Nuclear Factor of Activated T Cells Is Activated in the Endothelium of Retinal Microvessels in Diabetic Mice

Hindawi Publishing Corporation Journal of Diabetes Research Volume 2015, Article ID 428473, 14 pages http://dx.doi.org/10.1155/2015/428473 Research Article Nuclear Factor of Activated T Cells Is Activated in the Endothelium of Retinal Microvessels in Diabetic Mice Anna V. Zetterqvist,1 Fabiana Blanco,1,2 Jenny Öhman,1 Olga Kotova,1 Lisa M. Berglund,1 Sergio de Frutos Garcia,3 Raed Al-Naemi,1 Maria Wigren,1 Paul G. McGuire,3 Laura V. Gonzalez Bosc,3 and Maria F. Gomez1 1 Department of Clinical Sciences in Malmö, Lund University, 20502 Malmö, Sweden Departamento de Biofı́sica, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay 3 Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA 2 Correspondence should be addressed to Maria F. Gomez; Received 28 November 2014; Revised 25 February 2015; Accepted 25 February 2015 Academic Editor: Ute Christine Rogner Copyright © 2015 Anna V. Zetterqvist et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The pathogenesis of diabetic retinopathy (DR) remains unclear but hyperglycemia is an established risk factor. Endothelial dysfunction and changes in Ca2+ signaling have been shown to precede the onset of DR. We recently demonstrated that high extracellular glucose activates the Ca2+ /calcineurin-dependent transcription factor NFAT in cerebral arteries and aorta, promoting the expression of inflammatory markers. Here we show, using confocal immunofluorescence, that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by high glucose. This was inhibited by the NFAT blocker A-285222 as well as by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Acute hyperglycemia induced by an IP-GTT (intraperitoneal glucose tolerance test) resulted in increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity in retinal vessels of NFAT-luciferase reporter mice. In both Akita (Ins2+/− ) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels. In vivo inhibition of NFAT with A-285222 decreased the expression of OPN and ICAM-1 mRNA in retinal vessels, prevented a diabetes driven downregulation of anti-inflammatory IL-10 in retina, and abrogated the increased vascular permeability observed in diabetic mice. Results identify NFAT signaling as a putative target for treatment of microvascular complications in diabetes. 1. Introduction Diabetic retinopathy (DR) is still one of the leading causes of vision loss worldwide. Even though the underlying pathogenesis is not clear, hyperglycemia is an important risk factor [1]. We have recently demonstrated that modest elevations of extracellular glucose activate the Ca2+ /calcineurindependent transcription factor NFAT (nuclear factor of activated T cells) in smooth muscle cells of conduit and resistance arteries [2, 3]. The effect of glucose involved the local release of extracellular nucleotides, such as ATP and UTP, acting on P2Y receptors, leading to increased intracellular Ca2+ ([Ca2+ ]i ) and subsequent activation of calcineurin and NFAT [2]. ATP and UTP are vasoactive signals able to increase [Ca2+ ]i in the retina, via stimulation of purinergic receptors including P2Y4 [4]. Also, high glucose has been shown to increase extracellular ATP in rat retinal cell cultures [5]. Therefore, we hypothesize that hyperglycemia may activate NFAT in retinal microvessels. Inflammation and endothelial activation are important early steps in the development of DR, leading to leukostasis, platelet activation, and upregulation of inflammatory cytokines [6]. The NFAT family (NFATc1–c4) plays a central role in the production of cytokines in immune cells and in the regulation of T-cell proliferation. We and others have shown that in conduit and resistance arteries and in cultured vascular cells NFAT regulates the expression of inflammatory genes, such as IL-6, allograft inflammatory factor 1 (AIF-1), tissue factor (TF), cyclooxygenase 2 (Cox-2), and osteopontin (OPN) [3, 7–9]. Expression of endothelial activation markers, such as VCAM-1 and E-selectin, is also dependent on NFAT signaling in cultured smooth muscle 2 and endothelial cells, respectively [10, 11]. More recently, we showed that in vivo inhibition of NFAT signaling reduces ICAM-1 mRNA expression in the aortas of diabetic Apoe−/− mice [9], a leukocyte adhesion molecule that is elevated in retinal vessels from diabetic mice and patients [6, 12]. Another early feature of DR is the breakdown of the blood-retinal barrier (BRB) [13], which results in vascular leakage and development of retinal edema. Earlier investigations focused on vascular endothelial growth factor (VEGF), shown to induce rapid phosphorylation of tight junction proteins and increased retinal permeability [14]. However, recent in vivo kinetic data show that the retinal barrier function is compromised before VEGF levels are increased and use of a neutralizing anti-VEGF antibody is not effective at reducing permeability at early stages of diabetes (8 weeks) [15]. In the context of angiogenesis [16, 17], VEGF appears to be an upstream activator of NFAT, but both VEGF and its receptor VEGFR2 are also downstream targets of NFAT in endothelial cells [18, 19]. Hence, a role of NFAT in the early changes of DR cannot be ruled out. Here, we investigated the effects of high glucose and diabetes on NFAT activation in a streptozotocin (STZ) model of diabetes and in hyperglycemic Akita (Ins2+/− ) mice. We also explored the effects of in vivo NFAT-signaling inhibition on the expression of inflammatory mediators, endothelial adhesion molecules, and vascular permeability in diabetic mice. 2. Research Design and Methods 2.1. Animals. All animal protocols in this study were reviewed and approved by Institutional Animal Care and Use Committees, University of New Mexico, School of Medicine and Lund University, Sweden. The following mice strains (number of animals per strain indicated) were bred in our animal facilities: FVBN 9x-NFAT-luciferase reporter (NFATluc [2, 7, 20]; 𝑁 = 133), Akita (Ins2+/− ), and wildtype (Ins2+/+ ) littermates (stock number 003548, C57Bl/6J background, Jackson Laboratories, Maine, here referred to as Akita and WT; 𝑁 = 31). We also generated Akita/NFATluc mice and WT/NFAT-luc littermates (𝑁 = 43), which were backcrossed at least four generations into the C57Bl/6J background. Wild-type adult BALB/c (𝑁 = 76), C57Bl/6 (Taconic, Europe; 𝑁 = 12), and ApoE−/− (B6.129P2Apoetm1Unc /J; Charles River, Sulzfeld, Germany; 𝑁 = 22) mice were also used. Animals had free access to tap water and were fed normal chow diet. Retinas, cerebral arteries, aortas, and plasma were used. Both eyes (...truncated)