Abstract Body: Background: Smooth muscle cells (SMCs) play a critical role in maintaining vascular integrity and regulating blood flow. Unlike many other cell types, SMCs can undergo phenotypic modulation in response to vascular injury and cardiovascular disease (CVD) pathologies. Recent in vitro studies indicate that reduced expression of Leiomodin 1 (LMOD1), a gene preferentially expressed in SMCs, promotes SMC proliferation, a key mechanism associated with phenotypic modulation. However, the contribution of this gene in regulating SMC proliferation in an in vivo mouse model and the underlying molecular mechanism(s) responsible for this association remains unknown.
Methods: Carotid artery ligation in wild-type (n=6) and Lmod1 heterozygous (Lmod1^+/-) mice (n=9) was implemented to assess the impact of reduced LMOD1 on SMC proliferation in vivo. RNA sequencing, followed by gene regulatory network analysis using PANDA and LIONESS, was performed on control (n=5) and LMOD1-deficient (n=4) cultured SMCs to determine the underlying transcriptional and regulatory changes as well as signaling pathways linking reduced LMOD1 with increased SMC proliferation.
Results: In vivo carotid artery ligation studies revealed that Lmod1^+/- mice displayed enhanced neointimal formation. Interestingly, immunohistochemistry analysis partly attributed this phenotype to increased SMC proliferation. To understand the underlying molecular mechanism involved, we performed transcriptomic analysis on control and LMOD1-deficient SMCs. We found that genes linked to cell cycle signaling and G1/S transition, key processes associated with SMC proliferation, were significantly dysregulated in LMOD1-deficient SMCs compared to control SMCs. Furthermore, gene regulatory network analysis corroborated these findings, identifying CDK6 as a key regulator of LMOD1-mediated SMC proliferation, which we subsequently validated through in vitro experiments. Lastly, simultaneous in vitro knockdown of LMOD1 and CDK6 demonstrating the restoration of SMC proliferation to baseline confirmed the reversibility of this proposed mechanism in vitro.
Conclusions: In conclusion, these findings reveal that reduced LMOD1 promotes SMC proliferation following vascular injury through CDK6, which in turn could serve as a potential therapeutic target in developing new strategies to effectively inhibit neointimal formation.