| BackgroundAt present, acute cardiovascular and cerebrovascular events (acute coronary syndrome and stroke) is a serious threat to the health and lives of the elderly, which has high morbidity and mortality. Cardiovascular disease is the main cause of death in developed countries, and is fast becoming the number one killer in developing countries. With the development of society, people’s lifestyles change and the arrival of the aging society, more than two thousand people died of atherosclerotic cardiovascular disease every year, mainly for coronary heart disease, and that number continues to increment in. Atherosclerosis (atherosclerosis, AS) is a common pathological basis of cardiovascular and cerebrovascular disease, and the formation of vulnerable plaque and the ensuing thrombosis is the real culprit and the most fundamental initiating factor of the fatal vascular disease, therefore the prevention and treatment of AS disease should be focused on stabilizing and repairing vulnerable plaque. Vulnerable plaque is not only the plaque easy to rupture, all ones are likely to have thrombotic complications and rapid progress should be considered vulnerable plaque. And vulnerable plaque is not the only crime factor for acute coronary syndrome (ACS), myocardial infarction (MI) and sudden cardiac death, the vulnerable blood (easy occurrence of thrombosis) and vulnerable myocardium (prone to fatal arrhythmia) also play an important role on prognosis of these diseases. In recent years, the international community prefers to use the term "vulnerable patients" instead of "vulnerable plaque" in order to better assess the risk of acute cardiovascular and cerebrovascular events in patients. Atherosclerosis is scattered, systemic arteries involved chronic disease, which have various clinical manifestation, usually presents clinical coronary atherosclerosis (ACS, MI), cerebral arteriosclerosis (cerebral infarction, rupture of cerebral blood vessels), carotid atherosclerosis (cerebral insufficiency, cerebral infarction), aortic lesions (aortic dissection, aortic aneurysm), renal artery atherosclerosis (kidney disease, hypertension) et al. Therefore, the assessment of the vulnerability of the plaque load should not be limited to a single unstable plaque, but should include the total atherosclerotic plaque load (aortic and carotid and femoral artery) and coronary vulnerable plaques, also should include blood and myocardial vulnerability risk assessment (vulnerability index).Although there have been a variety of treatment applied successfully to control or reduce the occurrence and development of coronary heart disease, mainly including medical treatment measures such as dissolving blood clots, lipid-lowering, suppression the inflammatory, intervention treatment and surgical coronary artery bypass surgery. The treatment plays a certain role but is extremely limited. Surgical intervention will continue to be one of the main technical therapies for ACS patients with more than70%coronary stenosis. However, a large number of studies showed that the formation of about70%-80%ACS is caused by rupture and secondary thrombosis of mild to moderate stenosis plaque, which is not suitable to adopt PCI, its treatment should focus on stabilize and repair vulnerable plaques. Because of the systemic and multi-distribution characteristics of atherosclerosis and vulnerable plaque, and the vulnerability characteristics of the blood, the treatment for a local vulnerable plaque is too partial and one-sided, and can not avoid acute cardio-cerebral vascular events occurred in a greatest degree. Current treatment/prevention trends for acute cardiovascular and cerebrovascular events tend to consolidated total atherosclerotic load and coronary vulnerable plaques and vulnerable patients, including a comprehensive assessment of the fragile blood and vulnerable myocardial of the patients, to prevent the occurrence of acute cardiovascular and cerebrovascular disease. Thus, the drug treatment should be the basis of stabilization of vulnerable plaque. However, there is still no effective drugs to stabilize vulnerable plaque. In animal experiments, statins can significantly reduce the level of LDL-c, inhibition of the inflammatory response, thereby increasing the thickness of the fibrous cap. However, liver toxicity of statins makes so many patients can not use such drugs.Cells (or organization) replacement therapy and gene therapy technology developed based on tissue engineering and gene engineering in recent years, and become the research focus and cutting-edge in the field of medicine and even in the entire field of life sciences, and provide a new way and hope for conquering many diseases. Bone marrow mesenchymal stem cells(MSCs), is a kind of the early cells from mesoderm, and widely exist in a variety of adult tissues. MSCs have a wide range of advantages relative to other stem cells, such as low immunogenicity, multi-differentiation characteristic, easy to cultivate and amplification in vitro and unique homing feature. Not only can MSCs differentiate into the hematopoietic and stromal cells, but also can differentiate into a variety of other organization, especially the organizations cells from mesoderm and neuroectoderm. In recent years, MSCs has a broad application prospects in clinical practice. There are a large number of experimental to check the transplantation of MSCs for the treatment of a variety of diseases, and have achieved a certain amount of research. Currently, MSCs showed a better therapeutic effect in a variety of clinical trials of disease, such as myocardial infarction, corneal damage, nervous system damage, diabetes, acute graft-versus-host reaction, some lung dieases, liver fibrosis, bone and joint injuries, and Crohn’s disease and other diseases. The study found that the MSCs have special biological characteristics, including:①MSCs as a "seed" cells can be planted in the damaged tissues and differentiate into tissue cells and play a role in tissue repair.②immunomodulatory effects (through the bypass/autocrine manner), play to the therapeutic effect by reducing the inflammatory response in the diseased tissue by inhibition of T lymphocytes and cytotoxic T lymphocytes and natural killer cells.③hematopoietic support function.④secretory ability of a variety of biologically active substances. Many of the clinical and laboratory studies have confirmed that MSCs cultured in vitro has some biological functions including immune regulation, hematopoietic support and the ability to repair a variety of tissue damage, which mostly dependent on the bioactive substances secreted by MSCs.However, domestic and foreign studies on MSCs in recent years did not involve the stabilizing effect of MSCs transplantation on AS vulnerable plaque. Taking into account vulnerable plaque is a multi-site multi-system disease, and current treatment methods can not effectively play a role in prevention, and can not avoid the occurrence of vascular events. Comprehensive multiple mechanisms that MSCs involved in tissue repair, in this experiment, we verify the stabilizing role of MSCs on the vulnerable plaque.Purpose1. To culture and amplified rabbit MSCs in vitro and check the osteogenesis and lipogenesis ability, and specific surface markers. And select a appropriate method to labeled MSCs between lentiviral transfection and fluorescent dye staining. And to preliminary explore the effects of MSCs on ox-LDL-induced vascular endothelial cell apoptosis in vitro.2. Based on the AS vulnerable plaque model, and MSCs’ unique "homing" function, multi-differentiation potential and strong secretory function, make use of MSCs transplantation, to explore the effect and mechanism of MSCs on stability and repair of vulnerable plaque, serum and plaque-inflammatory cytokines and anti-inflammatory protein from the organizational level, the protein level and gene level, and to provide theoretical and experimental evidence for the clinical application of MSCs to combat acute coronary syndrome.Methods and ResultsPart Ⅰ:Methods:MSCs was extracted from the bone marrow of New Zealand white rabbits, and the whole bone marrow adherence method were used to isolate and culture rabbit MSCs. P3generation cells were induced to osteoblasts and lipoblasts by culturing for21days in osteogenic medium and14days in adipogenic medium. And the specific surface markers of MSCs CD29, CD44, CD45were identified by flow cytometry. Lentiviral transfection and CFSE fluorescence dye staining were used to MSCs labeled, and select the appropriate one. In addition, establish human umbilical vein endothelial cells (hUVECs) injury model by Oxidized Low-density lipoprotein (ox-LDL) in vitro, then co-cultured MSCs and hUVECs, apoptosis rate was detected using the flow cytometry, tumor necrosis factor a (TNF-a) and vascular endothelial growth factor (VEGF) of cell culture medium were measured by ELISA, and rt-PCR was used to detect the expression of apoptosis-related genes bcl-2and Bax. The data was analyzed by SPSS13.0statistical software. Quantitative data was showed by (x±s), the independent sample t test was used to compare the two samples, one-way ANOVA and Welch F were used to compare three or more samples according to homogeneity of variance, repeat measurement data used repeated measure ANOVA, P<0.05was considered statistically significant.The results show that:(1) The MSCs cultured and amplified in vitro is uniformity spindle cells with great proliferation ability. After21days’osteogenic induction, mineralized nodule formation is visible by alizarin red staining; and after14days’adipogenic induction, lipid droplets is visible by oil red O staining. The results show that rabbit MSCs was successfully cultured and amplified in vitro, which can differentiate into osteoblasts and lipoblasts under certain conditions. Flow cytometry results showed the3rd generation cells have specific surface markers of MSCs:above98%of cultured cells express surface markers CD29and CD44, but CD45, which illustrate the3rd generation of adherent cells are MSCs. All of these provide the cellular basis for subsequent experiments. Compared to lentiviral transfection to MSCs can reduce the proliferation of the cells, CFSE staining as a fast, convenient and safe method of dyeing, not only do not affect the proliferation of rabbit MSCs, but also not affecting its function of differentiation and secretion, is more suitable for labeling a large dose of transplanted cells.The efficiency of lentiviral transfected MSCs was up to80～90%, but it had a great impact on the proliferation of MSCs, the proliferation of transfected cells was significantly lower than the control group (F=2970.659, P<0.001). CFSE staining was a fast, convenient, and safe method for dyeing. A final concentration of10μmol/L and10minutes stained was ideal conditions for CFSE labeled MSCs, which not only do not affect the proliferation of MSCs (F=0.693, P=0.425), but aslo not affect its conversion function and secretion of VEGF (t=2.070, P=0.065). It’s more suitable for large doses of transplanted cells using.(2) The normal hUVECs have different forms, the round nucleus, rich and clear cytoplasm, near the nucleus of cells surrounding seen clearly Halo, forming a typical "cobblestone structure". After24h’100μg/ml ox-LDL, hUVECs began to appear obvious morphological changes, the cells were shrunken, cell gap increases, more particles and vacuoles were see in intracytoplasmic. Flow cytometry results showed that hUVECs apoptosis rate of the three groups were significantly different (F=105.229, P<0.001), ox-LDL group is significantly higher than that of control group (P=0.001); the MSCs co-culture group can significantly inhibited the ox-LDL-induced hUVECs damage, and apoptosis rate significantly reduced (P=0.008). VEGF and TNF-a content of three groups are significant differences (F=50.957, P<0.001and F=61.578, P<0.001). VEGF and TNF-a levels were significantly increased compared with the blank control group(P=0.001, P<0.001), VEGF in MSCs co-culture group supernatant was significantly higher than the control group and the ox-LDL group(P=0.001, P=0.008), and TNF-a level was significantly reduced compared with ox-LDL group (P=0.002), but still higher than the control group ((P=0.005). Bcl-2and Bax mRNA expression of three groups are significant different (F=13.381, P=0.002and F=14.633, P<0.001). Bcl-2mRNA expression in ox-LDL group had a significant reduction (P=0.041) and bax mRNA expression significant increased compared with the control group (P=0.001). Compared with ox-LDL group, MSCs co-culture group had increased bcl-2mRNA expression (P=0.004) and reduced bax mRNA expression (P<0.001). The Bcl-2/Bax ratios of three groups were significant difference (F=42.433, P<0.001). And comparing with the control group, the ratio of ox-LDL group was significantly decreased (P<0.001), MSCs co-culture group had no significant difference(P=0.269). Compared with ox-LDL group, the Bcl-2/Bax ratio of MSCs co-culture group increased, the difference was statistically significant (P=0.001).Part II:Methods:34healthy male New Zealand white rabbits were randomly divided into three groups, the MSC group (14) and (10) VP group rabbit first underwent a high fat diet for one week, and then cold-induced endothelial injury with liquid nitrogen, postoperative continued high fat diet fed for7weeks, and again in the8th week to2nd liquid nitrogen frostbite surgery, immediately following transplantation of1×107MSCs (approximately1ml) or lml PBS via ear vein, continue to change ordinary feed fed for four weeks. SP group (10) rabbit first underwent the high fat diet for one week, and then the liquid nitrogen frostbite surgery, postoperative were fed high-fat diet for7weeks and followed normal diet fed for four weeks. Before the experiment and after the high-fat diet3d,1w,2w to12w once every2weeks, triglyceride (TG), total cholesterol (TC) and low-density lipoprotein (LDL) levels of serum were detected. After6h and24h cell transplantation respectively, whole blood film was produced and MSCs changes in blood was observed under fluorescence microscope.3Days and2weeks after transplantation, two rabbit in the MSCs group were taken the right common carotid artery, cells’ homing was observed under confocal microscope. The high sensitivity C reactive protein (hs-CRP), tumor necrosis factor a (TNF-a), interleukin6(IL-6), interleukin10(IL-10) and VEGF level in serum was determined respectively at Id,2d,3d,1w,2w,4w end after transplant by ELISA method. Rabbits were sacrificed4weeks after cell transplantation, the blood vessels were removed to make HE and Masson staining, measure cap nuclear ratio, and detected the nuclear factor κB (NF-κB), matrix metalloproteinase1,2,9(MMP-1, MMP-2, MMP-9) and tissue inhibitor of metalloproteinase1(TIMP-1) level in plaque using immunohistochemical study, the number of apoptotic cells in the plaque was measured by TUNEL. Rt-PCR was to test the level TSG6mRNA expression and Western blot was to test the level TSG6protein expression. The data were analyzed by SPSS13.0statistical software. Quantitative data was showed by (x±s), the independent sample t test was used to compare the two samples, one-way ANOVA and Welch F were used to compare three or more samples according to homogeneity of variance, repeat measurement data used repeated measure ANOVA, P<0.05was considered statistically significant.Results:(1) After high fat diet for3days, TG, TC and LDL significantly increased (P=0.037, P=0.041,P=0.048), and increased fastest in one week, the first2to8W, lipids increased slowly. After replaced normal diet, rabbit serum lipid levels declined rapidly. All rabbits in MSC group and VP group had multiple yellow-white plaques in right carotid artery at8weeks, and the left common carotid artery had no obvious and visible plaque. And at12weeks, SP group showed localized thickening and fibrotic intima, and integrity morphology of the atherosclerotic plaque with thick fibrous cap, fewer inflammatory cells in the plaque, no plaque rupture. VP group showed a large lipid core in plaque center and a thinner fibrous cap, and residual foam cells and a large number of inflammatory cells (such as macrophages and lymphocytes) infiltrated in the plaque shoulder. In addition, rupture (or) thrombosis can be seen in some plaques. The MSC group showed a moderate morphology between VP and SP group. The endothelial cells and smooth muscle cells of left common carotid artery have a normal morphology and structure, with occasional mild intimal hyperplasia, a very small part of the intima showed a small amount of lipid deposition, but no prominent plaque formation. We can see more fluorescent cells in whole blood film under a fluorescence microscope after6hours transplantation, and after24hours, the fluorescent cells were significantly reduced. Fluorescence laser scanning confocal microscope showed after3days transplantation, more fluorescent cell aggregation can be found in the plaque intimal surface, but the cells decreased significantly after2weeks transplantation. And no fluorescent cell was found in the surface of left common carotid artery in the MSCs group. The Masson staining showed SP group plaque contains a large number of smooth muscle cells and elastic fibers, and followed by MSC group. Compared with MSC and VP group, the VP group plaque had decreased smooth muscle cells and elastic fibers, and disordered arrangement of collagen fibers. There are significant differences between the three groups of cap/core ratio (F=28.298, P<0.001). The plaque cap/nuclear ratio of MSC and SP group were significantly higher than the VP group (P<0.001).(2) There were significant differences of serum hs-CRP in different times (F=21.670, P<0.001). The serum hs-CRP level in three groups were significant difference (F=428.245, P<0.001), and at each time point was P<0.001. Hs-CRP in VP group was significantly higher than MSC and SP group, MSC was significantly higher than SP group. Interaction between different times with different groups were significantly (F=11.857, P<0.001). There were significant differences of TNF-a in different times (F=8.056, P<0.001). The TNF-a level in three groups were significant difference (F=245.006, P<0.001), and at each time point was P<0.001. TNF-α in VP group was significantly higher than the MSC and SP group. Interaction between different times with different groups were significantly (F=9.362, P<0.001). There were significant differences of IL-6in different times (F=5.852, P<0.001); The IL-6level in three groups were significant differences (F=49.492, P=0.015), and at each time point was P<0.05. IL-6in VP group was significantly higher than the SP group; the VP group was significantly higher than the MSC group at D2, D3, W1and W2. Interaction between different times with different groups were significantly (F=1.951, P=0.044). There were significant differences of VEGF in different times (F=15.241, P<0.001); The VEGF level in three groups were significant differences (F=132.641, P<0.001), and all were P<0.001. While MSC group had no significant difference with VP group at D2, but in the rest of the time points the VEGF level of MSC group were significantly higher the VP and SP group. And VEGF of VP was significantly higher than SP group at W1, W2and W4. Interaction between different times with different groups were significantly (F=10.702, P<0.001).(3) The content of NF-κB, MMP-1, MMP-2, MMP-9and TIMP-1among the three groups were significant differences (F=44.953, P<0.001; F=46.530, P<0.001; F=34.292, P<0.001; F=27.108, P<0.001; F=63.138, P<0.001). The content of NF-κB, MMP-1, MMP-2, MMP-9in MSC group were significantly lower than that in the VP group, and the differences were statistically significant (P<0.001, P<0.001, P<0.001, P<0.001), and TIMP-1level was significantly higher than that in VP group(P<0.001). The NF-κB, MMP-1, MMP-2, MMP-9and TIMP-1content in the plaques of SP group were significantly lower than VP group, the differences were statistically significant (P<0.001, P<0.001, P<0.001, P<0.001, P=0.002); the NF-κB, MMP-1, MMP-2, MMP-9and TIMP-1expression in MSC group were also significantly higher than the SP group (P=0.005, P=0.018, P=0.046, P=0.005, P<0.001). There were significant differences of TSG6mRNA and protein expression among three groups (F=106.082, P<0.001; F=41.484, P<0.001), TSG6mRNA and protein expression in MSCs group were significant higher than the VP and SP groups (P=0.017, P=0.007; P<0.001, P<0.001); while TSG6expression in VP group increased significantly than SP group (P<0.001; P=0.008).TUNEL results showed that apoptotic cells were seen in the plaques of three groups, and AIs of three groups were significant differences (F=96.153, P<0.001).AI of VP group was significantly higher than MSCs and SP group (P<0.001, P<0.001), and AI of MSC group was also significantly higher than SP group (P<0.001). The apoptotic cells were mainly distributed in the lipid nucleus and medial smooth muscle area in the plaques of MSC and VP group, and were mainly distributed in the area of medial smooth muscle of SP group, there were little of apoptotic cells in the minimal lipid core area and intimal of SP group.The content of caspase-9among the three groups were significant differences (F=14.391, P=0.002), the caspase-9expression of VP group was significantly higher than those of MSCs and SP group (P<0.001, P<0.001). There were not statistically differences between MSC group and SP group(P=0.335).Conclusion1We successfully extracted and amplified a sufficient number and vigorous MSCs through whole bone marrow adherent culture method. And through a series of identifications in vitro, we proved the cells extracted were MSCs. Compared to the lentivirus transfection, the CFSE fluorescence staining has no effect on the biological activities, was more applicable to large doses of transplanted cells labeled. And we found that MSCs could reduce the level of inflammatory factors, increased the secretion of VEGF, and through a series of mechanisms to regulate the expression of apoptosis-related genes, thereby improving the ox-LDL-induced vascular endothelial cell apoptosis.2Transplation of allogeneic bone marrow mesenchymal stem cells (MSCs) in vein can stabilize and repair the vulnerable plaque. MSCs in the body can "homing" to the vulnerable plaques, can reduce inflammatory factors and increase anti-inflammatory protein in serum and local plaque, thereby stabilizing the vulnerable plaque. The specific mechanisms may be that the transplanted MSCs can secrete TSG-6, which can inhibits macrophage releasing NF-κB, thereby inhibiting the release of a series of inflammatory cytokines in the signaling pathway. In addition, MSCs can inhibit local cells apoptosis in plaque, which may be associated with MSCs by suppressing inflammation reduce the damage and secreting growth factors such as VEGF to promote the repair and proliferation of local cells and inhibite mitochondrial apoptotic pathway. |
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