Reduced expression of a novel peptide, prostacyclin-stimulating factor, in the kidneys of streptozotocin-induced diabetic rats.

Prostacyclin (PGI2) produced by vascular endothelial cells (ECs) is a potent vasoactive prostanoid involved in maintenance of vessel wall homeostasis. Reduced PGI2 synthesis by vascular ECs could be a mechanism of pathogenesis in the development of vascular lesions such as diabetic angiopathy. Recently, we purified and cloned a novel bioactive peptide, PGI2-stimulating factor (PSF), which stimulates PGI2 production by vascular ECs. PSF may act on vascular ECs in a paracrine and/or autocrine fashion to regulate PGI2 synthesis. Decreased PSF production in the vessel wall may result in an imbalance of prostanoid synthesis, leading to the development of vascular lesions such as diabetic angiopathy. Our immunohistochemical study demonstrated that PSF is located in vascular resident cells such as vascular smooth muscle cells (SMCs) and ECs, as well as in bronchial SMCs. Moreover, PSF mRNA was found to be expressed in various tissues in Wistar rats, particularly in the kidneys and lungs. The present study demonstrated that streptozotocin (STZ)-induced diabetic rats showed less PSF mRNA expression in the kidneys (PSF mRNA/28S rRNA ratio; STZ versus control; 1.7+/-0.2 versus 2.5+/-0.2, p < 0.05) and reduced immunohistochemical staining for PSF in arteries in the kidney. However, in the lungs, there were no changes in tissue PSF mRNA expression (STZ versus control; 10.9+/-0.9 versus 11.5+/-1.0, NS) or in the extent of PSF staining in bronchial SMCs of STZ-induced diabetic rats. These findings suggest that decreased expression of PSF in renal vessels of STZ-induced diabetic rats may cause an imbalance of prostanoid synthesis, leading to the development and progression of vascular damage in the kidney.

Increased mRNA expression of a novel prostacyclin-stimulating factor in human colon cancer.

We recently cloned a prostacyclin (PGl2)-stimulating factor (PSF), which stimulates PGl2 production by cultured vascular endothelial cells. Immunohistochemistry and Northern blot analysis demonstrated that PSF was highly expressed in colon cancer sites compared with normal colon mucosa obtained from the same patient, as well as in cultured adenocarcinoma cell lines compared with cultured normal colon mucosal cell lines. Increased levels of the PSF protein were detected in the culture media of these adenocarcinoma cells compared with levels in the culture media of normal mucosal cells. These results suggest that PSF is closely associated with carcinogenesis of colon mucosa.

Immunohistochemical study of prostacyclin-stimulating factor (PSF) in the diabetic and atherosclerotic human coronary artery.

Prostacyclin (PGI2) synthesis by vascular endothelial cells (ECs) decreases in diabetic subjects, possibly leading to the development of diabetic angiopathy, such as that seen in atherosclerosis. We recently found a novel bioactive peptide, prostacyclin-stimulating factor (PSF), which stimulates PGI2 synthesis by cultured aortic ECs. Our previous studies demonstrated that PSF is dominantly expressed by arterial smooth muscle cells (SMCs). In the present study, we found PSF to exist in the SMCs of human coronary arteries by means of immunohistochemical methods. Human coronary arteries obtained from autopsies were divided into four subgroups, with or without NIDDM and/or myocardial infarction. Immunostaining for PSF was performed by the avidin-biotin peroxidase complex method using a purified anti-PSF antibody, and the immunostaining for PSF was assessed semiquantitatively. PSF staining was markedly reduced in coronary arterial SMCs from patients with NIDDM and/or myocardial infarction. In addition, the effect of a high glucose culture on PSF mRNA expression and PSF production in bovine aortic SMCs was examined by immunocytochemical staining and both Western and Northern blot analyses. The immunostaining and immunoblot band for PSF also significantly decreased when bovine aortic SMCs were cultured with high concentrations of glucose. Furthermore, as compared with the SMCs cultured with a physiological glucose concentration, the density ratio of PSF mRNA to 28S rRNA expression significantly decreased when the SMCs were cultured with high concentrations of glucose. These results strongly suggest that the decreased PSF production may thus results in a decreased production of PGI2 in the coronary artery, thus leading to the development of both diabetic macroangiopathy and atherosclerosis.

Difference in serum-induced prostacyclin production by cultured aortic and capillary endothelial cells.

Prostacyclin (PGl2) generated by vascular endothelial cells play an important role in the maintenance of vessel wall homeostasis. Human plasma-derived serum (PDS) stimulated PGl2 synthesis by both cultured bovine aortic endothelial cells (BAEC) and adrenal capillary endothelial cells (BCEC), but the PGl2 response of the latter cells was far smaller. When BAEC were cultured with a high concentration of glucose (400 mg/dl), the PGl2 synthesis induced by 20% PDS was significantly lower than in the culture with a physiological concentration of glucose (100 mg/dl) (258 +/- 45 pg/10(4) cells/h vs. 402 +/- 52 pg/10(4) cells/h, n = 4, P < 0.05). On the other hand, there was no significant difference in the PDS-induced PGl2 synthesis between BCEC cultured with high and physiological concentrations of glucose. Additionally, 10% PDS obtained from patients with non-insulin dependent diabetes mellitus (n = 6) stimulated significantly less PGl2 synthesis than that from healthy subjects (n = 4) in the case of both BAEC (133 +/- 27 pg/10(4) cells/h vs. 402 +/- 38 pg/10(4) cells/h, P < 0.05) and BCEC (72 +/- 15 pg/10(4) cells/h vs. 118 +/- 12 pg/10(4) cells/h, P < 0.05), with the difference in PGl2 synthesis being smaller for BCEC. These findings indicate that the PDS-induced PGl2 synthesis differs between cultured vascular endothelial cells from large and small vessels with the decrease in PGl2 by diabetic PDS and high glucose being more marked for BAEC than BCEC.

Human diploid fibroblast cell culture medium contains a factor that increases cytosolic Ca2+ and stimulates prostaglandin synthesis by cultured bovine aortic endothelial cells.

Previously, we demonstrated that conditioned medium (CM) from cultures of human diploid fibroblast cells contains a factor that stimulates the production of prostacyclin (PGI2) by cultured bovine aortic endothelial cells (BAEC). To study the mechanism by which CM stimulates PGI2 production, we measured the effect of removal of extracellular calcium (Ca2+) on the concentration of cytosolic Ca2+ and on the production of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), a stable metabolite of PGI2. The CM-induced production of 6-keto-PGF1 alpha was dependent on extracellular Ca2+ and did not require nascent protein synthesis. Application of CM to BAEC induced a transient increase in cytosolic Ca2+ concentration that was dependent on extracellular Ca2+. Bradykinin induced the production of 6-keto-PGF1 alpha by BAEC. However, bradykinin induced an increase in cytosolic Ca2+ concentration in the presence or absence of extracellular Ca2+. Voltage dependent Ca2+ channel blocker (verapamil, diltiazem) did not inhibit either the CM-induced increase in cytosolic Ca2+ or the production of 6-keto-PGF1 alpha by BAEC. These data suggest that CM increases the cytosolic Ca2+ concentration and stimulates PGI2 production by BAEC. The increase in cytosolic Ca2+ concentration occurred via the influx of extracellular Ca2+ independent of L-type Ca2+ channels blocked by verapamil or diltiazem.

Prostacyclin-stimulating factor, novel protein, and diabetic angiopathy.

We recently purified and cloned a new protein that stimulates the synthesis of prostacyclin (PGI2) by the vascular endothelial cells (ECs). We have termed this protein "PGI2-stimulating factor" (PSF). The present study evaluated the expression of PSF mRNA in tissues of Wistar rats, including the kidneys of rats with streptozotocin-induced diabetes, and in cultured cells. Furthermore, we evaluated the presence of PSF in human sera and the immunohistochemical localization of PSF in tissues of patients obtained at autopsy. The latter included a coronary atherosclerotic lesion of a patient who died of acute myocardial infarction. PSF was observed by Northern blot analysis to be expressed in all rat tissues examined (brain, lung, liver, kidney, skeletal muscle, and fat tissue) and was expressed in cultured vascular ECs, smooth muscle cells (SMCs), and fibroblast cells (FCs). A decreased expression of PSF was observed in the kidneys of diabetic rats versus those of normal rats. The presence of PSF in human serum was confirmed by Western blot analysis. In humans, PSF was mainly localized in vascular ECs and SMCs of arterial media and in SMCs of bronchi. Reduced staining for PSF was found in an atherosclerotic versus a normal coronary artery of humans. PSF may be involved in the production of PGI2 in the vessel wall and may participate in the maintenance of vascular homeostasis. PSF abnormalities may be involved in the development of such vascular lesions as atherosclerosis and diabetic angiopathy.

Immunohistochemical localization of a novel peptide, prostacyclin-stimulating factor (PSF), in human tissues.

In a recent study, we purified and cloned a newly identified bioactive factor that stimulates prostacyclin (PGI2) production by vascular endothelial cells (ECs) using conditioned medium (CM) from cultured human diploid fibroblast cells. The present study was undertaken to clarify whether PSF is expressed in ECs and vascular smooth muscle cells (SMCS) of human tissues at a protein level. In an immunohistochemical study of seven human autopsy cases, all arteries in all lungs (n = 4) and kidneys (n = 6) examined stained positive to variable extents for PGI2-stimulating factor (PSF). PSF was predominantly expressed by SMCs in the media of small arteries. Staining for PSF was weaker in SMCs of aortic media (n = 3) and strong in SMCs of vaso vasorum (n = 3). PSF staining was also found in the SMCs of human bronchi (n = 4). Immunoblot analysis confirmed that PSF is present in CM from cultured bovine aortic SMCS.

Metabolism of oxidized glycated low-density lipoprotein in cultured bovine aortic endothelial cells.

The serum concentration of glycated low-density lipoprotein (gLDL) is increased in individuals with diabetes mellitus, which may be a contributing factor to the increased incidence of atherosclerosis in this population. Given the importance of oxidized LDL (oLDL) in atherosclerosis and that vascular endothelial cells express receptors for oLDL, oxidized glycated LDL (ogLDL) was prepared in vitro and its binding and degradation by cultured bovine aortic endothelial cells were examined. Glycation of native LDL (nLDL) isolated from normal human subjects was performed by incubation with 20 mM glucose at 37 degrees C for 3 days, and ogLDL was prepared by oxidation of gLDL with 1 microM CuSO4 at 37 degrees C for 12 hours. The electrophoretic mobility and thiobarbituric acid reactive substance (TBARS) value of ogLDL were greater than those of nLDL and gLDL. Both binding and degradation of ogLDL by cultured endothelial cells also were significantly greater than for nLDL and gLDL. Degradation of nLDL by endothelial cells was completely inhibited by ogLDL, whereas degradation of acetylated LDL was not inhibited by nLDL or ogLDL. Thus, the binding and degradation of ogLDL by endothelial cells do not appear to be mediated by the scavenger receptor. Although the exact mechanism is not clear, it appears that vascular endothelial cells may play a protective role against atherosclerosis by removing potential atherogenic lipoproteins.

Enhancement of prostacyclin production in cultured bovine aortic endothelial cells by oxidized glycated low-density lipoprotein.

Oxidized low-density lipoprotein (oLDL) is implicated in the pathogenesis of atherosclerosis. The serum concentration of glycated LDL (gLDL) is increased in diabetics, and it is possible that oxidative modification of gLDL contributes to the increased incidence of atherosclerosis associated with diabetes. The mechanism and effect on prostacyclin (PGI2) production by cultured bovine aortic endothelial cells of oxidized glycated LDL (ogLDL) prepared in vitro have now been examined. Glycation of LDL was performed by incubating LDL with 20 mM glucose for 3 days. ogLDL was then prepared by incubation of gLDL with 1 microM CuSO4 for 12 h. Both the electrophoretic mobility and the thiobarbituric acid reactive substance content of ogLDL were greater than those of native LDL (nLDL) or gLDL. Binding, cell-association, and degradation of ogLDL in endothelial cells were significantly greater than those of nLDL and gLDL. The stimulatory effect of ogLDL on PGI2 production was significantly greater than that of nLDL or gLDL; this effect was dose dependent. Both cell-association and the stimulatory effect on PGI2 production of oLDL were dependent on the extent of oxidation in a biphasic manner. Endothelial cells thus appear to protect against atherosclerosis by removing atherogenic lipoproteins and by producing PGI2.

Human fibroblast cells produce a factor that stimulates prostacyclin synthesis by vascular endothelial cells.

The prostacyclin (PGI2) produced by vascular endothelium plays a key role in maintaining vascular homeostasis. The present study demonstrated that the conditioned medium (CM) of human diploid fibroblast cells contained PGI2-stimulatory activity (PSA) for bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC). CM significantly stimulated the production of 6-keto-PGF1 alpha, a stable PGI2 metabolite, by both cultured BAEC and HUVEC in a concentration-dependent manner. Since the factor responsible for the PSA seemed to be negatively charged, PSA was partially purified using a DEAE-5PW high performance liquid chromatography column. The partially purified PSA was completely inhibited by preincubation with 15 microM indomethacin, a cyclooxygenase inhibitor. However, partially purified PSA was partially inhibited by preincubation with 50 microM mepacrine, a phospholipase A2 inhibitor. These findings suggest that PSA stimulates preferentially cyclooxygenase relative to phospholipase A2 in vascular endothelial cells. The partially purified PSA showed no effect on thromboxane A2 production by human washed platelets, and had no growth-promoting activity on BAEC. We conclude that cultured human fibroblast cells produce factor that stimulate the synthesis of prostaglandin by vascular endothelial cells but not by platelets.

Purification and molecular cloning of prostacyclin-stimulating factor from serum-free conditioned medium of human diploid fibroblast cells.

We attempted to identify the factor that stimulated prostacyclin (PGI2) production using conditioned medium from cultured human diploid fibroblast cells subjected to a series of purification steps using h.p.l.c. on DEAE-5PW, Heparin-5PW, Protein-Pak 300, and an insulin-like growth factor-1 ligand affinity column. The purified prostacyclin-stimulating factor (PSF) ran as a single band with a molecular mass of 31 kDa by SDS/PAGE. Analysis of the purified PSF by C4 reversed-phase h.p.l.c. showed a single sharp peak in 31% (v/v) acetonitrile. The material was purified 8000-fold with an overall yield of about 18%. The purified PSF stimulated PGI2 production by cultured bovine aortic endothelial cells at a concentration of about 10 ng/ml; maximal stimulation was achieved at a concentration of 25 ng/ml. A cDNA coding for PSF was cloned and sequenced, revealing an apparently novel protein with no obvious sequence similarity to known proteins.

Expression of prostacyclin-stimulating factor, a novel protein, in tissues of Wistar rats and in cultured cells.

We recently purified and cloned a newly identified PGI2-stimulating factor (PSF). In the present study, we examined the PSF expression in the tissues of Wistar rats and in cultured cells, such as fibroblast cells (FCs), endothelial cells (ECs), and smooth muscle cells (SMCs). The expression of PSF was observed in many tissues of Wistar rats, such as brain, lung, liver, kidney, skeletal muscle, and fat tissue. Especially, lung and kidney showed a greater expression than the other tissues. PSF was also expressed in cultured FCs, ECs, and SMCs. These results indicate that PSF is conserved over species, suggesting that PSF plays a significant role in regulating PGI2 production.

Effects of a new oral hypoglycaemic agent (CS-045) on metabolic abnormalities and insulin resistance in type 2 diabetes.

The effects of a thiazolidinedione antidiabetic agent (CS-045) on diabetic metabolic abnormalities were studied in a double-blind clinical trial. Fourteen patients with Type 2 diabetes were selected according to study criteria. Eight were treated with oral CS-045 at 400 mg daily, and six were given placebo. A multi-step, hyperinsulinaemic, euglycaemic clamp study, with simultaneous plasma free fatty acid study, and glucagon tolerance test were performed before and after administration of drug. Following 3 months of treatment with CS-045, there were significant decreases in the mean levels of fasting plasma glucose (from 9.18 +/- 0.95 to 7.78 +/- 0.44 mmol l-1), postprandial plasma glucose (from 11.8 +/- 1.23 to 10.36 +/- 1.06 mmol l-1), and haemoglobin A1c (from 9.3 +/- 0.4 to 6.8 +/- 0.4%). Insulin sensitivity also improved (1st step: from 3.12 +/- 0.33 to 4.70 +/- 0.47 mg kg-1 min-1 (p < 0.01); 2nd step: from 5.61 +/- 0.63 to 7.54 +/- 0.58 mg kg-1 min-1 (p < 0.01); 3rd step: from 9.21 +/- 0.67 to 11.10 +/- 0.87 mg kg-1 min-1). The fasting free fatty acid level decreased significantly from 0.28 +/- 0.04 to 0.22 +/- 0.02 g l-1. The residual free fatty acid level (%) under insulin infusion clamp conditions decreased significantly from 63.7 +/- 9.7 to 45.0 +/- 9.2%. CS-045 treatment was associated with decrease in total cholesterol, total triglycerides, and increase in HDL cholesterol. Basal C-peptide immunoreactivity level decreased, but there was no change in the peak C-peptide immunoreactivity value.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum-free conditioned medium of human diploid fibroblast cells contains an activity that stimulates prostacyclin production by cultured bovine aortic endothelial cells.

Serum-free culture medium conditioned by human diploid fibroblast cells stimulated prostacyclin production by cultured bovine aortic endothelial cells. Gel filtration chromatography using Sephacryl S-100HR revealed at least three peaks of prostacyclin-stimulating activity. This factor was relatively heat-stable, acid-labile, trypsin-sensitive and bound to heparin Sepharose CL-6B. This factor was completely inhibited by indomethacin.

High glucose reduces specific binding for D-alpha-tocopherol in cultured aortic endothelial cells.

We investigated the effects of glucose on specific D-alpha-tocopherol binding to cultured bovine aortic endothelial cells. Our results confirmed that cultured bovine aortic endothelial cells have specific binding sites for D-alpha-tocopherol. These binding sites exhibited time- and temperature-dependent saturation. The specific binding affinity of D-alpha-tocopherol was significantly lower in endothelial cells cultured in high concentrations of glucose (16.8 or 22.4 mM) for > 7 days compared with cells cultured in a physiological glucose concentration (5.6 mM). No significant reduction occurred in D-alpha-tocopherol binding when 11.1 mM mannitol was added to cells cultured in 5.6 mM glucose. The addition of an aldose reductase inhibitor (ICI-128436, Statil) did not significantly affect the high-glucose-induced reduction of D-alpha-tocopherol binding, although it reduced sorbitol levels in the cells compared with those from cells cultured in high concentrations of glucose. Moreover, significantly higher amounts of lipid peroxides were produced in aortic endothelial cells cultured in high concentrations of glucose (16.8 or 22.4 mM) for > 3 days compared with cells cultured in a physiological concentration of glucose. These results indicate that high concentrations of glucose reduce D-alpha-tocopherol binding through mechanisms independent of putative osmotic effects of sorbitol accumulation in the cells. Possible mechanisms include glycation of protein or oxidative damage of cells and/or redox and metabolic imbalances associated with increased flux of glucose via the sorbitol pathway. A glucose-mediated reduction in D-alpha-tocopherol binding could diminish the beneficial effects of D-alpha-tocopherol to vascular endothelial cells and thereby may increase the vascular toxicity of hyperglycemia in diabetes mellitus.

Apolipoprotein E polymorphism affects the response to pravastatin on plasma apolipoproteins in diabetic patients.

In the present study, we examined the levels of plasma lipids and apolipoproteins in patients with non-insulin dependent diabetes mellitus (NIDDM) with hypercholesterolemia in different apolipoprotein E (apo E) phenotypes. We also examined the influences of apo E polymorphism on the response to pravastatin. The patients were divided into three groups, E4/E3, E3/E3, and E3/E2. There were no differences in the baseline levels of plasma lipids and apolipoproteins, except that the level of triglycerides in E3/E2 heterozygotes was significantly higher than E3/E3 homozygotes. Three months of pravastatin administration significantly reduced plasma levels of total cholesterol and low-density lipoprotein cholesterol in each group to the same degree. We observed a significant reduction of apo B both in the E4/E3 and E3/E3 groups and apo E in the E3/E3 group. Such reduction was not observed in the E3/E2 group. We conclude that pravastatin is a potent drug to correct lipid abnormalities, particularly in NIDDM patients with apo E4/E3 and E3/E3. In the E3/E2 group, its effectiveness may be diminished.

[A case of hypopituitarism associated with empty sella and agenesis of corpus callosum, a variant form of septo-optic-pituitary dysplasia].

A 41-year-old man was referred to Kyushu University Hospital for evaluation of hypothyroidism and hypocortisolemia. Pituitary function test revealed the deficiency of GH(growth hormone), ACTH(adrenocorticotropic hormone), prolactin and TSH(thyroid stimulating hormone). MRI showed empty sella and agenesis of corpus callosum. Clinical diagnosis was hypopituitarism with midline brain anomaly. Septo-optic-pituitary dysplasia (SOPD) is a syndrome characterized by agenesis of septum pellucidum or corpus callosum, optic nerve hypoplasia and congenital hypothalamic-pituitary insufficiency. Our case had no ocular anomalies, but today it is regarded as a variant form of SOPD. Evaluation of the integrity of midline brain structures in patients with congenital hypopituitarism is thus thought to be important for their etiology.