Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS

We hereby provide the initial portrait of lincNORS, a spliced lincRNA generated by the MIR193BHG locus, entirely distinct from the previously described miR-193b-365a tandem. While inducible by low O2 in a variety of cells and associated with hypoxia in vivo, our studies show that lincNORS is subject to multiple regulatory inputs, including estrogen signals. Biochemically, this lincRNA fine-tunes cellular sterol/steroid biosynthesis by repressing the expression of multiple pathway components. Mechanistically, the function of lincNORS requires the presence of RALY, an RNA-binding protein recently found to be implicated in cholesterol homeostasis. We also noticed the proximity between this locus and naturally occurring genetic variations highly significant for sterol/steroid-related phenotypes, in particular the age of sexual maturation. An integrative analysis of these variants provided a more formal link between these phenotypes and lincNORS, further strengthening the case for its biological relevance

Assessment of Angular Spectral Distributions of Laser Accelerated Particles for Simulation of Radiation Dose Map in Target Normal Sheath Acceleration Regime of High Power Laser-Thin Solid Target Interaction—Comparison with Experiments

An adequate simulation model has been used for the calculation of angular and energy distributions of electrons, protons, and photons emitted during a high-power laser, 5-µm thick Ag target interaction. Their energy spectra and fluencies have been calculated between 0 and 360 degrees around the interaction point with a step angle of five degrees. Thus, the contribution of each ionizing species to the total fluency value has been established. Considering the geometry of the experimental set-up, a map of the radiation dose inside the target vacuum chamber has been simulated, using the Geant4 General Particle Source code, and further compared with the experimental one. Maximum values of the measured dose of the order of tens of mGy per laser shot have been obtained in the direction normal to the target at about 30 cm from the interaction point

3D Matrices for Enhanced Encapsulation and Controlled Release of Anti-Inflammatory Bioactive Compounds in Wound Healing

Current trends in the development of wound dressings are oriented towards the use of biopolymer-based materials, due to their unique properties such as non-toxicity, hydrophilicity, biocompatibility and biodegradability, properties that have advantageous therapeutic characteristics. In this regard, the present study aims to develop hydrogels based on cellulose and dextran (CD) and to reveal their anti-inflammatory performance. This purpose is achieved by incorporating plant bioactive polyphenols (PFs) in CD hydrogels. The assessments include establishing the structural characteristics using attenuated total reflection Fourier transformed infrared (ATR-FTIR) spectroscopy, the morphology by scanning electron microscopy (SEM), the swelling degree of hydrogels, the PFs incorporation/release kinetics and the hydrogels’ cytotoxicity, together with evaluation of the anti-inflammatory properties of PFs-loaded hydrogels. The results show that the presence of dextran has a positive impact on the hydrogel’s structure by decreasing the pore size at the same time as increasing the uniformity and interconnectivity of the pores. In addition, there is an increased degree of swelling and of the encapsulation capacity of PFs, with the increase of the dextran content in hydrogels. The kinetics of PFs released by hydrogels was studied according to the Korsmeyer–Peppas model, and it was observed that the transport mechanisms depend on hydrogels’ composition and morphology. Furthermore, CD hydrogels have been shown to promote cell proliferation without cytotoxicity, by successfully culturing fibroblasts and endothelial cells on CD hydrogels (over 80% viability). The anti-inflammatory tests performed in the presence of lipopolysaccharides demonstrate the anti-inflammatory properties of the PFs-loaded hydrogels. All these results provide conclusive evidence on the acceleration of wound healing by inhibiting the inflammation process and support the use of these hydrogels encapsulated with PFs in wound healing applications

The Specific Molecular Changes Induced by Diabetic Conditions in Valvular Endothelial Cells and upon Their Interactions with Monocytes Contribute to Endothelial Dysfunction

Aortic valve disease (AVD) represents a global public health challenge. Research indicates a higher prevalence of diabetes in AVD patients, accelerating disease advancement. Although the specific mechanisms linking diabetes to valve dysfunction remain unclear, alterations of valvular endothelial cells (VECs) homeostasis due to high glucose (HG) or their crosstalk with monocytes play pivotal roles. The aim of this study was to determine the molecular signatures of VECs in HG and upon their interaction with monocytes in normal (NG) or high glucose conditions and to propose novel mechanisms underlying valvular dysfunction in diabetes. VECs and THP-1 monocytes cultured in NG/HG conditions were used. The RNAseq analysis revealed transcriptomic changes in VECs, in processes related to cytoskeleton regulation, focal adhesions, cellular junctions, and cell adhesion. Key molecules were validated by qPCR, Western blot, and immunofluorescence assays. The alterations in cytoskeleton and intercellular junctions impacted VEC function, leading to changes in VECs adherence to extracellular matrix, endothelial permeability, monocyte adhesion, and transmigration. The findings uncover new molecular mechanisms of VEC dysfunction in HG conditions and upon their interaction with monocytes in NG/HG conditions and may help to understand mechanisms of valvular dysfunction in diabetes and to develop novel therapeutic strategies in AVD

P-Selectin Targeted Dexamethasone-Loaded Lipid Nanoemulsions: A Novel Therapy to Reduce Vascular Inflammation

Inflammation is a common process associated with numerous vascular pathologies. We hypothesized that targeting the inflamed endothelium by coupling a peptide with high affinity for P-selectin to the surface of dexamethasone-loaded lipid nanoemulsions will highly increase their specific binding to activated endothelial cells (EC) and reduce the cell activation. We developed and characterized dexamethasone-loaded lipid nanoemulsions directed towards P-selectin (PLN-Dex) and monitored their anti-inflammatory effects in vitro using cultured EC (EA.hy926 cells) and in vivo using a mouse model of acute inflammation [lipopolysaccharides (LPS) intravenously administered in C57BL/6 mice]. We found that PLN-Dex bound specifically to the surface of activated EC are efficiently internalized by EC and reduced the expression of proinflammatory genes, thus preventing the monocyte adhesion and transmigration to/through activated EC. Given intravenously in mice with acute inflammation, PLN-Dex accumulated at a significant high level in the lungs (compared to nontargeted nanoemulsions) and significantly reduced mRNA expression level of key proinflammatory cytokines such as IL-1β, IL-6, and MCP-1. In conclusion, the newly developed nanoformulation, PLN-Dex, is functional in vitro and in vivo, reducing selectively the endothelium activation and the consequent monocyte infiltration and diminishing significantly the lungs’ inflammation, in a mouse model of acute inflammation

Advances in Spectral Distribution Assessment of Laser Accelerated Protons using Multilayer CR-39 Detectors

We show that a spectral distribution of laser-accelerated protons can be extracted by analyzing the proton track diameters observed on the front side of a second CR-39 detector arranged in a stack. The correspondence between the proton track diameter and the incident energy on the second detector is established by knowing that protons with energies only higher than 10.5 MeV can fully deposit their energy in the second CR-39 detector. The correlation between the laser-accelerated proton track diameters observed on the front side of the second CR-39 detector and the proton incident energy on the detector stack is also presented. By calculating the proton number stopped in the CR-39 stack, we find out that its dependence on the proton energy in the 1−15 MeV range presents some discontinuities at energies higher than 9 MeV. Thus, we build a calibration curve of the track diameter as a function of the proton incident energy within the 1−9 MeV range, and we infer the associated analytical function as the calculations performed indicate best results for proton spectra within the 1−9 MeV range. The calibration curve is used as a tool to ascertain the pits identified on the surfaces of both CR-39 detectors to proton tracks. The proton tracks spatial distribution analyzed by optical and atomic force microscopy is correlated with the peculiarity of the used targets

Cross talk between smooth muscle cells and monocytes/activated monocytes via CX3CL1/CX3CR1 axis augments expression of pro-atherogenic molecules

AbstractObjectiveIn atherosclerotic lesions, fractalkine (CX3CL1) and its receptor (CX3CR1) expressed by smooth muscle cells (SMC) and monocytes/macrophages, mediate the heterotypic anchorage and chemotaxis of these cells. We questioned whether, during the close interaction of monocytes with SMC, the CX3CL1/CX3CR1 pair modulates the expression of pro-atherogenic molecules in these cells.Methods and resultsSMC were co-cultured with monocytes or LPS-activated monocytes (18h) and then the cells were separated and individually investigated for the gene and protein expression of TNFα, IL-1β, IL-6, CX3CR1 and metalloproteinases (MMP-2, MMP-9). We found that SMC–monocyte interaction induced, in each cell type, an increased mRNA and protein expression of TNFα, IL-1β, IL-6, CX3CR1, MMP-2 and MMP-9. Blocking the binding of fractalkine to CX3CR1 (by pre-incubation of monocytes with anti-CX3CR1 or by CX3CR1 siRNA transfection) before cell co-culture decreased the production of TNFα, CX3CR1 and MMP-9. Monocyte–SMC interaction induced the phosphorylation of p38MAPK and activation of AP-1 transcription factor. Silencing the p65 (NF-kB subunit) inhibited the IL-1β and IL-6 and silencing c-jun inhibited the TNFα, CX3CR1 and MMP-9 induced by SMC–monocyte interaction.ConclusionsThe cross-talk between SMC and monocytes augments the inflammatory response in both cell types as revealed by the increased expression of TNFα, IL-1β, IL-6, CX3CR1 and MMPs. Up-regulation of TNFα, CX3CR1 and MMP-9 is further increased upon interaction of SMC with activated monocytes and is dependent on fractalkine/CXRCR1 pair. These data imply that the fractalkine/CX3RCR1 axis may represent a therapeutic target to impede the inflammatory process associated with atherosclerosis

Chronic High Glucose Concentration Induces Inflammatory and Remodeling Changes in Valvular Endothelial Cells and Valvular Interstitial Cells in a Gelatin Methacrylate 3D Model of the Human Aortic Valve

Calcific aortic valve disease (CAVD), a degenerative disease characterized by inflammation, fibrosis and calcification, is accelerated in diabetes. Hyperglycemia contributes to this process by mechanisms that still need to be uncovered. We have recently developed a 3D model of the human aortic valve based on gelatin methacrylate and revealed that high glucose (HG) induced osteogenic molecules and increased calcium deposits in a pro-osteogenic environment. To further understand the events leading to calcification in diabetic conditions in CAVD, we analyzed here the inflammatory and remodeling mechanisms induced by HG in our 3D model. We exposed valvular endothelial cells (VEC) and interstitial cells (VIC) to normal glucose (NG) or HG for 7 and 14 days, then we isolated and separated the cells by anti-CD31 immunomagnetic beads. The changes induced by HG in the 3D model were investigated by real-time polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. Our results showed that HG induced expression of different cytokines, cell adhesion molecules and matrix metalloproteinases in VEC and VIC. In addition, protein kinase C was increased in VEC and VIC, indicating molecular mechanisms associated with HG induced inflammation and remodeling in both valvular cells. These findings may indicate new biomarkers and targets for therapy in diabetes associated with CAVD

Parathyroid Hormone Induces Human Valvular Endothelial Cells Dysfunction That Impacts the Osteogenic Phenotype of Valvular Interstitial Cells

Parathyroid hormone (PTH) is a key regulator of calcium, phosphate and vitamin D metabolism. Although it has been reported that aortic valve calcification was positively associated with PTH, the pathophysiological mechanisms and the direct effects of PTH on human valvular cells remain unclear. Here we investigated if PTH induces human valvular endothelial cells (VEC) dysfunction that in turn could impact the switch of valvular interstitial cells (VIC) to an osteoblastic phenotype. Human VEC exposed to PTH were analyzed by qPCR, western blot, Seahorse, ELISA and immunofluorescence. Our results showed that exposure of VEC to PTH affects VEC metabolism and functions, modifications that were accompanied by the activation of p38MAPK and ERK1/2 signaling pathways and by an increased expression of osteogenic molecules (BMP-2, BSP, osteocalcin and Runx2). The impact of dysfunctional VEC on VIC was investigated by exposure of VIC to VEC secretome, and the results showed that VIC upregulate molecules associated with osteogenesis (BMP-2/4, osteocalcin and TGF-β1) and downregulate collagen I and III. In summary, our data show that PTH induces VEC dysfunction, which further stimulates VIC to differentiate into a pro-osteogenic pathological phenotype related to the calcification process. These findings shed light on the mechanisms by which PTH participates in valve calcification pathology and suggests that PTH and the treatment of hyperparathyroidism represent a therapeutic strategy to reduce valvular calcification