Mechanism Research On AD-SMCs Promoting Functional Activity Of Vascular Endothelial Cells

Objective:This sdudy is to explicit the the biology effects of vascularization by adipose-derived mesenchymal stem cells (AD-MSCs) through promoting functional activity of Endothelial cells(ECs). And in turn we study and ascertain the molecular mechanism of this promotion of AD-MSCs, to access the pathway by which the AD-MSCs regulate the functional activity of HUVECs.The performences will lay the foundation for future clinical application of AD-MSCs and cell-free therapy system.Methods:Part 1 Isolation, culture and identification of AD-MSCs ①The adipose tissue was collected from healthy females who had liposuction (we have been given the permission to use the liposuction aspirates in the experiment.). ②As AD-MSCs retain the potential of multiple-directional differentiation, we assessed the ability of AD-MSCs to differentiate into adipocytes and osteoblasts with the use of the appropriate medium. Oill-red dye and Alizarin red-S staining were used for the estimation of adipogenesis and osteogenesis separately. ③ The percentage of CD 31+HUVES were analysed with flow cytometry.Part 2 AD-MSCs of passage 3-6 were used in the subsequent experiments. The cells were then cultured by DMEM-HG with FBS-free instead of the H-DMEM with 10%FBS.The supernatant were extracted after throughout the night, followed by centrifugation. After adding 3% FBS and 5% FBS,the supernatant were reserved for the experimental groups, while the DMEM-HG with corresponding concentration of FBS were used as the control group.The cell proliferation assay, cell wound healing assay, Matrigel cell tubule formation assay, Matrigel 3D culture of sprouting experiments were carried respectively, results were expressed as mean± standard deviation (χ±SD).Part 3 Molecular mechanism study①The quantities of VEGF in the supernatant harvest from the 24-well plates were measured using a sandwich ELISA kit.②HUVECs and AD-MSCs were followed indirectly cocultured in the Transwell system, to observe the migratory activity of HUVECs after AD-MSCs treatment.③Then the VEGF level were detected with ELISA kit in the groups. ④On previous basis, the difference of VEGF paracrine by HUVECs and AD-MSCs were examined. ⑤HUVECs were cultured in the MSC-CM and the counterpart, respectively. Then total protein and total RNA were extracted to analyse the protein expression as well as mRNA expression of HIF-1α VEGF and Cdk5Rap3, witn Western blot and real-time RT-PCR, respectively.Results:Partl ①HUVECs and AD-MSCs grew well in high glucose DMEM with 10% FBS.② Immunohistochemistry demonstrated that HUVECs were CD31 positive, which located in the cell membrane. ③Flow cytometry analysis showed that HUVECs were detected positive for CD31 (98.3%), and negative for CD34 with only 0.016% CD34(+). ④AD-MSCs can differentiate into adipocytes and osteoblasts with Oill-red dye and Alizarin red-S staining positive,respectively.Part 2 ①the viability assay with CCK-8 kits showed the absolute optic density values of MSC-CM-treated HUVECs after 24-hour(P<0.01) and 36 hours (P<0.05), repectively.② Wound healing assay:the migration rate of the MSC-CM experiment group is higher than that of the control group at the first 6 hours. To 12 hours, the migration area of the MSC-CM experiment group had reached 42%, while only 10% of which for the control group.③Tube formation assay The tube structure is 21 per field on average for the MCM-CM group versus 12 per field for the control group, P<0.001, which was of predominantly statistical significance. ④The sprouting assay also demonstrated a significant result:the number of the vascular sprouting in the Matrigel for MSC-CM cultured HUVESCs was 18.80±1.07 (n=5), versus 10.60+0.93 (n=5) for the Control group (P=0.004).Part 3 ①The VEGF expression in the AD-MSCs supernatant as compared with the HUVECs supernatant was 18.57±2.38 pg/ml (n= 4) versus 11.54±1.48 pg/ml (n= 4) (P<0.01).②Transwell migration assay:the migrated cells of the AD-MSCs group was also great differencewith the control group (P<0.001). ③VEGF expression differences in Transwell: the VEGF expression in the AD-MSCs supernatant as compared with the HUVECs supernatant was 26.87±2.77pg/mL, versus 7.65+1.23pg/mL (Table 3-6) (P<0.001).④ Every 1 X 104 cells as a unit, the VEGF expression of MSC-CM and HUCVEC-CM was of predominantly statistical significance (P<0.01), ⑤The protein expression of VEGF、HIF-la and Cdk5Rap3 were markedly increased when HUVECs co-cultured with MSC-CM. ⑥As ame as the result of Western Blot, mRNA expression of VEGF、HIF-la and Cdk5Rap3 were also significantly increased, respectively.Conclusion:AD-MSCs can promote the biology function of HUVEs, such as proliferation, migration, tube formation. On one hand, AD-MSCs promote the functional activity of HUVECs through paracrine factors such as VEGF; on the other hand, AD-MSCs probablly regulate HUVECs to enhance the expression of VEGF, HIF-1α and CDK5RAP3, thereby modulating and further strengthening the biological behavior of endothelial cells. Ehanced neovascularization was observed by HUVECs co-culturing with AD-MSCs.