Adipose derived stem cell extracellular vesicles modulate primary human macrophages to an anti-inflammatory phenotype in vitro.

EVs released by adipose derived stem cells (ADSCs) have shown promise as a therapeutic for tissue repair because of their purported immune-regulatory properties. Extracellular vesicles (EVs) from ADSCs could be beneficial in improving graft retention rates for autologous fat grafting (AFG) post-mastectomy as, currently, grafted tissue rates are variable. Enriching grafted tissue with ADSC-EVs may improve retention rates by modulating macrophages resident within both the breast and lipoaspirate. We aimed to identify key macrophage phenotypes that are modulated by ADSC-EVs in vitro. ADSCs were isolated from lipoaspirates of women undergoing AFG and characterised by flow cytometry and differentiation potential. ADSC-EVs were isolated from culture media and characterised by tuneable resistive pulse sensing, transmission electron microscopy and Western blot. Primary monocyte-derived macrophages were polarized to an M1-like (GM-CSF, IFNγ), M2-like phenotype (M-CSF, IL-4) or maintained (M0-like; M-CSF) and ADSC-EVs were co-cultured with macrophages for 48 h. Flow cytometry and high-dimensional analysis clustered macrophages post co-culture. A manual gating strategy was generated to recapitulate these clusters and was applied to a repeat experimental run. Both runs were analysed to examine the prevalence of each cluster, representing a unique macrophage phenotype, with and without ADSC-EVs. Following the addition of ADSC-EVs, M0-like macrophages demonstrated a reciprocal shift of cell distribution from a cluster with a 'high inflammatory profile' (CD36+++CD206+++CD86+++; 16.5 ± 7.0%; p < 0.0001) to a cluster with a 'lower inflammatory profile' (CD36+CD206+CD86+; 35  ± 21.5%; p < 0.05). M1-like macrophages shifted from a cluster with a 'high inflammatory profile' (CD206++CD11b++CD36++CD163++; 26.1 ± 9.4%; p = 0.0024) to a 'lower inflammatory profile' (CD206+CD11b+CD36+CD163+; 72.8  ± 8.7%; p = 0.0007). There was no shift in M2-like clusters following ADSC-EV treatment. ADSC-EVs are complex regulators of macrophage phenotype that can shift macrophages away from a heightened pro-inflammatory state.

Circulating Extracellular Vesicle MicroRNA as Diagnostic Biomarkers in Early Colorectal Cancer-A Review.

Colorectal cancer (CRC) is one of the most common malignancies in the developed world, with global deaths expected to double in the next decade. Disease stage at diagnosis is the single greatest prognostic indicator for long-term survival. Unfortunately, early stage CRC is often asymptomatic and diagnosis frequently occurs at an advanced stage, where long-term survival can be as low as 14%. Circulating microRNAs encapsulated in extracellular vesicles (EVs) have recently come to prominence as novel diagnostic markers for cancer. EV-miRNAs are dysregulated in the circulation of CRC patients compared to healthy controls, and several specific miRNA candidates have been posited as diagnostic markers, including miR-21, miR-23a, miR-1246, and miR-92a. This review outlines the current landscape of EV-miRNAs as potential diagnostic markers for CRC, with a specific focus on those able to detect early stage disease.

exRNA Atlas Analysis Reveals Distinct Extracellular RNA Cargo Types and Their Carriers Present across Human Biofluids.

To develop a map of cell-cell communication mediated by extracellular RNA (exRNA), the NIH Extracellular RNA Communication Consortium created the exRNA Atlas resource (https://exrna-atlas.org). The Atlas version 4P1 hosts 5,309 exRNA-seq and exRNA qPCR profiles from 19 studies and a suite of analysis and visualization tools. To analyze variation between profiles, we apply computational deconvolution. The analysis leads to a model with six exRNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4), each detectable in multiple biofluids (serum, plasma, CSF, saliva, urine). Five of the cargo types associate with known vesicular and non-vesicular (lipoprotein and ribonucleoprotein) exRNA carriers. To validate utility of this model, we re-analyze an exercise response study by deconvolution to identify physiologically relevant response pathways that were not detected previously. To enable wide application of this model, as part of the exRNA Atlas resource, we provide tools for deconvolution and analysis of user-provided case-control studies.

Plasma Circulating Extracellular RNAs in Left Ventricular Remodeling Post-Myocardial Infarction.

Despite substantial declines in mortality following myocardial infarction (MI), subsequent left ventricular remodeling (LVRm) remains a significant long-term complication. Extracellular small non-coding RNAs (exRNAs) have been associated with cardiac inflammation and fibrosis and we hypothesized that they are associated with post-MI LVRm phenotypes. RNA sequencing of exRNAs was performed on plasma samples from patients with "beneficial" (decrease LVESVI ≥ 20%, n = 11) and "adverse" (increase LVESVI ≥ 15%, n = 11) LVRm. Selected differentially expressed exRNAs were validated by RT-qPCR (n = 331) and analyzed for their association with LVRm determined by cardiac MRI. Principal components of exRNAs were associated with LVRm phenotypes post-MI; specifically, LV mass, LV ejection fraction, LV end systolic volume index, and fibrosis. We then investigated the temporal regulation and cellular origin of exRNAs in murine and cell models and found that: 1) plasma and tissue miRNA expression was temporally regulated; 2) the majority of the miRNAs were increased acutely in tissue and at sub-acute or chronic time-points in plasma; 3) miRNA expression was cell-specific; and 4) cardiomyocytes release a subset of the identified miRNAs packaged in exosomes into culture media in response to hypoxia/reoxygenation. In conclusion, we find that plasma exRNAs are temporally regulated and are associated with measures of post-MI LVRm.

The immunomodulatory role of tumor-derived extracellular vesicles in colorectal cancer.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide with rising mortality rates predicted in the coming decades. In light of this, there is a continued need for improvement in our understanding of CRC biology and the development of novel treatment options. Tumor-derived extracellular vesicles (tEVs) have emerged as both novel cancer biomarkers and functional mediators of carcinogenesis. tEVs are released by tumor cells in abundance and play an important role in mediating tumor cell-immune cell interactions in the tumor microenvironment. Furthermore, tEVs are released into the circulation in humans where they could also interact with circulating immune cells. This review aims to describe CRC-specific tEVs and what is currently known about their role in immunomodulation. In particular, we discuss the ability of CRC-derived tEVs to affect monocyte differentiation into macrophages and dendritic cells, and their effects on T-cell viability and activity. Finally, the potential for tEVs in the development of immunotherapies will be discussed.

Extracellular RNA Isolation from Cell Culture Supernatant.

Extracellular RNAs are emerging as novel biomarkers and mediators of intercellular communication. Various methods to isolate RNA from biofluids and cell culture supernatants have been previously used by investigators. Here, we describe several standardized protocols for the isolation of RNAs from cell culture supernatants that utilize commercially available kits and reagents.

miRNA Signatures of Insulin Resistance in Obesity.

OBJECTIVE: Extracellular microRNAs (miRNAs) represent functional biomarkers for obesity and related disorders; this study investigated plasma miRNAs in insulin resistance phenotypes in obesity. METHODS: One hundred seventy-five miRNAs were analyzed in females with obesity (insulin sensitivity, n = 11; insulin resistance, n = 19; type 2 diabetes, n = 15) and without obesity (n = 12). Correlations between miRNA level and clinical parameters and levels of 15 miRNAs in a murine obesity model were investigated. RESULTS: One hundred six miRNAs were significantly (adjusted P ≤ 0.05) different between controls and at least one obesity phenotype, including miRNAs with the following attributes: previously reported roles in obesity and altered circulating levels (e.g., miR-122, miR-192); known roles in obesity but no reported changes in circulating levels (e.g., miR-378a); and no current reported role in, or association with, obesity (e.g., miR-28-5p, miR-374b, miR-32). The miRNAs in the latter group were found to be associated with extracellular vesicles. Forty-eight miRNAs showed significant correlations with clinical parameters; stepwise regression retained let-7b, miR-144-5p, miR-34a, and miR-532-5p in a model predictive of insulin resistance (R2 = 0.57, P = 7.5 × 10-8 ). Both miR-378a and miR-122 were perturbed in metabolically relevant tissues in a murine model of obesity. CONCLUSIONS: This study expands on the role of extracellular miRNAs in insulin-resistant phenotypes of obesity and identifies candidate miRNAs not previously associated with obesity.

High Throughput Sequencing of Extracellular RNA from Human Plasma.

The presence and relative stability of extracellular RNAs (exRNAs) in biofluids has led to an emerging recognition of their promise as 'liquid biopsies' for diseases. Most prior studies on discovery of exRNAs as disease-specific biomarkers have focused on microRNAs (miRNAs) using technologies such as qRT-PCR and microarrays. The recent application of next-generation sequencing to discovery of exRNA biomarkers has revealed the presence of potential novel miRNAs as well as other RNA species such as tRNAs, snoRNAs, piRNAs and lncRNAs in biofluids. At the same time, the use of RNA sequencing for biofluids poses unique challenges, including low amounts of input RNAs, the presence of exRNAs in different compartments with varying degrees of vulnerability to isolation techniques, and the high abundance of specific RNA species (thereby limiting the sensitivity of detection of less abundant species). Moreover, discovery in human diseases often relies on archival biospecimens of varying age and limiting amounts of samples. In this study, we have tested RNA isolation methods to optimize profiling exRNAs by RNA sequencing in individuals without any known diseases. Our findings are consistent with other recent studies that detect microRNAs and ribosomal RNAs as the major exRNA species in plasma. Similar to other recent studies, we found that the landscape of biofluid microRNA transcriptome is dominated by several abundant microRNAs that appear to comprise conserved extracellular miRNAs. There is reasonable correlation of sets of conserved miRNAs across biological replicates, and even across other data sets obtained at different investigative sites. Conversely, the detection of less abundant miRNAs is far more dependent on the exact methodology of RNA isolation and profiling. This study highlights the challenges in detecting and quantifying less abundant plasma miRNAs in health and disease using RNA sequencing platforms.

Diurnal Variations of Circulating Extracellular Vesicles Measured by Nano Flow Cytometry.

The identification of extracellular vesicles (EVs) as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM) to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions) and concentration (proportions) of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus further inform their use as biomarkers for disease.

Extracellular Vesicles in Heart Disease: Excitement for the Future ?

Extracellular vesicles (EV), including exosomes, microvesicles and apoptotic bodies, are released from numerous cell types and are involved in intercellular communication, physiological functions and the pathology of disease. They have been shown to carry and transfer a wide range of cargo including proteins, lipids and nucleic acids. The role of EVs in cardiac physiology and heart disease is an emerging field that has produced intriguing findings in recent years. This review will outline what is currently known about EVs in the cardiovascular system, including cellular origins, functional roles and utility as biomarkers and potential therapeutics.