The Role of Apoptosis During Intestinal Adaptation After Small Bowel Resection

Transcription

1 The Role of Apoptosis During Intestinal Adaptation After Small Bowel Resection By Carlo F.M. Welters, Femke E. Piersma, David M. Hockenbery, and Erik Heineman Maastricht, The Netherlands; Seattle, Washington; and Aukland, New Zealand Background/Purpose: Adaptation after small bowel resection (SBR) is characterised by a new set point in the balance of enterocyte proliferation and apoptosis. Apoptosis is gene directed. The authors hypothesised that the adaptive response is influenced positively by antiapoptotic gene products (eg, bcl-2 gene-produced protein). The authors tested this hypothesis by studying the effect of bcl-2 overexpression on intestinal adaptation after SBR. Methods: Male bcl-2 transgenic mice, overexpressing bcl-2 in the small intestinal epithelium, and wild type control mice underwent either a 75% mid-sbr, or a sham operation. The 4 experimental groups consisted of resection wild type (n 8), transection wild type (n 6), resection bcl-2 transgenic (n 8), and transection bcl-2 transgenic (n 8). Seven days postoperatively small bowel was harvested; total weight, mucosal weight, and mucosal protein, DNA, and RNA content in jejunal and ileal tissue were determined to quantitate the hyperplastic response. Results: Compared with sham-operated animals, SBR resulted in increased total jejunal weight; mucosal weight; and mucosal protein, DNA, and RNA content. Furthermore, in the SBR groups, the jejunal mucosal weight and mucosal protein and DNA content were significantly higher in the bcl-2 transgenic mice compared with the wild-type mice. No differences were observed between any of these parameters in the transection wild-type and transgenic mice. In the ileum, similar changes were observed. The differences between resected and transected wild-type mice were less pronounced, and only total ileal weight and mucosal protein content reached statistical significance. In the transgenic animals, all ileal variables, with the exception of mucosal RNA content, were significantly higher in the SBR group than in the transected group. SBR in the transgenic mice resulted in higher ileal mucosal weight and mucosal protein, DNA, and RNA content compared with the wild-type mice. Conclusions: The results show that the murine SBR model is a true representation of the process of adaptation after SBR. Furthermore, major components of the adaptive response, both in the jejunum and in the ileum, are significantly more pronounced in the bcl-2 transgenic mice than in the wild-type control animals. Thus, it can be concluded that intestinal hyperplasia after SBR is significantly enhanced by overexpression of the anti-apoptotic bcl-2 gene product. This finding should prompt further research on the effects of antiapoptotic interventions on adaptation after SBR. J Pediatr Surg 35: Copyright 2000 by W.B. Saunders Company. INDEX WORDS: Bowel resection, adaptation, apoptosis, bcl-2 gene. THE MAMMALIAN small intestine contains a rapidly proliferating and constantly differentiating epithelium. 1 In neonates and children, loss of functional small bowel surface area after surgical resection for necrotising enterocolitis, intestinal atresia, gastroschisis, and midgut volvulus leads to an adaptive response in the residual intestine. 2,3 Depending on the magnitudes of the loss of small bowel surface area and epithelial response, From the Department of Surgery, University Hospital Maastricht, The Netherlands; the Fred Hutchinson Cancer Research Center, Seattle, WA; and the Departments of Surgery and Pediatrics, Starship Children s Hospital, Auckland, New Zealand. This study was supported by a grant from the Schumacher Kramer Foundation. Presented at the 46th Annual International Congress of the British Association of Paediatric Surgeons, Liverpool, England, July 21-24, Address reprint requests to Carlo F.M. Welters, MD, Department of Surgery, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands. Copyright 2000 by W.B. Saunders Company /00/ $3.00/0 patients may continue to be enterally fed or will suffer from short bowel syndrome and require short- or longterm total parenteral nutrition for survival. 4 In the process of adaptation, the intestinal remnant will undergo compensatory growth involving all layers of the bowel wall, leading to dilatation, lengthening, and thickening. 5-7 Most prominent is the hyperplasia of the mucosa. There is an increase in both the epithelial cell proliferation rate in the crypts and migration rate onto the villi, resulting in elongated villi with increased cellularity and deeper crypts. This villous hyperplasia is characterised by an increase in mucosal cell mass and in DNA, RNA, and protein content. 8,9 The absorption of specific nutrients per unit area of mucosa increases, although the absorption per cell decreases, probably as a result of less mature cells populating the villi. 10,11 The adaptive change is proportional to the amount of resected intestine, with most marked changes occurring in the ileum. 12,13 The process of adaptation is driven by multiple factors. Generally, 3 different groups have been identified: pancreatico-biliary secretions, nonnutritive factors, and nutri- 20 Journal of Pediatric Surgery, Vol 35, No 1 (January), 2000: pp 20-24

2 APOPTOSIS AND INTESTINAL ADAPTATION 21 tive factors. 7,12,14-19 Although many studies have been undertaken to stimulate intestinal adaptation, little information is available on the complicated cellular processes underlying the mechanism of adaptation. 4,16 Concerning intestinal adaptation and apoptosis in the gut, there is hardly any information at all. Apoptosis (a combination of the Greek words apo, off, and ptosis, falling) is a highly regulated form of programmed cell death defined by distinct morphological and biochemical features. 20 Apoptosis plays a major role in tissue homeostasis. 21 In complex differentiating epithelia, like the intestinal mucosa, apoptosis is believed to be crucial in the maintenance of the balance between cell proliferation and cell loss. 22 The process of apoptosis is gene directed, and cellular products regulate the selfdestruction of the cell. 23 One of these products, bcl-2, is a mitochondrial membrane protein that blocks programmed cell death. 24 Bcl-2 transgenic mice with targeted intestinal overexpression of bcl-2 are resistant to ischemiareperfusion injury and radiation, with diminished apoptotic cell death. 25,26 The unstressed intestinal epithelium in these animals is hyperplastic compared with wild-type littermates. 25 However, bcl-2 deficient mice are found to have small intestinal defects, characterised by retarded development, accelerated exfoliation of enterocytes, and very few mitotic progenitor cells in the crypts. 27 On the basis of the morphological characteristics of bcl-2 overexpressing and bcl-2 deficient mice we hypothesised that the bcl-2 gene product would play a significant role in the hyperplastic response after small intestinal resection. Ideally one would test this hypothesis in both types of transgenic mice with the wild type as a control. However, the significant extraintestinal abnormalities in bcl-2 deficient mice 27 make a small intestinal resection experiment not feasible, thus, we tested our hypothesis in the bcl-2 overexpressing mice and the wild type controls. Animals MATERIALS AND METHODS Male bcl-2 transgenic and wild-type mice (strain C57Bl/6 C3H/ HeJ; Animal Health Resources, Fred Hutchinson Cancer Research Center, Seattle, WA) were used (n 16, 30 1 g and n 14, 28 1 g, respectively). The bcl-2 transgenic line was created on the C57Bl/6 C3H/HeJ genetic background and will be described in a separate report. The transgene consists of human bcl-2 cdna fused to the liver fatty acid binding protein promotor. Animals were housed under standard conditions with free access to chow and water. The Institutional Animal Care and Use Committee of the Fred Hutchinson Cancer Research Center in Seattle approved these experiments. Experimental Groups Transgenic and wild-type mice were assigned randomly to undergo resection or transection. This led to 8 wild-type mice with a small bowel resection, 6 wild types with a transection, 8 transgenics with a resection, and 8 transgenics with a transection. Surgical Procedures The surgical procedure is a modification of a technique for small bowel resection in the rat. 28 After an overnight fast, anaesthesia was induced with an intraperitoneal injection of 10 mg ketamine per 100 g body weight and a subcutaneous injection of 0.5 mg xylazine per 100 g body weight. Hereafter, mice received a subcutaneous injection of 4 ml normal saline per 100 g body weight to compensate for peroperative fluid losses. A midenterectomy was performed by removing 75% of the small intestine, leaving approximately 6 cm of the terminal ileum and 6 cm of the proximal jejunum. An anastomosis was made between the remaining jejunum and ileum with a single running Prolene 7-0 (Ethicon, Hamburg, Germany), and the abdomen was closed with Vicryl 3-0 (Ethicon). After this procedure, animals were allowed to recover and transferred back to their metabolic cages. Postoperatively, animals were fed with liquid feeding (liquid rat diet, Bio-Serv, Frenchtown, NY; 1.5 g N/kg/d and 185 kcal/kg/d). After 7 days, animals were anaesthetised again, and a laparotomy was performed. The proximal part of the jejunum and the distal part of the ileum were excised, freed of mesentery, and measured. Hereafter, the parts of intestine were opened longitudinally, rinsed in ice cold saline, blotted dry, and weighed. The mucosa was scraped off, using a glass slide. After weighing, the mucosa was snap-frozen in liquid nitrogen and stored at 70 C until further processing. Biochemical Analysis The protein content of the jejunal and ileal mucosa was determined by the folin reagent method as described by Lowry et al, 29 using a commercially available protein assay (DC Protein Assay, Bio-Rad, Hercules, CA). Mucosal samples were homogenised in Tri-reagent (Sigma Chemical Company, St Louis, MO), and DNA and RNA were isolated using acid guanidinium thiocyanate-phenol-chloroform extraction. 30,31 The DNA and RNA content of the isolates was measured spectrophotometrically. Statistical Analysis Results are expressed as mean SEM. Differences between groups were tested using the Mann-Whitney U test. 32 Differences were considered significant at P less than.05. RESULTS Weight of Mice At resection, the weight of wild-type mice was not significantly different from the weight of transgenics (28 1 v 30 1 g). At the time of death, 1 week after resection, differences in weight increased, but still did not reach significance (wild-type transection, 23 1; wild type resection, 24 1; transgenic transection, 27 2; transgenic resection, 27 2). Jejunum After 1 week, resection resulted in both wild-type and transgenic animals, in a higher weight of the jejunum, compared with transected animals (Table 1). This was accompanied by a higher mucosal weight with a higher protein content of the mucosa (Table 1), in combination with a higher DNA and RNA content of the mucosa (Fig 1). No differences could be observed between the transected groups of wild-type and transgenic animals. This was not the case for the resected groups, in which transgenic animals had a significant higher mucosal weight and

3 22 WELTERS ET AL Table 1. Total and Mucosal Weight and Mucosal Protein Content of Jejunum and Ileum Wild Type Transgenic Transection Resection Transection Resection Jejunum Total weight a b Mucosal weight a bc Mucosal protein a bc Ileum Total weight a b Mucosal weight bc Mucosal protein a b Total weight, mucosal weight and mucosal protein in mg/cm intestine. Statistics: Mann-Whitney U, P 0.05: a Wild type resection versus wild type transection; b Transgenic resection versus transgenic transection: c Transgenic resection versus wild type resection. protein content (Table 1), together with a higher mucosal DNA content (Fig 1). Ileum The changes observed in the ileum after small bowel resection were very similar to the changes in the jejunum. Fig 1. Mucosal DNA and RNA content in jejunum (upper panel) and ileum (lower panel). Statistics: Mann-Whitney U, P F.05; a, Wild-type resection versus wild-type transection; b, transgenic resection versus transgenic transection; c, transgenic resection versus wild-type resection. However, the differences between the resected and the transected groups of the wild type mice were not as prominent: it was only statistically significant for the weight of the ileum and for the mucosal protein content (Table 1). In transgenic animals, all variables, with the exception of mucosal RNA content (Fig 1), were significantly higher in the resected group than in the transected group. Again, when comparing transected wild-type and transgenic mice, no significant differences could be observed. Small intestinal resection in transgenic mice resulted in a higher ileal mucosal weight (Table 1) and higher levels of ileal mucosal DNA and RNA compared with wild-type mice (Fig 1). DISCUSSION In animal and human studies intestinal adaptation after small bowel resection is characterised by a hyperplastic response of the mucosa. It is widely accepted that this hyperplastic response is the result of an enhanced enterocyte proliferation. Very few studies have focussed on the role of apoptosis and cell survival in the process of adaptation after small bowel resection. Apoptosis is the name given by Kerr et al in to the process of physiological programmed cell death. In general, there has been an underestimation of the quantitative importance of apoptosis in the dynamic control of cell populations, because of both short duration of the apoptotic process and the rapid clearance of apoptotic debris. 34,35 It is now recognised that apoptosis has broad regulatory roles in health and disease. Tissue atrophy and proliferation, disease processes, and malignant transformation all have been associated with apoptotic and antiapoptotic gene activity. These concepts have led to several therapeutic strategies focused on apoptosis, such as the induction of apoptosis for treatment of malignancies or enhancing tissue repair by inhibiting apoptosis. 20 The role of apoptosis in the turnover of the gastrointestinal epithelium is controversial. 36 The bcl-2 protooncogene has the unique effect of blocking apoptosis without increasing cell proliferation. 24,37,38 The observation that bcl-2 deficient mice have shortened and bizarreshaped villi, 27 indicates that apoptosis plays an important role in the mucosal architecture of the intestine and possibly also mucosal function. What does this mean for the process of adaptation after small bowel resection? The place of enterocyte apoptosis after small bowel resection has been studied by Helmrath et al. 39 They showed in mice that adaptation after small bowel resection increases the rates of both enterocyte proliferation and apoptosis. Our hypothesis was that overexpression of the antiapoptotic gene bcl-2 would provide an additive effect to the hyperplastic response after small bowel resection. First,

4 APOPTOSIS AND INTESTINAL ADAPTATION 23 we showed that in the employed model of small bowel resection in both wild-type and bcl-2 transgenic mice, a true small bowel resection and adaptation pattern could be observed. The significant rise in total bowel weight, mucosal weight, mucosal protein, DNA, and RNA content after small bowel resection are well-known characteristics of a hyperplastic response. 8,9 Second, and even more importantly, we found that the hyperplastic response after small bowel resection was significantly more pronounced in the bcl-2 transgenic mice compared with the wild-type control mice. This leads to the conclusion that the overexpression of the bcl-2 gene has a positive effect on small bowel adaptation after small bowel resection. We would have liked to test this conclusion in bcl-2 deficient mice to determine whether hyperplastic adaptation is possible in the absence of such a critical survival factor. The significant other abnormalities in these mice made such an experiment impossible. The crucial question is whether apoptosis blockers will be useful in the treatment of short bowel syndrome. It has been suggested that inhibitors of apoptosis might be useful in the treatment of a number of conditions like heart attacks and strokes. 23,40 They also might be useful in the treatment of short bowel syndrome. There is evidence for a role of IGF-1, a recognised survival factor, on the balance between proliferation and apoptosis in intestinal adaptation after small bowel resection Recently, it has been reported that epidermal growth factor (EGF), which augments both proliferation and adaptation, attenuates enterocyte apoptosis. 45 The adaptation-enhancing effects of fibre, short chain fatty acids, and other so-called nutriceuticals have to be revisited in light of their possible antiapoptotic properties. We propose that there is a need to direct adaptation promoting strategies after small bowel resection toward research into the role of antiapoptotic-agents. 1. Wright NA, Alison M: The biology of epithelial cell populations. New York, NY, Oxford University Press, Weber TR, Tracy T, Connors RH: Short-bowel syndrome in children. Arch Surg 126: , Sondheimer JM, Cadnapaphornchai M, Sontag M, et al: Predicting the duration of dependence on parenteral nutrition after neonatal intestinal resection. J Pediatr 132:80-84, Rubin DC, Swietlicki EA, Wang JL, et al: Enterocyte gene expression in intestinal adaptation: Evidence for a specific cellular response. Am J Physiol 270:G143-G152, Rombeau JL, Rolandelli RH: Enteral and parenteral nutrition in patients with enteric fistulas and short bowel syndrome. Surg Clin North Am 67: , Scott RB, Sheehan A, Chin BC, et al: Hyperplasia of the muscularis propria in response to massive intestinal resection in rat. 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Endocrinology 139: , Helmrath MA, Shin CE, Erwin CR, et al: The EGFEGF-receptor axis modulates enterocyte apoptosis during intestinal adaptation. J Surg Res 77:17-22, 1998 Discussion G. Gittes (New York, NY): Thank you for that very clear presentation. I have a couple of questions about the role of bcl-2 in relation to animals and how these data may be interpreted. For example, do you have any evidence of what the expression of bcl-2 is or what its role may be in normal adaptation as opposed to following transection? What is the differential role of bcl-2 in the epithelium as it divides migrates and undergoes apoptosis versus the lamina propria, which must enlarge in an enlarged villus. C.F.M. Welters (response): As far as I know it is not known whether bcl-2 is expressed more after resection compared with animals undergoing a transection. We are currently studying this question in our wild-type groups. It is known that in normal intestinal tissue, bcl-2 is only expressed in the crypts. Up on the villus, enterocytes loose their bcl-2 properties. This could be the reason why higher up on the villus, enterocytes go into apoptosis and are being shed into the lumen of the intestine. The relationship between an enhanced bcl-2 expression in enterocytes, enlarged villi, and the lamina propria has to be elucidated further. H. Ford (Pittsburgh, PA): I take issue with your conclusion advocating treatment with antiapoptosis factors for short bowel syndrome. Number one, you never really showed us any evidence that there is accelerated enterocyte apoptosis in small bowel syndrome, and number two, the process of apoptosis or homeostasis of the enterocyte in the intestine is a complex and highly regulated process that involves not only bcl-2 but a number of other proapoptotic and antiapoptotic genes. For instance, it is not just bcl-2 alone but the balance between bcl-2 and bax, or bcl-2 and bad protein, or bcl-x long and short that influence apoptosis. These are some of the factors that intermingle to determine the fate of the enterocyte, whether it is going to survive or undergo apoptosis. To look simply at bcl-2 transgenic mice, which really have nothing to do with intestinal adaptation in humans and advocate that we should avoid using fiber in the diet and so forth is really a long stretch that does not really stick with the data that you presented. C.F.M. Welters (response): Of course, it is very worthwhile looking at all these other factors and their roles in this process, and we are currently investigating our histological material for changes in the expression of bcl-2, bax and bcl-x. However, we were not interested in the role of bcl-2 on other bcl-2 family members. We wanted to test if an antiapoptotic strategy would lead to an increased adaptation after a resection, and the bcl-2 transgenic mouse model gave us the opportunity to answer this question. I totally disagree with you on the subject of using fibre. It is generally accepted that fibre, supplemented enterally, stimulates intestinal adaptation. Also, we did not suggest that we studied the effect of fibre on intestinal adaptation. We merely suggested, as future research projects, that it would be very worthwhile to study the effect of wellknown adaptation enhancing factors, like IGF, EGF, glutamine, and fibre, on apoptosis of enterocytes during the process of adaptation.