Changes in vitreous concentrations of human hepatocyte growth factor ...

Clinical Science (2000) 98, 9–14 (Printed in Great Britain)

Changes in vitreous concentrations of human hepatocyte growth factor (hHGF) in proliferative diabetic retinopathy: implications for intraocular hHGF production Masato NISHIMURA*, Tsunehiko IKEDA†, Masaji USHIYAMA*, Shigeru KINOSHITA† and Manabu YOSHIMURA* *Department of Clinical and Laboratory Medicine, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602-8566, Japan, and †Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602-8566, Japan

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We measured human hepatocyte growth factor (hHGF) concentrations in the original vitreous and in the artificial vitreous after vitrectomy in 13 patients with proliferative diabetic retinopathy (PDR) undergoing repeated pars plana vitrectomy, in order to investigate whether the vitreous hHGF concentration is related to the recurrence of PDR after vitrectomy as well as to the original occurrence of PDR. We also examined the relationship between vitreous concentrations of hHGF and transforming growth factor-β2 (TGF-β2), the predominant TGF-β isoform in the vitreous, in 14 patients with PDR. For the original vitreous, mean hHGF concentrations were higher (P 0.05) in that from patients with severe PDR (vitreous haemorrhage, fibrovascular proliferation and tractional retinal detachment) than in that from patients with vitreous haemorrhage alone. In the artificial vitreous, mean vitreous hHGF concentrations were higher (P 0.05) in that from patients with severe PDR than in that from patients with vitreous haemorrhage alone or with vitreous haemorrhage plus fibrovascular proliferation. No correlation was found between the hHGF concentration in the artificial vitreous and time between vitrectomies. Vitreous hHGF concentrations were directly proportional to vitreous concentrations of latent TGF-β2 (r l 0.831 ; P l 0.0002), but inversely proportional to vitreous concentrations of active TGF-β2 (r l 0.495 ; P l 0.072), which inhibits hHGF production. A decreased conversion of latent into active TGF-β2 in ocular disorders such as PDR is likely to result in an increased concentration of hHGF in the vitreous. Thus intraocular hHGF may be involved in pathological mechanisms causing not only the occurrence, but also the recurrence, of PDR.

INTRODUCTION Proliferative diabetic retinopathy (PDR) is a major cause of adult blindness, but the precise mechanisms causing the retinal fibrovascular proliferative changes, which may

result in vitreous haemorrhage or tractional retinal detachment, have not been determined [1]. Numerous mitogenic and angiogenic factors have been reported to be involved in PDR, including insulin-like growth factors, transforming growth factor-β (TGF-β), fibro-

Key words : hepatocyte growth factor, neovascularization, pars plana vitrectomy, proliferative diabetic retinopathy, retinal detachment, transforming growth factor-β . # Abbreviations : hHGF, human hepatocyte growth factor ; PDR, proliferative diabetic retinopathy ; TGF, transforming growth factor. Correspondence : Dr Masato Nishimura (e-mail nishim!labmed.kpu-m.ac.jp).

# 2000 The Biochemical Society and the Medical Research Society

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blast growth factor, tumour necrosis factor and vascular endothelial growth factor [2–6]. Recently we showed that mean vitreous concentrations of human hepatocyte growth factor (hHGF) are higher in subjects with PDR than in non-diabetic control subjects, non-diabetic subjects with proliferative vitreoretinopathy or diabetic subjects without PDR [7]. Previous studies indicate that hHGF is a powerful inducer of angiogenesis [8,9], and this growth factor may contribute to the genesis of AIDS-associated Kaposi’s sarcoma, a cytokine-dependent neoplasm characterized by a major component of neovascularization [10]. These findings suggest that vitreous hHGF plays a key role in neovascularization in PDR. The diffusable factors associated with pathological changes in the retinal region should be present within the vitreous [3]. Intraocular hHGF may be involved in the recurrence of PDR after vitrectomy, as well as in the original occurrence of PDR. The aim of the present study was to investigate whether the vitreous hHGF concentration, not only in the original vitreous obtained at first vitrectomy but also in the artificial vitreous obtained at repeated vitrectomy, is related to the severity of PDR, by measuring vitreous hHGF concentrations in PDR patients undergoing repeated pars plana vitrectomy. We also investigated the relationship between the vitreous concentrations of hHGF and TGF-β , which is a pre# dominant isoform of TGF-β in the posterior segment of the eye [11,12] and has an inhibitory effect on hHGF production [13–16].

METHODS Subjects Vitreous samples were collected from diabetic patients with PDR undergoing pars plana vitrectomy, which was performed in the Ophthalmology Department, Kyoto Prefectural University of Medicine. No patients enrolled in the study had severe hepatic or renal disorders which could affect the serum concentration of hHGF. The study was approved by the Ethical Committee for Human Research of Kyoto Prefectural University of Medicine, and all subjects provided informed consent for participation.

Changes in vitreous hHGF concentrations in PDR In total, 13 diabetic patients with PDR, who had undergone pars plana vitrectomy at least twice, were studied [nine females and four males ; age 56p8 years (meanpS.D.)]. Samples of original or artificial vitreous were collected at each vitrectomy, and hHGF concentrations were measured in 32 vitreous samples in all (Table 1). The indications for the vitrectomy were # 2000 The Biochemical Society and the Medical Research Society

vitreous haemorrhage, vitreo-retinal fibrovascular proliferation or tractional retinal detachment. All subjects suffered from type II diabetes mellitus, and had been treated with either insulin or anti-diabetic drugs. The mean serum haemoglobin A1c value at the time of the first vitrectomy was 7.5p0.2 %. The detailed characteristics of the participants are described in Table 1.

Vitreous samples Before intraocular infusion, the vitreous core was cut and aspirated via the pars plana with a vitreous cutter, and collected undiluted. After removal of the vitreous, irrigating solution (OPEGUARD-MA : glucose 1.5 mg\ml, NaCl 6.6 mg\ml, KCl 0.36 mg\ml, CaCl 0.18 mg\ # ml, MgCl 0.3 mg\ml, NaHCO 2.1 mg\ml, pH 6.7–8.2 ; # $ Senju Pharmaceutical Co. Ltd., Osaka, Japan) was used to filled the intraocular space as the artificial vitreous. Vitreous samples were spun for 15 min at 13 000 g in a refrigerated centrifuge at 4 mC to remove particles, and then stored in aliquots in polypropylene tubes at k80 mC until assay.

Vitreous concentration of hHGF Vitreous concentrations of hHGF were measured using a specific ELISA kit (Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan). The intra- and inter-assay variation was 2.9 % and 2.6 % respectively.

Vitreous concentration of TGF-β2 Vitreous concentrations of active and latent TGF-β were # measured using a specific-capture ELISA kit (Amersham Life Science) in samples from 14 subjects with PDR (eight females and six males, age 54p10 years). The original vitreous obtained at first vitrectomy was used in the measurement of vitreous TGF-β concentrations. # Because this ELISA kit only detects active TGF-β , # vitreous samples were diluted 1 : 2 (v\v) with the standard diluent provided in the kit. Half the volume of each vitreous sample was used for assay of active TGF-β , and # the other half was activated by acidification with 150 mM HCl for 30 min at room temperature, followed by neutralization with NaOH [17]. Concentrations of latent TGF-β were determined by subtracting the former # (active) TGF-β concentration from the latter (total) # TGF-β concentration. Vitreous hHGF concentrations # were also measured in these samples, and the correlation between vitreous concentrations of hHGF and TGF-β # was analysed.

Statistical analysis Data are expressed as meanspS.E.M. The significance of differences between groups was evaluated by analysis of variance followed by Duncan’s multiple-range test. The criterion for statistical significance was P 0.05. Simple regression analyses were performed to assess the re-

Vitreous hepatocyte growth factor in proliferative diabetic retinopathy

Table 1

Clinical characteristics of 13 study participants who had undergone repeated vitrectomies due to PDR

Abbreviations : F, female ; M, male ; VH, vitreous haemorrhage ; RD, tractional retinal detachment ; P, vitreo-retinal fibrovascular proliferation. Serum concentrations of haemoglobin A1c are the values measured at the last vitrectomy.

Subject (years, sex)

Duration of diabetes (years)

Serum concn. of haemoglobin A1c (%)

Treatment

1 (48, F)

6

7.8

Insulin

2 (49, M)

5

6.5

Insulinjsulphonylurea

3 (54, M)

8

8.1

Insulin

4 (54, F)

6

7.1

Sulphonylurea

5 (54, F)

8

6.9

6 (70, F)

16

8.2

Sulphonylureaj α-glucosidase inhibitor Sulphonylurea

7 (56, F)

6

8.4

8 (69, M)

11

6.8

9 (43, F)

3

7.5

10 (54, F)

10

6.6

11 (56, M)

6

7.7

Sulphonylureaj α-glucosidase inhibitor Insulinjsulphonylurea

12 (65, F)

8

7.3

Sulphonylurea

13 (57, F)

6

7.9

Insulin

Insulinj sulphonylurea Sulphonylureaj α-glucosidase inhibitor Insulin

lationship between vitreous hHGF concentration and other parameters.

RESULTS Vitreous hHGF concentrations in subjects with PDR The mean hHGF concentration in vitreous samples from PDR patients was 5.12p0.41 ng\ml (n l 32 measurements). No difference was found between the mean hHGF concentrations in the original (5.63p0.72 ng\ml ; n l 13) and artificial (4.76p2.15 ng\ml ; n l 19) vitreous. Vitreous hHGF concentrations were measured

Time from previous vitrectomy (days)

Causes of vitrectomy

Vitreous hHGF concn. (ng/ml)

First vitrectomy 7 First vitrectomy 245 3 73 160 First vitrectomy 98 86 First vitrectomy 15 First vitrectomy 43 First vitrectomy 7 First vitrectomy 8 First vitrectomy 15 First vitrectomy 28 First vitrectomy 35 First vitrectomy 17 35 First vitrectomy 17 First vitrectomy 9 37

VHjPjRD VHjPjRD VHjPjRD P VHjP VHjP VHjPjRD VHjPjRD VH VH VHjPjRD VH VHjPjRD PjRD PjRD VH VH VH VHjPjRD VHjP VHjPjRD VH PjRD VHjP VH VH VH PjRD VH PjRD VHjP VHjP

4.43 10.05 4.23 5.23 4.53 2.33 8.37 4.15 3.21 4.05 9.97 4.45 6.13 5.11 5.07 4.85 2.45 4.71 10.42 3.88 8.77 1.82 4.35 1.87 2.21 5.12 3.13 5.23 4.37 5.81 8.20 5.18

five times in one patient (no. 2), three times in three patients (nos. 3, 11 and 13) and twice in each of the other nine patients (Table 1). The interval between successive measurements of vitreous hHGF in any one subject ranged from 3 days to 8 months. Vitreous hHGF concentrations ranged from 1.82 ng\ml to 10.42 ng\ml (Table 1). In some patients, the vitreous hHGF concentration showed a rapid recovery after vitrectomy (patient no. 2, 4.53 ng\ml at 3 days after vitrectomy ; patient no. 1, 10.05 ng\ml at 7 days after vitrectomy ; patient no. 6, 4.85 ng\ml at 7 days after vitrectomy ; patient no. 7, 4.71 ng\ml at 8 days after vitrectomy ; patient no. 13, 8.20 ng\ml at 9 days after vitrectomy). Because hHGF concen# 2000 The Biochemical Society and the Medical Research Society

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Figure 1 Vitreous hHGF concentrations and PDR lesions in the original vitreous obtained at first vitrectomy (A) and in the artificial vitreous obtained at repeated vitrectomy (B) in 13 subjects with PDR

Figure 2 Correlations between vitreous concentrations of hHGF and of active TGF-β2 (A) or latent TGF-β2 (B) in 14 subjects with PDR

Panel (B) includes hHGF concentrations measured in the artificial vitreous obtained at different vitrectomies in the same patients (see Table 1 ; patient nos. 2, 3, 11 and 13). Abbreviations : VH, vitreous haemorrhage ; P, fibrovascular proliferative changes in the retina and vitreous ; RD, tractional retinal detachment. Significance of differences : *P 0.05.

subgroup with vitreous haemorrhage, vitreo-retinal fibrovascular proliferation and tractional retinal detachment (6.87p1.05 ng\ml ; n l 7) than in that with vitreous haemorrhage alone (2.33p0.12 ng\ml ; n l 2) (Figure 1A). For the artificial vitreous, the mean vitreous hHGF concentration was higher (P 0.05) in the subgroup with vitreous haemorrhage, fibrovascular proliferation and tractional retinal detachment (9.12p0.84 ng\ml ; n l 2) than in those with vitreous haemorrhage alone (3.97p0.35 ng\ml ; n l 9) or with vitreous haemorrhage plus fibrovascular proliferation (4.33p0.93 ng\ml ; n l 6). However, the number of patients with vitreous haemorrhage, fibrovascular proliferation and tractional retinal detachment was lower in the artificial vitreous group than in the original vitreous group (Figure 1).

trations in the artificial vitreous were not related to the time from the previous vitrectomy (r l 0.059, P l 0.812, n l 19), there seemed to be no correlation between the hHGF concentration in the artificial vitreous and the time between vitrectomies. Subgroups of patients were defined by the degree of PDR, based on the presence of vitreous haemorrhage, vitreo-retinal fibrovascular proliferation and tractional retinal detachment ; vitreous hHGF concentrations were compared between these subgroups for both the original vitreous (Figure 1A) and the artificial vitreous (Figure 1B). Separate analyses were carried out because the differences in vitreous composition between the original and artificial vitreous could have major effects on the distribution, synthesis and breakdown of hHGF molecules. For the original vitreous, the mean vitreous hHGF concentration was higher (P 0.05) in the # 2000 The Biochemical Society and the Medical Research Society

Vitreous TGF-β2 and hHGF concentrations Vitreous concentrations of TGF-β were 0.28p0.05 ng\ # ml for the active form (n l 14) and 2.61p0.24 ng\ml for the latent form (n l 14). Vitreous hHGF concentrations were directly proportional to vitreous concentrations of total TGF-β (r l 0.780, P l 0.001, n l 14) and of latent

#

Vitreous hepatocyte growth factor in proliferative diabetic retinopathy

TGF-β (Figure 2B), but inversely proportional to # vitreous concentrations of active TGF-β (Figure 2A), in # subjects with PDR.

DISCUSSION In the present study, changes in the vitreous hHGF concentration were not related to the time between vitrectomies, but appeared to be associated with the degree of PDR. Mean hHGF concentrations in either the original vitreous or the artificial vitreous after repeated vitrectomy were higher in the vitreous of subjects with severe PDR, which showed vitreous haemorrhage, fibrovascular proliferative changes and tractional retinal detachment, than in the vitreous of subjects not suffering from these conditions. These findings suggest that vitreous hHGF is involved in the pathological mechanism causing severe PDR. The mean vitreous hHGF concentrations seen in the present study were approx. 24-fold higher than the reported mean serum hHGF concentration of 0.213 p0.025 ng\ml in subjects with PDR [18]. The concentrations of hHGF found in the vitreous of patients with PDR are within the range shown previously to cause endothelial cell proliferation ; concentrations of hHGF ranging from 1 to 10 ng\ml are sufficient to stimulate DNA synthesis in human aortic endothelial cells [19], and hHGF at concentrations between 1 and 5 ng\ml elicits proliferation of human endothelial cells in a dosedependent manner [8]. Therefore hHGF in the vitreous is likely to play a significant role in retinal neovascularization in PDR. Concentrations of vitreous hHGF that are higher than serum concentrations indicate that vitreous hHGF does not represent leakage from the serum to the vitreous, but rather that hHGF is produced endogenously in the human eye. In the present study, the vitreous was completely removed and a fresh irrigating solution was used to fill up the intraocular space at each pars plana vitrectomy ; therefore the concentrations of vitreous hHGF obtained at repeated vitrectomy are not thought to be affected by the vitreous hHGF concentration at the previous vitrectomy. In addition, vitreous hHGF concentrations showed rapid recovery after vitrectomy in some patients. Thus vitreous hHGF is likely to be produced endogenously in the eye, although further investigation is required to determine the mechanism of the intraocular production of hHGF. In humans, there are three known TGF-β isoforms (TGF-β , -β and -β ), which are localized to both the " # $ anterior and posterior segments of the eye [17,20–22]. Of the three isoforms, TGF-β is the predominant form # in the posterior segment of the eye [11,12]. TGF-β is known to inhibit HGF production [13–16]. In the present study, the vitreous concentration of hHGF in

PDR patients was directly proportional to the concentrations of both total and latent TGF-β , but inversely # proportional to the concentration of active TGF-β . # These findings suggest that a decrease in the conversion of latent TGF-β into active TGF-β may be involved in # # increasing the concentration of vitreous hHGF in PDR patients. TGF-β is presumed to be involved in the # pathological mechanisms of PDR, such as synthesis of extracellular matrix, enhancement of fibrosis and contraction of collagen gels [12,23,24], and hHGF is likely to be involved in the neovascularization of PDR, as described above. Thus the relationship between intraocular TGF-β and hHGF may be important in the pathogenesis # of PDR. In the present study, high concentrations of hHGF were found in the original vitreous and also in the artificial vitreous after vitrectomy of patients with severe PDR (with fibrovascular proliferation, retinal detachment and vitreous haemorrhage). Increased vitreous hHGF is presumed to play a role not only in the occurrence of PDR, but also in its recurrence after vitrectomy. Decreased conversion of latent into active TGF-β is likely to be involved in the increased intra# ocular production of hHGF in PDR, although direct evidence for this has not been obtained in the present study. Clarifying the precise role of intraocular hHGF will be of significance for understanding the complex pathological mechanisms causing PDR.

ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science and Culture of Japan, and by the Charitable Trust Clinical Pathology Research Foundation of Japan.

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Received 10 May 1999/6 July 1999; accepted 10 September 1999
