Masataka Kudo

Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells1

Angiotensin (A) II plays a critical role in vascular remodeling, and its action is mediated by type 1 AII receptor (AT1R). Recently, 15-deoxy-Δ12,14-prostaglandin J2 and thiazolidinediones have been shown to be ligands for peroxisome proliferator-activated receptor (PPAR)-γ and activate PPAR-γ. In the present work, we have studied the effect of PPAR-γ on AT1R expression in rat vascular smooth muscle cells (VSMCs). We observed that: 1) endogenous AT1R expression was significantly decreased by PPAR-γ ligands both at messenger RNA and protein levels, whereas AT1R messenger RNA stability was not affected; 2) AII-induced increase of 3H-thymidine incorporation into VSMCs was inhibited by PPAR-γ ligands; 3) rat AT1R gene promoter activity was significantly suppressed by PPAR-γ ligands, and PPAR-γ overexpression further suppressed the promoter activity; 4) transcriptional analyses using AT1R gene promoter mutants revealed that a GC-box-related sequence within the −58/−34 region of the AT1R gene promoter was respo...

Transcription suppression of thromboxane receptor gene by peroxisome proliferator-activated receptor-gamma via an interaction with Sp1 in vascular smooth muscle cells

Thromboxane (TX) A2exerts contraction and proliferation of vascular smooth muscle cells (VSMCs) via its specific membrane TX receptor (TXR), possibly leading to the progression of atherosclerosis. A nuclear hormone receptor, peroxisome proliferator-activated receptor (PPAR)-γ, has recently been reported to be expressed in VSMCs. Here we examined a role of PPAR-γ in TXR gene expression in VSMCs. PPAR-γ ligands 15-deoxy-Δ12,14-prostaglandin J2 and troglitazone reduced TXR mRNA expression levels as well as cell growth as assessed by [3H]thymidine incorporation. Transcriptional activity of the TXR gene promoter was suppressed with PPAR-γ ligands, and the suppression was augmented further by PPAR-γ overexpression. By deletion and mutation analyses, the transcription suppression was shown to be the result of a −22/−7 GC box-related sequence (upstream of transcription start site). Electrophoretic mobility shift assays also showed that the sequence was bound by Sp1 but not by PPAR-γ, and the formation of a Sp1·DNA complex was inhibited either by coincubation with PPAR-γ or PPAR-γ ligand treatment of VSMCs. Moreover, glutathione S-transferase pull-down assays demonstrated a direct interaction between PPAR-γ and Sp1. In conclusion, PPAR-γ suppresses TXR gene transcription via an interaction with Sp1. PPAR-γ may possibly have an antiatherosclerotic action by inhibiting TXR gene expression in VSMCs.

Upregulation of Nitric Oxide Production in Vascular Endothelial Cells by All-trans Retinoic Acid Through the Phosphoinositide 3-Kinase/Akt Pathway

Background—A natural retinoid all-trans retinoic acid (ATRA) contains various beneficial effects on vasculature, including suppression of neointima formation after balloon injury. However, little is known about the effects of ATRA on vascular endothelial function. We therefore studied its role in nitric oxide (NO) production of vascular endothelial cells (ECs). Methods and Results—Human dermal microvascular ECs, human umbilical vein ECs, and SV40-transformed rat lung vascular ECs were incubated with or without ATRA (1 &mgr;mol/L) for 48 hours. Their NO production was determined with the use of a fluorescent NO indicator, diaminofluorescein-2 diacetate. ATRA significantly increased their basal as well as acetylcholine-induced NO production. Treatment with N&ohgr;-nitro-l-arginine methyl ester or carboxy-PTIO suppressed their fluorescence. Increase of NO production was also observed by incubation with retinoic acid receptor (RAR) agonist Am580. ATRA-induced NO increase was abolished by coincubation with RAR antagonist LE540. Moreover, the NO increase was completely inhibited by the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and LY294002. ATRA as well as Am580 enhanced endothelial NO synthase (eNOS) phosphorylation at Ser-1177 as well as Akt phosphorylation at Ser-473 without changing their protein expression. Overexpression of dominant-negative Akt inhibited the eNOS phosphorylation. Moreover, ATRA increased PI3K activity as well as PI3K catalytic subunit p110β protein expression, which was completely inhibited by LE540 treatment. Real-time polymerase chain reaction analyses demonstrated that ATRA increased PI3K catalytic subunit p110β mRNA expression without affecting its stability. Finally, ATRA-induced NO increase was observed in COS-1 cells transfected with wild-type eNOS and RARα, but not with mutated eNOS whose Ser-1177 was substituted. Conclusions—ATRA increases NO production by eNOS phosphorylation through RAR-mediated PI3K/Akt pathway activation in vascular ECs and possibly plays beneficial roles in vascular endothelium. Retinoids may therefore be candidates as novel therapeutic agents against vascular disorders with endothelial damage.

Measurement of Peripheral Plasma 18-Oxocortisol Can Discriminate Unilateral Adenoma From Bilateral Diseases in Patients With Primary AldosteronismNovelty and Significance

Adrenal venous sampling is currently the only reliable method to distinguish unilateral from bilateral diseases in primary aldosteronism. In this study, we attempted to determine whether peripheral plasma levels of 18-oxocortisol (18oxoF) and 18-hydroxycortisol could contribute to the clinical differentiation between aldosteronoma and bilateral hyperaldosteronism in 234 patients with primary aldosteronism, including computed tomography (CT)–detectable aldosteronoma (n=113) and bilateral hyperaldosteronism (n=121), all of whom underwent CT and adrenal venous sampling. All aldosteronomas were surgically resected and the accuracy of diagnosis was clinically and histopathologically confirmed. 18oxoF and 18-hydroxycortisol were measured using liquid chromatography tandem mass spectrometry. Receiver operating characteristic analysis of 18oxoF discrimination of adenoma from hyperplasia demonstrated sensitivity/specificity of 0.83/0.99 at a cut-off value of 4.7 ng/dL, compared with that based on 18-hydroxycortisol (sensitivity/specificity: 0.62/0.96). 18oxoF levels above 6.1 ng/dL or of aldosterone >32.7 ng/dL were found in 95 of 113 patients with aldosteronoma (84%) but in none of 121 bilateral hyperaldosteronism, 30 of whom harbored CT-detectable unilateral nonfunctioning nodules in their adrenals. In addition, 18oxoF levels below 1.2 ng/dL, the lowest in aldosteronoma, were found 52 of the 121 (43%) patients with bilateral hyperaldosteronism. Further analysis of 27 patients with CT-undetectable micro aldosteronomas revealed that 8 of these 27 patients had CT-detectable contralateral adrenal nodules, the highest values of 18oxoF and aldosterone were 4.8 and 24.5 ng/dL, respectively, both below their cut-off levels indicated above. The peripheral plasma 18oxoF concentrations served not only to differentiate aldosteronoma but also could serve to avoid unnecessary surgery for nonfunctioning adrenocortical nodules concurrent with hyperplasia or microadenoma.

Risk factors for persistent low bladder compliance after radical hysterectomy.

Introduction: Bladder compliance deteriorates immediately after radical hysterectomy (RH), and low bladder compliance causes upper urinary tract dysfunctions such as progressive hydronephrosis. The aims of this study were to clarify risk factors for persistent low bladder compliance after RH and to propose a postsurgical management protocol for improved recovery of bladder function. Methods: A total of 113 consecutive patients who underwent RH with the intention to preserve the pelvic autonomic nerve system were included in this prospective study. Urodynamic studies were performed according to a planned schedule: presurgery and 1, 3, 6, and 12 months after surgery. Autonomic nerves were preserved at least unilaterally in 95 (84.1%) of the 113 patients, but this was not possible in the remaining 18 patients (15.9%). Postoperative adjuvant radiation therapy (RT) was performed in 14 patients. The relationships between bladder compliance and various clinical factors were investigated using logistic regression analysis. Covariates included age, nerve-sparing procedure, adjuvant RT, and maximum abdominal pressure during the voiding phase. Bladder compliance at 12 months after surgery was used as the dependent variable. Results: Radical hysterectomy with a non-nerve-sparing procedure (odds ratio [OR], 3.4; 95% confidence interval [CI], 1.1-11.0), adjuvant RT (OR, 10.3; 95% CI, 2.5-43.5), and voiding with abdominal pressure at 3 months after surgery (OR, 2.9; 95% CI, 1.1-7.2) were risk factors for persistent low bladder compliance. Conclusions: A nerve-sparing procedure and prohibition of voiding with abdominal strain during the acute and subacute phases after RH resulted in improved recovery of bladder compliance. Adjuvant RT should be avoided in patients who undergo nerve-sparing RH if an alternative postoperative strategy is possible.

Stimulatory Effects of Low-Dose 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitor Fluvastatin on Hepatocyte Growth Factor-Induced Angiogenesis: Involvement of p38 Mitogen-Activated Protein Kinase

Therapeutic angiogenesis has received much attention for its potential benefits in ischemic vascular disorders. Recently, the clinical application of hepatocyte growth factor (HGF) for therapeutic angiogenesis has become well known. Statins have also been reported to promote angiogenesis and ameliorate ischemic conditions. In the present study, we examined the effects of fluvastatin on HGF-induced angiogenesis using a human umbilical vein endothelial cell (HUVEC)/normal human dermal fibroblast (NHDF) co-culture system. The HGF-induced angiogenesis was augmented by fluvastatin at low dose, but it was decreased at high dose. Although fluvastatin increased vascular endothelial growth factor expression in NHDFs, it was observed only at a high dose. Low-dose fluvastatin decreased the HGF-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation (Thr-180/Tyr-182) and HUVEC apoptosis in the presence of HGF. SB203580, a p38 MAPK inhibitor, ameliorated anisomycin (a p38 MAPK activator)-induced angiogenesis suppression in the presence of HGF. Moreover, the augmentation of the HGF-induced angiogenesis by fluvastatin was abrogated by the p38 MAPK inhibitors, SB203580, SB202190, and FR167653. High-dose fluvastatin decreased Akt phosphorylation (Ser-473) and HUVEC proliferation, and it increased p27kip1 in HUVECs. Interestingly, fluvastatin decreased the mRNA expression of integrins and tissue inhibitor of metalloproteinases (TIMPs) in HUVECs. Our data therefore indicate that the stimulatory effects of low-dose fluvastatin on the HGF-induced angiogenesis are mediated by its inhibitory effects on p38 MAPK phosphorylation induced by HGF, which may result in the suppression of EC apoptosis. High-dose fluvastatin inhibits Akt phosphorylation and HUVEC proliferation, and it increases p27kip1, which may result in its inhibitory effects on angiogenesis. In addition, integrins and TIMPs are candidates for angiogenesis regulation by fluvastatin.

PPARγ Agonist Beyond Glucose Lowering Effect

The nuclear hormone receptor PPARγ is activated by several agonists, including members of the thiazolidinedione group of insulin sensitizers. Pleiotropic beneficial effects of these agonists, independent of their blood glucose-lowering effects, have recently been demonstrated in the vasculature. PPARγ agonists have been shown to lower blood pressure in animals and humans, perhaps by suppressing the renin-angiotensin (Ang)-aldosterone system (RAAS), including the inhibition of Ang II type 1 receptor expression, Ang-II-mediated signaling pathways, and Ang-II-induced adrenal aldosterone synthesis/secretion. PPARγ agonists also inhibit the progression of atherosclerosis in animals and humans, possibly through a pathway involving the suppression of RAAS and the thromboxane A2 system, as well as the protection of endothelial function. Moreover, PPARγ-agonist-mediated renal protection, especially the reduction of albuminuria, has been observed in diabetic nephropathy, including animal models of the disease, and in non-diabetic renal dysfunction. The renal protective activities may reflect, at least in part, the ability of PPARγ agonists to lower blood pressure, protect endothelial function, and cause vasodilation of the glomerular efferent arterioles. Additionally, anti-neoplastic effects of PPARγ agonists have recently been described. Based on the multiple therapeutic actions of PPARγ agonists, they will no doubt lead to novel approaches in the treatment of lifestyle-related and other diseases.

Difficult-to-control hypertension due to bilateral aldosterone-producing adrenocortical microadenomas associated with a cortisol-producing adrenal macroadenoma

The patient was a 54-year-old woman who developed a right adrenal tumour, Cushingoid features, elevated levels of cortisol that were not suppressed by 1 nor 8 mg of dexamethasone, and suppression of adrenocorticotropin (ACTH) during treatment for severe hypertension. Computed tomography (CT) revealed a right adrenal tumour and an atrophic left adrenal gland. In addition, elevated plasma aldosterone concentration (PAC) and suppressed plasma renin activity (PRA) with an aldosterone-to-renin ratio of 128 (ng per 100 ml per ng ml–1 h−1) suggested aldosterone excess. Urinary excretion of aldosterone was relatively high, and the captopril and rapid ACTH tests resulted in no response of PRA and exaggerated increase in PAC, respectively. ACTH-loaded adrenal venous sampling showed bilateral excess of aldosterone with right predominance of cortisol. Right laparoscopic partial adrenalectomy (ADX) and immunohistochemical analysis showed both a cortisol-producing adenoma and an aldosterone-producing microadenoma (microAPA) within the attached adrenal, which had not been detected by CT preoperatively. After the right partial ADX, her blood pressure, aldosterone level and suppressed PRA remained unchanged. Subsequently, laparoscopic total left ADX was performed. Two microAPAs with paradoxical hyperplasia were revealed within the apparently atrophic left adrenal gland. Soon after the second surgery, her blood pressure normalized without requiring any anti-hypertensive medication.

8D.04: CLINICAL BENEFITS OF ADMINISTERING SUPER-SELECTIVE SEGMENTAL ADRENAL VENOUS SAMPLING AND PERFORMING ADRENAL SPARING SURGERY IN THE PATIENTS WITH PRIMARY ALDOSTERONISM.

Objective: Adrenal venous sampling (AVS) has been well known to play pivotal roles in clinical differential diagnosis of unilateral aldosterone producing adenoma (APA) from bilateral idiopathic hyperaldosteronism (IHA). However, it is also true that a central vein AVS or c-AVS which collects the blood from right and left central adrenal veins can by no means discriminate bilateral APA from BHA. There have been no published studies reporting the reliable clinical differential diagnosis between bilateral APA and IHA, especially IHA cases with bilateral non-functioning adenomas (NFA), which has been considered practically impossible in clinical differential diagnosis. As an attempt to this clinical dilemma, segmental AVS (S-AVS), which could evaluate segmental effluents from adrenal tributary veins, has been recently developed. Design and method: We have performed S-AVS in these patients above following C-AVS, via the insertion of a microcatheter in up to three intra-adrenal first-degree tributary veins on bilateral adrenals. Results: S-AVS did enable us to evaluate the intra-adrenal localization of corticosteroidogenesis. These data did indicate that S-AVS should be performed in the PA patients who had increased aldosterone levels in bilateral central vein and demonstrated space occupying lesions in the bilateral adrenals in order to avoid bilateral adrenalectomy or long lasting medical treatment toward persistent PA. In addition to the situations above, we have administere S-AVS to the following patients; those who had clinically suspected APA but not sufficiently high lateralization indexes according to the results of C-AVS, very young ones with higher clinical probability of recurrence and those who could benefit from partial adrenalectomy by demonstrating the sites of specific steroidogenesis. However, it is also entirely true that S-AVS is more expensive, time-consuming and labor-intensive compared to C-AVS. Figure. No caption available. The angiography during S-AVS (A, B), the coronal CT image (C), and the data in external iliac vein (EIV), each central vein (1, 4) and each tributary vein (2, 3, 5, 6) of 66 year-old male patient with bilateral APAs. Conclusions: We should carefully select the candidate patients who should undergo S-AVS, which will give a benefit to themselves by demonstrating intra-adrenal steroidogenesis for a safer preserving adrenalectomy.

Rapid Screening of Primary Aldosteronism by a Novel Chemiluminescent Immunoassay

Measurement of plasma aldosterone and renin concentration, or activity, is useful for selecting antihypertensive agents and detecting hyperaldosteronism in hypertensive patients. However, it takes several days to get results when measured by radioimmunoassay and development of more rapid assays has been long expected. We have developed chemiluminescent enzyme immunoassays enabling the simultaneous measurement of both aldosterone and renin concentrations in 10 minutes by a fully automated assay using antibody-immobilized magnetic particles with quick aggregation and dispersion. We performed clinical validation of diagnostic ability of this newly developed assay-based screening of 125 patients with primary aldosteronism from 97 patients with essential hypertension. Results of this novel assay significantly correlated with the results of radioimmunoassay (aldosterone, active renin concentration, and renin activity) and liquid chromatography–tandem mass spectrometry (aldosterone). The analytic sensitivity of this particularly novel active renin assay was 0.1 pg/mL, which was better than that of radioimmunoassay (2.0 pg/mL). The ratio of aldosterone-to-renin concentrations of 6.0 (ng/dL per pg/mL) provided 92.0% sensitivity and 76.3% specificity as a cutoff for differentiating primary aldosteronism from essential hypertension. This novel measurement is expected to be a clinically reliable alternative for conventional radioimmunoassay and to provide better throughput and cost effectiveness in diagnosis of hyperaldosteronism from larger numbers of hypertensive patients in clinical settings.