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BACKGROUND: Elevated levels of prokineticin-2 (PK2), regarded as a protein involved in modulating immune/inflammatory responses, have been detected in the substantia nigra, serum, and olfactory neurons of Parkinson's disease (PD) patients. Of note, emerging evidence suggests that gut alterations, including dysbiosis and enteric inflammation, play a role in PD via the gut-brain axis. OBJECTIVES: Our goal was to investigate the expression of PK2 in colonic biopsies of PD patients. METHODS: Mucosal biopsies from the descending colon were obtained in 11 PD patients and five asymptomatic subjects. Biopsy samples were processed for PK2 immunofluorescence and western blot. RESULTS: We revealed an increased PK2 expression in colonic mucosa from PD patients in the early stages compared to controls. In addition, we found that PK2 was expressed by activated enteric glial cells and macrophages. CONCLUSIONS: PK2 is highly expressed within neurogenic/inflammatory cells of colonic mucosa from early PD patients, suggesting a potential role of PK2 in gut inflammation, especially in the early stages of PD. © 2024 International Parkinson and Movement Disorder Society.
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BACKGROUND: Experimental evidence suggests a key role of SIRT1 (silent information regulator 1) in age- and metabolic-related vascular dysfunction. Whether these effects hold true in the human microvasculature is unknown. We aimed to investigate the SIRT1 role in very early stages of age- and obesity-related microvascular dysfunction in humans. METHODS: Ninety-five subjects undergoing elective laparoscopic surgery were recruited and stratified based on their body mass index status (above or below 30 kg/m2) and age (above or below 40 years) in 4 groups: Young Nonobese, Young Obese, Old Nonobese, and Old Obese. We measured small resistance arteries' endothelial function by pressurized micromyography before and after incubation with a SIRT1 agonist (SRT1720) and a mitochondria reactive oxygen species (mtROS) scavenger (MitoTEMPO). We assessed vascular levels of mtROS and nitric oxide availability by confocal microscopy and vascular gene expression of SIRT1 and mitochondrial proteins by qPCR. Chromatin immunoprecipitation assay was employed to investigate SIRT1-dependent epigenetic regulation of mitochondrial proteins. RESULTS: Compared with Young Nonobese, obese and older patients showed lower vascular expression of SIRT1 and antioxidant proteins (FOXO3 [forkhead box protein O3] and SOD2) and higher expression of pro-oxidant and aging mitochondria proteins p66Shc and Arginase II. Old Obese, Young Obese and Old Nonobese groups endothelial dysfunction was rescued by SRT1720. The restoration was comparable to the one obtained with mitoTEMPO. These effects were explained by SIRT1-dependent chromatin changes leading to reduced p66Shc expression and upregulation of proteins involved in mitochondria respiratory chain. CONCLUSIONS: SIRT1 is a novel central modulator of the earliest microvascular damage induced by age and obesity. Through a complex epigenetic control mainly involving p66Shc and Arginase II, it influences mtROS levels, NO availability, and the expression of proteins of the mitochondria respiratory chain. Therapeutic modulation of SIRT1 restores obesity- and age-related endothelial dysfunction. Early targeting of SIRT1 might represent a crucial strategy to prevent age- and obesity-related microvascular dysfunction.
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Arginasa , Obesidad , Sirtuina 1 , Enfermedades Vasculares , Adulto , Arginasa/metabolismo , Epigénesis Genética , Humanos , Proteínas Mitocondriales/metabolismo , Óxido Nítrico/metabolismo , Obesidad/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismo , Enfermedades Vasculares/etiologíaRESUMEN
Intestinal barrier alterations represent a primum movens in obesity and related intestinal dysfunctions. However, whether gut barrier remodeling represents prodromal events in obesity before weight gain, metabolic alterations, and systemic inflammation remains unclear. Herein, we examined morphologic changes in the gut barrier in a mouse model of high-fat diet (HFD) since the earliest phases of diet assumption. C57BL/6J mice were fed with standard diet (SD) or HFD for 1, 2, 4, or 8 weeks. Remodeling of intestinal epithelial barrier, inflammatory infiltrate, and collagen deposition in the colonic wall was assessed by histochemistry and immunofluorescence analysis. Obese mice displayed increased body and epididymal fat weight along with increased plasma resistin, IL-1ß, and IL-6 levels after 8 weeks of HFD. Starting from 1 week of HFD, mice displayed (1) a decreased claudin-1 expression in lining epithelial cells, (2) an altered mucus in goblet cells, (3) an increase in proliferating epithelial cells in colonic crypts, (4) eosinophil infiltration along with an increase in vascular P-selectin, and (5) deposition of collagen fibers. HFD intake is associated with morphologic changes in the large bowel at mucosal and submucosal levels. In particular, the main changes include alterations in the mucous layer and intestinal epithelial barrier integrity and activation of mucosal defense-enhanced fibrotic deposition. These changes represent early events occurring before the development of obesity condition that could contribute to compromising the intestinal mucosal barrier and functions, opening the way for systemic dissemination.
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Dieta Alta en Grasa , Obesidad , Animales , Ratones , Dieta Alta en Grasa/efectos adversos , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/metabolismo , Aumento de Peso , ColágenoRESUMEN
BACKGROUND AND PURPOSE: Changes in gut microbiota composition, enteric inflammation, impairments of the intestinal epithelial barrier and neuroplastic changes in the enteric nervous system have been reported in Parkinson's disease (PD) patients and could contribute to the onset of both neurological and gastrointestinal symptoms. However, their mutual interplay has rarely been investigated. This study evaluated, in an integrated manner, changes in faecal microbiota composition, morphofunctional alterations of colonic mucosal barrier and changes of inflammatory markers in blood and stools of PD patients. METHODS: Nineteen PD patients and nineteen asymptomatic subjects were enrolled. Blood lipopolysaccharide binding protein (LBP, marker of altered intestinal permeability) and interleukin-1ß (IL-1ß) levels, as well as stool IL-1ß and tumour necrosis factor (TNF) levels, were evaluated. Gut microbiota analysis was performed. Epithelial mucins, collagen fibres, claudin-1 and S100-positive glial cells as markers of an impairment of the intestinal barrier, mucosal remodelling and enteric glial activation were evaluated on colonic mucosal specimens collected during colonoscopy. RESULTS: Faecal microbiota analysis revealed a significant difference in the α-diversity in PD patients compared to controls, while no differences were found in the ß-diversity. Compared to controls, PD patients showed significant chenags in plasma LBP levels, as well as faecal TNF and IL-1ß levels. The histological analysis showed a decrease in epithelial neutral mucins and claudin-1 expression and an increased expression of acidic mucins, collagen fibres and S100-positive glial cells. CONCLUSIONS: Parkinson's disease patients are characterized by enteric inflammation and increased intestinal epithelial barrier permeability, as well as colonic mucosal barrier remodelling, associated with changes in gut microbiota composition.
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Mood alterations, anxiety, and cognitive impairments associated with adult-onset hypothyroidism often persist despite replacement treatment. In rodent models of hypothyroidism, replacement does not bring 3-iodothyronamine (T1AM) brain levels back to normal. T1AM is a thyroid hormone derivative with cognitive effects. Using a pharmacological hypothyroid mouse model, we investigated whether augmenting levothyroxine (L-T4) with T1AM improves behavioural correlates of depression, anxiety, and memory and has an effect on hippocampal neurogenesis. Hypothyroid mice showed impaired performance in the novel object recognition test as compared to euthyroid mice (discrimination index (DI): 0.02 ± 0.09 vs. 0.29 ± 0.06; t = 2.515, p = 0.02). L-T4 and L-T4+T1AM rescued memory (DI: 0.27 ± 0.08 and 0.34 ± 0.08, respectively), while T1AM had no effect (DI: -0.01 ± 0.10). Hypothyroidism reduced the number of neuroprogenitors in hippocampal neurogenic niches by 20%. L-T4 rescued the number of neuroprogenitors (mean diff = 106.9 ± 21.40, t = 4.99, pcorr = 0.003), while L-T4+T1AM produced a 30.61% rebound relative to euthyroid state (mean diff = 141.6 ± 31.91, t = 4.44, pcorr = 0.004). We performed qPCR analysis of 88 genes involved in neurotrophic signalling pathways and found an effect of treatment on the expression of Ngf, Kdr, Kit, L1cam, Ntf3, Mapk3, and Neurog2. Our data confirm that L-T4 is necessary and sufficient for recovering memory and hippocampal neurogenesis deficits associated with hypothyroidism, while we found no evidence to support the role of non-canonical TH signalling.
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Hipotiroidismo , Tiroxina , Ratones , Animales , Tiroxina/metabolismo , Hipotiroidismo/tratamiento farmacológico , Hipotiroidismo/metabolismo , Hipocampo/metabolismo , Suplementos Dietéticos , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismoRESUMEN
Connexins (Cxs) are transmembrane proteins involved in the formation of hemichannels and gap junctions (GJs). GJs are involved in various physiological functions, including secretion in glandular tissue. It has been demonstrated that Cx26, Cx32, and Cx43 are mainly expressed in glands, but no data are available in human salivary glands to date. The aim of our study was to investigate the presence and the localization of Cxs in human minor labial salivary glands. Immunofluorescence and immunoelectron microscopy were employed to evaluate the Cx26, Cx32, and Cx43 protein in human labial salivary gland biopsies (hLSGBs). RT-PCR was also used to detect their mRNA expression. Cx expression was found at both the mRNA and protein levels in all hLSGBs analysed. Cxs were observed at the level of the duct and acinar cells, as well as in myoepithelial cells. The localization of the three Cx types was very similar, suggesting colocalization of these Cxs in the same connexons. These results demonstrated the presence of Cxs in human salivary glands for the first time. Moreover, the few samples with primary Sjögren's Syndrome analysed only by immunofluorescence showed an alteration of the Cx expression, indicating that these proteins could be involved in salivary gland dysfunctions.
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Conexina 43 , Conexinas , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Humanos , Microscopía , ARN Mensajero/metabolismo , Glándulas Salivales Menores/química , Glándulas Salivales Menores/metabolismoRESUMEN
BACKGROUND INFORMATION: Planarians are a sound, well-established model system for molecular studies in the field of stem cells, cell differentiation, developmental biology and translational research. Treated stem cell-less planarians produced by X-ray treatment are commonly used to study stem cell transcriptional profile and their role in planarian biological processes. X-ray induces oxidative and DNA damage to differentiated cells, requires expensive radiation machines that are not available in most of the research centres and demand rigorous risk management and dedicated staff. RESULTS: We tested the use of the well-known antimetabolite genotoxic drug 5-fluorouracil which mainly affects proliferating cells in way to demonstrate its use in replacing X-ray treatment. We succeeded in demonstrating ability of high doses of 5-fluorouracil to deplete Dugesia japonica stem cells and in identifying a 5-fluorouracil transiently resistant population of lineage committed stem cells. CONCLUSIONS AND SIGNIFICANCE: Our results encourage the use of 5-fluorouracil-treated planarians as a model system for studying mechanisms of resistance to genotoxicants, planarian stem cell heterogeneity and molecular cascades of tissue aging.
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Envejecimiento , Diferenciación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Fluorouracilo/farmacología , Planarias/efectos de los fármacos , Células Madre/efectos de los fármacos , AnimalesRESUMEN
To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt-Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development.
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Corazón/crecimiento & desarrollo , MicroARNs/genética , Proteínas de Dominio T Box/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Calcio/metabolismo , Línea Celular , Regulación hacia Abajo/genética , Femenino , Regulación de la Expresión Génica/genética , Factor 4 Similar a Kruppel , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Embarazo , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba/genética , Pez Cebra/metabolismoRESUMEN
BACKGROUND AND AIM: Patients with Parkinson's disease (PD) are often characterized by functional gastrointestinal disorders. Such disturbances can occur at all stages of PD and precede the typical motor symptoms of the disease by many years. However, the morphological alterations associated with intestinal disturbances in PD are undetermined. This study examined the remodelling of colonic wall in 6-hydroxydopamine (6-OHDA)-induced PD rats. METHODS: 8 weeks after 6-OHDA injection animals were sacrificed. Inflammatory infiltrates, collagen deposition and remodelling of intestinal epithelial barrier and tunica muscularis in the colonic wall were assessed by histochemistry, immunohistochemistry, immunofluorescence and western blot analysis. RESULTS: 6-OHDA rats displayed significant alterations of colonic tissues as compared with controls. Signs of mild inflammation (eosinophil infiltration) and a transmural deposition of collagen fibres were observed. Superficial colonic layers were characterized by severe morphological alterations. In particular, lining epithelial cells displayed a reduced claudin-1 and transmembrane 16A/Anoctamin 1 (TMEM16A/ANO1) expression; goblet cells increased their mucin expression; colonic crypts were characterized by an increase in proliferating epithelial cells; the density of S100-positive glial cells and vimentin-positive fibroblast-like cells was increased as well. Several changes were found in the tunica muscularis: downregulation of α-smooth muscle actin/desmin expression and increased proliferation of smooth muscle cells; increased vimentin expression and proliferative phenotype in myenteric ganglia; reduction of interstitial cells of Cajal (ICCs) density. CONCLUSIONS: A pathological remodelling occurs in the colon of 6-OHDA rats. The main changes include: enhanced fibrotic deposition; alterations of the epithelial barrier; activation of mucosal defense; reduction of ICCs. These results indicate that central nigrostriatal denervation is associated with histological changes in the large bowel at mucosal, submucosal and muscular level. These alterations might represent morphological correlates of digestive symptoms in PD.
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Colon/patología , Neuronas Dopaminérgicas/patología , Animales , Anoctamina-1 , Colon/metabolismo , Dopamina/metabolismo , Fibrosis , Enfermedades Gastrointestinales/metabolismo , Motilidad Gastrointestinal , Masculino , Oxidopamina , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Ratas , Ratas Sprague-Dawley , Sustancia NegraRESUMEN
Increasing evidence suggests that intestinal dysfunctions may represent early events in Alzheimer's disease and contribute to brain pathology. This study examined the relationship between onset of cognitive impairment and colonic dysfunctions in a spontaneous AD model before the full development of brain pathology. SAMP8 mice underwent Morris water maze and assessment of faecal output at four, six and eight months of age. In vitro colonic motility was examined. Faecal and colonic Aß, tau proteins, α-synuclein and IL-1ß were assessed by ELISA. Colonic citrate synthase activity was assessed by spectrophotometry. Colonic NLRP3, caspase-1 and ASC expression were evaluated by Western blotting. Colonic eosinophil density and claudin-1 expression were evaluated by immunohistochemistry. The effect of Aß on NLRP3 signalling and mitochondrial function was tested in cultured cells. Cognitive impairment and decreased faecal output occurred in SAMP8 mice from six months. When compared with SAMR1, SAMP8 animals displayed: (1) impaired in vitro colonic contractions; (2) increased enteric AD-related proteins, IL-1ß, active-caspase-1 expression and eosinophil density; and (3) decreased citrate synthase activity and claudin-1 expression. In THP-1 cells, Aß promoted IL-1ß release, which was abrogated upon incubation with caspase-1 inhibitor or in ASC-/- cells. Aß decreased mitochondrial function in THP-1 cells. In SAMP8, enteric AD-related proteins deposition, inflammation and impaired colonic excitatory neurotransmission, occurring before the full brain pathology development, could contribute to bowel dysmotility and represent prodromal events in AD.
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Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Colon/patología , Colon/fisiopatología , Motilidad Gastrointestinal , Inflamación/patología , Proteínas del Tejido Nervioso/metabolismo , Síntomas Prodrómicos , Péptidos beta-Amiloides/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/metabolismo , Claudina-1/metabolismo , Cognición , Eosinófilos/patología , Heces , Conducta Alimentaria , Humanos , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Mucosa Intestinal/patología , Ratones , Mitocondrias/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Agregado de Proteínas , Células THP-1 , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMEN
BACKGROUND: The murine model of high fat diet (HFD)-induced obesity is characterized by an increment of intestinal permeability, secondary to an impairment of mucosal epithelial barrier and enteric inflammation, followed by morphofunctional rearrangement of the enteric nervous system. The present study investigated the involvement of abdominal macrophages in the mechanisms underlying the development of enteric dysmotility associated with obesity. METHODS: Wild type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD, 18% kcal from fat) for 8 weeks. Groups of mice fed with NCD or HFD were treated with clodronate encapsulated into liposomes to deplete abdominal macrophages. Tachykininergic contractions, elicited by electrical stimulation or exogenous substance P (SP), were recorded in vitro from longitudinal muscle colonic preparations. Substance P distribution was examined by confocal immunohistochemistry. The density of macrophages in the colonic wall was examined by immunohistochemical analysis. Malondialdehyde (MDA, colorimetric assay) and IL-1ß (ELISA assay) levels were also evaluated. RESULTS: MDA and IL-1ß levels were increased in colonic tissues from HFD-treated animals. In colonic preparations, electrically evoked tachykininergic contractions were enhanced in HFD mice. Immunohistochemistry displayed an increase in substance P immunoreactivity in myenteric ganglia, as well as in the muscular layers of colonic cryosections from obese mice. Macrophage depletion in HFD mice was associated with a significant reduction of colonic inflammation. In addition, the decrease in macrophage density attenuated the morphofunctional alterations of tachykininergic pathways observed in obese mice. CONCLUSION: Obesity elicited by HFD determines a condition of colonic inflammation, followed by a marked rearrangement of motor excitatory tachykininergic enteric nerves. Macrophage depletion counteracted the morphofunctional changes of colonic neuromuscular compartment, suggesting a critical role for these immune cells in the onset of enteric dysmotility associated with obesity.
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Colon , Dieta Alta en Grasa/efectos adversos , Inflamación/fisiopatología , Obesidad , Animales , Peso Corporal , Colon/citología , Colon/patología , Colon/fisiopatología , Enfermedades del Colon/fisiopatología , Motilidad Gastrointestinal/fisiología , Interleucina-1beta/análisis , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Masculino , Malondialdehído/análisis , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/fisiopatologíaRESUMEN
Objective- Arginase can reduce NO availability. In this study, we explored arginase as a determinant of endothelial dysfunction in small arteries from obese patients and its relationship with aging and microvascular remodeling. Approach and Results- Small arteries were dissected after subcutaneous fat biopsies and evaluated on a pressurized micromyograph. Endothelium-dependent vasodilation was assessed by acetylcholine, repeated under L-NAME ( N G-nitro-L-arginine-methyl ester), N(ω)-hydroxy-nor-l-arginine (arginase inhibitor) and gp91ds-tat (NADPH [nicotinamide adenine dinucleotide phosphate oxidase] oxidase inhibitor) in vessels from young (age <30 years) control and obese and old (>30 years) control and obese subjects. Media-lumen ratio and amount of vascular wall fibrosis were used as markers of vascular remodeling. Amount of vascular superoxide anions and NO production were determined with immunofluorescence, whereas arginase expression was quantified using Western blot and quantitative polymerase chain reaction. Obese and older age groups had lower vascular NO, as well as higher media-lumen ratio, wall fibrosis, intravascular superoxide, and blunted inhibitory effect of L-NAME on acetylcholine versus controls and younger age groups. N(ω)-hydroxy-nor-l-arginine restored the acetylcholine-induced vasodilation in young and, to a lesser extent, in old obese subjects. This effect was abolished by addition of L-NAME. Gp91ds-tat increased the vasodilatory response to N(ω)-hydroxy-nor-l-arginine in old obese. Superoxide anions and arginase I/II levels were higher in the vascular wall of obese versus controls. Conclusions- Arginase contributes to microvascular endothelial dysfunction in obesity. Its impact is reduced by aging because of higher levels of vascular oxidative stress. Obesity is accompanied by accelerated microvascular remodeling, the extent of which is related to the amount of arginase in the vascular wall.
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Envejecimiento/metabolismo , Arginasa/metabolismo , Arterias/enzimología , Óxido Nítrico/metabolismo , Obesidad/enzimología , Grasa Subcutánea/irrigación sanguínea , Vasodilatación , Adulto , Factores de Edad , Arginasa/antagonistas & inhibidores , Arterias/efectos de los fármacos , Arterias/fisiopatología , Estudios de Casos y Controles , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , NADPH Oxidasas/metabolismo , Obesidad/diagnóstico , Obesidad/fisiopatología , Estrés Oxidativo , Transducción de Señal , Superóxidos/metabolismo , Remodelación Vascular , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Adulto JovenRESUMEN
Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.
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Neoplasias Cardíacas/patología , Captura por Microdisección con Láser/métodos , Microscopía Confocal/métodos , Miocardio/patología , Mixoma/patología , Actinas/biosíntesis , Actinas/genética , Adulto , Anciano , Anciano de 80 o más Años , Calbindina 2/biosíntesis , Calbindina 2/genética , Diferenciación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Neoplasias Cardíacas/genética , Neoplasias Cardíacas/cirugía , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Miocardio/metabolismo , Mixoma/genética , Mixoma/cirugía , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , ARN Neoplásico/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Adenosine A2B receptors (A2BR) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A2BR in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A2BR localization was examined by immunofluorescence. The role of A2BR in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A2BR were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A2BR antagonist MRS1754 enhanced electrically induced NK1-mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A2B receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A2BR ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A2BR expression. A2BR, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.
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Dieta Alta en Grasa/efectos adversos , Motilidad Gastrointestinal/fisiología , Obesidad/metabolismo , Receptor de Adenosina A2B/metabolismo , Animales , Colon/metabolismo , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/complicacionesRESUMEN
BACKGROUND: Parkinson's disease (PD) is frequently associated with gastrointestinal (GI) symptoms, including constipation and defecatory dysfunctions. The mechanisms underlying such disorders are still largely unknown, although the occurrence of a bowel inflammatory condition has been hypothesized. This study examined the impact of central dopaminergic degeneration, induced by intranigral injection of 6-hydroxydopamine (6-OHDA), on distal colonic excitatory tachykininergic motility in rats. METHODS: Animals were euthanized 4 and 8 weeks after 6-OHDA injection. Tachykininergic contractions, elicited by electrical stimulation or exogenous substance P (SP), were recorded in vitro from longitudinal muscle colonic preparations. SP, tachykininergic NK1 receptor, and glial fibrillary acidic protein (GFAP) expression, as well as the density of eosinophils and mast cells in the colonic wall, were examined by immunohistochemical analysis. Malondialdehyde (MDA, colorimetric assay), TNF, and IL-1ß (ELISA assay) levels were also examined. The polarization of peritoneal macrophages was evaluated by real-time PCR. RESULTS: In colonic preparations, electrically and SP-evoked tachykininergic contractions were increased in 6-OHDA rats. Immunohistochemistry displayed an increase in SP and GFAP levels in the myenteric plexus, as well as NK1 receptor expression in the colonic muscle layer of 6-OHDA rats. MDA, TNF, and IL-1ß levels were increased also in colonic tissues from 6-OHDA rats. In 6-OHDA rats, the number of eosinophils and mast cells was increased as compared with control animals, and peritoneal macrophages polarized towards a pro-inflammatory phenotype. CONCLUSIONS: The results indicate that the induction of central nigrostriatal dopaminergic degeneration is followed by bowel inflammation associated with increased oxidative stress, increase in pro-inflammatory cytokine levels, activation of enteric glia and inflammatory cells, and enhancement of colonic excitatory tachykininergic motility.
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Dopamina/metabolismo , Enfermedades Gastrointestinales/etiología , Motilidad Gastrointestinal/fisiología , Enfermedades Neurodegenerativas/complicaciones , Receptores de Neuroquinina-1/metabolismo , Animales , Benzoxazoles/farmacología , Modelos Animales de Enfermedad , Sistema Nervioso Entérico/efectos de los fármacos , Sistema Nervioso Entérico/fisiología , Eosinófilos/patología , Motilidad Gastrointestinal/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Indoles/farmacología , Masculino , Mastocitos/patología , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/patología , Oxidopamina/toxicidad , Piperidinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Neuroquinina-1/genética , Sustancia P/metabolismo , Sustancia P/farmacología , Simpaticolíticos/toxicidad , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Parkinson's disease is frequently associated with gastrointestinal symptoms, mostly represented by constipation and defecatory dysfunctions. This study examined the impact of central dopaminergic denervation, induced by injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle, on distal colonic excitatory cholinergic neuromotor activity in rats. Animals were euthanized 4 and 8 weeks after 6-OHDA injection. In vivo colonic transit was evaluated by radiologic assay. Electrically induced and carbachol-induced cholinergic contractions were recorded in vitro from longitudinal and circular muscle colonic preparations, whereas acetylcholine levels were assayed in the incubation media. Choline acetyltransferase (ChAT), HuC/D (pan-neuronal marker), muscarinic M2 and M3 receptors were assessed by immunohistochemistry or western blot assay. As compared with control rats, at week 4, 6-OHDA-treated animals displayed the following changes: decreased in vivo colonic transit rate, impaired electrically evoked neurogenic cholinergic contractions, enhanced carbachol-induced contractions, decreased basal and electrically stimulated acetylcholine release from colonic tissues, decreased ChAT immunopositivity in the neuromuscular layer, unchanged density of HuC/D immunoreactive myenteric neurons, and increased expression of colonic muscarinic M2 and M3 receptors. The majority of such alterations were also detected at week 8 post 6-OHDA injection. These findings indicate that central nigrostriatal dopaminergic denervation is associated with an impaired excitatory neurotransmission characterized by a loss of myenteric neuronal ChAT positivity and decrease in acetylcholine release, resulting in a dysregulated smooth muscle motor activity, which likely contributes to the concomitant decrease in colonic transit rate.
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Acetilcolina/metabolismo , Colon/diagnóstico por imagen , Colon/metabolismo , Sistema Nervioso Entérico/diagnóstico por imagen , Sistema Nervioso Entérico/metabolismo , Motilidad Gastrointestinal/fisiología , Trastornos Parkinsonianos/diagnóstico por imagen , Animales , Neuronas Colinérgicas/diagnóstico por imagen , Neuronas Colinérgicas/metabolismo , Masculino , Técnicas de Cultivo de Órganos , Radiografía , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/fisiologíaRESUMEN
AIMS: We assessed whether acute intra-arterial infusion of exogenous ghrelin can improve endothelial dysfunction by restoring nitric oxide (NO) availability in the forearm microcirculation of essential hypertensive patients. The effect of ghrelin on endothelial dysfunction (pressurized myograph), vascular oxidative stress generation (fluorescent dihydroethidium), and phosphorylation of p47phox (western blot), an index of NAD(P)H oxidase activation, in isolated small arteries taken from essential hypertensive patients (subcutaneous biopsy) were also investigated. METHODS AND RESULTS: In 18 normotensive control subjects and 18 essential hypertensive patients, we studied the forearm blood flow (strain-gauge plethysmography) response to intra-arterial acetylcholine, repeated under NO synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA) or the antioxidant ascorbic acid. The protocol was repeated at the end of exogenous ghrelin intra-arterial infusion. In hypertensive patients, ghrelin normalized the blunted response to acetylcholine, restored the inhibiting effect of l-NMMA and abrogated the potentiating effect of ascorbic acid on acetylcholine. In controls, ghrelin failed to modify these vascular responses. In hypertensive patients, ghrelin decreased venous levels of malondialdehyde, lipoperoxide, and interleukin-6, and concomitantly increased endogenous antioxidant capacity. Small vessels from hypertensive patients showed an enhanced intravascular oxidative stress, which was strongly and similarly decreased by incubation with ghrelin, the NAD(P)H oxidase inhibitor gp91 ds-tat, or both. Ghrelin also normalized the overexpression of p47 phosphorylation and restored the NO availability in small vessels from hypertensive patients. CONCLUSIONS: Exogenous ghrelin increases endothelial dysfunction by restoring NO availability in the forearm microcirculation of essential hypertensive patients, an effect ascribable to an antioxidant effect via inhibition of NAD(P)H oxidase activation.
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Ghrelina/farmacología , Hipertensión/fisiopatología , NADPH Oxidasas/fisiología , Óxido Nítrico/biosíntesis , Vasodilatadores/farmacología , Análisis de Varianza , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Estudios de Casos y Controles , Endotelio Vascular , Inhibidores Enzimáticos/farmacología , Femenino , Antebrazo/irrigación sanguínea , Ghrelina/administración & dosificación , Humanos , Hipertensión/enzimología , Infusiones Intraarteriales , Masculino , Persona de Mediana Edad , NADPH Oxidasas/metabolismo , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Vasodilatadores/administración & dosificación , omega-N-Metilarginina/farmacologíaRESUMEN
Bowel inflammatory fibrosis has been largely investigated, but an integrated assessment of remodelling in inflamed colon is lacking. This study evaluated tissue and cellular changes occurring in colonic wall upon induction of colitis, with a focus on neuromuscular compartment. Colitis was elicited in rats by 2,4-dinitrobenzenesulfonic acid (DNBS). After 6 and 21 days, the following parameters were assessed on paraffin sections from colonic samples: tissue injury and inflammatory infiltration by histology; collagen and elastic fibres by histochemistry; HuC/D, glial fibrillar acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), nestin, substance P (SP), von Willebrand factor, c-Kit and transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) by immunohistochemistry. TMEM16A/ANO1 was also examined in isolated colonic smooth muscle cells (ICSMCs). On day 6, inflammatory alterations and fibrosis were present in DNBS-treated rats; colonic wall thickening and fibrotic remodelling were evident on day 21. Colitis was associated with both an increase in collagen fibres and a decrease in elastic fibres. Moreover, the neuromuscular compartment of inflamed colon displayed a significant decrease in neuron density and increase in GFAP/PCNA-positive glia of myenteric ganglia, enhanced expression of neural SP, blood vessel remodelling, reduced c-Kit- and TMEM16A/ANO1-positive interstitial cells of Cajal (ICCs), as well as an increase in TMEM16A/ANO1 expression in muscle tissues and ICSMCs. The present findings provide an integrated view of the inflammatory and fibrotic processes occurring in the colonic neuromuscular compartment of rats with DNBS-induced colitis. These morphological alterations may represent a suitable basis for understanding early pathophysiological events related to bowel inflammatory fibrosis.
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Colitis/patología , Miocitos del Músculo Liso/patología , Animales , Colon/patología , Inflamación/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Starvation resistance is a life-saving mechanism for many organisms facing food availability fluctuation in the natural environment. Different strategies have been episodically identified for some model organisms, the first of which was the ability to suppress metabolic rate. Among the identified strategies, the ability of planarians to shrink their body under fasting conditions and revert the process after feeding (the growth-degrowth process) represents a fascinating mechanism to face long periods of fasting. The growth-degrowth process is strictly related to the capability of planarians to continuously maintain tissue homeostasis and body proportions even in challenging conditions, thanks to the presence of a population of pluripotent stem cells. Here, we take advantage of several previous studies describing the growth-degrowth process and of recent progress in the understanding of planarian homeostasis mechanisms, to identify tissue-selective transcriptional downregulation as a driving strategy for the development of a thrifty phenotype, and the p53 transcription factor as a player in adjusting tissue homeostasis in accordance with food availability.
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Planarias , Animales , Planarias/genética , Ayuno , Regulación hacia Abajo , Fenotipo , Factores de TranscripciónRESUMEN
BACKGROUND: Empagliflozin (EMPA), a selective sodium-glucose cotransporter type 2 (SGLT2) inhibitor, has been shown to reduce major adverse cardiovascular events in patients with heart failure of different etiologies, although the underlying mechanism still remains unclear. Ponatinib (PON) is a multi-tyrosine kinase inhibitor successfully used against myeloid leukemia and other human malignancies, but its cardiotoxicity remains worrisome. Cardiac connexins (Cxs) are both substrates and regulators of autophagy and responsible for proper heart function. Alteration in connexin expression and localization have been described in patients with heart failure. AIMS: To assess whether EMPA can mitigate PON-induced cardiac dysfunction by restoring the connexin 43-autophagy pathway. METHODS AND RESULTS: Male C57BL/6 mice, randomized into four treatment groups (CNTRL, PON, EMPA, PON+EMPA) for 28 days, showed increased autophagy, decreased Cx43 expression as well as Cx43 lateralization, and attenuated systo-diastolic cardiac dysfunction after treatment with EMPA and PON compared with PON alone. Compared with CNTRL (DMSO), cardiomyocyte-differentiated H9c2 (dH9c2) cells treated with PON showed significantly reduced cell viability to approximately 20â¯%, decreased autophagy, increased cell senescence and reduced DNA binding activity of serum response factor (SRF) to serum response elements (SRE), which were paralleled by reduction in cardiac actin expression. Moreover, PON induced a significant increase of Cx43 protein and its S368-phosphorylated form (pS368-Cx43), as well as their displacement from the plasma membrane to the perinuclear and nuclear cellular region. All these effects were reverted by EMPA. CONCLUSION: EMPA attenuates PON-induced cardiotoxicity by reducing senescence, enhancing the SRE-SRF binding and restoring the connexin 43-autophagy pathway. This effect may pave the way to use of SGLT2 inhibitors in attenuating tyrosine-kinase inhibitor cardiotoxicity.