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P093. Circulating microvesicles in Crohn's disease

E. Gaetani, M. Marcantoni, I. Giarretta, I. Gatto, F. Scaldaferri, F. Del Zompo, L. Laterza, A. Tortora, R. Landi, A. Gasbarrini, R. Pola, Catholic University of Sacred Heart, Internal Medicine and Gastroenterology, Rome, Italy

Background

Circulating microvesicles (cMVs) are small membrane bound fragments released by a number of cell types, including endothelial cells (ECs), platelets, leukocytes, macrophages, and smooth muscle cells. Though initially dismissed as cellular debris, cMVs are now considered important mediators of cell signaling and molecular communication between cells. Indeed, cMVs are enriched with nucleic acids and proteins, shuttle specific mRNAs and miRNAs, and transfer biological information between cells, thus conferring new function to the target cell. In this study, we analyzed number, immunophenotype, and content of cMVs in subjects with active Crohn's disease.

Methods

MVs were isolated from the peripheral blood of 10 subjects with active CD and 10 control individuals. Fluorescence activated cell sorting was used to assess the immunophenotypical characteristics of cMVs and determine whether they were derived from ECs, platelets, monocytes, or apoptotic cells. To analyze the content of cMVs, both RNAs and proteins were extracted. RNA was used for gene expression profiling using specific profiler PCR arrays for angiogenic pathways. Proteins were analzyed using specific angiogenic antibody arrays. To determine the functional activity of the angiogenic message carried by cMVs, human umbilical ECs (HUVECs) were seeded into plates coated with Matrigel and stimulated with cMVs isolated from the blood of either subjects with active CD or control individuals. Number of newly-formed tubes and branching points were determined.

Results

Compared to controls, subjects with active CD have a significantly higher number of cMVs derived from ECs, activated ECs, platelets, activated platelets, monocytes, and apoptotic cells. In individuals with CD, cMVs are significantly enriched with pro-angiogenic mRNAs, including EGF, FGF2, IGF1, PDGFA, TGFB2, VEGFC, and ANG1. They also contain significantly higher levels of prototypical angiogenic proteins, such as Angiogenin, Platelet factor-4, SERPINF1, PAI-1, TIMP-1 and TSP-1. When used to stimulate ECs in matrigel, cMVs isolated from subjects with active CD have the ability to induce a robust angiogenic response, in terms of increased tube formation and enhanced EC branching.

Conclusion

In CD, angiogenesis is a hallmark of active disease and is closely related to disease severity. The endothelium in newly formed or inflamed vessels differs from that in normal vessels in the production of and response to inflammatory cytokines, growth factors, and adhesion molecules. Our findings demonstrate that, in active CD, cMVs carry a potent and functionally active angiogenic message, with novel and potentially important implications on the pathophysiological mechanisms that underlie development and progression of the disease.