Molecular control of vascular smooth muscle reprogramming in arteriovenous fistula maturation

项目摘要 通过手术在动脉和静脉之间建立动静脉瘘（AVF）现在是首选的方法， 为慢性肾脏病（CKD）患者的血液透析提供血管通路。但近 由于向外重塑和血流不足，60%的AVF未能成熟为临床有用的管道 容量、闭塞性新生内膜增生和/或纤维化狭窄。目前，没有治疗方法可以 通过增强AVF外向重塑改善AVF早期成熟失败，这主要是由于我们的新生 了解静脉适应AVF血流动力学应激的机制。急剧增加 静脉中的剪切应力和脉动压力通过快速扩张随后壁增厚而标准化。 静脉平滑肌细胞（vSMC）是响应于血管收缩而感知血管壁拉伸的主要细胞。 增加流量和血压。进步的一个重大障碍是我们对人类社会的认识不足。 在AVF适应早期vSMC对动脉血流动力学的反应机制。有力证据来自 AVF小鼠模型和人类样本研究都首次证明了分化的 AVF外向重塑和成熟中的vSMC。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肾切除术CKD AVF小鼠模型。我们进一步表明，AVF的早期成熟涉及 vSMC从静止重编程为以前未表征的增殖合成状态， 令人惊讶地保留了差异化的收缩特性。心肌素相关转录因子（TF）A和B （MRTFA和B，MRTFs）通过反式激活多个基因程序来响应循环拉伸。VSMC缺陷 MRTFs的减少损害AVF成熟，AVF壁厚度减少。除了收缩基因程序， MRTFA上调新的靶基因（MMP 2和ATF 3），以促进基质重塑和细胞增殖。 这表明MRTF充当vSMC重编程的节点TF。CAMK 2是一种主要的信号转换器 准备好整合牵张诱导的血管重塑初步数据显示，生长因子诱导核 在培养的vSMC中CAMK 2和MRTFA的相互作用。主要VSMC CAMK 2同种型的VSMC缺陷， CAMK 2D，表型模拟VSMC MRTF的损失，表明CAMK 2D从AVF壁转导信号 应激触发MRTF反式作用。这些初步数据支持我们的中心假设，成功的AVF 适应和成熟涉及CAMK 2/MRTF依赖性vSMC重编程为增殖性， 基质组织和收缩表型。Aim 1将阐明vSMC依赖性AVF的机制 使用Itga 8 CreERT 2Confetti报告基因的适应性重塑和成熟，单核（sn）ATAC/RNA-seq， 和空间组学以确定AVF成熟的基础vSMC克隆扩增和转录组学。目的2 将使用新的VSMC特异性MRTF和CAMK 2D敲除小鼠来阐明 AVF成熟中的CAMK 2D/MRTF。这些研究的成功完成将提供新的见解， 潜在的治疗靶点，AVF成熟失败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.