NGF modulates trkANGFR/p75NTR in αsMA-expressing conjunctival fibroblasts from human ocular cicatricial pemphigoid (OCP)

OBJECTIVE: In a previous study, we reported the upregulation of Nerve Growth Factor (NGF) and trkANGFR expression in Ocular Cicatricial Pemphigoid (OCP), an inflammatory and remodeling eye disease. Herein, we hypothesize a potential NGF-driven mechanism on fibroblasts (FBs) during OCP remodeling events. To verify, human derived OCP-FBs were isolated and characterized either at baseline or after NGF exposure. MATERIALS AND METHODS: Conjunctival biopsies were obtained from 7 patients having OCP and 6 control subjects (cataract surgery). Both conjunctivas and primary FB cultures were characterised for αSMA, NGF and trkANGFR/p75NTR expression. Subcultures were exposed to NGF and evaluated for αSMA, NGF, trkANGFR/p75NTR expression as well as TGFβ1/IL4 release. For analysis, early and advanced subgroups were defined according to clinical parameters. RESULTS: OCP-conjunctivas showed αSMA-expressing FBs and high NGF levels. Advanced OCP-FBs showed higher αSMA expression associated with higher p75NTR and lower trkANGFR expression, as compared to early counterparts. αSMA expression was in keeping with disease severity and correlated to p75NTR. NGF exposure did not affect trkANGFR levels in early OCP-FBs while decreased both αSMA/p75NTR expression and TGFβ1/IL4 release. These effects were not observed in advanced OCP-FBs. CONCLUSIONS: Taken together, these data are suggestive for a NGF/p75NTR task in the potential modulation of OCP fibrosis and encourages further studies to fully understand the underlying mechanism occurring in fibrosis. NGF/p75NTR might be viewed as a potential therapeutic target

Nerve growth factor has a modulatory role on human primary fibroblast cultures derived from vernal keratoconjunctivitis-affected conjunctiva

Purpose: To evaluate the role of nerve growth factor (NGF) in remodeling processes of vernal keratoconjunctivitis (VKC). VKC is a chronic inflammatory disorder of the conjunctiva and is characterized by marked tissue remodeling. NGF, a pleiotrophic factor with documented profibrogenic activities, is produced by inflammatory and structural cells populating the VKC conjunctiva and is increased in the serum and tears of VKC patients.Methods: Primary cultures of VKC-derived fibroblasts (VKC-FBs) were exposed to increasing NGF concentrations (1500 ng/ml) to evaluate and compare the expression of alpha-smooth muscle actin (alpha SMA, a defining myofibroblast marker), collagens (types I and IV), and metalloproteinases and tissue inhibitors (MMP9/TIMP1, MMP2/TIMP2) at the biochemical as well as molecular levels.Results: Endogenous NGF was increased in the VKC-FB supernatant, as compared to healthy-FB supernatant. VKC-FBs expressed aSMA and increased types I and IV collagens. VKC-FBs, and in particular all aSMA positive cells, expressed both trkA(NGFR) and p75(NTR), while healthy-FBs only expressed trkA(NGFR). Exogenous NGF did not change aSMA expression, while aSMA expression was enhanced by specific neutralization of p75(NTR). NGF (10 ng/ml) exposure significantly decreased type I collagen expression, without affecting type IV collagen, and increased MMP9mRNA and protein.Conclusions: The autocrine modulation of differentiation and response of VKC-FBs to NGF exposure with downregulation of type I collagen and upregulation of MMP9 expression supports a relevant role for NGF in tissue remodeling of VKC

Potenziale coinvolgimento dell’NGF nell’infiammazione e nel rimodellamento tissutale della superficie oculare

L'infiammazione è per definizione una reazione locale ad un danno tissutale, che tenta di risolvere gli effetti del trauma con esiti fisiologici o in rari casi patologici. I fibroblasti (FBs) sono le cellule chiave del processo di remodelling, interagendo sia con cellule infiammatorie che strutturali. I FBs attivati differenziano in myofibroblasti (myoFBs), caratterizzati dall'espressione del marker alpha-muscle actin (alpha-SMA). Tra i fattori pro-fibrogenici il Nerve Growth Factor (NGF), sembra essere associato al processo di remodelling tissutale attraverso i due recettori trkA e p75. Abbiamo studiato due patologie oculari con un differente decorso cronico infiammatorio associato a fibrosi: la Cheratocongiuntivite primaverile (VKC, un'infiammazione di tipo allergico, Th2) e il Pemfigoide Cicartriziale Oculare (OCP, una malattia autoimmune, Th1). Entrambe le patologie hanno in comune l'aumentata deposizione di matrice extracellulare (ECM). In questo studio in particolare abbiamo approfondito in vitro i seguenti punti: 1. gli effetti del NGF sul fenotipo FBs/myoFBs di VKC e OCP; 2. rapporto tra NGF e fenotipo FBs/myoFBs; 3. la possibile modulazione del NGF in questo processo di remodelling, in relazione al TGF-beta1 ed altre citochine profibrogeniche; 4. il potenziale contributo dell'NGF nell'immunità innata (TLRs). A tale fine sono state analizzate le biopsie congiuntivali di VKC, OCP e di soggetti sani. Sono state quindi sviluppate colture primarie di FBs, caratterizzate, esposte ad NGF ed infine valutati a livello cellulare (confocale), biochimico (Western Blot, ELISA, FACS) e molecolare (Real-Time PCR). I dati ottenuti su FBs sia di VKC che di OCP hanno evidenziato che una buona percentuale di queste cellule sono myoFBs, che esprimono entrambi i recettori dell'NGF e sono in grado di produrre alte quantità di NGF. In VKC l'esposizione ad NGF risulta in un aumento della produzione dell'ECM, ma non nell'espressione di alpha-SMA. In FBs di OCP l'espressione di alpha-SMA invece è strettamente collegata allo stadio della malattia. In accordo con i dati sulla VKC, questi FBs risultano anche p75 positivi, con una maggior espressione negli stadi avanzati della patologia. Diversamente trkA è presente nei primi stadi della malattia e cala in quelli avanzati. Il trattamento con NGF inverte questa tendenza nei FBs ottenuti da pazienti ad uno stadio precoce della patologia. E' stato quindi valutato l'effetto dell'NGF sulla sintesi ed il rilascio di citochine pro/anti-infiammatorie della risposta Th1 da parte dei Fbs in studio. I livelli di IL-2, IL-4 e il TGF-beta1, alti nella prima fase patologica diminuiscono in condizioni più severe della patologia e sono significativamente modulati dal NGF. Probabilmente l'NGF potrebbe agire sia sulla sopravvivenza/apoptosi dei linfociti che sull'attivazione dei FBs/myoFBs. Per valutare quindi se l'effetto dell'NGF potesse esser dipendente dal TGF-beta1 abbiamo sottoposto FBs non patologici a stimoli con NGF e/o TGF-beta1 o con anticorpi neutralizzanti, ed osservato che entrambi i fattori si stimolano reciprocamente sul fenotipo alpha-SMA. Si è valutato anche un possibile coinvolgimento nel processo di remodelling della microflora (biofilm) e quindi dei TLRs. La valutazione dell'espressione dei TLR4 e TLR9 in cellule di FBs/myoFBs di VKC ed OCP dimostra che l'espressione è diversa nelle due patologie: mentre nei FBs di OCP entrambi Toll risultano aumentati in quelli di VKC sono "down" regolati rispetto i normali controlli. Questo probabilmente dovuto ad una diversa storia immunitaria nelle due malattie. Il trattamento con NGF sembra modulare l'espressione dei suddetti recettori rendendo le cellule più o meno reattive nel riconoscimento dei batteri. Da questi studi è emerso che l'effetto pro-fibrogenico dell'NGF potrebbe esser parzialmente dipendente dal rapporto di espressione tra i recettori trkA e p75 sui FBs/myoFBs, dall'espressione del TGF-beta1, dei TLRs e dallo stadio patologico della malattia. In particolare nello studio dell'OCP è possibile che questi rapporti siano alterati e ciò spiegherebbe il diverso comportamento dei FBs/myoFBs sia ai diversi stimoli dell'NGF che ai diversi stadi della patologia.Inflammation is by definition a local reaction against a tissue damage, trying to solve the traumatic effects with physiological or rarely pathological behaviours. The fibroblasts (FBs) are the key cells of tissue remodelling process, interacting both with inflammatory and structural cells. The activated FBs differentiate into myofibroblasts (myoFbs), characterized by the expression of the alpha-Smooth Muscle Actin (alpha-SMA) marker. In the fibrogenic factors set, the Nerve Growth Factor (NGF), seems to be associated with the tissue remodelling process via the two receptors trkA and p75. We have studied two ocular pathologies with different chronic inflammatory courses associated with fibrosis: the Vernal Keratocongiuntivitis (VKC, an allergic inflammation, Th2) and the Ocular Cicatricial Pemphigoid (OCP, an auto-immune disorder). Both these pathologies share the common feature of an increased deposition of Extra-Cellular Matrix (ECM). In this study in particular we have analyzed the following points: 1. the NGF effects on FBs/myoFBs phenotype of VKC and OCP; 2. the relation between NGF and FBs/myoFBs phenotypes; 3. the possible modulation effects by NGF in this remodelling process in relation with TGF-beta1 and other inflammatory cytokines (Th1/Th2); 4. The possible involvement of NGF in the innate immunity (TLRs). To this aim the we have analysed conjunctival biopsies of VKC, OCP and of healthy subjects. Primary FBs cultures have been created, characterized, NGF treated and finally evaluated at cellular (confocal), biochemical (Western Blot, ELISA, FACS) and molecular (Real-Time PCR) levels. Both VKC and OCP FBs data showed that a large part of these cells are myoFBs, expressing the NGF receptors and releasing high NGF levels. In VKC, the NGF treatment results in an increase of the ECM production but does not influence alpha-SMA expression. On the other hand, in OCP FBs the alpha-SMA expression is strictly connected with the pathological status of the disease. In accordance with VKC data, these FBs result also p75 positive, with a larger expression in the advanced stage of the pathology. Differently, TrkA is present in the in initial stage of the disease and decreases in the advanced one. The NGF treatment inverts this trend in FBs from patients at early stages of the disease. Moreover the NGF effect on synthesis and release of the pro/anti-inflammatory cytokines of the Th1 response, by FBs under exam, has been evaluated. The IL-2, IL-4 and TGF-beta1 levels, which were high in first pathological phase, decreased in the more severe conditions of the disease and resulted to be relevantly modulated by NGF. Probably, NGF might act both on survival/apoptosis of lymphocytes and on the activation of FBs/myoFBs. In order to evaluate if the NGF effect could be independent by TGF-beta1, we have stimulated non pathologic FBs with NGF and/or TGF-beta1 or neutralizing antibodies, and we have observed that both these factors stimulate each other on alpha-SMA phenotype. A possible role of microflora (biofilm) and TLRs in the remodelling process has been also studied. The evaluation of TLR4 and TLR9 expression in VKC and OCP FBs/myoFBs demonstrates that the expression is different in these two pathologies: while in FBs with OCP, being both Toll, these are augmented, in those of VKC these are down regulated with respect to normal controls. This fact is probably due to a different immunological history in the two pathologies. The NGF treatment seems to modulate the expression of these receptors making the cells more or less reactive in recognizing bacteria. From these studies it appeared that the NGF pro-fibrogenic effect could be partially dependent by the expression ratio of trkA and p75 on the FBs/myoFBs, by the TGF-beta1 and TLRs. In particular in the study of OCP it is possible that these ratio could be altered and this would explain the different FBs/myoFBs behaviour in relation with different NGF stimuli and with the different levels of the pathology

In vivo characterization of doxycycline effects on tear metalloproteinases in patients with chronic blepharitis

Matrix metalloproteinases (MMPs) have a role in the pathogenesis of rosacea-associated chronic blepharitis. Doxycycline is largely used as a treatment for recalcitrant chronic blepharitis. It has been shown in vitro that doxycycline inhibits MMPs activation. The aim of this study was to investigate in vivo the effect of doxycycline in modulating MMPs in patients with chronic idiopathic blepharitis

Preliminary evidence of the efficacy of probiotic eye-drop treatment in patients with vernal keratoconjunctivitis

Probiotics have been shown to improve allergic inflammation. The aim of this study was to evaluate the efficacy of Lactobacillus Acidophilus eye-drops in controlling signs and symptoms of vernal keratoconjunctivitis (VKC)

Nerve growth factor modulates toll-like receptor (TLR) 4 and 9 expression in cultured primary VKC conjunctival epithelial cells

To investigate if nerve growth factor (NGF) might modulate toll-like receptor (TLR) 4 and 9 expression in primary cultures of vernal keratoconjunctivitis (VKC)-derived conjunctival epithelial cells (VKC-ECs)

Nerve growth factor and tissue repair remodeling: trkA(NGFR) and p75(NTR), two receptors one fate

This review deals with the role of nerve growth factor (NGF) in healing process as a result of injury. The role of both trkA(NGFR) and p75(NTR) specific NGF receptors and their contribution in the complex network of tissue repair process, is discussed and highlighted in view of recent findings. In fact, NGF represents a significant advance in the treatment of etiologically different ulcers (corneal ulcers, pressure ulcers, post-viral infections, chemical burns) and might shorten the recovery process. For these diseases, no specific treatment is actually available. It is reasonable that apart from NGF and/or neurotrophins a different time-course of trkA(NGRF)/p75(NTR) expression, might regulate the final process. In summary, these novel findings on the potential pro-healing capacity of NGF might open new possibilities for this growth factor in modulating the healing processes in several pathological conditions

Human idiopathic epiretinal membranes express NGF and NGF receptors

Glial cells and fibroblasts (FBs) play a key role in epiretinal membrane (ERM) development and progression. Myofibroblasts (myoFBs), arising from these cells, can lead to the hypertrophic scars and tissue contraction observed in ERMs. Nerve growth factor (NGF) and transforming growth factor-beta1 (TGF-beta1) play a crucial role in FB activities. Therefore, the authors evaluated myoFBs in ERMs and NGF, trkA(NGFR and p75(NTR) expression, as well as TGF-beta1/TGF-betaRII levels in both ERMs and vitreous

Nerve growth factor effect on human primary fibroblastic-keratocytes: possible mechanism during corneal healing

In response to corneal injury, cytokines and growth factors play a crucial role by influencing epithelial-stromal interaction during the healing and reparative processes which may resolve in tissue remodeling and fibrosis. While transforming growth factor-beta1 (TGF-beta1) is considered the main profibrogenic modulator of these process, recently the nerve growth factor (NGF) appears as a pleiotropic modulator of wound-healing and inflammatory responses. Interestingly in the cornea, where NGF, trkA(NGFR) and p75(NTR) are expressed by epithelial cells and keratocytes, the NGF eye-drop induces the healing of neurotrophic or autoimmune corneal ulcers. During corneal healing, quiescent keratocytes are replaced by active fibroblast-like keratocytes/myofibroblasts. While the NGF effect on epithelial cells has been investigated, no data are reported for NGF effects on fibroblastic-keratocytes, during corneal healing. NGF, trkA(NGFR) and p75(NTR) were found expressed by fibroblastic-keratocytes. NGF was able to induce fibroblastic-keratocyte differentiation into myofibroblasts, migration, Metalloproteinase-9 expression/activity and contraction of a 3D collagen gel, without affecting their proliferation and collagen production. These data also show a two-directional control of fibroblastic-keratocytes by NGF and TGF-beta1. To sum up, the findings of this study indicate that NGF can modulate some functional activities of fibroblastic-keratocytes, thus substantiating the healing effects of NGF on corneal wound-healing