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ANNEXIN V belongs to a family of phospholipid binding proteins which is able to bind to negatively charged phospholipids such as phosphatidylserine (PS) in the presence of a high affinity Ca 2+ ion. When apoptosis occurs, even at early stage, PS will be exposed to the outside of the cell surface from the cytoplasm side of membrane leaflets., Therefore ANNEXIN V has been suggested as a bio-marker for imaging early apoptotic events of various cell death including those in disease conditions. However, most ANNEXIN V-based apoptotic detecting techniques were in vitro approaches. Here, we presented a new BRET (Bioluminescence Resonance Energy Transfer) based genetic coded biosensor by fusing ANNEXIN V and a BRET version of NanoLuc (teLuc) for both in vitro and in vivo apoptosis detection. The BRET feature of this new sensor makes it convenient to be applied to both conventional fluorescent-based in vitro apoptosis detection and bioluminescence-based animal live imaging for in vivo study. Because of its robust bioluminescence signal, it is possible to perform the evaluation of the disease-induced apoptotic damage and recovery process directly at deep tissue level in live animal., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Annexin A5 (anxA5) is a marker for apoptosis, but has also therapeutic potential in cardiovascular diseases, cancer, and, due to apoptotic mimicry, against dangerous viruses, which is limited by the short blood circulation. An 864-amino-acid XTEN polypeptide was fused to anxA5. XTEN864-anxA5 was expressed in Escherichia coli and purified using XTEN as tag. XTEN864-anxA5 was coupled with DTPA and indium-111. After intravenous or subcutaneous injection of 111 In-XTEN864-anxA5, mouse blood samples were collected for blood half-life determination and organ samples for biodistribution using a gamma counter. XTEN864-anxA5 was labeled with 6S-IDCC to confirm binding to apoptotic cells using flow cytometry. To demonstrate targeting of atherosclerotic plaques, XTEN864-anxA5 was labeled with MeCAT(Ho) and administered intravenously to atherosclerotic ApoE -/- mice. MeCAT(Ho)-XTEN864-anxA5 was detected together with MeCAT(Tm)-MAC-2 macrophage antibodies by imaging mass cytometry (CyTOF) of aortic root sections. The ability of anxA5 to bind apoptotic cells was not affected by XTEN864. The blood half-life of XTEN864-anxA5 was 13 h in mice after IV injection, markedly longer than the 7-min half-life of anxA5. 96 h after injection, highest amounts of XTEN864-anxA5 were found in liver, spleen, and kidney. XTEN864-anxA5 was found to target the adventitia adjacent to atherosclerotic plaques. XTEN864-anxA5 is a long-circulating fusion protein that can be efficiently produced in E. coli and potentially circulates in humans for several days, making it a promising therapeutic drug., (© 2021. The Author(s).)

Annexin V (ANXV), mostly characterized by its ability to interact with biological membranes in a calcium-dependent manner. ANXV interacts mainly with phosphatidylserine (PS), for that fluorescent ANXV widely produced and used as a sensitive and specific probe to mark apoptotic cells or any PS-containing bilayers membranes. Many reports described the prokaryotic expression of recombinant human ANXV. To overcome some of E. coli expression limitations, we aimed in this work to investigate unconventional alternative expression system in mammalian cells for producing secreted human ANXV in fusion with the super folder green fluorescent protein (sfGFP). HEK239T cells were transfected using polyethylenimine (PEI) and pcDNA-sfGFP-ANXV plasmid. Forty-eight hours post transfection, direct fluorescence measurement, immunoblotting and ELISA confirmed the presence of secreted sfGFP-ANXV in cells supernatant. The yield of secreted 6 × His-tagged sfGFP-ANXV after affinity purification was estimated to be around 2 µg per 1 ml of cells supernatant. The secretion system was proper to produce a fully functional sfGFP-ANXV fusion protein in quantities enough to recognize and bind PS-containing surfaces or liposomes. Besides, biological assays such as flow cytometry and fluorescent microscopy confirmed the capacity of the secreted sfGFP-ANXV to detect PS exposure on apoptotic cells. Taken together, we present mammalian expression as a quick, affordable and endotoxin-free system to produce sfGFP-ANXV fusion protein. The secreted sfGFP-ANXV in eukaryotic system is a promising biotechnological tool, it opens up new horizons for additional applications in the detection of PS bearing surfaces and apoptosis in vitro and in vivo assays.

Objective: This study was carried out to determine the potential role of the M2/ANXA5 haplotype as a risk factor for recurrent implantation failure (RIF). Carriage of the M2/ANXA5 haplotype that induces prothrombotic changes has been implicated in failure of early pregnancies and placenta-mediated complications (preeclampsia, IUGR, preterm birth)., Material and Methods: In the present case control study, 63 couples (females and males) with RIF presenting for IVF/ICSI to the Fertility Center of [masked] were analyzed. RIF was defined as ≥ 4 consecutive failed ART-transfers of ≥ 4 blastocysts or ≥ 8 cleavage-stage embryos of optimal quality and maternal age ≤ 41. Fertile female controls (n = 90) were recruited from the same center. Population controls (n = 533) were drafted from the PopGen biobank, UKSH Kiel., Results: Couples carrying the M2/ANXA5 haplotype turned out to have a significantly increased relative risk (RR) for RIF. Compared with female fertile controls, RR was 1.81 with p = 0.037 (OR 2.1, 95%CI 1.0-4.3) and RR was 1.70, with p = 0.004 (OR 2.0, 95%CI 1.2-3.1) compared with population controls (15.4% M2 carriers). Male partners were comparable with RIF females for M2/ANXA5 haplotypes (28.6% vs. 23.8%, p = 0.54). RIF females compared with population controls had a RR of 1.55 (p = 0.09) and RIF males compared with population controls had a RR of 1.9 (p = 0.01). Couples with ≥ 7 failed transfers showed a RR of 1.82 (p = 0.02) compared with population controls., Conclusion: Our findings suggest that maternal as well as paternal M2/ANXA5 haplotype carriages are risk factors for RIF. These results allow new insights into the pathogenesis of RIF and might help to identify relevant risk groups.

Purpose: To investigate the effect of the anticoagulation factor annexin A5 on male fertility and to provide perspective on the influence of members of the coagulation cascade on fertility., Methods: Patients with normozoospermia and with unexplained severe oligozoospermia were retrospectively selected and their genomic DNA sequenced for the promoter region of ANXA5. The genotypes proportions and the odds ratio for carriership of the haplotype M2 were compared between the groups and population control. The clinical data used were gathered from parameters determined during routine clinical assessment and were compared between carriers and non-carriers within the patient groups., Results: The carrier rates for the haplotype M2/ANXA5 were of 25.73%, 20.81%, and 15.3% in the severe oligozoospermic, the normozoospermic, and the general population control groups, respectively. The OR between patients groups was of 1.31 (95% CI 0.88 to 1.96 p = 0.176). Oligozoospermic and normozoospermic patients compared with the control group had an OR of 1.9 (95% CI 1.33 to 2.73 p < 0.001) and 1.45 (95% CI 0.99 to 2.10 p = 0.054) respectively. The clinical parameters that differed between the carriers and non-carriers of the haplotype M2/ANXA5 were prolactin, α-glucosidase, and fructose. The differences were only statistically significant in the normozoospermic group., Conclusions: Athough the infertile patient groups had a higher prevalence of promoter variants, we could not demonstrate any biologically relevant effect of lower levels of annexin A5 on most male fertility parameters. A deficiency in an anticoagulation factor does not seem to impact male fertility.

Background: Glioma, the most common primary malignant brain tumor, is highly malignant with a poor prognosis. We aimed to clarify the relevance of ANXA5 polymorphisms to glioma risk and prognosis among the Chinese Han population., Method: Six single-nucleotide polymorphisms (SNPs) of ANXA5 were genotyped by Agena MassARRAY in 593 glioma patients and 589 healthy controls. Logistic regression model was used to calculate odds ratios (OR) and 95% confidence intervals (CI). The association between polymorphisms and survival were evaluated using the log-rank test, Kaplan-Meier analysis and Cox regression model., Results: We found that rs117677079 polymorphism was strongly associated with an increased risk of glioma (OR 1.64, p = 0.003) and a worse prognosis for glioma, especially in high-grade glioma (HR 1.76, p = 0.005). Whereas, rs145619195 CT genotype might weaken the susceptibility (OR 0.63, p = 0.024) and prognosis (HR 0.20, p = 0.025) of glioma. Haplotype analysis showed that haplotype ″GACCG″ in the block (rs41278075, rs2306420, rs117677079, rs2306415 and rs1131239) significantly decreased the susceptibility of glioma (OR 0.61, p = 0.003). Furthermore, we also found that age, extent of resection and chemotherapy were key prognostic factors in glioma patients., Conclusion: This study firstly provided evidence for the impact of ANXA5 polymorphism on the susceptibility and prognosis of glioma, suggesting ANXA5 variants might have potential roles in the etiology of glioma.

Deficient expression of the placental anticoagulant annexin A5 (ANXA5) has been associated with thrombophilia-related pregnancy complications and ultimately with recurrent pregnancy loss (RPL). Carrier status of M2/ANXA5 ( RPRGL3), common ANXA5 promoter variant, has been identified as genetic cause of reduced ANXA5 levels and proposed as biomarker for successful anticoagulant treatment of RPL women. A murine model of AnxA5 loss of function displayed characteristic placental pathology and fetal loss that was alleviated through anticoagulant intervention. This study identified an alternative means of supplementing anticoagulation, through elevated ANXA5 expression. Physiological micromolar Zn 2+ stimulated ANXA5 transcription, raising ANXA5 protein expression and surface abundance on BeWo and human umbilical vein endothelial cells (HUVEC), thus resulting in prolonged coagulation times. Zn 2 -fed AnxA5 functionally deficient pregnant mice showed a trend to increase litter size when primiparous that grew comparable to wild-type progeny in subsequent pregnancies. Elevated AnxA5 signal upon Zn 2+ treatment was confirmed in murine placentae. Micromolar Zn 2+ stimulated ANXA5 expression in cell culture directly and alleviated RPL in AnxA5 genetically deficient mice, without notable toxicity effects.

As an important member of the Annexins, AnnexinA5 has been attributed important functions in trophoblast membrane repair, anticoagulation and cellular signal transduction. Accumulated studies show that AnnexinA5 is closely associated with various types of carcinomas. However, the potential contribution of AnnexinA5 to glioma cancer progression remains unclear. In this study, we report that AnnexinA5 is significantly upregulated in both high-grade glioma samples and glioma cell lines. Moreover, overexpression of AnnexinA5 promotes cell migration and invasion in vitro and tumorigenicity of glioma cells in nude mice, while knockdown of AnnexinA5 manifests a repressive function during these cellular processes. Importantly, mechanistic studies further reveal that AnnexinA5 is an essential transcriptional target of Snail via activating the PI3K/Akt/NF-κB signaling pathway. Taken together, these findings suggest that AnnexinA5 or the PI3K/Akt/NF-κB pathway may be promising therapeutic molecules to eradicate glioma metastases.

Purpose: Annexin A5 (ANXA5) is a protein abundantly expressed in normal placenta where it contributes to the healthy outcome of a pregnancy. Lower ANXA5 levels have been observed in M2/ANXA5 haplotype carrying chorion. Consequently, this study aimed to assess the potential association of M2 maternal carrier status with the risk of recurrent pregnancy loss (RPL), the timing of miscarriages, and other obstetric complications, for the first time in a population from Latin America., Methods: This study was designed as a prospective recruitment of RPL patients with post hoc analysis. The distribution of the M2/ANXA5 haplotype was compared between a group of 229 Argentine women with RPL and 100 parous controls, and was further analyzed in subgroups of patients stratified according to the timing of miscarriages and in relation to other obstetric complications., Results: No significant differences were found in the distribution of M2 haplotype among either RPL patients or the subgroups with embryonic, early fetal, or late fetal losses compared to parous controls. Notwithstanding, maternal M2/ANXA5 was found to be independently associated with a higher risk of suffering intrauterine growth restriction (IUGR) and/or preeclampsia (PE). Simultaneously, the presence of inherited and/or acquired thrombophilia also proved to be an independent risk factor for these., Conclusions: The association found between the maternal carriage of the M2/ANXA5 haplotype and an elevated risk of IUGR and/or PE supports the hypothesis that carrier status of this haplotype and the consequently reduced placental ANXA5 expression might be responsible, at least partially, for the onset of these gestational vascular complications.

Purpose: The aim of this study was to confirm the associated M2/ANXA5 carrier risk in women with placenta-mediated pregnancy complications (PMPC) and to test their male partners for such association. Further analysis evaluated the influence of maternal vs. paternal M2 alleles on miscarriage., Methods: Two hundred eighty-eight couples with preeclampsia (PE), intrauterine growth restriction (IUGR), or premature birth (PB) were recruited (n = 96 of each phenotype). The prevalence of the M2 haplotype was compared to two control cohorts. They included a group of women with a history of normal pregnancy without gestational pathology (Munich controls, n = 94) and a random population sample (PopGen controls, n = 533)., Results: Significant association of M2 haplotype and pregnancy complications was confirmed for women and for couples, where prevalence was elevated from 15.4 to 23.8% (p < 0.001). Post hoc analyses demonstrated an association for IUGR and PB individually. A strong link between previous miscarriages and M2 carrier status was identified which may explain the predisposition to placental pregnancy complication. M2/ANXA5 appears to be a risk factor for adverse pregnancy outcomes related, but not limited to miscarriages, with similar prevalence in women and their male partners., Conclusion: These findings support the proposed physiological function of ANXA5 as an embryonic anticoagulant that appears deficient in contiguous specter of thrombophilia-related pregnancy complications culminating more frequently in miscarriage in a maternal M2 carrier background.

Purpose: The aim of this study was to evaluate a new predisposition factor, M2/ANXA5 (RPRGL3), in recurrent pregnancy loss (RPL) patients of Malay origin, since it was previously known that the prevalence of this condition is relatively high among the Malay population of Malaysia, where conventional hereditary thrombophilia factors have been generally ruled out., Methods: A total of 232 women who had experienced ≥2 unexplained RPL and 141 available male partners were recruited, with 360 healthy Malay and 166 parous female controls. Prevalence of M2 carriage and RPL odds ratios were calculated in (a) control and patient groups; (b) clinically defined subgroups in categories of pregnancy loss, primary, secondary, and tertiary; and (c) timing of pregnancy loss in early, ≤15th gestation week and "late" fetal losses, and >15th gestation week subgroups., Results: Both male and female subjects had similar M2/ANXA5 allele frequencies. The carrier rate of M2/ANXA5 for the general Malay population was 42.2 and 34.9% for parous controls. These carrier rates compared to Malay RPL subjects (52% M2 carriers) resulted in elevated odds ratios (95% confidence interval) of 1.53 (1.1 to 2.1) and 1.97 (1.3 to 3.1) accordingly for early fetal losses. Moreover, exceeding copy numbers of M2/ANXA5 alleles seemed to afflict a greater chance of RPL in couples, especially when both partners were M2 carriers., Conclusion: This study confirmed the proposed role of M2/ANXA5 as embryonic, genetically associated thrombophilia predisposition factor for early RPL among ethnic Malay of Malaysia.

Imaging agents that enable direct detection of apoptosis are highly desirable in the field of monitoring chemotherapeutic response as well as early diagnosis and disease monitoring. Previous work demonstrated that the dansyled amino acid DNSBA is used to specifically and selectively detect apoptotic cancer cells at the both early and late stages, but the mechanism remains unclear. In this work, we evaluated DNSBA as a tool for monitoring cell apoptosis in CNE1 tumor cell models both in vitro and ex vivo after its in vivo administration, which was confirmed by other assays. The ability of DNSBA to detect multiple pathways and different stages of apoptosis leading to cell death may be advantageous in the evaluation of cancer treatment indicative of a positive therapeutic outcome. The uptake change of molecular probes DNSBA in CNE1 cells represented the changes of apoptotic rate in a caspase-dependent manner. However, the accumulation of DNSBA in apoptotic cells did not increase with the enhanced membrane permeability. Furthermore, ex vivo study demonstrated DNSBA has a similar pattern as the TUNEL-positive cells. In conclusion, DNSBA cellular imaging is useful for the early assessment of treatment-induced apoptosis, and thus may act as a substitute for Annexin V for assessing treatment response.

Purpose: The M2 haplotype in ANXA5 as well as antitrophoblast antibodies predispose to recurrent pregnancy loss (RPL). Since M2/ANXA5 can be a factor for development of antiphospholipid antibodies (aPL), this study aimed to trace a possible association of M2 with antitrophoblast antibodies., Methods: One hundred patients with two or more consecutive, idiopathic RPLs were divided in two subgroups, JEG-3(+) (n = 42) and JEG-3(-) (n = 58), according to the anti-JEG-3 reactivity measured in subjects' sera. Both subgroups were genotyped for ANXA5 promoter haplotypes and genetic frequencies were compared to available fertile and control populations, as well as within the subgroups., Results: M2/ANXA5 was generally enriched in the JEG-3 screened cohort of RPL patients in comparison to fertile and population controls. Despite the relatively higher abundance of the haplotype in the JEG-3(-) sample as compared to JEG-3(+) patients and in the JEG-3(-) primary RPL subset in particular, compared to the rest of patients, there was no statistically significant difference between both, JEG-3(-) and JEG-3(+) subgroups., Conclusion: It appears that the haplotype M2/ANXA5 is not associated with the presence of anti-trophoblast antibodies. Our finding indicates that anti-trophoblast antibodies are a class of molecules that differ from aPL and from anti-b2-GPI antibodies, apparently not directed to same or similar epitopes that aPL and anti-b2-GPI would recognize.

The β-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-β-D: -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues.