Study On The Changes Of Clock Genes, Apoptosis-related Genes And Fibrinolytic-related Genes Expressions In Human Plaque-derived Vascular Smooth Muscle Cells

Atherosclerosis (AS) is one of the most serious diseases that threaten human beings today. Vascular remodeling and plaque vulnerability have been the research focus. The studies find the circadian clock genes are closely related to the function of cardiovascular system. In mammals, many behavioral and physiological processes exhibit circadian (approximately 24 h) rhythms that are controlled by a clock system. This system includes the central circadian clock residing in the hypothalamic suprachiasmatic nucleus (SCN) and the peripheral clock located in many peripheral tissues. It is considered that circadian rhythmicity of peripheral tissues is uniquely controlled by SCN via neural and humoral signals. However, recent research demonstrates that peripheral tissues and cells also contain a similar clock system to that in the SCN. The core clock genes include Bmall, Clock, Cry, Per and Rev-erba etc., which form a negative feedback loop involving a positive limb (Bmall and Clock) and a negative limb (Per and Cry). The heterodimer of BMAL1/CLOCK binds to the E-boxes located within the promoters of Cry and Per genes and activates their transcription. Then, the proteins of PER and CRY form a complex and inhibit the positive limb, resulting in rhythmic oscillation.By regulating the clock-control genes, the clock genes involved in the regulation of several cardiovascular disease risk factors, such as metabolic disorders, inflammation and coagulation abnormalities. It has found that the rhythmic expression of clock genes changes in process of various cardiovascular diseases, which implies that the clock genes as well as its downstream signal pathway may play important roles in the process of atherosclerosis and the change of plaque vulnerability. The studies have illustrated:(1)clock genes involved in the regulation of cell proliferation and apoptosis. (2)The clock genes knockout mice are prone to generate hyperlipidemia and obesity syndrome. (3) The vascular of clock genes knockout mice would be pathologic remodeling, aging, weakened vasorelaxation, increased expression of proinflammatory cytokines, decreased synthesis of vasodilatory factors, and these changes together would promote the atherosclerosis. (4) The coagulation function of clock genes mutation mice are changed in vivo, as the rhythmic expression of PAI-1 and t-PA are disordered, which may result in thrombosis. Our previous studies found that the clock genes expression in cardiovascular system was changed in apoE knockout mice. We also found the rhythms of apoptosis-related genes (c-myc and p53) and fibrinolytic-related genes (PAI-1 and t-PA) expressions were disordered. However, the current research in this area is mainly focus on the animal experiment, little is known about the circadian clock system in human VSMCs, especially the VSMCs in human plaques. In the present study, we established a model of primary cultured human plaque-derived VSMCs and human normal carotid VSMCs in vitro that both possess circadian oscillators by the serum shock method to compare the rhythm changes of clock genes, apoptosis-related genes and fibrinolytic-related genes in human plaque-derived VSMCs with that in the human normal carotid VSMCs.Part Ⅰ :Rhythm changes of clock genes in human plaque-derived VSMCsPurpose:To establish a model of primary cultured human plaque-derived VSMCs and observe its physicochemical and morphological characteristics. Detect changes of clock genes rhythmic expressions in human plaque-derived VSMCs.Methods:We used the explant culture method to obtain human VSMCs. Human plaque VSMCs were cultured from carotid plaques of patients who had undergone carotid endarterectomy. Donors from the Zhongshan Hospital Transplant Program provided the sections of carotid to culture the normal human carotid VSMCs. We used the inverted phase contrast microscope, immunofluorescence staining, oil red O staining and transmission electron microscopy to observe cell morphology and physicochemical characteristics. We induced the rhythmic expression of clock genes in VSMCs by serum shock method. The timing of the beginning serum shock was defined as Zeitgeber time 0 (ZTO), and cells were harvested for RNA extraction at ZTO, ZT4, ZT8, ZT12, ZT16 and ZT20. The mRNA levels of the target genes were measured by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).Result:Fifty-six patients underwent carotid endarterectomy between May 2012 and July 2013 in Zhongshan Hospital (Shanghai, China), and 21 of them were successfully cultured plaque-derived VSMCs. Seven of the total 10 donors yielded viable cultured normal VSMCs. Cells started to migrate from the explanted tissues within 7 to 12 days and formed typical "hills and valleys" in about 4 weeks. VSMCs cultured from normal carotid had a typical fusiform shape, while cells cultured from plaques had two distinctly different morphologies:fusiform and a big, flattened shape. The VSMC marker, smooth muscle cell protein α-smooth muscle actin (a SMA), was expressed in all phenotypes. But the Oil-red-O staining demonstrated that the lipid content within human plaque-derived VSMCs was much richer than that in normal human carotid VSMCs. And beside the different shapes observed in human plaque-derived VSMCs, the cells with a big, flattened shape contain much more lipid than the fusiform ones, whose lipid content was quite similar to normal human carotid VSMCs. Transmission electron microscopy revealed the fusiform ones had abundant myofilament bundles and distinct dense bodies, while the cells with a big, flattened shape were full of rough endoplasmic reticulum (RER) and large lipid droplets. According to their morphologies, content of lipid and ultrastructure, the fusiform cells would be the contractile VSMCs, and the phenotype of big shape ones was switching to the synthetic type. After serum shock, both types of cells showed clear circadian expressions of Bmall, Cry1, Cry2, Perl, Per2, Per3 and Rev-erba mRNA; meanwhile the Clock mRNA show a rhythmic expression in plaque-derived VSMCs but not in normal carotid VSMCs. The expression levels of these main clock genes were significantly attenuated in human plaque-derived VSMCs compared with normal human carotid VSMCs. The rhythm of Bmall mRNA in plaque-derived VSMCs was changed. After adjustment for age factors, we found the expression levels of clock genes were still significant higher in normal human carotid VSMCs than in human plaque-derived VSMCsPart Ⅱ:The changes of apoptosis-related genes and fibrinolytic-related genes expressions in human plaque-derived vascular smooth muscle cellsPurpose:Observe the changes of apoptosis-related genes and fibrinolytic-related genes expressions in human plaque-derived vascular smooth muscle cells. Explore the potential pathways between clock genes and downstream clock-control genes.Methods:Using the serum shock method to induce the rhythmic expression of clock genes in VSMCs. The timing of the beginning serum shock was defined as Zeitgeber time 0 (ZTO), and cells were harvested for RNA extraction at ZTO, ZT4, ZT8, ZT12, ZT16 and ZT20. The mRNA levels of poptosis-related genes (p53, Fas and Bax) and fibrinolytic-related genes (PAI-1 and t-PA) were measured by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).Result:After serum shock, apoptosis-related genes (Fas and p53) and fibrinolytic-related genes (PAI-1 and t-PA) exhibited rhythmic expression in human carotid VSMCs. While the expression rhythms and levels of these genes (except t-PA) were disordered in human plaque-derived VSMCs. Bax did not show circadian rhythm in either type of cells. The expression level of PAI-1 was higher in plaque-derived VSMCs. For the mRNA of t-PA, not only the peak time moved forward to ZT12, but the expression level also reduced in human plaque-derived VSMCs, compared with normal carotid VSMCs.Conclusion:We established a model of primary cultured human plaque-derived VSMCs using explant culture method. The phenotypes switched in human plaque-derived VSMCs, which divided into two distinctly different phenotypes:the contractile type and the synthetic type. We used the serum shock method to induce the rhythmic expression of clock genes in VSMCs. The expression levels of clock genes were significantly attenuated in human plaque-derived VSMCs compared with normal human carotid VSMCs. While the expression rhythms and levels of apoptosis-related genes and fibrinolytic-related genes were disordered in human plaque-derived VSMCs. These changes together may be involved in the progression of atherosclerosis and its subsequent complications. Further research should be conducted to reveal how circadian genes regulate these clock-controlled genes and affect the diurnal variations of cardiovascular function.