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1.
PLoS One ; 10(3): e0120770, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25790110

RESUMEN

Endothelial dysfunction contributes to the development of acute kidney injury (AKI) in animal models of ischemia reperfusion injury and sepsis. There are limited data on markers of endothelial dysfunction in human AKI. We hypothesized that Protein C (PC) and soluble thrombomodulin (sTM) levels could predict AKI. We conducted a multicenter prospective study in 80 patients to assess the relationship of PC and sTM levels to AKI, defined by the AKIN creatinine (AKI Scr) and urine output criteria (AKI UO). We measured marker levels for up to 10 days from intensive care unit admission. We used area under the curve (AUC) and time-dependent multivariable Cox proportional hazard model to predict AKI and logistic regression to predict mortality/non-renal recovery. Protein C and sTM were not different in patients with AKI UO only versus no AKI. On intensive care unit admission, as PC levels are usually lower with AKI Scr, the AUC to predict the absence of AKI was 0.63 (95%CI 0.44-0.78). The AUC using log10 sTM levels to predict AKI was 0.77 (95%CI 0.62-0.89), which predicted AKI Scr better than serum and urine neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C, urine kidney injury molecule-1 and liver-fatty acid-binding protein. In multivariable models, PC and urine NGAL levels independently predicted AKI (p=0.04 and 0.02) and PC levels independently predicted mortality/non-renal recovery (p=0.04). In our study, PC and sTM levels can predict AKI Scr but are not modified during AKI UO alone. PC levels could independently predict mortality/non-renal recovery. Additional larger studies are needed to define the relationship between markers of endothelial dysfunction and AKI.


Asunto(s)
Lesión Renal Aguda/sangre , Proteína C/metabolismo , Trombomodulina/sangre , Trombomodulina/química , Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/mortalidad , Biomarcadores/sangre , Enfermedad Crítica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Solubilidad
2.
Blood ; 119(3): 874-83, 2012 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-22117049

RESUMEN

The cytoprotective effects of activated protein C (aPC) are well established. In contrast, the receptors and signaling mechanism through which aPC conveys cytoprotection in various cell types remain incompletely defined. Thus, within the renal glomeruli, aPC preserves endothelial cells via a protease-activated receptor-1 (PAR-1) and endothelial protein C receptor-dependent mechanism. Conversely, the signaling mechanism through which aPC protects podocytes remains unknown. While exploring the latter, we identified a novel aPC/PAR-dependent cytoprotective signaling mechanism. In podocytes, aPC inhibits apoptosis through proteolytic activation of PAR-3 independent of endothelial protein C receptor. PAR-3 is not signaling competent itself as it requires aPC-induced heterodimerization with PAR-2 (human podocytes) or PAR-1 (mouse podocytes). This cytoprotective signaling mechanism depends on caveolin-1 dephosphorylation. In vivo aPC protects against lipopolysaccharide-induced podocyte injury and proteinuria. Genetic deletion of PAR-3 impairs the nephroprotective effect of aPC, demonstrating the crucial role of PAR-3 for aPC-dependent podocyte protection. This novel, aPC-mediated interaction of PARs demonstrates the plasticity and cell-specificity of cytoprotective aPC signaling. The evidence of specific, dynamic signaling complexes underlying aPC-mediated cytoprotection may allow the design of cell type specific targeted therapies.


Asunto(s)
Apoptosis , Citoprotección , Podocitos/metabolismo , Proteína C/metabolismo , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Animales , Anticoagulantes/metabolismo , Comunicación Celular , Células Cultivadas , Humanos , Glomérulos Renales/citología , Glomérulos Renales/metabolismo , Lipopolisacáridos/farmacología , Microdominios de Membrana , Ratones , Podocitos/efectos de los fármacos , Podocitos/patología , Multimerización de Proteína , Transducción de Señal , Trombina
3.
Am J Physiol Renal Physiol ; 302(6): F703-12, 2012 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-22129968

RESUMEN

Chronic kidney disease pathogenesis involves both tubular and vascular injuries. Despite abundant investigations to identify the risk factors, the involvement of chronic endothelial dysfunction in developing nephropathies is insufficiently explored. Previously, soluble thrombomodulin (sTM), a cofactor in the activation of protein C, has been shown to protect endothelial function in models of acute kidney injury. In this study, the role for sTM in treating chronic kidney disease was explored by employing a mouse model of chronic vascular activation using endothelial-specific TNF-α-expressing (tie2-TNF) mice. Analysis of kidneys from these mice after 3 mo showed no apparent phenotype, whereas 6-mo-old mice demonstrated infiltration of CD45-positive leukocytes accompanied by upregulated gene expression of inflammatory chemokines, markers of kidney injury, and albuminuria. Intervention with murine sTM with biweekly subcutaneous injections during this window of disease development between months 3 and 6 prevented the development of kidney pathology. To better understand the mechanisms of these findings, we determined whether sTM could also prevent chronic endothelial cell activation in vitro. Indeed, treatment with sTM normalized increased chemokines, adhesion molecule expression, and reduced transmigration of monocytes in continuously activated TNF-expressing endothelial cells. Our results suggest that vascular inflammation associated with vulnerable endothelium can contribute to loss in renal function as suggested by the tie2-TNF mice, a unique model for studying the role of vascular activation and inflammation in chronic kidney disease. Furthermore, the ability to restore the endothelial balance by exogenous administration of sTM via downregulation of specific adhesion molecules and chemokines suggests a potential for therapeutic intervention in kidney disease associated with chronic inflammation.


Asunto(s)
Albuminuria/prevención & control , Inflamación/tratamiento farmacológico , Fallo Renal Crónico/metabolismo , Trombomodulina/uso terapéutico , Albuminuria/tratamiento farmacológico , Albuminuria/genética , Albuminuria/metabolismo , Animales , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Regulación de la Expresión Génica/fisiología , Inflamación/genética , Inflamación/metabolismo , Fallo Renal Crónico/genética , Ratones , Ratones Transgénicos , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor TIE-2 , Técnicas de Cultivo de Tejidos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo
4.
Thromb Haemost ; 106(6): 1189-96, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21901240

RESUMEN

Recombinant human activated protein C (APC), which has both anticoagulant and anti-inflammatory properties, improves survival of patients with severe sepsis. This beneficial effect is especially apparent in patients with pneumococcal pneumonia. Earlier treatment with APC in sepsis has been associated with a better therapeutic response as compared to later treatment. In a mouse model it was recently confirmed that recombinant murine (rm-)APC decreases coagulation activation and improves survival in pneumococcal pneumonia; however, APC did not impact on the inflammatory response. The aim of this study was to determine the effect of APC treatment instigated early in infection on activation of coagulation and inflammation after induction of pneumococcal pneumonia. Mice were infected intranasally with viable S. pneumoniae . Mice were treated with rm-APC (125 µg) or vehicle intraperitoneally 12 hours after infection and were sacrificed after 20 hours, after which blood and organs were harvested for determination of bacterial outgrowth, coagulation activation and inflammatory markers. In this early treatment model, rm-APC treatment inhibited pulmonary and systemic activation of coagulation as reflected by lower levels of thrombin-antithrombin complexes and D-dimer. Moreover, rm-APC reduced the levels of a large number of cytokines and chemokines in the lung. When administered early in pneumococcal pneumonia, rm-APC inhibits systemic and pulmonary activation of coagulation and moreover exerts various anti-inflammatory effects in the lung.


Asunto(s)
Pulmón/efectos de los fármacos , Neumonía Neumocócica/inmunología , Proteína C/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Streptococcus pneumoniae/inmunología , Animales , Antiinflamatorios/administración & dosificación , Antitrombina III/metabolismo , Biomarcadores/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Humanos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Péptido Hidrolasas/metabolismo , Neumonía Neumocócica/sangre , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/patogenicidad
5.
J Infect Dis ; 202(10): 1600-7, 2010 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-20925531

RESUMEN

BACKGROUND: Recombinant human activated protein C (APC) improves survival of patients with severe sepsis; this beneficial effect is especially apparent in patients with pneumococcal pneumonia. The aim of this study was to determine the effect of APC treatment initiated after induction of pneumococcal pneumonia on pulmonary coagulation, inflammation, and survival, with or without concurrent antibiotic therapy. METHODS: Mice were infected intranasally with viable Streptococcus pneumoniae and were treated intraperitoneally after 24 h of infection with vehicle, recombinant mouse (rm) APC (125 µg), ceftriaxone (500 µg), or rm-APC plus ceftriaxone. Treatment with rm-APC or vehicle was repeated every 8 h for a maximum of 96 h. Animals were either killed 48 h after infection or were monitored in a survival study (with an extra dose of ceftriaxone given after 72 h). RESULTS: Rm-APC treatment inhibited pulmonary activation of coagulation, as reflected by lower levels of thrombin-antithrombin complexes and D-dimer. Rm-APC did not affect the pulmonary levels of 55 inflammatory mediators in the context of antibiotic therapy. Rm-APC added to ceftriaxone markedly improved survival, compared with ceftriaxone treatment alone. CONCLUSIONS: Rm-APC inhibits pulmonary activation of coagulation and, when added to antibiotic therapy, improves survival in murine pneumococcal pneumonia.


Asunto(s)
Trastornos de las Proteínas de Coagulación/tratamiento farmacológico , Trastornos de las Proteínas de Coagulación/microbiología , Neumonía Neumocócica/tratamiento farmacológico , Proteína C/uso terapéutico , Animales , Antibacterianos/uso terapéutico , Factores de Coagulación Sanguínea/antagonistas & inhibidores , Ceftriaxona/administración & dosificación , Ceftriaxona/uso terapéutico , Evaluación Preclínica de Medicamentos , Quimioterapia Combinada , Humanos , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos C57BL , Neumonía Neumocócica/complicaciones , Proteína C/administración & dosificación , Proteína C/farmacología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico
6.
Crit Care ; 14(2): R65, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20398279

RESUMEN

INTRODUCTION: Influenza accounts for 5 to 10% of community-acquired pneumonias and is a major cause of mortality. Sterile and bacterial lung injuries are associated with procoagulant and inflammatory derangements in the lungs. Activated protein C (APC) is an anticoagulant with anti-inflammatory properties that exert beneficial effects in models of lung injury. We determined the impact of lethal influenza A (H1N1) infection on systemic and pulmonary coagulation and inflammation, and the effect of recombinant mouse (rm-) APC here on. METHODS: Male C57BL/6 mice were intranasally infected with a lethal dose of a mouse adapted influenza A (H1N1) strain. Treatment with rm-APC (125 microg intraperitoneally every eight hours for a maximum of three days) or vehicle was initiated 24 hours after infection. Mice were euthanized 48 or 96 hours after infection, or observed for up to nine days. RESULTS: Lethal H1N1 influenza resulted in systemic and pulmonary activation of coagulation, as reflected by elevated plasma and lung levels of thrombin-antithrombin complexes and fibrin degradation products. These procoagulant changes were accompanied by inhibition of the fibrinolytic response due to enhanced release of plasminogen activator inhibitor type-1. Rm-APC strongly inhibited coagulation activation in both plasma and lungs, and partially reversed the inhibition of fibrinolysis. Rm-APC temporarily reduced pulmonary viral loads, but did not impact on lung inflammation or survival. CONCLUSIONS: Lethal influenza induces procoagulant and antifibrinolytic changes in the lung which can be partially prevented by rm-APC treatment.


Asunto(s)
Anticoagulantes/antagonistas & inhibidores , Anticoagulantes/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/sangre , Proteína C/antagonistas & inhibidores , Proteína C/uso terapéutico , Animales , Anticoagulantes/metabolismo , Regulación hacia Abajo/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Pulmón/efectos de los fármacos , Pulmón/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Infecciones por Orthomyxoviridae/fisiopatología , Proteína C/genética , Proteína C/metabolismo , Proteínas Recombinantes , Carga Viral
7.
Curr Drug Targets ; 10(12): 1212-26, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19715537

RESUMEN

Protein C is a plasma serine protease that when activated plays a central role in modulating the function of the vascular endothelium and its interface with the innate immune system. Activated protein C (APC) has a dual mechanism of action via the feedback inhibition of thrombin generation, and as an agonist of protease activated receptor-1 (PAR-1). Through different cofactor interactions, this dual mechanism of antithrombotic and cytoprotective activity results in the ability of APC to modulate endothelial dysfunction by blocking cytokine signaling, functional cell adhesion expression, vascular permeability, apoptosis, and modulating leukocyte migration and adhesion. Deficiency in protein C, which occurs during systemic inflammatory activation, is highly associated with organ dysfunction. APC has shown efficacy in a number of preclinical models of thrombosis and ischemia, and the recombinant human APC drotrecogin alfa (activated), reduces mortality in patients with high-risk severe sepsis. The ability of APC to suppress pro-inflammatory pathways and enhance cellular survival suggests that APC plays a key role in the adaptive response to protect the vessel wall from insult and to enhance endothelial, cellular, and organ survival. The focus of this review will be to summarize the emerging data suggesting the potential therapeutic benefit of APC and related members of the pathway in the prevention and treatment of acute kidney injury.


Asunto(s)
Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/metabolismo , Proteína C/fisiología , Receptor PAR-1/efectos de los fármacos , Resistencia a la Proteína C Activada/patología , Lesión Renal Aguda/prevención & control , Animales , Humanos , Riñón/fisiología , Sustancias Protectoras/uso terapéutico , Proteína C/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Transducción de Señal/efectos de los fármacos
8.
J Am Soc Nephrol ; 20(3): 524-34, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19176699

RESUMEN

Altered coagulation and inflammation contribute to the pathogenesis of ischemic renal injury. Thrombomodulin is a necessary factor in the anticoagulant protein C pathway and has inherent anti-inflammatory properties. We studied the effect of soluble thrombomodulin (sTM) in a hypoperfusion model of ischemic kidney injury. To markedly reduce infrarenal aortic blood flow and femoral arterial pressures, we clamped the suprarenal aorta of rats, occluding them 90%, for 60 min. Reversible acute kidney injury (AKI) occurred at 24 h in rats subjected to hypoperfusion. Histologic analysis at 24 h revealed acute tubular necrosis (ATN), and intravital two-photon microscopy showed flow abnormalities in the microvasculature and defects of endothelial permeability. Pretreatment with rat sTM markedly reduced both I-R-induced renal dysfunction and tubular histologic injury scores. sTM also significantly improved microvascular erythrocyte flow rates, reduced microvascular endothelial leukocyte rolling and attachment, and minimized endothelial permeability to infused fluorescence dextrans, assessed by intravital quantitative multiphoton microscopy. Furthermore, sTM administered 2 h after reperfusion protected against ischemia-induced renal dysfunction at 24 h and improved survival. By using an sTM variant, we also determined that the protective effects of sTM were independent of its ability to generate activated protein C. These data suggest that sTM may have therapeutic potential for ischemic AKI.


Asunto(s)
Isquemia/prevención & control , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Trombomodulina/administración & dosificación , Animales , Secuencia de Bases , Permeabilidad Capilar/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Cartilla de ADN/genética , Fibrinolíticos/administración & dosificación , Variación Genética , Riñón/lesiones , Necrosis Tubular Aguda/patología , Necrosis Tubular Aguda/fisiopatología , Necrosis Tubular Aguda/prevención & control , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Masculino , Proteína C/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Circulación Renal/efectos de los fármacos , Solubilidad , Trombomodulina/genética , Trombomodulina/fisiología
9.
J Am Soc Nephrol ; 20(2): 267-77, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19092124

RESUMEN

Administration of activated protein C (APC) protects from renal dysfunction, but the underlying mechanism is unknown. APC exerts both antithrombotic and cytoprotective properties, the latter via modulation of protease-activated receptor-1 (PAR-1) signaling. We generated APC variants to study the relative importance of the two functions of APC in a model of LPS-induced renal microvascular dysfunction. Compared with wild-type APC, the K193E variant exhibited impaired anticoagulant activity but retained the ability to mediate PAR-1-dependent signaling. In contrast, the L8W variant retained anticoagulant activity but lost its ability to modulate PAR-1. By administering wild-type APC or these mutants in a rat model of LPS-induced injury, we found that the PAR-1 agonism, but not the anticoagulant function of APC, reversed LPS-induced systemic hypotension. In contrast, both functions of APC played a role in reversing LPS-induced decreases in renal blood flow and volume, although the effects on PAR-1-dependent signaling were more potent. Regarding potential mechanisms for these findings, APC-mediated PAR-1 agonism suppressed LPS-induced increases in the vasoactive peptide adrenomedullin and infiltration of iNOS-positive leukocytes into renal tissue. However, the anticoagulant function of APC was responsible for suppressing LPS-induced stimulation of the proinflammatory mediators ACE-1, IL-6, and IL-18, perhaps accounting for its ability to modulate renal hemodynamics. Both variants reduced active caspase-3 and abrogated LPS-induced renal dysfunction and pathology. We conclude that although PAR-1 agonism is solely responsible for APC-mediated improvement in systemic hemodynamics, both functions of APC play distinct roles in attenuating the response to injury in the kidney.


Asunto(s)
Enfermedades Renales/metabolismo , Riñón/lesiones , Proteína C/fisiología , Animales , Humanos , Inflamación , Interleucina-18/metabolismo , Interleucina-6/metabolismo , Riñón/metabolismo , Lipopolisacáridos/metabolismo , Masculino , Microcirculación , Proteína C/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor PAR-1/metabolismo , Transducción de Señal
10.
Adv Exp Med Biol ; 614: 83-91, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18290317

RESUMEN

The protein C (PC) pathway plays an important role in vascular function, and acquired deficiency during sepsis is associated with increased mortality. We have explored the role of PC suppression in modulating early inflammatory events in a model of polymicrobial sepsis. We show that increased levels of organ damage and dysfunction are associated with decreased levels of endogenous PC. Notably, animals with low PC had correspondingly high levels of pulmonary iNOS expression, which correlated with chemokines KC/Gro and MIP2, previously shown to predict outcome in this model. Treatment with activated protein C (aPC) not only reduced the pathology score, leukocyte infiltration and markers of organ dysfunction, but also suppressed the induction of iNOS, and the chemokine response (including KC/Gro, MIP2, IP-10, RANTES, GCP-2 and lymphotactin), and increased apoA1. aPC treatment also suppressed the induction of VEGF, a marker recently suggested to play a pathophysiological role in sepsis. These data demonstrate a clear link between low protein C and degree of organ damage and dysfunction in sepsis, as well as the early reversal with aPC treatment. Moreover, our data show a direct role of aPC in broadly modulating monocyte and T-cell chemokines following systemic inflammatory response.


Asunto(s)
Anticoagulantes/uso terapéutico , Quimiocinas/metabolismo , Proteína C/fisiología , Proteína C/uso terapéutico , Sepsis/tratamiento farmacológico , Animales , Biomarcadores/sangre , Ciego/cirugía , Modelos Animales de Enfermedad , Inducción Enzimática/efectos de los fármacos , Ligadura , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteína C/genética , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/uso terapéutico , Sepsis/sangre , Sepsis/etiología , Sepsis/patología , Índice de Severidad de la Enfermedad , Estadística como Asunto , Resultado del Tratamiento
11.
Nature ; 451(7182): 1076-81, 2008 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-18278032

RESUMEN

Understanding the neuropathology of multiple sclerosis (MS) is essential for improved therapies. Therefore, identification of targets specific to pathological types of MS may have therapeutic benefits. Here we identify, by laser-capture microdissection and proteomics, proteins unique to three major types of MS lesions: acute plaque, chronic active plaque and chronic plaque. Comparative proteomic profiles identified tissue factor and protein C inhibitor within chronic active plaque samples, suggesting dysregulation of molecules associated with coagulation. In vivo administration of hirudin or recombinant activated protein C reduced disease severity in experimental autoimmune encephalomyelitis and suppressed Th1 and Th17 cytokines in astrocytes and immune cells. Administration of mutant forms of recombinant activated protein C showed that both its anticoagulant and its signalling functions were essential for optimal amelioration of experimental autoimmune encephalomyelitis. A proteomic approach illuminated potential therapeutic targets selective for specific pathological stages of MS and implicated participation of the coagulation cascade.


Asunto(s)
Perfilación de la Expresión Génica , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Proteómica , Adulto , Animales , Coagulación Sanguínea , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Humanos , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Persona de Mediana Edad , Esclerosis Múltiple/clasificación , Esclerosis Múltiple/tratamiento farmacológico , Proteína C/genética , Proteína C/metabolismo , Proteína C/farmacología , Células TH1/inmunología , Células Th2/inmunología , Trombina/antagonistas & inhibidores , Trombina/metabolismo
12.
J Pharmacol Exp Ther ; 325(1): 17-26, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18182560

RESUMEN

The protein C (PC) pathway plays an important role in vascular and immune function, and acquired deficiency during sepsis is associated with increased mortality in both animal models and in clinical studies. However, the association of acquired PC deficiency with the pathophysiology of lung injury is unclear. We hypothesized that low PC induced by sepsis would associate with increased pulmonary injury and that replacement with activated protein C (APC) would reverse the activation of pathways associated with injury. Using a cecal ligation and puncture (CLP) model of polymicrobial sepsis, we examined the role of acquired PC deficiency on acute lung injury assessed by analyzing changes in pulmonary pathology, chemokine response, inducible nitric-oxide synthase (iNOS), and the angiotensin pathway. Acquired PC deficiency was strongly associated with an increase in lung inflammation and drivers of pulmonary injury, including angiotensin (Ang) II, thymus and activation-regulated chemokine, plasminogen activator inhibitor (PAI)-1, and iNOS. In contrast, the protective factor angiotensin-converting enzyme (ACE)-2 was significantly suppressed in animals with acquired PC deficiency. The endothelial protein C receptor, required for the cytoprotective signaling of APC, was significantly increased post-CLP, suggesting a compensatory up-regulation of the signaling receptor. Treatment of septic animals with APC reduced pulmonary pathology, suppressed the macrophage inflammatory protein family chemokine response, iNOS expression, and PAI-1 activity and up-regulated ACE-2 expression with concomitant reduction in AngII peptide. These data demonstrate a clear link between acquired PC deficiency and pulmonary inflammatory response in the rat sepsis model and provide support for the concept of APC as a replacement therapy in acute lung injury associated with acquired PC deficiency.


Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Peptidil-Dipeptidasa A/efectos de los fármacos , Deficiencia de Proteína C/tratamiento farmacológico , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Sepsis/complicaciones , Síndrome de Respuesta Inflamatoria Sistémica/tratamiento farmacológico , Enzima Convertidora de Angiotensina 2 , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Inflamatorias de Macrófagos/genética , Óxido Nítrico Sintasa de Tipo II/genética , Inhibidor 1 de Activador Plasminogénico/genética , Deficiencia de Proteína C/etiología , Ratas
13.
Nat Med ; 13(11): 1349-58, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17982464

RESUMEN

Data providing direct evidence for a causative link between endothelial dysfunction, microvascular disease and diabetic end-organ damage are scarce. Here we show that activated protein C (APC) formation, which is regulated by endothelial thrombomodulin, is reduced in diabetic mice and causally linked to nephropathy. Thrombomodulin-dependent APC formation mediates cytoprotection in diabetic nephropathy by inhibiting glomerular apoptosis. APC prevents glucose-induced apoptosis in endothelial cells and podocytes, the cellular components of the glomerular filtration barrier. APC modulates the mitochondrial apoptosis pathway via the protease-activated receptor PAR-1 and the endothelial protein C receptor EPCR in glucose-stressed cells. These experiments establish a new pathway, in which hyperglycemia impairs endothelial thrombomodulin-dependent APC formation. Loss of thrombomodulin-dependent APC formation interrupts cross-talk between the vascular compartment and podocytes, causing glomerular apoptosis and diabetic nephropathy. Conversely, maintaining high APC levels during long-term diabetes protects against diabetic nephropathy.


Asunto(s)
Apoptosis , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/prevención & control , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/prevención & control , Endotelio Vascular/patología , Podocitos/patología , Proteína C/fisiología , Sustitución de Aminoácidos/genética , Animales , Apoptosis/genética , Línea Celular Transformada , Células Cultivadas , Citoprotección/genética , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/enzimología , Nefropatías Diabéticas/genética , Endotelio Vascular/enzimología , Activación Enzimática/genética , Humanos , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/enzimología , Glomérulos Renales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Microcirculación/enzimología , Microcirculación/patología , Podocitos/enzimología , Proteína C/biosíntesis , Proteína C/genética , Transducción de Señal/genética , Trombomodulina/fisiología
14.
Arterioscler Thromb Vasc Biol ; 27(12): 2634-41, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17932312

RESUMEN

UNLABELLED: Background- APC is an antithrombotic and antiinflammatory serine protease that plays an important role in vascular function. We report that APC can suppress the proapoptotic mediator TRAIL in human umbilical vein endothelial cells, and we have investigated the signaling mechanism. METHODS AND RESULTS: APC inhibited endothelial TRAIL expression and secretion and its induction by cell activation. To explore the mechanism, we examined factors associated with TRAIL regulation and demonstrated that APC increased the level of EGR-1, a transcriptional factor known to suppress the TRAIL promoter. APC also induced a significant increase in phosphorylation of ERK-1/2, required to activate EGR-1 expression. Activation of ERK-1/2 was dependent on the protease activated receptor-1 (PAR-1), but independent of the endothelial protein C receptor (EPCR). Using siRNA, we found that the effect of APC on the EGR-1/ERK signaling required for TRAIL inhibition was dependent on the S1P1 receptor and S1P1 kinase. CONCLUSIONS: Our data suggest that APC may provide cytoprotective activity by activating the ERK pathway, which upregulates EGR-1 thereby suppressing the expression of TRAIL. Moreover, we provide evidence that APC can induce a cell signaling response through a PAR-1/S1P1-dependent but EPCR-independent mechanism.


Asunto(s)
Antígenos CD/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Células Endoteliales/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína C/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Antígenos CD/genética , Células Cultivadas , Citoprotección , Células Endoteliales/enzimología , Receptor de Proteína C Endotelial , Activación Enzimática , Humanos , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Receptor PAR-1/metabolismo , Receptores de Superficie Celular/genética , Receptores de Lisoesfingolípidos/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Factores de Tiempo , Transcripción Genética , Factor de Necrosis Tumoral alfa/metabolismo
15.
J Biol Chem ; 282(51): 36837-44, 2007 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-17928287

RESUMEN

Inducible nitric-oxide synthase (iNOS) plays a central role in the regulation of vascular function and response to injury. A central mediator controlling iNOS expression is transforming growth factor-beta (TGF-beta), which represses its expression through a mechanism that is poorly understood. We have identified a binding site in the iNOS promoter that interacts with the nuclear heterodimer TCF11/MafG using chromatin immunoprecipitation and mutation analyses. We demonstrate that binding at this site acts to repress the induction of iNOS gene expression by cytokines. We show that this repressor is induced by TGF-beta1 and by Smad6-short, which enhances TGF-beta signaling. In contrast, the up-regulation of TCF11/MafG binding could be suppressed by overexpression of the TGF-beta inhibitor Smad7, and a small interfering RNA to TCF11 blocked the suppression of iNOS by TGF-beta. The binding of TCF11/MafG to the iNOS promoter could be enhanced by phorbol 12-myristate 13-acetate and suppressed by the protein kinase C inhibitor staurosporine. Moreover, the induction of TCF11/MafG binding by TGF-beta and Smad6-short could be blocked by staurosporine, and the effect of TGF-beta was blocked by the selective protein kinase C inhibitor calphostin C. Consistent with the in vitro data, we found suppression of TCF11 coincident with iNOS up-regulation in a rat model of endotoxemia, and we observed a highly significant negative correlation between TCF11 and nitric oxide production. Furthermore, treatment with activated protein C, a serine protease effective in septic shock, blocked the down-regulation of TCF11 and suppressed endotoxin-induced iNOS. Overall, our results demonstrate a novel mechanism by which iNOS expression is regulated in the context of inflammatory activation.


Asunto(s)
Regulación Enzimológica de la Expresión Génica , Factor Nuclear 1-beta del Hepatocito/metabolismo , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Elementos de Respuesta , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Carcinógenos/farmacología , Células Cultivadas , Dimerización , Modelos Animales de Enfermedad , Endotoxemia/enzimología , Endotoxemia/genética , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Factor Nuclear 1-beta del Hepatocito/antagonistas & inhibidores , Factor Nuclear 1-beta del Hepatocito/genética , Humanos , Inflamación/enzimología , Inflamación/genética , Factor de Transcripción MafG/genética , Factor de Transcripción MafG/metabolismo , Masculino , Mutación , Naftalenos/farmacología , Óxido Nítrico Sintasa de Tipo II/genética , Proteína Quinasa C/antagonistas & inhibidores , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Elementos de Respuesta/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína smad6/genética , Proteína smad6/metabolismo , Proteína smad7/genética , Proteína smad7/metabolismo , Estaurosporina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factor de Crecimiento Transformador beta1/farmacología
16.
Biomaterials ; 28(28): 4047-55, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17597201

RESUMEN

Multi-functional bilayer polymeric coatings are prepared with both controlled nitric oxide (NO) release and surface-bound active thrombomodulin (TM) alone or in combination with immobilized heparin. The outer-layer is made of CarboSil, a commercially available copolymer of silicone rubber (SR) and polyurethane (PU). The CarboSil is either carboxylated or aminated via an allophanate reaction with a diisocyanate compound followed by a urea-forming reaction between the generated isocyanate group of the polymer and the amine group of an amino acid (glycine), an oligopeptide (triglycine) or a diamine. The carboxylated CarboSil can then be used to immobilize TM through the formation of an amide bond between the surface carboxylic acid groups and the lysine residues of TM. Aminated CarboSil can also be employed to initially couple heparin to the surface, and then the carboxylic acid groups on heparin can be further used to anchor TM. Both surface-bound TM and heparin's activity are evaluated by chromogenic assays and found to be at clinically significant levels. The underlying NO release layer is made with another commercial SR-PU copolymer (PurSil) mixed with a lipophilic NO donor (N-diazeniumdiolated dibutylhexanediamine (DBHD/N(2)O(2))). The NO release rate can be tuned by changing the thickness of top coatings, and the duration of NO release at physiologically relevant levels can be as long as 2 weeks. The combination of controlled NO release as well as immobilized active TM and heparin from/on the same polymeric surface mimics the highly thromboresistant endothelium layer. Hence, such multifunctional polymer coatings should provide more blood-compatible surfaces for biomedical devices.


Asunto(s)
Materiales Biocompatibles Revestidos , Endotelio/metabolismo , Heparina/metabolismo , Óxido Nítrico/metabolismo , Polímeros , Trombomodulina/metabolismo , Sangre/metabolismo , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/metabolismo , Humanos , Ensayo de Materiales , Estructura Molecular , Cemento de Policarboxilato/química , Polímeros/química , Polímeros/metabolismo , Poliuretanos/química , Unión Proteica , Siliconas/química , Propiedades de Superficie
17.
Shock ; 28(4): 468-76, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17558353

RESUMEN

Activated protein C (APC) is an important modulator of vascular function that has antithrombotic and anti-inflammatory properties. Studies in humans have shown modulation of endotoxin-induced hypotension by recombinant human APC, drotrecogin alfa (activated), however, the mechanism for this effect is unclear. We have found that APC suppresses the induction of the potent vasoactive peptide adrenomedullin (ADM) and could downregulate lipopolysaccharide (LPS)-induced ADM messenger RNA (mRNA) and nitrite levels in cell culture. This effect was dependent on signaling through protease-activated receptor 1. Addition of 1400W, an irreversible inducible nitric oxide synthase (iNOS) inhibitor, inhibited LPS-induced ADM mRNA, suggesting that ADM induction is NO mediated. Furthermore, in a rat model of endotoxemia, APC (100 microg/kg, i.v.) prevented LPS (10 mg/kg, i.v.)-induced hypotension, and suppressed ADM mRNA and protein expression. APC also inhibited iNOS mRNA and protein levels along with reduction in NO by-products (NOx). We also observed a significant reduction in iNOS-positive leukocytes adhering to vascular endothelium after APC treatment. Moreover, we found that APC inhibited the expression of interferon-gamma (IFN-gamma), a potent activator of iNOS. In a human study of LPS-induced hypotension, APC reduced the upregulation of plasma ADM levels, coincident with protection against the hypotensive response. Overall, we demonstrate that APC blocks the induction of ADM, likely mediated by IFN-gamma and iNOS, and suggests a mechanism that may account for ameliorating LPS-induced hypotension. Furthermore, our data provide a new understanding for the role of APC in modulating vascular response to insult.


Asunto(s)
Adrenomedulina/metabolismo , Hipotensión/prevención & control , Lipopolisacáridos/toxicidad , Proteína C/farmacología , Adrenomedulina/sangre , Adrenomedulina/genética , Animales , Western Blotting , Línea Celular , Movimiento Celular/efectos de los fármacos , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Hipotensión/inducido químicamente , Interferón gamma/genética , Interferón gamma/metabolismo , Leucocitos/citología , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/metabolismo , Óxidos de Nitrógeno/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
18.
J Clin Invest ; 117(7): 1951-60, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17557119

RESUMEN

Endothelial protein C receptor (EPCR) and thrombomodulin (TM) are expressed at high levels in the resting microvasculature and convert protein C (PC) into its activated form, which is a potent anticoagulant and antiinflammatory molecule. Here we provide evidence that in Crohn disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel disease (IBD), there was loss of expression of endothelial EPCR and TM, which in turns caused impairment of PC activation by the inflamed mucosal microvasculature. In isolated human intestinal endothelial cells, administration of recombinant activated PC had a potent antiinflammatory effect, as demonstrated by downregulated cytokine-dependent cell adhesion molecule expression and chemokine production as well as inhibited leukocyte adhesion. In vivo, administration of activated PC was therapeutically effective in ameliorating experimental colitis as evidenced by reduced weight loss, disease activity index, and histological colitis scores as well as inhibited leukocyte adhesion to the inflamed intestinal vessels. The results suggest that the PC pathway represents a new system crucially involved in governing intestinal homeostasis mediated by the mucosal microvasculature. Restoring the PC pathway may represent a new therapeutic approach to suppress intestinal inflammation in IBD.


Asunto(s)
Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Microcirculación/patología , Proteína C/metabolismo , Transducción de Señal , Animales , Antígenos CD/metabolismo , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Quimiocinas/biosíntesis , Regulación hacia Abajo , Receptor de Proteína C Endotelial , Endotelio/patología , Humanos , Inmunohistoquímica , Inflamación/metabolismo , Inflamación/patología , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/citología , Ratones , Microcirculación/metabolismo , Proteína C/farmacología , Receptores de Superficie Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Trombomodulina/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/metabolismo
19.
Am J Physiol Renal Physiol ; 293(1): F245-54, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17409278

RESUMEN

Endothelial dysfunction contributes significantly to acute renal failure (ARF) during inflammatory diseases including septic shock. Previous studies have shown that activated protein C (APC) exhibits anti-inflammatory properties and modulates endothelial function. Therefore, we investigated the effect of APC on ARF in a rat model of endotoxemia. Rats subjected to lipopolysaccharide (LPS) treatment exhibited ARF as illustrated by markedly reduced peritubular capillary flow and increased serum blood urea nitrogen (BUN) levels. Using quantitative two-photon intravital microscopy, we observed that at 3 h post-LPS treatment, rat APC (0.1 mg/kg iv bolus) significantly improved peritubular capillary flow [288 +/- 15 microm/s (LPS) vs. 734 +/- 59 microm/s (LPS+APC), P = 0.0009, n = 6], and reduced leukocyte adhesion (P = 0.003) and rolling (P = 0.01) compared with the LPS-treated group. Additional experiments demonstrated that APC treatment significantly improved renal blood flow and reduced serum BUN levels compared with 24-h post-LPS treatment. Biochemical analysis revealed that APC downregulated inducible nitric oxide synthase (iNOS) mRNA levels and NO by-products in the kidney. In addition, APC modulated the renin-angiotensin system by reducing mRNA expression levels of angiotensin-converting enzyme-1 (ACE1), angiotensinogen, and increasing ACE2 mRNA levels in the kidney. Furthermore, APC significantly reduced ANG II levels in the kidney compared with the LPS-treated group. Taken together, these data suggest that APC can suppress LPS-induced ARF by modulating factors involved in vascular inflammation, including downregulation of renal iNOS and ANG II systems. Furthermore, the data suggest a potential therapeutic role for APC in the treatment of ARF.


Asunto(s)
Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/prevención & control , Angiotensina II/biosíntesis , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/toxicidad , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Proteína C/farmacología , Lesión Renal Aguda/patología , Angiotensina II/antagonistas & inhibidores , Animales , Nitrógeno de la Urea Sanguínea , Adhesión Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Endotoxemia/metabolismo , Endotoxemia/patología , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Leucocitos/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Circulación Renal/efectos de los fármacos , Sistema Renina-Angiotensina/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Urea/sangre , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos
20.
J Am Soc Nephrol ; 18(3): 860-7, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17301189

RESUMEN

Protein C (PC) plays an important role in vascular function, and acquired deficiency during sepsis is associated with increased mortality in both animal models and in clinical studies. This study explored the consequences of PC suppression on the kidney in a cecal ligation and puncture model of polymicrobial sepsis. This study shows that a rapid drop in PC after sepsis is strongly associated with an increase in blood urea nitrogen, renal pathology, and expression of known markers of renal injury, including neutrophil gelatinase-associated lipocalin, CXCL1, and CXCL2. The endothelial PC receptor, which is required for the anti-inflammatory and antiapoptotic activity of activated PC (APC), was significantly increased after cecal ligation and puncture as well as in the microvasculature of human kidneys after injury. Treatment of septic animals with APC reduced blood urea nitrogen, renal pathology, and chemokine expression and dramatically reduced the induction of inducible nitric oxide synthase and caspase-3 activation in the kidney. The data demonstrate a clear link between acquired PC deficiency and renal dysfunction in sepsis and suggest a compensatory upregulation of the signaling receptor. Moreover, these data suggest that APC treatment may be effective in reducing inflammatory and apoptotic insult during sepsis-induced acute renal failure.


Asunto(s)
Lesión Renal Aguda/etiología , Riñón/metabolismo , Proteína C/metabolismo , Sepsis/metabolismo , Proteínas de Fase Aguda/metabolismo , Animales , Apoptosis , Biomarcadores/metabolismo , Caspasa 3/metabolismo , Ciego/cirugía , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas CXC/metabolismo , Modelos Animales de Enfermedad , Riñón/patología , Lipocalina 2 , Lipocalinas , Óxido Nítrico Sintasa/metabolismo , Deficiencia de Proteína C/complicaciones , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba
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