Glyoxalase I Is Critical for Human Retinal Capillary Pericyte Survival under Hyperglycemic Conditions
2006; Elsevier BV; Volume: 281; Issue: 17 Linguagem: Inglês
10.1074/jbc.m513813200
ISSN1083-351X
AutoresAntonia G. Miller, Dawn Smith, Manjunatha B. Bhat, Ram H. Nagaraj,
Tópico(s)Alcohol Consumption and Health Effects
ResumoRetinal capillary pericytes undergo premature death, possibly by apoptosis, during the early stages of diabetic retinopathy. The α-oxoaldehyde, methylglyoxal (MGO), has been implicated as a cause of cell damage in diabetes. We have investigated the role of MGO and its metabolizing enzyme, glyoxalase I, in high glucose-induced apoptosis (annexin V binding) of human retinal pericyte (HRP). HRP incubated with high glucose (30 mm d-glucose) for 7 days did not undergo apoptosis despite accumulation of MGO. However, treatment with a combination of high glucose and S-p-bromobenzylglutathione cyclopentyl diester, a competitive inhibitor of glyoxalase I, resulted in apoptosis along with a dramatic increase in MGO. Overexpression of glyoxalase I in HRP protected against S-p-bromobenzylglutathione cyclopentyl diester-induced apoptosis under high glucose conditions. Incubation of HRP with high concentrations of MGO resulted in an increase of apoptosis relative to untreated controls. We found an elevation of nitric oxide (NO·) in HRP that was incubated with high glucose when compared with those incubated with either the l-glucose or untreated controls. When HRP were incubated with an NO· donor, DETANONOATE ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), we observed both decreased glyoxalase I expression and activity relative to untreated control cells. Further studies showed that HRP underwent apoptosis when incubated with DETANONOATE and that apoptosis increased further on co-incubation with high glucose. Our findings indicate that glyoxalase I is critical for pericyte survival under hyperglycemic conditions, and its inactivation and/or down-regulation by NO· may contribute to pericyte death by apoptosis during the early stages of diabetic retinopathy. Retinal capillary pericytes undergo premature death, possibly by apoptosis, during the early stages of diabetic retinopathy. The α-oxoaldehyde, methylglyoxal (MGO), has been implicated as a cause of cell damage in diabetes. We have investigated the role of MGO and its metabolizing enzyme, glyoxalase I, in high glucose-induced apoptosis (annexin V binding) of human retinal pericyte (HRP). HRP incubated with high glucose (30 mm d-glucose) for 7 days did not undergo apoptosis despite accumulation of MGO. However, treatment with a combination of high glucose and S-p-bromobenzylglutathione cyclopentyl diester, a competitive inhibitor of glyoxalase I, resulted in apoptosis along with a dramatic increase in MGO. Overexpression of glyoxalase I in HRP protected against S-p-bromobenzylglutathione cyclopentyl diester-induced apoptosis under high glucose conditions. Incubation of HRP with high concentrations of MGO resulted in an increase of apoptosis relative to untreated controls. We found an elevation of nitric oxide (NO·) in HRP that was incubated with high glucose when compared with those incubated with either the l-glucose or untreated controls. When HRP were incubated with an NO· donor, DETANONOATE ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), we observed both decreased glyoxalase I expression and activity relative to untreated control cells. Further studies showed that HRP underwent apoptosis when incubated with DETANONOATE and that apoptosis increased further on co-incubation with high glucose. Our findings indicate that glyoxalase I is critical for pericyte survival under hyperglycemic conditions, and its inactivation and/or down-regulation by NO· may contribute to pericyte death by apoptosis during the early stages of diabetic retinopathy. Diabetic retinopathy is one of the leading causes of blindness in the working age population (1Stitt A.W. Exp. Mol. Pathol. 2003; 75: 95-108Crossref PubMed Scopus (185) Google Scholar). Loss of pericytes from the retinal microvas-culature is considered one of the earliest hallmarks of diabetic retinopathy (2Addison D.J. Garner A. Ashton N. Br. Med. J. 1970; 1: 264-266Crossref PubMed Scopus (77) Google Scholar, 3Engerman R.L. Diabetes. 1989; 38: 1203-1206Crossref PubMed Google Scholar), and it is thought to occur via an apoptotic pathway (4Li W. Yanoff M. Jian B. He Z. Cell. Mol. Biol. (Noisy-le-grand). 1999; 45: 59-66PubMed Google Scholar, 5Podesta F. Romeo G. Liu W.H. Krajewski S. Reed J.C. Gerhardinger C. Lorenzi M. Am. J. Pathol. 2000; 156: 1025-1032Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Once pericytes are eliminated from retinal capillaries, endothelial cell death follows with the subsequent formation of acellular capillaries (6Hammes H.P. Horm. Metab. Res. 2005; 37: 39-43Crossref PubMed Scopus (188) Google Scholar). In the proliferative form of the disease, areas containing acellular capillaries become ischemic and subsequently non-perfused. Non-perfused regions of the retina suffer hypoxia, which causes the expression of a number of angiogenic factors including vascular endothelial growth factor (7Aiello L.P. Pierce E.A. Foley E.D. Takagi H. Chen H. Riddle L. Ferrara N. King G.L. Smith L.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10457-10461Crossref PubMed Scopus (1172) Google Scholar, 8Caldwell R.B. Bartoli M. Behzadian M.A. El-Remessy A.E. Al-Shabrawey M. Platt D.H. Caldwell R.W. Diabetes Metab. Res. Rev. 2003; 19: 442-455Crossref PubMed Scopus (241) Google Scholar). New blood vessels that grow toward the vitreous are fragile and prone to rupture, resulting in retinal detachment. In both diabetic humans and animal models of diabetes, retinal capillary pericytes and endothelial cells die, possibly by apoptosis, as indicated by observations of terminal dUTP nick-end labeling-positive cells in capillaries (9Mizutani M. Kern T.S. Lorenzi M. J. Clin. Investig. 1996; 97: 2883-2890Crossref PubMed Scopus (596) Google Scholar) and activation of key apoptotic proteins and caspases in the retina (10Mohr S. Xi X. Tang J. Kern T.S. Diabetes. 2002; 51: 1172-1179Crossref PubMed Scopus (172) Google Scholar). One recent study suggested that mitochondrial dysfunction causes apoptosis of both endothelial cells and pericytes in retinas of diabetics (11Kowluru R.A. Abbas S.N. Investig. Ophthalmol. Vis. Sci. 2003; 44: 5327-5334Crossref PubMed Scopus (234) Google Scholar). Other studies documented activation of pro-apoptotic caspases and other apoptotic pathway proteins in the diabetic retina (12Kusner L.L. Sarthy V.P. Mohr S. Investig. Ophthalmol. Vis. Sci. 2004; 45: 1553-1561PubMed Google Scholar, 13Kowluru R.A. Koppolu P. Free Radic. Res. 2002; 36: 993-999Crossref PubMed Scopus (129) Google Scholar). Some investigators proposed that the frequency of early apoptosis in retinal pericytes was the major determinant for development of diabetic retinopathy (14Kern T.S. Tang J. Mizutani M. Kowluru R.A. Nagaraj R.H. Romeo G. Podesta F. Lorenzi M. Investig. Ophthalmol. Vis. Sci. 2000; 41: 3972-3978PubMed Google Scholar). Exactly how pericytes undergo apoptosis is still uncertain, although evidence suggests various biochemical mechanisms (5Podesta F. Romeo G. Liu W.H. Krajewski S. Reed J.C. Gerhardinger C. Lorenzi M. Am. J. Pathol. 2000; 156: 1025-1032Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 13Kowluru R.A. Koppolu P. Free Radic. Res. 2002; 36: 993-999Crossref PubMed Scopus (129) Google Scholar, 15Yamagishi S. Okamoto T. Amano S. Inagaki Y. Koga K. Koga M. Choei H. Sasaki N. Kikuchi S. Takeuchi M. Makita Z. Mol. Med. 2002; 8: 179-184Crossref PubMed Google Scholar, 16Romeo G. Liu W.-H. Asnaghi V. Kern T.S. Lorenzi M. Diabetes. 2002; 51: 2241-2248Crossref PubMed Scopus (323) Google Scholar), including triggering of a pro-apoptotic program by activation of NF-κB in response to hyperglycemia (16Romeo G. Liu W.-H. Asnaghi V. Kern T.S. Lorenzi M. Diabetes. 2002; 51: 2241-2248Crossref PubMed Scopus (323) Google Scholar). Cultured pericytes undergo apoptosis in the presence of high concentrations of glucose (11Kowluru R.A. Abbas S.N. Investig. Ophthalmol. Vis. Sci. 2003; 44: 5327-5334Crossref PubMed Scopus (234) Google Scholar), implying that glucose-driven processes cause or enhance their death. MGO 2The abbreviations used are: MGO, methylglyoxal; glu, glucose; AGE, advanced glycation end product; BBGC, bromobenzylglutathione cyclopentyl diester; DAPI, 4′,6-diamidino-2-phenylindole; DETANONOATE, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; HRP, human retinal pericytes; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. is an α-oxoaldehyde that is highly reactive with lysine or arginine residues in proteins, modifying them at their amine moieties to form AGEs (17Nagaraj R.H. Shipanova I.N. Faust F.M. J. Biol. Chem. 1996; 271: 19338-19345Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 18Shamsi F.A. Partal A. Sady C. Glomb M.A. Nagaraj R.H. J. Biol. Chem. 1998; 273: 6928-6936Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Elevated levels of both MGO and AGEs have been observed in serum and various tissues of diabetic patients, respectively (19Beisswenger P.J. Howell S.K. Touchette A.D. Lal S. Szwergold B.S. Diabetes. 1999; 48: 198-202Crossref PubMed Scopus (351) Google Scholar, 20Monnier V.M. Sell D.R. Nagaraj R.H. Miyata S. Grandhee S. Odetti P. Ibrahim S.A. Diabetes. 1992; 41: 36-41Crossref PubMed Google Scholar), and both AGEs and MGO cause pericyte apoptosis (21Denis U. Lecomte M. Paget C. Ruggiero D. Wiernsperger N. Lagarde M. Free Radic. Biol. Med. 2002; 33: 236-247Crossref PubMed Scopus (120) Google Scholar, 22Kim J. Son J.W. Lee J.A. Oh Y.S. Shinn S.H. J. Korean Med. Sci. 2004; 19: 95-100Crossref PubMed Scopus (53) Google Scholar). Apoptosis prompted by this mechanism is probably due to enhanced oxidative stress within the cells. In fact, antioxidants inhibit apoptosis caused by MGO and AGEs (22Kim J. Son J.W. Lee J.A. Oh Y.S. Shinn S.H. J. Korean Med. Sci. 2004; 19: 95-100Crossref PubMed Scopus (53) Google Scholar, 23Kowluru R.A. Life Sci. 2005; 76: 1051-1060Crossref PubMed Scopus (72) Google Scholar). We showed that extracellular matrix proteins modified by dicarbonyls induce pericytes apoptosis as well (24Liu B. Bhat M. Padival A.K. Smith D.G. Nagaraj R.H. Investig. Ophthalmol. Vis. Sci. 2004; 45: 1983-1995Crossref PubMed Scopus (41) Google Scholar). In addition, activation of the polyol pathway is implicated in pericyte apoptosis (25Murata T. Ishibashi T. Khalil A. Hata Y. Yoshikawa H. Inomata H. Ophthalmic Res. 1995; 27: 48-52Crossref PubMed Scopus (204) Google Scholar, 26Dagher Z. Park Y.S. Asnaghi V. Hoehn T. Gerhardinger C. Lorenzi M. Diabetes. 2004; 53: 2404-2411Crossref PubMed Scopus (171) Google Scholar). Retinal capillary cells produce insulin-like growth factor-1, and a recent study related loss of retinal pericytes to excessive levels of insulin-like growth factor-1 (27Ruberte J. Ayuso E. Navarro M. Carretero A. Nacher V. Haurigot V. George M. Llombart C. Casellas A. Costa C. Bosch A. Bosch F. J. Clin. Investig. 2004; 113: 1149-1157Crossref PubMed Scopus (159) Google Scholar). Until now, studies were concerned with damage of pericytes by high glucose, but there was little emphasis on how high glucose stress influences enzymes within these cells. Glyoxalase I is an integral part of the cellular machinery for removal of MGO. The glyoxalase system is composed of two enzymes; glyoxalase I, which metabolizes MGO to S-d-lactoylglutathione, and glyoxalase II, which converts S-d-lactoylglutathione to d-lactate. Work by Shinohara et al. (28Shinohara M. Thornalley P.J. Giardino I. Beisswenger P. Thorpe S.R. Onorato J. Brownlee M. J. Clin. Investig. 1998; 101: 1142-1147Crossref PubMed Scopus (433) Google Scholar) demonstrated that overexpression of glyoxalase I in bovine endothelial cells reduced intracellular AGEs when the cells were cultured in the presence of high glucose. In addition, a series of studies indicated that certain tumor primary cultures and cell lines overexpress glyoxalase I (29Davidson S.D. Milanesa D.M. Mallouh C. Choudhury M.S. Tazaki H. Konno S. Urol. Res. 2002; 30: 116-121Crossref PubMed Scopus (30) Google Scholar, 30Rulli A. Carli L. Romani R. Baroni T. Giovannini E. Rossi G. Talesa V. Breast Cancer Res. Treat. 2001; 66: 67-72Crossref PubMed Scopus (119) Google Scholar), suggesting that increased amounts of this enzyme prevent tumor cell apoptosis (31Sakamoto H. Mashima T. Kizaki A. Dan S. Hashimoto Y. Naito M. Tsuruo T. Blood. 2000; 95: 3214-3218Crossref PubMed Google Scholar), possibly by limiting MGO production. These findings spurred us to determine whether impaired function of glyoxalase I could result in HRP apoptosis in diabetes. Materials—Dulbecco's modification of Eagle's medium (with 5 mmol/liter d-glucose), 0.25% trypsin, 0.1% EDTA, and Hanks' balanced salt solution were purchased from Mediatech (Herndon, VA). Fetal bovine serum and antibiotic/mycotic solution were purchased from Invitrogen. Endothelial growth supplement, insulin-transferrin-sodium selenite (ITS), porcine esterase, nitrate reductase, and MGO (40% solution) were purchased from Sigma. MGO was purified by distilling twice under low pressure and temperature. The TACS-annexin apoptosis detection kit was purchased from R&D Systems Inc. (Minneapolis, MN). Annexin-647 was obtained from Invitrogen. Bromobenzylglutathione cyclopentyl diester (BBGC) was synthesized as previously described (32Thornalley P.J. Edwards L.G. Kang Y. Wyatt C. Davies N. Ladan M.J. Double J. Biochem. Pharmacol. 1996; 51: 1365-1372Crossref PubMed Scopus (148) Google Scholar). The NO· donor DETANONOATE was purchased from Cayman Chemical Co. (Ann Arbor, MI). Isolation, Culture, and Characterization of HRP—HRP were isolated by the method of Grant and Guay (33Grant M. Guay C. Investig. Ophthalmol. Vis. Sci. 1991; 32: 53-64PubMed Google Scholar), with minor modifications. Two sets of eyes from two non-diabetic donors (aged 41 and 72) were received 23 h after death from the Cleveland Eye Bank. Retina were detached from the pigmented layer, placed on 55-μm nylon mesh, and macerated while flooding with 2% bovine serum albumin in Hanks' balanced salt solution. The vasculature was transferred to a flask containing 5 mg/ml collagenase (Type I, Worthington Biochemical, Lakewood, NJ) and stirred for 30-45 min at 37 °C. Collagenase was inactivated by the addition of growth media (1:1 Dulbecco's modified Eagle's medium/Ham's F-12 containing 10% fetal bovine serum, 15 μg/ml endothelial cell growth supplement, 1× ITS, antibiotic/antimycotic (10 units of penicillin, 10 μg of streptomycin, 25 μg of amphotericin)), and the homogenate was centrifuged at 1300 rpm for 5 min. The pellet was washed once with 5 ml of growth medium and centrifuged again. The final pellet was re-suspended in 10 ml of growth medium and seeded onto 2 × 25 cm2 flasks pre-coated with gelatin. The cultures were maintained at 37 °C in 5% CO2 with a change of medium every 2-4 days until the cells attained near confluence. Cells were detached by washing with PBS then covered with 0.25% trypsin, 0.1% EDTA for 60-90 s. Fresh medium was added, and the cells were centrifuged at 1300 rpm for 5 min. The pellet was resuspended in growth media, and cells were plated in 10-cm2 dishes. Positive staining for both smooth muscle actin a subunit (Dako, Carpinteria, CA) and NG-2 (Chemicon, Temecula, CA) identified the cultured cells as pericytes (Fig. 1). Once they were successfully characterized as HRP, the cells were maintained in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 IU penicillin, and 100 μg/ml streptomycin. They were used between passages 3-7 for all experiments detailed below. Cell Experiments—HRP were incubated at 37 °C in an atmosphere of 5% CO2 in either 30 mm d-or 25 mm l-glucose (d-glu or l-glu) until the cultures reached 80% confluence (typically 7 days). The medium was replaced every 2 days. For apoptosis experiments with BBGC, 80% confluent HRP were treated with BBGC and incubated for a further 24 h. BBGC dissolved in Me2SO was added to achieve final concentrations of 25, 50, 100, and 200 μm. The final concentration of Me2SO in the media was estimated to be <5 mm. In later experiments cells were incubated for 6 days with either 30 mm d- or 25 mm l-glu. The inclusion of 5 mm d-glu in our normal medium brought the final glucose concentration to 30 mm in these experiments. Along with the addition of glucose, the cells were also treated with either 35 μm BBGC or Me2SO alone (3.9 μl/10 ml media) and incubated 24 h longer. For apoptosis and quantitative PCR experiments, 80% confluent HRP were incubated for 24 h with the NO· donor, DETANONOATE, at final concentrations of 100, 250, 500 μm, 1 mm, and 1.2 mm. In another experiment cells were incubated with either 30 mm d- or 25 mm l-glu for 6 days and then treated with 1 mm DETANONOATE for the final 24 h. For measurement of apoptosis, the media serum concentration was reduced to 2% 24 h before further treatment. MGO or glyoxal (175 μm) in 2% serum-containing media was then added to 80% confluent cultures. After 24 h the cells were supplied with fresh media containing either MGO or glyoxal and 35 μm BBGC in Me2SO or Me2SO alone. The cultures were then incubated for 24 h longer. Measurement of Glyoxalase I Activity—Cells were detached with trypsin-EDTA for 5 min, washed with PBS, and harvested by centrifugation at 13,000 rpm for 5 min at 4 °C. The pellet was suspended in 10 mm Tris-HCl, pH 7.4, containing 1:100 diluted protease inhibitor mixture (Sigma), subjected to 3 freeze/thaw cycles in liquid nitrogen, and sonicated for 20 s on ice. The lysate was centrifuged at 20,000 × g for 20 min at 4 °C and extensively dialyzed against PBS overnight at 4 °C, and the supernatant was tested for glyoxalase I activity as previously described (28Shinohara M. Thornalley P.J. Giardino I. Beisswenger P. Thorpe S.R. Onorato J. Brownlee M. J. Clin. Investig. 1998; 101: 1142-1147Crossref PubMed Scopus (433) Google Scholar). One unit is defined as the amount of enzyme required to produce 1 mmol of S-d-lactoylglutathione/min/mg of protein. Protein concentration of lysates was measured using the Bio-Rad protein assay solution (Bio-Rad). For BBGC de-esterification, BBGC dissolved in 10% Me2SO was added to 0.5 units of esterase in 50 mm Tris-HCl, pH 8.0, and incubated for 2 h at room temperature. Controls contained esterase or BBGC alone in either 10% Me2SO or 50 mm Tris-HCl. After incubation for 2 h at room temperature, 50 μg of lysate was added, and the mixtures were incubated 1 h longer. Glyoxalase I activity was measured as described above. Flow Cytometry—After exposure of cells to BBGC and DETANONOATE, the incubation media along with any detached HRP was removed and retained. The adherent cells were detached with trypsin, mixed with the retained media, and centrifuged at 1000 rpm for 5 min. The cell pellet was washed with Hanks' balanced salt solution and then incubated with fluoroisothiocyanate-conjugated annexin V and propidium iodide according to the manufacturer's instructions. Apoptosis was quantified using a Beckman Coulter XL flow cytometer (Fullerton, CA) equipped with an argon-ion laser for excitation at 488 nm. Fifteen thousand cells were analyzed per sample. HRP treated with 1 μm staurosporine (Kamiya Biomedical Co., Seattle, WA) for 1.5 h were positive controls for apoptosis. We used the same procedure for transfected cells (see below), except annexin V-647 was substituted for annexin V-fluoroisothiocyanate. The samples were analyzed with a BD Biosciences LSR I flow cytometer (San Jose, CA) using 2 laser excitations; a 488-nm argon laser for propidium iodide and a 633 nm helium/neon laser for annexin V-647 fluorescence. At least 20,000 events were collected for transfection experiments. Quantification of MGO in HRP—HRP cultures were washed with PBS, the cells were detached with trypsin, pooled with the media in which they had been incubated, and collected by centrifugation at 13,000 rpm for 5 min. The cell pellets were resuspended in 10% trichloroacetic acid and centrifuged at 13,000 rpm for 5 min at 4 °C. The resulting supernatant was derivatized with 7 mm 6-hydroxy-2,4,5-triaminopyrimidine at 60 °C for 45 min. The subsequent pterin adduct (6-methylpterin) was measured by high pressure liquid chromatography as previously described (34Padayatti P.S. Jiang C. Glomb M.A. Uchida K. Nagaraj R.H. Curr. Eye Res. 2001; 23: 106-115Crossref PubMed Scopus (40) Google Scholar). Glyoxalase I Expression after Treatment with DETANONOATE—Cells were treated with various concentrations of DETANONOATE as described for the apoptosis experiments. Total RNA was extracted using Trizol reagent (Invitrogen), and 0.5 μg of RNA was subjected to reverse transcription-PCR using the SuperScript First Strand Synthesis system for reverse transcription-PCR (Invitrogen), according to the manufacturer's instructions. The resulting cDNA was diluted 10× in distilled H2O. Ten μl of cDNA was added to the appropriate primers (final concentration of primers was 1 μm). The human glyoxalase I forward primer was 5-CCGCCATGATTCACATTTGA-3, and the reverse primer was 5-GTTGGCATGGCCTTTCCA-3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used to normalize expression. Human GAPDH M33197 forward primer (5-ACCCACTCCTCCACCTTTGA-3) and reverse primer (5-CTGTTGCTGTAGCCAAATTCGT-3) were added to cDNA in parallel. Twelve μl of SYBR Green Master Mix (Applied Biosystems, Foster City, CA) was added to these mixtures, and quantitative PCR was done with an ABI PRISM 7000 sequence detection system (Applied Biosystems). The relative -fold difference in expression was calculated using the 2-ΔΔCT method (35Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (127074) Google Scholar). Transient Transfection of HRP with Vector Harboring Glyoxalase I—The human glyoxalase I sequence (accession number NM_006708) was PCR-amplified from a pUC 19 vector harboring the gene (gift from Dr. Sulabha Ranganathan, Fox Chase Cancer Center, Philadelphia, PA). We introduced restriction sites NheI and KpnI and subcloned the sequence into pCMS-EGFP (Clontech, Mountain View, CA) to form pCMS-EGFP-glyI. Next, we used 10 μg of either pCMS-EGFP or pCMS-EGFP-glyI to transfect 8 × 105 HRP in 100 μl of Basic Nucleofector™ kit for Primary Smooth Muscle Cell solution (Amaxa, Gaithersburg, MD). Mock-transfected cells were prepared by substituting sterile distilled H2O for DNA. The remainder of the transfection procedure was done according to the manufacturer's instructions. Electroporation was carried out with a Nucleofector II apparatus (Amaxa) using the P-13 program. After electroporation, each sample containing 8 × 105 cells was incubated in 500 μl of RPMI (Cambrex, Pittsburgh, PA) for 15 min and then diluted 1:7 in HRP media in a 60-mm dish. Transfected cells were incubated between 48-72 h with a change of medium every day. The percentage of cells transfected was estimated by counting green fluorescent protein-expressing cells through a Nikon Eclipse TS100 light microscope (Melville, NY) fitted with an epifluorescence attachment and camera (Digital Sight DS-L1, Nikon). For apoptosis experiments immediately after transfection, the cells were incubated for 48 h in media supplemented with either 30 mm d-glu or 25 mm l-glu media alone. Controls were incubated in media without additions. Then all samples were treated for 24 h with either 10 μm BBGC or 50% Me2SO in the presence of the appropriate glucose isomer or H2O. Measurement of NO· Concentration in HRP—Cell lysates were prepared as described for the glyoxalase I assay, except that the culture media from the cells was pooled with the trypsinized cells. After sonication and centrifugation, the samples were passed through 10-kDa cutoff filters (Millipore, Billerica, MA). Samples (125 μl) were treated with nitrate reductase to reduce nitrates to nitrite (36Hevel J.M. Marletta M.A. Methods Enzymol. 1994; 233: 250-258Crossref PubMed Scopus (424) Google Scholar). Twenty μl of Griess reagent (Griess reagent kit for nitrite determination, Invitrogen) was then added, and absorbance of the resulting azo compound was measured at 570 nm in a microplate reader (Dynex Technologies, Chantilly, VA). The concentration was determined by comparison with a standard curve constructed from known standards of sodium nitrite. Statistical Analyses—All experiments were performed independently at least twice, with triplicate samples for each intervention within the experiment. Data were assessed for normal distribution and then compared by either Student's t test (parametric) or a Mann-Whitney U test (non-parametric). A p value of less than 0.05 was considered statistically significant. Pericytes isolated from human retina by the procedure described were 90-95% positive for the purported pericyte markers, smooth muscle α-actin and NG-2 proteoglycan (Fig. 1). This procedure was used previously to isolate human retinal endothelial cells (33Grant M. Guay C. Investig. Ophthalmol. Vis. Sci. 1991; 32: 53-64PubMed Google Scholar). The absence of endothelial growth factors in the culture medium allowed us to select for pericytes over endothelial cells. Our first experiments sought to determine whether incubation in a high glucose environment increased glyoxalase I activity in HRP. Glyoxalase I activity was assessed by measuring the product, S-d-lactoylglutathione. Control cell lysates had 1.64 units of glyoxalase I activity (Fig. 2A). The enzyme activities in cells incubated with either d-glu or l-glu (final concentration = 30 mm) were not significantly different from controls (Fig. 2A). To determine whether glyoxalase I protects HRP from apoptosis, we used BBGC, a competitive inhibitor of glyoxalase I. We first needed to confirm that BBGC inhibits the HRP glyoxalase I under our experimental conditions. We showed that incubation of HRP with BBGC results in a concentration-dependent increase in the un-metabolized substrate, MGO. Fig. 2B shows that MGO increases ∼4-fold compared with untreated controls at the highest concentration (200 μm) of BBGC. These data confirm inhibition of HRP glyoxalase I activity by BBGC, as determined by increased intracellular MGO. We confirmed that BBGC inhibited glyoxalase I in a second experiment. HRP cell lysates were incubated with de-esterified BBGC, the form required for its inhibitory capacity (37Creighton D.J. Zheng Z.-B. Holewinski R. Hamilton D.S. Eiseman J.L. Biochem. Soc. Trans. 2003; 31: 1378-1382Crossref PubMed Google Scholar). Fig. 2C shows that glyoxalase I activity in cell lysates incubated with de-esterified BBGC decreased ∼45% compared with lysates treated with esterase or BBGC alone. Because we found an increase in MGO with increasing concentrations of BBGC, we wanted to determine whether elevation of MGO caused pericyte apoptosis. We defined early apoptosis by quantifying cells that bind annexin V-fluoroisothiocyanate. In the process of early apoptosis, phosphatidylserine is exposed to the outer leaflet of the plasma membrane, which then binds annexin V-fluoroisothiocyanate in the presence of Ca2+. Late apoptosis is defined by the binding of propidium iodide; this reagent intercalates with DNA only after permeabilization of the cell membrane. Treatment of HRP with BBGC results in a concentration-dependent increase in both early and late apoptosis (up to 50 μm, Fig. 3). Fifty μm BBGC appears to be the limit for early apoptosis (22%), with little increase at either 100 or 200 μm. However, 100 μm BBGC increased the late apoptotic population of HRP as much as 45% relative to the untreated control. Our results, thus, link inhibition of HRP glyoxalase I to pericyte apoptosis. To our knowledge this is the first report of such a correlation. We wanted to determine whether glyoxalase I contributes to pericyte dropout during diabetic retinopathy. Accordingly, we investigated whether a high glucose environment increased apoptosis in cultures of retinal pericytes and whether the addition of BBGC exacerbates this effect. A high glucose environment can elevate MGO in a number of tissues (38Thornalley P.J. Mol. Aspects Med. 1993; 14: 287-371Crossref PubMed Scopus (465) Google Scholar), apparently by increasing the metabolic flux of glucose through the glycolytic pathway (38Thornalley P.J. Mol. Aspects Med. 1993; 14: 287-371Crossref PubMed Scopus (465) Google Scholar) (which is the major route) and also through glucose autoxidation, degradation of Amadori products, acetone and threonine metabolism (a minor route). Experiments with HRP cultured for as long as 7 days in a high concentration of glucose (30 mm) failed to show statistically significant increases in early apoptosis relative to either the l-glu control or the untreated control (Fig. 4). We noted a statistically significant increase in late apoptosis compared with the l-glu control, but not the untreated control. A dramatic increase in both early and late apoptosis occurred when cells cultured in media containing 30 mm d-glu were also exposed to BBGC. Both early and late apoptosis increased in both the osmotic control co-incubated with BBGC (l-glu plus BBGC) and cells treated with BBGC alone, although not to the same degree as the d-glu plus BBGC samples (Fig. 4). These findings indicate that as long as glyoxalase I remains functional, HRP remain viable, even in a high glucose environment. However, once this enzyme is inhibited, apoptosis ensues. To address whether apoptosis was due to an elevation of intracellular MGO due to glyoxalase I blockade and additional synthesis of MGO as a result of the high glucose environment, we measured MGO levels in BBGC and d-glu-treated cells. After 7 days of culture in media containing 30 mm d-glu, the intracellular MGO of HRP increased relative to untreated controls (to 89 pmol of MGO/mg of protein). There was a slight but significant increase in MGO concentration in BBGC-treated cells (
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