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

Atherosclerosis is the leading cause of mortality worldwide. However, regrettably, there are no treatment options that target the root causes of the disease, as its pathogenic mechanisms largely remain to be defined. Atherosclerosis has been viewed as a chronic inflammatory and unresolvable condition of all three arterial wall layers: Plaques develop in the inner intima layer of arteries leading to lumen narrowing and reduction of blood flow to downstream tissues, and when critical thresholds are breached, they cause heart attacks and strokes; smooth muscle cells in the media layer transdifferentiate into a synthetic and migratory phenotype and participate in artery remodelling by changing their transcriptome profile and secreting cytokines; and immune cells accumulate in the outer connective tissue coat of arteries, that is, the adventitia, and adopt roles in atherosclerosis-specific immune responses. Since atherosclerotic plaques are not innervated, until recently, the hardwired connections between the peripheral nervous system (PNS), the central NS (CNS) and the arterial wall remained unknown. However, since the NS uses the adventitia of all arteries as its main conduit to reach distant targets and indeed all parenchymal cells, we speculated that PNS axons may crosstalk to adventitia immune cells. Unexpectedly, we found that widespread neuroimmune cardiovascular interfaces (NICIs) arise in atherosclerosis-diseased adventitia segments and that both the sensory NS and the sympathetic NS (SYNS) establish a structural artery–brain circuit (ABC): its sensory arm enters the CNS via dorsal root ganglia, and from there, polysynaptic artery-brain projections connect to higher brain regions including the parabrachial nucleus and the central amygdala; and the efferent arm of the ABC project from hypothalamic neurons back to the adventitia via bifurcated projections involving the parasympathetic NS and the SYNS. Moreover, celiac ganglionectomy reduces disease progression and stabilises plaque vulnerability. Thus, the PNS uses NICIs to assemble a structural ABC, and therapeutic intervention into the ABC attenuates atherosclerosis. Here, we outline how this previously unknown hardwired connection of arteries with the CNS may lead to new classes of therapeutics. Our recent data in experimental mice and human cardiovascular tissues1, 2 have provided information on a series of unexpected mechanistic insights into the pathogenesis of atherosclerosis.3-5 This body of evidence suggests a new disease paradigm of atherosclerosis progression. We propose that adventitia NICIs are proxy sentinel sensors and effectors of atherosclerosis created by long-lasting interactions of the PNS with both the immune and vascular systems in tripartite tissue interactions.1 Using virus tracing techniques, we reconstructed a disease-specific polysynaptic hard-wired PNS-CNS connectivity network to form a structural ABC (Figure 1). The initiating event to establish this ABC appears to originate in plaques throughout the arterial tree: here, aggregates of immune cells accumulate in adventitia segments adjacent to plaques but not in disease-free segments. The adventitia NICI and its connection to the brain therefore appear to be a systemic phenomenon encompassing the entire arterial tree. Over time, a multisynaptic ABC emerges during adulthood and ageing, including a sensory NS (SENS) arm, and bifurcated SYNS and parasympathetic NS (PANS) effector arms (Figure 1). Moreover, therapeutic intervention into the SYNS attenuates atherosclerosis (Figure 2). Although neuroimmune interactions6 have been described previously including those in cancer,7 obesity,8 thermoregulation,9 brain diseases10 and inflammatory bowel diseases11, 12 the identification of the ABC may establish a new disease paradigm for atherosclerosis. It addresses key features of neuroimmunology13-15 in atherosclerosis pathogenesis and integrates the vascular system as a core third systemic participant.16 Indeed, the vascular system qualifies for a dual role in tripartite rather than bidirectional tissue interactions in atherosclerosis: adventitia segments adjacent to plaques speak with the SENS and the SYNS by providing a biological platform to sense plaque-derived molecular information via neuroimmune junction formation to transient receptor potential (TRP)- type nociceptors capable of translating inflammatory cytokine-derived signals into action potentials at axon endings which are ultimately projected to the brain.6, 17 On the intima side of the arterial wall, lumenal endothelial cells receive signals from the circulation resulting in plaque growth and, eventually, trigger clinically significant diseases such as heart attacks and strokes.3, 16 As none of the multiple interactions between the vascular system, the immune system and the NS have been known until recently, the possibility of new therapeutic strategies deserves careful consideration. Adventitia NICIs so far studied are characterised by marked axon neogenesis and restructuring involving axons of both the SENS and the SYNS but we did not observe axons in the adventitia PANS until now. At a cellular level, multiple immune cells in the adventitia have been identified: they include myeloid cells including macrophage subtypes, dendritic cell subtypes, and both types of adaptive immune cells and their subtypes, that is, B cells and T cells. Adventitia NICIs markedly alter signalling molecules of cells involved in the interaction of a large number of participating cells with axon tips of the SYNS and the SENS as indicated by whole genome transcriptome and single cell RNA sequencing (scRNA-seq) profiling.1 Using whole genome sequencing profiling of ganglia of the PNS has yielded a large number of potential targets for future therapeutic approaches including molecules and transcripts released at axon endings such as epinephrine, cytokines, synaptic proteins, regulators of axon neogenesis and inflammatory mediators.18 Many of these newly identified molecules are apparent target candidates for future treatments of late stage atherosclerosis. Yet, defining atherosclerosis-associated molecules and studies of their impact on atherosclerosis progression is a daunting task because of their sheer number. Such studies will involve extensive further usage of preclinical mouse models as well as translational studies in human patients. Translational studies will need to focus not only on leukocytes in the adventitia adjacent to atherosclerotic plaques but they should seek to examine paraarterial ganglia, the nodose ganglia (a major SENS ganglion of the PNS) and DRGs to identify neuronal genes that may be altered during human atherosclerosis progression (Figure 1). Moreover, delineation of the cell-cell crosstalk within NICIs has uncovered previously less known mediators of atherosclerosis progression at axon endings including axon-derived neurotransmitters of the SENS such as calcitonin gene-related peptide and the SYNS such as high tissue concentrations of norepinephrine but also those of Schwann cells such as nerve growth factor and many more. Many of these molecules have previously not been tied to atherosclerosis progression including regulators of axon homeostasis and inflammatory cytokines and their receptors. For example, we observed marked increases in TRPV1 expression at axon endings of SENS axons that are in close proximity to various types of adventitia immune cells most likely forming neuroimmune junctions. Axon tips expressing TRPV1 and possibly other sensory potential channels now need to be studied at functional levels. These include potential channels mediating signals of stretch, pain, heat, cold and inflammation. Our observation of apparent TRPV1 smooth muscle cell junctions indicates that the SENS may sense the inflammatory environment and transmit signals via the pain pathway in the brain raising the important possibility that TRP family members may be involved in atherosclerosis progression.13, 17, 19-25 We noticed earlier that there is also aberrant lymph vessel neogenesis, angiogenesis and de novo synthesis of high endothelial venules in ATLOs.26 It will be of interest to examine whether the altered lymph vessels in the adventitia adjacent to atherosclerotic plaques interact with components of the PNS and whether TRP channels are expressed in lymph vessels and newly formed blood vessels as well as in high endothelial venules. All these cells, molecules and structural components may turn out to be candidates for future therapeutic approaches. It is apparent, however, that a more detailed molecular and cellular characterization of NICIs—not to speak about the epigenetic landscape—will require major efforts in the years to come. As surgical therapeutic intervention into the SYNS of aged hyperlipidemic mice was shown to attenuate atherosclerosis progression and stabilise plaque erosion,1 we obtained proof-of-concept evidence that interference into components of the PNS can impact atherosclerosis progression (Figure 2). These observations call for a comprehensive delineation of other components of the PNS as well as the CNS that may affect disease progression. While such a proof-of-concept is conceptually important, there are caveats when considering the development of future therapeutic strategies. Some of these caveats need to be taken into consideration early to avoid flawed approaches going forward: that is, removal of the celiac ganglion and its relation to the function of adventitia axon networks is difficult to interpret in the sense that its spatial microanatomy is complex: removal of the celiac ganglion not only removes all axons within the adventitia but also takes out the splenic nerve which originates in the celiac ganglion and in addition it removes the innervation of the gastrointestinal tract. Thus, it has been shown that the spleen is an important regulator within the immune system to affect healing of the myocardium after experimentally introducing a myocardial infarct.27, 28 All the experimental evidence so far obtained regarding therapies in future human trials is by far not sufficient to justify the initiation of clinical trials any time soon. Much more information on mice has to be obtained to even outline potential strategies. From future preclinical studies in mice, much more has to be done to translate the most significant studies to humans. This translational work will require a large number of patients to stratify any concept to the more complex risk factor profile of humans with atherosclerosis versus atherosclerosis mouse models. However, human studies should be initiated soon and should involve the design of separate clinical cohorts relative to gender, age and risk factor profiles. Provide a connectivity map of the atherosclerotic PNS and the CNS using virus tracing methodologies.39 Identify atherosclerosis-associated transcriptome modifications of PNS and CNS neuronal genes of crucial ABC territories in the atherosclerotic CNS in mouse models and human brains. Test activation and inactivation strategies using DREADDs in mouse models of atherosclerosis Use tissue clearing approaches to define the spatial relation of the immune system, the NS and the cardiovascular system The discovery of a NICI-triggered ABC opens the field of neurobiology to study atherosclerosis progression, define the underlying molecular mechanisms in the PNS and CNS and design and develop new classes of therapeutics in the hope to treat the root causes of clinically significant disease as well as develop therapeutics in primary prevention approaches. This work has been supported by Deutsche Forschungsgemeinschaft (DFG): MO 3054/1-1 and grant SFB1123-Z1 to Sarajo Kumar Mohanta; YI 133/3-5 to Changjun Yin; HA 1083/15-5 to Andreas J. R. Habenicht; European Research Area Network on Cardiovascular Diseases (ERA-CVD): PLAQUEFIGHT (01KL1808) to Andreas J. R. Habenicht; and Easemedcontrol R & D Gmbh & Co KG to Sarajo Kumar Mohanta, Changjun Yin and Andreas J. R. Habenicht. There is no conflict of interest.