Peripheral macrophages in the development and progression of structural cerebrovascular pathologies

Abstract

The human cerebrovascular system is responsible for maintaining neural function through oxygenation, nutrient supply, filtration of toxins, and additional specialized tasks. While the cerebrovascular system has resilience imparted by elaborate redundant collateral circulation from supportive tertiary structures, it is not infallible, and is susceptible to developing structural vascular abnormalities. The causes of this class of structural cerebrovascular diseases can be broadly categorized as 1) intrinsic developmental diseases resulting from genetic or other underlying aberrations (arteriovenous malformations and cavernous malformations) or 2) extrinsic acquired diseases that cause compensatory mechanisms to drive vascular remodeling (aneurysms and arteriovenous fistulae). Cerebrovascular diseases of both types pose significant risks to patients, in some cases leading to death or disability. The drivers of such diseases are extensive, yet inflammation is intimately tied to all of their progressions. Central to this inflammatory hypothesis is the role of peripheral macrophages; targeting this critical cell type may lead to diagnostic and therapeutic advancement in this area. Here, we comprehensively review the role that peripheral macrophages play in cerebrovascular pathogenesis, provide a schema through which macrophage behavior can be understood in cerebrovascular pathologies, and describe emerging diagnostic and therapeutic avenues in this area.

Introduction

The targeting of inflammation-related cellular mediators revolutionized the treatment of conditions such as cancer, asthma, dementia, and inflammatory bowel disease.^{1 –5} In diseases of the central nervous system (CNS), broad or selective modulation of immune-mediated pathways has yielded benefits for patients suffering from conditions including certain metastatic brain malignancies and multiple sclerosis.^{6 –8} While such advances are encouraging, these conditions account for a small proportion of all-cause CNS-related morbidity and mortality when compared to cerebrovascular diseases, an area where such therapies have made smaller strides. ⁹ Cerebrovascular diseases including brain arteriovenous malformations (bAVMs), cerebral cavernous malformations (CCMs), intracranial aneurysms (IAs), and dural arteriovenous fistulae (dAVFs) contribute to this high morbidity given their risk of hemorrhage and other symptomatic events.^{10 –12} Improved imaging and screening for high-risk patients has enabled earlier identification of cerebrovascular pathologies, extending the window for potential intervention prior to symptom development. ¹³ Medical therapy is an appealing standalone or adjunct strategy to mitigate risk of symptomatic events. ¹⁴

Though bAVMs, CCMs, IAs, and dAVFs each carry unique risk factors, mounting evidence implicates inflammation and immune cell infiltration as contributors to their pathogenesis, shifting our understanding of these conditions from diseases of genetic abnormalities and artifacts of hemodynamic stress to diseases with pathology-promoting microenvironments rich with complex cellular interactions.^{15 –18} Macrophages in particular play a critical role in the pathophysiology of these diseases and have significant therapeutic potential. ¹⁹ In-vivo animal studies have implicated macrophages in upregulation of proteinases (including major metalloproteinases, MMPs), extracellular matrix (ECM) degradation, hemoglobin phagocytosis, leukocyte recruitment, angiogenesis, vascular permeability regulation, and induction of pro-inflammatory cytokines in the behavior of vascular abnormalities and malformations.^{20 –25} In human studies, serum levels of macrophage-derived cytokines (interleukin (IL)-1β, IL-6, and others) have been found to prognosticate rupture risk, and macrophage-based imaging biomarkers facilitate risk stratification of these diseases.^{26 –28}

Macrophage recruitment patterns and behaviors are variable across cerebrovascular conditions. The heterogeneity in macrophage functions is due to assorted macrophage polarization states, varying disease etiologies, and conflicting disease-associated and steady-state cues.^23,29 Even within a given vascular lesion, spatial and temporal variation in macrophage activity impacts growth, rupture, and recurrence.^30,31 Therefore, while similar events may trigger macrophage involvement in these diseases and modulate the progression to symptomatic lesions, each disease carries unique hallmark macrophage phenotypes (Figure 1). ²⁹

Figure 1.

Macrophage functions in cerebrovascular lesions. In aneurysms, stress from aberrant flow can recruit macrophages that promote rupture when polarized to inflammatory states. In dAVFs, macrophages facilitate processes that lead to the formation of vessel fistulae. In bAVMs, macrophages promote vascular instability and may be involved in regrowth of these lesions. In CCMs, macrophages may promote the progression from simple cavernous lesions to complex multicavernous lesions.

In this work, we review peripheral macrophages in cerebrovascular pathologies. We first comment on macrophage lineages in the CNS and describe the origin of these cells. We then elaborate on monocyte recruitment to the site of cerebrovascular pathology and the role macrophages have in the development of each class of disease. This assessment allows for comparisons of macrophage function across cerebrovascular diseases. Finally, we consider the diagnostic and therapeutic potential of macrophages in cerebrovascular pathologies, proposing future directions to serve as a guide for subsequent study in this area.

Macrophage populations in the CNS and beyond

Historical perspectives of monocytes and macrophages

Mononuclear phagocytic immune cells were discovered in the late 19th century and coined macrophages (“big eaters”) in contrast to microphages (“small eaters”, or neutrophils).^32,33 Macrophages serve diverse functions in tissue development, homeostasis, and host defense throughout the body. ³⁴ For decades, these cells were believed to be derived from the tissue in which they were found. ³⁵ Later studies demonstrated that most macrophages originate from bone marrow hematopoietic progenitors that produce circulating monocytic intermediates that then migrate to diseased tissues in response to disease-associated signals. ³² These monocytes differentiate into macrophages upon arrival at diseased tissues they infiltrate. Differentiation from circulating monocytes reflects the origin of most macrophages, but several organ systems, including the central nervous system (CNS), contain unique lineages of yolk sac-derived macrophages that become restricted to an organ or tissue during embryogenesis and development. ³⁶ The resident macrophages of the CNS include microglia and 3 additional subtypes of non-parenchymal macrophages. ³⁷

Resident phagocytes of the CNS

The best-studied phagocytes of the CNS are the microglia, a plastic glial cell type derived from the embryonic yolk sac involved in synaptic pruning, neurogenesis, debris clearance, myelin homeostasis, and defense from various insults.^{38 –41} The complexity of phagocytes in the CNS is illustrated by the presence of three classes of macrophages with roles exclusive to their CNS niche. These are perivascular macrophages, meningeal macrophages, and choroid plexus macrophages, which serve sentinel-like surveillance roles between the periphery and the brain parenchyma. ⁴¹ Like microglial cells, perivascular and meningeal macrophages are exclusively of yolk sac origin, while choroid plexus macrophages have dual ontogeny from hematopoietic progenitors and yolk sac progenitors.^37,42 Some studies have noted that CNS macrophages have regional differences in their transcriptomic profiles, suggesting an added layer of context-dependent function beyond simple surveillance. ⁴³ A recent line of evidence implicates perivascular and meningeal macrophages – often referred to as “border zone macrophages” of the brain – as crucial mediators of a variety of CNS pathologies, because they partake in a series of complex interactions between the CNS and periphery. ⁴⁴ Border zone macrophages can communicate between the brain and the meninges freely by passing through specialized meningeal vasculature that facilitates immune surveillance. ⁴⁴ Importantly, perivascular and meningeal macrophages have been demonstrated to play a pivotal role in erythrocyte breakdown following SAH. ⁴⁵

Defining the peripheral macrophage

Past studies of neural immunity were obscured by the widely-held, yet false notion that the brain is a completely immune-privileged site, leading to a limited scope of early studies examining the role of peripheral cells in CNS pathology. ⁴¹ It is now accepted that myeloid cells influence CNS disease processes, including cerebrovascular pathologies. An important consideration of cerebrovascular pathologies is that the site of disease is proximal to or inclusive of the blood-brain-barrier (BBB). Therefore, monocyte-derived macrophages can infiltrate the diseased vessels and participate in signaling with glial cells to drive these diseases.^19,46 Key to analyzing cerebrovascular pathogenesis is the function of macrophages from myeloid lineages, which are the focus of our discourse. Microglia, perivascular macrophages, meningeal macrophages, and choroid plexus macrophages are discussed when pertinent. We define macrophages that differentiate from circulating monocytes following arrival to a site of pathology as “peripheral” macrophages. Macrophages can be distinguished from circulating monocytes via identification of surface markers including CD86, CD68, CD80, CD163 and CD136 that are present in differentiated macrophages.^47,48 Gene products can also be assessed, with the metabolic state in macrophages favoring different combinations of ornithine, polyamines, NO, and citrulline. ⁴⁹ Peripheral macrophages are distinguished from microglia with the markers CD11b^{hi /} CD45^hi as a signature for peripheral macrophages and CD11b^hi/CD45^low as a signature for microglia, though several other markers are expressed by peripheral macrophages and not microglia including CD44, CD45, CD169, and CD206.^46,50 Microglia-specific markers include TMEM119, P2RY12, and SALL1. Additional markers for identifying intrinsic CNS resident macrophages are also available. ⁴¹ Experimental techniques including immunohistochemistry for in-situ characterization of macrophage populations or flow cytometry allow for characterization of monocytic and microglial lineages.^51,52 More recent work has also included the use of single cell RNA sequencing to further characterize these populations in cerebrovascular lesions. ⁵³ Similar advances using single cell techniques have clarified the distinct subtypes of microglia in ischemic cerebrovascular processes. ⁵⁴

Alternate sources of peripheral macrophages

Recent evidence suggests that the peripheral origin of monocytes influences their behavior. ⁴⁴ Generally, monocytes are derived from bone marrow throughout the body and migrate to the circulatory system following loss of bone marrow retention cues, at which point they are permanently in the circulation until either senescence or tissue entry and differentiation into macrophages. ⁵⁵ In contradistinction to this dogma of monocyte behavior, myeloid cells derived from the skull bone marrow travel through direct fenestrations and conduits to the CNS border zones and CNS, which permits bidirectional transport from the myeloid cell reservoir rather than unidirectional transport seen in other organ systems.^44,56 Studies assessing macrophages in bAVMs, CCMs, IAs, and dAVFs have not distinguished between skull or vertebral marrow origin and other peripheral sources.^14,57 Future studies of both pre- and post-rupture cerebrovascular pathology should employ lineage tracing techniques to characterize the contributions of each myeloid cell reservoir in a given pathology.

Macrophage functions in the CNS and beyond

Monocyte migration and localization

Timely and facile monocyte localization to sites of vascular pathology is necessary for macrophages to have a mechanistic role in disease development. Monocyte access to cerebrovascular lesions is more obvious for IAs or dAVFs supplied by larger arteries branching from the circle of Willis, while the penetration of peripheral macrophages in deep-seated CCMs and bAVMs is explained by the complex network of perforating arterioles present throughout cerebral gray matter and white matter alike.^58,59 Circulating monocytes pass through this extensive CNS vascular network and localize to disease sites in response to homing chemokines and cytokines. ⁴¹ Newly described channels facilitate easy migration from the skull bone marrow or meningeal borders and may permit additional CNS access for myeloid populations. ⁵⁶

Macrophage polarization

After monocytes differentiate into macrophages in target tissues, condition-dependent responses are activated in a process called polarization, which dictate the expression profiles of macrophages. These polarization responses range from pro-inflammatory and deleterious to restorative and regenerative, with the final balance dictated by both locally and temporally mediated signals.^60,61 The current paradigm describing macrophage behavior is analogous to the Th1/Th2 paradigm in T lymphocytes: classically activated “M1” macrophage phenotypes are induced by interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), or lipopolysaccharide (LPS) paired with autocrine interferon beta (IFN-β), while alternatively activated “M2” macrophages are induced by interleukin 4 (IL-4) and interleukin 10 (IL-10).^48,49 M1 macrophages are involved in inflammation and have functions that include producing reactive nitrogen species/reactive oxygen species (RNS/ROS) through inducible nitric oxide synthase (iNOS), up-regulating proteases such as matrix metalloproteinase 2 and 9 (MMP-2/MMP-9), and producing pro-inflammatory cytokines. ²⁰ M2 macrophages have functions that include resolving inflammation via inactivation of the M1 response, promoting tissue healing, secreting anti-inflammatory cytokines that inactivate local responses, and angiogenesis. ⁴⁹ Importantly, macrophages of both the M1 and M2 phenotype have been observed together in cerebrovascular lesions.^14,30 Notably, CNS macrophages can exhibit unique polarization profiles: in non-CNS tissues, macrophage responses are characterized by M1 predominance followed by later M2 predominance, but the inverse is seen in some CNS injuries and pathologies. ⁶² This has primarily been observed in acute cerebral injuries, and therefore may not be representative of how macrophage polarization states impact cerebrovascular lesion development on a more chronic timescale. ⁶² M1 macrophages express the markers CD16, CD32, CD64, CD80, and CD86 while M2 macrophages express CD163 and CD206.^47,63 For details regarding inducers of macrophage polarization and hallmark expression by these respective macrophage types, see Figure 2.

Figure 2.

Depiction of molecular factors promoting macrophage polarization to the M1 and M2 state. M2 macrophages have named subtypes including M2a, M2b, M2c, and M2d that serve various disease-related functions. Macrophages are capable of expressing any phenotype from strongly inflammatory to strongly anti-inflammatory. Antagonistic functions are observed across the spectrum of macrophage polarization states. M1 macrophages synthesize NO from arginine while M2 macrophages synthesize reparative ornithine from arginine. Factors promoting the M2 state serve as antagonists of the M1 state.

While the M1/M2 paradigm of macrophage polarization facilitates discussion of macrophage behavior in disease processes, it is an oversimplification of the transcriptomic repertoire possessed by these cells. Environmental cues or epigenetic changes can conflict, resulting in a spectrum of macrophage responses and intermediate phenotypes rather than true terminal speciation into exclusively M1 or M2 phenotypes.^27,64 Verily, a single macrophage can express essentially the entire range of macrophage polarity phenotypes over its lifetime. ³¹ The absence of terminal differentiation states during macrophage polarization makes their involvement in cerebrovascular pathology more compelling. ⁶⁵ The pathogenesis of many cerebrovascular lesions is dynamic, with periods of growth and regression, which may reflect the contributions of differential macrophage phenotypes to these processes.^66,67 Additionally, known functions of M1 and M2 macrophages advance the idea that the presence of these cells in cerebrovascular lesions is more than a by-product of unrelated processes. For example, improper and untimely M1 macrophage responses are known to disrupt vessel homeostasis and impair vascular repair processes, while macrophages polarized towards the M2 phenotype carry significant angiogenic potential.^68,69

Macrophages in intrinsic pathologies of vascular development

Appropriate development of the cerebral vasculature to ensure appropriate neural function is contingent upon several key embryonic events. ⁷⁰ Major alterations in cerebral vascular development result in embryonic lethality, but focal or regional alterations result in structural malformations including bAVMs and CCMs.^71,72 In these diseases, several genetic mutations have been identified as drivers of abnormal development.^71,73

Macrophages have early roles in vascular development including anastomosis of endothelial tip cells in a VEGF-mediated fashion. ⁷⁴ Additional investigation is necessary to characterize the role of macrophages in pathologic vascular development. Assessment of bAVMs and CCMs together allows for comparison of macrophage behavior and extrapolation of potential functions across lesion types. ¹⁸ Discussion of these pathologies together highlights important features given their anatomic locations and thinner vascular walls, which may portend greater cross-talk between peripheral immune cells and glial cells in addition to promoting similar bleeding compartmentalization in hemorrhage. ⁷⁵

Brain arteriovenous malformations

Underlying abnormalities in bAVMs

Brain AVMs are masses of abnormal vessels characterized by direct connections between arteries and veins without intervening capillaries that aggregate into a nidus and associated perinidal networks. ⁷⁶ These lesions can be composed of single or multiple compartments and include numerous feeding arteries and draining veins. ⁷⁷ The majority of bAVMs are congenital and are thought to arise in the 4th–8th week of embryonic development – the period during which the potential boundary of the vascular malformation is defined – with the ultimate angioarchitecture of the bAVM determined by cellular and molecular interactions throughout develpoment.^{78 –80} Others have posited that bAVMs are at least partially driven by reactive angiopathic processes following hemorrhage or ischemia. ⁸¹ Genetic diseases such as hereditary hemorrhagic telangiectasia (HHT) and Sturge-Weber syndrome associated with bAVMs implicate various molecular factors including transforming growth factor-β (TGF-β), mitogen-activated protein kinase (MAPK), activin-like kinase (Alk1), and endoglin (Eng) in heritable bAVM pathogenesis, while VEGF and KRAS variants are observed in sporadic bAVMs.^73,82 The VEGF-KRAS pathway in particular has garnered significant attention as a central mediator of bAVM growth, with Nikolaev et al. finding that KRAS signaling in bAVMs specifically functions through MAPK-ERK and not Protein Kinase B (also called Akt) or p38. ⁸² The growth and progression of bAVMs involves chronic interplay between endothelial cells, smooth muscle cells, pericytes, and peripheral leukocytes. ⁷⁶ Interactions between leukocytes and the vessel wall during bAVM growth and hemorrhage have recently provided new insights into bAVM biology. ⁸³ Clinically, approximately 50% of symptomatic bAVMs present hemorrhagically, 20–45% with seizures, and the remaining with headaches, neurological deficits, or incidentally. ⁸⁴

Inflammatory phenotype of bAVMs

Genetic polymorphisms predicting bAVM development and rupture are present in the promoter sequences for inflammatory factors including TNF-α, IL-1β, and IL-6.^18,85 Building on this evidence supporting inflammatory drivers of bAVM progression, next-generation RNA sequencing performed on human bAVM tissue identified that genes of inflammation and immune cell localization are up-regulated. ²⁹ For example, chitotriosidase, an enzyme produced by macrophages that is involved in pathogen clearance and fibrosis, was one of the genes with the highest differential expression in bAVM tissue.^29,86 These findings comport with histopathological studies that observe macrophages as the dominant inflammatory cell type throughout human bAVM tissue and occasionally in the adjacent parenchyma.^18,87 Within bAVM vessel walls, macrophages localize to the outer vascular walls and adventitia. ⁸⁸ High-dimensionality profiling techniques and transcriptomic assessment of human bAVMs have identified three perivascular macrophage subpopulation clusters and three peripheral monocyte subpopulation clusters, indicating both lineage and spatial diversity amongst macrophages within bAVMs. ⁵³

Monocyte recruitment to bAVMs

Mendelian diseases associated with bAVM development largely exclude inflammatory or macrophage-related genes. ⁸⁹ However, the arrival of macrophages in bAVMs following the development of pathologic angiogenesis promotes secondary lesional growth. Prior hypotheses purported that the presence of macrophages was in response to hemorrhage-induced inflammatory signals produced by ruptured bAVMs. ⁹⁰ This theory was revised when robust macrophage infiltration was also noted in unruptured bAVM specimens. ⁸⁷ Notably, bAVMs without overt clinical hemorrhage have been observed to contain hemosiderin-laden macrophages and hemosiderin deposits in vessel walls; these hallmarks of prior bleeding indicate that subclinical microhemorrhages may influence the recruitment and activation of peripheral macrophages in bAVMs.^22,90 This recruitment can be explained by the scavenging role of macrophages. CD163 is a cell surface protein highly expressed by M2 macrophages and serves an important role in post-hemorrhagic physiology as the primary mediator of hemoglobin complex degradation, which mitigates the ability of hemoglobin to induce oxidative damage.^51,91,92 The arrival of CD163-expressing macrophages to facilitate clearance of hemoglobin products represents an important function of macrophages in these lesions. CD163 agonism also promotes IL-10 signaling through phosphatidylinositol-3 kinase (PI3K) and Akt, which favors the anti-inflammatory M2 state in certain disease states. ⁹³ The subclinical microhemorrhage hypothesis, however, does not explain why macrophages are also present in hemosiderin-negative AVM samples, implying that additional processes must also attract macrophages to bAVMs. ²¹ Prior to the first instance of hemorrhage or microhemorrhage, VEGFs that drive angiogenesis also increase regional vascular permeability and serve as chemotactic agents that promote monocyte localization to angiogenic bAVM foci.^22,94 This may contribute to the early presence of macrophages in bAVMs in hemosiderin-negative samples. In-vivo studies have demonstrated that the number of peripheral macrophages in VEGF-induced brain angiogenic regions peaks before angiogenesis does. This observation suggests that peripheral macrophages potentiate angiogenic processes. ⁹⁵ This may occur via the supplying of MMP-9, which is cleaved from pro-MMP-9 by activators including other MMPs, cathepsin G, and plasmin, and degrades the ECM.^96,97 Finally, a component of local inflammatory signaling is induced by pathologic hemodynamic forces present as a consequence of the dysfunctional angioarchitecture of the bAVM, implying that larger bAVMs involving more vessels may stimulate greater inflammation, and that hemodynamic stress itself is may be sufficient to recruit monocytes in this disease. ⁸⁹

Macrophage activity and functions in bAVMs

Regardless of the initial polarization state of macrophages, their lability and absence of a terminal phenotype allows for both polarization states to coexist in bAVMs. ⁴³ Macrophages may arrive at the bAVM, perform the functions of one polarization state, and as their milieu evolves, polarize towards another phenotype. Peripheral macrophages in bAVMs contribute to vascular instability by promoting inflammation, weakening the ECM, secreting inflammatory cytokines that recruit additional leukocytes, increasing basement membrane degradation through MMP secretion, and producing ROSs.^89,98 These functions are hallmarks of the M1 polarization state, which may be partially induced by the iron overload in post-hemorrhagic states and by the pro-inflammatory environment of an enlarging bAVM. ⁸⁷ Increased inflammatory macrophages accelerates disease progression towards rupture and symptomatic presentation, with macrophage-associated products predicting bAVM rupture, and bAVM hemorrhages correlating with focal inflammation within vessel walls.^18,79 Importantly, GPNMB⁺ monocyte derived populations were recently identified to induce smooth muscle cell depletion and are associated with bAVM rupture. ⁵³ Another important mediator of macrophage function in bAVMs is macrophage inhibitory factor (MIF), which serves important roles in macrophage aggregation, MMP-9 induction, and vessel regulation within bAVMs. ⁹⁹ M2-polarized macrophages also explain many aspects of bAVM evolution and development. Studies examining the activity of leukocytes in the perinidal dilated capillary network (PDCN) identified an elevated level of M2-polarized CD163⁺ macrophages. ¹⁰⁰ Though these alternatively-activated macrophages are often restorative or anti-inflammatory in nature, they may contribute to pathogenesis by serving as misguided chaperones for aberrant ECM remodeling and vascular anastomosis.^100,101 In the PDCN, M2 macrophages can stimulate the proliferation of brittle vessels that are prone to hemorrhage. ¹⁰² Due to abnormal BBB, lack of basement membranes, and absence of astrocytic foot processes, the PDCN also provides an environment promoting leukocyte migration and frequent microhemorrhages ¹⁰³ (Figure 3). However, the role of peripheral macrophages in recurrent bAVMs is mostly speculatory, and future studies should capitalize on the availability of endoluminal biopsies or liquid biopsies to better-profile recurrent bAVMs and profile the populations of macrophages present.^104,105 This may clarify the mechanisms of recurrence in these lesions that are frequently very challenging to treat.

Figure 3.

Schematic of macrophage behavior in bAVM growth and post-treatment responses.

Cerebral cavernous malformations

Underlying abnormalities in CCMs

CCMs are composed of abnormal capillary clusters without intervening brain parenchyma and involve slow or stagnant blood flow that promotes clotting and thrombosis. ⁴⁶ Like bAVMs, CCMs are dynamic and can enlarge, regress, and rupture.^106,107 Histologic studies have demonstrated that CCM vessels are composed of thick hyalinized walls and contain encapsulated hematomas. ¹⁰⁸ Approximately 80% of CCMs are associated with somatic mutations, while 20% are secondary to inherited germline mutations. ¹⁰⁹ In patients that develop CCMs from somatic mutations, lesions are typically solitary, while patients with germline mutations commonly develop multiple CCMs. ⁴⁶ Contemporary studies indicate that CCMs progress through two characteristic phases in their maturation. ²⁴ Initially, these lesions take on an “angiogenic” phenotype of isolated caverns formed by diseased capillary networks that demonstrate disrupted cell-cell junctions in the vessel wall. ²⁴ Some CCMs then proceed to complex multicavernous lesions with aggressive clinical behavior characterized by hemosiderin deposition, induction of pro-inflammatory pathways, and peripheral leukocyte infiltration. ²⁴ In this biphasic model, the first phase of CCM development is driven by hypoxic angiogenesis, while the second is driven by inflammation in response to induction of cytoplasmic NLR family pyrin domain containing 3 (NLRP3) inflammasome (Figure 4). ²⁴ Mutations in the Krev1 interaction trapped gene 1 (KRIT1; CCM1), malcavernin (CCM2), or programmed cell death protein 10 (PDCD10; CCM3) underly the pathologic vascular remodeling that drives the first phase of CCM development. ¹⁰⁹ The second phase is driven by NLRP3 inflammasome signaling in endothelial cells secondary to reactive astrogliosis, which promotes inflammatory cytokine production to recruit both peripheral leukocytes (including macrophages) and intrinsic microglia to CCMs. ²⁴ A comprehensive understanding of CCM progression is still lacking, as CCMs exhibit heterogeneous behaviors and disease progression, even amongst patients with familial types that have shared mutations. ⁴⁶ Clinically, 40% of CCM patients remain asymptomatic, while the remainder experience recurrent headaches, intracerebral hemorrhages, seizures, and neurological deficits most commonly presenting between the second and fifth decades of life.^110,111

Figure 4.

Schematic of CCM progression. Early CCM development is primarily driven by angiogenesis in a hypoxic environment, leading to isolated caverns. As the CCM grows, inflammatory signaling leads to induction of the NLRP3 inflammasome, resulting in astrocyte-endothelium signaling that promotes release of inflammatory factors. Macrophages are subsequently recruited to the CCM. Macrophage recruitment and activation is driven by chemokines including MCP-1, CCL5, and CXCL4. Sustained inflammation within CCMs leads to chronic CCMs, characterized by thrombosed blood, hemosiderin, infiltrating leukocytes, and production of antibodies.

Inflammatory polymorphisms influence CCM development

Genetic polymorphisms may explain the heterogenous disease progression in patients with shared driver mutations. ¹¹² A recent study conducted in patients carrying a common CCM1 mutation identified associations between TGF-β2R, CD14, IL-6R, MSR1, IGH, VEGF, and TLR4 variants with clinical presentation of their disease. ¹¹² In addition to variants in polymorphisms, inflammation has been implicated in the development of CCMs from their initial angiogenic phase to their complex multicavernous phase. TGF-β, a multifunctional cytokine that induces M2 macrophage polarization, is associated with CCM-related ICH and size and number of cavernous lesions, suggesting that TGF-beta is a critical mediator of CCM pathogenesis. Granulocyte-macrophage colony-stimulating factor (GM-CSF), produced by various cell lines to promote granulocytes and monocyte production, was identified as a diagnostic biomarker to predict symptomatic presentation in patients with CCMs. ¹¹³ GM-CSF is a key promoter of macrophage survival, activation, differentiation, and mobilization. ¹¹⁴ While macrophages have long been recognized as a major infiltrating cell type in CCMs, they are accompanied by CD20⁺ B-lymphocytes, CD138⁺ plasma cells, CD3⁺ T-lymphocytes, and high concentrations of oligoclonal immunoglobulins synthesized in-situ. ¹¹⁵

Monocyte recruitment to CCMs

The arrival of monocytes to CCMs shares common features with monocyte recruitment in bAVMs. In CCMs, the presence of macrophages is canonically viewed as a response to acute bleeding, with MRI studies noting large clusters of hemosiderin-laden macrophages in human CCMs, and transgenic CCM1 animal models confirming this.^116,117 Because of the low-flow state of CCMs, accumulation of thrombosed encapsulated blood occurs within the malformation independent of intra- or perilesional hemorrhage. ¹⁰⁸ This results in chronic iron deposition and blood degradation products that generate an inflammatory environment. ¹¹⁸ While the need for CD163⁺ macrophages to clear heme-containing blood products and conduct phagocytosis is evident, macrophages may additionally contribute to blood accumulation by facilitating immunothrombosis via interactions between macrophage CD11b/CD18 and integrin with platelet P-selectin.^24,119 Activated platelets then further recruit monocytes to the site of thrombosis by stimulating MCP-1 secretion and intercellular adhesion molecule-1 (ICAM-1) expression on endothelial cells in an NF-kB mediated pathway, leading to macrophage activation through CCL5 and CXCL4. ¹¹⁹ Monocyte recruitment to CCMs also occurs independently of blood accumulation. ¹⁷ Indeed, CCMs have a “leaky capillary” phenotype partially induced by VEGF, which facilitates monocyte migration. ¹²⁰

Endothelium-derived disease signals provide an additional mechanism for monocyte recruitment to CCMs. Pro-inflammatory signaling between reactive astrocytes and the CCM endothelium is orchestrated by the NLRP3 inflammasome, which then modulates the progression from the early stage of isolated caverns to the later stage of multicavernous inflammatory disease. ²⁴ Once activated, astrocytes stimulate MCP-1 and CX3CL1 secretion to potentiate leukocyte recruitment to CCMs. The function of astrocytes and other glial cells in this circuit of cross-talk resulting in cerebrovascular dysfunction and monocyte recruitment is an area of active interest in CCM research.

Macrophage activity and functions in CCMs

Macrophages present in CCMs behave similarly to those in bAVMs, with functions that include hemoglobin scavenging, promoting of vascular instability, and angiogenesis. These functions correlate with the dynamic nature of CCMs. In addition, CCMs contain high levels of B- and T-lymphocytes, which are typically absent in bAVMs, suggesting an additional component of adaptive immunity in these lesions. ¹¹⁸ Adaptive immune cells including CD20⁺ B lymphocytes, CD138⁺ plasma cells, and CD3⁺ T lymphocytes have all been observed in CCMs alongside CD68⁺ macrophages.^118,121 Isoelectric focusing of immunoglobulins present in CCMs has revealed oligoclonal patterns of IgGs that suggest in-situ synthesis rather than systemic production, though it remains unknown if these immunoglobulins are targeted against shared antigens across patients or if there is variation in antibody specificity. ¹¹⁸ Locally-produced IgGs form immune complexes that amplify local inflammation and endothelial disruption through apoptotic signaling and other activity. ¹¹⁸ Macrophages contribute to this adaptive immune response by serving an organizational role as antigen presenting cells (APCs) in CCMs. This active microenvironment of autoimmunity and innate immunity warrants additional investigation, as macrophages function at the intersection of these converging systems, and the degree of macrophage contribution to this milieu has not been clearly measured.

Macrophages in acquired cerebrovascular pathologies

Injury to the cerebrovascular system, whether acute or chronic, initiates compensatory mechanisms and alters homeostasis in a manner that can proceed to outright pathology. For example, chronic, sustained hemodynamic stress in patients with underlying vascular damage secondary to cigarette smoking, atherosclerosis, or hypertension can lead to dilatation of large artery walls and development of brain aneurysms. Similarly, acute injuries to the meninges and cerebral vasculature such as traumatic brain injury, surgical manipulation, or venous sinus thrombosis sometimes leads to the development of dAVFs. ¹²² In acute and chronic exogenous stress to the cerebral vasculature, remodeling activates inflammatory signaling, which can result in the recruitment of monocytes to the site of injury and may play a role in the pathogenesis of these acquired cerebrovascular lesions. ¹⁴

The related etiologies of extrinsic disruptions in IAs and dAVFs provides a lens through which the role of peripheral macrophages can be examined. These lesions affect larger vessels the CNS, and result in similar hemorrhage patterns when ruptured.

Intracranial aneurysms

Underlying abnormalities in IAs

Once viewed as congenital vessel dilations present at birth, IAs are now understood to form as a consequence of chronic hemodynamic stress and inflammatory responses.^{123 –125} The majority of IAs have saccular morphologies and form at branch points in the circle of Willis. ¹²⁶ Risk factors include smoking, alcohol use, and atherosclerosis. ¹²⁷ A minority of IAs develop in association with heritable connective tissue disorders including autosomal dominant polycystic kidney disease, Ehlers-Danlos Syndrome, and Loeys-Dietz syndrome. ¹²⁸ Twin-based studies have estimated the heritability of IA rupture at 40%, suggesting a strong familial predisposition to this pathology beyond mendelian diseases.^128,129 Histological studies have demonstrated that disruption of the internal elastic lamina is the hallmark vascular change in IAs, and is one of the first events to initiate the formation of IAs. ¹³⁰ Additional findings include irregular luminal surfaces, myointimal hyperplasia, disorganization of smooth muscle cells, hypocellularity in the media, inflammatory cell infiltration, and stretching of the adventitia layer. ¹³¹ Clinically, the majority of IAs are identified incidentally, while some are discovered following rupture or development of other symptoms. ¹³² Once detected, some IAs remain relatively stable in size while others grow secondary to sustained hemodynamic stress and persistent inflammation, at which point intervention is pursued to prevent rupture. ¹³³

Hemodynamic stress in IAs

Prior work has firmly established inflammation as a critical mediator in the progression of IAs, which is activated by hemodynamic stress and luminal turbulence. ¹⁴ Hemodynamic forces responsible for early degradation of the internal elastic lamina concurrently activate the pro-inflammatory pathway of prostaglandin E₂ (PGE₂) and PGE receptor subtype 2 (EP2) in endothelial cells, promoting local inflammation and leukocyte infiltration via up-regulation of inflammatory cytokines, cellular adhesion molecules, and selectins.^134,135 Therefore, the arrival of macrophages to IA appears to be due to flow-induced stress. MCP-1 and GM-CSF facilitate monocyte localization and macrophage infiltration to the site of stress, while IL-1β and TNF-α promote inflammation and vessel degradation.^{14,136 –138} NF-κB is the critical transcription factor orchestrating these inflammatory processes, with its deletion resulting in reduced formation of IAs in a murine model. ¹³⁹ Many leukocytes including mast cells, neutrophils, B-lymphocytes, and T-lymphocytes are present in IAs, however, macrophages demonstrate the most pronounced infiltration in histopathologic studies, and are invariably observed in human IAs.^16,140 The number of macrophages present in IAs corresponds with aneurysm growth suggesting that these cells are more than passive spectators.^14,137

Imbalances in macrophage polarization in IAs

Follow-up studies on NF-κB in IAs demonstrated that NF-κB activation first takes place in macrophages in the adventitial and endothelial cell layer, and subsequently occurs throughout the entire vessel wall, mediated by EP2-COX2 signaling, in a spatially and temporally-regulated manner. ¹³⁴ The pro-inflammatory functions of macrophages in IAs involve remodeling and degrading the ECM via MMP-2, MMP-9, ROSs, and secreting cytokines that recruits additional inflammatory cells.^131,141 Macrophages present in the IA serve as a reservoir for local proliferation and differentiation of more macrophages, supplementing the growing population of infiltrating monocyte-derived macrophages. ¹⁴² Local inflammation and degeneration induced by infiltrating macrophages occurs via positive feedback with macrophages promoting additional iNOS and MMP induction, alongside cytokine and chemokine production. ¹⁴³ When polarized towards the M1 phenotype, macrophages increase aneurysm rupture risk, but can be suppressed by M2 macrophages.^14,23 M1 and M2-polarized macrophages are present in relatively equal proportions in unruptured IAs, however, M1 macrophage expression is significantly elevated in IAs at the time of rupture.^23,144 It is possible that periods of rapid growth leading to rupture are catalyzed by macrophage polarity imbalance (Figure 5). This concept is supported by the observation that reduced M2 macrophage levels correlate with impaired IA structural integrity. ¹⁴⁵ An anabolic/catabolic-like inflammatory balance in quiescent IAs could prevent enlargement and progression towards rupture, and disturbances of this balance may lead to wall weakening and hemorrhage. Ultimately, the M1/M2 classification system represents an oversimplification in IAs as it does in other conditions. A single cell transcriptome analysis in murine IAs identified 6 unique monocyte/macrophage clusters with genetic profiles representing both pro- and anti-inflammatory states. ¹⁴² This reflects highly diverse macrophage populations in IAs, and similar studies in humans may reveal additional macrophage functions. Nevertheless, the idea that aneurysm rupture can be avoided by maintaining a balanced population of macrophages may serve as a possible therapeutic strategy for preventing aneurysmal subarachnoid hemorrhage.

Figure 5.

Role of macrophages in IA rupture. Hemodynamic stress and other sustained intravascular pathology lead to activation of the PGE-EP2 signaling axis within endothelial cells, which drives early inflammation within IAs. This is followed by NF-kB-mediated signaling pathways that potentiate leukocyte recruitment and promote macrophage polarization. Once present in the aneurysm walls, M1 and M2 macrophages coexist in relatively equal proportions. When this balance is disrupted, unopposed inflammatory activity is mediated by the M1-polarized macrophages and promotes rupture.

Organizational role of macrophages in IAs

Macrophage behavior in IAs is highly dependent on signals from surrounding immune cells, perhaps even more than in CCMs, bAVMs, and dAVFs. ¹⁴ Predominance of the M1 phenotype depends on neutrophil infiltration, while inhibition of neutrophils via selective CXCL1 blockade increases the M2/M1 ratio, implicating pro-inflammatory signals from neutrophils in aneurysm rupture. Mechanisms of neutrophilic inflammation are also involved in aneurysm progression, with neutrophilic extracellular trap (NET) activity and other neutrophil-derived factors favoring IA growth, partially by promoting the polarization of macrophages to a pro-inflammatory state as they participate in NET uptake and breakdown.^146,147 The initial recruitment of neutrophils occurs as a consequence of macrophage-mediated chemokine secretion.^148,149 Mast cells are also observed in ruptured aneurysms, but they localize to IAs in response to macrophage signaling.^23,150 When present in experimentally-induced IAs, mast cells increase chronic inflammatory processes in aneurysm walls and augment local activation of MMP2 and iNOS through degranulation. ¹⁵¹ It is speculated that cross-talk between M1-polarized macrophages and mast cells within IAs accelerates instability and promotes rupture. ²³ T-lymphocytes are also noted in IAs, where they play an important role in ECM remodeling and smooth muscle damage. ¹⁵² However, T-lymphocytes are neither necessary nor sufficient for IA formation, so IAs should continue to be viewed as the product of a macrophage-mediated chronic inflammatory process.^131,153

Dural arteriovenous fistulae

Underlying abnormalities in dAVFs

Pathological anastomoses between meningeal arteries and venous sinuses or cortical veins characterize dAVFs. These abnormal connections are generally idiopathic but can be precipitated by venous sinus thrombosis, coagulopathies, traumatic brain injury, or intracranial surgery.^{154 –156} Anatomically, these fistulized vessels commonly reside within the dural leaflets that surround a venous sinus channel. ¹⁵⁵ They arise from either re-canalization and enlargement of intrinsic arteriovenous connections that perforate the dura or from reactive angiogenesis and neovascularization. ¹⁵⁷ While often described alongside bAVMs, dAVFs are a distinct entity because unlike bAVMs, dAVFs have their arterial supply from dural vessels and do not have a parenchymal nidus.^154,155 In contrast to bAVMs, CCMs, and IAs, minimal evidence suggests a familial or genetic component to the development of dAVFs, with supporting data only present in the context of coagulopathy-inciting mutations promoting secondary dAVF development. ¹⁵⁸ Clinically, dAVFs may present with an array of symptoms ranging from the more benign pulsatile tinnitus, ophthalmologic symptoms, and headaches to severe manifestations including intracranial hemorrhage. ¹⁵⁹

Prevailing hypotheses of dAVF formation

Compared to the majority of intracranial cerebrovascular lesions, far less work has implicated inflammation in the development of dAVFs. Hypotheses from the 1970s proposed that localized inflammation associated with venous sinus thrombosis induced neovascularization and fistula formation, and sinus thrombosis models continue to be used in experiments assessing dAVFs.^160,161 Mechanistically, sinus thrombosis initiates a pathologic sequence from clot organization to development of dural blood supply to the patent portion of the venous sinus, leading to progressive hypertrophy and enlargement of a fistula. ¹⁶¹ Subsequent studies revised this hypothesis and provided several lines of evidence indicating that ischemia induced by cerebral venous hypertension rather than inflammation was the main driver of dAVF formation. ¹⁵⁷ In histological analyses, macrophages and other leukocytes are not noted in experimentally-induced dAVFs.^156,162 Though the mechanisms of dAVF development remain controversial, many suggest that hypoxia secondary to chronic local hypoperfusion from elevated sinus pressure drives the formation of dAVFs via angiogenic factors despite limitations in the animal models used to generate these findings. ¹⁶³

Macrophage involvement in dAVF initiation

Though a body of work implicating ischemia-driven angiogenesis in the development of dAVFs, recent evidence supports revisiting early hypotheses of inflammation. ¹⁵⁴ Inflammatory mediators are known to induce cerebral venous thrombosis, leading to focal hypercoagulable states and endothelial injury.^164,165 The primary innate immune mediators of this process are neutrophils and microglial cells, with peripheral macrophages contributing by providing microparticles with thrombogenic tissue factors that induce immunothrombosis. ¹⁶⁶ Once present, macrophage signaling and activity is sustained for 1 to 2 weeks in rodent models of venous sinus thrombosis, which aligns with the timeframe of experimental dAVF induction and development.^157,167 Another line of evidence that has brought the inflammatory hypothesis of dAVF development to the fore of discussion is the influence of the meningeal immune system. ¹⁵ Now recognized as an essential tertiary structure heavily involved in CNS disease, the role of this complex immune landscape is contingent upon innate populations of CNS macrophages and communications with peripheral immune cells. ¹⁶⁸ The abnormal vascular anastomoses characterizing dAVFs may reflect compensatory consequences of immune surveillance throughout the meningeal immune system. ¹⁵ While these findings suggest that macrophage involvement in dAVFs may have been overlooked and that timepoint-specific investigations are warranted, it is unlikely that macrophages serve a central role in these lesions as is seen in IAs, CCMs, and bAVMs, especially given the heterogeneous etiologies of dAVFs. Further studies in this area are necessary to distinguish intrinsic CNS macrophage involvement in these lesions from peripheral macrophages.

Macrophage-based diagnostics in cerebrovascular diseases

The presence of macrophages in structural cerebrovascular diseases allows for their use in diagnosis and prognostication using both imaging and non-imaging methods of macrophage identification.

Imaging

Catheter-based digital subtraction angiography (DSA) remains the gold standard for anatomic characterization of IAs, dAVFs, and bAVMs, while MRI with susceptibility weighted imaging (SWI) or gradient echo sequences (GRE) is the optimal imaging modality for CCMs.^169,170 Though risk stratification is possible with these imaging techniques by assessing lesion size, location, morphology, and growth between serial imaging sessions, these modalities are often ineffective at identifying unstable lesions at high risk of hemorrhage or symptomatic presentation with a single study. ¹⁷¹ Given the correlation between inflammation and rupture risk in IAs, DeLeo et al. synthesized myeloperoxidase (MPO)-specific MRI contrast (di-5-hydroxytryptamide of gadopentetate dimeglumine) that allows for indirect measurement of neutrophil and macrophage activity in experimentally-induced IAs using T1-weighted MRI sequences. ¹⁷² This technique revealed that IAs with LPS-induced inflammation had substantially elevated enhancement compared to controls. Building on these findings, later studies refined this imaging technique to compensate for irregular blood flow and improve imaging sensitivity. ¹⁷³ While this imaging strategy was developed for IAs, the presence of active inflammation in bAVMs and CCMs suggest that broader application of this technique can be considered.

Capitalizing on the role of macrophages in cerebrovascular lesions, a macrophage-specific imaging technology was subsequently developed using ferumoxytol. ¹⁷⁴ Ferumoxytol is a negatively-charged iron nanoparticle with a carbohydrate coating cleared primarily by the reticuloendothelial system of macrophages, and provides a reliable surrogate metric for inflammation and aneurysm instability.^174,175 Pilot studies of this technology identified that aneurysms with ferumoxytol uptake in the first 24 hours resulting in T2 GRE signal changes had an elevated probability of rupture within 6 months, indicating that this technique was able to identify IAs requiring prompt treatment. ¹⁷⁴ With a growing body of research supporting the use of non-steroidal anti-inflammatory (NSAID) therapy such as Aspirin to slow aneurysm growth, another benefit of this imaging technology is its use to measure response to NSAID therapy.^176,177 Despite the clinical potential of ferumoxytol, there are limitations to its widespread application. First, this imaging strategy is limited in the workup of small lesions, which comprise the majority of IAs detected in the population, and large IAs are likely to be treated irrespective of findings. ²⁶ Cellular effects of ferumoxytol must also be considered, especially in the context of inflammatory cerebrovascular lesions. In tumor-associated macrophages, therapeutic ferumoxytol is phagocytosed by M2 tumor-associated macrophages and promotes polarization to the M1 state to enable targeting and neutralization of malignant cells.^178,179 However, as discussed in this review, polarization towards the M1 phenotype is harmful. ²³ Because the effect of ferumoxytol in macrophage polarization has not been studied specifically in cerebrovascular diseases, the effect of this compound on macrophage polarization should be studied prior to widespread clinical implementation of this technique. Finally, like MPO-specific imaging, the overwhelming majority of studies supporting the use of ferumoxytol-based MRI have been conducted in IAs, though some studies have explored its use in other cerebrovascular conditions. In a proof-of-concept study, the feasibility of imaging macrophages in bAVMs with ferumoxytol-enhanced MRI was performed in 5 patients, and demonstrated that accurately measuring macrophage presence requires a later time point compared to IAs (5 days vs 24 hours) to compensate for high nidal blood volume and surrounding vascular density to accurately measure macrophage presence. ¹⁸⁰ Ferumoxytol has also been used to monitor residual and recurrent pediatric bAVMs, but was only employed to compare visualization to CTA and DSA.^181,182 In studies applying post-ferumoxytol MRI imaging to CCMs, no signal changes were seen across selected timepoints, though the investigated timepoints of 25 minutes and 24 hours limit the detection of late signal change noted in bAVMs. ¹⁷⁵ Nevertheless, ferumoxytol carries tremendous potential for monitoring in situ inflammation as well as detecting response to medical therapy.

Plasma and serum biomarkers

Circulating chemokines or cytokines associated with peripheral monocytes have previously been suggested as biomarkers to evaluate injury severity and response to therapy. ¹⁸³ In bAVMs, secretory products of macrophages including VEGF, pro-inflammatory cytokines, and S100 proteins are highly expressed in the nidus of bAVMs and have corresponding elevated levels in the plasma.^102,184,185 Noncoding RNAs (NcRNAs) such as MiR-18a, which downregulates MMP expression, also deserve consideration as clinical biomarkers relating to macrophage-mediated functions. ¹⁸⁶ Similarly, CCMs also have plasma and serum products that can be utilized as clinical biomarkers. These include VEGF, pro-inflammatory cytokines (namely TNF-α, IFN-γ, IL-1β, IL-2, IL-6), and MMPs for monitoring progression and hemorrhage risk.^28,185,187

Macrophage-related biomarkers may also help estimate the natural history of pathologies acquired secondary to cerebrovascular stressors, whether sustained or acute/subacute. In IAs, complement cascade proteins, complement activation end products, MMPs, IL-6 quotient (ratio of cerebrospinal fluid IL-6 levels to serum levels), MCP-1, TNF-α, and VEGF are biomarkers that may aid in either initial detection of IAs or in prediction of rupture.^{188 –191} Interestingly, the use of VEGF as a biomarker for IAs is only predictive in male patients and not in females. ¹⁹¹ Due to the apparent lack of macrophages in intracranial dAVFs, measurement of macrophage-related products is not useful for these lesions, however, C4BPA and C1QA have been demonstrated as biomarkers for spinal dAVFs and future studies should identify similar biomarkers for intracranial dAVFs. ¹⁹²

Therapeutic targeting of macrophages

Standalone medical therapy or medical therapy as adjuncts to treat cerebrovascular lesions currently includes non-steroidal anti-inflammatory (NSAID) therapy, statin therapy, or other medications to promote lesional stability and minimize the risk of rupture.^193,194 Building on these treatments, we explore medical therapies targeting macrophages involved in these diseases, focusing on pre-rupture intervention to mitigate disease progression and hemorrhage.

Anti-inflammatory agents

Medications with anti-inflammatory mechanisms of action provide the most obvious manner of modulating macrophages. Aspirin and statins provide a convenient starting point for anti-inflammatory intervention in these diseases since these are well-tolerated medications with low risk profiles and are already in wide-spread clinical use for a variety of other conditions. Aspirin is known to prevent IA rupture in animal models via modulation of the COX-2 pathway and the microsomal PGE2 synthase-1 pathway, which has generated momentum supporting trials of aspirin to reduce IA progression and rupture.^195,196 Aspirin also lowers the macrophage burden in a manner that can be followed using ferumoxytol-based imaging.^{197 –199} Because macrophage activity promotes progression of bAVMs and CCMs towards a hemorrhagic phenotype, aspirin may also provide stabilizing effects on these other cerebrovascular lesions. Statins, widely used as lipid lowering drugs due to their inhibition of HMG-CoA reductase, have pleiotropic effects on other cell types including smooth muscle cells, endothelial cells, and macrophages. In macrophages, statins abrogate MMP expression, reduce VEGF production, and mitigate local inflammation.^200,201

Aside from using currently available pharmacologic agents with broad mechanisms of action, selective targeting of macrophage-mediated inflammation with biologic or molecular therapies has tremendous promise. Previous studies indicate that knockdown of the well-studied inflammatory transcription factor NF-κB reduces IA formation in rats, and similar approaches may reduce macrophage-mediated inflammation in bAVMs and CCMs.^139,202 Blockade of cell surface receptors including TNFR, IL1R, and TLR upstream of NF-κB provide indirect reduction of macrophage-mediated inflammation.^202,203 Another therapeutic strategy involves targeting downstream effects of macrophage-mediated inflammation. For example, selective MMP inhibition is achievable using drugs including tolylsam, tetracyclines, and imidapril.^16,125,204 Scavenging the ROS produced by M1 macrophages with the superoxide anion scavenger thymoquinone may also reduce the harmful effects of classically-activated macrophages. ²⁰⁵

Polarization state modulation

Macrophage reprogramming has been well-described in tumor-associated macrophages, where M2 macrophages are reprogrammed to the M1 phenotype to appropriately recognize and attack malignant cells. ²⁰⁶ Selective macrophage polarization may provide therapeutic benefits in cerebrovascular diseases depending on the underlying pathology. In IAs, it is well described that reducing the M1/M2 ratio minimizes the risk of IA progression and rupture. ²³ In contradistinction, decreasing the M1/M2 ratio in CCMs and bAVMs may have harmful effects given that M2 macrophages promote aberrant angiogenesis and are associated with hemorrhagic events.^18,100 The proper M1/M2 balance in bAVMs and CCMs remains unclear and experimental characterization remains necessary. Nevertheless, available exogenous agents including IL-10 DNA plasmid-based nanoparticles, miRNA-181a, and other aptamers can reprogram macrophages from an M1-polarized state to an M2-polarized state.^14,207

Inhibition of recruitment

Outright prevention of macrophage recruitment and infiltration provides an additional interventional strategy. Deletion of MCP-1 or complete depletion of macrophages using clodronate liposome treatment hinders disease progression in preclinical studies, particularly in IAs where the infiltration of macrophages through the endothelium into the vessel wall is correlated with lesion growth.^136,208 Activation of sphingosine-1-phosphate receptor type 1 (S1P₁) may further inhibit infiltration and recruitment of macrophages by strengthening the endothelial barrier and preventing trans-migration. ²⁰⁹ Blockade of CXCL1 prevents neutrophil infiltration, yet results in reduced IA development in preclinical models by promoting the M2 phenotype in local macrophages. ²¹⁰ Inhibition of migratory factors for B-lymphocytes and T-lymphocytes may have therapeutic value, particularly in CCMs where their role is most influential to macrophages. More broadly, phosphoiesterase-4 (PDE-4), a cyclic adenosine monophosphate-specific enzyme, can be targeted with ibudilast to both downregulate local inflammation and suppress macrophage infiltration. ²¹¹ Though these techniques of recruitment inhibition have been promising preclinically, they carry off-target effects due to their systemic delivery and must be modified prior to human applications. ²¹²

Blood product scavenging

The presence of hemorrhagic blood products and their derivatives influences the recruitment of peripheral macrophages to bAVMs and CCMs. The arrival of macrophages into the tissue mitigates free heme toxicity in a CD163-mediated fashion. ²¹³ However, reducing free heme and blood products by administering exogenous agents may prevent macrophage infiltration into the site of hemorrhage. For example, haptoglobin has been shown to effectively scavenge free heme and blood products in preclinical subarachnoid hemorrhage models. ²¹⁴ The use of iron chelators including deferoxamine, similarly studied in subarachnoid hemorrhage models, may assist in reducing post-hemorrhage recruitment of peripheral macrophages to bAVMs and CCMs.

Preconditioning-based modulation

Preconditioning is limited exposure to a sublethal stimulus that increases resilience to later exposures to the same stimulus, and studies suggest this process alters macrophage polarization. ²¹⁵ Methods of preconditioning include exposure to transient hypoxia, administering volatile anesthetics, and using LPS-based mechanisms. All of these preconditioning techniques promote a series of cascades that induce neuroprotective post-translational modifications of proteins.^216,217 While preconditioning responses occur in virtually all cells of the central nervous system, immune cells seem to play a particularly critical role because depleting microglia abolishes preconditioning responses.^218,219 Additionally, reduced inflammation and initiation of vascular remodeling have been described as components of the phenotypic response to preconditioning. ²¹⁸ A recent study assessing the role of ischemic preconditioning on peripheral macrophages identified an elevated M2/M1 macrophage ratio in the brain following experimental ischemic injury, as well as augmented expression of M2-associated genes. ²¹⁸

Endovascular therapy adjuncts

Historically, cerebrovascular lesions were treated with open surgical resection or repair when intervention was necessary. ²²⁰ However, for bAVMs, IAs, and dAVFs, endovascular treatment comprises the majority of modern intervention, while CCMs continue to be treated surgically or with stereotactic radiosurgery. Coiling is a common treatment for IAs, and is sometimes used in dAVFs depending on morphological features. One drawback of coil embolization in IAs is the high rate of lesion recurrence. ²²¹ Interestingly, specimen analyses of previously-coiled brain aneurysms revealed macrophages interspersed within the intra-aneurysmal organizing thrombus. ²²² The evolution of macrophage populations in treated aneurysms contributes to their healing. Initially, a higher M1 macrophage population is present in the organizing thrombus. This is followed by an M2 predominance in the subsequent months, which accelerates healing. ²²³ While previous efforts to develop biologically-active coils yielded underwhelming results and led their abandonment in clinical practice, recent efforts have explored augmenting macrophage recruitment to promote aneurysmal healing.^224,225 Indeed, studies have demonstrated that MCP-1 coated coils increase the rate of aneurysm healing through monocyte recruitment in experimental aneurysms, with IL-6 and osteopontin serving as key regulators.^226,227

Concluding remarks

Sustained investigation into the role of macrophages and other inflammatory mediators in cerebrovascular pathologies may reveal diagnostic and therapeutic strategies that revolutionize clinical approaches to these diseases.^6,14 The current evidence supporting cerebrovascular diseases as dynamic, immune-mediated conditions heavily influenced by peripheral macrophages should galvanize the next wave of studies to harness modern techniques of high-throughput sequencing, gene editing, and advanced imaging strategies to characterize the pathophysiology of these lesions in unprecedented detail. ⁵³ Despite the potential future for macrophage-based diagnostics and therapeutics in clinical practice, current limitations of these methods must be addressed. Functional differences between peripheral macrophages, non-parenchymal CNS macrophages, and microglia must be carefully considered in future studies by using cell-specific markers, given our growing understanding of the different ontogeny and respective niches of these macrophage subtypes. Similarly, study of peripheral macrophages should consider the role differing peripheral monocyte sources, namely the skull marrow, have on the development of CNS disease in contrast to other sites of hematopoiesis. ⁴⁴ Another limitation resides in the fact that most experimental studies of peripheral macrophages in cerebrovascular structural abnormalities have been performed in experimental IAs. This may reflect the relative population burden of ruptured aneurysms, or the paucity of suitable animal models for some cerebrovascular lesions, especially dAVFs. ²²⁸ Developing high-quality animal models for each cerebrovascular lesion should be prioritized.

In the context of our discussion, we deliberately focused on the most prevalent forms of each disease in our characterization and description of macrophage activity. While this addresses the overwhelming majority of patients suffering from these diseases, it precludes consideration of scenarios where macrophage behavior deviates from the norm. For example, bAVMs in patients with HHT may have different patterns of monocyte recruitment and macrophage activation given the impaired monocyte migration seen in patients with this disease. ²²⁹ Further, overlap between diseases, such as prenidal aneurysms in bAVMs, suggests common mechanisms of progression between diseases of cerebrovascular development and acquired cerebrovascular diseases. The need for additional research in these subgroups of patients provides exciting avenues for potential therapies in high-risk populations.