Effects of bone marrow-derived cells on monocrotaline- and hypoxia-induced pulmonary hypertension in mice

Abstract

Background: Bone marrow -derived cells (BMDCs) can either limit or contribute to the process of pulmonary vascular remodeling. Whether the difference in their effects depends on the mechanism of pulmonary hypertension (PH) remains unknown.

Objectives: We investigated the effect of BMDCs on PH induced in mice by either monocrotaline or exposure to chronic hypoxia.

Methods: Intravenous administration of the active monocrotaline metabolite (monocrotaline pyrrole, MCTp) to C57BL/6 mice induced PH within 15 days, due to remodeling of small distal vessels. Three days after the MCTp injection, the mice were injected with BMDCs harvested from femurs and tibias of donor mice treated with 5-fluorouracil (3.5 mg IP/animal) to deplete mature cells and to allow proliferation of progenitor cells.

Results: BMDCs significantly attenuated PH as assessed by reductions in right ventricular systolic pressure (20 +/- 1 mmHg vs. 27 +/- 1 mmHg, P < or = 0.01), right ventricle weight/left ventricle+septum weight ratio (0.29 +/- 0.02 vs. 0.36 +/- 0.01, P < or = 0.03), and percentage of muscularized vessels (26.4% vs. 33.5%, P < or = 0.05), compared to control animals treated with irradiated BMDCs. Tracking cells from constitutive GFP-expressing male donor mice with anti-GFP antibodies or chromosome Y level measurement by quantitative real-time PCR showed BMDCs in the lung. In contrast, chronically hypoxic mice subjected to the same procedure failed to show improvement in PH.

Conclusion: These results show that BMDCs limit pulmonary vascular remodeling induced by vascular injury but not by hypoxia.

Figure 1

A. Ratio of right ventricle to left ventricle + septum weight [RV/(LV+S)] on day 15 in control untreated mice, MCTp-treated mice, MCTp-treated mice injected with bone marrow-derived cells (BMDCs), and MCTp-treated mice injected with irradiated BMDCs. Values are means ± SEM for 6 animals in each group except control mice (n = 15). B. Right ventricular systolic pressure (RVSP) on day 15 in control untreated mice, MCTp-treated mice, MCTp-treated mice injected with BMDCs, and MCTp-treated mice injected with irradiated BMDCs. Values are means ± SEM for 5 animals in each group except control mice (n = 8). C. Percentage of muscularized intraacinar vessels in lungs on day 15 in control untreated mice, MCTp-treated mice, MCTp-treated mice injected with BMDCs, and MCTp-treated mice injected with irradiated BMDCs. The percentage of pulmonary vessels in each muscularization category was determined by dividing the number of vessels in that category by the total number counted in the same experimental group. Values are means ± SEM for 5 animals in each group except control mice (n = 11). D. Representative panels of proximal artery remodeling. D1) normal artery in a control mouse; D2) MCTp-treated mouse with smooth muscle cell proliferation; D3) MCTp-treated mouse injected with bone marrow-derived cells (BMDCs) showing recovery of a nearly normal arterial morphology; D4) MCTp-treated mice injected with irradiated BMDCs showing no amelioration of remodeling or SMC proliferation. Sections were stained with hematoxylin-phloxine-saffron. Original magnification ×500.

Figure 2

Effect of the number of bone marrow-derived cells on the increases in right ventricular systolic pressure (RVSP) and right ventricle to left ventricle + septum weight [RV/(LV+S)] after MCTp injection: 12·10⁶ cells (2 male donor mice for 1 female recipient), 2.5·10⁶ cells (1 male donor mouse for 1 female recipient), or 3.5·10⁵ cells (1/2 male donor mouse sample for 1 female recipient). Values are means ± SEM for 5 animals in each group.

Figure 3

A. Ratio of right ventricle to left ventricle + septum weight [RV/(LV+S)] on day 15 in control untreated mice, MCTp-treated mice, MCTp-treated mice injected with Lin-/CD45- selected cells, and MCTp-treated mice injected with Lin-/CD45+ selected cells. Values are means ± SEM for 6 animals in each group. B. Right ventricular systolic pressure (RVSP) on day 15 in control untreated mice, MCTp-treated mice, MCTp-treated mice injected with Lin-/CD45- selected cells, and MCTp-treated mice injected with Lin-/CD45+ selected cells. Values are means ± SEM for 5 animals in each group. C. Percentage of muscularized intraacinar vessels in lungs on day 15 in control untreated mice, MCTp-treated mice, MCTp-treated mice injected with Lin-/CD45- selected cells, and MCTp-treated mice injected with Lin-/CD45+ selected cells. The percentage of pulmonary vessels in each muscularization category was determined by dividing the number of vessels in that category by the total number counted in the same experimental group. Values are means ± SEM for 6 animals in each group.

Figure 4

A. Ratio of right ventricle to left ventricle + septum weight [RV/(LV+S)] on day 15 in control untreated mice, hypoxia-exposed mice, hypoxia-exposed mice injected with bone marrow-derived cells (BMDCs), and hypoxia-exposed mice injected with irradiated BMDCs. Values are means ± SEM for 6 animals in each group except control mice (n = 11). B. Right ventricular systolic pressure (RVSP) on day 15 in control untreated mice, hypoxia-exposed mice, hypoxia-exposed mice injected with BMDCs, and hypoxia-exposed mice injected with irradiated BMDCs. Values are means ± SEM for 6 animals in each group. C. Percentage of muscularized intraacinar vessels in lungs on day 15 in control untreated mice, hypoxia-exposed mice, hypoxia-exposed mice injected with BMDCs, and hypoxia-exposed mice injected with irradiated BMDCs. The percentage of pulmonary vessels in each muscularization category was determined by dividing the number of vessels in that category by the total number counted in the same experimental group. Values are means ± SEM for 5 animals in each group except control mice (n = 10). D. Representative panels of proximal artery remodeling. D1) normal artery from a control mouse; D2) hypoxia-exposed mouse with SMC proliferation; D3) hypoxia-exposed mouse injected with BMDCs, with no evidence of recovery of normal arterial morphology; D4) hypoxia-exposed mouse injected with irradiated BMDCs, with no improvement in remodeling or SMC proliferation. Sections were stained with hematoxylin-phloxine-saffron. Original magnification ×500.

Figure 5

Panels A and B: Quantification of bone marrow-derived cells (BMDCs) engraftment in recipient mouse lung by real-time quantitative PCR. A) Relationship between the threshold cycle number and the percentage of male genomic DNA in the samples. The standard curve was generated by using samples containing 100 ng to 0.01 ng of male genomic DNA (losanges). Triangles: DNA from MCTp-treated mice injected with BMDCs 3 days later; squares: DNA from MCTp-treated mice injected with irradiated BMDCs 3 days later; stars : DNA from hypoxia-treated mice injected with BMDCs 3 days later. B) Histogram of the above-reported data showing the percentage of male genomic DNA in lung tissue from female mice treated with MCTp, MCTp followed by BMDCs (black bar), or MCTp followed by irradiated BMDCs (open bar) and hypoxia followed by BMDCs. Values are means ± SEM for 5 animals in each group.

Figure 6

eGFP staining in representative lung panels. Representative panel of a control mouse lung (A and B) constitutively expressing GFP and wild-type mouse lung (C) injected with eGFP bone marrow-derived cells (BMDCs). Panels D, E, F and G are representative panels of recipient mouse lung 12 days after injection with eGFP BMDCs, which occurred 3 days after acute lung injury by a single administration of MCTp. Staining was detected in few cells in alveolar space, bronchial and distal arteries. Panel H. representative panel of recipient mouse lung exposed to hypoxia for 15 days and 12 days after injection with eGFP BMDCs: no staining was detected. Original magnification ×125 (A, D and H) ×500 (B, C, E, F and G).

Figure 7

Western blot analysis of eNOS in lung tissues. A) Representative blot of eNOS protein. B) Quantification of eNOS immunoreactivity in lung homogenates from control untreated mice (vehicle), MCTp-treated mice, MCTp-treated mice injected with bone marrow-derived cells (BMDCs), hypoxia-exposed mice, and hypoxia-exposed mice injected with BMDCs. Values are means ± SEM for 5 animals in each group.