Analysis of single-cell RNA-Seq reveals dynamic changes during macrophage state transition in atherosclerosis mouse model

Background: Atherosclerosis is an arterial inflammation that causes ischemic heart disease, which is the first leading cause of death worldwide. Macrophages play major roles during disease development by having pro-inflammatory and anti-inflammatory functions. Lack of effective treatment is mainly due to incomplete understanding of the molecular mechanisms underlying disease progression and regression. Materials and methods: The transcripts of the macrophages from two aortic samples from atherosclerotic region during disease progression and regression were analyzed using previously published dataset (GEO Accession GSE123587). Pre-processing, clustering of cells and identification of unique markers for each cluster were done using Seurat package implemented in R programming language. Monocle package was used to order the cells in pseudotime and to detect the key molecules that changed dramatically during comparison between distinct macrophages states (pro-inflammatory and anti-inflammatory). Ingenuity Pathway Analysis (IPA) software was used to analyze the pathways activity across macrophage states along the trajectory and to retrieve the transcriptional regulatory network between the genes determining the final states. Prediction of the miRNAs that might be involved in the disease progression was performed using TargetScan and GSEA (Gene Set Enrichment Analysis). Cytoscape application was used to visualize the regulatory network between the differentially regulated genes across macrophages states. Results: Clustering analysis of macrophages revealed their presence in distinct 11 states. In addition, Two states were found to be dominant in the progression group macrophages, and one state was found to be dominant in the regression group macrophages. Moreover, trajectory analysis showed a bifurcation point near the end of the trajectory, where macrophages fates were destined to be either pro-inflammatory or anti-inflammatory. Macrophages unique to the disease progression branch were found to activate STAT cascade, induce acute inflammatory response and upregulate inflammatory cytokines, denoting M1 polarization. In contrast, regression-branch specific macrophages were found to activate cholesterol efflux pathways and upregulate anti-inflammatory cytokines such as TSLP and CCL24. The transcription regulatory network between differentially regulated genes in both branches revealed changes in the transcriptional dynamics acquired during macrophage states transition. STAT1 (Signal transducer and activator of transcription 1) and IRF7 (Interferon Regulatory Factor 7) were found to be upregulated in the progression branch to maintain an inflammatory module resulting in production of distinct inflammatory cytokines. On the other hand, MAFB (MAF BZIP Transcription Factor B) and IGF1 (Insulin-like growth factor 1) were found to be upregulated in the regression branch to interrupt the inflammatory module at different levels. In addition, 10 miRNAs were predicted to be unregulated in progression-branch specific macrophages such as miR-344, miR-346 and miR-485. Conclusion: Inflammatory sites in atherosclerosis lesions contain both pro-inflammatory and anti-inflammatory macrophages. Each subset of macrophage activates unique transcriptional program. Certain transcription factors and growth factors have potential to alter the whole transcriptional regulatory network, thereby shifting the macrophages from inflammatory to anti-inflammatory state. Understanding how macrophage state transition occurs from inflammatory to anti-inflammatory state will be a key step to better understanding and treating atherosclerosis

Functional outcome and mortality prediction after decompressive craniectomy in patients with malignant middle cerebral artery infarction

Abstract Background Although it is well known that performing decompressive craniectomy (DC) in cases of swollen middle cerebral artery infarction (SMCAI) improves patient outcomes, limited evidence presently exists on the likelihood of mortality and achieving functional outcome following DC. The aim of the present study was to identify the predictors of early in-hospital mortality and functional status six months after performing DC in a cohort of patients with SMCAI. Material and methods All patients that had suffered SMCAI and underwent DC were included in the study sample and their demographic information, along with clinical and radiological findings, and risk factors were recorded for further analyses, which were conducted using the commercial software STATA. Modified Rankin Scale score (mRS) served as an outcome measure at 6-month follow-up. Results DC was performed on 50 patients with SMCAI (72% of whom had left hemisphere infractions) aged 45.2 ± 10.2 years (range 24–67 years), 32 (64%) of whom were female and 18 (36%) were male. While the initial median Glasgow Coma Scale (GCS) score was 8 (range 4–13), clinical deterioration mostly occurred within the first five days, whereby DC was performed within 9 ± 7.2 h (range 2–36 h). Following surgery, 16 (32%) patients died while in hospital, whereas the remaining 34 were discharged after 135 days on average, and attended the 6-month follow-up, when the median mRS score of 3 (range 2–6) was recorded for this subsample. Conclusions Younger age, higher GCS score, presence of isochoric pupils, history of prior treatment (e.g., embolectomy or tissue plasminogen activator), and shorter interval between clinical deterioration and surgery are associated with a lower likelihood of mortality and a greater odd of a favorable functional outcome

Proteomic Pathway Analysis of Monocyte-Derived Exosomes during Surgical Sepsis Identifies Immunoregulatory Functions

Background: Patients with sepsis exhibit significant long-term immunosuppressive sequelae. Monocyte dysfunction is a hallmark of this damage. Circulating exosomes are an important mediator of the systemic signaling events that occur during the septic response; thus, we sought to characterize the contribution of circulating exosomes to the inflammatory process induced during sepsis Methods: Monocyte-derived exosomes were isolated from cultured monocytes from healthy adult donors via stimulation with lipopolysaccharide (LPS) or phosphate-buffered saline (PBS). The proteome was determined by capillary-liquid chromatography-nanospray tandem mass spectrometry (capillary-LC/NT/MS). Using pathway analysis, proteomic networks of exosomes derived from LPS-stimulated monocytes were compared with those isolated from patients with surgical sepsis. Naïve monocytes were then treated with these exosomes and stimulated with LPS to determine the effects on recipient-cell immune function. Results: Proteomic analysis demonstrated 18 differentially expressed proteins (17 down-regulated, one up-regulated) in sepsis-derived exosomes, with 15 differentially expressed proteins (14 down-regulated, one up-regulated) in the LPS-stimulated exosomes. Functional enrichment analysis demonstrated several down-regulated processes, including localization, biogenesis, and metabolic and cellular processes in addition to immune system processes. In LPS-stimulated macrophages, similar down-regulated processes were seen, including metabolic and cellular processes, as well as the response to stimulus. Cells treated with sepsis-derived exosomes or exosomes from LPS-stimulated monocytes demonstrated significant reductions in tumor necrosis factor (TNF)-α generation in response to LPS stimulation. Conclusions: Proteomic analysis of sepsis-derived exosomes and LPS-stimulated, macrophage-derived exosomes exhibited down-regulation of several important protein networks, including the immune response. In addition, human monocytes treated with exosomes from patients with sepsis or LPS-stimulated monocytes demonstrated significant reductions in TNF-α generation in response to LPS stimulation. These data suggest the contribution of circulating exosomes to systemic signaling and immunomodulation during sepsis

Identification of a physiologic vasculogenic fibroblast state to achieve tissue repair

Tissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds