Role of Neuropilin-1/Semaphorin-3A signaling in the functional and morphological integrity of the cochlea.

Neuropilin-1 (Nrp1) encodes the transmembrane cellular receptor neuropilin-1, which is associated with cardiovascular and neuronal development and was within the peak SNP interval on chromosome 8 in our prior GWAS study on age-related hearing loss (ARHL) in mice. In this study, we generated and characterized an inner ear-specific Nrp1 conditional knockout (CKO) mouse line because Nrp1 constitutive knockouts are embryonic lethal. In situ hybridization demonstrated weak Nrp1 mRNA expression late in embryonic cochlear development, but increased expression in early postnatal stages when cochlear hair cell innervation patterns have been shown to mature. At postnatal day 5, Nrp1 CKO mice showed disorganized outer spiral bundles and enlarged microvessels of the stria vascularis (SV) but normal spiral ganglion cell (SGN) density and presynaptic ribbon body counts; however, we observed enlarged SV microvessels, reduced SGN density, and a reduction of presynaptic ribbons in the outer hair cell region of 4-month-old Nrp1 CKO mice. In addition, we demonstrated elevated hearing thresholds of the 2-month-old and 4-month-old Nrp1 CKO mice at frequencies ranging from 4 to 32kHz when compared to 2-month-old mice. These data suggest that conditional loss of Nrp1 in the inner ear leads to progressive hearing loss in mice. We also demonstrated that mice with a truncated variant of Nrp1 show cochlear axon guidance defects and that exogenous semaphorin-3A, a known neuropilin-1 receptor agonist, repels SGN axons in vitro. These data suggest that Neuropilin-1/Semaphorin-3A signaling may also serve a role in neuronal pathfinding in the developing cochlea. In summary, our results here support a model whereby Neuropilin-1/Semaphorin-3A signaling is critical for the functional and morphological integrity of the cochlea and that Nrp1 may play a role in ARHL.

Activation of Toll-like receptor 3 amplifies mesenchymal stem cell trophic factors and enhances therapeutic potency.

Clinical trials of bone marrow mesenchymal stem cell (MSC) therapy have thus far demonstrated moderate and inconsistent benefits, indicating an urgent need to improve therapeutic efficacy. Although administration of sufficient cells is necessary to achieve maximal therapeutic benefits, documented MSC clinical trials have largely relied on injections of ∼1 × 10(6) cells/kg, which appears too low to elicit a robust therapeutic response according to published preclinical studies. However, repeated cell passaging necessary for large-scale expansion of MSC causes cellular senescence and reduces stem cell potency. Using the RNA mimetic polyinosinic-polycytidylic acid [poly(I:C)] to engage MSC Toll-like receptor 3 (TLR3), we found that poly(I:C), signaling through multiple mitogen-activated protein kinase pathways, induced therapeutically relevant trophic factors such as interleukin-6-type cytokines, stromal-derived factor 1, hepatocyte growth factor, and vascular endothelial growth factor while slightly inhibiting the proliferation and migration potentials of MSC. At the suboptimal injection dose of 1 × 10(6) cells/kg, poly(I:C)-treated MSC, but not untreated MSC, effectively stimulated regeneration of the failing hamster heart 1 mo after cell administration. The regenerating heart exhibited increased CD34(+)/Ki67(+) and CD34(+)/GATA4(+) progenitor cells in the presence of decreased inflammatory cells and cytokines. Cardiac functional improvement was associated with a ∼50% reduction in fibrosis, a ∼40% reduction in apoptosis, and a ∼55% increase in angiogenesis, culminating in prominent cardiomyogenesis evidenced by abundant distribution of small myocytes and a ∼90% increase in wall thickening. These functional, histological, and molecular characterizations thus establish the utility of TLR3 engagement for enabling the low-dose MSC therapy that may be translated to more efficacious clinical applications.