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Background:  There is some evidence indicating that inflammation of the aneurysmal wall is related to aneurysmal growth and rupture. The presence of CD68 may indicate greater inflammatory activity. The objective of this study is to evaluate CD68 immunoexpression in surgically resected brain aneurysms and its association with smoking., Methods:  The resected brain aneurysmal walls after microsurgical clipping were envoyed to immunohistochemistry investigation. The objective was to evaluate the expression of CD68 and CD34 antibodies. The associations between inflammatory markers, smoking, and rupture were tested using Fischer's exact test., Results:  CD68 immunoexpression in the tunica media was associated with larger aneurysms: 7.0 mm (7.0-9.0 mm) versus 5.0 mm (3.5-5 mm; p  = 0.011). There was no statistically significant association between smoking and CD68 expression in the tunica media ( p  = 0.234) or in either the tunica media or the tunica intima ( p  = 0.628). There was also no statistically significant association between hemorrhagic presentation of the aneurysm and CD68 expression in the tunica media ( p  = 0.689) or in either the tunica media or the tunica intima ( p  = 0.348). Therefore, the presence of CD68-positive cells in the aneurysmal walls indicates an association with size, especially if the tunica media is exclusively compromised ( p  = 0.011)., Conclusion:  Immunohistochemistry investigation for CD68 antibodies was used to determine histiocytic infiltration. Adequately powered studies are necessary to further investigate the association between CD68-positive cells and both smoking history and hemorrhagic presentation of aneurysms., Competing Interests: None declared., (Thieme. All rights reserved.)

BACKGROUNDDisease of the aorta varies from atherosclerosis to aneurysms, with complications including rupture, dissection, and poorly characterized limited tears. We studied limited tears without any mural hematoma, termed intimomedial tears, to gain insight into aortic vulnerability to excessive wall stresses. Our premise is that minimal injuries in aortas with sufficient medial resilience to prevent tear progression correspond to initial mechanisms leading to complete structural failure in aortas with significantly compromised medial resilience.METHODSIntimomedial tears were macroscopically identified in 9 of 108 ascending aortas after surgery and analyzed by histology and immunofluorescence confocal microscopy.RESULTSNonhemorrhagic, nonatheromatous tears correlated with advanced aneurysmal disease and most lacked distinctive symptoms or radiological signs. Tears traversed the intima and part of the subjacent media, while the resultant defects were partially or completely filled with neointima characterized by differentiated smooth muscle cells, scattered leukocytes, dense fibrosis, and absent elastic laminae despite tropoelastin synthesis. Healed lesions contained organized fibrin at tear edges without evidence of plasma and erythrocyte extravasation or lipid accumulation.CONCLUSIONThese findings suggest a multiphasic model of aortic wall failure in which primary lesions of intimomedial tears either heal if the media is sufficiently resilient or progress as dissection or rupture by medial delamination and tear completion, respectively. Moreover, mural incorporation of thrombus and cellular responses to injury, two historically important concepts in atheroma pathogenesis, contribute to vessel wall repair with adequate conduit function, but even together are not sufficient to induce atherosclerosis.FUNDINGNIH (R01-HL146723, R01-HL168473) and Yale Department of Surgery.

Hypertension-induced tunica media thickening (TMT) is the most important fundamental for the subsequent complications like stroke and cardiovascular diseases. Pathogenically, TMT originates from both vascular smooth muscle cells (VSMCs) hypertrophy due to synthesizing more amount of intracellular contractile proteins and excess secretion of extracellular matrix. However, what key molecules are involved in the pathogenesis of TMT is unknown. We hypothesize that formin homology 2 domain-containing protein 1 (FHOD1), an amply expressed mediator for assembly of thin actin filament in VSMCs, is a key regulator for the pathogenesis of TMT. In this study, we found that FHOD1 expression and its phosphorylation/activation were both upregulated in the arteries of three kinds of hypertensive rats. Ang-II induced actin filament formation and hypertrophy through activation and upregulation of FHOD1 in VSMCs. Active FHOD1-mediated actin filament assembly and secretions of collagen-1α/collagen-3α played crucial roles in Ang-II-induced VSMCs hypertrophy in vitro and hypertensive TMT in vivo. Proteomics demonstrated that activated FL-FHOD1 or its C-terminal diaphanous-autoregulatory domain significantly upregulated RNF213 (ring finger protein 213), a 591-kDa cytosolic E3 ubiquitin ligase with its loss-of-functional mutations being a susceptibility gene for Moyamoya disease which has prominent tunica media thinning in both intracranial and systemic arteries. Mechanistically, activated FHOD1 upregulated its downstream effector RNF213 independently of its classical pathway of decreasing G-actin/F-actin ratio, transcription, and translation, but dependently on its C-terminus-mediated stabilization of RNF213 protein. FHOD1-RNF213 signaling dramatically promoted collagen-1α/collagen-3α syntheses in VSMCs. Our results discovered a novel signaling axis of FHOD1-RNF213-collagen-1α/collagen-3α and its key role in the pathogenesis of hypertensive TMT., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Diabetic macroangiopathy is characterized by increased extracellular matrix deposition, including excessive hyaluronan accumulation, vessel thickening and stiffness, and endothelial dysfunction in large arteries. We hypothesized that the overexpression of hyaluronan in the tunica media also led to endothelial cell (EC) dysfunction. To address this hypothesis, we investigated the following in the aortas of mice with excessive hyaluronan accumulation in the tunica media (HAS-2) and wild-type mice: EC dysfunction via myograph studies, nitric oxide (NO) bioavailability via diaminofluorescence, superoxide formation via dihydroethidium fluorescence, and the distances between ECs via stereological methods. EC dysfunction, characterized by blunted relaxations in response to acetylcholine and decreased NO bioavailability, was found in the aortas of male HAS-2 mice, while it was unaltered in the aortas of female HAS-2 mice. Superoxide levels increased and extracellular superoxide dismutase (ecSOD) expression decreased in the aortas of male and female HAS-2 mice. The EC-EC distances and LDL receptor expression were markedly increased in the HAS-2 aortas of male mice. Our findings suggest hyaluronan increases oxidative stress in the vascular wall and that together with increased EC distance, it is associated with a sex-specific decrease in NO levels and endothelial dysfunction in the aorta of male HAS-2 transgenic mice.

Arterial media calcification (AMC) is predominantly regulated by vascular smooth muscle cells (VSMCs), which transdifferentiate into pro-calcifying cells. In contrast, there is little evidence for endothelial cells playing a role in the disease. The current study investigates cellular functioning and molecular pathways underlying AMC, respectively by, an ex vivo isometric organ bath set-up to explore the interaction between VSMCs and ECs and quantitative proteomics followed by functional pathway interpretation. AMC development, which was induced in mice by dietary warfarin administration, was proved by positive Von Kossa staining and a significantly increased calcium content in the aorta compared to that of control mice. The ex vivo organ bath set-up showed calcified aortic segments to be significantly more sensitive to phenylephrine induced contraction, compared to control segments. This, together with the fact that calcified segments as compared to control segments, showed a significantly smaller contraction in the absence of extracellular calcium, argues for a reduced basal NO production in the calcified segments. Moreover, proteomic data revealed a reduced eNOS activation to be part of the vascular calcification process. In summary, this study identifies a poor endothelial function, next to classic pro-calcifying stimuli, as a possible initiator of arterial calcification.

Background: Arterial medial calcification (AMC) is associated with a high incidence of cardiovascular risk in patients with type 2 diabetes and chronic kidney disease. Here, we tested whether hydrogen sulfide (H 2 S) can prevent AMC in rats with diabetic nephropathy (DN)., Methods: DN was induced by a single injection of streptozotocin and high-fat diet (45% kcal as fat) containing 0.75% adenine in Sprague-Dawley rats for 8 weeks., Results: Rats with DN displayed obvious calcification in aorta, and this was significantly alleviated by Sodium Hydrosulfide (NaHS, a H 2 S donor, 50 μmol/kg/day for 8 weeks) treatment through decreasing calcium and phosphorus content, ALP activity and calcium deposition in aorta. Interestingly, the main endogenous H 2 S generating enzyme activity and protein expression of cystathionine-γ-lyase (CSE) were largely reduced in the arterial wall of DN rats. Exogenous NaHS treatment restored CSE activity and its expression, inhibited aortic osteogenic transformation by upregulating phenotypic markers of smooth muscle cells SMα-actin and SM22α, and downregulating core binding factor α-1 (Cbfα-1, a key factor for bone formation), protein expressions in rats with DN when compared to the control group. NaHS administration also significantly reduced Stat3 activation, cathepsin S (CAS) activity and TGF-β1 protein level, and improved aortic elastin expression., Conclusions: H 2 S may have a clinical significance for treating AMC in people with DN by reducing Stat3 activation, CAS activity, TGF-β1 level and increasing local elastin level., (© 2021. The Author(s).)

Objective: Atheromatous fibrous caps are produced by smooth muscle cells (SMCs) that are recruited to the subendothelial space. We tested whether the recruitment mechanisms are the same as in embryonic artery development, which relies prominently on Notch signaling to form the subendothelial medial SMC layers., Approach and Results: Notch elements were expressed in regions of fibrous cap in human and mouse plaques. To assess the causal role of Notch signaling in cap formation, we studied atherosclerosis in mice where the Notch pathway was inactivated in SMCs by conditional knockout of the essential effector transcription factor RBPJ (recombination signal-binding protein for immunoglobulin kappa J region). The recruitment of cap SMCs was significantly reduced without major effects on plaque size. Lineage tracing revealed the accumulation of SMC-derived plaque cells in the cap region was unaltered but that Notch-defective cells failed to re-acquire the SMC phenotype in the cap. Conversely, to analyze whether the loss of Notch signaling is required for SMC-derived cells to accumulate in atherogenesis, we studied atherosclerosis in mice with constitutive activation of Notch signaling in SMCs achieved by conditional expression of the Notch intracellular domain. Forced Notch signaling inhibited the ability of medial SMCs to contribute to plaque cells, including both cap SMCs and osteochondrogenic cells, and significantly reduced atherosclerosis development., Conclusions: Sequential loss and gain of Notch signaling is needed to build the cap SMC population. The shared mechanisms with embryonic arterial media assembly suggest that the cap forms as a neo-media that restores the connection between endothelium and subendothelial SMCs, transiently disrupted in early atherogenesis.

Coronary ectasia is a rare vessel defect that represents a pathological and incidental finding in routine coronary angiography performed for other coronary syndromes. This defect exposes to the risk of intra-coronary thrombosis by blood stasis due to the turbulent blood flow in those dilated areas that can lead to sudden death. We report an autopsy case of a male subject suddenly deceased. A medico-legal autopsy concluded an ischemic heart failure due to a vascular thrombosis by a blood clot in a coronary ectasia. Our case report aimed to discuss the mechanisms of sudden death attributed to coronary artery ectasia., (Copyright © 2021 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Biomineralisation, the deposition of mineral onto a matrix, can be both a physiological and pathological process. Bone formation involves the secretion of an extracellular matrix (ECM) by osteoblasts and subsequent mineralisation of that matrix. It is regulated by a number of local and systemic factors and is necessary for maintenance of normal bone health. Conversely, mineralisation (or calcification) of soft tissues, including the vasculature, is detrimental to that tissue, leading to diseases such as arterial medial calcification (AMC). The mechanisms underlying AMC development are not fully defined, though it is thought that vascular smooth muscle cells (VSMCs) drive this complex, cell-mediated process. Similarly, AMC is regulated by a variety of enzymes and molecules, many of which have already been implicated in the regulation of bone mineralisation. This review will provide an overview of the similar, and sometimes opposing effects of these signalling molecules on the regulation of bone mineralisation and AMC.

Background: Clinical studies have revealed a greater risk of pulmonary autograft dilation after the Ross procedure in patients with preoperative aortic insufficiency (AI). The present study examined whether the morphologic, biomechanical, and cellular properties of the pulmonary artery (PA) from patients with AI were phenotypically different compared with patients diagnosed with aortic stenosis (AS)., Methods: PA segments were harvested from patients undergoing the Ross procedure for AS (n = 16) and AI (n = 6). Preoperative aortic annulus was significantly larger (P < 0.05) in patients with AI (28.5 ± 1.8 mm) vs AS (22.8 ± 1.2 mm). Morphologic, biomechanical, and cellular phenotypes of the PA were analyzed., Results: Collagen and elastin content in the media of the PA wall were similar in patients with AS and AI. Elastic modulus and energy loss of the PA were not significantly different between the groups. In the media of the PA, expression of a panel of vascular smooth muscle cell-specific proteins were similar in patients with AS and AI. In contrast, nonmuscle myosin IIB protein levels in the PA of AS patients were significantly higher compared with AI patients, and immunofluorescence identified staining in α-smooth muscle actin-positive vascular smooth muscle cells., Conclusions: Despite similar morphological and biomechanical properties, the disparate expression of nonmuscle myosin IIB protein distinguishes the PA of patients with AI from patients with AS. The biological role in vascular smooth muscle cells and the potential contribution of nonmuscle myosin IIB to pulmonary autograft dilation in a subset of AI patients after the Ross procedure remain to be determined., (Copyright © 2020 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Objective: There is no medical treatment to prevent abdominal aortic aneurysm (AAA) growth and rupture, both of which are linked to smoking. Our objective was to map the tunica-specific pathophysiology of AAA with consideration of the intraluminal thrombus, age, and sex, and to subsequently identify which mechanisms were linked to smoking and diameter growth rate. Approach and Results: Microarray analyses were performed on 246 samples from 76 AAA patients and 13 controls. In media and adventitia, there were 5889 and 2701 differentially expressed genes, respectively. Gene sets related to adaptive and innate immunity were upregulated in both tunicas. Media-specific gene sets included increased matrix disassembly and angiogenesis, as well as decreased muscle cell development, contraction, and differentiation. Genes implicated in previous genome-wide association studies were dysregulated in media. The intraluminal thrombus had a pro-proteolytic and proinflammatory effect on the underlying media. Active smoking resulted in increased inflammation, oxidative stress, and angiogenesis in all tissues and enriched lipid metabolism in adventitia. Processes enriched with active smoking in control aortas overlapped to a high extent with those differentially expressed between AAAs and controls. The AAA diameter growth rate (n=24) correlated with T- and B-cell expression in media, as well as lipid-related processes in the adventitia., Conclusions: This tunica-specific analysis of gene expression in a large study enabled the detection of features not previously described in AAA disease. Smoking was associated with increased expression of aneurysm-related processes, of which adaptive immunity and lipid metabolism correlated with growth rate.

Background and Aim: We previously showed that 12-month high-fat diet (HFD) in pigs led to fattening and increased artery intima-media-thickness, which were partly reversed after 3-month return to control diet (CD). The aim of this study was to decipher underlying mechanism of action by using transcriptomic analyses of intima and media of aorta., Methods and Results: Thirty-two pigs were divided into three groups: CD for 12 months; HFD for 12 months; switch diet group (regression diet; RD): HFD for 9 months followed by CD for 3 months. After 12 months, RNA was isolated from aorta intima and media for nutrigenomic analyses. HFD significantly affected gene expression in intima, while RD gene expression profile was distinct from the CD group. This suggests that switch to CD is not sufficient to correct gene expression alterations induced by HFD but counteracted expression of a group of genes. HFD also affected gene expression in media and as for intima, the expression profile of media of pigs on RD differed from that of these on CD., Conclusions: This study revealed nutrigenomic modifications induced by long-term HFD consumption on arterial intima and media. The return to CD was not sufficient to counteract the genomic effect of HFD., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2020 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Glucagon-like peptide 1 receptor (GLP-1R) belongs to the family B of G protein-coupled receptors (GPCRs) and has antidiabetic and cardioprotective effects. Classical GLP-1R at the plasma membrane undergoes desensitization and internalization and is recycled back to the plasma membrane under the control of GLP-1 in islet β-cells. However, the subcellular localization of GLP-1R in the vascular system remains unclear. Here, we find that GLP-1R is localized in the nucleus of rat aortic smooth muscle cells (RASMCs) and in the tunica media. We identify a functional nuclear localization signal (NLS; 412-442aa) at the C-terminal region of GLP-1R. Nuclear import of GLP-1R is mediated by an importin-α-dependent pathway and regulated by phosphorylation of Ser416 in the NLS. Upon leaving the nucleus, GLP-1R promotes cell proliferation in RASMCs. These findings may provide insights into the cardiovascular functions of GLP-1R., (© 2020 Federation of European Biochemical Societies.)

This review seeks to provide an update of preclinical findings and available clinical data on the chronic persistent inflammation and its direct role on the pulmonary arterial hypertension (PAH) progression. We reviewed the different mechanisms by which the inflammatory and immune pathways contribute to the structural and functional changes occurring in the three vascular compartments: the tunica intima, tunica media, and tunica adventitia. We also discussed how these inflammatory mediator changes may serve as a biomarker of the PAH progression and summarize unanswered questions and opportunities for future studies in this area., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Objectives: Brain arteriovenous malformations (bAVM) are rare vascular lesions. Recent observations challenge the congenital nature of these lesions. The underlying cellular and molecular mechanisms associated with dynamic changes of bAVM still remain unclear. The objective of this study was to explore the potential role of COL4A2 (Collagen alpha-2(IV)) in the pathophysiology of bAVM., Patients and Methods: Expression and localization of COL4A2 were analyzed on tissue microarrays from bAVM patients (n = 60) by immunohistochemistry. Correlations between COL4A2 levels and clinical parameters were examined with Pearson's test for normally- distibuted or Spearman's Rho for not normally distributed data. Comparison between different clinical parameters was performed using t-test, non-parametric Mann-Whitney U test or Kruskal- Wallis test. Fisher's exact test was used for categorical data., Results: COL4A2 was mainly expressed beneath the endothelium of vessels in the tunica media of bAVM. This pattern of expression indicates the basement membrane of the vessel walls. High levels of COL4A2 expression correlated with the age at surgery of patients (p = 0.005; R = 0.393; Spearman's Rho). The age at surgery in young (17-25 years) and old patients (55-76 years) showed a linear correlation; a greater variance of COL4A2 expression was observed in the age group between 26-54 years., Conclusion: This study reports for the first time the expression of COL4A2 in bAVM and suggests a potential role of COL4A2 in bAVM pathophysiology. These findings contribute to a better understanding of the microenvironment of bAVM and may foster the development of improved therapeutic strategies for this disease., (Copyright © 2019. Published by Elsevier B.V.)

Arterial media calcification and arterial stiffness are independent predictors of cardiovascular mortality. Both processes reinforce one another, creating a vicious cycle in which transdifferentiation of endothelial cells and vascular smooth muscle cells play a central role. Physiological functioning of vascular smooth muscle cells in the arterial medial layer greatly depends on normal endothelial cell behavior. Endothelial or intimal layer cells are the primary sensors of pathological triggers circulating in the blood during, for example, ageing or inflammation, and often can be seen as initiators of this vicious cycle. As such, the search for treatment of arterial media calcification, which until now has been mainly concentrated at the level of the vascular smooth cell, may need to be expanded to intimal layer targets., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The body of a 43-year-old African woman with a history of aortic aneurysm and hypertension was forensically investigated after her sudden death. The cause of death was related to a cardiac tamponade due to a ruptured aneurysm of the ascending aorta. Post-mortem gross examination showed an abnormal whitish discoloration of the intima with fibrous thickening of the aortic wall. Several arteries (left main and circumflex coronaries, carotid, renal and iliac arteries) showed similar features. Upon histological examination, the aortic aneurysm as well as the other arteries sampled showed mucoid degeneration, excess mucopolysaccharides and pools of mucin inside the intima and the media associated with collagen and elastic fiber destruction and loss of smooth muscle cells. This pattern strongly suggested the diagnosis of intimomedial mucoid degeneration (IMMD), a rare arterial disorder consisting of a progressive deposition of mucin into the intima and media, with a strong prevalence in middle-aged black African females with high blood pressure. In addition to the typical features of IMMD, histological examination of the ascending aorta showed a thickening of the adventita with sparse mixed inflammatory infiltrates and fibrosis, suggesting an additional chronic infectious aortitis. No infectious agent was detected. The body of literature on IMMD is reviewed and the origin of death is discussed in this case report.

Context: Crataegus aronia (Willd.) Bosc (Rosaceae) (syn. Azarolus L) is traditionally used to treat cardiovascular disorders., Objectives: To investigate C. aronia protection against a high-fat diet (HFD)-induced vascular inflammation in rats., Materials and Methods: Wistar Male rats (180-220 g) were divided (n = 10/group) as control fed a standard diet (STD), STD + C. aronia (200 mg/kg, orally), HFD, HFD + C. aronia and HFD post-treated with C. aronia. Simvastatin (20 mg/kg) was co- or post-administered as a positive control drug. HFD was given for 8 weeks, and all other treatments were administered for 4 weeks., Results: Most significantly, co-administration of C. aronia to HFD-fed rats reduced the thickness of aorta tunica media (90 ± 5 vs. 160 ± 11.3 µm) and adventitia (54.3 ± 3.8 vs. 93.6 ± 9.4 µm). It also lowered protein levels of TNF-α (0.51 ± 0.15 and 0.15 ± 0.16 vs. 0.1 ± 0.09%) and IL-6 (0.52 ± 0.19 vs. 1.0 ± 0.2%) in their aorta or serum (5.9 ± 0.91 vs. 12.98 ± 1.3 ng/mL and 78.1 ± 6.7 vs. 439 ± 78 pg/mL, respectively). It also lowered all serum lipids and increased aorta levels of GSH levels (70.4 ± 4.0 vs. 40.7 µM) and activity of SOD (5.7 ± 0.7 vs. 2.9 ± 0.6 U/mg) and decreased serum levels of ox-LDL-c (566.7 ± 46 vs. 1817 ± 147 ng/mL). Such effects were more profound than all other treatments., Conclusions: C. aronia inhibits the HFD-induced vascular inflammation and its use in clinical trials is recommended.

Aortic aneurysms are associated with fatal aortic rupture. Current therapeutic approaches are limited to implantation of aortic prostheses and stent-grafts; no effective drugs are available because the pathogenic mechanisms of aortic aneurysms remain unclear. Here, we aimed to elucidate the molecular mechanisms of the initiation and progression of aortic aneurysm by lipidomics. We performed lipidomics analyses of lipids in the aortic media of normal, border, and aneurysm areas from patients with thoracic atherosclerotic aortic aneurysm (N = 30), thoracic nonatherosclerotic aortic aneurysm (N = 19), and abdominal atherosclerotic aortic aneurysm (N = 11) and from controls (N = 8) using liquid chromatography and mass spectrometry. Significant alterations were observed in the lipid profiles of patients with atherosclerotic aortic aneurysms and to a lesser extent in those with nonatherosclerotic aneurysms. Increased triacylglycerols (TGs) and decreased ether-type phosphatidylethanolamines (ePEs) were observed throughout the normal, border, and aneurysm areas of thoracic and abdominal atherosclerotic aortic aneurysms. Prostaglandin D 2 increased, but ePEs and TGs decreased in normal areas of thoracic atherosclerotic aortic aneurysms and thoracic nonatherosclerotic aortic aneurysms compared with the control tissues. These findings expand our knowledge of metabolic changes in aortic aneurysms and provide insights into the pathophysiology of aortic aneurysms.

Arterial medial calcification (AMC) is the deposition of calcium phosphate mineral, often as hydroxyapatite, in the medial layer of the arteries. AMC shares some similarities to skeletal mineralisation and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) towards an osteoblast-like phenotype. This study used primary mouse VSMCs and calvarial osteoblasts to directly compare the established and widely used in vitro models of AMC and bone formation. Significant differences were identified between osteoblasts and calcifying VSMCs. First, osteoblasts formed large mineralised bone nodules that were associated with widespread deposition of an extracellular collagenous matrix. In contrast, VSMCs formed small discrete regions of calcification that were not associated with collagen deposition and did not resemble bone. Second, calcifying VSMCs displayed a progressive reduction in cell viability over time (≤7-fold), with a 50% increase in apoptosis, whereas osteoblast and control VSMCs viability remained unchanged. Third, osteoblasts expressed high levels of alkaline phosphatase (TNAP) activity and TNAP inhibition reduced bone formation by to 90%. TNAP activity in calcifying VSMCs was ∼100-fold lower than that of bone-forming osteoblasts and cultures treated with β-glycerophosphate, a TNAP substrate, did not calcify. Furthermore, TNAP inhibition had no effect on VSMC calcification. Although, VSMC calcification was associated with increased mRNA expression of osteoblast-related genes (e.g. Runx2, osterix, osteocalcin, osteopontin), the relative expression of these genes was up to 40-fold lower in calcifying VSMCs versus bone-forming osteoblasts. In summary, calcifying VSMCs in vitro display some limited osteoblast-like characteristics but also differ in several key respects: 1) their inability to form collagen-containing bone; 2) their lack of reliance on TNAP to promote mineral deposition; and, 3) the deleterious effect of calcification on their viability., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Ribosome biogenesis is a crucial biological process related to cell proliferation, redox balance, and muscle contractility. Aortic smooth muscle cells (ASMCs) show inhibition of proliferation and apoptosis, along with high levels of oxidative stress in aortic dissection (AD). Theoretically, ribosome biogenesis should be enhanced in the ASMCs at its proliferative state but suppressed during apoptosis and oxidative stress. However, the exact status and role of ribosome biogenesis in AD are unknown. We therefore analyzed the expression levels of BOP1, a component of the PeBoW complex which is crucial to ribosome biogenesis, in AD patients and a murine AD model and its influence on the ASMCs. BOP1 was downregulated in the aortic tissues of AD patients compared to healthy donors. In addition, overexpression of BOP1 in human aortic smooth muscle cells (HASMCs) inhibited apoptosis and accumulation of p53 under hypoxic conditions, while knockdown of BOP1 decreased the protein synthesis rate and motility of HASMCs. The RNA polymerase I inhibitor cx-5461 induced apoptosis, ROS production, and proliferative inhibition in the HASMCs, which was partly attenuated by p53 knockout. Furthermore, cx-5461 aggravated the severity of AD in vivo , but a p53-/- background extended the life-span and lowered AD incidence in the mice. Taken together, decreased ribosome biogenesis in ASMCs resulting in p53-dependent proliferative inhibition, oxidative stress, and apoptosis is one of the underlying mechanisms of AD.

Atherosclerosis is an inflammatory disease involving dysfunction of endothelial cells (EC) and enhanced permeability of the endothelium to oxidized low-density lipoprotein and the transmigration of monocytes from the blood to the intima where they are transformed into foam cells after lipid engulfment. Changes in the composition of the basement membrane leading to increased fibronectin deposition also occur and modify EC-extracellular matrix (ECM) mechanotransduction. The release of lipids due to foam cell apoptosis, as well as the migration of vascular smooth muscle cells from the media to the intima and their proliferation, increase the stiffness of arteries at later stages of atherosclerosis. EC dysfunction also involves other factors, including soluble cytokines and growth factors (GF) such as bone morphogenetic proteins (BMP). BMP-9 is a potent circulatory GF which has been shown to affect EC behavior. However, to date, few studies have investigated its role in atherosclerosis. The present review describes the histology and homeostasis of arteries by explaining EC function/dysfunction and discusses BMP-9 effect on EC behavior, considering factors engaged in the development of atherosclerosis.

Changes in extracellular matrix proteins may contribute significantly to the adaptation of vein grafts to the arterial circulation. We examined the production and distribution of versican and hyaluronan in intact human vein rings cultured ex vivo, veins perfused ex vivo, and cultured venous adventitial and smooth muscle cells. Immunohistochemistry revealed higher levels of versican in the intima/media compared to the adventitia, and no differences in hyaluronan. In the vasa vasorum, versican and hyaluronan associated with CD34+ progenitor cells. Culturing the vein rings for 14 days revealed increased versican immunostaining of 30-40% in all layers, with no changes in hyaluronan. Changes in versican accumulation appear to result from increased synthesis in the intima/media and decreased degradation in the adventitia as versican transcripts were increased in the intima/media, but unchanged in the adventitia, and versikine (the ADAMTS-mediated cleavage product of versican) was increased in the intima/media, but decreased in the adventitia. In perfused human veins, versican was specifically increased in the intima/media in the presence of venous pressure, but not with arterial pressure. Unexpectedly, cultured adventitial cells express and accumulate more versican and hyaluronan than smooth muscle cells. These data demonstrate a differential regulation of versican and hyaluronan in human venous adventitia vs. intima/media and suggest distinct functions for these extracellular matrix macromolecules in these venous wall compartments during the adaptive response of vein grafts to the arterial circulation., Competing Interests: The authors have declared that no competing interests exist.

Arterial media calcification is associated with diabetes mellitus. Previous studies have shown that advanced glycation end products (AGEs) are responsible for vascular smooth muscle cell (VSMC) calcification, but the underlying mechanisms remain unclear. Hypoxia-inducible factor-1α (HIF-1α), one of the major factors during hypoxia, and pyruvate dehydrogenase kinase 4 (PDK4), an important mitochondrial matrix enzyme in cellular metabolism shift, have been reported in VSMC calcification. The potential link among HIF-1α, PDK4, and AGEs-induced vascular calcification was investigated in this study. We observed that AGEs elevated HIF-1α and PDK4 expression levels in a dose-dependent manner and that maximal stimulation was attained at 24 h. Two important HIF-1α-regulated genes, vascular endothelial growth factor A (VEGFA) and glucose transporter 1 (GLUT-1), were significantly increased after AGEs exposure. Stabilization or nuclear translocation of HIF-1α increased PDK4 expression. PDK4 inhibition attenuated AGEs-induced VSMC calcification, which was evaluated by measuring the calcium content, alkaline phosphatase (ALP) activity and runt-related transcription factor 2 (RUNX2) expression levels and by Alizarin red S staining. In addition, the glucose consumption, lactate production, key enzymes of glucose metabolism and oxygen consumption rate (OCR) were decreased during AGEs-induced VSMC calcification. In conclusion, this study suggests that AGEs accelerate vascular calcification partly through the HIF-1α/PDK4 pathway and suppress glucose metabolism.

Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are the predominant gelatinases in the developing lung. Studies have shown that the expression of MMP-2 and MMP-9 is upregulated in hypoxic fibroblasts, 15-hydroxyeicosatetraenoic acid (15-HETE) regulated fibroblasts migration via modulating MMP-2 or MMP-9, and that hypoxia/15-HETE is a predominant contributor to the development of pulmonary arterial hypertension (PAH) through increased angiogenesis. However, the roles of MMP-2 and MMP-9 in pulmonary arterial endothelial cells (PAECs) angiogenesis as well as the molecular mechanism of hypoxia-regulated MMP-2 and MMP-9 expression have not been identified. The aim of this study was to investigate the role of MMP-2 and MMP-9 in PAEC proliferation and vascular angiogenesis and to determine the effects of hypoxia-induced 15-HETE on the expression of MMP-2 and MMP-9. Western blot, immunofluorescence, and real-time PCR were used to measure the expression of MMP-2 and MMP-9 in hypoxic PAECs. Immunohistochemical staining, flow cytometry, and tube formation as well as cell proliferation, viability, scratch-wound, and Boyden chamber migration assays were used to identify the roles and relationships between MMP-2, MMP-9, and 15-HETE in hypoxic PAECs. We found that hypoxia increased MMP-2 and MMP-9 expression in pulmonary artery endothelium both in vivo and in vitro in a time-dependent pattern. Moreover, administration of the MMP-2 and MMP-9 inhibitor MMI-166 significantly reversed hypoxia-induced increases in right ventricular systemic pressure (RVSP), right ventricular function, and thickening of the tunica media. Furthermore, up-regulation of MMP-2 and MMP-9 expression was induced by 15-HETE, which regulates PAEC proliferation, migration, and cell cycle transition that eventually leads to angiogenesis. Our study demonstrated that hypoxia increases the expression of MMP-2 and MMP-9 through the 15-lipoxygenase/15-HETE pathway, and that MMP-2 and MMP-9 promote PAEC angiogenesis. These findings suggest that MMP-2 and MMP-9 may serve as new potential therapeutic targets for the treatment of PAH., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: In the development of hypertensive microvascular remodeling, a relevant role may be played by changes in extracellular matrix proteins. Aim of this study was the to evaluate some extracellular matrix components within the tunica media of subcutaneous small arteries in 9 normotensive subjects and 12 essential hypertensive patients, submitted to a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region., Patients and Methods: Subcutaneous small resistance arteries were dissected and mounted on an isometric myograph, and the tunica media to internal lumen ratio was measured. In addition, fibronectin, laminin, transforming growth factor-beta-1 (TGF-β1) and emilin-1 contents within the tunica media were evaluated by immunofluorescence and relative immunomorphometrical analysis (immunopositivity % of area). The total collagen content and collagen subtypes within the tunica media were evaluated using both Sirius red staining (under polarized light) and immunofluorescence assay., Results: Normotensive controls had less total and type III collagen in respect with hypertensive patients. Fibronectin and TGF-β1 tunica media content was significantly greater in essential hypertensive patients, compared with normotensive controls, while laminin and emilin-1 tunica media content was lesser in essential hypertensive patients, compared with normotensive controls. A significant correlation was observed between fibronectin tunica media content and media to lumen ratio., Conclusions: Our results indicate that, in small resistance arteries of patients with essential hypertension, a relevant fibrosis may be detected; fibronectin and TGF-β1 tunica media content is increased, while laminin and emilin-1 content is decreased; these changes might be involved in the development of small resistance artery remodeling in humans.

Though the composition of the three layers constituting the blood vessel wall varies among the different types of blood vessels, and some layers may even be missing in capillaries, certain basic components, and properties are shared by all blood vessels, though each histologically distinct layer contains a unique complement of extracellular components, growth factors and cytokines, and cell types as well. The structure and composition of vessel layers informs and is informed by the function of the particular blood vessel. The adaptation of the composition and the resulting function of the extracellular matrix (ECM) to changes in circulation/blood flow and a variety of other extravascular stimuli can be characterized as remodeling spearheaded by vascular cells. There is a surprising amount of cell traffic among the three layers. It starts with endothelial cell mediated transmigration of inflammatory cells from the bloodstream into the subendothelium, and then into tissue adjoining the blood vessel. Smooth muscle cells and a variety of adventitial cells reside in tunica media and tunica externa, respectively. The latter cells are a mixture of progenitor/stem cells, fibroblasts, myofibroblasts, pericytes, macrophages, and dendritic cells and respond to endothelial injury by transdifferentiation as they travel into the two inner layers, intima and media for corrective mission in the ECM composition. This chapter addresses the role of various vascular cell types and ECM components synthesized by them in maintenance of normal structure and in their contribution to major pathological processes, such as atherosclerosis, organ fibrosis, and diabetic retinopathy., (© 2018 Elsevier Inc. All rights reserved.)

Background and Aims: In-stent restenosis (ISR) remains a major limitation of percutaneous coronary intervention despite improvements in stent design and pharmacological agents, whereas the mechanism of ISR has not been fully clarified. In the present study, we sought to investigate the potential association of serum soluble TREM-1 (sTREM-1) levels with the incidence of ISR. The role of TREM-1 was evaluated in cultured vascular smooth muscle cells (VSMCs)., Methods: Out of 1683 patients undergoing coronary intervention and follow-up coronary angiography after approximately one year, 130 patients were diagnosed with ISR, and 150 gender- and age-matched patients with no ISR were randomly included as controls. Levels of sTREM-1 were determined by ELISA. The role of TREM-1 signaling in the activation of VSMCs was tested., Results: Serum sTREM-1 concentrations were significantly elevated in patients with than without ISR. Multivariable logistic regression analysis showed that sTREM-1, besides conventional factors, was independently associated with the incidence of ISR. Evident expression of TREM-1 in VSMCs was detected in the neointimal and medial layers of stenotic lesions of mouse carotid ligation models. In cultured VSMCs, expression of TREM-1 was significantly induced upon exposure to lipopolysaccharide. Blocking of TREM-1 with a synthetic inhibitory peptide LP17 dramatically inhibited, whereas TREM-1-activating antibody promoted cellular inflammation, proliferation and migration in VSMCs., Conclusions: These data suggest that TREM-1 is a predictive biomarker of ISR and an important mediator of cellular inflammation, migration, and proliferation in VSMCs. Pharmacological inhibition of TREM-1 may serve as a promising approach to attenuate the progression of ISR., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: Congenital bicuspid aortic valve (BAV) is distinctly associated with the development of ascending aortopathy in adulthood, portending risk of both ascending aortic aneurysm and dissection. Our previous work implicated deficiency in oxidative stress response as a mediator of the BAV-associated aortopathy. We hypothesize that reactive oxygen species generation invokes elevated local oxidative tissue damage in ascending aorta of patients with BAV., Methods: Ascending aortic specimens were obtained from patients undergoing elective aortic replacement and/or aortic valve replacement and during heart transplant operations. Levels of superoxide anion were measured via high-pressure liquid chromatography-based detection of 2-hydroxyethidium in aortic specimens. Lipid peroxidation and enzymatic activity of superoxide dismutase and peroxidase were quantified in aortic specimens., Results: Superoxide anion production was elevated in aortic specimens from patients with nonaneurysmal BAV (n = 59) compared with specimens from patients with the morphologically normal tricuspid aortic valve (TAV, n = 38). Total superoxide dismutase activity was similar among aortic specimens from patients with TAV versus BAV (n = 27 and 26, respectively), whereas peroxidase activity was increased in aortic specimens from patients with BAV compared with specimens from patients with TAV (n = 14 for both groups). Lipid peroxidation was elevated in aortic specimens from BAV patients compared with TAV patients (n = 14 and 11, respectively)., Conclusions: Superoxide anion accumulation and increased lipid peroxidation demonstrate that, despite increased peroxidase activity, the ascending aortopathy of patients with BAV involves oxidative stress. In addition, the absence of increased superoxide dismutase activity in BAV specimens indicates a deficiency in antioxidant defense. This suggests that the characteristic smooth muscle cell loss observed in BAV aortopathy may be a consequence of superoxide-mediated cell damage., (Copyright © 2017. Published by Elsevier Inc.)

Objective: The role of TGF-β (transforming growth factor-β) signaling in abdominal aortic aneurysm (AAA) formation is controversial. Others reported that systemic blockade of TGF-β by neutralizing antibodies accelerated AAA development in angiotensin II-infused mice. This result is consistent with other studies suggesting that TGF-β signaling prevents AAA. Development of a therapy for AAA that exploits the protective actions of TGF-β would be facilitated by identification of the mechanisms through which TGF-β prevents AAA. We hypothesized that TGF-β signaling prevents AAA by its actions on aortic medial smooth muscle cells., Approach and Results: We compared the prevalence, severity, and histopathology of angiotensin II-induced AAA among control mice (no TGF-β blockade), mice with antibody-mediated systemic neutralization of TGF-β, and mice with genetically based smooth muscle-specific loss of TGF-β signaling. Surprisingly, we found that systemic-but not smooth muscle-specific-TGF-β blockade significantly increased the prevalence of AAA and tended to increase AAA severity, adventitial thickening, and aortic wall macrophage accumulation. In contrast, abdominal aortas of mice with smooth muscle-specific loss of TGF-β signaling differed from controls only in having a thinner media. We examined thoracic aortas of the same mice. Here we found that smooth muscle-specific loss of Tgfbr2 -but not systemic TGF-β neutralization-significantly accelerated development of aortic pathology, including increased prevalence of intramural hematomas, medial thinning, and adventitial thickening., Conclusion: Our results suggest that TGF-β signaling prevents both abdominal and thoracic aneurysmal disease but does so by distinct mechanisms. Smooth muscle extrinsic signaling protects the abdominal aorta and smooth muscle intrinsic signaling protects the thoracic aorta., (© 2017 American Heart Association, Inc.)

Objective: Smooth muscle cells (SMCs) of the proximal thoracic aorta are embryonically derived from the second heart field (SHF) and cardiac neural crest (CNC). However, distributions of these embryonic origins are not fully defined. The regional distribution of SMCs of different origins is speculated to cause region-specific aortopathies. Therefore, the aim of this study was to determine the distribution of SMCs of SHF and CNC origins in the proximal thoracic aorta., Approach and Results: Mice with repressed LacZ in the ROSA26 locus were bred to those expressing Cre controlled by either the Wnt1 or Mef2c (myocyte-specific enhancer factor 2c) promoter to trace CNC- and SHF-derived SMCs, respectively. Thoracic aortas were harvested, and activity of β-galactosidase was determined. Aortas from Wnt1- Cre mice had β-galactosidase-positive areas throughout the region from the proximal ascending aorta to just distal of the subclavian arterial branch. Unexpectedly, β-galactosidase-positive areas in Mef2c- Cre mice extended from the aortic root throughout the ascending aorta. This distribution occurred independent of sex and aging. Cross and sagittal aortic sections demonstrated that CNC-derived cells populated the inner medial aspect of the anterior region of the ascending aorta and transmurally in the media of the posterior region. Interestingly, outer medial cells throughout anterior and posterior ascending aortas were derived from the SHF. β-Galactosidase-positive medial cells of both origins colocalized with an SMC marker, α-actin., Conclusions: Both CNC- and SHF-derived SMCs populate the media throughout the ascending aorta. The outer medial cells of the ascending aorta form a sleeve populated by SHF-derived SMCs., (© 2017 American Heart Association, Inc.)

Purpose: Bone metabolism disorder is often associated with cardiovascular calcification in patients with chronic kidney disease (CKD). Sclerostin, a novel candidate protein, has been identified to be involved in the bone-vascular axis. The aims of the current investigation were to assess vessel sclerostin expression and its relationship with circulating sclerostin levels., Methods: A cross-sectional observational study was conducted from January 2012 to December 2014. Thirty-two predialysis patients with CKD stage 5 who received arteriovenous fistula (AVF) operations were enrolled in this study. Radial arteries were collected and paraffin-embedded during the AVF operation, followed by immunohistochemical staining for sclerostin expression. In addition, serum sclerostin levels were measured by the enzyme-linked immunosorbent assay., Results: The prevalence of positive sclerostin staining in the radial arteries was 56.25%. Sclerostin expression was localized in the artery media layer. Serum sclerostin levels in patients with positive sclerostin expression were much higher than in those with negative expression (p = 0.018). Multivariate logistic regression analyses including potential confounders as age, gender, systolic blood pressure (BP), diastolic BP, serum sclerostin, corrected calcium (Ca), phosphate (P), Ca × P product, alkaline phosphatase, intact parathyroid hormone, and estimated glomerular filtration rate showed that only serum sclerostin levels were closely related to vessel sclerostin expression (p = 0.025). The area under the curve of serum sclerostin levels for predicting positive vessel sclerostin expression was 0.742 with 61.1% sensitivity and 85.7% specificity (p  = 0.008). The cutoff point for vessel sclerostin expression of serum sclerostin was 1591.53 pg/mL., Conclusions: Positive expression of sclerostin in the radial artery media layer was related to high serum sclerostin levels. Sclerostin may act as both a local and systemic regulator involved in vascular calcification.

Background: Vascular endothelium induces smooth muscle cell (SMC) relaxation mainly mediated by endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF). It has previously been reported that functions of these endothelium factors have been greatly impaired in vein grafts. The present study was undertaken to determine whether the functions of EDNO and EDHF might be altered in artery graft.Methods and Results:In rabbits, the right carotid artery was excised and implanted in its original position as an autogenous graft ("artery graft") and the non-operated left carotid artery served as the "control artery". Histochemical changes, acetylcholine (ACh)-induced effects on the intracellular concentration of Ca 2+ ([Ca 2+ ] i ) in endothelial cells, endothelium-dependent SMC hyperpolarization and relaxation, and tissue cGMP content were examined on post-operative day 28. "Artery graft" displayed a minimal amount of intimal hyperplasia. When compared with the "control artery", it exhibited greater ACh-induced, endothelium-dependent relaxation, but the reverse was true when EDNO production was blocked. In the "artery graft" (vs. the "control artery"), basal cGMP content was greater, whereas the [Ca 2+ ] i increase in endothelial cells and the endothelium-dependent SMC-hyperpolarization induced by ACh were less., Conclusions: It is suggested that the [Ca 2+ ] i -independent EDNO production covers the loss of function of endothelium-dependent SMC hyperpolarization and minimizes intimal hyperplasia caused by surgical operation in autogenous carotid artery graft.

Elastin and collagen fibers play important roles in the mechanical properties of aortic media. Because knowledge of local fiber structures is required for detailed analysis of blood vessel wall mechanics, we investigated 3D microstructures of elastin and collagen fibers in thoracic aortas and monitored changes during pressurization. Using multiphoton microscopy, autofluorescence images from elastin and second harmonic generation signals from collagen were acquired in media from rabbit thoracic aortas that were stretched biaxially to restore physiological dimensions. Both elastin and collagen fibers were observed in all longitudinal-circumferential plane images, whereas alternate bright and dark layers were observed along the radial direction and were recognized as elastic laminas (ELs) and smooth muscle-rich layers (SMLs), respectively. Elastin and collagen fibers are mainly oriented in the circumferential direction, and waviness of collagen fibers was significantly higher than that of elastin fibers. Collagen fibers were more undulated in longitudinal than in radial direction, whereas undulation of elastin fibers was equibiaxial. Changes in waviness of collagen fibers during pressurization were then evaluated using 2-dimensional fast Fourier transform in mouse aortas, and indices of waviness of collagen fibers decreased with increases in intraluminal pressure. These indices also showed that collagen fibers in SMLs became straight at lower intraluminal pressures than those in EL, indicating that SMLs stretched more than ELs. These results indicate that deformation of the aorta due to pressurization is complicated because of the heterogeneity of tissue layers and differences in elastic properties of ELs, SMLs, and surrounding collagen and elastin.

Objective: Vascular calcification is the consequence of the complex interaction between genetic, environmental, and vascular factors, which ultimately lead to the deposition of calcium in the tunica intima (atherosclerotic calcification) or tunica media (Mönckenberg's sclerosis). Vascular calcification is also closely related to other pathologies, such as diabetes mellitus, dyslipidemia, and chronic kidney disease. It has been concluded that the degree of vascular calcification may vary from person to person, even if the associated pathologies and environmental factors are the same. Therefore, this suggests an important genetic contribution to the development of vascular calcification. This review aimed to find the most recent evidence about vascular calcification pathophysiology regarding the genetic aspects and molecular pathways., Data Sources: We conducted an exhaustive search in Scopus, EBSCO, and PubMed with the keywords "genetics and vascular calcification", "molecular pathways, genetic and vascular calcification" and included the main articles from January 1995 up to August 2016. We focused on the most recent evidence about vascular calcification pathophysiology regarding the genetic aspects and molecular pathways., Study Selection: The most valuable published original and review articles related to our objective were selected., Results: Vascular calcification is a multifactorial disease; thus, its pathophysiology cannot be explained by a single specific factor, rather than by the result of the association of several genetic variants, molecular pathway interactions, and environmental factors that promote its development., Conclusion: Although several molecular aspects of this mechanism have been elucidated, there is still a need for a better understanding of the factors that predispose to this disease.

The purpose. In the study we investigated the impact of the partial blood flow shutdown on structural changes in the rabbit vena cava posterior wall after exposure to high-intensity focused ultrasound (HIFU). Methods. Ultrasound Exposure: frequency of 1.65 MHz, the ultrasound intensity in the focus of 13.6 kW/cm2, the area of the focal spot 1 mm2, continuous ultrasound, exposure for 3 seconds. Results. Immediately after HIFU exposure all layers of the vein wall showed characteristic signs of thermal damage. A week after exposure structural changes in the intima, media and adventitia was minimal in the part of vessel with preserved blood flow, and after 4 weeks the changes were not revealed. A week after HIFU exposure partial endothelium destruction, destruction of myocytes, disorganization and consolidation of collagen fibers of the adventitia were observed in an isolated segment of the vessel, and in 4 weeks endothelium restored and signs of damage in media and adventitia persisted, but were less obvious than in a week after exposure. Conclusion. The shutdown of blood flow after exposure to HIFU promotes persistent changes in the vein wall. Vein compression appears to be necessary for the obliteration of the vessel, when using HIFU-technology.

Hypertension and diabetes are multifactorial diseases that frequently coexist and exacerbate each another. During the development of diabetes, the impairment of noradrenergic and renin-angiotensin systems has been reported in the response mediated by α 1 -AR and AT 1 receptors. Although their participation in the development of cardiovascular complications is still controversial, some studies have found increased or diminished response to the vasoconstrictive effect of noradrenaline or angiotensin II in a time-dependent manner of diabetes. Thus, the aim of this work was to investigate the possible changes in the expression or localization of α 1 -AR (α 1A and α 1D ) and angiotensin II receptors (AT 1 and AT 2 ) in aorta of rats after 4 weeks of the onset of diabetes. In order to be able to examine the expression of these receptors, immunofluorescence procedure was performed in tunica intima and tunica media of histological sections of aorta. Fluorescence was detected by a confocal microscopy. Our results showed that the receptors are expressed in both tunics, where adrenergic receptors have a higher density in tunica intima and tunica media of SHR compared with WKY; meanwhile, the expression of angiotensin II receptors is not modified in both groups of rats. On the other hand, the results showed that diabetes produced an increase or a decrease in the expression of receptors that is not associated to a specific type of receptor, vascular region, or strain of rat. In conclusion, diabetes and hypertension modify the expression of the receptors in tunica intima and tunica media of aorta in a different way.

Background and Objectives: Arteriovenous fistula maturation requires an increase in the diameter and blood flow of the feeding artery and the draining vein after its creation. The structural properties of the native vessels may affect the magnitude of these changes. We hypothesized that an increase in the collagen content of the vascular media (medial fibrosis) preoperatively would impair vascular dilation and thereby, limit the postoperative increase in arteriovenous fistula diameter and blood flow and clinical arteriovenous fistula maturation., Design, Setting, Participants, & Measurements: We enrolled 125 patients undergoing arteriovenous fistula creation between October of 2008 and April of 2012 and followed them prospectively. Any consenting subject was eligible. Arterial and venous specimens were sampled during arteriovenous fistula surgery. Masson's trichrome-stained samples were used to quantify medial fibrosis. Arteriovenous fistula diameter and blood flow were quantified using 6-week postoperative ultrasound. Clinical arteriovenous fistula maturation was assessed using a predefined protocol. The association of preexisting vascular medial fibrosis with arteriovenous fistula outcomes was evaluated after controlling for baseline demographics, comorbidities, and the preoperative venous diameter., Results: The mean medial fibrosis was 69%±14% in the arteries and 63%±12% in the veins. Arterial medial fibrosis was associated with greater increases in arteriovenous fistula diameter (Δdiameter =0.58 mm; 95% confidence interval [95% CI], 0.27 to 0.89 mm; P<0.001) and arteriovenous fistula blood flow (Δblood flow =85 ml/min; 95% CI, 19 to 150 ml/min; P=0.01) and a lower risk of clinical arteriovenous fistula nonmaturation (odds ratio, 0.71; 95% CI, 0.51 to 0.99; P=0.04), all per 10% absolute difference in medial fibrosis. In contrast, venous medial fibrosis was not associated with the postoperative arteriovenous fistula diameter, blood flow, or clinical maturation., Conclusions: Preoperative arterial medial fibrosis was associated with greater arteriovenous fistula diameter and blood flow and a lower risk of clinical arteriovenous fistula nonmaturation. This unexpected observation suggests that medial fibrosis promotes arteriovenous fistula development by yet undefined mechanisms or alternatively, that a third factor promotes both medial fibrosis and arteriovenous fistula maturation., (Copyright © 2016 by the American Society of Nephrology.)

Objective: To investigate the expression of Oct4 in human thoracic aortic dissection (TAD) and the regulation mechanisms of Oct4 on phenotype transition of human aortic smooth muscle cells (HASMCs)., Methods: Aortic samples from TAD patients (n = 12) and organ donors (n = 6) were collected. qRT-PCR, western blot, and immunohistochemistry were performed to identify Oct4 expression in aortic media. Immunofluorescence was performed to analyze Oct4 expression in primary HASMCs. Oct4A and Oct4B isoforms were detected. Gain-of-function experiments were performed to determine the effects of Oct4 on HASMC phenotype transition. Chromatin immunoprecipitation, luciferase assay, and rescue experiments were performed to analyze mechanisms of Oct4 on HASMC phenotype transition., Results: Oct4 expression levels, especially the Oct4A isoform, were significantly higher in TAD patients compared with normal controls. Notably, Oct4 presented a strong and strict nuclear localization in primary HASMCs of TAD patients but a mild and diffuse distribution in both cytoplasm and nucleus in the control group. Overexpression of Oct4 induced dedifferentiation of HASMCs characterized by decreased contractile proteins and elevated migration capability. Krüppel-like factor 5 (KLF5) was found to be a directly regulated target gene of Oct4 in HASMCs. Furthermore, downregulation of KLF5 significantly alleviated the effects of Oct4 on phenotype transition of HASMCs., Conclusions: Oct4 expression was significantly upregulated in aortic tissues and primary HASMCs of TAD patients. The increased Oct4 induced phenotype transition of HASMCs from the contractile type to the synthetic type by directly upregulating KLF5., (Copyright © 2016 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Hypertension, which is a risk factor of heart failure, provokes adaptive changes at the vasculature and cardiac levels. Notch3 signaling plays an important role in resistance arteries by controlling the maturation of vascular smooth muscle cells. Notch3 deletion is protective in pulmonary hypertension while deleterious in arterial hypertension. Although this latter phenotype was attributed to renal and cardiac alterations, the underlying mechanisms remained unknown. To investigate the role of Notch3 signaling in the cardiac adaptation to hypertension, we used mice with either constitutive Notch3 or smooth muscle cell-specific conditional RBPJκ knockout. At baseline, both genotypes exhibited a cardiac arteriolar rarefaction associated with oxidative stress. In response to angiotensin II-induced hypertension, the heart of Notch3 knockout and SM-RBPJκ knockout mice did not adapt to pressure overload and developed heart failure, which could lead to an early and fatal acute decompensation of heart failure. This cardiac maladaptation was characterized by an absence of media hypertrophy of the media arteries, the transition of smooth muscle cells toward a synthetic phenotype, and an alteration of angiogenic pathways. A subset of mice exhibited an early fatal acute decompensated heart failure, in which the same alterations were observed, although in a more rapid timeframe. Altogether, these observations indicate that Notch3 plays a major role in coronary adaptation to pressure overload. These data also show that the hypertrophy of coronary arterial media on pressure overload is mandatory to initially maintain a normal cardiac function and is regulated by the Notch3/RBPJκ pathway., (© 2016 American Heart Association, Inc.)

Incorporation of collagen structural information into the study of biomechanical behavior of ascending thoracic aortic (ATA) wall tissue should provide better insight into the pathophysiology of ATA. Structurally motivated constitutive models that include fiber dispersion and recruitment can successfully capture overall mechanical response of the arterial wall tissue. However, these models cannot examine local microarchitectural features of the collagen network, such as the effect of fiber disruptions and interaction between fibrous and non-fibrous components, which may influence emergent biomechanical properties of the tissue. Motivated by this need, we developed a finite element based three-dimensional structural model of the lamellar units of the ATA media that directly incorporates the collagen fiber microarchitecture. The fiber architecture was computer generated utilizing network features, namely fiber orientation distribution, intersection density and areal concentration, obtained from image analysis of multiphoton microscopy images taken from human aneurysmal ascending thoracic aortic media specimens with bicuspid aortic valve (BAV) phenotype. Our model reproduces the typical J-shaped constitutive response of the aortic wall tissue. We found that the stress state in the non-fibrous matrix was homogeneous until the collagen fibers were recruited, but became highly heterogeneous after that event. The degree of heterogeneity was dependent upon local network architecture with high stresses observed near disrupted fibers. The magnitude of non-fibrous matrix stress at higher stretch levels was negatively correlated with local fiber density. The localized stress concentrations, elucidated by this model, may be a factor in the degenerative changes in aneurysmal ATA tissue., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Deposition of amyloid β (Aβ) in the walls of cerebral arteries as cerebral amyloid angiopathy (CAA) suggests an age-related failure of perivascular drainage of soluble Aβ from the brain. As CAA is associated with Alzheimer's disease and with intracerebral haemorrhage, the present study determines the unique sequence of changes that occur as Aβ accumulates in artery walls. Paraffin sections of post-mortem human occipital cortex were immunostained for collagen IV, fibronectin, nidogen 2, Aβ and smooth muscle actin and the immunostaining was analysed using Image J and confocal microscopy. Results showed that nidogen 2 (entactin) increases with age and decreases in CAA. Confocal microscopy revealed stages in the progression of CAA: Aβ initially deposits in basement membranes in the tunica media, replaces first the smooth muscle cells and then the connective tissue elements to leave artery walls completely or focally replaced by Aβ. The pattern of development of CAA in the human brain suggests expansion of Aβ from the basement membranes to progressively replace all tissue elements in the artery wall. Establishing this full picture of the development of CAA is pivotal in understanding the clinical presentation of CAA and for developing therapies to prevent accumulation of Aβ in artery walls. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Scavenging of nitric oxide (NO) often interferes with studies on NO signaling in cell-free preparations. We observed that formation of cGMP by NO-stimulated purified soluble guanylate cyclase (sGC) was virtually abolished in the presence of cytosolic preparations of porcine coronary arteries, with the scavenging activity localized in the tunica media (smooth muscle layer). Electrochemical measurement of NO release from a donor compound and light absorbance spectroscopy showed that cytosolic preparations contained a reduced heme protein that scavenged NO. This protein, which reacted with anti-human hemoglobin antibodies, was efficiently removed from the preparations by haptoglobin affinity chromatography. The cleared cytosols showed only minor scavenging of NO according to electrochemical measurements and did not decrease cGMP formation by NO-stimulated sGC. In contrast, the column flow-through caused a nearly 2-fold increase of maximal sGC activity (from 33.1 ± 1.6 to 54.9 ± 2.2 μmol × min(-1) × mg(-1)). The proteins retained on the affinity column were identified as hemoglobin α and β subunits. The results indicate that hemoglobin, presumably derived from vasa vasorum erythrocytes, is present and scavenges NO in preparations of porcine coronary artery smooth muscle. Selective removal of hemoglobin-mediated scavenging unmasked stimulation of maximal NO-stimulated sGC activity by a soluble factor expressed in vascular tissue., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Vascular calcification (VC) is a critical complication in patients with chronic kidney disease (CKD). The effects of spironolactone (SPL), a mineralocorticoid receptor (MR) antagonist, on VC have not been fully investigated in CKD. The present in vivo study determined the protective effects of SPL on VC in CKD rats. Rats were divided into a control group and four groups of rats with adenine-induced CKD. Three groups were treated with 0, 50, and 100 mg·kg(-1)·day(-1) SPL for 8 wk, and one group was treated with 100 mg·kg(-1)·day(-1) SPL for the last 2 wk of the 8-wk treatment period. After 8 wk, CKD rats developed azotemia and hyperphosphatemia, with increases in the expression of serum and glucocorticoid-regulated kinase-1 and sodium-phosphate cotransporter, in inflammation and oxidative stress level, in osteogenic signaling and apoptosis, and in aortic calcification, compared with control rats. SPL dose dependently decreased these changes in the aortas, concomitant with improvements in renal inflammation, tubulointerstitial nephritis, and kidney function. SPL neither lowered blood pressure level nor induced hyperkalemia. Treatment of CKD rats for the last 2 wk with 100 mg·kg(-1)·day(-1) SPL attenuated VC compared with CKD rats with the same degree of kidney function and hyperphosphatemia. In conclusion, SPL dose dependently inhibits the progression of VC by suppressing MR signaling, local inflammation, osteogenic transition, and apoptosis in the aortas of CKD rats., (Copyright © 2015 the American Physiological Society.)

Objective: Angiotensin II (Ang II) infusion causes aortic medial thickening via stimulation of angiotensin II type 1a (AT1a) receptors. The purpose of this study was to determine the cellular loci of AT1a receptors that mediate this Ang II-induced aortic pathology., Approach and Results: Saline or Ang II was infused into AT1a receptor floxed mice expressing Cre under control of cell-specific promoters. Initially, AT1a receptors were depleted in aortic smooth muscle cell and endothelium by expressing Cre under control of SM22 and Tie2 promoters, respectively. Deletion of AT1a receptors in either cell type had no effect on Ang II-induced medial thickening. To determine whether this effect was related to neural stimulation, AT1a receptors were depleted using an enolase 2-driven Cre. Depletion of AT1a receptors in neural cells attenuated Ang II-induced medial thickening of the ascending, but not descending aorta. Lineage tracking studies, using ROSA26-LacZ, demonstrated that enolase 2 was also expressed in adventitial cells adjacent to the region of attenuated thickening. To determine whether adventitial fibroblasts contributed to this attenuation, AT1a receptors in fibroblasts were depleted using S100A4 driven Cre. Similar to enolase 2-Cre, Ang II-induced medial thickening was attenuated in the ascending, but not the descending aorta. Lineage tracking demonstrated an increase of S100A4-LacZ positive cells in the media of the ascending region during Ang II infusion., Conclusions: AT1a receptor depletion in fibroblasts attenuates Ang II-induced medial hyperplasia in the ascending aorta., (© 2015 American Heart Association, Inc.)

MicroRNA (miR)-145 is the most abundant miR in vascular smooth muscle cells (VSMCs). However, the effect of hyperglycemia on the regulation of miR-145 is unknown. We hypothesized that the hyperglycemic condition activates a proinflammatory response that mediates the expression of miR-145 in VSMCs. We investigated whether miR-145 serves as a critical regulator to regulate the downstream proliferation factors (including Kruppel-like factor 4 [Klf4] and myocardin) in VSMCs under hyperglycemic conditions. Human coronary artery smooth muscle cells (HCASMCs) were cultured under high glucose conditions. Sustained high glucose at 25 mmol/L significantly decreased the expression of miR-145 in HCASMCs. High glucose significantly increased angiotensin II (Ang II) secretion from HCASMCs and Ang II suppressed miR-145 expression in HCASMCs. Ang II repression of miR145 expression resulted in increased Klf4 and decreased myocardin expression under conditions of high glucose. Overexpression of miR-145 significantly decreased Klf4 and increased myocardin expression and inhibited HCASMC proliferation and migration induced by a high glucose state. Balloon injury of the carotid artery in diabetic rats was performed to investigate miR-145, Klf and myocardin expression. The expression of miR-145 was maximally increased at 7 d after carotid injury and gradually declined thereafter. Overexpression of miR-145 and treatment with valsartan reversed Klf4 and myocardin protein expression induced by balloon injury and improved vascular injury. In conclusion, our study reveals that Ang II downregulates miR-145 to regulate Klf4 and myocardin expression in HCASMCs under high glucose conditions. Ang II plays a critical role in the regulation of miR-145 under hyperglycemic conditions.

Drug-eluting stents (DES), which release anti-proliferative drugs into the arterial wall in a controlled manner, have drastically reduced the rate of in-stent restenosis and revolutionized the treatment of atherosclerosis. However, late stent thrombosis remains a safety concern in DES, mainly due to delayed healing of the endothelial wound inflicted during DES implantation. We present a framework to optimize DES design such that restenosis is inhibited without affecting the endothelial healing process. To this end, we have developed a computational model of fluid flow and drug transport in stented arteries and have used this model to establish a metric for quantifying DES performance. The model takes into account the multi-layered structure of the arterial wall and incorporates a reversible binding model to describe drug interaction with the cells of the arterial wall. The model is coupled to a novel optimization algorithm that allows identification of optimal DES designs. We show that optimizing the period of drug release from DES and the initial drug concentration within the coating has a drastic effect on DES performance. Paclitaxel-eluting stents perform optimally by releasing their drug either very rapidly (within a few hours) or very slowly (over periods of several months up to one year) at concentrations considerably lower than current DES. In contrast, sirolimus-eluting stents perform optimally only when drug release is slow. The results offer explanations for recent trends in the development of DES and demonstrate the potential for large improvements in DES design relative to the current state of commercial devices.

Objective: The dynamic expressions of monocyte chemo attractant protein-1 (MCP-1) and CC chamomile receptor 2 (CCR2) after balloon injury and their effects in intimal proliferation were discussed. In this study, the expression of MCP-1 and its receptor during the intimal proliferation in rat artery after balloon injury were studied., Methods: Using the model of balloon injury of rats' arteries, the changes of intimal proliferation were observed with optical microscopy and the expressions of MCP-1 and CCR2 at different times were examined with the methods of RT-PCR and immunohistochemistry. The expressions of MCP-1 and CCR2 in the arterial tissues were detected using reverse transcription polymerase chain reaction (RT-PCR) and analyzed by semi-quantitative method., Results: The expressions of MCP-1 and CCR2 mRNA began to gradually increase after balloon injury. The MCP-1 reached to the peak on the first day, but decreased gradually later on. Expressions of CCR2 mRNA began to increase on the first day and reached to the peak on the 7th day, but then started to decrease gradually until 28th day when we can still detect it. The expressions of MCP-1 proteins began to increase gradually after balloon injury and were obviously detected in the VSMC on the 4th and 7th day, until 14th day when we can still detect it clearly in the proliferating intima., Conclusion: The dynamic expressions of MCP-1, MCP-1 proteins and CCR2 mRNA after balloon injury were shown to play an important role in intimal proliferation.