Targeting mechanisms activating ion-channel for preventing acute lung injury

激活离子通道的靶向机制预防急性肺损伤

• 批准号: 10659781
• 负责人: DOLLY MEHTA
• 金额: $ 59.96万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-15 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659781
• 关键词: ATAC-seq; Acute Lung Injury; Acute Respiratory Distress Syndrome; Adopted; Animal Model; Ankyrin Repeat; Bacterial Infections; Binding; Binding Sites; Blood Vessels; Cell physiology; Cells; ChIP-seq; Chromatin; Coupled; DNA; Data; Development; Disease; Endothelium; Enhancers; Epigenetic Process; Fluid Balance; Generations; Genes; Genetic Transcription; Homeostasis; Impairment; Induced Mutation; Infection; Inflammatory; Injury; Ion Channel; Isoleucine; Isomerism; Leucine; Ligand Binding; Liquid substance; Lung; Lung infections; Mediating; Molecular Conformation; Mus; Mutate; Mutation; Mutation Analysis; Natural regeneration; Nuclear Magnetic Resonance; Outcome; Pathology; Patients; Peptides; Phenotype; Play; Pre-Clinical Model; Proliferating; Proteins; Pulmonary Inflammation; Resolution; Role; Signal Transduction; Sorting; Supportive care; Surface; Systemic infection; TRP channel; Testing; Therapeutic; Tissues; Transcriptional Activation; Trauma; Vascular Permeabilities; Virus Diseases; antagonist; cellular imaging; epigenome; gain of function; intravital imaging; lung imaging; lung injury; lung repair; lung vascular injury; molecular modeling; mouse model; multiple omics; mutant; novel; prevent; programs; promoter; receptor; recruit; regenerative; repaired; restoration; trafficking; transcription factor; two-photon; vascular injury

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.

抽象的 细菌和病毒感染期间发生的内皮损伤导致蛋白质不受控制的积累 底层组织中富含液体和炎症细胞，这是急性肺损伤 (ALI) 的标志， 呼吸窘迫综合征（ARDS）。尽管支持性护理取得了显着进步，但患者的生存率 ALI 和 ARDS 的设置仍然接近 40%。我们已经证明了瞬时受体电位的关键作用 通道 6 (TRPC6) 介导 Ca2+ 进入引发导致 ALI 的炎症信号。然而，我们也 结果表明，TRPC6 中的异亮氨酸 (I)111 突变为其异构体亮氨酸 (L)111 或 TRPC6 的阻断 使用新型肽的异亮氨酸 111 允许通道获得独立于 Ca2+ 进入的新功能 将 EC 从炎症细胞编程为再生细胞系，从而解决肺损伤。因此， 了解 I111 诱导通道活性的作用机制以及阻断的治疗价值 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 TRPC6 的 Ist 锚蛋白重复结构域 (ARD) 中的异构体 L111 会阻止 Ca2+ 进入； 2) I111L突变启动 根据核磁共振 (NMR)，TRPC6 中的变构转变导致通道功能丧失 研究； 3) I111L-TRPC6 在损伤过程中诱导 EC 再生信号传导，ERG 的表达证明了这一点。 转录因子维持 EC 稳态和 EC 增殖，导致损伤后肺快速修复； 4） 在 Trpc6-/- 小鼠 EC 中拯救 WT-TRPC6 但不恢复 I111L-TRPC6 突变体可恢复 LPS 诱导的肺血管 通过抑制 ERG 表达但增加 NFκB 表达和炎症来抑制通透性过高 信号发送； 5)诱导EC中ERG的条件性缺失，损害EC增殖并诱导肺损伤，并且， 6) 跨越 I111-TRPC6 的 TRPC6 阻断肽抑制 EC 中的 Ca2+ 进入，但促进 EC 增殖 和肺部炎症损伤的解决。染色质可及性的表观遗传变化使信号 结合基因启动子和增强子以决定细胞功能的转录因子的依赖性激活。 有趣的是，从对照肺与受损肺中分选的 EC 的 ATAC-seq 和 Chip-seq 表明 WT 或突变 通道选择性激活 EC 表观基因组，有利于 NF+B 或 ERG 转录活性进行切换 EC 表型，从而决定肺损伤的结果。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在调节TRPC6结构组织中诱导的新机制和 功能。在目标#2中，我们将测试以下假设：与 WT-TRPC6 相比，I111L TRPC6 突变体增益 独立于通道活动的新功能对 EC 表观基因组进行编程以采用再生谱系 因此，治疗性阻断这种残留功能将修复临床前模型中的血管损伤 的肺损伤。研究将采用多管齐下的方法，包括分子建模、多组学和 2- 肺 EC 的光子成像以及 I111-TRPC6 阻断肽可实现这些目标。我们相信 这些研究可转化为开发特异性 TRPC6 拮抗剂来预防 ARDS。