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 maturatio...