https://scholars.lib.ntu.edu.tw/handle/123456789/144468
標題: | 脂泌素及心血管危險因子對一氧化氮合成酵素的調控:分子與臨床的研究 Adiponectin and cardiovascular risk factors on the regulation of nitric oxide synthase: Molecular and clinical studies |
作者: | 林亮宇 Lin, Lian-Yu |
關鍵字: | 一氧化氮合成酵素;一氧化氮;內皮細胞功能失常;熱激蛋白;脂泌素;細胞凋亡;第二型血管張力素;高糖;家族性高膽固醇血症;肥胖;endothelial nitric oxide synthase;nitric oxide;endothelial cell dysfunction;heat shock protein;adiponectin;apoptosis;angiotensin II;hyperglycemia;familial hypercholesterolemia;obesity | 公開日期: | 2005 | 摘要: | 二十世紀心血管醫學最重要的進展之一,就是發現血管內皮細胞功能失常(endothelial cell dysfunction, ECD) 是心血管疾病最早期的變化, 也就是說, 血管內皮功能失常是血管產生粥狀硬化(atherosclerosis)的許多細胞生物機制中的第一步。”血管內皮細胞功能失常” 這個名詞,最早是用來描述血管發生 ”內皮細胞相依的血管舒張” (endothelium-dependent vasorelaxation) 失常。 但近來研究發現,血管內皮功能不僅只是調節血管張力,還參與其他作用, 如抗凝血作用,發炎反應,血管生成,以及平滑肌細胞的移行與增生等複雜生物機制的調控。近來對於引發血管內皮細胞功能失常的分子機轉的研究,顯示內皮細胞eNOS (endothelial nitric oxide synthase, eNOS) 的失調扮演關鍵性的角色。因此,內皮細胞eNOS調控的研究對於血管內皮細胞功能失常發生的機轉之瞭解,是相當重要的。 一氧化氮 (NO) 是L-arginine被內皮細胞內的eNOS轉換成L-citrulline所產生。這個過程需要許多的輔助酵素包括,FAD、FMN、tetrahydrobiopterin (BH4)等。血管受到shear stress及血液中bradykinin或acetylchoine等的刺激時,內皮細胞的eNOS即可穩定的分泌NO。所有動脈粥狀硬化的危險因子包括高血脂,糖尿病,高血壓及抽煙等都被證實和內皮細胞相依的血管舒張失常有關。雖然引起血管內皮功能失常的原因很多,但NO和內皮細胞eNOS的失調,包括內皮細胞eNOS的表現減少,血管對NO產生抗性及NO的清除增加還是扮演最重要的角色。許多引起內皮細胞功能失常的機制中,NO受到超氧化物的結合是相當重要的,研究也證實粥狀動脈硬化的血管會產生大量的超氧化物。近來,研究顯示在某些狀況下,內皮細胞eNOS會失調而產生超氧化物而非NO。內皮細胞eNOS失調的機轉逐漸受到研究者的重視。 近來研究發現,eNOS必須被Akt或AMPK燐酸化後才能穩定而有效的發揮作用。更進一步的研究顯示,eNOS必須和熱激蛋白90 (heat shock protein, HSP90)結合,才能有效的發生燐酸化。原因是HSP90能讓Akt與eNOS的結合更穩固。未受活化的時候,eNOS與caveolin於細胞膜結合,當受到荷爾蒙如血管內皮增生因子 (vascular endothelial growth factor, VEGF) 刺激的時候,與eNOS結合的caveolin會被鈣離子-calmodulin所取代,並進一步促進熱激蛋白90與eNOS結合。HSP90在和eNOS結合後,就可吸引被活化的Akt蛋白來和這個複合體結合,並將eNOS燐酸化。HSP90與eNOS的結合,將eNOS從早期的鈣離子相依 (calcium-dependent) 變成晚期的燐酸化相依(phosphorylation-dependent),這對於eNOS的有效運作相當重要。實驗顯示,在給予HSP90與eNOS結合的阻斷劑 (如geldanamycin或radicicol)後,eNOS會產生去耦合現象(uncoupling)而產生大量的超氧化物,進而導致內皮細胞的損傷。雖然研究顯示熱激蛋白90與eNOS的結合對內皮細胞功能的正常運作很重要,但諸如高血糖等心臟血管疾病危險因子對這個結合的影響之相關研究卻付之闕如。 本研究的細胞實驗部分 (研究1及研究2) 即透過在實驗室中細胞培養的實驗,探討高血糖、第二型血管張力素 (angiotensin II) 等引起內皮細胞損害的因子是否會影響或改變eNOS的調控。 研究1探討高血糖如何影響eNOS的調控。高血糖是糖尿病的一個重要的表徵,血糖控制不好和心血管疾病死亡率的增加有強烈的相關性。實驗研究顯示,高血糖會透過抑制eNOS的功能而引起內皮細胞的凋亡 (apoptosis)。 然而高血糖是否會影響eNOS與HSP90結合則未知。我們將人類臍靜脈內皮細胞(human umbilical vein endothelial cell, HUVEC) 培養於含33mM高濃度或5.5mM正常濃度糖的培養皿內,分別培養一段不同的時間 (2, 4, 6及24小時)。並利用免疫沉澱法 (immunoprecipitation) 測量eNOS/HSP90 及eNOS/Akt等酵素之間的結合情形。結果顯示,在高濃度糖培養的前2到4個小時,人類臍靜脈內皮細胞的eNOS/HSP90及eNOS/Akt的結合都明顯的增加,燐酸化的eNOS也伴隨增加。我們也注意到,隨著培養的時間增長,這個結合及燐酸化的效應卻漸漸減少。為了探討內皮細胞凋亡與eNOS調控的關係,我們在高糖培養的前幾個小時,給予HSP90抑制劑geldanamycin。結果發現eNOS/HSP90及eNOS/Akt的結合被抑制。同時內皮細胞凋亡的現象也明顯增加。而這個凋亡增加的現象可以透過NO的供應而將其抑制。我們也設計實驗,探討Akt活化所扮演的角色。在高糖培養的前幾個小時給予內皮細胞phosphatidylinositol 3 (PI3) kinase的抑制劑LY294002,結果發現eNOS與Akt的結合及eNOS的燐酸化都被抑制。但eNOS和HSP90的結合不受影響。 從研究1我們得知,人類臍靜脈內皮細胞在暴露於高糖培養溶液的早期,eNOS與HSP90的結合會增強,Akt也被活化而與eNOS/HSP90複合體結合並燐酸化eNOS。這些分子機制的運轉可以使內皮細胞免於凋亡。但隨著暴露的時間增長,eNOS調控機制漸漸失靈而引起內皮細胞的凋亡。這個研究的結果和許多動物實驗的結果類似。這些實驗均證實在糖尿病的早期,血管內皮細胞相依的血管舒張功能是增加的。但到了比較晚期,血管內皮相依的血管舒張功能卻減退。臨床的觀察也發現,早期糖尿病病人的血管阻力下降而血流量上升。對非糖尿病病人給予糖份注射,也會透過促進NO的分泌作用而促進血管的舒張。我們過去的細胞實驗也顯示,人類臍靜脈內皮細胞的eNOS在高糖的暴露下會產生雙相(biphasic) 反應,即早期增加而晚期減少,使得晚期NO和自由基的產生發生失調。我們的研究顯示,血管內皮細胞eNOS的調節也是雙相的。這個結果,有助於我們對糖尿病病患血管併發症病理生成機轉的瞭解。 研究2在探討angiotensin II對eNOS調控的影響。Angiotensin II起初被認為是控制血壓及調節鹽分、水分代謝、中樞神經活性、渴覺及血管平滑肌張力的賀爾蒙。後來發現,angiotensin II長期作用能改變組織的結構,包括心肌的肥厚、血管的重形塑 (remodeling) 及腎臟的纖維化。近來臨床的研究也發現,ACE抑制劑及angiotensin II受體阻斷劑均能減少中風、糖尿病及末期腎病變。研究更證實,angiotensin II能引發內皮細胞的凋亡,而引發內皮細胞功能失常。 在研究2中我們也想探討一種新發現的蛋白質脂泌素 (adiponectin) 能否預防angiotensin II對內皮細胞的傷害。過去認為脂肪僅是儲存及分解三酸甘油脂 (triglyceride, TG) 的組織。近來發現脂肪組織能分泌許多調控能量代謝的物質,包括游離脂肪酸、adipsin、leptin、plasminogen activator inhibitor-1、resistin及TNFα等。脂肪組織已被視為內分泌器官之一。脂泌素亦由脂肪組織分泌,最近受到廣泛的重視。實驗及臨床的研究發現,脂泌素能促進骨骼肌及肝臟的脂肪代謝而降低胰島素抗性(insulin resistance)。近來許多研究發現,除了代謝的作用外,脂泌素尚可透過許多分子機轉來保護血管。其中,脂泌素已被證實能促進eNOS的作用。故我們假設,脂泌素能透過強化eNOS與HSP90的結合而防止angiotensin II所引發的內皮細胞損害。 我們將人類臍靜脈內皮細胞培養於2µM濃度的angiotensin II中18小時,以引發細胞凋亡。如果在給予angiotensin II前,細胞先培養於不同濃度 (2, 5, 10µg/ml) 的合成脂泌素球狀片段(含globular domain) 中一個小時,可以發現,隨著培養液中的脂泌素濃度增加,內皮細胞凋亡的狀況明顯的減少。angiotensin II引發的內皮細胞凋亡,也可以透過NO的供給及給予angiotensin II接受體的阻斷劑而將其抑制。這個抑制細胞凋亡的效應,在給予eNOS的抑制劑NG-nitro-L-arginine methyl ether (L-NAME) 後就消失。相同的,eNOS的燐酸化,在angiotensin II存在的情形下會被抑制,而在同時給予angiotensin II受體阻斷劑或脂泌素後,eNOS的燐酸化就可以恢復。這個實驗的結果顯示,angiotensin II是經由angiotensin II受體影響eNOS,而引發細胞凋亡。脂泌素也可以透過對eNOS的調控,抑制angiotensin II的影響。們進一步探討HSP90是否參與其中。利用免疫沉澱法,我們發現,和控制組比較,NO和HSP90的結合及NO的燐酸化在給予angiotensin II後明顯的被抑制。如果在給予angiotensin II前,提早一個小時培養於含脂泌素的培養液,angiotensin II對eNOS的效應就被抑制,而eNOS的燐酸化也會增加。如果同時給予HSP90的抑制劑radicicol,那麼脂泌素的保護效應又消失。我們的研究顯示,透過穩定eNOS與HSP90的結合,脂泌素可以預防angiotensin II引發的人類臍靜脈內皮細胞凋亡。 這個研究在angiotensin II與NO的調節及脂泌素保護血管受損的機轉上,提供了一個新的解釋及未來可能的研究方向。這個研究另一個新的發現,即angiotensin II能透過第二型血管張力受體,抑制eNOS與HSP90的結合。過去的研究發現,angiotensin II的受體可能以直接和eNOS結合的方式抑制它的活性。是否因為angiotensin II受體受到angiotensin II的刺激而和eNOS結合,進而阻斷HSP90形成複合體,是一個有趣而值得研究的題目。至於脂泌素如何促進兩者的結合,機制目前還不清楚,值得進一步探討。 本研究的臨床研究部分 (研究3及研究4),在探討兩個心血管疾病高危險群: 家族原發性高膽固醇血症 (familial primary hypercholesterolemia) 及嚴重肥胖(morbid obesity) 病人,內皮細胞功能失調的原因及調控機轉。家族原發性高膽固醇血症患者有相當高的比例在中年之前就罹患心血管疾病。動物及細胞實驗已證明脂泌素可以預防及修補血管的損傷。人體的研究也顯示,脂泌素過低與冠狀動脈心臟病及動脈硬化有顯著的相關性。雖然低脂泌素和血脂失常 (dyslipidemia)的相關性已被許多研究證實,且胰島素抗性被認為是潛在的原因,然而針對家族原發性高膽固醇血症此高危險群的研究卻付之闕如。研究3即探討此一高危險群病人血清中脂泌素的狀態及與胰島素抗性的關係。為了單純化,我們研究的對象是一群年輕(<30歲)而沒有代謝症候群 (metabolic syndrome) 的患者。因為沒有做基因分析以確定高膽固醇血症的病因,我們稱這些病人罹患 ”家族相關的嚴重原發性高膽固醇血症” (familial related severe primary hypercholesterolemia, FRSPH)。 研究3一共收羅了23位罹患 ”家族相關的嚴重原發性高膽固醇血症” 的年輕病人。這些參與者必須符合以下兩個條件: 1. 病人本身及至少兩個一等親其低密度脂蛋白 (low density lipoprotein, LDL) 超過4.92mM,並/或在小時候即有肌腱黃色瘤 (tendinous xanthomas)。 2. 血清中的高密度脂蛋白(high density lipoprotein, HDL) 及三酸甘油酯 (triglyceride, TG) 必須在正常範圍內。此外,這些病人沒有同時罹患其它能引起次發性高血脂症的病因,如腎病症候群(nephrotic syndrome)、阻塞性膽道疾病、甲狀腺功能低下、糖尿病、或服用可引起高血脂的藥物。另外我們收羅46個年紀和性別相仿的健康人當作控制組。這些病人接受空腹抽血並測量脂泌素、血糖、胰島素及血脂。我們同時也測量他們的血壓、體重、腹圍及身高。我們以空腹血糖和胰島素計算HOMA (homeostasis model assessment) 當作是評估胰島素抗性的指標。結果顯示,罹患 ”家族相關的嚴重原發性高膽固醇血症” 的病人脂泌素明顯較控制組病人低 (7.7±1.8 μg/ml vs. 10.1±4.3 μg/ml, p=0.013)。而兩組的胰島素抗性沒有差異。利用多重線性回歸分析 (multiple linear regression) 調整其它相關的變數後,結果仍然顯示罹病組和低脂泌素有顯著的相關性。我們的研究顯示,罹患 ”家族相關的嚴重原發性高膽固醇血症” 的病人有較低的脂泌素。 這可能是這群高危險病人罹患心血管疾病的機轉之一,也為治療提供一個新的可能方向。此外,這個研究也指出,低脂泌素不盡然和胰島素抗性及代謝症候群有關。這個觀察表示,高膽固醇血症和低脂泌素的相關性是透過獨立於胰島素抗性之外的其它途徑。目前尚不清楚這條途徑背後的機轉,但或許和極度高的膽固醇所誘發的細胞激素分泌有關。過去的研究已知細胞激素如TNFα或Interleukin-6能抑制脂肪細胞分泌脂泌素。高膽固醇所誘發產生的細胞激素,能抑制脂肪細胞脂泌素的分泌,此極有可能是家族相關的嚴重原發性高膽固醇血症患者脂泌素降低的機轉。 研究4在探討嚴重肥胖病人接受減重手術前後,NO產量的變化及其調控的機制。成人甚至是小孩肥胖的盛行率在開發中國家正快速的增加。肥胖被證實是冠狀動脈疾病、心臟功能受損、中風及心律不整等的重要危險因子。研究也證實肥胖和血管內皮細胞功能受損有獨立的相關性。近來,針對肥胖的成人給予低卡路里飲食來減重也被發現能改善血管內皮細胞受損。NO生体可使用性(bioavailability) 的減少據信是肥胖引發的血管內皮細胞功能失調的重要因素。NO生体可使用性的減少主要有兩個機制: 一種是NO產生的減少,另一種是能將NO轉換成能引發發炎反應的媒介物的反應性氧化產物 (reactive oxygen species) 的增加。經由血液中NO濃度的測量,能分辨出哪一種機制參與其中。由於NO產生後,很快就被人體代謝,故測量血液中NO的代謝產物,已經被證實為是評估NO產生的一個可靠的方法。故我們研究一群嚴重肥胖的病人在接受胃間隔術前後NO產生量的變化情形,藉以瞭解NO的調控如何受到肥胖的影響。我們也想透過這個研究探討那些因素是影響NO分泌的重要因子。一群年紀和性別相仿的健康人也加入研究當做對照組。除了NO外,其它能影響血管內皮細胞的因子包括血脂型態、血糖、胰島素、脂泌素、發炎指標、氧化壓力也一併測量。我們一共收羅了69個嚴重肥胖者當為實驗組,69個健康的人當為控制組。我們的研究結果顯示,減重手術前,嚴重肥胖組病人的血脂、血糖、C反應蛋白 (C-reactive protein, CRP) 、8-iso-prostaglandin F2α(8-iso-PGF2α)及胰島素抗性指標HOMA均較控制組為高。然而血液中NO代謝產物的濃度,在兩組並無統計上的差別 (肥胖組比上控制組, 43.4±22.8 vs. 48.9±22.2μM/L, p=0.148)。我們也以多變項線性回歸的方式分析,找出影響NO分泌的因子。結果發現,NO的分泌和男性、脂泌素及三酸甘油酯有統計上有意義的正相關。如果個別以實驗組和控制組來進行分析,結果顯示NO的分泌在實驗組僅和三酸甘油酯成正相關,而在控制組則和脂泌素成正相關,和HOMA成負相關。在接受減重手術3至6 個月後,體重、腰圍、舒張壓明顯的下降。肥胖病人血清中NO代謝物的濃度自43.3±22.8μM/L下降成為24.4±12.5μM/L(p<0.001),其它的血清指標除脂泌素上升外 (4.5±3.6上升至6.4±3.4μg/mL),C反應蛋白、8-iso-PGF2α、血糖、胰島素、總膽固醇、三酸甘油酯等均呈統計上有意義的下降。如果以減重手術前後的變化量來分析相關性,可以發現NO代謝物的濃度變化僅和體重及三酸甘油酯的濃度變化成正相關。 這個研究顯示,嚴重肥胖病人的NO的產生在減重手術後明顯減少。這表示肥胖病人的NO過度的產生,內皮細胞功能的功能失常主要是因為NO去活化而非生產不足所引起。從這個研究中我們推論,其1,肥胖病人血清NO產量的減少或許反應的是減重後iNOS的減少及NO過度產生的減緩。其2,在正常的狀態下,NO的產量和胰島素抗性成反比而和血清脂泌素成正比。其3,在肥胖的狀態下,血清NO的產量和血清三酸甘油酯而非氧化壓力有關,這顯示肥胖病人NO的調節,也和氧化壓力以外的其它因素有關。 臨床價值及意義 綜言之,我們的細胞研究顯示,eNOS與HSP90的結合,能確保eNOS的燐酸化及正常運作。過長的高糖暴露及angiotensin II等心血管危險因子,能干擾eNOS與HSP90的結合並引發內皮細胞損傷。我們也發現,脂泌素能透過穩定eNOS與HSP90的結合而保護內皮細胞。這些都表示eNOS與熱激蛋白結合的失調,在內皮細胞的損傷上扮演重要的角色。我們的研究,為往後eNOS的調控及內皮細胞功能失常的相關研究,開發一個可能的新方向。 臨床上,我們進一步探討兩個心臟血管疾病高危險群,其內皮細胞損傷的可能機轉。我們發現,家族相關的嚴重原發性高膽固醇血症的年輕病人,既使在臨床上沒有胰島素抗性的表現,血清中的脂泌素仍然較控制組為低。這個發現除了為這個族群過多的心血管疾病提供一個新的病理機轉及可能的治療方法,也指出脂泌素代謝另一個可能的代謝途徑。 最後我們探討嚴重肥胖病人在接受減重手術前後NO產生的調節。我們發現,肥胖病人在減重後NO的產生明顯的減少,顯示肥胖病人的內皮細胞功能失常是起因於NO的去活化,而非產量減少。我們也發現,NO的變化和三酸甘油酯有密切的相關性。這個研究,為肥胖引發的內皮細胞功能失常的成因提出可能的機轉。 One of the most important progresses of cardiovascular medicine in the twentieth century is to uncover that endothelial cell dysfunction (ECD) develops in the very early phase of cardiovascular diseases. ECD is the first step in the cellular mechanisms leading to atherosclerosis. ECD, initially introduced to describe defective endothelium-dependent vasorelaxation, has now been broadened to encompass impaired anti-thrombogenic and anti-inflammatory properties; perturbed angiogenic capacity; inappropriate regulation of smooth muslce proliferative capacity and migratory properties. Since recent studies have pointed to the pivotal role of dysregulation of nitric oxide (NO) in the pathophysiology of ECD, it is important to investigate the molecular mechanisms that regulate endothelial nitric oxide synthase (eNOS). Nitric oxide is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of an NADPH-dependent NO synthase (NOS), which requires Ca2+/calmodulin, FAD, flavin mononucleotide (FMN), and tetrahydrobiopterin (BH4) as the cofactors. In the blood vessels, NO is produced from the endothelium by constitutive expression of the endothelial isoform of eNOS, which can be activated by mechanical stresses, such as shear-stress, and stimulation with agonists, such as bradykinin and acetylcholine. All major risk factors for atherosclerosis, such as hyperlipidemia, diabetes, hypertension, and smoking, are associated with impaired endothelium-dependent vasorelaxation. Although the underlining mechanisms of ECD are divergent, the most important one is the derangement of eNOS/NO pathway, which may be produced by impaired eNOS (either reduced activity or reduced expression of eNOS), decreased sensitivity to NO of the tissues, or increased degradation of NO by superoxide. Among the various mechanisms responsible for ECD, increased NO breakdown by superoxide is especially important as there is augmented production of superoxide in atherosclerotic vessels. Recently, it has been revealed that, under certain circumstances, eNOS would become dysfunctional and produce superoxide rather than NO. The pathophysiological role of eNOS dysfunction in vascular disorders, including atherosclerosis, has attracted much attention in past decades. Recent studies indicate that eNOS is highly regulated by post-translational modifications, such as Akt- or AMPK-induced phosphorylation and interaction with several regulatory proteins, such as heat shock protein 90 (HSP90). The association between eNOS and HSP90 has recently been shown to be critical in the regulation of eNOS function. In the quiescent state, eNOS is associated with caveolin and remains cell membrane-bound and inactive. When stimulated with vascular endothelial growth factor (VEGF), the eNOS-caveolin complex would be disrupted by Ca(2+)/calmodulin and the association between eNOS and HSP90 promoted. The eNOS-bound HSP90 can then recruit VEGF-activated Akt to the complex to induce phosphorylation of eNOS. The binding of HSP90 to eNOS ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of eNOS. Failure of this binding can cause eNOS uncoupling and increase eNOS-dependent superoxide anion production, leading to endothelial cell injury. Even though these observations suggest that the association of HSP90 with eNOS is critical in eNOS-associated NO production, few studies have focused on the effects of cardiovascular risk factors, such as hyperglycemia, on the eNOS/HSP90 protein interaction. The cell experiment part of present investigation (Studies #1 and #2) is intended to investigate the effects of endothelial cell injuring factors, such as hyperglycemia and angiotensin II, on the regulation of eNOS. Study #1 was designed to investigate the effects of hyperglycemia on the regulation of eNOS. Hyperglycemia is the hallmark of diabetes mellitus. Poor glycemic control is correlated with increased cardiovascular morbidity and mortality. High glucose can trigger endothelial cell apoptosis by de-activation of eNOS. Yet, little is known about the molecular mechanisms that regulate eNOS activity during high glucose exposure. The present study was designed to determine the involvement of protein interaction between eNOS and HSP90 by immunoprecipitation in high glucose-induced endothelial cell apoptosis. The protein interactions of eNOS/HSP90 and eNOS/Akt were studied in cultured human umbilical vein endothelial cells (HUVECs) exposed to either control-level (5.5mM) or high-level (33mM) glucose for different durations (2, 4, 6 and 24 h). The results showed that the protein interactions between eNOS and HSP90 and between eNOS and Akt and the phosphorylation of eNOS were all up-regulated by high glucose exposure for 2 to 4 h. With longer exposures, these effects decreased gradually. During early hours of exposure, the protein interactions of eNOS/HSP90 and eNOS/Akt and the phosphorylation of eNOS were all inhibited by geldanamycin, an HSP90 inhibitor. High glucose-induced endothelial cell apoptosis was also enhanced by geldanamycin and was reversed by NO donors. LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor, inhibited the association of eNOS/Akt and the phosphorylation of eNOS but had no effect on the interaction between eNOS and HSP90 during early hours of exposure. From our results we proposed that, during early phase of high glucose exposure, apoptosis in HUVECs can be prevented by enhancement of eNOS activity through augmentation of protein interaction between eNOS and HSP90 with recruitment of activated Akt. With longer high glucose exposure, dysregulation of eNOS activity would result in enhanced apoptosis. This finding can be correlated with the observations in animal studies in which endothelial-dependent vasorelaxation is evident in the early stage of diabetes mellius but it deteriorates in the later stage. Clinically, it has been shown that, in humans, the peripheral resistance is decreased and the blood flow increased in early diabetes. In nondiabetic individuals, acute exposure to high glucose also induces vasodilatation through increase in the secretion of endothelium-derived nitric oxide. In our previous study in which cultured human endothelial cells were exposed to high glucose, we demonstrated a biphasic response of eNOS expression, an early up-regulation and a later down-regulation, resulted in an imbalance between NO production and free radical generation. The present study showed that the association between eNOS and HSP90 is also biphasic. This finding provides a molecular basis for the effects of eNOS in the prevention of endothelial cells apoptosis during early phase of high glucose exposure. These observations may contribute to the understanding of the pathogenesis of vascular complications in diabetes mellitus. In study #2, we investigated the effects of angiotensin II on the regulation of eNOS. In the past, angiotensin II was recognized as a hormone that control blood pressure through the regulation of renal salt and water metabolism, central nervous system mechanisms (thirst and sympathetic outflow), and vascular smooth muscle cell tone. More recently, angiotensin II was found to exert long-term effects on tissue structure, including cardiac hypertrophy, vascular remodeling, and renal fibrosis. Recent clinical observations indicated that the wide-spread use of angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) have resulted in remarkable clinical benefits in decreasing the incidence of stroke, diabetes mellitus, and end-stage renal disease in high risk patients. Studies also demonstrated that angiotensin II can induce endothelial cell apoptosis and ECD. Furthermore, we tested whether a newly found protein, adiponectin, can protect endothelial cells from angiotensin II-induced injury. It has long been well known that adipose tissue serves as a structure for triglyceride (TG) storage and free fatty acid/glycerol release in response to changes in energy demands. In recent years, adipose tissue has been found to participate in the regulation of energy homeostasis and also serves as an important endocrine organ in secreting a number of biologically active adipokines, such as free fatty acid, adipsin, leptin, plasminogen activator inhibitor-1, resistin, and TNFα. Adiponectin is one such adipokine that has recently attracted much attention. Adiponectin is abundantly expressed in adipose tissue. Clinical and animal studies have shown that adiponectin can promote liver and skeletal muscle fat metabolism and reduce insulin resistance. In addition to the metabolic effects, adiponectin was found to protect vessels from damage through various mechanisms. In the present study, we tested the hypothesis that adiponectin can prevent endothelial cell apoptosis induced by angiotensin II through promotion of the association between eNOS and HSP90. Cultured HUVECs were treated with angiotensin II (2µM) to induce apoptosis. In the presence of globular adiponectin, apoptosis was inhibited in a dose-response manner. Angiotensin II- induced apoptosis was also inhibited by treatment with an NO donor and by combined treatment with both angiotensin II type 1 and type 2 receptor blockers. eNOS phosphorylation was markedly inhibited by angiotensin II treatment and this effect could be prevented by pre-treatment of the cells with either globular adiponectin or combined angiotensin II receptor blockers. These findings indicated that angiotensin II-induced HUVEC apoptosis is proceeded through angiotensin II receptor. HUVECs were pre-treatment with adiponectin or radicicol for 1 hour, and then subjected to angiotensin II exposure for 18 hours. Proteins from the cell lysates were subjected to Western blotting and immunoprecipitation for eNOS and HSP90. The results showed that the association between eNOS and HSP90 were significantly inhibited by angiotensin II. Pre-treatment of the cells with globular adiponectin could restore the eNOSHSP90 association and the phosphorylation of eNOS while radicicol, an HSP90 inhibitor, reversed the effects of adiponectin on eNOS and HSP90. In our study, we showed that angiotensin II-induced human endothelial cell apoptosis can be prevented by adiponectin through promotion and stabilization of the association between eNOS and HSP90. From our study, we disclosed an interesting novel finding that the inhibitory effect of angiotensin II on the formation of eNOSHSP90 complex is mediated through angiotensin II receptor. One previous in vitro study has demonstrated that angiotensin II receptor can directly bind eNOS to exert its subsequent inhibitory activity. Although the molecular mechanisms of the detrimental effect of angiotensin II is still unclear, it is possible that, by stabilizing the eNOS/HSP90 complex, binding of angiotensin II to its receptor can be interfered and the angiotensin II-induced de-activation of eNOS activity can be prevented. This study provided new mechanisms for angiotensin II in its effects on endothelial cell injury and for adiponectin in the protective effect against vascular injury. The mechanism of adiponectin is not clear and further study is warranted (Difficult in understanding this sentence.). The clinical observation part of present investigation (Studies #3 and #4) were designed to investigate the mechanisms of ECD in two groups of patients with high cardiovascular risk: familial primary hypercholesterolemia and morbid obesity. Familial primary hypercholesterolemia is associated with excessive premature cardiovascular mortality and morbidity. Animal and cell studies have proved that adiponectin can prevent and reverse vascular injuries. Clinical observations also have shown that low adiponectin level is associated with coronary artery disease and atherosclerosis. Although several studies have proved that decreased serum adiponectin is associated with dyslipidemia and its underling etiology has been attributed to insulin resisitance, the serum adiponectin status has never been studied in patients with familial primary hypercholesterolemia. The aim of our Study #3 was to measure serum adiponectin level in a group of young patients with primary familial hypercholesterolemia and determine its correlation with insulin resistant status. For simplicity, we choice a group of young patients (<30 year-old) without clinical manifestations of metabolic syndrome. Since genetic mutations were not confirmed in these patients, we called our study subjects as “familial related severe primary hypercholesterolemia” (FRSPH). We included 23 young patients with FRSPH in Study #3. The inclusion criteria were as follows: 1. In the proband and at least 2 first-degree relatives, the low-density lipoprotein cholesterol (LDL-C) should be above 4.921mM, with the presence of tendinous xanthomas. 2. The serum high density lipoprotein (HDL-C ) and triglycerides (TG) should be in normal ranges. Subjects with any condition which may cause secondary hyperlipidemia, such as nephritic syndrome, obstructive liver disease, hypothyroidism, diabetes mellitus (DM) and subjects who were taking drugs which may affect lipid metabolism were excluded. Forty-six age- and- sex matched healthy controls were included for comparison. The serum adiponectin, fasting sugar, insulin , lipids, systolic and diastolic blood pressure (SBP and DBP) and anthropometrical indices, such as body weight, waist circumference and body height were obtained. The homeostasis model assessment (HOMA) was calculated to estimate the insulin resistant status. The results showed that compared with healthy controls, patients with FRSPH had a significantly lower mean serum adiponectin level (7.7±1.8 μg/ml vs. 10.1±4.3 μg/ml, p=0.013). The HOMA score was not different between theses two groups. After adjustment for HOMA and associated covariates, multiple linear regression analysis showed that patients with FRSPH are significantly associated with hypoadiponectinemia. Our study showed that, the serum adiponectin levels are lower in the young FRSPH patients than in the age and sex-matched control subjects. This may contribute to the high incidence of cardiovascular diseases in this patient population and it is possible that increase in serum adiponectin may reduce the development of cardiovascular diseases. Further more, we found that in young FRSPH subjects with extremely high serum LDL-C levels, the serum adiponectin levels were even lower after adjustment of other insulin sensitivity markers. These observations indicate that the correlation between hypercholesterolemia and hypoadiponectinemia may be mediated through certain pathways independent of insulin resistance. It is well known that cytokines such as TNFα and Interleukin-6 can inhibit the secretion of adiponectin from adipocytes. It is postulated that extreme hypercholesterolemia in FRSPH patients may elicit secretion of cytokines which in turn decrease the secretion of adiponectin. Study #4 investigated the status of nitric oxide production and the regulating mechanisms involved in patients with morbid obesity both before and after weight reduction surgery. In the developing countries, the prevalence of obesity is increasing rapidly in adults as well as in children. Obesity is demonstrated as an important risk factor for coronary heart disease, ventricular dysfunction, stroke and cardiac arrhythmias. Studies have demonstrated that, in children, obesity is independently associated with ECD. Weight reduction with very-low-calorie diet in obese adults was recently demonstrated to be able to reverse ECD. Reduced bioavailability of vascular NO is believed to contribute to obesity-associated ECD. Processes that can decrease NO bioavailability include impaired synthesis of NO and increased production of reactive oxygen species that convert NO to a pro-inflammatory mediator. Insights regarding these processes can be obtained by measuring metabolites of NO in blood. The plasma level of stable metabolites of NO (NOx), mostly contributed by vascular endothelial cells, have been adopted as a reliable measurement of NO production in the body. The present study was designed to measure the changes of NO production before and after gastric partition surgery in a group of morbidly obese patients. Our aims were to investigate the effects of obesity on the regulation of nitric oxide production and to delineate possible factors which might be associated with nitric oxide production. A group of age-and-sex matched health controls were also included for comparison. Body mass index, blood pressure, serum levels of lipids, high sensitivity C-reactive protein (hs-CRP), HOMA, adiponectin, total nitrite and nitrate (NOx), and 8-iso-prostaglandin F2α (8-iso-PGF2α, the lipid peroxidation product), were obtained in the healthy controls and in the obese subjects both before and after gastric partition surgery. We included 69 mobidly obese patients and 69 health controls. The results showed that at baseline, the serum levels of lipids, glucose, insulin, hs-CRP and 8-iso-PGF2α, and HOMA were all higher in the obese group than in the control group. The serum NOx levels were not different between the two groups (43.4±22.8 vs. 48.9±22.2μM/L, p=0.148). For all subjects, the baseline serum NOx levels were positively associated with male gender, serum triglyceride (TG) and adiponectin levels, as analyzed by multiple linear regression analysis. In obese patients, the baseline serum NOx was only positively associated with the serum TG levels. For healthy controls, the serum NOx levels were positively associated with serum adiponectin levels but negatively associated with HOMA. Three to six months after weight reduction surgery, the body weight, waist circumference and diastolic blood pressure decreased significantly. The serum NOx levels decreased from 43.3±22.8μM/L to 24.4±12.5μM/L(p<0.001). Other serum markers, including hs-CRP, 8-iso-PGF2α, fasting sugar, insulin, total cholesterol and triglyceride levels, decreased significantly while the serum adiponectin levels increased (4.5±3.6 vs. 6.4±3.4μg/mL, p<0.001). The changes of serum NOx levels after weight reduction surgery were positively associated with the changes of body mass index and serum TG levels. This study showed that, in severely obese subjects, the NO production was down-regulated after weight reduction surgery, suggesting an excessive production and/or inactivation of NO in these patients before surgery. We proposed that, in the present study, the decreased NO production after weight reduction surgery might reflect the weight loss-associated down-regulation of iNOS and the reversal of NO overproduction. Our results also suggested that, in healthy subjects, the NO production is negatively associated with the insulin resistant status and positively associated with the serum adiponectin levels, while in obese state, the NO production is positively associated with the serum TG levels rather than the oxidative stress or inflammatory markers. These findings imply that mechanisms other than oxidative stress might contribute to the regulation of NO production in severely obese patients. Significance and clinical implication In conclusion, our cell studies showed that the protein association between eNOS and HSP90 can enhance the phosphorylation and efficiency of eNOS. Cardiovascular injuring factors such as long exposure to high glucose and angiotensin II can interfere with this association and lead to endothelial cell injury. Furthermore, we found that adiponectin can protect endothelial cells from injury through stabilization of the eNOS/HSP90 complex. These observations all pointed to the pivotal role of the dysregulation of eNOS/HSP90 association in the pathogenesis of ECD. Our studies provide a new direction for the associated studies on the regulation of eNOS and ECD. We also investigated the pathogenesis of two groups of patients with high cardiovascular risk. We found that FRSPH patients without clinical evidence of insulin resistance have lower serum adiponectin levels. This finding not only provides a possible mechanism and treatment for the excessive mortality and morbidity of the patients, but also disclosed a new alternative pathway for the development of hypoadiponectinemia in these patients. Finally, we studied the regulation of eNOS in the extremely obese patients before and after weight reduction surgery. We found that the production of NO decreased significantly after weight reduction, indicating that ECD in obese patients is contributed by de-activation of NO rather than decreased production. Also, we found that the changes of NO production after weight reduction are associated with changes of serum TG. This study discovered a possible novel mechanism for the pathogenesis of ECD in extremely obese patients. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/55516 | 其他識別: | zh-TW |
顯示於: | 臨床醫學研究所 |
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