RESUMEN
We determined the epigenetic mechanisms regulating mean arterial pressure (MAP) and renal dysfunction in guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene-targeted mice. The Npr1 (encoding NPRA) gene-targeted mice were treated with class 1 specific histone deacetylase inhibitor (HDACi) mocetinostat (MGCD) to determine the epigenetic changes in a sex-specific manner. Adult male and female Npr1 haplotype (1-copy; Npr1+/-), wild-type (2-copy; Npr1+/+), and gene-duplicated heterozygous (3-copy; Npr1++/+) mice were intraperitoneally injected with MGCD (2 mg/kg) for 14 days. BP, renal function, histopathology, and epigenetic changes were measured. One-copy male mice showed significantly increased MAP, renal dysfunction, and fibrosis than 2-copy and 3-copy mice. Furthermore, HDAC1/2, collagen1alpha-2 (Col1α-2), and alpha smooth muscle actin (α-SMA) were significantly increased in 1-copy mice compared with 2-copy controls. The expression of antifibrotic microRNA-133a was attenuated in 1-copy mice but to a greater extent in males than females. NF-κB was localized at significantly lower levels in cytoplasm than in the nucleus with stronger DNA binding activity in 1-copy mice. MGCD significantly lowered BP, improved creatinine clearance, and repaired renal histopathology. The inhibition of class I HDACs led to a sex-dependent distinctive stimulation of acetylated positive histone marks and inhibition of methylated repressive histone marks in Npr1 1-copy mice; however, it epigenetically lowered MAP, repaired renal fibrosis, and proteinuria and suppressed NF-kB differentially in males versus females. Our results suggest a role for epigenetic targets affecting hypertension and renal dysfunction in a sex-specific manner.
Asunto(s)
Presión Sanguínea , Epigénesis Genética , Receptores del Factor Natriurético Atrial , Animales , Femenino , Masculino , Receptores del Factor Natriurético Atrial/genética , Receptores del Factor Natriurético Atrial/metabolismo , Ratones , Presión Sanguínea/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Inhibidores de Histona Desacetilasas/farmacología , Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Enfermedades Renales/patologíaRESUMEN
Corneal collagen cross-linking (CXL) is widely used to treat keratoconus and ecstatic corneal disorders. The present studies were carried out to investigate the distribution of glycosaminoglycans (GAGs) and collagen fibril (CF) at different depths of the normal and CXL treated corneal stroma of four week old rats 7 days after standard CXL application. Ten Wistar rats' corneas were used for the study. The epithelium of the cornea from the left eye of each rat was removed and treated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The cornea from the right eye was used as the control cornea. The cornea was removed from the eye and processed for transmission electron microscopy. A bottom mounted Quemesa camera was used to capture digital images and these images were analysed using iTEM software. In the control cornea, the GAGs area size was not significantly different in the anterior, middle, and posterior stroma. In the CXL treated rats the GAGs area size gradually increased from the anterior to the posterior stroma whereas the spacing between the GAGs gradually decreased. There were very large GAGs present in the posterior stroma of the CXL treated rats. When comparing the control and CXL cornea, the GAGs area in the CXL cornea was significantly higher and inter-GAGs-spacing was smaller than in the control cornea. In the control cornea, the collagen fibrils diameter was higher in the anterior stroma and lowest in the posterior stroma. In the CXL treated cornea, the CF diameter and the interfibrillar spacing gradually decreased from the anterior to the posterior stroma. On comparison between the control and the CXL treated cornea, the interfibrillar spacing was significantly smaller in the CXL treated cornea than the control cornea in the anterior, middle, and posterior stroma but there was no difference in the diameter. The CXL treatment significantly increased the GAGs area and decreased the inter-GAGs-spacing, and inter-CF-spacing. This could be due to the gradual decline in the availability of riboflavin, UVA, and oxygen in the middle and posterior stroma. Further studies are required to investigate the role of keratan sulphate and chondroitin sulphate by using monoclonal antibodies with immunogold technique.
Asunto(s)
Sustancia Propia , Queratocono , Animales , Ratas , Glicosaminoglicanos , Colágeno , Reactivos de Enlaces Cruzados , Ratas Wistar , Córnea , Riboflavina/uso terapéutico , Rayos Ultravioleta , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
Corneal collagen cross-linking (CXL) is a treatment that is widely applied to halt the progression of ectatic diseases such as keratoconus by creating biomechanical strength in the cornea. Most of the studies assessed the effect of the CXL on the cornea without any differentiation of its effect between periphery and the center of the untreated control cornea especially after the 7 days of CXL application. We investigate the ultrastructural changes in the architecture of the center and periphery of rat corneas, 7 days after standard CXL application. Five Wistar rats (10 corneas) were used in the present study. The left eye corneas (5 mm area) were de-epithelialized and irradiated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The right eye corneas were used as a control. The sclera-cornea button was removed and processed for electron microscopy. Digital images were captured with a bottom mounted Quemesa camera and analyzed using the iTEM software. The ultrastructure of epithelium, hemi-desmosomes, Bowman's layer and stroma were organized in both untreated control and CXL rat cornea in both untreated control and CXL rat cornea. Within the same CXL cornea, both the collagen fibril (CF) diameter and interfibrillar spacing at the center were significantly smaller compared to the peripheral diameter and spacing of the cornea. When comparing the untreated control and CXL cornea, the central interfibrillar spacing of the CXL cornea was significantly smaller than the central spacing the untreated control cornea. In the CXL cornea the peripheral spacing was significantly higher compared to the peripheral interfibrillar spacing of the untreated control cornea. Within the CXL cornea, the proteoglycans (PGs) area and density of the periphery was significantly higher compared to the area and density of the center of the cornea. It suggests that CXL was more effective at the periphery of the cornea. This could be due to the higher amount of leucine rich PG lumican and higher diffusion of oxygen and riboflavin at the periphery cornea.
Asunto(s)
Colágeno , Queratocono , Animales , Colágeno/farmacología , Córnea , Sustancia Propia/ultraestructura , Reactivos de Enlaces Cruzados/farmacología , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Ratas , Ratas Wistar , Riboflavina/farmacología , Rayos UltravioletaRESUMEN
As the prevalence of microbial keratitis increases, it creates an environment conducive to genotoxicity response. A potential connection between growth arrest and DNA-damage-inducible 45 gamma (GADD45G) gene expression has not been proven in the corneal epithelial cells. The aim of this study was to determine whether lipopolysaccharide (LPS) enhances genotoxicity, DNA damage, and inflammatory responses in human corneal epithelial cells (HCECs) in vitro. In a set of parameters, cytotoxicity, reactive oxygen species, mitochondrial membrane potential, DNA damage, inflammatory response, and apoptosis were assessed. LPS (1, 5, and 10 µg/mL) treated HCECs were increased reactive oxygen species formation, mitochondrial membrane depolarization, and genotoxicity in a concentration-dependent manner. Similarly, NF-κB, PARP1, and TP53 were also overexpressed in the LPS treated HCECs. 24 hours after LPS induction, micronucleus scoring, and proapoptotic factors were also increased. Among them, the GADD45G, NF-κB, and γH2AX were overexpressed both on the mRNA and protein levels in LPS (10 µg/mL) treated HCECs. In our study, we show that the GADD45G signaling can trigger genotoxic instability in HCECs exposed to LPS. Therefore, understanding the factors contributing to infectious keratitis, such as GADD45G, NF-κB, and γH2AX signaling, may help to develop antigenotoxic and anti-inflammatory therapies for corneal dystrophy and epithelial cell remodeling.
Asunto(s)
Daño del ADN/efectos de los fármacos , Epitelio Corneal/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/efectos adversos , FN-kappa B/metabolismo , Apoptosis , HumanosRESUMEN
This study was conducted to investigate the ultrastructure of a unique structures at the anterior side of the endothelium of the posterior peripheral cornea and compare their inner fibers to those of the limbus and sclera. The unique structures at the anterior side of endothelium was referred as a pre-endothelial (PENL) structures in the present manuscript. Ten anonymous-donor human corneoscleral rims (leftover after corneal transplants) were processed for electron microscopy. Semi-thin sections were examined using an Olympus BX53 microscope, and ultrathin sections were studied using a JOEL 1400 transmission electron microscope. A unique PENL structures was identified at the posterior peripheral cornea at a radial distance of approximately 70-638 µm, from the endpoint of Descemet's membrane. The PENL thinned out gradually and disappeared in the center. The contained an electron-dense sheath with periodic structures (narrow-spacing fibers), wide-spacing fibers, and numerous microfibrils. Typical elastic fibers were present in the sclera and limbus but were not observed in the PENL. This study revealed the existence of a new acellular PENL, containing unique fibrillar structures that were unseen in the corneal stroma. From the evidence describe in this paper we therefore suggest that PENL is a distinct morphological structure present at the corneal periphery.
Asunto(s)
Córnea/ultraestructura , Sustancia Propia/ultraestructura , Endotelio/ultraestructura , Microscopía Electrónica/métodos , Adulto , Anciano , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
To investigate the effects of iontophoresis-ultraviolet A (UVA) cross-linking (CXL) with hypotonic riboflavin solution on the ultrastructural changes in the lamellae, collagen fibrils (CFs), and proteoglycans (PGs) in the central and peripheral stroma of the human corneal buttons. The iontophoresis method was used for the trans-epithelial application of hypotonic riboflavin in ex vivo corneal culture for 5 min. The corneas were irradiated using three methods: Group 1 (G1) , a UVA irradiance of 3 mW/cm2 for 30 min; Group 2 (G2) , a UVA irradiance of 10 mW/cm2 for 9 min; Group 3 (G3) , without UVA irradiation. Three untreated corneas were used as controls ( G0 ). After the CXL procedure, the corneas were processed for electron microscopy. The CF diameter and PGs in each sample were analyzed using the iTEM program. The keratocyte organelles and stromal architecture in the peripheral cornea were better preserved than those in the central cornea. In G1 and G2, the mean CF diameter in the peripheral cornea was significantly higher than that in the central cornea. In G3, the CF diameter in the central cornea was significantly larger than that in the peripheral cornea. Furthermore, differences in PG area size were observed between the central and peripheral corneas in all groups. Riboflavin + UVA application at 3 mW/cm2 for 30 min and 10 mW/cm2 for 9 min was a suitable method of CXL; however, 3 mW/cm2 for 30 min improved the organization and size of the collagen fibrils. CXL treatment applied at the periphery was more effective than that applied at the center.
RESUMEN
The two vasoactive hormones, angiotensin II (ANG II; vasoconstrictive) and atrial natriuretic peptide (ANP; vasodilatory) antagonize the biological actions of each other. ANP acting through natriuretic peptide receptor-A (NPRA) lowers blood pressure and blood volume. We tested hypothesis that ANG II plays critical roles in the transcriptional repression of Npr1 (encoding NPRA) and receptor function. ANG II significantly decreased NPRA mRNA and protein levels and cGMP accumulation in cultured mesangial cells and attenuated ANP-mediated relaxation of aortic rings ex vivo. The transcription factors, cAMP-response element-binding protein (CREB) and heat-shock factor-4a (HSF-4a) facilitated the ANG II-mediated repressive effects on Npr1 transcription. Tyrosine kinase (TK) inhibitor, genistein and phosphatidylinositol 3-kinase (PI-3K) inhibitor, wortmannin reversed the ANG II-dependent repression of Npr1 transcription and receptor function. ANG II enhanced the activities of Class I histone deacetylases (HDACs 1/2), thereby decreased histone acetylation of H3K9/14ac and H4K8ac. The repressive effect of ANG II on Npr1 transcription and receptor signaling seems to be transduced by TK and PI-3K pathways and modulated by CREB, HSF-4a, HDACs, and modified histones. The current findings suggest that ANG II-mediated repressive mechanisms of Npr1 transcription and receptor function may provide new molecular targets for treatment and prevention of hypertension and cardiovascular diseases.
Asunto(s)
Angiotensina II/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción del Choque Térmico/metabolismo , Histona Desacetilasas/metabolismo , Receptores del Factor Natriurético Atrial/genética , Receptores del Factor Natriurético Atrial/metabolismo , Acetilación , Angiotensina II/farmacología , Animales , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Factores de Transcripción del Choque Térmico/genética , Histonas/metabolismo , Ratones , Modelos Biológicos , Unión Proteica , Activación Transcripcional/efectos de los fármacosRESUMEN
Ultraviolet radiation is an environmental carcinogenic agent that enhances inflammation and immunological reactions in the exposed human skin cells leading to oxidative photoaging of the epidermal and dermal segment. In the present study, we investigated the protective role of ursolic acid (UA) against ultraviolet B (UVB) radiation- induced photoaging an in vitro model of human skin dermal fibroblasts. UA-pretreated human skin dermal fibroblast (HDF) cells were exposed to UVB radiation to evaluated cell viability, reactive oxygen species (ROS), mitochondrial membrane potential, lipid peroxidation, antioxidant status, DNA damage, proinflammatory response, apoptotic induction, and matrix metalloproteinase (MMP) alteration. The UA pretreatment of HDFs mitigated the UVB irradiation-induced cytotoxicity, ROS generation, and mitochondrial membrane potential alteration and lipid peroxidation, depletion of antioxidant status, DNA damage, and apoptotic induction. UA pretreatment of HDFs also attenuated the UVB-induced expression of inflammatory (TNF-α and NF-κB) and apoptotic (p53, Bax, and caspase-3) and MMPs (MMP-2 and MMP-9) and enhanced the Bcl-2 protein levels in 20 µM UA treatment, when compared to concentrations. Hence, these results revealed that UA has the potential to mitigate UVB-induced extracellular damage by interfering with the ROS-mediated apoptotic induction and photoaging senescence and thus is a potential therapeutic agent to protect the skin against UVB-irradiation induced photooxidative damage.
Asunto(s)
Senescencia Celular , Dermis/patología , Fibroblastos/patología , Inflamación/patología , Estrés Oxidativo/efectos de los fármacos , Triterpenos/farmacología , Rayos Ultravioleta , Caspasa 3/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Senescencia Celular/efectos de la radiación , Daño del ADN , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Triterpenos/química , Factor de Necrosis Tumoral alfa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Ácido UrsólicoRESUMEN
Transient receptor potential vanilloid 1 (TRPV1), which has been identified as a molecular target for the activation of sensory neurons by various painful stimuli, was reported to regulate the signaling and activation of CD4+ T cells. However, the role of TRPV1 in CD4+ T cell in allergic rhinitis remains poorly understood. In this study, TRPV1 expression was localized in CD4+ T cells. Both knockout and chemical inhibition of TRPV1 suppressed Th2/Th17 cytokine production in CD4 T cells and Jurkat T cells, respectively, and can suppress T cell receptor signaling pathways including NF-κB, MAP kinase, and NFAT. In TRPV1 knockout allergic rhinitis (AR) mice, eosinophil infiltration, Th2/Th17 cytokines in the nasal mucosa, and total and ova-specific IgE levels in serum decreased, compared with wild-type AR mice. The TRPV1 antagonists, BCTC or theobromine, showed similar inhibitory immunologic effects on AR mice models. In addition, the number of TRPV1+/CD4+ inflammatory cells increased in the nasal mucosa of patients with AR, compared with that of control subjects. Thus, TRPV1 activation on CD4+ T cells is involved in T cell receptor signaling, and it could be a novel therapeutic target in AR.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Inflamación/inmunología , Rinitis Alérgica/inmunología , Canales Catiónicos TRPV/inmunología , Adolescente , Adulto , Animales , Western Blotting , Linfocitos T CD4-Positivos/metabolismo , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Eosinófilos/inmunología , Eosinófilos/metabolismo , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunohistoquímica , Inflamación/genética , Inflamación/metabolismo , Células Jurkat , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Mucosa Nasal/inmunología , Mucosa Nasal/metabolismo , Ovalbúmina/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rinitis Alérgica/genética , Rinitis Alérgica/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Although several studies have claimed that mesenchymal stem cells (MSC) derived from human tissues can ameliorate allergic airway inflammation, the immunomodulatory mechanism of MSCs remains unclear. OBJECTIVE: We aimed to determine the effects and the underlying mechanism of tonsil-derived MSCs (T-MSC) on allergic inflammation compared with adipose tissue-derived stem cells (ASC) in a mouse model of allergic rhinitis (AR). METHODS: MSCs were isolated from human palatine tonsil (T-MSC) and the surface markers were analyzed. The effect of T-MSCs was evaluated in 24 BALB/c mice that were randomly divided into four groups (negative control group, positive control group, T-MSC group, and ASC group). MSCs were administered intravenously to ovalbumin (OVA) sensitized mice (T-MSC and ASC groups) on days 18 to 23, and subsequent OVA challenge was conducted daily from days 24 to 28. Several parameters of allergic inflammation were assessed. RESULTS: T-MSC and ASC had similar characteristics in surface markers. Intravenous injection of T-MSC significantly reduced allergic symptoms, eosinophil infiltration, serum total, and OVA-specific immunoglobulin E (IgE), and the nasal and systemic T-helper (Th) 2 cytokine profile. Further analysis revealed that nasal innate cytokines, such as interleukin (IL) 25 and IL-33, and chemokines, such as CCL11, CCL24, induction was suppressed in T-MSCs injected groups, which explained their underlying mechanism. In addition, the T-MSC group had more inhibition of allergic inflammation than did the ASC group, which might be attributed to the more proliferative activity of T-MSC. CONCLUSION: Administration of T-MSC effectively reduced allergic symptoms and inflammatory parameters in the mouse model of AR. T-MSC treatment reduced Th2 cytokines and OVA-specific IgE secretion from B cells. In addition, innate cytokine (IL-25 and IL-33) expression and eotaxin messenger RNA expression was inhibited in the nasal mucosa, which is suggestive of the mechanism of reduced allergic inflammation. Therefore, T-MSC treatment is potentially an alternative therapeutic modality in AR.
Asunto(s)
Inmunomodulación , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/inmunología , Rinitis Alérgica/cirugía , Tejido Adiposo/inmunología , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal , Tonsila Palatina/inmunología , Distribución Aleatoria , Rinitis Alérgica/inmunología , Resultado del TratamientoRESUMEN
BACKGROUND: Exposure to cigarette smoking (CS) is a major risk factor for airway inflammation. However, little is known about the effects of CS exposure on eosinophilic rhinosinusitis with nasal polyps (ERSwNPs). Histopathological and molecular studies were performed to investigate its effects using a murine model of ERSwNPs. METHODS: Mice were assigned to one of the following four groups (n = 8 for each group): control group, CS exposure (CS group), ERSwNP (ERS group), and ERSwNPs exposed to CS (ERS + CS group). Histopathological changes were investigated using various stains, including hematoxylin and eosin for inflammation and polyp-like lesions, Sirius red for eosinophils, toluidine blue for mast cells, Alcian blue for goblet cells, and Masson's trichrome stain for collagen fibers. mRNA expression of cytokines from nasal mucosae was measured. Serum IgE and systemic cytokine levels were measured by enzyme-linked immunosorbent assays. The expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF) 1-alpha was evaluated by immunohistochemical staining. RESULTS: The ERS + CS group showed more severe symptoms, increased the number of polyp-like lesions, infiltration of eosinophils, goblet cell hyperplasia, and subepithelial fibrosis, compared with the ERS group. Additionally, mRNA expressions of IL-4 and IL-17A were up-regulated in ERS + CS group and higher levels of IL-4, IL-6, IL-17A, and interferon gamma from splenocytes were observed significantly in the ERS + CS group compared with the ERS group. In the ERSwNP murine model, exposure to CS enhanced the expression of VEGF and HIF-1-alpha in nasal epithelial cells. CONCLUSION: Chronic exposure to CS aggravated eosinophilic inflammation and promoted airway remodeling and nasal polyp formation in a murine model of ERSwNPs. The underlying mechanism might involve up-regulated expression of VEGF and HIF-1-alpha.