Formation of new blood vessels during gastric ulcer healing. Role of bone marrow derived endothelial progenitor cells.

Regeneration of blood vessels (neovascularization) is critical for gastric ulcer (GU) healing. The contributions of bone marrow-derived endothelial progenitor cells (BMD-EPCs) to neovascularization during GU healing are not fully elucidated. Our specific aims were to determine whether in GU, BMD-EPCs are incorporated into blood vessels of GU granulation tissue jointly with ECs, thus forming hybrid vessels; or, form separate vessels consisting of only BMD-EPCs. GUs were induced in rats by serosal application of acetic acid. Vascular cast studies were performed at 7, 21 and 60 days after GU induction and tissue specimens were immunostained for CD34, CD133, VEGFR2, and SDF-1 at 14 days. Human relevance was determined using archival human GU specimens. In rat GU granulation tissue BMD-EPCs constituted 28 ± 3% of all cells lining newly formed blood vessels, and were nested between fully differentiated ECs. In rat GU granulation tissue, expression of stromal derived factor-1 (SDF-1) - the major chemoattractant for BMD-EPCs was strongly upregulated. In human GU specimens, BMD-EPCs were also present in granulation tissue constituting 34 ± 3% of all cells lining blood vessels and jointly formed hybrid vessels with differentiated ECs. Our study uncovered that BMD-EPCs incorporate into newly formed blood vessels in GU granulation tissue; and, together with ECs of pre-existing vessels, contribute to and support neovascularization through vasculogenesis. This study is the first demonstration that vasculogenesis occurs during GU healing in both humans and in rats.

In vitro model of vasculo-angiogenesis: demonstration that bone marrow derived endothelial progenitor cells form new hybrid capillary blood vessels jointly with gastric endothelial cells.

Regeneration of blood vessels (neovascularization) is critical for tissue injury healing. The contribution of bone marrow-derived endothelial progenitor cells (BMD-EPCs) to neovascularization during tissue injury healing is not fully elucidated and it is not clear whether BMD-EPCs can form new capillary blood vessels independently or jointly with fully differentiated endothelial cells (ECs). The aim of this study was to establish an in vitro model of vasculogenesis/angiogenesis by co-culture of BMD-EPCs and gastric endothelial cells (GECs) on Matrigel, examine direct interactions of these cells; and, identify the mechanisms involved. We isolated BMD-EPCs and GECs from bone marrow and stomach of rats, respectively. In these cells, we examined the expression of CD34, CD133, CD31, VEGF-R2, stromal derived factor 1 (SDF-1) and CXCR4, and, their ability to form capillary-like tubes when cultured separately or when co-cultured (1:5 ratio) on growth factor-reduced Matrigel. Fluorescence-labeled BMD-EPCs seeded alone on Matrigel formed capillary-like tubes reflecting in vitro vasculogenesis, and when co-cultured with GECs on Matrigel, formed 'hybrid' tubes containing BMD-EPCs nested between GECs thus reflecting in vitro angio-vasculogenesis. These 'hybrid' tubes were 1.5-fold wider (P < 0.001) and had more extensive (5.1-fold increase) loops (P < 0.01) at the junctions of BMD-EPCs and GECs versus tubes formed by GECs alone. GECs expressed SDF-1 that likely mediated homing of BMD-EPCs (which expressed the SDF-1 receptor, CXCR4) and their incorporation during neovascularization. BMD-EPCs can independently form capillary-like tubes on Matrigel, and when co-cultured with fully differentiated ECs on Matrigel, form capillary-like 'hybrid' tubes comprised of both cell types. Both BMD-EPCs and GECs express SDF-1 and CXCR4, which indicate direct paracrine interactions between these cells during neovascularization.

Expression of nerve growth factor in rat stomach. Implications for interactions between endothelial, neural and epithelial cells.

This study was aimed to determine the expression and localization of nerve growth factor (NGF) in the gastric mucosa. Transmural gastric specimens were obtained from euthanized rats. STUDIES: 1) expression of NGF and TrkA receptor by Western blotting; 2) histological evaluation of gastric wall architecture; 3) expression of NGF using immunostaining. Immunostaining showed strong and differential expression of NGF in neural elements of gastric myenteric and submucosal plexuses; in epithelial cells: mainly in chief and progenitor cells, in enterochromaffin-like (ECL) cells; and, in endothelial cells (ECs) lining blood vessels. We concluded that NGF expression in neural elements, epithelial cells and endothelial cells of blood vessels indicated a complex local interaction between neural, epithelial and endothelial cells that regulated gastric mucosal homeostasis and, likely, the protection against gastric injury and ulcer healing.

Nerve growth factor injected into the gastric ulcer base incorporates into endothelial, neuronal, glial and epithelial cells: implications for angiogenesis, mucosal regeneration and ulcer healing.

A previous study has demonstrated that locally administered growth factors such as epidermal growth factor, basic fibroblast growth factor and hepatocyte growth factor can accelerate healing of experimental gastric ulcers in rats. That study indicates that locally administered growth factors can exert potent biological effects resulting in enhanced gastric ulcers healing. However, the fate of injected growth factors, their retention and localization to specific cellular compartments have not been examined. In our preliminary study, we demonstrated that local injection of nerve growth factor to the base of experimental gastric ulcers dramatically accelerates ulcer healing, increases angiogenesis - new blood vessel formation, and improves the quality of vascular and epithelial regeneration. Before embarking on larger, definitive and time sequence studies, we wished to determine whether locally injected nerve growth factor is retained in gastric ulcer's tissues and taken up by specific cells during gastric ulcer healing. Gastric ulcers were induced in anesthetized rats by local application of acetic acid using standard methods; and, 60 min later fluorescein isothiocyanate-labeled nerve growth factor was injected locally to the ulcer base. Rats were euthanized 2, 5 and 10 days later. Gastric specimens were obtained and processed for histology. Unstained paraffin sections were examined under a fluorescence microscope, and the incorporation of fluorescein isothiocyanate-labeled nerve growth factor into various gastric tissue cells was determined and quantified. In addition, we performed immunostaining for S100ß protein that is expressed in neural components. Five and ten days after ulcer induction labeled nerve growth factor (injected to the gastric ulcer base) was incorporated into endothelial cells of blood vessels, neuronal, glial and epithelial cells, myofibroblasts and muscle cells. This study demonstrates for the first time that during gastric ulcer healing locally administered exogenous nerve growth factor is retained in gastric tissue and is taken up by endothelial, neural, muscle and epithelial cells. This is likely the basis for the therapeutic action of locally administered nerve growth factor and its stimulation of angiogenesis, tissue regeneration and gastric ulcer healing.

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.

UNLABELLED: This study was aimed to determine the expression and localization of nerve growth factor (NGF) and several neural peptides in porcine esophagus. Transmural esophageal specimens were obtained from euthanized pigs. STUDIES: 1) histologic evaluation, 2) expressions of NGF and its tropomyosin receptor kinase A (TrkA) receptor, calcitonin generelated peptide (CGRP), neuronal nitric oxide synthase (nNOS), and neuronal enolase using immunostaining and quantification of signal distribution and intensity. Immunostaining for NGF, CGRP, nNOS and neuronal specific enolase (NSE) showed their strong and differential expression and localization in the neuronal network. NGF was strongly expressed in the majority of neurons and nerves, distribution of TrkA was complementary; its signal was 1.5-fold weaker P < 0.001 than NGF). Quantitatively the signal intensity was: CGRP > nNOS > NGF > NES > TrkA. In addition to neural structures, nNOS, NGF and TrkA were expressed in keratinocyte progenitor cells of esophageal mucosa and in endothelial cells of blood vessels. We conclude that a strong expression of NGF in majority of esophageal neurons and nerves indicates important, but previously unrecognized regulatory roles in the esophagus; 2) This study showed expression of NGF and some of the neuropeptides in neural elements, keratinocyte progenitor cells and endothelial cells of blood vessels, which indicates local interactions between neural, epithelial and endothelial cells.

Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival.

In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.

Molecular imaging of epidermal growth factor-receptor and survivin in vivo in porcine esophageal and gastric mucosae using probe-based confocal laser-induced endomicroscopy: proof of concept.

UNLABELLED: Confocal laser-induced endomicroscopy (CLE) enables in vivo, real time visualization of the subsurface cells and tissue structures in gastrointestinal mucosa at a subcellular resolution of ≈1000x magnification. The aims of this pilot study were to establish a principle of molecular imaging and determine in vivo expression of epidermal growth factor receptor (EGF-R) and survivin in porcine esophageal and gastric mucosa using probe-based CLE (pCLE) and topically applied FITC-labeled antibodies. Studies were performed in anesthetized pigs. During endoscopy FITC-labeled antibodies against EGF-R and survivin were either sprayed onto esophageal and gastric mucosa in preselected areas or administered via submucosal injection. Thirty minutes later pCLE was performed using a through-the-scope probe (GastroFlex UHD, Cellvizio, Mauna Kea Technologies, Paris, France) to determine cellular and tissue localization of EGF-R and survivin. Then the pigs were euthanized and esophageal and gastric walls from the areas sprayed or injected with antibodies were collected for histologic examination under epifluorescence microscopy. RESULTS: CLE enabled visualization of EGF-R and survivin in esophageal and gastric mucosa and this was confirmed by histology. In the esophagus both EGF-R and survivin were localized predominantly to the keratinocyte progenitor cells. In the stomach, EGF-R was localized to progenitor zone cells and some epithelial cells. Localization of survivin was similar, but involved more surface epithelial cells. This study demonstrated feasibility of using CLE and topical administration of FITC labeled antibodies for in vivo localization of EGF-R and survivin in esophageal and gastric mucosa.

The mechanisms of gastric mucosal injury: focus on microvascular endothelium as a key target.

This paper reviews and updates current views on gastric mucosal injury with a focus on the microvascular endothelium as the key target and the role of the anti-apoptosis protein survivin. Under normal conditions, mucosal integrity is maintained by well structured and mutually amplifying defense mechanisms, which include pre-epithelial "barrier"--the first line of defense; and, an epithelial "barrier". Other important defense mechanisms of gastric mucosa include: continuous epithelial cell renewal, blood flow through mucosal microvessels (providing oxygen and nutrients), an endothelial microvascular "barrier," sensory innervation, and generation of PGs, nitric oxide and hydrogen sulfide. The microvascular endothelium lining gastric mucosal blood microvessels severs not only as a barrier but is a biologically active tissue involved in many synthetic and metabolic functions. It allows transport of oxygen and nutrients, and produces prostaglandins and leukotriens, procoagulant factors, nitric oxide, endothelin, ghrelin, HSP, growth factors such VEGF, bFGF, angiopoietin 2 and others, specific types of collagen, plasminogen activator, and can also actively contract. Accumulating evidence indicates that the gastric microvascular endothelium is a critical target for injury by ethanol, NSAIDs, free radicals, ischemia-reperfusion and other damaging factors. The injury--microvessel rupture, plasma and erythrocyte extravasation, platelet aggregation and fibrin deposition caused by these damaging factors--occurs early (1-5 min), precedes glandular epithelial cell injury and results in cessation of blood flow, ischemia, hypoxia and impaired oxygen and nutrient transport. As a consequence, mucosal necrosis develops. One of the main reasons for the increased susceptibility of gastric microvascular endothelial (vs. epithelial) cells to injury is reduced expression levels of survivin, an anti-apoptosis protein, which is a regulator of both proliferation and cell survival.

Molecular mechanisms of epithelial regeneration and neovascularization during healing of gastric and esophageal ulcers.

In this paper we reviewed and updated current views on the cellular and molecular mechanisms of gastric and esophageal ulcer healing. Gastric ulcer healing encompasses inflammation, cell proliferation, epithelial regeneration, gland reconstruction, formation of granulation tissue, neovascularization (new blood vessel formation), interactions between various cells and the matrix and tissue remodeling, resulting in scar formation. All these events are controlled by the cytokines and growth factors, GI hormones including gastrin, CCK, and orexigenic peptides such as ghrelin, orexin-A and obestatin as well as Cox2 generated prostaglandins. These growth factors and hormones trigger cell proliferation, migration, and survival utilizing Ras, MAPK, PI-3K/AKT, PLC-γ and Rho/Rac/actin signaling pathways. Hypoxia triggers activation of some of these genes (e.g., VEGF) via hypoxia inducible factor (HIF). Growth factors: EGF, HGF, IGF-1, their receptors and Cox2 are important for epithelial cell proliferation, migration, re-epithelialization and regeneration of gastric glands during gastric ulcer healing. Serum response factor (SRF) is also essential for re-epithelialization and muscle restoration. VEGF, bFGF, angiopoietins, nitric oxide, endothelin, prostaglandins and metalloproteinases are important for angiogenesis, vascular remodeling and mucosal regeneration within gastric ulcer scar. SRF is critical limiting factor for VEGF-induced angiogenesis. Esophageal ulcer healing follows similar pattern to gastric ulcer, but KGF and its receptor are the key players in regeneration of the epithelium. In addition to local mucosal cells from viable mucosa bordering necrosis, circulating bone marrow derived stem and progenitor cells are potentially important for ulcer healing, contributing to the regeneration of epithelial and connective tissue components and neovascularization.

Critical role of hypoxia sensor--HIF-1α in VEGF gene activation. Implications for angiogenesis and tissue injury healing.

Vascular injury of esophageal and gastrointestinal mucosa caused by injurious and ulcerogenic factors leads to the cessation of blood flow, ischemia, and hypoxia and tissue necrosis in form of erosions or ulcers. The re-establishment of blood vessels through the process of angiogenesis--formation of new blood vessels--is critical for healing of tissue injury because is essential for delivery of oxygen and nutrients to the healing site. Hypoxia increases expression of hypoxia inducible factor (HIF-1), which serves as hypoxia sensor and activates compensatory and adaptive mechanisms. However, the molecular mechanisms and the role of HIF-1α in hypoxia-driven cellular and molecular events of angiogenesis in gastrointestinal injury healing have not been fully explored. The review discusses the novel molecular mechanisms of angiogenesis in gastric and esophageal mucosa with focus on HIF1α and VEGF interactions during healing of gastric mucosal injury and esophageal ulcers. HIF-1α is upregulated by gastric mucosal injury and esophageal ulcers; this upregulation correlates with VEGF gene activation and initiation of angiogenesis. The non-steroidal anti-inflammatory drugs (NSAIDs) interfere with hypoxia-induced HIF-1α accumulation, VEGF gene activation and angiogenesis through upregulation of von Hippel- Lindau (VHL) tumor suppressor, which activates degradation of HIF-1α protein. HIF-1α is a transcription factor that under hypoxic conditions, accumulates in endothelial cells and can bind to VEGF gene promoter and induce VEGF gene expression. In order to activate the VEGF gene, HIF-1α must be transported to the nucleus. Recent evidence implicates importins as key mechanism in this process.

Colonic mucosal biopsies obtained during confocal endomicroscopy are pre-stained with fluorescein in vivo and are suitable for histologic evaluation.

BACKGROUND AND STUDY AIMS: Confocal laser endomicroscopy (CLE) with intravenous infusion of fluorescein allows noninvasive, real-time in vivo visualization of gastrointestinal mucosa at ~ × 1000 magnification ("virtual biopsy"). Conventional biopsies obtained during these procedures serve as the reference and established diagnostic standard. The aim of the present study was to assess whether the standard histologic biopsies that are obtained during CLE retain fluorescein in the tissues and allow the visualization of mucosal structures without any additional staining. PATIENTS AND METHODS: CLE optical imaging of the mucosa was performed in 16 patients who were undergoing CLE colonoscopy. Standard conventional biopsies were also obtained from both normal colonic mucosa and colonic polyps. De-paraffinized mucosal sections were examined under a fluorescence microscope for the presence and distribution of fluorescein, and then underwent immunostaining for expression of vascular endothelial growth factor (VEGF). RESULTS: Standard mucosal biopsy sections from patients undergoing CLE displayed a strong fluorescence and showed well-delineated mucosal structures. In colonic adenomas, there was a 4.6-fold increased vascular permeability compared with normal mucosa (P<0.001), indicated by fluorescein leakage to the extravascular space. Immunostaining demonstrated an aberrantly increased expression of VEGF in the epithelium of colonic adenomas but not in the epithelium of normal mucosa or hyperplastic polyps. CONCLUSIONS: This study shows for the first time that standard colonic biopsies obtained during CLE retain fluorescein, show excellent delineation of mucosal structures without additional staining, allow the evaluation of mucosal microvasculature and vascular permeability, and are suitable for immunostaining.

In vivo visualization of epidermal growth factor receptor and survivin expression in porcine pancreas using endoscopic ultrasound guided fine needle imaging with confocal laser-induced endomicroscopy.

UNLABELLED: The aims of this pilot study were to establish a principle of molecular imaging of the pancreas and determine in vivo expression of epidermal growth factor receptor (EGF-R) and survivin using a novel endoscopic ultrasound-guided fine needle imaging (EUS-FNI) technique, which incorporates needle based confocal laser-induced endomicroscopy (nCLE) after intrapancreatic injection of FTIC-labeled antibodies. Studies were performed in anesthetized pigs. FITC-labeled specific antibodies against EGF-R and survivin were injected into the tail and neck of the pancreas using a 19 gauge needle introduced under EUS guidance. Thirty minutes later, nCLE was performed using a prototype needle-based confocal laser-induced endomicroscopy probe (Cellvizio AQ-Flex-19, Mauna Kea Technologies, Paris, France) to determine cellular and tissue localization of EGF-R and survivin in the pancreas. Then pigs were euthanized and specimens of pancreas from areas injected with antibodies were obtained for histologic examination under epifluorescence microscope. RESULTS: EUS-guided nCLE enabled visualization of EGF-R and survivin in pancreatic tissue. Expression of EGF-R and survivin in pancreas was confirmed by histology. EGF-R immunoreactivity was localized to majority of duct-lining cells and to the surface and cytoplasm of many acinar cells. Survivin was localized mainly to the acinar cells. This study demonstrated the feasibility of in vivo, real time visualization of EGF-R and survivin in the pancreas by local injection of FITC-labeled antibodies via EUS-guided fine needle injection, followed by EUS-guided needle based confocal laser-induced endomicroscopy.

Activation of the metabolic sensor-AMP activated protein kinase reverses impairment of angiogenesis in aging myocardial microvascular endothelial cells. Implications for the aging heart.

Impairment of angiogenesis - new capillary blood vessel formation from pre-existing vessels, is frequent in aging tissues and cells. Reduced angiogenesis in aging individuals is associated with increased incidence of myocardial infarctions and other cardiovascular diseases. Therefore there is a need to develop novel strategies to enhance angiogenesis in aging individuals. Our previous study demonstrated aging-related impairment of angiogenesis in aging (vs. young) rat myocardial microvascular endothelial cells (MMEC), and identified reduced activation of the vascular endothelial growth factor (VEGF, the most potent stimulator of angiogenesis) gene as the main underlying mechanism. In the present study we examined the possibility of increasing angiogenesis and activating VEGF gene expression in aging MMECs using a chemical activator of the metabolic sensor - AMP activated protein kinase (AMPK). We hypothesized that activation of VEGF gene in aging MMECs by AMPK would stimulate angiogenesis and reverse the impairment in angiogenesis seen in these cells. We used MMECs isolated from aging (24 months old) Fisher F-344 rats and treated them with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a specific pharmacological stimulator of AMPK. We examined: 1) in vitro angiogenesis; and 2) the expression of phosphorylated AMPK, VEGF, and P-MAPK/Erk1/2. Treatment of aging MMECs with AICAR increased in vitro angiogenesis and VEGF mRNA expression by 2.1-fold and 3.7-fold, respectively. Furthermore, AICAR treatment resulted in phosphorylation of MAPK/Erk1/2. This study demonstrated the successful use AICAR to reverse aging-related impairment of angiogenesis in aging MMECs by enhancing VEGF gene expression and also identified phosphorylation of MAPK/Erk1/2 as a likely mechanism of these changes.

Impaired angiogenesis in aging myocardial microvascular endothelial cells is associated with reduced importin alpha and decreased nuclear transport of HIF1 alpha: mechanistic implications.

Aging is associated with increased incidence of myocardial infarctions and impaired angiogenesis - new capillary blood vessel formation from preexisting vessels. The molecular mechanism(s) of aging-related impairment of angiogenesis are unknown. In the present study we focused on the mechanism of activation of the gene for vascular endothelial growth factor (VEGF - the most potent stimulator of angiogenesis) in young and aging myocardial microvascular endothelial cells (MMEC). Activation of VEGF gene in the cell nucleus is mediated in part by the transcription factor hypoxia-inducible factor 1 alpha (HIF1 alpha). In order to activate VEGF gene, HIF1 alpha must first be transported to the nucleus, but the mechanisms of this transport are unknown. We hypothesized that reduced VEGF gene activation and impaired angiogenesis in myocardium during aging can result from downregulation of the nuclear transport receptor - importin alpha that leads to decreased transport of HIF1 alpha to the nucleus. We examined in MMEC isolated from young (3 months of age) and aging (24 months old) Fisher F-344 rats: 1) in vitro angiogenesis; and 2) the expression of VEGF, importin alpha and HIF1 alpha. Aging MMEC exhibited a 3.7-fold reduction in angiogenesis and a corresponding reduction in VEGF (by 3-fold) and importin alpha (by 1.9-fold) levels compared to young MMEC. Aging MMEC also exhibited cytoplasmic accumulation (by 1.8-fold) of HIF1 alpha protein, reduced HIF1 alpha transport to the nucleus and decreased binding of HIF1 alpha protein to the VEGF gene promoter. This study is the first demonstration of the downregulation of importin alpha in aging MMEC and reduced nuclear transport of HIF1 alpha, which likely lead to decreased VEGF gene activation and impaired angiogenesis.

Reduced ghrelin in endothelial cells plays important mechanistic role in aging-related impairment of angiogenesis.

Ghrelin, a hormone produced mainly by gastric mucosal cells stimulates growth hormone (GH) release. Ghrelin is also expressed in the endothelial cells of blood vessels suggesting its physiological role and a function in these cells. We recently demonstrated that ghrelin induces angiogenesis--new capillary blood vessel formation- in neonatal human microvascular endothelial cells (HMVECs). Angiogenesis is impaired in aging individuals both in vitro and in vivo, but the precise mechanism(s) of this phenomenon is unknown. We examined whether HMVECs derived from aging individuals (66 years and 90 years old), 66-HMVECs and 90-HMVECs have reduced ghrelin levels vs. neonatal (Neo) HMVECs and whether treatment with exogenous ghrelin can restore impaired in vitro angiogenesis on matrigel in aged HMVECs. Ghrelin levels were reduced in the aged HMVECs by 3.2-fold (p<0.05) compared to Neo-HMVECs. Angiogenesis was significantly decreased in the aged 66- and 90-HMVECs by 39.7% (p = 0.003) and 62.4% (p = 0.003), respectively compared to Neo-HMVECs. Treatment with exogenous ghrelin significantly reversed impaired angiogenesis in aged HMVECs with the EC(50) 0.05 nM. Ghrelin induced angiogenesis in Neo-HMVECs mainly through ERK2 activation. This study is the first demonstration that reduced ghrelin is one of the factors responsible for aging-related impairment of angiogenesis.

Mesalazine downregulates c-Myc in human colon cancer cells. A key to its chemopreventive action?

BACKGROUND: Dysplasia and malignant transformation of colonocytes in ulcerative colitis are associated with overexpression of c-Myc and genes regulating cell survival. 5-Aminosalicylates such as mesalazine may reduce the development of colorectal cancer in ulcerative colitis, but the mechanisms of its chemopreventive action are not clear. AIMS: To examine whether mesalazine affects the expression of c-Myc in human colon cancer cell lines. METHODS: Human colon cancer cells were treated with vehicle or mesalazine (4 mm or 40 mm). We examined: (i) mRNA expression by gene array, (ii) protein expression by Western blotting and immunohistochemistry and (iii) apoptosis by Annexin V labelling. RESULTS: Mesalazine significantly reduced expression of c-Myc mRNA and protein. CONCLUSIONS: Mesalazine downregulates gene and protein expression of c-Myc. The apoptotic and growth inhibitory effects of mesalazine are dose-dependent. Expression of c-Myc is significantly reduced by mesalazine 40 mm.

Altered angiogenic balance in ulcerative colitis: a key to impaired healing?

Angiogenesis is an essential component of ulcer healing since it assures delivery of oxygen and nutrients to the healing site. Previous studies demonstrated increased serum and tissue levels of vascular endothelial growth factor (VEGF, the most potent angiogenic growth factor) in patients with active ulcerative colitis (UC) and animal models of UC. However, there is no explanation why the healing of UC-related mucosal injury is impaired despite increased expression of VEGF. Expression of angiogenesis inhibitors, angiostatin and/or endostatin, in UC has not been determined before. We examined expression of VEGF, angiostatin, and endostatin in two models of experimental UC. The results revealed that in addition to increased VEGF, both endostatin and angiostatin levels were markedly (2-3-folds) increased in colonic mucosa at early stage of experimental UC. This is the first demonstration that colitis triggers increase in angiostatin and endostatin levels. The results may explain why mucosal lesions heal slowly despite increased VEGF levels, and may provide a novel and mechanistic insight into UC.

Comparison of prostaglandin and cimetidine in protection of isolated gastric glands against indomethacin injury.

Prostaglandins can protect the in vivo gastric mucosa against necrosis produced by a variety noxious agents. Cimetidine has also been shown to have protective properties in humans and in some models of experimental injury. Whether prostaglandins or cimetidine may protect gastric mucosal cells directly in the absence of systemic factors remains controversial. In the present study, the potential protective actions of prostaglandin and cimetidine against indomethacin injury were assessed in isolated rat gastric glands. Gastric glands were pre-incubated in oxygenated medium with either placebo, 16,16 dimethyl prostaglandin E(2) (dm PGE(2)) or cimetidine and incubated at 37 degrees C in medium containing 0.5 mg/ml of indomethacin for 2, 4 and 6 hrs. Cell injury and protection was assessed by the Fast Green exclusion test (viability test), leakage of lactate dehydrogenase (LDH) into the medium, and by scanning and transmission electron microscopy. In addition, the generation of PGE(2) by the gland cells was determined using RIA assay. Indomethacin by itself significantly reduced the viability of gastric glands, increased LDH release into the medium and produced prominent ultrastructural damage. In contrast to cimetidine, co-incubation of gastric glands with dm PGE(2) added to indomethacin, significantly reduced indomethacin-induced injury, increased the number of viable cells, reduced LDH leakage and diminished the extent of ultrastructural damage. The dose of indomethacin (5 microg/ml) which significantly inhibited the generation of PGE(2) (up to 90% inhibition) had no effect on cell viability nor LDH release. We conclude that 1) exogenous PGE2 exerts a potent protective activity in vitro which is independent on neural, vascular and hormonal factors; 2) inhibition of endogenous PGs may not the primary mechanism in the deleterious action of indomethacin against damage to gastric glandular cells and 3) indomethacin can exert a direct cytotoxic effect on the mucosal cells in gastric glands.

Rebamipide inhibits gastric cancer growth by targeting survivin and Aurora-B.

Rebamipide accelerates healing of gastric ulcers and gastritis but its actions on gastric cancer are not known. Survivin, an anti-apoptosis protein, is overexpressed in stem, progenitor, and cancer cells. In gastric cancer, increased and sustained survivin expression provides survival advantage and facilitates tumor progression and resistance to anti-cancer drugs. Aurora-B kinase is essential for chromosome alignment and mitosis progression but surprisingly its role in gastric cancer has not been explored. We examined in human gastric cancer AGS cells: (1) survivin expression, (2) localization of survivin and Aurora-B, (3) cell proliferation, and (4) effects of specific survivin siRNA and/or rebamipide (free radical scavenging drug) on survivin and Aurora-B expression and cell proliferation. Survivin and Aurora-B are strongly expressed in human AGS gastric cancer cells and co-localize during mitosis. Survivin siRNA significantly reduces AGS cell viability. Rebamipide significantly downregulates in AGS cell survivin expression, its association with Aurora-B and cell proliferation. Rebamipide-induced downregulation of survivin is at the transcription level and does not involve ubiquitin-proteasome pathway.