Preexisting smooth muscle cells contribute to neointimal cell repopulation at an incidence varying widely among individual lesions

Abstract

Background: With the diverse origin of neointimal cells, previous studies have documented differences of neointimal cell lineage composition across models, but the animal-to-animal difference has not attracted much attention, although the cellular heterogeneity may impact neointimal growth and its response to therapeutic interventions.

Methods: R26R(+);Myh11-CreER(+), and R26R(+);Scl-CreER(+) mice were used to attach LacZ tags to the preexisting smooth muscle cells (SMCs) and endothelial cells (ECs), respectively. Neointimal lesions were created via complete ligation of the common carotid artery (CCA) and transluminal injury to the femoral artery (FA).

Results: LacZ-tagged SMCs were physically relocated from media to neointima and changed to a dedifferentiated phenotype in both CCA and FA lesions. The content of SMCs in the neointimal tissue, however, varied widely among specimens, ranging from 5 to 70% and 0 to 85%, with an average at low levels of 27% and 29% in CCA (n = 15) and FA (n = 15) lesions, respectively. Bone marrow cells, although able to home to the injured arteries, did not differentiate fully into SMCs after either type of injury. Preexisting ECs were located in the subendothelial region and produced mesenchymal marker α-actin, indicating endothelial-mesenchymal transition (EndoMT); however, EC-derived cells represented only 7% and 3% of the total neointimal cell pool of CCA (n = 7) and FA (n = 7) lesions, respectively. ECs located on the luminal surface exhibited little evidence of EndoMT.

Conclusion: Neointimal hyperplasia proceeds with a wide range of variation in its cellular composition between individual lesions. Relative to ECs, SMCs are major contributors to the lesion-to-lesion heterogeneity in neointimal cell lineage composition.

Fig 1

SMCs from the tunica media repopulate neointimal lesions via a process of physical relocation and de-differentiation. A–D: x-gal staining of CCA (A–B) and FA (C–D) lesions. B and D, magnified view of the boxed area in A and C; Blue, LacZ-tagged SMCs; Purple, nuclear counterstain. Arrows point to internal elastic lamina (IEL). The residual media of FA lesions was torn apart during frozen sectioning due to the weak mechanical strength of this layer. Note the accumulation of LacZ-tagged SMCs in the neointimal layer of both CCA and FA lesions. E–H: immunofluorescent images representative of 15 CCA and FA lesions. Green, β-gal (LacZ gene product); Red, α-actin or SMMHC. White dash lines delineate IEL. Note the loss of α-actin (E and G) and SMMHC (F and H, arrows) in the majority of the SMC-derived neointimal cells (arrows). Scale bars, 50 μm.

Fig 2

SMCs from the tunica media repopulate neointimal lesions with a degree varying widely between animals. A–C: x-gal staining of CCA (top row) and FA (bottom row) lesions (d38). Blue, LacZ-tagged SMCs; Purple, nuclear counterstain. Red dash lines delineate IEL. Note the remarkable variation in the number of LacZ-tagged SMCs in both CCA and FA lesions. Scale bars, 100 μm. D: accumulation of LacZ-tagged SMCs in FA and CCA lesions of individual animals. Regression analysis detected a significant individual dependency (top panel) though the averaged content of LacZ-tagged SMCs was not significantly different between CCA and FA lesions (lower panel).

Fig 3

Bone marrow cells home to neointimal lesions but cannot acquire Myh11 promoter activity. A: x-gal staining of the CCA and FA neointimal lesions created in R26R⁺;Mhy11-CreER⁺ bone marrow chimeras. Samples were assayed at time points specified on the left of the panel. Purple, nuclear counterstain. Note the absence of β-gal positive cells in both the CCA and FA lesions at various time points. B: fluorescent imaging of an FA lesion (d38) created in an EGFP bone marrow chimera. Green, bone marrow derived EGFP positive cells; Blue, nuclear counterstain; L, lumen; M, media. C–D: x-gal staining of the R26R⁺;Myh11-CreER⁺ bone marrow cells treated with (C) and without (D) PDGF. Note that LacZ tags (blue) are attached to a subset of bone marrow cells. Scale bars, 50 μm.

Fig 4

ECs repopulate the neointima with a very low frequency. A: x-gal (left panels) and immunofluorescent (right panels) staining images representing CCA (n=6) and FA (n=6) lesions on d38. Dash lines, IEL. L, lumen; Blue, LacZ tagged ECs; Purple, nuclear counterstain; Green, β-gal; Red, a-actin. Red arrows point to EC-derived neointimal cells, white arrows denote cells stained positive for both β-gal and α-actin, and arrow heads point to LacZ-tagged luminal ECs. B: Fraction of EC-derived cells in the sub-endothelial region of CCA (7±1%, n=6) and FA (3±1%, n=6) lesions. P=0.061, by unpaired t-test.; Scale bars, 50 μm.