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Tropomyosin (TM) is a regulatory protein of actomyosin system. Muscle type-specific expression of TM isoforms is generated from different genes and by alternative splicing. beta-TM isoforms in chicken skeletal and smooth muscles are encoded by a single gene and transcribed from the same promoter. We previously reported a smooth muscle cell (SMC) phenotype-dependent change in beta-TM expression (Kashiwada, K., Nishida, W., Hayashi, K., Ozawa, K., Yamanaka, Y., Saga, H., Yamashita, T., Tohyama, M., Shimada, S., Sato, K., and Sobue, K. (1997) J. Biol. Chem. 272, 15396-15404), and identified beta-TM as an SMC-differentiation marker. Here, we characterized the transcriptional machinery of the beta-TM gene in SMCs. Promoter and gel mobility shift analyses revealed an obligatory role for serum response factor and its interaction with the CArG box sequence in the SMC-specific transcription of the beta-TM gene in differentiated SMCs. We further isolated a novel homologue of the Barx homeoprotein family, Barx1b, from chicken gizzard. Barx1b was exclusively localized to SMCs of the upper digestive organs and their attached arteries and to craniofacial structures. Serum response factor and Barx1b bound each other directly, coordinately transactivated the beta-TM gene in differentiated SMCs and heterologous cells, and formed a ternary complex with a CArG probe. Taken together, these results suggest that SRF and Barx1b are coordinately involved in the SMC-specific transcription of the beta-TM gene in the upper digestive organs and their attached arteries.

CCAAT/enhancer-binding protein (C/EBP)-binding motifs have been identified in the promoter regions of interleukin (IL)-6, tumor necrosis factor-alpha, and platelet-derived growth factor-alpha receptor (PDGFalphaR). Recently, peroxisome proliferator-activated receptors (PPARs) have been suggested to be important immunomodulatory mediators. Although many studies have demonstrated that the interaction between C/EBPs and PPARs plays a central role in lipid metabolism, expression and function of these factors are unknown in vascular smooth muscle cells (VSMCs). In the present study, we clarified a functional relationship between C/EBPs and PPARgamma in the regulation of IL-1beta-induced PDGFalphaR expression in VSMCs. PPARgamma activators, troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2), inhibited IL-1beta-induced PDGFalphaR expression and suppressed PDGF-induced proliferation activity of VSMCs. Electromobility shift and supershift assays for a C/EBP motif in the PDGFalphaR promoter region revealed that PPARgamma activators suppressed IL-1beta-induced DNA binding activity of C/EBPdelta and beta. PPARgamma activators also suppressed IL-1beta-induced C/EBPdelta expression. In contrast, overexpression of C/EBPdelta reversed the suppressive effect of PPARgamma activators on PDGFalphaR expression almost completely. From these results, we conclude that the inhibitory effect of PPARgamma activators on PDGFalphaR expression is mainly mediated by C/EBPdelta suppression. Regulation of C/EBPdelta by PPARgamma activators probably plays critical roles in modulating inflammatory responses in the arterial wall.

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-1beta (IL-1beta) 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, PDGFalpha-receptor (PDGFalphaR), very little is known about the regulatory mechanism of PDGFalphaR gene expression in VSMC. To understand the mechanism, we studied the transcriptional control of the PDGFalphaR gene in VSMC after treatment with IL-1beta. IL-1beta (10 ng/ml) drastically increased both PDGFalphaR and CCAAT/enhancer-binding protein delta (C/EBPdelta) mRNA levels in a time dependent manner. A rapid induction of C/EBPdelta mRNA within 30 min was followed by slower emergence of PDGFalphaR mRNA, which reached the maximum level in 12 h, whereas C/EBPdelta mRNA was detectable at 30 min and reached the maximum level at 3 h. Electromobility shift and supershift assays revealed that IL-1beta markedly increased DNA-protein complex, which was mainly composed of C/EBPbeta and/or -delta. Both Western blotting and immunohistochemistry demonstrated that either C/EBPbeta or -delta expression was induced by IL-1beta exclusively in nuclei of VSMC. On the other hand, overexpression of C/EBPdelta specifically transactivated the promoter activity of the PDGFalphaR gene and significantly enhanced VSMC proliferation in PDGF-treated cells. We conclude that induction of PDGFalphaR expression is mainly mediated by C/EBPdelta expression in VSMC, and a high level of C/EBPdelta expression may be involved in the pathogenesis of atherosclerosis and restenosis.

Angiotensin converting enzyme(ACE) activities of broncho-alveolar lavage fluid(BALF) and serum in patients with sarcoidosis and with silicosis were measured. Serum ACE was measured by enzymic assay and radioimmunoassay. There was a close relationship between ACE activity and content (r=0.78). Serum ACE activities in patients with active sarcoidosis (37.5 ± 11.1 nmol/min/ml, mean ± SD) and with silicosis (25.5 ± 9.3) were significantly elevated from the control (18.6 ± 6.0). BALF ACE activities in the control, patients with active sarcoidosis and with silicosis were 0.23 ± 0.19 nmol/min/ml, 0.94 ± 0.97 and 0.38 ± 0.05, respectively. BALF ACE in patients with active sarcoidosis and with silicosis were significantly different from the control. When BALF ACE was corrected by the cell count of alveolar macrophage (per 106 cells), activity was significantly different from control only in the patients with sarcoidosis. Moreover, only the alveolar macrophages in sarcoidosis were stained by immunofluorescence and immunocytochemistry using rabbit anti-human ACE antibody. Induction of ACE in alveolar macrophage might have an important role for the activity or progression of sarcoidosis.

To investigate the relationship between alterations of the autonomic nervous activity and left ventricular mass index in essential hypertensive patients, 24-h power spectral analysis of R-R intervals was performed using Holter electrocardiography. Fifty-three patients (mean age, 58.0 +/- 13.1 years; 30 men and 23 women) participated. The urinary excretions and plasma levels of catecholamines were also determined. Power spectral analysis of R-R interval was performed every 10 min by the maximum entropy method to obtain the low frequency band (LFB; 0.04 to 0.15 Hz), which is an index of both parasympathetic and sympathetic nervous activities, and the high frequency band (HFB; 0.15 to 0.4 Hz), which reflects parasympathetic nervous activity. LFB and HFB were averaged every hour to obtain hourly LFB and HFB levels. Total LFB and HFB were calculated as the summation of 24-h LFBs and HFBs. Left ventricular mass index showed a significant negative correlation with total LFB (r = -0.466, P < .001) and total HFB (r = -0.319, P < .02). These findings suggest that the level of end-organ damage correlates with neuronal alteration in essential hypertension.

The nucleotide and deduced amino acid sequences of human basic and acidic calponins were determined. The basic calponin cDNA from human aorta (1496 bp) contained a single open reading frame (ORF) which encodes 297 amino acids (33,169 Da). The acidic calponin cDNA from human kidney (1607 bp) contained a single ORF which encodes 329 amino acids (36,412 Da). Basic calponin mRNA was expressed in only smooth muscle tissues, but acidic calponin mRNA was expressed in non-smooth muscle tissues as well as smooth muscle tissues. Fluorescent in situ hybridization revealed that basic and acidic calponin genes localize in 19p13.1-13.2 and 1p21-22 of human chromosomes, respectively.

The role of brain glutathione metabolism in hypertensive animals was studied. In spontaneously hypertensive rats (SHR) from prehypertension to established hypertension, the content of oxidized glutathione (GSSG) and the ratio of GSSG to GSH in the hypothalamus were significantly (p < 0.05) higher than those in age -matched normotensive Wistar Kyoto rats (WKY). Hypothalamic glutathione reductase (GR) activities in prehypertensive and established hypertensive SHR were significantly (p< 0.05) lower than those in WKY. DOCA-salt hypertensive rats (DSR) also had a significantly (p < 0.05) higher content of GSSG and GSSG/GSH ratio and a significantly (p <0.05) lower GR activity in the hypothalamus than the normotensive control. There were no significant differences in these values in the brain stem between hypertensive and normotensive rats. These results suggest that the increased GSSG/GSH ratio due to reduced activity of GR in the hypothalamus may have an important role in the development of hypertension in SHR and BSR.

Setting:: KL-6, a human MUC-1 mucin preferentially expressed on type II pneumocytes, is a sensitive serum marker for evaluating alveolar damage of interstitial pneumonia and pulmonary fibrosis. Some patients with pulmonary tuberculosis develop severe respiratory dysfunction caused by extensive pulmonary fibrosis, compensatory emphysema and fibrous pleural thickening. Objective:: To evaluate the clinico-pathological significance of KL-6 in pulmonary tuberculosis. Design:: Serum KL-6 levels were measured in sera from 57 patients with active pulmonary tuberculosis and 38 healthy controls by a sandwich-type enzyme-linked immunosorbent assay. Immunohistochemistry was performed by an avidin-biotin-peroxidase complex method. Results:: KL-6 levels were significantly higher in the patients than in the healthy controls (518 +/- 693 [SD] vs 227 +/- 91 U/ml, P < 0.001) and increased significantly according to the extent of pulmonary lesions evaluated by chest X-ray (P < 0.001). There was a significant negative correlation between serum KL-6 levels and % vital capacity (VC) (r = 0.642, P < 0.05). KL-6 was strongly expressed on proliferated type II pneumocytes and cuboidal epithelial cells adjacent to thickened intralobular septa and pleura. Conclusions:: In pulmonary tuberculosis, serum KL-6 originates from proliferated type II pneumocytes and cuboidal epithelial cells, and is a useful marker presenting the degree and extent of pulmonary fibroproductive lesions.

Dipeptide and tripeptide derivatives containing a statine residue were synthesized as human renin inhibitors. ES-305. his(I-naphthyl)methyl acetyl-histidyl-statine-2(S)-methylbutylamide, was found to he a highly potent human renin inhibitor that is species-specific and enzyme-specific. The replacement of the methylhutylamide of ES-305 with the Ieucyl-lysinol (ES-1005) showed similar high potency against human renin (kivalue of 2.4 x 10-9M) and monkey renin (Kivalue of 7.9 x 10-9M) as ES-305. ES-1005 competitively inhibited human renin. The compound was about one order of magnitude less potent against pig. dog, and rahhit renins. It had moderate inhibitory potencies against cathepsin D and pepsin (ICof cathepsin D) and pepsin of 1.6 x 10-6and 8.0 x 10-6, M, respectively). ES-1005, a newly synthesized tripeptide derivative containing statine, is a highly potent inhibitor of not only primate renin hut also a wide variety of nonprimate renins.

PDGF a-receptor gene activation induced by inflammatory cytokines such as IL-1ß and IL-6 is negatively regulated by PPAR? activator, troglitazone (TRO) or 15?-PGJ2, in vascular smooth muscle cells (VSMCs). This inhibitory effect is mainly regulated by a specific nuclear factor, CCAAT/enhancer-binding protein (C/EBP)d. However, underling molecular mechanisms of the C/EBPd gene regulation by PPAR? activator are still unknown. To identify a key factor that mainly regulates the C/EBPd transcription after treatment with PPAR? activator, IL-6-induced C/EBPd expression were evaluated by Western or Northern blotting in the VSMCs pretreated with TRO or 15?-PGJ2. Protein or mRNA levels of C/EBPd induced by treatment with IL-6 (5 ng/mL) were significantly reduced by the pretreatment with TRO (0.1–10 µM) or 15?-PGJ2 (0.1–5 µM) in a dose-dependent manner. Functional promoter analyses revealed that TRO or 15?-PGJ2 suppressed C/EBPd gene promoter activity through the inhibition of DNA-protein complex formation, STAT3 and acute-phase response element (APRE). Furthermore, TRO or 15?-PGJ2 induced dephosphorylation of STAT3. These results indicate that IL-6-induced transcriptional activation of the C/EBPd gene is negatively regulated by PPAR? activator through the dephosphorylation of STAT3.

In vitro studies demonstrated adhesion molecules could connect low shear stress and atherosclerosis. In the present study, we investigated the relationship between circulating adhesion molecules and carotid atherosclerosis and wall shear stress in essential hypertensive patients. Seventy subjects with risk factors for atherosclerosis, free from the medication for at least a week, were enrolled in the study. Intimal-media thickness (IMT) was evaluated by high-resolution ultrasonography. Shear rate was obtained from Poiseuilleans parabolic model: shear rate=4×blood velocity / carotid internal diameter. Shear stress was obtained by shear stress × blood viscosity. Plasma levels of ICAM-1 and VCAM-1 were measured by ELISA. There were no relationship between IMT and plasma levels of ICAM-1 and VCAM-1. VCAM-1 showed a significant negative correlation with peak shear stress in common carotid artery (r=0.39, p<0.001), while there was no relationship between ICAM-1 and wall shear stress. These findings indicate that low shear stress may promote atherosclerosis through in part activation of soluble form of adhesion molecules.

Objective: This study was designed to elucidate the association between left ventricular geometric adaptation and insulin resistance in essential hypertension. Design and Methods: 73 untreated hypertensive patients were subdivided into 4 groups based on left ventricular mass index (LVMI) and relative wall thickness (RWT). With respect to insulin resistance, homeostasis model assessment (HOMA) was performed. HOMA-R = Fasting blood sugar (mmol/L) × Immunoreactive insulin (µU/ml)/22.5. Results: 1) Among hypertensive patients, LVMI and RWT were normal in 29 patients (NG), whereas 9 patients had increased RWT with normal LVMI (CR), 12 patients had concentric hypertrophy (CH, increase both LVMI and RWT) and 23 patients had increased LVMI and normal in RWT (EH). 2) There were no significant differences in office blood pressure and body mass index (BMI) among 4 groups. 3) HOMA-R was 1.8 ± 1.2 in NG, 1.4 ± 0.9 in CR, 2.1 ± 0.8 in EH and 4.2 ± 1.8 in CH (p < 0.001 vs other 3 groups). In multiple regression analyses, there was a strongly significant correlation between the degree of LVMI and the severity of insulin resistance independent of office blood pressure and BMI. Conclusion: Pattern of left ventricular geometric adaptation to hypertension is closely related to the severity of insulin resistance.