| BackgroundIncreasing studies have shown that vasa vasorum (VV) proliferation is associated with atherogenesis, plaque progression and instability, and its extent and distribution are regulated by the balance between pro-angiogenic factors and anti-angiogenic factors. The vascular endothelial growth factor (VEGF) members and angiopoietin (ANGPT) family play pivotal roles in the regulation of angiogenesis. VEGF-A, the major VEGF subtype, could induce activation, adhesion and migration of monocyte to subendothelium and increase vascular permeability, leading to intimal hyperplasia and augmentation of atherosclerosis. Immnohistochemical staining demonstrated that, VEGF-A is detected at all stages of human coronary atherosclerosis and quantitative analysis revealed larger VEGF+ staining percentage in atheromatous lesions than that in arteries with diffuse intimal thickening. Moreover, in human carotid endarterectomy samples, reduced ANGPT-1 and increased ANGPT-2 levels were observed in unstable plaques. In addition, the ratio of ANGPT-1 to ANGPT-2 was in favor of ANGPT-2 in haemorrhagic plaques and was negatively correlated with microvessels density.The expression of angiogenic factors are regulated by some transcription factors, such as hypoxia-inducible factor la (HIF-la), E26 transformation specific sequence 1 (Ets-1). The stabilization and transcriptional activity of HIF-la is regulated through both hypoxia-dependent and hypoxia-independent signaling pathways.Increased intimal thickness exceeding the maximum oxygen diffusion distance and the high metabolic demand of inflammatory environment in atherosclerosis could lead to accumulation of HIF-la protein. Many inflammatory cytokines and growth factors could induce the expression of angiogenic factors (VEGF-A, ANGPT-2, ANGPT-4, et al.) via PI3K/Akt/HIF-1α signaling pathway. Moreover, the Ets transcription factor member Ets-1 could bind to the Ets binding sites on ANGPT-2 and activate its promoter activity, leading to accumulation of ANGPT-2.Traditional Chinese medicine Tongxinluo (TXL) is registered for treatment of angina pectoris and ischemic stroke by the State Food and Drug Administration of China in 1996. Moreover, in murine myocardial infarction and heart failure model, TXL was reported to promote angiogenesis in ischemic myocardium, reduce ventricular remodeling and improve ischemic myocardial function. So here come the problems, whether VV proliferation influences early atherogenesis? If yes, what’re the potential mechanisms? Would TXL enhance angiogenesis in atherosclerosis and further promote atherogenesis and plaque destabilization when applicate in anti-myocardial ischemic therapy?In light of these findings, we sought to observe the effect of VV proliferation on early atherogenesis, and further explore the effect and underlying mechanisms of TXL on VV neovascularization and early atherogenesis in apolipoprotein E deficient (apoE-/-) mice to provide more experimental basis for its clinical application.Objectives1. To observe the effect of TXL on VV neovascularization and early atherogenesis in apoE-/-mice;2. To explore the potential mechanisms of TXL on angiogenesis.Methods1. Preparation of TXL ultrafine powder and solutionTXL ultrafine powder with the diameter≤10μm was prepared. In vivo study, TXL was dissolved in saline and given to apoE-/-mice by gavage. In vitro study, TXL ultrafine powder was dissolved in basal culture medium and was adjusted to certain concentrations before treatment of cultured cells.2. Animal models and experimental designOne hundred apoE-/-mice (male,12 weeks old) were first fed a high fat diet for one week, then the mice were randomly divided into saline control group (Control, n=25) and TXL treatment groups (n=75). To illustrate the dose-response of TXL in mice, TXL treatment groups were divided into three subgroups:low-dose TXL (TXL-L), medium-dose TXL (TXL-M) and high-dose TXL (TXL-H), with TXL given at 0.38, 0.75,1.5 g/kg/d by gavage, respectively. And the Control group was given an equal volume of saline. After five weeks’treatment, all mice underwent euthanasia. Blood and tissues were collected.3. Serum biochemical measurementSerum triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and serum glucose concentration were measured through enzymatic assay.4. Aortic ring assay ex vivoTo observe the effect of TXL on neovessels sprouting, thoracic aortas rings of four treatment groups were embedded in rat type I collagen.5. Histopathological measurementEn face measurement was performed with Oil-Red O staining. Hematoxylin and eosin (H&E) staining, Oil-Red O staining, Sirius red staining and immunohistochemical staining were performed in serial cryosections of aortic roots. The plaque burden, cross-sectional atherosclerotic area, maximal intima thickness, the ratios of positive staining areas of lipids, collagen, MOMA-2, a-SMA, VEGF-A, ANGPT-1 and ANGPT-2 to the cross-sectional areas of aortic roots, VV number and the ratio of ANGPT-1/ANGPT-2 were analyzed.6. Correlation analysisCorrelation analysis was analyzed between the number of VV and the cross-sectional atherosclerotic area, the intraplaque expression of VEGF-A, ANGPT-1, ANGPT-2 and the ratio of ANGPT-1/ANGPT-2.7. Molecular biological analysisProteins were isolated from mice aortas. The protein expression level of VEGF-A, ANGPT-1 and ANGPT-2 in plaques was analyzed by Western Blot assay.8. Cell cultureRAW 264.7 cells were obtained from American Type Culture Collection. RAW 264.7 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with supplement of 10% fetal bovine serum (FBS).9. Experimental design in vitroTo explore the effect of TXL on regulation of angiogenic factors and potential mechanisms under inflammatory condition, RAW 264.7 cells were divided into the tumor necrosis factor alpha (TNF-α) control group and TXL treatment groups.10. Quantitative real time PCR (qRT-PCR) analysisRNA was extracted from treated RAW264.7 cells and the mRNA expression level of VEGF-A, ANGPT-1 and ANGPT-2 was analyzed by qRT-PCR. The relative mRNA expression between treatments was analyzed by 2-△△CT method. Targeted gene expression was normalized against GAPDH expression.11. Western Blot analysisProteins were isolated from treated RAW 264.7 cells. The protein expression level of VEGF-A, ANGPT-1, ANGPT-2, PI3K, p-Akt, Akt, HIF-1α and Ets-1 was analyzed by Western Blot assay. The densitometry was analyzed by the Photoshop software. Targeted protein expression was normalized against GAPDH expression.12. Statistical analysisAll data are shown as mean±SEM and analyzed by use of GraphPad Prism 5 software. Differences between two groups were analyzed by independent t test and differences among groups were analyzed by one-way ANOVA followed by Tukey post-hoc test. P<0.05 was defined as statistical significance.Results1. General conditions of apoE-/-miceNo mice were died at the end of the experiment. No change in body weight was found among the four treatment groups. Biochemical measurement demonstrated no significant difference in serum TG, TC, LDL-C, HDL-C and glucose concentration.2. Effect of TXL on aortic ring sprouting ex vivo and VV proliferation in vivoThe aortic ring sprouting assay demonstrated that, compared to those in the Control group, TXL treatment significantly reduced the sprouting number and length. Consistently, the VV number, identified by Tomato lectin perfusion, was significantly reduced in the three doses of TXL group.3. Effect of TXL on expression of angiogenic factors in atherosclerotic plaquesImmunohistochemical staining and Western Blot analysis revealed that, compared with the Control group, TXL treatment could dose dependently reduce the expression of VEGF-A and increase the expression of ANGPT-1, whereas, have no significant effect on ANGPT-2 expression in atherosclerotic plaques of apoE-/-mice. Therefore, the ratios of ANGPT-1/ANGPT-2 in TXL treatment groups are significantly increased.4. Effect of TXL on atherosclerotic plaque burden, area and maximal intima thicknessPlaque burden in en face aortas was detected by Oil Red O staining. Compared to that of Control group, plaque burden was significantly reduced in TXL treatment groups.H&E staining was performed to analyze the morphology of cross-sectional plaque, plaque area and the maximal intima thickness. Compared to that of Control group, the cross-sectional plaque area in aortic roots was significantly reduced in TXL treatment groups. However, no statistical difference in the maximal intima thickness of atherosclerotic plaques was found in the four treatment groups.5. Effect of TXL on atherosclerotic plaque compositionsHistopathological analysis demonstrated that, compared with those in the Control group, the plaque content of lipids and macrophages were much lower and the plaque content of smooth muscle cells and collagen were much higher in TXL treatment groups.6. Correlation analysisA positive correlation was found between VV number and cross-sectional plaque area, the intraplaque expression of VEGF-A (r=0.900, p<0.01; r=0.891,p<0.01); while, a negative correlation was observed between VV number and the intraplaque expression of ANGPT-1 (r=-0.821,p<0.01), and VV number was positively correlated with the intraplaque expression of ANGPT-2 (r=0.805,p<0.01). However, a negative correlation was observed between VV number and the ratio of ANGPT-1/ANGPT-2 (r=-0.849,p<0.01).7. Effect of TXL on expression of angiogenic factors in treated RAW 264.7 cellsqRT-PCR and Western Blot analysis revealed that, compared to the TNF-a stimulation group, pretreatment with TXL could dose-dependently inhibit the expression of VEGF-A and increase the expression of ANGPT-1 in RAW 264.7 cells both in the mRNA and protein level. No significant effect in ANGPT-2 mRNA and protein expression was found by TXL treatment. However, the protein ratio of ANGPT-1/ANGPT-2 was significantly increased in TXL treatment groups.8. Effect of TXL on PI3K/Akt/HIF-la signaling pathway and ANGPT-2 transcription factor Ets-1 protein expression in RAW 264.7 cellsWestern Blot analysis demonstrated that the protein level of PI3K, p-Akt, HIF-1α and Ets-1 was elevated by TNF-a stimulation, pretreatment with TXL could significantly inhibit TNF-a induced activation of the PI3K/Akt/HIF-la signaling pathway, while had no significant effect on Ets-1 expression.Conclusions1. Suppression of VV proliferation might be another mechanism of TXL contributing to inhibition of early atherogenesis and amelioration of plaque composition;2. The inhibition of TXL on VV proliferation may attribute to the regulation of angiogenic factors expression in plaques;3. The effect of TXL on angiogenic factors expression may attribute to the inhibition of PI3K/Akt/HIF-1α signaling pathway.BackgroundThe concept that atherosclerosis (AS) being a chronic, systemic inflammatory disease of large and medium-size arterial wall has been well accepted nowadays. Inflammation plays a central role at all stages of atherosclerotic development. It is involved in the initiation of early fatty streaks, when the activated endothelial cells secretes chemokines and expresses high level of adhesion molecules contributing to circulating monocytes recruitment and then infiltration into the intima to differentiate to resident macrophages. It is also implicated in the triggering of adverse clinical vascular events by plaque rupture and thrombus formation, when the activated inflammatory cells within the atherosclerotic plaques produce matrix metalloproteinases (MMPs) leading to degradation of extracellular matrix proteins and weakness of the fibrous cap.For years, the studies on the role of inflammation to atherosclerotic development have been mainly focused on its directed immune response. However, studies regarding the association between inflammation and angiogenesis in atherosclerosis have been arising recent years. Atherosclerotic vulnerable plaques are characterized by large eccentric lipid-rich cores, thin fibrous cap, activated inflammatory response, positively remodeling, enhanced vasa vasorum (VV) neovascularization and intraplaque neovessels. However, in late imaging studies reported that plaques progress rapidly before the occurence of acute cardiovascular sydromes. The potential mechanisms for rapid plques progression may attribute to 1) subclinically repeated plque rupture and healing; 2) intraplaque neovessels which are leaky of red blood cells membranes, containing abundant cholesterol, and inflammatory factors; 3) intraplaque hemmorahage leading to enlargement of plaque volume, accumulation of cholesterol and augment of oxidative stress. The secreted inflammatory cytokines and MMPs are reported to have directed or in-directed pro-angiogenic effects, while the newly formed neovessels are considered to be a new conduit for inflammatory cells, erythrocytes and lipid components to entre atherosclerotic lesions, as the much more prevalent expression of adhesion molecules on intraplaque microvessel endothelial cells, lacking of coverage of supporting cells and deteriorated junctions between extracellular matrix leading to increased vascular permeability. The intraplaque inflammation and angiogenesis act reciprocally and forms a "positive feedback" loop, thereby further promoting atherosclerotic progression and destabilization. Regarding the intimate relationship between inflammation and angiogenesis, the researchers in this field invent the term "inflammatory angiogenesis" in order to describe more precisely on the neovascularization process associated with inflammatory diseases.Tumor necrosis factor a (TNF-a) is a pleiotropic cytokine which is a potent stimulator of adhesion molecules, MMPs and plasminogen activator inhibitor-1, and modulate cell death. TNF-a is thought to play an important role at the initiation and progression of atherosclerosis. Although high level of TNF-a inhibits proliferation of endothelial cells in vitro, low level of TNF-a is proved to promote endothelial cells migration and tube formation via activating signaling pathways involved bone marrow kinase in chromosome X (BMX), nuclear factor Kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs). Moreover, in chick chorioallantoic membrane model, rabbit cornea model and mouse ischemic hind limb model, TNF-a is proved to be a potent inducer of angiogenesis in vivo.In the first part of this thesis, we found that traditional Chinese medication Tongxinluo (TXL) could ameliorate early atherogenesis through regulating expression of angiogenic factors and inhibition of VV proliferation. However, the effects of TXL on pre-existing VV neovascularization and intraplaque angiogenesis in advanced atherosclerosis and inflammatory angiogenesis in vitro are still uncertain. Thus, in the present study, we sought to observe the effect of TXL on pre-existing inflammatory angiogenesis in advanced atherosclerotic plaques of apolipoprotein E deficient (apoE-/-) mice and plaque stabilization, and further explore the effect of TXL on TNF-a induced angiogenesis in vitro and its potential mechanisms.Objectives1. To observe the effect of TXL on pre-existing VV neovascularization, intraplaque angiogenesis and plaque stabilization in advanced plaques of apoE-/-mice;2. To observe the effect of TXL on angiogenic sprouting of endothelial cells under inflammatory condition;3. To explore the potential mechanisms of TXL on inflammatory angiogenesis.Methods1. Animal models and experimental protocolOne hundred male apoE-/-mice aged 10 weeks were first fed a high fat diet for 20 weeks, then the mice were randomly divided into saline control group (Control, n=25), low-dose TXL (TXL-L,0.38 g/kg/d, n=25), medium-dose TXL (TXL-M,0.75g/kg/d, n=25) and high-dose TXL (TXL-H,1.5g/kg/d, n=25); and the control group was given an equal volume of saline. After 16 weeks’treatment, all mice underwent euthanasia by overdose of phenobarbital and blood samples and tissues were collected.2. Serum biochemical measurementAt the end of the experiment, serum lipid profiles, including triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), and serum glucose were measured.3. Pathological analysis of tissue samplesPlaque burden was determined through Oil-Red O staining of lipids in aorta en face preparations. Special staining of serial cryosections of aortic roots were performed with hematoxylin and eosin (H&E), Oil-Red O, Sirius red to observe the morphology of atherosclerotic plaques and analyze the cross-sectional atherosclerotic plaque area, the ratios of positive staining areas of lipids and collagen. Immunohistochemical staining with specific antibodies against macrophages (Monocyte/Macrophage Marker, MOMA-2), smooth muscle cells (a-smooth muscle actin, a-SMA),, endothelial cells of VV and intraplaque neovessels (Tomato lectin), chemokine monocyte chemotactic protein-1 (MCP-1), inflammatory cytokines TNF-a, interleukin 1β (IL-1β) and IL-6 and angiogenic factors vascular endothelial growth factor A (VEGF-A) and MMP-2 were performed. Vulnerable index is calculated as the following formula:vulnerable index= (lipids staining % +MOMA-2 staining%)/(α-SMA staining%+collagen staining%).4. Correlation analysisCorrelation analysis was performed between the number of VV, intraplaque neovessels and the cross-sectional plaque area, the intraplaque expression of MCP-1, TNF-a, IL-1β, IL-6, VEGF-A and MMP-2, respectively.5. Molecular biological analysisProteins were isolated from mice aortas. The protein expression level of MCP-1, TNF-a, IL-1β, IL-6, VEGF-A and MMP-2 in plaques was analyzed by Western Blot assay.6. In vitro experimental designPrimary human umbilical vein endothelial cells (HUVECs) and THP-1 cell line were obtained. To observe the effect of TXL on inflammatory angiogenesis in vitro, cells were divided into several groups:the blank control (no TNF-a, no TXL), TNF-a control (only TNF-a, no TXL) and TXL treatment groups (TNF-a+different concentration of TXL). The ultimate concentration of TNF-a in medium was lng/ml.7. CCK-8 assayCCK-8 assay was performed to evaluate the effect of TXL solutions (0.25、50、100、 200、500、1OOOμg/ml) on cell viability.8. Cell migration assayScratch assay and Transwell assay were performed to observe the effect of TXL on TNF-a induced cell migration.9. Tube formation assayIn the growth factor reduced Matrigel, the effect of TXL on TNF-a induced capillary-like tube formation was analyzed.10. Endothelium-monocyte adhesion assayBCECF-AM labeled THP-1 cells were used to analyze the effect of TXL on TNF-a induced endothelium-monocyte adhesion.11. Immunocytochemistry analysisThe effect of TXL on TNF-a induced NF-κB p65 translocation was performed in immunocytochemical analysis.12. Western Blot analysisThe effect of TXL on TNF-a induced protein expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), BMX, NF-κB and MAPK signaling pathway was analyzed through Western Blot assay.13. Statistical analysisQuantitative data are expressed as mean±SEM, and analyzed by use of GraphPad Prism 5 software. Differences among groups were analyzed by one-way ANOVA followed by Tukey post-hoc test. P value of<0.05 was considered to be statistically significant.Results1. General conditions of miceAt the end of the experiment, totally 10 mice were died, with 3 in the Control group,3 in TXL-L group,2 in TXL-M and TXL-H group, respectively. No significant difference in body weight was found among the four groups. And the levels of serum TG, TC, LDL-C, HDL-C and glucose were did not differ among the four groups.2. Effect of TXL treatment on VV proliferation and intraplaque neovascularizationCompared with that of the Control group, the number of neovesseles in adventitia and intraplaque was significantly reduced in TXL treatment groups in a dose-dependent manner.3. Effect of TXL treatment on plaque burden and cross-sectional plaque area in aortic rootsOil-Red O staining demonstrated that, compared with the Control group, all three TXL treatment groups significantly reduced the plaque burden in a dose-dependent manner. H&E staining demonstrated that, compared to Control group, TXL treatment could dose-dependently reduce the cross-sectional plaque area in aortic roots.4. Effect of TXL treatment on plaque composition and stabilizationCompared with that of the Control, the content of plaque lipids and macrophages were significantly decreased, while the content of collagen and smooth muscle cells were significantly increased in TXL treatment groups; thus, the calculated vulnerable index was significantly reduced in TXL treatment groups.5. Effect of TXL treatment on intraplaque expression levels of chemokines and inflammatory cytokinesImmunohistochemical and Western Blot analysis demonstrated that, compared with the Control group, all three TXL treatment groups could significantly reduce the expression levels of intraplaque MCP-1, inflammatory cytokines TNF-a, IL-1β and IL-6, thus ameliorate the inflammatory response in palques.6. Effect of TXL treatment on intraplaque expression levels of angiogenic factorsImmunohistochemical and Western Blot analysis demonstrated that, compared to Control group, all three TXL treatment groups could significantly reduce the expression levels of intraplaque VEGF-A and MMP-2.7. Correlation analysisA positive correlation was found between VV number and cross-sectional plaque area (r=0.891,/?<0.01), the intraplaque expression of MCP-1 (r=0.952,p<0.01), TNF-a (r=0.915, p<0.01), IL-1β(r=0.841, p<0.01), IL-6 (r=0.870,p<0.01) and angiogenic factors VEGF-A (r=0.931,p<0.01) and MMP-2 (r=0.940,p<0.01), respectively.Similarily, a positive correlation was found between the number of intraplaque neovessels and cross-sectional plaque area (r=0.903, p<0.01), the intraplaque expression of MCP-1 (r=0.955, p<0.01), TNF-α (r=0.930, p<0.01), IL-1β (r=0.853, p<0.01), IL-6 (r=0.899, p<0.01) and angiogenic factors VEGF-A (r=0.924, p<0.01) and MMP-2 (r=0.935,p<0.01), respectively.8. Cell viability assayCCK-8 assay demonstrated that the viability of HUVEC cells was decreased along with the increase of TXL solution concentration. Cell viability was decreased to 49.79% when incubated with 1000μg/ml of TXL solution.9. Effect of TXL treatment on TNF-α induced inflammatory angiogenesisBoth scratch assay and Transwell migration assay demonstrated that pretreatment with TXL could significantly inhibit TNF-α induced endothelial cell migration.Matrigel tube formation assay revealed that, compared with the TNF-α control group, pretreatment with TXL could significantly reduce TNF-α induced angiogenic sprouting of endothelial cells.TNF-α stimulates the expression of adhesion molecules in endothelial cell surface which leading to increased recruitment of monocyte. Fluorescent images analysis revealed that pretreatment with TXL could significantly reduce TNF-α induced endothelium-monocyte adhesion. Moreover, Western Blot analysis demonstrated that the expression level of ICAM-1 and VCAM-1 was significantly decreased in TXL treatment groups.10. Mechanisms of TXL on inhibition of TNF-α induced inflammatory angiogenesisWestern Blot analysis demonstrated that, TNF-stimulation could activate BMX in endothelial cells, leading to elevated expression level of p-BMX; however, TXL pretreatment could significantly inhibit TNF-α induced BMX phosphorylation.Immunocytochemistry analysis revealed that TNF-stimulation obviously promoted NF-κB translocation from the cytoplasm to the nucleus, which was significantly inhibited by TXL pretreatment. In consistent with the immunocytochemical results, Western Blot analysis shown that pretreatment with TXL could decrease TNF-a induced NF-κB p65 phosphorylation in a time-dependent manner.TNF-stimulation could activate MAPK signaling pathway, leading to elevated phosphorylation of MAPK family members JNK, p38 and ERK1/2. Pretreatment with TXL could significantly inhibit TNF-a induced phosphorylation of JNK and p38, while have no effect on ERK1/2 phosphorylation.Conclusions1. Highly expressed inflammatory factors and angiogenic factors leading to angiogenesis in advanced plaques;2. TXL treatment could inhibit inflammatory angiogenesis in advanced atherosclerotic plaques which contribute to the reduced plaque burden and enhanced plaque stabilization;3. TXL treatment could inhibit TNF-a induced endothelial cell migration, tube formation and endothelium-monocyte adhesion.4. The effect of TXL on inhibition of inflammatory angiogenesis may attribute to the inhibition of BMX, NF-κB and partial MAPK signaling pathway. |
PDF Full Text Request