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1.
J Integr Neurosci ; 21(4): 118, 2022 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-35864769

RESUMO

Chondroitin sulfate proteoglycans (CSPGs) present a formidable barrier to regrowing axons following spinal cord injury. CSPGs are secreted in response to injury and their glycosaminoglycan (GAG) side chains present steric hindrance preventing the growth of axons through the lesion site. The enzyme chondroitinase has been proven effective at reducing the CSPG GAG chains, however, there are issues with direct administration of the enzyme specifically due to its limited timeframe of activity. In this perspective article, we discuss the evolution of chondroitinase-based therapy in spinal cord injury as well as up-to-date advances on this critical therapeutic. We describe the success and the limitations around use of the bacterial enzyme namely issues around thermostability. We then discuss current efforts to improve delivery of chondroitinase with a push towards gene therapy, namely through the use of lentiviral and adeno-associated viral vectors, including the temporal modulation of its expression and activity. As a chondroitinase therapy for spinal cord injury inches nearer to the clinic, the drive towards an optimised delivery platform is currently underway.


Assuntos
Traumatismos da Medula Espinal , Regeneração da Medula Espinal , Axônios/fisiologia , Condroitina ABC Liase/metabolismo , Condroitina ABC Liase/uso terapêutico , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteoglicanas de Sulfatos de Condroitina/uso terapêutico , Condroitinases e Condroitina Liases/metabolismo , Condroitinases e Condroitina Liases/uso terapêutico , Humanos , Regeneração Nervosa/fisiologia , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo
2.
Enzyme Microb Technol ; 160: 110073, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35689963

RESUMO

Regarding the existence of similar helices on the structure of different proteins, recently, novel variants of Chondroitinase ABC I (cABC I) have been constructed, where a representative helix between two structural motifs in Chondroitinase ABC I from Proteus vulgaris has been replaced by similar versions of helices found in other proteins. The previous study has revealed that the structural features and the activity of double mutants M886A/G887E (inspired by the 30 S ribosomal protein S1 from Geminocystis herdmanii) and M889I/Q891K (inspired by the chondroitin lyase from Proteus mirabilis) is comparable with that of wild-type (WT) cABC I. Here, the kinetic parameters of the enzyme activity for the WT and double mutants were determined. Of the recombinant double mutants, M889I/Q891K gave the highest catalytic efficiency with the kcat/Km value of approximately 2.3-fold increase, as compared with the WT and M886A/G887E. Modeling of experimental data showed that the mechanism of the heat-induced structural alteration, and the enzyme-substrate complex formation, changed upon mutation. These natural versions of the connecting helix can be used as an efficient linker in protein engineering studies as well as those investigations involving the use of biological linkers.


Assuntos
Condroitina ABC Liase , Proteus vulgaris , Catálise , Condroitina ABC Liase/química , Cinética , Engenharia de Proteínas , Proteus vulgaris/genética
3.
Adv Healthc Mater ; 11(10): e2102101, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35112508

RESUMO

Among the many molecules that contribute to glial scarring, chondroitin sulfate proteoglycans (CSPGs) are known to be potent inhibitors of neuronal regeneration. Chondroitinase ABC (ChABC), a bacterial lyase, degrades the glycosaminoglycan (GAG) side chains of CSPGs and promotes tissue regeneration. However, ChABC is thermally unstable and loses all activity within a few hours at 37 °C under dilute conditions. To overcome this limitation, the discovery of a diverse set of tailor-made random copolymers that complex and stabilize ChABC at physiological temperature is reported. The copolymer designs, which are based on chain length and composition of the copolymers, are identified using an active machine learning paradigm, which involves iterative copolymer synthesis, testing for ChABC thermostability upon copolymer complexation, Gaussian process regression modeling, and Bayesian optimization. Copolymers are synthesized by automated PET-RAFT and thermostability of ChABC is assessed by retained enzyme activity (REA) after 24 h at 37 °C. Significant improvements in REA in three iterations of active learning are demonstrated while identifying exceptionally high-performing copolymers. Most remarkably, one designed copolymer promotes residual ChABC activity near 30%, even after one week and notably outperforms other common stabilization methods for ChABC. Together, these results highlight a promising pathway toward sustained tissue regeneration.


Assuntos
Condroitina ABC Liase , Traumatismos da Medula Espinal , Axônios/metabolismo , Teorema de Bayes , Condroitina ABC Liase/química , Condroitina ABC Liase/metabolismo , Condroitina ABC Liase/farmacologia , Humanos , Regeneração Nervosa
4.
BMC Gastroenterol ; 21(1): 473, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34911454

RESUMO

BACKGROUND: Photobiomodulation with low-intensity laser (LIL) and chondroitinase ABC (ChABC) can repair damaged muscle tissue, so the aim of this study was to investigate the effect of co-administration of these two factors on anal sphincter repair in rabbits. METHODS: Male rabbits were studied in 5 groups (n = 7): Control (intact), sphincterotomy, laser, ChABC and laser + ChABC. 90 days after intervention were evaluated resting and maximum squeeze pressures, number of motor units, collagen amount, markers of muscle regeneration and angiogenesis. RESULTS: Resting pressure in the Laser + ChABC group was higher than the sphincterotomy, laser and ChABC groups (p < 0.0001). Maximum squeeze pressure in the all study groups was higher than sphincterotomy group (p < 0.0001). In the laser + ChABC and ChABC groups, motor unit numbers were more than the sphincterotomy group (p < 0.0001). Collagen content was significantly decreased in the laser (p < 0.0001) and laser + ChABC groups. ACTA1 (p = 0.001) and MHC (p < 0.0001) gene expression in the Laser + ChABC group were more than the laser or ChABC alone. VEGFA (p = 0.009) and Ki67 mRNA expression (p = 0.01) in the Laser + ChABC group were more than the laser group, But vimentin mRNA expression (p < 0.0001) was less than the laser group. CONCLUSION: Co-administration of ChABCs and photobiomodulation with LIL appears to improve the tissue structure and function of the anal sphincter in rabbits more than when used alone.


Assuntos
Canal Anal , Condroitina ABC Liase , Animais , Colágeno , Lasers , Masculino , Coelhos
5.
Glycobiology ; 31(11): 1571-1581, 2021 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-34392362

RESUMO

Chondroitinase ABC I (cABC-I) is the enzyme which cleaves the ß-1,4 glycosidic linkage of chondroitin sulfate (CS) by ß-elimination. To elucidate more accurately the substrate specificity of cABC-I, we evaluated the kinetic parameters of cABC-I and its reactivity with CS isomers displaying less structural heterogeneity as substrates, e.g., approximately 90 percent of disaccharide units in Chondroitin sulfate A (CSA) or Chondroitin sulfate C (CSC) is D-glucuronic acid and 4-O-sulfated N-acetyl galactosamine (GalNAc) (A-unit) or D-glucuronic acid and 6-O-sulfated GalNAc (C-unit), respectively. cABC-I showed the highest reactivity to CSA and CSC among all CS isomers, and the kcat/Km of cABC-I was higher for CSA than for CSC. Next, we determined the crystal structures of cABC-I in complex with CS disaccharides, and analyzed the crystallographic data in combination with molecular docking data. Arg500 interacts with 4-O-sulfated and 6-O-sulfated GalNAc residues. The distance between Arg500 and the 4-O-sulfate group was 0.8 Å shorter than that between Arg500 and the 6-O-sulfated group. Moreover, it is likely that the 6-O-sulfated group is electrostatically repulsed by the nearby Asp490. Thus, we demonstrated that cABC-I has the highest affinity for the CSA richest in 4-O-sulfated GalNAc residues among all CS isomers. Recently, cABC-I was used to treat lumbar disc herniation. The results provide useful information to understand the mechanism of the pharmacological action of cABC-I.


Assuntos
Condroitina ABC Liase/metabolismo , Sulfatos de Condroitina/metabolismo , Dissacarídeos/metabolismo , Simulação de Acoplamento Molecular , Configuração de Carboidratos , Condroitina ABC Liase/química , Sulfatos de Condroitina/química , Cristalografia por Raios X , Dissacarídeos/química , Humanos , Cinética , Especificidade por Substrato
6.
J Neural Eng ; 18(4)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34082409

RESUMO

Objective. Chondroitinase ABC (ChABC) has emerged as a promising therapeutic agent for central nervous system regeneration. Despite multiple beneficial outcomes for regeneration, translation of this enzyme is challenged by poor pharmacokinetics, localization, and stability.Approach. This study explored the function andin vitroapplication of engineered ChABC fused to galectin-3 (Gal3). Two previously developed ChABC-Gal3 oligomers (monomeric and trimeric) were evaluated for functionality and kinetics, then applied to anin vitrocellular outgrowth model using dorsal root ganglia (DRGs). The fusions were combined with two formulations of hyaluronan (HA)-based scaffolds to determine the extent of active enzyme release compared to wild type (WT) ChABC.Main Results. Monomeric and trimeric ChABC-Gal3 maintained digestive capabilities with kinetic properties that were substrate-dependent for chondroitin sulfates A, B, and C. The fusions had longer half-lives at 37 °C on the order of seven fold for monomer and twelve fold for trimer compared to WT. Both fusions were also effective at restoring DRG outgrowthin vitro. To create a combination approach, two triple-component hydrogels containing modified HA were formulated to match the mechanical properties of native spinal cord tissue and to support astrocyte viability (>80%) and adhesion. The hydrogels included collagen-I and laminin mixed with either 5 mg ml-1of glycidyl methacrylate HA or 3 mg ml-1Hystem. When combined with scaffolds, ChABC-Gal3 release time was lengthened compared to WT. Both fusions had measurable enzymatic activity for at least 10 d when incorporated in gels, compared to WT that lost activity after 1 d. These longer term release products from gels maintained adequate function to promote DRG outgrowth.Significance. Results of this study demonstrated cohesive benefits of two stabilized ChABC-Gal3 oligomers in combination with HA-based scaffolds for neural applications. Significant improvements to ChABC stability and release were achieved, meriting future studies of ChABC-Gal3/hydrogel combinations to target neural regeneration.


Assuntos
Condroitina ABC Liase , Traumatismos da Medula Espinal , Animais , Galectina 3 , Ácido Hialurônico , Hidrogéis , Ratos , Ratos Sprague-Dawley
7.
Mol Vis ; 27: 300-308, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34035644

RESUMO

Purpose: Migration and integration remain critical challenges for stem cell replacement therapy. Glial barriers play an important role in preventing cell migration and integration. The purpose of this study was to investigate the effect and mechanisms of chondroitinase ABC on the migration of murine retinal progenitor cells (mRPCs) transplanted into the subretinal space of B6 mice. Methods: mRPCs were harvested from the neural retinas of P1 enhanced green fluorescent protein (GFP) B6 mice. Two µl containing 2 × 105 expanded RPCs alone or combined with chondroitinase ABC in suspension were injected into the subretinal space of the recipient B6 mice. Immunohistochemistry was performed on the recipient B6 retinas to evaluate the glial barrier formation and migration of the mRPCs. Western blotting was also used to check the expression of the glial barriers. Results: Glial fibrillary acidic protein (GFAP) and vimentin could be seen around the transplanted mRPCs in the B6 mice. Formation of glial barriers prevented the migration of donor cells into the retinal layers. Chondroitinase ABC promoted the migration and survival rates of the engrafted retinal progenitor cells in the retinal layers of recipient B6 mice. Injection induced upregulation of GFAP, chondroitin, and CD44 expression. Chondroitinase ABC disrupted the glial barriers. The CD44 around the mRPCs was much lower in the chondroitinase group. However, the CD44 in the retinal layers was considerably higher in the chondroitinase group. With the employment of chondroitinase ABC, more cells migrated into the outer nuclear layer or inner nuclear layer. The chondroitin and CD44 expression decreased 3 weeks after transplantation in the chondroitinase ABC group. Conclusions: Chondroitinase ABC degraded glial barriers and enhanced the migration of transplanted mouse retinal progenitor cells. Chondroitinase ABC may also have induced activation of the CD44 signaling pathway to exert the effect.


Assuntos
Movimento Celular/efeitos dos fármacos , Neuroglia/metabolismo , Retina/citologia , Células-Tronco/citologia , Animais , Western Blotting , Sobrevivência Celular , Células Cultivadas , Condroitina ABC Liase/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Células-Tronco , Vimentina/metabolismo
8.
Stem Cell Res Ther ; 12(1): 10, 2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33407795

RESUMO

BACKGROUND: Spinal cord injury (SCI) presents a significant challenge for the field of neurotherapeutics. Stem cells have shown promise in replenishing the cells lost to the injury process, but the release of axon growth-inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs) by activated cells within the injury site hinders the integration of transplanted cells. We hypothesised that simultaneous application of enteric neural stem cells (ENSCs) isolated from the gastrointestinal tract, with a lentivirus (LV) containing the enzyme chondroitinase ABC (ChABC), would enhance the regenerative potential of ENSCs after transplantation into the injured spinal cord. METHODS: ENSCs were harvested from the GI tract of p7 rats, expanded in vitro and characterised. Adult rats bearing a contusion injury were randomly assigned to one of four groups: no treatment, LV-ChABC injection only, ENSC transplantation only or ENSC transplantation+LV-ChABC injection. After 16 weeks, rats were sacrificed and the harvested spinal cords examined for evidence of repair. RESULTS: ENSC cultures contained a variety of neuronal subtypes suitable for replenishing cells lost through SCI. Following injury, transplanted ENSC-derived cells survived and ChABC successfully degraded CSPGs. We observed significant reductions in the injured tissue and cavity area, with the greatest improvements seen in the combined treatment group. ENSC-derived cells extended projections across the injury site into both the rostral and caudal host spinal cord, and ENSC transplantation significantly increased the number of cells extending axons across the injury site. Furthermore, the combined treatment resulted in a modest, but significant functional improvement by week 16, and we found no evidence of the spread of transplanted cells to ectopic locations or formation of tumours. CONCLUSIONS: Regenerative effects of a combined treatment with ENSCs and ChABC surpassed either treatment alone, highlighting the importance of further research into combinatorial therapies for SCI. Our work provides evidence that stem cells taken from the adult gastrointestinal tract, an easily accessible source for autologous transplantation, could be strongly considered for the repair of central nervous system disorders.


Assuntos
Células-Tronco Neurais , Traumatismos da Medula Espinal , Animais , Axônios , Condroitina ABC Liase/farmacologia , Proteoglicanas de Sulfatos de Condroitina , Regeneração Nervosa , Células-Tronco Neurais/transplante , Ratos , Medula Espinal , Traumatismos da Medula Espinal/terapia
9.
Cartilage ; 13(2_suppl): 672S-683S, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-32441107

RESUMO

OBJECTIVE: To enhance the in vitro integration of self-assembled articular cartilage to native articular cartilage using chondroitinase ABC. DESIGN: To examine the hypothesis that chondroitinase ABC (C-ABC) integration treatment (C-ABCint) would enhance integration of neocartilage of different maturity levels, this study was conducted in 2 phases. In phase I, the impact on integration of 2 treatments, TCL (TGF-ß1, C-ABC, and lysyl oxidase like 2) and C-ABCint, was examined via a 2-factor, full factorial design. In phase II, construct maturity (2 levels) and C-ABCint concentration (3 levels) were the factors in a full factorial design to determine whether the effective C-ABCint dose was dependent on neocartilage maturity level. Neocartilages formed or treated per the factors above were placed into native cartilage rings, cultured for 2 weeks, and, then, integration was studied histologically and mechanically. Prior to integration, in phase II, a set of treated constructs were also assayed to provide a baseline of properties. RESULTS: In phase I, C-ABCint and TCL treatments synergistically enhanced interface Young's modulus by 6.2-fold (P = 0.004) and increased interface tensile strength by 3.8-fold (P = 0.02) compared with control. In phase II, the interaction of the factors C-ABCint and construct maturity was significant (P = 0.0004), indicating that the effective C-ABCint dose to improve interface Young's modulus is dependent on construct maturity. Construct mechanical properties were preserved regardless of C-ABCint dose. CONCLUSIONS: Applying C-ABCint to neocartilage is an effective integration strategy with translational potential, provided its dose is calibrated appropriately based on implant maturity, that also preserves implant biomechanical properties.


Assuntos
Cartilagem Articular , Condrócitos , Condroitina ABC Liase , Resistência à Tração , Engenharia Tecidual
10.
Enzyme Microb Technol ; 143: 109701, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33375969

RESUMO

Chondroitinase ABC can be used to prepare chondroitin sulfate (CS) oligosaccharides efficiently and environmentally. It also promotes nerve recovery through enzymatic degradation of glycosaminoglycan chains in damaged nerve tissue. In this study, two new chondroitin sulfate ABC lyases were expressed and characterized from Edwardsiella tarda LMG2793, with molecular weight of 116.8 kDa and 115.9 kDa, respectively. Two lyases ChABC I and ChABC II belonged to the polysaccharide lyase (PL) family 8. ChABC I and ChABC II showed enzyme activity towards chondroitin sulfate A (CS-A), CS-B, CS-C and CS-D, but had no activity towards hyaluronan (HA). The optimal temperature for ChABC I to exhibit the highest activity against CS-A was 40 °C and the optimal pH was 7.0. ChABC II showed the highest activity to CS-A at optimal temperature of 40 °C and pH of 9.0. ChABC I and ChABC II were stable at 37 °C and remained about 90 % of activity after incubation at 37 °C for 3 h. Many metal ions had no effect on the activity of ChABC I and ChABC II. These properties were beneficial to their further basic research and application. ChABC I was an endo-type enzyme while ChABC II was an exo-type enzyme. A group of amino acids were selected for further study by evaluating the sequence homology with other CS degradation lyases. Mutagenesis studies speculated that the catalytic residues in ChABC I were His522, Tyr529 and Arg581. The catalytic residues of ChABC II were His498, Tyr505 and Arg558. This work will contribute to the structural and functional characterization of biomedically relevant CS and promote the application of CS lyase in further basic research and therapeutics.


Assuntos
Condroitina ABC Liase , Sulfatos de Condroitina , Condroitina ABC Liase/genética , Clonagem Molecular , Edwardsiella tarda/genética , Íons
11.
Biotechnol J ; 16(5): e2000321, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33350041

RESUMO

Chondroitinase ABC I (csABC I) has attracted intensive attention because of its great potential in heparin refining and the enzymatic preparation of low-molecular-weight chondroitin sulfate (LMW-CS). However, low thermal resistance (<30℃) restricts its applications. Herein, structure-guided and sequence-assisted combinatorial engineering approaches were applied to improve the thermal resistance of Proteus vulgaris csABC I. By integrating the deletion of the flexible fragment R166-L170 at the N-terminal domain and the mutation of E694P at the C-terminal domain, variant NΔ5/E694P exhibited 247-fold improvement of its half-life at 37℃ and a 2.3-fold increase in the specific activity. Through batch fermentation in a 3-L fermenter, the expression of variant NΔ5/E694P in an Escherichia coli host reached 1.7 g L-1 with the activity of 1.0 × 105 U L-1 . Finally, the enzymatic approach for the preparation of LMW-CS was established. By modulating enzyme concentration and controlling depolymerization time, specifically distributed LMW-CS (7000, 3400, and 1900 Da) with low polydispersity was produced, demonstrating the applicability of these processes for the industrial production of LMW-CS in a more environmentally friendly way.


Assuntos
Condroitina ABC Liase , Sulfatos de Condroitina , Condroitina ABC Liase/genética , Condroitinases e Condroitina Liases , Peso Molecular , Proteus vulgaris/genética
12.
Neurobiol Learn Mem ; 177: 107358, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33285318

RESUMO

The perineuronal net (PNN) is a specialized type of extracellular matrix found in the central nervous system. The PNN forms on fast spiking neurons during postnatal development but the ontogeny of PNN development has yet to be elucidated. By studying the development and prevalence of the PNN in the juvenile and adult rat brain, we may be able to understand the PNN's role in development and learning and memory. We show that the PNN is fully developed in the deep cerebellar nuclei (DCN) of rats by P18. By using enzymatic digestion of the PNN with chondroitinase ABC (ChABC), we are able to study how digestion of the PNN affects cerebellar-dependent eyeblink conditioning in vivo and perform electrophysiological recordings from DCN neurons in vitro. In vivo degradation of the PNN resulted in significant differences in eyeblink conditioning amplitude and area. Female animals in the vehicle group demonstrated higher levels of conditioning as well as significantly higher post-probe conditioned responses compared to males in that group, differences not present in the ChABC group. In vitro, we found that DCN neurons with a disrupted PNN following exposure to ChABC had altered membrane properties, fewer rebound spikes, and decreased intrinsic excitability. Together, this study further elucidates the role of the PNN in cerebellar learning in the DCN and is the first to demonstrate PNN degradation may erase sex differences in delay conditioning.


Assuntos
Cerebelo/fisiologia , Condicionamento Clássico/fisiologia , Neurônios/fisiologia , Animais , Piscadela/fisiologia , Núcleos Cerebelares/efeitos dos fármacos , Núcleos Cerebelares/fisiologia , Cerebelo/anatomia & histologia , Cerebelo/efeitos dos fármacos , Condroitina ABC Liase/farmacologia , Condicionamento Clássico/efeitos dos fármacos , Eletrofisiologia , Feminino , Masculino , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Long-Evans , Fatores Sexuais
13.
J Biomed Mater Res A ; 109(7): 1232-1246, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33040470

RESUMO

Intervertebral disc (IVD) degeneration (IVDD) leads to structural and functional changes. Biomaterials for restoring IVD function and promoting regeneration are currently being investigated; however, such approaches require validation using animal models that recapitulate clinical, biochemical, and biomechanical hallmarks of the human pathology. Herein, we comprehensively characterized a sheep model of chondroitinase-ABC (ChABC) induced IVDD. Briefly, ChABC (1 U) was injected into the L1/2 , L2/3 , and L3/4 IVDs. Degeneration was assessed via longitudinal magnetic resonance (MR) and radiographic imaging. Additionally, kinematic, biochemical, and histological analyses were performed on explanted functional spinal units (FSUs). At 17-weeks, ChABC treated IVDs demonstrated significant reductions in MR index (p = 0.030) and disc height (p = 0.009) compared with pre-operative values. Additionally, ChABC treated IVDs exhibited significantly increased creep displacement (p = 0.004) and axial range of motion (p = 0.007) concomitant with significant decreases in tensile (p = 0.034) and torsional (p = 0.021) stiffnesses and long-term viscoelastic properties (p = 0.016). ChABC treated IVDs also exhibited a significant decrease in NP glycosaminoglycan: hydroxyproline ratio (p = 0.002) and changes in microarchitecture, particularly in the NP and endplates, compared with uninjured IVDs. Taken together, this study demonstrated that intradiscal injection of ChABC induces significant degeneration in sheep lumbar IVDs and the potential for using this model in evaluating biomaterials for IVD repair, regeneration, or fusion.


Assuntos
Condroitina ABC Liase/metabolismo , Modelos Animais de Doenças , Degeneração do Disco Intervertebral/patologia , Disco Intervertebral/patologia , Ovinos , Animais , Materiais Biocompatíveis/uso terapêutico , Fenômenos Biomecânicos , Condroitina ABC Liase/administração & dosagem , Feminino , Disco Intervertebral/diagnóstico por imagem , Disco Intervertebral/enzimologia , Degeneração do Disco Intervertebral/diagnóstico por imagem , Degeneração do Disco Intervertebral/enzimologia , Degeneração do Disco Intervertebral/terapia , Imageamento por Ressonância Magnética , Masculino , Teste de Materiais , Ovinos/fisiologia
14.
J Control Release ; 330: 1208-1219, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33229053

RESUMO

Activated microglia/macrophages infiltration, astrocyte migration, and increased production of inhibitory chondroitin sulfate proteoglycans (CSPGs) are standard harmful events taking place after the spinal cord injuries (SCI). The gliotic scar, viz. the outcome of chronic SCI, constitutes a long-lasting physical and chemical barrier to axonal regrowth. In the past two decades, various research groups targeted the hostile host microenvironments of the gliotic scar at the injury site. To this purpose, biomaterial scaffolds demonstrate to provide a promising potential for nervous cell restoration. We here focused our efforts on two self-assembling peptides (SAPs), featuring different self-assembled nanostructures, and on different methods of drug loading to exploit the neuroregenerative potential of Chondroitinase ABC (ChABC), a thermolabile pro-plastic agent attenuating the inhibitory action of CSPGs. Enzymatic activity of ChABC (usually lasting less than 72 hours in vitro) released from SAPs was remarkably detected up to 42 days in vitro. ChABC was continuously released in vitro from a few days to 42 days as well. Also, injections of ChABC loaded SAP hydrogels favored host neural regeneration and behavioral recovery in chronic SCI in rats. Hence, SAP hydrogels showed great promise for the delivery of Chondroitinase ABC in future therapies targeting chronic SCI.


Assuntos
Condroitina ABC Liase , Traumatismos da Medula Espinal , Animais , Condroitina ABC Liase/uso terapêutico , Preparações de Ação Retardada/uso terapêutico , Hidrogéis/uso terapêutico , Regeneração Nervosa , Peptídeos/uso terapêutico , Ratos , Medula Espinal , Traumatismos da Medula Espinal/tratamento farmacológico
15.
Eur J Neurosci ; 53(12): 4005-4015, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33220084

RESUMO

Current methods of experimentally degrading the specialized extracellular matrix (ECM), perineuronal nets (PNNs) have several limitations. Genetic knockout of ECM components typically has only partial effects on PNNs, and knockout of the major ECM component aggrecan is lethal in mice. Direct injection of the chondroitinase ABC (ChABC) enzyme into the mammalian brain is effective at degrading PNNs in vivo but this method typically lacks consistent, localized spatial targeting of PNN degradation. PNNs also regenerate within weeks after a ChABC injection, thus limiting the ability to perform long-term studies. Previous work has demonstrated that viral delivery of ChABC in mammalian neurons can successfully degrade PNNs for much longer periods, but the effects are similarly diffuse beyond the injection site. In an effort to gain cell-specific targeting of ChABC, we designed an adeno-associated virus encoding ChABC under the control of the Cre-LoxP system. We show that this virus is effective at targeting the synthesis of ChABC to Cre-expressing mouse neurons in vivo. Although ChABC expression is localized to the Cre-expressing neurons, we also note that ChABC is apparently trafficked and secreted at projection sites, as was previously reported for the non-Cre dependent construct. Overall, this method allows for cell-specific targeting of ChABC and long-term degradation of PNNs, which will ultimately serve as an effective tool to study the function of cell-autonomous regulation of PNNs in vivo. This novel approach may also aid in determining whether specific, long-term PNN loss is an appropriate strategy for treatment of neurodevelopmental disorders associated with PNN pathology.


Assuntos
Condroitina ABC Liase , Dependovirus , Animais , Dependovirus/genética , Matriz Extracelular , Integrases , Camundongos , Neurônios
16.
Sci Rep ; 10(1): 22422, 2020 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-33380731

RESUMO

The glycocalyx is thought to perform a potent, but not yet defined function in cellular adhesion and signaling. Since 95% of cancer cells have altered glycocalyx structure, this role can be especially important in cancer development and metastasis. The glycocalyx layer of cancer cells directly influences cancer progression, involving the complicated kinetic process of cellular adhesion at various levels. In the present work, we investigated the effect of enzymatic digestion of specific glycocalyx components on cancer cell adhesion to RGD (arginine-glycine-aspartic acid) peptide motif displaying surfaces. High resolution kinetic data of cell adhesion was recorded by the surface sensitive label-free resonant waveguide grating (RWG) biosensor, supported by fluorescent staining of the cells and cell surface charge measurements. We found that intense removal of chondroitin sulfate (CS) and dermatan sulfate chains by chondroitinase ABC reduced the speed and decreased the strength of adhesion of HeLa cells. In contrast, mild digestion of glycocalyx resulted in faster and stronger adhesion. Control experiments on a healthy and another cancer cell line were also conducted, and the discrepancies were analysed. We developed a biophysical model which was fitted to the kinetic data of HeLa cells. Our analysis suggests that the rate of integrin receptor transport to the adhesion zone and integrin-RGD binding is strongly influenced by the presence of glycocalyx components, but the integrin-RGD dissociation is not. Moreover, based on the kinetic data we calculated the dependence of the dissociation constant of integrin-RGD binding on the enzyme concentration. We also determined the dissociation constant using a 2D receptor binding model based on saturation level static data recorded at surfaces with tuned RGD densities. We analyzed the discrepancies of the kinetic and static dissociation constants, further illuminating the role of cancer cell glycocalyx during the adhesion process. Altogether, our experimental results and modelling demonstrated that the chondroitin sulfate and dermatan sulfate chains of glycocalyx have an important regulatory function during the cellular adhesion process, mainly controlling the kinetics of integrin transport and integrin assembly into mature adhesion sites. Our results potentially open the way for novel type of cancer treatments affecting these regulatory mechanisms of cellular glycocalyx.


Assuntos
Adesão Celular/fisiologia , Glicocálix/metabolismo , Glicocálix/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Fenômenos Biofísicos , Técnicas Biossensoriais , Condroitina ABC Liase/metabolismo , Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/metabolismo , Adesões Focais/metabolismo , Adesões Focais/patologia , Células HeLa , Humanos , Integrinas/metabolismo , Cinética , Modelos Biológicos , Oligopeptídeos/metabolismo
17.
Medicina (Kaunas) ; 56(11)2020 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-33228119

RESUMO

Background and Objectives: Chondroitin sulfate ABC endolyase (condoliase) was launched as a new drug for chemonucleolysis in 2018. Few studies assessed its clinical outcomes, and many important factors remain unclear. This study aimed to clarify the preoperative conditions in which condoliase could be highly effective. Materials and Methods: Of 47 patients who received condoliase, 34 were enrolled in this study. The mean age of the patients was 33 years. The average duration since the onset of disease was 8.6 months. We evaluated patients' low back and leg pain using a numerical rating scale (NRS) score at two time points (before therapy and 3 months after therapy). We divided the patients into two groups (good group (G): NRS score improvement ≥ 50%, poor group (P): NRS score improvement < 50%). The parameters evaluated were age, disease duration, body mass index (BMI), and positive or negative straight leg raising test results. In addition, the loss of disc height and preoperative radiological findings were evaluated. Results: In terms of low back and leg pain, the G group included 9/34 (26.5%) and 21/34 (61.8%) patients, respectively. Patients' age (low back pain G/P, 21/36.5 years) was significantly lower in the G group for low back pain (p = 0.001). High-intensity change in the protruded nucleus pulposus (NP) and spinal canal occupancy by the NP ≥ 40% were significantly high in those with leg pain in the G groups (14/21, p = 0.04; and 13/21, p = 0.03, respectively). Conclusions: The efficacy of improvement in leg pain was significantly correlated with high-intensity change and size of the protruded NP. Condoliase was not significantly effective for low back pain but could have an effect on younger patients.


Assuntos
Quimiólise do Disco Intervertebral , Deslocamento do Disco Intervertebral , Dor Lombar , Adulto , Condroitina ABC Liase/uso terapêutico , Humanos , Deslocamento do Disco Intervertebral/diagnóstico por imagem , Deslocamento do Disco Intervertebral/tratamento farmacológico , Dor Lombar/tratamento farmacológico , Vértebras Lombares/diagnóstico por imagem , Prognóstico , Resultado do Tratamento , Adulto Jovem
18.
J Biotechnol ; 324: 83-90, 2020 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-32979433

RESUMO

The bacterial enzyme chondroitinase ABC, which digests extracellular chondroitin sulfate proteoglycans, has been shown to enhance axonal regeneration. However, the utilization of this enzyme as therapeutics is notably restricted due to its thermal instability. Therefore, red luminescent porous silicon that hold promise for potential applications in biological/medical imaging was used as a carrying matrix for chondroitinase with the aim of enhancing its stability. Porous Si nanoparticles were prepared by electrochemical etching of silicon wafers in ethanolic HF solution. The size of nanoparticles (210 nm) and the mean pore diameter (8 -20 nm) were determined using dynamic light scattering and scanning electron microscopy. Purified chondroitinase was then incorporated into the silicon pores. Results revealed similar Km and lower Vmax value for the immobilized enzyme when compared with the free enzyme. The immobilized chondroitinase exhibited about a 4 fold increase in stability at 37 °C after 50 min. It is likely possible that, the enzyme was protected inside the pores resulted in higher stability. Moreover, porous silicon was seen to be capable of holding the chondroitinase for repeated cyclic tests for three times. The cell viability assay exhibited no significant cytotoxicity for Psi-chondroitinase up to 24 h.


Assuntos
Nanopartículas , Silício , Condroitina ABC Liase , Condroitinases e Condroitina Liases , Porosidade
19.
Int J Biol Macromol ; 164: 3762-3770, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32871123

RESUMO

Chondroitinases degrade chondroitin sulfate (CS) into oligosaccharides, of which the biological activities have vital roles in various fields. Some chondroitinases in polysaccharide lyase family 8 (PL8) have been classified into four subfamilies (PL8_1, PL8_2, PL8_3, and PL8_4) based on their sequence similarity and substrate specificities. In this study, a gene, vpa_0049, was cloned from marine bacterium Vibrio sp. QY108. The encoded protein, Vpa_0049, did not belong to the four existing subfamilies in PL8 based on phylogenetic analysis. Vpa_0049 could degrade various glycosaminoglycans (CS-A, CS-B, CS-C, CS-D, and HA) into unsaturated disaccharides in an endolytic manner, which was different from PL8 lyases of four existing subfamilies. The maximum activity of Vpa_0049 on different glycosaminoglycan substrates appeared at 30-37 °C and pH 7.0-8.0 in the presence of NaCl. Vpa_0049 showed approximately 50% of maximum activity towards CS-B and HA at 0 °C. It was stable in alkaline conditions (pH 8.0-10.6) and 0-30 °C. Our study provides a new broad-substrate chondroitinase and presents an in-depth understanding of PL8.


Assuntos
Condroitina ABC Liase/genética , Clonagem Molecular , Polissacarídeo-Liases/genética , Vibrio/genética , Condroitina Liases/genética , Sulfatos de Condroitina/genética , Glicosaminoglicanos/genética , Oligossacarídeos/genética , Filogenia , Especificidade por Substrato , Vibrio/enzimologia
20.
Biochem Biophys Res Commun ; 532(3): 420-426, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-32888649

RESUMO

The efficiency of cell therapy after spinal cord injury (SCI) depend on the survival of transplanted cells. However, sterile microenvironment and glial scar hyperplasia extremely reduce their numbers. Our previous study found overexpression of ChABC gene is positively correlated to migration ability. Expression of PTEN gene is closely associated with proliferation. However, whether manipulation of PTEN and ChABC on adipose-derived mesenchymal stem cells (ADSCs) promote motor recovery is unknown. This study aimed to promote hindlimb function recovery in SCI rats by enhancing proliferation and migration ability of ADSCs, transiently silencing expression of PTEN following overexpression of ChABC (double-gene modified ADSCs, DG-ADSCs). After PTEN silencing, we observed strong proliferation and accelerated G1-S transition in DG-ADSCs using CCK8 assay and flow cytometry. In addition, we demonstrated that migration numbers of DG-ADSCs were higher than control group using Transwell assay. The protein and mRNA levels of MAP2 and ßⅢ-tubulin in DG-ADSCs were increased compared with ADSCs. These results were further confirmed in SCI rats. Increased survival cells and reduction of glial scars were quantitatively analyzed in DG-ADSCs groups, which is definitely correlated to function recovery. Recovery of motor function was observed in DG-ADSCs treatment rats using BBB score, which emphasized that improved viability of transplanted cells and reduction of glial scars were an effective strategy for enhancing recovery of neurological function after SCI.


Assuntos
Condroitina ABC Liase/genética , Condroitina ABC Liase/metabolismo , Transplante de Células-Tronco Mesenquimais , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , Traumatismos da Medula Espinal/terapia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Feminino , Inativação Gênica , Células-Tronco Mesenquimais/patologia , Células-Tronco Mesenquimais/fisiologia , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/metabolismo , Neurônios/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/fisiopatologia , Regulação para Cima
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