Annexin A5 Expression Research Articles

Postbiotic-based recombinant receptor activator of NF-κB ligand enhanced oral vaccine efficiency in chicken

Functional M cells are differentiated by receptor activator of NF-κB ligand (RANKL) and capture of luminal antigens to initiate immune responses. We aimed to use postbiotic-based recombinant chicken RANKL (cRANKL) to promote M cell differentiation and test the efficacy of oral vaccines. Chicks were divided into three groups that were administered phosphate-buffered saline (PBS), cell extracts of wild-type Lactococcus lactis subsp. lactis IL1403 (WT_CE), or cell extracts of recombinant L. lactis expressing cRANKL (cRANKL_CE). The expression of the M cell marker was measured, and the gut microbiome was profiled. The efficiency of the infectious bursal disease (IBD) vaccine was tested after 12 consecutive days of administering cRANKL_CE. The chickens that were administered cRANKL_CE (p = 0.038) had significantly higher Annexin A5 (ANXA5) mRNA expression levels than those in the PBS group (PBS vs. WT_CE, p = 0.657). In the gut microbiome analysis, no significant changes were observed. However, the relative abundance of Escherichia-Shigella was negatively correlated (r = − 0.43, p = 0.019) with ANXA5 mRNA expression in Peyer’s patches. cRANKL_CE/IBD (p = 0.018) had significantly higher IBD-specific faecal IgA levels than PBS/IBD (PBS/IBD vs. WT_CE/IBD, p = 0.217). Postbiotic-based recombinant cRANKL effectively improved the expression of M cell markers and the efficiency of oral vaccines. No significant changes were observed in the gut microbiome after administration of postbiotic-based recombinant cRANKL. This strategy can be used for the development of feed additives and adjuvants.Key points• Postbiotic-based recombinant cRANKL enhanced the expression of ANXA5 in chicken.• The relative abundance of Escherichia-Shigella was negatively correlated with ANXA5 expression.• Postbiotic-based recombinant cRANKL effectively improved the efficiency of oral vaccine.

Augmentation of hepatocellular carcinoma malignancy by annexin A5 through modulation of invasion and angiogenesis

ABSTRUCT Background Hepatocellular carcinoma (HCC) continues to play a substantial role in cancer-related morbidity and mortality, largely owing to its pronounced tumor heterogeneity and propensity for recurrence. This underscores the pressing need for in-depth examination of its highly malignant mechanisms. Annexin A5 (ANXA5), recognized as a hallmark tumor protein, has emerged as a focal point of interest because of its ambiguous function and mechanism in HCC prognosis. This study aimed to provide a comprehensive understanding of the role of ANXA5 in the malignant progression of human HCC cells by employing an integrative approach that combines conventional experimental methods with RNA sequencing. Methods Differences in ANXA5 expression between HCC tissues and corresponding nontumor tissues were evaluated using immunofluorescence (n = 25). Correlation analysis was subsequently performed to assess the association between ANXA5 expression and clinicopathological features (n = 65). The role of ANXA5 in human HCC cell lines with ANXA5 gene knockout and overexpression was explored in vitro using migration and invasion assays and Ki-67 indices and in vivo based on node mice xenograft model. A tube formation assay using human umbilical vein endothelial cells (HUVECs) was conducted to demonstrate the angiogenic effects of ANXA5 in HCC. Single-cell and bulk RNA sequencing was used to further investigate the underlying mechanisms involved. Results This study revealed that ANXA5 is highly expressed in patients with HCC and correlates with poor prognosis. Assays for migration, invasion, and proliferation based on ANXA5 gene knockout and overexpression systems in human HCC cell lines have demonstrated that ANXA5 enhances HCC malignancy in vitro and in vivo. Tube formation assays of HUVECs indicated that ANXA5 facilitates angiogenesis and recruits endothelial cells to HCC cells. Single-cell and bulk RNA sequencing data analysis further confirmed that ANXA5 expression in HCC is associated with hepatocyte metabolism, immune response activation, and various oncogenic signaling pathways. Conclusions This study revealed a meaningful association between elevated ANXA5 expression in tumor tissues and an unfavorable prognosis in patients with HCC. In addition, ANXA5 promotes HCC malignancy by promoting invasion and angiogenesis. Thus, ANXA5 has emerged as a promising therapeutic target for HCC and has the potential to improve patient outcomes.

Investigation of the combined cytotoxicity induced by sodium butyrate and a flavonoid quercetin treatment on MCF-7 breast cancer cells

ABSTRACT Quercetin (QUE) belonging to the flavonoid class is a common phytochemical present in the daily diet of some individuals. Quercetin is an important source of free radical scavengers. This property makes this flavonoid a reliable antioxidant with the following properties: anti-inflammatory, anti-diabetic, antimicrobial and anti-carcinogenic. Sodium butyrate (NaBu) acts as a histone deacetylase inhibitor (HDACi) and is known to regulate apoptosis in cancer cells. Combining natural flavonoids such as QUE with different substances may synergistically enhance their anti-carcinogenic capacity. Thus, the aim of this study was to examine the combined treatment effects of QUE and NaBu in hormone-sensitive breast cancer cells in vitro. MCF-7 breast cancer cells were treated with QUE alone, NaBu alone, as well as QUE and NaBu combined to determine the following: cell proliferation, levels of protein annexin A5 (ANXA5) and reactive oxygen species (ROS), mRNA protein expression, as well as cell and nuclear morphology. Data demonstrated that either QUE or NaBu alone inhibited cell proliferation, and reduced levels protein ANXA5, ROS and mRNA protein expression, The combination of QUE and NaBu produced a significant synergistic inhibitory effect compared to treatment groups of QUE or NaBu alone. In conclusion, our findings showed that the combination treatment of QUE and NaBu may constitute a promising therapeutic approach to breast cancer treatment but this needs further molecular and in vivo investigations

Increased ANXA5 expression in stomach adenocarcinoma infers a poor prognosis and high level of immune infiltration.

The prognostic role of annexin A5 (ANXA5) in stomach adenocarcinoma (STAD) has not been studied, and its relationship with immune infiltration is still unclear. This investigation aimed at exploring the role of ANXA5 in STAD using an integrated bioinformatics analysis. The expression of ANXA5 in STAD and the correlations between the effect of ANXA5 and survival of STAD patients were investigated using database. The clusterProfiler package in R software was used to perform enrichment analysis on the top 100 co-expressed genes of ANXA5 from the COXPRESdb online database. Correlations between ANXA5 and immune cell infiltrates were analyzed using the TIMER database. In STAD, ANXA5 expression was significantly upregulated and increased ANXA5 expression was significantly correlated with poor overall survival (P< 0.05). In multivariate analysis, upregulated ANXA5 expression was an independent predictive factors of poor prognosis (P< 0.05). The co-expressed genes were involved in extracellular matrix (ECM)-related processes. In STAD, ANXA5 expression was significantly correlated with various infiltrating immune cells (P< 0.05). Together with our findings, ANXA5 could serve as a potential biomarker to assess prognosis and immune infiltration level in STAD.

Annexin A5 suppression promotes the progression of cervical cancer.

Cervical cancer is a common malignant gynecological disease that threatens the health of women all over the world. The abnormal expression of Annexin A5 (ANXA5) is closely related to the biological behavior of various malignant tumors, however, the relationship between ANXA5 and cervical cancer is still unclear. Therefore, the effects of low expression of ANXA5 on the proliferation, apoptosis, migration and invasion of cervical cancer cells (HeLa) and its related mechanism were explored. The cells were divided into three groups: ANXA5-si group, negative control group and blank group. RNA interference was used to suppress ANXA5 expression. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, flow cytometry and propidium iodide (PI) staining, wound healing assay and transwell assay were employed to detect cell proliferation, apoptosis, migration and invasion respectively. Meanwhile, gene expression was detected by qPCR and Western blotting. ANXA5 suppression lead to the increase of proliferation, migration, invasion and the decrease of apoptosis of cervical cancer HeLa cells. Furthermore, the expression of both pPI3K and pAkt increased. ANXA5 might inhibit Hela cells proliferation and metastasis by regulating PI3K/Akt signal pathway.

Association between ANXA5 haplotypes and the risk of recurrent pregnancy loss

BackgroundAnnexin A5 (ANXA5) haplotypes can increase the risk of recurrent pregnancy loss (RPL). This study aimed to investigate the effect of ANXA5 haplotypes on ANXA5 expression in patients with RPL.MethodsFemale subjects with RPL, parous controls (those who intentionally aborted without medical conditions or complications), and population controls (normal delivery) were studied. Real-time polymerase chain reaction was carried out to evaluate ANXA5 expression in the placenta and peripheral blood. Western blotting and immunohistochemistry were used to assess ANXA5 protein expression. The luciferase assay was performed to detect the effect of M1 and M2 haplotypes on transcription efficiency of the ANXA5 promoter.ResultsWe found that the percentage of the M2 carrier was highest in the RPL group. ANXA5 expression in the placenta and peripheral blood in subjects with RPL was significantly inhibited. Furthermore, ANXA5 expression in subjects carrying the M2 haplotype was remarkably suppressed compared with that in carriers of other haplotypes. Finally, the M2 haplotype decreased the transcription efficiency of the ANXA5 promoter.ConclusionOur findings show that ANXA5 expression is decreased in carriers of the M2 haplotype and that M1/M2 haplotypes in the ANXA5 gene are associated with an increased risk of RPL.

Clinicopathological study and immunohistochemical analysis of expression of annexin A5 and apelin in human placentae of gestational diabetes mellitus

Background and aimsGestational diabetes mellitus (GDM) is one of the commonest medical complications of pregnancy. Annexin A5 (ANXA5) is a protein, found in apical surfaces of syncytiotrophoblasts, which prevents fetal and placental vascular thrombosis in GDM. Apelin is a bioactive peptide which has been linked to GDM. The aim of the present study was to correlate macroscopic as well as microscopic changes and immunohistochemical expression of ANXA5 and apelin in placentae of GDM with maternal and neonatal clinical features and also to compare the results with those in matched controls. MethodsThis prospective observational study was undertaken for a period of one year from April 2020 to March 2021. It comprised of 42 patients of GDM. Gross features, microscopic features and intensity and grade of expression of ANXA5 and Apelin were analyzed in placentae of GDM. ResultsMorphological changes detected in GDM placentae included increased immature villi (16 cases, 38%), increased syncytial knots (36, 86%), perivillous fibrin deposition (20, 48%), fibrosis of villous stroma (20, 48%), presence of nucleated red blood cells (12, 28.5%) and hypervascularity (34, 81%). The extent of histopathological changes noted in GDM placentae was significantly higher than that in matched controls. GDM placentae showed significantly reduced expression of ANXA5 and Apelin in terms of grade and intensity when compared with matched controls. Reduced expression (mild intensity) of ANXA5 was noted in 22 GDM cases (52.3%) whereas apelin expression was of weak intensity in 26 (61.9%) cases. Among GDM patients, statistically significant association was noted between ANXA5 intensity and neonatal resuscitation, apelin grade and preterm birth as well as low birth weight and apelin intensity and requirement of treatment in sick neonatal care unit. ConclusionThe placental expression of the proteins, ANXA5 and Apelin, is altered in GDM though their exact pathogenetic mechanisms are yet to be understood. They can be targets for development of prophylactic and therapeutic agents in future.

Gonadotropin-releasing hormone stimulation of annexin A5 expression in the thymus of male rats.

As gonadotropin-releasing hormone (GnRH) is expressed in the thymus, its direct action on thymic cells, including thymic involution, has been suggested. Annexin A5 (ANXA5), a biomarker of GnRH, was used to determine whether GnRH affects the thymus of male rats. Immunohistochemistry showed positive reactions for ANXA5 in large medullary epithelial cells at 30 days of age, and the expression continued until 180 days of age. Organ culture of thymus pieces was performed to examine the direct action of a GnRH agonist (GnRHa) on the expression of Anxa5 and Gnrh mRNA. Thymus tissues obtained from male rats (40–60 days old) were cut into small pieces (2–3 mm3) and incubated for 3 hr with the GnRHa. The expression levels of Anxa5 and Gnrh mRNA were augmented by the GnRHa. Immunohistochemistry of these tissue fragments showed that ANXA5 expression was enhanced, especially in medullary epithelial cells. These results revealed that GnRH synthesis in the thymus could affect thymic epithelial cells after puberty.

Clinicopathological study of annexin A5 & apelin in pre-eclamptic placentae with emphasis on foetal outcome.

Background & objectives:Pre-eclampsia has remained an elusive disease with serious impacts on both maternal and foetal health. Two novel markers, annexin A5 (ANXA5) and apelin are currently of considerable interest. The present study aimed to determine the placental expression of ANXA5 and apelin in pre-eclamptic placentae and also to elucidate if there is any correlation between the expression of these markers and the clinical features of both, mother and neonate. The comparison between gross and histopathological features of pre-eclamptic placentae and controls was another objective.Methods:A prospective, observational study was undertaken for one year. Placentae of pre-eclamptic patients and matched controls (matched for age, ethnic and socio-economic background) were collected along with the clinical data. Gross and histopathological analyses were done and immunohistochemical study of placental sections with ANXA5 and apelin was also undertaken.Results:79 pre-eclamptic patients and equal numbers of matched controls were included in the study. The difference in weight and dimensions of placentae between the pre-eclampsia group and matched controls was significant. Histopathological features noted in the pre-eclamptic placentae included decidual vasculopathy, infarction, perivillous fibrin deposition, increased syncytial knots and distal villous hypoplasia. There was a significant reduction in the expression of both ANXA5 and apelin in pre-eclamptic placentae compared to controls. Among pre-eclamptic patients, the intensity of ANXA5 and apelin expression showed a significant association with respect to neonatal resuscitation. Furthermore, the intensity of apelin showed expression a significant correlation with the requirement of sick neonatal care unit treatment.Interpretation & conclusions:The results of the present study suggest that both ANXA5 and apelin levels are reduced in pre-eclamptic placentae. Hence, it is recommended to further explore the impact of these markers on pregnancy outcomes by undertaking randomized controlled trials.

Identification of proteins associated with development of metastasis from cutaneous squamous cell carcinomas (cSCCs) via proteomic analysis of primary cSCCs.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common cancers capable of metastasizing. Proteomic analysis of cSCCs can provide insight into the biological processes responsible for metastasis, as well as future therapeutic targets and prognostic biomarkers. To identify proteins associated with development of metastasis in cSCC. A proteomic-based approach was employed on 105 completely excised, primary cSCCs, comprising 52 that had metastasized (P-M) and 53 that had not metastasized at 5years post-surgery (P-NM). Formalin-fixed, paraffin-embedded cSCCs were microdissected and subjected to proteomic profiling after one-dimensional (1D), and separately two-dimensional (2D), liquid chromatography fractionation. A discovery set of 24 P-Ms and 24 P-NMs showed 144 significantly differentially expressed proteins, including 33 proteins identified via both 1D and 2D separation, between P-Ms and P-NMs. Several differentially expressed proteins were also associated with survival in SCCs of other organs. The findings were verified by multiple reaction monitoring on six peptides from two proteins, annexin A5 (ANXA5) and dolichyl-diphosphooligosaccharide-protein glycosyltransferase noncatalytic subunit (DDOST), in the discovery group and validated on a separate cohort (n=57). Increased expression of ANXA5 and DDOST was associated with reduced time to metastasis in cSCC and decreased survival in cervical and oropharyngeal cancer. A prediction model using ANXA5 and DDOST had an area under the curve of 0·93 (confidence interval 0·83-1·00), an accuracy of 91·2% and higher sensitivity and specificity than cSCC staging systems currently in clinical use. This study highlights that increased expression of two proteins, ANXA5 and DDOST, is significantly associated with poorer clinical outcomes in cSCC.

Overexpression of annexin A5 might guide the gemtuzumab ozogamicin treatment choice in patients with pediatric acute myeloid leukemia

Background:Acute myeloid leukemia (AML) is a common hematological malignancy. Gemtuzumab ozogamicin (GO), a humanized anti-CD33 antibody conjugated with the potent anti-tumor antibiotic calicheamicin, represents a promising targeted therapy for AML. Annexin A5 (ANXA5) is a proposed marker for the clinical prognosis of AML to guide treatment choice.Methods:In total, 253 patients with pediatric AML were enrolled and divided into two treatment groups: conventional chemotherapy alone and conventional chemotherapy in combination with GO. Univariate, multivariate, and Kaplan–Meier survival analyses were conducted to assess risk factors and clinical outcomes, and to estimate hazard ratios (HRs) and their 95% confidence interval. The level of statistical significance was set at p < 0.05.Results:In the GO treatment group, high ANXA5 expression was considered a favorable prognostic factor for overall survival (OS) and event-free survival (EFS). Multivariate analysis showed that high ANXA5 expression was an independent favorable factor for OS (HR = 0.629, p = 0.084) and EFS (HR = 0.544, p = 0.024) distinct from the curative effect of GO treatment. When all patients were again divided into two groups, this time based on the median expression of ANXA5, patients undergoing chemotherapy combined with GO had significantly better OS (p = 0.0012) and EFS (p = 0.0003) in the ANXA5 high-expression group. Gene set enrichment analysis identified a relevant series of pathways associated with glutathione metabolism, leukocyte transendothelial migration, and hematopoietic cell lineage.Conclusion:The expression level of ANXA5 can help optimize the treatment regimen for individual patients, and patients with overexpression of ANXA5 may circumvent poor outcomes from chemotherapy combined with GO.

Genetic variations and haplotypes in the annexin A5 gene are associated with the risk of recurrent pregnancy loss.

The expression of annexin A5 (ANXA5) was shown to affect the pathogenesis of recurrent pregnancy loss (RPL). In this study, the effects of two haplotypes, M1 and M2, on the transcription efficiency of ANXA5 promoter were explored. Correlation analysis was used to investigate the association between the single-nucleotide polymorphism haplotypes in ANXA5 promoter and the risk of RPL. And a luciferase reporter assay was carried out to study the effects of haplotypes M1 and M2 on the transcription efficiency of the ANXA5 promoter. To study the association between ANXA5 haplotypes and the risk of RPL, real-time polymerase chain reaction, western blot analysis, and immunohistochemistry assays were conducted to observe the expressions of ANXA5 messenger RNA (mRNA) and protein. Compared to M1 haplotype carriers, M2 haplotype carriers were associated with a higher risk of RPL. Additionally, compared to GATGTC haplotype carriers, GATGGC haplotype carriers were associated with a higher risk of RPL. Compared with RPL cases, the incidences of M2 haplotype were lower in both the population control and parous control cases. Furthermore, M2 carriers showed more significantly decreased activity of ANXA5 promoter compared to the carriers of other haplotypes, indicating that the haplotypes of ANXA5 promoter may be used as a potential biomarker to predict the prognosis of RPL. Moreover, the activity of ANXA5 as well as the mRNA/protein expression of ANXA5 was significantly downregulated in RPL patients, indicating that the M2 haplotype significantly increased the risk of RPL. Therefore, haplotype M2 increased the risk of RPL by inhibiting the expression of ANXA5.

ANXA5 Enhances Malignancy of Murine Hepatocarcinoma Hca-P Cells &amp;lt;i&amp;gt;via&amp;lt;/i&amp;gt; ERK Activation and E-cadherin Suppression

The invasion and metastasis are linked to the rapid progression and poor prognosis of hepatocarcinoma patients. Lymphatic metastasis is potentially involved in above pathogenesis with unclear mechanism. Previously, we found the deregulation of annexin A5 (ANXA5), a member of Ca²⁺-regulated phospholipid- and membrane-binding annexin family protein, mediated the <i>in vivo</i> malignancy, lymph node metastasis (LNM) rate and level of mice transplanted with Hca-P, a murine hepatocarcinoma cell line with the LNM potential rate of ~25%. Current work aimed to investigate the influence with action mechanism of ANXA5 overexpression on the <i>in vitro</i> malignant behaviours of Hca-P cells. For overexpressing ANXA5, the <i>Anxa5</i> gene was ligated into pCDNA3.1 (+) vector and transfected into Hca-P named as Hca-P-ANXA5up. Hca-P transfected with empty pCDNA3.1 (+) vector was named as Hca-P-mock and used as the control. The monoclonal Hca-P-mock and Hca-P-ANXA5up cell lines were obtained against G418 screening using limiting dilution method. Compared with the Hca-P-mock cells, Western blotting assay indicated ANXA5 expression level was increased by 50.1% (<i>p</i>=0.025) in the Hca-P-ANXA5up cells. Transwell chamber assays indicated that the migration and invasion capacities of Hca-P-ANXA5up cells were increased by 150.2% (<i>p</i>=0.001) and 94.8% (<i>p</i>=0.003) than Hca-P-mock cells. ANXA5 overexpression enhanced the levels of p-MEK (Ser217/221), ERK1, ERK2, p-ERK1 (Thr202/Tyr204) and p-ERK2 (Thr185/Tyr187), and suppressed the levels of E-Cadherin, Snail and Slug in Hca-P cells. Current work shows ANXA5 overexpression enhances the malignant behaviours of hepatocarcinoma Hca-P cells through activating p-MEK-ERK pathway and suppressing E-Cadherin, Snail and Slug. It is of potential value in tumor malignancy and lymphatic metastasis of hepatocarcinoma.

Annexin A5 overexpression might suppress proliferation and metastasis of human uterine cervical carcinoma cells.

Annexin A5 (ANXA5) is a kind of Ca2+-dependent phospholipid binding protein which is involved in cell membrane dynamics and organization. Recent data showed that ANXA5 might involve in tumorigenesis. To explore what role ANXA5 play in human uterine cervical carcinoma. In this study, a recombined ANXA5 plasmid was constructed and uterine cervical carcinoma cell lines HeLa and SiHa were transfected with it. After ANXA5 overexpression was determined by Western Blot, cell proliferation test was detected by MTT assay and colony formation assay respectively. FACS assay and Hochest33258 staining methods were employed to detect cell apoptosis. To further investigate whether ANXA5 influence cell migration and invasion, wound healing assay and transwell assay were applied. At the same time, the relative mechanism was investigated. When ANXA5 expression increased, cell proliferation was inhibited by regulating the expression of bcl-2 and bax while cell metastasis was suppressed by regulating E-cadherin and MMP-9 expression. ANXA5 overexpression in the uterine cervical carcinoma might play important roles in cell proliferation and metastasis of uterine cervical cancer cells and act as an anti-cancer gene in uterine cervical cancer.

Annexin A1 is a novel target gene of gonadotropin-releasing hormone in LβT2 gonadotrope cells.

Gonadotropin-releasing hormone (GnRH) regulates gonadotropin secretion. We previously demonstrated that the expression of annexin A5 (ANXA5) is stimulated by GnRH in gonadotropes and has a significant role in gonadotropin secretion. It is therefore of interest to know whether other members of the ANXA family, which consists of twelve structurally related members, are also regulated by GnRH. Therefore, the expression of all annexins was examined in LβT2 gonadotrope cells. ANXA4, A5, A6, A7 and A11 were detected in LβT2 cells. The expression of ANXA5 and A1 mRNA was stimulated by a GnRH agonist. An increase in ANXA1 protein by this agonist was demonstrated by western blotting. Immunohistochemistry showed that ANXA1 was present in the nucleus and to a lesser extent in the cytoplasm of some rat pituitary cells. The GnRH agonist induced translocation of ANXA1 to the periphery of LβT2 cells. The presence of ANXA1 in gonadotropes and its increase upon GnRH agonist treatment were confirmed in a primary pituitary cell culture. ANXA1 expression was also demonstrated in the ovary, the testis, the thyroid gland and the pancreas in a different manner to that of ANXA5. These data suggest that ANXA1 is a novel GnRH target gene in gonadotropes. ANXA1 also may be a target of local GnRH in peripheral tissues and may have a different role than that of ANXA5.

The association between annexin A5 (ANXA5) gene polymorphism and left ventricular hypertrophy (LVH) in Chinese endogenous hypertension patients.

Left ventricular hypertrophy (LVH) is common in endogenous hypertension (EH). We evaluated annexin A5 (ANXA5) promoter polymorphism in a cross-sectional study with a total of 850 EH patients, including 337 EH patients with LVH.Genotyping of ANXA5 promoter single nucleotide polymorphisms (SNPs) was conducted by SNaPshot assays and statistical analyses were performed to quantify its association with LVH.Of all potential SNPs, rs1050606 showed significant association with LVH (P = .008 in dominant and P = .006 in codominant models, respectively). During further analysis of SNPs on ANXA5 promoter region, rs1050606 had the most prominent effect. Furthermore, haplotypes M2 had higher risk of inducing LVH in EH patients compared with M1 (P = .032, OR = 1.42, 95%CI = 1.03–1.94). Patients with ANXA5 promoter haplotype GATGTC were also more susceptible to LVH (P = .022, OR = 1.35, 95%CI = 1.04–1.74). In the luciferase experiment, ANXA5 rs1050606 had the most promoter activity in myocardial cells (P < .001).These results showed that ANXA5 rs1050606 was significantly associated with LVH in Chinese EH patients, likely via influencing ANXA5 expression in serum and in myocardial cells.

Annexin A5 suppresses cyclooxygenase-2 expression by downregulating the protein kinase C-ζ-nuclear factor-κB signaling pathway in prostate cancer cells.

Annexin A5 (ANXA5) is a member of the annexin protein family. Previous studies have shown that ANXA5 is involved in anti-inflammation and cell death. However, the detailed mechanism of the role of ANXA5 in cancer cells is not well understood. In this study, we investigated the inhibitory effect of ANXA5 on cyclooxygenase-2 (COX-2) in prostate cancer cells. Expression of COX-2 induced by TNF-α was inhibited by overexpression of ANXA5 and inhibition of COX-2 expression by auranofin, which could induce ANXA5 expression, was restored by ANXA5 knockdown. In addition, ANXA5 knockdown induces phosphorylation of NF-κB p65 in prostate cancer cells, indicating that ANXA5 causes COX-2 downregulation through inhibition of p65 activation. We also found that protein kinase C (PKC)-ζ protein levels were upregulated by the inhibition of ANXA5, although the mRNA levels were unaffected. We have shown that upregulated COX-2 expression by inhibition of ANXA5 is attenuated by PKC-ζ siRNA. In summary, this study demonstrates that downregulation of PKC-ζ-NF-κB signaling by ANXA5 may inhibit COX-2 expression in prostate cancer.

Prolactin Suppression of Gonadotropin-Releasing Hormone Initiation of Mammary Gland Involution in Female Rats.

It has been demonstrated that mammary gland involution after lactation is initiated by accumulation of milk in alveoli after weaning. Here, we report that involution is also dependent on mammary GnRH expression that is suppressed by PRL during lactation. Reduction of plasma prolactin (PRL) by the withdrawal of suckling stimuli increased GnRH and annexin A5 (ANXA5) expression in the mammary tissues after lactation with augmentation of epithelial apoptosis. Intramammary injection of a GnRH antagonist suppressed ANXA5 expression and apoptosis of epithelial cells after forcible weaning at midlactation, whereas local administration of GnRH agonist (GnRHa) caused apoptosis of epithelial cells with ANXA5 augmentation in lactating rats. The latter treatment also decreased mammary weight, milk production, and casein accumulation. Mammary mast cells were strongly immunopositive for GnRH and the number increased in the mammary tissues after weaning. GnRHa was shown to be a chemoattractant for mast cells by mammary local administration of GnRHa and Boyden chamber assay. PRL suppressed the mammary expression of both ANXA5 and GnRH mRNA. It also decreased mast cell numbers in the gland after lactation. These results are the first to demonstrate that GnRH, synthesized locally in the mammary tissues, is required for mammary involution after lactation. GnRH is also suggested to introduce mast cells into the regressing mammary gland and would be in favor of tissue remodeling. The suppression of these processes by PRL is a novel physiological function of PRL.

Gonadotropin-releasing hormone is prerequisite for the constitutive expression of pituitary annexin A5.

Annexin A5 (ANXA5), a member of the structurally related family of annexin proteins, is expressed in pituitary gonadotropes. We previously reported that ANXA5 expression is stimulated by gonadotropin-releasing hormone (GnRH). In the present study, we investigated ANXA5 expression in the anterior pituitary gland of GnRH-deficient mutant hypogonadal (hpg) mice. RT-PCR demonstrated that luteinizing hormone β subunit (LHβ) and ANXA5 mRNA levels were both lower in the pituitary gland of hpg mice than in wild-type mice. Immunohistochemistry showed that ANXA5 expression throughout the pituitary gland was very low in hpg mice, suggesting that ANXA5 is diminished in gonadotropes and also in other cell types. Subcutaneous administration of a GnRH analogue, des-gly10 (Pro9)-GnRH ethylamide (1 μg/day for 7 days), augmented the expression of LHβ and ANXA5 in the pituitary gland in hpg mice. However, LHβ- and ANXA5-positive cells did not show exactly matched spatial distributions. These findings suggest that GnRH is necessary for constitutive ANXA5 expression in the pituitary gland, not only in gonadotropes but also in other pituitary gland cell types. A close relationship between ANXA5 and LHβ expression was confirmed. It is suggested that a significant role of ANXA5 in the physiologic secretion of LH.

Reduced ANXA5 mRNA and protein expression in pregnancies complicated by preeclampsia

The placental anticoagulant protein Annexin A5 (ANXA5) is a multifunctional protein that is highly expressed on the apical surfaces of syncytiotrophoblasts, and plays an important role in haemostatic regulations, maintaining blood fluidity of the placenta. The aim of this study was to investigate the expression of ANXA5 in pregnancies complicated by preeclampsia (PE). Placental tissue samples were collected from 23 pregnancies with PE and 34 normal pregnancies. ANXA5 mRNA levels were measured by quantitative Real-Time PCR (qPCR), while ANXA5 protein expression was measured by Western Blot (WB) and immunohistochemistry. ANXA5 mRNA expression in PE samples was lower than 1% of its expression in normal samples (mean ± SD: 0.002 ± 0.004 vs. 0.55 ± 0.38, p < 0.001), while ANXA5 protein levels in PE samples were approximately at 65% of the average normal expression (mean ± SD: 0.53 ± 0.30 vs. 0.81 ± 0.25, p=0.001). Immunohistochemical analysis also verified the above results, since PE placentas tended to have low labelling indexes (LIs), in contrast to controls which demonstrated high LIs (p=0.020). Statistical analysis of the WB data revealed that ANXA5 protein expression was increased in PE smokers vs. PE non-smokers (mean ± SD: 0.64 ± 0.23 vs. 0.41 ± 0.33, p=0.027). These results suggest that ANXA5 downregulation could be part of the pathophysiology of PE and the possible impairment in coagulation processes, which are seen in pregnancies that demonstrate PE. Further studies may investigate whether ANXA5 could be used as a biomarker for the early detection of PE and for the prediction of its severity.