AKAP12 in astrocytes induces barrier functions in human endothelial cells through protein kinase Cf Yoon Kyung Choi and Kyu-Won Kim NeuroVascular Coordination Research Center, College of Pharmacy and Research, Institute of Pharmaceutical Sciences, Seoul National University, Korea Keywords AKAP12 (A-kinase anchor protein 12); blood–neural barrier; protein kinase Cf; thrombospondin-1; vascular endothelial growth factor Correspondence K.-W Kim, NeuroVascular Coordination Research Center, College of Pharmacy, Seoul National University, Seoul 151-742, Korea Fax: +82 872 1795 Tel: +82 880 6988 E-mail: qwonkim@plaza.snu.ac.kr (Received 12 November 2007, revised March 2008, accepted March 2008) doi:10.1111/j.1742-4658.2008.06387.x Interactions between astrocytes and blood vessels are essential for the formation and maintenance of the blood–neural barrier (BNB) Astrocytederived A-kinase anchor protein 12 (AKAP12) influences BNB formation, but the mechanism of regulation of BNB functions by AKAP12 is not fully understood We have defined a new pathway of barriergenesis in human retina microvascular endothelial cells (HRMECs) involving astrocytic AKAP12 Treatment of HRMECs with conditioned media from AKAP12overexpressing astrocytes reduced phosphorylation of protein kinase Cf (PKCf), decreased the levels of vascular endothelial growth factor (VEGF) mRNA and protein, and increased thrombospondin-1 (TSP-1) levels, which led to antiangiogenesis and barriergenesis Transfection of a small interference RNA targeting PKCf decreased VEGF levels and increased TSP-1 levels in HRMECs Rho is a putative downstream signal of PKCf, and inhibition of Rho kinase with a specific inhibitor, Y27632, decreased VEGF levels and increased TSP-1 levels We therefore suggest that AKAP12 in astrocytes differentially regulates the expression of VEGF and TSP-1 via the inhibition of PKCf phosphorylation and Rho kinase activity in HRMECs The interaction of astrocytes and blood vessels plays an important role in retinal vascular development and angiogenesis [1] Astrocytes act as a sensor and guide for the developing retinal vasculature by detecting hypoxia, and responding by increasing the expression of hypoxia-inducible angiogenic factors, such as hypoxia-inducible factor-1a (HIF-1a) and vascular endothelial growth factor (VEGF) [2,3] Astrocytederived VEGF is the most potent angiogenic factor, and promotes endothelial cell proliferation, migration and permeability during hypoxia [3–6] Thrombospondin-1 (TSP-1) is an antiangiogenic factor that inhibits angiogenesis in vivo [7] TSP-1 mRNA and protein levels are significantly reduced by oxygen ⁄ glucose deprivation in cerebral endothelial cells [8] TSP-1 belongs to a family of secreted glycoproteins and is a constitutive component of the basement membrane, which plays an integral role in the differentiation and migration of endothelial cells during angiogenesis [9,10] Src-suppressed C kinase substrate is the rodent ortholog of human gravin Src-suppressed C kinase substrate and gravin have been redesignated as A-kinase anchor protein 12 (AKAP12) [11] AKAP12 acts as a multivalent scaffold protein, and has been shown to associate with protein kinase C (PKC), protein kinase A, calmodulin, cyclins, F-actin, and b-adrenergic receptors [11–13] Thus, AKAP12 functions as a dynamic platform for signal transduction AKAP12 is Abbreviations AKAP12, A-kinase anchor protein 12; Ang1, angiopoietin-1; CM, conditioned medium; COMP, cartilage oligomeric matrix protein; DAPI, 4¢, 6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H, hypoxia; HIF-1a, hypoxia-inducible factor-1a; HRMEC, human retina microvascular endothelial cell; N, normoxia; NC, negative control; PKC, protein kinase C; RITC, rhodamine B isothiocyanate; si, small interfering; TSP-1, thrombospodin-1; VEGF, vascular endothelial growth factor 2338 FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS Y K Choi and K.-W Kim involved in the regulation of the actin cytoskeleton, cell morphology, cell adhesion, and cell spreading [11,12,14] Cytoskeletal remodeling is regulated by intracellular signaling pathways that involve PKC, calcium-regulated signaling, and the Rho family GTPases [15–18] In addition, Rho has been shown to induce HIF-1a transactivation and VEGF expression [19] Previously, we showed that AKAP12 in astrocytes is important for regulating the formation of the mouse blood–brain and human blood–retinal barriers by downregulating HIF-1a-mediated VEGF expression [20,21] The effects of PKC activation on endothelial cell permeability are of great interest PKC belongs to a family of serine ⁄ threonine kinases that are involved in signal transduction Isoforms of PKC are classified according to their structure, activity, and substrate requirements, and comprise three main classes: classic PKCs (a, bI, bII, c), novel PKCs (d, e, g, h), and atypical PKCs (f, k) [22] The activity of PKCs is regulated by localization to the plasma membrane and phosphorylation status [22] Several studies have shown that PKCs increase vascular permeability [23–27] One of the atypical PKCs, PKCf, upregulates HIF-1a activity and VEGF expression in renal cell carcinoma cells [28], and increases thrombin-induced vascular permeability in human umbilical vein endothelial cells [25] In the current study, we investigated the mechanism of reduced vascular permeability and angiogenesis caused by astrocytic AKAP12 We show that astrocytic AKAP12 inhibits phosphorylation of PKCf in neighboring human retina microvascular endothelial cells (HRMECs), which leads to a decrease in VEGF levels and an increase in TSP-1 levels in HRMECs, resulting in reduced vascular permeability, decreased endothelial cell migration, and upregulation of tight junction proteins In addition, VEGF165 treatment of HRMECs induced VEGF mRNA levels via a positive feedback mechanism of regulation and decreased TSP1 levels, suggesting that the differential regulation of VEGF and TSP-1 in HRMECs by astrocytic AKAP12 could stem from the astrocyte-secreted factor VEGF Results Effects of AKAP12-overexpressing astrocytes on endothelial cell migration, vascular permeability, and the levels of tight junction proteins In the unvascularized retina during eye development, astrocytes detect hypoxia, and respond by inducing the expression of VEGF, which stimulates new vessel Regulation of the blood–neural barrier by AKAP12 formation [2,3] To better understand the effect of astrocytic AKAP12 on the retinal vasculature, we treated HRMECs with conditioned medium (CM) from mock-transfected astrocytes that were exposed to normoxia (N-mock-CM) or hypoxia (H-mock-CM), or Akap12-transfected astrocytes that were exposed to hypoxia (H-AKAP12-CM) After the treatment with CM, we examined endothelial cell migration, vascular permeability, and the levels of tight junction proteins and an adhesion molecule H-mock-CM markedly increased HRMEC migration as compared to N-mockCM (Fig 1A,B) When cells were incubated with H-AKAP12-CM, reduced migration in comparison with that of cells incubated with H-mock-CM was observed, and this reduced migration was similar to that in N-mock-CM-treated cells (Fig 1A,B) We also examined the passage of rhodamine B isothiocyanate (RITC)–dextran through monolayers of HRMECs as a measure of permeability, and found that vascular permeability was increased by H-mock-CM, and significantly reduced by H-AKAP12-CM (Fig 1C) We next investigated whether AKAP12 regulated the expression of tight junction proteins and an adhesion molecule, vascular endothelial (VE)-cadherin H-mock-CM strongly decreased the expression of the junction proteins ZO-1 and ZO-2, as well as VE-cadherin, whereas H-AKAP12-CM significantly increased the expression of ZO-1, ZO-2 and VE-cadherin in HRMECs (Fig 1D) The increased levels of the junction proteins in the H-AKAP12- CM-treated cells were almost same as the levels in the N-mock-CM-treated cells (Fig 1D) In our previous study, transfection of Akap12 into human astrocytes increased angiopoietin-1 (Ang1) levels in CM under hypoxic conditions, and this played a role in barrier properties in HRMECs [20] Therefore, we examined whether the junction proteins claudin-1 and VE-cadherin were also regulated by Ang1 in CM from hypoxic astrocytes H-AKAP12-CM strongly increased the expression of claudin-1 and VE-cadherin as compared to H-mock-CM in HRMECs (Fig 1E) These effects were blocked when the H-AKAP12-CM was pretreated with an antibody to Ang1 (Fig 1E) These results suggest that astrocytic AKAP12 plays a role in endothelial barrier function through astrocytederived secretion factor(s), including Ang1 AKAP12 in astrocytes differentially regulates the expression of VEGF and TSP-1 in HRMECs VEGF is an important signal for retinal vessel migration and permeability [29] TSP-1 is involved in endothelial cell differentiation and migration [30,31] Observing that CM from AKAP12-overexpressing FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS 2339 Regulation of the blood–neural barrier by AKAP12 A N-mock-CM Y K Choi and K.-W Kim H-mock-CM B –1 RITC passed (µg·mL ) Migrated area (%) C 125 100 75 # * 50 25 CM : N-mock H-mock H-AKAP12 # CM : N-mock H-mock H-AKAP12 Cytosol ZO-2 Actin VE-cadherin Membrane H-M Relative expression of proteins (%) CM : N-mock H-mock H-AKAP12 ZO-1 E * H-A H-A/Ang1 H-A/IgG : CM Claudin-1 VE-cadherin Actin 150 ZO-1 ZO-2 VE-cadherin *# 100 ** * ** * 50 Actin CM : Relative expression of proteins (%) D H-AKAP12-CM 150 N-mock H-mock H-AKAP12 Claudin-1 VE-cadherin * ** 100 * 50 # ** CM : H-M H-A H-A/Ang1 H-A/IgG Fig AKAP12 in astrocytes regulates endothelial cell migration, vascular permeability, and the expression of tight junction proteins in HRMECs (A) CM from mock-transfected astrocytes exposed to normoxia (N-mock-CM) or hypoxia (H-mock-CM) and CM from Akap12-transfected astrocytes exposed to hypoxia (H-AKAP12-CM) were collected and concentrated (4·) HRMECs were treated with N-mock-CM, H-mock-CM or H-Akap12-CM for 24 h, and migration was observed (B) HRMECs were marked with an injury line and the distance of the injury line was measured After 24 h, the quantification of the area of migration was performed (n = 4) The area was set to 100% in the H-mock-CM treatment condition #P < 0.001 as compared to the N-mock-CM condition; *P < 0.05 as compared with the H-mock-CM condition (C) RITC– dextran permeability as a marker for vascular permeability was analyzed in HRMECs *P < 0.005 as compared to the N-mock-CM condition; # P < 0.005 as compared to the H-mock-CM condition (n = 4) (D) HRMECs were treated for 24 h with N-mock-CM, H-mock-CM or H-AKAP12CM The expression of ZO-1 and ZO-2 in cytosolic fractions and the expression of VE-cadherin in membrane fractions were analyzed by western blot The quantification of these protein levels from three independent experiments is shown on the right The expression was set to 100% in the N-mock-CM treatment condition *P < 0.05 as compared to control; #P < 0.01 as compared to control; **P < 0.005 as compared to control In the case of the H-mock-CM condition, the control is the N-mock-CM condition; in the case of the H-AKAP12-CM condition, the control is the H-mock-CM condition (E) The expression of claudin-1 and VE-cadherin was detected in the membrane fraction in the H-M (H-mock-CM), H-A (H-AKAP12-CM), H-A ⁄ Ang1 (H-AKAP12-CM neutralized by Ang1 antibody) and H-A ⁄ IgG (H-AKAP12-CM neutralized by IgG antibody) treatment conditions by western blot assay RMECs were treated for 24 h with these CMs The quantification of these protein levels from three independent experiments is shown on the right The expression was set to 100% in the H-AKAP12-CM treatment condition *P < 0.05 as compared to control; #P < 0.01 as compared to control; **P < 0.005 as compared to control In the case of H-A-CM, the control is H-M-CM; in the case of H-A ⁄ Ang1-CM, the control is H-A-CM; in the case of H-A ⁄ IgG-CM, the control is H-A ⁄ Ang1-CM 2340 FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS Y K Choi and K.-W Kim Regulation of the blood–neural barrier by AKAP12 hypoxic astrocytes reduced endothelial cell migration and vascular permeability (Fig 1A–C), we investigated the effects of AKAP12 on VEGF and TSP-1 expression in endothelial cells HRMECs were treated with A N-mock-CM H-mock-CM CM from mock-transfected or Akap12-transfected hypoxic astrocytes, and fluorescence immunohistochemistry was carried out to assess the levels of VEGF and TSP-1 in cells The level of VEGF was increased and H-AKAP12-CM Relative expression of proteins (%) 150 VEGF TSP-1 * N-mock H-mock H-AKAP12 Relative expression of mRNA (%) CM : VEGF165 VEGF121 RT-PCR TSP-1 C CM : N H H/VEGF H/IgG VEGF165 VEGF121 RT-PCR TSP-1 GAPDH Relative expression of mRNA (%) GAPDH 150 ** 100 # 50 CM : B VEGF TSP-1 N-mock H-mock H-AKAP12 VEGF TSP-1 * 100 ** * # 50 CM : 150 N-mock H-mock H-AKAP12 VEGF TSP-1 * 100 * * * 50 CM : N * * H H/VEGF H/IgG Fig AKAP12 in astrocytes regulates the expression of VEGF and TSP-1 in HRMECs (A) The expression of VEGF and TSP-1 (green) was analyzed by fluorescence immunocytochemistry Nuclei (blue) were stained with DAPI Scale bar, 50 lm Data from four independent experiments were quantified, and are presented on the right Quantification of immunohistochemical staining area was analyzed using IMAGE-PRO PLUS (Media Cybernetics) Each stained area is presented relative to the area with the highest staining intensity The level of VEGF in H-mock-CM-treated cells was set at 100% The level of TSP-1 in H-AKAP12-CM-treated cells was set at 100% *P < 0.05 as compared to N-mock-CM-treated cells; #P < 0.005 as compared to H-mock-CM-treated cells; **P < 0.05 as compared to H-mock-CM-treated cells (B) The mRNA levels of VEGF, TSP-1 and GAPDH were analyzed by RT-PCR The quantification of VEGF and TSP-1 mRNA levels from three independent experiments is shown on the right The level of VEGF in H-mock-CM-treated cells was set at 100% The level of TSP-1 in N-mock-CM-treated cells was set at 100% *P < 0.05 as compared to N-mock-CM-treated cells; #P < 0.001 as compared to H-mock-CMtreated cells; **P < 0.05 as compared to H-mock-CM-treated cells (C) The mRNA levels of VEGF, TSP-1 and GAPDH were analyzed in the N (CM from astrocytes exposed to normoxia), H (CM from astrocytes exposed to hypoxia), H ⁄ VEGF (H-CM neutralized by an antibody to VEGF) and H ⁄ IgG (H-CM neutralized by an antibody to IgG) treatment conditions by RT-PCR The quantification of VEGF and TSP-1 mRNA levels from three independent experiments is shown on the right The level of VEGF in H ⁄ IgG-treated cells was set at 100% The level of TSP-1 in N-treated cells was set at 100% *P < 0.05 as compared to control In the case of H, the control is N; in the case of H ⁄ VEGF, the control is H; in the case of H ⁄ IgG, the control is H ⁄ VEGF GAPDH served as an internal control FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS 2341 Regulation of the blood–neural barrier by AKAP12 Y K Choi and K.-W Kim the level of TSP-1 was decreased in the H-mock-CMtreated cells as compared to the levels in the N-mockCM-treated cells (Fig 2A) H-AKAP12-CM resulted in decreased VEGF levels and increased TSP-1 levels, and these levels were similar to the levels in N-mockCM-treated cells (Fig 2A) These results indicate that there is an inverse relationship between the levels of VEGF and TSP-1 in endothelial cells, and that the relationship is regulated by AKAP12-overexpressing astrocytes We further examined VEGF and TSP-1 mRNA levels in HRMECs, using RT-PCR H-mockCM induced a marked increase in VEGF mRNA levels as compared to N-mock-CM, whereas H-AKAP12CM decreased VEGF mRNA levels as compared to H-mock-CM (Fig 2B) TSP-1 mRNA levels exhibited an inverse pattern of expression as compared to VEGF (Fig 2B) These results were consistent with the immunocytochemical analysis (Fig 2A), and indicated that AKAP12 might regulate the secretion of astrocytespecific signal(s) that could reduce VEGF expression and increase TSP-1 expression in endothelial cells Because astrocytes secrete a high level of VEGF during hypoxia [20], we hypothesized that astrocyte-derived VEGF functions as a stimulator of its own expression in HRMECs CM from hypoxic astrocytes (H-CM) induced a marked increase in VEGF mRNA level as compared to N-CM, whereas VEGF-neutralizing H-CM (H ⁄ VEGF) decreased VEGF mRNA levels as compared to H-CM (Fig 2C) TSP-1 mRNA levels exhibited an inverse pattern of expression as compared to VEGF (Fig 2C) These results suggest that the dif- ferential regulation of VEGF and TSP-1 in HRMECs by astrocytic AKAP12 could stem from the astrocytesecreted factor VEGF VEGF regulates the expression of VEGF and TSP-1 in HRMECs To confirm the effects of VEGF on the differential regulation of VEGF and TSP-1 in HRMECs, we treated HRMECs with recombinant VEGF165 As shown in Fig 3A, VEGF mRNA levels in HRMECs were increased following treatment with recombinant VEGF165 in a concentration-dependent manner We investigated whether treatment of HRMECs with VEGF165 influenced cell survival, because treatment with VEGF165 increased endothelial VEGF expression (Fig 3A) As shown in Fig 3B, treatment with VEGF165 induced a 10% increase in cell viability (P < 0.005) However, hypoxia did not increase cell viability as compared to normoxia (Fig 3B), although hypoxia induces endothelial VEGF expression Next, we pretreated HRMECs with an antibody to Flk-1 (VEGF receptor 2), and followed this with VEGF165 treatment Treatment of HRMECs with VEGF165 strongly increased the VEGF mRNA level, and this effect was abolished by antibody blockade of VEGF165–Flk-1 interactions (Fig 3C) These results suggest that treatment of HRMECs with VEGF165 increased the expression of VEGF mRNA in part via Flk-1 in HRMECs We observed that treatment of HRMECs with VEGF165 induced intracellular VEGF Fig VEGF regulates the expression of VEGF and TSP-1 in HRMECs (A) HRMECs were treated with recombinant VEGF165 (0, 10, 50 ngỈmL)1) for 24 h, and VEGF mRNA levels were analyzed by RT-PCR Data from four independent experiments were quantified, and are presented on the right The level of VEGF in cells treated with 50 ngỈmL)1 VEGF was set as 100% *P < 0.05 as compared to cells treated with ngỈmL)1 VEGF; ##P < 0.0001 as compared to cells treated with ngỈmL)1 VEGF (B) HRMECs were treated with recombinant VEGF165 (50 ngỈmL)1) or subjected to hypoxia for 24 h, and viable cells were evaluated by a cell viability assay (n = 5) The levels of viable cells among cells treated with 50 ngỈmL)1 VEGF or normoxic cells were set as 100% **P < 0.005 as compared to cells treated with ngỈmL)1 VEGF (C) The VEGF mRNA level was detected by RT-PCR in the conditions with or without VEGF165 treatment Cells were pretreated with an antibody to Flk-1 or an antibody to IgG for h, and this was followed by VEGF165 treatment for 24 h The quantification of VEGF mRNA level from three independent experiments is shown on the right The expression of VEGF mRNA in cells treated with VEGF165 (50 ngỈmL)1) was set at 100% **P < 0.005 as compared to control; *P < 0.05 as compared to control In the case of the VEGF165 treatment condition, the control is no treatment condition; in the case of the Flk-1 neutralizing condition, the control is the VEGF165 treatment condition; in the case of the IgG neutralizing condition, the control is the Flk-1 neutralizing condition (D) HRMECs were treated with a combination of recombinant VEGF165 (50 ngỈmL)1) and PKC inhibitors such as GF109203X (GF; 0.1 lM or lM) and chelerythrine chloride (C; lM) for 24 h The VEGF mRNA level was detected by RT-PCR (E) HRMECs were treated with recombinant VEGF165 (50 ngỈmL)1) for or 24 h, and the level of TSP-1 secretion was analyzed by western blot The data from five independent experiments were quantified, and are presented on the right The level of TSP-1 in the ngỈmL)1 VEGF treatment condition (control) was set at 100% **P < 0.005 as compared to control (F) HRMECs were pretreated with an antibody to Flk-1 or an antibody to IgG for h, and this was followed by VEGF165 treatment for 24 h The TSP-1 secretion level was analyzed by western blot (n = 3) b-Actin, GAPDH and Ponceau Red staining served as the internal controls (G,H) The levels of PKCf and phosphorylated PKCf in HRMECs treated without (control) or with recombinant VEGF165 (+VEGF) for 15 were analyzed by fluorescence immunocytochemistry (G) and western blot (H) Scale bar, 50 lm Nuclei (blue) were stained with DAPI Data from four independent experiments were quantified, and are presented on the right Expression levels in control cells were set at 100% **P < 0.005 as compared to control 2342 FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS Y K Choi and K.-W Kim Regulation of the blood–neural barrier by AKAP12 VEGF165 (ng·mL–1) 10 50 VEGF165 VEGF121 β-actin 120 ## * 40 + + Flk-1 IgG VEGF165 VEGF121 GAPDH Relative expression of VEGF mRNA (%) + VEGF165 (50 ng·mL–1) 120 80 VEGF C Viable cells (relative ratio to control %) B Relative expression of VEGF mRNA (%) A 10 ** 80 40 /+ /+ 50 VEGF hypoxia D 120 * ** 80 ** + + 0.1 + + GF (μM) 40 C (μM) VEGF VEGF VEGF165 β-actin + + + E 4h 24 h VEGF165 (50 ng·mL–1) CM TSP-1 Ponceau Relative expression of TSP-1 protein (%) Flk-1 IgG F 80 Nuclei PKCζ ** ** + Flk-1 CM + IgG VEGF165 (50 ng·mL–1) TSP-1 40 Ponceau VEGF G + 120 h 24 h p-PKCζ Nuclei Merge Merge Control H + VEGF PKCζ p-PKCζ Actin Relative expression of proteins (%) +VEGF 200 150 PKCζ p-PKCζ ** 100 50 VEGF FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS + 2343 Regulation of the blood–neural barrier by AKAP12 Y K Choi and K.-W Kim mRNA levels (Fig 3A), and this increase was blocked by PKC inhibitors such as bisindolylmaleimide I (GF109203X) and chelerythrine chloride (Fig 3D) Chelerythrine chloride, an inhibitor of all PKC isoforms, decreased VEGF165-stimulated VEGF mRNA levels in HRMECs (Fig 3D) GF109203X has an indolylmaleimide structure that inhibited all PKC isoforms in cells treated with lm, and inhibited classic and novel PKCs in cells treated with 0.1 lm [25] VEGF mRNA levels were more reduced in cells treated with lm GF109203X than in cells treated with with 0.1 lm GF109203X (Fig 3D) These results suggest that atypical PKC isoforms may play a role in VEGF165-stimulated VEGF induction We also examined whether VEGF treatment influenced the expression of TSP-1, as it has been shown that the expression of VEGF and TSP-1 are inversely regulated [32,33] We observed a significant reduction in the levels of secreted TSP-1 at h after VEGF treatment, followed by a partial recovery at 24 h after VEGF treatment (Fig 3E) In addition, treatment of HRMECs with VEGF165 decreased TSP-1 secretion levels, and this effect was blocked when cells were pretreated with an antibody to Flk-1 (Fig 3F) These results suggest that astrocyte-derived VEGF differentially regulates VEGF and TSP-1 levels via Flk-1 in HRMECs VEGF induces phosphorylation of PKCf in HRMECs We examined whether VEGF is involved in the activation of phosphorylated PKCf in HRMECs, because vascular permeability has been linked to PKCf activa- tion [25], and the increase in VEGF mRNA levels after VEGF165 treatment could be mediated by atypical PKCs (Fig 3D) We treated HRMECs with recombinant VEGF165 and examined the effect on phosphorylated PKCf levels using fluorescence immunocytochemistry The level of PKCf was unchanged by VEGF165, whereas that of phosphorylated PKCf was remarkably increased by VEGF165 treatment (Fig 3G) Western blot analysis confirmed these results (Fig 3H), and also showed that Flk-1 coimmunoprecipitates with PKCf (data not shown) These results suggest that VEGF secreted from astrocytes binds to its cognate receptor on HRMECs and induces the phosphorylation of PKCf AKAP12-overexpressing astrocytes inhibit phosphorylation of PKCf in HRMECs We next investigated whether PKCf activation is affected by astrocytic AKAP12 When cells were incubated with N-mock-CM, H-mock-CM or H-AKAP12CM, the levels and distribution of PKCf were unchanged (Fig 4A) Thereafter, we examined the levels of phosphorylated PKCf in HRMECs using an antibody that specifically recognizes phosphorylated Thr410 within the activation loop of PKCf When cells were incubated with H-mock-CM, phosphorylated PKCf levels were significantly increased as compared to N-mock-CM-treated cells (Fig 4A) H-AKAP12CM decreased phosphorylated PKCf levels, in a similar pattern to that seen with N-mock-CM (Fig 4A) These results were confirmed by western blot analysis (Fig 4B) When H-CM was pretreated with an antibody to VEGF, this effect was abolished (Fig 4C) Fig AKAP12 from astrocytes regulates the level of phosphorylated PKCf in HRMECs (A) Levels of PKCf and phosphorylated PKCf in HRMECs treated with N-mock-CM, H-mock-CM, and H-AKAP12-CM for 15 were analyzed by fluorescence immunocytochemistry (green) Nuclei (blue) were stained with DAPI Scale bar, 50 lm (B) PKCf (7 lg) and phosphorylated PKCf (25 lg) levels under the indicated conditions were analyzed by western blot Data from four independent experiments were quantified, and are presented on the right The expression in N-mock-CM-treated cells was set at 100% *P < 0.05 as compared to N-mock-CM-treated cells; #P < 0.05 as compared to H-mock-CM-treated cells (C) PKCf and phosphorylated PKCf levels were detected in the N (CM from normoxic astrocytes), H (CM from hypoxic astrocytes), H ⁄ VEGF (H-CM neutralized by an antibody to VEGF) and H ⁄ IgG (H-CM neutralized by an antibody to IgG) treatment conditions by western blot HRMECs were treated for 24 h with these CMs The quantification of protein levels from three independent experiments is shown on the right The levels of PKCf and phosphorylated PKCf in N-CM-treated cells were set at 100% *P < 0.05 as compared to control; #P < 0.001 as compared to control In the case of H, the control is N; in the case of H ⁄ VEGF, the control is H; in the case of H ⁄ IgG, the control is H ⁄ VEGF (D–E) Human astrocytes were transfected with NC RNA or siRNA targeting Akap12 (siAkap12) and incubated for 36 h, and CM then was collected and concentrated (4·) HRMECs were treated with CM for 15 min, and the levels of phosphorylated PKCf and PKCf were analyzed by immunocytochemistry (D) and western blot (E) Scale bar, 50 lm Nuclei (blue) were stained with DAPI Data from four independent experiments were quantified, and are presented on the right The expression in NC-CM-treated cells was set at 100% *P < 0.05 as compared to control Actin was used as an internal control (F) HRMECs were pretreated with or without COMP-Ang1 (100 ngỈmL)1) for 15 min, and this was followed by treatment with NC-CM or siAkap12-CM for 15 PKCf and phosphorylated PKCf immunoblots were analyzed by western blot The quantification of the immunoblots from three independent experiments is shown on the right The expression in NC-CM-treated cells was set at 100% #P < 0.01 as compared to cells treated with NC-CM only; *P < 0.05 as compared to siAkap12-CM-treated cells 2344 FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS Y K Choi and K.-W Kim A Regulation of the blood–neural barrier by AKAP12 N-mock-CM H-mock-CM H-AKAP12-CM D NC-CM siAkap12-CM PKCζ PKCζ p-PKCζ p-PKCζ B N-mock H-mock H-AKAP12 : CM E + siAkap12-CM PKCζ PKCζ p-PKCζ p-PKCζ Actin Relative expression of proteins (%) Relative expression of proteins (%) Actin PKCζ p-PKCζ 200 * 150 # 100 50 CM : N-mock H-mock H-AKAP12 C N H H/VEGF H/IgG : CM 200 PKCζ p-PKCζ 150 * 100 50 + siAkap12-CM F + + PKCζ + + Ang1 siAkap12-CM PKCζ p-PKCζ p-PKCζ Actin 200 PKCζ p-PKCζ # 150 * Relative expression of proteins (%) Relative expression of proteins (%) Actin * 100 50 CM : N H H/VEGF H/IgG 200 150 PKCζ p-PKCζ # * 100 50 Ang1 siAkap12-CM FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS + + + + 2345 Regulation of the blood–neural barrier by AKAP12 Y K Choi and K.-W Kim We next treated HRMECs with CM from small interfering (si)Akap12-transfected cells (siAkap12-CM) and examined phosphorylated PKCf levels by fluorescence immunocytochemistry The levels of PKCf were not changed in siAkap12-CM-treated cells, whereas phosphorylated PKCf levels were strongly increased as compared to negative control-CM (NC-CM)-treated cells (Fig 4D) The result was confirmed by western blot analysis (Fig 4E) A chimeric form of Ang1 is soluble and more potent than native Ang1 in Tie2 (an Angl receptor on endothelial cells) phosphorylation, and its N-terminal portion has the short coiled-coil domain of cartilage oligomeric matrix protein (COMP) (COMP-Ang1) [34] When HRMECs were pretreated with COMP-Ang1 and then treated with siAkap12CM, phosphorylation of PKCf was significantly decreased as compared to what was seen with siAkap12-CM treatment alone (Fig 4F), suggesting that + + 150 CM VEGF * 50 ** TSP-1 Ponceau + + VEGF TSP-1 100 Actin Hypoxia siPKCζ VEGF RT-PCR partly siPKCζ p-PKCζ + is Hypoxia PKCζ B activation Astrocytic AKAP12 downregulates phosphorylation of PKCf (Fig 4), decreases the level of VEGF (Fig 2A,B) and increases the level of TSP-1 in HRMECs (Fig 2A,B) We next examined the relationship between PKCf activation and the expression of VEGF and TSP-1 in HRMECs We transfected HRMECs with an siRNA that targets PKCf (siPKCf), and found that VEGF and TSP-1 secretion and mRNA levels were markedly downregulated and upregulated, respectively (Fig 5A,B) Therefore, we propose that AKAP12 in astrocytes differentially regulates the secretion levels of VEGF and TSP-1 in HRMECs via PKCf Relative expression of proteins (%) + PKCf PKCf regulates the expression of VEGF and TSP-1 in HRMECs TSP-1 β-actin Hypoxia siPKCζ 150 Relative expression of mRNA (%) A siAkap12-CM-induced blocked by Ang1 + + + VEGF TSP-1 * 100 ** 50 Hypoxia siPKCζ + + + Fig PKCf regulates the expression of VEGF and TSP-1 in HRMECs (A,B) HRMECs were transfected with an siRNA targeting PKCf (siPKCf), incubated for 24 h, and then exposed to hypoxia for 24 h CM was collected and concentrated (4·) (A) The levels of PKCf and phosphorylated PKCf in cell lysates, and the levels of VEGF and TSP-1 in CM, were analyzed by western blot The data from four independent experiments were quantified, and are presented on the right The level of VEGF in cells transfected with an NC RNA was set at 100% The level of TSP-1 in siPKCf-transfected cells was set at 100% *P < 0.05 as compared to control; **P < 0.005 as compared to control (B) VEGF and TSP-1 mRNA levels were analyzed by RT-PCR The data from three independent experiments were quantified, and are presented on the right The level of VEGF in cells transfected with a negative control RNA was set at 100% The level of TSP-1 in siPKCf-transfected cells was set as 100% *P < 0.05 as compared to control; **P < 0.005 as compared to control b-Actin and Ponceau Red staining served as the internal controls 2346 FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS Y K Choi and K.-W Kim Rho kinase is involved in the regulation of VEGF and TSP-1 levels in HRMECs Recent studies have shown that Rho family GTPases are activated by atypical PKCf during cell motility [35,36] As our data indicated that PKCf regulates both VEGF and TSP-1 in HRMECs (Fig 5), we examined the effect of a specific Rho kinase inhibitor, Y27632, on VEGF and TSP-1 levels VEGF and TSP1 levels are differentially regulated by hypoxia [8,37] Cells were grown under normoxic or hypoxic conditions, and treated or not treated with Y27632 Under both normoxic and hypoxic conditions, Y27632 treatment decreased VEGF and increased TSP-1 mRNA and secretion levels as compared to untreated cells (Fig 6A,B) These results show that differential regulation of VEGF and TSP-1 levels in response to oxygen levels may result from Rho kinase activity Because treatment of HRMECs with siAkap12-CM activated PKCf (Fig 4D,E), we examined whether a similar pattern of regulation of VEGF and TSP-1 occurred siAkap12-CM upregulated VEGF expression and downregulated TSP-1 levels in HMRECs (Fig 6C,D) When cells were treated with a combination of Y27632 and siAkap12-CM, VEGF levels were significantly decreased and TSP-1 levels were increased (Fig 6C,D) These results suggest that AKAP12 in astrocytes inversely regulates VEGF and TSP-1, and that this regulation is mediated by Rho kinase activity Discussion Astrocytes are very complex cells, capable of responding to hypoxia in the developing retina [2], and secreting the angiogenic factor VEGF, which subsequently induces angiogenesis [3–5] The initial event in angiogenesis is the loosening of cell–cell contacts, and this is followed by the migration of endothelial cells to form a capillary tube network [38] Oxygen delivered by the developing brain and retinal vasculature stops angiogenesis, and the blood–neural barrier is formed [20,21,37,39] Elevated oxygen levels (reoxygenation) upregulate AKAP12 expression in astrocytes, resulting in decreased VEGF secretion and increased Ang1 secretion [20,21] In the current study, we demonstrated that AKAP12-overexpressing hypoxic astrocytes induce endothelial tightening and antiangiogenesis (Fig 1) We showed that AKAP12 in astrocytes decreases VEGF levels and increases TSP-1 levels in HRMECs (Fig 2A,B) VEGF is a key factor in the induction of vascular permeability during angiogenesis and in barrier disruption [4–6,40–43] TSP-1 is an angiostatic factor that is upregulated during reoxy- Regulation of the blood–neural barrier by AKAP12 genation [8,37], and that plays a key role in vessel differentiation and migration [7,30,31] These data show that AKAP12 plays a critical role in mediating important interactions between retinal endothelial cells and astrocytes during retinal angiogenesis and barriergenesis Our previous studies revealed that AKAP12 in astrocytes reduces vascular permeability [20,21] However, the mechanism by which AKAP12 regulates vascular permeability is unclear In this study, we demonstrated that AKAP12 from astrocytes inversely regulates VEGF and TSP-1 levels via PKCf activation in HRMECs We showed that AKAP12 in astrocytes decreases VEGF levels and increases TSP-1 levels in HRMECs (Fig 2A,B), and that these effects are blocked in the VEGF-neutralizing condition (Fig 2C) We found that VEGF165 treatment of HRMECs induces VEGF expression and reduces TSP-1 expression (Fig 3A,E), suggesting that the differential regulation of VEGF and TSP-1 in HRMECs by astrocytic AKAP12 could stem from the astrocyte-secreted factor VEGF These results show that development of new blood vessels during retinal development may be triggered not only by secreted VEGF from hypoxic astrocytes, but also by autocrine-stimulated VEGF from retinal endothelial cells The increase in VEGF levels and the decrease in TSP-1 levels after VEGF165 treatment could be mediated by Flk-1 (Fig 3C,F) VEGF165 treatment of HRMECs increased cell viability by 10% (Fig 3B) However, we did not observe a significant increase in cell viability in the hypoxic condition as compared to the normoxic condition (Fig 3B), although hypoxia induces endothelial VEGF expression (Fig 6A) Considering that both VEGF165 treatment and hypoxia increase endothelial VEGF expression, further studies are necessary to clarify the differential effects of VEGF and hypoxia on endothelial cell survival We observed that treatment of HRMECs with recombinant VEGF165 induced intracellular VEGF mRNA levels via a positive feedback mechanism of regulation (Fig 3A) These effects were reduced when HRMECs were treated with a combination of VEGF165 and PKC inhibitors such as GF109203X and chelerythrine chloride (Fig 3D) According to our data, VEGF mRNA levels were more reduced in cells treated with lm GF109203X than in cells treated with 0.1 lm GF109203X, suggesting that atypical PKCs may be involved in this VEGF induction We also found that recombinant VEGF165 treatment increased the levels of phosphorylated PKCf, one of the atypical PKCs, in HRMECs (Fig 3G,H), which suggests that the secretion of VEGF from astrocytes is involved in the activation and phosphorylation of PKCf in HRMECs FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS 2347 Regulation of the blood–neural barrier by AKAP12 + A Y K Choi and K.-W Kim + + + Hypoxia Y27632 VEGF RT-PCR TSP-1 + + + + Hypoxia Y27632 VEGF CM TSP-1 Ponceau 120 * NC-CM NC-CM+Y siAkap12-CM # 40 Hypoxia Y27632 C * 80 siAkap12-CM+Y VEGF TSP-1 + + + + Relative expression of proteins (%) B Relative expression of TSP-1 protein (%) β-actin 150 VEGF TSP-1 ## 100 ** ** 50 * siAkap12-CM + + + siAkap12-CM Y27632 VEGF CM TSP-1 Ponceau Relative expression of TSP-1 protein (%) + + 2348 + + 120 * 80 # # 40 + siAkap12-CM Y27632 (Fig 3G,H) Interestingly, our data showed that PKCf activation by VEGF in HRMECs is induced by the phosphorylation of PKCf (Fig 3F), whereas PKCf translocation to the membrane was observed either following treatment with thrombin in endothelial cells [25], or following treatment with VEGF in retinal pigment epithelial cells [44] To determine whether the ** + Y27632 D ## + + + expression of VEGF and TSP-1 was regulated by PKCf, we transfected HRMECs with siPKCf, and observed that the expression of VEGF was decreased and that the expression of TSP-1 was increased in siPKCf-transfected cells (Fig 5) PKCf had a significant effect on the levels of secreted VEGF and TSP-1 (Fig 5) as compared with intracellular VEGF and FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS Y K Choi and K.-W Kim Regulation of the blood–neural barrier by AKAP12 Fig The involvement of Rho kinase in the expression of VEGF and TSP-1 in HRMECs (A) HRMECs were treated with 10 lM Y27632 for 24 h under both normoxic and hypoxic conditions VEGF and TSP-1 mRNA levels were detected by RT-PCR (B) VEGF and TSP-1 secretion levels were detected by western blot The data from five independent experiments were quantified, and are shown on the right The level of expression of TSP-1 in Y27632-treated cells under normoxia was set at 100% *P < 0.05 as compared to control; #P < 0.005 as compared to control The control for Y27632 treatment under normoxia was no Y27632 under normoxia; the control for no Y27632 treatment under hypoxia was no Y27632 under normoxia; the control for Y27632 treatment under hypoxia was no Y27632 under hypoxia (C) HRMECs were treated with NC-CM, NC-CM plus 10 lM Y27632 (NC-CM + Y), siAkap12-CM or siAkap12-CM plus Y27632 (siAkap12-CM + Y) for 24 h The levels of VEGF and TSP-1 (green) were detected by immunocytochemistry Scale bar, 50 lm Nuclei (blue) were stained with DAPI Quantification of the immunohistochemical staining area was performed using IMAGE-PRO PLUS The expression levels from four independent experiments were quantified, and are shown on the right The level of VEGF in siAkap12-CM-treated cells was set at 100% The level of TSP-1 in NC-CM + Y27632-treated cells was set at 100% *P < 0.05 as compared to control; **P < 0.005 as compared to control; ##P < 0.001 as compared to control The control for NC-CM + Y was NC-CM; the control for siAkap12-CM was NC-CM; the control for siAkap12-CM + Y was siAkap12-CM (D) Secretion levels of VEGF and TSP-1 were detected by western blot under the same conditions as for (C) The data from four independent experiments were quantified, and are shown on the right The level of TSP-1 in NC-CM + Y27632-treated cells was set at 100% *P < 0.05 as compared to control; #P < 0.01 as compared to control The control for NC-CM + Y was NC-CM; the control for siAkap12-CM was NC-CM; the control for siAkap12-CM + Y was siAkap12-CM b-actin and Ponceau Red staining served as the internal controls TSP-1 levels (data not shown), because the signal transduction requires the binding of secreted VEGF and TSP-1 to endothelial cell receptors [45,46] On the basis of these data, we suggest that astrocyte-derived VEGF activates PKCf, resulting in differential regulation of VEGF and TSP-1 in HRMECs A recent study showed that PKCa induced angiogenesis via induction of VEGF in human umbilical vein endothelial cells (HUVECs) [47] Therefore, we propose that PKCf, as well as PKCa, can induce VEGF in endothelial cells As astrocytic AKAP12 inhibited phosphorylation of PKCf in HRMECs (Fig 4) and increased Ang1 secretion levels [20], we examined whether PKCf activation could be reduced by Ang1 COMP-Ang1 induced partial inhibition of phosphorylation of PKCf induced by siAkap12-CM (Fig 4F) In addition, when HRMECs were incubated with H-AKAP12-CM, claudin-1 and VE-cadherin levels were upregulated as compared to those seen with H-mock-CM (Fig 1E), and this effect was blocked by pretreatment of H-AKAP12-CM with an antibody to Ang1 (Fig 1E) These results indicate that Ang1 derived from astrocytes may induce junction proteins by inhibition of phosphorylation of PKCf in HRMECs Several studies have shown that atypical PKCf regulates Rho activity [35,36] In the current study, we investigated the effect of a specific Rho kinase inhibitor, Y27632, on VEGF and TSP-1 levels in HRMECs We demonstrated that hypoxia increases the secretion of VEGF and decreases the secretion of TSP-1, and that this effect is blocked by Y27632 (Fig 6A,B) The same effect was also observed in HRMECs when these cells were treated with CM from siAkap12-transfected astrocytes (Fig 6C,D) siAkap12-CM activated PKCf in HRMECs (Fig 4D,E), leading to increased VEGF levels and decreased TSP-1 levels, and this effect was blocked by Y27632 (Fig 6C,D) Our results suggest that Rho kinase activity plays a key role in the differential regulation of VEGF and TSP-1 by AKAP12 or oxygen tension In summary, we showed that VEGF and TSP-1 levels in HRMECs are differentially regulated by AKAP12 in astrocytes (Fig 2A,B), leading to reduced vascular permeability, decreased endothelial cell migration, and upregulation of tight junction proteins We also demonstrated that the regulation of VEGF and TSP-1 by astrocytic AKAP12 is mediated by phosphorylation of PKCf and Rho kinase activity The most prominent features of the blood–neural barrier are the presence of complex tight junctions, and the interaction of adhesion molecules of central nervous system endothelial cells, which together form an endothelial barrier [48–51] Our results may elucidate a pathway to restoring barrier function in central nervous system diseases that are associated with increased VEGF expression and decreased TSP-1 expression Experimental procedures Immunofluorescence staining HRMECs were incubated overnight at °C with the indicated primary antibodies, and this was followed by incubation with Alexa Fluor antibodies as secondary antibodies Nuclei were stained with 4¢,6-diamidino-2-phenylindole (DAPI) (Sigma, St Louis, MO, USA) Images were obtained with an Axiovert M200 (Carl Zeiss, Oberkochen, Germany) microscope, and analyzed using image-pro plus (Media Cybernetics, Bethesda, MD, USA) We counted the area stained with green fluorescence according to the FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS 2349 Regulation of the blood–neural barrier by AKAP12 Y K Choi and K.-W Kim manufacturer’s instructions Each stained area was presented relative to the area with the highest staining intensity Cell culture Primary human brain astrocyte cells, dissociated from normal human brain cortex tissue, were purchased from the Applied Cell Biology Research Institute (Kirkland, WA, USA) Primary human brain astrocyte cells were cultured in DMEM supplemented with 10% fetal bovine serum (Invitrogen, San Diego, CA, USA) and antibiotics HRMECs were purchased from the Applied Cell Biology Research Institute and grown in M199 medium supplemented with 20% fetal bovine serum, ngỈmL)1 basic fibroblast growth factor (Invitrogen) and 10 mL)1 heparin (Sigma) For hypoxia experiments, astrocytes were incubated in a hypoxic chamber (Forma Scientific, San Bruno, CA, USA), which maintained the cells under low oxygen tension (5% CO2 with 1% O2, balanced with N2) Migration assay HRMECs were seeded on gelatin-coated 12-well culture dishes At 90% confluence, the endothelial monolayers were marked with an injury line and wounded with the end of a 200-lL tip Plates were rinsed with serum-free medium to remove cellular debris CM from human astrocytes transiently transfected with mock or Akap12 vectors under normoxic or hypoxic conditions was then added HRMECs were allowed to migrate for 24 h, rinsed with NaCl ⁄ Pi, fixed with absolute methanol for min, and stained with Giemsa (Sigma) Western blot analysis Cellular protein and CM protein were analyzed by western blot assay Western blot analysis was performed as described previously [52] We used antibodies specific for Ang1, VEGF, VE-cadherin, ZO-2, Flk-1, PKCf and phosphorylated PKCf (Snata Cruz Biotechnology, Santa Cruz, CA, USA), ZO-1 (Zymed, San Francisco, CA, USA), TSP1 (Neomarkers), and b-actin (Sigma) Recombinant VEGF165 was purchased from R&D Systems (Minneapolis, MN, USA), and COMP-Ang1 was a generous gift from G Y Koh (Korea Advanced Institute of Science and Technology) Y27632, a specific Rho kinase inhibitor, was purchased from Calbiochem (La Jolla, CA, USA) Ponceau S solution was purchased from Sigma Cell fractionation Cell fractionation was performed as described previously [25] with minor modifications Cells were washed with NaCl ⁄ Pi and harvested by scraping into 50 lL of homoge- 2350 nization buffer (20 mm Tris ⁄ HCl, pH 7.4, 0.5 mm EDTA, 0.5 mm EGTA, 10 mm b-mercaptoethanol, 5% glycerol, mm NaF, mm Na3VO4, and proteinase inhibitor mixture) Cells were mechanically homogenized and centrifuged for 15 at 1200 g at °C The supernatant was further centrifuged for 80 at 28 700 g at °C The resulting supernatant containing the cytosolic components was removed, and the pellet containing the membrane components were resuspended in 50 lL of homogenized buffer supplemented with 0.5% Triton X-100 and 100 mm NaCl Proteins in 20 lL of each fraction were separated by SDS ⁄ PAGE Transient transfection and CM preparation The full-length rat Akap12 cDNA (from I H Gelman, Roswell Park Cancer Institute, Buffalo, NY, USA) was subcloned into pcDNA3 Transient transfections were performed using Lipofectamine and Plus reagent (Invitrogen) For the preparation of CMs for treating HRMECs, medium from transfected human astrocytes was changed to M199 medium containing 1% fetal bovine serum for 24 h, collected and filtered through a 0.22 lm pore membrane (Millipore, Beverly, MA, USA), and then concentrated four times using centrifugal filters (Millipore) For preparation of CM for western blot analysis, M199 medium containing 1% fetal bovine serum from transfected cells was collected and concentrated through Ultra-4 centrifugal filters (Millipore) Cell viability assay [2,3-bis(2-methoxy-4-nitro5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt assay] Cell viability was determined using CellTiter 96 Aqueous One Solution (Promega) Cells were seeded into gelatincoated 96-well plates, and incubated with or without reagents for 24 h Each culture condition was analyzed in triplicate The absorbance values at 492 nm were corrected by subtracting the average absorbance from the control wells containing ‘no cells’ Permeability assay Permeability across the endothelial cell monolayer was measured by using type I collagen-coated transwell units (6.5 mm diameter, 3.0 lm pore spolycarbonate filter; Corning, Corning, NY, USA) After HRMECs become confluent, CM was treated for 24 h Permeability was measured by adding 0.1 mg of RITC-labeled dextran (relative molecular mass  10 000) ⁄ mL to the upper chamber After incubation for 15 min, 100 lL of sample from the lower compartment was diluted with 100 lL of NaCl ⁄ Pi and measured for fluorescence at 635 nm when excited at 540 nm FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS Y K Choi and K.-W Kim with a spectrophotometer (Tecan Spectra Fluor; Tecan Durham, NC, USA) Regulation of the blood–neural barrier by AKAP12 tion Research Center, College of Pharmacy, Seoul National University) for a useful discussion We declare that we have no competing financial interests RNA interference Astrocytes and HRMECs were grown to 80% confluence, and siRNAs (siAkap12 50 nm; siPKCf 150 nm) were transfected into the cells using Lipofectamine and Plus reagent (Invitrogen) All transfections were performed according to the manufacturer’s instructions siRNAs and the control nonsilencing RNAs were designed by Dharmacon (Lafayette, CO, USA) The human Akap12 target sequence used was: 5¢-AGACGGATGTAGTGTTGAA-3¢ The siRNA targeting PKCf was designed by Dharmacon (Catalog number, M-003526-04) RT-PCR Total RNA was isolated from the indicated cells using Trizol reagent (Invitrogen) RT-PCR analysis was performed as described previously [53] The following sets of primers were used: VEGF, 5¢-GAGAATTCGGCCTCCGAAA CCATGAACTTTCTGT-3¢ (forward) and 5¢-GAGCATG CCCTCCTGCCCGGCTCACCGC-3¢ (reverse); TSP-1, 5¢-CGTCCTGTTCCTGATGCATG-3¢ (forward) and 5¢-GGCCCTGTCTTCCTGCACAA-3¢ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5¢-CAGGG CTGCTTTTAACTCTG-3¢ (forward) and 5¢-TAGAGG CAGGGATGATGTTC-3¢ (reverse); and b-actin, 5¢-GACTA CCTCATGAAGATC-3¢ (forward) and 5¢-GATCC ACATCTGCTGGAA-3¢ (reverse) The PCR products were separated on 1.2% agarose gels and visualized by ethidium bromide staining under a transilluminator (LAS 3000; Fujifilm, Tokyo, Japan) Data analysis and statistics Quantification of band intensity was analyzed using imagej (http://rsb.info.nih.gov/ij/) and normalized to the density of the b-actin or Ponceau staining band All data are presented as mean ± SD changed into relative percentage Statistical comparisons between groups were done using Student’s t-test P < 0.05 was considered to be statistically significant Acknowledgements This work was supported by the Creative Research Initiatives (NeuroVascular Coordination Research Center) of the Ministry of Science and Technology We thank Dr G Y Koh (Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea) for providing COMP-Ang1 Y K Choi is grateful to Kyu Han Kim (NeuroVascular Coordina- References Abbott NJ, Ronnback L & Hansson E (2006) Astrocyte–endothelial interactions at the blood–brain barrier Nat Rev Neurosci 7, 41–53 Zhang Y, Porat RM, Alon T, Keshet E & Stone J (1999) Tissue oxygen levels control astrocyte movement and differentiation in developing retina Brain Res Dev Brain Res 118, 135–145 Stone J, Itin A, Alon T, Pe’er J, Gnessin H, Chan-Ling T & Keshet E (1995) Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia J Neurosci 15, 4738–4747 Provis JM, Leech J, Diaz CM, Penfold PL, Stone J & Keshet E (1997) Development of the human retinal vasculature: cellular relations and VEGF expression Exp Eye Res 65, 555–568 Dvorak HF, Nagy JA, Feng D, Brown LF & Dvorak AM (1999) Vascular permeability factor ⁄ vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis Curr Top Microbiol Immunol 237, 97–132 Alon T, Hemo I, Itin A, Pe’er J, Stone J & Keshet E (1995) Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity Nat Med 1, 1024–1028 Vailhe B & Feige JJ (2003) Thrombospondins as antiangiogenic therapeutic agents Curr Pharm Des 9, 583– 588 Hu CJ, Chen SD, Yang DI, Lin TN, Chen CM, Huang TH & Hsu CY (2006) Promoter region methylation and reduced expression of thrombospondin-1 after oxygen– glucose deprivation in murine cerebral endothelial cells J Cereb Blood Flow Metab 26, 1519–1526 Kalluri R (2003) Basement membranes: structure, assembly and role in tumour angiogenesis Nat Rev Cancer 3, 422–433 10 Rastinejad F, Polverini PJ & Bouck NP (1989) Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene Cell 56, 345–355 11 Gelman IH & Gao L (2006) SSeCKS ⁄ Gravin ⁄ AKAP12 metastasis suppressor inhibits podosome formation via RhoA- and Cdc42-dependent pathways Mol Cancer Res 4, 151–158 12 Gelman IH, Lee K, Tombler E, Gordon R & Lin X (1998) Control of cytoskeletal architecture by the srcsuppressed C kinase substrate, SSeCKS Cell Motil Cytoskeleton 41, 1–17 FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS 2351 Regulation of the blood–neural barrier by AKAP12 Y K Choi and K.-W Kim 13 Lin X, Tombler E, Nelson PJ, Ross M & Gelman IH (1996) A novel src- and ras-suppressed protein kinase C substrate associated with cytoskeletal architecture J Biol Chem 271, 28430–28438 14 Cheng C, Liu H, Ge H, Qian J, Qin J, Sun L & Shen A (2007) Essential role of Src suppressed C kinase substrates in endothelial cell adhesion and spreading Biochem Biophys Res Commun 358, 342–348 15 Kaibuchi K, Kuroda S & Amano M (1999) Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells Annu Rev Biochem 68, 459–486 16 Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N, Matsuura Y & Kaibuchi K (1997) Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase Science 275, 1308–1311 17 Park JH, Okayama N, Gute D, Krsmanovic A, Battarbee H & Alexander JS (1999) Hypoxia ⁄ aglycemia increases endothelial permeability: role of second messengers and cytoskeleton Am J Physiol 277, C1066– C1074 18 Chapline C, Cottom J, Tobin H, Hulmes J, Crabb J & Jaken S (1998) A major, transformation-sensitive PKCbinding protein is also a PKC substrate involved in cytoskeletal remodeling J Biol Chem 273, 19482–19489 19 Hayashi M, Sakata M, Takeda T, Tahara M, Yamamoto T, Minekawa R, Isobe A, Tasaka K & Murata Y (2005) Hypoxia up-regulates hypoxia-inducible factor1alpha expression through RhoA activation in trophoblast cells J Clin Endocrinol Metab 90, 1712–1719 20 Choi YK, Kim JH, Kim WJ, Lee HY, Park JA, Lee SW, Yoon DK, Kim HH, Chung H, Yu YS et al (2007) AKAP12 regulates human blood–retinal barrier formation by downregulation of hypoxia-inducible factor-1alpha J Neurosci 27, 4472–4481 21 Lee SW, Kim WJ, Choi YK, Song HS, Son MJ, Gelman IH, Kim YJ & Kim KW (2003) SSeCKS regulates angiogenesis and tight junction formation in blood– brain barrier Nat Med 9, 900–906 22 Parekh DB, Ziegler W & Parker PJ (2000) Multiple pathways control protein kinase C phosphorylation EMBO J 19, 496–503 23 Wu HM, Yuan Y, Zawieja DC, Tinsley J & Granger HJ (1999) Role of phospholipase C, protein kinase C, and calcium in VEGF-induced venular hyperpermeability Am J Physiol 276, H535–H542 24 Haller H, Ziegler W, Lindschau C & Luft FC (1996) Endothelial cell tyrosine kinase receptor and G proteincoupled receptor activation involves distinct protein kinase C isoforms Arterioscler Thromb Vasc Biol 16, 678–686 25 Li X, Hahn CN, Parsons M, Drew J, Vadas MA & Gamble JR (2004) Role of protein kinase Czeta in thrombin-induced endothelial permeability changes: inhibition by angiopoietin-1 Blood 104, 1716–1724 2352 26 Haller H, Hempel A, Homuth V, Mandelkow A, Busjahn A, Maasch C, Drab M, Lindschau C, Jupner A, Vetter K et al (1998) Endothelial-cell permeability and protein kinase C in pre-eclampsia Lancet 351, 945–949 27 Pal S, Datta K, Khosravi-Far R & Mukhopadhyay D (2001) Role of protein kinase Czeta in Ras-mediated transcriptional activation of vascular permeability factor ⁄ vascular endothelial growth factor expression J Biol Chem 276, 2395–2403 28 Datta K, Li J, Bhattacharya R, Gasparian L, Wang E & Mukhopadhyay D (2004) Protein kinase C zeta transactivates hypoxia-inducible factor alpha by promoting its association with p300 in renal cancer Cancer Res 64, 456–462 29 Ferrara N, Gerber HP & LeCouter J (2003) The biology of VEGF and its receptors Nat Med 9, 669–676 30 Li Z, Wang C, Jiao X, Lu Y, Fu M, Quong AA, Dye C, Yang J, Dai M, Ju X et al (2006) Cyclin D1 regulates cellular migration through the inhibition of thrombospondin and ROCK signaling Mol Cell Biol 26, 4240–4256 31 Weinstat-Saslow DL, Zabrenetzky VS, VanHoutte K, Frazier WA, Roberts DD & Steeg PS (1994) Transfection of thrombospondin complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis Cancer Res 54, 6504–6511 32 Zhang YW, Su Y, Volpert OV & Vande Woude GF (2003) Hepatocyte growth factor ⁄ scatter factor mediates angiogenesis through positive VEGF and negative thrombospondin regulation Proc Natl Acad Sci USA 100, 12718–12723 33 Greenaway J, Gentry PA, Feige JJ, LaMarre J & Petrik JJ (2005) Thrombospondin and vascular endothelial growth factor are cyclically expressed in an inverse pattern during bovine ovarian follicle development Biol Reprod 72, 1071–1078 34 Cho CH, Kammerer RA, Lee HJ, Steinmetz MO, Ryu YS, Lee SH, Yasunaga K, Kim KT, Kim I, Choi HH et al (2004) COMP-Ang1: a designed angiopoietin-1 variant with nonleaky angiogenic activity Proc Natl Acad Sci USA 101, 5547–5552 35 Kuribayashi K, Nakamura K, Tanaka M, Sato T, Kato J, Sasaki K, Takimoto R, Kogawa K, Terui T, Takayama T et al (2007) Essential role of protein kinase C zeta in transducing a motility signal induced by superoxide and a chemotactic peptide, fMLP J Cell Biol 176, 1049–1060 36 Van Kolen K & Slegers H (2006) Atypical PKCzeta is involved in RhoA-dependent mitogenic signaling by the P2Y(12) receptor in C6 cells FEBS J 273, 1843–1854 37 Song HS, Son MJ, Lee YM, Kim WJ, Lee SW, Kim CW & Kim KW (2002) Oxygen tension regulates the maturation of the blood–brain barrier Biochem Biophys Res Commun 290, 325–331 FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS Y K Choi and K.-W Kim 38 Jackson C (2002) Matrix metalloproteinases and angiogenesis Curr Opin Nephrol Hypertens 11, 295–299 39 West H, Richardson WD & Fruttiger M (2005) Stabilization of the retinal vascular network by reciprocal feedback between blood vessels and astrocytes Development 132, 1855–1862 40 Provis JM (2001) Development of the primate retinal vasculature Prog Retin Eye Res 20, 799–821 41 Qaum T, Xu Q, Joussen AM, Clemens MW, Qin W, Miyamoto K, Hassessian H, Wiegand SJ, Rudge J, Yancopoulos GD et al (2001) VEGF-initiated blood– retinal barrier breakdown in early diabetes Invest Ophthalmol Vis Sci 42, 2408–2413 42 Witmer AN, Vrensen GF, Van Noorden CJ & Schlingemann RO (2003) Vascular endothelial growth factors and angiogenesis in eye disease Prog Retin Eye Res 22, 1–29 43 Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C et al (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele Nature 380, 435–439 44 Young TA, Wang H, Munk S, Hammoudi DS, Young DS, Mandelcorn MS & Whiteside CI (2005) Vascular endothelial growth factor expression and secretion by retinal pigment epithelial cells in high glucose and hypoxia is protein kinase C-dependent Exp Eye Res 80, 651–662 45 Millauer B, Wizigmann-Voos S, Schnurch H, Martinez R, Moller NP, Risau W & Ullrich A (1993) High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis Cell 72, 835–846 Regulation of the blood–neural barrier by AKAP12 46 Murphy-Ullrich JE & Mosher DF (1987) Interactions of thrombospondin with endothelial cells: receptor-mediated binding and degradation J Cell Biol 105, 1603– 1611 47 Xu H, Czerwinski P, Hortmann M, Sohn HY, Forstermann U & Li H (2008) Protein kinase C {alpha} promotes angiogenic activity of human endothelial cells via induction of vascular endothelial growth factor Cardiovasc Res, doi:10.1093/cvr/cvm/085 48 Morcos Y, Hosie MJ, Bauer HC & Chan-Ling T (2001) Immunolocalization of occludin and claudin-1 to tight junctions in intact CNS vessels of mammalian retina J Neurocytol 30, 107–123 49 Staddon JM & Rubin LL (1996) Cell adhesion, cell junctions and the blood–brain barrier Curr Opin Neurobiol 6, 622–627 50 Gumbiner B & Simons K (1986) A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of a uvomorulin-like polypeptide J Cell Biol 102, 457–468 51 Gavard J & Gutkind JS (2006) VEGF controls endothelial-cell permeability by promoting the beta-arrestindependent endocytosis of VE-cadherin Nat Cell Biol 8, 1223–1234 52 Moon EJ, Jeong CH, Jeong JW, Kim KR, Yu DY, Murakami S, Kim CW & Kim KW (2004) Hepatitis B virus X protein induces angiogenesis by stabilizing hypoxia-inducible factor-1alpha FASEB J 18, 382–384 53 Lee MS, Moon EJ, Lee SW, Kim MS, Kim KW & Kim YJ (2001) Angiogenic activity of pyruvic acid in in vivo and in vitro angiogenesis models Cancer Res 61, 3290–3293 FEBS Journal 275 (2008) 2338–2353 ª 2008 The Authors Journal compilation ª 2008 FEBS 2353 ... astrocytic AKAP12 inhibits phosphorylation of PKCf in neighboring human retina microvascular endothelial cells (HRMECs), which leads to a decrease in VEGF levels and an increase in TSP-1 levels in HRMECs,... Ziegler W, Lindschau C & Luft FC (1996) Endothelial cell tyrosine kinase receptor and G proteincoupled receptor activation involves distinct protein kinase C isoforms Arterioscler Thromb Vasc... transfection of Akap12 into human astrocytes increased angiopoietin-1 (Ang1) levels in CM under hypoxic conditions, and this played a role in barrier properties in HRMECs [20] Therefore, we examined whether