Yukio Hirata

Salusins: newly identified bioactive peptides with hemodynamic and mitogenic activities

The discovery of endogenous bioactive peptides has typically required a lengthy identification process. Computer-assisted analysis of cDNA and genomic DNA sequence information can markedly shorten the process. A bioinformatic analysis of full-length, enriched human cDNA libraries searching for previously unidentified bioactive peptides resulted in the identification and characterization of two related peptides of 28 and 20 amino acids, which we designated salusin-α and salusin-β. Salusins are translated from an alternatively spliced mRNA of TOR2A, a gene encoding a protein of the torsion dystonia family. Intravenous administration of salusin-α or salusin-β to rats causes rapid, profound hypotension and bradycardia. Salusins increase intracellular Ca2+, upregulate a variety of genes and induce cell mitogenesis. Salusin-β stimulates the release of arginine-vasopressin from rat pituitary. Expression of TOR2A mRNA and its splicing into preprosalusin are ubiquitous, and immunoreactive salusin-α and salusin-β are detected in many human tissues, plasma and urine, suggesting that salusins are endocrine and/or paracrine factors.

Aldosterone-Stimulated SGK1 Activity Mediates Profibrotic Signaling in the Mesangium

Several recent reports support the hypothesis that aldosterone contributes to the progression of renal injury. Mineralocorticoids increase the expression of serum- and glucocorticoid-inducible protein kinase 1 (SGK1), which is upregulated in several fibrotic diseases. It was hypothesized that SGK1 may mediate the effects of aldosterone on glomerular fibrosis and inflammation. In primary cultures of rat mesangial cells, aldosterone stimulated the expression, phosphorylation, and kinase activity of SGK1, as well as SGK1-dependent NF-kappaB activity. Furthermore, aldosterone augmented the promoter activity and protein expression of intercellular adhesion molecule-1 (ICAM-1), which modulates the inflammatory response, and the profibrotic cytokine connective tissue growth factor (CTGF) in an SGK1- and NF-kappaB-dependent manner. Similar to the in vitro results, uninephrectomized rats that were treated with aldosterone demonstrated increased glomerular expression of SGK1, ICAM-1, and CTGF proteins than untreated rats; these changes were accompanied by hypertension, glomerulosclerosis, and inflammation. In conclusion, these findings suggest that aldosterone stimulates ICAM-1 and CTGF transcription via the activation of SGK1 and NF-kappaB, effects that may contribute to the progression of aldosterone-induced mesangial fibrosis and inflammation.

Systemic Distribution of Salusin Expression in the Rat

Salusin-α and salusin-β are multifunctional bioactive peptides with hypotensive and bradycardic effects. They were originally identified from full-length human cDNAs by bioinformatics analyses. Salusin peptides are expressed in human tissues at the mRNA level, but no information is available about their systemic distributions in any species. We examined the distributions of preprosalusin mRNA and the salusin peptides in a variety of normal rat organs. Whereas preprosalusin mRNA was expressed ubiquitously, immunoreactive salusin-β was detected most strongly in the hypothalamus and posterior pituitary, and less abundantly in the anterior pituitary and gastrointestinal, immune, and hematopoietic systems. Salusin-β−positive cells appeared to be of either hematopoietic or endocrine origin, and many hematopoietic cells were also stained with anti-CD68, which specifically recognizes macrophages. Salusin-α−like immunoreactivity was not detected in any of the rat tissues. These results indicate that rat salusin is immunologically similar to human salusin-β and widely expressed, especially in the immune, gastrointestinal, and central nervous systems and mainly in endocrine- and hematopoietic-derived cells.

Presence of immunoreactive salusin-α in human serum and urine

Salusins, identified from a full-length enriched human cDNA library by bioinformatics analyses, show mitogenic, neuromodulatory and hemodynamic activities in rats. They are expressed in a wide variety of human tissues, but their precise structures and levels in human body fluids remain unknown. We developed a radioimmunoassay suitable for the detection of immunoreactive human salusin-alpha and characterized the molecular forms and concentrations of salusin-alpha in human serum and urine. The assay allowed for measurement of immunoreactive salusin-alpha concentrations as low as 1 fmol/tube after extraction of serum with an octyl-silica column, and the concentration required for 50% inhibition of binding was 40 fmol/tube. Cross-reactivities with salusin-beta and other bioactive peptides were negligible. Salusin-alpha-like immunoreactivity in normal human serum and urine ranged from 11.0 to 40.4 pmol/l (mean+/-S.D., 23.3+/-8.1 pmol/l, n=31) and from 18.6 to 367.3 pmol/l (mean+/-S.D., 156.8+/-95.8 pmol/l), respectively. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection revealed a major immunoreactive component that coeluted with authentic salusin-alpha. These data indicate the presence of salusin-alpha in human serum and urine, thereby verifying the initially predicted processing sites for salusin-alpha in humans.

Improvement of endothelial function in patients with hypertension and type 2 diabetes after treatment with telmisartan

Telmisartan, a selective antagonist for angiotensin type1 receptor and a partial agonist for peroxisome proliferator-activated receptor-γ, decreases blood pressure and has been shown to improve glucose and lipid metabolism, suggesting potential cardiovascular protective effects. In this study, we investigated whether long-term treatment with telmisartan improved endothelial function in 35 hypertensive patients with type 2 diabetes mellitus (T2DM). Office and home early morning blood pressure levels and flow-mediated vasodilation (FMD) were evaluated before and after 12 months of treatment with telmisartan. Blood samples were also obtained for measurement of several biochemical parameters and of adiponectin (AN) and highly sensitive C-reactive protein (hs-CRP) before and after treatment. After 12 months of treatment, office and morning blood pressure levels had significantly decreased, and levels of plasma glucose, glycosylated hemoglobin, total cholesterol, triglyceride and low-density lipoprotein cholesterol had also significantly decreased. Plasma AN and high-density lipoprotein cholesterol levels increased, but hs-CRP levels decreased. Furthermore, FMD significantly increased; changes in percent FMD showed a significant negative correlation with changes in systolic and diastolic blood pressure and a significant positive correlation with changes in AN. Stepwise multivariate regression analysis revealed that changes in plasma AN and office systolic blood pressure were both independent determinants for endothelial function after telmisartan treatment. In conclusion, this study shows that long-term treatment with telmisartan improves not only blood pressure and glucose and lipid metabolism but also endothelial function in hypertensive patients with T2DM, possibly by increased circulating AN and decreased blood pressure.

Transcriptome analysis of aldosterone-regulated genes in human vascular endothelial cell lines stably expressing mineralocorticoid receptor.

A series of studies have demonstrated that endothelial cell is one of the target tissues of aldosterone. Here, we have conducted a transcriptome analysis of aldosterone-inducible genes in human endothelial cell lines stably expressing human mineralocorticoid receptor (MR) by retroviral system (MR-EAhy). We found that aldosterone in physiologic concentrations robustly induced MR-dependent transcriptional response in MR-EAhy. By DNA microarray analysis, we validated 12 aldosterone-up-regulated genes among which at least seven were concomitantly associated with increased protein expression. We also found five aldosterone-down-regulated genes. Among 11 aldosterone-up-regulated genes tested, mRNA expressions of three (ESM1, SNF1LK, ANGPTL4) were significantly up-regulated in aortic tissue from aldosterone-induced hypertensive rats compared to those from control rats, suggesting their potential pathophysiologic significance in vivo. In conclusion, using MR stably expressed human endothelial cell lines, we identified a variety of aldosterone-inducible genes, suggesting their possible roles in the development and/or the protection for aldosterone-induced vascular injury.

Vasculoprotective effect of cilostazol in aldosterone-induced hypertensive rats

Cilostazol (CILO), a selective inhibitor of phosphodiesterase 3 with potent antithrombotic property, has been shown to have a vasculoprotective effect in atherosclerosis animal models due to its potential anti-inflammatory and antioxidant actions. This study was undertaken to investigate whether CILO has in fact any vasculoprotective effects in aldosterone-induced hypertensive rats (Aldo-rats), and whether CILO affects Aldo-induced oxidative stress, nitric oxide (NO) production and pro-inflammatory gene expression. Treatment with CILO markedly ameliorated perivascular inflammatory changes in the coronary arterioles of Aldo-rats without affecting the systolic blood pressure and left ventricular weight. Treatment with CILO also prevented the increase in plasma levels of thiobarbituric acid-reactive substances, an oxidative stress marker, as well as decreased urinary NOx excretion in Aldo-rats. Furthermore, CILO almost completely inhibited a set of upregulated proinflammatory genes (ICAM-1, MCP-1, PDGF-A, osteopontin, MMP-2 and ACE), as well as NAD(P)H oxidase components (p22phox, gp91phox, p47phox) and Aldo-inducible genes (SGK-1 and NHE-1) in the aortic tissues from Aldo-rats. Taken together, this study showed for the first time that CILO prevented Aldo-induced vascular inflammation and injury without affecting the blood pressure, suggesting its vasculoprotective effect on Aldo-induced vascular injury independent of blood pressure.