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Regulation of Angiogenesis in Diabetic Retinopathy
Possible Balance Between Vascular Endothelial Growth Factor and Endostatin
Hidetaka Noma, MD;
Hideharu Funatsu, MD;
Hidetoshi Yamashita, MD;
Shigehiko Kitano, MD;
Hiromu K. Mishima, MD;
Sadao Hori, MD
Arch Ophthalmol. 2002;120:1075-1080.
ABSTRACT
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Objective To investigate the mechanisms of regulation between vascular endothelial
growth factor (VEGF) as a stimulator and endostatin as an inhibitor of angiogenesis
in diabetic retinopathy (DR).
Methods One hundred fifty-nine eyes of 120 diabetic patients were studied. Concentrations
of VEGF and endostatin in vitreous fluid and aqueous humor, obtained from
the eyes during ocular surgery, were measured by enzyme-linked immunosorbent
assay. The severity of DR was quantified according to the Early Treatment
Diabetic Retinopathy Study retinopathy severity scale; fundus findings, including
soft exudates, intraretinal microvascular abnormalities, venous abnormalities,
new vessels elsewhere, new vessels on the disc, vitreous hemorrhage, and retinal
detachment, were graded and evaluated. Concentrations of VEGF and endostatin
in plasma were also measured by enzyme-linked immunosorbent assay.
Main Outcome Measures Concentrations of VEGF and endostatin in vitreous fluid and plasma.
The correlations among the clinical records and the levels of VEGF and endostatin
were analyzed statistically.
Results The concentrations of VEGF in aqueous humor and vitreous fluid were
significantly correlated with the severity of DR ( = 0.447, P<.001 and = 0.363, P = .007, respectively).
The concentrations of endostatin in aqueous humor and vitreous fluid were
also significantly correlated with the severity of DR ( = 0.302, P<.001 and = 0.344, P
= .009, respectively). The slope of the regression line between the VEGF and
endostatin concentrations in vitreous fluid differed significantly between
active DR and quiescent DR (P = .04). The concentrations
of VEGF and endostatin in the eyes were not correlated with those in the plasma.
Conclusions These results show that both VEGF and endostatin are correlated with
angiogenesis in DR. Our study suggests that the regulation mechanism between
VEGF and endostatin is associated with the activity of DR and may be a good
candidate to develop useful therapeutic agents for proliferative DR.
INTRODUCTION
NEW VESSEL formation in diabetic retinopathy (DR) causes visual loss
with vitreous hemorrhage, retinal detachment, and neovascular glaucoma.1-2 Angiogenesis is a complex multistep
process. Various cytokines and growth factors are considered to be involved
in these processes and the pathogenesis of angiogenesis.3-5
However, not all the mechanisms involved in the process of neovascularization
in proliferative DR can be explained in terms of only angiogenic stimulators.
It is hypothesized that the net balance between angiogenic stimulators and
inhibitors regulates the switching of the angiogenic process.6-8
That is, a net balance of inhibitors over activators would maintain the switch
in the off position, whereas a shift to an excess of activating stimuli would
turn on angiogenesis. Vascular endothelial growth factor (VEGF) acts as an
endothelial cell mitogen9-10 in
vitro and induces increased vascular permeability11
and angiogenesis in vivo. Intraocular VEGF concentrations are increased during
the periods of active intraocular neovascularization in patients with proliferative
DR.12
Many endogenous inhibitors of angiogenesis, including endostatin,13 thrombospondin,14-15
interferon  and  , prolactin, platelet factor 4,16-18
and angiostatin,19 have been reported. Endostatin
is an angiogenic inhibitor produced by hemangioendothelioma.13
Endostatin specifically inhibits endothelial cell proliferation and potently
inhibits angiogenesis and tumor growth.13 Endostatin
inhibits the VEGF-stimulated migration of umbilical vein endothelial cells.20 Based on these findings of previous studies, we investigated
the roles of endostatin in the pathogenesis of DR.
In the present study, we examined the regulation mechanism between VEGF
as an angiogenic stimulator and endostatin as an angiogenic inhibitor in DR
and showed that endostatin may be related to angiogenesis activity in cooperating
with VEGF in DR.
PARTICIPANTS AND METHODS
STUDY PARTICIPANTS
Samples of aqueous humor and vitreous fluid were obtained from 159 eyes
of 120 diabetic patients whose mean (SD) age was 62.0 (12.5) years (range,
24-88 years). The patients included 77 men and 43 women. The mean (SD) duration
of diabetes mellitus was 15.7 (9.5) years (range, 9-30 years). Patients were
excluded if they had undergone previous intraocular surgery or had a history
of branch retinal vein occlusion and uveitis. All procedures conformed to
the Declaration of Helsinki for research involving human subjects. Ethics
committee approval was obtained, and all participants gave informed consent.
The activity of DR is classified into active and quiescent. If there
are extensive changes, including active neovascularization and proliferative
membrane, fresh vitreous hemorrhage, and progressive retinal detachment, it
is classified as active. If it becomes silent on photocoagulation, even with
remaining neovascularization and proliferative membrane, it is classified
as quiescent.21-22
SAMPLE COLLECTION
At the start of eye surgery, a sample of undiluted aqueous humor (100-200
µL) was manually aspirated into a disposable tuberculin syringe, and
a sample of undiluted vitreous fluid (300-500 µL) was aspirated using
a vitreous cutter (under air flow), transferred immediately to a sterile tube,
frozen immediately at -5°C in the operating room, and then stored
in a deep freezer at -80°C. Blood samples were collected simultaneously
and centrifuged at 3000g for 5 minutes to obtain
serum and then aliquoted and stored at -80°C until they were assayed.
MEASUREMENT OF VEGF AND ENDOSTATIN
Concentrations of VEGF and endostatin were measured by an enzyme-linked
immunosorbent assay using immunoassay kits for human VEGF (R&D Systems,
Minneapolis, Minn) and human endostatin (Cytimmune Sciences, Baltimore, Md),23-24 respectively, according to the manufacturer's
standard protocol. The results ensured that the levels of factors in intraocular
fluid and plasma samples were within the detectable range using these assays.
The minimum detectable concentrations (sensitivity) using the assay kits were
15.6 pg/mL for VEGF and 0.95 ng/mL for endostatin.
FINDINGS AND GRADING PROCEDURES OF DR
The preoperative and operative findings were recorded. Clinical data,
including the severity of DR, were obtained by the surgeon using standardized
forms at the time of surgery and were confirmed by standardized fundus color
photography and fluorescein angiography performed within 3 days after the
operation. The severity of DR was graded according to the modified Early Treatment
Diabetic Retinopathy Study (ETDRS) retinopathy severity scale.25-26
In particular, the severity of soft exudates, intraretinal microvascular abnormalities
(IRMA), venous beading, venous loops, new vessels elsewhere (NVE), new vessels
on the disc (NVD), fibrous proliferation elsewhere (FPE), vitreous hemorrhage,
and retinal detachment were graded according to the ETDRS system.25
STATISTICAL ANALYSIS
Statistical analysis was performed with SAS/STAT statistical software
(SAS Institute Inc, Cary, NC; changes and enhancements through release 6.12).
Results are presented as mean ± SD or geometric mean ± SD for
data shown on the logarithmic scale. To determine the relationship between
angiogenic factors and the ETDRS retinopathy severity, Spearman rank-order
correlation coefficient was applied. To test the heterogeneity of slopes of
2 linear regression lines, analysis of covariance with interaction was used.
In this model, variables were analyzed on the logarithmic scale because of
skewed distribution. Two-tailed P values of less
than .05 were considered to indicate statistical significance.
RESULTS
ANGIOGENIC FACTORS AND SEVERITY OF DR
The concentrations of VEGF in the aqueous humor (geometric mean, 223.3
pg/mL) and vitreous fluid (geometric mean, 1291 pg/mL) were significantly
correlated with the severity of DR ( = 0.447, P<.001
and = 0.363, P = .007, respectively) (Figure 1A and B). The concentrations of endostatin
in the aqueous humor (mean, 2.6 ng/mL) and vitreous fluid (mean, 4.0 ng/mL)
were also significantly correlated with the severity of DR ( = 0.302, P<.001 and = 0.344, P
= .009, respectively) (Figure 2A
and B).
ANGIOGENIC FACTORS AND SEVERITY OF FUNDUS FINDINGS
The concentration of VEGF in vitreous fluid was significantly correlated
with the grades of soft exudate, IRMA, venous beading, venous loops, NVE,
NVD, FPE, and vitreous hemorrhage (Table
1). The concentration of endostatin in vitreous fluid was significantly
correlated with the grades of NVE, FPE, and retinal detachment (Table 1).
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Correlation Between the Concentration of VEGF and Endostatin in Vitreous
Fluid and Grade of Fundus Findings*
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VEGF CORRELATIONS
VEGF and Endostatin in Eyes With Active or Quiescent DR
The VEGF levels were higher in the vitreous fluid of active DR than
in the vitreous fluid of quiescent DR. The endostatin levels were lower in
the vitreous fluid of active DR than in the vitreous fluid of quiescent DR.
On the other hand, in the vitreous fluid of quiescent DR, the concentration
of VEGF was low but the concentration of endostatin was high.
The recurrence equations were as follows: endostatin =1.538754 + 0.003985
x VEGF for quiescent DR and endostatin = 1.619433 + 0.001151 x
VEGF for active DR. The slope of the regression line between the VEGF and
endostatin concentrations in vitreous fluid differed significantly between
active DR and quiescent DR (P = .04) (Figure 3).
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Figure 3. The relationship between the vitreous
concentrations of vascular endothelial growth factor (VEGF) and endostatin
in patients with active diabetic retinopathy (DR) or quiescent DR. In the
vitreous fluid of active DR, the concentration of VEGF was high, but the concentration
of endostatin was low (thick line). On the other hand, in the vitreous fluid
of quiescent DR, the concentration of VEGF was low but the concentration of
endostatin was high (thin line). Recurrence equation (quiescent): endostatin
= 1.538754 + 0.003985 x VEGF. Recurrence equation (active): endostatin
= 1.619433 + 0.001151 x VEGF. The slope of the regression line between
the VEGF and endostatin concentrations in vitreous fluid differed significantly
between active DR and quiescent DR (n = 42, P = .04).
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Concentrations of VEGF and Endostatin
There were significant correlations between the concentration of VEGF
and endostatin in the aqueous humor and vitreous fluid ( = 0.320, P = .004 and = 0.374, P
= .006, respectively) (Figure 4A and B).
Plasma Concentrations of VEGF and the Severity of DR
There was no significant correlation between the plasma concentration
of VEGF or endostatin and the severity of DR (data not shown).
Concentrations of VEGF and Endostatin in the Ocular Fluid and Plasma
The concentration of VEGF in neither the aqueous humor (223.3 pg/mL)
nor the vitreous fluid (1291 pg/mL) was significantly correlated with that
in plasma (66.3 pg/mL). As for endostatin, no significant relationship was
found between its concentration in aqueous humor (2.6 ng/mL) and plasma (7.4
ng/mL), but a significant relationship was found between its concentration
in vitreous fluid (4.0 ng/mL) and plasma ( = 0.054, P = .72 and = 0.377, P<.05, respectively).
COMMENT
We obtained the following findings in this study. First, in patients
with DR, endostatin was detected in aqueous humor and vitreous fluid. Second,
the endostatin and VEGF concentrations in aqueous humor and vitreous fluid
were correlated with the severity of DR. Third, some of the patients with
severe DR showed a high endostatin concentration in vitreous fluid, but others
showed a low concentration. Fourth, the slope of the regression line between
the VEGF and endostatin concentrations in vitreous fluid differed significantly
between active DR and quiescent DR.
There was a significant positive correlation between the severity of
DR and the VEGF concentration in the aqueous humor and vitreous fluid in the
present study. These results were consistent with the findings of previous
reports.27-29
The VEGF concentration in the vitreous fluid was positively correlated with
the grades of soft exudates, IRMA, venous beading, venous loops, NVE, NVD,
FPE, and vitreous hemorrhage (Table 1).
These results suggest that VEGF stimulates angiogenesis in the pathology of
DR. Our results and the previous reports suggest that VEGF is associated with
enhanced vascular permeability, vascular occlusion, and angiogenesis.27-31
The severity of DR was also positively correlated with the endostatin
concentrations in both aqueous humor and vitreous fluid. Of the fundus findings,
the grades of NVE, FPE, and retinal detachment were positively correlated
with the endostatin concentration in vitreous fluid (Table 1). In the aqueous humor and vitreous fluid, the VEGF concentration
was positively correlated with the endostatin concentration. These results
suggest that endostatin expression is correlated with VEGF expression.
The theory that the balance between stimulators and inhibitors is critical
in the process of tumor angiogenesis has been proposed.6-8
We consider endostatin to be an angiogenic inhibitor associated with the pathogenesis
of proliferative DR (PDR). Endostatin is an angiogenic inhibitor produced
by hemanigioendothelioma.13 Endostatin specifically
inhibits endothelial cell proliferation, potently inhibiting angiogenesis
and tumor growth. Endostatin has also been reported to inhibit VEGF-stimulated
endothelial cell proliferation and migration.20, 32
Thus, there is a possibility that endostatin inhibits angiogenesis in PDR,
but this possibility has not yet been evaluated. In this study, the endostatin
concentration in intraocular fluid and blood samples was evaluated.
Although the endostatin concentration in both aqueous humor and vitreous
fluid showed a significant positive correlation with the severity of DR, the
endostatin concentration in aqueous humor was high even in some patients with
mild DR and varied widely in patients with severe DR, showing a uniform distribution
independent of the severity of DR. The endostatin concentration in vitreous
fluid varied more widely as the severity of DR increased. This indicates that
the endostatin concentration in vitreous fluid is high in some patients with
severe DR but low in others with severe DR. To clarify differences between
patients with a high endostatin concentration and those with a low concentration,
the net balance between VEGF and endostatin was evaluated in association with
the activity of DR. The activity of DR was classified as active (highly active
neovascularization and proliferative membrane, fresh vitreous hemorrhage,
and progression to retinal detachment) or quiescent (silent on photocoagulation
even with remaining neovascularization and proliferative membrane).21-22 In addition, DR was active in patients
with a high VEGF concentration and a low endostatin concentration in vitreous
fluid. However, DR was quiescent in patients with a low VEGF concentration
and a high endostatin concentration in vitreous fluid. Retinopathy was frequently
active in patients with high VEGF and high endostatin concentrations but frequently
quiescent in those with low VEGF and low endostatin concentrations. The slope
of the regression line between the VEGF and endostatin concentrations in vitreous
fluid differed significantly between active DR and quiescent DR (P<.05, Figure 3). These
findings suggest that the activity of DR rather than the severity of DR more
accurately reflects the effects of angiogenic stimulators and inhibitors on
DR. In addition, 4 patients with a high endostatin concentration had quiescent
DR despite a high VEGF concentration, and 1 patient with a low endostatin
concentration had active DR despite a low VEGF concentration. Although these
results suggest the promotion of neovascularization by VEGF and its inhibition
by endostatin, their direct mechanisms could not be clarified in this study.
However, in DR and tumors, the net balance of factors seems to affect the
eyes.
It was reported that other angiogenesis stimulators and inhibitors possibly
play a role in angiogenesis in PDR.33-36
The lower vitreous levels of pigment epithelium–derived factor or angiostatin
may be related to the angiogenesis in PDR and result in active PDR.34-36 It is possible that
not only endostatin but also other angiogenesis inhibitors play a role in
antiangiogenesis in PDR. Further investigation is needed to resolve the mechanisms
of angiogenesis in PDR.
There was no significant correlation between the plasma VEGF or endostatin
concentration and the severity of DR or between the VEGF concentration in
the aqueous humor or vitreous fluid and the plasma VEGF concentration. The
VEGF concentration in vitreous fluid (1291 pg/mL) was much higher than in
plasma (66.3 pg/mL). These results suggest that VEGF is produced in the eyes.12, 27-28,37
The endostatin concentration in plasma was not significantly correlated
with that in the aqueous humor but was positively correlated with that in
vitreous fluid. The mean endostatin concentration in plasma (7.4 ng/mL) was
higher than that in vitreous fluid (4.0 ng/mL). Also, VEGF has been reported
to be involved in the destruction of the blood-retinal barrier.12, 27-28,37
These results suggest that leakage of endostatin from the blood to the vitreous
body occurs because of VEGF-induced destruction of the blood-retinal barrier.
Since no difference was observed in the endostatin concentration according
to the presence or absence of retinal photocoagulation, endostatin may not
be produced exclusively in the eyes (data not shown). However, the range of
the plasma endostatin concentration was narrow, but that of the vitreous endostatin
concentration was wide. In addition, the intraocular endostatin concentration
was higher than the plasma endostatin concentration in 10 patients, suggesting
that endostatin is also produced in the eyes. Whether endostatin is produced
in the eyes could not be determined in this study. Further studies will be
necessary to evaluate the tissue origin of endostatin.
In conclusion, we found that the levels of endostatin and VEGF concentrations
are correlated with the severity of DR, and the slope of the regression line
between the VEGF and endostatin concentrations in vitreous fluid differs significantly
between active DR and quiescent DR.
AUTHOR INFORMATION
Submitted for publication July 10, 2001; final revision received March
28, 2002; accepted April 24, 2002.
This study was supported by Health Science Research grants (10060101)
from the Ministry of Health and Welfare, Research on Eye and Ear Sciences,
Immunology, Allergy and Organ Transplantation in Japan (Tokyo).
We thank Erika Shimizu, MD, Yasuhiro Konno, MD, Kozue Ohara, MD, Kaori
Sekimoto, MD, Rie Takeda, MD, Koji Makita, MD, Kensuke Haruyama, MD, Shinko
Nakamura, MD, and Maho Hirai, MD, for assistance in the collection of vitreous
samples and ophthalmological examinations. We thank Katsunori Shimada (Department
of Biostatistics, STATZ Corporation, Tokyo) for assistance with statistical
analysis.
Corresponding author and reprints: Hideharu Funatsu, MD, Department
of Ophthalmology, Diabetes Center, Tokyo Women's Medical University, 8-1,
Kawada-cho, Shinuku-ku, Tokyo 162-8666, Japan (e-mail: tfunatsu{at}nifty.com).
From the Departments of Ophthalmology, Diabetes Center, Tokyo Women's
Medical University, Tokyo (Drs Noma, Funatsu, and Kitano), Hiroshima University
Medical School, Hiroshima (Drs Noma and Mishima), and Yamagata University
Medical School, Yamagata (Dr Yamashita), and Tokyo Women's Medical University
(Dr Hori), Japan.
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