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Extreme Diversity of the Human Vascular Mesenchymal Cell Landscape

Background Human mesenchymal cells are culprit factors in vascular (patho)physiology and are hallmarked by phenotypic and functional heterogeneity. At present, they are subdivided by classic umbrella terms, such as “fibroblasts,” “myofibroblasts,” “smooth muscle cells,” “fibrocytes,” “mesangial cells,” and “pericytes.” However, a discriminative marker‐based subclassification has to date not been established. Methods and Results As a first effort toward a classification scheme, a systematic literature search was performed to identify the most commonly used phenotypical and functional protein markers for characterizing and classifying vascular mesenchymal cell subpopulation(s). We next applied immunohistochemistry and immunofluorescence to inventory the expression pattern of identified markers on human aorta specimens representing early, intermediate, and end stages of human atherosclerotic disease. Included markers comprise markers for mesenchymal lineage (vimentin, FSP‐1 [fibroblast‐specific protein‐1]/S100A4, cluster of differentiation (CD) 90/thymocyte differentiation antigen 1, and FAP [fibroblast activation protein]), contractile/non‐contractile phenotype (α‐smooth muscle actin, smooth muscle myosin heavy chain, and nonmuscle myosin heavy chain), and auxiliary contractile markers (h1‐Calponin, h‐Caldesmon, Desmin, SM22α [smooth muscle protein 22α], non‐muscle myosin heavy chain, smooth muscle myosin heavy chain, Smoothelin‐B, α‐Tropomyosin, and Telokin) or adhesion proteins (Paxillin and Vinculin). Vimentin classified as the most inclusive lineage marker. Subset markers did not separate along classic lines of smooth muscle cell, myofibroblast, or fibroblast, but showed clear temporal and spatial diversity. Strong indications were found for presence of stem cells/Endothelial‐to‐Mesenchymal cell Transition and fibrocytes in specific aspects of the human atherosclerotic process. Conclusions This systematic evaluation shows a highly diverse and dynamic landscape for the human vascular mesenchymal cell population that is not captured by the classic nomenclature. Our observations stress the need for a consensus multiparameter subclass designation along the lines of the cluster of differentiation classification for leucocytes.


Bruijn et al
Diversity of Human Vascular Mesenchymal Panorama exploration of potential relevant class-specific marker sets.
To address this point, we performed a systematic literature search to identify candidate mesenchymal cell-specific markers, and evaluated the expression pattern and the expression dynamics of the identified markers in different stages of the human atherosclerotic process.

METHODS
This study is based on a 2-step approach. First, we conducted a systematic literature search to map the reported markers for vascular mesenchymal cell subpopulation characterization and classification. Subsequently, we applied immunohistochemistry and immunofluorescence to evaluate the specificity and expression pattern of the identified markers in a representative sample of early, intermediate, and (stabilized) end stages of the human aortic atherosclerotic process (Virmani classification, 10 respectively: adaptive intimal thickening [AIT], late fibroatheroma [LFA], and fibrocalcific plaque [FCP]) ( Figure 1).
The authors declare that all supporting data are available within the article (and its online supplementary files).

Systematic Literature Review of Phenotypical Immunohistochemical Markers
A systematic literature review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Studies were identified by searching PubMed and Embase. The search strategy (outlined in Data S1 and S2 [Systematic Review Protocol]) was based on 3 search themes, combined in the search by AND. The first theme was created for vascular remodeling and phenotypic heterogeneity. The second theme included descriptions of fibroblasts, myofibroblasts, and SMCs. The final, third theme consisted of terms for atherosclerosis, aortic aneurysmal disease, and fibrosis. Because the focus of the study was on the classic supportive mesenchymal vascular cell type, we considered aspects of osteogenic, adipogenic, and pericyte differentiation beyond the scope of the literature review.
The search was most recently updated in December 2019. First, 2 authors (J.L. and L.B.) independently reviewed the titles and abstracts for eligibility. Thereafter, full-text articles were assessed.
In parallel to the above phenotypic markers, we mapped reported markers of a synthetic and proinflammatory phenotype for functional subclassification, as these functions are considered independent of the cell phenotype (ie, SMCs, myofibroblasts, and fibroblasts can be synthetic and/ or inflammatory).

Human Atherosclerotic Tissue Sampling
Formalin-fixed, paraffin-embedded aortic wall samples were selected from the Vascular Tissue Repository at the Department of Vascular Surgery, Leiden, the Netherlands. These human perirenal aortic patches were obtained during clinical organ transplantation CLINICAL PERSPECTIVE What Is New?
• A classification scheme for the vascular mesenchymal cell population is missing. • This study provides a first framework for a systematic marker-based classification of human vascular mesenchymal cells, and implies an underappreciated, extremely diverse spectrum of human mesenchymal cells within the aortic wall.
What Are the Clinical Implications?
• Mesenchymal cells play a central role in vascular pathological conditions, such as atherosclerosis and abdominal aortic aneurysms. • This systematic evaluation indicates an extreme diverse and dynamic mesenchymal cell landscape, but also implies an unappreciated cellular flexibility with indications for both Endothelial-to-Mesenchymal cell Transition as well as Leucocyte-to-Mesenchymal cell Transition (fibrocytes) as common events. • This study provides a first step in a better understanding of the role of vascular mesenchymal cells in human disease.

Nonstandard Abbreviations and Acronyms
AIT adaptive intimal thickening FAP fibroblast activation protein FCP fibrocalcific plaque FSP-1 fibroblast-specific protein-1 HR healed rupture LFA late fibroatheroma P4HB prolyl 4-hydroxylase β SM22α smooth muscle protein 22α SMC smooth muscle cell Smemb nonmuscle myosin heavy chain SM-MHC smooth muscle myosin heavy chain Thy-1 thymocyte differentiation antigen 1 αSMA α-smooth muscle actin with grafts derived from cadaveric donors. Histologic sections were prepared for each tissue block, sections were Movat pentachrome stained (for protocol, see Data S3), and the extent of atherosclerosis was classified (modified American Heart Association classification, according to Virmani et al 10 ) The tissue block showing the highest degree of atherosclerosis was used as the reference block. For this evaluation, we randomly selected preclassified tissue blocks representative for AIT, LFA, and FCP ( Figure 1). All stainings were performed on sequential tissue sections from the selected tissue blocks.
To evaluate mesenchymal cell presence in respectively progressive and stabilizing atherosclerotic lesions, representative sections of the unstable lesion thin cap fibroatheroma 10 in addition to the stable lesion LFA and healed rupture (HR) 10 were selected. HR was selected as well because of a suspected enrichment of the mesenchymal cell subtype fibrocytes. 11 Immunohistochemical Staining on Atherosclerotic Lesions

Single-Labeling Immunohistochemistry
Consecutive (4-μm) sections were immunostained for the 28 immunohistochemistry markers (Table 1) identified in the literature review. All single stainings were performed by immunohistochemistry, because immunohistochemistry allows for direct clear overview, provides superior contextual information, and is not interfered by background staining (mainly caused by elastin) when assessed by immunofluorescence. A, AIT is characterized by a thickening intima, consisting of smooth muscle cells (SMCs) in a proteoglycan-rich matrix (B) and a normal media and adventitia (C). D, LFA is characterized by a necrotic core of cellular debris and cholesterol crystals that is covered by a multilayered fibrous cap, consisting of SMCs in a collagenous proteoglycan-rich matrix with infiltration of inflammatory cells (F). E, Shoulder regions. G, FCP is characterized by extensive fibrosis, a condensated (former) necrotic core, and ample calcification (H) and neointimal formation (I). Legend to the Movat staining: red, SMCs/fibrin; violet, leukocytes; black, elastin; blue, proteoglycans/mucins; yellow, collagen. Various shades of green reflect colocalization of collagen (yellow) and proteoglycans (blue).
Heat-induced (Tris/EDTA, pH 9.2/citrate, pH 6) or enzyme-induced antigen retrieval was performed if required (Table 1). All primary antibodies were diluted in 1% BSA/PBS and were incubated overnight at 4°C. Endogenous peroxidase activity was blocked with a 20-minute incubation of 0.3% hydrogen peroxide. The Envision/3,3'diaminobenzidine (Dako, Glostrup, Denmark) system was used for visualization. Nuclei were counterstained by Mayer hematoxylin (Merck Millipore, Amsterdam, the Netherlands). Slides stained for phosphorylated nuclear factor-κB were washed with Triton X-100 (Abcam, Cambridge, UK) 0.1% in PBS for 10 minutes. All stainings for a given antibody were processed in a single batch.

Imaging of Immunohistochemistry Slides
Immunohistochemistry images were captured by means of a digital microscope (Philips IntelliSite Pathology Solution Ultra-Fast Scanner; Philips Eindhoven, the Netherlands).

Evaluation of Marker Expression on Atherosclerotic Tissue
For all markers, expression patterns were inventoried for 6 separate aspects of the aortic wall (see Figure S1 for an outline): intima, inner media, middle media, outer media, adventitia, as well as at the level of the arterioletype (thick-walled) vasa vasorum and venule-type (thinwalled) vasa vasorum in the adventitia. In addition, we evaluated the mesenchymal populations in the areas adjacent to shoulders of and covering (multilayered fibrous cap) the necrotic core of the LFA-type lesion. For the FCP lesion type, the cells in the newly formed intima overlying the fibrous lesion, rather than the remnants of the former fibrous cap, were appreciated.

Multilabeling Immunohistochemistry
Double-labeling stainings were primarily performed by immunohistochemistry, for the same reasons single immunohistochemistry stainings were preferred over single immunofluorescence stainings. Double-labeling immunohistochemistry stainings were performed by sequential single-labeling immunohistochemistry. A second heat-induced antigen retrieval after the first chromogen staining was used to inactivate the previous signal. All epitopes resisted the second heat retrieval. Vulcan red (10 minutes, dilution 1:50) and Ferangi blue (5 minutes, dilution 1:50; both from BioCare Medical, Pacheco, CA; both alkaline phosphatase enzymatic chromogens) were combined in the double staining because these chromogens provide optimal color separation and a clear colocalization signal (purple). Double-stained slides were not counterstained.

Multilabeling Immunofluorescence
Colocalization of >2 markers was visualized by immunofluorescence, as no triple chromogen panel could be established that provided adequate color differentiation.
All primary (Table 2) and Alexa Fluor secondary antibodies (dilution 1:200; Thermofisher, Waltham, MA) were diluted in 1% PBS/BSA and incubated overnight at 4°C and 60 minutes at room temperature, respectively. Negative controls were created by omitting the primary antibodies, and antigen stability was checked after the first heat retrieval.
In the triple-labeling immunofluorescence stainings, cluster of differentiation (CD) 45 staining was first performed as a single staining: the CD45 antibody was incubated overnight and visualized using goat anti-mouse (MACH2 AP-Polymer; Biocare Medical) as a secondary antibody (30 minutes incubation at room temperature) and visualized using Vulcan Fast Red (10 minutes, dilution 1:50; Biocare Medical) fluorescence. After a second heat retrieval, the other 2 antibodies of different isotypes were incubated overnight and corresponding fluorescent-labelled seondary antibodies were applied.

Imaging of Immunofluorescence Slides
Digital images were acquired using the Panoramic MIDI Digital Slide Scanner (3D HISTECH Ltd, Budapest, Hungary) and analyzed with CaseViewer software (3D HISTECH Ltd). Minor linear adjustments (brightness and contrast) were performed. Nonlinear adjustments were not performed. Because (partial) overlapping cells may result in pseudocolocalization in widefield optical microscopy, the anticipated pseudocolocalization of CD31 and respectively FSP-1 (fibroblast-specific protein-1)/thymocyte differentiation antigen 1 (Thy-1)/FAP (fibroblast activation protein), as well as the anticipated genuine colocalization of CD45 and vimentin, was validated by confocal microscopy (Zeiss LSM 800 CLSM, Oberkochen, Germany). Image analysis was performed with ZEN Lite (Zeiss).

Identification of Phenotypical Immunohistochemistry Markers
The search strategy identified 3246 articles after removal of duplicates, 655 of which were considered of potential relevance ( Figure 2 [Preferred Reporting Items for Systematic Reviews and Meta-Analyses diagram]). Potentially relevant articles mainly addressed SMC differentiation (n=559 articles), and to a lesser extent aspects of (myo)fibroblastic or mesenchymal differentiation (96 articles). The abstracts of these latter 96 articles were all assessed for potential relevance. Given the large number of publications on SMC  differentiation (n=559), it was decided for an alternative approach by focusing on all review articles for assessment of the abstracts (n=69). Because the most recent review was published in March 2018, we additionally screened abstracts of articles published after January 2018 (n=44). On exclusion of articles deemed not relevant for this study, the abstract screening resulted in 190 potentially relevant articles, of which 180 full-text articles were included for the qualitative synthesis. Motivation for noneligibility of full-text articles is provided in Table S1. The identified markers were included for further evaluation (Table 3) Table S2. All in all, this strategy yielded 16 candidate markers (either lineage or differentiation specific), which are summarized in Table 3.

Cell Identity Markers
On the basis of the literature, identified markers were classified as (mesenchymal) lineage or (sub)class specific (ie, potentially discriminating between fibroblasts, myofibroblasts, or SMCs). The literature synopsis did not indicate a discriminatory marker(set) for myofibroblasts versus SMCs (Table 3), nor a single fibroblastspecific marker. In this context, it was decided to categorize the identified markers along the following lines: we first defined a group of 4 markers that are reported as lineage specific (eg, vimentin) and a second group consisting of 3 markers associated with the principal force generating machinery (α-smooth muscle actin [αSMA], the 2 smooth muscle myosin heavy chain [SM-MHC] isoforms [SM1 and SM2], and nonmuscle myosin heavy chain [Smemb]). This cluster may allow differentiation between contractile (expressed in both SMC-like and myofibroblastic classes) and noncontractile mesenchymal cells.
A third group constituted of 7 molecules that are accessory to the contractile machinery (eg, tropomyosin). The final 2 markers (final fourth subset) are associated with cell-cell and/or cell-matrix interactions (eg, vinculin).

Markers of Functional Status
The literature review for candidate functional markers (ie, proinflammatory and synthetic markers) in the context of vascular biology research identified 222 full-text articles ( Figure 3). Motivation for noneligibility of the reviewed full-text articles is provided in Table S3. Again, a threshold of at least 3 independent references for each functional marker was adopted to include the marker for immunohistochemistry evaluation (Tables S4 and S5).
On the basis of this search strategy, 8 synthetic and 4 proinflammatory markers were selected for further evaluation (Table 4).  Histological Validation

Interference by Rabbit Polyclonal Antibodies
A particular point of concern that emerged from the histological evaluation was an apparent interference when using rabbit polyclonal antibodies on formalin-fixed, paraffin-embedded vessel wall samples. Interference was consistent for different sources and batches of isotype controls, and found for rabbit serum ( Figure S2). In fact, all rabbit immunoglobulins in concentrations beyond 1 μg/mL produced a characteristic staining pattern on the arterial wall samples. This phenomenon was rabbit IgG/serum specific. Consequently, we avoided the use of rabbit polyclonal antibodies requiring working dilutions of ≥1 μg/mL in this evaluation.

Vascular Distribution and Specificity
We validated the expression patterns and staining specificity of the phenotypical and functional cell markers identified in the literature review on the vessel wall samples from the biobank. All markers selected in the review process were stained (single staining) on consecutive slides of the reference tissue block. Results from the evaluation (summarized as semiquantitative scores for the different aspects of the arterial wall) are summarized in Figure 4.

Lineage (mesenchymal) markers
Vimentin, FSP-1/S100A4, Thy-1/CD90, and FAP were identified as mesenchymal lineage-specific markers ( Figure 5A). All 4 markers were diffusely expressed throughout the vessel wall and vasa vasorum in the early atherosclerotic (AIT) reference sample. However, a notable inconsistent expression was found for these markers in the media, with subsets of spindle-shaped cells being vimentin + and FAP + , but negative for both FSP-1 and Thy-1, challenging Thy-1 and FSP-1 as generic mesenchymal lineage markers. Indeed, validation of this observation in triple immunofluorescence stainings of (Thy-1/FSP-1/FAP)/vimentin/αSMA showed that up to 10% of the spindle-shaped αSMA + /vimentin + or αSMA + /FAP + cells in the media were negative for both Thy-1 and FSP-1 ( Figure S3).
This dissociation between vimentin and Thy-1 expression became even more pronounced in the more advanced atherosclerotic stages by an apparent inverse association between Thy-1 expression and disease progression, with a particularly low expression of Bruijn  Restricted to mesenchymal cells 72 10. Desmin 13,34,35 Maintenance of myofibril, myofiber, and whole muscle tissue structural and functional integrity 73,74 Restricted to mesenchymal cells (in vasculature) 75 11. SM22α 22,28,29 Involved in the reorganization of the actin cytoskeleton and cell contractility 76,77 Restricted to mesenchymal cells 78 12.
Smoothelin-B 28,36,37 Associates with stress fibers and constitutes part of the cytoskeleton 79 Restricted to mesenchymal cells 80 13. a-Tropomyosin 27,38,39 Regulation of smooth muscle contraction 81 Restricted to mesenchymal cells 82 14. Telokin 40-42 Involved in initiation or maintenance of smooth muscle relaxation as well as in contraction 83,84 Restricted to mesenchymal cells 85 Focal adhesion 15. Paxillin 43-45 Focal adhesion protein involved in cell-matrix adhesion and presumably coordination of transmission of downstream signals in cell movement and migration 86 Expressed in leukocytes, endothelial cells, and epithelial cells as well 87 16. Vinculin 38,46,47 F-actin binding protein involved in cell-matrix adhesion and cell-cell interactions 88 Expressed in leukocytes, endothelial cells, and epithelial cells as well 89 The selected phenotypical markers are divided in 3 main groups, of which the contractile markers are subdivided on the basis of whether they are part of the contractile apparatus or they are accessory proteins that regulate actin-myosin interaction. Fibroblastic cells, myofibroblastic cells, and C-SMCs were evaluated on their reported discriminative power: dark gray represents controversy over discriminating ability of the marker, and light gray represents consensus that the marker is nondiscriminative. CD indicates cluster of differentiation; C-SMC, contractile smooth muscle cell; ECM, extracellular membrane; FAP, fibroblast activation protein; FSP-1, fibroblast-specific protein-1; αSMA, α-smooth muscle actin; SM22α, smooth muscle protein 22α; Smemb, nonmuscle myosin heavy chain; SM-MHC, smooth muscle myosin heavy chain; and Thy-1, thymocyte differentiation antigen 1. *References from systematic review. † Supplementary references (not from systematic review).
Thy-1 in the neointima. Further discrepancies were observed for vimentin and FAP. Although medial vimentin expression remained stable in advanced-stage (LFA) atherosclerotic disease samples, FAP expression varied ( Figure S4). For explorative purposes, we also evaluated vimentin and αSMA coexpression in the cap of progressive lesions (ie, LFA and the unstable progressive atherosclerotic lesion [thin cap fibroatheroma]), as well as a stabilized lesion type (HR) ( Figure S5). The cap in LFA was rich in mesenchymal cells (elongated vimentin + cells). Transition to a thin cap fibroatheroma was associated with a clear decrease in cap cell density. In both LFA and thin cap fibroatheroma, ≈80% of the vimentin + cells were double vimentin + /αSMA + . Similarly, 80% of the vimentin + cells in the cell-rich/proteoglycan-rich luminal granulation tissue associated with healing of a ruptured atherosclerotic lesion (HR) were double vimentin + /αSMA + .
On the basis of these observations, vimentin classified as the most inclusive lineage marker. However, we did observe a small population of spindle-shaped FAP + /vimentincells in the cap of LFA ( Figure S4.2), defying vimentin as an all-inclusive panmesenchymal marker.
Specificity of vimentin as a mesenchymal lineage marker was challenged by the diffuse presence of round, vimentin + cells in the vicinity of the vasa vasorum in the adventitia. Validation studies that included triple immunofluorescence staining for the panleucocyte marker CD45, the macrophage marker CD68, and vimentin showed subsets of triple-positive cells in the adventitia ( Figure S6.1/2). This colocalization was observed for different vimentin antibodies, and confirms the expression of vimentin in subsets of macrophages. Along similar lines, we identified (small) subsets CD45 + /CD68 + and FSP-1 + , Thy-1 + , or FAP + triple-positive cells in the adventitia, consistent with subsets of FSP-1 + , Thy-1 + , and FAP + macrophages ( Figure S6.3-5).
Moreover, indications were found for vimentin expression in subsets of endothelial cells. Endothelial cell-specific expression was confirmed by CD31/vimentin double staining ( Figure S7). Confocal microscopy characterized the apparent spatial associations between CD31 and FSP-1, Thy-1, and FAP as pseudocolocalization. Distinct, small populations of solitary vimentin + /CD31 + and vimentin + /CD34 + were observed in the vicinity of the adventitial vasa vasorum ( Figure S8).
A third nonclassic population of vimentin + cells was observed in the granulation tissue of HR. Approximately 10% of these spindle-shaped cells were double Vim + / CD45 + (Figure S9.1). Incidental (<5% of the population)   double Vim + /CD45 + cells were also observed in the cap and neointima of LFA and FCP reference sections. Distinct (spindle-shaped and round) morphological features may imply distinct subpopulations ( Figure S9.2/3).

Contractile/noncontractile phenotype markers
αSMA, SM-MHC (isoforms SM1 and SM2), and Smemb (embryonic form of SM-MHC) are principle parts of the contractile machinery that is characteristic for SMCs and myofibroblasts ( Figure 5B). αSMA was expressed in virtually all spindle-shaped cells in the intima, media, and adventitia. In mesenchymal cells covering the vasa vasorum, αSMA was consistently expressed. Expression of the SM-MHC isoforms and Smemb was more variable: SM-MHC (SM1) expression was notably less in the middle section of the media than in the inner and outer segments of the media, and expression of the second isoform (SM2) was limited to the outer segment of the media. Smemb expression was more pronounced in the outer medial segment than in other medial segments. A discriminatory expression profile was seen for SM-MHC/ Smemb expression in the vasa vasorum with parallel expression in the thick-walled arteriole-like vessels, but Smemb single positivity was found in the thin-walled venule-like vessels.

Bruijn et al
Diversity of Human Vascular Mesenchymal Panorama contractile markers. Smemb expression has been linked to a synthetic phenotype. Indeed, most, but not all, of the Smemb+ cells in the cap and shoulder regions expressed the synthetic marker prolyl 4-hydroxylase β (P4HB) ( Figure S10).

Auxiliary contractile markers
The third group of markers identified in the review consisted of a group of auxiliary molecules to the contractile machinery (SM22α [smooth muscle protein 22α], h1-Calponin, h-Caldesmon, Telokin, Tropomyosin, Desmin, and Smoothelin) ( Figure 5C). Spatial expression of these markers was variable: subsets of spindle-shaped cells in the intima, media, and vaso vasora were positive for SM22α and h-Caldesmon. Desmin and Smoothelin expression were both selectively expressed in the medioadventitial border zone. Desmin was selectively expressed in arteriole-type vasa vasorum, whereas Smoothelin was specifically expressed in venule-type vasa vasorum.
Although h1-Calponin was expressed in virtually all spindle-shaped cells in the intima and media in the early stages of atherosclerosis, spatial expression of h1-Calponin was more pronounced in LFA, shown by h1-Calponin − /αSMA + cells in the cap ( Figure S11). Telokin and Tropomyosin were not fully contractile cell specific, as round triple Telokin + /vimentin + /CD45 + and Tropomyosin + /vimentin + /CD45 + cells were present in the adventitia ( Figure S12.1/2).
Heterogeneous responses were seen for the auxiliary contractile markers in the context of the atherosclerotic disease progression: Smoothelin and Desmin expression related inversely to disease progression, whereas medial expression of h1-Calponin, h-Caldesmon, SM22α, Tropomyosin, and Telokin remained stable. H1-Calponin+, Tropomyosin+, Telokin+, and SM22α+ were present in subsets of mesenchymal cells in the cap/shoulder, and in the neointima regions in LFA and FCP. A subpopulation (<10%) of spindle-shaped Tropomyosin + αSMAcells was observed in the cap of the LFA lesion ( Figure S13).

Focal adhesion proteins: vinculin and paxillin
The fourth cluster of markers included Vinculin and Paxillin, molecules involved in cell-cell and cell-matrix interactions ( Figure 5D). In AIT, they were both abundantly expressed in the intimal and outer medial zone and to a lesser extent in the inner and middle media. Although Paxillin expression was also observed in double CD45 + /vimentin + cells in the adventitia ( Figure S12.3), Vinculin expression was absent in the adventitia. Although Paxillin expression remained stable during atherogenic progression, Vinculin expression decreased during disease progression.

Functional markers
Apart from their phenotypical identities, mesenchymal cells can be actively involved in matrix deposition and homeostasis (synthetic phenotype), and may adapt an inflammatory phenotype. We evaluated several markers for a synthetic or an inflammatory phenotype (results are summarized in Table 4).
On the basis of an evaluation of compatibility with immunohistochemistry-based subtyping, which involved a preferably intracellular staining pattern and availability of antibodies compatible with paraffin-embedded material, the signal/background ratio, and specificity, it was decided for P4HB and interleukin 6 (IL-6) or interleukin 8 (IL-8; aka, CXCL8) as preferred markers for a secretory inflammatory phenotype. Motivations for refraining from the other candidate markers are provided in Figure S14.
IL-6 and IL-8 expression was used to visualize inflammatory status of the mesenchymal cell population ( Figure 5E): in AIT, IL-6 and IL-8 expression was observed for infiltrating mesenchymal cells in the intima, and in subsets of adventitial mesenchymal cells. IL-8 was expressed in the medioadventitial border as well. Increased medial IL-6 and IL-8 expression, and significant expression of IL-6 and IL-8 in the cap and shoulder regions, was observed during atherogenic progression.
Expression of these inflammatory markers in the mesenchymal cell population may reflect the inflammatory character of atherosclerosis, an aspect that is    Figure S15). P4HB, an enzyme involved in (pre) collagen processing, was identified as preferred marker for a synthetic phenotype ( Figure 5F). Expression of P4HB was confined to the intima and, with the exception of some vasa vasorum absent in the media and adventitia, in early-stage atherosclerosis. In more advanced stages of atherosclerosis, P4HB expression was observed in the cap and shoulder regions, as well as in the adventitial venule-like and arteriole-like vasa vasorum. P4HB remained absent in the entire media.
On the basis of the tentative results of the review and the inventory, a proposed marker set was compiled for an explorative evaluation of the vascular mesenchymal landscape (Tables 5 and 6).

DISCUSSION
The vascular mesenchymal landscape appears to be highly dynamic, diverse, and complex. It extends far beyond the classic tripartite classification scheme of fibroblasts, myofibroblasts, and SMCs. Furthermore, there is no evidence for a clear separation along the lines of myofibroblast and SMC populations. These Mesenchymal cells are the pivotal cellular component of load-bearing structures and organs. They are the principle component of blood vessels, where they modulate vascular tone and maintain vascular integrity through deposition and maintenance of the extracellular matrix. 131,132 As a consequence, mesenchymal cells are in the center of vascular pathological conditions, such as atherosclerosis and aneurysmal disease. 133,134 In fact, mesenchymal cell activation and migration in response to intimal lipoprotein deposition is the initiating step in the human atherosclerotic process. 135 Data from murine atherosclerotic models suggest that SMCs contribute the majority of foam cells. 136 In the more advanced stages of atherosclerotic disease, mesenchymal cells critically contribute to plaque stability, as well as to aspects such as vascular calcification and intimal hyperplasia. 137,138 Indeed, an exploratory inventory implied clear qualitative changes in the cap during plaque progression with a nadir in cell density in thin cap lesions, but recovery of mesenchymal cell density (elongated vimentin + cells) in the cell-rich/matrix-rich granulation tissue of a healed rupture.
Along similar lines, mesenchymal cells are key players in both genetic (eg, thoracic aneurysms associated with bicuspid valve disease 139 ) and degenerative aneurysms, such as the abdominal aortic aneurysm. 140 For the latter, impaired mesenchymal differentiation has been directly linked to aneurysm rupture. 141 The vascular mesenchymal landscape is particularly complex, not only as a reflection of the heterogeneous embryological origin of the vascular tree, 142 and the vascular layers, 143 but also because SMCs are nonterminally differentiated, 144,145 thus allowing a high degree of phenotypical plasticity. Moreover, it is now clear that processes, such as endothelial-to-mesenchymal transition, 146 contribute to the vascular mesenchymal population.
Evidence was also found for the presence of fibrocytes. Ample elongated double CD45 + /vimentin + cells were observed in the process of cap healing following plaque rupture, and in the neointima overlaying a fibrous lesions. Moreover, we observed subpopulations of round and spindle-shaped double CD45 + /vimentin + cells in the cap of LFA. This observation is consistent with the (co)existence of distinct subpopulations of double CD45 + /vimentin + cells in the atherosclerotic process: spindle-shaped fibrocytes, which could be consistent with the phenomenon of leukocyte-mesenchymal transition, 147 and the round cells possibly representing a subclass of macrophages. 148 The immunological field has benefited enormously from the introduction of the consensus classification of leukocyte subtypes, based on well-defined marker sets (CD markers); such classification system does not exist for mesenchymal cells. In a first attempt toward a mesenchymal cell classification for the vasculature, we inventoried candidate subtype markers through a literature review, and mapped the identified markers on a set human aorta specimens with successive stages of the atherosclerotic process.
On the basis of its performance in this evaluation, and on the assumption that most spindle-shaped cells are mesenchymal cells, vimentin classified as the preferred mesenchymal lineage marker for the vasculature because this was the most inclusive marker for spindle-shaped cells in the media. However, histological stainings identified small subsets of vimentin -/FAP + cells in specific niches, suggesting that vimentin may not be fully inclusive and that a comprehensive appreciation of the full mesenchymal spectrum may rely on the vimentin/FAP dual-marker set.
On the same token, vimentin expression was seen in subset(s) of vascular macrophages, as well as the endothelial lining of vasa vasorum, indicating that vimentin is not fully mesenchymal cell specific, and that full specificity relies on costaining of exclusion markers (eg, CD31 and CD68). This study also identified Construction scheme of proposed mesenchymal marker set selection. Suggestions for well-working antibodies are provided in Table 1. To acquire ≈99% inclusivity for mesenchymal cells, a dual-marker set of vimentin + /FAP + (possibly as costaining, stained by the same chromogen) is needed. Likewise, for contractile mesenchymal cells, the dual-marker set αSMA/Tropomyosin reaches ≈99% inclusivity. CD indicates cluster of differentiation; EndoMT, Endothelialto-Mesenchymal cell Transition; FAP, fibroblast activation protein; IL-6, interleukin 6; IL-8, interleukin 8; LeucoMT, Leuccyte-to-Mesenchymal cell Transition; P4HB, prolyl 4-hydroxylase β; and αSMA, α-smooth muscle actin.
*For a list of accessory contractile and focal adhesion markers, see Table 3. † OR,/ AND.

Bruijn et al Diversity of Human Vascular Mesenchymal Panorama
solitary double vimentin + /CD31 + and vimentin + /CD34 + cells in the vicinity of adventitial vasa vasorum, possibly identifying vascular stem cells or cells in Endothelial-to-Mesenchymal cell Transition. 149 Next to the lineage markers, the review identified several subclass markers. Markers for contractile phenotype were subdivided in 2 closely related subgroups: principal constituents of the contractile apparatus (αSMA, SM-MHC, and Smemb) and its auxiliary molecules (ie, actin/myosin interaction regulating [h1-Calponin, Desmin, h-Caldesmon, Tropomyosin, Telokin, Smoothelin, and SM22α]).
αSMA classified as the most inclusive marker for presence of a professional contractile machinery. However, coverage of the full spectrum of contractile mesenchymal cells may require a dual-marker set of αSMA/Tropomyosin, as the histological evaluation identified small specific niches in the cap of LFA that contained spindle-shaped αSMA − /Tropomyosin + cells.
Smemb has been linked to a synthetic phenotype. Indeed, a subset of elongated Smemb + in the shoulder and cap of progressive atherosclerotic lesions also expressed P4HB. Yet, Smemb + /P4HB − elongated cells were abundantly present in the media of early-stage atherosclerosis. These observations characterize Smemb as a mere differentiation marker.
A considerable degree of coexpression was observed for the auxiliary contractile markers in early atherosclerotic disease (AIT). However, increased heterogeneity was observed for the progressive stages. Clear spatial distribution of these subpopulations implies some form of synchronization in the processes of subdifferentiation. Exploration of underlying molecular synchronization pathways and functional diversity of the subdifferentiated cells is beyond the scope of this inventorying exploration.
The literature review further identified the focal adhesion proteins Vinculin and Paxillin, a binding partner of Vinculin, 150 as markers of mesenchymal differentiation. These proteins do not associate with the contractile apparatus, but are involved in environmental sensing, 151 and are abundantly expressed by mesenchymal cells in the normal vessel wall. 152 We observed downregulation of Vinculin in spindle-shaped cells in the media during atherogenic progression, a phenomenon that has been interpreted as an indication of disturbed intermesenchymal or mesenchymal-extracellular matrix interaction. 153 The identification of functional markers set for histological phenotyping came with several technical challenges. The inflammatory spectrum is notably broad, thus interfering with the identification of a generic marker. Moreover, by virtue of the responsive and adaptive nature of the inflammatory, protein expression can be extremely low and volatile, thus creating suboptimal conditions for immunohistochemistry. The cytokines/chemokines IL-6 and IL-8 (both essentially controlled by nuclear factor-κB activity) can be present as intracellular stores, and thus are well identifiable by immunohistochemistry staining. On this basis, we evaluated their potential as markers of (aspects of) an inflammatory phenotype. Indeed, IL-6 and IL-8 were both particularly upregulated in lesional intimas, such as the cap and shoulder regions of LFA. The dynamics of the innate and adaptive cellular immune response in human atherosclerosis have been extensively reported previously. 154,155 Along similar lines, challenges exist for markers of a synthetic, secretory phenotype. Histological staining of deposited matrix products results in a profound extracellular staining pattern that interferes with the interpretation of intracellular stainings. Our evaluation identified the (pre)collagen processing enzyme PH4B as the optimal marker for mapping a synthetic phenotype in immunohistochemistry. In AIT, P4HB expression was confined to the intima, with the exception of some vasora, and showed upregulation during atherogenic progression in lesional intimas, such as the cap and shoulder regions in LFA.
Because our literature review did not provide conclusive evidence with respect to a discriminatory marker set identifying the classic smooth muscle phenotype and myofibroblast phenotype, it was reasoned that the arteriolar smooth muscle cell of the vasa vasorum in the adventitia constitutes the best reference to the classic, functionally contractile SMC phenotype. On basis of this premise, we could not establish a clear separation along the lines of myofibroblastic and SMC populations based on the (auxillary) contractile markers.
As (myo)fibroblastic cells are characterized by their ability to synthesize collagen, P4HB was explored as discriminative factor. However, spindle-shaped P4HB + cells covering the vasa vasorum were found as well.
This may suggest that myofibroblasts are rather cell states of fibroblastic cells or SMCs than a discrete cell type.
This evaluation of the mesenchymal landscape on the basis of a parallel evaluation of >25 markers indicates a spatially diverse, highly dynamic, and heterogeneous panorama. The spatial diversity and extreme granularity, and the relative long protein half-lives for most markers, pose particular challenges to RNAbased analysis, and to techniques relying on tissue dissection and clustering, such as single-cell analysis. Although immunohistochemistry has a clear advantage to these challenges, this explorative study has some limitations as well. First, the study is based on the results of a literature review. As such, the evaluation may be incomplete and findings from in vitro Bruijn et al Diversity of Human Vascular Mesenchymal Panorama studies may not apply to the in vivo context (eg, we did not encounter a clear myofibroblast phenotype).
Although specific cell isolation studies may add a further level of information about the in vivo context, studies on isolated cells were considered outside the scope of this inventory. Moreover, immunohistochemistry is semiqualitative at best, and heavily relies on the quality of the antibodies. Although quality control was performed, the specificity of antibodies for formalin-fixed, paraffin-embedded samples cannot be guaranteed. For this reason, we performed validation studies with alternative antibodies for the potentially controversial positive findings of vimentin positivity in nonmesenchymal cell lineages (fibrocytes and macrophages). Other stainings were not validated by staining with a different antibody. However, decisions to refrain from a candidate marker were only taken when multiple clones produced negative or nonspecific staining. The impact of nonspecific staining on data interpretation is clearly illustrated by the consistently observed nonspecific staining pattern when using rabbit polyclonal antibodies in concentrations beyond 1 µg/mL (1:1000 for most antibodies) on formalin-fixed, paraffin-embedded vessel sections. The purpose of the study was to establish a marker set for mapping the mesenchymal landscape. The extreme granularity and spatial variation were unexpected. The full extent of the landscape can only be appreciated through systematic cataloging of the phenotypical diversity through a process that will rely on multiparameter imaging of samples covering the full disease spectra, targeted expression profiling, and functional evaluation. We consider this aspect beyond the scope of this inventorying study. However, this study provides the groundwork for a consensus cluster classification.