RESUMO
One of the most outstanding scientific achievements in the thrombolysis is the development and administration of fibrinolysin - the first Soviet drug that lyses blood clots. Intracoronary administration of fibrinolysin reduced the mortality of patients with myocardial infarction by almost 20%. For his work in this field Yevgeny Chazov was awarded the Lenin Prize in 1982. Over the next decades, under his leadership, the Cardiology Center established scientific and clinical laboratories that created new generations of drugs based on fibrinolytics for treating patients with myocardial infarction, restoration of blood flow in ischemic tissue, and also studying the mechanisms of remodeling of blood vessels involving the fibrinolysis system. It have been found new mechanisms of regulation of the navigation of blood vessels and nerves growth, tumor growth and its metastasis with the participation of the fibrinolysis system proteins. The review reports the role of the fibrinolysis system in the thrombolysis, blood vessels growth and remodeling, neurogenesis, carcinogenesis and fibrosis. The article is dedicated to the 90th anniversary of academician E.I. Chazov.
Assuntos
Fibrinólise , Terapia Trombolítica , Carcinogênese , Fibrose , Humanos , NeurogêneseRESUMO
BACKGROUND: Endothelial and smooth muscle cells are considered promising resources for regenerative medicine and cell replacement therapy. It has been shown that both types of cells are heterogeneous depending on the type of vessels and organs in which they are located. Therefore, isolation of endothelial and smooth muscle cells from tissues relevant to the area of research is necessary for the adequate study of specific pathologies. However, sources of specialized human endothelial and smooth muscle cells are limited, and the search for new sources is still relevant. The main goal of our study is to demonstrate that functional endothelial and smooth muscle cells can be obtained from an available source-post-surgically discarded cardiac tissue from the right atrial appendage and right ventricular myocardium. METHODS: Heterogeneous primary cell cultures were enzymatically isolated from cardiac explants and then grown in specific endothelial and smooth muscle growth media on collagen IV-coated surfaces. The population of endothelial cells was further enriched by immunomagnetic sorting for CD31, and the culture thus obtained was characterized by immunocytochemistry, ultrastructural analysis and in vitro functional tests. The angiogenic potency of the cells was examined by injecting them, along with Matrigel, into immunodeficient mice. Cells were also seeded on characterized polycaprolactone/chitosan membranes with subsequent analysis of cell proliferation and function. RESULTS: Endothelial cells isolated from cardiac explants expressed CD31, VE-cadherin and VEGFR2 and showed typical properties, namely, cytoplasmic Weibel-Palade bodies, metabolism of acetylated low-density lipoproteins, formation of capillary-like structures in Matrigel, and production of extracellular matrix and angiogenic cytokines. Isolated smooth muscle cells expressed extracellular matrix components as well as α-actin and myosin heavy chain. Vascular cells derived from cardiac explants demonstrated the ability to stimulate angiogenesis in vivo. Endothelial cells proliferated most effectively on membranes made of polycaprolactone and chitosan blended in a 25:75 ratio, neutralized by a mixture of alkaline and ethanol. Endothelial and smooth muscle cells retained their functional properties when seeded on the blended membranes. CONCLUSIONS: We established endothelial and smooth muscle cell cultures from human right atrial appendage and right ventricle post-operative explants. The isolated cells revealed angiogenic potential and may be a promising source of patient-specific cells for regenerative medicine.
Assuntos
Prótese Vascular , Células Endoteliais/citologia , Miocárdio/citologia , Miócitos de Músculo Liso/citologia , Neovascularização Fisiológica , Desenho de Prótese , Animais , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quitosana/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Humanos , Camundongos SCID , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/ultraestrutura , Neovascularização Fisiológica/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Poliésteres/farmacologia , Corpos de Weibel-Palade/metabolismoRESUMO
The effect of substances known as inducers of neuronal differentiation on cultured human and mouse adipose-derived mesenchymal stem cells (ASCs) and their fate after transplantation into the injured and ischemic mouse brains were studied. ASCs were isolated from the human and mouse adipose tissue. Inducers of neuronal differentiation included ß-mercaptoethanol, glial cell line-derived neurotrophic factor (GNDF), brain-derived neurotrophic factor (BDNF), retinoic acid (RA), 5-azacytidine, as well as their combinations. Three days after the induction, the phenotype of the induced cells was analyzed using immunocytochemistry and real-time PCR assay for differential expression of specific genes. The induction efficiency was evaluated by the increased transcription of neuronal differentiation markers: nestin, ß-III-tubulin (Tub-B), microtubule-associated protein 2 (MAP2), and neuron-specific enolase (ENO2). The expression of marker genes was tested by immunocytochemical analysis. ASC cultivation in the medium with RA or BDNF in combination with 5- azacytidine for a week increased the mRNA and protein levels of nestin, Tub-B, and ENO2. The transplantation of induced mouse ASCs into the mouse brain increased the lifespan of the cells relative to control uninduced cells and promoted their migration from the transplantation site to the recipient cerebral parenchyma. The transplantation of the induced cells into the mouse brain pre-exposed to endothelin- 1 promoted a more active cell migration into the surrounding ischemic brain tissue. Thus, ASC exposure to RA or BDNF in combination with 5-azacytidine elevated the transcription of the neuronal differentiation markers and improved the viability and integration of ASCs grafted into the mouse brain.
Assuntos
Azacitidina/administração & dosagem , Fator Neurotrófico Derivado do Encéfalo/administração & dosagem , Fator Neurotrófico Derivado de Linhagem de Célula Glial/administração & dosagem , Mercaptoetanol/administração & dosagem , Células-Tronco Mesenquimais/efeitos dos fármacos , Tretinoína/administração & dosagem , Tecido Adiposo/citologia , Adulto , Animais , Lesões Encefálicas/metabolismo , Diferenciação Celular , Proteínas do Domínio Duplacortina , Humanos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Receptor trkB/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
In the present study, expression of T-cadherin was shown to induce intracellular signaling in NIH3T3 fibroblasts: it activated Rac1 and Cdc42 (p < 0.01) but not RhoA. T-Cadherin overexpression in human umbilical vein endothelial cells (HUVEC) using adenoviral constructs induced disassembly of microtubules and polymerization of actin stress fibers, whereas down-regulation of endogenous T-cadherin expression in HUVEC using lentiviral constructs resulted in microtubule polymerization and a decrease in the number of actin stress fibers. Moreover, suppression of the T-cadherin expression significantly decreased the endothelial monolayer permeability as compared to the control (p < 0.001).
Assuntos
Caderinas/metabolismo , Permeabilidade da Membrana Celular , Células Endoteliais/metabolismo , Ativação Transcricional , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Caderinas/genética , Células Cultivadas , Regulação para Baixo , Células Endoteliais/química , Humanos , Camundongos , Microtúbulos/metabolismo , Células NIH 3T3 , Transdução de Sinais , Fibras de Estresse/metabolismo , Veias Umbilicais/química , Veias Umbilicais/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Perivascular application of urokinase to the ballooned artery promoted the growth of neointima and constrictive remodeling of the vessel and stimulated the inflammatory response in the damaged vascular wall in vivo. Recombinant tissue plasminogen activator did not induce these changes. Our results indicate that urokinase is involved in the regulation of the inflammatory response during in vivo remodeling of the damaged vascular wall.
Assuntos
Artérias/efeitos dos fármacos , Artérias/lesões , Inflamação/induzido quimicamente , Neovascularização Fisiológica/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Angioplastia com Balão , Animais , Artérias/anatomia & histologia , Artérias/fisiologia , Inflamação/imunologia , Masculino , Ratos , Ratos Wistar , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
The results presented in this paper suggest the presence of an interaction between the kringle- and the growth-factor-like urokinase domains. This interaction regulates chemotactic properties of urokinase. We also show that interaction of urokinase with its "classical" receptor (uPAR) has a "permissive" effect on the interactions between the kringle domain and other targets on the cell surface. On the basis of our data we can suggest that uPAR serves as an "adaptor" for urokinase, and the binding of urokinase kringle domain to its receptor causes immediate activation of intracellular signaling and induction of cell migration.
Assuntos
Quimiotaxia , Kringles , Ativador de Plasminogênio Tipo Uroquinase/química , Sequência de Aminoácidos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
Urokinase plasminogen activator (uPA) has been implicated in the healing responses of injured arteries, but the importance of its various properties that influence smooth muscle cell (SMC) proliferation and migration in vivo is unclear. We used three recombinant (r-) forms of uPA, which differ markedly in their proteolytic activities and abilities to bind to the uPA receptor (uPAR), to determine, which property most influences the healing responses of balloon catheter injured rat carotid arteries. After injury, uPA and uPAR expression increased markedly throughout the period when medial SMCs were rapidly proliferating and migrating to form the neointima. Perivascular application of uPA neutralizing antibodies immediately after injury attenuated the healing response, significantly reducing neointima size and neointimal SMC numbers. Perivascular application of r-uPAwt (wild type uPA) or r-uPA/GDF (r-uPA with multiple mutations in its growth factor-like domain) doubled the size of the neointima. Four days after injury these two uPAs nearly doubled neointimal and medial SMC numbers in the vessels, and induced greater reductions in lumen size than injury alone. Proteolytically inactive r-uPA/H/Q (containing glutamine rather than histidine-204 in its catalytic site) did not affect neointima or lumen size. Also, in contrast to the actions of proteolytically active uPAs, tissue plasminogen activator (tPA) did not affect the rate of neointima development. We conclude that uPA is an important factor regulating the healing responses of balloon catheter injured arteries, and its proteolytic property, which cannot be mimicked by tPA, greatly influences SMC proliferation and early neointima formation.
Assuntos
Lesões das Artérias Carótidas/enzimologia , Lesões das Artérias Carótidas/patologia , Divisão Celular/efeitos dos fármacos , Regeneração/fisiologia , Túnica Íntima/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Análise de Variância , Angioplastia com Balão , Animais , Sequência de Bases , Divisão Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Probabilidade , Ratos , Ratos Endogâmicos WKY , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Túnica Íntima/citologia , Túnica Íntima/enzimologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
OBJECTIVES: Increases in urokinase plasminogen activator (uPA) have been reported in tissues undergoing remodelling, but its effects on the vessel intima formation are not known. We investigated its effects on carotid artery intima, media and lumen size, as well as smooth muscle cell (SMC) proliferation and migration in vivo. DESIGN AND METHODS: Carotid arteries of rats were distended with an inflated balloon catheter and uPA, or uPA-neutralizing antibodies were applied perivascularly in pluronic gel; control rats received vehicle. Carotid artery structure, cell migration and proliferation were assessed after 4 days by quantitative morphometry and immunohistochemistry. RESULTS: Four days after increasing vessel uPA, the intima/media ratio was double compared to that in control rats (both P < 0.05). The size of the lumen reduced by 75%, compared to the vehicle-treated vessels (P < 0.05). The elevation in uPA also increased SMC numbers in the intima and media, compared to the vehicle-treated vessels (both P < 0.05). Antibody neutralizing endogenous uPA attenuated the growth responses in the distended arteries, reduced neointimal SMC numbers by approximately 50% and prevented much of the reduction in lumen size. CONCLUSIONS: Thus, local increases in uPA in distended, injured arteries augment SMC migratory and proliferative responses, leading to increases in the thickness of the carotid artery intima and media and a reduction in lumen size; effects at least partially attributable to its proteolytic properties.
Assuntos
Artérias Carótidas/crescimento & desenvolvimento , Lesões das Artérias Carótidas/patologia , Ativadores de Plasminogênio/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Animais , Artérias Carótidas/efeitos dos fármacos , Cateterismo , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Ratos , Ratos Endogâmicos WKY , Proteínas Recombinantes/farmacologiaRESUMO
1. The present study compares plasma urokinase plasminogen activator (uPA) peptide levels, plasma plasminogen inhibitor (PAI-1) activity and urokinase receptors (uPAR) on peripheral blood monocytes of patients with stable coronary artery disease (SCAD) and healthy volunteers. 2. Urokinase plasminogen activator levels were analysed by ELISA and PAI-1 activity was determined by a plasmin generation method using the chromogenic substrate S2390. Relative uPAR numbers and the adhesion molecules CD11b/CD18 on peripheral blood monocytes were estimated using specific antibodies and flow cytometry. 3. Patients with SCAD were found to have higher plasma uPA peptide levels than age-matched healthy subjects (10.40 +/- 0.99 vs 8.25 +/- 0.53 pmol/L, respectively; P < 0.05). 4. Plasma PAI-1 activity was also higher in patients with SCAD than in healthy subjects (13.6 +/- 2.5 vs 5.2 +/- 1.0 IU/mL, respectively; P < 0.05). 5. Relative uPAR and CD11b/CD18 adhesion molecules were similar on peripheral blood monocytes of patients with SCAD and in healthy subjects. 6. The data indicate a pattern of expression/activity of uPA and PAI-1 in patients with SCAD suggestive of an impaired fibrinolytic ability.
Assuntos
Doença das Coronárias/sangue , Inibidor 1 de Ativador de Plasminogênio/sangue , Ativador de Plasminogênio Tipo Uroquinase/sangue , Adulto , Doença das Coronárias/etiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ativadores de Plasminogênio/sangue , Receptores de Superfície Celular/sangue , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Regulação para CimaRESUMO
The aim of the study was to evaluate how platelet aggregation relates to left ventricular hypertrophy, presence of transient myocardial ischemia and coronary atherosclerosis in essential hypertensive patients. For that purpose platelet aggregation, symptom-limited treadmill-test, echocardiography and coronary angiography were performed in 65 male patients with uncomplicated essential hypertension. Aggregation was monitored both by a turbidometric method and by a method based on real-time estimation of mean aggregate size. Platelet function was also studied in 18 male normotensive subjects. Hypertensive patients with transient myocardial ischemia were characterized by increased platelet sensitivity to adenosine diphosphate, thromboxane A2 mimetic-U46619, serotonin, platelet activating factor and by increased spontaneous aggregability of platelets compared with normotensive subjects and to patients without myocardial ischemia. Platelet aggregation induced by low doses of agonists positively correlated with the degree of left ventricular hypertrophy. This study provides evidence that platelets play an important role in myocardial ischemia. Besides, it was found that platelet activity positively correlated with the degree of left ventricular hypertrophy. This may provide an additional link between left ventricular hypertrophy and coronary heart disease in hypertensive patients.