molecular and cellular basis of ulcer healing
TRANSCRIPT
Molecular and Cellular Basis of Ulcer Healing S. SZABO, S. KUSSTATSCHER, Z. SANDOR & G. SAKOULAS Dept. of Pathology, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
Szabo S, Kusstatscher Z, Sandor Z, Sakoulas G. Molecular and cellular basis of ulcer healing. Scand J Gastroenterol 1995;30 Suppl208:>8.
Background: The high ulcer recurrence rates after treatment with antacids or antisecretory drugs illustrate the need for direct treatment of GI ulcers by stimulating repair and healing mechanisms. The molecular regulators of ulcer healing include polyamines and growth factors such as EGF, TGF-8, bFGF and PDGF. Methods and results: Oral treatment of rats with bFGF or PDGF accelerated the healing of chronic cysteamine-induced duodenal ulcers without decreasing gastric secretion. We found that suc- ralfate binds bFGF in vitro and in vivo, and the elevated local concentration of this growth factor may contribute to the ulcer healing properties of sucralfate. Parallel treatment with bFGF + sucralfate resulted in synergistic healing of chronic duodenal ulcers and chronic gastritis. Conclusions: Rapid changes in mucosal concentration of bFGF and EGF receptors during ulceration suggest that these peptides play a role in the natural history of GI ulcers. Thus, treatment based on molecular and cellular mechanisms of ulcer healing allows a direct and efficient ulcer therapy.
Key words: Angiogenesis; bFGF; chronic gastritis; cysteamine; duodenal ulcer; EGF; growth factors; iodoacetamide; PDGF; sucralfate
Sandor Szabo, M. D . , Ph.D., Dept. of Pathology, Brigham & Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA
Recent advances in basic sciences such as molecular physi- ology, pharmacology and pathology, as well as cell and molecular biology, provide new tools and yield new results in the study complex and multifactorial disorders such as ulcer disease of the gastrointestinal (GI) tract. Unfor- tunately, the etiology and pathogenesis of GI ulceration is poorly understood, despite intensive investigative work during the last few decades. By implication, etiologic pre- vention and treatment, the primary goal in medical practice, is rarely possible with GI ulceration. Even if the cause is known, e.g., with the GI side effects on non-steroidal anti- inflammatory drugs (NSAID) or Helicobacter pylori injec- tion, the culprit can rarely be eliminated because of the medical need of the beneficial effect of NSAID, or the contribution of antibiotic therapy to ulcer healing.
As a next option, the pathogenesis-based prophylaxis and therapy may be attempted in any stage of development of mucosal injury, repair and healing. Since the recurrence of gastroduodenal ulcers after treatment with antacids or histamine H2 receptor antagonists is unusually high, the quality of ulcer healing is a major concern (1,2). Direct treatment of ulcer crater instead of, or in addition to indirectly modulating the primary or secondary secretory changes should be a major goal (Table I). With the recent availability of growth factors which exert direct ulcer healing properties that are about one million times more potent on molecular basis than the H2 antagonist cimetidine ( 3 3 , the direct treatment of ulcers in both the upper and lower GI tracts is now feasible. We review here the cellular and molecular mechanisms of GI mucosal repair and ulcer
Table I. Therapy of gastroduodenal ulcers
Indirect Neutralization of aggressive factors (i.e., pH, Hp) Decreased gastric acid and/or pepsin secretion Locally acting antiulcer drugs via endogenous mediators Direct Stimulation of ulcer healing by enhancing
angiogenesis granulation tissue re-epithelialization smooth muscle regeneration neural regeneration
healing, as well as our recent data on the role of growth factors and angiogenesis in the healing of experimental duo- denal and gastric ulcers, and chronic erosive gastritis.
THE CELLULAR MECHANISMS OF REPAIR AND HEALING
The cellular mechanisms of mucosal repair and ulcer healing depend on the depth of lesions, i.e., erosion versus ulcer (Table 11). The mucosal repair can be divided into restitution and regeneration.
Epithelial restitution refers to rapid cell migration (e.g., from surviving gastric mucous neck cells) to cover the super- ficial mucosal defect caused by intraluminal damaging agents (7). Restitution is part of the natural defense mechanisms. It cannot be directly stimulated by gastroprotective agents such as prostaglandins (PG) and sulfhydryls (SH), but if
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Table 11. Lesions and their repair in the GI-tract
Lesions Defect Repair ~
Erosion Epithelial/mucosal Epithelial restitution
Ulcer Mucosal and smooth Angiogenesis and regeneration
muscle Granulation tissue Connective tissue Smooth muscle Neural elements
these compounds maintain microvascular integrity and mucosal blood flow (&lo), the energy-dependent epithelial restitution will passively but rapidly ensue. Although res- titution has been extensively investigated only in epithelial cells, ultrastructural studies of gastric microvascular injury indicate that rapid cellular restitution may take place in vascular endothelial damage as well (11).
Regeneration refers to cell division or multiplication and may involve epithelial, endothelial and mesenchymal cells (Table 111). If the mucosal injury is superficial (e.g., erosion which does not reach the muscularis mucosae), the lost tissue is usually replaced by the original (e.g., epithelial) cells. If, however, the damage is extensive, in the form of deep ulcer, the lost tissue is first replaced by granulation tissue which consists of rapidly proliferating fibroblasts and capillaries, deposited collagen, and chronic inflammatory cells (e.g., lymphocytes, plasma cells). Eventually, the loose granu- lation tissue becomes a dense fibrous framework which pro- vides the basis for re-epithelization by restitution and regeneration. Certain growth factors (see below), on the other hand, directly stimulate cell-specific tissue regen- eration (e.g., smooth muscle) and neural regeneration, mini- mizing the role of granulation tissue and achieving complete restoration of gut architecture.
One of the key elements in granulation tissue production and ulcer healing is angiogenesis or neovascularization.
Table 111. Molecular and cellular basis of ulcer healing ~
Molecules Cells Tissue
Peptides EGF
(scar) TGF-t9
bFGF PDGF
Hormones Gastrin Somatostatin Angiosteroids
Others Polyamines Collagen
Constituent Epithelial Granulation tissue Endothelid Connective tissue
Fibroblasts Smooth muscle Neurons Inflammatory Neutrophils Lymphocytes Plasma cells Macrophages
Namely, poorly vascularized loose granulation tissue is not a good basis for re-epithelization, and we believe this could be one of the major reasons for poor quality of ulcer healing and recurrence. If, however, the ulcer crater is treated directly by angiogenic factors, the quality of ulcers healed should be superior to those healed spontaneously or after administration of antacids or antisecretory drugs.
THE MOLECULAR REGULATORS OF ULCER HEALING
The main endogenous regulators of cell regeneration and proliferation are polyamines, cytokines 0,- growth factors and certain hormones (e.g., steroids). Po,yamines (e.g., spermine, putrescine) stimulate the growth (e.g., DNA syn- thesis) and proliferation of virtually any cell type, while growth factors and hormones are usually group or cell specific. Among the growth factors, the most relevant are epidermal growth factor (EGF) and transforming growth factor-p (TGF-/3), which essentially uses the common EGF receptor as well as basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) (Table 111).
EGF is a 53 aminoacid polypeptide which stimulates mess- enger RNA, DNA and protein synthesis mainly in epithelial cells (12,13). It stimulates keratinocyte division in vitro and epidermal regeneration in vivo. Parental administration decreases the secretion of gastric acid and stimulates the DNA synthesis of the rat gastric mucosa (14,15). Later, it was shown that this peptide is produced by the Brunner’s glands of the duodenum (16,17), and it can be found not only in the salivary glands but also in the duodenal juice. The concentration of EGF in the saliva of patients with duodenal ulcer is lower compared to the normal healthy controls (18).
The bFGF family consists of at least seven structurally related polypeptides, of which bFGF and acidic FGF are the best characterized. bFGF is an 18 kDa polypeptide which was first isolated from brain as a fibroblast stimulator (19); it turned out to be identical to the most potent angiogenic stimulator (20). Indeed, it is a direct mitogen for vascular endothelial cells, fibroblasts, smooth muscle cells, certain epithelial cells and neural cells. bFGF has a high affinity for heparin and proteoglycans, which are components of the extracellular matrix. Its strong affinity to heparin is used to purify this angiogenic peptide. Recently, different molecular forms of bFGF were identified: a low molecular weight form (18 kDa) which consists of 140-154 amino acids and several higher molecular weight forms (21, 22,25 kDa) containing a specific aminoterminal characteristic of the nuclear proteins
PDGF was originally described as a product of platelets, but it is also synthesized and secreted by activated macro- phages. This polypeptide is a potent mitogen for fibroplasts, osteoblasts, arterial smooth muscle cells and glial cells. It consists of two disulfide-linked polypeptides: A chain
(2 1-25).
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Mechanisms of UIcer Healing 5
(14 kDa) and B chain (17 kDa) sharing approximately 60% sequence homology (26). The biological action of PDGF is initiated at the cellular level by binding to a high affinity receptor and stimulation of tyrosine-specific kinase capable of phosphorylation of tyrosine residues (27).
Pretreatment of rats with polyamines or EGF resulted in an SH-dependent acute gastroprotection, while parenteral administration of EGF also accelerated the healing of chronic cysteamine-induced duodenal ulcers (28-30). The antiulcer effect of EGF was ascribed to inhibition of gastric acid secretion and stimulation of epithelial cell proliferation (29). Indeed, EGF is virtually specific for epithelial cells, with very little if any influence on fibroblast proliferation and angiogenesis.
As a follow-up to our focus on vascular factors in mucosal injury, protection and healing, we have been testing growth factors and angiogenic steroids which stimulate angiogenesis and/or granulation tissue production. We now briefly review our recent results with bFGF, which is the most potent angiogenic endothelial mitogen but also stimulates the growth of other cell types and enhances the production of granulation tissue. These studies with animal models of chronic duodenal ulcers and gastritis have been performed in collaboration with Drs J. Folkman, H. Satoh and P. Vattay (3-6).
ULCER HEALING BY GROWTH FACTORS ALONE AND IN COMBINATION WITH SUCRALFATE
Growth factors may not only have a physiologic role in ulcer healing, they could also be new pharmacologic agents directly stimulating the healing of necrotic and inflammatory lesions in the GI tract. Because of the localization of EGF receptors on the basolateral side of epithelial cells, EGF can be administered only parenterally. Indeed, twice a week intravenous injections of EGF accelerated the healing of gastric ulcers in patients (31). bFGF, especially its acid resistant mutein bFGF-CS23, and PDGF, which is acid- resistant in its natural form, can be given per 0s both in animal models of GI ulcers and patients.
A helpful tool in investigating the pharmacologic role of growth factors is the locally acting antiulcer drug sucralfate. During the last few years we have been testing the hypothesis that vascular factors are important in the mechanisms of acute and chronic actions of sucralfate (32, 33). We found that in subcutaneously (s.c.) implanted sponges sucralfate (Marion Merrel Dow) stimulates angiogenesis and granu- lation tissue production in the rat (33). Subsequent in vitro studies revealed that sucralfate and its water soluble com- ponent sucrose octasulfate, which structurally resemble hep- arin, bind bFGF more avidly than heparin (34). In rats with cysteamine-induced chronic duodenal ulcers, per 0s administration of sucralfate elevated local levels of bFGF in the ulcer crater. We thus postulate that the locally accumu- lated angiogenic bFGF may contribute to the ulcer healing
properties of sucralfate, and this peptide might be one of the key endogenous mediators of the pharmacologic actions of sucralfate (32,34).
CHRONIC DUODENAL ULCERS
In these experiments the modified model of cysteamine- induced duodenal ulcers was used (30, 35). Sucralfate was tested as a positive control at 50 mg/100 g twice daily, that is, an effective antiulcer dose in rats, and at an ineffective dose of 10 mg/100 g alone and in combination with bFGF- wild at 50 ng/100 g. Briefly, on the third day after induction of duodenal ulcer, the rats were randomized into groups treated with either vehicle or sucralfate and/or bFGF by gavage twice daily for 3 weeks. Additional groups of rats received only bFGF-wild at 50 ng/100 g, which is a partially effective antiulcer dose of this growth factor based on our previous dose-response studies. When the combination was used, sucralfate and bFGF solutions were mixed on the day of the administration. Autopsy was performed on the 21st day, when the sue of the duodenal ulcer was evaluated by measuring the diagonals of the ulcer crater in millimeters and subsequently by stereomicroscopic planimetry.
The preliminary results indicate (Fig. 1) that indeed suc- ralfate at 10mg was not effective, as predicted, while the drug at 50 mg/ and bFGF at 50 ng/100 g alone accelerated the healing of experimental chronic duodenal ulcers. How- ever, the combination of low doses of sucralfate + bFGF was also effective, since sucralfate at 10 mg + bFGF was significantly better than sucralfate 10 mg alone.
Thus, bFGF alone but especially after combination with sucralfate markedly accelerated the healing of experimental chronic duodenal ulcers. The acid-resistant bFGF-CS23 was also tested in the model of acetic-acid induced chronic gastric ulcer healing. Although the doses of the peptide had to be elevated (5 ) , it nevertheless accelerated the healing of experimental chronic gastric ulcers as well. Furthermore, daily injections of neutralizing antibodies against bFGF delayed the healing of both chronic gastric and duodenal ulcers (36,37). Hence, bFGF may play a role not only in the new pharmacology of ulcer disease, but endogenous bFGF the like natural EGF may have a physiologic role in ulcer healing.
CHRONIC GASTRITIS
In certain countries, e.g., Japan, gastric ulcer and gastritis are more prevalent than duodenal ulcer disease. Unfor- tunately, in contrast to the plethora of models of acute gastric mucosal injury and the few animal models of chronic gastric and duodenal ulcers, virtually no rat model of chronic diffuse erosive gastritis existed until recently. We found, as a follow-up to our investigations (38) on the gastroprotective role of SH compounds, that ingestion of low concentrations of SH alkylators (e.g., 0.1% iodoacetamide) in drinking
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Controls Suor. 1Omg Suor. 60mg bFQF-w Suor. lOmg + bFQF-w
Fig. 1. Effect of natural-bFGF and sucralfate on cysteamine-induced duodenal ulcer in rats after 3 weeks’ treatment.
water induced severe, diffuse acute and chronic erosive gastritits in rats (6, 39). The lesions in the mucosa of the glandular stomach initially contain acute inflammatory cells such as leukocytes, mostly replaced by lymphocytes, plasma cells and macrophages in the chronic stage of gastritis, which resembles the human alcoholic gastritis. We used this new animal model of gastritis to investigate the additive or syn- ergistic therapeutic interaction of bFGF and sucralfate
In these experiments, after induction of gastritis by the SH alkylator 0.1% iodoacetamide in drinking water for 1 week, 60 rats were randomized to receive: (a) native bFGF- w (25 ng/100 g); (b) a site-specific mutated acid stable bFGF- CS23 (25 ng/lOO g); (c) sucralfate in low dose ( 5 mg/100 g), or (d) combinations of these agents by gavage twice daily. Untreated rats received vehicle only. All the rats except normal controls remained on drinking water containing iodoacetamide for 1 week more. At autopsy on the 14th day, macroscopic and histologic involvement of gastric glandular mucosa was quantified, and wet and dry stomach weights were obtained.
The results (Table IV) demonstrate that only acid-resistant bFGF was effective (p < 0.05). Neither native bFGF-w nor sucralfate had any effect at low dose on chronic gastritis. Both native bFGF and the acid-resistant mutein in com- bination with sucralfate at low dose were more efficient in the repair of mucosal injury and significantly more effective than either of these agents alone (p < 0.001). Thus, bFGF and sucralfate may act synergistically in healing chronic erosive gastritis.
(6739).
MECHANISTIC CONSIDERATIONS
The mechanism of potent ulcer healing by bFGF alone and in combination with sucralfate is poorly understood, although a few possible pathways are emerging on the basis of intensive research work.
The results of several studies indicate that endogenous peptide growth factors are involved in the natural ulcer healing. Such an implication was already inescapable from the above discussed rat ulcer model studies, in which daily injections of neutralizing anti-bFGF monoclonal antibodies delayed the healing of experimental gastric and duodenal ulcers (36,37). Furthermore, the elevated concentration on bFGF in the ulcer crater of sucralfate-treated rats was also a strong implication for a luminal role of bFGF (34).
Our more recent biochemical and immunohistochemical studies also imply a role for endogenous bFGF in ulcer healing (41,42). Namely, Western blot analysis of rat duo- denal mucosa revealed a rapid depletion of mucosal cyto- plasmic 18 kDa bFGF and increased synthesis of nuclear 21- 25 kDa bFGF in cysteamine-induced duodenal ulceration in rats. At 12 h we found a 2-3-fold increase in the nuclear forms of bFGF with a 50% decrease in the cytoplasmic form, while 48 h after administration of cysteamine the samples showed a trend toward the pattern seen in the control rats. Preliminary experiments using Western blot analysis demon- strated that sucralfate prevents bFGF depletion induced by cysteamine.
Endogenous bFGF was also visualized by immunostaining techniques in the normal rat duodenum. After the adminis-
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Mechanisms of Ulcer Healing I
Table IV. Effect of oral administration of bFGF and sucralfate on the chronic gastritis induced by iodoacetamide in rats
Wet weight of stomach Groups Treatment Chronic gastritis (g/100 g)
1 Control Absent 0.66 f 0.01 2 Vehicle Extensive 0.94 f 0.01 3 Sucralfate (5 mg/IOO g) Extensive 0.82 f 0.03 4 bFGF-CS23 (25 ng) Mild/healed 0.77 ? 0.02** 5 bFGF-wild (25 ng) Extensive 0.82 f 0.02 6 bFGF-wild + sucralfate Mild/healed 0.70 * 0.02* 7 bFGF-CS23 + sucralfate Hardly detectable/healed 0.64 * 0.02*
~
* p < 0.001 versus vehicle; * * p < 0.005 versus vehicle. Rats of groups 2-7 ingested 0.1% iodoacetamide to induce gastritis. Reproduced with permission from Szabo et al. Role of bFGF and angiogenesis in ulcer healing and the treatment of gastritis. In:
Domschke W, Konturek SJ, editors. The stomach: physiology, pathophysiology and treatment. Berlin Heidelberg, New York, London, Paris, Tokyo, Hong Kong, Barcelona, Budapest: Springer-Verlag, 1993:193-7.
tration of the duodenal ulcerogen cysteamine, bFGF immunoreactivity was time-dependently reduced in the mucosa, submucosa and the muscularis propria.
Other recent new studies have revealed an increased expression of EGF receptors in the gastric mucosa in the healing of ethanol-induced gastric mucosal lesions in rats (43), and the gastric mucosa of gastric ulcer patients treated with omeprazole (44).
Since we recently found a potentiation of ulcer healing by sucralfate with a parallel treatment of rats with bFGF, we examined whether bFGF could potentiate the effect of suc- ralfate on angiogenesis, which is a key element in ulcer healing. Six rounded sterile sponges (8x3 mm), which con- tained vehicle, sucralfate and/or bFGF (added in 100 pl to the sponges before implantation) were placed under the abdominal skin of Sprague-Dawley female rats (45). The rats were killed on the 7th day, the sponges removed and fixed in 10% buffered formalin, cut and stained with hema- toxylin and eosin and for the endothelial specific factor VIII. The number of newly developed blood vessels was counted in the ingrowing part of the sponges in at least seven areas where dense vessels (no./mm2) were seen: vehicle 40.0 * 4.3, sucralfate (5 mg) 59.4 ? 7.0*, bFGF (25 ng) 162.2 2 lo**(++), sucralfate + bFGF (25 ng) 281.1 * 12.2**(++), * = p < 0.05, ** = p < 0.001 versus vehicle, (++) = 0.001 versus sucralfate.
We concluded that sucralfate alone accelerated angio- genesis, which was significantly enhanced by combination with an ineffective dose of bFGF. The same dose of suc- ralfate combined with an angiogenic dose of bFGF also resulted in synergistic stimulation (e.g., more than 5-fold) of angiogenesis. We postulate that this synergistic enhance- ment of angiogenesis and granulation tissue production by sucralfate and bFGF may have a role in the mechanism of ulcer healing by sucralfate.
technical contributions to these studies. These investigations were supported in part by grants from Chugai Pharma- ceutical Co., Ltd, and Takeda Chemical Industries.
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