Exploring the relationship between hemodynamics and the immune microenvironment in carotid atherosclerosis: Insights from CFD and CyTOF technologies.

Carotid atherosclerosis is a major cause of stroke. Hemodynamic forces, such as shear stress and oscillatory shear, play an important role in the initiation and progression of atherosclerosis. The alteration of the immune microenvironment is the fundamental pathological mechanism by which diverse external environmental factors impact the formation and progression of plaques. However, Current research on the relationship between hemodynamics and immunity in atherosclerosis still lack of comprehensive understanding. In this study, we combined computational fluid dynamics (CFD) and Mass cytometry (CyTOF) technologies to explore the changes in the immune microenvironment within plaques under different hemodynamic conditions. Our results indicated that neutrophils were enriched in adverse flow environments. M2-like CD163+CD86+ macrophages were predominantly enriched in high WSS and low OSI environments, while CD163-CD14+ macrophages were enriched in low WSS and high OSI environments. Functional analysis further revealed T cell pro-inflammatory activation and dysregulation in modulation, along with an imbalance in M1-like/M2-like macrophages, suggesting their potential involvement in the progression of atherosclerotic lesions mediated by adverse flow patterns. Our study elucidated the potential mechanisms by which hemodynamics regulated the immune microenvironment within plaques, providing intervention targets for future precision therapies.

Somatic PDGFRB activating variants promote smooth muscle cell phenotype modulation in intracranial fusiform aneurysm.

BACKGROUND: The fusiform aneurysm is a nonsaccular dilatation affecting the entire vessel wall over a short distance. Although PDGFRB somatic variants have been identified in fusiform intracranial aneurysms, the molecular and cellular mechanisms driving fusiform intracranial aneurysms due to PDGFRB somatic variants remain poorly understood. METHODS: In this study, single-cell sequencing and immunofluorescence were employed to investigate the phenotypic changes in smooth muscle cells within fusiform intracranial aneurysms. Whole-exome sequencing revealed the presence of PDGFRB gene mutations in fusiform intracranial aneurysms. Subsequent immunoprecipitation experiments further explored the functional alterations of these mutated PDGFRB proteins. For the common c.1684 mutation site of PDGFRß, we established mutant smooth muscle cell lines and zebrafish models. These models allowed us to simulate the effects of PDGFRB mutations. We explored the major downstream cellular pathways affected by PDGFRBY562D mutations and evaluated the potential therapeutic effects of Ruxolitinib. RESULTS: Single-cell sequencing of two fusiform intracranial aneurysms sample revealed downregulated smooth muscle cell markers and overexpression of inflammation-related markers in vascular smooth muscle cells, which was validated by immunofluorescence staining, indicating smooth muscle cell phenotype modulation is involved in fusiform aneurysm. Whole-exome sequencing was performed on seven intracranial aneurysms (six fusiform and one saccular) and PDGFRB somatic mutations were detected in four fusiform aneurysms. Laser microdissection and Sanger sequencing results indicated that the PDGFRB mutations were present in smooth muscle layer. For the c.1684 (chr5: 149505131) site mutation reported many times, further cell experiments showed that PDGFRBY562D mutations promoted inflammatory-related vascular smooth muscle cell phenotype and JAK-STAT pathway played a crucial role in the process. Notably, transfection of PDGFRBY562D in zebrafish embryos resulted in cerebral vascular anomalies. Ruxolitinib, the JAK inhibitor, could reversed the smooth muscle cells phenotype modulation in vitro and inhibit the vascular anomalies in zebrafish induced by PDGFRB mutation. CONCLUSION: Our findings suggested that PDGFRB somatic variants played a role in regulating smooth muscle cells phenotype modulation in fusiform aneurysms and offered a potential therapeutic option for fusiform aneurysms.

Metabolic alterations of peripheral blood immune cells and heterogeneity of neutrophil in intracranial aneurysms patients.

BACKGROUND: Intracranial aneurysms (IAs) represent a severe cerebrovascular disease that can potentially lead to subarachnoid haemorrhage. Previous studies have demonstrated the involvement of peripheral immune cells in the formation and progression of IAs. Nevertheless, the impact of metabolic alterations in peripheral immune cells and changes in neutrophil heterogeneity on the occurrence and progression of IAs remains uncertain. METHODS: Single-cell Cytometry by Time-of-Flight (CyTOF) technology was employed to profile the single-cell atlas of peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) in 72 patients with IAs. In a matched cohort, metabolic shifts in PBMC subsets of IA patients were investigated by contrasting the expression levels of key metabolic enzymes with their respective counterparts in the healthy control group. Simultaneously, compositional differences in peripheral blood PMNs subsets between the two groups were analysed to explore the impact of altered heterogeneity in neutrophils on the initiation and progression of IAs. Furthermore, integrating immune features based on CyTOF analysis and clinical characteristics, we constructed an aneurysm occurrence model and an aneurysm growth model using the random forest method in conjunction with LASSO regression. RESULTS: Different subsets exhibited distinct metabolic characteristics. Overall, PBMCs from patients elevated CD98 expression and increased proliferation. Conversely, CD36 was up-regulated in T cells, B cells and monocytes from the controls but down-regulated in NK and NKT cells. The comparison also revealed differences in the metabolism and function of specific subsets between the two groups. In terms of PMNs, the neutrophil landscape within patients group revealed a pronounced shift towards heightened complexity. Various neutrophil subsets from the IA group generally exhibited lower expression levels of anti-inflammatory functional molecules (IL-4 and IL-10). By integrating clinical and immune features, the constructed aneurysm occurrence model could precisely identify patients with IAs with high prediction accuracy (AUC = 0.987). Furthermore, the aneurysm growth model also exhibited superiority over ELAPSS scores in predicting aneurysm growth (lower prediction errors and out-of-bag errors). CONCLUSION: These findings enhanced our understanding of peripheral immune cell participation in aneurysm formation and growth from the perspectives of immune metabolism and neutrophil heterogeneity. Moreover, the predictive model based on CyTOF features holds the potential to aid in diagnosing and monitoring the progression of human IAs.

Single-Cell Atlas of Atherosclerosis Patients by Cytof: Circulatory and Local Immune Disorders.

Atherosclerosis (AS) is a common underlying pathology of coronary artery disease, peripheral artery disease, and stroke. The characteristics of immune cells within plaques and their functional relationships with blood are crucial in AS. In this study, Mass cytometry (CyTOF), RNA-sequencing and immunofluorescence were combined to comprehensively analyze plaque tissues and peripheral blood from 25 AS patients (22 for Mass cytometry and 3 for RNA-sequencing), as well as blood from 20 healthy individuals. The study identified a complexity of leukocytes in the plaque, including both defined anti-inflammatory and pro-inflammatory subsets such as M2-like CD163+ macrophages, Natural killer T cells (NKT), CD11b+ CD4+ T effector memory cells (Tem), and CD8+ terminally differentiated effector memory cells (TEMRA). Functionally activated cell subsets were also found in peripheral blood in AS patients, highlighting the vivid interactions between leukocytes in plaque and blood. The study provides an atlas of the immune landscape in atherosclerotic patients, where pro-inflammatory activation was found to be a major feature of peripheral blood. The study identified NKT, CD11b+ CD4+ Tem, CD8+ TEMRA and CD163+ macrophages as key players in the local immune environment.

RNA sequencing analysis between ruptured and un-ruptured brain AVM.

BACKGROUND: A brain arteriovenous malformation (BAVM) is a tangle of abnormal blood vessels connecting the arteries and veins in the brain and is associated with a higher risk for intracerebral hemorrhage (ICH). RNA sequencing technology has been recently used to investigate the mechanism of diseases owing to its ability to identify the gene changes on a transcriptome-wide level. This study aims to gain insights into the potential mechanism involved in BAVM rupture. METHODS: Sixty-five BAVM nidus samples were collected, among which 28 were ruptured and 37 were un-ruptured. Then, next-generation RNA sequencing was performed on all of them to obtain differential expressed genes (DEGs) between the two groups. In addition, bioinformatics analysis was performed to evaluate the involved biological processes and pathways by GO and KEGG analysis. Finally, we performed a univariate Cox regression analysis to obtain the early rupture-prone DEGs. RESULTS: A total of 951 genes were differentially expressed between the ruptured and un-ruptured BAVM groups, of which 740 genes were upregulated and 211 genes were downregulated in ruptured BAVMs. Then, bioinformatics analysis showed the biological processes and pathways related to the inflammatory processes and extracellular matrix organization were significantly enriched. Meanwhile, some downregulated genes are involved in cell adhesion and genes participating in response to muscle activity and the terms of nervous system development. Finally, one hundred twenty-five genes, many were involved in inflammation, were correlated with the early rupture of BAVMs. CONCLUSIONS: The upregulated genes in the ruptured BAVM group were involved in inflammatory processes and extracellular matrix organization. Some of the downregulated genes participated in cell adhesion and myofibril assembly, indicating the role of enhanced inflammation and reduced inflammation vessel strength in BAVMs rupture.

Single-cell atlas reveals different immune environments between stable and vulnerable atherosclerotic plaques.

Introduction: Regardless of the degree of stenosis, vulnerable plaque is an important cause of ischemic stroke and thrombotic complications. The changes of the immune microenvironment within plaques seem to be an important factor affecting the characteristics of the plaque. However, the differences of immune microenvironment between stable and vulnerable plaques were remained unknown. Methods: In this study, RNA-sequencing was performed on superficial temporal arteries from 5 traumatic patients and plaques from 3 atherosclerotic patients to preliminary identify the key immune response processes in plaques. Mass cytometry (CyTOF) technology was used to explore differences in immune composition between 9 vulnerable plaques and 12 stable plaques. Finally, immunofluorescence technique was used to validate our findings in the previous analysis. Results: Our results showed that more CD86+CD68+ M1 pro-inflammatory macrophages were found in vulnerable plaques, while CD4+T memory cells were mainly found in stable plaques. In addition, a CD11c+ subset of CD4+T cells with higher IFN-r secretion was found within the vulnerable plaque. In two subsets of B cells, CD19+CD20-B cells in vulnerable plaques secreted more TNF-a and IL-6, while CD19-CD20+B cells expressed more PD-1 molecules. Conclusion: In conclusion, our study suggested that M1-like macrophages are the major cell subset affecting plaque stability, while functional B cells may also contribute to plaque stability.

The Relationship Between Smoking and Delayed Cerebral Ischemia After Intracranial Aneurysm Rupture: A Systematic Review and Meta-Analysis.

Background: Delayed cerebral ischemia (DCI) is the main cause of death and disability after intracranial aneurysm rupture. Previous studies have shown that smoking can lead to DCI after intracranial aneurysm rupture. However, some recent studies have shown that nicotine, as the main ingredient of tobacco, can cause cerebral vasodilation. This view has led to a debate about the relationship between smoking and DCI. This study aims to determine the relationship between smoking and DCI. Methods: A systematic literature search was performed according to PRISMA guidelines. The Cochrane Library, Web of Science, PubMed, and Embase online databases were searched for studies published up to September 2020. All studies related to smoking and DCI were included in the analysis. The R and RevMan software were used for data analysis, and random or fixed model analysis was selected depending on the degree of heterogeneity. Publication bias was examined by using the Begg-Mazumdar test and using contour-enhanced funnel plots with trim method. Results: A total of eight original articles (12 cohorts) with 10,722 patients were included in this meta-analysis. There were statistically significant higher rates of DCI in the smoking group than in the non-smoking group (RRtotal = 1.16, 95%CI: 1.05-1.27). After heterogeneity among cohorts was removed by sensitivity analysis, there was still a statistically significant difference in the incidence of DCI between the smoking and non-smoking groups (RRtotal = 1.13, 95%CI: 1.07-1.20). Conclusions: Although the effects of nicotine as the main component of tobacco are unclear in terms of cerebral vessels, the present study suggests that smoking is a risk factor for DCI in patients with ruptured aneurysm.

Risk Factors for Higher Volume of Hemorrhage in Ruptured Anterior Circulation Intracranial Aneurysms.

Purpose: To explore the influencing factors of volume hemorrhage in ruptured anterior circulation aneurysms, so as to identify the characteristics of anterior circulation aneurysms with high volume of hemorrhage, and to provide advice for clinical diagnosis and treatment for those aneurysms. Methods: We retrospectively reviewed 437 cases of ruptured anterior intracranial aneurysms in our center between the years 2012 and 2017. According to the inclusion criteria, a total of 100 qualified patients were screened out. We collected demographic characteristics, environmental exposure, and admission status of enrolled patients. In addition, morphological parameters and location of aneurysms were also included. The semiautomatic threshold method was used to measure the volume of hemorrhage. According to the results, the patients were divided into the group with high blood volume and low blood volume. Univariate and multivariate logistic regression analyses were used to discover the related factors affecting the bleeding volume. Results: In univariable analysis, pulse pressure (P = 0.014) showed a significant difference at the P < 0.05 test level. In terms of aneurysm morphology, the irregular shape (P < 0.001), calcification (P = 0.001), and flow angle (P = 0.006) showed significant statistical differences between the two groups at the P < 0.01 level (P < 0.01). Multivariate logistic regression analysis showed that irregular shape (OR = 5.370 P = 0.002 < 0.05), large flow angle (OR = 1.033 P = 0.016 < 0.05), and calcification (OR = 5.460 P = 0.003 < 0.05) were risk factors for volume of hemorrhage in ruptured anterior circulation aneurysms. The influence of hypertension history was at critical state (OR = 2.877 P = 0.051 > 005). Conclusions: According to our analysis results, intracranial anterior circulation aneurysms with irregular shapes, calcifications, and large flow angle are more dangerous. Aneurysms with these characteristics often have a large amount of hemorrhage, requiring timely treatment in clinical practice.