Profiling Hypoxia Markers in Pediatric Inflammatory Bowel Disease

Abstract

Background: Inflammation-associated hypoxia has been studied extensively in murinemodels of inflammatory bowel diseases (IBD), and to a lesser extent in human IBD. Results from studies strongly implicate the potential that circulating or tissue markers of hypoxia could serve as important biomarkers of disease. A systematic analysis of hypoxia markers associated with IBD has not been pursued. Aims: To characterize markers of hypoxia in serum and colon biopsies from patients with IBD colitis and controls without colitis, and to correlate hypoxia markers with severity of disease assessed by histology, pro-inflammatory cytokines, questionnaires, and other laboratory data. Methods: Samples of blood and colon biopsies were obtained from 10 pediatric patients (mean age 13.9±2.7) with active IBD colitis and 8 controls (mean age 14.6±2.1), one of which had quiescent IBD. Protein levels of three hypoxia markers were measured in serum: erythropoietin (EPO), vascular endothelial growth factor (VEGF), and insulin-like growth factor binding protein-1 (IGFBP-1); and four proinflammatory cytokines were measured in tissue: IL-1 beta, IL-6, IL-8, and TNF alpha via an electrochemiluminescence-based ELISA platform. Pediatric Ulcerative Colitis Activity Index (PUCAI) and puberty scores were also collected. A single pathologist, blinded to diagnosis, graded biopsy histology based on a standardized scoring system. Differences by study group or PUCAI category were tested with two-sample t-tests or exact chi-square tests, and differences across histology grade by ANOVA. The monotonic relationship between hypoxia markers and pro-inflammatory cytokines and puberty score was tested with Spearman's correlation coefficient. Results: Patient demographics did not significantly differ by gender (p=0.06), race (p=0.7) or age (p=0.6). Children with active colitis had higher levels of EPO than controls (9.4 vs. 3.4, p=0.03). Notably, serum EPO levels did not correlate with hemoglobin. No significant differences were noted in total circulating VEGF (562 vs. 256, p=0.12) or IGFBP-1 (6716 vs. 3812, p=0.45), however all three serum hypoxia markers differed across histology grade (VEGF p=0.002, EPO p<0.0001, IGFBP-1=0.018). Serum VEGF was significantly correlated with tissue IL-6, IL-8, and TNF alpha (0.62, p=0.006; 0.57, p=0.014; 0.60, p=0.008), as was EPO with IL-6, IL-8, and TNF alpha (0.62, p=0.006; 0.63, p=0.005; 0.72, p=0.0008). VEGF and IGFBP-1 did not significantly differ by puberty score or across PUCAI score. Conclusions: These studies identify at least three hypoxia targets which could be pursued as potential biomarkers for disease activity. We conclude that markers of hypoxia strongly correlate with severity of colitis and that peripheral blood hypoxia markers may be useful surrogates to assess tissue inflammation.