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patients (nondiabetic) had VH (n 10). None of the

MH patients had associated proliferative vitreoreti-nopathy and diabetes. All the cases had no history of

ocular surgery.The PDR patients (21 male; 4 female) were aged

54.9 8.9 years (mean SD) had diabetes duration

of 13.6 8.3 years (mean SD) which included bothactive (n 16) and inactive PDR (n 9). Sixteen ofthese PDR patients had undergone panretinal photo-

coagulation (PRP). The ED patients (10 male; 0 fe-male) were aged 29.3 6.1 years. The control group

MH patients (6 male; 19 female) were aged 61.8 8.1years.

Sample Collection

At the beginning of vitrectomy, undiluted vitreous(200700 L) was aspirated via pars plana with a

vitreous cutter, before opening the infusion port.These undiluted vitreous samples were immediately

frozen in aliquots in polypropylene tube at 80Cuntil assay.

Cytokine Assay

The concentrations of IL-6, IL-8, IL1-, MCP-1,VEGF, and PEDF were determined in the vitreous

samples using sandwich enzyme linked immunosor-bent assay (ELISA) (BD, R&D systems, Chemicon

international) according to the manufacturers instruc-tions. The standard curve was prepared using recom-

binant human cytokines. A calibration run on a fewvitreous samples showed that a dilution of 1:5 was

found to be appropriate for IL-6, IL-8, MCP-1, VEGF;

1:4 for PEDF; and 1:2 for IL1-. The minimum de-

tectable concentrations were found to be 7.8 (IL-1and MCP-1), 15.0 (IL-6), 3.1 (IL-8), and 31.3 (VEGF)

pg/mL and 0.98 ng/mL for PEDF, respectively.

Statistical Analysis

Mann-Whitney U-test was used to analyze thedifference between PDR, ED, and control groups.

Values are reported as medians with ranges. Toexamine the correlations between VEGF and other

cytokines, Spearmans rank correlation test wasused and graphically represented by means of Pear-

sons correlations. The results were considered sig-nificant at P 0.05.

Results

Vitreous Levels of IL-6, IL-8, IL-1, and MCP-1

The median levels of IL-6 and IL-8 were signifi-

cantly higher in PDR (80.7 pg/mL, P 0.001; 59.6pg/mL, P 0.001) and ED patients (201.9 pg/mL,

P 0.001; 148.2 pg/mL, P 0.001) when comparedwith the MH patients (0, 0 pg/mL; 0, 137.1 pg/mL).

However, no significant difference was observed inIL-1 (Figure 1). In PDR (1812.9 pg/mL, P 0.0001)

and ED (1754 pg/mL, P 0.001) the median vitreousMCP-1 concentration was significantly higher when

compared to MH patients (0, 1168.3 pg/mL) (Figure 2).

Vitreous Levels of VEGF and PEDFThe median VEGF concentration in vitreous was

significantly higher in PDR (1123.3 pg/mL; P

Fig. 1. IL-6, IL-8, and IL-1levels in the vitreous of patients

with proliferative diabetic reti-nopathy (PDR), Eales disease(ED), and macular hole (MH).

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tients. The vitreous levels of cytokines (IL-6, IL-8),

VEGF, and PEDF showed no significant difference inPDR patients, who had undergone PRP treatment be-

fore vitrectomy, when compared with patients whohad no PRP treatment. Concentration of all the cyto-

kines, angiogenic and antiangiogenic factors in thevitreous of PDR, ED, and control subjects are men-

tioned in Table 1 as median ranges. We have analyzedthe proinflammatory cytokines and growth factors in

serum of PDR, ED, and MH patients. The cytokineIL-6 and antiangiogenic factor PEDF were below the

detectable levels in the serum of PDR, ED, and MH

patients. The serum levels of IL-8 (25.1 142.2;

median SD), VEGF (165.9 145.7), and MCP-1(319.9 92.6) were below the vitreous levels of PDR

patients.

Discussion

Earlier studies on PDR involved the detection of

the levels of angiogenic stimulator and inhibitorsalone22,24 or any one cytokine and angiogenic factor in

the vitreous.2527 However, the present report is acomprehensive study to estimate the inflammatory

cytokines (IL-6, IL-8, IL1-), chemokines (MCP-1),angiogenic factor (VEGF), and antiangiogenic factor

(PEDF) all together in each of the vitreous samples ofPDR, ED, and MH patients. This was carried out to

elucidate whether neovascularization in PDR and EDare mediated by inflammatory cytokines and growth

factors and further to understand the relationship be-tween the cytokines and growth factors. To our

knowledge, this is the first report to demonstrate thatthe vitreous levels of VEGF, IL-6, IL-8, and MCP-1

were significantly and simultaneously increased inboth PDR and ED when compared with MH patients.

ED is associated with localized inflammation ofretinal blood vessel walls and is suggested to be an

immune-mediated disease, as shown by the increase inthe levels of -1 acid glycoprotein.28 DR may not

display most of the macroscopic attributes of inflam-mation, but all the microscopic signs of inflammation,

Fig. 4. Levels of PEDF inthe vitreous of proliferative

diabetic retinopathy (PDR),Eales disease (ED), and mac-ular hole (MH) patients..

Fig. 5. Correlation between VEGF and IL-6 in vitreous of Eales

disease patients; r 0.698, P 0.025.

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i.e., vasodilatation, altered flow, fluid exudation, and

leukocyte migration/accumulation, are observed in

DR.8 This path-breaking information has overseen a

surge of myriad studies evaluating corticosteroids in

the treatment of DR, particularly macular edema, cul-

minating into the National Eye Institutesponsored

trials on the role of steroids in the treatment of DR(http://www.nei.nih.gov/neitrials). Gao et al have

demonstrated the elevated levels of carbonic anhy-

drase-1 (CA-1) in vitreous of DR patients. They

proved that injection of CA-1 into rats induced alka-

linization of vitreous which increased kallikrein activ-

ity and its generation of factor XIIa, revealing that

inhibition of extracellular CA-1 and kallikrein-medi-

ated innate inflammation could provide new therapeu-

tic opportunities for the treatment of DR.29 Though

PDR and ED differ in their etiology and early patho-

genic mechanisms the result in uncontrolled disease is

neovascularization.The role of cytokines in the pathogenesis of PDR

and ED is not completely understood. However, there

are reports suggesting that cytokine IL-6 can increase

endothelial cell permeability in vitro by rearranging

actin filaments and by changing the shape of endothe-

lial cells.30 Further, the increased vitreous levels of

VEGF and IL-6 correlated with the progression of

PDR in the outcome of vitreous surgery.31 Even

though there are reports on high levels of IL-6, IL-8,

and MCP-1 in the vitreous of PDR,25,3234 we demon-

strate for the first time the increased levels of IL-6,

IL-8, MCP-1, and VEGF in the vitreous of ED pa-tients. The source of the high levels of cytokines and

chemokine detected within the vitreous of PDR and

ED patients remains unclear. A possibility is that cells

in the vitreous could be the main cause accounting for

the high levels of these cytokines and chemokine. The

vitreous of MH patients are largely devoid of inflam-

matory cells.35 Further, El-Ghrably and associates re-

ported that macrophages, monocytes, retinal pigment

epithelial cells, and glial cells are found in the vitreous

of patients with PDR and the majority of these cells

are capable of producing cytokines in vitro.35 We

suggest that increased levels of IL-6, IL-8, and MCP-1are involved in the pathogenesis of inducing neovas-

cularization in PDR and ED. Further studies on this

issue are needed to understand the mechanism of PDR

and ED.

There are varying reports on the status of PEDF and

VEGF in diabetic retinopathy. The PEDF level in MH

is equal to the normal bovine eyes.36 We confirm the

earlier report that higher concentration of VEGF and

lower concentration of PEDF in the vitreous of PDR

may be related to angiogenesis.21,22 On the other hand,

Table1.

VitreousLevelso

fCytokines,

AngiogenicandAnti-AngiogenicFactorsinProliferativeDia

beticRetinopathy,

EalesDisease,a

ndMacular

HolePatients

Cytokines

Prolifera

tiveDiabetic

Retinopathy(PDR)

Eales

Disease(ED)

MacularHole(MH)

PDR

and

MH

P

Value

ED

and

M

H

P

V

alue

PDR

andED

P

Value

Sample

No.

Median

(Minimum,

Maximum)

Sample

No.

Median(Minimum,

Maximum)

Sample

No.

Median

(Minimum,

Maximum)

IL-6(pg/mL)

25

80.7

(0,

1645.9

)

10

201.9

(0,

1200)

25

0.0

(0)

0.0

001

0

.0001

0.5

46

IL-8(pg/mL)

25

59.6

(0,

777.2

)

10

148.2

(13,

748.7

)

25

0.0

(0,

137.0

8)

0.0

001

0

.0001

0.3

22

IL1-(pg/mL)

20

0.0

(0,

57.9

)

9

0.0

(0,

23.8

7)

25

0.0

(0,

103.7

6)

0.1

27

0

.127

0.7

53

MCP-1(pg/mL)

25

1812.9

(0,

5830.9

)

10

1754.0

(0,

4104.1

)

25

277.7

(0,

1168.3

)

0.0

001

0

.001

0.6

74

VEGF(pg/mL)

25

1123.3

(0,

8092.8

)

10

1219.9

(121.8,

9900.2

)

25

0.0

(0,

139.7

5)

0.0

001

0

.0001

0.4

75

PEDF(ng/mL)

25

0.0

(0,

21.8

)

10

13.6

(0,

93.8

)

25

17.3

(0,

298.7

)

0.0

001

0

.432

0.0

26

Valuesarepresentedasmedianranges(minimum,maximum).

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similar decrease in PEDF was not observed in ED.

Duh et al demonstrated a significant increase rather

than a decrease in the vitreous of PEDF levels in PDR

patients.24 Furthermore, Ogata and coworkers sug-

gested that significantly elevated levels of PEDF in the

plasma of PDR patients may be related to the progres-

sion of diabetic retinopathy.37 Moreover, a concentra-tion of 50 ng/mL of PEDF completely inhibits VEGF-

induced migration of cultured microvascular endothelial

cells.36 The lowest baseline vitreous concentration of

PEDF reported among all the studies is 20-fold higher

than the concentration that was maximally effective in

vitro and a 1:20 dilution of human vitreous completely

inhibited VEGF-induced migration of vascular endo-

thelial cells. This inhibitory activity was also neutral-

ized by anti-PEDF antibody.36 The mechanism of

increase and decrease in PEDF levels is not clear in

the modulation of ocular neovascularization. The de-

creased level of PEDF in PDR when compared with

ED and MH suggests that the regulation of the inhib-

itory effect of PEDF may differ in ED. Further, the

significant correlation between VEGF and IL-6 in ED

suggests that the roles and induction mechanism of

VEGF also differ between PDR and ED.

In conclusion, we suggest that increased levels of

IL-6, IL-8, MCP-1, and VEGF may act as a key

regulator of neovascularization in PDR and ED. The

regulatory role of VEGF and the inhibitory effect of

PEDF may differ in the modulation of neovascular-

ization in PDR and ED. Even though the etiology isdifferent in both diseases, we demonstrated the levels

of cytokines and growth factors in PDR and ED.

Moreover, the regulation of VEGF and the inhibitory

role of PEDF in inducing the neovascularization can-

not be clearly explained. Further, investigation is re-

quired to understand the regulatory role of neovascu-

larization in PDR and ED.

Key words: cytokines, Eales disease, neovasculariza-

tion, proliferative diabetic retinopathy, pigment epithe-

lium derived factor, vascular endothelial growth factor.

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