Melatonin ameliorates aging-related impaired angiogenesis in gastric endothelial cells via local actions on mitochondria and VEGF-survivin signaling.

Tissue injury healing is impaired in aging, and this impairment is caused in part by reduced angiogenesis. Melatonin, a neuroendocrine hormone that regulates sleep and circadian rhythm, is also produced in the gastrointestinal tract. The expression of melatonin receptors MT1 and MT2 in gastric endothelial cells and their roles in aging-related impairment of gastric angiogenesis have not been examined. We hypothesized that MT1 and MT2 expression is reduced in gastric endothelial cells of aging rats and that melatonin treatment can upregulate their expression and improve angiogenesis. We examined the expression of MT1 and MT2 in gastric endothelial cells (GECs) isolated from young and aging rats. We also examined the effects of melatonin treatment on angiogenesis, GEC mitochondrial function, expression of vascular endothelial growth factor (VEGF), its signaling receptor (VEGFR-2), and the inhibitor of apoptosis protein, survivin. Young and aging GECs expressed MT1 (in the cytoplasm and mitochondria) and MT2 (in nucleus and mitochondria). In aging GECs, MT1 and MT2 levels, in vitro angiogenesis, and mitochondrial membrane potential were significantly reduced (by 1.5-fold, 1.9-fold, 3.1-fold, and 1.63-fold, respectively) compared with young GECs. Melatonin treatment of aging GECs significantly increased MT1 and MT2 expression compared with the controls, induced nuclear translocation of MT1, and significantly ameliorated the aging-related impairment of angiogenesis and mitochondrial function. Aging GECs have significantly reduced MT1 and MT2 expression, angiogenesis, and mitochondrial membrane potential compared with young GECs. Treatment of aging GECs with melatonin increases expression of VEGF receptor and survivin and ameliorates aging-related impaired angiogenesis and mitochondrial function.NEW & NOTEWORTHY This study showed reduced expression of melatonin receptors MT1 and MT2, angiogenesis, and mitochondrial function in gastric endothelial cells (GECs) isolated from aging rats. Treatment of aging GECs with melatonin increases expression of VEGF receptor and survivin and ameliorates aging-related impaired angiogenesis and mitochondrial function. These studies provide new insight into the mechanisms of the aging-related impairment of angiogenesis and delayed tissue injury healing and provide a rationale for melatonin treatment to reverse these abnormalities.

Mitochondria in gastric epithelial cells are the key targets for NSAIDs-induced injury and NGF cytoprotection.

Gastric epithelial cells are important components of mucosal protection and targets of nonsteroidal anti-inflammatory drugs (NSAIDs)-induced injury. Diclofenac (DFN) is one of the most widely used NSAIDs; however, even its short-term use can induce gastric erosions and ulcers. Nerve growth factor (NGF) has been reported to act not only on neuronal cells but also on endothelial cells; however, its action on gastric epithelial cells is unknown. This study was aimed to determine, whether NGF can protect gastric epithelial cells against DFN-induced injury, and to determine the underlying molecular mechanisms with a focus on mitochondria, survivin, and insulin-like growth factor 1 (IGF-1). Cultured normal rat gastric mucosal epithelial cells 1 (RGM1) were treated with phosphate-buffered saline (PBS; control), NGF (100 ng/mL) and/or DFN (0.25-1.00 mM) for 4 hours. We examined: (1) cell injury by confocal microscopy; (2) cell death/survival using Calcein AM live cell tracking dye; (3) mitochondrial structure and membrane potential function using MitoTracker in live cells; and (4) expression of NGF, its receptor - tropomyosin receptor kinase A (TrkA), survivin and IGF-1 by immunostaining. DFN treatment of RGM1 cells for 4 hours caused extensive cell injury, mitochondrial disintegration, reduced cell viability (from 94 ± 3% in controls to 14 ± 4% in 0.5 mM DFN-treated cells; P < 0.001), and expression of survivin and IGF-1. NGF treatment significantly increased survivin and IGF-1 expression by 41% and 75%, respectively versus PBS controls. Pretreatment with NGF before DFN treatment reduced mitochondrial damage and cell death by 73% and 82%, respectively versus treatment with DFN alone (all P < 0.001). This study also showed the presence of high-affinity TrkA receptors in the plasma membrane and mitochondria of RGM1 cells indicating novel actions of NGF.

NSAID-induced injury of gastric epithelial cells is reversible: roles of mitochondria, AMP kinase, NGF, and PGE2.

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DFN) and indomethacin (INDO) are extensively used worldwide. Their main side effects are injury of the gastrointestinal tract, including erosions, ulcers, and bleeding. Since gastric epithelial cells (GEPCs) are crucial for mucosal defense and are the major target of injury, we examined the extent to which DFN- and INDO-induced GEPC injury can be reversed by nerve growth factor (NGF), 16,16 dimethyl prostaglandin E2 (dmPGE2), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), the pharmacological activator of the metabolic sensor AMP kinase (AMPK). Cultured normal rat gastric mucosal epithelial (RGM1) cells were treated with PBS (control), NGF, dmPGE2, AICAR, and/or NSAID (DFN or INDO) for 1-4 h. We examined cell injury by confocal microscopy, cell death/survival using calcein AM, mitochondrial membrane potential using MitoTracker, and phosphorylation of AMPK by Western blotting. DFN and INDO treatment of RGM1 cells for 2 h decreased mitochondrial membrane potential and cell viability. NGF posttreatment (initiated 1 or 2 h after DFN or INDO) reversed the dissipation of mitochondrial membrane potential and cell injury caused by DFN and INDO and increased cell viability versus cells treated for 4 h with NSAID alone. Pretreatment with dmPGE2 and AICAR significantly protected these cells from DFN- and INDO-induced injury, whereas dmPGE2 and AICAR posttreatment (initiated 1 h after NSAID treatment) reversed cell injury and significantly increased cell viability and rescued the cells from NSAID-induced mitochondrial membrane potential reduction. DFN and INDO induce extensive mitochondrial injury and GEPC death, which can be significantly reversed by NGF, dmPGE2, and AICAR.NEW & NOTEWORTHY This study demonstrated that mitochondria are key targets of diclofenac- and indomethacin-induced injury of gastric epithelial cells and that diclofenac and indomethacin injury can be prevented and, importantly, also reversed by treatment with nerve growth factor, 16,16 dimethyl prostaglandin E2, and 5-aminoimidazole-4-carboxamide ribonucleotide.

Nerve growth factor is critical requirement for in vitro angiogenesis in gastric endothelial cells.

Angiogenesis is critical for the healing of gastric mucosal injury and is considered to be primarily regulated by vascular endothelial growth factor (VEGF), the fundamental proangiogenic factor. The role of nerve growth factor (NGF) in gastric angiogenesis is unknown. We examined the expression of NGF and its TrkA receptor in endothelial cells (ECs) isolated from gastric mucosa of rats (GMECs), the effect of NGF treatment on in vitro angiogenesis in GMECs, and, the mechanisms underlying NGF's proangiogenic actions. Isolated GMECs from Fisher rats were treated with vehicle, NGF (10-1,000 ng/ml), VEGF (20 ng/ml), or NGF+VEGF. To determine whether and to what extent NGF is critical for angiogenesis in GMECs, we silenced NGF expression using specific siRNA and examined in vitro angiogenesis with and without treatment with exogenous NGF and/or VEGF. Treatment of GMECs with NGF significantly increased in vitro angiogenesis similar to that seen in GMECs treated with VEGF. Silencing of NGF in GMECs abolished angiogenesis, and this effect was reversed only by exogenous NGF but not VEGF, which indicates a direct proangiogenic action of NGF on GMECs that is, at least in part, distinct and independent of VEGF. NGF's proangiogenic action on GMECs was mediated via PI3-K/Akt signaling. This study showed for the first time that gastric mucosal ECs express NGF and its receptor TrkA and that NGF is critical for angiogenesis in these cells.

In vivo imaging of porcine gastric enteric nervous system using confocal laser endomicroscopy &molecular neuronal probe.

BACKGROUND AND AIM: The gastric enteric nervous system (GENS) is organized into the submucosal plexus and the myenteric plexus that regulate muscle activity and mucosal functions, respectively. A non-invasive, in vivo visualization of GENS was not possible until recent introduction of needle-based confocal laser endomicroscopy (nCLE). Our aim was to determine the feasibility of in vivo visualization of GENS in the porcine stomach using endoscopic ultrasound (EUS) guided nCLE and local injection of molecular neuronal probe NeuroTrace. METHODS: In anesthetized pigs during endoscopy, NeuroTrace was injected into the submucosa and muscularis propria of distal, and proximal stomach under EUS guidance and nCLE imaging was performed using the Cellvizio AQ Flex probe. After euthanasia, transmural gastric specimens from the areas of NeuroTrace injection were obtained for histology. We performed quantitative analysis of nCLE images recorded during in vivo studies: histologic evaluation of unstained specimens under fluorescence microscope for NeuroTrace localization. We also performed immunostaining of these specimens for nerve growth factor (NGF). In in vitro studies, we examined the uptake of NeuroTrace by glial cells. RESULTS: The nCLE imaging successfully visualized neuronal cells and nerve fibers in distinctive image patterns. Fluorescence microscopy of mucosal sections showed that in vivo-injected NeuroTrace was retained in GENS components. NGF was strongly expressed in neural and glial cells, and the pattern of NGF staining was similar to that of NeuroTrace staining. CONCLUSIONS: This study demonstrates for the first time that combined use of EUS-guided nCLE and NeuroTrace is capable to visualize GENS.

EUS-guided in vivo imaging of the porcine esophageal enteric nervous system by using needle-based confocal laser endomicroscopy.

BACKGROUND AND AIMS: The GI tract is innervated by the autonomic enteric nervous system, mainly composed of submucosal Meissner's plexus and myenteric Auerbach's plexus, which is essential for motility, blood flow regulation, and secretory functions. In vivo visualization of the esophageal enteric nervous system (EENS) during endoscopy has not been possible without invasive mucosal resection. This study aimed to visualize the EENS without mucosal resection, in vivo by using the novel probe, needle-based confocal laser-induced endomicroscopy (nCLE) with a fluorescence neuronal probe, NeuroTrace, under EUS guidance and to evaluate the feasibility of ex vivo imaging of the neuronal network in submucosal biopsy samples acquired at endoscopy. METHODS: Four Yorkshire pigs were anesthetized and examined. In vivo experiment: During endoscopy, NeuroTrace was injected into the submucosa and muscularis propria of the middle and distal esophagus under EUS guidance, and nCLE imaging was performed. Ex vivo experiment: Submucosal tissue biopsy specimens from the porcine esophagus were obtained for ex vivo evaluation by using a "through-the-needle" forceps technique. After incubation of the samples in NeuroTrace solution, pCLE was used to visualize the EENS elements in the tissue. RESULTS: Imaging of the EENS network by using EUS-guided nCLE was successful, both within the submucosa and the muscularis propria, and clearly visualized neuronal cells, glial cells, nerve bundles, and nerve fibers provided distinctive image patterns with excellent imaging quality. The use of the "through-the-needle" forceps technique achieved ex vivo images similar to those acquired in vivo. CONCLUSIONS: EUS-guided in vivo imaging of the enteric nervous system is feasible without mucosal resection and provides a novel ex vivo imaging alternative for human application. These novel, minimally invasive imaging approaches could be of tremendous diagnostic value to better characterize and explore the EENS of the GI tract.

Confocal laser endomicroscopy: a new gold standard for the assessment of mucosal healing in ulcerative colitis.

BACKGROUND AND AIM: Endoscopic assessment of mucosal healing in ulcerative colitis (UC) is increasingly accepted as a measure of disease activity, therapeutic goal, and the key prognostic indicator. While regular endoscopy evaluates appearance of the mucosal surface, confocal laser endomicroscopy (CLE) enables in vivo visualization of subepithelial mucosa at 1000× magnification during ongoing endoscopy. Our aims were to determine using CLE whether endoscopically normal appearing colonic mucosa in patients with UC in remission (UC-IR) has fully regenerated mucosal structures, resolved inflammation, and to identify the mechanisms. METHODS: Twelve patients (six controls and six with UC-IR) underwent colonoscopy using CLE and intravenous fluorescein infusion. During colonoscopy, CLE images of colonic mucosa and conventional mucosal biopsies were obtained and evaluated using image-analysis systems. We quantified; (i) regeneration of colonic crypts and blood microvessels; (ii) cyclooxygenase 2 (COX2) expression; (iii) mitochondrial DNA (mtDNA) mutations; (iv) inflammatory infiltration; and (v) vascular permeability (VP). RESULTS: In control subjects, CLE demonstrated normal colonic crypts and microvasculature. COX2 expression was minimal, and < 7% crypts showed mtDNA mutations. Colonic mucosa of UC-IR patients had impaired and distorted crypt regeneration, increased COX2, 69% crypts with mtDNA mutations, persistent inflammation, and abnormal vascular architecture with increased VP (all P < 0.001 vs normal mucosa). CONCLUSIONS: (i) Endoscopically normal appearing colonic mucosa of patients with UC-IR remains abnormal: CLE demonstrates impaired crypt regeneration, persistent inflammation, distinct abnormalities in angioarchitecture and increased vascular permeability; molecular imaging showed increased COX2 and mtDNA mutations; (ii) CLE may serve as a new gold standard for the assessment of mucosal healing in UC.

Key role of endothelial importin-α in VEGF expression and gastric angiogenesis: novel insight into aging gastropathy.

Recent in vivo studies demonstrated that aging gastric mucosa has impaired angiogenesis and reduced expression of vascular endothelial growth factor (VEGF). Angiogenesis is triggered by hypoxia and VEGF gene activation, and the latter requires transport of transcription factor(s) into endothelial cell nuclei. We focused on gastric mucosal endothelial cells (GMEC), which are key targets and effectors of gastric angiogenesis, and determined whether and to what extent importin-α, a nuclear transport protein, regulates VEGF gene activation and gastric angiogenesis and the possible role of importin-α in aging gastropathy. GMEC were isolated from rats 3 and 24 mo of age, young (YGEC) and aging (AGEC), respectively. We examined in these cells 1) in vitro angiogenesis, 2) expression of VEGF and importin-α, 3) nuclear transport of hypoxia-inducible factor (HIF)-1α by importin-α, 4) binding of HIF-1α to the VEGF gene promoter, and 5) effects of importin-α silencing in YGEC and its upregulation in AGEC on angiogenesis and VEGF expression. AGEC exhibited significantly impaired in vitro angiogenesis by fourfold and decreased expression of VEGF, importin-α, and nuclear HIF-1α by 1.4-fold, 1.6-fold, and 2.9-fold, respectively, vs. YGEC. Upregulation of importin-α in AGEC significantly reversed all these abnormalities. In YGEC, knockdown of importins-α1 and -α3 significantly reduced in vitro angiogenesis by 93% and 73% and VEGF expression by 48% and 52%, respectively. The above findings demonstrate that importin-α is a novel and critical regulator of gastric angiogenesis. Its reduced expression in AGEC is the key mechanism for impaired angiogenesis and reduced VEGF.

Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB.

Clinical studies indicate that prostaglandins of E class (PGEs) may promote healing of tissue injury e.g., gastroduodenal and dermal ulcers. However, the precise roles of PGEs, their E-prostanoid (EP) receptors, signaling pathways including cAMP and cAMP response element-binding protein (CREB), and their relation to VEGF and angiogenesis in the tissue injury healing process remain unknown, forming the rationale for this study. Using an esophageal ulcer model in rats, we demonstrated that esophageal mucosa expresses predominantly EP2 receptors and that esophageal ulceration triggers an increase in expression of the EP2 receptor, activation of CREB (the downstream target of the cAMP signaling), and enhanced VEGF gene expression. Treatment of rats with misoprostol, a PGE1 analog capable of activating EP receptors, enhanced phosphorylation of CREB, stimulated VEGF expression and angiogenesis, and accelerated esophageal ulcer healing. In cultured human esophageal epithelial (HET-1A) cells, misoprostol increased intracellular cAMP levels (by 163-fold), induced phosphorylation of CREB, and stimulated VEGF expression. A cAMP analog (Sp-cAMP) mimicked, whereas an inhibitor of cAMP-dependent protein kinase A (Rp-cAMP) blocked, these effects of misoprostol. These results indicate that the EP2/cAMP/protein kinase A pathway mediates the stimulatory effect of PGEs on angiogenesis essential for tissue injury healing via the induction of CREB activity and VEGF expression.

Angiogenesis in gastric mucosa: an important component of gastric erosion and ulcer healing and its impairment in aging.

Angiogenesis (also referred to as neovascularization-formation of new blood vessels from existing vessels) is a fundamental process essential for healing of tissue injury and ulcers because regeneration of blood microvessels is a critical requirement for oxygen and nutrient delivery to the healing site. This review article updates the current views on angiogenesis in gastric mucosa following injury and during ulcer healing, its sequential events, the underlying mechanisms, and the impairment of angiogenesis in aging gastric mucosa. We focus on the time sequence and ultrastructural features of angiogenesis, hypoxia as a trigger, role of vascular endothelial growth factor signaling (VEGF), serum response factor, Cox2 and prostaglandins, nitric oxide, and importin. Recent reports indicate that gastric mucosa of aging humans and experimental animals exhibits increased susceptibility to injury and delayed healing. Gastric mucosa of aging rats has increased susceptibility to injury by a variety of damaging agents such as ethanol, aspirin, and other non-steroidal anti-inflammatory drugs because of structural and functional abnormalities including: reduced gastric mucosal blood flow, hypoxia, reduced expression of vascular endothelial growth factor and survivin, and increased expression of early growth response protein 1 (egr-1) and phosphatase and tensin homolog (PTEN). Until recently, postnatal neovascularization was assumed to occur solely through angiogenesis sprouting of endothelial cells and formation of new blood vessels from pre-existing blood vessels. New studies in the last decade have challenged this paradigm and indicate that in some tissues, including gastric mucosa, the homing of bone marrow-derived endothelial progenitor cells to the site of injury can also contribute to neovascularization by a process termed vasculogenesis.

Editor-in-Chief articles of choice and comments at the year-end of 2023.

As the Editor-in-Chief of World Journal of Gastroenterology, every week prior to a new issue's online publication, I perform a careful review of all encompassed articles, including the title, clinical and/or research importance, originality, novelty, and ratings by the peer reviewers. Based on this review, I select the papers of choice and suggest pertinent changes (e.g., in the title) to the Company Editors responsible for publication. This process, while time-consuming, is very important for assuring the quality of publications and highlighting important articles that Readers may revisit.

An imbalance between VEGF and endostatin underlies impaired angiogenesis in gastric mucosa of aging rats.

Gastric mucosa of aging individuals exhibits increased susceptibility to injury and delayed healing. Our previous studies in young rats showed that healing of mucosal injury depends on and is critically dependent on VEGF and angiogenesis. Since angiogenesis in aging gastric mucosa has not been examined before, in this study we examined the extent to which angiogenesis is impaired in gastric mucosa of aging vs. young rats and determined the underlying mechanisms with a focus on mucosal expression of VEGF (proangiogenic factor) and endostatin (antiangiogenic factor). Aging rats had significantly impaired gastric angiogenesis by ~12-fold, 5-fold, 4-fold, and 3-fold, respectively (vs. young rats; all P < 0.001) at 24, 48, 72, and 120 h following ethanol-induced gastric injury and reduced and delayed healing of mucosal erosions. In gastric mucosa of aging (vs. young) rats at baseline, VEGF expression was significantly reduced, whereas endostatin levels were significantly increased (P < 0.05 and P < 0.01, respectively). In contrast to young rats, gastric mucosal VEGF levels did not increase following ethanol-induced injury in aging rats. MMP-9 enzyme activity was significantly higher in gastric mucosa of aging vs. young rats both at baseline (2.7-fold) and 24 h (3.8-fold) after ethanol injury (both P < 0.001). Since endostatin is generated from collagen XVIII by MMP-9, this finding can explain the mechanism of increased endostatin expression in aging gastric mucosa. The above findings demonstrate that reduced VEGF and increased endostatin result in the impaired angiogenesis and delayed injury healing in gastric mucosa of aging rats.

Aberrant, ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism.

UNLABELLED: Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy. MATERIAL AND METHODS: We examined and quantified expression of VEGF, VEGF-R1 and VEGF-R2 in three different human colonic tissue arrays containing sections of adenocarcinoma (n=43) and normal mucosa (n = 41). In human colon cancer cell lines HCT116 and HT29 and normal colon cell lines NCM356 and NCM460, we examined expression of VEGF, VEGF-R1 and VEGF-R2 mRNA and protein, VEGF production and secretion into the culture medium; and, the effect of a potent, selective inhibitor of VEGF receptors, AL-993, on cell proliferation. RESULTS: Human colorectal cancer specimens had strong expression of VEGF in cancer cells and also expressed VEGF-R1 and VEGF-R2.In vitro studies showed that human colon cancer cell lines, HCT116 and HT29, but not normal colonic cell lines, express VEGF, VEGF-R1 and VEGF-R2 and secrete VEGF into the medium up to a concentration 2000 pg/ml within 48 h. Furthermore, we showed that inhibition of VEGF receptors using a specific VEGF-R inhibitor significantly reduced proliferation (by >50%) of cultured colon cancer cell lines. CONCLUSIONS: Our findings support the contention that VEGF generated by colon cancer cells stimulates their growth directly through an autocrine mechanism that is independent of its primary function in the induction of angiogenesis.

Endothelial cells and blood vessels are major targets for COVID-19-induced tissue injury and spreading to various organs.

The coronavirus disease 2019 (COVID-19) infected so far over 250 million people and caused the death of over 5 million worldwide. Aging, diabetes, and cardiovascular diseases, conditions with preexisting impaired endothelial functions predispose to COVID-19. While respiratory epithelium is the main route of virus entry, the endothelial cells (ECs) lining pulmonary blood vessels are also an integral part of lung injury in COVID-19 patients. COVID-19 not only affects the lungs and respiratory system but also gastrointestinal (GI) tract, liver, pancreas, kidneys, heart, brain, and skin. Blood vessels are likely conduits for the virus dissemination to these distant organs. Importantly, ECs are also critical for vascular regeneration during injury/lesions healing and restoration of vascular network. The World Journal of Gastroenterology has published in last two years over 67 outstanding papers on COVID-19 infection with a focus on the GI tract, liver, pancreas, etc., however, the role of the endothelial and vascular components as major targets for COVID-19-induced tissue injury, spreading to various organs, and injury healing have not been sufficiently emphasized. In the present article, we focus on these subjects and on current treatments including the most recent oral drugs molnupiravir and paxlovid that show a dramatic, significant efficacy in controlling severe COVID-19 infection.

Gastric mucosal injury activates bFGF gene expression and triggers preferential translation of high molecular weight bFGF isoforms through CUG-initiated, non-canonical codons.

Basic fibroblast growth factor (bFGF or FGF-2) is a pleiotropic growth factor that promotes growth of mesenchymal and epithelial cells, stimulates angiogenesis and neuroprotection. Moreover, exogenous bFGF by stimulating angiogenesis promotes healing of gastroduodenal ulcers and cardiac and brain injury. All these actions were demonstrated in regard to 18kDa bFGF isoform that is secreted by cells via an ER/Golgi-independent pathway and activates FGF receptors. However in some transformed and stressed cells and in some tissues (e.g. brain) the single copy bFGF gene encodes multiple gene products: 18 kDa and also higher molecular weight (HMW) bFGF isoforms: ∼21 and ∼22 kDa in rodents, and ∼22, ∼23 and ∼24 kDa in humans. The biologic roles of these HMW bFGF isoforms in vivo remain unknown. In this study we demonstrated that in normal, uninjured gastric mucosa, bFGF is almost exclusively expressed as 18kDa isoform translated through a classical AUG (methionine) codon. In contrast, in injured gastric mucosa of rat, bFGF gene is preferentially translated to HMW bFGF isoforms through alternative CUG (leucine) initiation codon. Gastric mucosal injury caused in rats a significant increase in bFGF mRNA at 8 and 24h vs. normal mucosa and a significant increase in bFGF protein at 24-72h, mainly due to increased expression of ∼21 and ∼22 kDa HMW bFGF isoforms. This is first demonstration that gastric mucosal injury and repair triggers local activation of bFGF gene with preferential translation of HMW bFGF isoforms through a non-canonical CUG codon. This study uncovered CUG-initiated HMW bFGF translation as a novel regulatory mechanism operating in vivo during gastric injury repair.

Prostaglandin E2 transactivates EGF receptor: a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophy.

Prostaglandins (PGs), bioactive lipid molecules produced by cyclooxygenase enzymes (COX-1 and COX-2), have diverse biological activities, including growth-promoting actions on gastrointestinal mucosa. They are also implicated in the growth of colonic polyps and cancers. However, the precise mechanisms of these trophic actions of PGs remain unclear. As activation of the epidermal growth factor receptor (EGFR) triggers mitogenic signaling in gastrointestinal mucosa, and its expression is also upregulated in colonic cancers and most neoplasms, we investigated whether PGs transactivate EGFR. Here we provide evidence that prostaglandin E2 (PGE2) rapidly phosphorylates EGFR and triggers the extracellular signal-regulated kinase 2 (ERK2)--mitogenic signaling pathway in normal gastric epithelial (RGM1) and colon cancer (Caco-2, LoVo and HT-29) cell lines. Inactivation of EGFR kinase with selective inhibitors significantly reduces PGE2-induced ERK2 activation, c-fos mRNA expression and cell proliferation. Inhibition of matrix metalloproteinases (MMPs), transforming growth factor-alpha (TGF-alpha) or c-Src blocked PGE2-mediated EGFR transactivation and downstream signaling indicating that PGE2-induced EGFR transactivation involves signaling transduced via TGF-alpha, an EGFR ligand, likely released by c-Src-activated MMP(s). Our findings that PGE2 transactivates EGFR reveal a previously unknown mechanism by which PGE2 mediates trophic actions resulting in gastric and intestinal hypertrophy as well as growth of colonic polyps and cancers.

The Critical Role of Growth Factors in Gastric Ulcer Healing: The Cellular and Molecular Mechanisms and Potential Clinical Implications.

In this article we review the cellular and molecular mechanisms of gastric ulcer healing. A gastric ulcer (GU) is a deep defect in the gastric wall penetrating through the entire mucosa and the muscularis mucosae. GU healing is a regeneration process that encompasses cell dedifferentiation, proliferation, migration, re-epithelialization, formation of granulation tissue, angiogenesis, vasculogenesis, interactions between various cells and the matrix, and tissue remodeling, all resulting in scar formation. All these events are controlled by cytokines and growth factors (e.g., EGF, TGFα, IGF-1, HGF, bFGF, TGFß, NGF, VEGF, angiopoietins) and transcription factors activated by tissue injury. These growth factors bind to their receptors and trigger cell proliferation, migration, and survival pathways through Ras, MAPK, PI3K/Akt, PLC-γ, and Rho/Rac/actin signaling. The triggers for the activation of these growth factors are tissue injury and hypoxia. EGF, its receptor, IGF-1, HGF, and COX-2 are important for epithelial cell proliferation, migration, re-epithelialization, and gastric gland reconstruction. VEGF, angiopoietins, bFGF, and NGF are crucial for blood vessel regeneration in GU scars. The serum response factor (SRF) is essential for VEGF-induced angiogenesis, re-epithelialization, and blood vessel and muscle restoration. Local therapy with cDNA of human recombinant VEGF165 in combination with angiopoietin1, or with the NGF protein, dramatically accelerates GU healing and improves the quality of mucosal restoration within ulcer scars. The future directions for accelerating and improving healing include local gene and protein therapies with growth factors, their combinations, and the use of stem cells and tissue engineering.

PTEN silencing reverses aging-related impairment of angiogenesis in microvascular endothelial cells.

Aging is associated with impaired angiogenesis (new blood vessels formation from the endothelial cells of pre-existing vessels) in a variety of tissues. The precise mechanisms of aging-related impairment of angiogenesis are not known. PTEN is a dual-specificity phosphatase that antagonizes in some cells the PI3K/Akt signaling pathway, important for cell survival, function and angiogenesis. PTEN's role in aging-related impairment of angiogenesis is not known. In this study, we investigated whether expression of PTEN in endothelial cells may play a mechanistic role in aging-related impairment of angiogenesis. We demonstrated that human microvascular endothelial cells (HMVEC) derived from aging individuals (Aged-HMVEC) have: (1) significantly increased PTEN mRNA and protein levels and (2) impaired in vitro angiogenesis vs. neonatal derived HMVEC (Neo-HMVEC), and that (3) downregulation of PTEN using specific siRNA restores angiogenesis in Aged-HMVEC to normal. This is the first demonstration of increased PTEN expression in human microvascular endothelial cells derived from aging tissues and that elevated PTEN is a major factor responsible for aging-related impairment of in vitro angiogenesis.

Long-term use of nonsteroidal anti-inflammatory drugs normalizes the kinetics of gastric epithelial cells in patients with Helicobacter pylori infection via attenuation of gastric mucosal inflammation.

BACKGROUND: Helicobacter pylori (H. pylori) is associated with chronic gastritis and gastric carcinogenesis. The effects of nonsteroidal anti-inflammatory drugs (NSAIDs), which exert chemopreventive effects on several cancers, on H. pylori-induced gastritis remain unknown. We investigated the effects of NSAIDs on gastric inflammation and the kinetics of gastric epithelial cells in H. pylori-induced gastritis. METHODS: Patients with rheumatoid arthritis or osteoarthritis who took NSAIDs for more than 1 month and complained of dyspeptic symptoms were recruited for this study. Patients not on any NSAIDs were included as non-NSAID user controls. All patients underwent diagnostic testing for H. pylori infection, esophagogastroduodenoscopy, and gastric biopsies. Neutrophil infiltration into gastric mucosa, expression of inducible nitric oxide synthase (iNOS), and apoptosis and proliferation of gastric epithelial cells were evaluated by immunohistochemistry. In an in vitro study, the effects of NSAIDs on production of interleukin (IL)-8 induced by H. pylori in a gastric epithelial cell line (AGS) were determined. RESULTS: Numbers of neutrophils infiltrating the gastric mucosa, iNOS-expressing inflammatory cells and apoptotic cells, and proliferating cells in gastric epithelium were higher in H. pylori-positive groups than H. pylori-negative groups. Among H. pyloripositive groups, these parameters were lower in NSAID users than in non-NSAID users. NSAIDs inhibited the production of IL-8 induced by H. pylori in AGS cells. CONCLUSIONS: These findings suggest that long-term use of NSAIDs normalizes the kinetics of gastric epithelial cells in patients with H. pylori infection by attenuating gastric mucosal inflammation, which may result in prevention of the gastric carcinogenesis associated with H. pylori infection.