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Serpins regulate coagulation and inflammation, binding serine proteases in suicide-inhibitory complexes. Target proteases cleave the serpin reactive center loop scissile P1-P1' bond, resulting in serpin-protease suicide-inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases, whereas mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast, Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon γ-receptor-deficient mice (IFNγR(-/-)). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the reactive center loop produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing Arg or Arg-Asn, not Arg-Met, with a 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking Arg or Arg-Asn with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b(+) monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in plasminogen activator inhibitor-1 serpin-deficient transplants. Plaque reduction correlated with mononuclear cell activation. In a separate study, Serp-1 peptide S-7 improved survival in the MHV68 vasculitis model, whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins.
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Coagulação Sanguínea/efeitos dos fármacos , Infecções por Herpesviridae/imunologia , Fatores Imunológicos , Proteínas de Membrana , Peptídeos , Rhadinovirus/imunologia , Vasculite/imunologia , Animais , Coagulação Sanguínea/imunologia , Modelos Animais de Doenças , Infecções por Herpesviridae/tratamento farmacológico , Humanos , Fatores Imunológicos/síntese química , Fatores Imunológicos/química , Fatores Imunológicos/imunologia , Células Jurkat , Proteínas de Membrana/síntese química , Proteínas de Membrana/química , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Knockout , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Vasculite/tratamento farmacológicoRESUMO
A biologically crucial natural product rapanone 1 was isolated from Embelia ribes at the gram scale with excellent purity. Semi-synthetic analogs of 1 semi-synthesized through reductive C-alkylation could increase the therapeutic value of the compounds. Herein, a new synthetic methodology was developed as a single-step reductive C-alkylation protocol using a metal-free, room-temperature-based reaction condition that can be scaled up to gram-scale synthesis with an excellent yield of up to 93%. A straightforward purification protocol was employed for the product obtained by this method. The derivatives of 1 showed antioxidant activity, which was evaluated using DPPH and ABTS scavenging assays. Compounds 5a-5ze showed an IC50 value of 2.48-3.37 µM and 1.81-3.12 µM. Substitution by electron-donating groups on the quinone moiety seems to play an essential role in the increased antioxidant activity of compounds 5a-5i, 5v, 5w, 5zc, and 5z. Further, the in vivo embryotoxicity of 1 and its derivatives was analyzed in a zebrafish-based aquatic toxicology model. Zebrafish embryos were exposed to 1 and 5a-5ze at 20 to 160 µM concentrations. They showed reduced toxicity and a survival rate of 90-98% after 96 hpf of treatment; similarly, the compounds 5a-5i, 5v, 5w, 5zc, and 5zd did not significantly affect the hatching rates of 75.66-85.33% or developmental abnormalities of the embryos after 48 hpf of treatment. In silico molecular docking studies for the parent compound, along with its derivatives 5a-5i, 5v-5w, 5zc-5zd, and standard l-ascorbic acid (l-Aa) indicated favorable interactions with the active site of the crystal structure, coupled with the assay protein PDB:1ZB6, which was responsible for the observed biological understanding and potential.
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Herein, we report the first-ever design strategy of modifying RAPTA-C into a self-reporting prodrug candidate based on Ru-coordinated polydiacetylene self-assembly. This nanosystem exhibits a dual lock strategy that responds to visible light and pH-stimuli sequentially one by one with a concomitant color change for controlled RAPTA-C release and real-time release monitoring in human gastric cancer cells.
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Polímero Poliacetilênico , Poli-Inos , Pró-Fármacos , Pró-Fármacos/química , Humanos , Polímero Poliacetilênico/química , Poli-Inos/química , Concentração de Íons de Hidrogênio , Liberação Controlada de Fármacos , Linhagem Celular Tumoral , Antineoplásicos/química , Antineoplásicos/farmacologia , Rutênio/química , Luz , Neoplasias Gástricas/tratamento farmacológico , Polímeros/química , Estrutura MolecularRESUMO
The urokinase-type plasminogen activator receptor (uPAR) is a unique protease binding receptor, now recognized as a key regulator of inflammation. Initially, uPA/uPAR was considered thrombolytic (clot-dissolving); however, recent studies have demonstrated its predominant immunomodulatory functions in inflammation and cancer. The uPA/uPAR complex has a multifaceted central role in both normal physiological and also pathological responses. uPAR is expressed as a glycophosphatidylinositol (GPI)-linked receptor interacting with vitronectin, integrins, G protein-coupled receptors, and growth factor receptors within a large lipid raft. Through protein-to-protein interactions, cell surface uPAR modulates intracellular signaling, altering cellular adhesion and migration. The uPA/uPAR also modifies extracellular activity, activating plasminogen to form plasmin, which breaks down fibrin, dissolving clots and activating matrix metalloproteinases that lyse connective tissue, allowing immune and cancer cell invasion and releasing growth factors. uPAR is now recognized as a biomarker for inflammatory diseases and cancer; uPAR and soluble uPAR fragments (suPAR) are increased in viral sepsis (COVID-19), inflammatory bowel disease, and metastasis. Here, we provide a comprehensive overview of the structure, function, and current studies examining uPAR and suPAR as diagnostic markers and therapeutic targets. Understanding uPAR is central to developing diagnostic markers and the ongoing development of antibody, small-molecule, nanogel, and virus-derived immune-modulating treatments that target uPAR.
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A suitable drug delivery strategy for metallodrugs is as significant as the strategies adopted for an efficient metallodrug design. In this study, piperlongumine, which is isolated from long pepper, is coordinated with a Ru(II)-p-cymene moiety to obtain an organoruthenated complex containing the natural product (Ru(pip)). The isolated complex shows higher cytotoxicity in MCF-7 breast cancer cells than in THP-1 leukemia and HepG2 liver cancer cells. The IC50 value of the complex in non-cancerous HEK-239 cells is also almost equal to that in MCF-7 cells. Next, with an aim to modulate the antiproliferative activity of Ru(pip) using a drug delivery strategy, the complex is loaded into mesoporous silica nanorods (MSNRs), which have a higher surface area than spherical silica nanoparticles. Furthermore, the outer surface of the loaded nanorods is covered with a polydiacetylene-lipid (PL) hybrid bilayer. Given the unique optical properties of polydiacetylene, the PL coating modifies non-fluorescent MSNRs into red-emissive particles (PL-Ru(pip)@MSNRs), which can be useful for diagnostic applications. The release profile studies reveal that the ene-yne conjugation in the PL coating ensures the sustained release of the complex from nanoparticles in both physiological and simulated cancer cell media. While Ru(pip) exhibits both necrotic and apoptotic modes of cell death, PL-Ru(pip)@MSNRs preferably induce the apoptotic mode of cell death in MCF-7 and THP-1 cells. Also, the nanoformulation exhibits a higher percentage of cell cycle arrest in the G0/G1 phase than Ru(pip), as measured by flow cytometry analysis. In contrast, the in vitro antioxidant potency of the complex is decreased after being loaded into PL-coated silica nanoparticles.
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Antineoplásicos , Benzodioxóis , Nanopartículas , Nanotubos , Polímero Poliacetilênico , Humanos , Linhagem Celular Tumoral , Dióxido de Silício , Preparações de Ação Retardada , Lipídeos , Antineoplásicos/farmacologiaRESUMO
This paper presents the synthesis of two new chitosan Schiff base (CSB) polymers, namely, 2PCT and 4MCT based on pyridin-2-ylmethyl-1H-indole-3-carbaldehyde and 1-(4-methyl-3,5-dimethylisoxazole)-1H-indole-3-carbaldehyde with chitosan (CT). The structural features of CSB polymers were confirmed by Fourier-transform infrared (FTIR) and proton nuclear magnetic resonance (1H NMR) spectroscopy and their antimicrobial activity was evaluated against Staphylococcus aureus, Escherichia coli and Candida albicans. The antioxidant studies found that both 2PCT and 4MCT presented significant free radical scavenging activity with IC50 at 169.01 and 372.84 µg/mL, respectively. The cell viability results obtained from in vitro cytotoxicity studies performed using human monocyte leukemia (THP-1) cells were found to be 75.6 ± 0.25 % and 79.1 ± 1.5 % for 2PCT and 4MCT, respectively, at a concentration of 10 mg/mL. Flow cytometry analysis demonstrated the reducing ability of CSB polymers on intracellular reactive oxygen species (ROS) in THP-1 cells. The overall results of antioxidant activity, in vitro biocompatibility and ability to reduce the intracellular ROS production emphasized that the CSB polymers prepared could serve as a potential biomaterial in biomedical applications, such as wound treatment process.
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Anti-Infecciosos , Quitosana , Humanos , Antioxidantes/farmacologia , Quitosana/química , Bases de Schiff/química , Células THP-1 , Espécies Reativas de Oxigênio , Anti-Infecciosos/química , Isoxazóis , Piridinas , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Two imine derivatives of chitosan (i-CTs), namely 2FCT and 5MCT, were synthesized by reacting chitosan (CT) with 2-(3-formyl-1H-indol-1yl)acetonitrile (2F), and 5-methoxyindole-3-carbaldehyde (5M), respectively. The antimicrobial evaluation of i-CTs exhibited stronger inhibition effect against Staphylococcus aureus, Escherichia coli and Candida albicans. The antioxidant activity of 2FCT and 5MCT showed strong scavenging ability with IC50 2.31 and 6.92 µg/mL, respectively. The results of in vitro cytotoxicity of 2FCT and 5MCT examined using human monocyte leukemia (THP-1) cells indicate no cytotoxic effect on host cells and the value of cell viability was found to be 87.08 and 84.47%, respectively. Measurement of intracellular Reactive Oxygen Species (ROS) production by flow cytometry analysis revealed that the 2FCT and 5MCT reduced the ROS generation by 83 and 43%, respectively. In summary, these findings show that i-CTs synthesized to be promising biomaterial for biomedical applications such as wound healing.
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Quitosana , Bases de Schiff , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Quitosana/farmacologia , Escherichia coli , Humanos , Indóis , Polímeros/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Bases de Schiff/farmacologia , Células THP-1RESUMO
The prominent characteristics of the biochar, high porosity, sorption capacity with low density improve the aeration, making it a desirable amendment material for composting process. The composting efficiency was analysed by the impact of rice husk biochar amendment (0, 2, 4, 6, 8 and 10%) in the presence of salts for the co-composting of food waste and swine manure, in composting reactors for 50 days. Results revealed that biochar amendment had improved the degradation rates by microbial activities in comparison with control. The final compost quality was improved by reducing the bulk density (29-53%), C/N ratio (29-57%), gaseous emissions (CO2, CH4, and NH3) and microbial pathogens (Escherichia coli and Salmonella sp.). However, 6% biochar amendment had significant improvement in compost quality, degradation rates and nutritional value which is recommended as the ideal ratio for obtaining mature compost from the feedstock, food waste and swine manure.
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Compostagem , Eliminação de Resíduos , Animais , Carvão Vegetal , Alimentos , Gases , Esterco , Nitrogênio/análise , Nutrientes , Sais , Solo , SuínosRESUMO
The present study proposes a system for co-composting food waste and poultry manure amended with rice husk biochar at different doses (0, 3, 5, 10%, w/w), saw dust, and salts. The effect of rice husk biochar on the characteristics of final compost was evaluated through stabilization indices such as electrical conductivity, bulk density, total porosity, gaseous emissions and nitrogen conservation. Results indicated that when compared to control, the biochar amendment extended the thermophilic stage of the composting, accelerated the biodegradation and mineralization of substrate mixture and helped in the maturation of the end product. Carbon dioxide, methane and ammonia emissions were reduced and the nitrogen conservation was achieved at a greater level in the 10% (w/w) biochar amended treatments. This study implies that the biochar and salts addition for co-composting food waste and poultry manure is beneficial to enhance the property of the compost.
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Compostagem , Eliminação de Resíduos , Animais , Carvão Vegetal , Poeira , Esterco , Minerais , Nitrogênio/análise , Aves Domésticas , Sais , SoloRESUMO
In this study agro-waste (Agwt) was aerobically composted using cow dung (CD) and mule dung (MD). Totally six different sets of compost treatments were prepared, as T1 (Agwt + CD, 1:1), T2 (Agwt + MD, 1:1), T3 (Agwt + CD, 1:3), T4 (Agwt + MD, 1:3), T5 (Agwt + CD, 3:1) and T6 (Agwt + MD, 3:1) in individual containers. All the compost treatments were degraded for 90 days. The organic wastes in the treatment containers were maintained with proper moisture level. All the final composts reached good manural stability and maturity index after 90 days. Among the six treatments, the T2 with Agwt + MD in 1:1 proportion attained a 10:1 C/N ratio and a near neutral pH (7.3). Indigenous microbes isolated and identified from the T2 compost sample showed protease, cellulase, amylase and lipase activities. The germination of Raphanus sativus L. seeds and vigorous plant growth parameters confirmed the non-pathogenic phytotoxic-free nature of finished composts. The radish crops supplied with T2 compost showed healthy tuber growth parameters (16.6 cm width, 35.6 cm length) compared with other treatments. The results from the experiments established that, the composts derived are eco-friendly amendment to plants and it has also improved the soil fertility due to its stability and maturity index. Thus, the present study concluded that composting agricultural crops waste with animal manure, especially mule dung promoted excellent biodegradation of organic complexes. It is a nature friendly solution for the management of solid waste such as agro-wastes utilizing mule dung.
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Compostagem , Animais , Biomassa , EquidaeRESUMO
Fluorescent carbon dots (C-dots) were fabricated from Anogeissus latifolia (Gum ghatti) gum extract using direct microwave pyrolysis method. The C-dots are fine-tuned concerning three parameters, viz., NaOH addition (presence and absence), microwave power, and irradiation time. C-dots optical properties were investigated through UV-visible (UV-Vis) and fluorescence spectroscopy. Using field emission scanning electron microscope (FESEM), high-resolution transmission electron microscopy (HRTEM), Fourier transform infrared (FT-IR), X-ray diffraction (XRD), and Raman Spectroscopy, physiochemical properties of synthesized C-dots were inspected. The average size of C-dots was estimated to be 4.8 ± 2 nm and is amorphous. These C-dots displayed high solubility in an aqueous medium due to oxygen functionality, and showed good fluorescence stability to high-ionic concentration and varied pH. The fluorescence spectra outcomes specified that C-dots exhibited excitation-dependent emission behavior. Furthermore, the C-dots biological function was tested for cell biocompatibility and bioimaging. The cytotoxicity studies were performed on Vero cell lines and compared with THP-1 human monocyte cell lines at different concentrations. The results revealed good biocompatibility app. 80 and 90% for Vero and THP-1 cell lines even after 24 h incubation with the C-dots. Finally, by employing C-dots as the fluorescent tool, THP-1 cells were imaged successfully via a Confocal Laser Scanning Microscope (CLSM) in a concentration-dependent manner.
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Lectins are a cluster of proteins which are capable of recognizing and binding to glycoconjugates and are extensively found in plants, animals, fungi and bacteria. Plant-derived lectins have been gaining importance over the years due to their innumerable biological activities and also have the added possibility of being compatible to the human system while simultaneously exhibiting properties like antimicrobial and antitumor activities. Abelmoschus esculentus (AE) commonly known as okra is a vegetable with medicinal properties. AE extracts are used to treat disorders such as constipation, microbial infection, urine retention, hypoglycemia and inflammation in humans. Previous studies showed that lectin isolated from AE exhibited anti inflammatory, anti nociceptive, anticancer, antioxidant and hemagglutinating activities. However, the antitumor effect of the lectin derived from this plant against neural cancer cells still remains unexplored. Glioblastoma is a malignant tumor of the nervous system. Treatment options for patients afflicted by glioblastoma is limited to surgical resection, preceded by radiation therapy and followed by chemotherapy. Hence it would be of interest to identify novel bio molecules with ability to selectively target glioblastoma with minimum side effects. In this aspect, lectins from vegetables that are commonly used as food products could offer a promising lead as anticancer molecules. The present study proves the anti-proliferative effect of lectin isolated from AE on human U87 glioma cells. MTT assay showed significant concentration dependent cytotoxic activity and the IC50 value was calculated as 21 µg/ml. Further, annexin V/FITC staining by FACS, the expression of caspase 3 and 7 and the circadian genes clock and Bmal1 using RT-PCR and the generation of intracellular ROS, cell cycle analysis by FACS revealed the ability of AEL to induce effective apoptosis.
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Abelmoschus , Relógios Circadianos , Glioblastoma , Animais , Apoptose , Caspases , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Humanos , LectinasRESUMO
Zaluzanin D (ZD) is a sesquiterpene lactone isolated from the leaves of Vernonia arborea. Earlier studies have highlighted the Sesquiterpene lactones (SLs) as molecules of medicinal value. The current study investigates the anti-inflammatory potential of ZD and its biotransformed derivatives in PMA differentiated human monocytic THP-1 cells. ZD and its fungal biotransformed derivatives Zaluzanin C (ZC) and 11,13- dihydrozaluzanin C (DZC) were screened for anti-inflammatory activity using their IC50 concentration. ZD showed significant ability to reduce PMA mediated THP-1 activation, while both ZC and DZC did not show any anti-inflammatory activity. Further studies revealed that ZD had ability to attenuate intracellular reactive oxygen species production in THP-1 cells as confirmed with FACS and fluorescence microscope experiments. Similarly, Oil red O (ORO) assay showed ability of ZD to inhibit lipid accumulation in monocytes. ZD also significantly reduced the expression of pro-inflammatory markers, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, MMP (Matrix metalloproteinases)-9 and MMP-2 as observed with RT-PCR and ELISA. Interestingly the molecule ZD also partially reversed DNA methylation levels in the PMA activated THP-1 cells. This indicated the ability of ZD to influence the epigenetic machinery of the cell. Overall the current study indicates that ZD has ability to attenuate inflammation in differentiated human THP-1 cells by regulating genes involved in the atherosclerosis inflammatory pathway. Thus ZD could potentially be used to modulate inflammation in atherosclerosis like disorders wherein monocytes play a key role.
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Anti-Inflamatórios/uso terapêutico , Aterosclerose/terapia , Inflamação/tratamento farmacológico , Metaloproteinase 9 da Matriz/genética , Monócitos/imunologia , Sesquiterpenos/uso terapêutico , Aterosclerose/imunologia , Diferenciação Celular , Metilação de DNA , Humanos , Concentração Inibidora 50 , Interleucina-1beta/metabolismo , Regiões Promotoras Genéticas/genética , Espécies Reativas de Oxigênio/metabolismo , Células THP-1 , Fator de Necrose Tumoral alfa/metabolismo , VernoniaRESUMO
BACKGROUND: To understand the mechanism underlying tamoxifen-induced multidrug resistance (MDR) and stem-like phenotypes in breast cancer cells, we treated the MCF-7 cells with 4-hydroxy-tamoxifen (TAM) for 6 months continuously and established MCF-7 tamoxifen resistance (TR) phenotypes. METHODS: In the present study, the following methods were used: cell viability assay, colony formation, cell cycle analysis, ALDEFLUOR assay, mammosphere formation assay, chromatin immunoprecipitation (ChIP) assay, PCR array, western blot analysis and quantitative reverse transcription polymerase chain reaction (QRT-PCR). RESULTS: The expression of ERα was significantly higher in MCF7-TR cells when compared with parental MCF-7 cells. MCF7-TR cells exposed to TAM showed a significant increase in the proliferation and rate of colony formation. The number of cancer stem cells was higher in MCF7-TR cells as observed by the increase in the number of ALDH+ cells. Furthermore, the number of mammospheres formed from the FACS-sorted ALDH+ cells was higher in MCF7-TR cells. Using PCR array analysis, we were able to identify that the long-term exposure of TAM leads to alterations in the epigenetic and MDR stem cell marker genes. Furthermore, western blot analysis demonstrated elevated levels of Notch-1 expression in MCF-TR cells compared with MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay revealed that Notch-1 enhanced the cyclin D1 expression significantly in these cells. In addition, we observed that MCF7-TR cells were resistant to doxorubicin but not the MCF-7 cells. CONCLUSIONS: In the present study, we conclude that the treatment with tamoxifen induces multiple epigenetic alterations that lead to the development of MDR and stem-like phenotypes in breast cancers. Therefore, our study provides better insights to develop novel treatment regime to control the progression of breast cancer.
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Thrombolytic serine proteases not only initiate fibrinolysis, but also are up-regulated in vascular disease and acute inflammatory responses. Although the serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is considered a main regulator of thrombolysis, PAI-1 is also associated with vascular inflammation. The role of other serpins that target thrombolytic proteases, PAI-2, PAI-3, and neuroserpin (NSP), in vascular inflammation is, however, less well defined. NSP is a mammalian serpin that, similar to PAI-1, inhibits urokinase- and tissue-type plasminogen activators (uPA and tPA, respectively) and has been most closely associated with the nervous system, with a demonstrated protective role after cerebral infarction in mouse models. However, the role of NSP in systemic arterial inflammation and plaque growth is not known. Serp-1 is a myxoma viral serpin that also inhibits tPA and uPA, as well as additionally inhibiting plasmin and factor Xa (fXa). Serp-1 has proven highly potent anti-inflammatory and anti-atherogenic activity. Here we assess the effects of NSP treatment on plaque growth and T-helper (Th) lymphocyte activity in a mouse aortic allograft transplant model, with comparison to Serp-1. NSP and Serp-1 both significantly reduced plaque growth and T-cell invasion. T-bet (a Th1 differentiation marker) was significantly reduced in transplanted aorta with associated reductions in Th1 and Th17, but not Th2, in splenocytes. NSP had additional Th modifying activity in non-transplanted mice. In summary, this is the first report that NSP possesses anti-inflammatory activity in systemic arteries, modifying Th cell responses and significantly reducing plaque growth in mouse aortic allografts.
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Aorta/transplante , Neuropeptídeos/farmacologia , Serpinas/farmacologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Animais , Anti-Inflamatórios , Doenças da Aorta/etiologia , Doenças da Aorta/prevenção & controle , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/uso terapêutico , Serpinas/uso terapêutico , Transplante/efeitos adversos , Doenças Vasculares/etiologia , Doenças Vasculares/prevenção & controle , NeuroserpinaRESUMO
BACKGROUND: Binding of chemokines to glycosaminoglycans (GAGs) is classically described as initiating inflammatory cell migration and creating tissue chemokine gradients that direct local leukocyte chemotaxis into damaged or transplanted tissues. While chemokine-receptor binding has been extensively studied during allograft transplantation, effects of glycosaminoglycan (GAG) interactions with chemokines on transplant longevity are less well known. Here we examine the impact of interrupting chemokine-GAG interactions and chemokine-receptor interactions, both locally and systemically, on vascular disease in allografts. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of GAG or CC chemokine receptor 2 (CCR2) deficiency were coupled with the infusion of viral chemokine modulating proteins (CMPs) in mouse aortic allograft transplants (n = 239 mice). Inflammatory cell invasion and neointimal hyperplasia were significantly reduced in N-deacetylase-N-sulfotransferase-1 (Ndst1(f/f)TekCre(+)) heparan sulfate (GAG)-deficient (Ndst1(-/-), p<0.044) and CCR2-deficient (Ccr2(-/-), p<0.04) donor transplants. Donor tissue GAG or CCR2 deficiency markedly reduced inflammation and vasculopathy, whereas recipient deficiencies did not. Treatment with three CMPs was also investigated; Poxviral M-T1 blocks CC chemokine receptor binding, M-T7 blocks C, CC, and CXC GAG binding, and herpesviral M3 binds receptor and GAG binding for all classes. M-T7 reduced intimal hyperplasia in wild type (WT) (Ccr2(+/+), p< or =0.003 and Ccr2(-/-), p=0.027) aortic allografts, but not in Ndst1(-/-) aortic allografts (p = 0.933). M-T1 and M3 inhibited WT (Ccr2(+/+) and Ndst1(+/+), p< or =0.006) allograft vasculopathy, but did not block vasculopathy in Ccr2(-/-) (p = 0.61). M-T7 treatment alone, even without immunosuppressive drugs, also significantly prolonged survival of renal allograft transplants (p< or =0.001). CONCLUSIONS/SIGNIFICANCE: Interruption of chemokine-GAG interactions, even in the absence of chemokine-receptor blockade, is a highly effective approach to reduction of allograft rejection, reducing vascular inflammation and prolonging allograft survival. Although chemokines direct both local and systemic cell migration, interruption of inherent chemokine responses in the donor tissue unexpectedly had a greater therapeutic impact on allograft vasculopathy.
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Quimiocinas/metabolismo , Glicosaminoglicanos/metabolismo , Rejeição de Enxerto/imunologia , Doenças Vasculares/imunologia , Doenças Vasculares/patologia , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Aorta/transplante , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocinas/farmacologia , Modelos Animais de Doenças , Rejeição de Enxerto/complicações , Rejeição de Enxerto/enzimologia , Hiperplasia , Inflamação/complicações , Inflamação/patologia , Transplante de Rim , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores CCR2/deficiência , Receptores CCR2/metabolismo , Sulfotransferases/deficiência , Sulfotransferases/metabolismo , Análise de Sobrevida , Doadores de Tecidos , Transplante Homólogo , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologia , Doenças Vasculares/complicações , Doenças Vasculares/enzimologia , Proteínas Virais/farmacologiaRESUMO
Chemokines are important for activation of a host of cellular immune and inflammatory responses including cell signaling, activation, and communication. M-T7, a myxoma virus protein, inhibits the activity of chemokines by direct binding to chemokines and/or with glycosaminoglycans (GAGs). To study the effects of this chemokine-modulating protein (CMP), we use a variety of in vitro and in vivo techniques to evaluate M-T7 inhibition of inflammatory cells. To quickly analyze the effects of M-T7, changes in cell adhesion and membrane fluidity are measured as well as cell migration in mouse ascites. For more physiological analyses, an aortic transplant model in rodents is used to assess change in inflammatory cell infiltrates and vascular plaque growth (rejection). Utilization of the combination of these in vitro and in vivo techniques allows for a more complete study of the chemokine-modulating activity of M-T7, and can be used to study other immune and inflammation-modulating proteins.
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Quimiocinas/farmacologia , Monócitos/efeitos dos fármacos , Myxoma virus/metabolismo , Proteínas Virais/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Humanos , Fluidez de Membrana/efeitos dos fármacos , Camundongos , Ligação Proteica/efeitos dos fármacos , Coelhos , RatosRESUMO
Serp-1 is a secreted myxoma viral serine protease inhibitor (serpin) with proven, highly effective, anti-inflammatory defensive activity during host cell infection, as well as potent immunomodulatory activity in a wide range of animal disease models. Serp-1 binds urokinase-type plasminogen activator (uPA) and the tissue-type PA, plasmin, and factor Xa, requiring uPA receptor (uPAR) for anti-inflammatory activity. To define Serp-1-mediated effects on inflammatory cell activation, we examined the association of Serp-1 with monocytes and T cells, effects on cellular migration, and the role of uPAR-linked integrins and actin-binding proteins in Serp-1 cellular responses. Our results show that Serp-1 associates directly with activated monocytes and T lymphocytes, in part through interaction with uPAR (P<0.001). Serp-1, but not mammalian serpin PA inhibitor-1 (PAI-1), attenuated cellular adhesion to the extracellular matrix. Serp-1 and PAI-1 reduced human monocyte and T cell adhesion (P<0.001) and migration across endothelial monolayers in vitro (P<0.001) and into mouse ascites in vivo (P<0.001). Serp-1 and an inactive Serp-1 mutant Serp-1(SAA) bound equally to human monocytes and T cells, but a highly proinflammatory mutant, Serp-1(Ala(6)), bound less well to monocytes. Serp-1 treatment of monocytes increased expression of filamin B actin-binding protein and reduced CD18 (beta-integrin) expression (P<0.001) in a uPAR-dependent response. Filamin colocalized and co-immunoprecipitated with uPAR, and short interference RNA knock-down of filamin blocked Serp-1 inhibition of monocyte adhesion. We report here that the highly potent, anti-inflammatory activity of Serp-1 is mediated through modification of uPAR-linked beta-integrin and filamin in monocytes, identifying this interaction as a central regulatory axis for inflammation.
Assuntos
Adesão Celular , Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/metabolismo , Monócitos/citologia , Myxoma virus/patogenicidade , Serpinas/fisiologia , Proteínas Virais/fisiologia , Filaminas , Humanos , Inflamação , Cadeias beta de Integrinas/metabolismo , Ligação Proteica/imunologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
OBJECTIVE: Apolipoprotein E (ApoE) is a lipid transport protein with expanded functions in cellular responses to tissue injury, immune regulation and cell growth. ApoE directs vascular changes that contribute to arterial protection as evidenced by the fact that isoforms of ApoE and ApoE deficiency correlate closely with accelerated plaque growth. The N-terminus of the ApoE protein has well-characterized functions, displaying lipid-binding and anti-atherogenic activity, whereas the function of the C-terminus is only partially defined. We have assessed the effects of a 14 amino acid C-terminal ApoE peptide, termed Ep1.B (239-252), on intimal neoplasia in animal models. This peptide is a fragment of a naturally processed peptide (236-252) of murine ApoE. METHODS AND RESULTS: Ep1.B injection reduced neointimal hyperplasia after vascular surgery in rats and mice. When given during early plaque progression in ApoE-deficient mice, Ep1.B injections also prevented plaque growth. Treatment with Ep1.B did not, however, reduce established plaque growth in older mice. Peptides with alanine substitution of amino acid 249, Ep1.N, and with complete sequence reversal, Ep1.R, did not consistently inhibit plaque growth. CONCLUSION: A naturally processed C-terminal ApoE peptide, Ep1.B, has anti-atherogenic activity indicating a role for this naturally metabolized peptide in vascular wound healing and lipid homeostasis.