The Role of NF-κB in Intracranial Aneurysm Pathogenesis: A Systematic Review

Intracranial aneurysms (IAs) are abnormal dilations of the cerebral vessels, which pose a persistent threat of cerebral hemorrhage. Inflammation is known to contribute to IA development. The nuclear factor “kappa-light-chain-enhancer” of activated B-cells (NF-κB) is the major driver of inflammation. It increases the expression of inflammatory markers and matrix metalloproteinases (MMPs), which contribute heavily to the pathogenesis of IAs. NF-κB activation has been linked to IA rupture and resulting subarachnoid hemorrhage. Moreover, NF-κB activation can result in endothelial dysfunction, smooth muscle cell phenotypic switching, and infiltration of inflammatory cells in the arterial wall, which subsequently leads to the initiation and progression of IAs and consequently results in rupture. After a systematic search, abstract screening, and full-text screening, 30 research articles were included in the review. In this systematic review, we summarized the scientific literature reporting findings on NF-κB’s role in the pathogenesis of IAs. In conclusion, the activation of the NF-κB pathway was associated with IA formation, progression, and rupture.


Introduction
Intracranial aneurysms are asymptomatic abnormal dilations of the intracranial blood vessels with an increased diameter compared to the parent artery.IAs occur in 3-5% of the population with a gender ratio of 1:1 and a mean age of 50, but after the age of 50 years, the gender ratio changes significantly with the increasing number of female cases [1,2].The rupture of IAs is a persistent risk for hemorrhage with an incidence rate of around 7.9 per 100,000 person-years [3].About 15% of intracranial hemorrhage patients die before reaching a hospital.Even after state-of-the-art medical intervention, the mortality rate of SAH patients is very high (~40%) [4,5], and the rupture of IAs can lead to a lifelong disability.
The pathophysiology of IAs is complex.Although many factors play together in the formation, progression, and rupture of IAs, the research hitherto suggests that inflammation heavily contributes to IAs from formation to rupture [6,7].The endothelial dysfunction, smooth muscle cells (SMCs) phenotypic switching, infiltration and accumulation of inflammatory cells in the arterial walls, and the expression and release of pro-inflammatory cytokines such as interleukin (IL) -1β, and tumor necrosis factor-alpha (TNF-α), chemokines such as monocyte chemoattractant protein-1 (MCP-1), and IL-8, cell adhesion molecules, namely, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and extracellular matrix remodeling proteinases such as MMPs including MMP-2 and MMP-9 have been implicated in IA formation and rupture [6,7].The expression of these pro-inflammatory markers and MMPs is regulated by NF-κB transcription activity [7].
Experimental studies have shown that NF-κB activation increases the expression of these markers, and by blocking NF-κB activation, the expression of these inflammatory markers and MMPs could be reduced [8][9][10].Previously, deficiency of NF-κB subunit P50 in mice has been shown to decrease the incidence of IA formation and reduced macrophage infiltration [8].Moreover, silencing the adenomatous polyposis coli (APC) gene in rats promoted IA formation with increased NF-κB protein expression and activation [11].It is worth noting that potential acquired risk factors for IA formation and rupture including smoking, alcohol abuse, obesity, and oxidative stress activate NF-κB and increase the expression of pro-inflammatory markers and MMPs [12][13][14][15].
In this systematic review, we focused on the contribution of NF-κB in the formation, progression, and rupture of IAs.

Systematic Literature Search
The PRISMA guidelines were followed for the scientific literature search.The search for research articles reporting findings on NF-κB's role in IA formation, progression, and rupture was conducted in June 2023 in PubMed, BASE, and Embase search engines.The detailed search strategy was "NF-κB" or "NF-kappaB" in the "intracranial aneurysm" and "cerebral aneurysms" and "ruptured cerebral aneurysm" and "intracranial aneurysm" and "Un-ruptured intracranial aneurysm" and "Unruptured intracranial aneurysm" and "Unruptured cerebral aneurysms" and "Unruptured cerebral aneurysms".The search results were deduplicated, and the irrelevant research articles were screened out after reading the titles.

Literature Screening Criteria
The original peer-reviewed research article reporting the findings, which included expression of NF-κB in IA walls in animal or human studies, targeting gene manipulation to block NF-κB activation, and using pharmacological compounds or other strategies that directly or indirectly manipulated NF-κB expression and/or activation were included in this review.The studies reporting NF-κB findings in human IAs with only mutated genomes without comparing them to normal genomes were excluded.

Results
The systematic search on search engines PubMed, Embase, and BASE for scientific literature reporting NF-κB's role in aneurysm biology delivered a total of 118 records.After deduplication and title screening, 92 unique research articles were left.The abstract screening delivered 38 research articles for full-text screening.The full-text screening delivered 30 scientific research articles.The flowchart for the systematic search is shown in Figure 1.Eight research articles reported findings on human samples, and 25 studies reported NF-κB findings using animal models.Three studies reported both experimental and clinical data.[16].A systematic search for scientific literature was performed in June 2023.

Clinical Studies
Seven clinical studies reported NF-κB expression and/or activation in IAs compared to normal cerebral or nonvascular diseased arteries.The findings of these studies are summarized in Table 1.One study provided findings on NF-κB genotype correlation to IA formation.According to this study, ATTG1/ATTG2 and ATTG2/ATTG2 genotypes compared with the ATTG1/ATTG1 genotype were linked to a significantly decreased risk of IAs, suggesting that the ATTG2 allele may be a protective factor against IA formation [17].

Clinical Studies
Seven clinical studies reported NF-κB expression and/or activation in IAs compared to normal cerebral or nonvascular diseased arteries.The findings of these studies are summarized in Table 1.One study provided findings on NF-κB genotype correlation to IA formation.According to this study, ATTG1/ATTG2 and ATTG2/ATTG2 genotypes compared with the ATTG1/ATTG1 genotype were linked to a significantly decreased risk of IAs, suggesting that the ATTG2 allele may be a protective factor against IA formation [17].

Animal Experimental Studies
Among the included studies, 16 studies reported findings on NF-κB's role in IA formation, progression, and rupture.In addition to NF-κB, these studies reported findings on the regulation of inflammatory markers, MMPs, internal elastic lamina (IEL) loss, media thinning, vascular smooth muscle cells (VSMCs) loss and apoptosis, and macrophage infiltration.The findings of these studies are summarized in Table 2. Three studies used rabbits, 18 studies used rats, and 8 studies used mouse models of IAs.In rabbit models, the common carotid artery was ligated to induce IAs.In rats, all studies used left common carotid artery and bilateral posterior renal arteries ligation with increased salt intake with or without β-aminopropionitrile (BAPN) to induce IAs.In mice, IAs were induced either by using the same IA model as that of rats or by using deoxycorticosterone acetate-salt hypertension combined with a single elastase injection with or without BAPN.Expression of TLR10 mRNA gradually increased with cerebral aneurysm progression.mRNA, protein expression, and staining intensity of TLR-4 increased in IA walls after one month and decreased after three months.Expression of TLR4 coincided well with NF-κB P65 activation.[34] In total, 21 studies used different approaches to decrease the incidence of aneurysm formation and rupture and to ameliorate aneurysmal changes.Among these studies, 2 studies used the rabbit IA model, 6 studies used the rat IA model, and 7 studies used the mouse model of IAs.In all studies, the NF-κB expression and/or activation was directly or indirectly inhibited, except in one study, where the protein expression and phosphorylation of NF-κB P65 were enhanced.The findings of these studies are summarized in Table 3.The mRNA expression of MCP-1 and Ptger2 was abolished.
Less IA formation and reduced macrophage infiltration [40] 16d Mice IkB mutant-Cdh5Cre Did not affect mRNA expression Ptger2 Did not affect IA formation and macrophage infiltration [40] 16e Mice F-04418948

Reduced phosphorylation of NF-κB P65
Reduced staining intensity for COX-2 and MCP-1 Decreased the size of IAs, reduced thinning of media, suppressed macrophage infiltration [40]

Discussion
The clinical studies provided ample convincing evidence that the NF-κB P65 subunit is overexpressed at mRNA and protein levels with increased phosphorylation in IAs compared to normal arteries and nonvascular diseased tissue (Table 1) [8,11,[19][20][21][22].Elevated NF-κB activation was linked to the increased diameter of IAs [11].In addition to that, polymorphism in the genotype of the NF-κB promoter was linked to a lower risk of IAs [17].
NF-κB activation was detected in macrophages, endothelial cells, and SMCs in IA walls of experimental animals [8,38,40].Macrophages were predominantly infiltrated and accumulated in IA walls compared to normal arteries in human and arteries in SHAM-operated animals, and their number increased with the progression of IAs in experimental animal studies (Table 2) [14,23,25,28,29].In addition to macrophages, infiltration and accumulation of neutrophils, mast cells, and T cells were also detected in IA walls (Table 2) [23,35].Blocking NF-κB expression and activation and the treatments reducing IA formation and rupture attenuated the infiltration and accumulation of these inflammatory cell types in IA tissue (Table 3) [8,23,25,28,29,32,33,[35][36][37][38][39][40].Blocking or suppressing cell-specific NF-κB activation provided interesting results.It has been shown that macrophage-specific NF-κB inhibition and suppression could potentially reduce the incidence of IA formation, while blocking and suppressing NF-κB activation in endothelial cells did not reduce the incidence of IA formation [40].The inhibition of NF-κB in macrophages reduced CCL2 expression and, consequently, ameliorated macrophage infiltration in IA walls [40].Contrary to these findings, macrophages do not seem to be the only culprit, as clodronate liposomes lowered the number of circulating and infiltrated macrophages in IA tissue, but it did not reduce aneurysmal damage in the rabbit model of IAs [24].The treatment with clodronate liposomes did not reduce the mRNA and protein levels of MMP-2 and MMP-9 in IA walls [24].Furthermore, immunofluorescence staining showed that SMCs were the source of increased MMP-2 and MMP-9 with increased NF-κB P65 and MCP-1 levels [24], suggesting the contribution of NF-κB expression and activation in SMCs to IA initiation.The vascular and inflammatory cells orchestrate the cellular and molecular events leading to IA initiation, progression, and rupture, thus development of IAs cannot be attributed to a single cell type.Moreover, different experimental animal models and time points of investigation after IA induction could affect the outcomes of the studies, which can explain the contradictory results discussed above.
Taken together, these studies suggest that the pharmacological drugs, which can block NF-κB activation and can suppress the consequent expression and release of cytokines, chemokines, cell adhesion molecules, and MMPs in macrophages and SMCs, can be suitable candidates that might reduce IA formation and rupture.Experimental animal studies will be needed to further explore the potential of these drug candidates.

Conclusions
Clinical and experimental studies have shown the activation of NF-κB in IA formation.NF-κB contributes to IA formation and rupture probably via increasing the transcription of inflammatory markers and MMPs.Blocking NF-κB expression and/or activation attenuated mRNA expression and protein levels of inflammatory markers and MMPs and reduced IA formation and rupture in different animal models.Limitations: For this systematic review, a meta-analysis was not performed.Moreover, the inclusion criteria were strictly limited to NF-κB regulation, and the studies reporting findings on NF-κB upstream/downstream signaling molecules without investigating NF-κB regulation were not included in the study.

Figure 1 .
Figure 1.Flowchart showing a systematic search of the literature[16].A systematic search for sci-

Figure 1 .
Figure 1.Flowchart showing a systematic search of the literature [16].A systematic search for scientific literature was performed in June 2023.

Table 1 .
Studies reporting NF-κB status in patients with IAs compared to control with nonvascular diseased arteries or normal cerebral arteries.

Table 2 .
Animal studies reporting NF-κB status in IA animals compared to control animals.

Table 3 .
Studies reporting the effects of pharmacological/other treatments or genetic manipulations on NF-κB status and IA formation/rupture in animals.