Matrix metalloproteinase (MMP) activity is tightly regulated by the endogenous tissue inhibitors (TIMPs), and dysregulated activity contributes to extracellular matrix remodelling. Accordingly, MMP/TIMP balance is associated with atherosclerotic plaque progression and instability, alongside adverse post-infarction cardiac fibrosis and subsequent heart failure. Here, we demonstrate that prolonged high-fat feeding of apolipoprotein (Apo)e-deficient mice triggered the development of unstable coronary artery atherosclerosis alongside evidence of myocardial infarction and progressive sudden death. Accordingly, the contribution of select MMPs and TIMPs to the progression of both interrelated pathologies was examined in Apoe-deficient mice with concomitant deletion of Mmp7, Mmp9, Mmp12, or Timp1 and relevant wild-type controls after 36-weeks high-fat feeding. Mmp7 deficiency increased incidence of sudden death, while Mmp12 deficiency promoted survival, whereas Mmp9 or Timp1 deficiency had no effect. While all mice harboured coronary disease, atherosclerotic burden was reduced in Mmp7-deficient and Mmp12-deficient mice and increased in Timp1-deficient animals, compared to relevant controls. Significant differences in cardiac fibrosis were only observed in Mmp-7-deficient mice and Timp1-deficient animals, which was associated with reduced capillary number. Adopting therapeutic strategies in Apoe-deficient mice, TIMP-2 adenoviral-overexpression or administration (delayed or throughout) of a non-selective MMP inhibitor (RS-130830) had no effect on coronary atherosclerotic burden or cardiac fibrosis. Taken together, our findings emphasise the divergent roles of MMPs on coronary plaque progression and associated post-MI cardiac fibrosis, highlighting the need for selective therapeutic approaches to target unstable atherosclerosis alongside adverse cardiac remodelling while negating detrimental adverse effects on either pathology, with targeting of MMP-12 seeming a suitable target.

Objective: Previously published work has indicated that transcripts encoding transglutaminase 2 (TG2) increase markedly in a rat model of abdominal aortic aneurysm. This study determines whether TG2 and the related TG, factor XIII-A (FXIII-A), protect against aortic aneurysm development in mice. Methods: C57BL/6J wild-type, Tgm2(-/-) knockout, F13a1(-/-) knockout, and Tgm2(-/-)/F13a1(-/-) double knockout mice were subjected to laparotomy and periaortic application of CaCl2. Results: Tgm2(-/-) mice showed slightly greater aortic dilatation at 6 weeks after treatment when compared with wild type. However, vessels from Tgm2(-/-) mice, but not wild-type mice, continued to dilate up to 6 months after injury and by 24 weeks, a greater number of Tgm2(-/-) mice had developed aneurysms (16/17 vs 10/19; P 1 / 4 . 008). Laparotomy resulted in a high death rate in F13a1-/- knockout mice, more frequently from cardiac complications than from hemorrhage, but among F13a1-/-mice that survived for 6 weeks after CaCl2 treatment, abdominal aortic aneurysm diameter was unaltered relative to wild-type mice. Laparotomy resulted in a higher death rate among Tgm2(-/-)/F13a1(-/-) double knockout mice, owing to an increased frequency of delayed bleeding. Surprisingly, Tgm2(-/-)/F13a1(-/-)double knockout mice showed a trend toward decreased dilatation of the aorta 6 weeks after injury, and this finding was replicated in Tgm2(-/-)/F13a1(-/-) mice subjected to carotid artery injury. Levels of transcripts encoding TG2 were not increased in the aortas of injured wild-type or F13a1(-/-) knockout mice relative to uninjured mice, although changes in the levels of other transcripts accorded with previous descriptions of the CaCl2 aneurysm model in mice. Conclusions: Knockout of Tgm2, but not F13a1 exacerbates aortic dilatation, suggesting that TG2 confers protection. However, levels of TG2 messenger RNA are not acutely elevated after injury. FXIII-A plays a role in preventing postoperative damage after laparotomy, confirming previous reports that it prevents distal organ damage after trauma. TG2 promotes wound healing after surgery and, in its absence, the bleeding diathesis associated with FXIII-A deficiency is further exposed.

Two new alloy compositions for possible disk rotor applications have been examined. Both were intended to have higher \( \gamma ^{{\prime }} \) content than the existing alloy, RR1000, and be produced using powder metallurgy and isothermal forging to enable forgings to show a consistent coarse grain microstructure. Small pancake forgings of the new alloys and RR1000 were made and from these, blanks were cut, solution heat treated, cooled at measured rates and aged. Results of screening tests to understand the tensile, creep and dwell crack growth behavior, oxidation resistance and phase stability of these new alloys and coarse grain RR1000 are reported. The development alloys were similar in composition but exhibited different tensile and creep properties, phase stability and resistance to oxidation damage. Despite attempts to minimize variation in microstructure from heat treatment, differences in \( \gamma ^{{\prime }} \) size distribution were found to influence tensile and creep behavior. One of the new alloys (Alloy 2) showed improved yield and tensile strength compared to RR1000. Alloy 2 displayed similar initial creep strain behavior to RR1000 but superior resistance to subsequent creep damage, producing longer creep rupture lives. All of the alloys showed crack retardation at low stress intensity factor ranges (ΔK) from 3600 s dwell cycles at 700 °C in air. This occurred whilst crack growth was intergranular. Alloy 1 was found to precipitate C14 Laves phase from long term exposure at 800 °C. Like RR1000, σ phase was not detected in the new alloys after 750 h at 800 °C.

Objective: Galectin-3 (formerly known as Mac-2), encoded by the LGALS3 gene, is proposed to regulate macrophage adhesion, chemotaxis, and apoptosis. We investigated the role of galectin-3 in determining the inflammatory profile of macrophages and composition of atherosclerotic plaques. Approach and Results: We observed increased accumulation of galectin-3-negative macrophages within advanced human, rabbit, and mouse plaques compared with early lesions. Interestingly, statin treatment reduced galectin-3-negative macrophage accrual in advanced plaques within hypercholesterolemic (apolipoprotein E deficient) Apoe(-/-) mice. Accordingly, compared with Lgals3(+/+):Apoe(-/-) mice, Lgals3(-/-):Apoe(-/-) mice displayed altered plaque composition through increased macrophage:smooth muscle cell ratio, reduced collagen content, and increased necrotic core area, characteristics of advanced plaques in humans. Additionally, macrophages from Lgals3(-/-) mice exhibited increased invasive capacity in vitro and in vivo. Furthermore, loss of galectin-3 in vitro and in vivo was associated with increased expression of proinflammatory genes including MMP (matrix metalloproteinase)-12, CCL2 (chemokine [C-C motif] ligand 2), PTGS2 (prostaglandin-endoperoxide synthase 2), and IL (interleukin)-6, alongside reduced TGF (transforming growth factor)-beta 1 expression and consequent SMAD signaling. Moreover, we found that MMP12 cleaves macrophage cell-surface galectin-3 resulting in the appearance of a 22-kDa fragment, whereas plasma levels of galectin-3 were reduced in Mmp12(-/-):Apoe(-/-) mice, highlighting a novel mechanism where MMP12-dependent cleavage of galectin-3 promotes proinflammatory macrophage polarization. Moreover, galectin-3-positive macrophages were more abundant within plaques of Mmp12(-/-):Apoe(-/-) mice compared with Mmp12(+/+):Apoe(-/-) animals. Conclusions: This study reveals a prominent protective role for galectin-3 in regulating macrophage polarization and invasive capacity and, therefore, delaying plaque progression.

Background and aims: Transglutaminase (TG) 2 and Factor (F) XIII-A have both been implicated in cardiovascular protection and repair. This study was designed to differentiate between two competing hypotheses: that TG2 and FXIII-A mediate these functions in mice by fulfilling separate roles, or that they act redundantly in this respect. Methods: Atherosclerosis was assessed in brachiocephalic artery plaques of fat-fed mixed strain apolipoprotein (Apo)e deficient mice that lacked either or both transglutaminases. Cardiac fibrosis was assessed both in the mixed strain mice and also in C57BL/6J Apoe expressing mice lacking either or both transglutaminases. Results: No difference was found in the density of buried fibrous caps within brachiocephalic plaques from mice expressing or lacking these transglutaminases. Cardiac fibrosis developed in both Apoe/F13a1 double knockout and F13a1 single knockout mice, but not in Tgm2 knockout mice. However, concomitant Tgm2 knockout markedly increased fibrosis, as apparent in both Apoe/Tgm2/F13a1 knockout and Tgm2/F13a1 knockout mice. Amongst F13a1 knockout and Tgm2/F13a1 knockout mice, the extent of fibrosis correlated with hemosiderin deposition, suggesting that TG2 limits the extravasation of blood in the myocardium, which in turn reduces the pro-fibrotic stimulus. The resulting fibrosis was interstitial in nature and caused only minor changes in cardiac function. Conclusions: These studies confirm that FXIII-A and TG2 fulfil different roles in the mouse myocardium. FXIII-A protects against vascular leakage while TG2 contributes to the stability or repair of the vasculature. The protective function of TG2 must be considered when designing clinical anti-fibrotic therapies based upon FXIII-A or TG2 inhibition.

The current paper describes TMF crack growth behaviour in an advanced nickel-based superalloy. Changes in behaviour are examined which occur as a function of the phase angle between applied stress and temperature. The fractography of the failed specimens reveals changes from transgranular to intergranular growth between high and low phase angle tests as a result of the onset of high temperature damage mechanisms. More targeted testing has also been undertaken to isolate the contributions of these mechanisms, with specific transitions in behaviour becoming clear in 90° diamond cycles, where dynamic crack growth and oxidation strongly interact.

Purpose: There is emerging evidence of an association between osteoarthritis (OA) and atherosclerosis, with an underlying metabolic syndrome potentially driving the progression of both diseases. Proteomic analysis of OA synovial fluid, degenerative cartilage secretome and chondrocyte membranome has recently identified lumican, chondroadherin and low-density lipoprotein (LRP-1) as potential exploratory markers of joint disease. These biomarker candidates have all been shown to be upregulated in the atherosclerotic plaque, and implicated in pathology. Statin, a classic treatment for atherosclerosis, has been shown to have disease modifying potential in OA, both in clinical and in vitro studies. The aim of this study was to confirm the presence of lumican, chondroadherin and LRP-1 with more targeted analysis, in an explant model of inflammatory cartilage degradation. In addition, we determined whether these proteins, both common to OA and atherosclerosis, are involved in the chondroprotective mechanism of statins; in order to identify novel pathways for a more refined therapeutic intervention. Methods: Macroscopically normal articular cartilage was obtained from the metacarpophalangeal joint of 18 month cows (n = 4). Bovine articular cartilage explants were cultured in serum-free media with or without species-specific pro-inflammatory cytokines, 10 ng/ml interleukin-1β and 50 ng/ml oncostatin M for 2, 4, 6, 8 and 14 days. Explants cultured for 8 and 14 days were also treated with 10, 100 or 1000 nM simvastatin and 500 μM mevalonate. Spent media (secretome) was collected, and explant proteins extracted with guanidine hydrochloride. Biomarker levels in secretome and explant extract were determined by western blotting. Sulphated glycosaminoglycan (sGAG) and collagen content of secretome was determined by dimethylmethylene blue analysis and hydroxyproline quantification, respectively. One-way analysis of variance, with Tukey's multiple comparison post-tests, was used to determine differences between means. Results: Release of sGAG and collagen into the secretome was elevated with cytokine treatment by day 4 and 14 respectively (P < 0.001). 100 nM and 1000 nM simvastatin inhibited collagen release by 57 and 85 % respectively (P < 0.001), and this effect was negated by the addition of mevalonate (downstream metabolite of statin inhibition). sGAG release was unaffected by simvastatin treatment. Lumican release into the secretome was elevated with cytokine treatment by day 14 (P < 0.01), and simvastatin treatment lowered cytokine-induced release by up to 60 % (P < 0.01). Chondroadherin and LRP-1 were elevated in the secretome by day 4 and 8 respectively (P < 0.01) and preliminary analysis showed that simvastatin treatment inhibited release of both. Chondoradherin was lost from explant extract with cytokine treatment, whereas lumican was elevated, and both of these effects were reversed with simvastatin treatment. Conclusions: Lumican is a marker of cartilage degradation and appears to parallel collagen release. Chondroadherin and LRP-1 are potential early markers of cytokine-induced degradation, preceding collagen release. Cytokine treatment increases lumican levels in both secretome and explant extract, suggesting an overall increase in lumican synthesis. Simvastatin inhibits collagen release and lumican levels suggesting an effect on collagenase activity, and lumican expression. Preliminary evidence shows that chondroadherin and LRP-1 release is inhibited by simvastatin, suggesting interruption of a collagenase-independent pathway. LRP-1, chondroadherin and lumican are all potential markers of different stages of cartilage degradation, each with potentially different mechanisms, but all inhibited by statin. Further investigation into these mechanisms may shed light on novel pathways, common to OA and atherosclerosis, and novel targets for therapeutic intervention. The research leading to these results was funded by the Medical Research Council, Arthritis Research UK and the European Commission Framework 7 programme (EU FP7; HEALTH.2012.2.4.5-2, project number 305815; Novel Diagnostics and Biomarkers for Early Identification of Chronic Inflammatory Joint Diseases).

We present a Data Centre Virtualisation architecture with an SDN-controlled all-optical data plane combining OCS and TSON. Orchestration dynamically translates and provisions Virtual Data Centres requests onto the optical layer. We describe an implementation and characterisation of the data plane.

Aims MMPs contribute to atherosclerotic plaque progression and instability, but the relative potency of their endogenous tissue inhibitors of metalloproteinases (TIMPs) as protective factors has not been defined. We therefore investigated the impact of TIMP-1 and TIMP-2 knockout on atherosclerotic plaque burden and composition in apolipoprotein E-knockout (Apoe(-/-)) mice and studied the underlying effects on monocyte/macrophage behaviour.Methods and results Analysis of brachiocephalic artery plaques revealed comparable atherosclerotic lesion areas between TIMP-1(-/-) Apoe(-/-) or TIMP-2(-/-) Apoe(-/-) double deficient mice and relevant age-matched, strain-matched Apoe(-/-) controls after 8 weeks of high-fat feeding. However, lesions from TIMP-2(-/-) Apoe(-/-) mice had higher levels of markers associated with plaque vulnerability, including increased macrophage: vascular smooth muscle cell ratios, larger necrotic core areas, reduced collagen contents, increased macrophage proliferation, and apoptosis frequencies, compared with TIMP-1(-/-)Apoe(-/-) and controls. In contrast, TIMP-1(-/-) Apoe(-/-) animals only had a significant reduction in vascular smooth muscle cell content compared with Apoe(-/-) controls. In vitro and in vivo findings implicated heightened monocyte/macrophage invasion in the detrimental effects observed on atherosclerotic plaque composition in TIMP-2(-/-) Apoe(-/-) mice. Moreover, TIMP-2 specifically decreased MMP-14-dependent monocyte/macrophage infiltration into sites of experimentally induced inflammation and established atherosclerotic lesions.Conclusion Our data demonstrate that TIMP-2 plays a greater protective role than TIMP-1 during the pathogenesis of atherosclerosis, in part by suppressing MMP-14-dependent monocyte/macrophage accumulation into plaques.

INTRODUCTION:The main limitation of coronary artery bypass grafting (CABG) is rapid neointimal hyperplasia leading to graft failure.AIM:To assess plaque formation in saphenous vein grafts (SVG) covered by an external Dacron stent in comparison with the classical technique.MATERIAL AND METHODS:In the study group vein grafts covered by external stent mesh made of Dacron were implanted. An intravascular ultrasonography (IVUS) study was performed in 35 aorto-coronary SVG covered by an external Dacron stent and in 64 normal SVG during the first year after CABG. In each SVG 25 mm of good quality IVUS image, volumes of lumen, plaque (neointima), outer border of the vein graft (external SVG) and adventitia were calculated in three time periods: 0-130 days, 130-260 days and 260-390 days.RESULTS:Between the first and second time period, lumen volume (mm3) was reduced from 10.33 ±4.4, to 6.80 ±2.23 in the second period and 5.69 ±1.26 in the third one. This effect was much less marked in normal grafts. The corresponding lumen volume (mm3) was: 10.90 ±3.9, 9.15 ±2.94 and 8.92 ±2.93 in consecutive time periods. Plaque volume (mm3) did not change in control grafts during the course of the study, but it increased very significantly in stented grafts from 0.86 ±1.24 in the first period to 2.70 ±1.58 in the second and 3.29 ±2.66 in the third one.CONCLUSIONS:The experimental technique of implanting SVG covered with an external elastic Dacron stent seems to be inferior to traditional ones. This is probably due to the more complicated process of vein implantation and higher micro-injury occurrence during the surgery.

We propose and demonstrate an algorithm that allows for automatic synchronization of SDN-controlled all-optical TDM switching nodes connected in a ring network. We experimentally show successful WDM-SDM transmission of data bursts between all ring nodes.

We demonstrate an all-optical dynamic DCN utilizing a fully SDN-enabled data-plane including programmable FPGA-based NICs, offering Network Function Virtualization OCS/OPS driven multicasting for Virtual DC applications enabling link recovery and VM migration.

We demonstrated an SDN-enabled optical DCN leveraging AoD, OPS, FPGA-based ToR with extended OpenDayLight controller and extended OF protocol. Experimental results show application-aware OCS/OPS connection provisioning (753/214ms), OPS end-to-end connections latency (252µs), and OCS/OPS switchover.

Galectin-3 (also known as mac-2) is a beta-galactoside binding lectin proposed to function as a novel mediator of inflammation and is involved in fibrosis, cell adhesion, apoptosis and chemotaxis. Galectin-3 is expressed in human atherosclerotic plaques, and has been suggested to modulate the inflammatory profile of macrophages. We therefore hypothesised that altered Galectin-3 expression may influence macrophage behaviour and consequently modulate plaque progression.

Transglutaminase activity has been widely implicated in bone deposition. A predominant role has been proposed for factor (F)XIII-A and a subsidiary role suggested for the homologous protein, transglutaminase 2. Full-length FXIII-A is an 83kDa protransglutaminase that is present both in plasma and also in haematopoietic and connective tissue lineages. Several studies have reported expression in murine cells, including osteocytes, of a 37 kDa protein that reacts with the monoclonal anti-FXIII-A antibody AC-1A1. This protein was presumed to be a catalytically active fragment of FXIII-A-83 and to play a major role in bone deposition. We detected a 37 kDa AC-1A1 reactive protein in FXIII-A mRNA negative cell lines and in tissues from FXIII-A(-/-) mice. By mass spectrometric sequencing of AC-1A1 immunoprecipitates, we identified this protein as transaldolase-1, and confirmed that recombinant transaldolase-1 is recognised by AC-1A1. We have also shown that bone deposition is normal in FXIII-A(-/-).TG2(-/-) double knockout mice, casting doubt on the role of transglutaminases in bone mineralisation. Various studies have used antibody AC-1A1 for immunohistochemistry or immunofluorescence. We observe strong FXIII-A dependent staining in paraffin embedded mouse heart sections, with relatively low background in non-expressing mouse cells. In contrast, FXIII-A independent staining predominates in cultured human cells using a standard immunofluorescence procedure. Immunofluorescence is present in membrane compartments that are expected to lack transaldolase, indicating that other off-target antigens are recognised by AC-1A1. This has significant implications for studies that have used this approach to define the subcellular trafficking of FXIII-A in osteocytes.

You are probably thinking ‘if this series is so straightforward, why does it need an article to guide me through it?’ and you would be right, it does not. The series is self-explanatory and will be useful to basic scientists and clinicians because the articles are written by international experts who have been selected for their ability to communicate clearly and enjoyably. However, there is a hidden purpose …

Rationale High-fat diet with obesity-associated co-morbidities triggers cardiac remodeling and renders the heart more vulnerable to ischemia/reperfusion injury. However, the effect of high-fat diet without obesity and associated co-morbidities is presently unknown. Objectives To characterize a non-obese mouse model of high-fat diet, assess the vulnerability of hearts to reperfusion injury and to investigate cardiac cellular remodeling in relation to the mechanism(s) underlying reperfusion injury. Methods and Results Feeding C57BL/6J male mice high-fat diet for 20 weeks did not induce obesity, diabetes, cardiac hypertrophy, cardiac dysfunction, atherosclerosis or cardiac apoptosis. However, isolated perfused hearts from mice fed high-fat diet were more vulnerable to reperfusion injury than those from mice fed normal diet. In isolated cardiomyocytes, high-fat diet was associated with higher diastolic intracellular Ca2+ concentration and greater damage to isolated cardiomyocytes following simulated ischemia/reperfusion. High-fat diet was also associated with changes in mitochondrial morphology and expression of some related proteins but not mitochondrial respiration or reactive oxygen species turnover rates. Proteomics, western blot and high-performance liquid chromatography techniques revealed that high-fat diet led to less cardiac oxidative stress, higher catalase expression and significant changes in expression of putative components of the mitochondrial permeability transition pore (mPTP). Inhibition of the mPTP conferred relatively more cardio-protection in the high-fat fed mice compared to normal diet. Conclusions This study shows for the first time that high-fat diet, independent of obesity-induced co-morbidities, triggers changes in cardiac oxidative state, calcium handling and mitochondria which are likely to be responsible for increased vulnerability to cardiac insults.