Naotaka Sekiguchi

Protein Kinase C–Dependent Increase in Reactive Oxygen Species (ROS) Production in Vascular Tissues of Diabetes: Role of Vascular NAD(P)H Oxidase

Hyperglycemia seems to be an important causative factor in the development of micro- and macrovascular complications in patients with diabetes. Several hypotheses have been proposed to explain the adverse effects of hyperglycemia on vascular cells. Both protein kinase C (PKC) activation and oxidative stress theories have increasingly received attention in recent years. This article shows a PKC-dependent increase in oxidative stress in diabetic vascular tissues. High glucose level stimulated reactive oxygen species (ROS) production via a PKC-dependent activation of NAD(P)H oxidase in cultured aortic endothelial cells, smooth muscle cells, and renal mesangial cells. In addition, expression of NAD(P)H oxidase components were shown to be upregulated in vascular tissues and kidney from animal models of diabetes. Furthermore, several agents that were expected to block the mechanism of a PKC-dependent activation of NAD(P)H oxidase clearly inhibited the increased oxidative stress in diabetic animals, as assessed by in vivo electron spin resonance method. Taken together, these findings strongly suggest that the PKC-dependent activation of NAD(P)H oxidase may be an essential mechanism responsible for increased oxidative stress in diabetes.

A possible target of antioxidative therapy for diabetic vascular complications-vascular NAD(P)H oxidase

A growing body of evidence has shown that oxidative stress may be involved in the development of vascular complications associated with diabetes. However, the molecular mechanism for increased reactive oxygen species (ROS) production in diabetes remains uncertain. Among various possible mechanisms, attention have increasingly been paid to NAD(P)H oxidase as the most important source of ROS production in vascular cells. High glucose level stimulates ROS production through protein kinase C (PKC)-dependent activation of vascular NAD(P)H oxidase. Furthermore, the expression of NAD(P)H oxidase components is increased in micro- and macrovascular tissues of diabetic animals in association with various functional disorders and histochemical abnormalities. These results suggest that vascular NAD(P)H oxidase-driven ROS production may contribute to the onset or development of diabetic micro- or macrovascular complications. In this point of view, the possible new strategy of antioxidative therapy for diabetic vascular complications is discussed in this review.

INCREASED MRNA EXPRESSION OF A NOVEL PROSTACYCLIN-STIMULATING FACTOR IN HUMAN COLON CANCER

Abstract: 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 semiquantita-tively. 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 result in a decreased production of PGI2 in the coronary artery, thus leading to the development of both diabetic macroangiopathy and atherosclerosis.

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.

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 flbroblast 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.

Effect of high glucose concentrations on prostacyclin-stimulating factor mRNA expression in cultured aortic smooth muscle cells

Summary Prostacyclin (PGI2) is a potent vasoactive prostanoid regulating vascular tone. We recently purified and cloned a PGI2-stimulating factor (PSF), which stimulates PGI2 production by vascular endothelial cells (ECs). Previous study demonstrated that PSF is predominantly located in vascular smooth muscle cells (SMCs) and present in serum. PSF may act on vascular ECs to regulate PGI2 synthesis for maintaining vessel wall homeostasis. 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. In the present study, to investigate the regulatory mechanisms of PSF gene expression, we examined the effect of high glucose concentrations on PSF mRNA expression in cultured bovine aortic SMCs. Expression of PSF mRNA was significantly decreased to 66 ± 6 % of control value (p < 0.01), when the glucose level was raised from 5.5 to 27.8 mmol/l. We also examined the effect of osmolarity on PSF mRNA expression by addition of an appropriate dose of mannitol to the culture medium. We confirmed that high glucose concentration itself reduced the expression of PSF mRNA and glucose had much more effect than the osmolarity control. The expression of PSF mRNA was significantly decreased to 72 ± 5 % of control value (p < 0.05) by a protein kinase C (PKC) activator, phorbol-12-myristate-13-acetate (PMA). The decreased expression of PSF mRNA in the presence of high glucose or PMA was restored by co-incubation with a PKC-specific inhibitor (GF109203X). These results suggest that PSF gene expression in vascular SMCs may be decreased via a specific effect of high glucose concentrations. High glucose-induced activation of PKC is suggested to participate partly in the regulation of PSF gene expression. [Diabetologia (1998) 41: 134–140]

Lysophosphatidylcholine inhibits the expression of prostacyclin stimulating factor in cultured vascular smooth muscle cells

We have cloned a prostacyclin (PGI2) stimulating factor (PSF), which stimulates PGI2 production by vascular endothelial cells. Previous study demonstrated the reduced PSF expression in the coronary arteries from the patients with ischemic heart disease. To clarify the mechanism of reduced PSF expression in atherosclerosis, we examined the effect of lysophosphatidylcholine (lysoPC), a main component of oxidized low density lipoprotein (LDL), on PSF expression in cultured vascular smooth muscle cells. LysoPC reduced PSF expression dose-dependently. Whereas neither phosphatidylcholine nor native LDL affects the PSF expression. Calphostin C, a protein kinase C (PKC) inhibitor, restored the reduction of PSF expression by lysoPC. These results suggest that lysoPC-induced reduction of PSF expression is mediated by PKC activation and is playing a role in the initiation and progression of atherosclerotic lesions.

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.

Expression of prostacyclin-stimulating factor (PSF) in mononuclear cells of human peripheral blood and THP-1 derived macrophage-like cells, and effects of high glucose concentration

Prostacyclin (PGI2) synthesis by vascular endothelial cells (ECs) decreases in diabetic subjects, possibly leading to development of diabetic angiopathies including that in atherosclerosis. We identified a bioactive peptide, prostacyclin-stimulating factor (PSF), which stimulates PGI2 synthesis in cultured aortic ECs. Our previous studies demonstrated that PSF was predominantly expressed by arterial smooth muscle cells (SMCs) and ECs. We immunohistochemically showed that PSF existed in SMCs of human coronary arteries, and PSF staining was markedly reduced in coronary arterial SMCs of patients with type 2 diabetes and/or myocardial infarction. In the present study, we investigated the existence of PSF in human serum, and effects of glucose on serum PSF levels in patients with type 2 diabetes. Immunoblot analysis revealed the presence of PSF in serum, and showed that serum PSF protein concentration was significantly decreased in type 2 diabetic patients. Moreover, there was a significant negative correlation between serum PSF and HbA1c levels in these patients. Using immunohistochemistry, we also showed that PSF was present in serum and in macrophages (Mφs). PSF mRNA was found in Mφs using reverse transcription-polymerase chain reaction (RT-PCR). In addition, effects of high glucose conditions on PSF production in Mφs were examined by Western blotting, and we showed that PSF significantly decreased when Mφs were cultured in high glucose conditions. These results strongly suggested that decreased PSF production might result in decreased production of PGI2 in atherosclerotic lesions, thus leading to development of diabetic macroangiopathy and atherosclerosis.