以蛋白質影像化研究探討亞硝酸鹽保護內皮細胞免於缺氧性傷害的機制

Abstract

當內皮細胞屏障失去功能時會使血管內環境失去平衡，導致許多病理情況發生，例如:微血管滲漏症候群、動脈粥狀硬化、腫瘤轉移等。其中缺氧是造成血管內皮通透性增加的主要原因，加速內皮細胞屏障功能的惡化，不過詳細機制尚未被發現。最近的研究發現一氧化氮可以透過蛋白質半胱胺酸亞硝基化修飾 (cysteine S-nitrosylation) 調控不同訊息路徑，至今有幾百個蛋白質被認為有潛力透過亞硝基化調控，但是詳細生理及病理機制尚未了解。在本篇的研究中主要探討內皮細胞產生的一氧化氮如何調控訊息間的平衡，對於維持內皮細胞屏障功能扮演非常重要的角色。因此，我們建立了一個新穎的偵測方法，利用化學標記並且利用自製特定的抗體偵測，使我們可以由免疫螢光染色看到細胞內經亞硝基化的蛋白質。由此方式偵測可以得知在動脈內皮細胞質中的蛋白質持續被亞硝基化的現象。令人興奮的是在低氧壓力下蛋白質的亞硝基化程度明顯地降低，並且伴隨內皮細胞屏障功能損傷。因此指出有些半胱胺酸水解蛋白酶 (cysteine-protease) 的受質與內皮細胞屏障功能有某種程度上的關聯，而且我們已經確認過caspase-3的去亞硝基化在缺氧導致內皮細胞傷害扮演關鍵角色。重點是當亞硝酸鹽於缺氧情況下加入內皮細胞中，具有生物活性的一氧化氮含量增促加使蛋白質的亞硝基化修飾提高，caspase-3失活同時也抑制內皮細胞屏障功能傷。而在這個過程中需要將亞硝酸鹽經由亞硝酸還原酶轉變成一氧化氮。因此我們的研究顯示亞硝酸鹽如何扮演一個細胞保護的角色對抗缺氧引發內皮細胞屏障的缺失，所以提出亞硝酸鹽的治療在解決缺氧於血管系統中造成的缺失及疾病具有極大的潛力。

Dysfunction of endothelial barrier causes problems in vascular homeostasis, leading to pathological conditions such as capillary leakage syndrome, atherosclerosis, and tumor metastasis. It has known that hypoxia is a main driving force that promotes endothelial permeability and accelerates the progress of barrier dysfunction. However, the precise underlying mechanism of this process remains elusive. Recent studies have suggested that nitric oxide (NO) acts as a modulator of diverse signaling pathways via protein Cys S-nitrosylation. To date hundreds of proteins are considered being potentially S-nitrosylated. However, the spatiotemporal control of S-nitrosylation under physiological and pathological settings is unknown. Here we present a case to demonstrate how endothelial NO regulates signaling homeostasis, which is essential for the maintenance of barrier function. For this, we have developed a novel method using the combination of chemical labeling and subsequent detection by house-made specific antibodies that allows microscopic visualization of S-nitrosylated proteins in cells. The application of this method indicated that cytosolic proteins in aortic endothelia were constitutively S-nitrosylated. Surprisingly, in response to hypoxic stress, protein S-nitrosylaion levels decreased dramatically, concomitant with the onset of endothelial barrier dysfunction. In addition, it suggested that some substrates of Cys-proteases were related to endothelial barrier function, and we have already identified caspase-3 in the denitrosylated form as a key player in hypoxia-induced endothelial injury. Importantly, when nitrite was supplied to endothelia under hypoxia, bioavailable levels of NO were increased, resulting in rebound of protein S-nitrosylation, inactivation of endogenous caspase-3 and inhibition of barrier dysfunction. Furthermore, this process required conversion of nitrite to bioactive nitric oxide in a nitrite reductase-dependent manner. Together, our study demonstrates how nitrite acts as a cytoprotector for endothelia against hypoxia-induced barrier dysfunction. We propose that nitrite treatment may have a great potential in resolving hypoxia-induced disorders and diseases in the vascular system.