John C. Angello

Formation of Hyaluronan- and Versican-Rich Pericellular Matrix Is Required for Proliferation and Migration of Vascular Smooth Muscle Cells

The accumulation of hyaluronan (HA) and the HA-binding proteoglycan versican around smooth muscle cells in lesions of atherosclerosis suggests that together these molecules play an important role in the events of atherogenesis. In this study we have examined the formation of HA- and versican-rich pericellular matrices by human aortic smooth muscle cells in vitro, using a particle-exclusion assay, and the role of the pericellular matrix in cell proliferation and migration. The structural dependence of the pericellular matrix on HA can be demonstrated by the complete removal of the matrix with Streptomyces hyaluronidase. The presence of versican in the pericellular matrix was confirmed immunocytochemically. By electron microscopy, the cell coat was seen as a tangled network of hyaluronidase-sensitive filaments decorated with ruthenium red-positive proteoglycan granules. Ninety percent of migrating cells in wounded cultures, and virtually all mitotic cells, displayed abundant HA- and versican-rich coats. Time-lapse video imaging revealed that HA- and versican-rich pericellular matrix formation is dynamic and rapid, and coordinated specifically with cell detachment and mitotic cell rounding. HA oligosaccharides, which inhibit the binding of HA to the cell surface and prevent pericellular matrix formation, significantly reduced proliferation and migration in response to platelet-derived growth factor, whereas larger HA fragments and high molecular weight HA had no effect. Treatment with HA oligosaccharides also led to changes in cell shape from a typical fusiform morphology to a more spread and flattened appearance. These data suggest that organization of HA- and versican-rich pericellular matrices may facilitate migration and mitosis by diminishing cell surface adhesivity and affecting cell shape through steric exclusion and the viscous properties of HA proteoglycan gels.

EGF and TGF-α stimulate retinal neuroepithelial cell proliferation in vitro

Abstract Peptide growth factors have been shown to have diverse effects on cells of the CNS, such as promoting neuronal survival, neurite outgrowth, and several other aspects of neuronal differentiation. In addition, some of these factors have been shown to be mitogenic for particular classes of glial cells within the brain and optic nerve, and recently two peptide growth factors, fibroblast growth factor and nerve growth factor, have been shown to have mitogenic activity on the CNS neuronal progenitors. We now report that two members of another peptide growth factor, epidermal growth factor and transforming growth factor-α, are mitogenic for retinal neuroepithelial cells in primary cultures and provide evidence for the presence of both of these factors in normal developing rat retina.

Organized type I collagen influences endothelial patterns during “spontaneous angiogenesis in vitro”: Planar cultures as models of vascular development

SummarySelected strains of vascular endothelial cells, grown as confluent monolayers on tissue culture plastic, generate flat networks of cellular cords that resemble beds of capillaries—a phenomenon referred to as “spontaneous angiogenesis in vitro”. We have studied spontaneous angiogenic activity by a clonal population (clone A) of bovine aortic endothelial cells to indentify processes that mediate the development of cellular networks. Confluent cultures of clone A endothelial cells synthesized type I collagen, a portion of which was incorporated into narrow, extracellular cables that formed a planar network beneath the cellular monolayer. The collagenous cables acted as a template for the development of cellular networks: flattened, polygonal cells of the monolayer that were in direct contact with the cables acquired spindle shapes, associated to form cellular cords, and became elevated above the monolayer. Networks of cables and cellular cords did not form in a strain of bovine aortic endothelial cells that did not synthesize type I collagen, or when traction forces generated by clone A endothelial cells were inhibited with cytochalasin D. In a model of cable development, tension applied by a confluent monolayer of endothelial cells reorganized a sheetlike substrate of malleable type I collagen into a network of cables via the formation and radial enlargement of perforations through the collagen sheet. Our results point to a general involvement of extracellular matrix templates in two-dimensional (planar) models of vascular development in vitro. For several reasons, planar models simulate invasive angiogenesis poorly. In contrast, planar models might offer insights into the growth and development of planar vascular systems in vivo.

Quantitative Proteomic Identification of Six4 as the Trex-Binding Factor in the Muscle Creatine Kinase Enhancer

ABSTRACT Transcriptional regulatory element X (Trex) is a positive control site within the Muscle creatine kinase (MCK) enhancer. Cell culture and transgenic studies indicate that the Trex site is important for MCK expression in skeletal and cardiac muscle. After selectively enriching for the Trex-binding factor (TrexBF) using magnetic beads coupled to oligonucleotides containing either wild-type or mutant Trex sites, quantitative proteomics was used to identify TrexBF as Six4, a homeodomain transcription factor of the Six/sine oculis family, from a background of ∼900 copurifying proteins. Using gel shift assays and Six-specific antisera, we demonstrated that Six4 is TrexBF in mouse skeletal myocytes and embryonic day 10 chick skeletal and cardiac muscle, while Six5 is the major TrexBF in adult mouse heart. In cotransfection studies, Six4 transactivates the MCK enhancer as well as muscle-specific regulatory regions of Aldolase A and Cardiac troponin C via Trex/MEF3 sites. Our results are consistent with Six4 being a key regulator of muscle gene expression in adult skeletal muscle and in developing striated muscle. The Trex/MEF3 composite sequence ([C/A]ACC[C/T]GA) allowed us to identify novel putative Six-binding sites in six other muscle genes. Our proteomics strategy will be useful for identifying transcription factors from complex mixtures using only defined DNA fragments for purification.

Fibroblast growth factor receptor-1 mediates the inhibition of endothelial cell proliferation and the promotion of skeletal myoblast differentiation by SPARC: A role for protein kinase A

The role of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) in modulation of vascular cell proliferation is believed to be mediated, in part, by its ability to regulate the activity of certain growth factors through direct binding. In this study, we demonstrate that SPARC does not bind to basic fibroblast growth factor (bFGF/FGF‐2) or interfere with complex formation between FGF‐2 and its high‐affinity FGF receptor‐1 (FGFR1), yet both native SPARC and a peptide derived from the C‐terminal high‐affinity Ca2+‐binding region of protein significantly inhibit ligand‐induced autophosphorylation of FGFR1 (>80%), activation of mitogen‐activated protein kinases (MAPKs) (>75%), and DNA synthesis in human microvascular endothelial cells (HMVEC) stimulated by FGF‐2 (>80%). We also report that in the presence of FGF‐2, a factor which otherwise stimulates myoblast proliferation and the repression of terminal differentiation, both native SPARC and the Ca2+‐binding SPARC peptide significantly promote (>60%) the differentiation of the MM14 murine myoblast cell line that expresses FGFR1 almost exclusively. Moreover, using heparan sulfate proteoglycan (HSPG)‐deficient myeloid cells and porcine aortic endothelial cells (PAECs) expressing chimeric FGFR1, we show that antagonism of FGFR1‐mediated DNA synthesis and MAPK activation by SPARC does not require the presence of cell‐surface, low‐affinity FGF‐2 receptors, but can be mediated by an intracellular mechanism that is independent of an interaction with the extracellular ligand‐binding domain of FGFR1. We also report that the inhibitory effect of SPARC on DNA synthesis and MAPK activation in endothelial cells is mediated in part (>50%) by activation of protein kinase A (PKA), a known regulator of Raf‐MAPK pathway. SPARC thus modulates the mitogenic effect of FGF‐2 downstream from FGFR1 by selective regulation of the MAPK signaling cascade. J. Cell. Biochem. 90: 408–423, 2003.

The relationship between the rate of entry into S phase, concentration of DNA polymerase α, and cell volume in human diploid fibroblast-like monokaryon cells☆

We have examined the kinetic relationship between the rate of entry into the S phase in human diploid fibroblast-like (HDFL) monokaryon cells and (1) the concentration of DNA polymerase alpha activity and (2) the cell volume. In the former studies, a first-order dependence between the rate of entry into the S phase and the concentration of DNA polymerase alpha activity was observed, consistent with the enzyme, or a coregulated factor, being rate limiting for this metabolic process. Examination of the nature of the dependence of the rate of entry into the S phase upon cell volume revealed a more complex relationship. The results obtained in studies with synchronized cultures are consistent with the presence of two to three rate-limiting reactants when cell volume is the independent variable. Studies with asynchronous HDFL cell cultures revealed that the smallest cells in the G1 population, presumably the early G1 cells, enter the S phase at an increasing rate as a function of cell volume up to a certain size, beyond which the cells enter at a decreasing rate similar to that observed in the studies with the synchronized cultures. Similar studies examining the relationship between cell volume and the rate of entry into S phase in three established immortal cell lines revealed positive correlation between the rate of entry into S phase and cell volume throughout the size range of the G1 population. This latter observation suggests that the factors involved in the initiation of the S phase may be present in concentrations that are not rate limiting in immortal cell lines.

Hyaluronic acid synthesis and turnover by myotubes in culture

Analysis of glycosaminoglycan (GAG) synthesis by high density and clonal cultures of chick myoblasts and fibroblasts, and by cultures enriched for myotubes, indicates that all culture types produce hyaluronic acid (HA) and sulfated polysaccharides (S-GAG). Of these cell types the myotube synthesizes the most HA-rich complement of polysaccharides and has the most HA-rich cell surface. It appears that this is the result of decreased S-GAG synthesis rather than increased HA synthesis. Myotube cell surface HA is qualitatively different from HA released to the medium; cell surface HA is predominantly a single high molecular weight species, whereas HA isolated from the medium comprises a heterogeneous mixture of lower molecular weight HA. Since HA molecules of similar size classes can be generated by treating high molecular weight HA with hyaluronidase, an endogenous hyaluronidase could be involved. Alternatively, myotube-enriched cultures may synthesize and secrete HA polymers of several intermediate lengths.

The relationship between cell size, the activity of DNA polymerase α and proliferative activity in human diploid fibroblast-like cell cultures

In kinetic studies with human diploid fibroblast-like (HDFL) cells carried out in heterokaryons and in monokaryons, we have observed a first-order relationship between the level or concentration of DNA polymerase alpha and the rate of initiation of new rounds of DNA synthesis. Because cell size is inversely proportional to the concentration of DNA polymerase alpha and presumably other replication factors, it is inversely related to the initiation of new rounds of DNA synthesis. An inverse relationship between cell size and clonogenic activity was also observed in both unsorted HDFL cells and in HDFL cells sorted on the basis of size. Experimental enlargement of cells by serum deprivation at low density resulted in changes in colony-forming ability that would be predicted by these studies. A causal relationship between the observed increase in cell size with advancing passage level and the loss of proliferative activity is suggested by these studies; in addition, cell size may be a useful biophysical marker for cellular aging.

Hyaluronate-cell interaction. Effects of exogenous hyaluronate on muscle fibroblast cell surface composition.

Abstract Evidence that exogenous hyaluronate (HA) binds to the surface of muscle fibroblast cultures was obtained by incubating confluent fibroblasts with 14C-HA purified from fibroblast cell surfaces. Surface-bound 14C-HA was operationally defined as material resistant to six saline washes and solubilized by brief trypsinization. All of the surface-bound radioactivity remains as authentic HA. Exposure of fibroblasts to 100 μg/ml cold HA caused a nearly 3-fold ‘reduction’ in incorporation of isotopic precursors into glycosaminoglycan (GAG); but when intracellular 14C precursors to GAG were quantitated, the entire ‘reduction’ could be accounted for by decreased precursor uptake. Exposure to exogenous HA altered the distribution of newly synthesized GAG by stimulating an increase in total GAG secreted to the medium at the expense of that bound to the culture surface. Qualitatively, the cell surface ratio of 14C-HA: 14C-sulfated GAG (SGAG) of HA-treated cells is about 2.5 times greater than that of untreated cells and the medium ratio is correspondingly reversed. This is primarily the result of stimulated 14C-SGAG release to the culture medium. Addition of cold HA to prelabeled cultures also stimulates the selective turnover of SGAG from the culture surface. Thus, exposure to HA alters the fibroblast surface by accumulation of exogenous HA as well as by stimulation of SGAG turnover.

BMP Induction of Cardiogenesis in P19 Cells Requires Prior Cell-Cell Interaction(s)

Mouse P19 embryonal carcinoma cells undergo cardiogenesis in response to high density and DMSO. We have derived a clonal subline that undergoes cardiogenesis in response to high density, but without requiring exposure to DMSO. The new subline retains the capacity to differentiate into skeletal muscle and neuronal cells in response to DMSO and retinoic acid. However, upon aggregation, these Oct 4‐positive cells, termed P19‐SI because they “self‐induce” cardiac muscle, exhibit increased mRNAs encoding the mesodermal factor Brachyury, cardiac transcription factors Nkx 2.5 and GATA 4, the transcriptional repressor Msx‐1, and cytokines Wnt 3a, Noggin, and BMP 4. Exposure of aggregated P19‐SI cells to BMP 4, a known inducer of cardiogenesis, accelerates cardiogenesis, as determined by rhythmic beating and myosin staining. However, cardiogenesis is severely inhibited when P19‐SI cells are aggregated in the presence of BMP 4. These results demonstrate that cell–cell interaction is required before P19‐SI cells can undergo a cardiogenic response to BMP 4. A concurrent increase in the expression of Msx‐1 suggests one possible process underlying the inhibition of cardiogenesis. The phenotype of P19‐SI cells offers an opportunity to explore new aspects of cardiac induction. Developmental Dynamics 235:2122–2133, 2006.