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1.
Thromb Res ; 209: 59-68, 2022 Jan.
Article in English | MEDLINE | ID: mdl-34871983

ABSTRACT

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.


Subject(s)
Collagen Type I , Thrombosis , Vascular Remodeling , Animals , Aorta , Collagen , Mice , Mice, Inbred C57BL , Thrombosis/chemically induced
2.
Mem Inst Oswaldo Cruz ; 114: e190088, 2019.
Article in English | MEDLINE | ID: mdl-31188952

ABSTRACT

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.


Subject(s)
Heparitin Sulfate/pharmacology , Merozoites/drug effects , Mollusca/chemistry , Plasmodium falciparum/drug effects , Animals , Cell Adhesion/drug effects , Erythrocytes/drug effects , Protozoan Proteins/drug effects , Reproducibility of Results , Time Factors
3.
Cytotherapy ; 21(4): 444-459, 2019 04.
Article in English | MEDLINE | ID: mdl-30904331

ABSTRACT

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.


Subject(s)
Endothelial Progenitor Cells/transplantation , Thrombosis/therapy , Animals , Arteries/pathology , Cell Differentiation , Cells, Cultured , Chemokine CXCL12/metabolism , Embolization, Therapeutic , Endothelial Progenitor Cells/metabolism , Gelatinases/metabolism , Male , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mice, Inbred C57BL , Nitric Oxide Synthase Type III/metabolism , Platelet Aggregation , Thrombosis/enzymology , Thrombosis/pathology , Vascular Endothelial Growth Factor A/metabolism
4.
Thromb Res ; 175: 21-31, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30685522

ABSTRACT

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 6 weeks with a high-fat diet and performed or not physical exercises for 2 weeks before the arterial injury. To verify endothelium recovery the animals were exercised or not 2 weeks before the injury, and 3 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.


Subject(s)
Atherosclerosis/therapy , Exercise/physiology , Neointima/therapy , Thrombosis/therapy , Vascular Remodeling/physiology , Animals , Atherosclerosis/pathology , Humans , Male , Mice
5.
Cell Biol Int ; 43(2): 136-146, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30362212

ABSTRACT

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.


Subject(s)
Cell Proliferation/drug effects , Endothelial Progenitor Cells/metabolism , Oxygen/pharmacology , Animals , Bone Marrow Cells/cytology , Cell Movement/drug effects , Cells, Cultured , Endothelial Progenitor Cells/cytology , Endothelial Progenitor Cells/drug effects , Intercellular Adhesion Molecule-1/metabolism , Male , Mice , Mice, Inbred C57BL , Nitric Oxide Synthase Type III/metabolism , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/pharmacology
6.
Mem. Inst. Oswaldo Cruz ; 114: e190088, 2019. graf
Article in English | LILACS | ID: biblio-1012679

ABSTRACT

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.


Subject(s)
Plasmodium falciparum , Malaria, Falciparum , Receptors, Cytoadhesin , Heparitin Sulfate , Mollusca
7.
Int Immunopharmacol ; 58: 160-165, 2018 May.
Article in English | MEDLINE | ID: mdl-29604489

ABSTRACT

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.


Subject(s)
Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , NF-kappa B p50 Subunit/metabolism , Neutrophils/immunology , Simvastatin/pharmacology , rho GTP-Binding Proteins/metabolism , Animals , CD18 Antigens/metabolism , Cell Adhesion , Cells, Cultured , Chemotaxis , Cytoskeleton/metabolism , Fibronectins/metabolism , Humans , Mice , Neutrophils/drug effects , Neutrophils/pathology , Protein Transport , Tumor Necrosis Factor-alpha/immunology , rhoA GTP-Binding Protein/metabolism
8.
FASEB J ; 32(8): 4470-4481, 2018 08.
Article in English | MEDLINE | ID: mdl-29558201

ABSTRACT

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.


Subject(s)
Brain/metabolism , Hypoxia/metabolism , Kynurenine/metabolism , Malaria, Cerebral/metabolism , Oxygen/metabolism , Animals , Cerebrovascular Circulation/physiology , Endothelial Cells/metabolism , Female , Hyperbaric Oxygenation/methods , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Mice, Inbred C57BL , Microcirculation/physiology
9.
J Leukoc Biol ; 103(1): 87-98, 2018 01.
Article in English | MEDLINE | ID: mdl-28798145

ABSTRACT

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.


Subject(s)
Actin Cytoskeleton/physiology , CD18 Antigens/metabolism , Cell Adhesion , Fetal Proteins/metabolism , Microfilament Proteins/metabolism , Neutrophils/physiology , Nuclear Proteins/metabolism , Tumor Necrosis Factor-alpha/pharmacology , rhoA GTP-Binding Protein/metabolism , Actin Cytoskeleton/drug effects , Adolescent , Adult , Animals , CD18 Antigens/genetics , Cells, Cultured , Fetal Proteins/genetics , Formins , Humans , Male , Mice , Mice, Inbred C57BL , Microfilament Proteins/genetics , Middle Aged , Neutrophils/cytology , Neutrophils/drug effects , Nuclear Proteins/genetics , Signal Transduction , Young Adult , rhoA GTP-Binding Protein/genetics
10.
Sci Rep ; 7(1): 6438, 2017 07 25.
Article in English | MEDLINE | ID: mdl-28743887

ABSTRACT

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.


Subject(s)
Exosomes/pathology , Mammary Neoplasms, Experimental/pathology , Neutrophils/pathology , Thrombosis/etiology , Animals , Cell Line, Tumor , Disease Models, Animal , Extracellular Traps , Female , Granulocyte Colony-Stimulating Factor/pharmacology , Mammary Neoplasms, Experimental/complications , Mice, Inbred BALB C , Thrombosis/drug therapy , Venous Thrombosis/drug therapy , Venous Thrombosis/etiology
11.
Thromb Res ; 138: 7-15, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26826502

ABSTRACT

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.


Subject(s)
Antihypertensive Agents/therapeutic use , Captopril/therapeutic use , Contractile Proteins/genetics , Extracellular Matrix Proteins/genetics , Losartan/therapeutic use , Thrombosis/drug therapy , Thrombosis/genetics , Animals , Carotid Arteries/drug effects , Carotid Arteries/metabolism , Carotid Arteries/physiopathology , Contractile Proteins/metabolism , Extracellular Matrix Proteins/metabolism , Gelatinases/metabolism , Gene Deletion , Male , Mice , Mice, Inbred C57BL , Platelet Aggregation/drug effects , Platelet Function Tests , RNA Splicing Factors , Thrombosis/metabolism , Thrombosis/physiopathology , Transforming Growth Factor beta/metabolism
12.
Cytotherapy ; 17(10): 1447-64, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26349001

ABSTRACT

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.


Subject(s)
Carotid Artery Injuries/therapy , Dermatan Sulfate/therapeutic use , Endothelial Progenitor Cells/transplantation , Fibrinolytic Agents/therapeutic use , Thrombosis/prevention & control , Vascular Remodeling/physiology , Animals , Anti-Inflammatory Agents/pharmacology , Bone Marrow Cells/cytology , Carotid Arteries/drug effects , Carotid Arteries/pathology , Carotid Artery Injuries/drug therapy , Carotid Artery Injuries/surgery , Cell Adhesion/physiology , Cell Adhesion Molecules/metabolism , Cell Movement/physiology , Cells, Cultured , Chemokine CXCL12/biosynthesis , Combined Modality Therapy , Intercellular Adhesion Molecule-1/biosynthesis , Male , Mice , Mice, Inbred C57BL , Nitric Oxide Synthase Type III/biosynthesis , P-Selectin/biosynthesis
13.
Blood ; 126(6): 711-20, 2015 Aug 06.
Article in English | MEDLINE | ID: mdl-26019278

ABSTRACT

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.


Subject(s)
Cyclic N-Oxides/pharmacology , Free Radical Scavengers/pharmacology , Hemoglobins/metabolism , Hydroxyurea/pharmacology , Imidazoles/pharmacology , Leukocytes/drug effects , Nitric Oxide/metabolism , Anemia, Sickle Cell/blood , Anemia, Sickle Cell/drug therapy , Anemia, Sickle Cell/pathology , Animals , Cell Movement/drug effects , Disease Models, Animal , Hemolysis/drug effects , Humans , Hydrazines/antagonists & inhibitors , Hydrazines/pharmacology , Inflammation/blood , Inflammation/drug therapy , Inflammation/pathology , Leukocytes/metabolism , Leukocytes/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nitric Oxide Donors/antagonists & inhibitors , Nitric Oxide Donors/pharmacology , Primary Cell Culture , Tumor Necrosis Factor-alpha/pharmacology , Viscosity , Water/pharmacology
14.
Cell Biol Int ; 39(10): 1138-50, 2015 Oct.
Article in English | MEDLINE | ID: mdl-25965197

ABSTRACT

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.


Subject(s)
Bone Marrow Cells/cytology , Cell Differentiation , Endothelial Progenitor Cells/cytology , Animals , Bone Marrow Cells/drug effects , Cell Culture Techniques , Intercellular Signaling Peptides and Proteins/pharmacology , Mice , Mice, Inbred C57BL
15.
Antimicrob Agents Chemother ; 58(4): 1862-71, 2014.
Article in English | MEDLINE | ID: mdl-24395239

ABSTRACT

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.


Subject(s)
Antimalarials/pharmacology , Chondroitin Sulfates/pharmacology , Merozoites/drug effects , Plasmodium falciparum/drug effects , Animals , Antimalarials/adverse effects , Cells, Cultured , Chondroitin Sulfates/adverse effects , Erythrocytes/drug effects , Erythrocytes/parasitology , Hep G2 Cells , Humans , Sea Cucumbers/chemistry
16.
Biomed Res Int ; 2013: 903292, 2013.
Article in English | MEDLINE | ID: mdl-24058917

ABSTRACT

We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) from Lachesis muta rhombeata venom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BA ρ NA, with a broad optimum pH (7-10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts, in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, "ex vivo", a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).


Subject(s)
Blood Coagulation/drug effects , Blood Platelets/drug effects , Crotalid Venoms/isolation & purification , Crotalid Venoms/pharmacology , Platelet Aggregation/drug effects , Serine Proteases/isolation & purification , Serine Proteases/pharmacology , Snake Venoms/enzymology , Amino Acid Sequence , Animals , Cattle , Chromatography, High Pressure Liquid , Crotalid Venoms/chemistry , Fibrinogen/metabolism , Kinetics , Male , Mice , Molecular Sequence Data , Molecular Weight , Partial Thromboplastin Time , Prothrombin Time , Serine Proteases/chemistry , Spectrometry, Mass, Electrospray Ionization , Viperidae/metabolism
17.
Cytotherapy ; 13(6): 695-704, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21250866

ABSTRACT

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.


Subject(s)
Bone Marrow Cells/cytology , Carotid Arteries/drug effects , Dermatan Sulfate/therapeutic use , Neointima/prevention & control , Neointima/therapy , Animals , Anticoagulants/therapeutic use , Bone Marrow Cells/physiology , Carotid Arteries/metabolism , Leukocyte Common Antigens/metabolism , Mice , Mice, Inbred C57BL , P-Selectin/metabolism , Thrombosis/prevention & control , Thrombosis/therapy
18.
J Neural Transm (Vienna) ; 117(6): 689-93, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20387083

ABSTRACT

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.


Subject(s)
Peripheral Nervous System Diseases/metabolism , Peripheral Nervous System Diseases/pathology , Proteome/metabolism , Spinal Cord/metabolism , Animals , Animals, Newborn , Axotomy/methods , Electrophoresis, Gel, Two-Dimensional/methods , Lumbar Vertebrae , Rats , Rats, Wistar
19.
J Biol Chem ; 283(37): 25533-25543, 2008 Sep 12.
Article in English | MEDLINE | ID: mdl-18625713

ABSTRACT

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight component of the fibrillin-rich microfibril. Gene-targeted inactivation of MAGP-1 reveals a complex phenotype that includes increased body weight and size due to excess body fat, an altered wound healing response in bone and skin, and a bleeding diathesis. Elastic tissues rich in MAGP-1-containing microfibrils develop normally and show normal function. The penetrance of MAGP-1-null phenotypes is highly variable and mouse strain-dependent, suggesting the influence of modifier genes. MAGP-1 was found to bind active transforming growth factor-beta (TGF-beta) and BMP-7 with high affinity, suggesting that it may be an important modulator of microfibril-mediated growth factor signaling. Many of the phenotypic traits observed in MAGP-1-deficient mice are consistent with loss of TGF-beta function and are generally opposite those associated with mutations in fibrillin-1 that result in enhanced TGF-beta signaling. Increased body size and fat deposition in MAGP-1-mutant animals are particularly intriguing given the localization of obesity traits in humans to the region on chromosome 1 containing the MAGP-1 gene.


Subject(s)
Contractile Proteins/genetics , Contractile Proteins/physiology , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/physiology , Animals , Body Weight , Bone Morphogenetic Protein 7 , Bone Morphogenetic Proteins/metabolism , Fibrillin-1 , Fibrillins , Genotype , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfibrils/metabolism , Microfilament Proteins/metabolism , Models, Biological , Mutation , Phenotype , RNA Splicing Factors , Transforming Growth Factor beta/metabolism
20.
Blood ; 111(8): 4137-44, 2008 Apr 15.
Article in English | MEDLINE | ID: mdl-18281502

ABSTRACT

Mice lacking the extracellular matrix protein microfibril-associated glycoprotein-1 (MAGP1) display delayed thrombotic occlusion of the carotid artery following injury as well as prolonged bleeding from a tail vein incision. Normal occlusion times were restored when recombinant MAGP1 was infused into deficient animals prior to vessel wounding. Blood coagulation was normal in these animals as assessed by activated partial thromboplastin time and prothrombin time. Platelet number was lower in MAGP1-deficient mice, but the platelets showed normal aggregation properties in response to various agonists. MAGP1 was not found in normal platelets or in the plasma of wild-type mice. In ligand blot assays, MAGP1 bound to fibronectin, fibrinogen, and von Willebrand factor, but von Willebrand factor was the only protein of the 3 that bound to MAGP1 in surface plasmon resonance studies. These findings show that MAGP1, a component of microfibrils and vascular elastic fibers, plays a role in hemostasis and thrombosis.


Subject(s)
Carotid Arteries/pathology , Contractile Proteins/deficiency , Extracellular Matrix Proteins/deficiency , Thrombosis/pathology , Animals , Bleeding Time , Blood Coagulation/drug effects , Blood Platelets/drug effects , Blood Pressure/drug effects , Carotid Arteries/drug effects , Carotid Arteries/physiopathology , Cattle , Contractile Proteins/metabolism , Extracellular Matrix Proteins/metabolism , Fibrinogen/metabolism , Fibronectins/metabolism , Humans , Immunohistochemistry , Injections , Mice , Mice, Inbred C57BL , Platelet Function Tests , Protein Binding/drug effects , RNA Splicing Factors , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Surface Plasmon Resonance , von Willebrand Factor/metabolism
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