巨噬细胞生物钟基因Bmal1在高血压血管重构中的作用

Misalignment of circadian rhythm, for example, shift work, has been associated with higher risk of cardiovascular disease. Central clock in the hypothalamus entrained by sunlight coordinates the daily cycle of physiology and behavior while the intrinsic molecular clock in peripheral tissues controls cellular function. Bmal1, the non-redundant transcription factor in the core molecular clock, has been implicated in cardio-metabolic diseases in mice and human. Bmal1 controls the cyclic trafficking of pro-inflammatory monocytes in acute and chronic inflammation. Previously, we and others showed that global or tissue-specific disruption of Bmal1, Clock, Per and Cry modulate the pathogenesis of atherosclerosis. .Vascular remodeling is consequence of a maladaptive repair mechanism in response to vascular inflammation and injury commonly occurs in hypertension. Vascular remodeling contributes to both the development and complications of hypertension. Recent studies suggested the importance of both innate and adaptive immune components in the pathogenesis of hypertension. Because Bmal1 is a critical regulator of inflammatory response, we hypothesize that deletion of Bmal1 in macrophages and monocytes promotes blood pressure increase in mouse model of hypertension, through increased vascular remodeling due to upregulation of MMP9 and MMP13. Preliminary results showed that macrophage-specific Bmal1 deletion further exacerbated blood pressure increase induced by angiotensin II, accompanied by increased vascular remodeling and upregulation of MMPs..We will induce hypertension using angiotensin II infusion and DOCA-salt induced hypertension in macrophage-specific Bmal1 knockout mice and wild type mice. We will examine whether Bmal1 deletion in macrophages further increases blood pressure, vessel thickness and stiffness; and promotes vascular dysfunction in hypertensive mice. Secondly, we will examine whether deletion of Bmal1 in macrophage upregulates MMP9 and MMP13, accompanied by heightened vascular smooth muscle cell migration proliferation and migration. Finally, we will explore the underlying molecular mechanisms of Bmal1 mediated suppression of MMP9 and MMP13. We hypothesize that BMAL1 interacts with transcriptional repressor EZH2, leading to increased methylation of histone mark H3K27me3 at the promoters of MMP9 and MMP13 E-box region. In addition, BMAL1 suppresses TGFb1-Smad2-MMP signaling by interacting with Smad3. The results will provide novel insight into understanding the temporal regulation of vascular responses to hypertension and anti-hypertension therapies, and explore the possibility of targeting peripheral circadian clock in cardiovascular diseases.

血管重构是引起高血压靶器官损伤的重要病理基础。巨噬细胞能分泌细胞因子和基质金属蛋白酶MMP，在血管炎症和重构中起了关键作用。我们既往研究发现生物钟基因Bmal1参与了维持血管稳态和抑制动脉粥样硬化的血管炎症。Bmal1是否作为调节巨噬细胞功能的关键分子和参与血管重构尚未明确。预实验中发现巨噬细胞特异性敲除Bmal1加剧高血压引起的血管重构。本项目将证明高血压时Bmal1敲除增加血管外膜的巨噬细胞浸润，产生更多MMP9和MMP13促进细胞外基质失衡，导致血管重构和功能障碍；BMAL1通过减少高血压中TGFb1-Smad2-MMP9/13信号上调；同时通过与EZH2结合，增加H3K27me3组蛋白修饰，抑制MMP9和MMP13表达。从表观+转录调控-细胞功能-疾病表现这个主线，深入探讨Bmal1调控巨噬细胞功能从而影响高血压血管重构的分子机制，提出血管重构发病机制的新切入点和研发药物的可能性。

项目背景：高血压是常见的慢性心血管疾病，也是导致冠心病、脑卒中等心血管疾病的首要危险因素。目前临床上尚没有针对高血压血管重构的理想药物，寻找新的发病机制和治疗靶点可为高血压血管重构的防治提供新的策略。巨噬细胞在高血压发病过程中发挥重要作用，除了促炎,细胞因子和趋化因子外，血管重构的病理变化是否受巨噬细胞的上游调控因子的调控尚未明确。.主要研究内容：在高血压引起的血管重构过程中，受促炎因子的诱导，巨噬细胞Bmal1的敲除在转录层面引起MMP上调，导致ECM失衡，血管中层平滑肌细胞增殖迁移增加，加剧血管重构和血管功能异常，导致外周阻力增加，进而使血压上升加剧和促进高血压引起的靶器官损害。.重要结果：利用LysM-Cre与Bmal1floxP小鼠杂交取得巨噬细胞特异性敲除小鼠(Bmal1-MKO)和野生型对照小鼠(Bmal1-MWT)，用血管紧张素AngII诱导高血压，Bmal-MKO鼠的AngII升高血压更快更高，特别是收缩压和脉压差与Bmal1-MWT鼠相比显著增加。组织学发现高血压的Bmal1-MKO鼠和Bmal1-MWT鼠相比，主动脉血管平滑肌增厚，外膜有更多的胶原沉积，且符合高血压大动脉外向肥厚性重塑的特点。在高血压Bmal1-MKO鼠的血管外膜有更多的M2巨噬细胞，血管组织表达M2标记物增加。几种基质金属蛋白酶特别是MMP9和MMP13的表达增加。体外培养的骨髓分化的细胞(BMDM) Bmal1-KO加强了白介素4 (IL-4)诱导的STAT6磷酸化，同时使下游的MMP13 的表达和活性增加，细胞水平上MMP13增加切割pro-MMP9为有活性的MMP9。BMDM中IL4Ra敲除能抑制IL-4诱导的STAT6磷酸化和MMP13的上调。Bmal1敲除能增加IL-4引起的p-STAT6入核，免疫共沉淀提示BMAL1蛋白与STAT6蛋白有直接的结合。这些结果提示Bmal1敲除加剧血管重构可能与促纤维化的IL4-STAT6-MMP13通路的活性增加有直接关系。 .科学意义：这些实验结果证实Bmal1通过调控巨噬细胞功能参与高血压血管重构，揭示巨噬细胞Bmal1通过与STAT6相互作用调控关键的作用因子MMP的分子机制和Bmal1在血管稳态中的关键作用，为血管重构的免疫机制提供新的信号通路和治疗靶点。