Clinical and Cytokine Profile in Patients with Early and Late Onset Meniere Disease

Background: Meniere disease (MD) is an inner ear disorder associated with comorbidities such as autoimmune diseases or migraine. This study describes clinical and cytokine profiles in MD according to the age of onset of the condition. Methods: A cross-sectional study including 83 MD patients: 44 with early-onset MD (EOMD, <35 years old), and 39 with late-onset MD (LOMD, >50 years old), 64 patients with migraine and 55 controls was carried out. Clinical variables and cytokines levels of CCL3, CCL4, CCL18, CCL22, CXCL,1 and IL-1β were compared among the different groups. Results: CCL18 levels were higher in patients with migraine or MD than in controls. Elevated levels of IL-1β were observed in 11.4% EOMD and in 10.3% LOMD patients and these levels were not dependent on the age of individuals. EOMD had a longer duration of the disease (p = 0.004) and a higher prevalence of migraine than LOMD (p = 0.045). Conclusions: Patients with EOMD have a higher prevalence of migraine than LOMD, but migraine is not associated with any cytokine profile in patients with MD. The levels of CCL18, CCL3, and CXCL4 were different between patients with MD or migraine and controls.ISCIII and European Regional Funds (Grants PI17/01644 and PI20/01126)Andalusian Health Government (Grant PE-0356-2018).Andalusian Health Government (Grant PI-0027-2020)

Efecto de la inhibición de PARP-1 sobre angiogénesis, metástasis y mimetismo vasculogénico en melanoma

La inhibición de PARP pueden inducir efectos anti tumorales cuando se usa como monoterapia o en combinación con la quimioterapia o la radioterapia en tumores diversos ajustes, sin embargo, la base para las actividades contra la metástasis como resultado de la inhibición de PARP sigue siendo una incógnita. El análisis proteómico de las células endoteliales reveló que la vimentina, un filamento de intermediarios involucrados en la angiogénesis y el marcador específico del EndMT (transición endotelio mesénquimal), disminuye bajo la pérdida de la función de PARP-1 en las células endoteliales. Asimismo, en este trabajo se observó que la VE-cadherina, un marcador endotelial vascular de la normalización, se reguló en HUVEC bajo tratamientos con inhibidores de PARP o el silenciamiento de PARP-1. En las células del melanoma, la inhibición de PARP fué capaz de reducir marcadores pro-angiogénicos, tanto en cultivos celulares como en modelos in vivo Hemos demostrado que la vimetina por sí sola es suficiente para inducir el aumento de los efectos fenotípicos mesenquimales / prometastasicos en las células del melanoma, incluyendo OLK/GSK 3¿b dependiente E-cadherina bajo regulada, Snail 1 junto a la activación y la motilidad celular y el aumentó de la migración. En un modelo murino de melanoma metastásico, la inhibición de PARP puede contrarrestar la capacidad de las células del melanoma para formar metástasis en el pulmón. Los ratones implantados con células de melanoma metastático, que fueron tratados también con el inhibidor de PARP DPQ, exhibieron una drástica disminución tanto en la metástasis de pulmón como en la angiogénesis tumoral en comparación con el grupo de ratones control. El grupo de ratones tratados también experimentó un aumento de la supervivencia en comparación con el grupo control. Un silenciamiento estable de PARP-1 en células de melanoma también dio lugar a un aumento significativo en la supervivencia. Estos resultados sugieren que la inhibición de PARP interfiere con los procesos clave que promueven la metástasis, como el cambio en el fenotipo que permite a las células de melanoma adquirir propiedades invasivas.Tesis Univ. Granada. Departamento de Bioquímica y Biología Molecular III e Inmunologí

ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy

In response to nutrient stress, cells start an autophagy program that can lead to adaptation or death. The mechanisms underlying the signaling from starvation to the initiation of autophagy are not fully understood. In the current study we show that the absence or inactivation of PARP-1 strongly delays starvation-induced autophagy. We have found that DNA damage is an early event of starvation-induced autophagy as measured by γ-H2AX accumulation and comet assay, with PARP-1 knockout cells displaying a reduction in both parameters. During starvation, ROS-induced DNA damage activates PARP-1, leading to ATP depletion (an early event after nutrient deprivation). The absence of PARP-1 blunted AMPK activation and prevented the complete loss of mTOR activity, leading to a delay in autophagy. PARP-1 depletion favors apoptosis in starved cells, suggesting a pro-survival role of autophagy and PARP-1 activation after nutrient deprivation. In vivo results show that neonates of PARP-1 mutant mice subjected to acute starvation, also display deficient liver autophagy, implying a physiological role for PARP-1 in starvation-induced autophagy. Thus, the PARP signaling pathway is a key regulator of the initial steps of autophagy commitment following starvation

PARP-1 regulates metastatic melanoma through modulation of vimentin-induced malignant transformation

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-β-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.This work was supported by Ministerio de Ciencia e Innovación SAF2006-01094, SAF2009-13281-C02-01, Fundación La Caixa BM06-219-0 and Junta de Andalucía P07-CTS-0239 and CTS-6602 to FJO, Ministerio de Educación y Ciencia SAF2007-64597; CICYT: SAF2009-13281-C02-02; Junta de Andalucía, P06-CTS-01385 to JMRdA and grants CEIC (P10-CTS5865) and FEDER-ISCIII (PI10/00883) to JCR-M. AGdH has been funded by grants from ‘‘Fundación Científica de la Asociación Española Contra el Cáncer’’, Ministerio de Ciencia y Tecnología SAF2010-16089, and ‘‘Fundación La Marató de TV3’’. JCR-M has been funded by Grants CEIC (P1=-CTS5865) and FEDER-ISCIII (PI10/00883). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptBaulida and García de Herreros' labs is supported by the Instituto de Salud Carlos III (PI12/ 00257 and D012/0036/0005, part of the Plan Nacional I+D+ I and cofounded by the ISCIII-Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional-FEDER), the Fundación Científica Asociación Española Contra el Cáncer (Ayudas a grupos estables de investigación, 2010-1) and the Ministerio de Economía y Competitividad (SAF2013-4889-C2-1R)

PARP inhibition down-regulates vimentin expression and inhibits endothelial-to-mesenchymal transition in HUVECs.

<p>Cell extracts from HUVEC either treated with vehicle or 40 µM DPQ were subjected to 2D electrophoresis as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003531#s4" target="_blank">Materials and Methods</a>. Image analysis software (DeCyder) indicated that seven proteins exhibited decreased expression in HUVEC treated with DPQ compared to untreated cells. Proteins were identified using MALDI-TOF. Spots labeled with arrows indicate proteins that were identified by mass spectrometry (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003531#pgen-1003531-g002" target="_blank">Figure 2</a>). (<b>A</b>) The spot with the arrow is vimentin. (<b>B</b>) PARP inhibition reduced the expression of both vimentin and Snail1 and up-regulated VE-cadherin in human endothelial cells (HUVEC) as determined by immunoblotting, indirect immunofluorescence (<b>C</b>), and mRNA levels (<b>D</b>). PARP inhibition decreased HUVEC cell migration (<b>E</b>). (**<i>P</i><0.01, ***<i>P<</i>0.001 PARP inhibitor groups <i>versus</i> DPQ).</p

PARP inhibition inhibits the acquisition of an EMT phenotype in malignant melanoma cells.

<p>Human melanoma G361 cells and murine B16- F10 melanoma cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003531#pgen.1003531.s003" target="_blank">Figure S3</a>) were used for these experiments. Cells were treated with either DPQ (40 µM), PJ-34 (10 µM) or KU0058948 (100 nM) for 22 hours. IF, western blot or qPCR assays were performed to evaluate the effects of PARP inhibition on EMT markers. PARP inhibition reduced the expression of vimentin and Snail1 and up-regulated E-cadherin in human melanoma cells as determined by immunoblotting (<b>A</b>), indirect immunofluorescence (<b>B</b>), and mRNA levels (<b>C</b>). (*<i>P<</i>0.05, ***<i>P<</i>0.001, PARP Inhibitor groups <i>versus</i> the control). β-actin was used as an internal control for protein loading. (<b>D</b>) Snail1 and E-cadherin promoter activity are regulated by PARP inhibitors. Luciferase activity was determined after transfecting the constructions into G361 cells. Firefly Luciferase was standarized to the levels of Renilla Luciferase. Cells were cotransfected with 0.5 µg renilla as a transfection control and 0.5 µg of Snail1 or E-cadherin using jetPEI cationic polymer transfection reagent according to the manufacturer's instructions. Cells were compared in the presence or absence of serum (***<i>P<</i>0.001 control <i>versus</i> PJ-34). The expression of both Firefly and Renilla luciferase was analyzed 48 h after transfection. Cloning of the human Snail1 promoter (−869/+59) into pGL3 basic (Promega) was described previously (41). The E-Cadherin promoter was cloned into pGL3-basic (Promega) to generate pGL3-E-cadherin (−178/+92). (<b>E</b>) Inhibitory effect of PARP on B16F10 motility. Treatment with the PARP inhibitor PJ-34 (10 µM) decreased cell migration in vitro. Migration was quantified as distance between Wound Healing limits (*** <i>P<</i>0.001 control <i>versus</i> DPQ).</p

Decreased melanoma-induced lung metastasis following PARP inhibition.

<p>(<b>A</b>) Mice were inoculated with the murine melanoma cell line B16-F10-luc. Localization and the intensity of luciferase expression were monitored by <i>in vivo</i> bioluminescence imaging (dpi, days post cells injection). At the bottom of Figure A two lungs from vehicle (left) or DPQ (right) treated mice are shown. Lungs were extracted to analyze the number of melanoma foci. Quantification of luciferase activity over time shows the average light (photons) emission in photons/s (<b>B</b>) (**P<0.01; ***P<0.001 <i>versus</i> DPQ). (<b>C</b>) The number of metastatic foci/lung were counted macroscopically (***<i>P<</i>0.001). (<b>D</b>) Angiogenesis was measured using a specific endothelial cell marker (tomato lectin) and measured as blood vessels per mm² in tumor sections of lung metastasis (Columns, mean ± SE. *<i>P<</i>0.05, with respect to control and DPQ–treated mice. (<b>E</b>) Immunohistochemistry staining of Snail1 and E-Cadherin in lung metastasis and quantitation using ImageJ , colour deconvolution plugin (<b>F and G</b>) Kaplan-Meyer survival curve shows the survival advantage of DPQ-treated mice following intravenous tail injection of melanoma cells as previously described in mice treated with DPQ (<b>F</b>) or injected with B16F10 stably silenced for PARP-1 (<b>G</b>) (** <i>P<</i>0. 01).</p

Proteins differentially expressed and identified by mass spectrometry analysis in HUVEC.

<p>The level of expression of various proteins in HUVEC was altered following PARP inhibition as determined by 2D-DIGE, and the proteins were positively identified using mass spectrometry analysis. Of particular interest for this study was vimentin, the major structural protein of intermediary filaments (spot 1). Expression of this protein was decreased in HUVEC following PARP inhibition. The proteins were identified by MALDI-TOF. Sequence coverage (%) and number of peptides were identified with  = 1% FDR (false discovery rate cut-off against decoy-concatenated randomized database). Coverage and score was determined using the MASCOT algorithm. The average ratio of protein expression between the control and cells treated with the PARP inhibitor DPQ was determined in HUVEC.</p

Interaction between vimentin over-expression and the activation of EMT signaling pathway.

<p>(<b>A</b>) Over-expression of vimentin in G361 cells. (<b>B</b>) Forced expression of vimentin drives human breast tumor epithelial cells (MCF7) to a mesenchymal phenotype through the integrin-linked-kinase/GSK-3β axis. 5 mM LiCl was used to inhibit GSK-3β, as detected by the accumulation of beta-catenin. (<b>C</b>) ILK was knocked down to analyze the significance of the interaction between vimentin and ILK in promoting the transition to a mesenchymal phenotype.</p

PARP-1 or vimentin is sufficient to reverse EMT and confer increased cell motility.

<p>(<b>A</b>) Melanoma (G361) and endothelial (HUVEC) (<b>B</b>) cells were silenced for PARP-1 or vimentin and the expression levels of Axl, E-/VE-cadherin, Snail1, ILK, β-catenin, GSK-3β, PARP-1, and vimentin were determined by immunoblot. (<b>C</b>) HUVEC were silenced for vimentin and wound healing was measured. After over-expression of vimentin wound healing closure was measured in HUVEC cells (<b>D</b>) or B16-F10 (<b>E</b>). (<b>F</b>) Cell migration was analyzed in epithelial cell line Madin Darby canine kidney (MDCK) cells transfected with either GFP or GFP-vimentin using video-microscopy and MetaMorph Image Analysis software. While vimentin was able to increase the length of the trajectories in the absence or presence of hepatocyte growth factor (HGF), treatment with PARP inhibitor resulted in a sustained reduction in cell motility (*<i>P<</i>0.05 PJ-34 or olaparib <i>versus</i> control).</p