Smooth Muscle Cells Derived from Second Heart Field and Cardiac Neural Crest

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 promoter to trace CNC and SHF-derived SMCs, respectively. Thoracic aortas were harvested and activity of β-galactosidase (β-gal) was determined. Aortas from Wnt1-Cre mice had β-gal positive areas throughout the region from the proximal ascending aorta to just distal of the subclavian arterial branch. Unexpectedly, β-gal 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 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-gal positive medial cells of both origins co-localized with a 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.

Introduction

Several forms of aortopathies localize to specific regions of the proximal thoracic aorta.¹ In addition, aortopathies frequently show a medial gradient which increases from lumen to the adventitial aspect.^{2, 3} A proposed contributor to this disease-prone location has been the association of heterogeneity of the embryonic origin of medial SMCs.⁴ SMCs populating this region originate from the second heart field (SHF) and the cardiac neural crest (CNC). It has been speculated that aortopathies in this region are a consequence of the interfaces of these developmentally distinctive cells. These developmental differences in SMCs have the potential for different biological behaviors.

Embryonic origins of SMCs in the proximal thoracic aorta were originally defined using chicken-quail chimeras to demonstrate that SHF-derived cells only populated the aortic root.^{5, 6} These finding were confirmed in subsequent studies using lineage tracing techniques in which SHF-derived cells were identified by expression of Cre driven by a Nkx2.5 promoter.⁷ These data were acquired from aortic roots in embryonic and P01 postnatal stages. Within the root, there was progressive localization to the adventitial side of the media, until transition into the ascending aorta where Nkx2.5 traced cells were absent.⁷ Lineage tracing studies of CNC cells, using Cre driven by a Wnt1 promoter, demonstrated that SMCs of this source populate the media from the ascending aorta to just distal of the branch of the left subclavian artery.^8-10 Collectively, these findings led to the concept of the proximal thoracic aorta being populated by SMCs derived from the SHF in the root and CNC in the ascending aorta.⁴

While whole tissue staining of lineage traced aortas indicates that CNC-derived SMCs populate the aorta from root to the interface of the descending aorta, the few published examples of tissue sections from these mice reflect non uniform staining across the media. In sections from the ascending aorta of ROSA26^LacZ mice expressing Wnt1-driven Cre, there was an absence of enzyme activity in the outer laminar layers.^8,9 SHF-derived cells populated the aortic root, but not the ascending aorta. Thus, embryonic origin of SMCs in the outer laminar layers has not been identified. Since the presence of SMCs of distinct embryonic origin could have a major impact on development of aortopathies, this study identified the distribution of SHF- versus CNC-derived cells in the proximal thoracic aorta.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

CNC- and SHF-derived cells in the proximal thoracic aorta

Regional heterogeneity of embryonic origin of SMCs in the proximal thoracic aorta was delineated by β-gal activity in tissue dissected from Wnt1 and Mef2c-Cre ROSA26R^LacZ mice at 12 weeks of age. Distinct distributions of enzyme activity were apparent between these two promoters. In Wnt1-Cre mice, β-gal positive areas were detected from the aortic root and ascending aorta with extension into the arch to just distal to the subclavian arterial branch. This distribution was similar in male and female mice and consistent among individual mice (Figure 1A, Figure IIA, IIIA in online-only Data Supplement). In Mef2c-Cre, β-gal activity was present from the aortic root and ascending aorta with termination just proximal of the branch of the innominate artery (Figure 1B, Figure IIB, IIIB in online-only Data Supplement). Again, there was no sex difference. The pulmonary artery was positive for β-gal in both Wnt1- and Mef2c-Cre male and female mice (Figure 1A, B, Figure IIA, B, IIIA, B in online-only Data Supplement). The ductus arteriosus was positive in Wnt1-Cre mice, but not in Mef2c-Cre mice (Figure 1A, B, Figure IIA, B, IIIA, B in online-only Data Supplement). As expected, litter mates not expressing Cre were devoid of β-gal staining (Figure IIC, D in online-only Data Supplement). These results indicate that the ascending aorta is composed of both CNC- and SHF-derived SMCs. To determine effects of aging, tissues were acquired from Mef2c-Cre mice in the early postnatal phase of 3 weeks and at 25 weeks of age. There was no discernable difference in the regions that stained for β-gal activity, compared to the 12 week old mice described above (Figure IV, V in online-only Data Supplement).

Medial distribution of CNC- and SHF-derived cells in the ascending aorta

To examine cellular distribution of CNC- and SHF-derived cells, both sagittal and cross-sections of ascending aortas were obtained. In sagittal sections from Wnt1-Cre mice, the β-gal positive area was detected in the media from the sinotubular junction to just distal of the subclavian artery (Figure 1C). However, in Mef2c-Cre mice, β-gal positive areas were observed in the media and extended from the aortic valve to just proximal innominate artery (Figure 1D). β-gal positive areas were also detected in the right ventricle and ventricular septum of Mef2c-Cre mice (Figure 1D). Next, we cross-sectioned the entire ascending aorta to examine distribution of these cells in the media. β-gal positive areas in Wnt1-Cre mice were detected in the inner medial aspect of the anterior (ventral) region and transmedial in the posterior (dorsal) region of the ascending aorta (Figure 1E, Figure VIA in online-only Data Supplement). Interestingly, in Mef2c-Cre mice, β-gal positive areas were located in the outer medial aspect of both anterior and posterior regions (Figure 1F, Figure VIB in online-only Data Supplement). In addition, histograms depicted β-gal positive areas in the inner part of media of the anterior region; while present in the whole media of the posterior region from Wnt1-Cre mice (Figure 1G, Figure VIA in online-only Data Supplement). In contrast, β-gal positive staining was consistently detected only in the outer medial aspect of ascending aortas from Mef2c-Cre mice (Figure 1F, Figure VIB in online-only Data Supplement). These results indicate that the anterior region of the ascending aorta has inner medial cells derived from the CNC, while cells in the outer medial sleeve are derived from the SHF (Table, Figure VII in online-only Data Supplement).

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.

Table. Distribution of CNC- and SHF-derived Cells in the Thoracic Aorta.

Embryonic Origin	Promoter	Distribution
		Thoracic Aorta		Media of Ascending Aorta
		Proximal	Distal	Anterior region	Posterior region
Cardiac Neural Crest (CNC)	Wnt1	Aortic Root	Subclavian Artery	Inner	Transmedial
Second Heart Field (SHF)	Mef2c	Aortic Root	Innominate Artery	Outer	Outer

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

Double immuno-fluorescent staining was performed to determine cellular localization of CNC- and SHF-derived cells. Although a few β-gal positive cells co-localized with CD31 and ER-TR7 positive cells of the ascending aorta from both Wnt1-Cre and Mef2c-Cre mice (Figure VIIIA & B in online-only Data Supplement), the majority of β-gal positive cells specifically co-localized with α-SMA positive cells (Figure 2A-C). These results demonstrated that most of the β-gal positive cells are SMCs, and CNC-derived SMCs present in the inner medial aspect are covered by multiple layers of outer medial SHF-derived SMCs.

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.

Discussion

Our study determined the distribution of CNC- and SHF-derived SMCs in the ascending aorta using Wnt1 and Mef2c promoters, respectively. Wnt1-Cre mice displayed localization of CNC-derived SMCs that extended from the aortic root to the end of aortic arch. SHF-derived SMCs were present throughout the ascending aorta in Mef2c-Cre mice. In addition, neither sexual dimorphism nor aging affected the distribution of SMC populations in the ascending aortic wall.

Distribution of CNC-derived SMCs is consistent with previous reports.^8-10 Unexpectedly, SHF distribution in our study is inconsistent with previous reports showing that SHF-derived cells were located only in the aortic root.^6,7 These studies used Cre under the control of an Nkx2.5 promoter and demonstrated SHF distribution in the aortic root of either chick embryos or fetal and early postnatal mice. Since our time course study demonstrated that distribution of SHF-derived cells was not changed by aging, the difference of SHF distribution is not caused by the difference of age. However, effects of species and promoters on the difference of SHF distribution are still unclear. Although the avian cardiovascular anatomy is quite similar to the mammalian anatomy, avian development of the aorta is different from mammalian development.¹¹ The aortic arch in birds is derived from right 4^th pharyngeal arch artery, whereas the mammalian aortic arch is derived from the left 4^th arch artery. On the other hand, both Nkx2.5 and Mef2c promoters have been used for tracking SHF-derived cells in cardiac tissue, with some disparities in distributions between them. Left ventricular myocardium is predominantly affected by Nkx2.5, while myocardium of the outflow tract is controlled by Mef2c.¹² Since cardiac Mef2c expression is generally later in developmental timing than Nkx2.5, we reasoned that the Mef2c promoter was more specific for tracking SHF-derived cells than Nkx2.5.¹³ However, there is a possibility that the difference of SHF distribution between the previous study and ours is attributable to induced by the difference in choice of species or promoter. The most important aspect of our study is that the use of these two promoters demonstrated that SMCs populating the outer laminar layers of the ascending aorta are distinct from SMCs populating the inner layers. These different Cre promoters might be useful tools to genetically dissect aortic SMCs in the media.

We determined that outer laminar layers of the ascending aorta have a sleeve of SMCs from a different embryonic origin than inner laminar regions. This is also the region that is prone to aneurysm and dissection in many animal models. For example, angiotensin II infusion induces medial thickening and intra-medial hemorrhage in the outer media in mice.^2,14 Genetic mouse models of thoracic aortic aneurysm also exhibited a gradient of these pathologies with major changes in the outer aspect of the media. These models include SMC-specific deletion of LRP1, Smad4, and TGF-β receptors.^15-19 This pathological gradient is reported in not only mouse tissues, but also in the human aortic tissues. Aortic α-SMA expression is decreased in outer medial layers of human thoracic aortic aneurysms.² In addition, aortic dissection occurs in the outer media.³ The present study determined that SMCs in the outer aspect of the media which is disease-prone are derived from the SHF in the ascending aorta. Therefore, distribution of SMCs of different embryonic origins may be associated with the location of aortic pathology and should be taken into consideration for studying aortopathies in the ascending aorta.

While CNC- and SHF-derived cells are located separately in the anterior region, CNC-derived cells overlap with SHF-derived cells in the posterior region of the ascending aorta in this study. These distributions were demonstrated in tissues acquired from mice that expressed Cre under the control of either Wnt1 or Mef2c promoters. Greater precision in lineage mapping in this region will require development of mice that can trace more than one cell type in a single tissue.

In conclusion, the proximal thoracic aorta in a mammalian system is composed of both CNC- and SHF-derived SMCs. In addition, the ascending aorta has an inner medial layer of CNC-derived SMCs covered by an outer sleeve of SHF-derived SMCs. Selective deletion of genes from SMCs that are hypothesized to modulate aortic pathologies would be helpful to elucidate molecular mechanisms and find novel therapeutic targets for aortic diseases.

Highlights.

• $Both CNC- and SHF-derived SMCs populate the media throughout the ascending aorta.
• $In the posterior region of the ascending aorta, CNC-derived SMCs populate transmedially, while SHF-derived SMCs are located in the outer media.
• $In the anterior region, CNC-derived SMCs are located in the inner media covered by an outer sleeve of SHF-derived SMCs.

Abbreviations

α-SMA

alpha smooth muscle actin

β-gal

beta galactosidase

CNC

cardiac neural crest

Mef2c

myocyte specific enhancer factor 2c

SHF

second heart field

SMCs

smooth muscle cells