Basic fibroblast growth factor accelerates gastric mucosal restoration in vitro by promoting mesenchymal cell migration and proliferation

It has been generally accepted that basic fibroblast growth factor is a potent stimulator of duodenal ulcer healing. However, the detailed mechanism and mode of action of growth factor on gastric ulcer healing is still controversial. Therefore, in the present study, the effects of basic fibroblast growth factor on gastric mucosal repair were studied using an in vitro cultured cell system. Artificial wounds were made in confluent monolayer rabbit gastric fibroblast and epithelial cell sheets by mechanical denudation. Changes in the size of the cell-free area were analysed quantitatively. Cell proliferation was assessed by BrdU staining. For both cell types, mucosal restoration involved cell migration and proliferation. Although the speed of restoration of epithelial cells was not affected by the addition of basic fibroblast growth factor, it was much faster for epithelial cells than for fibroblasts. Basic fibroblast growth factor accelerated wound repair of fibroblasts but not epithelial cells. Basic fibroblast growth factor accelerated wound repair by stimulating both cell migration and proliferation. Therefore, the effects of basic fibroblast growth factor in peptic ulcer diseases may be mainly due to the stimulation of mesenchymal cells.     

Analysis of the cellular basis of keratinocyte growth factor overexpression in inflammatory bowel disease

BACKGROUND: Keratinocyte growth factor (KGF) stimulates gastrointestinal epithelial cells in vivo, and is protective against gastrointestinal injury and colitis. Endogenous KGF is increased in inflammatory bowel disease (IBD), and may be an important mediator of mucosal repair. KGF is expressed by mesenchymal cells and activated intraepithelial lymphocytes (IEL). AIMS: To investigate the relative contributions of these cellular sources of KGF expression in IBD. METHODS: IELs and lamina propria lymphocytes (LPL) were isolated from inflamed and uninflamed IBD tissues. mRNA expression was determined by ribonuclease protection assay. In situ hybridisation was combined with immunohistochemistry to determine whether KGF mRNA was expressed by specific cell types in vivo. RESULTS: Low levels of KGF mRNA expression were detected in three of five IEL samples derived from inflamed tissue, but not in two IEL samples from uninflamed tissue. No KGF expression was detected in LPLs from either inflamed or uninflamed tissue. In contrast, KGF was expressed by primary cultures of human intestinal fibroblasts, and was induced by treatment with interleukin 1. CONCLUSIONS: The major source of KGF expression in IBD was lamina propria cells of non-immune origin, most likely fibroblasts and/or smooth muscle cells. Compared with these cell types, relatively little KGF synthesis was associated with IELs in inflamed IBD tissue.     

Erythropoietin-induced proliferation of gastric mucosal cells

AIM: To analyze the localization of erythropoietin receptor on gastric specimens and characterize the effects of erythropoietin on the normal gastric epithelial proliferation using a porcine gastric epithelial cell culture model. METHODS: Erythropoietin receptor was detected by RT-PCR, Western blotting and immunohistochermistry. Growth stimulation effects of erythropoietin on cultured gastric mucosal cells were determined by ELISA using bromodeoxyuridine (BrdU). RESULTS: Erythropoietin receptor was detected on cultured porcine gastric mucosal epithelial cells. Erythropoietin receptor was also detected histochemically at the base of gastric mucosal epithelium. BrdU assay demonstrated a dose-dependent increase in growth potential of cultured porcine gastric mucosal epithelial cells by administration of erythropoietin, as well as these effects were inhibited by administration of anti-erythropoietin antibody (P<0.01). CONCLUSION: These findings indicate that erythropoietin has a potential to proliferate gastric mucosal epithelium via erythropoietin receptor.     

Expression of keratinocyte growth factor and its receptor in adaptive changes of ileorectal pouch mucosa

Objective: Total proctocolectomy with formation of an ileo-anal pouch is a well-established surgical procedure for patients with ulcerative colitis (UC) or familiar adenomatous polyposis (FAP). The pouch mucosa undergoes adaptive changes, with inflammation of the ileal reservoir being the most common complication. The aetiology is unknown. The keratinocyte growth factor (KGF) has not only been shown to promote intestinal wound healing and re-epithelialization but also to have some immunomodulatory properties. This study was designed to investigate a putative involvement of KGF in observed histomorphological changes in the pouch mucosa. Material and methods. Multiple biopsies were obtained from age-matched and sex-matched patients. Biopsies were stained with H&E and scored for inflammation and morphological changes. mRNA expression levels of KGF and KGF-receptor (KGFR) were determined using competitive RT-PCR, protein expression and phosphorylation was analyzed by Western blotting. KGF and KGFR were localized in tissue samples by immunohistochemistry. Results. Expression of KGF and KGFR was significantly increased in inflamed and adapting mucosa. Patterns of expression did not significantly differ in pouch mucosa from UC or FAP patients. Protein expression correlated with the mRNA results and KGFR was shown to be activated in adapting pouch mucosa. KGF was detected on subepithelial cells, mainly on fibroblasts, whereas expression of KGFR was restricted to epithelial cells. Conclusion. Expression of KGF and KGFR is significantly increased in the pouch mucosa, suggesting an involvement of this growth factor in tissue repair and adaptive changes. Topical application of KGF might alleviate the inflammatory and promote the adaptive process in the ileo-anal pouch mucosa.     

Regulation of Keratinocyte Growth Factor (KGF) and KGF Receptor mRNAs by Nutrient Intake and KGF Administration in Rat Intestine

The aim of this study was to investigate the regulation of keratinocyte growth factor (KGF) and KGF receptor mRNAs by diet and KGF treatment in rat intestine. Fasting for three days up-regulated KGF and KGF receptor mRNA levels in ileum and increased KGF receptor mRNA expression in colon. KGF and KGF receptor mRNA levels returned toward control values with ad libitum refeeding but remained elevated when refeeding was limited to 25% of ad libitum intake. KGF treatment during nutrient repletion did not alter intestinal KGF mRNA levels but increased KGF receptor mRNA abundance in ileum and colon. We conclude that the increase in KGF and KGF receptor mRNAs induced by malnutrition may be an adaptive response to attenuate gut mucosal atrophy in this setting. The gut-trophic effects of KGF treatment may be mediated, in part, by up-regulation of the KGF receptor mRNA in small bowel and colon.     

Alveolar epithelial cells: differentiation and lung injury

Abstract:   Re-epithelialization of alveolar epithelial cells is one of the important repair processes in many types of lung injury. The major functions of alveolar type II cells are synthesis and secretion of surfactant, hyperplasia in reaction to alveolar epithelial injury, and serving as progenitor cells for alveolar type I cells. The authors have examined the effects of several soluble factors on cultured alveolar type II cells in vitro , and also examined the histopathology and gene expression of surfactant proteins in the rat lungs with LPS, bleomycin and/or treated with keratinocyte growth factor. The authors next examined the effects of bone marrow stromal cells (BMSC) implanted transvenously into bleomycin-induced lungs. The authors found that keratinocyte growth factor (KGF) is a strong growth factor for alveolar type II cells, and that KGF instillation prevents bleomycin-induced lung injury. Furthermore, the authors showed the possibility of differentiation of implanted BMSC into alveolar epithelial cells. KGF and BMSC may play an important role in maintaining the alveolar epithelium and repairing the damaged epithelium after injury, and may well provide potential therapeutic alternatives.     

Expression of keratinocyte growth factor and its receptor in adaptive changes of ileorectal pouch mucosa

OBJECTIVE: Total proctocolectomy with formation of an ileo-anal pouch is a well-established surgical procedure for patients with ulcerative colitis (UC) or familiar adenomatous polyposis (FAP). The pouch mucosa undergoes adaptive changes, with inflammation of the ileal reservoir being the most common complication. The aetiology is unknown. The keratinocyte growth factor (KGF) has not only been shown to promote intestinal wound healing and re-epithelialization but also to have some immunomodulatory properties. This study was designed to investigate a putative involvement of KGF in observed histomorphological changes in the pouch mucosa. MATERIALS AND METHODS: Multiple biopsies were obtained from age-matched and sex-matched patients. Biopsies were stained with H&E and scored for inflammation and morphological changes. mRNA expression levels of KGF and KGF-receptor (KGFR) were determined using competitive RT-PCR, protein expression and phosphorylation was analyzed by Western blotting. KGF and KGFR were localized in tissue samples by immunohistochemistry. RESULTS: Expression of KGF and KGFR was significantly increased in inflamed and adapting mucosa. Patterns of expression did not significantly differ in pouch mucosa from UC or FAP patients. Protein expression correlated with the mRNA results and KGFR was shown to be activated in adapting pouch mucosa. KGF was detected on subepithelial cells, mainly on fibroblasts, whereas expression of KGFR was restricted to epithelial cells. CONCLUSION: Expression of KGF and KGFR is significantly increased in the pouch mucosa, suggesting an involvement of this growth factor in tissue repair and adaptive changes. Topical application of KGF might alleviate the inflammatory and promote the adaptive process in the ileo-anal pouch mucosa.     

Sensory neuropeptides and epithelial cell restitution: the relevance of SP- and CGRP-stimulated mast cells

Background Calcitonin-gene-related peptide (CGRP) and substance P (SP) are neurotransmitters of extrinsic primary afferent neurons located within the dorsal root ganglia. In experimental models of colitis in rats and rabbits, a protective role of SP and CGRP on intestinal mucosa was presumed. The mucosal protection partly depends on a CGRP-mediated modulation of mucosal blood flow. Limited data are available regarding CGRP- or SP-mediated effects on epithelial cell restitution. Having shown earlier that SP-stimulated fibroblasts but not CGRP-stimulated fibroblasts induce epithelial cell migration in vitro, the aim of this study was to explore whether mast cells mediate effects of SP and CGRP on epithelial cell restitution in vitro. Methods Mast cells (C57) were exposed to SP [10⁻¹²–10^{−6 M}] and CGRP [10⁻¹²–10^{−7 M}]. After a 24-h incubation period, the cell supernatants (conditioned media, CDM) were taken from mast cell cultures and directly applied to rat intestinal epithelial cell lines-18 or Caco-2 monolayers, which had been wounded with a razor blade 24 h prior to the experiments. Epithelial cell migration was assessed by counting cells across the wound edge and epithelial cell proliferation was measured using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide test. Results CGRP significantly induced epithelial cell migration and proliferation via mast cells when supernatants were directly applied to epithelial cells in vitro. The effects on epithelial cell migration were abolished after neutralizing anti-transforming growth factor-beta (TGF-β) had been added to the cell cultures. SP had no effects on epithelial cells following stimulation of mast cells. Conclusion CGRP modulates epithelial cell restitution in vitro mediated by mast cells. The CGRP- and mast-cell-induced epithelial cell migration is TGF-β dependent. This observation underlines an important role for extrinsic primary afferent neurons in mucosal defence and repair and in keeping the mucosal homeostasis. This knowledge leads to a better understanding of the interaction of the enteric nervous system and wound healing and may, in the future, lead to new therapeutic approaches to inflammatory diseases of the intestine. Kerem Bulut and Peter Felderbauer contributed equally.     

Protection of the intestinal mucosa by intraepithelial gamma delta T cells

gammadelta intraepithelial T lymphocytes (IEL) represent a major T cell population within the intestine of unclear functional relevance. The role of intestinal gammadelta IEL was evaluated in the dextran sodium sulfate (DSS) induced mouse colitis model system. Large numbers of gammadelta T cells, but not alphabeta T cells, were localized at sites of DSS-induced epithelial cell damage. gammadelta IEL in DSS treated mice expressed keratinocyte growth factor (KGF), a potent intestinal epithelial cell mitogen. gammadelta cell-deficient mice (TCRdelta(-/-)) and KGF-deficient mice (KGF(-/-)), but not alphabeta cell-deficient mice (TCRalpha(-/-)), were more prone than wild-type mice to DSS-induced mucosal injury and demonstrated delayed tissue repair after termination of DSS treatment. Termination of DSS treatment resulted in vigorous epithelial cell proliferation in wild-type mice but not in TCRdelta(-/-) mice or KGF(-/-) mice. These results suggest that gammadelta IEL help preserve the integrity of damaged epithelial surfaces by providing the localized delivery of an epithelial cell growth factor.     

Substance P induces intestinal wound healing via fibroblasts—evidence for a TGF-β-dependent effect

Background Substance P (SP) and calcitonin gene-related peptide (CGRP) are neurotransmitters of the afferent sensory nervous system. In experimental models of colitis in rats and rabbits, a protective role of SP and CGRP on the intestinal mucosa was presumed. In part, mucosal protection depends on a SP-mediated and CGRP-mediated modulation of mucosal blood flow after injury. We thought to explore whether there is a fibroblast-mediated effect of SP and CGRP on epithelial cell restitution in vitro. Materials and methods Rat kidney fibroblast (NRK-49F) cell lines were exposed to CGRP or SP in various concentrations. After incubation, the cell culture supernatants were taken from the fibroblast cultures and were directly applied to IEC-18 or Caco-2 monolayers, which had been wounded with a razor blade 24 h before the experiments. Epithelial cell migration was assessed by counting cells across the wound edge. Epithelial cell proliferation was assessed using the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) test. Results SP significantly induced epithelial cell migration and inhibited epithelial cell proliferation via stimulation of fibroblasts when supernatants were directly applied to epithelial cells in vitro. The effects on epithelial cell migration were abolished after neutralising anti-transforming growth factor-β (TGF-β) was added to the cell cultures. CGRP had no effect on epithelial cells via stimulation of fibroblasts. Neither CGRP nor SP had any effect on epithelial cell migration or proliferation when directly applied to epithelial cells. Conclusion SP modulates epithelial cell restitution in vitro mediated by fibroblasts. The epithelial cell migration depends on a TGF-β release from SP-stimulated fibroblasts. This observation underlines an important role for the sensory nervous system in mucosal defence and repair and in keeping mucosal homeostasis. Modulation of SP may be potentially useful for the treatment of various intestinal disorders characterised by injury and ineffective repair of the intestinal mucosa. P. Felderbauer and K. Bulut are equally first contributing authors.     

Purification and characterization of a newly identified growth factor specific for epithelial cells.

A growth factor specific for epithelial cells was identified in conditioned medium of a human embryonic lung fibroblast cell line. The factor, provisionally termed keratinocyte growth factor (KGF) because of its predominant activity on this cell type, was purified to homogeneity by a combination of ultrafiltration, heparin-Sepharose affinity chromatography, and hydrophobic chromatography on a C4 reversed-phase HPLC column. KGF was both acid and heat labile and consisted of a single polypeptide chain of approximately 28 kDa. Purified KGF was a potent mitogen for epithelial cells, capable of stimulating DNA synthesis in quiescent BALB/MK epidermal keratinocytes by greater than 500-fold with activity detectable at 0.1 nM and maximal at 1.0 nM. Lack of mitogenic activity on either fibroblasts or endothelial cells indicated that KGF possessed a target cell specificity distinct from any previously characterized growth factor. Microsequencing revealed an amino-terminal sequence containing no significant homology to any known protein. The release of this growth factor by human embryonic fibroblasts raises the possibility that KGF may play a role in mesenchymal stimulation of normal epithelial cell proliferation.     

Hepatocyte growth factor accelerates restitution of intestinal epithelial cells

Many cytokines are involved in the repair of damaged tissue, and one of these, hepatocyte growth factor (HGF), is involved not only with liver regeneration but also in the repair of other tissues. To investigate the importance of HGF in the repair of the small intestine, we evaluated its effect and that of other growth factors in IEC-6 cells, an intestinal epithelial cell line derived from normal rat small intestine. Round "wounds" were made in confluent monolayers of IEC-6 by silicon rubber-tipped steel rods and various cytokines; transforming growth factor α (TGF-α), transforming growth factor β1 (TGF-β1), keratinocyte growth factor (KGF), and HGF, were added. We photographed the repaired monolayers every 24 h and calculated the ratios of areas not covered by cells to initial areas. Cell proliferation with TGF-α, TGF-β, KGF, or HGF was examined in terms of [3H]-thymidine uptake. Finally, we determined c-met (the HGF receptor) mRNA in the IEC-6 cells by Northern blot hybridization. HGF was the most potent of the cytokines in accelerating repair of the damaged monolayer of IEC-6. HGF was also 1.34 times more effective than control the medium for inducing cell proliferation of IEC-6. By Northern blot hybridization, three bands of mRNA bound to c-met cDNA. These results suggest that HGF is important in the repair of the small intestine. (Received Feb. 21, 1997; accepted Aug. 22, 1997)     

Fibroblast growth factor 7, secreted by breast fibroblasts,is an interleukin-1beta-induced paracrine growth factor for human breast cells

Keratinocyte growth factor/fibroblast growth factor 7 (KGF/FGF7) is known to be a potent growth factor for mammary cells but its origin, cellular targets and mode of action in the breast are unclear. In this study, we carried out studies to determine the localisation of FGF7 and its receptor, and the related growth factor FGF10. We also determined the factors that regulate FGF7 release from stromal cells and the effects of FGF7 on normal and neoplastic breast cells. Using an FGF7-specific antibody which does not react with the FGF7 heparan sulphate proteoglycan (HSPG)-binding site, we showed epithelial and myoepithelial immunohistochemical staining in normal breast sections, and epithelial staining in breast carcinomas. Stromal staining was also detected in some lobular carcinomas as well as a subset of invasive ductal carcinomas. FGF10 and FGF receptor (FGFR)2 immunostaining showed a similar epithelial expression pattern, whereas no stromal staining was observed. We purified normal breast stromal, epithelial and myoepithelial cells and showed that FGF7 stimulated proliferation of both epithelial cell types, but not stromal fibroblasts. We also examined the effects of FGF7 on Matrigel-embedded organoids, containing both epithelial and myoepithelial cells, and showed FGF7 induced an increase in cellular proliferation. Furthermore, conditioned medium derived from stromal cells was shown to increase the proliferation of normal and neoplastic breast epithelial cells, which could be abolished by a neutralising antibody to FGF7. Finally, we showed that interleukin-1beta, but not oestradiol or other oestrogen receptor ligands, caused a dose-related FGF7 release. Further results also indicate that the epithelial localisation of FGF7 and FGF10 in breast tissue sections is likely to be due to their binding to their cognate receptor. In summary, our findings suggest that FGF7 is a paracrine growth factor in the breast. FGF7 is produced by the breast stromal fibroblasts and has profound proliferative and morphogenic roles on both epithelial and myoepithelial cells.     

Protection from thymic epithelial cell injury by keratinocyte growth factor: a new approach to improve thymic and peripheral T-cell reconstitution after bone marrow transplantation

Decreased thymopoietic capacity contributes to the severe and clinically significant immune deficiency seen after bone marrow transplantation (BMT). One mechanism for thymopoietic failure is damage to the interleukin 7 (IL-7)-producing thymic epithelial cells (TECs) by irradiation and chemotherapy, which can be partially treated by IL-7 administration. Pretreatment of BMT recipients with keratinocyte growth factor (KGF, or Fgf7), an epithelial cell-specific growth factor, protects mucosal, cutaneous, and pulmonary epithelial cells from cytotoxic therapy-induced damage in experimental murine models. Like other epithelial cells, TECs specifically express KGF receptors. Because KGF specifically protects KGF receptor-bearing epithelial cells and post-BMT immune deficiency is caused by loss of TECs, we hypothesized that KGF pretreatment would improve post-BMT thymic function. To test the hypothesis, BMT recipient mice were given KGF or placebo prior to congenic or allogeneic BMT. Administration of KGF before murine BMT significantly increased the capacity of the thymus to generate donor-derived thymocytes. KGF pretreatment also normalized the proportion of thymic subpopulations, increased the number of naive T cells in the periphery, and improved the response to neoantigen immunization. KGF treatment caused increased production of intrathymic IL-7, and the thymopoietic effects of KGF required an intact IL-7 signaling pathway. These results demonstrate that KGF may have immunomodulatory effects by a unique mechanism of protection of TECs. Furthermore, thymic injury and prolonged posttransplantation immune deficiency in BMT recipients can be prevented by KGF administration.     

Parathyroid hormone-related protein is a positive regulator of keratinocyte growth factor expression by normal dermal fibroblasts.

Parathyroid hormone-related protein (PTHrP), an important factor in the pathogenesis of humoral hypercalcemia of malignancy, is produced by many normal tissues, including the skin, where it regulates keratinocyte growth and differentiation and dermal fibroblast function. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, is a secretory product of stromal cells and functions as a mediator of epithelial cell growth and differentiation. Phenotypes of the skin in several transgenic mouse models, in which the KGF and PTHrP genes have been overexpressed or disrupted, suggest that these two factors interact in vivo to regulate homeostasis of the skin. In this study, we investigated the effects of KGF on PTHrP secretion and expression by normal human foreskin keratinocytes (NHFK) and the effects of PTHrP on KGF secretion and expression by normal human dermal fibroblasts (NHDF) in vitro. N-terminal PTHrP(1-36) increased KGF secretion, protein expression and mRNA expression by NHDF in a dose-dependent manner, however, KGF did not regulate PTHrP expression and secretion by NHFK. By flow cytometry, PTHrP also increased the percentage of NHDF producing KGF. Our results indicate that PTHrP produced by keratinocytes is a potential paracrine regulator of KGF expression by dermal fibroblasts in vivo. This paracrine regulation may explain, in part, the epidermal atrophy seen in the PTHrP null mice and epidermal hyperplasia seen in transgenic mice overexpressing PTHrP in their basal keratinocytes. Our results also suggest that PTHrP is an important mediator for the healing of skin wounds and growth of neoplasms of squamous origin.