Yukichi Okuda

Ocular vascular endothelial growth factor levels in diabetic rats are elevated before observable retinal proliferative changes

Summary Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. VEGF levels in ocular tissue of 6-, 12-, 18- and 28-week-old Goto-Kakizaki (GK) rats, a well-known model of non-insulin-dependent diabetes, were evaluated by highly sensitive ELISA. VEGF concentrations in the GK rat as well as in non-diabetic Wistar rat significantly decreased from the age of 6 weeks to 18 weeks. However, although VEGF concentrations in the Wistar rat continued to fall significantly from 18 to 28 weeks of age, the levels were maintained between 18 and 28 weeks of age in GK rats. Levels were significantly different between the GK and Wistar rats at 28 weeks of age. Results of immunohistochemical studies of the eyes of Wistar and GK rats at 28 weeks of age suggest diffuse distribution of this cytokine in cells of neural origin. Weak to moderate VEGF immunoreactivity was exhibited mainly in the ganglion cell layer, inner plexiform layer and inner/outer nuclear layers in rats with and without diabetes. However, in the retinal optic nerve fiber layer, retinal pigment epithelium and choroid, strong VEGF immunoreactivity was noted only in the GK rat. In conclusion, increased VEGF production in certain ocular tissue, similar to that in humans, is observed quite early, at least before the appearance of observable retinal changes in the diabetic GK rat. This also suggests that the GK rat can be used as a model of initial or latent phase diabetic retinopathy. [Diabetologia (1997) 40: 726–730]

Long-term effects of eicosapentaenoic acid on diabetic peripheral neuropathy and serum lipids in patients with type II diabetes mellitus

The present study was undertaken to investigate the efficacy of a new, highly purified (purity greater than 91%), ethyl esterification product from natural eicosapentaenoic acid (EPA-E, C20:5 omega 3) in patients with type II diabetes mellitus (NIDDM). Hemodynamic changes were assessed at the level of the dorsalis pedis artery using an ultrasonic color Doppler duplex system before and after oral administration of EPA-E at a dose of 1800 mg/day for 48 weeks. The cross-sectional area of the dorsalis pedis artery increased significantly from 2.5 +/- 0.2 to 3.9 +/- 0.4 mm2 (48 weeks, mean +/- SE, p < 0.05). Moreover, EPA-E improved the clinical symptom (coldness, numbness) as well as the vibration perception threshold sense of the lower extremities [from 32.1 +/- 8.5 to 16.1 +/- 4.8 (48 weeks) microns]. A significant decrease of serum triglycerides was also noted by EPA-E administration. Furthermore, significant decrease of the excretion of albumin in urine [from 24.4 +/- 3.3 to 13.9 +/- 1.8 (48 weeks) mg/g.Cr, p < 0.05]. The results of this study suggest that EPA-E has significant beneficial effects on diabetic neuropathy and serum lipids as well as other diabetic complications such as nephropathy and macroangiopathy.

Hypoxia and endothelin-1 induce VEGF production in human vascular smooth muscle cells

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a secreted mitogen for vascular endothelial cells, and it promotes vascular permeability and neovascularization in vivo. We investigated the mechanisms by which low oxygen tension modulates the expression of VEGF in human aortic vascular smooth muscle cells (h-SMC) in vitro. Moreover, we measured VEGF levels in the cultured medium with or without endothelin-1 (ET-1) using a newly developed, highly sensitive, enzyme-linked immunosorbent assay. Hypoxia resulted in a substantial induction of VEGF transcripts at 3 and 24 hr. VEGF levels were significantly higher when h-SMC were cultured in medium containing ET-1 than when cultured in medium without ET-1. In conclusion, hypoxia and ET-1 constitute potent stimuli for VEGF production in h-SMC.

Bile acids increase intracellular Ca2+ concentration and nitric oxide production in vascular endothelial cells

The effects of bile acids on intracellular Ca2+ concentration [Ca2+]i and nitric oxide production were investigated in vascular endothelial cells. Whole‐cell patch clamp techniques and fluorescence measurements of [Ca2+]i were applied in vascular endothelial cells obtained from human umbilical and calf aortic endothelial cells. Nitric oxide released was determined by measuring the concentration of NO2−. Deoxycholic acid, chenodeoxycholic acid and the taurine conjugates increased [Ca2+]i concentration‐dependently, while cholic acid showed no significant effect. These effects resulted from the first mobilization of Ca2+ from an inositol 1,4,5‐triphosphate (IP3)‐sensitive store, which was released by ATP, then followed by Ca2+ influx. Both bile acids and ATP induced the activation of Ca2+‐dependent K+ current. Oscillations of [Ca2+]i were occasionally monitored with the Ca2+‐dependent K+ current in voltage‐clamped cells and Ca2+ measurements of single cells. The intracellular perfusion of heparin completely abolished the ATP effect, but failed to inhibit the bile acid effect. Deoxycholic acid and chenodeoxycholic acid enhanced NO2− production concentration‐dependently, while cholic acid did not enhance it. The bile acids‐induced nitric oxide production was suppressed by NG‐nitro‐L‐arginine methyl ester, exclusion of extracellular Ca2+ or N‐(6‐aminohexyl)‐5‐chloro‐l‐naphthalenesulphonamide hydrochloride (W‐7) and calmidazolium, calmodulin inhibitors. These results provide novel evidence showing that bile acids increase [Ca2+]i and subsequently nitric oxide production in vascular endothelial cells. The nitric oxide production induced by bile acids may be involved in the pathogenesis of circulatory abnormalities in liver diseases including cirrhosis.

Effects of intraocular or systemic administration of neutralizing antibody against vascular endothelial growth factor on the murine experimental model of retinopathy

Vascular endothelial growth factor (VEGF), the strongest known angiogenic cytokine and also a potent enhancer of vascular permeability, is closely associated with diabetic ocular complications and other intraocular neovascular diseases. The therapeutic effect of VEGF-neutralizing antibody on oxygen-induced retinopathy in an experimental murine model of proliferative retinopathy was investigated. Intraocular and systemic injection of the antibody resulted in 46% and 18% reductions in the number of nuclei of newly formed vessels of this model, respectively. The results demonstrated that a neutralizing antibody against VEGF was highly effective in the treatment of intraocular neovascularization and suggested possible modes of therapy in human intraocular neovascular diseases, including diabetic proliferative retinopathy.

Antioxidants and an inhibitor of advanced glycation ameliorate death of retinal microvascular cells in diabetic retinopathy

Pericyte ghosts and acellular capillaries are well known as early histological changes resulting from diabetic retinopathy. These histological changes mean that the cell death of retinal microvessels has accelerated. It was reported that apoptosis of retinal microvascular cells (RMCs) was increased in diabetic patients. Therefore, we investigated apoptosis of RMCs in Goto‐Kakizaki (GK) rats, a type 2 diabetic model, and involvement with antioxidants (a combination of vitamins C and E) or a novel inhibitor of advanced glycation, OPB‐9195.

PPARγ ligands, troglitazone and pioglitazone, up-regulate expression of HMG-CoA synthase and HMG-CoA reductase gene in THP-1 macrophages

Recently it has been reported that macrophages express a nuclear receptor, peroxisome proliferator‐activated receptor γ (PPARγ). Using a ligand of PPARγ, troglitazone or pioglitazone, we have shown that the expression of two genes involved in cholesterol biosynthesis, 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) synthase and HMG‐CoA reductase, were increased by activation of PPARγ through a PPAR response element (PPRE) in THP‐1 macrophages. In addition, treatment with troglitazone significantly increased the activity of HMG‐CoA reductase and the amount of intracellular cholesterol. Thus, we conclude that PPARγ and its agonists increase the cholesterol content of macrophages by the increased expression of genes involved in cholesterol biosynthesis. These findings suggest that PPARγ may play a role in cholesterol metabolism in macrophages.

Troglitazone Inhibits Voltage-Dependent Calcium Currents in Guinea Pig Cardiac Myocytes

BACKGROUND It has been suggested that intracellular Ca2+ overload in cardiac myocytes leads to the development of diabetic cardiomyopathy. Troglitazone, an insulin-sensitizing agent, is a promising therapeutic agent for diabetes and has been shown to prevent diabetes-induced myocardial changes. To elucidate the underlying mechanism of troglitazone action on cardiac myocytes, the effects of troglitazone on voltage-dependent Ca2+ currents were examined and compared with classic Ca2+ antagonists (verapamil and nifedipine). METHODS AND RESULTS Whole-cell voltage-clamp techniques were applied in single guinea pig atrial myocytes. Under control conditions with CsCl internal solution, the voltage-dependent Ca2+ currents consisted of both T-type (ICa,T) and L-type (ICa,L) Ca2+ currents. Troglitazone effectively reduced the amplitude of ICa,L in a concentration-dependent manner. Troglitazone also suppressed ICa,T, but the effect of troglitazone on ICa,T was less potent than that on ICa,L. The current-voltage relationships for ICa,L and the reversal potential for ICa,L were not altered by troglitazone. The half-maximal inhibitory concentration of troglitazone on ICa,L measured at a holding potential of -40 mV was 6.3 micromol/L, and 30 micromol/L troglitazone almost completely inhibited ICa,L. Troglitazone 10 micromol/L did not affect the time courses for inactivation of ICa,L and inhibited ICa,L mainly in a use-independent fashion, without shifting the voltage-dependency of inactivation. This effect was different from those of verapamil and nifedipine. Troglitazone also reduced isoproterenol- or cAMP-enhanced ICa,L. CONCLUSIONS These results demonstrate that troglitazone inhibits voltage-dependent Ca2+ currents (T-type and L-type) and then antagonizes the effects of isoproterenol in cardiac myocytes, thus possibly playing a role in preventing diabetes-induced intracellular Ca2+ overload and subsequent myocardial changes.

Inhibitory effects of ω‐3 polyunsaturated fatty acids on receptor‐mediated non‐selective cation currents in rat A7r5 vascular smooth muscle cells

The effects of ω‐3 polyunsaturated fatty acids on receptor‐mediated non‐selective cation current (Icat) and K+ current were investigated in aortic smooth muscle cells from foetal rat aorta (A7r5 cells). The whole‐cell voltage clamp technique was employed. With a K+‐containing solution, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA, 30 μm) produced an outward current at a holding potential of −40 mV. This response was inhibited by tetraethylammonium (20 mm) or Cs+ in the patch pipette solution, and the reversal potential of the EPA‐induced current followed the K+ equilibrium potential in a near Nernstian manner. Under conditions with a Cs+‐containing pipette solution, both vasopressin and endothelin‐1 (100 nm) induced a long‐lasting inward current at a holding potential of −60 mV. The reversal potential of these agonist‐induced currents was about +0 mV, and was not significantly altered by the replacement of the extracellular or intracellular Cl− concentration, suggesting that the induced current was a cation‐selective current (Icat). La3+ and Cd2+ (1 mm) completely abolished these agonist‐induced Icat, but nifedipine (10 μm) failed to inhibit it significantly. ω‐3 polyunsaturated fatty acids (3100 μm), EPA, DHA and docosapentaenoic acids (DPA), inhibited the agonist‐induced Icat in a concentration‐dependent manner. The potency of the inhibitory effect was EPA>DHA>DPA, and the half maximal inhibitory concentration (IC50) of EPA was about 7 μm. Arachidonic and linoleic acids (10, 30 μm) showed a smaller inhibitory effect compared to ω‐3 fatty acids. Also, oleic and stearic acids (30 μm) did not show a significant inhibitory effect on Icat. A similar inhibitory action of EPA was observed when Icat was activated by intracellularly applied GTPγS in the absence of agonists, suggesting that the site of action of ω‐3 fatty acids is not located on the receptor. These results demonstrate that ω‐3 polyunsaturated fatty acids can activate a K+ current and also effectively inhibit receptor‐mediated non‐selective cation currents in rat A7r5 vascular smooth muscle cells. Thus, the data suggest that ω‐3 fatty acids may play an important role in the regulation of vascular tone.

Troglitazone and pioglitazone attenuate agonist‐dependent Ca2+ mobilization and cell proliferation in vascular smooth muscle cells

The effects of troglitazone and pioglitazone on agonist‐induced Ca2+ mobilization and cell proliferation were studied using fluorescent Ca2+ indicator fura‐2 AM and incorporation of [3H]‐thymidine in rat aortic smooth muscle cells. The patch clamp techniques were also employed. Vasopressin and platelet‐derived growth factor‐BB (PDGF) caused a transient elevation in [Ca2+]i by Ca2+ mobilization from intracellular stores, followed by a sustained rise due to Ca2+ entry. Nicardipine partly inhibited the sustained phase, but La3+ completely abolished it. Troglitazone and pioglitazone did not significantly affect the transient rise elicited by these agonists, but preferentially inhibited the sustained phase of [Ca2+]i. Under voltage clamp conditions, troglitazone and pioglitazone inhibited voltage‐dependent L‐type Ca2+ current (ICa·L). They also inhibited nonselective cation channels (Icat) elicited by vasopressin in a concentration‐dependent manner. The half maximal inhibitory concentrations of troglitazone on ICa·L and Icat were 4.6 and 5.7 μM, respectively. On the other hand, nifedipine and nicardipine did not inhibit Icat. Vasopressin and PDGF increased incorporation of [3H]‐thymidine, and nifedipine and nicardipine partly suppressed it. However, the inhibitory effects of La3+ and exclusion of extracellular Ca2+ were more potent than the Ca2+ blocking agents. Troglitazone and pioglitazone also inhibited it concentration‐dependently. These results suggest that troglitazone and pioglitazone preferentially inhibited agonist (vasopressin and PDGF)‐induced Ca2+ entry and proliferation in rat vascular smooth muscle cells, where the inhibitory effects of thiazolidinediones on ICa·L and Icat might be partly involved. Thus, thiazolidinediones may exert hypotensive and antiatherosclerotic effects.