| BackgroundWith the development of social economy and rising living standards, cardiovascular and cerebrovascular diseases have become the major diseases that threaten human health. Atherosclerosis is the common pathological basis of cardiovascular and cerebrovascular diseases. So far, the pathogenesis of atherosclerosis is still unclear. The current studies found that atherosclerotic plaque formation occurs preferentially in areas such as the vicinity of branch points, the outer wall of bifurcations and the inner wall of curvatures, where the co mmon hemodynamic characteristic is low shear stress or oscillatory shear stress. The vascular endothelial injury is initiating factor of atherosclerosis, which has got consensus by scholars. Vascular endothelial injury cause s endothelial structure changes and dysfunction, which could induce the occurrence of atherosclerosis. Shear stress plays an important role in endothelial structure changes and dysfunction.Peroxisome proliferator activated receptors(PPARs) belong to a family of ligand-activated transcription factors of the nuclear hormone receptor superfamily that comprise three subtypes: PPAR-α, PPAR-β and PPAR-γ. In the past, the major physiological role of PPAR-α, PPAR-γ and their agonists was regarded to correct and improve lipid and carbohydrate metabolism. Recent studies have shown t hat it also has a role in cardiovascular protection by inhibiting the expression of inflammatory cytokines, inhibiting the proliferation and migration of smooth muscle cells and endothelial cell, reversing cardiac hypertrophy, improving insulin resistance and correcting lipid metabolism, and improving cardiovascular pathological remodeling. These results indicate that PPAR-α, PPAR-γ and agonists may play a negative regulation of metabolic syndrome and cardiovascular disease.T-helper 17(TH17) cells and regulatory T(Treg) cells are two subsets of CD4+ T cells distinguished from TH1 and TH2 cells. TH17 cells play critical roles in inflammation by producing IL-17, IL-6, IL-21, IL-22 and TNF-α, while Treg cells have important effects on the ma intenance of immune tolerance and immune homeostasis by the release of anti- inflammatory cytokines, such as IL-10 and TGF-β. TH17/Treg cells are balanced under normal circumstances and jointly maintain immune homeostasis. TH17/Treg cells imbalance was found in carotid atherosclerosis plaque of Apo E-/- mice and peripheral blood of patients with acute coronary syndrome(ACS). CCR6 is expressed on the surface of TH17 cell and Treg cell. The unique ligand of CCR6 was CCL20, which expressed in variety of tissues and organs.Whether local PPAR signaling pathways and TH17/Treg imbalance mediated by CCL20 involved in the AS induced by local flow shear stress changes is unclear. ObjectivesThe present study was designed to investigate whether flow shear stress affects local PPAR signaling pathways and TH17/Treg imbalance mediated by CCL20, promotes the occurrence and development of AS, and thus provides experimental evidence for the mechanism of AS. Methods 1. Establishment of BALB/c mice model with AS induced by shear stress changesSix-week-old male inbred BALB/c mice(n = 30) were used in present study. The mice were randomly divided into three groups. Ten mice were taken as the sham-operated group and fed with a normal diet. The group model one and model two were established by rapid perivascular collar placement at left common carotid artery(LCCA) and fed a normal and high- fat diet, respectively. The body weight was measured periodically. The alterations of peak blood flow, end-diastolic diameter of vessels and shear stress of carotid artery were assessed by carotid artery Doppler ultrasound on the third day and eighth week post-operation. 2. Serum lipid measurementThe levels of serum triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C) and high-density lipoprotein cholesterol(HDL-C) were measured by automatic biochemical analyzer. 3. Histological analysisSerial paraffin sections of carotid artery were stained with hematoxylin and eosin. The carotid artery media thickness was measured by using image analysis software. The ultrastructural changes of carotid artery were evaluated by electron microscope. The CCL20 expression of endothelial cells and distribution of TH17/Treg cells in the local blood vessels were detected by immunohistochemical staining. 4. Microarray and QRT- PCR validationThe gene expression profile in the low shear stress blood vessels and the self-controlled normal blood vessels were compared by microarray. GO and KEGG signaling pathway were analyzed. We focused on the mi RN As related to PPARG gene, the genes related to cell migration and proliferation and downstream signaling pathways negative regulated by PPARs(such as mitogen activated protein kinase(MAPK), nuclear factor kappa B(NF-κB), transforming growth factor-β(TGF-β), phosphatidylinositol 3 kinase-serine/threonine protein kinase B(PI3K-Akt)). The gene expressions of cytokines related to TH17/Treg immune cells and chemotactic factor CCR6/CCL20 were also compared. The partial results were verified by QRT-PCR(quantificational real time- polymerase chain reaction). 5. Serum levels of IL-17 and IL-10The levels of serum IL-17 and IL-10 were detected by ELISA. Results1. Compared with the bilateral common carotid artery of sham-operated group and self right common carotid artery, the proximal part of left common carotid artery(LCCA-P) in group model one and two presented low shear stress changes.2. Compared with those of sham-operated group, the body weight and serum lipids levels had no significant differences in group model one fed with normal diet at the eighth week post-operation. However, the body weight and serum lipids(TC, TG, LDL-C and HDL-C) significantly increased in group model two. Compared with sham-operated group and self right common carotid artery, endothelial cell damages, media thickening and endothelial cell vacuolation occurred in the LCCA-P of both group model one and group model two. The lesions in group two were more serious than those in group model one. But no fatty streaks, plaque formations and lipid accumulations were observed by light microscope and electron microscope.3. Microarray results suggested that compared with the self- controlled blood vessels from normal shear stress, the blood vessels from low shear stress elevated mi R-27a(> 2.0-fold), which is one of the micro RNAs that regulate the expression of PPAR-γ expression. Meanwhile, PPARG gene expression decreased to 0.46- fold. PPAR signaling pathway down-regulated. However, MAPK, TGF-β, PI3K/Akt and NF-κB signaling pathways up-regulated. The genes related to cell migration and / or proliferation, such as TGFB2, END1 and CTGF, up-regulated. The results of QRT-PCR were consistent with microarray. The expressions of PPARG decreased to 0.14-fold(P < 0.05, n = 4), TGFB2 and EDN1 increased to 2.51- fold and 1.83- fold respectively(P < 0.05, n = 4) in the blood vessels from low shear stress.4. No endothelial cells expressed with CCL20 and Treg cells infiltration were detected at the blood vessels from low shear stress by immunohistochemical staining, which was similar to the results of the self-controlled artery from normal shear stress. Microarray results also suggested that the blood vessels from low shear stress presented VCAM-1 and ICAM-1 up-regulation. No significant differences were found in the gene expressions of c ytokines related to TH17/Treg immune cells and the chemotactic factor CCR6/CCL20. Compared with sham-operated group and group model two, the serum levels of IL-17 significantly increased in group model one. There were no significant differences in serum concentrations of IL-10 among three groups. Conclusions1. Compared with the blood vessels from normal shear stress, the blood vessels from low shear stress showed the damage of local endothelial cells and media thickening after eight weeks post-operation in BALB/c mice.2. Down-regulation of local PPAR signaling pathways in the blood vessels from low shear stress promoted the genes expression and signaling pathways related to cell proliferation and migration, and might therefore involved in low shear stress induced AS early lesions.3. Endothelial cells expressed with CCL20 and Local TH17/Treg cells imbalance were not detected in the pathological processes of endothelial AS early lesions induced by low shear stress after eight weeks operation. |
PDF Full Text Request