Original Paper
Digestion 1992;53:17-27
Leo R. Fitzpatrick Anna Jakubom ka Gregory E. Martin Marjorie Davis Michael C. Jaye Craig A. Dionne
Acidic Fibroblast Growth Factor Accelerates the Healing of Acetic-Acid-Induced Gastric Ulcers in Rats
Pharmacology and Molecular Biology Departments, Rhône-Poulenc Rorer Central Research, Collegeville, Pa., USA
Growth factors like epidermal growth fac tor, platelet-derived growth factor, transform ing growth factor and fibroblast growth fac tors (FGFs) are potential therapeutic agents for wound healing [1], FGF is present in a wide variety of tissues [1-3] and is found in macrophages [4] as well as endothelial cells
Received: January 6, 1992 Received in revised form: June 11,1992
[2], At very low concentrations, FGF can stimulate the angiogenic process [1-4], which is thought to play an important role in wound healing. Increasing experimental evidence supports a role for FGF in the healing of gastrointesti nal ulcers. Nakamura et al. [5] demonstrated
Leo R. Fitzpatrick, PhD Pharmacology and Molecular Biology Departments Rhône-Poulenc Rorer Central Research 500 Areola Road, PO Box 1200 Collegeville, PA 19426-0107 (USA)
©1992 S. Karger AG, Basel 0012-2823/92/ 0532-0017S2.75/0
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Keywords Acidic fibroblast growth factor Gastric ulcer Angiogenesis Rats
Abstract Acidic fibroblast growth factor (aFGF) was evaluated for the healing of acetic-acid-induced gastric ulcers in rats. The effect of aFGF on angiogenesis in the gastric ulcer bed was deter mined by the carmine dye infusion method, while its effect on gastric acid secretion was assessed in chronic gastric fistula rats. Oral treatment with aFGF, in the presence of heparin, reduced (ED5o value = 30.2 pg/kg/day) the acetic-acid-induced gastric ulcer area, when assessed 1 week later. aFGF was about 1.333-fold more potent than famotidine for healing such ulcers. At a dose of 200 pg/kg/day, aFGF increased the car mine density 3-fold and correspondingly reduced (80%) the gastric ulcer area. Thus, the ulcer healing effect of this agent involves angiogenesis in the gastric ulcer bed. This effect of aFGF appears to be unrelated to an inhibition of gastric acid secretion, as it was ineffective in chronic gastric fistula rats. In summary, oral aFGF significantly accelerates the healing of experimental gastric ulcers in rats. It may be a potent and effective agent for the treatment of peptic ulcers in humans.
Materials and Methods Acetic-Acid-Induced Gastric Ulcers Female Sprague-Dawley rats (Taconic Farms, Ger mantown, N.Y.. USA), weighing 180-200 g. were used in these studies. Animals were fasted for 24 h and anes thetized with ether prior to gastric ulcer induction. Gastric ulcers were produced according to the method described by Konturek et al. [ 11]. with slight modifica tion. Briefly. 50 pi of 100% acetic acid were applied through a plastic mold of 4 mm inner diameter to the serosal surface of the rat stomach (anterior corpus area) for 30 s. Subsequently, this area was rinsed with
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0.9% saline, and the abdomen was closed with suture and surgical staples. Starting the day after ulcer induc tion. rats were maintained on the dietary and treat ment regimen originally described by Ito et al. [ 12]. which was used previously in our laboratory [ 13], Ani mals were fed twice daily, for I h (6:30-7:30 a.m. and 2:00-3:00 p.m.). The control vehicles (0.5% methylcellulose, phosphate-buffered saline) or drugs (famoti dine. 20 mg/kg. heparin. 300 pg/kg. Maalox. 425 mg/kg. aFGF. 1-100 pg/kg and aFGF. 100 pg/kg + Maalox, 425 mg/kg) were then administered orally. 30 min after each feeding period, at a dose volume of 10 ml/kg. aFG F was always kept cool on ice prior to oral administration by gavage and was given in the presence of heparin (5 U/ml, 300 pg/kg). Treatment began the day after ulcer induction with acetic acid, and rats were sacrificed on day 7 except in the initial time course studies. The stomachs were removed, opened along the greater curvature and pinned flat for the determination of an ulcer index as well as the severity of gastric ulceration. This was done with the aid of a magnifying glass (X2.5). The gastric ulcer severity was evaluated according to the following scale: 0 = no visible damage: 1 = erosion; 2 = penetrating ulcer: 3 = perforating ulcer, and 4 = severe, perforating ulcer. A severe, perforating ulcer (grade 4) was distin guished by the following features: large size, presence of inflammation in the ulcerated area and severe/tight adhesions of the liver to the gastric ulcer. The ulcer index was determined as the product of ulcer length X width (expressed in mm2). Angiogenesis in Acetic-Acid-Induced Gastric Ulcers Angiogenesis in the experimental gastric ulcer bed of aFGF- or vehicle-treated animals was measured on day 7 after ulcer induction by the carmine dye infusion method of Hase et al. [ 14], Briefly, rats were injected through the tail vein with 2 g/kgofa 10% carmine sus pension containing 5% gelatin. After the injection of carmine, animals were sacrificed w ith carbon dioxide and cooled at 4 °C for 2 h. Upon analyzing the gastriculcer for size and severity, it was excised from the sur rounding normal tissue along its margin. Any adhering liver w'as scraped from the gastric tissue by blunt abla tion. The resected ulcer was weighed and placed in 4 ml of 3 N NaOH for 30 min at 30 °C. After adding 1 ml of ION HC1. the solution w'as filtered. Subse quently, absorbance of the filtrate at 530 nm was mea sured with a Beckman model 25 spectrophotometer (Beckman Instruments, Cedar Grove, N.J. USA). To tal carmine content of the samples was determined by regression analysis from a standard calibration curve
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that FGF binding sites are present on myofi broblasts in the granulation tissue of experi mental gastric ulcers. More recently, other investigators have shown the presence of acidic FGF (aFGF) and the FGF receptor, fig, at the wound margin of acetic-acid-induced gastric ulcers in rats [6], Moreover, oral treat ment with an acid-resistant mutein of basic FGF (TGP-580) accelerated the healing of both acetic-acid-induccd gastric ulcers [7] and cysteamine-induced duodenal ulcers [8] in rats. The duodenal ulcer healing effect was linked to increased angiogenesis [8], Finally. Tarnawski et al. [9] have demonstrated that aFGF stimulates the restoration of the rat gas tric mucosa following ethanol administration. To date, however, no studies have been re ported on the efficacy of aFGF for healing chronic experimental gastric ulcers. There fore, the purposes of the present study were: (1) to assess the capability of human recombi nant aFGF [10], when administered orally, to heal acetic-acid-induced gastric ulcers in rats: (2) to compare the healing effect of aFGF to that of standard antiulcer agents (famotidine, Maalox): (3) to determine whether combined aFGF and Maalox treatment would enhance the healing of experimental gastric ulcers, and (4) to assess whether orally administered aFGF stimulated angiogenesis in the aceticacid-induced gastric ulcer bed.
Gastric A d d Secretion in Rats Sprague-Dawley rats (300-400 g) were fitted with chronically implanted gastric cannulas, as described previously [ 16], Animals were not used for 2 weeks fol lowing surgery. Subsequently, these rats were acclima tized to the experimental procedure for a 2-week peri od. before being used in actual studies. The same 24 chronic gastric fistula rats were then used in basal and pentagastrin-stimulated acid secretion experiments. Rats were fasted for 24 h prior to their use in exper iments. In the basal acid secretion studies, the gastric cannula was opened, and the stomach was thoroughly rinsed. Subsequently, fluid was allowed to drain from the stomach for a 30-min period. The cannula was closed, and rats were dosed orally with the following compounds: ( I ) PBS + 0 .1% BSA (vehicle): (2) vehicle + heparin (300 pg/kg); (3) vehicle, heparin + aFGF (100 pg/kg); (4) vehicle, heparin + Maalox (425 mg/kg): (5) vehicle, heparin + aFG F (100 pg/kg) + Maalox (425 mg/kg). and (6) vehicle, heparin + famotidine (20 mg/kg). The cannula was kept closed for 1 h to allow time for drug absorption. During this time period, ani mals were given subcutaneously 5 ml of warm saline to account for subsequent fluid loss upon opening the cannula. Following this 60-ntin period, the cannula was opened, and gastric juice collections were made continuously 30. 60. 120 and 180 min later. These time points correspond to 90. 120. 180 and 240 min after compound administration. The pentagastrin-stimulated acid secretion studies were done as described above with only slight modifi cation. One hour after oral administration of the com pounds listed above, the cannula was opened. Subse quently. animals were immediately injected subcuta neously with pentagastrin (10 pg/kg) or vehicle (in one group of PBS-BSA pretreated rats). Gastric collections were made 30. 60 and 120 min later, as described above. The gastric juice was collected in a graduated test tube and measured to the nearest 0 .1 ml. Acid concen tration was determined by titration of gastric juice with 0.1 N N aO H using a Brinkmann 702 SM titrator (Brinkmann Instruments, Westbury, N.Y., USA). To tal acid output w'as calculated as the product of volume X acid concentration and is expressed in micro equivalents.
Drugs Famotidine, carmine, pentagastrin and gelatin (from bovine skin) were obtained from Sigma (St. Louis, Mo., USA). Maalox was provided by RhônePoulenc Rorer (Fort Washington. Pa.. USA). The dose of Maalox refers to the active ingredients per 5 ml (225 mg aluminum hydroxide and 200 mg magnesium hy droxide). Heparin (from beef lung) was obtained via U pjohn (Kalamazoo, Mich.. USA). Human recombi nant aFGF was synthesized by the method of Jaye et al. [10] and provided by Dr. Mark Ravera (Molecular Biology Department. Rhône-Poulenc Rorer Central Research, King of Prussia. Pa., USA). Statistical Analysis The effects of drugs on the gastric ulcer index and carmine density were compared to vehicle-treated con trols by Student’s t test for unpaired data. Ulcer sever ity scores were evaluated statistically by the Wilcoxon rank sum test. Gastric acid secretion data were evalu ated by one-way analysis of variance and Duncan’s multiple range test. For these methods, statistical sig nificance compared to vehicle treatment is reported at the p < 0.05 confidence level. The ED50 value and 95% confidence limits to the dose-response curve of aFGF for gastric ulcer healing w'as obtained by inverse regression analysis.
Results Time Course o f Acetic-Acid-Induced Gastric Ulcers
Initially, the time course of acetic-acidinduced gastric ulceration in rats was evaluat ed. As shown in Figure 1, the ulcerated area (15.4 mm2), 5 h after acetic acid administra tion. conforms nicely to the diameter (4 mm) of the plastic application tube used in these experiments. There was no change in the gas tric ulcer index for the next 5 days after the induction of ulceration. On day 5, the ulcer severity score was 2.8, indicating that the gas tric ulcers were generally perforating in na ture. On day 7, there was only a slight reduc tion in the ulcer index to a value of 12.8 mm2 (Fig. 1). The mean ulcer severity score (n = 4 experiments) on this day was 2.6 (i.e. both
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for carmine (25-200 gg/ml). Angiogenesis in the gas tric ulcer was expressed as the carmine density [total carmine content (pg) ulcer index (mm2)] as suggested byTsuchida etal. [15].
Table 1. Effect of various drugs on acetic-acid-induced gastric ulcers in rats
Drug (dose)
Ulcer severity
Ulcer index, mm2
Vehicle Famotidine (40 mg/kg/day) Maalox (850 mg/kg/day) Heparin (600 gg/kg/day)
3.0 ±0.2 1.8 ±0.3* 2.2 ±0.3* 3.1 ±0.3
13.9 ±2.7 6.5 ±2.1** 7.9± 1.7 13.3 ±2.6
Drugs or vehicle (0.5% methylcellulose) were given orally twice daily, starting on day 1 after the induction of gastric ulceration. Animals were sacrificed on day 7. n = 9-12 per treatment group. * p < 0.05 vs. vehicle by Wilcoxon rank sum test. ** p < 0.05 vs. vehicle by Student’s t test.
Table 2. Effect of aFGF ± Maalox on acctic-acid-induccd gastric ulcers in rats
Drug (dose)
Ulcer severity
Ulcer index mm2
Carmine density gg/ntm2
Vehicle aFG F (200 gg/kg/day) aFG F + Maalox (200 gg/kg/day + 850 mg/kg/day)
2.6 ± 0.2 1.1 ± 0. 1* 1.1 ± 0. 1*
12.2 ± 1.1 2.5 ±0.6** 2.4 ±0.5**
17.4 ±5.4 52.2 ±17.9** 35.6 ±7.9**
Drugs or vehicle (PBS + 0.1 % BSA) were given orally twice daily, star ting on day 1 after induction of gastric ulceration. Animals were sacrificed on day 7. Angiogenesis was determined in the gastric ulcer base by the carmine dye infusion method, n = 12-18 per treatment group. * p < 0.05 vs. vehicle by Wilcoxon rank sum test, ** p < 0.05 vs. vehicle by Student's t test.
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Effect o f Drugs on the Healing o f Acetic-Acid-Induced Gastric Ulcers
Oral treatment (twice daily) with the Hy receptor antagonist, famotidine, significantly reduced (53%) the gastric ulcer index (ta ble 1). Maalox also reduced (43%) the gastric ulcer index, however, statistical significance compared to vehicle treatment was not at tained. Heparin, when given orally, had vir tually no effect on the size of acetic-acidinduced gastric ulcers. Similar effects were observed for these drugs with regard to the
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penetrating/perforating ulceration was pres ent). However, by 10 days after acetic acid administration, there was a singificant attenu ation in the ulcer index to 4.8 mm2, indicating that significant endogenous healing had oc curred. On day 10, the ulcer severity score was 1.9. Thus, at this time, gastric ulcers were mainly penetrating in nature. 15 days after the induction of ulceration, the gastric ulcers were usually small erosions (mean ulcer sever ity = 1.1), which had almost completely healed (mean ulcer index = 2.9 mm2).
Time, days
aFG F aFG F aFG F Famotidine 2 ag/kg/day 20 ng/kgfaay 200 (ig/kg/day 40 mg/kg/day
1
2a
severity of acetic-acid-induced gastric ulcers. Both famotidine and Maalox significantly re duced (40 and 27%. respectively) the gastric ulcer severity, while heparin was completely ineffective. We next assessed the efficacy of oral aFGF treatment, either alone or in combination with Maalox, for healing acetic-acid-induced gastric ulcers. As shown in table 2, aFGF treatment (200 pg/kg/day) resulted in dra matic attenuations of both the ulcer index (by 80%) and ulcer severity (by 58%). When ad
aFGFiEDso value = 30.2 jig/kg/day
aFG F aFG F aFG F Famotidine 2 ng/kg/day 20 ng/kg/day 200 ng/kg/day 40 mg/kg/day
2b
ministered at doses of 20 or 2 pg/kg/day, aFGF reduced the gastric ulcer index by 49 and 0%. respectively (fig. 2a). The calculated ED50 value and 95% confidence limits of aFGF for reducing the acetic-acid-induced gastric ulcer index in rats were 30.2 (13. 70) pg/kg/day. A similar trend was evident (fig. 2b) with regard to the effectiveness of aFGF for reducing the gastric ulcer severity. As illustrated in figure 3, aFGF (2-200 pg/kg/day) increased the carmine density in a dose-related fashion. When administered
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Fig. 1. Time course of acetic-acid-induced gastric ulcer development in rats. Acetic acid (100%. 50 pi) was applied to the serosal surface of the rat stomach. The area of gastric ulceration (ulcer index) was deter mined 5 h later (day 0) and subsequently 2. 5. 7 , 10 and 15 days after application of acetic acid, n = 3-6 per time point. Fig. 2. Effect of aFGF or famotidine on the gastric ulcer index (area in mm2. a) and gastric ulcer severity (0—4 scale, b) induced by acetic acid. Drugs were administered at the doses indicated for 6 days, and rats were sacrificed on day 7 after the induction of ulcer ation. Data are expressed as a percent of the vehicle control values. Mean (n = 4 experiments) vehicle ulcer index = 13.9 mm2, while mean ulcer severity = 2.60. n = 9 - 16 per treatment group. * p < 0.05 vs. vehicle by t test (a), or Wilcoxon rank sum test (b).
orally at a dose of 200 pg/kg/day. the carmine density was significantly enhanced (3-fold) by aFGF. Finally, as shown in figure 4, with increasing doses of aFGF there was a very good inverse correlation (r = -0.993) between enhanced carmine density in the gastric ulcer base and the corresponding decrease in the gastric ulcer area. Interestingly, combined treatment with aFGF plus Maalox did not result in enhanced ulcer healing or increased angiogenesis (carmine density) in the gastric ulcer bed, compared to treatment with aFGF alone (table 2). Effect o f Drugs on Gastric Acid Secretion in Rats
As shown in table 3, the total (3-hour) gas tric acid output in vehicle-treated rats was 267.1 pEq. Heparin did not significantly af fect basal acid secretion in chronic gastric fis tula rats. In contrast, famotidine (20 mg/kg) almost completely inhibited gastric acid se-
Fig.3. Effect of aFGF on angiogenesis in aceticacid-induced gastric ulcers. On day 7 after the induc tion of ulceration, angiogenesis (carmine density) was determined in the gastric ulcer bed of rats by the car mine dye method. Data are expressed as a percent of the vehicle-treated control values. Mean (n = 3 experi ments) vehicle carmine density = 26.7 pg carmine/mm2. n = 9-16 per treatment group. * p < 0.05 vs. vehicle by Student’s t test.
Fig. 4. Correlation between angiogenesis (carmine density) and the healing of acetic-acid-induced gastric ulcers in rats on day 7. Values for each dose of aFGF arc expressed as a percent of vehicle-treated controls (n = 3 experiments) and are the means of 9-16 observations. The slope (m), y intercept (b) and correlation coefficient (r) were determined by inverse linear regression analysis.
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Carm ine density. % control
Table 3. Effect of drugs on gastric acid secretion in chronic gastric fistula rats
T reatment group
1 2 3 4 5 6
Vehicle Heparin aFGF Maalox aFGF + Maalox Famotidine
Total acid output, pEq 2h
3h
4h
total
73.6+15.7 113.5 ±27.4 78.3 ±21.8 46.5 ±22.4* 41.4 ± 19.2* 0.2 ± 0. 1*
73.8 ±18.7 129.9 ±27.1 71.4 ±14.3* 55.3 ±21.9* 45.5 ±14.9* 1.4 ± 0.8*
117.4 ±29.4 102.1 ±27.1 109.3 ±27.1 91.1 ±28.9 46.0 ± 15.1* 1.5 ± 1.1*
267.1 ±48.4 345.5 ±71.2 259.3 ±42.9 193.0 ±58.6 132.1 ±44.3* 3.1 ±1.8*
Animals were pretreated orally with vehicle (0.5% methylcellulose), heparin (300 pg/kg) famotidine (20 mg/kg) or aFGF (100 pg/kg) ± Maalox (425 mg/kg). Rats in groups 3-6 also received heparin. Total gastric output was determined at the times indicated following drug administration, n = 6-8 per treatment group. * p < 0.05 vs. group 2 by Anova and Duncan’s multiple range test.
Table 4. Effect of drugs on pentagastrin-stimulatcd gastric acid secretion in chronic gastric
fistula rats Treatment group
1 2 3 4 5 6 7
No pentagastrin Pentagastrin Heparin aFGF Maalox aFGF + Maalox Famotidine
Total acid output. pEq 2h
3h
total
91.7 ± 19.7 315.7 ± 55.9** 339.0 ±69.9 253.8 ± 18.7 143.7 ±55.9* 12.1 ±5.1* 0.6 ±0.4*
99.3 ±29.2 89.8 ± 15.5 72.4 ± 15.9 84.8 ±5.9 30.4 ± 14.3 3.0±2.0* 0 ± 0*
190.9 ±45.1 405.5 ±62.7** 411.5 ± 80.5 338.3 ±21.1 174.1 ±64.3* 15.1 ± 6. 1* 0.6 ±0.4*
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Animals were pretreated orally with 0.5% methylcellulose (groups 1 and 2), heparin (300 pg/kg). famotidine (20 mg/kg) or aFG F ( 100 pg/kg) ± Maalox (425 mg/kg). Drugs were given I h prior to the subcutaneous injection of 2% DMSO vehicle (group l ) or 10 pg/kg pentagastrin (groups 2-7). Rats in groups 4-7 also received heparin. The total gastric acid output was determined at the limes indicated following oral drug administration, n = 6-7 per treatment group. * p < 0.05 vs. group 3 or ** p < 0.05 vs. group 1 by Anova and Duncan's multiple range test.
Discussion Acetic-acid-induced gastric ulceration in rats is the current prototype gastric ulcer mod el, and it has been used by many investigators to study the efficacy of antiulcer drugs [7, 1115], Tarnawski et al. [17, 18] found that such chronic experimental ulcers are similar gross
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ly, histologically and ultrastructurally to hu man gastric ulcers. In this study, we initially examined the time course of ulcers induced by topical application of acetic acid to the serosal gastric surface of rats. Our data (fig. 1) indicate a significant decrease in the gastric ulcer index (i.e. area) between weeks 1 and 2 after the induction of ulceration. These results are similar to those reported previously by Konturek et al. [II]. In our experiments, we utilized the dietary regimen described by Ito et al. [12], in which rats were maintained on a limited food intake (i.e. fed twice daily). With such a restricted dietary regimen, the area of gastric ulceration would be more exposed to hydrochloric acid and pepsin, which is more akin to the situa tion that exists in humans [12], Therefore, this regimen makes the gastric ulcer area more sensitive to the action of antisecretory agents like Hi receptor antagonists [12. 13], In fact, we previously found that ranitidine signifi cantly reduced (59%) the acetic-acid-induced gastric ulcer area on day 5, when rats were maintained on the limited food intake dietary regimen [13], In the present study, we exam ined the efficacy of another Hi receptor antag onist (famotidine) for healing acetie-acid-induced gastric ulcers. Twice daily treatment with a dose of famotidine (20 mg/kg) that decreased basal acid secretion in gastric fis tula rats by 99% (table 3) significantly re duced both the gastric ulcer area (53%) and severity (40%). Maalox (425 mg/kg X 2) also reduced these indices, but not to the extent as with famotidine treatment. In summary, these initial experiments established the effi cacy of two common antiulcer drugs for heal ing chronic gastric ulcers in rats. Although heparin had no effect on gastric ulcer healing (table 1) at the dose (300 pg/kg) we used, it was included in subsequent experi ments which assessed the efficacy of aFGF. This was done because heparin binds with
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crction. Maalox (425 mg/kg) significantly re duced basal acid secretion only during the ini tial two collection periods (i.e. 2 and 3 h after its administration). When administered in combination with aFGF, Maalox attenuated basal secretion over the entire duration of the study. However. Maalox reduced the total (3hour) acid output to approximately the same degree (44-62%), whether administered alone or in combination with aFGF. When administered by itself. aFGF (100 |ig/kg) did not consistently demonstrate any significant antisecretory activity in chronic gastric fistula rats (table 3). Pentagastrin (10 pg/kg, s.c.) increased acid secretion 3.4-fold in chronic gastric fistula rats, 1 h after its administration (see 2-hour column in table 4). This effect was transient in nature, as gastric acid output returned to the nontreated (group 1) level during the next hour. As was the case in the basal acid secre tion studies, neither heparin nor aFGF (25% reduction) significantly attenuated pentagastrin-stimulated acid secretion. Maalox re duced the initial phase of the pentagastrininduced secretory response by 57%. while famotidine completely abolished acid secre tion to a level below that found in nontreated rats. Combined treatment with aFGF plus Maalox also dramatically attenuated (96.4%) pentagastrin-stimulated gastric acid output, to a value of 12.1 pEq. suggesting a possible synergistic interaction between the two drugs.
acetic-acid-induced gastric ulcers, when Maa lox 70 was combined with another growth fac tor (epidermal growth factor). However. Kusstatscher et al. [20] have recently reported an additive effect for bFGF and cimetidine on chronic duodenal ulcer healing in rats. It is possible that the lack of enhanced ulcer heal ing in our study is due to the relatively short acid-neutralizing time of Maalox (table 3) in the fasted state. In this regard, our ulcer heal ing studies were conducted with rats main tained on a restricted feeding regimen. There fore, a basal (i.e. nonstimulated) gastric acid secretory state was predominantly present in these animals. Moreover, the results in table 3 also suggest that combined treatment with aFGF plus Maalox did not significantly en hance antisecretory activity compared to treatment with Maalox alone. Interestingly, however, combined administration of aFGF plus Maalox resulted in a significant enhance ment in antisecretory action against pentagastrin-stimulated acid secretion (table 4). Al though the mechanism/s of this apparent synergistic interaction remain to be deter mined, it is doubtful whether such an interac tion had physiologic significance in our ulcer healing studies because of the dietary regimen utilized. Finally, as suggested by other investi gators [20], it is also likely that combined treatment of aFGF with longer acting antise cretory' agents (H2 receptor antagonists, hy drogen-potassium ATPase inhibitors) could result in a significant enhancement of experi mental ulcer healing. Various studies [14, 15, 21] have utilized the carmine dye technique to quantify angio genesis. Previously. Hase et al. [14] confirmed histologically that injected carmine dye was distributed exclusively within the vascular lu men. Moreover, using this technique, these investigators demonstrated the appropriate antiangiogenic activity in the experimental gastric ulcer bed of rats treated with predniso
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high affinity to aFGF and protects this growth factor from acidic/proteolytic degradation [3]. Under this experimental condition, oral treatment with aFGF (200 pg/kg/day) signifi cantly decreased (58%) the gastric ulcer sever ity score from 2.6 in vehicle-treated rats to a value of 1.1 (table 2). aFGF also attenuated the gastric ulcer area from 12.2 mm2 in vehi cle-treated animals to 2.5 mm2 (80% reduc tion). To put this data in perspective, the gas tric ulcers in vehicle-treated animals on day 7 were typically penetrating/perforating in na ture, while in aFGF-lreated rats only small erosions were present. In addition, the ulcer index on day 7 in aFGF-treated rats was smaller than the value (2.9 mm2) obtained in nontreated animals on day 15 (fig. 1). Thus, aFGF decreased the severity and accelerated the healing time of acetic-acid-induced gastric ulcers in rats. Interestingly, the ulcer healing effect of aFGF appears to be unrelated to an inhibition of gastric acid secretion, as it was ineffective against either basal or pentagastrin-stimulated acid secretion in chronic gas tric fistula rats (table 3). The calculated ED50 value (fig. 2a) of aFGF for reducing the acetic-acid-induced gastric ulcer area is 30.2 pg/kg/day. Because famotidine (40 mg/kg/day) reduced the gas tric ulcer index by about 50% (table 1). aFGF is approximately 1,333-fold more potent than this H2 receptor antagonist for healing such experimental gastric ulcers. Our results re semble those reported previously for the acidresistant bFGF mutein. TGP-580. Satoh et al. [7] found that at a dose of 10 pg/kg it reduced a calculated gastric ulcer index by 55%, while cimetidine (100 mg/kg) was only moderately effective (43% reduction). In our study, animals treated with aFGF plus Maalox demonstrated the same degree of gastric ulcer healing as rats treated only with aFGF (table 2). Konturek et al. [19] also found no additive effect on the healing of
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omental adhesions, which could have in fluenced the ulcer healing process by enhanc ing angiogenesis. The capability of aFGF to increase angio genesis in the gastric ulcer bed (fig. 3) may improve the quality of ulcer healing and also might have an impact on ulcer recurrence [17], Indeed. Satoh et al. [25] recently re ported that bFGF prevented indomethacininduced gastric ulcer relapse in rats. An inves tigation of such potential actions for aFGF should be the focus of future studies. It should also be noted that Folkman et al. [26] have presented substantial experimental evidence suggesting that endogenous bFGF may be the primary factor involved in ulcer healing. Ac cording to their hypothesis, all commonly used antiulcer drugs (e.g. sucralfate, antacids. Hi receptor antagonists) prevent the degrada tion of endogenous bFGF by gastric acid. Thus, the angiogenic and other growth-pro moting activities of bFGF would be preserved for utilization in ulcer healing. Finally, Hannson and Norstrom [6] have recently proposed that endogenous aFGF may also play a role in ulcer healing. In summary, orally administered aFGF significantly accelerated the healing of aceticacid-induccd gastric ulcers in rats. It was > 1,000-fold more potent than famotidine for healing such ulcers. The ulcer healing effect of aFGF involves increased angiogenesis in the gastric ulcer base, but appears to be unrelated to an inhibition of gastric acid secretion. aFGF may be a potent and effective agent for the treatment of gastric ulcers in man.
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lone. A recent study from our laboratory also found that treatment with another corticoste roid. hydrocortisone, significantly decreased the carmine density in the gastric ulcer bed of rats [22], As a whole, these studies provide good evidence that the carmine dye method is an acceptable method for measuring angio genesis. Finally, Tsuchida et al. [15] demon strated a very good correlation between vas cular density in acetic-acid-induced gastric ul cers as measured by direct video image analy sis and carmine dye infusion. Therefore, as suggested by these investigators [ 15], we used the carmine density (pg carmine/ulcer index) as a measurement of angiogenesis in the gas tric ulcer bed. As shown in table 2, aFGF treatment (200 gg/kg/day) increased (3-fold) the carmine density (i.e. angiogenesis) in the gastric ulcer bed on day 7 after the initiation of ulceration. Moreover, the effect of aFGF on angiogenesis was dose related (fig. 3). With increasing doses of this growth factor, a very good inverse correlation (r = -0.993) was found between enhanced angiogenesis in the gastric ulcer bed and decreased ulcer size (fig. 4). Therefore, the stimulation of new mi crovessel formation by aFGF appears to en hance the ulcer healing process. Such a mech anism of action was also reported by Folkman et al. [8] for the healing of cysteamineinduced duodenal ulcers by TGP-580, and more recently by other investigators [23, 24] for the healing of acetic-acid-induced gastric ulcers by bFGF. In this regard. Konturek et al. [23] reported that either exogenous bFGF or omental attachment resulted in a similar in crease in experimental gastric ulcer healing in rats. Moreover, in both instances, there was a stimulation of angiogenesis in the acetic-acidinduced gastric ulcer bed. These data may also have relevance to our ulcer healing re sults. In our studies, experimental gastric ul cers were often penetrating/perforating in na ture. Such penetration of the ulcer resulted in
References 10 Jaye M. Burgess WH, Shaw AB. Drohan WN: Biological equivalence of natural bovine and recombinant alpha-endothelial cell growth fac tors. .1 Biol Chern 1987:262; 1612— 1617. 11 Konturek SJ. Stachura J. Radek T, Drodowicz D, Brzozowski T: Cytoprotective and ulcer healing proper ties of prostaglandin Ev. colloidal bismuth and sucralfate in rats. Di gestion 1987:38:103-117. 12 Ito M. Tsukahara T. Suzuki Y: Marked heali ng-promoting action of cimetidine on acetic acid-induced ulcer in rats with a limited food intaking-time. Jpn J Pharmacol 1986:41:260-262. 13 Fitzpatrick LR. Decktor DL: Gastroprotective and ulcer healing pro file o f the mast cell stabilizer quazolast in rats. Agents Actions 1991:33: 331-336. 14 Hase S. Nakazawa S. Tsukamoto Y, Segawa K: Effects of prednisolone and human epidermal growth factor on angiogenesis in granulation tis sue of gastric ulcer induced by acetic acid. Digestion 1989:38:135-142. 15 Tsuchida T. Tsukamoto Y. Segawa K, Goto H. Hase S: Effects of cimet idine and omeprazole on angiogene sis in granulation tissue of acetic acid-induced gastric ulcers in rats. Digestion 1990:47:8-14. 16 Decktor DL. Pendleton RG. Kellner AT. Davis MA: Acute effects of ran itidine. famotidine and omeprazole on plasma gastrin in the rat. J Phar macol Exp Thcr 1989:249:1-5. 17 Tarnawski A. Hollander D. Sta chura J. Krause WJ. Eltorai M. Dabros W'. Gergiev H: Vascular and microvascular changes - key factors in the development of acetic acidinduced gastric ulcer in rats. J Clin Gastroenterol 1990; 12:S 148-SI 57.
18 Tarnawski A, Stachura J. Krause WJ. Douglass TG, Gergley H: Qual ity of gastric ulcer healing: A new emerging concept. J Clin Gastroen terol 1991:13:S42-S4 7. 19 Konturek SJ. Brzozowski T. Drozdowicz D. Dembinski A. Nauerl C: Healing of chronic gastroduodenal ulcerations by antacids: Role of prostaglandins and epidermal growth factor. Dig Dis Sci 1990:35: 1121-1129. 20 Kusslatscher S. Folkman J. Nagy L. Szabo S: Additive effect of fibro blast growth factor (bFGF) and cimetidinc on chronic duodenal ulcer healing in rats. Gastroenterology 1992:102: A 104. 21 Kimura M. Amemiya K. YamadaT, Suzuki J: Quantitative method for measuring adjuvant-induced granu loma angiogenesis in insulin-treated diabetic mice. J Pharmacobiodyn 1986:9:442-446. 22 Fitzpatrick I.R. Pendley CE. Jakubowska A: Delayed healing of gas tric ulcers in rats by hydrocortisone and cysteamine. Gastroenterology 1992:102:A69. 23 Konturek SJ. Brzozowski T. Pawlik W', Stachura J: Omentum and bFGF in stimulation of healing of chronic gastric ulcerations. Digestion 1991: 49(suppl l):20. 24 Satoh H. Shino A. Sato F. Inatomi N, Kozai Y. Kato K. Szabo S. Folkman J: Role of endogenous and ex ogenous bFGF in the healing of gas tric ulcers in the rat. Gastroenterol ogy 1992; 102: A159. 25 Satoh H. Shino A. Murakami I, Asano S. Sato F. Inatomi N: New ani mal model for gastric ulcer relapse in the rat: bFGF may play a role in the prevention of ulcer relapse. Gas troenterology 1992:102: A 159. 26 Folkman J. Szabo S. Stovroff M. McNeil P. Shing Y: Experimental duodenal ulcer: Role of basic fibro blast growth factor (bFGF) in accel erated healing. Digestion 1991; 49(suppl l):3.
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1 Lobb RR: Clinical applications of heparin-binding growth factors. Eur J Clin Invest 1988:18:321-326. 2 Schweigerer L. Ncufcld G. Fried man J. Abraham J. Fiddcs J, Gospodarowicz D: Capillars' endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature 1987:325: 257-259. 3 Burgess WH. MaciagT: The heparin binding (fibroblast) growth factor family of proteins. Annu Rev Bioehem 1989:58:575-606. 4 Baird A. Mormede P, Bolder P: Immunoreactive fibroblast growth fac tor in cells of peritoneal exudates suggests its identity with macro phage-derived growth factor. Biochcm Biophys Res Commun 1985: 126:358-364. 5 Nakamura M. Oda M. Inoue J. Ito T. Tsuchiya M: Interaction of fibro blast growth factor to the myofibro blast induced by acetic acid-induced ulcer. Gastroenterology 1991:100: A 13 1. 6 Hannson HA. Norstrom E: aFGF, EGF and their receptors in the nor mal and healing rat gastric mucosa. Digestion I99l:49(suppl 1):17—18. 7 Satoh H, Shino A. Inatomi N, Nagaya H. Sato F. Szabo S: Effects of rhbFGF mutein CS23 (TGP-580) on the healing of gastric ulcers in duced by acetic acid in rats. Gastro enterology 19 9 1:100:A 155. 8 Folkman J, Szabo S. Vattav P. Mo rales R. Pinkus G. Kalo K: Effect of orally administered bFGF on heal ing of chronic duodenal ulcers, gas tric secretion, and acute mucosal le sions in rats. Gastroenterology 1990:98:A45. 9 Tarnawski A, Stachura J, Sarfeh IJ. Jekhon S, Krause WJ. Gergley H: Prostacyclin, endothelial cell growth factor and antacid stimulate angio genesis in injured gastric mucosa. Gastroenterology 1991; 100: A174.
Digestion 1992;53:17-27
Leo R. Fitzpatrick Anna Jakubom ka Gregory E. Martin Marjorie Davis Michael C. Jaye Craig A. Dionne
Acidic Fibroblast Growth Factor Accelerates the Healing of Acetic-Acid-Induced Gastric Ulcers in Rats
Pharmacology and Molecular Biology Departments, Rhône-Poulenc Rorer Central Research, Collegeville, Pa., USA
Growth factors like epidermal growth fac tor, platelet-derived growth factor, transform ing growth factor and fibroblast growth fac tors (FGFs) are potential therapeutic agents for wound healing [1], FGF is present in a wide variety of tissues [1-3] and is found in macrophages [4] as well as endothelial cells
Received: January 6, 1992 Received in revised form: June 11,1992
[2], At very low concentrations, FGF can stimulate the angiogenic process [1-4], which is thought to play an important role in wound healing. Increasing experimental evidence supports a role for FGF in the healing of gastrointesti nal ulcers. Nakamura et al. [5] demonstrated
Leo R. Fitzpatrick, PhD Pharmacology and Molecular Biology Departments Rhône-Poulenc Rorer Central Research 500 Areola Road, PO Box 1200 Collegeville, PA 19426-0107 (USA)
©1992 S. Karger AG, Basel 0012-2823/92/ 0532-0017S2.75/0
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Keywords Acidic fibroblast growth factor Gastric ulcer Angiogenesis Rats
Abstract Acidic fibroblast growth factor (aFGF) was evaluated for the healing of acetic-acid-induced gastric ulcers in rats. The effect of aFGF on angiogenesis in the gastric ulcer bed was deter mined by the carmine dye infusion method, while its effect on gastric acid secretion was assessed in chronic gastric fistula rats. Oral treatment with aFGF, in the presence of heparin, reduced (ED5o value = 30.2 pg/kg/day) the acetic-acid-induced gastric ulcer area, when assessed 1 week later. aFGF was about 1.333-fold more potent than famotidine for healing such ulcers. At a dose of 200 pg/kg/day, aFGF increased the car mine density 3-fold and correspondingly reduced (80%) the gastric ulcer area. Thus, the ulcer healing effect of this agent involves angiogenesis in the gastric ulcer bed. This effect of aFGF appears to be unrelated to an inhibition of gastric acid secretion, as it was ineffective in chronic gastric fistula rats. In summary, oral aFGF significantly accelerates the healing of experimental gastric ulcers in rats. It may be a potent and effective agent for the treatment of peptic ulcers in humans.
Materials and Methods Acetic-Acid-Induced Gastric Ulcers Female Sprague-Dawley rats (Taconic Farms, Ger mantown, N.Y.. USA), weighing 180-200 g. were used in these studies. Animals were fasted for 24 h and anes thetized with ether prior to gastric ulcer induction. Gastric ulcers were produced according to the method described by Konturek et al. [ 11]. with slight modifica tion. Briefly. 50 pi of 100% acetic acid were applied through a plastic mold of 4 mm inner diameter to the serosal surface of the rat stomach (anterior corpus area) for 30 s. Subsequently, this area was rinsed with
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0.9% saline, and the abdomen was closed with suture and surgical staples. Starting the day after ulcer induc tion. rats were maintained on the dietary and treat ment regimen originally described by Ito et al. [ 12]. which was used previously in our laboratory [ 13], Ani mals were fed twice daily, for I h (6:30-7:30 a.m. and 2:00-3:00 p.m.). The control vehicles (0.5% methylcellulose, phosphate-buffered saline) or drugs (famoti dine. 20 mg/kg. heparin. 300 pg/kg. Maalox. 425 mg/kg. aFGF. 1-100 pg/kg and aFGF. 100 pg/kg + Maalox, 425 mg/kg) were then administered orally. 30 min after each feeding period, at a dose volume of 10 ml/kg. aFG F was always kept cool on ice prior to oral administration by gavage and was given in the presence of heparin (5 U/ml, 300 pg/kg). Treatment began the day after ulcer induction with acetic acid, and rats were sacrificed on day 7 except in the initial time course studies. The stomachs were removed, opened along the greater curvature and pinned flat for the determination of an ulcer index as well as the severity of gastric ulceration. This was done with the aid of a magnifying glass (X2.5). The gastric ulcer severity was evaluated according to the following scale: 0 = no visible damage: 1 = erosion; 2 = penetrating ulcer: 3 = perforating ulcer, and 4 = severe, perforating ulcer. A severe, perforating ulcer (grade 4) was distin guished by the following features: large size, presence of inflammation in the ulcerated area and severe/tight adhesions of the liver to the gastric ulcer. The ulcer index was determined as the product of ulcer length X width (expressed in mm2). Angiogenesis in Acetic-Acid-Induced Gastric Ulcers Angiogenesis in the experimental gastric ulcer bed of aFGF- or vehicle-treated animals was measured on day 7 after ulcer induction by the carmine dye infusion method of Hase et al. [ 14], Briefly, rats were injected through the tail vein with 2 g/kgofa 10% carmine sus pension containing 5% gelatin. After the injection of carmine, animals were sacrificed w ith carbon dioxide and cooled at 4 °C for 2 h. Upon analyzing the gastriculcer for size and severity, it was excised from the sur rounding normal tissue along its margin. Any adhering liver w'as scraped from the gastric tissue by blunt abla tion. The resected ulcer was weighed and placed in 4 ml of 3 N NaOH for 30 min at 30 °C. After adding 1 ml of ION HC1. the solution w'as filtered. Subse quently, absorbance of the filtrate at 530 nm was mea sured with a Beckman model 25 spectrophotometer (Beckman Instruments, Cedar Grove, N.J. USA). To tal carmine content of the samples was determined by regression analysis from a standard calibration curve
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Gastric Ulcer Healing by aFGF
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that FGF binding sites are present on myofi broblasts in the granulation tissue of experi mental gastric ulcers. More recently, other investigators have shown the presence of acidic FGF (aFGF) and the FGF receptor, fig, at the wound margin of acetic-acid-induced gastric ulcers in rats [6], Moreover, oral treat ment with an acid-resistant mutein of basic FGF (TGP-580) accelerated the healing of both acetic-acid-induccd gastric ulcers [7] and cysteamine-induced duodenal ulcers [8] in rats. The duodenal ulcer healing effect was linked to increased angiogenesis [8], Finally. Tarnawski et al. [9] have demonstrated that aFGF stimulates the restoration of the rat gas tric mucosa following ethanol administration. To date, however, no studies have been re ported on the efficacy of aFGF for healing chronic experimental gastric ulcers. There fore, the purposes of the present study were: (1) to assess the capability of human recombi nant aFGF [10], when administered orally, to heal acetic-acid-induced gastric ulcers in rats: (2) to compare the healing effect of aFGF to that of standard antiulcer agents (famotidine, Maalox): (3) to determine whether combined aFGF and Maalox treatment would enhance the healing of experimental gastric ulcers, and (4) to assess whether orally administered aFGF stimulated angiogenesis in the aceticacid-induced gastric ulcer bed.
Gastric A d d Secretion in Rats Sprague-Dawley rats (300-400 g) were fitted with chronically implanted gastric cannulas, as described previously [ 16], Animals were not used for 2 weeks fol lowing surgery. Subsequently, these rats were acclima tized to the experimental procedure for a 2-week peri od. before being used in actual studies. The same 24 chronic gastric fistula rats were then used in basal and pentagastrin-stimulated acid secretion experiments. Rats were fasted for 24 h prior to their use in exper iments. In the basal acid secretion studies, the gastric cannula was opened, and the stomach was thoroughly rinsed. Subsequently, fluid was allowed to drain from the stomach for a 30-min period. The cannula was closed, and rats were dosed orally with the following compounds: ( I ) PBS + 0 .1% BSA (vehicle): (2) vehicle + heparin (300 pg/kg); (3) vehicle, heparin + aFGF (100 pg/kg); (4) vehicle, heparin + Maalox (425 mg/kg): (5) vehicle, heparin + aFG F (100 pg/kg) + Maalox (425 mg/kg). and (6) vehicle, heparin + famotidine (20 mg/kg). The cannula was kept closed for 1 h to allow time for drug absorption. During this time period, ani mals were given subcutaneously 5 ml of warm saline to account for subsequent fluid loss upon opening the cannula. Following this 60-ntin period, the cannula was opened, and gastric juice collections were made continuously 30. 60. 120 and 180 min later. These time points correspond to 90. 120. 180 and 240 min after compound administration. The pentagastrin-stimulated acid secretion studies were done as described above with only slight modifi cation. One hour after oral administration of the com pounds listed above, the cannula was opened. Subse quently. animals were immediately injected subcuta neously with pentagastrin (10 pg/kg) or vehicle (in one group of PBS-BSA pretreated rats). Gastric collections were made 30. 60 and 120 min later, as described above. The gastric juice was collected in a graduated test tube and measured to the nearest 0 .1 ml. Acid concen tration was determined by titration of gastric juice with 0.1 N N aO H using a Brinkmann 702 SM titrator (Brinkmann Instruments, Westbury, N.Y., USA). To tal acid output w'as calculated as the product of volume X acid concentration and is expressed in micro equivalents.
Drugs Famotidine, carmine, pentagastrin and gelatin (from bovine skin) were obtained from Sigma (St. Louis, Mo., USA). Maalox was provided by RhônePoulenc Rorer (Fort Washington. Pa.. USA). The dose of Maalox refers to the active ingredients per 5 ml (225 mg aluminum hydroxide and 200 mg magnesium hy droxide). Heparin (from beef lung) was obtained via U pjohn (Kalamazoo, Mich.. USA). Human recombi nant aFGF was synthesized by the method of Jaye et al. [10] and provided by Dr. Mark Ravera (Molecular Biology Department. Rhône-Poulenc Rorer Central Research, King of Prussia. Pa., USA). Statistical Analysis The effects of drugs on the gastric ulcer index and carmine density were compared to vehicle-treated con trols by Student’s t test for unpaired data. Ulcer sever ity scores were evaluated statistically by the Wilcoxon rank sum test. Gastric acid secretion data were evalu ated by one-way analysis of variance and Duncan’s multiple range test. For these methods, statistical sig nificance compared to vehicle treatment is reported at the p < 0.05 confidence level. The ED50 value and 95% confidence limits to the dose-response curve of aFGF for gastric ulcer healing w'as obtained by inverse regression analysis.
Results Time Course o f Acetic-Acid-Induced Gastric Ulcers
Initially, the time course of acetic-acidinduced gastric ulceration in rats was evaluat ed. As shown in Figure 1, the ulcerated area (15.4 mm2), 5 h after acetic acid administra tion. conforms nicely to the diameter (4 mm) of the plastic application tube used in these experiments. There was no change in the gas tric ulcer index for the next 5 days after the induction of ulceration. On day 5, the ulcer severity score was 2.8, indicating that the gas tric ulcers were generally perforating in na ture. On day 7, there was only a slight reduc tion in the ulcer index to a value of 12.8 mm2 (Fig. 1). The mean ulcer severity score (n = 4 experiments) on this day was 2.6 (i.e. both
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for carmine (25-200 gg/ml). Angiogenesis in the gas tric ulcer was expressed as the carmine density [total carmine content (pg) ulcer index (mm2)] as suggested byTsuchida etal. [15].
Table 1. Effect of various drugs on acetic-acid-induced gastric ulcers in rats
Drug (dose)
Ulcer severity
Ulcer index, mm2
Vehicle Famotidine (40 mg/kg/day) Maalox (850 mg/kg/day) Heparin (600 gg/kg/day)
3.0 ±0.2 1.8 ±0.3* 2.2 ±0.3* 3.1 ±0.3
13.9 ±2.7 6.5 ±2.1** 7.9± 1.7 13.3 ±2.6
Drugs or vehicle (0.5% methylcellulose) were given orally twice daily, starting on day 1 after the induction of gastric ulceration. Animals were sacrificed on day 7. n = 9-12 per treatment group. * p < 0.05 vs. vehicle by Wilcoxon rank sum test. ** p < 0.05 vs. vehicle by Student’s t test.
Table 2. Effect of aFGF ± Maalox on acctic-acid-induccd gastric ulcers in rats
Drug (dose)
Ulcer severity
Ulcer index mm2
Carmine density gg/ntm2
Vehicle aFG F (200 gg/kg/day) aFG F + Maalox (200 gg/kg/day + 850 mg/kg/day)
2.6 ± 0.2 1.1 ± 0. 1* 1.1 ± 0. 1*
12.2 ± 1.1 2.5 ±0.6** 2.4 ±0.5**
17.4 ±5.4 52.2 ±17.9** 35.6 ±7.9**
Drugs or vehicle (PBS + 0.1 % BSA) were given orally twice daily, star ting on day 1 after induction of gastric ulceration. Animals were sacrificed on day 7. Angiogenesis was determined in the gastric ulcer base by the carmine dye infusion method, n = 12-18 per treatment group. * p < 0.05 vs. vehicle by Wilcoxon rank sum test, ** p < 0.05 vs. vehicle by Student's t test.
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Effect o f Drugs on the Healing o f Acetic-Acid-Induced Gastric Ulcers
Oral treatment (twice daily) with the Hy receptor antagonist, famotidine, significantly reduced (53%) the gastric ulcer index (ta ble 1). Maalox also reduced (43%) the gastric ulcer index, however, statistical significance compared to vehicle treatment was not at tained. Heparin, when given orally, had vir tually no effect on the size of acetic-acidinduced gastric ulcers. Similar effects were observed for these drugs with regard to the
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Gastric Ulcer Healing by aFGF
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penetrating/perforating ulceration was pres ent). However, by 10 days after acetic acid administration, there was a singificant attenu ation in the ulcer index to 4.8 mm2, indicating that significant endogenous healing had oc curred. On day 10, the ulcer severity score was 1.9. Thus, at this time, gastric ulcers were mainly penetrating in nature. 15 days after the induction of ulceration, the gastric ulcers were usually small erosions (mean ulcer sever ity = 1.1), which had almost completely healed (mean ulcer index = 2.9 mm2).
Time, days
aFG F aFG F aFG F Famotidine 2 ag/kg/day 20 ng/kgfaay 200 (ig/kg/day 40 mg/kg/day
1
2a
severity of acetic-acid-induced gastric ulcers. Both famotidine and Maalox significantly re duced (40 and 27%. respectively) the gastric ulcer severity, while heparin was completely ineffective. We next assessed the efficacy of oral aFGF treatment, either alone or in combination with Maalox, for healing acetic-acid-induced gastric ulcers. As shown in table 2, aFGF treatment (200 pg/kg/day) resulted in dra matic attenuations of both the ulcer index (by 80%) and ulcer severity (by 58%). When ad
aFGFiEDso value = 30.2 jig/kg/day
aFG F aFG F aFG F Famotidine 2 ng/kg/day 20 ng/kg/day 200 ng/kg/day 40 mg/kg/day
2b
ministered at doses of 20 or 2 pg/kg/day, aFGF reduced the gastric ulcer index by 49 and 0%. respectively (fig. 2a). The calculated ED50 value and 95% confidence limits of aFGF for reducing the acetic-acid-induced gastric ulcer index in rats were 30.2 (13. 70) pg/kg/day. A similar trend was evident (fig. 2b) with regard to the effectiveness of aFGF for reducing the gastric ulcer severity. As illustrated in figure 3, aFGF (2-200 pg/kg/day) increased the carmine density in a dose-related fashion. When administered
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Fig. 1. Time course of acetic-acid-induced gastric ulcer development in rats. Acetic acid (100%. 50 pi) was applied to the serosal surface of the rat stomach. The area of gastric ulceration (ulcer index) was deter mined 5 h later (day 0) and subsequently 2. 5. 7 , 10 and 15 days after application of acetic acid, n = 3-6 per time point. Fig. 2. Effect of aFGF or famotidine on the gastric ulcer index (area in mm2. a) and gastric ulcer severity (0—4 scale, b) induced by acetic acid. Drugs were administered at the doses indicated for 6 days, and rats were sacrificed on day 7 after the induction of ulcer ation. Data are expressed as a percent of the vehicle control values. Mean (n = 4 experiments) vehicle ulcer index = 13.9 mm2, while mean ulcer severity = 2.60. n = 9 - 16 per treatment group. * p < 0.05 vs. vehicle by t test (a), or Wilcoxon rank sum test (b).
orally at a dose of 200 pg/kg/day. the carmine density was significantly enhanced (3-fold) by aFGF. Finally, as shown in figure 4, with increasing doses of aFGF there was a very good inverse correlation (r = -0.993) between enhanced carmine density in the gastric ulcer base and the corresponding decrease in the gastric ulcer area. Interestingly, combined treatment with aFGF plus Maalox did not result in enhanced ulcer healing or increased angiogenesis (carmine density) in the gastric ulcer bed, compared to treatment with aFGF alone (table 2). Effect o f Drugs on Gastric Acid Secretion in Rats
As shown in table 3, the total (3-hour) gas tric acid output in vehicle-treated rats was 267.1 pEq. Heparin did not significantly af fect basal acid secretion in chronic gastric fis tula rats. In contrast, famotidine (20 mg/kg) almost completely inhibited gastric acid se-
Fig.3. Effect of aFGF on angiogenesis in aceticacid-induced gastric ulcers. On day 7 after the induc tion of ulceration, angiogenesis (carmine density) was determined in the gastric ulcer bed of rats by the car mine dye method. Data are expressed as a percent of the vehicle-treated control values. Mean (n = 3 experi ments) vehicle carmine density = 26.7 pg carmine/mm2. n = 9-16 per treatment group. * p < 0.05 vs. vehicle by Student’s t test.
Fig. 4. Correlation between angiogenesis (carmine density) and the healing of acetic-acid-induced gastric ulcers in rats on day 7. Values for each dose of aFGF arc expressed as a percent of vehicle-treated controls (n = 3 experiments) and are the means of 9-16 observations. The slope (m), y intercept (b) and correlation coefficient (r) were determined by inverse linear regression analysis.
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Carm ine density. % control
Table 3. Effect of drugs on gastric acid secretion in chronic gastric fistula rats
T reatment group
1 2 3 4 5 6
Vehicle Heparin aFGF Maalox aFGF + Maalox Famotidine
Total acid output, pEq 2h
3h
4h
total
73.6+15.7 113.5 ±27.4 78.3 ±21.8 46.5 ±22.4* 41.4 ± 19.2* 0.2 ± 0. 1*
73.8 ±18.7 129.9 ±27.1 71.4 ±14.3* 55.3 ±21.9* 45.5 ±14.9* 1.4 ± 0.8*
117.4 ±29.4 102.1 ±27.1 109.3 ±27.1 91.1 ±28.9 46.0 ± 15.1* 1.5 ± 1.1*
267.1 ±48.4 345.5 ±71.2 259.3 ±42.9 193.0 ±58.6 132.1 ±44.3* 3.1 ±1.8*
Animals were pretreated orally with vehicle (0.5% methylcellulose), heparin (300 pg/kg) famotidine (20 mg/kg) or aFGF (100 pg/kg) ± Maalox (425 mg/kg). Rats in groups 3-6 also received heparin. Total gastric output was determined at the times indicated following drug administration, n = 6-8 per treatment group. * p < 0.05 vs. group 2 by Anova and Duncan’s multiple range test.
Table 4. Effect of drugs on pentagastrin-stimulatcd gastric acid secretion in chronic gastric
fistula rats Treatment group
1 2 3 4 5 6 7
No pentagastrin Pentagastrin Heparin aFGF Maalox aFGF + Maalox Famotidine
Total acid output. pEq 2h
3h
total
91.7 ± 19.7 315.7 ± 55.9** 339.0 ±69.9 253.8 ± 18.7 143.7 ±55.9* 12.1 ±5.1* 0.6 ±0.4*
99.3 ±29.2 89.8 ± 15.5 72.4 ± 15.9 84.8 ±5.9 30.4 ± 14.3 3.0±2.0* 0 ± 0*
190.9 ±45.1 405.5 ±62.7** 411.5 ± 80.5 338.3 ±21.1 174.1 ±64.3* 15.1 ± 6. 1* 0.6 ±0.4*
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Animals were pretreated orally with 0.5% methylcellulose (groups 1 and 2), heparin (300 pg/kg). famotidine (20 mg/kg) or aFG F ( 100 pg/kg) ± Maalox (425 mg/kg). Drugs were given I h prior to the subcutaneous injection of 2% DMSO vehicle (group l ) or 10 pg/kg pentagastrin (groups 2-7). Rats in groups 4-7 also received heparin. The total gastric acid output was determined at the limes indicated following oral drug administration, n = 6-7 per treatment group. * p < 0.05 vs. group 3 or ** p < 0.05 vs. group 1 by Anova and Duncan's multiple range test.
Discussion Acetic-acid-induced gastric ulceration in rats is the current prototype gastric ulcer mod el, and it has been used by many investigators to study the efficacy of antiulcer drugs [7, 1115], Tarnawski et al. [17, 18] found that such chronic experimental ulcers are similar gross
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ly, histologically and ultrastructurally to hu man gastric ulcers. In this study, we initially examined the time course of ulcers induced by topical application of acetic acid to the serosal gastric surface of rats. Our data (fig. 1) indicate a significant decrease in the gastric ulcer index (i.e. area) between weeks 1 and 2 after the induction of ulceration. These results are similar to those reported previously by Konturek et al. [II]. In our experiments, we utilized the dietary regimen described by Ito et al. [12], in which rats were maintained on a limited food intake (i.e. fed twice daily). With such a restricted dietary regimen, the area of gastric ulceration would be more exposed to hydrochloric acid and pepsin, which is more akin to the situa tion that exists in humans [12], Therefore, this regimen makes the gastric ulcer area more sensitive to the action of antisecretory agents like Hi receptor antagonists [12. 13], In fact, we previously found that ranitidine signifi cantly reduced (59%) the acetic-acid-induced gastric ulcer area on day 5, when rats were maintained on the limited food intake dietary regimen [13], In the present study, we exam ined the efficacy of another Hi receptor antag onist (famotidine) for healing acetie-acid-induced gastric ulcers. Twice daily treatment with a dose of famotidine (20 mg/kg) that decreased basal acid secretion in gastric fis tula rats by 99% (table 3) significantly re duced both the gastric ulcer area (53%) and severity (40%). Maalox (425 mg/kg X 2) also reduced these indices, but not to the extent as with famotidine treatment. In summary, these initial experiments established the effi cacy of two common antiulcer drugs for heal ing chronic gastric ulcers in rats. Although heparin had no effect on gastric ulcer healing (table 1) at the dose (300 pg/kg) we used, it was included in subsequent experi ments which assessed the efficacy of aFGF. This was done because heparin binds with
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crction. Maalox (425 mg/kg) significantly re duced basal acid secretion only during the ini tial two collection periods (i.e. 2 and 3 h after its administration). When administered in combination with aFGF, Maalox attenuated basal secretion over the entire duration of the study. However. Maalox reduced the total (3hour) acid output to approximately the same degree (44-62%), whether administered alone or in combination with aFGF. When administered by itself. aFGF (100 |ig/kg) did not consistently demonstrate any significant antisecretory activity in chronic gastric fistula rats (table 3). Pentagastrin (10 pg/kg, s.c.) increased acid secretion 3.4-fold in chronic gastric fistula rats, 1 h after its administration (see 2-hour column in table 4). This effect was transient in nature, as gastric acid output returned to the nontreated (group 1) level during the next hour. As was the case in the basal acid secre tion studies, neither heparin nor aFGF (25% reduction) significantly attenuated pentagastrin-stimulated acid secretion. Maalox re duced the initial phase of the pentagastrininduced secretory response by 57%. while famotidine completely abolished acid secre tion to a level below that found in nontreated rats. Combined treatment with aFGF plus Maalox also dramatically attenuated (96.4%) pentagastrin-stimulated gastric acid output, to a value of 12.1 pEq. suggesting a possible synergistic interaction between the two drugs.
acetic-acid-induced gastric ulcers, when Maa lox 70 was combined with another growth fac tor (epidermal growth factor). However. Kusstatscher et al. [20] have recently reported an additive effect for bFGF and cimetidine on chronic duodenal ulcer healing in rats. It is possible that the lack of enhanced ulcer heal ing in our study is due to the relatively short acid-neutralizing time of Maalox (table 3) in the fasted state. In this regard, our ulcer heal ing studies were conducted with rats main tained on a restricted feeding regimen. There fore, a basal (i.e. nonstimulated) gastric acid secretory state was predominantly present in these animals. Moreover, the results in table 3 also suggest that combined treatment with aFGF plus Maalox did not significantly en hance antisecretory activity compared to treatment with Maalox alone. Interestingly, however, combined administration of aFGF plus Maalox resulted in a significant enhance ment in antisecretory action against pentagastrin-stimulated acid secretion (table 4). Al though the mechanism/s of this apparent synergistic interaction remain to be deter mined, it is doubtful whether such an interac tion had physiologic significance in our ulcer healing studies because of the dietary regimen utilized. Finally, as suggested by other investi gators [20], it is also likely that combined treatment of aFGF with longer acting antise cretory' agents (H2 receptor antagonists, hy drogen-potassium ATPase inhibitors) could result in a significant enhancement of experi mental ulcer healing. Various studies [14, 15, 21] have utilized the carmine dye technique to quantify angio genesis. Previously. Hase et al. [14] confirmed histologically that injected carmine dye was distributed exclusively within the vascular lu men. Moreover, using this technique, these investigators demonstrated the appropriate antiangiogenic activity in the experimental gastric ulcer bed of rats treated with predniso
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high affinity to aFGF and protects this growth factor from acidic/proteolytic degradation [3]. Under this experimental condition, oral treatment with aFGF (200 pg/kg/day) signifi cantly decreased (58%) the gastric ulcer sever ity score from 2.6 in vehicle-treated rats to a value of 1.1 (table 2). aFGF also attenuated the gastric ulcer area from 12.2 mm2 in vehi cle-treated animals to 2.5 mm2 (80% reduc tion). To put this data in perspective, the gas tric ulcers in vehicle-treated animals on day 7 were typically penetrating/perforating in na ture, while in aFGF-lreated rats only small erosions were present. In addition, the ulcer index on day 7 in aFGF-treated rats was smaller than the value (2.9 mm2) obtained in nontreated animals on day 15 (fig. 1). Thus, aFGF decreased the severity and accelerated the healing time of acetic-acid-induced gastric ulcers in rats. Interestingly, the ulcer healing effect of aFGF appears to be unrelated to an inhibition of gastric acid secretion, as it was ineffective against either basal or pentagastrin-stimulated acid secretion in chronic gas tric fistula rats (table 3). The calculated ED50 value (fig. 2a) of aFGF for reducing the acetic-acid-induced gastric ulcer area is 30.2 pg/kg/day. Because famotidine (40 mg/kg/day) reduced the gas tric ulcer index by about 50% (table 1). aFGF is approximately 1,333-fold more potent than this H2 receptor antagonist for healing such experimental gastric ulcers. Our results re semble those reported previously for the acidresistant bFGF mutein. TGP-580. Satoh et al. [7] found that at a dose of 10 pg/kg it reduced a calculated gastric ulcer index by 55%, while cimetidine (100 mg/kg) was only moderately effective (43% reduction). In our study, animals treated with aFGF plus Maalox demonstrated the same degree of gastric ulcer healing as rats treated only with aFGF (table 2). Konturek et al. [19] also found no additive effect on the healing of
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omental adhesions, which could have in fluenced the ulcer healing process by enhanc ing angiogenesis. The capability of aFGF to increase angio genesis in the gastric ulcer bed (fig. 3) may improve the quality of ulcer healing and also might have an impact on ulcer recurrence [17], Indeed. Satoh et al. [25] recently re ported that bFGF prevented indomethacininduced gastric ulcer relapse in rats. An inves tigation of such potential actions for aFGF should be the focus of future studies. It should also be noted that Folkman et al. [26] have presented substantial experimental evidence suggesting that endogenous bFGF may be the primary factor involved in ulcer healing. Ac cording to their hypothesis, all commonly used antiulcer drugs (e.g. sucralfate, antacids. Hi receptor antagonists) prevent the degrada tion of endogenous bFGF by gastric acid. Thus, the angiogenic and other growth-pro moting activities of bFGF would be preserved for utilization in ulcer healing. Finally, Hannson and Norstrom [6] have recently proposed that endogenous aFGF may also play a role in ulcer healing. In summary, orally administered aFGF significantly accelerated the healing of aceticacid-induccd gastric ulcers in rats. It was > 1,000-fold more potent than famotidine for healing such ulcers. The ulcer healing effect of aFGF involves increased angiogenesis in the gastric ulcer base, but appears to be unrelated to an inhibition of gastric acid secretion. aFGF may be a potent and effective agent for the treatment of gastric ulcers in man.
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lone. A recent study from our laboratory also found that treatment with another corticoste roid. hydrocortisone, significantly decreased the carmine density in the gastric ulcer bed of rats [22], As a whole, these studies provide good evidence that the carmine dye method is an acceptable method for measuring angio genesis. Finally, Tsuchida et al. [15] demon strated a very good correlation between vas cular density in acetic-acid-induced gastric ul cers as measured by direct video image analy sis and carmine dye infusion. Therefore, as suggested by these investigators [ 15], we used the carmine density (pg carmine/ulcer index) as a measurement of angiogenesis in the gas tric ulcer bed. As shown in table 2, aFGF treatment (200 gg/kg/day) increased (3-fold) the carmine density (i.e. angiogenesis) in the gastric ulcer bed on day 7 after the initiation of ulceration. Moreover, the effect of aFGF on angiogenesis was dose related (fig. 3). With increasing doses of this growth factor, a very good inverse correlation (r = -0.993) was found between enhanced angiogenesis in the gastric ulcer bed and decreased ulcer size (fig. 4). Therefore, the stimulation of new mi crovessel formation by aFGF appears to en hance the ulcer healing process. Such a mech anism of action was also reported by Folkman et al. [8] for the healing of cysteamineinduced duodenal ulcers by TGP-580, and more recently by other investigators [23, 24] for the healing of acetic-acid-induced gastric ulcers by bFGF. In this regard. Konturek et al. [23] reported that either exogenous bFGF or omental attachment resulted in a similar in crease in experimental gastric ulcer healing in rats. Moreover, in both instances, there was a stimulation of angiogenesis in the acetic-acidinduced gastric ulcer bed. These data may also have relevance to our ulcer healing re sults. In our studies, experimental gastric ul cers were often penetrating/perforating in na ture. Such penetration of the ulcer resulted in
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