Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.825
Filtrar
1.
PLoS One ; 15(1): e0218494, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935212

RESUMO

Inhibiting vascular endothelial growth factor (VEGF) is a therapeutic option in diabetic microangiopathy. However, VEGF is needed at physiological concentrations to maintain glomerular integrity; complete VEGF blockade has deleterious effects on glomerular structure and function. Anti-VEGF therapy in diabetes raises the challenge of reducing VEGF-induced pathology without accelerating endothelial cell injury. Heparan sulfate (HS) act as a co-receptor for VEGF. Calcium dobesilate (CaD) is a small molecule with vasoprotective properties that has been used for the treatment of diabetic microangiopathy. Preliminary evidence suggests that CaD interferes with HS binding sites of fibroblast growth factor. We therefore tested the hypotheses that (1) CaD inhibits VEGF signaling in endothelial cells, (2) that this effect is mediated via interference between CaD and HS, and (3) that CaD ameliorates diabetic nephropathy in a streptozotocin-induced diabetic mouse model by VEGF inhibition. We found that CaD significantly inhibited VEGF165-induced endothelial cell migration, proliferation, and permeability. CaD significantly inhibited VEGF165-induced phosphorylation of VEGFR-2 and suppressed the activity of VEGFR-2 mediated signaling cascades. The effects of CaD in vitro were abrogated by heparin, suggesting the involvement of heparin-like domain in the interaction with CaD. In addition, VEGF121, an isoform which does not bind to heparin, was not inhibited by CaD. Using the proximity ligation approach, we detected inhibition of interaction in situ between HS and VEGF and between VEGF and VEGFR-2. Moreover, CaD reduced VEGF signaling in mice diabetic kidneys and ameliorated diabetic nephropathy and neuropathy, suggesting CaD as a VEGF inhibitor without the negative effects of complete VEGF blockade and therefore could be useful as a strategy in treating diabetic nephropathy.


Assuntos
Dobesilato de Cálcio/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Sítios de Ligação/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Células Endoteliais/efeitos dos fármacos , Heparitina Sulfato/metabolismo , Humanos , Cinética , Camundongos , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos NOD/crescimento & desenvolvimento , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
2.
J Surg Res ; 246: 274-283, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31614325

RESUMO

BACKGROUND: Fluid therapy influences glycocalyx shedding; however, the effect of this intervention on glycocalyx shedding in patients with glioma remains unclear. In this study, we have investigated glycocalyx shedding and cerebral metabolism during colloid loading in patients with and without glioma. METHODS: Forty patients undergoing general anesthesia were assigned to the glioma brain group (n = 20) or the normal brain group (n = 20); patients in the normal brain group were undergoing partial hepatectomy to treat liver cancer. All patients were subjected to 15 mL/kg hydroxyethyl starch (HES) loading after the induction of anesthesia. Glycocalyx shedding, reflected by syndecan-1 and heparan sulfate levels at the jugular venous bulb, was measured in both groups. We also evaluated cerebral metabolism parameters, including jugular venous oxygen saturation (SjvO2), arterial-jugular venous differences in oxygen (CajvO2), glucose (A-JvGD), lactate (A-JvLD), the cerebral extraction ratio for oxygen (CERO2), and the oxygen-glucose index. RESULTS: Our results showed that patients in the glioma brain group had lower preoperative basal syndecan-1 shedding in plasma than patients in the normal brain group. The hematocrit (Hct)-corrected syndecan-1 level was significantly increased after 15 mL/kg HES fluid administration (19.78 ± 3.83 ng/mL) compared with the Hct-correct baseline syndecan-1 level (15.67 ± 2.35 ng/mL) in patients in the glioma brain group. Similarly, for patients in the normal brain group, Hct-corrected syndecan-1 level was significantly increased after HES loading (34.71 ± 12.83 ng/mL) compared with the baseline syndecan-1 level (26.07 ± 12.52 ng/mL). However, there were no intergroup or intragroup differences in Hct-corrected heparan sulfate levels at any time point. Our study also showed that the SjvO2 was lower and CajvO2 and CERO2 were higher in the glioma brain group at 30 min after HES loading. Intragroup analysis showed that CERO2 and CajvO2 increased after general anesthesia compared with the baseline values in the glioma brain group. In contrast, cerebral metabolism in the normal brain group was unchanged during perioperative period. There were no significant differences in oxygen-glucose index between the two groups throughout the study period. CONCLUSIONS: Preoperative 15 mL/kg HES loading had similar effects on systemic glycocalyx shedding in both the glioma brain and normal brain groups, although patients in the normal brain group had higher levels of plasma syndecan-1. Furthermore, the intraoperative anesthetic management may substantially influence cerebral metabolism in patients with glioma.


Assuntos
Encéfalo/metabolismo , Hidratação/efeitos adversos , Glicocálix/efeitos dos fármacos , Derivados de Hidroxietil Amido/efeitos adversos , Cuidados Pré-Operatórios/efeitos adversos , Adulto , Encéfalo/efeitos dos fármacos , Encéfalo/cirurgia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/cirurgia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Hidratação/métodos , Glioma/metabolismo , Glioma/cirurgia , Glicocálix/metabolismo , Heparitina Sulfato/análise , Heparitina Sulfato/metabolismo , Humanos , Derivados de Hidroxietil Amido/administração & dosagem , Cuidados Intraoperatórios/efeitos adversos , Cuidados Intraoperatórios/métodos , Veias Jugulares/química , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Cuidados Pré-Operatórios/métodos , Estudos Prospectivos , Sindecana-1/sangue , Sindecana-1/metabolismo
3.
J Photochem Photobiol B ; 202: 111671, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731076

RESUMO

As a molecular chaperone, ß-casein is difficult to form amyloid fibrils under physiological conditions due to its chaperone activity. Heparan sulfate (HS) has drawn attention of technologists all over the word because of its relation to amyloid deposits in some amyloidosis diseases. For better understanding the relationship between the ß-casein and HS, the multi-spectroscopic studies were employed. The data of thioflavin T (ThT) binding assay, transmission electron microscopy (TEM) and circular dichroism (CD) demonstrated that HS promoted fibril formation by ß-casein in the amount and the growth speed. The results of steady-state UV-vis absorption spectra, fluorescence spectroscopy and fluorescence lifetime revealed that the ß-casein-HS complexes were formed and HS quenched the fluorescence of ß-casein by a static quenching mechanism. On the basis of fluorescence analysis, the value of binding constant was equal to 1.17 × 107 L mol-1 at 338.15 K and there was about one binding site between them. According to thermodynamic parameters obtained, it was deduced that a spontaneous reaction happened, and protein-ligand complex was stabilized by hydrogen bonds and hydrophobic interaction. Furthermore, using fluorescence resonance energy transfer (FRET) assay, the value of binding distance between HS and Trp143 of ß-casein was calculated to be 0.93 nm. Finally, on the basis of synchronous fluorescence experiment, the polarity increasing and hydrophobicity decreasing around Trp143 occurred during the period of fibril formation by ß-casein.


Assuntos
Amiloide/metabolismo , Caseínas/química , Heparitina Sulfato/química , Amiloide/química , Animais , Sítios de Ligação , Caseínas/metabolismo , Bovinos , Dicroísmo Circular , Transferência Ressonante de Energia de Fluorescência , Heparitina Sulfato/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Transmissão , Ligação Proteica , Termodinâmica
4.
Int J Mol Sci ; 20(20)2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614727

RESUMO

Vascular endothelial growth factor-A (VEGF) is critical for the development, growth, and survival of blood vessels. Retinal pigmented epithelial (RPE) cells are a major source of VEGF in the retina, with evidence that the extracellular matrix (ECM)-binding forms are particularly important. VEGF associates with fibronectin in the ECM to mediate distinct signals in endothelial cells that are required for full angiogenic activity. Hypoxia stimulates VEGF expression and angiogenesis; however, little is known about whether hypoxia also affects VEGF deposition within the ECM. Therefore, we investigated the role of hypoxia in modulating VEGF-ECM interactions using a primary retinal cell culture model. We found that retinal endothelial cell attachment to RPE cell layers was enhanced in cells maintained under hypoxic conditions. Furthermore, we found that agents that disrupt VEGF-fibronectin interactions inhibited endothelial cell attachment to RPE cells. We also found that hypoxia induced a general change in the chemical structure of the HS produced by the RPE cells, which correlated to changes in the deposition of VEGF in the ECM, and we further identified preferential binding of VEGFR2 over VEGFR1 to VEGF laden-fibronectin matrices. Collectively, these results indicate that hypoxia-induced HS may prime fibronectin for VEGF deposition and endothelial cell recruitment by promoting VEGF-VEGFR2 interactions as a potential means to control angiogenesis in the retina and other tissues.


Assuntos
Endotélio Vascular/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Heparitina Sulfato/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Adesão Celular , Hipóxia Celular , Linhagem Celular , Células Cultivadas , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Humanos , Oxigênio/metabolismo , Ratos , Epitélio Pigmentado da Retina/citologia
5.
Elife ; 82019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31436532

RESUMO

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.


Assuntos
Plaquetas/fisiologia , Heparitina Sulfato/metabolismo , Megacariócitos/fisiologia , Receptores Imunológicos/metabolismo , Animais , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Multimerização Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Transdução de Sinais
6.
Mar Drugs ; 17(6)2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31212795

RESUMO

Heparin or highly sulfated heparan sulfate (HS) has been described in different invertebrates. In ascidians (Chordata-Tunicata), these glycosaminoglycans occur in intracellular granules of oocyte accessory cells and circulating basophil-like cells, resembling mammalian mast cells and basophils, respectively. HS is also a component of the basement membrane of different ascidian organs. We have analyzed an HS isolated from the internal organs of the ascidian Phallusia nigra, using solution 1H/13C NMR spectroscopy, which allowed us to identify and quantify the monosaccharides found in this glycosaminoglycan. A variety of α-glucosamine units with distinct degrees of sulfation and N-acetylation were revealed. The hexuronic acid units occur both as α-iduronic acid and ß-glucuronic acid, with variable sulfation at the 2-position. A peculiar structural aspect of the tunicate HS is the high content of 2-sulfated ß-glucuronic acid, which accounts for one-third of the total hexuronic acid units. Another distinct aspect of this HS is the occurrence of high content of N-acetylated α-glucosamine units bearing a sulfate group at position 6. The unique ascidian HS is a potent inhibitor of the binding of human colon adenocarcinoma cells to immobilized P-selectin, being 11-fold more potent than mammalian heparin, but almost ineffective as an anticoagulant. Thus, the components of the HS structure required to inhibit coagulation and binding of tumor cells to P-selectin are distinct. Our results also suggest that the regulation of the pathway involved in the biosynthesis of glycosaminoglycans suffered variations during the evolution of chordates.


Assuntos
Adenocarcinoma/metabolismo , Anticoagulantes/metabolismo , Neoplasias do Colo/metabolismo , Glucuronatos/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Selectina-P/metabolismo , Urocordados/metabolismo , Animais , Anticoagulantes/química , Linhagem Celular Tumoral , Colo/metabolismo , Ácido Glucurônico/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Humanos
7.
Can J Physiol Pharmacol ; 97(8): 746-752, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31084580

RESUMO

The shear-stress sensor function of vascular glycocalyx heparan sulphate and hyaluronic acid was investigated in vivo by assessing flow-mediated dilation before and after their removal. Heparinase III exposure (100 mU·mL-1 for 20 min;n = 6) did not significantly affect flow-mediated dilation of the iliac, from 0.42 ± 0.08 mm (mean ± SEM) to 0.34 ± 0.07 mm after (P = 0.12; paired Student's t test) for a statistically similar increase in shear stress; 18.24 ± 4.2 N·m-2 for the control and 15.8 ± 3.6 N·m-2 for the heparinase III experiment (P = 0.18). Hyaluronidase exposure (0.14-1.4 mg·mL-1 for 20 min; n = 8) also did not significantly reduce flow-mediated dilation of the iliac, which averaged 0.39 ± 0.08 mm before and 0.38 ± 0.09 mm after (P = 0.11) for a statistically similar increase in shear stress; 11.90 ± 3.20 N·m-2 for the control and 9.8 ± 3.33 N·m-2 for the hyaluronidase experiment (P = 0.88). Removal of both heparan sulphate and hyaluronic acid was confirmed using immunohistochemistry. Neither the heparan sulphate nor the hyaluronic acid components of the glycocalyx mediate shear-stress-induced vasodilation in conduit arteries in vivo.


Assuntos
Glicocálix/metabolismo , Heparitina Sulfato/metabolismo , Ácido Hialurônico/metabolismo , Artéria Ilíaca/fisiologia , Vasodilatação , Anestesia , Animais , Suínos
8.
PLoS Pathog ; 15(5): e1007760, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31071193

RESUMO

Enterovirus A71 (EV-A71) is a non-polio neurotropic enterovirus with pandemic potential. There are no antiviral agents approved to prevent or treat EV-A71 infections. We here report on the molecular mechanism by which a novel class of tryptophan dendrimers inhibits (at low nanomolar to high picomolar concentration) EV-A71 replication in vitro. A lead compound in the series (MADAL385) prevents binding and internalization of the virus but does not, unlike classical capsid binders, stabilize the particle. By means of resistance selection, reverse genetics and cryo-EM, we map the binding region of MADAL385 to the 5-fold vertex of the viral capsid and demonstrate that a single molecule binds to each vertex. By interacting with this region, MADAL385 prevents the interaction of the virus with its cellular receptors PSGL1 and heparan sulfate, thereby blocking the attachment of EV-A71 to the host cells.


Assuntos
Antivirais/farmacologia , Capsídeo/metabolismo , Infecções por Enterovirus/metabolismo , Enterovirus/efeitos dos fármacos , Heparitina Sulfato/metabolismo , Glicoproteínas de Membrana/metabolismo , Triptofano/farmacologia , Antivirais/química , Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Dendrímeros/química , Dendrímeros/farmacologia , Infecções por Enterovirus/tratamento farmacológico , Infecções por Enterovirus/virologia , Células HeLa , Heparitina Sulfato/antagonistas & inibidores , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Conformação Proteica , Triptofano/química , Replicação Viral/efeitos dos fármacos
9.
Glycoconj J ; 36(2): 165-174, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30963354

RESUMO

Retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are major causes of blindness worldwide. Humans cannot regenerate retina, however, axolotl (Ambystoma mexicanum), a laboratory-bred salamander, can regenerate retinal tissue throughout adulthood. Classic signaling pathways, including fibroblast growth factor (FGF), are involved in axolotl regeneration. Glycosaminoglycan (GAG) interaction with FGF is required for signal transduction in this pathway. GAGs are anionic polysaccharides in extracellular matrix (ECM) that have been implicated in limb and lens regeneration of amphibians, however, GAGs have not been investigated in the context of retinal regeneration. GAG composition is characterized native and decellularized axolotl and porcine retina using liquid chromatography mass spectrometry. Pig was used as a mammalian vertebrate model without the ability to regenerate retina. Chondroitin sulfate (CS) was the main retinal GAG, followed by heparan sulfate (HS), hyaluronic acid, and keratan sulfate in both native and decellularized axolotl and porcine retina. Axolotl retina exhibited a distinctive GAG composition pattern in comparison with porcine retina, including a higher content of hyaluronic acid. In CS, higher levels of 4- and 6- O-sulfation were observed in axolotl retina. The HS composition was greater in decellularized tissues in both axolotl and porcine retina by 7.1% and 15.4%, respectively, and different sulfation patterns were detected in axolotl. Our findings suggest a distinctive GAG composition profile of the axolotl retina set foundation for role of GAGs in homeostatic and regenerative conditions of the axolotl retina and may further our understanding of retinal regenerative models.


Assuntos
Sulfatos de Condroitina/análise , Heparitina Sulfato/análise , Ácido Hialurônico/análise , Sulfato de Ceratano/análise , Retina/química , Ambystoma mexicanum , Animais , Sulfatos de Condroitina/metabolismo , Heparitina Sulfato/metabolismo , Ácido Hialurônico/metabolismo , Sulfato de Ceratano/metabolismo , Retina/metabolismo , Suínos
10.
Bioanalysis ; 11(8): 727-740, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30994022

RESUMO

Aim: Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by a deficiency of the iduronate-2-sulfatase enzyme leading to the accumulation of heparan sulfate (HS) and dermatan sulfate (DS) in organs and biological fluids. enzyme-replacement therapy is available for affected patients. Results/methodology: A 6-min UPLC-MS/MS method was developed/validated for HS and DS quantification in mouse tissues and biological fluids with high accuracy and precision. In MPS II mice, HS was more abundant than DS. 8-week enzyme-replacement therapy significantly reduced HS and DS levels in all matrices, except the brain. These reduced levels were maintained over a 16-week extended treatment period. Conclusion: The devised method is sensitive, robust and useful for the evaluation of biomarker distribution in MPS II mice.


Assuntos
Cromatografia Líquida/métodos , Dermatan Sulfato/metabolismo , Terapia de Reposição de Enzimas/métodos , Heparitina Sulfato/metabolismo , Mucopolissacaridose II/genética , Mucopolissacaridose II/metabolismo , Espectrometria de Massas em Tandem/métodos , Animais , Modelos Animais de Doenças , Humanos , Camundongos
11.
Biochimie ; 166: 173-183, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30981871

RESUMO

Formation of new blood vessels from preexisting ones, a process known as angiogenesis, is one of the limiting steps for success in treatment of ischemic disorders. Therefore, efforts to understanding and characterize new agents capable to stimulate neovascularization are a worldwide need. Crataeva tapia bark lectin (CrataBL) has been shown to have chemoattractant properties for endothelial cells through the stimulation of migration and invasiveness of human umbilical vein endothelial cells (HUVEC) because it is a positively charged protein with high affinity to glycosaminoglycan. In addition, CrataBL increased the production of chondroitin and heparan sulfate in endothelial cells. These findings orchestrated specific adhesion on collagen I and phosphorylation of tyrosine kinase receptors, represented by vascular endothelial growth factor receptor-2 (VEGFR-2) and fibroblast growth factor receptor (FGFR), whose downstream pathways trigger the angiogenic cascade increasing cell viability, cytoskeleton rearrangement, cell motility, and tube formation. Moreover, CrataBL inhibited the activity of matrix metalloproteases type 2 (MMP-2), a protein related to tissue remodeling. Likewise, CrataBL improved wound healing and increased the number of follicular structures in lesioned areas produced in the dorsum-cervical region of C57BL/6 mice. These outcomes altogether indicate that CrataBL is a pro-angiogenic and healing agent.


Assuntos
Indutores da Angiogênese/farmacologia , Condroitina/metabolismo , Heparitina Sulfato/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Animais , Capparaceae/metabolismo , Movimento Celular/efeitos dos fármacos , Fatores Quimiotáticos/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cicatrização/efeitos dos fármacos
12.
Mar Drugs ; 17(3)2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30818840

RESUMO

In tumor development, the degradation of heparan sulfate (HS) by heparanase (HPSE) is associated with cell-surface and extracellular matrix remodeling as well as the release of HS-bound signaling molecules, allowing cancer cell migration, invasion and angiogenesis. Because of their structural similarity with HS, sulfated polysaccharides are considered a promising source of molecules to control these activities. In this study, we used a depolymerisation method for producing λ-carrageenan oligosaccharides (λ-CO), with progressive desulfation over time. These were then used to investigate the influence of polymeric chain length and degree of sulfation (DS) on their anti-HPSE activity. The effects of these two features on λ-CO anticoagulant properties were also investigated to eliminate a potential limitation on the use of a candidate λ-CO as a chemotherapeutic agent. HPSE inhibition was mainly related to the DS of λ-CO, however this correlation was not complete. On the other hand, both chain length and DS modulated λ-CO activity for factor Xa and thrombin IIa inhibition, two enzymes that are involved in the coagulation cascade, and different mechanisms of inhibition were observed. A λ-carrageenan oligosaccharide of 5.9 KDa was identified as a suitable anticancer candidate because it displayed one of the lowest anticoagulant properties among the λ-CO produced, while showing a remarkable inhibitory effect on MDA-MB-231 breast cancer cell migration.


Assuntos
Anticoagulantes/farmacologia , Antineoplásicos/farmacologia , Carragenina/farmacologia , Glucuronidase/antagonistas & inibidores , Oligossacarídeos/farmacologia , Anticoagulantes/química , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Carragenina/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios Enzimáticos , Feminino , Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Oligossacarídeos/química
13.
mSphere ; 4(1)2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760613

RESUMO

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that can cause severe disease following in utero exposure, during primary infection, or latent virus reactivation in immunocompromised populations. These complications lead to a 1- to 2-billion-dollar economic burden, making vaccine development and/or alternative treatments a high priority. Current treatments for HCMV include nucleoside analogues such as ganciclovir (GCV), foscarnet, and cidofovir. Recently, letermovir, a terminase complex inhibitor, was approved for prophylaxis after stem cell transplantation. These treatments have unwanted side effects, and HCMV is becoming resistant to them. Therefore, we sought to develop an alternative treatment that targets a different stage in viral infection. Currently, small antiviral peptides are being investigated as anti-influenza and anti-HIV treatments. We have developed heparan sulfate-binding peptides as tools for preventing CMV infections. These peptides are highly effective at stopping infection of fibroblasts with in vitro-derived HCMV and murine cytomegalovirus (MCMV). However, they do not prevent MCMV infection in vivo Interestingly, these peptides inhibit infectivity of in vivo-derived CMVs, albeit not as well as tissue culture-grown CMVs. We further demonstrate that this class of heparan sulfate-binding peptides is incapable of inhibiting MCMV cell-to-cell spread, which is independent of heparan sulfate usage. These data indicate that inhibition of CMV infection can be achieved using synthetic polybasic peptides, but cell-to-cell spread and in vivo-grown CMVs require further investigation to design appropriate anti-CMV peptides.IMPORTANCE In the absence of an effective vaccine to prevent HCMV infections, alternative interventions must be developed. Prevention of viral entry into susceptible cells is an attractive alternative strategy. Here we report that heparan sulfate-binding peptides effectively inhibit entry into fibroblasts of in vitro-derived CMVs and partially inhibit in vivo-derived CMVs. This includes the inhibition of urine-derived HCMV (uCMV), which is highly resistant to antibody neutralization. While these antiviral peptides are highly effective at inhibiting cell-free virus, they do not inhibit MCMV cell-to-cell spread. This underscores the need to understand the mechanism of cell-to-cell spread and differences between in vivo-derived versus in vitro-derived CMV entry to effectively prevent CMV's spread.


Assuntos
Antivirais/farmacologia , Infecções por Citomegalovirus/prevenção & controle , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/fisiologia , Peptídeos/farmacologia , Animais , Células Cultivadas , Infecções por Citomegalovirus/tratamento farmacológico , Modelos Animais de Doenças , Fibroblastos/virologia , Heparitina Sulfato/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
14.
J Clin Invest ; 129(4): 1779-1784, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30720464

RESUMO

Septic patients frequently develop cognitive impairment that persists beyond hospital discharge. The impact of sepsis on electrophysiological and molecular determinants of learning is underexplored. We observed that mice that survived sepsis or endotoxemia experienced loss of hippocampal long-term potentiation (LTP), a brain-derived neurotrophic factor-mediated (BDNF-mediated) process responsible for spatial memory formation. Memory impairment occurred despite preserved hippocampal BDNF content and could be reversed by stimulation of BDNF signaling, suggesting the presence of a local BDNF inhibitor. Sepsis is associated with degradation of the endothelial glycocalyx, releasing heparan sulfate fragments (of sufficient size and sulfation to bind BDNF) into the circulation. Heparan sulfate fragments penetrated the hippocampal blood-brain barrier during sepsis and inhibited BDNF-mediated LTP. Glycoarray approaches demonstrated that the avidity of heparan sulfate for BDNF increased with sulfation at the 2-O position of iduronic acid and the N position of glucosamine. Circulating heparan sulfate in endotoxemic mice and septic humans was enriched in 2-O- and N-sulfated disaccharides; furthermore, the presence of these sulfation patterns in the plasma of septic patients at intensive care unit (ICU) admission predicted persistent cognitive impairment 14 days after ICU discharge or at hospital discharge. Our findings indicate that circulating 2-O- and N-sulfated heparan sulfate fragments contribute to septic cognitive impairment.


Assuntos
Disfunção Cognitiva/metabolismo , Heparitina Sulfato/metabolismo , Hipocampo/metabolismo , Transtornos da Memória/metabolismo , Sepse/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Disfunção Cognitiva/patologia , Feminino , Hipocampo/patologia , Potenciação de Longa Duração , Masculino , Transtornos da Memória/patologia , Camundongos , Sepse/patologia
15.
Vitam Horm ; 110: 157-188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30798810

RESUMO

Hepcidin is considered the major regulator of systemic iron homeostasis in human and mice, and its expression in the liver is mainly regulated at a transcriptional level. Central to its regulation are the bone morphogenetic proteins, particularly BMP6, that are heparin binding proteins. Heparin was found to inhibit hepcidin expression and BMP6 activity in hepatic cell lines and in mice, suggesting that endogenous heparan sulfates are involved in the pathway of hepcidin expression. This was confirmed by the study of cells and mice overexpressing heparanase, the enzyme that hydrolyzes heparan sulfates, and by cellular models with altered heparan sulfates. The evidences supporting the role of heparan sulfate in hepcidin expression are summarized in this chapter and open the way for new understanding in hepcidin expression and its control in pathological condition.


Assuntos
Glucuronidase/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Hepcidinas/metabolismo , Animais , Regulação da Expressão Gênica , Hepcidinas/genética , Humanos
16.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626687

RESUMO

Merkel cell polyomavirus (MCPyV) is a small, nonenveloped tumor virus associated with an aggressive form of skin cancer, Merkel cell carcinoma (MCC). MCPyV infections are highly prevalent in the human population, with MCPyV virions being continuously shed from human skin. However, the precise host cell tropism(s) of MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. However, MCPyV appears different from other polyomaviruses, as it requires sulfated polysaccharides, such as heparan sulfates and/or chondroitin sulfates, for initial attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co)receptor. To explore the infectious entry process of MCPyV, we analyzed the cell biological determinants of MCPyV entry into A549 cells, a highly transducible lung carcinoma cell line, in comparison to well-studied simian virus 40 and a number of other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis, or glycosphingolipid-enriched carriers. The viruses were internalized in small endocytic pits that led the virus to endosomes and from there to the endoplasmic reticulum (ER). Similar to other polyomaviruses, trafficking required microtubular transport, acidification of endosomes, and a functional redox environment. To our surprise, the virus was found to acquire a membrane envelope within endosomes, a phenomenon not reported for other viruses. Only minor amounts of viruses reached the ER, while the majority was retained in endosomal compartments, suggesting that endosome-to-ER trafficking is a bottleneck during infectious entry.IMPORTANCE MCPyV is the first polyomavirus directly implicated in the development of an aggressive human cancer, Merkel cell carcinoma (MCC). Although MCPyV is constantly shed from healthy skin, the MCC incidence increases among aging and immunocompromised individuals. To date, the events connecting initial MCPyV infection and subsequent transformation still remain elusive. MCPyV differs from other known polyomaviruses concerning its cell tropism, entry receptor requirements, and infection kinetics. In this study, we examined the cellular requirements for endocytic entry as well as the subcellular localization of incoming virus particles. A thorough understanding of the determinants of the infectious entry pathway and the specific biological niche will benefit prevention of virus-derived cancers such as MCC.


Assuntos
Poliomavírus das Células de Merkel/patogenicidade , Infecções por Polyomavirus/virologia , Células A549 , Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/virologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Fibroblastos/virologia , Células HEK293 , Células HeLa , Heparitina Sulfato/metabolismo , Humanos , Poliomavírus das Células de Merkel/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Pele/virologia , Neoplasias Cutâneas/virologia , Infecções Tumorais por Vírus/virologia , Tropismo Viral/fisiologia
17.
J Biol Chem ; 294(12): 4412-4424, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670588

RESUMO

Human interleukin-12 (hIL-12) is a heparin-binding cytokine whose activity was previously shown to be enhanced by heparin and other sulfated glycosaminoglycans. The current study investigated the mechanisms by which heparin increases hIL-12 activity. Using multiple human cell types, including natural killer cells, an IL-12 indicator cell line, and primary peripheral blood mononuclear and T cells, along with bioactivity, flow cytometry, and isothermal titration calorimetry assays, we found that heparin-dependent modulation of hIL-12 function correlates with several of heparin's biophysical characteristics, including chain length, sulfation level, and concentration. Specifically, only heparin molecules longer than eight saccharide units enhanced hIL-12 activity. Furthermore, heparin molecules with three sulfate groups per disaccharide unit outperformed heparin molecules with one or two sulfate groups per disaccharide unit in terms of enhanced hIL-12 binding and activity. Heparin also significantly reduced the EC50 value of hIL-12 by up to 11.8-fold, depending on the responding cell type. Cytokine-profiling analyses revealed that heparin affected the level, but not the type, of cytokines produced by lymphocytes in response to hIL-12. Interestingly, although murine IL-12 also binds heparin, heparin did not enhance its activity. Using the gathered data, we propose a model of hIL-12 stabilization in which heparin serves as a co-receptor enhancing the interaction between heterodimeric hIL-12 and its receptor subunits. The results of this study provide a foundation for further investigation of heparin's interactions with IL-12 family cytokines and for the use of heparin as an immunomodulatory agent.


Assuntos
Heparina/farmacologia , Interleucina-12/farmacologia , Animais , Fenômenos Biofísicos , Calorimetria , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Células HEK293 , Heparina/química , Heparitina Sulfato/metabolismo , Humanos , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
18.
PLoS One ; 14(1): e0207836, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30657762

RESUMO

Sanfilippo syndrome type B (Sanfilippo B; Mucopolysaccharidosis type IIIB) occurs due to genetic deficiency of lysosomal alpha-N-acetylglucosaminidase (NAGLU) and subsequent lysosomal accumulation of heparan sulfate (HS), which coincides with devastating neurodegenerative disease. Because NAGLU expressed in Chinese hamster ovary cells is not mannose-6-phosphorylated, we developed an insulin-like growth factor 2 (IGF2)-tagged NAGLU molecule (BMN 250; tralesinidase alfa) that binds avidly to the IGF2 / cation-independent mannose 6-phosphate receptor (CI-MPR) for glycosylation independent lysosomal targeting. BMN 250 is currently being developed as an investigational enzyme replacement therapy for Sanfilippo B. Here we distinguish two cellular uptake mechanisms by which BMN 250 is targeted to lysosomes. In normal rodent-derived neurons and astrocytes, the majority of BMN250 uptake over 24 hours reaches saturation, which can be competitively inhibited with IGF2, suggestive of CI-MPR-mediated uptake. Kuptake, defined as the concentration of enzyme at half-maximal uptake, is 5 nM and 3 nM in neurons and astrocytes, with a maximal uptake capacity (Vmax) corresponding to 764 nmol/hr/mg and 5380 nmol/hr/mg, respectively. Similar to neurons and astrocytes, BMN 250 uptake in Sanfilippo B patient fibroblasts is predominantly CI-MPR-mediated, resulting in augmentation of NAGLU activity with doses of enzyme that fall well below the Kuptake (5 nM), which are sufficient to prevent HS accumulation. In contrast, uptake of the untagged recombinant human NAGLU (rhNAGLU) enzyme in neurons, astrocytes and fibroblasts is negligible at the same doses tested. In microglia, receptor-independent uptake, defined as enzyme uptake resistant to competition with excess IGF2, results in appreciable lysosomal delivery of BMN 250 and rhNAGLU (Vmax = 12,336 nmol/hr/mg and 5469 nmol/hr/mg, respectively). These results suggest that while receptor-independent mechanisms exist for lysosomal targeting of rhNAGLU in microglia, BMN 250, by its IGF2 tag moiety, confers increased CI-MPR-mediated lysosomal targeting to neurons and astrocytes, two additional critical cell types of Sanfilippo B disease pathogenesis.


Assuntos
Acetilglucosaminidase/metabolismo , Endocitose , Fator de Crescimento Insulin-Like II/uso terapêutico , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/patologia , Proteínas Recombinantes de Fusão/uso terapêutico , Acetilglucosaminidase/farmacocinética , Acetilglucosaminidase/uso terapêutico , Animais , Astrócitos/metabolismo , Axônios/metabolismo , Cátions , Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Hipocampo/patologia , Humanos , Fator de Crescimento Insulin-Like II/farmacocinética , Lisossomos/enzimologia , Microglia/metabolismo , Ratos , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética
19.
Virology ; 529: 91-100, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30684694

RESUMO

Zika virus (ZIKV) is an emerging arbovirus and its infection associates with neurologic diseases. Whether heparan sulfate (HS), an attachment factor for many viruses, plays a role in the ZIKV infection remains controversial. Our study generated several HS biosynthesis-deficient cell clones by disrupting SLC35B2, B3GAT3, or B4GALT7 gene using the CRISPR/Cas9 system. The HS deficiency did not affect the viral attachment and internalization of ZIKV, but reduced the attachment of Dengue virus (DENV) 2. The early RNA and protein levels of ZIKV and DENV2 were impaired in the HS deficient cells, while the viral yields were not accordingly reduced. Our data further showed that HS promoted the cell death induced by virus infection, and inhibition of cell death significantly increased the viral replication of ZIKV and DENV2. Collectively, our study described an unexpected role of HS in the viral attachment, replication and cell death induced by ZIKV.


Assuntos
Morte Celular , Heparitina Sulfato/metabolismo , Internalização do Vírus , Replicação Viral/fisiologia , Zika virus/fisiologia , Animais , Linhagem Celular , Humanos , Interferon beta , Regulação para Cima , Zika virus/genética
20.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30602608

RESUMO

Porcine circovirus 2 (PCV2) is the smallest pathogenic virus capable of autonomous replication within its host. Infections result in immunosuppression and subsequent death of the host and are initiated via the attachment of the PCV2 icosahedral capsid to heparan sulfate (HS) and chondroitin sulfate B (CSB) glycosaminoglycans on the cell surface. However, the underlying mechanism of structural recognition remains to be explored. Using heparin, a routinely used analog of heparan sulfate, we demonstrate that increasing lengths of heparin exhibit a greater affinity toward PCV2. Our competition assays indicate that dextran sulfate (8 kDa) has a higher affinity for PCV2 than heparin (12 kDa), chondroitin sulfate B (41 kDa), hyaluronic acid (1.6 MDa), and dextran (6 kDa). This suggests that polymers high in sulfate content are capable of competing with the PCV2-heparan sulfate interaction and, thus, have the potential to inhibit PCV2 infection. Finally, we visualized the interaction between heparin and the PCV2 capsid using cryo-electron microscopy single-particle analysis, symmetry expansion, and focused classification. The image reconstructions provide the first example of an asymmetric distribution of heparin on the surface of an icosahedral virus capsid. We demonstrate that each of the 60 capsid subunits that generate the T=1 capsid can bind heparin via one of five binding sites. However, not all of the binding sites were occupied by heparin, and only one-third to two-thirds of the binding sites were occupied. The binding sites are defined by arginine, lysine, and polar amino acids. Mutating the arginine, lysine, and polar amino acids to alanine diminished the binding capacity of PCV2 to heparin.IMPORTANCE It has been demonstrated that porcine circovirus 2 (PCV2) attaches to cells via heparan sulfate (HS) and chondroitin sulfate B (CSB) glycosaminoglycans; however, the underlying structural mechanism describing the HS/CSB recognition by PCV2 remains to be explored. We used cryo-electron microscopy with single-particle analysis, symmetry expansion, and focused classification to visualize the interaction between the PCV2 capsid and heparin, an analog of heparan sulfate, to better than 3.6-Å resolution. We observed that the interaction between PCV2 and heparin does not adhere to the icosahedral symmetry of the capsid. To the best of our knowledge, this is the first example where the interaction between heparin and an icosahedral capsid does not follow the symmetry elements of the capsid. Our findings also suggest that anionic polymers, such as dextran sulfate, may act to inhibit PCV2 infection.


Assuntos
Sítios de Ligação/fisiologia , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Infecções por Circoviridae/metabolismo , Infecções por Circoviridae/virologia , Circovirus/patogenicidade , Heparitina Sulfato/metabolismo , Animais , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Suínos , Vírion/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA