Periodic Reporting for period 1 - Athero MPh proliferation (Macrophage proliferation and ontogeny in murine models of atherosclerosis)
Berichtszeitraum: 2016-02-01 bis 2018-01-31
Atherosclerosis is a slowly progressing inflammatory disease of the large arteries. During its chronic phase it is largely symptomless, but its acute clinical manifestation after atherosclerotic plaque rupture is responsible for severe cardiovascular events like stroke and myocardial infarction, the most common causes of death in Western society. Most alarmingly, its incidence is expected to increase over the next decade, mainly due to the aging population and the ever-increasing prevalence of obesity. Current gold-standard treatments, including lipid-lowering by statins, have been proven effective in preventing primary or recurrent CVD events in ≈30% of patients, but fail in the remaining 70% of cases. This prompts the design of more effective and and better targted supplementary therapies to enable improved individualized treatment.
The atherosclerotic plaque is a highly inflammatory milieu in which macrophages represent the most prevalent immune cell type. They play a central role in all stages of pathogenesis, exerting a wide range of functions. Not only will these professional scavengers ingest (modified) lipoproteins and cell debris, and thus become large, lipid-laden foam cells, they also produce cytokines and chemokines fostering the local inflammation, as well as lytic proteases that help increasing plaque vulnerability to rupture. At the same time however, macrophages can temper inflammation by producing anti-inflammatory cytokines and mediators that resolve inflammation as well as promote the plaque-stabilizing growth of vascular smooth muscle cells and the formation of a fibrous cap. These central, varied but ambiguous roles of macrophages throughout atherogenesis makes them both an obvious and refractory target for therapeutic strategies to interfere with plaque growth and stability.
Macrophages have long been seen as a cell type that somehow can exert this spectacular range of different functions simultaneously, but we now know that these highly plastic cells will rather differentiate into subsets that each exhibit their own characteristics and functions. These phenotypes are often classified into the comprehensible but oversimplified conceptual framework of the M1-M2 dichotomy, which is easily applicable in many other inflammatory diseases. But even though a lot of effort was put in applying this paradigm in atherosclerosis as well, identifying pro-atherosclerotic M1s and anti-atherosclerotic M2s, its prototypical inducers and readouts often only have limited relevance in the context of the plaque. This very specific and highly complex milieu rather offers its own stimuli and requires different activation states and functional specializations, which already prompted publications on additional phenotypes such as M4 and Mhem. This poor translatability, the semantic confusion and the lack of a unified model warrant the development of a more relevant classification of atherosclerotic plaque macrophages. But despite earlier calls to map this intraplaque heterogeneity in an unbiased way using modern approaches such as single cell transcriptomics, surprisingly little efforts have yet been undertaken to do so.
Macrophage heterogeneity has been described to be associated with the microenvironment. Local clues and stimuli affect the cells’ orientation while factors produced by the cells will in turn add to the distinct milieu. During atherogenesis, local stimuli can highly differ with the disease stage and plaque phenotype. But also within an atherosclerotic plaque, the plaque region, the proximity of other cell types and the accumulation of lipids will shape local differences in phenotype-promoting milieus.
While single-cell RNAseq and flow cytometry approaches reveal an enormous amount of information on the identity of the cell types present in the plaque, it’s exactly this information on the cells’ location that is lost. We therefore decided to reveal intraplaque macrophage heterogeneity through alternative methods that allow us to retain the contextual information as much as possible.
The atherosclerotic plaque is a highly inflammatory milieu in which macrophages represent the most prevalent immune cell type. They play a central role in all stages of pathogenesis, exerting a wide range of functions. Not only will these professional scavengers ingest (modified) lipoproteins and cell debris, and thus become large, lipid-laden foam cells, they also produce cytokines and chemokines fostering the local inflammation, as well as lytic proteases that help increasing plaque vulnerability to rupture. At the same time however, macrophages can temper inflammation by producing anti-inflammatory cytokines and mediators that resolve inflammation as well as promote the plaque-stabilizing growth of vascular smooth muscle cells and the formation of a fibrous cap. These central, varied but ambiguous roles of macrophages throughout atherogenesis makes them both an obvious and refractory target for therapeutic strategies to interfere with plaque growth and stability.
Macrophages have long been seen as a cell type that somehow can exert this spectacular range of different functions simultaneously, but we now know that these highly plastic cells will rather differentiate into subsets that each exhibit their own characteristics and functions. These phenotypes are often classified into the comprehensible but oversimplified conceptual framework of the M1-M2 dichotomy, which is easily applicable in many other inflammatory diseases. But even though a lot of effort was put in applying this paradigm in atherosclerosis as well, identifying pro-atherosclerotic M1s and anti-atherosclerotic M2s, its prototypical inducers and readouts often only have limited relevance in the context of the plaque. This very specific and highly complex milieu rather offers its own stimuli and requires different activation states and functional specializations, which already prompted publications on additional phenotypes such as M4 and Mhem. This poor translatability, the semantic confusion and the lack of a unified model warrant the development of a more relevant classification of atherosclerotic plaque macrophages. But despite earlier calls to map this intraplaque heterogeneity in an unbiased way using modern approaches such as single cell transcriptomics, surprisingly little efforts have yet been undertaken to do so.
Macrophage heterogeneity has been described to be associated with the microenvironment. Local clues and stimuli affect the cells’ orientation while factors produced by the cells will in turn add to the distinct milieu. During atherogenesis, local stimuli can highly differ with the disease stage and plaque phenotype. But also within an atherosclerotic plaque, the plaque region, the proximity of other cell types and the accumulation of lipids will shape local differences in phenotype-promoting milieus.
While single-cell RNAseq and flow cytometry approaches reveal an enormous amount of information on the identity of the cell types present in the plaque, it’s exactly this information on the cells’ location that is lost. We therefore decided to reveal intraplaque macrophage heterogeneity through alternative methods that allow us to retain the contextual information as much as possible.
Three sequential frozen sections of murine atherosclerotic plaques were cut and the first and third were used for MS imaging. They respectively revealed lipidomic and the peptidomic profiles at a 10µm resolution. This way, characterizing lipid or peptide species could be identified for each area, plaque region or even cell. Or inversely, the tissue could be computationally subdivided into regions with similar profiles.
The middle, second section was first subjected to Raman spectroscopy to reveal differences in molecular composition between the plaque regions. Subsequently, tissue autofluorescence was overcome through a custom-designed and home-built bleaching apparatus and the section was stained with a multiplex immunofluorescence strategy that was designed, optimized and validated during this project. This allows for the simultaneous imaging of fourteen markers through spectral microscopy, and thus detects more cell surface markers than most flow cytometry facilities would be capable of, all while retaining the tissue structure and thus the individual cells’ location.
After the respective measurements, all three sections were stained with haematoxylin and eosin, allowing to align them and thus to combine the data from the different techniques to characterize the plaque regions, or even at the level of single cells after the computational segmentation of the H&E pictures.
This high-dimensional information can then be used to cluster cells with similar profiles and thus reveal intraplaque heterogeneity based on their surface marker expression, molecular composition, lipid content and proteomics. Detailed information on the different cell phenotypes will also help to further identify them and reveal their function(s) and role in the pathogenesis of atherosclerosis, when compared to other disease stages. Single-cell RNAseq on laser capture microdissected plaque regions may add further detail to this information and point in the direction of valid markers but also targetable signaling pathways or markers to be deployed in novel therapeutic approaches that focus on the deletion or phenotypical skewing of specific detrimental macrophage phenotypes, as part of a follow-up project that currently is being initiated.
In parallel, a similar combination of techniques is applied to human plaques at different disease stages, increasing the chance of translationability of the findings. Meanwhile, also other disease models with a central role for macrophages and their heterogeneity are being investigated using the same methodology.
The middle, second section was first subjected to Raman spectroscopy to reveal differences in molecular composition between the plaque regions. Subsequently, tissue autofluorescence was overcome through a custom-designed and home-built bleaching apparatus and the section was stained with a multiplex immunofluorescence strategy that was designed, optimized and validated during this project. This allows for the simultaneous imaging of fourteen markers through spectral microscopy, and thus detects more cell surface markers than most flow cytometry facilities would be capable of, all while retaining the tissue structure and thus the individual cells’ location.
After the respective measurements, all three sections were stained with haematoxylin and eosin, allowing to align them and thus to combine the data from the different techniques to characterize the plaque regions, or even at the level of single cells after the computational segmentation of the H&E pictures.
This high-dimensional information can then be used to cluster cells with similar profiles and thus reveal intraplaque heterogeneity based on their surface marker expression, molecular composition, lipid content and proteomics. Detailed information on the different cell phenotypes will also help to further identify them and reveal their function(s) and role in the pathogenesis of atherosclerosis, when compared to other disease stages. Single-cell RNAseq on laser capture microdissected plaque regions may add further detail to this information and point in the direction of valid markers but also targetable signaling pathways or markers to be deployed in novel therapeutic approaches that focus on the deletion or phenotypical skewing of specific detrimental macrophage phenotypes, as part of a follow-up project that currently is being initiated.
In parallel, a similar combination of techniques is applied to human plaques at different disease stages, increasing the chance of translationability of the findings. Meanwhile, also other disease models with a central role for macrophages and their heterogeneity are being investigated using the same methodology.
The here described research features a unique combination of several state-of-the-art techniques, some of them developed in the context of this project. The information gathered from them will not only enhance our understanding of the pathogenesis of atherosclerosis and the role that macrophages and their phenotypic heterogeneity play in it. It will also lead to the detection of specific macrophage subsets that can be related to detrimental functions in disease progression and thereby identify useful targets for future therapeutic strategies. Thanks to the parallel study of animal models and human material, translation of experimental treatments to the patient will be facilitated.
Meanwhile, the application of the in this project developed methodology to other disease models will further increase its scientific impact and output.
Meanwhile, the application of the in this project developed methodology to other disease models will further increase its scientific impact and output.