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MEF2C Ablation in Endothelial Cells Reduces Retinal Vessel Loss and Suppresses Pathologic Retinal Neovascularization in Oxygen-Induced Retinopathy

Open AccessPublished:April 20, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.02.021
      Ischemic retinopathies, including retinopathy of prematurity and diabetic retinopathy, are major causes of blindness. Both have two phases, vessel loss and consequent hypoxia-driven pathologic retinal neovascularization, yet relatively little is known about the transcription factors regulating these processes. Myocyte enhancer factor 2 (MEF2) C, a member of the MEF2 family of transcription factors that plays an important role in multiple developmental programs, including the cardiovascular system, seems to have a significant functional role in the vasculature. We, therefore, generated endothelial cell (EC)–specific MEF2C-deficient mice and explored the role of MEF2C in retinal vascularization during normal development and in a mouse model of oxygen-induced retinopathy. Ablation of MEF2C did not cause appreciable defects in normal retinal vascular development. However, MEF2C ablation in ECs suppressed vessel loss in oxygen-induced retinopathy and strongly promoted vascular regrowth, consequently reducing retinal avascularity. This finding was associated with suppression of pathologic retinal angiogenesis and blood-retinal barrier dysfunction. MEF2C knockdown in cultured retinal ECs using small-interfering RNAs rescued ECs from death and stimulated tube formation under stress conditions, confirming the endothelial-autonomous and antiangiogenic roles of MEF2C. HO-1 was induced by MEF2C knockdown in vitro and may play a role in the proangiogenic effect of MEF2C knockdown on retinal EC tube formation. Thus, MEF2C may play an antiangiogenic role in retinal ECs under stress conditions, and modulation of MEF2C may prevent pathologic retinal neovascularization.
      Retinal neovascularization (NV) is a major cause of blindness in the working-age and pediatric populations owing to ischemic retinopathies, including retinopathy of prematurity, diabetic retinopathy, and retinal vein occlusions. In these conditions, blood vessel loss [vaso-obliteration (VO)] results in hypoxic retina, which secretes growth factors that stimulate pathologic retinal angiogenesis. This destructive angiogenesis could be prevented either by direct inhibition of the pathologic NV or by reducing retinal vessel loss, thus decreasing the hypoxic stimulus that drives the NV. Current therapeutic strategies focus largely on the former approach of inhibiting the late stage of pathologic NV. There is great potential in the alternative strategy of reducing VO or promoting normal vascular repair, thereby reducing the hypoxic stimulus that drives the late stage of NV.
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      The effects of oxygen stresses on the development of features of severe retinopathy of prematurity: knowledge from the 50/10 OIR model.
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      Retinopathy of prematurity: understanding ischemic retinal vasculopathies at an extreme of life.
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      Through the eyes of a child: understanding retinopathy through ROP the Friedenwald lecture.
      It would, therefore, be highly beneficial to learn more about the factors governing the extent of VO and normal vascular regrowth in the ischemic retinopathies.
      Animal models of oxygen-induced retinopathy (OIR) have been highly useful in providing insights into retinal vascular pathobiology.
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      • Penn J.S.
      Animal models of oxygen-induced retinopathy.
      The mouse model of OIR
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      • Sullivan R.
      • D'Amore P.A.
      Oxygen-induced retinopathy in the mouse.
      has been widely used in part because of the potential for genetic manipulation
      • Stahl A.
      • Connor K.M.
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      • Chen J.
      • Dennison R.J.
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      • Guerin K.I.
      • Hua J.
      • Lofqvist C.
      • Hellstrom A.
      • Smith L.E.
      The mouse retina as an angiogenesis model.
      and has enabled the assessment of various factors on retinal VO, vascular regrowth after injury, and pathologic angiogenesis.
      • Sapieha P.
      • Joyal J.S.
      • Rivera J.C.
      • Kermorvant-Duchemin E.
      • Sennlaub F.
      • Hardy P.
      • Lachapelle P.
      • Chemtob S.
      Retinopathy of prematurity: understanding ischemic retinal vasculopathies at an extreme of life.
      • Smith L.E.
      • Wesolowski E.
      • McLellan A.
      • Kostyk S.K.
      • D'Amato R.
      • Sullivan R.
      • D'Amore P.A.
      Oxygen-induced retinopathy in the mouse.
      • Stahl A.
      • Connor K.M.
      • Sapieha P.
      • Chen J.
      • Dennison R.J.
      • Krah N.M.
      • Seaward M.R.
      • Willett K.L.
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      • Guerin K.I.
      • Hua J.
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      The mouse retina as an angiogenesis model.
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      • Sennlaub F.
      • Lachapelle P.
      • Chemtob S.
      Understanding ischemic retinopathies: emerging concepts from oxygen-induced retinopathy.
      This model has yielded great insights into paracrine factors in the retinal milieu that regulate the retinal vasculature.
      • Sapieha P.
      • Joyal J.S.
      • Rivera J.C.
      • Kermorvant-Duchemin E.
      • Sennlaub F.
      • Hardy P.
      • Lachapelle P.
      • Chemtob S.
      Retinopathy of prematurity: understanding ischemic retinal vasculopathies at an extreme of life.
      • Stahl A.
      • Connor K.M.
      • Sapieha P.
      • Chen J.
      • Dennison R.J.
      • Krah N.M.
      • Seaward M.R.
      • Willett K.L.
      • Aderman C.M.
      • Guerin K.I.
      • Hua J.
      • Lofqvist C.
      • Hellstrom A.
      • Smith L.E.
      The mouse retina as an angiogenesis model.
      • Kermorvant-Duchemin E.
      • Sapieha P.
      • Sirinyan M.
      • Beauchamp M.
      • Checchin D.
      • Hardy P.
      • Sennlaub F.
      • Lachapelle P.
      • Chemtob S.
      Understanding ischemic retinopathies: emerging concepts from oxygen-induced retinopathy.
      Relatively less is known regarding endothelial intrinsic factors and genes dictating VO, vascular regrowth, and pathologic retinal NV in OIR, including relevant transcription factors.
      The transcription factor myocyte enhancer factor 2 (MEF2) C is known to play a critical role in cardiovascular development.
      • Bi W.
      • Drake C.J.
      • Schwarz J.J.
      The transcription factor MEF2C-null mouse exhibits complex vascular malformations and reduced cardiac expression of angiopoietin 1 and VEGF.
      • Lin Q.
      • Lu J.
      • Yanagisawa H.
      • Webb R.
      • Lyons G.E.
      • Richardson J.A.
      • Olson E.N.
      Requirement of the MADS-box transcription factor MEF2C for vascular development.
      • Lin Q.
      • Schwarz J.
      • Bucana C.
      • Olson E.N.
      Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C.
      MEF2C is one of four MEF2 proteins, which compose a distinct class of MADS (MCM1-agamous-deficiens serum response factor)-box family of transcription factors.
      • Shore P.
      • Sharrocks A.D.
      The MADS-box family of transcription factors.
      MEF2 proteins are central regulators of multiple developmental programs and have been demonstrated to play a pivotal role in the morphogenesis and myogenesis of skeletal, cardiac, and smooth muscle cells.
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      • Perry M.
      • Schulz R.A.
      Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors.
      • Potthoff M.J.
      • Olson E.N.
      MEF2: a central regulator of diverse developmental programs.
      Global deletion of mef2c leads to early embryonic lethality due to cardiac defects.
      • Lin Q.
      • Schwarz J.
      • Bucana C.
      • Olson E.N.
      Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C.
      Of particular interest to us, MEF2C seems to have a significant functional role in the vasculature. MEF2C is expressed in endothelial cells (ECs) in vivo
      • Lin Q.
      • Lu J.
      • Yanagisawa H.
      • Webb R.
      • Lyons G.E.
      • Richardson J.A.
      • Olson E.N.
      Requirement of the MADS-box transcription factor MEF2C for vascular development.
      and in vitro.
      • Hosking B.M.
      • Wang S.C.
      • Chen S.L.
      • Penning S.
      • Koopman P.
      • Muscat G.E.
      SOX18 directly interacts with MEF2C in endothelial cells.
      • Maiti D.
      • Xu Z.
      • Duh E.J.
      Vascular endothelial growth factor induces MEF2C and MEF2-dependent activity in endothelial cells.
      Targeted deletion of mef2c in mice leads to severe vascular defects.
      • Bi W.
      • Drake C.J.
      • Schwarz J.J.
      The transcription factor MEF2C-null mouse exhibits complex vascular malformations and reduced cardiac expression of angiopoietin 1 and VEGF.
      • Lin Q.
      • Lu J.
      • Yanagisawa H.
      • Webb R.
      • Lyons G.E.
      • Richardson J.A.
      • Olson E.N.
      Requirement of the MADS-box transcription factor MEF2C for vascular development.
      Our laboratory previously found that MEF2C expression and MEF2-dependent activity in ECs are activated by vascular endothelial growth factor (VEGF).
      • Maiti D.
      • Xu Z.
      • Duh E.J.
      Vascular endothelial growth factor induces MEF2C and MEF2-dependent activity in endothelial cells.
      To address whether MEF2C has a role in retinal vascular regulation, we generated EC-specific MEF2C knockout mice and investigated the function of MEF2C in OIR. We found that inactivation of MEF2C reduces retinal blood vessel loss and promotes retinal revascularization in OIR, thereby suppressing pathologic retinal NV. In addition, knockdown of MEF2C promotes angiogenesis of cultured retinal ECs. Together, the results of these studies suggest that MEF2C has an important inhibitory role in regulating retinal vascularization under stress conditions.

      Materials and Methods

      Animals

      To generate mice that lack mef2c in ECs, Tie2-Cre transgenic (Tie2-Cre+) mice
      • Koni P.A.
      • Joshi S.K.
      • Temann U.A.
      • Olson D.
      • Burkly L.
      • Flavell R.A.
      Conditional vascular cell adhesion molecule 1 deletion in mice: impaired lymphocyte migration to bone marrow.
      were first crossed with mice that carry the conventional exon 2–deleted allele of mef2c (mef2cΔ2)
      • Lin Q.
      • Schwarz J.
      • Bucana C.
      • Olson E.N.
      Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C.
      to generate Tie2-Cre+/mef2c+/Δ2 mice. These genes are on the same chromosome, making the Tie2-Cre and mef2c deletion linked in subsequent breeding. These mice were then mated with mef2cloxp/loxp mice
      • Vong L.H.
      • Ragusa M.J.
      • Schwarz J.J.
      Generation of conditional Mef2cloxP/loxP mice for temporal- and tissue-specific analyses.
      • Vong L.
      • Bi W.
      • O'Connor-Halligan K.E.
      • Li C.
      • Cserjesi P.
      • Schwarz J.J.
      MEF2C is required for the normal allocation of cells between the ventricular and sinoatrial precursors of the primary heart field.
      to generate Tie2-Cre+/mef2cloxp/Δ2 EC-specific mef2c-deficient mice (referred to as mef2cΔEC) and littermate mice mef2c+/loxp (referred to as control). The genotypes of mef2cΔEC mice and control mice were identified by PCR analysis of DNA isolated from tail samples. The PCR primers were as follows: Tie2-Cre forward: 5′-ACCAGCCAGCTATCAACTCG-3′, Tie2-Cre reverse: 5′-TTACATTGGTCCAGCCACC-3′; MEF2C lox forward: 5′-TTCAGGTGACCTCATTTGAACC-3′, MEF2C lox reverse: 5′-GGAGCCATTGCTCATAAGAAAG-3′; and MEF2C neomycin forward: 5′-GGCATGCTGGGGATGCGGTGGGC-3′, MEF2C neomycin reverse: 5′-GTCACCTTAAGACATAAAGCACCCTCC-3′. ROSA 26 reporter (R26R) mice [B6.129S4-Gt(ROSA)26Sortm1Sor/J; The Jackson Laboratory, Bar Harbor, ME]
      • Soriano P.
      Generalized lacZ expression with the ROSA26 Cre reporter strain.
      were used to cross with Tie2-Cre transgenic mice or Tie2-Cre+/mef2c+/Δ2 mice to monitor the specific expression of Cre recombinase under the control of Tie2 promoter. All the animal procedures were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University School of Medicine and were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research.

      Mouse Model of OIR

      The OIR mouse model was described previously by Smith et al.
      • Smith L.E.
      • Wesolowski E.
      • McLellan A.
      • Kostyk S.K.
      • D'Amato R.
      • Sullivan R.
      • D'Amore P.A.
      Oxygen-induced retinopathy in the mouse.
      Briefly, mef2cΔEC mice and control littermates at postnatal day (P)7 along with their nursing mothers were put into an oxygen chamber and exposed to 75% oxygen for 5 days. At P12, mice were removed from the chamber and were maintained in room air for 5 days until P17. Mice were euthanized at different time points, and retinas were harvested for whole-mount staining or were processed for immunohistochemical staining.

      Retinal Whole-Mount Preparations and Staining

      Retinal whole-mount staining was performed as previously described.
      • Connor K.M.
      • Krah N.M.
      • Dennison R.J.
      • Aderman C.M.
      • Chen J.
      • Guerin K.I.
      • Sapieha P.
      • Stahl A.
      • Willett K.L.
      • Smith L.E.
      Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis.
      Briefly, whole eyes were fixed in 4% paraformaldehyde for 30 minutes at room temperature. After the corneas and lenses were removed, the eyecups were fixed in 4% paraformaldehyde for 10 more minutes, and the retinas were carefully dissected. After 1 hour of blocking in 10% normal goat serum in PBST (PBS plus 3% Triton X-100; Roche Diagnostics GmbH, Mannheim, Germany), the retinas were incubated with Alexa Fluor 594–conjugated isolectin GS-IB4 from Griffonia simplicifolia (Invitrogen, Carlsbad, CA) or rat anti–platelet endothelial cell adhesion molecule 1 (PECAM-1) antibody (BD Biosciences, San Jose, CA) at 4°C overnight. For PECAM immunostaining, retinas were washed in PBST and then were incubated with anti-rat IgG conjugated with Alexa Fluor 488 or 594 antibodies (Invitrogen) at 4°C overnight. Anti-NG2 chondroitin sulfate proteoglycan (Millipore, Billerica, MA) was used for immunostaining of pericytes.
      • Ozerdem U.
      • Grako K.A.
      • Dahlin-Huppe K.
      • Monosov E.
      • Stallcup W.B.
      NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis.
      After four 1-hour washes in PBST, retinas were flat mounted on slides in Fluoromount-G (Electron Microscopy Sciences, Hatfield, PA). Retina whole-mount images were taken using a Zeiss AxioVision microscope (Carl Zeiss Microscopy LLC, Thornwood, NY) at ×50 magnification. For LacZ staining, Tie2-Cre/R26R and R26R mice at P3 and P30 were euthanized, and eyes were fixed in 0.5% glutaraldehyde for 5 minutes. After lens removal, eye cups were fixed in 0.5% glutaraldehyde for 10 minutes. Retinas were isolated and washed in PBS three times and then were stained in X-Gal staining solution (0.1% w/v X-Gal, 5 mmol/L K3Fe(CN)6, 5 mmol/L K4Fe(CN)6.6H2O, and 2 mmol/L MgCl2) at 37°C overnight. After staining, retinas were mounted on slides in Aqua-Poly/Mount solution (Polysciences Inc., Warrington, PA).

      Quantitation of Retinal VO and Pathologic NV

      Retinal VO and NV were quantitated as previously described.
      • Connor K.M.
      • Krah N.M.
      • Dennison R.J.
      • Aderman C.M.
      • Chen J.
      • Guerin K.I.
      • Sapieha P.
      • Stahl A.
      • Willett K.L.
      • Smith L.E.
      Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis.
      Briefly, images of each of the four quadrants of retina were imported into Adobe Photoshop CS2 (Adobe Systems Inc., San Jose, CA). Retinal segments were merged to produce one image of each entire retina. VO and pathologic NV areas were quantified by comparing the number of pixels in the VO or NV area with the total number of pixels in the retina.

      Immunohistochemical and Immunofluorescence Staining for Retinal Frozen Sections

      Mice were sacrificed on P9, and eyes were enucleated and directly embedded in optimal cutting temperature medium (Electron Microscopy Sciences) at −80°C. Sections (10 μm) were cut and fixed in cold 4% paraformaldehyde. For immunohistochemical staining, sections were first blocked with 10% normal swine serum in 0.05 mol/L Tris-buffered saline and then were incubated with biotinylated lectin (1:40; Vector Laboratories, Burlingame, CA) for 2 hours. After washes in Tris-buffered saline, a 1:100 dilution of horseradish peroxidase avidin D (Vector Laboratories) was added. Diaminobenzidine substrate solution was applied later to visualize antibody binding. For immunofluorescence staining, sections were fixed as described previously herein and were blocked in 5% normal goat serum for 1 hour. After overnight incubation with anti-MEF2C antibody (Cell Signaling Technology Inc., Beverly, MA) and anti–PECAM-1 antibody (BD Biosciences) at 4°C, sections were washed in Tris-buffered saline plus 0.1% Tween 20 and then were stained with anti-rabbit IgG conjugated with Alexa Fluor 488 (Invitrogen) and anti-rat IgG conjugated with Cy3 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Hoechst 33258 (Invitrogen) was used to stain nuclei. Photographs were taken using a Zeiss LSM 710 confocal microscope (Carl Zeiss Microscopy LLC).

      Measurement of Apoptosis in Retina Vessels

      To detect EC apoptosis in mouse retina under hyperoxic conditions, pups were exposed to 75% oxygen for 16 hours, and retinal cryosections were prepared as described previously herein. Ten-micrometer serial sections were cut and collected from the iris root to the optic nerve. The apoptotic cells were labeled using the ApopTag Plus fluorescein in situ apoptosis detection kit (Millipore). After washing in PBS for 10 minutes, the sections were incubated with anti–PECAM-1 antibody to stain ECs. The retinal sections were also counterstained with Hoechst 33258 for nuclei staining. For each section, images were taken using a Zeiss LSM 710 confocal microscope. To quantify the apoptotic ECs, cells with both PECAM- and fluorescein-positive staining were counted. The data are presented as the mean number of apoptotic ECs per section.

      Blood-Retinal Barrier Assay

      The blood-retinal barrier assay was performed as previously described.
      • Derevjanik N.L.
      • Vinores S.A.
      • Xiao W.H.
      • Mori K.
      • Turon T.
      • Hudish T.
      • Dong S.
      • Campochiaro P.A.
      Quantitative assessment of the integrity of the blood-retinal barrier in mice.
      • Huang H.
      • Van de Veire S.
      • Dalal M.
      • Parlier R.
      • Semba R.D.
      • Carmeliet P.
      • Vinores S.A.
      Reduced retinal neovascularization, vascular permeability, and apoptosis in ischemic retinopathy in the absence of prolyl hydroxylase-1 due to the prevention of hyperoxia-induced vascular obliteration.
      Mice at P17 with OIR were given an i.p. injection of 1 μCi/g body weight of [3H]-mannitol. One hour after injection, the mice were sedated and retinas rapidly removed and dissected to free the lens, vitreous, and any retinal pigment epithelium. The retinas were placed into preweighed scintillation vials. The thoracic cavity was opened, and the left superior lobe of the lung was removed, blotted to remove excess blood, and placed in another preweighed scintillation vial. A left dorsal incision was made, and the retroperitoneal space was entered without entering the peritoneal cavity. The renal vessels were clamped, and the left kidney was removed, cleaned of all fat, blotted, and placed into a preweighed scintillation vial. The remaining droplets on the tissues were allowed to evaporate for 20 minutes. The vials were weighed, and the tissue weights were calculated. One milliliter of NCSII solubilizing solution (Amersham, Chicago, IL) was added to each vial, and the vials were incubated overnight in a 50°C water bath. Solubilized tissue was brought to room temperature and decolorized with 20% benzoyl peroxide in toluene in a 50°C water bath. After reequilibration to room temperature, 5 mL of scintillation fluid CytoScint ES (Fisher Scientific, Pittsburgh, PA) and 30 μL of glacial acetic acid were added. The vials were stored for several hours in darkness at 4°C to eliminate chemiluminescence. Radioactivity was counted using a liquid scintillation counter (LS 6500; Beckman Coulter Inc., Brea, CA). The counts per minute per milligram tissue measured for lung, kidney, and retina from mef2cΔEC mice and control mice were used to calculate the retina/lung and retina/kidney ratios.

      Cell Culture

      Human retinal ECs (HRECs; Cell Systems, Kirkland, WA) were cultured in EGM-2MV medium (Lonza Inc., Walkersville, MD) in a humidified 5% CO2 incubator at 37°C, and the medium was changed every 2 to 3 days. HRECs were grown in fibronectin (Invitrogen)-coated dishes and were used at passages 6 to 10.

      siRNA Transfection

      HRECs were transfected with 25 nmol/L negative control small-interfering RNA (siRNA) (AM4611; Ambion, Austin, TX) and MEF2C siRNA (#1, 106791; #2, 143535; Ambion) or heme oxygenase-1 (HO-1) siRNA (s6673; Ambion) using siPORT amine transfection reagent (Ambion) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were treated with or without H2O2 for 15 minutes and then were used for further assays. Specific knockdown of MEF2C and HO-1 were confirmed by real-time PCR and Western blot analysis.

      Western Blot Analysis

      After treatment, cells were washed with PBS and lysed in Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA). The protein samples from total cell lysates were subjected to 10% SDS-PAGE and were transferred to Hybond ECL nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). After incubating with appropriate primary and secondary antibodies, the blots were detected using the SuperSignal West Pico or Femto chemiluminescent substrates (Pierce Biotechnology, Rockford, IL). For reprobing, the blots were washed in Western blot stripping buffer (Thermo Fisher Scientific Inc, Rockford, IL) for 10 minutes before proceeding with new blotting. Rabbit monoclonal MEF2C antibody (1:1000) was purchased from Cell Signaling Technologies. Rabbit polyclonal anti–HO-1 antibody (1:1000) was from Enzo Life Sciences International Inc. (Plymouth Meeting, PA). Monoclonal glyceraldehyde-3-phosphate dehydrogenase antibody (1:2000; Abcam Inc., Cambridge, MA) was used for loading control.

      Apoptosis Assay

      For the measurement of in vitro apoptosis, siRNA-transfected HRECs were treated with 300 μmol/L H2O2 in endothelial basal medium 2 (EBM2) + 2% fetal bovine serum for 6 hours, and caspase-3/7 activity was measured using a Caspase-Glo 3/7 assay kit (Promega Corp., Madison, WI) according to the manufacturer's instructions.

      Tube Formation Assay

      EC tube formation was evaluated using a two-layer collagen gel mixture assay, as previously described.
      • Im E.
      • Venkatakrishnan A.
      • Kazlauskas A.
      Cathepsin B regulates the intrinsic angiogenic threshold of endothelial cells.
      • Im E.
      • Kazlauskas A.
      Regulating angiogenesis at the level of PtdIns-4,5-P2.
      • Im E.
      • Motiejunaite R.
      • Aranda J.
      • Park E.Y.
      • Federico L.
      • Kim T.I.
      • Clair T.
      • Stracke M.L.
      • Smyth S.
      • Kazlauskas A.
      Phospholipase Cγ activation drives increased production of autotaxin in endothelial cells and lysophosphatidic acid-dependent regression.
      • Watanabe D.
      • Takagi H.
      • Suzuma K.
      • Suzuma I.
      • Oh H.
      • Ohashi H.
      • Kemmochi S.
      • Uemura A.
      • Ojima T.
      • Suganami E.
      • Miyamoto N.
      • Sato Y.
      • Honda Y.
      Transcription factor Ets-1 mediates ischemia- and vascular endothelial growth factor-dependent retinal neovascularization.
      Forty-eight hours after siRNA transfection, HRECs were trypsinized, and 7 × 104 cells were seeded on top of the lower collagen gel layer in each well of the 24-well plate. After overnight culture, cells were then treated with different doses of H2O2 in EBM2 + 2% fetal bovine serum for 5 hours. Medium was then removed, and 150 μL of collagen gel mixture was added to each well. After 2 hours of gel polymerization at 37°C, 500 μL of medium containing H2O2 was added to the upper layer of collagen gel. Eighteen hours later, six random fields in each well were chosen and photographed using an Axiovert 200M microscope (Carl Zeiss Microscopy LLC), and total tube length in each field was measured using ImageJ version 1.41o (NIH, Bethesda, MD). Each experimental condition was assayed in triplicate, and at least three independent experiments were performed.

      Cell Proliferation Assay

      EC proliferation activity was measured using the CellTiter 96 AQueous One Solution Cell proliferation assay (Promega Corp.).
      • Xu Z.
      • Maiti D.
      • Kisiel W.
      • Duh E.J.
      Tissue factor pathway inhibitor-2 is upregulated by vascular endothelial growth factor and suppresses growth factor-induced proliferation of endothelial cells.
      Twenty-four hours after siRNA transfection, cells were trypsinized and seeded into each well of a 96-well plate at a cell density of 2500 cells. The cells were serum starved in EBM2 supplemented with 2% fetal bovine serum for 2 to 3 hours, and the medium was replaced with 100 μL of fresh EBM2 with 2% fetal bovine serum containing 10 ng/mL VEGF. After 72 hours of incubation, 20 μL of MTS reagent from the kit was added to each well and was incubated at 37°C in 5% CO2 for 2 hours. The plates were shaken thoroughly, and absorbance was measured using a microplate reader (FLUOstar OPTIMA; BMG Labtech GmbH, Ortenberg, Germany) at 492 and 620 nm.

      Cell Migration Assay

      Modified Boyden chambers containing polycarbonate membranes (Transwell, 8-μm pore size; Corning Inc., Lowell, MA) coated with 10 μg/mL collagen were used for migration assay.
      • Xu Z.
      • Maiti D.
      • Kisiel W.
      • Duh E.J.
      Tissue factor pathway inhibitor-2 is upregulated by vascular endothelial growth factor and suppresses growth factor-induced proliferation of endothelial cells.
      Forty-eight hours after siRNA transfection, cells were washed twice with PBS and serum starved in EBM2 + 0.1% bovine serum albumin overnight. The next day, cells were trypsinized and seeded onto a collagen I–coated Transwell plate at 6 × 104 per well, and 10 ng/mL VEGF was added to the lower chamber. After incubation at 37°C for 4 hours, the stationary cells on the top of the membrane were removed by a cotton swab, and the migrated cells on the bottom of the membrane were stained with DAPI. Ten photographs for each well were randomly taken under a microscope (Zeiss Axiovert 200) at 20× objective, and cells were counted using ImageJ.

      Recombinant Adenovirus Infection

      Adenovirus encoding green fluorescent protein and constitutively active MEF2C were gifts from Dr. Jeffery Molkentin (University of Cincinnati, Cincinnati, OH).
      • Xu J.
      • Gong N.L.
      • Bodi I.
      • Aronow B.J.
      • Backx P.H.
      • Molkentin J.D.
      Myocyte enhancer factors 2A and 2C induce dilated cardiomyopathy in transgenic mice.
      The cDNA for constitutively active MEF2C encodes amino acids 1 to 143 of MEF2C fused to the VP16 transcriptional activation domain. Adenoviruses were used to infect HRECs at multiplicities of infection of 10 and 25. Forty-eight hours after infection, cells were washed twice with PBS, and proteins were extracted using Laemmli buffer.

      Microarray Analysis

      HRECs were transfected with either control or MEF2C siRNA for 24 hours. For profiling of mRNA, total RNA was extracted using the RNeasy mini kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. RNA amplification and labeling procedures were performed by using the Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies, Palo Alto, CA). RNA spike-in controls (Agilent Technologies) are added to RNA samples before amplification and are labeled according to the manufacturer's protocol. Amplified RNA (from control versus MEF2C siRNA transfection) was labeled with either Cy3 or Cy5, and the two labeled pools were mixed for microarray analysis. Whole human genome microarrays (G4112F; Agilent Technologies) were used that contain 41,000 probes measuring approximately 21,000 unique transcripts. Microarrays were scanned using an Agilent G2505B scanner controlled by Agilent Scan Control software version 7.0 (Agilent Technologies). Data were extracted using Agilent Feature Extraction software version 9.1 (Agilent Technologies). Differentially expressed targets were identified by using processed data and PValueLog ratios generated by software.

      Statistical Analysis

      Data are presented as mean ± SD or mean ± SEM. Student's t-test was used to perform statistical analysis. Multiple groups were compared by using one-way analysis of variance. A value of P < 0.05 was considered statistically significant.

      Results

      Expression of MEF2C in Mouse Retina

      MEF2C is known to be expressed in ECs systemically. We investigated the localization of MEF2C in the mouse retina at P9 and P35. Immunofluorescence staining analysis demonstrated nuclear localization of MEF2C (Figure 1), consistent with its role as a transcription factor. Co-immunostaining with endothelial marker PECAM-1 revealed MEF2C expression in ECs from superficial and deep retinal vessels at P9 (Figure 1). At P35, retinal vessels continued to exhibit MEF2C staining. Aside from retinal ECs, MEF2C was also strongly expressed in some non-EC types distributed in the ganglion cell layer and inner nuclear layer (Figure 1).
      Figure thumbnail gr1
      Figure 1Expression of MEF2C in retinal ECs. Frozen eye sections from P9 mice (top and middle panels) and P35 mice (bottom panel) were labeled with MEF2C antibody (green), PECAM-1 antibody (red) to visualize vessels, and Hoechst 33258 (blue) for nuclear staining. MEF2C was expressed in ECs from superficial and deep vascular layers and was also evident in choroidal vasculature. Besides ECs, there was prominent staining of MEF2C in non-ECs in the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL). Hoechst counterstaining clearly demonstrated MEF2C localization primarily in the cell nucleus. Scale bars: 100 μm (top and bottom panels); 20 μm (middle panel).

      EC-Specific Ablation of mef2c

      Since knockout of the mef2c gene is embryonic lethal due to severe cardiac defects, we selectively knocked out mef2c in vascular ECs using Tie2-driven Cre expression. A similar approach has previously been used to study selective mef2c ablation in multiple other cell types.
      • Vong L.
      • Bi W.
      • O'Connor-Halligan K.E.
      • Li C.
      • Cserjesi P.
      • Schwarz J.J.
      MEF2C is required for the normal allocation of cells between the ventricular and sinoatrial precursors of the primary heart field.
      • Khiem D.
      • Cyster J.G.
      • Schwarz J.J.
      • Black B.L.
      A p38 MAPK-MEF2C pathway regulates B-cell proliferation.
      • Li H.
      • Radford J.C.
      • Ragusa M.J.
      • Shea K.L.
      • McKercher S.R.
      • Zaremba J.D.
      • Soussou W.
      • Nie Z.
      • Kang Y.J.
      • Nakanishi N.
      • Okamoto S.
      • Roberts A.J.
      • Schwarz J.J.
      • Lipton S.A.
      Transcription factor MEF2C influences neural stem/progenitor cell differentiation and maturation in vivo.
      • Verzi M.P.
      • Agarwal P.
      • Brown C.
      • McCulley D.J.
      • Schwarz J.J.
      • Black B.L.
      The transcription factor MEF2C is required for craniofacial development.
      • Wilker P.R.
      • Kohyama M.
      • Sandau M.M.
      • Albring J.C.
      • Nakagawa O.
      • Schwarz J.J.
      • Murphy K.M.
      Transcription factor Mef2c is required for B cell proliferation and survival after antigen receptor stimulation.
      EC-specific mef2c-deficient mice were generated by crossing Tie2-Cre+/mef2c+/Δ2 mice with mef2cloxp/loxp mice. Heterozygous mef2c+/loxp mice were indistinguishable from wild-type mice and served as littermate controls. Retinal vascular endothelial targeting of Tie2-Cre was evaluated by crossing Tie2-Cre transgenic mice with R26R mice.
      • Claxton S.
      • Kostourou V.
      • Jadeja S.
      • Chambon P.
      • Hodivala-Dilke K.
      • Fruttiger M.
      Efficient, inducible Cre-recombinase activation in vascular endothelium.
      X-Gal staining of whole-mount retina from Tie2-Cre/R26R mice revealed Cre recombination activity exclusively in the retinal vascular network (see Supplemental Figure S1 at http://ajp.amjpathol.org). In contrast, no staining was found in Tie2-Cre–negative R26R retinas.

      Normal Retinal Vascular Development in Mice with EC-Specific Ablation of MEF2C

      We first investigated the impact of EC-specific ablation of mef2c on retinal vascular development in mice. At P5, there was no significant difference in vascularized area between mef2cΔEC and control mice (Figure 2, A–C). At P17, the ganglion cell layer, inner nuclear layer, and photoreceptor layers were histologically similar between mef2cΔEC and control mice (Figure 2, D and E). Superficial and deep retinal vessels were present and similar in both groups of mice (Figure 2, D and E). Therefore, EC-specific ablation of MEF2C did not cause abnormalities in normal retinal vascular development.
      Figure thumbnail gr2
      Figure 2mef2cΔEC mice display no significant difference in normal retinal vascular development compared with control mice. Retina whole mounts from control mice (A) and mef2cΔEC mice (B) at P5 were stained with anti-PECAM (red) antibody for retinal vasculature. C: Quantification of the retinal vascularized area. No significant difference was observed (n = 3 to 7; P = 0.59). Data are given as mean ± SD. Histochemical staining of lectin for vasculature and counterstaining of hematoxylin in control (D) and mef2cΔEC (E) retinas at P17. GCL, ganglion cell layer; INL, inner nuclear layer; and ONL, outer nuclear layer. Scale bars: 500 μm (A and B); 50 μm (D and E).

      Attenuation of Retinal VO and Reduced EC Apoptosis in Endothelial-Specific mef2c-Deficient Mice

      We then proceeded to investigate the possible role of MEF2C on retinal vascular health in a pathologic setting using the OIR model.
      • Smith L.E.
      • Wesolowski E.
      • McLellan A.
      • Kostyk S.K.
      • D'Amato R.
      • Sullivan R.
      • D'Amore P.A.
      Oxygen-induced retinopathy in the mouse.
      • Stahl A.
      • Connor K.M.
      • Sapieha P.
      • Chen J.
      • Dennison R.J.
      • Krah N.M.
      • Seaward M.R.
      • Willett K.L.
      • Aderman C.M.
      • Guerin K.I.
      • Hua J.
      • Lofqvist C.
      • Hellstrom A.
      • Smith L.E.
      The mouse retina as an angiogenesis model.
      In this widely used model for the study of ischemic retinopathies, including retinopathy of prematurity, hyperoxic exposure is used to induce retinal VO in neonatal mice.
      • Smith L.E.
      • Wesolowski E.
      • McLellan A.
      • Kostyk S.K.
      • D'Amato R.
      • Sullivan R.
      • D'Amore P.A.
      Oxygen-induced retinopathy in the mouse.
      • Stahl A.
      • Connor K.M.
      • Sapieha P.
      • Chen J.
      • Dennison R.J.
      • Krah N.M.
      • Seaward M.R.
      • Willett K.L.
      • Aderman C.M.
      • Guerin K.I.
      • Hua J.
      • Lofqvist C.
      • Hellstrom A.
      • Smith L.E.
      The mouse retina as an angiogenesis model.
      Retinal ischemia, in turn, results in the up-regulation of multiple proangiogenic growth factors, stimulating pathologic retinal NV. In this model, physiologic retinal revascularization does occur, albeit at a relatively slow rate. Therefore, therapeutic strategies that accelerate retinal revascularization (and thereby reduce retinal ischemia) in this model significantly inhibit pathologic NV.
      • Connor K.M.
      • SanGiovanni J.P.
      • Lofqvist C.
      • Aderman C.M.
      • Chen J.
      • Higuchi A.
      • Hong S.
      • Pravda E.A.
      • Majchrzak S.
      • Carper D.
      • Hellstrom A.
      • Kang J.X.
      • Chew E.Y.
      • Salem Jr, N.
      • Serhan C.N.
      • Smith L.E.
      Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis.
      • Joyal J.S.
      • Sitaras N.
      • Binet F.
      • Rivera J.C.
      • Stahl A.
      • Zaniolo K.
      • Shao Z.
      • Polosa A.
      • Zhu T.
      • Hamel D.
      • Djavari M.
      • Kunik D.
      • Honore J.C.
      • Picard E.
      • Zabeida A.
      • Varma D.R.
      • Hickson G.
      • Mancini J.
      • Klagsbrun M.
      • Costantino S.
      • Beausejour C.
      • Lachapelle P.
      • Smith L.E.
      • Chemtob S.
      • Sapieha P.
      Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A.
      We studied the expression of MEF2C in the hyperoxic (P8) and hypoxic (P14) phases of the OIR model. MEF2C immunostaining was observed in a subset of ECs at P8. Significantly increased MEF2C expression in ECs was apparent at P14 (see Supplemental Figure S2 at http://ajp.amjpathol.org). Hoechst counterstaining clearly demonstrated MEF2C localization primarily in the cell nucleus. We investigated the effect of EC-specific mef2c ablation on hyperoxia-induced retinal VO. Hyperoxia-induced VO is known to be rapid, reaching a peak level of VO within 48 hours after the onset of oxygen exposure (ie, P9).
      • Stahl A.
      • Connor K.M.
      • Sapieha P.
      • Chen J.
      • Dennison R.J.
      • Krah N.M.
      • Seaward M.R.
      • Willett K.L.
      • Aderman C.M.
      • Guerin K.I.
      • Hua J.
      • Lofqvist C.
      • Hellstrom A.
      • Smith L.E.
      The mouse retina as an angiogenesis model.
      • Lange C.
      • Ehlken C.
      • Stahl A.
      • Martin G.
      • Hansen L.
      • Agostini H.T.
      Kinetics of retinal vaso-obliteration and neovascularisation in the oxygen-induced retinopathy (OIR) mouse model.
      No differences in the retinal vasculature between mef2cΔEC and control mice were noted at P9 in room air (Figure 3, A and B), consistent with the observations of normal retinal vascular development at P5 and P17 (Figure 2). At P9 in OIR, however, there was significantly reduced retinal VO in mef2cΔEC mice (Figure 3, C and D), with approximately 37% less VO in mef2cΔEC mice compared with in littermate controls (Figure 3E).
      Figure thumbnail gr3
      Figure 3Decreased VO and EC apoptosis in mef2cΔEC mice during the hyperoxia phase in the mouse OIR model. There is no vessel loss in mef2cΔEC mice or their control littermates at P9 in room air (RA) (A and B) as demonstrated by staining vasculature with GS lectin–Alexa Fluor 549. Retinal VO at P9 (C and D) is shown by staining vasculature with GS lectin–Alexa Fluor 549, as described in Materials and Methods. Avascular areas are highlighted with white dashed lines. Scale bars: 500 μm. E: mef2cΔEC mice had significantly less avascular retinal area than did control mice at P9 (n = 5 to 11 per group). Data are given as mean ± SD. ***P < 0.0001. After 16-hour exposure to 75% oxygen, apoptosis of ECs in control (F) and mef2cΔEC (G) retinas was detected using the TUNEL staining (green) method and co-immunostaining with anti-PECAM antibody (red). Nuclei are stained with Hoechst 33258 (blue). Arrows show apoptotic ECs. Scale bars: 50 μm. GCL, ganglion cell layer; INL, inner nuclear layer; and ONL, outer nuclear layer. H: The number of retinal ECs undergoing apoptosis is significantly decreased in mef2cΔEC mice (n = 7, 12.0 ± 1.8) compared with in their control littermates (n = 9, 16.6 ± 2.3). Data are given as mean ± SD. **P < 0.001.
      It is known that hyperoxia-induced EC apoptosis contributes to vessel regression.
      • Alon T.
      • Hemo I.
      • Itin A.
      • Pe'er J.
      • Stone J.
      • Keshet E.
      Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity.
      We studied EC apoptosis 16 hours after initiation of the hyperoxia phase (P7 + 16 hours) since the maximal degree of VO (and retinal avascularity) is known to occur at P9 in OIR.
      • Stahl A.
      • Connor K.M.
      • Sapieha P.
      • Chen J.
      • Dennison R.J.
      • Krah N.M.
      • Seaward M.R.
      • Willett K.L.
      • Aderman C.M.
      • Guerin K.I.
      • Hua J.
      • Lofqvist C.
      • Hellstrom A.
      • Smith L.E.
      The mouse retina as an angiogenesis model.
      There was significant reduction (approximately 28%) in TUNEL-positive ECs in mef2cΔEC mice compared with in control mice (P = 0.007; Figure 3, F–H). We also found prominent TUNEL-positive cells distributed in the inner nuclear layers of control mice and mef2cΔEC mice (Figure 3, F and G).

      Marked Improvement in Retinal Revascularization Associated with Reduced Pathologic NV in mef2cΔEC Mice

      After the initial phase of retinal VO, induced by the hyperoxic stimulus, the retina undergoes a second phase characterized by a degree of “physiologic” retinal revascularization (or vascular recovery) and pathologic preretinal NV.
      • Stahl A.
      • Connor K.M.
      • Sapieha P.
      • Chen J.
      • Dennison R.J.
      • Krah N.M.
      • Seaward M.R.
      • Willett K.L.
      • Aderman C.M.
      • Guerin K.I.
      • Hua J.
      • Lofqvist C.
      • Hellstrom A.
      • Smith L.E.
      The mouse retina as an angiogenesis model.
      The amount of pathologic preretinal NV is inversely related to the degree of vascular recovery since the latter dictates the extent of retinal hypoxia/ischemia that drives NV.
      • Stahl A.
      • Connor K.M.
      • Sapieha P.
      • Chen J.
      • Dennison R.J.
      • Krah N.M.
      • Seaward M.R.
      • Willett K.L.
      • Aderman C.M.
      • Guerin K.I.
      • Hua J.
      • Lofqvist C.
      • Hellstrom A.
      • Smith L.E.
      The mouse retina as an angiogenesis model.
      As shown in Figure 4, A and B, there was a dramatic reduction in avascular retinal area in mef2cΔEC mice compared with in littermate controls at P12 and P17. Specifically, avascular area was significantly decreased ∼50% in endothelial-specific MEF2C knockout mice compared with in littermate controls at P12. At P17, littermate control mice exhibited an almost fourfold increase in avascular retina compared with mef2cΔEC mice (Figure 4B), indicating a markedly higher degree of retinal revascularization in mef2cΔEC mice compared with in controls. Associated with this reduction in avascular retina, mef2cΔEC mice exhibited significantly less (2.3-fold decrease) pathologic retinal NV (Figure 4C). The vessels in the revascularized area in mef2cΔEC mice were covered by pericytes, as demonstrated by the expression of NG2 (Figure 4D).
      Figure thumbnail gr4
      Figure 4Ablation of MEF2C in ECs stimulates vascular recovery and inhibits pathologic NV. A: Retinal VO and NV of mef2cΔEC mice and control mice at P12 and P17 are shown by staining retinal vasculature with GS lectin–Alexa Flour 549. Avascular areas are highlighted with white dashed lines. Scale bars: 500 μm. B: Quantification of VO area at P12 and P17. mef2cΔEC retinas show significantly enhanced vascular recovery compared with control retinas (n = 3 to 4 for P12; n = 5 to 10 for P17). *P < 0.05, ***P < 0.001. C: mef2cΔEC mice have reduced pathologic NV areas at P17 (n = 5, 4.4% ± 1.9%) compared with littermate control mice (n = 10, 10.3% ± 2.7%). ***P < 0.001. D: The staining of pericytes in mef2cΔEC and control mice at P17 in the OIR model. Whole-mount retinas were stained with NG2 antibody (green) for pericytes and with PECAM-1 antibody (red) to visualize vessels. Scale bars: 200 μm. Data are given as mean ± SD.
      In addition to the structural end point of revascularization, we also looked at the functional end point of ischemia-induced vascular leakage in OIR. At OIR P17, increased blood-retina barrier breakdown was found in control mice, an approximately fivefold increase for the leakage ratio of retina to lung and a sixfold increase for the leakage ratio of retina to kidney (Figure 5). In contrast, endothelial-specific MEF2C knockout mice showed a significant decrease of the blood-retinal barrier compared with control mice at P17 in the OIR model.
      Figure thumbnail gr5
      Figure 5EC-specific MEF2C knockout mice displayed decreased retinal vascular leakage at P17 in the OIR model. A: The leakage ratio of retina to lung. B: The leakage ratio of retina to kidney. Data are given as mean ± SEM (n = 4 to 10). *P < 0.01. RA, room air.

      MEF2C Knockdown Rescues Retinal ECs from Oxidative Stress–Induced Cell Death

      The present results in the mouse model of OIR indicate that MEF2C plays an antiangiogenic role in ECs in this setting since ablation of the mef2c gene results in reduced VO and EC apoptosis and in a dramatic increase in revascularization. To gain further insights into MEF2C function, we investigated the effect of MEF2C knockdown in cultured HRECs. In particular, we were interested in the response to oxidative stimuli, as oxidative stress is known to be a major regulator of the retinal vascular response in the OIR model.
      • Sapieha P.
      • Joyal J.S.
      • Rivera J.C.
      • Kermorvant-Duchemin E.
      • Sennlaub F.
      • Hardy P.
      • Lachapelle P.
      • Chemtob S.
      Retinopathy of prematurity: understanding ischemic retinal vasculopathies at an extreme of life.
      Since EC-specific ablation of mef2c reduced EC apoptosis (Figure 3H), we first investigated the effect of MEF2C knockdown on retinal EC survival. MEF2C protein levels in HRECs were significantly reduced by two different MEF2C siRNA species compared with control siRNA (Figure 6A). Exposure to H2O2 significantly increased EC apoptosis, as expected (Figure 6B). However, EC apoptosis was dramatically reduced by MEF2C knockdown compared with control siRNA (Figure 6B). These data indicate that inhibiting MEF2C expression might help retinal ECs survive under oxidative stress conditions.
      Figure thumbnail gr6
      Figure 6MEF2C regulates H2O2-induced apoptosis in HRECs. A: HRECs were transfected with negative control siRNA (siCTL) or one of two different MEF2C siRNAs (siMEF2C #1 and #2) for 48 hours, and knocking down of MEF2C was confirmed by Western blot analysis to detect MEF2C protein level. B: After siRNA transfection, cells were treated with 300 μmol/L H2O2 for 6 hours. Knocking down of MEF2C significantly decreases H2O2-induced apoptosis in HRECs. Data are from the average values of five independent experiments. Data are given as mean ± SD. *P < 0.05, **P < 0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

      MEF2C Knockdown in Retinal ECs Stimulates Tube Formation under Basal Conditions and Oxidative Stress

      In the OIR model, mice with EC-specific ablation displayed significantly increased retinal revascularization (Figure 4B). We investigated the effect of MEF2C knockdown on in vitro angiogenesis. We used a tube formation assay that has previously been used for retinal ECs.
      • Im E.
      • Venkatakrishnan A.
      • Kazlauskas A.
      Cathepsin B regulates the intrinsic angiogenic threshold of endothelial cells.
      • Im E.
      • Kazlauskas A.
      Regulating angiogenesis at the level of PtdIns-4,5-P2.
      • Im E.
      • Motiejunaite R.
      • Aranda J.
      • Park E.Y.
      • Federico L.
      • Kim T.I.
      • Clair T.
      • Stracke M.L.
      • Smyth S.
      • Kazlauskas A.
      Phospholipase Cγ activation drives increased production of autotaxin in endothelial cells and lysophosphatidic acid-dependent regression.
      • Watanabe D.
      • Takagi H.
      • Suzuma K.
      • Suzuma I.
      • Oh H.
      • Ohashi H.
      • Kemmochi S.
      • Uemura A.
      • Ojima T.
      • Suganami E.
      • Miyamoto N.
      • Sato Y.
      • Honda Y.
      Transcription factor Ets-1 mediates ischemia- and vascular endothelial growth factor-dependent retinal neovascularization.
      Under basal conditions and oxidative stress, knockdown of MEF2C using two different siRNAs resulted in significantly increased retinal EC tube formation compared with control siRNA (Figure 7 and see Supplemental Figure S3 at http://ajp.amjpathol.org). The degree of stimulation of tube formation by MEF2C knockdown was comparable with the stimulation of tube formation by VEGF (10 ng/mL) in control siRNA-transfected HRECs (see Supplemental Figure S4A at http://ajp.amjpathol.org). Although MEF2C knockdown resulted in significant stimulation of tube formation, it had no significant effect on VEGF-induced cell proliferation or migration (see Supplemental Figure S4, B and C, at http://ajp.amjpathol.org). These data suggest an inhibitory role of MEF2C in angiogenesis under basal and stress conditions.
      Figure thumbnail gr7
      Figure 7MEF2C regulates the tube formation ability of HRECs with or without H2O2 treatment. A: Representative images of tube formation assay with HRECs after transfection with control siRNA (siCTL) and MEF2C siRNA (siMEF2C). Scale bar = 100 μm. B: After treatment with 300 μmol/L H2O2, cells with decreased MEF2c expression exhibit stronger tube formation ability than do control cells. Data are from the average values of four independent experiments. Data are given as mean ± SD. *P < 0.01 compared with siCTL.

      HO-1 Induction Is Involved in the Proangiogenic Effect of MEF2C Knockdown on EC Tube Formation

      Given the role of MEF2C as a transcription factor, we looked at possible changes in gene expression resulting from the inhibition of MEF2C. We found that knockdown of MEF2C resulted in significant up-regulation of multiple angiogenesis-related genes through cDNA array analysis, including HO-1, angiopoietin 2, angiopoietin-like 4, fibroblast growth factor 2, and transforming growth factor β 2 (Table 1). There was no change in VEGF-A expression in this microarray study (data not shown). Of these genes, induction of HO-1 was especially significant and interesting. HO-1 is an essential enzyme in heme catabolism and plays a major protective role in cells against stress condition. We confirmed by Western blot analysis that MEF2C knockdown resulted in significant up-regulation of HO-1 (Figure 8A). Consistent with this finding, overexpression of constitutively active MEF2C resulted in significant down-regulation of HO-1 (see Supplemental Figure S5 at http://ajp.amjpathol.org).
      Table 1Multiple Angiogenesis-Related Genes Up-Regulated in MEF2C siRNA-Treated HRECs
      Gene symbolAccession numberGene nameFold enrichmentP value
      HMOX1NM_002133Heme oxygenase (decycling) 12.753.55 × 10−09
      PLAUNM_002658Plasminogen activator, urokinase2.188.91 × 10−07
      TGFB2NM_003238Transforming growth factor beta 22.313.97 × 10−07
      ANGPTL4NM_139314Angiopoietin-like 42.32.55 × 10−07
      FGF18NM_003862Fibroblast growth factor 182.852.97 × 10−05
      FGF2NM_002006Fibroblast growth factor 2 (basic)1.952.61 × 10−05
      ANGPT2NM_001147Angiopoietin 21.648.94 × 10−04
      S100A7NM_002963S100 calcium binding protein A72.127.43 × 10−04
      HS6ST1NM_004807Heparan sulfate 6-O-sulfotransferase 11.659.62 × 10−03
      JUNNM_002228Jun oncogene1.55.88 × 10−03
      CEACAM1NM_001024912Carcinoembryonic antigen-related cell adhesion molecule 11.575.77 × 10−03
      LAMA5NM_005560Laminin, alpha 51.534.31 × 10−03
      ARHGAP22NM_021226Rho GTPase activating protein 221.611.22 × 10−03
      Figure thumbnail gr8
      Figure 8HO-1 mediates the inhibitory effect of MEF2C in regulating EC tube formation under oxidative stress conditions. A: Down-regulation of MEF2C increases HO-1 protein expression in HRECs. HO-1 protein was detected by Western blot analysis 48 hours after HRECs were transfected with MEF2C siRNA (siMEF2C). B: HO-1 siRNA (siHO-1) is effective in knocking down HO-1 expression in HRECs. MEF2C and HO-1 protein were detected by Western blot analysis 48 hours after siRNA transfection. C: Knockdown of HO-1 significantly abrogates increased tube formation in siMEF2C-transfected HRECs with or without H2O2 treatment. Representative images for tube formation assay are shown. Scale bar = 100 μm. D: Quantitation of tube formation in siRNA-transfected HRECs. Data are from the average values of four independent experiments. Data are given as mean ± SD. *P < 0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siCTL, control siRNA.
      To determine the functional importance of HO-1 in MEF2C action, we performed experiments with siRNA-mediated knockdown of HO-1 and MEF2C. HO-1 siRNA was effective at reducing HO-1 protein expression (Figure 8B). The beneficial effect of MEF2C knockdown on HREC tube formation observed in Figure 7 was abrogated when there was concomitant HO-1 knockdown. We observed this under basal conditions and in the presence of oxidative stress stimuli (Figure 8, C and D). This finding indicates that induction of HO-1 plays an important role in the beneficial effects of MEF2C knockdown on HREC, although there are likely to be other genes that are also involved in this beneficial effect.

      Discussion

      Although current therapies for ischemic retinopathies focus largely on the late stage of pathologic retinal NV, there is great rationale for the strategy of reducing the retinal avascularity that provides the driving force for retinal NV. Such a strategy could result in reducing VO, increasing retinal revascularization, or a combination of the two. It is, therefore, of great benefit to learn more about the mechanisms that govern these processes. A great deal of insight has been gained using the OIR model, especially regarding the paracrine factors in the retinal milieu that regulate the retinal vasculature in this model. In contrast, relatively less is known about the EC intrinsic factors that play a role, including transcription factors that regulate ECs in OIR.
      MEF2C is expressed in ECs,
      • Lin Q.
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      Requirement of the MADS-box transcription factor MEF2C for vascular development.
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      • Chen S.L.
      • Penning S.
      • Koopman P.
      • Muscat G.E.
      SOX18 directly interacts with MEF2C in endothelial cells.
      • Maiti D.
      • Xu Z.
      • Duh E.J.
      Vascular endothelial growth factor induces MEF2C and MEF2-dependent activity in endothelial cells.
      and global deletion of mef2c leads to severe vascular defects in mice, suggesting an important functional role of MEF2C in ECs.
      • Bi W.
      • Drake C.J.
      • Schwarz J.J.
      The transcription factor MEF2C-null mouse exhibits complex vascular malformations and reduced cardiac expression of angiopoietin 1 and VEGF.
      • Lin Q.
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      • Yanagisawa H.
      • Webb R.
      • Lyons G.E.
      • Richardson J.A.
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      Requirement of the MADS-box transcription factor MEF2C for vascular development.
      We were, therefore, interested in the possible role that MEF2C might play in ECs in the retina in developmental and pathologic settings. We first confirmed the expression pattern of MEF2C in the retina and found specific nuclear-localized MEF2C in vascular ECs and other cell types in the retinal ganglion cell layer and inner nuclear layer at P9 and P35 (Figure 1). MEF2C expression was not detected in photoreceptors at this early time point, although a recent report found MEF2C to be expressed in interneurons in the inner nuclear layer and rod photoreceptors in adult retinas.
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      we found no effect of EC-specific ablation of mef2c on normal retinal vascular development (Figure 2). Because mice with global deletion of MEF2C also exhibit severe cardiac defects,
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      the vascular defects observed in these mice may be secondary to the circulatory problems resulting from the severely malformed heart.
      We next investigated the possible role of MEF2C in regulating retinal ECs under pathologic circumstances using the OIR model. We found that EC-specific ablation of mef2c reduced retinal VO and dramatically promoted retinal revascularization in OIR. We found a significant decrease in retinal vessel loss in mice with EC-specific ablation of mef2c during the hyperoxia phase at P9 (Figure 3, C–E), which is known to be the point at which peak VO (and retinal avascularity) is reached in OIR.
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      The mouse retina as an angiogenesis model.
      This was associated with a significant decrease in retinal EC apoptosis in mutant mice.
      After P9 in the OIR model, mice undergo a relatively slow process of retinal revascularization, first during the hyperoxia phase from P9 to P12 and subsequently during the phase of relative hypoxia that commences when mice are returned to a room air environment.
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      The mouse retina as an angiogenesis model.
      In mice with EC-specific ablation of mef2c, we found substantial enhancement of revascularization during the hyperoxia and hypoxia phases (Figure 4, A and B). Consequently, the degree of retinal avascularity was dramatically reduced in mutant mice by P17. This was associated with a significant reduction in pathologic retinal NV in these mice at P17, as expected (Figure 4C), since the degree of retinal avascularity is a major driver of pathologic NV.
      • Stahl A.
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      • Chen J.
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      • Hellstrom A.
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      The mouse retina as an angiogenesis model.
      The physiologic revascularization in the knockout was associated with pericytes (Figure 4D), which are implicated in vessel stabilization.
      • Kielczewski J.L.
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      • Grant M.B.
      Novel protective properties of IGFBP-3 result in enhanced pericyte ensheathment, reduced microglial activation, increased microglial apoptosis, and neuronal protection after ischemic retinal injury.
      In addition, the knockout mice exhibited a significant reduction in blood-retinal barrier dysfunction (Figure 5), supporting the conclusion that endothelial ablation of MEF2C promotes physiologic revascularization and normalization of the retinal vasculature.
      We complemented the studies in mice with in vitro studies in cultured HRECs, using an siRNA approach to suppress MEF2C expression. We exposed HRECs to conditions of oxidative stress since this is thought to be a major factor in vessel loss in OIR.
      • Sapieha P.
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      • Rivera J.C.
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      • Sennlaub F.
      • Hardy P.
      • Lachapelle P.
      • Chemtob S.
      Retinopathy of prematurity: understanding ischemic retinal vasculopathies at an extreme of life.
      Consistent with the findings in mouse retina during OIR, MEF2C deficiency promoted angiogenesis in retinal ECs in vitro. Specifically, MEF2C knockdown reduced apoptosis in retinal ECs exposed to oxidative stress (Figure 6) and enhanced EC angiogenesis in a tube formation assay (Figure 7). These in vitro findings are in line with the results in OIR, which demonstrate a reduction in retinal EC apoptosis and a dramatic enhancement in retinal revascularization in mice with EC ablation of mef2c. These results confirm an EC-autonomous role for MEF2C in regulating angiogenesis.
      To gain further insights into the antiangiogenic actions of MEF2C in vitro, we used microarray analysis to investigate gene expression changes after siRNA-mediated MEF2C knockdown in retinal ECs. HO-1 expression was significantly increased and was of particular interest as a possible mechanism for inhibition of EC apoptosis and enhancement of angiogenesis, especially under oxidative stress conditions. HO-1 is well-known to play an important protective role for blood vessels, in large part by mitigating the effects of oxidative stress.
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      We, therefore, directed additional studies toward the possible role of HO-1 in the HREC culture system. We confirmed that knockdown of MEF2C significantly increased HO-1 expression at the protein level in HRECs (Figure 8A). In addition, siRNA-mediated knockdown of HO-1 suppressed retinal EC tube formation in vitro under normal or stress conditions (Figure 8, C and D). In addition, concomitant knockdown of HO-1 significantly abrogated the effect of MEF2C knockdown on enhancing retinal EC tube formation (Figure 8, C and D). This finding indicates that the induction of HO-1 is an important mechanism of the proangiogenic effects of knocking down MEF2C, at least in vitro, although there are likely to be other genes involved in this beneficial effect. Whether MEF2C has a direct or indirect effect on HO-1 expression requires further investigation. It will be of interest to investigate the role of HO-1 in the regulation of retinal ECs in OIR.
      Overall, these findings in vivo and in vitro indicate that MEF2C plays an important role in retinal ECs in regulating cellular response to stress conditions. These findings in mice were performed using a Tie2-Cre system, which is widely used to ablate gene expression in ECs. Of note, this system has also been found to be active in some hematopoietic stem cells.
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      Therefore, since macrophages, myeloid progenitor cells, and endothelial progenitor cells, which are derived from hematopoietic stem cells, could also affect revascularization in OIR, we cannot exclude a contribution from these other cell types to the effects we observed.
      It is becoming increasingly appreciated that reducing retinal avascularity may be an important therapeutic strategy for ischemic retinopathies, therefore making it critical to gain a better understanding of retinal VO and revascularization in OIR. Much has been discovered about the paracrine factors in the retinal milieu that regulate the retinal vascular response to OIR. Research into retinal avascularity in animal models of OIR has identified molecules that can either reduce VO or enhance revascularization in OIR, including insulin-like growth factor binding protein-3,
      • Kielczewski J.L.
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      • Hellstrom A.
      • Smith L.E.
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      erythropoietin,
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      • Smith L.E.
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      • Bosl M.R.
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      In addition, pathogenic molecules have been identified in OIR, including thrombospondin-1,
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      tumor necrosis factor-α,
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      • Gibson D.S.
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      • de la Cruz V.F.
      • McDonald D.M.
      • Stitt A.W.
      Inhibition of tumor necrosis factor-α improves physiological angiogenesis and reduces pathological neovascularization in ischemic retinopathy.
      NADPH oxidase,
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      peroxynitrite/nitrosative stress,
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      trans-arachidonic acids,
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      • Andelfinger G.
      • Kooli A.
      • Germain S.
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      • d'Orleans-Juste P.
      • Gobeil Jr, F.
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      • Falck J.R.
      • Balazy M.
      • Chemtob S.
      Trans-arachidonic acids generated during nitrative stress induce a thrombospondin-1-dependent microvascular degeneration.
      and semaphorin 3A.
      • Joyal J.S.
      • Sitaras N.
      • Binet F.
      • Rivera J.C.
      • Stahl A.
      • Zaniolo K.
      • Shao Z.
      • Polosa A.
      • Zhu T.
      • Hamel D.
      • Djavari M.
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      • Picard E.
      • Zabeida A.
      • Varma D.R.
      • Hickson G.
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      • Costantino S.
      • Beausejour C.
      • Lachapelle P.
      • Smith L.E.
      • Chemtob S.
      • Sapieha P.
      Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A.
      • Duh E.J.
      Sema 3A resists retinal revascularization.
      However, less is known about the molecular determinants of the retinal EC response to OIR, which affects vascular degeneration and revascularization. The present study indicates that MEF2C is an important transcription factor that modulates the response of retinal ECs to stress conditions. Inhibiting MEF2C in retinal ECs protects the retinal vasculature against degeneration and enhances normal revascularization in OIR. Modulation of MEF2C in retinal ECs could, therefore, be a therapeutic strategy to reduce retinal avascularity in ischemic retinopathies.

      Acknowledgments

      We thank Jeremy Nathans, Gerard Lutty, and Imran Bhutto for helpful suggestions, Yanshu Wang for technical assistance regarding retinal whole-mount preparation and staining, Rhonda Grebe for confocal microscope training, and Wayne Yu (Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University) for help with the microarray study.

      Supplementary data

      • Supplemental Figure S1

        Tie2-Cre/R26R mice robustly express active Cre recombinase in retinal vasculature. Tie2-Cre transgenic mice were crossed with R26R mice. X-Gal staining of retinas at P3 (A and B) and P30 (C and D) from Tie2-Cre/R26R (A and C) and R26R (B and D) mice. Scale bars: 100 μm.

      • Supplemental Figure S2

        Expression of MEF2C in retinal ECs at P8 (hyperoxia phase) and P14 (hypoxic phase) in the OIR model. Frozen eye sections from OIR P8 (top panel) and P14 (bottom panel) mice were labeled with MEF2C antibody (green), PECAM-1 antibody (red) to visualize vessels, and Hoechst 33258 (blue) for nuclear staining. GCL, ganglion cell layer; INL, inner nuclear layer; and ONL, outer nuclear layer. Scale bars: 100 μm.

      • Supplemental Figure S3

        MEF2C regulates tube formation in HRECs. A: Representative images of tube formation assay with HRECs are shown after transfection with control siRNA (siCTL) and MEF2C siRNAs #1 and #2 (si-MEF2C). Scale bar = 100 μm. B: Cells transfected with si-MEF2C compared with siCTL cells. Data are from the average values of three independent experiments. Data are given as mean ± SD. **P < 0.001 compared with siCTL.

      • Supplemental Figure S4

        Knocking down MEF2C in HRECs increased tube formation but did not affect cell migration and proliferation. A: Tube formation assay with control siRNA (siCN) and MEF2C siRNA (siMEF2C)–transfected HRECs treated with or without 10 ng/mL VEGF. *P < 0.05, ***P < 0.001. B: Analysis of HREC migration by the modified Boyden chamber assay. siCN or siMEF2C-transfected cells were seeded in migration chambers and were allowed to migrate toward 10 ng/mL VEGF for 4 hours. The migrated cells were counted in 10 different fields (20× objective). **P < 0.01. C: Cell proliferation assay with siRNA-transfected cells treated with or without 10 ng/mL VEGF for 5 days. Data are given as mean ± SD.

      • Supplemental Figure S5

        Overexpression of constitutively active MEF2C suppressed HO-1 expression in HRECs. Adenovirus encoding GFP (AdGFP) and constitutively active MEF2C (AdMEF2-VP16) were used to infect HRECs at multiplicities of infection (MOIs) 10 and 25. HO-1 protein was detected by Western blot analysis 48 hours after infection. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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