The Study Of NPR-C Gene On Stabilizing Atherosclerotic Plaque Of Carotid Artery In ApoE-/-Mice

Background and objective:Atherosclerosis(AS) is a multi-factorial process and is thought to be an infectious, inflammatory and autoimmune disease. Previous research shows that the rupture of atherosclerotic plaques followed by thromosis formation is the main cause of the acute myocardial infarction, stroke and sudden death. In fact, the instable plaques are easy to rupture. Rupture-prone atherosclerotic plaque usually has a thin fibrous cap, a large lipid core, enriched inflammatory cells and reduced contents of smooth muscle cells and collagen. As more and more person are suffered from cardia-cerebrovascular diseases, plaque rupture is of great interest of researchers all over the world.Natriuretic Peptide C receptor(NPR-C), at the time of its discovery, is considered as a clearance receptor committed to remove natriuretic peptides(NPs) from the circulation without any physilogical functions. NPs bind with NPR-C leading to internalization and degradation of the peptides. However, an increasing number of evidence suggests that NPR-C may have additional functions on cardiovascular system and other organs. Recent studies intimate C-type Natriuretic Peptide(CNP) has the potential to prevent the development of atherosclerosis, and this effect can be contributed to its ability to modulate smooth muscle cell growth, leukocyte recruitment and platelet aggregation, and blood vessel tone in the resistance vasculature. Of note, more and more evidence suggests the cardioprotective effects of CNP are mainly mediated via CNP/NPR-C signaling. Furthermore, a previous research shows that NPR-C is strongly expressed in neointimal SMCs from 1 to 9 months after PCI. This suggests that NPR-C may be important in controlling neointimal growth after PCI in humans. Besides, some researchers find that C-ANP4-23 and small cytoplamic domain peptides of NPR-C could modulate vasoactive peptide-stimulated protein synthesis in A10 vascular smooth muscle cells. In our previous study we identified NPR-C gene could attenuate progression of atherosclerosis using gene over-expression and silencing in ApoE-/-mice. All the information above indicates that NPR-C is widely involved in the process of cardiovascular diseases. Instability of plaque plays an indispensable role in the occurrence of acute cardiovascular events. However, the biological function of NPR-C in instable plaque of carotid artery is still not clear. Based on these evidence, we hypothesized NPR-C may increase the stability of atherosclerotic plaque. In order to investigate the relationship between NPR-C and plaque stability, we established vulnerable plaque model in the right carotid artery in ApoE-/-mice, and successfully delivered the lentivirus carrying mouse NPR-C into carotid artery. Mice received a local infusion of normal saline(NS) or lentivirus carring enhanced green fluorescent protein (EGFP) gene served as vehicle controls. Further studies were conducted using the methods of histopathology and molecular biology.Method:1. Establish carotid vulnerable plaque model in ApoE-/-miceA total of 70 male ApoE-/-mice aged 8 weeks old with genetic background of C57BL/6 were purchased from Beijing Hua Fu Kang Company and caged in animal center of Key Laboratory of Cardiovascular Remodeling and Function Research in Qilu Hospital, Shandong University. The mice were housed under standard conditions of humidity, room temperature and dark-light cycles and had free access to water and food. After a two-week high-fat diet, all mice underwent constrictive collar (inner diameter,0.30mm; length,2.5mm) placement around the right common carotid artery after anesthesia with an intraperitoneal injection of pentobarbital sodium. Finally, the skin wound was closed with silk sutures and all the mice were injected with penicillin sodium. Another five weeks of high-fat diet was maintained.2. Local infusion of lentiviral suspension into carotid arteriesFive weeks after the surgery of constrictive collar placement, all mice were divided into three groups randomly:NS group(n=20), LV-EGFP group(n=20), LV-NPR-C group(n=30). After the mice were anaesthetized, a sagittal anterior neck incision was performed and common carotid arteries were dissected. The mice of three groups received a local infusion of 100μL lentiviral suspension(2×10^7 TU) or NS via the vascular adventitia of the right common carotid arteries. Subsequently the skin wound was closed with silk sutures. Two weeks later, two mice of LV-EGFP group were selected to observe the efficiency of lentivirus transfection in carotid atherosclerotic plaque. GFP expression was viewed on fluorescence microscopy through cryosections. All mice were sacrificed after an another four-week high-fat diet.3. Body weight and biological analysisThe concentration of plasma glucose, triglycerides(TG), low-density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C) and total cholesterol (TC) were measured before lentivirus transfection and the end of the whole experiment. Simultaneously, body weights were measured.4. Blood pressure and heart rate analysisThe systolic, mean, and diastolic blood pressure(SBP, MBP, DBP) and heart rate(HR) were measured by a programmable sphygmomanometer using the noninvasive tail-cuff method before the lentivirus transfection and the end of the whole experiment. Daily means were calculated from the means of the 3 trials per day. The BP of each mouse was averaged by 3 consecutive days.5. Histological AnalysisAll mice were euthanized and the right carotid arteries were embedded in opti-mum cutting temperature compound(O.C.T Compound) or paraffin and then sectioned. Hematoxylin and eosin (HE) staining was used for histological analysis of the plaque. Oil Red O staining and Sirius Red were performed to visualize the contents of lipids and collagen in the plaque respectively.6. ImmunohistochemistryTo detect mocrophages(MAC), a-Smooth Muscle Actin(a-SMA), NPR-C, Atrial Natriuretic Peptide(ANP), CNP expression in the carotid plaque, tissue sections were incubated with the appropriate primary antibodies according to the protocols. The vulnerability index(VI) of the plaque was calculated as:VI=(macrophages stained%+ lipids stained%)/(smooth muscle cells stained% +collagen stained%).7. Real-time quantitative-polymerase chain reaction(RT-PCR)Total RNA was extracted from the right carotid arteries using trizol reagent and then reverse-transcribed. The resulting cDNA samples were then amplified. Relative quantitation of target genes such as NPR-C, Matrix Metalloproteinase-9(MMP-9), and Vascular cell adhesion molecule-1(VCAM-1) was calculated by the 2-ΔΔCt method using P-actin as an internal control. Each experiment was repeated at least 3 times in 3 samples.8. Western blotThe total protein of the right carotid arteries were extracted and then separated by electrophoresis,transferred to nitrocellulose membranes, blocked with 5% nonfat milk, washed with Tris buffered saline-Tween 20(TBST) and incubated with the respective primary antibodies. The antibody-antigen complexes were detected by second antibody, and the immunoreactive bands were visualized using enhanced chemiluminescent HRP Substrate. Quantitative analysis of NPR-C、ANP、CNP Tumor Necrosis Factor-a(TNF-a)Monocyte Chemotactic Protein-1(MCP-1) was performed using Image J and normalized by that of β-actin.Statistical Analysis:All data are expressed as mean±standard deviation(SD). SPSS 18.0 was used for all data analysis. Data were compared using t test or one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.Results:1. General condition of ApoE-/-miceA total of 70 male ApoE-/-mice aged 8 weeks old were given constrictive collar placement and local infusion of lentivirus or NS. During the whole period of eleven-week high-fat diet, all the mice keep healthy and stay alive.2. Successful local transfection of Ientivirus in carotid plaqueFirst, obvious fluorescence could be seen in cryosections of carotid plaques obtained from two of the LV-EGFP-transfected mice two weeks after local Ientivirus transfection. Then immunohistochemical staining showed that LV-NPR-C group exhibited significantly a higher level of NPR-C (p<0.05) than NS and LV-EGFP group. No significant differences was found between NS group and LV-EGFP group(p>0.05). Compared with NS group and LV-EGFP group, the mRNA and protein levels of NPR-C gene in LV-NPR-C group were also found to be upregulated using RT-PCR and western blot analysis(p<0.05). In Contrast, there was no significant difference between the NS group and LV-EGFP group(p>0.05).3. NPR-C overexpression has no effect on body weight and biological analysis in ApoE-/-miceNo significant difference in body weight, Glucose, TG, LDL-C, HDL-C and TC was found among NS group, LV-EGFP group and LV-NPR-C group (p>0.05).4. NPR-C overexpression has no effect on heart rate and blood pressure in ApoE-/-miceThere was no significant difference in heart rate, SBP, DBP and MBP among the three groups(p>0.05).5. NPR-C overexpression can significantly decrease the contents of lipids in carotid plaqueOil red O staining showed that compared with NS and LV-EGFP group, LV-NPR-C group can significantly decrease the contents of lipids in carotid plaque(p<0.05). In Contrast, there was no significant difference between the NS group and LV-EGFP group(p>0.05).6. NPR-C overexpression can markedly reduce the contents of macrophage in carotid plaqueThe relative contents of macrophages in the LV-NPR-C group was apparently lower than NS and LV-EGFP group using immunohistochemical staining(p<0.05). However, no significant difference was found between the NS group and LV-EGFP group(p>0.05).7. NPR-C overexpression can significantly decrease the contents of a-SMA in carotid plaqueLV-NPR-C group showed markedly reduced contents of a-SMA in carotid plaque(p<0.05) in contrast with NS and LV-EGFP group, but there was no significant difference between the NS group and LV-EGFP group(p>0.05).8. NPR-C overexpression can markedly increase the contents of collagen in carotid plaqueSirius Red staining showed that compared with NS and LV-EGFP group, the contents of collagen in carotid plaque in LV-NPR-C group (p<0.05) was significantly elevated. In contrast, no significant difference was found between the NS group and LV-EGFP group(p>0.05).9. NPR-C overexpression can significantly decrease the vulnerability indexThe vulnerability index(VI) of plaque of carotid artery from LV-NPR-C group was significantly lower than NS and LV-EGFP group (p<0.05) while there was no significant difference between the NS group and LV-EGFP group(p>0.05).10. NPR-C overexpression can significantly decrease the expression of ANP and CNP in carotid plaqueCompared with NS and LV-EGFP group, the contents of ANP and CNP in carotid plaque significantly reduced in LV-NPR-C group (p<0.05), detected by immunohistochemical staining. However, there was no significant difference between the NS group and LV-EGFP group(p>0.05).Compared with NS and LV-EGFP group, LV-NPR-C group showed markedly decrease of the protein expression of ANP and CNP in carotid plaque (p<0.05) using western blot analysis. However, no significant difference was found between the NS group and LV-EGFP group(p>0.05).11. NPR-C overexpression can significantly decrease the expression of TNF-a, MCP-1, MMP-9 and VCAM-1 in carotid plaqueCompared with NS and LV-EGFP group, LV-NPR-C group showed the lower protein level of TNF-a and MCP-1 in carotid plaque (p<0.05) by western blot. In contrast, there was no significant difference between the NS group and LV-EGFP group(p>0.05).Compared with NS and LV-EGFP group, LV-NPR-C group also showed the lower mRNA level of MMP-9 and VCAM-1 in carotid plaque (p<0.05) using PCR analysis. However, no significant difference was found between the NS group and LV-EGFP group(p>0.05).Conclusion:NPR-C gene can stabilize atherosclerosis plaque via improving the components of plaque and inhibiting the expression of inflammatory cytokines.