RESUMO
The gradual loss of kidney function due to increasing age is accompanied by structural changes such as fibrosis of the tissue. The underlying molecular mechanisms are complex, but not yet fully understood. Non-fibrillar collagen type VIII (COL8) could be a potential factor in the fibrosis processes of the aging kidney. A pathophysiological significance of COL8 has already been demonstrated in the context of diabetic kidney disease, with studies showing that it directly influences both the development and progression of renal fibrosis occurring. The aim of this study was to investigate whether COL8 impacts age-related micro-anatomical and functional changes in a mouse model. The kidneys of wild-type (Col8-wt) and COL8-knockout (Col8-ko) mice of different age and sex were characterized with regard to the expression of molecular fibrosis markers, the development of nephrosclerosis and renal function. The age-dependent regulation of COL8 mRNA expression in the wild-type revealed sex-dependent effects that were not observed with collagen IV (COL4). Histochemical staining and protein analysis of profibrotic cytokines TGF-ß1 (transforming growth factor) and CTGF (connective tissue growth factor) in mouse kidneys showed significant age effects as well as interactions of the factors age, sex and Col8 genotype. There were also significant age and Col8 genotype effects in the renal function data analyzed by urinary cystatin C. In summary, the present study shows, for the first time, that COL8 is regulated in an age- and sex-dependent manner in the mouse kidney and that the expression of COL8 influences the severity of age-induced renal fibrosis and function.
Assuntos
Envelhecimento , Colágeno Tipo VIII , Fator de Crescimento do Tecido Conjuntivo , Fibrose , Rim , Animais , Feminino , Masculino , Camundongos , Envelhecimento/metabolismo , Colágeno Tipo VIII/metabolismo , Colágeno Tipo VIII/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Rim/metabolismo , Rim/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/genéticaRESUMO
A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early-onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.
Assuntos
Sistemas CRISPR-Cas/genética , Códon de Iniciação/genética , Distrofia Endotelial de Fuchs , Edição de Genes/métodos , Animais , Colágeno Tipo VIII/genética , Modelos Animais de Doenças , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Psoriasis (Ps) and Psoriatic Arthritis (PsA) are characterized by a multifactorial etiology, involving genetic and environmental factors. The present study aimed to investigate polymorphisms (SNPs) within genes involved in extracellular matrix and cell homeostasis and microRNA genes as susceptibility biomarkers for Ps and PsA. Bioinformatic analysis on public RNA-seq data allowed for selection of rs12488457 (A/C, COL6A5), rs13081855 (G/T, COL8A1), rs3812111 (A/T, COL10A1) and rs2910164 (C/G, MIR146A) as candidate biomarkers. These polymorphisms were analyzed by Real-Time PCR in a cohort of 1417 Italian patients (393 Ps, 424 PsA, 600 controls). Statistical and bioinformatic tools were utilized for assessing the genetic association and predicting the effects of the selected SNPs. rs12488457, rs13081855 and rs2910164 were significantly associated with both Ps (p = 1.39 × 10-8, p = 4.52 × 10-4, p = 0.04, respectively) and PsA (p = 5.12 × 10-5, p = 1.19 × 10-6, p = 0.01, respectively). rs3812111, instead, was associated only with PsA (p = 0.005). Bioinformatic analysis revealed common and differential biological pathways involved in Ps and PsA. COL6A5 and COL8A1 take part in the proliferation and angiogenic pathways which are altered in Ps/PsA and contribute to inflammation together with MIR146A. On the other hand, the exclusive association of COL10A1 with PsA highlighted the specific involvement of bone metabolism in PsA.
Assuntos
Colágeno Tipo VIII/metabolismo , Colágeno Tipo VI/metabolismo , Colágeno Tipo X/metabolismo , Predisposição Genética para Doença/genética , MicroRNAs/metabolismo , Psoríase/metabolismo , Adulto , Idoso , Artrite Psoriásica/genética , Artrite Psoriásica/metabolismo , Biomarcadores/sangue , Estudos de Coortes , Colágeno Tipo VI/genética , Colágeno Tipo VIII/genética , Colágeno Tipo X/genética , Bases de Dados Genéticas , Feminino , Genótipo , Humanos , Itália , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Psoríase/genética , RNA-SeqRESUMO
Diabetic retinopathy is a common complication of diabetes mellitus that causes pathogenic damage to the retina. Particularly, the proliferative diabetic retinopathy (PDR) state can cause abnormal angiogenesis in the retina tissues and trigger the retina destruction in advanced stage. In the clinic, the symptoms during the initiation and progression of PDR are relatively unrecognizable. Therefore, various studies have focused on the pathogenesis of PDR. According to published literature, genetic contributions play an irreplaceable role in the initiation and progression of PDR. Although many computational methods, such as shortest path- and random walk with restart-based methods, have been applied in screening the potential pathogenic factors of PDR, advanced computational methods, which may provide essential supplements for previous ones, are still widely needed. In this study, a novel computational method was presented to infer novel PDR-associated genes. Different from previous methods, the method used in this work employed a different network algorithm, that is, the Laplacian heat diffusion algorithm. This algorithm was applied on the protein-protein interaction network reported in the STRING database. Three screening tests were performed to filter the most likely inferred genes. A total of 26 genes were accessed using the proposed method. Compared with the two previous predictions, most of the identified genes were novel, and only one gene was shared. Several inferred genes, such as CSF3, COL18A1, CXCR2, CCR1, FGF23, CXCL11, and IL13, were related to the pathogenesis of PDR.
Assuntos
Algoritmos , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/genética , Predisposição Genética para Doença/genética , Temperatura Alta , Mapas de Interação de Proteínas/fisiologia , Quimiocina CXCL11/genética , Colágeno Tipo VIII/genética , Colágeno Tipo XVIII , Progressão da Doença , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Estudos de Associação Genética , Humanos , Receptores CCR1/genética , Receptores de Interleucina-8B/genética , RetinaRESUMO
BACKGROUND: The composition and function of the adipose tissue covering the heart are poorly known. In this study, we have investigated the epicardial adipose tissue (EAT) covering the cardiac ventricular muscle and the EAT covering the left anterior descending artery (LAD) on the human heart, to identify their resident stem cell functional activity. METHODS: EAT covering the cardiac ventricular muscle was isolated from the apex (avoiding areas irrigated by major vessels) of the heart (ventricular myocardium adipose tissue (VMAT)) and from the area covering the epicardial arterial sulcus of the LAD (PVAT) in human hearts excised during heart transplant surgery. Adipose stem cells (ASCs) from both adipose tissue depots were immediately isolated and phenotypically characterized by flow cytometry. The different behavior of these ASCs and their released secretome microvesicles (MVs) were investigated by molecular and cellular analysis. RESULTS: ASCs from both VMAT (mASCs) and the PVAT (pASCs) were characterized by the expression of CD105, CD44, CD29, CD90, and CD73. The angiogenic-related genes VEGFA, COL18A1, and TF, as well as the miRNA126-3p and miRNA145-5p, were analyzed in both ASC types. Both ASCs were functionally able to form tube-like structures in three-dimensional basement membrane substrates. Interestingly, pASCs showed a higher level of expression of VEGFA and reduced level of COL18A1 than mASCs. Furthermore, MVs released by mASCs significantly induced human microvascular endothelial cell migration. CONCLUSION: Our study indicates for the first time that the resident ASCs in human epicardial adipose tissue display a depot-specific angiogenic function. Additionally, we have demonstrated that resident stem cells are able to regulate microvascular endothelial cell function by the release of MVs.
Assuntos
Tecido Adiposo/citologia , Expressão Gênica , Células-Tronco/metabolismo , Movimento Celular , Micropartículas Derivadas de Células/metabolismo , Colágeno Tipo VIII/genética , Colágeno Tipo VIII/metabolismo , Colágeno Tipo XVIII , Vasos Coronários/citologia , Meios de Cultivo Condicionados/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Pericárdio/citologia , Células-Tronco/citologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Objective: The aim of this study was to investigate genetic factors associated with idiopathic choroidal neovascularization (ICNV). Methods: We conducted a case-control study including 69 cases with ICNV and 114 controls who underwent cataract surgery. Single nucleotide polymorphisms (SNPs) from genes reported to be related to AMD, CNV and uveitis were selected for this study. Results: In an univariate analysis, the rs669676 SNP located in the COL8A1 gene was associated with the proportion of people who has idiopathic CNV ( X2 = 9.3453, corrected p-value = 0.1). For the rs669676 SNP, minor allele homozygotes, in the dominant model of genotype analysis (GG versus AA-GA), it showed significant differences in the ICNV group vs controls (p = .01, OR = 1.219 (95%CI: 1.04-1.429)). Conclusions: The rs669676 SNP located in the COL8A1 gene may contribute to a genetic susceptibility for ICNV.
Assuntos
Neovascularização de Coroide/genética , Neovascularização de Coroide/patologia , Colágeno Tipo VIII/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Seguimentos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Epithelial keratinization involves complex cellular modifications that provide protection against pathogens and chemical and mechanical injuries. In the oral cavity, keratinized mucosa is also crucial to maintain healthy periodontal or peri-implant tissues. In this study, we investigated the roles of type XVIII collagen, a collagen-glycosaminoglycan featuring an extracellular matrix component present in the basement membrane, in oral mucosal keratinization. Histological analysis of keratinized and non-keratinized oral mucosa showed that type XVIII collagen was highly expressed in keratinized mucosa. Additionally, a 3D culture system using human squamous carcinoma cells (TR146) was used to evaluate and correlate the changes in the expression of type XVIII collagen gene, COL18A1, and epithelial keratinization-related markers, e.g., keratin 1 (KRT1) and 10 (KRT10). The results showed that the increase in COL18A1 expression followed the increase in KRT1 and KRT10 mRNA levels. Additionally, loss-of-function analyses using silencing RNA targeting COL18A1 mRNA and a Col18-knockout (KO) mouse revealed that the absence of type XVIII collagen induces a dramatic decrease in KRT10 expression as well as in the number and size of keratohyalin granules. Together, the results of this study demonstrate the importance of type XVIII collagen in oral mucosal keratinization.
Assuntos
Colágeno Tipo XVIII/metabolismo , Grânulos Citoplasmáticos/metabolismo , Queratinas/metabolismo , Mucosa Bucal/metabolismo , Animais , Biomarcadores , Diferenciação Celular/genética , Colágeno Tipo VIII/genética , Colágeno Tipo VIII/metabolismo , Colágeno Tipo XVIII/genética , Imunofluorescência , Humanos , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: Increased collagen expression and deposition are associated with cancer progression and poor prognosis in breast cancer patients. However, function and regulation of membrane-associated collagen in breast cancer have not been determined. Collagen XIII is a type II transmembrane protein within the collagen superfamily. Experiments in tissue culture and knockout mouse models show that collagen XIII is involved in cell adhesion and differentiation of certain cell types. In the present study, we determined roles of collagen XIII in breast cancer progression and metastasis. METHODS: We analyzed the association of collagen XIII expression with breast cancer development and metastasis using published gene expression profiles generated from human breast cancer tissues. Utilizing gain- and loss- of function approaches and 3D culture assays, we investigated roles of collagen XIII in regulating invasive tumor growth. Using the tumorsphere/mammosphere formation assay and the detachment cell culture assay, we determined whether collagen XIII enhances cancer cell stemness and induces anoikis resistance. We also inhibited collagen XIII signaling with ß1 integrin function-blocking antibody. Finally, using the lung colonization assay and the orthotopic mammary tumor model, we investigated roles of collagen XIII in regulating breast cancer colonization and metastasis. Cox proportional hazard (log-rank) test, two-sided Student's t-test (two groups) and one-way ANOVA (three or more groups) analyses were used in this study. RESULTS: Collagen XIII expression is significantly higher in human breast cancer tissue compared with normal mammary gland. Increased collagen XIII mRNA levels in breast cancer tissue correlated with short distant recurrence free survival. We showed that collagen XIII expression promoted invasive tumor growth in 3D culture, enhanced cancer cell stemness, and induced anoikis resistance. Collagen XIII expression induced ß1 integrin activation. Blocking ß1 integrin activation significantly reduced collagen XIII-induced invasion and mammosphere formation. Importantly, silencing collagen XIII in MDA-MB-231 cells reduced lung colonization and metastasis. CONCLUSIONS: Our results demonstrate a novel function of collagen XIII in promoting cancer metastasis, cell invasion, and anoikis resistance.
Assuntos
Anoikis , Neoplasias da Mama/metabolismo , Colágeno Tipo VIII/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Linhagem Celular , Linhagem Celular Tumoral , Colágeno Tipo VIII/genética , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Proteínas de Membrana/genética , Camundongos SCID , Interferência de RNA , Terapêutica com RNAi/métodos , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Collagen type VIII alpha 1 chain (COL8A1) is a component of the extracellular matrix. Our previous studies suggested that COL8A1 is associated with the proliferation of muscle-derived satellite cells (MDSCs). Additionally, it has been demonstrated that COL8A1 promotes the proliferation of smooth muscle cells and liver cancer cells. Therefore, we predicted that COL8A1 is associated with the proliferation of bovine MDSCs, which have potential applications in research. In this study, we constructed vectors to activate and repress COL8A1 in bovine MDSCs using the CRISPR/Cas9 technique and determined the effects of COL8A1 modulation by EdU labeling, Western blotting, and dual-luciferase reporter assays. The results showed that activation of COL8A1 increased the number of EdU-positive cells and expression of the proliferation markers cyclin B1 (CCNB1) and P-AKT. The expression of P-Akt was unchanged after addition of LY294002 (a protein kinase inhibitor capable of blocking the signal transduction pathway of the phosphoinositide 3-kinase). In contrast, repression of COL8A1 reduced the number of EdU-positive cells and expression of CCNB1 and P-AKT. We also observed upregulation and downregulation of COL8A1 following the overexpression and repression of EGR1, respectively. The dual-luciferase reporter assay revealed that EGR1 regulates the promoter activity of COL8A1. To our knowledge, this is the first study demonstrating that EGR1 positively regulates the expression of COL8A1, which in turn promotes the proliferation of bovine MDSCs via the PI3 K/AKT signaling pathway.
Assuntos
Colágeno Tipo VIII/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Animais , Sistemas CRISPR-Cas , Bovinos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Colágeno Tipo VIII/genética , Ciclina B1/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Edição de Genes/métodos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
BACKGROUNDS AND AIMS: Human colon adenocarcinoma is one of the major causes of tumor-induced death worldwide. A complicated gene interconnection network significantly regulates its progression and prognosis. The aim of our study was to find hub genes associated with the progression and prognosis of colon adenocarcinoma and to illustrate the underlying mechanisms. METHODS: A weighted gene co-expression network analysis was performed in our study to identify significant gene modules and hub genes associated with the TNM stage of colon adenocarcinoma (n = 441). RESULTS: In the turquoise module of interest, 23 hub genes were initially selected, and 10 of them were identified as "real" hub genes with high connectivity in the protein-protein interaction network. In the terms of validation, COL8A1 had the highest correlation with clinical traits among all of the hub genes. Data obtained from the Oncomine and GEPIA databases showed a higher expression of COL8A1 in colon adenocarcinoma tissues compared with normal colon tissues. Kaplan-Meier survival curves showed that higher expression of COL8A1 resulted in a shorter overall survival time and disease-free survival time. Univariate and multivariate Cox proportional hazards analyses indicated that the COL8A1 expression was an independent prognostic factor for survival in colon adenocarcinoma patients. Finally, gene set enrichment analysis indicated that the gene sets associated with focal adhesion were significantly enriched in colon adenocarcinoma samples with COL8A1 highly expressed. CONCLUSIONS: COL8A1 was identified and proved to be correlated with the progression and prognosis of human colon adenocarcinoma, probably through regulating focal adhesion-related pathways.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Colágeno Tipo VIII/genética , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Adenocarcinoma/diagnóstico , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/mortalidade , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Análise de SobrevidaRESUMO
BACKGROUND: Antiangiogenic therapies are considered promising for the treatment of glioblastoma (GB). The non-collagenous C-terminal globular NC1 domain of type VIII collagen a1 chain, Vastatin, is an endogenous antiangiogenic polypeptide. Sustained enhanced expression of Vastatin was shown to inhibit tumour growth and metastasis in murine hepatocellular carcinoma models. In this study, we further explored the efficacy of Vastatin in the treatment of GB xenografts. METHOD: Treatment of Vastatin was carried out using a nanopolymer gene vector PEI600-CyD-Folate (H1). Antiangiogenic effect of Vastatin was tested in vitro by using co-culture system and conditioned medium. An orthotopic GB murine model was established to examine the in vivo therapeutic effect of Vastatin alone treatment and its combination with temozolomide. RESULTS: Vastatin gene transfection mediated by H1 could target tumour cells specifically and suppress the proliferation of microvessel endothelial cells (MECs) through a paracrine inhibition manner. Enhancing Vastatin expression by intracerebral injection of H1-Vastatin significantly prolonged animal survival from 48 to 75 days in GB murine model, which was comparable to the effect of Endostatin, the most studied endogenous antiangiogenic polypeptide. The diminished presence of CD34 positive cells in the GB xenografts suggested that Vastatin induced significant antiangiogenesis. Moreover, a synergistic effect in extending survival was detected when H1-Vastatin was administered with temozolomide (TMZ) in GB chemoresistant murine models. CONCLUSION: Our results suggest, for the first time, that Vastatin is an antiangiogenic polypeptide with significant potential therapeutic benefit for GB. H1-Vastatin gene therapy may have important implications in re-sensitizing recurrent GB to standard chemotherapeutic agents.
Assuntos
Neoplasias Encefálicas/mortalidade , Proliferação de Células , Colágeno Tipo VIII/metabolismo , Glioblastoma/mortalidade , Neovascularização Patológica/prevenção & controle , Animais , Apoptose , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/prevenção & controle , Colágeno Tipo VIII/genética , Feminino , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/prevenção & controle , Humanos , Camundongos , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) is an aromatic, long-lived environmental contaminant. While the pathogenesis of TCDD-induced toxicity is poorly understood, it has been shown that the aryl hydrocarbon receptor (AHR) is required. However, the specific transcriptomic changes that lead to toxic outcomes have not yet been identified. We previously identified a panel of 33 genes that respond to TCDD treatment in two TCDD-sensitive rodent species. To identify genes involved in the onset of hepatic toxicity, we explored 25 of these in-depth using liver from two rat strains: the TCDD-resistant Han/Wistar (H/W) and the TCDD-sensitive Long-Evans (L-E). Time course and dose-response analyses of mRNA abundance following TCDD insult indicate that eight genes are similarly regulated in livers of both strains of rat, suggesting that they are not central to the severe L-E-specific TCDD-induced toxicities. The remaining 17 genes exhibited various divergent mRNA abundances between L-E and H/W strains after TCDD treatment. Several genes displayed a biphasic response where the initial response to TCDD treatment was followed by a secondary response, usually of larger magnitude in L-E liver. This secondary response was most often an exaggeration of the original TCDD-induced response. Only cytochrome b5 type A (microsomal) (Cyb5a) had equivalent TCDD sensitivity to the prototypic AHR-responsive cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1), while six genes were less sensitive. Four genes showed an early inter-strain difference that was sustained throughout most of the time course (atypical chemokine receptor 3 (Ackr3), collagen, type XVIII, alpha 1 (Col18a1), Cyb5a and glutamate dehydrogenase 1 (Glud1)), and of those genes examined in this study, are most likely to represent genes involved in the pathogenesis of TCDD-induced hepatotoxicity in L-E rats.
Assuntos
Carcinógenos Ambientais/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , RNA Mensageiro/metabolismo , Animais , Animais não Endogâmicos , Carcinógenos Ambientais/administração & dosagem , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Colágeno Tipo VIII/agonistas , Colágeno Tipo VIII/antagonistas & inibidores , Colágeno Tipo VIII/genética , Colágeno Tipo VIII/metabolismo , Citocromos b5/antagonistas & inibidores , Citocromos b5/química , Citocromos b5/genética , Citocromos b5/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Feminino , Perfilação da Expressão Gênica , Glutamato Desidrogenase , Cinética , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Dibenzodioxinas Policloradas/administração & dosagem , Ratos Long-Evans , Receptores CXCR/agonistas , Receptores CXCR/antagonistas & inibidores , Receptores CXCR/genética , Receptores CXCR/metabolismo , Receptores de Glutamato/química , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismoRESUMO
Collagen XVIII is a ubiquitous basement membrane (BM) proteoglycan produced in three tissue-specific isoforms that differ in their N-terminal non-collagenous sequences, but share collagenous and C-terminal non-collagenous domains. The collagenous domain provides flexibility to the large collagen XVIII molecules on account of multiple interruptions in collagenous sequences. Each isoform has a complex multi-domain structure that endows it with an ability to perform various biological functions. The long isoform contains a frizzled-like (Fz) domain with Wnt-inhibiting activity and a unique domain of unknown function (DUF959), which is also present in the medium isoform. All three isoforms share an N-terminal laminin-G-like/thrombospondin-1 sequence whose specific functions still remain unconfirmed. The proteoglycan nature of the isoforms further increases the functional diversity of collagen XVIII. An anti-angiogenic domain termed endostatin resides in the C-terminus of collagen XVIII and is proteolytically cleaved from the parental molecule during the BM breakdown for example in the process of tumour progression. Recombinant endostatin can efficiently reduce tumour angiogenesis and growth in experimental models by inhibiting endothelial cell migration and proliferation or by inducing their death, but its efficacy against human cancers is still a subject of debate. Mutations in the COL18A1 gene result in Knobloch syndrome, a genetic disorder characterised mainly by severe eye defects and encephalocele and, occasionally, other symptoms. Studies with gene-modified mice have elucidated some aspects of this rare disease, highlighting in particular the importance of collagen XVIII in the development of the eye. Research with model organisms have also helped in determining other structural and biological functions of collagen XVIII, such as its requirement in the maintenance of BM integrity and its emerging roles in regulating cell survival, stem or progenitor cell maintenance and differentiation and inflammation. In this review, we summarise current knowledge on the properties and endogenous functions of collagen XVIII in normal situations and tissue dysregulation. When data is available, we discuss the functions of the distinct isoforms and their specific domains.
Assuntos
Membrana Basal/efeitos dos fármacos , Colágeno Tipo VIII/genética , Encefalocele/genética , Neoplasias/genética , Neovascularização Patológica/prevenção & controle , Descolamento Retiniano/congênito , Animais , Membrana Basal/metabolismo , Membrana Basal/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo VIII/metabolismo , Colágeno Tipo XVIII , Encefalocele/metabolismo , Encefalocele/patologia , Endostatinas/farmacologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Domínios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise , Proteínas Recombinantes/farmacologia , Degeneração Retiniana , Descolamento Retiniano/genética , Descolamento Retiniano/metabolismo , Descolamento Retiniano/patologiaRESUMO
Hepatocellular carcinoma (HCC) is a hypervascular cancer without effective treatment. Here we report that polypeptide of NC1 domain of type VIII collagen (Vastatin) is an endogenous polypeptide expressed in normal liver tissue but lost in the liver of most HCC patients (73.1%). Its expression level is negatively associated with tumor size (P = 0.035) and metastasis (P = 0.016) in HCC patients. To evaluate its potential use as a therapeutic, we constructed a recombinant adeno-associated virus carrying Vastatin (rAAV-Vastatin) to treat HCC in an orthotopic Buffalo rat model. rAAV-Vastatin treatment significantly prolonged the median survival, inhibited tumor growth, and completely prevented metastasis in HCC-bearing rats by decreasing microvessel density and increasing tumor necrosis. No detectable toxicity in nontumor-bearing mice was observed. To investigate its molecular mechanisms, we performed DNA microarray, western blotting assays, and bioinformatic analysis to determine its effect on global gene expression patterns and signal transduction pathways. Our results indicated that rAAV-Vastatin significantly reduced the expressions of Pck1, JAG2, and c-Fos, thus inhibiting the cellular metabolism, Notch and AP-1 signaling pathways, respectively. Hence, we demonstrated for the first time that Vastatin is a novel, safe, and effective antiangiogenic therapeutic and a potential biomarker for HCC.
Assuntos
Inibidores da Angiogênese/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Colágeno Tipo VIII/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Adulto , Idoso , Inibidores da Angiogênese/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Colágeno Tipo VIII/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Necrose , Gradação de Tumores , Metástase Neoplásica , Neovascularização Patológica/genética , Ratos , Receptores Notch/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Transdução Genética , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We conducted a case-control study to investigate the association of COL18A1 D104N polymorphism in the development of osteosarcoma in a Chinese population. Between May 2012 and May 2014, 141 patients with pathologically proven osteosarcoma and 341 were selected into this study. Genotyping of COL18A1 D104N was analyzed using polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP). By logistic regression analysis, we found that individuals with the NN genotype of COL18A1 D104N were significantly associated with an increased risk of osteosarcoma when compared with the DD genotype (OR=20.97, 95% CI=2.74-933.42). In dominant model, the NN+DN genotype of COL18A1 D104N had a 1.99 fold risk of osteosarcoma when compared with the DD genotype. Moreover, the NN genotype was correlated with a 20.45 fold risk of osteosarcoma when compared with the DN+DD genotype in recessive model. However, we did not find significant interaction between COL18A1 D104N polymorphism and Enneking stage, histological subtype, tumor metastasis and tumor location of patients with osteosarcoma. In conclusion, our study suggests that the homozygous DN and NN genotypes of COL18A1 D104N were associated with the risk of osteosarcoma.
Assuntos
Povo Asiático/genética , Neoplasias Ósseas/genética , Colágeno Tipo VIII/genética , Colágeno Tipo XVIII/genética , Osteossarcoma/genética , Polimorfismo Genético , Adolescente , Adulto , Neoplasias Ósseas/patologia , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Genótipo , Homozigoto , Humanos , Masculino , Osteossarcoma/patologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Risco , Adulto JovemRESUMO
PURPOSE: Age-related macular degeneration (AMD) is the main cause of visual impairment and legal blindness in older individuals. COL8A1 rs13095226 variants have recently been implicated associated with neovascular age-related macular degeneration (nAMD) and Polypoidal Choroidal Vasculopathy (PCV) in American studies. The aim of this study was to investigate the association between the COL8A1 rs13095226 Polymorphism and neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) in Chinese people. METHODS: 900 Chinese subjects-300 cases with nAMD, 300 cases with PCV and 300 controls, were enrolled in a cross-sectional observational study. The diagnoses of nAMD and PCV were confirmed by Fundus photography, Fluorescence Fundus Angiography (FFA) and Indocyanine Green Angiography (ICGA). Genomic DNA was extracted from venous blood leukocytes and genotypes of rs13095226 were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Differences in allele distribution between cases and controls were tested by chi-square tests, with age and gender adjusted by logistic regression analysis. RESULT: The COL8A1 rs13095226 polymorphism was not statistically significantly different from the nAMD or PCV to the normal controls (P>0.05) in Chinese Population. The association remained insignificant after adjustment for age and gender differences (P>0.05). CONCLUSIONS: This case-control study indicated that the COL8A1 rs13095226 polymorphism is not associated with nAMD or PCV, which suggesting this gene maybe not a susceptibility gene locus for nAMD or PCV in Chinese subjects.
Assuntos
Colágeno Tipo VIII/genética , Polimorfismo Genético/genética , Degeneração Macular Exsudativa/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Povo Asiático/genética , Estudos de Casos e Controles , Corioide/patologia , Colágeno Tipo XVIII , Estudos Transversais , Feminino , Angiofluoresceinografia , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Degeneração Macular Exsudativa/diagnósticoRESUMO
Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet's Stripping Endothelial Keratoplasty (DSEK) and Descemet's Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type.
Assuntos
Proteínas de Transporte de Ânions/genética , Antiporters/genética , Colágeno Tipo VIII/genética , Células Endoteliais/metabolismo , Endotélio Corneano/metabolismo , Expressão Gênica , Proteínas de Membrana/genética , Adulto , Idoso , Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Autopsia , Biomarcadores/metabolismo , Colágeno Tipo VIII/metabolismo , Ceratócitos da Córnea/citologia , Ceratócitos da Córnea/metabolismo , Substância Própria/citologia , Substância Própria/metabolismo , Células Endoteliais/citologia , Endotélio Corneano/citologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Especificidade de Órgãos , Cultura Primária de CélulasRESUMO
As a therapeutic or chemopreventative agent for various cancers, all-trans retinoic acid (atRA) has been reported to inhibit growth, induce apoptosis or cause differentiation. It was found that atRA could protect hepatocellular carcinoma (HCC) cells against cell death induced by serum starvation. Furthermore, it was found that atRA could enhance cell adhesion, but had no effect on the cell cycle and apoptosis. Using an Illumina Human HT-12 v4 expression microarray, 207 upregulated and 173 downregulated genes were identified in HepG2 cells treated with atRA. The most upregulated genes are cytochrome P450 family 26 subfamily A polypeptide 1 (CYP26A1), histidine triad nucleotide binding protein 3 (HINT3), miR-1282 and cytochrome P450 family 26 subfamily B polypeptide 1 (CYP26B1), which showed more than fivefold greater expression. Using Gene Ontology analysis, the greatest significance was found in extracellular-matrix-related molecular functions and the cellular component in upregulated genes. The upregulation of collagen 8A2 (COL8A2) was further confirmed using quantitative RT-PCR and western blotting. Knockdown of COL8A2 blocked enhancement in the early stage of cell adhesion by atRA treatment. Re-expression of COL8A2 in COL8A2-knocked-down HCC cells reversed the effect of small interfering RNA-COL8A2. In addition, COL8A2 could increase HCC cell migration and invasion. Thus, COL8A2 was identified as the key protein involved in the enhancement of cell adhesion of atRA under serum-free conditions. In conclusion, atRA protects HCC cells against serum-starvation-induced cell death by enhancing cell adhesion, and COL8A2 plays an important role in HCC cell migration and invasion.
Assuntos
Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/tratamento farmacológico , Colágeno Tipo VIII/genética , Meios de Cultura Livres de Soro/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Tretinoína/farmacologia , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Colágeno Tipo VIII/metabolismo , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inanição , Células Tumorais Cultivadas , Regulação para CimaRESUMO
PURPOSE: To characterize the morphology and gene expression of transformed murine corneal endothelial cells. METHODS: Primary immortomouse corneal endothelial cells were continuously cultured before and after cryopreservation. Morphologic assessment, real time-reverse transcriptase polymerase chain reaction ((RT)-PCR) and immunofluorescence studies were performed using newly cultured cells, cells that had been continuously in culture for 1 year, and cryopreserved cells, to assess for structural and functional integrity. The expression of corneal endothelial markers zonula occludens-1 (ZO1), NaK-ATPase and collagen VIII (α2) (COL8A2), and myofibroblast markers Desmin, alpha smooth muscle actin (αSMA), and Vimentin was assessed and compared by both RT-PCR and immunofluorescence. RESULTS: Cells in culture formed a monolayer, and exhibited a polygonal shape after reaching confluence. Cells retained this morphology during the full observation time of 12 months and when reused after cryopreservation. Immunofluorescence experiments exhibited positive staining for NaK-ATPase and COL8A2 with low variability between the three groups. In RT-PCR experiments, ZO1, COL8A2 and Desmin were increased in fresh and thawed cells, αSMA was decreased, and NaK-ATPase and Vimentin remained unchanged, compared to 12-month-old cells. Comparing fresh and thawed cells, COL8A2 was increased in thawed cells, while Desmin was increased in fresh cells. CONCLUSIONS: Using the immortomouse strain, murine corneal endothelial cells can be propagated over a long time period and be used after cryopreservation. Cells retain the expression of NaK-ATPase, but show some decline in ZO1 and COL8A2 over time and after cryopreservation. The expression of myofibroblast markers suggests an endothelial-to-mesenchymal transformation process in culture.
Assuntos
Criopreservação , Células Endoteliais , Endotélio Corneano/citologia , Actinas/genética , Actinas/metabolismo , Animais , Biomarcadores , Técnicas de Cultura de Células , Linhagem Celular Transformada , Colágeno Tipo VIII/genética , Colágeno Tipo VIII/metabolismo , Desmina/genética , Desmina/metabolismo , Endotélio Corneano/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Vimentina/genética , Vimentina/metabolismo , Proteína da Zônula de Oclusão-1RESUMO
Fuchs' endothelial corneal dystrophy is the most common corneal endotheliopathy, and a leading indication for corneal transplantation in the US. Relatively little is known about its underlying pathology. We created a cellular model of the disease focusing on collagen VIII alpha 2 (COL8A2), a collagen which is normally present in the cornea, but which is found in abnormal amounts and distribution in both early and late-onset forms of the disease. We performed cellular transfections using COL8A2 cDNAs including both wild-type and mutant alleles which are known to result in early-onset FECD. We used this cell model to explore the cellular production of wild-type and mutant monomeric and trimeric collagen VIII and measured production levels and patterns using Western blotting and immunofluorescence. We studied the thermal stability of the mutated collagen VIII helices using computer modeling, and further investigated these differences using collagen mimetic peptides. The Western blots demonstrated that similar amounts of wild-type and mutant collagen VIII monomers were produced in the cells. However, the levels of trimeric collagen peptide in the mutant-transfected cells were elevated. Intracellular accumulation of trimeric collagen VIII was confirmed on immunofluorescence studies. Both the computer model and the collagen mimetic peptides demonstrated that the L450W mutant was less thermally stable than either the Q455K or wild-type collagen VIII. Thus, although both mutant collagen VIII peptides were retained intracellularly, the biochemical reasons for the retention varied between genotypes. Collagen VIII mutations, which clinically result in Fuchs' dystrophy, are associated with abnormal cellular accumulation of collagen VIII. Different collagen VIII mutations may act via distinct biochemical mechanisms to produce the FECD phenotype.