ICAM-1 Mediated Adhesion Between Endothelial Cell And Endothelial Progenitor Cell Through Pulse Electrical Stimulation

Background and objectiveBioelectricity is the inherent propertiy of human body along with the completion of vital movement. Different types and concentration ion could exchange between cells and microenvironment due to the selective permeability of cell membranes. This is so called condition of membrane potential, also origin of bioelectricity. Under normal circumstance, tight junction between cells maintains an equal distribution of membrane potential. Once sufferring from wound, intact tight junction no longer exits, resulting in a weak or collapse local potential. Therefore, the potential gradient between normal region and wound will establish an endogenous electric field, which could induce cells to repair around the lesion. According to this, it is important to deepen our understanding and make good use of this electrical field.Based on the bioelectricity and the effect of endogenous electric field, electrical stimulation in vascular repair has been reported in the past decade and two main strategies will be the right direction. First, an external electrical stimulation is used to promote vascular regeneration directly, equivalent to adjust or strengthen the endogenous electrical effect. Second, taking vascular tissue engineering into consideration, integration of seed cells and the biomaterial through arrangement,adhesion and resistance is also being studied under electrical stimulation. Because vascular endothelial cell covers all of the blood vessel, intimately contacts with the blood flow and presents as the first step of angiogenesis, preliminary studies have pay more attention to the function of endothelial cell under electric field. However, recent researches show that it is difficult to meet the need of vascular reconstruction only depending on endothelial cell proliferation or sprouting in situ. Endothelial progenitor cell (EPC) has been proven to be irreplaceable in the adult neovascularization. It is indicated that research direction has been changed from traditional angiogenesis to vasculogenesis mediated by EPC. Therefore, to discuss how EPC participates in neovascularization through electrical stimulation is very meaningful.With a rapid development of EPC study, many researchers have been focus on EPC homing instead of EPC induction,proliferation and differentiation. There are several steps in EPC homing, including cell mobilization and direct migration due to cytokines secreted from ischemia, rolling and firm adhesion with endothelium, transendothelial transfer and finally direct differentiation or emiocytosis to complete the repair process. In these steps, adhesion between EPC and endothelial cell is the most important part in the homing process and three pair adhesion molecules are involved, making a close link between EPC chemotaxis and functioning. Intercellular adhesion molecule-1 (ICAM-1 or CD54) belongs to immunoglobulin superfamily and responses for maintenance of physiological function, pathogenesis and wound healing. Its uniform ligand is integrinαvβ2, which is also called CD 11 a/CD 18 compounds. Expression of ICAM-1 in endothelial cell do mediates EPC adhesion, but more importantly, under combination with the integrinαvβ2 in EPC, especially integrinβ2, the external stimulation signal will be passed from vascular lesion to EPC, and then switch on the EPC repair process. Consequently, our research is going to find out whether electrical stimulation could do well in the adhesion between endothelial cell and EPC? And ICAM-1 up regulation could be related to the mechanism of endothelial cell capturing more EPC under electrical stimulation?To investigate the change and mechanism of adhesion between endothelial cell and EPC under electrical stimulation, our research is divided into three parts.(1)We are going to make the suitable electrical stimulator by ourselves and establish a stimulate culture model. In order to find out a safe and effective stimulate interval, we first conduct the evaluation including stability of culture system, morphologic and proliferation of HUVEC.(2)We induce EPC from periphery blood and make phenotype identification. To assess the change of HUVEC-EPC adhesion, we label the EPC with fluorchrome and quantify by fluorescence intensity after co-culture with HUVEC monolayer under stimulation.(3)To explore the possible mechanism of HUVEC capturing EPC under stimulation, we detect the expression of ICAM-1 in HUVEC which plays an important role in HUVEC-EPC adhesion.Methods(1)Development and evaluation of electrical stimulatorElectrical stimulator with a pulse output was made by our cooperation with school of biomedical engineering, Southern Medical University. With a fixed and maximum pulse and frequency, we assessed medium resistance using voltammetry and detected temperature of culture system under different voltage stimulation within 2h.(2)Morphology and proliferation changes of HUVEC under different stimulationHUVEC was digested by collagenase from human umbilical cord and identified with the morphology andⅧfactor related antigen. Then they were divided into different stimulation groups and conventional culture group. After 24h stimulation, morphology of HUVEC was observed and the proliferation activity was analyzed by MTS.(3)egulation on HUVEC-EPC adhesion under different stimulationPeriphery blood mononuclear cells were isolated from periphery blood using density gradient centrifugation. They were seed in the special endothelial culture medium and would be induced to EPC after 9 day. Then EPC identification was determined by immunofluorescence, with a staining of Ulex europaeus lectin I and acetylated low-density lipoprotein. Phenotypic analyses of the EPC were performed by flow cytometry, including CD34, CD133, CD31, VEGFR2 and CD14.We collected EPC at day 9 and labeled them with fluorochrome CFSE. The labeled EPC were seed in the HUVEC monolayer and stimulated for 24h under different pulse. To measure adhesion between HUVEC and EPC, CFSE-EPC adhered to monolayer was discriminated in fluorescence microscopy and quantified by fluorescence micro plate reader. An index of fluorescence ratio was used to indicate the adhesion intensity.(4)Regulation on ICAM-1 expression of HUVEC under different stimulationTotal RNA was collected from different groups where HUVEC had been stimulated for 24h and then ICAM-1 mRNA was detected by SYBR Green staining, one of the methods about real time PCR. Finally, ICAM-1 mRNA relative expression was analyzed using 2-ΔΔCt.Statistical analysisData was showed as mean±standard deviation, surface markers between two group were compared using paired Sample T test, resistance at different time points between groups were compared using repeated measurement of variance analysis, temperature and fluorescence ratio between different groups were compared using one-way ANOVA, proliferation index and relative gene expression were compared using two-way ANOVA. Once there was significant difference between groups, two methods of multiple comparison were selectively used, Bonferroni for equal varicances assumed while Dunnett T3 for not assmed. P<0.05 was consider to be significant difference and all the data was analyzed by SPSS13.0 software.Results(1)Development and evaluation of electrical stimulatorOur self-made stimulator could provide an biphasic pulse, including adjustable voltage(0-40V),frequency(0.01～10Hz) and pulse width (0.1-24ms). The simulated model was set up with the combination of C-Dish electrode. With a fixed and maximum pulse width and frequency, there was a significant difference in medium resistance detected by voltammetry among different voltage groups(0V,5V,10V, 20V and 40V)(F=174.14, P=0.000). Moreover, there was a significant interaction between voltage and stimulation time, suggesting a different trend of different time under different voltage. According to the analysis, both the resistance and temperature in 10V group was relative stable, indicating a stable electrolysis reaction during our stimulation process indirectly.(2)Morphology and proliferation changes of HUVEC under different stimulationTo further select the suitable parameters, we first chose the passage 2 or 3 of HUVEC for stimulation within 10V. After various selection, typical morphological changes of HUVEC was present in 5V/5Hz/9ms, including flat and fusiform shape, deeply stained peri-nuclei zone stretched to the cell long axis. Then we fixed the frequency and voltage at 5V and 5Hz, utilized MTS to detect the proliferation of HUVEC under different pulse width. The OD values in 0ms,1ms,3ms,6ms,9ms group were 1.457,1.471,1.469,1.461 and 1.458 respectively, showing that no significant difference was among them (F=1.822, P=0.218). (3)Regulation on HUVEC-EPC adhesion under different stimulationEPC induced from periphery blood was present as spindle shape. They could uptake acLDL and exhibit UEA lectin binding capability simultaneously at day 9, also with an enhanced synthesis ofⅧfactor related antigen. The expression profile of EPC at day 9 were CD34(0.19%±0.06%), CD133(1.67%±0.52%), CD14(89.31%±4.11%), CD31(61.56%±5.57%) and VEGFR2(70.29%±7.37%). All these showed that adhesion cells presented as differentiating early EPC.EPC from day 9 was collected for CFSE labeling. Fluorometric method was used to detect the fluorescence intensity of adhesion CFSE-EPC in different pulse width. Results showed that fluorescence ratio were 0.441,0.470,0.489,0.557 and 0.370 in 0ms,1ms,3ms,6ms and 9ms group respectively. There was a significant difference among these groups and 6ms group was the best of all, indicating that suitable electrical stimulation could benefit for the HUVEC-EPC adhesion to some extent. In contrast, the ratio of 9ms group decreased significantly, even lower than the control group.(4)Regulation on ICAM-1 expression of HUVEC under different stimulationCompared with control group by real time PCR, ICAM-1 mRNA expression of HUVEC in different groups, including lms,3ms,6ms and 9ms were 1.23,2.42,3.79 and 1.90 respectively. Significant difference was present among them (F=17.706, P =0.000), indicating that up regulation expression was follow with an extension of pulse width. Taking 6ms group in particular, its expression was the significantly highest while there was a decreasing trend in 9ms group.Conclusion(1)It was successful to make an electrical stimulator with a biphasic pulse and set up a safe stimulated model. There is no change in proliferation of HUVEC but a typical change of morphology from 0ms to 9ms. HUVEC presented as more flat and spindle. (2)EPC was induced from PBMC successfully and it belonged to differentiating early EPC according to the phenotype.(3)Biphasic electrical stimulation was benefit for the HUVEC-EPC adhesion and 5V/5Hz/6ms would be the best.(4)Electrical stimulation could up regulate the mRNA expression of ICAM-1. Its change was consistent with the trend of HUVEC-EPC adhesion. It is suggest that electrical stimulation contributing to HUVEC-EPC adhesion should be related to the up regulation of ICAM-1 expression.Point of innovation(1)Regulation on the endothelial cell using direct current have been reported in previous studies and there were few literatures about pulse stimulation. Our research is successful to regulate HUVEC under biphasic pulse stimulation. It will provide a novel method to the future studies about vascular regeneration through electrical stimulation.(2)We are the first to make integration between EPC and endothelial cell under 5V/5Hz/6ms electrical stimulation. The stimulated strengthen HUVEC-EPC adhesion is probably a new theory about electrical stimulation contributing to neovascularization. So far, there is no report about this concept.(3)Our research has suggested that up regulation of ICAM-1 may be responsible for the mechanism of endothelial cell capturing more EPC under electrical stimulation. These will help us to reaffirm the importance of ICAM-1 for EPC homing and provide a good reference for EPC homing under other stimulation methods.