Promoters to Study Vascular Smooth Muscle

Abstract

Smooth muscle cells (SMCs) are a critical component of blood vessel walls that provide structural support, regulate vascular tone, and allow for vascular remodeling. These cells also exhibit a remarkable plasticity that contributes to vascular growth and repair but also to cardiovascular pathologies, including atherosclerosis, intimal hyperplasia and restenosis, aneurysm, and transplant vasculopathy. Mouse models have been an important tool for the study of SMC functions. The development of smooth muscle-expressing Cre-driver lines has allowed for exciting discoveries, including recent advances revealing the diversity of phenotypes derived from mature SMC transdifferentiation in vivo using inducible CreER ^T2 lines. We review SMC-targeting Cre lines driven by the Myh11, Tagln, and Acta2 promoters, including important technical considerations associated with these models. Limitations that can complicate study of the vasculature include expression in visceral SMCs leading to confounding phenotypes, and expression in multiple nonsmooth muscle cell types, such as Acta2-Cre expression in myofibroblasts. Notably, the frequently employed Tagln/ SM22α- Cre driver expresses in the embryonic heart but can also confer expression in nonmuscular cells including perivascular adipocytes and their precursors, myeloid cells, and platelets, with important implications for interpretation of cardiovascular phenotypes. With new Cre-driver lines under development and the increasing use of fate mapping methods, we are entering an exciting new era in SMC research.

Keywords: atherosclerosis; gene expression; mice, transgenic; muscle, smooth; myeloid cells; neointima.

Figure 1. Flow cytometric analysis reveals Tagln-Cre mediated switch from tdTomato to GFP in a majority of platelets.

FACS analysis of platelets isolated from platelets from WT (A), mTmG (B) and Tagln-Cre x mTmG F1 (C) mice for tdTomato red (left) and GFP (right). The cutoff for positive signal is based on analysis of WT (A) nonfluorescent platelets. In 8 week old Tagln-Cre x mTmG mice (C), 33% of platelets are positive for tdTomato expression (solid arrow) while 58% are positive for GFP expression (dashed arrow) after Cre-mediated recombination.

Figure 2. Tagln-Cre-mTmG labels the aortic media and perivascular adipose tissue.

Confocal spinning disk microscopy of a section of aorta from a male Tagln-Cre-mTmG (Tg(Tagln-cre)1Her/J) mouse that was immunostained with an antibody to mouse Adiponectin (#1119 R&D Systems, 1:10 dilution) and secondary antibody (Cy5, Cyan). Merged image at top left, individual channels for GFP, tdTomato, and Cyan also shown. Scale bar = 50 mm. GFP (green) indicates cells in which Tagln-Cre is active, while tdTomato (red) indicates cells in which there is no Cre-induced recombination. DAPI (dark blue) staining indicates nuclei. A negative control with no secondary antibody is shown at bottom left. Note that adiponectin, a marker of differentiated adipocytes, strongly costains the perivascular adipocytes, as well as some medial SMCs, which we have shown to express adiponectin .