RESUMEN
The lysine methyltransferase Smyd1 with its characteristic catalytic SET-domain is highly enriched in the embryonic heart and skeletal muscles, participating in cardiomyogenesis, sarcomere assembly and chromatin remodeling. Recently, significant Smyd1 levels were discovered in endothelial cells (ECs) that responded to inflammatory cytokines. Based on these biochemical properties, we hypothesized that Smyd1 is involved in inflammation-triggered signaling in ECs and therefore, investigated its role within the LPS-induced signaling cascade. Human endothelial cells (HUVECs and EA.hy926 cells) responded to LPS stimulation with higher intrinsic Smyd1 expression. By transfection with expression vectors containing gene inserts encoding either intact Smyd1, a catalytically inactive Smyd1-mutant or Smyd1-specific siRNAs, we show that Smyd1 contributes to LPS-triggered expression and secretion of IL-6 in EA.hy926 cells. Further molecular analysis revealed this process to be based on two signaling pathways: Smyd1 increased the activity of NF-κB and promoted the trimethylation of lysine-4 of histone-3 (H3K4me3) within the IL-6 promoter, as shown by ChIP-RT-qPCR combined with IL-6-promoter-driven luciferase reporter gene assays. In summary, our experimental analysis revealed that LPS-binding to ECs leads to the up-regulation of Smyd1 expression to transduce the signal for IL-6 up-regulation via activation of the established NF-κB pathway as well as via epigenetic trimethylation of H3K4.
Asunto(s)
Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Células Endoteliales/metabolismo , Interleucina-6/genética , Proteínas Musculares/genética , Factores de Transcripción/genética , Metilación de ADN/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Células Endoteliales/efectos de los fármacos , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación/inducido químicamente , Lipopolisacáridos/farmacología , Proteínas Musculares/antagonistas & inhibidores , FN-kappa B/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
Promyelocytic leukemia protein (PML) is a constitutive component of PML nuclear bodies (PML-NBs), which function as stress-regulated SUMOylation factories. Since PML can also act as a regulator of the inflammatory and fibroproliferative responses characteristic of atherosclerosis, we investigated whether PML is implicated in this disease. Immunoblotting, ELISA and immunohistochemistry showed a stronger expression of PML in segments of human atherosclerotic coronary arteries and sections compared with non-atherosclerotic ones. In particular, PML was concentrated in PML-NBs from α-smooth muscle actin (α-SMA)-immunoreactive cells in plaque areas. To identify possible functional consequences of PML-accumulation in this cell type, differentiated human coronary artery smooth muscle cells (dHCASMCs) were transfected with a vector containing the intact PML-gene. These PML-transfected dHCASMCs showed higher levels of small ubiquitin-like modifier (SUMO)-1-dependent SUMOylated proteins, but lower levels of markers for smooth muscle cell (SMC) differentiation and revealed more proliferation and migration activities than dHCASMCs transfected with the vector lacking a specific gene insert or with the vector containing a mutated PML-gene coding for a PML-form without SUMOylation activity. When dHCASMCs were incubated with different cytokines, higher PML-levels were observed only after interferon γ (IFN-γ) stimulation, while the expression of differentiation markers was lower. However, these phenotypic changes were not observed in dHCASMCs treated with small interfering RNA (siRNA) suppressing PML-expression prior to IFN-γ stimulation. Taken together, our results imply that PML is a previously unknown functional factor in the molecular cascades associated with the pathogenesis of atherosclerosis and is positioned in vascular SMCs (VSMCs) between upstream IFN-γ activation and downstream SUMOylation.
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Miocitos del Músculo Liso/patología , Placa Aterosclerótica/metabolismo , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Vasos Coronarios/metabolismo , Femenino , Humanos , Interferón gamma , Masculino , Fragmentos de Péptidos , Fenotipo , Placa Aterosclerótica/patología , SumoilaciónRESUMEN
Smyd1 is an epigenetic modulator of gene expression that has been well-characterized in muscle cells. It was recently reported that Smyd1 levels are modulated by inflammatory processes. Since inflammation affects the vascular endothelium, this study aimed to characterize Smyd1 expression in endothelial cells. We detected Smyd1 in human endothelial cells (HUVEC and EA.hy926 cells), where the protein was largely localized in PML nuclear bodies (PML-NBs). By transfection of EA.hy926 cells with expression vectors encoding Smyd1, PML, SUMO1, active or mutant forms of the SUMO protease SuPr1 and/or the SUMO-conjugation enzyme UBC9, as well as Smyd1- or PML-specific siRNAs, in the presence or absence of the translation blocker cycloheximide or the proteasome-inhibitor MG132, and supported by computational modeling, we show that Smyd1 is SUMOylated in a PML-dependent manner and thereby addressed for degradation in proteasomes. Furthermore, transfection with Smyd1-encoding vectors led to PML up-regulation at the mRNA level, while PML transfection lowered Smyd1 protein stability. Incubation of EA.hy926 cells with the pro-inflammatory cytokine TNF-α resulted in a constant increase in Smyd1 mRNA and protein over 24â h, while incubation with IFN-γ induced a transient increase in Smyd1 expression, which peaked at 6â h and decreased to control values within 24â h. The IFN-γ-induced increase in Smyd1 was accompanied by more Smyd1 SUMOylation and more/larger PML-NBs. In conclusion, our data indicate that in endothelial cells, Smyd1 levels are regulated through a negative feedback mechanism based on SUMOylation and PML availability. This molecular control loop is stimulated by various cytokines.
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Citocinas/farmacología , Proteínas de Unión al ADN/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Proteínas Musculares/metabolismo , Proteína de la Leucemia Promielocítica/metabolismo , Sumoilación/efectos de los fármacos , Factores de Transcripción/metabolismo , Núcleo Celular/metabolismo , Cicloheximida/farmacología , Proteínas de Unión al ADN/genética , Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interferón gamma/farmacología , Leupeptinas/farmacología , Proteínas Musculares/genética , Proteína de la Leucemia Promielocítica/genética , Inhibidores de Proteasoma/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , ARN Interferente Pequeño , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Sumoilación/genética , Factores de Transcripción/genética , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia ArribaRESUMEN
It is unclear how microangiopathic changes in skeletal muscle vary among systemic vascular pathologies. We therefore analyzed the capillary fine structure in skeletal muscle from patients with arterial hypertension (HYPT), diabetes mellitus type 2 (T2DM) or intermittent claudication - peripheral arterial disease (IC/PAD). Tablet-based image analysis (TBIA) was carried out to largely re-evaluate 5,000 transmission electron micrographs of capillaries from 126 vastus lateralis biopsies of 75 individuals (HYPT, T2DM or IC/PAD patients as well as healthy individuals before and after endurance exercise training) used in previous morphometric studies, but assessed using stereological counting grids of different sizes. Serial block-face scanning electron microscopy (SBFSEM) of mouse skeletal muscle was used for validation of the particular fine structural events observed in human biopsies. The peri-capillary basement membrane (BM) was 38.5 and 45.5% thicker (P < 0.05) in T2DM and IC/PAD patients than in the other groups. A 17.7-39.6% lower (P < 0.05) index for intraluminal endothelial cell (EC) surface enlargement by projections was exclusively found in T2DM patients by TBIA morphometry. The proportion of capillaries with disrupted BM between pericytes (PC) and EC was higher (P < 0.05) in HYPT (33.2%) and T2DM (38.7%) patients than in the control group. Empty EC-sockets were more frequent (P < 0.05) in the three patient groups (20.6% in HYPT, 27.1% in T2DM, 30.0% in IC/PAD) than in the healthy individuals. SBFSEM confirmed that EC-sockets may exhibit close proximity to the capillary lumen. Our comparative morphometric analysis demonstrated that structural arrangement of skeletal muscle capillaries is more affected in T2DM than in HYPT or IC/PAD, although some similar elements of remodeling were found. The increased frequency of empty EC-sockets in the three patient groups indicates that the PC-EC interaction is commonly disturbed in these systemic vascular pathologies.
RESUMEN
BACKGROUND: Zinc finger protein 580 (ZNF580) was reported to modulate angiogenesis, endothelial homeostasis and blood pressure control. ZNF580 regulated genes include VEGF-A and IL-8. However, it is unknown if ZNF580 could play a role during inflammation. The aim of this study was to find out if ZNF580 affects the expression of IL-6, if it occurs in monocytic cells and responds to inflammatory mediators. RESULTS: Overexpression of ZNF580 reduced LPS-induced promotor activity of IL-6. Consistently, overexpression of ZNF580 reduced by half the LPS-induced expression of IL-6. ZNF580 was strongly expressed in the nucleus of MonoMac6, a human monocytic cell line. LPS-stimulated IL-6 secretion increased when ZNF580 was suppressed with siRNA. After stimulation of MonoMac6 with LPS for 24 h, ZNF580 negatively correlated with the amount of secreted IL-6. In response to LPS, ZNF580 was increased within the first 8 h, followed by a marked decrease after 16 h. This decrease coincided with sustained IL-6 production. CONCLUSION: This study demonstrated that ZNF580 inhibits LPS-induced expression of IL-6. ZNF580 was highly expressed in monocytic cells and therefore may contribute to the modulation of its IL-6 production, at least in response to LPS. This suggests cooperation between ZNF580 and NFκB, which could play a role during sepsis.
RESUMEN
The expression of neuronal NO synthase (nNOS) alpha- and beta-isoforms in skeletal muscle is well documented but only little information is available about their regulation/functions. Using different mouse models, we now assessed whether the expression of nNOS-isoforms in muscle fibers is related to mitochondria content/activity and regulated by peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha). Catalytic histochemistry revealed highest nNOS-concentrations to be present in type-2 oxidative muscle fibers. Differences in mitochondrial density between nNOS-KO-mice and WT-littermates established by morphometry after transmission electron microscopy were significant in the oxidative portion of the tibialis anterior muscle (TA) but not in rectus femoris muscle (RF) indicating an nNOS-dependent mitochondrial pool in TA. Quantitative immunoblotting displayed the nNOS alpha-isoform to preponderate in those striated muscles of C57BL/6-mice that comprise of many type-2 oxidative fibers, e.g. TA, while roughly even levels of the two nNOS-isoforms were expressed in those muscles that mainly consist of type-2 glycolytic fibers, e.g. RF. Differences in citrate synthase-activity in muscle homogenates between nNOS-KO-mice and WT-littermates were positively related to nNOS alpha-isoform levels. In transgenic-mice over-expressing muscular PGC-1alpha compared to WT-littermates, immunoblotting revealed a significant shift in nNOS-expression in favor of the alpha-isoform in six out of eight striated muscles (exceptions: soleus muscle and tongue) without consistent relationship to changes in the expression of mitochondrial markers. In summary, our study demonstrated the nNOS alpha-isoform expression to be related to mitochondrial content/activity and to be up-regulated by up-stream PGC-1alpha in striated muscles, particularly in those enriched with type-2 oxidative fibers implying a functional convergence of the two signaling systems in these fibers.
Asunto(s)
Mitocondrias/metabolismo , Músculo Estriado/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/biosíntesis , Animales , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismoRESUMEN
OBJECTIVE: Significant sex differences exist in cardiovascular diseases. Although an impact of gonadal hormones is presumed, it is largely unknown whether sexually dimorphic gene expression also plays a role and whether cells themselves show intrinsic sex differences. METHODS: We performed whole genome expression analyses in human umbilical vein endothelial cells (HUVEC) from 20 male and 20 female donors and compared levels of gene transcription between the sexes. To further assess whether there is a sex-specific response to stress, we subjected male and female HUVEC to shear for 24 h and analysed changes in gene expression. RESULTS: Genes indicative for greater immune responsiveness were stronger expressed in female compared to male HUVEC. There was a significant enrichment of 77 immune-related genes in female HUVEC. These increased transcriptional levels in female cells were verified for 20 genes by real-time RT-PCR. 6.7% of all mRNAs were regulated by shear stress. Female HUVEC showed a more pronounced transcriptional response to shear than did their male counterparts. In addition to quantitative differences, a number of genes were regulated in the opposite direction between the two sexes by shear stress. Functionally, female HUVEC showed a higher cell viability after serum starvation and an increased tube formation capacity compared to male cells. CONCLUSION: These findings underscore the importance for differentiation between male and female cells in cell culture experiments. This may apply not only to endothelial cells but might be generalized to other cell types as well. The observed sexual dimorphisms in gene expression in endothelial cells may contribute to sex differences between males and females in endothelial function.
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Células Endoteliales de la Vena Umbilical Humana/metabolismo , Caracteres Sexuales , Transcripción Genética , Supervivencia Celular , Células Cultivadas , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Marcadores Genéticos , Humanos , Masculino , Neovascularización Fisiológica , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores Sexuales , Estrés Mecánico , Estrés Fisiológico , Factores de TiempoRESUMEN
Transient receptor potential canonical (TRPC) channels type 6 play an important role in the function of human podocytes. Diabetic nephropathy is characterized by altered TRPC6 expression and functions of podocytes. Thus, we hypothesized that high glucose modifies TRPC6 channels via increased oxidative stress and syndecan-4 (SDC-4) in human podocytes. Human podocytes were exposed to control conditions (5.6 mmol/L D-glucose), high glucose (30 mmol/L D-glucose or L-glucose), 100 µmol/L peroxynitrite, or high glucose and the superoxide dismutase mimetic tempol (100 µmol/L). TRPC6 and SDC-4 transcripts and protein expression were measured using RT-PCR and in-cell Western assay. Intracellular reactive oxygen species (ROS) and cytosolic calcium were measured using fluorescent dye techniques. High D-glucose increased TRPC6 transcripts to 8.66±4.08 (p<0.05) and TRPC6 protein expression to 1.44±0.07 (p<0.05) without altering SDC-4 transcripts or protein expression. The D-glucose induced increase of TRPC6 expression was blocked by tempol. Increased oxidative stress using peroxynitrite significantly increased TRPC6 transcripts to 4.29±1.26 (p<0.05) and TRPC6 protein expression to 1.28±0.05 (p<0.05) without altering SDC-4 transcripts or protein expression. In human podocytes transfected with scrambled siRNA, high D-glucose increased ROS after 90 min to 3.55±0.08 arbitrary units while 5.6 mmol/L D-glucose increased ROS to 2.49±0.09 (p<0.001) only. The increase in ROS was inhibited by tempol and by SDC-4 knockdown. High glucose modifies TRPC6 channels and ROS production via SDC-4 in human podocytes.
Asunto(s)
Señalización del Calcio/fisiología , Glucosa/administración & dosificación , Estrés Oxidativo/fisiología , Oxígeno/metabolismo , Podocitos/metabolismo , Sindecano-4/metabolismo , Canales Catiónicos TRPC/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Estrés Oxidativo/efectos de los fármacos , Podocitos/efectos de los fármacos , Canales Catiónicos TRPC/efectos de los fármacos , Canal Catiónico TRPC6RESUMEN
The aim of the study was to establish a user-friendly approach for single fluorescence particle 3D localization and tracking with nanometre precision in a standard fluorescence microscope using a point spread function (PSF) approach, and to evaluate validity and precision for different analysis methods and optical conditions with particular application to microcirculatory flow dynamics and cell biology. Images of fluorescent particles were obtained with a standard fluorescence microscope equipped with a piezo positioner for the objective. Whole pattern (WP) comparison with a PSF recorded for the specific set-up and measurement of the outermost ring radius (ORR) were used for analysis. Images of fluorescent particles were recorded over a large range (about 7µm) of vertical positions, with and without distortion by overlapping particles as well as in the presence of cultured endothelial cells. For a vertical range of 6.5µm the standard deviation (SD) from the predicted value, indicating validity, was 9.3/8.7 nm (WP/ORR) in the vertical and 8.2/11.7 nm in the horizontal direction. The precision, determined by repeated measurements, was 5.1/3.8 nm in the vertical and 2.9/3.7 nm in the horizontal direction. WP was more robust with respect to underexposure or overlapping images. On the surface of cultured endothelial cells, a layer with 2.5 times increased viscosity and a thickness of about 0.8µm was detected. With a validity in the range of 10 nm and a precision down to about 3-5 nm obtained by standard fluorescent microscopy, the PSF approach offers a valuable tool for a variety of experimental investigations of particle localizations, including the assessment of endothelial cell microenvironment.
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Fluorescencia , Nanopartículas , Reología , Viscosidad , EndocitosisRESUMEN
skNAC (skeletal and heart muscle specific variant of nascent polypeptide-associated complex α) is a skeletal and heart muscle-specific protein known to be involved in the regulation of sarcomerogenesis. The respective mechanism, however, is largely unknown. In the present paper, we demonstrate that skNAC regulates calpain activity. Specifically, we show that inhibition of skNAC gene expression leads to enhanced, and overexpression of the skNAC gene to repressed, activity of calpain 1 and, to a lesser extent, calpain 3 in myoblasts. In skNAC siRNA-treated cells, enhanced calpain activity is associated with increased migration rates, as well as with perturbed sarcomere architecture. Treatment of skNAC-knockdown cells with the calpain inhibitor ALLN (N-acetyl-leucyl-leucyl-norleucinal) reverts both the positive effect on myoblast migration and the negative effect on sarcomere architecture. Taken together, our data suggest that skNAC controls myoblast migration and sarcomere architecture in a calpain-dependent manner.
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Calpaína/metabolismo , Chaperonas Moleculares/genética , Proteínas Musculares/metabolismo , Mioblastos/metabolismo , Sarcómeros , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
Alternative splicing is a key regulatory mechanism for cellular metabolism controlling cell proliferation and angiogenesis, both of which are crucial processes for tumorigenesis under hypoxia. Human cells express two tissue factor (TF) isoforms, alternatively spliced TF (asTF) and 'full length' TF (flTF). flTF is the major source of thrombogenicity whereas, the function of soluble asTF, particularly in cancer, is widely unknown. In the present study, we examined the impact of alternative splicing on the pro-angiogenic potential and the TF expression pattern of A549 cells under hypoxia. We focused our efforts toward alternative splicing factors, such as Clk1, and pro-angiogenic proliferation-regulating factors, such as Cyr61. We further examined the influence of asTF overexpression on the expression of MCP-1, Cyr61 and VEGF, as well as on cell number and pro-angiogenic properties of A549 cells. Notably, we found hypoxia to induce the expression of alternative splicing factors (Clk1 and Clk4) as well as proliferation- and angiogenesis-promoting factors (Cyr61 and flTF). asTF overexpression in A549 cells also increased both cell number and tube formation. These effects were mediated by the induction of Cyr61, MCP-1 and VEGF, as well as by integrin α(v)ß(3). Taken together, our results suggest that the pro-angiogenic potential of A549 lung cancer cells is modulated under hypoxic conditions via modulation of TF isoform expression which in turn is controlled by alternative splicing.
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Empalme Alternativo/genética , Hipoxia de la Célula , Neoplasias Pulmonares/metabolismo , Neovascularización Patológica/metabolismo , Tromboplastina/metabolismo , Línea Celular Tumoral , Proliferación Celular , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/metabolismo , Proteína 61 Rica en Cisteína/biosíntesis , Proteína 61 Rica en Cisteína/metabolismo , Humanos , Integrina alfaVbeta3/biosíntesis , Integrina alfaVbeta3/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/biosíntesis , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: The impact of smoking on the local innate immune response in the oral cavity, and, commonly, on oral health is actively discussed in the scientific literature. The aim of the present study was to evaluate possible effects of smoking on gene expression of human beta-defensin-1 and -2 in human gingival tissue. MATERIAL AND METHODS: Biopsies of keratinized gingival tissues were taken from donors (with written informed consent) undergoing routine surgical treatment. Prior to the sample collection, participants with clinically healthy periodontium were classified as smokers (n=9) or non-smokers (n=9). Gingival tissue was homogenized, and total RNA was extracted and analysed by real-time RT-PCR for human beta-defensins-1-, -2-, and interleukins IL-1ß- and IL-6-, as well as GAPDH-mRNA. The data obtained were analysed for significant differences using the Mann-Whitney-U test. RESULTS: hBD-1- and hBD-2-, as well as IL-1ß- and IL-6-mRNA were detected in all gingival samples. Expression of hBD-1 and -2 was significantly reduced by nearly 2.5-fold (p<0.05; Mann-Whitney) in gingival samples of smokers compared to control group specimens (non-smokers). In contrast, no significant differences of the gene expression of IL-6 and IL-1ß were observed in human gingival tissue of smokers and non-smokers. CONCLUSION: The results presented here suggest that expression of human beta-defensins hBD-1 and -2, and, thus, the basal levels of innate immune defense reactions in the oral cavity are reduced by smoking.
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Encía/metabolismo , Inmunidad Innata/genética , Fumar/efectos adversos , beta-Defensinas/biosíntesis , beta-Defensinas/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/biosíntesis , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/genética , Humanos , Interleucina-1/biosíntesis , Interleucina-1/genética , Interleucina-6/biosíntesis , Interleucina-6/genética , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas , Adulto JovenRESUMEN
The goal of the study was to assess whether pulsatile atheroprone shear stress modulates the expression of transient receptor potential (TRP) channels, TRPC3, TRPC6, TRPM7, and TRPV1 mRNA, in human umbilical vascular endothelial cells. Exposure of cultured vascular endothelial cells to defined shear stress, producing a constant laminar flow (generating a shear stress of 6 dyne/cm(2)), laminar pulsatile atheroprotective flow (with a mean shear stress of 20 dyne/cm(2)), or laminar atheroprone bidirectional flow (with a mean shear stress of 0 dyne/cm(2)) differentially induced TRPC6 and TRPV1 mRNA as measured by quantitative real-time RT-PCR and normalized to GAPDH expression. Thereby, TRPC6 and TRPV1 mRNA expressions were significantly increased after 24 hours of exposure to an atheroprone flow profile compared with an atheroprotective flow profile. Furthermore, the expression of transcription factors GATA1 and GATA4 was significantly correlated with the expression of TRPC6 mRNA. In contrast, after 24 hours of constant laminar flow, the expression of TRPC6 and TRPV1 mRNA was unchanged, whereas the expression of TRPC3 and TRPM7 was significantly higher in endothelial cells exposed to shear stress in comparison with endothelial cells grown under static conditions. There was a significant association between the expression of TRPC6 and tumor necrosis factor-α mRNA in human vascular tissue. No-flow and atheroprone flow conditions are equally characterized by an increase in the expression of tumor necrosis factor-α; however, inflammation-associated endothelial cell reactions may be further aggravated at atheroprone flow conditions by the increase of TRPV1 and TRPC6, as observed in our study.
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Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Flujo Pulsátil/fisiología , Canales de Potencial de Receptor Transitorio/genética , Arterias/metabolismo , Arterias/patología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/fisiopatología , Secuencia de Bases , Sitios de Unión/genética , Western Blotting , Células Cultivadas , Femenino , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés Mecánico , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6 , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: The impact of nicotine on the local innate immune response in the oral cavity is unclear. The aim of the present study was to evaluate the possible effects of nicotine on the gene expression of human beta-defensin-1 and -2 in HaCaT keratinocytes. MATERIALS AND METHODS: HaCaTs were cultured in six-well plates in Dulbecco's minimum essential medium (DMEM) supplemented with 10% FBS at a density of ×10(6). Cells were pretreated with 10 µg/ml nicotine (12 h), and then stimulated with 50 ng/ml TNF-α (during the following 12 h); or were pretreated with 50 ng/ml TNF-α, and then stimulated with 10 µg/ml nicotine; or were not pretreated but only stimulated with either nicotine or TNF-α, or a combination of both. Total RNA was extracted and analysed by real-time RT-PCR for human beta-defensins-1-, -2-, and interleukins IL-1ß- and IL-6-, as well as GAPDH-mRNA. The obtained data were analysed using Tukey's B multiple comparison test for post hoc analysis. RESULTS: Pretreatment with nicotine caused a significant 2.5-fold inhibition of TNF-α-stimulated hBD-2 mRNA expression compared to TNF-α alone (p = 0.004). Simultaneous treatment with TNF-α and nicotine caused a significant 2-fold inhibition of hBD-2 mRNA compared to TNF-α alone (p = 0.041). CONCLUSION: The present results suggest that the pre-exposition to nicotine seems to reduce a stimulating effect of TNF-α on the gene expression of hBD-2.
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Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Nicotina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , beta-Defensinas/efectos de los fármacos , beta-Defensinas/genética , Análisis de Varianza , Línea Celular , Células Cultivadas , Defensinas/metabolismo , Perfilación de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Both, increased plasma concentrations of vascular endothelial growth factor (VEGF) and increased expression of transient receptor potential canonical type 6 (TRPC6) channels in podocytes have been associated with proteinuric kidney diseases. Now, we investigated the hypothesis that VEGF regulates TRPC6 in podocytes. METHODS: TRPC6 messenger RNA (mRNA) and TRPC6 protein expression were analyzed in cultured podocytes after administration of VEGF165 using quantitative real-time reverse transcription-polymerase chain reaction and immunoblotting, respectively. YFP-tagged TRPC6 in podocytes was analyzed using confocal laser scanning microscopy. TRPC6-associated calcium influx was measured fluorometrically. Both, immunofluorescence and immunohistochemistry were performed in renal tissue from patients with diabetes mellitus and controls. RESULTS: Administration of VEGF165 to podocytes significantly increased TRPC6 mRNA expression and TRPC6 protein levels. The effects of VEGF165 were dose dependent and could be blocked by phosphoinositide-3-kinase inhibitors. In the presence of cycloheximide, an inhibitor of protein biosynthesis, we did not observe an effect of VEGF on TRPC6 protein levels, indicating the requirement of de novo protein synthesis. VEGF165 significantly increased TRPC6-mediated calcium influx in podocytes. Calcium influx was significantly lower in podocytes after gene knockdown using siRNA against TRPC6. Immunohistochemistry showed both increased TRPC6 channel protein and VEGF receptor type 2 (VEGFR-2) protein in podocytes from patients with diabetic nephropathy compared to control subjects. There was a significant association between VEGFR-2 mRNA and TRPC6 mRNA (n = 48; r(2) = 0.585; P < 0.0001) in human renal cortex. CONCLUSION: VEGF regulates TRPC6 in podocytes.
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Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Regulación de la Expresión Génica , Podocitos/metabolismo , Canales Catiónicos TRPC/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/patología , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Podocitos/citología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6 , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial VascularRESUMEN
Endothelial connexin (Cx)40 plays an important role in signal propagation along blood vessel walls, modulating vessel diameter and thereby blood flow. Blood flow, in turn, has been shown to alter endothelial Cx40 expression. However, the timing and shear stress dependence of this relationship have remained unclear, as have the signal transduction pathways involved and the functional implications. Therefore, the aim of this study was to quantify the effects of shear stress on endothelial Cx40 expression, to analyze the role of phosphoinositide 3-kinase (PI3K)/Akt signaling involved, and to assess the possible functional consequences for the adaptation of microvascular networks. First-passage human umbilical vein endothelial cells were exposed to defined shear stress conditions and analyzed for Cx40 using real-time RT-PCR and immunoblot analysis. Shear stress caused long-term induction of Cx40 protein expression, with two short-term mRNA peaks at 4 and 16 h, indicating the dynamic nature of the adaptation process. Maximum shear stress-dependent induction was observed at shear levels between 6 and 10 dyn/cm(2). Simulation of this pattern of shear-dependent Cx expression in a vascular adaptation model of a microvascular network led to an improved fit for the simulated results to experimental measurements. Cx40 expression was greatly reduced by inhibiting PI3K or Akt, with PI3K activity being required for basal Cx40 expression and Akt activity taking part in its shear stress-dependent induction.
Asunto(s)
Conexinas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Mecanotransducción Celular , Microvasos/metabolismo , Animales , Western Blotting , Células Cultivadas , Simulación por Computador , Conexinas/genética , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Mecanotransducción Celular/efectos de los fármacos , Microcirculación , Modelos Cardiovasculares , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Flujo Sanguíneo Regional , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés Mecánico , Factores de Tiempo , Regulación hacia Arriba , Proteína alfa-5 de Unión ComunicanteRESUMEN
In skeletal muscles, the expression of neuronal NO synthase (nNOS) isoforms is uncharacterized at the protein level. We therefore conducted epitope mapping with anti-peptide-antibodies. Antibodies specific for the nNOS N-terminus recognized the 160-kDa alpha-isoform. In contrast, antibodies against the middle portion or the C-terminus of nNOS bound additionally to the truncated 140-kDa beta-isoform which lacks the PDZ-domain present in the alpha-isoform. All nNOS immunohistochemical reactivity was confined to the sarcolemma. Consistently, immunoblotting disclosed both nNOS-isoforms to be co-enriched in the membrane-associated fractions. The beta-isoform was co-immunoprecipitated with alpha-isoform antibodies in muscle extracts indicating an association of both nNOS-isoforms to direct the beta-variant to the sarcolemma.
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Proteínas Musculares/metabolismo , Músculo Esquelético/enzimología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Sarcolema/enzimología , Animales , Inmunohistoquímica , Isoenzimas , Ratones , Ratones Noqueados , Proteínas Musculares/genética , Óxido Nítrico Sintasa de Tipo I/genética , Estructura Terciaria de Proteína , Ratas , Sarcolema/genéticaRESUMEN
Recent studies revealed that in vivo the inner blood vessel surface is lined with an endothelial surface layer at least 0.5 µm thick, which serves as an aegis, protecting the vessel wall from arteriosclerosis. Hyaluronan seems to be a constitutive component in regard to the atheroprotective properties of this surface structure. It has been shown that arterial pulsatile laminar blood flow increases the thickness of this surface layer in vivo, while it is significantly reduced at atheroprone regions with disturbed flow. This study was undertaken to reveal whether endothelial hyaluronan synthesis via hyaluronan synthase 2 (HAS2) can be changed by different shear stress conditions in vitro, especially in regard to an undisturbed, arterial-like pulsatile flow profile. Human umbilical vein endothelial cells, exposed to constant or pulsatile shear stress in a cone-and-plate system, were analysed for HAS2 expression by real-time RT-PCR and immunoblotting, and for hyaluronan by ELISA. Hyaluronan synthase 2 mRNA and protein were found to be transiently increased in a shear stress-dependent manner via the phosphatidylinositol 3-kinase-Akt pathway. Especially pulsatile, arterial-like shear stress conditions induced enzyme and hyaluronan effectively, while lower shear stress that continuously changed its direction did not induce any differences in comparison with control cultures not exposed to shear stress. These experiments provide a link between the production of a constitutive component of the endothelial surface layer by endothelial cells and blood flow.
Asunto(s)
Arteriosclerosis/prevención & control , Glucuronosiltransferasa/biosíntesis , Ácido Hialurónico/biosíntesis , Flujo Pulsátil , Estrés Mecánico , Cromonas/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hialuronano Sintasas , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3RESUMEN
OBJECTIVE: The Interleukin 8 (IL-8) response of endothelial cells to lipoproteins has well known implications for the development and progression of atherosclerosis. In this study we sought for the role of zinc finger protein 580 (ZNF580) in the endothelial IL-8 response to lipoproteins. METHODS: In human umbilical vein endothelial cells (HUVEC) ZNF580 and IL-8 levels were examined by real-time-RT-PCR, immunoblotting and immunostaining or ELISA, respectively. RESULTS: ZNF580 is located in the nucleus and regulated by LDL and HDL depending on the oxLDL/LDL-ratio but not by TNFα. IL-8 expression profiles are inversely influenced by the oxLDL/LDL-ratio, both in vitro and in vivo. Knock down of ZNF580 enhances the expression and release of IL-8 and increases monocyte arrest under flow conditions in vitro. CONCLUSIONS: ZNF580 is a novel factor in the lipoprotein-dependent regulation of IL-8 and monocyte arrest. Therefore it may be a new potential target for intervention in atherosclerosis.
Asunto(s)
Células Endoteliales/metabolismo , Interleucina-8/metabolismo , Lipoproteínas LDL/metabolismo , Factores de Transcripción/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Western Blotting , Adhesión Celular , Células Cultivadas , Técnicas de Cocultivo , Regulación hacia Abajo , Células Endoteliales/inmunología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Interleucina-8/genética , Lipoproteínas HDL/metabolismo , Monocitos/metabolismo , Reacción en Cadena de la Polimerasa , Interferencia de ARN , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3-kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP-1. The anti-angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70-kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress-treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress-treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non-perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress-dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis.