Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
1.
J Biol Chem ; 292(2): 732-747, 2017 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-27913625

RESUMO

Podocyte injury is an early event in diabetic kidney disease and is a hallmark of glomerulopathy. MicroRNA-146a (miR-146a) is highly expressed in many cell types under homeostatic conditions, and plays an important anti-inflammatory role in myeloid cells. However, its role in podocytes is unclear. Here, we show that miR-146a expression levels decrease in the glomeruli of patients with type 2 diabetes (T2D), which correlates with increased albuminuria and glomerular damage. miR-146a levels are also significantly reduced in the glomeruli of albuminuric BTBR ob/ob mice, indicating its significant role in maintaining podocyte health. miR-146a-deficient mice (miR-146a-/-) showed accelerated development of glomerulopathy and albuminuria upon streptozotocin (STZ)-induced hyperglycemia. The miR-146a targets, Notch-1 and ErbB4, were also significantly up-regulated in the glomeruli of diabetic patients and mice, suggesting induction of the downstream TGFß signaling. Treatment with a pan-ErbB kinase inhibitor erlotinib with nanomolar activity against ErbB4 significantly suppressed diabetic glomerular injury and albuminuria in both WT and miR-146a-/- animals. Treatment of podocytes in vitro with TGF-ß1 resulted in increased expression of Notch-1, ErbB4, pErbB4, and pEGFR, the heterodimerization partner of ErbB4, suggesting increased ErbB4/EGFR signaling. TGF-ß1 also increased levels of inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and MCP-1 induced protein-1 (MCPIP1), a suppressor of miR-146a, suggesting an autocrine loop. Inhibition of ErbB4/EGFR with erlotinib co-treatment of podocytes suppressed this signaling. Our findings suggest a novel role for miR-146a in protecting against diabetic glomerulopathy and podocyte injury. They also point to ErbB4/EGFR as a novel, druggable target for therapeutic intervention, especially because several pan-ErbB inhibitors are clinically available.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , MicroRNAs/metabolismo , Podócitos/metabolismo , Receptor ErbB-4/biossíntese , Receptor Notch1/biossíntese , Regulação para Cima , Animais , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Cloridrato de Erlotinib/farmacologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , Podócitos/patologia , Receptor ErbB-4/genética , Receptor Notch1/genética , Ribonucleases/genética , Ribonucleases/metabolismo , Fatores de Risco , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
2.
J Am Soc Nephrol ; 27(11): 3308-3319, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27020855

RESUMO

Gain-of-function mutations of classic transient receptor potential channel 6 (TRPC6) were identified in familial FSGS, and increased expression of wild-type TRPC6 in glomeruli is observed in several human acquired proteinuric diseases. Synaptopodin, an actin binding protein that is important in maintaining podocyte function, is downregulated in various glomerular diseases. Here, we investigated whether synaptopodin maintains podocyte function by regulating podocyte surface expression and activity of TRPC6. We show indirect interaction and nonrandom association of synaptopodin and TRPC6 in podocytes. Knockdown of synaptopodin in cultured mouse podocytes increased the expression of TRPC6 at the plasma membrane, whereas overexpression of synaptopodin decreased it. Mechanistically, synaptopodin-dependent TRPC6 surface expression required functional actin and microtubule cytoskeletons. Overexpression of wild-type or FSGS-inducing mutant TRPC6 in synaptopodin-depleted podocytes enhanced TRPC6-mediated calcium influx and induced apoptosis. In vivo, knockdown of synaptopodin also caused increased podocyte surface expression of TRPC6. Administration of cyclosporin A, which stabilizes synaptopodin, reduced LPS-induced proteinuria significantly in wild-type mice but to a lesser extent in TRPC6 knockout mice. Furthermore, administration of cyclosporin A reversed the LPS-induced increase in podocyte surface expression of TRPC6 in wild-type mice. Our findings suggest that alteration in synaptopodin levels under disease conditions may modify intracellular TRPC6 channel localization and activity, which further contribute to podocyte dysfunction. Reducing TRPC6 surface levels may be a new approach to restoring podocyte function.


Assuntos
Proteínas dos Microfilamentos/fisiologia , Podócitos/metabolismo , Proteinúria/metabolismo , Canais de Cátion TRPC/biossíntese , Animais , Membrana Celular/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Podócitos/ultraestrutura , Canal de Cátion TRPC6
3.
N Engl J Med ; 369(25): 2416-23, 2013 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-24206430

RESUMO

Abatacept (cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin fusion protein [CTLA-4-Ig]) is a costimulatory inhibitor that targets B7-1 (CD80). The present report describes five patients who had focal segmental glomerulosclerosis (FSGS) (four with recurrent FSGS after transplantation and one with primary FSGS) and proteinuria with B7-1 immunostaining of podocytes in kidney-biopsy specimens. Abatacept induced partial or complete remissions of proteinuria in these patients, suggesting that B7-1 may be a useful biomarker for the treatment of some glomerulopathies. Our data indicate that abatacept may stabilize ß1-integrin activation in podocytes and reduce proteinuria in patients with B7-1-positive glomerular disease.


Assuntos
Antígeno B7-1/metabolismo , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Imunoconjugados/farmacologia , Abatacepte , Adolescente , Adulto , Antígeno B7-1/antagonistas & inibidores , Biomarcadores/metabolismo , Criança , Feminino , Glomerulosclerose Segmentar e Focal/complicações , Glomerulosclerose Segmentar e Focal/imunologia , Humanos , Imunoconjugados/uso terapêutico , Masculino , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Proteinúria/tratamento farmacológico , Proteinúria/etiologia , Adulto Jovem
4.
J Am Soc Nephrol ; 26(11): 2741-52, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25858967

RESUMO

Podocyte injury and loss mark an early step in the pathogenesis of various glomerular diseases, making these cells excellent targets for therapeutics. However, cell-based high-throughput screening assays for the rational development of podocyte-directed therapeutics are currently lacking. Here, we describe a novel high-content screening-based phenotypic assay that analyzes thousands of podocytes per assay condition in 96-well plates to quantitatively measure dose-dependent changes in multiple cellular features. Our assay consistently produced a Z' value >0.44, making it suitable for compound screening. On screening with >2100 pharmacologically active agents, we identified 24 small molecules that protected podocytes against injury in vitro (1% hit rate). Among the identified hits, we confirmed an ß1-integrin agonist, pyrintegrin, as a podocyte-protective agent. Treatment with pyrintegrin prevented damage-induced decreases in F-actin stress fibers, focal adhesions, and active ß1-integrin levels in cultured cells. In vivo, administration of pyrintegrin protected mice from LPS-induced podocyte foot process effacement and proteinuria. Analysis of the murine glomeruli showed that LPS administration reduced the levels of active ß1 integrin in the podocytes, which was prevented by cotreatment with pyrintegrin. In rats, pyrintegrin reduced peak proteinuria caused by puromycin aminonucleoside-induced nephropathy. Our findings identify pyrintegrin as a potential therapeutic candidate and show the use of podocyte-based screening assays for identifying novel therapeutics for proteinuric kidney diseases.


Assuntos
Hidroxiquinolinas/química , Integrina beta1/metabolismo , Glomérulos Renais/metabolismo , Podócitos/citologia , Sulfonamidas/química , Actinas/metabolismo , Albuminúria/metabolismo , Animais , Movimento Celular , Células Epiteliais/efeitos dos fármacos , Adesões Focais/metabolismo , Ensaios de Triagem em Larga Escala , Nefropatias/metabolismo , Lipopolissacarídeos/química , Camundongos , Microscopia Confocal , Fenótipo , Proteinúria/patologia , Puromicina Aminonucleosídeo/química , Ratos
5.
Am J Respir Cell Mol Biol ; 53(6): 793-801, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25909334

RESUMO

Lung inflammation plays a key role in the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants. The challenge in BPD management is the lack of effective and safe antiinflammatory agents. Leukadherin-1 (LA1) is a novel agonist of the leukocyte surface integrin CD11b/CD18 that enhances leukocyte adhesion to ligands and vascular endothelium and thus reduces leukocyte transendothelial migration and influx to the injury sites. Its functional significance in preventing hyperoxia-induced neonatal lung injury is unknown. We tested the hypothesis that administration of LA1 is beneficial in preventing hyperoxia-induced neonatal lung injury, an experimental model of BPD. Newborn rats were exposed to normoxia (21% O2) or hyperoxia (85% O2) and received twice-daily intraperitoneal injection of LA1 or placebo for 14 days. Hyperoxia exposure in the presence of the placebo resulted in a drastic increase in the influx of neutrophils and macrophages into the alveolar airspaces. This increased leukocyte influx was accompanied by decreased alveolarization and angiogenesis and increased pulmonary vascular remodeling and pulmonary hypertension (PH), the pathological hallmarks of BPD. However, administration of LA1 decreased macrophage infiltration in the lungs during hyperoxia. Furthermore, treatment with LA1 improved alveolarization and angiogenesis and decreased pulmonary vascular remodeling and PH. These data indicate that leukocyte recruitment plays an important role in the experimental model of BPD induced by hyperoxia. Targeting leukocyte trafficking using LA1, an integrin agonist, is beneficial in preventing lung inflammation and protecting alveolar and vascular structures during hyperoxia. Thus, targeting integrin-mediated leukocyte recruitment and inflammation may provide a novel strategy in preventing and treating BPD in preterm infants.


Assuntos
Anti-Inflamatórios/farmacologia , Benzoatos/farmacologia , Displasia Broncopulmonar/prevenção & controle , Hiperóxia/tratamento farmacológico , Tioidantoínas/farmacologia , Animais , Animais Recém-Nascidos , Anti-Inflamatórios/uso terapêutico , Benzoatos/uso terapêutico , Displasia Broncopulmonar/etiologia , Avaliação Pré-Clínica de Medicamentos , Hiperóxia/complicações , Hipertensão Pulmonar/tratamento farmacológico , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Infiltração de Neutrófilos , Ratos Sprague-Dawley , Tioidantoínas/uso terapêutico , Resultado do Tratamento , Remodelação Vascular
6.
J Biol Chem ; 289(25): 17454-67, 2014 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24817115

RESUMO

Podocytes are highly differentiated cells and critical elements for the filtration barrier of the kidney. Loss of their foot process (FP) architecture (FP effacement) results in urinary protein loss. Here we show a novel role for the neutral amino acid glutamine in structural and functional regulation of the kidney filtration barrier. Metabolic flux analysis of cultured podocytes using genetic, toxic, and immunologic injury models identified increased glutamine utilization pathways. We show that glutamine uptake is increased in diseased podocytes to couple nutrient support to increased demand during the disease state of FP effacement. This feature can be utilized to transport increased amounts of glutamine into damaged podocytes. The availability of glutamine determines the regulation of podocyte intracellular pH (pHi). Podocyte alkalinization reduces cytosolic cathepsin L protease activity and protects the podocyte cytoskeleton. Podocyte glutamine supplementation reduces proteinuria in LPS-treated mice, whereas acidification increases glomerular injury. In summary, our data provide a metabolic opportunity to combat urinary protein loss through modulation of podocyte amino acid utilization and pHi.


Assuntos
Podócitos/metabolismo , Proteinúria/metabolismo , Animais , Transporte Biológico Ativo/genética , Transporte Biológico Ativo/imunologia , Células Cultivadas , Citoesqueleto/genética , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Knockout , Podócitos/imunologia , Podócitos/patologia , Proteinúria/genética , Proteinúria/imunologia , Proteinúria/patologia
7.
J Biol Chem ; 288(13): 9077-83, 2013 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-23386618

RESUMO

Toll-like receptor (TLR) signaling is an important component in the inflammatory response generated in diseases characterized by autoantibody reactivity to proteins such as SSA/Ro in complex with endogenous nucleic acids. Complement receptor 3 (CR3), a genetic variant of which has been identified as a risk factor in systemic lupus erythematosus, has been shown to induce tolerogenic responses in dendritic cells and suppress TLR4 responses in a murine sepsis model. Accordingly, this study addressed the hypothesis that activation of CR3, influenced by genotype of CD11b, negatively regulates TLR7/8-dependent effector function. Allosteric activation of CD11b via pretreatment with the small molecule, leukadhedrin 1 (LA1), significantly attenuated TLR7/8-induced (hY3 RNA, R848) secretion of TNFα in THP-1 cells and human macrophages isolated from donors homozygous for the ancestral common ITGAM allele at rs1143679. This inhibition was accompanied by profound degradation of the adaptor protein MyD88, an effect not observed with direct inhibition of TLR ligation by an antagonist oligonucleotide. In contrast, the addition of LA1 after incubation with the TLR agonists did not result in MyD88 degradation and subsequent attenuation of TNFα secretion. In TLR7/8-stimulated macrophages isolated from donors heterozygous for the CD11b variant, pretreatment with LA1 did not down-regulate TNFα release. These novel findings support a negative cross-talk between CR3 and TLR pathways likely to be induced by antibodies reactive with ribonucleoproteins and point to the development of CR3-specific agonists as potential therapeutics for diseases such as neonatal lupus.


Assuntos
Doenças Autoimunes/metabolismo , Antígeno CD11b/biossíntese , Regulação da Expressão Gênica , Antígeno de Macrófago 1/metabolismo , Macrófagos/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Sítio Alostérico , Anti-Inflamatórios/farmacologia , Adesão Celular , Regulação para Baixo , Heterozigoto , Humanos , Inflamação , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Macrófagos/citologia , Modelos Genéticos , Fator 88 de Diferenciação Mieloide/metabolismo , RNA/metabolismo , Transdução de Sinais
8.
Biochim Biophys Acta ; 1830(6): 3696-710, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23454649

RESUMO

BACKGROUND: CD11b/CD18 is a key adhesion receptor that mediates leukocyte adhesion, migration and immune functions. We recently identified novel compounds, leukadherins, that allosterically enhance CD11b/CD18-dependent cell adhesion and reduce inflammation in vivo, suggesting integrin activation to be a novel mechanism of action for the development of anti-inflammatory therapeutics. Since a number of well-characterized anti-CD11b/CD18 activating antibodies are currently available, we wondered if such biological agonists could also become therapeutic leads following this mechanism of action. METHODS: We compared the two types of agonists using in vitro cell adhesion and wound-healing assays and using animal model systems. We also studied effects of the two types of agonists on outside-in signaling in treated cells. RESULTS: Both types of agonists similarly enhanced integrin-mediated cell adhesion and decreased cell migration. However, unlike leukadherins, the activating antibodies produced significant CD11b/CD18 macro clustering and induced phosphorylation of key proteins involved in outside-in signaling. Studies using conformation reporter antibodies showed that leukadherins did not induce global conformational changes in CD11b/CD18 explaining the reason behind their lack of ligand-mimetic outside-in signaling. In vivo, leukadherins reduced vascular injury in a dose-dependent fashion, but, surprisingly, the anti-CD11b activating antibody ED7 was ineffective. CONCLUSIONS: Our results suggest that small molecule allosteric agonists of CD11b/CD18 have clear advantages over the biologic activating antibodies and provide a mechanistic basis for the difference. GENERAL SIGNIFICANCE: CD11b/CD18 activation represents a novel strategy for reducing inflammatory injury. Our study establishes small molecule leukadherins as preferred agonists over activating antibodies for future development as novel anti-inflammatory therapeutics.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Anticorpos Monoclonais Murinos/farmacologia , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Artéria Ilíaca/lesões , Transdução de Sinais/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Anti-Inflamatórios não Esteroides/química , Antígeno CD11b/genética , Antígenos CD18/genética , Adesão Celular/efeitos dos fármacos , Humanos , Artéria Ilíaca/metabolismo , Artéria Ilíaca/patologia , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Células K562 , Camundongos , Estrutura Quaternária de Proteína , Ratos , Ratos Sprague-Dawley
9.
Am J Physiol Heart Circ Physiol ; 306(5): H641-53, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24414074

RESUMO

Aging has been associated with pathological vascular remodeling and increased neointimal hyperplasia. The understanding of how aging exacerbates this process is fundamental to prevent cardiovascular complications in the elderly. This study proposes a mechanism by which aging sustains leukocyte adhesion, vascular inflammation, and increased neointimal thickness after injury. The effect of aging on vascular remodeling was assessed in the rat balloon injury model using microarray analysis, immunohistochemistry, and LINCOplex assays. The injured arteries in aging rats developed thicker neointimas than those in younger animals, and this significantly correlated with a higher number of tissue macrophages and increased vascular IL-18. Indeed, IL-18 was 23-fold more abundant in the injured vasculature of aged animals compared with young rats, while circulating levels were similar in both groups of animals. The depletion of macrophages in aged rats with clodronate liposomes ameliorated vascular accumulation of IL-18 and significantly decreased neointimal formation. IL-18 was found to inhibit apoptosis of vascular smooth muscle cells (VSMC) and macrophages, thus favoring both the formation and inflammation of the neointima. In addition, injured arteries of aged rats accumulated 18-fold more fibrinogen-γ than those of young animals. Incubation of rat peritoneal macrophages with immobilized IL-18 increased leukocyte adhesion to fibrinogen and suggested a proinflammatory positive feedback loop among macrophages, VSMC, and the deposition of fibrinogen during neointimal hyperplasia. In conclusion, our data reveal that concentration changes in vascular cytokine and fibrinogen following injury in aging rats contribute to local inflammation and postinjury neointima formation.


Assuntos
Envelhecimento/metabolismo , Fibrinogênio/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-18/metabolismo , Macrófagos/metabolismo , Músculo Liso Vascular/metabolismo , Neointima , Comunicação Parácrina , Lesões do Sistema Vascular/metabolismo , Fatores Etários , Envelhecimento/imunologia , Envelhecimento/patologia , Animais , Apoptose , Adesão Celular , Células Cultivadas , Quimiotaxia , Ácido Clodrônico/farmacologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hiperplasia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Monócitos/imunologia , Monócitos/metabolismo , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/patologia , Comunicação Parácrina/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Transdução de Sinais , Fatores de Tempo , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/imunologia , Lesões do Sistema Vascular/patologia , Lesões do Sistema Vascular/prevenção & controle
10.
Biophys J ; 105(11): 2517-27, 2013 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-24314082

RESUMO

Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn(2+) additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1(-/-) neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn(2+)-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn(2+) induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.


Assuntos
Benzoatos/farmacologia , Membrana Celular/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno de Macrófago 1/metabolismo , Tioidantoínas/farmacologia , Citoesqueleto de Actina/metabolismo , Sequência de Aminoácidos , Animais , Benzoatos/química , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/química , Leucócitos/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/química , Manganês/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Neutrófilos/metabolismo , Ligação Proteica , Tioidantoínas/química
11.
Biochem Biophys Res Commun ; 394(1): 194-9, 2010 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-20188705

RESUMO

Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high-throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique.


Assuntos
Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas , Animais , Ligantes , Camundongos , Camundongos Endogâmicos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia
12.
Bioorg Med Chem Lett ; 19(24): 6902-6, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19879752

RESUMO

We report the identification of novel small molecule agonists of integrin CD11b/CD18, which increased, in a dose-dependent manner, the adhesion of the integrin CD11b/CD18 expressing cells to two physiologically relevant ligands: Fibrinogen and iC3b. Compound 6 showed an ex vivo EC(50) of 10.5 microM and in vitro selectivity for binding to the recombinant alphaA-domain of CD11b/CD18. In silico docking experiments suggest that the compounds recognized a hydrophobic cleft in the ligand-binding alphaA-domain, implying an allosteric mechanism of modulation of integrin affinity by this novel compound.


Assuntos
Anti-Inflamatórios não Esteroides/química , Antígeno CD11b/efeitos dos fármacos , Antígenos CD18/efeitos dos fármacos , Furanos/química , Tiazolidinedionas/química , Anti-Inflamatórios não Esteroides/farmacologia , Antígeno CD11b/química , Antígeno CD11b/metabolismo , Antígenos CD18/química , Antígenos CD18/metabolismo , Células CACO-2 , Adesão Celular/efeitos dos fármacos , Furanos/farmacologia , Humanos , Conformação Proteica , Relação Estrutura-Atividade , Tiazolidinedionas/farmacologia
13.
J Intensive Care ; 6: 19, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29568527

RESUMO

BACKGROUND: Multi-organ failure occurs during critical illness and is mediated in part by destructive neutrophil-to-endothelial interactions. The ß2 integrin receptor, CR3 (complement receptor 3; Mac-1; CD11b/CD18), which binds endothelial intercellular adhesion molecule-1 (ICAM-1), plays a key role in promoting the adhesion of activated neutrophils to inflamed endothelia which, when prolonged and excessive, can cause vascular damage. Leukadherin-1 (LA-1) is a small molecule allosteric activator of CR3 and has been shown to promote adhesion of blood neutrophils to inflamed endothelium and restrict tissue infiltration. Therefore, LA-1 offers a novel mechanism of anti-inflammatory action by activation, rather than inhibition, of the neutrophil CR3 integrin. However, whether promotion of neutrophil-to-endothelial interaction by this novel therapeutic is of benefit or detriment to endothelial barrier function is not known. METHODS: Critically ill septic and trauma patients were prospectively enrolled from the surgical and the trauma ICU. Blood was collected from these patients and healthy volunteers. Neutrophils were isolated by dextran sedimentation and adhered to TNF-α (tumor necrosis factor-α)-activated human umbilical vein endothelial (HUVEC) monolayers in the presence or absence of fMLP (formylmethionine-leucine-phenylalanine) and/or LA-1. Electric cell-substrate impedance sensing (ECIS) and exposure of underlying collagen were used to quantify endothelial barrier function and permeability. RESULTS: Neutrophils from critically ill trauma and septic patients caused similar degrees of endothelial barrier disruption which exceeded that caused by cells obtained from healthy controls both kinetically and quantitatively. LA-1 protected barrier function in the absence and presence of fMLP which served as a secondary stimulant to cause maximal loss of barrier function. LA-1 protection was also observed by quantifying collagen exposure underlying endothelial cells challenged with fMLP-stimulated neutrophils. LA-1 treatment resulted in decreased migration dynamics of neutrophils crawling on an endothelial monolayer with reduced speed (µm/s = 0.25 ± 0.01 vs. 0.06 ± 0.01, p < 0.05), path length (µm = 199.5 ± 14.3 vs. 42.1 ± 13.0, p < 0.05), and displacement (µm = 65.2 ± 4.7 vs. 10.4 ± 1.3; p < 0.05). CONCLUSION: Neutrophils from patients with trauma or sepsis cause endothelial barrier disruption to a similar extent relative to each other. The CR3 agonist LA-1 protects endothelial barrier function from damage caused by neutrophils obtained from both populations of critically ill patients even when exposed to secondary stimulation.

14.
J Clin Invest ; 127(4): 1271-1283, 2017 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-28263189

RESUMO

Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-ß, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.


Assuntos
Antígeno CD11b/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Macrófagos/imunologia , Receptores Toll-Like/imunologia , Animais , Antígeno CD11b/genética , Feminino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/imunologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos MRL lpr , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores Toll-Like/genética
15.
Front Med (Lausanne) ; 1: 45, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25593918

RESUMO

Kidney allograft rejection is associated with infiltration of inflammatory CD11b+ leukocytes. A CD11b agonist leukadherin-1 (LA1) increases leukocyte adhesion, preventing their transmigration and tissue recruitment in vivo. Here, we test the extent to which LA1-mediated activation of CD11b/CD18 enhances kidney allograft survival in a mouse model of fully MHC-mismatched orthotopic kidney transplantation, where C57BL/6J (H-2(b)) recipients received kidney allografts from Balb/c mice (H-2(d)). Isograft control recipients received a kidney from a littermate. Control isograft and allograft recipients were treated daily with cyclosporine (CsA) for 2 weeks, while the test group received CsA therapy and daily LA1 injections during week 1 and alternate days during weeks 2-8. LA1 treatment reduced interstitial leukocyte infiltration in the allograft, reduced neointimal hyperplasia and glomerular damage, and prolonged graft survival from 48.5% (CsA only) to 100% (CsA and LA1) on day 60. Serum creatinine levels showed significantly improved kidney function in LA1-treated mice compared to CsA-treated allograft controls [0.52 ± 0.18 mg/dL vs 0.24 ± 0.07 mg/dL (n = 5), respectively]. Furthermore, combination therapy reduced macrophage infiltration and increased the frequency of FoxP3 + Tregs in the allograft. These findings indicate a crucial role for CD11b/CD18 in the control of leukocyte migration to the transplanted kidney and identify integrin agonist LA1 as a novel potential therapeutic agent for kidney transplantation.

16.
Biophys Chem ; 179: 12-25, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23714425

RESUMO

The BclXL apoptotic repressor bears the propensity to associate into megadalton oligomers in solution, particularly under acidic pH. Herein, using various biophysical methods, we analyze the effect of temperature on the oligomerization of BclXL. Our data show that BclXL undergoes irreversible aggregation and assembles into highly-ordered rope-like homogeneous fibrils with length in the order of mm and a diameter in the µm-range under elevated temperatures. Remarkably, the formation of such fibrils correlates with the decay of a largely α-helical fold into a predominantly ß-sheet architecture of BclXL in a manner akin to the formation of amyloid fibrils. Further interrogation reveals that while BclXL fibrils formed under elevated temperatures show no observable affinity toward BH3 ligands, they appear to be optimally primed for insertion into cardiolipin bicelles. This salient observation strongly argues that BclXL fibrils likely represent an on-pathway intermediate for insertion into mitochondrial outer membrane during the onset of apoptosis. Collectively, our study sheds light on the propensity of BclXL to form amyloid-like fibrils with important consequences on its mechanism of action in gauging the apoptotic fate of cells in health and disease.


Assuntos
Temperatura Alta , Proteína bcl-X/química , Apoptose , Cardiolipinas/metabolismo , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Estrutura Secundária de Proteína
17.
Sci Signal ; 4(189): ra57, 2011 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-21900205

RESUMO

The integrin CD11b/CD18 (also known as Mac-1), which is a heterodimer of the α(M) (CD11b) and ß(2) (CD18) subunits, is critical for leukocyte adhesion and migration and for immune functions. Blocking integrin-mediated leukocyte adhesion, although beneficial in experimental models, has had limited success in treating inflammatory diseases in humans. Here, we used an alternative strategy of inhibiting leukocyte recruitment by activating CD11b/CD18 with small-molecule agonists, which we term leukadherins. These compounds increased the extent of CD11b/CD18-dependent cell adhesion of transfected cells and of primary human and mouse neutrophils, which resulted in decreased chemotaxis and transendothelial migration. Leukadherins also decreased leukocyte recruitment and reduced arterial narrowing after injury in rats. Moreover, compared to a known integrin antagonist, leukadherins better preserved kidney function in a mouse model of experimental nephritis. Leukadherins inhibited leukocyte recruitment by increasing leukocyte adhesion to the inflamed endothelium, which was reversed with a blocking antibody. Thus, we propose that pharmacological activation of CD11b/CD18 offers an alternative therapeutic approach for inflammatory diseases.


Assuntos
Adesão Celular/fisiologia , Inflamação/tratamento farmacológico , Leucócitos/fisiologia , Antígeno de Macrófago 1/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Cálcio , Adesão Celular/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/fisiologia , Humanos , Células K562 , Leucócitos/citologia , Antígeno de Macrófago 1/uso terapêutico , Magnésio , Manganês , Camundongos , Ratos
18.
PLoS One ; 4(10): e7627, 2009 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-19859549

RESUMO

During atherogenesis and vascular inflammation quiescent platelets are activated to increase the surface expression and ligand affinity of the integrin alphaIIbbeta3 via inside-out signaling. Diverse signals such as thrombin, ADP and epinephrine transduce signals through their respective GPCRs to activate protein kinases that ultimately lead to the phosphorylation of the cytoplasmic tail of the integrin alphaIIbbeta3 and augment its function. The signaling pathways that transmit signals from the GPCR to the cytosolic domain of the integrin are not well defined. In an effort to better understand these pathways, we employed a combination of proteomic profiling and computational analyses of isolated human platelets. We analyzed ten independent human samples and identified a total of 1507 unique proteins in platelets. This is the most comprehensive platelet proteome assembled to date and includes 190 membrane-associated and 262 phosphorylated proteins, which were identified via independent proteomic and phospho-proteomic profiling. We used this proteomic dataset to create a platelet protein-protein interaction (PPI) network and applied novel contextual information about the phosphorylation step to introduce limited directionality in the PPI graph. This newly developed contextual PPI network computationally recapitulated an integrin signaling pathway. Most importantly, our approach not only provided insights into the mechanism of integrin alphaIIbbeta3 activation in resting platelets but also provides an improved model for analysis and discovery of PPI dynamics and signaling pathways in the future.


Assuntos
Plaquetas/metabolismo , Regulação da Expressão Gênica , Integrinas/metabolismo , Proteômica/métodos , Motivos de Aminoácidos , Biologia Computacional , Citometria de Fluxo/métodos , Humanos , Espectrometria de Massas/métodos , Fosforilação , Agregação Plaquetária , Proteoma , Transdução de Sinais
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA