[The impact of LDL-C/HDL-C ratio on severity of coronary artery disease and 2-year outcome in patients with premature coronary heart disease: results of a prospective, multicenter, observational cohort study].

Objective: To explore the relationship between low density lipoprotein cholesterol (LDL-C)/high density lipoprotein cholesterol (HDL-C) ratio with the severity of coronary artery disease and 2-yeat outcome in patients with premature coronary heart disease. Methods: This prospective, multicenter, observational cohort study is originated from the PROMISE study. Eighteen thousand seven hundred and one patients with coronary heart disease (CHD) were screened from January 2015 to May 2019. Three thousand eight hundred and sixty-one patients with premature CHD were enrolled in the current study. According to the median LDL-C/HDL-C ratio (2.4), the patients were divided into two groups: low LDL-C/HDL-C group (LDL-C/HDL-C≤2.4, n=1 867) and high LDL-C/HDL-C group (LDL-C/HDL-C>2.4, n=1 994). Baseline data and 2-year major adverse cardiovascular and cerebrovascular events (MACCE) were collected and analyzed in order to find the differences between premature CHD patients at different LDL-C/HDL-C levels, and explore the correlation between LDL-C/HDL-C ratio with the severity of coronary artery disease and MACCE. Results: The average age of the low LDL-C/HDL-C ratio group was (48.5±6.5) years, 1 154 patients were males (61.8%); the average age of high LDL-C/HDL-C ratio group was (46.5±6.8) years, 1 523 were males (76.4%). The number of target lesions, the number of coronary artery lesions, the preoperative SNYTAX score and the proportion of three-vessel coronary artery disease in the high LDL-C/HDL-C group were significantly higher than those in the low LDL-C/HDL-C group (1.04±0.74 vs. 0.97±0.80, P=0.002; 2.04±0.84 vs. 1.85±0.84, P<0.001; 13.81±8.87 vs. 11.70±8.05, P<0.001; 36.2% vs. 27.4%, respectively, P<0.001). Correlation analysis showed that there was a significant positive correlation between LDL-C/HDL-C ratio and preoperative SYNTAX score, the number of coronary artery lesions, the number of target lesions and whether it was a three-vessel coronary artery disease (all P<0.05). The 2-year follow-up results showed that the incidence of MACCE was significantly higher in the high LDL-C/HDL-C group than that in the low LDL-C/HDL-C group (6.9% vs. 9.1%, P=0.011). There was no significant difference in the incidence of all-cause death, cardiac death, myocardial infarction, stroke, revascularization and bleeding between the two groups. Cox multivariate regression analysis showed that the LDL-C/HDL-C ratio has no correlation with 2-year MACCE, death, myocardial infarction, revascularization, stroke and bleeding events above BARC2 in patients with premature CHD. Conclusion: High LDL-C/HDL-C ratio is positively correlated with the severity of coronary artery disease in patients with premature CHD. The incidence of MACCE of patients with high LDL-C/HDL-C ratio is significantly higher during 2 years follow-up; LDL-C/HDL-C ratio may be an indicator for evaluating the severity of coronary artery disease and long-term prognosis in patients with premature CHD.

[Impact of prolonging dual antiplatelet therapy on long-term prognosis of elderly patients with coronary heart disease complicated with diabetes mellitus undergoing drug-eluting stent implantation].

Objective: To explore and compare the effect of standard or prolonged dual antiplatelet therapy (DAPT) on the long-term prognosis of elderly patients with coronary heart disease complicated with diabetes mellitus after drug-eluting stent (DES) implantation. Methods: Consecutive patients with diabetes mellitus, ≥65 years old, underwent DES implantation, and had no adverse events within 1 year after operation underwent percutaneous coronary intervention (PCI) from January to December 2013 in Fuwai Hospital were enrolled in this prospective cohort study. These patients were divided into three groups according to DAPT duration: standard DAPT duration group (11 ≤ DAPT duration≤ 13 months) and prolonged DAPT duration group (1324 months). All the patients were followed up at 1, 6 months, 1, 2 and 5 years in order to collect the incidence of major adverse cardiovascular and cerebrovascular events (MACCE), and type 2 to 5 bleeding events defined by the Federation of Bleeding Academic Research (BARC). MACCE were consisted of all cause death, myocardial infarction, target vessel revascularization or stroke. The incidence of clinical adverse events were compared among 3 different DAPT duration groups, and Cox regression model were used to analyze the effect of different DAPT duration on 5-year long-term prognosis. Results: A total of 1 562 patients were enrolled, aged (70.8±4.5) years, with 398 female (25.5%). There were 467 cases in standard DAPT duration group, 684 cases in 1324 months group. The patients in standard DAPT duration group and the prolonged DAPT duration groups accounted for 29.9% (467/1 562) and 70.1% (1 095/1 562), respectively. The 5-year follow-up results showed that the incidence of all-cause death in 1324 month group(4.1%(17/411) vs. 8.6%(40/467),P=0.008) were significantly lower than in standard DAPT group. The incidence of myocardial infarction in 1324 month group were 19.3% (90/467), 12.3% (84/684), 20.2% (83/411), respectively, P<0.001). There was no significant difference in the incidence of stroke and bleeding events among the three groups (all P>0.05). Multivariate Cox analysis showed that compared with the standard DAPT group, prolonged DAPT to 13-24 months was negatively correlated with MACCE (HR=0.601, 95%CI 0.446-0.811, P=0.001), all-cause death (HR=0.568, 95%CI 0.357-0.903, P=0.017) and myocardial infarction (HR=0.353, 95%CI 0.179-0.695, P=0.003). DAPT>24 months was negatively correlated with all-cause death (HR=0.687, 95%CI 0.516-0.913, P=0.010) and positively correlated with revascularization (HR=1.404, 95%CI 1.116-1.765, P=0.004). There was no correlation between prolonged DAPT and bleeding events. Conclusions: For elderly patients with coronary heart disease complicated with diabetes mellitus underwent DES implantation, and had no MACCE and bleeding events within 1 year after operation, appropriately prolonging of the DAPT duration is related to the reduction of the risk of cardiovascular adverse events. Patients may benefit the most from the DAPT between 13 to 24 months. In addition, prolonging DAPT duration does not increase the incidence of bleeding events in this patient cohort.

[Effect of platelet reactivity on clinical events in patients using bivalirudin in selective percutaneous coronary intervention].

Objective: To investigate the effect of platelet reactivity and other clinical factors on the postoperative 1-year adverse clinical events in patients who underwent selective percutaneous coronary intervention (PCI) anticoagulated with bivalirudin. Methods: This is a multicenter, retrospective and observational study, enrolling 632 patients at high risk of bleeding adjudicated by operators who underwent selective PCI anticoagulated with bivalirudin and had preoperative thrombelastography (TEG) test results in Fuwai Hospital, Northern Theater General Hospital and Xinxiang Central Hospital between January 2017 and August 2018. Platelet reactivity was tested by TEG and adenosine-induced maximal amplitude (MAADP) was recorded. According to MAADP patients were divided into three groups: low on-treatment platelet reactivity (LTPR) group (MAADP<31 mm, n=229), normal on-treatment platelet reactivity (NTPR) group (31 mm≤MAADP≤47 mm, n=207) and high on-treatment platelet reactivity (HTPR) group (MAADP>47 mm, n=196). The endpoints consisted of major adverse cardiovascular and cerebrovascular events (MACCE) and bleeding events. The definition of MACCE was the composite of all-cause mortality, myocardial infarction, intrastent thrombosis, stroke and revascularization. Bleeding events were defined by bleeding academic research consortium (BARC) type 2, 3 and 5 bleeding. Using multivariate Cox regression to analyze the factors of MACCE and bleeding events in patients underwent selective PCI anticoagulated with bivalirudin. Results: A total of 632 patients were finally enrolled in the study with age of (68.3±10.0) years and there were 423 (66.9%) males. All of 632 patients finished one-year follow-up, and 48 (7.6%) patients occurred MACCE and 11 (1.7%) patients occurred bleeding events. There was not statistically significant difference in the incidence of MACCE (8.3% (19/229) vs. 6.3% (13/207) vs.8.2% (16/196), P=0.68) and bleeding events (1.8% (4/229) vs. 2.9% (6/207) vs. 0.5% (1/196), P=0.17) in LTPR, NTPR and HTPR group. Multivariate Cox regression showed that HTPR was not the independent factor of MACCE (HR=1.25, 95%CI 0.67-2.30, P=0.49), and the history of peripheral vessel disease was the independent risk factor of MACCE (HR=2.47, 95%CI 1.19-5.11, P=0.02). LTPR was not the independent factor of bleeding events (HR=1.35, 95%CI 0.39-4.66, P=0.64), and the independent factors of bleeding events were history of peripheral vessel disease (HR=3.95, 95%CI 1.03-15.22, P=0.05) and hemoglobin (HR=0.96, 95%CI 0.93-0.99, P=0.01). Conclusions: In patients undergoing selective PCI anticoagulated with bivalirudin, there is no significant association between platelet reactivity and postoperative 1-year MACCE or bleeding events. History of peripheral vessel disease is an independent risk factor of MACCE, and history of peripheral vessel disease and decreased hemoglobin are independent risk factors of bleeding events.

[Related factors and the long-term outcome after percutaneous coronary intervention of premature acute myocardial infarction].

Objective: To explore the related factors of premature acute myocardial infarction(AMI), and to compare the the long-term outcomes in patients with and without premature AMI after percutaneous coronary intervention (PCI). Methods: This study was a prospective cohort study.From January 2013 to December 2013, 10 724 consecutive patients with coronary heart disease undergoing PCI in Fuwai Hospital were enrolled. Among them 1 920 patients with the diagnosis of AMI were divided into two groups: premature AMI (man≤50 years old, woman≤60 years old) and non-premature AMI. The baseline characteristics were collected, and multivariate logistic regression was uesed to analysis the related factors of premature AMI. The clinical outcomes, including the major adverse cardiovascular and cerebrovascular events(MACCE) which was the composite of cardiac death, myocardial infarction, revascularization, stroke and stent thrombosis, as well as bleeding events, during hospitalization, at 2 years and 5 years follow-up were analyzed. Results: A total of 1 920 AMI patiens were included(age was (56.5±11.3) years old)，with 1 612(84.0%) males. There were statistically significant differences between the two groups in gender, body mass index, blood lipid, complications, inflammatory markers, etc (all P<0.05). Multivariate logistic regression analysis showed body mass index(OR=1.06, 95%CI 1.01-1.10, P<0.01), triglyceride(OR=1.47, 95%CI 1.14-1.90, P<0.01), serum uric acid level(OR=1.02, 95%CI 1.01-1.04, P<0.01), high density lipoprotein cholesterol level(OR=0.33, 95%CI 0.14-0.78, P=0.01) and history of hypertension(OR=0.72, 95%CI 0.56-0.93, P=0.01) were independent related factors of premature AMI. The incidence of all-cause death and cardiac death were lower during hospitalization, at 2 years and 5 years follow-up in the premature AMI group than in non-premature AMI group(all P<0.05). In the premature AMI group, the incidence of MACCE and stroke was lower, with more bleeding events in 5 years follow-up(all P<0.05). Conclusions: Metabolic abnormalities, including high BMI, high triglyceride level and high serum uric acid, low high-density lipoprotein cholesterol level are the related factor of premature AMI. The incidence of ischemic events in patients with premature AMI is lower, while the incidence of bleeding events is higher than non-premature AMI patients.

Zinc-regulated genes in Saccharomyces cerevisiae revealed by transposon tagging.

The biochemistry of human nutritional zinc deficiency remains poorly defined. To characterize in genetic terms how cells respond to zinc deprivation, zinc-regulated genes (ZRG's) were identified in yeast. Gene expression was probed using random lacZ reporter gene fusions, integrated by transposon tagging into a diploid genome as previously described. About half of the genome was examined. Cells exhibiting differences in lacZ expression on low or moderate ( approximately 0. 1 vs. 10 microm) zinc media were isolated and the gene fusions were sequenced. Ribonuclease protection assays demonstrated four- to eightfold increases for the RNAs of the ZAP1, ZRG17 (YNR039c), DPP1, ADH4, MCD4, and YEF3B genes in zinc-deficient cells. All but YEF3B were shown through reporter gene assays to be controlled by a master regulator of zinc homeostasis now known to be encoded by ZAP1. ZAP1 mutants lacked the flocculence and distended vacuoles characteristic of zinc-deficient cells, suggesting that flocculation and vacuolation serve homeostatic functions in zinc-deficient cells. ZRG17 mutants required extra zinc supplementation to repress these phenotypes, suggesting that ZRG17 functions in zinc uptake. These findings illustrate the utility of transposon tagging as an approach for studying regulated gene expression in yeast.

Spectral and kinetic properties of the Fet3 protein from Saccharomyces cerevisiae, a multinuclear copper ferroxidase enzyme.

High affinity iron uptake in Saccharomyces cerevisiae requires Fet3p. Fet3p is proposed to facilitate iron uptake by catalyzing the oxidation of Fe(II) to Fe(III) by O2; in this model, Fe(III) is the substrate for the iron permease, encoded by FTR1. Here, a recombinant Fet3p has been produced in yeast that, lacking the C-terminal membrane-spanning domain, is secreted directly into the growth medium. Solutions of this Fet3p at >1 mg/ml have the characteristic blue color of a type 1 Cu(II)-containing protein, consistent with the sequence homology that placed this protein in the class of multinuclear copper oxidases that includes ceruloplasmin. Fet3p has an intense absorption at 607 nm (epsilon = 5500 M-1 cm-1) due to this type 1 Cu(II) and a shoulder in the near UV at 330 nm (epsilon = 5000 M-1 cm-1) characteristic of a type 3 binuclear Cu(II) cluster. The EPR spectrum of this Fet3p showed the presence of one type 1 Cu(II) and one type 2 Cu(II) (A parallel = 91 and 190 x 10(-4) cm-1, respectively). Copper analysis showed this protein to have 3.85 g atom copper/mol, consistent with the presence of one each of the three types of Cu(II) sites found in multinuclear copper oxidases. N-terminal analysis demonstrated that cleavage of a signal peptide occurred after Ala-21 in the primary translation product. Mass spectral and carbohydrate analysis of the protein following Endo H treatment indicated that the preparation was still 15% (w/w) carbohydrate, probably O-linked. Kinetic analysis of the in vitro ferroxidase reaction catalyzed by this soluble Fet3p yielded precise kinetic constants. The Km values for Fe(II) and O2 were 4.8 and 1.3 microM, respectively, while kcat values for Fe(II) and O2 turnover were 9.5 and 2.3 min-1, consistent with an Fe(II):O2 reaction stoichiometry of 4:1.

The yeast CLC chloride channel functions in cation homeostasis.

A defect in the yeast GEF1 gene, a CLC chloride channel homolog leads to an iron requirement and cation sensitivity. The iron requirement is due to a failure to load Cu2+ onto a component of the iron uptake system, Fet3. This process, which requires both Gef1 and the Menkes disease Cu2+-ATPase yeast homolog Ccc2, occurs in late- or post-Golgi vesicles, where Gef1 and Ccc2 are localized. The defects of gef1 mutants can be suppressed by the introduction of Torpedo marmorata CLC-0 or Arabidopsis thaliana CLC-c and -d chloride channel genes. The functions of Gef1 in cation homeostasis provide clues to the understanding of diseases caused by chloride channel mutations in humans and cation toxicity in plants.

Restriction of copper export in Saccharomyces cerevisiae to a late Golgi or post-Golgi compartment in the secretory pathway.

The CCC2 gene in the yeast Saccharomyces cerevisiae encodes a P-type ATPase (Ccc2p) required for the export of cytosolic copper to the extracytosolic domain of a copper-dependent oxidase, Fet3p. Ccc2p appears to be both a structural and functional homolog of ATPases impaired in two human disorders of intracellular copper transport, Menkes disease and Wilson disease. In the present work, three approaches were used to determine the locus of Ccc2p-dependent copper export within the secretory pathway. First, like ccc2 mutants, sec mutants blocked in the secretory pathway at steps prior to and including the Golgi complex failed to deliver radioactive copper to Fet3p. Second, also like ccc2 mutants, vps33 and certain other mutants with defects in post-Golgi sorting exhibited phenotypes traceable to deficient copper delivery to Fet3p. These findings were sufficient to explain the respiratory deficiency of these mutants. Third, immunofluorescence microscopy revealed that Ccc2p was distributed among several punctate foci within wild-type cells, consistent with late Golgi or post-Golgi localization. Thus, copper export by Ccc2p appears to be restricted to a late or post-Golgi compartment in the secretory pathway.

Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae.

Wilson disease is a disorder of copper metabolism characterized by hepatic cirrhosis and neuronal degeneration due to inherited mutations in a gene encoding a putative copper-transporting P-type ATPase. Polyclonal antisera generated against the amino terminus of the Wilson protein detected a specific 165-kDa protein in HepG2 and CaCo cell lysates. Further analysis revealed that this protein is synthesized as a single-chain polypeptide and localized to the trans-Golgi network under steady state conditions. An increase in the copper concentration resulted in the rapid movement of this protein to a cytoplasmic vesicular compartment. This copper-specific cellular redistribution of the Wilson protein is a reversible process that occurs independent of a new protein synthesis. Expression of the wild-type but not mutant Wilson protein in the ccc2Delta strain of Saccharomyces cerevisiae restored copper incorporation into the multicopper oxidase Fet3p, providing direct evidence of copper transport by the Wilson protein. Taken together these data reveal a remarkable evolutionary conservation in the cellular mechanisms of copper metabolism and provide a unique model for the regulation of copper transport into the secretory pathway of eucaryotic cells.

Identification and functional expression of HAH1, a novel human gene involved in copper homeostasis.

To search for a mammalian homologue of ATX1, a human liver cDNA library was screened and a cDNA clone was isolated, which encodes a protein with 47% amino acid identity to Atx1p including conservation of the MTCXGC copper-binding domain. RNA blot analysis using this cDNA identified an abundant 0.5-kilobase mRNA in all human tissues and cell lines examined. Southern blot analysis using this same clone indicated that the corresponding gene exists as a single copy in the haploid genome, and chromosomal localization by fluorescence in situ hybridization detected this locus at the interface between bands 5q32 and 5q33. Yeast strains lacking copper/zinc superoxide dismutase (SOD1) are sensitive to redox cycling agents and dioxygen and are auxotrophic for lysine when grown in air, and expression of this human ATX1 homologue (HAH1) in these strains restored growth on lysine-deficient media. Yeast strains lacking ATX1 are deficient in high affinity iron uptake and expression of HAH1 in these strains permits growth on iron-depleted media and results in restoration of copper incorporation into newly synthesized Fet3p. These results identify HAH1 as a novel ubiquitously expressed protein, which may play an essential role in antioxidant defense and copper homeostasis in humans.

A permease-oxidase complex involved in high-affinity iron uptake in yeast.

Iron must cross biological membranes to reach essential intracellular enzymes. Two proteins in the plasma membrane of yeast--a multicopper oxidase, encoded by the FET3 gene, and a permease, encoded by the FTR1 gene--were shown to mediate high-affinity iron uptake. FET3 expression was required for FTR1 protein to be transported to the plasma membrane. FTR1 expression was required for apo-FET3 protein to be loaded with copper and thus acquire oxidase activity. FTR1 protein also played a direct role in iron transport. Mutations in a conserved sequence motif of FTR1 specifically blocked iron transport.

The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake.

The CCC2 gene of the yeast Saccharomyces cerevisiae is homologous to the human genes defective in Wilson disease and Menkes disease. A biochemical hallmark of these diseases is a deficiency of copper in ceruloplasmin and other copper proteins found in extracytosolic compartments. Here we demonstrate that disruption of the yeast CCC2 gene results in defects in respiration and iron uptake. These defects could be reversed by supplementing cells with copper, suggesting that CCC2 mutant cells were copper deficient. However, cytosolic copper levels and copper uptake were normal. Instead, CCC2 mutant cells lacked a copper-dependent oxidase activity associated with the extracytosolic domain of the FET3-encoded protein, a ceruloplasmin homologue previously shown to be necessary for high-affinity iron uptake in yeast. Copper restored oxidase activity both in vitro and in vivo, paralleling the ability of copper to restore respiration and iron uptake. These results suggest that the CCC2-encoded protein is required for the export of copper from the cytosol into an extracytosolic compartment, supporting the proposal that intracellular copper transport is impaired in Wilson disease and Menkes disease.

The Saccharomyces cerevisiae copper transport protein (Ctr1p). Biochemical characterization, regulation by copper, and physiologic role in copper uptake.

The CTR1 gene of Saccharomyces cerevisiae encodes a protein required for high affinity copper uptake. The protein is expressed on the plasma membrane, is heavily glycosylated with O-linkages, and exists as an oligomer in vivo. The transcript abundance is strongly regulated by copper availability, being induced by copper deprivation and repressed by copper excess. Regulation occurs at very low, nontoxic levels of available copper and is independent of ACE1, the trans-inducer of yeast metallothionein. Expression of Ctr1p is limiting for copper uptake, since overexpression from a 2 mu high copy number plasmid increases copper uptake. Mutations in CTR1 result in altered cellular responses to extracellular copper, demonstrating a physiologic role for CTR1 in the delivery of copper to the cytosol. A copper-dependent reporter gene construct, CUP1-lacZ, is not expressed in CTR1 mutants to the same level as in wild-type strains, and Cu,Zn superoxide dismutase activity is deficient in these mutants. The growth arrest that occurs in CTR1 mutants grown aerobically in copper-deficient media is attributable to the defect in Cu,Zn superoxide dismutase activity.

Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for copper in iron transport.

We report the identification and characterization of CTR1, a gene in the yeast S. cerevisiae that encodes a multispanning plasma membrane protein specifically required for high affinity copper transport into the cell. The predicted protein contains a methionine- and serine-rich domain that includes 11 examples of the sequence Met-X2-Met, a motif noted in proteins involved in bacterial copper metabolism. CTR1 mutants and deletion strains have profound deficiency in ferrous iron uptake, thus revealing a requirement for copper in mediating ferrous transport into the cell. Genetic evidence suggests that the target for this requirement is the FET3 gene (detailed in a companion study), predicted to encode a copper-containing protein that acts as a cytosolic ferro-oxidase. These findings provide an unexpected mechanistic link between the uptake of copper and iron.

Cibacron blue-induced enhancement of agonist binding to cholecystokinin (CCK) receptors in solubilized pancreatic membranes.

The pancreatic receptor for cholecystokinin (CCK) typifies many G protein-coupled receptors in that its ability to bind agonist can be reduced by GTP or the solubilization of membranes. We found, however, that a dye, cibacron blue, caused up to a 6-fold increase in binding of the CCK receptor agonist, 125I-CCK-8, to rat pancreatic membranes solubilized with digitonin. Binding optimally enhanced in this manner was comparable to binding of 125I-CCK-8 to native membranes with respect to time-course, maximal amount bound, reversibility, and sensitivity to inhibition by various CCK receptor ligands. Increases in affinity of the CCK receptor for CCK-8 accounted fully for the enhancement of binding of 125I-CCK-8. Cibacron blue did not enhance binding of 125I-CCK-8 to native membranes, and also failed to enhance binding of the CCK receptor antagonist, [3H]L-364,718, to solubilized or native membranes. The ability of cibacron blue to enhance binding of agonist but not that of antagonist suggests that this dye may mimic or perhaps stimulate the effects of G protein on CCK receptors. Such a phenomenon may provide new insights into the mechanisms by which receptors distinguish agonists from antagonists.