Mary R. Dusing

Epithelial lineages of the small intestine have unique patterns of GATA expression.

The ability of the GATA family of factors to interact with numerous other factors, co-factors, and repressors suggests that they may play key roles in tissues and cells where they are expressed. Adult mouse small intestine has been shown to express GATA-4, GATA-5, and GATA-6, where they have been implicated in the activation of a number of intestinal genes. Determination of which GATA factor(s) are involved in a specific function in tissues expressing multiple family members has proven difficult. The immunohistochemical analysis presented here demonstrate that within the mouse small intestine GATA-4/-5/-6 are found to be uniquely distributed among the various differentiated lineages of the intestinal epithelium. Among differentiated cells GATA-4 is found only in the villous enterocytes. GATA-5 is absent from enterocytes, but was found in the remaining lineages: goblet, Paneth and enteroendocrine. Additionally, high levels of GATA-6 are found in only one of these differentiated cell types, the enteroendocrine lineage. The observed distribution suggests that the GATA factors may have distinct roles in lineage allocation, lineage maintenance, and/or terminal differentiation events in small intestine.

Onecut-2 knockout mice fail to thrive during early postnatal period and have altered patterns of gene expression in small intestine

Ablation of the mouse genes for Onecut-2 and Onecut-3 was reported previously, but characterization of the resulting knockout mice was focused on in utero development, principally embryonic development of liver and pancreas. Here we examined postnatal development of these Onecut knockout mice, especially the critical period before weaning. Onecut-3 knockout mice develop normally during this period. However, Onecut-2 knockout mice fail to thrive, lagging behind their littermates in size and weight. By postnatal day (d)19, they are consistently 25-30% smaller. Onecut-2 knockout mice also have a much higher level of mortality before weaning, with only approximately 70% survival. Interestingly, Onecut-2 knockout mice that are heterozygous for the Onecut-3 knockout allele are diminished even further in their ability to thrive. They are approximately 50-60% as large as their normal-sized littermates at d19, and less than half of these mice survive to weaning. As reported previously, the Onecut-2/Onecut-3 double knockout is a perinatal lethal. Microarray technology was used to determine the effect of Onecut-2 ablation on gene expression in duodenum, whose epithelium has among the highest levels of Onecut-2. A subset of intestinally expressed genes showed dramatically altered patterns of expression. Many of these genes encode proteins associated with the epithelial membrane, including many involved in transport and metabolism. Previously, we reported that Onecut-2 was critical to temporal regulation of the adenosine deaminase gene in duodenum. Many of the genes with altered patterns of expression in Onecut-2 knockout mouse duodenum displayed changes in the timing of gene expression.

Mouse strain influences angiogenic response to dextran sodium sulfate–induced colitis

BACKGROUND Angiogenesis is a known pathologic factor in chronic inflammatory diseases. Regarding the murine dextran sodium sulfate (DSS) colitis model, different mouse strains produce variable clinical and inflammatory responses. We hypothesize that DSS colitis applied to diverse mouse strains will similarly elevate colonic microvessel density in parallel with inflammation, but will do so with different angiogenic profiles. MATERIALS AND METHODS We induced DSS colitis in 129S2/SvPas, BALB/c, and C57BL/6 mice, then performed histologic and molecular analysis at day 7 to evaluate colonic inflammation and angiogenesis. RESULTS Inflammation and microvessel density were similarly increased in DSS groups. The C57BL/6 cohort mounted a more severe colitis with 25% weight loss and greater colonic ulceration. Gene expression of angiogenic factors at baseline and in colitis groups were widely variable among strains. BALB/c mice exhibited higher angiogenic gene expression in control and DSS groups compared with other strains, specifically platelet-derived growth factor, angiopoietin-1, angiopoietin-1 (Ang-2), vascular endothelial growth factor receptor, and PDGF receptor. When evaluating the effect of DSS relative to controls, BALB/c mice were not significantly affected. 129S2/SvPas mice exhibited broad suppression of growth factors, significantly platelet-derived growth factor, Ang-2, and PDGF receptor. In contrast, C57BL/6 mice displayed increased gene expression, especially for angiopoietin-1 and Ang-2. CONCLUSIONS Genetic heterogeneity influences the angiogenic profile elicited by DSS colitis. We demonstrate that within a model of murine colitis, mouse strain significantly affects inflammation-associated angiogenesis. These results may impact strain selection when using a colitis model focusing on inflammation and angiogenesis. Future studies to further define the angiogenesis pathway and potentially alter the disease course with targeted antiangiogenics are warranted.

Vascular endothelial growth factor receptor-2 inhibition in experimental murine colitis

BACKGROUND In the setting of inflammatory bowel disease, inflammation is associated with a simultaneous increase in angiogenesis; moreover, elevated vascular endothelial growth factor (VEGF) levels implicate angiogenesis as a pathologic contributor to disease severity. We hypothesize that selectively inhibiting vascular endothelial growth factor receptor-2 (VEGFR2) in a model of murine colitis will reduce angiogenesis, resulting in decreased inflammation and disease severity, providing mechanistic insight into the role of pathologic angiogenesis in IBD. MATERIALS AND METHODS In a dextran sodium sulfate model of murine colitis, anti-VEGFR2 monoclonal antibody (DC101) or placebo was administered. Clinical assessments followed by histologic and molecular tissue analysis were performed to quantify inflammation, microvessel density (MVD), VEGF and VEGFR2 gene expression, and phosphorylated mitogen-activated protein kinase protein expression. RESULTS Weight loss began after d 6 with the treatment group demonstrating a more favorable percent weight change. Inflammation and MVD were similar between cohorts, both increasing in parallel toward a plateau. VEGF and VEGFR2 messenger RNA expression were not significantly different, but phosphorylated mitogen-activated protein kinase was elevated in the DC101 cohort (P = 0.03). CONCLUSIONS Despite a more favorable weight change profile in the treated group, no difference was observed between cohorts regarding clinical disease severity. However, a parallel rise in inflammation and MVD was observed coinciding with weight loss, suggesting their relationship in IBD. VEGFR2 downstream signaling was significantly elevated in the treated cohort, possibly by VEGF-independent signal transduction. Early and effective inhibition of angiogenesis by limiting downstream VEGF signaling or targeting multiple angiogenic pathways may block angiogenesis, thereby reducing disease severity and provide evidence toward the mechanism and clinical benefit of antiangiogenics in the setting of IBD.

Angiogenesis and Vascular Endothelial Growth Factor-A Expression Associated with Inflammation in Pediatric Crohn’s Disease

Background and AimsAngiogenesis is a component of chronic inflammatory diseases including inflammatory bowel disease. Some studies describe increased angiogenesis associated with acute disease in adult Crohn’s disease and ulcerative colitis, while animal models aid investigations of mechanism and pathophysiology of angiogenesis. We aim to explore the role of angiogenesis and its pathways in pediatric Crohn’s disease.MethodsSurgical specimens were obtained from pediatric Crohn’s disease (both inflamed and non-inflamed regions of ileum) and control patients. Samples were examined for inflammation, microvessel density, and molecular expression of vascular endothelial growth factor-A, platelet-derived growth factor-β, angiopoietin-1, and angiopoietin-2.ResultsAngiogenesis and inflammation were increased in parallel in Crohn’s disease compared to controls. We also discovered increased angiogenesis in Crohn’s disease tissue that was relatively free of inflammatory disease. Vascular endothelial growth factor-A gene expression (P = 0.034) was elevated in Crohn’s disease over controls, while gene expression of platelet-derived growth factor-β (P = 0.069), angiopoietin-1 (P = 0.206), and angiopoietin-2 (P = 0.082) was not significantly elevated.ConclusionsWe confirm that inflammation-associated angiogenesis is upregulated in pediatric Crohn’s disease. This population also exhibits elevated mucosal angiogenesis at the surgical margin with limited inflammation. This suggests that angiogenesis is an additional pathologic characteristic to potentially identify normal mucosa and margins of surgical resection that are uninvolved with disease and, furthermore, may have implications for monitoring complete disease remission. We further identify the vascular endothelial growth factor-A pathway involvement in the disease process, which may serve as a future molecular target for anti-angiogenic therapy in inflammatory bowel disease.

Murine colitis treated with multitargeted tyrosine kinase inhibitors

BACKGROUND Angiogenesis, a known pathogenic component of neoplastic and nonneoplastic diseases, serves as a therapeutic target. Vascular endothelial growth factor (VEGF) and angiogenesis are clinically elevated in inflammatory bowel disease. By targeting vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) with receptor tyrosine kinase inhibitors in a murine model of colitis, we hypothesize that angiogenesis will be suppressed and disease severity improved. METHODS AND METHODS Sorafenib, sunitinib, and axitinib were administered by oral gavage in a murine model of dextran sodium sulfate (DSS) colitis. Inflammation score, microvessel density (MVD), and gene expression of VEGF, VEGFR, platelet-derived growth factor, PDGFR, Ang-2, and epidermal growth factor receptor was assessed. RESULTS Inflammation and MVD were elevated in groups receiving DSS, but were similar between DSS-only and treatment cohorts. Unexpected weight loss was present in the gavaged groups versus DSS only. In treated groups, VEGFR was significantly decreased (P = 0.002) and VEGF gene expression trended down (P = 0.213) versus DSS only. Neither the platelet-derived growth factor/PDGFR pathway nor the alternative pathways, Ang-2 and epidermal growth factor receptor, were significantly changed from DSS control in treatment cohorts. CONCLUSIONS This study confirms the association between inflammation and MVD. Antiangiogenic receptor tyrosine kinase inhibitors suppressed the VEGF/VEGFR pathway but the expected decrease in colonic MVD did not follow, suggesting possible involvement of other angiogenic pathway(s). In the DSS model of colitis, vehicle selection and mouse strain can impact disease response.

Temporal Regulation of Enhancer Function in Intestinal Epithelium A ROLE FOR ONECUT FACTORS

An intestine-specific gene regulatory region was previously identified near the second exon of the human adenosine deaminase (ADA) gene. In mammalian intestine, ADA is expressed at high levels only along the villi of the duodenal epithelium, principally if not exclusively in enterocytes. Within the ADA intestinal regulatory region, a potent duodenum-specific enhancer was identified that controls this pattern of expression. This enhancer has been shown to rely on PDX-1, GATA factors, and Cdx factors for its function. Upstream of the enhancer, a separate temporal regulatory region was identified that has no independent enhancer capability but controls the timing of enhancer activation. DNase I footprinting and electrophoretic mobility shift assays were used to begin to characterize temporal region function at the molecular level. In this manner, binding sites for the Onecut (OC) family of factors, YY1, and NFI family members were identified. Identification of the OC site was especially interesting, because almost nothing is known about the function of OC factors in intestine. In transgenic mice, mutation of the OC site to ablate binding resulted in a delay of 2–3 weeks in enhancer activation in the developing postnatal intestine, a result very similar to that observed previously when the entire temporal region was deleted. In mammals, the OC family is comprised of OC-1/HNF-6, OC-2, and OC-3. An examination of intestinal expression patterns showed that all three OC factors are expressed at detectable levels in adult mouse duodenum, with OC-2 predominant. In postnatal day 2 mice only OC-2 and OC-3 were detected in intestine, with OC-2 again predominant.