Role of inflammatory response in aortic valve lesions

描述（由申请人提供）：外科瓣膜置换术是钙化性主动脉瓣狭窄的唯一可用疗法。尽管慢性炎症被认为在主动脉瓣病变的发病机制和进展中起主要作用，但在瓣膜细胞中启动和维持炎症过程的信号传导机制仍不清楚。了解主动脉瓣细胞的炎症和成骨反应对于预防主动脉瓣病变及其进展具有重要意义。我们证明，刺激Toll样受体4（TLR 4）与内毒素上调骨形态发生蛋白2（BMP-2）在人主动脉瓣肌成纤维细胞（HAVMFs）的表达和BMP-2在TLR 4诱导的成骨反应，其特征在于Runx 2，碱性磷酸酶（ALP）和骨桥蛋白的表达中起着重要作用。此外，我们发现TLR 4的慢性刺激促进老年小鼠的主动脉瓣钙化和增厚。初步研究表明，TLR 4信号上调HAVMFs中Notch 1胞内结构域（NICD 1）的水平。中和INF 2，TRIF调节的1型干扰素，消除细胞NICD 1的增加，并降低TLR 4刺激后的BMP-2水平。此外，Notch 1的抑制也降低了TLR 4刺激后BMP-2的表达。总之，这些新的发现表明，INF 2激活Notch 1，NICD 1增强TLR 4诱导的BMP-2表达。有趣的是，在TLR 4刺激后24小时，人AVMF中ILCD 1和BMP-2水平升高。NICD 1可能不仅在早期阶段调节BMP-2表达，而且在调节BMP-2信号传导中起作用。事实上，初步研究表明，Notch 1的激活增强了BMP-2诱导的Runx 2表达。因此，TLR 4信号传导诱导人AVMF中的成骨反应，并且机制涉及BMP-2和Notch 1通路之间的相互作用。我们制定了三个相互关联的目的来确定TLR 4诱导的瓣膜成骨反应的机制：1）测试TLR 4信号传导通过BMP-2介导的Runx 2上调诱导主动脉瓣成骨反应的假设，2）测试NICD 1通过调节NF-：B活性增强BMP-2表达的假设，3）测试NICD 1通过与Smad 1/5/8相互作用增强BMP-2信号传导的假设。这些研究将为了解促炎信号介导AVMF成骨反应和主动脉瓣病变的分子机制提供重要的见解。 公共卫生相关性：主动脉瓣病变（钙化和增厚）影响大量人群，并经常进展为钙化性主动脉瓣狭窄，导致发病率和死亡率。由于对机制的了解有限，目前无法进行药物干预以预防钙化或其进展为钙化性狭窄。拟议的研究将推进我们的知识，并将确定抑制主动脉瓣病变过程的治疗靶点。

DESCRIPTION (provided by applicant): Surgical valve replacement is the only available therapy for calcific aortic valve stenosis. Although chronic inflammation is believed to play a major role in the pathogenesis and progression of aortic valve lesions, the signaling mechanisms that initiate and sustain the inflammatory process in valvular cells remain unclear. Understanding of the inflammatory and osteogenic responses in valvular cells is important for prevention aortic valve lesions and their progression. We demonstrate that stimulating Toll-like receptor 4 (TLR4) with endotoxin up-regulates the expression of bone morphogenetic protein 2 (BMP-2) in human aortic valve myofibroblasts (HAVMFs) and that BMP-2 plays a major role in TLR4-induced osteogenic responses, characterized by expression of Runx2, alkaline phosphatase (ALP) and osteopontin. Further, we found that chronic stimulation of TLR4 promotes aortic valve calcification and thickening in old mice. Preliminary studies show that TLR4 signaling up-regulates the levels of Notch1 intracellular domain (NICD1) in HAVMFs. Neutralizing INF2, a TRIF-regulated type 1 interferon, abrogates the increase in cellular NICD1 and reduces BMP-2 levels following stimulation of TLR4. Further, inhibition of Notch1 also reduces BMP-2 expression following TLR4 stimulation. Together, these novel findings indicate that INF2 activates Notch1 and that NICD1 enhances TLR4-induced BMP-2 expression. Interestingly, elevated levels of lNICD1 and BMP-2 coexist in human AVMFs at 24 h following TLR4 stimulation. It is likely that NICD1 plays a role not only in regulating BMP-2 expression in the early phase but also in modulating BMP-2 signaling. Indeed, preliminary studies show that activation of Notch1 enhances BMP-2-induced expression of Runx2. Thus, TLR4 signaling induces the osteogenic responses in human AVMFs, and mechanisms involve the interplay between the BMP-2 and Notch1 pathways. We formulated three interrelated aims to determine the mechanisms of TLR4-induced valvular osteogenic responses: 1) to test the hypothesis that TLR4 signaling induces aortic valve osteogenic responses through BMP-2-mediated up-regulation of Runx2, 2) to test the hypothesis that NICD1 enhances BMP-2 expression through modulation of NF-:B activity and 3) to test the hypothesis that NICD1 enhances BMP-2 signaling via interaction with Smad1/5/8. These studies will provide important insights into the molecular mechanisms by which pro-inflammatory signaling mediates AVMF osteogenic responses and aortic valve lesions. PUBLIC HEALTH RELEVANCE: Aortic valve lesions (calcification and thickening) affects a large number of people and frequently progresses to calcific aortic stenosis that causes morbidity and mortality. Due to limited knowledge of the mechanisms, pharmacological intervention to prevent calcification or its progression to calcific stenosis is currently unavailable. The proposed studies will advance our knowledge and would identify therapeutic targets for suppressing the process of aortic valve lesions.