Smooth Muscle Cells Derived From Second Heart Field and Cardiac Neural Crest Reside in Spatially Distinct Domains in the Media of the Ascending Aorta-Brief Report

Abstract

Objective: Smooth muscle cells (SMCs) of the proximal thoracic aorta are embryonically derived from the second heart field (SHF) and cardiac neural crest (CNC). However, distributions of these embryonic origins are not fully defined. The regional distribution of SMCs of different origins is speculated to cause region-specific aortopathies. Therefore, the aim of this study was to determine the distribution of SMCs of SHF and CNC origins in the proximal thoracic aorta.

Approach and results: Mice with repressed LacZ in the ROSA26 locus were bred to those expressing Cre controlled by either the Wnt1 or Mef2c (myocyte-specific enhancer factor 2c) promoter to trace CNC- and SHF-derived SMCs, respectively. Thoracic aortas were harvested, and activity of β-galactosidase was determined. Aortas from Wnt1-Cre mice had β-galactosidase-positive areas throughout the region from the proximal ascending aorta to just distal of the subclavian arterial branch. Unexpectedly, β-galactosidase-positive areas in Mef2c-Cre mice extended from the aortic root throughout the ascending aorta. This distribution occurred independent of sex and aging. Cross and sagittal aortic sections demonstrated that CNC-derived cells populated the inner medial aspect of the anterior region of the ascending aorta and transmurally in the media of the posterior region. Interestingly, outer medial cells throughout anterior and posterior ascending aortas were derived from the SHF. β-Galactosidase-positive medial cells of both origins colocalized with an SMC marker, α-actin.

Conclusions: Both CNC- and SHF-derived SMCs populate the media throughout the ascending aorta. The outer medial cells of the ascending aorta form a sleeve populated by SHF-derived SMCs.

Keywords: aorta; cell tracking; neural crest; second heart field; smooth muscle.

Figure 1. Distribution of CNC- and SHF-derived cells in the proximal thoracic aorta

Representative ventral views of β-gal activity in proximal thoracic aortas from Wnt1- (A) and Mef2c- (B) Cre mice in tissues acquired at 12 weeks of age, n = 3 - 4 for each group. Representative images of β-gal activity and eosin staining from sagittal sections of the aortic root and arch in Wnt1- (C) and Mef2c-Cre (D) male mice, n = 3 for each group. Magnified images were taken from the anterior (blue box) and posterior region (green box). L = lumen, Ao = aorta, ST-J = sinotubular junction, PA = pulmonary artery, IA = innominate artery, CA = common carotid artery, SA = subclavian artery, DA = ductus arteriosus, LV = left ventricle, AR = anterior region, PR = posterior region. Cross sections of mid-ascending aortas from Wnt1- (E) and Mef2c- (F) Cre mice were stained with X-gal and eosin B, n = 3 for each group. Magnified images were taken from the anterior region (blue box). Representative histograms measured β-gal activity from internal to external elastic lamina in the anterior region of ascending aortas from Wnt1- (G) and Mef2c-Cre (H) mice, n = 3 for each group. Blue color is positive staining for distribution of Cre excision. IEL = internal elastin lamina, EEL = external elastin lamina, L = lumen, M = media, A = adventitia. Yellow dotted lines depict location of IEL and EEL.

Figure 2. Cellular localization of CNC- and SHF-derived cells in the ascending aorta

Representative images of double immuno-fluorescence for β-gal and α-SMA in ascending aortas from Wnt1- (A) and Mef2c- (B) Cre mice acquired from mice at 12 weeks of age, n = 3 for each group. Green color indicates positivity for β-gal and red color indicates positivity for α-SMA. Yellow color in merged images indicates that cells are both β-gal and α-SMA positive. Yellow lines indicate internal and external elastic laminae. Negative controls for immunostaining are counterstained blue with DAPI for nuclear detection and the boundary of the media is shown by the green autofluorescence of elastin fibers (C). L = lumen, IEL = internal elastin lamina, EEL = external elastin lamina. Images are orientated lumen up.