SVEP1 influences monocyte to macrophage differentiation via integrin α4ß1/α9ß1 and Rho/Rac signalling.

BACKGROUND: The large extracellular matrix protein SVEP1 mediates cell adhesion via integrin α9ß1. Recent studies have identified an association between a missense variant in SVEP1 and increased risk of coronary artery disease (CAD) in humans and in mice Svep1 deficiency alters the development of atherosclerotic plaques. However how SVEP1 functionally contributes to CAD pathogenesis is not fully understood. Monocyte recruitment and differentiation to macrophages is a key step in the development of atherosclerosis. Here, we investigated the requirement for SVEP1 in this process. METHODS: SVEP1 expression was measured during monocyte-macrophage differentiation in primary monocytes and THP-1 human monocytic cells. SVEP1 knockout THP-1 cell lines and the dual integrin α4ß1/α9ß1 inhibitor, BOP, were utilised to investigate the effect of these proteins in THP-1 cell adhesion, migration and cell spreading assays. Subsequent activation of downstream integrin signalling intermediaries was quantified by western blotting. RESULTS: SVEP1 gene expression increases in monocyte to macrophage differentiation in human primary monocytes and THP-1 cells. Using two SVEP1 knockout THP-1 cells we observed reduction in monocyte adhesion, migration, and cell spreading compared to control cells. Similar results were found with integrin α4ß1/α9ß1 inhibition. We demonstrate reduced activity of Rho and Rac1 in SVEP1 knockout THP-1 cells. CONCLUSIONS: SVEP1 regulates monocyte recruitment and differentiation phenotypes through an integrin α4ß1/α9ß1 dependent mechanism. GENERAL SIGNIFICANCE: These results describe a novel role for SVEP1 in monocyte behaviour relevant to CAD pathophysiology.

A novel electrospun biphasic scaffold provides optimal three-dimensional topography for in vitro co-culture of airway epithelial and fibroblast cells.

Conventional airway in vitro models focus upon the function of individual structural cells cultured in a two-dimensional monolayer, with limited three-dimensional (3D) models of the bronchial mucosa. Electrospinning offers an attractive method to produce defined, porous 3D matrices for cell culture. To investigate the effects of fibre diameter on airway epithelial and fibroblast cell growth and functionality, we manipulated the concentration and deposition rate of the non-degradable polymer polyethylene terephthalate to create fibres with diameters ranging from nanometre to micrometre. The nanofibre scaffold closely resembles the basement membrane of the bronchiole mucosal layer, and epithelial cells cultured at the air-liquid interface on this scaffold showed polarized differentiation. The microfibre scaffold mimics the porous sub-mucosal layer of the airway into which lung fibroblast cells showed good penetration. Using these defined electrospinning parameters we created a biphasic scaffold with 3D topography tailored for optimal growth of both cell types. Epithelial and fibroblast cells were co-cultured onto the apical nanofibre phase and the basal microfibre phase respectively, with enhanced epithelial barrier formation observed upon co-culture. This biphasic scaffold provides a novel 3D in vitro platform optimized to mimic the different microenvironments the cells encounter in vivo on which to investigate key airway structural cell interactions in airway diseases such as asthma.

Human airway smooth muscle maintain in situ cell orientation and phenotype when cultured on aligned electrospun scaffolds.

Human airway smooth muscle (HASM) contraction plays a central role in regulating airway resistance in both healthy and asthmatic bronchioles. In vitro studies that investigate the intricate mechanisms that regulate this contractile process are predominantly conducted on tissue culture plastic, a rigid, 2D geometry, unlike the 3D microenvironment smooth muscle cells are exposed to in situ. It is increasingly apparent that cellular characteristics and responses are altered between cells cultured on 2D substrates compared with 3D topographies. Electrospinning is an attractive method to produce 3D topographies for cell culturing as the fibers produced have dimensions within the nanometer range, similar to cells' natural environment. We have developed an electrospun scaffold using the nondegradable, nontoxic, polymer polyethylene terephthalate (PET) composed of uniaxially orientated nanofibers and have evaluated this topography's effect on HASM cell adhesion, alignment, and morphology. The fibers orientation provided contact guidance enabling the formation of fully aligned sheets of smooth muscle. Moreover, smooth muscle cells cultured on the scaffold present an elongated cell phenotype with altered contractile protein levels and distribution. HASM cells cultured on this scaffold responded to the bronchoconstrictor bradykinin. The platform presented provides a novel in vitro model that promotes airway smooth muscle cell development toward a more in vivo-like phenotype while providing topological cues to ensure full cell alignment.

A two-site ELISA can quantify upregulation of SMN protein by drugs for spinal muscular atrophy.

OBJECTIVES: Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons during early or postnatal development. Severity is variable and is inversely related to the levels of survival of motor neurons (SMN) protein. The aim of this study was to produce a two-site ELISA capable of measuring both the low, basal levels of SMN protein in cell cultures from patients with severe SMA and small increases in these levels after treatment of cells with drugs. METHODS: A monoclonal antibody against recombinant SMN, MANSMA1, was selected for capture of SMN onto microtiter plates. A selected rabbit antiserum against refolded recombinant SMN was used for detection of the captured SMN. RESULTS: The ratio of SMN levels in control fibroblasts to levels in SMA fibroblasts was greater than 3.0, consistent with Western blot data. The limit of detection was 0.13 ng/mL and SMN could be measured in human NT-2 neuronal precursor cells grown in 96-well culture plates (3 x 10(4) cells per well). Increases in SMN levels of 50% were demonstrable by ELISA after 24 hours treatment of 10(5) SMA fibroblasts with valproate or phenylbutyrate. CONCLUSION: A rapid and specific two-site, 96-well ELISA assay, available in kit format, can now quantify the effects of drugs on survival of motor neurons protein levels in cell cultures.

Level of ex vivo interleukin 6 expression in human peripheral fat compared with other tissues.

OBJECTIVES: Adipose tissue is able to secrete a variety of active mediators into the circulation. One of these is Interleukin 6 (IL6). IL6 may play a causal role in the development of atherosclerosis. It has therefore been suggested that IL6 may form part of the link between obesity and vascular disease. The aim of this study was to quantify the relative IL6 expression in adipose tissue compared to other tissues. METHODS: Tissue (vein, fat, muscle, blood) was collected from 32 patients undergoing varicose vein surgery. RNA was extracted and mRNA measured using RT-PCR relative quantification. The mean relative IL6 mRNA levels were compared between tissues using the Mann Whitney U test and the independent t-test. Tissue levels were compared for individuals using the Wilcoxon signed rank test. RESULTS: Mean relative IL6 mRNA levels (mean+/-SEM) were significantly greater in adipose tissue 44.8+/-16.1 than in other tissues (leukocytes 1.1+/-0.3, vein 2.0+/-0.8, muscle 0.06+/-0.03: p<0.001). mRNA expression levels were also significantly higher in fat than in all other tissue types in individuals (p<0.001). CONCLUSIONS: IL6 mRNA expression is significantly higher in adipose than in many other tissues known to express IL6.

Aortic endograft infection: open surgical management with endograft preservation.

INTRODUCTION: We report successful management of aortic endograft infection without graft explantation or extra-anatomic bypass. REPORT: A 66 year-old male who had undergone endovascular repair of an aortic aneurysm presented with abdominal pain and raised inflammatory markers following embolisation of a type-2 'endoleak'. CT scanning revealed a left psoas fluid collection. Endograft infection was diagnosed. Following failure of CT-guided drainage and conservative management, surgical drainage with irrigation drain placement was undertaken with preservation of the endograft. There was no evidence of recurrent infection after follow-up at 30 months. DISCUSSION: Aortic endograft infection may be managed without endograft removal and extra-anatomic bypass.

Multi centre study to assess the feasibility of a new covered stent and delivery system in combination with remote superficial femoral artery endarterectomy (RSFAE).

OBJECTIVES: To evaluate the feasibility and efficacy of an innovative new covered stent and adjustable deployment system (aSpire Covered Stent, Vascular Architects Inc., San Jose, CA, USA) in combination with remote superficial femoral artery endarterectomy (RSFAE) for the treatment of long segment femoropopliteal occlusive disease. DESIGN: Prospective multi-centre trial. MATERIALS AND METHODS: Sixty-two limbs in 61 patients (41 men; median age 69 years, range 40-88) with severe disabling claudication (n=56) or critical limb ischaemia (n=6) were treated in five European centres with aSpire stenting after RSFAE for long segment occlusions (mean length 25 cm). Follow-up was by duplex scanning at 1-, 6-, 12- and 18-months. Primary, primary-assisted and secondary patency rates were analysed. RESULTS: The median follow-up was 17 (range 2-34) months. A mean of 1.3 stents (range 1-3) were deployed with a median stent diameter of 7 mm (range 6-9). There were one early and 24 late failures. At 18-months the cumulative primary, primary-assisted and secondary patency rates were 60, 70 and 72%, respectively. There were no device related adverse events, such as kinking or fracturing and no stent migrations. CONCLUSIONS: The aSpire stent and the delivery system are both safe and feasible in combination with RSFAE. The mid term follow-up appears favourable in view of the long segment occlusions treated. Further follow-up is required to compare the mid- and long-term outcomes with current stents and conventional femoropopliteal bypass.

Implications of screening for abdominal aortic aneurysms on surgical workload.

BACKGROUND: The Multicentre Aneurysm Screening Study (MASS) provided strong evidence for both the clinical benefit and the cost-effectiveness of a screening programme for abdominal aortic aneurysms (AAAs) in men. If a national screening programme for AAA were adopted in the UK, it would be expected to increase the elective and decrease the emergency surgical workload. METHODS: The MASS trial randomized 67,800 men aged 65-74 years to be invited to attend for ultrasonographic screening for AAA or to a control group that received no invitation. Predictions of elective and emergency surgical workload were made for a 20-year interval after the introduction of a screening programme for 65-year-old men, based on surgical rates observed in the MASS trial and national mortality statistics. RESULTS: For a district general hospital serving a population of 400,000, there was an estimated reduction from nine emergency operations per year before introduction of the screening programme to three emergency operations annually in men aged 65 years and over by the end of the 20-year interval, and an increase from 24 to 43 AAA operations overall. The corresponding estimated annual costs for all AAA surgery increased by 47 per cent, from pound 209,000 to pound 308,000. These results were not affected by changes in the underlying assumptions. CONCLUSION: The results support the expectation of very few emergency operations, and principally elective operations, being performed following the introduction of a screening programme. For a typical district general hospital, a screening programme would be expected to lead to two additional elective AAA operations per month, and to save 11 AAA-related deaths per year.

C-reactive protein is elevated in symptomatic compared with asymptomatic patients with carotid artery disease.

OBJECTIVES: to investigate the level of inflammatory markers between symptomatic and asymptomatic carotid stenosis patients. DESIGN: cross-sectional study. MATERIALS AND METHODS: a prospective study of 137 consecutive patients, admitted electively for carotid endarterectomy during 1997-2000, was conducted. 125 patients had cerebrovascular symptoms: either stroke (neurological deficit >24 h), Transient ischaemic attack (neurological deficit<24 h) or amaurosis fugax. Twelve patients were asymptomatic. A medical history and a fasting venous blood sample were taken from each patient around 6 weeks before surgery. The plasma concentrations of cholesterol and of inflammatory markers; (high sensitivity C-reactive protein (hs-CRP), sICAM-1, sVCAM-1, sE-selectin) were determined. RESULTS: the concentration of hs-CRP in the symptomatic group (3.9 mg/L) was significantly higher than in the asymptomatic group (2.1 mg/L; p = 0.04). These concentrations were within normal range (<10 mg/L). sICAM-1, sVCAM-1, sE-selectin and total cholesterol concentrations were not different between the two groups. CONCLUSION: plasma hs-CRP was elevated in symptomatic compared to asymptomatic patients with carotid artery disease. High sensitivity C-reactive protein has been shown to be of prognostic value in a number of cardiovascular conditions and this study suggests it may be of value to identify patient at high risk of developing neurological deficits.

Autosomal dominant Emery-Dreifuss muscular dystrophy: a new family with late diagnosis.

Emery-Dreifuss muscular dystrophy is characterized by the clinical triad of early onset contractures of elbows, Achilles tendons and spine, wasting and weakness with a predominantly humero-peroneal distribution and life-threatening cardiac conduction defects and/or cardiomyopathy. Two main types of inheritance have been described: the X-linked form is caused by mutations in the STA gene on locus Xq28 and the gene for the autosomal dominant form (LMNA gene) has been localized on chromosome 1q11-q23. Recently, mutations in this LMNA gene have been also found to be responsible for the less frequent autosomal recessive form of the disease. Although all forms share a similar clinical presentation, some differences appear to exist between them as has been described recently in a large number of patients. We present the first documented Spanish family genetically confirmed to have autosomal dominant Emery-Dreifuss muscular dystrophy. Clinical, pathological and genetic data are described. We emphasize the difficulties in diagnosis, especially in sporadic cases or young patients in whom the clinical picture is not completely established.

The role of the nuclear envelope in Emery-Dreifuss muscular dystrophy.

The X-linked form of Emery-Dreifuss muscular dystrophy (X-EDMD) is caused by absence, or greatly reduced amounts, of the inner nuclear-membrane protein, emerin. The autosomal dominant form (AD-EDMD) is caused by missense mutations in lamins A and C, two components of the nuclear lamina that interact directly with emerin. Lamin A/C mutations also cause one form of dilated cardiomyopathy (CMD1A) and one form of limb-girdle muscular dystrophy (LGMD1B), both of which have clinical features in common with EDMD, as well as a rare, unrelated form of lipodystrophy (FPLD). Evidence is now emerging that defective assembly of the nuclear lamina is a feature of all these diseases, although not necessarily the direct cause. Why only heart and skeletal muscle, and possibly connective tissue, are affected in EDMD and why expression of the disease is so extremely variable between individuals remains to be explained.

Both emerin and lamin C depend on lamin A for localization at the nuclear envelope.

Physical interactions between lamins and emerin were investigated by co-immunoprecipitation of in vitro translated proteins. Emerin interacted with in vitro translated lamins A, B1 and C in co-immunprecipitation reactions. Competition reactions revealed a clear preference for interactions between emerin and lamin C. Structural associations between lamins and emerin were investigated in four human cell lines displaying abnormal expression and/or localisation of lamins A and C. In each cell line absence of lamins A and C from the nuclear envelope (NE) was correlated with mis-localisation of endogenous and exogenous emerin to the ER. In two cell lines that did not express lamin A but did express lamin C, lamin C as well as emerin was mis-localised. When GFP-lamin A was expressed in SW13 cells (which normally express only very low levels of endogenous lamin A and mis-localise endogenous emerin and lamin C), all three proteins became associated with the NE. When GFP-lamin C was expressed in SW13 cells neither the endogenous nor the exogenous lamin C was localised to the NE and emerin remained in the ER. Finally, lamins A and C were selectively eliminated from the NE of HeLa cells using a dominant negative mutant of lamin B1. Elimination of these lamins from the lamina led to the accumulation of emerin as aggregates within the ER. Our data suggest that lamin A is essential for anchorage of emerin to the inner nuclear membrane and of lamin C to the lamina.

How does a g993t mutation in the emerin gene cause Emery-Dreifuss muscular dystrophy?

X-linked Emery-Dreifuss muscular dystrophy is usually caused by absence of the nuclear membrane protein, emerin, due to nonsense mutations or deletions, but a few missense mutations also exist. A pathogenic g993t mutation causes a Q133H change in the nuclear targeting region of emerin, but it may also reduce emerin levels by affecting mRNA splicing. We have introduced the g993t mutation by in vitro mutagenesis and studied the effect of Q133H on nuclear targeting by transfection of COS-7 cells. No qualitative or quantitative differences in nuclear targeting were observed between normal and mutant emerin. Quantitative BIAcore analysis showed no significant change in lamin A binding to emerin when the mutation was present. We conclude that Q133 is not essential for nuclear targeting of emerin or its interaction with lamin A. Reduced emerin levels due to altered splicing or defective interaction with an unidentified binding partner remain possible pathogenic mechanisms.

Skeletal muscle pathology in autosomal dominant Emery-Dreifuss muscular dystrophy with lamin A/C mutations.

We present our observations on the skeletal muscle pathology of nine cases from seven families of autosomal dominant Emery-Dreifuss muscular dystrophy (ADEDMD) with identified mutations in the lamin A/C gene, aged 2-35 years at the time of biopsy. The severity of pathological change was moderate and the most common features were variation in fibre size (hypertrophy and atrophy), an increase in internal nuclei and smaller diameter fibres with high oxidative enzyme activity. Only one case showed necrosis, which was present in two separate samples taken from the quadriceps and tibialis anterior, at different ages. Immunocytochemistry detected an age-related reduction of laminin beta1 on the muscle fibres in adolescent and adult cases. Antibodies to lamins A and A/C, and emerin did not reveal any detectable differences from controls. Electron microscopy of two out of three cases showed an abnormal distribution of heterochromatin in many fibre nuclei. Our results show that dystrophic changes in skeletal muscle are not a major feature of ADEDMD, and that nuclear abnormalities may be detected with electron microscopy. Immunodetection of reduced laminin beta1 may be a useful secondary marker in adults with this disorder, as immunocytochemistry of lamins is not yet of diagnostic use.

Dystrophin in adult zebrafish muscle.

Mutations in the human dystrophin gene are implicated in the fatal muscle wasting disease Duchenne Muscular Dystrophy (DMD). This gene expresses a sarcolemmal-associated protein that is evolutionarily conserved, underpinning its important role in the architecture of muscle. In terms of DMD modelling, the mouse has served as a suitable vertebrate species but the pathophysiology of the disease in the mouse does not entirely mimic human DMD. We have examined the zebrafish in order to expand the repertoire of vertebrate species for muscle disease modelling, and to dissect further the functional interactions of dystrophin. We report here the identification of an apparent zebrafish orthologue of the human dystrophin gene that expresses a 400-kDa protein that is localised to the muscle membrane surface. These data suggest that the zebrafish may prove to be a beneficial vertebrate model to examine the role and functional interactions of dystrophin in disease and development.

Epitopes in the interacting regions of beta-dystroglycan (PPxY motif) and dystrophin (WW domain).

The dystroglycan gene produces two products from a single mRNA, the extracellular alpha-dystroglycan and the transmembrane beta-dystroglycan. The Duchenne muscular dystrophy protein, dystrophin, associates with the muscle membrane via beta-dystroglycan, the WW domain of dystrophin interacting with a PPxY motif in beta-dystroglycan. A panel of four monoclonal antibodies (MANDAG1-4) was produced using the last 16 amino acids of beta-dystroglycan as immunogen. The mAbs recognized a 43 kDa band on Western blots of all cells and tissues tested and stained the sarcolemma in immunohistochemistry of skeletal muscle over a wide range of animal species. A monoclonal antibody (mAb) against the WW domain of dystrophin, MANHINGE4A, produced using a 16-mer synthetic peptide, recognized dystrophin on Western blots and also stained the sarcolemma. We have identified the precise sequences recognized by the mAbs using a phage-displayed random 15-mer peptide library. A 7-amino-acid consensus sequence SPPPYVP involved in binding all four beta-dystroglycan mAbs was identified by sequencing 17 different peptides selected from the library. PPY were the most important residues for three mAbs, but PxxVP were essential residues for a fourth mAb, MANDAG2. By sequencing five different random peptides from the library, the epitope on dystrophin recognized by mAb MANHINGE4A was identified as PWxRA in the first beta-strand of the WW domain, with the W and R residues invariably present. A recent three-dimensional structure confirms that the two epitopes are adjacent in the dystrophin-dystroglycan complex, highlighting the question of how the two interacting motifs can also be accessible to antibodies during immunolocalization in situ.

Absence of utrophin in intercalated discs of human cardiac muscle.

Utrophin is the autosomal homologue of dystrophin. In normal skeletal muscle it is localised only to neuromuscular and myotendinous junctions, nerves and vascular tissue. In Xp21 muscular dystrophies, utrophin is also detected on the sarcolemma of skeletal and cardiac muscle, while dystrophin is absent or reduced. In normal cardiac muscle, some reports have demonstrated utrophin at intercalated discs and T-tubules. We have re-examined the distribution of utrophin in normal human cardiac muscle using a panel of eight monoclonal antibodies against different epitopes in N- and C-terminal domains. In contrast to previous studies, utrophin was not detected at the intercalated discs or T-tubules, although labelling of blood vessels was strong. We conclude that the primary location of utrophin in normal heart is in the vascular system. In addition, our results show that the utrophin on cardiac blood vessels is full length, similar to that of skeletal muscle blood vessels.