The universal stem cell.

Current dogma is that there exists a hematopoietic pluripotent stem cell, resident in the marrow, which is quiescent, but with tremendous proliferative and differentiative potential. Furthermore, the hematopoietic system is essentially hierarchical with progressive differentiation from the pluripotent stem cells to different classes of hematopoietic cells. However, results summarized here indicate that the marrow pluripotent hematopoietic stem cell is actively cycling and thus continually changing phenotype. As it progresses through cell cycle differentiation potential changes as illustrated by sequential changes in surface expression of B220 and GR-1 epitopes. Further data indicated that the potential of purified hematopoietic stem cells extends to multiple other non-hematopoietic cells. It appears that marrow stem cells will give rise to epithelial pulmonary cells at certain points in cell cycle. Thus, it appears that the marrow "hematopoietic" stem cell is also a stem cell for other non-hematopoietic tissues. These observations give rise to the concept of a universal stem cell. The marrow stem cell is not limited to hematopoiesis and its differentiation potential continually changes as it transits cell cycle. Thus, there is a universal stem cell in the marrow which alters its differentiation potential as it progresses through cell cycle. This potential is expressed when it resides in tissues compatible with its differentiation potential, at a particular point in cell cycle transit, or when it interacts with vesicles from that tissue.

How do face masks impact communication amongst deaf/HoH people?

Face coverings have been key in reducing the spread of COVID-19. At the same time, they have hindered interpersonal communication, particularly for those who rely on speechreading to aid communication. The available research indicated that deaf/hard of hearing (HoH) people experienced great difficulty communicating with people wearing masks and negative effects on wellbeing. Here we extended these findings by exploring which factors predict deaf/HoH people's communication difficulties, loss of information, and wellbeing. We also explored the factors predicting perceived usefulness of transparent face coverings and alternative ways of communicating. We report the findings from an accessible survey study, released in two written and three signed languages. Responses from 395 deaf/HoH UK and Spanish residents were collected online at a time when masks were mandatory. We investigated whether onset and level of deafness, knowledge of sign language, speechreading fluency, and country of residence predicted communication difficulties, wellbeing, and degree to which transparent face coverings were considered useful. Overall, deaf/HoH people and their relatives used masks most of the time despite greater communication difficulties. Late-onset deaf people were the group that experienced more difficulties in communication, and also reported lower wellbeing. However, both early- and late-onset deaf people reported missing more information and feeling more disconnected from society than HoH people. Finally, signers valued transparent face shields more positively than non-signers. The latter suggests that, while seeing the lips is positive to everyone, signers appreciate seeing the whole facial expression. Importantly, our data also revealed the importance of visual communication other than speechreading to facilitate face-to-face interactions.

Differentiation Epitopes Define Hematopoietic Stem Cells and Change with Cell Cycle Passage.

Hematopoietic stem cells express differentiation markers B220 and Gr1 and are proliferative. We have shown that the expression of these entities changes with cell cycle passage. Overall, we conclude that primitive hematopoietic stem cells alter their differentiation potential with cell cycle progression. Murine derived long-term hematopoietic stem cells (LT-HSC) are cycling and thus always changing phenotype. Here we show that over one half of marrow LT-HSC are in the population expressing differentiation epitopes and that B220 and Gr-1 positive populations are replete with LT-HSC after a single FACS separation but if subjected to a second separation these cells no longer contain LT-HSC. However, with second separated cells there is a population appearing that is B220 negative and replete with cycling c-Kit, Sca-1 CD150 positive LT-HSC. There is a 3-4 h interval between the first and second B220 or GR-1 FACS separation during which the stem cells continue to cycle. Thus, the LT-HSC have lost B220 or GR-1 expression as the cells progress through cell cycle, although they have maintained the c-kit, Sca-1 and CD150 stem cells markers over this time interval. These data indicate that cycling stem cells express differentiation epitopes and alter their differentiation potential with cell cycle passage.

A Randomized Controlled Trial to Evaluate the Safety and Efficacy of a Novel Inhaled Biologic Therapeutic in Adults with Respiratory Distress Secondary to COVID-19 Infection.

INTRODUCTION: Inhaled therapeutics may act to directly target and attenuate lung inflammation due to COVID-19. An inhalation form of a novel biologic drug, AMP5A, is being developed as an immunomodulatory agent to treat dysregulated immune responses and is being studied in hospitalized patients to treat respiratory complications due to COVID-19. METHODS: A randomized, controlled, phase I trial was conducted to evaluate hospitalized adults with respiratory distress secondary to COVID-19. Patients received the standard care (SOC) for COVID-19, including respiratory therapy, corticosteroids, and antiviral therapies such as remdesivir. Patients were randomized 1:1 to inhalation treatment with AMP5A as an adjunct to SOC or to SOC alone (control). AMP5A was administered via inhalation daily for 5 days via hand-held nebulizer, non-invasive ventilator, or mechanical ventilation. Safety and clinical efficacy endpoints were evaluated. RESULTS: Forty subjects were enrolled and randomized (n = 19 AMP5A, n = 21 control). Remdesivir was used in fewer AMP5A subjects (26%) than control (52%), and dexamethasone was administered for most subjects (84% AMP5A, 71% control). The study met its primary endpoint with no AMP5A treatment-related adverse events (AEs), and the incidence and severity of AEs were comparable between groups: 18 AEs for control (8 mild, 1 moderate, 9 severe) and 19 AEs for AMP5A (7 mild, 7 moderate, 5 severe). Notably, subjects treated with AMP5A had fewer deaths (5% vs. 24%), shorter hospital stay (8 days vs. 12 days), fewer ICU admissions (21% vs. 33%), and a greater proportion with improved clinical outcomes than control. CONCLUSION: The phase I clinical results indicate inhaled AMP5A is safe, is well tolerated, and could lead to fewer patients experiencing deterioration or death. Based on the treatment effect (i.e., reduced mortality), a phase II trial has been initiated. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT04606784.

Extracellular Vesicles and Hematopoietic Stem Cell Aging.

Extracellular vesicles (EVs), important mediators of intercellular communication, play a critical role in modulating hematopoiesis within the bone marrow microenvironment. Although few studies have explicitly examined the connections between EVs and hematopoietic stem cell (HSC) aging, there is a growing body of evidence that implicates EVs in numerous age-related biologic processes and diseases. This, coupled with their tremendous capacity to influence hematopoiesis, suggests EVs may be key mediators of HSC aging. This review provides an overview of the effects of aging on HSCs, the role of EVs in aging in general, and then details key work in EV modulation of normal and malignant hematopoiesis, with a particular focus on how these effects may translate into the ability of EVs to drive HSC aging. Finally, it describes an exciting emerging literature that provides direct evidence for EV modulation of HSC phenotypes during natural aging and highlights their potential in HSC rejuvenation. Taken collectively, this body of research has profound implications for the future of HSC aging studies. More clearly defining how EVs modify HSC function in an age-dependent fashion and determining the molecular mechanisms by which they drive these age-related HSC phenotype changes will undoubtedly yield innovative strategies to delay or even reverse age-related hematologic dysfunction.

The novel immunomodulatory biologic LMWF5A for pharmacological attenuation of the "cytokine storm" in COVID-19 patients: a hypothesis.

BACKGROUND: A common complication of viral pulmonary infections, such as in the ongoing COVID-19 pandemic, is a phenomenon described as a "cytokine storm". While poorly defined, this hyperinflammatory response results in diffuse alveolar damage. The low molecular weight fraction of commercial human serum albumin (LMWF5A), a novel biologic in development for osteoarthritis, demonstrates beneficial in vitro immunomodulatory effects complimentary to addressing inflammation, thus, we hypothesize that LMWF5A could improve the clinical outcomes of COVID-19 by attenuating hyperinflammation and the potential development of a cytokine storm. PRESENTATION OF THE HYPOTHESIS: A variety of human in vitro immune models indicate that LMWF5A reduces the production of pro-inflammatory cytokines implicated in cytokine storm associated with COVID-19. Furthermore, evidence suggests LMWF5A also promotes the production of mediators required for resolving inflammation and enhances the barrier function of endothelial cultures. TESTING THE HYPOTHESIS: A randomized controlled trial, to evaluate the safety and efficacy of nebulized LMWF5A in adults with Acute Respiratory Distress Syndrome (ARDS) secondary to COVID-19 infection, was developed and is currently under review by the Food and Drug Administration. IMPLICATIONS OF HYPOTHESIS: If successful, this therapy may attenuate the cytokine storm observed in these patients and potentially reduce mortality, increase ventilation free days, improve oxygenation parameters and consequently lessen the burden on patients and the intensive care unit. CONCLUSIONS: In conclusion, in vitro findings suggest that the immunomodulatory effects of LMWF5A make it a viable candidate for treating cytokine storm and restoring homeostasis to the immune response in COVID-19.

Language Experience Impacts Brain Activation for Spoken and Signed Language in Infancy: Insights From Unimodal and Bimodal Bilinguals.

Recent neuroimaging studies suggest that monolingual infants activate a left-lateralized frontotemporal brain network in response to spoken language, which is similar to the network involved in processing spoken and signed language in adulthood. However, it is unclear how brain activation to language is influenced by early experience in infancy. To address this question, we present functional near-infrared spectroscopy (fNIRS) data from 60 hearing infants (4 to 8 months of age): 19 monolingual infants exposed to English, 20 unimodal bilingual infants exposed to two spoken languages, and 21 bimodal bilingual infants exposed to English and British Sign Language (BSL). Across all infants, spoken language elicited activation in a bilateral brain network including the inferior frontal and posterior temporal areas, whereas sign language elicited activation in the right temporoparietal area. A significant difference in brain lateralization was observed between groups. Activation in the posterior temporal region was not lateralized in monolinguals and bimodal bilinguals, but right lateralized in response to both language modalities in unimodal bilinguals. This suggests that the experience of two spoken languages influences brain activation for sign language when experienced for the first time. Multivariate pattern analyses (MVPAs) could classify distributed patterns of activation within the left hemisphere for spoken and signed language in monolinguals (proportion correct = 0.68; p = 0.039) but not in unimodal or bimodal bilinguals. These results suggest that bilingual experience in infancy influences brain activation for language and that unimodal bilingual experience has greater impact on early brain lateralization than bimodal bilingual experience.

Mechanical stretch regulates the expression of specific miRNA in extracellular vesicles released from lung epithelial cells.

The underlying mechanism of normal lung organogenesis is not well understood. An increasing number of studies are demonstrating that extracellular vesicles (EVs) play critical roles in organ development by delivering microRNAs (miRNA) to neighboring and distant cells. miRNAs are important for fetal lung growth; however, the role of miRNA-EVs (miRNAs packaged inside the EVs) during fetal lung development is unexplored. The aim of this study was to examine the expression of miRNA-EVs in MLE-12, a murine lung epithelial cell line subjected to mechanical stretch in vitro with the long-term goal to investigate their potential role in the fetal lung development. Both cyclic and continuous mechanical stretch regulate miRNA differentially in EVs released from MLE-12 and intracellularly, demonstrating that mechanical signals regulate the expression of miRNA-EVs in lung epithelial cells. These results provide a proof-of-concept for the potential role that miRNA-EVs could play in the development of fetal lung.

Inflammation-related gene expression profiles of salivary extracellular vesicles in patients with head trauma.

At present, there is no reliable biomarker for the diagnosis of traumatic brain injury (TBI). Studies have shown that extracellular vesicles released by damaged cells into biological fluids can be used as potential biomarkers for diagnosis of TBI and evaluation of TBI severity. We hypothesize that the genetic profile of salivary extracellular vesicles in patients with head trauma differs from that in uninjured subjects. Findings from this hypothesis would help investigate the severity of TBI. This study included 19 subjects, consisting of seven healthy controls who denied history of head trauma, six patients diagnosed with concussion injury from an outpatient concussion clinic, and six patients with TBI who received treatment in the emergency department within 24 hours after injury. Real-time PCR analysis of salivary extracellular vesicles in participants was performed using TaqMan Human Inflammation array. Gene expression analysis revealed nine upregulated genes in emergency department patients (LOX5, ANXA3, CASP1, IL2RG, ITGAM, ITGB2, LTA4H, MAPK14, and TNFRSF1A) and 13 upregulated genes in concussion clinic patients compared with healthy participants (ADRB1, ADRB2, BDKRB1, HRH1, HRH2, LTB4R2, LTB4R, PTAFR, CYSLTR1, CES1, KLK1, MC2R, and PTGER3). Each patient group had a unique profile. Comparison between groups showed that 15 inflammation-related genes had significant expression change. Our results indicate that inflammation biomarkers can be used for diagnosis of TBI and evaluation of disease severity. This study was approved by the Institutional Review Board on December 18, 2015 (approval No. 0078-12) and on June 9, 2016 (approval No. 4093-16).

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.

Biodistribution of Mesenchymal Stem Cell-Derived Extracellular Vesicles in a Radiation Injury Bone Marrow Murine Model.

We have previously shown that injury induced by irradiation to murine marrow can be partially or completely reversed by exposure to human or murine mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). Investigation of the biodistribution of EVs in vivo is essential for understanding EV biology. In this study, we evaluated the DiD lipid dye labeled MSC-EV biodistribution in mice under different conditions, including different MSC-EV doses and injection schedules, time post MSC-EV injection, and doses of radiation. DiD-labeled MSC-EVs appeared highest in the liver and spleen; lower in bone marrow of the tibia, femur, and spine; and were undetectable in the heart, kidney and lung, while a predominant EV accumulation was detected in the lung of mice infused with human lung fibroblast cell derived EVs. There was significantly increased MSC-EV accumulation in the spleen and bone marrow (tibia and femur) post radiation appearing with an increase of MSC-EV uptake by CD11b+ and F4/80+ cells, but not by B220 cells, compared to those organs from non-irradiated mice. We further demonstrated that increasing levels of irradiation caused a selective increase in vesicle homing to marrow. This accumulation of MSC-EVs at the site of injured bone marrow could be detected as early as 1 h after MSC- EV injection and was not significantly different between 2 and 24 h post MSC-EV injection. Our study indicates that irradiation damage to hematopoietic tissue in the spleen and marrow targets MSC-EVs to these tissues.

Heuristic bias in stem cell biology.

When studying purified hematopoietic stem cells, the urge for mechanisms and reductionist approaches appears to be overwhelming. The prime focus of the field has recently been on the study of highly purified hematopoietic stem cells using various lineage and stem cell-specific markers, all of which adequately and conveniently fit the established hierarchical stem cell model. This methodology is tainted with bias and has led to incomplete conclusions. Much of our own work has shown that the purified hematopoietic stem cell, which has been so heavily studied, is not representative of the total population of hematopoietic stem cells and that rather than functioning within a hierarchical model of expansion the true hematopoietic stem cell is one that is actively cycling through various differentiation potentials within a dynamic continuum. Additional work with increased emphasis on studying whole populations and direct mechanistic studies to these populations is needed. Furthermore, the most productive studies may well be mechanistic at the cellular or tissue levels. Lastly, the application of robust machine learning algorithms may provide insight into the dynamic variability and flux of stem cell fate and differentiation potential.

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.

Potential biomarkers to detect traumatic brain injury by the profiling of salivary extracellular vesicles.

Traumatic brain injury (TBI) is a common cause of death and acquired disability in adults and children. Identifying biomarkers for mild TBI (mTBI) that can predict functional impairments on neuropsychiatric and neurocognitive testing after head trauma is yet to be firmly established. Extracellular vesicles (EVs) are known to traffic from the brain to the oral cavity and can be detected in saliva. We hypothesize the genetic profile of salivary EVs in patients who have suffered head trauma will differ from normal healthy controls, thus constituting a unique expression signature for mTBI. We enrolled a total of 54 subjects including for saliva sampling, 23 controls with no history of head traumas, 16 patients enrolled from an outpatient concussion clinic, and 15 patients from the emergency department who had sustained a head trauma within 24 hr. We performed real-time PCR of the salivary EVs of the 54 subjects profiling 96 genes from the TaqMan Human Alzheimer's disease array. Real-time PCR analysis revealed 57 (15 genes, p < 0.05) upregulated genes in emergency department patients and 56 (14 genes, p < 0.05) upregulated genes in concussion clinic patients when compared with controls. Three genes were upregulated in both the emergency department patients and concussion clinic patients: CDC2, CSNK1A1, and CTSD ( p < 0.05). Our results demonstrate that salivary EVs gene expression can serve as a viable source of biomarkers for mTBI. This study shows multiple Alzheimer's disease genes present after an mTBI.

Language experience influences audiovisual speech integration in unimodal and bimodal bilingual infants.

Infants as young as 2 months can integrate audio and visual aspects of speech articulation. A shift of attention from the eyes towards the mouth of talking faces occurs around 6 months of age in monolingual infants. However, it is unknown whether this pattern of attention during audiovisual speech processing is influenced by speech and language experience in infancy. The present study investigated this question by analysing audiovisual speech processing in three groups of 4- to 8-month-old infants who differed in their language experience: monolinguals, unimodal bilinguals (infants exposed to two or more spoken languages) and bimodal bilinguals (hearing infants with Deaf mothers). Eye-tracking was used to study patterns of face scanning while infants were viewing faces articulating syllables with congruent, incongruent and silent auditory tracks. Monolinguals and unimodal bilinguals increased their attention to the mouth of talking faces between 4 and 8 months, while bimodal bilinguals did not show any age difference in their scanning patterns. Moreover, older (6.6 to 8 months), but not younger, monolinguals (4 to 6.5 months) showed increased visual attention to the mouth of faces articulating audiovisually incongruent rather than congruent faces, indicating surprise or novelty. In contrast, no audiovisual congruency effect was found in unimodal or bimodal bilinguals. Results suggest that speech and language experience influences audiovisual integration in infancy. Specifically, reduced or more variable experience of audiovisual speech from the primary caregiver may lead to less sensitivity to the integration of audio and visual cues of speech articulation.

Impact of Language Experience on Attention to Faces in Infancy: Evidence From Unimodal and Bimodal Bilingual Infants.

Faces capture and maintain infants' attention more than other visual stimuli. The present study addresses the impact of early language experience on attention to faces in infancy. It was hypothesized that infants learning two spoken languages (unimodal bilinguals) and hearing infants of Deaf mothers learning British Sign Language and spoken English (bimodal bilinguals) would show enhanced attention to faces compared to monolinguals. The comparison between unimodal and bimodal bilinguals allowed differentiation of the effects of learning two languages, from the effects of increased visual communication in hearing infants of Deaf mothers. Data are presented for two independent samples of infants: Sample 1 included 49 infants between 7 and 10 months (26 monolinguals and 23 unimodal bilinguals), and Sample 2 included 87 infants between 4 and 8 months (32 monolinguals, 25 unimodal bilinguals, and 30 bimodal bilingual infants with a Deaf mother). Eye-tracking was used to analyze infants' visual scanning of complex arrays including a face and four other stimulus categories. Infants from 4 to 10 months (all groups combined) directed their attention to faces faster than to non-face stimuli (i.e., attention capture), directed more fixations to, and looked longer at faces than non-face stimuli (i.e., attention maintenance). Unimodal bilinguals demonstrated increased attention capture and attention maintenance by faces compared to monolinguals. Contrary to predictions, bimodal bilinguals did not differ from monolinguals in attention capture and maintenance by face stimuli. These results are discussed in relation to the language experience of each group and the close association between face processing and language development in social communication.

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.

A New Stem Cell Biology: Transplantation and Baseline, Cell Cycle and Exosomes.

Hematopoietic stem cell biology has focused on stem cell purification and the definition of the regulation of purified stem cells in a hierarchical system. Work on the whole unpurified murine marrow cell population has indicated that a significant number of hematopoietic stem cells, rather than being dormant, are actively cycling, always changing phenotype and therefore resistant to purification efforts by current approaches. The bulk of cycling marrow stem cells are discarded with the standard lineage negative, stem cell marker positive separations. Therefore, the purified stem cells do not appear to be representative of the total hematopoietic stem cell population. In addition, baseline hematopoiesis does not appear to be determined by the transplantable stem cells but rather by many short-lived clones of varying differentiation potential. These systems appear to be impacted by tissue derived extracellular vesicles and a number of other variables. Thus hematopoietic stem cell biology is now at a fascinating new beginning with great promise.

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.