Control Of Signal Propagation Research Articles

The effect of CGRP and SP and the cell signaling dialogue between sensory neurons and endothelial cells

Increasing evidences demonstrate the role of sensory innervation in bone metabolism, remodeling and repair, however neurovascular coupling in bone is rarely studied. Using microfluidic devices as an indirect co-culture model to mimic in vitro the physiological scenario of innervation, our group demonstrated that sensory neurons (SNs) were able to regulate the extracellular matrix remodeling by endothelial cells (ECs), in particular through sensory neuropeptides, i.e. calcitonin gene-related peptide (CGRP) and substance P (SP). Nonetheless, still little is known about the cell signaling pathways and mechanism of action in neurovascular coupling. Here, in order to characterize the communication between SNs and ECs at molecular level, we evaluated the effect of SNs and the neuropeptides CGRP and SP on ECs. We focused on different pathways known to play a role on endothelial functions: calcium signaling, p38 and Erk1/2; the control of signal propagation through Cx43; and endothelial functions through the production of nitric oxide (NO). The effect of SNs was evaluated on ECs Ca2+ influx, the expression of Cx43, endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production, p38, ERK1/2 as well as their phosphorylated forms. In addition, the role of CGRP and SP were either analyzed using respective antagonists in the co-culture model, or by adding directly on the ECs monocultures. We show that capsaicin-stimulated SNs induce increased Ca2+ influx in ECs. SNs stimulate the increase of NO production in ECs, probably involving a decrease in the inhibitory eNOS T495 phosphorylation site. The neuropeptide CGRP, produced by SNs, seems to be one of the mediators of this effect in ECs since NO production is decreased in the presence of CGRP antagonist in the co-culture of ECs and SNs, and increased when ECs are stimulated with synthetic CGRP. Taken together, our results suggest that SNs play an important role in the control of the endothelial cell functions through CGRP production and NO signaling pathway.

Polarity Induced in Human Stem Cell Derived Motoneurons on Patterned Self-Assembled Monolayers.

The control of polarized human neurite/axon development at the single neuron level is critical in geographically directing signal propagation in engineered neural networks, for both in vitro and in vivo applications. While there is an increasing need to exert control over axonal growth for the successful development and establishment of integrative and functional in vitro systems, controlled, polarized distribution of either human-derived neurons or motoneurons in vitro has yet to be reported. In this study, we established the polarized distribution of stem cell derived human motoneurons, using a patterned surface, and maintained the cells in a serum-free system. A surface pattern with defined polarity was developed using self-assembled monolayers (SAMs). A cell permissive SAM, DETA (trimethoxysilyl propyldiethylenetri-amine), combined with photolithography and a nonpermissive fluorinated silane, 13F (tridecafluoro-1,1,2,2-tetrahydroctyl-1-dimethylchloro-silane), generated a surface where neurons only adhered to the designed attachment sites and did so with preferred orientation. In addition, 75% of the cells attached to the patterns were motoneurons compared to their percentage in the standard unpatterned surface which was used as a control condition (20%), demonstrating the preference of these human motoneurons in adhering to the patterns. The ability to dictate the distribution and polarity of human motoneurons will be essential to the engineering of human-based functional in vitro systems in which the control of signal propagation is necessary but more importantly for cell implantation studies. Such systems will greatly benefit the study of motor function as well as aid the development of high-throughput systems for drug screening and test beds for use in preclinical studies related to conditions such as spinal cord injury, ALS, and muscular dystrophy.

Oral abstract presentations

cAMP generated in response to β-adrenoceptor stimulation modulates excitation-contraction coupling via activation of PKA. It is now widely accepted that cAMP signalling is compartmentalised in the heart and that PDEs, the only cAMP degrading enzyme, play a central role in spatial control of signal propagation. We have previously demonstrated that PKA-I and PKA-II isoforms are selectively activated by spatially confined pools of cAMP leading to phosphorylation of specific downstream targets. It has been suggested that selective deregulation of cAMP/PKA signals in specific subcellular compartment may lead to cardiac pathology. Here we investigate cAMP signalling in an in vitro model of cardiomyocyte hypertrophy and explore the role of different PDE families in the control of the local cAMP response to β-adrenoceptor stimulation. We used a real-time FRET-based imaging approach to monitor cAMP dynamics in adult myocytes. Adenoviral vectors carrying RI or RII Epac were generated by fusing the N-terminus of the cAMP FRET reporter Epac-1 and the dimerisation-docking (DD) domain of the regulatory subunit of PKA-I or PKA-II. Adult rat cardiac myocytes expressing the cAMP reporters were treated overnight with 1μM norepinephrine (NE) as a model of in vitro cardiomyocyte hypertrophy. In control cells, upon stimulation with 100nM isoproterenol, we detected a significantly higher cAMP response in the PKA-II compartment compared to the PKA-I compartment. In NE-treated myocytes, the differences between these compartments were abolished. To investigate which PDE family may be responsible for these changes in cAMP compartmentalisation, experiments were performed in the presence of selective PDEs inhibitors. We found that when PDE2 was inhibited with BAY60-7550 in control cells the resulting increase in cAMP was significantly greater in the PKA-I than in the PKA-II compartment. In hypertrophic myocytes, PDE2 inhibition resulted in a much greater increase in cAMP in the PKA-II compartment compared to control cells. Similar results were found with selective PDE4 inhibition with rolipram. Selective inhibition of PDE3 with cilostamide showed a small but significant rise in cAMP response in the PKA-I compartment in control cells. This was not replicated in hypertrophic myocytes. Our findings indicate that spatial control of cAMP/PKA signalling is altered in this in vitro model of hypertrophy, suggesting that in hypertrophic myocytes mechanisms may be activated resulting in altered localisation and/or activity of PDEs. Further studies will be necessary to fully determine the mechanism responsible for the observed changes.