RESUMEN
The HS3ST1 gene controls endothelial cell production of HSAT+ - a form of heparan sulfate containing a specific pentasaccharide motif that binds the anticoagulant protein antithrombin (AT). HSAT+ has long been thought to act as an endogenous anticoagulant; however, coagulation was normal in Hs3st1-/- mice that have greatly reduced HSAT+ (HajMohammadi et al., 2003). This finding indicates that HSAT+ is not essential for AT's anticoagulant activity. To determine if HSAT+ is involved in AT's poorly understood inflammomodulatory activities, Hs3st1-/- and Hs3st1+/+ mice were subjected to a model of acute septic shock. Compared with Hs3st1+/+ mice, Hs3st1-/- mice were more susceptible to LPS-induced death due to an increased sensitivity to TNF. For Hs3st1+/+ mice, AT treatment reduced LPS-lethality, reduced leukocyte firm adhesion to endothelial cells, and dilated isolated coronary arterioles. Conversely, for Hs3st1-/- mice, AT induced the opposite effects. Thus, in the context of acute inflammation, HSAT+ selectively mediates AT's anti-inflammatory activity; in the absence of HSAT+, AT's pro-inflammatory effects predominate. To explore if the anti-inflammatory action of HSAT+ also protects against a chronic vascular-inflammatory disease, atherosclerosis, we conducted a human candidate-gene association study on >2000 coronary catheterization patients. Bioinformatic analysis of the HS3ST1 gene identified an intronic SNP, rs16881446, in a putative transcriptional regulatory region. The rs16881446G/G genotype independently associated with the severity of coronary artery disease and atherosclerotic cardiovascular events. In primary endothelial cells, the rs16881446G allele associated with reduced HS3ST1 expression. Together with the mouse data, this leads us to conclude that the HS3ST1 gene is required for AT's anti-inflammatory activity that appears to protect against acute and chronic inflammatory disorders.
Asunto(s)
Antitrombinas/fisiología , Aterosclerosis/genética , Enfermedades de las Arterias Carótidas/genética , Sulfotransferasas/genética , Animales , Antitrombinas/farmacología , Aterosclerosis/enzimología , Aterosclerosis/inmunología , Enfermedades de las Arterias Carótidas/enzimología , Enfermedades de las Arterias Carótidas/inmunología , Femenino , Estudios de Asociación Genética , Genotipo , Humanos , Inmunomodulación , Desequilibrio de Ligamiento , Lipopolisacáridos/farmacología , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Polimorfismo de Nucleótido Simple , Factor de Necrosis Tumoral alfa/fisiología , VasodilataciónRESUMEN
The exquisite control of growth factor function by heparan sulfate (HS) is dictated by tremendous structural heterogeneity of sulfated modifications. How specific HS structures control growth factor-dependent progenitor expansion during organogenesis is unknown. We isolated KIT+ progenitors from fetal salivary glands during a stage of rapid progenitor expansion and profiled HS biosynthetic enzyme expression. Enzymes generating a specific type of 3-O-sulfated-HS (3-O-HS) are enriched, and fibroblast growth factor 10 (FGF10)/FGF receptor 2b (FGFR2b) signaling directly regulates their expression. Bioengineered 3-O-HS binds FGFR2b and stabilizes FGF10/FGFR2b complexes in a receptor- and growth factor-specific manner. Rapid autocrine feedback increases 3-O-HS, KIT, and progenitor expansion. Knockdown of multiple Hs3st isoforms limits fetal progenitor expansion but is rescued with bioengineered 3-O-HS, which also increases adult progenitor expansion. Altering specific 3-O-sulfated epitopes provides a mechanism to rapidly respond to FGFR2b signaling and control progenitor expansion. 3-O-HS may expand KIT+ progenitors in vitro for regenerative therapy.
Asunto(s)
Factor 10 de Crecimiento de Fibroblastos/metabolismo , Regulación Enzimológica de la Expresión Génica , Heparitina Sulfato/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Células Madre/citología , Sulfotransferasas/metabolismo , Animales , Comunicación Autocrina , Western Blotting , Proliferación Celular , Feto/citología , Feto/metabolismo , Factor 10 de Crecimiento de Fibroblastos/genética , Técnica del Anticuerpo Fluorescente , Heparitina Sulfato/química , Inmunoprecipitación , Hibridación in Situ , Ratones , Técnicas de Cultivo de Órganos , Proteínas Proto-Oncogénicas c-kit/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándulas Salivales/citología , Glándulas Salivales/metabolismo , Células Madre/metabolismo , Sulfotransferasas/genéticaRESUMEN
Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Capilares/fisiología , Capilares/ultraestructura , Permeabilidad Capilar , Proteínas Portadoras/metabolismo , Endotelio Vascular/fisiología , Endotelio Vascular/ultraestructura , Proteínas de la Membrana/metabolismo , Animales , Proteínas Portadoras/genética , Caveolas/fisiología , Membrana Celular/metabolismo , Endotelio Vascular/citología , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Enteropatías Perdedoras de Proteínas/fisiopatologíaRESUMEN
The genes encoding the cytochrome P450 2C9 enzyme (CYP2C9) and vitamin K-epoxide reductase complex unit 1 (VKORC1) are major determinants of anticoagulant response to warfarin. Together with patient demographics and clinical information, they account for approximately one-half of the warfarin dose variance in individuals of European descent. Recent prospective and randomized controlled trial data support pharmacogenetic guidance with their use in warfarin dose initiation and titration. Benefits from pharmacogenetics-guided warfarin dosing have been reported to extend beyond the period of initial dosing, with supportive data indicating benefits to at least 3 months. The genetic effects of VKORC1 and CYP2C9 in African and Asian populations are concordant with those in individuals of European ancestry; however, frequency distribution of allelic variants can vary considerably between major populations. Future randomized controlled trials in multiethnic settings using population-specific dosing algorithms will allow us to further ascertain the generalizability and cost-effectiveness of pharmacogenetics-guided warfarin therapy. Additional genome-wide association studies may help us to improve and refine dosing algorithms and potentially identify novel biological pathways.
Asunto(s)
Anticoagulantes/efectos adversos , Hidrocarburo de Aril Hidroxilasas/genética , Oxigenasas de Función Mixta/genética , Warfarina/efectos adversos , Anticoagulantes/farmacología , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2C9 , Variación Genética , Genotipo , Humanos , Relación Normalizada Internacional , Oxigenasas de Función Mixta/metabolismo , Vitamina K Epóxido Reductasas , Warfarina/farmacologíaRESUMEN
BACKGROUND: Obesity is a growing worldwide problem with genetic and environmental causes, and it is an underlying basis for many diseases. Studies have shown that the toxicant-activated aryl hydrocarbon receptor (AHR) may disrupt fat metabolism and contribute to obesity. The AHR is a nuclear receptor/transcription factor that is best known for responding to environmental toxicant exposures to induce a battery of xenobiotic-metabolizing genes. OBJECTIVES: The intent of the work reported here was to test more directly the role of the AHR in obesity and fat metabolism in lieu of exogenous toxicants. METHODS: We used two congenic mouse models that differ at the Ahr gene and encode AHRs with a 10-fold difference in signaling activity. The two mouse strains were fed either a low-fat (regular) diet or a high-fat (Western) diet. RESULTS: The Western diet differentially affected body size, body fat:body mass ratios, liver size and liver metabolism, and liver mRNA and miRNA profiles. The regular diet had no significant differential effects. CONCLUSIONS: The results suggest that the AHR plays a large and broad role in obesity and associated complications, and importantly, may provide a simple and effective therapeutic strategy to combat obesity, heart disease, and other obesity-associated illnesses.
Asunto(s)
Grasas de la Dieta/metabolismo , Hígado/metabolismo , Obesidad/genética , Receptores de Hidrocarburo de Aril/genética , Tejido Adiposo/metabolismo , Animales , Peso Corporal , Dieta , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Modelos Animales , Obesidad/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Little is known of the environmental factors that initiate and promote disease. The aryl hydrocarbon receptor (AHR) is a key regulator of xenobiotic metabolism and plays a major role in gene/environment interactions. The AHR has also been demonstrated to carry out critical functions in development and disease. A qualitative investigation into the contribution by the AHR when stimulated to different levels of activity was undertaken to determine whether AHR-regulated gene/environment interactions are an underlying cause of cardiovascular disease. We used two congenic mouse models differing at the Ahr gene, which encodes AHRs with a 10-fold difference in signaling potencies. Benzo[a]pyrene (BaP), a pervasive environmental toxicant, atherogen, and potent agonist for the AHR, was used as the environmental agent for AHR activation. We tested the hypothesis that activation of the AHR of different signaling potencies by BaP would have differential effects on the physiology and pathology of the mouse cardiovascular system. We found that differential AHR signaling from an exposure to BaP caused lethality in mice with the low-affinity AHR, altered the growth rates of the body and several organs, induced atherosclerosis to a greater extent in mice with the high-affinity AHR, and had a huge impact on gene expression of the aorta. Our studies also demonstrated an endogenous role for AHR signaling in regulating heart size. We report a gene/environment interaction linking differential AHR signaling in the mouse to altered aorta gene expression profiles, changes in body and organ growth rates, and atherosclerosis.
Asunto(s)
Aterosclerosis/metabolismo , Benzo(a)pireno/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Longevidad/efectos de los fármacos , Miocardio/metabolismo , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Aorta/metabolismo , Apolipoproteínas E/genética , Peso Corporal , Crecimiento , Corazón/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Tamaño de los Órganos , Reacción en Cadena de la Polimerasa , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Heparan sulfate (HS) is a structurally complex polysaccharide that interacts with a broad spectrum of extracellular effector ligands and thereby is thought to regulate a diverse array of biologic processes. The specificity of HS-ligand interactions is determined by the arrangement of sulfate groups on HS, which creates distinct binding motifs. Biologically important HS motifs are expected to exhibit regulated expression, yet there is a profound lack of tools to identify such motifs; consequently, little is known of their structures and functions. We have identified a novel phage display-derived antibody (NS4F5) that recognizes a highly regulated HS motif (HS(NS4F5)), which we have rigorously identified as (GlcNS6S-IdoA2S)(3). HS(NS4F5) exhibits a restricted expression in healthy adult tissues. Blocking HS(NS4F5) on cells in culture resulted in reduced proliferation and enhanced sensitivity to apoptosis. HS(NS4F5) is up-regulated in tumor endothelial cells, consistent with a role in endothelial cell activation. Indeed, TNF-α stimulated endothelial expression of HS(NS4F5), which contributed to leukocyte adhesion. In a mouse model of severe systemic amyloid protein A amyloidosis, HS(NS4F5) was expressed within amyloid deposits, which were successfully detected by microSPECT imaging using NS4F5 as a molecularly targeted probe. Combined, our results demonstrate that NS4F5 is a powerful tool for elucidating the biological function of HS(NS4F5) and can be exploited as a probe to detect novel polysaccharide biomarkers of disease processes.
Asunto(s)
Amiloidosis/metabolismo , Anticuerpos Monoclonales/farmacología , Células Endoteliales/metabolismo , Heparitina Sulfato/metabolismo , Neoplasias/metabolismo , Anticuerpos de Cadena Única/farmacología , Proteínas Amiloidogénicas/inmunología , Proteínas Amiloidogénicas/metabolismo , Amiloidosis/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Biomarcadores/metabolismo , Células CHO , Secuencia de Carbohidratos , Proliferación Celular/efectos de los fármacos , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Células Endoteliales/inmunología , Femenino , Heparitina Sulfato/antagonistas & inhibidores , Heparitina Sulfato/inmunología , Humanos , Masculino , Ratones , Neoplasias/inmunología , Ratas , Ratas Wistar , Anticuerpos de Cadena Única/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Vascular endothelial cells (ECs) produce anticoagulant heparan sulfate (HSAT+)-a small subpopulation of heparan sulfate (HS) containing a specific pentasaccharide motif with high affinity for plasma antithrombin (AT). This pentasaccharide is responsible for the anticoagulant action of therapeutic heparin, which dramatically catalyzes AT neutralization of coagulation proteases. Consequently, HSAT+ has been designated as "anticoagulant HS," and has long been thought to convey antithrombotic properties to the blood vessel wall. The Hs3st1 gene encodes HS 3-O-sulfotransferase-1, whose rate limiting action regulates EC production of HSAT+. To elucidate the biologic role of HSAT+, we generated Hs3st1-/- knock-out mice that have undetectable EC HSAT+. Despite long held historic expectations, hemostasis was unaffected in Hs3st1-/- mice. In light of this surprising finding, herein we evaluate historic, biochemical, kinetic, physiologic, and molecular genetic studies of AT, heparin, and HSAT+. We find that a hemostatic role for HSAT+ cannot presently be excluded; however, HSAT+ may well not be essential for AT's anticoagulant function. Specifically, in the absence of glycosaminoglycans, physiologic levels of AT can neutralize coagulation proteases at a sufficiently high throughput to account for most of AT's anticoagulant function. Moreover, at the vessel wall surface, glycosaminoglycans distinct from HSAT+ may be the predominant catalysts of AT's anticoagulant activity. We then explore the possibility that HSAT+ regulates a less well known function of AT, anti-inflammatory activity. We find that Hs3st1-/- mice exhibit a strong proinflammatory phenotype that is unresponsive to AT's anti-inflammatory activity. We conclude that the predominant function of HSAT+ is to mediate AT's anti-inflammatory activity.
Asunto(s)
Anticoagulantes/metabolismo , Coagulación Sanguínea , Endotelio Vascular/citología , Heparitina Sulfato/metabolismo , Sulfotransferasas/fisiología , Animales , Endotelio Vascular/metabolismo , Ratones , Ratones NoqueadosRESUMEN
A high frequency ultrasound-coupled fluorescence tomography system, primarily designed for imaging of protoporphyrin IX production in skin tumors in vivo, is demonstrated for the first time. The design couples fiber-based spectral sampling of the protoporphyrin IX fluorescence emission with high frequency ultrasound imaging, allowing thin-layer fluorescence intensities to be quantified. The system measurements are obtained by serial illumination of four linear source locations, with parallel detection at each of five interspersed detection locations, providing 20 overlapping measures of subsurface fluorescence from both superficial and deep locations in the ultrasound field. Tissue layers are defined from the segmented ultrasound images and diffusion theory used to estimate the fluorescence in these layers. The system calibration is presented with simulation and phantom validation of the system in multilayer regions. Pilot in-vivo data are also presented, showing recovery of subcutaneous tumor tissue values of protoporphyrin IX in a subcutaneous U251 tumor, which has less fluorescence than the skin.
Asunto(s)
Protoporfirinas/análisis , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/metabolismo , Espectrometría de Fluorescencia/instrumentación , Ultrasonografía/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
There is an emerging interest in the extracellular matrix (ECM) of the lung, especially in the role it plays in development and disease. There is a rapid change from the classical view of the ECM as a supporting structure towards a view of the ECM as a regulatory entity with profound effects on proliferation, migration, and differentiation of pulmonary cells. In the ECM, a variety of molecules is present in a highly organized pattern. Next to the abundant fiber-forming molecules such as collagens and elastin, a large number of less abundant molecules are part of the ECM, including proteoglycans. In this review, we will focus on one class of proteoglycans, the heparan sulfate proteoglycans. We will particularly address the structure, biosynthesis, and function of their saccharide moiety, the heparan sulfates, including their role in development and (patho)physiology.
Asunto(s)
Heparitina Sulfato/química , Pulmón/anatomía & histología , Pulmón/química , Alveolos Pulmonares/anatomía & histología , Alveolos Pulmonares/química , Enfisema Pulmonar/metabolismo , Mucosa Respiratoria/anatomía & histología , Mucosa Respiratoria/química , Animales , Heparitina Sulfato/metabolismo , Heparitina Sulfato/fisiología , Humanos , Pulmón/metabolismo , Pulmón/fisiología , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/fisiología , Enfisema Pulmonar/patología , Enfisema Pulmonar/fisiopatología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/fisiologíaRESUMEN
Anticoagulant heparan sulfate proteoglycans bind and activate antithrombin by virtue of a specific 3-O-sulfated pentasaccharide. They not only occur in the vascular wall but also in extravascular tissues, such as the ovary, where their functions remain unknown. The rupture of the ovarian follicle at ovulation is one of the most striking examples of tissue remodeling in adult mammals. It involves tightly controlled inflammation, proteolysis, and fibrin deposition. We hypothesized that ovarian heparan sulfates may modulate these processes through interactions with effector proteins. Our previous work has shown that anticoagulant heparan sulfates are synthesized by rodent ovarian granulosa cells, and we now have set out to characterize heparan sulfates from human follicular fluid. Here we report the first anticoagulant heparan sulfate purified from a natural human extravascular source. Heparan sulfate chains were fractionated according to their affinity for antithrombin, and their structure was analyzed by 1H NMR and MS/MS. We find that human follicular fluid is a rich source of anticoagulant heparan sulfate, comprising 50.4% of total heparan sulfate. These antithrombin-binding chains contain more than 6% 3-O-sulfated glucosamine residues, convey an anticoagulant activity of 2.5 IU/ml to human follicular fluid, and have an anti-Factor Xa specific activity of 167 IU/mg. The heparan sulfate chains that do not bind antithrombin surprisingly exhibit an extremely high content in 3-O-sulfated glucosamine residues, which suggest that they may exhibit biological activities through interactions with other proteins.
Asunto(s)
Anticoagulantes/química , Heparitina Sulfato/química , Anticoagulantes/metabolismo , Cromatografía en Gel , Cromatografía por Intercambio Iónico/métodos , Femenino , Líquido Folicular/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Inflamación , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Modelos Biológicos , Modelos Químicos , Ovario/metabolismo , Azufre/química , Ésteres del Ácido Sulfúrico/químicaRESUMEN
The extracellular membrane surface contains a substantial amount of negatively charged sialic acid residues. Some of the sialic acids are located close to the pore of voltage-gated channel, substantially influencing their gating properties. However, the role of sialylation of the extracellular membrane in modulation of neuronal and network activity remains primarily unknown. The level of sialylation is controlled by neuraminidase (NEU), the key enzyme that cleaves sialic acids. Here we show that NEU treatment causes a large depolarizing shift of voltage-gated sodium channel activation/inactivation and action potential (AP) threshold without any change in the resting membrane potential of hippocampal CA3 pyramidal neurons. Cleavage of sialic acids by NEU also reduced sensitivity of sodium channel gating and AP threshold to extracellular calcium. At the network level, exogenous NEU exerted powerful anticonvulsive action both in vitro and in acute and chronic in vivo models of epilepsy. In contrast, a NEU blocker (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid) dramatically reduced seizure threshold and aggravated hippocampal seizures. Thus, sialylation appears to be a powerful mechanism to control neuronal and network excitability. We propose that decreasing the amount of extracellular sialic acid residues can be a useful approach to reduce neuronal excitability and serve as a novel therapeutic approach in the treatment of seizures.
Asunto(s)
Potenciales de Acción/fisiología , Líquido Extracelular/fisiología , Hipocampo/fisiología , Ácido N-Acetilneuramínico/fisiología , Red Nerviosa/fisiología , Neuronas/fisiología , Animales , Potenciales de la Membrana/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Within the nervous system, heparan sulfate (HS) of the cell surface and extracellular matrix influences developmental, physiologic and pathologic processes. HS is a functionally diverse polysaccharide that employs motifs of sulfate groups to selectively bind and modulate various effector proteins. Specific HS activities are modulated by 3-O-sulfated glucosamine residues, which are generated by a family of seven 3-O-sulfotransferases (3-OSTs). Most isoforms we herein designate as gD-type 3-OSTs because they generate HS(gD+), 3-O-sulfated motifs that bind the gD envelope protein of herpes simplex virus 1 (HSV-1) and thereby mediate viral cellular entry. Certain gD-type isoforms are anticipated to modulate neurobiologic events because a Drosophila gD-type 3-OST is essential for a conserved neurogenic signaling pathway regulated by Notch. Information about 3-OST isoforms expressed in the nervous system of mammals is incomplete. Here, we identify the 3-OST isoforms having properties compatible with their participation in neurobiologic events. We show that 3-OST-2 and 3-OST-4 are principal isoforms of brain. We find these are gD-type enzymes, as they produce products similar to a prototypical gD-type isoform, and they can modify HS to generate receptors for HSV-1 entry into cells. Therefore, 3-OST-2 and 3-OST-4 catalyze modifications similar or identical to those made by the Drosophila gD-type 3-OST that has a role in regulating Notch signaling. We also find that 3-OST-2 and 3-OST-4 are the predominant isoforms expressed in neurons of the trigeminal ganglion, and 3-OST-2/4-type 3-O-sulfated residues occur in this ganglion and in select brain regions. Thus, 3-OST-2 and 3-OST-4 are the major neural gD-type 3-OSTs, and so are prime candidates for participating in HS-dependent neurobiologic events.
Asunto(s)
Sistema Nervioso Central/enzimología , Sistema Nervioso Periférico/enzimología , Sulfotransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/enzimología , Encéfalo/metabolismo , Células CHO , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Cricetinae , Cricetulus , Femenino , Regulación Enzimológica de la Expresión Génica , Heparitina Sulfato/metabolismo , Herpesvirus Humano 1/fisiología , Humanos , Hibridación in Situ , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neuronas/enzimología , Neuronas/metabolismo , Sistema Nervioso Periférico/citología , Sistema Nervioso Periférico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad por Sustrato , Sulfotransferasas/genética , Internalización del VirusRESUMEN
Endothelial and other select cell types synthesize a subpopulation of heparan sulfate (HS) proteoglycans (HSPGs), anticoagulant HSPGs (aHSPGs) that bear aHS-HS chains with the cognate 3-O-sulfated pentasaccharide motif that can bind and activate anti-thrombin (AT). Endothelial cells regulate aHSPG production by limiting levels of HS 3-O-sulfotransferase-1 (3-OST-1), which modifies a non-limiting pool of aHS-precursors. By probing kidney cryosections with (125)I-AT and fluorescently tagged AT we found that the glomerular basement membrane contains aHSPGs, with the staining pattern implicating synthesis by glomerular epithelial cells (GECs). Indeed, cultured GECs synthesized aHS with high AT affinity that was comparable with the endothelial product. Disaccharide analyses of human GEC (hGEC) HS in conjunction with transcript analyses revealed that hGECs express predominantly 3-OST-1 and 3-OST-3(A). aHS production has not been previously examined in cells expressing multiple 3-OST isoforms. This unusual situation appears to involve novel mechanisms to regulate aHS production, as HS structural analyses suggest hGECs exhibit excess levels of 3-OST-1 and an extremely limiting pool of aHS-precursor. A limiting aHS-precursor pool may serve to minimize aHS synthesis by non-3-OST-1 isoforms. Indeed, we show that high in vitro levels of 3-OST-3(A) can efficiently generate aHS. Non-3-OST-1 isoforms can generate aHS in vivo, as the probing of kidney sections from 3-OST-1-deficient mice revealed GEC synthesis of aHSPGs. Surprisingly, Hs3st1(-/-) kidney only expresses 3-OST isoforms having a low specificity for aHS synthesis. Thus, our analyses reveal a cell type that expresses multiple 3-OST isoforms and produces minimal amounts of aHS-precursor. In part, this mechanism should prevent aHS overproduction by non-3-OST-1 isoforms. Such a role may be essential, as 3-OST isoforms that have a low specificity for aHS synthesis can generate substantial levels of aHSPGs in vivo.
Asunto(s)
Anticoagulantes/metabolismo , Células Epiteliales/enzimología , Proteoglicanos de Heparán Sulfato/biosíntesis , Glomérulos Renales/citología , Sulfotransferasas/metabolismo , Animales , Células Cultivadas , Células Epiteliales/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Isoenzimas/metabolismo , Glomérulos Renales/enzimología , Masculino , Ratones , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sulfotransferasas/genéticaRESUMEN
Heparan sulfate (HS) regulates the activity of various ligands and is involved in molecular recognition events on the cell surface and in the extracellular matrix. Specific binding of HS to different ligand proteins depends on the sulfation pattern of HS. For example, the interaction between antithrombin and a particular 3-O sulfated HS motif is thought to modulate blood coagulation. However, a recent study of mice defective for this modification suggested that 3-O sulfation plays other biological roles. Here, we show that Drosophila melanogaster HS 3-O sulfotransferase-b (Hs3st-B), which catalyzes HS 3-O sulfation, is a novel component of the Notch pathway. Reduction of Hs3st-B function by transgenic RNA interference compromised Notch signaling, producing neurogenic phenotypes. We also show that levels of Notch protein on the cell surface were markedly decreased by loss of Hs3st-B. These findings suggest that Hs3st-B is involved in Notch signaling by affecting stability or intracellular trafficking of Notch protein.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Heparitina Sulfato/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Sulfotransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases/genética , Membrana Celular/genética , Membrana Celular/metabolismo , ADN Complementario/análisis , ADN Complementario/genética , Regulación hacia Abajo/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/aislamiento & purificación , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Fenotipo , Unión Proteica/genética , Transporte de Proteínas/genética , Interferencia de ARN , Receptores Notch , Transducción de Señal/genética , Sulfotransferasas/genética , Sulfotransferasas/aislamiento & purificaciónRESUMEN
PURPOSE OF REVIEW: Neovascularization is a recognized feature of many valvular diseases and is established by numerous angiogenic modulators. Less known is that angiogenic modulators are multifunctional and have additional roles in valve development and disease. Recent advancements in this area are described. RECENT FINDINGS: Initiation of epithelial to mesenchymal transformation, a developmental induction that specifies primordial interstitial cells (mesenchymal cells), requires vascular endothelial growth factor A, which stimulates matrix metalloproteinase 2 production and the invasive migration of mesenchymal cells. Epithelial to mesenchymal transformation also requires the matrix component hyaluronan to facilitate signaling through ErbB2/ErbB3 receptors and then is terminated by an increase in vascular endothelial growth factor A expression. Fibroblast growth factor 4 has been implicated in stimulating the following stage of proliferative expansion. Subsequently, in the remodeling phase, heparin-binding epidermal growth factor-like growth factor limits mesenchymal cell proliferation by signaling through the EGFR/ErbB1 receptor. Many adult valvular lesions appear similar to the embryonic proliferative expansion phase as they exhibit accumulations of extracellular matrix and myofibroblasts (a mesenchyme-like interstitial cell). The origins of such lesions may involve transforming growth factor beta 1. Similar to epithelial to mesenchymal transformation, tumor growth factor beta1 can induce cultured valvular endothelial cells to transdifferentiate to a myofibroblast-like phenotype. This scenario may occur in carcinoid valve disease because serotonin can induce interstitial cell expression of tumor growth factor beta1. Additionally, prolonged tumor growth factor beta1 activity may predispose to calcific degeneration. Calcific leaflets also exhibit tenascin-C, which may facilitate inflammatory cell migration through upregulation of pro-matrix metalloproteinase 2. SUMMARY: Numerous angiogenic modulators control multiple stages of valvulogenesis and in the context of adult valvular disease may recapitulate their embryonic roles. Thus, lessons learned from valvulogenesis may provide insights into the molecular basis of adult valvular disease.
Asunto(s)
Enfermedades de las Válvulas Cardíacas/patología , Válvulas Cardíacas/crecimiento & desarrollo , Neovascularización Patológica , Transducción de Señal/fisiología , Adulto , Diferenciación Celular/fisiología , Endotelio Vascular/patología , Factor de Crecimiento Epidérmico/fisiología , Epitelio/embriología , Epitelio/fisiología , Factor 4 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/fisiología , Válvulas Cardíacas/embriología , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Ácido Hialurónico/fisiología , Péptidos y Proteínas de Señalización Intercelular , Mesodermo/citología , Mesodermo/fisiología , Fenotipo , Proteínas Proto-Oncogénicas/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Endothelial cell production of anticoagulant heparan sulfate (HS(act)) is controlled by the Hs3st1 gene, which encodes the rate-limiting enzyme heparan sulfate 3-O-sulfotransferase-1 (3-OST-1). In vitro, HS(act) dramatically enhances the neutralization of coagulation proteases by antithrombin. The in vivo role of HS(act) was evaluated by generating Hs3st1(-/-) knockout mice. Hs3st1(-/-) animals were devoid of 3-OST-1 enzyme activity in plasma and tissue extracts. Nulls showed dramatic reductions in tissue levels of HS(act) but maintained wild-type levels of tissue fibrin accumulation under both normoxic and hypoxic conditions. Given that vascular HS(act) predominantly occurs in the subendothelial matrix, mice were subjected to a carotid artery injury assay in which ferric chloride administration induces de-endothelialization and occlusive thrombosis. Hs3st1(-/-) and Hs3st1(+/+) mice yielded indistinguishable occlusion times and comparable levels of thrombin.antithrombin complexes. Thus, Hs3st1(-/-) mice did not show an obvious procoagulant phenotype. Instead, Hs3st1(-/-) mice exhibited genetic background-specific lethality and intrauterine growth retardation, without evidence of a gross coagulopathy. Our results demonstrate that the 3-OST-1 enzyme produces the majority of tissue HS(act). Surprisingly, this bulk of HS(act) is not essential for normal hemostasis in mice. Instead, 3-OST-1-deficient mice exhibited unanticipated phenotypes suggesting that HS(act) or additional 3-OST-1-derived structures may serve alternate biologic roles.
Asunto(s)
Anticoagulantes/farmacología , Heparitina Sulfato/farmacología , Animales , Antitrombinas/metabolismo , Arterias Carótidas/patología , Cruzamientos Genéticos , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Exones , Femenino , Genotipo , Hemostasis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Genéticos , Retroviridae/genética , Sulfotransferasas/metabolismo , Trombina/metabolismo , Distribución TisularRESUMEN
Binding of basic fibroblast growth factor (FGF2) to its high affinity receptors requires the presence of specific heparan sulfate (HS) moieties on the cell surface that act as coreceptors. To determine the contribution of cell-surface HS to modulation of FGF2-dependent cell growth, we studied the changes in the cell mass and FGF2 binding of endothelial cell HS under normoxic and hypoxic conditions in vitro. Both large vein and cardiac microvascular endothelial cells cultured under hypoxic conditions demonstrated an increase in the ratio of cell-surface HS to chondroitin sulfate (CS), as well as an increase in the number of low affinity (HS-associated) binding sites for FGF2 with no change in the apparent K(d). This increase in the number of HS-FGF2 binding sites, in the absence of a significant change in FGF receptor expression, resulted in enhanced responsiveness of hypoxic, compared with normoxic, endothelial cells to FGF2 stimulation. Gene expression studies demonstrated increased expression of the key regulatory enzyme responsible for HS chain synthesis, 1,4 GlcNAc transferase (GlcNAcT-I), as well as increased expression of 2-O sulfotransferase (HS2ST), the enzyme responsible for sulfation of IdoA, a crucial part of the HS-FGF2 binding site. Transduction of cells with an adenovirus encoding a HIF-1alpha expression construct resulted in a similar increase in GlcNAcT-I and HS2ST expression. We conclude that hypoxia increases endothelial cell responsiveness to FGF2 by promoting preferential synthesis of HS rather than CS chains and increasing the number of FGF2-binding sites on HS chains. Both of these events are mediated by a HIF-1alpha-dependent increase in expression of the enzymes GlnNAcT-I and HS2ST. This shift in cell-surface HS composition results in enhanced cell sensitivity to FGF2-induced growth stimulation.
Asunto(s)
Endotelio Vascular/enzimología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparitina Sulfato/biosíntesis , Hipoxia/enzimología , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Sulfatos de Condroitina/biosíntesis , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/crecimiento & desarrollo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Glicosaminoglicanos/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Heparan sulfate that contains antithrombin binding sites is designated as anticoagulant heparan sulfate (HS(act)) since, in vitro, it dramatically enhances the neutralization of coagulation proteases by antithrombin. Endothelial cell production of HS(act) is controlled by the Hs3st1 gene, which encodes the rate limiting enzyme-heparan sulfate 3-O-sulfotransferase-1 (Hs3st1). It has long been proposed that levels of endothelial HS(act) may tightly regulate hemostatic tone. This potential in vivo role of HS(act) was assessed by generating Hs3st1(-/-) knockout mice. Hs3st1(-/-) and Hs3st1(+/+) mice were evaluated with a variety of methods, capable of detecting altered hemostatic tone. However, both genotypes were indistinguishable. Instead, Hs3st1(-/-) mice exhibited lethality on a specific genetic background and also showed intrauterine growth retardation. Neither phenotypes result from a gross coagulopathy. So although this enzyme produces the majority of tissue HS(act), Hs3st1(-/-) mice do not show an obvious procoagulant phenotype. These results suggest that the bulk of HS(act) is not essential for normal hemostasis and that hemostatic tone is not tightly regulated by total levels of HS(act). Moreover, the unanticipated non-thrombotic phenotypes suggest structure(s) derived from this enzyme might serve additional/alternative biologic roles.