Integrative single cell RNA and spatial profiling identify mechanisms of neonatal brain hemorrhage pathophysiology and repair

Precise control of cell-cell communication networks within brain neurovascular units (NVUs) promotes normal tissue physiology, and dysregulation of these networks can lead to pathologies including intracerebral hemorrhage (ICH). The cellular and molecular mechanisms underlying ICH development and subsequent tissue repair processes remain poorly understood. Here we employed quantitative single cell RNA sequencing coupled with spatial in situ gene expression profiling to characterize NVU signaling pathways associated with ICH in neonatal mouse brain tissue. The initial stages of ICH pathogenesis are characterized by downregulation of extracellular matrix (ECM)-associated signaling factors (Adamtsl2, Htra3, and Lama4) that functionally connect to canonical TGFβ activation and signaling in vascular endothelial cells. Conversely, the progressive resolution of ICH involves upregulation of neuroinflammatory signaling networks (Gas6 and Axl) alongside activation of iron metabolism pathway components (Hmox1, Cp, and Slc40a1) in astrocytes and microglial cells. Integrated computational modeling identifies additional ligand-receptor signaling networks between perivascular glial cells and endothelial cells during both ICH pathogenesis and resolution. Collectively, these findings illuminate the molecular signaling networks that promote NVU maturation and provide novel mechanistic insights into the pathways controlling ICH pathogenesis and repair.

Intracerebral hemorrhage (ICH) is a severe stroke subtype with high morbidity, disability, and mortality rates. Currently, no biomarkers for ICH are available for use in clinical practice. We aimed to explore the roles of RNAs in ICH pathogenesis and identify potential diagnostic biomarkers. We collected 233 individual blood samples from two independent cohorts, including 64 patients with ICH, 59 patients with ischemic stroke (IS), 60 patients with hypertension (HTN) and 50 healthy controls (CTRL) for RNA sequencing. Differentially expressed genes (DEGs) analysis, gene set enrichment analysis (GSEA), and weighted correlation network analysis (WGCNA) were performed to identify ICH-specific modules. The immune cell composition was evaluated with ImmuneCellAI. Multiple machine learning algorithms to select potential biomarkers for ICH diagnosis, and further validated by quantitative real-time polymerase chain reaction (RT-PCR). Receiver operating characteristic (ROC) curve analysis and decision curve analysis (DCA) were performed to evaluate the diagnostic value of the signature for ICH. Finally, we generated M1 and M2 macrophages to investigate the expression of candidate genes. In both cohorts, 519 mRNAs and 131 lncRNAs were consistently significantly differentially expressed between ICH patients and HTN controls. Gene function analysis suggested that immune system processes may be involved in ICH pathology. ImmuneCellAI analysis revealed that the abundances of 11 immune cell types were altered after ICH in both cohorts. WGCNA and GSEA identified 18 immune-related DEGs. Multiple algorithms identified an RNA panel (CKAP4, BCL6, TLR8) with high diagnostic value for discriminating ICH patients from HTN controls, CTRLs and IS patients (AUCs: 0.93, 0.95 and 0.82; sensitivities: 81.3%, 84.4% and 75%; specificities: 100%, 96% and 79.7%, respectively). Additionally, CKAP4 and TLR8 mRNA and protein levels decreased in RAW264.7 M1 macrophages and increased in RAW264.7 M2 macrophages, while BCL6 expression increased in M1 macrophages but not in M2 macrophages, which may provide potential therapeutic targets for ICH. This study demonstrated that the expression levels of lncRNAs and mRNAs are associated with ICH, and an RNA panel (CKAP4, BCL6, TLR8) was developed as a potential diagnostic tool for distinguishing ICH from IS and controls, which could provide useful insight into ICH diagnosis and pathogenesis.

The circumventricular organs (CVOs) in the central nervous system (CNS) lack a vascular blood-brain barrier (BBB), creating communication sites for sensory or secretory neurons, involved in body homeostasis. Wnt/β-catenin signaling is essential for BBB development and maintenance in endothelial cells (ECs) in most CNS vessels. Here we show that in mouse development, as well as in adult mouse and zebrafish, CVO ECs rendered Wnt-reporter negative, suggesting low level pathway activity. Characterization of the subfornical organ (SFO) vasculature revealed heterogenous claudin-5 (Cldn5) and Plvap/Meca32 expression indicative for tight and leaky vessels, respectively. Dominant, EC-specific β-catenin transcription in mice, converted phenotypically leaky into BBB-like vessels, by augmenting Cldn5+vessels, stabilizing junctions and by reducing Plvap/Meca32+ and fenestrated vessels, resulting in decreased tracer permeability. Endothelial tightening augmented neuronal activity in the SFO of water restricted mice. Hence, regulating the SFO vessel barrier may influence neuronal function in the context of water homeostasis.

The pathogenesis of intracerebral hemorrhage (ICH) can be divided into two distinct injury phases. Almost immediately following ICH, mechanical tissue disruption and mass effect induces primary brain injury, followed by an activated immune response to damaged brain cells and products of red blood cell lysis that drives a secondary wave of brain injury. Secondary brain injury causes increased intracranial pressure and worse pathological outcomes in both pre-clinical models and in ICH patients (Wang, 2010). Modulating the immune response represents a realistic target for therapeutics after ICH, however to date a precise understanding of the specific neuroinflammatory landscape in ICH is currently lacking. Although anti-inflammatory trials for ICH have started, these are based on observations from ischemic stroke and subarachnoid hemorrhage trials (Galea et al., 2017; BLOC-ICH, 2018; Smith et al., 2018). Using transcriptomic profiling to improve our fundamental understanding of the molecular regulation of inflammation after ICH could reveal new therapeutic strategies for patients to improve clinical outcomes after stroke. Leukocytes can contribute to both neuroprotection and neurodegeneration after ICH-induced brain injury. Following primary injury, extravasation of leukocytes into the brain parenchyma contributes to a rise in intracranial pressure and oedema. Activated innate immune cells, such as macrophages and neutrophils, are recruited from the periphery to the brain and generate free radicals that contribute to oxidative damage of neurons (Zhao et al., 2007). Conversely, activated microglia and brain macrophages, are also essential for haematoma clearance and recovery after ICH (Zhao et al., 2015). Studying the transcriptomic profile in these cell types will contribute to our understanding of how these cells are regulated following ICH and may highlight specific molecular targets to modulate the inflammatory response to benefit patients. Here, we employed a zebrafish larval model of spontaneous ICH (bubblehead; bbh) to identify a transcriptomic signature of the innate immune response in the secondary injury phase following ICH. The bbh mutant has a hypomorphic mutation in βpix, resulting in leaky neurovasculature at a critical developmental timepoint (Liu et al., 2007). Zebrafish larvae represent a useful alternative in vivo system for studying ICH pre-clinically. Unlike more commonly used rodent models, ICH in bbh mutant zebrafish larvae can occur without the need for invasive surgical procedures and thereby mimic the spontaneous nature of the human condition more closely. We have previously characterized disease outcomes in the zebrafish bbh model which recapitulates key pathological outcomes observed in mammalian models and patients (Crilly et al., 2018, 2019). The secondary injury response in zebrafish larvae is identified by an increase in macrophage recruitment and activation within the head at 24 h post injury, whilst neutrophils are the most numerous leukocyte population at this developmental stage. Using a double transgenic zebrafish reporter line crossed onto the mutant bbh background, we isolated macrophages and neutrophils from this period of secondary injury 24 h after ICH and performed transcriptomic profiling using RNA-Seq analyses (Figure 1). Data acquired in this study showed differential gene expression in leukocytes between ICH– and ICH+ groups, in addition to the differences between the two cell types (Figure 2). Open in a separate window Figure 1 Experimental workflow and quality control steps for transcriptomic analysis. (A) Diagrammatical representation of the sample acquisition and preparation. Bbh, Bubblehead mutants; mpo, myeloperoxidase; GFP, green fluorescent protein; mpeg1, macrophage expressed gene 1; mCherry, red fluorescent protein; ICH+, hemorrhaged larvae; ICH–, non-hemorrhaged sibling controls; hpf, hours post fertilization; FACS, fluorescence-activated cell sorting; RNA-Seq, RNA sequencing. (B) Representative images of ICH– and ICH+ mpo:mpeg larvae at 72 hpf. Larvae were anesthetized briefly and images were acquired using the Leica M205 FA Stereo fluorescence microscope. Larvae were confirmed to have expression of green mpo positive neutrophils and red mpeg1 positive macrophages, and separated according to presence of ICH (*). Images at higher magnification (top panels) shows an increase in mpeg1 positive cells within the brain as previously observed. (C) Sort strategy to isolate macrophages and neutrophils from ICH+ and ICH– control zebrafish larvae at 72 hpf. From a single cell suspension, neutrophils were sorted based on their expression of GFP and macrophages by their expression of mCherry. Cells expressing both (P5) were excluded from all analyses. Numbers represent frequencies of macrophages and neutrophils as a proportion of viable cells. Plots are representative from three independent replicates. (D) FastQC plots for the quality across all bases from all 12 samples. Graphical representation of the quality for each base pair position in the reads. Green region equates to very good quality calls, orange is reasonable and red poor quality. Box plots show the median value and interquartile range, whiskers include 10 and 90% points. Blue line is overall mean quality.

BACKGROUND: Adipokines and inflammatory factors play an important role in disease progression. Two cardiovascular diseases which have important contributions to mortality and morbidity in China are in-tracerebral hemorrhage (ICH) and myocardial infarction (MI). Acylation stimulating protein has been shown in North American populations to have strong associations with risk factors for MI. Complement C3 (C3) a component of the innate complement immune system is the precursor protein to ASP; C3 has been impli-cated in the pathogenesis of ICH. OBJECTIVE: In this case-control study we examined the association be-tween BMI, lipoproteins adiponectin, C3 and ASP) in a Chinese population. METHODS AND RESULTS: Three groups of subjects were studied: ICH group (N = 41), MI group (N = 60) and a control group (N = 44). There was no difference in BMI for either ICH or MI compared to controls (Control: 22.3 ± 0.3 kg/m2; ICH: 21.3 ± 0.4 vs MI: 22.5 ± 0.2, ICH and MI versus control pNS). The ICH group had lower LDL-C (Control: 3.21 ± 0.13 mmol/L; ICH: 2.54 ± 0.13; MI: 2.99 ± 0.13; ICH vs control p < 0.05), total cholesterol (Control: 5.06 ± 0.16 mmol/L; ICH: 4.40 ± 0.15 ; MI: 4.51 ± 0.14 ; ICH and MI vs control p < 0.05),, HDL-C (Control: 1.34 ± 0.05 mmol/L; ICH: 1.22 ± 0.06; MI: 0.95 ± 0.04; ICH and MI vs control p < 0.05), and C3 (Control: 2.58 ? 0.21 g/L; ICH: 1.85 ? 0.19; MI: 2.87 ? 0.16; ICH vs control p < 0.05), and higher TG (Control: 1.10 ± 0.07 mmol/L; ICH: 1.77 ± 0.17; MI: 1.61 ± 0.10, ICH and MI vs control p < 0.05), compared to the controls. The MI group had lower total cholesterol and HDL-C and higher TG and ASP (Control: 33.70 ? 2.07 nM; ICH: 35.10 ? 2.33; MI: 41.50 ? 1.81; MI vs control p < 0.05) compared to control. CONCLUSION: Chinese men and women who had an MI displayed elevated ASP unrelated to an increase in the precursor protein, C3. Chinese men and women with ICH had ASP levels similar to controls yet lower C3 suggesting that C3, and the regulation of C3 conversion to ASP may be important in ICH disease pathology.

Expression profile of circular RNAs in blood samples of Northern Chinese males with intracerebral hemorrhage shows downregulation of hsa-circ-0090829

Intracerebral hemorrhage (ICH) has features of high disability and high lethality, which is still lack for effective preventions and treatments. Therefore, it is urgent to focus on the key scientific problems facing the clinical treatment of ICH, and carry out basic clinical translational research from the key mechanism of occurrence and development, providing new ideas for ICH preventions and treatments. The previous theories about the pathogenesis of ICH are still need to be further explored. The use of modern gene sequencing and multi-omics methods is expected to reveal the whole picture of the pathogenesis of ICH. Hematoma formation and secondary brain damage are important to the development of ICH. Minimally invasive hematoma removal should be the first choice for ICH treatment in the future, but it is still necessary to push innovative clinical research from multiple disciplines such as treatment time window, treatment methods and radiography. Key words: Intracerebral hemorrhage; Endothelial cell; Minimal invasive surgery

Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes both in normal conditions and in pathological conditions. However, the signaling pathways involved in VEGF-induced angiogenesis are not well defined. Protein kinase D (PKD), a newly described serine/threonine protein kinase, has been implicated in many signal transduction pathways and in cell proliferation. We hypothesized that PKD would mediate VEGF signaling and function in endothelial cells. Here we found that VEGF rapidly and strongly stimulated PKD phosphorylation and activation in endothelial cells via VEGF receptor 2 (VEGFR2). The pharmacological inhibitors for phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) significantly inhibited VEGF-induced PKD activation, suggesting the involvement of the PLCgamma/PKC pathway. In particular, PKCalpha was critical for VEGF-induced PKD activation since both overexpression of adenovirus PKCalpha dominant negative mutant and reduction of PKCalpha expression by small interfering RNA markedly inhibited VEGF-induced PKD activation. Importantly, we found that small interfering RNA knockdown of PKD and PKCalpha expression significantly attenuated ERK activation and DNA synthesis in endothelial cells by VEGF. Taken together, our results demonstrated for the first time that VEGF activates PKD via the VEGFR2/PLCgamma/PKCalpha pathway and revealed a critical role of PKD in VEGF-induced ERK signaling and endothelial cell proliferation.

Introduction: Combining biologically related traits in genome-wide association studies has been demonstrated to increase the power for genetic discovery and to identify new genes associated with intracerebral hemorrhage (ICH). Given the pathological relationship between lobar ICH and cerebral amyloid angiopathy (CAA), and between the latter and levels of CSF amyloid-β 42 (CSF-Aβ 42 ), we leveraged genetic predisposition for lower CSF Aβ 42 levels, as a proxy phenotype for CAA, in pleiotropy analysis for lobar ICH. Methods: We used publicly available GWAS data for Aβ 42 levels (n=3,146) and for lobar ICH (n=1,813) from the International Stroke Genetics Consortium. To evaluate the association between lobar ICH risk and CSF-Aβ 42 , we computed a polygenic risk score (PRS) for CSF-Aβ 42 in lobar ICH patients. We then applied multi-trait analysis of GWAS (MTAG) for pleiotropy analysis of lobar ICH and CSF-Aβ 42 . Gene-level associations based on the MTAG result were further tested using S-Predixcan. Results: We found a significant association between CSF-Aβ 42 PRS and ICH risk (β = -0.35; P = 0.04). MTAG analyses identified one novel near-genome-wide significant association within CDH9 (rs1007589; minor allele frequency=0.09; MTAG β = -0.12 and P = 5.4x10 -8 ; lobar ICH β = 0.34 and P = 2.4x10 -3 ; CSF-Aβ 42 β = -0.03 and P = 4.5x10 -6 ). According to the BRAINEAC brain tissue gene expression database, rs1007589 is significantly associated with increased expression of CDH9 in both temporal and occipital cortices. Discussion: This pleiotropy analysis was designed to identify genetic determinants of lobar ICH driven by amyloid-related mechanisms. We identified a variant in one novel gene associated with both traits, CDH9 , which causes differential expression in two brain regions characteristically affected by vascular amyloid pathology. CDH9 encodes for one subtype of the cadherin superfamily, which regulates intercellular adhesion. Cadherins have been involved in blood-brain barrier integrity and are elevated in AD patients. Further analyses are warranted to understand the effects of the variant on the pathogenesis of ICH and its clinical significance.

To assess the interaction of cerebral amyloid angiopathy (CAA) and arterial hypertension as cofactors for intracerebral hemorrhage (ICH). The authors investigated 129 postmortem brains of hypertensive patients with and without ICH. Sixty-four patients had had deep (n = 40) or lobar (n = 24) ICH. Sixty-five patients without ICH served as controls. Established risk factors for ICH (age, gender, severity of hypertension, bleeding disorders, intake of anticoagulants, and chronic alcoholism) were identified from medical records. Four specimens per brain were stained with hematoxylin-eosin and Congo red. The entire ICH cohort and subgroups were compared with controls using single-factor and multiple logistic regression analyses. CAA was found in 15 of 64 subjects (23%) with ICH and in five of 65 controls (8%; p = 0.026). In single-factor analysis, CAA was more prevalent in lobar ICH compared with controls (p = 0.007) but not in deep ICH. Poor control of hypertension was more prevalent in the entire ICH group (p = 0.01) and in deep ICH (p = 0.016) but not in lobar ICH. ICH was predictive of the presence of CAA (odds ratio: 5.6, 95% CI: 1.8 to 19.5, p = 0.003), and CAA was more likely to be found in lobar ICH in multivariable-adjusted analysis. After adjustment for conventional risk factors, there was a weak association between CAA and deep ICH. Cerebral amyloid angiopathy plays a major role in the pathogenesis of intracerebral hemorrhage even in patients with more evident risk factors.

Intracerebral hemorrhage (ICH) is a major cause of maternal morbidity, but its pathophysiology is poorly characterized. We investigated characteristics of pregnancy-associated ICH (P-ICH), compared with ICH in similar aged nonpregnant adults of both sexes. We performed a retrospective analysis of 134 adults aged 18 to 44 years admitted to our center with nontraumatic ICH from January 1, 2012, to December 31, 2021. We compared ICH characteristics among 3 groups: those with P-ICH (pregnant or within 12 months of end of pregnancy); nonpregnant women; and men. We categorized ICH pathogenesis according to a modified scheme, SMASH-UP (structural, medications, amyloid angiopathy, systemic, hypertension, undetermined, posterior reversible encephalopathy syndrome/reversible cerebral vasoconstriction syndrome), and calculated odds ratios and 95% CIs for primary (spontaneous small-vessel) ICH versus secondary ICH (structural lesions or coagulopathy related), using nonpregnant women as the reference. We also compared specific ICH pathogenesis by SMASH-UP criteria and functional outcomes between groups. Of 134 young adults with nontraumatic ICH, 25 (19%) had P-ICH, of which 60% occurred postpartum. Those with P-ICH had higher odds of primary ICH compared with nonpregnant women (adjusted odds ratio, 4.5 [95% CI, 1.4-14.7]). The odds of primary ICH did not differ between men and nonpregnant women. SMASH-UP pathogenesis for ICH differed significantly between groups (P<0.001). While the in-hospital mortality rate was lowest in the P-ICH group (4%) compared with nonpregnant women (13%) and men (24%), 1 in 4 patients with P-ICH were bedbound and dependent at the time of discharge. In our cohort of young adults with ICH, 1 in 5 was pregnancy related. P-ICH differed in pathogenesis compared with non-pregnancy-related ICH in young adults, suggesting unique pathophysiology.

Nuclear factor-κB and apoptosis in patients with intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is a major cause of death and disability worldwide. Despite treatment advances, reliable prognostic biomarkers are still lacking. While phagocytosis regulation is implicated in ICH pathogenesis, its potential for diagnosis and prognosis remains underexplored. Identifying phagocytic regulatory factors-related genes (PRGs) as biomarkers could provide new insights into ICH. We used multiple datasets from the Gene Expression Omnibus (GEO) database to investigate the role of phagocytic regulatory factors in ICH. We performed Gene Set Variation Analysis (GSVA) on transcriptomic data to evaluate phagocytic regulation in ICH tissues. Single-cell RNA sequencing (scRNA-seq) was used to explore cell-specific PRGs' activity. We then used machine learning for feature selection. Immunoinfiltration levels were assessed using the CIBERSORT algorithm. The biological functions of identified biomarkers were further analyzed through Gene Set Enrichment Analysis (GSEA) and a competing endogenous RNA (ceRNA) network was constructed to reveal interactions between mRNAs, miRNAs, and lncRNAs. GSVA analysis revealed significantly elevated phagocytic regulatory activity in ICH compared to normal brain tissues. Using machine learning methods, four key biomarkers-ANXA2, ANXA5, MGAT1, and VASP-were identified as potential biomarkers with diagnostic and prognostic relevance in ICH. Immunoinfiltration analysis showed a correlation between immune cell infiltration and the identified biomarkers. GSEA highlighted the involvement of these biomarkers in various biological processes. Additionally, the ceRNA network uncovered complex regulatory interactions, where miRNAs and lncRNAs modulated the expression of the identified biomarkers. The four biomarkers offer promising candidates for early detection and may also provide insights into prognosis during post-ICH recovery. These biomarkers are linked to immune cell infiltration and phagocytic regulation, offering new insights into ICH pathogenesis and potential therapeutic targets.

Bioinformatics analysis of potential pathogenesis and risk genes of neuroinflammation-promoted brain injury in intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is a severe neurological disorder with substantial societal implications. Cellular senescence plays a critical role in ICH pathogenesis. This study aims to identify senescence-related biomarkers in ICH for diagnostic and therapeutic purposes. Raw data from GSE24265 in Gene Expression Omnibus was downloaded. Senescence-related genes were acquired from CellAge. Differential gene analysis was done between patients with ICH and controls. The intersection of ICH differentially expressed genes and senescence-related genes for senescence-related ICH genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed. Protein-protein interaction network was constructed through the Search Tool for the Retrieval of Interacting Genes. Single sample gene set enrichment analysis was done for immune cell infiltration and function evaluation in control and ICH groups. miRWalk2.0 database was used for microRNA predictions targeting ICH biomarkers. Transcriptional regulatory relationships unraveled by sentence-based text mining database was employed to predict transcription factors regulating identified biomarkers. Thirteen senescence-related ICH genes were identified. They were primarily enriched in the positive regulation of angiogenesis and the Advanced Glycation End Product -Receptor for AGE signaling pathway in diabetic complications. Validation in the GSE149317 data set and receiver operating characteristic analysis highlighted Caveolin 1, C-X-C Motif Chemokine Ligand 1, ETS proto-oncogene 1, transcription factor, and Serpin Family E Member 1 as potential ICH biomarkers. Single sample gene set enrichment analysis revealed increased Type 2 T helper cell 2_cells, Treg cells, and immune functions like Antigen-presenting cells_co_stimulation in patients with ICH. Fourteen microRNA, including has-miR-6728-3p, were predicted to regulate these biomarkers. transcription factors such as PPARG, RARA, HMGA1, and NFKB1 were identified as potential regulators of the ICH biomarkers. Caveolin 1, C-X-C Motif Chemokine Ligand 1, ETS proto-oncogene 1, transcription factor, and Serpin Family E Member 1 may serve as valuable biomarkers in ICH. Targeting these genes could contribute to ICH prevention and treatment.