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Vascular endothelial growth factor up-regulates the expression of intracellular adhesion molecule-1 in retinal endothelial cells via reactive oxygen species, but not nitric oxide

ZHANG, Xiao-ling; WEN, Liang; CHEN, Yan-jiong; ZHU, Yi

Section Editor(s): GUO, Li-shao

doi: 10.3760/cma.j.issn.0366-6999.2009.03.019
Original article
Free
SDC

Background The vascular endothelial growth factor (VEGF) is involved in the initiation of retinal vascular leakage and nonperfusion in diabetes. The intracellular adhesion molecule-1 (ICAM-1) is the key mediator of the effect of VEGFs on retinal leukostasis. Although the VEGF is expressed in an early-stage diabetic retina, whether it directly up-regulates ICAM-1 in retinal endothelial cells (ECs) is unknown. In this study, we provided a new mechanism to explain that VEGF does up-regulate the expression of ICAM-1 in retinal ECs.

Methods Bovine retinal ECs (BRECs) were isolated and cultured. Immunohistochemical staining was performed to identify BRECs. The cultured cells were divided into corresponding groups. Then, VEGF (100 ng/ml) and other inhibitors were used to treat the cells. Cell lysate and the cultured supernatant were collected, and then, the protein level of ICAM-1 and phosphorylation of the endothelial nitric oxide synthase (eNOS) were detected using Western blotting. Griess reaction was used to detect nitric oxide (NO).

Results Western blotting showed that the VEGF up-regulated the expression of ICAM-1 protein and increased phosphorylation of the eNOS in retinal ECs. Neither the block of NO nor protein kinase C (PKC) altered the expression of ICAM-1 or the phosphorylation of eNOS. The result of the Western blotting also showed that inhibition of phosphatidylinositol 3-kinase (PI3K) or reactive oxygen species (ROS) significantly reduced the expression of ICAM-1. Inhibition of PI3K also reduced phosphorylation of eNOS. Griess reaction showed that VEGF significantly increased during NO production. When eNOS was blocked by L-NAME or PI3K was blocked by LY294002, the basal level of NO production and the increment of NO caused by VEGF could be significantly decreased.

Conclusion ROS-NO coupling in the retinal endothelium may be a new mechanism that could help to explain why VEGF induces ICAM-1 expression and the resulting leukostasis in diabetic retinopathy.

Edited by

Department of Ophthalmology, First Affiliated Hospital, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061, China (Zhang XL and Wen L)

Department of Immunology and Microbiology, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061, China (Chen YJ)

Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100083, China (Zhu Y)

Correspondence to: Dr. ZHANG Xiao-ling, Department of Ophthalmology, First Affiliated Hospital, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061, China (Tel and Fax: 86–29–82655564. Email: zhangxle@mail.xjtu.edu.cn)

This study was supported in part by grants from the National Natural Science Foundation of China (No. 30571994, No. 30570713 and No. 30630032).

(ReceivedAugust 13, 2008)

Different stages of diabetic retinopathy are characterized by increased vascular permeability and leakage, thickening of the basement membrane, capillary occlusion, retinal ischaemia, and angiogenesis. New vessel growth in proliferative diabetic retinopathy appears to be associated with the ischaemic area of the retina in patients with diabetes. Ischaemia or hypoxia is an initial angiogenic stimulus that may alter the expression of specific growth factors, including the vascular endothelial growth factor (VEGF), a potent endothelial cell (EC) mitogen.1,2 The injection of VEGF into normal nondiabetic eyes recapitulates many of the retinal vascular changes triggered by diabetes, including intercellular adhesion molecule-1 (ICAM-1) up-regulation, leukocyte adhesion, vascular permeability, and capillary nonperfusion.3,4 The causes and consequences of this phenomenon are largely unknown. Diabetic retinopathy is generally not considered an inflammatory disease, but the retinal vasculature of humans and rodents with diabetes mellitus contains an increased number of leukocytes.5 Therefore, the adhesion of leukocytes to the retinal vasculature may be one of the earliest events of experimental diabetes.6 Enhanced vascular permeability, EC damage, and capillar nonperfusion are some of the potential pathological consequences of diabetic retinal leukocyte adhesion.6,7 Although VEGF is expressed in an early-stage diabetic retina8, whether it directly up-regulates ICAM-1 in retinal ECs is unknown.

ICAM-1, an 80- to 114-kD member of the immunoglobulin gene superfamily, mediates leukocyte adhesion and transmigration.9,10 In both humans and rodents, the diabetic retinal vasculature shows up-regulated ICAM-1, increased leukocyte adhesion, blood-retinal barrier breakdown and capillary nonperfusion. 5,6,11,12 Neutralizing anti-ICAM-1 antibodies can greatly reduce the leukocyte-related abnormalities in newly diabetic animals. 6,12 The exact mechanisms governing ICAM-1 induction in diabetic retinopathy remain to be determined. VEGF triggers an increase in the leukocyte adhesion to retinal capillaries, which mediates the pathogenesis of vascular leakage4. In line with these observations, the blockage of ICAM-1 can abrogate the stimulatory effect of VEGF on the monocyte adhesion to retinal ECs, thus supporting the notion that ICAM-1 is the key mediator of the effect of VEGF on retinal leukostasis.

An oxidation-sensitive mechanism has been proposed as being mainly responsible for the ICAM-1 induction in ECs through cytokine-induced reactive oxygen species (ROS) and the activation of the transcription factor nuclear factor-κB (NF-κB). In brain endothelium, VEGF-induced ICAM-1 up-regulation is mediated by nitric oxide (NO), a molecule with both cytotoxic and signaling capabilities.13 NO can be generated by various NO synthases (NOS), but the endothelial isoform, eNOS, is generally considered a cardiovascular protective molecule.14 The role of eNOS activation of VEGF in ICAM-1 induction in the diabetic retina remains unknown.

In the current study, we examined the role of VEGF in inducing retinal endothelial ICAM-1 and the underlying mechanism. Our results indicate that VEGF up-regulates ICAM-1 through a mechanism requiring reactive oxygen species but not nitric oxide.

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METHODS

Isolation and culture of bovine retinal ECs (BRECs)

Primary cultures of BRECs were isolated and cultured as described previously.15,16 The retina was gently peeled from fresh bovine eyes and briefly transferred into Dulbecco’s modified Eagle's medium (DMEM, Gibco; Grand Island, NY, USA) containing antibiotics. BRECs were isolated by homogenization and underwent a series of filtration steps. The resulting cells were seeded onto fibronectin-coated 24-well tissue culture plates in DMEM supplemented with 10 mmol/L sodium bicarbonate, 10 ng/ml epidermal growth factor (Sigma; St. Louis, MO, USA), and 10% fetal bovine serum (FBS, Hyclone; Logan, UT, USA); the media was changed every 3 days. The endothelial homogeneity was ascertained by applying the von Willebrand factor (vWF) immunofluorescence analysis. All the experiments were performed with cells from passages 2 to 8. A mouse capillary EC line, bEnd.3 from ATCC, was cultured with DMEM, supplemented with 10% FBS, and maintained in a humidified 95% air/5% CO2 incubator at 37°C.

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Immunohistochemical staining

We used a standard two-stage indirect immunofluo-rescence technique with an anti-vWF factor (Santa Cruz Biotechnology; Santa Cruz, CA, USA). After the slides with cultured cells were fixed, they were incubated with anti-vWF antibodies overnight at 4°C, followed by FITC-conjugated secondary antibodies. Human umbilical vein ECs were used to specify the reaction of the antibodies. Negative controls were species-matched IgG. The results were obtained under fluorescence microscopy (Leica, Germany).

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Western blotting

Cultured ECs were lysed and protein concentrations were measured using the BCA protein assay kit. Fifty micrograms of cell lysates were resolved by a 10% SDS-PAGE and transferred to a nitrocellulose membrane. ICAM-1, eNOS, and β-actin proteins were detected using monoclonal anti-ICAM-1, anti-phosphorylated eNOS (anti-P-eNOS), anti-eNOS (Santa Cruz Biotechnology), and anti-β-actin (Bioss; Beijing, China), followed by an HRP-conjugated secondary antibody. The protein bands were visualized with the ECL detection system (Amersham; Arlington Heights, IL, USA), and the densities of the bands were quantified with the Scion Image software (Scion Corp., Frederick, MD, USA).

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NO measurement

Confluent ECs in 6-well plates were serum-starved for 18 hours and then treated with VEGF in a phenol red-free medium for various times. The cell culture supernatants were collected at 6 hours after treatment. NO production was detected by measuring their final stable equimolar degradation products: nitrite and nitrate. All of the nitrite was quantified after reduction with the use of nitrate reductase and was determined spectrophotometrically at 540 nm by the Griess reaction. The total nitrite concentration was calculated from a standard curve constructed over the linear range of the assay and expressed as nanomoles per liter milligram protein. The amount of NO product was normalized against that of total cellular protein.

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Statistical analysis

The results are expressed as mean ± standard deviation (SD) from at least three independent experiments. Data were analyzed by applying the unpaired two-tailed Student's t test or analysis of variance (ANOVA). Each experiment involved three measurements for each condition tested, unless indicated otherwise. A P <0.05 was considered statistically significant.

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RESULTS

VEGF induced ICAM-1 up-regulation and NO production in BRECs

Primary cultured cells at passage two were composed of approximately 90% ECs according to the vWF immunofluorescence analysis (data not shown). After an 8-hour treatment with 100 ng/ml VEGF, ICAM-1 protein expression increased to levels similar to those induced by 10 ng/ml lipopolysaccharide (LPS) (Figure 1A). Interestingly, treatment with VEGF, but not LPS significantly, induced NO generation, which suggests a unique pattern of EC activation by VEGF in microvascular ECs (Figure 1B). In the bEnd.3 cell line, VEGF induced a time-dependent up-regulation of ICAM-1, beginning at 2 hours and peaking at 6 hours (Figure 2A). Since NO is produced by eNOS to catalyze its substrate L-arginine, eNOS phosphorylation was determined by the use of an antibody recognizing the 1179 P-eNOS. Also, VEGF induced eNOS phosphorylation, which peaked at 2 hours (Figure 2B).

Figure 1.

Figure 1.

Figure 2.

Figure 2.

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Up-regulation of ICAM-1 by VEGF is not mediated by NO generation or PKC activation

We treated bEnd.3 cells with the eNOS substrate L-arginine and L-NAME, an arginine analog, to inhibit the function of eNOS. VEGF increased NO production in bEnd.3 cells (Figure 3) in the same pattern as in BRECs (Figure 1B). Pre-treatment with L-NAME greatly reduced both basal and VEGF-induced NO production, and L-arginine significantly increased NO generation. However, neither L-NAME nor L-Arginine altered VEGF-induced ICAM-1 up-regulation (Figure 4).

Figure 3.

Figure 3.

Figure 4.

Figure 4.

ICAM-1 induction in arterial ECs occurs through a cytokine-induced PKC pathway.17 We further detected the role of PKC in micro-vascular ECs. Pretreatment with calphostin C, an inhibitor of all isozymes of PKC, did not significantly alter VEGF-induced expression of ICAM-1 or phosphorylation of eNOS (Figure 5).

Figure 5.

Figure 5.

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ICAM-1 up-regulation by VEGF in microvascular ECs is PI3K and ROS dependent

VEGF receptors are receptor-tyrosine kinases, with PI3K as their downstream kinase. The binding of VEGF can activate its receptors and the PI3K-Akt pathway, which in turn, phosphorylates eNOS and increases NO production. In bEnd.3 cells, we confirmed that LY294002, a PI3K inhibitor, can significantly attenuate a VEGF-induced phosphorylation of eNOS and production of NO (Figure 6). Surprisingly, inhibition of PI3K by LY294002 also markedly blocked both basal and VEGF-induced ICAM-1 expression in both primary BRECs and bEnd.3 cells (Figure 7). To explain the paradox that PI3K but not NO is involved in ICAM-1 regulation, we examined the role of PKC and ROS in the VEGF induction of ICAM-1 and found that PKC is not involved in the VEGF/eNOS/NO pathway (Figures 3–5). In both BRECs and bEnd.3 cells, pre-treatment with MnTMPyP largely blocked both basal and VEGF-induced ICAM-1 expressions (Figure 7).

Figure 6.

Figure 6.

Figure 7.

Figure 7.

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DISCUSSION

VEGF was reported to be increased in both the vitreous and the ocular fluids of patients with diabetic retinopathy.18 VEGF is currently viewed as a major effector for retinal neovascularization in all proliferative retinopathies.19 VEGF may also be involved in the genesis of retinal vascular leakage and nonperfusion, two major complications of diabetes. In diabetic rats, the rates of retinal endothelial barrier breakdown and VEGF activity were increased in proportion to the duration of the diabetes.20 VEGF can trigger many of the retinal vascular changes caused by diabetes, including ICAM-1 up-regulation, leukocyte adhesion to endothelial cells, vascular permeability, and capillary nonperfusion.3,4 To study the role of VEGF in the induction of retinal endothelial ICAM-1 and the underlying mechanism, we first isolated and cultured BRECs and then treated the cells with VEGF. The results showed that VEGF increased ICAM-1 expression and induced eNOS phosphorylation and NO generation in BRECs. Previous studies found that the up-regulation of ICAM-1 and VEGF coincides in experimental diabetes and that the inhibition of VEGF suppresses ICAM-1 expression and leukostasis.21 ICAM-1 participates in different types of cell-cell interactions and transendothelial migration; the process leads to blood-retinal barrier breakdown and capillary nonperfusion.22 Thus, the up-regulation of ICAM-1 seems to play a crucial role in the development of diabetic retinopathy.23 Endothelial NO is known to play a role in the regulation of retinal vascular functions and is postulated to contribute to the pathophysiology of retinopathy.23 VEGF has been shown to stimulate endothelial NO production by activating eNOS, 24,25 which may mediate the angiogenic and inflammatory activities of VEGF.7,26,27 We showed that VEGF increased eNOS activation and NO generation, as well as ICAM-1 production in microvascular ECs, which differs from the effects of other pro-inflammatory stimuli such as LPS.

Our results revealed that although VEGF increased NO production and ICAM-1 expression, the up-regulation of ICAM-1 was not directly mediated by NO. In order to examine whether NO is involved in the signal transduction of ICAM-1 induction by VEGF, we used L-Arginine and its analogy L-NAME to block NO production. It is significant to note that in accordance with our findings, Murohara and colleagues28 found that L-NAME did not alter the expression of multiple angiogenesis-related cell adhesion molecules, including ICAM-1. ECs contain at least two high affinity receptors for VEGF or for Flk-1/KDR and Flt-1; both belong to the family of receptor-tyrosine kinases. Autophosphorylation of these receptors leads to the activation of PI3K and the subsequent phosphorylation of PKC.29,30 In our experiment, we used Calphostin C to block PKC and revealed that neither of them altered ICAM-1 expression or eNOS phosphorylation, indicating that PKC did not participate in the up-regulation of ICAM-1 by VEGF. Interestingly, blocking PI3K with LY294002 could significantly decrease protein expression of ICAM-1 and phosphorylation of eNOS. Furthermore, LY294002 also attenuated NO production. Thus, PI3K, but not PKC, is involved in the up-regulation of ICAM-1 by VEGF.

ROS, including superoxide (O2-) and H2O2, have been shown to mediate cellular responses to cytokines and growth factors and to induce changes in gene expression, cell differentiation, and apoptosis.31,32 We further dissected the mechanism of ICAM-1 production by VEGF with the use of MnTMPyP, a cell-permeant mimic of superoxide dismutase (SOD) to catalyze the ROS. In diabetic retinas, mellitus, ischemia and hypoxia can induce the expression of VEGF and superoxide and other ROS. Indeed, ROS has been implicated in the initiation and progression of several pathophysiological states. Importantly, ROS reacts with NO, which results in the loss of the anti-atherogenic properties of NO. High glucose was reported to up-regulate eNOS in aortic ECs, albeit insufficiently to compensate for decreased NO bioavailability because of the increased O2- production, which breaks down NO.33 Several mechanisms cause eNOS to produce vascular O2- rather than NO; this is termed as the ‘uncoupling’ of eNOS. First, PKC may cause the phosphorylation of eNOS at Thr495, which may uncouple oxygen reduction from L-arginine oxidation so that eNOS produces O2-, Then, O2- may react with NO to form peroxynitrite, a more potent ROS. Finally, peroxynitrite may release Zn2- from the Zn2-thiolate complex, thus breaking the eNOS homodimer and uncoupling eNOS.12,34 When ECs are under oxidative stress, the transcriptional factors NF-κB and AP-1 can be activated to induce certain redox-sensitive genes, including ICAM-1.35,36

In summary, retinal ischemia can induce that the expression of VEGF in retinal ECs in diabetes mellitus. VEGF activates its downstream effector PI3K, then through the VEGF/PI3K/AKT/eNOS pathway, promotes NO production. However, VEGF can also increase ROS production and eNOS uncoupling; ROS induces ICAM-1 expression by activating the transcriptional factors NF-κB and AP-1, and O2- combines with NO to form peroxynitrite, an effect that is stronger than the addition of O2-, to promote ICAM-1 expression.

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Keywords:

vascular endothelial growth factor; intercellular adhesion molecule-1; reactive oxygen species; endothelial nitric oxide synthase; phosphatidylinositol 3-kinase

© 2009 Chinese Medical Association