Progeny of single VSMCs can take on different phenotypes

Given that plaques are typically mono/oligo-clonal regarding their VSMC-derived component it seems logical to suggest that the progeny a single VSMC can trans-differentiate into the multiple VSMC- derived cells which can occupy a plaque (section 1.5). To test the plasticity of single VSMCs, sequential cryo-sections were stained for either the VSMC marker aSma or Mac3, a marker upregulated in VSMCs which adopt a macrophage-like state (Figure 26 and 28). aSma+ cells were frequently located within the cap and shoulder regions of an atherosclerotic plaque and less so within core of the plaque (Figure 26), similar to other studies (Shankman et al. 2015).

Figure 26: VSMC-derived aSma+ cells locate to the cap of an atherosclerotic plaque Immunostaining for aSma of a plaque cryo-section from a high density-labelled animal (10x 1 mg tamoxifen), containing RFP-expressing VSMC-derived cells. Signals for fluorescent proteins, nuclear DAPI (white) and aSma (magenta) are shown as indicated on each image. The region outlined in A are magnified in B, C, D. Arrows in B, C, D point to RFP+ aSma+ cells, arrow heads point to RFP- aSma- cells. Scale bars are

150 µm. Adapted from Chappell et al.,2016

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Quantification of all stained plaque sections (Appendix B) showed that 72% (±26) and 64% (±25) of Confetti+ cells within the shoulder and cap regions were aSma+. In contrast only 15% (±21) of Confetti+ cells within the core were aSma+ (Figure 27). Others who have lineage traced VSMCs in atherosclerosis using a single coloured reporter suggest that 18% (Shankman et al. 2015) and 30% (Albarrán-Juárez et al. 2016) of VSMC-derived cells are aSma+, these studies do not sub-categorise the plaque into multiple regions. Within this study, without plaque region sub-categorisation, on average 50% (±15) of all VSMC-derived cells were aSma+, which is 32% and 20% higher than the studies by Shankman et al. (2015) and Albarrán-Juárez et al. (2016). This varitation might be due to differences between mice in different facilities, the details with which positive cells were defined/scored and/or the fact that two regions (cap and core) which are highly aSma+ were scored here, compared to one region (core) which contains less aSma+ cells. No significant difference between vascular region and individual mice regarding the porpotion of Confetti+ cells or all cells (DAPI) which were aSma+ was observed (Figure 27 C-F), suggesting that variation is between individual plaques. The majority of aSma+ cells (97% ±7) were Confetti+ and therefore VSMC-derived, this result is consistent with studies which have shown that bone marrow-derived cells can, but rarely, upregulate aSma(Yu et al. 2011). Others have, however, suggested that up to 31% of aSma+ cells are not VSMC-derived and that 17% of aSma+ cells are derived from macrophage cells (Albarrán-Juárez et al. 2016). The former result is hard to reconcile, but again may be due to differences in how positive cells were scored or perhaps differences in labelling frequencies. One possible reason for the latter claim is that Albarrán-Juárez et al. (2016) use a constitutively active Cre recombinase (rather than a tamoxifen inducible Cre), driven by the myeloid marker gene LysM, which might be induced in VSMC-derived cells which have entered the plaque.

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Mac3-staining of cryo-sections sequential to those stained for aSma identify that Mac3+ cells are predominately located within the core of the plaque (Figure 28), which is similar to what was shown in other studies using macrophage markers such as Lgals3 (Shankman et al. 2015).

Figure 27: Quantification of Confetti+ aSma+ cells in plaques

Box plots and bar charts showing the proportion of cells that express the Confetti reporter and stain positive for aSma (Confetti+ Stain+), relative to all cells expressing the Confetti reporter (A, C, E) and all cells which

stain positive for DAPI (B, D, F). A and B are stratified by plaque region. C and D are stratified by vascular region, Arch = aortic arch, CA = carotid arteries, DA = descending aorta. E and F are stratified by individual mouse. A red star indicates a significant difference (p<0.05) determined by a two-way ANOVA. Data are from

23 plaques from 6 animals. All data is from animals labelled at high density (10x 1 mg tamoxifen). Adapted from Chappell et al., 2016.

A B C D E F

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On average 48% (±28) of Confetti+ and 29% (±19) of all (DAPI) core cells were Mac3+, whereas only a small proportion of Confetti+ cap (15% ±19) and shoulder (26% ±20) cells were Mac3+ (Figure 29 A, B). These results suggest that VSMC-derived cells within the plaque core are more likely to trans- differentiate into macrophage-like cells compared to cap and shoulder cells. Similarly, Shankman et al. (2015) found that 30% of all VSMC-derived cells were positive for the macrophage marker Lgals3. Of all the Mac3+ cells counted 72% (±20) were Confetti+, and therefore VSMC derived. Previous studies have shown that only 16% of plaques cells expressing CD68 were VSMC-derived (Albarrán- Juárez et al. 2016), this difference may be due to the fact that CD68 is a stricter method of staining for macrophage-like cells. The proportion of Confetti+ Mac3+ cells relative to Confetti+ or all cells (DAPI) did not differ over vascular region or mice, except in one case between mice 1 and 3 regarding the proportion of Confetti+ cells which are also Mac3+ (Figure 29 C-F). Which, again, shows the trend that inter-plaque differences accounts for most of the variation observed.

Figure 28: VSMC-derived Mac3+ cells locate to the core of an atherosclerotic plaque Immunostaining for Mac3 of a plaque cryo-section from a high density-labelled animal (10x 1 mg tamoxifen), containing RFP-expressing VSMC-derived cells. Signals for fluorescent proteins, nuclear DAPI (white) and Mac3 (magenta) are shown as indicated on each image. The region outlined in A are magnified

in B, C, D. Arrows in B, C, D point to RFP+ Mac3+ cells, arrow heads point to RFP- Mac3+ cells. Scale bars are 150 µm. Adapted from Chappell et al., 2016.

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As Confetti+, Mac3+ cells and aSma+ cells were detected in all regions of the plaque (Figures 27 and 29), questions regarding the plasticity of a single VSMC-derived cell were raised. For example, can one cell express both proteins at once or can, for example, Mac3 only be expressed following the downregulation of aSma. To test this, plaques were co-stained for both proteins, however, as the entirety of the useable light spectrum is already being used to image all Confetti colours, DAPI and a single immuno-stain stain, this proved problematic. To overcome this, only red or blue- monochromatic plaques were selected and the aSma stain was visualised in the channel normally reserved for GFP (488 laser). Co-staining revealed that a proportion of Confetti+ cells express both

Figure 29: Quantification of Confetti+ Mac3+ cells in plaques

Box plots and bar charts showing the proportion of cells that express the Confetti reporter and stain positive for Mac3 (Confetti+ Stain+), relative to all cells expressing the Confetti reporter (A, C, E) and all cells which

satin positive for DAPI (B, D, F). A and B are stratified by plaque region. C and D are stratified by vascular region, Arch = aortic arch, CA = carotid arteries, DA = descending aorta. E and F are stratified by individual mouse. A red star indicates a significant difference (p<0.05) determined by a two-way ANOVA. Data are from

23 plaques from 6 animals. All data is form animal labelled at high density (10x 1 mg tamoxifen). Adapted from Chappell et al., 2016.

A C E

D F

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aSma and Mac3 (26% ±20, Figure 30). The majority of these VSMC-derived cells located to the plaque core, however, were generally within the region bordering the core and cap (Figure 30). This is in agreement with recent single cell RNA data suggesting that co-expression of inflammatory and VSMC- markers is present in VSMC-derived plaque cells(Albarrán-Juárez et al. 2016). Of the 1100 Confetti- negative cells counted less than 0.03% were double positive for both Mac3 and aSma, suggesting that co-expression of VSMC and macrophage markers are typically restricted to VSMC-derived plaque cells.

Figure 30: VSMC-derived plaque cells can express both aSma and Mac3

A, Arterial cryo-section containing RFP-expressing VSMC-derived cells, co-stained for aSma and Mac3. The region outlined in (i) is magnified in (ii-iv). Arrows point to RFP+ Mac3+ aSma+ cells. Scale bars are 150 µm

(i) and 50 µm (ii-vi). Signals for fluorescent proteins, nuclear DAPI (white), aSma (green) and Mac3 (magenta) are shown as indicated on each image. B, Box plot showing the proportion of cells expressing the Confetti reporter (Confetti+) which co-stain for aSma and Mac3 (Confetti+Sma+Mac3+) within different

plaque regions (7 plaques from 6 mice). Only red and blue plaques were used for this analysis as the 488 channel was required for aSma imaging. All data is from animals labelled at high density (10x 1 mg

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In total, 27/37 monochromatic regions in 23 plaques contained single cells expressing aSma or Mac3. Given that monochromatic plaque regions will generally arise from clonal proliferation of a single VSMC this suggest that, at least a proportion of, individual VSMCs have the propensity to generate plaques cells of different phenotypes. Several plaques, however, contained monochromatic regions which occupied only the core or the cap (for example Figure 22 C, D) raising the question whether particular VSMCs will preferentially occupy a particular plaque region and therefore either regional phenotype (aSma+ cap and Mac3+ core). To test whether VSMCs are preferentially biased towards either the aSma+ cap or Mac3+ core, the positions of 126 monochromatic regions in 82 plaques form 16 high density-labelled animals were scored with respect to the plaques cap and core (Figure 31).

The majority of monochromatic regions (96/126) spanned both plaque domains with a few restricted to either the cap (11%) or core (13%) (Figure 31). The frequency at which single monochromatic regions occupied both plaque regions was significantly larger than expected by independent chance labelling of two proximate uni-potent clones of the same colour (2_{=228, p<0.01, 3 degrees of}

freedom, n=96; statistical analysis for each colour is detailed in the Methods Chapter, section 3.17). Furthermore, 14/17 plaques from the animals labelled at a ~40% labelling efficiency (example shown in Figure 25) contained monochromatic regions which spanned both the cap and core plaque regions. This data therefore suggests that VSMC-derived cells originating from the clonal expansion of a single VSMC do not preferentially locate to either plaque region nor associated phenotype. One hypothesis explaining the observed occurrence of monochromatic regions locating to either the cap or the core,

Figure 31: Monochromatic regions occupy both the cap and core regions of the plaque more frequently than just the cap or core

Bar chart showing the proportion of monochromatic regions which occupy both the cap and core or only a single region within an atherosclerotic plaque (126 regions in 82 plaques from 16 high

density-labelled animals). A red star indicates a significant difference (p<0.05) determined by a t-test. Adapted from Chappell

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but not both, may be due to the way in which a plaque grows. For example, a process whereby one cell grows underneath or over the top of a pre-existing plaque, creating a layered effect.