Marsico, G. | Search bioRxiv

bioRxiv2020

Temporal Changes Guided by Mesenchymal Stem Cells on a 3D Microgel Platform Enhances Angiogenesis In Vivo at a Low-Cell Dose

Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival and the non-maintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behaviour including migration, proliferation and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of pre-conditioning human MSCs for 96 hours on a three-dimensional ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the pro-angiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and outside-in integrin signaling. The microgels tested at a low-cell dose (5x104 cells) in a pre-clinical hindlimb ischemia model showed accelerated formation of new blood vessels with a reduced inflammatory response impeding progression of tissue damage. Molecular analysis revealed that several key mediators of angiogenesis were upregulated in the low-cell dose microgel group, providing a mechanistic insight of pathways modulated in vivo. Our research adds to current knowledge in cell encapsulation strategies by highlighting the importance of preconditioning or priming the capacity of biomaterials through cell-material interactions. Obtaining therapeutic efficacy at a low-cell dose in the microgel platform is a promising clinical route that would aid faster tissue repair and reperfusion in no-option patients suffering from peripheral arterial diseases such as Critical Limb Ischemia (CLI).

bioengineering↗

bioRxiv2017

RNA G-quadruplexes mark repressive upstream open reading frames in human mRNAs

RNA secondary structures in the 5 untranslated regions (UTRs) of mRNAs have been characterised as key determinants of translation initiation. However the role of non-canonical secondary structures, such as RNA G-quadruplexes (rG4s), in modulating translation of human mRNAs and the associated mechanisms remain largely unappreciated. Here we use a ribosome profiling strategy to investigate the translational landscape of human mRNAs with structured 5 untranslated regions (5-UTR). We found that inefficiently translated mRNAs, containing rG4-forming sequences in their 5-UTRs, have an accumulation of ribosome footprints in their 5-UTRs. We show that rG4-forming sequences are determinants of 5-UTR translation, suggesting that the folding of rG4 structures thwarts the translation of protein coding sequences (CDS) by stimulating the translation of repressive upstream open reading frames (uORFs). To support our model, we demonstrate that depletion of two rG4s-specialised DEAH-box helicases, DHX36 and DHX9, shifts translation towards rG4-containing uORFs reducing the translation of selected transcripts comprising proto-oncogenes, transcription factors and epigenetic regulators. Transcriptome-wide identification of DHX9 binding sites using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) demonstrate that translation regulation is mediated through direct physical interaction between the helicase and its rG4 substrate. Our findings unveil a previously unknown role for non-canonical structures in governing 5-UTR translation and suggest that the interaction of helicases with rG4s could be considered as a target for future therapeutic intervention.

genomics↗