Masayoshi Shichiri

Insulin-like growth factor-I induces hypertrophy with enhanced expression of muscle specific genes in cultured rat cardiomyocytes.

BackgroundCardiac hypertrophy is commonly observed in acromegalic patients, in whom serum insulinlike growth factor-I (IGF-I) levels are elevated. In the present study, we examined whether IGF-I induces hypertrophy in cultured neonatal rat cardiomyocytes through its specific receptor and whether IGF binding protein-3 (IGFBP-3), which is a major circulating carrier protein for IGF-I, inhibits IGF-I-induced cardiac hypertrophy in vitro. Methods and ResultsBecause the response of cardiac hypertrophy is characterized by the induction of expression for muscle-specific genes, the effect of IGF-I on steady-state levels of mRNA for myosin light chain-2 (MLC-2) and troponin I and for skeletal and cardiac a-actin isoforms was evaluated by Northern blot analysis. IGF-I (10-7 M) increased mRNA levels for MLC-2 and troponin I as early as 60 minutes with maximum levels by 6 hours, which were maintained for as long as 24 hours. IGF-I (10-7 M) also increased transcripts for skeletal a-actin but not for cardiac a-actin. The cell size as evaluated morphometrically was almost doubled after 48-hour treatment with IGF-I. IGF-I induction of protein synthesis was dose dependent (10-10 to 10-7 M) with a maximal 2.2-fold increase seen at 10-8 M. In contrast to the hypertrophic effect of IGF-I, growth hormone affected neither protein synthesis nor expression for muscle-specific genes. Binding study using125IIGF-I revealed the presence of specific binding sites for IGF-I in rat cardiomyocytes. IGFBP-3 induced a dose-dependent inhibition of protein synthesis stimulated by IGF-I; IGFBP-3 (10-7 M) completely inhibited the [3HIleucine uptake stimulated by IGF-I 10-8 M). IGFBP-3 similarly inhibited the IGF-I-stimulated gene expressions for MLC-2 and troponin I. ConclusionsThese results suggest that IGF-I directly causes cardiac hypertrophy and that its effect can be blocked by IGFBP-3.

Plasma endothelin levels in hypertension and chronic renal failure.

Endothelin-1 is a novel endothelium-derived vasoconstrictive peptide. Using a highly specific and sensitive radioimmunoassay for endothelin-1, plasma levels of immunoreactive endothelin-1 were measured in 32 research subjects with normal renal function (21 normal subjects and 11 patients with essential hypertension), 24 patients with nondialyzed chronic renal failure, and 51 patients undergoing maintenance hemodialysis. Although there was no significant difference in plasma immunoreactive endothelin-1 levels among the three groups, patients with essential hypertension had significantly higher plasma endothelin-1 levels than normal subjects (2.29 +/- 1.09 vs. 1.41 +/- 0.50 pg/ml, p less than 0.025). When nondialyzed and hemodialyzed patients were divided into hypertensive and normotensive groups, the nondialyzed hypertensive group (n = 17) had higher plasma endothelin-1 levels than the comparable normotensive group (n = 7) (3.08 +/- 3.43 vs. 0.73 +/- 0.34 pg/ml, p less than 0.05), and the hemodialyzed hypertensive group (n = 18) had higher plasma endothelin-1 levels than the comparable normotensive group (n = 33) (2.66 +/- 1.92 vs. 1.35 +/- 0.73 pg/ml, p less than 0.005). Plasma atrial natriuretic factor, arginine vasopressin, renin activity, and aldosterone concentration did not show significant differences between hypertensive and normotensive individuals or a correlation with plasma endothelin-1 levels. These data suggest that circulating endothelin-1 may be partly involved in the development or maintenance of hypertension in humans.

Secretory mechanism of immunoreactive endothelin in cultured bovine endothelial cells

To elucidate the cellular mechanism by which endothelin (ET) is secreted, we have studied the effects of a variety of vasoactive agents on the secretion of immunoreactive (IR)-ET from cultured bovine endothelial cells (EC). Confluent bovine EC cultured in serum-free medium secreted IR-ET as a function of time. Not only thrombin, but also vasoconstrictive hormones, such as arginine-vasopressin (AVP) and angiotensin (ANG) II, dose-dependently stimulated IR-ET secretion, and these effects were completely abolished by V1-receptor antagonist and [Sar1,Ala8]-ANG II, respectively. Protein kinase C (PKC)-activating phorbol ester and Ca2+ ionophore ionomycin had stimulatory effects on IR-ET secretion, and the combination of both compounds had a synergistic effect. These data suggest that AVP and ANG II, like thrombin, stimulate ET secretion from EC by a mechanism possibly involving receptor-mediated mobilization of intracellular Ca2+ and activation of PKC.

Presence of immunoreactive endothelin in human plasma

A highly specific and sensitive radioimmunoassay has been established for measurement of human endothelin (hET) in human plasma. After extraction of plasma with an octyl‐silica column, this assay allowed for detection of immunoreactive (IR) hET as low as 0.2 fmol/ml. In 16 healthy subjects, the mean concentration of plasma IR‐hET was 0.6 fmol/ml. Reverse‐phase HPLC coupled with radioimmunoassay revealed two major IR‐hET components, one corresponding to authentic hET(1–21) and another with more hydrophilicity than hET(1–21). These data indicate that ET is a circulating vasoconstrictor hormone in man.

Co-expression of urotensin II and its receptor (GPR14) in human cardiovascular and renal tissues.

Background Urotensin-II (UII), a cyclic dodecapeptide originally isolated from fish urophysis that has potent cardiovascular effects, has recently been identified as an endogenous ligand for the orphan G protein-coupled receptor, GPR14. The physiological roles of endogenous UII and its receptor in humans remain unknown. Objective To investigate the presence of human (h) UII-like immunoreactivity (hUII-LI) in human biological fluids, and the expression of hUII and GPR14 genes in human tissues. Methods We have established a specific radioimmunoassay for hUII and the real-time quantitative reverse transcriptase polymerase chain reaction method using LightCycler for the quantification of hUII and GPR14 mRNAs. Results Gel filtration and reverse-phase high performance liquid chromatography of human urine extracts revealed a single major peak of hUII-LI co-eluting with known hUII. The concentrations of hUII-LI in urine from normal individuals were 7.4 ± 0.9 μg/g creatinine, whereas its plasma concentration was undetectable (< 50 pg/ml). Urinary hUII concentrations from patients with essential hypertension and those with renal tubular abnormality, but not with glomerular diseases, were significantly greater than those from normal individuals. The resulting fractional excretion of hUII, exceeding the glomerular filtration rate, suggests a renal origin of urinary UII-LI. hUII mRNAs were abundantly expressed in the kidney and the right atrium, but far less so in the vasculature, whereas GPR14 mRNAs were equally and abundantly expressed in both cardiovascular and renal tissues. Conclusions These data suggest that urinary hUII is derived mainly from a renal source, and that hUII functions as an autocrine/paracrine vasoactive factor not only in the cardiovascular system, but also in the kidney, with an as yet unspecified function.

Cellular mechanism of endothelin-1 release by angiotensin and vasopressin.

Release of endothelin-1, a novel potent vasoconstrictor peptide originally isolated from endothelial cells, from cultured bovine endothelial cells has been shown to be stimulated by arginine vasopressin and angiotensin II. To elucidate the cellular mechanism by which endothelin-1 is released by these vasoconstrictors, we tested the effects of several compounds on the agonist-induced endothelin-1 release and studied the changes of cytosolic free Ca2+ concentrations and phosphoinositide breakdown by these agonists in cultured bovine endothelial cells. Protein kinase C inhibitors (H-7, staurosporine), an intracellular Ca2+ chelator, and an inhibitor of phospholipase C (neomycin), all abolished the agonist-induced endothelin-1 release, whereas the Ca2+ channel blocker nicardipine was ineffective. Although synthetic 1,2-diglyceride (diolein) dose dependency stimulated endothelin-1 release, downregulation of protein kinase C after pretreatment with phorbol ester resulted in decreased effects to increase endothelin-1 release by the agonists. Both arginine vasopressin and angiotensin II induced immediate and transient increases in intracellular Ca2+ levels of fura-2-loaded endothelial cells as well as formation of inositol trisphosphate; the agonist-induced intracellular Ca2+ increases were not affected either by nicardipine or by chelating extracellular Ca2+. The arginine vasopressin- and angiotensin U-induced intracellular Ca2+ increases, inositol trisphosphate formation, and endothelin-1 release were completely abolished by V,-receptor antagonist and saralasin, respectively. It is concluded that arginine vasopressin and angiotensin II stimulate the release of endothelin-1 by a common mechanism, involving receptormediated mobilization of intracellular Ca2+ and activation of protein kinase C in endothelial cells.

Endothelin-1 is an autocrine/paracrine growth factor for human cancer cell lines.

We studied whether a novel vasoconstrictor peptide, endothelin-1 (ET-1), is synthesized by and released from human carcinoma cell lines, and whether ET-1 stimulates proliferation of these tumor cells. ET-1-like immunoreactivity was released from both HeLa and HEp-2 cells as a function of time. Reverse-phase HPLC of the conditioned media from HeLa cells revealed a major peak coeluting with standard ET-1. Northern blot analysis demonstrated the expression of mRNA for ET-1 precursor in both tumor cell lines. Both cell lines contained a single class of specific binding sites for ET-1. ET-1 dose-dependently induced increases in cytosolic free Ca2+ concentration in fura-2-loaded tumor cells, whose effect was completely abolished by chelating extracellular Ca2+ or by Ca(2+)-channel blocker. ET-1 stimulated proliferation of the quiescent cell lines in a dose-dependent manner, whose effect was inhibited by Ca(2+)-channel blocker. Polyclonal antibody for ET-1 inhibited proliferation of these cell lines, whereas nonimmune serum had no effect. These results demonstrate that ET-1 is synthesized by and released from human epithelial carcinoma cell lines, and that exogenous and endogenous ET-1 stimulates proliferation of the cells possibly through Ca2+ influx, suggesting its role as an autocrine/paracrine growth factor for certain tumor cells.

Endothelin-1 as an Autocrine/Paracrine Apoptosis Survival Factor for Endothelial Cells

Endothelin-1 (ET-1), an endothelium-derived vasoactive peptide, functions as a potent vasoconstrictor as well as mitogen. We show here a novel role for ET-1 as an apoptosis survival factor for cultured rat endothelial cells. When we rendered endothelial cells obtained from rat aorta quiescent by serum starvation, significant portions of cultured cells underwent apoptotic death as demonstrated by nucleosomal laddering on agarose gel electrophoresis, flow cytometry analysis with FACS, and the TdT-mediated dUTP biotin nick-end labeling (TUNEL) method. ET-1 dose-dependently (10[-12] to 10[-6] mol/L) suppressed the apoptosis induced by serum starvation. The ET(B) receptor antagonist (BQ788; 10[-6] mol/L) and ET(A/B) receptor antagonists (PD142893 and PD145065; 10[-6] mol/L), but not the ET(A) receptor antagonist (BQ123; 10[-6] mol/L), blocked the apoptosis protective effect of 10[-7] mol/L ET-1. Nonimmune rabbit serum reduced the apoptotic event induced by serum deprivation, whereas neutralization of endogenous ET-1 by polyclonal anti-ET-1 antiserum abrogated this protective effect. The ET(B) receptor antagonist (BQ788; 10[-8] to 10[-6] mol/L), but not the ET(A) receptor antagonist (BQ123; 10[-8] to 10[-6] mol/L), significantly inhibited proliferation of endothelial cells. These data suggest that ET-1, as well as mitogen, functions as an apoptosis survival factor for endothelial cells in an autocrine/paracrine manner via the ET(B) receptor.

Th2-predominant inflammation and blockade of IFN-γ signaling induce aneurysms in allografted aortas

Abdominal aortic aneurysms (AAAs) cause death due to complications related to expansion and rupture. The underlying mechanisms that drive AAA development remain largely unknown. We recently described evidence for a shift toward T helper type 2 (Th2) cell responses in human AAAs compared with stenotic atheromas. To evaluate putative pathways in AAA formation, we induced Th1- or Th2-predominant cytokine environments in an inflammatory aortic lesion using murine aortic transplantation into WT hosts or those lacking the receptors for the hallmark Th1 cytokine IFN-gamma, respectively. Allografts in WT recipients developed intimal hyperplasia, whereas allografts in IFN-gamma receptor-deficient (GRKO) hosts developed severe AAA formation associated with markedly increased levels of MMP-9 and MMP-12. Allografts in GRKO recipients treated with anti-IL-4 antibody to block the characteristic IL-4 Th2 cytokine or allografts in GRKO hosts also congenitally deficient in IL-4 did not develop AAA and likewise exhibited attenuated collagenolytic and elastolytic activities. These observations demonstrate an important dichotomy between cellular immune responses that induce IFN-gamma- or IL-4-dominated cytokine environments. The findings establish important regulatory roles for a Th1/Th2 cytokine balance in modulating matrix remodeling and have important implications for the pathophysiology of AAAs and arteriosclerosis.

NO Inhibits Cytokine-Induced iNOS Expression and NF-κB Activation by Interfering With Phosphorylation and Degradation of IκB-α

Nitric oxide (NO) is known to have antiatherogenic and anti-inflammatory properties, but its effects on the cytokine-induced nuclear factor-kappa B (NF-kappaB) activation pathway in relation to the regulation of inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs) remain elusive. To elucidate the roles of NO in the regulation of cytokine-induced NF-kappaB activation and consequent iNOS gene expression, we studied the effects of NO donors [(+/-)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide (NOR3) and sodium nitroprusside] on interleukin (IL)-1beta-induced NF-kappaB activation and IkappaB-alpha degradation and subsequent iNOS expression in rat VSMCs. Northern blot and Western blot analyses demonstrated that NO donors decreased IL-1beta-induced iNOS mRNA and protein expression. Electrophoretic mobility shift assay using synthetic oligonucleotide corresponding to the downstream NF-kappaB site of rat iNOS promoter as a probe showed that NOR3 inhibited IL-1beta-induced NF-kappaB activation and its nuclear translocation, as demonstrated with immunocytochemical study. These effects were independent of guanylate cyclase activation; an inhibitor of soluble guanylate cyclase (1H-oxadiazolo-1,2,4-[4,3-alpha]quinoxaline-1-one) had no effect on NOR3-induced inhibition of NF-kappaB activation or iNOS mRNA expression by IL-1beta, and a cGMP derivative (8-bromo-cGMP) failed to mimic the effects of NO donors. Western blot analysis using anti-IkappaB-alpha and anti-phospho-IkappaB-alpha antibodies revealed that IL-1beta induced a transient degradation of IkappaB-alpha preceded by a rapid appearance of phosphorylated IkappaB-alpha, both of which were completely blocked by NOR3. A proteasome inhibitor (MG115) blocked IL-1beta-induced transient degradation of IkappaB-alpha and stabilized the appearance of phosphorylated IkappaB-alpha stimulated by IL-1beta. NOR3 inhibited the appearance of IL-1beta-induced phosphorylated IkappaB-alpha even in the presence of MG115. Our results indicate that an inhibitory action by NO on cytokine-induced NF-kappaB activation and iNOS gene expression is due to its direct blockade on phosphorylation and subsequent degradation of IkappaB-alpha via the cGMP-independent pathway in rat VSMCs.