In vivo transfection of cis element "decoy" against nuclear factor-kappaB binding site prevents myocardial infarction.

The transcriptional factor nuclear factor-kappaB (NFkappaB) plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes that might be involved in myocardial damage after ischemia and reperfusion. Therefore, we hypothesized that synthetic double-stranded DNA with high affinity for NFkappaB could be introduced in vivo as "decoy" cis elements to bind the transcriptional factor and to block the activation of genes mediating myocardial infarction, thus providing effective therapy for myocardial infarction. Treatment before and after infarction by transfection of NFkappaB decoy, but not scrambled decoy, oligodeoxynucleotides before coronary artery occlusion or immediately after reperfusion had a significant inhibitory effect on the area of infarction. Here, we report the first successful in vivo transfer of NFkappaB decoy oligodeoxynucleotides to reduce the extent of myocardial infarction following reperfusion, providing a new therapeutic strategy for myocardial infarction.

Engineered metalloregulation in enzymes.

Protein engineering of metal-dependent enzyme activity is now possible due to the wealth of information available about metalloproteins. The results emerging from these studies provide insight into our understanding of the chemistry of metals in macromolecular environments as well as the biology of metal-protein interactions.

Ciliary neurotrophic factor prevents neuronal degeneration and promotes low affinity NGF receptor expression in the adult rat CNS.

Recombinant human ciliary neurotrophic factor (CNTF) was infused for 2 weeks into the lateral ventricle of fimbria-fornix transected adult rats, and its effects were compared with those of purified mouse nerve growth factor (NGF). We provide evidence that CNTF can prevent degeneration and atrophy of almost all injured medial septum neurons (whereas NGF protects only the cholinergic ones). CNTF is also involved in up-regulation of immunostainable low affinity NGF receptor (LNGFR) in cholinergic medial septum and neostriatal neurons and in a population of lateral septum neurons. In contrast to NGF, CNTF did not stimulate choline acetyltransferase in the lesioned septum and normal neostriatum (pointing to different mechanisms for the regulation of choline acetyltransferase and LNGFR), cause hypertrophy of septal or neostriatal cholinergic neurons, or cause sprouting of LNGFR-positive (cholinergic) septal fibers.

Inhibition of beta-amyloid formation identifies proteolytic precursors and subcellular site of catabolism.

Cerebral deposition of beta-amyloid protein is a pathological feature central to Alzheimer's disease. Production of beta-amyloid by proteolytic processing of the beta-amyloid precursor protein (beta APP) is a critical initial step in beta-amyloidogenesis. We use an inhibitor of beta APP processing to block beta-amyloid peptide formation. Application of the inhibitor to cultured cells results in an accumulation of proteolytic intermediates of beta APP, enabling a precursor-product relationship between beta APP carboxy-terminal fragments and beta-amyloid peptides to be demonstrated directly. In the presence of inhibitor, these amyloidogenic carboxy-terminal fragments can be degraded to nonamyloidogenic products. The catabolism of beta APP carboxy-terminal intermediates and the formation of beta-amyloid peptides are likely to involve an early endosomal compartment as the subcellular site of processing.

Usefulness of the renal resistive index to predict an increase in urinary albumin excretion in patients with essential hypertension.

Microalbuminuria is a risk factor for cardiovascular events and death in hypertensive patients. Patients who are expected to increase albuminuria need strict blood pressure control. In the present study, we assessed the association between the renal resistive index (RI) and future increases in albuminuria in patients with essential hypertension. Sixty-six patients with essential hypertension were included in the study. Univariate and multivariate logistic regression analyses were used to identify the factors, including renal RI, that were significant independent determinants of increased in urinary albumin excretion (UAE), defined as an increase of >50% in the urinary albumin-to-creatinine ratio over 2 years. Receiver operator characteristics curve analysis was used to select the optimal cut-off point that predicted an increase in UAE. RI was the only significant variable that predicted the increase in UAE, with the optimal cut-off value of renal RI that predicted this increase being 0.71 (sensitivity 52.4% and specificity 84.4%). Renal RI is associated with the future increase in albuminuria in patients with essential hypertension.

Hypoxia-induced endothelial apoptosis through nuclear factor-kappaB (NF-kappaB)-mediated bcl-2 suppression: in vivo evidence of the importance of NF-kappaB in endothelial cell regulation.

The transcription factor nuclear factor-kappaB (NF-kappaB) plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes involved in endothelial activation. Although recent reports have documented the contribution of NF-kappaB to apoptosis, it is still controversial. Especially, the role of NF-kappaB in endothelial apoptosis is largely unknown. Hypoxia significantly induced human aortic endothelial cell death and apoptosis in a time-dependent manner (P<0.01), accompanied by NF-kappaB activation. Decrease in total cell number and increase in apoptotic cells induced by hypoxia were significantly attenuated by NF-kappaB decoy, but not by scrambled decoy, oligodeoxynucleotides (ODNs) (P<0.01). Increase in DNA fragmentation induced by hypoxia was also significantly inhibited by NF-kappaB decoy ODNs as compared with scrambled decoy ODNs (P<0.01). Moreover, transfection of NF-kappaB decoy ODNs resulted in a significant decrease in caspase-3-like activity, which is a common pathway for apoptosis, compared with scrambled decoy ODNs. Importantly, transfection of NF-kappaB decoy ODNs significantly increased protein of bcl-2, an inhibitor of apoptosis, and did not alter bax, a promoter of apoptosis, thereby resulting in a significant increase in the ratio of bcl-2 to bax (P<0.01). bcl-2 mRNA was also decreased by hypoxia, whereas transfection of NF-kappaB decoy ODNs significantly attenuated decrease in bcl-2 mRNA. These results demonstrate that activation of NF-kappaB by hypoxia induced endothelial apoptosis in a bcl-2-dependent manner. The importance of NF-kappaB in endothelial apoptosis was confirmed by the observation that pyrrolidine dithiocarbamate, a potent NF-kappaB inhibitor, prevented endothelial apoptosis, caspase 3-like activity, and bcl-2 downregulation induced by hypoxia. To test this hypothesis in vivo, we transfected NF-kappaB decoy ODNs into rat intact carotid artery after reperfusion injury. Reperfusion injury was associated with a significant increase in endothelial apoptosis at 24 hours, whereas NF-kappaB decoy ODN treatment markedly decreased terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive endothelial cells at 24 hours after reperfusion (P<0.01). Here, using synthetic double-stranded DNA with high affinity for NF-kappaB as a decoy approach, we demonstrated that activation of NF-kappaB by hypoxia caused aortic endothelial cell death and apoptosis through the suppression of bcl-2. NF-kappaB-mediated endothelial apoptosis induced by hypoxia may be involved in the pathogenesis of endothelial dysfunction observed in cardiovascular ischemic diseases.

Deletion allele of angiotensin-converting enzyme gene increases risk of essential hypertension in Japanese men : the Suita Study.

BACKGROUND: The Framingham Study recently revealed that the homozygous deletion polymorphism of the angiotensin-converting enzyme gene (ACE DD) is associated with increased risk for essential hypertension in a male-specific manner. However, this association has not been confirmed in races other than whites. METHODS AND RESULTS: Using a large number of Japanese subjects (n=5014) that were randomly selected from the general population (the Suita Study), we examined the association between ACE DD and hypertension. The frequency of DD (17.1%) in hypertensive men was significantly higher (P<0.0015) than that (11.8%) in other mildly hypertensive or normotensive men, and the estimated odds prevalence for hypertension (DD vs II) was 1.75 (95% CI 1.21 to 2.53). In contrast, no significant association was confirmed in women (OR 1.17, 95% CI 0.79 to 1.72). CONCLUSIONS: Despite the lower frequency of the DD genotype in Japanese than in whites, the ACE gene polymorphism was associated with increased risk for hypertension, suggesting that this polymorphism is a mild but certain genetic risk factor for essential hypertension in men.

Ribozyme oligonucleotides against transforming growth factor-beta inhibited neointimal formation after vascular injury in rat model: potential application of ribozyme strategy to treat cardiovascular disease.

BACKGROUND: Because the mechanisms of atherosclerosis or restenosis after angioplasty have been postulated to involve an increase in transforming growth factor (TGF)-beta, a selective decrease in TGF-beta may have therapeutic value. Thus, we used the ribozyme strategy to actively cleave the targeted gene to selectively inhibit TGF-beta expression. METHODS AND RESULTS: We constructed ribozyme oligonucleotides (ONs) targeted to the sequence of the TGF-beta gene that shows 100% homology among the human, rat, and mouse species. The specificity of ribozyme against TGF-beta gene was confirmed by selective inhibition of TGF-beta mRNA in cultured vascular smooth muscle cells as well as balloon-injured blood vessels in vivo. Importantly, the marked decrease in TGF-beta resulted in significant inhibition of neointimal formation after vascular injury in a rat carotid artery model (P:<0.01), whereas DNA-based control ONs and mismatched ribozyme ONs did not have any inhibitory effect on neointimal formation. Inhibition of neointimal formation was accompanied by (1) a reduction in collagen synthesis and mRNA expression of collagen I and III and (2) a significant decrease in DNA synthesis as assessed by proliferating cell nuclear antigen staining. Moreover, we modified ribozyme ONs containing phosphorothioate DNA and RNA targeted to the TGF-beta gene. Of importance, modified ribozyme ONs showed a further reduction in TGF-beta expression. CONCLUSIONS: Overall, this study provides the first evidence that selective blockade of TGF-beta resulted in inhibition of neointimal formation, accompanied by a reduction in collagen synthesis and DNA synthesis in a rat model. We anticipate that modification of ribozyme ON pharmacokinetics will facilitate the potential clinical utility of the ribozyme strategy.

Potential contribution of a novel antifibrotic factor, hepatocyte growth factor, to prevention of myocardial fibrosis by angiotensin II blockade in cardiomyopathic hamsters.

BACKGROUND: Because hepatocyte growth factor (HGF) prevented and/or regressed fibrosis in liver and pulmonary injury models, HGF may play an important role in the pathogenesis of fibrotic cardiovascular disease. Because angiotensin (Ang) II significantly decreased local HGF production, we performed (1) in vitro experiments using fibroblasts and (2) administration of an ACE inhibitor (temocapril) and an Ang II type 1 receptor antagonist (CS-866) to cardiomyopathic hamsters. METHODS AND RESULTS: In human fibroblasts, HGF significantly increased the production of matrix metalloprotease-1 (MMP-1) and urokinase plasminogen activator, whereas HGF also significantly attenuated the reduction of MMP-1 activity induced by Ang II. In contrast, HGF significantly decreased transforming growth factor (TGF)-beta mRNA stimulated by Ang II, whereas HGF also decreased basal TGF-beta protein level without affecting growth. Similarly, in rat cardiac fibroblasts, HGF inhibited the expression and production of TGF-beta, whereas HGF upregulated its specific receptor, c-met. Conversely, in vivo experiments revealed that administration of temocapril and CS-866 to cardiomyopathic hamsters resulted in a significant decrease in fibrotic area and increase in cardiac HGF concentration and mRNA (P<0.01), whereas cardiac concentration and mRNA of HGF were significantly decreased in cardiomyopathic hamsters. In contrast, mRNA expression of collagen III was markedly decreased by treatment with temocapril and CS-866. CONCLUSIONS: Here, we demonstrated that Ang II blockade prevented myocardial fibrosis in the cardiomyopathic hamster, accompanied by a significant increase in cardiac HGF. Overall, increase in local HGF expression may participate in the prevention of myocardial injury by Ang II blockade through its antifibrotic action.

Transfection of antisense p53 tumor suppressor gene oligodeoxynucleotides into rat carotid artery results in abnormal growth of vascular smooth muscle cells.

BACKGROUND: Although loss of activity of an antioncogene, the p53 tumor suppressor gene product, has been postulated in the pathogenesis of human restenosis, little is known about the role of p53 in the regulation of vascular smooth muscle cell (VSMC) growth. In this study, to clarify the role of p53 in the pathogenesis of restenosis, we examined transfection of antisense p53 oligodeoxynucleotides (ODN) into VSMC in vitro and rat carotid artery in vivo. METHODS AND RESULTS: The specificity of antisense p53 ODN was confirmed by a significant decrease in p53 protein. Transfection of antisense p53 ODN into VSMC resulted in a significant increase in DNA synthesis and cell number as compared with sense and scrambled ODN (P<0.01). Importantly, transfection of antisense p53 ODN into rat intact carotid artery resulted in a significant increase in the ratio of neointima to medial area at 2 and 4 weeks after transfection, accompanied by a significant decrease in p53 protein (P<0.01). Moreover, cotransfection of wild-type p53 plasmid completely abolished neointimal formation induced by antisense p53 ODN. The sustained effect of a single antisense ODN administration was confirmed by the kinetics of ODN in the vessel wall with the use of FITC-labeled ODN. CONCLUSIONS: Overall, the present study demonstrated that loss of p53 by antisense p53 ODN resulted in an abnormal VSMC growth in vitro and in vivo. These results demonstrated the potential contribution of p53 to the pathogenesis of restenosis.

Quantitative ultrasonic tissue characterization can identify high-risk atherosclerotic alteration in human carotid arteries.

BACKGROUND: Recently, ultrasonic tissue characterization of the composition of plaques has been performed in a quantitative fashion on the basis of integrated backscatter (IBS) analysis, but most of those studies have used high-frequency ultrasound to obtain microscopic images. METHODS AND RESULTS: We performed B-mode measurement and IBS signal analysis with acoustic densitometry with a 7.5-MHz linear-array transducer in freshly excised human aortas (n=58) (normal, atheromatous, and fibrous tissue) obtained at autopsy. Atheromatous and fibrous tissue had a similar intima-media thickness (IMT), but the IBS value in atheromatous specimens was lower than that in fibrous specimens. We further applied this method to human carotid ultrasonography. The subjects were young (80 regions), middle aged with 1 or no coronary risk factors (low risk) (120 regions), middle aged with >/=2 coronary risk factors (high risk) (240 regions), or elderly (80 regions) or were patients with myocardial infarction (MI) with multivessel disease (90 regions). The IMT was similar in middle-aged, elderly, and MI subjects. In contrast, the IBS value was significantly higher in elderly subjects and lower in high-risk middle-aged and MI subjects compared with that in low-risk middle-aged subjects. The percent of regions diagnosed as atheromatous (IBS less than mean minus 2-SD value of IBS in young subjects) was 11% in low-risk middle-aged subjects, 29% in high-risk middle-aged subjects, and 63% in the MI group. CONCLUSIONS: In conjunction with conventional B-mode imaging, IBS analysis with carotid ultrasonography appeared to provide prognostic information to identify a high-risk group with systemic atherosclerosis, which could lead to coronary heart disease in individuals with early-stage disease.

Human prostacyclin synthase gene and hypertension : the Suita Study.

BACKGROUND: Prostacyclin (prostaglandin I(2)) is a strong vasodilator that inhibits the growth of vascular smooth muscle cells and is also the most potent endogenous inhibitor of platelet aggregation. Therefore, it has been considered to play an important roles in cardiovascular disease. On the basis of the hypothesis that variations of the prostacyclin synthase gene may also play an important role in human cardiovascular disease, we performed a screening for variations in the human prostacyclin synthase gene. METHODS AND RESULTS: We have detected a repeat polymorphism in the promoter region of the human prostacyclin synthase gene. The number of 9-bp (CCGCCAGCC) repeats in the promoter region, which encodes a tandem repeat of Sp1 transcriptional binding sites, varied between 3 and 7 in Japanese subjects. Luciferase reporter analysis indicated that the alleles of 3 and 4 repeats (R3 and R4, respectively) had less promoter activity in cultured human umbilical vein endothelial cells. We then investigated the possible association of this repeat polymorphism with blood pressure in a large population-based sample (the Suita Study), which consisted of 4971 Japanese participants. Multivariate models indicated that participants with the R3R3, R3R4, or R4R4 genotype (SS genotype, n=80) had significantly higher systolic pressure (P=0.0133) and pulse pressure (P=0.0005). The odds ratio of hypertension (140/90 mm Hg) for the SS genotype was 1.942 (95% confidence interval 3.20 to 1.19, P=0.0084). CONCLUSIONS: Repeat polymorphism of the human prostacyclin synthase gene seems to be a risk factor for higher pulse pressure and is consequently a risk factor for systolic hypertension in the Japanese population.

Potential role of an endothelium-specific growth factor, hepatocyte growth factor, on endothelial damage in diabetes.

Endothelial cells are known to secrete various antiproliferative and vasodilating factors. Although injury of endothelial cells has been postulated as an initial trigger of the progression of atherosclerosis in patients with diabetes, the mechanisms of endothelial injury in diabetes are not yet clarified. Therefore, it is important to know the effects of high glucose on the factors that may influence endothelial cell growth. A novel member of endothelium-specific growth factors, hepatocyte growth factor (HGF), is produced in vascular cells. To investigate the effects of high glucose on vascular cells, we examined 1) the effects of high glucose on endothelial cell and vascular smooth muscle cell (VSMC) growth and 2) the effects of high glucose on local HGF production in endothelial cell and VSMC. Treatment of human aortic endothelial cell with a high concentration of D-glucose, but not mannitol and L-glucose, resulted in a significant decrease in cell number. Interestingly, addition of recombinant HGF attenuated high D-glucose-induced endothelial cell death. Therefore, we measured local HGF secretion of endothelial cell. Importantly, local HGF production was significantly decreased by high D-glucose treatment. In contrast, high D-glucose treatment resulted in a significant increase in the number of human aortic VSMCs, whereas local HGF production was significantly decreased in accordance with increase in D-glucose concentration. No significant changes in numbers were observed in VSMC treated with high mannitol and L-glucose. We also studied the mechanisms of local HGF suppression by high D-glucose. High D-glucose treatment stimulated transforming growth factor-beta (TGF-beta) concentration in endothelial cell and VSMC. Decreased local vascular HGF production was abolished by addition of anti-TGF-beta antibody. As TGF-beta inhibited local HGF production in endothelial cell and VSMC, increased TGF-beta induced by high D-glucose may suppress local HGF production. This study demonstrated that high D-glucose induced endothelial cell death, stimulated VSMC growth, and decreased local HGF production through the stimulation of TGF-beta production both in endothelial cell and VSMC. Overall, decrease in a local endothelial stimulant, HGF, by high D-glucose may be a trigger of endothelial injury in diabetes, potentially resulting in the progression of atherosclerosis.

Additive effects of nicorandil on coronary blood flow during continuous administration of nitroglycerin.

OBJECTIVES: We examined whether patients with ischemic heart disease (IHD) should be treated with nicorandil, an adenosine triphosphate-sensitive potassium channel opener, in addition to the regular use of nitrates. BACKGROUND: It has been reported that nicorandil possibly has additive effects on nitroglycerin (NTG) treatment for angina, but the mechanism is not clear. METHODS: We directly measured anterograde coronary blood flow (CBF) with a Doppler guide wire to examine the effects of intravenous administration of NTG (0.3 mg) and nicorandil (6 mg) during continuous administration of NTG at a sufficient dose (25 microg/min) in subjects with normal and stenotic coronary arteries. RESULTS: Additional systemic administration of NTG decreased anterograde CBF (normal -19.7%; stenotic -21.2%). In contrast, nicorandil increased anterograde CBF in both normal (54.6%) and stenotic (89.6%) coronary arteries, without the coronary steal phenomenon. There was a tendency toward nicorandil-dilated diameters in the patients with stenotic arteries (p = 0.06). There were no effects of additional administration on pulmonary artery wedge pressure. There was no difference in changes in heart rate and mean aortic blood pressure between NTG and nicorandil therapy. CONCLUSIONS: These results suggest that in patients treated with nitrates, additional administration of nicorandil is more useful, in terms of increasing CBF, than additional administration of nitrates. Adjunctive use of nicorandil with nitrates may provide the further benefit of myocardial protection and may improve the prognosis of patients with IHD.

Crystallographic analysis of trypsin-G226A. A specificity pocket mutant of rat trypsin with altered binding and catalysis.

The crystal structure of trypsin-G226A has been determined, in the presence of benzamidine, to a resolution of 1.75 A with an R-factor of 14.6%. The mutation was designed to alter substrate specificity by disrupting arginine binding, but was previously found to disrupt catalysis to a greater extent than binding. The arginine analog, benzamidine, has rotated 40 degrees and 49 degrees and translated 1.1 A in the specificity pocket, relative to the position in wild-type trypsin. The salt-bridge between the amidinium group of benzamidine and the carboxylate of D189 as well as four other hydrogen bonds have been replaced by a set of six new hydrogen bonds. Based on these interactions, computer modeling of an arginine substrate demonstrates that arginine terminal nitrogen atoms can occupy the new benzamidine nitrogen positions with torsion angle adjustments and without short contacts. In the secondary orientation, arginine substrates appear to be forced out of alignment with the active site. This may account for the larger drop in kcat with arginine relative to lysine substrates. A second possible cause of the altered activity is a change of the enzyme structure with concomitant loss of activity. No evidence of such a change is seen in the co-ordinates or temperature factors of the trypsin-G226A-benzamidine complex. A226 disrupts mainly the co-ordinates of amino acids with which it has direct contacts such that the effects of the mutation are absorbed locally.

Crystal structure of rat trypsin-S195C at -150 degrees C. Analysis of low activity of recombinant and semisynthetic thiol proteases.

The X-ray crystal structure of trypsin-S195C, a rat anionic trypsin mutant in which the active site serine has been replaced by cysteine, was determined at -150 degrees C and room temperature to 1.6 A resolution, R = 15.4% and 1.8 A resolution, R = 15.0%, respectively. Cryo-crystallography was employed to improve the quality of the diffraction data and the resulting structure by eliminating radiation damage and decreasing atomic thermal motion. The average temperature factor decreased by 10 A2 relative to that of the room temperature structure. No radiation-induced decay of the data was detected. The side-chains of the catalytic cysteine and histidine of trypsin-S195C are found with 25% occupancy in secondary orientations rotated 104 degrees and 90 degrees out of the active site, respectively. These alterations, as well as more subtle changes in the active site may be caused by the oxidation of the catalytic sulfur to sulfenic acid. The position of the carbonyl carbon of the tetrahedral intermediate analog, p-amidinophenylpyruvic acid, modeled into trypsin-S195C, is 1.1 A from the catalytic sulfur. The large size and altered approach of the catalytic sulfur to substrates could account for the observed low catalytic activity relative to wild-type trypsin. In addition to the benzamidine in the specificity pocket, two additional binding sites for benzamidine are characterized. One of these mediates an intermolecular contact that appears to maintain the crystal lattice.

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.

Angiotensin I-converting enzyme gene polymorphism is associated with myocardial infarction, but not with retinopathy or nephropathy, in NIDDM.

OBJECTIVE: To clarify the relationship between the angiotensin I-converting enzyme (ACE) gene polymorphism and diabetic micro- and macroangiopathy in patients with non-insulin-dependent diabetes mellitus (NIDDM). RESEARCH DESIGN AND METHODS: We examined 267 NIDDM patients with various stages of diabetic retinopathy, 61 patients with myocardial infarction (MI), and 136 patients without MI. An insertion/deletion polymorphism of the ACE gene was typed by polymerase chain reaction. RESULTS: Although no association was found between ACE gene polymorphism and diabetic retinopathy or nephropathy, this polymorphism was associated with MI in the patients with NIDDM. Homozygotes for the deletion polymorphism (DD genotype) were found more frequently in diabetic patients with MI (31.1%) than in diabetic patients without ischemic heart disease (16.9%), with a relative risk of 2.22 (95% confidence interval 1.11-4.46, P = 0.024). CONCLUSION: These data indicate that ACE gene polymorphism is associated with MI, but not with retinopathy or nephropathy, in patients with NIDDM and suggest that the ACE gene confers susceptibility to diabetic macroangiopathy but not to microangiopathy.

Low potentiality of angiotensin-converting enzyme gene insertion/deletion polymorphism as a useful predictive marker for carotid atherogenesis in a large general population of a Japanese city: the Suita study.

BACKGROUND AND PURPOSE: Some previous studies, almost all western, have investigated whether there is a relationship between the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) and carotid atherosclerosis. The results, however, have not been consistently positive. Further, there have been few investigations based on a large, general population. Therefore, the present study aimed to clarify whether ACE gene deletion polymorphism was associated with carotid atherosclerosis in a large Japanese general population with a more homogeneous genetic background than Caucasian populations. METHODS: Subjects aged 30 to 86 years were randomly selected from Suita City, located in Osaka, the second largest urban area of Japan, and included 1894 men and 2137 women. With the aid of high-resolution ultrasonography, carotid atherosclerosis was evaluated using our atherosclerotic indexes of intimal-medial thickness (IMT), plaque number (PN), plaque score (PS), and percentage of stenosis of the carotid artery assessed using high-resolution B-mode ultrasonography. ACE gene I/D polymorphism was detected by polymerase chain reaction. RESULTS: There were no significant differences among the ACE genotypes for age and conventional cardiovascular risk factors, except for systolic blood pressure (SBP) and the percentage of hypertension in men. The values of IMT, PN, and PS as carotid atherosclerotic indexes were not significantly different among genotypes for either sex. After adjusting for age, body mass index, smoking habit, high-density lipoprotein cholesterol, triglycerides, presence of hypertension, presence of diabetes mellitus, and presence of hyperlipidemia, the estimated ORs for the presence of IMT >/=1.10 mm (defined as thickened IMT), according to ACE genotype (DD versus II, DD+ID versus II, and DD versus ID+II), for men were 0.80 (95% CI 0.60 to 1.23), 0.89 (0.62 to 1.29), and 0.89 (0.70 to 1.28), respectively. On the other hand, the ORs for women after the same adjustment were 0.92 (95% CI 0.58 to 1.35), 0.93 (0.59 to 1.45), and 0.91 (0.59 to 1.27), respectively. CONCLUSIONS: Our present data suggest that ACE I/D polymorphism is not potentially a useful predictive marker for carotid atherogenesis when investigated in a large and homogeneous general Japanese population of 4031 subjects, a finding similar to that in a Caucasian population study, the Perth Carotid Ultrasound Disease Assessment Study, an Australian study based on a general population using 1111 subjects.