Yi xing zhuan hua sheng zhang yin zi tiao kong de ju shi xi bao-ji xian wei mu xi bao zhuan fen hua zai xue guan zai xia zhai de zuo yong he chuan xiong qin zhi liao de ji zhi tan suo

Ph.D.Restenosis is the narrowing of blood vessels leading to restricted blood flow by proliferative process similar to wound healing after procedures of angioplasty and stenting. The pathophysiology underlies dynamic responses across vascular layers to cause negative remodelling and neointimal hyperplasia (NIH). Macrophage-to-myofibroblast transition (MMT), a transdifferentiation process mediated by TGF-β/Smad3 signalling, may contribute to NIH with roles to be elucidated. The mechanisms through which Smad3 mediates MMT in restenosis and their respective therapeutic potentials remain to be explored. Being a major active ingredient of Chinese herb Chuanxiong, tetramethylpyrazine (TMP) is used extensive for cardiovascular diseases such as restenosis, but it is limited in clinics by inadequate mechanistic studies. Whether TMP antagonizes MMT and whether regulation of Smad3 is involved remain uninvestigated. The present study therefore aimed to investigate whether MMT participates in animal model of femoral artery endothelial denudation and whether anti-stenotic effect of TMP is acted through MMT inhibition by the regulation of Smad3 signaling.Mouse restenosis model of femoral artery denudation was used for the presence of MMT with Lyz-Cre/Rosa-Tomato mice for confirmational fate mapping studies by fluorescence confocal microscopy and flow cytometry. Smad3 WT/KO mice, together with mice receiving specific inhibitor of Smad3 (SIS3) or TMP were used to study roles of TGF-β/Smad3 signalling in MMT and restenosis by histological measurements, fluorescence/CT-angiography, and PW-doppler ultrasonography. Bone marrow-derived macropages (BMDMs) were isolated from mouse tibia and primary cell cultures were used for western blot, reverse-transcription PCR analysis, ChIP.MMT is found to dock on denuded endothelium at 1 week, elongate and peak at 2 weeks, and continue to contribute to neointima at 4 weeks post-injury with loss of macrophage markers, and macrophage fate mapping studies reveal a substantial proportion of MMT in restenotic lesion 2 and 4 weeks post-injury. Platelet-derived growth factor-BB (PDGF-BB) works synergistically with TGF-β for MMT induction via Smad3 signalling, while knockout of Smad3 abolishes most transdifferentiation process. Smad3 knockout mice after injury experiences less MMT and neointima in the restenotic lesion but with impaired ECM integrity, hence leading to disturbed luminal patency and blood flow. Similarly, targeting MMT by modest Smad3 inhibition attenuates restenosis, but increased inhibition paradoxically disrupts ECM and compromises blood flow. While TMP possesses moderate Smad3 inhibition capacity, it dose-dependently inhibits MMT via KLF5-Smad3 axis and decreases the expression of MMPs, while promoting endothelial recovery.The present study suggests that Smad3 signaling-mediated MMT contributes to NIH in vascular remodeling of restenosis. TMP has modest Smad3 inhibition capacity through which TMP normalizes KLF5 expression for MMT inhibition and amelioration of NIH, and it confers protective effect by encouraging endothelial recovery and MMP inhibitions. These findings added new mechanistic insight into the cardiovascular benefits of this traditional Chinese medicine that may have potential clinical implications.血管再狹窄是血管中的重要疾病。常見於血管介入手術如支架植入和血管成形術後，或出現於患有動脈粥樣硬化，缺血性中風，周邊血管病變，和經常接受血液透析的病人身上。但是其病理性增生的機制至今不明，尤其是當中病理性增生的細胞來源及其作用，以致限制了治療的成效。乙型轉化生長因子調控的巨噬細胞-肌纖維母細胞轉分化在炎症和纖維化成形都有很重要的已知病理功能，當中可能參與了Smad3份子蛋白的訊息通路調控。傳統中草藥川芎的最主要活性成分四甲基吡嗪 (Tetramethypyrazine, TMP) 因其抗炎特性和抗氧化功效而廣泛應用於心血管疾病治療。然而，對於其保護機制的認識仍留有很多空白，機理研究尚有不少不足。故此，本研究旨在探討 1) 乙型轉化生長因子 （TGF-b）調控的巨噬細胞-肌纖維母細胞轉分化 （MMT）有否參與血管再狹窄的病理性增生及其作用，及2) Smad3份子蛋白的訊息通路在轉分化和在血管再狹窄中的功能，以及 2) TMP治療血管再狹窄的機制有否透過MMT細胞的抑制和Smad3蛋白的調控。本研究選用血管再狹窄的小鼠模型，以共聚焦顯微鏡和高端細胞流式儀檢測MMT細胞，配合自帶螢光蛋白的轉基因小鼠作譜系追蹤以確定MMT細胞來源。Smad3基因敲除小鼠則用以研究MMT轉分化和血管再狹窄在Smad3缺失下的影響。Smad3特異性抑制劑（SIS3）和四甲基吡嗪 （TMP）用作小鼠術後腹腔注射治療，以研究治療背後的完整機制藥理。骨髓分離巨噬細胞（BMDMs）以原代細胞培養，採用免役螢光及共聚焦顯微鏡，西方免疫印跡法，細胞流式儀和染色質免疫沉澱法測量檢析。血管再狹窄則透過組織病理學，電腦斷層掃瞄血管造影重組，活體共聚焦顯微鏡實時螢光血管造影，和脈衝波式多普勒超音波血液流速定量分析等技術以定性血管狹窄的結構和功能相關性。乙型轉化生長因子 （TGF-b）調控的巨噬細胞-肌纖維母細胞轉分化 （MMT）參與了血管再狹窄的病理性增生並有著病理性的作用。Smad3蛋白對血管再狹窄有複雜性影響，其基因敲除有效減弱內膜增生厚度，完全抑制MMT細胞的轉分化，但同時卻影響了細胞外基質的完整性，導致阻礙血流正常運行。而低劑量Smad3特異性抑制劑更能成功對抗相當程度的內膜增生，同時保有細胞外基質的完整性和保持部分血流。四甲基吡嗪則具劑量依賴性效用以緩和血管再狹窄及抑制MMT細胞的轉分化，同時又備有劑量依賴性功能來平衡Smad3和Smad7的表達量失衡，及帶來血液流速功能性的進步。上述結果表明:1) 巨噬細胞-肌纖維母細胞轉分化參與了血管再狹窄的病理性增生。2) Smad3蛋白對血管再狹窄有複雜性影響，一方面其敲除或抑制可減弱內膜增生厚度和MMT細胞的轉分化，但另一方面會影響細胞外基質的完整性。低劑量抑制的最佳劑量可望從中取得平衡，達致治療效果。3) 四甲基吡嗪的劑量依賴性效用成功對抗血管再狹窄可能是透過平衡Smad3和Smad7表達量以抑制MMT細胞的轉分化。這些研究結果豐富及增加了對於川芎嗪用作保護心血管的機制認知，為這一傳統中醫藥更廣泛及全面應用于更多臨床實踐提供了一定的科學理論基礎和論證。Mak, Shiu Kwong Thomas.Thesis Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 180-220).Abstracts also in Chinese.Title from PDF title page (viewed on 1, April 2021).Mak, Shiu Kwong Thomas