Effect of chemokine receptor CXCR4 on hypoxia-induced pulmonary hypertension and vascular remodeling in rats

Abstract

Background: CXCR4 is the receptor for chemokine CXCL12 and reportedly plays an important role in systemic vascular repair and remodeling, but the role of CXCR4 in development of pulmonary hypertension and vascular remodeling has not been fully understood.

Methods: In this study we investigated the role of CXCR4 in the development of pulmonary hypertension and vascular remodeling by using a CXCR4 inhibitor AMD3100 and by electroporation of CXCR4 shRNA into bone marrow cells and then transplantation of the bone marrow cells into rats.

Results: We found that the CXCR4 inhibitor significantly decreased chronic hypoxia-induced pulmonary hypertension and vascular remodeling in rats and, most importantly, we found that the rats that were transplanted with the bone marrow cells electroporated with CXCR4 shRNA had significantly lower mean pulmonary pressure (mPAP), ratio of right ventricular weight to left ventricular plus septal weight (RV/(LV+S)) and wall thickness of pulmonary artery induced by chronic hypoxia as compared with control rats.

Conclusions: The hypothesis that CXCR4 is critical in hypoxic pulmonary hypertension in rats has been demonstrated. The present study not only has shown an inhibitory effect caused by systemic inhibition of CXCR4 activity on pulmonary hypertension, but more importantly also has revealed that specific inhibition of the CXCR4 in bone marrow cells can reduce pulmonary hypertension and vascular remodeling via decreasing bone marrow derived cell recruitment to the lung in hypoxia. This study suggests a novel therapeutic approach for pulmonary hypertension by inhibiting bone marrow derived cell recruitment.

Figure 1

Effect of CXCR4 inhibitor on pulmonary artery pressure and right ventricular hypertrophy induced by chronic hypoxia in rats. (A) mPAP, showing representative tracings of pulmonary artery pressure (upper panel) and quantitative data (lower panel). (B-E) Right ventricular hypertrophy, showing data on RV/(LV+S) (B), right ventricular weight (C), left ventricular plus septal weight (D) and whole heart weight (E). *p < 0.05 as compared with other groups and # p > 0.05 as compared with normoxic control rats. n = 5 rats for each group.

Figure 2

Effect of CXCR4 inhibitor on wall thickness of pulmonary arteries induced by chronic hypoxia in rats. (A) Wall thickness showing quantitative data on percent wall thickness (%WT) (left panel) and representative microphotographs (right panel). TA = terminal bronchial arterioles; I A = intra-acinous arterioles. (B) Body weight change. *p < 0.05 as compared with other groups. (C) hematocrit. *p < 0.05 as compared with normoxia. n = 5 rats for each group.

Figure 3

Effect of electroporation of bone marrow cells with CXCR4 shRNA on hypoxia-induced pulmonary hypertension and right ventricular hypertrophy in rats: (A) mPAP and (B) RV/(LV+S). *p < 0.05 as compared with other groups; #p < 0.05 as compared with normoxia and BMC+CXCR4; $p < 0.05 as compared with normoxia. n = 5 rats for each group. BMC = transplantation of bone marrow cells without shRNA, BMC+S-RNA = transplantation of bone marrow cells with scrambled shRNA, BMC+ CXCR4 = transplantation of bone marrow cells with CXCR4 shRNA.

Figure 4

Effect of electroporation of bone marrow cells with CXCR4 shRNA on hypoxia-induced pulmonary hypertension and vascular remodeling in rats: (A) Percent wall thickness. *p < 0.05 as compared with normoxia and hypoxic controls. # p < 0.05 as compared with normoxia and BMC+CXCR4. $p < 0.05 as compared with normoxia. (B) Body weight change. # p < 0.05 as compared with normoxia. *p < 0.05 as compared with normoxia and hypoxic controls. (C) hematocrit. *p < 0.05 as compared with normoxia control. n = 5 rats for each group. BMC = transplantation of bone marrow cells without shRNA, BMC+S-RNA = transplantation of bone marrow cells with scrambled shRNA, BMC+ CXCR4 = transplantation of bone marrow cells with CXCR4 shRNA.

Figure 5

Effect of CXCR4 shRNA delivery on CXCR4 expression in bone marrow cells: Western blot on proteins isolated from rat bone marrow cells was performed to analyze CXCR4 expression. Quantitative data (upper panel) and representative images (lower panel). C = control, D2 = day 2, W3 = week 3 and W5 = week 5. *p < 0.05 as compared with control. n = 3 for each groups.

Figure 6

Effect of CXCR4 shRNA delivery on bone marrow cell migration to rat lung: (A) GFP expression. Proteins were isolated form rat lungs and Western blot was performed for analysis of GFP protein expression. Quantitative data (upper panel), setting hypoxia BMC as 1, and representative images (lower panel). *p < 0.05 as compared with other groups. (B) c-kit expression. Proteins were isolated form rat pulmonary artery and Western blot was performed for analysis of c-kit expression. Quantitative data (upper panel), setting normoxia control as 1, and representative images (lower panel). *p < 0.05 as compared with other hypoxia groups. # p > 0.05 as compared with normoxia control. n = 3 for each groups. BMC = transplantation of bone marrow cells without shRNA, BMC+S-RNA = transplantation of bone marrow cells with scrambled shRNA, BMC+ CXCR shRNA = transplantation of bone marrow cells with CXCR4 shRNA.