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1.
Nat Commun ; 11(1): 4520, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908154

RESUMO

Tumor extracellular matrix has been associated with drug resistance and immune suppression. Here, proteomic and RNA profiling reveal increased collagen levels in lung tumors resistant to PD-1/PD-L1 blockade. Additionally, elevated collagen correlates with decreased total CD8+ T cells and increased exhausted CD8+ T cell subpopulations in murine and human lung tumors. Collagen-induced T cell exhaustion occurs through the receptor LAIR1, which is upregulated following CD18 interaction with collagen, and induces T cell exhaustion through SHP-1. Reduction in tumor collagen deposition through LOXL2 suppression increases T cell infiltration, diminishes exhausted T cells, and abrogates resistance to anti-PD-L1. Abrogating LAIR1 immunosuppression through LAIR2 overexpression or SHP-1 inhibition sensitizes resistant lung tumors to anti-PD-1. Clinically, increased collagen, LAIR1, and TIM-3 expression in melanoma patients treated with PD-1 blockade predict poorer survival and response. Our study identifies collagen and LAIR1 as potential markers for immunotherapy resistance and validates multiple promising therapeutic combinations.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos Imunológicos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Colágeno/metabolismo , Resistencia a Medicamentos Antineoplásicos/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Receptores Imunológicos/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Animais , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/imunologia , Matriz Extracelular/patologia , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Pulmão/imunologia , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA-Seq , Receptores Imunológicos/genética
2.
Nat Commun ; 11(1): 4659, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938936

RESUMO

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.


Assuntos
Butiratos/farmacologia , Fibrose Pulmonar Idiopática/tratamento farmacológico , Integrinas/antagonistas & inibidores , Naftiridinas/farmacologia , Pirazóis/farmacologia , Pirrolidinas/farmacologia , Administração por Inalação , Animais , Antígenos de Neoplasias/metabolismo , Bleomicina/toxicidade , Butiratos/administração & dosagem , Butiratos/metabolismo , Butiratos/farmacocinética , Colágeno/metabolismo , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/patologia , Integrinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Naftiridinas/administração & dosagem , Naftiridinas/metabolismo , Naftiridinas/farmacocinética , Pirazóis/administração & dosagem , Pirazóis/metabolismo , Pirazóis/farmacocinética , Pirrolidinas/administração & dosagem , Pirrolidinas/metabolismo , Pirrolidinas/farmacocinética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tomografia Computadorizada de Emissão de Fóton Único , Fator de Crescimento Transformador beta/metabolismo , Pesquisa Médica Translacional
3.
BMC Infect Dis ; 20(1): 580, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32762658

RESUMO

BACKGROUND: Dengue virus (DENV) causes the hospitalisation of an estimated 500,000 people every year. Outbreaks can severely stress healthcare systems, especially in rural settings. It is difficult to discriminate patients who need to be hospitalized from those that do not. Earlier work identified thrombocyte count and subsequent function as a promising prognostic marker of DENV severity. Herein, we investigated the potential of quantitative thrombocyte function tests in those admitted in the very early phase of acute DENV infections, using Multiplate™ multiple-electrode aggregometry to explore its potential in triage. METHODS: In this prospective cohort study all patients aged ≥13 admitted to Universitas Airlangga Hospital in Surabaya, Indonesia with a fever (≥38 °C) between 25 January and 1 August 2018 and with a clinical suspicion of DENV, were eligible for inclusion. Exclusion criteria were a thrombocyte count below 100 × 109/L and the use of any medication with a known anticoagulant effect, nonsteroidal anti-inflammatory drugs and acetyl salicylic acid. Clinical data was collected and blood was taken on admission, day 1 and day 7. Samples were tested for acute DENV, using Panbio NS1 ELISA. Platelet aggregation using ADP-, TRAP- and COL-test were presented as Area Under the aggregation Curve (AUC). Significance was tested between DENV+, probably DENV, fever of another origin, and healthy controls (HC). RESULTS: A total of 59 patients (DENV+ n = 10, DENV probable n = 25, fever other origin n = 24) and 20 HC were included. We found a significantly lower thrombocyte aggregation in the DENV+ group, compared with both HCs and the fever of another origin group (p < .001). Low ADP AUC values on baseline correlated to a longer hospital stay in DENV+ and probable DENV cases. CONCLUSION: Thrombocyte aggregation induced by Adenosine diphosphate, Collagen and Thrombin receptor activating peptide-6 is impaired in human DENV cases, compared with healthy controls and other causes of fever. This explorative study provides insights to thrombocyte function in DENV patients and could potentially serve as a future marker in DENV disease.


Assuntos
Plaquetas/metabolismo , Vírus da Dengue/imunologia , Dengue/diagnóstico , Dengue/epidemiologia , Sistemas Automatizados de Assistência Junto ao Leito , Testes Imediatos , Difosfato de Adenosina/sangue , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Colágeno/metabolismo , Dengue/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Febre/diagnóstico , Humanos , Indonésia/epidemiologia , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/sangue , Agregação Plaquetária , Contagem de Plaquetas , Estudos Prospectivos , Índice de Gravidade de Doença , Adulto Jovem
4.
Int J Nanomedicine ; 15: 4943-4956, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764927

RESUMO

Background: Hydroxyapatite (HA) [Ca5(PO4)3(OH)] is a naturally occurring calcium phosphate which makes up 60-70% of the dry weight of human bones. Nano-scale HA particles are increasingly being used as carriers for controlled and targeted delivery of bioactive agents like drugs, proteins, and nucleic acids due to their high porosity, negative charge, and biodegradability. Purpose: Although much effort has been devoted to understanding the delivery kinetics and effects of the payloads in such carriers, a thorough understanding of the influence of the carriers themselves is lacking. Methods: HA particles (300 µg/mL) were administered to primary human dermal fibroblasts (HDFs). The uptake and intracellular localization of the particles were determined by flow cytometry, confocal imaging, and transmission electron microscopy (TEM). Immunological assays and PCR were performed to determine the levels of pro-inflammatory cytokines and collagens in cell lysates and media supernatant. Results: The current study explores the effects of poly-dispersed HA particles on primary HDFs as a model system. The majority of the particles were determined to range between 150 and 200 nm in diameter. Upon exposure to HA suspensions, primary HDFs internalized the particles by endocytosis within 6 hours of exposure, showing maximum uptake at 72 hours following which the particles were exocytosed by 168 hours. This correlated to reduced secretion of various pro-inflammatory and pro-collagenic cytokines. Biochemical analysis further revealed a reduction in Type I collagen expression and secretion. Conclusion: HA particles have an immune-modulatory effect on dermal fibroblasts and reduce collagen production, which may impact the integrity of the extracellular matrix (ECM). This study demonstrates the need to consider the secondary effects of particulate carriers like HA, beyond basic cytotoxicity, in the specific tissue environment where the intended function is to be realized.


Assuntos
Colágeno/metabolismo , Durapatita/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Pele/citologia , Colágeno Tipo I/metabolismo , Citocinas/metabolismo , Durapatita/química , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Humanos
5.
Int J Nanomedicine ; 15: 4991-5004, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764931

RESUMO

Introduction: Various materials and approaches have been used to reduce the mesh-induced inflammatory response and modify the mesh with tissue-matched mechanical properties, aiming to improve the repair of abdominal wall defects. Materials and Methods: In this study, we fabricated a polycaprolactone (PCL)/silk fibroin (SF) mesh integrated with amoxicillin (AMX)-incorporating multiwalled carbon nanotubes (MWCNTs) via electrospinning, grafting and crosslinking, developing a sustainable antibiotic and flexible mesh. AMX was loaded into the hollow tubular MWCNTs by physical adsorption, and a nanofibrous structure was constructed by electrospinning PCL and SF (40:60 w/w). The AMX@MWCNTs were then chemically grafted onto the surfaces of the PCL/SF nanofibers by treating with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) solution for simultaneous crosslinking and coating. The incorporation of AMX into the MWCNTs (AMX@MWCNTs) and the integration of the AMX@MWCNTs with the PCL/SF nanofibers were characterized. Then, the functional mesh was fabricated and fully evaluated in terms of antibacterial activity, mechanical properties and host response. Results: Our results demonstrated that the PCL/SF nanofibrous structure was fabricated successfully by electrospinning. After integrating with AMX@MWCNT by grafting and crosslinking, the functional mesh showed undeformed structure, modified surface hydrophilicity and biocompatible interfaces, abdominal wall-matched mechanical properties, and a sustained-release antibiotic profile in E. coli growth inhibition compared to those of PCL/SF mesh in vitro. In a rat model with subcutaneous implantation, the functional mesh incited less mesh-induced inflammatory and foreign body responses than PCL/SF mesh within 14 days. The histological analysis revealed less infiltration of granulocytes and macrophages during this period, resulting in the loosely packed collagen deposition on the functional mesh and prominent collagen incorporation. Discussion: Therefore, this designed PCL/SF-AMX@MWCNT nanofibrous mesh, functionalized with antibacterial and tissue-matched mechanical properties, provides a promising alternative for the repair of abdominal wall defects.


Assuntos
Amoxicilina/química , Antibacterianos/química , Nanofibras/química , Nanotecnologia/métodos , Telas Cirúrgicas , Amoxicilina/farmacocinética , Amoxicilina/farmacologia , Animais , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Colágeno/química , Colágeno/metabolismo , Reagentes para Ligações Cruzadas/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Fibroínas/química , Inflamação/etiologia , Masculino , Teste de Materiais , Camundongos , Nanotubos de Carbono/química , Poliésteres/química , Ratos Sprague-Dawley , Telas Cirúrgicas/efeitos adversos
6.
Gene ; 761: 145024, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32755659

RESUMO

Understanding how various pathologies of breast cancer respond to their environment may be imperative in the creation of novel therapeutic targets. Central to the organisation and behaviour of cells within the tumour microenvironment is the extracellular matrix (ECM), a meshwork of fibrous proteins and glycoproteins that directly influences cell behaviour and the bioavailability of signalling molecules. Our appreciation on how the composition of the ECM can influence cancer behaviour has evolved significantly and although we are highly cognisant of the dramatic impact the ECM can have on cancer cell behaviour, we continue to neglect this during diagnosis and treatment. In the following study, we aimed to identify how three breast cancer cell lines respond functionally and genetically to common components of the ECM. Using real time and end point assays we have identified similar patterns of behaviour among the three breast cancer cell lines in response to commonly found ECM components of the breast. Using a selected gene panel, we have been able to identify cell line specific changes in gene differentiation when breast cancer cells are in contact with these elements. Although the response of our cells to these elements differ at the genetic level, their functional responses are consistent. This work adds to the growing arguments that highlight a need for histologically assessing ECM composition of breast tumours. In particular monitoring of fibrous protein deposition at the site of malignancy could provide critical information during clinical assessment influencing disease prognosis and treatment decisions for breast cancer patients.


Assuntos
Neoplasias da Mama/genética , Colágeno/genética , Fibronectinas/genética , Mama/patologia , Linhagem Celular Tumoral , Colágeno/metabolismo , Colágeno Tipo I/genética , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Feminino , Fibronectinas/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Genótipo , Glicoproteínas/genética , Humanos , Fenótipo , Prognóstico , Transdução de Sinais , Microambiente Tumoral
7.
PLoS One ; 15(8): e0234672, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764753

RESUMO

Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein/proteoglycan (SLRP) family found in vitreous humour and cartilage. It was first identified associated with the surface of vitreous collagen fibrils and several other SLRPs are also known to bind collagen fibrils and it some cases alter fibril morphology. The purpose of this study was to investigate the binding of opticin to the collagen II-containing fibrils found in vitreous and cartilage. Electron microscopic studies using gold labelling demonstrated that opticin binds vitreous and thin cartilage collagen fibrils specifically at a single site in the gap region of the collagen D-period corresponding to the e2 stain band; this is the first demonstration of the binding site of a class III SLRP on collagen fibrils. Opticin did not bind thick cartilage collagen fibrils from cartilage or tactoids formed in vitro from collagen II, but shows high specificity for thin, heterotypic collagen fibrils containing collagens II, and XI or V/XI. Vitreous collagen fibrils from opticin null and wild-type mice were compared and no difference in fibril morphology or diameter was observed. Similarly, in vitro fibrillogenesis experiments showed that opticin did not affect fibril formation. We propose that when opticin is bound to collagen fibrils, rather than influencing their morphology it instead hinders the binding of other molecules to the fibril surfaces and/or act as an intermediary bridge linking the collagen fibrils to other non-collagenous molecules.


Assuntos
Colágeno/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteoglicanas/metabolismo , Animais , Sítios de Ligação , Bovinos , Colágeno/química , Colágeno/ultraestrutura , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/deficiência , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Ligação Proteica , Proteoglicanas/química , Proteoglicanas/deficiência , Corpo Vítreo/química , Corpo Vítreo/metabolismo , Corpo Vítreo/ultraestrutura
8.
PLoS One ; 15(8): e0237116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857787

RESUMO

Bone metastases are a frequent complication in prostate cancer, and several studies have shown that vitamin D deficiency promotes bone metastases. However, while many studies focus on vitamin D's role in cell metabolism, the effect of chronically low vitamin D levels on bone tissue, i.e. insufficient mineralization of the tissue, has largely been ignored. To investigate, whether poor tissue mineralization promotes cancer cell attachment, we used a fluorescence based adhesion assay and single cell force spectroscopy to quantify the adhesion of two prostate cancer cell lines to well-mineralized and demineralized dentin, serving as biomimetic bone model system. Adhesion rates of bone metastases-derived PC3 cells increased significantly on demineralized dentin. Additionally, on mineralized dentin, PC3 cells adhered mainly via membrane anchored surface receptors, while on demineralized dentin, they adhered via cytoskeleton-anchored transmembrane receptors, pointing to an interaction via exposed collagen fibrils. The adhesion rate of lymph node derived LNCaP cells on the other hand is significantly lower than that of PC3 and not predominately mediated by cytoskeleton-linked receptors. This indicates that poor tissue mineralization facilitates the adhesion of invasive cancer cells by the exposure of collagen and emphasizes the disease modifying effect of sufficient vitamin D for cancer patients.


Assuntos
Calcificação Fisiológica , Adesão Celular , Neoplasias da Próstata/metabolismo , Animais , Materiais Biomiméticos/química , Linhagem Celular Tumoral , Colágeno/metabolismo , Citoesqueleto/metabolismo , Dentina/química , Elefantes , Humanos , Masculino , Receptores de Superfície Celular/metabolismo , Tecidos Suporte/química , Vitamina D/metabolismo
9.
PLoS One ; 15(8): e0236516, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32776970

RESUMO

Breast cancer is the most common cause of cancer-related deaths in women worldwide. Identification of reliable prognostic indicators and therapeutic targets is critical for improving patient outcome. Cancer in companion animals often strongly resembles human cancers and a comparative approach to identify prognostic markers can improve clinical care across species. Feline mammary tumors (FMT) serve as models for extremely aggressive triple negative breast cancer (TNBC) in humans, with high rates of local and distant recurrence after resection. Despite the aggressive clinical behavior of most FMT, current prognostic indicators are insufficient for accurately predicting outcome, similar to human patients. Given significant heterogeneity of mammary tumors, there has been a recent focus on identification of universal tumor-permissive stromal features that can predict biologic behavior and provide therapeutic targets to improve outcome. As in human and canine patients, collagen signatures appear to play a key role in directing mammary tumor behavior in feline patients. We find that patients bearing FMTs with denser collagen, as well as longer, thicker and straighter fibers and less identifiable tumor-stromal boundaries had poorer outcomes, independent of the clinical variables grade and surgical margins. Most importantly, including the collagen parameters increased the predictive power of the clinical model. Thus, our data suggest that similarities with respect to the stromal microenvironment between species may allow this model to predict outcome and develop novel therapeutic targets within the tumor stroma that would benefit both veterinary and human patients with aggressive mammary tumors.


Assuntos
Colágeno/metabolismo , Neoplasias Mamárias Animais/cirurgia , Prognóstico , Neoplasias de Mama Triplo Negativas/cirurgia , Animais , Gatos , Colágeno/genética , Modelos Animais de Doenças , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Animais/cirurgia , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/genética
10.
Gene ; 758: 144946, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32649978

RESUMO

Hepatic injury is one of the most challenging diseases in clinical medicine. Hepatic injury is accompanied by hepatocyte apoptosis and leads to hepatic fibrosis and cirrhosis, which may cause liver cancer and increased mortality. Therefore, it is essential to investigate the regulation mechanism and therapeutic strategies for hepatic injury. In the study, the effects of Thymosin ß4 (Tß4) on Long intergenic noncoding RNA-p21 (lincRNA-p21)-mediated liver injury were investigated. Results showed that lincRNA-p21 overexpression promoted hepatocytes apoptosis, which was blocked by Tß4. Besides, Tß4 reversed the levels of cleaved caspase-3 and caspase-9 induced by lincRNA-p21. LincRNA-p21 overexpression also caused the pathological injury and fibrosis in hepatic tissues and increased the levels of fibrosis-related proteins (Collagen I, α-SMA and TIMP-1), and induced hydroxyproline and ALT production. However, Tß4 reversed the effects of overexpression of lincRNA-p21 on hepatic injury and fibrosis. In vitro experiments, after lincRNA-p21 was overexpressed in hepatic stellate cells (HSCs), the proliferation ability and the levels of HSCs markers α-SMA and Desmin were increased. However, Tß4 reversed the effects of lincRNA-p21 on HSCs. Furthermore, the PI3K-AKT-NF-κB pathway was activated by lincRNA-p21, which was then reversed by the Tß4 administration. After the mice treated by insulin-like growth factor-1 (IGF-1) (the activator of PI3K-AKT), the inhibitory effect of Tß4 on activated the PI3K-AKT-NF-κB pathway was abrogated. Besides, IGF-1 abolished the protective effects of Tß4 on hepatic apoptosis and fibrosis induced by lincRNA-p21. Therefore, Tß4 reversed. lincRNA-p21-mediated liver injury through inhibiting PI3K-AKT-NF-κB pathway. Tß4 may be a promising drug for fibrosis therapy.


Assuntos
Apoptose/efeitos dos fármacos , Cirrose Hepática/prevenção & controle , Fígado/lesões , RNA Longo não Codificante/genética , Timosina/farmacologia , Actinas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Hepatócitos/patologia , Proteínas I-kappa B/antagonistas & inibidores , Fígado/metabolismo , Cirrose Hepática/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-1/metabolismo
11.
J Nippon Med Sch ; 87(3): 110-117, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32655090

RESUMO

BACKGROUND: Cutaneous wound healing is a complex, dynamic physiological process. Traditional methods of promoting wound healing are not always effective. Consequently, alternative modalities, such as photodynamic therapy (PDT), are needed. We examined the effectiveness and underlying mechanisms of PDT in a murine model of acute wound healing. METHODS: Two excisional wounds were produced, one on each side of the midline, in C57bL/6J mice. Methyl 5-aminolevulinate hydrochloride (MAL) was applied to the right-side wound. After 1 to 3 hours of incubation, the wound was irradiated with red light. The left-side wound was not treated with MAL or red light. On Day 14, the wounds were excised and subjected to histological and immunohistochemical analysis. RESULTS: During the first week, no difference was seen between the two sides. However, at week 2, PDT-treated wounds exhibited delayed re-epithelialization. On Day 14, hematoxylin and eosin (HE) staining showed a continuous epithelial lining in untreated wounds. In contrast, PDT-treated wounds partially lacked epithelium in the wound bed. Masson's Trichrome (MTC) staining showed a thicker dermis and more collagen fibers and inflammatory cells in PDT-treated wounds than in untreated wounds. Immunohistochemical analyses showed significantly fewer CD31+ blood vessels and greater collagen III density in PDT-treated wounds than in untreated wounds. However, treated and untreated wounds did not differ in collagen I density. CONCLUSIONS: PDT delayed acute wound healing in a murine model of secondary intention wound healing.


Assuntos
Fotoquimioterapia/efeitos adversos , Fenômenos Fisiológicos da Pele , Cicatrização/efeitos da radiação , Animais , Colágeno/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Pele/metabolismo , Fatores de Tempo , Cicatrização/fisiologia , Infecção dos Ferimentos
12.
Adv Exp Med Biol ; 1250: 3-13, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32601934

RESUMO

Collagen is an important component that makes 25-35% of our body proteins. Over the past decades, tissue engineers have been designing collagen-based biocompatible materials and studying their applications in different fields. Collagen obtained from cattle and pigs has been mainly used until now, but collagen derived from fish and other livestock has attracted more attention since the outbreak of mad cow disease, and they are also used as a raw material for cosmetics and foods. Due to the zoonotic infection using collagen derived from pigs and cattle, their application in developing biomaterials is limited; hence, the development of new animal-derived collagen is required. In addition, there is a religion (Islam, Hinduism, and Judaism) limited to export raw materials and products derived from cattle and pig. Hence, high-value collagen that is universally accessible in the world market is required. Therefore, in this review, we have dealt with the use of duck's feet-derived collagen (DC) as an emerging alternative to solve this problem and also presenting few original investigated bone regeneration results performed using DC.


Assuntos
Regeneração Óssea , Colágeno , Patos , Engenharia Tecidual , Animais , Materiais Biocompatíveis , Regeneração Óssea/fisiologia , Colágeno/química , Colágeno/metabolismo , Engenharia Tecidual/métodos , Tecidos Suporte
13.
PLoS One ; 15(7): e0235965, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701960

RESUMO

Secondary lymphedema (SL)is a frequent and devastating complication of modern oncological therapy and filarial infections. A lack of a reliable preclinical model to investigate the underlying mechanism of clinical stage progression has limited the development of new therapeutic strategies. Current first line treatment has shown to be merely symptomatic and relies on lifetime use of compression garments and decongestive physiotherapy. In this study, we present the development of a secondary lymphedema model in 35 rats using pre- and intraoperative fluorescence-guided mapping of the lymphatics and microsurgical induction. In contrast to the few models reported so far, we decided to avoid the use of radiation for lymphedema induction. It turned out, that the model is nearly free of complications and capable of generating a statistically significant limb volume increase by water displacement measurements, sustained for at least 48 days. A translational, accurate lymphatic dysfunction was visualized by a novel VIS-NIR X-ray ICG-Clearance-Capacity imaging technology. For the first-time SL stage progression was validated by characteristic histological alterations, such as subdermal mast cell infiltration, adipose tissue deposition, and fibrosis by increased skin collagen content. Immunofluorescence confocal microscopy analysis suggested that stage progression is related to the presence of a characteristic α SMA+/HSP-47+/vimentin+ fibroblast subpopulation phenotype. These findings demonstrate that the in-vivo model is a reliable and clinically relevant SL model for the development of further secondary lymphedema therapeutic strategies and the analysis of the veiled molecular mechanisms of lymphatic dysfunction.


Assuntos
Corantes Fluorescentes/química , Linfedema/patologia , Microcirurgia/efeitos adversos , Actinas/metabolismo , Animais , Colágeno/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Verde de Indocianina/química , Linfedema/etiologia , Microscopia de Fluorescência , Ratos , Ratos Endogâmicos Lew , Pele/patologia , Vimentina/metabolismo
14.
Life Sci ; 257: 118138, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32712298

RESUMO

AIMS: Hypertension is a relevant sex and sex hormones-dependent risk factor where the cardiovascular and renal health of the population are concerned. Men experience greater losses of renal function (RF) than women, but the mechanisms remain somewhat unclear. Our goal was to evaluate the relationship between oxidative stress (OS), angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) activities and RF in male and female SHR. MAIN METHODS: Twelve-week-old spontaneously hypertensive rats (SHR) were submitted to either castration or SHAM surgery and divided into 4 groups, SHAM or Castrated (CAST) males or females. After 51 days we evaluated RF (inulin and sodium para-aminohippurate), ACE and ACE2 activities (fluorimetry), OS (flow cytometry), collagen deposition (picrosirius red) and protein expression (western blot). KEY FINDINGS: Males presented lower RF than females and castration impaired this parameter in both groups. Sexual dimorphism was not observed regarding OS and inflammation; however, castration increased this parameter more severely in males than in females. SHAM males exhibited higher collagen deposition than females, though castration increased it in both sexes, eliminating the difference. We found sexual dimorphism regarding renal ACE and ACE2 activities, which were lower in males than in females. Although castration did not alter ACE activity, it reduced ACE2 activity in females and increased it in males. SIGNIFICANCE: These results indicate that sex hormones affect RF in SHR. As alterations in the oxidative system were capable of promoting podocyte injury, inflammation, and collagen deposition, we put forward that these effects are differently modulated by ACE and ACE2.


Assuntos
Hormônios Esteroides Gonadais/metabolismo , Nefropatias/etiologia , Estresse Oxidativo , Peptidil Dipeptidase A/metabolismo , Ratos Endogâmicos SHR/metabolismo , Animais , Western Blotting , Colágeno/metabolismo , Feminino , Rim/enzimologia , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Nefropatias/patologia , Nefropatias/fisiopatologia , Masculino , Orquiectomia , Ovariectomia , Estresse Oxidativo/fisiologia , Ratos , Ratos Endogâmicos SHR/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fatores Sexuais
15.
Prostate ; 80(13): 1071-1086, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32687633

RESUMO

BACKGROUND: The emergence of reactive stroma is a hallmark of prostate cancer (PCa) progression and a potential source for prognostic and diagnostic markers of PCa. Collagen is a main component of reactive stroma and changes systematically and quantitatively to reflect the course of PCa, yet has remained undefined due to a lack of tools that can define collagen protein structure. Here we use a novel collagen-targeting proteomics approach to investigate zonal regulation of collagen-type proteins in PCa prostatectomies. METHODS: Prostatectomies from nine patients were divided into zones containing 0%, 5%, 20%, 70% to 80% glandular tissue and 0%, 5%, 25%, 70% by mass of PCa tumor following the McNeal model. Tissue sections from zones were graded by a pathologist for Gleason score, percent tumor present, percent prostatic intraepithelial neoplasia and/or inflammation (INF). High-resolution accurate mass collagen targeting proteomics was done on a select subset of tissue sections from patient-matched tumor or nontumor zones. Imaging mass spectrometry was used to investigate collagen-type regulation corresponding to pathologist-defined regions. RESULTS: Complex collagen proteomes were detected from all zones. COL17A and COL27A increased in zones of INF compared with zones with tumor present. COL3A1, COL4A5, and COL8A2 consistently increased in zones with tumor content, independent of tumor size. Collagen hydroxylation of proline (HYP) was altered in tumor zones compared with zones with INF and no tumor. COL3A1 and COL5A1 showed significant changes in HYP peptide ratios within tumor compared with zones of INF (2.59 ± 0.29, P value: .015; 3.75 ± 0.96 P value .036, respectively). By imaging mass spectrometry COL3A1 showed defined localization and regulation to tumor pathology. COL1A1 and COL1A2 showed gradient regulation corresponding to PCa pathology across zones. Pathologist-defined tumor regions showed significant increases in COL1A1 HYP modifications compared with COL1A2 HYP modifications. Certain COL1A1 and COL1A2 peptides could discriminate between pathologist-defined tumor and inflammatory regions. CONCLUSIONS: Site-specific posttranslational regulation of collagen structure by proline hydroxylation may be involved in reactive stroma associated with PCa progression. Translational and posttranslational regulation of collagen protein structure has potential for new markers to understand PCa progression and outcomes.


Assuntos
Colágeno/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-Traducional , Idoso , Sequência de Aminoácidos , Autoantígenos , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Colágeno Tipo IV/metabolismo , Colágeno Tipo VIII/metabolismo , Progressão da Doença , Colágenos Fibrilares/metabolismo , Humanos , Hidroxilação , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Colágenos não Fibrilares , Prolina/metabolismo , Próstata/metabolismo , Prostatectomia , Neoplasias da Próstata/diagnóstico por imagem , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
16.
Arterioscler Thromb Vasc Biol ; 40(9): 2159-2170, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32673527

RESUMO

OBJECTIVE: Evidence from preclinical and clinical studies has demonstrated that myocardial infarction promotes atherosclerosis progression. The impact of focal vascular inflammation on the progression and phenotype of remote atherosclerosis remains unknown. Approach and Results: We used a novel ApoE-/- knockout mouse model of sustained arterial inflammation, initiated by mechanical injury in the abdominal aorta. Using serial in vivo molecular MRI and ex vivo histology and flow cytometry, we demonstrate that focal arterial inflammation triggered by aortic injury, accelerates atherosclerosis in the remote brachiocephalic artery. The brachiocephalic artery atheroma had distinct histological features including increased plaque size, plaque permeability, necrotic core to collagen ratio, infiltration of more inflammatory monocyte subsets, and reduced collagen content. We also found that arterial inflammation following focal vascular injury evoked a prolonged systemic inflammatory response manifested as a persistent increase in serum IL-6 (interleukin 6). Finally, we demonstrate that 2 therapeutic interventions-pravastatin and minocycline-had distinct anti-inflammatory effects at the plaque and systemic level. CONCLUSIONS: We show for the first time that focal arterial inflammation in response to vascular injury enhances systemic vascular inflammation, accelerates remote atheroma progression and induces plaques more inflamed, lipid-rich, and collagen-poor in the absence of ischemic myocardial injury. This inflammatory cascade is modulated by pravastatin and minocycline treatments, which have anti-inflammatory effects at both plaque and systemic levels that mitigate atheroma progression.


Assuntos
Aortite/complicações , Aterosclerose/etiologia , Tronco Braquiocefálico/metabolismo , Mediadores da Inflamação/sangue , Placa Aterosclerótica , Animais , Anti-Inflamatórios/farmacologia , Aortite/sangue , Aortite/patologia , Aterosclerose/sangue , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Tronco Braquiocefálico/efeitos dos fármacos , Tronco Braquiocefálico/patologia , Colágeno/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Interleucina-6/sangue , Metabolismo dos Lipídeos , Masculino , Camundongos Knockout para ApoE , Minociclina/farmacologia , Necrose , Pravastatina/farmacologia , Fatores de Tempo
17.
PLoS One ; 15(7): e0235692, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32697783

RESUMO

Nowadays, opportunistic small predators, such as foxes (Vulpes vulpes and Vulpes lagopus), are well known to be very adaptable to human modified ecosystems. However, the timing of the start of this phenomenon in terms of human impact on ecosystems and of the implications for foxes has hardly been studied. We hypothesize that foxes can be used as an indicator of past human impact on ecosystems, as a reflection of population densities and consequently to track back the influence of humans on the Pleistocene environment. To test this hypothesis, we used stable isotope analysis (δ13C, δ15N) of bone collagen extracted from faunal remains from several archaeological sites located in the Swabian Jura (southwest Germany) and covering a time range over three important cultural periods, namely the Middle Palaeolithic (older than 42,000 years ago) attributed to Neanderthals, and the early Upper Palaeolithic periods Aurignacian and Gravettian (42,000 to 30,000 years ago) attributed to modern humans. We then ran Bayesian statistic systems (SIBER, mixSIAR) to reconstruct the trophic niches and diets of Pleistocene foxes. We observed that during the Middle Palaeolithic period, when Neanderthals sparsely populated the Swabian Jura, the niches occupied by foxes suggest a natural trophic behavior. In contrast, during the early Upper Palaeolithic periods, a new trophic fox niche appeared, characterized by a restricted diet on reindeer. This trophic niche could be due to the consumption of human subsidies related to a higher human population density and the resulting higher impact on the Pleistocene environment by modern humans compared to Neanderthals. Furthermore, our study suggests that, a synanthropic commensal behavior of foxes started already in the Aurignacian, around 42,000 years ago.


Assuntos
Dieta/veterinária , Ecossistema , Raposas/fisiologia , Animais , Arqueologia , Teorema de Bayes , Osso e Ossos/metabolismo , Isótopos de Carbono/análise , Colágeno/química , Colágeno/metabolismo , Fósseis/história , História Antiga , Humanos , Homem de Neandertal , Isótopos de Nitrogênio/análise
18.
PLoS One ; 15(6): e0235116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32569321

RESUMO

Here, we examine known GTPase regulators of vesicle trafficking events to assess whether they affect endothelial cell (EC) lumen and tube formation. We identify novel roles for the small GTPases Rab3A, Rab3B, Rab8A, Rab11A, Rab27A, RalA, RalB and caveolin-1 in co-regulating membrane trafficking events that control EC lumen and tube formation. siRNA suppression of individual GTPases such as Rab3A, Rab8A, and RalB markedly inhibit tubulogenesis, while greater blockade is observed with combinations of siRNAs such as Rab3A and Rab3B, Rab8A and Rab11A, and RalA and RalB. These combinations of siRNAs also disrupt very early events in lumen formation including the formation of intracellular vacuoles. In contrast, knockdown of the endocytosis regulator, Rab5A, fails to inhibit EC tube formation. Confocal microscopy and real-time videos reveal that caveolin-1 strongly labels intracellular vacuoles and localizes to the EC apical surface as they fuse to form the luminal membrane. In contrast, Cdc42 and Rab11A localize to a perinuclear, subapical region where intracellular vacuoles accumulate and fuse during lumen formation. Our new data demonstrates that EC tubulogenesis is coordinated by a series of small GTPases to control polarized membrane trafficking events to generate, deliver, and fuse caveolin-1-labeled vacuoles to create the apical membrane surface.


Assuntos
Caveolina 1/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Colágeno/metabolismo , Exocitose , Proteínas de Fluorescência Verde/metabolismo , Humanos , Modelos Biológicos , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(25): 14102-14109, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32522867

RESUMO

The spatial-temporal relationship between cells, extracellular matrices, and mineral deposits is fundamental for an improved understanding of mineralization mechanisms in vertebrate tissues. By utilizing focused ion beam-scanning electron microscopy with serial surface imaging, normally mineralizing avian tendons have been studied with nanometer resolution in three dimensions with volumes exceeding tens of micrometers in range. These parameters are necessary to yield sufficiently fine ultrastructural details while providing a comprehensive overview of the interrelationships between the tissue structural constituents. Investigation reveals a complex lacuno-canalicular network in highly mineralized tendon regions, where ∼100 nm diameter canaliculi emanating from cell (tenocyte) lacunae surround extracellular collagen fibril bundles. Canaliculi are linked to smaller channels of ∼40 nm diameter, occupying spaces between fibrils. Close to the tendon mineralization front, calcium-rich deposits appear between the fibrils and, with time, mineral propagates along and within them. These close associations between tenocytes, tenocyte lacunae, canaliculi, small channels, collagen, and mineral suggest a concept for the mineralization process, where ions and/or mineral precursors may be transported through spaces between fibrils before they crystallize along the surface of and within the fibrils.


Assuntos
Biomineralização , Matriz Extracelular/ultraestrutura , Tendões/ultraestrutura , Tenócitos/ultraestrutura , Animais , Cálcio/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Imageamento Tridimensional , Extremidade Inferior/diagnóstico por imagem , Masculino , Tenócitos/metabolismo , Perus
20.
Am J Physiol Heart Circ Physiol ; 319(1): H222-H234, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32530752

RESUMO

Protection against increased vascular stiffness in young women is lost after menopause. However, little is known about vascular stiffness in older, premenopausal females, because most of the prior work has been conducted in rodents, which live for only 1-3 yr and do not go through menopause. The goal of the current investigation was to quantitate differences in stiffness down the aortic tree and the mechanisms mediating those differences in older, premenopausal (24 ± 0.7 yr) versus young adult (7 ± 0.7 yr) female nonhuman primates. Aortic stiffness (ß), calculated from direct and continuous measurements of aortic diameter and pressure in chronically instrumented, conscious macaque monkeys, increased 2.5-fold in the thoracic aorta and fivefold in the abdominal aorta in old premenopausal monkeys. The aortic histological mechanisms mediating increased vascular stiffness, i.e., collagen/elastin ratio, elastin, and collagen disarray, and the number of breaks in elastin and collagen fibers were greater in the old premenopausal versus young monkeys and greater in the abdominal versus the thoracic aorta and greatest in the iliac artery. In addition, more immature and less cross-linked fibers of collagen were found in the aortas of young females. Aortic stiffness increased in old premenopausal female monkeys, more so in the abdominal aorta than in the thoracic aorta. Histological mechanisms mediating the increased aortic stiffness were augmented in the old premenopausal females, greater in the abdominal versus the thoracic aorta, and greatest in the iliac artery.NEW & NOTEWORTHY This is the first study to examine vascular stiffness down the aortic tree in aging premenopausal females (24 ± 0.7 yr old), whereas prior work studied mainly rodents, which are short-lived and do not undergo menopause. Histological mechanisms mediating vascular stiffness in older premenopausal females increased progressively down the aortic tree, with greater increases in the abdominal aorta compared with the thoracic aorta and with the greatest increases and differences observed in the iliac artery.


Assuntos
Envelhecimento/patologia , Aorta/patologia , Rigidez Vascular , Animais , Aorta/crescimento & desenvolvimento , Aorta/metabolismo , Colágeno/metabolismo , Elastina/metabolismo , Feminino , Macaca fascicularis , Macaca mulatta
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