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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.

Atherosclerosis is a complex process with strong inflammatory component. We developed a straightforward sevenfold staining protocol for simultaneous assessment of dominant leukocyte classes, vascularization, and expression of the putative foam cell maker CD36. The method was applied on human coronaries covering the full spectrum of atherosclerotic disease. Results confirm the progressive association of macrophages and T cells with the process and a global presence of mast cells. B cells are exclusively present in adventitial follicles that accompany the process plaque destabilization (thin cap and ruptured lesions) and are otherwise absent. Neutrophils are only present as part of the hemorrhage that accompanies plaque rupture. This study does not classify CD36 as a key factor in foam cell formation. Observed macrophage accumulation in the neointima of stabilized fibrous calcified plaques is consistent with a process of neoatherosclerosis. This study on human coronaries shows a progressive association of macrophage and T-cell abundance with plaque progression. Follicle-like structures are transiently present during the process of plaque destabilization. Plaque healing is accompanied by cessation of the inflammatory response but followed by a new cycle of atherosclerosis., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background/objectives: Carotid intimal media thickness (IMT) and coronary calcium scores (CCS) are thought to reflect atherosclerotic burden. The validity of this assumption for IMT is challenged by recent meta-analyses; for CCS by absence of a relationship between negative scores, and freedom of future events. As such, we considered evaluation of the relationship between tissue IMT and CCS, and extend of atherosclerotic disease relevant., Methods: Analyses were performed on donor aortas obtained during renal graft procurement, and on coronary arteries collected during heart valve procurement for tissue donation. Movat pentachrome and Hematoxylin staining was performed, and the degree of atherosclerosis histologically graded. IMT and presence of calcium deposits were quantified on graded tissue sections., Results: 304 aortas and 185 coronary arteries covering the full atherosclerotic spectrum were evaluated. Aortas and coronaries showed similar relationships between tissue IMT and degree of atherosclerosis, with gradual increase in tissue IMT during earlier phases of atherosclerosis (r=0.68 and r=0.30, P<0.00001 for aorta and coronaries respectively), followed by plateauing of the curve in intermediate and advanced stages. Results for tissue IMT reveal high variability, resulting in wide confidence intervals. Results for CCS are similar for aorta and coronaries, with calcium depositions limited to advanced lesions., Conclusions: Histological IMT measurements for the aorta and coronaries show large variations around the trend and plateauing of, and possibly reductions in IMT in late stage atherosclerotic disease. These observations for the aorta and coronaries may (partly) explain the limited benefit of including carotid IMT in risk prediction algorithms., (Copyright © 2016 The Author(s). Published by Elsevier Ireland Ltd.. All rights reserved.)

Background: The concept of innate immunity is well recognized within the spectrum of atherosclerosis, which is primarily dictated by macrophages. Although current insights to this process are largely based on murine models, there are fundamental differences in the atherosclerotic microenvironment and associated inflammatory response relative to humans. In this light, we characterized the cellular aspects of innate immune response in normal, nonprogressive, and progressive human atherosclerotic plaques., Methods and Results: A systematic analysis of innate immune response was performed on 110 well-characterized human perirenal aortic plaques with immunostaining for specific macrophage subtypes (M1 and M2 lineage) and their activation markers, neopterin and human leukocyte antigen-antigen D related (HLA-DR), together with dendritic cells (DCs), natural killer (NK) cells, mast cells, neutrophils, and eosinophils. Normal aortae were devoid of low-density lipoprotein, macrophages, DCs, NK cells, mast cells, eosinophils, and neutrophils. Early, atherosclerotic lesions exhibited heterogeneous populations of (CD68(+)) macrophages, whereby 25% were double positive "M1" (CD68(+)/ inducible nitric oxide synthase [iNOS](+)/CD163(-)), 13% "M2" double positive (CD68(+)/iNOS(-)/CD163(+)), and 17% triple positive for (M1) iNOS (M2)/CD163 and CD68, with the remaining (≈40%) only stained for CD68. Progressive fibroatheromatous lesions, including vulnerable plaques, showed increasing numbers of NK cells and fascin-positive cells mainly localized to the media and adventitia whereas the M1/M2 ratio and level of macrophage activation (HLA-DR and neopterin) remained unchanged. On the contrary, stabilized (fibrotic) plaques showed a marked reduction in macrophages and cell activation with a concomitant decrease in NK cells, DCs, and neutrophils., Conclusions: Macrophage "M1" and "M2" subsets, together with fascin-positive DCs, are strongly associated with progressive and vulnerable atherosclerotic disease of human aorta. The observations here support a more complex theory of macrophage heterogeneity than the existing paradigm predicated on murine data and further indicate the involvement of (poorly defined) macrophage subtypes or greater dynamic range of macrophage plasticity than previously considered., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Objectives: Unstable atherosclerotic lesions in carotid arteries require surgical endarterectomy to reduce the risk of ischemic stroke. We aimed to identify microRNAs that exert a broad effect on atherosclerotic plaque formation and stability in the carotid artery., Background: We made a selection of 164 genes involved in atherosclerosis. Using www.targetscan.org, we determined which microRNAs potentially regulate expression of these genes. We identified multiple microRNAs from the 14q32 microRNA cluster, which is highly involved in vascular remodeling. In human plaques, collected during carotid endarterectomy surgery, we found that 14q32 microRNA (miR-494) was abundantly expressed in unstable lesions., Methods: We induced atherosclerotic plaque formation in hypercholesterolemic ApoE mice by placing semiconstrictive collars around both carotid arteries. We injected "Gene Silencing Oligonucleotides" against miR-494 (GSO-494) or negative control (GSO-control). Using fluorescently labeled GSOs, we confirmed uptake of GSOs in affected areas of the carotids, but not elsewhere in the vasculature., Results: After injection of GSO-494, we observed significant downregulation of miR-494 expression in the carotid arteries, although miR-494 target genes were upregulated. Further analyses revealed a 65% decrease in plaque size after GSO-494 treatment. Plaque stability was increased in GSO-494-treated mice, determined by an 80% decrease in necrotic core size and a 50% increase in plaque collagen content. Inhibition of miR-494 also resulted in decreased cholesterol levels and decreased very low-density lipoprotein (VLDL) fractions., Conclusions: Treatment with GSO-494 results in smaller atherosclerotic lesions with increased plaque stability. Inhibition of miR-494 may decrease the risk of surgical complications or even avert endarterectomy surgery in some cases.

Objective: The role of toll-like receptors (TLRs) in vascular remodeling is well established. However, the involvement of the endosomal TLRs is unknown. Here, we study the effect of combined blocking of TLR7 and TLR9 on postinterventional remodeling and accelerated atherosclerosis., Methods and Results: In hypercholesterolemic apolipoprotein E*3-Leiden mice, femoral artery cuff placement led to strong increase of TLR7 and TLR9 presence demonstrated by immunohistochemistry. Blocking TLR7/9 with a dual antagonist in vivo reduced neointimal thickening and foam cell accumulation 14 days after surgery by 65.6% (P=0.0079). Intima/media ratio was reduced by 64.5% and luminal stenosis by 62.8%. The TLR7/9 antagonist reduced the arterial wall inflammation, with reduced macrophage infiltration, decreased cytoplasmic high-mobility group box 1 expression, and altered serum interleukin-10 levels. Stimulation of cultured macrophages with TLR7 and TLR9 ligands enhanced tumor necrosis factor-α expression, which is decreased by TLR7/9 antagonist coadministration. Additionally, the antagonist abolished the TLR7/9-enhanced low-density lipoprotein uptake. The antagonist also reduced oxidized low-density lipoprotein-induced foam cell formation, most likely not via decreased influx but via increased efflux, because CD36 expression was unchanged whereas interleukin-10 levels were higher (36.1 ± 22.3 pg/mL versus 128.9 ± 6.6 pg/mL; P=0.008)., Conclusions: Blocking TLR7 and TLR9 reduced postinterventional vascular remodeling and foam cell accumulation indicating TLR7 and TLR9 as novel therapeutic targets.

Animal studies implicate the AP-1 (activator protein-1) pro-inflammatory pathway as a promising target in the treatment of atherosclerotic disease. It is, however, unclear whether these observations apply to human atherosclerosis. Therefore we evaluated the profile of AP-1 activation through histological analysis and tested the potential benefit of AP-1 inhibition in a clinical trial. AP-1 activation was quantified by phospho-c-Jun nuclear translocation (immunohistochemistry) on a biobank of aortic wall samples from organ donors. The effect of AP-1 inhibition on vascular parameters was tested through a double blind placebo-controlled cross-over study of 28 days doxycycline or placebo in patients with symptomatic peripheral artery disease. Vascular function was assessed by brachial dilation as well as by plasma samples analysed for hs-CRP (high-sensitivity C-reactive protein), IL-6 (interleukin-6), IL-8, ICAM-1 (intercellular adhesion molecule-1), vWF (von Willebrand factor), MCP-1 (monocyte chemoattractant protein-1), PAI-1 (plasminogen activator inhibitor-1) and fibrinogen. Histological evaluation of human atherosclerosis showed minimal AP-1 activation in non-diseased arterial wall (i.e. vessel wall without any signs of atherosclerotic disease). A gradual increase of AP-1 activation was found in non-progressive and progressive phases of atherosclerosis respectively (P<0.044). No significant difference was found between progressive and vulnerable lesions. The expression of phospho-c-Jun diminished as the lesion stabilized (P<0.016) and does not significantly differ from the normal aortic wall (P<0.33). Evaluation of the doxycycline intervention only revealed a borderline-significant reduction of circulating hs-CRP levels (-0.51 μg/ml, P=0.05) and did not affect any of the other markers of systemic inflammation and vascular function. Our studies do not characterize AP-1 as a therapeutic target for progressive human atherosclerotic disease.

Background and aims: With the intention to gain support for the hypothesis that incident ischemic complications of atherosclerotic disease involve a stochastic aspect, we performed a histological, qualitative evaluation of the epidemiology of coronary atherosclerotic disease in a cohort of aortic valve donors. Patients and methods: Donors (n = 695, median age 54, range 11–65 years) were dichotomized into a non-cardiovascular (non-CVD) and a cardiovascular disease death (CVD) group. Consecutive 5 mm proximal left coronary artery segments were Movat stained, and the atherosclerotic burden for each segment was graded (revised AHA-classification). Results: Non-CVD and CVD groups showed steep increase of atherosclerosis severity beyond the age of 40, resulting in an endemic presence of advanced atherosclerosis in men over 40 and women over 50 years. In fact, only 19% of the non-CVD and 6% of the CVD donors over 40 years were classified with a normal LCA or a so called non-progressive lesion type. Fibrous calcified plaques (FCP), the consolidated remnants of earlier ruptured lesions, dominated in both non-CVD and CVD donors. Estimates of the atherosclerosis burden (i.e. average lesion grade, proportion of FCPs, and average number of FCPs per cross-section) were all higher in the CVD group (p<1.10−16, p<0.0001, and p<0.05, respectively). Conclusions: Dominance of consolidated FCP lesions in males over 40 and females over 50 years, show that plaque ruptures in the left coronary artery are common. However, the majority of these ruptures remain asymptomatic. This implies that the atherosclerotic process is repetitive. A relative difference in disease burden between CVD and non-CVD donors supports the concept that complications of atherosclerotic disease involve a stochastic element. [ABSTRACT FROM AUTHOR]

Aims Failure of vein graft conduits due to vein graft thickening, accelerated atherosclerosis, and subsequent plaque rupture is applicable to 50% of all vein grafts within 10 years. New potential therapeutic targets to treat vein graft disease may be found in components of the innate immune system, such as mast cells and complement factors, which are known to be involved in atherosclerosis and plaque destabilization. Interestingly, mast cells can be activated by complement factor C5a and, therefore, a direct role for C5a-mediated mast cell activation in vein graft disease is anticipated. We hypothesize that C5a-mediated mast cell activation is involved in the development and destabilization of vein graft lesions. Methods and results Mast cells accumulated in time in murine vein graft lesions, and C5a and C5a-receptor (CD88) expression was up-regulated during vein graft disease in apolipoprotein E-deficient mice. Mast cell activation with dinitrophenyl resulted in a profound increase in vein graft thickening and in the number of plaque disruptions. C5a application enhanced vein graft lesion formation, while treatment with a C5a-receptor antagonist resulted in decreased vein graft disease. C5a most likely exerts its function via mast cell activation since the mast cell inhibitor cromolyn totally blocked C5a-enhanced vein graft disease. Conclusion These data provide evidence that complement factor C5a-induced mast cell activation is highly involved in vein graft disease, which identifies new targets to prevent vein graft disease. [ABSTRACT FROM AUTHOR]