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.

摘要 在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- 下层组织中富含液体和炎症细胞，是急性肺损伤（ALI）和急性肺损伤的标志 呼吸窘迫综合征（ARDS）。尽管在支持性治疗方面取得了显著进展， ALI和ARDS的发生率仍接近40%。我们已经证明了瞬时受体电位的重要作用 通道6（TRPC 6）介导的Ca 2+内流在引发引起ALI的炎症信号传导中起作用。但是我们也 显示在TRPC 6中异亮氨酸（I）111突变为其异构体亮氨酸（L）111或阻断TRPC 6， 异亮氨酸111使用一种新的肽允许通道获得新的功能，不依赖于Ca 2+进入， 将EC从炎性编程为再生谱系，从而解决肺损伤。因此，在本发明中， 了解I111在诱导通道活性中的作用机制以及阻断I111的治疗价值。 促进EC再生是预防这些疾病的关键。我们证明：1）I111取代 TRPC 6的第一锚蛋白重复结构域（ARD）中的其异构体L111阻断Ca 2+进入; 2）I111 L突变启动 TRPC 6中的变构转换导致通道功能丧失，基于核磁共振（NMR） 研究; 3）I111 L-TRPC 6在损伤期间诱导EC再生信号传导，如通过ERG表达所证明的， 维持EC稳态和EC增殖的转录因子，导致损伤后的快速肺修复; 4） 在Trpc 6-/-小鼠的EC中拯救WT-TRPC 6而不是I111 L-TRPC 6突变体恢复LPS诱导的肺血管 通过抑制ERG的表达但增加NF κ B的表达和炎性 5）在EC中诱导ERG的条件性缺失损害EC增殖并诱导肺损伤，以及， 6)跨I111-TRPC 6的TRPC 6阻断肽抑制EC中的Ca 2+内流但促进EC增殖 和肺部炎性损伤的消退。染色质可及性的表观遗传变化使信号- 转录因子的依赖性激活，结合基因启动子和增强子以决定细胞功能。 有趣的是，从对照与损伤肺中分选的EC的ATAC-seq和Chip-seq表明WT或突变的EC与对照相比具有更高的表达水平。 通道选择性地激活EC表观基因组，有利于NF κ B B或ERG转录活性， 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TRPC 6结构组织中诱导的新机制， 功能协调发展的在目标#2中，我们将检验以下假设：与WT-TRPC 6相比，I111 L TRPC 6突变体获得 独立于通道活性的新功能，以编程EC表观基因组以采用再生谱系 因此在治疗上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。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，沿着I111-TRPC 6阻断肽以实现这些目的。我们认为 这些研究对于开发特异性TRPC 6拮抗剂以预防ARDS具有重要意义。