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Antiangiogenic and Antineuroinflammatory Effects of Kallistatin Through Interactions with the Canonical Wnt Pathway Xiaochen Liu,1,2 Bin Zhang,3 Jeffrey D

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Antiangiogenic and Antineuroinflammatory Effects of Kallistatin Through Interactions with the Canonical Wnt Pathway Xiaochen Liu,1,2 Bin Zhang,3 Jeffrey D ORIGINAL ARTICLE Antiangiogenic and Antineuroinflammatory Effects of Kallistatin Through Interactions With the Canonical Wnt Pathway Xiaochen Liu,1,2 Bin Zhang,3 Jeffrey D. McBride,4 Kevin Zhou,2 Kyungwon Lee,4 Yueping Zhou,1 Zuguo Liu,1 and Jian-xing Ma2 Kallistatin is a member of the serine proteinase inhibitor super- also in the heart, kidney, retina, blood vessels, and many family. Kallistatin levels have been shown to be decreased in the other tissues and cell types (6). The broad tissue distribution vitreous while increased in the circulation of patients with diabetic of kallistatin suggests that it may confer multiple functions retinopathy (DR). Overactivation of the Wnt pathway is known to such as regulation of cardiovascular function and blood play pathogenic roles in DR. To investigate the role of kallistatin vessel development (7). The retina is a specialized, vascu- in DR and in Wnt pathway activation, we generated kallistatin larized tissue and direct extension of the central nervous transgenic (kallistatin-TG) mice overexpressing kallistatin in multi- system. We previously found that the vitreous levels of ple tissues including the retina. In the oxygen-induced retinopathy kallistatin were significantly reduced in patients with di- (OIR) model, kallistatin overexpression attenuated ischemia- abetic retinopathy (DR), compared with that in nondiabetic induced retinal neovascularization. In diabetic kallistatin-TG mice, fi kallistatin overexpression ameliorated retinal vascular leakage, leuko- control patients (8). This nding suggests that decreased stasis, and overexpression of vascular endothelial growth factor and vitreous levels of kallistatin may contribute to a proangiogenic intracellular adhesion molecule. Furthermore, kallistatin overexpres- and proneuroinflammatory environment in DR. Interestingly, sion also suppressed Wnt pathway activation in the retinas of the OIR similar to other SERPINs, such as PEDF and plasminogen and diabetic models. In diabetic Wnt reporter (BAT-gal) mice, activator inhibitor-1 (SERPINE1), serum kallistatin levels are kallistatin overexpression suppressed retinal Wnt reporter activity. increased in type 1 diabetic patients and correlate with the In cultured retinal cells, kallistatin blocked Wnt pathway activation presence of microvascular complications (9). This paradox- induced by high glucose and by Wnt ligand. Coprecipitation and ical trend reflects the need to study the local tissue effects of ligand-binding assays both showed that kallistatin binds to a Wnt various SERPINs, since circulation levels of a protein do not coreceptor LRP6 with high affinity (K = 4.5 nmol/L). These obser- d always parallel vitreous levels in diabetes. Taken together, vations suggest that kallistatin is an endogenous antagonist of fi LRP6 and inhibitor of Wnt signaling. The blockade of Wnt signaling these ndings suggest a potential role of kallistatin in mod- may represent a mechanism for its antiangiogenic and antineuro- ulation of microvasculature in diabetic tissues. inflammatory effects. Diabetes 62:4228–4238, 2013 Recent evidence suggests that the Wnt pathway medi- ates inflammatory responses and modulates angiogenesis during development and under disease conditions (10,11). The roles of Wnt signaling in some pathological conditions number of natural antiangiogenic factors or associated with abnormal neovascularization have been angiogenic inhibitors have been identified (1). reported. Mutations in components of the Wnt signaling These endogenous angiogenic inhibitors include pathway are known to cause several hereditary vascular A several proteins in the serine proteinase in- disorders and defective retinal vascular development (12,13). hibitor (SERPIN) superfamily, such as pigment epithelium- Further, Wnt signaling upregulates expression of a number of derived factor (PEDF), a1-antitrypsin (AAT), and kallistatin angiogenic and inflammatory factors. Our recent study (SERPINA4) (2–4). Kallistatin was first identified as a se- showed that overactivation of Wnt signaling plays pathogenic creted, specific inhibitor of tissue kallikrein (5). Later, roles in diabetic microvascular complications (14). kallistatin displayed novel functions independent of its In the current study, we generated transgenic mice that interaction with tissue kallikrein, including inhibition of overexpress and secrete human kallistatin in multiple tis- angiogenesis and tumor growth (4). Like other SERPIN sues, with high levels in the retina, and investigated the members, kallistatin is expressed not only in the liver but impacts of kallistatin on ischemia-induced retinal neo- vascularization, diabetes-induced retinal neuroinflammation, and Wnt signaling activation. From the 1Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, China; the 2Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; the 3Department of Molecular and Cellular Biology, Baylor Col- RESEARCH DESIGN AND METHODS lege of Medicine, Houston, Texas; and the 4Department of Cell Biology, Uni- Generation and characterization of kallistatin-transgenic mice. The versity of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma. transgenic mice were generated through a contracted service at Transgenic Corresponding authors: Zuguo Liu, [email protected], and Jian-xing Ma, Animal Facility at Stanford University and confirmed by genotyping with PCR [email protected]. using a forward primer (59-AGG GAA GAT TGT GGA TTT GG-39) and a reverse Received 11 December 2012 and accepted 20 July 2013. 9 9 fi DOI: 10.2337/db12-1710 primer (5 -ATG AAG ATA CCA GTG ATG CTC-3 ) speci c for the human This article contains Supplementary Data online at http://diabetes kallistatin cDNA. Retinal structure and function were examined by histology .diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1710/-/DC1. and electroretinography (ERG) analysis. Ó 2013 by the American Diabetes Association. Readers may use this article as Retinal angiography. Retinal angiography was performed as previously de- long as the work is properly cited, the use is educational and not for profit, scribed (15). Briefly, mice were perfused with 20 mg/mL high–molecular and the work is not altered. See http://creativecommons.org/licenses/by weight (2 3 106) fluorescein isothiocyanate (FITC)-dextran (Sigma, St. Louis, -nc-nd/3.0/ for details. MO). The eyes were fixed and the retina flat mounted. The nonperfused area See accompanying commentary, p. 3993. was measured in the retina using ImageJ software. 4228 DIABETES, VOL. 62, DECEMBER 2013 diabetes.diabetesjournals.org X. LIU AND ASSOCIATES Quantification of preretinal vascular cells. Preretinal vascular cells were For determination of whether kallistatin overexpression quantified as previously described (16). Retinal sections from mice with in the retina affects retinal development, ocular sections oxygen-induced retinopathy (OIR) were stained with hematoxylin-eosin and from WT and kallistatin-TG mice were examined by mi- examined under a light microscope. The preretinal vascular nuclei were counted in 8 discontinuous sections per eye. croscopy. At 4 months of age, there were no detectable Vascular permeability assay. Retinal vascular permeability was measured differences in the histological structure or the nuclear using Evans blue as tracer as previously described (17). Briefly, Evans blue was layers of the retinas between WT and kallistatin-TG mice injected through the femoral vein (10 mg/kg body wt) under microscopic in- (Supplementary Fig. 2A). spection. After 2 h circulation, the mice were perfused and the retina was As measured by optical coherence tomography, the dissected. Evans blue dye in the retina was measured and normalized by total average retinal thickness in kallistatin-TG mice was not protein concentrations. fi Leukostasis assay. The assay was performed following a documented protocol signi cantly different from that in age-matched WT mice (18). Briefly, mice were perfused through the left ventricle to remove circulating (Supplementary Fig. 2B). Furthermore, there were no sig- leukocytes in blood vessels. The adherent leukocytes in the vasculature were nificant differences in the dark-adapted (scotopic or rod stained by perfusion with FITC-conjugated concanavalin-A (40 mg/mL; Vector associated) or light-adapted (photopic or cone associated) Laboratories, Burlingame, CA) and counted in retinal flat mounts. ERG responses between WT and kallistatin-TG mice ELISA, Western blot analysis, and immunoprecipitation. Intracellular C adhesion molecule (ICAM)-1, vascular endothelial growth factor (VEGF), and (Supplementary Fig. 2 ). Overall, these results suggest human kallistatin were measured using ELISA kits purchased from R&D that kallistatin-TG mice do not manifest structural or Systems (Minneapolis, MN), and an ELISA kit for both mouse and human functional defects in the retina under normal conditions. kallistatin was purchased from Sino Biological (Beijing, China) according to Kallistatin overexpression inhibits retinal neovascula- the manufacturer’s instructions. rization in the OIR model. The impact of kallistatin over- Western blot analysis was performed as described previously (19). For expression on ischemia-induced retinopathy was evaluated immunoprecipitation assays, purified protein, human serum, supernatants of cell lysates, and conditioned media were mixed. Protein G agarose resin using the OIR model (16). After kallistatin-TG and WT mice (Roche Applied
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