Requirement of Site-Specific Tyrosine Phosphorylation of Cortactin in Retinal Neovascularization and Vascular Leakage
Arteriosclerosis, Thrombosis, and Vascular Biology
Abstract
BACKGROUND:
Retinal neovascularization is a major cause of vision impairment. Therefore, the purpose of this study is to investigate the mechanisms by which hypoxia triggers the development of abnormal and leaky blood vessels.
METHODS:
A variety of cellular and molecular approaches as well as tissue-specific knockout mice were used to investigate the role of Cttn (cortactin) in retinal neovascularization and vascular leakage.
RESULTS:
We found that VEGFA (vascular endothelial growth factor A) stimulates Cttn phosphorylation at Y421, Y453, and Y470 residues in human retinal microvascular endothelial cells. In addition, we observed that while blockade of Cttn phosphorylation at Y470 inhibited VEGFA-induced human retinal microvascular endothelial cell angiogenic events, suppression of Y421 phosphorylation protected endothelial barrier integrity from disruption by VEGFA. In line with these observations, while blockade of Cttn phosphorylation at Y470 negated oxygen-induced retinopathy–induced retinal neovascularization, interference with Y421 phosphorylation prevented VEGFA/oxygen-induced retinopathy–induced vascular leakage. Mechanistically, while phosphorylation at Y470 was required for its interaction with Arp2/3 and CDC6 facilitating actin polymerization and DNA synthesis, respectively, Cttn phosphorylation at Y421 leads to its dissociation from VE-cadherin, resulting in adherens junction disruption. Furthermore, whereas Cttn phosphorylation at Y470 residue was dependent on Lyn, its phosphorylation at Y421 residue required Syk activation. Accordingly, lentivirus-mediated expression of shRNA targeting Lyn or Syk levels inhibited oxygen-induced retinopathy–induced retinal neovascularization and vascular leakage, respectively.
CONCLUSIONS:
The above observations show for the first time that phosphorylation of Cttn is involved in a site-specific manner in the regulation of retinal neovascularization and vascular leakage. In view of these findings, Cttn could be a novel target for the development of therapeutics against vascular diseases such as retinal neovascularization and vascular leakage.
Graphical Abstract

Highlights
•
VEGFA (vascular endothelial growth factor A) phosphorylates cortactin at Y421, Y453, and Y470 residues in endothelial cells.
•
Cttn (cortactin) phosphorylation at Y470 residue is required for its interaction with Arp2/3 and CDC6 in enhancing actin polymerization and DNA synthesis, respectively, in promoting the angiogenic responses of endothelial cells.
•
Cttn phosphorylation at Y421 residue leads to disruption of its interaction with VE-cadherin and AJs, resulting in vascular leakage.
•
Whereas Lyn activation is required for cortactin phosphorylation at Y470 residue mediating retinal neovascularization, activation of Syk is needed for cortactin phosphorylation at Y421 residue in the modulation of vascular leakage.
Diabetic retinopathy (DR) is a leading cause of visual impairment and vision loss worldwide.1,2 Epidemiology data indicate that among the 34.4 million US adults with diagnosed diabetes, almost 12% of these individuals were reported with vision disability.3 In addition, the number of people with diagnosed diabetes is predicted to rise to 63 million by 2045 in the United States, which is an alarming factor for an increased incidence of DR.4 Based on the progression of microvascular lesions and changes in the nonvascular cell types in the lesions, DR is classified into an early stage of non–proliferative DR (PDR) and advanced stage of PDR.5 Non-PDR is characterized by microaneurysms and blockage of blood vessels, which restricts blood supply to the retinal tissue resulting in the development of hypoxia.6 The hypoxic tissue then stimulates the secretion of several growth factors, including VEGF (vascular endothelial growth factor), which influence the development of new blood vessels in that area.7 On the other hand, PDR is an advanced stage of non-PDR where newly developed blood vessels grow along the inner surface of the retina into the vitreous.8 Thus, PDR is characterized by retinal neovascularization comprising fragile and leaky blood vessels that leads to the development of a scar retina that in a later stage can result in the retinal detachment from the underlying sclera.8 Furthermore, the leaky blood vessels allow the passage of circulating cells and proteins into the neural retina.9 Together, all these changes could damage retinal neurons and contribute to vision loss.10 It seems that VEGF produced by hypoxic retina triggers the development of nonproductive pathological angiogenesis instead of reparative angiogenesis.11,12 Accordingly, many multicenter randomized clinical trials have been focused on anti-VEGF therapies in treating retinal neovascularization in DR patients.13 However, anti-VEGF therapy was found to be effective in some, but not all patients.14,15
See cover image
The formation of new blood vessels through vasculogenesis and further expansion of the network by angiogenesis appears to be dependent on coordinated endothelial cell (EC) migration, proliferation, and sprouting, which are critically dependent on cytoskeleton remodeling.16 Actin, which is one of the most abundant intracellular cytoskeletal proteins in mammalian cells, through its reorganization plays an important role in the regulation of cell shape, cell migration, cell division, and transcriptional responses.17 As total actin content per cell was not affected during these events, they are achieved by modulation of the ratio of filamentous (F-)actin to monomeric, also called globular (G-)actin.18,19 The helical F-actin filaments are continuously produced in the cytoskeleton by incorporating Mg2+/ATP-bound G-actin monomers into the growing filaments at their fast-growing, so-called barbed ends.20 The resulting F-actin cytoskeleton organized in structures such as lamellipodia, filopodia, or stress fibers mediates various cellular functions.17 However, F-actin cytoskeleton remodeling is dynamically regulated by actin interactions with a plethora of actin-binding proteins.21 The actin-binding proteins can cross-link actin filaments and thus stabilize or enforce given cytoskeletal structures or modulate their interactions with other cytoskeletal components and regulators.22 Cttn (cortactin) is 1 member of a family of F-actin-binding proteins playing important roles in cellular functions.23,24 Mechanistically, Cttn can bind both actin filaments and one of its prominent nucleators, the actin-related protein 2/3 (Arp2/3) complex, and is thereby thought to stabilize actin filament branches formed by this prominent complex.25,26 Aside from this, many reports indicate that Cttn constitutes a key player in aggressive cancers.27 Cttn was found overexpressed in breast cancer,28 colorectal cancer,29 gliosarcoma,30 lung squamous cell carcinoma,31 melanoma,32 and neck squamous cell carcinoma.33 In carcinoma, Cttn was not only observed to be overexpressed but also to undergo posttranslational modifications, such as phosphorylation thereby mediating enhanced cancer cell migration.34,35 In addition, deacetylation of Cttn by HDAC6 was described to be linked to EC migration and angiogenesis.36 Thus, Cttn function seems to be dependent on its induction of expression as well as posttranslational modifications. However, the role of Cttn and its posttranslational modifications in pathological angiogenesis such as retinal neovascularization is unknown. To this end, our results show for the first time that VEGFA phosphorylates Cttn at tyrosine residues 421, 453, and 470 in human retinal microvascular ECs (HRMVECs). Furthermore, while Cttn phosphorylation at Y470 was required for retinal neovascularization, phosphorylation at Y421 was involved in vascular leakage. Besides that, while Cttn phosphorylation at Y470 requires Lyn activation, its phosphorylation at Y421 was dependent on Syk activation in mediating retinal neovascularization and vascular leakage, respectively.
MATERIALS AND METHODS
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Reagents
Recombinant human VEGF165a (catalog no. 293-VE-010/CF) and Mouse VEGFA 164a (catalog no. 493-MV-025/CF) were bought from R&D Systems (Minneapolis, MN). Growth factor–reduced Matrigel (catalog no. 354230) was purchased from BD Biosciences (Bedford, MA). Anti-Btk (sc-1696), anti-CDC6 (sc-9964), anti-Cttn (sc-55579), anti-Fak (sc-1688), anti-Frk (sc-166478), anti-Fyn (sc-365913), anti-Lyn (sc-7274), anti-MEK1 (sc-6250), anti-p53 (sc-126), anti-Syk (sc-573), anti-Yes (sc-8403), and anti-β-tubulin (sc-5274) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-CD31 antibody (catalog no. 550274) was bought from BD Pharmingen (Palo Alto, CA). Anti-pFyn (Y530; ab192172), anti-Ki67 (ab15580), anti-pLyn (Y507; ab33914), and anti-Pyk2 (ab32571) antibodies were obtained from Abcam (Cambridge, MA). Anti-Src (catalog no. 05-184) and anti-pTyr antibody (PY20; catalog no. 05-777) were purchased from EMD Millipore (Burlington, MA). [3H]-Thymidine (catalog no. NET027E001MC, SA 20 Ci/mmole) was bought from Perkin Elmer (Boston, MA). Syk inhibitor (BAY61-3606; catalog no. ALX-270-479) was obtained from Enzo Life Sciences (Farmingdale, NY). Endothelial basal medium (EBM; CC-3133), and endothelial growth medium (EGM) SingleQuot kit supplements and growth factors (CC-4133) were bought from Lonza (Walkersville, MD). Cytodex microcarrier beads (catalog no. C3278) and thrombin (catalog no. T8885) were obtained from Sigma-Aldrich (St Louis, MO). Fibrinogen (catalog no. 820224) was procured from MP Biomedicals LCC (Solon, OH). Anti-Arp2 (catalog no. 3128), anti-Arp3 (catalog no. 4738), anti-pBtk (Y223; catalog no. 5082), anti-pFak (Y397; catalog no. 3291), anti-pPyk2 (Y402; catalog no. 3291), anti-pSrc (Y416; catalog no. 2101), and anti-pSyk (Y323; catalog no. 2715) were obtained from Cell Signaling Technology (Danvers, MA). Anti-p Cttn (Y421; catalog no. 44-854G), anti-p Cttn (Y466/Y470; catalog no. 44-856), and anti-pYes (Y537; catalog no. PA5-12698) antibodies, human Lyn siRNA (Id: s8358), human Rac1 siRNA (Id: s11711), human RhoA siRNA (Id: s759), human Src siRNA (Id: s13411), invivofectamine 3.0 (catalog no. IVF3001), Nuclear and cytoplasmic extraction kit (catalog no. 78835), phalloidin (catalog no. A12380), and lipofectamine 3000 transfection reagent (catalog no. L-3000-015) were purchased from Thermo Fisher Scientific (Waltham, MA). Arp2/3 inhibitor (CK666; catalog no. 3950), Evans blue (EB; eb catalog no. 0845), and Pyk2 inhibitor (PF431396; catalog no. 4278) were obtained from Tocris Bioscience (Minneapolis, MN). Cell tracker green (C7025), goat anti-rabbit IgG-AlexaFluor 488 (A11034), goat anti-rat IgG-AlexaFluor 568 (A11077), goat anti-rat IgG-AlexaFluor 350 (A21093), Hoechst 3342, isolectin B4-594 (I21413), and Prolong Gold antifade reagent (P36930) were bought from Molecular Probes (Eugene, OR).
Plasmids
Cloning of human Cttn Y421F, Y446F, Y453F, Y470F, and Y486F mutant plasmids were described previously.37
Lentiviral shRNA Particles
Nonmammalian nontarget control (catalog no. SHC0002V), Lyn (catalog no. SHCLNV; Clone ID no. TRCN0000023664), and Syk (catalog no. SHCLNV; Clone ID no. TRCN0000023569) MISSON shRNA lentiviral particles were obtained from Sigma-Aldrich (St Louis, MO). For transduction into retina, 106 viral particles were intravitreally injected once into mouse pups at P10.
Mice
C57BL/6J (WT; strain code, 027) pregnant mice at E16 were bought from Charles River Laboratories (Wilmington, MA). Cdh5-CreERT2 mice were obtained from Dr Luisa Iruela-Arispe at the University of California in Los Angeles, CA. The generation of Cttnflox/flox mice was described previously.38 To delete Cttn in endothelium during postnatal development, we crossbred Cttnflox/flox mice with Cdh5-CreERT2 mice to generate Cttnflox/−:Cdh5-CreERT2 mice. These mice were then backcrossed with Cttnflox/flox mice to generate Cttnflox/flox:Cdh5-CreERT2 litters. To induce Cre activity, 2 consecutive intraperitoneal injections of tamoxifen (50 μL; Sigma-Aldrich, T5648; 2 mg/mL in 10% ethanol and 90% corn oil) were given to mice pups at P9 and P10 to delete Cttn in the endothelium (CttniΔEC). Tamoxifen was also injected into Cttnflox/flox mice pups as a control. The mice strains used in this study were free of RD1 and RD8 mutations,39,40 as revealed by genotyping (Figure S1). Mice were fed with Tekland irradiated LM-485 diet (catalog no. 7912), bred, and maintained at the University of Tennessee Health Science Center’s vivarium. All experiments involving animals were approved by the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center (Memphis, TN).
Cell Culture
HRMVECs (catalog no. ACBRI 181) were purchased from Cell Systems (Kirkland, WA) and grown in EGM containing growth supplements, 10 μg/mL gentamycin, and 0.25 μg/mL amphotericin B. Human fibroblasts (catalog no. PCS-201-013) were obtained from American Type Culture Collection (Manassas, VA) and grown in EGM containing growth supplements, 10 μg/mL gentamycin, and 0.25 μg/mL amphotericin B. Cultures were maintained at 37 °C in a humidified 95% air and 5% CO2 atmosphere. HRMVECs with passage numbers between 5 and 10 were synchronized by maintaining cells in growth factor–free EBM for 24 hours and were used to perform the experiments unless otherwise indicated.
Cell Migration
HRMVEC migration was measured by wound-healing assays essentially as described previously.41 Briefly, HRMVECs were plated at 2×105 cells/mL in each chamber of the ibidi culture inserts and grown to full confluency. Following a 24-hour growth arrest period in EBM, the inserts were removed using sterile tweezers, and 1 mL of EBM containing 5 mmol/L hydroxyurea was added. Cells were treated with and without VEGFA (40 ng/mL) for 24 hours at which time the migrated cells were observed under a Nikon Eclipse TS100 microscope with a 4×/0.13 NA objective, and the images were captured with a Nikon Digital Sight DS-L1 camera. Cell migration was expressed as percentage of wound closure (total area minus area not occupied by the cells/total area×100). Wherever plasmids were used, cells were transfected with either pCMV empty vector or expression vector for the indicated gene and then allowed to grow to 70% to 80% confluence. Cells were then trypsinized, plated at 2×105 cells/mL in each chamber of the ibidi culture inserts, and subjected to migration assay.
DNA Synthesis
DNA synthesis was measured by [3H]-thymidine incorporation as described previously.42 Briefly, cells were transfected with either pCMV or pCMV-Cttn plasmids. Cells were then plated onto 6-well plates, allowed to grow to 70% to 80% confluence, synchronized for 24 hours, and then treated with or without VEGFA (40 ng/mL) for 24 hours. After 6 hours of VEGFA addition, cells were pulse-labeled with 1 μCi/mL of [3H]-thymidine for 18 hours. After a 24-hour incubation period, cells were washed with cold PBS, trypsinized, and pelleted by centrifugation. The cell pellet was resuspended in 3 mL of 20% (w/v) cold TCA and vortexed vigorously to lyse cells. The mixture was then kept on ice for 30 minutes and passed through a GF/F glass microfiber filter. The filter was washed first with 3 mL of 5% cold TCA and then 3 mL of cold ethanol, then dried, placed in a liquid scintillation vial containing 5 mL of scintillation fluid, and the radioactivity counted in a liquid scintillation counter (Beckman LS 3801). DNA synthesis was expressed as fold changes in cost per thousand per dish over control.
Lamellipodia Formation
Lamellipodia formation was measured as described previously.43 Cells were transfected with either pCMV or pCMV-Cttn (Y470F) expression plasmids, trypsinized, pelleted, and seeded onto coverslips in 6-well plates. At 70% to 80% confluency, cells were synchronized for 24 hours and treated with or without VEGFA (40 ng/mL) for 2 hours. Cells were then washed with cold PBS, fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X100, blocked with 3% BSA, and incubated with anti-Arp3 antibody (1:100) overnight followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody and Alexa Fluor 568 Phalloidin. Cells were observed under a Zeiss inverted microscope (Axiovision Observer.z1; 40×/NA 0.6), and the fluorescence images were captured by a Zeiss AxioCam MRm camera using the microscope operating and image analysis software Zen 2.6 (blue edition; Carl Zeiss Imaging Solutions GmbH). Lamellipodia were identified by visualizing F-actin/Arp3 colocalization at the edges of each cell.
Three-Dimensional Sprouting Assay
A 3-dimensional sprouting assay was performed as described previously.42 Briefly, cells were transfected with either pCMV or pCMV-Cttn plasmids. Cells were labeled with a cell tracker, trypsinized, pelleted, and then, an equal number of cells was coated onto Cytodex beads for 6 hours. Nonbinding cells were washed with PBS, and the beads embedded in fibrin gel. Human fibroblasts suspended in EGM with and without VEGFA (40 ng/mL) were seeded onto the top of the fibrin gel at a concentration of 2×104 cells/well and incubated at 37 °C for 6 hours, at which time the medium was replaced with fresh EGM with and without VEGFA, and incubation continued for 3 days. Sprouting was examined on day 3 under a Zeiss inverted fluorescence microscope (AxioVision Observer.z1; 10×/NA 0.45), and the fluorescence images were captured using a Zeiss AxioCam MRm camera, with microscope and camera operated by image analysis software Zen 2.6 (blue edition; Carl Zeiss Imaging Solutions GmbH) (https://www.zeiss.com/content/dam/Microscopy/Downloads/Pdf/zenreleasenotes/zen-2_6_blue_edition-release-notes.pdf). Sprouting was expressed as the number of sprouts per bead.
Tube Formation Assay
Tube formation was measured as described previously.42 Briefly, cells were transfected with either pCMV or pCMV-Cttn plasmids. Cells were synchronized for 24 hours and plated in a 24-well culture plate coated with growth factor–reduced Matrigel. Cells were added with or without VEGFA (40 ng/mL), and incubation continued for 6 hours at 37 °C. Tube formation was observed under an inverted phase contrast microscope (Eclipse TS100; Nikon, Tokyo, Japan), and the images were captured with a CCD color camera (KP-D20AU; Hitachi, Ibaraki, Japan) using Apple iMovie 7.1.4 software. The tube length was calculated using NIH ImageJ version 1.53 software (http://imagej.nih.gov/ij) and expressed in micrometers.
Stress Fiber (F-Actin) Formation
Stress fiber formation was measured as described previously.44 Cells were transfected with either pCMV or pCMV-Cttn plasmids, plated on coverslips placed into 6-well plates, allowed to grow to 70% to 80% confluence, synchronized for 24 hours, and then treated with or without VEGFA (40 ng/mL) for 2 hours. After the incubation period, cells were washed with cold PBS, fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X100, and blocked with 3% BSA. Cells were then incubated with Alexa Fluor 568 Phalloidin (1:500) for 2 hours, washed, counter stained with DAPI, and mounted onto slides. The cells were then observed under a Zeiss inverted microscope (Axiovision Observer.z1; 40×/NA 0.6 or 10×/NA 0.45), and the fluorescence images captured by a Zeiss AxioCam MRm camera using the microscope operating and image analysis software Zen 2.6 (blue edition). Stress fibers were quantified by measuring the intensity of F-actin using ImageJ Version 1.53 software and expressed as fold changes over control.
FITC-Dextran Flux Assay
HRMVECs were grown to a confluent monolayer on the apical side of a 12-well transwell (polycarbonate membrane with 0.4 µm pore size). The cells were growth arrested for 6 hours in EBM. The monolayer was treated with and without VEGFA (40 ng/mL) for the indicated time periods. Wherever plasmids were used, cells were transfected with the indicated plasmid and 36 hours later, these cells were seeded onto the transwell. Fluorescein isothiocyanate-conjugated dextran (MW ≈70 kDa) was added to the top chamber (apical surface) at 100 μg/mL concentration and incubated at 37 °C for 2 hours. The fluorescence intensity of the medium from each chamber (both apical and basal) was measured using SpectraMax Gemini XPS Spectrofluorometer (Molecular Devices). The permeability coefficient (Po) of dextran flux was calculated by using the following equation: Po (flux/h/cm2)=([F1/ΔT]×VA]/(FA×A), where F1 is basal fluorescence; FA is apical fluorescence; ΔT, is change in time (hours); A is surface area (1.22 cm2); VA is the volume of the medium in the basal chamber. The flux was expressed as % dextran flux/h per cm2.45
Transendothelial Electrical Resistance
HRMVECs were seeded onto the apical chamber of a 12-well transwell (polycarbonate membrane with 0.4 µm pore size) at a density of 105 cells/cm2. The cells were allowed to grow to a confluent monolayer and then growth arrested for 6 hours in EBM. The cells were treated with and without VEGFA (40 ng/mL) for the indicated time periods. Wherever plasmid was used, cells were transfected with the indicated plasmid, and 36 hours later, these cells were seeded onto a transwell. Resistance, as an index of barrier function, to current flow between cells and beneath the cell layer, created by cell-cell and cell-matrix components, was measured at various time periods using a Millicell ERS-2 Volt-Ohm Meter (MERS00002, EMD Millipore). Transendothelial resistance was calculated using the following equation: transendothelial electrical resistance=(Rsample–Rblank)×surface area of the transwell. The surface area of 12-mm inserts, which were used in this study, is ≈1.33 cm2. Resistance was expressed as ohm×square centimeter.45
Actin Polymerization Assay
Actin assembly was measured by using the pyrene-actin polymerization kit (BK003, Cytoskeleton, Inc) following the manufacturer’s instructions. Briefly, cells were transfected with pCMV-Cttn (WT) or pCMV-Cttn (mutant) plasmids, and 36 hours later, cells were growth arrested in EGM and treated with VEGFA for 30 minutes. Cell extracts were prepared, immunoprecipitated with anti-Cttn antibody, followed by incubation with Sepharose A beads (40 µL of 50 mg/mL slur), and the immunocomplexes eluted from the beads with 0.2 M glycine (pH 2.6) and neutralized by addition of an equal volume of 20 mmol/L Tris-HCl (pH 8.5). To measure actin polymerization, stock pyrene-actin was diluted to 2.3 µmol/L with actin buffer (5 mmol/L Tris-HCl, pH 8.0, and 0.2 mmol/L CaCl2) containing 0.2 mmol/L ATP and 0.5 mmol/L DTT and stored on ice for 60 minutes to depolymerize the actin oligomers. The actin monomers were then collected by centrifugation at 18 800g for 30 minutes at 4 °C. The pyrene-actin monomers were mixed with the eluted Cttn in a 96-well plate containing polymerization buffer (25 mmol/L KCl and 1 mmol/L MgCl2, pH 7.0). The kinetics of actin polymerization were measured by the fluorescence intensity emitted by pyrene-actin copolymerized into filaments for 1 hour in a SpectraMax Gemini XS spectrofluorometer (Molecular Devices) at 355 nm excitation and 405 nm emission.44
Adherens Junction Staining
Adherens junction (AJ) staining was performed as described previously.45 Briefly, HRMVECs were grown to a confluent monolayer on cell culture-grade glass coverslips, quiesced, and treated with and without VEGFA (40 ng/mL) for 4 hours. After treatments, cells were washed with cold PBS, fixed with 95% ethanol for 30 minutes at 4 °C, permeabilized in TBS (10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl) containing 0.1% Triton ×100 for 10 minutes at room temperature and blocked with 2% BSA in TBS containing 10 mmol/L CaCl2, 5 mmol/L MgCl2, and 0.1% saponin overnight at 4 °C. After incubation with the appropriate primary antibody (1:200 dilution) overnight, Alexa Flour-conjugated secondary antibody was added (1:500 dilution) and incubation continued for 1 hour at room temperature, counterstained with Hoechst 33342 (1:1000 dilution in PBS) for 4 minutes, and mounted onto glass slides with Prolong Gold antifade mounting medium. Wherever plasmids were used, cells were transfected with the indicated plasmid, and 36 hours later, these cells were seeded onto the coverslips. Fluorescence images of the cells were observed under a Zeiss inverted microscope (Axiovision Observer.z1; 40×/NA 0.6 or 10×/NA 0.45), and the fluorescence images captured by a Zeiss AxioCam MRm camera using the microscope operating and image analysis software Zen 2.6 (blue edition).
Western Blotting
After appropriate treatments, cell or retinal extracts were prepared and equal amounts of protein from control, and each treatment were resolved by electrophoresis on SDS-polyacrylamide gels. The proteins were transferred electrophoretically onto a nitrocellulose membrane. After blocking in 5% (w/v) nonfat dry milk or BSA for 1 hour, the membranes were incubated with indicated primary antibodies overnight at 4 °C. Membranes were then washed with TBST 3×, 10 minutes each, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 hour. After washing again with TBST for 3× for 10 minutes each, membranes were incubated with enhanced chemiluminescence reagents (Amersham Biosciences), and the antigen-antibody complexes detected by exposure to X-ray film and developed in an X-ray film developer.
Transfections
HRMVECs were transfected with plasmid DNA (1 μg/mL) using Lipofectamine 3000 transfection reagent according to the manufacturer’s instructions. After 6 hours of incubation, cells were recovered in EGM for 30 hours, synchronized overnight in EBM, and used as needed.
Oxygen-Induced Retinopathy
Oxygen-induced retinopathy (OIR) was performed as per the method of Smith et al46 and quantified as per the protocol of Connor et al47 and as described by us previously.45 C57BL/6J and Cttnflox/flox:Cdh5-CreERT2 mice pups at postnatal day 7 (P7) along with their dams were exposed to 75% oxygen for 5 days (P7–P12) and then returned to room air at P12 to develop relative hypoxia (post-OIR). Mice pups of the same age kept in room air were used as controls. All the pups were euthanized at P17, and eyes were enucleated and fixed in 4% (w/v) paraformaldehyde for 1 hour at room temperature. Retinas were isolated, stained with isolectin B4, flat mounts were made, placed on a coverslip, and examined under a Zeiss inverted fluorescence microscope (Axiovision Observer.z1). Retinal neovascularization was quantified by first setting a scale with a tolerance point of 50 based on the fluorescence intensity in the screenshot using Nikon NIS-Elements software version AR31. Neovascularity was highlighted in red (pseudo color) and then quantified by dividing the fluorescence intensity in the highlighted area by the total fluorescence intensity in the screenshot. When eyes were transfected with plasmid DNA, the pups’ eyes were injected intravitreally with the indicated plasmid DNA at 1 μg/0.5 μL/eye using a 33G needle. Plasmid DNAs were prepared by mixing an equal volume of plasmid DNA (4 μg/μL) with an equal volume of complexation buffer, which was then mixed with an equal volume of Invivofectamine 3.0 transfection reagent (Thermo Fisher). The invivofectamine and plasmid mixtures were then incubated at 50 °C for 30 minutes and diluted 6× with PBS. The resulting mixture was then concentrated using Amicon Ultra-15 Ultracel-50K filters to achieve a final concentration of plasmid DNA of 2.0 μg/μL. Unless stated, mice pups with almost similar body weight (±5%) were used in the study and both eyes of a pup received the same treatment.
Immunofluorescence Staining
Normoxic and hypoxic (post-OIR) eyes were enucleated, fixed in 4% PFA, placed into OCT compound, and cut 8-μm sections from the central part of the retina. After blocking in normal goat serum, the retinal sections were incubated with an indicated primary antibody raised in rabbit or mouse in combination with a rat anti-CD31 antibody at 1:100 dilution followed by incubation with Alexa Fluor 568-conjugated goat anti-rabbit/mouse and Alexa Fluor 488-conjugated goat anti-rat secondary antibodies. The sections were then observed under a Zeiss inverted microscope (Axiovision Observer.z1; 40×/NA 0.6 or 10×/NA 0.45) and the fluorescence images captured by a Zeiss AxioCam MRm camera using the microscope operating and image analysis software Zen 2.6 (blue edition).
Avascular Area
The avascular area was calculated as per the method of Connor et al.47 Briefly, images of post-OIR retinal flat mounts stained with isolectin B4 were obtained using Zeiss AxioObserver.Z1 microscope and AxioCam MRm camera were opened in Adobe Photoshop V. 23.4. Polygonal Lasso tool and the Add To Selection function tool was used to obtain total retinal area. Using the same tools, the avascular area (ie, vaso-obliterated area) was highlighted in white color. Once the total and avascular areas were outlined, with the help of a Refresh key, the number of pixels in those areas was recorded. The avascular area was calculated by dividing the pixels in the avascular area by pixels in the total area and multiplying by 100.
Retinal EC Actin Assembly
After blocking in 5% normal goat serum, the cryosections of P15 normoxic and 72 hours of post-OIR eyes were incubated with rat anti-CD31 antibody (1:100) overnight at 4 °C followed, washed 4× with PBS and incubated with Alexa Fluor 488-conjugated goat anti-rat secondary antibody in combination with Alexa Fluor 568 Phalloidin (1:500). The sections were then observed under a Zeiss inverted microscope (Axiovision Observer.z1; 40×/NA 0.6 or 10×/NA 0.45) and the fluorescence images captured by a Zeiss AxioCam MRm camera using the microscope operating and image analysis software Zen 2.6 (blue edition; Carl Zeiss Imaging Solutions GmbH). The actin polymerization as observed by Phalloidin staining in EC was expressed as CD31/phalloidin-positive cells/section.
Retinal EC Filopodia-Like Protrusions
P15 normoxic and 72 hours of post-OIR retinal flat mounts stained with isolectin B4 were observed under a Zeiss inverted microscope (Axiovision Observer.z1; 40×/NA 0.6 or 10×/NA 0.45) and the fluorescence images captured using a Zeiss AxioCam MRm camera and the microscope operating and image analysis software Zen 2.6 (blue edition; Carl Zeiss Imaging Solutions GmbH). The filopodia-like extensions from ECs at the leading edge of the growing superficial vascular network (vascular front) were counted using NIH ImageJ version 1.53 software and expressed as filopodia-like protrusions/100 µm vessel length.
Retinal Vascular Permeability Assay
Retinal vascular permeability assay was performed as per the method of Vähätupa et al.48 Briefly, C57BL/6J and Cttnflox/flox:Cdh5-CreERT2 mice pups were subjected to OIR, and at 96 hours post-OIR (P16), pups were injected with 100 μL of 1% EB intraperitoneally or FITC-dextran (200 μg/mL) intravenously via tail vein. Twenty-four hours later (ie, at P17), pups were euthanized, eyes enucleated, fixed and retinas isolated. Retinas from EB-injected pups were photographed and weighed. Then, EB was eluted from the retinas by incubation in 200 μL of N, N-dimethylformamide overnight at 78 °C with gentle shaking. The supernatants were collected after centrifugation at 17 000g for 45 minutes at 4 °C, and the absorbance was measured in a spectrophotometer at 620 nm. Using a standard curve for EB, the EB concentration in the retina was calculated and normalized to the weight of the retina.49 The extravasation of the EB was presented in μg/retina. In the case of FITC-dextran extravasation assay, eyes were enucleated, fixed, retinas isolated, and stained with Hoechst 33342 (1:1000 dilution in PBS) for 30 minutes at room temperature. Subsequently, flat mounts were made, placed on a coverslip, examined under a Zeiss inverted fluorescence microscope (Axiovision Observer.z1), and the images were captured using a Zeiss AxioCam MRm camera. FITC-dextran extravasation was quantified by measuring the fluorescence intensity in the images by NIH ImageJ v1.53 software and presented as fold changes in the fluorescence intensity compared with normoxic retinas.
Miles’ Assay
Vascular permeability was performed as per the method of Brash et al.50 Briefly, 8 weeks of Cttn+/+ and CttniΔEC mice were injected by IP with histamine inhibitor pyrilamine maleate (40 μg/g body weight in saline) followed by IV injection of 100 μL of 1% (w/v in saline) EB via tail vein. The mice were placed on a 37 °C heat plate for 30 minutes to circulate the dye. The mice were anesthetized, and 20 μL of VEGFA (2.5 μg/mL) was injected intradermally into the left flank at 3 sites, 1 cm apart from each other. PBS was injected into the right flanks of the same mice to serve as a vehicle control. After 20 minutes of intradermal injections of PBS or VEGFA, the mice were euthanized, pinned onto a board, subjected to a vertical incision of ≈3 to 4 cm from the lower abdomen up to the chest, followed by teasing away the skin from both flanks with photos taken using a camera to visualize the EB leakage into the skin. Then, equally sized skin regions where PBS or VEGFA were injected were cut and dried overnight at 55 °C. The EB was extracted from the skin samples with 250 μL of formamide and the absorbance measured as described above.
Statistics
Since mouse pups from P6 to P17 were used in the OIR experiments, and the pups are sexually immature, sex differences were not expected to influence the observations. Therefore, OIR experiments were not performed in a sex-specific manner. Regarding Miles’ assay, which was performed in adult mice, no sex differences were observed as analyzed by 2-tailed Student t test at P<0.05 and, therefore, the data were combined and presented. All the cell culture experiments were repeated 3×, and the data presented as mean±SD. D’Agostino-Pearson and F tests were used to determine the normality of the data and the equality of group variance, respectively, with GraphPad Prism v 8.00 software (https://www.graphpad.com/features) and the present data satisfied these 2 measures. Normally distributed data with similar variance were analyzed by 1-way ANOVA followed by Fisher least significant difference post hoc test or 2-tailed Student t test with P<0.05 considered as statistically significant. The group size for mice was determined by priori power analysis, where α=0.05, β=0.20, and power=0.80.
RESULTS
Cttn Phosphorylation at Y470 Is Required for VEGFA-Induced HRMVEC Angiogenic Events In Vitro and OIR-Induced Retinal Neovascularization In Vivo
Cttn plays an important role in F-actin formation and its stabilization, which is a critical factor in actin cytoskeletal remodeling.26,51 Many studies have reported that cytoskeletal remodeling is required for cell migration, proliferation, and differentiation.52–54 Since VEGFA plays a pivotal role in physiological and pathological angiogenesis by stimulating EC migration, proliferation, and differentiation,55,56 we asked whether Cttn has any role in VEGFA-induced EC angiogenic responses. To this end, first, we studied the time-course effect of VEGFA on Cttn tyrosine phosphorylation. We found that VEGFA induces tyrosine phosphorylation of Cttn in a time-dependent manner with maximum effect at 30 minutes and declining thereafter in HRMVECs (Figure 1A). Previously, we and others have reported the presence of several tyrosine phosphorylation sites in human Cttn.37,51,57,58 In consideration of these observations, we next wanted to identify the tyrosine residue(s) that was phosphorylated in HRMVECs by VEGFA. Using mutants for the potential phosphorylation sites,37 we found that VEGFA induces Cttn phosphorylation at Y421, Y453, and Y470 residues (Figure 1B). Since Cttn is an actin-binding protein and involved in F-actin formation,26 we tested whether phosphorylation of Cttn at these sites has any link to actin assembly. We found that VEGFA induces actin polymerization and that Cttn Y470F but not Y421F or Y453F mutant attenuates this effect (Figure 1C). Thus, Cttn appeared to promote the formation of stress fibers in HRMVECs upon treatment with VEGFA. Interestingly, expression of Cttn Y470F mutant suppressed VEGFA-induced F-actin stress fiber formation by ≈65% (Figure 1D). Previous studies have reported that Cttn interacts with Arp2/3 complex in the regulation of actin polymerization.59 Based on these observations, we tested whether phosphorylation of Cttn at Y470 was required for its interaction with Arp2/3 complex. We found that in response to VEGFA, Cttn interacts with Arp2/3 and that expression of Cttn Y470F mutant blocks this interaction by >70% (Figure 1E). In addition, inhibition of Arp2/3 complex by CK66660 blocked VEGFA-induced actin polymerization completely (Figure 1F). These observations indicated that Cttn phosphorylation at Y470 residue is required for VEGFA-induced actin polymerization and stress fiber formation. Cttn has been reported to play a role in cell migration and proliferation.23,24,37 In lieu of these observations, we next studied the role of Cttn tyrosine phosphorylation in VEGFA-induced HRMVEC migration and proliferation. While expression of Cttn variants deficient for phosphorylation at Y421 or Y453 had little effect, the mutant that prevents phosphorylation at Y470 residue suppressed VEGFA-induced HRMVEC migration by ≈75% and proliferation by ≈55% (Figure 2A and 2B). Because lamellipodia formation is required for cell migration,41 we next tested the effect of Cttn Y470F mutant on VEGFA-induced HRMVEC lamellipodia formation. As expected, overexpression of Cttn Y470F mutant attenuated VEGFA-induced HRMVEC lamellipodia formation substantially, a finding that confirms the role of Cttn Y470 phosphorylation in VEGFA-induced HRMVEC migration (Figure S2). Regarding the mechanism by which Cttn modulates VEGFA-induced EC proliferation, previously we reported that CDC6 interacts with Cttn.61 CDC6 is an essential component of prereplication complex that plays an indispensable role in DNA replication.62 Therefore, we first studied a time-course effect of VEGFA on Cttn interaction with CDC6. VEGFA induced Cttn interaction with CDC6 in a manner that correlates with its tyrosine phosphorylation (Figure S3A; Figure 1A). Furthermore, coimmunofluorescence staining revealed that Cttn interaction with CDC6 occurs in the cytoplasm from 30 to 60 minutes, and later, the CDC6 appears only in the nucleus leaving Cttn in the cytoplasm, mostly perinuclear (Figure S3B). Next, we found that Cttn Y470F mutant blocks VEGFA-induced Cttn interaction with CDC6 (Figure S3C). Not only overexpression of Cttn Y470F mutant blocked Cttn interaction with CDC6 but also prevented VEGFA-induced CDC6 translocation from the cytoplasm to the nucleus (Figure S3D). Corroborating the role of Cttn phosphorylation at Y470 in VEGFA-induced EC migration and proliferation, blockade of Cttn phosphorylation at Y470 also inhibited VEGFA-induced HRMVEC sprouting and tube formation (Figure 2C and 2D). To extrapolate these observations to an in vivo setting, we used an OIR model.46,47 Mouse pups with dams (mothers) were either left at room air (normoxia) or in a hyperoxic chamber from postnatal day 7 (P7) to P12 and returned to room air at P12 to develop relative hypoxia (hereon referred to as post-OIR). To study the role of Cttn tyrosine phosphorylation in OIR-induced retinal neovascularization, normoxic and hyperoxic mouse pups were injected intravitreally with pCMV, pCMV-Cttn (WT) or pCMV-Cttn (Y470F) expression plasmids at P10 and P11 for determination of Cttn phosphorylation, and at P10, P11, and P13 to examine retinal neovascularization. As shown in Figure 3A (bottom), phosphorylation of Cttn at Y466, a murine equivalent to human Cttn Y470 residue,63 was found to be induced very robustly in mouse retinas at 24 hours post-OIR as compared with normoxic retinas. In line with the in vitro observations in HRMVECs, overexpression of Cttn Y470F mutant also attenuated OIR-induced Cttn tyrosine phosphorylation in mouse retinas (Figure 3A, bottom). Furthermore, overexpression of Y470F mutant suppressed OIR-induced actin assembly by ≈60% in retinal ECs as measured by fluorescence microscopy of retinal cross sections stained with phalloidin and CD31 (Figure 3B). In addition, overexpression of Cttn Y470F mutant inhibited OIR-induced EC filopodia-like protrusions and proliferation (Figure 3C and 3D). Similarly, overexpression of Cttn Y470F mutant decreased vascular density and retinal neovascularization with an increase in avascular area (Figure 3E through 3G). It was interesting to note that overexpression of WT Cttn enhanced the effect of OIR on retinal neovascularization with an additive decrease in the avascular area (Figure 3B through 3G). In addition, expression of Cttn Y470F mutant delayed revascularization of OIR retinas at least until P23 (11 days post-OIR) as compared with pCMV control (Figure S4). In other words, at P23 (ie, 11 days post-OIR), there was almost ≈80% retinal revascularization in pCMV control, whereas in pCMV-Cttn (Y470F) transfected retinas, the revascularization reached only to 30%. Together, these results indicated that Cttn phosphorylation at Y470 is required for OIR-induced formation of EC filopodia-like protrusions, proliferation, and retinal neovascularization.



Cttn Phosphorylation at Y421 Residue Is Required for Disruption of AJs Leading to Vascular Leakage
ECs by virtue of developing intercellular junctions such as AJs play an important role in the maintenance of vascular permeability.64–67 AJs are composed of protein complexes between VE-cadherin, a transmembrane protein, and its intracellular binding partners, namely α-, β-, γ-, and p120-catenins.64,67 Besides its association with catenins, VE-cadherin also interacts with actin-binding proteins directly or via α-catenin in the maintenance of cytoskeletal stability.67,68 Based on these clues and the fact that Cttn is an actin-binding protein, we wanted to find whether Cttn interacts with VE-cadherin. Interestingly, under resting conditions, Cttn was found to be in a complex with VE-cadherin in HRMVECs, and in response to VEGFA, it dissociated from VE-cadherin in a time-dependent manner (Figure 4A). Many studies have reported a role for posttranslational modifications such as phosphorylation of VE-cadherin and catenins in the regulation of AJ stability.69–71 As VEGFA disrupts AJs72 and phosphorylates Cttn at Y421, Y453, and Y470 residues (present study), we next studied the role of Cttn tyrosine phosphorylation in EC barrier function. As expected, VEGFA decreased transendothelial resistance of the HRMVEC monolayer in a time-dependent manner (Figure 4B). In addition, overexpression of Cttn Y421F but not Y453F or Y470F mutant prevented the decrease in transendothelial electrical resistance in response to VEGFA (Figure 4B). In line with these observations, VEGFA increased dextran flux through the HRMVEC monolayer in a time-dependent manner as compared with vehicle control, and overexpression of Cttn Y421F mutant prevented this effect (Figure 4C). To find whether Cttn phosphorylation at Y421 residue was involved in the disruption of its association with VE-cadherin, next, we tested the effect of Cttn Y421F mutant on Cttn-VE-cadherin interactions. Overexpression of Cttn Y421F mutant protected Cttn and VE-cadherin complex from their disruption by VEGFA in HRMVECs (Figure 4D). In line with these observations, overexpression of Cttn Y421F mutant also protected AJs from VEGFA-induced disruption by >70% (Figure 4E). Together, these results indicated that Cttn phosphorylation at Y421 is involved in VEGFA-induced disruption of HRMVEC AJs and its barrier function. To extrapolate the involvement of Cttn Y421 phosphorylation in EC barrier function in vivo, we examined its role in vascular leakage. WT mouse pups with dams were housed in normoxia or in a hyperoxia chamber from P7 to P12 and at P10, P11, and P13, the pups were given intravitreal injections of pCMV, pCMV-Cttn (WT), or pCMV-Cttn (Y421F) expression plasmids. The pups that were exposed to hyperoxia were then returned to room air at P12 to develop relative hypoxia. At P13, retinas were isolated and examined for Cttn phosphorylation at Y421. We found that in pups that received pCMV alone and subjected to OIR, the retinas showed increased phosphorylation of Cttn at Y421, and this response was completely negated in the retinas of pups that received pCMV-Cttn (Y421F) mutant (Figure 5A and 5B). To test the role of Cttn Y421 phosphorylation in vascular leakage, at P16, the pups were injected by IP with EB, and 24 hours later, the eyes from the pups were enucleated, fixed, retinas isolated, and EB extravasation measured. OIR-induced retinal vascular leakage as observed by EB extravasation (Figure 5A and 5C). While overexpression of Cttn Y421F mutant suppressed retinal vascular leakage by >55%, overexpression of WT Cttn showed a 50% additive effect on vascular leakage (Figure 5C). It is noteworthy that overexpression of Cttn-(WT) also caused a small increase in vascular leakage in normoxic retinas as compared with vector control (Figure 5D). To further confirm the role of Cttn Y421 phosphorylation in retinal vascular leakage, we also used a FITC-dextran (70 kDa) extravasation approach. In line with the observations obtained using EB, the FITC-dextran approach also yielded similar results in normoxic and OIR-induced retinal vascular leakage (Figure 5E). Consistent with its lack of a role in VEGFA-induced HRMVEC migration, proliferation, sprouting, and tube formation, forced expression of Cttn (Y421F) mutant had no effect on OIR-induced retinal neovascularization (Figure S5). Together, these observations indicated a role for Cttn Y421 phosphorylation in retinal vascular leakage.


Lyn and Syk via Mediating Cttn Y470 and Y421 Phosphorylation Modulate Retinal Neovascularization and Vascular Leakage, Respectively
Previous studies have shown that small G proteins such as Rac1 and RhoA play a crucial role in cytoskeletal remodeling.73,74 Since Cttn is an actin-binding cytoskeletal protein, we first studied the role of Rac1 and RhoA in VEGFA-induced Cttn tyrosine phosphorylation. Depletion of either Rac1 or RhoA levels by their target siRNAs had no effect on VEGFA-induced Cttn tyrosine phosphorylation in HRMVECs (Figure S6). Due to the lack of a role for Rac1 and RhoA in VEGFA-induced Cttn tyrosine phosphorylation, we next examined the role of tyrosine kinases. We found that VEGFA stimulates tyrosine phosphorylation of Src, Lyn, Syk, and Pyk2 more robustly and Yes and Fak moderately in a time-dependent manner with maximum effects at 30 minutes and declining thereafter in HRMVECs (Figure 6A). No apparent changes were observed in the phosphorylation of Btk, Fyn, and Frk between control and VEGFA-treated cells (Figure 6A). As VEGFA activated Src, Lyn, Syk, and Pyk2 more robustly than Yes and Fak, we next studied their role in VEGFA-induced Cttn Y421 and Y470 phosphorylation. We found that siRNA-mediated depletion of Lyn but not Src completely blocked VEGFA-induced Cttn Y470 phosphorylation (Figure 6B). Inhibition of either Syk or Pyk2 had no effect on VEGFA-induced Cttn Y470 phosphorylation (Figure 6C). On the other hand, inhibition of Syk but not Pyk2 attenuated Cttn Y421 phosphorylation by VEGFA (Figure 6C). Downregulation of either Lyn or Src had no effect (Figure 6B). These findings revealed that different tyrosine kinases are involved in the phosphorylation of Cttn at different tyrosine residues in response to VEGFA. Consistent with these observations, OIR also induced tyrosine phosphorylation of Lyn and Syk in the mouse pup retinas in a time-dependent manner with maximum effects at 12 hours post-OIR and decreasing thereafter (Figure 6D). In addition, while lentivirus-mediated expression of shRNA targeting Lyn depletion blocked Cttn phosphorylation at Y466, an equivalent of Y470 residue in humans,63 shRNA targeting Syk downregulation suppressed OIR-induced Cttn phosphorylation at Y421 residue (Figure 6E). Based on the role of Lyn and Syk in Cttn Y470 and Y421 phosphorylation, respectively, we next studied their roles in retinal neovascularization and vascular leakage. OIR-induced EC filopodia-like protrusions and retinal neovascularization in mouse pup retinas were reduced substantially (>60%) upon shRNA-mediated depletion of Lyn levels, coinciding with increased avascular areas as compared with control pup retinas (Figure 7A through 7C). On the other hand, OIR-induced retinal vascular leakage was attenuated by shRNA-mediated depletion of Syk levels as compared with their respective controls (Figure 7D and 7E). These observations thus supported a role for Lyn and Syk in the phosphorylation of Cttn at residues Y470 and Y421 in mediating retinal neovascularization and vascular leakage, respectively.


EC-Specific Deletion of Cttn Suppresses OIR-Induced Retinal Neovascularization and Vascular Leakage
To relate that Cttn tyrosine phosphorylation at different sites in ECs was involved in retinal neovascularization and vascular leakage, we used EC-specific Cttn knockout mice. Cttnflox/flox:Cdh5-CreERT2 mouse pups with dams were housed in normoxia or a hyperoxia chamber from P7 to P12 and returned to room air at P12 to develop relative hypoxia. Cre was activated by tamoxifen injection at P9 and P11 to generate EC-specific Cttn knockout pups (now onward referred to as CttniΔEC pups). The eyes from normoxic and 72 and 120 hours post-OIR pups were enucleated, fixed, retinas isolated, and analyzed for neovascularization. At the same time, tail clips (2 mm) were collected from these pups and genotyped for Cttnflox/flox and Cdh5-CreERT2 alleles. Mouse pups that were positive for both Cttnflox/flox and Cdh5-CreERT2 alleles were considered as CttniΔEC and those which were positive for Cttnflox/flox and negative for Cdh5-CreERT2 alleles were considered as Cttnflox/flox pups. Coimmunostaining for Cttn along with CD31 showed that Cttn levels were completely depleted only in the ECs of both normoxic and post-OIR retinas of CttniΔEC mouse pups as compared with retinas of Cttnflox/flox mouse pups (Figure 8A, bottom). We next examined the role of EC-specific Cttn deletion in OIR-induced actin assembly in the retinal ECs of these mouse pups. As expected, coimmunostaining of retinal cross sections for phalloidin and CD31 showed that actin assembly was induced prominently in ECs of 72 hours post-OIR Cttnflox/flox mouse pup retinas, and this effect was blunted in CttniΔEC mouse pup retinas (Figure 8B). Consistent with these observations, EC-specific deletion of Cttn also reduced EC filopodia-like protrusions, EC proliferation, and retinal neovascularization resulting in increased avascular area as compared with Cttnflox/flox mouse pup retinas (Figure 8C through 8G). Regarding vascular leakage, we used 2 approaches. In the first approach, Cttnflox/flox:Cdh5-CreERT2 mouse pups with dams were housed in normoxia or a hyperoxia chamber from P7 to P12 and returned to room air at P12. Cre was induced in pups by tamoxifen injections at P9 and P11. Normoxic (P16) and 96 hours post-OIR mouse pups (P16) were injected with EB by IP or FITC-dextran by IV and 24 hours later the eyes from these pups were enucleated, fixed, retinas isolated, and examined for retinal vascular leakage. EC-specific deletion of Cttn blunted OIR-induced vascular leakage by >60% as observed by reduced EB/FITC-dextran extravasation (Figure 8H and 8I). In the second approach, we used adult mice to evaluate Cttn’s role in VEGFA-induced vascular hyperpermeability in the skin by Mile’s assay. VEGFA induced EB hyperpermeability in the skin of Cttnflox/flox mice and EC-specific deletion of Cttn blunted this effect by >75% (Figure 8J). Thus, these results demonstrate the involvement of EC-specific Cttn in mediating OIR-induced retinal neovascularization and vascular leakage.

DISCUSSION
DR negatively impacts the quality of life, mental health, and accelerates the fear of vulnerability with loss of independence, self-care, and mobility.75,76 Vision loss in DR is primarily due to hypoxia, retinal hyperpermeability, and neovascularization, which eventually leads to anatomic and functional alterations in retinal cells.9,11,13,48,77 In treating DR, medical interventions such as pan-retinal photocoagulation surgeries and anti-VEGF therapies have emerged as effective approaches.78 In contrast to pan-retinal photocoagulation, which seems to be a 1-time procedure in patients with DR,79 the anti-VEGF therapy requires near-monthly follow-up and injection regimen for adequate treatment for prevention of PDR recurrency.13 Therefore, new therapies that are more targeted, potent, and long-lasting are required for treating PDR.
To this end, our results demonstrate for the first time that Cttn, a key scaffold actin-binding protein, plays a crucial role in OIR-induced retinal neovascularization. In validating a full-blown role of Cttn in retinal angiogenesis, we found that Cttn was phosphorylated on tyrosine residues both in VEGFA-treated HRMVECs and in post-OIR retinas. These findings may infer that Cttn tyrosine phosphorylation was involved in VEGFA and OIR-induced angiogenesis. It is also noteworthy that VEGFA induces tyrosine phosphorylation of Cttn at Y421, Y453, and Y470 residues, perhaps in mediating different aspects of angiogenic events. Previous studies have reported that tyrosine phosphorylation of Cttn was required for promoting actin polymerization.80 In this context, our findings reveal that among the 3 tyrosine residues phosphorylated by VEGFA, phosphorylation of Cttn at Y470 residue was required for its interaction with Arp2/3 complex in the modulation of VEGFA-induced actin polymerization and stress fiber formation. Increased actin assembly and stress fiber formation have been associated with cell stiffness, an important factor in the modulation of cell migration, whereas a reduction in its polymerization correlates with reduced cell stiffness.81 The canonical, so-called ventral stress fibers are not only directly connected to and thus anchored in cell-matrix adhesions, allowing cells to stabilize their protrusions but also able to drag themselves forward during directional migration.82 However, excess stress fiber formation can also counteract efficient migration due to unproductive, overemphasized spatial immobilization of migrating cells, so these processes must be well balanced. In any case, our observations reveal that among the 3 sites of Cttn phosphorylated by VEGFA, blockade of phosphorylation at Y470 residue by Cttn Y470F mutant blocked VEGFA-induced HRMVEC migration. In fact, expression of Cttn Y470F mutant also blocked VEGFA-induced lamellipodia formation in HRMVECs, a finding that confirms the involvement of Cttn phosphorylation at Y470 in EC migration. Stress fiber formation has also been shown to be involved in cell proliferation and differentiation.82–84 Indeed, our findings show that phosphorylation of Cttn at Y470 residue that promoted actin assembly and stress fiber formation was also involved in VEGFA-induced HRMVEC proliferation, sprouting, and tube formation. Regarding the possible mechanism by which Cttn modulates VEGFA-induced EC proliferation, our results revealed that Cttn interacts with CDC6 and promotes its nuclear import and that these effects were dependent on phosphorylation of Cttn at Y470 residue. Cttn phosphorylation at Y470 residue was not only required for VEGFA-induced HRMVEC stress fiber formation but also involved in OIR-induced F-actin formation in retinal ECs, suggesting the role of Cttn Y470 phosphorylation in angiogenic responses during OIR. Notably, since the formation of filopodia-like protrusions is one of the characteristic features of tip cells,85 we also studied the effect of Cttn Y470F mutant on these processes in OIR-induced retinal ECs. Our results suggest that OIR-induced formation of filopodia-like protrusions in ECs of pup’s retinas also requires Cttn phosphorylation at Y470. EC migration and proliferation are essential for tip cell formation in the development of new blood vessels.56 In this regard, our observations show that forced expression of Cttn Y470F mutant blunts OIR-induced EC proliferation and neovascularization with increased avascular area in the retinas of these pups. On the other hand, forced expression of WT Cttn had additive effects on EC proliferation and retinal neovascularization with decreased avascular area. This additive effect could be due to heightened Cttn phosphorylation at Y466 (Y470 in humans), which in turn, increases stress fiber formation and neovascularization. Aside from this, the involvement of Cttn in OIR-induced retinal neovascularization was further supported by the findings that EC-specific knockout of Cttn substantially reduced F-actin and formation of filopodia-like protrusions in OIR-induced retinal ECs, EC proliferation, as well as neovascularization. Thus, the avascular area was also increased in the retinas of these mouse pups. Since blockade of Cttn Y466/Y470 phosphorylation in OIR retinas delayed retinal revascularization at least by 120 hours as compared with vector control, it appears that Cttn phosphorylation at this site is crucial for retinal neovascularization. Together, these observations clearly suggest that Cttn phosphorylation at Y470 residue is required for both VEGFA and OIR-induced EC angiogenic responses.
EC can form lamellipodia as initial contacts with the extracellular matrix and, frequently combined with filopodia-like bridges, develop nascent, VE-cadherin-based junctions.86 Previous studies reported that Cttn is involved in E-cadherin-mediated cell-cell contact formation via its recruitment into cell adhesion sites and involvement in actin reorganization.87 Moreover, Cttn accumulates preferentially at the extending regions of cadherin-adhesive contact zones and by interaction with Arp2/3 was proposed to participate in actin remodeling at these contacts.87 Thus, Cttn can play important roles in the biogenesis of adherens junctions87 and thus in barrier function.38 To this end, our findings reveal that Cttn interacts with VE-cadherin in the resting state and operates in the maintenance of AJ integrity and EC barrier function, but in response to VEGFA, it dissociates from VE-cadherin coinciding with disruption of AJs and barrier integrity. Since the Y421F mutant of Cttn prevented these effects, it seems that the phosphorylation of Cttn at this residue is required for VEGFA-induced disruption of VE-cadherin-Cttn interactions, affecting AJ integrity and EC barrier function. The observation that phosphorylation at Y453 or Y470 residues had no effect on VEGFA-induced VE-cadherin interactions, AJ integrity, and EC barrier function confirmed the site-specificity of Cttn phosphorylation in these events. These conclusions were further supported by our findings that Cttn Y421F mutant also restored OIR-induced retinal vascular leakage in WT mice pups. Similarly, EC-specific deletion of Cttn in mice attenuated OIR-induced retinal vascular leakage as well as VEGFA-induced vascular hyperpermeability. In addition, the role of Cttn in vascular leakage seems to be mediated by its site-specific phosphorylation, as blockade of its phosphorylation at Y421 residue had no effect on OIR-induced neovascularization. Thus, the present observations suggested that Cttn phosphorylation at Y421 leads to its dissociation from VE-cadherin, affecting AJ integrity and thereby increasing vascular permeability in response to VEGFA/OIR. Regarding the role of Cttn Y453 phosphorylation, it had no effect on VEGFA-induced EC angiogenic responses such as migration, proliferation, sprouting, tube formation, or EC monolayer barrier function. It might be possible that Cttn phosphorylation at Y453 is involved in other EC responses such as cell-to-cell interactions that need to be investigated.
In elucidating the upstream mechanisms by which VEGFA triggers Cttn phosphorylation at different sites, we found that without a role for small G proteins such as Rac1 and RhoA, Lyn mediates Cttn phosphorylation at Y470 residue and Syk mediates Cttn phosphorylation at Y421 residue. A large body of literature suggests that Cttn is a substrate for Src.88,89 Besides this, a few studies also showed that Cttn can be a substrate for Lyn and Syk.90–92 However, these studies did not identify the specific phosphorylation sites of Cttn by Lyn or Syk. To this end, our observations show that Lyn and Syk not only phosphorylate Cttn at different sites but also reveal that these phosphorylation sites have differential roles in mediating retinal neovascularization and vascular leakage. In line with their role in the phosphorylation of Cttn at Y470 and Y421, the depletion of Lyn levels attenuated OIR-induced retinal neovascularization, and depletion of Syk levels blunted OIR-induced vascular leakage. As Cttn phosphorylation at Y421 is required for disruption of Cttn-VE-cadherin interactions and AJs, and Syk mediates Cttn phosphorylation at Y421, we conclude that Syk activation will mediate the disruption of AJ integrity leading to barrier dysfunction and vascular leakage. Together with the involvement of site-specific phosphorylation of Cttn in retinal neovascularization and vascular leakage, EC-specific Cttn deletion also suppressed OIR-induced retinal neovascularization and vascular leakage. Thus, all these observations support a role for EC-specific Cttn on VEGFA/OIR-induced retinal angiogenesis and vascular leakage.
CONCLUSIONS
As depicted in Figure 8K, the above observations suggest that while phosphorylation of Cttn at Y470 by Lyn is required for VEGFA/OIR-induced retinal neovascularization, its phosphorylation at Y421 residue by Syk is involved in contributing to vascular leakage. Therefore, Cttn could be a novel target for the development of therapeutics targeting pathological retinal angiogenesis. Since 2 different phosphorylation sites were involved in retinal neovascularization and vascular leakage and both the events are characteristic features of PDR, use of decoy peptides targeting blockade of phosphorylation at both sites could be considered in the development of effective therapeutic agents against PDR.
ARTICLE INFORMATION
Supplemental Material
Figures S1–S6
Data Set
Major Resources Table
Footnote
Nonstandard Abbreviations and Acronyms
- Cttn
- cortactin
- DR
- diabetic retinopathy
- EB
- Evans blue
- EBM
- endothelial basal medium
- EC
- endothelial cell
- EGM
- endothelial growth medium
- HRMVEC
- human retinal microvascular endothelial cell
- NPDR
- nonproliferative diabetic retinopathy
- OIR
- oxygen-induced retinopathy
- PDR
- proliferative diabetic retinopathy
- VEGF
- vascular endothelial growth factor
- VEGFA
- vascular endothelial growth factor A
- VEGFR
- vascular endothelial growth factor receptor
- WT
- wild type
Supplemental Material
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Received: 16 October 2023
Accepted: 6 December 2023
Published online: 21 December 2023
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This work was supported by the National Eye Institute grants EY014856 and EY034425 from the National Institutes of Health to G.N. Rao and by intramural funding from the Helmholtz Society to K. Rottner.
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