Previously differentiated medial vascular smooth muscle cells contribute to neointima formation following vascular injury
© Herring et al.; licensee BioMed Central Ltd. 2014
Received: 11 August 2014
Accepted: 10 September 2014
Published: 1 October 2014
The origins of neointimal smooth muscle cells that arise following vascular injury remains controversial. Studies have suggested that these cells may arise from previously differentiated medial vascular smooth muscle cells, resident stem cells or blood born progenitors. In the current study we examined the contribution of the previously differentiated vascular smooth muscle cells to the neointima that forms following carotid artery ligation.
We utilized transgenic mice harboring a cre recombinase-dependent reporter gene (mTmG). These mice express membrane targeted tandem dimer Tomato (mTomato) prior to cre-mediated excision and membrane targeted EGFP (mEGFP) following excision. The mTmG mice were crossed with transgenic mice expressing either smooth muscle myosin heavy chain (Myh11) or smooth muscle α-actin (Acta2) driven tamoxifen regulated cre recombinase. Following treatment of adult mice with tamoxifen these mice express mEGFP exclusively in differentiated smooth muscle cells. Subsequently vascular injury was induced in the mice by carotid artery ligation and the contribution of mEGFP positive cells to the neointima determined.
Analysis of the cellular composition of the neointima that forms following injury revealed that mEGFP positive cells derived from either Mhy11 or Acta2 tagged medial vascular smooth muscle cells contribute to the majority of neointima formation (79 ± 17% and 81 ± 12%, respectively).
These data demonstrate that the majority of the neointima that forms following carotid ligation is derived from previously differentiated medial vascular smooth muscle cells.
Vascular smooth muscle cells (VSMCs) are the major contractile components of the vascular system. They are critically important for regulating blood pressure and flow throughout the vascular system. Unlike skeletal and cardiac muscle cells, VSMCs are remarkably plastic and modulate their phenotype in response to extracellular cues during the development and progression of a variety of diseases including atherosclerosis, hypertension, stenosis following injury and restenosis following vascular interventions. Cardiovascular disease is the leading cause of death among the US population, yet despite intense research efforts a number of basic questions regarding the etiology of cardiovascular disease remain elusive. Classically these diseases are described as being associated with dedifferentiated VSMCs that have decreased expression of proteins required for the normal contractile function, increased expression of extracellular matrix proteins and increased cell proliferation . These proliferating dedifferentiated VSMCs are a major component of neointimal lesions and atherosclerotic plaques. Neointimal VSMCs have been proposed to arise from several sources, including blood and bone marrow derived precursor cells, dedifferentiated medial VSMCs, resident progenitor cells and adventitial fibroblasts. However, recent definitive studies showed a relatively minor contribution of blood and bone marrow derived cells to the neointima or atherosclerotic plaque VSMC population [2–5]. The most widely accepted paradigm that neointimal VSMCs arise from the dedifferentiation and migration of medial VSMCs has been recently challenged . This finding has stimulated much controversy in the field  and has prompted us to further investigate the origin of these cells. Using a genetic fate mapping approach with tamoxifen regulated smooth muscle-specific cre recombinase and a dual color cre-dependent reporter gene we unequivocally show that the neointimal SMCs that arise following carotid artery ligation are largely derived from the previously differentiated medial VSMCs.
Transgenic mice and carotid ligation
Histological and immuno-staining
OCT was removed from sections by 3, 5 minute washes in Tris buffered saline (TBS; 100 mM Tris pH7.6, 150 mM NaCl). Slides were mounted in Prolong Gold containing DAPI (Invitrogen) and visualized by confocal microscopy (Olympus Fluoview FV1000). For immuno-fluorescent staining cryosections were permeabilized in 0.2% triton in TBS for 5 minutes, washed in TBS for 5 minutes them blocked in 5% goat serum diluted in TBS at room temperature for 1 hour. Blocked sections were incubated for 4-5 hours at 37° with primary antibodies to, the SM2 isoform of smooth muscle myosin heavy chain (1:500) , CD31 (1:100, clone 390, Affymetrix, eBioscience), CD68 (1:200, clone FA-11, AbD Serotec) diluted in 5% goat serum/TBS. Some sections were incubated without primary antibody as a negative control. Following washing in TBS primary antibodies were detected by incubation with anti-rabbit or anti-rat Alexa Fluor 647 (1:10,000, Jackson ImmunoResearch). After washing slides were mounted in Prolong Gold containing DAPI (Invitrogen) and visualized by confocal microscopy.
Quantitation of mEGFP and mTomato positive cells in lesions
In order to count the number of mEGFP and mTomato positive cells within the neointima of each vessel, a Z-stack series of 1 μm optical sections were obtained from each vessel. mEGFP and mTomato positive cells were counted in each of the optical sections through the entire Z-stack of images, each DAPI positive nuclei was scored as being associated with either an mEGFP or mTomato positive cell. For some sections it was possible to obtain 2 different fields of Z-stack images.
Fluorescence activated cell sorting (FACS)
For FACS blood was harvested from mice immediately prior to tissue collection. Blood collected into heparinized tubes was diluted 10 fold into red blood cell lysis buffer (168 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH7.3). Cells were collected by brief centrifugation (5 min, 400×g), washed in the same buffer and recentrifuged. Pelleted cells were resuspended in phosphate buffered saline containing 0.5% bovine serum albumen, 0.5 mM EDTA and subjected to FACS analysis. Red blood cell fragments were excluded based on side scatter analysis and the remaining cells sorted based on their red and green fluorescence. A minimum of 10,000 cells were counted in each sample. For each analysis, cells obtained from a mouse that expresses EGFP ubiquitously (CAG-EGFP: C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ) were used as a positive control for EGFP-expressing cells. Cells obtained from a cre negative mTmG mouse were used as a positive control for mTomato expressing cells and cells obtained from a nontransgenic mouse were used as negative controls. Gates were established based on the distribution of the control cell groups and the numbers of cells in the experimental mice that fall into each group were determined.
Results of a recent study suggest that neointimal cells that form following vascular injury are derived from a stem cell population resident within the vascular wall rather than from previously differentiated VSMCs . In that study fate mapping experiments were performed in which differentiated VSMCs were tagged using a cre-dependent EGFP reporter strain crossed with transgenic mice expressing cre recombinase exclusively in smooth muscle cells (B6.Cg-Tg(Myh11-cre,-EGFP)2Mik/J) . The conclusion that previously differentiated VSMCs do not contribute to neointima formation was thus, based largely on the inability to detect EGFP-positive cells in the neointima in these mice. As there are many factors that can contribute to a negative result in these experiments, including the reported silencing of the ROSA 26 promoter that was used to drive EGFP expression in neointimal cells , we reevaluated these findings using an alternative fate mapping strategy. We utilized a dual color reporter transgenic mouse line (mTmG: B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J) in which all cells express a membrane localized mTomato tandem dimer in the absence of cre recombinase activity. Following cre-mediated excision of the mTomato cassette, cells express membrane localized EGFP  (Figure 1). In this reporter mouse line, both mTomato and mEGFP are driven by the same CMV/β-actin (CAG) promoter that has been shown to be active in neointimal cells . This dual color reporter strain obviates the need to interpret negative data, as all neointimal cells should be either red or green depending on whether they express mTomato of mEGFP, respectively. To specifically identify differentiated VSMCs we crossed the mTmG mice with mice expressing a tamoxifen regulated cre recombinase directed by the smooth muscle-specific Myh11  or Acta2  promoters. The ability to temporally control cre recombinase activity also permitted us to distinguish previously differentiated VSMCs from newly differentiated VSMCs. Mice were treated with tamoxifen to activate the cre recombinase 2 weeks prior to vascular injury. As tamoxifen is rapidly metabolized in mice, at the time of surgical injury and subsequently, any cre recombinase expressed in VSMCs will be inactive and thus unable to switch cells from mTomato positive to mEGFP positive. Thus only VSMCs that were differentiated during the period of tamoxifen treatment, prior to injury will be mEGFP positive. As cre-mediated recombination results in a permanent heritable change in a cell’s genome these cells and any of their progeny will remain mEGFP positive even if they dedifferentiate and loose expression of cre or Myh11 or Acta2.
Quantitation of mEGFP positive neointimal cells 28 days following ligation
% mEGFP +
76 ± 9
1827 cells (18 stacks, 10 sections)
86 ± 4
600 cells (5 stacks, 3 sections)
80 ± 11
773 cells (12 stacks, 8 sections)
90 ± 5
653 cells (6 stacks, 4 sections)
92 ± 4
227 cells (5 stacks, 3 sections)
42 ± 10
517 cells (5 stacks, 3 sections)
85 ± 7
463 cells (10 stacks, 8 sections)
79 ± 17
5060 cells (61 stacks, 39 sections)
91 ± 4
731 cells (5 stacks, 3 sections)
72 ± 13
208 cells (8 stacks, 6 sections)
63 ± 16
189 cells (10 stacks, 6 sections)
74 ± 4
261 cells (6 stacks, 3 sections)
81 ± 7
561 cells (5 stacks, 4 sections)
94 ± 7
759 cells (8 stacks, 8 sections)
92 ± 3
190 cells (4 stacks, 4 sections)
81 ± 12
2899 cells (46 stacks, 34 sections)
Results from the current studies support the widely accepted paradigm that following vascular injury medial VSMCs dedifferentiate and migrate into the lumen of vessels forming a neointima. As we utilized a tamoxifen regulated cre recombinase and waited 2 weeks after tamoxifen treatment before performing carotid ligation, given the 12 hour half life of tamoxifen in serum, it is highly unlikely that mEGFP positive cells seen following injury are derived from newly differentiated cells. Moreover, the use of a dual color reporter system avoided any artifacts that may arise due to promoter or reporter silencing as all cells should be either mTomato or mEGFP positive thus no conclusions need to be drawn that are based on negative staining data. Our data also suggest that unlike the ROSA-LACz reporter gene  the CMV enhancer/chicken beta-actin core promoter (CAG) driven mTmG reporter gene is not down-regulated in neointimal SMCs (compare the mEGFP intensity of control and injured vessels in Figures 3,8 and 10). The use of the mTmG reporter strain has the additional advantage that the reporter proteins are membrane localized and thus better retained during sample processing. Moreover, this is a single copy, targeted transgene, hence, it is also not subject to complications that may arise from partial recombination of multicopy transgenes, such that in each cell’s nucleus either the mTomato gene is present or it is excised. Cells will thus express either mTomato or mEGFP. Anecdotally we have noted, that the mTomato protein is relatively stable such that after tamoxifen treatment smooth muscle cells can have detectable expression of both mTomato and mEGFP for 3-4 days before the mTomato protein is turned over and degraded. We speculate that one or more of these advantages of the mTmG reporter system and tamoxifen regulated cre transgenes used in our study may explain why we were able to detect mEGFP positive neointimal cells whereas they were not detected in a previous study .
Our data are consistent with and extend previous fate mapping studies using a cre-dependent LacZ reporter . In this study a cre-dependent ROSA-LacZ reporter was used together with Myh11-creER(T2) transgenic mice to show that following femoral artery wire injury the neointima that formed contained LacZ positive cells . Together these studies suggest that in both the small concentric lesions that form following femoral wire injury and the more complex large lesions that form following carotid ligation, neointimal cells arise from differentiated medial VSMCs. Our rigorous quantitative analysis using 1 μm optical sections to obtain Z-stack series through the entire thickness of each 8 μm section, revealed some heterogeneity between mice, with mEGFP positive cells comprising 42-94% of total neointimal cells (Table 1). Although most mice have between 70-90% mEGFP positive neointimal cells, this may perhaps account for some of confusion in the literature related to the contribution of different cell types to the neointima. Despite this variability, the data indicate that, on average, the majority (~80%) of neointimal cells arise from the previously differentiated medial SMCs. This number would be consistent with previous studies that showed that about 20% of neointimal cells are derived from blood borne cells . In further support of the contribution of blood or endothelial derived cells to the neointima some of the mTomato positive, mEGFP negative neointimal cells observed in our study, stained positive for endothelial and monocyte markers (CD31 and CD68, respectively; Figures 6,7 and 9). It is perhaps a little surprising that in some lesions there were more CD31 positive endothelial cells within the lesions than CD68 positive monocytes/macrophages. Some of these CD31 positive endothelial cells may be present in new vessels that are growing within the neointima (e.g. Figure 6, lower panels) and some may be miscounted lumen endothelial cells in which the plane of the section has obscured a luminal invagination. We also speculate that the proliferating neointimal smooth muscle cells may trap endothelial cells within the lesion as they extend out into the vessel lumen.
Although our studies do not rule out the possibility that under appropriate, in vitro, culture conditions the expansion of a progenitor cell population may be favored, the current studies provide compelling evidence that, in vivo, the majority of neointimal cells that arise following carotid ligation are derived from differentiated medial VSMCs. The lack of detectable mEGFP positive cells circulating in the blood, further suggests that the neointimal cells likely arise from the dedifferentiation and migration of locally derived medial VSMCs.
This research was supported by research support funds from IUPUI.
We would like to thank Dr. Joseph Miano, Dr. Richard Karas and Dr. Matthew Distasi for providing the Myh11 creER(T2), Acta2 creER(T2) and CAG-EGFP mice, respectively. We thank Drs. Daniel Metzger and Pierre Chambon for generating the creER(T2) construct and Acta2 creER(T2) mice and we would also like to thank Dr. Julian Albarran Juarez for sharing his unpublished data and for advice on the study.
- Dandre F, Owens GK: Platelet-derived growth factor-BB and Ets-1 transcription factor negatively regulate transcription of multiple smooth muscle cell differentiation marker genes. Am J Physiol Heart Circ Physiol. 2004, 286: H2042-H2051. 10.1152/ajpheart.00625.2003.View ArticlePubMedGoogle Scholar
- Hu Y, Davison F, Ludewig B, Erdel M, Mayr M, Url M, Dietrich H, Xu Q: Smooth muscle cells in transplant atherosclerotic lesions are originated from recipients, but not bone marrow progenitor cells. Circulation. 2002, 106: 1834-1839. 10.1161/01.CIR.0000031333.86845.DD.View ArticlePubMedGoogle Scholar
- Bentzon JF, Sondergaard CS, Kassem M, Falk E: Smooth muscle cells healing atherosclerotic plaque disruptions are of local, not blood, origin in apolipoprotein E knockout mice. Circulation. 2007, 116: 2053-2061. 10.1161/CIRCULATIONAHA.107.722355.View ArticlePubMedGoogle Scholar
- Iwata H, Manabe I, Fujiu K, Yamamoto T, Takeda N, Eguchi K, Furuya A, Kuro-o M, Sata M, Nagai R: Bone marrow-derived cells contribute to vascular inflammation but do not differentiate into smooth muscle cell lineages. Circulation. 2010, 122: 2048-2057. 10.1161/CIRCULATIONAHA.110.965202.View ArticlePubMedGoogle Scholar
- Nemenoff RA, Horita H, Ostriker AC, Furgeson SB, Simpson PA, VanPutten V, Crossno J, Offermanns S, Weiser-Evans MC: SDF-1alpha induction in mature smooth muscle cells by inactivation of PTEN is a critical mediator of exacerbated injury-induced neointima formation. Arterioscler Thromb Vasc Biol. 2011, 31: 1300-1308. 10.1161/ATVBAHA.111.223701.PubMed CentralView ArticlePubMedGoogle Scholar
- Tang Z, Wang A, Yuan F, Yan Z, Liu B, Chu JS, Helms JA, Li S: Differentiation of multipotent vascular stem cells contributes to vascular diseases. Nat Commun. 2012, 3: 875-PubMed CentralView ArticlePubMedGoogle Scholar
- Nguyen AT, Gomez D, Bell RD, Campbell JH, Clowes AW, Gabbiani G, Giachelli CM, Parmacek MS, Raines EW, Rusch NJ, Speer MY, Sturek M, Thyberg J, Towler DA, Weiser-Evans MC, Yan C, Miano JM, Owens GK: Smooth muscle cell plasticity: fact or fiction?. Circ Res. 2013, 112: 17-22. 10.1161/CIRCRESAHA.112.281048.PubMed CentralView ArticlePubMedGoogle Scholar
- Wirth A, Benyo Z, Lukasova M, Leutgeb B, Wettschureck N, Gorbey S, Orsy P, Horvath B, Maser-Gluth C, Greiner E, Lemmer B, Schutz G, Gutkind JS, Offermanns S: G12-G13-LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension. Nat Med. 2008, 14: 64-68. 10.1038/nm1666.View ArticlePubMedGoogle Scholar
- Wendling O, Bornert JM, Chambon P, Metzger D: Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse. Genesis. 2009, 47: 14-18. 10.1002/dvg.20448.View ArticlePubMedGoogle Scholar
- Kumar A, Lindner V: Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow. Arterioscler Thromb Vasc Biol. 1997, 17: 2238-2244. 10.1161/01.ATV.17.10.2238.View ArticlePubMedGoogle Scholar
- Gallagher PJ, Jin Y, Killough G, Blue EK, Lindner V: Alterations in expression of myosin and myosin light chain kinases in response to vascular injury. Am J Physiol Cell Physiol. 2000, 279: C1078-C1087.PubMed CentralPubMedGoogle Scholar
- Xin HB, Deng KY, Rishniw M, Ji G, Kotlikoff MI: Smooth muscle expression of Cre recombinase and eGFP in transgenic mice. Physiol Genomics. 2002, 10: 211-215.View ArticlePubMedGoogle Scholar
- Cuttler AS, LeClair RJ, Stohn JP, Wang Q, Sorenson CM, Liaw L, Lindner V: Characterization of Pdgfrb-Cre transgenic mice reveals reduction of ROSA26 reporter activity in remodeling arteries. Genesis. 2011, 49: 673-680. 10.1002/dvg.20769.PubMed CentralView ArticlePubMedGoogle Scholar
- Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L: A global double-fluorescent Cre reporter mouse. Genesis. 2007, 45: 593-605. 10.1002/dvg.20335.View ArticlePubMedGoogle Scholar
- Owens GK, Kumar MS, Wamhoff BR: Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004, 84: 767-801. 10.1152/physrev.00041.2003.View ArticlePubMedGoogle Scholar
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