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1.
Cancer Immunol Immunother ; 68(12): 1959-1969, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31641797

RESUMO

Hepatic stellate cells (HSCs) are important stromal cells and pivotal mediators involved in the pathogenesis and immunosuppression of hepatocellular carcinoma (HCC). The liver has been demonstrated to be a site for accumulation of tumor-induced myeloid-derived suppressor cells (MDSCs). We previously reported that HSCs induced an increase in the number of MDSCs in HCC. However, how MDSCs are recruited in HCC remains largely unclear. In the present study, we found that HSC-conditioned medium (HSC-CM) induced bone marrow-derived cell and splenocyte migration, especially MDSC migration. Using chemokine-neutralizing antibodies and chemokine receptor inhibitors, we found that HSCs promoted MDSC migration through the SDF-1/CXCR4 axis. Subsequently, we used an orthotopic mouse liver tumor model to determine how HSCs mediated MDSC migration to HCC in vivo. The in vivo results indicated that pretreatment of MDSCs with a CXCR4 inhibitor or injection with SDF-1-knocked down HSCs inhibited MDSC migration to the spleen and liver of the tumor-bearing mice. Together, our findings indicate a central role for HSCs in MDSC migration mediated by the SDF-1/CXCR4 axis, thus revealing a potentially effective approach for modulating the tumor microenvironment by targeting HSCs in HCC.


Assuntos
Carcinoma Hepatocelular/imunologia , Quimiocina CXCL12/metabolismo , Células Estreladas do Fígado/patologia , Neoplasias Hepáticas/imunologia , Células Supressoras Mieloides/patologia , Receptores CXCR4/metabolismo , Animais , Movimento Celular , Quimiocina CXCL12/genética , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Interferente Pequeno/genética , Transdução de Sinais
2.
Int J Mol Sci ; 20(19)2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31554271

RESUMO

In tumor cells of more than 20 different cancer types, the CXCR4-CXCL12-axis is involved in multiple key processes including proliferation, survival, migration, invasion, and metastasis. Since data on this axis in diffuse large B cell lymphoma (DLBCL) are inconsistent and limited, we comprehensively studied the CXCR4-CXCL12-axis in our DLBCL cohort as well as the effects of CXCR4 antagonists on lymphoma cell lines in vitro. In DLBCL, we observed a 140-fold higher CXCR4 expression compared to non-neoplastic controls, which was associated with poor clinical outcome. In corresponding bone marrow biopsies, we observed a correlation of CXCL12 expression and lymphoma infiltration rate as well as a reduction of CXCR4 expression in remission of bone marrow involvement after treatment. Additionally, we investigated the effects of three CXCR4 antagonists in vitro. Therefore, we used AMD3100 (Plerixafor), AMD070 (Mavorixafor), and WKI, the niacin derivative of AMD070, which we synthesized. WK1 demonstrated stronger pro-apoptotic effects than AMD070 in vitro and induced expression of pro-apoptotic genes of the BCL2-family in CXCR4-positive lymphoma cell lines. Finally, WK1 treatment resulted in the reduced expression of JNK-, ERK1/2- and NF-κB/BCR-target genes. These data indicate that the CXCR4-CXCL12-axis impacts the pathogenesis of DLBCL and represents a potential therapeutic target in aggressive lymphomas.


Assuntos
Apoptose/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/farmacologia , Biomarcadores , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12/genética , Éxons , Feminino , Expressão Gênica , Compostos Heterocíclicos com 1 Anel/farmacologia , Humanos , Linfoma Difuso de Grandes Células B/mortalidade , Linfoma Difuso de Grandes Células B/patologia , Masculino , Mutação , Estadiamento de Neoplasias , Prognóstico , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética
3.
Int Immunopharmacol ; 75: 105778, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31494340

RESUMO

We previously demonstrated that decreased expression of miR-140-5p was associated with the progression of multiple sclerosis (MS) and miR-140-5p targeted STAT1 and interfered with the expression of IFN-γ. However, the underlying mechanisms how miR-140-5p regulated the differentiation of encephalomyelitic CD4+T cell remained unclear. In this study, we analyzed the levels of miR-140-5p in a mouse model of experimental autoimmune encephalomyelitis (EAE). We also analyzed the outcomes in response to either over- or under-expression of miR-140-5p. We found that the expression of miR-140-5p was inversely related to the progression of EAE. With the remission of the disease, the expression of miR-140-5p was restored to levels comparable to the control. The expression of miR-140-5p was downregulated in the encephalomyelitic CD4+T cells whereas enhanced expression of miR-140-5p inhibited the development of T helper type 1 (Th1) cell and significantly attenuated EAE. MiR-140-5p also caused hypermethylation of STAT1 and demethylation of GATA3. Furthermore, we found that miR-140-5p enhanced mitochondrial glycolysis in CD4+T cells with simultaneous activation of ATP activity. By blockage of the respiratory electron transport chain with the inhibitors of complex I and III, the effect of miR-140-5p on Th1 differentiation was blocked, which suggested a role for mitochondrial respiratory pathway in miR-140-5p-mediated inhibition of Th1 differentiation. In summary, our results demonstrated that the expression of miR-140-5p was negatively correlated with the progression of EAE and that miR-140-5p regulated Th1 differentiation via DNA methylation and mitochondrial respiratory pathway.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Metilação de DNA , Encefalomielite Autoimune Experimental/imunologia , MicroRNAs/imunologia , Animais , Diferenciação Celular , Quimiocina CXCL12/genética , Regulação para Baixo , Encefalomielite Autoimune Experimental/genética , Feminino , Lentivirus/genética , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Fator de Transcrição STAT1/genética , Proteína Smad3/genética
4.
BMC Cancer ; 19(1): 802, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412792

RESUMO

BACKGROUND: To validate the utility of the chemokine ligand 12 (CXCL12) as prognostic marker in patients with localized and metastatic germ cell tumors (GCT). METHODS: CXCL12 expression was analyzed on a tissue microarray consisting of 750 tissue cores of different histological tumor components, Germ cell neoplasia in situ (GCNIS) and adjacent normal tissue of 263 testicular cancer patients using a semi-quantitative score. The association between CXCL12 expression and recurrence-free survival (RFS) as well as overall survival (OS) was assessed using Kaplan-Meier curves with log-rank tests. RESULTS: CXCL12 expression was absent in all seminomas but was found in 52 of 99 (52.5%) non-seminomas. Follow-up was available for 260 patients of which 36 (13.8%) recurred. In patients with stage 1 non-seminoma GCT, CXCL12 expression was not associated with higher risk of disease recurrence (p = 0.270). In contrast, post chemotherapy RFS of patients with metastatic non-seminoma and positive CXCL12 expression was significantly shorter compared to CXCL12 negative patients (p = 0.003). OS differences were not statistically different between patients with CXCL12 positive or negative tumors for either localized or metastatic disease. CONCLUSIONS: CXCL12 is almost exclusively expressed in non-seminoma. Pure seminoma, GCNIS and adjacent normal testicular tissue are CXCL12 negative. Our analysis suggests that patients with metastatic disease and a CXCL12-positive non-seminoma are at higher risk for disease recurrence after first-line chemotherapy and might thus be candidates for more intensive treatment and/or closer follow-up.


Assuntos
Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Recidiva Local de Neoplasia , Neoplasias Embrionárias de Células Germinativas/fisiopatologia , Neoplasias Testiculares/fisiopatologia , Adolescente , Adulto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Humanos , Masculino , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Prognóstico , Seminoma/diagnóstico , Seminoma/fisiopatologia , Seminoma/terapia , Análise de Sobrevida , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Adulto Jovem
5.
Mol Cells ; 42(7): 530-545, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31362469

RESUMO

Tumor cells can vary epigenetically during ionizing irradiation (IR) treatment. These epigenetic variegations can influence IR response and shape tumor aggressiveness. However, epigenetic disturbance of histones after IR, implicating in IR responsiveness, has been elusive. Here, we investigate whether altered histone modification after IR can influence radiation responsiveness. The oncogenic CXCL12 mRNA and protein were more highly expressed in residual cancer cells from a hepatoma heterotopic murine tumor microenvironment and coculture of human hepatoma Huh7 and normal IMR90 cells after radiation. H3K4 methylation was also enriched and H3K9 methylation was decreased at its promoter region. Accordingly, invasiveness and the subpopulation of aggressive CD133+/CD24- cells increased after IR. Histone demethylase inhibitor IOX1 attenuated CXCL12 expression and the malignant subpopulation, suggesting that responses to IR can be partially mediated via histone modifications. Taken together, radiation-induced histone alterations at the CXCL12 promoter in hepatoma cells are linked to CXCL12 upregulation and increased aggressiveness in the tumor microenvironment.


Assuntos
Carcinoma Hepatocelular/genética , Quimiocina CXCL12/genética , Histonas/metabolismo , Neoplasias Hepáticas/genética , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Microambiente Tumoral/genética , Regulação para Cima/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimiocina CXCL12/metabolismo , Epigênese Genética/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Compostos Heterocíclicos/farmacologia , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Proteínas Recombinantes/farmacologia , Transcrição Genética/efeitos da radiação , Microambiente Tumoral/efeitos da radiação , Raios X
6.
Int J Mol Med ; 44(3): 927-938, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31257476

RESUMO

The present study aimed to further investigate the effects of high glucose on the function of circulating fibrocytes and its underlying mechanisms. The total peripheral blood mononuclear cells were obtained from normal glucose tolerance patients and type 2 diabetic mellitus patients. Circulating fibrocytes were stimulated with different glucose concentrations for different time periods (24, 48 and 72 h). Cell proliferation was determined by Cell Counting Kit­8 assay. The expression of connective tissue growth factor (CTGF) was detected by western blotting. The expression of COL­I was detected by flow cytometry. The apoptotic bodies of cells were detected by fluorescence microscopy after Hoechst33258 staining. The invasive and migration abilities of fibrocytes were detected by Transwell chamber assay. Secretion of stromal cell­derived factor 1 (SDF­1) was measured by ELISA. The circulating fibrocytes showed a typical spindle­shape and were double­positive for cluster of differentiation 45 (green) and COL­I (red). Compared with the 5.5 mmol/l glucose group, a high glucose concentration significantly promoted the proliferation of circulating fibrocytes and showed the most significant effects at 30 mmol/l after treatment for 48 h. AMD3100 showed no effects on the proliferation of circulating fibrocytes. Flow cytometry revealed that 30 mmol/l glucose significantly promoted the expression of COL­I vs. 5.5 mmol/l glucose group (P<0.01), while AMD3100 reversed this (P<0.05). Hoechst33258 staining showed no differences in the apoptotic bodies between experimental groups (P>0.05). Western blotting revealed that the expression of CTGF was decreased significantly by AMD3100 pretreatment (P<0.01). Transwell chamber assay showed that 30 mmol/l glucose significantly promoted the invasive and transfer abilities (P<0.01) of fibrocytes when compared with the 5.5 mmol/l glucose group. While AMD3100 reversed the cell migratory effects induced by high glucose (P<0.01). In addition, the secretion of SDF­1 stimulated by 30 mmol/l glucose DMEM showed no differences compared with 5.5 mmol/l glucose DMEM (P>0.05). High glucose stimulated the expressions of CTGF and COL­I, and promoted migration of circulating fibrocytes via the CXC chemokine receptor 4/SDF­1 axis.


Assuntos
Glicemia , Células do Tecido Conjuntivo/metabolismo , Glucose/metabolismo , Idoso , Apoptose , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células do Tecido Conjuntivo/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Regulação da Expressão Gênica , Glucose/farmacologia , Compostos Heterocíclicos/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Receptores CXCR4/metabolismo , Transdução de Sinais
7.
Genet Test Mol Biomarkers ; 23(7): 435-441, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31294628

RESUMO

Aims: To discover possible relationships between CXCL12 single nucleotide polymorphisms (SNPs) and type 2 diabetes mellitus (T2DM) and its risk factors. Methods: The present sib-pair study was conducted in a rural community of Beijing, China. SNPs rs2297630, rs1746048, and rs1801157 located within or nearby the CXCL12 gene were genotyped using the allele-specific polymerase chain reaction method. Haseman-Elston regression was used to investigate linkages between these SNPs and T2DM. A generalized estimating equation logistic regression model was used to discover associations between the SNPs, T2DM, and its risk factors. Results: A total of 3171 participants were recruited, comprising 2277 sib pairs. After Bonferroni correction (α = 0.016), rs2297630 was found to be significantly linked to (p = 0.003) and associated with T2DM (AA vs. GG/GA: OR = 2.26, 95% CI: 1.31-3.88, p = 0.003). There were interactions between rs2297630 and dyslipidemia (p < 0.001) and between rs1746048 and hypertension (p = 0.011). Compared to dyslipidemia-free subjects with rs2297630 GG/GA genotypes, dyslipidemia patients with rs2297630 AA had a higher risk of T2DM (OR = 4.15, 95% CI: 2.24-7.67, p < 0.001). Compared to hypertension-free subjects with rs1746048 CC genotypes, hypertension-free subjects with rs1746048 CT/TT had a decreased risk of T2DM (OR = 0.77, 95% CI: 0.60-0.99, p = 0.045). Conclusions: A novel linkage and association was found between rs2297630 and T2DM. Moreover, novel interactions were found between rs2297630 and dyslipidemia as well as rs1746048 and hypertension. These findings will help identify individuals at higher risk of developing T2DM.


Assuntos
Quimiocina CXCL12/genética , Diabetes Mellitus Tipo 2/genética , China , Dislipidemias/genética , Feminino , Interação Gene-Ambiente , Estudos de Associação Genética , Humanos , Estilo de Vida , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de Risco
8.
Cancer Sci ; 110(10): 3197-3203, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31361379

RESUMO

Intrahepatic cholangiocarcinoma is a rare malignant biliary neoplasm that causes a poor prognosis even after curative hepatectomy. Liver metastasis is the major recurrence pattern of intrahepatic cholangiocarcinoma; therefore, the prevention of liver metastasis is a desirable objective. The aim of this study is to identify gene(s) related to liver metastasis of intrahepatic cholangiocarcinoma and to examine the inhibitory effects on metastasis of intrahepatic cholangiocarcinoma by controlling such gene(s). We collected 3 pairs of intrahepatic cholangiocarcinoma frozen samples, and 36 pairs (primary and metastatic lesions) of intrahepatic cholangiocarcinoma formalin-fixed paraffin-embedded samples, from patients who underwent surgical resection at hospitals related to the Kyushu Study Group of Liver Surgery between 2002 and 2016. We carried out cDNA microarray analyses and immunohistochemistry to identify candidate genes, and evaluated one of them as a therapeutic target using human cholangiocarcinoma cell lines. We identified 4 genes related to liver metastasis using cDNA microarray, and found that CXCL12 was the only gene whose expression was significantly higher in liver metastasis than in primary intrahepatic cholangiocarcinoma by immunohistochemistry (P = .003). In prognosis, patients in the high CXCL12 group showed a significantly poor prognosis in disease-free (P < .0001) and overall survival (P = .0004). By knockdown of CXCL12, we could significantly suppress the invasive and migratory capabilities of 2 human cholangiocarcinoma cell lines. Therefore, CXCL12 might be associated with metastasis and poor prognosis in intrahepatic cholangiocarcinoma.


Assuntos
Neoplasias dos Ductos Biliares/patologia , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Colangiocarcinoma/patologia , Neoplasias Hepáticas/secundário , Idoso , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Prognóstico , Regulação para Cima
9.
Anticancer Res ; 39(6): 2891-2902, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31177127

RESUMO

BACKGROUND/AIM: Long-term exposure to betel quid (BQ)-, cigarette-, and alcohol-induced chronic inflammation is a crucial risk factor for oral and pharyngeal squamous cell carcinoma (OPSCC) progression. We analyzed the genotypes of stromal-cell-derived factor-1 (SDF-1) and CXC-chemokine receptor-4 (CXCR4) and determined the association between their polymorphisms and the risk of OPSCC. MATERIALS AND METHODS: This study consisted of 452 patients with pathologically proved OPSCC and 424 sex- and age-matched cancer-free controls. The genotypes of SDF-1 and CXCR4 were detected through the TaqMan real-time polymerase chain reaction (PCR) method. RESULTS: Our data indicated that the C allele and C/C genotypes of CXCR4 were significantly associated with OPSCC [adjusted odds ratio (AOR)=1.41, 95% confidence interval (CI):1.02-1.96, p=0.037 and AOR=1.51, 95% CI:1.05-2.17, p=0.028, respectively] and OSCC (AOR=1.41, 95%CI:1.00-2.00, p=0.049 and AOR=1.49, 95%CI:1.01-2.20, p=0.044, respectively) risk. Patients with genetic polymorphisms of the genotype combination SDF-1/CXCR4 had a higher risk of OSCC (p trend=0.033). We analyzed the effects of CXCR4 genetic variants on susceptibility to OPSCC in patients with different risk habits of BQ chewing, tobacco smoking and alcohol consumption, and revealed that C/T+T/T genotypes exerted an increased risk only in patients with one (AOR=2.68, p=0.036) or two risk habits (AOR=2.02, p=0.027) compared to patients with the C/C genotype. CONCLUSION: We concluded that CXCR4 C>T can be used as a genetic marker of susceptibility to OPSCC, particularly in OPSCC patients with one or two types of risk habits with a synergistic effect.


Assuntos
Carcinoma de Células Escamosas/genética , Quimiocina CXCL12/genética , Neoplasias Bucais/genética , Neoplasias Faríngeas/genética , Polimorfismo Genético , Receptores CXCR4/genética , Progressão da Doença , Etanol/efeitos adversos , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/induzido quimicamente , Neoplasias Faríngeas/induzido quimicamente , Polimorfismo de Nucleotídeo Único , Taiwan , Tabaco sem Fumaça/efeitos adversos
10.
BMB Rep ; 52(7): 463-468, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31186083

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic diseases (frequency of 1/1000-1/400), is characterized by numerous fluid-filled renal cysts (RCs). Inactivation of the PKD1 or PKD2 gene by germline and somatic mutations is necessary for cyst formation in ADPKD. To mechanistically understand cyst formation and growth, we isolated RCs from Korean patients with ADPKD and immortalized them with human telomerase reverse transcriptase (hTERT). Three hTERT-immortalized RC cell lines were characterized as proximal epithelial cells with germline and somatic PKD1 mutations. Thus, we first established hTERT-immortalized proximal cyst cells with somatic PKD1 mutations. Through transcriptome sequencing and Gene Ontology (GO) analysis, we found that upregulated genes were related to cell division and that downregulated genes were related to cell differentiation. We wondered whether the upregulated gene for the chemokine CXCL12 is related to the mTOR signaling pathway in cyst growth in ADPKD. CXCL12 mRNA expression and secretion were increased in RC cell lines. We then examined CXCL12 levels in RC fluids from patients with ADPKD and found increased CXCL12 levels. The CXCL12 receptor CXC chemokine receptor 4 (CXCR4) was upregulated, and the mTOR signaling pathway, which is downstream of the CXCL12/CXCR4 axis, was activated in ADPKD kidney tissue. To confirm activation of the mTOR signaling pathway by CXCL12 via CXCR4, we treated the RC cell lines with recombinant CXCL12 and the CXCR4 antagonist AMD3100; CXCL12 induced the mTOR signaling pathway, but the CXCR4 antagonist AMD3100 blocked the mTOR signaling pathway. Taken together, these results suggest that enhanced CXCL12 in RC fluids activates the mTOR signaling pathway via CXCR4 in ADPKD cyst growth. [BMB Reports 2019; 52(7): 463-468].


Assuntos
Quimiocina CXCL12/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Células Cultivadas , Quimiocina CXCL12/genética , Humanos , Mutação , Rim Policístico Autossômico Dominante/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/genética , Telomerase/metabolismo , Regulação para Cima
11.
Int J Mol Sci ; 20(9)2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31071921

RESUMO

Activation of multiple pathways is associated with cardiac hypertrophy and heart failure. We previously published that CXCR4 negatively regulates ß-adrenergic receptor (ß-AR) signaling and ultimately limits ß-adrenergic diastolic (Ca2+) accumulation in cardiac myocytes. In isolated adult rat cardiac myocytes; CXCL12 treatment prevented isoproterenol-induced hypertrophy and interrupted the calcineurin/NFAT pathway. Moreover; cardiac specific CXCR4 knockout mice show significant hypertrophy and develop cardiac dysfunction in response to chronic catecholamine exposure in an isoproterenol-induced (ISO) heart failure model. We set this study to determine the structural and functional consequences of CXCR4 myocardial knockout in the absence of exogenous stress. Cardiac phenotype and function were examined using (1) gated cardiac magnetic resonance imaging (MRI); (2) terminal cardiac catheterization with in vivo hemodynamics; (3) histological analysis of left ventricular (LV) cardiomyocyte dimension; fibrosis; and; (4) transition electron microscopy at 2-; 6- and 12-months of age to determine the regulatory role of CXCR4 in cardiomyopathy. Cardiomyocyte specific-CXCR4 knockout (CXCR4 cKO) mice demonstrate a progressive cardiac dysfunction leading to cardiac failure by 12-months of age. Histological assessments of CXCR4 cKO at 6-months of age revealed significant tissue fibrosis in knockout mice versus wild-type. The expression of atrial naturietic factor (ANF); a marker of cardiac hypertrophy; was also increased with a subsequent increase in gross heart weights. Furthermore, there were derangements in both the number and the size of the mitochondria within CXCR4 cKO hearts. Moreover, CXCR4 cKO mice were more sensitive to catocholamines, their response to ß-AR agonist challenge via acute isoproterenol (ISO) infusion demonstrated a greater increase in ejection fraction, dp/dtmax, and contractility index. Interestingly, prior to ISO infusion, there were significant differences in baseline hemodynamics between the CXCR4 cKO compared to littermate controls. However, upon administering ISO, the CXCR4 cKO responded in a robust manner overcoming the baseline hemodynamic deficits reaching WT values supporting our previous data that CXCR4 negatively regulates ß-AR signaling. This further supports that, in the absence of the physiologic negative modulation, there is an overactivation of down-stream pathways, which contribute to the development and progression of contractile dysfunction. Our results demonstrated that CXCR4 plays a non-developmental role in regulating cardiac function and that CXCR4 cKO mice develop a progressive cardiomyopathy leading to clinical heart failure.


Assuntos
Cardiomiopatias/genética , Insuficiência Cardíaca/genética , Receptores CXCR4/genética , Animais , Fator Natriurético Atrial/genética , Cardiomiopatias/fisiopatologia , Quimiocina CXCL12/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Isoproterenol/administração & dosagem , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Receptores Adrenérgicos beta/genética , Transdução de Sinais/genética
12.
Nucleic Acids Res ; 47(11): 5465-5479, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31034558

RESUMO

Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.


Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Oligonucleotídeos Antissenso/química , Organofosfonatos/química , Oligonucleotídeos Fosforotioatos/química , Células 3T3-L1 , Animais , Caspases/metabolismo , Linhagem Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células HeLa , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Fosforotioatos/administração & dosagem , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo
13.
Cancer Biomark ; 25(1): 115-126, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31006667

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies, and its global morbidity and mortality are increasing. Previous studies confirmed that miR-342 was involved in the development and progression of malignant tumors. However, the relationship between miR-342 and Wnt/ß-catenin signaling pathway in HCC remains unknown. MATERIALS AND METHODS: Cell viability was detected by MTT assay. Immunofluorescence staining was used to detect Brdu-positive cells and Western blot was used to detect the apoptotic proteins. Furthermore, linear correlation analysis was used to investigate the possible relationship between miR-342 and the downstream genes of Wnt/ß-catenin signaling pathway in the progression of HCC. RESULTS: Over-expression of miR-342 significantly reduced cell proliferation and obviously increased apoptosis in HCC, while silencing of miR-342 showed an opposite effect on HCC cell proliferation and apoptosis. In addition, we found that the CXCL12 was the target gene of miR-342. This study also demonstrated that miR-342 up-regulation suppressed Wnt/ß-catenin signaling pathway by inhibiting CXCL12 expression. CONCLUSION: Up-regulation of miR-342 inhibited cell proliferation and induced cell apoptosis in HCC by inhibiting Wnt/ß-catenin signaling pathway, suggesting that miR-342 might act as a promising tumor gene therapeutic target for HCC patients.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , MicroRNAs/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade
14.
Arterioscler Thromb Vasc Biol ; 39(6): 1113-1124, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31018661

RESUMO

Objective- To determine the role of the oncofetal protein TPBG (trophoblast glycoprotein) in normal vascular function and reparative vascularization. Approach and Results- Immunohistochemistry of human veins was used to show TPBG expression in vascular smooth muscle cells and adventitial pericyte-like cells (APCs). ELISA, Western blot, immunocytochemistry, and proximity ligation assays evidenced a hypoxia-dependent upregulation of TPBG in APCs not found in vascular smooth muscle cells or endothelial cells. This involves the transcriptional modulator CITED2 (Atypical chemokine receptor 3 CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich tail) and downstream activation of CXCL12 (chemokine [C-X-C motif] ligand-12) signaling through the CXCR7 (C-X-C chemokine receptor type 7) receptor and ERK1/2 (extracellular signal-regulated kinases 1/2). TPBG silencing by siRNA transfection downregulated CXCL12, CXCR7, and pERK (phospho Thr202/Tyr204 ERK1/2) and reduced the APC migratory and proangiogenic capacities. TPBG forced expression induced opposite effects, which were associated with the formation of CXCR7/CXCR4 (C-X-C chemokine receptor type 4) heterodimers and could be contrasted by CXCL12 and CXCR7 neutralization. In vivo Matrigel plug assays using APCs with or without TPBG silencing evidenced TPBG is essential for angiogenesis. Finally, in immunosuppressed mice with limb ischemia, intramuscular injection of TPBG-overexpressing APCs surpassed naïve APCs in enhancing perfusion recovery and reducing the rate of toe necrosis. Conclusions- TPBG orchestrates the migratory and angiogenic activities of pericytes through the activation of the CXCL12/CXCR7/pERK axis. This novel mechanism could be a relevant target for therapeutic improvement of reparative angiogenesis.


Assuntos
Movimento Celular , Glicoproteínas de Membrana/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Pericitos/metabolismo , Veia Safena/metabolismo , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Membro Posterior , Humanos , Isquemia/genética , Isquemia/metabolismo , Isquemia/fisiopatologia , Isquemia/cirurgia , Masculino , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Nus , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Pericitos/transplante , Fosforilação , Receptores CXCR/genética , Receptores CXCR/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo
15.
Transpl Immunol ; 55: 101206, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31009686

RESUMO

BACKGROUND: Hematological abnormalities after transplantation are complications that may arise after renal transplantation, of which thrombocytopenia is associated with increased risk of bleeding and other complications. The development of thrombocytopenia is affected by various clinical conditions, and the stromal-derived factor 1 (SDF1) and platelet factor 4 (PF4) genes are known to be involved in the production or destruction of platelets. The purpose of this study was to investigate the prevalence of posttransplant thrombocytopenia and its association with other clinical conditions and genetic polymorphisms of SDF1 and PF4 genes a long time after transplantation. METHODS: This is a retrospective study that includes a total of 305 kidney transplant (KT) recipients between 2008 and 2012 at St. Vincent Medical Center, Los Angeles, CA. In this study, posttransplant thrombocytopenia was defined as a 30% reduction in platelet count from the baseline in the first week or a decrease of <100 (×103/µL) within 1 year after KT. The subjects were divided into posttransplant thrombocytopenia and control groups. The chi-square test, t-test, and logistic regression were used for the analyses. RESULTS: In the first week, 65 patients had a 30% reduction in platelet count (21.3%). Gender, simultaneous kidney-pancreas transplantation, induction therapy (IT), and only alleles of rs2297630 of SDF1, among the SDF1 and PF4 genes, showed statistically significant differences. The rs2297630 alleles were consistently significant risk factors (non G vs. G: odds ratio = 0.445; 95% confidence interval, 0.224-0.884; p = .021) in the multiple logistic regression. In the 1-year study, 61 patients (20.0%) had platelet counts of <100 × 103/µL and had statistically significant differences in patients who had delayed graft function and induction therapy. CONCLUSIONS: In this study, non-G group of rs2297630 in SDF1 significantly increased the risk of post-transplant thrombocytopenia in the first week of kidney transplantation.


Assuntos
Alelos , Quimiocina CXCL12/genética , Transplante de Rim , Fator Plaquetário 4/genética , Polimorfismo Genético , Trombocitopenia/genética , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Fatores Sexuais , Trombocitopenia/epidemiologia , Trombocitopenia/etiologia , Fatores de Tempo
16.
J Orthop Res ; 37(6): 1429-1439, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30977556

RESUMO

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous population of cells that yields a homogeneous population of plastic-adherent adipose tissue-derived stromal cells (ASC) when culture-expanded. SVF and ASC have been used clinically to improve tendon healing, yet their mechanism of action is not fully elucidated. The objective of this study was to investigate the potential for ASC to act as trophic mediators for tendon healing. Flexor digitorum superficialis tendons and adipose tissue were harvested from adult horses to obtain SVF, ASC, and tenocytes. Growth factor gene expression was quantified in SVF and ASC in serial passages and growth factors were quantified in ASC-conditioned medium (CM). Microchemotaxis assays were performed using ASC-CM. Tenocytes were grown in co-culture with autologous ASC or allogeneic SVF. Gene expression for insulin-like growth factor 1 (IGF-1), stromal cell-derived factor-1α (SDF-1α), transforming growth factor-ß1 (TGF-ß1) and TGF-ß3 was significantly higher in SVF compared to ASC. Concentrations were significantly increased in ASC-CM compared to controls for IGF-1 (4-fold) and SDF-1α (6-fold). Medium conditioned by ASC induced significant cell migration in a dose-dependent manner. Gene expression for collagen types I and III, decorin, and cartilage oligomeric matrix protein was modestly, but significantly increased following co-culture of tenocytes with autologous ASC. Our findings support the ability of SVF and ASC to act as trophic mediators in tendon healing, particularly through chemotaxis, which stands to critically impact the intrinsic healing response. In vivo studies to further delineate the potential for SVF and/or ASC to improve tendon healing are warranted. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1429-1439, 2019.


Assuntos
Tecido Adiposo/citologia , Células Estromais/fisiologia , Tendões/fisiologia , Animais , Células Cultivadas , Quimiocina CXCL12/análise , Quimiocina CXCL12/genética , Técnicas de Cocultura , Cavalos , Fator de Crescimento Insulin-Like I/análise , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/genética
17.
Diabetes ; 68(6): 1303-1314, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30936144

RESUMO

Diabetes impairs the mobilization of hematopoietic stem/progenitor cells (HSPCs) from the bone marrow (BM), which can worsen the outcomes of HSPC transplantation and of diabetic complications. In this study, we examined the oncostatin M (OSM)-p66Shc pathway as a mechanistic link between HSPC mobilopathy and excessive myelopoiesis. We found that streptozotocin-induced diabetes in mice skewed hematopoiesis toward the myeloid lineage via hematopoietic-intrinsic p66Shc. The overexpression of Osm resulting from myelopoiesis prevented HSPC mobilization after granulocyte colony-stimulating factor (G-CSF) stimulation. The intimate link between myelopoiesis and impaired HSPC mobilization after G-CSF stimulation was confirmed in human diabetes. Using cross-transplantation experiments, we found that deletion of p66Shc in the hematopoietic or nonhematopoietic system partially rescued defective HSPC mobilization in diabetes. Additionally, p66Shc mediated the diabetes-induced BM microvasculature remodeling. Ubiquitous or hematopoietic restricted Osm deletion phenocopied p66Shc deletion in preventing diabetes-associated myelopoiesis and mobilopathy. Mechanistically, we discovered that OSM couples myelopoiesis to mobilopathy by inducing Cxcl12 in BM stromal cells via nonmitochondrial p66Shc. Altogether, these data indicate that cell-autonomous activation of the OSM-p66Shc pathway leads to diabetes-associated myelopoiesis, whereas its transcellular hematostromal activation links myelopoiesis to mobilopathy. Targeting the OSM-p66Shc pathway is a novel strategy to disconnect mobilopathy from myelopoiesis and restore normal HSPC mobilization.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Mielopoese/genética , Oncostatina M/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Adulto , Idoso , Animais , Transplante de Medula Óssea , Quimiocina CXCL12/genética , Diabetes Mellitus/metabolismo , Feminino , Fator Estimulador de Colônias de Granulócitos , Mobilização de Células-Tronco Hematopoéticas , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Oncostatina M/metabolismo , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Células-Tronco
18.
J Biol Chem ; 294(20): 8023-8036, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30936203

RESUMO

G protein-coupled receptor (GPCR) signaling is regulated by members of the protein kinase C (PKC) and GPCR kinase (GRK) families, although the relative contribution of each to GPCR function varies among specific GPCRs. The CXC motif receptor 4 (CXCR4) is a member of the GPCR superfamily that binds the CXC motif chemokine ligand 12 (CXCL12), initiating signaling that is subsequently terminated in part by internalization and lysosomal degradation of CXCR4. The purpose of this study is to define the relative contribution of PKC and GRK to CXCR4 signaling attenuation by studying their effects on CXCR4 lysosomal trafficking and degradation. Our results demonstrate that direct activation of PKC via the phorbol ester phorbol 12-myristate 13-acetate (PMA) mimics CXCL12-mediated desensitization, internalization, ubiquitination, and lysosomal trafficking of CXCR4. In agreement, heterologous activation of PKC by stimulating the chemokine receptor CXCR5 with its ligand, CXCL13, also mimics CXCL12-mediated desensitization, internalization, ubiquitination, and lysosomal degradation of CXCR4. Similar to CXCL12, PMA promotes PKC-dependent phosphorylation of serine residues within CXCR4 C-tail that are required for binding and ubiquitination by the E3 ubiquitin ligase AIP4 (atrophin-interacting protein 4). However, inhibition of PKC activity does not alter CXCL12-mediated ubiquitination and degradation of CXCR4, suggesting that other kinases are also required. Accordingly, siRNA-mediated depletion of GRK6 results in decreased degradation and ubiquitination of CXCR4. Overall, these results suggest that PKC and GRK6 contribute to unique aspects of CXCR4 phosphorylation and lysosomal degradation to ensure proper signal propagation and termination.


Assuntos
Lisossomos/metabolismo , Proteólise , Receptores CXCR4/metabolismo , Transdução de Sinais , Ubiquitinação , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Quimiocina CXCL13/genética , Quimiocina CXCL13/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Quinases de Receptores Acoplados a Proteína G/genética , Quinases de Receptores Acoplados a Proteína G/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Receptores CXCR4/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
19.
Biosci Biotechnol Biochem ; 83(6): 1072-1076, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30870116

RESUMO

Diabetes induced a serious of complications including diabetic retinopathy. Our study aimed to investigate the role of Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 in diabetic retinopathy. A mice model of diabetic retinopathy was established, and expression of SDF-1 and CXCR4 in retina was examined by Real-time quantitative PCR (qRT-PCR). Cells of human retinal pigment epithelial cell line ARPE-19 were treated with CXCR4 siRNAs and expression vector, and cell viability was detected by MTT assay. We found that expression of SDF-1 and CXCR4 in retina was significantly downregulated in mice with diabetic retinopathy than in normal healthy mice. High glucose treatment downregulated the expression of SDF-1 and CXCR4 in ARPE-19 cells at both mRNA and protein levels. Transfection with CXCR4 siRNAs decreased, while transfection with CXCR4 expression vector increased cell viability under high glucose treatment. We concluded that SDF-1/CXCR4 pathway improved diabetic retinopathy possibly by increasing cell viability. Abbreviations: SDF-1: Stromal cell-derived factor 1; CXCL12: C-X-C motif chemokine 12; qRT-PCR: Real-time quantitative PCR.


Assuntos
Quimiocina CXCL12/fisiologia , Retinopatia Diabética/fisiopatologia , Receptores CXCR4/fisiologia , Animais , Linhagem Celular , Sobrevivência Celular , Quimiocina CXCL12/genética , Retinopatia Diabética/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Inativação Gênica , Glucose/administração & dosagem , Humanos , Masculino , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores CXCR4/genética , Epitélio Pigmentado da Retina/citologia
20.
DNA Cell Biol ; 38(5): 457-467, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864829

RESUMO

Degenerative disc disease (DDD) is the main cause of low back pain, and the ingrowth of new blood vessels is one of its pathological features. The stromal cell-derived factor 1 (SDF1)/CXCR7 signaling axis plays a role in these physiological and pathological activities. The aims of this study were to explore whether this signaling axis participates in the angiogenesis of degenerated intervertebral discs (IVDs) and to define its underlying mechanism. In this study, we cocultured human nucleus pulposus cells (NPCs) and vascular endothelial cells (VECs) and regulated the expression of SDF1/CXCR7 to investigate the effect of VEC angiogenesis by NPCs. The results revealed that angiogenesis was enhanced with increased SDF1 and that angiogenesis was weakened with the inhibition of CXCR7. We found that PI3K/AKT was involved in the downstream pathway in the coculture. VEC angiogenesis induction by NPCs was enhanced with an increase in pAKT or a decrease in PTEN. We conclude that the SDF1/CXCR7 signaling axis plays a role in the angiogenesis of degenerated IVD through the PI3K/AKT pathway.


Assuntos
Quimiocina CXCL12/metabolismo , Degeneração do Disco Intervertebral/patologia , Neovascularização Patológica/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR/metabolismo , Idoso , Células Cultivadas , Quimiocina CXCL12/genética , Técnicas de Cocultura , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Receptores CXCR/genética , Transdução de Sinais
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