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

Abstract

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.

Keywords: Endothelial progenitor cells; Extracellular vesicles; Mesenchymal stem cells; Monocrotaline; Pulmonary hypertension.

Figure 1

Mice transplanted with bone marrow cells from mice with MCT‐induced pulmonary hypertension develop RV hypertrophy and pulmonary vascular remodeling. (A): RV/LV+S ratio, (B): WT/D ratio of MCT‐injured (red boxes) and vehicle‐injected (red diamonds) mice and ratios 4 weeks after transplantation of bone marrow cells from MCT‐injured (blue boxes) or vehicle‐injected (blue diamonds) mice. n = 5 mice/cohort; *, p < .05 versus vehicle‐injected mice; **, p < .05 versus transplant recipients of bone marrow from vehicle‐treated mice. Data presented as means with 95% confidence intervals. Hematoxylin and eosin stained lung sections from (C): MCT‐injured mice, (D): vehicle‐injected mice, (E): mice transplanted with WBM from MCT‐injured mice, and (F): mice transplanted with WBM from vehicle‐injected mice. (Red arrow pointing to a pulmonary blood vessel, Red bar = 20 μm). Abbreviations: MCT, monocrotaline; RV, right ventricular; RV/LV+S, RV‐to‐left ventricle+septum; WBM, whole bone marrow cells; WT/D, wall thickness‐to‐diameter.

Figure 2

Mice transplanted with bone marrow cells from mice with EV‐induced pulmonary hypertension develop RV hypertrophy and pulmonary vascular remodeling. (A): RV/LV+S ratio, (B): WT/D ratio of MCT‐injured (red boxes) and vehicle‐injected (red diamonds) mice, ratios 4 weeks after infusion of lung EVs isolated from MCT‐injured (blue boxes) or vehicle‐injected (blue diamonds) mice, ratios 4 weeks after transplantation of WBM isolated from mice infused with EVs from MCT‐injured mice (black boxes) or vehicle‐injected mice (black diamonds). n = 5 mice/cohort; *, p < .05 versus vehicle‐injected mice; **, p < .05 versus mice injected with EVs from vehicle‐injected mice; †, p < .05 versus mice transplanted with WBM from mice injected with EVs from vehicle‐injected mice; ‡, p < .05 versus MCT‐injured mice. Data presented as means with 95% confidence intervals. Hematoxylin and eosin stained lung sections from (C): mice transplanted with WBM from mice infused with EVs from MCT‐injured mice, (D): mice transplanted with WBM from mice infused with EVs from vehicle‐injected mice. (Red arrow pointing to a pulmonary blood vessel, Red bar = 20 μm). Abbreviations: EV, extracellular vesicles; MCT, monocrotaline; RV, right ventricular; RV/LV+S, RV‐to‐left ventricle+septum; WBM, whole bone marrow cells; WT/D, wall thickness‐to‐diameter.

Figure 3

Transplantation of EPCs and non‐EPC cells from MCT‐injured and vehicle‐injected mice. (A): RV/LV+S ratio, (B): WT/D ratio 4 weeks after transplantation of bone marrow‐derived EPCs (sca‐1+/c‐kit+/VEGFR2+ cells, diamonds) and non‐EPC cells (sca‐1−/c‐kit−/VEGFR2−, boxes) isolated from MCT‐injured (blue) and vehicle‐injected (red) mice. n = 5 mice/cohort, *, p < .05 versus mice transplanted with non‐EPC cells from MCT‐injured mice. **, p < .05 versus mice transplanted with EPCs from vehicle‐injected mice. †, p < .05 versus mice transplanted with non‐EPC cells from vehicle‐injected mice. Data presented as means with 95% confidence intervals. (C): Hematoxylin and eosin staining of lung sections from each cohort. (Red arrow pointing to a pulmonary blood vessel, Red bar = 20 μm). Abbreviations: EPC, endothelial progenitor cells; MCT, monocrotaline; RV/LV+S, right ventricular‐to‐left ventricle+septum; WT/D, wall thickness‐to‐diameter.

Figure 4

MSC‐EV treatment prevents RV hypertrophy and pulmonary vascular remodeling from being transferred via bone marrow transplantation. (A): RV/LV+S ratio, (B): WT/D ratio of MCT‐injured (red boxes) or vehicle‐injected (red diamonds) mice, MCT‐injured or vehicle‐injected mice 4 weeks after treatment with MSC‐EVs or vehicle (blue boxes, diamonds), recipients 4 weeks after bone marrow transplant using WBM isolated from the MCT‐injured and vehicle‐injected mice that were treated with MSC‐EVs or vehicle (black boxes, diamonds), n = 5 mice/cohort; *, p < .05 versus vehicle‐injected mice; **, p <.05 versus MCT‐injured mice treated with MSC‐EVs; †, p < .05 versus mice transplanted with WBM from MCT‐injured mice treated with MSC‐EVs; ‡, p < .05 versus MCT‐injured mice. Data presented as means with 95% confidence intervals. Hematoxylin and eosin stained lung sections from (C): MCT‐injured mice, (D): vehicle‐injected mice, (E): MCT‐injured mice treated with vehicle, (F): MCT‐injured mice treated with MSC‐EVs, (G): mice transplanted with WBM isolated from MCT‐injured mice treated with vehicle, (H): mice transplanted with WBM isolated from MCT‐injured mice treated with MSC‐E versus (Red arrow pointing to a pulmonary blood vessel, Red bar = 20 μm). Abbreviations: MCT, monocrotaline; MSC‐EV mesenchymal stem cell‐extracellular vesicles; RV, right ventricular; RV/LV+S, RV‐to‐left ventricle+septum; WBM, whole bone marrow cells; WT/D, wall thickness‐to‐diameter.

Figure 5

Peripheral blood EPCs in MCT‐injured and vehicle‐injected mice. (A): Peripheral blood EPCs (sca‐1+/c‐kit+/VEGFR2+ cells) per 50,000 peripheral blood leukocytes in MCT‐injured and (dark grey) and vehicle‐injected (white) mice. Box and whisker plots show the median (line), mean (X), 25th and 75th percentiles and 10th and 90th percentiles (error bars). n = 20 mice/cohort, *, p < .01 versus vehicle‐injected mice. Linear regression analysis (B): RV/LV+S, (C): WT/D ratio versus peripheral blood EPCs per 50,000 peripheral blood leukocytes in MCT‐injured (black squares) and vehicle‐injected mice (white circles). n = 20 mice/cohort; R ² = 0.609, 0.026 respectively, RV/LV+S, p < .05; R ² = 0.636, 0.048 respectively, WT/D, p < .05. Abbreviations: EPC, endothelial progenitor cells; MCT, monocrotaline; RV, right ventricular; RV/LV+S, RV‐to‐left ventricle+septum; WT/D, wall thickness‐to‐diameter.

Figure 6

Bone marrow EPCs in MCT‐injured and vehicle‐injected mice. (A): Bone marrow EPCs (sca‐1+/c‐kit+/VEGFR2+ cells) per 50,000 bone marrow leukocytes in MCT‐injured (dark grey) and vehicle‐injected (white) mice. Box and whisker plots show the median (line), mean (X), 25th and 75th percentiles and 10th and 90th percentiles (error bars). n = 10 mice/cohort, * p < .01 versus vehicle‐injected mice. Linear regression analysis (B): RV/LV+S, (C): WT/D ratio versus bone marrow EPCs per 50,000 bone marrow leukocytes in MCT‐injured (black squares) and vehicle‐injected mice (white circles). n = 10 mice/cohort; R ² = 0.616, 0.059 respectively, RV/LV+S, p < .05; R ² = 0.567, 0.026 respectively, WT/D, p < .05. Abbreviations: EPC, endothelial progenitor cells; MCT, monocrotaline; RV, right ventricular; RV/LV+S, RV‐to‐left ventricle+septum; WT/D, wall thickness‐to‐diameter.

Figure 7

Mice transplanted with EPCs from MCT‐injured mice cultured with MSC‐EVs prior to transplantation do not develop RV hypertrophy, pulmonary vascular remodeling. (A): RV/LV+S ratio, (B): WT/D ratio of mice injected with MCT‐injured (red boxes) or vehicle‐injected (red diamonds) mice, mice transplanted with non‐EPC cells from vehicle‐injected and MCT‐injured mice cultured with or without MSC‐EVs prior to transplantation (blue boxes, diamonds), mice transplanted with EPCs from vehicle‐injected and MCT‐injured mice cultured with or without MSC‐EVs prior to transplantation (black boxes, diamonds). Analysis performed 4 weeks after transplantation. Data presented as means with 95% confidence intervals. n = 6–15 mice/cohort; *, p < .05 versus vehicle‐injected mice; mice transplanted with EPCs from MCT‐injured mice that were not cultured with MSC‐EVs versus MCT‐injured mice (**, p < .05) or versus mice transplanted with non‐EPC cells from MCT‐injured mice that were not cultured with MSC‐EVs († p < .05) or versus mice transplanted with EPCs from MCT‐injured mice that were cultured with MSC‐EVs (††, p < .05). Abbreviations: EPC, endothelial progenitor cells; MCT, monocrotaline; MSC‐EV mesenchymal stem cell‐extracellular vesicles; RV, right ventricular; RV/LV+S, RV‐to‐left ventricle+septum; WT/D, wall thickness‐to‐diameter.