RESUMO
Thoracic aortic aneurysm/dissection (TAAD) is a lethal vascular disease, and several pathological factors participate in aortic medial degeneration. We previously discovered that the complement C3a-C3aR axis in smooth muscle cells promotes the development of thoracic aortic dissection (TAD) through regulation of matrix metalloproteinase 2. However, discerning the specific complement pathway that is activated and elucidating how inflammation of the aortic wall is initiated remain unknown. We ascertained that the plasma levels of C3a and C5a were significantly elevated in patients with TAD and that the levels of C3a, C4a, and C5a were higher in acute TAD than in chronic TAD. We also confirmed the activation of the complement in a TAD mouse model. Subsequently, knocking out Cfb (Cfb) or C4 in mice with TAD revealed that the alternative pathway and Cfb played a significant role in the TAD process. Activation of the alternative pathway led to generation of the anaphylatoxins C3a and C5a, and knocking out their receptors reduced the recruitment of inflammatory cells to the aortic wall. Moreover, we used serum from wild-type mice or recombinant mice Cfb as an exogenous source of Cfb to treat Cfb KO mice and observed that it exacerbated the onset and rupture of TAD. Finally, we knocked out Cfb in the FBN1C1041G/+ Marfan-syndrome mice and showed that the occurrence of TAA was reduced. In summary, the alternative complement pathway promoted the development of TAAD by recruiting infiltrating inflammatory cells. Targeting the alternative pathway may thus constitute a strategy for preventing the development of TAAD.NEW & NOTEWORTHY The alternative complement pathway promoted the development of TAAD by recruiting infiltrating inflammatory cells. Targeting the alternative pathway may thus constitute a strategy for preventing the development of TAAD.
Assuntos
Aneurisma da Aorta Torácica , Dissecção Aórtica , Azidas , Desoxiglucose/análogos & derivados , Humanos , Camundongos , Animais , Via Alternativa do Complemento , Metaloproteinase 2 da Matriz , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/patologia , Dissecção Aórtica/genética , InflamaçãoRESUMO
RESEARCH QUESTION: Does vitamin D affect the pregnancy rate of patients with polycystic ovary syndrome (PCOS) receiving ovulation-induction therapy? DESIGN: The retrospective study included 200 patients with PCOS and 200 healthy women. The prospective study included 160 patients with PCOS receiving vitamin D or placebo supplementation. Pregnancy rates were assessed after a maximum of three cycles of ovulation induction. Serum concentrations of 25-hydroxycalciferol [25-(OH)D3], LH, FSH, progesterone, oestradiol, testosterone and fasting insulin; LH/FSH ratio; and body mass index were evaluated. RESULTS: In the retrospective study, patients with PCOS had lower 25-(OH)D3 concentrations than healthy women, pregnant patients with PCOS had higher 25-(OH)D3 concentrations than non-pregnant patients with PCOS (both Pâ¯=â¯0.000), and the pregnancy rate was lower in the vitamin-D-deficient group compared with the non-vitamin-D-deficient group (Pâ¯=â¯0.022). In the prospective study, compared with placebo supplementation, vitamin D supplementation increased the serum concentration of 25-(OH)D3 (Pâ¯=â¯0.000), and reduced the LH/FSH ratio, and concentrations of LH and testosterone significantly (all P ≤ 0.049). After the intervention, it was found that the LH/FSH ratio, and concentrations of LH and testosterone were significantly lower in both groups compared with pre-intervention (Pâ¯=â¯0.000). After ovulation induction, the pregnancy rate was higher in patients in the vitamin D supplementation group compared with the placebo supplementation group (Pâ¯=â¯0.049). CONCLUSIONS: Vitamin D deficiency is common in patients with PCOS, and vitamin-D-deficient patients with PCOS have lower pregnancy rates after ovulation induction compared with non-vitamin-D-deficient patients with PCOS. Vitamin D supplementation can improve the pregnancy rate and mitigate basic hormone disorders. Therefore, monitoring vitamin D supplementation and checking vitamin D concentrations before and during interventions are essential for patients with PCOS.
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Síndrome do Ovário Policístico , Taxa de Gravidez , Vitamina D , Humanos , Feminino , Síndrome do Ovário Policístico/sangue , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/complicações , Gravidez , Estudos Retrospectivos , Adulto , Estudos Prospectivos , Vitamina D/sangue , Vitamina D/análogos & derivados , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/tratamento farmacológico , Testosterona/sangue , Hormônio Luteinizante/sangue , Indução da Ovulação/métodos , Suplementos Nutricionais , Hormônio Foliculoestimulante/sangue , Progesterona/sangue , Adulto JovemRESUMO
BACKGROUND: Pulmonary hypertension (PH) seriously affects the health of patients. We have found in clinical studies that PH has adverse effects on both maternal and offspring. OBJECTIVE: To establish a animal model of PH induced by hypoxia/SU5416 and observe the effects of PH on pregnant mice and their fetuses. METHODS: Twenty-four C57 mice aged 7-9 weeks were selected and divided into 4 groups with 6 mice in each group. â Female mice with normal oxygen; â¡ Female mice with hypoxia/SU5416; ⢠Pregnant mice with normal oxygen; ⣠Pregnant mice with hypoxia/SU5416. After 19 days, weight, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were compared in each group. Lung tissue and right ventricular blood were collected. The number and weight of fetal mice were also compared between the two pregnant groups. RESULTS: There was no significant difference in RVSP and RVHI between female and pregnant mice under the same condition. Compared with normal oxygen condition, two groups of mice in hypoxia/SU5416 had poor development, RVSP and RVHI were significantly increased, the number of fetal mice was small, hypoplasia, degeneration and even abortion. CONCLUSION: The model of mice PH was successfully established. PH affects the development and health of female and pregnant mice, and seriously affects the fetuses.
Assuntos
Hipertensão Pulmonar , Feminino , Camundongos , Animais , Gravidez , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Oxigênio/efeitos adversos , Modelos Animais de Doenças , Hipertrofia Ventricular Direita , Artéria PulmonarRESUMO
BACKGROUND: Neutrophils consume a large amount of energy when performing their functions. Compared with other white blood cells, neutrophils contain few mitochondria and mainly rely on glycolysis and gluconeogenesis to produce ATP. The inflammatory site is hypoxic and nutrient poor. Our aim is to study the role of abnormal adenosine metabolism of neutrophils in the asthmatic airway inflammation microenvironment. METHOD: In this study, an asthma model was established by intratracheal instillation of Aspergillus fumigatus extract in Ecto-5'-Nucleotidase (CD73) gene-knockout and wild-type mice. Multiple analyses from bronchoalveolar lavage fluid (BALF) were used to determine the levels of cytokines and chemokines. Immunohistochemistry was used to detect subcutaneous fibrosis and inflammatory cell infiltration. Finally, adenosine 5'-(α, ß-methylene) diphosphate (APCP), a CD73 inhibitor, was pumped subcutaneously before Aspergillus attack to observe the infiltration of inflammatory cells and subcutaneous fibrosis to clarify its therapeutic effect. RESULT: PAS staining showed that CD73 knockout inhibited pulmonary epithelial cell proliferation and bronchial fibrosis induced by Aspergillus extract. The genetic knockdownof CD73 significantly reduced the production of Th2 cytokines, interleukin (IL)-4, IL-6, IL-13, chemokine (C-C motif) ligand 5 (CCL5), eosinophil chemokine, neutrophil IL-17, and granulocyte colony-stimulating factor (G-CSF). In addition, exogenous adenosine supplementation increased airway inflammation. Finally, the CD73 inhibitor APCP was administered to reduce inflammation and subcutaneous fibrosis. CONCLUSION: Elevated adenosine metabolism plays an inflammatory role in asthma, and CD73 could be a potential therapeutic target for asthma.
Assuntos
Asma , Neutrófilos , Animais , Camundongos , Neutrófilos/metabolismo , Aspergillus fumigatus/metabolismo , Adenosina/metabolismo , Asma/terapia , Citocinas/metabolismo , Inflamação , Quimiocinas/metabolismo , Líquido da Lavagem Broncoalveolar , Extratos Vegetais , Remodelação das Vias AéreasRESUMO
Though the PD-L1 checkpoint inhibitor avelumab has shown efficacy in the treatment of some types of cancer, improved treatment strategies are desperately needed. We evaluated whether combined treatment with avelumab and adoptively transferred T-NK cells can provide enhanced anti-cancer effects for treating PD-L1-expressing tumours. Our results demonstrate that avelumab specifically targets tumour cells with high PD-L1 expression, and that cytolytic effects are mediated by T-NK effector cells cultured from patient peripheral blood monocytic cell populations. The effects were dependent on CD16 and the perforin/granzyme pathway, supporting a role for the T-NK subpopulation. In vivo assays verified the efficacy of T-NK cells in combination with avelumab in reducing tumour growth. Furthermore, T-NK + avelumab prolonged survival in a mouse orthotopic xenograft model. Collectively, our findings provide a basis for the combined use of adoptively transferred T-NK cells with avelumab as a novel strategy for cancer treatment.
Assuntos
Anticorpos Monoclonais , Linfócitos T , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antígeno B7-H1 , Linhagem Celular Tumoral , Granzimas/genética , Humanos , Células Matadoras Naturais , Camundongos , Perforina/genética , Fenótipo , Linfócitos T/metabolismoRESUMO
BACKGROUND: Mutations in bone morphogenetic protein type II receptor (BMPR2) have been found in patients with congenital heart disease-associated pulmonary arterial hypertension (CHD-PAH). Our study aimed to clarify whether deficient BMPR2 signalling acts through downstream effectors, inhibitors of DNA-binding proteins (IDs) during heart development to contribute to the progress of PAH in CHD patients. METHODS: To confirm that IDs are downstream effectors of BMPR2 signalling in cardiac mesoderm progenitors (CMPs) and contribute to PAH, we generated cardiomyocyte-specific Id 1/3 knockout mice (Ids cDKO), and 12 out of 25 developed mild PAH with altered haemodynamic indices and pulmonary vascular remodelling. Moreover, we generated ID1 and ID3 double-knockout (IDs KO) human embryonic stem cells that recapitulated the BMPR2 signalling deficiency of CHD-PAH induced pluripotent stem cells (iPSCs). RESULTS: Cardiomyocytes differentiated from iPSCs derived from CHD-PAH patients with BMP receptor mutations exhibited dysfunctional cardiac differentiation and reduced calcium (Ca2+) transients, as evidenced by confocal microscopy experiments. Smad1/5 phosphorylation and ID1 and ID3 expression were reduced in CHD-PAH iPSCs and in Bmpr2 +/- rat right ventricles. Moreover, ultrasound revealed that 33% of Ids cDKO mice had detectable defects in their ventricular septum and pulmonary regurgitation. Cardiomyocytes isolated from mouse right ventricles also showed reduced Ca2+ transients and shortened sarcomeres. Single-cell RNA sequencing analysis revealed impaired differentiation of CMPs and downregulated USP9X expression in IDs KO cells compared with wild-type cells. CONCLUSION: We found that BMPR2 signals through IDs and USP9X to regulate cardiac differentiation, and the loss of ID1 and ID3 expression contributes to cardiomyocyte dysfunction in CHD-PAH patients with BMPR2 mutations.
Assuntos
Cardiopatias Congênitas , Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cardiopatias Congênitas/genética , Camundongos , Mutação , Artéria Pulmonar , Ratos , Análise de Sequência de RNA , Função Ventricular DireitaRESUMO
Objectives To test the antitumor potential of lymphocytes transferred via adoptive cell therapy (ACT) in a mouse model of human gastric cancer (GC), and to evaluate the clinical efficacy and safety of combining lymphocytes as adjuvant therapy with first-line chemotherapy in patients with GC. Methods We constructed a human GC xenograft model in sublethally irradiated 6-8-week-old male NCG mice. MKN-45 cells (1 × 106 cells/mouse) were subcutaneously injected into mice's flanks. After tumors had become palpable, we randomized the mice into control, ACTIL-2, and ACTIL-15 groups. Human lymphocytes were then injected into mouse tail veins. In addition, 63 human patients with histologically or cytologically confirmed stage III-IV GC randomly received S-1 + oxaliplatin + ACTIL-15 (combination therapy group) or S-1 + oxaliplatin (chemotherapy group). Results In the mouse study, treatment with ACTIL-15 cells inhibited tumor growth on adoptive transfer, and mice that received ACTIL-15 cells had significantly longer survival rates (p < 0.05, ACTIL-15 vs. ACTIL-2). In the human study, the median survival rate of patients in the combination therapy group was 472 days (95% confidence interval [CI], 276-668 days), whereas that of patients in the chemotherapy group was 266 days (95% CI, 200-332 days; p < 0.05). Eleven percent (7/63) of patients had adverse reactions, but these reactions did not interfere with treatment. Conclusion Adoptive transfer of ACTIL-15 cells in a mouse model of GC and in patients with advanced GC treated with S1 + oxaliplatin improved survival rates in both, with an acceptable safety profile.
Assuntos
Imunoterapia Adotiva/métodos , Interleucina-15/farmacologia , Linfócitos/efeitos dos fármacos , Oxaliplatina/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Adulto , Idoso , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Quimioterapia Adjuvante , Feminino , Humanos , Interleucina-15/administração & dosagem , Masculino , Camundongos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Oxaliplatina/administração & dosagem , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adoptive T cell transfer therapy (ACT) has emerged as a promising approach to cancer immunotherapy; however, the efficacy of ACT is limited by the T-cell suppressive activity of myeloid-derived suppressor cells (MDSCs), which accumulate in the tumor microenvironment after ACT. We sought to determine whether the efficacy of ACT could be enhanced by co-treatment with docetaxel, a taxane chemotherapy agent that has been shown previously to inhibit MDSC function. Using a mouse tumor model, we demonstrated that ACT and docetaxel synergistically inhibit the growth either of engrafted CT26 colon cancer or 4T1 mammary carcinoma cells. While ACT mediated an increase in the recruitment of MDSCs to the site of the tumor, docetaxel reversed this increase. Furthermore, ex vivo cultures of tumor-associated MDSCs suppressed the cytotoxic activity of tumor-specific T cells, and this suppressive activity was abolished by docetaxel treatment. These results suggest that docetaxel inhibits both the tumor recruitment and T cell suppressive activity of MDSCs. Inhibitors of iNOS and arginase partially inhibited ex vivo MDSC activity, and combined inhibition of iNOS and arginase had a similar effect as docetaxel, which supports the possibility that docetaxel may function by inhibiting ACT-associated activation of these pathways. Furthermore, docetaxel mediated inhibition of the T cell suppressive activity of MDSCs from human blood, which supports the potential clinical applicability of these findings. On the basis of these findings, docetaxel treatment may represent an effective therapeutic approach for reversing immunosuppression by MDSCs subsequent to ACT-based therapy.
Assuntos
Transferência Adotiva/métodos , Antineoplásicos/farmacologia , Terapia Combinada/métodos , Docetaxel/farmacologia , Neoplasias Experimentais , Linfócitos T/transplante , Animais , Modelos Animais de Doenças , Humanos , Terapia de Imunossupressão/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologiaRESUMO
Thoracic aortic dissection (TAD), once ruptured, is devastating to patients, and no effective pharmaceutical therapy is available. Anaphylatoxins released by complement activation are involved in a variety of diseases. However, the role of the complement system in TAD is unknown. We found that plasma levels of C3a, C4a, and C5a were significantly increased in patients with TAD. Elevated circulating C3a levels were also detected in the developmental process of mouse TAD, which was induced by ß-aminopropionitrile monofumarate (BAPN) treatment, with enhanced expression of C1q and properdin in mouse dissected aortas. These findings indicated activation of classical and alternative complement pathways. Further, expression of C3aR was obviously increased in smooth muscle cells of human and mouse dissected aortas, and knockout of C3aR notably inhibited BAPN-induced formation and rupture of TAD in mice. C3aR antagonist administered pre- and post-BAPN treatment attenuated the development of TAD. We found that C3aR knockout decreased matrix metalloproteinase 2 (MMP2) expression in BAPN-treated mice. Additionally, recombinant C3a stimulation enhanced MMP2 expression and activation in smooth muscle cells that were subjected to mechanical stretch. Finally, we generated MMP2-knockdown mice by in vivo MMP2 short hairpin RNA delivery using recombinant adeno-associated virus and found that MMP2 deficiency significantly reduced the formation of TAD. Therefore, our study suggests that the C3a-C3aR axis contributes to the development of TAD via regulation of MMP2 expression. Targeting the C3a-C3aR axis may represent a strategy for inhibiting the formation of TAD.
Assuntos
Dissecção Aórtica/metabolismo , Complemento C3a/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Receptores de Complemento/metabolismo , Anafilatoxinas/metabolismo , Animais , Células Cultivadas , Ativação do Complemento/fisiologia , Complemento C5a/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Tissue damage and its associated-inflammation act as tumour initiators or propagators. AMP-activated protein kinase (AMPK) is activated by environmental or nutritional stress factors, such as hypoxia, glucose deprivation, and other cell injury factors, to regulate cell energy balance and differentiation. We previously have reported that AMPKα2 deficiency resulted in the energy deprivation in tumour-bearing liver and the enhanced-hepatocyte death. In this study, AMPKα2 knockout mice and the liver metastasis model of colon cancer cells were used to address the role of AMPKα isoforms in tumour inflammation. First, we found that the AMPKα2 deficiency exacerbated the liver injury and recruitment of macrophages. Meanwhile, although compensatory expression of AMPKα1 was not significant after AMPKα2 knockout, AMPKα1 phosphorylation was elevated in remnant liver in AMPKα2 knockout mice, which was positively associated with the enhanced energy deprivation in the AMPKα2 deficient mice. Furthermore, the activated AMPKα1 in macrophage contributed to its polarizing to tumour-associated phenotype. Thus, the enhanced tumour-associated inflammation and activation of AMPKα1 in the AMPKα2 deficient mice may exacerbate the tumour development by affecting the tumour inflammatory microenvironment. Our study suggests that the two isoforms of AMPKα, AMPKα1 and AMPKα2 play different roles in controlling tumour development.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Neoplasias do Colo/etiologia , Modelos Animais de Doenças , Inflamação/etiologia , Neoplasias Hepáticas/etiologia , Macrófagos/patologia , Animais , Diferenciação Celular , Células Cultivadas , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Metabolismo Energético , Inflamação/metabolismo , Inflamação/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Microambiente TumoralRESUMO
Dendritic cell (DC)-based immunotherapies have been created for a broad expanse of cancers, and DC vaccines prepared with Wilms' tumor protein 1 (WT1) peptides have shown great therapeutic efficacy in these diseases. In this paper, we report the results of a phase I/II study of a DC-based vaccination for advanced breast, ovarian, and gastric cancers, and we offer evidence that patients can be effectively vaccinated with autologous DCs pulsed with WT1 peptide. There were ten patients who took part in this clinical study; they were treated biweekly with a WT1 peptide-pulsed DC vaccination, with toxicity and clinical and immunological responses as the principal endpoints. All of the adverse events to DC vaccinations were tolerable under an adjuvant setting. The clinical response was stable disease in seven patients. Karnofsky Performance Scale scores were enhanced, and computed tomography scans revealed tumor shrinkage in three of seven patients. Human leukocyte antigen (HLA)/WT1-tetramer and cytoplasmic IFN-γ assays were used to examine the induction of a WT-1-specific immune response. The immunological responses to DC vaccination were significantly correlated with fewer myeloid-derived suppressor cells (P = 0.045) in the pretreated peripheral blood. These outcomes offered initial clinical evidence that the WT1 peptide-pulsed DC vaccination is a potential treatment for advanced cancer.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Imunoterapia/métodos , Neoplasias/terapia , Proteínas WT1/imunologia , Adulto , Idoso , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/imunologia , Neoplasias Ovarianas , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/terapia , Resultado do Tratamento , Vacinação/métodosRESUMO
We developed a simple and efficient method to construct 3D and 2D opal and inverse opal cellulose photonic crystal films (CPCF) by embedding 3D or 2D polymethyl methacrylate (PMMA) colloidal arrays into carboxymethyl cellulose (CMC), respectively. The morphology and optical performance of CPCFs were characterized by SEM, diffraction spectra, Debye rings, and structural color. The brilliant structural colors of CPCFs are visible to the eye in the entire visible spectrum, and can be tuned by changing the particle diameters or the pore sizes. Attributed to decreased particle spacing and lower average refractive index caused by air spheres instead of polymer spheres, the stopbands of the inverse opal CPCFs blue-shifted. To the contrary, the particle spacing of 2D inverse opal CPCFs increased due to the losing of the connection force of 2D arrays, along with decreasing of Debye ring diameters. By alternately being exposed to organic solvents of methanol, acetonitrile, butanol, dioxane, and carbon tetrachloride, the 3D inverse opal CPCFs displayed an excellent sensing performance with instantaneously reversible color changes from violet to red. Their high stability and flexibility, efficient visual detection, and wide range of analytes promises a new opportunity for optical switching and sensing applications.
RESUMO
Inflammatory cells such as macrophages can play a pro-tumorigenic role in the tumor stroma. Tumor-associated macrophages (TAMs) generally display an M2 phenotype with tumor-promoting activity; however, the mechanisms regulating the TAM phenotype remain unclear. Complement 5a (C5a) is a cytokine-like polypeptide that is generated during complement system activation and is known to promote tumor growth. Herein, we investigated the role of C5a on macrophage polarization in colon cancer metastasis in mice. We found that deficiency of the C5a receptor (C5aR) severely impairs the metastatic ability of implanted colon cancer cells. C5aR was expressed on TAMs, which exhibited an M2-like functional profile in colon cancer liver metastatic lesions. Furthermore, C5a mediated macrophage polarization and this process relied substantially on activation of the nuclear factor-kappa B (NF-κB) pathway. Finally, analysis of human colon carcinoma indicated that C5aR expression is negatively associated with tumor differentiation grade. Our results demonstrate that C5aR has a central role in regulating the M2 phenotype of TAMs, which in turn, contributes to hepatic metastasis of colon cancer through NF-κB signaling. C5a is a potential novel marker for cancer prognosis and drugs targeting complement system activation, specifically the C5aR pathway, may offer new therapeutic opportunities for colon cancer management.
Assuntos
Neoplasias do Colo/patologia , Complemento C5a/metabolismo , Neoplasias Hepáticas/secundário , Macrófagos/patologia , Receptor da Anafilatoxina C5a/metabolismo , Receptor da Anafilatoxina C5a/fisiologia , Animais , Carcinogênese , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Complemento C5a/genética , Citocinas/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor da Anafilatoxina C5a/genética , Transdução de Sinais , Células Tumorais Cultivadas , Microambiente TumoralRESUMO
Ribonucleotide reductase small subunit p53R2 is a member of the ribonucleotide reductase family that supplies dNTPs for nuclear and mitochondrial DNA replication and repair. Here, we have identified a mitochondrial thioredoxin reductase 2 (TrxR2) as a novel p53R2-binding protein. We demonstrated a direct interaction between the two, and observed that p53R2 stimulated the enzymatic activity of TrxR in vitro. Moreover, TrxR2 activity was significantly lower in p53R2 knockdown cells, and increased when p53R2 was overexpressed, effects that were independent of p53. Furthermore, p53R2 knockdown suppressed UV-induced TrxR activity. These findings suggest that p53R2 acts as a positive regulator of TrxR2 activity in mitochondria both under normal physiological conditions and during the cellular response to DNA damage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação Enzimológica da Expressão Gênica , Ribonucleotídeo Redutases/metabolismo , Tiorredoxina Redutase 2/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , DNA Mitocondrial/metabolismo , Vetores Genéticos , Humanos , Mitocôndrias/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Raios Ultravioleta , Regulação para CimaRESUMO
The degeneration of vascular smooth muscle cell(s) (SMC) is one of the key features of thoracic aortic aneurysm and dissection (TAAD). We and others have shown that elevated endoplasmic reticulum (ER) stress causes SMC loss and TAAD formation, however, the mechanism of how SMC dysfunction contributes to intimal damage, leading to TAAD, remains to be explored. In the present study, in vitro assay demonstrated that elevated mechanical stretch (18% elongation, 3600 cycles/h) stimulated the ER stress response and microparticle(s) (MP) production from both SMC and endothelial cell(s) (EC) in a time-dependent manner. Treatment of EC with isolated MP led to anoikis, which was determined by measuring the fluorescence of the ethidium homodimer (EthD-1) and Calcein AM cultured in hydrogel-coated plates and control plates. MP stimulation of EC also up-regulated the mRNA levels of inflammatory molecules (i.e. Vascular cellular adhesion molecular-1 (VCAM-1)), intercellular adhesion molecular-1 (ICAM-1), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6)). Use of an ER stress inhibitor or knockout of CHOP decreased mechanical stretch-induced MP production in SMC. In vivo, administration of an ER stress inhibitor or knockout of CHOP suppressed both apoptosis of EC and the infiltration of inflammatory cells. Moreover, TAAD formation was also suppressed by the administration of an ER stress inhibitor. In conclusion, our study demonstrates that elevated mechanical stretch induces MP formation in SMC leading to endothelial dysfunction, which is ER stress dependent. The inhibition of ER stress suppressed EC apoptosis, inflammation in the aorta, and TAAD development.
Assuntos
Aneurisma da Aorta Torácica/metabolismo , Dissecção Aórtica/metabolismo , Micropartículas Derivadas de Células/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Aminopropionitrilo , Dissecção Aórtica/induzido quimicamente , Dissecção Aórtica/genética , Dissecção Aórtica/patologia , Animais , Anoikis , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Aneurisma da Aorta Torácica/induzido quimicamente , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/patologia , Micropartículas Derivadas de Células/patologia , Células Cultivadas , Modelos Animais de Doenças , Retículo Endoplasmático/patologia , Células Endoteliais/patologia , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Mecanotransdução Celular , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Comunicação Parácrina , Fenótipo , RNA Mensageiro/genética , Estresse Mecânico , Fatores de Tempo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
Thoracic aortic aneurysm/dissection (TAAD) is characterized by excessive smooth muscle cell (SMC) loss, extracellular matrix (ECM) degradation and inflammation. However, the mechanism whereby signaling leads to SMC loss is unclear. We used senescence-associated (SA)-ß-gal staining and analysis of expression of senescence-related proteins (p53, p21, p19) to show that excessive mechanical stretch (20% elongation, 3600cycles/h, 48h) induced SMC senescence. SMC senescence was also detected in TAAD specimens from both mice and humans. High-performance liquid chromatography and luciferin-luciferase-based assay revealed that excessive mechanical stretch increased adenosine diphosphate (ADP) release from SMCs both in vivo and in vitro. Elevated ADP induced SMC senescence while genetic knockout of the ADP receptor, P2Y G protein-coupled receptor 12 (P2ry12), in mice protected against SMC senescence and inflammation. Both TAAD formation and rupture were significantly reduced in P2ry12-/- mice. SMCs from P2ry12-/- mice were resistant to senescence induced by excessive mechanical stretch or ADP treatment. Mechanistically, ADP treatment sustained Ras activation, whereas pharmacological inhibition of Ras protected against SMC senescence and reduced TAAD formation. Taken together, excessive mechanical stress may induce a sustained release of ADP and promote SMC senescence via P2ry12-dependent sustained Ras activation, thereby contributing to excessive inflammation and degeneration, which provides insights into TAAD formation and progression.
Assuntos
Difosfato de Adenosina/metabolismo , Aneurisma da Aorta Torácica/metabolismo , Dissecção Aórtica/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Transdução de Sinais , Dissecção Aórtica/diagnóstico por imagem , Dissecção Aórtica/etiologia , Dissecção Aórtica/patologia , Animais , Aneurisma da Aorta Torácica/diagnóstico por imagem , Aneurisma da Aorta Torácica/etiologia , Aneurisma da Aorta Torácica/patologia , Biópsia , Senescência Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Knockout , Receptores Purinérgicos P2Y12/deficiência , Receptores Purinérgicos P2Y12/genética , Estresse Mecânico , UltrassonografiaRESUMO
Complement 5a (C5a), a potent immune mediator generated by complement activation, promotes tumor growth; however, its role in tumor metastasis remains unclear. We demonstrate that C5a contributes to tumor metastases by modulating tumor inflammation in hepatic metastases of colon cancer. Colon cancer cell lines generate C5a under serum-free conditions, and C5a levels increase over time in a murine syngeneic colon cancer hepatic metastasis model. Furthermore, in the absence of C5a receptor or upon pharmacological inhibition of C5a production with an anti-C5 monoclonal antibody, tumor metastasis is severely impaired. A lack of C5a receptor in colon cancer metastatic foci reduces the infiltration of macrophages, neutrophils, and dendritic cells, and the role for C5a receptor on these cells were further verified by bone marrow transplantation experiments. Moreover, C5a signaling increases the expression of the chemokine monocyte chemoattractant protein-1 and the anti-inflammatory molecules arginase-1, interleukin 10, and transforming growth factor ß, but is inversely correlated with the expression of pro-inflammatory molecules, which suggests a mechanism for the role of C5a in the inflammatory microenvironment required for tumor metastasis. Our results indicate a new and potentially promising therapeutic application of complement C5a inhibitor for the treatment of malignant tumors.
Assuntos
Quimiocina CCL2/metabolismo , Neoplasias do Colo/imunologia , Complemento C5a/metabolismo , Neoplasias Hepáticas Experimentais/imunologia , Neoplasias Hepáticas Experimentais/secundário , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Ativação do Complemento , Feminino , Inflamação/imunologia , Inflamação/patologia , Neoplasias Hepáticas Experimentais/patologia , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Invasividade Neoplásica/imunologia , Invasividade Neoplásica/patologia , Infiltração de Neutrófilos , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Microambiente Tumoral/imunologiaRESUMO
Adiponectin is an adipocyte-specific adipocytokine with proliferative and pro-angiogenic effects that regulates many biological processes, including immunity, insulin resistance, and inflammation. The oncogenic role of adiponectin has been implicated in several cancer types. Stromal cells within tumor contribute tumor growth and angiogenesis; however, it is not clear that how adiponectin regulates stromal cell-mediated tumorigenesis. In this study, using the tumor xenograft models, we demonstrated that tumor development was severely impaired in mouse subcutaneous cancer tissue and metastasis tumor tissue in adiponectin knockout mice. Our results indicated adiponectin deficiency resulted in decrease of blood vessel and stromal senescent fibroblasts in subcutaneous and metastasis tumor tissue. These observations were confirmed in vitro, in which co-cultured tumor cells and fibroblasts treated with adiponectin promoted ECs tube formation. A secretion of CXCL1 by adiponectin-treated tumor cells was observed during the process of inducing stromal fibroblast senescence. Furthermore, stromal cells senescence was through p53 and p16 pathways. Taken together, our results indicate that adiponectin promotes stromal cell senescence within invasive colon cancer contributing to angiogenesis and tumor growth in part through the production of CXCL1 and may serve as a therapeutic target for tumor patients. © 2015 Wiley Periodicals, Inc.
Assuntos
Adiponectina/genética , Adiponectina/metabolismo , Quimiocina CXCL1/metabolismo , Fibroblastos/patologia , Neoplasias Experimentais/patologia , Animais , Linhagem Celular Tumoral , Quimiocina CXCL1/genética , Técnicas de Cocultura , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Metástase Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Transdução de Sinais , Células Estromais/citologia , Células Estromais/patologiaRESUMO
Pulmonary artery hypertension (PAH) is characterized as sustained elevation of pressure in the pulmonary vascular system that is attributable to a variety of causes. More than a dozen genes have previously been proposed as being associated with PAH. To examine potential mutations of these genes in patients with PAH, we developed a targeted exome kit containing 22 PAH-associated genes for genetic screens of 80 unrelated patients with PAH. As a result, we identified 16 different mutations in the BMPR2 gene and four different mutations in ACVRL1, the gene for activin receptor-like kinase-1 (ACVRL1). However, no deleterious mutations were found in the remaining 20 genes. In the present study, we provided detailed characterization of the ACVRL1 mutations in four pedigrees, including two novel missense mutations (c.676G>A, p.V226M; c.955G>C, p.G319R) and two recurrent mutations (c.1231C>T, p.R411W; c.1450C>T, p.R484W). Furthermore, we showed that markedly reduced Smad1/5 phosphorylation levels and reduced activities of luciferase reporters in each of the four ACVRL1 mutant-transfected NIH-3T3 cells. Therefore, our findings demonstrated that missense mutations of ACVRL1 identified in the present study significantly affected the bone morphogenetic protein 9 (BMP-9) pathway, implicating PAH pathogenesis. Detailed genotype-phenotype correlation analysis revealed initial symptoms of hereditary haemorrhagic telangiectasia (HHT) in some of the patients, suggesting the importance of sequencing molecular markers for early identification and intervention of individuals at risk for PAH and potential HHT. We developed a customized exome sequencing system to identify mutations in these PAH-associated genes, and found two novel missense mutations and two recurrent mutations in the ACVRL1 gene in four unrelated Chinese families; we also determined hypomorphic alleles using functional studies.