探討肺動脈高壓治療的機制: 第一型血基質氧化酶和內皮激素阻斷劑的角色

Abstract

摘要 肺動脈高血壓是一種罕見的肺血管疾病，其主要致病機轉為肺血管收縮,肺血管壁重塑和血栓形成，導致肺動脈血管阻力增加與肺動脈壓力升高，若不積極治療，會因右心衰竭而終至死亡。肺高壓的傳統治療方式包括利尿劑,抗凝血劑,毛地黃,氧氣治療或給予血管舒張劑(例如鈣離子阻斷劑或前列腺素合成劑)等，但因傳統治療方式的療效不佳，病患的生活品質與長期存活率不甚良好。為了治療此罕見疾病，現有許多學者專注在研究新興的肺高壓治療藥物，並探究其相關的作用機轉，期盼能開啟另一扇治療之窗。本論文主要探討四個主題,在第一個主題中我們探討第一型血基質氧化酶(HO-1)在肺高壓中所扮演的角色; 第二部分則是研究辛伐他汀(simvastatin)在治療肺動脈高壓的角色與機轉，第三部分則在觀察辛伐他汀對肺動脈高壓大鼠肺部C神經纖維敏感性以及呼吸反射作用的影響; 第四個主題則是在探討使用喘可利錠(Bosentan)，一種口服劑型之內皮激素接受阻斷劑(Endothelin Receptor Antagonist, 簡稱ERA)藥物，治療嚴重型原發性肺動脈高壓病患長期的安全性、療效及預後分析。以下簡述本論文的主要內容。 1. 為了探討第一型血基質氧化酶在肺高壓可能扮演的角色，我們利用已知的第一型血基質氧化酶誘發物Cobalt - protoporphyrin IX (CoPP) 應用在肺高壓大鼠中。在本實驗中，使用三種不同的肺高壓動物模式，一種是經由慢性低氧，另一種則是注射野百合鹼(monocrotaline, MCT)，第三種則是合併兩種處理方法來引發肺高壓。在動物手術進行的前48小時與96小時給予CoPP (20 μmole/kg) 來誘導第一型血基質氧化酶的產生。Copp可以有效的在三種不同的動物模式中降低肺動脈壓; 同時也可改善動脈血氧分壓。並增加大鼠肺部第一血基質氧化酶的蛋白質表現量以及其酵素活性。而經由免疫染色分析顯示第一型血基質氧化酶的蛋白質表現在肺部血管周圍。綜合以上結果，我們可以推論經由誘發第一型血基質氧化酶的產生與降低肺高壓有關。 2. 辛伐他汀是一種3-羥基-3-甲基戊二酰輔酶A (3-hydroxy-3-methylglutaryl coenzyme A, HMG-CoA)還原酶抑制劑，是現今常使用之降血脂藥物，而辛伐他汀除了降血脂外，還有心血管的保護效果。在本篇論文中, 我們假設辛伐他汀治療肺高壓的效果主要是透過活化與增加第一型血基質氧化酵素產生的作用。我們將辛伐他汀(10 mg/kg w/day)應用在治療兩種肺動脈高壓的動物模式上(1)野百合鹼處理, (2)慢性低氧處理。 測量其肺動脈壓力以及大鼠肺組織中的第一型血基質氧化酶表現量和酵素活性, 同時利用第一型血基質氧化酶抑制劑(SnPP, 20 μmol/kg w/day), 來拮抗辛伐他汀的作用，觀察加入第一型血基質氧化酶抑制劑後是否會抑制辛伐他汀的療效。實驗結果指出, 在此兩種肺動脈高壓動物模式中，辛伐他汀治療可以顯著改善大鼠的肺高壓以及右心室肥大。 同時投與SnPP則會抑制辛伐他汀的效用。辛伐他汀會顯著增加大鼠肺中第一型血基質氧化酶蛋白質的表現量以及其酵素活性; 然而在同時投與SnPP 只會降低第一型血基質氧化酶的酵素活性。以上觀察指出, 辛伐他汀在降低肺高壓的療效中主要是經由第一型血基質氧化酵素活性而不是其表現量, 證明了辛伐他汀改善肺高壓的機制主要是由第一型血基質氧化酶所調控的。 3. 第三部分則是利用野百合鹼所引發的肺高壓動物模式來研究肺高壓以及肺部C神經纖維之間的關聯性。經由化學刺激物刺激肺部C神經纖維並觀察其所誘發之呼吸反射作用；接續觀察辛伐他汀的治療是否能改變肺部C神經纖維敏感度。在本實驗中我們利用三種不同的化學刺激物: 辣椒素(capsaicin), α,β-亞甲基三磷酸腺苷(α,β-methylene-ATP) 和苯基双胍(phenylbiguanide) 所引發的呼吸中止程度來測試大鼠肺部C神經纖維敏感度；實驗結果指出大鼠在經由野百合鹼處理21天後其肺部C神經纖維敏感度有顯著增加的情形；而將大鼠的雙側迷走神經切斷或利用高濃度辣椒素處理後，皆可阻斷由野百合鹼所引起的肺部C神經纖維高敏感情形。而肺高壓大鼠給予辛伐他汀之後，可明顯的降低肺高壓以及肺部C神經纖維高敏感情形。此外我們研究辛伐他汀的療效對肺部C神經纖維的影響，並探討其相關機制,主要是針對自由基以及第一血基質氧化酶之間的相關性。實驗結果顯示在野百合鹼處理後的大鼠支氣管肺泡灌洗液以及血液中活性氧屬(ROS)的含量明顯增加；然而在給予辛伐他汀的大鼠組別則有顯著降低情形。辛伐他汀可以明顯的增加大鼠肺中第一血基質氧化酶的蛋白表現量以及酵素活性。而在同時給予第一血基質氧化酶抑制劑的組別中，大鼠肺中第一血基質氧化酶的酵素活性有顯著降低情形，而辛伐他汀所造成的降低肺動脈、肺部C神經纖維敏感度以及活性氧屬的產生則被阻斷。綜合以上結果，更加進一步的證明了辛伐他汀在改善大鼠肺高壓是經由第一血基質氧化酶所調控的路徑。 4. 原發性肺動脈高壓(Idiopathic pulmonary arterial hypertension，簡稱IPAH)的發生率約為百萬分之一至二，台灣地區粗估每年約有40位新增病例。如果不治療，IPAH病患平均存活年數約為2.8年，一年存活率為68％，能夠存活超過五年以上者不到35％。病患預後不佳的主要原因，是由於持續升高之肺動脈壓及肺血管阻力，導致負責供應肺血液循環之右心室負荷過重，病患終會因右心衰竭而死亡。，肺高壓病患肺部與血液中之內皮激素(Endothelin) 濃度較正常人高；而內皮激素是由血管內皮細胞所分泌，它會和血管平滑肌細胞上的二種接受體(ETA及ETB)接合，引起血管收縮，內皮細胞增生及纖維化。因此減少身體內內皮激素之濃度，應可改善肺高壓病患的症狀。內皮激素接受阻斷劑(Endothelin Receptor Antagonist, 簡稱ERA)可與ETA與ETB二種接受體結合，使內皮激素無法再與此二種接受體接合，便可達到舒張血管，抑制內皮細胞生長與增生的效果。喘可利錠是第一個獲美國食物暨藥物管理局(FDA)核准上市之ERA，它於西元2001年獲准用於治療第三期及第四期之肺高壓病患；台灣地區則由衛生署於2003年公告為治療原發性肺動脈高壓之罕見疾病用藥。因喘可利錠主要是由肝臟代謝，因此有些長期服用喘可利錠的病患會出現肝功能異常(如GOT及GPT數值大幅昇高)的現象，嚴重時須要停止喘可利錠之使用，改以其他藥物治療。因為迄今喘可利錠對黃種人的長期療效與相關副作用仍無文獻報告，所以我們嘗試探討嚴重型原發性肺動脈高壓病患接受喘可利錠治療之效果。自2003年10月至2005年2月，有十五位年齡大於十二歲以上之病患使用喘可利錠做為第一線治療肺動脈高壓之藥物，其中有三位病患在四星期內因肝功能在服藥後 異常(兩位)或肺動脈高壓持續惡化(一位)而改用其他藥物治療。十二位病患經過了一年的喘可利錠治療後，其生活品質、運動能力、肺功能、與右心室收縮功能皆獲得顯著的改善，同時減緩了疾病惡化的程度，提升高了病患的長期存活率。

Pulmonary hypertension (PH) is a rare disease that caused by three major pathogenic processes, vasoconstriction, remodeling of the pulmonary vessel wall and thrombosis, resulted in a progressive increase in pulmonary vascular resistance, high pulmonary arterial pressure (PAP) and right ventricular hypertrophy (RVH). Traditional treatment strategies for PH include diuretics, anticoagulation, digoxin, supplemental oxygen, and nonspecific vasodilating agents. Because these traditional therapeutic treatments were not effective, the mortality rate of this rare disease was still high and the long-term outcome was very unsatisfactory in the past. Recently, many investigators continued to study the pathogenesis of PH and explored the new therapeutic methods to treat this rare disease. In the basic parts of our studies, we focused on the protective role of heme oxygenase-1 (HO-1) in PH, the possible treatment mechanism of simvastatin in this rare disease and the effects of simvastatin on pulmonary C fiber (PCF) sensitivity in PH rats. Clinically, we surveyed the the long-term safety and efficacy of endothelin receptor antagonist in Taiwanese patients with advanced idiopathic pulmonary arterial hypertension. We summarized our researches in the following paragraphs. 1. To investigate the role of HO-1 in PH, we applied HO-1 inducer, Cobalt-protoporphyrin IX (CoPP), in pulmonary hypertensive rats. In this study, we used three PH-Rat models which were chronic hypoxia (CH)-induced monocrotaline (MCT)-induced, and combined. HO-1 was induced in lung by given CoPP (20 μmole/kgw) at the 96 and 48 hours before study. CoPP could significantly decrease the PAP in three PH rat models. PaO2 level were also improved after CoPP treatment. CoPP could significantly increased HO-1 protein expression and activity in rat lungs. Immunohistochemistry (IHC) stain revealed the HO-1 protein expression around the pulmonary vessels. In summary, these results suggest that the induction of HO-1 expression may involve in amelioration of PH. 2. Simvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor that confers cardiovascular benefits beyond its cholesterol-lowering effect. In this study, we demonstrated that the major benefits of simvastatin in PH occur via the HO-1 pathway. Simvastatin (10 mg/kg w/day) was tested in two rat models of PH: MCT administration and CH exposure. We also used Tin-protoporphyrin (SnPP, 20 μmol/kg w/day), a potent inhibitor of HO activity, to confirm the role of HO-1. Simvastatin significantly ameliorated pulmonary arterial hypertension and right ventricular hypertrophy in both rat models. Co-administration with SnPP could significantly abolish the benefits of simvastatin. Simvastatin significantly increased HO-1 protein expression and HO activity in the lungs of rats with PH; however co-administration of SnPP reduced HO-1 activity only. These observations indicated that the simvastatin-induced amelioration of PH was directly related to the activity of HO-1, rather than its expression. In conclusion, this study demonstrated that simvastatin treatment ameliorates established PH primarily through an HO-1-dependent pathway. 3. We examined the relationship between the pulmonary C fiber (PCF) sensitivity and PH in MCT-induced PH rats and whether the therapeutic effects of simvastatin would involve in the PCFs sensitivity or not. The PCF sensitivity was investigated by measuring the apneic durations evoked by three chemical stimulants: capsaicin, α,β-methylene-ATP (α,β-meATP) and phenylbiguanide (PBG), in rats. The sensitivity of PCFs was significantly increased after MCT application for 21 days. Bilateral vagatomy and high dose perivagal capsaicin (250 μg/ml) treatment both blocked the PCF hypersensitivity induced by MCT. Simvastatin treatment could significantly attenuate PH and reduced the hypersensitive status of PCFs in PH rats. Besides, we also examined the production of reactive oxygen species (ROS) and the expression of HO-1 protein to elucidate possible mechanisms of the therapeutic effect of simvastatin. In MCT-induced PH rats, the ROS productions were both significantly elevated in the blood and bronchoalveolar lavage; but both of them significantly decreased after simvastatin treatment. Simvastatin could significantly elevated HO-1 protein expression and the activity in rat lungs. Application of HO-1 inhibitor, SnPP, significantly attenuated the elevated HO-1 activity induced by simvastatin. Meanwhile, the effect of simvastatin on attenuating the PH, PCF hypersensitivity, and ROS production were all abolished after co-application of SnPP. In summary, these evidences further demonstrated that the effect of simvastatin in attenuating PH was mainly dependent on HO-1. 4. Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disease of the pulmonary arterioles that can rapidly progress to right heart failure and death without treatment. It belongs to one of five classes of PH defined through the diagnostic classification system for PH at the Third World Symposium held in Venice. The incidence of IPAH has been estimated to occur in 1-2 cases per million and the cumulative survival at 1 and 5 years were 68% and 35%, respectively. In the last few years, a number of novel treatments for this devastating disease have been developed and lots of clinical trials were undergoing to investigate their clinical efficacy. Bosentan, a non-peptide antagonist blocking both endothelin A and B receptors, is the first oral therapy approved for the treatment of pulmonary arterial hypertension in North America and Europe. However, the long-term benefits of bosentan therapy among the Asian populations are largely unknown. Since October 2003, oral bosentan received approval from the National Institutes of Health for the treatment of advanced IPAH in Taiwan. Given the ease of administration and well documented clinical benefits of bosentan, first-line treatment with bosentan for advanced IPAH patients (functional class III or IV) is considered both reasonable and feasible. In this part of our studies, we wanted to survey the long-term safety and effects of bosentan treatment in Taiwanese IPAH patients. Fifteen patients with advanced IPAH met eligibility requirements and were enrolled into our study. In the first four weeks, three of them were excluded due to abnormal elevations of liver enzymes or PAH disease deterioration. The other 12 patients tolerated the long-term bosentan treatment well with significant improvements in their heart and lung functions, exercise capacity, and long-term survival.