內皮PPARδ在血管穩態中的作用

Peripheral arterial disease (PAD) is a common vascular disease occurs in the lower limb caused by ischemia due to atherosclerotic occlusion. Accumulation of fatty deposits in the vessel wall narrows the artery and reduce blood flow to limbs, causing ischemia, tissue damage and may eventually leads to critical limb ischemia, tissue necrosis, and amputation. It is a common vascular complication in patients with hyperlipidemia and hyperglycemia. Restoration of vascular perfusion in PAD involves the functional recovery of the vascular endothelium. Emerging experimental and clinical evidence show that the peroxisome proliferator-activated receptor delta (PPARδ), participates in enhancing angiogenesis and inhibiting inflammation, based mostly on studies using ligand-dependent activation of PPARδ. However, how PPARδ may be triggered by hypoxic stress and its downstream effects during post-ischemic vascular repair and muscle regeneration were not well understood.In the first part of this thesis, we investigated the role of PPARδ in hindlimb ischemia mouse model. By constructing two in vivo mouse models: endothelial cell-specific knockout PPARδ mice and endothelial cells-specific overexpressing PPARδ in mice, I found that endothelial PPARδ effectively restored reperfusion of ischemic hindlimbs, improved regeneration of injured muscles, inhibited vascular inflammation, improved recovery endothelial function, and enhanced endothelial integrity.In the second part of this thesis, the results of single-cell transcriptome sequencing (scRNA-seq) revealed that selective deletion of PPARδ in endothelial cells caused significant changes in hypoxia-related signaling pathways and gene sets related to angiogenesis, endothelial permeability, and inflammatory response after ischemia. The results of in vivo mouse model and in vitro cell culture proved that deletion of PPARδ in endothelial cells hindered angiogenesis caused by hypoxia.In addition, after ischemia, loss of endothelial PPARδ caused a stronger inflammatory response, including the upregulation of molecules involved in endothelial activation (vascular adhesion molecules Vcam1, E-selectin, Icam1), the production of inflammatory cytokines and chemokines, (such as IL-6, Ccl2, etc.), accompanied with more immune cells infiltration (T Lymphocytes and macrophages). In addition, endothelial barrier function, as an important aspect of vascular homeostasis, is also compromised by selective knockout of endothelial PPARδ, which is mainly manifested by hyperpermeability surrounding the capillaries, and increased extravasation in the injured muscles, accompanied with downregulation of cell junction molecules.In the third part of this thesis, because scRNA-seq showed that PPARδ regulated several functionally important pathways including HIF1α signaling, I found that hypoxia upregulated and activated PPARδ in endothelial cells (EC), whereas PPARδ reciprocally stabilized HIF1α protein through its ligand binding domain direct binding to oxygen dependent degradation (ODD) domain of HIF1α to prevent its degradation. I also showed that such effect was independent of PPARδ ligand. Endothelial selective overexpression of stable HIF1α improved vascular repair impaired by Ppard knockout.In summary, hypoxia-induced, ligand-independent activation of PPARδ in ECs reciprocally stabilizes HIF1α and serves as a critical regulator for HIF1α activation, regulating endothelial gene expression to facilitate the post-ischemic restoration of vascular homeostasis.外周動脈疾病（PAD）是一種常見的外周血管疾病，通常發生在下肢，由動脈粥樣硬化引起缺血。血管壁中脂肪沉積物的不斷積累使動脈血管變窄，並減少了通向四肢的血流，從而導致下肢局部缺血，組織損傷，並可能最終導致嚴重的肢體缺血，組織壞死和截肢。它是高脂血症和高血糖患者的常見血管併發症。PAD中血管灌注的恢復與血管內皮的功能恢復關係密切。新興的實驗和臨床證據表明，過氧化物酶體增殖物激活受體δ（PPARδ）參與增強血管生成和抑制炎症的過程，而這些過程主要是基於使用配體或者激動劑激活PPARδ進行的研究。然而，對於缺氧應激如何觸發PPARδ及其在缺血後血管修復和肌肉再生過程中的下游分子機理尚不清楚。在本文的第一部分，我們研究了PPARδ在後肢缺血小鼠模型中的作用。通過構建兩種體內小鼠模型：內皮細胞特異性敲除PPARδ小鼠和內皮細胞特異性過表達PPARδ的小鼠，我發現內皮PPARδ有效地恢復了缺血後肢的再灌注，改善了受傷肌肉的再生，抑制了血管炎症，改善了內皮的恢復功能，並增強內皮完整性。在本文的第二部分中，單細胞轉錄組測序的結果表明，內皮細胞中PPARδ的特異性缺失導致與缺氧相關的信號通路與血管生成，內皮通透性和缺血後的炎症反應發生明顯變化。小鼠體內模型和體外細胞培養的結果證明，內皮細胞中PPARδ的缺失阻礙了由缺氧引起的血管生成。此外，缺血後，內皮細胞PPARδ的丟失會引起更強的炎症反應，包括上皮細胞活化相關分子（血管黏附分子Vcam1，選擇素E，細胞間粘附分子Icam1）的上調，炎症細胞因子和趨化因子（如IL-6，Ccl2等）的產生，並且伴隨著更多的免疫細胞浸潤（T淋巴細胞和巨噬細胞）。此外，作為血管穩態的重要方面的內皮屏障功能也因為內皮細胞選擇性敲除PPARδ而受到損害，這主要表現為毛細血管周圍的通透性高，受傷肌肉中的血管外滲增加，並伴有細胞連接分子的mRNA水平下調。在論文的第三部分中，由於單細胞轉錄組測序分析顯示PPARδ調節了HIF1α信號轉導等功能上重要的途徑，因此我發現缺氧會上調內皮細胞（EC）中的PPARδ的表達並使其活化，而PPARδ通過其配體結合域使HIF1α蛋白相互作用穩定HIF1α，並直接與HIF1α的氧依賴性降解（ODD）域結合，以防止其降解。我還發現，這種作用與PPARδ配體激活無關。內皮細胞選擇性過表達穩定的HIF1α可改善因Ppard基因敲除而損害的缺血後血管修復。綜上所述，缺氧誘導的EC中PPARδ的活化獨立地穩定了HIF1α，並作為HIF1α活化的關鍵調節劑，調節內皮基因的表達以促進缺血後血管穩態的恢復。WU, Yalan.Ph.D. Chinese University of Hong Kong 2021.Includes bibliographical references (leaves )Abstracts in English and Chinese.Title from PDF title page (viewed on ...