| Background and ObjectiveCardiovascular diseases caused by atherosclerosis have serious impact on human health and quality of life. Although the vascular intervention technology has largely enhanced the blood-flow perfusion in the ischemic tissue and improved the prognosis of the patients in recent years, the excessive repair response of the local vascular wall to the damages has always been the problem of bothering interventional therapy. The aging has aggravated the excessive repair for the blood vessel; nowadays, human society is gradually entering into the era of aging, it is more valuable to strengthen the research on excessive repair of injured vessel.With the increase of the age, the somatic cell is gradually being dysfunctional. For a long time, researches about aging promoting the atherosclerosis and neointimal hyperplasia mainly focus on the aging of cells in the blood vessel wall, but there are also some phenomena which cannot simply be explained by theories of cell aging, such as the abnormal smooth muscle cell proliferation is the main pathology variance of the atherosclerosis and injured vesse repair; it is generally considered that the aging will promote the proliferation of the smooth muscle cell, but there are also some materials shown the proliferation capacity of the smooth muscle cell is decreased in the aging individuals; and in addition, the studies on the measurement of cell aging index have shown that the?-galactosidase staining separated by the aging individuals is positive, however, some studies also found it is negative. These contradictory phenomena point out that aging promoting the generation of the atherosclerosis and the neointimal hyperplasia may not completely be caused by cell aging.It is generally considered that the aging individual has stronger response to the inflammatory and growth factors, which will promote the proliferation of smooth muscle cell. But in fact, the treatments simply for the inflammatory factor and growth factor cannot completely and effectively control the excessive proliferation of the injured vessel. Organs and tissues are composed of virous cells. The cell-cell adjustment plays a critical role in the cell function. At present, it has found that endothelial cell can affect the biological characteristics of the smooth muscle cell; however, many of the related studies are restricted to the ordinary adult, therefore, it is not clear whether the abnormal proliferation of the smooth muscle cell is related with the abnormal adjustment of the endothelial cell or not.It is reported that the age-related abnormal tissue repair capacity may be related with Notch signal pathway. Notch signal molecules distributed in the blood vascular system includes receptors (Notch1,3,4), ligands (Delta-4,Jagged1,2) and effectors(HERP1,2,3), which are closely related with the formation, development and reconfiguration of the blood vessel. Notch signal is evolutionarily conserved and it presents in many tissues for controlling the cell proliferation, differentiation and adherence and other cell fate, and the adjustment of cell proliferation in the blood vessel wall is realized through Jagged1/Notch1 pathways. Liu et al have found that Notch1 signal is related with MAPK and PI3K/Akt signal systems; moreover, studies established on the other cell model shows that the Notch also plays an important role in the cross talk of proliferation related signal of the NFkappaB, Wnt/TCF and so on. But, recent studies on the cell proliferation controlled by Notch signal are simply aims to endothelial cells or smooth muscle cells, and the effect of mutual adjustment among cells in the blood vessel wall is not clear. Notch signal can adjust the proliferation of adjacent cell through the lateral inhibition mechanism. Therefore, aging related abnormal smooth muscle cell proliferation may be related with the cross talk of related signals in the smooth muscle proliferation. Besides, apoptosis also plays an important role in cell growth; and Notch1 signal not only participates in the control of the cell proliferation but also affects the apoptosis of cells. Robert et al have cultured red blood cells together with Jagged1 over-expression cells and found that the Jagged1 over-expression cells can induce the apoptosis of red blood cells, which suggests that Jagged1/Notch1 can affect the apoptosis of cells through the cell to cell adjustment. The growing status of the cell is result of the balance of proliferation and apoptosis; we conjectured that the abnormal smooth muscle proliferation of the aging individual may be caused by unbalanced adjustment of smooth muscle cell proliferation and apoptosis through Jagged1/Notch1 signal in the endothelial cell.In summary, we intend to discuss whether the endothelial cell Jagged1 will participate in aging related abnormal smooth muscle cell proliferation through the adjustment of related signals in smooth muscle proliferation, and hope to reveal the pathogenesis of the aging-excessive neointimal formation and atherosclerosisl.Methods1.Construction of interference and over-expression vectorTo construct an adenovirus vector carrying rat Jagged1 siRNA. To design and combine three shRNA sequences targeting the rat Jagged1 gene, using the pGensil-1.1 to restructure three expression plasmids with different shRNA. SacI restriction endonuclease was used to confirm whether the connection was right, and the sequencing was used to detecte mutation. The Realtime PCR detected and screens the sequence of the highest inhibition efficiency, connecting the sequence to the shuttle plasmid PGSadeno, packaging recombination adenovirus through the LR homologous recombination in vitro. XbaI restriction endonuclease was used to identify the recombination of clone. The transfection of 293 cells is used to augmente culture. Finally, we detected the titer of virus by the plaque assay. For construction of the recombining expression plasmid, we used Xba I restriction endonuclease to cut pBOS–SN3T, and obtained the whole rat Jagged1 gene, which would directionally be connected to the linear eukaryotic expression plasmid pCMV-IRES-GFP after the glue recovery, renaming the obtained recombinant plasmid as p-rJagged1. After transforming and augmenting, restriction endonuclease NheI cutting for identifying and recovering gel, and then further transforming and augmenting.2. The section of in vivo experiments.Angioplasty of the rat carotid artery was performed by using a balloon embolectomy catheter. Animals were subjected to anesthesia with 2% pentobarbital sodium, then, exposing the left common carotid artery, internal carotid, external artery, 1.5F catheter was inserted to the initial part of the carotid artery from the external carotid artery. To expand the saccule for 30 seconds with two atmospheric pressures, then slowly pull the catheter back to the bifurcation of the external carotid artery three times. Exiting the catheter, closing the artery about 10min after injecting the virus suspension. Ligaturing the external carotid artery, conventional breeding after suturing layer by layer. Fetch the carotid artery at seven, fourteen and twenty-one days after the operation, HE staining, and image analysis software calculated the ratio between the neointima and media area. The immunofluorescence and western blot were used to detect whether the transfection in the blood vessel tissue was successful or not. The expression of the PCNA protein in the blood vessel tissue was detected by the western blot. The expression of the Jagged1 mRNA was detected by the Realtime PCR. And the rate of apoptosis was detected by the tissue Tunel staining.3. The section of in vitro experiments.The rat aortic endothelial cells (RAECs) were cultured by digestive method and identified by the vWF immunofluorescence staining. The vascular smooth muscle cells(VSMCs) were cultured by tissue explants method and identified by the anti-?-SM-actin immunofluorescence staining. To build RAECs and VSMCs co-cultured system, the Transwell insert was used. The GFP immunofluorescence was used to observe and detect the transfection efficiency, MTT method and 3H-TdR assay were used to detect proliferation capacity of the VSMCs. The apoptosis rate of the VSMCs was detected by the Hoechst33342 staining and Annexin V-APC/7-AAD. The protein expression of the Jagged1, NICD, Hey1, Bcl-2 and?-SM-actin was detected by the western blot. The expression of the Jagged1, Notch1 and Hey1 mRNA was detected by the Realtime PCR.Result1. Establishment and identification of the rat Jagged1 gene interfering adenovirusThree shRNA plasmids targeting Jagged1 gene were connected with the pGenesil-1.1 liner vector. After cutting with SacI restriction endonuclease, there was a DNA segment about 900bp, which indicated the successful construction of shuttle plasmid. Meanwhile the sequence determination proven that the target sequence was right inserted and no mutations. Respectively transfected the shuttle plasmids with different shRNA into endothelial cells (ECs) and the Real-time PCR screened out the sequence with highest inhibition efficiency, which was sequence GGAGAGTTACAGAATGGGA. The recombination adenovirus carrying this sequence was renamed Ad-si/rJagged1. Cutting Ad-si/rJagged1with restriction endonuclease XbaI obtained a small DNA segment with about 3K and a long segment more than 15kb, which suggest that the clone was correct. Ad-si/rJagged1 was transfected to 293 cells and augmented repeatedly, then collected the crude virus lysis solution. The titer of the virus was about 6×109 pfu/ml measured by the plaque test.2.The effect of Jagged1 in the aging-related neointimal formation after vessel injury.There were GFP proteins in the blood vessel wall after adenoviral vector transfection detected by Western blot analysis at 2?7?21 days after injury?The level of GFP expression was high at day 2 and day 7,and remain presention until day 21. There was no GFP expression in the lateral vessel without transfection.Respectively use the aging rats and young rats to establish the model of balloon injured carotid artery. The ratio of neointima and media areas in aging group was obviously increased compared with that in the young group. The level of Jagged1 mRNA in two groups was both increased after injury; however, compared with the young rats, the increase in Jagged1 expression of old rats after the injury was delayed and weakened. In contrast, the increase in the expression of proliferating cell nuclear antigen (PCNA) was increased in old rats than that in the young ones. Meanwhile, the index of apoptosis in neointima was decreased in old rats than that in the young ones.To transfect the balloon injured carotid artery with the interfering adenovirus Ad-si/rJagged1 and negative comparing adenovirus Ad-NSC. The increase of Jagged1 mRNA in Ad-si/rJagged1 group after injury was obviously reduced and delayed, compared with control group. Meanwhile, the increase in the expression of PCNA was increased in Ad-si/rJagged1 group, compared with control group. What`s more, the index of apoptosis in neointima was decreased in Ad-si/rJagged1 group than that in the control group. Finally, the value of I/M in Ad-si/rJagged1 group was more than that in control group after injury at day 21. However, there were no statistical difference of above indexes between the Ad-NSC group and control group.3. The effect and mechanism of the ECs Jagged1 in the aging-related proliferation and apoptosis of VSMCs.3.1 The aging reduced the Jagged1expression in the ECsDividing the ECs into young group and aging group according to the different age of the rats; in normal culture conditions, the levels of Jagged1 mRNA and protein in the aging group were both lower than that in the young group. Detecting the expression of notch1 receptor and its downstream gene Hey1, the result showed that the levels of Notch1 and Hey1 mRNA in the aging group were both lower than that in the young group. The intracellular domain NICD protein of the activated notch1, and the Hey1 protein in the aging group were both lower than that in the young group. It suggest that the aging can reduce the expression of Jagged1 in the EC and inhibit the activation of the Jagged1/Notch1 signal pathway partly at least.3.2 The aging ECs promote the proliferation and protect the apoptosis of the VSMCsTo co-culture the VSMCs with ECs obtained from rats with different age, which was divided into aging ECs co-culture group and young ECs co-culture group. In normal co-culture conditions, there was no statistical difference of the VSMCs proliferation between the aging ECs co-culture group and the young ECs co-culture group; however, when adding 10?g/mL platelet-derived growth factor (PDGF) into the co-culture system, the proliferation of VSMCs was enhanced in the aging ECs co-culture group obviously, compared to the young ECs co-culture group.Without H2O2 induction, the apoptotic rate of the VSMCs in the two group was similar, however, when using the H2O2 induction(50?mol/L), the apoptotic rate of the VSMCs in aging ECs co-culture group is obviously lower than that in the young ECs co-culture group(8.42±1.02% vs. 17.61±2.16% , P<0.01). The result showed that the aging ECs can promote the proliferation and protect the H2O2-induced apoptosis of the VSMCs.3.3 ECs Jagged1 inhibit the proliferation and promotes the apoptosis of the VSMCsThe young rat ECs decreased Jagged1 expression by 72% after Ad-si/rJagged1 transfection, and the aging rat ECs increased Jagged1 expression by 5 folds after over-expression plasmid p-rJagged1 transfection.The VSMCs were co-cultured with the young ECs transfected with Ad-si/rJagged1 and Ad-NSC respectively. The proliferation of VSMCs in the Ad-si/rJagged1 group was increased, compared with the control group (32434±8322cpm/well vs. 16643±1122cpm/well, P<0.01), however, the proliferation of VSMCs in the Ad-NSC group and control group was similar. In contrast, the rate of VSMCs apoptosis in the Ad-si/rJagged1 group was reduced compared with the control group (5.24±0.35% vs. 16.85±1.66%, P<0.01), however, there was no statistical difference of VSMCs apoptotic rate in the Ad-NSC group and the control group. It indicated that down-regulate the expression of the ECs Jagged1 can promote the proliferation and protect the apoptosis of the VSMCs.The VSMCs were co-cultured with the aging ECs transfected with p-rJagged1 and vector. The proliferation of the VSMCs in the p-rJagged1 group was reduced compared with the control group(11654±845cpm/well vs. 26350±1478cpm/well, P<0.01),however, the proliferation of VSMCs in the vector group and control group was similar. In contrast, the rate of VSMCs apoptosis in the p-rJagged1 group was reduced compared with the control group(42.55±3.81% vs. 8.92±0.86%, P<0.01), however, there was no statistical difference of VSMCs apoptotic rate in the vector group and the control group. It indicated that up-regulation the expression of the ECs Jagged1 can suppress the proliferation and protect the apoptosis of the VSMCs.3.4 The effect of the ECs Jagged1 on the expression of a-SM-actin protein in co-cultured VSMCsAfter co-culturing VSMCs with different age rats ECs for 48h(with PDGF, 10?g/mL), the a-SM-actin protein in the aging ECs co-cultured group was lower than that in the young ECs co-cultured group. The VSMCs were co-cultured with the aging ECs transfected with p-rJagged1 and vector for 48h(with PDGF, 10?g/mL), the levels of a-SM-actin protein in the p-rJagged1 group was higher than that in the vector and control group (0.87±0.05 vs 0.57±0.03 vs. 0.56±0.04,P<0.01) . Then, the VSMCs were co-cultured with the young ECs transfected with Ad-si/rJagged1 and Ad-NSC. for 48h (with PDGF, 10?g/mL), the level of a-SM-actin protein in the Ad-si/rJagged1 group was obvious lower than that in the Ad-NSC and control group (0.24±0.02 vs 0.61±0.03 vs. 0.62±0.03,P<0.01).It suggest that up-regulation the expression of the Jagged1 in the ECs can increase the a-SM-actin expression in the co-cultured VSMCs. Otherwise, it would reduce a-SM-actin protein expression.3.5 The effect of the Bcl-2 protein in the VSMCs apoptosis adjusted by the ECs Jagged1After co-culturing VSMCs with different age rats ECs for 24h, then add the H2O2 (50?mol/L, 24h). Analyses of the anti-apoptosis protein Bcl-2 expression in the VSMCs. The protein level of VSMCs in the aging ECs co-cultured group was lower than that in the young ECs co-cultured group. Then, the VSMCs were co-cultured with the aging ECs transfected with p-rJagged1 and vector, the Bcl-2 protein level of VSMCs in the p-rJagged1 group was obviously reduced, which indicated that the ECs Jagged1 can reduce the anti-apoptosis protein Bcl-2 expression in the VSMCs. The over-expression plasmid p-rBcl-2 transfection obviously raised the Bcl-2 protein level in the VSMCs, moreover, when these VSMCs were co-cultured with the ECs over-expressed Jagged1, the rate of apoptosis in VSMCs transfected with p-rBcl-2 was reduced, compared with that transfeceted with vector or without transfection. It suggest that EC Jagged1 may play the apoptotic effect through decrease the Bcl-2 protein expression of the co-cultured VSMC partly at least.Conclusion1. Jagged1 is increased after vessel injury. However, compared with the young rats, the increase in Jagged1 expression of old rats after the injury was delayed and weakened. The increase in the expression of PCNA was increased and the apoptosis index in neointima was decreased in old rats than that in the young ones.2. Down-regulation of Jagged1 expression in artery promotes the neointimal hyperplasia.3. The aging reduces the EC Jagged1expression and inhibits the Jagged1/Notch1 signal pathway partly at least.4. Aging EC can promote the proliferation and reduce the apoptosis of the co-cultured VSMC.5. The Jagged1 in EC can inhibit the proliferation and promote the apoptosis of the cocultured VSMC.6. EC Jagged1 increases the level of a-SM-actin protein in co-cultured VSMC, and may play the apoptotic effect through decrease the Bcl-2 protein expression of the co-cultured VSMC partly at least. |
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