RESUMEN
AIMS: Circulating levels of sphingosine 1-phosphate (S1P), an HDL-associated ligand for endothelial cell (EC) protective S1P receptor-1 (S1PR1), are reduced in disease states associated with endothelial dysfunction. Yet as S1PR1 has high affinity for S1P and can be activated by ligand-independent mechanisms and EC-autonomous S1P production, it is unclear if relative reductions in circulating S1P impact endothelial function. It is also unclear how EC S1PR1 insufficiency, whether induced by ligand deficiency or by S1PR1-directed immunosuppressive therapy, affects different vascular subsets. METHODS AND RESULTS: We here fine-map the zonation of S1PR1 signalling in the murine blood and lymphatic vasculature, superimpose cell type-specific and relative deficiencies in S1P production to define ligand source- and dose-dependence, and correlate receptor engagement to essential functions. In naïve blood vessels, despite broad expression, EC S1PR1 engagement was restricted to resistance-size arteries, lung capillaries and high-endothelial venules (HEV). Similar zonation was observed for albumin extravasation in EC S1PR1 deficient mice, and brain extravasation was reproduced with arterial EC-selective S1pr1 deletion. In lymphatic EC, S1PR1 engagement was high in collecting vessels and lymph nodes and low in terminal capillaries that drain tissue fluids. While EC S1P production sustained S1PR1 signaling in lymphatics and HEV, hematopoietic cells provided â¼90% of plasma S1P and sustained signaling in resistance arteries and lung capillaries. S1PR1 signaling and endothelial function were both surprisingly sensitive to reductions in plasma S1P with apparent saturation around 50% of normal levels. S1PR1 engagement did not depend on sex or age, but modestly increased in arteries in hypertension and diabetes. Sphingosine kinase (Sphk)-2 deficiency also increased S1PR1 engagement selectively in arteries, which could be attributed to Sphk1-dependent S1P release from perivascular macrophages. CONCLUSIONS: This study highlights vessel subtype-specific S1PR1 functions and mechanisms of engagement and supports the relevance of S1P as circulating biomarker for endothelial function.
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Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.
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Células Endoteliales , Infarto del Miocardio , Humanos , Autofagia , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Arterias Mesentéricas/metabolismo , Infarto del Miocardio/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Transducción de Señal , Vasodilatación , Animales , RatonesRESUMEN
Sphingosine-1-phosphate (S1P) is a potent lipid mediator that is secreted by several cell types. We recently showed that Mfsd2b is an S1P transporter from hematopoietic cells that contributes approximately 50% plasma S1P. Here we report the characterization of compound deletion of Mfsd2b and Spns2, another S1P transporter active primarily in endothelial cells. Global deletion of Mfsd2b and Spns2 (global double knockout [gDKO]) results in embryonic lethality beyond embryonic day 14.5 (E14.5), with severe hemorrhage accompanied by defects of tight junction proteins, indicating that Mfsd2b and Spns2 provide S1P for signaling, which is essential for blood vessel integrity. Compound postnatal deletion of Mfsd2b and Spns2 using Mx1Cre (ctDKO-Mx1Cre) results in maximal 80% reduction of plasma S1P. ctDKO-Mx1Cre mice exhibit severe susceptibility to anaphylaxis, indicating that S1P from Mfsd2b and Spns2 is indispensable for vascular homeostasis. Our results show that S1P export from Mfsd2b and Spns2 is essential for developing and mature vasculature.
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Anafilaxia , Proteínas de la Membrana/metabolismo , Anafilaxia/metabolismo , Animales , Proteínas de Transporte de Anión/metabolismo , Transporte Biológico , Células Endoteliales/metabolismo , Homeostasis , Lisofosfolípidos/metabolismo , Ratones , Esfingosina/metabolismoRESUMEN
Background: Innate immune responses to influenza A virus (IAV) infection are initiated in part by toll-like receptor 3 (TLR3). TLR3-dependent signaling induces an antiviral immune response and an NFκB-dependent inflammatory response. Protease-activated receptor 2 (PAR2) inhibits the antiviral response and enhances the inflammatory response. PAR2 deficiency protected mice during IAV infection. However, the PAR2 expressing cell-types contributing to IAV pathology in mice and the mechanism by which PAR2 contributes to IAV infection is unknown. Methods: IAV infection was analyzed in global (Par2-/- ), myeloid (Par2fl/fl;LysMCre+) and lung epithelial cell (EpC) Par2 deficient (Par2fl/fl ;SPCCre+) mice and their respective controls (Par2+/+ and Par2fl/fl). In addition, the effect of PAR2 activation on polyinosinic-polycytidylic acid (poly I:C) activation of TLR3 was analyzed in bone marrow-derived macrophages (BMDM). Lastly, we determined the effect of PAR2 inhibition in wild-type (WT) mice. Results: After IAV infection, Par2-/- and mice with myeloid Par2 deficiency exhibited increased survival compared to infected controls. The improved survival was associated with reduced proinflammatory mediators and reduced cellular infiltration in bronchoalveolar lavage fluid (BALF) of Par2-/- and Par2fl/fl;LysMCre+ 3 days post infection (dpi) compared to infected control mice. Interestingly, Par2fl/fl;SPCCre+ mice showed no survival benefit compared to Par2fl/fl . In vitro studies showed that Par2-/- BMDM produced less IL6 and IL12p40 than Par2+/+ BMDM after poly I:C stimulation. In addition, activation of PAR2 on Par2+/+ BMDM increased poly I:C induction of IL6 and IL12p40 compared to poly I:C stimulation alone. Importantly, PAR2 inhibition prior to IAV infection protect WT mice. Conclusion: Global Par2 or myeloid cell but not lung EpC Par2 deficiency was associated with reduced BALF inflammatory markers and reduced IAV-induced mortality. Our study suggests that PAR2 may be a therapeutic target to reduce IAV pathology.
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Virus de la Influenza A , Infecciones por Orthomyxoviridae/mortalidad , Receptor PAR-2/fisiología , Animales , Citocinas/análisis , Citocinas/biosíntesis , Femenino , Interferón beta/biosíntesis , Pulmón/patología , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Células Mieloides/fisiología , Neutrófilos/inmunología , Infecciones por Orthomyxoviridae/inmunología , Receptor PAR-2/deficienciaRESUMEN
Aims: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis. Methods: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times. Results: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα. Conclusions: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology.
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Granzimas/antagonistas & inhibidores , Peritonitis/tratamiento farmacológico , Peritonitis/enzimología , Sepsis/tratamiento farmacológico , Sepsis/enzimología , Anciano , Anciano de 80 o más Años , Animales , Citocinas/sangre , Modelos Animales de Enfermedad , Femenino , Granzimas/sangre , Granzimas/deficiencia , Granzimas/genética , Humanos , Mediadores de Inflamación/sangre , Interleucina-6/biosíntesis , Células Asesinas Naturales/enzimología , Macrófagos/enzimología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Terapia Molecular Dirigida , Peritonitis/etiología , Medicina de Precisión , Sepsis/etiología , Serpinas/farmacología , Receptor Toll-Like 4/metabolismoRESUMEN
RATIONALE: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. OBJECTIVE: To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. METHODS AND RESULTS: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. CONCLUSIONS: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.
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Barrera Hematoencefálica/metabolismo , Arterias Cerebrales/metabolismo , Células Endoteliales/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Ataque Isquémico Transitorio/metabolismo , Accidente Cerebrovascular Isquémico/metabolismo , Lisofosfolípidos/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Esfingosina/análogos & derivados , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/fisiopatología , Arterias Cerebrales/efectos de los fármacos , Arterias Cerebrales/patología , Arterias Cerebrales/fisiopatología , Circulación Cerebrovascular , Modelos Animales de Enfermedad , Células Endoteliales/patología , Femenino , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Infarto de la Arteria Cerebral Media/prevención & control , Ataque Isquémico Transitorio/patología , Ataque Isquémico Transitorio/fisiopatología , Ataque Isquémico Transitorio/prevención & control , Accidente Cerebrovascular Isquémico/patología , Accidente Cerebrovascular Isquémico/fisiopatología , Accidente Cerebrovascular Isquémico/prevención & control , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microcirculación , Fármacos Neuroprotectores/farmacología , Transducción de Señal , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/agonistas , Receptores de Esfingosina-1-Fosfato/genética , Grado de Desobstrucción VascularRESUMEN
Background: Activation of protease-activated receptor-2 (PAR2) has been implicated in inflammation, pruritus, and skin barrier regulation, all characteristics of atopic dermatitis (AD), as well as Netherton syndrome which has similar characteristics. However, understanding the precise role of PAR2 on neuro-immune communication in AD has been hampered by the lack of appropriate animal models. Methods: We used a recently established mouse model with epidermal overexpression of PAR2 (PAR2OE) and littermate WT mice to study the impact of increased PAR2 expression in epidermal cells on spontaneous and house dust mite (HDM)-induced skin inflammation, itch, and barrier dysfunction in AD, in vivo and ex vivo. Results: PAR2OE newborns displayed no overt abnormalities, but spontaneously developed dry skin, severe pruritus, and eczema. Dermatological, neurophysiological, and immunological analyses revealed the hallmarks of AD-like skin disease. Skin barrier defects were observed before onset of skin lesions. Application of HDM onto PAR2OE mice triggered pruritus and the skin phenotype. PAR2OE mice displayed an increased density of nerve fibers, increased nerve growth factor and endothelin-1 expression levels, alloknesis, enhanced scratching (hyperknesis), and responses of dorsal root ganglion cells to non-histaminergic pruritogens. Conclusion: PAR2 in keratinocytes, activated by exogenous and endogenous proteases, is sufficient to drive barrier dysfunction, inflammation, and pruritus and sensitize skin to the effects of HDM in a mouse model that mimics human AD. PAR2 signaling in keratinocytes appears to be sufficient to drive several levels of neuro-epidermal communication, another feature of human AD.
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Dermatitis Atópica/metabolismo , Epidermis/inervación , Ganglios Espinales/metabolismo , Queratinocitos/metabolismo , Prurito/metabolismo , Receptor PAR-2/metabolismo , Animales , Animales Modificados Genéticamente , Señalización del Calcio , Dermatitis Atópica/genética , Dermatitis Atópica/inmunología , Modelos Animales de Enfermedad , Endotelina-1/metabolismo , Queratinocitos/inmunología , Factor de Crecimiento Nervioso/metabolismo , Prurito/genética , Prurito/inmunología , Pyroglyphidae/inmunología , Receptor PAR-2/genéticaRESUMEN
If not properly regulated, the inflammatory immune response can promote carcinogenesis, as evident in colorectal cancer (CRC). Aiming to gain mechanistic insight into the link between inflammation and CRC, we perform transcriptomics analysis of human CRC, identifying a strong correlation between expression of the serine protease granzyme A (GzmA) and inflammation. In a dextran sodium sulfate and azoxymethane (DSS/AOM) mouse model, deficiency and pharmacological inhibition of extracellular GzmA both attenuate gut inflammation and prevent CRC development, including the initial steps of cell transformation and epithelial-to-mesenchymal transition. Mechanistically, extracellular GzmA induces NF-κB-dependent IL-6 production in macrophages, which in turn promotes STAT3 activation in cultured CRC cells. Accordingly, colon tissues from DSS/AOM-treated, GzmA-deficient animals present reduced levels of pSTAT3. By identifying GzmA as a proinflammatory protease that promotes CRC development, these findings provide information on mechanisms that link immune cell infiltration to cancer progression and present GzmA as a therapeutic target for CRC.
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Carcinogénesis/patología , Colon/patología , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Espacio Extracelular/enzimología , Granzimas/metabolismo , Inflamación/patología , Enfermedad Aguda , Animales , Azoximetano , Carcinogénesis/genética , Enfermedad Crónica , Neoplasias Colorrectales/genética , Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Sulfato de Dextran , Progresión de la Enfermedad , Granzimas/antagonistas & inhibidores , Granzimas/genética , Humanos , Inflamasomas/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-6/biosíntesis , Ratones Noqueados , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Despite the medical importance of G protein-coupled receptors (GPCRs), in vivo cellular heterogeneity of GPCR signaling and downstream transcriptional responses are not understood. We report the comprehensive characterization of transcriptomes (bulk and single-cell) and chromatin domains regulated by sphingosine 1-phosphate receptor-1 (S1PR1) in adult mouse aortic endothelial cells. First, S1PR1 regulates NFκB and nuclear glucocorticoid receptor pathways to suppress inflammation-related mRNAs. Second, S1PR1 signaling in the heterogenous endothelial cell (EC) subtypes occurs at spatially-distinct areas of the aorta. For example, a transcriptomically distinct arterial EC population at vascular branch points (aEC1) exhibits ligand-independent S1PR1/ß-arrestin coupling. In contrast, circulatory S1P-dependent S1PR1/ß-arrestin coupling was observed in non-branch point aEC2 cells that exhibit an inflammatory gene expression signature. Moreover, S1P/S1PR1 signaling regulates the expression of lymphangiogenic and inflammation-related transcripts in an adventitial lymphatic EC (LEC) population in a ligand-dependent manner. These insights add resolution to existing concepts of endothelial heterogeneity, GPCR signaling and S1P biology.
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Aorta/metabolismo , Endotelio Linfático/metabolismo , Endotelio Vascular/metabolismo , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Transcriptoma , Animales , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Transgénicos , Análisis de Secuencia de ARN/métodos , Transducción de Señal , Análisis de la Célula Individual/métodos , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , beta-Arrestinas/metabolismoRESUMEN
Factor VII activating protease (FSAP) is a circulating serine protease implicated in thrombosis, atherosclerosis, stroke, and cancer. Using an overexpression strategy, we have systematically investigated the role of protease activated receptors (PAR)-1, -2, -3, and -4 on FSAP-mediated signaling in HEK293T and A549 cells. Cleavage of PAR-reporter constructs and MAPK phosphorylation was used to monitor receptor activation. FSAP cleaved PAR-2 and to a lesser degree PAR-1, but not PAR-3 or PAR-4 in both cell types. Robust MAPK activation in response to FSAP was observed after PAR-2, but not PAR-1 overexpression in HEK293T. Recombinant serine protease domain of wild type FSAP, but not the Marburg I isoform of FSAP, could reproduce the effects of plasma purified FSAP. Canonical cleavage of both PARs was suggested by mass spectrometric analysis of synthetic peptide substrates from the N-terminus of PARs and site directed mutagenesis studies. Surprisingly, knockdown of endogenous PAR-1, but not PAR-2, prevented the apoptosis-inhibitory effect of FSAP, suggesting that PAR1 is nevertheless a direct or indirect target in some cell types. This molecular characterization of PAR-1 and -2 as cellular receptors of FSAP will help to define the actions of FSAP in the context of cancer and vascular biology.
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Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Serina Endopeptidasas/metabolismo , Apoptosis , Línea Celular Tumoral , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas , Mutagénesis Sitio-Dirigida , Péptidos/química , Fosforilación , Isoformas de Proteínas , Transducción de Señal , TrombosisRESUMEN
Hepatocyte growth factor activator inhibitor-1 (HAI-1), encoded by the SPINT1 gene, is a membrane-bound protease inhibitor expressed on the surface of epithelial cells. Hepatocyte growth factor activator inhibitor-1 regulates type II transmembrane serine proteases that activate protease-activated receptor-2 (PAR-2). We previously reported that deletion of Spint1 in ApcMin/+ mice resulted in accelerated formation of intestinal tumors, possibly through enhanced nuclear factor-κB signaling. In this study, we examined the role of PAR-2 in accelerating tumor formation in the ApcMin/+ model in the presence or absence of Spint1. We observed that knockout of the F2rl1 gene, encoding PAR-2, not only eliminated the enhanced formation of intestinal tumors caused by Spint1 deletion, but also reduced tumor formation in the presence of Spint1. Exacerbation of anemia and weight loss associated with HAI-1 deficiency was also normalized by compound deficiency of PAR-2. Mechanistically, signaling triggered by deregulated protease activities increased nuclear translocation of RelA/p65, vascular endothelial growth factor expression, and vascular density in ApcMin/+ -induced intestinal tumors. These results suggest that serine proteases promote intestinal carcinogenesis through activation of PAR-2, and that HAI-1 plays a critical tumor suppressor role as an inhibitor of matriptase, kallikreins, and other PAR-2 activating proteases.
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Proteína de la Poliposis Adenomatosa del Colon/genética , Neoplasias Intestinales/genética , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Receptor PAR-2/genética , Animales , Carcinogénesis/genética , Modelos Animales de Enfermedad , Células Epiteliales/patología , Humanos , Neoplasias Intestinales/patología , Calicreínas/genética , Ratones , FN-kappa B/genética , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Transducción de Señal/genética , Factor de Transcripción ReIA/genéticaRESUMEN
BACKGROUND: Atopic dermatitis (AD) is a complex skin disease involving causative effects from both intrinsic and extrinsic sources. Murine models of the disease often fall short in one of these components and, as a result, do not fully encapsulate these disease mechanisms. OBJECTIVE: We aimed to determine whether the protease-activated receptor 2 over-expressor mouse (PAR2OE) with topical house dust mite (HDM) application is a more comprehensive and clinically representative AD model. METHODS: Following HDM extract application to PAR2OE mice and controls, AD clinical scoring, itching behaviour, skin morphology and structure, barrier function, immune cell infiltration and inflammatory markers were assessed. Skin morphology was analysed using haematoxylin and eosin staining, and barrier function was assessed by transepidermal water loss measurements. Immune infiltrate was characterised by histological and immunofluorescence staining. Finally, an assessment of AD-related gene expression was performed using quantitative RT-PCR. RESULTS: PAR2OE mice treated with HDM displays all the characteristic clinical symptoms including erythema, dryness and oedema, skin morphology, itch and inflammation typically seen in patients with AD. There is a significant influx of mast cells (P < .01) and eosinophils (P < .0001) into the dermis of these mice. Furthermore, the PAR2OE + HDM mice exhibit similar expression patterns of key differentially expressed genes as seen in human AD. CONCLUSION: The PAR2OE + HDM mouse presents with a classic AD pathophysiology and is a valuable model in terms of reproducibility and overall disease representation to study the condition and potential therapeutic approaches.
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Dermatitis Atópica/etiología , Modelos Animales de Enfermedad , Pyroglyphidae/inmunología , Receptor PAR-2/fisiología , Animales , Dermatitis Atópica/patología , Piel/inmunología , Piel/patologíaRESUMEN
The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how parietal epithelial cells (PECs) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PECs in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PECs prevents glomerular damage in CGN and FSGS mouse models. Mechanistically, CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and ß1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions.
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Enfermedades Renales/patología , Tetraspanina 29/fisiología , Animales , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Femenino , Glomerulonefritis/genética , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Enfermedades Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Tetraspanina 29/genética , Tetraspanina 29/metabolismoRESUMEN
The bioactive lipid mediator sphingosine 1-phosphate (S1P) was recently assigned critical roles in platelet biology: whereas S1P1 receptor-mediated S1P gradient sensing was reported to be essential for directing proplatelet extensions from megakaryocytes (MKs) toward bone marrow sinusoids, MK sphingosine kinase 2 (Sphk2)-derived S1P was reported to further promote platelet shedding through receptor-independent intracellular actions, and platelet aggregation through S1P1 Yet clinical use of S1P pathway modulators including fingolimod has not been associated with risk of bleeding or thrombosis. We therefore revisited the role of S1P in platelet biology in mice. Surprisingly, no reduction in platelet counts was observed when the vascular S1P gradient was ablated by impairing S1P provision to plasma or S1P degradation in interstitial fluids, nor when gradient sensing was impaired by S1pr1 deletion selectively in MKs. Moreover, S1P1 expression and signaling were both undetectable in mature MKs in situ, and MK S1pr1 deletion did not affect platelet aggregation or spreading. When S1pr1 deletion was induced in hematopoietic progenitor cells, platelet counts were instead significantly elevated. Isolated global Sphk2 deficiency was associated with thrombocytopenia, but this was not replicated by MK-restricted Sphk2 deletion and was reversed by compound deletion of either Sphk1 or S1pr2, suggesting that this phenotype arises from increased S1P export and S1P2 activation secondary to redistribution of sphingosine to Sphk1. Consistent with clinical observations, we thus observe no essential role for S1P1 in facilitating platelet production or activation. Instead, S1P restricts megakaryopoiesis through S1P1, and can further suppress thrombopoiesis through S1P2 when aberrantly secreted in the hematopoietic niche.
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Plaquetas/metabolismo , Lisofosfolípidos/metabolismo , Megacariocitos/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Nicho de Células Madre , Trombopoyesis , Animales , Plaquetas/citología , Lisofosfolípidos/genética , Megacariocitos/citología , Ratones , Ratones Noqueados , Esfingosina/genética , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMEN
Impaired activated protein C (aPC) generation is associated with atherosclerosis and diabetes mellitus. Diabetes-associated atherosclerosis is characterized by the hyperglycaemic memory, e.g., failure of disease improvement despite attenuation of hyperglycaemia. Therapies reversing the hyperglycaemic memory are lacking. Here we demonstrate that hyperglycaemia, but not hyperlipidaemia, induces the redox-regulator p66Shc and reactive oxygen species (ROS) in macrophages. p66Shc expression, ROS generation, and a pro-atherogenic phenotype are sustained despite restoring normoglycemic conditions. Inhibition of p66Shc abolishes this sustained pro-atherogenic phenotype, identifying p66Shc-dependent ROS in macrophages as a key mechanism conveying the hyperglycaemic memory. The p66Shc-associated hyperglycaemic memory can be reversed by aPC via protease-activated receptor-1 signalling. aPC reverses glucose-induced CpG hypomethylation within the p66Shc promoter by induction of the DNA methyltransferase-1 (DNMT1). Thus, epigenetically sustained p66Shc expression in plaque macrophages drives the hyperglycaemic memory, which-however-can be reversed by aPC. This establishes that reversal of the hyperglycaemic memory in diabetic atherosclerosis is feasible.
RESUMEN
Coagulation and fibrinolytic system deregulation has been implicated in the development of idiopathic pulmonary fibrosis, a devastating form of interstitial lung disease. We used intratracheal instillation of bleomycin to induce pulmonary fibrosis in mice and analyzed the role of serine protease inhibitor E2 (serpinE2)/protease nexin-1 (PN-1), a tissue serpin that exhibits anticoagulant and antifibrinolytic properties. PN-1 deficiency was associated, after bleomycin challenge, with a significant increase in mortality, as well as a marked increase in active thrombin in bronchoalveolar lavage fluids, an overexpression of extracellular matrix proteins, and an accumulation of inflammatory cells in the lungs. Bone marrow transplantation experiments showed that protective PN-1 was derived from hematopoietic cell compartment. A pharmacological strategy using the direct thrombin inhibitor argatroban reversed the deleterious effects of PN-1 deficiency. Concomitant deficiency of the thrombin receptor protease-activated receptor 4 (PAR4) abolished the deleterious effects of PN-1 deficiency in hematopoietic cells. These data demonstrate that prevention of thrombin signaling by PN-1 constitutes an important endogenous mechanism of protection against lung fibrosis and associated mortality. Our findings suggest that appropriate doses of thrombin inhibitors or PAR4 antagonists may provide benefit against progressive lung fibrosis with evidence of deregulated thrombin activity.