Macrophage in Sporadic Thoracic Aortic Aneurysm and Dissection: Potential Therapeutic and Preventing Target

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening cardiovascular disorder lacking effective clinical pharmacological therapies. The underlying molecular mechanisms of TAAD still remain elusive with participation of versatile cell types and components including endothelial cells (ECs), smooth muscle cells (SMCs), fibroblasts, immune cells, and the extracellular matrix (ECM). The main pathological features of TAAD include SMC dysfunction, phenotypic switching, and ECM degradation, which is closely associated with inflammation and immune cell infiltration. Among various types of immune cells, macrophages are a distinct participator in the formation and progression of TAAD. In this review, we first highlight the important role of inflammation and immune cell infiltration in TAAD. Furthermore, we discuss the role of macrophages in TAAD from the aspects of macrophage origination, classification, and functions. On the basis of experimental and clinical studies, we summarize key regulators of macrophages in TAAD. Finally, we review how targeting macrophages can reduce TAAD in murine models. A better understanding of the molecular and cellular mechanisms of TAAD may provide novel insights into preventing and treating the condition.


Introduction
Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening cardiovascular disease with a high mortality rate.Although, the annual incidence of TAAD remains as low as 6 to 16 per 100,000, and it accounts for 1%-2% of all death according to population-based studies [1].The degradation of elastic fibers and medial degeneration lead to progressive weakening and dilation of the thoracic aorta, which increases the risk of acute aortic dissection or rupture [2].Surgical repair still remains a guideline-recommended therapy for TAAD [1].However, open surgical repair for TAAD is a challenge for both medical staff and patients themselves.An effective drug is still lacking to prevent or even reverse TAAD in clinical practice.
The etiology for TAAD is still elusive, with the participation of both genetic and acquired risk factors [3].According to the latest guidelines, TAAD is classified into three main categories including hereditary, sporadic, and bicuspid aortic valve (BAV)-associated TAAD [1].Hereditary TAAD accounts for 20% of TAAD and refers to those TAAD patients with clear genetic mutations including Marfan's syndrome, Loyes-Ditez syndrome, and others [4].Genetic disorders involve genes encoding various components of the TGF-β signaling cascade (FBN1, TGFBR1, TGFBR2, TGFB2, TGFB3, SMAD2, SMAD3 and SKI) and the smooth muscle contractile apparatus (ACTA2, MYH11, MYLK, and PRKG1) [3].These genetic dysfunctions could directly lead to vasculopathies.In addition, BAV-associated TAAD is a compounding pathological process with both participation of hereditary (i.e., NOTCH1, ACTA2, MAT2A, SMAD6, and LOX ) and acquired hemodynamics factors [5].Hereditary and BAV-associated TAAD have previously been reviewed and thus not considered in this review [4,6].The TAAD referred to in this article focuses on sporadic TAAD without a hereditary basis or BAV.
The molecular mechanisms of TAAD are complicated biological processes with the involvement of various cell types including immune cells [7].Macrophages are one of the major immune cells increased in TAAD both in human and murine models.Aggregation of macrophages is critical for thoracic aortic weakening, dilation, aneurysm, and dissection.This review will first introduce the important role of inflammation and immune cell infiltration in the pathogenesis of TAAD.Then, we will systematically summarize the role of macrophages in thoracic aortic dilation, aneurysm, and dissection.We further focus on how to regulate macrophages in TAAD.Finally, we will discuss the translational prospect of targeting macrophages pharmacologically to reduce TAAD.mainly composed of endothelial cells (ECs).Tunica media forms the main structure of thoracic aortic walls, comprising smooth muscle cells (SMCs), elastic fibers (fibrillin-1 and elastin) and many other extracellular matrix (ECM) proteins.The tunica adventitia is mainly composed of thick collagen fibers with fibroblasts to maintain its integrity, which form the external cuff.The adventitia also contains arterioles, nerves, and is a major source of aortic immune cells.Of note, the vasa vasorum is mainly found in the adventitia, but also exists in the outer layers of the media to feed the aorta.This vascular bed is actually the main source of the inflammatory cells in the outer wall media/adventitia [9].Crosslinks of elastic fiber-ECM, SMC-ECM and SMC-SMC maintains normal strength of thoracic aortic walls, suppress immune cell infiltration, and prevent the thoracic aorta from weakening.
The pathogenesis of TAAD is a progressive biological process with the involvement of various aortic cell types and components including SMCs, ECs, myofibroblasts, immune cells and the ECM.Medial degeneration and degradation of elastic fibers are basic morphological and pathological characteristics of TAAD.Elastic fiber-ECM, SMC-ECM, and SMC-SMC crosstalk are destroyed due to SMC dysfunction, phenotypic switching, and ECM degradation.During these pathological processes, immune cells play a pivotal role.When immune cells from the adventitia infiltrate into the media-intima, it may lead to medial inflammation, degeneration, SMC phenotypic switching, dysfunction, and ECM disruption.In turn, inflammatory cells could recruit secondary to ECM disruption and initiate a positive feedback loop (Fig. 2).These biological processes further contribute to weakness and vulnerability of thoracic aortic walls, leading to progressive thoracic aortic dilation, aneurysm, and dissection.
Parallel with findings in humans, thoracic aortic specimens of TAAD mice models are also characterized by inflammation and immune cell infiltration.Currently, TAAD mice models are established mostly by βaminopropionitrile (BAPN) administration, direct elastase treatment to thoracic aorta, angiotensin Ⅱ (Ang II) or combined administration of BAPN and infusion of Ang II [28].The inflammatory response is significantly activated in thoracic aortic specimens of BAPN induced TAAD mice, marked by an increase of IL-1β, IL-3, IL-5 and IL-18 [29][30][31][32], with significant recruitment of immune cell in the adventitia [33,34].In addition, inflammatory response and immune cell recruitment were also observed in Ang II induced TAAD [35][36][37].

Origin of Macrophage in the Aortic Wall
Although different types of immune cells were increased in thoracic specimens of TAAD, macrophages are one of the most abundant types [27,34,38,39].
Macrophages found in the thoracic aortic wall are mainly derived from two main sources.Most macrophages in the aneurysmal thoracic aortic wall derive from circulating monocytes, which are produced from bone barrow and mobilized from peripheral reservoirs such as the spleen [40][41][42].Transplantation of bone marrow cells expressing green fluorescent protein revealed that a proportion of macrophages in the aortic adventitia originated from bone marrow-derived monocytes [43].Besides circulating monocytes, aortic macrophages might also develop from embryonic precursors and early postnatal circulating monocytes.This group of macrophages are known as tissue-resident macrophages, which are independent from bone-marrow progenitors and are self-maintained and selfdeveloped during adulthood [41,42].Updated investigations have added evidence that tissue-resident macrophages might also originate from multipotent stem cells, which was identified in the media and adventitia of TAAD thoracic aorta expressing macrophage marker CD68 [44,45].

Classification of Macrophage in TAAD
Macrophages have been classified into several different phenotypes over the past two decades, mainly including M1 and M2 macrophages [46].M1 macrophages are also known as pro-inflammatory macrophages with production of proteolytic enzymes and pro-inflammatory cytokines.In comparison, M2 macrophages have an anti-inflammatory role through promoting ECM remodeling and tissue repair.
Macrophage subgroups are also identified in the thoracic aorta of BAPN administrated TAAD mice.A total of three macrophage subgroups were identified including 'resident-like', 'antigen-presenting' and 'proinflammatory' subsets [34].In detail, 'resident-like' macrophages highly expressed Lyve1, Cx3cr1, F13a1, Lyve1, and Gas6.This group of macrophages were thought to be located residentially in the adventitia and may develop and self-renew independently of CCR2-mediated monocyte recruitment [49,50].'Antigen-presenting' macrophages highly expressed Cd74, MHC II genes.This may indicate that this macrophage subcluster is involved in presenting antigens.'Pro-inflammatory' macrophages highly expressed pro-inflammatory chemokines and M1-polarized macrophage markers.This subcluster is similar to M1 macrophages and may infiltrate and drive inflammatory responses.Subclusters, classifications and definitions of these new macrophage subgroups might provide novel insights into understanding the pathogenesis of TAAD.

Infiltration into the Media Layer of Thoracic Aortic Wall
In the pathological process of TAAD, macrophages infiltrate from the adventitia into the media and participate in the inflammatory response, ECM degradation and medial degeneration [51] (Fig. 4).With the assistance of splenic B lymphocytes, monocytes mobilize from the spleen and infiltrate into the aortic walls [52,53].Subsequently, CCL2 and IL-6 produced by adventitia fibroblasts initiate monocyte recruitment and differentiation into macrophages, followed by stimulation of fibroblast proliferation and more production of CCL2 and IL-6 to form a positive feedback loop [54].In the inflammatory microenvironment of the aortic media, macrophages further accumulate and activate with the assistance of inflammatory cytokines, chemokines, and other biochemical factors such as reactive oxygen species [55].Although tissue-resident macrophages only occupy a small proportion, their role should not be overlooked and requires future investigation.

Inflammatory Response
Macrophage infiltration and accumulation initiates an amplified inflammatory response and ECM degradation, leading to disintegration and destruction of elastic fiber-ECM, SMC-ECM, and SMC-SMC crosslinks.Macrophages are prominent inflammatory signal senders on SMC in TAAD through CXCL, CCL, TNF, IFN-II, IL-16 and complement pathways [56,57].Consolidated evidence has shown that SMC is a major target cell of macrophages in TAAD.Cell-interaction analysis has also highlighted increased communication between macrophages and T cells in TAAD tissues, indicating that macrophages might drive further inflammatory responses on SMCs through other intermediary cells [58].
Pro-inflammatory macrophages could both produce and respond to inflammatory mediators, therefore further invade into thoracic aortic walls, and trigger inflammatory response through positive feedback loops [59][60][61].One possible theory proposes that aneurysmal and dissected thoracic aortic walls are the main sources of CXCL1, which could further induce neutrophils to produce IL-6 [62].Increased IL-6 might then induce pro-inflammatory macrophages to secrete CCL2, which further promotes macrophage activation and invasion [63].

ECM Degradation
ECM in the aortic media plays a distinct role in preventing TAAD as well.When ECM degrades, thoracic aortic walls may lose their integrity, and begin to weaken and dilate.Matrix metalloproteinases (MMPs) and ADAM metallopeptidase with thrombospondin type 1 motifs (ADAMTS) are known to have the capability to degrade various components of ECM for aortic vulnerability [64].Importantly, MMPs and ADAMTSs were mainly expressed in macrophages [32,38,[65][66][67].This evidence supports the idea that macrophage-derived MMPs and ADAMTSs may exert an important effect on ECM degradation.

Regulators of Macrophage Accumulation and Infiltration
Since macrophages play such a pivotal role in the pathogenesis of TAAD, understanding the regulatory mechanism for macrophages might provide novel insights into macrophage-based therapy for TAAD [68].Over the decades, a series of regulators of macrophages have been identified as being closely associated with the pathogenesis of TAAD.These regulators are mainly classified into three categories.The first type of regulators affects macrophage accumulation and infiltration, which initiate further biological process.The remaining two types of regulators influence macrophage functions through modulating proinflammatory phenotype and MMPs expression (Table 1, Ref. [11,29,32,34,35,63,65,).
Previous investigations have uncovered some mechanisms that are involved in macrophage accumulation, infiltration, driving the inflammatory response.Macrophage infiltration was more significant in TAAD patients with atherosclerosis [94].Elevated plasma levels of LDL cholesterol promoted Ang II-induced TAAD through enhancing macrophage infiltration [95].Nicotine free base promoted TAAD progression in SMC specific TGFBR2 knockout mice, whose effect was sensitized by BAPN co-stimulation [96].These results might partly explain why atherosclerosis, hypercholesterolemia and tobacco use might serve as established risk factors for TAAD [97].However, these studies only provided observations on this phenomenon and did not provide in-depth mechanisms of how macrophages differentiate and infiltrate into thoracic aortic walls.
Exogenous administration of specific drugs could also reduce the pathologic changes in different mouse models of TAAD through reducing macrophage infiltration and inflammation.Pretreatment of IL-1R antagonist anakinra reduced macrophage infiltration and attenuated TAAD formation induced by elastase [29].Similarly, calcium channel blocker azelnidipine reduced BAPN-induced TAAD through anti-inflammatory effects [107].Several investigations focus on in-depth mechanisms of how macrophage infiltration is reduced.The glycolytic enzyme pyruvate kinase M2 activator TEPP-46 markedly attenuated the progression of TAAD induced by BAPN through inhibition of macrophage infiltration associated with the NOD-like receptor family and NLRP3 inflammasome [55].

Regulators of Pro-Inflammatory Phenotype and Inflammatory Response
When macrophages accumulated and infiltrate, the pro-inflammatory phenotype and associated inflammatory response are essential to participate in the pathological process of TAAD.Hypertension is the most important risk factor for TAAD [108,109].A previous study has shown that hypertensive TAAD patients tended to have more proinflammatory macrophages in the adventitia and media of thoracic aorta [110].This result may indicate that hypertension might promote a pro-inflammatory phenotype of macrophages, with underlying mechanisms needing further investigation.
A pro-inflammatory phenotype of macrophages in TAAD is mainly regulated through affecting production of inflammatory factors and the capability of migration and invasion of macrophages [111].Several pro-inflammatory regulators have been identified over the past two decades.Single-nuclear RNA sequencing and genome-wide association studies have identified LRP1 as a key potential regu-lator of inflammation in macrophage of TAAD [75].In experimental investigations, overexpression of core circadian clock gene BMAL1 induced a pro-inflammatory response in cultured macrophages [76].TGF-β induced severe inflammatory response through enhancing macrophage invasion [77].Silencing of ANGPTL8 and AT1R decreased inflammatory factors in macrophages including IL-1β, IL-6, MCP-1 and TNF-α [11,78].In vivo, myeloid-specific knockout of TGF-β or NEU1 reduced macrophage proinflammatory functions and ameliorated TAAD induced by BAPN [79,80].ADAMTS1-deficient macrophages exhibited low activity of the inflammatory response through abrogated migration capacity in BAPN mice [81].
Notably, metabolic reprogramming is required for the proper polarization and function of activated macrophages [112].Similar to the Warburg effect of tumor cells, proinflammatory M1 pro-inflammatory macrophages increase glucose consumption, lactate release and decreased oxygen consumption rate.However, M2 anti-inflammatory macrophages are characterized by the employment of oxidative glucose metabolism pathways [113].Besides, fatty acid, vitamin and iron metabolisms are also closely associated with macrophage polarization [114].Using untargeted metabolomics of clinical TAAD specimens and BAPNinduced mice model, C18-ceramide was identified to be increased through the de novo synthesis pathway and promoted macrophage pro-inflammatory phenotype through the NLRP3-caspase 1 pathway [86].Similarly, succinate, tryptophan, kynurenine, quinolinic acid and kynurenineto-tryptophan ratio were also identified to be increased in TAAD [87,88].Knockdown of the key synthetic enzyme of succinate OGDH or kynurenine pathway enzyme kynureninase could reduce the expression of inflammatory factors in macrophages.
Regulation of the pro-inflammatory phenotype in macrophages through pharmacological intervention has been proven effective in attenuating TAAD in vivo.Targeting pro-inflammatory Il1rn + /Trem1 + macrophage subpopulations through mLR12 could significantly reduce thoracic aortic rupture rate in BAPN-administrated mice [34].Dexamethasone treatment suppressed NF-κB signaling pathway in macrophages and further reduced the inflamma-tory response, immune cell infiltration and incidence of TAAD in BAPN mice [115].Selective mineralocorticoid receptor antagonist eplerenone protected mice from BAPN-induced TAAD through decreasing TNFα and IL-6 in macrophages [116].An in-depth understanding of proinflammatory macrophages might broaden our horizon on anti-inflammatory therapy for TAAD.

Regulators of Macrophage-Based ECM Degradation
ECM degradation is another critical mechanism involved in TAAD development, which has been reported to be under the control of specific molecules.For example, macrophage-derived legumain is essential for ECM degradation.Macrophage-specific deletion of legumain (LGMN) alleviated BAPN-induced thoracic aortic dilation, aneurysm, and dissection in mice [89].Deficiency of urokinase-generated plasmin protected against media destruction and aneurysm formation by reducing the activation of pro-MMPs [90].Inhibition of endogenous ceramide synthesis in macrophages by myriocin, attenuated BAPNinduced TAAD in mice through reducing macrophage inflammation and expression of MMPs [86].

Future Prospective: Targeting Macrophage to Reduce TAAD
Currently, no pharmacological therapy has proven effective in preventing and treating TAAD patients in clinical practice.Since macrophages play an essential role in TAAD and could be regulated through versatile mechanisms, it is worthwhile to investigate whether targeting macrophages could reduce TAAD.As mentioned above, several anti-inflammatory drugs may have protective effects against murine TAAD by reducing macrophage infil-

Conclusions
Activation of the inflammatory response and immune cell infiltration are essential hallmarks of TAAD.Macrophages play a pivotal role in the formation and progression of TAAD.Accumulation and infiltration of macrophages might drive inflammatory responses in the aortic media, produce MMPs to degrade ECM and further lead to thoracic aortic dilation, aneurysm, and dissection.Importantly, macrophages are regulated through accumulation, pro-inflammatory response, and ECM degradation.Targeting macrophages has promising translational value for preventing and treating TAAD in the future.

Fig. 1 .
Fig. 1.Normal structure of thoracic aortic wall.Normal thoracic aortic walls are composed of three layers including tunica intima, tunica media and tunica adventitia.The tunica intima is mainly composed of endothelial cells.The tunica media mostly contains smooth muscle cells, elastic fibers, and other ECM components.The tunica adventitia is mainly composed of thick collagen fibers, fibroblast, arterioles, nerves, and various types of immune cells.Crosslinks of elastic fiber-ECM, SMC-ECM and SMC-SMC maintains normal strength of thoracic aortic walls and prevent thoracic aorta from dilation, aneurysm, and dissection.ECM, extracellular matrix; SMC, smooth muscle cell.

Fig. 2 .
Fig. 2. The basic pathophysiology of thoracic aortic aneurysm and dissection (TAAD).Medial degeneration and degradation of elastic fibers are basic hallmarks of TAAD.Elastic fiber-ECM, SMC-ECM, and SMC-SMC crosstalk are destroyed due to SMC dysfunction, phenotypic switching, and ECM degradation.When immune cells infiltrate into the media, it may lead to medial inflammation, SMC phenotypic switching, SMC dysfunction and ECM disruption.These biological processes form a positive feedback loop.SMC, smooth muscle cell; ECM, extracellular matrix.

Fig. 3 .
Fig. 3. Origin and classification of macrophages in TAAD thoracic aortic wall.Macrophages within thoracic aortic walls originate mainly from two sources.One main origin of macrophages is circulating monocytes.Another source is embryonic precursors and early postnatal circulating monocytes.Macrophages from human aneurysmal thoracic aortas are classified into eight subclusters including M1 like 1, M1 like 2, M1 like 3, M2 like 1, M2 like 2, IFN-response, remodeling, and proliferating macrophage.Macrophages of mice aneurysmal thoracic aorta induced by BAPN are divided into three subgroups including resident-like, antigen-presenting and proinflammatory macrophage.TAAD, thoracic aortic aneurysm and dissection; IFN, interferon; TNF, tumor necrosis factor; BAPN, β-aminopropionitrile; MHC, major histocompatibility complex; TAAD, thoracic aortic aneurysm and dissection.

Fig. 4 .
Fig. 4. The Role of Macrophages in the Pathogenesis of TAAD.Macrophages play a pivotal role in the pathogenesis of TAAD both through the inflammatory response and ECM degradation.Circulating monocytes first differentiate into macrophages, infiltrate, and accumulate in the media layer of the thoracic aorta wall.They further exert an inflammatory response through pro-inflammatory phenotypes and produce MMPs and ADAMTSs for ECM degradation.TAAD, thoracic aortic aneurysm and dissection; ECM, extracellular matrix; SMC, smooth muscle cell; MMP, matrix metalloproteinase; ADAMTS, ADAM metallopeptidase with thrombospondin type 1 motif.