RESUMO
At labor, the myometrium is infiltrated by a massive influx of macrophages that secrete high levels of pro-inflammatory cytokines inducing the expression of specific labor-associated markers. However, the interactions between myocytes and macrophages and the role of macrophages in the myometrium at labor remain to be elucidated. In this work, we studied the role of myometrium-infiltrated macrophages and their interaction with myocytes in lipopolysaccharide-induced preterm labor. A co-culture model of human primary myometrial cells and macrophages was developed and validated. Collagen lattices were used to evaluate myocyte contraction. Differentiation steps were assessed by (i) phalloidin and vinculin staining for cytoskeleton reorganization, (ii) gap junction protein alpha 1 expression and scrape loading/dye transfer with Lucifer Yellow for gap junction intercellular communication, and (iii) calcium imaging for cell excitability. We demonstrated that macrophages favored lipopolysaccharide-induced contraction and early differentiation of myometrial cells. Transwell assays showed that previous activation of macrophages by lipopolysaccharide was essential for this differentiation and that macrophage/myocyte interactions involved macrophage release of reactive oxygen species (ROS). The effects of macrophage-released ROS in myometrial cell transactivation were mimicked by H2O2, suggesting that superoxide anion is a major intermediate messenger in macrophage/myocyte crosstalk during labor. These novel findings provide the foundation for innovative approaches to managing preterm labor, specifically the use of antioxidants to inhibit the initial stages of labor before the contractile phenotype has been acquired. In addition, the co-culture model developed by our team could be used in future research to decipher pathophysiological signaling pathways or screen/develop new tocolytics.
Assuntos
Macrófagos/fisiologia , Miométrio/citologia , Parto/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Contração Uterina/fisiologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Lipopolissacarídeos/farmacologia , Contração Uterina/efeitos dos fármacosRESUMO
Thoracic aortic aneurysm (TAA) is a complex life-threatening disease characterized by extensive extracellular matrix (ECM) fragmentation and persistent inflammation, culminating in a weakened aorta. Although evidence suggests defective canonical signaling pathways in TAA, the full spectrum of mechanisms contributing to TAA is poorly understood, therefore limiting the scope of drug-based treatment. Here, we used a sensitive RNA sequencing approach to profile the transcriptomic atlas of human TAA. Pathway analysis revealed upregulation of key matrix-degrading enzymes and inflammation coincident with the axonal guidance pathway. We uncovered their novel association with TAA and focused on the expression of Semaphorins and Netrins. Comprehensive analysis of this pathway showed that several members were differentially expressed in TAA compared to controls. Immunohistochemistry revealed that Semaphorin4D and its receptor PlexinB1, similar to Netrin-1 proteins were highly expressed in damaged areas of TAA tissues but faintly detected in the vessel wall of non-diseased sections. It should be considered that the current study is limited by its sample size and the use of internal thoracic artery as control for TAA for the sequencing dataset. Our data determines important neuronal regulators of vascular inflammatory events and suggest Netrins and Semaphorins as potential key contributors of ECM degradation in TAA.
Assuntos
Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/patologia , Netrinas/metabolismo , Semaforinas/metabolismo , Aneurisma da Aorta Torácica/genética , Matriz Extracelular/metabolismo , Humanos , Netrinas/genética , Semaforinas/genética , Análise de Sequência de RNA , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Remodelação VascularRESUMO
The beta3 adrenergic receptor (ß3-AR) stimulation plays a protective role against preterm labor by blocking myometrial contraction, cytokine production, remodeling and apoptosis. We previously demonstrated that macrophage-induced ROS production in the myometrium was a key element leading to the induction of all these labor-associated features. We thus aimed to investigate if the ß3-AR could be expressed in human macrophages and could trigger its protective role in the myometrium by directly inhibiting ROS production. Using lipopolysaccharide (LPS)-stimulated myometrial samples and cell co-culture experiments, we demonstrated that ß3-AR stimulation inhibits the activation of the NADPH oxidase, leading to the subsequent inhibition of ROS production by macrophages. This antioxidant effect was associated with a potent anti-inflammatory response in macrophages. Furthermore, we observed that ß3-AR leads to the expression of catalase not only in macrophages but also in myometrial cells, thereby preventing the transactivation of myometrial cells by hydrogen peroxide. Pharmacological experiments allowed us to demonstrate that these effects were driven by an Erk1/2-mediated activation of the antioxidant transcription factor PPARγ. These results suggest that ß3-AR protective effects in the myometrium could be due to its dual antioxidant properties. Further, the effects observed in a macrophage could highlight new applications in chronic inflammatory diseases.
Assuntos
Apoptose/genética , Macrófagos/metabolismo , PPAR gama/genética , Receptores Adrenérgicos beta 3/genética , Antioxidantes/administração & dosagem , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Catalase/metabolismo , Técnicas de Cocultura , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Miométrio/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , PPAR gama/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Adrenérgicos beta 3/administração & dosagem , Transdução de Sinais/efeitos dos fármacosRESUMO
Preterm birth is an inflammatory process resulting from the massive infiltration of innate immune cells and the production of proinflammatory cytokines in the myometrium. However, proinflammatory cytokines, which induce labor in vivo, fail to induce labor-associated features in human myometrial cells (MCs). We thus aimed to investigate if reactive oxygen species (ROS) production could be the missing step between immune cell activation and MC response. Indeed, we found that ROS production is increased in the human preterm laboring myometrium (27% ROS producing cells, respectively, versus 2% in nonlaboring controls), with 90% ROS production in macrophages. Using LPS-stimulated myometrial samples and cell coculture experiments, we demonstrated that ROS production is required for labor onset. Furthermore, we showed that ROS are required first in the NADPH oxidase (NADPHox)-2/NF-κB-dependent macrophage response to inflammatory stimuli but, more importantly, to trigger macrophage-induced MCs transactivation. Remarkably, in a murine model of LPS-induced preterm labor (inducing delivery within 17 hours, with no pup survival), cotreatment with glutathione delayed labor onset up to 94 hours and prevented in utero fetal distress, allowing 46% pups to survive. These results suggest that targeting ROS production with the macrophage-permeable antioxidant glutathione could constitute a promising strategy to prevent preterm birth.
Assuntos
Morte Fetal/prevenção & controle , Glutationa/farmacologia , Macrófagos/metabolismo , Miométrio/efeitos dos fármacos , Trabalho de Parto Prematuro/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Adulto , Animais , Animais Recém-Nascidos , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Expressão Gênica , Glutationa/administração & dosagem , Humanos , Recém-Nascido , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Miométrio/citologia , Miométrio/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Trabalho de Parto Prematuro/induzido quimicamente , Trabalho de Parto Prematuro/metabolismo , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Adulto JovemRESUMO
The beta3 adrenergic receptor (B3-AR) reportedly induces cell proliferation, but the signaling pathways that were proposed, involving either Gs or Gi coupling, remain controversial. To further investigate the role of G protein coupling in B3-AR induced proliferation, we stimulated primary human myometrial smooth muscle cells with SAR150640 (B3-AR agonist) in the absence or presence of variable G-protein inhibitors. Specific B3-AR stimulation led to an Erk1/2 induced proliferation. We observed that the proliferative effects of B3-AR require two Erk1/2 activation peaks (the first after 3min, the second at 8h). Erk1/2 activation at 3min was mimicked by forskolin (adenylyl-cyclase activator), and was resistant to pertussis toxin (Gi inhibitor), suggesting a Gs protein signaling. This first signaling also required the downstream Gs signaling effectors PKA and Src. However, Erk1/2 activation at 8h turned out to be pertussis toxin-dependent, and PKA-independent, indicating a Gi signaling pathway in which Src and PI3K were required. The pharmacological inhibition of both the Gs and Gi pathway abolished B3-AR-induced proliferation. Altogether, these data indicate that B3-AR-induced proliferation depends on the biphasic activation of Erk1/2 sequentially induced by the Gs/PKA/Src and Gi/Src/PI3K signaling pathways.
Assuntos
Proliferação de Células/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Miócitos de Músculo Liso , Receptores Adrenérgicos beta/metabolismo , Células Cultivadas , Colforsina/farmacologia , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miométrio/metabolismo , Toxina Pertussis/farmacologiaRESUMO
OBJECTIVES: To study the influence of pregnancy and labor on the proportion and level of activation of monocyte subpopulations in human pregnancy. STUDY DESIGN: Peripheral blood samples were obtained from healthy nonpregnant women (n = 6); women in the third-trimester of healthy pregnancies (n = 18) and women with preterm premature rupture of membranes (n = 46), just before delivery for the last 2 groups. Monocyte subpopulations were characterized by flow cytometry using CD14, CD16, and activation level using macrophage chemoattractant protein-1 (MCP-1) and CCR2 antibodies. RESULTS: The relative proportion of each monocyte subset in nonpregnant women was similar to that in women with healthy or complicated pregnancies. However, pregnancy was associated with a significant decrease in MCP-1 expressing monocytes (79.5% ± 19.8% vs 9.3% ± 6.8% and 11.9% ± 8.3% for nonpregnant, healthy pregnancy, and preterm premature rupture of membranes (respectively, P < .05). Spontaneous labor was associated with a return to nonpregnant values for the proportion of MCP-1 expressing monocytes in both normal (74.4% ± 16.9) and preterm premature rupture of membranes pregnancy (68.4% ± 35.6), irrespective of the mode of delivery (vaginal or cesarean section). This was not observed in women who delivered without spontaneous labor onset. CCR-2 (MCP-1 receptor) expression was not modified in monocytes at the time of labor, but was significantly increased in granulocytes (3646 ± 1080 vs 7338 ± 2718 for nonlaboring and laboring preterm premature rupture of membranes, respectively, P < .05) CONCLUSION: In light of previous reports of a role for MCP-1 in labor, our results suggest the downregulation of activation levels of monocytes, via MCP-1 expression might be involved in maternofetal immune tolerance. Monocyte reactivation might be associated with labor.
Assuntos
Biomarcadores/sangue , Quimiocina CCL2/sangue , Ruptura Prematura de Membranas Fetais/sangue , Trabalho de Parto/sangue , Receptores de Lipopolissacarídeos/sangue , Monócitos/metabolismo , Trabalho de Parto Prematuro/sangue , Terceiro Trimestre da Gravidez/sangue , Receptores de IgG/sangue , Adolescente , Adulto , Feminino , Citometria de Fluxo , Humanos , Gravidez , Estudos Prospectivos , Adulto JovemRESUMO
Reorganization of myometrial extracellular matrix (ECM) is essential for the uterus to achieve powerful synchronous contractions during labor. Remodeling of the ECM has been implicated in membrane rupture and cervical ripening. Because maternal obesity is associated with both delivery disorders and elevated circulating leptin levels, this study aimed to assess the ability of leptin to interfere with lipopolysaccharide (LPS)-induced myometrial ECM remodeling. Myometrial biopsy samples were obtained from women undergoing cesarean delivery before labor onset. Myometrial explants were incubated for 48 h with LPS and leptin. LPS challenge was associated with a marked decrease in collagen content and in heat shock protein (HSP) 47 expression, reflecting a disruption in collagen synthesis and an increase in matrix metalloproteinase (MMP) 2 and MMP9 activity and in MMP2, MMP9, and MMP13 expression. Leptin prevented an LPS-induced decrease in myometrial collagen content in a concentration-dependent manner. This effect was associated with an increase in HSP47 expression and a decrease in MMP2 and MMP9 activity and expression. These results show that leptin prevents LPS-induced myometrial remodeling through collagen synthesis stimulation and inhibition of MMP2 and MMP9. Our study strengthens the hypothesis that leptin plays a role in the development of obesity-related delivery disorders.
Assuntos
Matriz Extracelular/metabolismo , Inflamação/metabolismo , Leptina/farmacologia , Lipopolissacarídeos/farmacologia , Miométrio/efeitos dos fármacos , Miométrio/metabolismo , Adulto , Biópsia , Colágeno/metabolismo , Relação Dose-Resposta a Droga , Matriz Extracelular/patologia , Feminino , Proteínas de Choque Térmico HSP47/metabolismo , Humanos , Técnicas In Vitro , Inflamação/patologia , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Miométrio/patologia , Obesidade/metabolismo , Obesidade/patologia , Gravidez , Complicações na Gravidez/metabolismo , Complicações na Gravidez/patologiaRESUMO
Tissue injury leads to the well-orchestrated mobilization of systemic and local innate and adaptive immune cells. During aging, immune cell recruitment is dysregulated, resulting in an aberrant inflammatory response that is detrimental for successful healing. Here, we precisely define the systemic and local immune cell response after femur fracture in young and aging mice and identify increased toll-like receptor signaling as a potential culprit for the abnormal immune cell recruitment observed in aging animals. Myd88, an upstream regulator of TLR-signaling lies at the core of this aging phenotype, and local treatment of femur fractures with a Myd88 antagonist in middle-aged mice reverses the aging phenotype of impaired fracture healing, thus offering a promising therapeutic target that could overcome the negative impact of aging on bone regeneration.
Assuntos
Fraturas Ósseas , Fator 88 de Diferenciação Mieloide , Imunidade Adaptativa , Envelhecimento , Animais , Regeneração Óssea , Consolidação da Fratura , Imunidade Inata , Camundongos , Fator 88 de Diferenciação Mieloide/genéticaRESUMO
Maternal obesity is associated with a wide spectrum of labour disorders, including preterm birth. Leptin, a pro-inflammatory adipokine and a key factor of obesity, is suspected to play a major role in these disorders. OB-R, its receptor, is expressed on macrophages and myocytes, two cell types critical for labour onset. Macrophages secrete reactive oxygen species/pro-inflammatory cytokines, responsible for myometrial differentiation while myocytes control uterine contractions. In this study, we assessed the effect of leptin on myometrial contraction and differentiation using our validated co-culture model of human primary macrophages and myocytes. We demonstrated that leptin had a different effect on myocytes and macrophages depending on the dose. A low leptin concentration induced a tocolytic effect by preventing myocytes' contraction, differentiation, and macrophage-induced ROS production. Additionally, leptin led to an increase in HLA-G expression, suggesting that the tocolytic effect of leptin may be driven by HLA-G, a tolerogenic molecule. Finally, we observed that recombinant HLA-G also prevented LPS-induced ROS production by macrophages. Altogether, these data provide a putative molecular mechanism by which leptin may induce immune tolerance and therefore interfere with labour-associated mechanisms. Therefore, HLA-G represents a potential innovative therapeutic target in the pharmacological management of preterm labour.
Assuntos
Nascimento Prematuro , Tocolíticos , Feminino , Antígenos HLA-G , Humanos , Recém-Nascido , Leptina/farmacologia , Gravidez , Nascimento Prematuro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Contração UterinaRESUMO
Mechanical overload of the vascular wall is a pathological hallmark of life-threatening abdominal aortic aneurysms (AAA). However, how this mechanical stress resonates at the unicellular level of vascular smooth muscle cells (VSMC) is undefined. Here we show defective mechano-phenotype signatures of VSMC in AAA measured with ultrasound tweezers-based micromechanical system and single-cell RNA sequencing technique. Theoretical modelling predicts that cytoskeleton alterations fuel cell membrane tension of VSMC, thereby modulating their mechanoallostatic responses which are validated by live micromechanical measurements. Mechanistically, VSMC gradually adopt a mechanically solid-like state by upregulating cytoskeleton crosslinker, α-actinin2, in the presence of AAA-promoting signal, Netrin-1, thereby directly powering the activity of mechanosensory ion channel Piezo1. Inhibition of Piezo1 prevents mice from developing AAA by alleviating pathological vascular remodeling. Our findings demonstrate that deviations of mechanosensation behaviors of VSMC is detrimental for AAA and identifies Piezo1 as a novel culprit of mechanically fatigued aorta in AAA.
Assuntos
Aneurisma Aórtico/metabolismo , Canais Iônicos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Aneurisma , Animais , Aorta Abdominal , Aneurisma Aórtico/patologia , Aneurisma da Aorta Abdominal/metabolismo , Engenharia Biomédica , Fenômenos Biofísicos , Modelos Animais de Doenças , Canais Iônicos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Netrina-1/metabolismo , Fenótipo , Estresse Mecânico , Remodelação VascularRESUMO
OBJECTIVE: This study was aimed at assessing the role of leptin on human myometrium, by studying its receptor expression in pregnant myometrium and the interaction of leptin with inflammation-induced apoptosis. STUDY DESIGN: Myometrial samples were obtained from women with uncomplicated pregnancies who underwent cesarean delivery at term before labor onset. The effect of leptin on apoptosis was assessed by the incubation of myometrial strips with leptin (10(-10) to 10(-8) mol/L; 48 hours) before lipopolysaccharide treatment (10 µg/mL; 48 hours). RESULTS: Long and short leptin receptor isoforms were expressed in myometrial cells of pregnant women. Leptin prevented lipopolysaccharide-induced apoptosis, in a concentration-dependent manner, by down-regulating cleaved caspase-3 and BCL2-associated X protein and up-regulating BCL2 expression. This effect was mediated specifically through leptin receptor stimulation, followed by ERK1/2 signaling pathway activation. CONCLUSION: These results suggest a new potential pathway that is involved in delivery disorders of obese women and propose a role for the leptin-induced inhibition of myometrial apoptosis in the development of such disorders.
Assuntos
Apoptose , Corioamnionite/etiologia , Leptina/fisiologia , Lipopolissacarídeos/fisiologia , Miométrio/citologia , Adulto , Feminino , Humanos , Técnicas In Vitro , Miométrio/química , Gravidez , Receptores para Leptina/análise , Receptores para Leptina/biossíntese , Adulto JovemRESUMO
PPM1D/Wip1 is a negative regulator of the tumor suppressor p53 and is overexpressed in several human solid tumors. Recent reports associate gain-of-function mutations of PPM1D in immune cells with worse outcomes for several human cancers. Here we show that mice with genetic knockout of Ppm1d or with conditional knockout of Ppm1d in the hematopoietic system, in myeloid cells, or in neutrophils all display significantly reduced growth of syngeneic melanoma or lung carcinoma tumors. Ppm1d knockout neutrophils infiltrate tumors extensively. Chemical inhibition of Wip1 in human or mouse neutrophils increases anti-tumor phenotypes, p53-dependent expression of co-stimulatory ligands, and proliferation of co-cultured cytotoxic T cells. These results suggest that inhibition of Wip1 in neutrophils enhances immune anti-tumor responses.
Assuntos
Dano ao DNA , Imunidade , Neutrófilos/metabolismo , Proteína Fosfatase 2C/genética , Proteína Fosfatase 2C/metabolismo , Animais , Antineoplásicos , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Linfócitos T , Microambiente Tumoral , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: We previously showed that supernatants of Lactobacillus biofilms induced an anti-inflammatory response by affecting the secretion of macrophage-derived cytokines, which was abrogated upon immunodepletion of the stress protein GroEL. METHODS: We purified GroEL from L. reuteri and analysed its anti-inflammatory properties in vitro in human macrophages isolated from buffy coats, ex vivo in explants from human biopsies and in vivo in a mouse model of DSS induced intestinal inflammation. As a control, we used GroEL purified (LPS-free) from E. coli. RESULTS: We found that L. reuteri GroEL (but not E. coli GroEL) inhibited pro-inflammatory M1-like macrophages markers, and favored M2-like markers. Consequently, L. reuteri GroEL inhibited pro-inflammatory cytokines (TNFα, IL-1ß, IFNγ) while favouring an anti-inflammatory secretome. In colon tissues from human biopsies, L. reuteri GroEL was also able to decrease markers of inflammation and apoptosis (caspase 3) induced by LPS. In mice, we found that rectal administration of L. reuteri GroEL (but not E. coli GroEL) inhibited all signs of haemorrhagic colitis induced by DSS including intestinal mucosa degradation, rectal bleeding and weight loss. It also decreased intestinal production of inflammatory cytokines (such as IFNγ) while increasing anti-inflammatory IL-10 and IL-13. These effects were suppressed when animals were immunodepleted in macrophages. From a mechanistic point of view, the effect of L. reuteri GroEL seemed to involve TLR4, since it was lost in TRL4-/- mice, and the activation of a non-canonical TLR4 pathway. CONCLUSIONS: L. reuteri GroEL, by affecting macrophage inflammatory features, deserves to be explored as an alternative to probiotics.
Assuntos
Chaperonina 60/farmacologia , Colo/efeitos dos fármacos , Inflamação/prevenção & controle , Lactobacillus/metabolismo , Animais , Chaperonina 60/uso terapêutico , Colo/fisiopatologia , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Limosilactobacillus reuteri/efeitos dos fármacos , Limosilactobacillus reuteri/metabolismo , Camundongos Endogâmicos BALB C , Estatísticas não ParamétricasRESUMO
During obesity, macrophages infiltrate the visceral adipose tissue and promote inflammation that contributes to type II diabetes. Evidence suggests that the rewiring of cellular metabolism can regulate macrophage function. However, the metabolic programs that characterize adipose tissue macrophages (ATM) in obesity are poorly defined. Here, we demonstrate that ATM from obese mice exhibit metabolic profiles characterized by elevated glycolysis and oxidative phosphorylation, distinct from ATM from lean mice. Increased activation of HIF-1α in ATM of obese visceral adipose tissue resulted in induction of IL-1ß and genes in the glycolytic pathway. Using a hypoxia-tracer, we show that HIF-1α nuclear translocation occurred both in hypoxic and non-hypoxic ATM suggesting that both hypoxic and pseudohypoxic stimuli activate HIF-1α and its target genes in ATM during diet-induced obesity. Exposure of macrophages to the saturated fatty acid palmitate increased glycolysis and HIF-1α expression, which culminated in IL-1ß induction thereby simulating pseudohypoxia. Using mice with macrophage-specific targeted deletion of HIF-1α, we demonstrate the critical role of HIF-1α-derived from macrophages in regulating ATM accumulation, and local and systemic IL-1ß production, but not in modulating systemic metabolic responses. Collectively, our data identify enhanced glycolysis and HIF-1α activation as drivers of low-grade inflammation in obesity.
Assuntos
Tecido Adiposo Branco/metabolismo , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Interleucina-1beta/biossíntese , Gordura Intra-Abdominal/metabolismo , Macrófagos/metabolismo , Obesidade/metabolismo , Tecido Adiposo Branco/patologia , Animais , Medula Óssea/patologia , Hipóxia Celular/genética , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica , Glicólise/efeitos dos fármacos , Glicólise/genética , Interleucina-1beta/genética , Gordura Intra-Abdominal/patologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/genética , Especificidade de Órgãos , Fosforilação Oxidativa , Palmitatos/farmacologiaRESUMO
In this work, we have explored natural unmodified low- and high-density lipoproteins (LDL and HDL, respectively) as selective delivery vectors in colorectal cancer therapy. We show in vitro in cultured cells and in vivo (NanoSPECT/CT) in the CT-26 mice colorectal cancer model that LDLs are mainly taken up by cancer cells, while HDLs are preferentially taken up by macrophages. We loaded LDLs with cisplatin and HDLs with the heat shock protein-70 inhibitor AC1LINNC, turning them into a pair of "Trojan horses" delivering drugs selectively to their target cells as demonstrated in vitro in human colorectal cancer cells and macrophages, and in vivo. Coupling of the drugs to lipoproteins and stability was assessed by mass spectometry and raman spectrometry analysis. Cisplatin vectorized in LDLs led to better tumor growth suppression with strongly reduced adverse effects such as renal or liver toxicity. AC1LINNC vectorized into HDLs induced a strong oxidative burst in macrophages and innate anticancer immune response. Cumulative antitumor effect was observed for both drug-loaded lipoproteins. Altogether, our data show that lipoproteins from patient blood can be used as natural nanocarriers allowing cell-specific targeting, paving the way toward more efficient, safer, and personalized use of chemotherapeutic and immunotherapeutic drugs in cancer.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Lipoproteínas HDL/farmacologia , Lipoproteínas LDL/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Humanos , Lipoproteínas/sangue , Lipoproteínas/química , Lipoproteínas HDL/sangue , Lipoproteínas HDL/química , Lipoproteínas LDL/sangue , Lipoproteínas LDL/química , Macrófagos/efeitos dos fármacos , Camundongos , Análise Espectral Raman/métodosRESUMO
Pulmonary disease increases the risk of developing abdominal aortic aneurysms (AAA). However, the mechanism underlying the pathological dialogue between the lungs and aorta is undefined. Here, we find that inflicting acute lung injury (ALI) to mice doubles their incidence of AAA and accelerates macrophage-driven proteolytic damage of the aortic wall. ALI-induced HMGB1 leaks and is captured by arterial macrophages thereby altering their mitochondrial metabolism through RIPK3. RIPK3 promotes mitochondrial fission leading to elevated oxidative stress via DRP1. This triggers MMP12 to lyse arterial matrix, thereby stimulating AAA. Administration of recombinant HMGB1 to WT, but not Ripk3-/- mice, recapitulates ALI-induced proteolytic collapse of arterial architecture. Deletion of RIPK3 in myeloid cells, DRP1 or MMP12 suppression in ALI-inflicted mice repress arterial stress and brake MMP12 release by transmural macrophages thereby maintaining a strengthened arterial framework refractory to AAA. Our results establish an inter-organ circuitry that alerts arterial macrophages to regulate vascular remodeling.
Assuntos
Lesão Pulmonar Aguda/complicações , Aneurisma da Aorta Abdominal/patologia , Proteína HMGB1/metabolismo , Macrófagos/metabolismo , Remodelação Vascular , Lesão Pulmonar Aguda/patologia , Animais , Aorta Abdominal/citologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/etiologia , Aneurisma da Aorta Abdominal/prevenção & controle , Células Cultivadas , Modelos Animais de Doenças , Dinaminas/antagonistas & inibidores , Dinaminas/metabolismo , Humanos , Macrófagos/citologia , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Dinâmica Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Cultura Primária de Células , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Estudos Retrospectivos , Regulação para CimaRESUMO
BACKGROUND: Peripheral artery disease (PAD), a diffuse manifestation of atherothrombosis, is a major cardiovascular threat. Although platelets are primary mediators of atherothrombosis, their role in the pathogenesis of PAD remains unclear. OBJECTIVES: The authors sought to investigate the role of platelets in a cohort of symptomatic PAD. METHODS: The authors profiled platelet activity, mRNA, and effector roles in patients with symptomatic PAD and in healthy controls. Patients with PAD and carotid artery stenosis were recruited into ongoing studies (NCT02106429 and NCT01897103) investigating platelet activity, platelet RNA, and cardiovascular disease. RESULTS: Platelet RNA sequence profiling mapped a robust up-regulation of myeloid-related protein (MRP)-14 mRNA, a potent calcium binding protein heterodimer, in PAD. Circulating activated platelets were enriched with MRP-14 protein, which augmented the expression of the adhesion mediator, P-selectin, thereby promoting monocyte-platelet aggregates. Electron microscopy confirmed the firm interaction of platelets with monocytes in vitro and colocalization of macrophages with MRP-14 confirmed their cross talk in atherosclerotic manifestations of PAD in vivo. Platelet-derived MRP-14 was channeled to monocytes, thereby fueling their expression of key PAD lesional hallmarks and increasing their directed locomotion, which were both suppressed in the presence of antibody-mediated blockade. Circulating MRP-14 was heightened in the setting of PAD, significantly correlated with PAD severity, and was associated with incident limb events. CONCLUSIONS: The authors identified a heightened platelet activity profile and unraveled a novel immunomodulatory effector role of platelet-derived MRP-14 in reprograming monocyte activation in symptomatic PAD. (Platelet Activity in Vascular Surgery and Cardiovascular Events [PACE]; NCT02106429; and Platelet Activity in Vascular Surgery for Thrombosis and Bleeding [PIVOTAL]; NCT01897103).
Assuntos
Plaquetas/fisiologia , Calgranulina B/imunologia , Monócitos/fisiologia , Doença Arterial Periférica , Adulto , Reprogramação Celular/imunologia , Feminino , Hemostasia , Humanos , Macrófagos/fisiologia , Masculino , Pessoa de Meia-Idade , Selectina-P/imunologia , Doença Arterial Periférica/sangue , Doença Arterial Periférica/fisiopatologia , Ativação Plaquetária/imunologiaRESUMO
Abdominal aortic aneurysms (AAA) are characterized by extensive extracellular matrix (ECM) fragmentation and inflammation. However, the mechanisms by which these events are coupled thereby fueling focal vascular damage are undefined. Here we report through single-cell RNA-sequencing of diseased aorta that the neuronal guidance cue netrin-1 can act at the interface of macrophage-driven injury and ECM degradation. Netrin-1 expression peaks in human and murine aneurysmal macrophages. Targeted deletion of netrin-1 in macrophages protects mice from developing AAA. Through its receptor neogenin-1, netrin-1 induces a robust intracellular calcium flux necessary for the transcriptional regulation and persistent catalytic activation of matrix metalloproteinase-3 (MMP3) by vascular smooth muscle cells. Deficiency in MMP3 reduces ECM damage and the susceptibility of mice to develop AAA. Here, we establish netrin-1 as a major signal that mediates the dynamic crosstalk between inflammation and chronic erosion of the ECM in AAA.
Assuntos
Aneurisma da Aorta Abdominal/metabolismo , Macrófagos/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Netrina-1/metabolismo , Animais , Cálcio/metabolismo , Deleção de Genes , Hematopoese , Humanos , Proteínas de Membrana , Camundongos Endogâmicos C57BL , Netrina-1/deficiênciaRESUMO
BACKGROUND AND PURPOSE: Leptin, an adipokine synthesized by the placenta during pregnancy, has been proposed for the management of preterm labour (PTL), as it is able to prevent in vitro uterine contractility and remodelling associated with labour onset. Another common feature of labour onset is the phenotypic switch of myometrial smooth muscle cells from a proliferative to a hypertrophic state. As proliferative effects have been demonstrated for leptin in other tissues, we aimed to investigate its ability to induce myometrial proliferation and thus to maintain uterine quiescence. EXPERIMENTAL APPROACH: We stimulated human primary myometrial smooth muscle cells with leptin in the presence or absence of receptor antagonists or signalling pathway inhibitors. KEY RESULTS: Leptin induced myometrial cell proliferation in a biphasic manner. At 6.25 ng · mL(-1), leptin-induced proliferation was mediated by the leptin receptor and required the early activation of ERK1/2. At a concentration above 25 ng · mL(-1), leptin induced direct non-specific stimulation of the IL-6 receptor, leading to NF-κB activation, and exerted anti-proliferative effects. However, at 50 ng · mL(-1), leptin re-induces proliferation via IL-6 receptor stimulation that requires STAT3 and delayed ERK1/2 activation. CONCLUSIONS AND IMPLICATIONS: These data bring new insights into leptin signalling-induced myometrial proliferation and its interrelationship with the IL-6/IL-6 receptor axis. In the light of our previous work, the present study emphasizes the potential value of leptin in the pharmacological management of PTL and it also strengthens the hypothesis that leptin might be a contributory factor in the parturition-related disorders observed in obese women.
Assuntos
Proliferação de Células/efeitos dos fármacos , Leptina/farmacologia , Miométrio/efeitos dos fármacos , Receptores para Leptina/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Interleucina-6/metabolismo , Leptina/administração & dosagem , Leptina/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miométrio/citologia , NF-kappa B/metabolismo , Gravidez , Receptores para Leptina/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The human inducible heat shock protein 70 (hHsp70), which is involved in several major pathologies, including neurodegenerative disorders and cancer, is a key molecular chaperone and contributes to the proper protein folding and maintenance of a large number of protein structures. Despite its role in disease, the current structural knowledge of hHsp70 is almost exclusively based on its Escherichia coli homolog, DnaK, even though these two proteins only share ~50 % amino acid identity. For the first time, we describe a complete heterologous production and purification strategy that allowed us to obtain a large amount of soluble, full-length, and non-tagged hHsp70. The protein displayed both an ATPase and a refolding activity when combined to the human Hsp40. Multi-angle light scattering and bio-layer interferometry analyses demonstrated the ability of hHsp70 to homodimerize. The role of the C-terminal part of hHsp70 was identified and confirmed by a study of a truncated version of hHsp70 that could neither dimerize nor present refolding activity.