心房中膈缺損(ASD)是第二常見的先天性心臟病，常見的臨床症狀有容易疲累、呼吸困難與運動耐受性降低，甚至在未經治療的病人中可能產生雖不常見但較嚴重的肺動脈高壓。Rho激酶是一種藉由調控細胞內骨架以使細胞收縮、形變、移行的細胞內激素。近來有一些研究報告顯示Rho激酶的活性與肺動脈高壓有關聯，甚至可作為肺動脈高壓的新興治療標的。雖然在先天性心臟病所引起的肺動脈高壓病人中發現Rho激酶的活性有上升，但這樣的研究報告多僅限於心室中膈缺損或是開放動脈導管的病人，對於心房中膈缺損的病人卻沒有特別的研究報告。此外，測量血中白血球內的Rho激酶活性已被應用在許多的研究中，然而目前依舊缺乏足夠的證據支持血中白血球的Rho激酶活性與研究標的組織中Rho激酶的活性有足夠的相關性，使得測量白血球中Rho激酶的活性具有足夠的代表性。因此，我們希望從本實驗的研究設計中去驗證上述的問題。
我們測量ASD病人的靜脈血中白血球Rho激酶的活性，並與對照組做比較。我們發現ASD病人的平均Rho激酶活性的確較對照組高，並且這樣的差異在肺動脈壓正常的病人中並不明顯，但在合併有肺動脈壓升高的ASD病人中則相當顯著。藉由分析實驗組的基本資料，我們發現年紀、肺動脈壓力的高低、與心房中膈缺損的大小與Rho激酶的活性呈現正相關。
膜邊型的ASD病人可以經由心導管置放心房中膈關閉器(atrial septal occluder, ASO)，這個術式可以矯正過多的肺動脈血流使其恢復正常血流量。藉由比較ASD病人接受心導管心房中膈閉合術前後血中白血球Rho激酶的活性，我們觀察到Rho激酶在術後會快速的下降，尤其在合併有肺動脈高壓的病人中，其Rho激酶活性的下降更為顯著。而術後病人的Rho激酶活性甚至相當接近對照組。對此我們提出一個假設，ASD的病人如果因肺動脈血流量快速改變，將對肺動脈組織產生應力作用並激發或減弱組織細胞中的Rho激酶活性，並經由未知的機轉將訊號傳遞給血液中的白血球引起其內Rho激酶活性的相對應變化。
在動物實驗中，我們在6週大的Sprague-Dawley (SD)老鼠上製造腹主動脈-下腔靜脈瘻管，以模擬高肺動脈血流引致肺高壓之模型。在製造瘻管6週後取其血液與肺動脈組織，並另外進行瘻管關閉術以模擬ASD病人接受ASO手術，之後同樣取其血液與肺動脈組織。藉由動物實驗，我們證實血中白血球的Rho激酶活性變化與肺動脈組織中的Rho激酶活性變化具有高度的相似性，並且Rho激酶活性的變化在模擬肺動脈血流增加引致的肺動脈高壓的動物模型中，也與ASD病人中發現的Rho激酶活性變化具有一致性。在關閉瘻管後，血中白血球與肺動脈組織的Rho激酶活性有一致性的下降。
藉由本實驗，我們證實了Rho激酶的活性在ASD的病人中，尤其是合併有肺動脈高壓的情況下將會上升，而經心導管心房中膈閉合術將使其活性下降。另外，在高肺血流引致肺動脈高壓的動物模型中，使用血中白血球的Rho激酶活性亦可良好的代表肺動脈組織中的Rho激酶活性變化。

Atrial septal defect (ASD) is the second common congenital heart defect, which can cause fatigue, dyspnea, exercise intolerance, and even pulmonary artery hypertension (PAH) if left untreated. Rho kinase (ROCK) has been reported to be involved in PAH and is a modern therapeutic target in severe PAH patients. However, the profiles of ROCK activity in ASD patients were not reported before. In addition, circulating leukocytes ROCK activity was used in clinical studies but the relationship of ROCK activity between the leukocytes and in-situ PA tissue was not studied. So, we designed a study to analyze the profiles of ROCK activity in ASD patients. In addition, an animal study was also conducted to test the association between peripheral leukocytes and pulmonary artery tissue expression of ROCK activity.
In the human study, we measured the peripheral leukocyte ROCK activity in ASD patients and compared it to the control subjects. We found that ROCK activity was higher in ASD patients, especially in ASD patients with PAH. ASD patients with normal pulmonary artery pressure had similar ROCK activity to the control subjects. Many factors were significantly correlated with the ROCK activity in ASD patients, including the PA pressure, ASD size, and age. Clinically, we fixed the ASD by using an atrial septal occluder (ASO). We thus measured the ROCK activity before and after ASD closure. By comparing the changes, ROCK activity would decrease after ASD closure and it was theoretically related to the change of pulmonary blood flow. The decrease of ROCK activity was more in PAH subgroup than in non-PAH subgroup and the post-ASO ROCK activity was similar to the control group. We hypothesized that the rapid flow change of pulmonary artery system may affect the expression of ROCK activity. We might conclude that Rho kinase pathway plays, at least in part, a role in the pathophysiologic mechanism of PAH under ASD condition.
The relationship of ROCK activity between leukocyte and local PA tissue has not been established yet. An animal model was successfully set to mimic the procedure of ASD occluder implantation in human heart in our lab. We created the aorto-caval fistula in 6 week-old rats to mimic the condition of flow-induced PAH in humans and measured the ROCK activity in leukocytes and in PA tissue 6 weeks after the fistula creation. Then, we closed the fistula surgically and measured the ROCK activity after fistula closure. We found a good correlation of ROCK activity between circulating leukocytes and PA tissue. In addition, the ROCK activity also decreased after we closed the fistula.
Our study results confirmed the enhancement of ROCK activity in ASD patients, especially in those with PAH, and the attenuation of ROCK activity after ASD closure. Furthermore, we provided the evidence of using peripheral leukocytes ROCK activity to predict PA ROCK activity in the condition of high pulmonary blood flow induced PAH.

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