內質網蛋白TXNDC5藉由調節eNOS導致內皮細胞功能異常及動脈粥狀硬化

Abstract

研究背景：動脈粥狀硬化(Atherosclerosis)是一種漸進性的發炎疾病，其病理特徵在於過度的脂肪堆積於血管內膜，並進一步形成動脈粥狀硬化斑塊(atherosclerotic plaques)。動脈粥狀硬化造成的心血管疾病在世界各國都是重要的死因之一。動脈粥狀硬化斑塊好發於血管彎曲、分枝及分岔的部位，主要是因為其幾何結構容易使血液產生擾流(disturbed flow)，造成血管內皮細胞的機械力感受器(mechanosensor)接收到的切應力(shear stress)降低，進而引發致病性的不良反應，包括促進血管內皮細胞發炎，破壞細胞連接(cell junction)，及刺激動脈硬化相關訊息傳遞而導致動脈粥狀硬化發生。然而，內皮細胞受血流切應力調節導致功能異常的分子機轉目前仍不清楚。透過結合生物資訊學與臨床及基礎的實驗，我們最近找到了對動脈粥狀硬化及血管內皮細胞受血流切應力調節具有重要性的新型媒介，是一個內質網蛋白TXNDC5，對於內皮細胞功能異常及動脈粥狀硬化發生具有潛在重要的影響力。 研究目的：探討TXNDC5在內皮細胞功能異常及動脈粥狀硬化中扮演的角色，評估是否能作為治療動脈粥狀硬化的標的。 實驗方法與結果：TXNDC5的基因及蛋白表達量在受到擾流刺激的人類主動脈內皮細胞中有明顯上升，同時在人類動脈粥狀硬化病灶上也有較高的表現量。透過在ApoE-/-小鼠施行頸動脈部分結紮(partial carotid artery ligation)手術以製造體內擾流動物模型，我們發現TXNDC5在內皮細胞中被大量表現，此外，在ApoE-/-小鼠餵食12週高脂飼料的動脈硬化動物模型中也發現同樣的結果。而這樣的現象，同時伴隨著具有血管保護效果的內皮一氧化氮合成酶(endothelial nitric oxide synthase, eNOS)的表現量下降，以及內皮細胞功能的異常，如一氧化氮(nitric oxide, NO)生成減少、內皮穿透性(permeability)及內皮細胞周轉率(turnover rate)增加。有趣的是，TXNDC5的敲弱(knockdown)或過度表達(overexpression)並不影響eNOS的基因表現。HSP90是一個調節eNOS功能重要的蛋白，在敲弱HSP90情況下，則可以反轉因TXNDC5缺乏造成eNOS上調的結果，說明了HSP90在TXNDC5調節的eNOS的過程中是關鍵的角色。重要的是，在 ApoE-/-小鼠中剔除(knockout) Txndc5，可以有效的減少高血脂刺激或是頸動脈部分結紮手術所導致的動脈粥狀硬化，同時也發現了減少eNOS下調的結果。這樣的血管保護效果則在給予了小鼠一氧化氮合成酶(nitric oxide synthase, NOS)抑制劑： L-NAME，而消失。這些實驗結果證實了TXNDC5引致的內皮細胞功能異常及動脈粥狀硬化是透過調節eNOS而發生的。我們更進一步發現在ApoE-/-小鼠中將內皮細胞專一性剔除Txndc5，也可以顯著的減少頸動脈部分結紮手術導致的動脈粥狀硬化，闡明TXNDC5特別在內皮細胞中對動脈粥狀硬化的發生具有格外的重要性。總結而論，這些實驗闡述TXNDC5透過影響eNOS，使得在血液擾流與高血脂刺激下造成動脈粥狀硬化，而扮演著舉足輕重的角色。 結論：目前的研究證明了內質網蛋白TXNDC5，是藉由調節eNOS而影響內皮細胞功能異常與動脈粥狀硬化。透過剔除Txndc5可以減少ApoE-/- 小鼠因血液擾流及高血脂所引致的動脈粥狀硬化斑塊的產生，因此，TXNDC5可能是一個具有預防或治療動脈粥狀硬化造成心血管疾病的新型治療標的。

Background: Atherosclerosis is a progressive inflammatory disease characterized by the accumulation of lipids in the arterial intima and build-up of atherosclerotic plaques. Atherosclerotic cardiovascular diseases (ASCVD) are the leading cause of death around the world. Interestingly, atherosclerotic lesions preferentially develop at the sites of curvature, branching and bifurcation, the areas under abnormal hemodynamics in the arteries, which leads to lower vessel wall shear stress detected by endothelium and trigger adverse reactions. However, the exact molecular mechanism of mechanoregulation in response to shear stress remains unclear. Exploiting the bioinformatic analysis and RNASeq dataset obtained from human aortic endothelial cells (HAECs) stimulated with atherogenic disturbed flow (DF), we identified thioredoxin domain containing 5 (TXNDC5), an endoplasmic reticulum protein with the enzyme activity of a protein disulfide isomerase, as a potentially important mediator of endothelial dysfunction and atherosclerosis. Purpose: To determine the role of TXNDC5 in endothelial dysfunction and atherosclerosis, and to further explore potential therapeutic approach against ASCVD. Methods & Results: TXNDC5 expression was significantly upregulated in HAECs exposed to DF, as well as in the human atherosclerotic lesions. Endothelial TXNDC5 level was markedly upregulated in the common carotid arteries (CCA) from ApoE-/- mice after partial carotid artery ligation (PCAL), a well-described method to generate DF in CCA, as well as from ApoE-/- mice fed with 12-week high-fat diet (HFD). The upregulation of endothelial TXNDC5 was accompanied by downregulation of endothelial nitric oxide synthase (eNOS), an atheroprotective factor in endothelial cells. Functional assay also demonstrated TXNDC5 induced-endothelial dysfunction, such as decreasing NO production, increasing cell permeability and turnover rate. Interestingly, neither knockdown nor overexpression of TXNDC5 affected NOS3 gene expression. Depletion of HSP90, a protective molecule for eNOS regulation, reversed TXNDC5 deficiency-induced upregulation of eNOS in HAECs, suggesting HSP90 was critical for TXNDC5 in regulating eNOS. Importantly, ApoE-/- x Txndc5-/-, compared with ApoE-/-, mice showed significantly reduced aortic atherosclerotic lesion areas after 12-week HFD treatment. Similarly, targeted deletion of Txndc5 prevented exacerbation of atheroma and eNOS downregulation resulted from 2-week PCAL and HFD in ApoE-/- mice. These atheroprotective effects of TXNDC5 deficiency were diminished after treatment with L-NAME, a NOS inhibitor. These data strongly suggest that TXNDC5 promotes endothelial dysfunction and atherosclerosis by regulating eNOS. Moreover, endothelium-specific knockout of Txndc5 (Tie2-Cre/ERT2 x Txndc5fl/fl) in ApoE-/- mice also showed significantly attenuated atherosclerotic lesion caused by PCAL, demonstrating the atherogenic role of endothelial TXNDC5. Taken together, the results from these experiments suggest an essential role of TXNDC5 in DF/dyslipidemia-induced endothelial dysfunction and atherosclerosis through eNOS regulation. Conclusion: The present study revealed a previously undiscovered role of ER protein TXNDC5 in modulating endothelial function in response to atherogenic stimuli by post-transcriptional regulation of eNOS. Targeted deletion of Txndc5 protects against endothelial dysfunction and atherosclerosis in ApoE-/- mice. Targeting TXNDC5, therefore, could be a novel therapeutic approach to treat or prevent atherosclerotic cardiovascular diseases.