EXPRESSION OF NERVE GROWTH FACTOR, ITS TRKA RECEPTOR, AND SEVERAL NEUROPEPTIDES IN PORCINE ESOPHAGUS. IMPLICATIONS FOR INTERACTIONS BETWEEN NEURAL, VASCULAR AND EPITHELIAL COMPONENTS OF THE ESOPHAGUS

The gastrointestinal tract, including the esophagus, is innervated by the autonomic enteric nervous system (ENS), mainly composed of the submucosal Meissner's plexus and myenteric Auerbach's plexus, the latter located between the circular and longitudinal muscle layers of the muscularis propria (1, 2). The ENS neural network is composed of neuronal cells, glial cells, interneurons, and nerve bundles and fibers. Normal structure and function of the esophageal enteric nervous system (EENS) network is essential for the motility, regulation of blood flow and secretory function of esophagus and coordination of these functions into organized patterns (1-5). Altered structure and/or malfunction of EENS are increasingly recognized as factors underlying functional gastrointestinal diseases and neuromuscular motility disorders often referred to as enteric neuropathies (2, 6-8).

The aim of this study was to determine the expression level and localization of nerve growth factor (NGF, a fundamental neurotrophic factor) and other neuropeptides: calcitonin gene related peptide (CGRP) - a marker of sensory nerves, neuronal nitric oxide synthase (nNOS) and neuronal specific enolase (NSE) in the porcine esophagus. NGF is an evolutionary conserved peptide, which is crucial for the growth, survival and maintenance of sensory and sympathetic neurons, septal cholinergic neurons in the brain and neurite outgrowth (9-11). Recent studies provide evidence of NGF's expression in endothelial cells and the possible involvement of NGF in vascular system. NGF has been shown to induce in vitro proliferation of HUVECs and brain capillary endothelial cells (12, 13) and can upregulate vascular endothelial growth factor (VEGF) production in some cells (14, 15). NGF also exerts a pro-angiogenic action on some endothelial cells (16). However, the expression of NGF in esophageal vasculature has not been examined before and existing data (from non-esophageal tissues) cannot predict whether NGF will affect angiogenesis and/or vasculogenesis in esophageal mucosa.

The biological actions of NGF are mediated through two types of specific receptors with distinct affinities (9, 10). NGF binding to its high affinity tropomyosin related kinase (TrkA) receptor signals neuroprotective and repair functions in physiological and pathological conditions of the nervous system (9, 10). The expression and the roles of NGF and its TrkA receptor protein in EENS and in esophageal wall are not known. Esophageal visceral hypersensitivity has been proposed to be a pathogenesis of heartburn in non-erosive reflux disease, but the underlying mechanisms are not clear. NGF, CGRP and nNOS may be involved in the sensitization of afferent neuronal pathways and visceral hpersensitivity (10, 11, 17, 18). It has been suggested that these neuropeptides can control sensory and pain mechanisms (17-22). A recent study demonstrated that chronic esophagitis increases the mRNA expressions of purinergic receptors P2X3 and P2X7 accompanied by an increased NGF mRNA expression (23).

In this study we demonstrate expression of NGF, CGRP, nNOS and NSE not only in neural elements of esophageal ENS but also in esophageal keratinocyte epithelial progenitor cells and in endothelial cells of blood vessels. This indicates previously unrecognized interactions between the submucosal and myenteric components of the autonomic ENS and the vascular and epithelial components of the esophagus.

MATERIAL AND METHODS

in vivo experiments

The experimental protocol was approved by the University of California Irvine's Institutional Animal Care and Use Committee (IACUC). Four Yorkshire pigs with body weights ranging between 30 and 40 kg were used. The animals were euthanized using a lethal dose of Euthasol (0.35 mL/Kg) - a solution containing pentobarbital sodium (390 mg/mL) and phenytoin sodium (50 mg/ml).

Sections of the distal and mid esophagus wall were resected, fixed in 10% buffered formalin and processed for histology and immunohistochemical staining.

Immunohistochemical studies

Five µm thin histologic sections of distal and mid esophagus were de-paraffinized and stained with H&E. Separate sections were immunostained for NGF, TrkA, CGRP, nNOS and NSE was performed. Antibodies used were: NGF - sc-548 (Santa Cruz Biotechnology, Santa Cruz, CA); TrkA - sc-14024 (Santa Cruz Biotechnology, Santa Cruz, CA); CGRP - T-4032 (Bachem, Peninsula labs, San Carlos, CA); nNOS - ab76067 (Abcam, Cambridge, MA); NSE - ab53025 (Abcam, Cambridge, MA). Tissue sections were incubated overnight at 4°C with respective antibodies (1:100 dilution). After washing, tissue sections were incubated with biotinylated secondary antibody (Dako, Carpinteria, CA; 1:500 dilution) for 30 min. Tissue sections were washed and incubated with streptavidin/HRP conjugate (Dako, Carpinteria, CA; 1:500 dilution) for 30 min and developed with AEC chromogen. Nuclei were counter stained using Mayer's hematoxylin. Immunostaining was evaluated on coded slides by two investigators unaware of the code and quantified by measuring staining signal intensity in each specimen using MetaMorph 7.0 analysis system (Molecular Devices Downington, PA).

RESULTS

Histological evaluation of the H&E stained esophageal specimens demonstrated normal esophageal structures. NGF was strongly expressed in the majority of neuronal and glial cells and nerves and in addition in esophageal keratinocyte progenitor cells at the basal layer of squamous epithelium and in endothelial cells of blood vessels (Fig. 1A and 1B). The distribution of TrkA was predominantly in some neurons and nerve fibers; its signal was 1.5-fold weaker (P < 0.001) than NGF (Fig. 2A). CGRP was expressed in the neuronal and glial cells and nerves, and in addition in esophageal keratinocyte progenitor cells and endothelial cells of blood vessels (Fig. 2B). The nNOS and NSE expression was strong and localized similar to NGF to majority of neuronal cells and nerves, and in addition in esophageal keratinocyte progenitor cells and in endothelial cells of blood vessels (Fig. 3A and 3B). All above neuronal peptides when evaluated quantitatively had similar signal intensity except TrkA, which had lower signal intensity (Table 1).

Fig. 1. Immunostaining for NGF protein in porcine esophagus. (Fig. 1A) and (Fig. 1B) NGF (brown staining) is expressed in neuronal cells (arrows), nerve fibers (arrowheads) and plexuses and also in endothelial cells (*) of blood vessels and keratinocyte progenitor cells (KPC).

Fig. 2. Immunostaining for TrkA and CGRP proteins in porcine esophagus. (Fig. 2A) TrkA, and (Fig. 2B) CGRP are expressed in neuronal cells (arrows), nerve fibers (arrowheads) and plexuses, and in endothelial cells (*) of blood vessels and keratinocyte progenitor cells (KPC).

Fig. 3. Immunostaining for nNOS and NSE proteins in porcine esophagus. (Fig. 3A) nNOS, and (Fig. 3B) NSE are expressed in neuronal cells (arrows), nerve fibers (arrowheads) and plexuses, and in endothelial cells (*) of blood vessels and keratinocyte progenitor cells (KPC).

Table 1. The signal intensity of respective immunostaining for NGF, TrkA CGRP, nNOS and NSE on scale 0 – 255.

DISCUSSION

These studies demonstrated for the first time a strong expression of NGF, TrkA, CGRP, nNOS and NSE in the majority of esophageal ENS components likely indicating their important, but previously unrecognized regulatory roles in the esophagus. Interestingly, in addition to neural structures, NGF and the other neural peptides examined were expressed in esophageal keratinocyte progenitor cells and endothelial cells of blood vessels, likely indicating previously unrecognized interactions between the submucosal and myenteric components of the autonomic ENS and the vascular and epithelial components of the esophagus. This is the first demonstration that NGF and TrkA receptor are expressed and co-localized in esophageal keratinocyte progenitor cells indicating that NGF can likely play an autocrine regulatory role in these cells's proliferation and survival.

Keratinocyte epithelial progenitor cells at the basal layer of squamous, stratified epithelium are critical for maintenance, renewal and healing of esophageal mucosa. While factors regulating integrity of gastric mucosa and gastric progenitor (e.g. prostaglandins, nitric oxide, growth factors) are well recognized, the mechanisms regulating function, survival and proliferation of keratinocyte epithelial progenitor cells in esophageal mucosa remain largely unknown. Our previous studies showed that keratinocyte epithelial progenitor cells in normal esophageal tissues and esophageal organ cultures strongly express VEGF (24, 25). In this study we showed the expression of NGF, CGRP, nNOS and NSE in esophageal keratinocyte epithelial progenitor cells. Since keratinocyte epithelial progenitor cells are important for epithelium integrity and healing, our findings indicate potential important roles of these peptides in the renewal of esophageal epithelium and maintaining its integrity.

Our present study demonstrated expression and cellular localization of NGF and several neuropeptides in the esophagus, including CGRP and eNOS that may play important roles in esophageal protection and healing. To this end a recent paper demonstrated esophagoprotective action of angiotensin in experimental model of acute reflux esophagitis and showed that this action is mediated in part by NOS and stimulation of sensory - CRGP nerves (26). Another recent study demonstrated that L-tryptophan and melatonin exert an esophagoprotective action via modulation of nitric oxide/NOS activity (27). In this context our present study provides target cellular sites for nNOS and CGRP.

Abbreviations: CGRP, calcitonin gene related peptide; ENS, enteric nervous system; EENS, esophageal enteric nervous system; NGF, nerve growth factor; nNOS, neuronal nitric oxide synthase; NSE, neuronal specific enolase; TrkA, tyrosine receptor kinase A - high affinity NGF receptor

Acknowledgements: Source of funding: H. H. Chao Family Foundation funding to KJC and VA Merit Review funding to AST.

Authors J.B. Samarasena and A. Ahluwalia contributed equally to the manuscript.

Conflict of interests: None declared.

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