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1.
Front Immunol ; 11: 575047, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33123154

RESUMO

Reports suggest a role of endothelial dysfunction and loss of endothelial barrier function in COVID-19. It is well established that the endothelial glycocalyx-degrading enzyme heparanase contributes to vascular leakage and inflammation. Low molecular weight heparins (LMWH) serve as an inhibitor of heparanase. We hypothesize that heparanase contributes to the pathogenesis of COVID-19, and that heparanase may be inhibited by LMWH. To test this hypothesis, heparanase activity and heparan sulfate levels were measured in plasma of healthy controls (n = 10) and COVID-19 patients (n = 48). Plasma heparanase activity and heparan sulfate levels were significantly elevated in COVID-19 patients. Heparanase activity was associated with disease severity including the need for intensive care, lactate dehydrogenase levels, and creatinine levels. Use of prophylactic LMWH in non-ICU patients was associated with a reduced heparanase activity. Since there is no other clinically applied heparanase inhibitor currently available, therapeutic treatment of COVID-19 patients with low molecular weight heparins should be explored.


Assuntos
Endotélio/patologia , Glucuronidase/antagonistas & inibidores , Glucuronidase/sangue , Antagonistas de Heparina/uso terapêutico , Heparina de Baixo Peso Molecular/uso terapêutico , Junções Íntimas/patologia , Idoso , Betacoronavirus , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Creatinina/sangue , Cuidados Críticos , Estudos Transversais , Feminino , Glucuronidase/metabolismo , Heparitina Sulfato/sangue , Humanos , Interleucina-6/sangue , L-Lactato Desidrogenase/sangue , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/patologia
2.
Am J Pathol ; 190(10): 2146-2154, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32745462

RESUMO

Patients with thalassemia exhibit an increased risk of thrombotic events that is augmented after splenectomy. Heparanase protein enhances cancer progression, angiogenesis, and inflammation; it also activates the coagulation system through direct interaction with tissue factor (TF). Additionally, erythropoietin, which is elevated in anemic patients, up-regulates heparanase expression via the Janus kinase 2 (JAK-2) pathway. This study aimed was to explore the heparanase profile in thalassemia. Coagulation factors were analyzed via immunostaining, enzyme-linked immunosorbent assay, and heparanase procoagulant activity assay. In spleen specimens of thalassemia major patients, a higher level of heparanase staining was observed compared with control spleens resected after trauma (P < 0.001). Higher heparanase levels, heparanase and TF procoagulant activity, and erythropoietin levels were found in the plasma of 67 thalassemia major patients compared with 29 control subjects. No difference was found in pediatric patients (23 of 67) compared with adults or splenectomized versus nonsplenectomized patients. Higher levels of heparanase, TF, TF pathway inhibitor, and TF pathway inhibitor-2 were observed in liver, spleen, heart, and kidney tissues of thalassemia intermedia mice (Hbbth3/+). These protein levels significantly reduced when mice were treated with the JAK-2 inhibitor ruxolitinib (P < 0.0001). In summary, heparanase levels are elevated in thalassemia, which may contribute to thrombotic phenomena in these patients. Inhibition of heparanase or the JAK-2 pathway may reduce thrombotic risk in thalassemia.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Glucuronidase/metabolismo , Janus Quinase 2/antagonistas & inibidores , Lipoproteínas/farmacologia , Trombose/tratamento farmacológico , Adulto , Animais , Humanos , Masculino , Camundongos Endogâmicos C57BL , Talassemia/tratamento farmacológico , Talassemia/metabolismo , Tromboplastina/metabolismo , Trombose/metabolismo , Adulto Jovem
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.
PLoS One ; 15(7): e0236943, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735612

RESUMO

Halophyte Lobularia maritima LmSAP encodes an A20AN1 zinc-finger stress-associated protein which expression is up-regulated by abiotic stresses and heavy metals in transgenic tobacco. To deepen our understanding of LmSAP function, we isolated a 1,147 bp genomic fragment upstream of LmSAP coding sequence designated as PrLmSAP. In silico analyses of PrLmSAP revealed the presence of consensus CAAT and TATA boxes and cis-regulatory elements required for abiotic stress, phytohormones, pathogen, and wound responses, and also for tissue-specific expression. The PrLmSAP sequence was fused to the ß-glucuronidase (gusA) reporter gene and transferred to rice. Histochemical GUS staining showed a pattern of tissue-specific expression in transgenic rice, with staining observed in roots, coleoptiles, leaves, stems and floral organs but not in seeds or in the root elongation zone. Wounding strongly stimulated GUS accumulation in leaves and stems. Interestingly, we observed a high stimulation of the promoter activity when rice seedlings were exposed to NaCl, PEG, ABA, MeJA, GA, cold, and heavy metals (Al3+, Cd2+, Cu2+ and Zn2+). These results suggest that the LmSAP promoter can be a convenient tool for stress-inducible gene expression and is a potential candidate for crop genetic engineering.


Assuntos
Regulação da Expressão Gênica de Plantas/genética , Regiões Promotoras Genéticas , Plantas Tolerantes a Sal/genética , Estresse Fisiológico/genética , Dedos de Zinco/genética , Produtos Agrícolas/genética , Engenharia Genética , Glucuronidase/metabolismo , Metais Pesados/metabolismo , Especificidade de Órgãos , Oryza/genética , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética
5.
Plant Mol Biol ; 104(1-2): 39-53, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32564178

RESUMO

Plants are exposed to various environmental cues that lead to reactive oxygen species (ROS) accumulation. ROS production and detoxification are tightly regulated to maintain balance. Although studies of glucose (Glc) are always accompanied by ROS in animals, the role of Glc in respect of ROS in plants is unclear. We isolated gsm2 (Glc-hypersensitive mutant 2), a mutant with a notably chlorotic-cotyledon phenotype. The chloroplast-localized GSM2 was characterized as a transaldolase in the pentose phosphate pathway. With 3% Glc treatment, fewer or no thylakoids were observed in gsm2 cotyledon chloroplasts than in wild-type cotyledon chloroplasts, suggesting that GSM2 is required for chloroplast protection under stress. gsm2 also showed evaluated accumulation of ROS with 3% Glc treatment and was more sensitive to exogenous H2O2 than the wild type. Gene expression analysis of the antioxidant enzymes in gsm2 revealed that chloroplast damage to gsm2 cotyledons results from the accumulation of excessive ROS in response to Glc. Moreover, the addition of diphenyleneiodonium chloride or phenylalanine can rescue Glc-induced chlorosis in gsm2 cotyledons. This work suggests that GSM2 functions to maintain ROS balance in response to Glc during early seedling growth and sheds light on the relationship between Glc, the pentose phosphate pathway and ROS.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Homeostase , RNA Helicases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transaldolase/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Clorofila/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Cotilédone/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação , Glucuronidase/metabolismo , Peróxido de Hidrogênio/metabolismo , Via de Pentose Fosfato/genética , Via de Pentose Fosfato/fisiologia , Fenótipo , RNA Helicases/genética , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , Plântula/genética , Plântula/metabolismo , Transaldolase/genética
6.
DNA Cell Biol ; 39(9): 1478-1485, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32584609

RESUMO

Combined antiretroviral therapy has improved quality and life expectancy of people living with human immunodeficiency virus (HIV). However, this therapy increases oxidative stress (OS), which in turn causes alterations in lipid and carbon metabolism, kidney disease, liver cirrhosis, and increased risk of cardiovascular disease. The Klotho gene has been implicated in cardiovascular risk increase. Klotho protein expression at X level decreases the risk of heart disease. HIV-positive people usually present low plasma levels of Klotho; thus, contributing to some extent to an increase in cardiovascular risk for these types of patients, mostly by favoring atherosclerosis. Therefore, our aim is to provide an overview of the effect of OS on Klotho protein and its consequent cardiometabolic alterations in HIV-positive patients on antiretroviral therapy.


Assuntos
Doenças Cardiovasculares/metabolismo , Glucuronidase/metabolismo , Infecções por HIV/metabolismo , Estresse Oxidativo , Animais , Terapia Antirretroviral de Alta Atividade/efeitos adversos , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/genética , Glucuronidase/genética , Infecções por HIV/tratamento farmacológico , Humanos
7.
Oncogene ; 39(24): 4636-4649, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32398866

RESUMO

Klotho is a transmembrane protein, which can be shed and act as a circulating hormone and is involved in regulating cellular calcium levels and inhibition of the PI3K/AKT pathway. As a longevity hormone, it protects normal cells from oxidative stress, and as a tumor suppressor it inhibits growth of cancer cells. Mechanisms governing these differential activities have not been addressed. Altered cellular metabolism is a hallmark of cancer and dysregulation of mitochondrial activity is a hallmark of aging. We hypothesized that klotho exerts its differential effects through regulation of these two hallmarks. Treatment with klotho inhibited glycolysis, reduced mitochondrial activity and membrane potential only in cancer cells. Accordingly, global metabolic screen revealed that klotho altered pivotal metabolic pathways, amongst them glycolysis and tricarboxylic acid cycle in breast cancer cells. Alteration of metabolic activity and increased AMP/ATP ratio lead to LKB1-dependent AMPK activation. Indeed, klotho induced AMPK phosphorylation; furthermore, inhibition of LKB1 partially abolished klotho's tumor suppressor activity. By diminishing deltapsi (Δψ) klotho also inhibited mitochondria Ca2+ shuttling thereby impairing mitochondria communication with SOCE leading to reduced Ca2+ influx by SOCE channels. The reduced SOCE was followed by ER Ca2+ depletion and stress. These data delineate mechanisms mediating the differential effects of klotho toward cancer versus normal cells, and indicate klotho as a potent regulator of metabolic activity.


Assuntos
Neoplasias da Mama/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Glucuronidase/metabolismo , Mitocôndrias/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Glucuronidase/genética , Humanos , Células MCF-7 , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas de Neoplasias/genética
8.
J Nutr ; 150(8): 2070-2076, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32470983

RESUMO

BACKGROUND: Dietary supplemental nicotinamide is used to treat hyperphosphatemia in humans. However, the mechanisms of its impact on body phosphorus homeostasis remain unclear. OBJECTIVE: This study was to determine effects and molecular mechanisms of 3 dietary nicotinamide concentrations on body phosphorus homeostasis in laying hens. METHODS: Hy-Line Brown layers (total = 21; 40 wk old; body weight: 1,876 ± 24 g) were individually housed (n = 7) and fed a corn-soybean meal-based diet supplemented with nicotinamide at 20 (N20), 140 (N140), and 1000 (N1000) mg/kg for 21 d. Serum phosphorus and fibroblast growth factor 23 (FGF23) concentrations, phosphorus and calcium excretion, and mRNA and/or protein of type II sodium-phosphate co-transporters (NPt2a, NPt2ab) and FGF23 and FGF23 receptors were measured in the intestines, calvaria, kidney, and liver. RESULTS: Hens in the N1000 group had a 16% lower serum phosphorus concentration and 22% greater phosphorus excretion than those in the N20 or N140 group (P ≤ 0.05). Compared with hens in the N20 group, hens in the N140 and N1000 groups, which did not differ, had 15-21% lower serum FGF23 concentrations, 19-22% greater calcium excretion, 43-56% lower ileum NPT2b protein production, and 1.5- to 1.6-fold greater kidney NPT2a protein production, respectively (all differences at P ≤ 0.05). CONCLUSIONS: Supplementing high concentrations of nicotinamide in diets for laying hens led to accelerated phosphorus and calcium excretions and decreased serum phosphorus and FGF23 concentrations, which were associated with downregulated intestinal NPt2b protein production. Our findings exclude kidney NPt2a protein production as a primary mechanism for the nicotinamide-induced body phosphorus loss.


Assuntos
Galinhas , Regulação da Expressão Gênica/efeitos dos fármacos , Niacinamida/farmacologia , Fósforo/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo II/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Dieta/veterinária , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/fisiologia , Rim/efeitos dos fármacos , Rim/metabolismo , Niacinamida/administração & dosagem , Oviposição , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo II/genética
9.
Adv Exp Med Biol ; 1221: 807-819, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274739

RESUMO

Urofacial syndrome (UFS) is a rare but potentially devastating autosomal recessive disease. It comprises both incomplete urinary bladder emptying and a facial grimace upon smiling. A subset of individuals with the disease has biallelic mutations of HPSE2, coding for heparanase-2. Heparanase-2 and the classical heparanase are both detected in nerves in the maturing bladder, and mice mutant for Hpse2 have UFS-like bladder voiding defects and abnormally patterned bladder nerves. Other evidence suggests that the heparanase axis plays several roles in the peripheral and central nervous systems, quite apart from UFS-related biology. Some individuals with UFS lack HPSE2 mutations and instead carry biallelic variants of LRIG2, encoding leucine-rich-repeats and immunoglobulin-like-domains 2. Like heparanase-2, LRIG2 is detected in bladder nerves, and mutant Lrig2 mice have urination defects and abnormal patterns of bladder nerves. Further work is now needed to define the precise roles of heparanase-2 and LRIG2 in normal and abnormal neural differentiation.


Assuntos
Glucuronidase/metabolismo , Doenças Urológicas/enzimologia , Doenças Urológicas/genética , Animais , Facies , Humanos
10.
Artigo em Inglês | MEDLINE | ID: mdl-32334391

RESUMO

A rapid procedure for the determination of amphenicol antibiotics in human urine by liquid chromatography with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) is proposed. The presence of thiamphenicol (TAP), florfenicol (FF) and chloramphenicol (CAP) in the human body can be attributed to their administration to treat certain diseases or by eating food of animal origin. The TAP, FF and CAP excreted in urine is mainly in the form of glucuronide conjugates, although their free forms may also be excreted to a lesser extent. In the procedure described, the enzymatic hydrolysis of amphenicol glucuronide forms in urine was carried out using ß-glucuronidase and sulfatase at pH 5 (37 °C, overnight) in order to discriminate the free and conjugated forms. Then, amphenicol antibiotics were submitted to dispersive liquid-liquid microextraction (DLLME) for preconcentration. All the parameters affecting DLLME efficiency were optimized, and the following conditions were selected: 0.9 g NaCl in 10 mL of urine, to which 1.2 mL methanol (as dispersant solvent) and 1 mL of 4-methyl-2-pentanone (as extractant solvent) were added. The absence of a matrix effect allowed quantification of the samples against aqueous standards. Detection limits were 29, 6 and 3 pg mL-1 for TAP, FF and CAP, respectively. Relative standard deviations were calculated to evaluate the intra- and inter-day precision and values lower than 10% were obtained in all cases. The trueness of the method was tested through recovery studies, obtaining satisfactory values (83-104%). Ten urine samples obtained from volunteers were analysed and all of them were free of the studied antibiotics.


Assuntos
Antibacterianos/urina , Cloranfenicol/urina , Glucuronídeos/urina , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão , Glucuronidase/metabolismo , Humanos , Hidrólise , Limite de Detecção , Microextração em Fase Líquida , Metabolômica , Metanol/química , Metil n-Butil Cetona/química , Padrões de Referência , Solventes/química , Sulfatases/metabolismo , Tianfenicol/análogos & derivados , Tianfenicol/urina
11.
Adv Exp Med Biol ; 1221: 747-757, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274735

RESUMO

Homeostasis and visual acuity of the surface of the eye are dependent on tears, a thin film comprising at least 1800 different extracellular proteins and numerous species of lipids through which 80% of entering light is refracted at the air interface. Loss of homeostasis in dry eye disease affects 5-7% of the world's population, yet little is known about key molecular players. Our story began as an unbiased screen for regulators of tearing that led to the discovery of homeostasis-restorative 'lacritin', a tear protein whose active form is selectively deficient in dry eye. Heparanase acts as a novel 'on-switch' for lacritin ligation of syndecan-1 necessary to trigger basal tearing, as well as pertussis toxin-sensitive and FOXO-dependent signaling pathways for healing of inflammation-damaged epithelia and restoring epithelial oxidative phosphorylation by mitochondrial fusion downstream of transiently accelerated autophagy. A phase 2 clinical trial has tested the applicability of this mechanism to the resolution of dry eye disease. Results are not yet available. With lacritin proteoforms detected in cerebral spinal fluid, plasma, and urine, the capacity of the lacritin-syndecan-1-heparanase axis to restore homeostasis might have wide systemic relevance to other organs.


Assuntos
Síndromes do Olho Seco/metabolismo , Glucuronidase/metabolismo , Glicoproteínas/metabolismo , Sindecana-1/metabolismo , Síndromes do Olho Seco/terapia , Homeostase , Humanos , Lágrimas/metabolismo
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.
Am J Physiol Renal Physiol ; 318(5): F1188-F1198, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32249611

RESUMO

Caloric restriction (CR) is known to have multiple beneficial effects on health and longevity. To study the effect of CR on phosphorus metabolism and vascular calcification (VC), rats were fed normal or restricted calories (67% of normal). The phosphorus content of the diets was adjusted to provide equal phosphorus intake independent of the calories ingested. After 50 days of CR, rats had negative phosphorus balance, lower plasma phosphorus, glucose, triglycerides, and leptin, and higher adiponectin than rats fed normal calories. Uremia was induced by 5/6 nephrectomy (Nx). After Nx, rats were treated with calcitriol (80 ng/kg ip every other day) and high-phosphorus diets (1.2% and 1.8%). No differences in aortic calcium content were observed between rats that ate normal or restricted calories before Nx in either rats that received 1.2% phosphorus (11.5 ± 1.7 vs. 10.9 ± 2.1 mg/g tissue) or in rats that received 1.8% phosphorus (12.5 ± 2.3 vs. 12.0 ± 2.9 mg/g of tissue). However, mortality was significantly increased in rats subjected to CR before Nx in both the 1.2% phosphorus groups (75% vs. 25%, P = 0.019) and 1.8% phosphorus groups (100% vs. 45%, P < 0.001). After calcitriol administration was stopped and phosphorus intake was normalized, VC regressed rapidly, but no significant differences in aortic calcium were detected between rats that ate normal or restricted calories during the regression phase (5.7 ± 2.7 and 5.2 ± 1.5 mg/g tissue). In conclusion, CR did not prevent or ameliorate VC and increased mortality in uremic rats.


Assuntos
Aorta/metabolismo , Doenças da Aorta/prevenção & controle , Restrição Calórica , Rim/metabolismo , Fósforo na Dieta/metabolismo , Uremia/dietoterapia , Calcificação Vascular/prevenção & controle , Animais , Aorta/patologia , Doenças da Aorta/etiologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Calcitriol , Modelos Animais de Doenças , Progressão da Doença , Metabolismo Energético , Feminino , Fatores de Crescimento de Fibroblastos/sangue , Glucuronidase/metabolismo , Rim/patologia , Nefrectomia , Ratos Wistar , Fatores de Tempo , Uremia/etiologia , Uremia/metabolismo , Calcificação Vascular/etiologia , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia
14.
Adv Exp Med Biol ; 1221: 685-702, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274732

RESUMO

Recent years have brought about fledgling realization of the role played by heparanase in the pathogenesis of diverse diseases including kidney diseases and, specifically, acute kidney injury. Human heparanase-1 is critically and uniquely engaged in cleavage of heparan sulfate, an integral part of glycocalyx and extracellular matrix where it harbors distinct growth factors, cytokines, and other biologically active molecules. The enzyme is induced and activated in acute kidney injury regardless of its causes, ischemic, nephrotoxic, septic or transplantation-related. This event unleashes a host of sequelae characteristic of the pathogenesis of acute kidney injury, such as induction and reinforcement of innate immune responses, predisposition to thrombosis, activation of monocytes/macrophages and remodeling of the extracellular matrix, thus setting up the stage for future fibrotic complications and development of chronic kidney disease. We briefly discuss the emerging therapeutic strategies of inhibiting heparanase, as well as the diagnostic value of detecting products of heparanase activity for prognostication and treatment.


Assuntos
Lesão Renal Aguda/enzimologia , Glucuronidase/metabolismo , Lesão Renal Aguda/tratamento farmacológico , Citocinas , Glucuronidase/antagonistas & inibidores , Heparitina Sulfato , Humanos
15.
Adv Exp Med Biol ; 1221: 61-69, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274706

RESUMO

Heparanase was discovered during a study of the heparin proteoglycan (serglycin) in mast cells. Newly synthesized polysaccharide chains, kDa 60-100 x 103, were rapidly degraded to fragments similar in size to commercially available heparin (averaging 15 x 103). Analysis of the degradation products identified reducing-terminal glucuronic acid residues, shown by studies of heparin biosynthesis to be of ßD-configuration in the intact polymer. Heparanase, thus identified as an endo-ßD-glucuronidase, was subsequently identified in a variety of tissues and cells. The enzyme was subsequently implicated with a variety of pathophysiological processes, including in particular cancer, inflammatory diseases, and amyloidosis, as detailed in subsequent chapters of this volume. The target for enzyme action in these settings is primarily extracellular heparan sulfate proteoglycans; furthermore, intracellular cleavage initiates degradation of heparan sulfate chains by exolytic hydrolases and sulfatases, as part of normal turnover of the polysaccharide. More unexpectedly, heparanase also influences heparan sulfate biosynthesis, such that overexpression of the enzyme results in generation of highly sulfated, heparin-like oligosaccharides. The mechanism behind this effect remains unclear - along with the overall design of the molecular machinery in control of proteoglycan biosynthesis.


Assuntos
Glucuronidase/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Oligossacarídeos/metabolismo , Especificidade por Substrato
16.
Adv Exp Med Biol ; 1221: 71-96, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274707

RESUMO

Heparanase is an endo-ß-glucuronidase that cleaves at a limited number of internal sites the glycosaminoglycan heparan sulfate (HS). Heparanase enzymatic activity was first reported in 1975 and by 1983 evidence was beginning to emerge that the enzyme was a facilitator of tumor metastasis by cleaving HS chains present in blood vessel basement membranes and, thereby, aiding the passage of tumor cells through blood vessel walls. Due to a range of technical difficulties, it took another 16 years before heparanase was cloned and characterized in 1999 and a further 14 years before the crystal structure of the enzyme was solved. Despite these substantial deficiencies, there was steady progress in our understanding of heparanase long before the enzyme was fully characterized. For example, it was found as early as 1984 that activated T cells upregulate heparanase expression, like metastatic tumor cells, and the enzyme aids the entry of T cells and other leukocytes into inflammatory sites. Furthermore, it was discovered in 1989 that heparanase releases pre-existing growth factors and cytokines associated with HS in the extracellular matrix (ECM), the liberated growth factors/cytokines enhancing angiogenesis and wound healing. There were also the first hints that heparanase may have functions other than enzymatic activity, in 1995 it being reported that under certain conditions the enzyme could act as a cell adhesion molecule. Also, in the same year PI-88 (Muparfostat), the first heparanase inhibitor to reach and successfully complete a Phase III clinical trial was patented.Nevertheless, the cloning of heparanase (also known as heparanase-1) in 1999 gave the field an enormous boost and some surprises. The biggest surprise was that there is only one heparanase encoding gene in the mammalian genome, despite earlier research, based on substrate specificity, suggesting that there are at least three different heparanases. This surprising conclusion has remained unchanged for the last 20 years. It also became evident that heparanase is a family 79 glycoside hydrolase that is initially produced as a pro-enzyme that needs to be processed by proteases to form an enzymatically active heterodimer. A related molecule, heparanase-2, was also discovered that is enzymatically inactive but, remarkably, recently has been shown to inhibit heparanase-1 activity as well as acting as a tumor suppressor that counteracts many of the pro-tumor properties of heparanase-1.The early claim that heparanase plays a key role in tumor metastasis, angiogenesis and inflammation has been confirmed by many studies over the last 20 years. In fact, heparanase expression is enhanced in all major cancer types, namely carcinomas, sarcomas, and hematological malignancies, and correlates with increased metastasis and poor prognosis. Also, there is mounting evidence that heparanase plays a central role in the induction of inflammation-associated cancers. The enzymatic activity of heparanase has also emerged in unexpected situations, such as in the spread of HS-binding viruses and in Type-1 diabetes where the destruction of intracellular HS in pancreatic insulin-producing beta cells precipitates diabetes. But the most extraordinary recent discoveries have been with the realization that heparanase can exert a range of biological activities that are independent of its enzymatic function, most notably activation of several signaling pathways and being a transcription factor that controls methylation of histone tails. Collectively, these data indicate that heparanase is a truly multifunctional protein that has the additional property of cleaving HS chains and releasing from ECM and cell surfaces hundreds of HS-binding proteins with a plethora of functional consequences. Clearly, there are many unique features of this intriguing molecule that still remain to be explored and are highlighted in this Chapter.


Assuntos
Glucuronidase/história , Glucuronidase/metabolismo , Animais , Glucuronidase/genética , Heparitina Sulfato/metabolismo , História do Século XX , História do Século XXI , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/enzimologia , Neoplasias/patologia , Neovascularização Patológica
17.
Adv Exp Med Biol ; 1221: 139-167, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274709

RESUMO

The retaining endo-ß-D-glucuronidase Heparanase (HPSE) is the primary mammalian enzyme responsible for breakdown of the glycosaminoglycan heparan sulfate (HS). HPSE activity is essential for regulation and turnover of HS in the extracellular matrix, and its activity affects diverse processes such as inflammation, angiogenesis and cell migration. Aberrant heparanase activity is strongly linked to cancer metastasis, due to structural breakdown of extracellular HS networks and concomitant release of sequestered HS-binding growth factors. A full appreciation of HPSE activity in health and disease requires a structural understanding of the enzyme, and how it engages with its HS substrates. This chapter summarizes key findings from the recent crystal structures of human HPSE and its proenzyme. We present details regarding the 3-dimensional protein structure of HPSE and the molecular basis for its interaction with HS substrates of varying sulfation states. We also examine HPSE in a wider context against related ß-D-glucuronidases from other species, highlighting the structural features that control exo/endo - glycosidase selectivity in this family of enzymes.


Assuntos
Glucuronidase , Animais , Matriz Extracelular , Glucuronidase/química , Glucuronidase/metabolismo , Glicosaminoglicanos , Heparitina Sulfato , Humanos , Especificidade por Substrato
18.
Adv Exp Med Biol ; 1221: 169-188, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274710

RESUMO

Heparanase is the principal enzyme that degrades heparan sulfate (HS) in both physiological (HS turnover) and pathological (tumor metastasis, inflammation) cell conditions, catalysing the hydrolysis of the ß-1-4 glycosidic bond in -GlcUA-ß(1-4)-GlcNX-. Despite efforts to define the minimum trisaccharide sequence that allows glycans to be recognized by heparanase, a rigorous "molecular code" by which the enzyme reads and degrades HS chains has not been identified. The X-ray diffraction model of heparanase, resolved by Wu et al (2015), revealed a complex between the trisaccharide GlcNS6S-GlcUA-GlcNS6S and heparanase. Efforts are ongoing to better understand how HS mimetics longer than three residues are recognized by heparanase before being hydrolyzed or inhibit the enzyme. It is also important to consider the flexibility of the enzyme active site, a feature that opens up the development of heparanase inhibitors with structures significantly different from HS or heparin. This chapter reviews the state-of-the-art knowledge about structural aspects of heparanase activities in terms of substrate recognition, mechanism of hydrolysis, and inhibition.


Assuntos
Glucuronidase , Glicóis , Heparina , Heparitina Sulfato , Glucuronidase/antagonistas & inibidores , Glucuronidase/química , Glucuronidase/metabolismo , Glicóis/química , Glicóis/metabolismo , Heparina/química , Heparina/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Hidrólise , Especificidade por Substrato
19.
Adv Exp Med Biol ; 1221: 189-229, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274711

RESUMO

In 2019, we mark the 20th anniversary of the cloning of the human heparanase gene. Heparanase remains the only known enzyme to cleave heparan sulfate, which is an abundant component of the extracellular matrix. Thus, elucidating the mechanisms underlying heparanase expression and activity is critical to understanding its role in healthy and pathological settings. This chapter provides a historical account of the race to clone the human heparanase gene, describes the intracellular and extracellular function of the enzyme, and explores the various mechanisms regulating heparanase expression and activity at the gene, transcript, and protein level.


Assuntos
Clonagem Molecular , Glucuronidase/genética , Glucuronidase/metabolismo , Matriz Extracelular , Heparitina Sulfato , Humanos , Transcrição Genética
20.
Adv Exp Med Biol ; 1221: 253-283, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274713

RESUMO

Two decades following the cloning of the heparanase gene, the significance of this enzyme for tumor growth and metastasis cannot be ignored. Compelling pre-clinical and clinical evidence tie heparanase with all steps of tumor formation namely, initiation, growth, metastasis, and chemo resistance, thus confirming and significantly expanding earlier observations that coupled heparanase activity with the metastatic capacity of tumor cells. This collective effort has turned heparanase from an obscure enzyme to a valid target for the development of anti-cancer drugs, and led basic researchers and biotech companies to develop heparanase inhibitors as anti-cancer therapeutics, some of which are currently examined clinically. As expected, the intense research effort devoted to understanding the biology of heparanase significantly expanded the functional repertoire of this enzyme, but some principle questions are still left unanswered or are controversial. For example, many publications describe increased heparanase levels in human tumors, but the mechanism underlying heparanase induction is not sufficiently understood. Moreover, heparanase is hardly found to be increased in many studies utilizing methodologies (i.e., gene arrays) that compare tumors vs (adjacent) normal tissue. The finding that heparanase exert also enzymatic activity-independent function significantly expands the mode by which heparanase can function outside, but also inside the cell. Signaling aspects, and a role of heparanase in modulating autophagy are possibly as important as its enzymatic aspect, but these properties are not targeted by heparanase inhibitors, possibly compromising their efficacy. This Book chapter review heparanase function in oncology, suggesting a somewhat different interpretation of the results.


Assuntos
Glucuronidase/metabolismo , Neoplasias , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Glucuronidase/antagonistas & inibidores , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Transdução de Sinais
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