Involvement of oxidative stress in atherosclerosis development in subjects with sarcopenic obesity.

INTRODUCTION: Co­existing decreased muscle mass and increased visceral fat, an age­associated change called sarcopenic obesity, results in fragility and cardiovascular disease. To assess the pathogenesis of sarcopenic obesity, we assessed the associations of clinical parameters with psoas muscle mass in elderly male subjects with obesity and type 2 diabetes. METHODS: The subjects were 55 patients, over 65 years of age and with a visceral fat area exceeding 100 cm2, with type 2 diabetes. The cross­sectional area of the psoas muscle is considered to provide an estimation of overall muscle mass. Sarcopenia was considered to be present when the total psoas muscle area was low, defined as a value below 500 mm2 m−2 on a computed tomographic scan. RESULTS: The maximum intima­media thickness (max IMT) and urinary 8­isoprostane values were significantly higher in the sarcopenic group. Multiple linear regression analysis revealed max IMT to be an independent variable related to muscle mass decline. In addition, logistic analysis showed max IMT and urinary 8­isoprostane to be variables independently contributing to total psoas muscle area <500 mm2 m−2. CONCLUSION: Worsening surrogate markers for systemic oxidative stress and atherosclerosis were associated with declining muscle mass in elderly subjects with obesity and type 2 diabetes. These results indicate that systemic oxidative stress is among the mechanisms underlying atherosclerosis development in subjects with sarcopenic obesity.

Positive association of free triiodothyronine with pancreatic ß-cell function in people with prediabetes.

AIM: To analyse the effects of thyroid hormones on ß-cell function and glucose metabolism in people with prediabetes who are euthyroid. METHODS: A total of 111 people who were euthyroid underwent 75-g oral glucose tolerance tests, of whom 52 were assigned to the normal glucose tolerance and 59 to the prediabetes groups. Homeostatic model assessment of ß-cell function, insulinogenic index and areas under the curve for insulin and glucose were evaluated as indices of pancreatic ß-cell function. RESULTS: In both groups, BMI, fasting insulin, homeostasis model assessment ratio and HDL cholesterol correlated significantly with all indices of pancreatic ß-cell function. Free triiodothyronine correlated positively with all insulin secretion indices in the prediabetes group. Multiple linear regression analysis showed that free triiodothyronine was an independent variable that had a positive correlation with all indices of ß-cell function in the prediabetes group. By contrast, no such correlation was found in the normal glucose tolerance group. CONCLUSIONS: Free triiodothyronine is associated with both basal and glucose-stimulated insulin secretion in people with prediabetes who are euthyroid; therefore, the regulation of insulin secretion by thyroid hormones is a potentially novel therapeutic target for the treatment of diabetes.

Tomographic fundus features in pseudoxanthoma elasticum: comparison with neovascular age-related macular degeneration in Japanese patients.

PURPOSE: To determine the retinal and subretinal features characteristic to pseudoxanthoma elasticum (PXE) compared with age-related macular degeneration by using spectral-domain optical coherence tomography (SD-OCT) in Japanese patients. METHODS: We reviewed colour fundus photographs, fluorescein angiograms, and SD-OCT images of 52 eyes (27 Japanese patients) with angioid streaks (AS) due to PXE. Then we compared the incidence of tomographic features between 24 eyes (24 patient) with choroidal neovascularization (CNV) secondary to AS and 44 eyes (44 patients) with CNV secondary to age-related macular degeneration (AMD). RESULTS: Secondary CNV was found in 44 eyes (84.6%) of 52 patients with PXE during follow-up. We found characteristic round or ovoid tubular structures with highly reflective annular lines (termed 'outer retinal tubulation' (ORT)) in 31 (70.5%) of 44 eyes with CNV, but none were found in eyes without CNV. We also found characteristic undulations of Bruch's membrane in 38 (73.1%) eyes with AS. The incidence of ORT was significantly greater in eyes with CNV secondary to AS (70.8%; P=0.005) compared with eyes with CNV secondary to AMD (34.1%). The incidence of Bruch's membrane undulation was significantly greater in eyes with CNV secondary to AS (70.8%; P<0.0001) than in eyes with CNV secondary to AMD (11.4%). CONCLUSION: SD-OCT imaging clearly revealed a greater incidence of unique lesions, including ORT and Bruch's membrane undulation, in eyes in PXE patients with CNV secondary to AS than in eyes with CNV secondary to AMD.

Retinal nerve fibre layer assessment in myopic glaucomatous eyes: comparison of GDx variable corneal compensation with GDx enhanced corneal compensation.

AIM: To compare the results of scanning laser polarimetry (GDx) with variable corneal compensation (VCC) and enhanced corneal compensation (ECC) when applied to myopic glaucomatous eyes. METHODS: Forty glaucoma eyes with moderate myopia (between -3 and -6 D) and 35 glaucoma eyes with high myopia (-8 D or greater) were enrolled in this study. GDx VCC, GDx ECC and standard automated perimetry (SAP) were performed. The prevalence of an atypical retardation pattern (ARP), the typical scan score (TSS) and retinal nerve fibre layer (RNFL) thickness were compared between VCC and ECC in both groups of myopic subjects. A correlation analysis between RNFL thickness and visual sensitivity was also conducted. RESULTS: In both myopic groups, the mean TSS is significantly lower (p<0.0001), and the prevalence of ARP was significantly higher (p<0.0001) by VCC scans than by ECC scans. Temporal, superior, nasal, inferior, temporal (TSNIT) average and temporal average thickness showed significantly higher values (p<0.001) by VCC than by ECC. A statistically significant association was observed between TSNIT average and mean deviation of SAP by ECC scan. CONCLUSIONS: ECC scans showed a better retardation pattern and structure-function relationship than did VCC, and ECC appeared to be more suitable for RNFL assessment in glaucomatous eyes that are moderately to highly myopic.

Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.

Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

Systemically expressed soluble Tie2 inhibits intraocular neovascularization.

Retinal and choroidal neovascularization are the most frequent causes of severe and progressive vision loss. Studies have demonstrated that Tie2, an endothelial-specific receptor tyrosine kinase, plays a key role in angiogenesis. In this study, we determined whether adenovirus-mediated gene delivery of extracellular domain of the Tie2 receptor (ExTek) could inhibit experimental retinal and choroidal neovascularization. Immunofluorescence histochemistry with a monoclonal antibody to human Tie2 showed that Tie2 expression is prominent around and within the base of newly formed blood vessels of retinal and choroidal neovascular lesions. A single intramuscular injection of adenovirus expressing ExTek genes achieved plasma levels of ExTek exceeding 500 microg/ml in mice for 10 days (in neonates) and 7 days (in adults). This treatment inhibited retinal neovascularization by 47% (p < 0.05) in a murine model of ischemia-induced retinopathy. The same treatment reduced the incidence and extent of sodium fluorescein leakage from choroidal neovascular lesions by 52% (p < 0.05) and 36% (p < 0.01), respectively, in a laser-induced murine choroidal neovascularization model. The same mice showed a 45% (p < 0.001) reduction of integrated area of the choroidal neovascularization. These findings indicate that Tie2 signaling is a common component of the angiogenic pathway in both retinal and choroidal neovascularization, providing a potentially useful target in the treatment of intraocular neovascular diseases.

Angiopoietin-1 upregulation by vascular endothelial growth factor in human retinal pigment epithelial cells.

PURPOSE: To determine whether vascular endothelial growth factor (VEGF) regulates angiopoietin (Ang)-1 and -2 expression in retinal pigment epithelial (RPE) cells. METHODS: Expression of VEGF, Ang1, and Ang2 in surgically removed human choroidal neovascular membranes (CNVMs) was analyzed by double-label confocal immunofluorescence microscopy. Total RNA was extracted from cultured human RPE cells treated with VEGF for mRNA analysis. Northern blot analysis was performed to examine the time course and dose response of Ang1 and Ang2 mRNA expression. mRNA stability and nuclear run-on analyses were performed. Secreted Ang1 and Ang2 protein levels in conditioned media from RPE cells were examined by Western blot analysis. RESULTS: Ang1 and Ang2 immunostaining colocalized with VEGF-positive stromal cells in human CNVMS: Ang1 and Ang2 mRNAs were expressed by cultured serum-starved RPE cells. VEGF upregulated Ang1 mRNA in a time- and dose-dependent manner without a significant change in Ang2 mRNA. Ang1 and Ang2 mRNAs in RPE cells were as stable as that of S18. VEGF stimulation further increased the half-life of Ang1 mRNA, but did not alter its transcription rate. VEGF increased the amount of Ang1, but not Ang2, protein secreted into the medium. CONCLUSIONS: The colocalization of Ang1 and Ang2 with VEGF in CNVM stromal cells and the upregulation of Ang1 expression by VEGF in cultured RPE cells suggest that VEGF may selectively modulate Ang expression during CNV.

Peroxisome proliferator-activated receptor-gamma ligands inhibit choroidal neovascularization.

PURPOSE: To determine the antiangiogenic effects of peroxisome proliferator-activated receptor (PPAR)-gamma agonists on ocular cells involved in the pathogenesis of choroidal neovascularization (CNV) in vitro and on experimental laser photocoagulation-induced CNV in vivo. METHODS: PPAR-gamma expression in human retinal pigment epithelial (RPE) cells and bovine choroidal endothelial cells (CECs) was determined using an RNase protection assay and Western blot analysis. Two PPAR-gamma ligands, troglitazone (TRO) and rosiglitazone (RSG; 0.1-20 microM), were used to assess effects on RPE and CEC proliferation and migration and CEC tube formation in response to vascular endothelial growth factor (VEGF). The effects of intravitreal injection of TRO on laser photocoagulation-induced CNV lesions in rat eyes (15 experimental, 15 control, nine burns per eye) and cynomolgus monkey eyes (two experimental, two control, seven paramacular burns per eye) was assessed by fluorescein angiography and histologic evaluation. RESULTS. PPAR-gamma1 was expressed in both RPE and CEC. PPAR-gamma ligands significantly inhibited VEGF-induced migration and proliferation in both cell types and tube formation of CEC in a dose-response manner. CNV in rats was markedly inhibited by intravitreous injection of TRO (P < 0.001). Lesions showed significantly less fluorescein leakage and were histologically thinner in the TRO-treated animals. Similar findings were present in the TRO-treated lesions in two monkey eyes. The drug showed no apparent adverse effects in the adjacent retina or in control eyes. CONCLUSIONS: The inhibition of VEGF-induced choroidal angiogenesis in vitro, and CNV in vivo by PPAR-gamma ligands suggests the potential application of these agents in the large group of patients with age-related macular degeneration complicated by CNV.

Hepatoma-specific antitumor activity of an albumin enhancer/promoter regulated herpes simplex virus in vivo.

Targeting viral vectors to appropriate cell types so that normal cells are not adversely affected is an important goal for gene therapy. Previously, we described a novel approach to viral gene therapy using a conditional, replication-competent herpes simplex virus (HSV), where replication and associated cytotoxicity are limited to a specific cell-type by the regulated expression of an essential immediate-early viral gene product. In this report we analyze the hepatoma-specific replication, cytotoxicity and anti-tumor effect of recombinant HSV G92A, regulated by the albumin enhancer/promoter. G92A efficiently replicated in vitro in two human hepatoma cell lines expressing albumin, but not in four human non-hepatoma, albumin-non-expressing tumor cell lines, while all cell lines were equally susceptible to a tissue nonspecific HSV recombinant, hrR3. In vivo, G92A replicated well in subcutaneous xenografts of human hepatoma cells (Hep3B) in athymic mice, but not in non-hepatoma subcutaneous tumors (PC3 and HeLa), whereas, hrR3 replicated well in both tumor types. Intratumoral inoculation of G92A inhibited the growth of established subcutaneous hepatoma tumors in nude mice, but not prostate tumors. Replication-competent viral vectors controlled by cell-specific transcriptional regulatory sequences provide a new therapeutic strategy for tumor therapy.

Expression of proteoglycan decorin in neural retina.

PURPOSE: To identify the expression of chondroitin/dermatan sulfate proteoglycan decorin in retina and to elucidate its changes during development and ischemia-reperfusion. METHODS: Expression of decorin in rat retina was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Distributional changes during development and transient ischemia in model eyes also were investigated by immunohistochemical experiments. RESULTS: Gene expression of decorin core protein was identified in rat retina by RT-PCR. Decorin immunoreactivities were shown throughout the retina, especially in the ganglion cell layer. In developing rat retinas, at embryonic stages (embryonic day 16), decorin was distributed uniformly throughout the retina. As retina matured, the intensity of decorin immunostaining in retinal inner layers and retinal pigment epithelium increased. Furthermore, in experimental transient retinal ischemia, after transient downregulation of the decorin core protein gene between 24 and 48 hours after the ischemia, recovered (or increased) expression was shown by semiquantitative RT-PCR experiments. Immunohistochemical studies revealed strong decorin immunoreactivities in the damaged inner layers 1 week later. CONCLUSIONS: The expression of decorin was identified in adult and developing rat retina. The distributional changes of decorin during the retinal development suggest that this proteoglycan may play a role in the differentiation of retinal ganglion cells. Moreover, in rat ischemia-reperfusion models, the alterations in gene expression and immunohistochemical localization showed the contribution of this proteoglycan to the damage and repair processes in diseased retina.

Mutant herpes simplex virus-mediated suppression of retinoblastoma.

PURPOSE: To test the ability of a mutant herpes simplex virus (HSV) hrR3 to inhibit growth of Y79 human retinoblastoma in vitro and in vivo. METHODS: Cultured Y79 cells were infected with multiplicities of infection (MOI) ranging from 0.004 to 0.1 of hrR3. Surviving cells were counted using trypan blue dye exclusion. Using X-gal staining, expression of the lacZ gene was examined in vitro on day 3 postinfection to evaluate viral replication. Nude mice harboring Y79 tumors subcutaneously received an intraneoplasmic injection of 5 x 10(7) plaque-forming units of hrR3. The tumor sizes were measured weekly. Expression of the lacZ gene was also examined on one week postinfection. RESULTS: There are 31% and 13% cells surviving in cultured Y79 cells infected by hrR3 at an MOI of 0.1 on days 3 and 5 postinfection respectively compared to those of mock-infected cells. Also more than 70% of Y79 cells were stained with X-gal at an MOI of 0.1 which demonstrated active viral replication in vitro. Virus-treated subcutaneous tumors were smaller than control tumors (p<<0.05, Student's t-test) on days 14, 21, and 28 postinfection. Positive X-gal staining was also observed in the tumor nodule which was challenged with this viral vector. CONCLUSIONS: We have demonstrated that hrR3 is capable of inhibiting Y79 tumor growth both in cell culture and in nude mice. These data suggest that gene therapy using this mutant HSV vector can be a new supplementary therapeutic modality for retinoblastoma.

Adenovirally expressed basic fibroblast growth factor rescues photoreceptor cells in RCS rats.

PURPOSE: To evaluate the abilities of recombinant adenovirus carrying the basic fibroblast growth factor (bFGF) gene to (1) produce bFGF protein in vitro and (2) rescue retinal photoreceptors in Royal College of Surgeons (RCS) rats in vivo. METHODS: Cultured human retinal pigment epithelial cells were infected with one of the following two replication-deficient adenoviral vectors that drive inserted genes by beta-actin promoter with cytomegalovirus enhancer: AxCAJSbFGF, which expresses the human bFGF gene, and AxCAlacZ, carrying the cDNA of bacterial beta-galactosidase as a viral control. These viruses and recombinant bFGF protein were also injected into the subretinal space of RCS rats at the age of 21 days. The production of bFGF was evaluated by an immunohistochemical method in vitro and in vivo. The secretion of bFGF produced in vitro was quantified by an enzyme-linked immunosorbent assay. The thickness of the outer nuclear layer (ONL) as a marker of photoreceptor cell rescue was estimated at 2, 28, and 56 days after the injections. RESULTS: AxCAJSbFGF produced human bFGF protein effectively both in vitro and in vivo. The semiquantitative analysis of ONL thickness revealed a significant protective effect of AxCAJSbFGF and the recombinant bFGF protein injection up to 56 days after injection. CONCLUSIONS: These results demonstrate that a recombinant adenoviral vector can achieve the transfer of bFGF gene in vitro and have a protective effect for photoreceptor cells in vivo. Gene therapy with a bFGF-expressing recombinant adenoviral vector may provide a new strategy with which to target retinal degenerative diseases.

Roles of constitutive nitric oxide synthase in postischemic rat retina.

PURPOSE: Nitric oxide is a reactive species that could be protective or destructive to the retina depending on the stage of the evolving ischemic process. This study was conducted to obtain a better understanding of the roles of constitutive nitric oxide synthase (cNOS) during reperfusion after ischemia in rat retina. METHODS: Retinal ischemia was induced for 60 minutes in Sprague-Dawley rats by ligating the optic nerve. Gene expression for endothelial and neuronal nitric oxide synthases (eNOS and nNOS) was studied by reverse transcription-polymerase chain reaction (RT-PCR). To inhibit cNOS, NG-nitro-L-arginine (L-NNA) was injected intraperitoneally four times (every 6 hours) beginning 2 hours after reperfusion, for a total dose of 80 mg/kg. Retinal damage was assessed by the rate of a- and b-wave recovery on electroretinograms and by the thickness of the retinal layers. Retinal circulation and vessel diameter were evaluated by the dye-dilution technique. RESULTS: After ischemia ended, eNOS mRNA initially decreased until 6 hours, then increased to a peak at 12 hours, and decreased progressively beyond 24 hours until the final measurement at 96 hours of reperfusion. nNOS mRNA decreased to nearly undetectable levels during the same measurement periods. L-NNA treatment enhanced reduction of a- and b-wave amplitudes and increased thinning of the inner retina in postischemic eyes. Retinal mean circulation time was markedly prolonged in L-NNA-treated postischemic eyes. Arterial mean transit times were 2.1-fold and 4.5-fold longer in L-NNA-treated postischemic eyes than in L-NNA-treated nonischemic eyes and in D-NNA-treated postischemic eyes, respectively. CONCLUSIONS: This study shows that postischemic inhibition of NOS worsens retinal damage after ischemia-reperfusion and alters postischemic retinal circulation. Nitric oxide may play an important role in protecting the retina from ischemic injury, possibly by preventing postischemic hypoperfusion.

Role of nitric oxide during the initial phase of reperfusion after retinal ischemia in the rat.

The onset of reperfusion and the recovery of the ERG b-wave following retinal ischemia was examined among three groups of rats: group 1 (n = 12) and group 2 (n = 6) received pretreatment with NG-nitro-L-arginine (20 mg/kg, i.p., 2 h before ischemia) followed by intravenous injection of saline (group 1) or of 200 mg/kg L-arginine (group 2) 5 min before the end of ischemia; group 3 (n = 7) received saline pretreatment followed by intravenous injection of saline as a control. Group 1 showed delayed onset of reperfusion compared to the other two groups and a reduction in the rate of the b-wave recovery compared to the control on the 1st day after reperfusion (group 1 vs. group 3; p = 0.0357). The L-arginine posttreatment significantly increased the b-wave recovery (group 2 vs. group 1; p = 0.0005 on day 1 and p < 0.0006 on day 3). The rate of the b-wave recovery in group 1 was inversely proportional to the time to establish complete reperfusion. Inhibition of nitric oxide synthase during the initial phase of reperfusion may worsen the recovery of the b-wave following retinal ischemia, at least in part, by inhibiting establishment of reperfusion.

Advanced glycation end products in age-related macular degeneration.

OBJECTIVE: To investigate the localization of N epsilon-(carboxymethyl)lysine (CML), a component and major immunologic epitope of advanced glycation end products, in aged eyes and choroidal neovascular membranes (CNVMs) surgically excised from eyes with age-related macular degeneration. METHODS: Immunohistochemistry for CML was performed using 8 snap-frozen, surgically excised CNVMs. Twelve eyes from patients aged 69 to 82 years and 2 donor eyes, 1 each from a 23-week-old fetus and 21-year-old patient, without age-related macular degeneration or diabetic retinopathy were also examined. To determine if retinal pigment epithelial cells in CNVMs accumulate advanced glycation end products, cytokeratin and CML were stained in paired serial sections. RESULTS: Soft, macular drusen and/or basal laminar and basal linear deposits were observed in 8 of 12 aged eyes. Each case showed CML accumulation, while overlying retinal pigment epithelial cells showed no accumulation in all 12 eyes. In CNVMs, however, retinal pigment epithelial cells showed CML accumulation in their cytoplasm. CONCLUSION: The additional accumulation of advanced glycation end products in soft, macular drusen and/or retinal pigment epithelial cells may play a role in the pathogenesis of CNVM formation in age-related macular degeneration. CLINICAL RELEVANCE: Recently, advanced glycation end products have been found to play a role both in aging changes and neovascularization. Localization of advanced glycation end products in the above-mentioned tissue may lead to a better understanding of the pathogenesis of age-related macular degeneration.

Growth inhibition of cultured human Tenon's fibroblastic cells by targeting the E2F transcription factor.

The transcription factor E2F regulates the expression of several genes concerned with cell growth. The ability to inhibit transcription by blocking E2F expression has great potential in the treatment of proliferative disorders. The effect of double-stranded phosphorothioate oligonucleotides containing E2F transcription factor cis element, a so called 'decoy' has examined on the growth of cultured human Tenon's fibroblastic cells. Human Tenon's fibroblastic cells were cultured and challenged by E2F decoy coated with the Hemagglutinating virus of Japan (HVJ) cationic liposomes (HVJ-CL). The outcome was evaluated using fluorescence microscopy, RT-PCR and growth assays. HVJ-CL facilitated the transfer of external oligonucleotides to cultured human Tenon's fibroblastic cells. The E2F decoy, transferred by HVJ-CL, inhibited simultaneously the expression of the mRNAs of several cell cycle related genes such as c-myc, cdc2, proliferative cell nuclear antigen, and dehydrofolate reductase. Entry into S phase was also reduced to 42.7% of the positive control by the E2F decoy. The total increase of DNA at four days was reduced to 59.7% of the positive control by 5 microM and 29.9% by 15 microM of E2F decoy. It is concluded that gene therapy using the E2F transcription factor offers a potential therapeutic modality for the treatment of proliferative disorders such as proliferative vitreoretinopathy and fibrosis following filtering surgery.