Abstract Body (Do not enter title and authors here): BACKGROUND: Acquisition and maintenance of vascular smooth muscle cell (VSMC) fate are important for vascular development and homeostasis; however, little is known about the key determinant for VSMC fate and vascular homeostasis.
METHODS: Lkb1^flox/flox;Tagln-Cre, Lkb1^flox/flox;Myh11-Cre/ERT2, and lineage tracing Lkb1^flox/flox;Myh11-Cre/ERT2;ROSA^mT/mG mice were generated to study the impact of Lkb1 on VSMC and vascular function in vivo. Single aortic cells isolated from wild-type and Lkb1^flox/flox;Myh11-Cre/ERT2 mice were analyzed by single-cell RNA sequencing (sc-RNA seq). The dynamic changes of vascular morphology and VSMC fate were investigated using high-frequency ultrasound and various histological techniques. In addition, VSMC phenotype and molecular mechanisms were further explored by mouse arterial ring assay ex vivo, in cultured human aortic VSMC in vitro, and in human aneurysmal aortic tissues.
RESULTS: We found that SMC-specific Lkb1 ablation in Lkb1^flox/flox;Tagln-Cre mice caused severe vascular abnormalities and embryonic lethality. SMC-specific deletion of Lkb1 in tamoxifen-inducible Lkb1^flox/flox;Myh11-Cre/ERT2 mice spontaneously and progressively induced aortic/arterial dilation, aneurysm, rupture, and premature death. Sc-RNA seq and imaging-based lineage tracing showed that Lkb1-deficient VSMCs transdifferentiated gradually from early modulated VSMCs to fibroblast-like and chondrocyte-like cells, leading to ossification and blood-vessel rupture. Moreover, we found that SMC-specific Lkb1 ablation resulted in decreased vascular contractility and hypotension in vivo. Mechanistically, Lkb1 regulates polypyrimidine tract binding protein 1 (Ptbp1) expression and controls alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2. Lkb1 loss in VSMC results in an increased PKM2/PKM1 ratio and alters the metabolic profile by promoting aerobic glycolysis. Treatment with PKM2 activator TEPP-46 rescues VSMC transformation and aortic dilation in Lkb1^flox/flox;Myh11-Cre/ERT2 mice. Furthermore, we found that Lkb1 expression decreased in human aortic aneurysm tissue compared to control tissue, along with changes in markers of VSMC fate.
CONCLUSIONS: Lkb1, via its regulation of Ptbp1-dependent alterative splicing of PKM, maintains VSMC in contractile states and sustains vascular homeostasis. Our findings have important implications for understanding the pathogenesis of aortic aneurysm.