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1.
Plast Reconstr Surg ; 145(2): 392-401, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31985629

RESUMO

BACKGROUND: Sequential compression devices are often considered a mainstay of prophylaxis against deep venous thromboses in surgical patients. The devices are believed to produce a milking action on the deep veins to prevent venous stasis. A systemic fibrinolytic effect has also been proposed, adding a second mechanism of action. The plasma levels of tissue plasminogen activator and plasminogen activator inhibitor-1 reflect fibrinolytic activity. METHODS: A randomized trial was conducted among 50 consecutive plastic surgery outpatients undergoing cosmetic surgery performed by the author under total intravenous anesthesia and without paralysis. Patients were randomized to receive calf-length sequential compression devices or no sequential compression devices during surgery. Blood samples were obtained from the upper extremity preoperatively and at hourly intervals until the patient was discharged from the postanesthesia care unit. Tissue plasminogen activator and plasminogen activator inhibitor-1 levels were measured. Ultrasound surveillance was used in all patients. There was no outside funding for the study. RESULTS: All patients agreed to participate (inclusion rate, 100 percent). No patient developed clinical signs or ultrasound evidence of a deep venous thrombosis. There were no significant changes in tissue plasminogen activator levels or plasminogen activator inhibitor-1 levels from the preoperative measurements at any hourly interval and no differences in levels comparing patients treated with or without sequential compression devices. CONCLUSIONS: No significant change in systemic fibrinolytic activity occurs during outpatient plastic surgery under total intravenous anesthesia. Sequential compression devices do not affect tissue plasminogen activator or plasminogen activator inhibitor-1 levels, suggesting no fibrinolytic benefit. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, I.


Assuntos
Fibrinólise/fisiologia , Dispositivos de Compressão Pneumática Intermitente , Adulto , Idoso , Técnicas Cosméticas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , Inativadores de Plasminogênio/metabolismo , Estudos Prospectivos , Procedimentos Cirúrgicos Reconstrutivos/métodos , Ativador de Plasminogênio Tecidual/metabolismo , Trombose Venosa/sangue , Trombose Venosa/prevenção & controle , Adulto Jovem
2.
Bioelectromagnetics ; 41(1): 52-62, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31802523

RESUMO

We established three types of thrombosis models to explore the effects of the static magnetic field (SMF) on thrombosis in rats and mice with three different MF intensities. In the carrageenan-induced thrombosis model in rats, the SMF treatments reduced the black tail length of rats, extracorporeal thrombus, and the mass of wet and dry thrombus, and improved the coagulation index value. In FeCl3 -induced arterial thrombosis model in rats, the SMF treatment showed some anti-thrombotic effects. More specifically, the SMF treatment affected rodent blood pressure, plasma plasminogen activator inhibitor, tissue-type plasminogen activator, thrombus mass, and thrombus protein content. In the adrenaline-induced thrombosis model in mice, the SMF treatment had certain effects on the diameter and blood flow velocity of mouse auricle microcirculation in fine veins and arteries. Overall, the highest MF intensities we tested, 20-150 mT, showed a trend of anti-thrombotic effect, indicating that the moderate-intensity SMF might serve as a potential treatment for clot-related diseases in the future. Bioelectromagnetics. 2020;41:52-62 © 2019 Bioelectromagnetics Society.


Assuntos
Campos Magnéticos/efeitos adversos , Trombose/prevenção & controle , Animais , Velocidade do Fluxo Sanguíneo , Pressão Sanguínea , Carragenina/metabolismo , Epinefrina/metabolismo , Frequência Cardíaca , Compostos de Ferro/metabolismo , Masculino , Camundongos , Microcirculação , Ativadores de Plasminogênio/metabolismo , Ratos , Ratos Sprague-Dawley , Ativador de Plasminogênio Tecidual/metabolismo
3.
Oxid Med Cell Longev ; 2019: 4591384, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31531183

RESUMO

Background: Among all the common complications that occur after abdominal surgery, intestinal adhesion is perhaps the most unpleasant one. However, current methods to treat and prevent intestinal adhesion are limited; thus, exploring new methods to prevent and treat intestinal adhesion is greatly needed. In this study, we demonstrated that Danhong injection (DHI) may be used as a promising method to prevent and treat intra-abdominal adhesion in a rat model. Materials and Methods: Forty-eight rats were randomly divided into six groups. Except for the sham-operated group, all rats underwent cecal abrasion to establish an adhesion model. After the operation, the rats in the DHI-treated groups received different doses of DHI via the tail vein daily, while the other group was treated with the same volume of saline solution. Seven days after the operation, all rats were sacrificed, and the degree of adhesion was evaluated by Nair's scoring system. The extent of inflammation in the adhesion tissue was detected by HE staining and the expression of tumor necrosis factor-α (TNF-α) and transforming growth factor-ß (TGF-ß). The collagen deposition was assessed by Sirius red staining and α-SMA, MMP9, t-PA, and PAI-1 levels. Oxidative stress was indicated by the level of reactive oxygen species (ROS) in adhesion tissues and by immunohistochemical labeling of Nrf2. Furthermore, rat primary peritoneal mesothelial cells (RPMCs) were treated with H2O2 and DHI, and NF-κB phosphorylation was detected to illustrate the effect of DHI on oxidative stress. Results: The intra-abdominal adhesion scores were significantly decreased in the groups treated with a high dose of DHI compared with the control groups, and the degree of inflammation, fibrosis, and oxidative stress was also significantly decreased. DHI treatment significantly reduced the levels of TNF-α, TGF-ß1, and PAI and increased the expression levels of MMP9, Nrf2, and t-PA in the adhesion tissues. ROS levels and NF-κB phosphorylation were significantly reduced in DHI-treated RPMCs compared with the control RPMCs. Conclusion: DHI alleviates the formation of postoperative intra-abdominal adhesions by inhibiting inflammation, collagen deposition, and oxidative stress in a rat model and may serve as a promising drug to prevent intra-abdominal adhesions.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Aderências Teciduais/tratamento farmacológico , Animais , Modelos Animais de Doenças , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Aderências Teciduais/metabolismo , Aderências Teciduais/patologia , Ativador de Plasminogênio Tecidual/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
4.
Inflammation ; 42(5): 1622-1629, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31165327

RESUMO

Periodontitis as a chronic inflammatory disease leads to the destruction of the supportive tissues of affected teeth. Crosstalk between periodontitis and the host immune system plays a crucial role in the pathogenesis of this disease. Since polyphenol components such as silymarin and resveratrol have anti-bacterial and anti-inflammatory effects on periodontal tissues, the purpose of this study was to investigate the anti-histaminic effects of silymarin and resveratrol on human gingival fibroblasts (HGFs). HGFs were treated with a concentration of silymarin or resveratrol (100 µg/ml) and a combination of these two polyphenols (50/100 or 100/200 µg/ml silymarin/resveratrol). The effect of silymarin and resveratrol on cell viability was assessed by MTT assay. Also, HGFs were treated with silymarin and/or resveratrol and were stimulated by histamine. The levels of interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), and tissue plasminogen activator 1 (TPA-1) were assessed by enzyme-linked immunosorbent assay (ELISA). After treatment with silymarin, the viability of fibroblast cells significantly increased, whereas treatment with resveratrol and combinations of these flavonoids (silymarin 50 µg/ml and resveratrol 100 µg/ml) did not have any significant effect on cell viability after 24 h. Treatment with 100/200 µg/ml silymarin/resveratrol significantly decreased the cell viability after 48 h. Resveratrol inhibited histamine-induced IL-6 secretion by HGFs significantly, whereas silymarin showed significant effect on TNF-α. A blend of silymarin and resveratrol displayed more valuable results. In conclusion, combination of resveratrol and silymarin could significantly inhibit inflammatory effects of histamine on cultured HGFs by reduction of IL-6, IL-8, TPA-1, and TNF-α.


Assuntos
Fibroblastos/efeitos dos fármacos , Gengiva/patologia , Resveratrol/farmacologia , Silimarina/farmacologia , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Histamina/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
5.
Thromb Haemost ; 119(7): 1094-1101, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31167251

RESUMO

There is a need to improve the efficacy and safety of catheter-directed thrombolysis (CDT) for thrombo-occlusive diseases, and ultrasound-assisted CDT (USAT) is a promising approach. We tested if thrombolysis efficacy of USAT can be improved by adding gaseous microbubbles (MB). We developed an in vitro dynamic overflow model for iliofemoral deep vein thrombosis, and added MB to an USAT system with ultrasound energy and dose of tissue plasminogen activator according to clinical practice. A total of 64 clots (mean baseline weight of 8.23 ± 1.12 g, generated from citrated human whole blood from 7 healthy male volunteers) were randomly assigned to 1 of 4 study protocols of 30 minutes' duration: negative control, CDT, USAT, and USAT + MB.Thrombolysis efficacy was assessed by measuring the change in D-dimer levels in the overflow liquid and the percentage of clot weight reduction. Compared to negative control, change in D-dimer increased by 62% (p = 0.017), 128% (p = 0.002), and 177% (p < 0.001) in the CDT, USAT, and USAT + MB groups, respectively. D-dimer increase was greater in the USAT than in the CDT group (p = 0.014), and greater in the USAT + MB than in the USAT group (p = 0.033). Compared to negative control, percentage of clot weight reduction increased by 123% (p = 0.016), 154% (p = 0.002), and 233% (p < 0.001) in the CDT, USAT, and USAT + MB groups, respectively. Percentage of clot weight reduction was greatest in the USAT + MB group (p < 0.05 compared with all other groups). In conclusion, our in vitro study suggests that the thrombolytic efficacy of USAT in human whole blood clots can be improved by local administration of MB.


Assuntos
Artéria Femoral/patologia , Fibrinolíticos/uso terapêutico , Terapia Trombolítica , Ultrassonografia de Intervenção/métodos , Trombose Venosa/tratamento farmacológico , Adulto , Cateteres , Artéria Femoral/diagnóstico por imagem , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Voluntários Saudáveis , Humanos , Masculino , Microbolhas , Ativador de Plasminogênio Tecidual/metabolismo , Resultado do Tratamento , Ondas Ultrassônicas
6.
Transplant Proc ; 51(3): 647-650, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30979447

RESUMO

BACKGROUND: Inflammation, coagulation, and fibrinolysis are tightly linked together. Reperfusion after transient ischemia activates both neutrophils, coagulation, and fibrinolysis. Experimental data suggest that tissue plasminogen activator (tPA) regulates renal neutrophil influx in kidney ischemia and reperfusion injury. METHODS: In 30 patients undergoing kidney transplantation, we measured renal neutrophil sequestration and tPA release from blood samples drawn from the supplying artery and renal vein early after reperfusion. tPA antigen levels were measured using a commercial enzyme-linked immunosorbent assay kit. For each parameter, transrenal difference (Δ) was calculated by subtracting the value of the arterial sample (ingoing blood) from the value of the venous sample (outgoing blood). RESULTS: Positive transrenal gradients of tPA antigen occurred at 1 minute [Δ = 14 (3-46) ng/mL, P < .01] and 5 minutes [Δ = 5 (-3 to 27) ng/mL, P < .01] after reperfusion. At 5 minutes after reperfusion, a negative transrenal gradient of neutrophils was observed [Δ = -0.17 (-1.45 to 0.24) x 10E9 cells/L, P < .001]. At 1 minute after reperfusion, neutrophil sequestration into the kidney (ie, negative transrenal neutrophil count) correlated significantly with tPA release from the kidney (ie, positive transrenal tPA concentration), (R = -0.513 and P = .006). CONCLUSIONS: The findings suggest a proinflammatory role for tPA in ischemia and reperfusion injury in human kidney transplantation.


Assuntos
Transplante de Rim , Rim/fisiopatologia , Neutrófilos/metabolismo , Traumatismo por Reperfusão/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Transplantes/fisiopatologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Traumatismo por Reperfusão/fisiopatologia
7.
Microbes Infect ; 21(8-9): 412-417, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31009808

RESUMO

Plasmin(ogen) acquisition is critical for invasive disease initiation by Streptococcus pyogenes (GAS). Host urokinase plasminogen activator (uPA) plays a role in mediating plasminogen activation for GAS dissemination, however the contribution of tissue-type plasminogen activator (tPA) to GAS virulence is unknown. Using novel tPA-deficient ALBPLG1 mice, our study revealed no difference in mouse survival, bacterial dissemination or the pathology of GAS infection in the absence of tPA in AlbPLG1/tPA-/- mice compared to AlbPLG1 mice. This study suggests that tPA has a limited role in this humanized model of GAS infection, further highlighting the importance of its counterpart uPA in GAS disease.


Assuntos
Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/patogenicidade , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Carga Bacteriana , Modelos Animais de Doenças , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Viabilidade Microbiana , Mutação , Infecções Estreptocócicas/patologia , Ativador de Plasminogênio Tecidual/genética , Virulência
8.
Sci Transl Med ; 11(482)2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842312

RESUMO

There is a major unmet clinical need to identify pathways in inflammatory bowel disease (IBD) to classify patient disease activity, stratify patients that will benefit from targeted therapies such as anti-tumor necrosis factor (TNF), and identify new therapeutic targets. In this study, we conducted global transcriptome analysis to identify IBD-related pathways using colon biopsies, which highlighted the coagulation gene pathway as one of the most enriched gene sets in patients with IBD. Using this gene-network analysis across 14 independent cohorts and 1800 intestinal biopsies, we found that, among the coagulation pathway genes, plasminogen activator inhibitor-1 (PAI-1) expression was highly enriched in active disease and in patients with IBD who did not respond to anti-TNF biologic therapy and that PAI-1 is a key link between the epithelium and inflammation. Functionally, PAI-1 and its direct target, the fibrinolytic protease tissue plasminogen activator (tPA), played an important role in regulating intestinal inflammation. Intestinal epithelial cells produced tPA, which was protective against chemical and mechanical-mediated colonic injury in mice. In contrast, PAI-1 exacerbated mucosal damage by blocking tPA-mediated cleavage and activation of anti-inflammatory TGF-ß, whereas the inhibition of PAI-1 reduced both mucosal damage and inflammation. This study identifies an immune-coagulation gene axis in IBD where elevated PAI-1 may contribute to more aggressive disease.


Assuntos
Colite/metabolismo , Colite/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Animais , Fatores Biológicos/farmacologia , Fatores Biológicos/uso terapêutico , Coagulação Sanguínea , Proliferação de Células/efeitos dos fármacos , Citrobacter/efeitos dos fármacos , Colite/imunologia , Colite/microbiologia , Colo/patologia , Citocinas/metabolismo , Inflamação/patologia , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/patologia , Interleucina-17/metabolismo , Camundongos , Índice de Gravidade de Doença , Bibliotecas de Moléculas Pequenas/farmacologia , Células Th17/imunologia , Ativador de Plasminogênio Tecidual/metabolismo , Transcrição Genética , Fator de Crescimento Transformador beta/metabolismo
9.
Int J Mol Sci ; 20(3)2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-30709025

RESUMO

Systemic sclerosis (SSc) is a connective tissue disease of autoimmune origin characterized by vascular dysfunction and extensive fibrosis of the skin and visceral organs. Vascular dysfunction is caused by endothelial cell (EC) apoptosis, defective angiogenesis, defective vasculogenesis, endothelial-to-mesenchymal transition (EndoMT), and coagulation abnormalities, and exacerbates the disease. Fibrinolytic regulators, such as plasminogen (Plg), plasmin, α2-antiplasmin (α2AP), tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) and its receptor (uPAR), plasminogen activator inhibitor 1 (PAI-1), and angiostatin, are considered to play an important role in the maintenance of endothelial homeostasis, and are associated with the endothelial dysfunction of SSc. This review considers the roles of fibrinolytic factors in vascular dysfunction of SSc.


Assuntos
Endotélio/citologia , Fibrinolíticos/metabolismo , Escleroderma Sistêmico/patologia , Angiostatinas/metabolismo , Apoptose , Endotélio/metabolismo , Endotélio/patologia , Fibrinolisina/metabolismo , Humanos , Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Escleroderma Sistêmico/metabolismo , Transdução de Sinais , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , alfa 2-Antiplasmina/metabolismo
10.
PLoS One ; 14(2): e0211646, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30811424

RESUMO

In the present study, we investigated the capabilities of a novel ultrasonic approach for real-time control of fibrinolysis under flow conditions. Ultrasonic monitoring was performed in a specially designed experimental in vitro system. Fibrinolytic agents were automatically injected at ultrasonically determined stages of the blood clotting. The following clots dissolution in the system was investigated by means of ultrasonic monitoring. It was shown, that clots resistance to fibrinolysis significantly increases during the first 5 minutes since the formation of primary micro-clots. The efficiency of clot lysis strongly depends on the concentration of the fibrinolytic agent as well as the delay of its injection moment. The ultrasonic method was able to detect the coagulation at early stages, when timely pharmacological intervention can still prevent the formation of macroscopic clots in the experimental system. This result serves as evidence that ultrasonic methods may provide new opportunities for real-time monitoring and the early pharmacological correction of thrombotic complications in clinical practice.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/administração & dosagem , Ultrassom/métodos , Humanos , Plasminogênio/metabolismo , Terapia Trombolítica/métodos , Trombose/tratamento farmacológico , Trombose/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo
11.
Blood Transfus ; 17(4): 312-320, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30747704

RESUMO

BACKGROUND: Increased systemic fibrinolytic activity can occur in liver transplant recipients after the donor graft is reperfused. However, it remains unclear whether this is related solely to tissue plasminogen activator (t-PA) levels or whether unique metabolic changes can alter t-PA activity and enhance fibrinolytic activity. We hypothesise that an increase in sensitivity to t-PA-mediated fibrinolysis (StF) following liver reperfusion is associated with specific metabolic abnormalities. MATERIALS AND METHODS: Liver transplant recipients had serial blood samples analysed with a modified thrombelastography assay using exogenous t-PA to measure sensitivity/resistance to fibrinolysis with the lysis 30 min after maximum clot strength (tLY30). Paired plasma samples were analysed with mass spectroscopy-based metabolomics. The tLY30 was correlated to metabolites using Spearman's rho. StF was defined as a tLY30 change of >8.5% from the anhepatic phase to 30 min after reperfusion based on the distribution of tLY30 in a healthy control population. RESULTS: StF occurred in 53% of patients. Cohorts had similar MELD scores (18 vs 16, p=0.876) and tLY30 at baseline (p=0.867) and anhepatic phase of surgery (p=0.463). Thirty min after reperfusion, the tLY30 was 73% in patient with StF vs 33% in those without StF 33% (p=0.006). StF was associated with increased red blood cell transfusions (p=0.035), during the first 2 hours of reperfusion. Nine metabolites demonstrated a correlation with tLY30 (p<0.05). DISCUSSION: StF is a transient event that resolves within 2 hours of graft reperfusion and is associated with increased blood product use. This phenomenon correlates with derangements in citric acid cycle, purine and amino acid metabolism. Future research is needed to determine whether these metabolites are biomarkers or mechanistically linked to increased sensitivity to t-PA-mediated fibrinolytic activity following graft reperfusion.


Assuntos
Fibrinólise , Transplante de Fígado , Ativador de Plasminogênio Tecidual/sangue , Adulto , Idoso , Transfusão de Sangue , Feminino , Humanos , Masculino , Metabolômica , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio/sangue , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Período Pré-Operatório , Ativador de Plasminogênio Tecidual/metabolismo , Adulto Jovem
12.
Methods Mol Biol ; 1938: 203-217, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30617982

RESUMO

Astrocytes are heterogeneous cells of the central nervous system whose uptake of neurotransmitters and neuromodulators can influence synaptic signaling. Any malfunction in this process can lead to serious defects in synaptic transmission found in, for example, neurodegenerative diseases like Alzheimer's or epilepsy.Here we describe how to visualize the uptake of an extracellularly located protein by in vitro cultured astrocytes on the example of tissue plasminogen activator, a serine protease tightly involved in long-term potentiation and seizure generation.


Assuntos
Astrócitos/metabolismo , Serina Proteases/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Transporte Biológico , Técnicas de Cultura de Células , Células Cultivadas , Imuno-Histoquímica , Camundongos , Transporte Proteico
13.
Cell Mol Life Sci ; 76(8): 1489-1506, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30656378

RESUMO

Endogenous protease tissue-type plasminogen activator (tPA) has highly efficient fibrinolytic activity and its recombinant variants alteplase and tenecteplase are established as highly effective thrombolytic drugs for ischemic stroke. Endogenous tPA is constituted of five functional domains through which it interacts with a variety of substrates, binding proteins and receptors, thus having enzymatic and cytokine-like effects to act on all cell types of the brain. In the past 2 decades, numerous studies have explored the clinical relevance of endogenous tPA in neurological diseases, especially in ischemic stroke. tPA is released from many cells within the brain parenchyma exposed to ischemia conditions in vitro and in vivo, which is believed to control neuronal fate. Some studies proved that tPA could induce blood-brain barrier disruption, neural excitotoxicity and inflammation, while others indicated that tPA also has anti-excitotoxic, neurotrophic and anti-apoptotic effects on neurons. Therefore, more work is needed to elucidate how tPA mediates such opposing functions that may amplify tPA from a therapeutic means into a key therapeutic target in endogenous neuroprotection after stroke. In this review, we summarize the biological characteristics and pleiotropic functions of tPA in the brain. Then we focus on possible hypotheses about why and how endogenous tPA mediates ischemic neuronal death and survival. Finally, we analyze how endogenous tPA affects neuron fate in ischemic stroke in a comprehensive view.


Assuntos
Neurônios/patologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Apoptose , Encéfalo/metabolismo , Encéfalo/patologia , Sobrevivência Celular , Modelos Animais de Doenças , Humanos
14.
Biotechnol Bioeng ; 116(3): 569-580, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30512193

RESUMO

Protein translation has emerged as a critical bottleneck for overall productivity of biological molecules. An augmentation of protein translation can be achieved by cell line engineering or by sophisticated vector design. However, for industrial process development purposes, identification of media additives that promote translation will be of great value, obviating the generation of new host platforms. Here, we examined the effect of low cadmium chloride concentrations on protein synthesis and cell line productivity. At low micromolar concentrations, cadmium chloride induced the mTOR pathway and promoted total protein synthesis in HEK 293T and CHO-K1 cells with minimal toxicity. In a parallel screening of kinase inhibitors for promoting protein expression, we identified the RSK1 inhibitor, BI-D1870, as having a transcription promoting activity on cytomegalovirus promoter-driven transgenes. Fed-batch analyses of CHO-K1 cells producing the anticoagulant factor tissue plasminogen activator (tPA) demonstrated that inclusion of cadmium chloride alone and particularly in combination with BI-D1870 improved overall yields of tPA by more than two-fold with minimal effect on cell growth. We, therefore, underscore the use of cadmium alone and in combination with BI-D1870 for improving bioproduction yields.


Assuntos
Cloreto de Cádmio/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Recombinantes , Animais , Células CHO , Cloreto de Cádmio/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Células HEK293 , Humanos , Pteridinas/farmacologia , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Ativador de Plasminogênio Tecidual/análise , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismo
15.
J Stroke Cerebrovasc Dis ; 28(3): 672-682, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30503681

RESUMO

BACKGROUND: High-intensity interval training (HIIT) improves functional and mental health in the patients with stroke. To investigate the potential mechanisms of HIIT on poststroke depression (PSD). METHODS: Wistar rats were randomly divided into control, Sham, PSD, moderate intensity continuous training (MICT), and HIIT groups. After PSD model was successful made, the maximum speed (Smax) and the blood lactate threshold corresponding speed (SLT) were measured. Different intensity training protocols were performed on the MICT and HIIT groups, respectively. The behavioral tests (open field, forced swimming, and sucrose preference tests) were performed before and after training. Nissl staining was used to observe the changes of neuronal cell morphology in the left hippocampus. The expression of mature brain-derived neurotrophic factor (mBDNF), tropomyosin receptor kinase B (TrkB), precursor BDNF (proBDNF), pan-neurotrophin receptor 75 (p75NTR), NR2A, NR2B proteins, and BDNF, tissue plasminogen activator (tPA) mRNA in the hippocampus were detected by Western blotting, immunohistochemistry or RT-PCR after training. RESULTS: After 28 days of training, higher center occupancy, immobility time, and level of proBDNF, p75NTR, and NR2B proteins, lower sucrose preference and level of mBDNF, TrkB, NR2A proteins, and BDNF, tPA mRNA were observed in the PSD group. Neuronal cells and Nissl body in the hippocampus were loosely arranged and lightly stained in the PSD group. The ethological findings, Nissl staining especially in the CA1 and dentate gyrus areas, expression of proteins and mRNA above in the MICT and HIIT rats were reversed. And the HIIT group changed more significantly compared with MICT. CONCLUSIONS: HIIT was superior to MICT in improving depression in the PSD rats might via increasing mBDNF/proBDNF ratio and further improving neural plasticity in the hippocampus.


Assuntos
Comportamento Animal , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Depressão/terapia , Treinamento Intervalado de Alta Intensidade , Hipocampo/metabolismo , Infarto da Artéria Cerebral Média/terapia , Plasticidade Neuronal , Precursores de Proteínas/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Depressão/metabolismo , Depressão/fisiopatologia , Depressão/psicologia , Modelos Animais de Doenças , Preferências Alimentares , Hipocampo/fisiopatologia , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Infarto da Artéria Cerebral Média/psicologia , Masculino , Atividade Motora , Precursores de Proteínas/genética , Ratos Wistar , Receptor trkB/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Recuperação de Função Fisiológica , Natação , Fatores de Tempo , Ativador de Plasminogênio Tecidual/metabolismo , Regulação para Cima
16.
Blood ; 133(7): 743-753, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30504459

RESUMO

Tissue-type plasminogen activator (tPA) is a major mediator of fibrinolysis and, thereby, prevents excessive coagulation without compromising hemostasis. Studies on tPA regulation have focused on its acute local release by vascular cells in response to injury or other stimuli. However, very little is known about sources, regulation, and fibrinolytic function of noninjury-induced systemic plasma tPA. We explore the role and regulation of hepatocyte-derived tPA as a source of basal plasma tPA activity and as a contributor to fibrinolysis after vascular injury. We show that hepatocyte tPA is downregulated by a pathway in which the corepressor DACH1 represses ATF6, which is an inducer of the tPA gene Plat Hepatocyte-DACH1-knockout mice show increases in liver Plat, circulating tPA, fibrinolytic activity, bleeding time, and time to thrombosis, which are reversed by silencing hepatocyte Plat Conversely, hepatocyte-ATF6-knockout mice show decreases in these parameters. The inverse correlation between DACH1 and ATF6/PLAT is conserved in human liver. These findings reveal a regulated pathway in hepatocytes that contributes to basal circulating levels of tPA and to fibrinolysis after vascular injury.


Assuntos
Fator 6 Ativador da Transcrição/fisiologia , Proteínas do Olho/fisiologia , Fibrinólise/fisiologia , Hepatócitos/patologia , Trombose/patologia , Ativador de Plasminogênio Tecidual/farmacologia , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Células Cultivadas , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/farmacologia , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Trombose/tratamento farmacológico , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Mol Biotechnol ; 61(2): 84-92, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30484145

RESUMO

The aim of this study is to assess the effect of methyl jasmonate (MeJA) and temperature on the valuable pharmaceuticals expression in a virus-mediated transient expression system, and so the Zuchini Yellow Mosaic Virus (ZYMV) based vector was used for transferring the GFP reporter gene and recombinant tissue plasminogen activator (rtPA) gene (K2S) to cucurbit (Cucurbita pepo L.). MeJA, temperature and time (days after inoculation), were evaluated as a factorial experiment in a completely randomized design (CRD). At first, the effect of all treatment combinations on GFP expression was assessed. At this step, the ELISA test was used to select the optimum treatment combination. ELISA method revealed the significant difference between applied treatments. The optimized treatment significantly increased the expression of rtPA compared to the control. The Real-Time PCR reaction for both GFP and rtPA genes showed no significant differences between optimum and control treatments, however, transcripts of the small subunit of RuBisCO were extremely down-regulated in optimum treatment condition. Reduction in RuBisCO expression at protein level was tangible under treatment condition based on the ELISA test. Therefore, it can be inferred that suppressing the expression of RuBisCO, probably resulted in higher access of expression system to free amino acids inside the cell. In this study, MeJA has been shown to be a positive factor, but the low temperature (17 °C), unlike previous studies, suppressed the expression of recombinant protein unexpectedly, probably due to the incompatibility of the viral construct with low temperature. In conclusion, the use of a suitable gene construct, which is not sensitive to temperature, is likely to result in a more favorable outcome.


Assuntos
Acetatos/farmacologia , Cucurbita/genética , Ciclopentanos/farmacologia , Expressão Gênica/efeitos dos fármacos , Oxilipinas/farmacologia , Reguladores de Crescimento de Planta/farmacologia , Ativador de Plasminogênio Tecidual/genética , Regulação da Expressão Gênica de Plantas , Genes Reporter/genética , Vetores Genéticos , Agricultura Molecular , Vírus do Mosaico/genética , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Temperatura , Ativador de Plasminogênio Tecidual/metabolismo
18.
Int J Cancer ; 144(9): 2074-2081, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30125343

RESUMO

ANX A2 is an important member of annexin family of proteins expressed on surface of endothelial cells (ECs), macrophages, mononuclear cells and various types of cancer cells. It exhibits high affinity binding for calcium (Ca++ ) and phospholipids. ANX A2 plays an important role in many biological processes such as endocytosis, exocytosis, autophagy, cell-cell communications and biochemical activation of plasminogen. On the cell surface ANX A2 organizes the assembly of plasminogen (PLG) and tissue plasminogen activator (tPA) for efficient conversion of PLG to plasmin, a serine protease. Proteolytic activity of plasmin is required for activation of inactive pro-metalloproteases (pro-MMPs) and latent growth factors for their biological actions. These activation steps are critical for degradation of extracellular matrix (ECM) and basement proteins (BM) for cancer cell invasion and metastasis. Increased expression of ANX A2 protein/gene has been correlated with invasion and metastasis in a variety of human cancers. Moreover, clinical studies have positively correlated ANX A2 protein expression with aggressive cancers and with resistance to anticancer drugs, shorter disease-free survival (DFS), and worse overall survival (OS). The mechanism(s) by which ANX A2 regulates cancer invasion and metastasis are beginning to emerge. Investigators used various technologies to target ANX A2 in preclinical model of human cancers and demonstrated exciting results. In this review article, we analyzed existing literature concurrent with our own findings and provided a critical overview of ANX A2-dependent mechanism(s) of cancer invasion and metastasis.


Assuntos
Anexina A2/metabolismo , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Neoplasias/patologia , Neovascularização Patológica/patologia , Anexina A2/genética , Ciclo Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Matriz Extracelular/metabolismo , Humanos , Neoplasias/genética , Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo
19.
Blood ; 133(11): 1233-1244, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30545831

RESUMO

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P < .05) and 149.3 ± 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA-plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.


Assuntos
Proteínas Sanguíneas/farmacologia , Tempo de Lise do Coágulo de Fibrina , Fibrinogênio/metabolismo , Fibrinólise/efeitos dos fármacos , Trombose/prevenção & controle , Humanos , Trombose/etiologia , Ativador de Plasminogênio Tecidual/metabolismo
20.
Reprod Fertil Dev ; 31(3): 433-442, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30304664

RESUMO

Besides its fibrinolytic function, the plasminogen-plasmin (PLG-PLA) system is also involved in fertilisation, where plasminogen activators bind to plasminogen to produce plasmin, which modulates sperm binding to the zona pellucida. However, controversy exists, depending on the species, concerning the role of the different components of the system. This study focused its attention on the role of the PLG-PLA system on fertilisation in the mouse with special attention to tissue plasminogen activator (tPA). The presence of exogenous plasminogen reduced invitro fertilisation (IVF) rates and this decline was attenuated by the presence of plasmin inhibitors in combination with plasminogen. The incubation of spermatozoa with either oocytes or cumulus cells together with plasminogen did not change the acrosome reaction but reduced the number of spermatozoa attached. When spermatozoa from tPA-/- mice were used, the IVF rate decreased drastically, although the addition of exogenous tPA during gamete co-incubation under invitro conditions increased fertilisation success. Moreover, fertility could not be restored after invivo insemination of tPA-/- spermatozoa in the female ampulla, although tPA-/- males were able to fertilise invivo. This study suggests a regulatory role of the PLG-PLA system during fertilisation in the mouse with possible implications in human reproduction clinics, such as failures in tPA production, which could be partially resolved by the addition of exogenous tPA during IVF treatment.


Assuntos
Fertilização In Vitro , Fertilização/fisiologia , Oócitos/metabolismo , Espermatozoides/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Reação Acrossômica/fisiologia , Animais , Células do Cúmulo/metabolismo , Feminino , Masculino , Camundongos , Motilidade Espermática/fisiologia
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