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1.
BMC Ophthalmol ; 19(1): 191, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438893

RESUMO

BACKGROUND: To investigate the efficacy and safety of repeated phototherapeutic keratectomies (PTKs) during long-term treatment for corneal dystrophy (CD) in a Chinese pedigree carrying the R124L mutation in TGFBI. METHODS: This was a retrospective review of 20-year medical and genetic records involving five CD patients (10 eyes) from one pedigree. During this period, PTK was conducted for an eye when best-corrected distance visual acuity (BCDVA) reached > 1.0 (LogMAR), due to either primary or recurrent opacities in the cornea. All PTKs were performed by 193-nm excimer laser with or without creation of epithelial flaps. For each eye, routine measurements were conducted for the number of PTKs during follow-up, mean time to recurrence, and BCDVA pre- and post- every PTK (measurements within 3 months from each PTK). Corneal thicknesses measured after the last PTK and at the last visit were analyzed, and subjective satisfaction was assessed. RESULTS: Gene testing revealed an R124L mutation in TGFBI. During 19.60 ± 1.78 years of follow-up, PTKs were conducted twice for three eyes, three times for six eyes, and four times for one eye. After each PTK, effective visual acuity was maintained for 3.60 ± 1.12 years before significant recurrence. BCDVA improved significantly postoperatively than preoperatively for the first PTK for each eye (p < 0.001), as well as the second (p < 0.001) and third one (p < 0.001). After the last PTK and at the final visit, the thinnest corneal thickness was 371.50 ± 56.47 µm and 358.40 ± 101.11 µm, respectively. The average subjective satisfaction score was 8.60 ± 0.89. CONCLUSIONS: Multiple repeated PTKs were effective and safe in a long-term study of CD patients with an R124L mutation in TGFBI.


Assuntos
Córnea/cirurgia , Distrofias Hereditárias da Córnea/cirurgia , Proteínas da Matriz Extracelular/genética , Previsões , Lasers de Excimer/uso terapêutico , Mutação , Ceratectomia Fotorrefrativa/métodos , Fator de Crescimento Transformador beta/genética , Adulto , Idoso , China/epidemiologia , Córnea/patologia , Distrofias Hereditárias da Córnea/epidemiologia , Distrofias Hereditárias da Córnea/genética , DNA/genética , Análise Mutacional de DNA , Proteínas da Matriz Extracelular/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Estudos Retrospectivos , Fator de Crescimento Transformador beta/metabolismo , Resultado do Tratamento , Acuidade Visual
2.
Medicine (Baltimore) ; 98(26): e15872, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31261495

RESUMO

Human epidermal growth factor receptor 2-positive (HER2+) breast cancer accounts for ∼20% of invasive breast cancers and is associated with poor prognostics. The recent outcome of HER2+ breast cancer treatment has been vastly improved owing to the application of antibody-targeted therapies. Trastuzumab (Herceptin) is a monoclonal antibody designed to target HER2+ breast cancer cells. In addition to improved survival in the adjuvant treatment of HER2+ breast cancer, trastuzumab treatment has also been associated with cardiotoxicity side effect. However, the molecular mechanisms of trastuzumab action and trastuzumab-mediated cardiotoxicity are still not fully understood. Previous research utilized bulk transcriptomics analysis to study the underlining mechanisms, which relied on averaging molecular signals from bulk tumor samples and might have overlooked key expression features within breast cancer tumor. In contrast to previous research, we compared the single cancer cell level transcriptome profile between trastuzumab-treated and nontreated patients to reveal a more in-depth transcriptome profile. A total of 461 significantly differential expressed genes were identified, including previously defined and novel gene expression signatures. In addition, we found that trastuzumab-enhanced MGP gene expression could be used as prognostics marker for longer patient survival in breast invasive carcinoma patients, and validated our finding using TCGA (The Cancer Genome Atlas) breast cancer dataset. Moreover, our study revealed a 48-gene expression signature that is associated with cell death of cardiomyocytes, which could be used as early biomarkers for trastuzumab-mediated cardiotoxicity. This work is the first study to look at single cell level transcriptome profile of trastuzumab-treated patients, providing a new understanding of the molecular mechanism(s) of trastuzumab action and trastuzumab-induced cardiotoxicity side effects.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Receptor ErbB-2/metabolismo , Transcriptoma/efeitos dos fármacos , Trastuzumab/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/epidemiologia , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/epidemiologia , Carcinoma Ductal de Mama/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Projetos Piloto , Prognóstico , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Análise de Sobrevida
3.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 37(3): 248-252, 2019 Jun 01.
Artigo em Chinês | MEDLINE | ID: mdl-31218856

RESUMO

OBJECTIVE: To verify the effect of the mutant gene vps4b on the expression of tooth development-related proteins, dentin sialophosphoprotein (DSPP) and collagenⅠ (COL-Ⅰ). METHODS: Paraffin tissue sections of the first molar tooth germ were obtained from the heads of fetal mice at the embryonic stages of 13.5, 14.5, and 16.5 days and from the mandibles of larvae aged 2.5 and 7 days after birth. The immunohistochemical method was used to detect the expression and location of DSPP and COL-Ⅰ in wild-type mouse and vps4b knockout mouse. RESULTS: DSPP and COL-Ⅰ were not found in the bud and cap stages of wild-type mouse molar germ. In the bell stage, DSPP was positively expressed in the inner enamel epithelium and dental papilla, whereas COL-Ⅰ was strongly expressed in the dental papilla and dental follicle. During the secretory and mineralized periods, DSPP and COL-Ⅰ were intensely observed in ameloblasts, odontoblasts, and dental follicles, but COL-Ⅰ was also expressed in the dental papilla. After vps4b gene knockout, DSPP was not expressed in the dental papilla of the bell stage and in the dental papilla and dental follicle of the secretory phase. The expression position of COL-Ⅰ in the bell and mineralization phase was consistent with that in the wild-type mice. Moreover, the expression of COL-Ⅰ in the dental papilla changed in the secretory stage. CONCLUSIONS: Gene vps4b plays a significant role in the development of tooth germ. The expression of DSPP and COL-Ⅰ may be controlled by gene vps4b and regulates the development of tooth dentin and cementum together with vps4b.


Assuntos
ATPases Associadas a Diversas Atividades Celulares , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas da Matriz Extracelular , Fosfoproteínas , Sialoglicoproteínas , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Colágeno/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Proteínas da Matriz Extracelular/metabolismo , Camundongos , Camundongos Knockout , Dente Molar , Odontoblastos , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Germe de Dente
4.
Cell Physiol Biochem ; 53(1): 87-100, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31204440

RESUMO

BACKGROUND/AIMS: Different components of the tumor microenvironment can be either tumor-promoting or tumor-suppressive agents depending on factors which are not fully understood. Fibulins are components of the extracellular matrix from different tissues and constitute a clear example of this dual function. In fact, fibulins may either support tumor growth or abolish progression of malignant cells depending on the crosstalk between tumor cells and their surrounding stroma through mechanisms that remain to be elucidated. Among all fibulins, fibulin-5 contains a particular structural hallmark which consists in the presence of a RGD motif within its architecture. Previous reports have highlighted the importance of the interaction of this motif with integrins, and not only in normal functions but also in a tumor context. METHODS: Site-Directed Mutagenesis technique was employed to introduce the change RGD to RGE (RGD-to-RGE) within Fbln5 cDNA sequence. Cell proliferation was measured using the MTT assay or by counting Ki-67 positive cell nuclei. Cell adhesion was analysed using culture plates coated with different extracellular matrix components. Cell invasion was evaluated using 24-well Matrigel-coated invasion chambers, and mammosphere formation was monitored using ultralow attachment culture plates. BALB/c mice were employed to induce subcutaneous tumors. RESULTS: The RGD-to-RGE change alters the capacity of breast cancer cells to adhere to different extracellular matrix proteins as well as to αvß3 and α5ß1 integrins, and promotes protumor effects using different cell-based assays. Moreover, 4T1 cells, a mouse breast cancer cell line model, shows an increased capacity to generate tumors when exogenously expresses fibulin-5 with a RGD-to-RGE change, and such capacity is similar to that shown for 4T1 cells with an interfered Fbln5 gene. CONCLUSION: These data highlight the importance of the RGD motif of fibulin-5 to induce antitumor effects and provide new insights into the involvement of fibulins in tumor processes.


Assuntos
Adesão Celular/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Oligopeptídeos/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/uso terapêutico , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Oligopeptídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Transplante Homólogo , Vimentina/metabolismo
5.
Nat Immunol ; 20(7): 915-927, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31110316

RESUMO

The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.


Assuntos
Perfilação da Expressão Gênica , Interferon Tipo I/metabolismo , Queratinócitos/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Transcriptoma , Biópsia , Linhagem da Célula/genética , Biologia Computacional/métodos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibrose , Perfilação da Expressão Gênica/métodos , Humanos , Nefrite Lúpica/patologia , Ligação Proteica , Transdução de Sinais , Análise de Célula Única , Pele/imunologia , Pele/metabolismo , Pele/patologia
6.
Cell Physiol Biochem ; 52(6): 1553-1568, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31135123

RESUMO

BACKGROUND/AIMS: Despite, several studies demonstrating pro-metastatic effects of the metalloproteinase ADAMTS1 in breast cancer, its role in facilitating the metastatic cascade remains unclear. To date there have been limited studies that have examined the expression of ADAMTS1 in primary and metastatic breast cancer tissues. METHODS: We assessed ADAMTS1 mRNA levels in publically available breast cancer sets and analysed ADAMTS1 protein levels by immunohistochemistry in breast tissue microarrays containing normal breast tissue (n=9), primary invasive ductal breast carcinomas (n=79) and metastatic lesions (n=58). To understand the underlying events influenced by ADAMTS1 and provide a mechanism by which tumors expressing this protease promote metastasis, we assessed the ability of PyMT/Adamts1+/+, PyMT/Adamts1+/- and PyMT/Adamts1-/- primary mammary cancer cells to adhere to matrigel and migrate or invade towards a chemoattractive environment. RESULTS: High ADAMTS1 expression was associated with reduced disease-free survival, distant metastasis free-survival and overall survival in breast cancer patients with node negative disease. Although ADAMTS1 expression was reduced in primary breast cancers compared to normal tissue and not elevated in metastatic lesions, high ADAMTS1 immunostaining was associated with a higher number of positive lymph nodes (p=0.006) and the presence of distant metastasis (p=0.023). Depletion of Adamts1 in mammary cancer cells impeded their adhesion to a biological matrix substratum and diminished cell migration but not invasion. CONCLUSION: The effects observed on cell adhesion and migration demonstrates a potential mechanism whereby ADAMTS1 promotes breast cancer metastasis.


Assuntos
Proteína ADAMTS1/metabolismo , Neoplasias da Mama/patologia , Proteínas da Matriz Extracelular/metabolismo , Proteína ADAMTS1/genética , Animais , Neoplasias da Mama/mortalidade , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Camundongos , Camundongos Transgênicos , Estadiamento de Neoplasias , Prognóstico
7.
Int J Mol Sci ; 20(7)2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970594

RESUMO

The cell microenvironment plays a pivotal role in mediating cell adhesion, survival, and proliferation in physiological and pathological states. The relevance of extracellular matrix (ECM) proteins in cell fate control is an important issue to take into consideration for both tissue engineering and cell biology studies. The glycosylation of ECM proteins remains, however, largely unexplored. In order to investigate the physio-pathological effects of differential ECM glycosylation, the design of affordable chemoselective methods for ECM components glycosylation is desirable. We will describe a new chemoselective glycosylation approach exploitable in aqueous media and on non-protected substrates, allowing rapid access to glyco-functionalized biomaterials.


Assuntos
Materiais Biocompatíveis/metabolismo , Técnicas de Cultura de Células/métodos , Proteínas da Matriz Extracelular/metabolismo , Materiais Biocompatíveis/química , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Colágeno/química , Colágeno/farmacologia , Glicosilação , Humanos
8.
Int J Mol Sci ; 20(7)2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987093

RESUMO

Advanced upper urinary tract urothelial carcinoma (UTUC) is often associated with poor oncologic outcomes. The secreted protein acidic and rich in cysteine-like 1 (SPARCL1) protein, belongs to the SPARC-related family of matricellular proteins. Much literature has been published describing the role of SPARCL1 in the prognosis many cancers. In this study, methylated promoter regions in high-grade and high-stage upper urinary urothelial tumours compared with normal urothelium were analyzed and revealed that SPARCL1 was the most significantly hypermethylated gene in UTUC tissues. Then we prospectively collected UTUC samples and adjacent normal urothelium for pyrosequencing validation, identifying significant CpG site methylation in UTUC tissues. In addition, SPARCL1 RNA levels were significantly lower in UTUC samples. Multivariate Cox regression analysis from 78 patients with solitary renal pelvic or ureteral pT3N0M0 urothelial carcinomas revealed that only negative SPARCL1 expression and nonpapillary tumour architecture were independently associated with systemic recurrence (p = 0.011 and 0.008, respectively). In vitro studies revealed that the behaviour of BFTC-909 cells was less aggressive and more sensitive to radiation or chemotherapy after SPARCL1 overexpression. Thus, SPARCL1 could be considered as a prognostic marker and help decision-making in clinical practice.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Metilação de DNA/genética , Proteínas da Matriz Extracelular/genética , Neoplasias Urológicas/genética , Neoplasias Urológicas/patologia , Urotélio/patologia , Idoso , Sequência de Bases , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Estudos de Coortes , Metilação de DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Regiões Promotoras Genéticas/genética , Análise de Regressão , Neoplasias Urológicas/tratamento farmacológico , Neoplasias Urológicas/radioterapia
9.
J Mol Neurosci ; 68(2): 261-274, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30949956

RESUMO

The level of miR-181a decreases rapidly in N2a cells following oxygen-glucose deprivation/reperfusion, but its role in this process is unclear. Reelin, a regulator of neuronal migration and synaptogenesis, is a predicted target of miR-181a. We hypothesized that miR-181a reduces neuronal apoptosis and protects neurons by targeting reelin. Second mitochondria-derived activator of caspases (Smac) is a protein located in mitochondria that regulates apoptosis. The pro-apoptotic effect of Smac is achieved by reversing the effects of apoptosis-inhibiting proteins (IAPs), particularly X-linked inhibitor of apoptosis (XIAP). We also evaluated the effect of miR-181a on the Smac/IAP signaling pathway after oxygen-glucose deprivation and reperfusion in N2a cells. The miR-181a level, apoptosis rate, and the levels of reelin mRNA and protein, Smac, and XIAP were assessed in N2a cells subjected to oxygen-glucose deprivation for 4 h and reperfusion for 0, 4, 12, or 24 h with/without an miR-181a mimic, or mismatched control. Direct targeting of reelin by miR-181a was assessed in vitro by dual luciferase assay and immunoblotting. Pre-treatment with miR-181a mimicked the increase in the miR-181a level in N2a cells after oxygen-glucose deprivation/reperfusion, resulting in a significant decrease in the apoptosis rate. Changes in the miR-181a level in N2a cells were inversely correlated with reelin protein expression. Direct targeting of the reelin 3' untranslated region by miR-181a was verified by dual luciferase assay, which showed that miR-181a significantly inhibited luciferase activity. The Smac level was significantly lower in the miR-181a mimics than the normal control and mimics-cont groups (P < 0.01), whereas the level of XIAP was increased slightly. These findings suggest that miR-181a protects neurons from apoptosis by inhibiting reelin expression and regulating the Smac/IAP signaling pathway after oxygen-glucose deprivation/reperfusion injury.


Assuntos
Apoptose , MicroRNAs/genética , Neurônios/metabolismo , Oxigênio/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Glucose/deficiência , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , MicroRNAs/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
10.
Nat Neurosci ; 22(5): 709-718, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988524

RESUMO

Disruption of the blood-brain barrier (BBB) is critical to initiation and perpetuation of disease in multiple sclerosis (MS). We report an interaction between oligodendroglia and vasculature in MS that distinguishes human white matter injury from normal rodent demyelinating injury. We find perivascular clustering of oligodendrocyte precursor cells (OPCs) in certain active MS lesions, representing an inability to properly detach from vessels following perivascular migration. Perivascular OPCs can themselves disrupt the BBB, interfering with astrocyte endfeet and endothelial tight junction integrity, resulting in altered vascular permeability and an associated CNS inflammation. Aberrant Wnt tone in OPCs mediates their dysfunctional vascular detachment and also leads to OPC secretion of Wif1, which interferes with Wnt ligand function on endothelial tight junction integrity. Evidence for this defective oligodendroglial-vascular interaction in MS suggests that aberrant OPC perivascular migration not only impairs their lesion recruitment but can also act as a disease perpetuator via disruption of the BBB.


Assuntos
Barreira Hematoencefálica/fisiopatologia , Encefalite/fisiopatologia , Esclerose Múltipla/fisiopatologia , Células Precursoras de Oligodendrócitos/fisiologia , Animais , Astrócitos/patologia , Astrócitos/fisiologia , Barreira Hematoencefálica/patologia , Movimento Celular , Células Cultivadas , Encefalite/patologia , Proteínas da Matriz Extracelular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Esclerose Múltipla/patologia , Células Precursoras de Oligodendrócitos/patologia , Junções Íntimas/metabolismo , Substância Branca/patologia
11.
Molecules ; 24(8)2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-31003546

RESUMO

ανß3 and α5ß1 are essential glycoproteins involved in the pathogenesis of rheumatoid arthritis (RA). Understanding of the role these integrins play in disease have been analyzed via description of cells-expressing ανß3 and α5ß1 and their mediators to trigger inflammation. ανß3 and α5ß1 facilitate cells-ECM and cell-cell communication, producing pro-inflammatory factors. Pro-inflammatory factors are essential for the building of undesirable new blood vessels termed angiogenesis which can further lead to destruction of bones and joints. Despite many attempts to target these glycoproteins, there are still some problems, therefore, there is still interest in understanding the synergistic role these integrins play in the pathogenesis of RA. The purpose of this review is to gain insights into the biological effects of ανß3 and α5ß1 in synovial tissues that are relevant to pathogenesis and therapy of RA.


Assuntos
Artrite Reumatoide/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/terapia , Comunicação Celular , Proteínas da Matriz Extracelular/metabolismo , Humanos , Terapia de Alvo Molecular
12.
Differentiation ; 106: 23-34, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30852470

RESUMO

The extracellular matrix (ECM) proteins play an important role in the establishment of the sex-dependent structure of developing gonads. The matrix metalloproteinases (MMPs) are the major players in the regulation of ECM. Our hypothesis was that the MMPs-dependent regulation of EMC is crucial for the establishment of the correct, either testis or ovary, structure of developing gonad. We cultured developing mouse gonads in vitro in the presence of the MMPs inhibitors (α-2-macroglobulin, leupeptin, phosphoramidon) or the MMPs activator, APMA (4-aminophenylmercuric acetate). These inhibitors and activator inhibit/activate, to a different degree, matrix metalloproteinases, but the exact mechanism of inhibition/activation remains unknown. We found that the MMP inhibitors increased accumulation of ECM in the developing gonads. The α-2-macroglobulin had the weakest, and the phosphoramidon the strongest effect on the ECM and the structure of the gonads. The α-2-macroglobulin caused a slight increase of ECM and did not disrupt the gonad structure. Leupeptin led to the strong accumulation of ECM, resulted in the formation of the structures resembling testis cords in both testes and ovaries, and caused increase of apoptosis and complete loss of germ cells. Phosphoramidon caused the strongest accumulation of ECM, which separated individual cells and completely prevented intercellular adhesion both in the testes and in the ovaries. As a result of aberrant morphology, the sex of the phosphoramidon-treated gonads was morphologically unrecognizable. The APMA - the activator of MMP caused ECM loss, which led to the loss of cell adhesion, cell dispersion and an aberrant morphology of the gonads. These results indicate that the ECM accumulation is MMPs-dependent and that the correct amount and distribution of ECM during gonad development plays a key role in the formation of the gonad structure.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Células Germinativas/citologia , Gônadas/citologia , Metaloproteinases da Matriz/metabolismo , Diferenciação Sexual , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Gônadas/metabolismo , Masculino , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL
13.
Adv Mater ; 31(16): e1900582, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30838715

RESUMO

Biogenesis and tissue development are based on the heterogenesis of multipotent stem cells. However, the underlying mechanisms of stem cell fate specification are unclear. Chirality is one of the most crucial factors that affects stem cell development and is implicated in asymmetrical cell morphology formation; however, its function in heterogeneous cell fate determination remains elusive. In this study, it is reported that the chirality of a constructed 3D extracellular matrix (ECM) differentiates mesenchymal stem cells to diverse lineages of osteogenic and adipogenic cells by providing primary heterogeneity. Molecular analysis shows that left-handed chirality of the ECM enhances the clustering of the mechanosensor Itgα5, while right-handed chirality decreases this effect. These differential adhesion patterns further activate distinct mechanotransduction events involving the contractile state, focal adhesion kinase/extracellular signal-regulated kinase 1/2 cascades, and yes-associated protein/runt-related transcription factor 2 nuclear translocation, which direct heterogeneous differentiation. Moreover, theoretical modeling demonstrates that diverse chirality mechanosensing is initiated by biphasic modes of fibronectin tethering. The findings of chirality-dependent lineage specification of stem cells provide potential strategies for the biogenesis of organisms and regenerative therapies.


Assuntos
Diferenciação Celular , Linhagem da Célula , Mecanotransdução Celular , Células-Tronco Mesenquimais/citologia , Resinas Acrílicas/química , Adipogenia , Animais , Células Cultivadas , Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Poliestirenos/química , Ratos , Estereoisomerismo , Propriedades de Superfície
14.
Invest Ophthalmol Vis Sci ; 60(4): 978-989, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30884524

RESUMO

Purpose: Lattice corneal dystrophy (LCD) is related to the denaturation of transforming growth factor-ß-induced protein (TGFBIp). Autophagic degradation of the denatured proteins by macrophages is one pathway to remove the denatured proteins. Thus, we investigated the role of autophagy in the degradation of mutant (MU) TGFBIp in macrophages. Methods: Corneas from participants were observed by slit-lamp photography and subjected to histopathologic and genetic analysis. Wild-type (WT) and MU TGFBIp were recombined and expressed. Macrophages from MU participants were isolated and cocultured with the recombinant TGFBIp. Colocalization of the two molecules was observed by immunofluorescent microscopy. Enzyme-linked immunosorbent assay, Western blotting, and flow cytometry were used to detect changes in molecule expression related to the phenotype and autophagy process. Results: Fourteen members from a family of 25 were identified as LCD sufferers. Significant TGFBIp aggregates and macrophage infiltration were found only in the corneas of LCD sufferers. Marker accumulation of TGFBIp was found in macrophages exposed to MU TGFBIp even at 5 hours after MU TGFBIp was withdrawn. High expressions of CD68 and CD36 were found in macrophages exposed to WT TGFBIp, but not to MU TGFBIp. Impaired autophagic flux due to defective autophagosome fusion to lysosomes was found in macrophages exposed to MU TGFBIp. Blockage of the autophagic process suppressed the expression of CD68 and CD36 in macrophages exposed to WT TGFBIp to levels similar to those found in macrophages exposed to MU TGFBIp. Conclusions: Our results suggested that reversion of the defective autophagic process in macrophages may be a therapeutic strategy for patients with LCD.


Assuntos
Autofagia , Distrofias Hereditárias da Córnea/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Macrófagos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Western Blotting , Antígenos CD36/metabolismo , Criança , Distrofias Hereditárias da Córnea/diagnóstico , Distrofias Hereditárias da Córnea/genética , Ensaio de Imunoadsorção Enzimática , Proteínas da Matriz Extracelular/genética , Feminino , Citometria de Fluxo , Humanos , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Linhagem , Fagocitose , Fator de Crescimento Transformador beta/genética
15.
Biol Pharm Bull ; 42(3): 354-356, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30828067

RESUMO

Reelin is a secreted protein that antagonizes the deposition and toxicity of amyloid ß peptide (Aß). Therefore, augmentation of Reelin activity may ameliorate Alzheimer's disease (AD). We have recently reported that a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS-3) cleaves and inactivates Reelin in the mouse brain. In the present study, we investigated the effect of reducing ADAMTS-3 on deposition of Aß by crossbreeding drug-inducible ADAMTS-3 conditional knock-out (cKO) mice with "next-generation" AD model mice. We found that reducing ADAMTS-3 inhibited deposition of Aß significantly in AppNL-F mice, which produce human wild-type Aß. On the other hand, reducing ADAMTS-3 had no effect in AppNL-G-F mice, which produce the Arctic mutant Aß (E22G) that forms protofibrils more efficiently than does wild-type Aß. Thus, the findings suggest that the administration of an inhibitor against ADAMTS-3 will prevent the progression of AD pathology caused by deposition of wild-type Aß.


Assuntos
Proteínas ADAMTS/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Proteínas ADAMTS/antagonistas & inibidores , Proteínas ADAMTS/genética , Doença de Alzheimer , Animais , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
16.
Methods Mol Biol ; 1944: 189-201, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30840244

RESUMO

Fibrosis is characterized by excessive deposition of collagen and additional extracellular matrix (ECM) components in response to chronic injuries. Liver fibrosis often results from chronic hepatitis C virus infection and alcohol abuse that can deteriorate to cirrhosis and liver failure. Current noninvasive diagnostic methods of liver fibrosis are limited in their ability to detect and differentiate between early and intermediate stages of fibrosis. New biomarkers of fibrosis that reflect ECM turnover are therefore badly needed. Procollagen C-proteinase enhancer 1 (PCPE-1), a connective tissue glycoprotein that functions as a positive regulator of C-terminal procollagen processing and subsequent collagen fibril assembly, is a promising candidate. Its tissue distribution and expression profile overlap those of collagen, and its expression in fibrosis is upregulated in parallel to the increase in collagen expression. The potential of PCPE-1 as a biomarker of liver fibrosis was recently established using a CCl4 mouse model of liver fibrosis by showing that the increase in collagen and PCPE-1 content in the fibrotic mouse liver was reflected by elevated plasma levels of PCPE-1. This was achieved using a newly developed highly sensitive, specific, accurate, and reproducible ELISA for mouse PCPE-1, which is based on commercially available antibodies and is offered as a new research tool in the field. A similar ELISA test was developed for human PCPE-1, and preliminary results with plasma from liver fibrosis patients revealed increased plasma concentrations of PCPE-1 in some patients. The protocols of both ELISA tests are outlined herein in great detail to permit their application by any laboratory with similar interests.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Cirrose Hepática/diagnóstico , Cirrose Hepática/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Humanos , Camundongos
17.
Int J Mol Med ; 43(5): 2103-2117, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864688

RESUMO

Family with sequence similarity 20­member C (FAM20C), a recently characterized Golgi kinase, performs numerous biological functions by phosphorylating more than 100 secreted proteins. However, the role of FAM20C in the salivary glands remains undefined. The present study demonstrated that FAM20C is mainly located in the cytoplasm of duct epithelial cells in the salivary glands. Fam20cf/f; Mmtv­Cre mice were created in which Fam20c was inactivated in the salivary gland cells and observed that the number of ducts and the ductal cross­sectional area increased significantly, while the number of acinar cells was reduced. The granular convoluted tubules (GCTs) exhibited an accumulation of aberrant secretory granules, along with a reduced expression and altered distribution patterns of ß nerve growth factor, α­amylase and bone morphogenetic protein (BMP) 4. This abnormality suggested that the GCT cells were immature and exhibited defects in developmental and secretory functions. In accordance with the morphological alterations and the reduced number of acinar cells, FAM20C deficiency in the salivary glands significantly decreased the salivary flow rate. The Na+, Cl- and K+ concentrations in the saliva were all significantly increased due to dysfunction of the ducts. Furthermore, Fam20c deficiency significantly increased BMP2 and BMP7 expression, decreased BMP4 expression, and attenuated the canonical and noncanonical BMP signaling pathways in the salivary glands. Collectively, the results of the present study demonstrate that FAM20C is a key regulator of acinar and duct structure and duct maturation and provide a novel avenue for investigating novel therapeutic targets for oral diseases including xerostomia.


Assuntos
Células Acinares/patologia , Proteínas de Ligação ao Cálcio/deficiência , Proteínas da Matriz Extracelular/deficiência , Glândulas Salivares/patologia , Células Acinares/metabolismo , Células Acinares/ultraestrutura , Animais , Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Camundongos Knockout , Reprodutibilidade dos Testes , Saliva/metabolismo , Glândulas Salivares/metabolismo , Glândulas Salivares/ultraestrutura , Salivação , Transdução de Sinais , Glândula Submandibular/patologia
18.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(2): 156-161, 2019 02 28.
Artigo em Chinês | MEDLINE | ID: mdl-30890502

RESUMO

OBJECTIVE: To explore the effect of telmisartan on the expression of metadherin in the kidney of mice with unilateral ureter obstruction. METHODS: Eighteen male C57 mice were randomized into sham-operated group, model group and telmisartan treatment group. In the latter two groups, renal interstitial fibrosis as the result of unilateral ureter obstruction (UUO) was induced by unilateral ureteral ligation with or without telmisartan intervention. Renal pathological changes of the mice were assessed using Masson staining, and immunohistochemistry and Western blotting were used to detect the expression of extracellular matrix proteins and metadherin in the kidney of the mice. In the in vitro experiment, cultured mouse renal tubular epithelial cells (mTECs) were stimulated with transforming growth factor-ß1 (TGF-ß1) and transfected with a siRNA targeting metadherin, and the changes in the expressions of extracellular matrix proteins and metadherin were detected using Western blotting. RESULTS: The expressions of extracellular matrix proteins and metadherin increased significantly in the kidney of mice with UUO (P < 0.05). Intervention with telmisartan significantly lowered the expressions of extracellular matrix proteins and metadherin and alleviated the pathology of renal fibrosis in mice with UUO (P < 0.05). In cultured mTECs, siRNA-mediated knockdown of metadherin obviously reversed TGF-ß1-induced increase in the expressions of extracellular matrix proteins and metadherin. CONCLUSIONS: Telmisartan can suppress the production of extracellular matrix proteins and the expression of metadhein to attenuate UUO-induced renal fibrosis in mice.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Rim/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Telmisartan/farmacologia , Obstrução Ureteral/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II , Animais , Anti-Hipertensivos , Fibrose , Rim/metabolismo , Rim/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno , Distribuição Aleatória , Fator de Crescimento Transformador beta1/farmacologia , Obstrução Ureteral/complicações
19.
eNeuro ; 6(1)2019 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30863790

RESUMO

CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system (CNS). Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. CRISPRa targeting unique promoters in the complex multi-transcript gene brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.


Assuntos
Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Técnicas Genéticas , Neurônios/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Cultura Primária de Células , Distribuição Aleatória , Ratos Sprague-Dawley , Serina Endopeptidases/metabolismo , Transcrição Genética , Transcriptoma
20.
Methods Mol Biol ; 1952: 1-20, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825161

RESUMO

Extracellular matrix (ECM) maintains the structural integrity of tissues and regulates cell and tissue functions. ECM is comprised of fibrillar proteins, proteoglycans (PGs), glycosaminoglycans, and glycoproteins, creating a heterogeneous but well-orchestrated network. This network communicates with resident cells via cell-surface receptors. In particular, integrins, CD44, discoidin domain receptors, and cell-surface PGs and additionally voltage-gated ion channels can interact with ECM components, regulating signaling cascades as well as cytoskeleton configuration. The interplay of ECM with recipient cells is enriched by the extracellular vesicles, as they accommodate ECM, signaling, and cytoskeleton molecules in their cargo. Along with the numerous biological properties that ECM can modify, autophagy and angiogenesis, which are critical for tissue homeostasis, are included. Throughout development and disease onset and progression, ECM endures rearrangement to fulfill cellular requirements. The main responsible molecules for tissue remodeling are ECM-degrading enzymes including matrix metalloproteinases, plasminogen activators, cathepsins, and hyaluronidases, which can modify the ECM structure and function in a dynamic mode. A brief summary of the complex interplay between ECM macromolecules and cells in tissues and the contribution of ECM in tissue homeostasis and diseases is given.


Assuntos
Autofagia , Comunicação Celular , Matriz Extracelular/metabolismo , Neovascularização Fisiológica , Animais , Proteínas da Matriz Extracelular/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Neoplasias/metabolismo
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