image: A. Experimental strategy of Myh11-Cre genetic tracing mice. B. Immunostaining for ZsGreen and Myh11 on aortas of Myh11 Cre mice. C. Experimental strategy of Myh11-CreER genetic tracing mice. D. Schematic showing the experiment design. E. Whole-mount bright-field and fluorescence view of mutant (Myh11-CreER;Smad4flox/flox;R26-tdT) and control (Myh11-CreER;Smad4flox/+;R26-tdT) aortas (female and male mice). F. Immunostaining for tdT, αSMA, and SMAD4 on aortic sections.
Credit: ©Science China Press
The research was led by Professor Wang Lixin (Department of Vascular Surgery, Zhongshan Hospital, Fudan University) and Professor Zhou Bin (Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences). The study reported two new mouse genetic tools, namely Myh11-Cre and Myh11-CreER. These innovative tools can specifically label smooth muscle cells (SMCs) irrespective of gender and surpass the limitations of sex-linked inheritance (Y chromosome). The introduction of Myh11-Cre and Myh11-CreER mice signifies a milestone in genetic research in the SMC field, furnishing the scientific community with robust resources for elucidating the fate transition dynamics of SMCs across physiological and pathological contexts within the cardiovascular system.
SMCs are the principal structural components of the arterial media, crucial for maintaining the architecture and functionality of arterial vessels. Characterized by an elongated spindle-shaped morphology, differentiated SMCs exhibit remarkable cellular specificity and express heightened contractile marker proteins including α-SMA, SM22α, and SM-MHC (encoded by Myh11). Under normal physiological conditions, mature SMCs display limited proliferative and synthetic activities, actively participating in the regulation of vascular contraction and relaxation, thus contributing to the control of blood pressure and distribution of blood flow. During the onset of pathological conditions, SMCs undergo dedifferentiation, exhibiting pronounced plasticity characterized by the upregulation of synthetic marker proteins such as OPN, and an increase in cell proliferation and migration capabilities. Concurrently, these cells acquire phenotypic traits reminiscent of mesenchymal stem cells and macrophages.
Aortic aneurysm is characterized by pathological dilation of the aortic vessel wall, exceeding 50% of the normal vessel diameter, primarily due to structural defects in the aortic vessel wall, with hypertension and atherosclerosis as major predisposing factors. As the aortic aneurysm grows, the weakened vessel wall becomes incapable of withstanding blood flow pressure, leading to sudden rupture of the aortic aneurysm and potentially fatal outcomes. Dysfunction of vascular smooth muscle cells (VSMCs) is crucial for the disruption of the structural integrity of the aorta, and the loss and phenotypical switching of VSMCs are important hallmarks of aortic aneurysms. Previous studies have implicated mutations in genes related to the transforming growth factor-beta (TGF-β) signaling pathway are associated with the formation of aneurysms in hereditary connective tissue disorders, including Loeys-Dietz and Marfan syndromes.
Currently, the Myh11-CreER-Off transgenic mouse, introduced by the Offermanns laboratory in 2008, stands as the predominant tool for specifically labeling SMCs in research endeavors. However, its utility is restricted to male mice due to the gene insertion site on the Y chromosome. Given that the incidence of cardiovascular diseases in females is lower than that in males, the results obtained from male mice may not necessarily be validated in females, thus lacking generalizability in clinical relevance. Considering the lower incidence of cardiovascular diseases in females compared to males, the findings derived from male mice may not universally extrapolate to females, thereby limiting clinical applicability and generalizability. To address this issue, researchers initially utilized CRISPR/Cas9 technology to insert Cre recombinase cDNA into the endogenous ATG of the mouse Myh11 gene locus, which was then crossed with a reporter mouse responding to Cre (Rosa26-loxP-Stop-loxP-ZsGreen), resulting in permanent expression of ZsGreen in Myh11+ SMCs. Immunofluorescence staining confirmed successful expression of ZsGreen in both male and female mouse Myh11+ SMCs. Furthermore, by inserting CreERT2 cDNA into the endogenous ATG of the Myh11 gene locus, a tamoxifen-inducible My11-CreER mouse model was constructed, which was crossed with the Rosa26-loxP-Stop-loxP-tdTomato (R26-tdT) reporter mouse, achieving successful labeling of Myh11+ SMCs in both male and female mice. Finally, utilizing My11-CreER mouse line, researchers investigated the role of Smad4 gene expression in aortic SMCs in the formation of aortic aneurysms, by developing Myh11-CreER;Smad4flox/flox;R26-tdT mutant mice, and using a murine model comprising Myh11-CreER;Smad4flox/+;R26-tdT mice as the control, wherein Smad4 expression remains intact. This model aimed to discern the gender-dependent variations in the progression of aortic aneurysms, thereby unveiling the intricate role of Smad4 in vascular pathophysiology.
Taken together, the research team has developed novel genetic tools, namely Myh11-Cre mice and Myh11-CreER mice, using gene knock-in technology. These innovative mouse models allow for the tracing of SMCs in both male and female mice. This study thus presents a robust experimental platform for investigating gender disparities in the structural and functional alterations of SMCs under both normal physiological conditions and disease states.
See the article:
Smooth Muscle Cell-Specific Genetic Targeting by Myh11-driven Cre(ER) Knockin Mice
Journal
Science China Life Sciences