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PDF file - 95KB, Supplementary Table S3-A. Human tumor pY proteome. S3-B. Comparison analysis of unique pTyr sites iden_fy from di_erent samples.

PDF file - 104KB, Supplementary Figure 1A-B: PC9GR cells acquired drug resistance to both reversible and irreversible EGFR-TKI. A: PC9 and PC9GR cells were treated for 72 h with increasing concentrations of erlotinib or CL387,785. Data generated by cell viability assay (CellTiter-Glo) are expressed as a percentage of the value for untreated cells. Determinations were done in triplicate. B: PC9 and PC9GR cells were incubated for 1 hour in the absence or presence of erlotinib (1 �M) as indicated. Cell lysates were subjected to protein expression analysis with antibodies to pEGFR, pSrc, pAkt, and pErk along with antibodies to β-actin as a loading control. C-D: Gene status in PC9 and PC9GR cells. C: PC9GR cells acquired T790M while retaining exon 19 deletion as determined by PCR-invader assay. Values of red bars above zero represent positive results with the detection probe for indicated mutation sequences of EGFR, while values of light blue bars are quality controls showing good PCR quality, as previously described (1). The value of red bars = (fluorescence of the samples with detection probe for mutation sequences) - (fluorescence of the normal control with the same probe � 2). The value of light blue bars = (fluorescence of the samples with detection probe for wild type sequences) - (fluorescence of the normal control with the same probe � 0.8). DEL1 represents E746-A750del type1 (2235-2249del GGAATTAAGAGAAGC). DEL2 represents E746-A750del type2 (2236-2250del GAATTAAGAGAAGCA). DEL3 represents L747-P753del insS. D: FISH analysis (CEP7 (green)/D7S522 (red)) was done in PC9 and PC9GR cells as previously described (2). No additional copy of D7S522 compared to CEP 7 was found in these cells, explaining that neither PC9 nor PC9GR cells has MET amplification.

PDF file - 139KB, Effects of ligands exposure with or without EGFR-TKI in PC9 and PC9GR cells. A-F: PC9 (A, C, and E) or PC9GR cells (B, D, and F) were incubated for 1 hour in the absence or presence of 100 nM erlotinib (A, C, and E) or afatinib (B, D, and F) with indicated concentrations of HGF (A and B), IGF1 (C and D), or Gas6 (E and F). Cell lysates were subjected to protein expression analysis with antibodies to pMET (A and B), IGF1R (C and D), or pAXL (E and F) along with pEGFR, pAkt, pErk, and antibodies to β-actin as a loading control.

PDF file - 54KB, A: Example of peak area (EIC) for EGFR pY 1172. B: Example of peak area (EIC) for MET pY 1234.

Purpose: Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non–small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M.Experimental Design: We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI–resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass spectrometry–based identification/quantification. Profiles of erlotinib perturbations were examined.Results: We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib–treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced antitumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M-mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib.Conclusions: Our results identified both codrivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in patients with NSCLC with acquired T790M. Clin Cancer Res; 20(15); 4059–74. ©2014 AACR.

PDF file - 91KB, A: Effects of dasatinib combined with afatinib on apoptosis in HCC4006-T790M and HCC827-T790M cells. Apoptosis assay was carried out using PE-conjugated caspase-3 antibody, following incubation of HCC4006-T790M or HCC827-T790M cells for 72 hours with DMSO, erlotinib (E), dasatinib (D), afatinib (A), and afatinib + dasatinib. Values are expressed as a percentage of caspase-3-positive cells. Determinations were done in triplicate. Bars, SD. *P < 0.001 versus DMSO or each single agent (Student's t-test). B: Validation of active pTyr sites from PC9 cell lines on lung cancer patient. Venn diagram was used to compare the pTyr sites identified from PC9 and human lung tumor tissue with positive EGFR mutation. Total 83 overlap active pTyr sites were obtained.

PDF file - 387KB, Effects of ligands exposure with or without EGFR-TKI in PC9 and PC9GR cells. A-F: PC9 (A, C, and E) or PC9GR cells (B, D, and F) were incubated for 1 hour in the absence or presence of 100 nM erlotinib (A, C, and E) or afatinib (B, D, and F) with indicated concentrations of HGF (A and B), IGF1 (C and D), or Gas6 (E and F). Cell lysates were subjected to protein expression analysis with antibodies to pMET (A and B), IGF1R (C and D), or pAXL (E and F) along with pEGFR, pAkt, pErk, and antibodies to β-actin as a loading control.

PDF file - 67KB, Supplementary Table S1: Quantitation of pY changes in between PC9 and PC9GR cell lines with and without erlotinib treatment. The cutoff was set as P value1.5; Supplementary Table S2: Cell viability IC50 in PC9GR cells and H1975 cells.

// Hisato Kawakami 1 , Isamu Okamoto 1, 2 , Kimio Yonesaka 1 , Kunio Okamoto 1 , Kiyoko Shibata 1 , Yume Shinkai 1 , Haruka Sakamoto 1 , Michiko Kitano 1 , Takao Tamura 1 , Kazuto Nishio 3 , Kazuhiko Nakagawa 1 1 Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-sayama, Osaka 589-8511, Japan 2 Center for Clinical and Translational Research, Kyushu University Hospital, Higashiku, Fukuoka 812–8582, Japan 3 Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka 589–8511, Japan Correspondence to: Isamu Okamoto, e-mail: okamotoi@kokyu.med.kyushu-u.ac.jp Keywords: colorectal cancer, heregulin, resistance, cetuximab, patritumab Received: September 21, 2014 Accepted: October 26, 2014 Published: December 19, 2014 ABSTRACT We previously showed that tumor-derived heregulin, a ligand for HER3, is associated with both de novo and acquired resistance to cetuximab. We have now examined whether patritumab, a novel neutralizing monoclonal antibody to HER3, is able to overcome such resistance. Human colorectal cancer (DiFi) cells that are highly sensitive to cetuximab were engineered to stably express heregulin by retroviral infection, and the effects of cetuximab and patritumab on the resulting DiFi-HRG cells were examined. DiFi-HRG cells released substantial amounts of heregulin and showed resistance to cetuximab. Cetuximab alone inhibited EGFR and ERK phosphorylation in DiFi-HRG cells, but it had no effect on the phosphorylation of HER2, HER3, or AKT, suggesting that sustained AKT activation by HER2 and HER3 underlies cetuximab resistance in these cells. In contrast, patritumab in combination with cetuximab markedly inhibited the phosphorylation of EGFR, HER2, HER3, ERK, and AKT. The combination therapy also inhibited the growth of DiFi-HRG tumor xenografts in nude mice to a greater extent than did treatment with either drug alone. Activation of HER2-HER3 signaling associated with the operation of a heregulin autocrine loop confers resistance to cetuximab, and patritumab is able to restore cetuximab sensitivity through inhibition of heregulin-induced HER3 activation.

Background: The role of the nitric oxide synthase (NOS) system in cardiac architecture and function remains unknown. This point was addressed in mice that lack all 3 NOS genes. Methods and Results: Morphological, echocardiographic, and hemodynamic analyses were performed in wild-type (WT), singly nNOS-/-, iNOS-/-, eNOS-/-, and triply n/i/eNOS-/- mice. At 5 months of age, but not at 2 months of age, significant left ventricular (LV) hypertrophy was noted in n/i/eNOS-/- mice and to a lesser extent in eNOS-/- mice, but not in nNOS-/- or iNOS-/- mice, compared with WT mice. Importantly, significant LV diastolic dysfunction (as evaluated by echocardiographic E/A wave ratio and hemodynamic -dP/dt and Tau), with preserved LV systolic function (as assessed by echocardiographic fractional shortening and hemodynamic +dP/dt), was noted only in n/i/eNOS-/- mice, and this was associated with enhanced LV end-diastolic pressure and increased lung wet weight, all of which are characteristics consistent with diastolic heart failure in humans. Finally, long-term oral treatment with an angiotensin II type 1 (AT1) receptor blocker, olmesartan, significantly prevented all these abnormalities of n/i/eNOS-/- mice. Conclusions: These results provide the first direct evidence that the complete disruption of all NOSs results in LV hypertrophy and diastolic dysfunction in mice in vivo through the AT1 receptor pathway, demonstrating a pivotal role of the endogenous NOS system in maintaining cardiac homeostasis. (Circ J 2010; 74: 2681-2692)

Background— The roles of nitric oxide (NO) in the cardiovascular system have been investigated extensively in pharmacological studies with NO synthase (NOS) inhibitors and in studies with NOS isoform–deficient mice. However, because of the nonspecificity of the NOS inhibitors and the compensatory interactions among NOS isoforms (nNOS, iNOS, and eNOS), the ultimate roles of endogenous NO derived from the entire NOS system are still poorly understood. In this study, we examined this point in mice deficient in all 3 NOS isoforms (triply n/i/eNOS −/− mice) that we have recently developed. Methods and Results— The triply n/i/eNOS −/− mice, but not singly eNOS −/− mice, exhibited markedly reduced survival, possibly due to spontaneous myocardial infarction accompanied by severe coronary arteriosclerotic lesions. Furthermore, the triply n/i/eNOS −/− mice manifested phenotypes that resembled metabolic syndrome in humans, including visceral obesity, hypertension, hypertriglyceridemia, and impaired glucose tolerance. Importantly, activation of the renin-angiotensin system was noted in the triply n/i/eNOS −/− mice, and long-term oral treatment with an angiotensin II type 1 receptor blocker significantly suppressed coronary arteriosclerotic lesion formation and the occurrence of spontaneous myocardial infarction and improved the prognosis of those mice, along with ameliorating the metabolic abnormalities. Conclusions— These results provide the first direct evidence that genetic disruption of the whole NOS system causes spontaneous myocardial infarction associated with multiple cardiovascular risk factors of metabolic origin in mice in vivo through the angiotensin II type 1 receptor pathway, demonstrating the critical role of the endogenous NOS system in maintaining cardiovascular and metabolic homeostasis.

Purpose: Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non–small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M. Experimental Design: We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI–resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass spectrometry–based identification/quantification. Profiles of erlotinib perturbations were examined. Results: We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib–treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced antitumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M-mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib. Conclusions: Our results identified both codrivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in patients with NSCLC with acquired T790M. Clin Cancer Res; 20(15); 4059–74. ©2014 AACR.

Nitric oxide (NO) is synthesized by three distinct NO synthase (NOS) isoforms (neuronal, inducible, and endothelial NOS), all of which are expressed in the human heart. The roles of NOSs in the pathogenesis of heart failure have been described in pharmacological studies with NOS inhibitors. Recently, genetically engineered animals have been used. We have generated mice in which all 3 NOS isoforms are completely disrupted (triple n/i/eNOS(-/-) mice). Morphological, echocardiographic, and hemodynamic analysis were performed in wild-type, singly nNOS(-/-), iNOS(-/-), eNOS(-/-), and triple n/i/eNOS(-/-) mice. Importantly, significant left ventricular (LV) hypertrophy and diastolic dysfunction was noted only in n/i/eNOS(-/-) mice, and those pathology was similar to diastolic heart failure in humans. Finally, treatment with an angiotensin II type 1 (AT1) receptor blocker, significantly prevented those abnormalities. These results provide the evidence that AT1 receptor pathway plays a center role in the pathogenesis of cardiac disorders in the n/i/eNOS(-/-) mice. Our studies with triple n/i/eNOS(-/-) mice provide pivotal insights into an understanding of the pathophysiology of NOSs in human heart failure.

Aims The precise role of the nitric oxide synthase (NOS) system in lipid metabolism remains to be elucidated. We addressed this point in mice that we have recently developed and that lack all three NOS isoforms. Methods and results Wild-type (WT), singly, doubly, and triply NOS−/− mice were fed either a regular or high-cholesterol diet for 3–5 months. The high-cholesterol diet significantly increased serum low-density lipoprotein (LDL) cholesterol levels in all the genotypes when compared with the regular diet. Importantly, when compared with the WT genotype, the serum LDL cholesterol levels in the high-cholesterol diet were significantly and markedly elevated only in the triply NOS−/− genotype, but not in any singly or doubly NOS−/− genotypes, and this was associated with remarkable atherosclerosis and sudden cardiac death, which occurred mainly in the 4–5 months after the high-cholesterol diet. Finally, hepatic LDL receptor expression was markedly reduced only in the triply NOS−/− genotype, accounting for the diet-induced dyslipidaemia in the genotype. Conclusion These results provide the first direct evidence that complete disruption of all NOS genes causes severe dyslipidaemia, atherosclerosis, and sudden cardiac death in response to a high-fat diet in mice in vivo through the down-regulation of the hepatic LDL receptor, demonstrating the critical role of the whole endogenous NOS system in maintaining lipid homeostasis.

We have recently succeeded in developing mice in which all three nitric oxide synthase isoforms (nNOS, iNOS, and eNOS) are completely disrupted ( PNAS 2005). In this study, we examined cardiac morphology and function in those mice. Cardiac echocardiography and left ventricular (LV) hemodynamic measurement were performed in male wild-type (WT), singly nNOS −/− , iNOS −/− , eNOS −/− , and triply n/i/eNOS −/− mice at 2 and 5 months of age (n=5–8). At 2 months of age, no significant cardiac morphological or functional changes were detected in any strains studied. However, at 5 months of age, significant LV hypertrophy (wall thickness, mm) were noted in the triply n/i/eNOS −/− mice (1.3±0.1, P −/− mice (1.1±0.1, P −/− (0.8±0.1) or iNOS −/− mice (1.0±0.1), as compared with the WT mice (1.0±0.2). Furthermore, significant LV diastolic dysfunction (as evaluated by echocardiographic E/A ratio and by hemodynamic peak negative dP/dt), with preserved LV systolic function (as assessed by echocardiographic ejection fraction and by hemodynamic peak positive dP/dt), was noted only in the 5-month-old triply n/i/eNOS −/− mice (2.7±0.1 and 2505±60, both P −/− (2.1±0.2 and 3833±402), iNOS −/− (2.0±0.1 and 3773±747), or eNOS −/− mice (2.0±0.3 and 2934±122), as compared with the WT mice (1.9±0.1 and 4038±344). In addition, significant cardiac fibrosis (fibrosis area, %, Masson-trichrome staining) was also detected only in the 5-month-old triply n/i/eNOS −/− mice (1.4±0.2, P −/− (143±3.1, P −/− mice (140±8.5). Thus, mechanism(s) other than hypertension appears to be involved in the cardiac abnormalities of the triply n/i/eNOS −/− mice. These results provide the first evidence that genetic disruption of all NOSs results in LV hypertrophy and diastolic dysfunction in mice in vivo, suggesting a pivotal role of the NOS system in maintaining cardiac homeostasis.

We have recently developed mice lacking all three nitric oxide synthase (NOS) isoforms: nNOS, iNOS, and eNOS ( PNAS 2005). In this study, we examined the effects of a high-cholesterol (HC) diet on lipid metabolism and vascular lesion formation in those mice. Experiments were performed in 2-month-old male wild-type (WT) and singly, doubly, and triply NOS −/− mice (n=6–9). They were maintained on either a regular diet or a HC diet for 3 months. The HC feeding significantly increased plasma levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL) in all the genotypes studied as compared on the regular diet (all P −/− genotypes as compared with the WT genotype (singly nNOS −/− [371±61 and 205±65], iNOS −/− [559±62 and 350±62], eNOS −/− [619±22 and 395±25], doubly n/iNOS −/− [518±77 and 328±72], n/eNOS −/− [635±56 and 458.8±42], e/iNOS −/− [480±38 and 260±40], triply n/i/eNOS −/− [2316±704 and 1588±715], and WT [326±43 and 244±54]) (all P −/− genotype among the NOS −/− genotypes, and intriguingly, the serum levels of TC and LDL in the triply n/i/eNOS −/− genotype were equivalent to those in apolipoprotein E −/− mice that exhibit severe hypercholesterolemia. Lipid accumulation in the aorta on the HC diet (lipid area, %, oil red O staining) was also significantly more accelerated in all the NOS −/− genotypes than in the WT genotype (singly nNOS −/− [6.6±1.5], iNOS −/− [6.7±2.2], eNOS −/− [5.5±2.3], doubly n/iNOS −/− [4.7±1.7], n/eNOS −/− [6.4±1.4], i/eNOS −/− [6.8±1.3], triply n/i/eNOS −/− [20.6±1.0], and WT [3.6±1.2]), while the extent of the aortic atherosclerosis was again by far severest in the triply n/i/eNOS −/− genotype (all P −/− mouse is a new experimental model of human hypercholesterolemia and atherosclerosis.

Background : We have recently succeeded in developing mice lacking all 3 nitric oxide synthase (NOS) isoforms (triply n/i/eNOS-KO mice) ( PNAS 2005). Our preliminary study has revealed that those mice spontaneously develop cardiovascular diseases, including hypertension, dyslipidemia, and myocardial infarction (MI), and that there seems to be a great gender difference in those disorders. Methods and Results : Experiments were performed in both genders of wild-type (WT) and triply-KO mice. In male mice, survival rate at 11 months of age was markedly more reduced in triply-KO mice than in WT mice (15% vs. 100%, P P P P P P P −8 M, 2 days) significantly enhanced prostacyclin production in isolated aortas of the triply-KO mice (3.07±0.44 to 6.56±0.56 nM/g, P Conclusions : These results provide the first evidence that the gender difference in the extent of arteriosclerotic cardiovascular diseases still exists even in the complete absence of NOSs system, in which estrogen may, at least in part, be involved.

Background : Nitric oxide (NO) synthase (NOS) system consists of 3 different isoforms, including neuronal (nNOS), inducible (iNOS), and endothelial NOSs (eNOS). We have recently succeeded in developing mice lacking all NOS genes (triply n/i/eNOS-KO mice) ( PNAS 2005). Our preliminary study has revealed that the triply-KO mice develop acute myocardial infarction (AMI). The present study was designed to investigate abnormalities of glucose metabolism, an important risk factor of MI, in those mice. Methods and Results : Experiments were performed in 3-month-old male wild-type (WT) and triply-KO mice (n=5–7). At 15 minutes after intravenous glucose administration (1 g/kg), plasma glucose levels (mg/dl) were significantly higher in the triply-KO (475 ± 44) than in the WT mice (241 ± 20) ( P< 0.05), and plasma insulin levels (ng/dl) were significantly lower in the triply-KO (0.50 ± 0.03) than in the WT mice (0.78 ± 0.03) ( P< 0.05). In the triply-KO mice, the size of pancreatic islets (islet/pancreatic area, 0.64 ± 0.16 vs. 2.81 ± 0.64%) was significantly smaller ( P< 0.05), and significant apoptosis of pancreatic islets (apoptotic/total islets, 19 ± 2 vs. 3 ± 1%) (TUNEL staining) was noted as compared with the WT mice ( P< 0.05). Furthermore, insulin sensitivity (as assessed by insulin-induced [ 3 H]-glucose uptake in isolated soleus muscle) (fold increase) was markedly reduced in the triply-KO mice (1.1 ± 0.03) as compared with the WT mice (2.1 ± 0.3) ( P< 0.05), associated with an impairment of insulin-induced translocation of glucose transporter-4 (GLUT-4) to the cellular membrane (immunofluorescent staining) ( P< 0.05). Importantly, supplementation of NO by long-term treatment with isosorbide dinitrate (0.4 mg/day, 6 weeks, dermal application) significantly reversed all these abnormalities of glucose metabolism in the triply-KO mice; plasma glucose (280 ± 28) and insulin levels (0.89 ± 0.10) after glucose tolerance test, insulin sensitivity (1.5 ± 0.08), and GLUT-4 translocation were all improved (all P< 0.05). Conclusions : These results indicate that disruption of whole NOSs system causes impaired glucose tolerance and insulin resistance in mice in vivo, suggesting a critical role of endogenous NO/NOSs system in maintaining glucose metabolism.

Background : We have recently succeeded in developing mice in which all three nitric oxide synthase (NOS) genes are completely disrupted (triply n/i/eNOS −/− [KO] mice) ( PNAS 2005). Our preliminary study has revealed that those mice manifest metabolic syndrome. In this study, we thus examined whether or not statin treatment ameliorates those metabolic phenotypes. Methods : Experiments regarding metabolic phenotypes were performed in 3-month-old male wild-type (WT) and triply-KO mice treated with or without atorvastatin (30 mg/kg/day, SC) for 1.5 months. Experiments regarding coronary vascular lesion formation were carried out in 5-month-old mice treated with or without atorvastatin for 2 months. Results : Hypertension (HT) (140 ± 3 vs. 110 ± 3 mmHg), hypertriglyceridemia (TG) (267 ± 15 vs. 179 ± 12 mg/dl), impaired glucose tolerance (IGT) (plasma glucose after intravenous glucose, 493 ± 38 vs. 238 ± 16 mg/dl), insulin resistance (IR) (insulin-induced [ 3 H]-glucose uptake in isolated soleus muscle, 106 ± 8 vs. 204 ± 30%), and visceral obesity (VO) (epididymal white adipose tissue weight, 0.25 ± 0.01 vs. 0.18 ± 0.01 g), were significantly noted in untreated triply-KO mice as compared with untreated WT mice (all P < 0.05, n = 6 –12). Long-term treatment with atorvastatin significantly alleviated all the metabolic phenotypes in the triply-KO mice; HT (129 ± 1), TG (176 ± 11), IGT (342 ± 47), IR (120 ± 6) and VO (0.17 ± 0.01) (all P < 0.05, n = 6 –12). Importantly, significant coronary vascular lesions, as characterized by neointimal formation (NF) (intima/media ratio, 0.15 ± 0.01 vs. 0.01 ± 0.01), medial thickening (MT) (media/vascular ratio, 0.43 ± 0.07 vs. 0.13 ± 0.01), and perivascular fibrosis (PF) (fibrosis/vascular ratio, 0.38 ± 0.05 vs. 0.18 ± 0.03) (all P < 0.05), were spontaneously developed in the untreated triply-KO mice as compared with the untreated WT mice, all of which changes were again significantly suppressed by the atorvastatin treatment; NF (0.06 ± 0.02), MT (0.32 ± 0.02), and PF (0.17 ± 0.02) (all P < 0.05, n = 6 each). Conclusions : These results provide the first evidence that statin treatment ameliorates metabolic syndrome as well as coronary arteriosclerosis in the presence of defective NOSs system, suggesting a novel therapeutic strategy for the disorder.

Large-scale clinical studies have indicated that angiotensin receptor blockers (ARBs) have beneficial effects against cardiovascular diseases. We designed this study to compare the effects of an ARB and a calcium channel blocker (CCB) on coronary flow velocity reserve (CFVR), a predictor of cardiovascular events, as estimated using transthoracic Doppler echocardiography. Sixteen hypertensive patients (63.1+/-9.6 years old; 10 males) were randomly allocated in a double-blind fashion to valsartan (n=8, 40-80 mg/day) or nifedipine (n=8, 20-40 mg/day) groups. Age- and gender-matched subjects without hypertension were enrolled as a control group (n=12). CFVR was calculated by dividing the adenosine triphosphate-induced hyperemic flow velocity by the basal flow velocity in the left anterior descending coronary artery. Baseline characteristics and reduction in systolic and diastolic blood pressure after 6 months were similar in both groups. CFVR in the valsartan group increased from 2.34+/-0.38 to 3.10+/-0.84 at 2 months (p0.05), and to 3.04+/-1.09 at 6 months (p0.01). Both values became comparable to that in the control group (2.81+/-0.60). CFVR in the valsartan group was significantly higher (p0.001) than that in the nifedipine group, which was little changed at 6 months. This discrepancy was derived from the significant increase of hyperemic velocity in the valsartan group, from 36.6+/-17.3 cm/s to 41.1+/-12.7 cm/s at 2 months, and to 48.1+/-20.2 cm/s at 6 months. We concluded that the ARB valsartan not only reduced high blood pressure but improved CFVR in hypertensive patients. However, these effects were not seen with the CCB nifedipine.

モノエタノールアミンあるいはジエタノ一ルアミンとプロムアセトアルデヒドージメチルアセタールおよびジエチルアセタールを原料として,種々の条件下にβ-オキシエチルアミノアセトアルデヒドージメチルアセタール(IVa),ビス-(β-オキシエチル)-アミノアセトアルデヒドージメチルアセタール(VIIIa)およびジエチルアセタール(VIIIb)を新しく合成し,さらにモルホリンの新しい誘導体,2-オキシモルホリン(V),2-メトキシモルホリン(VIa),2-エトキシ-4-ニトロソモルホリン(VIIb),2-メトキシ-4-(β-オキシエチル)-モルホリン(IXa),2-エトキシ-4-(β-オキシエチル)-モルホリン(IXb),2-メトキシ-4-(β-クロルエチル)-モルホリン(Xa),2-エトキシ-4-(β-クロルエチル)-モルホリン(Xb)を合成するとともに既知化合物であるβ-オキシエチルアミノァセトアルデヒドージエチルアセタール(IVb)と2-エトキシモルホリン(VIb)を従来と異なる方法で合成した。