Activation of ATM-c-IAP1 Pathway Mediates the Protective Effects of Estradiol in Human Vascular Endothelial Cells Exposed to Intermittent Hypoxia.

PURPOSE: Chronic intermittent hypoxia (CIH) contributes to the increased risk of cardiovascular diseases in obstructive sleep apnea (OSA). We previously reported the anti-apoptotic effects of estradiol (E2) on IH-exposed human umbilical vein endothelial cells (HUVECs). Herein, we employed a proteomic analysis to elucidate the mechanisms of the protective effects of E2 under IH exposure. METHODS: HUVECs were divided into three groups: control, IH, and IH+E2 group. Isobaric tags for relative and absolute quantification (iTRAQ) were performed to compare protein profiles among the groups. Some of the identified proteins were validated by Western blotting. RESULTS: A total of 185 proteins were differentially expressed in the IH+E2 group compared to the IH group. Bioinformatics analysis indicated that the effects of E2 may be linked to the regulation of cellular stress response. Among the differentially expressed proteins, we identified that serine-protein kinase ataxia telangiectasia mutated (ATM) and its downstream target, cellular inhibitor of apoptosis protein 1 (c-IAP1), were up-regulated by E2. We also observed that E2 decreased the level of cleaved caspase-3 and inhibited cell apoptosis in IH-exposed HUVECs. The inhibition of ATM abolished the anti-apoptotic effect of E2. CONCLUSION: The ATM-c-IAP1 pathway is involved in the cardioprotective effects of E2 in HUVECs exposed to IH.

Estradiol Regulates Txnip and Prevents Intermittent Hypoxia-Induced Vascular Injury.

Chronic intermittent hypoxia (IH) contributes to obstructive sleep apnea (OSA)-related cardiovascular diseases through increasing oxidative stress. It has been widely recognized that estradiol decreases the risk for cardiovascular disease, but the estrogen replacement therapy is limited for its side effects. Thioredoxin (Trx) and its endogenous inhibitor, thioredoxin-interacting protein (Txnip), are associated with the protective effect of estradiol in some conditions. In this study, we aimed to explore whether estradiol could protect against IH-induced vascular injury, and the possible effect of Trx-1/Txnip in this process. Forty-eight adult female C57/BL6J mice were randomly divided into 4 groups, ovariectomy combined with IH group, sham operation combined with IH group, IH group and the control group. The mice treated with IH for 8 hrs/day, and 28 days. IH induced the injury of aorta, and ovariectomized mice were more prone to the IH-induced aortic injury, with higher level of oxidative stress. In vitro, estradiol increased Trx-1 level, but decreased the level of Txnip and oxidative stress in human umbilical vein endothelial cells (HUVECs) treated with IH for 16 hrs. Knock-down of Txnip by specific siRNA rescued oxidative stress and apoptosis. In conclusion, estradiol protects against IH-induced vascular injury, partially through the regulation of Trx-1/Txnip pathway.

[Analysis of Human Platelet Antigen-1 System Alloantibodies Using Recombinant GPIIIa Fragments Coupled to Luminex Beads].

OBJECTIVE: To detect platelet anti-HPA-1a and -1b antibodies using recombinant GPIIIa fragments coupled to Luminex beads. METHODS: The sensitivity of 2 techniques, monoclonal antibody specific immobilization of platelet antigen (MAIPA) and Luminex bead assay, was compared using 12 twofold-serial dilutions (from neat to 1 in 2048) of an anti-HPA-1a WHO international standard. The specificity of Luminex assay to identify anti-HPA-1a and -1b antibodies was assessed using 8 negative or positive controls and 36 blinded samples provided by WHO Platelet Workshop. RESULTS: The sensitivity of MAIPA and Luminex bead assay to detect anti-HPA-1a was dilution 1/64 (i.e. 1.56 IU/ml) and far more than dilution 1/2048 (i.e. 0.049 IU/mL), respectively. The Luminex bead assay could specifically identify negative and positive controls of anti-HPA-1a and -1b. All results of 36 blinded samples by Luminex assay were accordant to reference results except one sample which contained high concentration antithetical antibody and resulted in false positive of anti-HPA-1b. Cross-reactivity was also not observed with the samples containing HLA, ABO or other platelet antibodies. CONCLUSION: The Luminex beads coupled with recombinant GPIIIa fragments can be used to detect HPA-1 system antibodies with sufficient sensitivity and specificity, that is suitable for the detection of platelet alloantibodies in clinical alloimmune thrombocytopenia.

Factors affecting blood pressure profile in pre and postmenopausal women with obstructive sleep apnea hypopnea syndrome.

PURPOSE: Obstructive sleep apnea hypopnea syndrome (OSAHS) is an independent risk factor for development of hypertension. Epidemiological surveys have found that risk of cardiovascular diseases increased in postmenopausal women. However, it is not well known about the profiles of hypertension of women with OSAHS in their different reproductive stages. This study aimed to investigate the difference of blood pressure profile between pre and postmenopausal women with OSAHS. METHODS: Women who were tested by polysomnography (PSG) (n = 207) in Ruijin Hospital were recruited in the study. The subjects were divided into two groups of premenopausal women (24 with and 30 without OSAHS), and two groups of postmenopausal women (116 with and 37 without OSAHS). Among the groups, the differences of demographic and PSG variables were compared. The factors influencing blood pressure were further studied. RESULTS: The prevalence of hypertension (56.9 %) in postmenopausal OSAHS patients was higher than the other three groups. Among OSAHS patients, both average systolic blood pressure (SBP) and diastolic blood pressure (DBP) of postmenopausal women were higher than those of premenopausal ones [(129.9 ± 16.16 mmHg vs. 123.7 ± 18.89), (84.96 ± 9.88 mmHg vs. 78.81 ± 10.34), P = 0.05, P < 0.01, respectively], with the elevation of DBP being more pronounced. For premenopausal women, body mass index (BMI) was the only factor affecting blood pressure (p < 0.05); in postmenopausal women, BMI was a factor affecting SBP, while BMI and apnea hypopnea index (AHI) affecting DBP (P < 0.05). CONCLUSION: Blood pressure profile of postmenopausal women with OSAHS was affected by both BMI and AHI. But those of premenopausal ones were predominantly related to BMI.

[Establishment of method detecting CD36 expression on human platelet and its application].

The individual with the deficiency of CD36 antigen on platelet displayed the risk of anti-CD36 immune reaction induced by transfusion, which is one of the reasons for platelet transfusion refractoriness (PTR). This study was purposed to detect the expression level of CD36 antigen on platelet by flow cytometry among apheresis platelet donors of Hangzhou area, and the frequency of CD36 deficiency was analyzed. Platelet-rich plasma (PRP) was separated from fresh anticoagulant whole blood by centrifugation, then the platelets were washed and adjusted to 1×10(6). The platelets were incubated with FITC-labeled CD36 and PE-labeled CD41 monoclonal antibodies, then the expression level of CD36 was detected by flow cytometry. The CD36 expression on monocytes for the samples of CD36-deficiency on the platelets was further analyzed. The results showed that 7 samples with CD36 antigen deficiency were found in 192 apheresis platelet donors. The frequency of CD36 deficiency was 3.6% and all of them were typeII deficiency. The significant difference of CD36 antigen expression was observed in the platelet donors of Hangzhou population, among them 59 individuals with low expressed CD36 antigen and 126 individuals with highly expressed CD36 antigen were found according to the geometric mean fluorescence intensity. It is concluded that the CD36 antigen deficient phenotype existed in the population, these data will provide the information for research of the CD36 antigen distribution and help to solve the platelet transfusion refractoriness.

[Rare blood group screening by serological and molecular methods in Zhejiang Han population].

This study was aimed to investigate the distribution of rare blood group in Zhejiang Han population. The H(-) (H system), GPA(-) and s(-) (MNS), Rhnull, Rhmod, D--, CCDEE, CCdEE (variations of Rh), GPC(-) (Gerbich), i(+) (I), Lu(b-) (Lutheran), Js(b-) and k(-) (Kell), Fy(a-) (Duffy), Ok(a-) (Ok), Di(b-) (Diego) phenotypes were screened by serological or molecular methods. Jk (a-b-) phenotype was detected by urea hemolytic test. The results showed that one Di (a+b-) individual was found in 1618 blood donors, three Fy (a-b+) individuals in 1007 donors and one CCdEE individual in 633 Rh negative donors. No Jk (a-b-), H(-), GPA(-), s(-), GPC(-), i(+) (adult), Lu(b-), k(-), Js(b-), Lu(b-) and Ok(a-) phenotypes were found in this large scale survey. It is concluded that Di (a+b-), Fy (a-b+), CCdEE phenotypes are confirmed in the blood donors and this study provides the distribution data of erythrocyte rare blood group in Zhejiang Han population.

[Identification of an ABx09 phenotype of ABO subtype].

OBJECTIVE: To analyze the molecular basis for an individual with ABx09 phenotype of ABO subtype. METHODS: The ABO group antigens on red blood cells of the proband were identified by monoclonal antibodies, and the ABO antibody in serum was detected by standard A, B, O cells. The exons 1 to 7 of ABO gene were amplified by polymerase chain reaction (PCR) respectively and the PCR products were sequenced directly. The amplified products for exons 5 to 7 were also cloned by TOPO TA cloning sequencing kit to split the two alleles apart, selected colonies were sequenced bidirectionally for exons 5 to 7 of the ABO gene. The samples of the proband's parents were collected, then serological test of the blood group and sequence analysis for exons 6 and 7 of ABO gene were preformed. RESULTS: Both A and B antigens were detected on red blood cells of the proband and there was anti-B antibody in the serum. There was no G deletion at position 261, while 297AG in exon 6, 467CT, 526CG, 657CT, 703GA, 796CA, 803GC, 889GA and 930GA heterozygote in exon 7 were detected by direct DNA sequencing, which can be assigned for A102Bx09 genotype. After cloning and sequencing, two alleles A102 and Bx09 were obtained. The sequence of Bx09 had one nucleotide changes (G to A) at position 889 compared with that of B101, which resulted in an amino acid change of Glu to Lys at 297 position. The Bx09 in the proband was inherited from her mother by family investigation. CONCLUSION: G to A at nt889 of alpha-1,3 galactosyltransferasegene can result in Bx09 phenotype, with the presence of anti-B antibody in serum.

[C742T mutation of α1, 3 N-acetyl-D-galactosaminyltransferase gene is responsible for A2 subgroup].

The objective of this study was to analyze the molecular genetic basis for 2 individuals with A2B phenotype of ABO subtype. The ABO group antigens on red blood cells were identified by monoclonal antibodies and the ABO antibodies in serum were detected by the standard A, B, O cells. The exon 5 to exon 7 coding region of ABO gene was amplified by polymerase chain reaction (PCR) and the PCR product was sequenced directly after the enzymes digested. The amplified product was also cloned by TOPO TA cloning sequencing kit to split the 2 alleles apart and chosen colonies were sequencing bidirectionally for exon 6 to 7 of ABO gene. The results showed that both A and B antigen were identified on red blood cells of the individuals and there was anti-A1 antibody in their serum. There was no 261G deletion and showed 297A/G, 467C/T, 526C/G, 657C/T, 703G/A, 742C/T, 796C/A, 803G/C, 930G/A heterozygotes by direct DNA sequencing. After cloning and sequencing, it was obtained the B101 and one novel A allele. The novel allele has one nucleotide change at 742 position C to T compared with A102, which results in an amino acid Arg to Cys at 248 position and was nominated as A213. It is concluded that C742T mutation of the α1, 3 N-acetyl-D-galactosaminyl-transferase gene can lead to A2 phenotype and with anti-A1 antibody in serum.

[Para-Bombay phenotype caused by combined heterozygote of two bases deletion on fut1 alleles].

This study was purposed to investigate the molecular basis of a para-Bombay phenotype for screening and identification of rare blood group. ABO and H phenotypes of the proband were identified by serological techniques. The exon 6 to exon 7 of ABO gene and full coding region of α-1,2-fucosyltransferase (fut1) gene of the proband were analyzed by polymerase chain reaction and direct sequencing of the amplified fragments. The haplotype of compound heterozygote of fut1 was also identified by cloning sequencing. The results indicated that a rare para-Bombay phenotype was confirmed by serological techniques. Two deletion or insertion variant sites near nucleotide 547 and 880 were detected in fut1 gene. The results of cloning sequence showed that one haplotype of fut1 gene was two bases deletion at 547-552 (AGAGAG→AGAG), and another one was two bases deletion at position 880-882 (TTT→T). Both two variants caused a reading frame shift and a premature stop codon. It is concluded that a rare para-Bombay phenotype is found and confirmed in blood donor population. The molecular basis of this individual is compound heterozygote of two bases deletion on fut1 gene which weaken the activity of α-1, 2-fucosyltransferase.

[Study on polymorphism of membrane glycoprotein genes related to human platelet alloantigens].

OBJECTIVE: To investigate the polymorphisms of platelet membrane glycoprotein genes related to human platelet alloantigen (HPA)-1 to 17w. METHODS: The DNA segments of platelet membrane glycoprotein genes related to HPA-1 to 17w were amplified using author's designed primers. The amplification products were purified and directly sequenced to identify the HPA genotype and glycoprotein gene polymorphisms. RESULTS: Thirteen new single nucleotide polymorphisms (SNPs) and a micro-satellite sequence were found in the glycoprotein genes from the 112 random samples, in which two SNPs (1333G/A and 1960G/A) in ITGB3 gene result in two amino acid change (V419M and E628K) on glycoprotein GPIIIa. CONCLUSION: New variants in platelet membrane glycoprotein genes were identified, which may lead to structure change of platelet membrane glycoprotein and affect the accuracy of partial HPA genotyping method.

[Cloning and sequence analysis of 5'untranslated region of human ABO gene].

OBJECTIVE: To clone and investigate the polymorphism of the 5'-untranslated region (5'-UTR) of the human ABO gene, in order to provide the basis for exploring the transcriptional regulation of the human ABO histo-blood group genes. METHODS: ABO phenotypes of 30 unrelated healthy blood donors were determined by serological technique, their genotypes were analyzed by sequencing the exons 6 and 7 of the ABO gene. Nearly 5 kb of the 5'-UTR of ABO gene was obtained by PCR amplification and sequencing was performed bidirectionally. Haplotypes of samples with heterozygous sites in the 5'-UTR of ABO gene were separated and analyzed after cloning. RESULTS: Twenty polymorphic sites were identified in these samples where ABO genotypes were consistent with serological phenotypes. It included sixteen nucleotide sequence variations, one 8 bp deletion, one 6 bp deletion/insertion, one 36 bp insertion and one 43 bp repeats. Among them, 11 were novel polymorphic sites. Seven different haplotypes of 5'-UTR of ABO gene were defined to the cis/trans linkage of those mutations. CONCLUSION: There were polymorphisms in the 5'-UTR of ABO gene and the nucleotide sequences near the proximal promoter were conservative.

[In vitro expression activity of α-(1,2) fucosyltransferase with 35C > T and 682A > G mutations].

In order to explore the effects of 35C > T and 682A > G mutations on the activity of alpha-(1,2) fucosyltransferase, the coding region of fut1 gene was amplified by polymerase chain reaction (PCR) from genomic DNA. PCR product was ligated into expression vector using TOPO TA cloning kit to obtain the recombinant plasmids. The recombinant plasmids were transfected into COS-7 cells by liposome method. After screening by using G418, H antigen expression on the COS-7 was tested by flow cytometry and fut1 mRNA was detected by real-time PCR. The results indicated that three kinds of recombinant plasmids pcDNA3.1/V5-His-wild (35C + 682A), pcDNA3.1/V5-His-35T and pcDNA3.1/V5-His-35T-682G were successfully constructed. After transfection, the H antigen expressed on membrane of COS-7 cells at the second day, with the maximum level of expression at the fourth day. When compared with pcDNA3.1/V5-His-wild transfected cells, the H antigen expression level of the 35T and 682G + 35T recombinant plasmids in the transfected cells was 52.7% and 13.3% on the fourth day, respectively. Although the level of fut1 mRNA decreased with prolonging of time, the mRNA expressed on the pcDNA3.1/V5-His-35T-682G transfected cells reached to 14% of the wild plasmids on the first day. It is concluded that 682A > G mutation obviously reduces the activity of alpha-(1,2) fucosyltransferase, while 35C > T mutation leads to partial reduction of H antigen in vitro expression.