SDF-1/CXCR4 Axis Mediates Bone Marrow-derived Smooth Muscle Progenitor Cells Contribute To Hypoxic Vascular Remodeling

AIM:To prove bone marrow-derived smooth muscle progenitor cells (SPCs) contribute to hypoxia-induced pulmonary vascular remodeling (PVR), which mediated by SDF-1/CXCR4 axis.METHODS:1. Isolation of bone marrow-derived SPCs.We constructed the smooth muscle specially promoted plasmid. Then transfered it to the bone marrow stromal cells and sorted the GFP positive ones (these were deemed to SPCs) by flow cytometry. SPCs were cultured 3 to 5 passages in vitro. Cell phenotype and growth characteristic were observed. Cell makers were identified by flow cytometry and immunofluorescence. SPCs were cultured with platelet derived growth factor-BB (PDGF-BB), and valuated whether those differentiate to smooth muscle-liked cells.2. SPCs involve in hypoxia-induced PVR.CM-Dil labeled SPCs (1×106 per rat) were injected into caudal vein of SD rats. Animals were placed in hypoxia cabin in 28 days, 8 hours per day, with setting barometric pressure to 380mmHg. Then SPCs in pulmonary arterial wall were measured. We established the models of SPCs adherent and penetrate to endothelial cells (ECs). Base on the models, the adherence and migration cof SPC were valued in the condition of hypoxia and presence of CoCl2.3. SDF-1/CXCR4 axis mediates SPCs directional migration.Through immunohistochemistry and ELISA assays, the SDF-1 expression was measured in pulmonary arterial wall or ECs under the condition of hypoxia. On cell models, the SPCs adherence and migration ability were valued with SDF-1 or CXCR4 neutral antibody. In vivo, SDF-1ab or SPCs (5×107 per rat) blocked CXCR4 binding site were injected to rats. Then the pulmonary artery pressure, indexes of vascular remodeling, and SPCs numbers in pulmonary arterial wall were measured.RESULT:1. In vitro, the cultured SPC was spindle-shape of single growth, and colony-liked in cells fusion.2. Both the precursor cell marker CXCR4 and SMC makerα-SM-actin were positive in SPC, but negative for the EC maker CD31 and the mature SMC maker CaM. 3. SPCs eliminated CXCR4, but expressed CaM gradually when cultured with PDGF-BB.4. Under the condition of hypoxia, more SPCs migrated into pulmonary arterial wall than normoxia, and most of those showed in media of the vascular.5. Contrast to normoxia, the adherence rate and migration quantity of SPCs were markedly increased under hypoxia or with CoCl2.6. SDF-1 expression rise in the pulmonary vascular endothelial and cultured PVECs supernatant the condition of hypoxia or cells cultured with CoCl2.7. On the cell models, inhibition of SDF-1 or blockade of CXCR4 could significantly decrease the adherence rate and migration quantity of SPCs.8. Blockade of SDF-1/CXCR4 axis could down regulate the pulmonary artery pressure, attenuate the PAs remodeling and reduce the number of SPCs in PAs wall.CONCLUSION:The PaO2 of pulmonary circulation is low under the hypoxic condition, which leads to the expression of SDF-1 in pulmonary endothelial, and homing the bone marrow–derived SPCs from blood. Once SDF-1 binding to CXCR4 on the SPC surface, cell adherents to pulmonary arterial ECs, migrate into vessel wall, and differentiate to matured SMCs, finally contribute to the vascular remodeling. These findings maybe proposal a new mechanism of hypoxia-induced PVR, and provide a new pharmacal target of HPAH.