Takafumi Okura

Differential gene expression and regulation of type-1 angiotensin II receptor subtypes in the rat.

A simplified and sensitive method for measuring expression levels of type-1 angiotensin II (AT1) receptor subtypes, AT1A and AT1B, was established. The two receptor cDNAs were co-amplified and measured by polymerase chain reaction using primers based on the corresponding receptor subtype genes. Both AT1A and AT1B mRNAs were widely expressed in the rat tissues including adrenal gland, kidney, heart, aorta, lung, liver, testis, pituitary gland, cerebrum and cerebellum. AT1A mRNA was predominantly expressed in the rat tissues examined except adrenal gland and pituitary gland where AT1B mRNA was predominantly expressed. Sodium depletion did not change mRNA levels of AT1A and AT1B in the all tissues. However, both AT1A and AT1B mRNA levels in the heart and aorta were down-regulated by treatment with AT1 specific antagonist, TCV 116. In contrast, AT1B mRNA in the adrenal gland was mainly reduced by the treatment. These results suggest that the expression level of AT1B mRNA in the adrenal gland depends on the activity of the renin-angiotensin-aldosterone system (RAAS) and both receptor subtypes mediate contraction and hypertrophy of the smooth and cardiac muscles via the RAAS.

C-Reactive Protein Induces Apoptosis in Human Coronary Vascular Smooth Muscle Cells

Background—Accumulating evidence suggests that C-reactive protein (CRP), in addition to being a predictor of coronary events, may have direct actions on the vessel wall in the evolution of atherosclerosis. Although accumulation of vascular smooth muscle cells (VSMCs) in the intima is a key event in the development of arterial lesions, apoptosis of VSMCs also plays an important role in progression of atherosclerotic lesions and contributes to increased plaque vulnerability. Methods and Results—In the present study we demonstrate that CRP induces caspase-mediated apoptosis of human coronary VSMCs. DNA microarray analysis was used to identify CRP-regulated genes. The growth arrest– and DNA damage–inducible gene 153 (GADD153) mRNA expression was prominently upregulated by CRP. As confirmed by Northern blot analysis, CRP induced a time- and dose-dependent increase of GADD153 mRNA expression. GADD153, a gene involved in growth arrest and apoptosis in vascular and nonvascular cells, is regulated at both transcriptional and posttranscriptional levels. CRP regulation of GADD153 mRNA expression in VSMCs occurs primarily at the posttranscriptional level by mRNA stabilization. Small interfering RNA (siRNA) specifically targeted to GADD153 reduced CRP-induced apoptosis. GADD153 also specifically colocalized to apoptotic VSMCs in human coronary lesions, further supporting a functional role for GADD153 in CRP-induced cell death. Conclusions—These results demonstrate that GADD153 is a CRP-regulated gene in human VSMCs and plays a causal role in CRP-induced apoptosis. Pharmacological targeting of CRP expression or action may provide a novel therapy for atherosclerosis.

Vascular Inflammation Is Negatively Autoregulated by Interaction Between CCAAT/Enhancer-Binding Protein-δ and Peroxisome Proliferator-Activated Receptor-γ

Abstract— CCAAT/enhancer-binding proteins (C/EBPs) upregulate transcription of various inflammatory cytokines and acute phase proteins, such as interleukin (IL)-1&bgr;, IL-6, tumor necrosis factor-&agr;, and cyclooxygenase-2. Recent studies have demonstrated that peroxisome proliferator-activated receptor (PPAR)-&ggr; is present in atherosclerotic lesions, and negatively regulates expression of these genes. Interestingly, PPAR-&ggr; gene promoter has tandem repeats of C/EBP-binding motif, and C/EBP-&dgr; plays a pivotal role in transactivation of PPAR-&ggr; gene. It has been well known that the interaction between C/EBPs and PPAR-&ggr; plays a central role in maintaining adipocyte differentiation and glucometabolism; however, the relationship between PPAR-&ggr; and C/EBPs in the vessel wall remains unclear. In the present study, we showed that a high level of C/EBP-&dgr; expression induced by inflammation positively regulated transcription and protein expression of PPAR-&ggr; in vascular smooth muscle cells (VSMCs). On the other hand, PPAR-&ggr; ligands troglitazone, pioglitazone, and 15-deoxy-&Dgr;12,14-prostaglandin J2 inhibited IL-1&bgr;-induced IL-6 expression at a transcriptional revel in VSMCs. Functional promoter analysis revealed that PPAR-&ggr; ligands inhibited IL-1&bgr;-induced transactivation of IL-6 gene via suppression of not only nuclear factor-&kgr;B but also C/EBP-DNA binding. Moreover, PPAR-&ggr; ligands suppressed protein expression and transcription of C/EBP-&dgr; through dephosphorylation of signal transducer and activator of transcription 3. These findings strongly suggest that C/EBP-&dgr; is negatively autoregulated via transactivation of PPAR-&ggr;. This feedback mechanism probably downregulates transcription of inflammatory cytokines and acute phase proteins, and modulates inflammatory responses in the early process of atherosclerosis.

Troglitazone induces apoptosis via the p53 and Gadd45 pathway in vascular smooth muscle cells

Thiazolidinediones, activators of peroxisome proliferator-activated receptor (PPAR)gamma, have been reported to induce apoptosis in many types of cells. In the present study, we investigated the effects of thiazolidinediones, troglitazone, and pioglitazone on the cell growth of vascular smooth muscle cells, and identified a specific effect of troglitazone in addition to PPARgamma activation. Subconfluent rat culture vascular smooth muscle cells were treated with or without PPARgamma activators, troglitazone (1-30 microM), or pioglitazone (1-30 microM) for 72 h. After treatment, cell viability was significantly reduced by troglitazone in concentrations of 5-30 microM but not by pioglitazone. Vascular smooth muscle cells appeared to float and shrink 48 h after treatment with 20 microM of troglitazone. In situ DNA labeling showed that the nuclei of these cells were positively stained, and genomic DNA extracted from the cells showed nucleosomal laddering. Messenger RNA expression levels of c-myc, p21, bax, bcl-2, and bcl-x were not changed by the treatment with troglitazone. In contrast, along with the induction of vascular smooth muscle cell apoptosis, both the mRNA and protein expression levels of p53 and Gadd45 markedly increased in response to troglitazone. These results strongly suggest that troglitazone can induce vascular smooth muscle cell apoptosis and that this effect is caused primarily by activation of the p53 and Gadd45 pathway but not by PPARgamma activation.

Effects of angiotensin II receptor blockade with valsartan on pro-inflammatory cytokines in patients with essential hypertension.

Chronic inflammation is common in hypertension and acts as an independent determinant of arterial blood pressure. Hypertensive patients are reported to have high circulating levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-reactive protein (CRP). Recently, angiotensin II receptor blockers (ARBs) have been shown to possess benefits in addition to their ability to lower blood pressure, including anti-inflammatory and antioxidative properties within the vasculature. We evaluated the effects of the angiotensin II receptor blocker, valsartan, on these inflammatory cytokines. Thirty-nine patients with essential hypertension participated. These subjects received valsartan, 40 to 80 mg/day. Serum TNF-α, IL-6, CRP, and serum amyloid A (SAA) were measured before and after 3 months of treatment with valsartan. Valsartan significantly decreased systolic and diastolic blood pressure (160 ± 16/92 ± 11 mm Hg to 147 ± 21/84 ± 11 mm Hg, P = 0.001/P = 0.001, respectively). Serum TNF-α (9.1 ± 8.6 pg/mL to 6.1 ± 1.0 pg/mL, P = 0.006) and IL-6 (9.3 ± 1.7 pg/mL to 8.9 ± 1.4 pg/mL, P = 0.005) were significantly reduced after treatment with valsartan. However, C-reactive protein and serum amyloid A did not change. The angiotensin II receptor blocker, valsartan, may inhibit the development of atherosclerosis by lowering serum pro-inflammatory cytokines.

Postprandial Hypotension Is Associated With Asymptomatic Cerebrovascular Damage in Essential Hypertensive Patients

offelucidate the relationship between postprandial hypotension (PPH) and asymptomatic cerebrovascular damage, we evaluated changes in blood pressure after a meal by 24-hour blood pressure monitoring in 70 hospitalized essential hypertensive patients aged >/=50 years. They received a diet containing standard nutritional ingredients with 120 mmol (7 g) NaCl and were free from medication for at least 1 week. PPH was defined as the mean reduction of systolic blood pressure during 2 hours after a meal. Patients were divided into three groups according to mean values of PPH after 3 meals: PPH-1 (n=16, 5 mm Hg</=PPH<10 mm Hg), PPH-2 (n=18, PPH>/=10 mm Hg), and normal (n=36, PPH<5 mm Hg). As asymptomatic cerebrovascular damage, lacunae and leukoaraiosis were evaluated by magnetic resonance imaging. PPH did not correlate with daytime or nighttime blood pressure or the nondipper phenomenon; however, PPH was significantly related to asymptomatic cerebrovascular damage. The prevalence of lacunae in the normal, PPH-1, and PPH-2 groups was 44%, 69%, and 83%, respectively (chi2=8.22, P<0.05). The number of lacunae in the normal, PPH-1, and PPH-2 groups was 1.0+/-1.3, 1.3+/-1.2, and 1. 9+/-1.4, respectively (F[2,67]=3.2, P<0.05). The prevalence of advanced leukoaraiosis in the normal, PPH-1, and PPH-2 groups was 44%, 50%, and 83%, respectively (chi2=7.63, P<0.05). Severity score of leukoaraiosis in the normal, PPH-1, and PPH-2 groups was 1.5+/-0. 7, 1.7+/-0.8, and 2.1+/-0.7, respectively (F[2,67]=4.3, P<0.05). These findings indicate that elderly hypertensive patients with marked PPH should be considered to have advanced cerebrovascular damage even in the absence of abnormal neurological findings.

Transcriptional Regulation of the Platelet-derived Growth Factor α Receptor Gene via CCAAT/Enhancer-binding Protein-δ in Vascular Smooth Muscle Cells

Inflammatory cytokines stimulate the proliferation of vascular smooth muscle cells (VSMC) and play a pivotal role in the pathogenesis of vascular diseases including atherosclerosis and restenosis. Mitogenic response of interleukin-1β (IL-1β) on VSMC is thought to be mediated by induction of endogenous platelet-derived growth factor (PDGF), especially PDGF-AA. Although the action of PDGF-AA is mediated by its specific receptor, PDGFα-receptor (PDGFαR), very little is known about the regulatory mechanism of PDGFαR gene expression in VSMC. To understand the mechanism, we studied the transcriptional control of the PDGFαR gene in VSMC after treatment with IL-1β. IL-1β (10 ng/ml) drastically increased both PDGFαR and CCAAT/enhancer-binding protein δ (C/EBPδ) mRNA levels in a time dependent manner. A rapid induction of C/EBPδ mRNA within 30 min was followed by slower emergence of PDGFαR mRNA, which reached the maximum level in 12 h, whereas C/EBPδ mRNA was detectable at 30 min and reached the maximum level at 3 h. Electromobility shift and supershift assays revealed that IL-1β markedly increased DNA-protein complex, which was mainly composed of C/EBPβ and/or -δ. Both Western blotting and immunohistochemistry demonstrated that either C/EBPβ or -δ expression was induced by IL-1β exclusively in nuclei of VSMC. On the other hand, overexpression of C/EBPδ specifically transactivated the promoter activity of the PDGFαR gene and significantly enhanced VSMC proliferation in PDGF-treated cells. We conclude that induction of PDGFαR expression is mainly mediated by C/EBPδ expression in VSMC, and a high level of C/EBPδ expression may be involved in the pathogenesis of atherosclerosis and restenosis.

Association between carotid haemodynamics and inflammation in patients with essential hypertension

Previous studies have shown that high blood pressure causes chronic inflammation. Hypertensive patients are reported to have high-circulating levels of proinflammatory cytokines such as interleukin-6 (IL-6) and high sensitive C-reactive protein (hs-CRP). The pulsatility index (PI) and resistive index (RI) are used as markers of peripheral vascular resistance. In the present study, we evaluated the relationship between carotid haemodynamics and the proinflammatory cytokines, IL-6 and hs-CRP. In all, 41 patients with essential hypertension participated. The intima-media thickness (IMT), peak systolic velocity (pVs), peak diastolic velocity (pVd) and mean velocity (mV) in the common carotid artery were measured using ultrasound Doppler flow methods, and PI [(pVs−pVd)/mV] and RI [(pVs−pVd)/pVs] were calculated. Serum IL-6 and hs-CRP concentrations were measured by an enzyme-linked immunosorbent assay. IMT was positively correlated with age and pulse pressure. Both PI and RI were positively correlated with pulse pressure, IL-6 and hs-CRP. A multiple regression analysis revealed that PI and RI were independently associated with hs-CRP. These results suggested that carotid haemodynamic parameters such as PI and RI are associated with atherosclerosis and inflammation in hypertensive patients.

SYMPLICITY HTN-Japan – First Randomized Controlled Trial of Catheter-Based Renal Denervation in Asian Patients –

BACKGROUND SYMPLICITY HTN-Japan is a prospective, randomized, controlled trial comparing renal artery denervation (RDN) with standard pharmacotherapy for treatment of resistant hypertension (systolic blood pressure [SBP] ≥160 mmHg on ≥3 anti-hypertensive drugs including a diuretic for ≥6 weeks). When SYMPLICITY HTN-3 failed to meet the primary efficacy endpoint, the HTN-Japan enrollment was discontinued before completion. METHODS AND RESULTS: The 6-month change in office and 24-h ambulatory SBP were compared between RDN (n=22) and control (n=19) subjects. Mean baseline office SBP was 181.0±18.0 mmHg and 178.7±17.8 mmHg for the RDN and control groups, respectively. The 6-month office SBP change was -16.6±18.5 mmHg for RDN subjects (P<0.001) and -7.9±21.0 mmHg for control subjects (P=0.117); the difference between the 6-month change in RDN and control subjects was -8.64 (95% CI: -21.12 to 3.84, P=0.169). Mean 24-h SBP was 164.7±18.3 (RDN group) and 163.3±17.2 mmHg (control group). The 24-h 6-month SBP change for the RDN group was -7.52±11.98 mmHg (P=0.008) and -1.38±10.2 mmHg (P=0.563) for control subjects; the between-group difference in SBP change was -6.15 (95% CI: -13.23 to 0.94, P=0.087). No major adverse events were reported. CONCLUSIONS SYMPLICITY HTN-Japan, the first randomized controlled trial of RDN in an Asian population, was underpowered for the primary endpoint analysis and did not demonstrate a significant difference in 6-month BP change between RDN and control subjects.

Osteopontin deficiency protects against aldosterone-induced inflammation, oxidative stress, and interstitial fibrosis in the kidney

Osteopontin (OPN) has been implicated in the pathology of several renal conditions. Recently, we demonstrated in vitro that aldosterone has important roles in collagen synthesis by inducing OPN (Irita J, Okura T, Kurata M, Miyoshi K, Fukuoka T, Higaki J. Hypertension 51: 507-513, 2008). The aim of the present study was to clarify the roles of OPN in aldosterone-mediated renal fibrosis by infusing aldosterone into either wild-type (WT) or OPN knockout mice (OPN(-/-)). We used uninephrectomized mice treated with aldosterone and high salt to exacerbate renal fibrosis. After 4 wk of treatment with aldosterone, we showed similar increases in systolic blood pressure in both strains of mice. Urine albumin excretion was greater in aldosterone-infused WT mice than in aldosterone-infused OPN(-/-) mice. Immunohistochemical analysis showed high levels of OPN expression in aldosterone-infused WT mice. Interstitial fibrosis and inflammatory infiltrations were increased in aldosterone-infused WT mice compared with either vehicle-infused WT or aldosterone-infused OPN(-/-) mice. These changes were ameliorated markedly by eplerenone treatment in aldosterone-infused WT mice. Aldosterone-infused WT mice also had increased expression of NADPH oxidase subunits compared with aldosterone-infused OPN(-/-) mice. We observed a marked increase in oxidative stress markers in aldosterone-infused WT mice compared with aldosterone-infused OPN(-/-) mice. These results indicate that OPN is a promoter of aldosterone-induced inflammation, oxidative stress, and interstitial fibrosis in the kidney and suggest that inhibition of OPN may be a potential therapeutic target for prevention of renal injury.