Safety evaluation of the use of calcineurin inhibitor to prenatal and postpartum women in Japan from a health administrative database.

BACKGROUND: To investigate the use of calcineurin inhibitors (CNIs) in pregnant Japanese women and to evaluate their safety in infants. METHODS: Data were extracted from the claims database of the Japan Medical Data Center. The prevalence of CNIs was evaluated 180 days before pregnancy onset, during pregnancy, and within180-days post partum. We investigated the characteristics of the infants, including the presence of major malformations and their diagnoses, for 1 year after birth. RESULTS: A total of 91,865 pregnancies in 80,049 women were included. Fifty-three women were prescribed CNIs between 180-day before pregnancy onset and 180-day postpartum; 35 of the 53 women were prescribed the drugs during pregnancy, and 10 of their infants were born preterm. Three were diagnosed with major congenital malformations, such as patent ductus arteriosus. Six preterm infants presented with infant respiratory distress syndrome. CONCLUSIONS: No congenital anomalies were clearly attributable to the use of CNIs during pregnancy.

Paternal height has an impact on birth weight of their offspring in a Japanese population: the Japan Environment and Children's Study.

This study examines the relationship between paternal height or body mass index (BMI) and birth weight of their offspring in a Japanese general population. The sample included 33,448 pregnant Japanese women and used fixed data, including maternal, paternal and infant characteristics, from the Japan Environment and Children's Study (JECS), an ongoing nationwide birth cohort study. Relationships between paternal height or BMI and infant birth weight [i.e., small for gestational age (SGA) and large for gestational age (LGA)] were examined using a multinomial logistic regression model. Since fetal programming may be a sex-specific process, male and female infants were analyzed separately. Multivariate analysis showed that the higher the paternal height, the higher the odds of LGA and the lower the odds of SGA in both male and female infants. The effects of paternal BMI on the odds of both SGA and LGA in male infants were similar to those of paternal height; however, paternal height had a stronger impact than BMI on the odds of male LGA. In addition, paternal BMI showed no association with the odds of SGA and only a weak association with the odds of LGA in female infants. This cohort study showed that paternal height was associated with birth weight of their offspring and had stronger effects than paternal BMI, suggesting that the impact of paternal height on infant birth weight could be explained by genetic factors. The sex-dependent effect of paternal BMI on infant birth weight may be due to epigenetic effects.

Comparison of pregnancy outcomes between women with gestational diabetes and overt diabetes first diagnosed in pregnancy: a retrospective multi-institutional study in Japan.

AIMS: To determine differences in pregnancy outcomes including diabetic complications, maternal and perinatal complications between gestational diabetes mellitus and overt diabetes in pregnancy in Japan. METHODS: A multi-institutional retrospective study compared pregnancy outcomes between gestational diabetes mellitus and overt diabetes in pregnancy. We examined pregnant women who met the former criteria for gestational diabetes mellitus and received dietary intervention with self-monitoring of blood glucose with or without insulin. Overt diabetes in pregnancy was defined as ≥2 abnormal values on 75-g oral glucose tolerance test, fasting glucose ≥126 mg/dl (7.0 mmol/l) and 2-h postprandial glucose ≥200 mg/dl (11.1 mmol/l), or glycated hemoglobin levels ≥6.5% (48 mmol/mol). RESULTS: Data were collected on 1267 women with gestational diabetes and 348 with overt diabetes in pregnancy. Pregestational body mass index was higher (26.2 ± 6.1 vs. 24.9 ± 5.7 kg, P<0.05) and gestational age at delivery was earlier (37.8 ± 2.5 weeks vs. 38.1 ± 2.1 weeks, P<0.05) in overt diabetes than in gestational diabetes. Glycated hemoglobin (6.8 ± 1.1% [51 mmol/mol] vs. 5.8 ± 0.5% [40 mmol/mol], P<0.05) and glucose on 75-g oral glucose tolerance test and prevalence of retinopathy (1.2% vs. 0%, P<0.05) and pregnancy-induced hypertension (10.1% vs. 6.1%, P<0.05) were higher in overt diabetes than in gestational diabetes. Pregnancy-induced hypertension was associated with pregestational body mass index, gestational weight gain, chronic hypertension, and nulliparity but not with 75-g oral glucose tolerance test. CONCLUSIONS: Overt diabetes in pregnancy is significantly associated with maternal complications such as retinopathy and pregnancy-induced hypertension.

Identification of protein phosphatase 2C and confirmation of other protein phosphatases in the ocular lenses.

The reversible phosphorylation of proteins plays essential roles in regulating various cellular events, and is regulated by the opposing actions of protein kinases and protein phosphatases. Protein kinases in the lens system have been well studied, but very little is known about lens protein phosphatases. Protein phosphatases can be divided several families, such as protein phosphatase types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C) and protein tyrosine phosphatases (PTP). In this study we evaluated what kinds of protein phosphatases are present in the lens by using various specific substrates and inhibitors. Samples were prepared from lenses of 17-day-old chick embryos, and fractionated by high-resolution gel permeation column chromatography, then the fractions were assayed for phosphatase activities. The results with 32P-labeled glycogen phosphorylase A, okadaic acid and inhibitor-1, which are a specific substrate and inhibitors of PP1 and/or PP2A, showed that PP1activities were present in the 500-, 115- and 45-kDa fractions of the lens protein. The 115-kDa fraction also contained PP2A activity. By using a phosphothreonine-containing peptide as a substrate, three peaks of phosphatase activities were found at around 115, 55 and 35 kDa. Based on their response to various phosphatase inhibitors and their metal dependency, the fractions of 115 and 35 kDa were concluded to contain PP2A, while the 55-kDa fraction contained PP2C. Immunoblot using specific antibodies against PP1, PP2A and PP2C confirmed that each fraction above contained corresponding protein phosphatases as proteins. When a phosphotyrosine-containing peptide substrate was examined at pH 7.4, we observed a major peak at 500 kDa, which was presumed to contain receptor-like PTP(s). On the other hand, at pH 5.5, we observed a peak of 18 kDa, which was confirmed to contain a low-molecular-weight PTP. These protein phosphatases have recently been suggested to be involved in stress response and apoptosis. Their physiological roles in the lens are of much interest.

Identification of protein phosphatase 2C and confirmation of other protein phosphatases in the ocular lenses.

The reversible phosphorylation of proteins plays essential roles in regulating various cellular events, and is regulated by the opposing actions of protein kinases and protein phosphatases. Protein kinases in the lens system have been well studied, but very little is known about lens protein phosphatases. Protein phosphatases can be divided several families, such as protein phosphatase types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C) and protein tyrosine phosphatases (PTP). In this study we evaluated what kinds of protein phosphatases are present in the lens by using various specific substrates and inhibitors. Samples were prepared from lenses of 17-day-old chick embryos, and fractionated by high-resolution gel permeation column chromatography, then the fractions were assayed for phosphatase activities. The results with 32P-labeled glycogen phosphorylase A, okadaic acid and inhibitor-1, which are a specific substrate and inhibitors of PP1 and/or PP2A, showed that PP1 activities were present in the 500-, 115- and 45-kDa fractions of the lens protein. The 115-kDa fraction also contained PP2A activity. By using a phosphothreonine-containing peptide as a substrate, three peaks of phosphatase activities were found at around 115, 55 and 35 kDa. Based on their response to various phosphatase inhibitors and their metal dependency, the fractions of 115 and 35 kDa were concluded to contain PP2A, while the 55-kDa fraction contained PP2C. Immunoblot using specific antibodies against PP1, PP2A and PP2C confirmed that each fraction above contained corresponding protein phosphatases as proteins. When a phosphotyrosine-containing peptide substrate was examined at pH 7.4, we observed a major peak at 500 kDa, which was presumed to contain receptor-like PTP(s). On the other hand, at pH 5.5, we observed a peak of 18 kDa, which was confirmed to contain a low-molecular-weight PTP. These protein phosphatases have recently been suggested to be involved in stress response and apoptosis. Their physiological roles in the lens are of much interest.

18 kDa protein tyrosine phosphatase in the ocular lens.

Protein tyrosyl phosphorylation and dephosphorylation play essential roles in regulating cellular events such as proliferation and differentiation, and their involvement in the lens development and transparency is also suggested. The level of tyrosine phosphorylation in a given protein is regulated by the opposing actions of protein-tyrosine kinases (Tyr kinases) and protein-tyrosine phosphatases (TyrPases). Recent studies have revealed that some Tyr kinases, such as platelet-derived growth factor receptor and fibroblast growth factor receptor, are present in the lens, however, little is known about TyrPases in the lens. In this study, we found a 18 kDa protein tyrosine phosphatase (18 kDa TyrPase) predominantly present in the ocular lens of various animals. We purified the phosphatase from the lens of chick embryo and characterized its activity.Phosphatase activity was determined in chick embryo, mouse, rabbit and bovine lenses using p -nitrophenyl phosphate (p NPP) as substrate. All lenses examined dephosphorylated p NPP under acidic conditions, and a large portion of the activity resided in a low molecular weight protein, ca. 18 kDa, following high-resolution gel permeation column chromatography. The brain and liver showed high dephosphorylation activities, but most of their activity was present in high molecular weight fractions, unlike that in the lens. The 18 kDa phosphatase was purified from the lens of 17 day old chick embryos to near-homogeneity with two-step rapid chromatography. This phosphatase showed strict substrate specificity for phosphotyrosine and phosphotyrosyl peptides, suggesting that it was a kind of protein tyrosine phosphatases (TyrPases). Several known inhibitors of TyrPases, such as SH blockers, vanadate and phenylarsine oxide, strongly inhibited the enzyme activity. The molecular weight, substrate specificity, and responses to various inhibitors and activators coincide well with those reported for the low molecular weight protein tyrosine phosphatase (LMW-TyrPase), belonging to the TyrPase superfamily. These results suggest that the 18 kDa phosphatase found in the lens is a LMW-TyrPase. The 18 kDa TyrPase is the predominant phosphatase in the ocular lens. It may be involved in regulation of lens cell proliferation, differentiation and/or lens transparency.

Suppressive effects of thyroxine on glucocorticoid (gc)-induced metabolic changes and cataract formation on developing chick embryos.

To prevent cataracts induced by glucocorticoids (GC) as a systemic disease, the suppression of oxidative stress caused by GC in the hepatic metabolism is of significant interest. In this study, to elucidate the formative mechanism of GC-induced cataracts, we examined the preventive effect and then analysed the mechanisms of thyroxine on GC-induced cataract formation. Fifteen day old chick embryos were administered with 0.25 micromol hydrocortisone succinate sodium (HC), and then 12-30 nmol of thyroxine 4 hr after HC administration. At the indicated time after HC treatment, we examined the incidence of cataract formation, the levels of serum glucose and lipids, lenticular and hepatic glutathione (GSH), and lipid peroxide (LPO) in the lens, blood and liver. Almost all lenses (96%) removed 48 hr after HC administration were opaque. Thyroxine prevented HC-induced cataract formation effectively, and suppressed the elevations of serum glucose and LPO in the lens, blood and liver. The treatment prevented the decreased lenticular GSH level at 48 hr, but the hepatic GSH level at 24 hr remained lowered in contrast to the results of previous studies using insulin. Moreover, thyroxine did not decrease the elevated serum lipid level (triglyceride and non-esterified fatty acid) caused by HC. Under thyroxine treatment, in constant to insulin, acceleration of GSH-GSSG cycle rather than GSH de novo synthesis keeps a certain level of hepatic GSH necessary for diminishing the elevation of LPO as a risk factor of GC-induced cataract formation. The regulation of metabolic changes ensured the maintenance of hepatic GSH level, which is necessary to reduce oxidative stress produced by GC and to protect the lens from oxidative stress leading to opacification.

Genetic variation in the hepatocyte nuclear factor (HNF)-3alpha gene does not contribute to maturity-onset diabetes of the young in Japanese.

Heterozygous mutations in the genes encoding transcription factors in the hepatocyte nuclear factor (HNF) cascade are associated with maturity-onset diabetes of the young (MODY), a monogenic form of diabetes mellitus. However, these genes are responsible for only approximately 20% of the cases of MODY in Japanese patients. Searching for a novel MODY gene in this population, we investigated a candidate for encoding the forkhead transcription factor HNF-3alpha, which also belongs to the HNF-transcription cascade. The human HNF-3alpha gene, which was assigned to the segment near microsatellites D14S75 and AFM200ZH4 on chromosome 14 by radiation hybrid mapping, spans approximately 5 kb and consists of two exons. Ninety-five Japanese subjects with MODY/early-onset non-ketotic diabetes were screened for mutations in this gene. Direct sequencing of the exons and flanking regions identified one missense mutation (Ala-83-Thr) in exon 2 and three nucleotide alterations in the non-coding regions. However, their frequencies were not significantly different between MODY and control subjects, indicating that mutations in the HNF-3alpha gene are not a major cause of MODY in Japanese patients.

Establishment of a chick embryo shell-less culture system and its use to observe change in behavior caused by nicotine and substances from cigarette smoke.

Early detection and removal of harmful factors are essential to the proper physical and psychological development of the fetus, presumably showing the effects during the prenatal period and after birth. As one procedure to aid in understanding such factors, we have established a shell-less culture system for video monitoring to observe change in behavior of 7-day-old chick embryos. Nicotine and aqueous cigarette smoke extract (ACSE) were selected for the present experiments, and the results showed a complete stoppage of swing-like movements by administrations of 10 microg nicotine and 1xACSE, possibly displaying paralytic symptoms. Quantitative analysis of nicotine in 1xACSE indicated that more than 10 microg of nicotine were contained in 100 microl of the extract. The present system, although in initial stage of development, may be a useful preliminary screening procedure for perhaps supervision and warning about the environment surrounding pregnant women.

Mutations in the small heterodimer partner gene are associated with mild obesity in Japanese subjects.

Mutations in several genes encoding transcription factors of the hepatocyte nuclear factor (HNF) cascade are associated with maturity-onset diabetes of the young (MODY), a monogenic form of early-onset diabetes mellitus. The ability of the orphan nuclear receptor small heterodimer partner (SHP, NR0B2) to modulate the transcriptional activity of MODY1 protein, the nuclear receptor HNF-4alpha, suggested SHP as a candidate MODY gene. We screened 173 unrelated Japanese subjects with early-onset diabetes for mutations in this gene and found five different mutations (H53fsdel10, L98fsdel9insAC, R34X, A195S, and R213C) in 6 subjects as well as one apparent polymorphism (R216H), all present in the heterozygous state. Interestingly, all of the subjects with the mutations were mildly or moderately obese at onset of diabetes, and analysis of the lineages of these individuals indicated that the SHP mutations were associated with obesity rather than with diabetes. Therefore, an additional group of 101 unrelated nondiabetic subjects with early-onset obesity was screened for mutations in the SHP gene. Two of the previously observed mutations (R34X and A195S) and two additional mutations (R57W and G189E) were identified in 6 subjects, whereas no mutations were identified in 116 young nondiabetic lean controls (P = 0.0094). Functional studies of the mutant proteins show that the mutations result in the loss of SHP activity. These results suggest that genetic variation in the SHP gene contributes to increased body weight and reveal a pathway leading to this common metabolic disorder in Japanese.

Stimulation of melanogenesis in murine melanoma cells by 2-mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB).

The effects of 2-mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB), one of the benzimidazole derivatives designed for a nucleic acid analogue, on melanogenesis of murine B16-F10 melanoma cell lines were investigated. MPB (40 microM) induced a striking dendricity in B16 melanoma cells within 12 h and maximal dendricity between 48 and 72 h. The stimulation of melanin synthesis was observed after only 2 days of treatment together with a dose-dependent growth inhibition. Moreover, MPB increased the activity of tyrosinase through the expression of tyrosinase mRNA without increasing the intracellular cyclic AMP content. MPB-induced melanogenesis was inhibited by novel protein kinase A inhibitors, KT-5720 and H-85. These findings indicate that MPB stimulated B16 cells to terminally differentiate and may be a useful drug in studying the regulation of melanogenesis.

Steroid-induced short term diabetes in chick embryo: reversible effects of insulin on metabolic changes and cataract formation.

PURPOSE: To determine the reversible effect of insulin on glucocorticoid (GC)-induced cataract formation in relation to systemic metabolic changes in the developing chick embryo. METHODS: Hydrocortisone sodium succinate (HC; 0.25 micromoles) was administered to 15-day-old embryos followed by administration of long-acting recombinant human insulin, 4 and 28 hours later. At the indicated time after HC administration, the incidence of cataractous lenses and any changes in the components of the lenses, liver, and blood were determined. RESULTS: At 48 hours after HC administration, the following observations were made: opacification of lenses; an elevation of glucose and lipids in the blood and lenses; an increase in lipid peroxide (LPO) in the blood, liver, and lenses; a decrease in glutathione (GSH) in the lens and liver (at 24 hours after HC administration); and a depletion of adenosine triphosphate (ATP) in the liver. These changes in response to HC administration were reversed by a double application of insulin. CONCLUSIONS: Insulin antagonizes GC-induced gluconeogenesis, stimulates glycolysis, and ultimately leads to recovery of decreased activity in the citric acid cycle. The restoration of ATP by the recovered citric acid cycle may facilitate de novo synthesis of GSH, which in turn may diminish GC-induced elevation of LPO in the liver. Thus, the metabolic changes in response to HC-accelerated gluconeogenesis in the liver, which can be reversed by insulin, are likely to produce oxidative stress that leads to cataract formation. GC-induced metabolic changes in the liver, which are antagonized by insulin, may relate to production of one of the risk factors for cataract formation.

Cloning of cDNA and the gene encoding human hepatocyte nuclear factor (HNF)-3 beta and mutation screening in Japanese subjects with maturity-onset diabetes of the young.

AIMS/HYPOTHESIS: Molecular defects of the genes for transcription factors, hepatocyte nuclear factor (HNF)-4 alpha, HNF-1 alpha, HNF-1 beta and insulin promoter factor-1 cause maturity-onset diabetes of the young (MODY1, 3, 5, and 4, respectively). This suggests the HNF-related transcription cascade is important in insulin secretion which is induced by glucose. These genes and the gene encoding glycolytic enzyme glucokinase (MODY2) are, however, responsible for only 15-20% of cases of MODY in the Japanese. Searching for a novel form of MODY in this population, we cloned a new candidate gene encoding human HNF-3 beta, a winged helix transcription factor, which also belongs to the same HNF-transcription cascade. METHODS: The cDNA clone for human HNF-3 beta was isolated from a liver cDNA library. The gene was also cloned from a genomic library and its organization and chromosomal localization were determined. We screened 68 Japanese subjects with MODY/early-onset diabetes for mutations in this gene. RESULTS: Human HNF-3 beta is composed of 457 amino acids. The human gene, which was mapped to the segment 30 cR from SHGC-37039 on chromosome 20p by radiation hybrid mapping, spans approximately 4.5 kb and consists of three exons. Direct sequencing of the exons and flanking regions identified one missense mutation A328 V and seven polymorphisms, although the functional significance of the mutation in the pathogenesis of diabetes is not known. CONCLUSION/INTERPRETATION: The characterization of the structure of the HNF-3 beta gene and its mapping in the framework of markers will be helpful in genetic studies of the various forms of diabetes mellitus.

ProMMP-9 (92 kDa gelatinase) in vitreous fluid of patients with proliferative diabetic retinopathy.

Matrix metalloproteinases (MMPs) are implicated in tissue destruction during various pathophysiologic conditions. The vitreous body is a gel-like extracellular matrix that undergoes liquefaction during aging and pathological processes. To investigate the pathogenic role of MMPs in proliferative diabetic retinopathy (PDR), we studied 73 eyes from PDR patients and 25 eyes from patients with non-diabetic ocular diseases. Vitreous MMPs were measured by zymography. Retinopathy was assessed by ophthalmoscopy and PDR was classified into 3 stages, 'naked', 'active', and 'quiescent'. Although proMMP-9 was expressed in only 8% (2/25) of non-diabetic patients, it was expressed in more than 80% (38/47) of 'active' PDR patients and still expressed in 60% (9/15) of those with 'quiescent' PDR. Vascular endothelial growth factor (VEGF) in vitreous fluids was undetectable (<0.16 ng/ml) in most of the non-diabetic patients, and was maximally elevated in the 'active' PDR patients (mean=2.20 ng/ml, range; 0.16-7.61), declining in patients with 'quiescent' PDR (1.04 ng/ml, 0.16-3.77). These results suggest that MMP-9 is one of the noteworthy factors in relation to the progress of PDR, as well as angiogenic cytokines such as VEGF.

Loss-of-function and dominant-negative mechanisms associated with hepatocyte nuclear factor-1beta mutations in familial type 2 diabetes mellitus.

Hepatocyte nuclear factor (HNF)-1beta, a homeodomain-containing transcription factor, regulates gene expression in a dimerized form in pancreas, liver, and some other tissues. Recent genetic studies have identified two HNF-1beta mutations, R177X and A263fsinsGG, in subjects with a monogenic form of type 2 diabetes. Despite the defects being in the same gene, diverse severities of disease are observed in the affected subjects. To investigate the molecular mechanism by which mutations might cause various phenotypic features, wild type and mutant proteins were transiently expressed in insulin-producing (MIN6) and hepatic (HepG2) cells. Luciferase reporter assay showed that both mutations resulted in a marked reduction of transactivation activity. Because their dimerization activity was found to be intact by the yeast two-hybrid system, it was possible that they were dominant-negative to wild type activity. When co-expressed with wild type, both of the mutants significantly decreased wild type activity in HepG2 cells. In contrast, although A263fsinsGG functioned similarly in MIN6 cells, R177X failed to affect wild type activity in this cell line. Immunohistochemical analysis of the mutants suggests that this functional divergence might be generated by the modification of nuclear localization. These results suggest that HNF-1beta mutations may impair pancreatic beta-cell function by loss-of-function and dominant-negative mechanisms.

Neutral and acidic glycosphingolipids in glucocorticoid-induced cataract in chick lens.

Administration of glucocorticoids induces transient cataract in 15-day-old chick embryos within 48 hr, and the opaque lens again becomes clear within the subsequent 48 hr. Oxidative stress is likely to be involved in the process of cataract formation, resulting in the appearance of numerous vacuoles around the perinuclear region. Chick lens contained low amounts of glycosphingolipids, which mainly consists of GM3, GD3, sialyl-LewisX gangliosides and glucosylceramide. Most lens gangliosides were immunohistochemically detected in lens epithelia, annular pads and developing fibers, but not in perinuclear and nuclear regions. Since cell surface gangliosides, for example GM3 and sialyl-LewisX gangliosides, are involved in cell adhesion, weak cell-to-cell interactions in the perinuclear and nuclear regions may allow vacuole formation in steroid-induced cataractogenesis.

Identification of active substances from Streptomyces culture fluids using p53-independent expression of p21/WAF1/Cip1 gene and their mode of action.

An assay system was constructed to identify chemicals that have a potential to induce p21/WAF1 gene, a target of the tumor suppressor p53 critical for negative growth regulation. Screening of about 1300 culture fluids of Streptomyces resulted in identification of active substances which induced the p21 gene in a p53-independent manner; one was a mixture of four members of the actinomycin group, and the other was trichostatin A. Transcriptional regulatory regions of p21 gene for induction by actinomycin D and trichostatin A were determined by transient expression of luciferase constructs in cells which are p53-deficient (Saos-2) or express a mutated form of p53 (TMK-1). The essential transcriptional elements for the response to these drugs localize within 210 bp of the 5'-upstream region of human p21 gene, and Sp1 elements were determined to be critical for the induction. DNA-binding activity of Sp1 was not increased in cells treated with these drugs, but kinase inhibitors such as staurosporin and wortmannin inhibited the induction.

Hepatocyte growth factor in vitreous fluid of patients with proliferative diabetic retinopathy and other retinal disorders.

OBJECTIVE: To determine whether hepatocyte growth factor (HGF) is elevated in the vitreous fluid of patients with proliferative diabetic retinopathy (PDR). RESEARCH DESIGN AND METHODS: Vitreous fluid samples were obtained at the time of vitreoretinal surgery from 73 eyes of PDR patients and from 17 eyes of nondiabetic patients (control subjects) who had macular hole, rhegmatogenous retinal detachment, or epiretinal membrane (9, 4, and 4 eyes, respectively) but no associated proliferative vitreoretinopathy Stages of PDR were classified as active or quiescent. Concentrations of HGF and vascular endothelial growth factor (VEGF) in vitreous fluid were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Intravitreous concentrations of HGF (median [range]) were significantly higher in diabetic patients with PDR (6.00 ng/ml [0.75-22.21) than in control patients (2.86 ng/ml [0.75-5.801). Intravitreous concentrations of VEGF were also higher in diabetic patients with PDR (1.62 ng/ml [0.15-7.91) than in control patients (0.16 ng/ml [0.160.29]). Both VEGF and HGF concentrations were significantly higher in patients with active retinopathy than in those with quiescent retinopathy However, vitreous concentrations of HGF were unrelated to those of VEGE CONCLUSIONS: We found that levels of HGF in vitreous fluid of PDR patients are significantly higher than in nondiabetic patients and that the levels of HGF are elevated in the active PDR stage. This suggests that HGF stimulates or perpetuates neovascularization in PDR.