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1.
Clin Appl Thromb Hemost ; 26: 1076029620954913, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33030036

RESUMO

INTRODUCTION: Sulodexide represents a mixture of fast-moving heparin (FMH) and dermatan sulfate (DS) and has been used for the management of venous diseases such as DVT and related disorders. The purpose of this study is to compare sulodexide and its components with unfractionated heparin (UFH) to determine its suitability for the indications in which UFH is used. MATERIALS AND METHOD: Active pharmaceutical ingredients (API) versions of sulodexide, FMH and DS were obtained from Alfasigma. API versions of UFH were obtained from Medefil Inc. Normal human citrated plasma was obtained from blood bank of the Loyola University Medical Center. Each of the individual agents were supplemented in plasma at a graded concentration of 0.0-10 µg/mL. Clotting assays (PiCT, aPTT, PT and TT), anti-Xa and anti-IIa and thrombin generation studies were carried out. Results were compiled as mean ± SD of 3 individual determination. RESULT: In the clot based (PiCT, aPTT and TT), anti-Xa and IIa assays, both the UFH and FMH produced stronger activities in these assays followed by sulodexide. DS did not show any anticoagulant activity. In the thrombin generation assay, FMH and UFH produced comparable inhibition of thrombin generation as measured by various parameters. Sulodexide was slightly weaker in this assay, whereas DS produced relatively weaker effects. CONCLUSION: In comparison to sulodexide, both UFH and FMH exhibit comparable anticoagulant activity despite differences in their molecular weight. These results suggest that sulodexide can be developed as a parenteral anticoagulant for indications in which UFH is used.


Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Glicosaminoglicanos/farmacologia , Trombina/farmacologia , Anticoagulantes/administração & dosagem , Antitrombinas/administração & dosagem , Antitrombinas/farmacologia , Glicosaminoglicanos/administração & dosagem , Heparina/administração & dosagem , Heparina/farmacologia , Humanos , Itália , Sensibilidade e Especificidade , Trombina/administração & dosagem
3.
EBioMedicine ; 59: 102969, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32853989

RESUMO

Coronavirus disease-2019 (COVID-19) is associated with severe inflammation in mainly the lung, and kidney. Reports suggest a beneficial effect of the use of heparin/low molecular weight heparin (LMWH) on mortality in COVID-19. In part, this beneficial effect could be explained by the anticoagulant properties of heparin/LMWH. Here, we summarise potential beneficial, non-anticoagulant mechanisms underlying treatment of COVID-19 patients with heparin/LMWH, which include: (i) Inhibition of heparanase activity, responsible for endothelial leakage; (ii) Neutralisation of chemokines, and cytokines; (iii) Interference with leukocyte trafficking; (iv) Reducing viral cellular entry, and (v) Neutralisation of extracellular cytotoxic histones. Considering the multiple inflammatory and pathogenic mechanisms targeted by heparin/LMWH, it is warranted to conduct clinical studies that evaluate therapeutic doses of heparin/LMWH in COVID-19 patients. In addition, identification of specific heparin-derived sequences that are functional in targeting non-anticoagulant mechanisms may have even higher therapeutic potential for COVID-19 patients, and patients suffering from other inflammatory diseases.


Assuntos
Anti-Inflamatórios/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Heparina/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Glucuronidase/antagonistas & inibidores , Glucuronidase/metabolismo , Heparina/metabolismo , Heparina/farmacologia , Heparina de Baixo Peso Molecular/metabolismo , Heparina de Baixo Peso Molecular/farmacologia , Heparina de Baixo Peso Molecular/uso terapêutico , Histonas/sangue , Histonas/metabolismo , Humanos , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Internalização do Vírus/efeitos dos fármacos
4.
Anal Chem ; 92(16): 10930-10934, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32678978

RESUMO

The emergence and rapid proliferation of the novel coronavirus (SARS-CoV-2) resulted in a global pandemic, with over 6,000,000 cases and nearly 400,000 deaths reported worldwide by the end of May 2020. A rush to find a cure prompted re-evaluation of a range of existing therapeutics vis-à-vis their potential role in treating COVID-19, placing a premium on analytical tools capable of supporting such efforts. Native mass spectrometry (MS) has long been a tool of choice in supporting the mechanistic studies of drug/therapeutic target interactions, but its applications remain limited in the cases that involve systems with a high level of structural heterogeneity. Both SARS-CoV-2 spike protein (S-protein), a critical element of the viral entry to the host cell, and ACE2, its docking site on the host cell surface, are extensively glycosylated, making them challenging targets for native MS. However, supplementing native MS with a gas-phase ion manipulation technique (limited charge reduction) allows meaningful information to be obtained on the noncovalent complexes formed by ACE2 and the receptor-binding domain (RBD) of the S-protein. Using this technique in combination with molecular modeling also allows the role of heparin in destabilizing the ACE2/RBD association to be studied, providing critical information for understanding the molecular mechanism of its interference with the virus docking to the host cell receptor. Both short (pentasaccharide) and relatively long (eicosasaccharide) heparin oligomers form 1:1 complexes with RBD, indicating the presence of a single binding site. This association alters the protein conformation (to maximize the contiguous patch of the positive charge on the RBD surface), resulting in a notable decrease in its ability to associate with ACE2. The destabilizing effect of heparin is more pronounced in the case of the longer chains due to the electrostatic repulsion between the low-pI ACE2 and the heparin segments not accommodated on the RBD surface. In addition to providing important mechanistic information on attenuation of the ACE2/RBD association by heparin, the study demonstrates the yet untapped potential of native MS coupled to gas-phase ion chemistry as a means of facilitating rational repurposing of the existing medicines for treating COVID-19.


Assuntos
Infecções por Coronavirus/patologia , Heparina/metabolismo , Espectrometria de Massas/métodos , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Betacoronavirus/isolamento & purificação , Betacoronavirus/metabolismo , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Gases/química , Heparina/farmacologia , Heparina/uso terapêutico , Humanos , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus/efeitos dos fármacos
5.
Nephron ; 144(8): 383-385, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32526763

RESUMO

In our opinion, the use of heparin could play a crucial role in these patients. In fact, recent studies have shown that heparin, the most commonly used anticoagulant during HD procedures, had anti-inflammatory properties and a direct antiviral action, due to its ability to prevent SARS-CoV-2 pseudovirus entry into host cells. These activities, together with its anticoagulant action, could explain the ability of heparin to ameliorate COVID-19 clinical course.


Assuntos
Anticoagulantes/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Heparina/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Diálise Renal , Anticoagulantes/farmacologia , Heparina/farmacologia , Humanos , Pandemias
6.
PLoS One ; 15(6): e0235168, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579611

RESUMO

OBJECTIVE: There is an increasing need for small diameter vascular grafts with superior host hemo- and cytocompatibilities, such as low activation of platelets and leukocytes. Therefore, we aimed to investigate whether the preparation of bacterial nanocellulose grafts with different inner surfaces has an impact on in vitro host cytocompatibility. METHODS: We have synthesized five different grafts in a bioreactor, namely open interface surface (OIS), inverted (INV), partially air dried (PAD), surface formed in air contact (SAC) and standard (STD) that were characterized by a different surface roughness. The grafts (length 55 mm, inner diameter 5 mm) were attached to heparinized polyvinyl chloride tubes, loaded with human blood and rotated at 37°C for 4 hours. Then, blood was analyzed for frequencies of cellular fractions, oxidative products, soluble complement and thrombin factors. The results were compared to clinically approved grafts made of polyethylene terephthalate and expanded polytetrafluoroethylene. Additionally, blood platelets were labelled with 111Indium-oxine to visualize the distribution of adherent platelets in the loop by scintigraphy. RESULTS: SAC nanocellulose grafts with the lowest surface roughness exhibited superior performance with <10% leukocyte and <50% thrombocyte loss in contrast to other grafts that exhibited >65% leukocyte and >90% thrombocyte loss. Of note, SAC nanocellulose grafts showed lowest radioactivity with scintigraphy analyses, indicating reduced platelet adhesion. Although the levels of reactive oxygen species and cell free DNA did not differ significantly, the levels of thrombin-antithrombin complexes were lowest in SAC grafts. However, all nanocellulose grafts exhibited enhanced complement activation. CONCLUSION: The systematic variation of the inner surfaces of BNC vascular grafts significantly improves biocompatibility. Especially, SAC grafts exhibited the lowest loss of platelets as well as leukocytes and additionally significantly diminished activation of the coagulation system. Further animal studies are needed to study in vivo biocompatibilities.


Assuntos
Materiais Biocompatíveis/química , Prótese Vascular , Celulose/química , Polissacarídeos Bacterianos/química , Grau de Desobstrução Vascular/fisiologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Implante de Prótese Vascular/métodos , Celulose/ultraestrutura , Oclusão de Enxerto Vascular/fisiopatologia , Oclusão de Enxerto Vascular/prevenção & controle , Heparina/farmacologia , Humanos , Teste de Materiais/métodos , Microscopia Eletrônica de Varredura , Adesividade Plaquetária/fisiologia , Polietilenotereftalatos/química , Politetrafluoretileno/química , Propriedades de Superfície , Grau de Desobstrução Vascular/efeitos dos fármacos
7.
Ann Vasc Surg ; 68: 88-92, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32589931

RESUMO

Heparin resistance is an uncommon phenomenon defined as the need for high-dose unfractionated heparin (UFH) of more than 35,000 IU/day to achieve the target activated partial-thromboplastin time ratio or the failure to achieve the desired activated clotting time after a full UFH dose. This rare phenomenon is being more commonly observed in Covid-19 patients in a hypercoagulable state. We describe a Covid-19 patient confirmed by reverse-transcriptase polymerase chain reaction assay, with acute limb ischemia, who developed heparin resistance. The patient was managed by the departments of vascular surgery, anesthesia and intensive care, and the Coagulation Service and Thrombosis Research from San Raffaele Scientific Institute, Milan, Italy.


Assuntos
Betacoronavirus , Infecções por Coronavirus/complicações , Resistência a Medicamentos , Heparina/farmacologia , Isquemia/tratamento farmacológico , Extremidade Inferior/irrigação sanguínea , Pneumonia Viral/complicações , Doença Aguda , Idoso , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Humanos , Isquemia/sangue , Isquemia/etiologia , Masculino , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Tomografia Computadorizada por Raios X
8.
PLoS One ; 15(5): e0233644, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469940

RESUMO

Cytotoxic and pro-inflammatory histones are present in neutrophil extracellular traps (NETs) and are elevated in blood in several inflammatory conditions, sepsis being a major example. Compounds which can attenuate activities of histones are therefore of interest, with heparin being one such material that has previously been shown to bind to histones. Heparin, a successful anticoagulant for nearly a century, has been shown experimentally to bind to histones and exhibit a protective effect in inflammatory conditions. In the present study carried out in whole blood, heparin and selectively desulfated heparin reduced histone induced inflammatory markers such as interleukin 6 (IL 6), interleukin 8 (IL 8) and tissue factor and C3a, a complement component. The selectively desulfated heparins, with reduced anticoagulant activities, retained a high degree of effectiveness as an anti-histone agent, whereas fully desulfated heparin was found to be ineffective. The results from this study indicate that the presence of sulfate and other specific structural features are required for heparin to attenuate the inflammatory action of histones in whole blood.


Assuntos
Anti-Inflamatórios/farmacologia , Anticoagulantes/farmacologia , Heparina/farmacologia , Histonas/imunologia , Inflamação/tratamento farmacológico , Anti-Inflamatórios/química , Anticoagulantes/química , Complemento C3a/análise , Complemento C3a/imunologia , Heparina/análogos & derivados , Histonas/sangue , Humanos , Inflamação/sangue , Inflamação/imunologia , Interleucina-6/sangue , Interleucina-6/imunologia , Interleucina-8/sangue , Interleucina-8/imunologia
9.
Adv Exp Med Biol ; 1221: 309-329, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274715

RESUMO

Tumor progression associated with hematogenous metastatic spread is a multistep process based on a cross-talk between tumor and stromal cells in a tumor microenvironment. In the blood circulation, tumor cells interact with blood cells through receptors such as selectin and integrins that promote tumor cells survival. At the metastatic sites, heparanase secreted by tumor or stromal cells is an important modifier of the tumor microenvironment while promoting tumor invasiveness and angiogenesis. Heparin, particularly low molecular weight heparin, is used for treatment of cancer patients with evidence of hypercoagulability. However, in preclinical studies heparins was shown to contain other biological activities that affect cancer progression including inhibition of heparanase, selectins and integrins. While ongoing clinical trials are assessing inhibition of heparanase on cancer progression, the remaining biological activities of heparins inhibiting cells adhesion, through selectins and integrins remains largely unexplored. This chapter addresses the potential role of heparins in oncology with respect to their anti-heparanase and anti-adhesive activities and aims to discuss aspects relevant for broader therapeutic application of heparins.


Assuntos
Moléculas de Adesão Celular/antagonistas & inibidores , Glucuronidase/antagonistas & inibidores , Glucuronidase/metabolismo , Heparina/farmacologia , Metástase Neoplásica , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Heparina/uso terapêutico , Heparina de Baixo Peso Molecular/farmacologia , Heparina de Baixo Peso Molecular/uso terapêutico , Humanos , Metástase Neoplásica/tratamento farmacológico
10.
Adv Exp Med Biol ; 1221: 493-522, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274724

RESUMO

The chapter will review early and more recent seminal contributions to the discovery and characterization of heparanase and non-anticoagulant heparins inhibiting its peculiar enzymatic activity. Indeed, heparanase displays a unique versatility in degrading heparan sulfate chains of several proteoglycans expressed in all mammalian cells. This endo-ß-D-glucuronidase is overexpressed in cancer, inflammation, diabetes, atherosclerosis, nephropathies and other pathologies. Starting from known low- or non-anticoagulant heparins, the search for heparanase inhibitors evolved focusing on structure-activity relationship studies and taking advantage of new chemical-physical analytical methods which have allowed characterization and sequencing of polysaccharide chains. New methods to screen heparanase inhibitors and to evaluate their mechanism of action and in vivo activity in experimental models prompted their development. New non-anticoagulant heparin derivatives endowed with anti-heparanase activity are reported. Some leads are under clinical evaluation in the oncology field (e.g., acute myeloid leukemia, multiple myeloma, pancreatic carcinoma) and in other pathological conditions (e.g., sickle cell disease, malaria, labor arrest).


Assuntos
Glucuronidase/antagonistas & inibidores , Heparina/análogos & derivados , Heparina/farmacologia , Animais , Glucuronidase/metabolismo , Heparina/química , Heparitina Sulfato/metabolismo , Humanos , Neoplasias/tratamento farmacológico
11.
Adv Exp Med Biol ; 1221: 567-603, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274727

RESUMO

Despite the enormous progress made in recent years with antibodies, vaccines, antisense oligonucleotides, etc., the so-called "biological" approaches for tackling the control of various diseases, medicinal chemistry remains a bulwark to refer to for the development of new drugs. Also in the case of heparanase, medicinal chemistry has always been in the forefront to identify new inhibitors, through modification of natural macromolecules, e.g., sulfated polysaccharides like heparin, or of natural compounds isolated from bacteria or plants, or through rational design. In this chapter, the reader will find a detailed description of the most relevant small-molecule heparanase inhibitors reported so far in the scientific literature and in patent applications, with mention to the design strategy and to structure-activity relationships. Starting from heparanase inhibitors of natural origin and the attempts to improve their potency and selectivity, the reader will be guided through the major chemical classes of synthetic inhibitors, with representation of the structure of the most relevant compounds. The last paragraph is dedicated to a brief description of inhibitors that have reached clinical trials, highlighting their structure, mechanism, and improved derivatives.


Assuntos
Glucuronidase/antagonistas & inibidores , Heparina/análogos & derivados , Heparina/química , Polissacarídeos/química , Polissacarídeos/farmacologia , Heparina/farmacologia , Humanos , Relação Estrutura-Atividade
12.
Adv Exp Med Biol ; 1221: 523-538, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274725

RESUMO

A growing interest around heparanase and its role in cancer, inflammation and other diseases prompted the identification of specific inhibitors of this enzyme and the exploration of their potential therapeutic role. Roneparstat, a 15-25 kDa N-acetylated and glycol split heparin, is one of the most potent and widely studied heparanase inhibitors. These studies generated a large body of data, which allowed to characterize Roneparstat properties and to endorse its potential therapeutic role. Multiple Myeloma represents the indication that most of the studies, including the phase I clinical trial, addressed. However, Roneparstat antitumor activity activity has been documented in other cancers, and in non-oncological conditions.In addition, assessing Roneparstat activity in different experimental models contributed to understanding heparanase role and the biological factors that may be affected by heparanase inhibition in more detail. Finally, some studies elucidated the molecular mechanisms regulating the enzyme-inhibitor kinetics, thus providing important data for the identification and design of new inhibitors.The objective of this chapter is to provide a comprehensive overview of the most significant studies involving Roneparstat and discuss its potential role in therapy.


Assuntos
Heparina/análogos & derivados , Mieloma Múltiplo/tratamento farmacológico , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Glucuronidase/antagonistas & inibidores , Glucuronidase/metabolismo , Heparina/química , Heparina/farmacologia , Heparina/uso terapêutico , Humanos
13.
Med Hypotheses ; 142: 109743, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32335456

RESUMO

Currently, our world is facing the 2019 Novel Coronavirus (COVID-19) outbreak and tremendous efforts are made for developing drugs to treat and vaccines to prevent the disease. At present, there is no specific antiviral drug or vaccine for COVID-19. The pathogenic infectivity of the virus requires the S1 subunit of the spike (S) protein to bind the host cell receptor, angiontensin converting enzyme (ACE2). While the binding to host cell receptor is the first step of infection, the entrance of the virus into the cell needs the cleavage of S1-S2 subunits to expose S2 for fusion to cell membrane via host proteases including cathepsins, cell surface transmembrane protease/serine (TMPRSS) proteases, furin, trypsin and factor Xa. Previous in vitro studies have shown that factor Xa inhibition can decrease viral infectivity. We suppose that host cell proteases including furin (as expressed highly in lungs), factor Xa and cathepsin are possible targets to decrease viral burden, therefore unfractioned heparin and low molecular weight heparin-LMWH (specifically dalteparin and tinzaparin for their anti inflammatory action) can be potential inhibitors of multiple endoproteases involved in virus infectivity. Our hypothesis needs to be tested in in vitro and clinical studies, however as we are in an urgent situation as the burden of SARS-CoV2 is increasing all around the world, we recommend the usage of unfractioned heparin or LMWH in intensive care unit (ICU) and non-ICU hospitalized patients with the risk-benefit judgement of the clinician. Whether our hypothesis is clinically applicable and successful in decreasing viral infection will be evaluated for further studies.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Heparina de Baixo Peso Molecular/farmacologia , Heparina/farmacologia , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Infecções por Coronavirus/sangue , Esquema de Medicação , Fator Xa/metabolismo , Humanos , Modelos Teóricos , Pandemias , Pneumonia Viral/sangue , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/química
14.
Invest Ophthalmol Vis Sci ; 61(3): 24, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32182331

RESUMO

Purpose: Elevated levels of transforming-growth-factor (TGF)-ß2 in the trabecular meshwork (TM) and aqueous humor are associated with primary open-angle glaucoma (POAG). The underlying mechanism includes alteration of extracellular matrix homeostasis through Smad-dependent and independent signaling. Smad4, an essential co-Smad, upregulates hepcidin, the master regulator of iron homeostasis. Here, we explored whether TGF-ß2 upregulates hepcidin, implicating iron in the pathogenesis of POAG. Methods: Primary human TM cells and human and bovine ex vivo anterior segment organ cultures were exposed to bioactive TGF-ß2, hepcidin, heparin (a hepcidin antagonist), or N-acetyl carnosine (an antioxidant), and the change in the expression of hepcidin, ferroportin, ferritin, and TGF-ß2 was evaluated by semiquantitative RT-PCR, Western blotting, and immunohistochemistry. Increase in reactive oxygen species (ROS) was quantified with dihydroethidium, an ROS-sensitive dye. Results: Primary human TM cells and bovine TM tissue synthesize hepcidin locally, which is upregulated by bioactive TGF-ß2. Hepcidin downregulates ferroportin, its downstream target, increasing ferritin and iron-catalyzed ROS. This causes reciprocal upregulation of TGF-ß2 at the transcriptional and translational levels. Heparin downregulates hepcidin, and reduces TGF-ß2-mediated increase in ferritin and ROS. Notably, both heparin and N-acetyl carnosine reduce TGF-ß2-mediated reciprocal upregulation of TGF-ß2. Conclusions: The above observations suggest that TGF-ß2 and hepcidin form a self-sustained feed-forward loop through iron-catalyzed ROS. This loop is partially disrupted by a hepcidin antagonist and an anti-oxidant, implicating iron and ROS in TGF-ß2-mediated POAG. We propose that modification of currently available hepcidin antagonists for ocular use may prove beneficial for the therapeutic management of TGF-ß2-associated POAG.


Assuntos
Glaucoma de Ângulo Aberto/metabolismo , Hepcidinas/metabolismo , Ferro/metabolismo , Malha Trabecular/efeitos dos fármacos , Fator de Crescimento Transformador beta2/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Carnosina/análogos & derivados , Carnosina/farmacologia , Proteínas de Transporte de Cátions/metabolismo , Bovinos , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Feminino , Ferritinas/metabolismo , Glaucoma de Ângulo Aberto/patologia , Heparina/farmacologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Doadores de Tecidos , Malha Trabecular/metabolismo , Malha Trabecular/patologia , Fator de Crescimento Transformador beta2/metabolismo , Regulação para Cima
15.
J Vis Exp ; (157)2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32202530

RESUMO

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body's circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 °C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (ß-thromboglobulin [ß-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.


Assuntos
Circulação Sanguínea/fisiologia , Prótese Vascular , Teste de Materiais/métodos , Modelos Biológicos , Biomarcadores/metabolismo , Contagem de Células Sanguíneas , Circulação Sanguínea/efeitos dos fármacos , Coleta de Amostras Sanguíneas , Proteínas do Sistema Complemento/metabolismo , Heparina/farmacologia , Humanos , Sistema Imunitário/metabolismo , Elastase Pancreática/metabolismo , Plasma , Stents , beta-Tromboglobulina/metabolismo
16.
J Med Chem ; 63(8): 4227-4255, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32216347

RESUMO

Heparanase cleaves polymeric heparan sulfate (HS) molecules into smaller oligosaccharides, allowing for release of angiogenic growth factors promoting tumor development and autoreactive immune cells to reach the insulin-producing ß cells. Interaction of heparanase with HS chains is regulated by specific substrate sulfation sequences. We have synthesized 11 trisaccharides that are highly tunable in structure and sulfation pattern, allowing us to determine how heparanase recognizes HS substrate and selects a favorable cleavage site. Our study shows that (1) N-SO3- at +1 subsite and 6-O-SO3- at -2 subsite of trisaccharides are critical for heparanase recognition, (2) addition of 2-O-SO3- at the -1 subsite and of 3-O-SO3- to GlcN unit is not advantageous, and (3) the anomeric configuration (α or ß) at the reducing end is crucial in controlling heparanase activity. Our study also illustrates that the α-trisaccharide having N- and 6-O-SO3- at -2 and +1 subsites inhibited heparanase and was resistant toward hydrolysis.


Assuntos
Ativação Enzimática/fisiologia , Glucuronidase/metabolismo , Glicosídeos/metabolismo , Heparitina Sulfato/metabolismo , Oligossacarídeos/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Glicosídeos/síntese química , Heparina/farmacologia , Heparitina Sulfato/antagonistas & inibidores , Humanos , Camundongos , Simulação de Acoplamento Molecular/métodos , Oligossacarídeos/síntese química
17.
Ann Vasc Surg ; 67: 449-460, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32179141

RESUMO

BACKGROUND: The use of unfractionated heparin in hypovolemic shock, aortic clamping, and visceral reperfusion is still not established, despite evidence of inhibition of early cell damage. This study investigated the potential protective effect of unfractionated heparin on hepatic and renal apoptosis in a porcine ischemia and reperfusion model. METHODS: Twenty-one male swine (Sus scrofa) were divided into 3 groups: sham (n = 5), heparin (n = 8), and nonheparin (n = 8). The heparin and nonheparin groups underwent hypovolemic shock for 30 min, supraceliac aortic clamping for 1 h and reperfusion for 3 h. Unfractionated heparin 200 mg/kg was administered to the heparin group during aortic clamping. Hemodynamic and laboratory parameters were monitored, including aminotransferase and serum urea. Histological lesion scores were applied to hematoxylin and eosin-stained liver and kidney sections. Apoptosis quantification was performed by caspase-3 immunohistochemistry. RESULTS: The proposed model caused a severe cardiocirculatory disturbance in the heparin and nonheparin groups, observed by the carotid-femoral pressure gradient and lactic acidosis. There was no significant difference in hemodynamic and laboratory parameters between these two groups. The mean values of liver and renal histological lesion scores did not present any significant differences. Caspase-3 immunoexpression was lower in the heparin than the nonheparin group for both liver and kidney. CONCLUSIONS: Attenuation of liver and kidney cell apoptosis in pigs undergoing systemic heparinization suggests a potential use for heparin in modulating cell death under critical hemodynamic conditions.


Assuntos
Apoptose/efeitos dos fármacos , Heparina/farmacologia , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Choque Hemorrágico/tratamento farmacológico , Animais , Biomarcadores/sangue , Caspase 3/metabolismo , Modelos Animais de Doenças , Hemodinâmica , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Choque Hemorrágico/sangue , Choque Hemorrágico/patologia , Choque Hemorrágico/fisiopatologia , Sus scrofa
18.
Diab Vasc Dis Res ; 17(1): 1479164119896975, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32000529

RESUMO

Advanced glycation end-products, especially toxic advanced glycation end-products derived from glyceraldehyde (advanced glycation end-product-2) and glycolaldehyde (advanced glycation end-product-3), are biologically reactive compounds associated with diabetic complications. We previously demonstrated that toxic advanced glycation end-products were internalised into macrophage-like RAW264.7 cells through scavenger receptor-1 class A (CD204). Toxic advanced glycation end-product uptake was inhibited by fucoidan, a sulphated polysaccharide and antagonistic ligand for scavenger receptors, suggesting that sulphated polysaccharides are emerging candidates for treatment of advanced glycation end-product-related diseases. In this study, we compared the effects of six types of sulphated and non-sulphated polysaccharides on toxic advanced glycation end-product uptake in RAW264.7 cells. Fucoidan, carrageenan and dextran sulphate attenuated toxic advanced glycation end-product uptake. Fucoidan and carrageenan inhibited advanced glycation end-product-2-induced upregulation of SR-A, while advanced glycation end-product-3-induced upregulation of scavenger receptor-1 class A was only suppressed by fucoidan. Dextran sulphate did not affect scavenger receptor-1 class A levels in toxic advanced glycation end-product-treated cells. Chondroitin sulphate, heparin and hyaluronic acid failed to attenuate toxic advanced glycation end-product uptake. Heparin and hyaluronic acid had no effect on scavenger receptor-1 class A levels, while chondroitin sulphate inhibited advanced glycation end-product-3-induced upregulation of scavenger receptor-1 class A. Taken together, fucoidan and carrageenan, but not the other sulphated polysaccharides examined, had inhibitory activities on toxic advanced glycation end-product uptake and toxic advanced glycation end-product-induced upregulation of scavenger receptor-1 class A, possibly because of structural differences among sulphated polysaccharides.


Assuntos
Carragenina/farmacologia , Produtos Finais de Glicação Avançada/metabolismo , Macrófagos/efeitos dos fármacos , Polissacarídeos/farmacologia , Receptores Depuradores Classe A/antagonistas & inibidores , Animais , Transporte Biológico , Sulfatos de Condroitina/farmacologia , Sulfato de Dextrana/farmacologia , Heparina/farmacologia , Ácido Hialurônico/farmacologia , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , Receptores Depuradores Classe A/metabolismo
19.
Carbohydr Polym ; 230: 115654, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31887958

RESUMO

A novel fluorescent heparanase assay based on hybrid nano-assembly of gold nanocluster and glycosaminoglycan is developed. The nanoparticle probes are fabricated through the co-assembly of positively charged gold nanoclusters with negatively charged heparin molecules, which is accompanied by a dramatic size change and a 2.5-fold fluorescence enhancement. It is demonstrated that the fluorescence enhancement is due to denser aggregation of Au-thiolate complexes in the hybrid nanoparticle and the fluctuation of the fluorescence intensity is an indicator of the variation in assembly efficiency. Experiments in solution and in cell lysis media showed that the heparanase could turn-off the fluorescence with a high selectivity, which could be utilized for the assessment of heparanase activity and the metastatic potentials of different tumour cells. This assay technique is low cost, easy to prepare, and showing good performance. The co-assembly strategy has potential to be transferable to construct other functional nanomaterial.


Assuntos
Técnicas Biossensoriais/métodos , Fluorescência , Ouro , Heparina , Nanopartículas Metálicas/química , Neoplasias/diagnóstico por imagem , Animais , Ouro/química , Ouro/farmacologia , Células HeLa , Heparina/química , Heparina/farmacologia , Humanos , Células MCF-7 , Camundongos , Células NIH 3T3
20.
Carbohydr Polym ; 230: 115592, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31887923

RESUMO

The complicated preparation procedure and carrier's suspicious biocompatibility are two major limitations for traditional drug carrier. In this manuscript, a novel polyion complex (PIC) was prepared by simply mixing two biocompatible components, thiolated heparin and doxorubicin (DOX), and subsequently crosslinking under atmosphere, so that it can overcome the above limitations. The PIC's particle size kept stable for one week storage in PBS, and the particles wouldn't decomposed by the dilution, indicating excellent storage and anti-dilution stability resulting from the crosslinking. The PIC can release the larger amount of DOX in acidic environment than psychological environment, and largest amount in acidic and glutathione (GSH) environment, showing the pH and GSH dual sensitive drug release behavior. Furthermore, the PIC exhibited obvious tumor inhibition effect in vivo as well as long circulation ability and low heart toxicity by anti-tumor tests on tumor-bearing mice. Consequently, as-prepared PIC shows promising potential in drug carrier application.


Assuntos
Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Glutationa/química , Heparina/farmacologia , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Glutationa/genética , Heparina/química , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Micelas , Neoplasias/genética , Compostos de Sulfidrila/química
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