VEGF-initiated blood-retinal barrier breakdown in early diabetes

PURPOSE: The objectives of this study were to (1) determine whether endogenous vascular endothelial growth factor (VEGF) triggers diabetic blood-retinal barrier breakdown, and (2) identify the site as well as phenotype of the hyperpermeable diabetic retinal vessels. METHODS: Retinal VEGF mRNA levels were quantified in 1-week diabetic rats using the RNase protection assay. VEGF bioactivity was blocked via the systemic administration of a highly specific VEGF-neutralizing soluble Flt/F(c) construct (VEGF TrapA(40)). An inactive IL6 receptor/F(c) construct (IL6R Trap) was used as an isotype control. Blood-retinal barrier breakdown was quantified using the Evans blue technique and was spatially localized with fluorescent microspheres. RESULTS: Retinal VEGF mRNA levels in 1-week diabetic animals were 3.2-fold higher than in nondiabetic controls (P < 0.0001). Similarly, retinal vascular permeability in 8-day diabetic animals was 1.8-fold higher than in normal nondiabetic controls (P < 0.05). Diabetes-induced blood-retinal barrier breakdown was dose-dependently inhibited with VEGF TrapA(40), with 25 mg/kg producing complete inhibition of the diabetes-induced increases (P < 0.05). Blood-retinal barrier breakdown in diabetic animals treated with solvent alone or IL6R Trap did not differ significantly from untreated diabetic animals (P > 0.05). Spatially, early blood-retinal barrier breakdown was localized to the retinal venules and capillaries of the superficial retinal vasculature. CONCLUSIONS: Early blood-retinal barrier breakdown in experimental diabetes is VEGF dependent and is restricted, in part, to the venules and capillaries of the superficial inner retinal vasculature. VEGF inhibition should prove a useful therapeutic approach in the treatment of early diabetic blood-retinal barrier breakdown.     

Corticosteroids inhibit VEGF-induced vascular leakage in a rabbit model of blood-retinal and blood-aqueous barrier breakdown

Purpose

Recent clinical studies show that a single intravitreal injection of the corticosteroid triamcinolone acetonide (TAA) may reduce edematous retinal swelling and improve visual acuity in patients with diabetic macular edema (DME). In addition, clinical and experimental studies strongly suggest that blood-retinal barrier breakdown in diabetes is induced by vascular endothelial growth factor (VEGF). These results suggest that corticosteroids may modulate VEGF-mediated responses in vivo. To test this hypothesis directly, the current study evaluated the effects of TAA and dexamethasone (DEX) in a newly developed rabbit model of VEGF-induced blood-retinal barrier and blood-aqueous (iris) barrier breakdown.

Methods

VEGF₁₆₅ or vehicle was injected intravitreally in female Dutch Belt rabbits, and scanning ocular fluorophotometry was used to non-invasively measure fluorescein leakage from retinal and iris vasculature. VEGF-induced retinal vasculopathy was further assessed with fundus imaging, fluorescein angiography, and ocular coherence tomography. For pharmacologic studies, rabbits were treated with either DEX (2 mg kg⁻¹ daily, s.c.), TAA (2 or 4 mg, intravitreal), indomethacin (20 mg kg⁻¹ daily, s.c.), or vehicle (s.c. or intravitreal). Human umbilical vein endothelial cells (HUVEC) were loaded with the fluorescent Ca²⁺ indicator dye fluo-4 and treated with dexamethasone (0·1-10 μm) or vehicle for either 2 or 24 hr prior to stimulation with 10 ng ml⁻¹ VEGF₁₆₅.

Results

VEGF injected intravitreally induced a time and dose-dependent breakdown of the blood-retinal and blood-aqueous barriers. Maximal vascular leakage was measured at 48 hr after intravitreal injection with a dose of 500 ng VEGF. Other effects of VEGF included prominent retinal vasodilation, vessel tortuousity, fluorescein leakage from retinal vessels, and inner retinal edema. These VEGF-mediated responses are transient and approach baseline by 1 week. VEGF-induced blood-retinal and blood-aqueous barrier breakdown was completely blocked by the corticosteroid DEX administered systemically for 3 days. In contrast, the non-steroidal anti-inflammatory drug, indomethacin, had no effect. In a separate study with VEGF injected intravitreally at six different time points over 5 months, a single intravitreal 2 mg dose of TAA completely blocked VEGF-induced retinal and iris leakage for 45 days. VEGF/VEGF receptor-2-mediated Ca²⁺ mobilization in endothelial cells was not affected by 2 or 24 hr pretreatment with dexamethasone.

Conclusion

These results indicate that one mechanism by which corticosteroids block blood-ocular barrier breakdown and edema is via their modulation of signaling or effector proteins downstream of the VEGF receptor.     

Hyperbaric oxygen therapy ameliorates the blood-retinal barrier breakdown in diabetic retinopathy

BACKGROUND: To study the effect of hyperbaric oxygen (HBO) therapy on diabetic retinopathy in a streptozotocin-induced diabetic rat model. METHODS: Sprague-Dawley albino male rats were divided into three groups. The three groups were as follow: (i) non-diabetic control group (non-DM control); (ii) diabetic control group (DM control); and (iii) diabetic rats receiving hyperbaric oxygen therapy (DM HBO). Rats in DM HBO group were incubated in an oxygen monoplace chamber. The HBO condition was set at 2.5 atmospheres and 100% oxygen. The duration of a single HBO treatment was 90 min. Rats in DM HBO groups received HBO three times per week for 3 months. Retinal vascular permeability was assessed by measuring fluorescein isothiocyanate-labelled bovine albumin and retinal Evans blue leakage into the retina. RESULTS: We found that the retinal parenchyma showed prominent thickening but not statistically significant in rats with DM, corresponding to the retinal oedema, compared with the control and DM HBO groups. fluorescein isothiocyanate relative fluorescence intensity (Mean+/-SE) in normal control animals, diabetic animals, and HBO-treated diabetic animals was 356+/-47, 865+/-78, and 518+/-49, respectively, demonstrating significant difference between the means of diabetic and HBO-treated diabetic animals, and between means of control and diabetic animals (n=8, P<0.05). Retinal Evans blue leakage in control animals, diabetic animals, and HBO-treated diabetic animals was 7.6+/-2.9, 18.5+/-4.2 and 10.2+/-3.1 microL plasma/g retinal dry weight/h, respectively, demonstrating significant difference between the means of diabetic and HBO-treated diabetic animals, and between means of control and diabetic animals (n=8, P<0.05). CONCLUSION: HBO therapy may diminish the extent of the increased blood-retinal barrier breakdown in diabetic animals.     

糖尿病血-视网膜屏障破坏的机制研究进展

血-视网膜屏障破坏所致的血管渗漏是糖尿病视网膜病变视力下降的主要原因,血管内皮细胞以及周围基质成分所构成的视网膜内屏障是受累的主要部位,血管内成分可以跨过受损的细胞间连接间隙,或通过细胞的吞饮囊泡的形态移动到组织间隙,这两种方式都在糖尿病视网膜病变中存在.内皮细胞间结构的破坏,吞饮方式的增加与高血糖异常代谢所致的氧化应激、炎症反应,以及周围其他细胞如周细胞、胶质细胞的凋亡,异常活化,以及由各种细胞所分泌的细胞因子如血管内皮生长因子(VEFG)和基质金属蛋白酶(MMP)等有密切的关系.     

曲安奈德对糖尿病鼠血-视网膜屏障破坏的治疗作用

目的探讨曲安奈德(TA)玻璃体注射在糖尿病Wistar鼠血-视网膜屏障(BRB)破坏方面的保护作用。方法选择健康成年Wistar鼠,链脲佐菌素(STZ)诱导大鼠糖尿病。分为正常对照组,糖尿病4个月(DM4)组和6个月(DM6)组,每组各分为形态学观察组和BRB测定组,BRB测定组再分为未行TA治疗(NT)组、TA治疗1周组和2周组。行消化铺片HE染色观察视网膜血管形态,利用视网膜标准化Evansblue(EB)含量测定大鼠BRB的变化。结果正常对照组各组间EB含量差异无统计学意义(P>0.05)。同正常对照组相比,DM4组大鼠出现毛细血管管径粗细不一,血管闭锁,内皮细胞增生,周细胞丢失等形态学改变;DM6组上述病变进一步加重,出现无细胞性毛细血管,糖尿病NT组EB含量明显升高(P>0.01),且6个月组高于4个月组,差异有统计学意义(P>0.01)。同糖尿病NT组相比,各糖尿病治疗组EB含量均显著降低(P>0.01),但除4个月治疗2周组EB含量基本恢复正常(P>0.05)外,余治疗组均仍高于正常对照组(P<0.05)。各糖尿病治疗组间EB含量差异无统计学意义(P>0.05)。结论糖尿病大鼠视网膜的形态学变化与BRB的破坏有关,TA对糖尿病大鼠BRB的破坏具有保护作用。     

Outward permeability of the blood-retinal barrier

To characterize quantitatively the active transport mechanism of the blood-retinal barrier (BRB), we estimated the inward (Pin) and outward permeability (P_out) of the BRB in monkey eyes using vitreous fluorophotometry. P_in values for fluorescein (F) and fluorescein glucuronide (FG) were simulated by computer in separate experiments following measurements of intraocular fluorescence at 1 h following the intravenous injection of F or FG. The estimated mean P_in values for F and FG were 4.7 ± 1.6 and 5.9 ± 1.7 × 10^–6 cm/min, respectively, in ten eyes of five monkeys. At 1 month thereafter, F was injected into the right vitreous cavity and the same amount of FG was injected into the left vitreous cavity. The intraocular kinetics of fluorescence were measured at 6–24 h postinjection. As estimated by the simulation model, the mean P_out values were 7.7 ± 2.6 and 1.7 ± 0.9 × 10^–4 cm/min for F and FG, respectively. The P_out/P_in ratio was 160±78 for F and 26±9 for FG. The intraperitoneal injection of probenecid resulted in a significant decrease in the P_out value for F (P < 0.005) but had no significant effect on that for FG, suggesting that F is excreted from the retina via an active transport mechanism; as characterized by the P_out/P_in ratio, the actual magnitude of the latter is far greater than that previously reported.This research was supported in part by a 1988 Grant-in-Aid for the Encouragement of Young Scientists (63771366, to A.Y.) from the Ministry of Education, Science and Culture, Japan Offprint requests to: A. Yoshida     

Oscillatory potentials and permeability of the blood-retinal barrier in noninsulin-dependent diabetic patients without retinopathy

Electroretinography and vitreous fluorophotometry were performed in 36 eyes of 36 noninsulin-dependent diabetic patients and in 32 eyes of 32 healthy control subjects between the ages of 30 and 59 years. Fluorescein fundus angiograms showed no abnormalities in either group. Peak implicit time of the first deflection of the oscillatory potential, interpeak interval between the first and second deflections, and the sum of the amplitudes of the upward deflections were analyzed. Inward permeability of the blood-retinal barrier was calculated by vitreous fluorophotometry and computer simulation. The peak implicit time of the first deflection and the interpeak interval between the first and second deflections of the oscillatory potential were significantly longer in diabetic patients than in control subjects (P less than 0.01). No significant difference in inward permeability of the blood-retinal barrier existed between the two groups. These results indicate that a selective delay in the peak implicit time of the oscillatory potential (neurosensory retinal abnormality) may be present in noninsulin-dependent diabetic patients, representing retinal functional changes before changes in blood-retinal barrier permeability are apparent.     

Effect of calcium dobesilate on the blood-retinal barrier in early diabetic retinopathy

Summary The effect of calcium dobesilate on the alteration of the blood-retinal barrier was studied in 41 adult-onset, non-insulin dependent diabetic patients with minimal or no retinopathy, randomly assigned to receive either oral calcium dobesilate (1000 mg twice daily) or a placebo for 12 months. The posterior vitreous value and the penetration ratio, determined by vitreous fluorophotometry, reflected stabilisation of blood-retinal barrier permeability in the calcium dobesilate patients and deterioration of blood retinal barrier in those given placebo. During the relatively short period of the study, one year, no significant change in microaneurysm and capillary closure gradings was observed. No side effects were associated with calcium dobesilate.     

Early breakdown of the blood-retinal barrier in diabetes.

The blood-retinal barrier plays an important part in the processes of retinal pathophysiology. A new clinical method for the study of the blood-retinal barrier by vitreous fluorophotometry appears to satisfy the necessary requirements in that it is quantitative and shows good reproducibility and high sensitivity. The application of this method to a series of diabetic patients with apparently "normal" fundi revealed the presence of a significant breakdown of the blood-retinal barrier in the early stages of retinal involvement in diabetes. The extent of the breakdown can be measured allowing for comparative and evolutionary evaluations. The disturbance of the blood-retinal barrier, as evidence by vitreous fluorophotometry, appears before microaneurysms or capillary closure can be demonstrated by fluorescein angiography.     

Inhibitory effects of bucillamine on increased blood-retinal barrier permeability in streptozotocin-induced diabetic rats

PURPOSE: To investigate the effect of bucillamine for prevention of increasing blood-retinal barrier (BRB) permeability in streptozotocin (STZ)-induced diabetic rats. METHODS: The groups included control and STZ-induced diabetic rats treated with or without bucillamine. Six months after intervention, the concentrations of reduced and oxidative glutathione (GSH and GSSG) in the retina were measured biochemically. In addition, vitreous fluorescein, which leaks from the vessels after intravenous injection of fluorescein sodium, was measured to evaluate BRB permeability. To evaluate the scavenging ability against the reactive oxygen species (ROS) in vitro, the second-order rate constant for the reaction of bucillamine with ROS was estimated from the kinetics based on the rate constant for the reaction of ROS. RESULTS: The BRB permeability was significantly higher (p = 0.01) in diabetic rats not treated with bucillamine, and bucillamine inhibited the BRB permeability. The GSH concentration and the GSH/GSSG ratio in the retinas decreased in diabetic rats not treated with bucillamine; bucillamine inhibited the decrease of the GSH concentrations. The ROS scavenging activity of bucillamine was similar with that of GSH. CONCLUSIONS: In diabetic retinas, oxidative stress might increase, which may be one of the causes of BRB breakdown. The antioxidant effects of bucillamine might take part in inhibition of increased permeability of the BRB in diabetes.     

Inward permeability of the blood-retinal barrier by fluorophotometry

A method for assessing the inward permeability of the blood-retinal barrier by fluorophotometry is presented. The permeability value is calculated with the fluorophotometer computer from fluorophotometric scan values in vitreous and non-protein-bound fluorescein concentration values in plasma. No diffusion coefficient of fluorescein in vitreous was required in the calculations. Corrections were performed for lens transmission, corneal transmission, and the spatial resolution of the apparatus. The method was applied to 58 healthy volunteers, aged 13-72 yr. An insignificant average increase of permeability values was found from 4.8 nm/s at 10 yr, up to 6.1 nm/s at 70 yr (P = 0.14; standard deviation: 1.8 nm/s). Permeability values showed an average increase of 10% between 30 min and 60 min after injection (P less than 0.001).     

Mouse model of post-surgical breakdown of the blood-retinal barrier

PURPOSE: Post-surgical macular edema is an important clinical problem resulting from breakdown of the blood-retinal barrier (BRB) after surgery. This study was designed to develop a mouse model of post-surgical BRB breakdown. METHODS: Two 25-gauge needles, one for infusion and one for aspiration, were inserted through the limbus and into the lens of one eye of adult male C57BL/6 mice. The anterior portion of the lens was aspirated and the fellow eye was untreated. At several time points after surgery, the integrity of the BRB was assessed quantitatively, using [3H]mannitol as a tracer, or qualitatively, using immunohistochemical staining for albumin. RESULTS: Eyes with partial lens extraction had a significant increase in retinal vascular leakage one day after surgery, which persisted two and three days after surgery, but by five days, was not significantly different from controls. Immunohistochemical staining for albumin demonstrated that the breech in the barrier was sufficient to allow passage of a 60kDa protein into the retina, and was localized predominantly to retinal vessels. CONCLUSIONS: Partial lens extraction in mice results in BRB breakdown (primarily the inner BRB) that is highly reproducible in the severity of leakage and its time course. This provides a valuable tool for investigation of the molecular pathogenesis and new treatment approaches for post-surgical breakdown of the BRB.     

Passive permeability and outward active transport of fluorescein across the blood-retinal barrier in early ARM

AIM: To study the passive and active transport of fluorescein across the blood-retina barrier in early age related maculopathy (ARM) (soft drusen > 63 microm, hyperpigmentation and/or hypopigmentation in patients above 50 years of age). METHODS: 15 patients and 10 healthy subjects were included. Morphological changes were graded from 30 degrees fundus photographs using a simplified version of the epidemiological ARM study group classification system. Differential vitreous spectrofluorophotometry was used to assess the transport properties of the blood-retina barrier (that is, passive permeability and unidirectional permeability caused by outward active transport from the vitreous to the blood). RESULTS: The passive permeability of the patient group was not significantly different from that of the control group. Four patients with passive permeability more than 3 SD above the mean of the control group (mean 1.8 (SD 0.7) nm/s, range 1.0-3.0 nm/s, data normally distributed) all had centrally located drusen > 500 microm and superjacent pigment clumps of 63-500 microm in diameter. There was no significant difference between the unidirectional permeabilities for the patient group and for the control group (mean 47.4 (29.3) nm/s, range 12.7-91.1 nm/s). CONCLUSION: There was no significant difference in the passive permeability and in the unidirectional permeability of fluorescein. However, the study may indicate that the combination of very large drusen and superjacent pigment clumps in ARM may be associated with a deterioration of the blood-retina barrier.     

Measurement of blood-retinal barrier permeability: a reproducibility study in normal eyes

Fluorescein penetration into the posterior vitreous depends on plasma-free fluorescein concentration and blood-retinal barrier (BRB) permeability. The reproducibility of two methods of deriving BRB permeability was studied in 19 normal eyes of 14 subjects using vitreous fluorophotometry on two separate occasions. Plasma-free fluorescence was measured at intervals over 1 hr and posterior vitreous fluorescence was measured before (background scan), within 6 min (bolus) and at 60 min (measurement) after intravenous fluorescein (14 mg X kg-1). A computer algorithm subtracted background fluorescence from the measurement scan which was then corrected for signal spread by using a "spread" function derived from the bolus scan. BRB permeability coefficient and vitreous diffusion coefficients were derived by fitting a mathematical model to the plasma and corrected vitreous fluorescence data. A permeability index was also calculated by dividing the area under the vitreous fluorescence by the area under the plasma fluorescence curve. There were no significant differences in the results between right and left eyes. Mean +/- SD values on first and second occasions for all eyes were permeability coefficient: (1.91 +/- 0.94) and (2.08 +/- 0.95) X 10(-7) cm X s-1; diffusion coefficient: (1.33 +/- 0.68) and (1.19 +/- 0.54) X 10(-5) cm2 X s-1; and permeability index: (2.05 +/- 1.03) and (2.11 +/- 1.02) X 10(-7) cm X s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy

PURPOSE: Nitric oxide (NO) is involved in leukostasis and blood-retinal barrier (BRB) breakdown in the early stages of diabetic retinopathy (DR), but it is unclear which NO synthase (NOS) isoforms are primarily involved. In this study, the authors aimed to clarify the involvement of constitutive (eNOS, nNOS) and inducible NOS (iNOS) isoforms and the mechanisms underlying NO-mediated leukostasis and BRB breakdown. METHODS: Diabetes was induced with streptozotocin for 2 weeks. Mice were treated with a NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), which shows a preference for constitutive isoforms over iNOS. Vessel leakage was assessed with Evans blue. Leukostasis was quantified in flat-mounted retinas with confocal microscopy, in vivo with a scanning laser ophthalmoscope, and in vitro in a retinal endothelial cell line. ICAM-1, occludin, and ZO-1 levels were assessed by Western blot, flow cytometry, or immunohistochemistry. Nitrotyrosine content was assessed by immunohistochemistry. RESULTS: Diabetes increased leukostasis within retinal vessels and BRB permeability, which were reduced by L-NAME. Similar effects were observed in diabetic iNOS knockout mice. In diabetic mouse retinas, ICAM-1 protein levels increased, whereas the immunoreactivity of tight junction proteins, occludin and ZO-1 decreased, in correlation with increased protein levels of all NOS isoforms. Those effects were prevented by L-NAME and also in diabetic iNOS knockout mice. High glucose and nitrosative/oxidative stress also increased leukostasis caused by ICAM-1 upregulation. CONCLUSIONS: These results indicate that the iNOS isoform plays a predominant role in leukostasis and BRB breakdown. The mechanism involves ICAM-1 upregulation and tight junction protein downregulation.     

环缩酚酞抑制血管内皮细胞生长因子诱导的血视网膜屏障破坏

目的 观察两种新的环缩酚酞（Hep）衍生物Hep-A、Hep-B对血管内皮细胞生长因子（VEGF）诱导的血视网膜屏障破坏的效应。 方法 将C57BL/6J小鼠分为正常对照组、阳性对照组、Hep-A、Hep-B治疗组，对照组皮下注射安慰剂，治疗组分别皮下注射Hep-A、Hep-B 10 mg/kg，2次/d，持续5d 。5d后，阳性对照组、Hep-A、Hep-B治疗组玻璃体内注射1μl 10^-6mol/L重组人类VEGF诱导血视网膜屏障破坏，6h后腹腔内注射3.7×10⁴Bq/g的³H-甘露醇。处死小鼠并测量视网膜、肺、肾组织的放射活性，分别计算各组的视网膜与肺、视网膜与肾放射性比率，用方差分析比较各组间差异。 结果 正常对照组的视网膜与肺和视网膜与肾放射性比率分别为0.38±0.04和0.21±0.03；阳性对照组为1.05±0.11和0.46±0.04，均较正常对照组显著升高（P值均<0.01）；Hep-A治疗组分别为0.59±0.06和0.32±0.03，均较阳性对照组显著降低（P值均<0.01）；Hep-B治疗组分别为0.54±0.04和0.35±0.03，均较阳性对照组显著降低（P值分别＝0.01和<0.01）。 结论 Hep-A和Hep-B可以抑制VEGF诱导的血视网膜屏障破坏。 （中华眼底病杂志，2004，20：352-354）     

Immunohistochemical detection of blood-retinal barrier breakdown in streptozotocin-diabetic rat

Immunohistochemical staining for albumin (69 kDa) and fibrinogen (340 kDa), as markers of blood-retinal barrier (BRB) breakdown of the retinas of streptozotocin-treated diabetic rats, was performed. The number of rats with BRB breakdown was three of nine at 6 months, four of seven at 12 months, and three of three at 18 months. Extravasation of albumin from the retinal vessels was detected in the retinas of rats maintained for 6, 12, and 18 months, while extravasation of fibrinogen was detected only in the retinas of rats maintained for 18 months. These findings suggested that the duration of diabetes has an influence on BRB breakdown and that each substance in the blood starts to permeate the vascular wall individually.     

Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions

Breakdown of the inner endothelial blood-retinal barrier (BRB), as occurs in diabetic retinopathy, age-related macular degeneration, retinal vein occlusions, uveitis and other chronic retinal diseases, results in vasogenic edema and neural tissue damage, causing loss of vision. The central mechanism of altered BRB function is a change in the permeability characteristics of retinal endothelial cells caused by elevated levels of growth factors, cytokines, advanced glycation end products, inflammation, hyperglycemia and loss of pericytes. Subsequently, paracellular but also transcellular transport across the retinal vascular wall increases via opening of endothelial intercellular junctions and qualitative and quantitative changes in endothelial caveolar transcellular transport, respectively. Functional changes in pericytes and astrocytes, as well as structural changes in the composition of the endothelial glycocalyx and the basal lamina around BRB endothelium further facilitate BRB leakage. As Starling's rules apply, active transcellular transport of plasma proteins by the BRB endothelial cells causing increased interstitial osmotic pressure is probably the main factor in the formation of macular edema. The understanding of the complex cellular and molecular processes involved in BRB leakage has grown rapidly in recent years. Although appropriate animal models for human conditions like diabetic macular edema are lacking, these insights have provided tools for rational design of drugs aimed at restoring the BRB as well as for design of effective transport of drugs across the BRB, to treat the chronic retinal diseases such as diabetic macular edema that affect the quality-of-life of millions of patients.