The Effects Of Atrovastatin On Interleukin-1β-induced Cathepsin S Expression And Rat Vascular Smooth Muscle Cell Migration

Background:Many cytokines and growth factors are activated in thedevelopment of restenosis after balloon angioplasty. The cross-talkbetween these cytokines and growrh factors amplifies the inflammationin the vascular wall, resulting in the neointima formation. Interleukin-1β(IL-1β) is a proinflammatory cytokine and a potent inducer for a numberof genes including other cytokines and growth factors. IL-1βisup-regulated after balloon angioplasty and play a distinct role in theneointima formation. Cathepsin S (Cat S) could induce vascular smoothcell (VSMC) migration and neointima formation. It is reported thatIL-1βstimulate Cat S expression in VSMC, but the time-response anddose-response effect on IL-1β-induced Cat S expression is not completeunderstood. It is not clear about the potential signaling pathwaymechanism for the effect.Objective:This study was aimed to explore the dose-response andtime-response effect of IL-1βon the expression of Cat S in rat VSMCand the effect of different specific signaling pathway inhibitors on Cat Sexpression stimulated by IL-1β, elucidating the underlying signaling pathway mechanism for the expression of Cat S stimulated by IL-1β.Methods:Rat vascular smooth muscle cells were isolated from Sprague-Dawley rat thoracic aortas using the enzyme digestion technique. VSMCbetween fourth and fifth generation were used in the test. The cells weretreated with different concentrations of IL-1βfor 24 hours or with10ng/ml IL-1βfor different duration of time. The cells were also treatedwith the ERK pathway inhibitor PD98059, the P38MAPK pathwayinhibitor SB203580, the JNK pathway inhibitor SP600125, the PI3Kpathway inhibitor LY294002 and the NF-κB inhibitor PDTC for 24h inthe presence of 10ng/ml IL-1β. Cat S expression was measured using thereverse transcription polymerase chain reaction (RT-PCR) and theimmunocytochemistry technique.Results:1. Normal VSMC had very little Cat S mRNA expression. VSMCtreated with different concentrations of IL-1βhad higher Cat S mRNAexpression. The concentration of IL-1βwas higher, the level of Cat Sexpression was higher. The Cat S mRNA expression was 0.15±0.02,0.38±0.04 and 0.88±0.07 respectively while IL-1βat the concentrationof 0.1 ng/ml, 1 ng/ml and 10ng/ml (all P＜0.01). The stimulating effect ofIL-1βon Cat S secretion was optimized at the concentration of 10ng/ml.The 100ng/ml IL-1βgroup had lower level of Cat S expression(0.32±0.03) compared with 10ng/ml IL-1βgroup, P＜0.01.2. Compared with normal VSMC, the VSMC treated with differentconcentrations of IL-1βhad higher Cat S immunocytochemistry score (all P＜0.01). The Cat S score was 4.25±0.30, 10.50±1.13 and18.94±1.50 respectively while IL-1βat the concentration of 0.1ng/ml,1ng/ml and 10ng/ml (all P＜0.01). The Cat S score in 100ng/ml IL-1βgroup (9.56±0.82) significantly decreased compared with that of10ng/ml IL-1β(P＜0.01).3. Compared with normal VSMC, the cells using 10ng/ml IL-1βtreatment with different durations of time had higher Cat S mRNAexpression (all P＜0.01). The IL-1βduration of time was longer, the levelof Cat S expression was higher. Cat S expression was 0.20±0.03,0.51±0.04 and 0.88±0.08 respectively while IL-1βduration of time at 6h,12h and 24h (all P＜0.01). The stimulating effect of IL-1βon Cat Ssecretion was optimized at a time of 24h. The Cat S expressionstimulated by 48h IL-1β(0.42±0.04) significantly decreased comparedwith that of 24h IL-1βgroup (P＜0.01).4. The Cat S score was 6.42±0.55, 12.67±1.05 and 18.94±1.50respectively while IL-1βduration of time at 6h, 12h and 24h (allP＜0.01). The Cat S score in 48h IL-1βgroup (10.73±0.94) decreasedcompared with that of 10ng/ml IL-1βgroup (P＜0.01).5. Compared with that of IL-1βgroup (0.89±0.07), Cat S mRNAexpression significantly decreased to 0.41±0.04, 0.45±0.04, 0.43±0.03,0.39±0.03 and 0.32±0.03 respectively when the cells were treated withPD98059, SB203580, SP600125, LY294002 and PDTC for 24h in thepresence of 1L-1β(all P＜0.01).6. Compared with that of 10ng/ml IL-1βgroup (18.94±1.50), theCat S score significantly decreased to 9.50±0.65, 9.45±0.73, 9.68±0.76, 9.03±0.81 and 8.85±0.67 respectively when the cells were treated withPD98059, SB203580, SP600125, LY294002 and PDTC for 24h in thepresence of IL-1β(all P＜0.01).ConclusionsIL-1βcan stimulate Cat S expression in VSMC in a dose-dependentand time-dependent manner. IL-1βmay stimulate Cat S expressionthrough the activation of ERK, P38, JNK, PI3K and NF-κB. Background:The migration of vascular smooth muscle cell (VSMC) is a keyevent in the vascular restenosis that frequently follows angioplasty.VSMC migration and neointimal hyperplasia are promoted bydegradation of extracelluar matrix by cathepsin S (Cat S). Nuclearfactor-κB (NF-κB) is an important transcription factor which canregulate a variety of gene products. Atorvastatin can inhibit VSMCmigration, but the underlying mechanism is not completely understood.It is not clear whether or not atorvastastin can down-regulate Cat Sexpression through inhibit NF-κB and then attenuate VSMC migration.Objective:The present study was aimed to investigate the effect of atorvastatinon IL-1β-induced rat VSMC migration, NF-κB and Cat S expression,trying to elucidate the mechanism of atorvastatin inhibiting cellmigration.Methods:Rat vascular smooth muscle cells were isolated from Sprague-Dawley rat thoracic aortas using the enzyme digestion technique.VSMC between fourth and fifth generation were used in the test. TheBoyden chamber test was used to measure the role of E64d and differentconcentrations of atorvastatin on the cell migration stimulated by IL-1β. The cells were treated with 10ng/ml IL-1βand then differentconcentrations of atorvastatin were added into it for 24 hours. NF-κBexpression was measured using the immunocytochemistry method. Theexpression of Cat S was evaluated using the immunocytochemistry andRT-PCR.Results:1. Atorvastatin could inhibit IL-1β-induced VSMC migration in adose-dependent manner. Compared with that of IL-1βgroup (27±4),VSMC migration decreased by 22.22%, and 55.56% respectively in1μmol/L and 10μmol/L atorvastatin group (all P＜0.01). There was nosignificant difference between 0.1μmol/L atorvastatin group (25±4) andIL-1βgroup (P＞0.05). VSMC migration in E64d group (10±2) alsosignificantly decreased compared with that of IL-1βgroup (P＜0.01).2. Atorvaststin could inhibit IL-1β-induced Cat SmRNA expressionin a dose-dependent manner. Compared with that of IL-1βgroup(0.88±0.08), Cat S expression in 1μmol/L and 10μmol/L atorvastatingroup decreased by 19.32% and 56.81% respectively (all P＜0.01).There was no significant difference between 0.1μmol/L atorvastatingroup (0.84±0.07) and IL-1βgroup (P＞0.05).3. Compared with that of IL-1βgroup (18.94±1.50), the Catimmunocytochemistry score of 1μmol/L and 10μmol/L atorvastatingroup decreased to 15.56±1.13 and 9.72±0.78 respectively (all P＜0.01).The effect of atorvastatin was dose-dependent. The Cat S score had nosignificant difference between 0.1μmol/L atorvastatin group (18.35±1.36)and IL-1βgroup (P＞0.05). 4. Atorvaststin could inhibit IL-1β-induced NF-κB expression in adose-dependent manner. Compared with that of IL-1βgroup(18.85±1.46), the NF-κB score of 1μmol/L and 10μmol/L atorvastatingroup decreased to 15.37±1.15 and 9.58±0.80 respectively (all P＜0.01).There was no significant difference in the expression of NF-κB between0.1μmol/L atorvastatin group (18.20±1.53) and IL-1βgroup (P＞0.05).Conclusion:The cathepsin play an important role in IL-1β-induced VSMCmigration, Atrovastatin could inhibit IL-1β-induced VSMC migration ina dose-dependent manner. Atorvastatin could decrease the expression ofNF-κB and Cat S stimulated by IL-1βin a dose-dependent manner.Atorvastatin may decrease Cat S expression through down-regulate theactivation of NF-κB, that might inhibit the VSMC migration. Background:Percutaneous transluminal coronary angioplasty is a majorprocedure for treating patients with ischemic coronary artery diseases;however, approximately 20-30% of patients suffer from vascularrestenosis within 6 months after the procedure. Neointima formationplays an important role in the pathogenesis of restenosis afterangioplasty. It has been believed that vascular smooth muscle cell(VSMC) migration from tunica media to the intima is a key step in thedevelopment of neointima lesion formation. Cathepsin S could degradethe compounds of extracelluar matrix (ECM), improving VSMCmigration, playing an important role in neointima formation.Atorvastatin can inhibit the neointima formation, but the underlyingmechanism is not completely understood. It is not clear whether or notatorvastastin can inhibit neointima formation through decrease Cat Sexpression.Objectives:The study was aimed to explore the effect of atrovastatin on theneointima formation, NF-κB and Cat S expression in rat balloon-injuredcarotid artery, elucidating the potential mechanisms of atorvastatin fordecreasing neointima formation. Methods:32 male Sprague-Dawley rats were randomly divided into controlgroup, sham operation group, operation group and atrovastatin group.Sham operation group inputed the balloon to the carotid artery withoutangioplasty. The rats of operation group and atrovastatin group wereperformed with balloon angioplasty to the left common carotid artery.After the operation, the rats of atrovastatin group were fed withatrovastatin 10mg·kg^-1·d^-1. The serum levels of IL-1βwere measuredbefore operation, first day and 28th days after operation usingenzyme-link-immunosorbent assay (ELISA). The carotid arteries wereexcised 28 days after operation. The NF-κB expression was measuredusing immunohistochemistry. The Cat S expression was measured usingthe RT-PCR and immunohistochemistry techniques.Results:1. There was no significantly difference in serum IL-1βlevels infour groups before operation, first day and 28th day after operation.2. Compared with control group and sham operation group,operation group had a significantly increase in intima area[(148.62±8.84)×10³Î¼m²] and the ratio of intima/media area (2.06±0.15),all P＜0.01. Atorvastatin group had a significantly decrease in intima area[(64.75±5.12)×10³Î¼m²] and the ratio of intima/media area (0.90±0.05)compared with operation group. The media area had no significantlydifference in four groups (P＞0.05).3. Compared with that in control and sham operation group, Cat SmRNA expression significantly increased in operation group (0.83±0.07), P＜0.01. The Cat S expression in atorvastatin group (0.48±0.04)significantly decreased compared with operation group, P＜0.01.4. There was no Cat S expression in control and sham operationgroup. The Cat S immunohistochemistry score in atorvastatin group(8.50±0.73) significantly decreased compared with operation group(15.28±1.42), P＜0.01.5. There was no NF-κB expression in control and sham operationgroup. The NF-κB score significantly decreased in atorvastatin group(6.74±0.54) compared with operation group (12.45±1.30), P＜0.01.6. The intima area was significantly positively correlated with theCat S mRNA, the Cat S score and NF-κB score (r=0.58, 0.56 and 0.53,all P＜0.05).Conclusions:Balloon angioplasty induced rat carotid artery neointimahyperplasia, increasing Cat S and NF-κB expression in intima.Atorvastatin might decrease Cat S expression through inhibiting NF-κB,that would attenuate the neointima formation. IL-1βlevels in serum hasnot direct relationship with Cat expression in vascular wall. Atorvastatindo not decrease Cat S expression through inhibiting serum IL-1βlevels.