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.

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.

Targeting RUNX1 as a novel treatment modality for pulmonary arterial hypertension.

AIMS: Pulmonary arterial hypertension (PAH) is a fatal disease without a cure. Previously, we found that transcription factor RUNX1-dependent haematopoietic transformation of endothelial progenitor cells may contribute to the pathogenesis of PAH. However, the therapeutic potential of RUNX1 inhibition to reverse established PAH remains unknown. In the current study, we aimed to determine whether RUNX1 inhibition was sufficient to reverse Sugen/hypoxia (SuHx)-induced pulmonary hypertension (PH) in rats. We also aimed to demonstrate possible mechanisms involved. METHODS AND RESULTS: We administered a small molecule specific RUNX1 inhibitor Ro5-3335 before, during, and after the development of SuHx-PH in rats to investigate its therapeutic potential. We quantified lung macrophage recruitment and activation in vivo and in vitro in the presence or absence of the RUNX1 inhibitor. We generated conditional VE-cadherin-CreERT2; ZsGreen mice for labelling adult endothelium and lineage tracing in the SuHx-PH model. We also generated conditional Cdh5-CreERT2; Runx1(flox/flox) mice to delete Runx1 gene in adult endothelium and LysM-Cre; Runx1(flox/flox) mice to delete Runx1 gene in cells of myeloid lineage, and then subjected these mice to SuHx-PH induction. RUNX1 inhibition in vivo effectively prevented the development, blocked the progression, and reversed established SuHx-induced PH in rats. RUNX1 inhibition significantly dampened lung macrophage recruitment and activation. Furthermore, lineage tracing with the inducible VE-cadherin-CreERT2; ZsGreen mice demonstrated that a RUNX1-dependent endothelial to haematopoietic transformation occurred during the development of SuHx-PH. Finally, tissue-specific deletion of Runx1 gene either in adult endothelium or in cells of myeloid lineage prevented the mice from developing SuHx-PH, suggesting that RUNX1 is required for the development of PH. CONCLUSION: By blocking RUNX1-dependent endothelial to haematopoietic transformation and pulmonary macrophage recruitment and activation, targeting RUNX1 may be as a novel treatment modality for pulmonary arterial hypertension.

SHP2 regulates the development of intestinal epithelium by modifying OSTERIX+ crypt stem cell self-renewal and proliferation.

The protein tyrosine phosphatase SHP2, encoded by PTPN11, is ubiquitously expressed and essential for the development and/or maintenance of multiple tissues and organs. SHP2 is involved in gastrointestinal (GI) epithelium development and homeostasis, but the underlying mechanisms remain elusive. While studying SHP2's role in skeletal development, we made osteoblast-specific SHP2 deficient mice using Osterix (Osx)-Cre as a driver to excise Ptpn11 floxed alleles. Phenotypic characterization of these SHP2 mutants unexpectedly revealed a critical role of SHP2 in GI biology. Mice lacking SHP2 in Osx+ cells developed a fatal GI pathology with dramatic villus hypoplasia. OSTERIX, an OB-specific zinc finger-containing transcription factor is for the first time found to be expressed in GI crypt cells, and SHP2 expression in the crypt Osx+ cells is critical for self-renewal and proliferation. Further, immunostaining revealed the colocalization of OSTERIX with OLFM4 and LGR5, two bona fide GI stem cell markers, at the crypt cells. Furthermore, OSTERIX expression is found to be associated with GI malignancies. Knockdown of SHP2 expression had no apparent influence on the relative numbers of enterocytes, goblet cells or Paneth cells. Given SHP2's key regulatory role in OB differentiation, our studies suggest that OSTERIX and SHP2 are indispensable for gut homeostasis, analogous to SOX9's dual role as a master regulator of cartilage and an important regulator of crypt stem cell biology. Our findings also provide a foundation for new avenues of inquiry into GI stem cell biology and of OSTERIX's therapeutic and diagnostic potential.

Effect of dose, dosing intervals, and hypoxic stress on the reversal of pulmonary hypertension by mesenchymal stem cell extracellular vesicles.

RATIONALE: Mesenchymal stem cell extracellular vesicles (MSC EVs) reverse pulmonary hypertension, but little information is available regarding what dose is effective and how often it needs to be given. This study examined the effects of dose reduction and use of longer dosing intervals and the effect of hypoxic stress of MSC prior to EV collection. METHODS: Adult male rats with pulmonary hypertension induced by Sugen 5416 and three weeks of hypoxia (SuHx-pulmonary hypertension) were injected with MSC EV or phosphate buffered saline the day of removal from hypoxia using one of the following protocols: (1) Once daily for three days at doses of 0.2, 1, 5, 20, and 100 µg/kg, (2) Once weekly (100 µg/kg) for five weeks, (3) Once every other week (100 µg/kg) for 10 weeks, (4) Once daily (20 µg/kg) for three days using EV obtained from MSC exposed to 48 h of hypoxia (HxEV) or MSC kept in normoxic conditions (NxEV). MAIN RESULTS: MSC EV reversed increases in right ventricular systolic pressure (RVSP), right ventricular to left ventricle + septum weight (RV/LV+S), and muscularization index of pulmonary vessels ≤50 µm when given at doses of 20 or 100 µg/kg. RVSP, RV/LV+S, and muscularization index were significantly higher in SuHx-pulmonary hypertension rats treated once weekly with phosphate buffered saline for five weeks or every other week for 10 weeks than in normoxic controls, but not significantly increased in SuHx-pulmonary hypertension rats given MSC EV. Both NxEV and HxEV significantly reduced RVSP, RV/LV+S, and muscularization index, but no differences were seen between treatment groups. CONCLUSIONS: MSC EV are effective at reversing SuHx-pulmonary hypertension when given at lower doses and longer dosing intervals than previously reported. Hypoxic stress does not enhance the efficacy of MSC EV at reversing pulmonary hypertension. These findings support the feasibility of MSC EV as a long-term treatment for pulmonary hypertension.

Sexual dimorphism in aging hematopoiesis: an earlier decline of hematopoietic stem and progenitor cells in male than female mice.

Adult hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) ensuring homeostasis of blood production and immune response throughout life. Sex differences in immunocompetence and mortality are well-documented in humans. However, whether HSPCs behave dimorphically between sexes during aging remains unknown. Here, we show that a significant expansion of BM-derived HSPCs occurs in the middle age of female but in the old age of male mice. We then show that a decline of HSPCs in male mice, as indicated by the expression levels of select hematopoietic genes, occurs much earlier in the aging process than that in female mice. Sex-mismatched heterochronic BM transplantations indicate that the middle-aged female BM microenvironment plays a pivotal role in sustaining hematopoietic gene expression during aging. Furthermore, a higher concentration of the pituitary sex hormone follicle-stimulating hormone (FSH) in the serum and a concomitant higher expression of its receptor on HSPCs in the middle-aged and old female mice than age-matched male mice, suggests that FSH may contribute to the sexual dimorphism in aging hematopoiesis. Our study reveals that HSPCs in the BM niches are possibly regulated in a sex-specific manner and influenced differently by sex hormones during aging hematopoiesis.

Mesenchymal Stem Cell Derived Extracellular Vesicles Ameliorate Kidney Injury in Aristolochic Acid Nephropathy.

Limitations in the current therapeutic strategies for the prevention of progression of chronic kidney disease (CKD) to end stage renal disease has been a drawback to improving patient recovery. It is therefore imperative that a solution is found to alleviate this problem and improve the health and well-being of patients overall. Aristolochic acid (AA) induced nephropathy, a type of nephrotoxic CKD is characterised by cortical tubular injury, inflammation, leading to interstitial fibrosis. Extracellular vesicles derived from human bone marrow mesenchymal stem cells (MSC-EVs) display therapeutic properties in various disease models including kidney injury. In the current study, we intended to investigate the ability of MSC-EVs on ameliorating tubular injury and interstitial fibrosis in a mouse model of aristolochic acid nephropathy (AAN). The chronic model of AAN is comprised of an intraperitoneal injection of AA in NSG mice, followed by a three-day incubation period and then inoculation of MSC-EVs intravenously. This routine was performed on a weekly basis for four consecutive weeks, accompanied by the monitoring of body weight of all mice. Blood and tissue samples were collected post sacrifice. All animals administered with AA developed kidney injury and renal fibrosis. A gradual loss of body weight was observed, together with a deterioration in kidney function. Although no significant recovery was observed in weight loss following treatment with MSC-EVs, a significant reduction in: blood creatinine and blood urea nitrogen (BUN), tubular necrosis, and interstitial fibrosis was observed. In addition, infiltration of CD45 positive immune cells, fibroblasts, and pericytes which were elevated in the interstitium post AA induced injury, were also significantly reduced by MSC-EVs. Kidneys were also subjected to molecular analyses to evaluate the regulation of pro-fibrotic genes. MSC-EVs significantly reduced AA induction of the pro-fibrotic genes α-Sma, Tgfb1 and Col1a1. A downregulation in pro-fibrotic genes was also observed in fibroblasts activated by AA injured mTECs in vitro. Furthermore, meta-analyses of miRNAs downregulated by MSC-EVs, such as miR21, revealed the regulation of multiple pathways involved in kidney injury including fibrosis, inflammation, and apoptosis. These results therefore suggest that MSC-EVs could play a regenerative and anti-fibrotic role in AAN through the transfer of biologically active cargo that regulates the disease both at a protein and genetic level.

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.

Prevalence and Effect on Survival of Pulmonary Hypertension in Myelofibrosis.

BACKGROUND: Myelofibrosis (MF), a rare disorder characterized by bone marrow fibrosis, has been implicated as a cause of pulmonary hypertension (PH). To date, studies examining this association have not looked at the impact of PH on survival in MF. We examined the relationship between MF and PH by echocardiogram (echo) using a retrospective patient database and examined the influence of PH on overall survival. PATIENTS AND METHODS: In this single-center retrospective chart review, we identified 65 patients with biopsy-proven primary and secondary MF, 31 of whom underwent transthoracic echo. After accounting for chronic obstructive pulmonary disease and left-sided or valvular heart dysfunction, which excluded 6 patients, we identified 14 patients (56%) who had echo evidence of group 5 PH (ie, PH due to unclear or multifactorial mechanisms), 8 with primary MF and 6 with secondary MF. MF patients with PH trended toward being predominantly female, being older, and less often having constitutional symptoms compared to the non-PH cohort. RESULTS: There was no effect of the presence of PH on overall survival in the entire MF cohort or in any subgroup analyzed, including primary MF versus secondary MF and primary MF intermediate risk patients. CONCLUSION: Given the high prevalence of MF-associated PH, there may be a larger role for routine echo screening in MF patients. Further, the underlying association between PH and MF may signify an endothelial plasticity or increased telomerase activity as part of the pathogenesis of MF.

Renal Regenerative Potential of Extracellular Vesicles Derived from miRNA-Engineered Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) possess pro-regenerative potential in different animal models with renal injury. EVs contain different molecules, including proteins, lipids and nucleic acids. Among the shuttled molecules, miRNAs have a relevant role in the pro-regenerative effects of EVs and are a promising target for therapeutic interventions. The aim of this study was to increase the content of specific miRNAs in EVs that are known to be involved in the pro-regenerative effect of EVs, and to assess the capacity of modified EVs to contribute to renal regeneration in in vivo models with acute kidney injuries. To this purpose, MSCs were transiently transfected with specific miRNA mimics by electroporation. Molecular analyses showed that, after transfection, MSCs and derived EVs were efficiently enriched in the selected miRNAs. In vitro and in vivo experiments indicated that EVs engineered with miRNAs maintained their pro-regenerative effects. Of relevance, engineered EVs were more effective than EVs derived from naïve MSCs when used at suboptimal doses. This suggests the potential use of a low amount of EVs (82.5 × 106) to obtain the renal regenerative effect.

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.

Bone marrow-specific loss of ABI1 induces myeloproliferative neoplasm with features resembling human myelofibrosis.

Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of Abi1 in a novel mouse model leads to development of an MPN-like phenotype resembling human PMF. Abi1 loss resulted in a significant increase in the activity of the Src family kinases (SFKs), STAT3, and NF-κB signaling. We also observed impairment of hematopoietic stem cell self-renewal and fitness, as evidenced in noncompetitive and competitive bone marrow transplant experiments. CD34+ hematopoietic progenitors and granulocytes from patients with PMF showed decreased levels of ABI1 transcript as well as increased activity of SFKs, STAT3, and NF-κB. In aggregate, our data link the loss of Abi-1 function to hyperactive SFKs/STAT3/NF-κB signaling and suggest that this signaling axis may represent a regulatory module involved in the molecular pathophysiology of PMF.

Exosomal Tat protein activates latent HIV-1 in primary, resting CD4+ T lymphocytes.

Replication competent HIV-1 persists in a subpopulation of CD4+ T lymphocytes despite prolonged antiretroviral treatment. This residual reservoir of infected cells harbors transcriptionally silent provirus capable of reigniting productive infection upon discontinuation of antiretroviral therapy. Certain classes of drugs can activate latent virus but not at levels that lead to reductions in HIV-1 reservoir size in vivo. Here, we show the utility of CD4+ receptor targeting exosomes as an HIV-1 latency reversal agent (LRA). We engineered human cellular exosomes to express HIV-1 Tat, a protein that is a potent transactivator of viral transcription. Preparations of exosomal Tat-activated HIV-1 in primary, resting CD4+ T lymphocytes isolated from antiretroviral-treated individuals with prolonged periods of viral suppression and led to the production of replication competent HIV-1. Furthermore, exosomal Tat increased the potency of selected LRA by over 30-fold in terms of HIV-1 mRNA expression, thereby establishing it as a potentially new class of biologic product with possible combinatorial utility in targeting latent HIV-1.

Marrow Hypocellularity, But Not Residual Blast Count or Receipt of Reinduction Chemotherapy, Is Prognostic on Day-14 Assessment in Acute Myeloid Leukemia Patients With Morphologic Residual Disease.

BACKGROUND: Induction chemotherapy for acute myeloid leukemia (AML) is based on the "7+3" cytarabine/anthracycline regimen. A nonhypocellular day 14 (D14) bone marrow sample with a blast count > 5% to 10% is suggestive of residual leukemia, for which a second course of induction chemotherapy has been recommended. Although the prognostic value of D14 bone marrow findings has been established, its use as a decision point is controversial because the benefit of repeat induction has been questioned. PATIENTS AND METHODS: In the present single-center retrospective study of 113 patients with newly diagnosed AML, we evaluated the role of cellularity on the clinical outcomes of patients with residual morphologic leukemia (blasts ≥ 5%). Among 64 patients with D14 bone marrow samples, 31 had residual morphologic leukemia. RESULTS: The complete remission (CR) rates were greater for the hypocellular (11 of 16) than for the nonhypocellular (4 of 15) patients (P = .03). The median overall survival (OS) for the hypocellular D14 patients was longer than that for the nonhypocellular patients (17 vs. 8 months; P = .02). No significant difference between the receipt of reinduction therapy and CR or OS was found on logistic or survival model analysis. The specificity for residual leukemia on D14 bone marrow samples was better for cellularity ≥ 20% and blasts ≥ 20% than for blasts ≥ 5%. CONCLUSION: The results of our study have shown that patients with < 20% cellularity and < 20% blasts on the D14 bone marrow assessment should continue observation until recovery rather than receive additional immediate therapy.

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.