Induzione di switch angiogenico e sua caratterizzazione mediante l'analisi di espressione di microRNA (miRNA signature)

I microRNA, brevi filamenti di acido ribonucleico lunghi 21-25 nucleotidi presenti in tutti gli organismi eucarioti, agiscono da silenziatori dell'espressione genica tramite un appaiamento imperfetto al 3'UTR di un mRNA bersaglio. Questo tipo di regolazione genica a livello post trascrizionale è estremamente fine e modulato, poiché i microRNA agiscono in concerto, al fine di mantenere una corretta omeostasi cellulare, in maniera combinatoria e cooperativa. A tutt'oggi si conoscono circa 500 microRNA umani che hanno un caratteristico profilo di espressione detto “miRNA signature”, specifico non solo di stadi di sviluppo e di differenziamento, ma anche di diversi tipi di tumori o di altre patologie. Lo scopo di questa tesi è stato quello di verificare se la miRNA signature può essere usata per caratterizzare la disfunzione endoteliale. Per fare questo abbiamo coltivato le cellule endoteliali umane HUVEC in presenza di concentrazioni crescenti di glucosio (range 5-40mM). Dalla curva di crescita è risultato che il glucosio riduce la proliferazione di HUVEC in maniera dose dipendente e che ad alte concentrazioni esso diventa citotossico. L’effetto dello stress da glucosio sulle proprietà angiogenetiche è stato studiato in HUVEC esposte a 30mM di glucosio (AG30). Abbiamo visto una riduzione della capacità di formare microtubi su matrice, come pure di saldare una discontinuità in colture di HUVEC confluenti. Per caratterizzare questo fenotipo complesso abbiamo estratto l’RNA totale da cellule di controllo e AG30. I due campioni di RNA sono stati prima marcati con fluorocromi diversi e poi ibridati insieme sul microarray miRCURY LNA Exiqon. La sovrapposizione dei segnali di ibridazione ottenuti ha messo in evidenza la sovraespressione di 13 miRNA e la sottoespressione di 3, suggerendo che i microRNA sono dei sensori in grado di misurare lo stress da glucosio nelle HUVEC. L’identificazione delle potenziali proteine target dei miRNA overespressi è stata fatta mediante software specifici, mentre la validazione sarà oggetto di studi futuri. Attualmente stiamo verificando se i miRNA trovati sotto o sovraespressi sono specifici dello stress da glucosio. Per far questo abbiamo preso in esame lo stress infiammatorio che nelle HUVEC può essere indotto dopo un’esposizione di 24 ore a lipoproteine a bassa densità (low density lipoprotein, LDL) ossidate alla concentrazione di 200μg/ml. Il fenotipo ottenuto è molto simile a quello indotto da stress da glucosio: ridotta capacità di migrazione e di angiogenesi. L’analisi del microarray è attualmente in corso. Dal confronto tra la miRNA signature che otterremo dopo trattamento con LDL ossidate e quella ottenuta dopo trattamento con glucosio si potrà capire se i due tipi di stress, che inducono però un fenotipo simile nelle cellule endoteliali, sono di fatto distinguibili l’uno dall’altro, gettando così le basi per uno studio sistematico delle proteine sotto il controllo dei microRNA e coinvolte nella risposta allo stress

MicroRNAs mediate metabolic stresses and angiogenesis

MicroRNAs are short endogenous RNA molecules that are able to regulate (mainly inhibiting) gene expression at the post-transcriptional level. The MicroRNA expression profile is cell-specific, but it is sensitive to perturbations produced by stresses and diseases. Endothelial cells subjected to metabolic stresses, such as calorie restriction, nutrients excess (glucose, cholesterol, lipids) and hypoxia may alter their functionality. This is predictive for the development of pathologies like atherosclerosis, diabetes, and hypertension. Moreover, cancer cells can activate a resting endothelium by secreting pro-angiogenic factors, in order to promote neoangiogenesis, which is essential for tumor growth. Endothelial altered phenotype is mirrored by altered mRNA, microRNA, and protein expression, with a microRNA being able to control pathways by regulating the expression of multiple mRNAs. In this review we will consider the involvement of microRNAs in modulating the response of endothelial cells to metabolic stresses and their role in promoting or halting angiogenesis

The RNA Activator ds-p21 Potentiates the Cytotoxicity Induced by Fludarabine in Dohh2 Cells

Recently, it has been reported that, in several tumor cell lines, short double-stranded RNAs tailored for promoter regions of specific genes are able to activate their transcription. Such molecules (named RNA activators) act opposite to other double-stranded RNA molecules (named RNA inhibitors) in that the overexpression instead of underexpression of a given gene is triggered. In Dohh2 non-Hodgkin lymphoma cells, the transcriptional repressor BCL6, which negatively controls both p53 and p21, is overexpressed, so that the cells can escape the check point governed by p53 and proliferate. The aim of this work was to investigate whether the RNA activator p21 can represent a tool to circumvent the transcriptional control of BCL6 and induce the blockage of cell proliferation in Dohh2 non-Hodgkin lymphoma cells. For that, Dohh2 cells were transfected with either a control RNA activator (ds-NC) or an RNA activator specific for human p21 promoter (ds-p21). At various time points after transfection, the cells were collected and p21 was measured. Dohh2 cells transfected with ds-p21 showed a slight but significant overexpression of p21 at both mRNA and protein levels. Nonetheless, cell proliferation, cell cycle, and apoptosis were not significantly modified. In contrast, the exposure of Dohh2 cells transfected with ds-p21 to fludarabine potentiates the cytotoxicity of the drug, suggesting the RNA activator p21 complements the fludarabine-dependent cell death pathways

In-depth proteomics identifies a role for autophagy in controlling reactive oxygen species mediated endothelial permeability

Endothelial cells (ECs) form the inner layer of blood vessels and physically separate the blood from the surrounding tissue. To support tissues with nutrients and oxygen, the endothelial monolayer is semipermeable. When EC permeability is altered, blood vessels are not functional, and this is associated with disease. A comprehensive knowledge of the mechanisms regulating EC permeability is key in developing strategies to target this mechanism in pathologies. Here we have used an in vitro model of human umbilical vein endothelial cells mimicking the formation of a physiologically permeable vessel and performed time-resolved in-depth molecular profiling using stable isotope labeling by amino acids in cell culture mass spectrometry (MS)-proteomics. Autophagy is induced when ECs are assembled into a physiologically permeable monolayer. By using siRNA and drug treatment to block autophagy in combination with functional assays and MS proteomics, we show that ECs require autophagy flux to maintain intracellular reactive oxygen species levels, and this is required to maintain the physiological permeability of the cells

The zebrafish/tumor xenograft angiogenesis assay as a tool for screening anti-angiogenic miRNAs

The zebrafish/tumor xenograft angiogenesis assay is used to approach tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Here, we evaluated whether the assay could allow the identification of microRNAs having an anti-angiogenic potential. For that, we transfected DU-145 prostate cancer cells with four microRNAs (miR-125a, miR-320, miR-487b, miR-492) responsive to both anti- and pro-angiogenic stimuli applied to human umbilical vein endothelial cells. After transfection, DU-145 cells were injected close to the developing subintestinal vessels of transgenic Tg(Kdrl:eGFP)s843 zebrafish embryos that express green fluorescent protein under the control of Kdrl promoter. At 72 h post-fertilization, we observed that green fluorescent protein–positive neo-vessels infiltrated the graft of DU-145 transfected with miR-125a, miR-320, and miR-487b. Vice versa, neo-vessel formation and tumor cell infiltration were inhibited when DU-145 cells transfected with miR-492 were used. These results indicated that the zebrafish/tumor xenograft assay was adequate to identify microRNAs able to suppress the release of angiogenic growth factors by angiogenic tumor cells

Development of an Electrochemical Sensor for Nitrate Analysis in Municipal Wastewaters Treated by Microalgae

Microalgae are photosynthetic microorganisms which may be employed in several fields. Amongst them, an emerging but promising sector of application is their usage for the remediation of wastewaters. They result particularly effective in treating municipal wastewaters, often resulting in nitrate concentrations exceeding the requirements for discharging treated wastewater into the sea. Furthermore, another advantage of using microalgae in civil wastewater treatment is the cooperation with heterotrophic bacteria which may naturally occur in the water or be introduced through sewage sludge in wastewater treatment plants (WTPs). Microalgae, in fact, produce oxygen which may be used by these bacteria reducing the overall operational costs of the WTP. The assessment of nitrogen compound concentrations in wastewater involves various techniques, with ionic chromatography (IC) and spectrometric methods being commonly used in laboratory settings. However, these methods have drawbacks such as the need for skilled personnel, time-consuming processes, and impracticality for in situ and real-time analysis. To address these issues, electrochemical sensors present a viable alternative. These sensors require portable instruments with low power requirements and can be miniaturized using nanotechnology. Electrochemical sensors operate by reducing/oxidizing the target analyte on the working electrode surface. The choice of electrode material is crucial for sensor sensitivity, and copper is found to exhibit excellent electrocatalytic properties for reducing nitrate ions in acidic media. In this study, a cost-effective electrochemical sensor made of copper was developed for quantifying nitrate in wastewater. The entire electrochemical cell was constructed from a common substrate used in Printed Circuit Boards (PCB). The whole fabrication procedure was optimized in order to obtain a reproducible fabrication procedure. The reference and counter electrodes were modified with graphite and Ag/AgCl paste, respectively. The results demonstrated that the sensor can effectively quantify nitrate ions in wastewater. Moreover, it can be employed during microalgal treatment to assess the in vivo reduction of nitrate, offering a practical and efficient solution for real-time monitoring of nitrogen compounds in wastewater treatment processes

Structure modeling hints at a granular organization of the Golgi ribbon

Funding Information: This work was funded by the Medical Research Council (grants MC_UU_12018/2 and MC_UU_00012/2 to D.F.C.) and by the British Heart Foundation (grant PG/14/76/31087 to D.F.C.). Publisher Copyright: © 2022, The Author(s).Background: In vertebrate cells, the Golgi functional subunits, mini-stacks, are linked into a tri-dimensional network. How this “ribbon” architecture relates to Golgi functions remains unclear. Are all connections between mini-stacks equal? Is the local structure of the ribbon of functional importance? These are difficult questions to address, without a quantifiable readout of the output of ribbon-embedded mini-stacks. Endothelial cells produce secretory granules, the Weibel-Palade bodies (WPB), whose von Willebrand Factor (VWF) cargo is central to hemostasis. The Golgi apparatus controls WPB size at both mini-stack and ribbon levels. Mini-stack dimensions delimit the size of VWF "boluses” whilst the ribbon architecture allows their linear co-packaging, thereby generating WPBs of different lengths. This Golgi/WPB size relationship suits mathematical analysis. Results: WPB lengths were quantized as multiples of the bolus size and mathematical modeling simulated the effects of different Golgi ribbon organizations on WPB size, to be compared with the ground truth of experimental data. An initial simple model, with the Golgi as a single long ribbon composed of linearly interlinked mini-stacks, was refined to a collection of mini-ribbons and then to a mixture of mini-stack dimers plus long ribbon segments. Complementing these models with cell culture experiments led to novel findings. Firstly, one-bolus sized WPBs are secreted faster than larger secretory granules. Secondly, microtubule depolymerization unlinks the Golgi into equal proportions of mini-stack monomers and dimers. Kinetics of binding/unbinding of mini-stack monomers underpinning the presence of stable dimers was then simulated. Assuming that stable mini-stack dimers and monomers persist within the ribbon resulted in a final model that predicts a “breathing” arrangement of the Golgi, where monomer and dimer mini-stacks within longer structures undergo continuous linking/unlinking, consistent with experimentally observed WPB size distributions. Conclusions: Hypothetical Golgi organizations were validated against a quantifiable secretory output. The best-fitting Golgi model, accounting for stable mini-stack dimers, is consistent with a highly dynamic ribbon structure, capable of rapid rearrangement. Our modeling exercise therefore predicts that at the fine-grained level the Golgi ribbon is more complex than generally thought. Future experiments will confirm whether such a ribbon organization is endothelial-specific or a general feature of vertebrate cells.publishersversionpublishe

Structure modeling hints at a granular organization of the Golgi ribbon

Background In vertebrate cells, the Golgi functional subunits, mini-stacks, are linked into a tri-dimensional network. How this “ribbon” architecture relates to Golgi functions remains unclear. Are all connections between mini-stacks equal? Is the local structure of the ribbon of functional importance? These are difficult questions to address, without a quantifiable readout of the output of ribbon-embedded mini-stacks. Endothelial cells produce secretory granules, the Weibel-Palade bodies (WPB), whose von Willebrand Factor (VWF) cargo is central to hemostasis. The Golgi apparatus controls WPB size at both mini-stack and ribbon levels. Mini-stack dimensions delimit the size of VWF "boluses” whilst the ribbon architecture allows their linear co-packaging, thereby generating WPBs of different lengths. This Golgi/WPB size relationship suits mathematical analysis. Results WPB lengths were quantized as multiples of the bolus size and mathematical modeling simulated the effects of different Golgi ribbon organizations on WPB size, to be compared with the ground truth of experimental data. An initial simple model, with the Golgi as a single long ribbon composed of linearly interlinked mini-stacks, was refined to a collection of mini-ribbons and then to a mixture of mini-stack dimers plus long ribbon segments. Complementing these models with cell culture experiments led to novel findings. Firstly, one-bolus sized WPBs are secreted faster than larger secretory granules. Secondly, microtubule depolymerization unlinks the Golgi into equal proportions of mini-stack monomers and dimers. Kinetics of binding/unbinding of mini-stack monomers underpinning the presence of stable dimers was then simulated. Assuming that stable mini-stack dimers and monomers persist within the ribbon resulted in a final model that predicts a “breathing” arrangement of the Golgi, where monomer and dimer mini-stacks within longer structures undergo continuous linking/unlinking, consistent with experimentally observed WPB size distributions. Conclusions Hypothetical Golgi organizations were validated against a quantifiable secretory output. The best-fitting Golgi model, accounting for stable mini-stack dimers, is consistent with a highly dynamic ribbon structure, capable of rapid rearrangement. Our modeling exercise therefore predicts that at the fine-grained level the Golgi ribbon is more complex than generally thought. Future experiments will confirm whether such a ribbon organization is endothelial-specific or a general feature of vertebrate cells

Development of an Electrochemical Sensor for Nitrate Analysis in Municipal Wastewaters Treated by Microalgae

Microalgae are photosynthetic microorganisms which may be employed in several fields. Amongst them, an emerging but promising sector of application is their usage for the remediation of wastewaters. They result particularly effective in treating municipal wastewaters, often resulting in nitrate concentrations exceeding the requirements for discharging treated wastewater into the sea. Furthermore, another advantage of using microalgae in civil wastewater treatment is the cooperation with heterotrophic bacteria which may naturally occur in the water or be introduced through sewage sludge in wastewater treatment plants (WTPs). Microalgae, in fact, produce oxygen which may be used by these bacteria reducing the overall operational costs of the WTP. The assessment of nitrogen compound concentrations in wastewater involves various techniques, with ionic chromatography (IC) and spectrometric methods being commonly used in laboratory settings. However, these methods have drawbacks such as the need for skilled personnel, time-consuming processes, and impracticality for in situ and real-time analysis. To address these issues, electrochemical sensors present a viable alternative. These sensors require portable instruments with low power requirements and can be miniaturized using nanotechnology. Electrochemical sensors operate by reducing/oxidizing the target analyte on the working electrode surface. The choice of electrode material is crucial for sensor sensitivity, and copper is found to exhibit excellent electrocatalytic properties for reducing nitrate ions in acidic media. In this study, a cost-effective electrochemical sensor made of copper was developed for quantifying nitrate in wastewater. The entire electrochemical cell was constructed from a common substrate used in Printed Circuit Boards (PCB). The whole fabrication procedure was optimized in order to obtain a reproducible fabrication procedure. The reference and counter electrodes were modified with graphite and Ag/AgCl paste, respectively. The results demonstrated that the sensor can effectively quantify nitrate ions in wastewater. Moreover, it can be employed during microalgal treatment to assess the in vivo reduction of nitrate, offering a practical and efficient solution for real-time monitoring of nitrogen compounds in wastewater treatment processes

Flexible electrode based on gold nanoparticles and reduced graphene oxide for uric acid detection using linear sweep voltammetry

In this work, an electrochemical sensor for uric acid determination is shown with a preliminary study for its validation in real samples (milk and urine). Uric acid can be electrochemically oxidized in aqueous solutions and thus it is possible to obtain electrochemical sensors for this chemical by means of this electrooxidation reaction. Indium tin oxide coated on flexible polyethylene terephthalate substrate, modified with reduced graphene oxide and gold nanoparticles by co-electrodeposition, was used. Electrodeposition was performed at -0.8V vs SCE for 200 s. All samples were characterized by electron scan microscopy and electron diffraction spectroscopy. A careful investigation on the effect of pH was performed to understand its influence on uric acid oxidation. The detection of uric acid was using the linear sweep voltammetry. Results show that the peak current increases linearly with uric acid concentration from 10 to 1000 μM with a limit of detection of about 7.1 μM. The sensor shows high selectivity towards different interferents that can be found in the milk and urine matrix, such as chloride, calcium, sodium and ammonium ions. To prove the applicability of the proposed sensor, uric acid was quantified in real milk and urine samples with excellent results comparable to those of conventional techniques