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

Abstract

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.

Keywords: exosomes; pulmonary vascular remodeling; right ventricular hypertrophy.

Fig. 1.

Effect of increasing doses of mesenchymal stem cell extracellular vesicles (EVs) compared to phosphate buffered saline vehicle (PBS) on (a) right ventricular systolic pressure (RVSP) and (b) right ventricle to left ventricle + septum ratio (RV/LV+S) in rats kept under normoxic conditions (Nx) or treated with Sugen 5416 and exposed to three weeks of hypoxia (SuHx). All doses are in micrograms of protein per kg. n = 3–13 per group. *P < 0.05, **P < 0.01, ****P < 0.0001, n.s.: not significant.

Fig. 2.

(a) Experimental protocol for reversal of pulmonary hypertension showing timing of administration of mesenchymal stem cell extracellular vesicles (MSC EVs) or phosphate buffered saline vehicle (PBS). (b) Right ventricular systolic pressure (RVSP) and (c) right ventricle to left ventricle + septum ratio (RV/LV+S) were measured five weeks after removal from three weeks of hypoxia (end of week 8 of experimental protocol). Nx PBS: normoxic PBS control; SuHx: rats treated with Sugen 5416 and exposed to three weeks of hypoxia. n = 5–9 per group. *P < 0.05, **P < 0.01, ***P < 0.001, n.s.: not significant.

Fig. 3.

Lung sections stained with an antibody against α-smooth muscle actin showing muscularization (dark brown) of peripheral pulmonary vessels from rats treated with phosphate buffered saline (PBS) or mesenchymal stem cell extracellular vesicles (MSC EVs) once weekly for five weeks following treatment with Sugen 5416 and three weeks of hypoxia (SuHx). (a) Normoxic controls (Nx) treated with PBS. (b) SuHx treated with PBS. (c) SuHx treated with MSC EV. (d) Muscularization of vessels ≤50 µm assessed as total area of vessel staining positive for α-smooth muscle actin divided by total cross-sectional area of the vessel. The NIH ImageJ program was used to assess vessel areas. n = 6–9 per group. ***P < 0.001, n.s. not significant.

Fig. 4.

(a) Experimental protocol for reversal of pulmonary hypertension showing timing of administration of mesenchymal stem cell extracellular vesicles (MSC EVs) or phosphate buffered saline vehicle (PBS). (b) Right ventricular systolic pressure (RVSP) and (c) right ventricle to left ventricle + septum ratio (RV/LV+S) were measured 10 weeks after removal from three weeks of hypoxia (end of week 13 of experimental protocol). Nx PBS: normoxic PBS control; SuHx: rats treated with Sugen 5416 and exposed to three weeks of hypoxia. n = 5–7 per group. *P < 0.05, **P < 0.01, n.s.: not significant.

Fig. 5.

Lung sections stained with an antibody against α-smooth muscle actin showing muscularization (dark brown) of peripheral pulmonary vessels from rats treated with phosphate buffered saline (PBS) or mesenchymal stem cell extracellular vesicles (MSC EVs) once every two weeks for 10 weeks following treatment with Sugen 5416 and three weeks of hypoxia (SuHx). (a) Normoxic controls (Nx) treated with PBS. (b) SuHx treated with PBS. (c) SuHx treated with MSC EV. (d) Muscularization of vessels ≤50 µm assessed as total area of vessel staining positive for α-smooth muscle actin divided by total cross-sectional area of the vessel. The NIH ImageJ program was used to assess vessel areas. n = 5–7 per group. *P < 0.05, n.s.: not significant.

Fig. 6.

(a) Experimental protocol for reversal of pulmonary hypertension showing timing of administration of mesenchymal stem cell extracellular vesicles (MSC EVs) or phosphate buffered saline vehicle (PBS). Rats were left in normoxic conditions (Nx) or treated with Sugen 5416 followed by three weeks of hypoxia (SuHx) before receiving PBS or EV isolated from normoxic or hypoxia-stressed MSCs (NxEV, HxEV). (b) Right ventricular systolic pressure (RVSP) and (c) right ventricle to left ventricle + septum ratio (RV/LV+S) measured two weeks after removal from three weeks of hypoxia (end of week 5 of experimental protocol). n = 3–6 per group. *P < 0.05, **P < 0.01, n.s.: not significant.

Fig. 7.

Lung sections stained with an antibody against α-smooth muscle actin showing muscularization (dark brown) of peripheral pulmonary vessels from rats treated with phosphate buffered saline (PBS) or EV isolated from normoxic or hypoxia-stressed MSCs (NxEV, HxEV) for three days following treatment with Sugen 5416 and three weeks of hypoxia (SuHx). Control rats were kept in normoxic conditions and treated with PBS or NxEV. (a) Normoxic controls (Nx) treated with PBS. (b) SuHx rats treated with PBS. (c) SuHx rats treated with NxEV. (d) SuHx rats treated with HxEV. (e) Muscularization of vessels ≤50 µm assessed as total area of vessel staining positive for α-smooth muscle actin divided by total cross-sectional area of the vessel. The NIH ImageJ program was used to assess vessel areas. n = 3–6 per group. *P < 0.05, n.s.: not significant.