Evaluation of rVEGF164b, a novel antiangiogenic VEGF-A isoform, in inflammatory bowel disease: Implications on VEGF-A regulation and potental therapeutic options.

摘要

Introduction: IBD is a complex disease that requires alterations in the immune system and physiology of the gastrointestinal tract for both initiation and progression of the disease. At the core of this alteration is the endothelium, which coordinates the interplay of inflammatory immune cells and the surrounding tissue as well as physiological factors such as tissue edema. Expansion of the endothelium in IBD through inflammatory angiogenesis in response to inflammatory cytokines, chemokines and growth factors is a hallmark of disease and drives increased disease severity. The primary growth factor that is responsible for the majority of inflammatory angiogenesis in IBD is VEGF-A, and over expression of VEGF-A and associated signaling leads to increased inflammation.;Methods: rVEGF164b inhibition of VEGF-A activity was measured in MVECs in vitro after treatment with VEGF-A, rVEGF164 b or a combination of both growth factors. VEGF-A induced proliferation was measured in sub-confluent cells and by direct cell counts and MTT assays while metabolic alterations were measured in confluent MVECs using MTT. TEER was measured in confluent MVEC monolayers using an epithelial voltmeter. Migration was measured using a Boyden chamber assay and wound healing was measured by percentage wound closure in MVEC monolayers. Cytoskeleton and barrier molecule alterations were observed by immunofluorescence. The activity of rVEGF164 b in vivo was measured in a model of TNBS induced UC. Disease activity, immune cell infiltration and macro and microscopic parameters of inflammation were scored. Angiogenesis and lymphangiogenesis were measured by imunohistochemistry. All data were analyzed for statistical significance.;Results: In vitro rVEGF164b blocked VEGF-A induced proliferation, metabolic alterations, barrier disruption, migration, wound healing, cytoskeleton rearrangement, and degradation of molecules that affect barrier function which suggested that this molecule was a potent inhibitor of VEGF-A. In vivo it was found that rVEGF164b reduced disease severity in an angiogenic model of UC. It was found that rVEGF164b reduced angiogenesis in the colons of colitic mice as well as reducing the infiltration of inflammatory cells from the circulation and reduced overall histopathological damage.;Conclusion: Our results confirm that rVEGF164b is a potent inhibitor of VEGF-A in endothelial cells in vitro and that it can reduce angiogenesis in a model of angiogenic UC. This data also confirms the importance of VEGF-A in the exacerbation of UC and that inhibition of VEGF-A signaling may represent a novel therapeutic mechanism for treating IBD. Most importantly this represents the first attempt to use an endogenous inhibitor of angiogenesis in a therapeutic manner and might lead the way for development of further treatments in inflammation and cancer.;Hypothesis: IBD promotes inflammatory vessel growth, permeability, and immune cell recruitment through VEGF-A signaling. Inhibition of VEGF-A signaling by rVEGF164b, an endogenous inhibitor of VEGF-A signaling would block inflammatory angiogenesis along with other inflammatory effects mediated by VEGF-A and reduce disease severity in a model of UC.