Plasmalemma Vesicle–Associated Protein Has a Key Role in Blood-Retinal Barrier Loss

Loss of blood-retinal barrier (BRB) properties induced by vascular endothelial growth factor (VEGF) and other factors is an important cause of diabetic macular edema. Previously, we found that the presence of plasmalemma vesicle–associated protein (PLVAP) in retinal capillaries associates with loss of BRB properties and correlates with increased vascular permeability in diabetic macular edema. In this study, we investigated whether absence of PLVAP protects the BRB from VEGF-induced permeability. We used lentiviral-delivered shRNA or siRNA to inhibit PLVAP expression. The barrier properties of in vitro BRB models were assessed by measuring transendothelial electrical resistance, permeability of differently sized tracers, and the presence of endothelial junction complexes. The effect of VEGF on caveolae formation was studied in human retinal explants. BRB loss in vivo was studied in the mouse oxygen-induced retinopathy model. The inhibition of PLVAP expression resulted in decreased VEGF-induced BRB permeability of fluorescent tracers, both in vivo and in vitro. PLVAP inhibition attenuated transendothelial electrical resistance reduction induced by VEGF in BRB models in vitro and significantly increased transendothelial electrical resistance of the nonbarrier human umbilical vein endothelial cells. Furthermore, PLVAP knockdown prevented VEGF-induced caveolae formation in retinal explants but did not rescue VEGF-induced alterations in endothelial junction complexes. In conclusion, PLVAP is an essential cofactor in VEGF-induced BRB permeability and may become an interesting novel target for diabetic macular edema therapy. Loss of blood-retinal barrier (BRB) properties induced by vascular endothelial growth factor (VEGF) and other factors is an important cause of diabetic macular edema. Previously, we found that the presence of plasmalemma vesicle–associated protein (PLVAP) in retinal capillaries associates with loss of BRB properties and correlates with increased vascular permeability in diabetic macular edema. In this study, we investigated whether absence of PLVAP protects the BRB from VEGF-induced permeability. We used lentiviral-delivered shRNA or siRNA to inhibit PLVAP expression. The barrier properties of in vitro BRB models were assessed by measuring transendothelial electrical resistance, permeability of differently sized tracers, and the presence of endothelial junction complexes. The effect of VEGF on caveolae formation was studied in human retinal explants. BRB loss in vivo was studied in the mouse oxygen-induced retinopathy model. The inhibition of PLVAP expression resulted in decreased VEGF-induced BRB permeability of fluorescent tracers, both in vivo and in vitro. PLVAP inhibition attenuated transendothelial electrical resistance reduction induced by VEGF in BRB models in vitro and significantly increased transendothelial electrical resistance of the nonbarrier human umbilical vein endothelial cells. Furthermore, PLVAP knockdown prevented VEGF-induced caveolae formation in retinal explants but did not rescue VEGF-induced alterations in endothelial junction complexes. In conclusion, PLVAP is an essential cofactor in VEGF-induced BRB permeability and may become an interesting novel target for diabetic macular edema therapy. Diabetic macular edema (DME) is the most frequent cause of vision loss among patients with diabetic retinopathy (DR). DME is a complex disease that has been associated with increased vascular permeability due to loss of the inner blood-retinal barrier (BRB).1Klaassen I. Van Noorden C.J. Schlingemann R.O. 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Van Noorden C.J. Schlingemann R.O. Rakoczy E.P. Molecular analysis of blood-retinal barrier loss in the Akimba mouse, a model of advanced diabetic retinopathy.Exp Eye Res. 2014; 122: 123-131Crossref PubMed Scopus (51) Google Scholar PLVAP expression in endothelium is triggered by VEGF6Wisniewska-Kruk J. Hoeben K.A. Vogels I.M. Gaillard P.J. Van Noorden C.J. Schlingemann R.O. Klaassen I. A novel co-culture model of the blood-retinal barrier based on primary retinal endothelial cells, pericytes and astrocytes.Exp Eye Res. 2012; 96: 181-190Crossref Scopus (77) Google Scholar, 13Klaassen I. Hughes J.M. Vogels I.M. Schalkwijk C.G. Van Noorden C.J. Schlingemann R.O. Altered expression of genes related to blood-retina barrier disruption in streptozotocin-induced diabetes.Exp Eye Res. 2009; 89: 4-15Crossref PubMed Scopus (80) Google Scholar, 26Strickland L.A. Jubb A.M. Hongo J.A. Zhong F. Burwick J. Fu L. Frantz G.D. Koeppen H. Plasmalemmal vesicle-associated protein (PLVAP) is expressed by tumour endothelium and is upregulated by vascular endothelial growth factor-A (VEGF).J Pathol. 2005; 206: 466-475Crossref PubMed Scopus (77) Google Scholar, 28Hofman P. Blaauwgeers H.G. Vrensen G.F. Schlingemann R.O. Role of VEGF-A in endothelial phenotypic shift in human diabetic retinopathy and VEGF-A-induced retinopathy in monkeys.Ophthalmic Res. 2001; 33: 156-162Crossref PubMed Scopus (35) Google Scholar, 33Witmer A.N. van Blijswijk B.C. van Noorden C.J. Vrensen G.F. Schlingemann R.O. In vivo angiogenic phenotype of endothelial cells and pericytes induced by vascular endothelial growth factor-A.J Histochem Cytochem. 2004; 52: 39-52Crossref PubMed Scopus (83) Google Scholar in a VEGF receptor (VEGFR)-2–dependent manner.26Strickland L.A. Jubb A.M. Hongo J.A. Zhong F. Burwick J. Fu L. Frantz G.D. Koeppen H. Plasmalemmal vesicle-associated protein (PLVAP) is expressed by tumour endothelium and is upregulated by vascular endothelial growth factor-A (VEGF).J Pathol. 2005; 206: 466-475Crossref PubMed Scopus (77) Google Scholar Therefore, PLVAP may be involved in VEGF-induced BRB loss in DR. Although PLVAP expression has been associated with BRB loss, a functional contribution of this protein to increased vascular permeability has not yet been found. This study examines the role of PLVAP in BRB loss in vivo and in vitro. To this end, we have assessed the changes that occur in permeability and in endothelial junctions and caveolae formation after knocking down PLVAP to elucidate the mechanisms that underlie the regulatory role of PLVAP in vascular permeability. For knockdown of PLVAP expression, shRNA lentiviral pLKO.1 constructs (SIGMA/TRC; Sigma-Aldrich, Zwijndrecht, the Netherlands) were used. Control cells were transduced with nontargeting shRNA (MISSION Non-Target shRNA Control Vector; Sigma-Aldrich). Virus particles were generated by co-transfecting the constructs with three packaging plasmids, pMDLg/pRRE, pMD2G, and RSV-Rev (Addgene, Cambridge, MA), into 293T cells. The sequences of PLVAP shRNA and Non-Target shRNA were 5′-CCGGCCCTTTCACACACACTTTCTACTCGAGTAGAAAGTGTGTGTGAAAGGGTTTTTTG-3′ and 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTT-3′, respectively. Cells were isolated as described previously.6Wisniewska-Kruk J. Hoeben K.A. Vogels I.M. Gaillard P.J. Van Noorden C.J. Schlingemann R.O. Klaassen I. A novel co-culture model of the blood-retinal barrier based on primary retinal endothelial cells, pericytes and astrocytes.Exp Eye Res. 2012; 96: 181-190Crossref Scopus (77) Google Scholar Three different models were assembled to study endothelial barrier permeability, including i) a bovine retinal endothelial cell (BREC) monolayer, ii) a triple co-culture model with BRECs, pericytes, and astrocytes, and iii) human umbilical vein endothelial cells (HUVECs). In the triple co-culture model, BRECs were seeded on top of the Transwell filter, primary rat astrocytes were seeded on the reverse side of the Transwell filters, and bovine primary pericytes were cultured on the bottom of a 24-well plate in which a Transwell filter was placed, as described previously.6Wisniewska-Kruk J. Hoeben K.A. Vogels I.M. Gaillard P.J. Van Noorden C.J. Schlingemann R.O. Klaassen I. A novel co-culture model of the blood-retinal barrier based on primary retinal endothelial cells, pericytes and astrocytes.Exp Eye Res. 2012; 96: 181-190Crossref Scopus (77) Google Scholar Three independent experiments were performed for each model (n ≥ 11). Three days after assembling each of the models, shRNA lentiviral particles (50 pg/mL) were added to the cells. After 24 hours, cells were stimulated apically with 200 ng/mL human recombinant VEGF-A (Sanquin, Amsterdam, the Netherlands). Permeability was measured 72 hours after stimulation by adding 766 Da Cy3 (GE Healthcare, Eindhoven, the Netherlands) and 70 kDa of fluorescein isothiocyanate (FITC)–dextran (FD; Sigma-Aldrich). Concentrations of the tracer molecules were determined using a fluorescence plate reader (BMG POLARstar; MTX Lab Systems, Vienna, VA), as described previously.6Wisniewska-Kruk J. Hoeben K.A. Vogels I.M. Gaillard P.J. Van Noorden C.J. Schlingemann R.O. Klaassen I. A novel co-culture model of the blood-retinal barrier based on primary retinal endothelial cells, pericytes and astrocytes.Exp Eye Res. 2012; 96: 181-190Crossref Scopus (77) Google Scholar Transendothelial electrical resistance (TEER) was measured in real time with the CellZscope system (NanoAnalytics, Münster, Germany) and expressed as Ω·cm2. In all experimental conditions, data were collected in quadruplicate. Three independent experiments were performed for each model. Total RNA was isolated using TRIzol reagent (Life Technologies, Bleiswijk, the Netherlands) following the manufacturer's protocol. Total RNA (1 μg) was treated with DNase I (Amplification Grade; Life Technologies) and reverse transcribed into first-strand cDNA using a Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, Roskilde, Denmark).6Wisniewska-Kruk J. Hoeben K.A. Vogels I.M. Gaillard P.J. Van Noorden C.J. Schlingemann R.O. Klaassen I. A novel co-culture model of the blood-retinal barrier based on primary retinal endothelial cells, pericytes and astrocytes.Exp Eye Res. 2012; 96: 181-190Crossref Scopus (77) Google Scholar Real-time PCR was performed using a CFX96 system (Bio-Rad; Hercules, CA) as described previously.13Klaassen I. Hughes J.M. Vogels I.M. Schalkwijk C.G. Van Noorden C.J. Schlingemann R.O. Altered expression of genes related to blood-retina barrier disruption in streptozotocin-induced diabetes.Exp Eye Res. 2009; 89: 4-15Crossref PubMed Scopus (80) Google Scholar Expression data were normalized by the geometric mean of the two most stable housekeeping genes (ACTG1 and GAPDH), as determined by NormFinder.34Andersen C.L. Jensen J.L. Orntoft T.F. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets.Cancer Res. 2004; 64: 5245-5250Crossref PubMed Scopus (5219) Google Scholar Three independent experiments were performed for each model (n ≥ 6). Primer sequences for bovine PLVAP were described previously.13Klaassen I. Hughes J.M. Vogels I.M. Schalkwijk C.G. Van Noorden C.J. Schlingemann R.O. Altered expression of genes related to blood-retina barrier disruption in streptozotocin-induced diabetes.Exp Eye Res. 2009; 89: 4-15Crossref PubMed Scopus (80) Google Scholar The primer sequences for ACTG1 and GAPDH were as follows: ACTG1, forward 5′-GATCTGGCACCACACCTTTT-3′, reverse 5′-CCACATACATGGCAGGAGTG-3′; GAPDH, forward 5′-GGCGTGAACCACGAGAAGTATAA-3′, reverse 5′-CCCTCCACGATGCCAAAGT-3′. Animal experiments were performed with the approval of the Animal Ethics Committee of the University of Amsterdam and in compliance with the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research. From postnatal day 7 (P7), C57BL/6 mice were exposed to 75% oxygen for 5 days.35Smith L.E. Wesolowski E. McLellan A. Kostyk S.K. D'Amato R. Sullivan R. D'Amore P.A. Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar At P12, pups were divided randomly, anesthetized, and injected intraocularly with 1 μL of 50 μM anti-Plvap siRNA (Accell SMARTpool; Thermo Scientific), treated with control siRNA (Accell Green Non-targeting siRNA; Thermo Scientific), or remained untreated. Pups were then returned to room air. At P17, fluorescein angiography was performed, and alternatively, eyes were analyzed for PLVAP expression and vascular leakage indicated by IgG extravasation into retinal tissue. Two independent experiments were performed (n ≥ 6). For evaluation of blood vessel tortuosity, retinas were flat mounted and stained with isolectin IB4 probe conjugated with Alexa647 (Life Technologies). A minimum total of four blood vessels (arterioles and venules) were measured per retina (n ≥ 8 per group) using ImageJ version 1.48v (NIH, Bethesda, MD; http://imagej.nih.gov/ij). The tortuosity index was expressed as a quotient: vessel curve length (mean length, 788 μm) over the line distance between the two ends. At P17, oxygen-induced retinopathy (OIR) mice were anesthetized. The pupils of the mouse eyes were dilated with tropicamide eye drops (Mydriacyl; Alcon Laboratories, Fort Worth, TX). A plano contact lens (Cantor + Nissel, Brackley, UK) was placed on the mouse eye to prevent dehydration of the cornea. Next, mice were injected intracardially with a mixture of 70 kDa FD (10 mg/mL; Sigma-Aldrich) and sodium fluorescein [approximately 4 kDa, 10% in phosphate-buffered saline (PBS); Sigma-Aldrich]. Immediately after injections, image acquisition was started using the scanning laser ophthalmoscope HRA2 (Heidelberg Engineering, Heidelberg, Germany). The HRA2 was operated in the fluorescence mode with the excitation light provided by a 488-nm Argon laser. All images were acquired by using the automatic real-time mode. Eyes were enucleated 20 minutes after perfusion of tracers, dissected, and fixed in 4% paraformaldehyde for 4 hours at 4°C. Next, retinas were washed in PBS to remove sodium fluorescein from the tissue and flat mounted. Images of retinas were recorded using a wide-field fluorescence microscope (Leica, Mannheim, Germany). The images were processed in GIMP software version 2.8v (GNU Image Manipulation Program; GNOME Foundation, Cambridge, MA; http://www.gimp.org). Mouse retinas were permeabilized with 0.5% Triton X-100 for 30 minutes and incubated for 1 hour with 2% normal goat serum (Dako, Glostrup, Denmark). Subsequently, retinas were incubated overnight at 4°C with isolectin IB4 (Alexa 546 conjugated; Invitrogen), anti-PLVAP antibody (rat monoclonal, MECA-32; Abcam, Cambridge, UK), or anti-mouse IgG antibody (Dako). Next, retinas were washed three times for 30 minutes in PBS, and Cy3-, Cy5-, FITC-, or Alexa488-conjugated secondary antibodies (Jackson ImmunoResearch, Suffolk, UK) were added. After 2 hours of incubation, retinas were washed three times for 30 minutes in PBS. Finally, retinas were flat mounted and covered with fluorescence mounting medium (Dako). BRECs or HUVECs were cultured on collagen- or gelatin-coated plastic coverslips (Nunc, Thermo Scientific), respectively. Cells were fixed and stained as described previously.6Wisniewska-Kruk J. Hoeben K.A. Vogels I.M. Gaillard P.J. Van Noorden C.J. Schlingemann R.O. Klaassen I. A novel co-culture model of the blood-retinal barrier based on primary retinal endothelial cells, pericytes and astrocytes.Exp Eye Res. 2012; 96: 181-190Crossref Scopus (77) Google Scholar The following antibodies were used: anti–claudin-5 (rabbit polyclonal; Life Technologies), anti–vascular endothelial cadherin (rabbit polyclonal; Abcam), anti–zonula occludens protein 1 (rabbit polyclonal; Life Technologies), anti-VEGFR2 (rabbit polyclonal; Abcam), phalloidin (Texas Red-X; Invitrogen, Life Technologies), and anti-PLVAP (174/2; Abcam). Nuclei were stained using 1 μg/mL Hoechst dye (Life Technologies). Images were recorded using a confocal laser scanning microscope SP8 (Leica). In all experiments, specificity of the staining was tested by omitting primary antibody. Three independent experiments were performed (n ≥ 8). To quantify actin stress fibers, GIMP software was used. Images of BRECs stained with phalloidin probe were converted to gray scale images with thr