Annexin A10 is a novel prognostic biomarker of papillary thyroid cancer.

BACKGROUND: The incidence of thyroid cancer (TC) is increasing rapidly worldwide. The target therapy for papillary TC (PTC) is limited, and the studies of PTC prognostic biomarkers are not common. As a new member of annexin A (ANXA) family, the function and clinical significance of ANXA10 in PTC have not been well investigated. METHODS: Expressions of all the 12 ANXA members were detected with qPCR in 12 PTC tissues, and the ANXA10 mRNAs in PTCs and their adjacent normal thyroid tissues were compared. The subcellular location and expression of ANXA10 in 121 PTC patients were investigated with immunohistochemistry, which further classified the patients into subgroups with low or high ANXA10. The clinical significance and prognostic value of ANXA10 were estimated by analyzing its correlation with clinical factors and overall survival rates by the chi-squared test, univariate analyses, and multivariate analyses. RESULTS: ANXA10 had the highest expression in PTCs among all the ANXA members. Moreover, ANXA10 was significantly upregulated in PTC compared with normal thyroid tissues. The PTC patients with low and high expression of ANXA10 took up 70.25% (85/121) and 29.75% (36/121), respectively. ANXA10 expression was associated with tumor size, differentiation, and overall survival rates of PTC. ANXA10 was an independent prognostic biomarker predicting the poor outcome of PTC. CONCLUSIONS: ANXA10 expression was upregulated in PTC, and it was an independent prognostic biomarker of PTC, suggesting that ANXA10 may be a promising target for individual treatment of ANXA10.

Endothelial vascular markers in coronary surgery.

Coronary heart disease is associated with high morbidity and mortality. Endothelial dysfunction in affected patients is linked to long-term atherosclerotic disease progression and cardiovascular event rates. The present paper reports on changes in the levels of endothelial progenitor cells (VEGFR2/CD133/CD34), essential for endothelial repair, and of endothelial microvesicles (CD31/annexin V) as indicators of endothelial lesion, in patients undergoing coronary bypass surgery with respect both to baseline levels and to counts in healthy subjects. In an observational descriptive study, 31 patients scheduled for coronary revascularization surgery were compared with those of 25 healthy controls. In a subsequent longitudinal study, patients undergoing surgery were monitored at 5 timepoints up until 48 h after surgery. Endothelial progenitor cell (VEGFR2/CD133/CD34) and endothelial microvesicle (CD31/annexin V) levels were quantified by flow cytometry. Baseline endothelial progenitor cell counts in coronary patients were significantly lower than those of healthy controls (p < 0.001); however, after surgery, levels rose steadily over all 5 timepoints to 48 h  with statistically significant differences (p < 0.001) between intra-operative and 48 h after surgery (T5). Endothelial microvesicle levels were significantly higher in coronary patients prior to surgery than in healthy controls (p < 0.001), and despite declining at 48 h remained significantly higher than those of controls (p < 0.001). Coronary surgery has had a positive impact on the endothelium in the patients, prompting a decrease in signs of endothelial dysfunction and a considerable improvement in the endothelial repair mechanisms involved in angiogenesis, playing an important role in the inflammatory response and the remodelling process of ischemic myocardium in postoperative period.

Annexin A10 is a candidate marker associated with the progression of pancreatic precursor lesions to adenocarcinoma.

Annexins are a multigene family of calcium and phospholipid-binding proteins that play important roles in calcium signaling, cell motility, differentiation and proliferation. Our previous mass spectrometry-based proteomics study revealed that annexin A10 (ANXA10) was uniquely overexpressed in pancreatic CD24+ adenocarcinoma cells that were dissected from clinical PDAC tissues but was absent in CD24- adjacent normal cells. The correlation between ANXA10 expression and the progression of pancreatic cancer remains unknown. In this study, we performed an immunostaining assay to evaluate ANXA10 expression in 155 primary human tissue specimens, including normal pancreas, chronic pancreatitis (CP), pancreatic adenocarcinoma (PDAC), pancreatic intraepithelial neoplasia (PanIN, the most important precursor of PDAC), and intraductal papillary mucinous neoplasm (IPMN). The immunostaining result showed that ANXA10 was significantly overexpressed in PanINs, IPMNs, and PDACs but negative in normal pancreas and the majority of chronic pancreatitis tissues. Statistical analysis revealed that ANXA10 expression was significantly associated with PDAC and its precursor lesions (p<0.0001). Abundant ANXA10 expression was predominantly present in pancreatic ductal epithelial cells of PanINs, IPMNs, and tumor cells of PDACs. Since PDAC develops through a series of PanINs which in turn arise from pancreatic ducts, the consistent overexpression of ANXA10 in ductal epithelial cells in PanINs and PDACs but negative in normal pancreatic ducts suggests that ANXA10 could serve as a potential marker indicating the presence of PDAC at its earliest precancerous stages. Double immunostaining of ANXA10 and CD24 showed that there was a large overlap between these two markers in PDAC and high-grade neoplasia lesions. The statistical analysis showed that the coexpression of ANXA10 and CD24 was significantly correlated with the progression of pancreatic precursor lesions towards PDACs.

Identification of natural peptides as a new class of antimalarial drugs by in silico approaches.

Malaria is one of the most widespread and serious parasitic diseases worldwide. Currently available antimalarial drugs have side effects, and many strains of Plasmodia have developed resistance to such drugs. The present review examines the use of annexins and of natural peptides from snake venom as a new class of anti-malarial agents, with the key property of reducing inflammation. Severe cases of malaria manifest elevated serum levels of liver enzymes, inflammation, fibrin deposition, apoptosis, and reduction in peripheral CD8+ T cells. The annexin-A1/5 proteins trigger inflammation via increased expression of diverse cytokines (tumor necrosis factor alpha, interleukin-1 beta, interleukin-10), however, by shielding microbial phospholipids they prevent injury via damage-associated molecular patterns (DAMPs). Here, we also review an in silico-based bioengineering approach that may allow for a better design, synthesis and characterization of novel peptides from snake venom as a more effective approach to treatment due to their improved antimalarial activity.

Detection of multiple annexin autoantibodies in a patient with recurrent miscarriages, fulminant stroke and seronegative antiphospholipid syndrome.

Anti-phospholipid syndrome (APS) is one of the main causes for recurrent miscarriages. The diagnosis of APS is based on the occurrence of clinical symptoms such as thrombotic events or obstetric complications as well as the detection of antiphospholipid antibodies directed against ß2-glycoprotein I and cardiolipin, or a positive lupus anticoagulant assay. However, there is a subpopulation of patients with clinical symptoms of APS, but the lack of serological markers (seronegative APS). In addition, a large proportion of patients with unexplained recurrent miscarriages exist. These cases may be attributed, at least in part, to a seronegative APS.The presence of autoantibodies against annexins is potentially associated with APS. Here we used immunoassays and immunoblots to detect autoantibodies directed against annexin A1-5, and A8, respectively, in a patient with a seronegative APS and a history of six recurrent pregnancy losses and fulminant stroke. We found strong IgM isotype antibody reactivity directed against annexin A2 and annexin A8, and moderate to weak IgM isotype antibody reactivity directed against annexin A1, A3, and A5. Further studies will evaluate the diagnostic value of IgM isotype antibodies against annexin A1-A5, and A8 for seronegative APS and recurrent miscarriages.

Exosomal microRNA Biomarkers: Emerging Frontiers in Colorectal and Other Human Cancers.

Diagnostic strategies, particularly non-invasive blood-based screening approaches, are gaining increased attention for the early detection and attenuation of mortality associated with colorectal cancer (CRC). However, the majority of current screening approaches are inadequate at replacing the conventional CRC diagnostic procedures. Yet, due to technological advances and better understanding of molecular events underlying human cancer, a new category of biomarkers are on the horizon. Recent evidence indicates that cells release a distinct class of small vesicles called 'exosomes', which contain nucleic acids and proteins that reflect and typify host-cell molecular architecture. Intriguingly, exosomes released from cancer cells have a distinct genetic and epigenetic makeup, which allows them to undertake their tumorigenic function. From a clinical standpoint, these unique cancer-specific fingerprints present in exosomes appear to be detectable in a small amount of blood, making them very attractive substrates for developing cancer biomarkers, particularly noninvasive diagnostic approaches.

Annexin A11 is associated with pulmonary fibrosis in African American patients with sarcoidosis.

Not available.

Eryptosis and oxidative damage in type 2 diabetic mellitus patients with chronic kidney disease.

It has been suggested that oxidative stress may participate in the progression of diabetes and its complications. Long-term complications of type 2 diabetes mellitus (T2DM) include retinopathy, atherosclerosis, shortened life span of erythrocytes, nephropathy, and chronic kidney disease (CKD). Oxidative damage has been associated with erythrocyte apoptosis induction in other pathological conditions. Our aim was to study the presence of eryptosis and its possible relationship with oxidative damage in patients with T2DM without CKD (T2DM/CKD(-)) and in patients with T2DM and CKD (T2DM/CKD(+)).Oxidative damage of lipids erythrocytes were increased in diabetic patients. The highest lipoperoxidation was found in T2DM/CKD(+). Likewise, the lower plasma total antioxidant capacity, GSH/GSSG ratio, and GSH in erythrocytes were found in T2DM/CKD(+) patients. A negative correlation was found between plasma total antioxidant capacity and oxidative damage. Phosphatidylserine (PS) externalization was measured in erythrocytes to evaluate eryptosis. Annexin binding in erythrocytes of T2DM/CKD(+) patients was higher than in healthy subjects and T2DM/CKD(-) patients. A positive correlation between lipoperoxidation and PS externalization in erythrocytes was found. This work showed that the erythrocytes of diabetic patients have increased oxidative damage, a reduction of antioxidant systems and more erythrocyte PS externalization. The duration of diabetes and the presence of CKD increase both oxidative damage and eryptosis. It is possible that a longer time of evolution induces an increase in erythrocyte oxidative damage and the consumption of blood antioxidant systems, adding to the osmotic stress in CKD and so contributes to an increase in PS externalization in diabetic patients.

Circulating cell-derived microparticles in women with pregnancy loss.

PROBLEM: To analyze cell-derived microparticles (cMP) in pregnancy loss (PL), both recurrent miscarriages (RM) and unexplained fetal loss (UFL). METHOD OF STUDY: Non-matched case-control study was performed at Vall d'Hebron Hospital. Cell-derived microparticles of 53 PL cases, 30 with RM, 16 with UFL, and 7 (RM + UFL), were compared to 38 healthy pregnant women. Twenty healthy non-pregnant women act as controls. Cell-derived microparticles were analyzed through flow cytometry. Results are given as total annexin (A5+), endothelial-(CD144+/CD31+ CD41-), platelet-(CD41+), leukocyte-(CD45+) and CD41- c-MP/µL of plasma. Antiphospholipid antibodies (aPLA) were analyzed according to established methods. RESULTS: Comparing PL versus healthy pregnant, we observed a significant endothelial cMP decrease in PL. When comparing RM subgroup with controls, we observed significant decreases in endothelial cMP. When comparing the PL positive for aPLA versus PL-aPLA-negative, no cMP numbering differences were seen. CONCLUSION: Pregnancy loss seems to be related to endothelial cell activation and/or consumption. A relationship between aPLA and cMP could not be demonstrated.

[Glomerular filtration rate is an indicator of endothelial function at early stages of chronic kidney disease].

AIM: To study effects of glomerular filtration rate (GFR) reduction on endothelial function in patients at early stages of chronic renal kidney (CKD). MATERIAL AND METHODS: Endothelial function of 101 patients with CKD of stage I-III was examined using reactive hyperemia test, dopplerography of skin vessels with ionophoresis of acetylcholin and nitroglycerin, lipidogram parameters, homocistein and annexin A5 levels, intima-media complex thickness of the common carotid artery, echocardiography findings. RESULTS: Cardiovascular complications risk factors were found in all the patients: increased body mass index, arterial hypertension, dyslipoproteinemia, hyperhomocysteinemia. Reduced GFR (under 90 ml/min) is an independent factor of atherosclerosis risk. CONCLUSION: GFR reduction corresponding to CRD of stage II is accompanied with enhancement of apoptosis and development of vasomotor endothelial dysfunction that in combination with risk factors contribute to development of a preclinical atherosclerosis phase.

Suicidal death of erythrocytes in recurrent hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia with fragmented erythrocytes, thrombocytopenia, and acute renal failure. Lack of complement inactivating factor H predisposes to the development of atypical HUS. Little is known about mechanisms linking complement activation with loss of erythrocyte integrity during HUS. Recent studies disclosed that increased cytosolic Ca2+ activity and cellular ceramide trigger programmed erythrocyte death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. In the present study, we investigated whether eryptosis occurs during the course of HUS. To this end, erythrocytes from healthy volunteers were exposed to plasma from a patient with severe idiopathic recurrent HUS secondary to factor H depletion. Phosphatidylserine exposure (Annexin binding), cell volume (forward scatter), cytosolic Ca2+ activity (Fluo3 fluorescence), and ceramide formation [anti-ceramide antibody and enzymatic (diacylgycerol kinase) analysis] were determined. Exposure of erythrocytes to plasma from the patient, but not to plasma from healthy individuals, triggered Annexin binding. The effect of plasma on erythrocyte Annexin binding was abolished by plasmapheresis or filtration at 30 kDa. It was paralleled by formation of ceramide and increase of cytosolic Ca2+ activity. Enhanced Annexin binding of erythrocytes from healthy individuals was observed after exposure to plasma from three other patients with HUS. The proeryptotic effect of patient plasma was mimicked by exposure to the Ca2+ ionophore ionomycin, and eryptosis was potentiated in the presence of cell membrane-permeable C6-ceramide. Furthermore, in vitro complement activation similarly triggered erythrocyte phosphatidylserine exposure, an effect which was blunted by the addition of factor H. In conclusion, our present observations disclose a novel, pathophysiological, factor-H dependent mechanism leading to injury of erythrocytes during the course of hemolytic uremic syndrome.

In vivo reduction of erythrocyte oxidant stress in a murine model of beta-thalassemia.

BACKGROUND AND OBJECTIVES: Oxidant damage is an important contributor to the premature destruction of erythrocytes and anemia in thalassemias. To assess the extent of oxidant damage of circulating erythrocytes and the effects of antioxidant therapy on erythrocyte characteristics and anemia, we used a mouse model of human beta-thalassemia intermedia (b1/b2 deletion). DESIGN AND METHODS: Several parameters indicative of oxidant damage were measured at baseline and following administration of the semi-synthetic flavonoid antioxidant, 7-monohydroxyethylrutoside (monoHER), to beta-thalassemic mice at a dose of either 500 mg/kg i.p. once a day (n=6) or 250 mg/kg i.p. twice a day (n=6) for 21 days. RESULTS: Significant erythrocyte oxidant damage at baseline was indicated by: (i) dehydration, reduced cell K content, and up-regulated K-Cl co-transport; (ii) marked membrane externalization of phosphatidylserine; (iii) reduced plasma and membrane content of vitamin E; and (iv) increased membrane bound IgG. MonoHER treatment increased erythrocyte K content, and markedly improved all cellular indicators of oxidant stress and of lipid membrane peroxidation. While anemia did not improve, monoHER therapy reduced reticulocyte counts, improved survival of a fraction of red cells, and reduced ineffective erythropoiesis with decreased total bilirubin, lactate dehydrogenase and plasma iron. INTERPRETATION AND CONCLUSIONS: Antioxidant therapy reverses several indicators of oxidant damage in vivo. These promising antioxidant effects of monoHER should be investigated further.

The presence of antiphospholipid antibodies is not related to increased levels of annexin A5 in plasma.

Annexin A5 has been proposed to be important for shielding of negatively charged phospholipids from blood, thereby preventing the binding of clotting factors. It has been suggested that antiphospholipid antibodies can disrupt the binding of annexin A5 from negatively phospholipid-containing surfaces, resulting in uncontrolled coagulation. If this hypothesis is correct, than the plasma levels of annexin A5 will be increased in patients with antiphospholipid antibodies. Therefore, we have measured plasma levels of annexin A5 of 175 patients with systemic lupus erythematosus (SLE), of which 104 had antiphospholipid antibodies and 23 patients had primary antiphospholipid syndrome. The annexin A5 levels were compared with the annexin A5 plasma levels measured in 23 patients with diabetes mellitus type 2 and 35 healthy volunteers. We found a significant increase of annexin A5 plasma levels in patients with SLE (median 6.7 ng mL(-1)) and primary antiphospholipid syndrome (median 7.1 ng mL(-1)) as compared to patients with diabetes mellitus type 2 (median 3.3 ng mL(-1)) and healthy volunteers (median 3.9 ng mL(-1)). However, no correlation was found with the presence of antiphospholipid antibodies or with a history of thromboembolic complications. Based on these observations, we conclude that displacement of annexin A5 from cellular surfaces by antiphospholipid antibodies is not a common mechanism in patients with antiphospholipid antibodies.

Altered membrane phospholipid organization and erythrophagocytosis in E beta-thalassemia.

The underlying cause behind the accelerated destruction of erythrocytes in the bone marrow and in the peripheral circulation, accompanying the beta-thalassemic syndromes is still not clearly understood. The present investigation demonstrates that increased phagocytosis of erythrocytes in E beta-thalassemia is inhibited by the presence of phosphatidylserine (PS) vesicles, suggesting a PS-'PS-receptor' type of interaction in premature recognition of these erythrocytes by macrophages. Increased exposure of both aminophospholipids phosphatidylethanolamine (PE) and PS was demonstrated by fluorescamine labeling and annexin binding, respectively. The slower rate of translocation of PS across the bilayer suggested that this contributed towards the increased exposure of PS in E beta-thalassemic erythrocytes.

Prolonged expression of procoagulant activity of human platelets degranulated by thrombin.

Platelets are exposed to thrombin when they take part in arterial thrombus formation, and they may return to the circulation when they are freed by fibrinolysis and dislodged by flowing blood. Thrombin causes the expression of procoagulant activity on platelets, and if this activity persists, the recirculating platelets may contribute to subsequent thrombosis. We have developed techniques to degranulate human platelets by treatment with thrombin, and recover then as single, discrete platelets that aggregate in response to both weak and strong agonists. In the present study we examined the duration of procoagulant activity on the surface of thrombin-degranulated platelets by two methods: a prothrombinase assay, and the binding of 125I-labeled annexin. Control platelets generated 0.9 +/- 0.4 U thrombin per 10(7) platelets in 15 min. Suspensions of thrombin-degranulated platelets formed 5.4 +/- 0.1 U thrombin per 10(7) platelets in this time. Binding of 125I-annexin V was also greater with thrombin-treated platelets than with control platelets (controls: 1.7 +/- 0.1 ng annexin/10(7) platelets; thrombin-degranulated platelets: 6.8 +/- 0.2 ng annexin/10(7) platelets). With thrombin-degranulated platelets, increased procoagulant activity and annexin binding persisted for at least 4 h after degranulation and resuspension, indicating that the catalytic activity for the prothrombinase complex is not reversed during this time. These platelets maintained their ability to aggregate for 4 h, even in response to the weak agonist, ADP. Thus, platelets that have taken part in thrombus formation and returned to the circulation may contribute to the promotion of further thrombotic events because of the persistence of procoagulant activity on their surface.

Virtual lack of annexins in human platelets argues against a role in phospholipase A2 regulation and platelet secretion.

Annexins are calcium-phospholipid binding proteins which share structural similarities and common biochemical properties. These proteins seem to be involved in different pathways of cell activation such as regulation of phospholipase A2 activity, membrane-cytoskeleton interaction, and exocytosis. The aim of this study was to attempt to characterize annexins in human platelets. Our results were based on specific EGTA extraction of proteins from platelet homogenates, immunodetection with specific antibodies raised against annexins I, II, V and VI, and measurement of phospholipase A2 inhibition. Antibodies raised against annexins I, V and VI revealed only trace amounts of these proteins in platelet EGTA extracts which did not promote phospholipase A2 inhibition in an in vitro assay. In addition, upon precipitation of membranes in the presence of calcium followed by EGTA extraction, the main substrate of protein kinase C (40-47-kDa protein) displayed a behaviour strictly different from that of annexins. Although one cannot exclude that a small amount of annexin(s) becomes phosphorylated in activated human platelets, these data argue against a role of annexins in the regulation of intracellular phospholipase A2 or in the processes of exocytosis in activated human platelets.

Evidence for the presence and location of annexins in human platelets.

In this study the identity of annexins in human platelets has been determined together with their ability to be released by agents which induce platelet degranulation. The presence of proteins cross-reacting to antibodies against annexins I and V was detected in human platelets. However, the study provided evidence that these annexins are not located on the surface of the plasma membrane in a Ca++ dependent manner. Moreover, activation of platelets with several agents which induced platelet degranulation did not cause release of annexins I or V as determined by both immunoblotting and ELISA.

Calelectrin in human blood cells.

Calelectrin is a new calcium-binding protein isolated from the cholinergic nerve terminals of the electric organ of Torpedo marmorata, which is widely distributed in nervous tissues and selectively binds to membranes, self-aggregates, and promotes calcium-induced membrane aggregation as a function of calcium concentration. We now show by immunofluorescence and immune blotting procedures that this protein is also present in human blood cells. Immunofluorescence demonstrates calelectrin in all human leucocytes, including mononuclear cells, but not in platelets or in erythrocytes. The immunofluorescence indicates an exclusively cytoplasmic location of calelectrin with a diffuse distribution and no primary association with the cytoskeleton or the cell membranes. SDS-polyacrylamide gel electrophoresis with immune blotting of fractionated blood cells (thrombocytes, mononuclear cells, granulocytes and erythrocytes) reveals the presence of a single protein crossreactive with calelectrin from Torpedo marmorata in the granulocyte and mononuclear cell fractions only. Human calelectrin has a molecular weight similar to Torpedo calelectrin (approximately 34-35 kD) and also binds to membranes in a Ca(2+)-dependent manner. Our results have several implications: (1) Calelectrin is conserved during evolution between the fish Torpedo marmorata and humans; (2) its expression in neural and mesenchymal cells points to an important functional role of the protein; (3) its absence from platelets excludes the hypothesis that it is a necessary participant in exocytosis per se and suggests some other function in Ca(2+)-triggered processes.