Study Of EphB4/EphrinB2and Its Reverse Signal Function On The Angiogenesis Regulation Of Xuefu Zhuyu Decoction On HMEC-1Celline

Angiogenesis is a multi-step process involving various molecular signals that simulate proliferation, migration and assembly to form new blood vessels from pre-existing vasculature. Receptor tyrosine kinases (RTK) are recognized as critical mediators of angiogenesis, and the family members, such as vascular endothelial growth factor(VEGF) and angiopoietins have already been well studied and been used in clinical treatment.The Eph family is the largest family of RTKs. Unlike other members of RTKs binding to soluble ligands, Eph receptors interact with ephrin ligands, which mediate cellular repulsion, adhesion and cell attachment. Eph receptors interacting with their ephrin ligands active bi-directional signalings and participate in many physical processes, especially the nervous system. Series of researches point out the essential role of Eph/ephrin in both embryonic and adult angiogenesis, particularly of EphB4/ephrinB2and their forward and reverse signals. Our previous researches have shown that XFZYD promoted angiogenesis and the promoting effect had a inverted U-curve relationship with serum concentration and action time, which avoided excessive growth of vessels as VEGF did. EphB4/ephrinB2have been paid more and more attention in regulating angiogenesis and our team observed various regulation factors of angiogenesis and found that the expression of ephrinB2was up-regulated. How does XFZYD promote angiogenesis through the EphB4/ephrinB2pathway? And how does it play the role of moderate regulation? All of these promote the exploration for EphB4/ephrinB2and the bidirectional signals between the pair of receptor and ligand.ObjectiveWe observed the effect of Xuefu Zhuyu Decoction containing serum (XFZY-CS) on human microvascular endothelial cell-1(HMEC-1) tube formation to pick up the best condition for promoting angiogengesis. And then studied on how XFZYD regulated the expression of EphB4/ephrinB2and its reverse signaling in cell functional, molecular and genetic levels, with the purpose of providing evident proofs for XFZYD in promoting angiogenesis.Methods1Preparation of XFZY-CSWe randomly distributed the healthy male volunteers’ into the XFZYD group and the control one. Collected the XFZY-containing serum after volunteers took two dose of Xuefu Zhuyu Decoction for a week. Both the XFZY-containing serum and the control serum were centrifuged at3000rpm for almost45min. they were inactivated at56℃for30min and then stored at-20℃after filtering through0.22um filters.2Groups divisionDivided the human microvascular endothelial cell-1(HMEC-1) randomly which cultured in routine into6groups, including the dose of1.25%%2.5%and5%XFZY-CS groups and their blank serum control ones. Dissolved different serums in MCDB-131culture media containing5%FBS.3Determination of tube formation In vitro angiogenesis assayBoth1.25%、2.5%and5%XFZY-CS groups and their blank serum control ones were cultured for24h、48h and72h. Compare the difference of tube formation of XFZYD-CS and their control group with the same serum content. Choose the best condition of XFZY-CS by the in vitro angiogenesis assay,4Determination of EphB4/ephrinB2mRNA levels by Real-time PCRTotal RNA was extracted from cells by Trizol, and the quantity and purity were detected. Then we transcribed the total RNA into complementary DNA (cDNA). The primers for both β-actin and target genes were designed by Primer5software and synthesized by Takara Company. Relative quantitative analysis method2-ΔΔCT was performed to determine the copy numbers of each sample.5Determination expression of EphB4/ephrinB2by Western Blot Total protein was extracted and tested by a bicinchoninic acid (BCA) assay kit.Samples were separated by electrophoresis in SDS-PAGE gels and blotted onto0.45um polyvinylidene difluoride (PVDF) membranes. The membranes were treated with5%non-fat milk or BSA in room temperature depending on weather they were used for phosphor-antibody or not. The primary antibodies were incubated at4℃overnight and the secondary antibodies were incubated at room temperature.6Activation and inhibition of EphB4/ephrinB2reverse signalingWe used EphB4/fc as the activator of reverse signaling and PP2as the inhibitor. Then compare their tube formation with2.5%XFZYD-CS group and blank group in order to evaluate the effect of XFZYD in moderately promoting angiogenensis.Results1. Different density of serum has different effects on cell condition(P<0.05). It is more objective to compare the results under the same serum condition between Xuefu Zhuyu Decoction-Contained Serum and the blank serum.2. In vitro angiogenesis assay, treatment of XFZY-CS in2.5%serum concentration for48h showes evident action in promoting angiogenesis(P<0.05), while5%serum concentration inhibits angiogenesis in48h and72h.3. Results of real-time Q-PCR show that under the2.5%serum concentration, XFZYD up-regulates the expression of EphB4-mRNA in the12th hour (P<0.05), and down-regulates its expression in the24th hour (P<0.01); while the changes of ephrinB2-mRNA have not been observed.4. Western Blot analysis of ephrinB2shows protein expression is apparent in24h with the treatment of2.5%serum concentration(P<0.01). XFZYD induces changes of ephrinB2phosphorylation in9h, which can not be tested in12h,24h and48h.5. Activating and repressing the reverse signaling of EphB4/ephrinB2with EphB4/Fc and PP2, then compare the effects of in vitro angiogenesis with2.5%serum concentration for48h. The results indicates that XFZYD plays a similar role as EphB4/fc (P>0.05).Conclusion1. XFZ YD plays a bi-directional regulation role in the process of angiogenesis, not only promotes angiogenesis, but also inhibits it.2. The effect of XFZ YD in promoting angiogenesis relates with reverse signaling.3. XFZ YD also actives forward signaling, and reverse signaling may have a shorter effect than the forward one. It may be the cause of why XFZYD promotes angiogenesis in a reasonable way.4. The expression of EphB4/ephrinB2changes as early as9h, which is earlier than the in vitro angiogenesis assay. That is to say, EphB4/ephrinB2is the upstream signals in the process of angiogenesis and XFZYD plays a complex role in regulation of angiogenesis.