Hiroshi Kosano

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 [0.75−22.2]) than in control patients (2.86 ng/ml [0.75−5.80]). Intravitreous concentrations of VEGF were also higher in diabetic patients with PDR (1.62 ng/ml [0.15−7.9]) than in control patients (0.16 ng/ml [0.16−0.29]). 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 VEGF 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.

Regulation of adipocytokine secretion and adipocyte hypertrophy by polymethoxyflavonoids, nobiletin and tangeretin

AIMS The polymethoxyflavonoids nobiletin and tangeretin possess several important biological properties such as neuroprotective, antimetastatic, anticancer, and anti-inflammatory properties. The present study was undertaken to examine whether nobiletin and tangeretin could modulate adipocytokine secretion and to evaluate the effects of these flavonoids on the hypertrophy of mature adipocytes. MAIN METHODS All experiments were performed on the murine preadipocyte cell line 3T3-L1. We studied the formation of intracellular lipid droplets in adipocytes and the apoptosis-inducing activity to evaluate the effects of polymethoxyflavonoids on adipocyte differentiation and hypertrophy, respectively. The secretion of adipocytokines was measured using ELISA. KEY FINDINGS We demonstrated that the combined treatment of differentiation reagents with nobiletin or tangeretin differentiated 3T3-L1 preadipocytes into adipocytes possessing less intracellular triglyceride as compared to vehicle-treated differentiated 3T3-L1 adipocytes. Both flavonoids increased the secretion of an insulin-sensitizing factor, adiponectin, but concomitantly decreased the secretion of an insulin-resistance factor, MCP-1, in 3T3-L1 adipocytes. Furthermore, nobiletin was found to decrease the secretion of resistin, which serves as an insulin-resistance factor. In mature 3T3-L1 adipocytes, nobiletin induced apoptosis; tangeretin, in contrast, did not induce apoptosis, but suppressed further triglyceride accumulation. SIGNIFICANCE Our results suggest that nobiletin and tangeretin are promising therapeutic candidates for the prevention and treatment of insulin resistance by modulating the adipocytokine secretion balance. We also demonstrated the different effects of nobiletin and tangeretin on mature adipocytes.

Genesis of breast cancer in Japanese: a possible relationship between sex hormone binding globulin (SHBG) and serum lipid components.

We investigated changes in the amount of sex hormone binding globulin (SHBG) and the percentage of free estradiol (% FE2) during normal menstrual cycles in the sera of 8 young Japanese controls, and also the relation between the lipid components of serum and the serum level of SHBG in 250 normal Japanese controls aged 18–77 years. The amount of SHBG measured by IRMA was significantly higher in the luteal phase than in the follicular phase, whereas the % FE2 values remained constant throughout the menstrual cycle in 8 healthy young controls. These results suggest that the estrogen status in serum is constantly controlled by SHBG. The amount of SHBG was significantly correlated with the serum level of triglycerides (TG) in 250 normal Japanese controls. From these results, we postulate that the decrease in serum SHBG, probably caused by increased level of serum TG, leads to elevation of % FE2 and later to a status with a high risk of breast cancer.

Enhancement of cyclic adenosine monophosphate content in bone cells by the factor extracted from a pancreatic cancer associated with hypercalcemia

A woman with exocrine pancreatic cancer presented a syndrome of humoral hypercalcemia of malignancy (HHM). Either urea extract or acid/ethanol extract of the tumor showed a dose-dependent activity to elevate cyclic adenosine monophosphate (AMP) level in rat bone cells in primary culture. When each population obtained by the sequential digestion of rat fetal calvaria was cultured individually and cyclic AMP responses to parathyroid hormone (PTH), calcitonin, and tumor extract were examined, tumor extract-sensitive cells showed a similar distribution to PTH-sensitive cells. Tumor extract and PTH, but not calcitonin, increased cyclic AMP in osteogenic cell line MC 3T3-E1. PTH receptor-mediated increase of cyclic AMP was indicated by an antagonistic action of PTH analogue, (3-34) hPTH, on increase of cyclic AMP in MC 3T3-E1 elicited by tumor extract. Human breast cancer derived cell line MCF-7 had calcitonin-sensitive adenylate cyclase, but neither PTH nor tumor extract increased cyclic AMP in the cells. On Bio-Gel P-60 column, the activity to stimulate bone cell cyclic AMP was eluted as a single peak at the molecular size between 6.5 K and 12.4 K. It was concluded that pancreatic cancer, although rather exceptional as a cause of HHM, produced a factor very similar to that reported in representative HHM tumors of human and animal models.

Polymethoxyflavones as agents that prevent formation of cataract: nobiletin congeners show potent growth inhibitory effects in human lens epithelial cells.

Posterior capsular opacification (PCO) is the most frequent complication and the primary reason for visual decrease after extracapsular cataract surgery. The proliferation and migration of leftover lens epithelial cells (LECs) after surgery may contribute to the development of PCO. To prevent PCO, a rational approach would be to inhibit both the proliferation and the migration of LECs using nontoxic xenobiotics. Nobiletin, one of the most abundant polymethoxyflavones (PMFs) in citrus peel, and its synthetic congeners displayed a potent inhibition of LEC proliferation. Structural features which enhance anti-proliferative activity have also been discussed.

Steroid-induced cataract: other than in the whole animal system, in the lens culture system, androgens, estrogens and progestins as well as glucocorticoids produce a loss of transparency of the lens.

PURPOSE To investigate the mechanism of glucocorticoid-induced cataract formation, the lenses of chick embryos were cultured with androgen, estrogen and mineralocorticoid as well as glucocorticoids. The incidence of loss of transparency induced by these steroids in the culture system and the whole body system was compared. METHODS In the culture system, clear lenses obtained from 16-day-old chick embryos were treated with various concentrations of steroid hormones for 48 h at 37 degrees C in a humidified atmosphere containing 5% CO2. In the whole body system, these steroids dissolved in 5% acetone in water were administered to 15-day-old embryos and the lenses were isolated and visually classified on day 17. RESULTS When 0.25 mumol of steroids were administered to 15-day-old chick embryos, only biologically active glucocorticoids such as hydrocortisone and prednisolone could cause cataract. Dexamethasone is approximately 25-fold stronger than hydrocortisone and prednisolone. Methyltestosterone as an androgen, estradiol and ethinylestradiol as estrogen, progesterone and 19-nor-ethisterone as progestin did not induce cataract formation. In the whole body system, the cataracts were caused with a dependence on the biological activity of glucocorticoids. However, other than in the whole body system, when the isolated chick lenses were cultured in the dishes, they could become opaque in the presence of testosterone, estradiol and aldosterone as well as dexamethasone and hydrocortisone at a similar dose (over 3 x 10(-5) M). CONCLUSION These results demonstrate that the loss of transparency of cultured lens can be induced independently from biological activities of steroids. Glucocorticoids have various physiological and pharmacological activities in the living system. We assume that the steroid-induced cataract is one of the adverse effects caused by synergic biological activities of glucocorticoids.

Purification and partial sequencing of inhibitory factor on renal membrane adenylate cyclase in pancreatic cancer extract: Identity with histones H1B or H1D

Inhibitory activity on renal membrane adenylate cyclase (AC) has previously been found in the extract of a pancreatic cancer associated with humoral hypercalcemia of malignancy (HHM). AC inhibitor was purified employing inhibition of AC activity of renal membrane stimulated by forskolin as its index. N-terminal 9 residues and a digested fragment of purified protein (14 residues) were completely consistent with that of histones H1b and H1d. Not only histone H1 but also histones H2A, H2B and H3 from calf thymus inhibited AC activity. These results indicate that the AC inhibitor in the pancreatic cancer extract is histone H1b or H1d and histones H2A, H2B and H3 also have an AC inhibitory activity.

The effect of PGF2α on parathyroid hormone-stimulated cyclic AMP production in mouse osteoblastic cell, MC3T3E1

The effect of prostaglandin F2 alpha (PGF2 alpha) was investigated in MC3T3E1 cells on the succeeding cAMP response to parathyroid hormone (PTH). PGF2 alpha increased the membrane-associated protein kinase C (PKC) activity, indicating the activation of this enzyme. The effect of PTH to increase cAMP production was enhanced by pretreatment with PGF2 alpha. Phorbol 12-myristate 13-acetate also enhanced cAMP production stimulated by PTH, and PKC inhibitor H7 attenuated the enhancement of PGF2 alpha. A23187 did not reproduce the PGF2 alpha effect, and this effect was not antagonized by the calmodulin antagonist W7. PGF2 alpha did not change the ED50 nor the maximally responsive dose of PTH in stimulating cAMP production. The effect of PGF2 alpha was not affected by pertussis toxin, and PGF2 alpha also enhanced cholera toxin- or forskolin-stimulated cAMP production. In accordance with the response of cAMP to PTH, the resorption of mouse limb bones stimulated submaximally by PTH was enhanced by the concomitant presence of PGF2 alpha. These results indicate that PGF2 alpha modulates cAMP response through the activation of PKC, the target of which might be the catalytic unit of adenylate cyclase. Such interaction between signal transduction systems may have significance in modulating the effect of PTH on bone, i.e., bone resorption.

Protein Kinase C-Mediated Regulation of Matrix Metalloproteinase and Tissue Inhibitor of Metalloproteinase Production in a Human Retinal Müller Cells

Purpose: Matrix metalloproteinases (MMPs) play an important role in the degradation of extracellular matrix (ECM) proteins in the retina. Breakdown of ECM proteins results in neovascularization and tractional retinal detachment, which eventually lead to the symptoms of proliferative diabetic retinopathy. Müller cells are reported to be one of the MMP-producing cells in the retina. However, the molecular mechanism of MMP production derived from Müller cells remains to be fully elucidated. Materials and Methods: The human retinal Müller cell line (MIO-M1) was continuously-subcultured in high glucose (25 mM) condition. After the cells reached confluence, they were treated for 24 h with phorbol ester and/or a protein kinase C (PKC) inhibitor, GF109203X in high (25 mM) or low (5 mM) glucose condition. Gelatinase activities in conditioned medium were assessed using gelatin zymography. RT-PCR was performed to analyze the mRNA expression level of MMP-9. Western blot analysis used to detect the protein expression of tissue inhibitors of metalloproteinases (TIMPs). Electrophoresis mobility shift assay was conducted to examine transcription factors involved in MMP-9 transcription. Results: We demonstrated the protein kinase C (PKC)-mediated regulation of proMMP-9 transcription and protein expression through the action of phorbol ester, an activator of PKC. Moreover, we demonstrated the expression of TIMPs, known as natural inhibitors of MMPs at the protein level in a human retinal Müller cell line for the first time, and report that production of these proteins was also regulated in a PKC-dependent manner. Conclusion: Our results suggest that imbalance between MMP and TIMP proteins may promote neovascularization by PKC activation in retinal Müller cells. In addition, the development of novel compounds with regulatory action on MMP and TIMP production through inhibiting PKC activity in retinal Müller cells may lead to new therapeutic approaches for the treatment and prevention of diabetic retinopathy.

Stimulation of melanogenesis in murine melanoma cells by 2-mercapto-1-(β-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.