Study Of The Protective Effects Of Osteoprotegrin On Vascular Smooth Muscle Cell And Its Mechanism

Part I:Effect of OPG on the expression of inflammatory factors in human aortic smooth muscle cellsObjective:Inflammatory responses throughout the entire process of AAA, which includes monocyte/macrophage infiltration as the main pathological features during early lesions. This study was designed to investigate the effect of OPG on the expression of inflammatory factors including MCP-1 and VCAM-1 in HVSMC to explore the possible role of OPG in inflammatory cell infiltration and the formation of AAA.Methods:HVSMC was cultured in vitro and was activated separately by TNF-a (5ng/ml), OPG (5ng/ml) and TNF-a (5ng/ml) combined with OPG (5ng/ml) in cultured media. Untreatment of HVSMC acted as the control. The expression of monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) were investigated with ELISA and RT-PCR at 6h, 12h and 24h after stimulation.Results:Based on mRNA and protein levels, an obvious increase in the expression of inflammatory factors including MCP-1 and VCAM-1 in HVSMC with the treatment of TNF-a (5ng/ml) was higher than control group, OPG (5ng/ml) group and TNF-a (5ng/ml) combined with OPG (5ng/ml) group, P?0.01. OPG could effectively inhibit the high expression of inflammatory factors in HVSMC induced by TNF-a (5ng/ml). There was no statistical difference between OPG treatment group and the control group.Conclusion:TNF-a plays a role as a contributing factor in occurrence and development of AAA. In vitro, OPG can effectively inhibit the pro-inflammotory effects of TNF-a, which induced inflammatory cell infiltration in AAA, and play a protective role in HVSMC. Part?:Effect of OPG on the apoptosis of human aortic smooth muscle cellsObjective:Relatively stable number of VSMC plays an important role to maintain the normal physiological function of aorta. As a typical example of vascular injury-reconstruction, the formation of AAA is related to the apoptosis of VSMC in the aorta medial. This study was designed to investigate the effect of OPG on the apoptosis of HVSMC induced by TNF-?in vitro to explore the possible role of OPG in the formation and development of AAA.Methods:HVSMC was cultured in vitro and was activated separately by TNF-?(5ng/ml), OPG (5ng/ml) and TNF-?(5ng/ml) combined with OPG (5ng/ml) in cultured media. Untreatment of HVSMC acted as the control. The apoptosis index (AI) was detected by terminal deoxynucleotidy transferase mediated dUTP nick end labeling (TUNEL) at 6h,12h and 24h after stimulation.Results:TNF-?(5ng/mL) stimulated the rise of AI in HVSMC. The AI in TNF-?treatment group was significantly higher than control group, OPG (5ng/ml) grouop and TNF-?(5ng/ml) combined with OPG (5ng/ml) group, P<0.01. The AI of TNF-?(5ng/ml) combined with OPG (5ng/ml) treatment was significantly higher than OPG (5ng/ml) treatment, P0.01.Conclusion:TNF-?plays a role as a contributing factor in apoptosis of VSMC. In vitro, OPG can inhibit the TNF-?-induced apoptosis of HVSMC, and play a protective role in the progess of vascular injury-reconstruction in aorta medial. Part?:Effect of downregulation OPG on the inflammatory activation characteristics of human aortic smooth muscle cellsObjective:Nuclear factor kappa?(NF-?B) is a major intracellular inflammatory response transcription factors. It involves in inflammation, immune response, cell proliferation, transformation, migration and apoptosis in the pathophysiology of a variety of important processes by regulating the transcription of multiple genes. This study was designed to investigate the impact of downregulating OPG in TNF-a-induced inflammatory activation in HVSMC.Methods:OPG-shRNA interference plasmid was successfully constructed. The transfection efficiency of HVSMC interference was detected by RT-PCR. Adenovirus vector construction and packet transfer rAd-OPG-shRNA and transfected it into HVSMC in vitro. Transfected OPG-shRNA HVSMC and control HVSMC were cultured in vitro and was activated separately by TNF-?(5ng/ml) and TNF-?(5ng/ml) combined with OPG (5ng/ml) in cultured media. The expression of NF-Kbp65 in different groups was investigated with Western-Blotting after treatment.Results:After transfection by rAd-OPG-shRNA, expression of OPG mRNA significantly decreased in HVSMC, and led the expression of NF-?Bp65 increased significantly in rAd-OPG-shRNA interference group compared to control group, which induced by TNF-?(5ng/ml), P<0.01. TNF-?(5ng/ml) combined with OPG treatment significantly inhibit the expression of NF-?Bp65 in the HVSMC after rAd-OPG-shRNA transfection, P<0.01 or P<0.05.Conclusion:HVSMC in vitro, OPG could exert its protective effect through inhibiting the function of active NF-?B to inhibit the production of inflammatory cytokines and apoptosis incuded by TNF-?. And the exogenous OPG also significantly inhibited the expression of NF-?B in rAd-OPG-shRNA transfected HVSMC. OPG play a protective role in TNF-?-induced NF-?Bp65 in HVSMC. Part IV:Changes of OPG serum concentration and their significance in AAA patientsObjective:Many studies indicate that the circulating OPG levels in patients are closely related to various cardiovascular diseases. The roles of OPG in vascular diseases and atherosclerosis still remain controversial. This study was designed to investigate the concentration of OPG in the AAA patients and its variation to explore the possible compensatory defense mechanism in the formation and development of AAA.Methods:66 patients were selected with inclusion criteria and exclusion criteria.36 AAA patients constituted the case group, which was identified by CTA (abdominal aorta diameter?30mm). The persons with normal diameter of abdominal aorta constituted the control group. The level of serum OPG was detected with ELISA. And the age, body mass index (BMI), cigarette use, history of hypertension, diabetes, hyperlipidemia was simultaneously collected.Results:The concentration of serum OPG of AAA group (5.79±0.44mmol/L) was significantly higher than the control group (3.36±0.43 mmol/L), P<0.01. Serum OPG levels in different diameter groups were statistically different (P<0.05), and the serum OPG levels had a positively correlation with the diameters of AAA, correlation coefecient R=0.844.Conclusion:The level of serum OPG was significantly higher in patients with AAA and had a partial correlation coefficient with the diameters of AAA. The high level of OPG could play a protective in the development of AAA as compensatory mechanisms.