three-dimensional-imaging-of-aortic-tissues-in-atherosclerosis

Three-Dimensional Imaging of Aortic Tissues in Atherosclerosis

Recent research has advanced the understanding of atherosclerosis as a transmural chronic inflammatory disease involving all three layers of the arterial ...

Ludwig-Maximilians-University

German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance

Tertiary lymphoid organs emerge in tissues in response to nonresolving inflammation. Recent research characterized artery tertiary lymphoid organs in the aorta adventitia of aged apolipoprotein E-deficient mice. The atherosclerosis-associated lymphocyte aggregates are organized into distinct compartments, including separate T-cell areas harboring conventional, monocyte-derived, lymphoid, and plasmacytoid dendritic cells, as well as activated T-cell effectors and memory cells; B-cell follicles containing follicular dendritic cells in activated germinal centers; and peripheral niches of plasma cells. Artery tertiary lymphoid organs show marked neoangiogenesis, aberrant lymphangiogenesis, and extensive induction of high endothelial venules. Moreover, newly formed lymph node-like conduits connect the external lamina with high endothelial venules in T-cell areas and also extend into germinal centers. Mouse artery tertiary lymphoid organs recruit large numbers of naïve T cells and harbor lymphocyte subsets with opposing activities, including CD4(+) and CD8(+) effector and memory T cells, natural and induced CD4(+) regulatory T cells, and memory B cells at different stages of differentiation. These data suggest that artery tertiary lymphoid organs participate in primary immune responses and organize T- and B-cell autoimmune responses in advanced atherosclerosis. In this review, we discuss the novel concept that pro- and antiatherogenic immune responses toward unknown arterial wall-derived autoantigens may be organized by artery tertiary lymphoid organs and that disruption of the balance between pro- and antiatherogenic immune cell subsets may trigger clinically overt atherosclerosis.

Artery tertiary lymphoid organs contribute to innate and adaptive immune responses in advanced mouse atherosclerosis.

Tertiary lymphoid organs (TLOs) emerge during nonresolving peripheral inflammation, but their impact on disease progression remains unknown. We have found in aged Apoe(-/-) mice that artery TLOs (ATLOs) controlled highly territorialized aorta T cell responses. ATLOs promoted T cell recruitment, primed CD4(+) T cells, generated CD4(+), CD8(+), T regulatory (Treg) effector and central memory cells, converted naive CD4(+) T cells into induced Treg cells, and presented antigen by an unusual set of dendritic cells and B cells. Meanwhile, vascular smooth muscle cell lymphotoxin β receptors (VSMC-LTβRs) protected against atherosclerosis by maintaining structure, cellularity, and size of ATLOs though VSMC-LTβRs did not affect secondary lymphoid organs: Atherosclerosis was markedly exacerbated in Apoe(-/-)Ltbr(-/-) and to a similar extent in aged Apoe(-/-)Ltbr(fl/fl)Tagln-cre mice. These data support the conclusion that the immune system employs ATLOs to organize aorta T cell homeostasis during aging and that VSMC-LTβRs participate in atherosclerosis protection via ATLOs.

Artery Tertiary Lymphoid Organs Control Aorta Immunity and Protect against Atherosclerosis via Vascular Smooth Muscle Cell Lymphotoxin β Receptors.

Atherosclerosis is a transmural chronic inflammatory disease of medium and large arteries. Though it is well recognized that immune responses contribute to atherosclerosis, it remains unclear whether these responses are carried out in secondary lymphoid organs such as the spleen and lymph nodes and/or within the arterial wall. Arteries are composed of three major layers, i.e., the laminae intima, media, and adventitia. However, each of these layers may play different roles in arterial wall biology and atherogenesis. We identified well-structured artery tertiary lymphoid organs (ATLOs) in the abdominal aorta adventitia but not in the intima of aged apolipoprotein E-null (ApoE(-/-)) mice. These observations suggested that disease-associated immune responses are highly territorialized within the arterial wall and that the adventitia may play distinct and hitherto unrecognized roles. Here, we set out to apply laser capture microdissection (LCM) to dissect plaque, media, adventitia, and adjacent aorta-draining lymph nodes (LN) in aged ApoE(-/-) mice in attempts to establish the territoriality of atherosclerosis immune responses. Using whole-genome mRNA expression microarrays of arterial wall tissues, we constructed robust transcript atlases of wild-type and ApoE(-/-) mouse aortas. Data were deposited in the National Center for Biotechnology Information's gene expression omnibus (GEO) and are accessible to the public through the Internet. These transcript atlases are anticipated to prove valuable to address a wide scope of issues ranging from atherosclerosis immunity and inflammation to the role of single genes in regulating arterial wall remodeling. This chapter presents protocols for LCM of mouse aorta and microarray expression analysis from LCM-isolated aorta laminae.

Generation of Aorta Transcript Atlases of Wild-Type and Apolipoprotein E-null Mice by Laser Capture Microdissection-Based mRNA Expression Microarrays.

Explore aorta B-cell immunity in aged apolipoprotein E-deficient (ApoE(-/-)) mice.

Artery Tertiary Lymphoid Organs Control Multilayered Territorialized Atherosclerosis B-Cell Responses in Aged ApoE-/- Mice.

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by lipid deposition, plaque formation, and immune cell infiltration. Innate and adaptive immune cells infiltrate the artery during development of the disease. Moreover, advanced disease leads to formation of artery tertiary lymphoid organs in the adventitia (Grabner ., 2009; Hu ., 2015). Various and diverse types of immune cells have been identified in the aorta adventitia vs atherosclerotic plaques (Elewa ., 2016; Galkina ., 2006; Lotzer ., 2010; Mohanta ., 2016; Mohanta ., 2014; Moos ., 2005; Srikakulapu ., 2016; Zhao ., 2004). There are conflicting reports on the number and subtypes of immune cells in the aorta depending on the age of the animals, the protocol that is used to obtain single cell suspensions, and the dietary conditions of the mice (Campbell ., 2012; Clement ., 2015; Galkina ., 2006; Kyaw ., 2012). The number of immune cells in the aorta differs as much as tenfold using different protocols (Butcher ., 2012; Galkina ., 2006; Gjurich ., 2015; Grabner ., 2009; Hu ., 2015). These discrepant results call for a protocol that robustly documents  aorta cells rather than those in the surrounding tissues or blood. Critical methodological hurdles include the removal of adjacent adipose tissue and small paraaortic lymph nodes lining the entire aortic tree that are not visible by the naked eye. A dissection microscope is therefore recommended. Moreover protocols of aorta preparations should ascertain that lymphocyte aggregates referred to as  (FALCs) (Benezech ., 2015; Elewa ., 2015) that are often present at the border between the adipose tissue and the adventitia are removed before enzyme digestion. We propose - besides other approaches (Hu ., 2015; Mohanta ., 2014) - a combination of immunohistochemical staining and fluorescence activated cell sorter (FACS) analyses from single cell suspensions to quantify the cells of interest. This protocol describes isolation of single cells from mouse aorta for FACS and other analysis.

Preparation of Single Cell Suspensions from Mouse Aorta.

Artery tertiary lymphoid organs (ATLOs) are atherosclerosis-associated lymphoid aggregates with varying degrees of complexity ranging from small T/B-cell clusters to well-structured lymph node-like though unencapsulated lymphoid tissues. ATLOs arise in the connective tissue that surrounds diseased arteries, i.e., the adventitia. ATLOs have been identified in aged atherosclerosis-prone hyperlipidemic apolipoprotein E-deficient (ApoE) mice: they are organized into distinct immune cell compartments, including separate T-cell areas, activated B-cell follicles, and plasma cell niches. Analyses of ATLO immune cell subsets indicate antigen-specific T- and B-cell immune reactions within the atherosclerotic arterial wall adventitia. Moreover, ATLOs harbor innate immune cells, including a large component of inflammatory macrophages, B-1 cells, and an aberrant set of antigen-presenting cells. There is marked neoangiogenesis, irregular lymphangiogenesis, neoformation of high endothelial venules, and  synthesis of lymph node-like conduits. Molecular mechanisms of ATLO formation remain to be identified though media vascular smooth muscle cells may adopt features of lymphoid tissue organizer-like cells by expressing lymphorganogenic chemokines, i.e., CXCL13 and CCL21. Although these data are consistent with the view that ATLOs participate in primary T- and B-cell responses against elusive atherosclerosis-specific autoantigens, their specific protective or disease-promoting roles remain to be identified. In this review, we discuss what is currently known about ATLOs and their potential impact on atherosclerosis and make attempts to define challenges ahead.

Artery Tertiary Lymphoid Organs: Powerhouses of Atherosclerosis Immunity.

Editorial: Tertiary Lymphoid Organs (TLOs): Powerhouses of Disease Immunity.

Apolipoprotein-E (ApoE) has been implicated in Alzheimer's disease, atherosclerosis, and other unresolvable inflammatory conditions but a common mechanism of action remains elusive. We found in ApoE-deficient mice that oxidized lipids activated the classical complement cascade (CCC), resulting in leukocyte infiltration of the choroid plexus (ChP). All human ApoE isoforms attenuated CCC activity via high-affinity binding to the activated CCC-initiating C1q protein (K~140-580 pM) in vitro, and C1q-ApoE complexes emerged as markers for ongoing complement activity of diseased ChPs, Aβ plaques, and atherosclerosis in vivo. C1q-ApoE complexes in human ChPs, Aβ plaques, and arteries correlated with cognitive decline and atherosclerosis, respectively. Treatment with small interfering RNA (siRNA) against C5, which is formed by all complement pathways, attenuated murine ChP inflammation, Aβ-associated microglia accumulation, and atherosclerosis. Thus, ApoE is a direct checkpoint inhibitor of unresolvable inflammation, and reducing C5 attenuates disease burden.

ApoE attenuates unresolvable inflammation by complex formation with activated C1q.

In the version of this article originally published, a sentence was erroneously included in the author contributions, and information regarding second shared authorship was missing from the author contributions. The following should not have been included in the author contributions: "C.W. and A.J.R.H. supervised the work presented in Figs. 1, 2, 5, 6; P.Z. and C.S. supervised the work presented in Figs. 3, 4." Additionally, this sentence should have appeared at the beginning of the author contributions: "These authors contributed equally: C.W., P.F.Z., C.S., and A.J.R.H." The errors have been corrected in the print, PDF and HTML versions of the article.

Publisher Correction: ApoE attenuates unresolvable inflammation by complex formation with activated C1q.

Vascular smooth muscle cells (VSMCs) constitute the major cells in the media layer of arteries, and are critical to maintain the integrity of the arterial wall. They participate in arterial wall remodeling, and play important roles in atherosclerosis throughout all stages of the disease. Studies demonstrate that VSMCs can adopt numerous phenotypes depending on inputs from endothelial cells (ECs) of the intima, resident cells of the adventitia, circulating immune cells, hormones, and plasma lipoproteins. This plasticity allows them to perform multiple tasks in physiology and disease. In this minireview, we focus on a previously underappreciated activity of VSMCs, i.e., their impact on atherosclerosis immunity via formation of artery tertiary lymphoid organs (ATLOs).

Vascular Smooth Muscle Cells Contribute to Atherosclerosis Immunity.

 The high expression of Galectin-3 (Gal3) in macrophages of atherosclerotic plaques suggests its participation in atherosclerosis pathogenesis, and raises the possibility to use it as a target to image disease severity . Here, we explored the feasibility of tracking atherosclerosis by targeting Gal3 expression in plaques of apolipoprotein E knockout (ApoE-KO) mice  PET imaging.  Targeting of Gal3 in M0-, M1- and M2 (M2a/M2c)-polarized macrophages was assessed  using a Gal3-F(ab') mAb labeled with AlexaFluor®488 and Zr- desferrioxamine-thioureyl-phenyl-isothiocyanate (DFO). To visualize plaques , ApoE-KO mice were injected i.v. with Zr-DFO-Gal3-F(ab') mAb and imaged via PET/CT 48 h post injection. Whole length aortas harvested from euthanized mice were processed for Sudan-IV staining, autoradiography, and immunostaining for Gal3, CD68 and α-SMA expression. To confirm accumulation of the tracer in plaques, ApoE-KO mice were injected i.v. with Cy5.5-Gal3-F(ab') mAb, euthanized 48 h post injection, followed by cryosections of the body and acquisition of fluorescent images. To explore the clinical potential of this imaging modality, immunostaining for Gal3, CD68 and α-SMA expression were carried out in human plaques. Single cell RNA sequencing (scRNA-Seq) analyses were performed to measure LGALS3 (i.e. a synonym for Gal3) gene expression in each macrophage of several subtypes present in murine or human plaques.  Preferential binding to M2 macrophages was observed with both AlexaFluor®488-Gal3-F(ab') and Zr-DFO-Gal3-F(ab') mAbs. Focal and specific Zr-DFO-Gal3-F(ab') mAb uptake was detected in plaques of ApoE-KO mice by PET/CT. Autoradiography and immunohistochemical analyses of aortas confirmed the expression of Gal3 within plaques mainly in macrophages. Moreover, a specific fluorescent signal was visualized within the lesions of vascular structures burdened by plaques in mice. Gal3 expression in human plaques showed similar Gal3 expression patterns when compared to their murine counterparts.  Our data reveal that Zr-DFO-Gal3-F(ab') mAb PET/CT is a potentially novel tool to image atherosclerotic plaques at different stages of development, allowing knowledge-based tailored individual intervention in clinically significant disease.

Imaging atherosclerotic plaques by targeting Galectin-3 and activated macrophages using (Zr)-DFO- Galectin3-F(ab') mAb.

Atherosclerotic plaques develop in the inner intimal layer of arteries and can cause heart attacks and strokes. As plaques lack innervation, the effects of neuronal control on atherosclerosis remain unclear. However, the immune system responds to plaques by forming leukocyte infiltrates in the outer connective tissue coat of arteries (the adventitia). Here, because the peripheral nervous system uses the adventitia as its principal conduit to reach distant targets, we postulated that the peripheral nervous system may directly interact with diseased arteries. Unexpectedly, widespread neuroimmune cardiovascular interfaces (NICIs) arose in mouse and human atherosclerosis-diseased adventitia segments showed expanded axon networks, including growth cones at axon endings near immune cells and media smooth muscle cells. Mouse NICIs established a structural artery-brain circuit (ABC): abdominal adventitia nociceptive afferents entered the central nervous system through spinal cord T-T dorsal root ganglia and were traced to higher brain regions, including the parabrachial and central amygdala neurons; and sympathetic efferent neurons projected from medullary and hypothalamic neurons to the adventitia through spinal intermediolateral neurons and both coeliac and sympathetic chain ganglia. Moreover, ABC peripheral nervous system components were activated: splenic sympathetic and coeliac vagus nerve activities increased in parallel to disease progression, whereas coeliac ganglionectomy led to the disintegration of adventitial NICIs, reduced disease progression and enhanced plaque stability. Thus, the peripheral nervous system uses NICIs to assemble a structural ABC, and therapeutic intervention in the ABC attenuates atherosclerosis.

Neuroimmune cardiovascular interfaces control atherosclerosis.

The dawn has come for new therapeutics to treat atherosclerosis: Targeting neuroimmune cardiovascular interfaces in artery brain circuits.

The cardiovascular system is hardwired to the brain via multilayered afferent and efferent polysynaptic axonal connections. Two major anatomically and functionally distinct though closely interacting subcircuits within the cardiovascular system have recently been defined: The artery-brain circuit and the heart-brain circuit. However, how the nervous system impacts cardiovascular disease progression remains poorly understood. Here, we review recent findings on the anatomy, structures, and inner workings of the lesser-known artery-brain circuit and the better-established heart-brain circuit. We explore the evidence that signals from arteries or the heart form a systemic and finely tuned cardiovascular brain circuit: afferent inputs originating in the arterial tree or the heart are conveyed to distinct sensory neurons in the brain. There, primary integration centers act as hubs that receive and integrate artery-brain circuit-derived and heart-brain circuit-derived signals and process them together with axonal connections and humoral cues from distant brain regions. To conclude the cardiovascular brain circuit, integration centers transmit the constantly modified signals to efferent neurons which transfer them back to the cardiovascular system. Importantly, primary integration centers are wired to and receive information from secondary brain centers that control a wide variety of brain traits encoded in engrams including immune memory, stress-regulating hormone release, pain, reward, emotions, and even motivated types of behavior. Finally, we explore the important possibility that brain effector neurons in the cardiovascular brain circuit network connect efferent signals to other peripheral organs including the immune system, the gut, the liver, and adipose tissue. The enormous recent progress vis-à-vis the cardiovascular brain circuit allows us to propose a novel neurobiology-centered cardiovascular disease hypothesis that we term the neuroimmune cardiovascular circuit hypothesis.

Cardiovascular Brain Circuits.

Atherosclerotic plaques form in the inner layer of arteries triggering heart attacks and strokes. Although T cells have been detected in atherosclerosis, tolerance dysfunction as a disease driver remains unexplored. Here we examine tolerance checkpoints in atherosclerotic plaques, artery tertiary lymphoid organs and lymph nodes in mice burdened by advanced atherosclerosis, via single-cell RNA sequencing paired with T cell antigen receptor sequencing. Complex patterns of deteriorating peripheral T cell tolerance were observed being most pronounced in plaques followed by artery tertiary lymphoid organs, lymph nodes and blood. Affected checkpoints included clonal expansion of CD4, CD8 and regulatory T cells; aberrant tolerance-regulating transcripts of clonally expanded T cells; T cell exhaustion; T-TH T cell conversion; and dysfunctional antigen presentation. Moreover, single-cell RNA-sequencing profiles of human plaques revealed that the CD8 T cell tolerance dysfunction observed in mouse plaques was shared in human coronary and carotid artery plaques. Thus, our data support the concept of atherosclerosis as a bona fide T cell autoimmune disease targeting the arterial wall.

Article	Year
Artery tertiary lymphoid organs contribute to innate and adaptive immune responses in advanced mouse atherosclerosis. Circulation research\| PubMed ID: 24855201	2014
Artery Tertiary Lymphoid Organs Control Aorta Immunity and Protect against Atherosclerosis via Vascular Smooth Muscle Cell Lymphotoxin β Receptors. Immunity\| PubMed ID: 26084025	2015
Generation of Aorta Transcript Atlases of Wild-Type and Apolipoprotein E-null Mice by Laser Capture Microdissection-Based mRNA Expression Microarrays. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 26445797	2015
Artery Tertiary Lymphoid Organs Control Multilayered Territorialized Atherosclerosis B-Cell Responses in Aged ApoE-/- Mice. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 27102965	2016
Preparation of Single Cell Suspensions from Mouse Aorta. Bio-protocol\| PubMed ID: 27335895	2016
Artery Tertiary Lymphoid Organs: Powerhouses of Atherosclerosis Immunity. Frontiers in immunology\| PubMed ID: 27777573	2016
Editorial: Tertiary Lymphoid Organs (TLOs): Powerhouses of Disease Immunity. Frontiers in immunology\| PubMed ID: 28321222	2017
ApoE attenuates unresolvable inflammation by complex formation with activated C1q. Nature medicine\| PubMed ID: 30692699	2019
Publisher Correction: ApoE attenuates unresolvable inflammation by complex formation with activated C1q. Nature medicine\| PubMed ID: 30718908	2019
Vascular Smooth Muscle Cells Contribute to Atherosclerosis Immunity. Frontiers in immunology\| PubMed ID: 31164888	2019
Imaging atherosclerotic plaques by targeting Galectin-3 and activated macrophages using (Zr)-DFO- Galectin3-F(ab') mAb. Theranostics\| PubMed ID: 33408786	2021
Neuroimmune cardiovascular interfaces control atherosclerosis. Nature\| PubMed ID: 35477759	2022
The dawn has come for new therapeutics to treat atherosclerosis: Targeting neuroimmune cardiovascular interfaces in artery brain circuits. Clinical and translational medicine\| PubMed ID: 36052959	2022
Cardiovascular Brain Circuits. Circulation research\| PubMed ID: 37228235	2023
Pairing of single-cell RNA analysis and T cell antigen receptor profiling indicates breakdown of T cell tolerance checkpoints in atherosclerosis. Nature cardiovascular research\| PubMed ID: 37621765	2023

Andreas J.R. Habenicht

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