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1.
Matrix Biol ; 120: 60-75, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37201729

RESUMEN

Multiple myeloma is a hematological neoplasm derived from plasma cells invariably developing in the bone marrow (BM). The persisting clinical challenge in MM resides in its high ability to resist drugs as shown by the frequent relapses observed in patients regardless of the treatment applied. In a mouse model of MM, we identified a subpopulation of cells harboring increased resistance to current MM drugs. These cells bound a proliferation inducing ligand (APRIL), a key MM promoting/survival factor. APRIL binding involved the heparan sulfate (HS) chain present on syndecan-1 (SDC-1), and correlated with reactivity to the anti-HS antibody 10e4. 10e4+cells had a high proliferation activity, and were able to form colonies in 3-D cultures. 10e4+ cells were the only cells able to develop in BM after intravenous injection. They also resisted drugs in vivo, since their number increased after treatment in BM. Notably, 10e4+ cells differentiated into 10e4- cells upon in vitro and in vivo expansion. Expression of one sulfotransferase, HS3ST3a1, allowed modification of syndecan-1 to confer reactivity to 10e4 and binding to APRIL. HS3ST3a1 deletion inhibited tumorigenesis in BM. Notably, the two populations coexisted at a variable frequency in the BM of MM patients at diagnosis. In total, our results indicate that 3-O-sulfation on SDC-1 carried out by HS3ST3a1 defines aggressive MM cells, and that targeting of this enzyme could possibly be used to better control drug resistance.


Asunto(s)
Mieloma Múltiple , Sindecano-1 , Animales , Ratones , Médula Ósea/metabolismo , Heparitina Sulfato/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Sulfotransferasas/genética , Sindecano-1/genética , Sindecano-1/metabolismo
2.
Sci Rep ; 10(1): 19114, 2020 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-33154448

RESUMEN

Heparan sulfate (HS) chains, covalently linked to heparan sulfate proteoglycans (HSPG), promote synaptic development and functions by connecting various synaptic adhesion proteins (AP). HS binding to AP could vary according to modifications of HS chains by different sulfotransferases. 3-O-sulfotransferases (Hs3sts) produce rare 3-O-sulfated HSs (3S-HSs), of poorly known functions in the nervous system. Here, we showed that a peptide known to block herpes simplex virus by interfering with 3S-HSs in vitro and in vivo (i.e. G2 peptide), specifically inhibited neural activity, reduced evoked glutamate release, and impaired synaptic assembly in hippocampal cell cultures. A role for 3S-HSs in promoting synaptic assembly and neural activity is consistent with the synaptic interactome of G2 peptide, and with the detection of Hs3sts and their products in synapses of cultured neurons and in synaptosomes prepared from developing brains. Our study suggests that 3S-HSs acting as receptors for herpesviruses might be important regulators of neuronal and synaptic development in vertebrates.


Asunto(s)
Proteoglicanos de Heparán Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Hipocampo/metabolismo , Sulfotransferasas/metabolismo , Sinapsis/metabolismo , Animales , Células Cultivadas , Ratones , Neurogénesis/fisiología , Neuronas/metabolismo
3.
Biomed Res Int ; 2018: 9873471, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30228991

RESUMEN

Glycosaminoglycans are important for cell signaling and therefore for proper embryonic development and adult homeostasis. Expressions of genes involved in proteoglycan/glycosaminoglycan (GAG) metabolism and of genes coding for growth factors known to bind GAGs were analyzed during skin development by microarray analysis and real time quantitative PCR. GAG related genes were organized in six categories based on their role in GAG homeostasis, viz. (1) production of precursor molecules, (2) production of core proteins, (3) synthesis of the linkage region, (4) polymerization, (5) modification, and (6) degradation of the GAG chain. In all categories highly dynamic up- and downregulations were observed during skin development, including differential expression of GAG modifying isoenzymes, core proteins, and growth factors. In two mice models, one overexpressing heparanase and one lacking C5 epimerase, differential expression of only few genes was observed. Data show that during skin development a highly dynamic and complex expression of GAG-associated genes occurs. This likely reflects quantitative and qualitative changes in GAGs/proteoglycans, including structural fine tuning, which may be correlated with growth factor handling.


Asunto(s)
Regulación de la Expresión Génica , Glicosaminoglicanos/metabolismo , Proteoglicanos/metabolismo , Piel/crecimiento & desarrollo , Animales , Dermis , Femenino , Ratones
4.
PLoS One ; 13(9): e0201560, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30248108

RESUMEN

INTRODUCTION: Proliferative glomerulonephritis manifests in a range of renal diseases and is characterized by the influx of inflammatory cells into the glomerulus. Heparan sulfate (HS) is an important (co-)receptor for binding of chemokines, cytokines and leukocytes to the endothelial glycocalyx, a thick glycan layer that covers the inside of blood vessels. During glomerulonephritis, HS in the glomerular endothelial glycocalyx plays a central role in chemokine presentation and oligomerization, and in binding of selectins and integrins expressed by leukocytes. We hypothesize that distinct endothelial HS domains determine the binding of different chemokines. In this study we evaluated the interaction of three pro-inflammatory chemokines (CXCL1, CXCL2 and CCL2) with mouse glomerular endothelial cells (mGEnC-1) in ELISA in competition with different HS preparations and anti-HS single chain variable fragment (scFv) antibodies specific for distinct HS domains. RESULTS: HS appeared to be the primary ligand mediating chemokine binding to the glomerular endothelial glycocalyx in vitro. We found differential affinities of CXCL1, CXCL2 and CCL2 for HS in isolated mGEnC-1 glycocalyx, heparan sulfate from bovine kidney or low molecular weight heparin in competition ELISAs using mGEnC-1 as a substrate, indicating that chemokine binding is affected by the domain structure of the different HS preparations. Blocking of specific HS domains with anti-HS scFv antibodies revealed a domain-specific interaction of the tested chemokines to HS on mGEnC-1. Furthermore, chemokines did not compete for the same binding sites on mGEnC-1. CONCLUSION: CXCL1, CXCL2 and CCL2 binding to the glomerular endothelial glycocalyx appears differentially mediated by specific HS domains. Our findings may therefore contribute to the development of HS-based treatments for renal and possibly other inflammatory diseases specifically targeting chemokine-endothelial cell interactions.


Asunto(s)
Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Células Endoteliales/metabolismo , Glicocálix/metabolismo , Heparitina Sulfato/metabolismo , Glomérulos Renales/metabolismo , Animales , Bovinos , Línea Celular Transformada , Células Endoteliales/citología , Glomérulos Renales/citología , Ratones
5.
J Neurovirol ; 23(3): 483-491, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28326469

RESUMEN

The molecular mechanism of herpes simplex virus (HSV) entry and the associated inflammatory response in the nervous system remain poorly understood. Using mouse-derived ex vivo dorsal root ganglia (DRG) explant model and single cell neurons (SCNs), in this study, we provided a visual evidence for the expression of heparan sulfate (HS) and 3-O-sulfated heparan sulfate (3-OS HS) followed by their interactions with HSV-1 glycoprotein B (gB) and glycoprotein D (gD) during cell entry. Upon heparanase treatment of DRG-derived SCN, a significant inhibition of HSV-1 entry was observed suggesting the involvement of HS role during viral entry. Finally, a cytokine array profile generated during HSV-1 infection in DRG explant indicated an enhanced expression of chemokines (LIX, TIMP-2, and M-CSF)-known regulators of HS. Taken together, these results highlight the significance of HS during HSV-1 entry in DRG explant. Further investigation is needed to understand which isoforms of 3-O-sulfotransferase (3-OST)-generated HS contributed during HSV-1 infection and associated cell damage.


Asunto(s)
Ganglios Espinales/metabolismo , Heparitina Sulfato/metabolismo , Herpesvirus Humano 1/genética , Interacciones Huésped-Patógeno , Neuronas/metabolismo , Animales , Biomarcadores/metabolismo , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/virología , Expresión Génica , Glucuronidasa/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/crecimiento & desarrollo , Herpesvirus Humano 1/metabolismo , Hidrólisis , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Ratones , Neuronas/efectos de los fármacos , Neuronas/virología , Cultivo Primario de Células , Análisis de la Célula Individual/métodos , Técnicas de Cultivo de Tejidos , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus/efectos de los fármacos
6.
Neuromuscul Disord ; 26(6): 378-85, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27133661

RESUMEN

Sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) pumps play the major role in lowering cytoplasmic calcium concentration in skeletal muscle by catalyzing the ATP-dependent transport of Ca(2+) from the cytosol to the lumen of the sarcoplasmic reticulum (SR). Although SERCA abnormalities have been hypothesized to contribute to the dysregulation of intracellular Ca(2+) homeostasis and signaling in muscle of patients with myotonic dystrophy (DM) and hypothyroid myopathy, the characterization of SERCA pumps remains elusive and their impairment is still unclear. We assessed the activity of SR Ca(2+)-ATPase, expression levels and fiber distribution of SERCA1 and SERCA2, and oligomerization of SERCA1 protein in muscle of patients with DM type 1 and 2, and with hypothyroid myopathy. Our data provide evidence that SR Ca(2+) ATPase activity, protein levels and muscle fiber distribution of total SERCA1 and SERCA2, and SERCA1 oligomerization pattern are similar in patients with both DM1 and DM2, hypothyroid myopathy and in control subjects. We prove that SERCA1b, the neonatal isoform of SERCA1, is expressed at protein level in muscle of patients with DM2 and, in lower amount, of patients with DM1. Our present study demonstrates that SERCA function is not altered in muscle of patients with DM and with hypothyroid myopathy.


Asunto(s)
Hipotiroidismo/enzimología , Músculo Esquelético/enzimología , Distrofia Miotónica/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Adulto , Femenino , Humanos , Hipotiroidismo/patología , Isoenzimas , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Distrofia Miotónica/patología , Adulto Joven
7.
Gene Ther ; 23(2): 135-43, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26583803

RESUMEN

Although most high-risk neuroblastomas are responsive to chemotherapy, relapse is common and long-term survival is < 40%, underscoring the need for more effective treatments. We evaluated the responsiveness of 12 neuroblastoma cell lines to the Δγ134.5 attenuated oncolytic herpes simplex virus (oHSV), Seprehvir (HSV1716), which is currently used in pediatric phase I trials. We found that entry of Seprehvir in neuroblastoma cells is independent of the expression of nectin-1 and the sum of all four known major HSV entry receptors. We observed varying levels of sensitivity and permissivity to Seprehvir, suggesting that the cellular anti-viral response, not virus entry, is the key determinant of efficacy with this virus. In vivo, we found significant anti-tumor efficacy following Seprehvir treatment, which ranged from 6/10 complete responses in the CHP-134 model to a mild prolonged median survival in the SK-N-AS model. Taken together, these data suggest that anti-tumor efficacy cannot be solely predicted based on in vitro response. Whether or not this discordance holds true for other viruses or tumor types is unknown. Our results also suggest that profiling the expression of known viral entry receptors on neuroblastoma cells may not be entirely predictive of their susceptibility to Seprehvir therapy.


Asunto(s)
Antineoplásicos/uso terapéutico , Herpesvirus Humano 1 , Neuroblastoma/terapia , Viroterapia Oncolítica , Virus Oncolíticos , Receptores Virales/metabolismo , Internalización del Virus , Animales , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Ratones , Ratones Desnudos , Neuroblastoma/inmunología , Virus Oncolíticos/genética , Virus Oncolíticos/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
8.
J Tissue Eng Regen Med ; 10(1): E34-44, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23468399

RESUMEN

Autologous skin grafts are the gold standard for the treatment of burn wounds. In a number of cases, treatment with autologous tissue is not possible and skin substitutes are used. The outcome, however, is not optimal and improvements are needed. Inspired by scarless healing in early embryonic development, we here set out a strategy for the design and construction of embryonic-like scaffolds for skin tissue engineering. This strategy may serve as a general approach in the construction of embryonic-like scaffolds for other tissues/organ. As a first step, key effector molecules upregulated during embryonic and neonatal skin formation were identified using a comparative gene expressing analysis. A set of 20 effector molecules was identified, from which insulin-like growth factor 2 (IGF2) and sonic hedgehog (SHH) were selected for incorporation into a type I collagen-heparin scaffold. Porous scaffolds were constructed using purified collagen fibrils and 6% covalently bound heparin (to bind and protect the growth factors), and IGF2 and SHH were incorporated either individually (~0.7 and 0.4 µg/mg scaffolds) or in combination (combined ~1.5 µg/mg scaffolds). In addition, scaffolds containing hyaluronan (up to 20 µg/mg scaffold) were prepared, based on the up- or downregulation of genes involved in hyaluronan synthesis/degradation and its suggested role in scarless healing. In conclusion, based on a comprehensive gene expression analysis, a set of effector molecules and matrix molecules was identified and incorporated into porous scaffolds. The scaffolds thus prepared may create an 'embryonic-like' environment for cells to recapitulate embryonic events and for new tissues/organs.


Asunto(s)
Embrión de Mamíferos/citología , Piel/metabolismo , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Western Blotting , Bovinos , Colágeno/farmacología , Colágeno Tipo I/farmacología , Embrión de Mamíferos/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Heparina/farmacología , Ácido Hialurónico/farmacología , Inmunohistoquímica , Factor II del Crecimiento Similar a la Insulina/farmacología , Ratones Endogámicos C57BL
9.
Organogenesis ; 11(3): 105-21, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26060888

RESUMEN

A persistent clinical demand exists for a suitable arterial prosthesis. In this study, a vascular conduit mimicking the native 3-layered artery, and constructed from the extracellular matrix proteins type I collagen and elastin, was evaluated for its performance as a blood vessel equivalent. A tubular 3-layered graft (elastin-collagen-collagen) was prepared using highly purified type I collagen fibrils and elastin fibers, resembling the 3-layered native blood vessel architecture. The vascular graft was crosslinked and heparinised (37 ± 4 µg heparin/mg graft), and evaluated as a vascular graft using a porcine bilateral iliac artery model. An intra-animal comparison with clinically-used heparinised ePTFE (Propaten®) was made. Analyses included biochemical characterization, duplex scanning, (immuno)histochemistry and scanning electron microscopy. The tubular graft was easy to handle with adequate suturability. Implantation resulted in pulsating grafts without leakage. One week after implantation, both ePTFE and the natural acellular graft had 100% patencies on duplex scanning. Grafts were partially endothelialised (Von Willebrand-positive endothelium with a laminin-positive basal membrane layer). After one month, layered thrombi were found in the natural (4/4) and ePTFE graft (1/4), resulting in occlusion which in case of the natural graft is likely due to the porosity of the inner elastin layer. In vivo application of a molecularly-defined tubular graft, based on nature's matrix proteins, for vascular surgery is feasible.


Asunto(s)
Arteriopatías Oclusivas/fisiopatología , Prótesis Vascular/efectos adversos , Colágeno/química , Elastina/química , Arteria Ilíaca/fisiología , Grado de Desobstrucción Vascular/fisiología , Animales , Arteriopatías Oclusivas/etiología , Bioprótesis , Análisis de Falla de Equipo , Proteínas de la Matriz Extracelular/química , Femenino , Rechazo de Injerto , Arteria Ilíaca/cirugía , Diseño de Prótesis , Porcinos , Resultado del Tratamiento , Injerto Vascular/efectos adversos , Injerto Vascular/instrumentación
10.
Gene Ther ; 20(7): 761-9, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23254370

RESUMEN

Epstein-Barr virus (EBV)-associated B-cell lymphoproliferative disease (LPD) after hematopoietic stem cell or solid organ transplantation remains a life-threatening complication. Expression of the virus-encoded gene product, EBER, has been shown to prevent apoptosis via blockade of PKR activation. As PKR is a major cellular defense against Herpes simplex virus (HSV), and oncolytic HSV-1 (oHSV) mutants have shown promising antitumor efficacy in preclinical models, we sought to determine whether EBV-LPD cells are susceptible to infection by oHSVs. We tested three primary EBV-infected lymphocyte cell cultures from neuroblastoma (NB) patients as models of naturally acquired EBV-LPD. NB12 was the most susceptible, NB122R was intermediate and NB88R2 was essentially resistant. Despite EBER expression, PKR was activated by oHSV infection. Susceptibility to oHSV correlated with the expression of the HSV receptor, nectin-1. The resistance of NB88R2 was reversed by exogenous nectin-1 expression, whereas downregulation of nectin-1 on NB12 decreased viral entry. Xenografts derived from the EBV-LPDs exhibited only mild (NB12) or no (NB88R2) response to oHSV injection, compared with a NB cell line that showed a significant response. We conclude that EBV-LPDs are relatively resistant to oHSV virotherapy, in some cases, due to low virus receptor expression but also due to intact antiviral PKR signaling.


Asunto(s)
Herpesvirus Humano 1/genética , Herpesvirus Humano 4/genética , Trastornos Linfoproliferativos/genética , Virus Oncolíticos/genética , Apoptosis/genética , Moléculas de Adhesión Celular/metabolismo , ADN Viral/genética , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 4/inmunología , Humanos , Trastornos Linfoproliferativos/patología , Trastornos Linfoproliferativos/virología , Nectinas , Viroterapia Oncolítica , Cultivo Primario de Células , Receptores Virales/genética
11.
Neuromuscul Disord ; 22(11): 944-54, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22704959

RESUMEN

Brody disease is a rare inherited myopathy due to reduced sarcoplasmic reticulum Ca(2+) ATPase (SERCA)1 activity caused by mutations in ATP2A1, which causes delayed muscle relaxation and silent cramps. So far the disease has mostly been diagnosed by measurement of SERCA1 activity. Since mutation analysis became more widely available, it has appeared that not all patients with reduced SERCA1 activity indeed have ATP2A1 mutations, and a distinction between Brody disease (with ATP2A1 mutations) and Brody syndrome (without ATP2A1 mutations) was proposed. We aim to compare the clinical features of patients with Brody disease and those with Brody syndrome and detect clinical features which help to distinguish between the two. In addition, we describe the Brody syndrome phenotype in more detail. We therefore performed a literature review on clinical features of both Brody disease and Brody syndrome and a cross-sectional clinical study consisting of questionnaires, physical examination, and a review of medical files in 17 Brody syndrome patients in our centre. The results showed that Brody disease presents with an onset in the 1st decade, a generalized pattern of muscle stiffness, delayed muscle relaxation after repetitive contraction on physical examination, and autosomal recessive inheritance. Patients with Brody syndrome more often report myalgia and experience a considerable impact on daily life. Future research should focus on the possible mechanisms of reduction of SERCA activity in Brody syndrome and other genetic causes, and on evaluation of treatment options.


Asunto(s)
Mutación/genética , Miotonía Congénita/genética , Adolescente , Adulto , Anciano , Niño , Estudios Transversales , Análisis Mutacional de ADN/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miotonía Congénita/diagnóstico , Fenotipo , Literatura de Revisión como Asunto , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Encuestas y Cuestionarios , Adulto Joven
12.
Inflamm Bowel Dis ; 18(6): 1112-26, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21987406

RESUMEN

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are considered important in maintaining physiological homeostasis in many systems. Their expression is altered greatly in several pathophysiological conditions. Herein, we assess the expression and cellular localization of HSPGs in two murine models of human inflammatory bowel disease (IBD). METHODS: Expression and localization of HSPGs, syndecans, and HS epitopes were examined in the colon of 129SvEv interleukin 10 knockout (IL10(-/-)), C3Bir IL10(-/-), and their genetic control (IL10(+/+)) counterparts (129SvEv; C3H/HeJ). mRNA expression of syndecans and heparan sulfate biosynthesis enzymes were evaluated by real-time polymerase chain reaction (PCR). Localization of HSPGs was determined by immunofluorescence. RESULTS: mRNA for all syndecans was detected and expression in colonic tissues altered in IL10(-/-) mice. Syndecan-1 protein was expressed in the intestinal epithelium and on lamina propria cells of IL10(-/-) and control mice but was significantly reduced on the intestinal epithelial cells of IL10(-/-), mice particularly with severe colitis. Syndecan-2 was not detected, whereas syndecan-3 immunoreactivity was localized in the lamina propria but did not differ between control and IL10(-/-) mice. Syndecan-4 was present on epithelial cells of all mice but was significantly reduced in IL10(-/-) mice. Differences in the expression of HS epitopes between control and IL10(-/-) mice were also confirmed. CONCLUSIONS: The study has revealed altered expression of syndecan-1 and -4 and HS epitopes in the gut of mice with an IBD-like gut disorder. The IL10(-/-) mouse is a useful model for further study of the functional role of HSPGs in chronic inflammation and in maintaining healthy gut barrier.


Asunto(s)
Colitis/metabolismo , Modelos Animales de Enfermedad , Proteoglicanos de Heparán Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Interleucina-10/fisiología , Sindecanos/metabolismo , Animales , Western Blotting , Células Cultivadas , Colitis/etiología , Colitis/patología , Colon/metabolismo , Colon/patología , Femenino , Técnica del Anticuerpo Fluorescente , Proteoglicanos de Heparán Sulfato/genética , Heparitina Sulfato/genética , Humanos , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sindecano-1/genética , Sindecano-1/metabolismo , Sindecano-2/genética , Sindecano-2/metabolismo , Sindecano-3/genética , Sindecano-3/metabolismo , Sindecano-4/genética , Sindecano-4/metabolismo , Sindecanos/genética
13.
Eur J Vasc Endovasc Surg ; 43(1): 66-72, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22001149

RESUMEN

In recent years, ample attention has been directed towards the mechanisms that play a major role in the process of vascular graft failure, especially graft thrombosis and intimal narrowing have been highlighted. In this article, a survey is conducted into the key mechanisms of the biological processes of intimal hyperplasia and ultimate graft failure. The sequence of biochemical events that lead to thrombosis of grafts is used as a guideline to describe possible counteracting prosthetic surface interventions in each separate phase of the process.


Asunto(s)
Implantación de Prótesis Vascular/instrumentación , Prótesis Vascular , Materiales Biocompatibles Revestidos , Oclusión de Injerto Vascular/prevención & control , Trombosis/prevención & control , Grado de Desobstrucción Vascular , Animales , Implantación de Prótesis Vascular/efectos adversos , Oclusión de Injerto Vascular/etiología , Oclusión de Injerto Vascular/patología , Oclusión de Injerto Vascular/fisiopatología , Humanos , Hiperplasia , Diseño de Prótesis , Trombosis/etiología , Trombosis/patología , Trombosis/fisiopatología
14.
Diabetologia ; 54(6): 1527-38, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21424539

RESUMEN

AIMS/HYPOTHESIS: The content of heparan sulphate is reduced in the endothelium under hyperglycaemic conditions and may contribute to the pathogenesis of atherosclerosis. Heparanase-1 (HPR1) specifically degrades heparan sulphate proteoglycans. We therefore sought to determine whether: (1) heparan sulphate reduction in endothelial cells is due to increased HPR1 production through increased reactive oxygen species (ROS) production; and (2) HPR1 production is increased in vivo in endothelial cells under hyperglycaemic and/or atherosclerotic conditions. METHODS: HPR1 mRNA and protein levels in endothelial cells were analysed by RT-PCR and Western blot or HPR1 enzymatic activity assay, respectively. Cell surface heparan sulphate levels were analysed by FACS. HPR1 in the artery from control rats and a rat model of diabetes, and from patients under hyperglycaemic and/or atherosclerotic conditions was immunohistochemically examined. RESULTS: High-glucose-induced HPR1 production and heparan sulphate degradation in three human endothelial cell lines, both of which were blocked by ROS scavengers, glutathione and N-acetylcysteine. Exogenous H(2)O(2) induced HPR1 production, subsequently leading to decreased cell surface heparan sulphate levels. HPR1 content was significantly increased in endothelial cells in the arterial walls of a rat model of diabetes. Clinical studies revealed that HPR1 production was increased in endothelial cells under hyperglycaemic conditions, and in endothelial cells and macrophages in atherosclerotic lesions. CONCLUSIONS/INTERPRETATION: Hyperglycaemia induces HPR1 production and heparan sulphate degradation in endothelial cells through ROS. HPR1 production is increased in endothelial cells from a rat model of diabetes, and in macrophages in the atherosclerotic lesions of diabetic and non-diabetic patients. Increased HPR1 production may contribute to the pathogenesis and progression of atherosclerosis.


Asunto(s)
Aterosclerosis/etiología , Aterosclerosis/metabolismo , Endotelio Vascular/metabolismo , Glucuronidasa/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Hiperglucemia/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular , Diabetes Mellitus/metabolismo , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Femenino , Glucosa/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Masculino , Persona de Mediana Edad , Ratas , Ratas Sprague-Dawley , Estreptozocina/efectos adversos
15.
Acta Biomater ; 7(3): 1063-71, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20965285

RESUMEN

Skin substitutes are of great benefit in the treatment of patients with full thickness wounds, but there is a need for improvement with respect to wound closure with minimal contraction, early vascularisation, and elastin formation. In this study we designed and developed an acellular double-layered skin construct, using matrix molecules and growth factors to target specific biological processes. The epidermal layer was prepared using type I collagen, heparin and fibroblast growth factor 7 (FGF7), while the porous dermal layer was prepared using type I collagen, solubilised elastin, dermatan sulfate, heparin, fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF). The construct was biochemically and morphologically characterised and evaluated in vivo using a rat full thickness wound model. The results were compared with the commercial skin substitute IntegraDRT and untreated wounds. The double-layered construct was prepared according to the design specifications. The epidermal layer was about 40 µm thick, containing 9% heparin and 0.2 µg FGF7 mg per layer, localised at the periphery. The dermal layer was 2.5 mm thick, had rounded pores and contained 10% dermatan sulfate+heparin, and 0.7 µg FGF2+VEGF mg per layer. The double-layered skin construct was implanted in a skin defect and on day 7, 14, 28 and 112 the (remaining) wound area was photographed, excised and (immuno) histologically evaluated. The double-layered skin construct showed more cell influx, significantly less contraction and increased blood vessel formation at early time points in comparison with IntegraDRT and/or the untreated wound. On day 14 the double-layered skin construct also had the fewest myofibroblasts present. On day 112 the double-layered skin construct contained more elastic fibres than IntegraDRT and the untreated wound. Structures resembling hair follicles and sebaceous glands were found in the double-layered skin construct and the untreated wound, but hardly any were found in IntegraDRT. The results provide new opportunities for the application of acellular skin constructs in the treatment of surgical wounds.


Asunto(s)
Vasos Sanguíneos/crecimiento & desarrollo , Piel Artificial , Piel/crecimiento & desarrollo , Animales , Vasos Sanguíneos/metabolismo , Colágeno/metabolismo , Evaluación Preclínica de Medicamentos , Elastina/metabolismo , Masculino , Microscopía Electrónica de Rastreo , Ratas , Ratas Wistar , Piel/metabolismo
16.
Acta Biomater ; 6(12): 4666-74, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20619367

RESUMEN

There is a consistent need for a suitable natural biomaterial to function as an arterial prosthesis in achieving arterial regeneration. Natural grafts are generally obtained by decellularization of native blood vessels, but batch to batch variations may occur and the nature/content of remaining contaminants is generally unknown. In this study we fabricated a molecularly defined natural arterial graft from scratch resembling the native three layered architecture from the fibrillar extracellular matrix components collagen and elastin. Using casting, moulding, freezing and lyophilization techniques, a triple layered construct was prepared consisting of an inner layer of elastin fibres, a middle (porous) film layer of collagen fibrils and an outer scaffold layer of collagen fibrils. The construct was carbodiimide cross-linked and heparinized. Characterization included biochemical/biophysical analyses, scanning electron microscopy, micro-computed tomography, (immuno)histology and haemocompatibility. Burst pressures were up to 400mm Hg and largely conferred by the intermediate porous collagen film layer. The highly purified type I collagen fibrils and elastin fibres used did not evoke platelet aggregation in vitro. Suturability of the graft in end to side anastomosis was successful and considered adequate for in vivo application.


Asunto(s)
Prótesis Vascular , Vasos Sanguíneos/fisiología , Colágeno/química , Elastina/química , Ensayo de Materiales/métodos , Andamios del Tejido/química , Animales , Bovinos , Colágeno/ultraestructura , Elastina/ultraestructura , Caballos , Humanos , Inmunohistoquímica , Fenómenos Mecánicos , Microscopía Electrónica de Rastreo , Microtomografía por Rayos X
18.
Biomaterials ; 30(31): 6213-20, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19699517

RESUMEN

Large-scale in vivo evaluation of biomaterials is time-consuming and limited by ethical considerations. The availability of a library of biomaterials would allow a fast and rational in vitro selection of those biomaterials to be evaluated in vivo. For this reason, we developed an array of 48 different, molecularly-defined films based on native fibrillar collagen. The films differed in the type and amount of extracellular matrix components (type I/IV collagens, fibrous/solubilised elastin, glycosaminoglycans, heparin, chondroitin sulfate or dermatan sulfate), method of preparation (homogenisation) and method and extent of crosslinking (carbodiimide (EDC/NHS) or glutaraldehyde). The array was evaluated by studying morphology, proliferation and differentiation of primary human keratinocytes/fibroblasts. Major differences were observed. Only a small selection of films (especially those containing elastin fibres) specifically stimulated the proliferation of keratinocytes, but not fibroblasts. Such films may be the biomaterials of choice for in vivo evaluation for skin tissue engineering and regenerative medicine.


Asunto(s)
Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Colágenos Fibrilares/química , Piel/citología , Ingeniería de Tejidos/métodos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Inmunohistoquímica , Microscopía Electrónica de Rastreo
19.
Neuromuscul Disord ; 18(11): 843-56, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18818079

RESUMEN

This review presents an overview of myopathies and inherited connective tissue disorders that are caused by defects in or deficiencies of molecules within the extracellular matrix (ECM). We will cover the myopathies caused by defects in transmembrane protein complexes (dystroglycan, sarcoglycan, and integrins), laminin, and collagens (collagens VI, XIII, and XV). Clinical characteristics of several of these myopathies imply skin and joint features. We subsequently describe the inherited connective tissue disorders that are characterized by mild to moderate muscle involvement in addition to the dermal, vascular, or articular symptoms. These disorders are caused by defects of matrix-embedded ECM molecules that are also present within muscle (collagens I, III, V, IX, lysylhydroxylase, tenascin, fibrillin, fibulin, elastin, and perlecan). By focussing on the structure and function of these ECM molecules, we aim to point out the clinical and molecular overlap between the groups of disorders. We argue that clinicians and researchers dealing with myopathies and inherited connective tissue disorders should be aware of this overlap. Only a multi-disciplinary approach will allow full recognition of the wide variety of symptoms present in the spectrum of ECM defects, which has important implications for scientific research, diagnosis, and for the treatment of these disorders.


Asunto(s)
Enfermedades del Tejido Conjuntivo/metabolismo , Enfermedades del Tejido Conjuntivo/patología , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Animales , Enfermedades del Tejido Conjuntivo/genética , Diagnóstico Diferencial , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Proteínas Musculares/metabolismo , Debilidad Muscular/metabolismo , Debilidad Muscular/patología
20.
Pediatr Neurosurg ; 44(1): 29-35, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18097188

RESUMEN

BACKGROUND/PURPOSE: The aim of the study was to evaluate whether a collagen biomatrix is useful for delayed intrauterine coverage of a surgically created spina bifida in a fetal lamb. METHODS: In 20 fetal lambs, surgery was performed at 72 or 79 days' gestation. In 15 lambs a spina bifida was created surgically. In 8 lambs it was covered with a collagen biomatrix 2 weeks later and in 7 lambs it was left uncovered. Five lambs served as sham operated controls. Neurological examination was performed at 1 week of age and afterwards the lambs were sacrificed for further histological evaluation. RESULTS: None of the 5 surviving lambs with the defect covered showed loss of spinal function and the architecture of the spinal cord was preserved in 4 of the 5 lambs. In the uncovered group, 1 of the 4 surviving lambs had loss of spinal function, 5 lambs were available for histological evaluation and 4 of them showed disturbance of the architecture of the spinal cord. CONCLUSIONS: Collagen biomatrices can be used for intrauterine coverage of an experimental spina bifida and can preserve the architecture of the spinal cord. Neurological outcome is not different between fetuses with their spinal cord covered and fetuses with uncovered cords.


Asunto(s)
Colágeno Tipo I/administración & dosificación , Modelos Animales de Enfermedad , Atención Prenatal/métodos , Disrafia Espinal/cirugía , Animales , Femenino , Embarazo , Oveja Doméstica , Disrafia Espinal/patología , Factores de Tiempo
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