| BackgroundAtherosclerotic plaque rupture is a major cause of acute cardiovascular events. Thus, stabilization of vulnerable plaques is of great clinical importance. Pathological studies have identified specific characteristics of atherosclerotic plaques that are associated with plaque instability and rupture, including the ongoing inflammatory response, matrix degradation, and cell death. These changes result in eventual thinning of the fibrous cap and an increase in the inflammatory and necrotic core content. Neovascularization is another crucial feature of atherosclerotic plaques. The number of neovessels increases with plaque progression, and such vessels are abundant in vulnerable plaques. Angiogenesis in plaques are characterized by high perfusion and fragility, and allowing for extravasation of lipoproteins and red blood cells that contribute to the formation of plaque lipids. This process results in intraplaque hemorrhage, increases the permeability of inflammatory cells, and leads to plaque destabilization. Ectopic angiogenesis within the intima and media is considered to be a hallmark of advanced vulnerable atherosclerotic lesions.Angiogenesis is induced by various growth-inducing and -inhibiting factors. Multiple complex signal transduction pathways are involved in intraplaque angiogenesis. Proteinases are required for degradation of the extracellular matrix (ECM), creating an avenue for migrating endothelial cells during angiogenesis. The specific MMPs necessary for endothelial cell migration and tube formation have attracted particular attention because they directly degrade ECM components. MMPs, also termed matrixins, are a family of more than 20 zinc-containing endopeptidases that degrade various components of the ECM. MMPs are subdivided into at least five groups based on their structure and/or substrate specificities. MMPs family members include collagenases (MMP-1,-8, -13, and-18), gelatinases (MMP-2 and -9), stromelysins (MMP-3,-10, and-11), and membrane-type MMPs (MMP-14 and-15).It has become clear that MMPs contribute more to angiogenesis than just degrading ECM components. Various MMPs, including MMP-1,-2,-3,-9, and-14, have been shown to enhance angiogenesis. Specific MMPs can also negatively contribute to angiogenesis. However, the predominant effects of MMPs in intraplaque angiogenesis at the advanced stages of atherosclerosis remain inconclusive. In the present study, we investigated the roles of different MMPs in angiogenesis in patients with atherosclerosis.Objective1. To establish an animal model of AS that is mimic to human pathological changes and observe the changes of specific MMPs at the advanced stage of atherosclerosis in rabbits.2. To elucidate the different roles of specific MMPs in intraplaque angiogenesis and plaque instability and the possible underlying mechanisms at the advanced stage of atherosclerosis in rabbits.Methods1. Animal ProtocolAdult male New Zealand White rabbits 3 months old (n= 27) weighing 1.7 to 2.1 kg underwent balloon injury in the abdominal aorta and then were fed with high-cholesterol diet (1% cholesterol). Five randomly chosen rabbits were euthanized at the end of weeks 4, 6, 8, 10, and 12, and their abdominal aortas were harvested.2. Blood Biochemical AnalysisBlood was drawn from the auricular artery after fasting overnight to measure lipid profile before euthanasia. The serum levels of TG, TC, HDL-C, and LDL-C were measured by enzymatic assays. The body weights of all rabbits were monitored throughout the experiment.3.IVUS AssayIVUS was accomplished to examine the morphological changes of the aortic plaques. LA and EEMA were measured on abdominal aortic cross-sectional images. PA was calculated by EEMA-LA, and PB was calculated by PA/EEMA × 100%.4. Histopathological Analysis:Analysis was assessed in perfusion and fixed tissues. The abdominal aorta (2 cm long) was fixed in 4% formaldehyde for 24 h, and 5-μm-thick segments were then serially sectioned. Frozen sections were stained with Oil Red O staining, and paraffin sections underwent Sirius red, hematoxylin and eosin (H&E).5. Immunohistochemical StainingParaffin sections were stained with immunohistochemical staining. The expression of RAM-11, α-actin, MMP-1,-2,-3,-9, and - 14, COL-Ⅰ,COL-Ⅲ and VEGF-A were determined. All the parameters were measured by two blinded observers with Image-Pro Plus 6.0.The intima-media thickness (IMT) of each aortic plaque was measured. The vulnerability index (Ⅵ) was calculated as follows: (macrophage staining% + lipid staining%)/(smooth muscle cell% + collagen fiber%)6. Microvessels DensityFor quantify the microvessels density (MVD), sections were stained with CD31, and the microvessels were then quantified by the plaque area.7. Western Blot AnalysisProtein of abdominal aorta was extracted and separated in 10% to 15% SDS-PAGE gels and then was transferred onto nitrocellulose membranes. After blocking with 5% nonfat milk for 2 h at room temperature, the membranes were incubated with the primary antibodies overnight at 4℃. After being washed in TBS-T, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Signals were detected by using of an enhanced chemiluminescence kit. The protein levels were normalized to β-actin.8. Statistical AnalysisAll data are presented as mean ± standard deviation of the mean. Intergroup comparisons involved one-way ANOVA followed by the least-squares difference Dunnett’s T3 test (equal variances not assumed) or test (with equal variances assumed). Spearman’s rank correlation coefficient was used for correlation analysis. A two-tailed P value of <0.05 was considered statistically significant. All data analyses were performed using Predictive Analysis Software 18.0.Results1. The General States of Experimental AnimalsAll 27 rabbits showed full recovery without complications after balloon injury. Administration of the atherogenic diet was well tolerated by all rabbits, and no adverse effects were observed. Two rabbits that underwent balloon injury died of diarrhea at weeks 6 and 10.2. Body WeightThe body weights of the rabbits gradually increased over time (P＜0.05).3. Blood Biochemical AnalysisThe serum levels of TC, HDL-C, LDL-C, and TG increased significantly after ingestion of the high-cholesterol diet (P<0.01). At the end of week 6, all four serum values were higher than at week 4 (P>0.05). At the end of weeks 8,10, and 12, the serum levels of TC, HDL-C, and LDL-C were significantly higher than those at week 6 (P<0.05); however, the TG level showed no significant difference (P>0.05). The TG level was significantly higher at the end of week 12 than at week 6 (P＜0.05). The body weights of the rabbits gradually increased over time (P＜0.05).4. IVUS MeasurementsThe LA, EEMA, PA, and PB values increased throughout the duration of the experiment. The LA in the abdominal aorta did not differ among the 5 weeks (P>0.05). However, the EEMA, PA, and PB values were higher at weeks 10 and 12 than at week 4 (P＜0.01), with no difference among weeks 4, 6, and 8 (P>0.05).5. Histopathological AnalysisThe H & E staining showed the plaque area gradually increased over time. Oil red O staining showed the lipid plaque content was higher at week 12 than at all other weeks (P＜0.01) with increasing plaque area. The lipid content was significantly higher at week 10 than 4 (P＜0.05), but no significant differences were observed among weeks 4, 6, and 8 (P>0.05). The positivity of Sirius red collagen staining did not differ among weeks 4, 6, and 8. Staining was more intense at weeks 10 and 12 than at any other weeks (P＜0.05), but no significant difference was observed between weeks 10 and 12 (P>0.05).6. Immunohistochemical ExaminationThe areas of a-actin-positive staining in the abdominal aortic SMCs gradually decreased over time. The areas of a-actin-positive staining within the abdominal aorta significantly decreased at all weeks with the exception of week 4 (P＜0.01). The SMC plaque content was significantly lower at week 8 than 6 and at week 12 than 10 (both P＜0.01), with no difference between weeks 8 and 10 (P>0.05). RAM-11 staining showed that the relative content of macrophages within plaques increased from weeks 4 to 12, with differences among all weeks (all P＜0.01). As a result, the VI gradually increased over time, with statistically significant differences among all weeks.The expression of MMP-1,-2,-3, and -9 significantly increased from weeks 4 to 12. The proportion of areas showing MMP-14-positive staining substantially decreased from weeks 4 to 12.The collagen I level in plaques did not significantly differ among any weeks (P>0.05), but was slightly lower at week 12 than at week 4 (P＜0.05). The relative content of collagen III in plaques increased from weeks 4 to 12, was higher at week 8 than at weeks 4 and 6 (P＜0.05), and exhibited a significant change from weeks 8 to 12 (P＜0.05). The expression of VEGF-A significantly increased from weeks 4 to 12 (P＜0.05).The vulnerability index gradually increased over time, with statistically significant differences among all weeks (all P＜0.01).The intima-media thickness of the abdominal aortic plaques increased until week 12 and was higher at week 6 than at week 4 (P＜0.01). The IMT was significantly thicker at weeks 8, 10, and 12 than at week 6 (P＜0.01).7. Microvessels DensityCD31 staining showed that plaque neovessels appeared at week 8. The MVD was higher at week 12 than 8 (P＜0.05), but no difference was observed between weeks 8 and 10 (both P>0.05).8. Western Blot AnalysisThe expression of MMP proteins exhibited the same trends as shown in the immunohistochemical results. The expression of MMP-1,-2,-3, and -9 significantly increased from weeks 4 to 12, while that of MMP-14 substantially decreased from weeks 4 to 12.9. Correlation AnalysisThe correlation analysis results showed that all correlations between the VI and MMP- 1,-2,-3, and -9 were positive (r= 0.767,0.809,0.890, and 0.887, respectively, all P＜0.01). The expression of MMP-1,-2,-3, and -9 was positively correlated with both the MVD in plaque (r= 0.762,0.813,0.884, and 0.769, respectively, all P＜0.01) and the VEGF-A in plaque (r= 0.760,0.762,0.858, and 0.789, respectively, all P＜0.01). The correlations between MMP-14 and the VI, MVD, and VEGF-A were all negative (r=-0.556,-0.424, and-0.525, respectively, P<0.05). The correlation between the VI and MVD was positive (r= 0.846, P＜0.01).Conclusions1. The expression of MMP-1,-2,-3,-9, VEGF-A and MVD gradually increased at the advanced stages of atherosclerosis, while that of MMP-14 gradually decreased. The Ⅵ also increased over time at the advanced stages of atherosclerosis.2. MMP-1,-2,-3, and -9 were positively correlated and MMP-14 was negatively correlated with intraplaque angiogenesis at the advanced stages of atherosclerosis. Upregulation of MMP-1,-2,-3, and -9 may enhance the development of angiogenesis partly through the role of activating VEGF-A and finally lead to intraplaque hemorrhage and plaque rupture at the advanced stages of atherosclerosis.BackgroundAcute cardiovascular events are commonly encountered and frequently-occurring disease that could seriously harm health. The morbidity and mortality. kept rising these years. The pathology change is atherosclerosis (AS). AS plaque rupture is a major cause of acute cardiovascular events. The pathologic processes of AS include many different factors and the AS pathogenesis is very complex. Researches about it are multiply. On account of the widespread incidence and the severity of outcome event, it has been realized that it is indispensable to prevent and cure the disease as soon as possible for the past years. Nevertheless the AS patients are common and the efficacious intervention about its treatment is still lacking. Therefore, stabilization of vulnerable plaques is of great clinical importance.Ectopic angiogenesis within the intima and media is considered to be a hallmark of advanced vulnerable atherosclerotic lesions. Neovessels within plaques are characterized by fragility and high perfusion, thus allowing for extravasation of lipoproteins and red blood cells that contribute to the formation of plaque lipids. This process results in intraplaque hemorrhage, increases the permeability of inflammatory cells, and leads to plaque destabilization. The number of neovessels increases with plaque progression, and such vessels are abundant in vulnerable plaques. Neovascularization is an crucial feature of AS plaques.Angiogenesis is induced by various growth-inducing and -inhibiting factors. Multiple complex signal transduction pathways are involved in intraplaque angiogenesis. Proteinases are required for degradation of the extracellular matrix (ECM), creating an avenue for migrating endothelial cells during angiogenesis. The specific MMPs necessary for endothelial cell migration and tube formation have attracted particular attention because they directly degrade ECM components. MMP-2 and -9 belong to gelatinases that can degrade basement membrane collagen, fibronectin and elastin and they also can degrade gelatin. The main component of the basement membrane is type IV collagen which has unique helical structure and most MMPs cannot degrade it. MMP-2 and MMP-9 have been shown to degrade type Ⅳ collagen and were known to be the "angiogenic switch" in tumor angiogenesis. Based on our studies part 1, we have observed the predominant effects of MMPs in intraplaque angiogenesis and the plaque instability in rabbits and have found that MMP-2 and MMP-9 can promote intraplaque angiogenesis and lead to AS plaque instability.Low density lipoprotein receptor-related protein (LRP) is a kind of cell surface protein which was discovered by Herz on 1988. It has been found that LRP is closely related to AS and is another risk factor of AS. In Heesang’s study, LRP1 promoted glioblastoma cell migration and invasion by regulating the expression and function of MMP-2 and-9 via an ERK-dependent signaling pathway. In another study, LRP1 regulated the expression and function of MMP2 and then promoted the SMCs migrating.Traditional Chinese medicines have the characteristic of more target points to stable AS plaque such as through protecting the vascular endothelial cell, downregulation of the lipid metabolism, and suppressing the inflammatory reaction. Moreover traditional Chinese medicines have potential therapy effect on preventing the development of AS plaque to vulnerable plaque and are appropriate for long-time use as a secondary treatment by reason of the moderate potency, less side-effect and the compatibility. Formerly, the research on traditional Chinese medicines about remedying AS aims directly at the simple cause or clinical manifestation, so the systemic research of mechanism is very requisite. Tongxinluo superfine is a typical Chinese drug that can remedy cardiovascular disease with the theory of collateral disorders and dredge collaterals herbs. Tongxinluo has effects of promoting blood flow. It has been used in CHD that show off imperfection of heart-energy and blood stasis. Many studies showed that Tongxinluo can prevent the plaque develop to instable plaque. The present study adopts the atorvastatin as positive control and aims to observe the therapeutic effects of Tongxinluo on intraplaque angiogenesis and plaque instability and elucidate the possible mechanism through the molecular biological and histological technology.Based on these findings, we proposed that Tongxinluo might have significant impacts on intraplaque angiogenesis; also, Tongxinluo might affect the expression of intraplaque angiogenesis though the expression of MMP-2 and -9 which were induced by LRP1.Objective1. To observe the effects of Tongxinluo on intraplaque angiogenesis and the plaque instability in rabbits;2. To elucidate the possible mechanism of Tongxinluo on inhibiting intraplaque angiogenesis and the plaque instability in rabbits.Methods1. Animal ProtocolAdult male New Zealand White rabbits 3 months old (n= 120) weighing 1.7 to 2.1 kg underwent balloon injury in the abdominal aorta and therewith were fed a high-cholesterol diet (1% cholesterol) for 12 weeks.2. Intervention MethodsSubsequently, the atherogenic diet was replaced by a regular diet for another 12 weeks. Then rabbits were randomly divided into 5 groups:Group A (control group):20 rabbits were fed with normal diet for 12 weeks after model established;Group B (Tongxinluo of low dose): 20 rabbits were fed with normal diet and Tongxinluo (0.15g/kg/d) for 12 weeks after model established;Group C (Tongxinluo of middle dose):20 rabbits were fed with normal diet and Tongxinluo (0.3g /kg/d) for 12 weeks after model established;Group D (Tongxinluo of high dose):20 rabbits were fed with normal diet and Tongxinluo (0.6g /kg/d) for 12 weeks after model established;Group E (atorvastatin: 20 rabbits were fed with normal diet and atorvastatin (5mg/kg/d) for 12 weeks after model established;Group F (combination group):20 rabbits were fed with normal diet and Tongxinluo (0.6g /kg/d), atorvastatin(5mg/kg/d) for 12 weeks after model established.3. Blood Biochemical AnalysisBlood was drawn from the auricular artery after fasting overnight to measure lipid profile before euthanasia. The serum levels of TG, TC, HDL-C, and LDL-C were measured by enzymatic assays. The body weights of all rabbits were measured at the end of 24th week.4.IVUS AssayIVUS was accomplished to examine the morphological changes of the aortic plaques. LA and EEMA were measured on abdominal aortic cross-sectional images. PA was calculated by EEMA- LA, and PB was calculated by PA/ EEMA × 100%.5. Histopathological Analysis:Histological analysis was assessed in perfusion/fixed tissues. The abdominal aorta (2 cm long) was fixed in 4% formaldehyde for 24 h, and 5-um-thick segments were then serially sectioned. Frozen sections were stained with Oil Red O staining, and paraffin sections underwent Sirius red, hematoxylin and eosin (H&E).6. Immunohistochemical StainingParaffin sections were stained with immunohistochemical staining. The expression of RAM-11, α-actin, LRP1, MMP -2, -9, and VEGF-A were determined. The morphologic parameters were measured using Image-Pro Plus 6.0, by two blinded observers.The thickness of fibrous cap, intima-media thickness and the thickness of fibrous cap / IMT ratio of each aortic plaque was measured. The vulnerability index (VI) was calculated as follows: (macrophage staining% + lipid staining%) / (smooth muscle cell% + collagen fiber %)7. Microvessels DensityFor quantify the microvessels density (MVD), sections were stained with CD31, and the microvessels were then quantified by the plaque area.8. Western Blot AnalysisProtein of abdominal aorta was extracted and separated on 10% to 15% SDS-PAGE gel and transferred onto nitrocellulose membranes. After blocking with 5% nonfat milk for 2 h at room temperature, the membranes were incubated with the primary antibodies overnight at 4℃. After being washed in TBS-T, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Signals were detected using an enhanced chemiluminescence kit. The protein levels were normalized to β-actin.9. Statistical AnalysisAll data are presented as mean ± standard deviation of the mean. Intergroup comparisons involved one-way ANOVA followed by the least-squares difference test (with equal variances assumed) or Dunnett’s T3 test (equal variances not assumed). Spearman’s rank correlation coefficient was used for correlation analysis. A two-tailed P value of <0.05 was considered statistically significant. All data analyses were performed using Predictive Analysis Software 18.0.Results1. General State of The Experimental AnimalsAll rabbits showed full recovery without complications after initial balloon injury. Five rabbits died of diarrhea or respiratory tract infection, and the other 112 rabbits finished the research: 19 in group A, 19 in group B, 18 in group C, 18 in group D, 19 in group E, and 19 in group F.2. Weight of the BodyThe body weights of the rabbits gradually increased over time and there were no difference among the groups after treatment (P>0.05).3. Blood Biochemical AnalysisCompared with group A, at the end of the research, all the lipid levels except the HDL-C in treatment groups reduced significantly especially in group E and F (P＜0.01), while the serum HDL-C level of drug groups increased. The level of TC, TG, and LDL-C in group E and F was lower than that in all Tongxinluo groups. And Tongxinluo lower TC, TG, and LDL-C level in a dose-dependent way.4. IVUS MeasurementsNo significant changes of LA in the abdominal aorta was found in treatment groups than that in control group (P>0.05). However, the levels of EEMA, PA, and PB in treatment groups were significantly lower than those without treatment (p<0.01).5. Histopathological AnalysisThe H&E staining showed that there were apparent plaque in all 6 groups and the plaque area in group A was significantly higher than that in all treatment groups.Oil red O staining showed the plaque content of lipid was higher in group A than other treatment groups.The positivity of Sirius red collagen staining showed the collagen content of plaque in group A was lower than that in group E, and F, with no discrepancy among group A, B, C, and D.6. Immunohistochemical ExaminationCompared with group A, the areas of α-actin-positive staining in the abdominal aortic SMCs was significantly higher in treatment groups. RAM-11 staining showed that the relative content of macrophages within plaques was lower in drug groups than that in control group. A statistically lower macrophage and lipid content and higher SMCs and collagen content were found in treatment groups than in control group, which led to a decreased vulnerability index in rabbits with drug treatment (P＜ 0.01).Similar to the expression of macrophage staining, the results of MMP-2, -9, LRP1 and VEGF-A immunohistochemical staining were less extensive in plaques of treatment groups than that in control group. The relative content of MMP-2,-9, LRP1 and VEGF-A in plaques were higher in group D, E, F than that in group B and C (P＜0.05), with no difference between group B and C (P>0.05).Compared with group A, the thickness of fibrous cap was significantly thicker in treatment groups (P＜0.01). The IMT of the abdominal aortic plaques decreased significantly after drugs treating compared with the control group (P＜0.01). As a result, the thickness of fibrous cap /IMT ratio was significantly higher in treatment groups than in group A (P＜0.01).7. Microvessels DensityCD31 staining showed that plaque neovessels was less in drug treatment groups than control group. The MVD was lower in treatment groups than group A (P＜0.05), but no difference was observed between group E and F (both P>0.05).8. Western Blot AnalysisThe expression of MMP-2, -9, and LRP1 proteins exhibited the same trends as shown in the immunohistochemical results. The expression of MMP-2,-9, LRP1 and p-ERK significantly decreased in group D, E, F than that in group B, C.9. Correlation AnalysisThe correlation analysis results showed that all correlations between the VI and LRP1, p-ERK, MMP-2, and -9 were positive (r = 0.568, 0.789, 0.834, and 0.638, respectively, all P＜0.01). The expression of LRP1, p-ERK, MMP-2, MMP-9 was positively correlated with both the MVD in plaque (r= 0.460, 0.469, 0.516, and 0.325, respectively, all P<0.01) and the VEGF-A in plaque (r= 0.492, 0.625,0.609, and 0.496, respectively, all P＜0.01). The correlation between the VI and MVD was positive (r= 0.424, P＜0.01).Conclusions1. Traditional Chinese medicine Tongxinluo can inhibit intraplaque angiogenesis, reduce the MVD and enhance the stability of plaque in the abdominal aortic of rabbits;2. Traditional Chinese medicine Tongxinluo could inhibit the expression of LRP1, regulate the expression and function of MMP2 and MMP9 via an ERK-dependent signaling pathway and finally enhance the stability of plaque in the abdominal aortic of rabbits. |
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