Helen J. S. Stewart

Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration

Cell therapy for degenerative muscle diseases such as the muscular dystrophies requires a source of cells with the capacity to participate in the formation of new muscle fibers. We investigated the myogenic potential of human fetal mesenchymal stem cells (hfMSCs) using a variety of stimuli. The use of 5‐azacytidine or steroids did not produce skeletal muscle differentiation, whereas myoblast‐conditioned medium resulted in only 1%–2% of hfMSCs undergoing muscle differentiation. However, in the presence of galectin‐1, 66.1% ± 5.7% of hfMSCs, but not adult bone marrow‐derived mesenchymal stem cells, assumed a muscle phenotype, forming long, multinucleated fibers expressing both desmin and sarcomeric myosin via activation of muscle regulatory factors. Continuous exposure to galectin‐1 resulted in more efficient muscle differentiation than pulsed exposure (62.3% vs. 39.1%; p < .001). When transplanted into regenerating murine muscle, galectin‐1‐exposed hfMSCs formed fourfold more human muscle fibers than nonstimulated hfMSCs (p = .008), with similar results obtained in a scid/mdx dystrophic mouse model. These data suggest that hfMSCs readily undergo muscle differentiation in response to galectin‐1 through a stepwise progression similar to that which occurs during embryonic myogenesis. The high degree of myogenic conversion achieved by this method has relevance for the development of therapies for muscular dystrophies.

Changes in DNA Synthesis Rate in the Schwann Cell Lineage In Vivo Are Correlated With the Precursor – Schwann Cell Transition and Myelination

During the development of the rat sciatic nerve extensive proliferation of glial cells occurs, and there is a very substantial rearrangement of the cytoarchitecture as axons and Schwann cells assume relationships which lead to the formation of the myelinated and unmyelinated axons characteristic of adult nerve. The maturation of Schwann cells from Schwann cell precursors and the matching of Schwann cell numbers to axons is an important part of this process. We have therefore studied the proliferation of Schwann cell precursors and Schwann cells during the development of the rat sciatic nerve from embryonic day 14 to postnatal day 28 by combining bromodeoxyuridine injections of rats with double‐label immunohistochemical techniques. The results reveal that DNA synthesis occurs in both Schwann cell precursors and Schwann cells throughout early nerve development. The labelling index is already substantial at embryonic day 14, but from embryonic day 17, when essentially all the glial cells have converted from precursor to Schwann cell phenotype, it rises sharply, peaking between embryonic day 19 and 20 before declining precipitously in the early postnatal period. This rapid decline in DNA synthesis coincides with the appearance of the myelin protein Po, and in individual cells DNA synthesis is incompatible with the expression of Po protein. Non‐myelin‐forming Schwann cells, which mature later in development, continue to synthesize DNA until at least postnatal day 15, but by day 28 essentially all Schwann cells in the nerve are quiescent.

Identification of circulating microRNAs as diagnostic biomarkers for use in multiple myeloma

Background:Multiple myeloma is a plasma cell disorder that is characterised by clonal proliferation of malignant plasma cells in the bone marrow, monoclonal paraprotein in the blood or urine and associated organ dysfunction. It accounts for approximately 1% of cancers and 13% of haematological cancers. Myeloma arises from an asymptomatic proliferation of monoclonal plasma cells termed monoclonal gammopathy of undetermined significance (MGUS).Methods:MicroRNA expression profiling of serum samples was performed on three patient groups as well as normal controls. Validation of the nine microRNAs detected as promising biomarkers was carried out using TaqMan quantitative reverse transcription PCR. MicroRNA levels in serum were normalised using standard curves to determine the numbers of microRNAs per μl of serum.Results:Three serum microRNAs, miR-720, miR-1308 and miR-1246, were found to have potential as diagnostic biomarkers in myeloma. Use of miR-720 and miR-1308 together provides a powerful diagnostic tool for distinguishing normal healthy controls, as well as patients with unrelated illnesses, from pre-cancerous myeloma and myeloma patients. In addition, the combination of miR-1246 and miR-1308 can distinguish MGUS from myeloma patients.Conclusion:We have developed a biomarker signature using microRNAs extracted from serum, which has potential as a diagnostic and prognostic tool for multiple myeloma.

Lack of Galectin-1 Results in Defects in Myoblast Fusion and Muscle Regeneration

Galectin‐1 has been implicated in the development of skeletal muscle, being maximally expressed at the time of myofiber formation. Furthermore, in the presence of exogenous galectin‐1, mononuclear myoblasts show increased fusion in vitro. In the current study, we have used the galectin‐1 null mouse to elucidate the role of galectin‐1 in skeletal muscle development and regeneration. Myoblasts derived from the galectin‐1 mutant showed a reduced ability to fuse in vitro. In galectin‐1 null mutants, there was evidence of a delay in muscle fiber development at the neonatal stage and muscle fiber diameter was reduced when compared with wild‐type at the adult stage. Muscle regeneration was also compromised in the galectin‐1 mutant with the process being delayed and a reduced fiber size being maintained. These results, therefore, show a definitive role for galectin‐1 in fusion of myoblasts both in vitro, in vivo, and in regeneration after recovery from induced injury. Developmental Dynamics 236:1014–1024, 2007.

Expression and regulation of α1β1 integrin in Schwann cells

The interaction of cells with the extracellular matrix plays a critical role in morphogenesis and cell differentiation. To define how Schwann cells might interact with the extracellular matrix, we chose to study the expression of the laminin/collagen receptor alpha1beta1 integrin during nerve development in the rat from embryonic day 14 to maturity. We found that this integrin is expressed predominantly on mature non-myelin-forming cells and only at very low levels on myelin-forming cells. Significant levels of this integrin were not detected on Schwann cell precursors or embryonic Schwann cells in vivo. Experiments using transected and crushed sciatic nerve showed that alpha1beta1 integrin expression is regulated at least in part by axonal contact. Furthermore, Schwann cell culture experiments showed that alpha1beta1 integrin levels are strongly upregulated by transforming growth factor-beta(s) and phorbol esters.

BRD4 associates with p53 in DNMT3A-mutated leukemia cells and is implicated in apoptosis by the bromodomain inhibitor JQ1

The bromodomain and extra terminal (BET) family protein bromodomain containing protein 4 (BRD4) is an epigenetic regulator recently identified as a therapeutic target for several hematological cancers, notably mixed lineage leukemia‐fusion acute myeloid leukemia (MLL‐AML). Here, we show that the BRD4 bromodomain inhibitor JQ1 is highly active against the p53‐wild‐type Ontario Cancer Institute (OCI)‐AML3 cell line which carries mutations in nucleophosmin (NPM1) and DNA methyltransferase 3 (DNMT3A) genes commonly associated with poor prognostic disease. We find that JQ1 causes caspase 3/7‐mediated apoptosis and DNA damage response in these cells. In combination studies, we show that histone deacetylase (HDAC) inhibitors, the HDM2 inhibitor Nutlin‐3, and the anthracycline daunorubicin all enhance the apoptotic response of JQ1. These compounds all induce activation of p53 suggesting that JQ1 might sensitize AML cells to p53‐mediated cell death. In further experiments, we show that BRD4 associates with acetylated p53 but that this association is not inhibited by JQ1 indicating that the protein–protein interaction does not involve bromodomain binding of acetylated lysines. Instead, we propose that JQ1 acts to prevent BRD4‐mediated recruitment of p53 to chromatin targets following its activation in OCI‐AML3 cells resulting in cell cycle arrest and apoptosis in a c‐MYC‐independent manner. Our data suggest that BET bromodomain inhibition might enhance current chemotherapy strategies in AML, notably in poor‐risk DNMT3A/NPM1‐mutated disease.

Galectin-1 stimulates monocyte chemotaxis via the p44/42 MAP kinase pathway and a pertussis toxin-sensitive pathway.

Galectin-1, the prototype of a family of beta-galactoside-binding proteins, has been implicated in a wide variety of biological processes. Data presented herein show that galectin-1 stimulates monocyte migration in a dose-dependent manner but is not chemotactic for macrophages. Galectin-1-induced monocyte chemotaxis is blocked by lactose and inhibited by an anti-galectin-1 antibody but not by nonspecific antibodies. Furthermore, galectin-1-mediated monocyte migration was significantly inhibited by MEK inhibitors in a rapid, time-dependent manner suggesting that MAP kinase pathways are involved in galectin-1. Migration was also almost completely blocked by pertussis toxin implying G-protein involvement in the galectin-1-induced chemotaxis. These results demonstrate a role for galectin-1 in monocyte chemotaxis which differs from galectin-3 in that macrophages are nonresponsive. Furthermore, our observations suggest that galectin-1 may be involved in chemoattraction at sites of inflammation in vivo and may contribute to disease processes such as atherosclerosis.

Developmental regulation and overexpression of the transcription factor AP-2, a potential regulator of the timing of Schwann cell generation

There is now evidence from in vivo and in vitro studies that the rate of Schwann cell generation is regulated by the balance of two opposing signals, β neuregulins and endothelins. The β neuregulins promote the development of precursors to Schwann cells whereas endothelins retard it through an action on endothelin‐B receptors. The present work has shown additional controls of this transition, and implicates AP‐2 transcription factors, in particular AP‐2α, as negative regulators of Schwann cell generation. We found that both AP‐2α and AP‐2γ are present in early embryonic nerves, whereas AP‐2β was not. Isoform‐specific analysis of AP‐2α showed that isoform 3 was most abundant with isoforms 1 and 2 present in lesser amounts; isoform 4 was absent. Maximal AP‐2α and AP‐2γ mRNA expression occurred at embryonic day (E) 12/13 in the mouse and at E14/15 in the rat, which correlates with the presence of Schwann cell precursors in the nerve. In both rats and in mice, in vivo and in vitro, downregulation of AP‐2α mRNA and protein coincided with one of the main steps in Schwann cell development, the precursor–Schwann cell transition. Moreover, Schwann cell generation was delayed if this downregulation was prevented by enforced expression of AP‐2α in precursors. These studies suggest that AP‐2 is involved in the control of the timing of Schwann cell development.

Substrate-induced phenotypical change of monocytes/macrophages into myofibroblast-like cells: a new insight into the mechanism of in-stent restenosis.

Stented coronary angioplasty is the procedure of choice to re-establish patency in obstructed coronary arteries. However, the stent implantation procedure often leads to in-stent restenosis, a process that is characterized by stent strut colonization by macrophages and smooth muscle cells and by neointima formation. The present in vitro study investigates the effect of stent materials on the phenotypical features of monocyte/macrophages. Human peripheral blood monocytes from healthy donors (n = 7) were cultured up to 7 days on substrates mimicking: (i) the stent surface (i.e., electropolished stainless steel), (ii) the de-endothelialized vessel wall (collagen-based extracellular matrix gel), and (iii) thrombus (i.e., fibrin gel). The cells were analyzed by immunocytochemistry for their ability to express alpha-actin, a typical myofibroblast marker, by ELISA to determine PDGF-BB and TGF-beta1 secretion and by PCR to evaluate hyaluronan synthase 1, 2, and 3 genes expression. Data were statistically analyzed by ANOVA (Dunnetts test) and data considered significantly different at p </= 0.05. The data demonstrated that mononuclear cells adhering to stainless steel acquire a phenotype capable of expressing alpha-actin while secreting significantly higher levels of PDGF-BB and TGF-beta. The expression of the three hyaluronan synthase isoforms was also altered by the metal substrate, where cells expressed genes only for the isoforms synthesizing high molecular weight hyaluronan. This study therefore suggests that mononuclear cells adhering on the stent metal surface undergo phenotypical transformation into myofibroblast-like cells that are able to contribute to neointimal tissue synthesis.

Nanog Requires BRD4 to Maintain Murine Embryonic Stem Cell Pluripotency and Is Suppressed by Bromodomain Inhibitor JQ1 Together with Lefty1

Embryonic stem cells (ESCs) are maintained in an undifferentiated state through expression of the core transcriptional factors Nanog, Oct4, and Sox2. However, the epigenetic regulation of pluripotency is poorly understood. Differentiation of ESCs is accompanied by a global reduction of panacetylation of histones H3 and H4 suggesting that histone acetylation plays an important role in maintenance of ESC pluripotency. Acetylated lysine residues on histones are read by members of the bromodomain family that includes BET (bromodomain and extraterminal domain) proteins for which highly potent and selective inhibitors have been developed. In this study we demonstrate that the pan-BET bromodomain inhibitor JQ1 induces rapid spontaneous differentiation of murine ESCs by inducing marked transcriptional downregulation of Nanog as well as the stemness markers Lefty1 and Lefty2, but not Myc, often used as a marker of BET inhibitor activity in cancer. We show that the effects of JQ1 are recapitulated by knockdown of the BET family member BRD4 implicating this protein in Nanog regulation. These data are also supported by chromatin immunoprecipitation experiments which confirm BRD4 binding at the Nanog promoter that is known to require acetylation by the histone acetyltransferase MOF for transcriptional activity. In further support of our findings, we show that JQ1 antagonizes the stem cell-promoting effects of the histone deacetylase inhibitors sodium butyrate and valproic acid. Our data suggest that BRD4 is critical for the maintenance of ESC pluripotency and that this occurs primarily through the maintenance of Nanog expression.