic rats, whereas it was undetectable in the others. Aortic calcium contents were significantly elevated in uremic and uremic/hyperglycemic rats, but they were not different between the two groups. Moreover, hyperglycemia had no effects on the reduced expression of SMC differentiation markers including smooth muscle α-actin and SM22α and on the increased expression of osteogenic markers, such as Runx2, in uremic rats. Second, cultured SMCs were incubated in the medium with various concentrations of phosphate (0.9-4.5 mmol/l) and glucose (5-50 mmol/l), and calcium deposition was measured. Although high phosphate dose-dependently increased calcium contents, they were unaffected by glucose concentration. Results suggest that glucose concentration does not directly modulate high phosphate-induced SMC phenotypic switching and arterial medial calcification., (© 2013 S. Karger AG, Basel.)

Detection of peripheral nerve tissues during surgery is required to avoid neural disturbance following surgery as an aspect of realizing better functional outcome. We provide a proof-of-principle demonstration of a label-free detection technique of peripheral nerve tissues, including myelinated and unmyelinated nerves, against adjacent tissues that employ spontaneous Raman microspectroscopy. To investigate the Raman spectral features of peripheral nerves in detail, we used unfixed sectioned samples. Raman spectra of myelinated nerve, unmyelinated nerve, fibrous connective tissue, skeletal muscle, tunica media of blood vessel, and adipose tissue of Wistar rats were analyzed, and Raman images of the tissue distribution were constructed using the map of the ordinary least squares regression (OLSR) estimates. We found that nerve tissues exhibited a specific Raman spectrum arising from axon or myelin sheath, and that the nerve tissues can be selectively detected against the other tissues. Moreover, myelinated and unmyelinated nerves can be distinguished by the intensity differences of 2,855 cm⁻¹, and 2,945 cm⁻¹, which are mainly derived from lipid and protein contents of nerve fibers. We applied this method to unfixed section samples of human periprostatic tissues excised from prostatic cancer patients. Myelinated nerves, unmyelinated nerves, fibrous connective tissues, and adipose tissues of the periprostatic tissues were separately detected by OLSR analysis. These results suggest the potential of the Raman spectroscopic observation for noninvasive and label-free nerve detection, and we expect this method could be a key technique for nerve-sparing surgery.

Background: Vein grafts fail as the result of wall maladaptations to surgical injury and hemodynamic perturbations. Interleukin-1 signaling has emerged as an important mediator of the vascular response to trauma and hemodynamically induced vascular lesions. We therefore hypothesized that interleukin-1 signaling drives early vein graft wall adaptations., Methods: Using interleukin-1 type I receptor knockout (IL-1RI(-/-)) and wild-type (B6129SF2/J) mice, we investigated morphologic changes 28 days after interposition isograft from donor inferior vena cava to recipient carotid artery, without (n = 19) or with (n = 13) outflow restriction. The impact of mouse strain on the response to vein arterialization also was evaluated between B6129SF2/J (n = 18) and C57BL/6J (n = 19) mice., Results: No differences were observed in the traditional end points of intimal thickness and calculated luminal area, yet media+adventitia thickness of the vein graft wall of IL-1RI(-/-) mice was 44% to 52% less than wild-type mice, at the both proximal (P < .01, P < .01) and distal (P = .054, P < .01) portions of vein grafts, for both normal flow and low flow, respectively. Compared with the C57BL/6J strain, B6129SF2/J mice exhibited no difference in vein graft intimal thickness but 2-fold greater media+adventitia thickness (P < .01)., Conclusion: When lacking IL-1 signaling, the vein graft wall adapts differently compared with the injured artery, showing typical intima hyperplasia although attenuated media+adventitia thickening. B6129SF2/J mice exhibit more media+adventitia response than C57BL/6J mice. The inflammatory networks that underlie the vein response to arterialization hold many roles in the adaptation of the total wall; thus, the utility of anti-inflammatory approaches to extend the durability of vein grafts comes into question., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Objective: Extracellular matrix dysregulation in the aortic media has been considered as the intrinsic factor for the formation of thoracic aortic dissection. However, the mechanisms of extracellular matrix disorders in the dissected aortic media remain unclear. This study was designed to investigate the relevance between smooth muscle cell phenotypes and extracellular matrix disorders in the dissected media. Their interaction may account for the pathogenesis of thoracic aortic dissection., Methods and Results: Thoracic aortic samples were collected from 10 patients with thoracic aortic dissection and 10 controls. Primary cultures of aortic medial smooth muscle cells were obtained with optimized explant technique. In this study, α-smooth muscle actin, smooth muscle myosin heavy chain 2, and smoothelin were applied as the contractile phenotypic markers and osteopontin was applied as the synthetic marker. Compared with controls, immunostaining and immunoblotting demonstrated that in vivo expression of α-smooth muscle actin, smooth muscle myosin heavy chain 2, and smoothelin were significantly decreased in the dissected media, whereas that of osteopontin was elevated (P<.01 for all). In vitro expression of the phenotypic markers showed the similar patterns. Furthermore, smooth muscle cells derived from the dissected media exhibited enhanced proliferation (P<.01), increased collagens I and III synthesis (2.6- and 4.4-fold, respectively; P<.01 for both), and elevated matrix metalloproteinase-2 production (4.2-fold; P<.01). Consistently, the protein levels of type I and III collagens and matrix metalloproteinase-2 in the dissected media were raised by 4.6-, 4.0-, and 3.7-fold, respectively (P<.01 for all). Collagen deposition was correspondingly increased and elastic fibers were decreased and disrupted., Conclusions: Smooth muscle cells in the dissected media exhibit phenotypic switching from the contractile to the synthetic type. The synthetic smooth muscle cells increase collagen synthesis and matrix metalloproteinase-2 production, both of which can promote collagen deposition and elastin degradation in thoracic aortic dissection., (Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

The distribution of two enzymes involved in H(2)S synthesis, cystationine β-synthase (CBS) and cystationine γ-liase (CSE), was studied in the walls of the internal carotid artery, order I-V branches of the middle cerebral artery basin, and intracerebral vessels of adult Wistar rats. Immunohistochemical staining showed the presence of CBS in the endothelium of small pial arteries (order IV-V branches) and intracerebral arterioles and in the capillary walls, neurons, and vascular nerves. As for CSE, in the internal carotid artery and large (order I-II) pial branches it was found mainly in the tunica media myocytes, in order III-IV vessels in myocytes and endothelium, and in smaller pial and intracerebral vessels in the endothelium. Along with enzyme-positive vessels, many pial and intracerebral arteries contained no these enzymes in the walls.

Coronary artery aneurysms are rare complications of autosomal dominant polycystic kidney disease (ADPKD), and their pathogenesis remains poorly understood. We report an autopsy case of a 64-year-old ADPKD patient with an asymptomatic, large (4 cm in diameter) saccular aneurysm arising from the left circumflex (LCX) branch of the coronary artery with only mild atherosclerotic changes. Autopsy also revealed small, focal defects of media with or without microaneurysm formation in the LCX, mesenteric and renal arteries, and a fibromuscular dysplasia-like lesion with microaneurysm in the common iliac artery. Since polycystin-1 and -2 are expressed in arterial smooth-muscle cells, these findings imply that abnormal polycystin expression in ADPKD initially causes the focal medial defects, some of which might later progress to microaneurysms and then overt aneurysms. To the best of our knowledge, this is the first description of the pathologic findings of an ADPKD-associated coronary aneurysm and its probable precursor lesions in arteries., (© 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

The concentrations of LPO products (including those present in LDL), oxidative modification of proteins, paraoxonase activity, concentrations of antioxidants, lipid values and biomarkers of endothelial dysfunction were studied in the blood and coronary artery intima/media of male patients with coronary atherosclerosis without acute coronary syndrome. Blood levels of LDL oxidized apolipoproteins and lipoprotein (a) were higher, while the content of NO metabolites, sVCAM endothelial adhesion molecules, and LDL oxidation resistance were lower in men with mainly unstable atherosclerotic plaques in the coronary arteries in comparison with men with mainly stable plaques in the coronary arteries. Of these blood biomarkers, only NO metabolites, oxidized proteins, and sVCAM correlated with the presence of unstable atherosclerotic plaques. A significant correlation between the levels of biomarkers in the vascular wall and blood was detected only for LPO parameters.

Introduction: Pulmonary artery root does not adapt properly when exposed to increased pressure stress, with progressive dilatation. The aim of this study was to evaluate, in an animal model, the histologic changes of the pulmonary root wall under increased pressure load., Methods and Results: To increase the systolic pressure in the pulmonary root, a banding of the pulmonary artery (PAB) was performed in 10 adult Sprague-Dawley rats and in 10 weanlings, using 7 adults and 8 weanlings as controls. We analyzed the structural changes of the pulmonary artery root after 30 days of increased pressure load. The mean pressure gradient across the banded pulmonary trunk was 53.57 ± 10 mmHg in the adult rats and 86.73 ± 15 mmHg in the weanlings. The pulmonary artery wall was significantly thicker in both age groups of PAB rats when compared to age-matched controls, showing also architectural structural changes, as a higher degree of mucoid degeneration, medionecrosis, and fibrosis as well as elastic fibers fragmentation. The apoptotic index was also increased in both PAB age groups. We also confirmed the physiologic higher degree of elastic fibers disarray in adult rats when compared to weanlings., Conclusions: The pulmonary artery wall seems to present maladaptive architectural changes in the media when exposed to systemic pressure. The PAB-related increase of the apoptotic index seems to reflect an accelerated involution of the pulmonary root's media. The physiologic higher degree of elastic fibers disarray in adult rats can possibly influence the worst adaptation of the pulmonary arterial wall to a systemic pressure load., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objectives: To investigate the influence of hypertension on large artery elasticity and the microstructure of the ascending aortic media in patients with coronary artery disease (CAD), and the association between arterial compliance and composition of the ascending aorta., Methods: 60 patients with CAD who underwent coronary artery bypass graft surgery were divided into two groups: 30 patients in a hypertension group and 30 patients in a non-hypertension group. Carotid-femoral pulse wave velocity (cfPWV) was measured by an automatic device (Complior, Artech, France). The severity of coronary atherosclerosis was assessed after selective coronary angiography using the Gensini score system. A quantitative study was conducted on ascending aorta specimens by histological and computer image analysis., Results: cfPWV of the hypertension group was higher than that of the non-hypertension group. The relative content of collagen in the ascending aortic media of the hypertension group was higher than that of the non-hypertension group, while the relative content of elastin in the ascending aortic media of the hypertension group was lower than that of the non-hypertension group. cfPWV showed a positive correlation with relative contents of collagen in the ascending aorta and a negative correlation with relative contents of elastin in the ascending aorta in the two groups., Conclusions: Hypertension may raise the contents of collagen and decrease the contents of elastin in the ascending aortic media of patients with CAD, which in turn may decrease the patients' large artery compliance. cfPWV may reflect the quantitative changes of collagen and elastin in the ascending aortic media in CAD patients independently of hypertension.

The microarchitecture of different components of the extracellular matrix (ECM) is crucial to our understanding of the properties of a tissue. In the study presented here, we used a top-down approach to understand how the interplay among different fibers determines the mechanical properties of real tissues. By selectively removing different elements of the arterial wall, we were able to measure the contribution of the different constituents of the ECM to the mechanical properties of the whole tissue. Changes in the network structure were imaged with the use of two-photon microscopy. We used an atomic force microscope to measure changes in the mechanical properties by performing nanoindentation experiments. We show that although the removal of a key element of the ECM reduced the local stiffness by up to 50 times, the remaining tissue still formed a coherent network. We also show how this method can be extended to study the effects of cells on real tissues. This new (to our knowledge) way of studying the ECM will not only help physicists gain a better understanding of biopolymers, it will be a valuable tool for biomedical researchers studying processes such as wound healing and cervix ripening., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Acid–base transport in the vascular wall remains incompletely understood. Here, we investigated (a) implications of Na(+)/H(+) exchanger NHE1 knockout for vascular smooth muscle (VSMC) and endothelial cell (EC) pH(i) regulation, mesenteric artery morphology, vasomotor function and blood pressure regulation, and (b) consequences of sustained EC and VSMC acidification for vasomotor function. Na(+)/H(+) exchange activity was abolished in VSMCs and ECs from NHE1 knockout mice, but with CO(2)/HCO(3)(−) present, steady-state pH(i) was unaffected. Active tension was 30% smaller in arteries from NHE1 knockout than wild-type mice, and media thickness equally reduced. Number of VSMCs per unit artery length was unchanged whereas volume and cross-sectional area of individual VSMCs were reduced. Media stress, force production per VSMC cross-sectional area and VSMC Ca(2+) responses were unaffected. Blood pressure was 25 mmHg lower in NHE1 knockout than wild-type mice. Omission of CO(2)/HCO(3)(−) caused VSMCs and ECs to acidify substantially more in NHE1 knockout (0.3–0.6 pH-units) than wild-type (0.02–0.1 pH units) mice. Removing CO(2)/HCO(3)(−) inhibited acetylcholine-induced NO-mediated relaxations in arteries from NHE1 knockout but not wild-type mice. Without CO(2)/HCO(3)(−), effects of NO synthase and rho kinase inhibition on noradrenaline-induced contractions were smaller in arteries from NHE1 knockout than wild-type mice whereas the EC Ca(2+) response to acetylcholine, VSMC Ca(2+) response to noradrenaline and vasorelaxation to S-nitroso-N-acetylpenicillamine were unaffected. In conclusion, NHE1 mediates the Na(+)/H(+) exchange in ECs and VSMCs. Under physiological conditions, CO(2)/HCO(3)(−)-dependent mechanisms mask the pH(i)-regulatory function of NHE1. NHE1 knockout causes hypotrophy of VSMCs, reduced artery tension and lower blood pressure. At acidic pH(i), NO-mediated vasorelaxation and rho kinase-dependent VSMC Ca(2+) sensitivity are reduced.

Background: Granzyme B, a proapoptotic serine protease, is abundant in advanced, unstable atherosclerotic plaques, and it is suggested to contribute to plaque instability by inducing vascular smooth muscle cells apoptosis and by degrading plaque extracellular matrix. Proteinase inhibitor 9, the only known endogenous inhibitor of granzyme B in humans, confers protection against granzyme-B-induced apoptosis. However, the role of proteinase inhibitor 9 in atherosclerotic lesion development has yet to be determined. We hypothesized that atherosclerotic lesions have lower proteinase inhibitor 9 expression levels that will increase their susceptibility to granzyme-B-induced apoptosis., Methods: Serial sections of human coronary arteries exhibiting different stages of lesion development were assessed by immunohistochemistry for proteinase inhibitor 9, α-smooth muscle cells actin, granzyme B, CD8, and active caspase-3. Frozen samples were analyzed by Western blot to evaluate total proteinase inhibitor 9 levels., Results: Vascular smooth muscle cells express less proteinase inhibitor 9 as disease severity increases, and a significant difference in proteinase inhibitor 9 expression is observed between medial and intimal smooth muscle cells. High granzyme B levels colocalize with CD8+ cells and foam cells in the shoulder region and necrotic core area of advanced lesions. In advanced lesions, increased expression of activated caspase-3 in intimal SMC was associated with reduced proteinase inhibitor 9 expression in the presence of granzyme B., Conclusion: Reduced proteinase inhibitor 9 expression in human vascular smooth muscle cells is associated with atherosclerotic disease progression and is inversely related to the extent of apoptosis within the intima. Reduced proteinase inhibitor 9 expression may contribute to increased smooth muscle cell susceptibility to granzyme-B-induced apoptosis within the plaque., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The present study was conducted to determine the morphological and morphometric features of the arterial wall structure at the bifurcation of blood vessels of the cerebral arterial circle (CAC) of Willis in people of different age (from birth till 65 years). Material obtained from 80 people was stained with hematoxylin-eosin, Van Gieson stain, with orcein by Unna-Taenzer's method and with sudan. The proliferative activity of the cells in tunica intima and tunica media at the site of bifurcation of the internal carotid and basilar arteries was studied immunohistochemically using monoclonal antibodies against Ki-67. It was found that intimal thickenings (cushions) appeared immediately after birth, initially only in a few places of CAC vessel branching; by 8-10 years they were detected in all the bifurcations of the circle. With aging, the thickening of intimal cushions with a thinning of the underlying tunica media was found. Ki-67 protein expression was noted in both the intimal cushions and underlying tunica media, indicating the activity of atherosclerosis process.

4,4'-Methylenedianiline (DAPM) is an aromatic diamine used directly in the production of polyurethane foams and epoxy resins, or as a precursor to MDI in the manufacture of some polyurethanes. In our prior experiments, we showed that chronic, intermittent treatment of female rats with DAPM resulted in vascular medial hyperplasia of pulmonary arteries. In addition, treatment of vascular smooth muscle cells (VSMC) in culture with DAPM increased the rates of proliferation in a manner that was inhibited by co-treatment with N-acetylcysteine but was not associated with oxidative stress. We thus hypothesized that NAC treatment inhibited DAPM toxicity by competing for binding reactive intermediates formed through DAPM metabolism. Because the peroxidase enzyme cyclooxygenase is constitutively expressed in VSMC, and because cyclooxygenase is known to metabolize similar aromatic amines to electrophilic intermediates, we further hypothesized that DAPM-induced VSMC proliferation was dependent upon COX-1/2-mediated bioactivation. To test this hypothesis, we treated VSMC with DAPM and measured cell proliferation, COX-2 expression, COX-1/2 activity, and levels of covalent binding. DAPM treatment resulted in a dose-dependent increase in proliferation that was abolished by co-treatment with the COX-2-selective inhibitor celecoxib. In addition, DAPM exposure increased the rates of proliferation in VSMC isolated from wild-type but not COX-2 (-/-) mice. Paradoxically, treatment with DAPM reduced the cellular production of PGE(2) and PGF(2α), but dose-dependently increased the COX-2 protein levels. Covalent binding of [(14)C]-DAPM to VSMC biomolecules was greater in wild-type than in COX-2 (-/-) cells. However, covalent binding of [(14)C]-DAPM was not altered by co-treatment with a nonselective inhibitor of cytochromes P450. These studies thus suggest that DAPM-induced VSMC proliferation may be due to bioactivation of DAPM, perhaps through the action of cyclooxygenase. The data furthermore suggest that DAPM's mechanism of action may possibly involve inhibition or suicide inactivation of COX-2. In addition, because we observed an increase in DAPM-induced VSMC proliferation in cells isolated from female compared to male rats, further studies into the potential interplay between DAPM, the estrogen receptor, and COX-2 seem warranted.

Background: Vascular calcifications (VC) are associated with cardiovascular (CV) morbidity and are independent predictors of CV mortality in end-stage renal disease (ESRD). This study aimed to investigate the presence of arterial intima calcification (AIC) and arterial media calcification (AMC) in nondiabetic patients on long-term hemodialysis, and to assess the association with CV risk factors., Methods: 34 ESRD patients (17 males) on hemodialysis for at least 5 years were evaluated for VC using B-mode ultrasonography., Results: AMC and AIC patterns were detected respectively in 62% and 59% of patients, and 21% had no VC. Patients with AIC were significantly older than those without AIC (p < 0.001). CRP levels (p < 0.001) were higher in patients with AMC. Using multivariate logistic analysis of regression, older age (> 50 years) and higher CRP levels (> 5 mg/l) were associated with AIC and AMC, respectively (p = 0.007 and p = 0.003). Logistic regression analysis showed that patients with CRP > 5 mg/l had a greater relative risk of having AMC (odds ratio 30, 95% confidence interval 27.041 - 32.959; p = 0.003)., Conclusions: Ultrasonography can be used to detect AIC and AMC, and could be useful for the early detection of VC. In nondiabetic patients who had been on hemodialysis for at least 5 years, older age was associated with AIC, and elevated CRP levels with AMC.

1. In endothelial cells, the major receptor for the binding and internalization of oxidized low-density lipoprotein (LDL) is the lectin-like oxidized LDL receptor (LOX-1). The aim of the present study was to investigate the effects of taurine on intimal thickening and LOX-1 expression under normal and oxidative conditions. 2. The iliac artery of rabbits were subjected to balloon injury and oxidative stress was induced by 14 days treatment of rabbits with 75 mg/kg, s.c., buthionine sulfoximine (BSO), a specific inhibitor of glutathione synthesis. Taurine was administered in drinking water (1%, w/v) for 14 days in the presence (BSO + Taurine group) and in the absence of BSO treatment (Taurine group). In taurine and placebo groups, rabbits were injected with 4 mL, s.c., 0.9% NaCl (vehicle for BSO) for 14 days. 3. Taurine (1% in drinking water, w/v) preserved plasma levels of anti-oxidants and lowered the increased blood pressure induced by BSO. The stenosis rate of 29.92% in the placebo group increased to 72.20% in the BSO group, which was significantly reduced to 42.21% by taurine (P < 0.001; n = 5). Localization of LOX-1 to the intima and media of the iliac artery was demonstrated in the present study. Taurine treatment reduced the BSO-induced increase in LOX-1 expression at both the protein and mRNA levels (P < 0.05 and P < 0.01, respectively). 4. The results demonstrate that the stenosis rate and LOX-1 expression correlate well with oxidative status. Manipulation of LOX-1 expression by taurine may have therapeutic benefits in preventing restenosis., (© 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.)

There is no good animal model of large artery injury-induced intimal hyperplasia (IH). Those available are reproducible, providing only a few layers of proliferating cells or have the disadvantage of the presence of a metallic stent that complicates histology evaluation. This study was designed to develop a new, simple model of accelerated IH based on balloon injury in conjunction with disruption of the Internal Elastic Lamina (IEL) in pig external iliac arteries. Iliac artery injury (n = 24) was performed in 12 Yorkshire pigs divided in two groups: Group I (n = 10), overdistention injury induced by an oversized non-compliant balloon; Group II (n = 14), arterial wall disruption by pulling back an isometric cutting balloon (CB) followed by stretching with a compliant Fogarty Balloon (FB). At two weeks, arteries were processed for morphometric analysis and immunohistochemistry (IHC) for smooth muscle cells (SMC) and proliferating cell nuclear antigen (PCNA). When comparing the two groups, at 2 weeks, arteries of group II had a higher incidence of IH (100%vs. 50%, P = 0.0059), increased intimal areas (2.54 ± 0.33 mm(2) vs. 0.93 ± 0.36 mm(2) , P = 0.004), increased intimal area/Media area ratios (0.95 ± 0.1 vs. 0.28 ± 0.05; P < 0.0001) and decreased lumen areas (6.24 ± 0.44 vs. 9.48 ± 1.56, P = 0.026). No thrombosis was noticed in Group II. Neointima was composed by proliferating SMC located with the highest concentration in the area of IEL disruption (IHC). Arterial injury by pulling back CB and FB induces significant IH in pig iliac arteries by two weeks without thrombosis. This model is superior to the classical overdistention non-compliant model and should be useful and cost-effective for preclinical testing of procedures designed to inhibit IH in large peripheral arteries., (© 2011 The Authors. International Journal of Experimental Pathology © 2011 International Journal of Experimental Pathology.)

Background: We recently reported that the efficacy of nitric oxide (NO) appears to be based on both sex and hormone status. The mechanism responsible for this differential efficacy is unknown. The aim of this study was to characterize the effect of sex, hormones, and NO on the extracellular signal-regulated kinase (ERK) and Akt signaling pathways after arterial injury., Methods: Male and female Sprague-Dawley rats underwent castration or sham surgery. Two weeks later, they underwent carotid artery balloon injury. Treatment groups included the following: control, injury, and injury + 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO) (n = 5 per group). Arteries were harvested 2 weeks after injury and assessed for phospho-ERK (pERK) and phospho-Akt (pAkt) expression., Results: After injury, more pERK and pAkt activity was seen in the adventitia than media in both sexes, regardless of hormone status (P < .05). In hormonally intact males, NO further increased pERK (44%) and pAkt (120%) after injury (P < .001). Castration attenuated the effects of NO. In hormonally intact females, NO caused the opposite pattern with pERK activity but did not affect pAkt activity., Conclusions: After arterial injury, ERK and Akt activity is significantly greater in the adventitia than the media, and depends on sex, hormone status, and NO. Understanding adventitial regulation of proliferative signaling pathways will allow the development of targeted therapies for neointimal hyperplasia., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Salviae Miltiorrhizae Radix (Danshen) and Puerariae Lobatae Radix (Gegen) are principal herbs have long been used in combination for treating cardiovascular disease., Aims of Study: Danshen and Gegen in the ratio of 7:3 (DGW) have significantly reduced the carotid intimal-media thickening (IMT) in patients in our previous clinical study. In the present study, we have demonstrated the mechanisms on IMT reduction by investigating its key processes on both vascular smooth muscle cell (vSMC) and endothelial cells., Materials and Methods: The anti-proliferative effects of DGW on platelet-derived growth factor (PDGF) induced vSMC proliferation were studied by cell proliferation, cell cycle distribution, p-ERK and cyclin D expression level. The anti-migratory effect of DGW was investigated by using transwell apparatus. For human umbilical endothelial cells (HUVEC), the inhibitory effects of DGW on TNF-alpha induced cell adhesion, cell adhesion molecules expression, MCP-1 and IL-6 production were investigated., Results: DGW significantly inhibited A7r5 proliferation and exhibited G1/S cell cycle arrest by suppressing both p-ERK and cyclin D expression. Moreover, DGW showed anti-migratory effect against PDGF-induced A7r5 migration. In addition, DGW inhibited the cell adhesion as well as the expression of ICAM-1 and VCAM-1, the production of MCP-1 but not IL-6 in TNF-α stimulated HUVECs., Conclusions: Our study provided strong scientific evidence on IMT reduction in patients by modulating the key atherogenic events in both vSMC and endothelial cells., (Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: The sequelae of aortic root dilation are the lethal consequences of Marfan syndrome. The root dilation is attributable to an imbalance between deposition of matrix elements and metalloproteinases in the aortic medial layer as a result of excessive transforming growth factor-beta signaling. This study examined the efficacy and mechanism of statins in attenuating aortic root dilation in Marfan syndrome and compared effects to the other main proposed preventative agent, losartan., Methods and Results: Marfan mice heterozygous for a mutant allele encoding a cysteine substitution in fibrillin-1 (C1039G) were treated daily from 6 weeks old with pravastatin 0.5 g/L or losartan 0.6 g/L. The end points of aortic root diameter (n=25), aortic thickness, and architecture (n=10), elastin volume (n=5), dp/dtmax (maximal rate of change of pressure) (cardiac catheter; n=20), and ultrastructural analysis with stereology (electron microscopy; n=5) were examined. The aortic root diameters of untreated Marfan mice were significantly increased in comparison to normal mice (0.161 ± 0.001 cm vs 0.252 ± 0.004 cm; P<0.01). Pravastatin (0.22 ± 0.003 cm; P<0.01) and losartan (0.221 ± 0.004 cm; P<0.01) produced a significant reduction in aortic root dilation. Both drugs also preserved elastin volume within the medial layer (pravastatin 0.23 ± 0.02 and losartan 0.29 ± 0.03 vs untreated Marfan 0.19 ± 0.02; P=0.01; normal mice 0.27 ± 0.02). Ultrastructural analysis showed a reduction of rough endoplasmic reticulum in smooth muscle cells with pravastatin (0.022 ± 0.004) and losartan (0.013 ± 0.001) compared to untreated Marfan mice (0.035 ± 0.004; P<0.01)., Conclusions: Statins are similar to losartan in attenuating aortic root dilation in a mouse model of Marfan syndrome. They appear to act through reducing the excessive protein manufacture by vascular smooth muscle cells, which occurs in the Marfan aorta. As a drug that is relatively well-tolerated for long-term use, it may be useful clinically.

Background and Purpose: Recent studies reported the differential effect of docosahexaenoic (DHA) and eicosapentaenoic acids (EPA). We examined the differential association of DHA and EPA with carotid intima-media thickness (IMT) in Japanese individuals in Japan and in U.S. white individuals and explored whether DHA or EPA contributes to the difference in IMT between the two groups., Methods: A population-based cross-sectional study in 608 Japanese and U.S. white men aged 40 to 49 was conducted to assess IMT, serum DHA, EPA, and other cardiovascular risk factors., Results: Japanese compared to U.S. whites had significantly lower IMT (mean±SD, 618±81 and 672±94 μm for Japanese and whites, respectively; P<0.001) and had >2-fold higher levels of DHA and EPA. DHA, but not EPA, had an inverse association with IMT in both Japanese and U.S. whites. The inverse association remained only in Japanese men after adjusting for risk and other factors. The significant difference in multivariable-adjusted IMT became nonsignificant after further adjusting for DHA (mean difference, 17 μm; 95% CI, -8 to 43; P=0.177) but not EPA. In this multivariable-adjusted model, DHA but not EPA was a significant predictor of IMT (P=-0.032 versus 0.863, respectively)., Conclusions: These data suggest that DHA may have a more potent antiatherogenic effect than EPA, especially in levels observed in the Japanese, independent of risk factors.