Andreia Gonçalves1, Luísa Almeida1, Ana Paula Silva1, Carlos Fontes-Ribeiro1, António F Ambrósio2, Armando Cristóvão3, Rosa Fernandes4. 1. Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, Coimbra, Portugal; CNC.IBILI Consortium, University of Coimbra, Coimbra, Portugal. 2. Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, Coimbra, Portugal; CNC.IBILI Consortium, University of Coimbra, Coimbra, Portugal; Association for Innovation and Biomedical Research on Light and Image (AIBILI), Coimbra, Portugal. 3. CNC.IBILI Consortium, University of Coimbra, Coimbra, Portugal; Department of Life Sciences, University of Coimbra, Coimbra, Portugal. 4. Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, Coimbra, Portugal; CNC.IBILI Consortium, University of Coimbra, Coimbra, Portugal. Electronic address: rcfernandes@fmed.uc.pt.
Abstract
Diabetic retinopathy is considered a low-grade chronic inflammatory disease and several inflammatory molecules, including tumor necrosis factor (TNF)-α, are known to play a major role in the degeneration of retinal capillaries. Previous studies have reported that sitagliptin, a DPP-4 inhibitor, prevents the increase in blood-retinal barrier (BRB) permeability and inhibits the tight junction disassembly induced by diabetes. AIM: Our goal was to investigate whether sitagliptin is able to prevent retinal endothelial cells (EC) dysfunction triggered by the pro-inflammatory cytokine TNF-α. MAIN METHODS: The effects of TNF-α and/or sitagliptin on primary cultures of bovine retinal EC were tested. The EC monolayer permeability was analyzed by using 70 kDa rhodamine isothiocyanate (RITC) dextran. The cellular distribution profile of claudin-5 was examined by immunofluorescence staining, and DPP-4 activity was evaluated by using a fluorogenic substrate. Cell viability was assessed by MTT assay, and cell proliferation by the BrdU incorporation assay. Retinal EC migration and angiogenesis were evaluated by a scratch assay and a capillary tube formation in matrigel assay, respectively. KEY FINDINGS: TNF-α increased the permeability of EC monolayer and induced the loss of claudin-5 immunostaining at the cell borders. This impairment was associated with decreased migration and capillary morphogenesis of retinal EC. Sitagliptin was unable to prevent the effect of TNF-α on EC permeability. However, it decreased DPP-4 activity in bovine retinal EC exposed to TNF-α, without affecting cell viability. Moreover, sitagliptin enhanced the migration and capillary morphogenesis in bovine retinal EC challenged with TNF-α. SIGNIFICANCE: These results suggest that sitagliptin is able to positively modulate vascular EC function under conditions of retinal inflammation.
Diabetic retinopathy is considered a low-grade chronic inflammatory disease and several inflammatory molecules, including tumor necrosis factor (TNF)-α, are known to play a major role in the degeneration of retinal capillaries. Previous studies have reported that sitagliptin, a DPP-4 inhibitor, prevents the increase in blood-retinal barrier (BRB) permeability and inhibits the tight junction disassembly induced by diabetes. AIM: Our goal was to investigate whether sitagliptin is able to prevent retinal endothelial cells (EC) dysfunction triggered by the pro-inflammatory cytokine TNF-α. MAIN METHODS: The effects of TNF-α and/or sitagliptin on primary cultures of bovine retinal EC were tested. The EC monolayer permeability was analyzed by using 70 kDa rhodamine isothiocyanate (RITC) dextran. The cellular distribution profile of claudin-5 was examined by immunofluorescence staining, and DPP-4 activity was evaluated by using a fluorogenic substrate. Cell viability was assessed by MTT assay, and cell proliferation by the BrdU incorporation assay. Retinal EC migration and angiogenesis were evaluated by a scratch assay and a capillary tube formation in matrigel assay, respectively. KEY FINDINGS: TNF-α increased the permeability of EC monolayer and induced the loss of claudin-5 immunostaining at the cell borders. This impairment was associated with decreased migration and capillary morphogenesis of retinal EC. Sitagliptin was unable to prevent the effect of TNF-α on EC permeability. However, it decreased DPP-4 activity in bovine retinal EC exposed to TNF-α, without affecting cell viability. Moreover, sitagliptin enhanced the migration and capillary morphogenesis in bovine retinal EC challenged with TNF-α. SIGNIFICANCE: These results suggest that sitagliptin is able to positively modulate vascular EC function under conditions of retinal inflammation.
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