RESUMEN
Heparan sulfate proteoglycans (HSPGs) occur in almost every tissue of the human body and consist of a protein core, with covalently attached glycosaminoglycan polysaccharide chains. These glycosaminoglycans are characterized by their polyanionic nature, due to sulfate and carboxyl groups, which are distributed along the chain. These chains can be modified by different enzymes at varying positions, which leads to huge diversity of possible structures with the complexity further increased by varying chain lengths. According to their location, HSPGs are divided into different families, the membrane bound, the secreted extracellular matrix, and the secretory vesicle family. As members of the extracellular matrix, they take part in cell-cell communication processes on many levels and with different degrees of involvement. Of particular therapeutic interest is their role in cancer and inflammation as well as in infectious diseases. In this review, we give an overview of the current status of medical approaches to antagonize HSPG function in pathology.
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Proteoglicanos de Heparán Sulfato/metabolismo , HumanosRESUMEN
The pro-inflammatory chemokine interleukin-8 (CXCL8) exerts its function by establishing a chemotactic gradient in infected or damaged tissues to guide neutrophil granulocytes to the site of inflammation via its G protein-coupled receptors (GPCRs) CXCR1 and CXCR2 located on neutrophils. Endothelial glycosaminoglycans (GAGs) have been proposed to support the chemotactic gradient formation and thus the inflammatory response by presenting the chemokine to approaching leukocytes. In this study, we show that neutrophil transmigration in vitro can be reduced by adding soluble GAGs and that this process is specific with respect to the nature of the glycan. To further investigate the GAG influence on neutrophil migration, we have used an engineered CXCL8 mutant protein (termed PA401) which exhibits a much higher affinity towards GAGs and an impaired GPCR activity. This dominant-negative mutant chemokine showed anti-inflammatory activity in various animal models of neutrophil-driven inflammation, i.e. in urinary tract infection, bleomycin-induced lung fibrosis, and experimental autoimmune uveitis. In all cases, treatment with PA401 resulted in a strong reduction of transmigrated inflammatory cells which became evident from histology sections and bronchoalveolar lavage. Since our CXCL8-based decoy targets GAGs and not GPCRs, our results show for the first time the crucial involvement of this glycan class in CXCL8/neutrophil-mediated inflammation and will thus pave the way to novel approaches of anti-inflammatory treatment.
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Glicosaminoglicanos/inmunología , Mediadores de Inflamación/inmunología , Neutrófilos/inmunología , Humanos , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/patología , Interleucina-8/inmunología , Interleucina-8/farmacología , Neutrófilos/patología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Migración Transendotelial y Transepitelial/inmunologíaRESUMEN
RATIONALE: Neutrophils play a fundamental role in a number of chronic lung diseases. Among the mediators of their recruitment to the lung, CXCL8 (IL-8) is considered to be one of the major players. CXCL8 exerts its chemotactic activity by binding to its GPCR receptors (CXCR1/R2) located on neutrophils, as well as through interactions with glycosaminoglycans (GAGs) on cell surfaces including those of the microvascular endothelium. Binding to GAG co-receptors is required to generate a solid-phase haptotactic gradient and to present IL-8/CXCL8 in a proper conformation to its receptors on circulating neutrophils. METHODS: We have engineered increased GAG-binding affinity into human CXCL8, thereby obtaining a competitive inhibitor that displaces wild-type IL-8/CXCL8 from GAGs. By additionally knocking-out the GPCR binding domain of the chemokine, we generated a dominant negative protein (dnCXCL8; PA401) with potent anti-inflammatory characteristics proven in vivo in a murine model of LPS-induced lung inflammation (Adage et al., 2015). Here we have further investigated PA401 activity in this pulmonary model by evaluating plasma changes induced by LPS on white blood cells (WBC) and a broad range of inflammatory markers, especially chemokines, by addressing immediate effects of PA401 on these parameters in healthy and LPS exposed mice. RESULTS: Aerosolized LPS induced a significant increase in bronchoalveolar lavage (BAL) neutrophils after 3 and 7h, as well as an increase in total WBC and changes in 21 of the 59 measured plasma markers, mostly belonging to the chemokine family. PA401 treatment in saline exposed mice didn't induce major changes in any of the measured parameters. When administered to LPS aerosolized mice, PA401 caused a significant normalization of KC/mCXCL1 and other inflammatory markers, as well as of blood WBC count. In addition, BAL neutrophils were significantly reduced, confirming the previously observed lung anti-inflammatory activity of PA401 in this experiment. CONCLUSIONS: PA401 is a new promising biologic therapeutic with a novel and unique mechanism of action for interfering with neutrophilic lung inflammation, that also normalizes plasma inflammatory markers.
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Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar , Modelos Animales de Enfermedad , Glicosaminoglicanos/metabolismo , Interleucina-8/metabolismo , Lipopolisacáridos/farmacología , Neutrófilos/efectos de los fármacos , Neumonía/inducido químicamente , Proteínas Recombinantes/metabolismo , Animales , Interleucina-8/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Neutrófilos/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Importance: Cerebral vasospasm largely contributes to a devastating outcome after aneurysmal subarachnoid hemorrhage (aSAH), with limited therapeutic options. Objective: To investigate the safety and efficacy of localized nicardipine release implants positioned around the basal cerebral vasculature at risk for developing proximal vasospasm after aSAH. Design, Setting, and Participants: This single-masked randomized clinical trial with a 52-week follow-up was performed between April 5, 2020, and January 23, 2023, at 6 academic neurovascular centers in Germany and Austria. Consecutive patients with World Federation of Neurological Surgeons grade 3 or 4 aSAH due to a ruptured anterior circulation aneurysm requiring microsurgical aneurysm repair participated. Intervention: During aneurysm repair, patients were randomized 1:1 to intraoperatively receive 10 implants at 4 mg of nicardipine each plus standard of care (implant group) or aneurysm repair alone plus standard of care (control group). Main Outcome and Measures: The primary end point was the incidence of moderate to severe cerebral angiographic vasospasm (aVS) between days 7 and 9 after aneurysm rupture as determined by digital subtraction angiography. Results: Of 41 patients, 20 were randomized to the control group (mean [SD] age, 54.9 [9.1] years; 17 female [85%]) and 21 to the implant group (mean [SD] age, 53.6 [11.9] years; 14 female [67%]). A total of 39 patients were included in the primary efficacy analysis. In the control group, 11 of 19 patients (58%) developed moderate or severe aVS compared with 4 of 20 patients (20%) in the implant group (P = .02). This outcome was paralleled by a lower clinical need for vasospasm rescue therapy in the implant group (2 of 20 patients [10%]) compared with the control group (11 of 19 patients [58%]; P = .002). Between days 13 and 15 after aneurysm rupture, new cerebral infarcts were noted in 6 of 19 patients (32%) in the control group and in 2 of 20 patients (10%) in the implant group (P = .13). At 52 weeks, favorable outcomes were noted in 12 of 18 patients (67%) in the control group and 16 of 19 patients (84%) in the implant group (P = .27). The adverse event rate did not differ between groups. Conclusions and Relevance: These findings show that placing nicardipine release implants during microsurgical aneurysm repair can provide safe and effective prevention of moderate to severe aVS after aSAH. A phase 3 clinical trial to investigate the effect of nicardipine implants on clinical outcome may be warranted. Trial Registration: ClinicalTrials.gov Identifier: NCT04269408.
RESUMEN
Biological functions of a variety of proteins are mediated via their interaction with glycosaminoglycans (GAGs). The structural diversity within the wide GAG landscape provides individual interaction sites for a multitude of proteins involved in several pathophysiological processes. This 'GAG angle' of such proteins as well as their specific GAG ligands give rise to novel therapeutic concepts for drug development. Current glycomic technologies to elucidate the glycan structure-function relationships, methods to investigate the selectivity and specificity of glycan-protein interactions and existing therapeutic approaches to interfere with GAG-protein interactions are discussed.
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Glicosaminoglicanos/metabolismo , Animales , Citocinas/metabolismo , Glicómica/métodos , Glicosaminoglicanos/química , Humanos , Polisacáridos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteómica/métodosRESUMEN
Introduction: Aneurysmal subarachnoid hemorrhage (aSAH) is associated with high morbidity and mortality. Post-hemorrhagic vasospasm with neurological deterioration is a major concern in this context. NicaPlant®, a modified release formulation of the calcium channel blocker nicardipine, has shown vasodilator efficacy preclinically and a similar formulation known as NPRI has shown anti-vasospasm activity in aSAH patients under compassionate use. Research question: The study aimed to assess pharmacokinetics and pharmacodynamics of NicaPlant® pellets to prevent vasospasm after clip ligation in aSAH. Material and methods: In this multicenter, controlled, randomized, dose escalation trial we assessed the safety and tolerability of NicaPlant®. aSAH patients treated by clipping were randomized to receive up to 13 NicaPlant® implants, similarly to the dose of NPRIs previous used, or standard of care treatment. Results: Ten patients across four dose groups were treated with NicaPlant® (3-13 implants) while four patients received standard of care. 45 non-serious and 13 serious adverse events were reported, 4 non-serious adverse events and 5 serious adverse events assessed a probable or possible causal relationship to the investigational medical product. Across the NicaPlant® groups there was 1 case of moderate vasospasm, while in the standard of care group there were 2 cases of severe vasospasm. Discussion and conclusion: The placement of NicaPlant® during clip ligation of a ruptured cerebral aneurysm raised no safety concern. The dose of 10 NicaPlant® implants was selected for further clinical studies.
RESUMEN
The recently identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, the cause of coronavirus disease (COVID-19) and the associated ongoing pandemic, frequently leads to severe respiratory distress syndrome and pneumonia with fatal consequences. Although several factors of this infection and its consequences are not completely clear, the presence and involvement of specific chemokines is undoubtedly crucial for the development and progression of COVID-19. Cytokine storm and the often-resulting cytokine release syndrome (CRS) are pathophysiological hallmarks in COVID-19 infections related to its most severe and fatal cases. In this hyperinflammatory event, chemokines and other cytokines are highly upregulated and are therefore not fulfilling their beneficial function in the host response anymore but causing harmful effects. Here, we present the recent views on the involvement of chemokines and selected cytokines in COVID-19 and the therapeutics currently in clinical development targeting or interfering with them, discussing their potentials in the treatment of COVID-19 infections.
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COVID-19/inmunología , Síndrome de Liberación de Citoquinas/inmunología , Citocinas/inmunología , SARS-CoV-2 , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Humanos , Tratamiento Farmacológico de COVID-19RESUMEN
OBJECTIVE: The management of patients with aneurysmal subarachnoid hemorrhage (aSAH) remains a highly demanding challenge in critical care medicine. Despite all efforts, the calcium channel antagonist nimodipine remains the only drug approved for improving outcomes after aSAH. However, in its current form of application, it provides less than optimal efficacy and causes dose-limiting hypotension in a substantial number of patients. Here, the authors tested in vitro the release dynamics of a novel formulation of the calcium channel blocker nicardipine and in vivo local tolerance and tissue reaction using a chronic cranial window model in mice. METHODS: To characterize the release kinetics in vitro, dissolution experiments were performed using artificial cerebrospinal fluid over a time period of 21 days. The excipients used in this formulation (NicaPlant) for sustained nicardipine release are a mixture of two completely degradable polymers. A chronic cranial window in C57BL/6 mice was prepared, and NicaPlant slices were placed in proximity to the exposed cerebral vasculature. Epifluorescence video microscopy was performed right after implantation and on days 3 and 7 after surgery. Vessel diameter of the arteries and veins, vessel permeability, vessel configuration, and leukocyte-endothelial cell interaction were quantified by computer-assisted analysis. Immunofluorescence staining was performed to analyze inflammatory reactions and neuronal alterations. RESULTS: In vitro the nicardipine release profile showed an almost linear curve with about 80% release at day 15 and full release at day 21. In vivo epifluorescence video microscopy showed a significantly higher arterial vessel diameter in the NicaPlant group due to vessel dilatation (21.6 ± 2.6 µm vs 17.8 ± 1.5 µm in controls, p < 0.01) confirming vasoactivity of the implant, whereas the venous diameter was not affected. Vessel dilatation did not have any influence on the vessel permeability measured by contrast extravasation of the fluorescent dye in epifluorescence microscopy. Further, an increased leukocyte-endothelial cell interaction due to the implant could not be detected. Histological analysis did not show any microglial activation or accumulation. No structural neuronal changes were observed. CONCLUSIONS: NicaPlant provides continuous in vitro release of nicardipine over a 3-week observation period. In vivo testing confirmed vasoactivity and lack of toxicity. The local application of this novel nicardipine delivery system to the subarachnoid space is a promising tool to improve patient outcomes while avoiding systemic side effects.
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Encéfalo/efectos de los fármacos , Bloqueadores de los Canales de Calcio/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Nicardipino/administración & dosificación , Hemorragia Subaracnoidea/tratamiento farmacológico , Animales , Encéfalo/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Preparaciones de Acción Retardada , Evaluación Preclínica de Medicamentos/métodos , Implantes de Medicamentos , Masculino , Ratones , Ratones Endogámicos C57BL , Nicardipino/metabolismo , Hemorragia Subaracnoidea/metabolismoRESUMEN
Non-competitive N-methyl-d-aspartate (NMDA) blockers induce schizophrenic-like behavior in healthy volunteers and exacerbate symptomatology in schizophrenic patients. Hence, a compound able to enhance NMDA neurotransmission by increasing levels of d-serine, an endogenous full agonist at the glycine site of the NMDA receptors, could have anti-psychotic activity. One way to increase d-serine levels is the inhibition of d-amino acid oxidase (DAAO), the enzyme responsible for d-serine oxidation. Indeed AS057278, a potent in vitro (IC(50)=0.91 microM) and ex vivo (ED(50)=2.2-3.95 microM) DAAO inhibitor, was able to increase d-serine fraction in rat cortex and midbrain (10 mg/kg i.v.). AS057278 was able to normalize phencyclidine (PCP)-induced prepulse inhibition after acute (80 mg/kg) and chronic (20 mg/kg b.i.d.) oral administration in mice. Finally, AS057278 after oral chronic treatment (10 mg/kg b.i.d.) was able to normalize PCP-induced hyperlocomotion. These results suggest that AS057278 has the potential to anti-psychotic action toward both cognitive and positive symptoms of schizophrenia.
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Aminoácido Oxidorreductasas/antagonistas & inhibidores , Antipsicóticos/farmacología , Inhibidores Enzimáticos/farmacología , Pirazoles/farmacología , Animales , Línea Celular , Corteza Cerebral/metabolismo , Colorimetría , D-Aspartato Oxidasa/antagonistas & inhibidores , D-Aspartato Oxidasa/genética , Inhibidores Enzimáticos/farmacocinética , Escherichia coli/enzimología , Glicina/metabolismo , Alucinógenos/farmacología , Inyecciones Intravenosas , Masculino , Mesencéfalo/metabolismo , Actividad Motora/efectos de los fármacos , Fenciclidina/farmacología , Plásmidos/genética , Pirazoles/farmacocinética , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Proteínas Recombinantes/química , Reflejo de Sobresalto/efectos de los fármacos , Serina/metabolismoRESUMEN
Multiple Sclerosis, a chronic inflammatory demyelinating disease of the central nervous system, involves an increased expression of monocyte chemotactic protein 1 MCP1-/CCL2. For exerting its chemotactic effects, chemokine binding to glycosaminoglycans (GAGs) is required and therefore this interaction represents a potential target for therapeutic intervention. We have designed an anti-inflammatory decoy variant, Met-CCL2 (Y13A S21K Q23R), embodying increased affinity for GAGs as well as knocked-out GPCR activation properties. This non-signalling dominant-negative mutant is shown here to be able to displace wild type CCL2 from GAGs by which it is supposed to interfere with the chemokine-related inflammatory response. In vivo, the anti-inflammatory properties were successfully demonstrated in a murine model of zymosan-induced peritonitis as well as in an experimental autoimmune encephalomyelitis, a model relevant for multiple sclerosis, where the compound lead to significantly reduced clinical scores due to reduction of cellular infiltrates and demyelination in spinal cord and cerebellum. These findings indicate a promising potential for future therapeutic development.
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Antiinflamatorios/administración & dosificación , Quimiocina CCL2/administración & dosificación , Encefalitis/prevención & control , Glicosaminoglicanos/química , Animales , Antiinflamatorios/farmacocinética , Cerebelo/efectos de los fármacos , Cerebelo/patología , Quimiocina CCL2/genética , Quimiocina CCL2/farmacocinética , Dexametasona/administración & dosificación , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/prevención & control , Femenino , Concentración 50 Inhibidora , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos , Peritonitis/inducido químicamente , Peritonitis/prevención & control , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , ZimosanRESUMEN
The CCL2-CCR2 chemokine axis has an important role in cancer progression where it contributes to metastatic dissemination of several cancer types (e.g., colon, breast, prostate). Tumor cell-derived CCL2 was shown to promote the recruitment of CCR2(+)/Ly6C(hi) monocytes and to induce vascular permeability of CCR2(+) endothelial cells in the lungs. Here we describe a novel decoy protein consisting of a CCL2 mutant protein fused to human serum albumin (dnCCL2-HSA chimera) with enhanced binding affinity to glycosaminoglycans that was tested in vivo. The monocyte-mediated tumor cell transendothelial migration was strongly reduced upon unfused dnCCL2 mutant treatment in vitro. dnCCL2-HSA chimera had an extended serum half-life and thus a prolonged exposure in vivo compared with the dnCCL2 mutant. dnCCL2-HSA chimera bound to the lung vasculature but caused minimal alterations in the leukocyte recruitment to the lungs. However, dnCCL2-HSA chimera treatment strongly reduced both lung vascular permeability and tumor cell seeding. Metastasis of MC-38GFP, 3LL, and LLC1 cells was significantly attenuated upon dnCCL2-HSA chimera treatment. Tumor cell seeding to the lungs resulted in enhanced expression of a proteoglycan syndecan-4 by endothelial cells that correlated with accumulation of the dnCCL2-HSA chimera in the vicinity of tumor cells. These findings demonstrate that the CCL2-based decoy protein effectively binds to the activated endothelium in lungs and blocks tumor cell extravasation through inhibition of vascular permeability.
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Quimiocina CCL2/metabolismo , Glicosaminoglicanos/metabolismo , Neoplasias/metabolismo , Receptores CCR2/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Quimiocina CCL2/antagonistas & inhibidores , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Expresión Génica , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Masculino , Ratones , Metástasis de la Neoplasia , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Sindecano-4/genética , Sindecano-4/metabolismoRESUMEN
Leptin gains access to the central nervous system where it influences activity of neuronal networks involved in ingestive behavior, neuroendocrine activity, and metabolism. In particular, the brain melanocortin (MC) system is important in leptin signaling and maintenance of energy balance. Although leptin or MC receptor insensitivity has been proposed to be associated with obesity, the present study compared central leptin and MC receptor stimulation on some of the above-mentioned parameters and investigated whether these treatments predict proneness to diet-induced obesity (DIO) in outbred Wistar rats. Third-cerebroventricular administration of equi-anorexigenic doses of leptin and of the MC agonist melanotan-II caused comparable increases in plasma ACTH and corticosterone levels and c-Fos-labeling in approximately 70% of paraventricular hypothalamic (PVN) neuronal cell bodies containing CRH. This reinforces involvement of paraventricular CRH neurons in the short-term neuroendocrine and ingestive effects of leptin and melanocortins. In the DIO prediction study, anorexigenic efficacy of melanotan-II was not correlated with any parameter linked to DIO but was highly correlated with MC in situ binding (with labeled [Nle(4),D-Phe(7)]alpha-MSH) as well as CRH immunoreactivity in the PVN of DIO rats. This suggests intricate relationships among MC signaling, the CRH system, and ingestive behavior unrelated to DIO. In the same animals, leptin's anorexigenic efficacy was not correlated with PVN MC in situ binding or CRH immunoreactivity but correlated inversely to post-DIO plasma leptin, liver weight, and abdominal adiposity, the latter being correlated to insulin resistance. Thus, differences in leptin but not MC signaling might underlie DIO, visceral obesity, and insulin resistance.
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Anorexia/inducido químicamente , Dieta , Leptina , Obesidad/etiología , Péptidos Cíclicos/administración & dosificación , Receptores de Melanocortina/agonistas , alfa-MSH/análogos & derivados , Animales , Ingestión de Alimentos/efectos de los fármacos , Humanos , Inyecciones Intraventriculares , Leptina/administración & dosificación , Leptina/farmacología , Masculino , Neuronas/efectos de los fármacos , Sistemas Neurosecretores/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/fisiopatología , Péptidos Cíclicos/farmacología , Valor Predictivo de las Pruebas , Ratas , Ratas Wistar , Receptores de Leptina , alfa-MSH/administración & dosificación , alfa-MSH/farmacologíaRESUMEN
We have engineered GPCR (G protein-coupled receptor) knock-out and high GAG-binding affinity into CXCL12α to inhibit CXCL12α-induced cell migration. Compared to wtCXCL12, the mutant CXCL12α (Δ8 L29K V39K) exhibited a 5.6-fold and a 2.2-fold affinity increase for heparin and heparan sulfate, respectively. From NaCl-based heparin displacement chromatography we concluded that more amino acid replacements would lead to altered GAG (glycosaminoglycan) ligand specificity. GAG silencing by this mutant was shown in a murine seeding model of human cancer cells, whereby a greatly reduced number of liver metastases was detected when the animals were treated intravenously with 1mg/kg CXCL12α (Δ8 L29K V39K) before cancer cell application.
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Quimiocina CXCL12/genética , Silenciador del Gen , Glicosaminoglicanos/deficiencia , Glicosaminoglicanos/genética , Mutación , Ingeniería de Proteínas , Animales , Línea Celular Tumoral , Quimiocina CXCL12/metabolismo , Femenino , Humanos , Hígado/patología , Ratones , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/genéticaRESUMEN
Chemokines like CCL2 mediate leukocyte migration to inflammatory sites by binding to G-protein coupled receptors on the target cell as well as to glycosaminoglycans (GAGs) on the endothelium of the inflamed tissue. We have recently shown that the dominant-negative Met-CCL2 mutant Y13A/S21K/Q23R with improved GAG binding affinity is highly bio-active in several animal models of inflammatory diseases. For chronic indications, we have performed here a fusion to human serum albumin (HSA) in order to extend the serum half-life of the chemokine mutant. To compensate a potential drop in GAG-binding affinity due to steric hindrance by HSA, a series of novel CCL2 mutants was generated with additional basic amino acids which were genetically introduced at sites oriented towards the GAG ligand. From this set of mutants, the Met-CCL2 variant Y13A/N17K/S21K/Q23K/S34K exhibited high GAG-binding affinity and a similar selectivity as wild type (wt) CCL2. From a set of different HSA-chemokine chimeric constructs, the linked HSA(C34A)(Gly)4Ser-Met-CCL2(Y13A/N17K/S21K/Q23K/S34K) fusion protein was found to show the best overall GAG-binding characteristics. Molecular modeling demonstrated an energetically beneficial fold of this novel protein chimera. This was experimentally supported by GdmCl-induced unfolding studies, in which the fusion construct exhibited a well-defined secondary structure and a transition point significantly higher than both the wt and the unfused CCL2 mutant protein. Unlike the wt chemokine, the quaternary structure of the HSA-fusion protein is monomeric according to size-exclusion chromatography experiments. In competition experiments, the HSA-fusion construct displaced only two of seven unrelated chemokines from heparan sulfate, whereas the unfused CCL2 mutant protein displaced five other chemokines. The most effective concentration of the HSA-fusion protein in inhibiting CCL2-mediated monocyte attachment to endothelial cells, as detected in the flow chamber, was 8.6 µg/ml. This novel HSA-fusion protein exhibits not only high affinity but also selective displacement of chemokines from GAGs binding. HSA is therefore proposed to be a highly promising scaffold candidate for therapeutic, GAG-targeting chemokine mutants.
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Sustitución de Aminoácidos , Quimiocina CCL2/química , Glicosaminoglicanos/química , Proteínas Recombinantes de Fusión/química , Albúmina Sérica/química , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Mutación Missense , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Albúmina Sérica/genética , Albúmina Sérica/metabolismoRESUMEN
It is broadly recognized that chemokine-activated neutrophils play a crucial role in the inflammation and disruption of lung tissue observed in several acute and chronic lung diseases. Since glycosaminoglycan side chains of proteoglycans act as chemokine co-receptors in inflammation, we have used a CXCL8-based dominant-negative mutant, dnCXCL8, to displace neutrophil-related chemokines in murine lungs using models of lung inflammation. Treatment with dnCXCL8 resulted in a dose-dependent reduction of neutrophil counts in bronchoalveolar lavage (BAL) of mice exposed to lipopolysaccharide after intravenous, subcutaneous and intratracheal administration. A strong and significant therapeutic effect was achieved already at a dose of 40 µg/kg of dnCXCL8. A similar dose response, but showing a broader spectrum of reduced inflammatory cells and soluble inflammatory markers, was observed in a murine model of tobacco smoke (TS)-induced lung inflammation. The broad spectrum of reduced inflammatory cells and markers can be due to the strong inhibition of neutrophil extravasation into the lung parenchyma, and/or to a relatively broad protein displacement profile of dnCXCL8 which may compete not only with wtCXCL8 for glycosaminoglycan-binding but possibly also with other related glycosaminoglycan-binding pro-inflammatory chemokines. Overall our results demonstrate that antagonizing CXCL8/glycosaminoglycan binding reduces lung inflammation as well as associated lung tissue damage due to LPS and TS and may therefore be a new therapeutic approach for lung pathologies characterized by a neutrophilic inflammatory phenotype.
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Glicosaminoglicanos/metabolismo , Interleucina-8/genética , Interleucina-8/farmacología , Pulmón/efectos de los fármacos , Terapia Molecular Dirigida/métodos , Neumonía/tratamiento farmacológico , Ingeniería de Proteínas , Animales , Biomarcadores/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-8/metabolismo , Interleucina-8/uso terapéutico , Lipopolisacáridos/farmacología , Pulmón/inmunología , Pulmón/metabolismo , Masculino , Ratones , Microvasos/citología , Infiltración Neutrófila/efectos de los fármacos , Neumonía/genética , Neumonía/inmunología , Neumonía/metabolismo , Humo/efectos adversos , Sindecano-4/genética , Nicotiana/químicaRESUMEN
BACKGROUND: The chemokine interleukin-8 (CXCL8) is a key mediator of inflammation in airways of patients with cystic fibrosis (CF). Glycosaminoglycans (GAGs) possess the ability to influence the chemokine profile of the CF lung by binding CXCL8 and protecting it from proteolytic degradation. CXCL8 is maintained in an active state by this glycan interaction thus increasing infiltration of immune cells such as neutrophils into the lungs. As the CXCL8-based decoy PA401 displays no chemotactic activity, yet demonstrates glycan binding affinity, the aim of this study was to investigate the anti-inflammatory effect of PA401 on CXCL8 levels, and activity, in CF airway samples in vitro. METHODS: Bronchoalveolar lavage fluid (BALF) was collected from patients with CF homozygous for the ΔF508 mutation (n=13). CXCL8 in CF BALF pre and post exposure to PA401 was quantified by ELISA. Western blot analysis was used to determine PA401 degradation in CF BALF. The ex vivo chemotactic activity of purified neutrophils in response to CF airway secretions was evaluated post exposure to PA401 by use of a Boyden chamber-based motility assay. RESULTS: Exposure of CF BALF to increasing concentrations of PA401 (50-1000pg/ml) over a time course of 2-12h in vitro, significantly reduced the level of detectable CXCL8 (P<0.05). Interestingly, PA401 engendered release of CXCL8 from GAGs exposing the chemokine susceptible to proteolysis. Subsequently, a loss of PA401 was observed (P<0.05) due to proteolytic degradation by elastase like proteases. A 25% decrease in neutrophil chemotactic efficiency towards CF BALF samples incubated with PA401 was also observed (P<0.05). CONCLUSION: PA401 can disrupt CXCL8:GAG complexes, rendering the chemokine susceptible to proteolytic degradation. Clinical application of a CXCL8 decoy, such as PA401, may serve to decrease the inflammatory burden in the CF lung in vivo.
Asunto(s)
Líquido del Lavado Bronquioalveolar , Fibrosis Quística/metabolismo , Interleucina-8/metabolismo , Proteínas Recombinantes/farmacología , Quimiotaxis/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Glicosaminoglicanos/metabolismo , Humanos , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , Proteolisis/efectos de los fármacos , Adulto JovenRESUMEN
IL (interleukin)-8 [CXCL8 (CXC chemokine ligand 8)] exerts its role in inflammation by triggering neutrophils via its specific GPCRs (G-protein-coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, for which additional binding to endothelial HS-GAGs (heparan sulphate-glycosaminoglycans) is required. We present here a novel approach for blocking the CXCL8-related inflammatory cascade by generating dominant-negative CXCL8 mutants with improved GAG-binding affinity and knocked-out CXCR1/CXCR2 activity. These non-signalling CXCL8 decoy proteins are able to displace WT (wild-type) CXCL8 and to prevent CXCR1/CXCR2 signalling thereby interfering with the inflammatory response. We have designed 14 CXCL8 mutants that we subdivided into three classes according to number and site of mutations. The decoys were characterized by IFTs (isothermal fluorescence titrations) and SPR (surface plasmon resonance) to determine GAG affinity. Protein stability and structural changes were evaluated by far-UV CD spectroscopy and knocked-out GPCR response was shown by Boyden chamber and Ca2+ release assays. From these experiments, CXCL8(Δ6F17KF21KE70KN71K) emerged with the most promising in vitro characteristics. This mutant was therefore further investigated in a murine model of mBSA (methylated BSA)-induced arthritis in mice where it showed strong anti-inflammatory activity. Based on these results, we propose that dominant-negative CXCL8 decoy proteins are a promising class of novel biopharmaceuticals with high therapeutic potential in inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , Interleucina-8/farmacología , Sustitución de Aminoácidos , Animales , Antiinflamatorios/química , Artritis Reumatoide/tratamiento farmacológico , Sitios de Unión , Bovinos , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Guanidina/química , Heparitina Sulfato/química , Humanos , Interleucina-8/química , Interleucina-8/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Unión Proteica , Desnaturalización Proteica , Receptores de Interleucina-8A/químicaRESUMEN
Glycosaminoglycans (GAGs) are a class of highly negatively charged, unbranched, O-linked polysaccharides that are involved in many diseases. Their role as a protein-binding matrix on cell surfaces has long been recognized, but therapeutic approaches to interfere with protein-GAG interactions have been limited due to the complex chemistry of GAGs, on one hand, and due to the lack of specific antibodies against GAGs, on the other hand. We have developed a protein engineering platform (the so-called CellJammer(®) technology), which enables us to introduce higher GAG-binding affinity into wild-type GAG-binding proteins and to combine this with impaired biological, receptor-binding function. Chemokines are among the prototypic GAG-binding proteins and here we present selected results of our CellJammer technology applied to several of these proinflammatory proteins. An overview is given of our lead decoy protein, PA401, which is a CXCL8-based mutant protein with increased GAG-binding affinity and decreased CXCR1/2 binding and activation. Major results from our CCL2 and CCL5 programmes are also summarized and the potential for clinical application of these decoy proteins is presented.
Asunto(s)
Quimiocinas/metabolismo , Glicosaminoglicanos/metabolismo , Animales , Quimiocinas/química , Glicosaminoglicanos/química , Humanos , Unión Proteica , Conformación ProteicaRESUMEN
OBJECTIVES: A nonagonist monocyte chemotactic protein-1 (MCP-1/CCL2) mutant (PA508) with increased affinity for glycosaminoglycans and thus competing with CCL2 was evaluated as a candidate for preventing neointima formation or myocardial ischemia/reperfusion injury. BACKGROUND: Myocardial infarction (MI) remains a major cause of death worldwide despite improved interventional and therapeutic options. Therefore, the discovery of drugs that limit restenosis after intervention and post-MI damage remains an important challenge. METHODS: The function of PA508 was assessed in functional assays in vitro and in mouse models of wire-induced neointima formation and experimental MI. RESULTS: PA508 was functionally inactive in CC chemokine receptor 2 (CCR2) binding and calcium influx but inhibited monocyte chemotaxis or transendothelial migration toward CCL2, suggesting that it interferes with CCL2 presentation. In wild-type but not CCR2-deficient mice, PA508 reduced inflammatory leukocyte recruitment without affecting differential leukocyte counts, CCL2 levels, organ function, or morphology, indicating that it specifically attenuates the CCL2-CCR2 axis. Compared with vehicle, daily intraperitoneal injection of PA508 significantly (p < 0.05, n = 5) reduced neointimal plaque area and mononuclear cell infiltration in carotid arteries of hyperlipidemic apolipoprotein E-deficient mice while increasing smooth muscle cell content. In C57Bl/6J mice that underwent myocardial ischemia/reperfusion, treatment with PA508 significantly reduced infarction size, monocyte infiltration, and collagen and myofibroblast content in the infarction area and preserved heart function compared with vehicle (p < 0.05, n = 4 to 8). CONCLUSIONS: Here we demonstrate that administration of a rationally designed CCL2 competitor reduced inflammatory monocyte recruitment, limited neointimal hyperplasia, and attenuated myocardial ischemia/reperfusion injury in mice and could therefore be envisioned as a combined therapeutic approach for restenosis and MI.
Asunto(s)
Quimiocina CCL2/fisiología , Daño por Reperfusión Miocárdica/complicaciones , Neointima , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BLRESUMEN
Receptor like protein tyrosine phosphatase zeta (RPTPz) (also known as RPTPbeta or PTPxi) is a tyrosine phosphatase widely expressed in the nervous system, thought to play a role in cell-cell communication. However, knocking out RPTPz does not induce major neural abnormalities in mice. In order to better assess the potential role of RPTPz in various neural functions, we performed a comprehensive behavioural characterization of CNS/PNS functions in knockout mice (RPTPz -/-) confirming previously observed impaired working memory functions and further demonstrating an altered motor coordination. Moreover, RPTPz -/- mice displayed reduced responses to moderate thermal and tactile stimuli, both in baseline and under inflammatory conditions. These findings assign novel functional role of RPTPz in motor coordination and nociception.