Presence Of Marker Proteins Research Articles

Exosome-mediated targeted delivery of NOX4 siRNA ameliorates angiotensin II-induced atrial fibrillation in mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research Foundation of Korea Background Exosomes have gained attention as nanocarrier; however, the poor targeting ability after systemic administration restrict exosomes' clinical application. The targeting ability of exosomes can be improved by surface modifications. Recently, the popular method is the cell engineering technique, but cell engineering processes are complex, high-cost, time-consuming, and labor-intensive. To resolve this problem, several previous reports demonstrated exosomes are directly modified by chemical reactions (e.g., biochemical conjugation or hydrophobic insertion). However, few studies have modified exosomes with targeting ligands for atrial fibrillation. Purpose This study aimed to develop exosomes endowed with heart-targeting properties using a fast and easy chemical modification method to serve as a promising therapeutic tool for atrial fibrillation. Methods We firstly generated heart-targeted delivery system (CTP-Exo) by engineering the surface of exosomes with cardiac targeting peptide (CTP) using bio-orthogonal copper-free click chemistry. Next, we loaded NOX4 siRNA into CTP-Exo, and NOX4 siRNA-mediated cardioprotective effects were examined in angiotensin II (Ang II)-treated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and mice. Results CTP-Exo had typical exosome properties, such as ~ 150 nm rounded or cup-shaped morphology, and the presence of marker proteins. Compared with unmodified-Exo, CTP-Exo specifically accumulated in the heart (P < 0.05). In addition, NOX4 siRNA-loaded CTP-Exo (siNOX4/CTP-Exo) exerted strong cardioprotective effects with excellent heart-targeting ability. In Ang II-treated iPSC-CMs, siNOX4/CTP-Exo exhibited the reduced the peak-to-peak interval variability, beating frequency, and Ca2+ transient amplitude. After intravenous injection of siNOX4/CTP-Exo in Ang II-treated mice, there was a significant improvement in cardiac function (P < 0.05) with reduced fibrosis and arrhythmia inducibility. Conclusion Taken together, our results suggest that CTP-Exo function as efficient vehicles for heart-targeted delivery of siRNA, which in turn may potentially be used for the treatment of atrial fibrillation.

Circulating Exosomes Isolated during Acute Chest Syndrome Disrupt Endothelial Integrity

Introduction: Acute chest syndrome (ACS) is a leading cause of morbidity and death among children with sickle cell disease (SCD). Aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of ACS. Endothelial integrity is partially regulated by circulating cell-derived extracellular vesicles (exosomes). We previously demonstrated that (even when at a healthy baseline) patients with SCD have increased numbers of circulating exosomes, and exosomes from SCD patients with a history of ACS cause increased disruption of endothelial integrity in vitro. The current study was designed to test the hypothesis that exosomes isolated from patients during an episode of ACS would induce much greater endothelial damage than those isolated from the same patient at their baseline.Methods: We identified 9 patients with SCD included in our biobank who had plasma isolated at baseline and at the beginning of an admission for ACS (prior to transfusion). Samples were considered baseline if the patient was more than 4 weeks since transfusion and had no new health-related complaints. ACS was defined by the presence of an infiltrate on chest x-ray combined with fever, pain, hypoxia or cough. Control plasma samples were obtained from children without SCD or known medical problems and a BMI < 95%ile. Exosomes were isolated from plasma using established methodologies. Assessment of vesicle size was performed by nanoparticle tracking analysis. To determine the effects on endothelium, exosomes were added to cultures of human microvascular endothelial cells (HMVEC), which were subsequently studied by fluorescence microscopy (after immunolocalization of VE-cadherin, reaction with TRITC-conjugated phalloidin and treatment with DAPI). Protein components of exosomes and cells were detected and quantified by immunoblotting. VE-cadherin mRNA expression was determined by qRT-PCR. Free heme was quantified using a colorimetric assay.Results: Nanoparticle tracking analysis showed that all exosome preparations (regardless of patient or disease status) had a size distribution with a sharp peak of between 90 - 100 nm. Immunoblotting showed the presence of exosomal marker proteins (CD63 and flotillin) in all samples. The abundance of these proteins was increased by 50% (on average) in the samples obtained during ACS. Immunoblots showed that the exosomes also contained hemoglobin, glycophorin A, and ferritin; of these, only hemoglobin was increased in patients with SCD (2-fold greater in SCD than control) and ACS (4-fold greater in ACS than control). Heme concentration in the exosome preparations was below the assay detection limit. Microscopy showed that application of exosomes to endothelial monolayers caused no immediate damage; in addition, most samples showed no significant damage after 24 hr. However, after 48 hr, ACS-exosomes caused alterations of the localization of VE-cadherin (with opening of gaps between cells) and of actin microfilaments (as detected by phalloidin staining) consistent with cell retraction. ACS exosomes caused significantly more disruption of the monolayers than baseline-exosomes (baseline: 0.36 ± 0.07 % cell-free space vs. ACS: 5.81±0.65 % cell-free space, p < 0.01). DAPI staining showed no differences in numbers of nuclei excluding cell death or detachment. Although VE-cadherin mRNA levels were unchanged, immunoblots showed a dramatic decrease (by 62%) in VE-cadherin protein (p <0.01), in cells treated with ACS-exosomes vs. baseline-exosomes.Conclusions: These results confirm and extend on our prior observations that circulating exosomes in SCD patients affect endothelial integrity. We confirmed that these patients have circulating vesicles consistent with designation as exosomes, based on size and the presence of marker proteins. Their composition shows that many of these exosomes must derive from red cell precursors, but they are not simply red cell-derived vesicles. ACS exosomes cause a disruption of endothelial monolayer integrity that involves rearrangement of the actin cytoskeleton and degradation of cadherin-based intercellular junctions. The time course suggests that it requires cellular signaling and perhaps new RNA/protein synthesis. We speculate that exosomes are central to the vascular pathology in all SCD patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 9992: Urine Proteome Analysis Allows Establishment of Polypeptide Patterns non-Invasively Reflecting Drug-Mediated Regression of Atherosclerosis in ApoE-/--Mice

Background: Recently, non-invasive urine proteome analysis established a multimarker proteome approach towards non-invasive detection of atherosclerosis in an ApoE-/--mouse model, and relevance of this proteome pattern was also confirmed in human atherosclerosis. Urine was used since it lacks peptidase activity, which is found in blood and could falsify proteome analysis. Additionally, non-invasive monitoring of anti-atherosclerotic therapy with statins is of high clinical interest, and could be an important tool for guiding medical therapy. We therefore aimed to identify urinary polypeptides reflecting statin therapy in the ApoE-/--mouse model. Methods and Results: Urine of ApoE-/--mice either on high fat diet (HFD) or chow diet (CD) was collected over 15 weeks; each group was divided into subgroups with or without statin therapy applied by oral gavaging (HFD+S and HFD-S). Capillary electrophoresis mass spectrometry (CE-MS) of urine samples identified 5 polypeptides specifically reflecting statin treatment, resulting in a shift towards a more “healthy” polypeptide pattern until week 12. Sequencing of the discovered polypeptides identified fragments of α1-antitrypsin, EGF, kidney androgen regulated protein (KAP) and napsin. In histology, plaque size was significantly reduced by statin treatment, and presence of marker proteins in atherosclerotic plaques was confirmed by immunohistochemistry. Furthermore, urinary excretion levels of α1-antitrypsin fragments significantly correlated with intraplaque α1-antitrypsin content, mirroring plaque protein expression in the urine proteome. Conclusions: Urine proteome analysis allows discovery of novel biomarkers for the non-invasive monitoring of an anti-atherosclerotic therapy with statins in an ApoE-/--mouse model. The established multimarker proteome pattern reflects regression of plaque size in histology, and marker excretion levels also correlate with their expression in immunohistochemistry. This approach allows monitoring of atherosclerosis progression and statin treatment in mice, and thus promises to be an important and clinically relevant tool for monitoring and guiding of established but even more so of novel developments of anti-atherosclerotic drugs.

Abstract 650: Response prediction to a multitargeted tyrosine kinase inhibitor by profiling serine/threonine kinase activity and inhibition

Abstract Introduction: Since treatment in oncology is rarely effective in all patients, stratification of patients with innate resistance or monitoring the emergence of acquired resistance will prevent patients being treated with ineffective drugs and will further the development of personal medicine. Several examples of such prediction methods have been reported, based on gene expression or presence of marker proteins. For kinase inhibitors, that target active kinases, it makes sense to monitor their effect on kinase activity ex vivo on a patients own tumor tissue. For the prediction of cell line response to a multityrosine kinase inhibitor (MTKI) in a proliferation assay, the feasibility of this approach has been shown, using tyrosine kinase activity profiling on a dynamic peptide microarray. Here we investigated the possibility of response prediction by profiling serine/threonine kinase activities. Methods 24 Cancer cell lines were cultured in the presence of a multi-tyrosine kinase inhibitor and the IC50 determined in a proliferation assay. Lysates from untreated cells were incubated in triplicate on a PamChip microarray comprising 140 ser/thr containing peptides in the presence and absence of hydroxystaurosporin or flavopiridol. Peptide phosphorylation by the lysates was monitored using a mixture of phosphoserine and phosphothreonine detecting antibodies. Ratios of inhibited/non-inhibited signal intensities were calculated for each peptide and correlated to the proliferation assay data. A peptide subset that gave ATP dependent signals (p&amp;lt;0.001) was used for classification by partial least square discriminant analysis (PLS-DA). Results. As shown previously, inhibition of tyrosine phosphorylation by MTKI was able to predict response or resistance of cells both in a proliferation assay and in xenografts1). Here, phosphorylation by ser/thr kinases was investigated. Basal phosphorylation profiles obtained on the PamChip peptide microarray did not correlate with sensitivity in the proliferation assay. Using ratios of phosphorylation in the presence and absence of inhibitor hydroxystaurosporin (a PKC/PDPK inhibitor), a peptide set able to predict response in the proliferation assay was identified. More inhibition was observed in cell lines expressing an activated Akt signaling pathway. When flavopiridol (a CDK inhibitor) was used, no profile predictive of MTKI response was found. Conclusion. Peptide microarrays can be used to predict response to treatment as exemplified for cell lines in a proliferation assay. Prediction of response can be based on inhibition profiles of both tyrosine kinases activity and ser/thr kinase activity. The multiparameter readout of kinase activity links response prediction to activity of relevant signaling pathways by the appropriate choice of kinase inhibitors. 1) Versele et al. Mol Cancer Therapeutics 2009; 8(7), 1846-1855. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 650.

Filamentous Actin Regulates Insulin Exocytosis through Direct Interaction with Syntaxin 4

Glucose-induced insulin exocytosis is coupled to associations between F-actin and SNARE proteins, although the nature and function of these interactions remains unknown. Toward this end we show here that both Syntaxin 1A and Syntaxin 4 associated with F-actin in MIN6 cells and that each interaction was rapidly and transiently diminished by stimulation of cells with d-glucose. Of the two isoforms, only Syntaxin 4 was capable of interacting directly with F-actin in an in vitro sedimentation assay, conferred by the N-terminal 39-112 residues of Syntaxin 4. The 39-112 fragment was capable of selective competitive inhibitory action, disrupting endogenous F-actin-Syntaxin 4 binding in MIN6 cells. Disruption of F-actin-Syntaxin 4 binding correlated with enhanced glucose-stimulated insulin secretion, mediated by increased granule accumulation at the plasma membrane and increased Syntaxin 4 accessibility under basal conditions. However, no increase in basal level Syntaxin 4-VAMP2 association occurred with either latrunculin treatment or expression of the 39-112 fragment. Taken together, these data disclose a new underlying mechanism by which F-actin negatively regulates exocytosis via binding and blocking Syntaxin 4 accessibility, but they also reveal the existence of additional signals and/or steps required to trigger the subsequent docking and fusion steps of exocytosis.

Mammalian Smaug Is a Translational Repressor That Forms Cytoplasmic Foci Similar to Stress Granules

Cytoplasmic events depending on RNA-binding proteins contribute to the fine-tuning of gene expression. Sterile alpha motif-containing RNA-binding proteins constitute a novel family of post-transcriptional regulators that recognize a specific RNA sequence motif known as Smaug recognition element (SRE). The Drosophila member of this family, dSmaug, triggers the translational repression and deadenylation of maternal mRNAs by independent mechanisms, and the yeast homologue Vts1 stimulates degradation of SRE-containing messengers. Two homologous genes are present in the mammalian genome. Here we showed that hSmaug 1, encoded in human chromosome 14, represses the translation of reporter transcripts carrying SRE motifs. When expressed in fibroblasts, hSmaug 1 forms cytoplasmic granules that contain polyadenylated mRNA and the RNA-binding proteins Staufen, TIAR, TIA-1, and HuR. Smaug 1 foci are distinct from degradation foci. The murine protein mSmaug 1 is expressed in the central nervous system and is abundant in post-synaptic densities, a subcellular region where translation is tightly regulated by synaptic stimulation. Biochemical analysis indicated that mSmaug 1 is present in synaptoneurosomal 20 S particles. These results suggest a role for mammalian Smaug 1 in RNA granule formation and translation regulation in neurons.