The Proliferation 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 outcome fundamentally, clarify the pathophysiology and molecular mechanisms of bone formation、reconstruction and remodeling are the base and starting point. Bone is a complex tissue with a dynamic processional of extracellular matrix mineralizing and the ability to adjust to its functional demands and self-healing.It is known that humoral factors, as well as communication amongst cells of the bone micro-environment, local bloody supply and neural factors between bone and nerve system, are all important in regulating bone metabolism.Calcitonin gene-related peptide (CGRP), a37-amino acid neuropeptide, is one of the most abundant neuropeptides in central and peripheral nervous system and participates in a variety of regulatory functions. CGRP is one of the most abundant neuropeptides in peripheral nervous system, has been demonstrated that play an important role in bone repair and bone remodeling. It is found that in the plasma of fracture rats, CGRP concentration was significantly increased and the number of nerve fibers containing CGRP were also increased, CGRP innervation coincides with bone formation during fracture healing and modeling [9]. It is suggest that CGRP may be transported by axoplasmic to the fracture site, and expand the local blood vessels to increase the blood supply of fracture, so as to promote fracture healing. Aoki’s research found that during the healing process of rat tibial fracture CGRP-immunofluorescent nerve fibres significantly increased in the periosteum, fibrous granulation tissue and new bone tissue, and the majority near the blood vessels. These sensory nerve fibers release much higher levels of CGRP in the local area of the fracture than the serum to heal the fracture.It is found that bone is a biological issue which is highly vascularized, neovascularization play an important role in bone repair and reconstruction process, angigonesis is essential in osteogenesis, which is the basis of bone formation, bone remodeling. The formation and development of an active microvasculature is an essential stage for bone remodeling and fracture healing, which could produce growth factor to regulate osteoblast activity and raise stem cells into bone cells in order to promote bone formation.. Increasing research has found that the key factor contributing to poor repair with tissue engineered bone is poor vascularization. The degradation, proliferation and migration of the basement membrane of vascular endothelial cells,is necessary for the formation of new blood vessels and a critical step in bone repair.In previous study, we found that CGRP promote the proliferation of HUVECs by AlamarBlue, and this effect was time-concentration dependent. However, In the former study, we did not use the antagonists from reverse to verify this effect, and the mechanism of this effect is not clear. In this study, we use CGRP antagonist CGRP8-37and MAPK pathway antagonists to further verify the proliferation effect of CGRP on HUVEC from both positive and negative side,preliminarily discuss the mechanism from signaling pathways and learn the role of MAPKs in CGRP induced HUVEC proliferation.OBJECTIVE1. To further verify the proliferation effect of CGRP on HUVEC from both positive and negative side.2. To discuss the mechanism of CGRP induced HUVEC proliferation from signaling pathways and learn the role of MAPKs in CGRP induced HUVEC proliferation.Part1. The isolation and identify of HUVECsObjective:To isolate and identify the HUVECsMethod:1. The isolation of HUVECsEndothelial cells were extracted from human umbilical veins essentially as described by Bordenave et al. according to the procedure of Jaffe et al. Endothelial cells were maintained in Endothelial Cell Medium (EGM) with10%(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,) Cells were used for the experiments after the first or the second passage.A sterile technique was utilized in all manipulations of the cord. The cord was severed from the placenta after birthplaced in2h in a sterile container filled with cord buffer. The vein was perfused with PBS buffer contain heparin to wash out the blood and allowed to drain.10ml of0.1%collagenase I was then infused into the umbilical vein and placed in a water bath containing cord buffer and incubated at370C for20 min. After incubationthe collagenase solution containing the endothelial cells was flushed from the cord by perfusion with PBS buffer contain heparin. The effiuent was collected in a sterile50ml conical centrifuge tube The cells were sedimented at800r/min for10min, discharge the supernatant, then add EGM and cultured at370C under5%CO2..in a1%gelatin-coated dish.The cells were fed twice a week with fresh culture medium..2. The identification of 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:HUVEC can be proliferated and passaged Successfully in vitro, HUVEC grew the fastest in a pattern of strict monolayer growth and show a cobblestone appearance under light microscopy,. VWF shown positive reation by immunohistochemistration.Conclusion:A large number of hish purified endothelial cells could be acquired by digestion of trypsin, so as to provide seed cells for the future study.Part2. The proliferation effect of calcitonin gene-related peptide(CGRP) on human umbilical vein endothelial cells(HUVECs) ObjectiveTo further verify the proliferation effect of CGRP on HUVEC from both positive and negative side. In order to reveal the mechanism of neutotized tissue-engineered bone promoting angiogenesis.Method:The experiment were divided into six groups:(A) control group, only add ECM;(B) add10-8mol/L CGRP;(C) cells were pretreated for1h with10"6mol/L CGRP8-37, prior to add10-8mol/L CGRP;(D) cells were pretreated for1h with10"5mol/L PD98059, prior to add10-8mol/L CGRP;(E) cells were pretreated for1h with10-5mol/L SB203580, prior to add10-8mol/L CGRP;(F) cells were pretreated for1h with10-5mol/L SP600125, prior to add10-8mol/L CGRP. To detect the proliferation of human umbilical vein endothelial cells (HUVECs) changes in each group with AlamarBlue.Result:Compared with the control group, CGRP can promote the proliferation of HUVECs. CGRP induced the HUVECs proliferation can be significantly inhibited by the CGRP antagonist CGRP8-37, ERK1/2antagonist PD98059, p38antagonist SB203580and JNK antangonist SP600125.Conculsion:CGRP promote the proliferation of HUVECs and ERK1/2, p-38, JNK antagonist inhibite this effect,suggesting that the MAPK pathway may play a role in this process..Part3. Calcitonin-gene-related peptide stimulates angiogenesis in human endothelial cells by VEGF-FLT1/KDR mechanismObjective: To discuss the mechanism of CGRP induced HUVEC proliferation from signaling pathways and learn the role of MAPKs in CGRP induced HUVEC proliferation.Method:Experiment were divided into six groups:(A) control group, only add ECM;(B) add10-8mol/L CGRP;(C) cells were pretreated for1h with10-6mol/L CGRP8-37, prior to add10-8mol/L CGRP;(D) cells were pretreated for1h with10-5mol/L PD98059, prior to add10-8mol/L CGRP;(E) cells were pretreated for1h with10-5mol/L SB203580, prior to add10-8mol/L CGRP;(F) cells were pretreated for1h with10-5mol/L SP600125, prior to add10-8mol/L CGRP. The expressions of total and phosphorylated ERK1/2, p38, and JNK induced by CGRP and with PD98059, SB203580, SP600125were determined by Western blot analysis.Result:The CGRP induced rapid increases in phospho-ERK1/2as early as5min after addition of CGRP and this was decreased gradually (Fig.2A). CGRP induced increases in phospho-p38(Fig.2B) and phospho-JNK (Fig.2C),just at20min and10min. Longer treatment (24h) with CGRP led to a increase in both phosphor-ERK1/2and phospho-p38. There was no change in the expression of total ERK1/2and p38, total JNK express a increase tendency.Conclusion:CGRP induced phosphorylation of were inhibited by PD98059, SB203580, SP600125, respectively (Fig.3). These data suggest that CGRP induced increase in proliferation may be mediated by the activation of ERK1/2, p38and MAPK.