Neuropeptides Substance P and Calcitonin Gene Related Peptide Accelerate the Development and Fibrogenesis of Endometriosis

Dingmin Yan,Xishi Liu,Sun-Wei Guo

doi:10.1038/s41598-019-39170-w

Abstract

Endometriotic lesions are known to be hyperinnervated, especially in lesions of deep endometriosis (DE), which are frequently in close proximity to various nerve plexuses. DE lesions typically have higher fibromuscular content than that of ovarian endometriomas (OE) lesions, but the underlying reason remains elusive. Aside from their traditional role of pain transduction, however, whether or not sensory nerves play any role in the development of endometriosis is unclear. Here, we show that, thorough their respective receptors neurokinin receptor 1 (NK1R), calcitonin receptor like receptor (CRLR), and receptor activity modifying protein 1 (RAMP-1), neuropeptides substance P (SP) and calcitonin gene related peptide (CGRP) induce epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT) and further turn stromal cells into smooth muscle cells (SMCs) in endometriotic lesions, resulting ultimately in fibrosis. We show that SP and CGRP, or the rat dorsal root ganglia (DRG) supernatant, through the induction of NK1R and CGRP/CRLR/RAMP-1 signaling pathways, promoted EMT, FMT and SMM in endometriosis, resulting in increased migratory and invasive propensity, cell contractility, production of collagen, and eventually to fibrosis. Neutralization of NK1R and/or CGRP/CRLR/RAMP-1 abrogated these processes. Extended exposure of endometriotic stromal cells to SP and/or CGRP or the DRG supernatant induced increased expression of α-SMA, desmin, oxytocin receptor, and smooth muscle myosin heavy-chain. Finally, we show that DE lesions had significantly higher nerve fiber density, increased staining levels of α-SMA, NK1R, CRLR, and RAMP-1, concomitant with higher lesional fibrotic content than that of OE lesions. The extent of lesional fibrosis correlated positively with the staining levels of NK1R, CRLR, and RAMP-1, as well as the nerve fiber density in lesions. Thus, this study provides another piece of evidence that sensory nerves play an important role in promoting the development and fibrogenesis of endometriosis. It explains as why DE frequently have higher fibromuscular content than that of OE, highlights the importance of lesional microenvironment in shaping the lesional fate, gives more credence to the idea that ectopic endometrium is fundamentally wounds that go through repeated tissue injury and repair, and should shed much needed light into the pathophysiology of endometriosis.

Highlights

Www.nature.com/scientificreports fibromuscular pelvic structures such as the uterosacral and utero-ovarian ligaments and the muscular wall of pelvic organs[6]
Consistent with the morphological changes, the protein expression levels of E-cadherin, an epithelial marker, were significantly decreased after 72 hours of substance P (SP) or calcitonin gene related peptide (CGRP) treatment (Fig. 1B), while the expression levels of genes involved in epithelial-mesenchymal transition (EMT), such as Snai[1] and Slug, and of markers of mesenchymal cells, such as vimentin and N-cadherin, as well as the gene involved in fibrosis such as PAI-1 (Serpine1) were significantly elevated in 11Z cells treated with SP and/or CGRP as compared with the controls
We have shown that, as with platelet-derived transforming growth factor (TGF)-β122, sensory nerve-derived neuropeptides SP and CGRP facilitate EMT, fibroblast-to-myofibroblast transdifferentiation (FMT) and differentiation to smooth muscle cells (SMCs) in endometriosis, yielding increased collagen production, elevated cellular contractility, and eventually fibrosis

Summary

Introduction

Www.nature.com/scientificreports fibromuscular pelvic structures such as the uterosacral and utero-ovarian ligaments and the muscular wall of pelvic organs[6]. One feature that DE stands out from other subtypes of endometriosis is its presence of smooth muscle metaplasia (SMM) and its high degree of fibrotic tissues[4,15,16,17], which explains the choice of the term adenomyosis externa, presumably because of its enriched fibromuscular content[15] akin to adenomyosis This is one of several reasons that prompted a recent proposal to re-define endometriosis to include the pro-fibrotic nature of endometriosis[18]. Extended exposure to TGF-β1 results in elevated expression of α-smooth muscle actin (α-SMA) and of markers of terminally differentiated smooth muscle cells (SMCs) in the stromal component of endometriotic lesions, accounting for SMM that is common in endometriotic lesions[23,24,25,26] This essentially depicts the natural history of endometriotic lesions[27]. Surgical denervation of sensory nerves significantly reduced lesional fibrosis, and the antagonism of neurokinin 1 receptor (NK1R), the SP receptor, achieved the same effect[35]

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Results

Conclusion