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1.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445791

RESUMO

Collagen is heavily hydroxylated. Experiments show that proline hydroxylation is important to triple helix (monomer) stability, fibril assembly, and interaction of fibrils with other molecules. Nevertheless, experiments also show that even without hydroxylation, type I collagen does assemble into its native D-banded fibrillar structure. This raises two questions. Firstly, even though hydroxylation removal marginally affects macroscopic structure, how does such an extensive chemical change, which is expected to substantially reduce hydrogen bonding capacity, affect local structure? Secondly, how does such a chemical perturbation, which is expected to substantially decrease electrostatic attraction between monomers, affect collagen's mechanical properties? To address these issues, we conduct a benchmarked molecular dynamics study of rat type I fibrils in the presence and absence of hydroxylation. Our simulations reproduce the experimental observation that hydroxylation removal has a minimal effect on collagen's D-band length. We also find that the gap-overlap ratio, monomer width and monomer length are minimally affected. Surprisingly, we find that de-hydroxylation also has a minor effect on the fibril's Young's modulus, and elastic stress build up is also accompanied by tightening of triple-helix windings. In terms of local structure, de-hydroxylation does result in a substantial drop (23%) in inter-monomer hydrogen bonding. However, at the same time, the local structures and inter-monomer hydrogen bonding networks of non-hydroxylated amino acids are also affected. It seems that it is this intrinsic plasticity in inter-monomer interactions that preclude fibrils from undergoing any large changes in macroscopic properties. Nevertheless, changes in local structure can be expected to directly impact collagen's interaction with extra-cellular matrix proteins. In general, this study highlights a key challenge in tissue engineering and medicine related to mapping collagen chemistry to macroscopic properties but suggests a path forward to address it using molecular dynamics simulations.


Assuntos
Colágeno Tipo I/metabolismo , Hidroxilação/fisiologia , Animais , Módulo de Elasticidade/fisiologia , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Prolina/metabolismo , Ratos
2.
Am J Hum Genet ; 108(9): 1710-1724, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34450031

RESUMO

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.


Assuntos
Osso e Ossos/metabolismo , Complexo I de Proteína do Envoltório/genética , Proteína Coatomer/genética , Deficiências do Desenvolvimento/genética , Deficiência Intelectual/genética , Osteoporose/genética , Animais , Ácido Ascórbico/farmacologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Criança , Pré-Escolar , Complexo I de Proteína do Envoltório/deficiência , Proteína Coatomer/química , Proteína Coatomer/deficiência , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/patologia , Embrião não Mamífero , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Complexo de Golgi , Haploinsuficiência , Humanos , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Masculino , Camundongos , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Osteoporose/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Índice de Gravidade de Doença , Peixe-Zebra
3.
Biomaterials ; 275: 120976, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34198162

RESUMO

Differentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic, shortcomings regarding their cellular and extracellular compositions, and their developmental plateau need to be overcome. We performed a proteomic analysis on kidney organoids cultured for a prolonged culture time and we found a specific change in the extracellular matrix composition with increased expression of types 1a1, 2 and 6a1 collagen. Such an excessive accumulation of specific collagen types is a hallmark of renal fibrosis that causes a life-threatening pathological condition by compromising key functions of the human kidney. Here we hypothesized the need for a three-dimensional environment to grow the kidney organoids, which could better mimic the in vivo surroundings of the developing kidney than standard culture on an air-liquid interface. Encapsulating organoids for four days in a soft, thiol-ene cross-linked alginate hydrogel resulted in decreased type 1a1 collagen expression. Furthermore, the encapsulation did not result in any changes of organoid structural morphology. Using a biomaterial to modulate collagen expression allows for a prolonged kidney organoid culture in vitro and a reduction of abnormal type 1a1 collagen expression bringing kidney organoids closer to clinical application.


Assuntos
Colágeno Tipo I/metabolismo , Matriz Extracelular , Hidrogéis , Organoides , Alginatos , Humanos , Rim , Proteômica , Compostos de Sulfidrila
4.
Chem Biol Interact ; 346: 109570, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34217686

RESUMO

Verapamil is reported to prevent scar formation. However, whether verapamil is involved in the ureteral stricture scar and the underlying mechanism need further investigation. Fibroblasts were isolated from ureteral scar tissues. TGF-ß1 stimulation was used to induce fibrosis of fibroblasts. Inhibition of CaMK II was achieved by shRNA transfection. CCK-8 was performed to evaluate cell viability. qRT-PCR was applied to determine the level of mRNA while western blotting was used to determine the level of proteins. Immunofluorescence was used to detect the level of vimentin, collagen I and collagen III. Primary fibroblasts was successfully isolated from ureteral scar tissues. TGF-ß1 stimulation was capable to induce collagen production and fibrosis in primary fibroblasts while inhibition of CaMK II attenuate collagen production. Overexpression of wild type CaMK II lead to further increase of collagen production upon TGF-ß1 stimulation while the mutated CaMK II did not exert this promotion. Treatment of verapamil inhibits TGF-ß1 induced collagen production via inhibiting CaMK II. In present study, we revealed a vital role of Verapamil and CaMK II in the formation of ureteral scar. Verapamil inhibited TGF-ß1 induced collagen fiber formation by regulating CaMK II. Our finding might provide new insight into mechanism of prevention and treatment of ureteral scar.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Verapamil/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Cicatriz/tratamento farmacológico , Cicatriz/metabolismo , Cicatriz/patologia , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Mutagênese , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima/efeitos dos fármacos , Verapamil/uso terapêutico , Vimentina/metabolismo
5.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208202

RESUMO

Chronic UVA exposure results in elevated reactive oxygen species in skin which leads to photoaging characterized as upregulated matrix metalloproteinase (MMP)-1 and loss of collagen. Therefore, natural antioxidants are hailed as promising agents to be utilized against photoaging. In the current study, reynosin and santamarine, two known sesquiterpene lactones isolated from Artemisia scoparia, were analyzed for their anti-photoaging properties in UVA-irradiated human dermal fibroblasts (HDFs). Results showed that UVA irradiation (8 J/cm2) upregulated the MMP-1 secretion and expression, and suppressed collagen production, which were significantly reverted by santamarine treatment (10 µM). Although both reynosin and santamarine exhibited ROS scavenging abilities, reynosin failed to significantly diminish UVA-stimulated MMP-1 release. UVA-irradiated HDFs showed increased collagen production when treated with santamarine. As a mechanism to suppress MMP-1, santamarine significantly suppressed the UVA-induced phosphorylation of p38 and JNK and nuclear translocation of p-c-Fos and p-c-Jun. Santamarine promoted collagen I production via relieving the UVA-induced suppression on TGF-ß and its downstream activator Smad2/3 complex. Antioxidant properties of santamarine were also shown to arise from stimulating Nrf2-dependent expression of antioxidant enzymes SOD-1 and HO-1 in UVA-irradiated HDFs. In conclusion, santamarine was found to be a promising natural antioxidant with anti-photoaging properties against UVA-induced damages in HDFs.


Assuntos
Fibroblastos/efeitos dos fármacos , Sesquiterpenos/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Proteína Smad4/agonistas , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Crescimento Transformador beta/agonistas , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Células Cultivadas , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Sistema de Sinalização das MAP Quinases , Metaloproteinase 1 da Matriz/metabolismo , Transdução de Sinais , Envelhecimento da Pele/patologia , Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta
6.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299372

RESUMO

Many biomaterials have been evaluated using cultured cells. In particular, osteoblast-like cells are often used to evaluate the osteocompatibility, hard-tissue-regeneration, osteoconductive, and osteoinductive characteristics of biomaterials. However, the evaluation of biomaterial osteogenesis-inducing capacity using osteoblast-like cells is not standardized; instead, it is performed under laboratory-specific culture conditions with different culture media. However, the effect of different media conditions on bone formation has not been investigated. Here, we aimed to evaluate the osteogenesis of MC3T3-E1 cells, one of the most commonly used osteoblast-like cell lines for osteogenesis evaluation, and assayed cell proliferation, alkaline phosphatase activity, expression of osteoblast markers, and calcification under varying culture media conditions. Furthermore, the various media conditions were tested in uncoated plates and plates coated with collagen type I and poly-L-lysine, highly biocompatible molecules commonly used as pseudobiomaterials. We found that the type of base medium, the presence or absence of vitamin C, and the freshness of the medium may affect biomaterial regeneration. We posit that an in vitro model that recapitulates in vivo bone formation should be established before evaluating biomaterials.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Osteogênese/efeitos dos fármacos , Células 3T3 , Fosfatase Alcalina/metabolismo , Animais , Ácido Ascórbico/farmacologia , Materiais Biocompatíveis/farmacologia , Biomarcadores/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo
7.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073146

RESUMO

Given the anatomical simplicity and the extraordinary ability to regenerate missing parts of the body, Cnidaria represent an excellent model for the study of the mechanisms regulating regenerative processes. They possess the mesoglea, an amorphous and practically acellular extracellular matrix (ECM) located between the epidermis and the gastrodermis of the body and tentacles and consists of the same molecules present in the ECM of vertebrates, such as collagen, laminin, fibronectin and proteoglycans. This feature makes cnidarians anthozoans valid models for understanding the ECM role during regenerative processes. Indeed, it is now clear that its role in animal tissues is not just tissue support, but instead plays a key role during wound healing and tissue regeneration. This study aims to explore regenerative events after tentacle amputation in the Mediterranean anemone Anemonia viridis, focusing in detail on the reorganization of the ECM mesoglea. In this context, both enzymatic, biometric and histological experiments reveal how this gelatinous connective layer plays a fundamental role in the correct restoration of the original structures by modifying its consistency and stiffness. Indeed, through the deposition of collagen I, it might act as a scaffold and as a guide for the reconstruction of missing tissues and parts, such as amputated tentacles.


Assuntos
Matriz Extracelular/metabolismo , Regeneração , Anêmonas-do-Mar/crescimento & desenvolvimento , Cicatrização , Animais , Colágeno Tipo I/metabolismo
8.
Am J Pathol ; 191(9): 1564-1579, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34119473

RESUMO

Although hepatocellular cancer (HCC) usually occurs in the setting of liver fibrosis, the causal relationship between liver fibrosis and HCC is unclear. in vivo and in vitro models of HCC involving Colr/r mice (that produce a collagenase-resistant type I collagen) or wild-type (WT) mice were used to assess the relationship between type I collagen, liver fibrosis, and experimental HCC. HCC was either chemically induced in WT and Colr/r mice or Hepa 1-6 cells were engrafted into WT and Colr/r livers. The effect of hepatic stellate cells (HSCs) from WT and Colr/r mice on the growth of Hepa 1-6 cells was studied by using multicellular tumor spheroids and xenografts. Collagen type I deposition and fibrosis were increased in Colr/r mice, but they developed fewer and smaller tumors. Hepa 1-6 cells had reduced tumor growth in the livers of Colr/r mice. Although Colr/r HSCs exhibited a more activated phenotype, Hepa 1-6 growth and malignancy were suppressed in multicellular tumor spheroids and in xenografts containing Colr/r HSCs. Treatment with vitronectin, which mimics the presence of degraded collagen fragments, converted the Colr/r phenotype into a WT phenotype. Although Colr/r mice have increased liver fibrosis, they exhibited decreased HCC in several models. Thus, increased liver type I collagen does not produce increased experimental HCC.


Assuntos
Carcinoma Hepatocelular/patologia , Colágeno Tipo I/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas Experimentais/patologia , Animais , Linhagem Celular Tumoral , Células Estreladas do Fígado/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL
9.
FASEB J ; 35(7): e21692, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34118087

RESUMO

For metastasis formation, individual cells from a primary tumor must migrate toward other tissues. The aim of this study was to determine if mesenchymal stromal cells (MSCs) from human bone marrow are able to emit signals that induce this migratory activity in cancer cells. We separated the supernatant of MSCs derived from human bone marrow by size-exclusion and ion-exchange chromatography and have subsequently studied the migratory behavior of the prostate cancer cell line PC3 and the breast cancer cell line MDA-MB-231 toward the respective fractions in a transwell migration assay. We identified the extracellular matrix (ECM) proteins type I collagen, type III collagen, fibronectin, and laminin 421 as potential drivers of cancer cell migration. These results could be reproduced using the corresponding isolated or recombinant ECM proteins. Knockdown of the gene encoding beta 1 integrin, an important cell surface receptor for fibronectin, has led to inhibition of cancer cell migration. This supports the hypothesis that beta 1 integrin signaling represents an initial event that leads to metastasis, and that signaling is triggered by binding of integrin heterodimers to ECM molecules. Further characterization of signaling factors and their respective receptors will have implications for anticancer drug development.


Assuntos
Movimento Celular , Colágeno Tipo III/metabolismo , Colágeno Tipo I/metabolismo , Fibronectinas/metabolismo , Laminina/metabolismo , Células-Tronco Mesenquimais/citologia , Neoplasias/patologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Colágeno Tipo I/genética , Colágeno Tipo III/genética , Fibronectinas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Laminina/genética , Células-Tronco Mesenquimais/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Células Tumorais Cultivadas
10.
Ann Agric Environ Med ; 28(2): 326-330, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34184518

RESUMO

INTRODUCTION: Estrogen deficiency found in postmenopausal women may lead to disturbances in the balance of bone metabolism. Study of the influence of estradiol on markers of bone turnover may help to understand the mechanisms of bone metabolism and to monitor osteoporosis therapy in postmenopausal women at high risk of fractures. The aim of the study was evaluation of the effect of estradiol on the basic markers of bone turnover in postmenopausal women. MATERIAL AND METHODS: The study was conducted in a group of 92 postmenopausal women, divided into two groups: Gr-1 with low estradiol levels ≤ 10 pg/ml and Gr-2 with reference estradiol levels ≥ 25 pg/ml). Basic markers of bone turnover were examined: Ctx (C-terminal cross-linked telopeptide of type I collagen alpha chain) and OC (osteocalcin); pro-resorptive cytokines: IL-6 and TNF-α; vitamin 25(OH)D3 and lipid profile. Women was also analyzed according to demographic and clinical data. RESULTS: A positive relationship was found between estradiol and the main bone formation marker - OC (p = 0.041, r = 0.213) and IL-6, TNF-α (p = 0.007, r = 0.281 and p = 0.018, r = 0.246, respectivly, but only in the group with a reference hormone level. Moreover, the main markers of bone turnover: Ctx and OC showed a mutual positive correlation (p = 0.013; r = 0.257) in women with reference estradiol levels. Relationships between markers of bone remodeling, pro-resorptive cytokines and vitamin D3 depending on the level of estradiol showed no statistically significant correlation. CONCLUSIONS: The study showed that only in women with the reference estradiol level (≥ 25 pg/ml) were the bone formation and resorption processes balanced.


Assuntos
Estradiol/administração & dosagem , Estrogênios/administração & dosagem , Osteoporose Pós-Menopausa/tratamento farmacológico , Idoso , Biomarcadores/metabolismo , Densidade Óssea/efeitos dos fármacos , Remodelação Óssea/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Feminino , Humanos , Interleucina-6/metabolismo , Pessoa de Meia-Idade , Osteocalcina/metabolismo , Osteoporose Pós-Menopausa/metabolismo , Osteoporose Pós-Menopausa/fisiopatologia , Pós-Menopausa/efeitos dos fármacos , Pós-Menopausa/metabolismo , Vitamina D/metabolismo
11.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069423

RESUMO

Endometrosis is a reproductive pathology that is responsible for mare infertility. Our recent studies have focused on the involvement of neutrophil extracellular traps enzymes, such as elastase (ELA), in the development of equine endometrosis. Noscapine (NOSC) is an alkaloid derived from poppy opium with anticough, antistroke, anticancer, and antifibrotic properties. The present work investigates the putative inhibitory in vitro effect of NOSC on collagen type I alpha 2 chain (COL1A2) mRNA and COL1 protein relative abundance induced by ELA in endometrial explants of mares in the follicular or mid-luteal phases at 24 or 48 h of treatment. The COL1A2 mRNA was evaluated by qPCR and COL1 protein relative abundance by Western blot. In equine endometrial explants, ELA increased COL 1 expression, while NOSC inhibited it at both estrous cycle phases and treatment times. These findings contribute to the future development of new endometrosis treatment approaches. Noscapine could be a drug capable of preventing collagen synthesis in mare's endometrium and facilitate the therapeutic approach.


Assuntos
Colágeno Tipo I/metabolismo , Endometriose/metabolismo , Noscapina/farmacologia , Animais , Colágeno/metabolismo , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/genética , Endometriose/tratamento farmacológico , Endometriose/veterinária , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Ciclo Estral , Armadilhas Extracelulares/metabolismo , Feminino , Fibrose , Doenças dos Cavalos/patologia , Cavalos , Noscapina/metabolismo , Elastase Pancreática/metabolismo , Inibidores de Proteases/farmacologia
12.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070506

RESUMO

Concentration of hyaluronic acid (HA) in the lungs increases in idiopathic pulmonary fibrosis (IPF). HA is involved in the organization of fibrin, fibronectin, and collagen. HA has been proposed to be a biomarker of fibrosis and a potential target for antifibrotic therapy. Hyaluronidase (HD) breaks down HA into fragments, but is a subject of rapid hydrolysis. A conjugate of poloxamer hyaluronidase (pHD) was prepared using protein immobilization with ionizing radiation. In a model of bleomycin-induced pulmonary fibrosis, pHD decreased the level of tissue IL-1ß and TGF-ß, prevented the infiltration of the lung parenchyma by CD16+ cells, and reduced perivascular and peribronchial inflammation. Simultaneously, a decrease in the concentrations of HA, hydroxyproline, collagen 1, total soluble collagen, and the area of connective tissue in the lungs was observed. The effects of pHD were significantly stronger compared to native HD which can be attributed to the higher stability of pHD. Additional spiperone administration increased the anti-inflammatory and antifibrotic effects of pHD and accelerated the regeneration of the damaged lung. The potentiating effects of spiperone can be explained by the disruption of the dopamine-induced mobilization and migration of fibroblast progenitor cells into the lungs and differentiation of lung mesenchymal stem cells (MSC) into cells of stromal lines. Thus, a combination of pHD and spiperone may represent a promising approach for the treatment of IPF and lung regeneration.


Assuntos
Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/farmacologia , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/efeitos dos fármacos , Espiperona/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Hialuronoglucosaminidase/administração & dosagem , Hialuronoglucosaminidase/farmacocinética , Hidroxiprolina/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/enzimologia , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Queratinas/metabolismo , Pulmão/enzimologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Poloxâmero/química , Receptores de IgG/metabolismo , Espiperona/administração & dosagem , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
13.
Biomed Pharmacother ; 140: 111700, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34044279

RESUMO

BACKGROUND: Frozen shoulders (FS) is a major clinical concern, where chronic synovial inflammation, abnormal angiogenesis, and fibrosis represent the critical pathologies in the glenohumeral capsule. However, no pharmacotherapy has been introduced to treat this pathology. Tetrandrine (TET) has been proposed as a treatment for many diseases due to its strong anti-inflammatory, anti-angiogenic, and anti-fibrotic effects. PURPOSE: To study the anti-inflammatory, anti-angiogenic, and anti-fibrotic effects of TET on FS, and identify whether TET can prevent the development of FS in rats. STUDY DESIGN: A controlled laboratory study. METHODS: Forty-eight male Sprague-Dawley (SD) rats were randomly divided into control, TET, and FS groups. The TET group was intraperitoneally injected with TET every 2 days. TET and saline treatment were started on the day of FS surgery. After 8 weeks, the animals were sacrificed, and samples were collected for X-ray examination, glenohumeral range of motion (ROM) evaluation, histology and immunohistochemistry analysis, transmission electron microscopy (TEM) observation, and profibrogenic factors as well as proinflammatory cytokines measurements. RESULTS: No significant difference in shoulder ROM was observed between the TET and control groups, but a significant difference was noted between these groups and the FS group (P < 0.01). Immunohistochemical staining showed no abnormal angiogenesis or fibrosis in the TET group or the control group. However, significant angiogenesis, collagen remodeling, and fibrosis were observed in the FS group, and the expression and proportion of type I and type III collagen in the FS group were significantly higher than those in the TET group or the control group (P < 0.01). TEM observation showed that TET protected the ultrastructure of collagen fibrous reticular arrangement of the articular capsule and prevented the formation of scar-like fibrotic structures, which are unique to FS. The significantly increased expression of Smad7 and the suppressed expression of Smad 2 in the TET group compared with that of the FS group indicated that TET also significantly inhibited the TGF-ß1 intracellular signal pathway. The expression of profibrogenic factors and proinflammatory cytokines in the TET group and the control group was significantly lower than that in the TET group (P < 0.01). CONCLUSION: The results demonstrated that TET protected the normal reticular structure of the capsule during the freezing period and prevented the development of FS by inhibiting inflammation, angiogenesis, and fibrosis in a rat FS model. CLINICAL RELEVANCE: TET may be a safe and effective clinical medication for preventing and treating FS.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Benzilisoquinolinas/uso terapêutico , Bursite/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Benzilisoquinolinas/farmacologia , Bursite/metabolismo , Bursite/patologia , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Fibrose , Cápsula Articular/efeitos dos fármacos , Cápsula Articular/metabolismo , Cápsula Articular/patologia , Cápsula Articular/ultraestrutura , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Ratos Sprague-Dawley , Inibidor Tecidual de Metaloproteinase-1/metabolismo
14.
Methods Mol Biol ; 2299: 147-156, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028741

RESUMO

Excessive deposition of type I collagen follows in the wake of chronic inflammation processes in dysregulated tissue healing and causes fibrosis that can ultimately lead to organ failure. While the development of antifibrotic drugs is targeting various upstream events in collagen matrix formation (synthesis, secretion, deposition, stabilization, remodeling), the evaluation of drug effects would use as net read-out of the above effects the presence of a deposited collagen matrix by activated cells, mainly myofibroblasts. Conventional methods comprise lengthy and labor-intensive protocols for the quantification of deposited collagen, some with sensitivity and/or specificity issues. Here we describe the Scar-in-a-Jar assay, an in vitro fibrosis model for anti-fibrotic drug testing that benefits from a substantially accelerated extracellular matrix deposition employing macromolecular crowding and a collagen-producing cell type of choice (e.g., lung fibroblasts like WI-38). The system can be aided by activating compounds such as transforming growth factor-ß1, a classical inducer of the myofibroblast phenotype in fibroblasts. Direct image analysis of the well plate not only eliminates the need for matrix extraction or solubilization methods, but also allows for direct imaging and monitoring of phenotypical markers and offers the option for high-content screening applications when adapted to well formats compatible with a screening format.


Assuntos
Colágeno Tipo I/metabolismo , Fibroblastos/citologia , Pulmão/patologia , Miofibroblastos/citologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Humanos , Pulmão/efeitos dos fármacos , Modelos Biológicos , Imagem Molecular , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Fenótipo , Fator de Crescimento Transformador beta1/farmacologia
15.
J Biol Chem ; 297(1): 100819, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34029590

RESUMO

Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal-regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active ß1-integrin-containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp-mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5ß1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine.


Assuntos
Movimento Celular/efeitos dos fármacos , Dipeptídeos/farmacologia , Homeostase/efeitos dos fármacos , Integrina beta1/metabolismo , Pseudópodes/metabolismo , Tendões/citologia , Envelhecimento , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Pseudópodes/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Tenócitos/citologia , Tenócitos/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
16.
Biochem Biophys Res Commun ; 560: 66-71, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-33975247

RESUMO

One major goal in tissue engineering is to create functional materials, mimicking scaffolds in native tissues, to modulate cell function for tissue repair. Collagen is the most abundant structural protein in human body. Though collagen I (COLI) and collagen III (COLIII) are the predominant collagen types in connective tissues and they form stable hybrid fibrils at varied ratios, cell responses to the hybrid matrices are underinvestigated. In this work, we aim to explicate the distinctive roles of COLI and COLIII in fibroblast activation. Unidirectionally aligned COLI, COLIII and COLI-COLIII hybrid nanofibrils were generated via epitaxial growth of collagen on mica. AFM analyses revealed that, with the increase of COLI/COLIII ratio, the fibril width and stiffness increased and the binding affinity of cells to the matrix decreased. A hybrid matrix was found to activate fibroblasts the most effectively, characterized by extensive cell polarization with rigid stress fiber bundles and high α-SMA expression, and by the highest-level of collagen synthesis. It is ascribed to the fine balance between biochemical and biophysical cues achieved on the hybrid matrix. Thus, matrices of aligned COLI-COLIII hybrid fibrils and their derived multifunctional composites can be good candidates of implantation scaffolds for tissue regeneration.


Assuntos
Colágeno Tipo III/fisiologia , Colágeno Tipo I/fisiologia , Fibroblastos/metabolismo , Polaridade Celular , Células Cultivadas , Colágeno/biossíntese , Colágeno/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo I/ultraestrutura , Colágeno Tipo III/metabolismo , Colágeno Tipo III/ultraestrutura , Citoesqueleto/ultraestrutura , Elasticidade , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/ultraestrutura , Expressão Gênica , Humanos , Integrina alfa1beta1/metabolismo , Microscopia de Força Atômica
17.
Biochem Biophys Res Commun ; 560: 87-92, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-33984769

RESUMO

Adult hearts have limited regenerative capacity. Hence, after acute myocardial infarction (MI), dead myocardial tissues are digested by immune cells and replaced by fibrosis, leading to ventricular remodeling and heart failure at the chronic stage. Direct reprogramming of the cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) with cardiac transcription factors, including Gata4, Mef2c, and Tbx5 (GMT), may have significant potential for cardiac repair. Sendai virus (SeV) vectors expressing GMT have been reported to reprogram the mouse cardiac fibroblasts into iCMs without any risk of insertional mutagenesis. In vivo reprogramming improved the cardiac function after acute MI in immunodeficient mice. However, it is unknown whether the newly generated iCMs could exist in infarct hearts for a prolonged period and SeV-GMT can improve cardiac function after MI at the chronic stage in immunocompetent mice. Here, we show that SeV vectors efficiently infect CFs in vivo and reprogram them into iCMs, which existed for at least four weeks after MI, in fibroblast-linage tracing mice. Moreover, SeV-GMT improved cardiac function and reduced fibrosis and collagen I expression at 12 weeks after MI in immunocompetent mice. Thus, direct cardiac reprogramming with SeV vectors could be a promising therapy for MI.


Assuntos
Reprogramação Celular , Vetores Genéticos , Infarto do Miocárdio/terapia , Vírus Sendai/genética , Animais , Doença Crônica , Colágeno Tipo I/metabolismo , Fibroblastos , Fibrose , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/citologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genética
18.
Food Chem ; 361: 130061, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34023689

RESUMO

Two collagens were made from giant salamander (Andrias davidianus) skin by using acid and pepsin extraction methods. The yields of acid-soluble and pepsin-soluble collagens were 26.9 and 58.7%, respectively. The results of spectrum, electrophoresis and amino acid analysis showed that they were type 1 collagen with two α and one ß peptides and high imino acid content. They had low solubility at a pH above 6 or salt concentration over 5%. The pepsin-soluble collagen had a better emulsion activity index. The odorants in raw skin and collagens were identified and evaluated using gas-chromatography mass-spectrometer and olfactometry methods and sensory analysis. The fishy and fatty off-odors in skin were not perceivable in the collagens. Sour, ammonia-like, and acrid off-odors were found in the collagens due to acid and enzymatic hydrolysis and protein degradation. The off-odor intensity of pepsin-soluble collagen was low. It could be considered a good and safe collagen material.


Assuntos
Aminoácidos/análise , Colágeno Tipo I/química , Urodelos/metabolismo , Ácidos , Animais , Colágeno Tipo I/isolamento & purificação , Colágeno Tipo I/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Concentração de Íons de Hidrogênio , Hidrólise , Iminoácidos/análise , Odorantes/análise , Olfatometria , Pepsina A/metabolismo , Proteólise , Pele/metabolismo , Microextração em Fase Sólida , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier
19.
Clin Nephrol ; 96(2): 96-104, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34042581

RESUMO

AIM: Bone mineral disorders are being increasingly seen among diabetic populations as the frequency of diabetes mellitus (DM) is rising at an alarming rate. Our aim is to examine the relationship between glycemic control and bone turnover markers like osteocalcin (OC), C-terminal carboxy telopeptide (CTX), and bone-specific alkaline phosphatase (ALP) in patients with type 2 diabetes, and the effects of anti-diabetic regimens on these markers. MATERIALS AND METHODS: A total of 80 newly diagnosed type 2 DM patients were enrolled into the study and divided into two groups according to glucose regulation (group 1 HbA1c < 7 and group 2 HbA1c ≥ 7). They were also classified into three groups according to antidiabetic regimen. Physical examination findings, demographic characteristics, and anti-diabetic regimens of the patients were recorded. Hemogram and biochemical parameters were studied after 12 hours of fasting. Serum levels OC and CTX were examined by ELISA method. Bone-specific ALP was examined by Chemiluminesence immuneassay (CLIA) method. Bone densitometry was performed on the 2016 model Stratos DR device of DMS brand, and T scores of the patients were recorded. All parameters were repeated at the 6th month of the study. RESULTS: Serum vitamin D and OC levels of group 1 were higher, while ALP was higher in group 2. However, we failed to determine a significant difference in CTX levels between the groups. OC levels were enhanced only in patients receiving metformin plus vildagliptin therapy. The CTX levels increased in all groups, whereas they decreased in the metformin plus DPP-4 group. CONCLUSION: Better glucose regulation is associated with better bone formation, and among three groups metformin plus vildagliptin therapy has a favorable effect on both bone formation and resorption.


Assuntos
Glicemia/metabolismo , Remodelação Óssea/fisiologia , Colágeno Tipo I/metabolismo , Diabetes Mellitus Tipo 2 , Hipoglicemiantes/uso terapêutico , Peptídeos/metabolismo , Fosfatase Alcalina/metabolismo , Biomarcadores , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Controle Glicêmico , Humanos , Osteocalcina/metabolismo
20.
Chem Commun (Camb) ; 57(42): 5131-5134, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33988188

RESUMO

Controlled release of oxygen from myoglobin was achieved by modulating autoxidation of oxymyoglobin using ascorbic acid as a reductant by temperature variation. Long-term storage, prompt release and re-storage of oxygen were also available with this system. Furthermore, 20 nm thick nanofilms composed of oxymyoglobin and type I collagen containing ascorbic acid could successfully show autoxidation of oxymyoglobin in response to environmental temperature. The ultrathin nanofilms will be useful as oxygen-controlled releasable scaffolds for tissue engineering application.


Assuntos
Mioglobina/metabolismo , Nanoestruturas/química , Oxigênio/metabolismo , Ácido Ascórbico/química , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Mioglobina/química , Oxirredução , Oxigênio/química , Técnicas de Microbalança de Cristal de Quartzo , Temperatura
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