原發性皮質醛酮症:內皮前驅幹細胞是皮質醛酮血管病變和預後的指標

Endothelial progenitor cells in primary aldosteronism:a biomarker of severity for aldosterone vasculopathy and prognosis

吳允升(Vin-Cent Wu)

指導教授 ： 吳寬墩

共同指導教授 ： 陳玉怜

國立臺灣大學/醫學院/臨床醫學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.02617

摘要

原發性皮質醛酮症（PA）是一個常見可以治癒的高血壓疾病。過去二十年來的研究發現，原發性皮質醛酮症的盛行率增加，有的研究報告發現在高血壓病人中盛行率達12%。原發性皮質醛酮症病人具有較一般高血壓的病人更高的心血管疾病。新進研究更發現受損的內皮細胞層可以藉由骨髓釋放至血液中的內皮前驅幹細胞(EPC)來修復，藉以維持血管內皮細胞層的功能性與完整性，而新生血管能提供缺氧組織所需要的血液循環，以減少因組織缺氧所造成的器官傷害，促進血管新生亦是近年來用來治療缺氧性心臟病或周邊血管疾病相當受重視的治療方式之一。但是皮質醛酮素對內皮前驅幹細胞的影響則尚屬未知，原發性皮質醛酮症較原發性高血壓（EH）可導致較高心血管事件發生率，很可能通過鹽皮質激素受體（MR）導致內皮細胞功能障礙的。在開刀前原發性皮質醛酮瘤病人有較高的血清皮質醛酮素，開完刀後血清皮質醛酮素明顯減少，因此原發性皮質醛酮瘤病人，提供了一個很好的機會，可以觀察皮質醛酮素影響內皮前驅幹細胞變化的機會。本計劃探討原發性皮質醛酮症病人術前術後，病人血管內皮幹細胞數目和功能是否隨著病人高血壓、皮質醛酮素和鉀離子的改善而改善；因而收集病人術前和術後內皮細胞功能、血管硬度和心血管指標等資料。並且在體外測試研究皮質醛酮素對血管內皮前驅幹細胞生長、增生及凋亡影響的方式。我們推測，原發性皮質醛酮症患者有缺陷的內皮前驅幹細胞，即可能造成動脈血管硬化和疾病的進展。因此整個研究計畫除了將臨床發現銜接到實驗室的研究之外，也包括從細胞株到病人實際的研究，探尋與疾病相關的生物指標與病理機制。希望藉由研究單一性的高血壓疾病，期待找出未明的心臟血管疾病調控機轉，進而尋找治療方式，減緩皮質醛酮素引起心臟血管疾病，未來能提供高血壓治療另一個方向。
我們進行了一項前瞻性研究，原發性皮質醛酮症患者，在接受單側腎上腺切除術或給予安達通(spironolactone)治療前及治療後6個月，給予完整評估。本研究是在多個轉介中心醫院進行，觀測治療後動脈血管硬度和內皮前驅幹細胞的改變，以及術後高血壓的恢復。共113例原發性皮質醛酮症患者（87例診斷皮質醛酮腺瘤，26例特異性皮質醛酮增生）和55例高血壓患者。 原發性皮質醛酮症患者比高血壓患者有較高的動脈血管硬度（P = 0.006）。然而，在接受單側腎上腺切除或給予安達通(spironolactone)治療後6個月，可以觀察到原發性皮質醛酮症患者比高血壓患者有較低的內皮前驅幹細胞和循環內皮細胞集落形成單位（CFU）（P<0.05）。內皮前驅幹細胞被證實表現鹽皮質激素受體（MR），用廣義加法模型（Generalized Additive Models, GAMs）分析。血漿皮質醛酮素濃度與循環的內皮前驅幹細胞數量呈負相關（p值= 0.021）。動脈血管硬度與循環的內皮前驅幹細胞數量呈負相關（P = 0.029）。體外高劑量皮質醛酮素（10-5和10 – 6M）抑制內皮前驅幹細胞的增殖和血管生成。血清高敏感C反應蛋白(hS-CRP)與循環的內皮前驅幹細胞數量呈負相關（p值= 0.03），並且在手術後降低。單側腎上腺切除六個月後，腎上腺切除術患者有顯著減少氧化活性（Log [RHOCL計數]％，1 ±0.112對比0.933±0.102，p值=0.047）。其中45例接受單側腎上腺切除術，32例（71％）高血壓獲得治癒。術前的循環內皮前驅幹細胞[Log CD34+ / KDR+(%)]可以預測腎上腺切除後，高血壓是否可治癒，（p值= 0.003）。皮質醛酮素對內皮前驅幹細胞的增殖有一個雙相(biphasic)作用。低劑量皮質醛酮素（10-9和10 -8M）加入早期和晚期內皮前驅幹細胞與的數量顯著增加內皮前驅幹細胞分化。與此相反，較高劑量皮質醛酮（10-5,10-6 M）則顯著減少內皮前驅幹細胞。這兩種效應被加入安達通(spironolactone)所減弱。在體外血管生成實驗以皮質醛酮素影響晚期的內皮前驅幹細胞新生血管效應。經過5天的培養，高皮質醛酮素較低皮質醛酮素導致管狀形成功能顯著降低。這種作用是劑量皮質醛酮依賴性（平均總長度和節點數，均p<0.001）。皮質醛酮素，無論是高或低劑量，並不會影響內皮前驅幹細胞凋亡。皮質醛酮素也不會改變衰老相關β-半乳糖苷酶陽性的內皮前驅幹細胞百分比。
原發性皮質醛酮症患者有相對不足的內皮前驅幹細胞，可以引起皮質醛酮症患者血管病變。可以以內皮前驅幹細胞的數量和能力缺損表示。這樣皮質醛酮素導致內皮前驅幹細胞的數量減少的機制在皮質醛酮素直接激活的鹽皮質激素受體會影響內皮前驅幹細胞的增殖和間接影響通過高皮質醛酮素引起的高C反應蛋白和氧化壓力。經過長期的後續追蹤，發現單側腎上腺切除或給予安達通(spironolactone) ，提供治療上改善內皮前驅幹細胞數量的策略。此外，長期高皮質醛酮素抑制內皮前驅幹細胞生成和增殖和減弱血管新生功能。循環的內皮前驅幹細胞可能是一個有價值的生物標誌物，其可用來評估原發性皮質醛酮症患者的發病率及較高的動脈硬度與心血管疾病風險，並預測術後高血壓恢復治癒率。這些發現可能將皮質醛酮素誘導血管病變提供新的見解，對新生血管增加新的意義，並確定新的治療目標，增加高血壓心血管疾病研究的進展。

關鍵字

原發性皮質醛酮症 ； 皮質醛酮素 ； 內皮幹細胞 ； 群落單位 ； 動脈血管硬度 ； C反應蛋白質 ； 氧化壓力

並列摘要

Growing evidence has shown that high plasma aldosterone level leads to a risk of cardiovascular diseases (CVD), including fatal stroke and sudden cardiac death. In addition, long-term exposure to increased aldosterone levels resulted in renal and metabolic sequelae independently of the blood pressure level. This phenomenon is best demonstrated in patients with primary aldosteronism (PA) who have excessive and inappropriate production of aldosterone. The excessive production of aldosterone in PA is associated with a high incidence of cardiovascular events, in comparison with essential hypertension (EH).
A total of 113 PA patients (87 patients with a diagnosis of aldosterone-producing adenoma, 26 with idiopathic hyperaldosteronism) and 55 patients with EH were enrolled. PA patients had higher arterial stiffness than EH patients (p = 0.006). However, lower numbers of circulating EPC and endothelial colony forming units (CFU) were observed in PA patients than in EH patients (p<0.05), which was ameliorated at six months after adrenalectomy or treatment with spironolactone. Expression of MR was identified in EPC. The plasma aldosterone concentration was inversely correlated with the number of circulating EPC (p =0.021) by the general additive model. The circulating number of EPC was inversely correlated with arterial stiffness (p=0.029) and the Framingham risk score (p=0.001). Serum high sensitively C-reactive protein was inversely correlated with the circulating EPC number (p=0.03) and decreased in patients after operation. Among the 45 patients who went through unilateral adrenalectomy, 32 (71%) were cured of hypertension. Preoperative number of circulation of EPC (Log[EPC number%] > -3.6) could predicted the curability of hypertension after adrenalectomy. (p=0.003).
There was a biphasic effect of aldosterone on EPC proliferation. Incubation of early EPC with a low aldosterone level (10-9 and 10-8 M) for 5 days significantly increased the number of early EPC. However, higher dosage of aldosterone (10-6M) decreased the EPC number. EPC also showed a biphasic response to aldosterone, i.e., an increase of cell proliferation at low aldosterone level (10-9 and 10-8M), and a decrease at high aldosterone level (10-5 and 10-6M). Both effects were blunted by adding spironolactone. Aldosterone, in either a low or high dose, did not affect the percentage of senescence-associated β-galactosidase-positive EPC or apoptosis of EPC.
After 5 days’ incubation with 10-6 M aldosterone, the functional capacity for tube formation of late EPC was significantly reduced (p<0.01). In contrast, a low dosages of aldosterone (10-8~10-9M) increased tube formation. This effect of aldosterone was dose-dependent (average total length and nodes number, all < 0.001).
Our study clearly showed that human EPC express MR both at gene and protein levels. The expression of MR and 11β-HSD2 in EPC, therefore, indicates that EPC have the capacity to modulate gene expression in response to aldosterone specifically. In accordance with the animal experiments, our study reveals a long-term in vivo effect of high aldosterone level on the number of circulating EPC in PA patients, with an inverse association between PAC and EPC numbers. A correlation of PAC with oxidative stress and endothelial inflammation was observed in PA patients, and the oxidative stress was attenuated after adrenalectomy. Increased intracellular ROS may result in EPC mobilization and impaired neovascularization capacity. Accordingly, CRP augments the effect of aldosterone on endothelial cell stiffness. In our study, a significant correlation between CRP level and aldosterone was noted in PA patients. Moreover, there was an inverse correlation between CRP and EPC numbers, and a reduction of CRP level accompanied by an increase of EPC number was noted in APA patients after adrenalectomy. Chronic aldosterone excess leads to vascular morphological change (wall thickening and carotid artery fibrosis) and vascular dysfunction (central stiffness), independent of blood pressure (BP). In the present study, we further demonstrate that the lower the preoperative EPC number, the higher the risk of residual hypertension after adrenalectomy. The finding indicates that EPC play an important role in both normal endothelial function and vasculature.
The inverse correlation between PAC and EPC number and the change of EPC numbers in response to the treatment of PA indicates that aldosterone contributes to the decreased EPC number in PA patients. The mechanisms accounting for low EPC numbers in PA patients are the direct activation of MR on EPC proliferation and an indirect effect through a high CRP level and oxidative stress caused by an excess of aldosterone. Low EPC numbers in PA plays a crucial role in the high incidence of arterial stiffness and in predicting residual hypertension in APA patients after adrenalectomy.

並列關鍵字

Primary aldosteronism ； endothelial progenitor cells ； colony forming units ； arterial stiffness ； CRP ； oxidative stress

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