RESUMO
BACKGROUND: Endothelial progenitor cells (EPCs) are circulating progenitor cells that can play an essential role in vascular remodelling. In this work, we compared the role of two EPCs cultivated with different mediums in the resolution of the arterial thrombus induced by FeCl3 lesion and in vessel re-endothelization in the mouse carotid artery. METHODS: Mice mononuclear cells were differentiated into EPCs using Dulbecco's Modified Eagle's Medium (DMEM) and vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and IGF (Insulin Growth Factor) called EPCs--M1) or with EGM2(endothelial growth medium) (media supplemented with growth factors from Lonza called (EPCs-M2) for 30days and characterized using flow cytometry. The animals received three EPC injections post-lesion, and we analyzed thrombosis time, vessel re-endothelization, metalloproteinases activities, eNOS (endothelial Nitric oxide synthase) presence and SDF-1(Stromal Derived Factor- 1) levels in circulation. RESULTS: EPC-M1 presented a more immature progenitor profile than EPC-M2 cells. The injection of EPC-M1 prolonged the thrombosis time, and the treatment with the different EPCs increased eNOS expression and MMP2 (Metalloproteinase 2) activity and decreased SDF-1 in plasma. Only EPC-M1 treatment increased both MMP2 and MMP9 and reduced thrombus after 7days. Also, both EPCs decreased platelet aggregation in vitro. CONCLUSIONS: EPCs-M1 were more efficient in all of the analyzed assays. EPCsM2 may be a more mature EPC, proliferating less and promoting a less significant matrix remodelling. EPCs can promote vascular remodelling by inhibiting thrombosis and stimulating vascular wall remodelling and the treatment with a more immature progenitor may be more efficient in this process.
Assuntos
Células Progenitoras Endoteliais/transplante , Trombose/terapia , Animais , Artérias/patologia , Diferenciação Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Embolização Terapêutica , Células Progenitoras Endoteliais/metabolismo , Gelatinases/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/metabolismo , Agregação Plaquetária , Trombose/enzimologia , Trombose/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cerebral malaria (CM) is a multifactorial syndrome involving an exacerbated proinflammatory status, endothelial cell activation, coagulopathy, hypoxia, and accumulation of leukocytes and parasites in the brain microvasculature. Despite significant improvements in malaria control, 15% of mortality is still observed in CM cases, and 25% of survivors develop neurologic sequelae for life-even after appropriate antimalarial therapy. A treatment that ameliorates CM clinical signs, resulting in complete healing, is urgently needed. Previously, we showed a hyperbaric oxygen (HBO)-protective effect against experimental CM. Here, we provide molecular evidence that HBO targets brain endothelial cells by decreasing their activation and inhibits parasite and leukocyte accumulation, thus improving cerebral microcirculatory blood flow. HBO treatment increased the expression of aryl hydrocarbon receptor over hypoxia-inducible factor 1-α (HIF-1α), an oxygen-sensitive cytosolic receptor, along with decreased indoleamine 2,3-dioxygenase 1 expression and kynurenine levels. Moreover, ablation of HIF-1α expression in endothelial cells in mice conferred protection against CM and improved survival. We propose that HBO should be pursued as an adjunctive therapy in CM patients to prolong survival and diminish deleterious proinflammatory reaction. Furthermore, our data support the use of HBO in therapeutic strategies to improve outcomes of non-CM disorders affecting the brain.-Bastos, M. F., Kayano, A. C. A. V., Silva-Filho, J. L., Dos-Santos, J. C. K., Judice, C., Blanco, Y. C., Shryock, N., Sercundes, M. K., Ortolan, L. S., Francelin, C., Leite, J. A., Oliveira, R., Elias, R. M., Câmara, N. O. S., Lopes, S. C. P., Albrecht, L., Farias, A. S., Vicente, C. P., Werneck, C. C., Giorgio, S., Verinaud, L., Epiphanio, S., Marinho, C. R. F., Lalwani, P., Amino, R., Aliberti, J., Costa, F. T. M. Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria.
Assuntos
Encéfalo/metabolismo , Hipóxia/metabolismo , Cinurenina/metabolismo , Malária Cerebral/metabolismo , Oxigênio/metabolismo , Animais , Circulação Cerebrovascular/fisiologia , Células Endoteliais/metabolismo , Feminino , Oxigenoterapia Hiperbárica/métodos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação/fisiologiaRESUMO
Hyperbaric oxygen is a clinical treatment that contributes to wound healing by increasing fibroblasts proliferation, collagen synthesis, and production of growth factors, inducing angiogenesis and inhibiting antimicrobial activity. It also has been shown that hyperbaric oxygen treatment (HBO), through the activation of nitric oxide synthase promotes an increase in the nitric oxide levels that may improve endothelial progenitor cells (EPC) mobilization from bone marrow to the peripheral blood and stimulates the vessel healing process. However, cellular mechanisms involved in cell proliferation and activation of EPC after HBO treatment remain unknown. Therefore, the present work aimed to analyze the effect of HBO on the proliferation of pre-treated bone marrow-derived EPC with TNF-alpha. Also, we investigated the expression of ICAM and eNOS by immunochemistry, the production of reactive species of oxygen and performed an in vitro wound healing. Although 1h of HBO treatment did not alter the rate of in vitro wound closure or cell proliferation, it increased eNOS expression and decreased ICAM expression and reactive oxygen species production in cells pre-treated with TNF-alpha. These results indicate that HBO can decrease the inflammatory response in endothelial cells mediated by TNF-alpha, and thus, promote vascular recovery after injury.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Oxigênio/farmacologia , Animais , Células da Medula Óssea/citologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Despite treatment with effective antimalarial drugs, the mortality rate is still high in severe cases of the disease, highlighting the need to find adjunct therapies that can inhibit the adhesion of Plasmodium falciparum-infected erythrocytes (Pf-iEs). OBJECTIVES: In this context, we evaluated a new heparan sulfate (HS) from Nodipecten nodosus for antimalarial activity and inhibition of P. falciparum cytoadhesion and rosetting. METHODS: Parasite inhibition was measured by SYBR green using a cytometer. HS was assessed in rosetting and cytoadhesion assays under static and flow conditions using Chinese hamster ovary (CHO) and human lymphatic endothelial cell (HLEC) cells expressing intercellular adhesion molecule-1 (ICAM1) and chondroitin sulfate A (CSA), respectively. FINDINGS: This HS inhibited merozoite invasion similar to heparin. Moreover, mollusk HS decreased cytoadherence of P. falciparum to CSA and ICAM-1 on the surface of endothelial cells under static and flow conditions. In addition, this glycan efficiently disrupted rosettes. CONCLUSIONS: These findings support a potential use for mollusk HS as adjunct therapy for severe malaria.
Assuntos
Heparitina Sulfato/farmacologia , Merozoítos/efeitos dos fármacos , Moluscos/química , Plasmodium falciparum/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Proteínas de Protozoários/efeitos dos fármacos , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Hemolysis and consequent release of cell-free hemoglobin (CFHb) impair vascular nitric oxide (NO) bioavailability and cause oxidative and inflammatory processes. Hydroxyurea (HU), a common therapy for sickle cell disease (SCD), induces fetal Hb production and can act as an NO donor. We evaluated the acute inflammatory effects of intravenous water-induced hemolysis in C57BL/6 mice and determined the abilities of an NO donor, diethylamine NONOate (DEANO), and a single dose of HU to modulate this inflammation. Intravenous water induced acute hemolysis in C57BL/6 mice, attaining plasma Hb levels comparable to those observed in chimeric SCD mice. This hemolysis resulted in significant and rapid systemic inflammation and vascular leukocyte recruitment within 15 minutes, accompanied by NO metabolite generation. Administration of another potent NO scavenger (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) to C57BL/6 mice induced similar alterations in leukocyte recruitment, whereas hemin-induced inflammation occurred over a longer time frame. Importantly, the acute inflammatory effects of water-induced hemolysis were abolished by the simultaneous administration of DEANO or HU, without altering CFHb, in an NO pathway-mediated manner. In vitro, HU partially reversed the Hb-mediated induction of endothelial proinflammatory cytokine secretion and adhesion molecule expression. In summary, pathophysiological levels of hemolysis trigger an immediate inflammatory response, possibly mediated by vascular NO consumption. HU presents beneficial anti-inflammatory effects by inhibiting rapid-onset hemolytic inflammation via an NO-dependent mechanism, independently of fetal Hb elevation. Data provide novel insights into mechanisms of hemolytic inflammation and further support perspectives for the use of HU as an acute treatment for SCD and other hemolytic disorders.
Assuntos
Óxidos N-Cíclicos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Hemoglobinas/metabolismo , Hidroxiureia/farmacologia , Imidazóis/farmacologia , Leucócitos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Anemia Falciforme/sangue , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/patologia , Animais , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Hemólise/efeitos dos fármacos , Humanos , Hidrazinas/antagonistas & inibidores , Hidrazinas/farmacologia , Inflamação/sangue , Inflamação/tratamento farmacológico , Inflamação/patologia , Leucócitos/metabolismo , Leucócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doadores de Óxido Nítrico/antagonistas & inibidores , Doadores de Óxido Nítrico/farmacologia , Cultura Primária de Células , Fator de Necrose Tumoral alfa/farmacologia , Viscosidade , Água/farmacologiaRESUMO
BACKGROUND AIMS: Dermatan sulfate (DS), an anticoagulant and antithrombotic glycosaminoglycan, also has anti-inflammatory activity. In this study, we investigated the effect of DS treatment in the presence or absence of bone marrow mononuclear cells (MNCs) or endothelial progenitor cells (EPCs) in the vascular response to carotid artery lesion in C57BL6 mice. METHODS: Thrombus formation, the expression of adhesion molecules and factors involved in vascular remodeling, inflammation or vascular tone were analyzed by histologic examination, Western blotting and enzyme-linked immunoassay 1 and 3 days after vascular injury. RESULTS: DS injections prevented thrombus formation and decreased P-selectin expression after 3 days of the injury. DS treatment also increased plasma SDF-1 levels but failed to rescue endothelial nitric oxide synthase (eNOS) expression, which is responsible for vascular tone. Treatment with MNCs alone failed to prevent thrombus formation 1 day after injury and increased intercellular adhesion molecule-1 expression, likely because of the inflammatory nature of these cells. Treatment with EPCs with DS was the most efficient among all therapies studied. Dual administration of EPCs and DS promoted an increase in the expression of adhesion molecules and, at the same time, induced a higher expression of eNOS at the injury site. Furthermore, it stimulated an elevated number of EPCs to migrate and adhere to the vascular wall. DISCUSSION: Simultaneous treatment with EPCs and DS increased the expression of adhesion molecules, prevented thrombosis, rescued the expression of eNOS and increased migration of EPCs to the site of injury, thereby affecting thrombus remodeling and inflammation and can be involved in vessel hemostasis.
Assuntos
Lesões das Artérias Carótidas/terapia , Dermatan Sulfato/uso terapêutico , Células Progenitoras Endoteliais/transplante , Fibrinolíticos/uso terapêutico , Trombose/prevenção & controle , Remodelação Vascular/fisiologia , Animais , Anti-Inflamatórios/farmacologia , Células da Medula Óssea/citologia , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/tratamento farmacológico , Lesões das Artérias Carótidas/cirurgia , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Movimento Celular/fisiologia , Células Cultivadas , Quimiocina CXCL12/biossíntese , Terapia Combinada , Molécula 1 de Adesão Intercelular/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/biossíntese , Selectina-P/biossínteseRESUMO
Endothelial progenitor cells (EPCs) can be isolated from bone marrow and characterized by the expression of cellular markers such as CD34, CD133, VEGFR2, CD31, and VE-Cadherin, by the uptake of acetylated low-density lipoprotein and by in vitro tube formation in tridimensional matrices. These cells are able to differentiate into mature endothelial cells and participate in the re-endothelization of damaged vessels. In this work, we tested different cultured media that can promote the proliferation and differentiation of mononuclear cells (MNCs) into early EPCs, with defined concentrations of growth factors and serum in order to establish a composition that may ensure us the reproducibility of our cultures. MNCs from mice bone marrow were cultivated using selective culture media containing DMEM or M199 supplemented with 10% FBS, VEGF, bFGF, and IGF, for 3, 7, and 14 days. Differentiation into early EPCs was analyzed using immunohistochemistry, FACS and western blotting and by functional parameters as uptake of ac-LDL, and formation of vessel-like structures. The cells cultivated with medium DMEM-M1 (DMEM plus VEGF, bFGF and IGF) expressed CD34, CD133, CD31, VEGFR2, and VE-Cadherin at all culture time-points with increased expression of these markers after 7 days. Only EPCs cultured for 30 days were able to form vessel-like structure. The uptake of ac-LDL was observed after 3, 7, 14, and 30 days, confirming the differentiation of mononuclear cells into early EPCs. DMEM-M1 was able to sustain MNCs proliferation and differentiation, increasing the expression of the characteristic EPC markers, allowing the expansion of early EPCs in culture in a similar way to that observed in commercial available media.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Células Progenitoras Endoteliais/citologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Técnicas de Cultura de Células , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Sequestration of Plasmodium falciparum-infected erythrocytes (Pf-iEs) in the microvasculature of vital organs plays a key role in the pathogenesis of life-threatening malaria complications, such as cerebral malaria and malaria in pregnancy. This phenomenon is marked by the cytoadhesion of Pf-iEs to host receptors on the surfaces of endothelial cells, on noninfected erythrocytes, and in the placental trophoblast; therefore, these sites are potential targets for antiadhesion therapies. In this context, glycosaminoglycans (GAGs), including heparin, have shown the ability to inhibit Pf-iE cytoadherence and growth. Nevertheless, the use of heparin was discontinued due to serious side effects, such as bleeding. Other GAG-based therapies were hampered due to the potential risk of contamination with prions and viruses, as some GAGs are isolated from mammals. In this context, we investigated the effects and mechanism of action of fucosylated chondroitin sulfate (FucCS), a unique and highly sulfated GAG isolated from the sea cucumber, with respect to P. falciparum cytoadhesion and development. FucCS was effective in inhibiting the cytoadherence of Pf-iEs to human lung endothelial cells and placenta cryosections under static and flow conditions. Removal of the sulfated fucose branches of the FucCS structure virtually abolished the inhibitory effects of FucCS. Importantly, FucCS rapidly disrupted rosettes at high levels, and it was also able to block parasite development by interfering with merozoite invasion. Collectively, these findings highlight the potential of FucCS as a candidate for adjunct therapy against severe malaria.
Assuntos
Antimaláricos/farmacologia , Sulfatos de Condroitina/farmacologia , Merozoítos/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/efeitos adversos , Células Cultivadas , Sulfatos de Condroitina/efeitos adversos , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Células Hep G2 , Humanos , Pepinos-do-Mar/químicaRESUMO
Impairment of pelvic organ support has been described in mice with genetic modifications of the proteins involved in elastogenesis, such as lysyl oxidase-like 1 (LOXL1) and fibulin 5. During pregnancy, elastic fiber-enriched pelvic tissues are modified to allow safe delivery. In addition, the mouse pubic symphysis is remodeled in a hormone-controlled process that entails the modification of the fibrocartilage into an interpubic ligament (IpL) and the relaxation of this ligament. After first parturition, recovery occurs to ensure pelvic tissue homeostasis. Because ligaments are the main supports of the pelvic organs, this study aimed to evaluate elastogenesis in the IpL during mouse pregnancy and postpartum. Accordingly, virgin, pregnant, and postpartum C57BL/6 mice were studied using light, confocal, and transmission electron microscopy as well as Western blots and real-time PCR. Female mice exhibited the separation of the pubic bones and the formation, relaxation, and postpartum recovery of the IpL. By the time the IpL was formed, the elastic fibers had increased in profile length and diameter, and they consisted of small conglomerates of amorphous material distributed among the bundles of microfibrils. Our analyses also indicated that elastin/tropoelastin, fibrillin 1, LOXL1/Loxl1, and fibulin 5 were spatially and temporally regulated, suggesting that these molecules may contribute to the synthesis of new elastic fibers during IpL development. Overall, this work revealed that adult elastogenesis may be important to assure the elasticity of the pelvic girdle during preparation for parturition and postpartum recovery. This finding may contribute to our understanding of pathological processes involving elastogenesis in the reproductive tract.
Assuntos
Tecido Elástico/metabolismo , Período Pós-Parto/metabolismo , Sínfise Pubiana/metabolismo , Aminoácido Oxirredutases/metabolismo , Animais , Tecido Elástico/citologia , Elasticidade , Elastina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibrilina-1 , Fibrilinas , Ligamentos/citologia , Ligamentos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Pelve , Gravidez , Sínfise Pubiana/citologia , Proteínas Recombinantes/metabolismoRESUMO
Matricryptins are collagen fragments proteolytically released from the extracellular matrix (ECM) with biological activity that can regulate several processes involved in ECM remodeling. Vessel wall matrix reorganization after lesion is important to the recovery of vascular function. This study aimed to analyze the effect of the peptide p1158/59 (Lindsey, 2015) on thrombosis, neointimal formation, and vascular remodeling of C57BL6 mice abdominal aorta. We used a FeCl3 induced vascular injury mice model and analyzed thrombus size, neointima formation, gelatinase activities in situ, re-endothelization, and collagen fibers organization on the arterial wall using polarization microscopy. As result, we observed that 2 days after injury the treatment with p1158/59 increased thrombus size and gelatinase activity, vascular lesion and it did not recover the endothelium loss induced by the chemical injury. We also observed that the peptide increased neointima growth and collagen birefringence, indicating collagen fibers reorganization. It also promoted increased re-endothelization and decreased activity of gelatinases 14 days after injury. Thus, we conclude that the peptide p1158/59 impaired the initial thrombosis recovery 2 days after injury but was able to induce vascular ECM remodeling after 14 days, improving vessel re-endothelization, collagen fibers deposition, and organization.
Assuntos
Colágeno Tipo I , Trombose , Remodelação Vascular , Animais , Aorta , Colágeno , Camundongos , Camundongos Endogâmicos C57BL , Trombose/induzido quimicamenteRESUMO
BACKGROUND AIMS: Previously, we have demonstrated that administration of dermatan sulfate (DS) suppresses neointima formation in the mouse carotid artery by activating heparin co-factor II. A similar suppressive effect was observed by increasing the number of progenitor cells in circulation. In this study, we investigated the combination of DS and bone marrow mononuclear cells (MNC), which includes potential endothelial progenitors, in neointima formation after arterial injury. METHODS: Arterial injury was induced by mechanical dilation of the left common carotid artery. We analyzed the extension of endothelial lesion, thrombus formation, P-selectin expression and CD45(+) cell accumulation 1 and 3 days post-injury, and neointima formation 21 days post-injury. Animals were injected with MNC with or without DS during the first 48 h after injury. RESULTS: The extension of endothelial lesion was similar in all groups 1 day after surgery; however, in injured animals treated with MNC and DS the endothelium recovery seemed to be more efficient 21 days after lesion. Treatment with DS inhibited thrombosis, decreased CD45(+) cell accumulation and P-selectin expression at the site of injury, and reduced the neointimal area by 56%. Treatment with MNC reduced the neointimal area by 54%. The combination of DS and MNC reduced neointima formation by more than 91%. In addition, DS promoted a greater accumulation of MNC at the site of injury. CONCLUSIONS: DS inhibits the initial thrombotic and inflammatory processes after arterial injury and promotes migration of MNC to the site of the lesion, where they may assist in the recovery of the injured endothelium.
Assuntos
Células da Medula Óssea/citologia , Artérias Carótidas/efeitos dos fármacos , Dermatan Sulfato/uso terapêutico , Neointima/prevenção & controle , Neointima/terapia , Animais , Anticoagulantes/uso terapêutico , Células da Medula Óssea/fisiologia , Artérias Carótidas/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Selectina-P/metabolismo , Trombose/prevenção & controle , Trombose/terapiaRESUMO
Every year traumatic peripheral nerve injuries (TPNI) result in considerable physical disability across the world; the mechanisms of plasticity and reorganization of spinal cord circuits following such injuries are complex and not completely understood. A comparative proteome analysis between neonatal rats submitted to peripheral lesion and controls was performed; a number of differentially expressed proteins involved in oxidative stress response, energy metabolism and cytoskeleton rearrangement were revealed, which may support future studies to help in the understanding and a posteriori the treatment of TPNI.
Assuntos
Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Proteoma/metabolismo , Medula Espinal/metabolismo , Animais , Animais Recém-Nascidos , Axotomia/métodos , Eletroforese em Gel Bidimensional/métodos , Vértebras Lombares , Ratos , Ratos WistarRESUMO
The practice of physical exercise is highly indicated to prevent cardiovascular diseases and is directly related to the improvement of endothelial function and the regulation of arterial blood pressure. The objective of this study was to analyze the effect of physical exercise in vascular remodeling after FeCl3 chemically induced arterial injury on atherosclerotic mice. To analyze the effect of exercises on thrombus formation, LDL receptor-deficient mice were fed for 6â¯weeks with a high-fat diet and performed or not physical exercises for 2â¯weeks before the arterial injury. To verify endothelium recovery the animals were exercised or not 2â¯weeks before the injury, and 3â¯weeks after it, when the vessels were analyzed. In this work, we observed that physical exercises done only before arterial injury reduced thrombosis time, protected the endothelial layer, promoted the recruitment of CD34 positive progenitor cells, increased the level of eNOS and gelatinases activities and decreased the number of inflammatory cells in the vessel, but do not avoid the growth of neointima. Otherwise exercises done before and continued after injury, increased gelatinase activities, reduced lipid deposition in the aortic arch and prevented neointima formation. Thus, we could conclude that physical exercises are done before and continued after endothelial injury stimulate endothelial recovery by promoting endothelial cell growth, matrix remodeling and decreasing inflammation in the vessel wall.
Assuntos
Aterosclerose/terapia , Exercício Físico/fisiologia , Neointima/terapia , Trombose/terapia , Remodelação Vascular/fisiologia , Animais , Aterosclerose/patologia , Humanos , Masculino , CamundongosRESUMO
Although essential for inflammatory responses, leukocyte recruitment to blood vessel walls in response to inflammatory stimuli, such as TNF-α, can contribute to vascular occlusion in inflammatory diseases, including atherosclerosis. We aimed to further characterize the mechanisms by which TNF stimulates adhesive and morphologic alterations in neutrophils. Microfluidic and intravital assays confirmed the potent effect that TNF has on human and murine neutrophil adhesion and recruitment in vitro and in vivo, respectively. Inhibition of actin polymerization by cytochalasin D significantly diminished TNF-induced human neutrophil adhesion in vitro and abolished TNF-induced membrane alterations and cell spreading. In contrast, TNF-induced increases in ß2-integrin (Mac-1 and LFA-1) expression was not significantly altered by actin polymerization inhibition. Consistent with a role for cytoskeletal rearrangements in TNF-induced adhesion, TNF augmented the activity of the Rho GTPase, RhoA, in human neutrophils. However, inhibition of the major RhoA effector protein, Rho kinase (ROCK), by Y-27632 failed to inhibit TNF-induced neutrophil adhesion. In contrast, the formin FH2 domain inhibitor, SMIFH2, abolished TNF-induced human neutrophil adhesion and diminished leukocyte recruitment in vivo. SMIFH2 also inhibited TNF-induced cytoskeletal reorganization in human neutrophils and abolished the alterations in ß2-integrin expression elicited by TNF stimulation. As such, Rho GTPase/mDia formin-mediated cytoskeletal reorganization appears to participate in the orchestration of TNF-induced neutrophil-adhesive interactions, possibly mediated by formin-mediated actin nucleation and subsequent modulation of ß2-integrin activity on the neutrophil surface. This pathway may represent a pharmacologic target for reducing leukocyte recruitment in inflammatory diseases.
Assuntos
Citoesqueleto de Actina/fisiologia , Antígenos CD18/metabolismo , Adesão Celular , Proteínas Fetais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neutrófilos/fisiologia , Proteínas Nucleares/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Adolescente , Adulto , Animais , Antígenos CD18/genética , Células Cultivadas , Proteínas Fetais/genética , Forminas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Proteínas Nucleares/genética , Transdução de Sinais , Adulto Jovem , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Neutrophil adhesion to the vasculature in response to potent inflammatory stimuli, such as TNF-α (TNF), can contribute to atheroprogression amongst other pathophysiological mechanisms. Previous studies have shown that simvastatin, a statin with known pleiotropic anti-inflammatory properties, can partially abrogate the effects of TNF-induced neutrophil adhesion, in association with the modulation of ß2-integrin expression. We aimed to further characterize the effects of this statin on neutrophil and leukocyte adhesive mechanisms in vitro and in vivo. A microfluidic assay confirmed the ability of simvastatin to inhibit TNF-induced human neutrophil adhesion to fibronectin ligand under conditions of shear stress, while intravital imaging microscopy demonstrated an abrogation of leukocyte recruitment by simvastatin in the microvasculature of mice that had received a TNF stimulus. This inhibition of neutrophil adhesion was accompanied by the inhibition of TNF-induced RhoA activity in human neutrophils, and alterations in cell morphology and ß2-integrin activity. Additionally, TNF augmented the activity of the p50 NFκB subunit in human neutrophils and TNF-induced neutrophil adhesion and ß2-integrin activity could be abolished using pharmacological inhibitors of NFκB translocation, BAY11-7082 and SC514. Accordingly, the TNF-induced elevation of neutrophil p50 activity was abolished by simvastatin. In conclusion, our data provide further evidence of the ability of simvastatin to inhibit neutrophil adhesive interactions in response to inflammatory stimuli, both in vivo and in vitro. Simvastatin appears to inhibit neutrophil adhesion by interfering in TNF-induced cytoskeletal rearrangements, in association with the inhibition of Rho A activity, NFκB translocation and, consequently, ß2-integrin activity.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Subunidade p50 de NF-kappa B/metabolismo , Neutrófilos/imunologia , Sinvastatina/farmacologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Antígenos CD18/metabolismo , Adesão Celular , Células Cultivadas , Quimiotaxia , Citoesqueleto/metabolismo , Fibronectinas/metabolismo , Humanos , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Transporte Proteico , Fator de Necrose Tumoral alfa/imunologia , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Cancer patients are at an increased risk of developing thromboembolic complications. Several mechanisms have been proposed to explain cancer-associated thrombosis including the release of tumor-derived extracellular vesicles and the activation of host vascular cells. It was proposed that neutrophil extracellular traps (NETs) contribute to the prothrombotic phenotype in cancer. In this study, we evaluated the possible cooperation between tumor-derived exosomes and NETs in cancer-associated thrombosis. Female BALB/c mice were orthotopically injected with 4T1 breast cancer cells. The tumor-bearing animals exhibited increased levels of plasma DNA and myeloperoxidase in addition to significantly increased numbers of circulating neutrophils. Mice were subjected to either Rose Bengal/laser-induced venous thrombosis or ferric chloride-induced arterial thrombosis models. The tumor-bearing mice exhibited accelerated thrombus formation in both models compared to tumor-free animals. Treatment with recombinant human DNase 1 reversed the prothrombotic phenotype of tumor-bearing mice in both models. Remarkably, 4T1-derived exosomes induced NET formation in neutrophils from mice treated with granulocyte colony-stimulating factor (G-CSF). In addition, tumor-derived exosomes interacted with NETs under static conditions. Accordingly, the intravenous administration of 4T1-derived exosomes into G-CSF-treated mice significantly accelerated venous thrombosis in vivo. Taken together, our observations suggest that tumor-derived exosomes and neutrophils may act cooperatively in the establishment of cancer-associated thrombosis.
Assuntos
Exossomos/patologia , Neoplasias Mamárias Experimentais/patologia , Neutrófilos/patologia , Trombose/etiologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Armadilhas Extracelulares , Feminino , Fator Estimulador de Colônias de Granulócitos/farmacologia , Neoplasias Mamárias Experimentais/complicações , Camundongos Endogâmicos BALB C , Trombose/tratamento farmacológico , Trombose Venosa/tratamento farmacológico , Trombose Venosa/etiologiaRESUMO
INTRODUCTION: MAGP1 is a glycoprotein present in the elastic fibers and is a part of the microfibrils components. MAGP1 interacts with von Willebrand factor and the active form of TGF-ß and BMP. In mice lacking MAGP1, thrombus formation is delayed, increasing the occlusion time of carotid artery despite presenting normal blood coagulation in vitro. MAGP1-containing microfibrils may play a role in hemostasis and thrombosis. In this work, we evaluated the function of MAGP1 and its relation to TGF-ß in the arterial thrombosis process. METHODS AND RESULTS: We analyzed thrombus formation time in wild type and MAGP1-deficient mice comparing Rose Bengal and Ferric Chloride induced arterial lesion. The potential participation of TGF-ß in this process was accessed when we treated both wild type and MAGP1-deficient mice with losartan (an antihypertensive drug that decreases TGF-ß activity) or captopril (an angiotensin converting enzyme inhibitor that was used as a control antihypertensive drug). Besides, we evaluated thrombus embolization and the gelatinolytic activity in the arterial walls in vitro and ex vivo. Losartan and captopril were able to recover the thrombus formation time without changing blood pressure, activated partial thromboplastin time (aPTT), PT (prothrombin time), platelet aggregation and adhesion, but decreased gelatinase activity. CONCLUSIONS: Our results suggest that both treatments are effective in the prevention of the sub-endothelial ECM degradation, allowing the recovery of normal thrombus formation.
Assuntos
Anti-Hipertensivos/uso terapêutico , Captopril/uso terapêutico , Proteínas Contráteis/genética , Proteínas da Matriz Extracelular/genética , Losartan/uso terapêutico , Trombose/tratamento farmacológico , Trombose/genética , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Artérias Carótidas/fisiopatologia , Proteínas Contráteis/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Gelatinases/metabolismo , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Agregação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , Fatores de Processamento de RNA , Trombose/metabolismo , Trombose/fisiopatologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Fibrillin-1 and -2 are large modular extracellular matrix glycoproteins found in many vertebrate organ systems and are known to be key components of the elastic fibre. In the present study, we identify a new heparin-binding region in fibrillin-2 between exons 18 and 24. Additionally, we have narrowed the location of heparin-binding activity previously identified in fibrillin-1 to the last 17 residues of the mature proteolytically processed protein. This domain demonstrated higher activity as a multimer than as a monomer. The fibrillin-1 C-terminal site supported cell attachment in each of nine cell types tested. Attachment was shown to be mediated by cell-surface heparan sulphate proteoglycans. Fibrillin-1 has been shown previously to have heparin-binding activity that is important for matrix deposition of the molecule by fibroblasts. This function in deposition was confirmed in two additional fibrillin-producing cell types (osteosarcoma and epithelial cells) for the deposition of both fibrillin-1 and -2 into the extracellular matrix.
Assuntos
Matriz Extracelular/metabolismo , Heparina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Células CHO , Células COS , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Dimerização , Células Epiteliais/citologia , Matriz Extracelular/efeitos dos fármacos , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Glicosaminoglicanos/farmacologia , Heparina/farmacologia , Humanos , Células Jurkat , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Células NIH 3T3 , Osteossarcoma/patologia , Proteoglicanas/farmacologiaRESUMO
We compared the disaccharide composition of dermatan sulfate (DS) purified from the ventral skin of three species of rays from the Brazilian seacoast, Dasyatis americana, Dasyatis gutatta, Aetobatus narinari and of Potamotrygon motoro, a fresh water species that habits the Amazon River. DS obtained from the four species were composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions C-2 of the uronic acid and at position C-4 or C-6 of GalNAc. However, DS from the skin of P. motoro presented a very low content of the disulfated disaccharides. The anticoagulant actions of ray skin DS, measured by both APTT clotting and HCII-mediated inhibition of thrombin assays, were compared to that of mammalian DS. DS from D. americana had both high APTT and HCII activities, whereas DS from D. gutatta showed activity profiles similar to those of mammalian DS. In contrast, DS from both A. narinari and P. motoro had no measurable activity in the APTT assay. Thus, the anticoagulant activity of ray skin DS is not merely a consequence of their charge density. We speculate that the differences among the anticoagulant activities of these three DS may be related to both different composition and arrangements of the disulfated disaccharide units within their polysaccharide chains.
Assuntos
Anticoagulantes/química , Coagulação Sanguínea/efeitos dos fármacos , Dermatan Sulfato/química , Rajidae , Animais , Anticoagulantes/isolamento & purificação , Anticoagulantes/farmacologia , Dermatan Sulfato/isolamento & purificação , Dermatan Sulfato/farmacologia , Estrutura Molecular , Especificidade da EspécieRESUMO
BACKGROUND: Low-molecular-weight dextran sulfate was tested on an experimental model of urolithiasis induced in rats. METHODS: Male Wistar rats weighing 250 g had a 15-mg calcium oxalate stone surgically placed into the bladder. A group was sham operated, another group was treated by daily intraperitoneal injection of low-molecular-weight dextran sulfate and the other by daily intraperitoneal saline injection. RESULTS: This treatment prevents the growth of exogenous calcium oxalate stone introduced into the bladder and also avoided the formation of secondary stones in the animals. In addition, low-molecular-weight dextran sulfate prevented the aggregation of other ions, such as ammonium, phosphate and magnesium to the calcium oxalate stone placed in the bladder. These effects of the low-molecular-weight dextran sulfate are associated with the presence of the sulfated polysaccharide in the urine. However, the polysaccharide did not adhere to the bladder stone. Possibly, dextran sulfate forms soluble complex with calcium ions dissolved in the urine and therefore prevented calcium salt crystallization. CONCLUSION: Dextran sulfate, 8000 Da, led to a decrease in calculi glycosaminoglycans in animals treated with dextran, and there was an inhibition in bladder-implanted stones growth.