RESUMEN
AIMS: Early identification of at-risk individuals for diabetic nephropathy would help in preventing or delaying end-stage renal failure. We measured the levels of circulating soluble tumor necrosis factor receptor 1 (sTNFR1) in various stages of proteinuria (MAC) to determine the association of this marker with diabetic nephropathy. MATERIALS AND METHODS: The study was performed on 160 subjects, and a case-control methodology was employed. Type 2 diabetic subjects were recruited based on albuminuria and were grouped as (1) normoalbuminuria (NA); (2) microalbuminuria (MIC); (3) MAC; (4) normal glucose tolerance (NGT) subjects who served as healthy controls. sTNFR1 levels were measured by quantitative enzyme-linked immunosorbent assay (ELISA). RESULTS: Soluble tumor necrosis factor receptor 1 (sTNFR1) levels were highest in the MAC group, followed by the microMAC group. The sTNFR1 levels were not statistically different between the NGT and NA groups. On regression models, sTNFR1 was associated with MIC [odds ratio (OR)- 6.491, 95% confidence interval (CI)-1.868-22.55] and MAC (OR per standard deviation-15.28; 95% CI-3.76-62.15; p < 0.001) even after controlling for all the possible confounding factors. Receiver operator curve (ROC) analysis revealed sTNFR1 cut-point of 1832 pg/mL had a C-statistic of 0.685 to discriminate MI from NA with 52% sensitivity. Whereas the sTNFR1 cut-point of 2050 pg/mL with a C-statistic of 0.8177 had 77% sensitivity for identifying MAC. CONCLUSION: Soluble tumor necrosis factor receptor 1 (sTNFR1) is significantly associated with MIC and MAC group in type 2 diabetes, and this suggests a potential early diagnostic biomarker role of sTNFR1 for MAC among Asian Indians.
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Diabetes Mellitus Tipo 2 , Nefropatías Diabéticas , Humanos , Receptores Tipo I de Factores de Necrosis Tumoral , Diabetes Mellitus Tipo 2/complicaciones , Nefropatías Diabéticas/complicaciones , Proteinuria/etiología , Albuminuria/diagnósticoRESUMEN
BACKGROUND: High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). METHODS AND RESULTS: A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC(-/-) mice. In RBCs from HFD-fed wild-type and DARC(-/-) mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. CONCLUSIONS: RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic.
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Aterosclerosis/metabolismo , Dieta Alta en Grasa/efectos adversos , Eritrocitos/metabolismo , Obesidad/metabolismo , Animales , Aterosclerosis/etiología , Aterosclerosis/patología , Eritrocitos/patología , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/etiología , Obesidad/patología , Fagocitosis/fisiologíaRESUMEN
The synthesis of a new library of 5-arylidenethiazolidinone compounds using an efficient three component reaction with thiazolidine-2,4-dione, piperidine and appropriate aldehydes is reported. This reaction is excellently high yielding, tolerant towards a variety of aldehydes and provides access to these compounds in a single step (in comparison to low yielding multistep syntheses reported in the literature). Once the reaction is complete, the desired product precipitates out of the reaction mixture and is isolated by filtration and purified by washing and recrystallization. These compounds revealed anti-proliferative activities against human breast cancer cells (MCF7 and MDA). Phenotypic profiling established the most active compound 17i (EC50 = 4.52 µM) as an apoptotic agent. A novel chemical proteomics approach identified ß-actin-like protein 2, γ-enolase and macrophage migration inhibitory factor (MMIF) as putative cellular binding partners of 17i.
Asunto(s)
Apoptosis/efectos de los fármacos , Piperidinas/química , Tiazolidinas/síntesis química , Tiazolidinas/farmacología , Técnicas de Química Sintética , Humanos , Indicadores y Reactivos/química , Células MCF-7 , Modelos Moleculares , Conformación Molecular , Tiazolidinas/químicaRESUMEN
OBJECTIVE: Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. APPROACH AND RESULTS: We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet-fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed α-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. CONCLUSIONS: These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1-dependent mechanisms.
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Tejido Adiposo/trasplante , Traumatismos de las Arterias Carótidas/metabolismo , Quimiocina CCL2/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima , Actinas/metabolismo , Adipocitos/metabolismo , Adipocitos/trasplante , Tejido Adiposo/metabolismo , Animales , Biomarcadores/metabolismo , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/etiología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Células Cultivadas , Quimiocina CCL2/deficiencia , Quimiocina CCL2/genética , Quimiotaxis , Técnicas de Cocultivo , Medios de Cultivo Condicionados/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Humanos , Hiperplasia , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/lesiones , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Neovascularización Patológica , Fenotipo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de Señal , Factores de Tiempo , Migración Transendotelial y TransepitelialRESUMEN
Alternatively spliced tissue factor (asTF) promotes neovascularization and monocyte recruitment via integrin ligation. While asTF mRNA has been detected in some pancreatic ductal adenocarcinoma (PDAC) cell lines and increased asTF expression can promote PDAC growth in a subcutaneous model, the expression of asTF protein in bona fide PDAC lesions and/or its role in metastatic spread are yet to be ascertained. We here report that asTF protein is abundant in lesional and stromal compartments of the five studied types of carcinoma including PDAC. Analysis of 29 specimens of PDAC revealed detectable asTF in >90% of the lesions with a range of staining intensities. asTF levels in PDAC lesions positively correlated with the degree of monocyte infiltration. In an orthotopic model, asTF-overexpressing high-grade PDAC cell line Pt45P1/asTF+ produced metastases to distal lymph nodes, which stained positive for asTF. PDAC cells stimulated with and/or overexpressing asTF exhibited upregulation of genes implicated in PDAC progression and metastatic spread. Pt45P1/asTF+ cells displayed higher coagulant activity compared to Pt45P1 cells; the same effect was observed for cell-derived microparticles (MPs). Our findings demonstrate that asTF is expressed in PDAC and lymph node metastases and potentiates PDAC spread in vivo. asTF elicits global changes in gene expression likely involved in tumor progression and metastatic dissemination, and it also enhances the procoagulant potential of PDAC cells and cell-derived MPs. Thus, asTF may comprise a novel therapeutic target to treat PDAC and, possibly, its thrombotic complications.
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Empalme Alternativo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Tromboplastina/genética , Animales , Coagulación Sanguínea/fisiología , Western Blotting , Citometría de Flujo , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica/genética , Análisis de Matrices TisularesRESUMEN
RATIONALE: Hemizygous deficiency of the transcription factor Krüppel-like factor 2 (KLF2) has been shown previously to augment atherosclerosis in hypercholesterolemic mice. However, the cell type responsible for the increased atherosclerosis due to KLF2 deficiency has not been identified. This study examined the consequence of myeloid cell-specific KLF2 inactivation in atherosclerosis. METHODS AND RESULTS: Cell-specific knockout mice were generated by Cre/loxP recombination. Macrophages isolated from myeloid-specific Klf2 knockout (myeKlf2(-/-)) mice were similar to myeKlf2(+/+) macrophages in response to activation, polarization, and lipid accumulation. However, in comparison to myeKlf2(+/+) macrophages, myeKlf2(-/-) macrophages adhered more robustly to endothelial cells. Neutrophils from myeKlf2(-/-) mice also adhered more robustly to endothelial cells, and fewer myeKlf2(-/-) neutrophils survived in culture over a 24-hour period in comparison with myeKlf2(+/+) neutrophils. When myeKlf2(-/-) mice were mated to Ldlr(-/-) mice and then fed a high fat and high cholesterol diet, significant increase in atherosclerosis was observed in the myeKlf2(-/-)Ldlr(-/-) mice compared with myeKlf2(+/+)Ldlr(-/-) littermates. The increased atherosclerosis in myeKlf2(-/-)Ldlr(-/-) mice was associated with elevated presence of neutrophils and macrophages, with corresponding increase of myeloperoxidase as well as chlorinated and nitrosylated tyrosine epitopes in their lesion areas compared with myeKlf2(+/+)Ldlr(-/-) mice. CONCLUSIONS: This study documents a role for myeloid KLF2 expression in modulating atherosclerosis. The increased neutrophil accumulation and atherosclerosis progression with myeloid-specific KLF2 deficiency also underscores the importance of neutrophils in promoting vascular oxidative stress and atherosclerosis. Collectively, these results suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of atherosclerosis.
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Aterosclerosis/inmunología , Adhesión Celular/inmunología , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Macrófagos/inmunología , Neutrófilos/inmunología , Animales , Aterosclerosis/patología , Muerte Celular/inmunología , Células Endoteliales/citología , Células Endoteliales/inmunología , Femenino , Hipercolesterolemia/inmunología , Hipercolesterolemia/patología , Recuento de Linfocitos , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/citología , Vasculitis/inmunología , Vasculitis/patologíaRESUMEN
Pre-eclampsia (PE) is a life-threatening complication that occurs during pregnancy, affecting a large number of pregnant women and newborns worldwide. Rapid, on-site and affordable screening of PE at an early stage is necessary to ensure timely treatment and minimize both maternal and neonatal morbidity and mortality rates. Placental growth factor (PlGF) is an angiogenic blood biomarker used for PE diagnosis. Herein, we report the plasmonic fiber optic absorbance biosensor (P-FAB) strategy for detecting PlGF at femtomolar concentration using polymethyl methacrylate (PMMA) based U-bent polymeric optical fiber (POF) sensor probes. A novel poly(amidoamine) (PAMAM) dendrimer based PMMA surface modification is established to obtain a greater immobilization of the bioreceptors compared to a linear molecule like hexamethylenediamine (HMDA). Plasmonic sandwich immunoassay was realized by immobilizing the mouse anti-PlGF (3H1) on the U-bent POF sensor probe surface and gold nanoparticles (AuNP) labels conjugated with mouse anti-PlGF (6H9). The POF sensor probes could measure PlGF within 30 min using the P-FAB strategy. The limit-of-detection (LoD) was found to be 0.19 pg/mL and 0.57 pg/mL in phosphate-buffered saline and 10× diluted serum, respectively. The clinical sample testing, with eleven positive and eleven negative preeclamptic pregnancy samples, successfully confirmed the accuracy, reliability, specificity, and sensitivity of the P-FAB based POF sensor platform, thereby paving the way for cost-effective technology for PlGF detection and its potential for pre-eclampsia diagnosis.
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Técnicas Biosensibles , Dendrímeros , Oro , Nanopartículas del Metal , Fibras Ópticas , Preeclampsia , Animales , Femenino , Humanos , Ratones , Embarazo , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Dendrímeros/química , Tecnología de Fibra Óptica/instrumentación , Oro/química , Inmunoensayo/métodos , Inmunoensayo/instrumentación , Límite de Detección , Nanopartículas del Metal/química , Factor de Crecimiento Placentario/sangre , Polimetil Metacrilato/química , Preeclampsia/diagnóstico , Preeclampsia/sangreRESUMEN
This study was performed to determine whether murine alternatively spliced tissue factor (masTF) acts analogously to human alternatively spliced tissue factor (hasTF) in promoting neovascularization via integrin ligation. Immunohistochemical evaluation of a spontaneous murine pancreatic ductal adenocarcinoma model revealed increased levels of masTF and murine full-length tissue factor (mflTF) in tumor lesions compared with benign pancreas; furthermore, masTF colocalized with mflTF in spontaneous aortic plaques of Ldlr(-/-) mice, indicating that masTF is likely involved in atherogenesis and tumorigenesis. Recombinant masTF was used to perform in vitro and ex vivo studies examining its integrin-mediated biologic activity. Murine endothelial cells (ECs) rapidly adhered to masTF in a ß3-dependent fashion. Using adult and embryonic murine ECs, masTF potentiated cell migration in transwell assays. Scratch assays were performed using murine and primary human ECs; the effects of masTF and hasTF were comparable in murine ECs, but in human ECs, the effects of hasTF were more pronounced. In aortic sprouting assays, the potency of masTF-triggered vessel growth was undistinguishable from that observed with hasTF. The proangiogenic effects of masTF were found to be Ccl2-mediated, yet independent of vascular endothelial growth factor. In murine ECs, masTF and hasTF upregulated genes involved in inflammatory responses; murine and human ECs stimulated with masTF and hasTF exhibited increased interaction with murine monocytic cells under orbital shear. We propose that masTF is a functional homolog of hasTF, exerting some of its key effects via ß3 integrins. Our findings have implications for the development of murine models to examine the interplay between blood coagulation, atherosclerosis and cancer.
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Empalme Alternativo , Integrinas/metabolismo , Transducción de Señal , Tromboplastina/genética , Tromboplastina/metabolismo , Animales , Adhesión Celular , Línea Celular , Movimiento Celular/genética , Análisis por Conglomerados , Células Endoteliales/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/metabolismo , Neovascularización Fisiológica/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Unión Proteica , Transporte de ProteínasRESUMEN
BACKGROUND AND AIMS: The burden of chronic kidney disease (CKD) in India is extremely high with the prevalent twin epidemic of diabetes and hypertension. Fast declining phenotype of renal function has yet not been reported in Indian context. Here, we report the prevalence of rapid decliners phenotype in Indian population. METHODS: Between the period 2014-2019, electronic records of 104636 subjects were reviewed. Subjects with serum creatinine values of at least one year apart were selected for further analysis. The study population was categorized based on eGFR, non-decliners < 1 mL/min/1.73 m2/year; progressive decliners 1-5 mL/min/1.73 m2/year and rapid decliners >5 mL/min/1.73 m2/year. Data on diabetes, hypertension, coronary artery disease and cerebrovascular disease were analyzed. RESULTS: During the mean follow up of 4 years, the prevalence of non-decliners, progressive and rapid decliners were 61%, 20% and 19% respectively. Diabetes was higher at 44% in rapid decliners when compared to non-decliners (35.1%); progressive decliners (39.2%). The progression of CKD to end stage renal disease (ESRD) was higher in rapid decliners (32%) in comparison to progressive decliners (19%) CONCLUSIONS: There is a high prevalence of rapid decliner phenotype in India and progression to ESRD is greater and probably is a risk factor for early progression to ESRD.
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Insuficiencia Renal Crónica , Progresión de la Enfermedad , Tasa de Filtración Glomerular , Humanos , India/epidemiología , Riñón/patología , Fenotipo , Prevalencia , Estudios Retrospectivos , Factores de RiesgoRESUMEN
BACKGROUND AND AIMS: Albuminuria is not an effective marker for early diagnosis of diabetic renal complication with several subjects progressing to chronic kidney disease without any albuminuria. A biomarker that can predict early changes of the diabetic kidney will be useful in effective management of type 2 diabetes. Mass spectrometry based metabolomics approach offers tremendous promise for the identification of novel metabolite biomarkers. METHODS: A case-control approach was carried out to identify renal biomarkers among Asian Indian subjects in a hospital setting. A total of 29 subjects were included in the study that included groups of diabetic controls, diabetic subjects with eGFR >90 ml/min/1.72 m2 and diabetic subjects with eGFR between 60 and 89 ml/min/1.72 m2 and eGFR between 15 and 30 ml/min/1.72 m2. We employed an un-targeted mass spectrometry method for the identification of plasma metabolites. RESULTS: A total of 1414 and 975 metabolites were identified in the positive and negative ion mode respectively. 19 metabolites were up regulated and 18 metabolites were down regulated in CKD2 and CKD4 groups when compared to control. Correlation analysis of the differential metabolites revealed Pregnenolone sulfate, creatinine and ganglioside GA1 to be negatively correlated and hexyl glucoside, all-trans-carophyll yellow and PG to be positively correlated with eGFR. CONCLUSION: We have identified Pregnenolone sulfate, GA1, PG and all-trans-Carophyll yellow as biomarkers for early identification of diabetic nephropathy. These markers could aid in better management of diabetic nephropathy that may result delaying the progression of the disease.
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Albuminuria/diagnóstico , Biomarcadores/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Nefropatías Diabéticas/diagnóstico , Espectrometría de Masas/métodos , Metaboloma , Albuminuria/etiología , Albuminuria/metabolismo , Estudios de Casos y Controles , Creatinina/sangre , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/metabolismo , Progresión de la Enfermedad , Diagnóstico Precoz , Femenino , Estudios de Seguimiento , Tasa de Filtración Glomerular , Humanos , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
One of the major complications of diabetes is the alteration of the blood-retinal barrier, leading to retinal edema and consequent vision loss. The aim of this study was to evaluate the role of the urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system in the regulation of retinal vascular permeability. Biochemical, molecular, and histological techniques were used to examine the role of uPA and uPAR in the regulation of retinal vascular permeability in diabetic rats and cultured retinal endothelial cells. The increased retinal vascular permeability in diabetic rats was associated with a decrease in vascular endothelial (VE) -cadherin expression in retinal vessels. Treatment with the uPA/uPAR-inhibiting peptide (A6) was shown to reduce diabetes-induced permeability and the loss of VE-cadherin. The increased permeability of cultured cells in response to advanced glycation end products (AGEs) was significantly inhibited with A6. Treatment of endothelial cells with specific matrix metalloproteinases or AGEs resulted in loss of VE-cadherin from the cell surface, which could be inhibited by A6. uPA/uPAR physically interacts with AGEs/receptor for advanced glycation end products on the cell surface and regulates its activity. uPA and its receptor uPAR play important roles in the alteration of the blood-retinal barrier through proteolytic degradation of VE-cadherin. The ability of A6 to block retinal vascular permeability in diabetes suggests a potential therapeutic approach for the treatment of diabetic macular edema.
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Barrera Hematorretinal/efectos de los fármacos , Diabetes Mellitus Experimental/fisiopatología , Fragmentos de Péptidos/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Animales , Antígenos CD/biosíntesis , Cadherinas/biosíntesis , Permeabilidad Capilar/efectos de los fármacos , Bovinos , Células Cultivadas , Endotelio Vascular/citología , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Ratas , Receptores de Superficie Celular/biosíntesis , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Retina/citología , Retina/metabolismo , Regulación hacia Arriba , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Activador de Plasminógeno de Tipo Uroquinasa/farmacologíaRESUMEN
The spiro[pyrrolidine-3, 3´-oxindole] moiety is present as a core in number of alkaloids with substantial biological activities. Here in we report design and synthesis of a library of compounds bearing spiro[pyrrolidine-3, 3´-oxindole] motifs that demonstrated exceptional inhibitory activity against the proliferation of MCF-7 breast cancer cells. The synthesis involved a one pot Pictet Spengler-Oxidative ring contraction of tryptamine to the desired scaffolds and occurred in 1:1 THF and water with catalytic trifluoroacetic acid and stoichiometric N-bromosuccinimide as an oxidant. Phenotypic profiling indicated that these molecules induce apoptotic cell death in MCF-7 cells. Target deconvolution with most potent compound 5l from the library, using chemical proteomics indicated histone deacetylase 2 (HDAC2) and prohibitin 2 as the potential cellular binding partners. Molecular docking of 5l with HDAC2 provided insights pertinent to putative binding interactions.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Compuestos de Espiro/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células COS , Chlorocebus aethiops , Diseño de Fármacos , Femenino , Humanos , Indoles/química , Células MCF-7 , Simulación de Dinámica Molecular , Estructura Molecular , Oxindoles , Pirrolidinas/química , Compuestos de Espiro/síntesis química , Compuestos de Espiro/químicaRESUMEN
Full-length Tissue Factor (flTF) - the obligatory co-factor for the serine protease (factor) VII/VIIa - serves as the initiator of blood coagulation. The flTF/VIIa complex triggers a sequence of proteolytic events that lead to the formation of a hemostatic plug. Aside from hemostatic maintenance, flTF can contribute to thrombogenesis in some settings. The proteolytic properties of the flTF/VIIa complex (as well as the flTF/VIIa/Xa complex) account for non-hemostatic functions of flTF, largely exerted through activation of intracellular signaling via Protease Activated Receptors (PARs). The flTF-PAR nexus impacts several kinases highly significant in the pathobiology of cancer and cardiovascular disease. Over the past decade, many advances have been made in the understanding of PAR-mediated functions of flTF, an important highlight of which was the finding that a sub-set of integrins - a diverse family of integral membrane proteins - cross-regulate flTF-elicited signaling. Concomitantly, an alternatively spliced TF form (asTF) was discovered in human and mouse. Initial studies characterizing asTF revealed that it is differentially expressed during development, continuously present in circulating blood and solid tissues, and possesses very low pro-coagulant activity. Hypomorphic nature of asTF's cofactor activity is the source of an ongoing controversy over whether asTF is pro-coagulant, and how it may contribute to hemostatic maintenance and/or its aberrations. Very recently, a novel concept emerged in asTF biology: asTF can evidently trigger intracellular signaling that promotes the formation of new vessels from the existing ones (angiogenesis) and monocyte-endothelial interactions, via interaction with integrins. We provide a brief overview of the fl/asTF-integrin nexus with an emphasis on asTF's non-proteolytic, integrin-mediated biological activity.
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Integrinas/metabolismo , Tromboplastina/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Expresión Génica , Humanos , Integrinas/genética , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas , Transducción de Señal , Tromboplastina/genéticaRESUMEN
Following the molecular cloning of human Tissue Factor (TF) in mid-1980's, great strides have been made in the understanding of TF biology, TF's crucial roles in the initiation of blood coagulation and embryonic development, and TF's contribution to the pathobiology of various disease states. The 21st century brought about a rather unexpected turn in the "TF journey"--a few years back it was reported that the TF gene produces not one, but two proteins with distinct structural and functional characteristics. The so-called "full-length TF" (flTF) - a much-studied integral membrane glycoprotein long presumed to be, and experimentally handled as "the TF" in hundreds of laboratories around the world - is now known to be one of the two TF forms naturally occurring in humans as well as mice. The other, recently discovered form is termed alternatively spliced TF (asTF) which, unlike flTF, lacks a transmembrane domain and can thus be secreted. In this review, we summarize the literature on asTF by discussing asTF's biologic roles as they are currently understood, tackling a number of questions pertaining to asTF's evident and proposed biologic properties, and briefly covering the emerging field of regulated TF pre-mRNA processing.
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Tromboplastina/genética , Empalme Alternativo , Animales , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/genética , Endotelio Vascular/fisiología , Ingeniería Genética , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Monocitos/fisiología , Neoplasias/sangre , Neoplasias/genética , Neovascularización Fisiológica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tromboplastina/química , Tromboplastina/deficiencia , Tromboplastina/metabolismoRESUMEN
PURPOSE: Although VEGF has been identified as an important mediator of the blood-retinal barrier alteration in diabetic retinopathy, the hypothesis for this study was that that other molecules, including the angiopoietins (Ang-1 and -2), may play a role. The expression of angiopoietins was analyzed in an animal model of diabetic retinopathy, and the role of Ang-2 in the regulation of diabetes-induced alterations of vascular permeability was characterized. METHODS: Diabetes was induced in rats, and human retinal endothelial cells (HRECs) were grown in media with 5.5 or 30.5 mM glucose. Levels of Ang-1 and -2 mRNA and protein were analyzed. Fluorescence-based assays were used to assess the effect of Ang-2 on vascular permeability in vivo and in vitro. The effect of Ang-2 on VE-cadherin function was assessed by measuring the extent of tyrosine phosphorylation. RESULTS: Ang-2 mRNA and protein increased in the retinal tissues after 8 weeks of diabetes and in high-glucose-treated cells. Intravitreal injection of Ang-2 in rats produced a significant increase in retinal vascular permeability. Ang-2 increased HREC monolayer permeability that was associated with a decrease in VE-cadherin and a change in monolayer morphology. High glucose and Ang-2 produced a significant increase in VE-cadherin phosphorylation. CONCLUSIONS; Ang-2 is upregulated in the retina in an animal model of diabetes, and hyperglycemia induces the expression of Ang-2 in isolated retinal endothelial cells. Increased Ang-2 alters VE-cadherin function, leading to increased vascular permeability. Thus, Ang-2 may play an important role in increased vasopermeability in diabetic retinopathy.
Asunto(s)
Angiopoyetina 2/fisiología , Barrera Hematorretinal/fisiología , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , Vasos Retinianos/metabolismo , Angiopoyetina 1/metabolismo , Animales , Antígenos CD/metabolismo , Western Blotting , Cadherinas/metabolismo , Permeabilidad Capilar , Células Cultivadas , Impedancia Eléctrica , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Glucosa/farmacología , Humanos , Hiperglucemia/metabolismo , Masculino , Fosforilación , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tirosina/metabolismo , Regulación hacia ArribaRESUMEN
Cellular inactivation through killer immunoglobulin-like receptors (KIRs) may allow neoplastic cells to evade host natural killer (NK) cell-mediated immunity. Recently, alloreactive NK cells were shown to mediate antileukemic effects against acute myelogenous leukemia (AML) after mismatched transplantation, when KIR ligand incompatibility existed in the direction of graft-versus-host disease (GVHD). Therefore, we investigated whether solid tumor cells would have similar enhanced susceptibility to allogeneic KIR-incompatible NK cells compared with their KIR-matched autologous or allogeneic counterparts. NK populations enriched and cloned from the blood of cancer patients or healthy donors homozygous for HLA-C alleles in group 1 (C-G1) or group 2 (C-G2) were tested in vitro for cytotoxicity against Epstein-Barr virus-transformed lymphoblastic cell lines (EBV-LCLs), renal cell carcinoma (RCC), and melanoma (MEL) cells with or without a matching KIR-inhibitory HLA-C ligand. Allogeneic NK cells were more cytotoxic to tumor targets mismatched for KIR ligands than their KIR ligand-matched counterparts. Bulk NK populations (CD3(-)/CD2(+)/CD56(+)) expanded 10(4)-fold from patients homozygous for C-G1 or C-G2 had enhanced cytotoxicity against KIR ligand-mismatched tumor cells but only minimal cytotoxicity against KIR ligand-matched targets. Further, NK cell lines from C-G1 or C-G2 homozygous cancer patients or healthy donors expanded but failed to kill autologous or KIR-matched MEL and RCC cells yet had significant cytotoxicity (more than 50% lysis at 20:1 effector-target [E/T] ratio) against allogeneic KIR-mismatched tumor lines. These data suggest immunotherapeutic strategies that use KIR-incompatible allogeneic NK cells might have superior antineoplastic effects against solid tumors compared with approaches using autologous NK cells.