The human malaria parasite Plasmodium falciparum is responsible for the deaths of more than a million people each year. Fosmidomycin has been proven to be efficient in the treatment of P. falciparum malaria by inhibiting 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an enzyme of the non-mevalonate pathway, which is absent in humans. However, the structural details of DXR inhibition by fosmidomycin in P. falciparum are unknown. Here, we report the crystal structures of fosmidomycin-bound complete quaternary complexes of PfDXR. Our study revealed that (i) an intrinsic flexibility of the PfDXR molecule accounts for an induced-fit movement to accommodate the bound inhibitor in the active site and (ii) a cis arrangement of the oxygen atoms of the hydroxamate group of the bound inhibitor is essential for tight binding of the inhibitor to the active site metal. We expect the present structures to be useful guides for the design of more effective antimalarial compounds.
Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis, suggesting that loss of PEDF contributes to proliferative diabetic retinopathy. However, the role of PEDF against retinal vascular hyperpermeability remains to be elucidated. We investigated here whether and how PEDF could inhibit the advanced glycation end product (AGE) signaling to vascular hyperpermeability. Intravenous administration of AGEs to normal rats not only increased retinal vascular permeability by stimulating vascular endothelial growth factor (VEGF) expression but also decreased retinal PEDF levels. Simultaneous treatments with PEDF inhibited the AGE-elicited VEGF-mediated permeability by down-regulating mRNA levels of p22phox and gp91phox, membrane components of NADPH oxidase, and subsequently decreasing retinal levels of an oxidative stress marker, 8-hydroxydeoxyguanosine. PEDF also inhibited the AGE-induced vascular hyperpermeability evaluated by transendothelial electrical resistance by suppressing VEGF expression. Furthermore, PEDF decreased reactive oxygen species (ROS) generation in AGE-exposed endothelial cells by suppressing NADPH oxidase activity via down-regulation of mRNA levels of p22PHOX and gp91PHOX. This led to blockade of the AGE-elicited Ras activation and NF-κB-dependent VEGF gene induction in endothelial cells. These results indicate that the central mechanism for PEDF inhibition of the AGE signaling to vascular permeability is by suppression of NADPH oxidase-mediated ROS generation and subsequent VEGF expression. Substitution of PEDF may offer a promising strategy for halting the development of diabetic retinopathy. Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis, suggesting that loss of PEDF contributes to proliferative diabetic retinopathy. However, the role of PEDF against retinal vascular hyperpermeability remains to be elucidated. We investigated here whether and how PEDF could inhibit the advanced glycation end product (AGE) signaling to vascular hyperpermeability. Intravenous administration of AGEs to normal rats not only increased retinal vascular permeability by stimulating vascular endothelial growth factor (VEGF) expression but also decreased retinal PEDF levels. Simultaneous treatments with PEDF inhibited the AGE-elicited VEGF-mediated permeability by down-regulating mRNA levels of p22phox and gp91phox, membrane components of NADPH oxidase, and subsequently decreasing retinal levels of an oxidative stress marker, 8-hydroxydeoxyguanosine. PEDF also inhibited the AGE-induced vascular hyperpermeability evaluated by transendothelial electrical resistance by suppressing VEGF expression. Furthermore, PEDF decreased reactive oxygen species (ROS) generation in AGE-exposed endothelial cells by suppressing NADPH oxidase activity via down-regulation of mRNA levels of p22PHOX and gp91PHOX. This led to blockade of the AGE-elicited Ras activation and NF-κB-dependent VEGF gene induction in endothelial cells. These results indicate that the central mechanism for PEDF inhibition of the AGE signaling to vascular permeability is by suppression of NADPH oxidase-mediated ROS generation and subsequent VEGF expression. Substitution of PEDF may offer a promising strategy for halting the development of diabetic retinopathy. Diabetic retinopathy is one of the miserable microvascular complications in diabetes and is a leading cause of acquired blindness among people of occupational age (1.Frank R.N. Ophthalmology. 1991; 98: 586-593Abstract Full Text PDF PubMed Scopus (146) Google Scholar). Chronic hyperglycemia is a major initiator of diabetic retinopathy. Two recent large prospective clinical studies have shown that intensive blood glucose control reduces microvascular complications among patients with diabetes (2.The Diabetes Control and Complications Trial Research GroupN. Engl. J. Med. 1993; 329: 977-986Crossref PubMed Scopus (22798) Google Scholar, 3.UK Prospective Diabetes Study GroupLancet. 1998; 352: 837-853Abstract Full Text Full Text PDF PubMed Scopus (19019) Google Scholar). However, strict control of hyperglycemia is often difficult to maintain and may increase the risk of severe hypoglycemia in diabetic patients. Therefore, the development of novel therapeutic strategies that specifically target diabetic retinopathy is desired for patients with diabetes. Various hyperglycemia-induced metabolic and hemodynamic derangements have been reported to contribute to the characteristic histopathological changes observed in diabetic retinopathy (4.Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7064) Google Scholar). Among them, advanced glycation end products (AGEs), 2The abbreviations used are: AGEs, advanced glycation end products; VEGF, vascular endothelial growth factor; ROS, reactive oxygen species; PEDF, pigment epithelium-derived factor; ECs, endothelial cells; DPI, diphenylene iodonium; Abs, antibodies; BSA, bovine serum albumin; BRB, blood retinal barrier; RT, reverse transcription; 8-OHdG, 8-hydroxydeoxyguanosine; TER, transendothelial electrical resistance; RAGE, receptor for AGEs; DN-Ras, dominant-negative human Ras mutant; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate. the senescent macroprotein derivatives, whose formation and accumulation occur at an accelerated rate in diabetes (5.Brownlee M. Cerami A. Vlassara H. N. Engl. J. Med. 1998; 318: 1315-1321Google Scholar, 6.Dyer D.G. Blackledgem J.A. Thorpe S.R. Baynes J.W. J. Biol. Chem. 1991; 266: 11654-11660Abstract Full Text PDF PubMed Google Scholar, 7.Grandhee S.K. Monnier V.M. J. Biol. Chem. 1991; 266: 11649-11653Abstract Full Text PDF PubMed Google Scholar), have been strongly implicated in the pathogenesis of diabetic vascular complications (8.Vlassara H. Bucala R. Diabetes. 1996; 3: S65-S66Crossref Google Scholar, 9.Yamagishi S. Fujimori H. Yonekura H. Yamamoto Y. Yamamoto H. Diabetologia. 1998; 41: 1435-1441Crossref PubMed Scopus (210) Google Scholar, 10.Wendt T. Bucciarelli L. Qu W. Lu Y. Yan S.F. Stern D.M. Schmidt A.M. Curr. Atheroscler. Rep. 2000; 4: 228-237Crossref Scopus (148) Google Scholar, 11.Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3671) Google Scholar, 12.Yamagishi S. Inagaki Y. Amano S. Okamoto T. Takeuchi M. Makita Z. Biochem. Biophys. Res. Commun. 2002; 296: 877-882Crossref PubMed Scopus (201) Google Scholar, 13.Yamagishi S. Inagaki Y. Okamoto T. Amano S. Koga K. Takeuchi M. Makita Z. J. Biol. Chem. 2002; 277: 20309-20315Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 14.Yamagishi S. Amano S. Inagaki Y. Okamoto T. Takeuchi M. Makita Z. Mol. Med. 2002; 8: 546-550Crossref PubMed Google Scholar, 15.Yamagishi S. Inagaki Y. Amano S. Okamoto T. Koga K. Takeuchi M. Kidney Int. 2003; 63: 464-473Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Indeed, we, along with others, have previously shown that AGEs could elicit a brisk angiogenic response, at least in part, by inducing autocrine production of vascular endothelial growth factor (VEGF), which is an important mediator in the development and progression of diabetic retinopathy (16.Yamagishi S. Yonekura H. Yamamoto Y. Katsuno K. Sato F. Mita I. Ooka H. Satozawa N. Kawakami T. Nomura M. Yamamoto H. J. Biol. Chem. 1997; 272: 8723-8730Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 17.Okamoto T. Yamagishi S. Inagaki Y. Amano S. Koga K. Abe R. Takeuchi M. Ohno S. Yoshimura A. Makita Z. FASEB J. 2002; 16: 1928-1930Crossref PubMed Scopus (259) Google Scholar, 18.Stitt A.W. Bhaduri T. McMullen T. Gardiner T.A. Archer D.B. Mol. Cell. Biol. Res. Commun. 2000; 3: 380-388Crossref PubMed Scopus (120) Google Scholar). AGEs exert pleiotropic actions on cells by inducing the generation of intracellular reactive oxygen species (ROS) (19.Yan S.D. Schmidt A.M. Anderson G.M. Zhang J. Brett J. Zou Y.S. Pinsky D. Stern D. J. Biol. Chem. 1994; 269: 9889-9897Abstract Full Text PDF PubMed Google Scholar). ROS in turn activate the Ras protooncogene and its downstream effectors that are important for both proliferative and differentiative responses. Pigment epithelium-derived factor (PEDF) is a glycoprotein that belongs to the superfamily of serine protease inhibitors (20.Tombran-Tink J. Chader C.G. Johnson L.V. Exp. Eye Res. 1991; 53: 411-414Crossref PubMed Scopus (561) Google Scholar). It was first purified from the conditioned media of human retinal pigment epithelial cells as a factor with potent neuronal differentiating activity (20.Tombran-Tink J. Chader C.G. Johnson L.V. Exp. Eye Res. 1991; 53: 411-414Crossref PubMed Scopus (561) Google Scholar). Recently, PEDF has been shown to be a highly effective inhibitor of angiogenesis in cell culture and animal models. PEDF inhibits the growth and migration of cultured endothelial cells (ECs), and it potently suppresses ischemia-induced retinal neovascularization (21.Dawson D.W. Volpert O.V. Gillis P. Crawford S.E. Xu H.J. Benedict W. Bouck N.P. Science. 1999; 285: 245-248Crossref PubMed Scopus (1401) Google Scholar, 22.Duh E.J. Yang H.S. Suzuma I. Miyagi M. Youngman E. Mori K. Katai M. Yan L. Suzuma K. West K. Davarya S. Tong P. Gehlbach P. Pearlman J. Crabb J.W. Aiello L.P. Campochiaro P.A. Zack D.J. Investig. Ophthalmol. Vis. Sci. 2002; 43: 821-829PubMed Google Scholar). PEDF levels in aqueous humor or vitreous are decreased in diabetic patients, especially with proliferative retinopathy (23.Spranger J. Osterhoff M. Reimann M. Mohlig M. Ristow M. Francis M.K. Cristofalo V. Hammes H.P. Smith G. Boulton M. Pfeiffer F.H. Diabetes. 2002; 50: 2641-2645Crossref Scopus (266) Google Scholar, 24.Ogata N. Tombran-Tink J. Nishikawa M. Nishimura T. Mitsuma Y. Sakamoto T. Matsumura M. Am. J. Ophthalmol. 2001; 132: 378-382Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 25.Boehm B.O. Lang G. Volpert O. Jehle P.M. Kurkhaus A. Rosinger S. Lang G.K. Bouck N. Diabetologia. 2003; 46: 394-400Crossref PubMed Scopus (132) Google Scholar). These observations suggest that the loss of PEDF activity in the eye may contribute to the pathogenesis of proliferative diabetic retinopathy. However, the protective role of PEDF against retinal vascular hyperpermeability, the characteristic feature of early diabetic retinopathy, remains to be elucidated. In this study, we have investigated whether PEDF could inhibit the AGE-induced retinal vascular hyperpermeability and the mechanism by which it might achieve this beneficial effect. Materials—Diphenylene iodonium (DPI), lucigenin, and NADPH were purchased from Sigma. Polyclonal antibodies (Abs) against rat VEGF were purchased from R & D Systems (Genzyme-Techne, Minneapolis, MN). Protease inhibitor mixtures were from Nakalai Tesque (Kyoto, Japan). Purification of PEDF Proteins—PEDF proteins were prepared and purified as described previously (12.Yamagishi S. Inagaki Y. Amano S. Okamoto T. Takeuchi M. Makita Z. Biochem. Biophys. Res. Commun. 2002; 296: 877-882Crossref PubMed Scopus (201) Google Scholar). SDS-PAGE analysis of purified PEDF proteins revealed a single band with a molecular mass of about 50 kDa, which showed positive reactivity with monoclonal Ab against human PEDF (Transgenic, Kumamoto, Japan). Preparations of AGEs—AGE-bovine serum albumin (BSA) was prepared as described previously (12.Yamagishi S. Inagaki Y. Amano S. Okamoto T. Takeuchi M. Makita Z. Biochem. Biophys. Res. Commun. 2002; 296: 877-882Crossref PubMed Scopus (201) Google Scholar). Briefly, BSA (50 mg/ml) was incubated under sterile conditions with 0.1 m d-glyceraldehyde in 0.2 m NaPO4 buffer, pH 7.4, for 7 days. Then unincorporated sugars were removed by dialysis against phosphate-buffered saline. Control nonglycated BSA was incubated in the same conditions except for the absence of reducing sugars. Preparations were tested for endotoxin using Endospecy ES-20S system (Seikagaku Co., Tokyo, Japan); no endotoxin was detectable. Preparations of AGE-rich Serum Fractions from Diabetic Patients on Hemodialysis—Serum AGE fractions were obtained from normal volunteers and diabetic patients with hemodialysis (DM-AGEs) as described previously (26.Takeuchi M. Watai T. Sasaki N. Choei H. Iwaki M. Ashizawa T. Inagaki Y. Yamagishi S. Kikuchi S. Riederer P. Saito T. Bucala R. Kameda Y. J. Neuropathol. Exp. Neurol. 2003; 62: 486-496Crossref PubMed Scopus (31) Google Scholar). Briefly, 10 ml of serum from each of 4 normal individuals and from 11 type 2 diabetic patients with end-stage renal disease on hemodialysis were concentrated by lyophilization and dissolved in 2 ml of distilled water. These solutions were applied to a Sephacryl S-200 column (1.5 × 110 cm), which was equilibrated with phosphate-buffered saline, pH 7.4, and eluted with the same buffer (fraction size, 1.5 ml; flow rate, 10 ml/h) in a cold room. Each fraction was monitored for absorbance at 280 nm, and the AGE concentration of each fraction was measured by a competitive ELISA as described below. Preparations were passed through Zeta-Pore filter to remove endotoxin. No endotoxin was detectable. AGE-rich serum fractions obtained from diabetic patients on hemodialysis and normal volunteers contained 176.1 and 28.5 μg/ml AGEs, respectively. Clinical characteristics of 11 type 2 diabetic patients are shown in Table 1.TABLE 1Characteristics of diabetic patients on hemodialysisNo. of patients11 (7 male/4 female)Age (years)51.7 ± 8.6Duration of diabetes (years)22.4 ± 9.1Duration of hemodialysis (months)85.8 ± 56.0Hemoglobin A1c (%)7.74 ± 1.04No. of patients with proliferative diabetic retinopathy11 Open table in a new tab Enzyme-linked Immunosorbent Assay (ELISA) for AGEs—Measurement of AGEs was performed with a competitive ELISA as described previously (27.Takeuchi M. Makita Z. Bucala R. Suzuki T. Koike T. Kameda Y. Mol. Med. 2000; 6: 114-125Crossref PubMed Google Scholar). Briefly, 96-well microtiter plates were coated with 0.1 μg/ml AGE-BSA. The test samples (50 μl) were then added to each well as a competitor for 50 μl of polyclonal Abs directed against AGE-BSA (1:1000), followed by incubation for 2 h at room temperature with gentle shaking on a horizontal rotary shaker. After incubating each well with alkaline phosphatase-conjugated anti-rabbit IgG, p-nitrophenyl phosphate was added as a colorimetric substrate. The plate was then read at 405 nm by using a microplate reader. AGE Treatments of Normal Rats Prepared in Vitro—Nine-week-old normoglycemic Sprague-Dawley rats were injected intravenously with 1 mg of AGE-BSA or nonglycated BSA in the presence or absence of 10 μg of PEDF proteins or 10 μgof Abs against rat VEGF every day for up to 10 days. The rats were sacrificed 1–2 h after injection on the final day. This AGE administration increases serum AGE levels by about 2-fold, compared with nonglycated BSA injection (33.4 ± 1.6 versus 18.7 ± 0.5 μg/ml). We have recently found that serum level of AGEs in diabetic rats (Goto-Kakizaki rats at 14 weeks old) was 32.8 ± 7.1 μg/ml. Therefore, the serum AGE concentrations obtained by the AGE injection were comparable with those of diabetic rats. All animal procedures were conducted according to the guidelines provided by the Kurume University Institutional Animal Care and Use Committee under an approved protocol. Leakage of FITC-conjugated Dextran from Retinal Vasculature—Leakage of FITC-conjugated dextran from retinal vasculature was determined by the method of Stitt et al. (18.Stitt A.W. Bhaduri T. McMullen T. Gardiner T.A. Archer D.B. Mol. Cell. Biol. Res. Commun. 2000; 3: 380-388Crossref PubMed Scopus (120) Google Scholar). Briefly, rats were deeply anesthetized, and then FITC-conjugated dextran (40 kDa, Sigma) was injected into the inferior vena cava. After the tracer was allowed to circulate, the eyes were enucleated and immediately fixed in 4% paraformaldehyde (Sigma). The retinas were imaged by a laser-scanning confocal microscope. Quantification of Blood Retinal Barrier (BRB) Breakdown—BRB breakdown quantification was determined by the method of Adamis and co-workers (28.Ishida S. Usui T. Yamashiro K. Kaji Y. Ahmed E. Carrasquillo K.G. Amano S. Hida T. Oguchi Y. Adamis A.P. Investig. Ophthalmol. Vis. Sci. 2003; 44: 2155-2162Crossref PubMed Scopus (345) Google Scholar). Briefly, after deep anesthesia, the rats received intravenous injection with FITC-conjugated dextran (4.4 kDa, Sigma). After 10–15 min, a blood sample was collected, and each rat was then perfused with phosphate-buffered saline. After perfusion, the retinas were carefully removed, weighed, and homogenized to extract the FITC-conjugated dextran. BRB breakdown was calculated by using Equation 1, retinal FITC−dextran (mg)/retinal weight (g)plasma FITC−dextran concentration (mg/ml)×circulation time (h) (Eq. 1) Effects of AGE-rich Serum Fractions on Retinal Vascular Permeability—Nine-week-old Sprague-Dawley rats were injected intravenously with AGE-rich serum fractions derived from normal volunteers or diabetic patients with hemodialysis (DM-AGEs) in the presence or absence of 10 μg of PEDF proteins or 77 μg of polyclonal anti-human PEDF Abs. After injection every day for 5 days, the rats were sacrificed, and retinal permeability and BRB breakdown were analyzed. Cells—Human adult skin microvascular ECs were cultured in endothelial basal medium supplemented with 5% fetal bovine serum, 0.4% bovine brain extracts, 10 ng/ml human epidermal growth factor, and 1 μg/ml hydrocortisone according to the supplier's instructions (Clonetics Corp., San Diego). AGE treatment was carried out in medium lacking epidermal growth factor and hydrocortisone. Cells at 3–5 passages were used for the experiments. Quantitative Real Time Reverse Transcription (RT)-PCR—Poly(A)+ RNAs were isolated from ECs or enucleated eyes as described previously (16.Yamagishi S. Yonekura H. Yamamoto Y. Katsuno K. Sato F. Mita I. Ooka H. Satozawa N. Kawakami T. Nomura M. Yamamoto H. J. Biol. Chem. 1997; 272: 8723-8730Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Quantitative real time RT-PCR was performed using Assay-on-Demand and TaqMan 5 fluorogenic nuclease chemistry (Applied Biosystems, Foster city, CA) according to the manufacturer's recommendations. Identifications of primers for human p22PHOX, human gp91PHOX, human VEGF, rat p22phox, rat gp91phox, and rat VEGF genes were Hs00164370_m1, Hs00166163_m1, Hs00173626_m1, Rn00577357_m1, Rn00576710_m1, and Rn00582935_m1, respectively. Immunohistochemistry—Rat eyes were removed and fixed for 1 day in 4% paraformaldehyde. The eyes were then embedded in paraffin wax for sectioning. Five-μm paraffin sections were incubated with polyclonal Abs raised against human PEDF (4 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) or monoclonal Abs raised against 8-hydroxydeoxyguanosine (8-OHdG) (10 μg/ml; Japan Institute for the Control of Aging, Shizuoka, Japan). After exposure to peroxidase-labeled secondary anti-rabbit Abs, the sections were incubated with 3,3′-diaminobenzidine solution (Nichirei, Tokyo, Japan) and then counterstained with methyl green for visualizing PEDF or 8-OHdG immunoreactivity. The immunoreactivity was measured with microcomputer-assisted NIH Image. Measurement of Transendothelial Electrical Resistance (TER)—Effects of PEDF on barrier function of ECs were assessed by measurement of TER using electric cell substrate impedance sensing (Applied Biophysics) according to the method of Becker et al. (29.Becker P.M. Waltenberger J. Yachechko R. Mirzapoiazova T. Sham J.S.K. Lee C.G. Elias J.A. Verin A.D. Circ. Res. 2005; 96: 1257-1265Crossref PubMed Scopus (108) Google Scholar). Briefly, the cells were seeded onto gold microelectrodes (Applied Biophysics) and grown to confluence for 2 days. Cells were washed three times with serum-free endothelial basal medium and then were connected to the impedance sensing system to measure the TER base line. The applied alternating current (1 μA) was clamped so that impedance (resistance) was directly related to changes in voltage, which was measured with a locked-in amplifier. Data from the electrical resistance experiments (ohms) were obtained over the experimental time course at 5-min intervals. Resistance values for each microelectrode were normalized as the ratio of measured resistance to base-line resistance and plotted as a function of time. NADPH Oxidase Assay—ECs were treated with 100 μg/ml AGE-BSA or nonglycated BSA in the presence or absence of 10 nm PEDF for 24 h, and the cells were then suspended in homogenization buffer (20 mm Hepes, pH 7.0, 100 mm KCl, and 1 mm EDTA containing protease inhibitor mixtures). NADPH oxidase activity of the cell homogenate was measured by luminescence assay in 50 mm phosphate buffer, pH 7.0, containing 1 mm EGTA, 150 mm sucrose, 5 μm lucigenin as the electron acceptor, and 100 μm NADPH as a substrate according to the methods of Griendling et al. (30.Griendling K.K. Minieri C.A. Ollerenshaw J.D. Alexander R.W. Circ. Res. 1994; 74: 1141-1148Crossref PubMed Scopus (2393) Google Scholar). Preparations of Abs Directed against AGE-BSA Prepared in Vitro—Polyclonal Abs directed against in vitro-modified AGE-BSA were prepared as described previously (27.Takeuchi M. Makita Z. Bucala R. Suzuki T. Koike T. Kameda Y. Mol. Med. 2000; 6: 114-125Crossref PubMed Google Scholar). We have shown previously that the Abs did not cross-react with several structurally identified AGE-modified BSAs, including pyrraline-BSA, pentosidine-BSA, argpyrimidine-BSA, 3-deoxyglucosone imidazolone-BSA, carboxymethyllysine-BSA, carboxyethyllysine-BSA, glyoxal-lysine dimer, or methyglyoxal-lysine dimer (13.Yamagishi S. Inagaki Y. Okamoto T. Amano S. Koga K. Takeuchi M. Makita Z. J. Biol. Chem. 2002; 277: 20309-20315Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 27.Takeuchi M. Makita Z. Bucala R. Suzuki T. Koike T. Kameda Y. Mol. Med. 2000; 6: 114-125Crossref PubMed Google Scholar). Preparations of Antiserum Directed against Receptor for AGEs (RAGE)—Antiserum directed against human RAGE for neutralizing assays, which recognizes the amino acid residues 167–180 of human RAGE protein, was prepared as described previously (31.Sasaki N. Takeuchi M. Chowei H. Kikuchi S. Hayashi Y. Nakano N. Ikeda H. Yamagishi S. Kitamoto T. Saito T. Makita Z. Neurosci. Lett. 2002; 326: 117-120Crossref PubMed Scopus (97) Google Scholar). Intracellular ROS Generation—ECs were treated with various concentrations of AGE-BSA or nonglycated BSA in the presence or absence of 10 nm PEDF, 50 nm DPI, 10 μg/ml Abs directed against AGEs, or 0.1% anti-RAGE serum for 24 h. The intracellular formation of ROS was detected by using the fluorescent probe CM-H2DCFDA (Molecular Probes Inc., Eugene, OR) as described previously (15.Yamagishi S. Inagaki Y. Amano S. Okamoto T. Koga K. Takeuchi M. Kidney Int. 2003; 63: 464-473Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Superoxide Generation—ECs were treated with serum AGE fractions in the presence or absence of 10 nm PEDF for 24 h, and the cells were then incubated with phenol red-free Dulbecco's modified Eagle's medium containing 3 μmol/liter dihydroethidium (Molecular Probes Inc., Eugene, OR). After 30 min, fluorescence intensity was measured, and the cells were imaged by a laser-scanning confocal microscope. Assay for Ras Activation—ECs were treated with 100 μg/ml AGE-BSA or nonglycated BSA in the presence or absence of 10 nm PEDF or 1 mm N-acetylcysteine for 24 h. Ras activity then was measured using a Ras activation assay kit (Upstate Biotechnology Inc., Lake Placid, NY) following the manufacturer's instructions. Transfection of Dominant-negative Mutant Vector—ECs were transiently transfected with a dominant-negative human Ras mutant (DN-Ras) vector or an empty vector (mock) as described previously (17.Okamoto T. Yamagishi S. Inagaki Y. Amano S. Koga K. Abe R. Takeuchi M. Ohno S. Yoshimura A. Makita Z. FASEB J. 2002; 16: 1928-1930Crossref PubMed Scopus (259) Google Scholar). Measurement of NF-κB Activity—ECs were transiently transfected with plasmids containing the NF-κB promoter attached upstream to the luciferase reporter gene. Luciferase activity was measured as described previously (17.Okamoto T. Yamagishi S. Inagaki Y. Amano S. Koga K. Abe R. Takeuchi M. Ohno S. Yoshimura A. Makita Z. FASEB J. 2002; 16: 1928-1930Crossref PubMed Scopus (259) Google Scholar). Statistical Analysis—All values were presented as means ± S.E. One-way analysis of variance followed by the Scheffe F test was performed for statistical comparisons. p < 0.05 was considered significant. Anti-vasopermeability Effects of PEDF in Vivo—We used glyceraldehyde-modified AGE-BSA for the present experiments as we have shown previously that this type of AGE could elicit a brisk angiogenic response by inducing autocrine production of VEGF, also known as a vascular permeability factor (17.Okamoto T. Yamagishi S. Inagaki Y. Amano S. Koga K. Abe R. Takeuchi M. Ohno S. Yoshimura A. Makita Z. FASEB J. 2002; 16: 1928-1930Crossref PubMed Scopus (259) Google Scholar, 32.Duh E. Aiello L.P. Diabetes. 1999; 48: 1899-1906Crossref PubMed Scopus (282) Google Scholar, 33.Dvorak H.F. Brown L.F. Detmar M. Dvorak A.M. Am. J. Pathol. 1995; 146: 1029-1039PubMed Google Scholar). We first examined whether intravenous administration of in vitro-prepared AGE-BSA to normal rats increased retinal vascular leakage in vivo. As shown in Fig. 1A, AGE-BSA increased retinal vascular permeability compared with nonglycated BSA. BRB function was also disturbed by the treatment with AGEs. The quantitative pooled data of BRB breakdown are shown in Fig. 1B. Moreover, to evaluate the pathophysiological relevance of the experiments using in vitro-prepared AGEs, we studied whether AGE-rich serum fractions derived from diabetic patients on hemodialysis (DM-AGEs) could elicit the same biological response. As shown in Fig. 1, C and D, DM-AGEs also increased retinal vascular permeability and induced BRB breakdown. Next, we examined the role of VEGF for the AGE-induced vascular hyperpermeability in two different ways, VEGF gene expression and effects of anti-VEGF Abs. VEGF mRNA levels were increased in the eye of AGE-treated rats (Fig. 1E), whereas anti-VEGF Abs completely suppressed the AGE-induced retinal vascular hyperpermeability (Fig. 1A). These observations indicate the central role of VEGF in the AGE-induced retinal vascular leakage. PEDF is a potent anti-angiogenic factor; it inhibits the VEGF-induced proliferation and migration of ECs (21.Dawson D.W. Volpert O.V. Gillis P. Crawford S.E. Xu H.J. Benedict W. Bouck N.P. Science. 1999; 285: 245-248Crossref PubMed Scopus (1401) Google Scholar, 22.Duh E.J. Yang H.S. Suzuma I. Miyagi M. Youngman E. Mori K. Katai M. Yan L. Suzuma K. West K. Davarya S. Tong P. Gehlbach P. Pearlman J. Crabb J.W. Aiello L.P. Campochiaro P.A. Zack D.J. Investig. Ophthalmol. Vis. Sci. 2002; 43: 821-829PubMed Google Scholar). Because VEGF is also known as a vascular permeability factor (32.Duh E. Aiello L.P. Diabetes. 1999; 48: 1899-1906Crossref PubMed Scopus (282) Google Scholar, 33.Dvorak H.F. Brown L.F. Detmar M. Dvorak A.M. Am. J. Pathol. 1995; 146: 1029-1039PubMed Google Scholar), we examined the involvement of PEDF and its therapeutic efficacy in the AGE-induced vascular hyperpermeability. Immunohistochemical analysis revealed that administration of AGE-BSA to normal rats decreased expression levels of PEDF in the retina, compared with that of nonglycated BSA; PEDF immunoreactivity in the ganglion cell layer and in the inner plexiform layer of AGE-injected rats was decreased to about 70% that of nonglycated BSA-treated rats (Fig. 1F). Furthermore, simultaneous treatments with PEDF inhibited the up-regulation of VEGF mRNA levels in the eye of AGE-injected rats (Fig. 1E). In addition, PEDF was found to block both the in vitro-prepared AGE-BSA- and DM-AGE-induced retinal vascular hyperpermeability and BRB breakdown (Fig. 1, A–D), and the effects of PEDF were reversed by the treatments with PEDF Abs (Fig. 1C). Taken together, our data indicate that PEDF could block the AGE-induced retinal hyperpermeability and BRB breakdown by suppressing VEGF expression. Because it has been demonstrated that NADPH oxidase plays an important role in the AGE-elicited ROS generation and subsequent gene expression in cultured ECs (34.Wautier M.P. Chappey O. Corda S. Stern D.M. Schmidt A.M. Wautier J.L. Am. J. Physiol. 2001; 280: E685-E694Crossref PubMed Google Scholar), we further examined whether intravenous administration of PEDF suppressed the up-regulation of mRNA levels for p22phox and gp91phox, key components of NADPH oxidase with respect to its enzymatic activity (35.Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Crossref PubMed Scopus (2612) Google Scholar), in the eye of AGE-treated rats. AGEs up-regulated mRNA levels of these membrane components of NADPH oxidase in the eye, which was suppressed by PEDF treatments (Fig. 1G). Moreover, immunohistochemistry of 8-OHdG, a sensitive indicator of oxidative damage to DNA, showed intense staining in the nuclei of cells in the inner and outer plexiform layers of AGE-treated retina, which was also blocked by simultaneous PEDF treatments (Fig. 1H). These observations suggest that PEDF could inhibit the AGE-induced retinal vascular hyperpermeability by suppressing VEGF induction via inhibition of NADPH oxidase expression and ROS generation. In addition, the present findings suggest that AGE infusion could down-regulate retinal PEDF levels, at least in part, via oxidative stress generation since we have recently found that AGE-BSA or H2O2 suppresses PEDF gene expression in microvascular ECs and that anti-oxidant N-acetylcys
The gangliosides in the brain of a cartilaginous fish, skate (Bathyraja smirnovi), have been isolated and characterized by means of methylation analysis, antibody binding, enzymatic hydrolysis and MALDI-TOF MS. In addition to gangliosides with known structures (GM2, fucosyl-GM1, GD3, GD2, GT3 and GT2), five polysialogangliosides were isolated and characterized as having the following structures. (1) IV 3 NeuAc, III 6 NeuAc, II 3 NeuAc-Gg4Cer; (2) IV 3 NeuAc2, III 6 NeuAc, II 3 NeuAc-Gg4Cer; (3) IV 3 NeuAc, III 6 NeuAc, II 3 NeuAc2-Gg4Cer; (4) IV 3 NeuAc, III 6 NeuAc, II 3 NeuAc3-Gg4Cer; and (5) IV 3 NeuAc2, III 6 NeuAc, II 3 NeuAc3Gg4Cer. These structures are ‘hybrid-type’ which comprise combinations of a-series and either a, b or c-series structures. Three gangliosides (2), (4) and (5), were novel. The main features of the ganglioside composition of skate brain were an abundance of gangliotriaosyl species, a lack of gangliotetraosyl species (except fucosylGM1), and an abundance of hybrid-types. These characteristics closely resemble those in shark brain which we reported previously [Nakamura, K., Tamai, Y. & Kasama, T. (1997) Neurochem. Int. 30, 593‐604]. Two of the hybrid-type gangliosides (1) and (4), were examined for their neuritogenic activity toward cultured neuronal cells (Neuro-2A), and were found to have more potent activity than nonhybrid-type gangliosides such as GM1.
