Characterization of PDGFRa+ cells in vascular walls

To investigate the contribution of PDGFRa+ adventitial cells in vascular injury response, we first examined PDGFRa expression in the adult aorta by immunostaining (Fig. 1A). While PDGFRa was strongly expressed in the adventitia, no expression was detected in the medial or intima layer, thus confirming its suitability as an adventitial marker. We used Pdgfra-CreER mice, in which green fluorescent protein (GFP) was fused with CreER and knocked into the first ATG of the Pdgfra locus (Fig. 1B); thus, the expression was driven by its own promoter20. Although > 95% of GFP-positive cells have been reported to be co-expressed with tdTomato cells in vitro20, GFP was not detected in the adult aorta or carotid arteries (Figure S1A).

Figure 1
Contribution of PDGFRα-positive cells in maintenance and
injury responses in mouse large vessels

Genetic labeling of PDGFRa+ cells in the carotid artery. (A) Immunostaining for PDGFRa in the mouse ascending aorta at 2 months of age. Scale bar: 100 µm. (B) Schematic showing genetic lineage tracing via Pdgfra-CreER. To express the fusion protein instead of endogenous PDGFRα, the cDNA of green fluorescent protein (GFP), Cre recombinase (Cre), and mutated estrogen receptor ligand-binding domain (ERT2) was inserted into the translational start site of the PDGFRα locus. (C) Schematic outlining the experimental strategy. (DE) Representative images of the initial labeling of tdTomato+ cells in the carotid artery (D) and ascending aorta (E) at 10-day-chase after tamoxifen administration. Asc Aorta: ascending aorta. Scale bars: 100 µm. (FI) Immunostaining for Sca1 (F), CD34 (G), αSMA (H), and SM-MHC (I) on carotid artery sections from 10-days-chase. The dotted box area is shown in the inset with higher magnification. Scale bars: 100 µm. See also Figures S1 and S2.

To verify inducible Cre expression in the adventitia in vivo, we crossed Pdgfra-CreER driver mice with R26-tdTomato mice for genetic labeling (Fig. 1B). Mice were treated with tamoxifen (100 ug/g body weight) or a vehicle at 2 months of age, and leakiness in the construct was tested 10 days after the final injection. No tdTomato expression was detected in the aortic wall without tamoxifen (Figure S1A). We then treated Pdgfra-CreER; R26 tdTomato mice with tamoxifen and harvested the carotid artery and ascending aorta on day 10 to examine the initial labeling (Figs. 1C–E). While labeled cells largely resided within the adventitia, some cells were also detected in the medial layers of both vessels at day 10.

To examine the detailed cell types of PDGFRa+ cells in the vessel walls, we performed immunostainings against cell lineage markers. The staining of carotid arteries for progenitor cell markers showed that tdTomato+ cells in the adventitial layer largely overlapped with Sca1+ and CD34+ at 10 days after tamoxifen administration (Figs. 1F, G). TdTomato+ cells with high and low Sca1 expression were observed in the adventitia. Additionally, we observed that tdTomato+ cells in the media expressed conventional SMC markers, alpha-smooth muscle actin (αSMA), and smooth muscle-myosin heavy chain (SM-MHC) (Figs. 1H, I, respectively) but not endothelial cell marker CD31 (Figure S1B). Similarly, we detected tdTomato+/Sca1+and tdTomato+/CD34+cells in the adventitia (Figures S2A, B) and tdTomato+/αSMA+ and tdTomato+/SM-MHC+ SMCs in the medial layers of the ascending aorta (Figures S2C, D), but not CD31 at 10-day-chase (Figure S2E). These observations suggest that PDGFRa labeled two distinct cell populations—one being adventitial cells that largely co-expressed progenitor markers, while the other is a subset of differentiated SMCs. It is less likely that PDGFRa+ adventitial cells differentiated into SMCs within a short period of time.

The turnover of SMCs in the large arteries was previously studied, and their half-life was determined to be approximately 270 ~ 400 days21. Another study also assessed SMC turnover in developing and adult femoral arteries in the context of injury response using the fate map of NG2+ and CD146+ immature SMCs22. In the present study, we sought to investigate the long-term survival of PDGFRa-labeled cells and their progeny. For this experiment, we injected tamoxifen over 5 days instead of 3 days to increase the labeling efficiency and maintained animals for 2 years in a standard housing condition (Fig. 2A). The initial labeling of 5-day tamoxifen injection was examined at 17 days after administration (Fig. 2B), and tdTomato+ cells were compared with a 2-year-chase in the aorta from ascending to descending as well as carotid arteries (Fig. 2C). At a 2-year-chase, Sca1 immunostaining showed Sca1+/tdTomato+ cells in the adventitia of ascending, arch, carotid arteries—even after 2 years of lineage tracing (see arrows, Fig. 2D). In contrast, the descending aorta contained much fewer tdTomato+ cells in the adventitia (see Des Aorta in Fig. 2D), which was already observed at the initial labeling (Fig. 2B). The medial layers of ascending, arch, and carotid arteries all exhibited a high number of tdTomato+ cells that co-expressed SM-MHC (Fig. 2E). However, in the descending aorta; tdTomato+ medial cells were much less commonly detected (Fig. 2E, Des Aorta). Staining for CD31 validated that labeled PDGFRa+ cells did not contribute to endothelial cells in any of the aortas (Figure S3). Taken together, the number of PDGFRa+ cells is maintained in the medial layers (even after 2 years) and are involved in the homeostasis of adventitial cells and SMCs in vessel walls. Additionally, the descending aorta—which is derived from the lateral mesoderm and has a distinct embryonic origin from the rest of the large arteries included in this study (i.e. neural crest)—exhibited less efficient labeling, which demonstrates the heterogeneity of PDGFRa-expressing cells among thoracic aortas.

Figure 2
Contribution of PDGFRα-positive cells in maintenance and
injury responses in mouse large vessels

The fate of of PDGFRa+ cells during a long-term homeostasis of the aortic wall. (A) Schematic outlining the experimental strategy. (B) Representative images of the initial labeling at 2 months of age in different portions of large arteries. L: lumen, Asc Aorta: ascending aorta, Des Aorta: descending aorta. Scale bars: 100 µm. (C) Representative images of 2-year-chase in different portions of larger arteries. L: lumen, Asc Aorta: ascending aorta, Des Aorta: descending aorta. Scale bars: 100 µm. (DE) Immunostaining for Sca1 (D) and SM-MHC (E) in the aortas at 2-year-chase. Arrows indicate tdTomato+ /Sca1+ adventitial cells. L: lumen, Asc Aorta: ascending aorta, Des Aorta: descending aorta. Scale bars: 100 µm. See also Figure S3.

PDGFRa+ cells contribute to neointima formation

Neointimal hyperplasia is a common result of atherosclerosis and atherosclerotic occlusion treatments (e.g., angioplasty) and has been extensively studied using different types of injury models23. In this study, we employed two types of neointima in the carotid artery: (a) a complete ligation that induces neointima due to flow cessation and resultant changes in flow shear stress; (b) wire injury in which the endothelial cell layer is denudated.

To determine whether PDGFRa-derived tdTomato+ cells contribute to neointima formation after carotid artery ligation, we injected tamoxifen at 2 months of age and performed ligation after 10 days (Fig. 3A). Tissues were analyzed at 28 days after ligation when a neointima was established and at 56 days when the long-term effects of cell contributions could be evaluated. Hematoxylin eosin staining displayed neointima formation after ligation (Fig. 3B). The sham-operated control showed no neointima and 91.8% of Sca1+ cells in the adventitia were tdTomato+ cells (n = 3, Figures S4A–C). Serial sections showed that tdTomato+ cells in the adventitia and media increased, whereas tdTomato+ cells were not detected in the neointima at 28 days after ligation, with a few exceptions (e.g., NI in Fig. 3C). Immunostaining for Sca1 revealed that a large number of adventitial tdTomato+ cells were Sca1+ and that Sca1 expression was also detected on endothelial cells in the neointima (Fig. 3D). αSMA—a marker for immature SMC—was positive in the medial layer and some parts of the neointima (Fig. 3E), whereas SM-MHC—a mature SMC marker—was downregulated after ligation injury in the media, as previously reported (Fig. 3F)24.

Figure 3
Contribution of PDGFRα-positive cells in maintenance and
injury responses in mouse large vessels

Contribution of PDGFRa+ cells to neointima formation following carotid artery ligation. (A) Schematic outlining the experimental strategy. (B) Hematoxylin eosin-stained carotid artery at 28 days after ligation. Scale bar: 100 µm. (C) Fluorescence image showing the distribution of tdTomato+ cells at 28 days after ligation. NI: neointima, M: media, A: adventitia. Scale bar: 100 µm. (DF) Immunostaining for Sca1 (D), αSMA (E), and SM-MHC (F) on carotid artery sections at 28 days after ligation. NI: neointima, M: media, A: adventitia, T: thrombus. Scale bars: 100 µm. (G) Hematoxylin eosin-stained carotid artery at 56 days after ligation. Scale bar: 100 µm. (H) Fluorescence image showing the contribution of tdTomato+ cells to neointima at 56 days after ligation. NI: neointima, M: media, A: adventitia. Scale bar: 100 µm. (I) Quantification of the contribution of tdTomato+ cells to neointima formation. Data are presented as the mean ± SEM, 28 days, N = 10, 56 days, N = 11, **p < 0.01, chi-square test. See also Figure S4.

At 56 days after carotid artery ligation, the neointima was still maintained (Fig. 3G) and a greater abundance of tdTomato+ cells was observed in the neointima (Fig. 3H) (contribution of tdTomato+ cells to neointima: 2 out of 10 mice at 28 days, 8 out of 11 mice at 56 days). The contribution of tdTomato+ cells within the neointima significantly increased at 56 days when compared to observation at 28 days (Fig. 3I). This observation suggests that the neointima is an unstable structure with a dynamic nature and constituents that can change as it matures. Interestingly, we observed two types of neointima at 56 days: in 8 out of 11 mice, the majority of neointimal cells were tdTomato+ (Fig. 4A), while nearly all neointimal cells were tdTomato- in the other 3 mice (Fig. 4B). We defined these two types of neointima as type I (abundant tdTomato+ cells) and type II (few tdTomato+ cells).

Figure 4
Contribution of PDGFRα-positive cells in maintenance and
injury responses in mouse large vessels

Two types of neointima formed by distinct SMCs after carotid artery ligation. (AB) Representative images of type I (A) and type II (B) neointima at 56 days after ligation. Scale bars: 100 µm. (CD) Immunostaining for Sca1 on carotid artery sections from type I (C) and type II (D) neointima. The dotted boxed area is shown on the right with higher magnification. Quantification of the percentage of Sca1 expressing cells in tdTomato+ cells within the vessel wall. Data are presented as the mean ± SEM, N = 3–8 per group. NI: neointima, M: media, A: adventitia. Scale bars: 50 µm. Scale bars in insets: 20 µm. (EF) Immunostaining for αSMA on carotid artery sections from type I (E) and type II (F) neointima. Arrowheads indicate tdTomato+/αSMA+ cells in the neointima. Arrows indicate tdTomato+/αSMA- cells. The dotted box area is shown on the right present an image with higher magnification. Quantification of the percentage of αSMA-expressing cells in tdTomato+ cells within the vessel wall. Data are presented as the mean ± SEM, N = 3–8 per group. NI: neointima, M: media, A: adventitia. Scale bars: 100 µm, Scale bars in insets: 20 µm. (GH) Immunostaining for SM-MHC on carotid artery sections from type I (G) and type II (H) neointima. The dotted box area is shown on the right with higher magnification. NI: neointima, M: media, A: adventitia. Scale bars: 100 µm, box, Scale bars in insets: 20 µm.

To further characterize two types of neointima, we stained the injured vessels with Sca1. In the adventitia, 68% of Sca-1+ cells were tdTomato+ around type I neointima (n = 8), while 78% of Sca-1+ cells were tdTomato+ around type II neointima (n = 3). The overall distribution pattern of Sca1-expressing tdTomato+ cells was similar between the two types (see graphs in Figs. 4C, D). Sca1+ cells were not observed in the neointima or media. On the other hand, approximately 76% of tdTomato+ cells were positive for αSMA in type I neointima (Fig. 4E). Similarly, tdTomato- cells in type II neointima were composed of αSMA+ cells (Fig. 4F), indicating that type II neointimal cells were derived from non-PDGFRa SMCs, although it is still possible that non-recombined PDGFRa+ cells may have contributed to type II neointima. The ratio of αSMA+/tdTomato+ cells in the media and adventitia was comparable between type I and type II neointima (graphs in Figs. 4D, E). Notably, SM-MHC was not observed in any tdTomato+ cells for both types (Figs. 4G, H). Finally, to examine the level of active PDGFRa expression in the neointima, we performed immunostaining using an anti-PDGFRa antibody. Neither type of neointima expressed PDGFRa, whereas tdTomato+ cells in the adventitia expressed PDGFRa in both types (Figures S4D, E). These results suggest that the type I eointima is comprised of PDGFRa progeny, and indicated that PDGFRa+ cells give rise to SMCs that contribute to neointima formation and that these cells are consistent with immature SMCs and distinct from Sca1+ cells.

PDGFRa+ cells generate SMCs after wire injury

To determine whether the contribution of the PDGFRa+ cells to neointima formation is context-dependent, we employed a wire injury model. Wire injury is a more severe type of injury performed by inserting a wire into the main carotid artery to remove endothelial cells. We injected tamoxifen into Pdgfra-CreER; R26-tdTomato mice at 2 months of age and performed wire injury after 10 days (Fig. 5A). At 14 days after injury, we observed that tdTomato+ cells contributed in parts of the neointima in all examined mice (n = 4, Figs. 5B, C). The sham-operated control showed no neointima and tdTomato+ cells were preferentially observed in the adventitia (n = 2, Figures S5A, B). Additionally, tdTomato+ cells showed no significant characteristic changes (Figures S5C–H). The ratio and number of tdTomato+ cells in the media remained after wire injury (Figs. 5D, E). Following the immunostaining of tissue sections, it was observed that tdTomato+ cells in the adventitia and outer medial layer were positive for Sca1 (arrows in Fig. 5F); however, the neointima was negative for Sca1 (NI in Fig. 5F). In contrast, neointimal tdTomato+ cells expressed the SMC markers αSMA (Fig. 5G) and SM-MHC (Fig. 5H), suggesting that the PDGFRa-derived cells were mature SMCs or premature SMCs that terminally differentiated within 14 days. SMCs in the media of injured arteries with neointima showed the downregulation of SM-MHC (M in Fig. 5H), whereas SMCs in the vessels free of neointima were SM-MHC+ (M in Fig. 5I). TdTomato+ cells in the neointima were negative for endothelial markers CD31 (Fig. 5J) and VE-cadherin (VE-cad, Fig. 5K) as well as hematopoietic markers CD45 (Fig. 5L) and CD68 (Fig. 5M), excluding the active contribution of endothelial cells or hematopoietic cells. Taken together, the carotid artery rapidly deployed tdTomato+ cells into the neointima and contributed to the accumulation of Sca1- mature SMCs after injury, whereas Sca1+ medial SMCs—which are also marked by PDGFRa+ cells—remained in the medial layers and exhibited differential responses to severe insults, even among PDGFRa+ progeny.

Figure 5
Contribution of PDGFRα-positive cells in maintenance and
injury responses in mouse large vessels

PDGFRa+ cells generate SMCs after wire injury. (A) Schematic outlining experimental strategy. (B) Hematoxylin eosin-stained carotid artery at 14 days after wire injury. The dotted box area is shown in the inset present an image with higher magnification. Scale bar: 100 µm. (C) Fluorescence image showing the distribution of tdTomato+ cells at 14 days after wire injury. Scale bar: 100 µm. (D) Quantification of the percentage of tdTomato+ cells in the media before and after wire injury. Data are presented as the mean ± SEM, day 0, N = 3, 14 days after wire injury, N = 4. Unpaired t-test, n.s.: not significant. (E) Quantification of the number of tdTomato+ cells in the media before and after wire injury. Data are presented as the mean ± SEM, day 0, N = 3, 14 days after wire injury, N = 4. Unpaired t-test, n.s.: not significant. (FM) Immunostaining for Sca1 (F), αSMA (G), SM-MHC (HI), CD31 (J), VE-cad (K), CD45 (L), and CD68 (M) on carotid artery sections at 14 days after wire injury. Arrows indicate tdTomato+ /Sca1+ cells in the media. L: lumen, I: intima, NI: neointima, M: media, A: adventitia. Scale bars: 20 µm. See also Figure S5.

PDGFRa-derived cells proliferated in response to pressure overload

Finally, we performed transverse aortic constriction (TAC) injury to induce aortic wall thickening via pressure overload (Fig. 6A). At 28 days after TAC, we observed a markedly thickened adventitia of the ascending aorta (Fig. 6B) and an abundance of tdTomato+ cells in the adventitia, indicating adventitial hyperplasia (Fig. 6C). In sham-operated animals, no adventitial hyperplasia was observed and tdTomato+ cells were preferentially located in the adventitia (Figures S5I, J). To further analyze the aortic remodeling with TAC, we performed immunostainings. TdTomato+ cells in the sham-operated control showed no characteristic changes (Figures S5K–M). In contrast, tdTomato+ cells in the adventitia were Sca1+ , and the cells at the boundary between the adventitia and media strongly expressed Sca1 (Fig. 6D). Additionally, tdTomato+ cells in the adventitia were αSMA+ but not SM-MHC+ (Figs. 6E, F), suggesting that these cells are most likely activated fibroblasts derived from PDGFRa+ cells.

Figure 6
Contribution of PDGFRα-positive cells in maintenance and
injury responses in mouse large vessels

Pressure overload increased PDGFRa-derived cells in the adventitia. (A) Schematic outlining the experimental strategy. (B) Hematoxylin eosin-stained ascending aorta at 28 days after TAC surgery. Asc Aorta: ascending aorta. Scale bar: 100 µm. (C) Fluorescence image showing the distribution of tdTomato+ cells at 28 days after TAC surgery. Asc Aorta: ascending aorta. Scale bar: 100 µm. (DF) Immunostaining for Sca1 (D), αSMA (E), SM-MHC (F) on ascending aorta sections at 28 days after TAC surgery. L: lumen, I: intima, M: media, A: adventitia, Asc Aorta: ascending aorta. Scale bars: 20 µm. See also Figure S5.

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