The Study Of Effects Of Natriuretic Peptide C Receptor (NPR-C) Gene On Atherosclerosis In ApoE-/-Mice Using Gene Over-expression And Silencing

Background and objective:Natriuretic peptide C receptor (NPR-C), without a guanylyl cyclase domain, has been described, at the time of its discovery, as a clearance receptor of natriuretic peptides devoiding of any physiological functions. However, evidences have been accumulated on a potential pathophysiology role of NPR-C in cardiovascular diseases. The epidemiology study and genotype with SNP have identified NPR-C gene SNPs associated with susceptibility for coronary artery disease (CAD). And NPR-C expression has been detected in many cellular component of blood vessel, such as vascular smooth muscle cells, endothelial cell and macrophage, especially left anterior descending artery(LAD). Recent study has identified increased atherosclerosis and smooth muscle cell hypertrophy in natriuretic peptide receptor A-/-/apolipoprotein E-/-mice. What’s more, the downregulation of natriuretic peptide A receptor(NPR-A) could cause upregulation of NPR-C in vascular smooth muscle cells. The above results deduce CNP/NPR-C signal pathway may be associated with the development of AS. At present, the role of NPR-C signal pathway in atherosclerosis remains unclear. To investigate the relationship between NPR-C and atherosclerosis, the recombinant adenovirus carrying mouse NPR-C (Ad-NPR-C) and SiNPR-C(Ad-SiNPR-C) will be respectively transfected into atherosclerosis-prone mice that lack apolipoprotein E knockout(ApoE-/-) by tail vein injection. Mice injected with green fluorescent protein (GFP) or phosphate-buffered saline (PBS) served as vehicle controls. Then histology characteristics of the atheromatous plaque of the mices from different groups were compared using immunohistochemistry and histopathology staining. Part one The study of effects of NPR-C gene on atherosclerosis in ApoE-/-mice using gene over-expressionMethod:1. Establish atherosclerosis ApoE-/-mice model Sixty male ApoE-/-mices aged8weeks were fed a full diet for2weeks, and then, a high-fat diet throughout the experimental procedure for10weeks with food and water freely available.2. Ad-NPR-C transfection in vivo Aftert full diet for2weeks and high-fat diet for4weeks, all mices were divided into three groups randomly. AdNPR-C(1×109pfu) or AdGFP(1×109pfu) diluted to a total volume of100ul was injected into the tail vein of each mouse in the two intervention groups (n=20each) and Mice(n=20) injected with PBS served as controls. Transfect adenovirus again with the same method2weeks later after the first transfection, and then,4weeks later all mice were euthanized and tissue specimens were left for immunohistochemistry and histopathology staining.3. Weight and serum biochemistry measurement The weight and serum levels of Total Cholesterol(TC), Triglycerides(TG), Low Density Lipoprotein Cholesterol(LDL-C), High Density Lipoprotein Cholesterol(HDL-C) and Glucose of each mouse were measured at week14before adenovirus transfection and at the end of the experiment respectively.4. Immunohistochemistry and histopathology staining The whole aortas were stained with oil Red O, the antigen of NPR-C, ANP (A-type natriuretic peptide), CNP(C-type natriuretic peptide), α-SMC(α-smooth muscle cell) and MAC(macrophage) were detected using appropriate primary antibodies. Sections of aortic root underwent routine staining with oil Red O staing, hematoxylin and eosin(H&E) staining. Collagen was depicted by Masson’s-trichrome and Sirius-red staining.Results:1. NPR-C over-expression attenuated atherosclerosis. The mice of AdNPR-C group showed significantly smaller atherosclerotic lesion in their aortas as shown by both enface(p<0.01) and aortic root section analysis(p<0.01), comparing with the mice of AdGFP group. In contrast, there was no significant difference in the atherosclerotic lesion area between the AdGFP-infected group and the PBS control group(p>0.05).2. NPR-C overexpression has on effect on weights, serum glucose and lipid.There was no significant difference in the weights and serum levels of TC, TG, LDL-C, HDL-C and glucose among the three groups before and after adenovirus transfection(p>0.05).3. NPR-C over-expression increased expression of NPR-C but reduced expression of ANP and CNP in aortic root.Cross-section of aortic root of AdNPR-C group exhibited significantly a higher level of NPR-C expression than those of AdGFP group(p<0.05). In contrast, there was no significant difference between the AdGFP group and the PBS control group(p>0.05). Compared with AdGFP-infected mice, AdNPR-C-infected mice significantly reduced the protein expression of ANP (p<0.05) and CNP(p<0.05). In contrast, there was no significant difference in the protein expression of ANP (p>0.05) and CNP(p>0.05) between the AdGFP-infected group and the PBS control group.4. NPR-C over-expression markedly reduced the protein expression of a-SMC in aortic root.Compared with AdGFP-infected mice AdNPR-C-infected mice significantly reduced the protein expression of α-SMC(p<0.01). In contrast, there was no significant difference between the AdGFP-infected group and the PBS control group(p>0.05).5. NPR-C over-expression have no effect on protein expression of MAC in aortic root.There was no significant difference in protein expression of MAC among the three groups(p>0.05)6. NPR-C over-expression markedly reduce cross-sectional collagen area in aortic root. Compared with AdGFP-infected mice, AdNPR-C-infected mice significantly reduced cross-sectional collagen in aortic root by Masson’s-trichrome staining (p<0.05).In contrast, there was no significant difference between the AdGFP-infected group and the PBS control group(p>0.05). Compared with AdGFP-infected mice AdNPR-C-infected mice showed significantly reduced cross-sectional type Ⅰ Collagen(p<0.05) and type Ⅲ Collagen(p<0.05) by sirius-red staining. In contrast, there was no significant difference in the type I Collagen(p>0.05) and type Ⅲ Collagen(p>0.05) in cross-sectional between the AdGFP-infected group and the PBS control group.Part two The study of effects of NPR-C gene on atherosclerosis in ApoE-/-mice using gene silencingMethods:1. Establish atherosclerosis ApoE-/-mice model Sixty male ApoE-/-mices aged8weeks were fed a full diet for2weeks, and then, a high-fat diet throughout the experimental procedure for10weeks with food and water freely available.2. Transfect the Ad-NPR-C adenovirus in vivo Aftert full diet for2weeks and high-fat diet for4weeks,4weeks feed, all mice were divided into three groups randomly. AdSiNPR-C(1×109pfu) or AdGFP(1×109pfu) diluted to a total volume of100ul was injected into the tail vein of each mouse in the two intervention groups (n=20each) and Mice (n=20) injected PBS served as controls. Transfect adenovirus again with the same method2weeks later after the first transfection, and then,4weeks later all animals were euthanized and tissue specimens were left for immunohistochemistry and histopathology staining.3. Weight and serum biocamistry measurement The weight and serum levels of TC, TG, LDL-C, HDL-C and glucose of each mouse were measured at week14before adenovirus transfection and at the end of the experiment respectively.4. Blood pressure measurement Use a noninvasive tail-cuff system to measure murine systolic blood pressure (SBP), diastolic blood pressure (DBP), Mean blood pressure(MBP) and Heart rate (HR) at week14before adenovirus transfeetion and at week20at the end of the experiment respectively.5. Immunohistochemistry and special staining The whole aortas were stained with oil Red O, and the antigen of NPR-C, ANP, CNP, a-SMC and MAC were identified using appropriate primary antibodies. Sections of aortic root underwent routine staining with oil Red O staing, HE staining. Collagen was depicted by Masson’s-trichrome and Sirius-red staining.Results:1. NPR-C silencing promoted atherosclerosis.The mice of AdSiNPR-C group showed significantly higher atherosclerotic lesion in their aortas as shown by both enface (p<0.01) and aortic root section analysis (p<0.01), comparing with the mice of AdGFP group. In contrast, there was no significant difference in the atherosclerotic lesion area between the AdGFP-infected group and the PBS control group (p>0.05)2. NPR-C silencing have on effect on weights, serum lipid and glucose.There was no significant difference in the weights and serum levels of TC, TG,LDL-C, HDL-C and glucose among the three groups before and after adenovirus transfection (p>0.05)3. NPR-C silencing increased the heart rate and has no effect on blood pressure.There was no significant difference in the level of SBP, DBP and MBP among the three groups before and after adenovirus transfection(p>0.05). However, compared with AdGFP-infected mice AdSiNPR-C-infected mice significantly increase the heart rate(p<0.05). In contrast, there was no significant difference between the AdGFP-infected group and the PBS control group(p>0.05).4. NPR-C silencing reduced expression of NPR-C but increased expression of ANP and CNP in aortic root.Cross-section of aortic root of AdSiNPR-C group exhibited significantly a lower level of NPR-C expression than those of the AdGFP group(p<0.05). In contrast, there was no significant difference between the AdGFP-infected group and the PBS control group(p>0.05). Compared with AdGFP-infected mice AdSiNPR-C-infected mice significantly increase the protein expression of ANP (p<0.05) and CNP(p<0.05). In contrast, there was no significant difference in the protein expression of ANP (p>0.05) and CNP(p>0.05) between the AdGFP-infected group and the PBS control group.5. NPR-C silencing increase the protein expression of α-SMC.Compared with AdGFP-infected mice AdSiNPR-C-infected mice significantly increase the protein expression of α-SMC (p<0.01).In contrast, there was no significant difference between the AdGFP-infected group and the PBS control group (p>0.05).6. NPR-C silencing have on effect on the protein expression of macrophage.There was no significant difference in protein expression of MAC among the three groups (p>0.05).7. NPR-C silencing increase cross-sectional collagen area in aortic root.Compared with AdGFP-infected mice AdSiNPR-C-infected mice significantly increase cross-sectional collagen in aortic root detected by Masson’s-trichrome staining(p<0.05). In contrast, there was no significant difference between the AdGFP-infected group and the PBS control group(p>0.05).Compared with AdGFP-infected mice AdSiNPR-C-infected mice showed significantly increase cross-sectional type Ⅰ Collagen (p<0.05)and type Ⅲ Collagen (p<0.05) detected by sirius-red staining. In contrast, there was no significant difference in type Ⅰ Collagen (p>0.05)and type Ⅲ Collagen (p>0.05) in cross-sectional between the AdGFP-infected group and the PBS control group.Conclusion:NPR-C gene can attenuate progression of AS by gene over-expression and silencing in ApoE-/-mice.