Yoshie Sawada

Upregulatory Expression of Furin and Transforming Growth Factor-β by Fluid Shear Stress in Vascular Endothelial Cells

Abstract—Furin, a yeast Kex2-family endoprotease, converts many vasoregulatory propeptides, including pro-transforming growth factor (TGF)-&bgr; to their mature forms. We examined whether furin expression is regulated by shear stress in vivo and in vitro. When an arteriovenous shunt was placed between the carotid artery and external jugular vein in rabbits, furin and TGF-&bgr; were highly expressed in shear stress-loaded endothelial cells. Exposure of bovine aortic endothelial cells in culture to shear stress induced furin and TGF-&bgr; expression in a similar manner. Molecular analysis of furin expression in bovine aortic endothelial cells revealed that shear stress increases the furin gene expression at transcriptional levels. Furthermore, TGF-&bgr; itself increased the furin mRNA levels. Shear-mediated furin expression was partly mediated by TGF-&bgr; because shear-induced furin mRNA levels were considerably decreased by overexpression of the truncated form of the TGF-&bgr; type II receptor. Likewise, blockade of furin activity by a furin inhibitor significantly decreased the endothelial production of mature TGF-&bgr;. Taken together, the results indicate that furin expression is induced and maintained by a coordination of shear stress and TGF-&bgr;. Increased furin expression may facilitate the formation of mature TGF-&bgr;, resulting in the enhanced effects of TGF-&bgr; on endothelial cells and vascular smooth muscle cells in the vasculature.

Release of nitric oxide in response to acetylcholine is unaltered in spontaneously hypertensive rats

Objective Although a decreased responsiveness to acetylcholine, an endothelium-dependent vasodilator, has been reported in arteries isolated from spontaneously hypertensive rats (SHR), the precise role of nitric oxide (NO) in the in vivo regulation of blood pressure is not clear. We investigated the effects of acetylcholine and of NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, on mean arterial pressure and the production of NO metabolites (nitrate and nitrite) in SHR and in Wistar-Kyoto (WKY) rats, their normotensive control strain. Design We determined serum levels of nitrate and nitrite before and after the intravenous injection of 40 μg/kg acetylcholine following the administration of L-NAME (30mg/kg) or its vehicle in adult SHR and WKY rats. Results Acetylcholine administration significantly reduced mean arterial pressure in both SHR and WKY rats, accompanied by a significant rise in serum nitrate and nitrite. Administration of L-NAME significantly increased the mean arterial pressure in SHR and in WKY rats. L-NAME inhibited the hypotension induced by acetylcholine and the rise in serum nitrate and nitrite both in SHR and in WKY rats. Conclusion The release of NO stimulated by acetylcholine was unaltered in SHR, supporting previous in vitro results.

Co‐elevation of brain natriuretic peptide and proprotein‐processing endoprotease furin after myocardial infarction in rats

We investigated the expression of the yeast Kex2 family endoproteases furin and PACE4, and brain natriuretic peptide (BNP) in the atrium and ventricle after infarction as well as the conversion of the BNP precursor γBNP to BNP‐45. In a rat heart failure model, plasma BNP rose in two phases – first at day 3, and again at day 14. BNP mRNA, as measured by Northern blot analysis, increased strongly at day 3, then at days 14 and 28 less strongly in the atrium, and in the ventricle it increased weakly at day 3, then strongly at days 14 and 28. Furin mRNA showed the same pattern of expression as that of BNP message, whereas PACE4 message stayed unchanged after the infarction. Both furin and BNP were immunostained in the myocardium adjacent to the infarcted tissue. We suggest that after myocardial infarction, furin is co‐expressed with BNP in both the atrium and ventricle, and that furin may be responsible for the conversion of γBNP to BNP‐45.

Proprotein-Processing Endoprotease Furin Controls Growth of Pancreatic β-Cells

We have previously reported that in the well-differentiated β-cell line MIN6 cells, theβ-cell–specific differentiated characteristics, such as insulin content, expression of prohormone convertases PC2 and PC3, and glucose-regulated insulin secretion, diminished when the proprotein-processing endoprotease furin was highly expressed. Since furin converts many growth-related protein precursors to their bioactive forms, we compared the four pancreatic islet cell lines RINm5F, βTC3, βHC9, and MIN6 with respect to cell growth rate, furin expression, endoprotease activity, and insulin content. RINm5F cells exhibited the strongest furin expression, higher furin-type endoprotease activity, and the fastest cell growth, but had the least insulin content. In contrast, MIN6 cells exhibited only a weak furin expression, little furin-type endoprotease activity, and the slowest cell growth, but had the highest insulin content. To test whether furin-expressing cells secrete growth-promoting factors cleaved by furin, we prepared conditioned media from RINmSF and furin cDNA–introduced MIN6 (MIN6-F) cells. The conditioned media from RINm5F and MIN6-F induced increased DNA synthesis and promoted the growth of normal MIN6 cells, compared with the medium from the empty vector-introduced MIN6-0 cells. We then examined the effect of the protease inhibitors α1-antitrypsin and its variants by infecting their vaccinia recombinants to the four cell lines. All conditioned media from each cell line expressing the furin-specific α1-antitrypsin variant exhibited the least DNA synthetic capacity on normal MIN6 cells. Furthermore, all three sublines of MIN6-F grew faster than MIN6-0 and MIN6. Thus, we suggest that the islet cells with higher furin expression may induce increased production of growth factors, which result in an increase in cell growth, through an autocrine/paracrine mechanism.

PTHrP increases pancreatic β-cell-specific functions in well-differentiated cells

Parathyroid hormone-related protein (PTHrP) is reportedly produced in normal islets and insulinomas. PTHrP induces differentiation in some cell-types and growth in others. We examined whether PTHrP production is greater in well-differentiated or growing beta cells and whether PTHrP induces differentiation or growth in beta cells. We used four groups of the well-differentiated mouse beta cell line MIN6 with 17, 25, 31 and 41 passages, and mouse pancreatic islets. With passage, insulin content diminished, whereas the expression of PTHrP, its activating enzyme furin and cell growth gradually increased. PTHrP increased insulin content and mRNA levels more in MIN6-17 cells than in MIN6-41 cells. In contrast, PTHrP increased DNA synthesis more extensively in MIN6-41 cells than in MIN6-17 cells. Dibutyryl cAMP reproduced PTHrPs effect on insulin content and DNA synthesis. We conclude that PTHrP increases insulin expression in well-differentiated beta cells through the cAMP pathway and stimulates growth in growing beta cells.

Visceral fat obesity contributes to the tortuosity of the thoracic aorta on chest radiograph in poststroke Japanese patients.

Tortuosity of the thoracic aorta on chest radiographs is characteristic of atherosclerotic disease. Aging and hypertension are associated with the tortuosity, but little is known about the influence of other atherosclerotic risk factors on this abnormality. The purpose of this study was to examine which atherosclerotic risk factors are determinants for tortuosity of the thoracic aorta. Forty-five poststroke Japanese patients (31 men and 14 women, age range 41-78 years and mean 60.5 ±8.6) were studied. The distance factor, ie, the ratio of meandering vessel length to the straight-line distance between its end points, was used to measure arterial tortuosity. The hospital records were reviewed for clinical and biochemical variables. Tortuosity of the thoracic aorta had a significant positive relationship with body mass index (BMI) (r =0.397, p<0.01), waist circumference (r =0.360, p<0.05), and the cardiothoracic ratio (CTR) (r =0.526, p<0.001), and a significant negative relationship with ankle-brachial pressure index (ABPI) (r =-0.360, p<0.05). Stepwise regression analysis showed that waist circumference and CTR were independently correlated with increased tortuosity, whereas ABPI was negatively correlated with it. These results suggest that visceral fat obesity is a novel contributor to tortuosity of the thoracic aorta, which may be as a shortening of the distance between aortic tethering points due to elevation of the diaphragm by excessive intraabdominal fat and as a consequence of aortic elongation due to arteriosclerosis caused by obesity-related metabolic disorders.

Effects of Conjugated Equine Estrogenand Medroxyprogesterone Acetate on Lipoprotein(a) and Other Lipoproteins in Japanese Postmenopausal Women with and without Dyslipidemia

Background/Aim: The cardiovascular effects of postmenopausal hormone replacement are controversially discussed. We investigated the effects of 12 months of treatment with conjugated equine estrogen and medroxyprogesterone acetate on lipoprotein(a) [Lp(a)] and other lipoproteins in Japanese postmenopausal women (PMW) with and without dyslipidemia. Methods: Forty-three normolipidemic and 17 dyslipidemic PMW [total cholesterol (TC) ≧220 mg/dl or triglyceride (TG) ≧150 mg/dl] received conjugated equine estrogen (0.625 mg) plus medroxyprogesterone acetate (2.5 mg) daily for 12 months, and the results were compared with those of 26 normolipidemic and 14 dyslipidemic subjects declining this treatment as controls. The fasting serum levels of Lp(a), TC, TG, high-density lipoprotein cholesterol, low- density lipoprotein cholesterol, apolipoprotein (Apo) AI, Apo AII, Apo B, Apo CII, and Apo E were measured in each subject at baseline and 12 months after this treatment initiation. Results: The treatment decreased Lp(a) similarly in normolipidemic and dyslipidemic PMW and decreased TC, low-density lipoprotein cholesterol, Apo CII, and Apo E and increased high-density lipoprotein cholesterol, Apo AI, and Apo AII in both groups. The therapy also significantly increased TG in normolipidemic but not dyslipidemic subjects. In controls, the levels of Lp(a) and other lipoproteins were unaltered. Conclusions: In PMW with or without dyslipidemia, improvement in Lp(a) and other lipoproteins may represent cardiovascular benefits of hormone replacement therapy. However, an elevation of the TG levels seen with the therapy warrants caution, especially in PMW without dyslipidemia.

Smooth Muscle Cell Outgrowth from Coronary Atherectomy Specimens in vitro Is Associated with Less Time to Restenosis and Expression of a Key Transcription Factor KLF5/BTEB2

Atherectomy specimens offer an opportunity to study the biology of coronary artery lesions. We cultured smooth muscle cells (SMCs) from specimens obtained from 24 patients with coronary restenosis after angioplasty to study the relationship between activity of SMCs (in vitro outgrowth) and the time course of restenosis. We also examined expression of a Kruppel-like zinc-finger transcription factor 5 (KLF; also known as BTEB2 and IKLF), which is markedly induced in activated SMCs, in the same specimens. SMC outgrowth was observed in 9 of 24 specimens (37.5%). Restenosis occurred sooner (p < 0.01) in patients whose specimens showed outgrowth compared to those whose specimens showed no outgrowth. Immunostaining for KLF5 was more common in specimens with outgrowth (89 vs. 20%, p < 0.01). These data suggest that the number of activated SMCs in lesions may determine in vitro outgrowth and also affect the time to restenosis.

Proprotein-processing endoprotease furin and its substrate parathyroid hormone-related protein are coexpressed in insulinoma cells

Parathyroid hormone-related protein (PTHrP) is frequently produced in pancreatic endocrine tumors. PTHrP is synthesized as the precursor pro-PTHrP and undergoes a series of posttranslational processing reactions, among which cleavage of a 12 amino acid sequence from its precursor is crucial for the biological activation of PTHrP. This cleavage is catalyzed by furin, a proprotein-processing endoprotease that cleaves the consensus sequence-Arg-X-(Lys/Arg)-Arg ↓ X-. We previously reported that furin is highly expressed in rat pancreatic islets during the perinatal stage and that the expression of furin in pancreatic β cells induces faster cell growth. From this, we postulated that furin may be co-expressed with PTHrP in insulinomas. We immunostained insulin, PTHrP, and furin in 21 human pancreatic endocrine tumors: 10 insulinomas, 5 VIPomas, 4 gastrinomas, and 2 somatostatinomas. Of these 21 endocrine tumors, furin was positively stained in all 10 insulinomas. Likewise, PTHrP was detected in the same insulinomas. We found one VIPoma and one gastrinoma contained a few insulin-positive cells scatteringly, which were also positive for furin and PTHrP. But other non-insulinoma endocrine tumors did not display furin and PTHrP positivity. We conclude that furin and its substrate pro-PTHrP are co-expressed specifically in insulinomas.

Effects of Red Blood Cells on Ultrasonic Wave Microbubble Trapping

One of the problems in microbubble trapping by ultrasonic waves when applied to in vivo experiments is the existence of red blood cells (RBCs). It is expected that the amount of microbubbles which are trapped by acoustic radiation forces decrease because the RBCs in the liquid act as obstacles when the bubbles produce the bubble clouds. In this study, the effects of RBCs on microbubble trapping are examined experimentally. The results for different RBCs concentrations are shown. The dependency of the ultrasonic wave frequency on ultrasonic wave microbubble trapping is also presented.