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Nerve Growth Factor (NGF) modulates in vitro induced myofibroblasts by highlighting a differential protein signature.

ABSTRACT: We previously described the profibrogenic effect of NGF on conjunctival Fibroblasts (FBs) and its ability to trigger apoptosis in TGF?1-induced myofibroblasts (myoFBs). Herein, cell apoptosis/signalling, cytokines' signature in conditioned media and inflammatory as well as angiogenic pathway were investigated. Experimental myoFBs were exposed to NGF (0.1-100 ng/mL), at defined time-point for confocal and biomolecular analysis. Cells were analysed for apoptotic and cell signalling activation in cell extracts and for some inflammatory and proinflammatory/angiogenic factors' activations. NGF triggered cJun overexpression and phospho-p65-NFkB nuclear translocation. A decreased Bcl2:Bax ratio and a significant expression of smad7 were confirmed in early AnnexinV-positive myoFBs. A specific protein signature characterised the conditioned media: a dose dependent decrease occurred for IL8, IL6 while a selective increase was observed for VEGF and cyr61 (protein/mRNA). TIMP1 levels were unaffected. Herein, NGF modulation of smad7, the specific IL8 and IL6 as well as VEGF and cyr61 modulation deserve more attention as opening to alternative approaches to counteract fibrosis.

SUBMITTER: Esposito G

PROVIDER: S-EPMC7814037 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Nerve Growth Factor (NGF) modulates in vitro induced myofibroblasts by highlighting a differential protein signature.

Esposito Graziana G Balzamino Bijorn Omar BO Stigliano Egidio E Biamonte Filippo F Urbani Andrea A Micera Alessandra A

Scientific reports 20210118 1

We previously described the profibrogenic effect of NGF on conjunctival Fibroblasts (FBs) and its ability to trigger apoptosis in TGFβ1-induced myofibroblasts (myoFBs). Herein, cell apoptosis/signalling, cytokines' signature in conditioned media and inflammatory as well as angiogenic pathway were investigated. Experimental myoFBs were exposed to NGF (0.1-100 ng/mL), at defined time-point for confocal and biomolecular analysis. Cells were analysed for apoptotic and cell signalling activation in c ...[more]

PMID: 33462282

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Project description:In the conjunctiva, repeated or prolonged exposure to injury leads to tissue remodeling and fibrosis associated with dryness, lost of corneal transparency and defect of ocular function. At the site of injury, fibroblasts (FB) migrate and differentiate into myofibroblasts (myoFB), contributing to the healing process together with other cell types, cytokines and growth factors. While the physiological deletion of MyoFB is necessary to successfully end the healing process, myoFB prolonged survival characterizes the pathological process of fibrosis. The reason for myoFB persistence is poorly understood. Nerve Growth Factor (NGF), often increased in inflamed stromal conjunctiva, may represent an important molecule both in many inflammatory processes characterized by tissue remodeling and in promoting wound-healing and well-balanced repair in humans. NGF effects are mediated by the specific expression of the NGF neurotrophic tyrosine kinase receptor type 1 (trkA(NGFR)) and/or the pan-neurotrophin glycoprotein receptor (p75(NTR)). Therefore, a conjunctival myoFB model (TGF?1-induced myoFB) was developed and characterized for cell viability/proliferation as well as ?SMA, p75(NTR) and trkA(NGFR) expression. MyoFB were exposed to acute and chronic NGF treatment and examined for their p75(NTR)/trkA(NGFR), ?SMA/TGF?1 expression, and apoptosis. Both NGF treatments significantly increased the expression of p75(NTR), associated with a deregulation of both ?SMA/TGF?1 genes. Acute and chronic NGF exposures induced apoptosis in p75(NTR) expressing myoFB, an effect counteracted by the specific trkA(NGFR) and/or p75(NTR) inhibitors. Focused single p75(NTR) and double trkA(NGFR)/p75(NTR) knocking-down experiments highlighted the role of p75(NTR) in NGF-induced apoptosis. Our current data indicate that NGF is able to trigger in vitro myoFB apoptosis, mainly via p75(NTR). The trkA(NGFR)/p75(NTR) ratio in favor of p75(NTR) characterizes this process. Due to the lack of effective pharmacological agents for balanced tissue repairs, these new findings suggest that NGF might be a suitable therapeutic tool in conditions with impaired tissue healing.

Project description:Corroborating data sustain the pleiotropic effect of nerve growth factor (NGF) in the protection of the visual system from dangerous stimuli, including ultraviolet (UV). Since UV exposure might promote ocular surface changes (conjunctival inflammation and matrix rearrangement), as previously reported from in vivo studies sustaining some protective NGF effects, in vitro cultures of human conjunctival fibroblasts (FBs) were developed and exposed to a single UV exposure over 15 min (0.277 W/m2), either alone or supplemented with NGF (1-10-100 ng/mL). Conditioned media and cell monolayers were collected and analyzed for protein release (ELISA, ELLA microfluidic) and transcript expression (real-time PCR). A specific "inflammatory to remodeling" pattern (IL8, VEGF, IL33, OPN, and CYR61) as well as a few epigenetic transcripts (known as modulator of cell differentiation and matrix-remodeling (DNMT3a, HDAC1, NRF2 and KEAP1)) were investigated in parallel. UV-exposed FBs (i), showed no proliferation or significant cytoskeleton rearrangement; (ii), displayed a trkANGFR/p75NTR phenotype; and (iii), synthesized/released IL8, VEGF-A, IL33, OPN, and CYR61, as compared to unexposed ones. NGF addition counteracted IL8, IL33, OPN, and CYR61 protein release merely at lower NGF concentrations but not VEGF. NGF supplementation did not affect DNMT3a or HDAC1 transcripts, while it significantly upregulated NRF2 at lowest NGF doses and did not change KEAP1 expression. Taken together, a single UV exposure activated conjunctival FBs to release pro-inflammatory/fibrogenic factors in association with epigenetic changes. The effects were selectively counteracted by NGF supplementation in a dose-dependent fashion, most probably accountable to the trkANGFR/p75NTR phenotype. Further in vitro studies are underway to better understand this additional NGF pleiotropic effect. Since UV-shield impairments represent a worldwide alert and UV radiation can slowly affect ocular surface homeostasis (photo-ageing, cataract) or might exacerbate ocular diseases with a preexisting fibrosis (pterygium, VKC), these findings on NGF modulation of UV-exposed FBs might provide additional information for protecting the ocular surface (homeostasis) from low-grade long-lasting UV insults.

Dataset Information

Nerve Growth Factor (NGF) modulates in vitro induced myofibroblasts by highlighting a differential protein signature.

Publications

Nerve Growth Factor (NGF) modulates in vitro induced myofibroblasts by highlighting a differential protein signature.

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