| Atherosclerosis is the leading cause of morbidity and mortality on a global scale. Despite familiarity with this disease, some of its pathogenesis remains poorly recognized and understood. Atherosclerosis lesions are asymmetric focal thickenings of the innermost layer of the artery, namely the intima. They are consisted of cells, connective-tissue elements, lipids, and debris. The lesions of atherosclerosis represent a series of highly specific cellular and molecular responses. Atherosclerosis develops in response to endothelial cell injury caused by many different stimulus including diabetes mellitus, hypertension, and dyslipidemia. After initial injury, different cell types, including endothelial cell, platelets and inflammatory cells release mediators, such as growth factors and cytokines which induce multiple effects. These growth factors and cytokines will promote the changes of vascular smooth muscle cells (VSMCs) from the quiescent contractile state to the active synthetic state, VSMCs proliferation and migration, and extracellular matrix protein deposition. In the center of atheroma, foam cells and extracellular lipid droplets form a core region, which is surrounded by a cap of VSMCs and collagen-rich matrix. The state of "fibrous cap" determines the stability of the plaque, while VSMCs is an important factor that affects the "fibrous cap" in good condition.Epidemiological studies’ have shown that the incidence of cardiovascular events in women increases after menopause and the morbidity of coronary heart disease have significant gender differences based on the evidence that the heart disease develops in women on average 10 years later in life compared with men, suggesting a beneficial action on cardiovascular system of endogenous estrogens. However, contradictory evidence also exists showing no cardiovascular benefit from estrogen replacement therapy(ERT).Currently the" windows of opportunity and timing hypothesis "is widely accepted to explain the controversy. This hypothesis was recently supported by the beneficial cardiovascular effects in postmenopausal women receiving ERT early after menopause.It was argued that estrogen had potentially beneficial effects on lipid parameters, such as reducing low-density lipoprotein cholesterol (LDLC) and increasing high-density lipoprotein cholesterol (HDLC), facilitating nitric oxide-mediated vasodilation, and inhibiting the response of blood vessels to injury and the development of atherosclerosis. It was reported that 17β-estradiol (E2) improves vascular endothelial functions and promotes endothelial migration. In addition, E2 inhibits lipopolysaccharide-promoted VSMCs migration. However, the mechanism underlying the regulatory effect of E2 on inflammatory molecules remains obscure.Recently it is reported that TNF-related apoptosis-inducing ligand (TRAIL) regulates cardiovascular functions. TRAIL, as a homologue of cytokines of the TNF family, was first discovered about 20 years ago, which is also known as Apo-2 ligand (Apo-2L) or TNF super family 10 (TNFSF10). TRAIL shows 23% homology to Fas ligand and 19% to TNF-α. Human TRAIL is a 281-amino acid (291 for murine TRAIL) type Ⅱ transmembrane protein. The membrane-bound TRAIL can be cleaved by cysteine proteases, and subsequently released into circulation, leading to formation of the soluble form of TRAIL. A major source of soluble TRAIL appears to be the activated leukocytes such as monocytes and neutrophils. Soluble TRAIL forms a homotrimer through interactions between the Cys230 residue and the bound zinc ion. This trimerization is essential for TRAIL binding to the cognate receptors and subsequent induction of biological responses. TRAIL has potent anti-tumor activities and has entered into clinical trials for cancer therapy. In addition to inducing tumor cell apoptosis, TRAIL shows broad other biological functions both in vitro and in vivo and these actions exert potential protective effects on cardiovascular disease. TRAIL gene deletion enhanced atherogenesis in hyperlipidemic mice, supporting that endogenous TRAIL has vascular protective effects. However, excessive TRAIL may stimulate endothelial cell apoptosis, smooth muscle proliferation and migration, foam cell formation. These results show that increased TRAIL production may also have potential detrimental effects on vascular inflammation and atherosclerosis.Recently there is evidence indicating that a significant inverse correlation existed between the serum levels of TRAIL and E2 and in vitro treatment with E2 decreased the TRAIL expression levels in peripheral blood mononuclear cells, which clearly indicates that TRAIL is the regulatory target of estrogen.Therefore, the aim of our study is to explore the effect of E2 on the expression of TRAIL in VSMCs, and further to illuminate the mechanism of E2-reduced VSMCs proliferation and migration.Chapter 1 The effect of E2 on the expression of TNF-a-induced TRAILIn order to explore the effect of E2 on the expression of TNF-a-induced TRAIL, we used ELISA and real-time PCR to detect the protein and mRNA level of TRAIL. Also, MTT and cell wound assay were used to observe the influence of E2 on TNF-a-induced VSMCs proliferation and migration. 1.1 TNF-a promoted VSMCs proliferation and migration via TRAILAdherent VSMCs were treated with different concentrations of TNF-a (1 ng/ml, 10 ng/ml,50 ng/ml,100 ng/ml) for 24 h. The TRAIL protein level in medium plus cell lysates was measured by ELISA and it was showed that compared with control, TNF-a (10 ng/ml,50 ng/ml,100 ng/ml) increased TRAIL protein level, with the increasing rate of (56.6±7.8)%, (80.9±9.2)%, (119.1±11.3)%(n=5,*p<0.01 vs. CON; **p<0.001 vs. CON), respectively. The TRAIL mRNA was measured by real-time PCR and it was shown that TNF-a (10 ng/ml,50 ng/ml,100 ng/ml) also increased TRAIL mRNA expression, with the increasing rate of (110±10.9)%, (180±13.1)%, (220±13.2)%, (n=5,**p<0.01 vs. CON). VSMCs were treated with different concentrations of TNF-a for 24 h and the proliferation rate was measured by MTT. As compared with the control, TNF-a at the concentration of 10 ng/ml,50 ng/ml,100 ng/ml all increased the proliferation rate, with the increasing rate of (48.6±6.7)%, (66.9±6.9)%, (98.6±8.6)%(n=5,*p<0.01 vs. CON;**p<0.001 vs. CON). Confluent VSMCs were scrapped to create a cell-free (wounded) area, as indicated by black lines. Cells were incubated with TNF-a (100 ng/mL), in the presence or absence of TRAIL neutralizing antibody (Ab,20 mg/mL) for 24 h. Migration was monitored. Cells were digitally imaged and representative images of cell migration are shown. The results showed that TNF-a significantly increased VSMCs migration, which was inhibited by the addition of neutralizing antibody.1.2 The dose-dependent effect of E2 on TNF-a-induced TRAIL expression in VSMCsVSMCs were pretreated with different concentration of E2 (10-9M,10-8M, 10-7M, 10-6M) for 24 h and then treated with TNF-a (100 ng/ml) for another 24 h. The TRAIL protein level in medium plus cell lysates was measured by ELISA and the TRAIL mRNA was measured by real-time PCR and normalized to TBP. The results showed that when compared with the control, TNF-a significantly increased TRAIL protein expression level, with the increasing rate of (164±12.5)%(n=5,*p<0.001 vs. CON). The pretreatment with different concentrations of E2 (10-9M,10-8M,10-7M, 10-6M) all inhibited TNF-a-increased TRAIL level, with the inhibitory rate of (10.3±3.1)%, (35.0±4.8)%, (21.7±4.6)%, (30.7±6.9)%(n=5, #p<0.05 vs. TNF-a; ##p<0.01vs. TNF-a), respectively. When compared with the control, TRAIL mRNA expression was significantly increased by TNF-a, with the increasing rate of (210±17.0)%(n=5,*p<0.001 vs. CON), which was inhibited by the different dose of E2 (10-9M,10-8M,10-7M,10-6M) with the inhibitory rate of (18.7±3.6)%, (51.6±3.3)%, (47.4±5.1)%, (44.2±3.8)% (n=5,#p<0.05 vs. TNF-a;**p<0.01 vs. TNF-a), respectively.1.3 The time-dependent effect of Ei on TNF-a-induced TRAIL expression in VSMCsVSMCs were pretreated with E2(10-8M) for 6 h,12 h,24 h,48 h and then treated with TNF-a (100 ng/ml) for 24 h. The TRAIL protein level in medium plus cell lysates was measured by ELISA and the TRAIL mRNA level was measured by real-time PCR.Our results showed that as compared with the control , TNF-a increased TRAIL protein and mRNA expression, with the increasing rate of (157.4±11.7)%, (230±16.9)%(n=5,**p<0.001 vs. CON), respectively. The pretreatment with E2 for 12 h， 24 h,48 h significantly reduced TRAIL expression level, with the inhibitory rate of (18.7±5.7)%, (30.3±6.5)%, (41.0±3.4)%(n=5, ##p<0.01 vs. TNF-a;###p<0.001 vs. TNF-a). Likewise, TNF-a-enhanced mRNA expression was also inhibited by E2 for different time, with the inhibitory rate of (54.5±6.1)%, (50.3±5.3)%, (48.9±4.2)%(n=5, ##p<0.01vs. TNF-a), respectively.1.4 E2 suppressed TNF-a-induced TRAIL expression via ERaVSMCs were pretreated with E2 (10-8M), PPT (10-9M) or DPN (10’9M) for 24 h, in the presence or absence of ICI 182,780 (10-6M). Then the cells were treated with TNF-a (100 ng/mL) for 24 h. TRAIL protein level and mRNA level were measured by ELISA and real-time PCR. Both E2 and PPT inhibited TNF-a-enhanced TRAIL protein expression, with the inhibitory rate of(32.7±4.7)%, (16.8±3.6)%, respectively. Meanwhile, TNF-a-increased mRNA was also inhibited by E2 or PPT, with the inhibitory rate of (52.9±5.6)%, (55.9±3.9)%(n=5,*p<0.001 vs. CON;#p<0.05 vs. TNF-a; ##p<0.01 vs. TNF-a). However, DPN had no effect on TNF-a-induced TRAIL expression while ICI 182,780 blocked the effect of E2, suggesting that estrogen inhibited TRAIL protein and mRNA expression via ERa.1.5 E2 suppressed TNF-a-induced VSMCs proliferation and migrationVSMCs were pretreated with E2 (10-8M), PPT (10-9M) for 24 h. Then the cells were treated with TNF-a (100ng/mL) for another 24h and the proliferation rate was measured by MTT. The results showed that as compared with the control, TNF-a significantly increased VSMCs proliferation rate, with the increasing rate of (88.4±6.5)%.This effect was inhibited by the addition of E2 or PPT, with the inhibitory rate of(23.1±3.5)%, (15.8±3.6)%(n=5,*P<0.001 vs. CON; #p<0.01 vs. TNF-a), respectively.Confluent VSMCs were scrapped and pretreated with E2 (10-8M) or PPT (10-9M) for 24 h and with TNF-a (100 ng/mL) for additional 24 h. Cells were digitally imaged and representative images of cell migration were shown. The results showed that TNF-a significantly increased VSMCs migration, which was inhibited by E2(10-8M) or PPT(10-9M).Summary:TNF-a increases the TRAIL expression in VSMCs and promotes VSMCs proliferation and migration. These effects are inhibited by E2 via ERa.Chapter 2 E2 suppressed TRAIL expression via NF-kB pathwayIn order to discuss the mechanism of E2 to inhibit the level of TRAIL, we used several kinase inhibitors to screen out the signal pathway that participated in this effect. Western blot and NF-kB activity assay kit were used to survey the influence of E2 on this signal pathway.2.1 TNF-a increased TRAIL expression via NF-kBVSMCs were pretreated with NF-kB inhibitor PDTC (20 μM), p38 inhibitor ML 3403 (50 μM), JNK inhibitor IQ 1S (20 μM), MEK inhibitor PD 98059 (5 μM) and ERK1/2 inhibitor FR 180204 (20 μM) for 24 h. Then the cells were treated with TNF-a (100 ng/mL) for 24 h.TRAIL protein level and mRNA level were measured by ELISA and real-time PCR respectively. Our results showed that when compared with the control, TNF-a significantly increased TRAIL protein and mRNA expression, which was exclusively inhibited by NF-kB inhibitor PDTC, with the inhibitory rate of(44.4±3.3)% and (59.1±5.6)% (n=5,*p<0.01 vs. CON;#p<0.01 vs. TNF-α), respectively, suggesting that TNF-a increases TRAIL protein and mRNA via NF-κB signaling, while it is irrelevant to MAPK signalings.2.2 E2 inhibited TNF-α-induced phosphorylation and translocation of NF-κB p65VSMCs were pretreated with E2 (10-8M) for 24 h and then treated with TNF-α (100 ng/mL) for 15 min. Western blot was used to detect the phosphorylation level of NF-κB subunit p65, the expression of NF-κB p65 in nuclear and in cytosol. The results showed that when compared with the control, TNF-α significantly increased p65 protein phosphorylation, with the increasing rate of (773.0± 13.9)%. Meanwhile 1 TNF-α increased the translocation of p65 to nucleus, with the increasing rate of (698.6±19.7)%. Treatment with E2 inhibited p65 protein phosphorylation and nuclear translocation, with the inhibitory rate of (89.8±12.9)% and (86.2±11.8)% (n=5, *p<0.001 vs. CON; #p<0.001 vs. TNF-α), respectively.2.3 E2 inhibited TNF-α-induced phosphorylation of IkBaVSMCs were pretreated with E2 (10-8M) for 24 h and then treated with TNF-α (100 ng/mL) for 15 min. Western blot was used to detect the phosphorylation level of IκBα and the total expression of IκBα. TNF-α significantly increased IkBa protein phosphorylation, with the increasing rate of (776.5±17.3)%. This effect was inhibited by E2, with the inhibitory rate of (86.2±6.2)%(n=5,*p<0.001 vs. CON, #p<0.001 vs. TNF-α). Meanwhile, TNF-α and E2 had no effect on the total IκBa protein expression.2.4 E2 inhibited TNF-α-induced phosphorylation and activity of IKKα/β/γVSMCs were pretreated with E2 (10-8M) for 24 h and then treated with TNF-α (100 ng/mL) for 15 min. The phosphorylation level of IKKα/β/γ was detected by Western blot. IKK activity assay kit was used to measure the activity of IKKα/β/γ. TNF-α significantly increased the phosphorylation of IKKa/p/y protein, with the increasing rate of (626.8±13.6)%, (701.6±19.6)%, (539.7±16.9)%, respectively. This effect was inhibited by E2, with the inhibitory rate of (85.0±7.3)%, (86.4±6.5)%, (80.8±5.4)% (n=5,*p<0.001 vs. CON,#p<0.001 vs. TNF-α), respectively. By using IKK’activity assay kit, we found that TNF-a increased IKKa/β/γ activity, with the increasing rate of (792.0±18.9)%. This effect was inhibited by E2, with the inhibitory rate of (86.4±6.1)%(n=5,*p<0.001 vs. CON,#p<0.001 vs. TNF-a).2.5 The overexpression of p65 enhanced VSMCs proliferation and migrationVSMCs were transfected with p65 plasmid for 48 h and then treated with TNF-a (100 ng/mL) or E2(10-8M) for 24 h. As the control, parallel cells were transfected with empty vector plasmid and treated as indicated. The proliferation rate was measured by MTT. In cells transfected with empty vector plasmid, TNF-a increased VSMCs proliferation, with the increasing rate of(122.2±12.4)%, which was inhibited by the addition of E2, with the inhibitory rate of(27.7±6.9)%, (n=5,*p<0.001 vs.CON; #p<0.01 vs. TNF-a). However, in cells transfected with p65 plasmid, TNF-a showed the greater ability to increase VSMCs proliferation, with the increasing rate of (164.7±5.6)%(n=5,*p<0.001 vs.CON), while the inhibitory effect of E2 dispeared. The wound healing experimental results showed that when p65 protein was overexpressed, VSMCs showed the greater ability to migration, while in this condition E2 was unable to antagonize the TNF-a-promoted VSMCs migration. Summary:E2 inhibits the NF-kB signal pathway, then suppresses the TNF-a-induced TRAIL expression.Chapter 3 E2 suppressed NF-kB pathway via the release of NOIn this part, we investigated the mechanism underlying estrogen’s effect on NF-kB signalings and we examined the role of NO in this event.3.1 Different doses of E2 increased the release of NO in VSMCsVSMCs were pretreated with different concentrations of E2 (10"9M,10"8M, 10-7M, 10-6M) for 24h and the NO release in medium was measured by assay kit. The results showed that E2(10-9M, 10-8M,10-7M, 10-6M) all increased NO release, with the increasing rate of (146.4±9.3)%, (185.2±11.6)%, (165.2±12.8)%, (137.2±10.5)% (n=5,*p<0.05 vs. CON;**p<0.01 vs. CON), respectively.3.2 The time-dependent effect of E2 on NO release in VSMCsVSMCs were pretreated with E2(10-8M) for 6 h,12 h,24 h,48 h and the NO release in medium was measured by assay kit. The results showed that at these time point, E2 increased NO release from VSMCs, with the increasing rate of (126.4±8.5)%, (165.2±10.4)%, (213.2±15.6)%, (180.2±12.6)%(n=5,*p<0.05 vs. CON;**p<0.01 vs. CON), repectively.3.3 NO donor SNP inhibited TNF-a-induced TRAIL expressionVSMCs were pretreated with NO donor SNP(10-4M) for 1 h, then treated with 10-8M E2 for 24 h and with TNF-a(100 ng/ml) for additional 24 h, The TRAIL protein in medium plus cell lysates was measured by ELISA. The results showed that SNP significantly inhibited TNF-a-induced TRAIL protein expression, with the inhibitory rate of (63.2±6.7)%(n=5,#p<0.01 vs. TNF-a).Likewise, E2(10-8M) also inhibited TNF-a-induced TRAIL protein expression, with the inhibitory rate of (60.1 ±7.3)% (n=5,#p<0.01 vs. TNF-a).While there was no additive effect with SNP, suggesting that estrogen regulates TRAIL protein via NO pathway.3.4 NO inhibitor L-NMMA inhibited E2 effect on TNF-a-induced TRAIL expressionVSMCs were pretreated with NO inhibitor L-NMMA(10-5M) for 1 h, then treated with 10-8M E2 for 24 h and with TNF-a(100 ng/ml) for additional 24 h, The TRAIL protein in medium plus cell lysates was measured by ELISA. The results showed that L-NMMA had no effect on TNF-a-induced TRAIL protein expression, but it significantly inhibited E2(10-8M) effect on TRAIL protein, with the inhibitory rate of(58.2±8.8)%(n=5,*p<0.01 vs. CON).3.5 SNP and L-NMMA regulated p65/IkBa phosphorylationVSMCs were pretreated with NO donor SNP(10-4M) or NO inhibitor L-NMMA(10-5M)for 1 h, then treated with 10-8M E2 for 24 h and with TNF-a(100 ng/ml) for additional 24 h. Western blot was used to detect the phosphorylation level of NF-kB subunit p65 and IKBa.The results showed that SNP significantly inhibited TNF-a-induced phosphorylation of p65, IkBa, while L-NMMA significantly inhibited E2 effects on these protein phosphorylation. Summary:E2 inhibits the NF-kB signal pathway in VSMCs via the release of NO> then suppresses the TNF-a-induced TRAIL expression.Conclusions:1. TNF-a increases TRAIL expression, and promotes VSMCs proliferation and migration.2. E2 suppresses the TNF-a-induced TRAIL expression in VSMCs. and inhibits VSMCs proliferation and migration.3. E2 inhibits the NF-kB signal pathway in VSMCs via the release of NO. then suppresses the TNF-a-induced TRAIL expression. |
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