Cell Type-Specific Targeting of Different Smooth Muscle Cell Populations by Intersectional Genetics

The Cre/loxP recombination system is the main tool for cell type-specific lineage tracing and gene targeting. Currently available smooth muscle cell (SMC)-specific Cre mouse lines show off-target activity outside the SMC lineage and are unable to distinguish among arterial SMCs (ASMCs), venous SMCs, and nonvascular SMCs (NVSMCs). These limitations prevent ASMC- and NVSMC-specific gene targeting, which is required to characterize the role of SMCs in different organs and diseases.

To achieve precise manipulation of ASMCs in vivo, we combined alleles for Cspg4-Dre and rox-Stop-containing Acta2-CreER (Acta2-rox-CreER), generating a mouse line with Cre activity exclusively in ASMCs. RNA sequencing of fluorescence-activated cell sorting-isolated ASMCs was used to reveal differences in ASMCs among multiple organs. To specifically target and characterize NVSMCs in different organs, a combination of Chrm2-Dre and Acta2-rox-CreER was used. Disease-specific transcriptional changes of pulmonary ASMCs and NVSMCs were determined in the Sugen 5416/hypoxia model of pulmonary arterial hypertension. Usefulness for functional studies was assessed by inactivation of the genes for the splicing factors RBPMS (RNA-binding protein with multiple splicing) and RBPMS2 (RNA-binding protein with multiple splicing 2) in ASMCs.

Intersectional genetic approaches using Cspg4-Dre and Acta2-rox-CreER mouse lines specifically targeted ASMCs within various organs. A combination of Chrm2-Dre with Acta2-rox-CreER achieved specific targeting of NVSMCs. Transcriptomic profiling revealed distinct gene expression signatures in ASMCs of different organs, indicating organ-dependent transcriptional adaptation of ASMCs. Transcriptional differences among NVSMCs were found in the lung, intestine, and bladder. Activation of distinct pathways was uncovered in pulmonary ASMCs and bronchial SMCs after induction of pulmonary arterial hypertension. Inactivation of Rbpms and Rbpms2 in ASMCs increased thickness of the muscular layer in pulmonary arteries, whereas inactivation in all SMCs abolished the contractile phenotype of NVSMCs in the intestine.

The successful generation of mouse lines specifically targeting different subtypes of SMCs enhances specificity, allowing distinction between vascular and nonvascular effects of diseased SMCs. The identification of vessel bed-specific gene signatures will pave the way for specific manipulation of SMCs in distinct diseased organs, such as the lung in pulmonary arterial hypertension.