Studies of Schwann cell proliferation. I. An analysis in tissue culture of proliferation during development, Wallerian degeneration, and direct injury

In this paper the stimuli for and pattern of Schwann cell proliferation are defined under various experimental conditions. We used a tissue culture system in which fetal rat dorsal root ganglia, treated to eliminate contaminating fibroblasts (Wood, P., 1976, Brain Res. 115:361--375), appear to recapitulate many aspects of the developing peripheral nervous system. We observed that: (a) proliferation of Schwann cells on neurites is initially rapid, but, as each neurite becomes fully ensheathed, division slows considerably and is confined to the periphery of the outgrowth; (b) during the period of rapid proliferation, excision of the ganglion causes a rapid decay in the number of dividing cells; (c) excision of the ganglion from more established cultures in which there was little ongoing proliferation resulted in a small increase in labeling at the site of excision for all Schwann cells and a substantial increase in labeling for myelin-related cells with a peak labeling period at 4 d; (d) direct mechanical injury during Wallerian degeneration is mitogenic for Schwann cells; (e) a variety of potential mitogens failed to stimulate Schwann cell proliferation, and (f) replated cells have a slightly higher level of proliferation and show a small and variable response to the addition of cAMP.     

Inhibition of transient receptor potential melastatin 7 (TRPM7) protects against Schwann cell trans-dedifferentiation and proliferation during Wallerian degeneration

ABSTRACT

Irreversible peripheral neurodegenerative diseases such as diabetic peripheral neuropathy are becoming increasingly common due to rising rates of diabetes mellitus; however, no effective therapeutic treatments have been developed. One of main causes of irreversible peripheral neurodegenerative diseases is dysfunction in Schwann cells, which are neuroglia unique to the peripheral nervous system (PNS). Because homeostasis of calcium (Ca²⁺) and magnesium (Mg²⁺) is essential for Schwann cell dynamics, the regulation of these cations is important for controlling peripheral nerve degeneration and regeneration. Transient receptor potential melastatin 7 (TRPM7) is a non-selective ion (Ca²⁺ and Mg²⁺) channel that is expressed in Schwann cells. In the present study, we demonstrated in an ex vivo culture system that inhibition of TRPM7 during peripheral nerve degeneration (Wallerian degeneration) suppressed dedifferentiable or degenerative features (trans-dedifferentiation and proliferation) and conserved a differentiable feature of Schwann cells. Our results indicate that TRPM7 could be very useful as a molecular target for irreversible peripheral neurodegenerative diseases, facilitating discovery of new therapeutic methods for improving human health.     

Somatostatin inhibits cell migration and reduces cell counts of human keratinocytes and delays epidermal wound healing in an ex vivo wound model

The peptide hormone somatostatin (SST) and its five G protein-coupled receptors (SSTR1-5) were described to be present in the skin, but their cutaneous function(s) and skin-specific signalling mechanisms are widely unknown. By using receptor specific agonists we show here that the SSTRs expressed in keratinocytes are functionally coupled to the inhibition of adenylate cyclase. In addition, treatment with SSTR4 and SSTR5/1 specific agonists significantly influences the MAP kinase signalling pathway. As epidermal hormone receptors in general are known to regulate re-epithelialization following skin injury, we investigated the effect of SST on cell counts and migration of human keratinocytes. Our results demonstrate a significant inhibition of cell migration and reduction of cell counts by SST. We do not observe an effect on apoptosis and necrosis. Analysis of signalling pathways showed that somatostatin inhibits cell migration independent of its effect on cAMP. Migrating keratinocytes treated with SST show altered cytoskeleton dynamics with delayed lamellipodia formation. Furthermore, the activity of the small GTPase Rac1 is diminished, providing evidence for the control of the actin cytoskeleton by somatostatin receptors in keratinocytes. While activation of all receptors leads to redundant effects on cell migration, only treatment with a SSTR5/1 specific agonist resulted in decreased cell counts. In accordance with reduced cell counts and impaired migration we observe delayed re-epithelialization in an ex vivo wound healing model. Consequently, our experiments suggest SST as a negative regulator of epidermal wound healing.     

Stromelysin generates a fibronectin fragment that inhibits Schwann cell proliferation

Our previous report (Muir, D., S. Varon, and M. Manthorpe. 1990. J. Cell Biol. 109:2663-2672) described the isolation and partial characterization of a 55-kD antiproliferative protein found in Schwann cell (SC) and schwannoma cell line-conditioned media and we concluded that SC proliferation is under negative autocrine control. In the present study the 55-kD protein was found to possess metalloprotease activity and stromelysin immunoreactivity. The SC-derived metalloprotease shares many properties with stromelysin isolated from other sources including the ability to cleave fibronectin (FN). Furthermore, limited proteolysis of FN by the SC-derived protease generated a FN fragment which itself expresses a potent antiproliferative activity for SCs. The active FN fragment corresponds to the 29-kD amino-terminal region of the FN molecule which was also identified as an active component in SC CM. Additional evidence that a proteolytic fragment of FN can possess antiproliferative activity for SCs was provided by the finding that plasmin can generate an amino-terminal FN fragment which mimicked the activity of the SC metalloprotease-generated antiproliferative FN fragment. Both the 55-kD SC metalloprotease and the 29-kD FN fragment could completely and reversibly inhibit proliferation of SCs treated with various mitogens and both were largely ineffective at inhibiting proliferation by immortalized or transformed SC lines. Normal and transformed SC types do secrete the proform of stromelysin, however, transformed cultures do not produce activated stromelysin and thus cannot generate the antiproliferative fragment of FN. These results suggest that, once activated, a SC-derived protease similar to stromelysin cleaves FN and generates an antiproliferative activity which can maintain normal SC quiescence in vitro.     

An ex vivo model of hematopoietic stem cell mobilization

BACKGROUND: Mobilization of hematopoietic stem cells to the circulation facilitates their collection, thereby providing a non-marrow source of these cells for transplantation. Hematopoietic cytokine administration induces mobilization for most, but not all, donors. Because the underlying biology of mobilization is not well understood, improving the process on a rational basis is difficult. The design of an in vitro mobilization model was pursued to facilitate investigations of the process. METHODS: MS5 murine stromal cell line cells were grown to confluence on microporous transwell membranes. Murine femoral marrow plugs were placed on top of the prepared transwell membranes. The transwells were then seated in wells containing media and hematopoietic growth factors. Cells that were released from the marrow plugs over time and migrated through the stromal layer into the wells were assayed for stem cell/progenitor cell characteristics. RESULTS: Few or no GM-CSF (progenitors) were found in wells containing media alone or media plus mobilizing cytokines after 24 h. After 120 h, the numbers of cells in the cytokine-containing wells increased, as did the numbers of CD34(+) cells. Cells in the wells at the time progenitor cells were most frequent were shown to include side population (SP) hematopoietic stem cells. After 120 h in the presence of cytokines, cells pooled from the wells were transplanted to lethally irradiated mice. Eighty per cent of the transplanted mice survived 30 days or more, demonstrating that radioprotective stem cells were present in the wells. DISCUSSION: An ex vivo model has been designed that may aid investigations of the various steps of stem cell mobilization.     

Radioprotective effects of ATP in human blood ex vivo

Damage to healthy tissue is a major limitation of radiotherapy treatment of cancer patients, leading to several side effects and complications. Radiation-induced release of pro-inflammatory cytokines is thought to be partially responsible for the radiation-associated complications. The aim of the present study was to investigate the protective effects of extracellular ATP on markers of oxidative stress, radiation-induced inflammation and DNA damage in irradiated blood ex vivo. ATP inhibited radiation-induced TNF-α release and increased IL-10 release. The inhibitory effect of ATP on TNF- α release was completely reversed by adenosine 5′-O-thiomonophosphate, indicating a P2Y₁₁ mediated effect. Furthermore, ATP attenuated radiation-induced DNA damage immediate, 3 and 6 h after irradiation. Our study indicates that ATP administration alleviates radiation-toxicity to blood cells, mainly by inhibiting radiation-induced inflammation and DNA damage.     

Extracellular ADP regulates lesion-induced in vivo cell proliferation and death in the zebrafish retina

Regeneration and growth that occur in the adult teleost retina by neurogenesis have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. In this report, we demonstrate that endogenous purinergic signals regulate cell proliferation induced by a cytotoxic injury of the adult zebrafish retina which mainly damages inner retinal layers. Particularly, we found that ADP but not ATP or adenosine significantly enhanced cell division as assessed by 5-bromo-2'-deoxyuridine incorporation following injury, during the degenerative and proliferative phase of the regeneration process. This effect of ADP occurs via P2Y1 metabotropic receptors as shown by intra-ocular injection of selective antagonists. Additionally, we describe a role for purinergic signals in regulating cell death induced by injury. Scavenging of extracellular nucleotides significantly increased cell death principally seen in the inner retinal layers. This effect is partially reproduced by blocking P2Y1 receptors suggesting a neuroprotective function for ADP, which is derived from extracellular ATP probably released by dying cells as a consequence of the ouabain treatment. This study demonstrates a crucial role for ADP as a paracrine signal in the repair of retinal tissue following injury.     

Apoptosis and neutrophils in the regulation of Ph-positive myeloid cell proliferation and differentiation ex vivo

Ex vivo proliferation and differentiation of Philadelphia chromosome-positive (Ph⁺) human myeloid cells (Ph+ cells) from chronic myeloid leukemia (CML) proceed via alternation stages of cell proliferation and neutrophil maturation. To regulate them, apoptosis is alternately blocked or induced with the help of neutrophils and expression of bcr/abl, bax, and bcl2. The regulation of apoptosis in main types of Ph⁺ cells depends on the alternation of (1) Ph⁺ cell proliferation and (2) neutrophil maturation and may follow two pathways. One consists in alternating blockages and inductions of apoptosis with initial maturation and subsequent proliferation under alternation stages as (2)-(1)-(2) and has not been described as yet. Neutrophil accumulation blocks apoptosis. As neutrophils are depleted, apoptosis is induced again. Its block accelerates proliferation with a new accumulation of neutrophils, which is followed by regular neutrophil death and a new induction of apoptosis. The way optimizes the proliferation efficiency (P/D index) with a regular alternation of maturation and proliferation, allowing the cycle of proliferation and differentiation to be completed. In another way, the alternation starts with proliferation as (1)-(2)-(1) at a lower neutrophil content) and leads to resistant decrease of the maximal apoptosis level by a factor of 3–8 as compared with (2)-(1)-(2) alternation. A stable block of apoptosis is observed in cells with prolonged stages of proliferation and maturation, leading to an accumulation of blasts and myelocytes with elevated bcr/abl expression and expression of bcl2 > bax. A stable block of apoptosis is associated with CML progression and in Ph⁺ cell lines. Cells follow the first pathway of the apoptotic regulation in chronic-phase CML. Ex vivo cultivation of Ph⁺ cells from individual CML patients was assumed to provide for a more exact diagnosis of the CML phase and optimizing the treatment.     

Astroglial injury in an ex vivo model: contributions to its analysis in enriched cell cultures

In vitro cell culture models have been proposed to analyze some of the complex structural and functional characteristics involved in astroglial changes after neural injury in vivo. This report contributes to analyze the proposed hypothesis that an experimentally induced discontinuity of a confluent cellular culture could represent a useful model for the analysis of the processes involved in a neural lesion. For this purpose, it was decided to characterize astroglial proliferation and dye coupling state after a “scratch wound” applied to confluent, astrocyte-enriched cell cultures, obtained from several rat brain regions. Proliferation was assessed in terms of bromodeoxyuridine nuclear incorporation as a function of lesion width, serum deprivation, time after confluence, brain region origin, postlesional culture medium changes and agitation, and after application of a gap-junction uncoupling agent. The proliferative reaction after injury was neither cell type-specific nor brain region specific, nor was significantly affected by neither of the above-mentioned variables. Furthermore, injury failed to significantly affect the astroglial dye coupling state. Results suggest that the proliferative response observed under present conditions would depend on the disruption of contact inhibition rather than on astroglial mitogenic signals released from the wound and operating by either extracellular or cell coupling mechanisms. Present results question the validity of astrocyte-enriched cell cultures as an experimental model of neural tissue injury in vivo, as they do not appear to reproduce fundamental characteristics expressed in situ.     

Canstatin-N fragment inhibits in vitro endothelial cell proliferation and suppresses in vivo tumor growth

Type IV collagen is one of the components of vascular basement involved in regulation of angiogenesis. Canstatin, the non-collagenous 1 (NC1) domain of alpha2 chain of type IV collagen, was identified as an inhibitor of angiogenesis and tumor growth by Kamphaus et al. Our previous studies showed that canstatin-N, the N-terminal 1-89 amino acid fragment of canstatin, inhibited the neovascularization in a dose-dependent manner as tested by CAM assay. In the present study, we demonstrate that canstatin-N produced in Escherichia coli specifically inhibited in vitro the proliferation of human umbilical vein endothelial cells (ECV304) and significantly induced apoptosis. The apoptosis-inducing activity of canstatin-N was much stronger than that of canstatin, indicating that the apoptosis-inducing activity of canstatin is likely located within its N-terminal 1-89 amino acid fragment. Canstatin-N also suppressed in vivo growth of B(16) murine melanoma in BALB/c mice at a dosage of 10mg/kg/day. These results suggest that canstatin-N is a useful candidate molecule for inhibition of tumor growth.     

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.