Adventitial Sca-1⁺CD45⁺ Cells Contain Macrophage Progenitors

To understand the context and specificity of the hematopoietic potential of aorta, short-term CFU assays were performed on freshly isolated, single-cell preparations of various hematopoietic and nonhematopoietic adult C57BL/6 murine tissues. As described previously,¹⁵ great care was taken to avoid peripheral blood contamination of aortic extracts, and this was verified in numerous ways, including the use of flow cytometry (Online Figure I).¹⁶ Enzymatic digestion of full-length aorta from 12-week-old C57BL/6 mice yielded 1.5 to 2×10⁶ cells. Although all tissues tested generated CFUs to a varying degree, aortic cells were distinguished by a robust hematopoietic CFU capacity, which was notable for their distinct predisposition to generate lineage-specific macrophage colonies (CFU-M) at a mean frequency of 13.7±1.4 per 10⁵ aortic cells plated (n=11) or ≈200 to 300 per aorta (Figure 1A). In addition to fulfilling conventional morphological criteria, the MPS-enriched content of aortic CFU assays was supported by their substantial upregulation of macrophage and dendritic cell marker expression after 14 days of culture in methylcellulose, with absence of granulocytes (Figure 1B). Unlike aorta, CFUs generated from BM, peripheral blood, and spleen comprised much higher proportions of multilineage CFU-GM (granulocyte–macrophage) and -GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte), along with CFU-G (granulocyte) and BFU-E (burst forming units-erythroid).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Enrichment of macrophage colonies within the aorta’s adventitial Sca-1⁺CD45⁺ compartment. A, Total and macrophage (M) colony-forming unit (CFU) counts from different tissues of 12-week-old C57BL/6 mice (n=8–13). B, Representative flow cytometry density plots of aortic CFU progeny after 14 days of methylcellulose culture showing absence of Gr-1 expression and abundance of CD11b⁺F4/80^Hi (macrophage) and CD11b⁺CD11c⁺ (dendritic cell) staining. C, Representative flow cytometry plots of aortic disaggregates for stem cell antigen-1 (Sca-1) and CD45 expression with IgG isotype control staining also shown. D, Immunofluorescent detection of Sca-1⁺CD45⁺ coexpression in the adventitia of 12-week-old C57BL/6 aortic root. The low magnification image is stained with Hoechst for nuclei, with the yellow dotted line indicating the external elastic lamina and the inset box corresponding to the higher magnification images. Adv indicates adventitia. Scale bars: 20 μm (white) and 5 μm (yellow). Pie chart shows mean expression of the different Sca-1(S)/CD45(45) subsets in C57BL/6 aortic root adventitia from tissue immunostaining of 30 sections, 5 mice. E, CFU frequency from different aortic Sca-1/CD45 fractions. Unf indicates unfractionated cells. n=4 to 8 different experiments per fraction, each performed from ≥6 C57BL/6 mice. *P<0.05, †P<0.01, ‡P<0.001 for comparison with Sca-1⁺CD45⁺ cells. Also see Online Figure III. F, Representative images of small (<100 cells) and large (>100 cells) CFU-M, with quantification of the proportion of large CFU-M from unfractionated and Sca-1⁺CD45⁺ cultures (n=7–10). G, Content of monocytes within the aortic Sca-1⁺CD45⁺ and Sca-1⁻CD45⁺ fractions as assessed by flow cytometry (n=11). H, Nuclear Ki67 expression determined for different aortic cell subsets by flow cytometry. Total indicates all aortic cells. n=8 C57BL/6 mice. §P<0.0001 for comparison of Sca-1⁺CD45⁺ cells vs each other population. I, Proportion of cells in different aortic subpopulations that were in S or G₂/M phases of cell cycle, assessed by flow cytometry of bromodeoxyuridine incorporation. n=8. P<0.0001. Also see Online Figure VI. For all graphical data, mean±SEM is shown, and *P<0.05, †P<0.01, ‡P<0.001, §P<0.0001.

Previously, we established that arterial capacity for CFU formation is predominantly contained within the adventitial subpopulation of cells expressing Sca-1.¹⁵ As others have reported that adventitial Sca-1⁺Lineage⁻CD45⁻ cells possess smooth muscle differentiation potential but lack hematopoietic clonogenicity,^17,18 we focused on the previously unstudied population of adventitial Sca-1⁺CD45⁺ cells. By flow cytometry, CD45 expression was present on 35.9±4.7% of Sca-1⁺ cells from C57BL/6 aortas (n=15; Figure 1C). Coexpression of Sca-1 and CD45 in healthy murine aorta was independently confirmed by tissue immunofluorescence and shown to be predominantly contained within the adventitia (Figure 1D, Online Figure II). Magnetic separation of the Sca-1⁺CD45⁺ subset of aortic cells resulted in 4-fold enrichment in hematopoietic CFU yield (Figure 1E, Online Figure III). These CFUs were exclusively CFU-M, which were more commonly of larger size than those from unfractionated cultures (Figure 1F). Exposure of Sca-1⁺CD45⁺ cells to either macrophage colony-stimulating factor (M-CSF) or interleukin-4 and GM colony-stimulating factor augmented CFU-M recovery (Online Figure IVA) and facilitated in vitro differentiation toward macrophage (CD115⁺F4/80⁺; Online Figure IVB) or dendritic cell (CD11c⁺) phenotypes (Online Figure IVC), respectively.

By comparison to aortic Sca-1⁺CD45⁺ cells, hematopoietic colony formation was nearly absent from the Sca-1⁺CD45⁻ fraction, consistent with previous depictions that these cells harbor smooth muscle and not hematopoietic progenitor capacity.^17,18 Critically, colony formation was also 16-fold less from resident Sca-1⁻CD45⁺ cells, with 25% of these colonies comprising CFU-GM and -GEMM. The much lower yield of CFU-M from Sca-1⁻CD45⁺ cells was in spite of this population containing a higher frequency of Lin⁻CD11b^HiF4/80^Lo monocytes than the Sca-1⁺CD45⁺ subset (Figure 1G). Along with the fact that CFU-M were generated at low frequency from peripheral blood (Figure 1A), this provided initial evidence that the cells responsible for arterial macrophage colonies are not monocytes, consistent with recent data showing the nonmonocyte origins of tissue-resident macrophages.^9,11,12 This was supported in separate experiments in which freshly disaggregated aortic cells were subjected to magnetic sorting on the basis of their expression of a panel of lineage markers, which included the monocyte/macrophage antigen CD11b, along with CD5, CD19, CD45R, Gr-1, Ter119. CFU-M were almost exclusively retrieved from the lineage-depleted fraction, with negligible CFU-M recovery from the lineage-positive cells which included CD11b⁺ monocytes and macrophages (Online Figure V).

Together with augmented CFU-forming ability, the adventitial Sca-1⁺CD45⁺ compartment also contained the highest proportion of actively cycling cells in aorta, as corroborated by Ki67 staining analyzed by flow cytometry of aortic disaggregates (Figure 1H) and tissue immunostaining (Online Figure VI), and bromodeoxyuridine incorporation (Figure 1I, Online Figure VI). The mean rate of apoptosis, as determined by annexin V and 7-amino-actinomycin D (7AAD) uptake, was ≈10% for aortic Sca-1⁺CD45⁺ cells and did not differ significantly with aging (Online Figure VII). Thus, aortic adventitia is enriched in macrophage progenitors that reside in the population of proliferative Sca-1⁺CD45⁺ cells.

Genomic and Phenotypic Characterization of Aortic Sca-1⁺CD45⁺ Cells

To further understand the innate differences among aortic cell populations according to their Sca-1 and CD45 expression, we performed, analyzed, and validated nonbiased transcriptional profiles of disaggregated aortic cells. Unsupervised clustering analysis of genome-wide expression arrays indicated that the combined presence of Sca-1 and CD45 was associated with extensive differences in the transcriptional profile of aortic cells (Figure 2A–2C, Online Table I). There were 44 genes that were upregulated in Sca-1⁺CD45⁺ cells compared with both the Sca-1⁻CD45⁺ (resident leukocytes) and Sca-1⁺CD45⁻ (smooth muscle progenitor) fractions, including those that encode Csf2 (granulocyte–macrophage colony-stimulating factor), Il5 (interleukin-5), Ccl1 (chemokine [C-C motif] ligand 1), Cxcl12 (chemokine [C–X–C motif] ligand 12), and Gata3. Many other genes were differentially regulated between Sca-1⁺CD45⁺ cells and each of the other 2 populations, with 879 and 552 genes upregulated by a factor of ≥2-fold compared with Sca-1⁻CD45⁺ and Sca-1⁺CD45⁻ cells, respectively (Figure 2C). These covered a broad range of biological pathways, including hematopoiesis, inflammation, innate and acquired immunity, and extracellular matrix metabolism, all of which are central to vascular maintenance and disease (Figure 2D and 2E). Quantitative reverse transcription polymerase chain reaction validation of 12 of these gene products provided a Spearman r coefficient of 0.712 (P<0.01) with the microarray (Online Table II).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

RNA microarray profiling of different stem cell antigen-1 (Sca-1)/CD45 fractions of aortic cells. Volcano plots (A) and heat map (B) from RNA microarray showing differential gene expression between Sca-1⁺CD45⁺, Sca-1⁻CD45⁺ and Sca-1⁺CD45⁻ aortic cells. C, Unsupervised clustering analysis of genome-wide expression arrays indicated that the combined presence of stem cell antigen-1 (Sca-1) and CD45 was associated with extensive differences in the genomic imprint of aortic cells compared with Sca-1⁻CD45⁺ and Sca-1⁺CD45⁻ cells, respectively. D and E, Histograms from Ingenuity analysis depict the top 10 biological pathways that had differential gene regulation between aortic Sca-1⁺CD45⁺ cells and Sca-1⁻CD45⁺ cells (D) and between aortic Sca-1⁺CD45⁺ cells and Sca-1⁺CD45⁻ cells (E).

We used flow cytometry to characterize the hematopoietic surface markers of these subpopulations. Compared with the other Sca-1/CD45 subpopulations in adult C57BL/6 aorta, a higher percentage of Sca-1⁺CD45⁺ cells expressed CX₃CR1, CD115 (c-fms), and c-Kit (CD117), which are established markers of MDPs and cMoPs in BM^7,8 (Figure 3A, Online Figure VIII). Further immunophenotypic characterization was then performed to examine for MDP-like (Lin⁻c-Kit⁺CD135⁺CD115⁺CX₃CR1⁺)⁷ and cMoP-like (Lin⁻c-Kit⁺CD135⁻CD115⁺CX₃CR1⁺Ly6C⁺CD11b⁻)⁸ surface marker profiles within the aortic Sca-1⁺CD45⁺ fraction, where the panel of lineage markers used was CD31, NK1.1, CD3, CD19, CD11c, Ly6G, Ter119, and a live-dead marker. Although expression of the MDP-like markers accounted for <5 per 100 000 aortic cells, the aortic Sca-1⁺CD45⁺ gate was found to contain a well-delineated subpopulation of cMoP-like cells (Figure 3B). The frequency of these Sca-1⁺CD45⁺Lin⁻c-Kit⁺CD135⁻CD115⁺CX₃CR1⁺Ly6C⁺CD11b⁻ cells in C57BL/6 aorta was in the order of 15 to 30 per 100 000 cells, which approximated the frequency of CFU-M from unfractionated aortic cell culture (see Figure 1A). This subpopulation contained a high proportion of Ki67⁺ cells (69.6±3.2%, n=9 mice), consistent with a proliferative progenitor (as distinct from quiescent stem cell) phenotype (Figure 3B).

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

Immunophenotypic profiling of aortic Sca-1⁺CD45⁺ cells for macrophage ancestral markers. Flow cytometry gating strategies used on C57BL/6 aortic disaggregates first involved selecting single cells and excluding cell debris. A, Representative flow cytometry density plots show the expression of c-Kit, CX₃CR1, and CD115 on gated Sca-1⁺CD45⁺ cells. Accompanying graphs compare expression of these markers on different stem cell antigen-1 (Sca-1)/CD45 gates from C57BL/6 aorta. n=6 mice. §P<0.0001. B, C57BL/6 aorta contained a subset of Lin⁻Sca-1⁺CD45⁺ cells with the c-Kit⁺CD135⁻CD115⁺CX₃CR1⁺Ly6C⁺CD11b⁻ phenotype recently used to identify common monocyte progenitors (cMoPs) in bone marrow.⁸ This subfraction contained a high percentage of proliferating Ki67⁺ cells (density plots are representative of n=9 mice). FSC-A indicates forward scatter-area; FSC-H, forward scatter-height; MDP, macrophage/dendritic cell progenitor; and SSC-A, side scatter-area. For other IgG isotype controls, see Online Figure VIII.

Collectively, the above data indicate the distinctive genotypic and phenotypic profiles portended by the dual expression of Sca-1 and CD45 on aortic cells. The CFU-M–enriched Sca-1⁺CD45⁺ fraction expresses immunophenotypic markers consistent with macrophage ancestry and includes a small subfraction of proliferative Lin⁻c-Kit⁺CD135⁻CD115⁺CX₃CR1⁺Ly6C⁺CD11b⁻ cells that has been identified as cMoP-like.

Maintenance of Aortic CFU-M Progenitors Is Not From Circulating Cells

Our data suggest that a key difference between macrophage ancestors in BM and spleen and CFU-M progenitors in aorta is that the latter express Sca-1, as this marker is known to be downregulated and ultimately lost with progression of BM hematopoiesis to lineage specification.⁷ This prompted us to investigate whether postnatal aortic Sca-1⁺CD45⁺ cells and their CFU-M progeny are maintained by circulatory trafficking from BM or spleen. In the first instance, we noted that isolation of Sca-1⁺CD45⁺ cells from BM and spleen yielded completely different CFU profiles that lacked the CFU-M specificity of the Sca-1⁺CD45⁺ fraction in aorta (Online Figure IX). Then BM or splenic cells from GFP donors were transferred intravenously to lethally irradiated C57BL/6 mice. In both sets of adoptive transfer experiments, there was rapid reconstitution of blood with GFP⁺ cells (85.8±2.8% at 2 weeks and 94.4±0.6% at 12 weeks; n=8). By 16 weeks, chimerism of GFP⁺ cells in whole aorta was 15.9±3.8%, contributing to 42.9±6.9% of the aorta’s Sca-1⁺CD45⁺ compartment but to few CD45⁻ cells (Figure 4A, Online Figure X). As consistent with our previous experience,¹⁵ recipient aortas demonstrated diminished CFU yield (total CFU at 16 weeks, 1.9±0.4 per 10⁵ unfractionated aortic cells) compared with the nonirradiated state (Figure 1A), indicating radiosensitivity. These aortic colonies remained predominantly CFU-M (920/982; 93.7%), with the majority still recovered from Sca-1⁺CD45⁺ cells (796/920; 86.5%). Despite nearly complete GFP reconstitution of blood monocytes, donor GFP⁺ cells contributed to only 3.3% (30/920) of all aortic CFU-M and 1.0% (8/796) of CFU-M generated from aortic Sca-1⁺CD45⁺ cells (n=8 experiments; Figure 4A, Online Figure XC). Therefore, although circulating cells contributed to the aorta’s Sca-1⁺CD45⁺ population, those Sca-1⁺CD45⁺ cells responsible for CFU-M capacity were of host origin and not the result of recruitment to the vessel wall from BM- or splenic-derived cells. In contrast, 90.3% (56/62) of nonmacrophage colonies, including CFU-GM and -GEMM that were mostly generated from aortic Sca-1⁻ cells, were of donor GFP⁺ source. These results indicate mixed sources of hematopoietic CFUs in aorta. Circulatory surveillance of BM and splenic hematopoietic stem cells/HPCs is largely responsible for the aorta’s rare content of CFU-granulocyte, -GM, -GEMM, and burst-forming units–erythroid, consistent with the previous model proposed by Massberg et al.¹⁹ In contrast, Sca-1⁺CD45⁺ CFU-M progenitors, which are much more prevalent in aorta, are inefficiently reconstituted from BM and spleen, suggesting that they may be maintained locally.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

Maintenance of aortic Sca-1⁺CD45⁺ macrophage colony-forming unit (CFU-M) progenitors occurs independently of bone marrow (BM) hematopoietic progenitor cells (HPCs), blood monocytes, and aortic macrophages. A, top left, Adoptive transfer of BM cells was performed from green fluorescent protein (GFP) donors to irradiated C57BL/6 recipients with aortic harvest at 16 weeks. Bottom left, Graphs show percentage of GFP⁺ chimerism in different aortic stem cell antigen-1 (Sca-1)(S)/CD45(45) fractions (left) and the total yield of CFUs from different aortic Sca-1/CD45 subpopulations (right). n=4 experiments, each performed using 6 recipient aortas. Top right, Table shows the numbers of total macrophage (M) and nonmacrophage (non-M) CFUs from different aortic Sca-1/CD45 fractions in these experiments, along with the number and percentage of GFP⁺ colonies for each Sca-1/CD45 fraction and CFU subtype. Bottom right, Representative flow cytometry density plot of CD11b and F4/80 staining in aorta 16 weeks after BM cell transfer, with CD11b⁺F4/80^Hi macrophages gated by red circle. Adjacent density plot depicts GFP⁺ (donor) chimerism for the gated macrophage population, with mean±SEM % donor chimerism shown from n=6. B, C57BL/6 mice were treated for 30 days with liposomal clodronate or vehicle control (n=6 each group). Despite resulting in marked reduction of circulating Lin⁻CD11b⁺ monocytes (density plots), this was accompanied by a small increase in aortic CFU-M. C and D, Aortas were harvested from osteopetrotic (op/op) mice and their wild-type (WT) littermates (age, 16–21 days). Graphs compare (C) their respective frequencies of monocytes, macrophages, and Sca-1⁺CD45⁺ cells (n=9 per group) and (D) their yield of total hematopoietic CFUs and different CFU subtypes (n=6 per group). Graphs summarize mean±SEM values. *P<0.05, †P<0.01, ‡P<0.001, §P<0.0001 (compared with Sca-1⁺CD45⁺ fraction in [A]). BFU-E indicates burst-forming units–erythroid; CFU-G, CFU-granulocyte; FSC, forward scatter; GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte; GM, granulocyte–macrophage; and SSC, side scatter.

Importantly, in these experiments we also observed that aortic CD11b⁺F4/80^Hi macrophages were equally of both donor and recipient origin 16 weeks after irradiation and adoptive transfer (49.4±5.5% GFP⁺; Figure 4A). This suggested the contribution of a local source to aortic macrophages, as has been shown recently for resident macrophages in other organs.¹² At the same time, it also provided additional evidence to dismiss the possibility that macrophages in the vessel wall were responsible for generating CFU-M, because 96.7% of these colonies were GFP⁻ (Figure 4A, Online Figure XC).

To further corroborate that aortic Sca-1⁺CD45⁺ CFU-M progenitors cannot be attributed to the recruitment of circulating monocytes or the presence of proliferative macrophages in the vessel wall, we studied 2 different models of monocyte/macrophage depletion: (1) liposomal clodronate treatment⁴ (Figure 4B) and (2) osteopetrotic (op/op) mice that are genetically deficient in M-CSF²⁰ (Figure 4C and 4D, Online Figure XI). Previously, clodronate treatment has been shown to result in virtual disappearance of circulating monocytes within 24 hours.⁴ We repeatedly administered liposomal clodronate to adult C57BL/6 mice for 30 days, because this time period exceeds the interval thought to be required for macrophage turnover in postnatal murine tissues, including atherosclerotic aorta.^4,12 Despite resulting in marked reduction of circulating monocytes, clodronate treatment was in fact associated with a small but significant increase in the frequency of CFU-M generated from aortic cell cultures (Figure 4B), providing more evidence that blood-derived monocytes are not the source of aortic CFU-M progenitors.

In comparison with wild-type littermates, aortas from weaning-age M-CSF–deficient op/op mice had preserved numbers of Sca-1⁺CD45⁺ cells (Figure 4C) and generated similar frequencies of CFU-M under standard colony outgrowth conditions (ie, without addition of M-CSF; Figure 4D), despite the expected reduction in their frequency of circulating monocytes, as well as monocytes and macrophages in spleen (Online Figure XI) and aorta (Figure 4C). These results affirm that the aorta’s capacity to produce CFU-M is independent of cells that are reduced in op/op mice, namely circulating monocytes and monocytes and macrophages that are resident in the aortic wall. Op/op mice also had markedly diminished CFU yield from BM attributable to osteopetrosis, but enhanced hematopoiesis in spleen, resulting from displacement of hematopoietic stem cells and HPCs from BM (Online Figure XI). Interestingly, yield of nonmacrophage colonies, especially CFU-GM and -GEMM, was increased from op/op aortas (Figure 4D), demonstrating that extramedullary hematopoiesis impacted the frequency of multilineage HPCs in aorta but not macrophage-specific progenitors.

Aortic Sca-1⁺CD45⁺ Macrophage Progenitors Are Upregulated in Atherosclerosis

Hyperlipidemia has been shown to have a stimulatory effect on BM hematopoietic stem cells/HPCs, providing potential mechanistic insight into leukocyte upregulation in atherosclerotic vessels.²¹ However, recent evidence has suggested a switch in the predominant source of macrophages in atheroma of ApoE^−/− mice from circulating monocytes in early lesion development to local macrophage proliferation in established intimal lesions.⁴ By comparison with age-matched C57BL/6 mice, we observed an increase in the frequency of Sca-1⁺CD45⁺ cells from disaggregates of whole aorta from 5-month-old ApoE^−/− mice, especially in those fed 12 weeks of Western diet (WD; Figure 5A, Online Figure XII). The aortas of these hyperlipidemic mice also contained increased adventitial levels of M-CSF, as determined by quantitative ELISA (Online Figure XIIB), along with increases in the generation of CFU-M (1.8-fold for chow diet ApoE^−/− relative to C57BL/6; 2.3-fold for WD ApoE^−/− relative to C57BL/6; Figure 5B), but not other CFU subtypes (data not shown). Similar results were observed for LDL-R^−/− mice (Online Figure XIII).

• Download figure
• Open in new tab
• Download powerpoint

Figure 5.

Sca-1⁺CD45⁺ macrophage progenitor cells in atherosclerosis. Frequencies of (A) Sca-1⁺CD45⁺ cells determined by flow cytometry and (B) macrophage colony-forming units (CFU-M) from disaggregates of whole aorta from 5-month-old C57BL/6, chow diet (CD)– and Western diet (WD)–fed ApoE^−/− mice, 12 weeks after splenectomy or sham surgery (n≥6 per group). C, Relative content of different leukocyte subsets in aortas of splenectomized WD ApoE^−/− mice (n=8) compared with sham counterparts (n=6). DCs indicates dendritic cells; Monos, monocytes; and Mϕs, macrophages. D, Immunostaining of WD ApoE^−/− aortic root showing at low magnification the presence of Sca-1⁺ cells within the media and atheroma plaque (Pl). Yellow dotted lines indicate the external and internal elastic lamina. Inset box corresponds to the adjacent high magnification images showing an Sca-1⁺CD45⁺ cell within plaque. Graph depicts quantitative summary of the prevalence of stem cell antigen-1 (Sca-1) and CD45 coexpression in atheroma, adventitia (Adv), and the combination of both from 22 aortic root sections, 5 mice. E, CFU-M yield from disaggregates of Adv or remaining vessel wall (non-Adv) after dissection of WD ApoE^−/− aortas (n=7). F, Low- and high-power images show predominantly adventitial distribution of cycling Ki67⁺ cells from WD ApoE^−/− aortic root, with (G) quantitative summary from n=14 sections, 3 mice. H, Percentage frequency of Ki67⁺ expression among all cells counted in atheroma and adventitia of these sections. I, Pie charts summarize the distribution of Ki67 staining among the different Sca-1/CD45 subsets in atheroma, adventitia, and both. Also see Online Movie I. J, Confirmation of the cycling nature of Sca-1⁺CD45⁺ cells in WD ApoE^−/− aorta by flow cytometry assessment of bromodeoxyuridine incorporation (n=10). For tissue immunostaining, nuclei are counterstained blue with Hoechst. Scale bars: 20 μm (white) and 5 μm (yellow). Graphs summarize mean±SEM values. *P<0.05, †P<0.01, ‡P<0.001, §P<0.0001.

Because the spleen has been implicated as an important reservoir from which proinflammatory monocytes are recruited to generate macrophages in atherosclerosis^22,23 and myocardial infarction,²⁴ we also interrogated the frequency of CFU-M progenitor cells in aortas of splenectomized mice. Neither CFU-M, Sca-1⁺CD45⁺, macrophage nor dendritic cell frequencies were found to be diminished in healthy, atheroprone or atherosclerotic aortas, 3 months after splenectomy, despite the anticipated reduction that splenectomy caused to aortic monocyte content (Figure 5A–5C). These data are consistent with those from our splenic cell adoptive transfer studies (Online Figure X) in verifying that the spleen is not a significant source of aortic macrophage progenitors via supply of circulating HPCs or monocytes.

We next set out to define the distribution of local macrophage progenitor cells during murine atherosclerosis development. In aortas from C57BL/6 and prelesional chow diet ApoE^−/−mice, Sca-1⁺CD45⁺ cells were almost exclusively contained within the adventitia. After 12 weeks of WD, ApoE^−/− and LDL-R^−/− aortas displayed transmural distribution of Sca-1⁺CD45⁺ cells, also involving media and lesional neointima (Figure 5D, Online Figures XII and XIII). Despite the presence of Sca-1⁺CD45⁺ cells and macrophages in neointimal plaque, the great majority of CFU-M from WD ApoE^−/− aortas were still derived from adventitia, as determined when the adventitia was separated by microscopic dissection from the remaining vessel wall before digestion and culture (Figure 5E). Quantification of Ki67 nuclear expression in sections of atheroma-containing aortic root revealed that almost 75% of proliferating (Ki67⁺) cells were located within the adventitia, the confines of which we defined as being within 5 cell layers from the external elastic lamina (Figure 5F and 5G). Of the 11.0±1.0% of adventitial cells that were Ki67⁺ (Figure 5H), a majority were Sca-1⁺CD45⁺ (63.7±5.4% from 14 sections, 3 mice; Figure 5I, Online Movie I). In contrast, only 5.3±1.3% of cells in atheroma itself were Ki67⁺ (P<0.001 versus adventitia), with 65.2±11.8% of these being Sca-1⁻CD45⁺. Overall, microscopic quantification of Ki67 staining for aortic root revealed that the highest proportion of actively cycling cells in WD ApoE^−/− aorta was contained within the Sca-1⁺CD45⁺ population, followed by Sca-1⁻CD45⁺ cells (Figure 5I). In keeping with the predominantly adventitial distribution of CFU-M progenitors in WD aorta, a higher percentage of adventitial Sca-1⁺CD45⁺ cells were proliferative than was the case for Sca-1⁺CD45⁺ cells in plaque (15.4±1.5% versus 1.8±0.6%; P<0.0001). By flow cytometry assessment of bromodeoxyuridine incorporation in disaggregates of whole aorta from WD ApoE^−/− mice, 19.8±1.2% of Sca-1⁺CD45⁺ cells were in the S or G₂/M phases of cycle (n=10; Figure 5J). Taken together, these observations reveal spatial diversity and heterogeneity of Sca-1⁺CD45⁺ cells within atherosclerotic aorta, highlighting that the majority of clonogenic and proliferative Sca-1⁺CD45⁺ macrophage progenitors do in fact remain in the adventitia.

Sca-1⁺CD45⁺ AMPCs Have Durable MPS Cell Fate

Finally, to interrogate the in vivo fate of Sca-1⁺CD45⁺ cells within atherosclerotic arteries, we isolated cells by Sca-1 and CD45 expression from aortas of GFP mice and injected each fraction into carotid artery adventitia of recipient 8-week-old ApoE^−/− mice (Figure 6A). WD was then administered for 16 weeks, during and after which GFP⁺ donor cells were not detectable systemically (Figure 6B). At study end, donor-derived cells were identified in the injected artery of all recipients of Sca-1⁺CD45⁺ cells (n=7 mice; Figure 6C) to a greater extent than was the case 48 hours after their administration (Figure 6A). From all carotid artery sections examined from GFP⁺Sca-1⁺CD45⁺ recipients, 89.4% (583/652) of GFP⁺ cells were located in adventitia and 10.6% (69/652) in plaque (Figure 6D), although the latter was increased to 18.6% (69/371) in sections that actually contained atheroma. Of the cells enumerated in atheroma, 7.5±3.5% of cells were GFP⁺, and 22.6±4.4% of all cells in adventitia were GFP⁺ (Figure 6E). Phenotypic analysis revealed active cycling (Ki67⁺) of some donor-derived cells (Online Figure XIV), as well as expression of macrophage (F4/80, monocyte/macrophage antibody-2 [MOMA-2]) and dendritic cell (CD11c) markers (Figure 6F). By comparison, GFP was absent after injection of cell-free matrigel and was rarely identified after transfer of Sca-1⁻CD45⁺ or Sca-1⁺CD45⁻ cells (Figure 6C). Although this in vivo model revealed the striking capacity of Sca-1⁺CD45⁺ cells to engraft after allogeneic transfer and provide long-term MPS progeny in their new environment, our findings suggest that the major contribution of these progenitors is to the maintenance of the adventitial, rather than neointimal, macrophage compartment, which is known to expand in atherosclerosis and play an important regulatory role in pathogenic processes, such as vasa vasorum formation.²

• Download figure
• Open in new tab
• Download powerpoint

Figure 6.

Fate of Sca-1⁺CD45⁺ cells in hyperlipidemic arteries. A, Confocal images show initial adventitial retention of GFP⁺Sca-1⁺CD45⁺ cells 2 days after injection to left carotid artery of 8-week-old ApoE^−/− mouse. Ad indicates adventitia. B, Flow cytometry histograms indicate absence of GFP⁺ chimerism in blood or bone marrow (BM) 12 weeks after adventitial delivery of GFP⁺Sca-1⁺CD45⁺ cells. Dotted histograms correspond to GFP⁻ controls. C, Immunostaining for green fluorescent protein (GFP) expression in carotid artery sections 16 weeks after Western diet and delivery of matrigel only, GFP⁺Sca-1⁻CD45⁺, GFP⁺Sca-1⁺CD45⁻, or GFP⁺Sca-1⁺CD45⁺ cells. In the 2 sections from GFP⁺Sca-1⁺CD45⁺ cell transfer, higher magnification images of the inset boxes show donor-derived cells in adventitia (Adv) in No. 1 and atheroma plaque (Pl) in No. 2. c/l indicates contralateral (right) carotid artery; and L, lumen. D, Pie chart depicts the percentage distribution of GFP⁺Sca-1⁺CD45⁺–derived cells between adventitia and atheroma in recipient carotid arteries (28 sections, 7 mice). E, Quantification of the percentage of cells in atheroma and adventitia that were donor-derived (GFP⁺). More than 3500 nuclei assessed in all. *P<0.05. F, Costaining for GFP with F4/80, MOMA-2, and CD11c after delivery of GFP⁺Sca-1⁺CD45⁺ cells, showing that these cells made a durable contribution to macrophage and dendritic cell progeny in recipient carotid adventitia. Representative IgG control staining is also shown. Nuclei are counterstained blue with Hoechst. Scale bars: 50 μm (orange), 20 μm (white), and 10 μm (yellow).