Two-ways Regulation Of QDTMT On VEGF And Its Protective Effects On HUVECs

一,Research background and aimThe role of vascular endothelial growth factor (VEGF, also referred to as VEGFA) in the regulation of angiogenesis is the object of intense investigation now. As the most critical and potent proangiogenic regulator, VEGF is a protein with antiapoptotic, mitogenic, and permeability -increasing activities specific for vascular endothelium. For one hand, VEGF is a survival factor for ECs, both in vitro and in vivo. But for the other hand, VEGF has been implicated in pathological angiogenesis associated with tumors, stroke,diabetes, coronary artery disease. The expression and secretion of VEGF is in ischemia and oxygen deficiency. There is much evidence that diverse biological effects of VEGF on ECs have much to do with its concentration. It has been an agreement that VEGF stimulates vascular endothelial cells mainly through VEGF receptor 2 ( VEGFR2,Flk-1/KDR ). VEGF-stimulated recycling of KDR regulates the sensitivity of endothelial cells to proangiogenic signals.It is well known that traditional Chinese medicine (TCM) is potential to the regulation of homeostasis. Recent studies have demonstrated that TCM of reinforcing Qi and promoting blood circulation exert two-ways regulation of VEGF expression and secretion. Some researchers have showed that TCM can upregulate the level of VEGF as an attractive approach in therapeutic angiogenesis. Conversely, a variety of studies suggest that TCM play an important role in antiangiogenic therapies.Previously, we have described Qidantongmai tablet (QDTMT) protect vascular endothelial cell (VEC), increase the VEGF expression in ventricles of myocardial ischemia rats and improve the microvessel density(MVD) in the ischemia zones. However, in hypoxic condition, whether QDTMT has regulation effects on VEGF and KDR is not known. What'more, a long time investigation is required of the complex pathophysiological process in animal experiments.In this study, the hypobaria hypoxia chamber is used for the rat hypoxic models. After QDTMT treatment, the concentrations of VEGF in rat serum is detected. The pathological changes of rat tissues and the expression of VEGF in blood vessel are observed in this study. In vitro, we have cultured and subcultured human umbilical endothelial cells(HUVECs), and have observed effects of serum containing QDTMT on endothelial proliferation and apoptosis in hypoxia. In addition, the expression of KDR is also investigated in HUVECs. We use this research to improve understanding of the balance between VEGF level and its biological activies,and of the relationship between the arterioprotection of endothelium and YiqiHuoxue principle in TCM.二,Regulation effect of QDTMT on VEGF expression in rats with hypoxic model1 Methods1.1 The establishment of animal modelMale Sprague-Dawley rats (n=18) were exposed to low pressure and low oxygen conditions in hypobaric and hypoxic chamber ,6 h /d, for the estab lishment of animal model. After observation of the general state of rats, the lung tissues of rats were collected with Hemotoxylin and Eosin dye, and investigated the proliferation of vascular.1.2 Effect of QDTMT on rats with hypoxic model Male Sprague-Dawley rats (n=30) were randomly divided into 3 groups: normal control, hypoxia+ QDTMT (QDTMT), hypoxia + normal saline(hypoxic control). With the animal model of hypobaric hypoxia (HBH) for 7d and 14d,we investigated the pathological changes. Then , as following:1) After hypoxia, the serum level of VEGF in rats were detected by ELISA kit.2) After hypoxia , lung tissue,heart tissue and liver tissue were fixed with 4% paraffin for about 24 h, and embedded, treated with Hemotoxylin and Eosin dye ,and observed with light microscope. The ultrastructure of liver tissue was observed with electron microscope too.3) VEGF expression Open the chest under the sterile condition and the thoracic aorta tissues were fixed with 4% paraform for 8 h .The VEGF positive cells were detected by immunofluorescence in aorta.2 Results2.1 ELISA After 7d of hypoxia, we found that the concentration of VEGF in serum of QDTMT group was higher than that of normal control and hypoxic control (P < 0.05); after 14d of hypoxia, the VEGF level of hypoxic control was higher than that of QDTMT group (P< 0.05).2.2 Pathological changes results Afer 7d of hypoxia, pathological changes of rats showed that blood vessel structural changes were slight in all the groups; afer 14d of hypoxia, pathological changes of vascullar structural were clear in hypoxic control under microscope. Pathological changes were decreased in QDTMT group. In addition , we found the endosome structure in QDTMT group with electron microscope.2.3 Immunofluorescence results After 14d of hypoxia, a lot of VEGF positive cells were seen in aorta of hypoxic control group.3 Conclusions3.1 The level of VEGF in rat serum changed in different pathological stages in this HBH hypoxia model.3.2 Two-ways regulation effect on VEGF level may be one of the mechanism for QDTMT to protect the endothelial tissue in hypoxia condition.三,Study on protective effects of QDTMT to anoxic HUVECs1 Methods1.1 Cell cultivation and identification Human umbilical vein endothelial cells(HUVECs) were got from infant umbilical cord for primary cultivation and subcultivation as previously described. HUVECs were identified by morphologic character and membrane antigenⅧfactor. HUVECs were subcultured for use at passage 3-5.1.2 Effects of serum containing QDTMT on HUVECs activities in hypoxia.1.2.1 preparation for serum containing QDTMT According to different treatment factors , male Sprague-Dawley rats (n=18) were randomly divided into three groups. After hypoxic treatment, serum containing QDTMT was collected and stored in -70℃(refer to study in vivo) .It was showed that the best dilution of serum containing QDTMT was 10%. After diluted in M199 media, the serum concentration of all groups was 10%.1.2.2 MTT for HUVECs cytoactiveHUVECs between passage 3 and 5 were cultured with M199 media for 24h, and were free of M199 media for 6h. Then cells were divided into three groups: (1)A, treated with 10% serum from normoxia control ; (2)B, treated with 10% serum from hypoxic control; (3) C, treated with 10% serum from QDTMT group. Except for normoxia control,cells in the other two groups were exposed in hypoxia condition for 24 h. The proliferation of HUVECs was observed in MTT.1.2.3 The apoptosis ratewas tested with FCM in all groups.1.3 Using immunofluorescence microscope, we examined the expression of VEGFR2 in HUVECs of all groups.1.4 After fixed with glutaraldehyde the ultrastructure of HUVECs was observed with electron microscope. 2 Results2.1 The morphology of cultured cells was obviously changed in the shape of cobble-stone under microscope. We also found the characteristic Weible-Palade bodies in cultured HUVECs. In addition , cells were seen stained with factorⅧby immunofluorescence microscope.2.2 The effect of serum containing QDTMT on hypoxic HUVECs.1) The MTT showed that the cell cytoactive of B ans C was lower than that of A. QDTMT promoted the proliferation of HUVECs in hypoxic condition, and the proliferation level was higher in C group treated with serum containing QDTMT than that of B group (P < 0.05).2) The FCM assay showed that the apoptosis rate was lower in C group than that of B group (P < 0.05).2.3 Result of immunofluorescence The VEGFR2 expression level of HUVECs was higher in C group than that of B.2.4 With electron microscope, we fund the endosome in C group. Endosome is an important intracellular pool for VEGFR2/KDR mobilization within endothelial cells.3 ConclusionsThe results demonstrated that serum containing QDTMT improved the activities of antiapoptosis and proliferation of cultured HUVECs .To enhance the expression of VEGFR2 may be one of the endothelial protective effects of QDTMT.