Human Annexin A5: from Biomarkers to Therapeutic Candidates / 中国生物化学与分子生物学报

Human annexin A5 (hAnxA5) as an important functional protein molecule in the human body, widely exists in human cells and body fluids. hAnxA5, a member of annexin group with complex structure, exhibits a variety of biological functions by reversibly and specifically binding phosphatidylserine (PS) in a calciumdependent manner and plays an important role in many human physiological processes. This paper has made induction and summary of the biochemical characteristics, mechanism, biological effects and important biomedical applications of hAnxA5. The hAnxA5 is present in the form of monomer and often exercise biological functions in a polymer. hAnxA5 affects the occurrence and development of pathological phenomena, such as vascular thrombosis disease, autoimmune disease, tumor disease, pulmonary fibrosis and lung injury, nonalcoholic fatty hepatitis, etc. As a biomarker, hAnxA5 has also been adopted in the study of diseases such as tumor, neurodegenerative diseases, heart failure, acute renal injury, asthma and so on. As novel drug candidates, hAnxA5 and its derivatives have been designed and applied to the therapeutic exploration of many kinds of diseases, especially for thrombotic diseases. There are also some gaps and shortcomings for hAnxA5 research. The in-depth of its research will not only expand the understanding of structure and functional relationships, but also promote its application in the field of biomedicine.

Regulatory effect of annexin A5 on glioma cell invasion andmigration and its mechanisms / 西安交通大学学报(医学版)

【Objective】 To investigate the regulatory effect of annexin A5 on glioma cell invasion and migration and its mechanism. 【Methods】 The expression of annexin A5 in 100 cases of glioma tissues and 20 cases of normal brain tissues was detected by immunohistochemistry. The expression of annexin A5 was downregulated by transfection with siRNA targeting annexin A5 (si-Annexin A5) in human glioma cell line (U251). The expression of annexin A5 was confirmed by RT-PCR and Western blot analysis. The proliferation ability of U251 cells was detected by MTT test and colony formation test, the apoptosis of U251 cells was detected by flow cytometry and Hoechst 33258 staining, and the migration and invasion ability of U251 cells was examined by wound healing test and Matrigel Transwell invasion test. The expressions of Raf, p-Raf, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, c-Myc and E-Cadherin in U251 cells were analyzed by Western blot. 【Results】 Compared with those of normal brain tissues, the mRNA and protein expression levels of Annexin A5 in glioma tissues increased by 2.45 times and 2.87 times, respectively (P<0.05). Pearson correlation analysis showed that with the increase of tumor grade, the positive rate of Annexin A5 gradually increased, and the tumor grade and positive rate were significantly positively correlated (r=1.000, P=0.000). The cell viability of U251 cells in the si-Annexin A5 group after 48 h and 72 h of culture was significantly reduced by 29.46% and 40.43%, respectively, compared with that in the control group (P<0.05). Compared with the control group, in the si-Annexin A5 group the colony formation rate was reduced by 68.58%, while the apoptosis rate was increased by 24.41 times (P<0.05); the cell migration rate and invasion rate were reduced by 65.35% and 68.80% (P<0.05). The protein expression of p-Raf, p-MEK1/2, p-ERK1/2 and c-Myc in the si-Annexin A5 group were significantly reduced by 54.67%, 70.37%, 60.26% and 54.95%, respectively, and that of E-Cadherin was increased by 3.58 times (P<0.05). 【Conclusion】 Downregulation of Annexin A5 inhibits the growth and motility of glioma cells and induces cell apoptosis by inhibiting the Raf/MEK/ERK signaling pathway.

Serum Annexin V and Anti-Annexin V levels and their relationship with metabolic parameters in patients with type 2 diabetes

SUMMARY BACKGROUND We investigated the serum annexin V and anti-annexin V levels and their relationship with metabolic parameters in patients recently diagnosed type 2 diabetic. METHODS A total of 143 patients recently diagnosed type 2 diabetes and 133 control subjects were included in the study. Body mass index (BMI), hs-CRP, HOMA-IR, carotid intima-media thickness, and serum levels of annexin V and anti-annexin V were investigated. RESULTS HOMA-IR, serum hs-CRP, and carotid intima-media thickness were found to be statistically significant. The Pearson correlation analysis revealed a statistically significant positive relationship between the carotid intima-media thickness and the annexin V level (r=0.29, p=0.006*). A statistically significant positive relationship was also detected between the Annexin V level and level of serum hs-CRP (r=0.29 p=0.006*). CONCLUSION A positive relationship was observed between the carotid intima-media thickness and annexin V at the end of our investigation. In this regard, we also believe that serum levels of annexin V may be increased for cardiovascular protection in the elevation of carotid intima-media thickness.

RESUMO OBJETIVO Investigar os níveis séricos de anexina V e antianexina V e sua relação com os parâmetros metabólicos em pacientes diabéticos tipo 2 recém-diagnosticados. MÉTODOS Foram incluídos no estudo 143 pacientes e 133 controles com diabetes tipo 2 recém-diagnosticado. O índice de massa corporal (IMC), PCR-as, Homa-IR, espessura íntima média carotídea e níveis séricos de anexina V e antianexina V foram investigados. RESULTADOS O Homa-IR, a PCR-s do soro e a espessura média da carótida foram estatisticamente significantes. A análise de correlação de Pearson revelou uma relação positiva estatisticamente significante entre a espessura média da carótida e anexina V (r=0,29; p=0,006 *). Foi também detectada uma relação positiva estatisticamente significativa entre o nível de anexina V e o nível sérico de PCR-as (r=0,29, p=0,006*). CONCLUSÃO Também foi observada uma relação positiva entre a espessura média da carótida e anexina V no final da nossa investigação. A esse respeito, também pensamos que os níveis séricos de anexina V podem ser aumentados para proteção cardiovascular na elevação da espessura média da carótida.

Niveles de anticuerpos anti-anexina A5 y manifestaciones de síndrome antifosfolípido primario obstétrico / Levels of anti-anexinal antibodies A5 and manifestations of obstetric primary antiphospholipid syndrome

El Síndrome Antifosfolípidos (SAF) describe un trastorno trombofílico autoinmune caracterizado por complicaciones obstétricas. La Anexina A5 (Anx A5) es una proteína que se estudia como un nuevo autoantígeno presente en el SAF, la presencia de autoanticuerpos frente a Anx A5 podría causar trombosis placentaria y pérdida del embarazo. El objetivo de este estudio fue analizar los niveles de IgG e IgM anti-Anx A5 en mujeres con SAF primario obstétrico y su asociación con diferentes complicaciones en una población de la ciudad de Córdoba. Se trabajó con muestras de pacientes puérperas que asistieron al Hospital Córdoba y al Hospital Materno Neonatal durante los años 2013-2017 con diagnóstico de SAF obstétrico y un grupo control formado por pacientes con embarazos normales. En la mayoría de las pacientes estudiadas, los niveles de IgG e IgM anti-Anx A5 se encontraron por debajo del rango de referencia, se mostró un aumento estadísticamente significativo de los niveles de IgG en pacientes con SAF respecto al grupo control. Pero no existieron asociaciones específicas entre los niveles de anticuerpo y los tres tipos de manifestaciones clínicas presentes en los criterios de clasificación. Estos hallazgos podrían sugerir una relación entre los anticuerpos anti-Anx A5 con el SAF obstétrico.

Antiphospholipid Syndrome (APS) describes an autoimmune thrombophilic disorder characterized by obstetric complications. Annexin A5 (Anx A5) is a protein that is studied as a new autoantigen present in APS, the presence of autoantibodies against Anx A5 could cause placental thrombosis and possibly pregnancy loss. The aim of this study was to analyze levels of IgG and IgM anti-Anx A5 in women with primary obstetric APS and its association with different complications in a population of the city of Córdoba. We worked with samples of puerperal patients who attended the Córdoba Hospital and the Maternal Neonatal Hospital during the years 2013-2017 with a diagnosis of obstetric APS and a control group formed by patients with normal pregnancies. In most of the patients studied, levels of IgG and IgM anti-Anx A5 were below the reference range, is demonstrate an increase statistically significant in the levels of the IgG in patients with APS compared with control group. But there were no specific associations between antibody levels and the three types of obstetric clinical manifestations present in the classification criteria. These findings could suggest a relationship between anti-Anx A5 antibodies and obstetric APS.

Efecto en los resultados reproductivos con la "magnetic-actived cell sorting (MACS)" para la eliminación de espermatozoides positivos a anexina V en parejas con infertilidad inexplicable / Effect on reproductive outcomes by using "magnetic-actived cell sorting (MACS)" for the elimination of sperm-positive annexin-V in couples with unexplained infertility

Resumen Objetivo: Determinar si la eliminación de espermatozoides positivos a marcadores tempranos de apoptosis en parejas con infertilidad inexplicable incrementa la tasa de nacidos vivos. Materiales y métodos: Ensayo piloto, con asignación al azar, controlado y triple ciego; y un estudio paralelo de dos grupos. Se incluyeron parejas con diagnóstico de infertilidad inexplicable que se asignaron en una proporción 1:1 al grupo A (método de capacitación espermática swim-up) o grupo B (método de capacitación espermática swim-up complementado con separación magnética de células activadas; magnetic-actived cell sorting; MACS). Posteriormente, a todas las muestras se les efectuó una inyección intracitoplasmática de espermatozoides, como técnica de fertilización. Por último, todos los embriones obtenidos se analizaron hasta la etapa de blastocisto y todas las transferencias se llevaron a cabo en la misma etapa. Resultados: Se incluyeron 40 parejas y no se encontraron diferencias en la tasa de fertilización. Con la aplicación de MACS se obtiene mayor porcentaje de embriones de buena calidad en día 3 (90.3 vs 99.5%; p = 0.03) y en día 5 (77.3 vs 90.1%; p = < 0.0001) disminuyó el porcentaje de embriones arrestados (16.3 vs 7.9%; p = 0.01). Por último, las tasas de implantación (42.1 vs 57.1%), embarazo clínico (60 vs 80%) y nacidos vivos (55 vs 80%) aumentaron, sin diferencias estadísticamente significativas. Conclusiones: La separación magnética de células activadas (MACS) en parejas con infertilidad inexplicable mejora el desarrollo embrionario. A pesar de no existir una diferencia significativa se observa una tendencia al incremento de embarazos clínicos y nacidos vivos.

Abstract Objective: To determine if the live births delivery rate with the eliminating sperm positive to early apoptotic events is higher in couples with unexplained infertility. Materials and methods: A pilot randomized controlled trialA pilot and triple-blinded; using a parallel study of two groups. We included a total of 40 couples with unexplained infertility assigned in a 1:1 proportion either to the group A (sperm training method swim-up) or to the group B (swim-up sperm training method supplemented with the use of "magnetic-actived cell sorting (MACS)"). Subsequently, all samples were submitted to intracytoplasmic sperm injection as a fertilization technique. Finally, all embryos obtained were analyzed until the blastocyst stage, and all the transfers were performed in the same stage. Results: There are no differences in the fertilization rate; however, with the use of "magnetic-actived cell sorting" there is a higher percentage of good quality embryos on day 3 (90.3% vs 99.5%, p = 0.03) and day 5 (77.3% vs 90.1%, p = <0.0001). In addition, a decrease in the percentage of arrested embryos was demonstrated (16.3% vs 7.9%, p = 0.01). Finally, implantation (42.1% vs 57.1%), clinical pregnancy (60% vs 80%) and live birth rates (55% vs 80%) increased; however, no statistically significant differences were reported. Conclusions: The use of "magnetic-actived cell sorting" in couples with unexplained infertility improves embryonic development. Although there is no significant difference, a trend is observed in relation to the increase in the number of clinical pregnancies and live births.

Changes of intraocular drug concentration and pharmacokinetics of bevacizumab with annexin A5-associated liposome topically applied in the rabbit eyes / 中华眼底病杂志

Objectives To explore intraocular drug concentration changes and the pharmacokinetics after topically applied of bevacizumab with armexin A5-associated liposome on rabbit eyes.Methods A total of 105 healthy New Zealand white rabbits were selected and divided randomly into 3 groups (group A,B and C),and each group had 35 rats.Bevacizumab with annexin A5-associated liposome,bevacizumab liposome and bevacizumab were topically applied 50 μl respectively on right eyes of rabbits in group A,B and C,respectively.Aqueous,vitreous body and retina/choroid were obtained at 5,15,30 minutes and 1,2,4,8 hours and the free bevacizumab concentrations in these ocular tissues were measured by ELISA (enzyme linked immunosorbent assay).DAS 2.1.1 software was used to fit the pharmacokinetic parameters.Results The peak drug concentrations in aqueous humor of the eyes in group A,B,C were at 15 minutes after topical administration and the difference was statistically significant (F=301.061,P＜ 0.01).The peak drug concentrations in vitreous of the eyes in the group A,B,C were at 2 hours after topical administration and the difference was statistically significant (F=885.997,P＜ 0.01).The peak drug concentrations in retina/choroid of the eyes in the group A,B,C were at 1 hour after topical administration and the difference was statistically significant (F=644.908,P＜0.01).Least significant difference pair-wise test found that the drug concentrations in aqueous humor,vitreous and retina/choroid of group A was higher than that of the group B and C respectively (P＜ 0.05),while that of the group B and C had no significant different (P＞ 0.05).Pharmacokinetic fitting analysis found that the half-life (t1/2) of bevacizumab in aqueous humor were 1.14,1.29,1.29 hours,the distribution t1/2 were 1.40,1.50,1.42 hours and the eliminated t1/2 were 2.62,2.84,2.73 hours in vitreous,the distribution t1/2 were 2.61,2.99,2.70hours and the eliminated t％ were 2.61,2.99,2.70 hours in retina/choroid respectively for the 3 groups.Changes of bevacizumab concentration in aqueous humor of rabbit eyes for 3 groups was complied with one compartment model,and that in vitreous body and retina/choroid complied with two compartment model.Conclusions Topically applied annexin A5-associated liposome has higher ocular concentrations of bevacizumab than those of controls.Changes of bevacizumab concentration in aqueous humor of rabbit eyes was complied with one compartment model,and that in vitreous body and retina/choroid complied with two compartment model.

Role of annexin A5 in tumor and basic research on the target of it / 国际肿瘤学杂志

As a key protein,annexin A5 involves in the process of invasion,metastasis and immune tolerance of tumors.And annexin A5 can not only promote the tumor,but also inhibit it.It has been widely concerned in the basic research targeting annexin AS.Because of the high affinity of annexin A5 for phosphatidylserine,annexin A5 is expected to be a new diagnostic marker and anti-tumor target.

Auranofin Suppresses Plasminogen Activator Inhibitor-2 Expression through Annexin A5 Induction in Human Prostate Cancer Cells

Auranofin has been developed as antirheumatic drugs, which is currently under clinical development for the treatment of chronic lymphocytic leukemia. Previous report showed that auranofin induced apoptosis by enhancement of annexin A5 expression in PC-3 cells. To understand the role of annexin A5 in auranofin-mediated apoptosis, we performed microarray data analysis to study annexin A5-controlled gene expression in annexin A5 knockdown PC-3 cells. Of differentially expressed genes, plasminogen activator inhibitor (PAI)-2 was increased by annexin A5 siRNA confirmed by qRT-PCR and western blot. Treatment with auranofin decreased PAI-2 and increased annexin A5 expression as well as promoting apoptosis. Furthermore, auranofin-induced apoptosis was recovered by annexin A5 siRNA but it was promoted by PAI-2 siRNA. Interestingly, knockdown of annexin A5 rescued PAI-2 expression suppressed by auranofin. Taken together, our study suggests that induction of annexin A5 by auranofin may enhance apoptosis through suppression of PAI-2 expression in PC-3 cells.

Effect of recombinant human annexin A5 on expression of p-PKCα and p120-catenin during endotoxin-induced damage to cardiomyocytes / 中华麻醉学杂志

Objective To evaluate the effect of recombinant human annexin A5 on the expression of phosphorylated protein kinase C alpha (p-PKCα) and p120-catenin during endotoxin-induced damage to cardiomyocytes.Methods H9c2 cells cultured in vitro were randomly divided into 3 groups (n=18 each) using a random number table:control group (group C),endotoxin group (group L),and recombinant human annexin A5 group (group A).Recombinant human annexin A5 (final concentration 5 ng/ml) was added,and the cells were incubated for 2 h in group A,and then lipopolysaccharide (final concentration 1 μg/ml) was added,and the cells were incubated for 4 h in L and A groups.At 4 h of incubation,cell apoptosis was detected using the cell apoptosis detection kit,the intercellular space was measured using the confocal microscopy,and the expression of p-PKCα and p120-catenin was determined by Western blot.The apoptosis rate was calculated.Results Compared with group C,the apoptosis rate was significantly increased,the intercellular space was significantly widened,the expression of p120-catenin was significantly down-regulated,and the expression of p-PKCα was significantly up-regulated in group L (P＜0.05).Compared with group L,the apoptosis rate and intercellular space were significantly decreased,the expression of p120-catenin was significantly up-regulated,and the expression of p-PKCα was significantly down-regulated in group A (P＜0.05).Conclusion Recombinant human annexin A5 can inhibit phosphorylation of PKCα and up-regulate the expression of p120-catenin,thus attenuating endotoxin-induced damage to cardiomyocytes.

The changes in expression of high-sensitivity C-reactive protein, glutathione S-transferase Pi and annexin A5 in elderly patients with old myocardial infarction and the clinical significance / 中华老年医学杂志

Objective To study the changes in expression of high-sensitivity C-reactive protein (hsCRP),glutathione S-transferase Pi(GSTPi)and annexin A5 (AnxA5)in elderly patients with old myocardial infarction and the clinical significance.Methods Serum levels of GSTPi and AnxA5 were measured by ELISA and the level of hs-CRP was measured by immunoturbidimetry in elderly patients with old myocardial infarction (n =185)from December 2012 to November 2015.Results Along with the increasing coronary artery stenosis,GSTPi level was decreased and AnxA5/hs-CRP levels were increased in elderly patients with old myocardial infarction.In comparison between coronary artery stenosis ＞ 95％ group versus stenosis of 55％-65％ group,GSTPi was(190.0±37.0)μg/L vs.(289.0 ±86.0)μg/L,AnxA5 was(33.9±4.0)μg/L vs.(8.1 ± 2.9) μg/L,and hs-CRP was (15.3 ± 1.3) mg/L vs.(5.9 ± 0.8) mg/L with statistically significant differences(all P＜0.01).There were significant differences between LVEF 30％ group[GSTPi(198.0±39.0) μg/L,AnxA5(38.9±5.1)μg/L and hs-CRP(17.9± 1.9)mg/L]and LVEF 40％-54％ group[GSTPi(219.0± 61.0)μg/L,AnxA5 (12.9±3.9)μg/L and hs-CRP(10.1 ± 1.0) mg/L] (all P＜0.01).There were significant differences between NYHA Ⅳ group [GSTPi (171.0 ± 43.0) μg/L,AnxA5 (18.1 ± 5.0) μg/L and hs-CRP (16.9±2.1)mg/L]and NYHAⅠgroup[GSTPi(295.0±91.0)μg/L,AnxA5(7.3±3.1)μg/L and hs-CRP (7.8± 1.3)mg/L](all P＜0.01).Conclusions The expression of GSTPi,AnxA5 and hs-CRP in elderly patients with old myocardial infarction may become the new indicators to forecast the degrees of coronary artery stenosis and heart failure.

Annexin A5 regulates Leydig cell testosterone production via ERK1/2 pathway / 亚洲男科学杂志(英文版)

This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig cells were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory (StAR), P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and 17α-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of StAR, P450scc, 3β-HSD, and 17β-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3β-HSD, and 17β-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 (50 μmol l-1 ) for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3β-HSD, and 17β-HSD were completely abrogated (P < 0.05). Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3β-HSD, and 17β-HSD in Leydig cells.

Progress in the mechanism of annexin A 5 in the related diseases / 基础医学与临床

Annexin A5 is a member of Ca 2+-dependent phospholipid binding proteins family .It exists in histocytes . And because of its unique structure , Annexin A5 plays an important role in many critical functions such as cell in-side/outside anticoagulation and pro-apoptosis .Moreover , Annexin A5 has certain relevance with many diseases such as systemic lupus erythematosus ,tumour and other diseases .

Role of Annexin A5 on Mitochondria-Dependent Apoptosis Induced by Tetramethoxystilbene in Human Breast Cancer Cells

We have previously shown that 2,4,3',5'-tetramethoxystilbene (TMS), a trans-stilbene analogue, induces apoptosis in human cancer cells. However, the detailed mechanisms of mitochondria-dependent apoptosis induced by TMS are not fully understood. In the present study, the possible roles of annexin A5 in TMS-mediated apoptosis were investigated in MCF7 human breast cancer cells. Quantitative real-time PCR analysis and Western blot analysis showed that the expression of annexin A5 was strongly increased in TMS-treated cells. TMS caused a strong translocation of annexin A5 from cytosol into mitochondria. Confocal laser scanning microscopic analysis clearly showed that TMS induced translocation of annexin A5 into mitochondria. TMS increased the expression and oligomerization of voltage-dependent anion channel (VDAC) 1, which may promote mitochondria-dependent apoptosis through disruption of mitochondrial membrane potential. When cells were treated with TMS, the levels of Bax, and Bak as well as annexin A5 were strongly enhanced. Moreover, we found that the cytosolic release of apoptogenic factors such as cytochrome c, or apoptosis-inducing factor (AIF) in mitochondria was markedly increased. Annexin A5 depletion by siRNA led to decreased proapoptotic factors such as Bax , Bak, and annexin A5. Taken together, our results indicate that annexin A5 may play an important role in TMS-mediated mitochondrial apoptosis through the regulation of proapoptotic proteins and VDAC1 expression.

Annexin A5 as a New Potential Biomarker for Cisplatin-Induced Toxicity in Human Kidney Epithelial Cells

Cisplatin is a member of platinum-containing anti-cancer drugs that causes cross-linking of DNA and ultimately cancer cell apoptosis. The therapeutic function of cisplatin on various types of cancers has been widely reported but the side effects have been discovered together and nephrotoxicity has been regarded as major side effect of cisplatin. To select candidates for new sensitive nephrotoxicity biomarker, we performed proteomic analysis using 2-DE/MALDI-TOF-MS followed by cisplatin treatment in human kidney cell line, HK-2 cells, and compared the results to the gene profi le from microarray composed of genes changed in expression by cisplatin from formerly reported article. Annexin A5 has been selected to be the most potential candidate and it has been identifi ed using Western blot, RT-PCR and cell viability assay whether annexin A5 is available to be a sensitive nephrotoxic biomarker. Treatment with cisplatin on HK-2 cells caused the increase of annexin A5 expression in protein and mRNA levels. Overexpression of annexin A5 blocked HK-2 cell proliferation, indicating correlation between annexin A5 and renal cell toxicity. Taken together, these results suggest the possibility of annexin A5 as a new biomarker for cisplatin-mediated nephrotoxicity.

Expression changes and clinical significance of annexin V in maternal blood and placenta in patients with preeclampsia / 中华妇产科杂志

Objective To evaluate the expression of annexin V in maternal blood and placenta,and to explore the relationship between annexin V and preeclampsia(PE). Methods 120 women with PE who delivered babies in the Second Hospital of Hebei Medical University from December 2007 to December 2009 were chosen as study groups. They were classified into four groups: early-onset mild group (n =30),early-onset severe group (n=30), late-onset mild group ( n = 30) and late-onset severe group (n=30). 30women without perinatal complications who accepted elective term cesarean section were chosen as control group. Western blot and immunohistochemistry were used to detect the expression and localization of annexin V in placenta and maternal blood. Flow cytometry was employed to detect the apoptosis of cytotrophoblast.Annexin V mRNA level was determined by reverse transcription (RT)PCR. Prothrombin time (PT),activated partial thromboplastin time (APTT), fibrinogen (FiB), international normalized ratio (INR) were detected in each group. Results (1) The expression of annexin V in placenta and maternal blood were:0.54±0.12 and 0.62±0.17 in early-onset mild group; 0.47±0.15 and 0.56±0.24 in early-onset severe group; 0.74±0.23 and 1.08±0.32 in late-onset mild group; 0.68±0.28 and 0.72±0.21 in late-onset severe group;1.73±0.35 and 1.55±0.27 in control group. They were significantly lower in PE groups than in control group (P＜0.05 ). However, there was no significant difference among PE groups (P＞0. 05). (2) The early apoptosis, late apoptosis percentage of trophoblast cells were: 3. 21%, 0. 86%, in early-onset mild group; 5.32%, 0. 72%, in early-onset severe group; 2. 43%, 0. 63%, in late-onset mild group;4. 28%, 0. 48% in late-onset severe group; 1.05%, 0. 59%, in control group. Early apoptosis percentage in each group of PE was higher than that in control group ( P ＜ 0. 05 ). However, there was no significant difference among PE groups (P＞0.05). (3) The annexin V mRNA levels in placenta were:25.0±3.0 in early-onset mild group; 24. 8 ± 3.0 in early-onset severe group; 25.4 ± 3. 9 in late-onset mildgroup; 25.1±2.7 in late-onset severe group, respectively. All were significantly higher than that in control group (30. 6±3.0, P＜ 0. 05), and no significant difference was found among PE groups (P＞0.05).(4) PT, APTT, FiB, INR levels were: (11.3±2.4), (25.6±2.9) s, (4.6±0.9) g/L and 0.9 ±0.2in early-onset mild group; (12.1±1.9), (27. 2 ±2. 1 ) s, (5.0 ± 1. 0) g/L and 0. 9 ±0. 2 in early-onset severe group; (11.7±2.3), (26.5±2.3)s,(5.0±0.7)g/L and 0.8±0.3 in late-onset mild group;(11.4±2.6), (27.3±3.0) s, (4.3 ±0.8) g/L and 0.8 ±0.3 in late-onset severe group; (12.4±2.7), ( 28.0±1.9) s, (5.1±1.2) g/L and 0.9 ± 0.2 in control group. There was no significant difference among PE groups and control group ( P ＞ 0.05 ). Conclusion The expression changes of annexin V in placenta and maternal blood were observed in patients with PE. This indicated that annexin V played an important role in the pathogenesis and progression of PE by affecting coagulation.

Targeted Ultrasound Imaging of Apoptosis with Annexin A5 Microbubbles in Acute Doxorubicin-Induced Cardiotoxicity

BACKGROUND: The aim of this study was to assess the feasibility of targeted ultrasound imaging on apoptosis with annexin A5 microbubbles (A5MB) in acute doxorubicin-induced cardiotoxicity. METHODS: Avidinated and octafluoropropan-filled phospholipid microbubbles were conjugated with biotinylated annexin A5. To confirm the specific binding of A5MB, flow cytometry was performed with hydrogen peroxide induced apoptosis in rat aorta smooth muscle cells incubated with fluorescein-5-isothiocyanate (FITC) labeled annexin A5 and A5MB. Adult male rats were injected intraperitoneally with 5 mg/kg doxorubicin weekly for 3 weeks (n = 5). Control rats were injected with normal saline (n = 5). At 24 hours after the final treatment, triggering imaging was performed 15 min after an intravenous bolus injection of A5MB for washout of freely circulating microbubbles. After echocardiography, the heart was isolated for histological detection of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. RESULTS: In the in vitro tests, fluorescence intensity was low for healthy cells and high for apoptotic cells when incubated with FITC-labeled annexin A5 and A5MB. Rats treated with doxorubicin showed significant contrast opacification of the myocardium on contrast echocardiography using A5MB. However, no opacification was observed in control rats. Apoptosis was confirmed by TUNEL assay in doxorubicin treated rats. CONCLUSION: Acute doxorubicin-induced cardiomyopathy based on early apoptosis can be assessed and imaged with targeted ultrasound imaging using A5MB in rats.