Human Mesenchymal Stem Cell Therapy Reverses Su5416/Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Abstract

Aims: Pulmonary arterial hypertension (PAH) is a disease characterized by an increase in pulmonary vascular resistance and right ventricular (RV) failure. We aimed to determine the effects of human mesenchymal stem cell (hMSC) therapy in a SU5416/hypoxia (SuH) mice model of PAH. Methods and Results: C57BL/6 mice (20-25 g) were exposure to 4 weeks of hypoxia combined vascular endothelial growth factor receptor antagonism (20 mg/kg SU5416; weekly s.c. injections; PAH mice). Control mice were housed in room air. Following 2 weeks of SuH exposure, we injected 5 × 10⁵ hMSCs cells suspended in 50 μL of vehicle (0.6 U/mL DNaseI in PBS) through intravenous injection in the caudal vein. PAH mice were treated only with vehicle. Ratio between pulmonary artery acceleration time and RV ejection time (PAAT/RVET), measure by echocardiography, was significantly reduced in the PAH mice, compared with controls, and therapy with hMSCs normalized this. Significant muscularization of the PA was observed in the PAH mice and hMSC reduced the number of fully muscularized vessels. RV free wall thickness was higher in PAH animals than in the controls, and a single injection of hMSCs reversed RV hypertrophy. Levels of markers of exacerbated apoptosis, tissue inflammation and damage, cell proliferation and oxidative stress were significantly greater in both lungs and RV tissues from PAH group, compared to controls. hMSC injection in PAH animals normalized the expression of these molecules which are involved with PAH and RV dysfunction development and the state of chronicity. Conclusion: These results indicate that hMSCs therapy represents a novel strategy for the treatment of PAH in the future.

Keywords: apoptosis; cell proliferation; human mesenchymal stem cell; inflammation; pulmonary arterial hypertension.

FIGURE 1

Schematic of the experimental design for the protocol of SuH-induced PAH. Echo Doppler images of the PA flow are showed for a normoxia and a SuH-induced PAH mice, with a mid-systolic notch being developed (red dashed line) in the PAH group at day 14 of protocol, as a sign of disease onset. SuH, SU5416/hypoxia; PA, pulmonary artery; PAH, pulmonary arterial hypertension.

FIGURE 2

Effects of the hMSC therapy in the pulmonary vascular changes observed in SuH-PAH mice. (A–C) Representative images of PA outflow profile (upper) are presented for all groups 28 days after protocol initiation. Lower images are representative tracings of RVSP at day 29 of protocol. (D) Ratio between PAAT and RVET; (E) RVSP, and (F) linear regression between PAAT-to-RVET ratio and RVSP. (G) Representative orcein and immunostaining for α-SMA of the distal PAs exposed to normoxia or SuH protocol for 4 weeks. (H) Vessel wall thickness expressed as a percent of the total area of the vessel ranging between 30 and 100 μm in external diameter, (I) total of fully muscularized vessels, and (J) lung weigh-to-body weight ratio. Data represent the mean ± SEM (n = 5–7 mice per group). ^∗P < 0.05 compared with normoxia group; ^†P < 0.05 compared with SuH group treated with vehicle. Ordinary one-way ANOVA with multiple comparisons. hMSC, human mesenchymal stem-cell; SuH, SU5416/hypoxia; PAH, pulmonary arterial hypertension; PA, pulmonary artery; RVSP, right ventricular systolic pressure; PAAT, pulmonary artery acceleration time; RVET, right ventricle ejection time; α-SMA, alpha smooth muscle actin.

FIGURE 3

Effects of the hMSC therapy in the pulmonary vascular extracellular matrix deposition. (A) Picrosirius red staining. (B) Shows the Western blot analyses of RAGE in lungs from all animal groups. GAPDH was used for normalization. (C) Perivascular collagen area, and (D) quantification of RAGE expression. Each column and bar represent the mean ± SEM (n = 5–7 mice per group). ^∗P < 0.05 compared with normoxia group; ^†P < 0.05 compared with SuH group treated with vehicle. Ordinary one-way ANOVA with multiple comparisons. hMSC, human mesenchymal stem-cell; SuH, SU5416/hypoxia; RAGE, receptor for advanced glycation end products; CSA, cross section area.

FIGURE 4

Effects of SuH model on the lung protein expression over 29 days of protocol and intravenous treatment with vehicle or hMSC at day 14 of protocol. (A) Shows the Western blot analyses of active caspase 3, TNF-α, p-p38 MAPK, p-38 MAPK, p-ERK5 and ERK 5 in lungs from normoxia or SuH mice, respectively. GAPDH was used for normalization. (B) Quantification of active caspase 3 expression. (C) Quantification of TNF-α. (D) Relative expression ratio of p-p-38 MAPK to p-38 MAPK and (E) relative expression of p-ERK5 to ERK5, respectively. (F) Linear regression between p-p-38 MAPK to p-38 MAPK ratio and TNF-α expression and (G) linear regression between p-ERK5 to ERK5 ratio and TNF-α expression. Each column and bar represent the mean ± SEM (n = 3–7 mice per group). P < 0.05 compared with normoxia group; ^†P < 0.05 compared with SuH group treated with vehicle. Ordinary one-way ANOVA with multiple comparisons. hMSC, human mesenchymal stem-cell; SuH, SU5416/hypoxia; Active casp 3, active caspase-3; TNF-α, tumor necrosis factor alpha; p-p-38 MAPK, phosphorylated P-38 MAPK; p-38 MAPK, p-38 mitogen-activated protein kinase; p-ERK5, phosphorylated extracellular-signal-regulated kinase 5; ERK5, extracellular-signal-regulated kinase 5.

FIGURE 5

Effects of the treatment with vehicle or hMSC on heart structure and function of SuH-PAH mice. (A) Representative images of parasternal short-axis views obtained by B-mode echocardiography (all end-diastolic), (B) right ventricle area, (C) left ventricle area, and (D) right ventricular cardiac output 28 days after protocol initiation. (E) Left ventricle ejection fraction and (F) heart rate. Each column and bar represent the mean ± SEM (n = 5–7 mice per group). ^∗P < 0.05 compared with normoxia group; ^†P < 0.05 compared with SuH group treated with vehicle. Ordinary one-way ANOVA with multiple comparisons. hMSC, human mesenchymal stem-cell; SuH, SU5416/hypoxia; RV, right ventricle; LV, left ventricle.

FIGURE 6

Effects of the therapy with vehicle or hMSC on RV free wall thickness, collagen volume fraction and RAGE expression in RV tissue from SuH-induced PAH or normoxia mice. (A, upper) Shows representative images of RV free wall obtained by M-mode echocardiography and (lower) picrosirius red staining under light microscopy (magnification 40×), showing collagen fibers in red in RVs from all animal groups. (B) RV free wall thickness, (C) RV-to-LV + septum ratio, (D) collagen volume fraction of RVs in relation to the tissue area. (E) Western blot analyses of RAGE in RV tissue from experimental groups. GAPDH was used for normalization. (F) Quantification of RAGE expression. Each column and bar represent the mean ± SEM (n = 3–7 mice per group). ^∗P < 0.05 compared with normoxia group; ^†P < 0.05 compared with SuH group treated with vehicle. Ordinary one-way ANOVA with multiple comparisons. hMSC, human mesenchymal stem-cell; SuH, SU5416/hypoxia; RAGE, receptor for advanced glycation end products; RV, right ventricle; LV, left ventricle; S, interventricular septum.

FIGURE 7

Effects of SuH model on the RV protein expression over 29 days of protocol and intravenous treatment with vehicle or hMSC at day 14 of protocol. (A) Shows the Western blot analyses of active caspase 3, TNF-α, p-p38 MAPK, p-38 MAPK, p-ERK5 and ERK 5 in RVs from normoxia or SuH mice, respectively. GAPDH was used for normalization. (B) Quantification of active caspase 3 expression. (C) Quantification of TNF-α. (D) Relative expression ratio of p-p-38 MAPK to p-38 MAPK and (E) relative expression of p-ERK5 to ERK5, respectively. (F) Linear regression between p-p-38 MAPK to p-38 MAPK ratio and TNF-α expression and (G) linear regression between p-ERK5 to ERK5 ratio and TNF-α expression. Each column and bar represent the mean ± SEM (n = 3–7 mice per group). ^∗P < 0.05 compared with normoxia group; ^†P < 0.05 compared with SuH group treated with vehicle. Ordinary one-way ANOVA with multiple comparisons. hMSC, human mesenchymal stem-cell; SuH, SU5416/hypoxia; Active casp 3, active caspase-3; TNF-α, tumor necrosis factor alpha; p-p-38 MAPK, phosphorylated P-38 MAPK; p-38 MAPK, p-38 mitogen-activated protein kinase; p-ERK5, phosphorylated extracellular-signal-regulated kinase 5; ERK5, extracellular-signal-regulated kinase 5.

FIGURE 8

Effects of SuH model on the cytokine levels measured by ELISA assessment in RVs from experimental groups. (A) Levels of TNF-α, (B) levels of IL-1β, and (C) levels of IL-6. Each column and bar represent the mean ± SEM (n = 3–7 mice per group). ^∗P < 0.05 compared with normoxia group; ^†P < 0.05 compared with SuH group treated with vehicle. Ordinary one-way ANOVA with multiple comparisons. hMSC, human mesenchymal stem-cell; SuH, SU5416/hypoxia; TNF-α, tumor necrosis factor alpha; IL-1β, interleukin-1 beta, IL-6, interleukin-6.