Supplementary Materials Datas S1CS3 Tables S1CS5 Figures S1CS15 References 2 , 156 , 157 , 311 JAH3-9-e017094-s001. following used immunohistochemistry and immunofluorescence to inventory the appearance design of discovered markers on individual 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 (\smooth muscle actin, smooth muscle myosin heavy chain, and nonmuscle myosin heavy chain), and auxiliary contractile markers (h1\Calponin, h\Caldesmon, Desmin, SM22 [smooth muscle protein 22], non\muscle myosin heavy chain, smooth muscle myosin heavy chain, Smoothelin\B, \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. reflect colocalization of collagen ( em yellow /em ) and proteoglycans ( em blue /em ). 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, TH 237A 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 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 Spi1 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\m) 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. TH 237A Heat\induced (Tris/EDTA, pH 9.2/citrate, pH 6) or enzyme\induced antigen retrieval was performed if required (Table?1). Table 1 Antibodies Used for Immunohistochemistry thead valign=”top” th align=”left” valign=”top” rowspan=”1″ colspan=”1″ Antibody, Clone or Catalog No. /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Abbreviation Used in TH 237A Study /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Host Isotype; Subclass /th th TH 237A align=”center” valign=”top” rowspan=”1″ colspan=”1″ Purification /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Cellular Localization /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Pretreatment /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Protein Block (Dako) /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Dilution /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Secondary Antibody /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Source /th /thead Vimentin, 3B4Vim Mouse IgG2a Purified from cell culture supernatantCytoskeleton (intermediate filament)Tris\EDTA (pH 9.2)No1:2000 DAKO EnVision+ System, anti\mouse MACH2 Biocare Medical, anti\mouse DakoFibroblast\specific protein\1/S100A4, D9F9DFSP\1Rabbit IgGNot specifiedNucleus, cytoplasm, and extracellular spaceTris\EDTA (pH 9.2)No1:6000DAKO EnVision+ System, anti\rabbitCell Signaling TechnologyCD90/thymocyte.