C-反應蛋白與機械性壓力於動脈粥狀硬化與心肌肥大中的致病機轉探討

動脈粥狀硬化與心肌肥大病變是常見的心血管疾病，病生理過程都有許多基因參與表現，相關聯的訊息傳遞路徑也相當複雜，病程演變接續造成的心肌梗塞與心臟衰竭也都會形成病患嚴重的生命威脅。本研究中試圖找出動脈粥狀硬化與心肌肥大初期參與調控的重要接受器，除了利用小片段干擾RNA確認此基因所扮演的角色外，也嘗試以小片段干擾RNA、藥物或拮抗劑阻斷基因表現，藉由調控接受器基因表現來達到抑制病程進行的目的。第一部份探討動脈粥狀硬化病程的初期，C-反應蛋白（CRP）是透過內皮細胞上哪個接受器來開啟發炎的訊息。實驗選用人類臍靜脈與大動脈內皮細胞，在經過加入CRP培養後，發現細胞中的NF-κB路徑會被活化，血管細胞黏附因子-1 (vascular cell adhesion molecule-1) 蛋白質表現增加，而CD32的mRNA與蛋白質都會向上調節。利用針對CD32的小片段干擾RNA、SB203580 (p38抑制劑) 都會有抑制CRP導致發炎的效果。此結果證明CRP在動脈與靜脈內皮細胞上的訊息傳遞都需要CD32這個接受器，並且有透過p38路徑。第二部份探討藥物simvastatin與CRP誘導CD32的關係，並比較CD32的小片段干擾RNA與simvastatin在阻斷發炎上的差別。實驗假設simvastatin抑制CRP導致的內皮細胞中發炎反應是透過mevalonate路徑過程產物。實驗以人類臍靜脈細胞加入CRP刺激後，檢測simvastatin、CD32的小片段干擾RNA的抑制效果。結果顯示simvastatin前處理明顯抑制CRP引起的CD32向上調節，也顯著抑制NF-κB活化、VCAM-1蛋白質的表現與單核球黏附在內皮細胞上的數目。這些抑制效果會在加入mevalonate與geranylgeranyl pyrophosphate而明顯回復，加入farnesyl pyrophosphate則沒有明顯影響。至於CD32的小片段干擾RNA所抑制的CRP相關發炎反應，則不會在加入mevalonate與geranylgeranyl pyrophosphate處理下而有所改變。這代表simvastatin抑制CRP在內皮細胞發炎的反應是透過mevalonate與其下游產物geranylgeranyl pyrophosphate而來，至於的小片段干擾RNA則不是經由此路徑達到抑制的效果。第三部份利用活體內與活體外的模式探討心肌肥大病程中血清素2B接受器（SR2BR）所扮演的角色。近期的研究發現血清素透過SR2BR可能有調控心臟病生理狀況的功能。實驗中選用Wistar大鼠進行動脈狹窄手術以及新生鼠心肌細胞機械性延展的過度壓力模擬模式，經過動脈狹窄手術兩週後的大鼠，血液中的血清素濃度、心臟組織中SR2BR的mRNA與蛋白質表現量都有明顯增加。以SR2BR抑制劑SB215505治療則可以明顯改善手術後老鼠的心臟功能並減少brain natriuretic peptide (BNP) 及增加nerve growth factor-β的表現，但不會抑制SR2BR的向上調節。在活體心肌細胞機械性延展加上1μM血清素培養的實驗中，所增加的NF-κB核內轉移與BNP蛋白質的增加都可以被針對SR2BR的小片段干擾RNA與caffeic acid phenethyl ester所抑制。根據這些結果都顯示SR2BR在壓力過度負荷的心肌病變當中扮演重要的角色，而且其在心肌細胞中的下游訊息有透過NF-κB來調控BNP的表現。總括來說，本研究中確認動脈粥狀硬化病程初期CRP造成內皮細胞發炎反應過程中，內皮細胞上的CD32接受器扮演重要的角色。壓力過度負荷引致的心肌肥大病程中，心肌細胞上的SR2BR表現甚為重要。藉由阻斷這兩個接受器表現都可在實驗中達到抑制病程進行的效果。期許對於接受器更深入的了解與研究後，能發展成為治療標的，以提供對於動脈粥狀硬化與心肌肥大疾病治療上另一更佳的藥物選擇。

關鍵字

動脈粥狀硬化；心肌肥大； C-反應蛋白； CD32接受器；血清素2B接受器；小片段干擾RNA

並列摘要

Atherosclerosis and pressure-overload cardiac hypertrophy are common diseases in cardiovascular medicine. A plethora of gene expressions and complicated signaling pathways are involved in the pathogenesis. In this study, we tried to identify the major receptor that was involved in the early stage of atherosclerosis and cardiac hypertrophy. We used siRNA for investigating the role of these receptors and downstream signaling pathways. We also experimentally attenuated the receptors up-regulation by specific siRNA, statins or antagonist. In the first part of the study, we investigated the relationship between C-reactive protein (CRP) and atherosclerosis and the exact receptor involved in CRP-induced endothelial changes. Human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) were used for the experiments. After incubation with CRP, immunobltting showed a significant NF-κB activation and vascular cell adhesion molecule-1 (VCAM-1) expression. The mRNA level of CD32, the major binding protein for CRP in HUVEC, increased significantly as measured by Northern blot. When these HUVEC were transfected with siRNA directed against CD32 (siCD32), the mRNA of CD32 decreased significantly. The IκB degradation, NF-κB nuclear translocation and VCAM-1 upregulation induced by CRP were all blocked by treatment with siRNA against CD32. SB203580, a P38 inhibitor, significantly attenuated the CRP induced responses while SP600125 (c-jun kinase inhibitor) did not. CRP induced IκB degradation, NF-κB nuclear translocation and VCAM-1 protein expression in HUVEC and HAEC. CRP also increased CD32 expression in HUVEC and HAEC. All the above changed were dependent on CD 32, which might serve as the receptor for CRP and mediated the effects of CRP on HUVEC and HAEC. In the second part of the study, we compared the effects of simvastatin and siRNA against CD32 in CRP-induced pro-inflammatory changes in endothelial cells. We tested the hypothesis that simvastatin inhibited CRP-induced pro-inflammatory changes in endothelial cells by decreasing mevalonate pathway products. HUVEC were incubated with CRP and measurement of CD32, NF-κB activation, VCAM-1 expression and monocyte adhesion were performed. The effects of simvastatin, siCD32 and mevalonate pathway products were also examined. After incubation with CRP, there was a significant increase of CD32 in HUVEC. IB degradation and NF-κB nuclear translocation were also observed. Pre-treatment with simvastatin significantly attenuated the CRP-induced CD32 expression and NF-κB activation. Simvastatin also decreased CRP-induced VCAM-1 expression and reduced monocyte adhesion on endothelial cells. The inhibitory effects of simvastatin were significantly reversed by adding mevalonate, geranylgeranyl pyrophosphate but not by adding farnesyl pyrophosphate. Pre-treatment with siCD32 also decreased CRP induced CD32 expression and IκB degradation. However, neither mevalonate nor geranylgeranyl pyrophosphate reversed the effects of siCD32. CRP induced CD32 expression, IκB degradation, NF-κB nuclear translocation and VCAM-1 protein expression in HUVEC. All the above changes were attenuated by simvastatin. A decrease of mevalonate and subsequent geranylgeranyl pyrophosphate contributes to the inhibitory effects of simvastatin. In the third part of the study, we identified the role of serotonin 2B receptors (SR2BR) in the pathogenesis of cardiac hypertrophy in vivo and in vitro. Recent data suggested that SR2BR might be involved in some cardiac physiopathological situations. Wistar rats of aortic banding and neonatal cardiomyocyte with mechanical stretch were used for cardiomyopathy models. After two weeks of aortic banding surgery, serum serotonin, mRNA and protein expression of SR2BR increased significantly. Selective SR2BR antagonist, SB215505 (SB), significantly reduced the increase in heart weight, the ratio of heart weight and body weight, interventricular septum thickness, left ventricular posterior wall thickness, left ventricular mass and brain natriuretic peptide (BNP) protein but did not significantly attenuate the up-regulation of SR2BR protein expression in rats after aortic banding for three weeks. Down-regulation of nerve growth factor-β in aortic banding rat was also reversed by SB. When cultured cardiomyocytes were subjected to mechanical stretch and serotonin 1 μM, the level of SR2BR and BNP protein increased time-dependently. When transfected with specific siRNA against SR2BR or pretreated with caffeic acid phenethyl ester, the increase of nuclear factor-κB (NF-κB) translocation and BNP protein in cardiomyocytes were both reversed. These results suggested that SR2BR expression was involved in excess of pressure-induced cardiomyopathy and its downstream signaling may through NF-κB to modulate BNP expression in cardiomyocyte. In conclusion, our study confirmed that CD32 mediate the CRP-induced inflammation in endothelial cells. Treatment with siRNA against CD32 and simvastatin both reduced the up-regulation of CD32 and the effects of CRP. On the other hand, SR2BR expression on cardiomyocyte played an important role in pressure-induced cardiac hypertrophy. Therefore, CD32 and SR2BR might serve as therapeutic targets for atherosclerosis and cardiac hypertrophy in the nearly future.

並列關鍵字

atherosclerosis ； cardiac hypertrophy ； C-reactive protein ； CD32 receptorserotonin 2B receptor ； small interference RNA

參考文獻

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Bang R, Marnell L, Mold C, Stein MP, Clos KT, Chivington-Buck C, et al. Analysis of binding sites in human C-reactive protein for Fcgamma RI, Fcgamma RIIa and C1q by site-directed mutagenesis. J Biol Chem 2005;280:25095-102.

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C-反應蛋白與機械性壓力於動脈粥狀硬化與心肌肥大中的致病機轉探討

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