HCl-induced and ATP-dependent upregulation of TRPV1 receptor expression and cytokine production by human esophageal epithelial cells

The pathogenesis of gastroesophageal reflux disease (GERD) remains elusive, but recent evidence suggests that early secretion of inflammatory cytokines and chemokines by the mucosa leads to influx of immune cells followed by tissue damage. We previously showed that exposure of esophageal mucosa to HCl causes ATP release, resulting in activation of acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT), the enzyme responsible for the production of platelet-activating factor (PAF). In addition, HCl causes release of IL-8 from the esophageal mucosa. We demonstrate that esophageal epithelial cells secrete proinflammatory mediators in response to HCl and that this response is mediated by ATP. Monolayers of the human esophageal epithelial cell line HET-1A were exposed to acidified cell culture medium (pH 5) for 12 min, a total of seven times over 48 h, to simulate the recurrent acid exposure clinically occurring in GERD. HCl upregulated mRNA and protein expression for the acid-sensing transient receptor potential cation channel, subfamily vanilloid member 1 (TRPV1), lyso-PAF AT, IL-8, eotaxin-1, -2, and -3, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1. The chemokine profile secreted by HET-1A cells in response to repeated HCl exposure parallels similar findings in erosive esophagitis patients. In HET-1A cells, the TRPV1 agonist capsaicin reproduced these findings for mRNA of the inflammatory mediators lyso-PAF AT, IL-8, and eotaxin-1. These effects were blocked by the TRPV1 antagonists iodoresiniferatoxin and JNJ-17203212. These effects were imitated by direct application of ATP and blocked by the nonselective ATP antagonist suramin. We conclude that HCl/TRPV-induced ATP release upregulated secretion of various chemoattractants by esophageal epithelial cells. These chemoattractants are selective for leukocyte subsets involved in acute inflammatory responses and allergic inflammation. The data support the validity of HET-1A cells as a model of the response of the human esophageal mucosa in GERD.     

Associations of Circulating Gut Hormone and Adipocytokine Levels with the Spectrum of Gastroesophageal Reflux Disease

Objective

The pathogenesis of gastroesophageal reflux disease (GERD) is complex and poorly understood. We aim to investigate the association of various circulating peptide hormones with heterogenous manifestations of GERD.

Methods

One hundred and four patients that had experienced typical GERD symptoms (heartburn and/or acid regurgitation) for at least 3 episodes per week in the past 3 months were enrolled. All patients received a baseline assessment of symptom severity and frequency with the Reflux Disease Questionnaire and an upper endoscopy to classify GERD into erosive esophagitis (EE, n = 67), non-erosive esophagitis (NE, n = 37), and Barrett’s esophagus (BE, n = 8). Fifty asymptomatic subjects with an endoscopically normal esophagus were recruited as the control group. Complete anthropometric measures and blood biochemistry were obtained and fasting serum levels of adipocytokines (adiponectin and leptin) and gut hormones (ghrelin and peptide YY (PYY)) were determined by enzyme-linked immunosorbent assay in all subjects.

Results

All circulating peptide hormone levels were not statistically different between the GERD and control groups. However, GERD patients appeared to have lower PYY levels [median (25^th-75^th percentile), 80.1 (49.8–108.3) vs. 99.4 (65.8–131.9) pg/ml, p = 0.057] compared with control subjects. Among the GERD patients, ghrelin levels were inversely associated with the frequency and severity of acid regurgitation. In male GERD patients, EE was associated with significantly higher PYY levels [107.0 (55.0–120.8) vs. 32.8 (28.7–84.5) pg/ml, p = 0.026] but lower adiponectin levels [6.7 (5.6–9.3) vs. 9.9 (9.6–10.6) μg/ml, p = 0.034] than NE. Patients with BE had significantly lower adiponectin levels [6.0 (5.1–9.2) vs. 9.2 (7.1–11.2) μg/ml, p = 0.026] than those without BE.

Conclusions

Humoral derangement of circulating peptide hormones might participate in inflammation and symptom perception in patients suffering from GERD. Further studies to clarify the exact role of these hormones in the pathogenesis of GERD are warranted.     

The extracellular calcium-sensing receptor (CaSR) on human esophagus and evidence of expression of the CaSR on the esophageal epithelial cell line (HET-1A)

Gastrointestinal reflux disease and eosinophilic esophagitis are characterized by basal cell hyperplasia. The extracellular calcium-sensing receptor (CaSR), a G protein-coupled receptor, which may be activated by divalent agonists, is expressed throughout the gastrointestinal system. The CaSR may regulate proliferation or differentiation, depending on cell type and tissue. The current experiments demonstrate the expression of the CaSR on a human esophageal epithelial cell line (HET-1A) and the location and expression of the CaSR in the human esophagus. CaSR immunoreactivity was seen in the basal layer of normal human esophagus. CaSR expression was confirmed in HET-1A cells by RT-PCR, immunocytochemistry, and Western blot analysis. CaSR stimulation by extracellular calcium or agonists, such as spermine or Mg(2+), caused ERK1 and 2 activation, intracellular calcium concentration ([Ca(2+)](i)) mobilization (as assessed by microspecfluorometry using Fluo-4), and secretion of the multifunctional cytokine IL-8 (CX-CL8). HET-1A cells transiently transfected with small interfering (si)RNA duplex against the CaSR manifested attenuated responses to Ca(2+) stimulation of phospho- (p)ERK1 and 2, [Ca(2+)](i) mobilization, and IL-8 secretion, whereas responses to acetylcholine (ACh) remained sustained. An inhibitor of phosphatidylinositol-specific phospholipase C (PI-PLC) (U73122) blocked CaSR-stimulated [Ca(2+)](i) release. We conclude that the CaSR is present on basal cells of the human esophagus and is present in a functional manner on the esophageal epithelial cell line, HET-1A.     

胃食管反流病的治疗进展

胃食管反流病(gastroesophaeal reflux disease,GERD)是医疗实践中的最常见的疾病之一,其发病率在世界范围内呈逐年上升趋势,且随年龄增长而增加,40-60岁为高发年龄[1]。GERD是一种由胃、十二指肠内容物反流入食管引起不适症状和(或)并发症的疾病,GERD在临床上大致可分为:糜烂性食管炎(EE)(反流性食管炎(RE))和非糜烂性食管炎(NERD)。其中NERD最多见,约占60%。GERD远期危害较小,但其病情漫长且极易复发,严重影响了生活质量。主要表现为食管症状(包括典型的烧心和反流)和食管外症状(包括咽部异物感、咳嗽、声嘶、哮喘、咽喉炎等表现),还有增加发展为Barrett食管及食管癌的危险[2,3]。GERD的治疗目的是愈合食管炎,快速缓解症状、减少复发、提高生活质量,治疗方法主要包括以下4个方面:一般治疗,药物治疗,内镜下治疗和外科治疗。近年来已成为国内外研究的热点,本文就近年来对GERD的治疗进展做一综述。     

Acid Reflux Directly Causes Sleep Disturbances in Rat with Chronic Esophagitis

Background & Aims

Gastroesophageal reflux disease (GERD) is strongly associated with sleep disturbances. Proton pump inhibitor (PPI) therapy improves subjective but not objective sleep parameters in patients with GERD. This study aimed to investigate the association between GERD and sleep, and the effect of PPI on sleep by using a rat model of chronic acid reflux esophagitis.

Methods

Acid reflux esophagitis was induced by ligating the transitional region between the forestomach and the glandular portion and then wrapping the duodenum near the pylorus. Rats underwent surgery for implantation of electrodes for electroencephalogram and electromyogram recordings, and they were transferred to a soundproof recording chamber. Polygraphic recordings were scored by using 10-s epochs for wake, rapid eye movement sleep, and non-rapid eye movement (NREM) sleep. To examine the role of acid reflux, rats were subcutaneously administered a PPI, omeprazole, at a dose of 20 mg/kg once daily.

Results

Rats with reflux esophagitis presented with several erosions, ulcers, and mucosal thickening with basal hyperplasia and marked inflammatory infiltration. The reflux esophagitis group showed a 34.0% increase in wake (232.2±11.4 min and 173.3±7.4 min in the reflux esophagitis and control groups, respectively; p<0.01) accompanied by a reduction in NREM sleep during light period, an increase in sleep fragmentation, and more frequent stage transitions. The use of omeprazole significantly improved sleep disturbances caused by reflux esophagitis, and this effect was not observed when the PPI was withdrawn.

Conclusions

Acid reflux directly causes sleep disturbances in rats with chronic esophagitis.     

Clinical epidemiology and natural history of gastroesophageal reflux disease

In the MUSE classification of gastroesophageal reflux disease (GERD), esophagitis is assessed by the presence of metaplasia, ulcer, stricture, or erosion, each being graded as absent, mild or severe. Daily reflux symptoms affect about 4 to 7 percent of the population; erosive esophagitis occurs in about 2 percent; the prevalence rate of Barrett's metaplasia is 0.4 percent; and esophageal adenocarcinoma leads to two deaths per million living population. In persons with GERD symptoms, about 20 percent are found to have erosive esophagitis, while ulcers or strictures are found in less than 5 percent of all patients with erosive esophagitis. No clear-cut temporal progression exists between successive grades of disease severity, as the most severe grade of GERD is reached at the onset of the disease. Mild forms of GERD tend to be more common in women than men, while severe GERD characterized by erosive esophagitis, esophageal ulcer, stricture or Barrett's metaplasia are far more common in men than women. All forms of GERD affect Caucasians more often than African Americans or Native Americans. The prevalence of GERD is high among developed countries in North America and Europe and relatively low in developing countries in Africa and Asia. During the past three decades, hospital discharges and mortality rates of gastric cancer, gastric ulcer and duodenal ulcer have declined, while those of esophageal adenocarcinoma and GERD have markedly risen. These opposing time trends suggest that corpus gastritis secondary to Helicobacter pylori infection protects against GERD. This hypothesis is consistent with the geographic and ethnic distributions of GERD. Case-control studies also indicate that cases with erosive esophagitis are less likely to harbor active or chronic corpus gastritis than controls without esophagitis.     

Value of heartburn for diagnosing gastroesophageal reflux disease in severely obese patients

Objective : To evaluate the prevalence of gastroesophageal reflux disease (GERD) in severely obese patients and the association between symptoms and objective data of GERD in this population. Research Methods and Procedures : A total of 158 consecutive severely obese patients (BMI ≥ 40 kg/m²) were prospectively evaluated. Symptoms were evaluated by a structured clinical questionnaire. Objective assessment was made by ambulatory 24‐hour esophageal pH monitoring and endoscopy. GERD was defined by the presence of symptoms or complications (esophagitis). The clinical criterion defining GERD was the presence of at least two episodes of heartburn per week. Results : The mean age of the 138 patients subjected to complete study was 42.6 ± 10.2 years, with a BMI of 50.1 ± 6.9 kg/m² (range, 40.6 to 69.4 kg/m²); 78% were women. The prevalence of GERD evaluated by symptoms and/or esophagitis was 33.3% (46/138). Clinical criteria of GERD were present in 31/138 cases (22.5%), and 26 (18.8%) had esophagitis. In 69/138 patients (50%), pHmetry was abnormal. Fifty‐three patients with esophagitis and/or abnormal pHmetry were asymptomatic. The sensitivity of heartburn as a diagnostic criterion of GERD in patients with severe obesity was 29.3%, with a specificity of 85.7%. No significant association was observed between severe obesity grade and the prevalence of symptoms and/or objective data. Discussion : Asymptomatic gastroesophageal reflux (abnormal esophageal acid exposure and/or reflux esophagitis) is more common than symptomatic gastroesophageal reflux in severely obese patients. Increased BMI is not associated with a greater prevalence of GERD in these patients.     

Human esophageal microvascular endothelial cells respond to acidic pH stress by PI3K/AKT and p38 MAPK-regulated induction of Hsp70 and Hsp27

The heat shock response maintains cellular homeostasis following sublethal injury. Heat shock proteins (Hsps) are induced by thermal, oxyradical, and inflammatory stress, and they chaperone denatured intracellular proteins. Hsps also chaperone signal transduction proteins, modulating signaling cascades during repeated stress. Gastroesophageal reflux disease (GERD) affects 7% of the US population, and it is linked to prolonged esophageal acid exposure. GERD is characterized by enhanced and selective leukocyte recruitment from esophageal microvasculature, implying activation of microvascular endothelium. We investigated whether phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK regulate Hsp induction in primary cultures of human esophageal microvascular endothelial cells (HEMEC) in response to acid exposure (pH 4.5). Inhibitors of signaling pathways were used to define the contribution of PI3K/Akt and MAPKs in the heat shock response and following acid exposure. Acid significantly enhanced phosphorylation of Akt and MAPKs in HEMEC as well as inducing Hsp27 and Hsp70. The PI3K inhibitor LY-294002, and Akt small interfering RNA inhibited Akt activation and Hsp70 expression in HEMEC. The p38 MAPK inhibitor (SB-203580) and p38 MAPK siRNA blocked Hsp27 and Hsp70 mRNA induction, suggesting a role for MAPKs in the HEMEC heat shock response. Thus acidic pH exposure protects HEMEC through induction of Hsps and activation of MAPK and PI3 kinase pathway. Acidic exposure increased HEMEC expression of VCAM-1 protein, but not ICAM-1, which may contribute to selective leukocyte (i.e., eosinophil) recruitment in esophagitis. Activation of esophageal endothelial cells exposed to acidic refluxate may contribute to GERD in the setting of a disturbed mucosal squamous epithelial barrier (i.e., erosive esophagitis, peptic ulceration). esophagus; esophagitis; gastroesophageal reflux disease; microvasculature; phosphatidylinositol 3-kinase/Akt; VCAM-1     

Impacts of Endoscopic Gastroesophageal Flap Valve Grading on Pediatric Gastroesophageal Reflux Disease

Background

Gastroesophageal flap valve (GEFV) endoscopic grading is reported to be associated with gastroesophageal reflux disease (GERD) in adults; however its role in pediatric groups remains unknown. This study aimed to investigate the significance of GEFV grading and the associations to multichannel intraluminal impedance and pH monitoring (MII-pH) in children with GERD.

Methods

A total of 48 children with GERD symptoms who received esophagogastroduodenoscopy and MII-pH monitoring were enrolled. The degree of GEFV was graded from I to IV according to the Hill classification, and classified into two groups: normal GEFV (Hill grades I and II), and abnormal GEFV (Hill grades III and VI). Endoscopic findings and MII-pH monitoring were analyzed among the groups.

Results

Thirty-six patients had normal GEFV while 12 had abnormal GEFV. The presence of erosive esophagitis was significantly more common in the patients with abnormal GEFV (p = 0.037, OR 9.84, 95% CI 1.15–84.42). Pathological acidic gastroesophageal reflux (GER) determined by MII-pH was more prevalent in the patients with loosened GEFV geometry (p = 0.01, OR 7.0, 95% CI 1.67–27.38). There were significant positive correlations between GEFV Hill grading I to IV and the severity of erosive esophagitis (r = 0.49, p<0.001), percentage of supine acid reflux (r = 0.37, p = 0.009), percentage of total acid reflux (r = 0.3284, p = 0.023), and DeMeester score (r = 0.36, p = 0.01) detected by pH monitoring. In the impedance study, GEFV Hill grading also positively correlated to median number of acid reflux events (r = 0.3015, p = 0.037).

Conclusions

GEFV dysfunction highly associated with acid GER and severe erosive esophagitis. An abnormal GEFV is a sign of acid GER in children.     

Does Helicobacter pylori infection contribute to gastroesophageal reflux disease?

Helicobacter pylori organisms that infect the stomach conceivably could contribute to esophageal inflammation in patients with gastroesophageal reflux disease (GERD) through any of at least three potential mechanisms: 1) by causing an increase in gastric acid secretion; 2) by spreading to infect the gastric-type columnar epithelium that occasionally can line the distal esophagus; and/or 3) by secreting noxious bacterial products into the gastric juice. Studies regarding these potential mechanisms are discussed in this report. Most investigations have found no apparent association between H. pylori infection and reflux esophagitis. Presently, infection with H. pylori does not appear to play an important role in the pathogenesis of GERD.     

Histomorphological and Immunophenotypic Features of Pill-Induced Esophagitis

The aim of this study was to investigate histomorphological and immunophenotypic features in pill-induced esophagitis. We comparatively evaluated the histomorphological, immunophenotypic features of pill-induced esophagitis vs. reflux esophagitis, as well as clinical information and endoscopic findings. Fifty-two tissue pieces from 22 cases of pill-induced esophagitis, 46 pieces from 20 reflux esophagitis, and 16 pieces from 14 control samples were subjected to immunohistochemistry for inflammatory infiltrates (CD3 for T lymphocyte, CD20 for B lymphocyte, CD56 for NK cell, CD68 for macrophage, CD117 for mast cell) and eosinophil chemotaxis-associated proteins (Erk, leptin, leptin receptor, pSTAT3, phospho-mTOR). As a result, Histomorphology showed that a diffuse pattern of dilated intercellular spaces was more frequently observed in pill-induced esophagitis, while reactive atypia and subepithelial papillary elongation were more often found in reflux esophagitis (P < 0.05, respectively). Interestingly, intraepithelial eosinophilic microabscess, intraepithelial pustule and diffuse pattern of dilated intercellular spaces were observed in 14% (3 cases), 9% (2 cases) and 32% (7 cases) of pill-induced esophagitis, respectively, but in no cases of reflux esophagitis. Regarding intraepithelial inflammatory infiltrates in pill-induced esophagitis, T lymphocytes were the most common cells, followed by eosinophil; 11 and 7 in one x400 power field, respectively. Intraepithelial pSTAT3-positive pattern was more frequently observed in pill-induced esophagitis than in reflux esophagitis, at 45% (10 cases) versus 10% (2 cases), respectively (P < 0.05). Considering the distal esophageal lesion only, intraepithelial pustule, diffuse dilated intercellular spaces and stromal macrophages were more frequently found in distal pill-induced esophagitis, whereas reactive atypia and intraepithelial mast cells in reflux esophagitis (P < 0.05, respectively). In conclusion, diffuse dilated intercellular spaces, intraepithelial eosinophil microabscess, pustule, T lymphocytes, eosinophils, and pSTAT3 positivity can be added to histopathological features of pill-induced esophagitis, other than non-specific ulcer. Besides, distal pill-induced esophagitis may be histopathologically differentiated from reflux esophagitis.     

Drugs, bugs, and esophageal pH profiles

Until relatively recently, gastroesophageal reflux disease (GERD) was thought to be a relatively trivial problem, and pharmaceutical companies initially had remarkably little interest in clinical trials for GERD. Over the last ten years, GERD therapy has become the subject of intense interest, since reflux disease is now recognized as a major market for antisecretory and prokinetic drugs. Even low-technology antacids are now known to effectively neutralize esophageal acid prevent acid reflux for up to 90 minutes. Esophageal pH profiling is known to be an excellent surrogate for clinical efficacy of GERD drugs, particularly in erosive esophagitis. Years ago, famotidine normalized esophageal mucosal exposure to pH < 4.0 only when administered in doses of 40 mg twice a day. Subsequent studies confirmed that multiple daily dosing of histamine-2 receptor antagonists (H2RAs) was mandatory for GERD treatment, with clear dose-response relationships for each agent. Proton pump inhibitors (PPIs) have each been carefully assessed in terms esophageal and gastric pH profiles. Omeprazole has a particularly flat dose response curve, making it difficult to differentiate pH or clinical effects of 20 vs. 40 mg doses. Improved rapidity of onset and/or enhanced potency is demonstrable in pH data obtained with lansoprazole, rabeprazole and pantoprazole. Such differences will translate to improved clinical efficacy, based on the meta-analyses of Richard Hunt and his group in Canada that correlate pH effects and symptom relief/healing. PPI's have dependably surpassed H2RAs and prokinetic drugs in management of the more severe grades of esophagitis. Helicobacter pylori has a peculiar relationship to GERD. There has been some concern that PPIs given to patients with H. pylori might accelerate development of severe atrophic gastritis. It is also now known that eradication of H. pylori may increase symptomatic GERD (possibly as a result of increased gastric acid secretion once the bacteria have been eliminated). New data confirm nocturnal breakthrough of acid secretion and esophageal acid exposure in three-fourths of patients on omeprazole 20 mg twice daily. This nocturnal acidity can be controlled more effectively with a nighttime dose of an H2RA than with a third dose of omeprazole. Control of acid secretion and improved gastric and esophageal pH profiles are goals of modern GERD therapy, and the product that most cost effectively normalizes esophageal acid exposure will have a substantial advantage in the ever-growing GERD marketplace.     

Gastroesophageal Reflux Disease and Atrial Fibrillation: A Nationwide Population-Based Study

Objectives

Precise mechanisms of atrial fibrillation (AF) are uncertain, but their association with esophageal disorders has been recently proposed. The association between gastroesophageal reflux disease (GERD), the most common gastroesophageal disorder, and AF remains undetermined. We therefore aimed to investigate the association between GERD and later development of AF.

Methods and Results

Patients with GERD were identified from the 1,000,000-person cohort dataset sampled from the Taiwan National Health Insurance database. The study cohort comprised 29,688 newly diagnosed adult GERD patients; 29,597 randomly selected age-, gender-, comobidity-matched subjects comprised the comparison cohort. Cox proportional hazard regressions were performed as a means of comparing the AF-free survival rate for the two cohorts. During a maximum three years of follow-up, a total of 351 patients experienced AF, including 184 (0.62%) patients in the GERD cohort and 167 (0.56%) in the control group. The log-rank test showed that patients with GERD had significantly higher incidence of AF than those without GERD (p = 0.024). After Cox proportional hazard regression model analysis, GERD was independently associated with the increased risk of AF (hazard ratio, 1.31; 95% confidence interval, 1.06–1.61, p = 0.013).

Conclusion

GERD was independently associated with an increased risk of future AF in a nationwide population-based cohort.     

Specific induction of a 72-kDa heat shock protein protects esophageal mucosa from reflux esophagitis

AimsThe aim of this study is to investigate the expression and cytoprotective function of a 72-kDa heat shock protein (HSP72) using a reflux esophagitis model in rats.Main methodsExpression of HSP60, HSP72, and HSP90 in rat esophageal mucosa was evaluated by Western blot analysis before and after hyperthermia (42.5 °C, 20 min). Rats received the operation to produce reflux esophagitis with or without pretreatment with hyperthermia to induce HSPs. The esophageal mucosal damage was evaluated 12 h after the operation.Key findingsExpression of HSP72 was significantly increased by hyperthermia in rat esophageal mucosa. Reflux esophagitis was dramatically prevented when HSP72 was preinduced by hyperthermia. Furthermore, activation of TNF-α and IL-1β in esophageal mucosa was also suppressed.SignificanceThese results suggested that hyperthermia protects the esophageal mucosa in reflux esophagitis model by inducing HSP72 and suppressing proinflammatory cytokine activation. These findings might suggest that HSP-inducing therapy could be a novel and unique therapy for reflux esophagitis.     

Regulatory volume decrease in human esophageal epithelial cells

In vivo human esophageal epithelial cells are regularly exposed to hyposmolal stress. This stress, however, only becomes destructive when the surface epithelial cell (barrier) layers are breached and there is contact of the hyposmolal solution with the basolateral cell membranes. The present investigation was designed to examine the effects of hyposmolal stress in the latter circumstance using as a model for human esophageal epithelial cells the noncancer-derived HET-1A cell line. Cell volume and the response to hyposmolal stress in suspensions of HET-1A cells were determined by cell passage through a Coulter Counter Multisizer II. HET-1A cells behaved as osmometers over the range of 280 to 118 mosmol/kg H(2)O with rapid increases in cell volume < or = 15-20% above baseline. Following swelling, the cells exhibited regulatory volume decrease (RVD), restoring baseline volume within 30 min, despite continued hyposmolal stress. With the use of pharmacologic agents and ion substitutions, RVD appeared to result from rapid activation of parallel K(+) and Cl(-) conductance pathways and this was subsequently joined by activation of a KCl cotransporter. Exposure to hyposmolal stress in an acidic environment, pH 6.6, inhibited, but did not abolish, RVD. These data indicate that human esophageal epithelial cells can protect against hyposmolal stress by RVD and that the redundancy in mechanisms may, to some extent, serve as added protection in patients with reflux disease when hyposmolal stress may occur in an acidic environment.     

The TRPV4 channel is a novel regulator of intracellular Ca2+ in human esophageal epithelial cells

The esophageal epithelium has sensory properties that enable it to sustain normal barrier function. Transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+)-permeable channel that is activated by extracellular hypotonicity, polyunsaturated fatty acids, phorbol esters, and elevated temperature. We found that TRPV4 is expressed in both human esophageal tissue and in HET-1A cells, a human esophageal epithelial cell line. Specific activation of TRPV4 by the phorbol ester 4α-phorbol 12,13-didecanoate (4α-PDD) increased intracellular Ca(2+) in a subset of HET-1A cells. Elevated temperature strongly potentiated this effect at low concentrations of 4α-PDD, and all of the responses were inhibited by the TRPV antagonist ruthenium red. TRPV4 activation differentially affected cell proliferation and cell viability; HET-1A cell proliferation was increased by 1 μM 4α-PDD, whereas higher concentrations (10 μM and 30 μM) significantly decreased cell viability. Transient TRPV4 activation triggered ATP release in a concentration-dependent manner via gap-junction hemichannels, including pannexin 1 and connexin 43. Furthermore, TRPV4 activation for 24 h did not increase the production of interleukin 8 (IL-8) but reduced IL-1β-induced IL-8 production. Small-interference RNA targeted to TRPV4 significantly attenuated all of the 4α-PDD-induced responses in HET-1A cells. Collectively, these findings suggest that TRPV4 is a novel regulator of Ca(2+)-dependent signaling pathways linked to cell proliferation, cell survival, ATP release, and IL-8 production in human esophageal epithelial cells.     

Effect of Silodosin,an Alpha1A-Adrenoceptor Antagonist,on Ventral Prostatic Hyperplasia in the Spontaneously Hypertensive Rat

Background

A decreased prostatic blood flow could be one of the risk factors for benign prostatic hyperplasia/benign prostatic enlargement. The spontaneously hypertensive rat (SHR) shows a chronic prostatic ischemia and hyperplastic morphological abnormalities in the ventral prostate. The effect of silodosin, a selective alpha_1A-adrenoceptor antagonist, was investigated in the SHR prostate as a prostatic hyperplasia model focusing on prostatic blood flow.

Methods

Twelve-week-old male SHRs were administered perorally with silodosin (100 μg/kg/day) or vehicle once daily for 6 weeks. Wistar Kyoto (WKY) rats were used as normotensive controls and were treated with the vehicle. The effect of silodosin on blood pressure and prostatic blood flow were estimated and then the prostates were removed and weighed. The tissue levels of malondialdehyde (MDA), interleukin-6 (IL-6), chemokine (C-X-C motif) ligand 1/cytokine-induced neutrophil chemoattractant 1 (CXCL1/CINC1), tumor necrosis factor-alpha (TNF-α), transforming growth factor beta 1 (TGF-β1), basic fibroblast growth factor (bFGF) and alpha-smooth muscle actin (α-SMA) were measured. The histological evaluation was also performed by hematoxylin and eosin staining.

Results

There was a significant increase in blood pressure, prostate weight, prostate body weight ratio (PBR), tissue levels of MDA, IL-6, CXCL1/CINC1, TNF-α, TGF-β1, bFGF and α-SMA in the SHR compared to the WKY rat. The ventral prostate in the SHR showed the morphological abnormalities compared to the WKY rat. Prostatic blood flow was decreased in the SHR. However, treatment with silodosin significantly restored the decreased prostatic blood flow in the SHR. Moreover, silodosin normalized tissue levels of MDA, IL-6, CXCL1/CINC1, TNF-α, TGF-β1, bFGF and α-SMA, and it ameliorated ventral prostatic hyperplasia in the SHR excluding blood pressure. Silodosin decreased PBR but not prostate weight in the SHR.

Conclusions

Silodosin can inhibit the progression of prostatic hyperplasia through a recovery of prostatic blood flow.     

Generation and Characterization of an Immortalized Human Esophageal Myofibroblast Line

Stromal cells with a myofibroblast phenotype present in the normal human esophagus are increased in individuals with gastro-esophageal reflux disease (GERD). We have previously demonstrated that myofibroblasts stimulated with acid and TLR4 agonists increase IL-6 and IL-8 secretion using primary cultures of myofibroblasts established from normal human esophagus. While primary cultures have the advantage of reflecting the in vivo environment, a short life span and unavoidable heterogeneity limits the usefulness of this model in larger scale in vitro cellular signaling studies. The major aim of this paper therefore was to generate a human esophageal myofibroblast line with an extended lifespan. In the work presented here we have generated and characterized an immortalized human esophageal myofibroblast line by transfection with a commercially available GFP-hTERT lentivirus. Immortalized human esophageal myofibroblasts demonstrate phenotypic, genotypic and functional similarity to primary cultures of esophageal myofibroblasts we have previously described. We found that immortalized esophageal myofibroblasts retain myofibroblast spindle-shaped morphology at low and high confluence beyond passage 80, and express α-SMA, vimentin, and CD90 myofibroblast markers. Immortalized human esophageal myofibroblasts also express the putative acid receptor TRPV1 and TLR4 and retain the functional capacity to respond to stimuli encountered in GERD with secretion of IL-6. Finally, immortalized human esophageal myofibroblasts also support the stratified growth of squamous esophageal epithelial cells in 3D organotypic cultures. This newly characterized immortalized human esophageal myofibroblast cell line can be used in future cellular signaling and co-culture studies.