Search

Background: In previous studies, we demonstrated the downregulation of several miRNAs from the DLK1-DIO3 genomic region in papillary thyroid carcinoma (PTC). Due to the large number of miRNAs within this region, the individual contribution of these molecules to PTC development and progression remains unclear., Objective: In this study, we aimed to clarify the contribution of DLK1-DIO3-derived miRNAs to PTC., Methods: We used different computational approaches and in vitro resources to assess the biological processes and signaling pathways potentially modulated by these miRNAs., Results: Our analysis suggests that, out of more than 100 mature miRNAs originated from the DLK1-DIO3 region, a set of 12 miRNAs accounts for most of the impact on PTC development and progression, cooperating to modulate distinct cancer-relevant biological processes, such as cell migration, extracellular matrix remodeling, and signal transduction. The restoration of the expression of one of these miRNAs (miR-485-5p) in a BRAFT199A-positive PTC cell line impaired proliferation and migration, suppressing the expression of GAB2 and RAC1, validated miR-485-5p targets., Conclusions: Overall, our results shed light on the role of the DLK1-DIO3 region, which harbors promising tumor suppressor miRNAs in thyroid cancer, and open prospects for the functional exploration of these miRNAs as therapeutic targets for PTC., Competing Interests: Declaration of competing interest All the following authors declare that there is no financial/personal interest or belief that could affect their objectivity, (Copyright © 2023. Published by Elsevier Inc.)

Background: Platelet-rich plasma (PRP) has a high concentration of growth factors (GFs), which present a therapeutic wound healing effect. Despite having been correlated with an immunomodulatory function, the administration of PRP has not yet been investigated in atherosclerosis models. Aim: Evaluate the effect of lyophilized PRP on atherosclerosis in mice models through serum analysis. Methods: Animals received a high-fat diet for disease induction and a weekly PRP retro-orbital application. Effectiveness was evaluated by measuring inflammatory markers in plasma following the treatment of mice with either PRP or saline solution. Results: PRP was well characterized for platelet and GF concentrations; the atherosclerotic profile was established. Cytokine concentrations were altered after PRP applications. Conclusion: PRP could modulate the inflammatory pattern in the early stages of atherosclerosis.

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 FeCl 3 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Wound healing materials to prevent blood loss are crucial during emergency medical treatment because uncontrolled bleeding can lead to patient death. Herein, bioabsorbable fibrous architectures of thrombin-loaded poly(ethylene oxide)-PEO/thrombin-are conceptualized and accomplished via electrospinning for faster wound clotting. Membranes with average fiber diameters ranging from 188 to 264 nm are achieved, where the active thrombin is entrapped within the nanofibers. The results of in vitro and in vivo wound healing activity tests revealed that when the nanofibers with thrombin-loaded capacity are in contact with the wound, the presence of water in the skin or blood catalyzes the degradation of the membranes, thus releasing thrombin. Thrombin then accelerates the wound clotting process. In contrast to other hemostatic materials, PEO/thrombin nanofibers do not require mechanical removal after application, and the viscoelastic nature of such biomaterials enables their conformation to a variety of wound topographies. Remarkably, PEO/thrombin membranes are promising functional materials and their use is a powerful strategy for hemostatic treatment, ranging from simple first aid and sealing to a wound to small surgical procedures.

Gliflozins (canagliflozin, dapagliflozin and empagliflozin) are the newest anti-hyperglycemic class and have offered cardiovascular and renal benefits. Because platelets are involved in the atherothrombosis process, this study is aimed to evaluate the direct effect of gliflozins on platelet reactivity. Platelet-rich plasma (PRP) or washed platelets (WP) were obtained from healthy volunteers. Aggregation, flow cytometry for glycoprotein IIb/IIIa, cyclic nucleotides and intracellular calcium levels, Western blot, thromboxane B 2 (TXB 2 ) measurement and COX-1 activity were performed in the presence of gliflozins (1-30 μM) alone or in combination with sodium nitroprusside (SNP, 10 or 100 nM) + iloprost (ILO, 0.1 or 1 nM). SGLT2 protein is not expressed on human platelets. Gliflozins produced little inhibitory effect in agonists-induced aggregation and this effect was greatly potentiated by ~10-fold in the presence of SNP + ILO, accompanied by lower levels of TXB 2 (58.1 ± 5.1%, 47.1 ± 7.2% and 43.4 ± 9.2% inhibition for canagliflozin, dapagliflozin and empagliflozin, respectively). The activity of COX-1 was not affected by gliflozins. Collagen increased Ca 2+ levels and α(IIb)β(3) activation, both of which were significantly reduced by gliflozins + SNP + ILO. The intracellular levels of cAMP and cGMP and the protein expression of p-VASPSer157 and p-VASPSer239 were not increased by gliflozins while the expression of the serine-threonine kinase, AktSer473 was markedly reduced. Our results showed that the antiplatelet activity of gliflozins were greatly enhanced by nitric oxide and prostacyclin, thus suggesting that the cardiovascular protection seen by this class of drugs could be in part due to platelet inhibition., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The objective of this study was to evaluate the effects of red Light Emiting Diode (red LED) irradiation on fibroblasts in adipose-derived mesenchymal stem cells (ASC) co-culture on the scratch assay. We hypothesized that red LED irradiation could stimulate paracrine secretion of ASC, contributing to the activation of genes and molecules involved in cell migration and tissue repair. ASC were co-cultured with NIH/3T3 fibroblasts through direct contact and subjected to red LED irradiation (1.45 J/cm 2 /5min6s) after the scratch assay, during 4 days. Four groups were established: fibroblasts (F), fibroblasts + LED (FL), fibroblasts + ASC (FC) and fibroblasts + LED + ASC (FLC). The analyzes were based on Ctgf and Reck expression, quantification of collagen types I and III, tenomodulin, VEGF, TGF-β1, MMP-2 and MMP-9, as well as viability analysis and cell migration. Higher Ctgf expression was observed in FC compared to F. Group FC presented higher amount of tenomodulin and VEGF in relation to the other groups. In the cell migration analysis, a higher number of cells was observed in the scratched area of the FC group on the 4 th day. There were no differences between groups considering cell viability, Reck expression, amount of collagen types I and III, MMP-2 and TGF-β1, whereas TGF-β1 was not detected in the FC group and the MMP-9 in none of the groups. Our hypothesis was not supported by the results because the red LED irradiation decreased the healing response of ASC. An inhibitory effect of the LED irradiation associated with ASC co-culture was observed with reduction of the amount of TGF-β1, VEGF and tenomodulin, possibly involved in the reduced cell migration. In turn, the ASC alone seem to have modulated fibroblast behavior by increasing Ctgf , VEGF and tenomodulin, leading to greater cell migration. In conclusion, red LED and ASC therapy can have independent effects on fibroblast wound healing, but the combination of both does not have a synergistic effect. Therefore, future studies with other parameters of red LED associated with ASC should be tested aiming clinical application for tissue repair., (© 2020 Published by Elsevier Ltd.)

Objectives: The cellular therapy using adipose-derived mesenchymal stem cells (ASCs) aims to improve tendon healing, considering that repaired tendons often result in a less resistant tissue. Our objective was to evaluate the effects of the ASCs combination with a low-level laser (LLL), an effective photobiostimulation for the healing processes., Materials and Methods: Rats calcaneal tendons were divided into five groups: normal (NT), transected (T), transected and ASCs (SC) or LLL (L), or with ASCs and LLL (SCL)., Results: All treated groups presented higher expression of Dcn and greater organization of collagen fibres. In comparison with T, LLL also up-regulated Gdf5 gene expression, ASCs up-regulated the expression of Tnmd, and the association of LLL and ASCs down-regulated the expression of Scx. No differences were observed for the expression of Il1b, Timp2, Tgfb1, Lox, Mmp2, Mmp8 and Mmp9, neither in the quantification of hydroxyproline, TNF-α, PCNA and in the protein level of Tnmd. A higher amount of IL-10 was detected in SC, L and SCL compared to T, and higher amount of collagen I and III was observed in SC compared to SCL., Conclusions: Transplanted ASCs migrated to the transected region, and all treatments altered the remodelling genes expression. The LLL was the most effective in the collagen reorganization, followed by its combination with ASCs. Further investigations are needed to elucidate the molecular mechanisms involved in the LLL and ASCs combination during initial phases of tendon repair., (© 2019 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

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 FeCl 3 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., (Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

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 FeCl 3 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (© 2018 International Federation for Cell Biology.)

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.

Tendon injuries represent a clinical challenge in regenerative medicine because their natural repair process is complex and inefficient. The high incidence of tendon injuries is frequently associated with sports practice, aging, tendinopathies, hypertension, diabetes mellitus, and the use of corticosteroids. The growing interest of scientists in using adipose-derived mesenchymal stem cells (ADMSC) in repair processes seems to be mostly due to their paracrine and immunomodulatory effects in stimulating specific cellular events. ADMSC activity can be influenced by GDF-5, which has been successfully used to drive tenogenic differentiation of ADMSC in vitro. Thus, we hypothesized that the application of ADMSC in isolation or in association with GDF-5 could improve Achilles tendon repair through the regulation of important remodeling genes expression. Lewis rats had tendons distributed in four groups: Transected (T), transected and treated with ADMSC (ASC) or GDF-5 (GDF5), or with both (ASC+GDF5). In the characterization of cells before application, ADMSC expressed the positive surface markers, CD90 (90%) and CD105 (95%), and the negative marker, CD45 (7%). ADMSC were also differentiated in chondrocytes, osteoblast, and adipocytes. On the 14th day after the tendon injury, GFP-ADMSC were observed in the transected region of tendons in the ASC and ASC+GDF5 groups, and exhibited and/or stimulated a similar genes expression profile when compared to the in vitro assay. ADMSC up-regulated Lox , Dcn , and Tgfb1 genes expression in comparison to T and ASC+GDF5 groups, which contributed to a lower proteoglycans arrangement, and to a higher collagen fiber organization and tendon biomechanics in the ASC group. The application of ADMSC in association with GDF-5 down-regulated Dcn , Gdf5 , Lox , Tgfb1 , Mmp2 , and Timp2 genes expression, which contributed to a lower hydroxyproline concentration, lower collagen fiber organization, and to an improvement of the rats' gait 24 h after the injury. In conclusion, although the literature describes the benefic effect of GDF-5 for the tendon healing process, our results show that its application, isolated or associated with ADMSC, cannot improve the repair process of partial transected tendons, indicating the higher effectiveness of the application of ADMSC in injured Achilles tendons. Our results show that the application of ADMSC in injured Achilles tendons was more effective in relation to its association with GDF-5.

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.

Tendinitis changes the biochemical and morphological properties of the tendon, promoting an increase of activity of metalloproteinases and disorganization of collagen bundles. Tenocytes, the primary cells in tendon, are scattered throughout the collagenic fibers, and are responsible of tendon remodeling and tissue repair in pathological condition. In vivo, glycine, component of the typical Gly-X-Y collagen tripeptide, showed beneficial effects in biochemical and biomechanical properties of Achilles tendon with tendinitis. In this study, we analyzed the effect of glycine in tenocytes subjected to inflammation. Tenocytes from Achilles tendon of rats were treated with TNF-α (10 ng/mL) with and without previous treatment with glycine (20 mM). Cell proliferation and migration were evaluated, as well as the expression of matrix molecules such as glycosaminoglycans, metalloproteinases (MMPs), TIMPs, and collagen I. Glycine can revert the inflammation due to the action of TNF-α by controlling the MMPs quantity and activity. These data indicated that the molecules involved to remodeling process of extracellular matrix are modulated both by TNF-α and the availability of collagen precursors; in fact, this study indicates the glycine can be useful for treatment of inflammation and for modulating tenocytes metabolism in tendons., (© 2018 International Federation for Cell Biology.)

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 β2-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 β2-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 β2-integrin activity on the neutrophil surface. This pathway may represent a pharmacologic target for reducing leukocyte recruitment in inflammatory diseases., (©2017 Society for Leukocyte Biology.)

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.

Human T-cell lymphotropic virus (HTLV) may affect the clinical course of human immunodeficiency virus 1 (HIV1). Both infections are common in endemic areas because these viruses share similar routes of transmission. The aim of this study was to estimate the seroprevalence of HTLV1/2 in a population of HIV1-infected patients in the state of Goiás, Midwestern Brazil. Of the 505 studied patients, four (0.79%) were positive for anti-HTLV1/2 by enzyme-linked immunosorbent assay (ELISA), with HTLV1 infection confirmed by line immunoassay (LIA) and polymerase chain reaction (PCR) in all of the ELISA-positive samples. No cases of HTLV2 infection were observed. The prevalence of HTLV1/HIV1 coinfection was 0.79% (4/505; 95% CI: 0.25-2.16). All the coinfected patients reported sexual risk behaviors and only one reported intravenous drug use. Sequencing of the viral long terminal repeat (LTR) region and phylogenetic analysis revealed that the four HTLV1 isolates belonged to the Transcontinental a subgroup of the Cosmopolitan (1a) subtype, the most frequent subgroup detected in Brazil. This study shows a low prevalence of HTLV1/2 in HIV1-infected patients in Midwestern Brazil.

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-β 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-β 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-β in this process was accessed when we treated both wild type and MAGP1-deficient mice with losartan (an antihypertensive drug that decreases TGF-β 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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (© 2015 International Federation for Cell Biology.)

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., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

The Bauhinia bauhinioides Kallikrein Inhibitor (BbKI) is a Kunitz-type serine peptidase inhibitor of plant origin that has been shown to impair the viability of some tumor cells and to feature a potent inhibitory activity against human and rat plasma kallikrein (Kiapp 2.4 nmol/L and 5.2 nmol/L, respectively). This inhibitory activity is possibly responsible for an effect on hemostasis by prolonging activated partial thromboplastin time (aPTT). Because the association between cancer and thrombosis is well established, we evaluated the possible antithrombotic activity of this protein in venous and arterial thrombosis models. Vein thrombosis was studied in the vena cava ligature model in Wistar rats, and arterial thrombosis in the photochemical induced endothelium lesion model in the carotid artery of C57 black 6 mice. BbKI at a concentration of 2.0 mg/kg reduced the venous thrombus weight by 65% in treated rats in comparison to rats in the control group. The inhibitor prolonged the time for total artery occlusion in the carotid artery model mice indicating that this potent plasma kallikrein inhibitor prevented thrombosis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Anthracyclines have various mechanisms of action that in the end lead to DNA double-strand breaks. Single-nucleotide polymorphisms (SNPs) in DNA repair genes may alter the protein function, affecting DNA repair proficiency and, therefore, the efficiency of DNA damaging chemotherapy. We have analysed whether SNPs in DNA repair genes (XRCC1, XRCC3 and XPD) could be useful to predict the response to anthracyclines in patients with early-stage breast cancer (EBC)., Methods: Peripheral blood samples from 150 patients with EBC were used for genotyping XRCC3Thr241Met, XRCC1Arg399Gln and XPDLys751Gln. Genotypes were correlated with survival outcomes., Results: Eighty-four patients received treatment with chemotherapy regimens containing anthracyclines. In this group, patients with XRCC1Arg399Arg had a significant improvement in 5-year Disease Free Survival (DFS) compared with those with the Arg/Gln and Gln/Gln variants (84 vs 46 %, p = 0.026). In the multivariate analysis, XRCC1Arg399Arg was reported as an independent prognostic factor for DFS (HR 0.4, CI-95 % 0.2-0.9, p = 0.035). Patients with the XRCC3 Met241Met genotype presented better 5-year OS than those carrying the Thr/Thr and Met/Thr variants (100 vs 70 %, p = 0.030). A multivariate analysis for OS confirmed the independent prognostic value of XRCC3 Met241Met (HR 0.15, CI-95 % 0.02-0.90, p = 0.048). These differences were not significant when patients receiving other chemotherapy treatments, different from anthracyclines, were also considered (n = 150). XPDLys751Lys was associated with older age at diagnosis than the Lys/Gln and Gln/Gln genotypes (65 vs 58 years, p < 0.0001)., Conclusions: XRCC3Thr241Met and XRCC1Arg399Gln may be predictive of survival outcome in EBC patients treated with anthracycline-based chemotherapy regimens.

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.

Introduction: Mature circulating endothelial cells (CEC) and circulating endothelial progenitor cells (EPC) have been described in several conditions associated with endothelial injury. Their role in deep vein thrombosis (DVT) has not been previously evaluated., Patients and Methods: In this pilot study we evaluated the time course of CEC and EPC release after vena cava experimental DVT in mice, using the FeCl3 model. We also evaluated their presence in patients with DVT at different phases of the disease (acute and chronic phase). CEC and EPC were evaluated by Flow Cytometry., Results: In mice, both CEC and EPC were increased 24 hours after DVT induction, peaking 48 hours thereafter. After 72 hours, CEC counts decreased sharply, whereas EPC counts decreased less substantially. In DVT patients we observed a significant increase in CEC counts immediately after DVT compared to healthy individuals. Patients with chronic disease also presented a significant elevation of these cell count. In a subgroup of patients for whom serial samples were available, CEC counts decreased significantly after 9-15 months of the acute event., Conclusions: Our results suggest the participation of these cells in the reparative processes that follows DVT, both at immediate and late time-points. The different kinetics of CEC and EPC release in experimental DVT suggests a heterogeneous role for these cells in the reparative events after DVT.

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).

Autophagy is the primary mechanism of degradation of cellular proteins and at least two functions can be attributed to this biological phenomenon: increased nutrient supply via recycling of the products of autophagy under nutrient starvation; and antimicrobial response involved in the innate immune system. Many microorganisms induce host cell autophagy and it has been proposed as a pathway by which parasites compete with the host cell for limited resources. In this report we provide evidence that the intracellular parasite Leishmania amazonensis induces autophagy in macrophages. Using western blotting, the LC3II protein, a marker of autophagosomes, was detected in cell cultures with a high infection index. Macrophages infected with L. amazonensis were examined by transmission electronic microscopy, which revealed enlarged myelin-like structures typical late autophagosome and autolysosome. Other evidence indicating autophagy was Lysotracker red dye uptake by the macrophages. Autophagy also occurs in the leishmaniasis skin lesions of BALB/c mice, detected by immunohistochemistry with anti-LC3II antibody. In this study, autophagy inhibitor 3-methyladenine (3MA) reduced the infection index, while autophagy inductors, such as rapamycin or starvation, did not alter the infection index in cultivated macrophages, suggesting that one aspect of the role of autophagy could be the provision of nutritive support to the parasite., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Introduction: The thrombin mutant W215A/E217A (WE thrombin) has greatly reduced procoagulant activity, but it activates protein C in the presence of thrombomodulin and inhibits binding of platelet glycoprotein Ib to von Willebrand factor and collagen under flow conditions. Both thrombomodulin-dependent protein C activation and inhibition of platelet adhesion could contribute to the antithrombotic activity of WE thrombin., Materials and Methods: To assess the role of thrombomodulin, we administered WE thrombin to thrombomodulin-deficient (TM(Pro/Pro)) mice and measured the time to occlusive thrombus formation in the carotid artery after photochemical injury of the endothelium., Results and Conclusions: Doses of WE thrombin ≥10μg/kg prolonged the thrombosis time of wild-type mice (>1.6-fold), while doses ≥100μg/kg only slightly prolonged the thrombosis time of TM(Pro/Pro) mice. We conclude that thrombomodulin plays a predominate role in mediating the antithrombotic effect of WE thrombin in the arterial circulation of mice after endothelial injury. Thrombomodulin-independent effects may occur only when high doses of WE thrombin are administered., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A basic phospholipase A₂ (LmrTX) isoform was isolated from Lachesis muta rhombeata snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-5 Discovery® Bio Wide column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of LmrTX was measured as 14.277.50 Da. The amino acid sequence showed a high degree of homology between PLA₂ LmrTX from L. muta rhombeata and other PLA₂ from snake venoms, like CB1 and CB2 from Crotalus durissus terrificus; LmTX-I and LmTX-II from Lachesis muta muta. LmrTX had PLA₂ activity in the presence of a synthetic substrate and alkylation of histidine residues significantly inhibited (P < 0.05) the enzymatic activity of LmrTX and its anticoagulant and antithrombotic activity. In this study, we examined the ability of the LmrTX in altering thrombus formation in living mouse, using a photochemically induced arterial thrombosis model. The control animals that did not receive protein injection showed a normal occlusion time, which was around 57 ± 7.8 min. LmrTX, the PLA₂ from L. muta rhombeata venom, caused a change in the occlusion time to 99 ± 10 min with doses of 7.5 μg/mice. Additionally, LmrTX showed the anticoagulant activity in vitro and ex vivo and prolonging the time aggregation in wash platelet induced by ADP and Thrombin., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Shedding of microvesicles (MVs) by cancer cells is implicated in a variety of biological effects, including the establishment of cancer-associated hypercoagulable states. However, the mechanisms underlying malignant transformation and the acquisition of procoagulant properties by tumour-derived MVs are poorly understood. Here we investigated the procoagulant and prothrombotic properties of MVs produced by a melanocyte-derived cell line (melan-a) as compared to its tumourigenic melanoma counterpart Tm1. Tumour cells exhibit a two-fold higher rate of MVs production as compared to melan-a. Melanoma MVs display greater procoagulant activity and elevated levels of the clotting initiator protein tissue factor (TF). On the other hand, tumour- and melanocyte-derived MVs expose similar levels of the procoagulant lipid phosphatidylserine, displaying identical abilities to support thrombin generation by the prothrombinase complex. By using an arterial thrombosis model, we observed that melanoma- but not melanocyte-derived MVs strongly accelerate thrombus formation in a TF-dependent manner, and accumulate at the site of vascular injury. Analysis of plasma obtained from melanoma-bearing mice showed the presence of MVs with a similar procoagulant pattern as compared to Tm1 MVs produced in vitro. Remarkably, flow-cytometric analysis demonstrated that 60% of ex vivo MVs are TF-positive and carry the melanoma-associated antigen, demonstrating its tumour origin. Altogether our data suggest that malignant transformation in melanocytes increases the production of procoagulant MVs, which may contribute for a variety of coagulation-related protumoural responses.

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.

Background: Previously, we have reported the presence of highly sulfated dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra) and Stolidobranchia (Halocynthia pyriformis and Styela plicata). Despite the identical disaccharide backbone, consisting of [→4IdoA(2S)β-1→3GalNAcβ-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi) and Phlebobranchia (Ciona intestinalis), aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units., Results: Disaccharide analyses indicated a high content of disulfated disaccharide units in the dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfate)β-1→3GalNAcβ-1→] modulate heparin cofactor II activity of dermatan sulfate polymers. Thus, high and low heparin cofactor II stimulating activity is observed in 2,4-sulfated dermatan sulfates and 2,6-sulfated dermatan sulfates, respectively, confirming the clear correlation between the anticoagulant activities of dermatan sulfates and the presence of 2,4-sulfated units., Conclusions: Our results indicate that in ascidian dermatan sulfates the position of sulfation on the GalNAc in the disaccharide [→4IdoA(2S)β-1→3GalNAcβ-1→] is directly related to the taxon and that the 6-O sulfation is a novelty apparently restricted to the Phlebobranchia. We also show that the increased content of [→4IdoA(2S)β-1→3GalNAc(4S)β-1→] disaccharide units in dermatan sulfates from Stolidobranchia accounts for the increased heparin cofactor II stimulating activity.

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.

Heparin cofactor II (HCII)-deficient mice form occlusive thrombi more rapidly than do wild-type mice following injury to the carotid arterial endothelium. Dermatan sulfate (DS) and heparan sulfate (HS) increase the rate of inhibition of thrombin by HCII in vitro, but it is unknown whether vascular glycosaminoglycans play a role in the antithrombotic effect of HCII in vivo. In this study, we found that intravenous injection of either wild-type recombinant HCII or a variant with low affinity for HS (K173H) corrected the abnormally short thrombosis time of HCII-deficient mice, while a variant with low affinity for DS (R189H) had no effect. When HCII was incubated with frozen sections of the mouse carotid artery, it bound specifically to DS in the adventitia. HCII was undetectable in the wall of the uninjured carotid artery, but it became concentrated in the adventitia following endothelial injury. These results support the hypothesis that HCII interacts with DS in the vessel wall after disruption of the endothelium and that this interaction regulates thrombus formation in vivo.

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin when bound to dermatan sulfate or heparin. HCII-deficient mice are viable and fertile but rapidly develop thrombosis of the carotid artery after endothelial injury. We now report the effects of HCII deficiency on atherogenesis and neointima formation. HCII-null or wild-type mice, both on an apolipoprotein E-null background, were fed an atherogenic diet for 12 weeks. HCII-null mice developed plaque areas in the aortic arch approximately 64% larger than wild-type mice despite having similar plasma lipid and glucose levels. Neointima formation was induced by mechanical dilation of the common carotid artery. Thrombin activity, determined by hirudin binding or chromogenic substrate hydrolysis within 1 hour after injury, was higher in the arterial walls of HCII-null mice than in wild-type mice. After 3 weeks, the median neointimal area was 2- to 3-fold greater in HCII-null than in wild-type mice. Dermatan sulfate administered intravenously within 48 hours after injury inhibited neointima formation in wild-type mice but had no effect in HCII-null mice. Heparin did not inhibit neointima formation. We conclude that HCII deficiency promotes atherogenesis and neointima formation and that treatment with dermatan sulfate reduces neointima formation in an HCII-dependent manner.

The anionic phospholipid, phosphatidyl-L-serine (PS), is sequestered in the inner layer of the plasma membrane in normal cells. Upon injury, activation, and apoptosis, PS becomes exposed on the surfaces of cells and sheds microparticles, which are procoagulant. Coagulation is initiated by formation of a tissue factor/factor VIIa complex on PS-exposed membranes and propagated through the assembly of intrinsic tenase (factor VIIIa/factor IXa), prothrombinase (factor Va/factor Xa), and factor XIa complexes on PS-exposed activated platelets. We constructed a novel series of recombinant anticoagulant fusion proteins by linking annexin V (ANV), a PS-binding protein, to the Kunitz-type protease inhibitor (KPI) domain of tick anticoagulant protein, an aprotinin mutant (6L15), amyloid beta-protein precursor, or tissue factor pathway inhibitor. The resulting ANV-KPI fusion proteins were 6- to 86-fold more active than recombinant tissue factor pathway inhibitor and tick anticoagulant protein in an in vitro tissue factor-initiated clotting assay. The in vivo antithrombotic activities of the most active constructs were 3- to 10-fold higher than that of ANV in a mouse arterial thrombosis model. ANV-KPI fusion proteins represent a new class of anticoagulants that specifically target the anionic membrane-associated coagulation enzyme complexes present at sites of thrombogenesis and are potentially useful as antithrombotic agents.

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. We previously reported that the time to thrombotic occlusion of the carotid artery after photochemical injury was shorter in HCII-deficient mice than in wild-type control animals. In this paper, we describe the antithrombotic activity of dermatan sulfate in wild-type and HCII-deficient mice. Intravenous administration of porcine skin dermatan sulfate induced a dose-dependent prolongation of the carotid artery occlusion time in HCII(+/+) mice that was not observed in HCII(-/-) animals. Pharmacokinetic studies suggested that porcine skin dermatan sulfate expresses antithrombotic activity after being transferred from the plasma to sites in the vessel wall. Using invertebrate dermatan sulfate preparations, we showed that N-acetylgalactosamine-4-O-sulfate residues are required for the HCII-dependent antithrombotic effect. Furthermore, the invertebrate dermatan sulfates, which have higher charge densities than mammalian dermatan sulfate, slightly prolonged the thrombotic occlusion time of HCII(-/-) mice. These results indicate that HCII mediates the antithrombotic effect of porcine skin dermatan sulfate after injury to the carotid arterial endothelium in mice, whereas more highly charged dermatan sulfates possess weak antithrombotic activity independent of HCII.

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate, heparan sulfate, or heparin. HCII has been proposed to regulate coagulation or to participate in processes such as inflammation, atherosclerosis, and wound repair. To investigate the physiologic function of HCII, about 2 kb of the mouse HCII gene, encoding the N-terminal half of the protein, was deleted by homologous recombination in embryonic stem cells. Crosses of F1 HCII(+/-) animals produced HCII(-/-) offspring at the expected mendelian frequency. Biochemical assays confirmed the absence of dermatan sulfate-dependent thrombin inhibition in the plasma of HCII(-/-) animals. Crosses of HCII(-/-) animals produced litters similar in size to those obtained from heterozygous matings. At 1 year of age, HCII-deficient animals were grossly indistinguishable from their wild-type littermates in weight and survival, and they did not appear to have spontaneous thrombosis or other morphologic abnormalities. In comparison with wild-type animals, however, they demonstrated a significantly shorter time to thrombotic occlusion of the carotid artery after photochemically induced endothelial cell injury. This abnormality was corrected by infusion of purified HCII but not ovalbumin. These observations suggest that HCII might inhibit thrombosis in the arterial circulation.

We compared the anticoagulant, antithrombotic and bleeding effects of highly sulfated dermatan sulfates from invertebrates and their mammalian counterpart. An invertebrate dermatan sulfate containing 2-O-sulfated alpha-L-iduronic acid and 4-O-sulfated N-acetyl-beta-D-galactosamine residues is a potent anticoagulant due to a high heparin cofactor II activity. It inhibits thrombin due to the formation of a covalent complex with heparin cofactor II, as in the case of mammalian dermatan sulfate, but the effect occurs at lower concentrations for the invertebrate polysaccharide. Surprisingly, the invertebrate dermatan sulfate has a lower potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the mammalian dermatan sulfate. In contrast, another invertebrate dermatan sulfate, also enriched in 2-O-sulfated alpha-L-iduronic acid, but in this case sulfated at O-6 position of the N-acetyl-beta-D-galactosamine units, has no in vitro or in vivo anticoagulant activity, does not prevent thrombus formation but shows a bleeding effect similar to the mammalian glycosaminoglycan. Overall, these results demonstrate unbalanced effects of dermatan sulfates with different sulfation patterns on coagulation, thrombosis and bleeding, and raise interesting questions concerning the relationship among these three biological actions of sulfated polysaccharides.

The antithrombotic activity of a fucosylated chondroitin sulfate extracted from the body wall of a sea cucumber was assessed using a stasis thrombosis model in rats. Intravenous administration of the polysaccharide reduced thrombosis in a dose-dependent manner. We also compared the antithrombotic action of the sea cucumber chondroitin sulfate with that of standard mammalian glycosaminoglycans, mainly heparin and dermatan sulfate. Intravascular injection of fucosylated chondroitin sulfate at the dose totally preventing thrombus formation produced a much more intense modification of the plasma anticoagulant activity than antithrombotic doses of unfractionated heparin, low-molecular-weight heparin and mammalian dermatan sulfate. Thus, it is possible that the mechanism of antithrombotic action of these polysaccharides are different. For fucosylated chondroitin sulfate, it depends mostly on modifications of the plasma anticoagulant activity, but it may involve additional effects in the case of mammalian glycosaminoglycans, perhaps modifications induced in the cells of the vessel wall. The anticoagulant and possibly the antithrombotic actions of fucosylated chondroitin sulfate are mostly dependent on heparin cofactor II activity, and both are markedly reduced with the decrease of the chain size of the polymer. Overall, the sulfated polysaccharide from the invertebrate revealed an unequivocal effect in preventing experimental venous thrombosis, is a useful tool to investigate the antithrombotic action in mammals and may offer an alternative for future development of a new therapeutic agent.

Liver is a major site of retinoid metabolism and storage, and more than 80% of the liver retinoids are stored in hepatic stellate cells. These cells represent less than 1% of the total liver protein, reaching a very high relative intracellular retinoid concentration. The plasma level of retinol is maintained close to 2 microM, and hepatic stellate cells have to be able both to uptake or to release retinol depending upon the extracellular retinol status. In view of their paucity in the liver tissue, stellate cells have been studied in primary cultures, in which they loose rapidly the stored lipids and retinol, and convert spontaneously into the activated myofibroblast phenotype, turning a long-term study of their retinol metabolism impossible. We have analyzed the retinol metabolism in the established GRX cell line, representative of stellate cells. We showed that this cell line behaves very similarly, with respect the retinol uptake and release, to primary cultures of hepatic stellate cells. Moreover, we showed that the cellular retinol binding protein (CRBP-I) expression in these cells, relevant for both uptake and esterification of retinol, responds to the extracellular retinol status, and is correlated to the retinol binding capacity of the cytosol. Its expression is not associated with the overall induction of the lipocyte phenotype by other agents. We conclude that the GRX cell line represents an in vitro model of hepatic stellate cells, and responds very efficiently to wide variations of the extracellular retinol status by autonomous controls of its uptake, storage or release.

Molecular mechanisms of lipid synthesis and their controls in hepatic stellate cells are not known. We have previously proposed that, in contrast to other fat storing cells, hepatic stellate cells are not involved in energy storage, but they represent a particular cell population specialized in storage of lipid-soluble substances, the major one being probably retinol. In agreement with this hypothesis, induction of the lipocyte phenotype in stellate cells is not under the control of insulin, but responds to retinoids and other molecules that modify the gene expression program in these cells. In the present study we have monitored the activity of the two major enzymes involved in lipid synthesis during the induction of the lipocyte phenotype in hepatic stellate cells: glycerol-3-phosphate dehydrogenase (GPDH) that mediates the de novo lipid synthesis, and lipoprotein lipase that mediates incorporation of plasma lipids. In early stages of lipocyte induction, both pathways of lipid synthesis are activated. When lipocytes have already constituted the lipid droplets, lipoprotein lipase pathway is downregulated, while GPDH activity remains high. Adult liver has been reported to lack lipoprotein lipase, but under stress, lipase activity was detected around and at the surface of the intrahepatic vasculature. We have now shown that the lipase activity can be induced in the hepatic stellate cells, located in the Disse's space. The high lipoprotein lipase activity under acute induction of lipocyte phenotype, followed by the low activity under conditions of metabolic equilibrium, are in compass with the increased activity of this enzyme under stress, and its low activity in adult liver parenchyma under normal conditions.

We propose an efficient procedure for constructing and training a feed-forward neural network. The network can perform binary classification for binary or analogue input data. We show that the procedure can also be used to construct feedforward neural networks with binary-valued weights. Neural networks with binary-valued weights are potentially straightforward to implement using microelectronic or optical devices and they can also exhibit good generalization.