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Extracellular vesicles (EVs) are intercellular communicators with key functions in physiological and pathological processes and have recently garnered interest because of their diagnostic and therapeutic potential. The past decade has brought about the development and commercialization of a wide array of methods to isolate EVs from serum. Which subpopulations of EVs are captured strongly depends on the isolation method, which in turn determines how suitable resulting samples are for various downstream applications. To help clinicians and scientists choose the most appropriate approach for their experiments, isolation methods need to be comparatively characterized. Few attempts have been made to comprehensively analyse vesicular microRNAs (miRNAs) in patient biofluids for biomarker studies. To address this discrepancy, we set out to benchmark the performance of several isolation principles for serum EVs in healthy individuals and critically ill patients. Here, we compared five different methods of EV isolation in combination with two RNA extraction methods regarding their suitability for biomarker discovery-focused miRNA sequencing as well as biological characteristics of captured vesicles. Our findings reveal striking method-specific differences in both the properties of isolated vesicles and the ability of associated miRNAs to serve in biomarker research. While isolation by precipitation and membrane affinity was highly suitable for miRNA-based biomarker discovery, methods based on size-exclusion chromatography failed to separate patients from healthy volunteers. Isolated vesicles differed in size, quantity, purity and composition, indicating that each method captured distinctive populations of EVs as well as additional contaminants. Even though the focus of this work was on transcriptomic profiling of EV-miRNAs, our insights also apply to additional areas of research. We provide guidance for navigating the multitude of EV isolation methods available today and help researchers and clinicians make an informed choice about which strategy to use for experiments involving critically ill patients.

Extracellular vesicles (EVs) are mediators of cell-to-cell communication in inflammatory lung diseases. They function as carriers for miRNAs which regulate mRNA transcripts and signaling pathways after uptake into recipient cells. We investigated whether miRNAs associated with circulating EVs regulate immunologic processes in COVID-19. We prospectively studied 20 symptomatic patients with COVID-19 pneumonia, 20 mechanically ventilated patients with severe COVID-19 (severe acute respiratory corona virus-2 syndrome, ARDS) and 20 healthy controls. EVs were isolated by precipitation, total RNA was extracted, profiled by small RNA sequencing and evaluated by differential gene expression analysis (DGE). Differentially regulated miRNAs between groups were bioinformatically analyzed, mRNA target transcripts identified and signaling networks constructed, thereby comparing COVID-19 pneumonia to the healthy state and pneumonia to severe COVID-19 ARDS. DGE revealed 43 significantly and differentially expressed miRNAs (25 downregulated) in COVID-19 pneumonia when compared to controls, and 20 miRNAs (15 downregulated) in COVID-19 ARDS patients in comparison to those with COVID-19 pneumonia. Network analysis for comparison of COVID-19 pneumonia to healthy controls showed upregulated miR-3168 (log2FC=2.28, p <0.001), among others, targeting interleukin-6 (IL6) (25.1, 15.2 - 88.2 pg/ml in COVID-19 pneumonia) and OR52N2, an olfactory smell receptor in the nasal epithelium. In contrast, miR-3168 was significantly downregulated in COVID-19 ARDS (log2FC=-2.13, p =0.003) and targeted interleukin-8 (CXCL8) in a completely activated network. Toll-like receptor 4 (TLR4) was inhibited in COVID-19 pneumonia by miR-146a-5p and upregulated in ARDS by let-7e-5p. EV-derived miRNAs might have important regulative functions in the pathophysiology of COVID-19: CXCL8 regulates neutrophil recruitment into the lung causing epithelial damage whereas activated TLR4, to which SARS-CoV-2 spike protein binds strongly, increases cell surface ACE2 expression and destroys type II alveolar cells that secrete pulmonary surfactants; both resulting in pulmonary-capillary leakage and ARDS. These miRNAs may serve as biomarkers or as possible therapeutic targets.

Cell‐free microRNAs (miRNAs) are transferred in disease state including inflammatory lung diseases and are often packed into extracellular vesicles (EVs). To assess their suitability as biomarkers for community‐acquired pneumonia (CAP) and severe secondary complications such as sepsis, we studied patients with CAP (n = 30), sepsis (n = 65) and healthy volunteers (n = 47) subdivided into a training (n = 67) and a validation (n = 75) cohort. After precipitating crude EVs from sera, associated small RNA was profiled by next‐generation sequencing (NGS) and evaluated in multivariate analyses. A subset of the thereby identified biomarker candidates was validated both technically and additionally by reverse transcription quantitative real‐time PCR (RT‐qPCR). Differential gene expression (DGE) analysis revealed 29 differentially expressed miRNAs in CAP patients when compared to volunteers, and 25 miRNAs in patients with CAP, compared to those with sepsis. Sparse partial‐least discriminant analysis separated groups based on 12 miRNAs. Three miRNAs proved as a significant biomarker signature. While expression levels of miR‐1246 showed significant changes with an increase in overall disease severity from volunteers to CAP and to sepsis, miR‐193a‐5p and miR‐542‐3p differentiated patients with an infectious disease (CAP or sepsis) from volunteers. Cell‐free miRNAs are potentially novel biomarkers for CAP and may help to identify patients at risk for progress to sepsis, facilitating early intervention and treatment.

Background: Open-heart surgery is one of the most commonly performed surgical procedures worldwide, but carries a substantial risk for adverse outcomes such as postoperative organ failure. Extracellular vesicle (EV)-based biomarkers for outcome prediction and risk-stratification may be useful to identify patients at risk for negative outcomes including mortality. Methods: We isolated serum EVs from patients (n = 19) prior to openheart surgery and from healthy volunteers (n = 20) by precipitation. EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblotting. Next-generation sequencing (NGS) was utilized to profile EV-associated miRNAs. Differential expression of miRNAs between patients and volunteers was assessed using DESeq2. Expression levels of dysregulated miRNAs were correlated to prospectively recorded outcome-relevant variables registered during and after surgery. Results: There were no significant differences in morphology or marker proteins in EV populations from patients and volunteers. In NGS data, however, a total of 86 and 77 miRNAs were significantly up- or downregulated, respectively, in patient EVs prior to surgery. Expression patterns of miRNAs separated cardiovascular disease patients from volunteers in principal component analysis. For a set of differentially regulated miRNAs, expression levels were found to correlate with intraoperative epinephrine dosing requirements (r = 0.52, p = 0.02), serum lactate levels (r = 0.47, p = 0.04) and low urine excretion (r = -0.48, p = 0.03), indicating a systemic low-perfusion state due to perioperative heart failure. These miRNAs include miR-125a-5p for epinephrine requirements (log2 fold change (FC) = 1.83, p-adj = 2.06E-06), let-7d3p (log2FC = 1.07, p-adj = 4.01E-08) for serum lactate and miR-30a-5p (log2FC = 1.04, padj = 2.06E-06) for low perioperative urine excretion. Clinical parameters more closely related to the surgical procedure than to organ dysfunction (e.g. duration of surgery and ICU therapy, inflammation) or demographic variables (e.g. age, sex or body mass index) did not significantly correlate with miRNA expression. Summary/Conclusion: Analysing EV-miRNAs prior to heart surgery might help to identify patients at risk for perioperative cardiovascular instability and adverse outcomes.

Background: The circulatory system entails arterial blood delivering nutritive substances to tissues, and venous blood removing metabolic waste. Although circulating extracellular vesicles (EVs) are crucial vehicles for intercellular communication, arteriovenous differences in EV composition still have to be revealed. Various biomarker studies use vesicular microRNA (miRNA) as target, and depending on the patient's state of health and the routinely used type of vascular access, blood samples may be either derived from arterial or venous origin. We compared EV-specific miRNA expression profiles of arterial and venous serum samples from cardiac surgical patients (CSPs) and further characterized the vesicles. Methods: We included 19 CSPs in our study. For each patient, arterial blood was drawn from the radial artery, while venous blood was sampled from the superior vena cava via indwelling vascular catheters. We isolated EVs from sera using a polymer-based precipitation method and extracted total vesicular RNA. EVs were characterized using nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Based on small RNA sequencing, differential expression analysis was performed using DESeq2. Results: EV size, concentration and morphology from arterial and venous blood samples were highly similar, and typical EV protein markers were present in all samples. The obtained next-generation sequencing data revealed no significantly regulated miRNAs between arterial and venous blood EVs (baseMean 50, log2 fold change >I1I, p-adj <0.05). High patient-specific intra-sample diversity was shown, while arteriovenous inter-sample variations were minimal (principal component analysis). Summary/Conclusion: Our data show that EVs from arterial and venous blood specimens of CSPs don't differ in size, morphology and miRNA content. It is likely that these results could be extended to other patient populations as well. Thus, it is probably feasible to use arterial or venous serum samples for EV biomarker studies with comparable results regarding miRNA expression profiles. This may not apply to all individuals and all disorders, however, and additional arteriovenous comparisons may have to be performed under different pathophysiologic situations (e.g. newborns or cardiogenic shock).

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (M pro ) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, M pro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood–brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the M pro -induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19. A novel study led by scientists in Lübeck, Germany, shows that SARS-CoV-2-infected brain endothelial cells undergo cell death due to the cleavage of NEMO by the viral protease M pro , potentially causing cerebral COVID-19 and ‘long COVID’ symptoms.

Background The availability of handheld, noninvasive sublingual video-microscopes allows for visualization of the microcirculation in critically ill patients. Recent studies demonstrate that reduced numbers of blood-perfused microvessels and increased penetration of erythrocytes into the endothelial glycocalyx are essential components of microvascular dysfunction. The aim of this study was to identify novel microvascular variables to determine the level of microvascular dysfunction in sepsis and its relationship with clinical variables. Methods This observational, prospective, cross-sectional study included 51 participants, of which 34 critically ill sepsis patients were recruited from intensive care units of a university hospital. Seventeen healthy volunteers served as controls. All participants underwent sublingual videomicroscopy by sidestream darkfield imaging. A new developed version of the Glycocheck™ software was used to quantify vascular density, perfused boundary region (PBR-an inverse variable of endothelial glycocalyx dimensions), red blood cell (RBC) velocity, RBC content, and blood flow in sublingual microvessels with diameters between 4 and 25 µm. Results A detailed analysis of adjacent diameter classes (1 µm each) of vessels between 4 and 25 µm revealed a severe reduction of vascular density in very small capillaries (5–7 µm), which correlated with markers of sepsis severity. Analysis of RBC velocity (V RBC ) revealed a strong dependency between capillary and feed vessel V RBC in sepsis patients ( R 2  = 0.63, p  < 0.0001) but not in healthy controls ( R 2  = 0.04, p  = 0.43), indicating impaired capillary (de-)recruitment in sepsis. This finding enabled the calculation of capillary recruitment and dynamic capillary blood volume (CBV dynamic ). Moreover, adjustment of PBR to feed vessel V RBC further improved discrimination between sepsis patients and controls by about 50%. By combining these dynamic microvascular and glycocalyx variables, we developed the microvascular health score (MVHS dynamic ™), which decreased from 7.4 [4.6–8.7] in controls to 1.8 [1.4–2.7] in sepsis patients ( p  < 0.0001) and correlated with sepsis severity. Conclusion We introduce new important diameter-specific quantification and differentiated analysis of RBC kinetics, a key to understand microvascular dysfunction in sepsis. MVHS dynamic , which has a broad bandwidth to detect microvascular (dys-) function, might serve as a valuable tool to detect microvascular impairment in critically ill patients.

A growing body of literature reports on the upregulation of C-X-C motif chemokine receptor 4 (CXCR4) in a variety of cancer entities, rendering this receptor as suitable target for molecular imaging and endoradiotherapy in a theranostic setting. For instance, the CXCR4-targeting positron emission tomography (PET) agent [ 68  Ga]PentixaFor has been proven useful for a comprehensive assessment of the current status quo of solid tumors, including adrenocortical carcinoma or small-cell lung cancer. In addition, [ 68  Ga]PentixaFor has also provided an excellent readout for hematological malignancies, such as multiple myeloma, marginal zone lymphoma, or mantle cell lymphoma. PET-based quantification of the CXCR4 capacities in vivo allows for selecting candidates that would be suitable for treatment using the theranostic equivalent [ 177 Lu]/[ 90 Y]PentixaTher. This CXCR4-directed theranostic concept has been used as a conditioning regimen prior to hematopoietic stem cell transplantation and to achieve sufficient anti-lymphoma/-tumor activity in particular for malignant tissues that are highly sensitive to radiation, such as the hematological system. Increasing the safety margin, pretherapeutic dosimetry is routinely performed to determine the optimal activity to enhance therapeutic efficacy and to reduce off-target adverse events. The present review will provide an overview of current applications for CXCR4-directed molecular imaging and will introduce the CXCR4-targeted theranostic concept for advanced hematological malignancies.

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages. Chumduri, Gurumurthy et al. show that cervical squamous and columnar epithelia derive from two stem cell populations, regulated by opposing Wnt signals, and that a Wnt-repressive environment can induce metaplasia.