Biology of Intracranial Aneurysms: Role of Inflammation

Pathogenesis, hemodynamics, and growth of intracranial aneurysms: Future directions.

Unruptured intracranial aneurysms (UIAs) are relatively common lesions that may cause devastating intracranial hemorrhage, thus producing considerable suffering and anxiety in those affected by the disease or an increased likelihood of developing it. Advances in the knowledge of the pathobiology behind intracranial aneurysm (IA) formation, progression, and rupture have led to preclinical testing of drug therapies that would prevent IA formation or progression. In parallel, novel biologically based diagnostic tools to estimate rupture risk are approaching clinical use. Arterial wall remodeling, triggered by flow and intramural stresses and mediated by inflammation, is relevant to both. This review discusses the basis of flow-driven vessel remodeling and translates that knowledge to the observations made on the mechanisms of IA initiation and progression on studies using animal models of induced IA formation, study of human IA tissue samples, and study of patient-derived computational fluid dynamics models. Blood flow conditions leading to high wall shear stress (WSS) activate proinflammatory signaling in endothelial cells that recruits macrophages to the site exposed to high WSS, especially through macrophage chemoattractant protein 1 (MCP1). This macrophage infiltration leads to protease expression, which disrupts the internal elastic lamina and collagen matrix, leading to focal outward bulging of the wall and IA initiation. For the IA to grow, collagen remodeling and smooth muscle cell (SMC) proliferation are essential, because the fact that collagen does not distend much prevents the passive dilation of a focal weakness to a sizable IA. Chronic macrophage infiltration of the IA wall promotes this SMC-mediated growth and is a potential target for drug therapy. Once the IA wall grows, it is subjected to changes in wall tension and flow conditions as a result of the change in geometry and has to remodel accordingly to avoid rupture. Flow affects this remodeling process. Flow triggers an inflammatory reaction that predisposes the arterial wall to IA initiation and growth and affects the associated remodeling of the UIA wall. This chronic inflammation is a putative target for drug therapy that would stabilize UIAs or prevent UIA formation. Moreover, once this coupling between IA wall remodeling and flow is understood, data from patient-specific flow models can be gathered as part of the diagnostic workup and utilized to improve risk assessment for UIA initiation, progression, and eventual rupture.

Studies have shown that chronic inflammatory response plays a key role in intracranial aneurysms (IA) formation and progression, and macrophages regulate the formation and progression of IA through a variety of pathways. Bone marrow monocyte-derived macrophages and resident-tissue macrophages infiltrate the vessel wall, after infiltration macrophages are polarized into various polarization phenotypes dominated by M1-like and M2-like cells. Polarized phenotypes of macrophages can regulate the formation and progression of intracranial aneurysms by releasing cytokines and regulating the inflammatory response of other immune cells, as well as release different cytokines to regulate the process of extracellular matrix remodeling. Some important progresses have been made in the clinical detection and treatment in targeting macrophages. This review provides a summary on the pathogenesis of IA and potential drug targets to prevent the formation and rupture of intracranial aneurysms.

BackgroundIntracranial aneurysm (IA) is a socially important disease due to its high incidence in the general public and the severity of resultant subarachnoid hemorrhage that follows rupture. Despite the social importance of IA as a cause of subarachnoid hemorrhage, there is no medical treatment to prevent rupture, except for surgical procedures, because the mechanisms regulating IA formation are poorly understood. Therefore, these mechanisms should be elucidated to identify a therapeutic target for IA treatment. In human IAs, the presence of inflammatory responses, such as an increase of tumor necrosis factor (TNF)-alpha, have been observed, suggesting a role for inflammation in IA formation. Recent investigations using rodent models of IAs have revealed the crucial role of inflammatory responses in IA formation, supporting the results of human studies. Thus, we identified nuclear factor (NF)-kappaB as a critical mediator of inflammation regulating IA formation, by inducing downstream pro-inflammatory genes such as MCP-1, a chemoattractant for macrophages, and COX-2. In this study, we focused on TNF-alpha signaling as a potential cascade that regulates NF-kappaB-mediated IA formation.ResultsWe first confirmed an increase in TNF-alpha content in IA walls during IA formation, as expected based on human studies. Consistently, the activity of TNF-alpha converting enzyme (TACE), an enzyme responsible for TNF-alpha release, was induced in the arterial walls after aneurysm induction in a rat model. Next, we subjected tumor necrosis factor receptor superfamily member 1a (TNFR1)-deficient mice to the IA model to clarify the contribution of TNF-alpha-TNFR1 signaling to pathogenesis, and confirmed significant suppression of IA formation in TNFR1-deficient mice. Furthermore, in the IA walls of TNFR1-deficient mice, inflammatory responses, including NF-kappaB activation, subsequent expression of MCP-1 and COX-2, and infiltration of macrophages into the IA lesion, were greatly suppressed compared with those in wild-type mice.ConclusionsIn this study, using rodent models of IAs, we clarified the crucial role of TNF-alpha-TNFR1 signaling in the pathogenesis of IAs by inducing inflammatory responses, and propose this signaling as a potential therapeutic target for IA treatment.

This study aims to identify characteristic genes linked to vascular smooth muscle cells (VSMCs) and intracranial aneurysm (IA) formation and rupture, providing insights for early diagnosis, risk prediction, and treatment of IA. We analyzed the GSE75436 and GSE13353 datasets from the GEO database, performing differential expression analysis with |log2FC|> 1 and adj p < 0.05. Gene Ontology (GO), KEGG, and Reactome pathway enrichment analyses were conducted. Using the GSE122897 dataset, Weighted Gene Co-expression Network Analysis (WGCNA) identified gene modules and hub genes associated with IA. Machine learning algorithms (LASSO, SVM-RFE, and Random Forest) were applied to select differentially expressed hub genes. The expression patterns of marker genes were explored using the GSE193533 single-cell RNA sequencing dataset. Lastly, we assessed the predictive value of these genes in IA formation and rupture by plotting Receiver Operating Characteristic (ROC) curves and calculating the Area Under the Curve (AUC), thereby validating their sensitivity and specificity in clinical diagnostics. We identified 1000 upregulated and 806 downregulated genes in IA compared to normal arteries, and 468 downregulated and 405 upregulated genes in ruptured IA tissue. WGCNA revealed key gene modules associated with IA. Machine learning identified genes such as COL5A2, CDH11, PLOD1, P3H4, PLIN2, and PLAUR. Single-cell analysis showed a phenotypic transition in VSMCs from contractile to synthetic, with these genes predominantly expressed in synthetic VSMCs. ROC analysis validated their excellent diagnostic performance for IA formation and rupture. COL5A2, CDH11, PLOD1, and P3H4 were identified as genes associated with IA formation, while PLIN2 and PLAUR were linked to IA rupture. These genes provide potential biomarkers for IA diagnosis and risk assessment.

Background: Intracranial aneurysm (IA) is a critical acquired cerebrovascular disease that may cause subarachnoid hemorrhage, and nuclear factor-κB (NF-κB)-mediated inflammation is involved in the pathogenesis of IA. Adenomatous polyposis coli (Apc) gene is a tumor suppressor gene associated with both familial and sporadic cancer. Herein, the purpose of our study is to validate effect of Apc gene on IA formation and rupture by regulating the NF-κB signaling pathway mediated inflammatory response. Methods: We collected IA specimens (from incarceration of IA) and normal cerebral arteries (from surgery of traumatic brain injury) to examine expression of Apc and the NF-κB signaling pathway related factors (NF-κB p65 and IκBα). ELISA was used to determine levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β (IL-1β), and IL-6. IA model was established in rats, and Apc-siRNA was treated to verify effect of Apc on IA formation and rupture. Next, regulation of Apc on the NF-κB signaling pathway was investigated. Results: Reduced expression of Apc and IκBα, and increased expression of NF-κB p65 were found in IA tissues. MCP-1, TNF-α, IL-1β, and IL-6 exhibited higher levels in unruptured and ruptured IA, which suggested facilitated inflammatory responses. In addition, the IA rats injected with Apc-siRNA showed further enhanced activation of NF-κB signaling pathway, and up-regulated levels of MCP-1, TNF-α, IL-1β, IL-6, MMP-2, and MMP-9 as well as extent of p65 phosphorylation in IA. Conclusion: Above all, Apc has the potential role to attenuate IA formation and rupture by inhibiting inflammatory response through repressing the activation of the NF-κB signaling pathway.

STAT3 Contributes to Intracranial Aneurysm Formation and Rupture by Modulating Inflammatory Response.

Vascular smooth muscle cell (VSMC) phenotypic modulation regulates the initiation and progression of intracranial aneurysm (IA). Dexmedetomidine (DEX) is suggested to play neuroprotective roles in patients with craniocerebral injury. Therefore, we investigated the biological functions of DEX and its mechanisms against IA formation and progression in the current study. The rat primary VSMCs were isolated from Sprague–Dawley rats. IA and superficial temporal artery (STA) tissue samples were obtained from patients with IA. Flow cytometry was conducted to identify the characteristics of isolated VSMCs. Hydrogen peroxide (H2O2) was used to mimic IA-like conditions in vitro. Cell viability was detected using CCK-8 assays. Wound healing and Transwell assays were performed to detect cell motility. ROS production was determined by immunofluorescence using DCFH-DA probes. Western blotting and RT-qPCR were carried out to measure gene expression levels. Inflammation responses were determined by measuring inflammatory cytokines. Immunohistochemistry staining was conducted to measure α2-adrenergic receptor levels in tissue samples. DEX alleviated the H2O2-induced cytotoxicity, attenuated the promoting effects of H2O2 on cell malignancy, and protected VSMCs against H2O2-induced oxidative damage and inflammation response. DEX regulated the GSK-3β/MKP-1/NRF2 pathway via the α2AR. DEX alleviates the inflammatory responses and oxidative damage of VSMCs by regulating the GSK-3β/MKP-1/NRF2 pathway via the α2AR in IA.

Despite the devastating consequences of aneurysmal subarachnoid hemorrhage (SAH), the mechanisms underlying the formation, progression, and rupture of intracranial aneurysms (IAs) are complex and not yet fully clear. In a real-world situation, continuously observing the process of aneurysm development in humans appears unrealistic, which also present challenges for the understanding of the underlying mechanism. We reported the relatively complete course of IA development in two real patients. On this basis, computational fluid dynamics simulation (CFD) was performed to evaluate the changes in hemodynamics and analyze the mechanism underlying the formation, progression, and rupture of IAs. Our results suggested that the formation and progression of IAs can be a dynamic process, with constantly changing hemodynamic characteristics. CFD analysis based on medical imaging provides the opportunity to study the hemodynamic conditions over time. From these two rare cases, we found that concentrated high-velocity inflow jets, flows with vortex structures, extremely high WSS, and a very steep WSSG were correlated with the formation of IAs. Complex multi-vortex flows are possibly related to IAs prior to growth, and the rupture of IAs is possibly related to low WSS, extreme instability and complexity of flow patterns. Our findings provide unique insight into the theoretical hemodynamic mechanism underlying the formation and progression of IAs. Given the small sample size the findings of this study have to be considered preliminary and exploratory.

Objective: Little is known about smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation or pathogenesis of intracranial aneurysms. TNF-α has been associated with aneurysms, but a direct role has not been established. Methods: Cultured cerebral SMC were treated with TNF-α for PCR, western blot, chromatin immune-precipitation (CHIP), and adenovirus promoter transfection. In vivo experiments were carried out in the following models: application of TNF-α to the surface of carotid arteries, cerebral model of hypertension and hemodynamic stress, and cerebral model of aneurysm formation and rupture. The TNF-α inhibitor 3,6’dithiothalidomide (DTH) was synthesized. Results: Cultured cerebral SMC over-expressing myocardin induced expression of key SMC contractile genes (SM-α-actin, SM-22α, SM-MHC), while dominant negative suppressed expression. TNF-α treatment inhibited this contractile phenotype and induced pro-inflammatory genes (MCP-1, MMPs, VCAM-1, IL-1β). TNF-α increased expression of KLF4 and KLF4 siRNA abrogated TNF-α induced phentotypic modulation. These mechanisms were confirmed in vivo following exposure of rat carotid arteries to TNF-α and early in a model of cerebral hypertension and hemodynamic stress prior to cerebral aneurysm formation. Treatment with DTH reversed these pathological vessel wall alterations. TNF-α knock-out mice and DTH pre-treatment decreased the incidence of aneurysm formation and rupture. As compared with sham mice, TNF-α expression was not significantly different in TNF-α knock-out mice or those pre-treated with DTH, but was elevated in unruptured and ruptured aneurysms. Initiation of DTH 7 days after aneurysm induction did not alter aneurysm incidence, but resulted in stabilization and decreased rupture. CHIP assays in vivo and in vitro demonstrated that TNF-α promotes epigenetic changes through KLF4 dependent alterations in promoter regions of myocardin, SMC’s, and inflammatory genes. Conclusion: TNF-α induces phenotypic modulation of cerebral SMC through myocardin and KLF4 regulated pathways. These results demonstrate a novel role for TNF-α in promoting a pro-inflammatory phenotype. These data suggests a critical role of TNF-α in the formation and rupture of aneurysms.

Inflammation plays a key role in formation and rupture of intracranial aneurysms. Because hepatocyte growth factor (HGF) protects against vascular inflammation, we sought to assess the role of endogenous HGF in the pathogenesis of intracranial aneurysms. Circulating HGF concentrations in blood samples drawn from the lumen of human intracranial aneurysms or femoral arteries were compared in 16 patients. Tissue from superficial temporal arteries and ruptured or unruptured intracranial aneurysms collected from patients undergoing clipping (n=10) were immunostained with antibodies to HGF and its receptor c-Met. Intracranial aneurysms were induced in mice treated with PF-04217903 (a c-Met antagonist) or vehicle. Expression of inflammatory molecules was also measured in cultured human endothelial, smooth muscle cells and monocytes treated with lipopolysaccharides in presence or absence of HGF and PF-04217903. We found that HGF concentrations were significantly higher in blood collected from human intracranial aneurysms (1076±656 pg/mL) than in femoral arteries (196±436 pg/mL; P<0.001). HGF and c-Met were detected by immunostaining in superficial temporal arteries and in both ruptured and unruptured human intracranial aneurysms. A c-Met antagonist did not alter the formation of intracranial aneurysms (P>0.05), but significantly increased the prevalence of subarachnoid hemorrhage and decreased survival in mice (P<0.05). HGF attenuated expression of vascular cell adhesion molecule-1 (P<0.05) and E-Selectin (P<0.05) in human aortic endothelial cells. In conclusion, plasma HGF concentrations are elevated in intracranial aneurysms. HGF and c-Met are expressed in superficial temporal arteries and in intracranial aneurysms. HGF signaling through c-Met may decrease inflammation in endothelial cells and protect against intracranial aneurysm rupture.

BackgroundOxidative stress and vascular smooth muscle cell (VSMC) phenotypic modulation influence intracranial aneurysm (IA) formation and progression. Oxidative stress plays an important role in phenotype switching, and nuclear factor erythroid 2-related factor 2 (Nrf-2) is one of the main antioxidant systems. Unfortunately, little is known about how Nrf-2 signaling influences VSMC phenotype switches during IA pathogenesis.MethodsWe examined the effect of Nrf-2 activation IA on formation and progression in an elastase-induced rat IA model. We also developed a hydrogen peroxide (H2O2)-induced VSMC oxidative damage model. Then, we analyzed VSMC phenotype changes in the setting of Nrf-2 activation or inhibition in vitro. The proliferation, migration ability, and apoptosis rate of VSMCs were tested. Lastly, we measured the expression levels of antioxidant enzymes and inflammatory cytokines downstream of Nrf-2.ResultsNrf-2 activation suppressed IA formation and progression in vivo. We confirmed Nrf-2 nuclear translocation and a VSMC switch from the contractile to synthetic phenotype. Nrf-2 activation inhibited the proliferation, migratory ability, and apoptosis rate enhanced by H2O2. Quantitative real-time polymerase chain reaction (PCR) and western blot analysis revealed that Nrf-2 activation promoted antioxidant enzymes and VSMC-specific marker gene expressions but decreased pro-inflammatory cytokine levels.ConclusionThese results suggest that Nrf-2 exerts protective effects against IA development by preventing VSMCs from changing to a synthetic phenotype.

BackgroundThe regulation of vascular smooth muscle cell (VSMC) phenotype plays an important role in intracranial aneurysm (IA) formation and progression. However, the underlying mechanism remains unclear. Metformin is a 5′ AMP-activated protein kinase (AMPK) agonist that has a protective effect on vasculature. The present study investigated whether metformin modulates VSMC phenotype switching via the AMPK/acetyl-CoA carboxylase (ACC) pathway during IA pathogenesis.MethodsAdult male Sprague-Dawley rats (n = 80) were used to establish an elastase-induced IA model. The effects of metformin on AMPK activation and VSMC phenotype modulation were examined. We also established a platelet-derived growth factor (PDGF)-BB-induced VSMC model and analyzed changes in phenotype including proliferation, migration, and apoptosis as well as AMPK/ACC axis activation under different doses of metformin, AMPK antagonist, ACC antagonist, and their combinations.ResultsMetformin decreased the incidence and rupture rate of IA in the rat model and induced a switch in VSMC phenotype from contractile to synthetic through activation of the AMPK/ACC pathway, as evidenced by upregulation of VSMC-specific genes and decreased levels of pro-inflammatory cytokines. AMPK/ACC axis activation inhibited the proliferation, migration, and apoptosis of VSMCs, in which phenotypic switching was induced by PDGF-BB.ConclusionsMetformin protects against IA formation and rupture by inhibiting VSMC phenotype switching and proliferation, migration, and apoptosis. Thus, metformin has therapeutic potential for the prevention of IA.

The role of inflammation and potential pharmacological therapy in intracranial aneurysms

Background and Purpose: Inflammation plays a key role in formation and rupture of intracranial aneurysms. Because hepatocyte growth factor (HGF) modulates vascular inflammation, we sought to assess the role of endogenous HGF in the pathogenesis of intracranial aneurysms. Methods: Circulating HGF concentrations in blood samples drawn from the lumen of human intracranial aneurysms or femoral arteries were compared in 16 patients. Intracranial aneurysm and superficial temporal artery tissue samples from patients undergoing clipping (n=10) were immunostained with antibodies to HGF and its receptor c-Met. Intracranial aneurysms were induced in mice treated with PF-04217903 (a c-Met antagonist) or vehicle. Expression of inflammatory molecules was also measured in cultured endothelial, smooth muscle cells and monocytes treated with LPS in presence or absence of HGF and PF-04217903. Results: HGF concentrations were significantly higher in blood collected from human intracranial aneurysm (1076 ± 656 pg/ml) than in femoral arteries (196 ± 436 pg/ml, p0.05), but significantly increased the prevalence of subarachnoid hemorrhage and decreased survival in mice (P&lt;0.05). HGF attenuated expression of markers of inflammation in human aortic endothelial cells (P&lt;0.05), but not in human aortic smooth muscle cells or monocytes. Conclusion: Plasma HGF concentrations are higher in intracranial aneurysms. HGF and c-Met are expressed in intracranial aneurysms. HGF signaling through c-Met may decrease inflammation in endothelium and protect from intracranial aneurysm rupture. The findings of this study may have significant therapeutic implications for prevention of intracranial aneurysm formation and rupture.