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The EGF\EGF-Receptor Axis Modulates Enterocyte Apoptosis during Intestinal Adaptation
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
77, 17–22 (1998)
JR985362
The EGF\EGF-Receptor Axis Modulates Enterocyte Apoptosis during Intestinal Adaptation1 Michael A. Helmrath, M.D., Cathy E. Shin, M.D., Christopher R. Erwin, Ph.D., and Brad W. Warner, M.D. Division of Pediatric Surgery, Children’s Hospital Medical Center, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039 Presented at the Annual Meeting of the Association for Academic Surgery, Dallas, Texas, November 6 – 8, 1997
increased rates of enterocyte proliferation that characterize adaptation are important for the genesis of taller villi and deeper crypts, which function to increase the overall mucosal surface area for digestion and absorption. For purposes of maintaining homeostasis, it is intuitive that the rates of enterocyte proliferation are balanced by the rates of enterocyte loss. Indeed, augmented recovery of DNA in the intestinal lumen after massive enterectomy probably reflects increased shedding of intestinal epithelial cells [3]. While the exact mechanism for enterocyte loss is currently unknown, our laboratory has recently identified increased numbers of apoptotic bodies in both crypts and villi during the adaptive response to SBR [4]. These findings suggest that the elevated rate of enterocyte shedding into the intestinal lumen during adaptation is due to increased rates of programmed cell death (apoptosis). Exogenous administration of epidermal growth factor (EGF) has been shown in several animal models of SBR to enhance intestinal adaptation [5–9]. Alternatively, adaptation is inhibited in mice with defective EGF receptor signaling function [10]. The specific mode by which EGF amplifies adaptation has not been well characterized. In addition to multiple nonmitogenic actions (reviewed in Ref. [11]), EGF provides a potent proliferative stimulus to the gastrointestinal tract [12]. While it has been demonstrated that EGF enhances enterocyte proliferation during adaptation [7], its effects on rates of apoptosis are not known. The purpose of this study, therefore, was to determine the influence of EGF on enterocyte apoptosis during intestinal adaptation following SBR.
Background. Adaptation after small bowel resection (SBR) is characterized by a new set point in the balance of enterocyte proliferation and apoptosis. Since epidermal growth factor (EGF) augments both proliferation and adaptation, we sought to determine the effect of EGF receptor manipulation on apoptosis following SBR. Materials and Methods. Male ICR mice underwent 50% SBR or sham operation (bowel transection with reanastomosis) and then were given EGF (50 mg/kg/day) or saline by orogastric gavage. At 1 week, a proliferation index (PI) was measured in the ileum by BrdU uptake and an apoptosis index in crypts (cAI) and villi (vAI) scored by counting apoptotic bodies in enterocytes. In other experiments, AI was scored after SBR in mice with defective receptors (waved-2). Results are expressed as means 6 SE and evaluated statistically using ANOVA. # denotes P < 0.001. Results. Following SBR, EGF increased PI (40 6 2% vs 50 6 1% BrdU 1 cells; #), villus height (252 6 4mm vs 401 6 15 mm; #), and crypt depth (77.3 6 1.5mm vs 120.8 6 5 mm; #). When compared with sham, SBR resulted in increased cAI (0.3 6 0.02 vs 2.0 6 0.1; #) and vAI (0.4 6 0.05 vs 1.1 6 0.1; #). EGF attenuated both cAI (0.5 6 0.04) and vAI (0.5 6 0.03) following SBR. In the waved-2 mice, the highest levels of cAI (3.1 6 0.2) and vAI (3.6 6 0.3) were noted after SBR. Conclusions. Enterocyte apoptosis during adaptation is attenuated by EGF and exaggerated when the EGF receptor is defective. In addition to enhancing proliferation, suppression of apoptosis may provide a previously unrecognized mechanism for the beneficial effect of EGF during intestinal adaptation. © 1998 Academic Press Key Words: EGF; apoptosis; adaptation; small bowel resection; waved-2 mice; proliferation; enterocyte.
MATERIALS AND METHODS Animals. This study was approved by the Institutional Animal Care and Use Committee of the Children’s Hospital Research Foundation (Children’s Hospital Medical Center, Cincinnati, OH). Male ICR (Jackson Laboratory, Bar Harbor, ME) or homozygous waved-2 mice (Jackson Laboratory) were housed in groups of four at 21°C on 12-h day/night cycles (06:00 –18:00). At approximately 8 –10 weeks of life, the mice underwent either a 50% proximal SBR with reanastomosis or sham operation (bowel transection with reanastomosis alone) as our laboratory has previously described [13].
INTRODUCTION
Following massive small bowel resection (SBR), a compensatory, mitogenic response occurs in the remnant intestinal mucosa termed adaptation [1, 2]. The 1
This study was supported by a Trustees Grant from the Children’s Hospital Research Foundation (Dr. Warner, grant recipient).
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0022-4804/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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JOURNAL OF SURGICAL RESEARCH: VOL. 77, NO. 1, JUNE 1998
Experimental design. The first experiment was designed to determine the effect of EGF on ileal mucosal architecture (villus height and crypt depth), enterocyte proliferation, and enterocyte apoptosis following SBR. Male ICR mice undergoing sham operation or SBR were then randomized to receive either EGF (50 mg/kg/day) or an equivalent volume of saline twice daily by orogastric gavage. This dosage and route of EGF administration were previously determined to be optimal for enhancing adaptation in our murine model of SBR [13a]. Mice were sacrificed by an overdose of inhaled methoxyfluorane after 7 days. The second experiment was designed to determine the effect of adaptation following SBR on rates of enterocyte apoptosis in the background of defective EGF receptor signaling. For this experiment, sham operation or SBR was performed on homozygous waved-2 mice. These mice harbor a spontaneous point mutation within the tyrosine kinase domain of the EGF receptor gene resulting in reduced (greater than sevenfold) tyrosine kinase activity in vivo [14, 15]. Despite the perturbed EGF receptor, homozygous waved-2 mice are healthy, fertile, and distinguished phenotypically from heterozygous littermates on the basis of wavy hair and curly whiskers. Since these mice do not survive beyond 5 days after SBR due to persistent weight loss and diarrhea [10], mice were sacrificed by an overdose of inhaled methoxyfluorane after 3 days. No differences in food intake were observed between mice undergoing SBR or sham operation. Rates of enterocyte apoptosis in the ileum were compared with those rates in the ICR mice as above. Operative procedure. Details of this technique have been described previously [13]. In brief, mice were anesthetized using a continuous flow of 2% isofluorane, 90% oxygen, and 4% carbon dioxide. The abdomen was clipped, prepped with povidine iodine solution, and draped sterilely. Perioperative antibiotics were not administered. Operations were performed with the aid of an operating microscope (10 –153 magnification). Through a midline abdominal incision, the small intestine was transected 12 cm proximal to the ileocecal valve. Sham mice underwent bowel reanastomosis at this point. In previous studies, we determined that the length of bowel in waved-2 mice was roughly 20% longer when compared with the intestinal length found in nonwaved-2 strains [10]. Therefore, in waved-2 mice undergoing SBR, 14.5 cm of proximal bowel was excised distal to the ligament of Trietz, whereas 12.5 cm of proximal bowel was excised in the ICR mice to ensure approximately a 50% SBR in both strains. Intestinal anastomosis in both sham and SBR groups was performed using an end-to-end, single-layer technique with an interrupted 9-O monofilament suture. The abdominal incision was closed with an interrupted 5-O silk suture incorporating all layers. Mice were resuscitated with an intraperitoneal injection of 0.9% saline (2 ml) and allowed to recover in a warmed incubator (33°C). Mice were provided with water ad libitum for the first 24 h, and then pair feeding with liquid diet (Micro-Stabilized Rodent Liquid Diet LD 101/101; Purina Mills Inc., Richmond, IN) was initiated and adjusted daily. Histology. After the mice were sacrificed, the ileum 2 cm distal to the anastomosis was immediately removed and fecal material was gently expressed with cotton swabs. The first centimeter of tissue was fixed in 10% neutral buffered formalin for evaluation of villus height and crypt depth. The remaining tissue was used for determination of proliferation and apoptosis indices as discussed below. Fixed specimens of ileum were embedded in paraffin and oriented to provide cut sections parallel with the longitudinal axis of the bowel. Five-micrometer-thick slices were mounted and stained with hematoxylin and eosin. Microscopic measurements were performed for villus height and crypt depth using a video-assisted integrated computer program (Image 1.57TV, National Institutes of Health). Villi were measured only if the entire central lymphatic channel was visualized and crypts based on the ability to identify the crypt–villus junction on both sides of the crypt. A minimum of 15 villi and crypts were counted per sample. Enterocyte proliferation. One hour prior to sacrifice, ICR mice in the first set of experiments received an intraperitoneal injection of 5-bromodeoxyuridine (BrdU; 1 ml/100 g body wt; Zymed Laboratories Inc., San Francisco, CA). Paraffin-embedded distal ileum was sectioned (5 mm), mounted on poly-L-lysine-coated slides, and depar-
affinized. Sections were then rehydrated (100, 95, and 75% ethanol) and endogenous tissue was peroxidase inactivated with 30% hydrogen peroxide in methanol (1:9). Incorporation of BrdU into proliferating crypt cells (S-phase) was detected using a biotinylated monoclonal anti-BrdU antibody system with steptavidin–peroxidase as a signal generator. The staining reagents and methods were provided in kit form (Zymed Laboratories Inc.). A proliferation index was derived by counting the number of enterocytes per crypt that incorporate BrdU divided by the total number of cells in the crypt. Ten representative crypts were counted from each mouse. The investigator was blinded as to the origin of the tissue during the scoring procedure. Enterocyte apoptosis. Apoptosis was quantitated by immunohistochemical labeling of DNA strand breaks in enterocytes and confirmed by propidium iodide staining of similar tissue sections. Labeling of DNA strand breaks was accomplished using the ApopTag kit (Oncor, Gaithersburg, MD). In short, formalin-fixed, paraffinembedded ileal specimens were deparaffinized and then rehydrated (100, 95, and 70% graded ethanol washes). Protein was digested with proteinase K and endogenous peroxidase activity quenched with 2% hydrogen peroxide. The slides were then incubated with a reaction mixture containing terminal deoxynucleotidyl transferase and its substrate digoxigenin–11-dUTP at 37°C for 1 h. The reaction was terminated and an anti-digoxigenin–peroxidase antibody was applied for 30 min. Hydrogen peroxide was added as a chromogenic substrate. The slides were then counterstained with methyl green, washed in 100% butanol, dehydrated with xylene, and mounted. Different sections from embedded ileum were then stained with propidium iodide to confirm the findings of the labeling procedure as above. A quantitative index of apoptosis was derived by counting the number of apoptotic bodies per crypt and villus that were both nick-end labeled and had abnormal morphology (pyknotic nuclei, condensed chromatin, and nuclear fragmentation). Blinded scoring of 50 crypts and villi per mouse was performed in triplicate. Statistical analysis. Results are presented as mean values 6 SE. A two-way ANOVA was used for comparisons of group means followed by a pairwise multiple comparison procedure (Student– Newman–Keuls method). A P value less than 0.05 was considered significant.
RESULTS
Following SBR or sham operation with or without EGF, the mice tolerated the procedures well with an overall survival of 82.5% (33/40) at 7 days. Survival in the waved-2 mice was equal (5/6) after either SBR or sham operation with an overall 83% survival at 3 days. Administration of EGF was trophic to the ileal mucosa as both villus height (Fig. 1) and crypt depth (Fig. 2) were significantly greater after either SBR or sham operation. Further, EGF significantly boosted the already increased enterocyte proliferation that occurred following SBR (Fig. 3). In the mice that received saline, apoptosis was significantly increased in both the crypts (Fig. 4) and villi (Fig. 5) following SBR. On the other hand, only a slight increase (P 5 NS) in apoptosis was detected in the mice that were given EGF after SBR. In contrast, crypt and villus rates of apoptosis were significantly greater in the sham waved-2 mice than in the mice that underwent SBR and were given EGF. The highest rates of apoptosis comparing all groups were identified in the waved-2 mice following SBR.
HELMRATH ET AL.: EGF INFLUENCES APOPTOSIS DURING ADAPTATION
FIG. 1. Ileal villus height (means 6 SE) at 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal SBR. Mice were then randomized to receive either human recombinant EGF (50 mg/kg/day) or saline twice daily by orogastric gavage. *P , 0.001 sham versus SBR; #P , 0.001 SBREGF versus SBR-saline.
DISCUSSION
In this study, we first confirmed an enhancing effect of EGF on intestinal adaptation following SBR with
FIG. 2. Ileal crypt depth (means 6 SE) at 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal SBR. Mice were then randomized to receive either human recombinant EGF (50 mg/kg/day) or saline twice daily by orogastric gavage. *P , 0.001 sham versus SBR; #P , 0.001 SBR-EGF versus SBR-saline.
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FIG. 3. Enterocyte proliferation at 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal SBR. Mice were then randomized to receive either human recombinant EGF (50 mg/kg/day) or saline twice daily by orogastric gavage. A proliferation index (means 6 SE) was derived by blinded counting of the number of enterocytes per crypt that incorporate BrdU divided by the total number of cells in the crypt. *P , 0.05 SBR versus sham; #P , 0.05 SBR-EGF versus SBR-saline.
regard to morphologic parameters (villus height and crypt depth) and rates of enterocyte proliferation. Further, we have validated our previously reported observation of increased rates of enterocyte apoptosis following SBR [4]. Finally, and most importantly, we have established a possible correlation between EGF receptor activity and rates of enterocyte apoptosis during intestinal adaptation. Administration of EGF substantially impeded the increase in apoptosis that occurs after SBR. Alternatively, rates of apoptosis were highest when SBR was performed in mice with defective EGF receptor signaling. Taken together, these results endorse a novel mechanism for the beneficial effect of exogenous EGF following SBR. Epidermal growth factor may serve a dual function of stimulating enterocyte proliferation and inhibiting enterocyte apoptosis. With this paradigm, newly produced enterocytes would have a longer halflife under the influence of exogenous EGF, thus contributing toward the genesis of taller villi and deeper crypts that characterize EGF-enhanced intestinal adaptation. Consistent with our observations, other investigators have demonstrated an inhibitory effect of EGF on apoptosis. In cultured rat ovarian granulosa cells, EGF appears to inhibit apoptosis via a tyrosine kinasedependent mechanism [16]. Further, in vivo administration of EGF has been demonstrated to retard the renal tubular cell apoptosis that follows experimental acute ureteral obstruction [17]. In malignant gliomas,
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FIG. 4. Apoptosis in ileal crypts at 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal SBR. Mice were then randomized to receive either human recombinant EGF (50 mg/kg/day) or saline twice daily by orogastric gavage. waved-2 mice did not receive either EGF or saline and were sacrificed at 3 days. An apoptosis index (means 6 SE) was determined by counting the number of apoptotic bodies per crypt that were both nick-end labeled and had abnormal morphology (pyknotic nuclei, condensed chromatin, and nuclear fragmentation). Blinded scoring of 50 crypts and villi per mouse was performed in triplicate. *P , 0.05; #P , 0.05 sham-waved-2 versus sham-saline; ¤P , 0.05 sham versus SBR.
in ileal DNA and protein content that we have previously observed [9]. Similarly, the enhancing effect of EGF on enterocyte proliferation after SBR has been reported in rats [7] and now for the first time in our murine model. Further delineation of parameters such as these will be necessary to elucidate a specific mechanism for the beneficial effect of EGF during adaptation using our mouse model of SBR. In this study, we monitored very high levels of apoptosis in the ileum of waved-2 mice that underwent sham operation. These levels were elevated even higher following SBR. We did not measure proliferation in these mice as we have previously reported that this parameter is reduced when compared with wildtype (non-waved-2) mice after SBR [10]. In that study, we also noted substantially shorter villi and deeper crypts in the waved-2 mice after either sham operation or SBR. Moreover, ileal DNA and protein content did not increase after SBR. The findings of the current and past experiments with waved-2 mice therefore champion a probable link between the EGF receptor activity and rates of enterocyte proliferation, apoptosis, migration, and/or differentiation during intestinal adaptation. It is important to appreciate the technical limitations associated with deriving rates of apoptosis by counting apoptotic bodies in histological sections [24]. Since apoptotic bodies represent fragmented remnants of a cell, the number of apoptotic bodies identified in a
a frequent spontaneous mutation within the coding region of the EGF receptor results in a truncated receptor that constitutively phosphorylates tyrosine residues independent of EGF stimulation [18]. This mutation is associated with enhanced tumorigenicity. The deregulated signaling by this mutant EGF receptor appears to drive both an increase in cell proliferation and a reduction in apoptosis [19]. Finally, apoptosis is enhanced in a human colorectal carcinoma cell line (DiFi) that expresses high numbers of EGF receptors following the addition of a monoclonal antibody directed toward the EGF receptor [20]. In contrast with the above studies, treatment of A431 epidermoid carcinoma cells (another cell line known to overexpress EGF receptors) with high dosages of EGF not only inhibits proliferation, but also enhances apoptosis [21]. Similar results have been observed in MDA-MB-468 human breast cancer cells [22]. The relationship between EGF receptor signaling, proliferation, and apoptosis therefore seems to vary depending on the specific cell type or tissue studied. Epidermal growth factor enhanced intestinal adaptation following SBR as monitored by increases in villus height, crypt depth, and enterocyte proliferation. Increases in various morphologic parameters such as intestinal mucosal thickness, villus height, or crypt depth with exogenous EGF have been reported in rat models of SBR [6, 23]. This study is the first report of morphologic changes in the ileum with EGF in our murine model of SBR and substantiates the increases
FIG. 5. Apoptosis in ileal villi at 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal SBR. Mice were then randomized to receive either human recombinant EGF (50 mg/kg/day) or saline twice daily by orogastric gavage. waved-2 mice did not receive either EGF or saline and were sacrificed at 3 days. An apoptosis index (means 6 SE) was determined by counting the number of apoptotic bodies per villus that were both nick-end labeled and had abnormal morphology (pyknotic nuclei, condensed chromatin, and nuclear fragmentation). Blinded scoring of 50 crypts and villi per mouse was performed in triplicate. *P , 0.05; #P , 0.05 sham-waved-2 versus sham-saline; ¤P , 0.05 sham versus SBR.
HELMRATH ET AL.: EGF INFLUENCES APOPTOSIS DURING ADAPTATION
tissue section could originate from a single or multiple cells. Consequently, the number of apoptotic bodies identified in a given crypt probably does not equate with the exact number of enterocytes undergoing apoptosis. Additionally, the interpretation of what constitutes an apoptotic body is somewhat subjective. Apoptotic bodies may be difficult to distinguish between other histologic findings such as intraepithelial lymphocytes, damaged DNA from conditions other than apoptosis, and enterocytes undergoing mitosis. In this regard, the increased number of apoptotic bodies that we discovered in the crypts of ICR mice after SBR could have been due to greater numbers of enterocytes undergoing mitosis. This potential confusion does not explain the greater number of apoptotic bodies that we observed after SBR in the villus tip, since this is not a site of mitosis. Further, the highest frequency of what we counted as apoptotic bodies was seen in the waved-2 mice. These mice have substantially lower rates of enterocyte proliferation in response to SBR when compared with non-waved-2 mice [10]. Determination of the apoptotic index is dependent upon the number of cells in the crypt and villus. The number of crypt and villus cells and hence crypt depth and villus height are substantially increased following SBR. If the rate of apoptosis was truly unchanged or decreased following SBR, then the number of apoptotic bodies per crypt or villus unit under both conditions should markedly decrease secondary to the expansion of the crypt depth and villus height alone. The increase in apoptosis that we observed in this study therefore may underestimate the true increase in apoptosis associated with SBR since we observed greater numbers of apoptotic bodies for each deeper crypt or taller villus due to SBR. This phenomenon insinuates an even greater effect of EGF on inhibiting apoptosis since the crypt depth and villus height were even further expanded after this treatment in mice following SBR. Since apoptosis is an active cellular process, it will be important in future studies to clarify changes in the transcription of several apoptosis-associated genes. While extremely complex, a few factors that have been identified in intestinal epithelial cells that believably play integral roles during apoptosis following SBR include bax, mcl-1, and bcl-x [25–27]. Expression of deoxyribonuclease I has been linked to apoptosis in the intestine [28]. Radiation-induced apoptosis in the gastrointestinal tract has been shown to synchronize with the expression of p53 [29] and is not present in mice that are p53 deficient [30]. The product of the protooncogene bcl-2 is known to play a role in cell survival, acting as an inhibitor of apoptosis [31]. The relationship between intestinal EGF receptor stimulation and the transcriptional regulation of these genes will be an important first step toward illuminating a mechanism for the therapeutic effect of exogenous EGF following massive intestinal loss.
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77, 17–22 (1998)
JR985362
The EGF\EGF-Receptor Axis Modulates Enterocyte Apoptosis during Intestinal Adaptation1 Michael A. Helmrath, M.D., Cathy E. Shin, M.D., Christopher R. Erwin, Ph.D., and Brad W. Warner, M.D. Division of Pediatric Surgery, Children’s Hospital Medical Center, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039 Presented at the Annual Meeting of the Association for Academic Surgery, Dallas, Texas, November 6 – 8, 1997
increased rates of enterocyte proliferation that characterize adaptation are important for the genesis of taller villi and deeper crypts, which function to increase the overall mucosal surface area for digestion and absorption. For purposes of maintaining homeostasis, it is intuitive that the rates of enterocyte proliferation are balanced by the rates of enterocyte loss. Indeed, augmented recovery of DNA in the intestinal lumen after massive enterectomy probably reflects increased shedding of intestinal epithelial cells [3]. While the exact mechanism for enterocyte loss is currently unknown, our laboratory has recently identified increased numbers of apoptotic bodies in both crypts and villi during the adaptive response to SBR [4]. These findings suggest that the elevated rate of enterocyte shedding into the intestinal lumen during adaptation is due to increased rates of programmed cell death (apoptosis). Exogenous administration of epidermal growth factor (EGF) has been shown in several animal models of SBR to enhance intestinal adaptation [5–9]. Alternatively, adaptation is inhibited in mice with defective EGF receptor signaling function [10]. The specific mode by which EGF amplifies adaptation has not been well characterized. In addition to multiple nonmitogenic actions (reviewed in Ref. [11]), EGF provides a potent proliferative stimulus to the gastrointestinal tract [12]. While it has been demonstrated that EGF enhances enterocyte proliferation during adaptation [7], its effects on rates of apoptosis are not known. The purpose of this study, therefore, was to determine the influence of EGF on enterocyte apoptosis during intestinal adaptation following SBR.
Background. Adaptation after small bowel resection (SBR) is characterized by a new set point in the balance of enterocyte proliferation and apoptosis. Since epidermal growth factor (EGF) augments both proliferation and adaptation, we sought to determine the effect of EGF receptor manipulation on apoptosis following SBR. Materials and Methods. Male ICR mice underwent 50% SBR or sham operation (bowel transection with reanastomosis) and then were given EGF (50 mg/kg/day) or saline by orogastric gavage. At 1 week, a proliferation index (PI) was measured in the ileum by BrdU uptake and an apoptosis index in crypts (cAI) and villi (vAI) scored by counting apoptotic bodies in enterocytes. In other experiments, AI was scored after SBR in mice with defective receptors (waved-2). Results are expressed as means 6 SE and evaluated statistically using ANOVA. # denotes P < 0.001. Results. Following SBR, EGF increased PI (40 6 2% vs 50 6 1% BrdU 1 cells; #), villus height (252 6 4mm vs 401 6 15 mm; #), and crypt depth (77.3 6 1.5mm vs 120.8 6 5 mm; #). When compared with sham, SBR resulted in increased cAI (0.3 6 0.02 vs 2.0 6 0.1; #) and vAI (0.4 6 0.05 vs 1.1 6 0.1; #). EGF attenuated both cAI (0.5 6 0.04) and vAI (0.5 6 0.03) following SBR. In the waved-2 mice, the highest levels of cAI (3.1 6 0.2) and vAI (3.6 6 0.3) were noted after SBR. Conclusions. Enterocyte apoptosis during adaptation is attenuated by EGF and exaggerated when the EGF receptor is defective. In addition to enhancing proliferation, suppression of apoptosis may provide a previously unrecognized mechanism for the beneficial effect of EGF during intestinal adaptation. © 1998 Academic Press Key Words: EGF; apoptosis; adaptation; small bowel resection; waved-2 mice; proliferation; enterocyte.
MATERIALS AND METHODS Animals. This study was approved by the Institutional Animal Care and Use Committee of the Children’s Hospital Research Foundation (Children’s Hospital Medical Center, Cincinnati, OH). Male ICR (Jackson Laboratory, Bar Harbor, ME) or homozygous waved-2 mice (Jackson Laboratory) were housed in groups of four at 21°C on 12-h day/night cycles (06:00 –18:00). At approximately 8 –10 weeks of life, the mice underwent either a 50% proximal SBR with reanastomosis or sham operation (bowel transection with reanastomosis alone) as our laboratory has previously described [13].
INTRODUCTION
Following massive small bowel resection (SBR), a compensatory, mitogenic response occurs in the remnant intestinal mucosa termed adaptation [1, 2]. The 1
This study was supported by a Trustees Grant from the Children’s Hospital Research Foundation (Dr. Warner, grant recipient).
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0022-4804/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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Experimental design. The first experiment was designed to determine the effect of EGF on ileal mucosal architecture (villus height and crypt depth), enterocyte proliferation, and enterocyte apoptosis following SBR. Male ICR mice undergoing sham operation or SBR were then randomized to receive either EGF (50 mg/kg/day) or an equivalent volume of saline twice daily by orogastric gavage. This dosage and route of EGF administration were previously determined to be optimal for enhancing adaptation in our murine model of SBR [13a]. Mice were sacrificed by an overdose of inhaled methoxyfluorane after 7 days. The second experiment was designed to determine the effect of adaptation following SBR on rates of enterocyte apoptosis in the background of defective EGF receptor signaling. For this experiment, sham operation or SBR was performed on homozygous waved-2 mice. These mice harbor a spontaneous point mutation within the tyrosine kinase domain of the EGF receptor gene resulting in reduced (greater than sevenfold) tyrosine kinase activity in vivo [14, 15]. Despite the perturbed EGF receptor, homozygous waved-2 mice are healthy, fertile, and distinguished phenotypically from heterozygous littermates on the basis of wavy hair and curly whiskers. Since these mice do not survive beyond 5 days after SBR due to persistent weight loss and diarrhea [10], mice were sacrificed by an overdose of inhaled methoxyfluorane after 3 days. No differences in food intake were observed between mice undergoing SBR or sham operation. Rates of enterocyte apoptosis in the ileum were compared with those rates in the ICR mice as above. Operative procedure. Details of this technique have been described previously [13]. In brief, mice were anesthetized using a continuous flow of 2% isofluorane, 90% oxygen, and 4% carbon dioxide. The abdomen was clipped, prepped with povidine iodine solution, and draped sterilely. Perioperative antibiotics were not administered. Operations were performed with the aid of an operating microscope (10 –153 magnification). Through a midline abdominal incision, the small intestine was transected 12 cm proximal to the ileocecal valve. Sham mice underwent bowel reanastomosis at this point. In previous studies, we determined that the length of bowel in waved-2 mice was roughly 20% longer when compared with the intestinal length found in nonwaved-2 strains [10]. Therefore, in waved-2 mice undergoing SBR, 14.5 cm of proximal bowel was excised distal to the ligament of Trietz, whereas 12.5 cm of proximal bowel was excised in the ICR mice to ensure approximately a 50% SBR in both strains. Intestinal anastomosis in both sham and SBR groups was performed using an end-to-end, single-layer technique with an interrupted 9-O monofilament suture. The abdominal incision was closed with an interrupted 5-O silk suture incorporating all layers. Mice were resuscitated with an intraperitoneal injection of 0.9% saline (2 ml) and allowed to recover in a warmed incubator (33°C). Mice were provided with water ad libitum for the first 24 h, and then pair feeding with liquid diet (Micro-Stabilized Rodent Liquid Diet LD 101/101; Purina Mills Inc., Richmond, IN) was initiated and adjusted daily. Histology. After the mice were sacrificed, the ileum 2 cm distal to the anastomosis was immediately removed and fecal material was gently expressed with cotton swabs. The first centimeter of tissue was fixed in 10% neutral buffered formalin for evaluation of villus height and crypt depth. The remaining tissue was used for determination of proliferation and apoptosis indices as discussed below. Fixed specimens of ileum were embedded in paraffin and oriented to provide cut sections parallel with the longitudinal axis of the bowel. Five-micrometer-thick slices were mounted and stained with hematoxylin and eosin. Microscopic measurements were performed for villus height and crypt depth using a video-assisted integrated computer program (Image 1.57TV, National Institutes of Health). Villi were measured only if the entire central lymphatic channel was visualized and crypts based on the ability to identify the crypt–villus junction on both sides of the crypt. A minimum of 15 villi and crypts were counted per sample. Enterocyte proliferation. One hour prior to sacrifice, ICR mice in the first set of experiments received an intraperitoneal injection of 5-bromodeoxyuridine (BrdU; 1 ml/100 g body wt; Zymed Laboratories Inc., San Francisco, CA). Paraffin-embedded distal ileum was sectioned (5 mm), mounted on poly-L-lysine-coated slides, and depar-
affinized. Sections were then rehydrated (100, 95, and 75% ethanol) and endogenous tissue was peroxidase inactivated with 30% hydrogen peroxide in methanol (1:9). Incorporation of BrdU into proliferating crypt cells (S-phase) was detected using a biotinylated monoclonal anti-BrdU antibody system with steptavidin–peroxidase as a signal generator. The staining reagents and methods were provided in kit form (Zymed Laboratories Inc.). A proliferation index was derived by counting the number of enterocytes per crypt that incorporate BrdU divided by the total number of cells in the crypt. Ten representative crypts were counted from each mouse. The investigator was blinded as to the origin of the tissue during the scoring procedure. Enterocyte apoptosis. Apoptosis was quantitated by immunohistochemical labeling of DNA strand breaks in enterocytes and confirmed by propidium iodide staining of similar tissue sections. Labeling of DNA strand breaks was accomplished using the ApopTag kit (Oncor, Gaithersburg, MD). In short, formalin-fixed, paraffinembedded ileal specimens were deparaffinized and then rehydrated (100, 95, and 70% graded ethanol washes). Protein was digested with proteinase K and endogenous peroxidase activity quenched with 2% hydrogen peroxide. The slides were then incubated with a reaction mixture containing terminal deoxynucleotidyl transferase and its substrate digoxigenin–11-dUTP at 37°C for 1 h. The reaction was terminated and an anti-digoxigenin–peroxidase antibody was applied for 30 min. Hydrogen peroxide was added as a chromogenic substrate. The slides were then counterstained with methyl green, washed in 100% butanol, dehydrated with xylene, and mounted. Different sections from embedded ileum were then stained with propidium iodide to confirm the findings of the labeling procedure as above. A quantitative index of apoptosis was derived by counting the number of apoptotic bodies per crypt and villus that were both nick-end labeled and had abnormal morphology (pyknotic nuclei, condensed chromatin, and nuclear fragmentation). Blinded scoring of 50 crypts and villi per mouse was performed in triplicate. Statistical analysis. Results are presented as mean values 6 SE. A two-way ANOVA was used for comparisons of group means followed by a pairwise multiple comparison procedure (Student– Newman–Keuls method). A P value less than 0.05 was considered significant.
RESULTS
Following SBR or sham operation with or without EGF, the mice tolerated the procedures well with an overall survival of 82.5% (33/40) at 7 days. Survival in the waved-2 mice was equal (5/6) after either SBR or sham operation with an overall 83% survival at 3 days. Administration of EGF was trophic to the ileal mucosa as both villus height (Fig. 1) and crypt depth (Fig. 2) were significantly greater after either SBR or sham operation. Further, EGF significantly boosted the already increased enterocyte proliferation that occurred following SBR (Fig. 3). In the mice that received saline, apoptosis was significantly increased in both the crypts (Fig. 4) and villi (Fig. 5) following SBR. On the other hand, only a slight increase (P 5 NS) in apoptosis was detected in the mice that were given EGF after SBR. In contrast, crypt and villus rates of apoptosis were significantly greater in the sham waved-2 mice than in the mice that underwent SBR and were given EGF. The highest rates of apoptosis comparing all groups were identified in the waved-2 mice following SBR.
HELMRATH ET AL.: EGF INFLUENCES APOPTOSIS DURING ADAPTATION
FIG. 1. Ileal villus height (means 6 SE) at 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal SBR. Mice were then randomized to receive either human recombinant EGF (50 mg/kg/day) or saline twice daily by orogastric gavage. *P , 0.001 sham versus SBR; #P , 0.001 SBREGF versus SBR-saline.
DISCUSSION
In this study, we first confirmed an enhancing effect of EGF on intestinal adaptation following SBR with
FIG. 2. Ileal crypt depth (means 6 SE) at 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal SBR. Mice were then randomized to receive either human recombinant EGF (50 mg/kg/day) or saline twice daily by orogastric gavage. *P , 0.001 sham versus SBR; #P , 0.001 SBR-EGF versus SBR-saline.
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FIG. 3. Enterocyte proliferation at 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal SBR. Mice were then randomized to receive either human recombinant EGF (50 mg/kg/day) or saline twice daily by orogastric gavage. A proliferation index (means 6 SE) was derived by blinded counting of the number of enterocytes per crypt that incorporate BrdU divided by the total number of cells in the crypt. *P , 0.05 SBR versus sham; #P , 0.05 SBR-EGF versus SBR-saline.
regard to morphologic parameters (villus height and crypt depth) and rates of enterocyte proliferation. Further, we have validated our previously reported observation of increased rates of enterocyte apoptosis following SBR [4]. Finally, and most importantly, we have established a possible correlation between EGF receptor activity and rates of enterocyte apoptosis during intestinal adaptation. Administration of EGF substantially impeded the increase in apoptosis that occurs after SBR. Alternatively, rates of apoptosis were highest when SBR was performed in mice with defective EGF receptor signaling. Taken together, these results endorse a novel mechanism for the beneficial effect of exogenous EGF following SBR. Epidermal growth factor may serve a dual function of stimulating enterocyte proliferation and inhibiting enterocyte apoptosis. With this paradigm, newly produced enterocytes would have a longer halflife under the influence of exogenous EGF, thus contributing toward the genesis of taller villi and deeper crypts that characterize EGF-enhanced intestinal adaptation. Consistent with our observations, other investigators have demonstrated an inhibitory effect of EGF on apoptosis. In cultured rat ovarian granulosa cells, EGF appears to inhibit apoptosis via a tyrosine kinasedependent mechanism [16]. Further, in vivo administration of EGF has been demonstrated to retard the renal tubular cell apoptosis that follows experimental acute ureteral obstruction [17]. In malignant gliomas,
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FIG. 4. Apoptosis in ileal crypts at 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal SBR. Mice were then randomized to receive either human recombinant EGF (50 mg/kg/day) or saline twice daily by orogastric gavage. waved-2 mice did not receive either EGF or saline and were sacrificed at 3 days. An apoptosis index (means 6 SE) was determined by counting the number of apoptotic bodies per crypt that were both nick-end labeled and had abnormal morphology (pyknotic nuclei, condensed chromatin, and nuclear fragmentation). Blinded scoring of 50 crypts and villi per mouse was performed in triplicate. *P , 0.05; #P , 0.05 sham-waved-2 versus sham-saline; ¤P , 0.05 sham versus SBR.
in ileal DNA and protein content that we have previously observed [9]. Similarly, the enhancing effect of EGF on enterocyte proliferation after SBR has been reported in rats [7] and now for the first time in our murine model. Further delineation of parameters such as these will be necessary to elucidate a specific mechanism for the beneficial effect of EGF during adaptation using our mouse model of SBR. In this study, we monitored very high levels of apoptosis in the ileum of waved-2 mice that underwent sham operation. These levels were elevated even higher following SBR. We did not measure proliferation in these mice as we have previously reported that this parameter is reduced when compared with wildtype (non-waved-2) mice after SBR [10]. In that study, we also noted substantially shorter villi and deeper crypts in the waved-2 mice after either sham operation or SBR. Moreover, ileal DNA and protein content did not increase after SBR. The findings of the current and past experiments with waved-2 mice therefore champion a probable link between the EGF receptor activity and rates of enterocyte proliferation, apoptosis, migration, and/or differentiation during intestinal adaptation. It is important to appreciate the technical limitations associated with deriving rates of apoptosis by counting apoptotic bodies in histological sections [24]. Since apoptotic bodies represent fragmented remnants of a cell, the number of apoptotic bodies identified in a
a frequent spontaneous mutation within the coding region of the EGF receptor results in a truncated receptor that constitutively phosphorylates tyrosine residues independent of EGF stimulation [18]. This mutation is associated with enhanced tumorigenicity. The deregulated signaling by this mutant EGF receptor appears to drive both an increase in cell proliferation and a reduction in apoptosis [19]. Finally, apoptosis is enhanced in a human colorectal carcinoma cell line (DiFi) that expresses high numbers of EGF receptors following the addition of a monoclonal antibody directed toward the EGF receptor [20]. In contrast with the above studies, treatment of A431 epidermoid carcinoma cells (another cell line known to overexpress EGF receptors) with high dosages of EGF not only inhibits proliferation, but also enhances apoptosis [21]. Similar results have been observed in MDA-MB-468 human breast cancer cells [22]. The relationship between EGF receptor signaling, proliferation, and apoptosis therefore seems to vary depending on the specific cell type or tissue studied. Epidermal growth factor enhanced intestinal adaptation following SBR as monitored by increases in villus height, crypt depth, and enterocyte proliferation. Increases in various morphologic parameters such as intestinal mucosal thickness, villus height, or crypt depth with exogenous EGF have been reported in rat models of SBR [6, 23]. This study is the first report of morphologic changes in the ileum with EGF in our murine model of SBR and substantiates the increases
FIG. 5. Apoptosis in ileal villi at 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal SBR. Mice were then randomized to receive either human recombinant EGF (50 mg/kg/day) or saline twice daily by orogastric gavage. waved-2 mice did not receive either EGF or saline and were sacrificed at 3 days. An apoptosis index (means 6 SE) was determined by counting the number of apoptotic bodies per villus that were both nick-end labeled and had abnormal morphology (pyknotic nuclei, condensed chromatin, and nuclear fragmentation). Blinded scoring of 50 crypts and villi per mouse was performed in triplicate. *P , 0.05; #P , 0.05 sham-waved-2 versus sham-saline; ¤P , 0.05 sham versus SBR.
HELMRATH ET AL.: EGF INFLUENCES APOPTOSIS DURING ADAPTATION
tissue section could originate from a single or multiple cells. Consequently, the number of apoptotic bodies identified in a given crypt probably does not equate with the exact number of enterocytes undergoing apoptosis. Additionally, the interpretation of what constitutes an apoptotic body is somewhat subjective. Apoptotic bodies may be difficult to distinguish between other histologic findings such as intraepithelial lymphocytes, damaged DNA from conditions other than apoptosis, and enterocytes undergoing mitosis. In this regard, the increased number of apoptotic bodies that we discovered in the crypts of ICR mice after SBR could have been due to greater numbers of enterocytes undergoing mitosis. This potential confusion does not explain the greater number of apoptotic bodies that we observed after SBR in the villus tip, since this is not a site of mitosis. Further, the highest frequency of what we counted as apoptotic bodies was seen in the waved-2 mice. These mice have substantially lower rates of enterocyte proliferation in response to SBR when compared with non-waved-2 mice [10]. Determination of the apoptotic index is dependent upon the number of cells in the crypt and villus. The number of crypt and villus cells and hence crypt depth and villus height are substantially increased following SBR. If the rate of apoptosis was truly unchanged or decreased following SBR, then the number of apoptotic bodies per crypt or villus unit under both conditions should markedly decrease secondary to the expansion of the crypt depth and villus height alone. The increase in apoptosis that we observed in this study therefore may underestimate the true increase in apoptosis associated with SBR since we observed greater numbers of apoptotic bodies for each deeper crypt or taller villus due to SBR. This phenomenon insinuates an even greater effect of EGF on inhibiting apoptosis since the crypt depth and villus height were even further expanded after this treatment in mice following SBR. Since apoptosis is an active cellular process, it will be important in future studies to clarify changes in the transcription of several apoptosis-associated genes. While extremely complex, a few factors that have been identified in intestinal epithelial cells that believably play integral roles during apoptosis following SBR include bax, mcl-1, and bcl-x [25–27]. Expression of deoxyribonuclease I has been linked to apoptosis in the intestine [28]. Radiation-induced apoptosis in the gastrointestinal tract has been shown to synchronize with the expression of p53 [29] and is not present in mice that are p53 deficient [30]. The product of the protooncogene bcl-2 is known to play a role in cell survival, acting as an inhibitor of apoptosis [31]. The relationship between intestinal EGF receptor stimulation and the transcriptional regulation of these genes will be an important first step toward illuminating a mechanism for the therapeutic effect of exogenous EGF following massive intestinal loss.
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