Identification of actin network proteins, talin-1 and filamin-A, in circulating extracellular vesicles as blood biomarkers for human myalgic encephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious, debilitating disorder with a wide spectrum of symptoms, including pain, depression, and neurocognitive deterioration. Over 17 million people around the world have ME/CFS, predominantly women with peak onset at 30-50 years. Given the wide spectrum of symptoms and unclear etiology, specific biomarkers for diagnosis and stratification of ME/CFS are lacking. Here we show that actin network proteins in circulating extracellular vesicles (EVs) offer specific non-invasive biomarkers for ME/CFS. We found that circulating EVs were significantly increased in ME/CFS patients correlating to C-reactive protein, as well as biological antioxidant potential. Area under the receiver operating characteristic curve for circulating EVs was 0.80, allowing correct diagnosis in 90-94% of ME/CFS cases. From two independent proteomic analyses using circulating EVs from ME/CFS, healthy controls, idiopathic chronic fatigue, and depression, proteins identified from ME/CFS patients are involved in focal adhesion, actin skeletal regulation, PI3K-Akt signaling pathway, and Epstein-Barr virus infection. In particular, talin-1, filamin-A, and 14-3-3 family proteins were the most abundant proteins, representing highly specific ME/CFS biomarkers. Our results identified circulating EV number and EV-specific proteins as novel biomarkers for diagnosing ME/CFS, providing important information on the pathogenic mechanisms of ME/CFS.

Modulation of ADAR mRNA expression in patients with congenital heart defects.

Adenosine (A) to inosine (I) RNA editing is a hydrolytic deamination reaction catalyzed by the adenosine deaminase (ADAR) enzyme acting on double-stranded RNA. This posttranscriptional process diversifies a plethora of transcripts, including coding and noncoding RNAs. Interestingly, few studies have been carried out to determine the role of RNA editing in vascular disease. The aim of this study was to determine the potential role of ADARs in congenital heart disease. Strong downregulation of ADAR2 and increase in ADAR1 expression was observed in blood samples from congenital heart disease (CHD) patients. The decrease in expression of ADAR2 was in line with its downregulation in ventricular tissues of dilated cardiomyopathy patients. To further decipher the plausible regulatory pathway of ADAR2 with respect to heart physiology, miRNA profiling of ADAR2 was performed on tissues from ADAR2-/- mouse hearts. Downregulation of miRNAs (miR-29b, miR-405, and miR-19) associated with cardiomyopathy and cardiac fibrosis was observed. Moreover, the upregulation of miR-29b targets COL1A2 and IGF1, indicated that ADAR2 might be involved in cardiac myopathy. The ADAR2 target vascular development associated protein-coding gene filamin B (FLNB) was selected. The editing levels of FLNB were dramatically reduced in ADAR2-/- mice; however, no observable changes in FLNB expression were noted in ADAR2-/- mice compared to wild-type mice. This study proposes that sufficient ADAR2 enzyme activity might play a vital role in preventing cardiovascular defects.

Gain-of-Function Mutation in Filamin A Potentiates Platelet Integrin αIIbß3 Activation.

OBJECTIVE: Dominant mutations of the X-linked filamin A (FLNA) gene are responsible for filaminopathies A, which are rare disorders including brain periventricular nodular heterotopia, congenital intestinal pseudo-obstruction, cardiac valves or skeleton malformations, and often macrothrombocytopenia. APPROACH AND RESULTS: We studied a male patient with periventricular nodular heterotopia and congenital intestinal pseudo-obstruction, his unique X-linked FLNA allele carrying a stop codon mutation resulting in a 100-amino acid-long FLNa C-terminal extension (NP_001447.2: p.Ter2648SerextTer101). Platelet counts were normal, with few enlarged platelets. FLNa was detectable in all platelets but at 30% of control levels. Surprisingly, all platelet functions were significantly upregulated, including platelet aggregation and secretion, as induced by ADP, collagen, or von Willebrand factor in the presence of ristocetin, as well as thrombus formation in blood flow on a collagen or on a von Willebrand factor matrix. Most importantly, patient platelets stimulated with ADP exhibited a marked increase in αIIbß3 integrin activation and a parallel increase in talin recruitment to ß3, contrasting with normal Rap1 activation. These results are consistent with the mutant FLNa affecting the last step of αIIbß3 activation. Overexpression of mutant FLNa in the HEL megakaryocytic cell line correlated with an increase (compared with wild-type FLNa) in PMA-induced fibrinogen binding to and in talin and kindlin-3 recruitment by αIIbß3. CONCLUSIONS: Altogether, our results are consistent with a less binding of mutant FLNa to ß3 and the facilitated recruitment of talin by ß3 on platelet stimulation, explaining the increased αIIbß3 activation and the ensuing gain-of-platelet functions.

Proteasome proteolysis supports stimulated platelet function and thrombosis.

OBJECTIVE: Proteasome inhibitors used in the treatment of hematologic cancers also reduce thrombosis. Whether the proteasome participates in platelet activation or function is unclear because little is known of the proteasome in these terminally differentiated cells. APPROACH AND RESULTS: Platelets displayed all 3 primary proteasome protease activities, which MG132 and bortezomib (Velcade) inhibited. Proteasome substrates are marked by ubiquitin, and platelets contained a functional ubiquitination system that modified the proteome by monoubiquitination and polyubiquitination. Systemic MG132 strongly suppressed the formation of occlusive, platelet-rich thrombi in FeCl3-damaged carotid arteries. Transfusion of platelets treated ex vivo with MG132 and washed before transfusion into thrombocytopenic mice also reduced carotid artery thrombosis. Proteasome inhibition reduced platelet aggregation by low thrombin concentrations and ristocetin-stimulated agglutination through the glycoprotein Ib-IX-V complex. This receptor was not appropriately internalized after proteasome inhibition in stimulated platelets, and spreading and clot retraction after MG132 exposure also were decreased. The effects of proteasome inhibitors were not confined to a single receptor as MG132 suppressed thrombin-stimulated, ADP-stimulated, and lipopolysaccharide-stimulated microparticle shedding. Proteasome inhibition increased ubiquitin decoration of cytoplasmic proteins, including the cytoskeletal proteins Filamin A and Talin-1. Mass spectrometry revealed a single MG132-sensitive tryptic cleavage after R1745 in an extended Filamin A loop, which would separate its actin-binding domain from its carboxy terminal glycoprotein Ibα-binding domain. CONCLUSIONS: Platelets contain a ubiquitin/proteasome system that marks cytoskeletal proteins for proteolytic modification to promote productive platelet-platelet and platelet-wall interactions.

Altered mucosal DNA methylation in parallel with highly active Helicobacter pylori-related gastritis.

BACKGROUND: Chronic inflammation triggered by Helicobacter pylori causes altered DNA methylation in stomach mucosae, which is deeply involved in gastric carcinogenesis. This study aimed to elucidate the correlation between altered mucosal DNA methylation levels and activity of H. pylori-related gastritis, because inflammatory activity shows particular correlations with the development of diffuse-type cancer. METHODS: Methylation levels in stomach mucosae of 78 healthy volunteers were determined by real-time methylation-specific PCR or bisulfite pyrosequencing. Examined loci were the promoter CpG islands of six genes (FLNc, HAND1, THBD, p41ARC, HRASLS, and LOX) and the CpG sites of non-coding repetitive elements (Alu and Satα) that are reportedly altered by H. pylori infection. Activity of H. pylori-related gastritis was evaluated using two serum markers: H. pylori antibody titer and pepsinogen II. RESULTS: Methylation levels of the six CpG islands were consistently increased, and those of the two repetitive elements were consistently decreased in a stepwise manner with the activity of gastric inflammation as represented by serum marker levels. Each serum marker level was well correlated with the overall DNA methylation status of stomach mucosa, and these two serologic markers were additive in the detection of the mucosa with severely altered DNA methylation. CONCLUSIONS: Alteration in mucosal DNA methylation level was closely correlated with activity of H. pylori-related gastritis as evaluated by serum markers. The observed correlation between altered DNA methylation levels and activity of H. pylori-related gastritis appears to be one of the relevant molecular mechanisms underlying the development of diffuse-type cancer.