The Biological Effects And Mechanisms Of Calcitonin Gene Related Peptide On Human Umbilical Vein Endothelial Cells

Despite the progress that has been made in bone regeneration, fracture caused by trauma、infection and a variety of reasons and Bone nonunion still represent the medical and socioeconomic challenge.In order to improve the treatment outcomefundamentally, clarify the pathophysiology and molecular mechanisms of boneformation、reconstruction and remodeling are the base and starting point. Bone is acomplex tissue with a dynamic processional of extracellular matrix mineralizing andthe ability to adjust to its functional demands and self-healing.lt is known thathumoral factors, as well as communication amongst cells of the bonemicro-environment, local bloody supply and neural factors between bone and nerve system, are all important in regulating bone metabolism。It is well known that CGRP signaling has anabolic effects in bone. CGRP-immunofluorescent fibres have been demonstrated in the vessels of the periosteum, Volkmann’s canals, bone marrow, osteochondral junction of the growth plate and the attachment of the synovial membrane. Bone tissue contains CGRP-immunoreactive nerve fibers whose increased concentrations during bone development and regeneration suggest they are directly involved in the local regulation of bone remodeling. Increase of CGRP-immunoreactive nerve fibers was also demonstrated in the repair process of experimental bone defects as well as bone grafts. The density of CGRP-ir fibers has been reported to be increased near the sites of postfracture osteogenesis (healing callus). Highest neuronal CGRP immunoreactivity on the concave side of angular fracture, where maximum bone formation took place during the healing and modeling in both angular and straight fractures. In an elegant model of angulated fracture healing, the presence of newly formed CGRP-ir nerve fibers is particularly abundant in areas with a high rate of new bone formation. The mouse in which a-CGRP gene selectively knocked out revealed that the rate of bone formation was substantially decreased. In line with this view, it was recently shown that capsaicin treatment destroys the unmyelinated sensory axons expressing CGRP and that the reduced CGRP signalling was associated with decreased bone mass, similar to patients with familial dysautomia which lacks unmyelinated sensory neurons and exhibit decreased bone mass and increased fracture rate.Also, angigonesis is essential in osteogenesis. Bone is formed by two distinct modes of ossification:by intramembranous ossification and by endochondral ossification. Despite the differences, the two types of ossification have as common feature:the pre-requisite of vascularization. In fact, the reconstruction of intraosseous circulation is one of the earliest events during bone repair. Lack of angiogenesis has been pointed out as one of the main reasons for non-healing bone. The rate of fracture healing is related to angiogenesis. The formation and development of an active microvasculature is an essential stage for bone remodeling and fracture healing Increasing research has found that the key factor contributing to poor repair with tissue engineered bone is poor vascularization.Previously we observed that β-tricalcium phosphate ((3-TCP) as the scaffold material either with sensory nerve tracts,vascular bundles or alone to repair a1.2cm femur defect in the rabbit. Better osteogenesis was observed by x-ray and histology in sensory nerve tracts group and vascular bundles group at4,8and12weeks. Implantation of sensory tract into tissue-engineered bone can significantly improve the CGRP contents and expression of CGRP1R.Considering the current understanding of the role of CGRP and that of Angiogenesis in bone remodeling. It is likely that CGRP could promote bone formation through angiogenesis. The present study was designed to investigate the effect of CGRP on angiogenesis and the mechanism of its effect. As we know,VEGF is the most important pro-angiogenic factor which is known to enhance proliferation, survival, and tube formation by endothelial cells. VEGF-A exists in five different isoforms:VEGF145, VEGF189and VEGF206which are able to bind to the extracellular matrix (ECM) through heparin, VEGF121which is soluble and VEGF165, which is the most abundant form and can be both soluble and bound to the ECM. Since VEGF165can be soluble and constitutes the predominant form of VEGF produced by the cells, we also decided to compare to role of VEGF to CGRP in angiogenesis.OBJECTIVE1. Clarify the the expression of CGRP receptors in HUVECs2. To investigate the effect and mechanism of CGRP on proliferation、migration of HUVECs.3. To investigate the effects and mechanisms of CGRP on angiogenensis of HUVECs4. To compare the effects on proliferation, migration and tube formation of HUVECs between CGRP and VEGF.Part1. The isolation and identify of HUVECs and clarify the CGRP receptors in HUVECsObjective: To isolate, and identify the HUVECs and clarify the CGRP receptors in HUVECs.Method:1. The isolation, and identification of HUVECsEndothelial cells were extracted from human umbilical veins essentially as described by Bordenave et al. according to the procedure of Jaffe et al.[35]. Endothelial cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) with20%(v/v) FCS(gibco),100units/ml penicillin,100mg/ml streptomycin,90mg/ml heparin,20mg/ml Endothelial Cell Growth Supplement (ECGS). Cell characterization was detected by immunocytochemical reaction using anti-von Willebrand factor (abeam,)and anti-CD31(abeam). Cells were used for the experiments after the first or the second passage.2. The identification of CGRP receptors in HUVECsHUVECs were cultured on cover slips. The cells were fixed for20min with3.7%(V/V) paraformaldehyde at room temperature and then permeabilized with ice-cold ethanol for5min. then treated with primary antibodies against human CGRP receptor overnight at4℃. Cells were then treated with the Dy Light594-conjugated secondary antibodies in1:200dilution for1h at room temperature with5μg/mL DAPI dying nuclear for10min. Immunostained cells were visualized with Inverted phase contrast microscopeResult:The primary cultured HUVECs started to grow attaching to flask about1h after inoculated. During3-4days, HUVEC grew the fastest in a pattern of strict monolayer growth and contact inhibition, showing a cobblestone or pitching stone-like appearance under light microscopy,. VWF and CD31shown positive reation by immunohistochemistration. Immunofluorescence showed CGRP receptors in HUVECs.Conclusion:A large number of hish purified endothelial cells could be acquired by digestion of trypsin. HUVEC can be proliferated and passaged Successfully in vitro and the CGRP receptors were expressed in HUVECs.Part2. The effect of calcitonin gene-related peptide(CGRP) on proliferation and migration of human umbilical vein endothelial cells(HUVECs)Objectiveneutotized tissue-engineered bone can effectively promote angiogenesis and repair of bone defects. To investigate the effects of calcitonin gene-related peptide (CGRP) on proliferation and migration of human umbilical vein endothelial cells (HUVECs),Further reveal the mechanism of neutotized tissue-engineered bone promoting angiogenesis.Method:HUVECs were collected from human umbilical core,and identified though von Willebrand factor(vWF) and CD31immunofluorescence.Experiment were divided CGRP(10-8-10-12M) group and control group.Immunoflurescence observed CGRP1R on HUVECs.The growth rate of HUVECs was detected through alarmarBlue at1,2,3,4and5day.Transwell chamber detected the ability of cell migration.ELISA assay detected VEGF secretion, the expression of FAK was examined using Western Blot and Q-PCR.Result:CGRP(10-12-10-8M) stimulated HUVECs proliferation, promoted HUVECs migrate and secreted VEGF. In this process, the expressions of FAK was up-regulated by CGRP(10-12-10-8M) at3,7and10d. Conculsion:CGRP had direct effect on the proliferation and migration of HUVECs.Increasing the secretion of VEGF and expression of FAK may be. the mechanism of action. This study further revealed the effect of CGRP on angiogenesis.Part3. Calcitonin-gene-related peptide stimulates angiogenesis in human endothelial cells by VEGF-FLT1/KDR mechanismObjective:To investigate the effects and mechanism of calcitonin gene-related peptide (CGRP) on angiogenesis of human umbilical vein endothelial cells (HUVECs) for revealing the theoretical basis on the role of sensory nerve in bone tissue engineering.Method:The human umbilical vein endothelial cells(HUVECs) were collected from human umbilical core. Experiment were divided CGRP(10-8-10-12-M) group and control group, totally six groups.Tube formation experiment detected the role of angiogenesis and VEGF ELISA detected the level of VEGF in supernatant.The mRNA expression levels of VEGF, CGRP receptor1, VEGF receptors1(FLT1) and VEGF receptors2(KDR)wrere detected through real-time flurescence quantitative PCR. Meanwhile, Western blot detected the expression of FLT1and KDR in HUVECs at3,7,10day.Result:CGRP can significently promote tube formation. VEGF ELISA showed CGRP-treated group can increase VEGF secretion in direct or indirect manner. The Q-PCR results showed that the mRNA expression level of VEGF and CGRP receptors1were significantly higher in CGRP-treated group at10day and mRNA level of FLT1and KDR were higher in CGRP-treated group at3,7,10days.Western blot showed the the expression of FLT1and KDR were significantly in (10-8-10-12M) CGRP-treated group at3,7,10day, too.Conclusion:CGRP can significantly promoting angiogenesis in vitro, maybe related to the secretion of VEGF and the expression of FLT1and KDR in HUVECs.Meanwhile,the expression of CGRP receptors increased in HUVECs treated with CGRP, further enhancing the effect on angeiogenesis of CGRP.Part4. The effect on angiogenesis of human umbilical vein endothelial cells(HUVECs):CGRP VS VEGFObjective:To compare the effects on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) between calcitonin gene-related peptide (CGRP) and Vascular endothelial growth factor (VEGF).Methods:Experiment were divided CGRP(10-12-10-8M) group, VEGF165(5-20ng/ml) and control group, totally9groups. The growth rate of HUVECs was detected through alarmarBlue at1,2,3,4and5day. Transwell chamber detected the ability of cell migration and tube formation experiment detected the role of angiogenesis. The mRNA expression levels of CGRP receptors were detected through real-time flurescence quantitative PCR.Results:CGRP and VEGF165stimulated the proliferation of HUVECs in a concentration-and time-dependent manner and the effect of VEGF165was stronger than CGRP. CGRP and VEGF165can also significantly promoted HUVECs migration and tube formation. The maximum effect of CGRP(10-1-10-10M) on migration and tube formation were equivalent with that of VEGF165(5-20ng/ml). The Q-PCR results showed that CGRP and VEGF165can significantly promoted the mRNA expression level of CGRP receptors. But, the level of CGRP receptors mRNA were significantly higher in CGRP-treated group.Conclusion:CGRP is a strong proangiogenic growth factor and the effect of CGRP on angiogenesis is equivalent or stronger than low dose VEGF165(5-20ng/ml).