Neural Differentiation of Human Dental Mesenchymal Stem Cells Induced by ATRA and UDP-4: A Comparative Study.

Human mesenchymal stem cells (MSC) are multipotent stem cells, which are isolated from various sources. Currently, there is a worldwide interest for dental MSC to be used against neurodegenerative diseases, since they derive from the neural crest and express embryonic stem cell markers. This fact prompted us to explore their potential for neural trans-differentiation in culture. We employed all-trans-retinoic acid (ATRA) and 2-(3-ethylureido)-6-methylpyridine (UDP-4) to induce neural differentiation of human MSC from the dental apical papilla (SCAP). The SCAP were exposed to either agent separately and assessed for proliferation, viability, morphology, and gene expression of the following neural-specific markers: neuron-specific enolase (ENO2), neurofibromin 1 (NF1), choline acetyltransferase (CHAT), tyrosine hydroxylase (TH), and the vesicular GABA transporter (SLC32A1). They were also assessed for the expression of glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen (NeuN) by immunofluorescence. ATRA or UDP-4 treatment inhibited the cell growth and promoted limited cell death, but to a different extent. The addition of the neuroprotective agent recombinant human erythropoietin-alpha (rhEPO-α) enhanced the UDP-4-inducing capacity for more than three weeks. ATRA or UDP-4 treatment significantly upregulated ENO2 and NF1 expression, indicating neuronal differentiation. Moreover, the ATRA treatment significantly induced the upregulation of the GABAergic-specific SLC32A1, while the UDP-4 treatment led to the significant upregulation of the adrenergic-specific TH. The UDP-4 treatment induced the expression of NeuN and GFAP after four and three weeks, respectively, while the ATRA-treatment did not. Our findings indicate that SCAP can be differentiated into neural-like cells after treatment with ATRA or UDP-4 by exhibiting a disparate pattern of differentiation. Therefore, UDP-4 is suggested here as a new potent neural-differentiation-inducing compound, which, when combined with rhEPO-α, could lay the foundation for robust stem-cell-based therapies of neurodegeneration.

Peptides Derived from Vascular Endothelial Growth Factor B Show Potent Binding to Neuropilin-1.

Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF-B growth factor is involved in cell survival, anti-apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin-1 (NRP1). We employed surface plasmon resonance technology and X-ray crystallography to analyse the molecular basis of the interaction between VEGF-B and the b1 domain of NRP1, and developed VEGF-B C-terminus derived peptides to be used as chemical tools for studying VEGF-B - NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF-B-derived peptides containing a C-terminal arginine show potent binding to NRP1-b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF-B peptides bind at the canonical C-terminal arginine binding site. VEGF-B C-terminus imparts higher affinity for NRP1 than the corresponding VEGF-A165 region. This tight binding may impact on the activity and selectivity of the full-length protein. The VEGF-B167 derived peptides were more effective than VEGF-A165 peptides in blocking functional phosphorylation events. Blockers of VEGF-B function have potential applications in diabetes and non-alcoholic fatty liver disease.

Discovery of a novel fluorescent chemical probe suitable for evaluation of neuropilin-1 binding of small molecules.

Neuropilin-1 (NRP1) is emerging as an important molecule in immune signaling where it has been shown to modulate the actions of TGF-ß1 in macrophages and regulatory T cells. The development of cost-effective and reliable assays for NRP1 binding is therefore important. We synthesized three new NRP1 small molecule fluorophores and examined their performance as fluorescent polarization probes. One molecule DS108 exhibited favorable binding and fluorescent characteristics and allowed us to establish a simple assay suitable for medium to high throughput screening of small molecules.