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1.
Arch Toxicol ; 96(7): 2003-2019, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35357534

RESUMEN

Hepatic sinusoidal obstruction disease (HSOS) is a rare but life-threatening vascular liver disease. However, its underlying mechanism and molecular changes in HSOS are largely unknown, thus greatly hindering the development of its effective treatment. Hepatic sinusoidal endothelial cells (HSECs) are the primary and essential target for HSOS. A tandem mass tag-based shotgun proteomics study was performed using primary cultured HSECs from mice with HSOS induced by senecionine, a representative toxic pyrrolizidine alkaloid (PA). Dynamic changes in proteome were found at the initial period of damage and the essential role of thrombospondin 1 (TSP1) was highlighted in PA-induced HSOS. TSP1 over-expression was further confirmed in human HSECs and liver samples from patients with PA-induced HSOS. LSKL peptide, a known TSP1 inhibitor, protected mice from senecionine-induced HSOS. In addition, TSP1 was found to be covalently modified by dehydropyrrolizidine alkaloids in human HSECs and mouse livers upon senecionine treatment, thus to form the pyrrole-protein adduct. These findings provide useful information on early changes in HSECs upon PA treatment and uncover TSP1 overexpression as a contributor in PA-induced HSOS.


Asunto(s)
Enfermedad Veno-Oclusiva Hepática , Trombospondina 1 , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/biosíntesis , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Enfermedad Veno-Oclusiva Hepática/inducido químicamente , Enfermedad Veno-Oclusiva Hepática/metabolismo , Enfermedad Veno-Oclusiva Hepática/patología , Humanos , Ratones , Proteómica , Alcaloides de Pirrolicidina/toxicidad , Trombospondina 1/biosíntesis , Trombospondina 1/genética
2.
Biomed Res ; 42(1): 1-11, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33563874

RESUMEN

We examined the effects of mild hyperbaric oxygen (mHBO) exposure on capillary rarefaction in skeletal muscles of rats with diabetes. Streptozotocin (100 mg/kg) was administered to male Wistar rats via the tail vein to prepare a diabetic model. These rats were divided into 2 groups: the group with mHBO exposure (1.25 atmospheres absolute (ATA) with 36% oxygen; 3 h/day) and the group without mHBO exposure. Age-matched rats were used as the control group. Eight weeks later, the soleus of the rats was removed and then analyzed. With the onset of diabetes mellitus, capillary number, diameter, and volume in the soleus of the rats with diabetes decreased compared with those of the rats in the control group. In addition, increased anti-angiogenic thrombospondin-1 (TSP-1) and decreased pro-angiogenic murine double minute 2 (MDM-2) protein expressions were observed in the rats with diabetes. Alternatively, mHBO exposure attenuated the decrease in capillary diameter and volume in skeletal muscles of rats with diabetes, suppressed the overexpression of TSP-1, and restored the MDM-2 expression. These results indicate the exposure of mHBO partially attenuates capillary rarefaction in diabetic soleus muscle.


Asunto(s)
Capilares/efectos de los fármacos , Diabetes Mellitus Experimental/terapia , Oxigenoterapia Hiperbárica/métodos , Músculo Esquelético/patología , Inhibidores de la Angiogénesis , Animales , Peso Corporal , Modelos Animales de Enfermedad , Masculino , Oxígeno/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/biosíntesis , Ratas , Ratas Wistar , Estreptozocina , Trombospondina 1/biosíntesis
3.
J Neurochem ; 158(4): 849-864, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33118159

RESUMEN

Lysophosphatidic acid (LPA), a brain membrane-derived lipid mediator, plays important roles including neural development, function, and behavior. In the present study, the effects of LPA on astrocyte-derived synaptogenesis factor thrombospondins (TSPs) production were examined by real-time PCR and western blotting, and the mechanism underlying this event was examined by pharmacological approaches in primary cultured rat cortical astrocytes. Treatment of astrocytes with LPA increased TSP-1 mRNA, and TSP-2 mRNA, but not TSP-4 mRNA expression. TSP-1 protein expression and release were also increased by LPA. LPA-induced TSP-1 production were inhibited by AM966 a LPA1 receptor antagonist, and Ki16425, LPA1/3 receptors antagonist, but not by H2L5146303, LPA2 receptor antagonist. Pertussis toxin, Gi/o inhibitor, but not YM-254890, Gq inhibitor, and NF499, Gs inhibitor, inhibited LPA-induced TSP-1 production, indicating that LPA increases TSP-1 production through Gi/o-coupled LPA1 and LPA3 receptors. LPA treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). LPA-induced TSP-1 mRNA expression was inhibited by U0126, MAPK/ERK kinase (MEK) inhibitor, but not SB202190, p38 MAPK inhibitor, or SP600125, JNK inhibitor. However, LPA-induced TSP-1 protein expression was diminished with inhibition of all three MAPKs, indicating that these signaling molecules are involved in TSP-1 protein production. Treatment with antidepressants, which bind to astrocytic LPA1 receptors, increased TSP-1 mRNA and protein production. The current findings show that LPA/LPA1/3 receptors signaling increases TSP-1 production in astrocytes, which could be important in the pathogenesis of affective disorders and could potentially be a target for the treatment of affective disorders.


Asunto(s)
Astrocitos/metabolismo , Corteza Cerebral/metabolismo , Lisofosfolípidos/farmacología , Trombospondina 1/biosíntesis , Animales , Astrocitos/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Femenino , Proteínas Quinasas JNK Activadas por Mitógenos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Trastornos del Humor/tratamiento farmacológico , Trastornos del Humor/genética , Embarazo , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Trombospondinas/biosíntesis
4.
Am J Physiol Cell Physiol ; 319(1): C45-C63, 2020 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-32374675

RESUMEN

Numerous age-dependent alterations at the molecular, cellular, tissue and organ systems levels underlie the pathophysiology of aging. Herein, the focus is upon the secreted protein thrombospondin-1 (TSP1) as a promoter of aging and age-related diseases. TSP1 has several physiological functions in youth, including promoting neural synapse formation, mediating responses to ischemic and genotoxic stress, minimizing hemorrhage, limiting angiogenesis, and supporting wound healing. These acute functions of TSP1 generally require only transient expression of the protein. However, accumulating basic and clinical data reinforce the view that chronic diseases of aging are associated with accumulation of TSP1 in the extracellular matrix, which is a significant maladaptive contributor to the aging process. Identification of the relevant cell types that chronically produce and respond to TSP1 and the molecular mechanisms that mediate the resulting maladaptive responses could direct the development of therapeutic agents to delay or revert age-associated maladies.


Asunto(s)
Envejecimiento/genética , Envejecimiento/metabolismo , Trombospondina 1/biosíntesis , Trombospondina 1/genética , Animales , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/terapia , Daño del ADN/fisiología , Humanos , Enfermedades Musculoesqueléticas/genética , Enfermedades Musculoesqueléticas/metabolismo , Enfermedades Musculoesqueléticas/terapia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Transducción de Señal/fisiología , Trombospondina 1/antagonistas & inhibidores , Cicatrización de Heridas/fisiología
5.
Sci Rep ; 10(1): 1175, 2020 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-31980715

RESUMEN

Thrombospondin-1 (TSP-1) is a multifunctional matrix protein with antitumor activities due in part to its ability to inhibit angiogenesis, which in turn contributes to determine the fate of many tumours. Previous studies have shown that TSP-1 expression supports normal kidney angiostasis, and decreased TSP-1 levels contribute to the angiogenic phenotype of renal cell carcinomas (RCC). The loss of the von Hippel-Lindau tumour suppressor gene (VHL) in these tumours favours stabilization of the Hypoxia Inducible Factors (HIF), which in turn contribute to adapt tumour cells to hostile environments promoting tumour progression. However, HIF-independent regulation of certain genes might also be involved. We have previously shown that TSP-1 is regulated in hypoxia in clear cell RCC (ccRCC) in a HIF-independent manner; however, the effect of VHL protein (pVHL) on TSP-1 expression has not been evaluated. Our results proved that pVHL loss or mutation in its alpha or beta domain significantly decreased TSP-1 levels in ccRCC in a HIF-independent manner. Furthermore, this regulation proved to be important for ccRCC cells behaviour showing that decreased TSP-1 levels rendered ccRCC cells more migratory. This data substantiates a unique regulation pattern for TSP-1 in a pVHL-dependent manner, which may be relevant in the aggressiveness of ccRCC.


Asunto(s)
Carcinoma de Células Renales/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/patología , Proteínas de Neoplasias/fisiología , Trombospondina 1/biosíntesis , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiología , Línea Celular Tumoral , Movimiento Celular , Medio de Cultivo Libre de Suero , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Uniones Intercelulares/metabolismo , Mutación Missense , Invasividad Neoplásica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Dominios Proteicos/genética , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , ARN Interferente Pequeño/genética , Trombospondina 1/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/antagonistas & inhibidores , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética
6.
Physiol Res ; 68(6): 893-900, 2019 12 30.
Artículo en Inglés | MEDLINE | ID: mdl-31647293

RESUMEN

Thrombospondins (TSPs) are matricellular glycoproteins expressed in response to vascular injury. TSP-1 and TSP-2 are promotors of arterial remodeling while TSP-5 is believed to be protective. The current study assessed the differential effect of TSPs on protein expression in vascular smooth muscle cells (VSMCs). We hypothesized that TSP-1, TSP-2 and TSP-5 would regulate VSMC proteins involved in arterial remodeling. Human VSMCs were exposed to TSP-1, -2, -5 or serum free media (24 hours). Cell lysates were used to assess the targets TSP-1, TSP-2, TSP-5 and CD44), while the culture media was used to detect TGF-ß1, PDGF-BB, ANGPTL-4 and IL-8. Statistical analysis was performed by t-test and p< 0.05 was considered significant. All TSPs increased their own expression and TSP-5 increased TSP-2. TSP-1 and TSP-2 increased production of ANGPTL-4 and PDGF-BB, while TSP-5 only increased ANGPTL-4. TSP-1 increased exclusively TGF-ß1 and CD44 production. TSP-2 increased TSP-1 expression. All TSPs decreased IL-8. The findings suggest that TSP-1 and TSP-2 may promote vascular remodeling, in part, by increasing ANGPTL-4, PDGF-BB and their own expression. TSP-5 did not upregulate the inflammatory mediators TSP-1, PDGF-BB or TGF-ß1, but upregulated its own expression, which could be a protective mechanism against the response to vascular injury.


Asunto(s)
Arterias/metabolismo , Músculo Liso Vascular/metabolismo , Trombospondinas/biosíntesis , Remodelación Vascular/fisiología , Proteína de la Matriz Oligomérica del Cartílago/biosíntesis , Células Cultivadas , Humanos , Miocitos del Músculo Liso/metabolismo , Trombospondina 1/biosíntesis
7.
Neuron ; 103(4): 642-657.e7, 2019 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-31255486

RESUMEN

Neuronal subtypes show diverse injury responses, but the molecular underpinnings remain elusive. Using transgenic mice that allow reliable visualization of axonal fate, we demonstrate that intrinsically photosensitive retinal ganglion cells (ipRGCs) are both resilient to cell death and highly regenerative. Using RNA sequencing (RNA-seq), we show genes that are differentially expressed in ipRGCs and that associate with their survival and axon regeneration. Strikingly, thrombospondin-1 (Thbs1) ranked as the most differentially expressed gene, along with the well-documented injury-response genes Atf3 and Jun. THBS1 knockdown in RGCs eliminated axon regeneration. Conversely, RGC overexpression of THBS1 enhanced regeneration in both ipRGCs and non-ipRGCs, an effect that was dependent on syndecan-1, a known THBS1-binding protein. All structural domains of the THBS1 were not equally effective; the trimerization and C-terminal domains promoted regeneration, while the THBS type-1 repeats were dispensable. Our results identify cell-type-specific induction of Thbs1 as a novel gene conferring high regenerative capacity.


Asunto(s)
Regeneración Nerviosa/fisiología , Células Ganglionares de la Retina/fisiología , Trombospondina 1/fisiología , Animales , Apoptosis , Axones/metabolismo , Línea Celular , Femenino , Perfilación de la Expresión Génica , Genes Reporteros , Factor I del Crecimiento Similar a la Insulina/deficiencia , Factor I del Crecimiento Similar a la Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Compresión Nerviosa , Traumatismos del Nervio Óptico/genética , Traumatismos del Nervio Óptico/fisiopatología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Opsinas de Bastones/deficiencia , Opsinas de Bastones/fisiología , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/fisiología , Trombospondina 1/biosíntesis , Trombospondina 1/genética , Transcripción Genética
8.
Can J Cardiol ; 35(1): 42-50, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30595182

RESUMEN

BACKGROUND: Previous studies have shown that thrombospondin 1 (TSP-1) is involved in cardiovascular diseases, such as atherosclerosis and abdominal aortic aneurysm. However, TSP-1 expression levels in human aortic dissection (AD) remain unknown. METHODS: TSP-1 levels were detected in aortas collected from control subjects and AD patients. The TSP-1, interleukin (IL) 6, matrix metalloproteinase (MMP) 2, and MMP9 levels in plasma from non-AD patients and AD patients were measured. In addition, the effects of recombinant mouse TSP-1 protein on macrophage differentiation and smooth muscle cell (SMC) apoptosis were investigated. RESULTS: Compared with the aortas from control subjects, aortas from AD patients showed a significant increase in TSP-1 expression, especially in the torn sections. SMCs and endothelial cells produced TSP-1, but SMCs were the main source. TSP-1, IL-6, MMP2, and MMP9 levels were higher in AD patients than in non-AD patients, and plasma IL-6, MMP2, and MMP9 levels were positively correlated with TSP-1 levels in AD patients. Simple linear regression analysis and multivariate linear regression analysis showed that TSP-1 levels were independently correlated with the onset of AD. In cultured cells, recombinant mouse TSP-1 further increased inducible nitric oxide synthase (iNOS) mRNA expression in angiotensin (Ang) II-treated macrophages, whereas it reduced B-cell lymphoma-2 (Bcl2) mRNA levels and increased Bcl2-associated X protein (Bax) mRNA levels in Ang II-treated SMCs. CONCLUSIONS: TSP-1 level is significantly increased in AD patients and might participate in AD via promoting classically activated macrophage (M1) macrophage differentiation and SMC apoptosis.


Asunto(s)
Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/genética , Disección Aórtica/genética , Regulación de la Expresión Génica , Músculo Liso Vascular/metabolismo , ARN/genética , Trombospondina 1/genética , Enfermedad Aguda , Adulto , Disección Aórtica/metabolismo , Disección Aórtica/patología , Animales , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Western Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Músculo Liso Vascular/patología , Reacción en Cadena de la Polimerasa , Trombospondina 1/biosíntesis
9.
Biochim Biophys Acta Mol Basis Dis ; 1864(8): 2633-2643, 2018 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-29684582

RESUMEN

Platelet microparticle (PMP)-induced angiogenesis plays a key role in tumour metastasis and has been proposed to contribute towards cardiovascular disease by enhancing atherosclerotic plaque vulnerability. However, the mechanisms underlying PMP induced angiogenesis are ill defined. Recent reports demonstrate that PMPs deliver micro-RNAs (miRNAs) to recipient cells, controlling gene expression. We therefore evaluated whether miRNA transfer was a key regulator of PMP-induced angiogenesis. Co-culturing PMPs with human umbilical vein endothelial cells (HUVEC) on extracellular matrix gel induced robust capillary like structure formation. PMP treatment altered the release of angiogenesis modulators from HUVEC, including significantly reducing production of anti-angiogenic thrombospondin-1 (THBS-1). Both functional responses were abrogated by treating PMPs with RNase, suggesting the transfer of PMP-derived RNA was a critical event. PMPs were an abundant source of miRNA Let-7a, which was transferred to HUVEC following co-incubation. Using luciferase reporter assays we have shown that Let-7a directly targets the 3'UTR of the THBS-1 mRNA. HUVEC transfection with a Let-7a anti-sense oligonucleotide reduced the ability of PMPs to inhibit THBS-1 release, and significantly decreased PMP induced in vitro angiogenesis. Antibody neutralisation of THBS-1 reversed the anti-angiogenic effect of let-7a inhibition in PMP treated HUVEC, highlighting Let-7a dependent translational repression of THBS-1 drives angiogenesis. Importantly, plasmid overexpression of Let-7a in HUVEC alone induced robust tubule formation on extracellular matrix gel. These data reveal a new role for Let-7a in promoting angiogenesis and show for the first time PMPs induced angiogenic responses occur through miRNA regulation of HUVEC.


Asunto(s)
Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , MicroARNs/metabolismo , Neovascularización Fisiológica , Regiones no Traducidas 3' , Plaquetas/citología , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Biosíntesis de Proteínas , Trombospondina 1/biosíntesis
10.
J Thromb Haemost ; 16(4): 791-801, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29442415

RESUMEN

Essentials It is unclear if platelet micro-RNAs can regulate de novo protein synthesis of platelets. Platelet de novo protein synthesis of thrombospondin-1 (TSP-1) was induced by thrombin. Thrombin stimulation in vitro altered platelet microRNA profiles, including decreased miR-27b. Decreased miR-27b hampers platelet angiogenic activities via enhancing de novo TSP-1 synthesis. SUMMARY: Background Platelets can synthesize proteins upon activation. Platelets contain a number of microRNAs (miRNA) and a fully functional miRNA effector machinery. It is, however, unclear if platelet miRNAs can regulate protein synthesis of platelets, and whether the regulation may produce a physiological impact. Objectives To investigate if and how platelet miRNAs regulate de novo syntheses of angiogenic regulators and subsequently modulate platelet angiogenic activities. Methods and Results Microarray-based miRNA profiling showed that thrombin stimulation in vitro down- or up-regulated a number of platelet miRNAs, both in the total platelet miRNAs and in Ago2-associated miRNAs. Among those altered miRNAs, miR-27b was down-regulated in both the total and Ago2-immunoprecipitated miRNA profiles of platelets, which was confirmed by reverse transcription-quantitative PCR (RT-qPCR). Using western blotting assays, we showed that thrombin induced platelet de novo synthesis of thrombospondin-1, and that the level of thrombospondin-1 synthesis could reach a level of 3-5-fold higher than that before thrombin stimulation. With either the platelet precursor megakaryocyte cell line MEG-01 cells or mature platelets, we demonstrated that transfection of miR-27b mimic, but not the negative control of miRNA mimic, markedly reduced thrombospondin-1 protein levels. The latter subsequently enhanced platelet-dependent endothelial tube formation on matrigel. Conclusions Thrombin stimulation in vitro reduces platelet miR-27b levels that may markedly enhance thrombin-evoked platelet de novo synthesis of thrombospondin-1. Elevation of platelet miR-27b by transfection inhibits thrombospondin-1 synthesis, and subsequently enhances platelet pro-angiogenic activities. Hence, platelet activation-dependent reduction of miR-27b levels may represent a novel negative regulatory mechanism of platelet angiogenic activities.


Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Plaquetas/efectos de los fármacos , MicroARNs/sangre , Neovascularización Fisiológica/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Trombina/farmacología , Trombospondina 1/biosíntesis , Adulto , Proteínas Argonautas/sangre , Plaquetas/metabolismo , Línea Celular , Células Progenitoras Endoteliales/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Transducción de Señal , Trombospondina 1/sangre , Trombospondina 1/genética , Adulto Joven
11.
J Neurochem ; 143(6): 722-735, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-29049855

RESUMEN

Stroke is a multi-factorial polygenic disease and is a major cause of death and adult disability. Administration of bone marrow stem cells protects ischemic rat brain by facilitating recovery of neurological functions. But the molecular mechanism of stem cells action and their effect on gene expression is not well explored. In this study, we have transplanted 1 × 106 human bone marrow mesenchymal stem cells (hBMMSCs) in middle cerebral artery occluded (MCAo) adult male Wistar rats through intracarotid artery route at 24 h after surgery. Motor behavioral tests (rotarod and open field) were performed to assess the changes in motor functions at day 0 and day1, 4, 8 and 14. The expression of studied genes at mRNA and protein level was quantified by using Q-PCR and western blotting, respectively. Further, we have assessed the methylation pattern of promoter of these genes by using methylation-specific PCR. Data were analyzed statistically and correlated. A significant improvement in behavioral deficits was observed in stem cells treated group after 14th day post stroke. Significantly (p < 0.05) increased mRNA and protein levels of brain derived neurotrophic factor and ANP genes in hBMMSCs treated group along with decrease in methylation level at their promoter was observed. On the other hand, significantly decreased mRNA and protein level of TSP1 and WNK1 in hBMMSCs treated group was observed. In conclusion, hBMMSCs administration significantly improves the behavioral deficits by improving motor and locomotor coordination. The promoter of TSP1 and WNK1 genes was found to be hyper-methylated in hBMMSCs group resulting in their decreased expression while the promoter of ANP and brain derived neurotrophic factor was found to be hypo-methylated. This study might shed a light on how hBMMSCs affect the gene expression by modulating methylation status.


Asunto(s)
Trasplante de Médula Ósea/métodos , Metilación de ADN , Trasplante de Células Madre Mesenquimatosas/métodos , Accidente Cerebrovascular/terapia , Transcriptoma , Animales , Conducta Animal , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratas Wistar , Trombospondina 1/biosíntesis , Proteína Quinasa Deficiente en Lisina WNK 1/biosíntesis
12.
Epilepsia ; 58(11): 1993-2001, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28913875

RESUMEN

OBJECTIVES: Thrombospondins, which are known to interact with the α2 δ subunit of voltage-sensitive calcium channels to stimulate the formation of excitatory synapses, have recently been implicated in the process of epileptogenesis. No studies have been so far performed on thrombospondins in models of absence epilepsy. We examined whether expression of the gene encoding for thrombospondin-1 was altered in the brain of WAG/Rij rats, which model absence epilepsy in humans. In addition, we examined the frequency of genetic variants of THBS1 in a large cohort of children affected by idiopathic/genetic generalized epilepsies (IGE/GGEs). METHODS: We measured the transcripts of thrombospondin-1 and α2 δ subunit, and protein levels of α2 δ, Rab3A, and the vesicular glutamate transporter, VGLUT1, in the somatosensory cortex and ventrobasal thalamus of presymptomatic and symptomatic WAG/Rij rats and in two control strains by real-time polymerase chain reaction (PCR) and immunoblotting. We examined the genetic variants of THBS1 and CACNA2D1 in two independent cohorts of patients affected by IGE/GGE recruited through the Genetic Commission of the Italian League Against Epilepsy (LICE) and the EuroEPINOMICS-CoGIE Consortium. RESULTS: Thrombospondin-1 messenger RNA (mRNA) levels were largely reduced in the ventrobasal thalamus of both presymptomatic and symptomatic WAG/Rij rats, whereas levels in the somatosensory cortex were unchanged. VGLUT1 protein levels were also reduced in the ventrobasal thalamus of WAG/Rij rats. Genetic variants of THBS1 were significantly more frequent in patients affected by IGE/GGE than in nonepileptic controls, whereas the frequency of CACNA2D1 was unchanged. SIGNIFICANCE: These findings suggest that thrombospondin-1 may have a role in the pathogenesis of IGE/GGEs.


Asunto(s)
Canales de Calcio/genética , Modelos Animales de Enfermedad , Epilepsia Tipo Ausencia/genética , Epilepsia Generalizada/genética , Trombospondina 1/genética , Animales , Canales de Calcio/biosíntesis , Estudios de Cohortes , Epilepsia Tipo Ausencia/metabolismo , Epilepsia Generalizada/metabolismo , Humanos , Masculino , Ratas , Ratas Wistar , Trombospondina 1/biosíntesis
13.
Cell Tissue Res ; 370(3): 441-449, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-28856432

RESUMEN

Kruppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays crucial roles during the development and maintenance of multiple organs. We and others have previously shown that KLF4 is involved in bone modeling and remodeling but roles played by KLF4 during skeletogenesis are still not fully understood. Here, we show that KLF4 is expressed in the epiphyseal growth plate and articular chondrocytes. Most articular chondrocytes expressed KLF4 in embryos but it localized only in a subset of superficial zone cells in postnatal mice. When KLF4 was overexpressed in chondrocytes in vitro, it severely repressed chondrocytic gene expressions. Global gene expression profiling of KLF4-transduced chondrocytes revealed matrix degrading proteinases of the matrix metalloproteinase and disintegrin and metalloproteinase with thrombospondin-1 domain families within the group of upregulated genes. Proteinase induction by KLF4 was alleviated by Trichostatin A treatment suggesting the possible involvement of epigenetic mechanisms on proteinase induction by KLF4. These results indicate the possible involvement of KLF4 in physiological and pathological aspects during cartilage development and maintenance.


Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Endopeptidasas/biosíntesis , Factores de Transcripción de Tipo Kruppel/metabolismo , Metaloproteinasas de la Matriz/biosíntesis , Trombospondina 1/biosíntesis , Animales , Células Cultivadas , Endopeptidasas/genética , Regulación del Desarrollo de la Expresión Génica , Ácidos Hidroxámicos/farmacología , Factor 4 Similar a Kruppel , Masculino , Metaloproteinasas de la Matriz/genética , Ratones , Ratones Endogámicos ICR , Inhibidores de la Síntesis de la Proteína/farmacología , Trombospondina 1/genética
14.
Transplantation ; 101(8): 1811-1819, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28737660

RESUMEN

BACKGROUND: Angiogenesis contributes to the repair process after renal ischemia/reperfusion (I/R) injury. In the present study, we tested the role of miR-21 in the angiogenesis induced by hypoxia inducible factor (HIF)-1α through inhibiting a predicted target gene thrombospondin 1 (TSP-1). METHODS: To stabilize HIF-1α, hypoxia (1% O2 for 24 hours) was performed in human umbilical vein endothelial cells and cobalt chloride (CoCl2) was pretreated intraperitoneally 24 hours before renal I/R in mice. Locked nucleic acid modified anti-miR-21 and scrambled control was transfected with hypoxic cells or delivered into the mice via tail vein 1 hour before CoCl2 injection. The kidneys and blood were collected at 24 hours after reperfusion. RESULTS: HIF-1α induced by hypoxia and CoCl2 upregulated vascular endothelial growth factor and miR-21, and increased angiogenesis. It was found that expression of TSP-1 was inversely related with miR-21 in vitro and in vivo. Targeting of TSP-1 by miR-21 was further confirmed in vitro. Furthermore, HIF-1α improved renal function, accompanied with increased angiogenesis after I/R injury in mice. The protective effect of HIF-1α was attenuated by inhibition of miR-21. CONCLUSIONS: HIF-1α induced angiogenesis by upregulating not only vascular endothelial growth factor but also miR-21 via inhibiting a novel target gene TSP-1. Both of them may contribute to the protective effect of HIF-1α on renal I/R injury.


Asunto(s)
Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Riñón/irrigación sanguínea , MicroARNs/genética , ARN/genética , Daño por Reperfusión/genética , Trombospondina 1/genética , Animales , Apoptosis , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Estudios de Seguimiento , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Inmunohistoquímica , Hibridación in Situ , Riñón/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/biosíntesis , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transducción de Señal , Trombospondina 1/biosíntesis , Factores de Tiempo
15.
Inflammation ; 40(5): 1606-1621, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28634844

RESUMEN

Thrombospondin-1 (TSP-1) is upregulated in several inflammatory diseases. Recent data have shown that macrophages from TSP-1-deficient mice have a reduced inflammatory phenotype, suggesting that TSP-1 plays a part in macrophage activation. DNA microarray approach revealed that Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) may induce the enhanced TSP-1 expression in human monocytes, suggesting a role of TSP-1-mediated pathogenesis in periodontitis. Until recently, the function of TSP-1 has been a matter of debate. In this study, we explored the role of TSP-1 in inflammatory cytokine secretions and its putative mechanism in pathogenesis of periodontitis. We demonstrated that TSP-1 expression was significantly upregulated in gingival tissues with periodontitis and in P. gingivalis LPS-stimulated THP-1 cells. Deficiency of TSP-1 by transfecting siRNAs decreased IL-6, IL-1ß, and TNF-α secretions in THP-1 cells, whereas overexpression of TSP-1 resulted in an upregulation of IL-6, IL-1ß, and TNF-α productions. Additional experiments showed that Pyrrolidine dithiocarbamate (PDTC) inhibited IL-6, IL-1ß, and TNF-α expression induced by overexpression of TSP-1, accompanying with downregulation of phosphorylated p65 and IκBα protein levels in response to P. gingivalis LPS. These results indicated that TSP-1 played a significant role in P. gingivalis LPS-initiated inflammatory cytokines (IL-6, IL-1ß, and TNF-α) secretions of THP-1 cells, and the NF-κB signaling is involved in its induction of expression. Thus, TSP-1 effectively elevated P. gingivalis LPS-induced inflammation mediated by the NF-κB pathway and may be critical for pathology of periodontitis.


Asunto(s)
Citocinas/metabolismo , Inflamación/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Trombospondina 1/fisiología , Humanos , Inflamación/inducido químicamente , Lipopolisacáridos , Periodontitis/etiología , Periodontitis/microbiología , Periodontitis/patología , Porphyromonas gingivalis/patogenicidad , Células THP-1 , Trombospondina 1/biosíntesis , Trombospondina 1/farmacología
16.
Int J Oncol ; 50(5): 1501-1512, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28339036

RESUMEN

Fibroblast growth factor 7 (FGF7) is a mesenchyme-specific heparin-binding growth factor that binds FGF receptor 2 (FGFR2) to regulate numerous cellular and physiological processes. FGF7/FGFR2 signal is associated with gastric cancer progression. In the present study, we investigated the molecular mechanism by which FGF7/FGFR2 promotes invasion and migration in human gastric cancer. We first demonstrated that increased FGFR2 expression in human gastric cancer tissues was significantly associated with tumor depth and clinical stage in human gastric cancer tissues. Thrombospondin 1 (THBS1) is an extracellular glycoprotein that plays multiple roles in cell-matrix and cell-cell interactions. Increased expression of THBS1 significantly correlated with tumor differentiation. FGFR2 and THBS1 expression were both increased in cancer tissues as compared with adjacent normal tissues and their expression was positively correlated. In vitro, FGF7 stimulation of cell invasion and migration was partially suppressed by the FGFR2 knockdown. In addition, FGF7/FGFR2 upregulated THBS1, and cell invasion and migration were decreased by knockdown of THBS1. Furthermore, the PI3K/Akt/mTOR signaling pathway was predominantly responsible for FGF7/FGFR2-induced THBS1 upregulation. Taken together, our data suggest that FGF7/FGFR2/THBS1 is associated with the regulation of invasion and migration in human gastric cancer.


Asunto(s)
Factor 7 de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Neoplasias Gástricas/genética , Trombospondina 1/biosíntesis , Anciano , Diferenciación Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Progresión de la Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Transducción de Señal , Neoplasias Gástricas/patología , Trombospondina 1/genética , Activación Transcripcional/genética
17.
J Neurochem ; 140(4): 645-661, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27735996

RESUMEN

Transactivating DNA-binding protein-43 (TDP-43) inclusions and the accumulation of phosphorylated and ubiquitinated tau proteins (p-tau) have been identified in postmortem brain specimens from patients with chronic traumatic encephalopathy (CTE). To examine whether these proteins contribute to the development of CTE, we utilized an in vitro trauma system known to reproduce many of the findings observed in humans and experimental animals with traumatic brain injury. Accordingly, we examined the role of TDP-43 and Tau in an in vitro model of trauma, and determined whether these proteins contribute to the defective neuronal integrity associated with CNS trauma. Single or multiple episodes of trauma to cultured neurons resulted in a time-dependent increase in cytosolic levels of phosphorylated TDP-43 (p-TDP-43). Trauma to cultured neurons also caused an increase in levels of casein kinase 1 epsilon (CK1ε), and ubiquitinated p-TDP-43, along with a decrease in importin-ß (all factors known to mediate the "TDP-43 proteinopathy"). Defective neuronal integrity, as evidenced by a reduction in levels of the NR1 subunit of the NMDA receptor, and in PSD95, along with increased levels of phosphorylated tau were also observed. Additionally, increased levels of intra- and extracellular thrombospondin-1 (TSP-1) (a factor known to regulate neuronal integrity) were observed in cultured astrocytes at early stages of trauma, while at later stages decreased levels were identified. The addition of recombinant TSP-1, conditioned media from cultured astrocytes at early stages of trauma, or the CK1ε inhibitor PF4800567 hydrochloride to traumatized cultured neurons reduced levels of p-TDP-43, and reversed the trauma-induced decline in NR1 subunit of the NMDA receptor and PSD95 levels. These findings suggest that a trauma-induced increase in TDP-43 phosphorylation contributes to defective neuronal integrity, and that increasing TSP-1 levels may represent a useful therapeutic approach for the prevention of the neuronal TDP-43 proteinopathy associated with CTE. Read the Editorial Highlight for this article on page 531.


Asunto(s)
Astrocitos/metabolismo , Encefalopatía Traumática Crónica/metabolismo , Neuronas/metabolismo , Biosíntesis de Proteínas/fisiología , Proteinopatías TDP-43/metabolismo , Trombospondina 1/biosíntesis , Animales , Animales Recién Nacidos , Células Cultivadas , Femenino , Masculino , Ratas , Ratas Endogámicas F344 , Trombospondina 1/metabolismo
18.
Exp Eye Res ; 153: 27-41, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27697548

RESUMEN

The purpose of this study was to investigate the changes that occur in the lacrimal glands (LGs) in female thrombospondin 1 knockout (TSP1-/-) mice, a mouse model of the autoimmune disease Sjogren's syndrome. The LGs of 4, 12, and 24 week-old female TSP1-/- and C57BL/6J (wild type, WT) mice were used. qPCR was performed to measure cytokine expression. To study the architecture, LG sections were stained with hematoxylin and eosin. Cell proliferation was measured using bromo-deoxyuridine and immunohistochemistry. Amount of CD47 and stem cell markers was analyzed by western blot analysis and location by immunofluorescence microscopy. Expression of stem cell transcription factors was performed using Mouse Stem Cell Transcription Factors RT2 Profiler PCR Array. Cytokine levels significantly increased in LGs of 24 week-old TSP1-/- mice while morphological changes were detected at 12 weeks. Proliferation was decreased in 12 week-old TSP1-/- mice. Three transcription factors were overexpressed and eleven underexpressed in TSP1-/- compared to WT LGs. The amount of CD47, Musashi1, and Sox2 was decreased while the amount of ABCG2 was increased in 12 week-old TSP1-/- mice. We conclude that TSP1 is necessary for maintaining normal LG homeostasis. Absence of TSP1 alters cytokine levels and stem cell transcription factors, LG cellular architecture, decreases cell proliferation, and alters amount of stem cell markers.


Asunto(s)
Citocinas/metabolismo , ADN/genética , Síndromes de Ojo Seco/metabolismo , Regulación de la Expresión Génica , Aparato Lagrimal/patología , Células Madre/patología , Trombospondina 1/genética , Animales , Western Blotting , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/patología , Femenino , Inmunohistoquímica , Aparato Lagrimal/metabolismo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Madre/metabolismo , Lágrimas/metabolismo , Trombospondina 1/biosíntesis , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética
19.
J Oral Pathol Med ; 45(10): 730-739, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26850833

RESUMEN

BACKGROUND: THBS1 (thrombospondin-1) is the extracellular matrix (ECM) protein that affects diverse cellular activities. It constitutes the tumor stroma, but the role of THBS1 in oral squamous cell carcinoma (OSCC) development is unclear. The aim of this study was to clarify the relevance of THBS1 in the pathogenesis of OSCC. MATERIALS AND METHODS: The expression of THBS1 was examined in 44 OSCC by immunohistochemical analysis and in 43 OSCC by cDNA microarray analysis. Cell culture experiments were conducted using human OSCC cell lines HSC3 and HO1N1 and mouse fibroblast ST2 cells to examine the effect of TGFB1 on THBS1 expression, and the effect of THBS1 on cellular behaviors. RESULTS: THBS1 was specifically induced in the tumor microenvironment of OSCC. THBS1 appeared to be produced mainly by the stromal cells, but also by OSCC cells. TGFB1 stimulated THBS1 expression in ST2, primary fibroblasts, and the OSCC cells. THBS1 promoted migration and invasion of HSC3 and HO1N1 in transwell migration assays. THBS1 stimulated the expression of MMP3 (matrix metalloprotease 3), MMP9, MMP11, and MMP13 in ST2 cells and MMP3, MMP11, and MMP13 in HO1N1 cells. The RGD peptide suppressed the THBS1-stimulated migration and upregulation of MMP11 and MMP13. CONCLUSIONS: THBS1 is a tumor-specific ECM protein that is induced by TGFB1 and promotes migration of cancer cells and stimulates the expression of MMPs partly through the integrin signaling, thereby favoring OSCC invasion.


Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Trombospondina 1/biosíntesis , Factor de Crecimiento Transformador beta1/farmacología , Animales , Carcinoma de Células Escamosas/enzimología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN Complementario/metabolismo , Neoplasias de Cabeza y Cuello/enzimología , Humanos , Inmunohistoquímica , Metaloendopeptidasas/biosíntesis , Ratones , Neoplasias de la Boca/enzimología , Carcinoma de Células Escamosas de Cabeza y Cuello , Células del Estroma/enzimología , Células del Estroma/metabolismo , Células del Estroma/patología , Regulación hacia Arriba
20.
Microcirculation ; 23(2): 146-56, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26660949

RESUMEN

Investigations into physiologically controlled capillary regression report the provocative finding that microvessel regression occurs in the face of persistent elevation of skeletal muscle VEGF expression. TSP-1, a negative angiogenic regulator, is increasingly being observed to temporally correlate with capillary regression, suggesting that increased TSP-1 (and not reduction in VEGF per se) is needed to initiate, and likely regulate, capillary regression. Based on evidence being gleaned from physiologically mediated regression of capillaries, it needs to be recognized that capillary regression (and perhaps capillary rarefaction with disease) is not simply the reversal of factors used to stimulate angiogenesis. Rather, the conceptual understanding that angiogenesis and capillary regression each have specific and unique requirements that are biologically constrained to opposite sides of the balance between positive and negative angioregulatory factors may shed light on why anti-VEGF therapies have not lived up to the promise in reversing angiogenesis and providing the cure that many had hoped toward fighting cancer. Emerging evidence from physiological controlled angiogenesis suggest that cases involving excessive or uncontrolled capillary expansion may be best treated by therapies designed to increase expression of negative angiogenic regulators, whereas those involving capillary rarefaction may benefit from inhibiting negative regulators (like TSP-1).


Asunto(s)
Capilares/metabolismo , Regulación de la Expresión Génica/fisiología , Músculo Esquelético/irrigación sanguínea , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Animales , Humanos , Trombospondina 1/biosíntesis
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