Paul A. Edwards

Activation of Vascular Endothelial Growth Factor through Reactive Oxygen Species Mediates 20-Hydroxyeicosatetraenoic Acid-Induced Endothelial Cell Proliferation

20-Hydroxyeicosatetraenoic acid (20-HETE) is formed by the ω-hydroxylation of arachidonic acid by cytochrome P450 4A and 4F enzymes, and it induces angiogenic responses in vivo. To test the hypothesis that 20-HETE increases endothelial cell (EC) proliferation via vascular endothelial growth factor (VEGF), we studied the effects of WIT003 [20-hydroxyeicosa-5(Z),14(Z)-dienoic acid], a 20-HETE analog on human macrovascular or microvascular EC. WIT003, as well as pure 20-HETE, stimulated EC proliferation by ∼40%. These proliferative effects were accompanied by increased VEGF expression and release that were observed as early as 4 h after 20-HETE agonist addition. This was accompanied by increased phosphorylation of the VEGF receptor 2. The proliferative effects of 20-HETE were markedly inhibited by a VEGF-neutralizing antibody. Polyethylene glycol-superoxide dismutase (PEG-SOD) markedly inhibited both the increases in VEGF expression and the proliferative effects of 20-HETE. In contrast, administration of the NAD(P)H oxidase inhibitor apocynin had no effect to the proliferative response to 20-HETE. The 20-HETE agonist markedly increased superoxide formation as reflected by an increase in dihydroethidium staining of EC, and this increase was inhibited by PEG-SOD but not by apocynin. 20-HETE also increased the phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) in EC, whereas an inhibitor of MAPK [U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] suppressed the proliferative and the VEGF changes but not the pro-oxidant effects of 20-HETE. These data suggest that 20-HETE stimulates superoxide formation by pathways other than apocynin-sensitive NAD(P)H oxidase, thereby activating MAPK and then enhancing VEGF synthesis that drives EC proliferation. Thus, 20-HETE may be involved in the regulation of EC functions, such as angiogenesis.

A comparative study between the PAM and the laser interferometer in cataracts

We conducted a prospective study on 35 consecutive eyes undergoing cataract extraction to compare the Guyton-Minkowski Potential Acuity Meter (PAM) and the Laser Interferometer (LI) in determining potential visual acuity. The eyes were divided into: group A, which had good visualization of optic disc details, and group B, whose media allowed only disc outline or less to be seen. We found that in group A eyes, both instruments were reliable (with accuracies of 94% for the PAM and 88% for the LI). However, in group B eyes (advanced cataracts), both instruments were unreliable (accuracies of 33% for the PAM and 53% for the LI). Interestingly, in a subgroup of high myopes (axial length >29 mm) with moderate cataracts and poor visual acuties, both instruments were very reliable and helpful in determining how much of the vision loss was due to the cataracts. We therefore strongly recommend potential acuity testing in high myopes with moderate cataracts and poor visual acuity.

Expression of CYP4A1 in U251 human glioma cell induces hyperproliferative phenotype in vitro and rapidly growing tumors in vivo.

Exogenous 20-hydroxyeicosatetraenoic acid (20-HETE) increases the growth of human glioma cells in vitro. However, glioma cells in culture show negligible 20-HETE synthesis. We examined whether inducing the expression of a 20-HETE synthase in a human glioma U251 cell line would increase proliferation. U251 cells transfected with CYP4A1 cDNA (termed U251 O) increased the formation of 20-HETE from less than 1 to over 60 pmol/min/mg proteins and increased their proliferation rate by 2-fold (p < 0.01). Compared with control U251, U251 O cells were rounded, smaller, showed a disorganized cytoskeleton, exhibited reduced vinculin staining, and were easily detached from the growing surface. They showed a marked increase in dihydroethidium staining, suggesting increased oxidative stress. The expression of phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1/2, and vascular endothelial growth factor was markedly elevated in U251 O. The hyperproliferative and signaling effects seen in U251 O cells are abolished by selective CYP4A inhibition of 20-HETE formation with HET0016 [N-hydroxy-N′-(4-butyl-2-methylphenyl)-formamidine], by small interfering RNA against the enzyme, and by the putative 20-HETE antagonist, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid. In vivo, implantation of U251O cells in the brain of nude rats resulted in a ∼10-fold larger tumor volume (10 days postimplantation) compared with animals receiving mock-transfected U251 cells. These data show that elevations in 20-HETE synthesis in U251 cells lead to an increased growth both in vitro and in vivo. This suggests that 20-HETE may have proto-oncogenic properties in U251 human gliomas. Further studies are needed to determine whether 20-HETE plays a role promoting growth of some human gliomas.

20-HETE can act as a nonhypoxic regulator of HIF-1α in human microvascular endothelial cells

20-HETE increases the expression of VEGF in human dermal microvascular endothelial cells (ECs). Since VEGF is regulated by hypoxia inducible factor (HIF)-1, we studied whether 20-HETE also upregulates HIF-1alpha using the stable 20-HETE analog 20-hydroxyeicosa-5(Z),14(Z)dienoic acid (WIT003; 1-10 microM) and found that it induced a marked increase in HIF-1alpha protein levels. The increases in VEGF after the addition of WIT003 preceded the changes in HIF-1alpha, and the increases in HIF-1alpha were prevented by a VEGF neutralizing antibody. This suggests that 20-HETE first causes increases in VEGF, which then, in turn, cause the upregulation of HIF-1alpha. Stimulation with exogenously added VEGF also led to an upregulation of HIF-1alpha. Incubation with the MEK1/ERK1/2 inhibitor U-0126 (10 microM) completely abolished the increases in VEGF and thus HIF-1alpha, suggesting the involvement of ERK1/2 activation. The addition of WIT003 resulted in a rapid and sustained increase in superoxide formation. When WIT003 was added in the presence of the nitric oxide (NO) synthase (NOS) inhibitor N-nitro-L-arginine, no changes in superoxide, VEGF, or HIF-1alpha were observed. This suggests that NOS is responsible for the early changes in superoxide induced by WIT003. Furthermore, WIT003 induced the expression of the NADPH oxidase subunit p47(phox) in ECs before the increases in HIF-1alpha. Incubation with polyethylene glycol-superoxide dismutase (400 U/ml), apocynin (100 microM), diphenylene iodonium (10 microM), or p47(phox) downregulation with small interfering (si)RNA all inhibited the increases in HIF-1alpha expression. This indicates that the early changes in superoxide lead to VEGF increases and thereby NADPH oxidase-dependent superoxide production, which is required for HIF-1alpha upregulation. We also found that the higher HIF-1alpha expression induced by WIT003 was accompanied by higher expression of erythropoietin receptor and angiopoietin-2 proteins. These increases were caused by HIF-1alpha because their levels were markedly decreased by siRNA downregulation of HIF-1alpha. 20-HETE may be a novel nonhypoxic regulator of HIF-1alpha and HIF-1alpha-regulated genes in ECs.

The Cytochrome P450 4A/F-20-Hydroxyeicosatetraenoic Acid System: A Regulator of Endothelial Precursor Cells Derived from Human Umbilical Cord Blood

Endothelial progenitor cells (EPCs) contribute to physiological and pathological neovascularization. Previous data have suggested that the cytochrome P450 4A/F (CYP4A/F)-20-hydroxyeicosatetraenoic acid (20-HETE) system regulates neovascularization. Therefore, we studied whether the angiogenic effects of the CYP4A/F-20-HETE system involve regulation of EPC function. We extracted human umbilical cord blood and isolated EPCs, which express AC133+CD34+ and kinase insert domain receptor (KDR) surface markers and contain mRNA and protein for CYP4A11 and CYP4A22 enzymes, as opposed to mesenchymal stem cells, which only express negligible amounts of CYP4A11/22. When EPCs were incubated with arachidonic acid, they produced 20-HETE, which stimulated the cells to proliferate and migrate, as did vascular endothelial growth factor. Incubation with 1 μM N-hydroxy-N′-(4-butyl-2-methylphenyl)formamidine (HET0016), a selective inhibitor of 20-HETE synthesis, reduced the proliferative and migratory effects of vascular endothelial growth factor and also significantly abolished EPC migration mediated by stroma-derived factor-1α, as did (6,15) 20-hydroxyeicosadienoic acid. Coculturing EPCs and endothelial cells on a Matrigel matrix led to tube formation, which in turn was inhibited by both HET0016 and 20-hydroxyeicosadienoic acid. We concluded that the CYP4A/F-20-HETE system is expressed in EPCs and can act as both an autocrine and a paracrine regulatory factor.

Inhibition of APE1/Ref-1 redox activity rescues human retinal pigment epithelial cells from oxidative stress and reduces choroidal neovascularization

The effectiveness of current treatment for age related macular degeneration (AMD) by targeting one molecule is limited due to its multifactorial nature and heterogeneous pathologies. Treatment strategy to target multiple signaling pathways or pathological components in AMD pathogenesis is under investigation for better clinical outcome. Inhibition of the redox function of apurinic endonuclease 1/redox factor-1 (APE1) was found to suppress endothelial angiogenesis and promote neuronal cell recovery, thereby may serve as a potential treatment for AMD. In the current study, we for the first time have found that a specific inhibitor of APE1 redox function by a small molecule compound E3330 regulates retinal pigment epithelium (RPEs) cell response to oxidative stress. E3330 significantly blocked sub-lethal doses of oxidized low density lipoprotein (oxLDL) induced proliferation decline and senescence advancement of RPEs. At the same time, E3330 remarkably decreased the accumulation of intracellular reactive oxygen species (ROS) and down-regulated the productions of monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF), as well as attenuated the level of nuclear factor-κB (NF-κB) p65 in RPEs. A panel of stress and toxicity responsive transcription factors that were significantly upregulated by oxLDL was restored by E3330, including Nrf2/Nrf1, p53, NF-κB, HIF1, CBF/NF-Y/YY1, and MTF-1. Further, a single intravitreal injection of E3330 effectively reduced the progression of laser-induced choroidal neovascularization (CNV) in mouse eyes. These data revealed that E3330 effectively rescued RPEs from oxidative stress induced senescence and dysfunctions in multiple aspects in vitro, and attenuated laser-induced damages to RPE–Bruch׳s membrane complex in vivo. Together with its previously established anti-angiogenic and neuroprotection benefits, E3330 is implicated for potential use for AMD treatment.

Reproducibility study on the scheimpflug cataract video camera

The Zeiss Scheimpflug Cataract Video Camera was designed to photograph, store and analyze cataracts in a semi-automated fashion for cross-sectional and longitudinal studies. We conducted a reproducibility study of this system. Twenty-four normal and 61 cataractous eyes were photographed twice by each of two of the authors in the 90 degree meridian and microdensitometry was performed on each of the stored images. Reproducibility was then determined using the intraclass correlation coefficient to determine whether or not the differences encountered were due to variability in the system or due to actual differences among the images. The intraclass correlation in the lens nucleus was 0.995 with 95% confidence limits of .992-.996. Therefore, reproducibility was 99.5%. In the anterior cortex, intraclass correlation was .941 with 95% confidence limits of .919-.959. In the posterior cortex intraclass correlation was .905 with 95% confidence limits of .870-.932. Reproducibility with this instrument was therefore excellent and with certain limitations, this may be a useful instrument in monitoring lens changes in certain diseases and the effects of anti-cataract agents.

MRI retinovascular studies in humans: research in patients with diabetes.

To review existing applications of MRI for detecting blood–retinal barrier (BRB) damage and retinal oxygenation response abnormalities in patients with diabetes and highlight new information available from such applications.

Bevacizumab prior to vitrectomy for diabetic traction retinal detachment.

PurposeTo assess the efficacy and safety of preoperative intravitreal bevacizumab (IVB) before vitrectomy for diabetic tractional retinal detachment (TRD).MethodsUsing ICD-9 codes, we located all patients with diabetic TRD who underwent 3-port 20-gauge vitrectomy primarily performed by one surgeon between January 2004 and January 2009. Eyes receiving IVB were compared with those not. The following outcomes were compared: visual acuity (VA), duration of surgery, and complication rates.ResultsA total of 99 eyes of 90 patients were included in the analysis. In all, 34 patients received IVB on an average of 11.5 (range, 3–30) days previtrectomy. Age was 46.5 and 51.6 in the IVB and non-IVB groups, respectively. VA was improved significantly in both groups: from 20/617 to 20/62 in the IVB group, and from 20/443 to 20/86 in the non-IVB group (P=0.11 between groups). Operating time and postoperative complications (glaucoma, RD, and revitrectomy rate) were similar in both groups. On comparing IVB and non-IVB eyes in younger patients (≤40), operating time was shorter (P=0.02) and a trend to better VA in the IVB group was seen.ConclusionsPreoperative IVB may be a useful adjunct to vitrectomy for severe PDR complicated by TRD, particularly in younger diabetics.

Evaluation of Visual Acuity Measurements After Autorefraction vs Manual Refraction in Eyes With and Without Diabetic Macular Edema

OBJECTIVE To compare visual acuity (VA) scores after autorefraction vs manual refraction in eyes of patients with diabetes mellitus and a wide range of VAs. METHODS The letter score from the Electronic Visual Acuity (EVA) test from the electronic Early Treatment Diabetic Retinopathy Study was measured after autorefraction (AR-EVA score) and after manual refraction (MR-EVA score), which is the research protocol of the Diabetic Retinopathy Clinical Research Network. Testing order was randomized, study participants and VA examiners were masked to refraction source, and a second EVA test using an identical supplemental manual refraction (MR-EVAsuppl score) was performed to determine test-retest variability. RESULTS In 878 eyes of 456 study participants, the median MR-EVA score was 74 (Snellen equivalent, approximately 20/32). The spherical equivalent was often similar for manual refraction and autorefraction (median difference, 0.00; 5th-95th percentile range, -1.75 to 1.13 diopters). However, on average, the MR-EVA scores were slightly better than the AR-EVA scores, across the entire VA range. Furthermore, the variability between the AR-EVA scores and the MR-EVA scores was substantially greater than the test-retest variability of the MR-EVA scores (P < .001). The variability of differences was highly dependent on the autorefractor model. CONCLUSIONS Across a wide range of VAs at multiple sites using a variety of autorefractors, VA measurements tend to be worse with autorefraction than manual refraction. Differences between individual autorefractor models were identified. However, even among autorefractor models that compare most favorably with manual refraction, VA variability between autorefraction and manual refraction is higher than the test-retest variability of manual refraction. The results suggest that, with current instruments, autorefraction is not an acceptable substitute for manual refraction for most clinical trials with primary outcomes dependent on best-corrected VA.