Extreme Diversity of the Human Vascular Mesenchymal Cell Landscape

Abstract

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.

Keywords: atherosclerosis; fibroblasts; myofibroblasts; vascular smooth muscle cells.

Figure 1. Histologic overview (Movat pentachrome staining) of selected representative sections of adaptive intimal thickening (AIT), late fibroatheroma (LFA), and fibrotic calcified plaque (FCP).

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).

Figure 2. Preferred Reporting Items for Systematic Reviews and Meta‐Analyses diagram for selection of articles on phenotypical mesenchymal markers.

SMC indicates smooth muscle cell.

Figure 3. Diagram for selection of articles on synthetic/proinflammatory markers.

SMC indicates smooth muscle cell; and LUMC, Leiden University Medical Center.

Figure 4. Semiquantitative evaluation of single immunohistochemistry (IHC) stainings.

The presence of immunohistochemical markers is appreciated in 6 zones of the vessel wall: intima (I), inner media underlying lesion (M1), middle media (M2), outer media (M3), adventitia (Ad), thin‐walled, venous‐type vasa vasorum (VV thin), and thick‐walled artery‐type vasa vasorum (VV thick). In late fibroatheroma (LFA), the intima is divided in a central cap region (Cap) and shoulder regions (Sh); and in fibrotic calcified plaque (FCP), the neointima (Neo) overlaying the fibrous cap is considered. AIT indicates adaptive intimal thickening; FAP, fibroblast activation protein; FSP, fibroblast‐specific protein; α‐SMA, α‐smooth muscle actin; SM22α, smooth muscle protein 22α; Smemb, nonmuscle myosin heavy chain; SM‐MHC, smooth muscle myosin heavy chain; and Thy1, thymocyte differentiation antigen 1.

Figure 5. Histological validation of the selected immunohistochemistry (IHC) markers.

A, Mesenchymal lineage IHC markers. Overview of staining patterns in the selected representative atherosclerotic sections of adaptive intimal thickening (AIT) (early), late fibroatheroma (LFA) (progressive), and fibrotic calcified plaque (FCP) (end stage). Close ups in LFA and FCP represent cap regions and neointima, respectively. B, Generic contractile IHC markers. Overview of staining patterns in the selected representative atherosclerotic sections of AIT (early), LFA (progressive), and FCP (end stage). Close ups in LFA and FCP represent cap regions and neointima, respectively. C, Accessory contractile IHC markers. Overview of staining patterns in the selected representative atherosclerotic sections of AIT (early), LFA (progressive), and FCP (end stage). Close ups in LFA and FCP represent cap regions and neointima, respectively. D, Focal adhesion IHC markers. Overview of staining patterns in the selected representative atherosclerotic sections of AIT (early), LFA (progressive), and FCP (end stage). Close ups in LFA and FCP represent cap regions and neointima, respectively. E, Interleukin 6 (IL‐6) and interleukin 8 (IL‐8) staining (activation markers). Overview of staining patterns in the selected representative atherosclerotic sections of AIT (early), LFA (progressive), and FCP (end stage). Close ups in LFA and FCP represent cap regions and neointima, respectively. F, Prolyl 4‐hydroxylase β (P4HB) staining (synthetic marker). Overview of staining patterns in the selected representative atherosclerotic sections of AIT (early), LFA (progressive), and FCP (end stage). Close ups in LFA and FCP represent cap regions and neointima, respectively. CD indicates cluster of differentiation; FAP, fibroblast activation protein; FSP, fibroblast‐specific protein; α‐SMA, α‐smooth muscle actin; SM22α, smooth muscle protein 22α; Smemb, nonmuscle myosin heavy chain; SM‐MHC, smooth muscle myosin heavy chain; and Thy1, thymocyte differentiation antigen 1.