Chitinase 3 like 1 contributes to the development of pulmonary vascular remodeling in pulmonary hypertension.

Chitinase 3 like 1 (CHI3L1) is the prototypic chitinase-like protein mediating inflammation, cell proliferation, and tissue remodeling. Limited data suggest CHI3L1 is elevated in human pulmonary arterial hypertension (PAH) and is associated with disease severity. Despite its importance as a regulator of injury/repair responses, the relationship between CHI3L1 and pulmonary vascular remodeling is not well understood. We hypothesize that CHI3L1 and its signaling pathways contribute to the vascular remodeling responses that occur in pulmonary hypertension (PH). We examined the relationship of plasma CHI3L1 levels and severity of PH in patients with various forms of PH, including group 1 PAH and group 3 PH, and found that circulating levels of serum CHI3L1 were associated with worse hemodynamics and correlated directly with mean pulmonary artery pressure and pulmonary vascular resistance. We also used transgenic mice with constitutive knockout and inducible overexpression of CHI3L1 to examine its role in hypoxia-, monocrotaline-, and bleomycin-induced models of pulmonary vascular disease. In all 3 mouse models of pulmonary vascular disease, pulmonary hypertensive responses were mitigated in CHI3L1-null mice and accentuated in transgenic mice that overexpress CHI3L1. Finally, CHI3L1 alone was sufficient to induce pulmonary arterial smooth muscle cell proliferation, inhibit pulmonary vascular endothelial cell apoptosis, induce the loss of endothelial barrier function, and induce endothelial-mesenchymal transition. These findings demonstrate that CHI3L1 and its receptors play an integral role in pulmonary vascular disease pathobiology and may offer a target for the treatment of PAH and PH associated with fibrotic lung disease.

Systolic Strain by Speckle-Tracking Echocardiography Is a Feasible and Sensitive Measure of Right Ventricular Dysfunction in Acute Respiratory Failure Patients on Mechanical Ventilation.

Right ventricular (RV) dysfunction is common in acute respiratory failure and associated with worse outcomes, but it can be difficult to detect in the ICU setting. Speckle-tracking echocardiography (STE) can identify early changes in RV systolic function and be quantified as systolic strain. We measured the feasibility of RV global longitudinal systolic strain (RV GLS) in respiratory failure patients and its association with clinical outcomes. DESIGN: Retrospective cohort. SETTING: Two tertiary hospital medical ICUs in Providence, RI, from March 2015 to January 2018. PATIENTS: Two hundred twenty-three patients with acute respiratory failure requiring mechanical ventilation (MV) with available echocardiograms. MEASUREMENTS AND MAIN RESULTS: Clinical data were extracted from medical records. RV GLS was measured via STE (TOMTEC, Chicago, IL), along with standard echocardiographic measurements by two independent readers blinded to outcomes. The average age was 65 years (range, 21-90 yr), 121 (54%) were men, and the most common etiology of respiratory failure was pneumonia (n = 83, 37%). The average RV GLS was -16% (sd ± 7). The intraobserver correlation coefficients were 0.78 and 0.94, whereas the interobserver correlation coefficient was 0.61 for RV GLS. In the majority of echocardiograms (n = 178, 80%), all wall segments were tracked appropriately by operator visual inspection. Worse RV GLS was associated with greater hospital mortality (odds ratio, 1.03; 95% CI, 1.00-1.07; p = 0.03), such that every 1% decrement in RV GLS was associated with up to a 7% increase in the risk of death. RV GLS was 90% sensitive for the detection of RV dysfunction compared with tricuspid annular plane systolic excursion. CONCLUSIONS: The measurement of RV GLS by STE in subjects on MV is feasible, reproducible, and sensitive for the detection of RV dysfunction. RV GLS may predict poor outcomes in acute respiratory failure.

Insights from the Menstrual Cycle in Pulmonary Arterial Hypertension.

Rationale: Sex hormones play a role in pulmonary arterial hypertension (PAH), but the menstrual cycle has never been studied.Objectives: We conducted a prospective observational study of eight women with stable PAH and 20 healthy controls over one cycle.Methods: Participants completed four study visits 1 week apart starting on the first day of menstruation. Relationships between sex hormones, hormone metabolites, and extracellular vesicle microRNA (miRNA) expression and clinical markers were compared with generalized linear mixed modeling.Results: Women with PAH had higher but less variable estradiol (E2) levels (P < 0.001) that tracked with 6-minute walk distance (P < 0.001), N-terminal prohormone of brain natriuretic peptide (P = 0.03) levels, and tricuspid annular plane systolic excursion (P < 0.01); the direction of these associations depended on menstrual phase. Dehydroepiandrosterone sulfate (DHEA-S) levels were lower in women with PAH (all visits, P < 0.001). In PAH, each 100-µg/dl increase in DHEA-S was associated with a 127-m increase in 6-minute walk distance (P < 0.001) and was moderated by the cardioprotective E2 metabolite 2-methoxyestrone (P < 0.001). As DHEA-S increased, N-terminal prohormone of brain natriuretic peptide levels decreased (P = 0.001). Expression of extracellular vesicle miRNAs-21, -29c, and -376a was higher in PAH, moderated by E2 and DHEA-S levels, and tracked with hormone-associated changes in clinical measures.Conclusions: Women with PAH have fluctuations in cardiopulmonary function during menstruation driven by E2 and DHEA-S. These hormones in turn influence transcription of extracellular vesicle miRNAs implicated in the pathobiology of pulmonary vascular disease and cancer.

Culture of pulmonary artery endothelial cells from pulmonary artery catheter balloon tips: considerations for use in pulmonary vascular disease.

Endothelial dysfunction is a hallmark of pulmonary arterial hypertension (PAH) but there are no established methods to study pulmonary artery endothelial cells (PAECs) from living patients. We sought to culture PAECs from pulmonary artery catheter (PAC) balloons used during right-heart catheterisation (RHC) to characterise successful culture attempts and to describe PAEC behaviour.PAECs were grown in primary culture to confluence and endothelial cell phenotype was confirmed. Standard assays for apoptosis, migration and tube formation were performed between passages three to eight. We collected 49 PAC tips from 45 subjects with successful PAEC culture from 19 balloons (39%).There were no differences in subject demographic details or RHC procedural details in successful versus unsuccessful attempts. However, for subjects who met haemodynamic criteria for PAH, there was a higher but nonsignificant (p=0.10) proportion amongst successful attempts (10 out of 19, 53%) versus unsuccessful attempts (nine out of 30, 30%). A successful culture was more likely in subjects with a lower cardiac index (p=0.03) and higher pulmonary vascular resistance (p=0.04). PAECs from a subject with idiopathic PAH were apoptosis resistant compared to commercial PAECs (p=0.04) and had reduced migration compared to PAECs from a subject with portopulmonary hypertension with high cardiac output (p=0.01). PAECs from a subject with HIV-associated PAH formed fewer (p=0.01) and shorter (p=0.02) vessel networks compared to commercial PAECs.Sustained culture and characterisation of PAECs from RHC balloons is feasible, especially in PAH with high haemodynamic burden. This technique may provide insight into endothelial dysfunction during PAH pathogenesis.

Mesenchymal Stem Cell Extracellular Vesicles Reverse Sugen/Hypoxia Pulmonary Hypertension in Rats.

Mesenchymal stem cell extracellular vesicles attenuate pulmonary hypertension, but their ability to reverse established disease in larger animal models and the duration and mechanism(s) of their effect are unknown. We sought to determine the efficacy and mechanism of mesenchymal stem cells' extracellular vesicles in attenuating pulmonary hypertension in rats with Sugen/hypoxia-induced pulmonary hypertension. Male rats were treated with mesenchymal stem cell extracellular vesicles or an equal volume of saline vehicle by tail vein injection before or after subcutaneous injection of Sugen 5416 and exposure to 3 weeks of hypoxia. Pulmonary hypertension was assessed by right ventricular systolic pressure, right ventricular weight to left ventricle + septum weight, and muscularization of peripheral pulmonary vessels. Immunohistochemistry was used to measure macrophage activation state and recruitment to lung. Mesenchymal stem cell extracellular vesicles injected before or after induction of pulmonary hypertension normalized right ventricular pressure and reduced right ventricular hypertrophy and muscularization of peripheral pulmonary vessels. The effect was consistent over a range of doses and dosing intervals and was associated with lower numbers of lung macrophages, a higher ratio of alternatively to classically activated macrophages (M2/M1 = 2.00 ± 0.14 vs. 1.09 ± 0.11; P < 0.01), and increased numbers of peripheral blood vessels (11.8 ± 0.66 vs. 6.9 ± 0.57 vessels per field; P < 0.001). Mesenchymal stem cell extracellular vesicles are effective at preventing and reversing pulmonary hypertension in Sugen/hypoxia pulmonary hypertension and may offer a new approach for the treatment of pulmonary arterial hypertension.

High-Risk Pulmonary Embolism: Current Evidence-Based Practices.

Acute pulmonary embolism (PE) causes significant morbidity and mortality, particularly for patients with subsequent right ventricular (RV) dysfunction. Once diagnosed, risk stratification is imperative for therapeutic decision making and centers on evaluation of RV function. Treatment includes supportive care, systemic anticoagulation, and consideration of reperfusion therapy. In addition to systemic anticoagulation, patients with high-risk PE should receive reperfusion therapy, typically with systemic thrombolysis. The role of reperfusion therapies, which include catheter-based interventions, systemic thrombolysis, and surgical embolectomy, are controversial in the management of intermediate risk PE. Catheter directed thrombolysis (CDT) can be considered in certain intermediate risk patients although prospective, comparative data for its use are lacking. Surgical or catheter embolectomy are viable treatment options for high-risk patients in whom reperfusion therapy is warranted but who have absolute contraindications to thrombolysis. Further research is needed to better elucidate which patients with PE would most benefit from advanced reperfusion therapies.

The Restrictive IV Fluid Trial in Severe Sepsis and Septic Shock (RIFTS): A Randomized Pilot Study.

OBJECTIVES: It is unclear if a low- or high-volume IV fluid resuscitation strategy is better for patients with severe sepsis and septic shock. DESIGN: Prospective randomized controlled trial. SETTING: Two adult acute care hospitals within a single academic system. PATIENTS: Patients with severe sepsis and septic shock admitted from the emergency department to the ICU from November 2016 to February 2018. INTERVENTIONS: Patients were randomly assigned to a restrictive IV fluid resuscitation strategy (≤ 60 mL/kg of IV fluid) or usual care for the first 72 hours of care. MEASUREMENTS AND MAIN RESULTS: We enrolled 109 patients, of whom 55 were assigned to the restrictive resuscitation group and 54 to the usual care group. The restrictive group received significantly less resuscitative IV fluid than the usual care group (47.1 vs 61.1 mL/kg; p = 0.01) over 72 hours. By 30 days, there were 12 deaths (21.8%) in the restrictive group and 12 deaths (22.2%) in the usual care group (odds ratio, 1.02; 95% CI, 0.41-2.53). There were no differences between groups in the rate of new organ failure, hospital or ICU length of stay, or serious adverse events. CONCLUSIONS: This pilot study demonstrates that a restrictive resuscitation strategy can successfully reduce the amount of IV fluid administered to patients with severe sepsis and septic shock compared with usual care. Although limited by the sample size, we observed no increase in mortality, organ failure, or adverse events. These findings further support that a restrictive IV fluid strategy should be explored in a larger multicenter trial.

Low dose 100 cGy irradiation as a potential therapy for pulmonary hypertension.

Pulmonary hypertension (PH) is an incurable disease characterized by pulmonary vascular remodeling and ultimately death. Two rodent models of PH include treatment with monocrotaline or exposure to a vascular endothelial growth factor receptor inhibitor and hypoxia. Studies in these models indicated that damaged lung cells evolve extracellular vesicles which induce production of progenitors that travel back to the lung and induce PH. A study in patients with pulmonary myelofibrosis and PH indicated that 100 cGy lung irradiation could remit both diseases. Previous studies indicated that murine progenitors were radiosensitive at very low doses, suggesting that 100 cGy treatment of mice with induced PH might be an effective PH therapy. Our hypothesis is that the elimination of the PH-inducing marrow cells by low dose irradiation would remove the cellular influences creating PH. Here we show that low dose whole-body irradiation can both prevent and reverse established PH in both rodent models of PH.

Daily rhythms influence the ability of lung-derived extracellular vesicles to modulate bone marrow cell phenotype.

Extracellular vesicles (EVs) are important mediators of intercellular communication and have been implicated in myriad physiologic and pathologic processes within the hematopoietic system. Numerous factors influence the ability of EVs to communicate with target marrow cells, but little is known about how circadian oscillations alter EV function. In order to explore the effects of daily rhythms on EV-mediated intercellular communication, we used a well-established model of lung-derived EV modulation of the marrow cell transcriptome. In this model, co-culture of whole bone marrow cells (WBM) with lung-derived EVs induces expression of pulmonary specific mRNAs in the target WBM. To determine if daily rhythms play a role in this phenotype modulation, C57BL/6 mice were entrained in 12-hour light/12-hour dark boxes. Lungs harvested at discrete time-points throughout the 24-hour cycle were co-cultured across a cell-impermeable membrane with murine WBM. Alternatively, WBM harvested at discrete time-points was co-cultured with lung-derived EVs. Target WBM was collected 24hrs after co-culture and analyzed for the presence of pulmonary specific mRNA levels by RT-PCR. In both cases, there were clear time-dependent variations in the patterns of pulmonary specific mRNA levels when either the daily time-point of the lung donor or the daily time-point of the recipient marrow cells was altered. In general, WBM had peak pulmonary-specific mRNA levels when exposed to lung harvested at Zeitgeber time (ZT) 4 and ZT 16 (ZT 0 defined as the time of lights on, ZT 12 defined as the time of lights off), and was most susceptible to lung-derived EV modulation when target marrow itself was harvested at ZT 8- ZT 12. We found increased uptake of EVs when the time-point of the receptor WBM was between ZT 20 -ZT 24, suggesting that the time of day-dependent changes in transcriptome modulation by the EVs were not due simply to differential EV uptake. Based on these data, we conclude that circadian rhythms can modulate EV-mediated intercellular communication.

Comparison of analogue and electronic stethoscopes for pulmonary auscultation by internal medicine residents.

BACKGROUND: Electronic stethoscopes are becoming more common in clinical practice. They may improve the accuracy and efficiency of pulmonary auscultation, but the data to support their benefit are limited. OBJECTIVE: To determine how auscultation with an electronic stethoscope may affect clinical decision making. METHODS: An online module consisting of six fictional ambulatory cases was developed. Each case included a brief history and lung sounds recorded with an analogue and electronic stethoscope. Internal medicine resident participants were randomly selected to hear either the analogue or electronic lung sounds. Numbers of correct answers, time spent on each case and numbers of times the recordings were played were compared between the groups who heard each mode of auscultation, with a p value of less than 0.05 indicating statistical significance. RESULTS: 61 internal medicine residents completed at least one case, and 41 residents completed all six cases. There were no significant differences in overall scores between participants who heard analogue and electronic lung sounds (3.14±0.10 out of 6 correct for analogue, 3.20±0.10 out of 6 for electronic, p=0.74). There were no significant differences in performance for any of the six cases (p=0.78), time spent on the cases (p=0.67) or numbers of times the recordings were played (p=0.85). CONCLUSION: When lung sounds were amplified with an electronic stethoscope, we did not detect an effect on performance, time spent on the cases or numbers of times participants listened to the recordings.

A modified Montpellier protocol for intubating intensive care unit patients is associated with an increase in first-pass intubation success and fewer complications.

BACKGROUND: The Montpellier protocol for intubating patients in the intensive care unit (ICU) is associated with a decrease in intubation-related complications. We sought to determine if implementation of a simplified version of the Montpellier protocol that removed selected components and allowed for a variety of pre-oxygenation modalities increased first-pass intubation success and reduced intubation-related complications. METHODS: A prospective pre/post-comparison of a modified Montpellier protocol in two medical and one medical/surgical/cardiac ICU within a hospital system. The modified eight-point protocol included: fluid administration, ordering sedation, two intubation trained providers, pre-oxygenation with non-invasive positive pressure ventilation, nasal high flow cannula or non-rebreather mask, rapid sequence intubation, capnography, sedation administration, and vasopressors for shock. RESULTS: Patient characteristics and indications for intubation were similar for the 275 intubations in the control (137) and intervention (138) periods. In the intervention vs. control periods, the modified Montpellier protocol was associated with a significant 16.2% [95% CI: 5.1-30.0%] increase in first-pass intubation success and a 12.6% [95% CI: 1.2-23.6%] reduction in all intubation-related complications. CONCLUSION: A simplified version of the Montpellier intubation protocol for intubating ICU patients was associated with an improvement in first-pass intubation success rates and a reduction in the rate of intubation-related complications.

Endothelial to haematopoietic transition contributes to pulmonary arterial hypertension.

AIMS: The pathogenic mechanisms of pulmonary arterial hypertension (PAH) remain unclear, but involve dysfunctional endothelial cells (ECs), dysregulated immunity and inflammation in the lung. We hypothesize that a developmental process called endothelial to haematopoietic transition (EHT) contributes to the pathogenesis of pulmonary hypertension (PH). We sought to determine the role of EHT in mouse models of PH, to characterize specific cell types involved in this process, and to identify potential therapeutic targets to prevent disease progression. METHODS AND RESULTS: When transgenic mice with fluorescence protein ZsGreen-labelled ECs were treated with Sugen/hypoxia (Su/Hx) combination to induce PH, the percentage of ZsGreen+ haematopoietic cells in the peripheral blood, primarily of myeloid lineage, significantly increased. This occurrence coincided with the depletion of bone marrow (BM) ZsGreen+ c-kit+ CD45- endothelial progenitor cells (EPCs), which could be detected accumulating in the lung upon PH-induction. Quantitative RT-PCR based gene array analysis showed that key transcription factors driving haematopoiesis were expressed in these EPCs. When transplanted into lethally irradiated recipient mice, the BM-derived EPCs exhibited long-term engraftment and haematopoietic differentiation capability, indicating these EPCs are haemogenic in nature. Specific inhibition of the critical haematopoietic transcription factor Runx1 blocked the EHT process in vivo, prevented egress of the BM EPCs and ultimately attenuated PH progression in Su/Hx- as well as in monocrotaline-induced PH in mice. Thus, myeloid-skewed EHT promotes the development of PH and inhibition of this process prevents disease progression in mouse models of PH. Furthermore, high levels of Runx1 expression were found in circulating CD34+ CD133+ EPCs isolated from peripheral blood of patients with PH, supporting the clinical relevance of our proposed mechanism of EHT. CONCLUSION: EHT contributes to the pathogenesis of PAH. The transcription factor Runx1 may be a novel therapeutic target for the treatment of PAH.

Bone Marrow Endothelial Progenitor Cells Are the Cellular Mediators of Pulmonary Hypertension in the Murine Monocrotaline Injury Model.

The role of bone marrow (BM) cells in modulating pulmonary hypertensive responses is not well understood. Determine if BM-derived endothelial progenitor cells (EPCs) induce pulmonary hypertension (PH) and if this is attenuated by mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). Three BM populations were studied: (a) BM from vehicle and monocrotaline (MCT)-treated mice (PH induction), (b) BM from vehicle-, MCT-treated mice that received MSC-EV infusion after vehicle, MCT treatment (PH reversal, in vivo), (c) BM from vehicle-, MCT-treated mice cultured with MSC-EVs (PH reversal, in vitro). BM was separated into EPCs (sca-1+/c-kit+/VEGFR2+) and non-EPCs (sca-1-/c-kit-/VEGFR2-) and transplanted into healthy mice. Right ventricular (RV) hypertrophy was assessed by RV-to-left ventricle+septum (RV/LV+S) ratio and pulmonary vascular remodeling by blood vessel wall thickness-to-diameter (WT/D) ratio. EPCs but not non-EPCs from mice with MCT-induced PH (MCT-PH) increased RV/LV+S, WT/D ratios in healthy mice (PH induction). EPCs from MCT-PH mice treated with MSC-EVs did not increase RV/LV+S, WT/D ratios in healthy mice (PH reversal, in vivo). Similarly, EPCs from MCT-PH mice treated with MSC-EVs pre-transplantation did not increase RV/LV+S, WT/D ratios in healthy mice (PH reversal, in vitro). MSC-EV infusion reversed increases in BM-EPCs and increased lung tissue expression of EPC genes and their receptors/ligands in MCT-PH mice. These findings suggest that the pulmonary hypertensive effects of BM are mediated by EPCs and those MSC-EVs attenuate these effects. These findings provide new insights into the pathogenesis of PH and offer a potential target for development of novel PH therapies. Stem Cells Translational Medicine 2017;6:1595-1606.

Exosomes induce and reverse monocrotaline-induced pulmonary hypertension in mice.

AIMS: Extracellular vesicles (EVs) from mice with monocrotaline (MCT)-induced pulmonary hypertension (PH) induce PH in healthy mice, and the exosomes (EXO) fraction of EVs from mesenchymal stem cells (MSCs) can blunt the development of hypoxic PH. We sought to determine whether the EXO fraction of EVs is responsible for modulating pulmonary vascular responses and whether differences in EXO-miR content explains the differential effects of EXOs from MSCs and mice with MCT-PH. METHODS AND RESULTS: Plasma, lung EVs from MCT-PH, and control mice were divided into EXO (exosome), microvesicle (MV) fractions and injected into healthy mice. EVs from MSCs were divided into EXO, MV fractions and injected into MCT-treated mice. PH was assessed by right ventricle-to-left ventricle + septum (RV/LV + S) ratio and pulmonary arterial wall thickness-to-diameter (WT/D) ratio. miR microarray analyses were also performed on all EXO populations. EXOs but not MVs from MCT-injured mice increased RV/LV + S, WT/D ratios in healthy mice. MSC-EXOs prevented any increase in RV/LV + S, WT/D ratios when given at the time of MCT injection and reversed the increase in these ratios when given after MCT administration. EXOs from MCT-injured mice and patients with idiopathic pulmonary arterial hypertension (IPAH) contained increased levels of miRs-19b,-20a,-20b, and -145, whereas miRs isolated from MSC-EXOs had increased levels of anti-inflammatory, anti-proliferative miRs including miRs-34a,-122,-124, and -127. CONCLUSION: These findings suggest that circulating or MSC-EXOs may modulate pulmonary hypertensive effects based on their miR cargo. The ability of MSC-EXOs to reverse MCT-PH offers a promising potential target for new PAH therapies.

Lung-derived exosome uptake into and epigenetic modulation of marrow progenitor/stem and differentiated cells.

BACKGROUND: Our group has previously demonstrated that murine whole bone marrow cells (WBM) that internalize lung-derived extracellular vesicles (LDEVs) in culture express pulmonary epithelial cell-specific genes for up to 12 weeks. In addition, the lungs of lethally irradiated mice transplanted with lung vesicle-modulated marrow have 5 times more WBM-derived type II pneumocytes compared to mice transplanted with unmanipulated WBM. These findings indicate that extracellular vesicle modification may be an important consideration in the development of marrow cell-based cellular therapies. Current studies were performed to determine the specific marrow cell types that LDEV stably modify. METHODS: Murine WBM-derived stem/progenitor cells (Lin-/Sca-1+) and differentiated erythroid cells (Ter119+), granulocytes (Gr-1+) and B cells (CD19+) were cultured with carboxyfluorescein N-succinimidyl ester (CFSE)-labelled LDEV. LDEV+ cells (CFSE+) and LDEV- cells (CFSE-) were separated by flow cytometry and visualized by fluorescence microscopy, analyzed by RT-PCR or placed into long-term secondary culture. In addition, murine Lin-/Sca-1+ cells were cultured with CFSE-labelled LDEV isolated from rats, and RT-PCR analysis was performed on LDEV+ and - cells using species-specific primers for surfactant (rat/mouse hybrid co-cultures). RESULTS: Stem/progenitor cells and all of the differentiated cell types studied internalized LDEV in culture, but heterogeneously. Expression of a panel of pulmonary epithelial cell genes was higher in LDEV+cells compared to LDEV - cells and elevated expression of these genes persisted in long-term culture. Rat/mouse hybrid co-cultures revealed only mouse-specific surfactant B and C expression in LDEV+ Lin-/Sca-1+cells after 4 weeks of culture, indicating stable de novo gene expression. CONCLUSIONS: LDEV can be internalized by differentiated and more primitive cells residing in the bone marrow in culture and can induce stable de novo pulmonary epithelial cell gene expression in these cells for several weeks after internalization. The gene expression represents a transcriptional activation of the target marrow cells. These studies serve as the basis for determining marrow cell types that can be used for cell-based therapies for processes that injure the pulmonary epithelial surfaces.

Potential functional applications of extracellular vesicles: a report by the NIH Common Fund Extracellular RNA Communication Consortium.

The NIH Extracellular RNA Communication Program's initiative on clinical utility of extracellular RNAs and therapeutic agents and developing scalable technologies is reviewed here. Background information and details of the projects are presented. The work has focused on modulation of target cell fate by extracellular vesicles (EVs) and RNA. Work on plant-derived vesicles is of intense interest, and non-mammalian sources of vesicles may represent a very promising source for different therapeutic approaches. Retro-viral-like particles are intriguing. Clearly, EVs share pathways with the assembly machinery of several other viruses, including human endogenous retrovirals (HERVs), and this convergence may explain the observation of viral-like particles containing viral proteins and nucleic acid in EVs. Dramatic effect on regeneration of damaged bone marrow, renal, pulmonary and cardiovascular tissue is demonstrated and discussed. These studies show restoration of injured cell function and the importance of heterogeneity of different vesicle populations. The potential for neural regeneration is explored, and the capacity to promote and reverse neoplasia by EV exposure is described. The tremendous clinical potential of EVs underlies many of these projects, and the importance of regulatory issues and the necessity of general manufacturing production (GMP) studies for eventual clinical trials are emphasized. Clinical trials are already being pursued and should expand dramatically in the near future.

Role of extracellular RNA-carrying vesicles in cell differentiation and reprogramming.

Growing evidence suggests that transcriptional regulators and secreted RNA molecules encapsulated within membrane vesicles modify the phenotype of target cells. Membrane vesicles, actively released by cells, represent a mechanism of intercellular communication that is conserved evolutionarily and involves the transfer of molecules able to induce epigenetic changes in recipient cells. Extracellular vesicles, which include exosomes and microvesicles, carry proteins, bioactive lipids, and nucleic acids, which are protected from enzyme degradation. These vesicles can transfer signals capable of altering cell function and/or reprogramming targeted cells. In the present review we focus on the extracellular vesicle-induced epigenetic changes in recipient cells that may lead to phenotypic and functional modifications. The relevance of these phenomena in stem cell biology and tissue repair is discussed.

Marrow Hematopoietic Stem Cells Revisited: They Exist in a Continuum and are Not Defined by Standard Purification Approaches; Then There are the Microvesicles.

Current concepts of hematopoiesis are encompassed in a hierarchical stem cell model. This developed initially from studies of colony-forming unit spleen and in vitro progenitors for different cell lineages, but then evolved into a comprehensive model of cells with different in vivo differentiative and proliferative potential. These cells were characterized and purified based largely on expression of a variety of lineage-specific and stem cell-specific surface epitopes. Monoclonal antibodies were bound to these epitopes and then used to physically and fluorescently separate different classes of these cells. The gold standard for the most primitive marrow stem cells was long-term multilineage repopulation and renewal in lethally irradiated mice. Progressive work seemed to have clonally defined a Lineage negative (Lin-), Sca-1+, c-kit+, CD150+ stem cell with great proliferative, differentiative, and renewal potential. This cell was stable and in the G0 phase of cell cycle. However, continued work in our laboratory indicated that the engraftment, differentiation, homing, and gene expression phenotype of the murine marrow stem cells continuously and reversibly changes with passage through cell cycle. Most recently, using cycle-defining supravital dyes and fluorescent-activated cell sorting and S-phase-specific tritiated thymidine suicide, we have established that the long-term repopulating hematopoietic stem cell is a rapidly proliferating, and thus a continually changing cell; as a corollary it cannot be purified or defined on a clonal single cell basis. Further in vivo studies employing injected and ingested 5-Bromodeoxyuridine (BrdU), showed that the G0 Lin-Sca-1, c-kit+ Flt3- cell was rapidly passing through cell cycle. These data are explained by considering the separative process: the proliferating stem cells are eliminated through the selective separations leaving non-representative dormant G0 stem cells. In other words, they throw out the real stem cells with the purification. This system, where the marrow stem cell continuously and reversibly changes with obligate cell cycle transit, is further complicated by the consideration of the impact of tissue microvesicles on the cell phenotypes. Tissue microvesicles have been found to alter the phenotype of marrow cells, possibly explaining the observations of "stem cell plasticity." These alterations, short-term, are due to transfer of originator cell mRNA and as yet undefined transcription factors. Long-term phenotype change is due to transcriptional modulation; a stable epigenetic change. Thus, the stem cell system is characterized by continuous cycle and microvesicle-related change. The challenge of the future is to define the stem cell population.

Cellular phenotype and extracellular vesicles: basic and clinical considerations.

Early work on platelet and erythrocyte vesicles interpreted the phenomena as a discard of material from cells. Subsequently, vesicles were studied as possible vaccines and, most recently, there has been a focus on the effects of vesicles on cell fate. Recent studies have indicated that extracellular vesicles, previously referred to as microvesicles or exosomes, have the capacity to change the phenotype of neighboring cells. Extensive work has shown that vesicles derived from either the lung or liver can enter bone marrow cells (this is a prerequisite) and alter their fate toward that of the originating liver and lung tissue. Lung vesicles interacted with bone marrow cells result in the bone marrow cells expressing surfactants A-D, Clara cell protein, and aquaporin-5 mRNA. In a similar vein, liver-derived vesicles induce albumin mRNA in target marrow cells. The vesicles contain protein, mRNA, microRNA, and noncoding RNA and variably some DNA. This genetic package is delivered to cells and alters the phenotype. Further studies have shown that initially the altered phenotype is due to the transfer of mRNA and a transcriptional modulator, but long-term epigenetic changes are induced through transfer of a transcriptional factor, and the mRNA is rapidly degraded in the cell. Studies on the capacity of vesicles to restore injured tissue have been quite informative. Mesenchymal stem cell-derived vesicles are able to reverse the injury to the damaged liver and kidney. Other studies have shown that mesenchymal stem cell-derived vesicles can reverse radiation toxicity of bone marrow stem cells. Extracellular vesicles offer an intriguing strategy for treating a number of diseases characterized by tissue injury.