Intestinal adaptation after massive small bowel resection (SBR) is an important compensatory response to the abrupt loss of absorptive and digestive mucosal surface area. Adaptation occurs in proportion to the amount of intestine removed and results in increased length and diameter of the bowel. Although all layers of the bowel wall are involved, the predominant changes occur within the mucosa and are monitored as increases in both villus height and crypt depth, and greater content of DNA, RNA and protein per unit length (1,2). Through these significant changes, the remaining intestine attempts to compensate for the loss of mucosal surface area by increasing the capacity for intestinal absorption and digestion.
A precise understanding of adaptation with therapy designed to optimize this response may be an important element toward a more explicit and effective management of the short gut syndrome. It has been estimated that there are roughly 10,000-20,000 patients with short gut syndrome who are maintained on home intravenous feedings in the United States (3). Assuming an annual cost of roughly $100,000 per patient (4,5), the economic impact simply for the provision of intravenous nutrition for this clinical entity is on the order of 1-2 billion dollars per year. If adaptation were able to be enhanced such that the need for intravenous nutrition was able to be eliminated in only 5% of patients with short gut syndrome, a minimum of $50,000,000 would be saved annually in intravenous infusion costs alone.
Despite the significance of this important response, the mechanisms and/or mediators of intestinal adaptation are not completely understood. Multiple, diverse factors have been implicated in the initiation and/or potentiation of adaptation including luminal nutrition (6,7), pancreaticobiliary secretions (8) and various trophic gastrointestinal hormones (9,10). Previous studies of adaptation using various animal models have provided much important information, but have generally relied upon the infusion of various candidate substances or measuring changes in levels of factors in the bowel or blood after intestinal resection.
Among the growing list of proposed enterotrophic hormones and peptides implicated to play a role in the genesis of adaptation, our laboratory has focused primarily on the role for epidermal growth factor (EGF) and its intestinal receptor (EGFR). This direction was taken as a result of an earlier study in which a significantly greater adaptation response was identified in rats administered EGF over a 28-day period after SBR (11). The purpose of this review is to summarize data that has been accrued over the past decade in support of the notion that the EGF/EGFR axis is critical for a robust adaptation response.
Murine Model for SBR
Although most studies of resection-induced adaptation have utilized multiple animal species, we initially focused on defining a mouse model of SBR (12). The primary advantage to using mice is the opportunity to employ varied transgenic, mutant and knockout strains to directly test the relevance of specific genes to the adaptation process. A liquid rodent diet (Micro-Stabilized Rodent Liquid Diet LD 101/101A; Purina Mills Inc, Richmond, IN) is provided to mice for 3 days before operation. Under general anesthesia, the abdomen is clipped, prepped with a povidone iodine solution and draped in a sterile fashion. Operations are performed with the aid of an operating microscope (10-15 × magnification). A sham operation consists of division and reanastomosis of the bowel approximately 15 cm proximal to the ileocecal junction. In mice undergoing SBR, the bowel is divided approximately 15 cm proximal to the ileocecal junction and again 2-3 cm distal to the ligament of Treitz. The mesentery of the resected intestine is ligated with a single 6-0 suture and approximately 15 cm of the intervening small intestine is removed to provide a 50% SBR. Intestinal continuity is restored by an end-to-end, single-layered anastomosis using interrupted 9-0 monofilament sutures (Fig. 1). Mice are provided 5% dextrose in water, ad lib for the first postoperative night. The following day, liquid rodent diet is provided until sacrifice.
We initially observed a substantial early postoperative mortality with this model until we recognized that the usual solid rodent pellets provided for food was associated with an anastomotic obstruction. Changing the feeding regimen to a liquid rodent diet was a critical maneuver to boost overall survival rates to over 85%. Significant adaptive changes were noted to occur as soon as 24 hours after SBR. A relative plateau in adaptation seems to be realized by 1 week.
EGFR Expression, Activity and Distribution are Increased in the Remnant Bowel After SBR
Early studies from our laboratory demonstrated a very modest increase in EGF receptor expression after intestinal resection (13). In these studies, EGFR protein expression was measured from whole bowel homogenates. Because critical changes in EGFR expression may be occurring in a relatively small proportion of cells (enterocytes) included in the whole bowel, an enterocyte isolation protocol was developed. The remnant ileum was everted over a glass capillary tube and using a calcium chelation and mechanical vibration technique as described by Weiser (14), crypt and villus enterocytes were isolated. Within these isolated enterocytes, the expression of EGFR and another EGFR family member ErbB2 (c-neu) were found to be much more elevated (roughly sixfold) (15). In addition, the activation of these receptors as indicated by probing for the phosphorylated moiety was increased by roughly twofold after SBR. Intestinal resection did not affect EGFR expression in other tissues including the liver or kidney. In a related study using a rabbit model for SBR, the Sax laboratory demonstrated a partial redistribution of the EGFR to the intestinal brush border membrane of the rabbits that underwent resection (16). Changes in affinity of the EGFR for the various ligands has not been previously studied, but would be of major interest.
More recently, laser capture microdissection microscopy was used to isolate specific cell fractions from the villus and crypt region of frozen intestinal sections. The mRNA was extracted from these captured cells and a reverse transcription reaction permitted the use of real-time polymerase chain reaction to fully quantitate changes in cDNA copy number. Using this technique, the greatest increase in EGFR mRNA expression not surprisingly took place in the crypt enterocytes after intestinal resection, the site of enterocyte production (17). We were surprised to identify significant postresection increases in EGFR mRNA within the smooth muscle layers of the bowel. These results suggest that growth factor signaling in the muscle layers during adaptation may play an important role in the genesis of a full adaptation response. As such, therapy targeted toward smooth muscle within the intestinal wall might provide a novel therapeutic approach to augmenting the mucosal response to SBR.
Distribution of Endogenous EGF After Intestinal Resection
In addition to localization signaling and expression alterations of the EGFR within the adapting intestine, several studies have documented enhanced alterations in the expression of a major EGFR ligand (EGF) after SBR. In mice, the major endogenous source of EGF is the bilateral submandibular glands. After intestinal resection, salivary EGF levels were found to be elevated (18). In addition to increased salivary EGF expression, there were no changes observed in serum EGF levels and urinary excretion of EGF was reduced. In regenerating intestinal neomucosa, Saxena et al. discovered increased uptake of radiolabeled EGF within the salivary gland and duodenal mucosa (19). These findings, coupled with the observation of increased EGFR activation within the remnant intestine suggest that increased salivary EGF might be one important mechanism for increased intestinal EGFR activation during adaptation.
Because salivary EGF concentrations are regulated in large part by androgens, and males are known to have greater concentrations of salivary EGF when compared with female mice, gender differences in adaptation, EGF expression and intestinal EGFR activation after SBR were interrogated (20). Both sexes responded to SBR with an increase in salivary EGF expression. The postresection expression of intestinal EGFR was more amplified in the female mice, whose salivary levels were lower. Taken together, these results strengthen the notion that the salivary-derived EGF is important for adaptation.
EGFR Stimulation Enhances Intestinal Adaptation
Because enhanced adaptation occurred as a result of exogenous EGF in our rat model (11), the most optimal dose and route of EGF administration in mice was then determined. Intraperitoneal versus orogastric gavage of EGF revealed that the orogastric route of administration induced a much more robust adaptation response (21). In addition, a lower dose of EGF (50 μg/kg/d range) was found to be associated with a much better adaptation response when compared with the higher dosages used in earlier studies. Finally, the timing of EGF administration relative to the adaptation period was studied. If EGF was given during the first postresection week, various parameters of adaptation were found to be augmented, as expected. On the other hand, if EGF was administered for 1 week after the full adaptation response had taken place (1 month), the additive effect of EGF did not occur. This observation is important because several clinical studies which have evaluated various growth factors as a means to enhance adaptation have been traditionally comprised of a heterogeneous population of patients who were well beyond the early phase of adaptation. Because there seems to be a window of effectiveness (the immediate postresection period) for growth factors, a greater therapeutic benefit might be realized if administered solely during the early postresection adaptation period.
In other models of resection-induced adaptation, the effects of EGF administration have been somewhat variable. Although administration of EGF has been shown to stimulate mucosal proliferation in the unperturbed bowel (22,23), O'Loughlin et al. found no significant enhancement of mucosal proliferation by oral EGF in young rabbits for 10 days after a 2/3 proximal enterectomy (24). These investigators did, however, identify a three- to fourfold increase in glucose uptake in the intestine of the EGF-treated animals. Similarly, Goodlad et al. found no trophic effect of EGF in rats subjected to 75% SBR (25). In this study, the animals were fed entirely by parenteral nutrition. In another report, EGF administration to rabbits in combination with SBR was associated with significantly greater mucosal wet weight, and enhanced maltase, aminooligopeptidase and glutamine uptake capacity when compared with the SBR group alone (26). The great variability in animal species studied multiple resection models (proximal versus distal enterectomy, magnitude of resection, etc.), postoperative feeding regimens and interval of time after surgery-all likely contribute to conflicting results.
Other ligands for the EGFR include transforming growth factor alpha (TGFα). This growth factor is produced directly by enterocytes in response to injury. Administration of TGFα to mice after SBR indeed resulted in an amplified adaptation response (27). On the other hand, adaptation was noted to occur normally in waved-1 mice that are deficient in TGFα. Although there are several known EGFR ligands and there is likely great overlap in their function, these data further endorse the notion that EGF is a critical ligand for EGFR signaling during adaptation.
It is known that the EGFR is comprised of 4 major subtypes-ErbB1-4. Although ErbB1 is the major receptor for EGF, homo- and heterodimerization of receptors follow ligand binding. It may therefore be important in future studies to delineate the most effective dimerization pattern to trigger proliferation of enterocytes after resection. Further, the specific pathway downstream from the EGFR that is most important for the trigger of proliferation is presently unknown. Candidate pathways known to be activated by the EGFR include the mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription (STAT) and the phosphotidylinositol-3 kinase (PI3K) pathway. Transactivation of the EGFR by other receptors such as the G-protein-coupled receptor system may be operative as well.
Because systemic administration of EGF may secondarily trigger the expression or secretion of other peptides or hormones to affect adaptation, a transgenic mouse line was created in which EGF overexpression was targeted to villus enterocytes by virtue of an intestinal fatty acid binding promoter (28). These mice demonstrated no particular phenotype and serum EGF levels were similar between transgenic and control mice. However, when subjected to SBR, the EGF transgenic mice had an exaggerated adaptation response.
EGFR Inhibition Attenuates Intestinal Adaptation
In a strain of mouse termed waved-2, a spontaneous point mutation causing the substitution of glycine for the highly conserved valine at residue 743 in the tyrosine kinase domain of the of the EGFR results in reduced (>sevenfold) tyrosine kinase activity in vivo (29,30). Despite the perturbed EGFR, homozygous waved-2 mice are healthy, fertile and distinguished from heterozygous littermates on the basis of wavy hair and whiskers. In response to SBR, waved-2 mice with impaired EGFR signaling demonstrated a markedly impaired adaptation response (31).
Inhibition of the EGFR was accomplished via several other experimental paradigms. In the first, the major endogenous sources of EGF (the submandibular glands) were removed by performing a bilateral sialoadenectomy before SBR (32). Intestinal adaptation was found to be markedly impaired after SBR in the mice undergoing sialoadenectomy. This impaired response was rescued by exogenous administration of EGF. In another study, administration of a pharmacologic EGFR inhibitor (Iressa; AstraZeneca Pharmaceuticals) to mice after SBR blunted several parameters of intestinal adaptation (33).
Possible Mechanisms for EGFR Augmentation of Intestinal Adaptation
It is known that the intestinal mucosa is in a constant state of rapid renewal with rates of enterocyte proliferation being matched by rates of enterocyte cell death by a process termed apoptosis. The postresection adaptation response is largely a mitogenic signal with increased rates of enterocyte proliferation. In addition, elevated rates of enterocyte apoptosis have been identified in the villus tip (34) and crypt locations (35). The mechanism(s) and contribution of enterocyte apoptosis to the adaptation response is under active investigation. It is possible that manipulation of the apoptotic response may be useful as a means to enhance adaptation.
In addition to stimulating enterocyte proliferation, EGFR stimulation through administration of exogenous EGF was also found to retard rates of enterocyte apoptosis (36). Alternatively, rates of enterocyte apoptosis were found in that study to be extremely high in waved-2 mice with perturbed EGFR function. Through a series of ribonuclease protection assays, the expression of several Bcl-2 family members was found to be altered within the adapting intestine. The proapoptotic family member whose expression increased to the greatest extent after SBR was bax simultaneous with a reduced expression of the anti-apoptotic bcl-2 family member bcl-w (37). Our current working hypothesis therefore is that postresection EGFR signaling regulates the bax:bcl-w ratio to control rates of apoptosis. Support for this theory was provided in a subsequent study in which exogenous EGF was found to reduce the bax:bcl-w ratio in favor of cell survival (38).
The significance of bax expression during resection-induced apoptosis was directly tested by performing intestinal resection procedures in bax-null mice (39). These mice demonstrated normal adaptation and enterocyte proliferation. However, rates of apoptosis remained unchanged after SBR. Despite prevention of the rise in apoptosis after SBR in bax-deficient mice, various parameters of adaptation were not enhanced (40). These studies would suggest that preventing the resection-induced rise in apoptosis alone probably does not contribute significantly to adaptation. On the other hand, it is possible that EGFR stimulation of proliferation, coupled with attenuation of apoptosis will provide a dual mechanism to maximally stimulate mucosal adaptation responses.
In additional studies, genetic crossing of bax-null mice with waved-2 mice rescued the significantly elevated rates of apoptosis in the waved-2 animals and improved adaptation (41). Additional studies testing the significance of bcl-w by performing SBR procedures on bcl-w-null mice are forthcoming.
In other studies of possible mediators of resection-induced apoptosis, a possible role for the extrinsic, death-receptor triggered pathway was evaluated. The rationale for testing this alternate pathway for the induction of apoptosis is the finding that bacterial translocation of luminal bacteria to mesenteric lymph nodes and spleen are increased after SBR in mice (42). Thus, it is plausible that increased bacterial and proinflammatory cytokine activity at the mucosa level would be an important inducer of enterocyte apoptosis. Despite this notion, increased apoptosis occurred normally after SBR in both tumor necrosis factor receptor (TNFR1)-null mice and in Fas-null mice (43). In further experiments, SBR procedures were performed in germ-free rats with the finding of normal postresection increases in apoptosis (44). Collectively, these data support the concept that the main mechanism for the induction of apoptosis after SBR is via an intrinsic (growth factor) regulated pathway. The contribution of the extrinsic death receptor-mediated pathway to this response does not seem to be significant.
Beyond a specific trophic effect, there seem to be other beneficial effects of EGF on enterocytes during adaptation. EGF is capable of redistributing the preformed microvillus plasma membrane to significantly increase brush border surface area 45). In addition to its mitogenic effects, absorption of various nutrients has been shown to be augmented by EGF (46-50).
There is compelling evidence to suggest a central role for the EGFR and its signaling in enterocytes to govern the adaptation response to massive SBR. Through the development of a mouse model, we have been able to manipulate the EGFR using several positive and negative paradigms, including transgenic, mutant and knockout mice. Future studies will further investigate the contribution of apoptosis to the adaptation response and how various Bcl-2 family members including Bax and Bcl-w are directly regulated by EGF receptor signaling after SBR. Finally, futher characterization of serum factors that are capable of inducing intestinal epithelial cell growth in vitro are warranted.
The work presented in this review was generated through the support of the National Institutes of Health (RO1 DK53234 and RO1 DK59288 - to BWW) and the Cincinnati Children's Hospital Medical Center Research Foundation. This laboratory effort would not have been possible without the outstanding residents in surgery who took time out from their clinical training to make major contributions and include: Michael Helmrath, MD, Cathy Shin, MD, Richard Falcone, Jr, MD, Lawrence Stern, MD, David O'Brien, MD, Andrew Knott, MD, Russell Juno, MD, Marcus Jarboe, MD, Nicole Bernal, MD, Wolfgang Stehr, MD, George Sheng, MD and Kathryn Bernabe, MD. Finally, past and current technicians have provided excellent support and include Christopher Kemp, Jodi Williams, Sherri Profitt, Yufang Zhang and Stephanie Schmidt.
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