Univ. Bordeaux, INSERM, BIOTIS, Bordeaux, U1026, F-33000, France.
Univ. Bordeaux, CNRS, UMR 5258, Solvay, Pessac, LOF, F-33006, France.
Biol Res. 2024 Sep 11;57(1):65. doi: 10.1186/s40659-024-00538-6.
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 Ca 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 Ca 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.
越来越多的证据表明感觉神经支配在骨代谢、重塑和修复中的作用，然而，骨中的神经血管耦联很少被研究。我们的研究小组使用微流控设备作为间接共培养模型，模拟神经支配的生理场景，证明了感觉神经元（SNs）能够通过内皮细胞（ECs）调节细胞外基质重塑，特别是通过感觉神经肽，即降钙素基因相关肽（CGRP）和 P 物质（SP）。尽管如此，对于神经血管耦联中的细胞信号通路和作用机制仍知之甚少。在这里，为了在分子水平上描述 SNs 和 ECs 之间的通讯，我们评估了 SNs 和神经肽 CGRP 和 SP 对 ECs 的影响。我们专注于已知在血管内皮功能中起作用的不同途径：钙信号、p38 和 ERK1/2；通过 Cx43 控制信号传播；以及通过产生一氧化氮（NO）来调节内皮功能。评估了 SNs 对 ECs Ca 内流、Cx43 表达、内皮型一氧化氮合酶（eNOS）和一氧化氮（NO）产生、p38、ERK1/2 及其磷酸化形式的影响。此外，通过在共培养模型中使用各自的拮抗剂分析 CGRP 和 SP 的作用，或者直接添加到 ECs 的单核培养物中分析它们的作用。我们表明，辣椒素刺激的 SNs 诱导 ECs 中 Ca 内流增加。SNs 刺激 ECs 中 NO 产生增加，可能涉及抑制性 eNOS T495 磷酸化位点减少。由 SNs 产生的神经肽 CGRP 似乎是 ECs 中这种作用的介导物之一，因为在 ECs 和 SNs 的共培养物中存在 CGRP 拮抗剂时，NO 产生减少，而当 ECs 受到合成 CGRP 刺激时，NO 产生增加。总之，我们的结果表明，SNs 通过 CGRP 产生和 NO 信号通路在控制内皮细胞功能方面发挥重要作用。
