The extracellular matrix dimension of skeletal muscle development.

Cells anchor to substrates by binding to extracellular matrix (ECM). In addition to this anchoring function however, cell-ECM binding is a mechanism for cells to sense their surroundings and to communicate and coordinate behaviour amongst themselves. Several ECM molecules and their receptors play essential roles in muscle development and maintenance. Defects in these proteins are responsible for some of the most severe muscle dystrophies at every stage of life from neonates to adults. However, recent studies have also revealed a role of cell-ECM interactions at much earlier stages of development as skeletal muscle forms. Here we review which ECM molecules are present during the early phases of myogenesis, how myogenic cells interact with the ECM that surrounds them and the potential consequences of those interactions. We conclude that cell-ECM interactions play significant roles during all stages of skeletal muscle development in the embryo and suggest that this "extracellular matrix dimension" should be added to our conceptual network of factors contributing to skeletal myogenesis.

TNF-alpha regulates the effects of irradiation in the mouse bone marrow microenvironment.

BACKGROUND: Secondary bone marrow (BM) myelodysplastic syndromes (MDS) are increasingly common, as a result of radio or chemotherapy administered to a majority of cancer patients. Patients with secondary MDS have increased BM cell apoptosis, which results in BM dysfunction (cytopenias), and an increased risk of developing fatal acute leukemias. In the present study we asked whether TNF-alpha, known to regulate cell apoptosis, could modulate the onset of secondary MDS. PRINCIPAL FINDINGS: We show that TNF-alpha is induced by irradiation and regulates BM cells apoptosis in vitro and in vivo. In contrast to irradiated wild type (WT) mice, TNF-alpha deficient (TNF-alpha KO) mice or WT mice treated with a TNF-alpha-neutralizing antibody were partially protected from the apoptotic effects of irradiation. Next we established a 3-cycle irradiation protocol, in which mice were sub-lethally irradiated once monthly over a 3 month period. In this model, irradiated WT mice presented loss of microsatellite markers on BM cells, low white blood cell (WBC) counts, reduced megakaryocyte (MK) and platelet levels (thrombocytopenia) and macrocytic anemia, phenoypes that suggest the irradiation protocol resulted in BM dysfunction with clinical features of MDS. In contrast, TNF-alpha KO mice were protected from the irradiation effects: BM cell apoptosis following irradiation was significantly reduced, concomitant with sustained BM MK numbers and absence of other cytopenias. Moreover, irradiated WT mice with long term (> or = 5 months) BM dysfunction had increased BM angiogenesis, MMPs and VEGF and NFkB p65, suggestive of disease progression. CONCLUSION: Taken together, our data shows that TNF-alpha induction following irradiation modulates BM cell apoptosis and is a crucial event in BM dysfunction, secondary MDS onset and progression.

Characterization and clinical relevance of circulating and biopsy-derived endothelial progenitor cells in lymphoma patients.

BACKGROUND AND OBJECTIVES: Endothelial progenitor cells (EPC) have been proven to be essential for tumor angiogenesis and growth in animal tumor models. However, the involvement and relevance of EPC in human cancers remain poorly studied. We, therefore, investigated the presence, differentiation potential and molecular characteristics of EPC in lymphoma patients. DESIGN AND METHODS: EPC (CD133+CD34+KDR+ cells) were detected in peripheral blood (PB) and lymph node (LN) biopsy samples of 70 lymphoma patients by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. Magnetically isolated EPC (PB and LN-derived) were tested, in vitro, for endothelial differentiation potential and RNA was collected to study their gene expression profiles by Affymetrix oligonucleotide arrays. Lymphoma patients were classified according to disease aggressiveness (indolent vs aggressive lymphoma) and their data (tumor angiogenesis, tumor stage and clinical treatment) were related to the presence or absence of EPC in the circulation or in tumor samples. RESULTS: Circulating EPC (CEPC) were more frequent in patients than in healthy controls and more frequent in younger patients than in older patients and in those with aggressive lymphomas. The levels of CEPC decreased in patients with complete response to treatment, but were sustained or increased in the non- or partial- responders to lymphoma therapy. Notably, EPC in the LN (LN-EPC) were more frequently detected than CEPC; LN-EPC were detected in vascular structures and also in the stroma, and after isolation differentiated into endothelial cells in vitro. The presence of LN-EPC correlated with lesion size and with increased angiogenesis in indolent lymphomas. CEPC and LN-EPC share endothelial markers but can be identified and quantified separately, since they express different CD133 isoforms. Gene expression profiling of isolated LN-EPC revealed the expression of pro-angiogenic and tumor growth factors that may influence lymphoma growth. INTERPRETATION AND CONCLUSIONS: EPC are present in the circulation and in tumor samples from patients with non-Hodgkin's lymphoma. Since there are relationships between EPC and various characteristics of lymphoma, our research has demonstrated the clinical and biological relevance of studying CEPC and LN-EPC in lymphoma patients.

Integrin repertoire on myogenic cells changes during the course of primary myogenesis in the mouse.

Cells interact with the extracellular matrix through receptors, most commonly of the integrin family. We (Cachaco et al. [2003] Development 130:1659-1671) and others (Schwander et al. [2003] Dev. Cell 4:673-685) have demonstrated a role for beta1 integrins in mouse primary myogenesis. However, it is unclear what alpha subunits pair with beta1 during this process in vivo. Here, we determined alpha subunit expression patterns at embryonic day (E) 11.5-E14.5. Differentiated myotomal myocytes express all alpha subunits studied. As the muscle masses form both in trunk (E12.5) and limbs (E11.5-E12.5), laminin receptors alpha6beta1 and alpha7beta1 are undetectable, and an assembled laminin matrix is absent. Instead alpha1beta1, alpha4beta1, alpha5beta1, and an alpha v-containing integrin are expressed and unassembled laminin and fibronectin are abundant around myogenic cells. At E13.5-E14.5, alpha6beta1 and alpha7beta1 are expressed, and a laminin matrix forms around individual myotubes. Thus, myogenic cells change their integrin expression pattern during the course of primary myogenesis in the mouse, suggesting different roles for fibronectin- and laminin-containing matrices in this process.

Knock-in of integrin beta 1D affects primary but not secondary myogenesis in mice.

Integrins are extracellular matrix receptors composed of alpha and beta subunits involved in cell adhesion, migration and signal transduction. The beta1 subunit has two isoforms, beta 1A ubiquitously expressed and beta 1D restricted to striated muscle. They are not functionally equivalent. Replacement of beta 1A by beta 1D (beta 1D knock-in) in the mouse leads to midgestation lethality on a 50% Ola/50% FVB background [Baudoin, C., Goumans, M. J., Mummery, C. and Sonnenberg, A. (1998). Genes Dev. 12, 1202-1216]. We crossed the beta 1D knock-in line into a less penetrant genetic background. This led to an attenuation of the midgestation lethality and revealed a second period of lethality around birth. Midgestation death was apparently not caused by failure in cell migration, but rather by abnormal placentation. The beta 1D knock-in embryos that survived midgestation developed until birth, but exhibited severely reduced skeletal muscle mass. Quantification of myotube numbers showed that substitution of beta 1A with beta 1D impairs primary myogenesis with no direct effect on secondary myogenesis. Furthermore, long-term primary myotube survival was affected in beta 1D knock-in embryos. Finally, overexpression of beta 1D in C2C12 cells impaired myotube formation while overexpression of beta 1A primarily affected myotube maturation. Together these results demonstrate for the first time distinct roles for beta1 integrins in primary versus secondary myogenesis and that the beta 1A and beta 1D variants are not functionally equivalent in this process.