Targeting mechanisms activating ion-channel for preventing acute lung injury

摘要 在细菌和病毒感染期间发生的内皮损伤会导致蛋白质的不受控制的积累- 基础组织中有丰富的液体和炎症细胞，是急性肺损伤(ALI)和急性肺损伤(ALI)的特征 呼吸窘迫综合征(ARDS)。尽管在支持性护理方面取得了显著进展，但患者在 ALI和ARDS的发病率保持在40%左右。我们已经证明了瞬时受体电位的关键作用。 通道6(TRPC6)介导钙离子内流，启动炎症信号，导致ALI。然而，我们也 发现TRPC6中的异亮氨酸(I)111与其异构体亮氨酸(L)111的突变或TRPC6的阻断 使用新肽的异亮氨酸111允许通道获得新的功能，而不依赖于钙离子进入 将EC从炎症转化为再生谱系，从而解决肺损伤。因此， 了解I111诱导通道活动的作用机制及阻断的治疗价值 I111促进EC再生是预防这些疾病的关键。我们证明：1)I111的替代 由于其在TRPC6的第一个Ankyrin重复结构域(ARD)上的异构体L111阻止了钙离子的进入；2)I111L突变引发 基于核磁共振的TRPC6中导致通道功能丧失的变构转变 研究：3)I111L-TRPC6在损伤过程中诱导内皮细胞再生信号，ERG，a 转录因子维持内皮细胞动态平衡，促进内皮细胞增殖，促进肺损伤后快速修复；4) 在TRPC6-/-小鼠EC中挽救WT-TRPC6而不是I111L-TRPC6突变体可恢复内毒素诱导的肺血管 通过抑制ERG的表达而增强NF-B的表达和炎症的高通透性 信号转导；5)在EC损伤EC增殖和诱导肺损伤中诱导ERG的条件缺失，以及， 6)跨I111-TRPC6的TRPC6封闭肽抑制内皮细胞钙内流，但促进内皮细胞增殖 肺组织炎性损伤的消退。染色质可及性的表观遗传变化使信号- 依赖于转录因子的激活，结合基因启动子和增强子来决定细胞功能。 有趣的是，从对照和受损肺中分离的EC的ATAC-SEQ和CHIP-SEQ表明WT或突变 通道选择性地激活EC表观基因组，使其有利于NFERGB或转录活性的转换 EC表型，从而决定肺损伤的结局。基于这些令人兴奋的发现，在目标1中，我们将 确定异亮氨酸111诱导的调节TRPC6结构组织和 功能。在目标2中，我们将测试与WT-TRPC6相反的假设，I111L TRPC6突变体获得 不依赖于通道活动的新功能来编程EC表观基因组以采用再生谱系 因此，通过治疗阻断这种残基功能将修复临床前模型中的血管损伤。 肺损伤的症状。研究将使用多管齐下的方法，包括分子建模、多组学和2- 肺内皮细胞的光子成像，以及I111-TRPC6封闭肽来实现这些目的。我们相信 这些研究对开发特异性的TRPC6拮抗剂以预防ARDS具有翻译意义。

ABSTRACT Endothelial injury occurring during bacterial and viral infections results in uncontrolled accumulation of protein- rich fluid and inflammatory cells in the underlying tissue, hallmarks of acute lung injury (ALI), and acute respiratory distress syndrome (ARDS). Despite remarkable advances in supportive care, patient survival in the setting of ALI and ARDS remains near 40%. We have demonstrated a crucial role of transient receptor potential channel 6 (TRPC6) mediated Ca2+ entry in initiating inflammatory signaling that causes ALI. However, we also showed that mutation of isoleucine (I)111 for its isomer leucine (L)111 in TRPC6 or block of TRPC6 at isoleucine111 using a novel peptide allows the channel to gain new functions independent of Ca2+ entry for programming EC from inflammatory into the regenerative lineage, thereby resolving lung injury. Thus, understanding the mechanisms of action of I111 in inducing channel activity and the therapeutic value of blocking I111 to promote EC regeneration hold the key to preventing these diseases. We show that: 1) substitution of I111 for its isomer L111 in the Ist ankyrin repeat domain (ARD) of TRPC6 blocks Ca2+ entry; 2) I111L mutation initiates allosteric transitions in TRPC6 leading to loss of channel function, based on nuclear magnetic resonance (NMR) studies; 3) I111L-TRPC6 induces EC regenerative signaling during injury as evidenced by expression of ERG, a transcription factor maintaining EC homeostasis, and EC proliferation, leading to rapid lung repair after injury; 4) rescue of WT-TRPC6 but not the I111L-TRPC6 mutant in EC of Trpc6-/- mice reinstates LPS-induced lung vascular hyperpermeability by suppressing the expression of ERG but augmenting NFB-expression and inflammatory signaling; 5) inducing conditional deletion of ERG in EC impaired EC proliferation and induced lung injury, and, 6) a TRPC6 blocking peptide spanning I111-TRPC6 suppresses Ca2+ entry in EC but promotes EC proliferation and resolution of lung inflammatory injury. Epigenetic changes in chromatin accessibility enable signal- dependent activation of transcription factors that bind gene promoters and enhancers to dictate cell functions. Intriguingly, ATAC-seq and Chip-seq of EC sorted from control versus injured lungs suggest that WT or mutated channel selectively activates the EC epigenome either in favor of NFB or ERG transcriptional activities to switch EC phenotype, thereby dictating the outcome of lung injury. Based on these exciting findings, in Aim#1, we will determine the novel mechanisms induced by isoleucine111 in regulating TRPC6 structural organization and functions. In Aim#2, we will test the hypothesis that in contrast to WT-TRPC6, the I111L TRPC6 mutant gains new functions independent of channel activity to program the EC epigenome to adopt a regenerative lineage and therapeutically blocking this residue function will therefore repair the vascular injury in the pre-clinical models of lung injury. Studies will use multipronged approaches, including molecular modeling, multi-omics, and 2- photon imaging of lung EC, along with an I111-TRPC6 blocking peptide to accomplish these aims. We believe these studies to be translational for developing specific TRPC6 antagonists to prevent ARDS.