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Introduction: We reported increased spleen tyrosine kinase (SYK) expression in kidney biopsies of patients with IgA nephropathy (IgAN) and that inhibition of SYK reduces inflammatory cytokines production from IgA stimulated mesangial cells. Methods: This study was a double-blind, randomized, placebo-controlled phase 2 trial of fostamatinib (an oral SYK inhibitor) in 76 patients with IgAN. Patients were randomized to receive placebo, fostamatinib at 100 mg or 150 mg twice daily for 24 weeks on top of maximum tolerated dose of renin-angiotensin system inhibitors. The primary end point was reduction of proteinuria. Secondary end points included change from baseline in estimated glomerular filtration rate (eGFR) and kidney histology. Results: Although we could not detect significant reduction in proteinuria with fostamatinib overall, in a predetermined subgroup analysis, there was a trend for dose-dependent reduction in median proteinuria (from baseline to 24 weeks by 14%, 27%, and 36% in the placebo, fostamatinib 100 mg, and 150 mg groups, respectively) in patients with baseline urinary protein-to-creatinine ratios (UPCR) more than 1000 mg/g. Kidney function (eGFR) remained stable in all groups. Fostamatinib was well-tolerated. Side effects included diarrhea, hypertension, and increased liver enzymes. Thirty-nine patients underwent repeat biopsy showing reductions in SYK staining associated with therapy at low dose (-1.5 vs. 1.7 SYK+ cells/glomerulus in the placebo group, P < 0.05). Conclusions: There was a trend toward reduction in proteinuria with fostamatinib in a predefined analysis of high risk patients with IgAN despite maximal care, as defined by baseline UPCR greater than 1000 mg/g. Further study may be warranted.
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The desmoplastic stroma in solid tumors presents a formidable challenge to immunotherapies that rely on endogenous or adoptively transferred T cells, however, the mechanisms are poorly understood. To define mechanisms involved, here we treat established desmoplastic pancreatic tumors with CAR T cells directed to fibroblast activation protein (FAP), an enzyme highly overexpressed on a subset of cancer-associated fibroblasts (CAFs). Depletion of FAP+ CAFs results in loss of the structural integrity of desmoplastic matrix. This renders these highly treatment-resistant cancers susceptible to subsequent treatment with a tumor antigen (mesothelin)-targeted CAR T cells and to anti-PD-1 antibody therapy. Mechanisms include overcoming stroma-dependent restriction of T cell extravasation and/or perivascular invasion, reversing immune exclusion, relieving T cell suppression, and altering the immune landscape by reducing myeloid cell accumulation and increasing endogenous CD8+ T cell and NK cell infiltration. These data provide strong rationale for combining tumor stroma- and malignant cell-targeted therapies to be tested in clinical trials.
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Terapia de Inmunosupresión , Neoplasias Pancreáticas , Humanos , Inmunoterapia , Movimiento Celular , Neoplasias Pancreáticas/terapia , Linfocitos T CD8-positivosRESUMEN
The desmoplastic stroma in solid tumors presents a formidable challenge to immunotherapies that rely on endogenous or adoptively transferred T cells, however, the mechanisms are poorly understood. To define mechanisms involved, we treat established desmoplastic pancreatic tumors with CAR T cells directed to fibroblast activation protein (FAP), an enzyme highly overexpressed on a subset of cancer-associated fibroblasts (CAFs). Depletion of FAP+CAFs results in loss of the structural integrity of desmoplastic matrix. This renders these highly treatment-resistant cancers susceptible to subsequent treatment with a tumor antigen (mesothelin)-targeted CAR and to anti-PD1 antibody therapy. Mechanisms include overcoming stroma-dependent restriction of T cell extravasation and/or perivascular invasion, reversing immune exclusion, relieving T cell suppression, and altering the immune landscape by reducing myeloid cell accumulation and increasing endogenous CD8+ T cell and NK cell infiltration. These data provide strong rationale for combining tumor stroma- and malignant cell-targeted therapies to be tested in clinical trials.
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PURPOSE: Despite the success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies, successful targeting of solid tumors with CAR T cells has been limited by a lack of durable responses and reports of toxicities. Our understanding of the limited therapeutic efficacy in solid tumors could be improved with quantitative tools that allow characterization of CAR T-targeted antigens in tumors and accurate monitoring of response. EXPERIMENTAL DESIGN: We used a radiolabeled FAP inhibitor (FAPI) [18F]AlF-FAPI-74 probe to complement ongoing efforts to develop and optimize FAP CAR T cells. The selectivity of the radiotracer for FAP was characterized in vitro, and its ability to monitor changes in FAP expression was evaluated using rodent models of lung cancer. RESULTS: [18F]AlF-FAPI-74 showed selective retention in FAP+ cells in vitro, with effective blocking of the uptake in presence of unlabeled FAPI. In vivo, [18F]AlF-FAPI-74 was able to detect FAP expression on tumor cells as well as FAP+ stromal cells in the tumor microenvironment with a high target-to-background ratio. We further demonstrated the utility of the tracer to monitor changes in FAP expression following FAP CAR T-cell therapy, and the PET imaging findings showed a robust correlation with ex vivo analyses. CONCLUSIONS: This noninvasive imaging approach to interrogate the tumor microenvironment represents an innovative pairing of a diagnostic PET probe with solid tumor CAR T-cell therapy and has the potential to serve as a predictive and pharmacodynamic response biomarker for FAP as well as other stroma-targeted therapies. A PET imaging approach targeting FAP expressed on activated fibroblasts of the tumor stroma has the potential to predict and monitor therapeutic response to FAP-targeted CAR T-cell therapy. See related commentary by Weber et al., p. 5241.
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Gelatinasas , Serina Endopeptidasas , Línea Celular Tumoral , Tomografía de Emisión de Positrones , Linfocitos T , Tomografía Computarizada por Tomografía de Emisión de Positrones , Radioisótopos de GalioRESUMEN
BACKGROUND: Patients with immune thrombocytopenia (ITP) are at risk of bleeding and, paradoxically, thromboembolic events (TEEs), irrespective of thrombocytopenia. The risk of thrombosis is increased by advanced age, obesity, and prothrombotic comorbidities: cancer, hyperlipidemia, diabetes, hypertension, coronary artery disease, and chronic kidney disease, among others. Certain ITP treatments further increase the risk of TEE, especially splenectomy and thrombopoietin receptor agonists. Spleen tyrosine kinase (SYK) is a key signaling molecule common to thromboembolic and hemostatic (in addition to inflammatory) pathways. Fostamatinib is an orally administered SYK inhibitor approved in the USA and Europe for treatment of chronic ITP in adults. METHODS: The phase III and extension studies included heavily pretreated patients with long-standing ITP, many of whom had risk factors for thrombosis prior to initiating fostamatinib. This report describes long-term safety and efficacy of fostamatinib in 146 patients with up to 5 years of treatment, a total of 229 patient-years, and assesses the incidence of thromboembolic events (by standardized MedDRA query). RESULTS: Platelet counts ⩾50,000/µL were achieved in 54% of patients and the safety profile was as described in the phase III clinical studies with no new toxicities observed over the 5 years of follow-up. The only TEE occurred in one patient (0.7%, or 0.44/100 patient-years), who experienced a mild transient ischemic attack. This is a much lower rate than might be expected in ITP patients. CONCLUSION: This report demonstrates durable efficacy and a very low incidence of TEE in patients receiving long-term treatment of ITP with the SYK inhibitor fostamatinib. CLINICALTRIALSGOV IDENTIFIERS: NCT02076399, NCT02076412, and NCT02077192.
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OBJECTIVE: Although the molecular components of circadian rhythms oscillate in discrete cellular components of the vasculature and many aspects of vascular function display diurnal variation, the cellular connections between the molecular clock and inflammatory cardiovascular diseases remain to be elucidated. Previously we have shown that pre- versus postnatal deletion of Bmal1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1), the nonredundant core clock gene has contrasting effects on atherogenesis. Here we investigated the effect of myeloid cell Bmal1 deletion on atherogenesis and abdominal aortic aneurysm formation in mice. Approach and Results: Mice lacking Bmal1 in myeloid cells were generated by crossing Bmal1 flox/flox mice with lysozyme 2 promoter-driven Cre recombinase mice on a hyperlipidemic low-density lipoprotein receptor-deficient background and were fed on a high-fat diet to induce atherosclerosis. Atherogenesis was restrained, concomitant with a reduction of aortic proinflammatory gene expression in myeloid cell Bmal1 knockout mice. Body weight, blood pressure, blood glucose, triglycerides, and cholesterol were unaltered. Similarly, myeloid cell depletion of Bmal1 also restrained Ang II (angiotensin II) induced formation of abdominal aortic aneurysm in hyperlipidemic mice. In vitro, RNA-Seq analysis demonstrated a proinflammatory response in cultured macrophages in which there was overexpression of Bmal1. CONCLUSIONS: Myeloid cell Bmal1 deletion retards atherogenesis and restrains the formation of abdominal aortic aneurysm and may represent a potential therapeutic target for inflammatory cardiovascular diseases.
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Factores de Transcripción ARNTL/deficiencia , Factores de Transcripción ARNTL/fisiología , Aneurisma de la Aorta Abdominal/prevención & control , Aterosclerosis/prevención & control , Hiperlipidemias/complicaciones , Células Mieloides/química , Factores de Transcripción ARNTL/genética , Angiotensina II/farmacología , Animales , Aneurisma de la Aorta Abdominal/inducido químicamente , Aterosclerosis/etiología , Aterosclerosis/patología , Células Cultivadas , Cruzamientos Genéticos , Dieta Alta en Grasa , Eliminación de Gen , Expresión Génica , Hiperlipidemias/etiología , Inflamación , Integrasas/genética , Macrófagos Peritoneales/química , Macrófagos Peritoneales/fisiología , Ratones , Ratones Noqueados , Muramidasa/genética , Regiones Promotoras Genéticas/genética , Receptores de LDL/deficiencia , Receptores de LDL/genéticaRESUMEN
Fibroblast activation protein (FAP) has been established as an inducible and mesenchymal cell-specific mediator of disease progression in cancer and fibrosis. Atherosclerosis is a fibroinflammatory disease, and FAP was previously reported to be up-regulated in human atherosclerotic plaques compared with normal vessel. We investigated the spatial and temporal distribution of Fap-expressing cells in a murine model of atherosclerosis and used a genetic approach to determine if and how Fap affected disease progression. Fap was found to be expressed predominantly on vascular smooth muscle cells in lesions of athero-prone Apoe-/- mice. Global deletion of Fap (Fap-/-) in Apoe-/- mice accelerated atherosclerotic disease progression in both males and females, with the effect observed earlier in males. Sex-specific effects on lesion morphology were observed. Relative levels of extracellular matrix, fibrotic, and inflammatory cell content were comparable in lesions in male mice regardless of Fap status. In contrast, lesions in Fap-/- female mice were characterized by a more fibrotic composition due to a reduction in inflammation, specifically a reduction in Mox macrophages. Combined, these data suggest that Fap restrains the progression of atherosclerosis and may contribute to the sexually dimorphic susceptibility to atherosclerosis by regulating the balance between inflammation (an indicator of vulnerability to plaque rupture) and fibrosis (an indicator of plaque stability).
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Aterosclerosis/metabolismo , Fibrosis/metabolismo , Gelatinasas/metabolismo , Inflamación/metabolismo , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Caracteres Sexuales , Animales , Apolipoproteínas E/deficiencia , Endopeptidasas , Femenino , Masculino , Ratones , Ratones Noqueados para ApoERESUMEN
Primary tumor-derived factors (TDFs) act upon normal cells to generate a pre-metastatic niche, which promotes colonization of target organs by disseminated malignant cells. Here we report that TDFs-induced activation of the p38α kinase in lung fibroblasts plays a critical role in the formation of a pre-metastatic niche in the lungs and subsequent pulmonary metastases. Activation of p38α led to inactivation of type I interferon signaling and stimulation of expression of fibroblast activation protein (FAP). FAP played a key role in remodeling of the extracellular matrix as well as inducing the expression of chemokines that enable lung infiltration by neutrophils. Increased activity of p38 in normal cells was associated with metastatic disease and poor prognosis in human melanoma patients whereas inactivation of p38 suppressed lung metastases. We discuss the p38α-driven mechanisms stimulating the metastatic processes and potential use of p38 inhibitors in adjuvant therapy of metastatic cancers.
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Neoplasias Pulmonares , Transducción de Señal , Fibroblastos/patología , Humanos , Pulmón/patología , Neoplasias Pulmonares/patología , Proteínas QuinasasRESUMEN
Fostamatinib demonstrated efficacy in phase 3 trials of adults with immune thrombocytopenia (ITP). Post hoc analysis compared patients who received fostamatinib as second-line therapy (after steroids ± immunoglobulins) versus third-or-later-line therapy (after ≥2 prior lines of therapy including a second-line agent). Platelet responses ≥50 000/µl were observed in 25/32 (78%) second-line and 54/113 (48%) third-or-later-line patients. Bleeding events were less frequent in second-line (28%) versus third-or-later-line (45%) patients. Responses once achieved tended to be durable in both groups. The safety profile was similar in both groups. In this post hoc analysis, fostamatinib was more effective as second-line than third-or-later-line therapy for ITP.
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Oxazinas/administración & dosificación , Púrpura Trombocitopénica Idiopática/sangre , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Piridinas/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Aminopiridinas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Morfolinas , Oxazinas/efectos adversos , Recuento de Plaquetas , Piridinas/efectos adversos , PirimidinasRESUMEN
Immune thrombocytopenia (ITP) is an autoimmune disease associated with substantial heterogeneity and varying outcomes. Significant bleeding, including intracranial hemorrhage, is a persistent risk for patients with ITP, along with cardiovascular disease. ITP has also been associated with decreased patient functionality and quality of life. The primary goal of ITP therapy is to lower the risk of bleeding and associated complications by raising platelet counts to levels that provide adequate hemostasis with minimal treatment-related toxicity. Current first-line treatments include corticosteroids, as well as intravenous and anti-D immunoglobulin. Despite the availability of several second-line options, the need for additional treatment options that can provide a stable, long-term response with few adverse effects is critical and ongoing. Fostamatinib disodium hexahydrate is an oral spleen tyrosine kinase inhibitor that produces a rapid, durable response in patients who have failed one or other treatments. Additionally, fostamatinib is well tolerated, and adverse effects can be actively mitigated through dose reduction, dose interruption, or standard therapeutic approaches.
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Inhibidores Enzimáticos/inmunología , Oxazinas/inmunología , Oxazinas/uso terapéutico , Proteínas Tirosina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/inmunología , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Piridinas/inmunología , Piridinas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Humanos , Masculino , Persona de Mediana Edad , Recuento de PlaquetasRESUMEN
Fibroblast activation protein (FAP), a cell surface serine protease, is upregulated on a subset of activated fibroblasts (often distinct from α-smooth muscle actin-expressing myofibroblasts) associated with matrix remodeling, including fibroblasts in idiopathic pulmonary fibrosis (Acharya PS, Zukas A, Chandan V, Katzenstein AL, Puré E. Hum Pathol 37: 352-360, 2006.). As FAP+ fibroblasts could be pivotal in either breakdown and/or production of collagen and other matrix components, the goal of this study was to define the role of FAP+ cells in pulmonary fibrosis in two established, but different, mouse models of chronic lung fibrosis: repetitive doses of intratracheal bleomycin and a single dose of an adenoviral vector encoding constitutively active TGF-ß1 (Ad-TGFß). To determine their role in fibrotic remodeling, FAP-expressing cells were depleted by injection of T cells expressing a chimeric antigen receptor specific for murine FAP in mice with established fibrosis. The contribution of FAP to the function of FAP-expressing cells was assessed in FAP knockout mice. Using histological analyses, quantification of soluble collagen content, and flow cytometry, we found that loss of FAP+ cells exacerbated fibrosis in the bleomycin model, a phenotype largely recapitulated by the genetic deletion of FAP, indicating that FAP plays a role in this model. In contrast, depletion of FAP+ cells or genetic deletion of FAP had little effect in the Ad-TGFß model highlighting the potential for distinct mechanisms driving fibrosis depending on the initiating insult. The role of FAP in human lung fibrosis will need to be well understood to guide the use of FAP-targeted therapeutics that are being developed.
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Diferenciación Celular/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosis/inducido químicamente , Factor de Crecimiento Transformador beta/metabolismo , Animales , Bleomicina/farmacología , Colágeno/metabolismo , Fibrosis Pulmonar Idiopática/patología , Pulmón/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Cutaneous wound healing consists of three main phases: inflammation, re-epithelialization, and tissue remodeling. During normal wound healing, these processes are tightly regulated to allow restoration of skin function and biomechanics. In many instances, healing leads to an excess accumulation of fibrillar collagen (the principal protein found in the extracellular matrix - ECM), and the formation of scar tissue, which has compromised biomechanics, tested using ramp to failure tests, compared to normal skin (Corr and Hart, 2013 [1]). Alterations in collagen accumulation and architecture have been attributed to the reduced tensile strength found in scar tissue (Brenda et al., 1999; Eleswarapu et al., 2011). Defining mechanisms that govern cellular functionality and ECM remodeling are vital to understanding normal versus pathological healing and developing approaches to prevent scarring. CD44 is a cell surface adhesion receptor expressed on nearly all cell types present in dermis. Although CD44 has been implicated in an array of inflammatory and fibrotic processes such as leukocyte recruitment, T-cell extravasation, and hyaluronic acid (the principal glycosaminoglycan found in the ECM) metabolism, the role of CD44 in cutaneous wound healing and scarring remains unknown. We demonstrate that in an excisional biopsy punch wound healing model, CD44-null mice have increased inflammatory and reduced fibrogenic responses during early phases of wound healing. At wound closure, CD44-null mice exhibit reduced collagen degradation leading to increased accumulation of fibrillar collagen, which persists after wound closure leading to reduced tensile strength resulting in a more severe scarring phenotype compared to WT mice. These data indicate that CD44 plays a previously unknown role in fibrillar collagen accumulation and wound healing during the injury response.
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Matriz Extracelular/genética , Receptores de Hialuranos/genética , Inflamación/genética , Cicatrización de Heridas/genética , Animales , Movimiento Celular/genética , Colágenos Fibrilares/genética , Colágenos Fibrilares/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis/genética , Fibrosis/patología , Humanos , Inflamación/patología , Ratones , Piel/crecimiento & desarrollo , Piel/metabolismo , Resistencia a la TracciónRESUMEN
Activated fibroblasts are key players in the injury response, tumorigenesis, fibrosis, and inflammation. Dichotomous outcomes in response to varied stroma-targeted therapies in cancer emphasize the need to disentangle the roles of heterogeneous fibroblast subsets in physiological and pathophysiological settings. In wound healing, fibrosis, and myriad tumor types, fibroblast activation protein (FAP) and alpha-smooth muscle actin (αSMA) identify distinct, yet overlapping, activated fibroblast subsets. Prior studies established that FAPHi reactive fibroblasts and αSMAHi myofibroblasts can exert opposing influences in tumorigenesis. However, the factors that drive this phenotypic heterogeneity and the unique functional roles of these subsets have not been defined. We demonstrate that a convergence of ECM composition, elasticity, and transforming growth factor beta (TGF-ß) signaling governs activated fibroblast phenotypic heterogeneity. Furthermore, FAPHi reactive fibroblasts and αSMAHi myofibroblasts exhibited distinct gene expression signatures and functionality in vitro, illuminating potentially unique roles of activated fibroblast subsets in tissue remodeling. These insights into activated fibroblast heterogeneity will inform the rational design of stroma-targeted therapies for cancer and fibrosis.
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Actinas/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/citología , Gelatinasas/metabolismo , Proteínas de la Membrana/metabolismo , Miofibroblastos/citología , Serina Endopeptidasas/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Endopeptidasas , Fibroblastos/metabolismo , Ratones , Miofibroblastos/metabolismo , Fenotipo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Inhibitors of cyclooxygenase-2 alleviate pain and reduce fever and inflammation by suppressing the biosynthesis of prostacyclin (PGI2) and prostaglandin E2. However, suppression of these prostaglandins, particularly PGI2, by cyclooxygenase-2 inhibition or deletion of its I prostanoid receptor also predisposes to accelerated atherogenesis and thrombosis in mice. By contrast, deletion of microsomal prostaglandin E synthase 1 (mPGES-1) confers analgesia, attenuates atherogenesis, and fails to accelerate thrombogenesis, while suppressing prostaglandin E2, but increasing biosynthesis of PGI2. METHODS: To address the cardioprotective contribution of PGI2, we generated mice lacking the I prostanoid receptor together with mPges-1 on a hyperlipidemic background (low-density lipoprotein receptor knockouts). RESULTS: mPges-1 depletion modestly increased thrombogenesis, but this response was markedly further augmented by coincident deletion of the I prostanoid receptor (n=10-18). By contrast, deletion of the I prostanoid receptor had no effect on the attenuation of atherogenesis by mPGES-1 deletion in the low-density lipoprotein receptor knockout mice (n=17-21). CONCLUSIONS: Although suppression of prostaglandin E2 accounts for the protective effect of mPGES-1 deletion in atherosclerosis, augmentation of PGI2 is the dominant contributor to its favorable thrombogenic profile. The divergent effects on these prostaglandins suggest that inhibitors of mPGES-1 may be less likely to cause cardiovascular adverse effects than nonsteroidal anti-inflammatory drugs specific for inhibition of cyclooxygenase-2.
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Aterosclerosis/enzimología , Epoprostenol/fisiología , Hiperlipidemias/genética , Prostaglandina-E Sintasas/deficiencia , Receptores de Prostaglandina/deficiencia , Animales , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/genética , Aterosclerosis/genética , Arteria Carótida Común/efectos de la radiación , Estenosis Carotídea/etiología , Hiperlipidemias/enzimología , Rayos Láser/efectos adversos , Ratones , Ratones Noqueados , Microsomas/enzimología , Polimorfismo de Nucleótido Simple , Prostaglandina-E Sintasas/genética , Prostaglandina-E Sintasas/fisiología , Receptores de Epoprostenol , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/fisiologíaRESUMEN
Microsomal prostaglandin E synthase-1 (mPGES-1) in myeloid and vascular cells differentially regulates the response to vascular injury, reflecting distinct effects of mPGES-1-derived PGE2 in these cell types on discrete cellular components of the vasculature. The cell selective roles of mPGES-1 in atherogenesis are unknown. Mice lacking mPGES-1 conditionally in myeloid cells (Mac-mPGES-1-KOs), vascular smooth muscle cells (VSMC-mPGES-1-KOs), or endothelial cells (EC-mPGES-1-KOs) were crossed into hyperlipidemic low-density lipoprotein receptor-deficient animals. En face aortic lesion analysis revealed markedly reduced atherogenesis in Mac-mPGES-1-KOs, which was concomitant with a reduction in oxidative stress, reflective of reduced macrophage infiltration, less lesional expression of inducible nitric oxide synthase (iNOS), and lower aortic expression of NADPH oxidases and proinflammatory cytokines. Reduced oxidative stress was reflected systemically by a decline in urinary 8,12-iso-iPF2α-VI. In contrast to exaggeration of the response to vascular injury, deletion of mPGES-1 in VSMCs, ECs, or both had no detectable phenotypic impact on atherogenesis. Macrophage foam cell formation and cholesterol efflux, together with plasma cholesterol and triglycerides, were unchanged as a function of genotype. In conclusion, myeloid cell mPGES-1 promotes atherogenesis in hyperlipidemic mice, coincident with iNOS-mediated oxidative stress. By contrast, mPGES-1 in vascular cells does not detectably influence atherogenesis in mice. This strengthens the therapeutic rationale for targeting macrophage mPGES-1 in inflammatory cardiovascular diseases.
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Aterosclerosis/enzimología , Aterosclerosis/etiología , Oxidorreductasas Intramoleculares/metabolismo , Células Mieloides/enzimología , Animales , Aterosclerosis/prevención & control , Movimiento Celular/fisiología , Células Endoteliales/enzimología , Femenino , Hiperlipidemias/enzimología , Oxidorreductasas Intramoleculares/deficiencia , Oxidorreductasas Intramoleculares/genética , Metabolismo de los Lípidos , Macrófagos/fisiología , Masculino , Ratones , Ratones Noqueados , Microsomas/enzimología , Miocitos del Músculo Liso/enzimología , Estrés Oxidativo , Prostaglandina-E Sintasas , Receptores de LDL/deficiencia , Receptores de LDL/genéticaRESUMEN
BACKGROUND: Placebo-controlled trials of nonsteroidal anti-inflammatory drugs selective for inhibition of cyclooxygenase-2 (COX-2) reveal an emergent cardiovascular hazard in patients selected for low risk of heart disease. Postnatal global deletion of COX-2 accelerates atherogenesis in hyperlipidemic mice, a process delayed by selective enzyme deletion in macrophages. METHODS AND RESULTS: In the present study, selective depletion of COX-2 in vascular smooth muscle cells and endothelial cells depressed biosynthesis of prostaglandin I2 and prostaglandin E2, elevated blood pressure, and accelerated atherogenesis in Ldlr knockout mice. Deletion of COX-2 in vascular smooth muscle cells and endothelial cells coincided with an increase in COX-2 expression in lesional macrophages and increased biosynthesis of thromboxane. Increased accumulation of less organized intimal collagen, laminin, α-smooth muscle actin, and matrix-rich fibrosis was also apparent in lesions of the mutants. CONCLUSIONS: Although atherogenesis is accelerated in global COX-2 knockouts, consistent with evidence of risk transformation during chronic nonsteroidal anti-inflammatory drug administration, this masks the contrasting effects of enzyme depletion in macrophages versus vascular smooth muscle cells and endothelial cells. Targeting delivery of COX-2 inhibitors to macrophages may conserve their efficacy while limiting cardiovascular risk.
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Aterosclerosis/metabolismo , Ciclooxigenasa 2/genética , Endotelio Vascular/enzimología , Hiperlipidemias/metabolismo , Músculo Liso Vascular/enzimología , Animales , Aorta Torácica/enzimología , Aorta Torácica/patología , Aterosclerosis/epidemiología , Aterosclerosis/patología , Presión Sanguínea/fisiología , Ciclooxigenasa 2/metabolismo , Dieta Aterogénica , Grasas de la Dieta/farmacología , Dinoprostona/biosíntesis , Endotelio Vascular/patología , Epoprostenol/biosíntesis , Femenino , Hiperlipidemias/epidemiología , Hiperlipidemias/patología , Macrófagos/enzimología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , Receptores de LDL/genética , Factores de RiesgoRESUMEN
There are two key processes underlying ligand-induced receptor endocytosis: receptor ubiquitylation and remodeling of the actin cytoskeleton. Tyrosine kinases play critical roles in both receptor endocytosis and actin reorganization. Interestingly, members of the Abl family are the only known tyrosine kinases that possess an actin-binding domain and thus have the potential to directly regulate the actin cytoskeleton. However, the role of non-transforming cAbl in receptor endocytosis remains undefined. We report that cAbl promotes ligand-induced antigen receptor endocytosis in B lymphocytes. We show that pharmacologic inhibition or genetic deletion of cAbl causes a defect in tyrosine phosphorylation of the cytoskeletal adapter CrkII. cAbl inhibition or ablation also impairs Rac activation downstream of CrkII, as well as antigen receptor capping and endocytosis. Although phosphorylation of CrkII has been suggested to maintain it in a closed inactive conformation, we demonstrate that it is in fact essential for the activation of Rac. On the other hand, association of CrkII with cCbl, a key mediator of receptor ubiquitylation, does not require CrkII phosphorylation and is cAbl-independent. Phosphorylation of cCbl itself is also cAbl-independent. Our results thus indicate that CrkII links receptor engagement to cytoskeletal remodeling by coupling cCbl- and cAbl-mediated signaling pathways that cooperatively regulate ligand-induced receptor endocytosis.
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Linfocitos B/inmunología , Endocitosis , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Proto-Oncogénicas c-crk/metabolismo , Actinas/metabolismo , Animales , Linfocitos B/metabolismo , Benzamidas , Pollos , Citoesqueleto , Mesilato de Imatinib , Piperazinas/farmacología , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-abl/deficiencia , Proteínas Proto-Oncogénicas c-abl/inmunología , Proteínas Proto-Oncogénicas c-cbl/inmunología , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Proto-Oncogénicas c-crk/inmunología , Pirimidinas/farmacología , Proteínas de Unión al GTP rac/inmunología , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Receptor endocytosis down-regulates ligand-induced signaling in a timely manner and depends on cytoskeletal remodeling. In B lymphocytes, internalization of B cell receptors (BCRs) is also critical to antigen presentation. However, the mechanisms underlying BCR endocytosis are not fully understood. Similarly, the molecular mechanisms linking endocytosis to cytoskeletal remodeling remain poorly defined. We used flow cytometry, pull-down assays, immunochemistry and fluorescence microscopy to investigate BCR internalization in the DT40 B cell line. We demonstrate that ablation of Cbl impacts BCR endocytosis and the underlying cytoskeletal dynamics. Specifically, we demonstrate that ligand-induced endocytosis is impaired in Cbl-/- cells and that the ubiquitin ligase activity is required for Cbl to promote endocytosis. We also show that phosphorylation of CrkII, activation of Rac downstream of CrkII and BCR capping require Cbl. Furthermore, we show that the association of Cbl and CrkII requires phosphorylation of Cbl, but not its ubiquitin ligase activity. Our data indicate that Cbl promotes BCR endocytosis and attenuates ligand-induced signaling by virtue of its ability to orchestrate receptor ubiquitylation and cytoskeletal dynamics.