Endothelial Progenitor Cells Are the Bone Marrow Cell Population in Mice with Monocrotaline-Induced Pulmonary Hypertension Which Induce Pulmonary Hypertension in Healthy Mice

Rationale: We have demonstrated that whole, undifferentiated bone marrow (WBM) cells isolated from mice with monocrotaline (MCT)-induced pulmonary hypertension (PH) cause pulmonary hypertensive changes when transplanted into lethally-irradiated mice (Aliotta et al., 2014 ATS abstract). In addition, mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs), sub-cellular particles shed by activated cells, can reverse features of MCT-induced PH (Aliotta et al., 2015 ATS abstract). Endothelial progenitor cells (EPCs) have been implicated in the pathogenesis of human PH as more have been noted to be present in the circulation and remodeled pulmonary vasculature of these patients compared with healthy humans. We wished to determine if bone marrow-derived EPCs isolated from mice with MCT PH were the cells responsible for inducing PH in healthy mice and if so, whether exposure of these EPCs to MSC EVs prevented the development of PH.

Methods: Sca-1+/ckit+/flk1+ cells (EPCs) and sca-1-/ckit-/flk1- cells (non-EPCs) were isolated from the bone marrow of MCT-injured and vehicle-injected mice. Cohorts of lethally-irradiated mice were transplanted with EPCs (2,000 EPCs + 300,000 helper WBM cells/recipient) or non-EPCs (200,000 non-EPCs + 200,000 helper WBM cells/recipient) from the bone marrow of MCT-injured and vehicle-injected mice. 28 days after transplantation, recipient mouse right ventricular (RV) hypertrophy was assessed by RV-to-left ventricle+septum (RV/LV+S) ratio (mg/g) and pulmonary vascular remodeling by blood vessel wall thickness-to-diameter (WT/D) ratio. In addition, WBM from MCT-injured and vehicle-injected mice was cultured with or without MSC EVs for 48 hours. EPCs (80-100 EPCs + 300,000 helper WBM cells/recipient) and non-EPCs (500,000-700,000 non- EPCs + 300,000 helper WBM cells/recipient) were separated by flow cytometry and infused into lethally-irradiated mice which were analyzed 28 days post-transplant.

Results: RV/LV+S ratios of mice transplanted with EPCs from MCT-injured mice (0.256+/-0.142 mg/g) were similar to those of MCT-injured mice (0.274+/-0.211 mg/g, p=NS, n=5/cohort) but elevated compared to mice transplanted with EPCs and non-EPCs from vehicle-injected mice (0.126+/-0.111 and 0.121+/-0.134 mg/g, p<0.05, n=5/cohort) and mice transplanted with non-EPCs from MCT-injured mice (0.139+/-0.218 mg/g, n=5/cohort) . WT/D ratios of mice transplanted with EPCs from MCT-injured mice (0.145+/-0.122) were similar to those of MCT-injured mice (0.156+/-0.232, p=NS, n=5/cohort) but elevated compared to mice transplanted with EPCs and non-EPCs from vehicle-injected mice (0.076+/-0.008 and 0.072+/-0.009, p<0.05, n=5/cohort) and mice transplanted with non-EPCs from MCT-injured mice (0.081+/-0.012, n=5/cohort). Mice transplanted with EPCs from MCT-injured mice that were treated with MSC EVs prior to transplantation had significantly lower RV/LV+S and WT/D ratios compared with mice transplanted with EPCs from MCT-injured mice that were not treated with MSC EVs prior to transplantation (0.119+/-0.164 mg/g, 0.072+/-0.008 vs. 0.188+/-0.201 mg/g, 0.122+/-0.011 p<0.05, n=5/cohort).

Conclusions: These findings suggest that EPCs are among the pathogenic bone marrow cells in MCT-injured mice that are responsible for inducing RV hypertrophy and pulmonary vascular remodeling, key features of PH, upon transplantation into healthy mice. MSC EVs appear to be capable of reversing this disease via interactions with EPCs.