The Role Of SDF-1/CXCR7 In Endothelial Progenitor Cells Participating In Angiogenesis

Emerging evidences show that Endothelial progenitor cells (EPCs) play a crucial role in physiological and pathological angiogenesis. The process of EPCs participating in neovascularization is a dynamic multi-step cascade process and regulated by a variety of factors in microenvironment. Previous studies confirmed that stromal cell-derived factor 1 (SDF-1) is a principal regulator of retention, migration, and mobilization of EPCs during steady-state homeostasis and injury. For many years, CXC chemokine receptor 4 (CXCR4) has been considered as the unique receptor of SDF-1 and the only mediator of SDF-1-induced biological effects. However, recent studies find that SDF-1 could bind to not only CXCR4 but also CXC chemokine receptor 7(CXCR7). CXCR7 has a significant higher binding affinity for SDF-1 than CXCR4. The evidence that SDF-1 binds to the CXCR7 raises a concern how to distinguish the potential contribution of the SDF-1/CXCR7 pathway from SDF-1/CXCR4 pathway in all the processes that were previously attributed to SDF-1/CXCR4. The role of CXCR7 in angiogenesis remains largely unclear, especially the role of CXCR7 in EPCs participating in angiogenesis has not been reported. To clarify the role of CXCR7 in angiogenesis, one of critical problem is to establish a more physiological in vitro angiogenesis model. Angiogenesis is a multi-step dynamic process involving complex interactions between vascular cells and the corresponding extracellular environment. However, many traditional models can not mimic this complex process. With development of microfluidic technologies, it has paved the way for new approaches to establish an in vitro anigogenesis model mimicking the in vivo conditions. In this study, we established an in-vitro angiogenesis model based on microfluidic technology, combining with cellular behavior experiments in vitro, to study the roles and effects mechanisms of CXCR7 in SDF-1-mediated EPCs homing and participating in angiogenesis, and further to explore the potentiality of CXCR7 as a target to promote or inhibit angiogenesis, which was expected to provide a new therapy for angiogenesis-related diseases. The main research and results were as follows:?Mononuclear cells (MNCs) were isolated from rat bone marrow by density gradient centrifugation and plated on 6-well plates. After cultured for 24 h, non-adhesion cells were collected and seeded on 6-well plates pre-coated with fibronectin. Bone marrow-derived MNCs cultured under endothelial-specific conditions developed a spindle-shape appearance and typical cell clusters at day 7.The appearance and organization of these cell clusters resembled the characteristic blood island-like cell clusters. Immunofluorescent staining assay demonstrated that most of the cells (up to 80%) were positive for CD133 and VEGFR2. And it was found that the cells were positive for Dil-Ac-LDL uptaking and lectin binding. The double positive cells were identified as differentiating EPCs. The results suggested that high-purity EPCs could be harvested by EBM-2 medium induced-culturing MNCs that were isolated by density gradient centrifugation combining with differential adhersion time methods.?Reverse transcriptase PCR (RT-PCR), western-blotting and flow cytometry assay demonstrated that both CXCR4 and CXCR7 were expressed highly in EPCs. We investigated the roles of chemokine receptor CXCR4 and CXCR7 in the adhesion, proliferation and tube formation of EPCs by blocking CXCR4 or CXCR7 with their antibodies or antagonists respectively. The results showed SDF-1 regulated the adhesion, transendothelial migration, proliferation and tube formation of EPCs through both of CXCR4 and CXCR7, and the chemotaxis of EPCs through CXCR4 alone, while SDF-1 regulated the survival of EPCs via CXCR7 not CXCR4.?It is essential for angiogenesis researches to establish a suitable in vitro angiogenesis model. In this study, we established a novel in vitro angiogenesis model based on microfluidic device, which can provide an in-vivo-like microenvironment for endothelial cell culture and monitor the response of endothelial cells to their microenvironment changes real-time. To evaluate the potential of this microfluidic device for angiogenesis model research, the effects of pro-angiogenic factors on endothelial cell proliferation, migration and tube-like structure formation were investigated. The results showed the proliferation rate of endothelial cells in 3D matrix was significantly promoted by the pro-angiogenic factors. With the stimulation of pro-angiogenic factors gradients, endothelial cells directly migrated into matrigel from low concentration to high concentration and consequently formed multi-cell chords and tube-like structures. The results suggested that the device could provide a suitable platform for elucidating the mechanism of angiogenesis and screening pro-angiogenic or anti-angiogenic drugs for‘angiogenesis-dependent'diseases.?By the angiogenesis model based on microfluidic device, we further investigated the role of CXCR7 and CXCR4 in SDF-1-induced EPCs incorporating into extracelluar matrix and forming tube-like structures in 3D matrix to explore their functional mechanisms. The results showed that both CXCR7 and CXCR4 were essential for EPCs incorporating into matrix under the induction of SDF-1 gradient. And it was also found that SDF-1 induced EPCs forming tube-like structures in 3D matrix via both CXCR7 and CXCR4. We further demonstrated the effect of MMP inhibitor (GM6001) as an agent against SDF-1-promoted EPCs incorporating into matrix and forming tube-like structures. The inhibition effects GM6001 were correspondent with CXCR7 or CXCR4 antagonist. Another research team in our lab demonstrated that SDF-1 could promote MMP2 secretion through its two receptors CXCR4 and CXCR7. Taking the results together, we deduced that SDF-1 promoted EPCs incorporating into matrix and forming tube-like structures partially through up-regulating the secretion of MMP to degrade extracellular matrix, which will facilitate EPCs migrating.In summary, CXCR7 and CXCR4 play essential, but differential roles in EPCs participating in angiogenesis. Both CXCR7 and CXCR4 are essential for SDF-1-mediated EPCs adhering, incorporating into matrix, transendothelial migrating and forming tube-like structures in 3D matrix. SDF-1 regulates the survival of EPCs via CXCR7 not CXCR4, while SDF-1 mediates the chemotaxis of EPCs through CXCR4 alone. These evidences indicate that CXCR7 plays a critical role in EPC homing and participating in angiogenesis, and CXCR7 may be another potential target molecule for angiogenesis-dependent diseases. In addition, a novel in vitro angiogenesis model was developed based on a microfluidic device, which could provide an in-vivo-like microenvironment for cell culture and monitor cell response in real-time. Thus this angiogenesis model will be a suitable platform for elucidating the mechanism of angiogenesis and testing pro-angiogenic or anti-angiogenic drugs for‘angiogenesis- dependent'diseases.