| Background:With the changes of habits and diets of modern people as well as the impact of the variational external environment, as a fatal disease the incidence rate of coronary artery disease (CAD) showed an increasing trend. Surgical or interventional treatment is highly effective, however, they could not satisfy the patients who eventually lost treatment opportunities in the long run. Therefore, it is an urgent requirement for a new and effective treatment for these patients. It has been confirmed that revascularization can improve the ischemia caused by cardiovascular or peripheral atherosclerosis based on the molecular biology research. The role of gene therapeutic angiogenesis can interfere with the process of ischemic disease, reconstruct the functional microcirculation network, and facilitate prevention and treatment.In the recent years, mature micro-vessel has become an important target of therapeutic neovascularization. There are kinds of angiogenic factor involved in angiogenesis. Vascular endothelial growth factor (VEGF) is widely perceived as a pro-angiogenic growth factor, which could promotes the survival, proliferation, and migration of endothelial cells (ECs), leads the extension of neo-vessels into the avascular area. In one hand, Angiopoietins family mediate activation of endothelium (Ang2), in another hand, they promote the stability of the endothelium (Angl). Under the stimulation of Angl, Platelet-derived growth factor (PDGF) mediated the recruitment of mural Cells (MCs). Apelin (APLN) could not only improve the proliferation and fusion of endothelial cells forming the new lumen, but also strengthen the connection between endothelial cells as well as endothelial cells to MCs.It is a very complex process related to more than one gene in angiogenesis. Due to limitations, single factor of treatment at times affect the structure and function of the new blood vessels. For instance, VEGF-A promotes the growth of neo-vessel but also leads the hyper-permebeality; Ang2induces angiogenesis while also causes the detachment of MCs.Aims: In this study, we adjust the balance between vascular destabilization and stabilization in the process of angiogenesis by selective combination of the four different genes VEGF-A, Ang2, Angl and APLN recombined with AAV. We explore the impact of multi-gene therapy for angiogenesis on microcirculation in ischemia disease, so as to develop the best strategy to intervene and treat ischemia disease.Methods:1. The effect of target genes on the tube formation of ECs and its surrounding PCs in vitro1) We selected human micro-vascular endothelial cells (HMECs), mouse brain endothelial cells3(bEnd.3) and murine pericytes (C3H/10T1/2), detected the Tie2, PDGFR receptor on cell surface by Fluorescence-activated cell sorting (FACS).2) We respectively or conjunctively transferred the plasmid of pCMV-VEGF-A, pCMV-Ang2, the pCMV-Angl and pCMV-APLN into HMECs and bEnd.3by application of transient transfection technique. The endothelial cells were cultured for48h.3) We applied the quantitative real-time polymerase chain reaction (qRT-PCR) to explicit the built over-expression of target gene.4) We assessed the tube formation of ECs promoted by over-expression of VEGF-A, Ang2, Angl and APLN with2D Matrigel model.5) We used co-culture of PCs with the tubes of ECs, and assess the recruitment of PCs induced by VEGF-A, Ang2, Angl and APLN.2. The effect of multi-gene combinations on promoting the mature angiogenesis in the microcirculation of mouse hindlimb ischemia1) We reconstructed, synthesized, produced and purified the virus of rAAV-CMV-hVEGF-A rAAV-CMV-mAng2, rAAV-CMV-mAPLN, rAAV-CMV-hAngl, rAAV-CMV-tetoff-mAng2and rAAV-CMV-LacZ with the adeno-associated virus vector (AAV) and the Tetoff System.2) We selected the C57BL/6J male mice to intramuscularly inject virus in right limb, and0.9%saline (NaCl) in left limb and fed them for14days. Virus concentration for each mouse as below:the low dose of VEGF-A is5×1011virus particles (vps), high dose of VEGF-A is3×1012vps, the remaining target gene virus is respectively3×1012vps.3) After14days, three mice of each group were executed by taking the muscle tissues of right limb for qRT-PCR detection and the muscle tissues of LacZ over-expressing mice were for β-galactosidase histological staining to clear the target gene expression level. The rest of mice were used for hindlimb ischemia model through the femoral artery ligation, which showed ligation on the right side, and pseudo-operated control on the left side. Raising them after the surgery.4) We applied laser Doppler imaging techniques (LDI) to detect the recovery of blood perfusion prior to ligation and after ligation d0, d7, d14.5) In rAAV-tetoff-mAng2group, three mice were taken the muscle tissue for qRT-PCR to detect the expression level of Ang2on d3after ligation, and the others were fed with food containing doxycycline (Dox). On d7, we took the muscle tissues of three mice again to do qRT-PCR for Ang2level. The rest were kept for the following experiments.6) The mice were sacrificed to take the hindlimb gastrocnemius muscle (GC) on d7and d14. The muscles were to do the frozen section. Respectively using the antibody of ECs marker PECAM-1and PCs marker NG2, we stained the tissue slice by immunofluorescence staining (IFS). The pictures were taken under a fluorescence microscope. Then we observed and counted the postitive cells.Results:In Vitro experiment1. Ang2itself does not induce tube formation, and has no effect on the significantly increased ECs tubes induced by VEGF-A and APLN, but competitively inhibit Ang1;2. Both of Ang1and APLN significantly increase the numbers of recruited PCs; by contrast, VEGF-A and Ang2could not have this effect;3. Ang2inhibited the recruitment of PCs by Ang1and APLN in either homologous co-culture or heterologous co-culture;In Vivo experiment1. Even though VEGF-A significantly increased the capillary density, it failed to promote the perfusion of mouse ischemic hind limb; Combining with Ang2, it increased the angiogenic effect of VEGF-A, but downregulated the recruitment of PCs, and cannot improved apparent recovery of reperfusion in ischemia, even worse at an early stage (d7); 2. APLN significantly restored the perfusion of ischemic hindlimb, increased capillary density and recruited PCs; sustained expression of APLN/Ang2increased capillary density, but reduced the recruitment of PCs. In the early stage (d7), it showed the apparent lower blood flow perfusion than APLN. Even though it was more significantly increased compared to control group in the late stage (d14), there was no statistical significance between APLN and APLN/Ang2.3. In the early stage, combination of APLN and transient expressed Ang2significantly up-regulated the recruitment of PCs compared to control group and APLN combined with persistent expressed Ang2. Furthermore, they apparently strengthened the density of capillaries induced by control and APLN, but there was a increased trend of the recovery of blood perfusion. In the late stage, combination of APLN and transient expressed Ang2still improved the capillaries, but had no apparent rise in the recruitment of PCs and blood perfusion.4. The co-application of the short Ang2-overexpression (d0-3) and persistent expression of Angl significantly increased the blood perfusion, up-regulated capillary density and the recruitment of PCs compared to control;5. Compared to control group, the combination of Angl and APLN apparently increased the surrounding PCs, but inhibit the density of capillaries so as to have no effect on the improvement of ischemic perfusion.Conclusions:1. In mouse ischemic hindlimb model, VEGF-A and Ang2acted as the growth factor of angiogenesis inducing destabilization. Co-application of both cannot promote the recovery of blood perfusion due to the detachment of the PCs.2. The combination of APLN and Angl as vascular maturation factor prematurely stabled vascular endothelium inhibited the sprouting and growth of new blood vessels, led the obstruction of blood perfusion.3. Co-application of the early Ang2-overexpression (d0-3) and late overexpression of Angl ensured that the reestablishment of mature micro-vascular in the ischemic region, and achieved the homeostasis of endothelial destabilization and stabilization. Innovations:1. We confirm the negative regulatory effect of VEGF-A and Ang2as destabilization factor of angiogenesis.2. This is the first time to combine APLN with Angl that proves that the over-maturation inhibited the process of angiogenesis by combining application of APLN and of Angl.3. This is the first time to use the combination of Angl and Ang2, and reveals a great strategy for therapy of mature angiogenesis in micro-vascular, which is stage-dependent overexpression of destabilization factor and maturation factor. It guides the direction for future gene therapy of ischemic heart disease. |
