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1.
Int J Mol Sci ; 22(5)2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800200

RESUMO

The identification of soluble fibroblast growth factor (FGF) receptors in blood and the extracellular matrix has led to the prediction that these proteins modulate the diverse biological activities of the FGF family of ligands in vivo. A recent structural characterization of the soluble FGF receptors revealed that they are primarily generated by proteolytic cleavage of the FGFR-1 ectodomain. Efforts to examine their biological properties are now focused on understanding the functional consequences of FGFR-1 ectodomain shedding and how the shedding event is regulated. We have purified an FGFR-1 ectodomain that is constitutively cleaved from the full-length FGFR-1(IIIc) receptor and released into conditioned media. This shed receptor binds FGF-2; inhibits FGF-2-induced cellular proliferation; and competes with high affinity, cell surface FGF receptors for ligand binding. FGFR-1 ectodomain shedding downregulates the number of high affinity receptors from the cell surface. The shedding mechanism is regulated by ligand binding and by activators of PKC, and the two signaling pathways appear to be independent of each other. Deletions and substitutions at the proposed cleavage site of FGFR-1 do not prevent ectodomain shedding. Broad spectrum inhibitors of matrix metalloproteases decrease FGFR-1 ectodomain shedding, suggesting that the enzyme responsible for constitutive, ligand-activated, and protein kinase C-activated shedding is a matrix metalloprotease. In summary, shedding of the FGFR-1 ectodomain is a highly regulated event, sharing many features with a common system that governs the release of diverse membrane proteins from the cell surface. Most importantly, the FGFR ectodomains are biologically active after shedding and are capable of functioning as inhibitors of FGF-2.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetulus , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Solubilidade
2.
DNA Cell Biol ; 40(3): 523-531, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33687273

RESUMO

Antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as or ciRS-7) is an important member of the circular RNA family and is involved in the regulation of numerous biological functions. Keratinocytes and fibroblasts (FBs) affect melanogenesis through paracrine effects. However, whether ciRS-7 is involved in melanogenesis by regulating paracrine effects remains unclear. This study demonstrates for the first time that ciRS-7 is highly expressed in keratinocytes, FBs, and melanocytes (MCs). Ultraviolet B (UVB) irradiation promotes ciRS-7 expression in keratinocytes and FBs. Following inhibition of ciRS-7 expression in keratinocytes and FBs, the culture supernatant from these cells inhibited melanogenesis of MCs. Further analyses revealed that the expression and secretion of fibroblast growth factor 2 (FGF2) and phosphorylation of STAT3 and AKT in keratinocytes and FBs were significantly downregulated following inhibition of ciRS-7 expression, whereas the level of miR-7 was increased. Overexpression of miR-7 in keratinocytes and FBs significantly inhibited the expression of FGF2. In conclusion, our findings demonstrate that UVB-induced ciRS-7 triggers melanogenesis in MCs through regulation of the miR-7/STAT3 and AKT/FGF2 paracrine axis in both keratinocytes and FBs. ciRS-7 may serve as a regulator in the development of pigmented skin diseases.


Assuntos
Regulação da Expressão Gênica/efeitos da radiação , Queratinócitos/metabolismo , Melaninas/biossíntese , Comunicação Parácrina/efeitos da radiação , RNA Longo não Codificante/metabolismo , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta , Linhagem Celular Transformada , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo
3.
J Vis Exp ; (167)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33491672

RESUMO

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials. One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Maleimidas/química , Compostos de Sulfidrila/química , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/química , Neoplasias/tratamento farmacológico , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Domínios Proteicos , Engenharia de Proteínas
4.
Exp Eye Res ; 203: 108402, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33326809

RESUMO

PURPOSE: To develop and characterize a new type of plasma rich in growth factors (PRGF) membrane for patients in which immune system is involved in the disease etiology. METHODS: Blood from three healthy donors was collected to obtain the different fibrin membranes by PRGF technology. PRGF obtained volumes were activated and divided into two groups: PRGF membrane (mPRGF) obtained after incubation at 37 °C for 30 min (control); and is-mPRGF: mPRGF obtained after incubation for 30 min at 56 °C. The concentration of several growth factors, proteins, immunoglobulin E and the complement activity was determined in the different mPRGF. The proliferative potential of heat-inactivated mPRGF were assayed on keratocytes (HK) and conjunctival fibroblasts (HConF). In addition, morphological and physical features of the inactivated mPRGF were evaluated in contrast to the control mPRGF. RESULTS: Heat-inactivation of the mPRGF preserves the content of most of the growth factors involved in the ocular wound healing while reducing drastically the content of IgE and the complement activity. The heat-inactivated mPRGF conserve the morphological and physical characteristics of the fibrin meshwork in comparison with the control mPRGF. Furthermore, no significant differences were found in the biological activity of the control mPRGF regarding the heat-inactivated mPRGF (is-mPRGF) in any of both ocular cell types evaluated. CONCLUSIONS: The heat-inactivation of the PRGF membranes (is-mPRGF) reduces drastically the content of IgE and complement activity while preserving the content of most of the proteins and morphogens involved in ocular wound healing. Furthermore, the morphological and physical features of the immunosafe mPRGF were also preserved after heat-inactivation.


Assuntos
Membrana Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fibrina Rica em Plaquetas/metabolismo , Tecnologia Biomédica , Doadores de Sangue , Células Cultivadas , Via Clássica do Complemento/fisiologia , Túnica Conjuntiva/citologia , Ceratócitos da Córnea/metabolismo , Ensaio de Imunoadsorção Enzimática , Fator de Crescimento Epidérmico/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunoglobulina E/imunologia , Microscopia Eletrônica de Varredura , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Gene ; 770: 145339, 2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33333220

RESUMO

Hair follicle (HF) development is characterized by periodic growth cycles regulated by numerous factors. We previously showed that SMAD2 might be involved in the HF growth cycle in Angora rabbits. However, its extra role in the HF growth and development remains obscure. In this study, we cloned the complete coding sequence (CDS) of the Angora rabbit SMAD2 gene. Within SMAD2 CDS, we identified the open reading frame (ORF) had a length of 1314 bp and encoding 437 amino acids. Bioinformatics analyses revealed that the SMAD2 protein is unstable and hydrophilic, and predominatelylocalizesin the cell nucleus. We identified that SMAD2 expression was elevated in the telogen phase of the during HF cycle. The knockdown and overexpression of SMAD2 could regulate HF growth and development related genes, such as WNT2, FGF2, and LEF1.Furthermore, SMAD2 may upregulate TGF-ß signaling pathway-related genes, including TFDP1, E2F4, and RBL1. In conclusion, our results indicate that SMAD2 plays a vital role in HF development by regulating the TGF-ß signaling pathway.


Assuntos
Folículo Piloso/metabolismo , Proteína Smad2/metabolismo , Animais , Fator 2 de Crescimento de Fibroblastos/metabolismo , Folículo Piloso/citologia , Masculino , Coelhos , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína Wnt2/metabolismo
6.
Mol Biol Rep ; 47(10): 8301-8304, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32920756

RESUMO

The interest on the role of angiogenesis in the pathogenesis and progression of human interstitial lung diseases is growing, with conventional sprouting (SA) and non-sprouting intussusceptive angiogenesis (IA) being differently represented in specific pulmonary injury patterns. The role of viruses as key regulators of angiogenesis is known for several years. A significantly enhanced amount of new vessel growth, through a mechanism of IA, has been reported in lungs of patients who died from Covid-19; among the angiogenesis-related genes, fibroblast growth factor 2 (FGF2) was found to be upregulated. These findings are intriguing. FGF2 plays a role in some viral infections: the upregulation is involved in the MERS-CoV-induced strong apoptotic response crucial for its highly lytic replication cycle in lung cells, whereas FGF2 is protective against the acute lung injury induced by H1N1 influenza virus, improving the lung wet-to-dry weight ratio. FGF2 plays a role also in regulating IA, acting on pericytes (crucial for the formation of intraluminal pillars), and endothelium, and FGF2-induced angiogenesis may be promoted by inflammation and hypoxia. IA is a faster and probably more efficient process than SA, able to modulate vascular remodeling through pruning of redundant or inefficient blood vessels. We can speculate that IA might have the function of restoring a functional vascular plexus consequently to extensive endothelialitis and alveolar capillary micro-thrombosis observed in Covid-19. Anti-Vascular endothelial growth factor (anti-VEGF) strategies are currently investigated for treatment of severe and critically ill Covid-19 patients, but also FGF2, and its expression and/or signaling, might represent a promising target.


Assuntos
Infecções por Coronavirus/patologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neovascularização Patológica/virologia , Pneumonia Viral/patologia , Antivirais/farmacologia , Antivirais/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/etiologia , Sistemas de Liberação de Medicamentos , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Humanos , Intussuscepção/virologia , Neovascularização Patológica/genética , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/etiologia
7.
Nat Commun ; 11(1): 4064, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792542

RESUMO

Regulation of the programming of tumour-associated macrophages (TAMs) controls tumour growth and anti-tumour immunity. We examined the role of FGF2 in that regulation. Tumours in mice genetically deficient in low-molecular weight FGF2 (FGF2LMW) regress dependent on T cells. Yet, TAMS not T cells express FGF receptors. Bone marrow derived-macrophages from Fgf2LMW-/- mice co-injected with cancer cells reduce tumour growth and express more inflammatory cytokines. FGF2 is induced in the tumour microenvironment following fractionated radiation in murine tumours consistent with clinical reports. Combination treatment of in vivo tumours with fractionated radiation and a blocking antibody to FGF2 prolongs tumour growth delay, increases long-term survival and leads to a higher iNOS+/CD206+ TAM ratio compared to irradiation alone. These studies show for the first time that FGF2 affects macrophage programming and is a critical regulator of immunity in the tumour microenvironment.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Radioterapia/métodos , Animais , Linhagem Celular Tumoral , Fator 2 de Crescimento de Fibroblastos/genética , Células HT29 , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/efeitos da radiação , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Arterioscler Thromb Vasc Biol ; 40(9): 2293-2309, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32757648

RESUMO

OBJECTIVE: Extracellular vesicles (EVs) have the potential to act as intercellular communicators. The aims were to characterize circulating EVs in patients with pulmonary arterial hypertension (PAH) and to explore whether these EVs contribute to endothelial activation and angiogenesis. Approach and Results: Patients with PAH (n=70) and healthy controls (HC; n=20) were included in this cross-sectional study. EVs were characterized and human pulmonary endothelial cells (hPAECs) were incubated with purified EVs. Endothelial cell activity and proangiogenic markers were analyzed. Tube formation analysis was performed for hPAECs, and the involvement of PSGL-1 (P-selectin glycoprotein ligand 1) was evaluated. The numbers of CD62P+, CD144+, and CD235a EVs were higher in blood from PAH compared with HC. Thirteen proteins were differently expressed in PAH and HC EVs, where complement fragment C1q was the most significantly elevated protein (P=0.0009) in PAH EVs. Upon EVs-internalization in hPAECs, more PAH compared with HC EVs evaded lysosomes (P<0.01). As oppose to HC, PAH EVs stimulated hPAEC activation and induced transcription and translation of VEGF-A (vascular endothelial growth factor A; P<0.05) and FGF (fibroblast growth factor; P<0.005) which were released in the cell supernatant. These proangiogenic proteins were higher in patient with PAH plasma compered with HC. PAH EVs induced a complex network of angiotubes in vitro, which was abolished by inhibitory PSGL-1antibody. Anti-PSGL-1 also inhibited EV-induced endothelial cell activation and PAH EV dependent increase of VEGF-A. CONCLUSIONS: Patients with PAH have higher levels of EVs harboring increased amounts of angiogenic proteins, which induce activation of hPAECs and in vitro angiogenesis. These effects were partly because of platelet-derived EVs evasion of lysosomes upon internalization within hPAEC and through possible involvement of P-selectin-PSGL-1 pathway.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Vesículas Extracelulares/metabolismo , Neovascularização Fisiológica , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , Idoso , Estudos de Casos e Controles , Células Cultivadas , Estudos Transversais , Células Endoteliais/ultraestrutura , Endotélio Vascular/fisiopatologia , Endotélio Vascular/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Selectina-P/metabolismo , Hipertensão Arterial Pulmonar/patologia , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/fisiopatologia , Artéria Pulmonar/ultraestrutura , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
9.
PLoS One ; 15(7): e0236050, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678832

RESUMO

Neurotrophic factors have been regarded having promising potentials for neuronal protection and regeneration, and thus promoting beneficial effects of kinesiological functions. They can be suspected to play important roles in cell/tissue grafting for various neural diseases. The clinical applications of such trophic factors to the central nervous system (CNS), however, have caused problematic side effects on account of the distinctive bioactive properties. In the course of developing synthetic compounds reflecting beneficial properties of basic fibroblast growth factor (bFGF), we conducted screening candidates that stimulate to trigger the intracellular tyrosine phosphorylation of FGF receptor and lead to the subsequent intracellular signaling in neurons. A small synthetic molecule SUN13837 was characterized by mimicking the beneficial properties of bFGF, which have been known as its specific activities when applied to CNS. What is more remarkable is that SUN13837 is eliminated the bioactivity to induce cell proliferation of non-neuronal somatic cells. On the bases of studies of pharmacology, behavior, physiology and histology, the present study reports that SUN13837 is characterized as a promising synthetic compound for treatment of devastating damages onto the rat spinal cord.


Assuntos
Materiais Biomiméticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Feminino , Crescimento Neuronal/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia
10.
Nat Commun ; 11(1): 3704, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709869

RESUMO

FGF-2 displays multifarious functions in regulation of angiogenesis and vascular remodeling. However, effective drugs for treating FGF-2+ tumors are unavailable. Here we show that FGF-2 modulates tumor vessels by recruiting NG2+ pricytes onto tumor microvessels through a PDGFRß-dependent mechanism. FGF-2+ tumors are intrinsically resistant to clinically available drugs targeting VEGF and PDGF. Surprisingly, dual targeting the VEGF and PDGF signaling produces a superior antitumor effect in FGF-2+ breast cancer and fibrosarcoma models. Mechanistically, inhibition of PDGFRß ablates FGF-2-recruited perivascular coverage, exposing anti-VEGF agents to inhibit vascular sprouting. These findings show that the off-target FGF-2 is a resistant biomarker for anti-VEGF and anti-PDGF monotherapy, but a highly beneficial marker for combination therapy. Our data shed light on mechanistic interactions between various angiogenic and remodeling factors in tumor neovascularization. Optimization of antiangiogenic drugs with different principles could produce therapeutic benefits for treating their resistant off-target cancers.


Assuntos
Inibidores da Angiogênese/farmacologia , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neoplasias/tratamento farmacológico , Fator de Crescimento Derivado de Plaquetas/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Animais , Biomarcadores Tumorais , Pressão Sanguínea , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Permeabilidade Capilar , Proliferação de Células , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Quimioterapia Combinada , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Transdução de Sinais/efeitos dos fármacos , Hipóxia Tumoral , Microambiente Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo
11.
Mol Carcinog ; 59(9): 1028-1040, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32557854

RESUMO

Cancer-associated fibroblasts (CAFs) constitute a major compartment of the tumor microenvironment. In the present study, we investigated the role for CAFs in breast cancer progression and underlying molecular mechanisms. Human breast cancer MDA-MB-231 cells treated with the CAF-conditioned media manifested a more proliferative phenotype, as evidenced by enhanced messenger RNA (mRNA) expression of Cyclin D1, c-Myc, and proliferating cell nuclear antigen. Analysis of data from The Cancer Genome Atlas revealed that fibroblast growth factor-2 (FGF2) expression was well correlated with the presence of CAFs. We noticed that the mRNA level of FGF2 in CAFs was higher than that in normal fibroblasts. FGF2 exerts its biological effects through interaction with FGF receptor 1 (FGFR1). In the breast cancer tissue array, 42% estrogen receptor-negative patients coexpressed FGF2 and FGFR1, whereas only 19% estrogen receptor-positive patients exhibited coexpression. CAF-stimulated MDA-MB-231 cell migration and invasiveness were abolished when FGF2-neutralizing antibody was added to the conditioned media of CAFs. In a xenograft mouse model, coinjection of MDA-MB-231 cells with activated fibroblasts expressing FGF2 dramatically enhanced tumor growth, and this was abrogated by silencing of FGFR1 in cancer cells. In addition, treatment of MDA-MB-231 cells with FGF2 enhanced expression of Cyclin D1, a key molecule involved in cell cycle progression. FGF2-induced cell migration and upregulation of Cyclin D1 were abolished by siRNA-mediated FGFR1 silencing. Taken together, the above findings suggest that CAFs promote growth, migration and invasion of MDA-MB-231 cells via the paracrine FGF2-FGFR1 loop in the breast tumor microenvironment.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/metabolismo , Proliferação de Células , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/patologia , Movimento Celular , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Comunicação Parácrina , Prognóstico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Medicine (Baltimore) ; 99(26): e20644, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32590739

RESUMO

The present study aimed to investigate the correlation between ultrasonographic features, basic fibroblast growth factor (bFGF), and the local invasiveness of papillary thyroid carcinoma (PTC).A total of 350 samples of thyroid nodules were collected. Routine ultrasonography was performed before the operation and routine pathological diagnosis and bFGF detection were performed after the operation.'These 350 samples of thyroid nodules included 90 samples of nodular goiter, 36 samples of focal thyroiditis, and 224 samples of PTC. A total of 326 thyroid nodules were examined for bFGF. The results revealed that the difference in the expression of bFGF between the benign and malignant groups was statistically significant (P < .05) and the difference in the positive expression of bFGF between the invasive and non-invasive PTC groups was statistically significant (P < .05).Whether the shape of PTC is regular or not and whether there is micro-calcification in PTC and other ultrasonographic features, the size and location of the lesions and the age of the patient help make a preliminary prediction of local invasiveness before the operation. Postoperative detection of bFGF is helpful for further risk assessments of PTC.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Câncer Papilífero da Tireoide/diagnóstico por imagem , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Neoplasias da Glândula Tireoide/metabolismo , Adulto , Fatores Etários , Idoso , Biomarcadores Tumorais/metabolismo , Feminino , Bócio/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estudos Prospectivos , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/metabolismo , Tireoidite/metabolismo , Ultrassonografia , Adulto Jovem
13.
Nucleic Acids Res ; 48(11): 6340-6352, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32383752

RESUMO

API5 (APoptosis Inhibitor 5) and nuclear FGF2 (Fibroblast Growth Factor 2) are upregulated in various human cancers and are correlated with poor prognosis. Although their physical interaction has been identified, the function related to the resulting complex is unknown. Here, we determined the crystal structure of the API5-FGF2 complex and identified critical residues driving the protein interaction. These findings provided a structural basis for the nuclear localization of the FGF2 isoform lacking a canonical nuclear localization signal and identified a cryptic nuclear localization sequence in FGF2. The interaction between API5 and FGF2 was important for mRNA nuclear export through both the TREX and eIF4E/LRPPRC mRNA export complexes, thus regulating the export of bulk mRNA and specific mRNAs containing eIF4E sensitivity elements, such as c-MYC and cyclin D1. These data show the newly identified molecular function of API5 and nuclear FGF2, and provide a clue to understanding the dynamic regulation of mRNA export.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Transporte de RNA , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Cristalografia por Raios X , Ciclina D1/metabolismo , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo
14.
Med Sci Monit ; 26: e920684, 2020 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-32283546

RESUMO

BACKGROUND Acute pancreatitis (AP) is a symptom of sudden pancreas inflammation, which causes patients severe suffering. In general, fibroblast growth factor (FGF) levels are increased and amylase and lipase activities are elevated during AP pathogenesis, but protein concentration are low. However, the mechanism through which FGF signaling regulates AP pathogenesis remains elusive. MATERIAL AND METHODS The concentrations of PGE2, TNF-alpha, sCRP, FGF1, and FGF2 in the serum samples of the AP group and healthy control group were detected by enzyme-linked immunosorbent assay. In addition, IkappaBalpha and p-IkappaBalpha levels were analyzed in the serum samples. Subsequently, the AP rat model was established, and FGF1, FGF2, anti-FGF1, and anti-FGF2 antibodies and Bay11-7082 were injected into AP rats. TNF-alpha, PAI-1 JNK, p-JNK, IkappaBalpha, and p-IkappaBalpha levels were also examined. RESULTS Results showed that levels of PGE2, TNF-alpha, sCRP, p-IkappaBalpha, FGF1, and FGF2, as well as amylase and lipase activity were increased in patients with AP compared with those in healthy people. In addition, protein concentrations were lower in patients with AP than in the healthy group. Activation of FGF signaling by injecting FGF1 or FGF2 also inhibited AP-induced inflammation response in the pancreas and increased amylase and lipase activities, as well as protein concentration. However, the injection of FGF1 and FGF2 antibodies accelerated AP-mediated inflammation responses in the serum. In addition, Bay11-7082 injection inhibited AP activation of inflammation response and amylase and lipase activities. Protein concentration were also increased in AP rats. CONCLUSIONS FGF signaling protects against AP-mediated damage by inhibition of AP-activating inflammatory responses.


Assuntos
Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Inflamação/metabolismo , Pancreatite/patologia , Transdução de Sinais , Doença Aguda , Adulto , Amilases/metabolismo , Animais , Proteína C-Reativa/análise , Estudos de Casos e Controles , Dinoprostona/sangue , Feminino , Fator 1 de Crescimento de Fibroblastos/sangue , Fator 2 de Crescimento de Fibroblastos/sangue , Humanos , Inflamação/patologia , Lipase/metabolismo , Masculino , Pessoa de Meia-Idade , Inibidor de NF-kappaB alfa/sangue , Nitrilos/farmacologia , Ratos , Ratos Sprague-Dawley , Sulfonas/farmacologia , Fator de Necrose Tumoral alfa/sangue
15.
Int J Mol Sci ; 21(2)2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-32284513

RESUMO

Cell-penetrating peptides (CPPs) are defined by their ability to deliver cargo into cells and have been studied and developed as a promising drug-delivery system (DDS). However, the issue of whether the CPPs that have already entered the cells can be re-released or reused has not been studied. The purpose of this research was to construct CPP-conjugated human fibroblast growth factor 2 (hFGF2) and investigate whether they can be re-released from the cell membrane for reuse. This study combined hFGF2 with Tat or Ara27, a newly developed CPP derived from the zinc knuckle (CCHC-type) family protein of Arabidopsis. Human dermal fibroblast (HDF) was treated with Tat-conjugated hFGF2 (tFGF2) and Ara27-conjugated hFGF2 (NR-FGF2) for both long and short durations, and the effects on cell growth were compared. Furthermore, tFGF2 and NR-FGF2 re-released from the cells were quantified and the effects were evaluated by culturing HDF in a conditioned medium. Interestingly, the proliferation of HDF increased only when NR-FGF2 was treated for 1 h in endocytosis-independent manner. After 1 h, NR-FGF2 was significantly re-released, reaching a maximum concentration at 5 h. Furthermore, increased proliferation of HDF cultured in the conditioned medium containing re-released NR-FGF2 was discovered. While previous studies have focused on the delivery of cargo and its associated applications, this study has revealed that combinations of superior CPPs and therapeutics can be expected to prolong both the retention time and the cell-penetrating capacity, even in the presence of external factors. Therefore, CPPs can be applied in the context of topical drugs and cosmetics as a new DDS approach.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Sistemas de Liberação de Medicamentos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Arabidopsis/genética , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/administração & dosagem , Endocitose , Humanos
16.
Carbohydr Polym ; 237: 116143, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32241440

RESUMO

A sulfated glucurono-xylo-rhamnan (EP-3-H) was purified from a green alga, Enteromorpha prolifera. EP-3-H and its oligomers were characterized by high performance liquid chromatography, mass spectrometry and one and two-dimensional nuclear magnetic resource spectroscopy. The structural analysis showed EP-3-H has a backbone of glucurono-xylo-rhamnan, branches with glucuronic acid and sulfated at C3 of rhamnose and/or C2 of xylose. The inhibition of EP-3-H on human lung cancer A549 cell proliferation in vitro and its therapeutic effects in BALB/c-nu mice in vivo were determined to evaluate the anti-lung cancer activity of EP-3-H. The tumor inhibition level was 59 %, suggesting that EP-3-H might be a good candidate for the treatment of lung cancer. Surface plasmon resonance (SPR) studies revealed the IC50 on the binding of fibroblast growth factors, (FGF1 and FGF2), to heparin were 0.85 and 1.47 mg/mL, respectively. These results suggest that EP-3-H inhibits cancer proliferation by interacting with these growth factors.


Assuntos
Antineoplásicos , Desoxiaçúcares , Neoplasias Pulmonares/tratamento farmacológico , Mananas , Células A549 , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Desoxiaçúcares/isolamento & purificação , Desoxiaçúcares/farmacologia , Desoxiaçúcares/uso terapêutico , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Mananas/isolamento & purificação , Mananas/farmacologia , Mananas/uso terapêutico , Camundongos Endogâmicos BALB C , Camundongos Nus , Sulfatos , Ulva/química
17.
Med Sci Monit ; 26: e920520, 2020 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-32188838

RESUMO

BACKGROUND Freshly isolated mouse embryonic fibroblasts (MEFs) have great proliferation capacity but quickly enter senescent state after several rounds of cell cycle, a process called premature senescence. Cellular senescence can be induced by various stresses such as telomere erosion, DNA damage, and oncogenic signaling. But the contribution of other molecules, such as growth factors, to cellular senescence is incompletely understood. This study aimed to compare the gene expression difference between non-senescent and senescent MEFs to identify the key molecule(s) involved in the spontaneous senescence of MEFs. MATERIAL AND METHODS Primary MEFs were isolated from E12.5 pregnant C57/BL6 mice. The cells were continuously cultured in Dulbecco's Modified Eagle Medium for 9 passages. SA-ß-Gal staining was used as an indicator of cell senescence. The supernatant from primary MEFs (P1 medium) or Passage 6 MEFs (P6 medium) were used to culture freshly isolated MEFs to observe the effects on cell senescence state. Gene expression profiles of primary and senescent MEFs were investigated by RNA-Seq to find the key genes involved in cell senescence. Adipocyte differentiation assay was used to evaluate the stemness of MEFs cultured in FGF2-stimulated medium. RESULTS The senescence of MEFs cultured in the P1 medium was alleviated when compared to the P6 medium. Downregulation of FGF2 expression was revealed by RNA-Seq and further confirmed by real-time quantitative polymerase chain reaction and western blot. FGF2-stimulated medium also had anti-senescence function and could maintain the differentiation ability of MEFs. CONCLUSIONS The premature senescence of MEFs was at least partially caused by FGF2 deficiency. Exogenous FGF2 could alleviate the senescent phenotype.


Assuntos
Senescência Celular/fisiologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Dano ao DNA , Embrião de Mamíferos/metabolismo , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais
18.
Sci Rep ; 10(1): 2939, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076044

RESUMO

Anti-vascular endothelial growth factor (VEGF) therapy shows antitumor activity against various types of solid cancers. Several resistance mechanisms against anti-VEGF therapy have been elucidated; however, little is known about the mechanisms by which the acquired resistance arises. Here, we developed new anti-VEGF therapy-resistant models driven by chronic expression of the mouse VEGFR2 extracellular domain fused with the human IgG4 fragment crystallizable (Fc) region (VEGFR2-Fc). In the VEGFR2-Fc-expressing resistant tumors, we demonstrated that the FGFR2 signaling pathway was activated, and pericytes expressing high levels of FGF2 were co-localized with endothelial cells. Lenvatinib, a multiple tyrosine kinase inhibitor including VEGFR and FGFR inhibition, showed marked antitumor activity against VEGFR2-Fc-expressing resistant tumors accompanied with a decrease in the area of tumor vessels and suppression of phospho-FGFR2 in tumors. Our findings reveal the key role that intercellular FGF2 signaling between pericytes and endothelial cells plays in maintaining the tumor vasculature in anti-VEGF therapy-resistant tumors.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Transdução de Sinais , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Modelos Biológicos , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismo
19.
Sci Rep ; 10(1): 2744, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066777

RESUMO

Hyperbaric oxygen (HBO) treatment promotes early recovery from muscle injury. Reactive oxygen species (ROS) upregulation is a key mechanism of HBO, which produces high O2 content in tissues through increased dissolution of oxygen at high pressure. Nitric oxide (NO), a type of ROS, generally stabilizes hypoxia-inducible factor (HIF) 1α and stimulates secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) from endothelial cells and macrophages, which then induces angiogenesis. The purpose of the present study was to investigate whether HBO could promote angiogenesis via induction of NO and induce muscle regeneration in contused rat skeletal muscles. The HBO protocol consisted of 2.5 atmospheres absolute (ATA) 100% oxygen for 120 minutes, once a day for 5 consecutive days. We also evaluated the effects of a ROS inhibitor (NAC) or NOS-specific inhibitor (L-NAME) on HBO. HBO significantly increased NO3-, VEGF, and bFGF levels and stabilized HIF1α within 1 day. HBO promoted blood vessel formation at 3-7 days and muscle healing at 5-7 days after contusion. Administration of both NAC and L-NAME before HBO suppressed angiogenesis and muscle regeneration even after HBO. HBO thus promoted angiogenesis and muscle regeneration mainly through generation of NO in the early phase after muscle contusion injury.


Assuntos
Contusões/terapia , Oxigenação Hiperbárica/métodos , Músculo Esquelético/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/biossíntese , Oxigênio/farmacologia , Acetilcisteína/farmacologia , Indutores da Angiogênese , Animais , Contusões/genética , Contusões/metabolismo , Contusões/patologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/agonistas , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Regeneração/efeitos dos fármacos , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Int J Mol Sci ; 21(5)2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-32106626

RESUMO

Liver ischaemia-reperfusion injury (IRI) is an intrinsic part of the transplantation process and damages the parenchymal cells of the liver including hepatocytes, endothelial cells and cholangiocytes. Many biomarkers of IRI have been described over the past two decades that have attempted to quantify the extent of IRI involving different hepatic cellular compartments, with the aim to allow clinicians to predict the suitability of donor livers for transplantation. The advent of machine perfusion has added an additional layer of complexity to this field and has forced researchers to re-evaluate the utility of IRI biomarkers in different machine preservation techniques. In this review, we summarise the current understanding of liver IRI biomarkers and discuss them in the context of machine perfusion.


Assuntos
Transplante de Fígado/métodos , Traumatismo por Reperfusão/diagnóstico , Animais , Biomarcadores/metabolismo , Endotelina-1/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Interleucinas/metabolismo , Transplante de Fígado/efeitos adversos , Transplante de Fígado/instrumentação , MicroRNAs/metabolismo , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo
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