| Background:Angiogenesis is a process characterized by the growth of new capillaries from pre-existing microvessels. Angiogenesis involves degradation of vessel basement membrane and extracellular matrix, endothelial proliferation, migration, tube formation, and the recruitment of mural cells and smooth muscle cells. Angiogenesis plays important roles in physiological processes for embryonic development and female reproductive cycles as well as in various pathological events such as rheumatoid arthritis, psoriasis, diabetic retinopathy, and cancer.Matrix mctalloprotcinases (MMPs) are a group of over20zinc-dependent proteinases, which was firstly discovered by Gross in a frog tail experiment in1962. In recent years, MMPs were thought as another important angiogenesis-associated protein family besides endothelial growth factors, which exerts important roles in both physiological and pathological angiogenesis. MMP14knockout mice showed severe defects in embryonic angiogenesis. MMP13knockout inhibited angiogenesis in mouse skin wound healing. Kxogeneous MMP7could induce the proliferation of Human umbilical vein endothelial cells (HUVECs), and promote angiogenesis in mice xenograft tumor model. Some MMPs were shown to inhibit angiogenesis such as MMP12, MMP27. Now it is well accepted that MMPs regulate angiogenesis by stimulating proliferation or migration of endothelial cells, inducing expressions of proangiogenic growth factors or cytokines, or mediating the release of angiogenic promoters or inhibitors from the extracellular matrix.MMP8, also known as neutrophil collagenase, is firstly discovered in neutrophils. With advanced studies in this area, it is found that MMP8is expressed in various kinds of cells such as endothelial cells, embryonic stem cells, smooth muscle cells, and macrophages. MMP8plays important roles in the proliferation or migration of some kinds of cells. MMP8could promote the migration of neutrophil in mice corneal stroma. MMP8could enhance the migration of mice embryonic stem cell though endothelial cells. MMP8could also induce the proliferation of mice smooth muscle cells. However, the role of MMP8in endothelial proliferation, migration, or angiogenesis remains unclear.Atherosclerosis is the pathological basis of various cardiovascular diseases and cerebrovascular diseases such as heart attack and stroke, which is the leading cause of human death in the world. Accumulating evidence show that intraplaque angiogenesis is associated with the progress of atherosclerosis. Intraplaque angiogenesis could exist in every stage of atherosclerosis and is positively associated with the severity of atherosclerosis. Intraplaque angiogenesis could enhance macrophage infiltration and transport of erytherocyte-derived cholesterol, increase intraplaque inflammation, and promote the transition from stable plaque to unstable plaque. On the other hand, new microvessels formed in atherosclerotic plaque are more fragile, and prone to rupture, leading to intraplaque hemorrhage and thrombosis. Once this happens, acute clinical events such as acute myocardial infarction and stroke may follow and become life-threatening.It has been shown that MMP8is expressed in human atherosclerotic lesions and its expression was elevated in rapidly progressing carotid plaques. Another study reports that MMP8knockout could inhibit the inflammatory reaction in atherosclerotic lesion, which suggests MMP8was involved in the progress of atherosclerosis. However, its role in intraplaque angiogenesis is still unknown.Objective:1. The study aimed to investigate the role of MMP8in endothelial proliferation, migration, angiogenesis as well as the related molecular mechanisms.2. By setting up animal models, we examined the role of MMP8in vivo angiogenesis.3. By using apoE-/-/MMP8+/+and apoE-/-/MMP8-/-mice, we established the atherosclerotic animal model and evaluated the role of MMP8in the development of atherosclerosis and intraplaque angiogenesis.Methods:1. HUVECs were infected with MMP8shRNA Lentivirus or Nontarget shRNA lentivirus. After ten days’ selection with Puromycin, HUVECs infected MMP8shRNA Lentivirus or Nontarget shRNA lentivirus were subjected to Roche BrdU Detection Kit and Ki67immumofluorescence staining for assessing proliferation, to Wound scratch assay for evaluating migration, and to In-vitro Matrigel tube formation assay for assessing tube number, tube length, and branch point number. Expressions of MMP8, CD31, and β-catenin in total protein of HUVECs infected with MMP8shRNA Lentivirus or Nontarget shRNA Lentivirus was then evaluated by Western blot. Cytoplasmic and nuclear protein were extracted from HUVECs infected with MMP8shRNA Lentivirus or Nontarget shRNA lentivirus. Western blot was performed to assess the levels of β-catenin in cytoplasmic protein or nuclear protein. To further confirm the effect of MMP8on β-catenin distribution in HUVECs, immunofluoresccnee staining was performed. Total RNA was also extracted from HUVECs infected with MMP8shRNA Lentivirus or Nontarget shRNA lentivirus and subjected to quantitative real-time PCR for determing the mRNA expressions of β-catenin targete genes. HUVECs infected with MMP8shRNA Lentivirus or Nontarget shRNA lentivirus were stimulated with indicated concentrations of Ang Ⅰ or Ang Ⅱ, Western blot was performed to examine the expression CD31in HUVECs. The Ang Ⅰ/Ang Ⅱ treatment experiments were designed to test the effects of MMP8-induced Ang Ⅱ from AngⅠ on HUVEC CD31expression.2. Two sets of in vivo Matrigel plug experiments were designed for this study. In the first set of Matrigel plug experiments, HUVECs infected with MMP8shRNA Lentivirus or Nontarget shRNA lentivirus were labeled with PKH26Red fluorescent reagent. Cells were then mixed with Matrigel, and subcutaneously injected into apoE-/-/MMPS8-/-mice. Under a fluorescence microscopy, number of PKH26labeled cells was counted to examine the survival of exogenous HUVECs in apoE-/-MMP8-/-mice. By using H&E staining, microvessel density and capillary density in Matrigel plug were calculated to assess the in vivo angiogenesis in MMP8-/-/apoE-/-mice. In the second set of Matrigel plug experiments, VEGF165was mixed with Matrigel and subcutaneously injected into apoE-/-/MMP8+/+or apoE-/-/MMP8-/-mice. By using double immunofluorescence staining, CD144positive cells and CD144/Ki67double positive cells were calculated to evaluate the migration and proliferation of mouse endothlelial cells in Matrigel plug. By using H&E staining, microvessel density were counted to assess the in vivo angiogenesis of mouse endothelial cells in apoE-/-/MMP8+/+and apoE-/-/MMP8-/-mice.3. Immunohistochemistry was used to detect the expression of MMP8in the microvessles of human aneurysm and atherosclerotic plaques. Both apoE-/-/MMP8+/and apoE-/-/MMP8-/-/mice were fed a high-fat diet for12weeks. The extent of aortic atherosclerosis and aortic root leision size were assessed by Oil red staining to evaluate the role of MMP8in the development of atherosclerosis. Immunohistochemistry was also conducted to detect the endothelial cell maker, CD31and CD144. The percentage of the positive staining area (the positive staining area/atherosclerotic area) and the positive subject intensity (the number of positive subjects/atherosclerotic area) were measured to examine the role of MMP8in intraplaque angiogenesis.Results:1. All of normal control HUVECs without lentivirus infection were killed after Puromycin selection for24h, but most cells of HUVECs infected with MMP8shRNA Lentivirus or Nontarget shRNA lentivirus remained alive. After selection with puromycin for ten days, HUVECs infected with MMP8shRNA Lentivirus or Nontarget shRNA lentivirus was under good growth condition. Western blot results showed that MMP8expression in HUVECs infected with MMP8shRNA lentivirus was significantly knocked down. MMP8knockdown significantly impaired endothelial proliferation, migration and in vitro tube formation. Western blot results showed that MMP8knockdown in HUVECs significantly downreuglated the CD31expression, inhibited β-catenin nuclear translocation, and reduced the expressions of β-catenin target genes such as TCP1B, TCP1E, FZD7, and CCND1. The effects of MMP8knockdown on β-catenin nuclear translocation were confirmed by immunofluorescence staining. Ten nM Ang Ⅱ could upregulate the expressions of CD31in both of HUVECs infected with MMP8shRNA Lentivirus and HUVECs infected with Nontarget shRNA lentivirus. However, Ten nM Ang I could only upregulate the expression of CD31in HUVECs infected with Nontarget shRNA lentivirus.2. In the first set of in vivo Matrigel plug experiments, more than90%cells in the Matrigel plugs were still PKH26positive, suggesting that these cells were exogenous HUVECs. Capillary density and microvessel density in Matrigel plug of apoE-/-/MMP8-/-mice were significantly lower than that in Matrigel plug of apoE-/-/MMP8+/mice. In the second set of Matrigel plug experiments, the number of CD144positive cells migrated into the Matrigel plug as well as the percentage of Ki67positive endothelial cells and microvessel desnsity in Matrigel plug of apoE-/-/MMP8-/-mice was significantly lower than that in Matrigel plug of apoE-/-/MMP8-/-mice.3. Microvessels were commonly observed in atherosclerotic lesions of human aneurysm and coronary artery. MMP-8was abundantly expressed in these microvessels and cells inside microvessels. The extent of aortic atherosclerosis and aortic root leision size in apoE-/-/MMP8-/-mice were significantly lower than that in apoE-/-/MMP8+/+mice. The percentage of the positive staining area and the positive subject intensity in apoE-/-/MMP8-/-/mice were significantly lower than that in apoE-MMP8+/+mice.Conclusion: 1. MMP8knockdown could inhibit the proliferation, migration, and tube formation in HUVECs, which is modulated by Ang Ⅱ/CD31/β-catenin signalling pathway.2. MMP8knockdown could inhibit in vivo angiogenesis of HUVECs in Matrigel plug subcutaneously implanted into apoE-/-/MMP8-/-mice. MMP8knockout could inhibit mouse endothelial cell proliferation, migration and in vivo angiogenesis in Matrigel plug.3. MMP8knockout could inhibit the development of atherosclerosis and intraplaque angiogenesis in mice.This study demonstrated for the first time that MMP8play important roles in angiogenesis from three levels of angiogenesis models, which was in vitro angiogenesis model (endothelial proliferation, migration, tube formation), in vivo angiogenesis model (Matrigel plug model) and Integrity angiogenesis model (intraplaque angiogenesis)... |
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