Experimental Studies On The Relationship Of TRIB3 And Diabetic Atherosclerosis

BackgroundDiabetes mellitus has been recognized as an equivalence of coronary heart disease. However, the mechanism of diabetes-accelerated CHD remains to be clarified. Therefore, it is important to choose an appropriate diabetic animal model for each type of diabetes when doing diabetic atherosclerosis research. In the present, diabetic atherosclerosis animal models were established through gene knockout combined with high fat diet diabetes and long-term high fat diet. The limitations of these models include four major parts. The first one is that mouse models cannot completely mimic human disease, so the extrapolation of relationships seen with human disease to mice is not straightforward. The second one is that the effect of metabolism-associated gene on atherosclerosis cannot be excluded in knockout mice which could not be intervened. The third one is that long-term high fat diet induced diabetic models take a long time, which makes it difficult to distinguish the effect of aging from diabetes mellitus on the development of atherosclerosis. The last one is that long-term high fat diet induced diabetic models precede hyperglycemia with dyslipidemia. None of the currently used models is a perfect diabetic atherosclerosis mouse model. Thus, the present study focuses on the establishment of diabetic atherosclerosis ApoE-/-/LDLR-/-mouse models by high-fat and high-sugar diet combined with a small dose of STZ. Then monitoring weight, blood pressure, blood glucose and glucose tolerance, detecting the initiation and development of atherosclerosis by ultrasound biomicroscopy(UBM) were performed to confirm the feasibility of establishing diabetic atherosclerosis model that closely resemble human diabetic atherosclerosis disease.Objectives1. To confirm the feasibility of establishing diabetic ApoE-/-/LDLR-/- mouse model by high-fat and high-sugar diet combined with a small dose of STZ;2. To confirm the feasibility of establishing diabetic atherosclerosis ApoE-/-/LDLR-/- mouse model by high-fat and high-sugar diet combined with a small dose of STZ.Methods1. Animal experiment:Thirty 3-week-old male ApoE-/-/LDLR-/- mice were randomized into 2 groups after intraperitoneal glucose tolerance test (IPGTT): chow(n=15) and diabetes(n=15) groups. Diabetes group were fed on high fat and sugar diet (20% fat,20% sugar and 1.25%cholesterol) for 6 weeks. IPGTT was performed to confirm the appearance of insulin resistance. Those insulin resistant mice were injected once with low dose of STZ (intraperitoneal at 75mg/kg). After 2 weeks, IPGTT was performed. Those with random blood glucose more than 11.1 mmol/l were regarded as diabetic mouse model.2. IPGTT test:Mice were fasted for 10-16h before glucose tolerance tests. Intraperitoneal injection glucose load was administered at 1.5 g/kg of body weight. Glucose levels were measured from tail bleeds with a glucometer at specified time points after glucose administration.3. Monitor body weight of ApoE-/-/LDLR-/- mice every week.4. Assessment of hemodynamics of ApoE-/-/LDLR-/- mice:All ApoE-/-/LDLR-/-mice underwent hemodynamic examination at weeks 20,22 and 24, including blood pressure and heart rate.5. Evaluation of atherosclerosis of ApoE-/-/LDLR-/- mice by UBM:All ApoE-/-/LDLR-/- mice underwent echocardiography at weeks 14,16,18,20,22 and 24 to monitor the initiation and development of atherosclerotic plaques in aortic, brachiocephalic and carotid arteries by measuring IMT.6. Serum parameters of ApoE-/-/LDLR-/- mice:At the end of experiment, blood glucose and serum insulin were measured to calculate the HOMA-IR. Results1. Comparison of body weight of ApoE-/-/LDLR-/- mice:At the age of 3 weeks, no significant difference was detected between the two groups. After high fat diet for 6 weeks, diabetic mice show significant increased body weight (P=0.010). However, no significant difference was detected between the two groups at two weeks after administration of STZ (P=0.081). At week 20, diabetic mice show significant increased body weight (P=0.0015). Then the body weight of mice kept stable. At week 24, diabetic mice still show significant increased body weight (P=0.0073).2. Hemodynamics alterations of ApoE-/-/LDLR-/- mice:Compared to the chow-diet mice, no significant difference was detected in the heart rate of diabetic mice (P=0.309). However, the heart rate went down with the age of mice increased (P=0.014). Compared to the chow-diet mice, diabetic mice show an increased systolic blood pressure (P=0.00004), while the age of mice had no effect on systolic blood pressure (P=0.212). No significant interactions were detected between the age and diabetes (P=0.390). Compared to the chow-diet mice, diabetic mice show an increased diastolic blood pressure (P=0.000003), while the age of mice had no effect on diastolic blood pressure (P=0.137). No significant interactions were detected between the age and diabetes (P=0.652). Compared to the chow-diet mice, diabetic mice show an increased mean arterial blood pressure (P=0.0001), while the age of mice had no effect on mean arterial blood pressure (P=0.212). No significant interactions were detected between the age and diabetes (P=0.263).3. Results of IPGTT of ApoE-/-/LDLR-/- mice:At the age of 3 weeks, no significant difference was detected between the two groups. After high fat diet for 6 weeks, diabetic mice showed significant increased blood glucose (P<0.05 for all). After administration of STZ, diabetic mice showed significant higher blood glucose than chow-diet mice (P<0.05 for all). No significant differences were detected among the chow-diet mice with different ages. Significant differences were detected among the diabetic mice with different ages.4. Parameters of glucose metabolism of ApoE-/-/LDLR-/- mice:At the age of 24 weeks, compared to the chow-diet mice, diabetic mice showed significantly increased blood glucose, serum insulin, and HOMA-IR (P=0.028, P=0.025, P=0.015, respectively), indicating that diabetic mice have hyperglycemia, hyperinsulinemia and insulin resistance.5. Monitoring IMT of ApoE-/-/LDLR-/- mice:Brachiocephalic IMT of ApoE-/-/LDLR-/- mice got thickened earlier than carotid IMT. At the age of 16 weeks, significant differences of brachiocephalic IMT were detected between the two groups (P=0.000), which will last till week 24. As for carotid IMT, it kept increased slowly. At week 22, significant differences of carotid IMT were detected between the two groups (P=0.022). At week 24, compared with the chow-diet mice, diabetic mice had significant thickened carotid IMT (P=0.001).Conclusions1. Male ApoE-/-/LDLR-/- mice induced by high fat and sugar diet combined with a small dose of STZ at the age of 3weeks would appear the stable state of diabetes mellitus.2. Diabetes mellitus induced by high fat and sugar diet combined with a small dose of STZ in male ApoE-/-/LDLR-/- mice would resemble human diabetes mellitus.3. Type 2 diabetic ApoE-/-/LDLR-/- mice would be attacked by atherosclerotic plaque. Furthermore, the atherosclerotic plaque formed ahead of time.4. Type 2 diabetic ApoE-/-/LDLR-/- mouse atherosclerosis model was appropriate for intervention. BackgroundAlthough diabetes mellitus is the most risk factors for atherosclerosis and CHD, the mechanism of more serious and vulnerable plaques in the diabetic patients remains to be understood. This has highlighted the importance and urgency of studying the mechanism of diabetic atherosclerosis and exploring therapeutic options.It is critical to find the crossroad of diabetes mellitus and acute coronary diseases to effectively deal with acute coronary syndromes-the most important cardiovascular complication. Up to date, studies on the mechanism of vulnerable plaque have showed that innate characteristics and the stress that the plaque bears with determine the vulnerability of the atherosclerotic plaque. It has been demonstrated that inflammation, oxidative stress, impaired glucose metabolism, dyslipidemia and dyshemodynamics would make the atherosclerotic plaque vulnerable. All these risk factors would cause more macrophages apoptosis which would make plaque vulnerable. However, the signal transduction involved in the macrophages apoptosis is a network, among which Akt signal transduction might play an essential role. TRIB3 has been confirmed to inhibit PI3K/Akt stimulated by insulin, causing insulin resistance. Furthermore, TRIB3, as a scaffold protein, was involved in the lipid metabolism. Epidemiological studies showed that TRIB3 Q84R polymorphism was associated with insulin resistance and the increase of cardiovascular diseases, especially atherosclerosis. However, whether TRIB3/Akt signal transduction was involved in diabetes cardiovascular complications remained to be clarified. In the present study, TRIB3-shRNA was transfected into type 2 diabetic ApoE-/-/LDLR-/- mice, silencing TRIB3, to investigate whether TRIB3 silence could stabilize the plaque and the underlying mechanism.Objectives1. To design and synthesize siRNA against mouse TRIB3 according to RNAi principle, then to construct the pAdxsi-TRIB3-shRNA;2. Transfection of pAdxsi-TRIB3-shRNA into type 2 diabetic ApoE-/-/LDLR-/ mice to investigate the vulnerability of atherosclerotic plaque;3. To explore the mechanism by which silence of TRIB3 would stabilize atherosclerotic plaque.Methods1. To design and synthesize 4 pieces of siRNA against mouse TRIB3 according to RNAi principle, then construct pGenesil-1.2-TRIB3-shRNA plasmid using the most effective siRNA. Subsequently, pShuttle-Basic-EGFP-TRIB3-shRNA plasmid would be synthesized. With the shuttle plasmid, the pAdxsi-TRIB3-shRNA was constructed.2. Animal experiment:Sixty 3-week-old male ApoE-/-/LDLR-/- mice were randomized into 2 groups after intraperitoneal glucose tolerance test (IPGTT): chow-diet (n=30) and diabetes (n=30) groups. Diabetes group were fed on high fat and sugar diet (20% fat,20% sugar and 1.25%cholesterol) for 6 weeks. IPGTT was performed to confirm the appearance of insulin resistance. Those insulin resistant mice were injected once with low dose of STZ(intraperitoneal at 75mg/kg). After 2 weeks, IPGTT was performed. Those with random blood glucose more than 11.1 mmol/l were regarded as diabetic mouse model.3. Transfection of pAdxsi-TRIB3-shRNA into type 2 diabetic ApoE-/-/LDLR-/ mice. At the age of 20 weeks, after echo examination, chow-diet with RNAi group (n=15) and diabetes with RNAi group (n=15) was injected by pAdxsi-TRIB3-shRNA through tail vein, while chow-diet group (n=15) and diabetes group (n=15) was injected by pAdxsi through tail vein. After 2 weeks, echo examination and transfection were performed again. At the age of 24 weeks, echo examination was performed before the animals were sacrificed.4. Serum parameters of ApoE-/-/LDLR-/- mice:At the end of experiment, blood glucose and serum insulin were measured to calculate the HOMA-IR. Furthermore, serum total cholesterol, triglycerides, HDL-C and LDL-C were also measured.5. Assessment of peritoneal macrophages'function:Isolate peritoneal macrophage, identified by MOMA-2, then migration, adhesion and phagocytosis were assessed.6. Pathology analyses:HE staining, Masson trichrome stain, oil red O, Sirius red, and Perl's staining were preformed on frozen sections from brachiocephalic plaque. Immunochemistry assay was performed to detect the macrophages, smooth muscle and TRIB3 in the brachiocephalic plaque. Plauqe area, fibrotic cap, collagen, lipid were measured to calculate cap/core ratio and vulnerability index.7. Real-time RT-PCR:The mRNA of TRIB3 was quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green method.8. Western bot:The proteins of TRIB3/Akt/Caspase-3 signal transduction were detected by western blotting.9. TUNEL:Apoptotic cells and apoptotic macrophages were evaluated in frozen sections by TUNEL.Results1. Construction of pAdxsi-TRIB3-shRNA:After transfection of TRIB3 siRNA for 24h, the mRNA levels of GAPDH and TRIB3 were quantified by real-time RT-PCR using SYBR Green method. Based upon the inhibition rate of TRIB3, one piece was selected, which could silence 99.98% TRIB3. The chosen sequence was used to construct pGenesil-1.2-TRIB3-shRNA plasmid. Then pShuttle-Basic-EGFP-TRIB3-shRNA plasmid would be synthesized. Subsequently, with the shuttle plasmid, the pAdxsi-TRIB3-shRNA was constructed.2. Alterations of ApoE-/-/LDLR-/- mice after transfection of pAdxsi-TRIB3-shRNA (1) Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/ mice showed significant increased body weight(P=0.001). Compared to the chow-diet ApoE-/-/LDLR-/- mice, chow-diet with RNAi group showed a decreased body weight (P=0.034); Compared to the diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi group showed a significantly decreased body weight (P=0.003). Therefore, diabetes could increase the body weight (P=0.001), while RNAi had no effect on body weight (P=0.112). No significant interactions were detected between the RNAi and diabetes (P=0.481).(2) Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/ mice showed significant increased systolic blood pressure(SBP), diastolic blood pressure (DBP) and mean arterial blood pressure (MABP) (P<0.001 for all). Compared to the chow-diet ApoE-/-/LDLR-/-mice, chow-diet with RNAi group showed decreased SBP, DBP and MABP (P<0.001 for all); Compared to the diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi group showed significantly decreased SBP, DBP and MABP (P<0.001 for all). Therefore, diabetes could increase SBP, DBP and MABP (P<0.001 for all), while RNAi could reduce SBP, DBP and MABP (P<0.05 for all). No significant interactions were detected between the RNAi and diabetes (P>0.05 for all).3. Serum parameters of ApoE-/-/LDLR-/- mice:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased blood glucose, serum insulin, HOMA-IR, serum TC, serum LDL-C and serum HDL-C (P<0.05 for all), with serum TG unchanged (P=0.768). Compared to the diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/-mice showed significantly decreased blood glucose and HOMA-IR (P<0.05 for both), but serum insulin, serum TC, serum TG, serum LDL-C and serum HDL-C remained unchanged.4. Comparison of peritoneal macrophages' function:Compared to the chow-diet ApoE-/-/LDLR-/- mice, peritoneal macrophages from diabetic ApoE-/-/LDLR-/-mice showed significantly increased migration, adhesion and phagocytosis functions. Compared to diabetic ApoE-/-/LDLR-/- mice, peritoneal macrophages from diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased migration function, enchanced phagocytosis function but unaltered adhesion function.5. UBM measurements of brachiocephalic artery:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed no significant differences in brachiocephalic IMT, diameters and blood velocity. Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- showed no decrease in brachiocephalic IMT, diameters and blood velocity (P>0.05 for all).6. Pathological analyses(1) Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/-mice showed significantly increased aortic atherosclerotic burden (17.00±3.12% vs 30.99±3.12%, P=0.000); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased aortic atherosclerotic burden (30.99±3.12% vs 22.54±2.13%, P=0.000).(2) Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/-mice showed significantly increased brachiocephalic plaque area, lipid content in plaque, and macrophages in plaque (P<0.05 for all); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed no significant differences in brachiocephalic plaque area, lipid content in plaque, and macrophages in plaque (P>0.05 for all).When compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/-mice showed significantly decreased thickness of fibrotic cap, cap/core ratio, and collagen content in brachiocephalic plaque (P<0.05 for all); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly increased thickness of fibrotic cap, cap/core ratio, and collagen content in brachiocephalic plaque (P<0.05 for all).Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed no significant differences in I/III collagen ratio and SMC content of brachiocephalic plaque (P>0.05 for both); Compared to diabetic ApoE-/-/LDLR-/-mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly increasedⅠ/Ⅲcollagen ratio and SMC content of brachiocephalic plaque (P<0.05 for both).When compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased vulnerability index of brachiocephalic plaque (1.75±0.45 vs 2.97±0.57, P=0.00001); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased vulnerability index of brachiocephalic plaque (2.97±0.57 vs 2.32±0.59,P=0.008).7. TUNEL analysis:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased apoptosis in brachiocephalic plaque (85.63±38.46 vs 201.00±61.01, P=0.00003); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased apoptosis in brachiocephalic plaque (201.00±61.01 vs 95.86±39.80, P=0.0001).Furthermore, compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased macrophages'apoptosis in brachiocephalic plaque (48.31±28.39 vs 108.80±33.44, P=0.0001); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased macrophages'apoptosis in brachiocephalic plaque (108.80±33.44 vs 46.20±15.21, P=0.00006).8. Real-time RT-PCR:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased TRIB3 mRNA in atherosclerotic plaque (1.69±0.76 vs 4.97±1.30, P=1×10-7); Compared to diabetic ApoE-/-/LDLR-/ mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased TRIB3 mRNA in atherosclerotic plaque (4.97±1.30 vs 1.59±0.52, P=1×10-7).9. Western bot:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased TRIB3 and caspase-3, but decreased phosphorylated Akt in atherosclerotic plaque; Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased TRIB3 and caspase-3, but increased phosphorylated Akt in atherosclerotic plaque.Conclusions1. Diabetic ApoE-/-/LDLR-/- mice showed significantly increased aortic atherosclerotic burden, which would be reversed by silencing TRIB3. 2. Brachiocephalic plaques of diabetic ApoE-/-/LDLR-/- mice were confirmed to be vulnerable, manifesting decreased fibrotic cap, reduced cap/core ratio, increased lipid in the plaque, decreased collagen content, incremental macrophages, increased apoptosis of macrophages, augmented vulnerability index and enhanced hemorrhage in the plaque.3. Silencing of TRIB3 would stabilize plaque by augmenting the thickness of fibrotic cap, increasing cap/core ratio, increasing collagen and smooth muscle contents, reducing apoptotic cells and decreasing vulnerability index.4. Silencing of TRIB3 would effectively increase the activity of Akt, improve insulin resistance, reduce apoptosis of macrophages, thereby protecting against the enchancement of lipid core, to stabilize plaques.5. Peritoneal macrophages from diabetic ApoE-/-/LDLR-/- mice showed significantly increased migration. In advanced atherosclerosis, silencing of TRIB3 would in part decrease migration of peritoneal macrophages.6. Peritoneal macrophages from diabetic ApoE-/-/LDLR-/- mice showed significantly increased adhesion. In advanced atherosclerosis, silencing of TRIB3 would enhance adhesion, reduce migration of peritoneal macrophages, thereby increasing local clearance. Diabetes mellitus and silencing of TRIB3 would enhance adhesion respectively.7. Peritoneal macrophages from diabetic ApoE-/-/LDLR-/- mice showed significantly increased phagocytosis of lipid, causing plaque size increased rapidly. In advanced atherosclerosis, silencing of TRIB3 would enhance phagocytosis of lipid, maintaining the ability of clearance, to avoid augmented lipid core due to apoptosis and necrosis. Diabetes mellitus and silencing of TRIB3 would enhance phagocytosis respectively.8. Silencing of TRIB3 would effectively improve insulin resistance and glucose metabolism, elevate HDL and lower blood pressure, therefore reducing risk factors for atherosclerosis.9. High resolution ultrasonic biomicroscopy could noninvasively monitor the initiation, development and prognosis of brachiocephalic plaque, highly identical with pathological analyses. BackgroundEarly identification of atherosclerosis and monitoring its development were essential for prevention of acute cardiovascular events in diabetic patients. Early identification to protect against atherosclerosis is an important method to prevent acute cardiovascular events, especially for vulnerable plaques. It has been demonstrated that spontaneous ruptures of atherosclerotic plaques mostly take place in brachiocephalic artery, which would be perfect for vulnerability of atherosclerotic plaques in type 2 diabetes mellitus. However, brachiocephalic artery was too deep to be detected. Therefore, the analyses nearly depend on the pathological analyses. Although pathological analyses could judge the development of atherosclerosis, it could not reflect the development of atherosclerosis in vivo. Furthermore, dynamic observations could not be realized, which make it difficult to choose the optimal opportunity and evaluate the effectiveness of interventions. Moreover, pathological analyses were restricted to the single sections without the integrity of atherosclerosis. Here, high resolution ultrasonic biomicroscopy UBM vevo770 was used to monitor the aortic, brachiocephalic and carotid atherosclerosis, to noninvasively evaluate diabetic atherosclerosis.Objectives1. To noninvasively evaluate diabetic atherosclerosis by monitoring atherosclerosis in diabetic animal model using UBM vevo770; 2. To compare the atherosclerosis in different arteries of diabetic ApoE-/-/LDLR-/- mice by UBM vevo770;3. To evaluate the application of UBM vevo770 on studying atherosclerosis in diabetic ApoE-/-/LDLR-/- miceMethods1. Animal experiment:Thirty 3-week-old male ApoE-/-/LDLR-/- mice were randomized into 2 groups after intraperitoneal glucose tolerance test (IPGTT): chow(n=15) and diabetes(n=15) groups. Diabetes group were fed on high fat and sugar diet (20% fat,20% sugar and 1.25%cholesterol) for 6 weeks. IPGTT was performed to confirm the appearance of insulin resistance. Those insulin resistant mice were injected once with low dose of STZ(intraperitoneal at 75mg/kg). After 2 weeks, IPGTT was performed. Those with random blood glucose more than 11.1 mmol/l were regarded as diabetic mouse model.2. Evaluation of atherosclerosis of ApoE-/-/LDLR-/- mice by UBM:All ApoE-/-/LDLR-/- mice underwent echocardiography at weeks 14,16,18,20,22 and 24 to monitor the initiation and development of atherosclerotic plaques in aortic, brachiocephalic and carotid arteries by measuring IMT, diameters and blood velocity.Results1. Comparison of body weight of ApoE-/-/LDLR-/- mice:At week 20, diabetic mice show significant increased body weight (P=0.0015). Then the body weight of mice kept stable. At week 24, diabetic mice still show significant increased body weight (P=0.0073).The heart rate went down with the age of mice increased (P=0.014). Compared to the chow-diet mice, diabetic mice show an increased blood pressure (P=0.00004), while the age of mice had no effect on blood pressure (P=0.212). No significant interactions were detected between the age and diabetes (P=0.390).2. At the age of 14 weeks, no significant differences of brachiocephalic IMT were detected between the two groups. The brachiocephalic IMT rose rapidly in diabetic mice, but slowed down at week 20. The brachiocephalic IMT rose slowly in chow-diet mice. The brachiocephalic IMT at week 24 in chow-diet mice was similar to that at week 20 in diabetic mice. At weeks 16,18 and 20, diabetic mice show significantly increased brachiocephalic IMT when compared with chow-diet mice (P<0.005 for all). At the age of 24 weeks, no significant differences of brachiocephalic IMT were detected between the two groups (P=0.656).At the age of 14 weeks, no significant differences of brachiocephalic IMT were detected between the two groups (P=0.656). At weeks 22 and 24, diabetic ApoE-/-/LDLR-/- mice show significantly increased brachiocephalic IMT when compared with chow-diet ApoE-/-/LDLR-/- mice (P<0.05 for both).3. At the age of 20 weeks, no significant differences of brachiocephalic blood velocity were detected between the two groups (P=0.503). Then brachiocephalic blood velocity decreased slowly. At week 24, diabetic ApoE-/-/LDLR-/- mice show significantly decreased brachiocephalic blood velocity when compared with chow-diet ApoE-/-/LDLR-/- mice (416.41±86.27 vs 310.05±175.99, P=0.045).At the age of 20 weeks, no significant differences of carotid blood velocity were detected between the two groups(42.84±136.52 vs 163.10±70.01, P=0.054). At the age of 22 weeks, diabetic ApoE-/-/LDLR-/- mice show significantly decreased carotid blood velocity when compared with chow-diet ApoE-/-/LDLR-/- mice (275.09±104.69 vs 188.45±80.92, P=0.017). Then carotid blood velocity decreased slowly. At week 24, no significant differences of carotid blood velocity were detected between the two groups (P=0.968).4. Alterations of carotid functions of diabetic ApoE-/-/LDLR-/- mice(1) At the age of 20 weeks, no significant differences of carotid stiffness, elasticity, and standardized elasticity were detected between the two groups. At the age of 22 weeks, diabetic ApoE-/-/LDLR-/- mice show significantly increased carotid stiffness, elasticity, and standardized elasticity when compared with chow-diet ApoE-/-/LDLR-/- mice. Then carotid blood velocity decreased slowly. At week 24, no significant differences of carotid stiffness, elasticity, and standardized elasticity were detected between the two groups.(2) At weeks 20,22 and 24, no significant differences of carotid expand coefficient and compliance were detected between the two groups. Conclusions1. Diabetic ApoE-/-/LDLR-/- mice had more atherosclerotic burden than chow-diet ApoE-/-/LDLR-/- mice.2. Diabetic ApoE-/-/LDLR-/- mice experience a rapid development of brachiocephalic atherosclerosis, which would slow down at the age of 20 weeks. Chow-diet ApoE-/-/LDLR-/- mice keep a slow development of brachiocephalic atherosclerosis. The brachiocephalic IMT at week 24 in chow-diet mice was comparable to that at week 20 in diabetic mice.3. Diabetic ApoE-/-/LDLR-/- mice's carotid atherosclerosis was preceded with brachiocephalic atherosclerosis.4. IMT is still an effective index to evaluate the initiation and development of atherosclerosis in diabetic ApoE-/-/LDLR-/- mice.5. High resolution ultrasonic biomicroscopy could noninvasively monitor the initiation, development and prognosis of atherosclerotic plaque, helping to select the optimal intervention opportunity.