Insulin enhances leukocyte–endothelial cell adhesion in the retinal microcirculation through surface expression of intercellular adhesion molecule-1

Microvascular Research 69 (2005) 135 – 141 www.elsevier.com/locate/ymvre

Insulin enhances leukocyte–endothelial cell adhesion in the retinal microcirculation through surface expression of intercellular adhesion molecule-1 Fumisato Hirataa,T, Munenori Yoshidaa, Yuji Niwaa, Masahiro Okouchib, Naotsuka Okayamab, Yoshiyuki Takeuchib, Makoto Itohb, Yuichiro Oguraa a

Department of Ophthalmology and Visual Science, Nagoya City University Graduate School of Medical Sciences, Mizuho-ku, Nagoya, Aichi 4678601, Japan b Department of Internal Medicine and Bioregulation, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan Received 19 August 2004 Available online 5 April 2005

Abstract The purpose of this study was to evaluate the effects of insulin on leukocyte–endothelial cell adhesion in the retinal microcirculation in vitro and in vivo. Human retinal endothelial cells (HRECs) were cultured in medium with or without insulin, and neutrophils allowed to adhere. Adherent neutrophils were quantified by measuring myeloperoxidase activity. Surface expression of endothelial adhesion molecules were studied with the use of an enzyme immunoassay. Insulin at concentrations of 50 and 100 AU/ml significantly increased neutrophil adhesion to HRECs compared with the control cells (P b 0.01, respectively). Surface expression of intercellular adhesion molecule-1 (ICAM-1) significantly increased when HRECs were exposed to 100 AU/ml insulin, as compared with the control cells (P b 0.05). Anti-ICAM-1 antibody significantly inhibited neutrophils adhesion to HRECs (P b 0.0001). Brown–Norway rats received subcutaneous injection of 0.2 U per 100 g body weight insulin three times. Control rats received the same amount of phosphate-buffered saline. Leukocyte entrapment in the retina was evaluated using acridine orange leukocyte fluorography. The number of leukocytes trapped in the retina of insulin-treated rats was significantly elevated compared with that in the control animals (P b 0.0001). These results suggested that insulin enhances leukostasis in retinal microcirculation. Hyperinsulinemia may be a risk factor of retinal microcirculatory disturbances. D 2005 Elsevier Inc. All rights reserved. Keywords: Insulin; Diabetic retinopathy; Hyperinsulinemia; Leukocyte; Early worsening

Introduction Clinical studies have demonstrated that acute intensive insulin therapy often causes an early worsening of diabetic retinopathy in patients with type 1 (Brinchmann-Hansen et al., 1992; Lauritzen et al., 1985; The Diabetes Control and Complications Trial Research Group, 1995; The Kroc Collaborative Study Group, 1988) or type 2 (Henricsson et al., 1995, 1997; Roysarkar et al., 1993) diabetes. Hyperinsulinemia is known as a risk factor of cardiovascular

T Corresponding author. Fax: +81 52 841 9490. E-mail address: [email protected] (F. Hirata). 0026-2862/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2005.03.002

disease (Ruige et al., 1998). Recently, vascular inflammation with atherosclerosis has been reported to be responsible for the onset of acute myocardial infarction (Ikeda et al., 1994). Adhesion of leukocyte to the endothelium, which is regulated by several endothelial adhesion molecules, is the crucial step in both vascular inflammation and atherosclerosis. There is also increasing evidence that leukocyte accumulation and enhanced expression of adhesion molecules in the retina are linked to the development of diabetic retinopathy (McLeod et al., 1995; Miyamoto and Ogura, 1999; Miyamoto et al., 1998, 1999). We have previously demonstrated that high levels of insulin exacerbate neutrophil–endothelial cell adhesion and increase the expression of endothelial intercellular adhesion molecule-1

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(ICAM-1) on human umbilical vein endothelial cells (HUVECs) (Okouchi et al., 2002a). Previously, we have also reported that high insulin enhances neutrophil transendothelial migration through increasing surface expression of platelet endothelial cell adhesion molecule-1 via activation of mitogen activated protein kinase on HUVECs (Okouchi et al., 2002b). However, the effect of hyperinsulinemia in the retinal microcirculation is poorly understood. The objective of this study is to evaluate the effect of insulin on leukocyte–endothelial interaction in the retinal microcirculation. We studied the effects of insulin on neutrophil adhesion to cultured human retinal endothelial cells (HRECs) and surface expression of adhesion molecules. We also investigated the in vivo effect of insulin on leukocyte accumulation in the retinal microcirculation of rats.

Materials and methods Cell culture This study was carried out in accordance with the tenets of the Declaration of Helsinki. HRECs (Cell Systems, Kirkland, WA) were cultured in collagen I-coated plates in CS-C medium (45% Dulbecco’s modified Eagles’ medium, 45% Ham’s F12 medium, 10% fetal bovine serum, 50 U/ml heparin, and 15 mmol/l HEPES; Cell Systems) with 10 ng/ ml fibroblast growth factor-basic (United States Biological, Swampscott, MA) at 378C in a 100% humidified atmosphere with 5% CO2. Cells were used for all experiments at passage 3. Neutrophil isolation Human neutrophilic polymorphonuclear leukocytes (PMN) were isolated from venous blood of healthy nonsmoking volunteers, after giving their informed consent, using standard dextran sedimentation and gradient separation on histopaque 1077 (Sigma, St. Lois, MO) (Yoshida et al., 1992). This procedure yielded a 98% pure PMN (acetic acid-crystal violet staining), and the PMN population was 95–98% viable (trypan blue exclusion). No platelet contamination was seen in isolated PMNs by examination using a microscope.

ical Co Ltd., Tokyo, Japan), and neutrophils were then added exactly at a concentration of 1  105 cells/well and allowed to adhere for 30 min at 378C. The monolayers were gently washed twice with 500 Al of warmed HBSS, and nonadhered neutrophils were then washed out. After washout, adhered neutrophils were quantified by myeloperoxidase (MPO) assay. MPO activity in neutrophils was determined using a method previously described (Grisham et al., 1990), with a minor modification, in which the H2O2-dependent oxidation of 3,3V,5,5V-tetramethylbenzine (Sigma) was measured. PMN adherence was expressed as the ratio of MPO activity in the adhered neutrophils to that in total neutrophils (1  105 cells/well). Surface expression assay of endothelial adhesion molecules Surface expression of ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), P-selectin and E-selectin was measured using a modified method of Khan et al. (1995), as previously reported (Okayama et al., 1998). Briefly, after incubation of cells with or without 100 AU/ml insulin for 48 h in 48-well collagen I-coated plates, cells were fixed with 1% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature. After 3 washes with 1 ml HBSS/PBS (1:1), cells were incubated with an anti-adhesion molecule antibody (primary antibodies; diluted 1:100; Serotec Ltd, Oxford, UK) and then with horseradish peroxidase-conjugated goat antimouse IgG (diluted 1:1000; Sigma) in HBSS/PBS containing 5% fetal calf serum for 60 min at 378C each. Cells were subsequently incubated with 0.25 ml 0.003% H2O2 plus 0.1 mg/ml 3,3V, 5,5V-tetramethylbenzidine (Sigma) as a substrate for 60 min at 378C in the dark. The color reaction was stopped by adding 75 Al 8 NH2SO4, and samples were transferred to 96-well plates for reading on a plate reader at 450 nm. Inhibitory protocols of PMN adhesion to HRECs after insulin exposure HRECs were incubated with following environments: (1) medium with 100 AU/ml insulin (Novolin R, Novo-Nordisk) for 48 h; (2) medium with 100 AU/ml insulin and 10 Ag/ml anti-ICAM-1 antibody (Sigma) for 48 h. A neutrophil–endothelial cell adhesion assay was done with both environments.

Neutrophil–endothelial cell adhesion assay

Animals

A neutrophil–endothelial cell adhesion assay was done according to a method described previously (Okayama et al., 1998). Briefly, HRECs were cultured on 48-well collagen I-coated plates and incubated for 48 h with or without human regular insulin (Novolin R, Novo-Nor-disk, Bagsvaard, Denmark). Culture medium was replaced with Hank’s balanced salt solution (HBSS; Nissui Pharmaceut-

All procedures performed in the present study were in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals. Male Brown–Norway rats, weighing approximately 200 g at 9 weeks of age, were used for the experiment. Thirty-six rats were divided into 4 groups: 16 rats received insulin or phosphate-buffered saline (PBS) for acridine orange leuko-

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cyte fluorography (AOLF), and 20 rats received insulin or PBS for the measurement of serum insulin concentration at 2 h after insulin treatment, as described next. Insulin treatment Eighteen rats received subcutaneous injection of 0.2 U per 100 g body weight insulin (Novolin R, Novo-Nor-disk) 3 times at 10-h intervals. Control rats (n = 18) received the same amount of phosphate-buffered saline. All rats were fed with 10% sucrose-containing rat chow and 5% sucrosesupplemented drinking water, as described in the literature (Solomon et al., 2000) only 2 days before AOLF or measurement of serum insulin concentration to prevent hypoglycemia. Measurement of serum insulin concentration Two hours after the third subcutaneous injection of 0.2 U per 100 g body weight insulin or PBS, 10 rats each were anesthetized intraperitoneally with a mixture (1:1) of xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (4 mg/kg). Blood samples were collected via an intracardiac puncture. Serum insulin concentrations were measured using a commercial kit (Rat Insulin [125I] Biotrak Assay System with Magnetic Separation, Amersham Biosciences, Buckinghamshire, England). Acridine orange leukocyte fluorography (AOLF) AOLF was performed as previously described elsewhere (Nishiwaki et al., 1996). This method allowed clear visualization of leukocytes and quantitative evaluation of their dynamics in the retinal microcirculation in vivo. In this technique, a scanning laser ophthalmoscope (SLO; Rodenstock Instrument, Munich, Germany), coupled with a computer-assisted image analysis system, made continuous high-resolution images of the fundus stained with the metachromatic fluorochrome acridine orange (Wako Pure Chemicals, Osaka, Japan). The dye emits a green fluorescence when it interacts with DNA. The argon laser (A9, 430 AW) was used for the illumination source, with a regular emission filter for fluorescein angiography because the spectral properties of leukocytes stained with acridine orange are similar to those of sodium fluorescein. A laser beam is focused at a spot on the retina only 100 ns. AOLF was performed 8 h after the third subcutaneous injection of insulin or PBS. Immediately before AOLF, rats were anesthetized, as described here. The pupils were dilated with 0.5% tropicamide and 2.5% phenylephrine. A contact lens was placed on the cornea to maintain transparency throughout the experiment. All whiskers were cut. Each rat had a 24-guage catheter inserted into the tail vein. The catheter was fixed by adhesive tape. Then, each rat was placed on a stereotaxic platform. Acridine orange (0.1%

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solution in saline) was injected continuously through the catheter for 1 min at a rate of 1 ml/min. At 30 min after injection, the fundus was observed to evaluate leukocytes accumulated in the retinal microcirculation with SLO. The obtained images were recorded on digital videotape for further analysis. After the experiment, the rat was killed with an overdose of anesthesia, and the eye was enucleated to determine a calibration factor for converting values measured on a computer monitor (in pixels) into real values (in micrometers). The calibration factor was the ratio between the actual size of each optic disk measured by microscopy and the apparent value on a computer monitor. Image analysis The digital video recordings were analyzed with an image analysis system, described in detail elsewhere (Nishiwaki et al., 1996), with a slight modification (Matsubara et al., 2000). In brief, we used a computer equipped with software (DVgate, SONY, Tokyo, Japan) that enters the digital images in real time (30 frames/s) to 640 horizontal and 480 vertical pixels with an intensity resolution of 256 steps into a personal computer. We evaluated diameters of major retinal vessels and the number of leukocytes accumulated in the retinal microcirculation through use of this system. Diameters of major retinal vessels were measured at 1 disc diameter from the center of the optic disc in images recorded just after acridine orange injection. The number of leukocytes was evaluated at 30 min after acridine orange injection. The number of fluorescent dots in the retina within 8 areas of 100 pixels square at a distance of 1 disk diameter from the edge of the optic disk was counted. Averages for individual areas were used as values for each rat. Statistical analysis All values were expressed as mean F SD. Two unpaired groups were compared using Student’s t test. ANOVA was used to compare 3 or more conditions, with post-hoc comparisons tested using Fisher’s protected least-significant difference test. Differences were considered statistically significant when the probability value was less than 0.05.

Results Effects of insulin on PMN adhesion to HRECs Incubation of HRECs with insulin significantly increased PMN adherence when the insulin concentrations were higher than 50 AU/ml for 48 h (Fig. 1). Adhered neutrophils on HRECs in the controls (without insulin) were 11.8 F 2.2% of total added neutrophils (1  105 cells/well). Insulin at concentrations of 50 and 100 AU/ml significantly increased neutrophil adhesion to HRECs (14.5 F 2.3%, P = 0.003 and

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control 0.048 F 0.004, n = 7, P N 0.05), P-selectin (high insulin 0.051 F 0.006, control 0.053 F 0.003, n = 7, P N 0.05) and E-selectin (high insulin 0.064 F 0.009, control 0.064 F 0.016, n = 7, P N 0.05) was not affected by the insulin treatment, respectively. Effect of an anti-ICAM-1 antibody on PMN adhesion to HRECs after insulin exposure Addition of an anti-ICAM-1 antibody attenuated the enhanced-PMN adhesion to HREC (high insulin 16.3 F 1.5%, high insulin with anti-ICAM-1 antibody 12.5 F 1.4%, n = 7, P b 0.0001). Serum insulin levels of rats

Fig. 1. Effect of various concentrations of insulin on PMN adherence to HRECs. Cells were treated without (control) or with 1, 10, 50 or 100 AU/ml (43.3, 432.9, 2164.5, 4329 pg/ml) insulin for 48 h. Values are expressed as mean F SD (n = 12). *P b 0.01 compared with controls.

16.6 F 2.1%, P = 0.0001, respectively). Furthermore, PMN adherence in 100 AU/ml of insulin concentration was significantly higher than with the 50 AU/ml concentration ( P = 0.016). Effect of insulin on surface expression of adhesion molecules of HRECs Surface expression of ICAM-1 significantly increased when HRECs were exposed to 100 AU/ml insulin for 48 h (high insulin 0.239 F 0.027, control 0.199 F 0.028, P = 0.018), as illustrated in Fig. 2. However, endothelial expression of VCAM-1 (high insulin 0.047 F 0.002,

Serum insulin levels in insulin-treated rats were higher than those in the controls at 2 h after three subcutaneous injections of 0.2 U per 100 g body weight insulin or PBS (insulin-treated 2331 F 836.1 pg/ml, control 1251.8 F 281.6 pg/ml, n = 10, P = 0.0011). Characteristics of insulin-treated and control rats for AOLF The comparison in body weight and blood glucose level at the time of dye injection between the insulin-treated rats and the controls is shown in Table 1. Insulin-treated rats had significantly lower blood glucose levels than the control rats ( P = 0.003). There were no significant differences in body weight between groups. Diameters of major retinal vessels In major retinal arteries, no significant differences in diameters were observed between insulin-treated and control rats (insulin-treated 27.8 F 1.9 Am, control 28.3 F 2.3 Am, n = 8, P N 0.05). Major retinal veins also showed no significant differences in diameters between insulin-treated and control rats (insulin-treated 40.1 F 1.9 Am, control 40.2 F 2.5 Am, n = 8, P N 0.05). Leukocyte accumulation in the retinal microcirculation of rats The accumulated leukocytes were easily observed 30 min after acridine orange injection. Figs. 3A and B show typical examples of late-phase retinal images of control and Table 1 Characteristics of insulin-treated and control rats

Fig. 2. Effect of 100 AU/ml insulin for 48 h on surface expression of ICAM1 on HRECs. Values are expressed as mean F SD (n = 7). *P b 0.05 compared with controls.

Number Body weight (g) Blood glucose (mg/dl)

Control

Insulin treated

8 202 F 7 191 F 19

8 208 F 9 156 F 18T

Values are mean F SD. T P b 0.01 compared with control rats.

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Fig. 3. Digitized images of ocular fundus obtained from a control (A) and an insulin-treated rat (B) 30 min after acridine orange injection. The trapped leukocytes are recognized as fluorescent dots. Scale bars = 100 Am.

insulin-treated rats. The density of leukocytes trapped in the retina of insulin-treated rats was 8.4 F 1.3 cells/mm2, which was significantly elevated ( P b 0.0001) compared with that in control animals (3.4 F 0.9 cells/mm2), as illustrated in Fig. 4.

Discussion We demonstrated that 50 and 100 AU/ml of insulin enhanced neutrophil–endothelial adhesion on cultured HRECs. A 100 AU/ml level of insulin also enhanced surface expression of ICAM-1. The insulin concentrations that we used (50 and 100 AU/ml) are thought to be around the physiological concentrations present in the insulinresistant condition because concentrations greater than 100 AU/ml can be found in insulin-resistant patients. Evidence of PMN activation in diabetic patients was reported (Wierusz-Wysocka et al., 1987). In this study, we used PMNs isolated from healthy volunteers, as in previous reports (Okayama et al., 1998; Okouchi et al., 2002a, 2003, 2004), to prove direct effect of insulin on HRECs. We also demonstrated that 3 times 0.2 U per 100 g body weight insulin injected subcutaneously induced leukocyte entrapment in the retina of rats. We confirmed that serum levels of insulin were actually increased by this method in the treated rats. King et al. (1983) reported that endothelial cells of microvessels and macrovessels showed differential responsiveness to insulin. The authors suggested that retinal capillary endothelium and retinal pericytes are both very

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insulin-sensitive tissues. We previously showed that 100 AU/ml insulin enhances neutrophil–endothelial adhesion on cultured HUVECs (Okouchi et al., 2002a). In this study, we demonstrated that lower concentration of insulin (50 AU/ml) also enhances neutrophil–endothelial adhesion on cultured HRECs. Our findings suggested that the retinal endothelial cells are more sensitive to insulin than the endothelial cells of other organs. In addition, we confirmed that hyperinsulinemia induced leukostasis in vivo. Abiko et al. (2003) showed that insulin resistance induced leukostasis in the retina using insulin-resistant rat. Thus, both hyperinsulinemia and insulin resistance exacerbate leukostasis in the retinal microcirculation. Leukostasis have been suggested to contribute to retinal vascular leakage and nonperfusion (Miyamoto et al., 1999). In addition, leukocyte retention has been shown to cause tissue injury by producing superoxide radicals (Werns et al., 1985). Several possible mechanisms are speculated for the enhanced leukocyte adhesion on retinal endothelium after insulin exposure. First, insulin directly acts on retinal endothelium to increase cell adhesion molecules. Our previous studies demonstrated that 100 AU/ml insulin enhanced surface expression of ICAM-1 but did not affect P-selectin or E-selectin on HUVECs (Okouchi et al., 2002a). In this report, 100 AU/ml insulin enhanced surface expression of ICAM-1 and did not affect VCAM-1, Pselectin or E-selectin on HRECs similarly. Furthermore, anti-ICAM-1 antibody decreased PMN adhesion to HRECs after insulin exposure. We previously investigated the intracellular signal transduction pathways on HUVECs after insulin exposure (Okouchi et al., 2002a). Enhancement of neutrophil adhesion and surface expression of ICAM-1 were attenuated by protein kinase C (PKC) inhibitors and a mitogen-activated protein (MAP) kinase inhibitor on HUVECs. The enhanced surface expression of ICAM-1 on the retinal endothelial cells by insulin may be caused via activation of PKC and MAP kinase.

Fig. 4. Density of leukocytes trapped in the retinal microcirculation. Values are expressed as mean F SD (n = 8). *P b 0.0001 compared with control rats.

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Lu et al. (1999) have recently reported that intravitreous injection of insulin increases vascular endothelial growth factor (VEGF) mRNA and secretes protein levels in retinal pigment epithelial cells through enhanced transcription of the VEGF gene. Miyamoto et al. (2000) has also demonstrated that intravitreous injection of VEGF increases leukostasis in the retina and that this phenomenon is blocked by anti-ICAM-1 antibody. A second possible mechanism is that insulin enhances leukocyte adhesion through up-regulation of VEGF. Iida et al. (2001) have reported that insulin up-regulates tumor necrosis factor-a (TNF-a) in macrophages. Murakami et al. (2000) have reported that TNF-a induced ICAM-1 mRNA in cultured HUVECs. Third possible pathway is that TNF-a produced by insulin-stimulated macrophages enhances ICAM-1 on retinal endothelium. In addition, insulin may cause vascular constriction or decrease blood flow. There are no direct data on how insulin affects retinal vascular hemodynamics (Lu et al., 1999). Although the blood flow was not measured, the present study shows that increased leukocyte accumulation in insulin-treated rat is not due to changes in diameters of major retinal vessels. Furthermore, Iwashita et al. (2001) reported insulin increase blood flow in the microvasculature of cremaster muscle. From these reasons, it is unlikely that vascular constriction or decreased blood flow leads to leukostasis in the retina. Further studies are needed to elucidate the exact mechanisms of enhanced leukocyte adhesion of the retinal endothelial cells by insulin. Early worsening of diabetic retinopathy has been observed in patients in response to a sudden and sustained reduction in the average blood glucose level after insulin treatment (Chantelau, 2001; The Diabetes Control and Complications Trial Research Group, 1995). It has been reported that insulin-induced VEGF gene expression may cause early worsening of diabetic retinopathy (Poulaki et al., 2002), yet the mechanism is still obscure. Our results suggest that enhanced leukostasis caused by insulin may be one explanation for the early worsening of diabetic retinopathy after intensive insulin treatment. In conclusion, the present study demonstrated the enhancement of leukocyte adhesion in the retinal microcirculation after insulin exposure in vitro and in vivo. Hyperinsulinemia may be a risk factor of retinal microcirculatory disturbances. Further investigations may show that our findings have contributed to the understanding of the pathogenic mechanism of retinal microvascular abnormality in diabetes.

Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research (No. 16390501) from the Ministry of Education, Science, and Culture of Japan. The authors thank Kenichi

Miyaki and Takeshi Kurachi for assistance with AOLF and Maxine A. Gere for editorial assistance.

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