PKC-β Inhibitor (LY333531) Attenuates Leukocyte Entrapment in Retinal Microcirculation of Diabetic Rats

Abstract

purpose. The activity of protein kinase C (PKC), preferentially β isoform of PKC, has been shown to be elevated in the diabetic retina. Recently, LY333531, a specific inhibitor of PKC-β, has been reported to improve the decrease of retinal blood flow in early diabetes. Increased leukocyte entrapment has been suggested to be involved in blood flow disturbances in the early diabetic retina. This study was designed quantitatively to evaluate leukocyte entrapment in the retinal microcirculation of diabetic rats and the effect of LY333531 on leukocyte entrapment.

methods. Diabetes was induced in male Long-Evans rats by intraperitoneal injection of streptozotocin (60 mg/kg). LY333531 (0.1, 1.0, or 10.0 mg/kg/d) was administered orally during a 4-week diabetic period. Leukocyte entrapment in the retinal microcirculation was quantitatively evaluated in vivo with acridine orange digital fluorography.

results. The number of leukocytes trapped in the retinal microcirculation of diabetic rats (mean ± SEM; 14.3 ± 1.3 cells/mm²) was significantly increased, compared with nondiabetic control rats (7.5 ± 0.3 cells/mm²; P < 0.0001). Oral administration of LY333531 significantly decreased the number of leukocytes trapped in the retinal microcirculation of diabetic rats (10.9 ± 0.6, 11.3 ± 0.7, and 10.4 ± 0.4 cells/mm² with LY333531 0.1, 1.0, and 10.0 mg/kg/d, respectively; P < 0.05).

conclusions. Treatment with LY333531 attenuated the increase of leukocyte entrapment in the retinal microcirculation during the period of early diabetes. This effect may contribute to the improvement of abnormal retinal blood flow in early diabetes with LY333531. LY333531 might have a therapeutic efficacy in preventing microcirculatory flow disturbances by trapped leukocytes in the early diabetic retina.

Among various hyperglycemia-induced metabolic changes that could lead to diabetic complications, activation of protein kinase C (PKC), especially β isoform, has been shown to contribute to abnormal retinal hemodynamics in diabetic animals. ¹ Recently, an inhibitor selective for the β isoform of PKC (LY333531) has been developed, which normalizes abnormal retinal blood flow of diabetic rats in parallel with its inhibition of PKC activities when orally administered or intravitreally injected. ^{2 3} Because abnormal retinal hemodynamics due to diabetes may be associated with the development of diabetic retinopathy, ⁴ LY333531 has attracted a great deal of attention as a potent agent for treatment of diabetic retinopathy. 

In contrast to their beneficial role with immunologic and antimicrobial activity, leukocytes are involved in the pathogenesis of various pathogenic conditions including diabetes. ⁵ Histologic evidence has suggested that microvascular occlusion and endothelial cell damage in the diabetic retina, which are primary events in the pathogenesis of diabetic retinopathy, were associated with the presence of leukocytes. ⁶ Recent in vivo studies have suggested that increased leukocyte entrapment in the early diabetic retina ⁷ may be associated with vascular nonperfusion and vascular leakage. ⁸ Therefore, leukocytes trapped in the diabetic retina may initiate a series of events leading to diabetic retinopathy. 

The purpose of this study was to evaluate quantitatively the effectiveness of LY333531 treatment on leukocyte entrapment in the retinal microcirculation of diabetic rats in vivo. 

Methods

Rat Model

All procedures performed in the present study were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male pigmented Long-Evans rats, weighing 200 to 250 g (n = 48), received an intraperitoneal injection of streptozotocin (STZ, 60 mg/kg; Sigma Chemical, St. Louis, MO) in physiologic saline after an overnight fast. We confirmed that the plasma glucose level in each rat was >250 mg/dl 48 hours after injection. Sixteen Long-Evans rats, which were injected with an equal volume of saline alone, served as nondiabetic controls. All rats were kept with free access to water and food in an air-conditioned room with a 12-hour light–12-hour dark cycle for 4 weeks before acridine orange fluorography was performed. 

To examine whether the administration of PKC-β inhibitor, LY333531 (Eli Lilly, Indianapolis, IN) can attenuate leukocyte entrapment in the retinal microcirculation of diabetic rats, LY333531 was administered orally at dosages of 0.1 (n = 8), 1.0 (n = 16), and 10.0 mg/kg/d (n = 8) for 4 weeks, from the time STZ was injected in the rats. 

Acridine Orange Digital Fluorography

Leukocyte entrapment in retinal microcirculation was evaluated with acridine orange digital fluorography, which has been previously described in detail elsewhere. ^{9 10} This technique uses a scanning laser ophthalmoscope (Rodenstock Instruments, Munich, Germany), coupled with a computer-assisted image analysis system, which makes continuous high-resolution images of fundus stained by acridine orange (Wako Pure Chemicals, Osaka, Japan). Acridine orange, a metachromatic fluorochrome, is a widely used probe in biochemical and cytochemical studies. The dye emits a green fluorescence when it interacts with DNA. The argon blue laser 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. 

Leukocyte entrapment was evaluated only once after a 4-week diabetic period in both groups of rats with and without LY333531 treatment, using one eye (right eye) of each rat. Immediately before acridine orange digital fluorography, rats were anesthetized with a mixture (1:1) of xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (10 mg/kg). The pupils were dilated with 0.5% tropicamide and 2.5% phenylephrine hydrochloride. A contact lens was placed on the cornea to maintain transparency throughout the experiments. Each rat had a catheter inserted into the tail vein and was placed on a movable platform. Body temperature was maintained between 37°C and 39°C throughout the experiment. 

Acridine orange (0.1% solution in saline) was injected continuously through the catheter for 1 minute at a rate of 1 ml/min. At 30 minutes after the injection, the fundus was observed to evaluate leukocytes accumulated in the retinal microcirculation with the scanning laser ophthalmoscope (SLO; Rodenstock Instruments). The obtained images were stored on an S-VHS videotape to replay and evaluate leukocyte entrapment in retinal microcirculation quantitatively. 

We analyzed the video recordings with an image analysis system, as described in detail elsewhere. ^{9 10} In brief, the system consists of a computer equipped with a video digitizer (Radius, San Jose, CA) that digitizes the video image in real time to 640 horizontal and 480 vertical pixels with an intensity resolution of 256 steps. We evaluated the number of leukocytes trapped in retinal microcirculation 30 minutes after acridine orange injection, as described previously. ¹⁰ Briefly, an observation area around the optic disc was determined by drawing a polygon surrounded by the adjacent major retinal vessels. The area was measured in pixels on a computer monitor, and the density of trapped leukocytes was calculated by dividing the number of trapped leukocytes that were recognized as fluorescent dots by the area of the observation region. The densities of leukocytes were calculated generally in eight peripapillary observation areas. The average density of individual areas was used as the number of leukocytes trapped in the retinal microcirculation for each rat. 

After the experiment, the rat was killed with an overdose of anesthesia. The eye was enucleated to determine a calibration factor to convert values measured on a computer monitor (in pixels) into real values (in μm). 

Statistical Analysis

Data are expressed as mean values ± SEM. The data were analyzed using an analysis of variance, 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

The plasma glucose levels were compared between the control and diabetic rats (Table 1) . The plasma glucose levels of STZ-induced diabetic rats were significantly higher than those of the control rats. There were no significant differences in plasma glucose levels among the groups of diabetic rats without and with treatment of LY333531 in each dose. 

Acridine orange easily infiltrates through the vessel wall and diffuses into the retina because of the permeability of the membrane. Accordingly, a few minutes after acridine orange injection was stopped, fluorescence of circulating leukocytes was faint because of the washout effect. In contrast, leukocytes trapped in the retina remained fluorescent for approximately 2 hours. These leukocytes were recognized as distinct fluorescent dots at 30 minutes after acridine orange injection, although no circulating leukocytes fluoresced (Fig. 1) . 

Figure 2 shows the number of leukocytes trapped in the retinal microcirculation in five groups: control rats, diabetic rats without treatment, and diabetic rats treated with 0.1, 1.0, and 10.0 mg/kg/d of LY333531. In the diabetic rats, the number of trapped leukocytes (14.3 ± 1.3 cells/mm²) increased significantly compared with the control rats (7.5 ± 0.3 cells/mm²; P < 0.0001). Oral administration of LY333531 resulted in a significant reduction of leukocyte entrapment in the diabetic retina (10.9 ± 0.6, 11.3 ± 0.7, and 10.4 ± 0.4 cells/mm² with LY333531 0.1, 1.0, and 10.0 mg/kg/d, respectively; P < 0.05). The increased entrapment of leukocytes due to a 4-week diabetic period was reduced by 50.6%, 44.1%, and 57.4% with treatment of LY333531 at doses of 0.1, 1.0, and 10.0 mg/kg/d, respectively. 

Discussion

In this study, we demonstrated the inhibitory effects of a novel specific inhibitor of PKC-β, LY333531, on leukocyte entrapment in the diabetic retina. Accumulating evidence would support the significance of this effect, suggesting that leukocytes play an important role in the pathogenesis of diabetic retinopathy. Recent histopathologic studies showed many capillary occlusions by leukocytes in retinas of chemically induced diabetic rats ⁶ and increased numbers of polymorphonuclear leukocytes in retinas of diabetic patients. ¹¹ Lutty et al. ¹² demonstrated that leukocytes would contribute to not only vascular occlusion but also endothelial cell injury in the choroid of diabetic patients. Moreover, leukocytes trapped in the diabetic retina could increase vascular permeability ⁸ by causing endothelial dysfunction and damaging the blood retinal barrier, because diabetic leukocytes were reported to be more activated ^{6 13} and to produce more oxygen-derived free radicals than normal leukocytes. ¹⁴ In addition, retinal hypoxia and neovascularization were suggested to be associated with the presence of leukocytes in the diabetic retina. ^{6 15} Thus, leukocytes trapped in retinal microcirculation have been shown to be associated with various pathologic changes observed in diabetic retinopathy. 

A growing body of evidence has suggested various abnormalities of microvascular rheology in diabetes. Leukocytes become less deformable in diabetes, in contrast to their natural property, which includes a larger volume and greater rigidity than erythrocytes. ¹⁶ This change would increase retinal microvascular occlusion by leukocytes. Moreover, leukocyte adhesion to retinal vascular endothelial cells would be increased in diabetes, supported by an in vitro examination showing increased adhesion of leukocytes after 24 hours’ exposure in a high-glucose condition. ¹⁷ Adhesion of leukocytes to the vascular endothelium is known to be mediated by the adhesion molecules, including intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1. Recent studies demonstrated that elevated number of leukocytes was accompanied by upregulation of ICAM-1 in the diabetic rat retina ⁸ and human retina. ¹¹ Expression of VCAM-1 was also significantly increased in cultured endothelial cells after high-glucose treatment for 24 hours. ¹⁸ These alterations due to diabetes would contribute to increased leukocyte entrapment in the diabetic retina. 

Recent reports have strongly enhanced the important role of PKC, especially β isoform, in alterations of retinal blood flow due to diabetes. ¹ PKC-β is preferentially activated in the retina, heart, aorta, and renal glomeruli of experimental diabetic animals. ^{19 20} Activation of the PKC-β has been shown to mimic the abnormal retinal blood circulation observed in early diabetes, ²⁰ and inhibition of PKC-β with LY333531 has been shown to normalize diabetic abnormal circulation of various organs in diabetic rats. ² Harris et al. ²¹ showed that microvascular flow resistance increased due to leukocyte plugging in the capillaries of skeletal muscle during experimental diabetes. Not only leukocytes plugged in the microcirculation, but also adhering leukocytes may cause a large increase of flow resistance. ²² Accordingly, the ability of LY333531 to reduce leukocyte entrapment in the diabetic retina would contribute to its beneficial effect to ameliorate abnormal diabetic circulation by reducing microvascular flow resistance. 

PKC is known to be a key regulatory protein with numerous substrates that affect nuclear and cytoplasmic events. Recent studies have suggested that PKC is involved in the expression of adhesion molecules on endothelial cells, such as ICAM-1 and VCAM-1. An in vitro study indicated that activation of PKC induces upregulation of ICAM-1 on human umbilical vein endothelial cells (HUVECs) and subsequent leukocyte adhesion to endothelial cells. ²³ Another study suggested that expression of VCAM-1 on HUVECs is also mediated by PKC. ²⁴ Furthermore, blocking PKC activity has been shown to inhibit glucose-induced leukocyte adhesion in in vitro experiments. ¹⁸ Because increased expression of ICAM-1 is reportedly involved in increased leukocyte entrapment in the diabetic retina, ⁸ the antiadhesive effect of inhibiting PKC activity would account for a reduction of leukocyte entrapment in diabetic retina with LY333531. Moreover, PKC activation is also involved in vasocontractility at microvessels. ¹ Therefore, LY333531 may improve lower perfusion in the retina in the early stages of diabetes by inhibiting vasocontraction. The increased blood flow causes higher shear stress at the retinal microcirculation, which may result in the reduction of the number of static leukocytes in the diabetic retina. ²⁵ However, the present study does not include any experiments to elucidate possible mechanisms explaining the inhibitory effect of LY333531 on leukocyte entrapment in the diabetic retina. Further examinations will be needed to investigate the mechanisms of this interesting phenomenon. 

In conclusion, the present study demonstrated the inhibitory effect of LY333531 on leukocyte entrapment in the diabetic retina. In light of the role leukocytes seem to play in diabetic retinopathy, treatment with LY333531 might have therapeutic efficacy in preventing the development of diabetic retinopathy. 

 Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture.

 Submitted for publication October 21, 1999; revised January 24, 2000 and April 3, 2000; accepted April 11, 2000.

 Commercial relationships policy: N.

 

Table 1.

 

Plasma Glucose in Peripheral Blood

LY333531	Control	DM
				No Treatment	0.1 mg/kg/day	1.0 mg/kg/day	10.0 mg/kg/day
Plasma glucose (mg/dl)	186.23± 17.9	439.1± 16.7*	446.0± 21.4*	450.0± 29.8*	420.0± 21.2*

 Values are mean ± SEM.

*   P < 0.001 compared with control rats. There were no significant differences among each group of diabetic rats without and with LY333531 treatment at three different doses.

Figure 1.

 

Leukocytes trapped in the retinal microcirculation were observed as fluorescent dots 30 minutes after acridine orange injection. A small number of leukocytes were found in the control rats (A). Leukocyte entrapment significantly increased after the 4-week diabetic period (B). Significant reduction of leukocyte entrapment was seen in rats treated with LY333531 for 4 weeks at dosages of 0.1 (C), 1.0 (D), and 10.0 mg/kg/d (E).

Figure 2.

 

Density of leukocytes trapped in the retinal microcirculation. Values are means ± SEM. *P < 0.01; ^† P < 0.05 compared with control rats; ^‡ P < 0.05 compared with diabetic rats without treatment.