Clinical Efficacy And Mechanism Of Action On Angiogenesis Of Thalidomide In Pediatric Patients With Crohn’s Disease

Crohn’s disease, as a clinical type of inflammatory bowel disease, is a chronic inflammatory disorder of gastrointestinal tract. The incidence of IBD in China showed a rising trend. Crohn’s disease is more common in pediatric patients than ulcerative colitis. Although there are many treatment options for IBD, it is still a therapeutic challenge for patients who are refractory to conventional agents.The etiology of IBD remains unclear. Recently, scientific evidences suggest that angiogenesis is an important process in the initiation and perpetuation of IBD. The expression of pro-angiogenic factors in IBD has become a hot issue in research. VEGF and angiopoietin/Tie system play a critical role in the angiogenesis of IBD.Our preliminary study presented thalidomide could be used for the treatment of moderate to severe refractory CD patients in children. And thalidomide has potent ability to anti-angiogenesis. According to the previous research, we aim to study in this thesis to evaluate the long-term efficacy of thalidomide for treatment of pediatric CD patients, to investigate the contribution of angiogenesis to the progression of pediatric CD and to elucidate the mechanism of action of thalidomide on angiogenesis.Part I Clinical efficacy and safety of thalidomide for treatment of pediatric Crohn’s diseaseObjective: To evaluate the long-term clinical efficacy and safety of thalidomide therapy in pediatric CD patients who are refractory to corticosteroids, or corticosteroids dependent, or those who are concomitant with tuberculosis infection.Methods:Pediatric patients with refractory Crohn’s disease were enrolled in this open-label study from Children’s Hospital of Fudan University between November 2006 and August 2012. Patients were administered thalidomide (2 mg/kg/d) orally at baseline. The clinical activity of disease was assessed with the Pediatric Crohn’s Disease Activity Index (PCDAI) at baseline and at 1,3,6,12 months. Weight-for-age Z score, laboratory evaluations and reduction in corticosteroid requirement were also used to assess clinical response. Adverse effects were recorded at each visit.Results:A total of 17 patients (9 boys and 8 girls) aged 11.8 ± 5.2 years with refractory CD diagnosed at the mean age of 10.2 ± 4.3 years were included in this study. All 15 patients completed 12 months follow-up. Mean PCDAI was 41.0± 11.9 at baseline, the values at 1,3,6 and 12 months decreased to 15.2±9.6,5.3±5.0,3.0 ±3.4 and 2.3±2.6, respectively. Eleven of 12 patients who required corticosteroids for management were withdrawn the medicine at 12 months. The mean period was 3.1±1.5 months. ESR, CRP and PLT levels decreased after thalidomide treatment, and there was an increase in hemoglobin level. Furthermore, a significant increase in weight-for-age Z score was observed 12 months after administration of thalidomide. Two patients experienced fatigue, and 2 drowsiness during therapy. One patient presented with transient liver function abnormity.Conclusions:Thalidomide long-term treatment appears to be clinically effective and well tolerated. It is a relatively safe drug in Crohn’s disease patients who were refractory to corticosteroids, or corticosteroids dependent, or those concomitant with tuberculosis infection.PartⅡ The role of angiogenesis in pediatric pediatric Crohn’s disease and the effect of thalidomide on angiogenesisObjective: To investigate the expression angiogenic factors in mucosal tissues of CD patients and in healthy controls, and to analyze changes of these levels after oral thalidomide treatment.Methods:Tissue samples were collected from 10 CD patients who received thalidomide before and after treatment and from 10 matched healthy controls. VEGF, CD31, Ang-1, Ang-2, Tie-2 were evaluated by immunohistochemistry. MVD was quantified by CD31 postive cells. Real-time PCR was used to detect the expression of Ang-1 and Ang-2 in healthy control group, before and after thalidomide treatment group. The protein expression of VEGF, Ang-1 and Ang-2 in the mucosa was measured by Western blot before and after treatment with thalidomide.Results:VEGF, CD31, Ang-2 levels in CD patients were significantly higher than in healthy controls (P<0.05), while levels of Ang-1, Tie-2 were not significantly different between the two groups. In CD patients who achieved clinical remission after thalidomide treatment, statistically significant lower mucosal levels of CD31, VEGF, Ang-2, Tie-2 were observed (P<0.05). Real-time PCR results showed Ang-2 mRNA levels were higher in CD patients compared to the healthy controls. After thalidomide treatment, VEGF and Ang-2 protein levels of mucosa tissues were downregulated.Conclusions: VEGF and Ang-2 might play a critical role in the pathogenesis of pediatric CD. And thalidomide may mediate angiogenesis by inhibiting the expression of mucosal levels of VEGF and Ang-2 in CD.Part III The mechanism of action of thalidomide on anti-angiogenesis in HUVECObjective: To determine the in vitro antiangiogenic activity of thalidomide in HUVEC.Methods: We examined the effect of thalidomide on human umbilical vein endothelial cell (HUVEC). The proliferation of HUVEC was measured by CCK8 under different concentrations of thalidomide stimulation. VEGF and Ang-2 induced migration and tube formation of endothelial cells were detected by thalidomide stimulation. LPS-induced expression of VEGF, Ang-1 and Ang-2 were measured by real-time PCR after thalidomide stimulation. And the effects of thalidomide on VEGF, Ang-2 and molecules on important signaling pathways related to angiogenesis were determined by Western blot.Results:It was shown significant effects of thalidomide on the proliferation of HUVEC at concentration larger than 10μM. Thalidomide inhibited endothelial cells migration and tube formation induced by VEGF and Ang-2. Real-time PCR showed thalidomide inhibited LPS-induced VEGF and Ang-2 expression in a dose-dependent manner. The expression of VEGF, Ang-2 and PI3K/Akt were downregulated with thalidomide interference as measured by Western blot.Conclusions:Thalidomide exerts inhibitory effects on endothelial cells proliferation, migration and tube formation. Thalidomide suppresses angiogenesis by inhibiting expression of VEGF and Ang-2, by inhibiting the activation of PI3K/AKT signaling pathway.