MiR-18a-5p Targets Connective Tissue Growth Factor Expression and Inhibits Transforming Growth Factor β2-Induced Trabecular Meshwork Cell Contractility

Increased trabecular meshwork (TM) cell and tissue contractility is a driver of the reduced outflow facility and elevation of intraocular pressure (IOP) associated with primary open-angle glaucoma (POAG). Connective tissue growth factor (CTGF) is an established mediator of TM cell contractility, and its expression is increased in POAG due to transforming growth factor β 2 (TGFβ2) signalling. Inhibiting CTGF upregulation using microRNA (miRNA) mimetics could represent a new treatment option for POAG. A combination of in silico predictive tools and a literature review identified a panel of putative CTGF-targeting miRNAs. Treatment of primary human TM cells with 5 ng/mL TGFβ2 for 24 h identified miR-18a-5p as a consistent responder, being upregulated in cells from five different human donors. Transfection of primary donor TM cells with 20 nM synthetic miR-18a-5p mimic reduced TGFβ2-induced CTGF protein expression, and stable lentiviral-mediated overexpression of this miRNA reduced TGFβ2-induced contraction of collagen gels. Together, these findings identify miR-18a-5p as a mediator of the TGFβ2 response and a candidate therapeutic agent for glaucoma via its ability to inhibit CTGF-associated increased TM contractility

MEF2 transcription factors are key regulators of sprouting angiogenesis

Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis

Vota ERC : cap a la independència l'esquerra puja :

OBJECTIVE: To detect and determine disease severity of osteoarthritis (OA) using a probe activated by matrix metalloproteinase-13 (MMP-13) in vivo in the murine destabilised medial meniscus (DMM) surgical model of OA. DESIGN: We have previously described MMP12ap and MMP13ap, internally quenched fluorescent peptide substrate probes that are activated respectively by MMP-12 and MMP-13. Here we used these probes to follow enzyme activity in vivo in mice knees 4, 6 and 8 weeks following DMM surgery. After in vivo optical imaging, disease severity was determined through traditional histological analysis. The amount of probe activation was analysed for discrimination between DMM, contralateral and sham operated knees, as well as for congruence between activity and histological damage. RESULTS: There was no specific activation of MMP12ap at the time points observed between sham operated and DMM operated, or their respective contralateral joints. The activation of the MMP13ap in the DMM model was highest 6 weeks after surgery, but was only specific compared against sham surgery 8 weeks after surgery (1.5-fold increase). The activation of MMP13ap correlated with histological damage 6 and 8 weeks after surgery, with correlations of 0.484 (P = 0.0032) and 0.478 respectively (P = 0.0049). This correlation dropped to 0.218 (P = 0.011) if all data were considered. CONCLUSION: The current MMP-13 activity probe is suitable for the discrimination between DMM and sham or contralateral knees 8 weeks after surgery, when cartilage loss is typified by the appearance of small fissures up to the tidemark, but not earlier. This activity correlates with the histological damage observed

Hammerhead ribozyme-mediated silencing of the mutant fibrillin-1 of tight skin mouse: insight into the functional role of mutant fibrillin-1.

The tight skin (Tsk/+) mouse is a model for fibrotic disorders. The genetic defect in the Tsk/+ is an in-frame duplication between exons 17 and 40 of the fibrillin-1 gene which gives rise to a large transcript and protein. Mice homozygous for the mutation die in utero, whereas heterozygotes survive and spontaneously develop connective tissue disease. In this study, we generated hammerhead ribozymes directed against the mutant fibrillin-1 transcript. A partially mispairing ribozyme was the most effective vehicle to cleave the mutant transcript without undesired cleavage of wild type transcripts, as shown by cell-free RNA cleavage and cleavage in cell lines harboring the ribozyme, by RT-PCR, Northern and Western Blotting. Global gene expression profiling using oligonucleotide microarrays showed the expected reduction in fibrillin-1 mRNA, and down-regulation of several gene cohorts in ribozyme harboring TskR1 cells compared to Tsk/+ cells. Two of the functional clusters included genes regulating extracellular matrix such as connective tissue growth factor, serpine-1 (plasminogen activator inhibitor-1) and TIMP-1 and TIMP-3, and those involved in cytoskeletal organization and myofibroblast formation including calponins and transgelin. Ribozyme-mediated inhibition was confirmed by Western Blot and functional analysis using cell-reporter systems and remodeling of three dimensional collagen gels. Our results underline the therapeutic potential of hammerhead ribozymes in dominant negative defects and suggest that changes in microfibril architecture brought about by fibrillin-1 mutation lead to a complex disease phenotype