Association of extracellular matrix proteins fibulin-1 and fibulin-2 with fibronectin in bone marrow stroma

Extracellular matrix (ECM) molecules, together with growth factors and stromal cells, regulate haematopoietic cell development in bone marrow (BM). We report here expression of ECM proteins fibulin-1 and fibulin-2 in mouse BM. In other tissues, fibulin-1 and fibulin-2 associate with fibronectin and other ECM proteins. Fibulin-2 has also been found to adhere to cells via beta3 integrins. We studied the association of fibulins with fibronectin in BM stroma. By confocal microscopy, fibulin-1 and fibulin-2 immunostainings were co-localized with fibronectin in the adherent layer of long-term BM cultures. In cell adhesion assays using recombinant proteins, mouse fibulin-2 adhered to human erythroid-megakaryocytic leukaemia cell line HEL. This adhesion was mediated by beta3 integrins. However, HEL cells did not adhere to human fibulin-2. We therefore studied a possible species-specific cell-adhesive activity of mouse fibulin-2 by using mouse megakaryocytes, obtained by culture of BM cells in the presence of thrombopoietin. These megakaryocytes did not adhere to mouse fibulin-2. Our findings suggested that the functional role of fibulin-1 and fibulin-2 in BM stroma is related to binding to the major cell adhesion protein fibronectin, whereas adhesion of mouse fibulin-2 to human cells containing the integrin beta3 chain is not related to an apparent physiological function of the protein.     

Proliferation of mouse mammary epithelial cells in vitro: interactions among epidermal growth factor, insulin-like growth factor I, ovarian hormones, and extracellular matrix proteins

The purpose of the present study was to investigate the role of extracellular matrix proteins (ECMs; collagens I and IV, fibronectin, and laminin) in modulating proliferative responses of normal mammary epithelial cells in serum-free culture to epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I). As EGF and IGF-I can alter steroid responses, the interactions among growth factors, estrogen, and R5020 were also investigated. We report the novel finding that all ECMs tested, but not a nonspecific attachment factor, poly-L-lysine (PL), promoted a highly synergistic proliferative response to EGF plus IGF-I. EGF receptors were significantly increased with culture time on all ECMs, but not on PL. IGF receptor expression was significantly 2- to 4-fold higher on all ECMs compared with PL. EGF decreased IGF-binding protein-2 (IGFBP-2) and IGFBP-3 by more than 50% in the presence of IGF-I on PL or collagen I. These results indicate that ECM-specific IGF-I/EGF synergism occurs in response to ECM up-regulation of growth factor receptors and EGF down-regulation of inhibitory IGFBPs. Growth factors did not synergize with estrogen and/or R5020. Instead, estrogen plus R5020 decreased EGF-plus IGF-I-induced proliferation in an ECM-dependent manner. These studies demonstrate that proliferation of normal mammary epithelial cells involves complex interactions among steroids, growth factors, binding proteins, and ECMs.     

MIG-17/ADAMTS controls cell migration by recruiting nidogen to the basement membrane in C. elegans

Mutations in the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) family of secreted proteases cause diseases linked to ECM abnormalities. However, the mechanisms by which these enzymes modulate the ECM during development are mostly unexplored. The Caenorhabditis elegans MIG-17/ADAMTS protein is secreted from body wall muscle cells and localizes to the basement membrane (BM) of the developing gonad where it controls directional migration of gonadal leader cells. Here we show that specific amino acid changes in the ECM proteins fibulin-1C (FBL-1C) and type IV collagen (LET-2) result in bypass of the requirement for MIG-17 activity in gonadal leader cell migration in a nidogen (NID-1)-dependent and -independent manner, respectively. The MIG-17, FBL-1C and LET-2 activities are required for proper accumulation of NID-1 at the gonadal BM. However, mutant FBL-1C or LET-2 in the absence of MIG-17 promotes NID-1 localization. Furthermore, overexpression of NID-1 in mig-17 mutants substantially rescues leader cell migration defects. These results suggest that functional interactions among BM molecules are important for MIG-17 control of gonadal leader cell migration. We propose that FBL-1C and LET-2 act downstream of MIG-17-dependent proteolysis to recruit NID-1 and that LET-2 also activates a NID-1-independent pathway, thereby inducing the remodeling of the BM required for directional control of leader cell migration.     

Implication of CD44 in adhesion-mediated overproduction of TGF-β and IL-1 in monocytes from patients with bone marrow fibrosis

Myelofibrosis (MF) is characterized by bone marrow (BM) fibrosis and excessive deposits of extracellular matrix (ECM) proteins which include fibronectin (FN), collagen type I and hyaluronic acid (HA). We have previously reported that adhesion to polystyrene overactivates MF monocytes. We now confirm their activation by increased CD25 expression and tyrosine phosphorylation. We hypothesize that ECM protein-adhesion molecule interactions induce overproduction of fibrogenic cytokines in MF monocytes leading to BM fibrosis. In this study we found that FN, collagen type I and HA induce 2–3-fold more TGF-β and 6–9-fold more interleukin (IL)-1 in MF monocytes than normal controls (NC). Since CD44 can function as the natural ligand for these proteins, its role was studied. We found that CD44 mediated most of the TGF-β and IL-1 produced. Immunoprecipitation of CD44 revealed three proteins at approximately 110 kD in MF monocytes and one in NC. Our results indicate that adhesion is important in overproduction of TGF-β and IL-1, and that their production is at least partly mediated by adhesion molecule–ECM protein interactions. These results implicate at least one adhesion molecule, CD44, in the pathophysiology of BM fibrosis.     

Varied low density lipoprotein binding property of proteoglycans synthesized by vascular smooth muscle cells cultured on extracellular matrix

Earlier we showed that the extracellular matrix (ECM) secreted by vascular cells modulated proteoglycan synthesis by vascular smooth muscle cells in culture and altered the proteoglycan characteristics. In this study, we tested the hypothesis that these ECM-mediated alterations increased the affinity of the proteoglycans for plasma low density lipoprotein (LDL). Newly synthesized proteoglycans were isolated from smooth muscle cells cultured on the ECMs secreted by vascular endothelial cells, smooth muscle cells, or THP-1 macrophages and their binding affinity for LDL determined. Proteoglycans from all cultures contained sub-fractions that bound LDL with low and high affinity. However, compared with the cells plated on the endothelial cell ECM, the cells plated on the smooth muscle cell ECM and macrophage ECM synthesized significantly more high affinity proteoglycans. Removal of collagen, elastin, and chondroitin sulfates from the smooth muscle cell ECM and chondroitin sulfates from the macrophage ECM increased the production of high affinity proteoglycans by 15-22%. However, neutralization of fibronectin from both ECMs decreased the high affinity proteoglycans by 20%. Removal of matrix-bound growth factors had no effect on the synthesis of high affinity proteoglycans. Compared with the low affinity proteoglycans, the high affinity proteoglycans were larger, more sulfated and contained higher proportions of chondritin sulfate, dermatan sulfate, and N-sulfated heparan sulfate chains. These results suggest that the ECM-mediated alterations in vascular smooth muscle cell proteoglycans may lead to increased deposition of LDL in the arterial wall.     

Extracellular matrix protein tenascin-C is required in the bone marrow microenvironment primed for hematopoietic regeneration

The BM microenvironment is required for the maintenance, proliferation, and mobilization of hematopoietic stem and progenitor cells (HSPCs), both during steady-state conditions and hematopoietic recovery after myeloablation. The ECM meshwork has long been recognized as a major anatomical component of the BM microenvironment; however, the molecular signatures and functions of the ECM to support HSPCs are poorly understood. Of the many ECM proteins, the expression of tenascin-C (TN-C) was found to be dramatically up-regulated during hematopoietic recovery after myeloablation. The TN-C gene was predominantly expressed in stromal cells and endothelial cells, known as BM niche cells, supporting the function of HSPCs. Mice lacking TN-C (TN-C(-/-)) mice showed normal steady-state hematopoiesis; however, they failed to reconstitute hematopoiesis after BM ablation and showed high lethality. The capacity to support transplanted wild-type hematopoietic cells to regenerate hematopoiesis was reduced in TN-C(-/-) recipient mice. In vitro culture on a TN-C substratum promoted the proliferation of HSPCs in an integrin α9-dependent manner and up-regulated the expression of the cyclins (cyclinD1 and cyclinE1) and down-regulated the expression of the cyclin-dependent kinase inhibitors (p57(Kip2), p21(Cip1), p16(Ink4a)). These results identify TN-C as a critical component of the BM microenvironment that is required for hematopoietic regeneration.     

Ovarian tissue remodeling: role of matrix metalloproteinases and their inhibitors

Follicular formation, growth or atresia, and ovulation as well as luteal formation and subsequent regression are dependent upon cyclical remodeling of the extracellular matrix (ECM). The proteinaceous and nonproteinaceous components of the ECM provide the tissue specific, extracellular architecture to which cells attach. Furthermore, the ECM modulates cellular activities through cellular surface receptors and serves as a reservoir for specific growth factors, cytokines, and binding proteins. The ability of the ECM to direct the proliferation, differentiation and function of cells implicates ECM remodeling in normal ovarian function. Specific components of the ECM are cleaved by matrix metalloproteinases (MMPs) whose activities are specifically inhibited by tissue inhibitors of metalloproteinases (TIMPs). MMPs are zinc- and calcium-dependent enzymes that collectively degrade proteinaceous components of the ECM. Controlled turnover of ECM by MMPs and TIMPs may be essential for creating and (or) preserving microenvironments conducive to follicular and luteal function and is likely dependent upon the ratio of enzyme to inhibitor. To date, most studies have focused upon correlating ovarian expression of MMPs and TIMPs with various stages of the reproductive cycle. From these studies, many potential key regulators of ovarian ECM remodeling have been identified. This review presents evidence for the involvement of MMPs and TIMPs in ECM remodeling associated with follicular and luteal function.     

Independent regulation of tumor cell migration by matrix stiffness and confinement

Tumor invasion and metastasis are strongly regulated by biophysical interactions between tumor cells and the extracellular matrix (ECM). While the influence of ECM stiffness on cell migration, adhesion, and contractility has been extensively studied in 2D culture, extension of this concept to 3D cultures that more closely resemble tissue has proven challenging, because perturbations that change matrix stiffness often concurrently change cellular confinement. This coupling is particularly problematic given that matrix-imposed steric barriers can regulate invasion speed independent of mechanics. Here we introduce a matrix platform based on microfabrication of channels of defined wall stiffness and geometry that allows independent variation of ECM stiffness and channel width. For a given ECM stiffness, cells confined to narrow channels surprisingly migrate faster than cells in wide channels or on unconstrained 2D surfaces, which we attribute to increased polarization of cell-ECM traction forces. Confinement also enables cells to migrate increasingly rapidly as ECM stiffness rises, in contrast with the biphasic relationship observed on unconfined ECMs. Inhibition of nonmuscle myosin II dissipates this traction polarization and renders the relationship between migration speed and ECM stiffness comparatively insensitive to matrix confinement. We test these hypotheses in silico by devising a multiscale mathematical model that relates cellular force generation to ECM stiffness and geometry, which we show is capable of recapitulating key experimental trends. These studies represent a paradigm for investigating matrix regulation of invasion and demonstrate that matrix confinement alters the relationship between cell migration speed and ECM stiffness.     

Neurite outgrowth on muscle cell surfaces involves extracellular matrix receptors as well as Ca2+-dependent and -independent cell adhesion molecules.

To identify the molecules on the neuronal surface that mediate axonal growth on myotubes, we have examined neurite formation by ciliary neurons grown on myotubes in the absence or presence of specific antibodies. Dramatic inhibition of neurite outgrowth was seen only when antibodies blocked simultaneously the functions of two cell adhesion molecules--neural cell adhesion molecule (N-CAM) and neural Ca2+-dependent CAM (N-Cal-CAM)--and the neuronal receptors for several extracellular matrix (ECM) proteins. Although the antibody used to block ECM receptors (JG22) has been shown to eliminate almost all neurite growth on ECMs, it had only small effects on neurite growth on myotubes, reducing somewhat the length of neurites. Similarly, antibodies to the two CAMs, when used alone, had no detectable effects on neurite length and, when used together, had only small inhibitory effects on neurite growth. Combination of anti-ECM receptor (JG22) with antibodies to either CAM, however, greatly shortened the length of neurites. These results imply that ECM receptors and the CAMs N-CAM and N-Cal-CAM are major macromolecules used by neuronal growth cones for interactions with myotubes. Each provides a distinct mechanism for regulating growth cone motility.     

Human bone marrow mesenchymal stem cells in vivo

Great confusion still exists amongst cell biologists, musculoskeletal and other specialists interested in regenerative medicine regarding the in vivo identity of human bone marrow (BM) mesenchymal stem cells (MSCs). Contrary to views held in some quarters, methods for the robust identification and purification of BM MSCs are now well established. Human BM MSCs represent a phenotypically homogeneous cell population that share an identical phenotype with marrow adventitial reticular cells (ARCs), which are stromal cells similar in nature to pericytes. When an extensive panel of markers is used to characterize BM MSCs, it appears that the diverse MSC markers described in different laboratories are expressed on the same cell population. Rare cell phenotypical analysis and in vitro colony forming unit-fibroblast (CFU-F) assays produce no compelling evidence that BM MSCs circulate in healthy man. Furthermore, although investigators speak of a number of specific MSC markers, a true marker of MSC 'stemness' and multipotentiality has not yet been defined since culture-expanded MSCs may lose some of these markers, but remain multipotential. This knowledge provides a platform for understanding MSCs in vivo leading to novel approaches for therapy development, including in situ tissue engineering.     

Excreted corticosterone metabolites co-vary with ambient temperature and air pressure in male Greylag geese (Anser anser)

In many species, seasonal activities such as reproduction or migration need to be fine-tuned with weather conditions. Air pressure and temperature changes are the best parameters for such conditions. Adapting to climatic changes invariably involves physiological and behavioral reactions associated with the adrenals. In the present study, we investigated the effects of ambient temperature and air pressure on excreted immuno-reactive metabolites of corticosterone (BM) and androgens (AM). Focal individuals were 14 paired male greylag geese (Anser anser) from a semi-tame, unrestrained flock. BM and AM were measured in individual fecal samples over 25 days in November and December. Two different ACTH-validated assays were used for the assessment of BM: the first one cross-reacting with 11beta,21-diol-20-one structures ("old assay") and the second one with 5beta,3alpha,11beta-diol structures ("new assay"). With the "new assay," BM correlated negatively with the minimum ambient temperature of the night before, which may reflect corticosterone involvement in thermoregulation. BM also correlated positively with the minimum air pressure of the previous afternoon, which supports the value of air pressure for predicting weather conditions. Together, these reactions suggest a role of the adrenals in responding behaviorally and physiologically to changes in weather. Preliminary analysis indicated a higher sensitivity to the excreted glucocorticosteroid metabolites in the "new assay." As expected for outside the mating season, no relationships were found between excreted AM and the weather parameters considered. The gradual changes in BM excretion in parallel with weather conditions may be part of the fine-tuning of physiology and behavior by environmental clues.     

Bone marrow cyclooxygenase-2 levels are elevated in chronic-phase chronic myeloid leukaemia and are associated with reduced survival

Increased angiogenesis is important in the pathophysiology of haematological malignancies. Cyclooxygenase-2 (Cox-2) converts arachidonic acid to prostaglandins, which induce expression of angiogenic factors, including vascular endothelial growth factor (VEGF), basic-fibroblast growth factor, transforming growth factor-beta and interleukin 6. Cox-2 may also reduce apoptosis and reduce cellular attachment to the extracellular matrix (ECM). Increased bone marrow (BM) vascularity, increased BM cellular and plasma VEGF levels, and decreased progenitor adherence to BM ECM have all been observed in chronic myeloid leukaemia (CML). We investigated the prognostic significance of levels of Cox-2 in BM cells from patients with CML. Western blot and solid-phase radioimmunoassay (RIA) were used to measure Cox-2 BM levels in 149 patients with chronic phase CML (CP CML). Results were compared with those of normal controls. Expression of Cox-2 was significantly higher in CML than in normal controls (P < 0.0001). Increasing levels of Cox-2 were significantly associated with shorter survival (P = 0.0002, Cox proportional hazard model). A multivariate model based on Cox-2 and degree of splenomegaly was developed for survival in patients with early CP CML. Agents that inhibit Cox-2 activity merit investigation in patients with CP CML.     

A Self-Healing Hierarchical Fiber Hydrogel That Mimics ECM Structure

Although there have been many studies on using hydrogels as substitutes for natural extracellular matrices (ECMs), hydrogels that mimic the structure and properties of ECM remain a contentious topic in current research. Herein, a hierarchical biomimetic fiber hydrogel was prepared using a simple strategy, with a structure highly similar to that of the ECM. Cell viability experiments showed that the hydrogel not only has good biocompatibility but also promotes cell proliferation and growth. It was also observed that cells adhere to the fibers in the hydrogel, mimicking the state of cells in the ECM. Lastly, through a rat skin wound repair experiment, we demonstrated that this hydrogel has a good effect on promoting rat skin healing. Its high structural similarity to the ECM and good biocompatibility make this hydrogel a good candidate for prospective applications in the field of tissue engineering.     

Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins

The sketches in Figures 11 and 12, necessarily simplified, summarize some of the points discussed in the previous paragraphs. It can be concluded that the hepatic ECM and the ECM in general is a complicated supramolecular assembly of numerous defined macromolecules, each of which is endowed with a specific potential for interactions with one another and with various cell types. This matrix not only adapts rapidly to slight metabolic changes of the cells producing it, but also itself modulates the biochemical and morphologic phenotype of the cells anchored to it. Since mesenchymal cells appear to be the major source of hepatic ECM proteins in health and disease, it follows that the mesenchyme determines to a significant degree the phenotype of liver epithelial cells. Based on the growing knowledge of the hepatic ECM, immunoassays for ECM proteins or peptides in serum may provide information about the dynamics of fibrogenesis or fibrolysis in individual patients on a day to day basis, or permit detection of derangements of the hepatic ECM before they become clinically apparent. The clinical value of such assays is already being studied. Finally, knowing the molecules and the cells involved in ECM pathologic states offers us the tools to develop a specific targeted antifibrogenic therapy.     

The phenotypic fate and functional role for bone marrow-derived stem cells in liver fibrosis

Liver fibrosis is an outcome of chronic liver injury of any etiology. It is manifested by extensive deposition of extracellular matrix (ECM) proteins that produce a fibrous scar in the injured liver. Bone marrow (BM) cells may play an important role in pathogenesis and resolution of liver fibrosis. BM cells contribute to the inflammatory response by TGF-β1 secretion and activation of liver resident myofibroblasts. Moreover, BM itself can serve as a source of collagen expressing cells, e.g. BM-derived fibrocytes and mesenchymal progenitors, which in turn, have a potential to in situ differentiate into fibrogenic myofibroblasts and facilitate fibrosis. Finally, BM cells play an active part in resolution of liver fibrosis after cessation of fibrogenic stimuli. While natural killer (NK) cells are implicated in apoptosis of activated hepatic stellate cells/myofibroblasts, cells of myelo-monocitic lineage secrete matrix metalloproteinases to actively degrade the fibrous scar. The focus of this review is on the current understanding of the role of different subsets of BM cells in the onset, development and resolution of liver fibrosis.     

In vivo diffusion of lactoferrin in brain extracellular space is regulated by interactions with heparan sulfate

The intercellular spaces between neurons and glia contain an amorphous, negatively charged extracellular matrix (ECM) with the potential to shape and regulate the distribution of many diffusing ions, proteins and drugs. However, little evidence exists for direct regulation of extracellular diffusion by the ECM in living tissue. Here, we demonstrate macromolecule sequestration by an ECM component in vivo, using quantitative diffusion measurements from integrative optical imaging. Diffusion measurements in free solution, supported by confocal imaging and binding assays with cultured cells, were used to characterize the properties of a fluorescently labeled protein, lactoferrin (Lf), and its association with heparin and heparan sulfate in vitro. In vivo diffusion measurements were then performed through an open cranial window over rat somatosensory cortex to measure effective diffusion coefficients (D*) under different conditions, revealing that D* for Lf was reduced approximately 60% by binding to heparan sulfate proteoglycans, a prominent component of the ECM and cell surfaces in brain. Finally, we describe a method for quantifying heparan sulfate binding site density from data for Lf and the structurally similar protein transferrin, allowing us to predict a low micromolar concentration of these binding sites in neocortex, the first estimate in living tissue. Our results have significance for many tissues, because heparan sulfate is synthesized by almost every type of cell in the body. Quantifying ECM effects on diffusion will also aid in the modeling and design of drug delivery strategies for growth factors and viral vectors, some of which are likely to interact with heparan sulfate.