Transitions towards either slow-oxidative or fast-glycolytic phenotype can be induced in the murine WTt myogenic cell line.

Contraction and energy metabolism are functions of skeletal muscles co-regulated by still largely unknown signals. To help elucidating these interconnecting pathways, we are developing new cellular models that will allow to control the switch from a neonatal to an adult slow-oxidative or fast-glycolytic phenotype of myofibers, during in vitro differentiation. Thus, our purpose was to direct the differentiation of the newly characterized WTt clone, from a mixed towards either fast or slow phenotype, by modifying amounts of two transcription factors respectively involved in control of glycolytic and oxidative energy metabolism, namely HIF-1alpha and PPARdelta. Our data support the idea that HIF-1alpha protein stabilization would favor expression of fast phenotypic markers, accompanied or not by a decreased expression of slow markers, depending on treatment conditions. Conversely, PPARdelta over-expression appears to enhance the slow-oxidative phenotype of WTt myotubes. Furthermore, we have observed that expression of PGC-1alpha, a coregulator of PPAR, is also modified in this cell line upon conditions that stabilize HIF-1alpha protein. This observation points to the existence of a regulatory link between pathways controlled by the two transcription factors HIF-1alpha and PPARdelta. Therefore, these cells should be useful to analyze the balance between oxidative and glycolytic energy production as a function of phenotypic transitions occurring during myogenic maturation. The newly characterized murine WTt clone will be a good tool to investigate molecular mechanisms implicating HIF-1alpha and PPARdelta in the coordinated metabolic and contractile regulations involved in myogenesis.

A unique family of endothelial cell polypeptide mitogens: the antigenic and receptor cross-reactivity of bovine endothelial cell growth factor, brain-derived acidic fibroblast growth factor, and eye-derived growth factor-II.

Bovine brain, hypothalamus, pituitary, and retina contain potent anionic polypeptide mitogens for endothelial cells. Immunological assays using murine monoclonal antibodies against bovine endothelial cell growth factor (ECGF) and radioreceptor assays using [125I]ECGF were performed to determine the cross-reactivity of ECGF with bovine acidic pI brain-derived fibroblast growth factor (acidic FGF) and bovine eye-derived growth factor-II [EDGF-II). We observed that acidic FGF and EDGF-II are recognized by anti-ECGF monoclonal antibodies and compete with [125I] ECGF for receptor occupancy. Furthermore, the biological activity of ECGF, acidic FGF, and EDGF-II is potentiated by the glycosaminoglycan, heparin. These results argue that ECGF, acidic FGF, and EDGF-II belong to a common family of polypeptide growth factors.

N6L pseudopeptide interferes with nucleophosmin protein-protein interactions and sensitizes leukemic cells to chemotherapy.

NPM1 is a multifunctional nucleolar protein implicated in several processes such as ribosome maturation and export, DNA damage response and apoptotic response to stress stimuli. The NPM1 gene is involved in human tumorigenesis and is found mutated in one third of acute myeloid leukemia patients, leading to the aberrant cytoplasmic localization of NPM1. Recent studies indicated that the N6L multivalent pseudopeptide, a synthetic ligand of cell-surface nucleolin, is also able to bind NPM1 with high affinity. N6L inhibits cell growth with different mechanisms and represents a good candidate as a novel anticancer drug for a number of malignancies of different histological origin. In this study we investigated whether N6L treatment could drive antitumor effect in acute myeloid leukemia cell lines. We found that N6L binds NPM1 at the N-terminal domain, co-localizes with cytoplasmic, mutated NPM1, and interferes with its protein-protein associations. N6L toxicity appears to be p53 dependent but interestingly, the leukemic cell line harbouring the mutated form of NPM1 is more resistant to treatment, suggesting that NPM1 cytoplasmic delocalization confers protection from p53 activation. Moreover, we show that N6L sensitizes AML cells to doxorubicin and cytarabine treatment. These studies suggest that N6L may be a promising option in combination therapies for acute myeloid leukemia treatment.

Targeting nucleolin for better survival in diffuse large B-cell lymphoma.

Anthracyclines have been a cornerstone in the cure of diffuse large B-cell lymphoma (DLBCL) and other hematological cancers. The ability of anthracyclines to eliminate DLBCL depends on the presence of topoisomerase-II-alpha (TopIIA), a DNA repair enzyme complex. We identified nucleolin as a novel binding partner of TopIIA. Abrogation of nucleolin sensitized DLBCL cells to TopIIA targeting agents (doxorubicin/etoposide). Silencing nucleolin and challenging DLBCL cells with doxorubicin enhanced the phosphorylation of H2AX (γH2AX-marker of DNA damage) and allowed DNA fragmentation. Reconstitution of nucleolin expression in nucleolin-knockdown DLBCL cells prevented TopIIA targeting agent-induced apoptosis. Nucleolin binding to TopIIA was mapped to RNA-binding domain 3 of nucleolin, and this interaction was essential for blocking DNA damage and apoptosis. Nucleolin silencing decreased TopIIA decatenation activity, but enhanced formation of TopIIA-DNA cleavable complexes in the presence of etoposide. Moreover, combining nucleolin inhibitors: aptamer AS1411 or nucant N6L with doxorubicin reduced DLBCL cell survival. These findings are of clinical importance because low nucleolin levels versus high nucleolin levels in DLBCL predicted 90-month estimated survival of 70% versus 12% (P<0.0001) of patients treated with R-CHOP-based therapy.

Cell Colonization Ability of a Commercialized Large Porous Alveolar Scaffold.

The use of filling biomaterials or tissue-engineered large bone implant-coupling biocompatible materials and human bone marrow mesenchymal stromal cells seems to be a promising approach to treat critical-sized bone defects. However, the cellular seeding onto and into large porous scaffolds still remains challenging since this process highly depends on the porous microstructure. Indeed, the cells may mainly colonize the periphery of the scaffold, leaving its volume almost free of cells. In this study, we carry out an in vitro study to analyze the ability of a commercialized scaffold to be in vivo colonized by cells. We investigate the influence of various physical parameters on the seeding efficiency of a perfusion seeding protocol using large manufactured bone substitutes. The present study shows that the velocity of the perfusion fluid and the initial cell density seem to impact the seeding results and to have a negative effect on the cellular viability, whereas the duration of the fluid perfusion and the nature of the flow (steady versus pulsed) did not show any influence on either the fraction of seeded cells or the cellular viability rate. However, the cellular repartition after seeding remains highly heterogeneous.

Effect of heparin affin regulatory peptide on the expression of vascular endothelial growth factor receptors in endothelial cells.

BACKGROUND: Heparin affin regulatory peptide (HARP) is an 18-kDa secreted protein that has been implicated in tumor growth and angiogenesis, although the mechanisms involved remain largely unknown. In the present work, the effect of human recombinant HARP on the expression of the vascular endothelial growth factor (VEGF) receptors KDR, Flt-1 and neuropilin-1 was studied in cultured human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: The mRNA and protein levels of VEGF receptors were estimated by semi-quantitative RT-PCR and Western blot, respectively. Cell proliferation and migration were measured by MTT, direct counting of the cells and modified Boyden chamber assays. RESULTS: HARP decreased the expression of KDR but increased the expression of Flt-1 and neuropilin-1 at both the mRNA and protein level. The effect reached a maximum 4 h after the addition of HARP into the cell culture medium and was reversed at later time-points. When HARP was added to the culture medium 4 h before the addition of VEGF165, it inhibited VEGF165-induced proliferation and migration of HUVEC. CONCLUSION: These data suggest that HARP affects the expression of VEGF receptors and inhibits VEGF165-induced activation of HUVEC.

Eye-derived growth factor isolated from bovine retina and used for epidermal wound healing in vivo.

Eye-derived growth factor (EDGF) has been found in several ocular tissues and shown to be able to stimulate the in vitro proliferation of cells from various tissues and organisms. It had already been shown that EDGF differs biochemically and biologically from other growth factors such as epidermal growth factor (EGF) and fibroblast growth factor in that it is the only one that can stimulate the in vitro growth of human adult keratinocytes. Moreover EDGF stimulates reepithelialization and neovascularization. In this paper we report data concerning the effect on the rate of epidermal wound healing in guinea pigs of different extracts obtained from adult bovine retina. Our results show that EDGF can significantly increase the rate of reepithelialization when epidermis is detached from dermis and removed after induction of a blister. The doses used were comparable to the ones used to obtain maximal increase of cell proliferation in vitro. However no attempt was made to further investigate the mechanism accounting for the observed wound healing. At 24 h, control wounds maintained under occlusive dressing had only about 50% of their surface covered with cells as opposed to EDGF-treated wounds which were covered up to about 80% (p = 0.05). On the other hand, EGF does not increase the rate of wound healing in this model even at 1000-fold higher doses than those used in in vitro bioassays. Although EDGF is still not purified to homogeneity and another 10- to 100-fold purification might be necessary to achieve homogeneity, our results suggest that EDGF may find therapeutic applications as a potent in vivo epidermal wound healing agent.

[In vitro properties of various growth factors and in vivo effects]. / Propriétés in vitro de quelques facteurs de croissance et effets in vivo.

This article summarizes some of the data that have been accumulated on several growth factors. Biochemical and biological properties of the Epidermal, Fibroblast, Astrocytes and Tumor growth factors (EGF, FGF, AGF, TGF) and those of growth factors derived from Platelets (PDGF), Brain (BDGF, ECGF), Eye (EDGF) and Cartilage (CDGF) are reviewed, as well as the in vitro mechanism of action of EGF and PDGF. The in vivo effects of these growth factors, particularly the experiments achieved to understand the physiological or physiopathological significance are described. The potential interest of these molecules in pharmacology and their use as wound healing agents is discussed.

Purification of basic fibroblast growth factor receptors from bovine brain.

Fibroblast growth factors are proteins which play a major role, in vitro and in vivo, in the control of cellular growth and differentiation of a large number of cells. Biological activities of these factors are mediated by the interaction with specific membrane receptors. Previous studies indicated that the apparent molecular weight of a family of these receptors for the basic form of Fibroblast Growth Factor (bFGF), ranges from 125 to 165 kDa according to cell species and types. We have purified this family of receptors from bovine brain. We first set up a radioreceptor assay to detect receptors throughout the purification by measuring its ability to inhibit the fixation of radiolabeled bFGF to insolubilized membranes from bovine brain. The purification was also monitored by using cross-linking reagents in order to allow the visualization of radiolabeled bFGF bound to its receptor. The first purification steps involved 2 anion-exchange chromatographic steps, DEAE Trysacryl and FPLC Mono Q, and yielded an enrichment over 500 fold. Affinity chromatography with bFGF immobilized on Sepharose 4B was then performed. Covalent fixation of bFGF to the Sepharose matrix was carried out in presence of N-acetylated heparin in order to protect the recognition site for bFGF on its receptor. These 3 chromatographic steps yielded only 2 bands of apparent molecular weight of 100 kDa and 135 kDa as detected by electrophoresis. These 2 bands are also detected after chromatography on immobilized wheat germ agglutinin hence confirming the presence of carbohydrates on bFGF receptors.

Purification of a heparin binding FGF receptor (HB-FGFR) from adult bovine brain membranes.

A new form of high affinity fibroblast growth factor receptor has been purified from adult bovine brain membranes. Purification was performed by chromatography on DEAE-Trisacryl and wheat germ agglutinin-agarose followed by FGF-2 affinity chromatography. Affinity labeling of purified fractions with 125I-FGF-2 showed after cross-linking a 170-kDa complex, suggesting the existence of a 150-kDa FGF receptor. No cross-reactivity with anti-FGF receptor 1 (FGFR-1 or flg) or with anti-receptor 2 (FGFR-2 or bek) antibodies could be detected with this partially purified receptor. Heparitinase treatment of the partially purified FGF receptor abolished the formation of the ligand receptor complex. The complex was restored in the presence of heparin in a dose dependent fashion, supporting the idea that heparin-like molecules are needed for proper binding. Further purification of the receptor was achieved by heparin-Sepharose affinity chromatography and yielded a purification of over 320,000-fold. The purified receptor fraction was radiolabeled and loaded on RPLC C4 column. Eluted fractions were analysed by SDS-PAGE. A major 150-kDa band was detected. These data show for the first time a new form of FGF receptor isolated from bovine brain membranes. This purified receptor displays affinity for heparin and was therefore named heparin binding FGF receptor (HB-FGFR). It remains unclear whether the receptor is a proteo-heparin sulfate or whether heparans are strongly associated and therefore are copurified. Large scale preparations are in progress for core protein structure studies.

Biochemical comparative studies between eye- and brain-derived growth factors.

Two bovine brain-derived growth factors, BDGF I and BDGF II, were isolated using the same extraction procedure as previously described for eye-derived growth factors (EDGF). The hypothesis that these growth factors were identical to EDGF I and EDGF II, respectively, was supported by their similar molecular weights (16,000 and 15,000, respectively) and isoelectric points (9.0 and 5.0, respectively), their identical retention behavior on reverse-phase chromatography and their similar amino acid compositions. From studies on their binding properties to cell surfaces, competition between EDGF I and BDGF I as well as competition between EDGF II and BDGF II to the same receptor was observed. The amino terminal sequence of EDGF II (1-16) was shown to be identical to the amino acid residues (7-22) of the acidic FGF, strongly confirming our observations on the identity of the factors isolated from bovine brain and retina.

Bovine retina contains three growth factor activities with different affinity to heparin: eye derived growth factor I, II, III.

Several ocular tissues have been shown to contain growth factor activity designated under a generic name as Eye Derived Growth Factor. Purification from bovine retina was undertaken and a fraction which could induce target cells to proliferate at doses of 5 ng per ml of culture medium was obtained. Using heparin sepharose chromatography we now show that this mitogenic activity can be fractionated into three different activities. Crude extract of bovine retina used as starting material was separated into two major fractions, one with no affinity for heparin and which was named Eye Derived Growth Factor III, and one with a strong affinity for heparin and eluted from the column with 1.4 M NaCl named Eye Derived Growth Factor I. This fraction EDGF I induces cell proliferation at doses of 100 pg/ml of culture medium. A 10(5) fold purification was achieved by this single chromatography step. Cibacron Blue purified EDGF was also further fractionated by heparin sepharose. All biological activity was found to bind to heparin. One fraction eluted at 1 M NaCl named Eye Derived Growth Factor II had a biological activity at doses of 1 ng while the other growth factor was the EDGF I with biological activity at 25 pg. At this step of purification EDGF I runs as a single band on SDS polyacrylamide gel at a molecular weight of 17 000 d. These data strongly suggest that Eye Derived Growth Factors I and II are respectively similar to Brain Fibroblast Growth Factor and to Endothelial Cell Growth Factor from hypothalamus.

Cellular distribution of the angiogenic factor heparin affin regulatory peptide (HARP) mRNA and protein in the human mammary gland.

The heparin affin regulatory peptide (HARP) growth factor, also known as pleiotrophin, is a developmentally regulated protein that displays biological functions during cell growth and differentiation. To study the physiological role of this protein, we investigated the cellular distribution of HARP mRNA and protein in the resting human mammary gland. In situ hybridization histochemistry revealed that HARP mRNA was localized in alveolar myoepithelial cells, whereas alveolar epithelial cells were negative. In the stroma, HARP mRNA was localized in endothelial cells and smooth muscle cells of blood vessels. Interestingly, HARP protein and mRNA were not always co-localized. HARP protein immunocytochemistry staining was observed in an area including both alveolar myoepithelial and epithelial cells, although epithelial cells do not express HARP transcript. In contrast, the distribution of HARP protein is parallel to that of HARP mRNA in endothelial and vascular smooth muscle cells. In the light of these results, the putative role of HARP in controlling the proliferation and/or differentiation of the different mammary cell types is proposed and discussed.

Upregulation of the angiogenic factor heparin affin regulatory peptide by progesterone in rat uterus.

Heparin affin regulatory peptide (HARP), also named pleiotropin, is a secreted polypeptide that belongs to a new family of heparin-binding growth/differentiation factors. In this study, we investigated the expression and distribution of HARP mRNA and protein in rat uterus. Semi-quantitative reverse transcriptase PCR experiments showed variations in HARP mRNA levels throughout the estrous cycle, with a maximum during diestrus, pointing to hormonal regulation of HARP mRNA expression. Uterine expression of HARP mRNA was studied in ovariectomized animals treated with 17 beta-estradiol, progesterone alone or progesterone and RU486. In these experiments, progesterone upregulated HARP mRNA expression. Induction was observed 6 h after progesterone injection and was inhibited by RU486 treatment. In contrast, after 17 beta-estradiol injection, a slight decrease in HARP mRNA expression was observed. In situ hybridization studies with digoxigenin-labeled DNA probe revealed that HARP mRNA was present in smooth muscle cells of both myometrium and blood vessels and also in endothelial cells from endometrium. Immunohistochemical studies showed that HARP expression was not limited to cells that expressed HARP mRNA, but also occurred in both the luminal and glandular epithelium even though its transcript was never detected. We conclude that HARP may mediate the effects of progesterone on the homeostasis and vascularization of uterine tissue.

Partial expression of monoaminergic (serotoninergic) properties by the multipotent hypothalamic cell line F7. An example of learning at the cellular level.

A serum-free medium supplemented with a glial conditioned medium, a brain extract from 8-to 10-day-old mice, hormones, and eye-derived growth factor has been devised which permitted the mouse primitive hypothalamic nerve cell line F7 to express some biochemical properties typical of monoaminergic neurons. Maximal expression was obtained when the culture conditions were applied for 2 days. Most (90-95%) cells then synthesized [(3)H]serotonin from [(4)H]5-hydroxytryptophan (but not from [(3)H]tryptophan). No synthesis was detected in the presence of carbidopa (20 ?M), therefore suggesting the involvement of l-aromatic-amino-acid decarboxylase in this process. In addition, F7 cells cultured in such serum-free medium exhibited the capacity of accumulating exogenous serotonin by an ouabain-sensitive mechanism. These data further supported that active molecules in the cell environment can induce, in a primitive cell line, some of the enzymatic activities associated with monoaminergic neurons. Since other well-defined culture conditions can promote the differentiation of the same clone into oligodendrocytes (De Vitry et al., 1983), it can be concluded that the F7 cell has the properties of an embryonic stem cell of the CNS which, depending on external signals, may switch into different alternative developmental neural pathways. We postulate that the stabilization of neuron-like properties due to repetitive cell stimulation by active signals in the environment may represent an example of learning at the cellular level.

High and low affinity membrane binding sites for fibroblast growth factors in the developing chick brain.

Acidic and basic fibroblast growth factors (aFGF and bFGF), two mitogenic, neurotrophic and angiogenic molecules, are present in the embryonic chick brain but their function remains unclear. In order to approach the biological activity of FGFs during brain development, we have looked for their receptors and studied their regulation through chick brain development. Competitive binding studies realized on brain membranes indicated the presence of two classes of FGF binding sites: high affinity binding sites (dissociation constant, Kd = 100 pM) and low affinity binding sites (Kd = 20 nM). Cross-competition experiments show that these two classes of binding sites both interact with aFGF and bFGF. The number of sites in these two classes of binding sites changes during embryogenesis. On the one hand, the membrane capacity of high affinity sites decreases from E7 (1 +/- 0.2 pmol/mg of protein) to E15 (0.5 +/- 0.2 pmol/mg of protein); on the other hand, the membrane capacity of low affinity sites increases from E15 (25 +/- 4 pmol/mg of protein) to P1 (75 +/- 20 pmol/mg of protein). Cross-linking experiments revealed the presence of two putative receptor forms of molecular masses of about 130 and 95 kDa. These results suggest that the biological activity of aFGF and bFGF during brain embryogenesis could be regulated by the expression of high and low affinity binding sites for these growth factors.

Comparison of the effects of EGF, pFGF and EDGF on corneal epithelium wound healing.

EDGF, a growth factor purified from bovine retina is able to increase the rate of wound healing of rabbit corneal epithelium in a dose dependent way. Two applications a day are enough to obtain the maximum rate. The most purified preparation of EDGF is as effective as EGF or pFGF in promoting this phenomenon and allows the epithelium to reach its normal organization earlier. Excess addition of EDGF did not adversely affect the normal or healed epithelium.

[Use of the eye-derived growth factor in the treatment of ulcer of the cornea. A veterinary medicine study in the dog]. / Utilisation du facteur de croissance dérivé de l'oeil dans le traitement des ulcères de cornée. Une étude en médecine vétérinaire chez le chien.

A growth factor purified from adult bovine retina and named Eye Derived Growth Factor (EDGF) has been shown to stimulate proliferation of target cells in vitro and in vivo, and to increase the rate of wound healing of experimentally induced corneal ulcers in rabbits. In the present report, dogs (mainly boxers) with chronic recurrent ulcers resistant to various treatments, including chloramphenicol and reputed wound healing drugs, were treated by morning and evening instillations of drops containing 10 stimulation units of EDGF in phosphate buffer saline. Stable healing of the ulcer was obtained in all cases after 12 days of treatment, and no sign of relapse was detected after three months. During the healing period, as well as during the period of increase in cellular proliferation, transitory inflammation accompanied by neovascularization was observed. EDGF represents a new drug for treatment of ulcer.

Intramyocardial protein therapy with vascular endothelial growth factor (VEGF-165) induces functional angiogenesis in rat senescent myocardium.

Myocardial capillary density and angiogenesis are impaired during aging but whether growth factor therapy is able to induce functional neovascularization in senescent heart have never been studied. In 3, 24, 28 and 32 mo male Wistar rats, cardiac hemodynamic measurements indicated heart failure at 28 and 32 mo, associated with left ventricular hypertrophy. VEGF/VEGF-R2, Ang-1/Ang-2/Tie-2 and PTN levels, quantitated in left ventricle by western blotting and immunohistochemistry, showed that VEGF and VEGF-R2 levels were specifically decreased during aging. In vitro angiogenesis ± rhVEGF-165 (5 and 50 ng/mL) was measured in aortic segments in 3D-collagen. Aortic sprouting was decreased during aging but restored by VEGF treatment (P<0.001), similarly in 3 and 24 mo with 50 ng/mLVEGF. Finally, 3 and 24 mo rats were submitted to in vivo intramyocardial rhVEGF-165 (10 micrograms) or saline solution injection and angiogenesis was measured by SPECT imaging of the alpha(v)beta(3) integrin-targeted tracer (99m)Tc-RAFT-RGD, capillary fluorescence staining in isolated perfused heart and vWF and alpha smooth muscle actin immunohistochemistry, 7 and 21 days later. VEGF administration increased capillary density in 3 but also in 24 mo rats at days 7 (+26%, P<0.01) and 21 (+41%, P<0.01) and arteriolar density at day 21 (+36%, P<0.01). Activity of (99m)Tc-RAFT-RGD and capillary fluorescence labeling indicated that new formed capillaries were functional. Cardiac aging was associated with strong VEGF/VEGF-R2 pathway downregulation. VEGF-165 protein therapy was able to induce in vitro and in vivo angiogenesis during aging. In 24 mo hearts, in vivo angiogenesis was functional, sustained and comparable to neovascularization observed in 3 mo hearts.