Molecular control of vascular smooth muscle reprogramming in arteriovenous fistula maturation

项目总结 动静脉之间的动静脉瘘(AVF)是目前手术治疗的首选方法 为慢性肾脏病(CKD)患者的救命血液透析提供血管通道。然而，几乎 60%的动静脉动静脉瘘未能成熟为临床有用的管道，原因是向外重塑和血流不足 容量、闭塞性新生内膜增生和/或纤维性狭窄。目前，还没有治疗方法可以 通过增强aVF的外向重塑改善aVF的早熟失败，这主要是由于我们的新生 了解静脉对动静脉动静脉血流动力学压力的适应机制。急剧增长 在切应力和脉动压方面，静脉通过快速扩张和管壁增厚来正常化。 静脉平滑肌细胞(VSMCs)是感觉血管壁伸展反应的主要细胞。 增加血流量和血压。进步的一个重要障碍是我们对 动静脉瘘适应早期血管平滑肌细胞对动脉血流动力学的反应机制。有力的证据来自 AVF小鼠模型和人类样本研究首次证明了分化的作用 AVF外向重构和成熟中的vSMCs。来自我们的新的初步数据进一步支持了这一点 临床相关的5/6肾切除CKD aVF小鼠模型。我们进一步表明，动静脉瘘的早期成熟包括 VSMC从静止状态重新编程到以前未表现出特征的增殖、合成状态 令人惊讶地保留了差异化的收缩特性。肌钙蛋白相关转录因子(Tf)A和B (MRTFA和B，MRTF)通过反式激活多个基因程序来响应循环拉伸。VSMC缺乏症 房室传导速度减慢，房室壁厚度减薄。在收缩基因程序之外， MRTFA上调新的靶基因(MMP2和ATF3)，以促进基质重塑和细胞增殖。 这表明MRTF作为vSMC重编程的节点TF。CaMK2是一种主要的信号转换器 准备整合拉伸诱导的血管重塑。初步数据显示生长因素导致核 CaMK2和MRTFA在培养的血管平滑肌细胞中的相互作用VSMC缺乏一个主要的VSMC CaMK2亚型， CAMK2D，表现为VSMC MRTF的丢失，提示CAMK2D从AVF壁传递信号(S) 应激以触发MRTF的反式反应。这些初步数据支持我们的中心假设，即成功的AVF 适应和成熟涉及依赖CaMK2/MRTFS的vSMC重新编程为增殖物， 基质组织，收缩表型。AIM1将阐明vSMC依赖的AVF的机制 使用ItGa8CreerT2 Confetti报告程序，单核(SN)ATAC/RNA-seq， 和空间组学来确定vSMC的克隆性扩张和AVF成熟背后的转录。目标2 将使用新的VSMC特异性MRTF和CAMK2D基因敲除小鼠来阐明VSMC特异性MRTF的机制作用 CAMK2D/MRTFS在AVF成熟过程中。这些研究的成功完成将为以下方面提供新的见解 动静脉瘘成熟失败的潜在治疗靶点，可归因于人类静脉适应不足。

PROJECT SUMMARY A surgically created arteriovenous fistula (AVF) between an artery and vein is now the preferred approach to provide a vascular access for life-saving hemodialysis in chronic kidney disease CKD) patients. However, nearly 60% of AVFs fail to mature into a clinically useful conduit due to insufficient outward remodeling and flow capacity, occlusive neointimal hyperplasia, and/or fibrotic stenosis. Currently, there are no therapies that can improve AVF early maturation failure by enhancing AVF outward remodeling, largely due to our nascent understanding of the mechanisms underlying vein adaptations to AVF hemodynamic stresses. Acute increases in shear stress and pulsatile pressure in the vein are normalized by rapid dilation followed by wall thickening. Venous smooth muscle cells (vSMCs) are the predominant cells sensing vessel wall stretch in response to increased flow volume and blood pressure. A significant barrier to progress is a deficit in our knowledge of the mechanisms by which vSMCs respond to arterial hemodynamics in early AVF adaptation. Strong evidence from both AVF mouse models and human sample studies demonstrate, for the first time, a role for differentiated vSMCs in AVF outward remodeling and maturation. This is further supported by new preliminary data from our clinically relevant 5/6-nephrectomy CKD AVF mouse model. We further show early AVF maturation involves vSMCs reprogramming from a quiescent to a previously uncharacterized proliferative, synthetic state that surprisingly retains differentiated contractile properties. Myocardin related transcription factor (TF) A and B (MRTFA and B, MRTFs) respond to cyclic stretch by transactivating multiple gene programs. VSMC-deficiency of MRTFs impairs AVF maturation with reduced AVF wall thickness. Beyond the contractile gene program, MRTFA upregulates novel target genes (MMP2 and ATF3) to facilitate matrix remodeling and cell proliferation. This suggests that MRTFs act as nodal TFs of vSMC reprogramming. CAMK2 is a major signal transducer poised to integrate stretch-induced vascular remodeling. Preliminary data show growth factors induce nuclear interaction of CAMK2 and MRTFA in cultured vSMCs. VSMC-deficiency of a major VSMC CAMK2 isoform, CAMK2D, phenocopies loss of VSMC MRTFs, suggesting that CAMK2D transduces a signal(s) from AVF wall stress to trigger MRTF transactivity. These preliminary data support our central hypothesis that successful AVF adaptation and maturation involves CAMK2/MRTFs-dependent vSMC reprogramming to a proliferative, matrix organizing, and contractile phenotype. Aim1 will elucidate mechanisms of vSMC-dependent AVF adaptive remodeling and maturation using Itga8CreERT2Confetti reporter, single nucleus (sn) ATAC/RNA-seq, and spatial omics to determine vSMC clonal expansion and transcriptomics underlying AVF maturation. Aim 2 will use novel VSMC-specific MRTFs and CAMK2D knockout mice to elucidate the mechanistic role of CAMK2D/MRTFs in AVF maturation. Successful completion of these studies will provide novel insights into, and potential therapeutic targets for, AVF maturation failure attributable to inadequate vein adaption in humans.