Biomedical subjects

D Hantaï

Publications and source records attributed to D Hantaï.

14 recordsLinked to original sources

PubMed1992

Thrombospondin, a platelet alpha-granule and matrix glycoprotein, is increased in muscle basement membrane of patients with amyotrophic lateral sclerosis.

In an attempt to obtain a biological marker for the enigmatic and fatal neurologic disorder, amyotrophic lateral sclerosis (ALS), several laboratories have explored alterations in various extracellular matrix components in both skeletal muscle and skin. We have studied the distribution of fibronectin, laminin, heparan sulfate proteoglycan (HSPG) and collagen types I, III and IV, along with the platelet alpha-granule glycoprotein, thrombospondin (TSP), by immunofluorescence in frozen sections of muscle from control denervating conditions and ALS patients. In ALS and control muscle, types I and III collagen were localized to the endomysium and the perimysium. Type IV collagen and laminin precisely delineated each muscle fiber (endomysium or basement membrane) but did not stain the perimysium. We found no marked quantitative or qualitative differences in the distribution of collagen types I, III and IV, laminin, fibronectin or HSPG in ALS patients compared to controls. However, when polyclonal antisera for TSP was used we found a marked increase in the deposition of this multi-domain glycoprotein in ALS patients' muscle compared to control muscle. Quantitative analysis of soluble extracts from control and ALS patients' muscle by ELISA also indicated that TSP was increased in ALS. TSP is released from platelet alpha-granules in response to thrombin stimulation. TSP elevation implies coagulation activity via the extravascular thrombolytic system in ALS and may correlate with regeneration. Other studies have indicated decreased circulating protease inhibitors and increased serine proteases in this disorder.

Amyotrophic Lateral Sclerosis↗

PubMed1991

Plasminogen activators and inhibitors in the neuromuscular system: III. The serpin protease nexin I is synthesized by muscle and localized at neuromuscular synapses.

Recent studies suggest that the nature of events leading to the formation, maintenance, and elimination of synapses may be regulated by cascade-type, locally expressed proteases and protease inhibitors acting on adhesive extracellular matrix components. We have identified a molecule in conditioned medium of murine skeletal muscle cells that in molecular weight, target protease inhibition, heparin-binding and cross-reactivity with authenic antisera is similar to the human serine proteinase inhibitor, protease nexin I. Protease nexin I is a 43-50 kDa glycoprotein of the serpin superfamily (arg-serpin class). Purified anti-protease nexin I antibody (anti-47 kDa) stains adult mouse skeletal muscle in discrete foci that precisely superimpose on synaptic neuromuscular junctions. Protease nexin I appears in patches on surfaces of cultured mouse skeletal myotubes, but not on myoblasts. These patches co-localize with acetylcholine receptor clusters and acetylcholinesterase staining during cellular maturation in culture. Evidence that protease nexin I is a synaptic, extracellular antigen is particularly intriguing since it has been shown to be identical, in structure and activity, with a factor released by glial cells, called glia-derived nexin that stimulates mouse neuroblastoma cell neurite outgrowth and inhibits granule cell migration. Protease nexin I inhibits both tumor cell and myoblast plasminogen activator-mediated destruction of extracellular matrix. Thus, such observations as presented in this report provide further evidence for involvement of cascade proteolytic systems, and their post-translational regulation by specific serpins, in the remodeling that occurs in synapse formation and elimination.

Amyloid beta-Protein Precursor↗

PubMed1991

Skeletal muscle involvement in human immunodeficiency virus infection.

In addition to muscle changes due to peripheral nervous system involvement, primary myopathic changes associated with the human immunodeficiency virus (HIV) have also been described. We studied seven cases: two had developed an acquired immunodeficiency syndrome (AIDS), four had seroconverted to HIV but were otherwise asymptomatic, one was HIV seronegative when the biopsy was performed and one was biopsied twice. Besides the HIV no other infectious agent was detected. Muscle biopsies showed: (a) muscle fiber necrosis and regeneration; (b) inflammatory changes with moderate perivascular infiltration; and (c) unusual myofibrillary disorganization. Immunocytochemical techniques using anti-HIV monoclonal antibodies showed the presence of the virus in one biopsy. HIV-RNA was detected by in situ hybridization in the same biopsy. With both techniques the HIV was detected in isolated mononuclear cells in the muscle endomysium and not within the muscle fibers. Muscle involvement associated with HIV infection may be related, at least in some cases, to the presence of the virus in interstitial cells.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Role of persisting basement membrane in the reorganization of myofibres originating from myogenic cell grafts in the rat.

Satellite cells grafted at the site of an irreversible muscle injury regenerate normal myofibres that become organized in fascicles. The role of the basement membrane in organization of the newly formed muscle fibres was investigated using polyclonal antibodies against laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan. In ungrafted muscles, original basement membranes were reactive to these antibodies at 7, 14 and 45 days after injury. Labelling of satellite cells with FITC-latex beads showed the labelled myoblasts and new myofibres within the remnants of old basement membranes at 7 days after cell implantation and thereafter. Electron microscopy of injured-ungrafted muscles showed persistence of electron dense material corresponding to thin layers of old basal laminae partially interrupted. After cell grafting, myotubes developed within these structures and were surrounded by redundant basal laminae. These results suggest that grafted cells are able to migrate inside the basement membranes which serve as scaffolding for their development.

Animals↗

PubMed1991

Developmental appearance of thrombospondin in neonatal mouse skeletal muscle.

Immunocytochemical localization of the adhesive glycoprotein thrombospondin made in comparison with other components of extracellular matrices shows its sequential appearance in the mouse muscle endomysium during postnatal development. Thrombospondin, absent at birth, in contrast to laminin, type IV collagen and fibronectin, is progressively detected during the first month of neonatal life in the whole muscle extracellular matrix. Immunoblotting of thrombospondin showed the appearance 14 days after birth of a band migrating at 180 kDa corresponding to thrombospondin. A fragment of thrombospondin at 110 kDa was already present at birth, as was also a lower molecular mass band at 70 kDa. Another band at 50 kDa also appeared during development in muscle extracts. Clonal muscle cells in culture were able to synthesize thrombospondin but only at the myotube stage, since little thrombospondin was detected at the myoblast stage. These data show a development regulation of thrombospondin expression in muscle which correlates with muscle differentiation.

Animals↗

PubMed1990

Plasminogen activators and their inhibitors in the neuromuscular system: I. Developmental regulation of plasminogen activator isoforms during in vitro myogenesis in two cell lines.

Plasminogen activators (PAs), were estimated qualitatively and quantitatively in two different clonal murine skeletal muscle cell lines. Both cell lines produced the two major types of PAs found in mammalian cells, urokinase-type (uPA) and tissue type (tPA). These two lines are models for the study of myogenesis in vitro, but differ in several growth and differentiation characteristics. Because of their possible involvement in these characteristics we assayed the expression of PAs in both cell systems during development in culture. Utilizing fibrin zymography two isoforms of tPA were detected. One co-migrated with human tPA at 75 kd and another may represent a tPA:inhibitor complex at 105 Kd. Several isoenzymes of uPA were detected and these changed depending on whether cell homogenates or conditioned medium was analyzed and whether myogenic cells were at single-cell myoblast or multi-nucleated myotube stage. Species-specific antisera to mouse uPA identified 4 uPA bands in muscle cell medium and 5 in cell layers. Antigenic uPA bands also varied depending on stage of myogenesis. Quantitative amidolytic studies using chromogenic substrates showed that maximal PA activity, both uPA and tPA, occurred at the time of myoblast fusion. Furthermore, uPA activity in membranes increased during myogenesis, while both uPA and tPA in medium decreased after fusion. These studies indicate that muscle PA expression is developmentally regulated and may correlate with growth and differentiation in skeletal muscle.

Animals↗

PubMed1990

Plasminogen activators and their inhibitors in the neuromuscular system: II. Serpins and serpin: protease complex receptors increase during in vitro myogenesis.

In the course of studies on the regulation of plasminogen activator-mediated extracellular matrix degradation in muscle we found the presence of a factor, a cellular inhibitor of serine proteases having features similar to the serpin protease nexin I (PNI). This factor was present in the medium and at maximum concentration following fusion of skeletal muscle cells in culture. The ability of the PNI homologue in mouse muscle to inhibit ECM degradation by urokinase in myoblast medium was compared to that of human PNI purified from human fibroblasts. Stable (to SDS) 1:1 molar ratio complex formation between PNI and proteases, the proposed means by which these enzymes are regulated and removed, was also detected. Cell surface receptors for protease:PNI complexes, the specific binding sites for inactive complex internalization, were found on multinucleated myotubes, while little or no receptor activity was detected on myoblasts. These data suggest that developmental regulation of a) increased PNI proteolytic inhibitory activity expression and b) the appearance of protease:inhibitor complex receptors on muscle cell surfaces during myogenesis may constitute important regulatory features of muscle surface proteolytic activity. They complement previous studies of proteoglycan metabolism in muscle, which itself contains molecules capable of regulating the activity of myotube surface proteases.

Amyloid beta-Protein Precursor↗

PubMed1990

Rapid neural regulation of muscle urokinase-like plasminogen activator as defined by nerve crush.

Muscle plasminogen activators (PAs), such as urokinase-like PA and, to a lesser extent, tissue PA, increase dramatically after denervation induced by axotomy. The PA/plasmin system has also been implicated in degradation of specific components of the muscle fiber basement membrane after local activation of plasminogen. These results suggest that neural regulation of muscle extracellular matrix metabolism accompanies or precedes regeneration after injury and is mediated by activation of PAs. In the present study, we have used nerve crush to explore the neural regulation of muscle PA activities directly after subtotal axon interruption and during the process of reinnervation. Muscle contraction after nerve stimulation and estimation of choline acetyltransferase activity were used to monitor reinnervation. Within 24 hr of nerve crush, muscle urokinase (but not tissue PA) activity rose in soluble and membrane-bound muscle fractions, as shown by an amidolytic assay and a fibrin zymography. Membrane-bound activity was 5-fold higher than cytosol activity, but there was no shift between cellular compartments during the time course of denervation. Coincident with the return of choline acetyltransferase activity and muscle contractility, muscle urokinase returned almost to baseline levels. These results show tight regulation of muscle urokinase levels by some neural influence.

Animals↗

PubMed1990

Slow and fast rat skeletal muscles differ in their plasminogen activator activities.

Slow and fast contracting muscles differ in their innervation and electrophysiological properties as well as in their regenerating potentialities. The purpose of the present work was to investigate the expression of plasminogen activators and its possible relation to each type of muscle. Slow (Soleus) and fast (Extensor Digitorum Longus) muscles were obtained from white Wistar rats. Before sectioning the muscles, the euthanized rats were perfused with cold phosphate buffer saline to avoid interference by circulating proteases and inhibitors. Muscle extracts were pounded in an ice-cold Potter tube. Plasminogen activators (PAs) were assayed by fibrin zymography and by both liquid and solid-phase fibrin spectrophotometric assays for the detection of PAs activity. Both urokinase (uPA) and tissue-type plasminogen activator (tPA) activities corresponding to proteins of 38 kDa and 65 kDa molecular masses, were detected in the extracts. Slow muscles contained higher amounts of both activators than fast muscles, but the relative amount of uPA was higher in both types of muscles. In addition, the characteristics of each type of extracts differed somewhat: the fast muscle activity curve was typical of an accelerating process, while the slow muscle curve showed an activity probably related to already formed plasmin or to some other trypsin-like enzyme. These results suggest that the amount of plasminogen activators could be a new criterion of discrimination between slow and fast skeletal muscles.

Animals↗

PubMed1989

Decrease in plasminogen activator correlates with synapse elimination during neonatal development of mouse skeletal muscle.

Previous studies have implicated proteases, acting extracellularly, in the mechanism of polyneuronal synapse elimination. Most studies have focused on mammalian, especially rodent, skeletal muscle, where retraction of subordinate nerve terminals occurs during a narrow time window 2-3 weeks after birth. To date no specific protease(s) has been detected that (i) coincides in time with maximal synapse elimination and (ii) is known to act extracellularly on specific extracellular matrix proteins. In previous studies of denervation in adult mouse muscle, rapid activation of urokinase-type plasminogen activator, a neutral serine protease, was detected. This enzyme, by activation of plasminogen to plasmin, specifically degrades matrix components such as fibronectin, type IV collagen, and laminin in muscle. We now present evidence for an initial increase and subsequent decrease in soluble urokinase-type PA--and, to a lesser extent, tissue PA--in developing muscle, suggesting postnatal developmental regulation of these enzymes during the period of maximal synapse elimination. Although considerably higher in specific activity, membrane-bound PA activity followed the wave of synapse elimination, possibly indicating a longer half-life of membrane-bound enzyme(s).

Aging↗

PubMed1988

Serine proteases and serpins: their possible roles in the motor system.

A review of the importance of the extravascular fibrinolytic enzymes and their inhibitors in the neuromuscular system is discussed. We present data showing the role of specific serine proteases, the plasminogen activators, during neuromuscular development as well as after nerve injury and regeneration through their action on muscle basement membrane components. We show the presence in muscle of protease nexin I, a member of the serine protease inhibitors (serpins) family, where it is highly concentrated in the neuromuscular junction. These data have lead us to propose a model where the extravascular components of the fibrinolytic cascade and cellular derived serpins, such as protease nexin I, would act in the formation and maintenance of the neuromuscular synapse. Our conclusion is that future studies with these molecules, including their regulation at several levels, are warranted in a number of neuromuscular and neuro-degenerative diseases.

Amyloid beta-Protein Precursor↗

PubMed1987

Degradation of muscle basement membrane zone by locally generated plasmin.

The turnover of basement membrane macromolecules in injured skeletal muscle has not been studied in contrast to other biologic systems undergoing remodeling. Plasminogen activators and other neutral proteases that are able to degrade these basement membrane macromolecules are secreted by cultured muscle cells. We sought to determine if locally released plasminogen activators could act on basement membrane components. Such degradation might be implicated in the disadhesion of nerve from muscle after motor nerve denervation. To test this hypothesis, we first undertook a study of the sensitivity of muscle extracellular matrix antigens following in vitro exposure to various proteases on frozen muscle sections. Fibronectin was found to be most sensitive, followed by type IV collagen and laminin. Of serine proteases, trypsin was the most active but was not selective, digesting matrix and sarcoplasmic components alike in less than 30 min. Purified urokinase was inactive unless plasminogen (also inactive alone) was previously added to tissue sections, at which time only matrix antigens were digested. Little if any observable degradation of sarcoplasmic proteins took place under these conditions. Using a highly sensitive and selective assay, we found that plasminogen activators were present in muscle tissue and increased 8- to 10-fold after 10 days of denervation. Using an extract of denervated muscle in the presence of plasminogen, we observed degradation of matrix antigens. No degradation was observed with control muscle extract. We next evaluated the degradation of these antigens in denervated muscle during a temporal study. The results, analyzed by quantitative image analysis, indicates that with increasing time after denervation a marked decrease of fibronectin and type IV collagen, followed by laminin occurred but, again, only in the present of plasminogen. These results indicate a selective sensitivity of basement membrane antigens of muscle and a role for plasminogen activators in the degradation of these adhesive basement membranes macromolecules after denervation.

Animals↗

PubMed1986

Plasminogen activator in mammalian skeletal muscle: characteristics of effect of denervation on urokinase-like and tissue activator.

Analyses were made of the fibrinolytic, plasminogen-activating system in skeletal muscle to determine if a regulating influence of the nerve could be detected on these enzymes. Young male mice underwent right sciatic neurectomy. Extracts were prepared from denervated muscle at 2-17 d after axotomy and compared with controls. Using a cascade-style biochemical assay (Rånby, M., B. Norrman, and P. Wallén, 1982, Thromb. Res., 27:743-748) we found that low levels of plasminogen activator (PA) were present in adult, innervated mouse muscle, but that denervation resulted in a marked time-dependent increase in enzyme activity. Qualitative separation showed an eightfold increase in urokinase-like PA with moderate elevation of tissue PA activity after 10 d. Fibrin zymography (Granelli-Piperno, A., and E. Reich, 1978, J. Exp. Med., 148:223-234) revealed clear zones of lysis corresponding to molecular masses of 48 kD for urokinase-like PA and 75 kD for tissue PA, consistent with the molecular masses found for these enzymes in other tissues of the mouse (Danø, K., P. A. Andreasen, J. Grøndahl-Hansen, P. Kristensen, L. S. Nielsen, and L. Skriver, 1985, Adv. Cancer Res., 44:139-266). In other studies we have shown that PA-activated plasmin readily attacks critical adhesive basement membrane molecules. The present results indicate that enzymes involved in plasminogen activation, particularly urokinase-like PA, rapidly increase after axotomy, suggesting they may have a role early in muscle denervation. Similar alterations in PA activity might underlie the elimination of polyneuronal innervation during mammalian muscle development. Certain neuromuscular diseases may also involve activation of these enzymes, resulting in degradation of basement membrane zone components and, therefore, warrant further study.

Animals↗

PubMed1985

Fibronectin, laminin, type I, III and IV collagens in Duchenne's muscular dystrophy, congenital muscular dystrophies and congenital myopathies: an immunocytochemical study.

The distribution pattern of fibronectin, laminin, type I, III and IV collagens in human skeletal muscle was studied by immunofluorescence. In normal muscle, as well as in congenital myopathies (CM), type I and III collagens were localized in the endomysium and the perimysium. Laminin and type IV collagen delineated precisely each muscle fiber but did not stain the perimysium. In Duchenne's muscular dystrophy (DMD) as well as in congenital muscular dystrophies (CMD) the extensive proliferation of connective tissue consisted mainly of fibronectin and type I and III collagens. Laminin and type IV collagen delineated principally the basal lamina but suprisingly were found to be distributed to some extent all over the extracellular matrix. No disease--specific accumulation of components of the extracellular matrix was found which would enable us to differentiate these last two diseases, though the immunofluorescence reactions for all components were stronger in DMD than in CMD.

Adolescent↗