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.

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.

Laminin-induced clustering of dystroglycan on embryonic muscle cells: comparison with agrin-induced clustering.

The effect of laminin on the distribution of dystroglycan (DG) and other surface proteins was examined by fluorescent staining in cultures of muscle cells derived from Xenopus embryos. Western blotting confirmed that previously characterized antibodies are reactive in Xenopus. In control cultures, alphaDG, betaDG, and laminin binding sites were distributed as microclusters (<1 microm2 in area) over the entire dorsal surface of the muscle cells. Treatment with laminin induced the formation of macroclusters (1-20 microm2), accompanied by a corresponding decline in the density of the microclusters. With 6 nM laminin, clustering was apparent within 150 min and near maximal within 1 d. Laminin was effective at 30 pM, the lowest concentration tested. The laminin fragment E3, which competes with laminin for binding to alphaDG, inhibited laminin-induced clustering but did not itself cluster DG, thereby indicating that other portions of the laminin molecule in addition to its alphaDG binding domain are required for its clustering activity. Laminin-induced clusters also contained dystrophin, but unlike agrin-induced clusters, they did not contain acetylcholine receptors, utrophin, or phosphotyrosine, and their formation was not inhibited by a tyrosine kinase inhibitor. The results reinforce the notion that unclustered DG is mobile on the surface of embryonic muscle cells and suggest that this mobile DG can be trapped by at least two different sets of molecular interactions. Laminin self binding may be the basis for the laminin-induced clustering.

Localization of the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating brain cell lines.

mAbs have been raised against different epitopes on the protein product of the DMDL gene, which is an autosomal homologue of the X-linked DMD gene for dystrophin. These antibodies provide direct evidence that DMDL protein is localized near acetylcholine receptors at neuromuscular junctions in normal and mdx mouse intercostal muscle. The primary location in tissues other than skeletal muscle is smooth muscle, especially in the vascular system, which may account for the wide tissue distribution previously demonstrated by Western blotting. The DMDL protein was undetectable in the nonjunctional sarcolemma of normal human muscle, but was observed in nonjunctional sarcolemma of Duchenne muscular dystrophy patients, where dystrophin itself is absent or greatly reduced. The expression of DMDL protein is not restricted to smooth and skeletal muscle, however, since relatively large amounts are present in transformed brain cell lines of both glial and Schwann cell origin. This contrasts with the low levels of DMDL protein in adult brain tissue.

Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibers by clonal expansion.

Conventionally, nonsense mutations within a gene preclude synthesis of a full-length functional protein. Obviation of such a blockage is seen in the mdx mouse, where despite a nonsense mutation in exon 23 of the dystrophin gene, occasional so-called revertant muscle fibers are seen to contain near-normal levels of its protein product. Here, we show that reversion of dystrophin expression in mdx mice muscle involves unprecedented massive loss of up to 30 exons. We detected several alternatively processed transcripts that could account for some of the revertant dystrophins and could not detect genomic deletion from the region commonly skipped in revertant dystrophin. This, together with exon skipping in two noncontiguous regions, favors aberrant splicing as the mechanism for the restoration of dystrophin, but is hard to reconcile with the clonal idiosyncrasy of revertant dystrophins. Revertant dystrophins retain functional domains and mediate plasmalemmal assembly of the dystrophin-associated glycoprotein complex. Physiological function of revertant fibers is demonstrated by the clonal growth of revertant clusters with age, suggesting that revertant dystrophin could be used as a guide to the construction of dystrophin expression vectors for individual gene therapy. The dystrophin gene in the mdx mouse provides a favored system for study of exon skipping associated with nonsense mutations.

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.

In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide.

In the canine model of Duchenne muscular dystrophy in golden retrievers (GRMD), a point mutation within the splice acceptor site of intron 6 leads to deletion of exon 7 from the dystrophin mRNA, and the consequent frameshift causes early termination of translation. We have designed a DNA and RNA chimeric oligonucleotide to induce host cell mismatch repair mechanisms and correct the chromosomal mutation to wild type. Direct skeletal muscle injection of the chimeric oligonucleotide into the cranial tibialis compartment of a six-week-old affected male dog, and subsequent analysis of biopsy and necropsy samples, demonstrated in vivo repair of the GRMD mutation that was sustained for 48 weeks. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of exons 5-10 demonstrated increasing levels of exon 7 inclusion with time. An isolated exon 7-specific dystrophin antibody confirmed synthesis of normal-sized dystrophin product and positive localization to the sarcolemma. Chromosomal repair in muscle tissue was confirmed by restriction fragment length polymorphism (RFLP)-PCR and sequencing the PCR product. This work provides evidence for the long-term repair of a specific dystrophin point mutation in muscle of a live animal using a chimeric oligonucleotide.

Molecular cloning of murine FLT and FLT4.

A wide range of growth and differentiation processes are regulated by the signalling of receptor tyrosine kinases (RTKs). We have developed a nested polymerase chain reaction (PCR) procedure with degenerate primers, and used it to identify RTKs expressed in murine fetal thymus. A novel RTK, called FLT4, and the murine homologue of FLT were found, and their PCR fragment sequences were used to isolate larger cDNA clones spanning the complete coding regions of these receptors. FLT4 was found to contain an extracellular region similar to the corresponding sequences of FLT and Flk-1, containing seven immunoglobulin domains.

Changes at the N-terminus of human brain creatine kinase during a transition between inactive folding intermediate and active enzyme.

CK-STAR, a monoclonal antibody against human brain creatine kinase (CK), can be shown by chemical cleavage mapping and peptide synthesis to recognize an epitope at the free N-terminus of the enzyme. The epitope could be largely reproduced by a synthetic peptide based on the first 18 amino acids and could be partly formed by the first 11 amino acids. The antibody did not bind to native CK, but it did bind to CK in various partially denatured forms and to an enzymically inactive intermediate in the refolding process. Competitive binding studies have shown that the N-terminal conformations of both the refolding intermediate and the free peptide resemble that of CK partially denatured by attachment to plastic. The results suggest that the final stages of CK refolding and reactivation involve a structural change at the N-terminus or its interaction with some other part of the CK molecule, thus masking the CK-STAR epitope.

Monoclonal antibody studies suggest a catalytic site at the interface between domains in creatine kinase.

We have located the epitopes recognized by four different monoclonal antibodies which bind to partially unfolded creatine kinase (CK) (ATP: creatine N-phosphotransferase, EC 2.7.3.2) but not to the native enzyme. The epitopes appear to be buried within the CK structure in its native, proteinase-resistant, state. When the epitopes are made accessible to antibody by mild denaturation, CK becomes enzymically-inactive and can be cleaved by proteinase V8 into two large fragments which retain the epitopes and may represent domains. Epitopes on each V8 fragment are associated with highly conserved sequences and are brought physically close to the active site of the enzyme during the later stages of CK refolding and reactivation. The results suggest a catalytic site formed at the interface between two domains which carry the epitopes on their interacting surfaces. Separation of loosely associated domains before or during immunization may account for the origin of antibodies against buried epitopes.

Structural changes in the C-terminal region of human brain creatine kinase studied with monoclonal antibodies.

Epitopes on human brain creatine kinase (B-CK) recognized by three monoclonal antibodies have been located by chemical cleavage methods, followed by peptide synthesis or analysis of specificity for natural variants (isoforms). One antibody, CK-HTB, recognizes a conformational, or assembled, surface epitope on native CK which is also present on partially unfolded forms. It requires an Asn residue at position 300 in the amino acid sequence and will not recognize variants with Lys or His in this position. This results in a striking specificity of the antibody, which binds to B-CK only in chicken and man, but to muscle-form (M-CK) only in the rat. The results suggest that Asn-300 is exposed on the enzyme surface as part of a relatively denaturation-resistant region. Two monoclonal antibodies, CK-END1 and CK-END2, recognise epitopes within 53 amino acids of the C-terminus and bind to a synthetic hexapeptide representing the last six amino acids of human B-CK (Leu-375-Lys-380). The two antibodies show overlapping, but distinct, specificities in their binding to CK variants. CK-END1 requires Met-376 and will not tolerate Ile in this position, whereas CK-END2 requires Leu-375 and will not tolerate Met. Neither antibody binds to native CK, though both will bind to a folding intermediate and to partially unfolded states. This shows that the C-terminus of CK becomes inaccessible to the antibodies during those later stages of protein folding associated with recovery of enzyme activity and suggests that the protein may 'tuck in its tail' during one of the final steps.

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.

The molecular basis for cross-reaction of an anti-dystrophin antibody with alpha-actinin.

The epitope recognised by the anti-dystrophin monoclonal antibodies MANDYS141 and MANDYS142 has been characterised using a phage display peptide library and a bacteriophage lambda cDNA library. Using a phage display library of random 15-mer peptides, the epitope recognised by the two antibodies was identified as EEXF. A lambda gt11 clone obtained by screening a human muscle cDNA library was shown to contain part of the out-of-frame human mitochondrial succinyl CoA synthetase (alpha-subunit) cDNA sequence which contains the sequence EEPL, suggesting a minimum requirement of EEXF/L for antibody binding. The sequence EEDF is located in the helical rod region of dystrophin and the N-terminal domain of alpha-actinin; this may explain why native dystrophin is not detected, since the alpha-helical, coiled-coil folding of the rod region of dystrophin may obscure the epitope in the native protein. The antibody cross-reaction between dystrophin and alpha-actinin is likely to be fortuitous and not due to any structural homology that exists between these two members of the spectrin superfamily.

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.

Interleukin-11 expression in donor bone marrow cells improves hematological reconstitution in lethally irradiated recipient mice.

Lethally irradiated mice were transplanted with syngeneic bone marrow cells infected with a recombinant retrovirus vector containing the human interleukin-11 (hIL-11) cDNA under the control of the human cytomegalovirus (CMV) immediate early promoter. The hIL-11 RNA transcript from the vector was detected in the spleen and bone marrow of the recipient mice, and hIL-11 protein accumulated in their serum. The hematological reconstitution of these mice was compared with recipient mice rescued with bone marrow infected with the parental retrovirus vector not containing the hIL-11 cDNA. The hIL-11-expressing mice had an accelerated recovery of circulating platelets and red and white blood cells. Three months after the transplantation, bone marrow was harvested from the mice and used to rescue other lethally irradiated recipients. The hIL-11 mRNA and protein were also detected in these secondary recipients, and the mice showed improved hematological reconstitution relative to a control group. No abnormal cell proliferation or other histopathology was observed in the hIL-11-expressing mice.

Localization and quantitation of the chromosome 6-encoded dystrophin-related protein in normal and pathological human muscle.

A dystrophin-related protein (DRP) encoded by a gene on chromosome 6 was studied in 14 normal and 79 pathological human skeletal muscle biopsies, as well as in cultured myotubes by light microscopic immunocytochemistry and quantitative immunoblots. In normal muscle immunoreactive DRP was present at the postjunctional surface membrane, at the surface of satellite cells, in the walls of blood vessels, in Schwann cells and in perineurium of intramuscular nerves. All of this produced a weak signal on immunoblots. In Duchenne/Becker dystrophy (DMD/BMD) and in polymyositis (PM) or dermatomyositis (DM) DRP was present throughout the extrajunctional surface membrane of extra- and intrafusal muscle fibers, particularly regenerating ones. This produced a 15-17-fold increase of DRP over normal in DMD/BMD and 4-10-fold increase over normal in PM and DM on immunoblots. In other pathological muscles, DRP localization pattern and quantity was about the same as in normals. Dystrophin-related protein was present in about the same amounts and distribution in normal and DMD cultured myoblasts and myotubes. The molecular stimulus for the marked upregulation of DRP in DMD/BMD and in the inflammatory myopathies is not known. In DMD/BMD the diffuse sarcolemmal DRP may partially compensate for dystrophin deficiency.

The R482Q lamin A/C mutation that causes lipodystrophy does not prevent nuclear targeting of lamin A in adipocytes or its interaction with emerin.

Most pathogenic missense mutations in the lamin A/C gene identified so far cause autosomal-dominant dilated cardiomyopathy and/or Emery-Dreifuss muscular dystrophy. A few specific mutations, however, cause a disease with remarkably different clinical features: FPLD, or familial partial lipodystrophy (Dunnigan-type), which mainly affects adipose tissue. We have prepared lamin A with a known FPLD mutation (R482Q) by in vitro mutagenesis. Nuclear targeting of lamin A in transfected COS cells, human skeletal muscle cells or mouse adipocyte cell cultures (pre- and post-differentiation) was not detectably affected by the mutation. Quantitative in vitro measurements of lamin A interaction with emerin using a biosensor also showed no effect of the mutation. The results show that the loss of function of R482 in lamin A/C in FPLD does not involve loss of ability to form a nuclear lamina or to interact with the nuclear membrane protein, emerin.

Utrophin, the autosomal homologue of dystrophin, is widely-expressed and membrane-associated in cultured cell lines.

Utrophin, the autosomal dystrophin-related protein (DRP), is expressed in HeLa cells, smooth muscle-like BC3H1 cells from mouse brain, COS monkey kidney cells, the P388D1 monocyte-macrophage cell line and untransformed human skin fibroblasts, as well as in rat C6 glioma and Schwannoma cells. It was undetectable, however, in the Sp2/O mouse myeloma cell line and in hybridoma lines derived from it. Dystrophin was not detected in any of these cell lines. Although all utrophin-containing cells were capable of forming monolayers in culture, no major effects of either attachment to substratum or length of time in culture (2-17 days) on utrophin levels were observed. After subcellular fractionation of BC3H1 or glioma cells, nearly all of the utrophin was found in the Triton-soluble fraction, suggesting an association with cell membranes.