Koichi Yuki

Ultrasonic assessment of vascular complications in coronary angiography and angioplasty after transradial approach.

The transradial approach has currently been accepted as an alternative entry method for coronary angiography and angioplasty. Vascular complications of this method were evaluated by 2-dimensional echo and color Doppler ultrasonic studies in 162 patients before, early (2+/-2 [mean+/-SD] days), and late (95+/-29 days) after catheterization. Mean age was 64+/-10 years, and 103 were men. Coronary angioplasty was performed in 59 patients (79 lesions) with angiographic success in 92%. Early after the procedure, segmental stenosis was noted in 35 patients (22%) and no flow in 15 patients (9%). Late after the procedure, segmental stenosis was noted in 2, diffuse stenosis in 36 (22%), and no flow in 8 (5%) patients. The cessation of radial artery pulse was unpalpable in only 2% of cases, whereas radial flow by color Doppler was undetectable in 9% early after the procedure. Late after the procedure, recanalization was observed in 60% of these occluded cases. Thirty-three of 86 patients (38%) with no flow or diffuse stenosis had radial artery diameters smaller than the sheath diameter, and 11 of 76 patients (14%) had radial artery diameters larger than the sheath diameter (p <0.01). Multivariate analysis revealed risk factors for vascular complications: (1) Radial artery diameter before the procedure was one of the significant and independent determinants of no flow both early (p = 0.06) and late (p = 0.004) after the procedure. (2) The difference in radial artery diameter and sheath size was related to the occurrence of diffuse stenosis late after the procedure (p = 0.003). (3) Diabetes mellitus was related to no flow (p = 0.05) or diffuse stenosis (p = 0.11) late after the procedure. Thus, ultrasonic evaluation of the radial artery was useful in selecting both an access route and an appropriate size of the sheath to determine early and late vascular complications.

Decreased serum concentrations of nitric oxide metabolites among Chinese in an endemic area of chronic arsenic poisoning in inner Mongolia

Prolonged exposure to arsenic results in peripheral and cardiovascular manifestations, as does impaired production of endothelial nitric oxide (NO). In vitro studies have indicated that endothelial cells undergo damage by arsenic. However, no information has been available on the relationship between NO synthesis and chronic arsenic poisoning in humans. The present study was designed to reveal this question. The subjects were 33 habitants who continued to drink well water containing high concentrations of inorganic arsenic (mean value = 0.41 microg/ml) for about 18 years in Inner Mongolia, China, and 10 other people who lived in this area but exposed to minimal concentrations of arsenic (mean value = 0.02 microg/ml) were employed as controls. Mean blood concentration of total arsenic was six times higher in exposed subjects than controls; 42.1 vs. 7.3 ng/ml, p <.001. Mean serum concentration of nitrite/nitrate, stable metabolites of endogenous NO, was lower in arsenic-exposed subjects than in controls: 24.7 vs. 51.6 microM, p<.001. In total samples, an inverse correlation with serum nitrite/nitrate levels was strong for blood inorganic arsenic (r = -0.52, p <.001) and less strong for its metabolites, monomethyl arsenic (r = -0.45, p<.005) and dimethyl arsenic (r = -0.37, p<.05). Furthermore, serum nitrite/nitrate concentration was significantly correlated with nonprotein sulfhydryl level in whole blood (r = 0.58, p<.001). In an in vitro study, we demonstrated that inorganic arsenite or arsenate suppresses the activity of endothelial NO synthase in human umbilical vein endothelial cells. These results suggest that long-term exposure to arsenic by drinking well water possibly reduces NO production in endothelial cells, resulting in a decrease in reduced nitrite/nitrate concentrations. Peripheral vascular disorders caused by arsenic may be attributable in part to impairment of NO production in vivo.

Novel molecular mechanism of increased myocardial endothelin-1 expression in the failing heart involving the transcriptional factor hypoxia-inducible factor-1α induced for impaired myocardial energy metabolism

Background—Hypoxia-inducible factor (HIF)-1&agr; is an important transcriptional factor that activates the gene expression of glycolytic enzymes, which are activated as compensation for impaired &bgr;-oxidation of fatty acid in the failing heart. We reported that cardiac endothelin (ET)-1 expression is markedly increased in heart failure. The mechanism, however, is unknown. Because we found an HIF-1&agr; binding site in the 5′-promoter region of the ET-1 gene, we hypothesized that HIF-1&agr; is involved in this mechanism. Methods and Results—In rat cardiomyocytes, luciferase assay and electrophoretic mobility shift assay showed that HIF-1&agr; transcriptionally activates ET-1 gene expression by direct interaction with the predicted DNA binding site in the 5′-promoter region. HIF-1&agr; mRNA and ET-1 mRNA in the failing heart increased during the aggravation of heart failure in vivo in animal models, ie, rats with myocardial infarction and hamsters with cardiomyopathy. In cultured cardiomyocytes treated with a mitochondrial inhibitor, HIF-1&agr; mRNA and ET-1 mRNA were markedly increased with activated glycolysis, and antisense oligonucleotide for HIF-1&agr; largely inhibited the increased gene expression of ET-1. Conclusions—The present study revealed a novel molecular mechanism of upregulation of myocardial ET-1 in heart failure, indicating that induction of HIF-1&agr; to stimulate glycolysis as an adaptation in heart failure against impaired energy metabolism alternatively causes an elevation of cardiac ET-1 gene expression as a maladaptation.

Sevoflurane binds and allosterically blocks integrin lymphocyte function-associated antigen-1.

Background:Volatile anesthetics have been shown to modify immune cell functions via several mechanisms, some of which have been only partially elucidated. We demonstrated that isoflurane inhibits primary leukocyte integrin lymphocyte function-associated antigen-1 (LFA-1) by binding to the allosteric cavity critical for conformational activation to its high-affinity form. It remains to be determined whether the allosteric inhibition of LFA-1 by isoflurane can be generalized to other anesthetics such as sevoflurane. Methods:The effects of sevoflurane on the ability of LFA-1 to bind to its counter-ligand, intercellular adhesion molecule-1, was studied in leukocytes by flow cytometry. To examine whether sevoflurane acts directly on LFA-1, we measured ligand-binding using beads coated with purified LFA-1 protein. To distinguish between competitive versus allosteric inhibition, we analyzed the effects of sevoflurane on both wild-type and mutant-locked high-affinity LFA-1. One-way analysis of variance was employed for statistical analysis of the data. Nuclear magnetic resonance spectroscopy was used to identify sevoflurane binding site(s). Results:Sevoflurane at clinically relevant concentrations inhibited the ligand-binding function of LFA-1 in leukocytes as well as in cell-free assays (P < 0.05). Sevoflurane blocked wild-type but not locked high-affinity LFA-1, thereby demonstrating an allosteric mode of inhibition. Nuclear magnetic resonance spectroscopy revealed that sevoflurane bound to the allosteric cavity, to which LFA-1 allosteric antagonists and isoflurane also bind. Conclusions:This study suggests that sevoflurane also blocks the activation-dependent conformational changes of LFA-1 to the high-affinity form. The allosteric mode of action exemplified by sevoflurane and isoflurane via LFA-1 might represent one of the underlying mechanisms of anesthetic-mediated immunomodulation.

Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.

The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein.

An internal ligand-bound, metastable state of a leukocyte integrin, αXβ2

The crystal structure of a metastable, internal ligand-bound conformation of the αXβ2 integrin suggests it enables rapid equilibration between the bent-closed and extended-open conformational states.

Polyunsaturated fatty acids are incorporated into maturating male mouse germ cells by lysophosphatidic acid acyltransferase 3

Long‐chain polyunsaturated fatty acids (PUFAs) accumulate in mammalian testis during puberty and are essential for fertility. To investigate whether lysophospholipid acyltransferases determine the PUFA composition of testicular phospholipids during pubertal development, we compared their mRNA expression, in vitro activity, and specificity with the lipidomic profile of major phospholipids. The accumulation of PUFAs in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine correlated with an induced lysophosphatidic acid acyltransferase (LPAAT)3 mRNA expression, increased microsomal LPAAT3 activity, and shift of LPAAT specificity to PUFA‐coenzyme A. LPAAT3 was induced during germ cell maturation, as shown by immunofluorescence microscopy. Accordingly, differentiation of mouse GC‐2spd(ts) spermatocytes into spermatides up‐regulated LPAAT3 mRNA, increased the amount of polyunsaturated phospholipids, and shifted the specificity for the incorporation of deuterium‐labeled docosahexaenoic acid toward phosphatidylcholine and phosphatidylethanolamine. Stable knockdown of LPAAT3 in GC‐2spd(ts) cells significantly decreased microsomal LPAAT3 activity, reduced levels of polyunsaturated phosphatidylethanolamine species, and impaired cell proliferation/survival during geneticin selection. We conclude that the induction of LPAAT3 during germ cell development critically contributes to the accumulation of PUFAs in testicular phospholipids, thereby possibly affecting sperm cell production.—Koeberle, A., Shindou, H., Harayama, T., Yuki, K., Shimizu, T. Polyunsaturated fatty acids are incorporated into maturating male mouse germ cells by lysophosphatidic acid acyltransferase 3. FASEB J. 26, 169–180 (2012). www.fasebj.org

The volatile anesthetic isoflurane perturbs conformational activation of integrin LFA-1 by binding to the allosteric regulatory cavity

The molecular and structural basis of anesthetic interactions with conformations and functionalities of cell surface receptors remains to be elucidated. We have demonstrated that the widely used volatile anesthetic isoflurane blocks the activation‐dependent conformational conversion of integrin lymphocyte function associated antigen‐1 (LFA‐1), the major leukocyte cell adhesion molecule, to a high‐affinity configuration. Perturbation of LFA‐1 activation by isoflurane at clinically relevant concentrations leads to the inhibition of T‐cell interactions with target cells as well as ligand‐triggered intracellular signaling. Nuclear magnetic resonance spectroscopy reveals that isoflurane binds within a cavity in the LFA‐1 ligand‐binding domain, which is a previously identified drug‐binding site for allosteric small‐molecule antagonists that stabilize LFA‐1 in a low‐affinity conformation. These results provide a potential mechanism for the immunomodulatory properties of isoflurane.— Yuki, K., Astrof, N. S., Bracken, C., Yoo, R., Silkworth, W., Soriano, S. G., Shimaoka, M. The volatile anesthetic isoflurane perturbs conformational activation of integrin LFA‐1 by binding to the allosteric regulatory cavity. FASEB J. 22, 4109–4116 (2008)

Post-Operative Outcomes in Children With and Without Congenital Heart Disease Undergoing Noncardiac Surgery

BACKGROUND Significant advances have been made in the diagnosis and treatment of children with congenital heart disease (CHD), allowing for longer life expectancies and an increasing number who will require noncardiac surgery. OBJECTIVES This study sought to compare the incidence of mortality and major adverse post-operative outcomes following noncardiac surgery in children with and without CHD. METHODS Data from the 2012 pediatric database of the American College of Surgeons National Surgical Quality Improvement Program were analyzed. After propensity score matching, and stratification by severity of CHD, mortality and adverse post-operative outcomes were compared between controls and children with CHD. RESULTS Among the 51,008 children included in the database, 4,520 children with CHD underwent noncardiac surgery. After propensity score matching, we included 2,805 children with minor CHD, 1,272 with major CHD, and 417 with severe CHD. Children in each subgroup were matched and compared with controls without CHD who underwent noncardiac surgery of comparable complexity. The incidence of overall mortality was significantly higher in children with moderate (3.9%) and severe (8.2%) CHD compared with their controls (respectively, 1.7% [p < 0.001] and 1.2% [p = 0.001]). Both 30-day and overall mortality were significantly increased in children with severe CHD (odds ratio [OR]: 8.43, 95% confidence interval [CI]: 2.52 to 28.21; p < 0.001; OR: 7.32, 95% CI: 2.83 to 18.90; p < 0.001) compared with their matched controls. Overall mortality was also significantly increased in children with major CHD compared with their controls (OR: 2.28; 95% CI: 1.37 to 3.79; p = 0.002), whereas no difference was observed between children with minor CHD and their matched controls. CONCLUSIONS Children with major and severe CHD, undergoing noncardiac surgery, have an increased risk of mortality compared with children without CHD. Further studies need to identify the optimal environment for surgical procedures, develop trained multidisciplinary teams to care for children with CHD, and define management strategies for improving outcomes in this high-risk population.

Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1

We previously demonstrated that isoflurane targets lymphocyte function‐associated antigen‐1 (LFA‐1), a critical adhesion molecule for leukocyte arrest. However, it remains to be determined how isoflurane interacts with the full ectodomain LFA‐1 and modulates its conformation and function. Isoflurane binding sites on the full ectodomain LFA‐1 were probed by photolabeling using photoactivatable isoflurane (azi‐isoflurane). The adducted residues were determined by liquid chromatography/mass spectrometry analysis. Separately, docking simulations were performed to predict binding sites. Point mutations were introduced around isoflurane binding sites. The significance of isofluranes effect was assessed in both intracellular adhesion molecule‐1 (ICAM‐1) binding assays and epitope mapping of activation‐sensitive antibodies using flow cytometry. Two isoflurane binding sites were identified using photolabeling and were further validated by the docking simulation: one at the hydrophobic pocket in the ICAM‐1 binding domain (the αI domain); the other at the βI domain. Mutagenesis of the α′1 helix showed that isoflurane binding sites at the βI domain were significantly important in modulating LFA‐1 function and conformation. Epitope mapping using activation‐sensitive antibodies suggested that isoflurane stabilized LFA‐1 in the closed conformation. This study suggested that isoflurane binds to both the αI and βI domains allosteric to the ICAM‐1 binding site, and that isoflurane binding stabilizes LFA‐1 in the closed conformation.—Yuki, K., Bu, W., Xi, J., Sen, M., Shimaoka, M., Eckenhof, R.G. Isoflurane binds and stabilizes a closed conformation of the leukocyte function‐associated antigen‐1. FASEB J. 26, 4408–4417 (2012). www.fasebj.org