Inflammatory bowel diseases (IBDs), including ulcerative colitis (UC) and Crohn disease (CD), are chronic inflammatory disorders of the gastrointestinal tract. The inflammation observed in UC is continuous and limited to the mucosa of the colon. In contrast, CD is characterized by discontinuous, transmural inflammation located in any region of the gastrointestinal tract. Both diseases, despite the multiple differences, share similar clinical symptoms, including weight loss, growth failure, and nutritional deficiencies (1). Nutritional deficiencies are more frequent in CD than in UC and in active disease versus in disease remission.
Pathophysiology of weight loss and growth failure is multifactorial. Chronic malnutrition is secondary to inadequate energy intake and increased energy expenditures, but a direct inhibiting effect of proinflammatory mediators secreted from the inflamed tissues also is essential (2).
Treatment options in IBD include pharmacological, nutritional, and surgical therapies. The aim of the treatment is the induction and maintenance of remission, correction of nutritional deficiencies, and prevention of complications.
Indications for enteral nutritional therapy in the treatment of IBD include exclusive enteral nutrition (EEN) in the active phase of the disease, supplemental enteral nutrition to maintain disease remission, and nutritional support to achieve adequate weight gain and growth. EEN is a more effective therapeutic method in achieving clinical remission in children with IBD than in adults (3,4). A recent meta-analysis of pediatric trials indicated that not only is the efficacy of EEN comparable to cortricosteroid therapy but also the former is a safer method of treatment, without adverse effects typical of corticosteroids, which are often observed in patients treated using this modality (5,6).
EEN decreases disease activity and promotes mucosal healing in patients with IBD, but the mechanism of action of this therapy is still poorly understood. Some reports suggest that the curative effect of this method of treatment is associated not only with improvement of nutritional status of the patients but also with stimulation of the anti-inflammatory mediators (7).
Vascular endothelial growth factor (VEGF) and tumor growth factor-beta 1 (TGF-β1) are pleiotropic and multifunctional growth factors. They play a crucial role in the early stage of inflammation, stimulating the healing process and angiogenesis (8,9). VEGF and TGF-β1 also have strong chemotactic attractions, which increases recruiting of circulating effector cells into the inflamed region and facilitates tissue repair (10). Data derived from the latest studies on patients with CD confirmed the influence of nutritional treatment with elemental diet on secretion of inflammatory mediators in the intestine (11). Additionally, enteral nutrition therapy with liquid diet containing TGF-β was shown to induce clinical remission associated with mucosal healing and decreased levels of proinflammatory cytokines in patients with CD (12).
Nevertheless, there are no data about VEGF and TGF-β1 concentrations during EEN in children with IBD. The objective of our study was to assess the influence of EEN on VEGF and TGF-β1 concentrations in the serum of children and adolescents with IBDs.
PATIENTS AND METHODS
Thirty-nine children and adolescents with IBD (20 boys, 19 girls; mean age 14.2 years, range 6.2–18 years) were enrolled in the study. The CD group consisted of 24 patients (13 boys, 11 girls; mean age 13.8 years, range 6.2–17.4 years) and the UC group included 15 patients (8 boys, 7 girls; mean age 14.3 years, range 7.5–18.0 years). The clinical, demographic, and serological characteristics of the CD and UC groups are summarized in Table 1. The control group consisted of 25 healthy children (14 boys, 11 girls; mean age 14.5 years, range 7.8–18.0 years). Children and adolescents from the control group were healthy, without any pathological clinical signs and complaints, and were not fed with EEN but with a normal oral diet, adequate for age, in keeping with protein and energy daily requirements.
The diagnosis of CD and UC was based on the Porto criteria (13). The clinical activity of CD was measured with the Pediatric Crohn Disease Activity Index (PCDAI), and the clinical remission was defined as a total PCDAI score <10 points. The clinical activity of UC was measured with the modified Truelove-Witts clinical index, and clinical remission was defined as a total score <2 points (14,15).
The improvement of nutritional status was defined as an increase of patient's weight, the Cole index, and BMI z score as compared with the baseline values. The Cole index was defined as weight/height2-for-age, and consisted of the squared weight/height ratio expressed as the percentage of the same ratio calculated for a reference child of the same age and sex (16).
All of the patients with IBD were newly diagnosed, in the active phase of the disease. None of the above patients was treated with pharmacological therapy before the enrollment in the study. Only 3 patients with CD underwent previous surgical procedures (2 appendectomies, 1 anal abscess operation). EEN was used for 6 weeks via a nasogastric tube for 24 hours/day, supplying 120% to 150% of protein and energy daily requirements in a semielemental, fiber-free diet (Peptisorb, Nutricia, Zoetermeer, the Netherlands). Additionally, for the first 2 to 3 weeks, in both the CD and UC group we used oral antibiotic therapy with metronidazole (15 mg/kg). The most recently published research studies showed that antibiotic therapy may be helpful in both patients with CD and patients with UC, decreasing clinical activity of the disease and the level of proinflammatory mediators (17–20).
We also used oral treatment with 5-aminosalicylates (50 mg/kg) in patients with CD and patients with UC (21). None of the patients with IBD was treated with antitumor necrosis factor (TNF) alpha therapy and systemic or local steroids. The antibiotic and 5-aminosalicylate therapy was introduced at the same time as EEN. VEGF and TGF-β1 concentrations were assessed 3 times in the CD and UC groups, at baseline, before starting and after 2 and 4 weeks of EEN, and once in the control group, using commercially available enzyme-linked immunosorbent assay (ELISA) immunoassays (R&D Systems, Minneapolis, MN). During week 6 of the study, we gradually decreased the dose of EEN and introduced a normal oral diet. Initially, we planned to assess serum VEGF and TGF-β1 concentrations also after 6 weeks of EEN, but because of the different levels of compliance of the patients and individual tolerance of the normal oral diet, we were unable to switch every patient from EEN to a normal oral diet at the same time and with the same dose. This is why, in our opinion, it was impossible to interpret and compare the VEGF and TGF-β1 results in week 6 of the study, and we finally disregarded these data in the further analysis. VEGF and TGF-β1 concentrations were expressed as median values and ranges.
Statistical analysis was performed with Statistica 7.0 software (StatSoft, Tulsa, OK) using the Mann-Whitney U test, Wilcoxon signed-rank test, and Spearman correlation rank test. P < 0.05 was considered statistically significant.
The study protocol was approved by the Jagiellonian University bioethical committee, and informed consent was obtained from all patients’ legal guardians and patients older than 16 years enrolled in the study.
Children and adolescents from both the CD and UC groups improved their nutritional status and decreased clinical activity of the disease after 4 and 6 weeks of EEN. Patients with CD achieved faster disease remission, and the improvement of their nutritional status was statistically significantly higher than in the case of patients with UC (Table 2). We also observed an improvement in clinical and laboratory parameters in both the CD and UC groups after 4 and 6 weeks of EEN (Table 3).
We found statistically significantly higher serum VEGF concentrations in the CD group at baseline before starting EEN as compared with the UC group and the controls, strongly decreased values after 2 weeks, and continuously slightly decreased after 4 weeks of EEN. There was also a statistically significant increase in serum VEGF concentrations at baseline in the UC group as compared with the controls; the values decreased after 2 weeks and slightly increased after 4 weeks of EEN. Serum VEGF concentrations decreased during EEN in both the CD and UC groups, but after 2 and 4 weeks of treatment, the relevant values were higher in the CD group as compared with the UC group (Fig. 1).
At baseline, serum TGF-β1 concentrations were comparable in the CD and control groups, but lower when compared with the UC group. Serum TGF-β1 concentrations significantly increased in CD group after 2 and 4 weeks of EEN when compared with the baseline values and the controls. We found in the UC group that serum TGF-β1 concentrations assessed at the baseline and after 2 weeks of enteral therapy were significantly increased as compared with the controls, and decreased after 4 weeks of EEN. Although TGF-β1 concentrations did not show statistically significant differences between the CD and UC groups after 2 weeks of EEN, they were significantly higher in CD after 4 weeks of the treatment. During the whole course of EEN, serum TGF-β1 concentrations showed the opposite trends in both groups, increasing in the CD group and decreasing in the UC group (Fig. 2). Moreover, serum TGF-β1 concentrations significantly correlated with protein and energy daily intake during EEN in the CD group (R = 0.95; P < 0.05), but not in the UC group.
We did not observe statistically significant differences of serum VEGF and TGF-β1 concentrations among subgroups of patients with CD and patients with UC with different disease location, as well as with different CD phenotypes. In both the CD and in UC groups, we noted a decrease of laboratory indices of inflammation (C-reactive protein [CRP], erythrocyte sedimentation rate [ESR]), and clinical activity of the disease, but they did not correlate with serum VEGF and TGF-β1 concentrations (Table 3). There were also no statistically significant differences among subgroups of patients with CD and patients with UC classified according to clinical and serological (perinuclear anti-neutrophil cytoplasmic autoantibodies, anti-Saccharomyces cerevisiae mannan antibodies) parameters.
Enteral nutrition is an effective treatment option with no adverse effects for patients with IBD, currently recommended as the first-line therapy in children with active CD and in adults when treatment with corticosteroids is not feasible (22). There is some evidence suggesting that this therapy may be more effective in children than in adults, especially in CD in contrast to UC (23,24).
Enteral nutrition therapy may be different with respect to composition of the enteral formulas and route of administration. A meta-analysis of clinical trials in adult patients with CD showed no statistical differences between elemental and nonelemental diets (24). Enteral nutrition was administered through a nasogastric tube in the majority of studies; giving the nutrients orally was followed by frequent intolerance of the prescribed volume (25).
The mechanism underlying a therapeutic response to enteral nutrition therapy remains unclear. There are a few theories that try to explain anti-inflammatory properties of enteral nutrition. Initially, low antigenic load was thought to be responsible for its curative effects, but the most recent studies indicated downregulation of mucosal proinflammatory cytokines as the possible factor responsible for inducing remission (7,26).
In our study, we assessed VEGF and TGF-β1 concentrations in the serum and not in the intestinal mucosa. Evaluation of inflammatory mediators in biopsy specimens of intestinal mucosa is more sensitive and allows for assessing their changes during EEN in the site of inflammation, yet to assess such changes we need to perform endoscopical examinations under sedation or general anesthesia, which is an invasive procedure in children and adolescents. Thus, a repeated assessment of inflammatory mediators in intestinal mucosal specimens during a few weeks of EEN is difficult to perform because of ethical considerations.
The results of the recent studies confirmed the secretion of the inflammatory mediators in the intestinal mucosa of patients with IBD.
Fell et al (7) found in children with CD a 10-fold increase of TGF-β1 mRNA in the terminal ileum but not in the colon after 8 weeks of enteral nutrition with an oral polymeric diet, associated with a decrease in proinflammatory mediators (interleukin-1β [IL-1β], IL-8, interferon-γ). This observation is in concordance with our findings of increased serum TGF-β1 concentrations during EEN.
The results of the study of Griga et al confirmed the intestinal mucosa of patients with IBD as a source of circulating VEGF (27). The authors found in the supernatants of cultured mucosal biopsies of patients with UC and CD a significantly higher spontaneous production of VEGF as compared with normal mucosa of the controls. Additionally, there was increased spontaneous VEGF production in inflamed compared with noninflamed mucosa of patients with CD (27).
Yamamoto et al (11) found that an elemental diet used for 4 weeks in patients with active CD decreased inflammation and promoted endoscopic and histopathologic mucosal healing in terminal ileum and colon. Also, mucosal concentrations of proinflammatory cytokines, which were increased before treatment, decreased to the values observed in the control group after enteral nutrition therapy.
Because the inflamed intestinal mucosa is a source of inflammatory mediators, different disease location, both in CD and in UC, may affect VEGF and TGF-β1 concentrations. The small intestine location of CD may be associated with different efficacy of EEN and different mediator concentrations from location in the colon. We did not observe statistically significant differences in the investigated mediators among the subgroups of patients with CD and patients with UC with different disease locations. In our study, the most frequent location was the large bowel in the CD group (46%) and pancolitis in the UC group (73%), which may have an impact on the results.
Angiogenesis, the growth and proliferation of new blood vessels, is essential in the early stage of mucosal healing and tissue repair process in the inflamed intestine. There are a number of angiogenic factors that may be important in the neovascularization, including VEGF and TGF-β (28).
Experimental studies on an animal model of IBD showed that TGF-β gene–deficient mice experienced a more extensive autoimmune process with inflammatory infiltrates, involving multiple organs, including the intestine (29). Hahm et al also reported that inactivation of TGF-β signaling in the mouse intestine led to the development of spontaneous colitis. This observation may confirm the participation of TGF-β signaling in regulating immune homeostasis in the intestine and its contribution to the development of IBD (30). Moreover, the blockade of TGF-β1 signaling maintained the chronic production of proinflammatory cytokines and increased the inflammatory process in IBD (31).
It is well known that VEGF is increased in serum and inflamed tissues and contributes to the chemotactic activity of endothelial cells, stimulating the formation of new vessels in autoimmune diseases, such as rheumatoid arthritis (32). There are some similarities in the pathogenesis and mediator-induced pathways in IBD and rheumatoid arthritis. Also, a similar positive effect of anti-inflammatory and immunosupressive drugs used in both diseases (steroids, methotrexate, infliximab) may suggest that VEGF and TGF-β play a similar role in mediating inflammation process in the tissue (32), but the role of VEGF and TGF-β in angiogenesis in IBD has not been established.
During inflammatory processes, VEGF is produced and released by both the inflamed intestinal mucosa and peripheral blood mononuclear cells (33–36). There are no data on serum VEGF concentrations in IBD children in the literature. Griga et al found increased serum VEGF concentrations in adults with active IBD, both in patients with CD and patients with UC, compared to inactive patients with IBD and healthy controls (35). Nevertheless, no significant differences were observed between inactive patients with IBD and the controls (37). Our results in children and adolescents with IBD are in concordance with these findings in adults with active CD and UC. Additionally, we found elevated serum VEGF concentrations in children and adolescents with IBD after 2 and 4 weeks of treatment, during recovery, which was not observed in adults.
Koch et al also found higher concentrations of VEGF in the acute phase of rheumatoid arthritis as compared with other types of arthritis (32). Clinical trials of anti-TNF-α antibody (infliximab) in rheumatoid arthritis showed a remarkable therapeutic efficacy in reducing both clinical and laboratory indices of disease activity (38). In addition to that some authors demonstrated that production and release of VEGF by synovial cells was reduced during anti-TNF-α therapy alone or combined with methotrexate (39,40).
Therapeutic efficacy of anti-TNF-α therapy, with both infliximab and methotrexate, is widely reported in acute and refractory IBD, especially in CD. The effect of these drugs also reduced clinical activity and decreased laboratory indices of inflammation (41–43).
In our study, in addition to EEN, 5-aminosalicylic acid and metronidazole were used in the pharmacological treatment of UC and CD, but there are no data on the effect of these drugs on serum angiogenic growth factors concentrations in IBD in children and adolescents. The confirmed high effectiveness in the treatment of IBD has been demonstrated for steroids and anti-TNF-α therapy, which were not used in our study. Berni Canani et al (4), in their study on children with newly diagnosed IBD, found higher effectiveness of enteral nutrition compared with steroid therapy, but they did not assess any inflammatory mediators.
Although there are no data on VEGF production in patients with IBD during anti-TNF-α therapy, the possible mechanism through a decrease in VEGF production may be responsible for inhibition of inflammation, as it was reported in rheumatoid arthritis (39,40).
Assessing TGF-β1, we found its concentrations increased in the active stage of the disease at baseline, before starting EEN in the UC group as compared to the CD group and the controls. In UC we did not note a significant increase of serum TGF-β1 during a 4-week course of EEN and its concentrations did not correlate to UC activity.
There are no studies in the literature on TGF-β1 concentration in children with IBD. In the published studies on adult patients with IBD, there are rather conflicting results related to the serum TGF-β concentrations.
Sturm et al did not find differences in plasma TGF-β concentrations among adult patients with CD and UC and healthy controls (42). On the other hand, Sambuelli et al, in their study of adult patients with UC, showed similar findings as we did in our children and adolescents (43). They found elevated serum TGF-β1 concentrations in active disease as compared with controls, which increased after 1 week of treatment in patients who responded to anti-inflammatory therapy. In their study, serum TGF-β1 concentrations negatively correlated to UC activity and decreased after 6 weeks of treatment. No changes of TGF-β1 concentrations were observed in nonresponding patients. The authors interpreted these data of increased production of TGF-β1 in affected intestine as participation in the injury-repair process (43).
Studies in which modified formulas enriched with immunological mediators such as TGF-β were used in enteral nutrition therapy. Fell et al (12), using 3 different polymeric formulas enriched with TGF-β, showed that enteral treatment induced clinical remission associated with mucosal healing. Mucosal healing was associated with a decrease in proinflammatory cytokines, such as IL-1, IL-8, and interferon-γ, in mucosal specimens of the terminal ileum and colon and an increase in TGF-β in the terminal ileum. These results suggest that clinical remission during enteral nutrition therapy is a result of a reduction in inflammatory process rather than an improvement in nutritional status, and confirm the crucial role of TGF-β in this process.
Bannerjee et al showed that the anti-inflammatory effect of EEN preceded the nutritional effect (44). Using EEN for 6 weeks in children with moderate and severe CD, the authors demonstrated an early decrease in PCDAI score and laboratory indices (CRP, ESR, IL-6) in the first 7 days of treatment, maintained until the end of the study. The specific enteral diet used in this study (AL 110, Nestle, Vevey, Switzerland) contained TGF-β, which was postulated for therapeutic activity of the formula used (7).
Hartman et al, in a retrospective study on 28 children with CD using enteral nutrition therapy with polymeric formula enriched with TGF-β 2 (Modulen IBD, Nestle), reported a decrease in clinical activity of disease and improvement in nutritional status of children with CD as compared with children fed with a non-TGF-β2–enriched formula (45).
We used a different formula (Peptisorb, Nutricia, Zoetermeer, the Netherlands) that did not contain TGF-β. We also observed both a clinical response for the therapy used and an increase in TGF-β1 in children and adolescents with CD after 2 and 4 weeks of EEN. Because we did not assess serum TGF-β1 concentrations during the first few days after starting EEN, it is not possible to state during which days of nutritional treatment this increase occurred.
Our observations confirm a potential role of TGF-β in the stimulation of healing and recovery in children and adolescents with CD. The time for clinical response in patients with CD treated with EEN is comparable to using TGF-β–enriched formulas and formulas stimulating TGF-β release.
In summary, our results showed in children and adolescents with active IBD an increased serum VEGF concentration in the CD group as compared with the UC group and controls, markedly decreasing during EEN. Serum TGF-β1 was higher before starting enteral nutrition in the UC group as compared with the CD group and controls. TGF-β1 increased in CD but not in the UC group, after 2 and 4 weeks of EEN. Children and adolescents from the CD group achieved disease remission faster than the UC group, and the weight gain of patients with CD during EEN was higher when compared with patients with UC. Additionally, TGF-β1 concentration correlated with protein and energy daily intake during EEN in the CD group.
Different effectiveness of EEN in achieving remission in CD and UC may result from a modification of growth factor production. EEN stimulated TGF-β1 production in patients with CD but not in patients with UC, which possibly resulted in the higher effectiveness of this method of treatment in this group of patients.
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