The Effect Of Different CD4+T Lymphocyte Subsets On The Vulnerability And Rupture Of Atherosclerotic Plaque And Its Mechanism

ObjectiveThe morbidity of acute coronary syndromes (ACS) has been rising recently along with the elevation of living standard and population aging in China. And the disease is precipitated by a rupture of the atherosclerotic plaque. The plaque prone to rupture can called instability plaque, whose character is a large necrosis core contained cholesterol clefts and thin fibrous cap infiltrated by less smooth muscle cells but numerous macrophages.foam cells and lymphocytes. Clinical study found the key factor of plaque unstable is inflammation. The plaques rupture and inflammation always coexist. The inflammation of unstable plaque always up-regulate in clinical. The role of CD4+T cells in the atherosclerotic plaque stability has aroused a great attention. CD4+T cell can be differentiated into different subsets after stimulated with antigen and different cytokines, including Th1cells, Th2cells, Th17cells and regulatory T cells (Treg). Nowadays, the function of different CD4+T cells on the stability of atherosclerotic plaque is not clear.To explore the relationship of different CD4+T lymphocyte subsets and the atherosclerotic plaque instability, we established an atherosclerotic plaque rupture model with ApoE-/-mice at first, then analyzed the changes of CD4+T lymphocyte subsets before and after plaque rupture. Based on the results, we investigated the relationship of Th17subsets/its effector molecule IL-17and atherosclerotic plaque instability. This study has important theoretic value and potential application value to clarify the immune mechanism of atherosclerotic plaque instability and plaques rupture, and to design the prevention projects of atherosclerotic plaque rupture. This project contains two parts, the first part is "The effect of different CD4+T lymphocyte subsets on the stability of atherosclerotic plaque", the second part is "The effect of Th17/IL17on the stability of atherosclerotic plaque and its mechanism"Part1The effect of different CD4+T lymphocyte subsets on the vulnerability and rupture of atherosclerotic plaqueMethod1. The establishment of atherosclerotic plaque rupture mice model.1) Animal Protocol. Male apoE-/-mice (n=75), were fed with high cholesterol diet from8weeks old, and collars were placed around the left common carotid arteries at10weeks old. The mice were randomly divided into five groups, including LPS group, Stress group, LPS+Stress group, LPS+Stress+Phenylephrine group and control group. At16weeks old (25) or24weeks old (50), the mice in the LPS, LPS+cold and LPS+cold+phenylephrine group were injected intraperitoneally with lmg/kg/day of lipopolysaccharide for two days. The mice in the cold, LPS+cold and LPS+cold+phenylephrine stimulated by cold in ice-water for5-10minutes for two days and the LPS+cold+phenylephrine group were injected8μg/kg of phenylephrine in a volume of0.2ml of phosphate-buffered saline (PBS) at the third day for further triggering the disruption of plaque. The mice in the control group were injected intraperitoneally with PBS without cold stimulation and phenylephrine.24hours after last challenge, status of plague was analyzed by histopathology.2) Micro-ultrasound Measurement3) Histological and Morphology Analyses. Separated the left carotid arteries and prepared continuous paraffin or frozen sections. Sections were stained with hematoxylin and eosin (H&E), Masson"s trichrome, Oil red O staining. Smooth muscle cells (SMCs) and macrophages were detected by immunostaining with anti-a-actin and MOMA-2antibody. Plaque area, vessel area and cap thickness were measured. Smooth muscle cell, macrophage, lipid and collagen positive areas were quantified and the ratios correlated to the intimal areas were calculated. Plaque rupture rate and vulnerable index were accounted.2. The change law of Th17, Thl and Treg cells before and after the atherosclerotic plaque rupture1) To prepare the splenocyte suspensions liquid from atherosclerotic stable plaque and atherosclerotic plaque rupture mice, then labeled with different antibody.2) To detect the percentage of Th17, Thl and Treg cells in spleen of mice model with FCM.3. The expression of transcript factors in the plaque.To prepare artery from stable plaque and rupture plaque, and detect the expression of T-bet and RORyt with RT-PCR4. The change of inflammatory factor (IL-17)The serum of ApoE-/-mice before and after the atherosclerotic plaque rupture detected by ELISAResult1. The establishment of atherosclerotic plaque rupture mice model.To research the role of CD4+T cells on atherosclerotic unstable plaque, we established a new atherosclerotic plaque rupture mice model.1) The establishment of atherosclerotic vulnerability plaqueThe size of plaque and necrotic core in24-week ApoE--mice was significantly larger than that at16-week mice (size of plaque:90625±4125μm2v.s.68750±6250μm2P<0.01; necrotic core:52340±14300μm2v.s.29883±3890μm2, P<0.05). In contrast to this, fibrous cap area and the ratio of necrotic core to fibrous cap in24-week ApoE-/-mice were markedly smaller than that16weeks (fibrous cap area:8690±1635μm2v.s.13850±4900μm2, P<0.05; ratio of necrotic core to fibrous cap:0.47±0.17v.s.0.12±0.034, P<0.01). Plaques rupture was confirmed by Micro-ultrasound Measurement. These data suggest that plaques of24-week ApoE-/-mice possess features of unstable plaque, prone to rupture. 2) The establishment of atherosclerotic plaque rupturesNo mice were dead in test. Body weight in LPS+cold and LPS+cold+phenylephrine groups decreased significantly compared with control group. There were no significant differences in the blood lipid between groups.The plaque disruption rate in the LPS+cold+phenylephrine group at24weeks old was significantly higher than that in other groups (70%,7/10). Among the5cases of ruptured plaques with luminal thrombus,12cases showed cap disrupture with intraplaque hemorrhages, and1case of ruptured plaques with Core extrusion. Vulnerable index from LPS+cold+phenylephrine group at16and24weeks old were1.67±0.19and3.05±0.25respectively, significantly greater than other groups. The results show the animal model which stimulated by LPS, cold and phenylephrine co-treatment could trigger high frequency of plaque rupture and thrombus, intraplaque hemorrhage are common in this kind of model.We first established an atherosclerotic plaque rupture mice model. The animal model was generated by perivascular constrictive collar placement around the carotid artery of high-fat diet apoE-/-mouse, followed by LPS, cold and phenylephrine co-treatment. It is high frequency and short time to trigger plaque rupture.2. The change of different Th subsets before and after the atherosclerotic plaque rupture1) The change of regulatory T cells before and after the atherosclerotic plaque ruptureTo research the change law of regulatory T cells before and after the atherosclerotic plaque rupture, we detect the percentage of LPS+cold+phenylephrine group and control group at16and24weeks old mice splenocytes with the flow cytometry. The results show that CD4+CD25+Foxp3+T cells are lower than control in mice at16weeks old. This suggests CD4+CD25+Foxp3+T cells can promote the stability of atherosclerotic plaque. And CD4+CD25+Foxp3+T cells are higher than control in mice at24weeks old. We suggest this is a protecting compensation when atherosclerotic plaques rupture. Furthermore, CD4+CD25-Foxp3+T cells significantly increase in mice at16and24weeks old that show CD4+CD25-Foxp3+T cells could reduce the stability of atherosclerotic plaque and promote the rupture of atherosclerotic plaque.2) The change of Thl before and after the atherosclerotic plaque ruptureTo research the change law of Th1before and after the atherosclerotic plaque rupture, we detect the percentage of LPS+cold+phenylephrine group and control group at16and24weeks old mice splenocytes with the flow cytometry. The percentage of Th1cells has no obviously change in mice at16weeks old, and significantly increases in mice at24weeks old. The result show Th1plays an important role in the rupture of atherosclerotic plaque.3) The change of Th17before and after the atherosclerotic plaque ruptureTo research the change law of Th17before and after the atherosclerotic plaque rupture, we detect the percentage of LPS+cold+phenylephrine group and control group at16and24weeks old mice splenocytes with the flow cytometry. The results show Th17significantly increase in mice at16and24weeks old, and correlated positively with the instability of atherosclerotic plaque. The result show Th17was closely related to the instability of atherosclerotic plaque.4) The change of IL-17+IFN-γ+CD4+T before and after the atherosclerotic plaque ruptureExcept Th1and Th17. we found another CD4+T cells subset, CD4+IL-17+FN-γ+T cells. CD4+IL-17+IFN-γ+T cells significantly increase in mice at16and24weeks old that show these cells could reduce the stability of atherosclerotic plaque and promote the rupture of atherosclerotic plaque.5) The ratio of Th1+Th17/Treg, Th1/Treg, Th17/Treg before and after the atherosclerotic plaque ruptureTo research the ratio of Th1+Th17/Treg,Th1/Treg, Th17/Treg before and after the atherosclerotic plaque rupture, statistically, we found Th1/Treg have no obviously change in mice at16and24weeks old, butTh1+Th17/Treg and Th17/Treg significantly increase in mice at16and24weeks old. The results suggest the imbalance of Th17/Treg play an important role in instability of atherosclerotic plaque.6) The change of IL-17before and after the atherosclerotic plaque rupture Furthermore, we found Th17high subset, which decreased after plaque rupture. We suggest these subsets to release IL-17. To demonstrate this suggestion, we measured Serum IL-17level by ELISA, and IL-17are higher than control group after plaque rupture (P=0.0004). The results suggest Th17play an important role in instability of atherosclerotic plaque.7) The expression of transcript factors in the plaqueTo determine the existence of Th1and Th17in the atherosclerotic plaque, we extract RNA from aortic arch of stable plaque and rupture plaque, and then we detect the expression of ROR t and T-bet in plaque with RT-PCR. The result shows that the expression of transcript factors in rupture plaque increase obviously than instable plaque. This proves that Th1and Th17both participate in the rupture of atherosclerotic plaque.Part2The role of IL17on the vulnerability and rupture of atherosclerotic plaque and its mechanismMethod1. The effect of IL-17on the vulnerability of atherosclerotic plaque1) Male apoE-/-mice (n=30), were fed with high cholesterol diet from8weeks old, and collars were placed around the left common carotid arteries at10weeks old. The mice were injected intraperitoneally with IL-17(lug/mice) for2weeks,4weeks or6weeks at8weeks after surgery. The mice in the control group were injected intraperitoneally with PBS.2) Separated the left carotid arteries and prepared continuous paraffin or frozen sections. Sections were stained with hematoxylin and eosin (H&E), Oil red O staining. Smooth muscle cells (SMCs) and macrophages were detected by immunostaining with anti-a-actin and MOMA-2antibody. Plaque area, vessel area and cap thickness were measured. Smooth muscle cell, macrophage, lipid and collagen positive areas were quantified and the ratios correlated to the intimal areas were calculated. Vulnerable index were accounted.2. The change law of Th17, Th1and Treg cells before and after intervened by IL-171) Male apoE-/-mice (n=30), were fed with high cholesterol diet from8weeks old, and collars were placed around the left common carotid arteries at10weeks old. The mice were injected intraperitoneally with IL-17(lug/mice) for2weeks,4weeks or6weeks at8weeks after surgery. The mice in the control group were injected intraperitoneally with PBS.2) To prepare the splenocyte suspensions liquid from different groups mice, then labeled with different antibody.3) To detect the percentage of Th17, Thl and Treg cells in spleen of mice model with FCM3. The apoptosis of Movas treated by IL-17The murine vascular smooth muscle cell Movas were cultured in vitro, were treated by IL-17with different concentration(25ug/ml,50ug/ml,100ug/ml) and different controlling time (24、48、72hours)1) The rate of apoptosis after treated by IL-17was tested by Hochest2) The rate of apoptosis after treated by IL-17was tested by FCM3) The apoptosis related proteins of cleaved caspase-3was tested by Western blotResult1. The effect of exogenous IL-17on the vulnerability of atherosclerotic plaqueTo confirm the role of JL-17on the atherosclerotic plaque, Plaque area, vessel area, cap thickness, necrotic core and vulnerable index were compared with control group. Plaque area, vessel area, cap thickness, necrotic core has no obviously change in IL-17treated-2weeks group, but vulnerable index was decreased (0.34±0.07v.s.0.55±0.14, P<0.05). The ratio of necrotic core to fibrous cap was decreased (0.34±0.07v.s.0.55±0.14, P<0.05) and vulnerable index was0.84+0.04v.s.1.53±0.12in IL-17treated-4weeks group and control group. The necrotic core was larger than control group and the ratio of necrotic core to fibrous cap was decreased (0.21±0.05v.s.0.67±0.11, P<0.01) and vulnerable index was1.02±0.03v.s.1.7810.51in IL-17treated-6weeks group and control group. The result shows IL-17promote the vulnerability of atherosclerotic plaque.2. The effect of exogenous IL-17on the atherosclerotic plaque ruptureIL-17treated-2weeks group have no plaque rupture; IL-17treated-4weeks group have2mice with buried fibrous cap; and IL-17treated-6weeks group have1mice with intra-plaque Hemorrhage, and3mice with buried fibrous cap. The result show IL-17could promote the vulnerability of atherosclerotic plaque, and induce plaque rupture.3. The change of different Th subsets after intervened by IL-17in different time.To confirm the relationship of Th17and the instability of atherosclerotic plaque, the mice were injected intraperitoneally with IL-17(lug/mice) for2weeks,4weeks or6weeks at18weeks old.1) The change of regulatory T cells after intervened by IL-17To research the change law of regulatory T cells before and after intervened by IL-17, we detect the percentage of IL-17treated-2weeks,-4weeks or-6weeks groups and control group mice splenocytes with the flow cytometry. The results show that CD4+CD25+Foxp3+T cells are all lower than control group, and CD4+CD25-Foxp3+T are all higher than control group. The result shows IL-17promote the vulnerability of atherosclerotic plaque.2) The change of Thl after intervened by IL-17To research the change law of Thl before and after intervened by IL-17, we detect the percentage of IL-17treated-2weeks,-4weeks or-6weeks groups and control group mice splenocytes with the flow cytometry. The results show Thl has no obviously change in all groups.3) The change of Th17after intervened by IL-17To research the change law of Th17before and after intervened by IL-17, we detect the percentage of IL-17treated-2weeks,-4weeks or-6weeks groups and control group mice splenocytes with the flow cytometry. The results show that Th-17were all higher than control group. The result shows IL-17promote the vulnerability of atherosclerotic plaque.4) The ratio of Th1+Th17/Treg, Th1/Treg,Th17/Treg after intervened by IL-17To research the ratio of Th1+Th17/Treg, Th1/Treg, Th17/Treg after intervened by IL-17, statistically, we found Th1/Treg, Th17/Treg significantly increase in mice at16and24weeks old. The results suggest IL-17could change the ratio of CD4+T cells subset, induced the imbalance of Th1/Treg, Th17/Treg.4. Exogenous IL-17treatments in vitro can increase the apoptosis of the murine vascular smooth muscle cell.To confirm the mechanism of IL-17to the instability of atherosclerotic plaque, we cultured the murine vascular smooth muscle cell Movas. After treated by IL-17with different concentration(25ug/ml,50ug/ml,100ug/ml) and different controlling time (24、48、72hours), The apoptosis of the cells were significantly increased. Apoptotic bodies were observed by Hochest. The apoptosis was significantly increased by FCM, and the apoptosis related proteins were significantly increased. The different concentrations and controlling time have significant dose and time dependent stimulatory effect. The results show the promote role of IL-17on atherosclerotic plaque vulnerability is through promoting the apoptosis of smooth muscle cell.Conclusion1. We established an atherosclerotic plaque rupture mice model. The animal model was generated by perivascular constrictive collar placement around the carotid artery of high-fat diet apoE-/-mouse, followed by LPS, cold and phenylephrine co-treatment. It is high frequency and short time to trigger plaque rupture, and with thrombus, intraplaque hemorrhage is common in this kind of model.2. Varieties of CD4+T lymphocyte subsets are participation in the induction of atherosclerotic plaque rupture. Different CD4+T lymphocyte subsets has obviously changes before and after the atherosclerotic plaque rupture. CD4+CD25+Foxp3+T cells and CD4+CD25-Foxp3+T cells significantly increase than control group, and Th1, Th17and CD4+IL-17+IFN-γ+T cells significantly increase than control group,and the imbalance of Treg/Th17was found.3. Th17plays an important role on the vulnerability of atherosclerotic plaque. The stability of atherosclerotic plaque was decreased by treated with IL-17which the effective molecule that Th17secrets. This confirms Th17/IL-17can promote the instability of atherosclerotic plaque.4. IL-17could increase the apoptosis of Movas which suggested Th17/IL-17could promote the vulnerability of atherosclerotic plaque by increased the apoptosis of smooth muscle cells.5. IL-17could upregulate Th17and downregulate Treg, and then can change the balance of Th17/Treg. The imbalance of Th17/Treg may be the one of the mechanism of atherosclerotic plaque rupture.Originality1. We established an atherosclerotic plaque rupture mice model which is high frequency and short time to trigger plaque rupture, and with thrombus, intraplaque hemorrhage is common in this kind of model.2. We found the change law of different T cell subsets before and after the atherosclerotic plaque rupture and demonstrated Th17significantly increase when atherosclerotic plaque ruptures. Then, we confirm, for the first time, Th17/IL-17could promote the vulnerability of atherosclerotic plaque. Provides a new perspective for further studies on the mechanism of atherosclerosis plaque rupture, and provides first-hand data to clinical therapy on Th17/IL-17.3. We found IL-17can increase the apoptosis of smooth muscle cells, and the imbalance of Th17/Treg which defined the mechanism of vulnerable atherosclerotic plaque and plaque rupture by Th17/IL-17.Limitations of this study1. The molecular mechanism of Th17in atherosclerotic plaque vulnerability and plaque rupture need to be further studied.2. It is still not satisfying with the present animal model. There are several limitations for further studies.