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• W2129081519 abstract "Vascular endothelial growth factors (VEGF) are a Janus-faced family of growth factors exerting both neuroprotective and maladaptive effects on the blood–brain barrier. For example, VEGFs are beneficial in promoting postischemic brain angiogenesis, but the newly formed vessels are leaky. We investigated the role of the naturally occurring murine inhibitory VEGF isoform VEGF165b in a mouse model of focal cerebral ischemia by middle cerebral artery occlusion and reperfusion (I/R) in male C57BL/6 mice. We investigated the roles of VEGF164/165 and VEGF165b in both brain and nonbrain endothelial barrier, angiogenesis, and neutrophil migration using oxygen glucose deprivation and reoxygenation as in vitro model. We investigated the role of VEGF165b in brain edema, neutrophil infiltration, ischemic brain damage, and neuronal death in vivo using an adenovirus encoding a recombinant VEGF164b isoform. Neither VEGF164/165 nor VEGF165b significantly altered brain endothelial barrier or angiogenesis in vitro. However, treatment of brain endothelial cells with VEGF165b increased neutrophil migration in vitro and exacerbated stroke injury by aggravating neutrophil infiltration and neurodegeneration in vivo. Our results indicate that alterations in the delicate balance in the relative levels of pro- and antiangiogenic VEGF isoforms can result in either adaptive or detrimental effects, depending on the VEGF isoform levels and on the duration and extent of injury. Vascular endothelial growth factors (VEGF) are a Janus-faced family of growth factors exerting both neuroprotective and maladaptive effects on the blood–brain barrier. For example, VEGFs are beneficial in promoting postischemic brain angiogenesis, but the newly formed vessels are leaky. We investigated the role of the naturally occurring murine inhibitory VEGF isoform VEGF165b in a mouse model of focal cerebral ischemia by middle cerebral artery occlusion and reperfusion (I/R) in male C57BL/6 mice. We investigated the roles of VEGF164/165 and VEGF165b in both brain and nonbrain endothelial barrier, angiogenesis, and neutrophil migration using oxygen glucose deprivation and reoxygenation as in vitro model. We investigated the role of VEGF165b in brain edema, neutrophil infiltration, ischemic brain damage, and neuronal death in vivo using an adenovirus encoding a recombinant VEGF164b isoform. Neither VEGF164/165 nor VEGF165b significantly altered brain endothelial barrier or angiogenesis in vitro. However, treatment of brain endothelial cells with VEGF165b increased neutrophil migration in vitro and exacerbated stroke injury by aggravating neutrophil infiltration and neurodegeneration in vivo. Our results indicate that alterations in the delicate balance in the relative levels of pro- and antiangiogenic VEGF isoforms can result in either adaptive or detrimental effects, depending on the VEGF isoform levels and on the duration and extent of injury. Cerebral ischemia occurs when any blood vessel supplying blood to the brain is occluded by a thrombus or embolus. This usually results in the formation of an ischemic infarct comprising a dead necrotic core surrounded by a salvageable apoptotic penumbra.1Xing C. Arai K. Lo E.H. Hommel M. Pathophysiologic cascades in ischemic stroke.Int J Stroke. 2012; 7: 378-385Crossref PubMed Scopus (242) Google Scholar The pathology of cerebral ischemia involves, but is not confined to, forms of apoptotic and/or necrotic cell death, severe inflammation, immune cell infiltration, and edema.1Xing C. Arai K. Lo E.H. Hommel M. Pathophysiologic cascades in ischemic stroke.Int J Stroke. 2012; 7: 378-385Crossref PubMed Scopus (242) Google Scholar, 2Chaitanya G.V. Eeka P. Munker R. Alexander J.S. Babu P.P. Role of cytotoxic protease granzyme-b in neuronal degeneration during human stroke.Brain Pathol. 2011; 21: 16-30Crossref PubMed Scopus (30) Google Scholar, 3Chaitanya G.V. Schwaninger M. Alexander J.S. Babu P.P. Granzyme-b is involved in mediating post-ischemic neuronal death during focal cerebral ischemia in rat model.Neuroscience. 2010; 165: 1203-1216Crossref PubMed Scopus (45) Google Scholar, 4Chaitanya G.V. Babu P.P. Multiple apoptogenic proteins are involved in the nuclear translocation of apoptosis inducing factor during transient focal cerebral ischemia in rat.Brain Res. 2008; 1246: 178-190Crossref PubMed Scopus (28) Google Scholar, 5Chaitanya G.V. Babu P.P. Activation of calpain, cathepsin-b and caspase-3 during transient focal cerebral ischemia in rat model.Neurochem Res. 2008; 33: 2178-2186Crossref PubMed Scopus (48) Google Scholar, 6Chaitanya G.V. Cromer W. Wells S. Jennings M. Mathis J.M. Minagar A. Alexander J.S. Metabolic modulation of cytokine-induced brain endothelial adhesion molecule expression.Microcirculation. 2012; 19: 155-165Crossref PubMed Scopus (19) Google Scholar Although several therapeutic options have been advanced for stroke treatment in recent decades, the tissue plasminogen activator protein tPA is the only drug shown to reduce the formation of ischemic infarcts. However, tPA is only modestly effective, and must be given within a 3-hour time window after stroke to be effective. Over the last few years, significant advances have been made in rescuing ischemic brain tissue by promoting brain angiogenesis and neurogenesis through mobilization of de novo stem cells from the circulation and from the subventricular zone.7Arai K. Jin G. Navaratna D. Lo E.H. Brain angiogenesis in developmental and pathological processes: neurovascular injury and angiogenic recovery after stroke.FEBS J. 2009; 276: 4644-4652Crossref PubMed Scopus (215) Google Scholar, 8Font M.A. Arboix A. Krupinski J. Angiogenesis, neurogenesis and neuroplasticity in ischemic stroke.Curr Cardiol Rev. 2010; 6: 238-244Crossref PubMed Scopus (151) Google Scholar, 9Xing C. Hayakawa K. Lok J. Arai K. Lo E.H. Injury and repair in the neurovascular unit.Neurol Res. 2012; 34: 325-330Crossref PubMed Scopus (81) Google Scholar, 10Zhang R.L. Zhang Z.G. Chopp M. Ischemic stroke and neurogenesis in the subventricular zone.Neuropharmacology. 2008; 55: 345-352Crossref PubMed Scopus (140) Google Scholar In addition to stem cell therapy, growth factor therapy aimed at protecting the neurovasculature and promoting ischemic brain angiogenesis is an area under active investigation.7Arai K. Jin G. Navaratna D. Lo E.H. Brain angiogenesis in developmental and pathological processes: neurovascular injury and angiogenic recovery after stroke.FEBS J. 2009; 276: 4644-4652Crossref PubMed Scopus (215) Google Scholar, 9Xing C. Hayakawa K. Lok J. Arai K. Lo E.H. Injury and repair in the neurovascular unit.Neurol Res. 2012; 34: 325-330Crossref PubMed Scopus (81) Google Scholar, 11Harms K.M. Li L. Cunningham L.A. Murine neural stem/progenitor cells protect neurons against ischemia by HIF-1alpha-regulated VEGF signaling.PLoS One. 2010; 5: e9767Crossref PubMed Scopus (79) Google Scholar, 12Hermann D.M. Kilic E. Therapeutic potential and possible risks of pleiotropic growth factors in ischemic stroke.Stroke. 2008; 39: e182Crossref PubMed Scopus (4) Google Scholar VEGFs are a family of growth factors currently under intense investigation in stroke research because of their involvement in neuronal survival and function, as well as in angiogenesis.11Harms K.M. Li L. Cunningham L.A. Murine neural stem/progenitor cells protect neurons against ischemia by HIF-1alpha-regulated VEGF signaling.PLoS One. 2010; 5: e9767Crossref PubMed Scopus (79) Google Scholar, 13Lee H.J. Kim K.S. Park I.H. Kim S.U. Human neural stem cells over-expressing VEGF provide neuroprotection, angiogenesis and functional recovery in mouse stroke model.PLoS One. 2007; 2: e156Crossref PubMed Scopus (213) Google Scholar, 14Taoufik E. Petit E. Divoux D. Tseveleki V. Mengozzi M. Roberts M.L. Valable S. Ghezzi P. Quackenbush J. Brines M. Cerami A. Probert L. TNF receptor I sensitizes neurons to erythropoietin- and VEGF-mediated neuroprotection after ischemic and excitotoxic injury.Proc Natl Acad Sci USA. 2008; 105: 6185-6190Crossref PubMed Scopus (91) Google Scholar, 15Wang Y. Jin K. Mao X.O. Xie L. Banwait S. Marti H.H. Greenberg D.A. VEGF-overexpressing transgenic mice show enhanced post-ischemic neurogenesis and neuromigration.J Neurosci Res. 2007; 85: 740-747Crossref PubMed Scopus (174) Google Scholar, 16Wang Y.Q. Cui H.R. Yang S.Z. Sun H.P. Qiu M.H. Feng X.Y. Sun F.Y. VEGF enhance cortical newborn neurons and their neurite development in adult rat brain after cerebral ischemia.Neurochem Int. 2009; 55: 629-636Crossref PubMed Scopus (67) Google Scholar Even though several VEGF isoforms with diverse functions exist, relatively few of them have received research attention, and some VEGF functions are still unknown. In stroke research, VEGF-A isoforms (VEGF164/165, mouse VEGF-A isoform VEGF164, human VEGF-A isoform VEGF165) are among the best investigated as potential targets in clinical applications. However, several reports point out that, although VEGF164/165 plays a major role in angiogenesis and neuroprotection, it also promotes endothelial barrier disturbances and hemorrhagic transformation.17Abumiya T. Yokota C. Kuge Y. Minematsu K. Aggravation of hemorrhagic transformation by early intraarterial infusion of low-dose vascular endothelial growth factor after transient focal cerebral ischemia in rats.Brain Res. 2005; 1049: 95-103Crossref PubMed Scopus (47) Google Scholar, 18Kanazawa M. Igarashi H. Kawamura K. Takahashi T. Kakita A. Takahashi H. Nakada T. Nishizawa M. Shimohata T. Inhibition of VEGF signaling pathway attenuates hemorrhage after tPA treatment.J Cereb Blood Flow Metab. 2011; 31: 1461-1474Crossref PubMed Scopus (67) Google Scholar, 19Valable S. Montaner J. Bellail A. Berezowski V. Brillault J. Cecchelli R. Divoux D. Mackenzie E.T. Bernaudin M. Roussel S. Petit E. VEGF-induced BBB permeability is associated with an MMP-9 activity increase in cerebral ischemia: both effects decreased by Ang-1.J Cereb Blood Flow Metab. 2005; 25: 1491-1504Crossref PubMed Scopus (173) Google Scholar Inhibition of VEGF164/165 in nonbrain pathologies (eg, inflammatory bowel diseases) has been shown to be beneficial, and several therapeutic monoclonal antibodies targeting VEGF164/165 are being developed.20Chidlow Jr., J.H. Glawe J.D. Pattillo C.B. Pardue S. Zhang S. Kevil C.G. VEGF164 isoform specific regulation of T-cell-dependent experimental colitis in mice.Inflamm Bowel Dis. 2011; 17: 1501-1512Crossref PubMed Scopus (12) Google Scholar, 21Taha Y. Raab Y. Larsson A. Carlson M. Loof L. Gerdin B. Thorn M. Vascular endothelial growth factor (VEGF)–a possible mediator of inflammation and mucosal permeability in patients with collagenous colitis.Dig Dis Sci. 2004; 49: 109-115Crossref PubMed Scopus (48) Google Scholar, 22Tolstanova G. Khomenko T. Deng X. Chen L. Tarnawski A. Ahluwalia A. Szabo S. Sandor Z. Neutralizing anti-vascular endothelial growth factor (VEGF) antibody reduces severity of experimental ulcerative colitis in rats: direct evidence for the pathogenic role of VEGF.J Pharmacol Exp Ther. 2009; 328: 749-757Crossref PubMed Scopus (67) Google Scholar In the intestine, the protection afforded by VEGF-A inhibition appears to mainly reflect inhibition of VEGF164/165–mediated inflammatory angiogenesis.23Cromer W.E. Mathis J.M. Granger D.N. Chaitanya G.V. Alexander J.S. Role of the endothelium in inflammatory bowel diseases.World J Gastroenterol. 2011; 17: 578-593Crossref PubMed Scopus (110) Google Scholar, 24Schweighofer B. Testori J. Sturtzel C. Sattler S. Mayer H. Wagner O. Bilban M. Hofer E. The VEGF-induced transcriptional response comprises gene clusters at the crossroad of angiogenesis and inflammation.Thromb Haemost. 2009; 102: 544-554PubMed Google Scholar In the brain, however, poststroke angiogenesis and vasodilatation are valuable in providing an adequate blood supply to metabolically deprived regions. Currently, it is not clear whether inflammation associated with this process is beneficial or detrimental.25Croll S.D. Ransohoff R.M. Cai N. Zhang Q. Martin F.J. Wei T. Kasselman L.J. Kintner J. Murphy A.J. Yancopoulos G.D. Wiegand S.J. VEGF-mediated inflammation precedes angiogenesis in adult brain.Exp Neurol. 2004; 187: 388-402Crossref PubMed Scopus (154) Google Scholar Given these mutually antagonistic functions of VEGF164/165, it remains unclear whether either administration or inhibition of VEGF164/165 is likely to be of any therapeutic benefit in stroke. We have recently shown that VEGF165b, a naturally occurring isoform of VEGF164/165, can effectively block VEGF164/165–induced inflammatory angiogenesis and attenuate VEGF164/165–induced endothelial barrier permeability in mouse colon endothelial cells.23Cromer W.E. Mathis J.M. Granger D.N. Chaitanya G.V. Alexander J.S. Role of the endothelium in inflammatory bowel diseases.World J Gastroenterol. 2011; 17: 578-593Crossref PubMed Scopus (110) Google Scholar, 26Cromer W. Jennings M.H. Odaka Y. Mathis J.M. Alexander J.S. Murine rVEGF164b, an inhibitory VEGF reduces VEGF-A-dependent endothelial proliferation and barrier dysfunction.Microcirculation. 2010; 17 ([Erratum appeared in Microcirculation 2010, 17:669]): 536-547PubMed Google Scholar Importantly, VEGF165b–mediated protection against barrier disturbances may mechanistically indicate suppression of VEGF164/165–dependent vascular remodeling of junctional molecules.26Cromer W. Jennings M.H. Odaka Y. Mathis J.M. Alexander J.S. Murine rVEGF164b, an inhibitory VEGF reduces VEGF-A-dependent endothelial proliferation and barrier dysfunction.Microcirculation. 2010; 17 ([Erratum appeared in Microcirculation 2010, 17:669]): 536-547PubMed Google Scholar The specific role of this inhibitory VEGF isoform in the pathophysiology of stroke is not known. More importantly, given that VEGF164/165 plays a critical role in ischemic protection of neurons, the outcome of VEGF164/165 antagonism by VEGF165b is not easy to predict. Because VEGF165b and VEGF164/165 can each exert beneficial effects in tissue injury, we hypothesized that VEGF165b might be either helpful in stroke (by decreasing barrier permeability and immune cell infiltration) or detrimental (by interfering with VEGF164/165–mediated angiogenesis after stroke). To understand the roles of VEGF165b, we initially characterized the cellular sources of VEGF165 and VEGF165b in the components of neurovascular unit after oxygen glucose deprivation and re-oxygenation (OGDR) in vitro. We next studied the effect of VEGF165b on brain endothelial barrier function and in vitro capillary tube formation (angiogenesis) assay in vitro. Lastly, to under the functional role of VEGF165b in vivo, we used an adenoviral vector to induce VEGF164b before stroke and assessed specific functional roles of VEGF165b in BBB damage, ischemic infarct sizes, behavioral deficit, neuronal survival, and immune responses in the pathophysiology of stroke. Human VEGF165 (293-VE), human VEGF165b (3045-VE), anti-human VEGF-A (MAB293), and anti-mouse VEGF165 (MAB493) antibodies were purchased from R&D Systems (Minneapolis, MN). Anti-human VEGF165b was prepared as described previously.27Woolard J. Wang W.Y. Bevan H.S. Qiu Y. Morbidelli L. Pritchard-Jones R.O. Cui T.G. Sugiono M. Waine E. Perrin R. Foster R. Digby-Bell J. Shields J.D. Whittles C.E. Mushens R.E. Gillatt D.A. Ziche M. Harper S.J. Bates D.O. VEGF165b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression.Cancer Res. 2004; 64: 7822-7835Crossref PubMed Scopus (389) Google Scholar Vascular growth factor receptor 1 (VEGFR-1) (sc-316) and VEGFR-2 (sc-505) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The human brain endothelial cell line HCMEC-D3 was provided by Dr. Pierre Couraud (CNRS, Paris). The human brain endothelial cell line HBMEC-3 and ECV-304 cell lines were provided by Dr. Anat Erdreich-Epstein (University of Southern California). The astrocyte cell line HFA was provided by Dr. Danica Stanimirovic (University of Ottawa). Mouse brain endothelial cell line bEnd.3 (provided by Dr. Eugene Butcher, Department of Pathology, Stanford University) and the ECV-304 cell lines were cultured as described previously.28Chaitanya G.V. Cromer W.E. Wells S.R. Jennings M.H. Couraud P.O. Romero I.A. Weksler B. Erdreich-Epstein A. Mathis J.M. Minagar A. Alexander J.S. Gliovascular and cytokine interactions modulate brain endothelial barrier in vitro.J Neuroinflammation. 2011; 8: 162Crossref PubMed Scopus (29) Google Scholar The human neuron-like cell line SHSY-5Y (ATCC, Manassas, VA) was cultured in 10% Dulbecco’s modified Eagle’s medium. Passage 1 to passage 3 primary human umbilical vein endothelial cells (HUVECs) isolated from normal umbilical veins were cultured in endothelial basal medium supplemented with a bulletkit (Cat No: CC-3124; Lonza, Basel, Switzerland). As an in vitro model of cerebral ischemia, HCMEC-D3, HFA, and SHSY-5Y cells were subjected to OGDR, as described previously.6Chaitanya G.V. Cromer W. Wells S. Jennings M. Mathis J.M. Minagar A. Alexander J.S. Metabolic modulation of cytokine-induced brain endothelial adhesion molecule expression.Microcirculation. 2012; 19: 155-165Crossref PubMed Scopus (19) Google Scholar In brief, at confluency, cells were incubated in medium without glucose and serum (D5030; Sigma-Aldrich, St. Louis, MO) supplemented with 3.7 g/L NaHCO3 and subjected to an atmosphere of approximately 95% N2, 4% CO2, and 1% O2 in a hypoxia chamber for 6 hours. After 6 hours of oxygen glucose deprivation (OGD), buffer was replaced with regular culture medium and cells were incubated for 24 hours in 5% CO2–enriched normoxic atmosphere, to reoxygenate. Human or mouse brain endothelial cells and HUVECs or ECV-304 (used as positive controls) were cultured in regular culture medium on 8.0-μm pore-size cell culture inserts (BD Falcon, San Jose, CA) with a surface area of 0.3cm2 in the upper chamber. At confluency, normal (without OGDR) or OGDR-challenged brain endothelial cells (or HUVECs or ECV-304 cells as controls) were treated with 100 ng/mL VEGF164/165 or VEGF165b applied on both apical and basal sides of the cell monolayer. Transendothelial electrical resistance (TEER) was then recorded as unit area resistance (Ω·cm2Chaitanya G.V. Eeka P. Munker R. Alexander J.S. Babu P.P. Role of cytotoxic protease granzyme-b in neuronal degeneration during human stroke.Brain Pathol. 2011; 21: 16-30Crossref PubMed Scopus (30) Google Scholar), using an EVOM voltohmmeter (World Precision Instruments, Sarasota, FL), at baseline (0 hours) and for 3 days at 24-hour intervals. C57BL/6 mice were bred and maintained in a specific-pathogen-free environment with a 12-hour light/dark cycle at the animal facility of Louisiana State University Health–Shreveport. All animal experiments were performed on male C57BL6 mice at 12 to 18 weeks of age, weighing approximately 25 to 30 g, according to Institutional Animal Care and Use Committee guidelines under an approved animal protocol. The adenovirus encoding recombinant VEGF164b (Ad-rVEGF164b) and its higher potential, compared with naturally occurring VEGF165b, have been described previously.26Cromer W. Jennings M.H. Odaka Y. Mathis J.M. Alexander J.S. Murine rVEGF164b, an inhibitory VEGF reduces VEGF-A-dependent endothelial proliferation and barrier dysfunction.Microcirculation. 2010; 17 ([Erratum appeared in Microcirculation 2010, 17:669]): 536-547PubMed Google Scholar Ad5-CMV-GFP, consisting of an adenovirus serotype 5 expressing GFP under the CMV promoter, was used as a relevant control. At 3 days before induction of cerebral ischemia, mice were injected intraperitoneally with 1 × 1011 viral particles, either Ad-GFP or Ad-rVEGF164b. We had earlier determined that Ad-rVEGF164b induces peak serum levels of VEGF164b by 72 hours after adenoviral infection (data not shown). Focal cerebral ischemia–reperfusion injury in mice was induced by transient middle cerebral artery occlusion. In brief, mice were anesthetized with a combination of 2.5 mg/kg xylazine and 50 mg/kg ketamine hydrochloride. After confirming that the animal was deeply anesthetized, a midline pharyngeal incision was made to expose the external and internal carotids and a 6-0 silicone-coated nylon monofilament (Doccol, Redlands, CA) was inserted from the cut end of the external carotid into the internal carotid and extended to a distance of 10 to 12 mm. The occlusion was detected by gentle resistance to further extension of the filament. The middle cerebral artery was occluded for 2 hours; the occluding filament was then removed and reperfusion was initiated. Sham animals were treated the same way as stroke animals except the nylon monofilament was not extended to occlude the middle cerebral artery. Twenty-four hours after reperfusion, sham, Ad-rVEGF164b treated, and Ad-GFP treated stroke mice were sacrificed by cardiac puncture or pneumothorax under deep xylazine–ketamine anesthesia and the brains were removed. Coronal slices (2 mm thick) were obtained from Ad-GFP–treated and Ad-rVEGF164b–treated stroke mouse brains. Stroke mouse brain slices were incubated in 2% tetrazolium trichloride at 37°C until the development of chromophore (approximately 5 to 7 minutes). After color development, brain slices were fixed in 10% formalin and were photographed. Infarct size was measured directly by quantifying infarct volume in the ipsilateral hemisphere using ImageJ analysis software version 1.46 (NIH, Bethesda, MD) as described previously.29Lin T.N. He Y.Y. Wu G. Khan M. Hsu C.Y. Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats.Stroke. 1993; 24: 117-121Crossref PubMed Scopus (629) Google Scholar Behavioral changes induced by stroke in mice were measured by grading behavioral deficits exhibited 24 hours after reperfusion.30Yilmaz G. Arumugam T.V. Stokes K.Y. Granger D.N. Role of T lymphocytes and interferon-gamma in ischemic stroke.Circulation. 2006; 113: 2105-2112Crossref PubMed Scopus (566) Google Scholar, 31Li X. Blizzard K.K. Zeng Z. DeVries A.C. Hurn P.D. McCullough L.D. Chronic behavioral testing after focal ischemia in the mouse: functional recovery and the effects of gender.Exp Neurol. 2004; 187: 94-104Crossref PubMed Scopus (263) Google Scholar, 32Kim J.H. Choi K.H. Jang Y.J. Kim H.N. Bae S.S. Choi B.T. Shin H.K. Electroacupuncture preconditioning reduces cerebral ischemic injury via BDNF and SDF-1alpha in mice.BMC Complement Altern Med. 2013; 13: 22Crossref PubMed Scopus (41) Google Scholar Scoring was on a six-point scale: 0, no noticeable behavioral deficit; 1, inability to extend forelimbs; 2, inability to move spontaneously; 3, contralateral spinning; 4, barrel rolling; 5, paralysis; and 6, dead. Twenty-four hours after ischemia–reperfusion injury, Ad-GFP–treated stroke animals and Ad-rVEGF164b–treated stroke animals were sacrificed. The brains were isolated, and the wet weight was recorded. Later, the brains were desiccated in an incubator at 60°C for 72 hours, and the dry weight was recorded. The percentage of brain water content as a measure of edema was calculated as (brain wet weight − dry weight/wet weight) × 100. Brain neutrophil content in stroke brains was measured in terms of myeloperoxidase (MPO) activity. In brief, Ad-GFP–treated and Ad-rVEGF164b–treated stroke mouse brains were isolated, homogenized in modified radioimmunoprecipitation assay buffer (0.25 mol/L sucrose, 50 mmol/L Tris base, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L MgCl2, 10 mmol/L KCl, 1% NP-40, and 1% Tween 20 supplemented with protease and phosphatase inhibitor cocktail; Sigma-Aldrich), and sonicated three times with −80°C freeze–thaw cycles. Later, the homogenate was centrifuged and the supernatant was collected. Protein in the supernatants was estimated by a bicinchoninic acid assay (Bio-Rad Laboratories, Hercules, CA), and equal quantities of supernatant protein were used to quantify MPO by colorimetric conversion of ortho-dianisidine at 450 nm. Twenty-four hours after reperfusion, sham and stroke mice were sacrificed as described above, and the brains were removed. Ipsilateral hemispheric tissues were homogenized in modified radioimmunoprecipitation assay buffer and equal amounts of protein were separated on SDS-PAGE, followed by immunoblotting with VEGF164 or VEGF165b antibodies. Equal volumes of serum obtained from sham-treatment and stroke mice were resolved on SDS-PAGE and immunoblotted with either VEGF164 or VEGF165b. Normal and OGDR-challenged HCMEC-D3, HFA, and SHSY-5Y cell lysates were immunoblotted with VEGF165 or VEGF165b. The ratio of VEGF164/165 to VEGF165b in mouse serum, brain lysates, and cell lysates was derived from scanning densitometry data. The VEGF165b/VEGF165 ratio in tissue lysates and cell lysates was normalized to actin, and then the ratio of normalized VEGF165 to VEGF165b was calculated. HCMEC-D3, bEnd.3, and HUVEC cells at confluency were subjected to OGD as described above for 6 hours (HCMEC-D3 and bEnd.3) or 3 hours (HUVEC). Control and OGD-treated cells were reoxygenated with treatment medium containing 100 ng of VEGF164/165 or VEGF165b for 24 hours. Later, cells from each well were trypsinized and equal numbers of cells were plated on growth factor–reduced Matrigel (BD Biosciences, San Jose, CA). After 5 to 6 hours, capillary tube-like structures formed on Matrigel were photographed, and their numbers were counted in each field. Neutrophils were isolated from the bone marrow of the mice. In brief, bone marrow was flushed from hindlimbs and forelimbs of mice with ice-cold PBS and then incubated with phycoerythrin-conjugated Ly6 antibody. Neutrophils from bone marrow suspension were enriched using an EasySep Mouse PE Positive selection kit (18554; STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer’s protocol. The isolated neutrophils were used for transendothelial migration assay across bEnd.3 cells. Neutrophils from healthy volunteers were isolated from venous blood. In brief, venous blood was allowed to sediment in 6% dextran. Supernatant from the sedimented blood was spun down, and the red blood cells were lysed using RBC lysis buffer prepared by dissolving 8.3 g NH4Cl, 1.0 g KHCO3, and 1.8 mL 5% EDTA in 1 L H2O. Later cells were layered on a Ficoll-Hypaque density gradient (1.077; GE Healthcare, Little Chalfont, UK), and the sedimented neutrophils were washed and used for neutrophil transendothelial migration across HCMEC-D3 cells. Neutrophils (1 × 107) isolated at 4°C were used as nonactivated controls, and 1 × 107 neutrophils were activated at 37°C for 24 hours with 100 nmol/L N-formyl-methionyl-leucyl-phenylalanine (FMLP). Later, cell lysates and culture supernatants were obtained and used for Western blotting with VEGF165 and VEGF165b antibodies. Mouse or human brain endothelial cells were cultured on cell culture inserts as described above and at confluency were subjected to OGD or normal conditions for 6 hours as described above. After treatment, the medium was replaced with regular culture medium and allowed to reoxygenate for 24 hours. At the 24-hour time point, 100 ng of VEGF (VEGF164 for bEnd.3 and VEGF165 for HCMEC-D3) and 100 ng of VEGF165b was added to the lower chamber, and mouse or human neutrophils were added to the insert (1 × 106/insert). FMLP (100 nmol/L) served as the positive control; PBS served as negative control. After 4 hours of incubation, cells migrated to the lower chamber were spun down and lysed in 0.5% hexadecylmethylammonium bromide. MPO was quantified by adding 3,3′-5,5′-tetramethylbenzidine–H2O2. Reaction was stopped using H2SO4, and the absorbance of the end products was measured spectrophotometrically at 450 nm. Two-tailed unpaired t-test was used to test the significance between two groups. Repeated-measures analysis of variance with Dunnett’s post hoc test was used to test for significance in endothelial barrier resistance studies. To evaluate the relative levels of VEGF164 and VEGF165b in mouse tissues after stroke, we performed Western blotting analysis on serum and brain lysates obtained from sham-treated and ischemia–reperfused stroke mice. Immunoblot analysis indicated a significant increase in levels of in VEGF165b in both serum and brain tissue after stroke, compared with sham-treatment controls (Figure 1, A and B). Interestingly, although VEGF164 levels in stroke mouse brain were significantly lower, compared with control, serum VEGF164 levels were significantly increased in stroke mice (Figure 1, A and B). More importantly, the relative level of VEGF165b compared with total VEGF (as determined by the VEGF165b/VEGF164 ratio) was significantly higher in brains of stroke mice, compared with controls (Figure 1A). Furthermore, expression levels of VEGFR-1 and VEGFR-2 in the brain and of soluble VEGFR-1 and VEGFR-2 in serum of stroke mice were also significantly increased, compared with controls (Figure 1, C and D). Because we observed a significant increase in the levels of VEGF165b relative to VEGF164 in the stroke brain, we investigated the cellular sources of VEGF165b production in the brain neurovasculature. Although densitometric analysis indicated significantly decreased VEGF165 levels in OGDR-challenged SHSY-5Y neurons (P = 0.003), the levels of VEGF165b were significantly increased (P = 0.04), compared with normal. Importantly, the VEGF165b/VEGF165 ratio was significantly increased, compared with the normal control, indicating that OGDR-challenged neurons have higher levels of VEGF165b (P = 0.004) (Figure 2A and Supplemental Figure S1A). Densitometric analysis revealed no significant difference in VEGF165 levels between control and OGDR-challenged HFA cells (P = 0.48), but there was a significant increase in VEGF165b levels (P = 0.01) and in the VEGF165b/VEGF165 ratio (P = 0.005) (Figure 2B and Supplementa" @default.
• W2129081519 created "2016-06-24" @default.
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• W2129081519 date "2013-09-01" @default.
• W2129081519 modified "2023-09-30" @default.
• W2129081519 title "A Recombinant Inhibitory Isoform of Vascular Endothelial Growth Factor164/165 Aggravates Ischemic Brain Damage in a Mouse Model of Focal Cerebral Ischemia" @default.
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