Elena Piazuelo

Effect of Growth Factors and Prostaglandin E2 on Restitution and Proliferation of Rabbit Esophageal Epithelial Cells

Factors and mechanisms involved in esophagealmucosal injury and repair are not well known. The aim ofthis study was to assess the effects of growth factorsand prostaglandins on esophageal mucosal cell repair activities. Rabbit esophageal cells wereisolated, cultured, and exposed to different growthfactors and prostaglandin E2. Subconfluentcell cultures were used to study proliferative responsesdetermined by [3H]thymidine incorporation intoDNA. Restitution was studied in confluent monolayerswounded by mechanical denudation. Restitution was themain mechanism involved in wound repair within the first24 hr. HGF, IGF-I, and EGF dose-dependentlystimulated cell proliferation but did not affectrestitution. TGF-β1 inhibited bothproliferation and restitution while PDGF-BB andprostaglandin E2 had no effect. Esophageal epithelial cell restitution andproliferation are affected by growth factors. HGF,IGF-I, EGF (stimulation), and TGF-β1(inhibition) are major growth factors affecting in vitroesophageal wound repair activities, which, unlike those ofother areas of the digestive tract, are not affected byprostaglandins.

Superoxide Anion and Nitric Oxide in High-Grade Esophagitis Induced by Acid and Pepsin in Rabbits

It has been proposed that free radicals are involved in the pathogenesis of esophageal mucosal damage induced by acid and pepsin. Recent data have suggested that nitric oxide (NO) is involved in the mucosal defense of the esophagus and that superoxide anion plays a minor role in low-grade esophagitis. To study the role and potential interaction of NO and superoxide anion in an experimental model of high-grade esophagitis, acidified pepsin was perfused (45 min/12 hr) for five days in rabbits with different agents to modulate the generation of these radicals. Measurements included both macroscopic and microscopic mucosal damage, superoxide anion generation, NO synthase mucosal activity, and peroxynitrite formation. High-grade esophagitis was associated with mucosal superoxide anion generation. Treatment with exogenous superoxide dismutase completely prevented mucosal damage. The perfusion of acidified pepsin in the lumen of the esophagus was initially associated with increased NO synthase mucosal activity but decreased with the progression of damage. Generation of peroxynitrites was present in those cases with severe damage. Treatment with NO-modifying agents did not induce consistent modification of mucosal damage. It is concluded that superoxide anion is involved in the induction of high-grade esophagitis and that it interacts with nitric oxide to generate peroxynitrite radicals in this model. Superoxide dismutase but not NO-donor-modifying agents might have a therapeutic role in preventing severe esophageal mucosal damage induced by acid and pepsin.

Platelet-Derived Growth Factor and Epidermal Growth Factor Play a Major Role in Human Colonic Fibroblast Repair Activities

Background and Aims: Ulceration is a common feature of inflammatory bowel diseases, where subepithelial cell growth is frequently necessary for resolution. In order to further understand the role of colonic fibroblasts in this process, we have used an in vitro model of wound repair to study the response of human colonic fibroblasts to several growth factors expressed in colonic tissues. Methods: Proliferation was determined by [3H]thymidine incorporation into DNA in subconfluent fibroblast cultures. In vitro wound repair was determined in confluent fibroblast monolayers after mechanical denudation. The presence of growth factors secreted by fibroblasts was studied in conditioned medium by heparin affinity chromatography and immunodetection with specific antibodies. Results: Serum and platelet-derived growth factor (PDGF-BB) induced a dramatic increase in both colonic fibroblast proliferation and closure of wounded cell monolayers. Epidermal growth factor (EGF) stimulated both fibroblast activities, but the effect was less potent. However, colonic fibroblasts did not respond to transforming growth factor-β1. Conditioned medium stimulated fibroblast proliferation and wound repair activity, which was reverted by the addition of suramin. Furthermore, a PDGF-like factor was isolated from colonic fibroblast-conditioned medium. Conclusions: EGF and PDGF-BB promote human colonic fibroblast-dependent wound repair activities. Human colonic fibroblasts may exert an autocrine regulation via the production of growth factors.

Effects of extracellular pH on restitution and proliferation of rabbit oesophageal epithelial cells.

: Exposure to luminal acid induces mucosal damage in the oesophagus. The extent and degree of interaction of acid with wound repair mechanisms of the oesophagus have not been explored.

Cyclooxygenase inhibitors decrease the growth and induce regression of human esophageal adenocarcinoma xenografts in nude mice

Cyclooxygenase (COX) inhibition has been shown to prevent the development of esophageal adenocarcinoma (EAC). However, the potential of this approach for treatment of established cancer has been poorly investigated. Our objective was to determine whether non-selective or selective inhibition of the COX pathway affects the growth of esophageal adenocarcinoma xenografts in nude mice. A human esophageal adenocarcinoma xenograft model was established by subcutaneous inoculation of OE33 cells in nude mice. Small tumor slices harvested from four OE33 xenografts were implanted in the flanks of new mice that were randomized to different treatments (6 animals per group): indomethacin (3 mg/kg/day), parecoxib (0.11 and 0.22 mg/kg/day) or a selective prostaglandin E₂ receptor antagonist (AH-23848B, 1 mg/kg/day). For each treatment, a control group of 6 animals (vehicle) carrying xenografts from the same OE33 tumor was included. Tumor growth was measured twice a week. After 8 weeks mice were euthanized. Tumors were assessed by histological analysis, mRNA expression of COX isoenzymes, PGE₂ receptors and PGE₂ content. All OE33 tumors were poorly differentiated esophageal adenocarcinomas. Tumors expressed COX-2, EP₁, EP₂ and EP₄ receptor mRNA. Treatment with parecoxib, higher dose or indomethacin significantly inhibited tumor growth. Furthermore, indomethacin induced tumor regression (74 vs 582% in control animals; p<0.01). However, AH-23848B or parecoxib low dose failed to affect tumor growth significantly. PGE₂ content in tumors was significantly decreased by high-dose parecoxib and indomethacin. Indomethacin and parecoxib inhibit the growth of human esophageal adenocarcinoma xenografts in nude mice, which suggests a potential role for NSAIDs or selective COX-2 inhibitors for EAC chemotherapy.

In vitro wound repair by human gastric fibroblasts: Implications for ulcer healing

Fibroblasts modulate epithelial biologicalactivities and play a key role in the ulcer healingprocess. There is no information regarding thebiological response of human gastric fibroblasts toregulatory compounds. The aim of this study was to assessthe effects of growth factors and prostaglandins on anin vitro model of human gastric fibroblast wound repair.Subconfluent fibroblast cultures were used to study proliferative responses, determined by[3H]thymidine incorporation into DNA. Invitro wound repair was determined in confluentfibroblast monolayers after mechanical denudation. Thepresence of putative growth factors secreted by fibroblastswas studied in conditioned medium by heparin-affinitychromatography and immunodetection with specificantibodies. Serum and platelet-derived growth factor (PDGF)-BB induced a dramatic increase in bothgastric fibroblast proliferation and closure of woundedcell monolayers, whereas these activities were inhibitedby both transforming growth factor(TGF)-β1 and prostaglandin E1. Basalactivities in unstimulated gastric fibroblasts werelower than those obtained in skin fibroblasts.Conditioned medium stimulated fibroblast proliferationand wound repair activity, which was inhibited by the addition of suramin,and was partially dependent on the presence of PDGF-likefactor. PDGF is a major, autocrine promotor of humangastric fibroblast-dependent wound repair activities, which are inhibited by prostaglandins andTGF-β. These findings might be important for futuretherapeutic ulcer healing approaches.

Collagen Secretion by Human Gastric and Skin Fibroblasts: Implications for Ulcer Healing

Fibroblasts (FIB) play an important role in the wound-healing process. It is not known whether human skin and gastric FIB show different responses to regulatory compounds. In this study, we have examined the collagen production by these FIB after different stimuli. In vitro release of collagen into the medium by steady-state confluent human FIB cultures was assessed over a 24-hour period by 3H-proline incorporation into collageneous protein. Serum and epidermal growth factor increased collagen secretion in both types of FIB, but gastric FIB produced less collagen than skin FIB. Prostaglandin E1 inhibited collagen production in both types of FIB, but nonsteroidal anti-inflammatory drugs and interleukin-1β, a cytokine involved in the wound-healing process, had opposite effects on gastric and skin FIB. The effects of lipoxygenase metabolites on collagen secretion was small, but different in both types of FIB. We conclude that, when compared to skin FIB, human gastric FIB produce less collagen and show pronounced different responses to different agents, which might be relevant to explain (in part) their clinical effects on ulcer healing. These data provide new insights into the wound-healing process.

Prostaglandin E2 Is the Major Arachidonic Acid Metabolite Secreted by Esophageal Mucosal Cells in Rabbits

Unlike gastric mucosa, it has been considered that lipoxygenase metabolites protect the esophageal mucosa and that prostaglandins are only secreted in the presence of esophageal inflammation. The aim of this study was to determine the profile of arachidonic acid metabolites and their response to regulatory compounds in rabbit esophageal mucosal cells in culture. Eicosanoids secreted into the medium were extracted and identified by HPLC and RIA. Esophageal mucosal cells in culture metabolized arachidonic acid mainly through the cycloxygenase pathway and PGE2 was the major arachidonic acid metabolite secreted. The addition of IL-1β and A23187 (calcium ionophore) stimulated PGE2 synthesis. In basal conditions neither leukotrienes nor HETEs were detected. However, the addition of the NDGA induced the secretion of lipoxygenase metabolites identified as 12–15 HETEs. In conclusion, rabbit esophageal epithelial cells in culture metabolize arachidonic acid via both cycloxygenase and lipoxygenase pathways. In our system, PGE2 was the main arachidonic acid metabolite.

Characterization of the Prostaglandin E2 Pathway in a Rat Model of Esophageal Adenocarcinoma

UNLABELLED Accumulating evidence indicates that the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway plays a key role in esophageal carcinogenesis. A better understanding of the pathway downstream of COX-2 may reveal novel targets for the prevention of esophageal adenocarcinoma (EAC). The objective of this study was to characterize the profile of genes involved in PGE2 metabolism and signaling in an experimental model of EAC. Esophagojejunostomy with gastric preservation was performed in wistar rats to induce gastroduodenal reflux. Rats were sacrificed 2 or 4 months after surgery. Nine non-operated rats were used to obtain normal (control) esophageal tissues. RESULTS All rats that underwent esophagojejunostomy developed inflammation. In addition, 90% of the animals showed intestinal metaplasia; of those, 40% progressed to AC. This process was accompanied by a significant increase in esophageal PGE2 levels and the induction of both mRNA and protein levels of COX-2, COX-1, prostaglandin E synthase, 15-hydroxyprostaglandin dehydrogenase, and PGE2 receptors EP3, EP4 and especially EP2, which rose to particularly high levels in experimental rats. In addition, exposure to a selective COX-2 inhibitor (SC58125) or an EP1/EP2 antagonist (AH6809), but not an EP4 antagonist (AH23848B), significantly reduced cell proliferation of esophageal explants in 24 hour-organ culture experiments. Our data suggest that, in addition to COX-2, other components of the PGE2 pathway, including COX-1, may play important roles in the development of EAC induced by gastroduodenal reflux in the rat. Although it must be confirmed in vivo, the EP2 receptor may represent a promising selective target in the prevention of Barretts associated AC.

Clinical Effects of NSAIDs and COXIBs in Colon Cancer Prevention

A large body of evidence from epidemiological, experimental and clinical studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors (COXIBs) have a chemopreventive effect on gastrointestinal cancers and, more specifically, in colorectal cancer (CRC). This chapter summarises scientific evidence derived from clinical studies assessing the role of NSAIDs and COXIBs in the prevention of both sporadic and hereditary CRC. Unfortunately, adverse side effects associated to both of them, mainly at gastrointestinal and cardiovascular level, make difficult their use for primary chemoprevention. Among NSAIDs, almost the unique agent with potential use as chemopreventive agent is aspirin at low dose since it has both no cardiovascular and low gastrointestinal risk. Therefore, currently, the use of nonaspirin NSAIDs (NA-NSAIDs) or COXIBs is restricted to hereditary colorectal cancer patients, to delay the progression of polyposis. In addition, the potential use of these drugs in established colorectal cancer is being tested in ongoing clinical trials. In the last years, different chemical modifications have been introduced in some conventional NSAIDs in an attempt to improve their potency and safety. Although some of these compounds have been evaluated in experimental studies with promising results, further studies are warranted before they can be prescribed in clinical setting to prevent CRC.