Science︱Breakthrough!

Written by ︱ Yuanjia Zheng and Editor ︱ Sizhen Wang The cerebrovascular system consists of a continuous dendritic circulatory vascular duct network, which ensures the delivery of oxygen, energy metabolites and nutrients to the brain, and removes metabolites from the brain, preventing the entry of toxins
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The cerebrovascular system is functionally divided into arteries, arterioles, capillaries, venules, and veins
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Each cerebral blood vessel is composed of endothelial cells, pericytes, smooth muscle cells (SMCs), and perivascular fibroblast-like cells (referred to as perivascular fibroblasts) [1-3]
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Coordinated molecular interactions between vascular cells and peripheral neurons, glial cells, and perivascular immune cells give cerebral blood vessels specific properties
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Single-cell mRNA sequencing (scRNA-seq) in mice suggested certain cellular variations and provided a molecular basis for the "zonations" of arteriovenous phenotypes [3]
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However, at present, the existing human brain cell atlas does not involve the cerebrovascular system, and many neurological diseases such as stroke, Alzheimer's disease, or brain aging are closely related to specific arteriovenous segments [4]
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Therefore, a large-scale single-cell atlas of the human cerebrovascular system will provide a reference to better understand the molecular basis of selective cellular vulnerability and aberrant gene expression patterns in human cerebrovascular disease
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 On January 27, 2022, the research group of Tomasz J.
Nowakowski, Daniel A.
Lim, and Adib A.
Abla from the Department of Neurosurgery at the University of California, San Francisco, USA, published online ahead of print in Science, entitled "A single- cell atlas of the normal and malformed human brain vasculature"
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The authors analyzed the transcriptomes of 181,388 cells to elucidate interactions between blood vessels and immune cells in targeting angiogenesis in vascular malformations and inflammatory programs
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 The research team performed lobectomy in epilepsy patients and obtained normal cerebral cortex tissue and microdissected smaller blood vessels (arterioles, capillaries, and venules) [6-8] (Fig.
1A), followed by scRNA-seq was performed to generate high-quality transcriptomic data from 74,535 cells from 5 individuals
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Major vascular cell types were identified: endothelial cells (CLDN5), pericytes (KCNJ8), SMCs (MYH11), and perivascular fibroblasts (DCN) (Fig.
1 BC) [3,9-10]
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The vascular cell diversity in the adult cerebral cortex was then spatially resolved using multiplex spatial transcriptomics (Fig.
2)
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Brain vascular cell subsets aggregated in various vascular cellular structures, such as arteries, capillaries, and veins (Fig.
2D)
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Combining spatial transcriptomics with information from cell subsets identified from scRNAseq can identify cell classes of major branches of cerebral vessels
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Figure 1 Cell typing of the human cerebrovascular system (Source: Ethan A.
Winkler, et al.
, Science, 2022) Endothelial cells (CLDN5+, PECAM1+) are mainly composed of six subpopulations (Figure 1 EF)
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Comparison of previously annotated mouse endothelial cell maps found that the six subpopulations corresponded to the gene expression signatures of four arteriovenous segments (arteries, capillaries, venules, and veins) [5] (Fig.
1G), and also found Three groups of endothelial cell subtypes within the arterial zoning include enrichment of the gene TXNIP (Fig.
1 EF), which encodes a regulator of glucose metabolism and oxidative stress [11], which may represent the metabolic state
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The authors also determined the location of VEGFC+, MFSD2A+, and ACKR1+ endothelial cells in arteries, capillaries, and veins (Fig.
2D) [3-5]
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Pericytes exist in capillaries, venules and some arterioles, and induce and maintain the blood-brain barrier [6]
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Here the authors propose a new marker to capture more pericytes: HIGD1B
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HIGD1B mRNA was detected in 91.
7% of pericytes (Fig.
1I) and also expressed in PDGFRB+ or KCNJ8+ pericytes (Fig.
2BC)
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SMCs are contractile cells in arteries, veins, and most arterioles [3], and these cells can be distinguished based on markers CNN1, TAGLN, and MYH11 (Fig.
1C,I)
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There may be additional variation in these cell subsets (Fig.
1HI), for example, these cells are enriched for the metallothionein-encoding genes MT1X, MT2A, MT1M, MT1E, and MT1A, which regulate SMC proliferation and migration [12], and coordinate brain response to The perivascular chemokine ligand gene CCL2 in response to systemic infection [13], and RGS16, which regulates sphingosine 1-phosphate signaling and SMC proliferation [14]
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Fibroblasts adhere loosely to arteries, arterioles, venules and veins around blood vessels, express extracellular matrix proteins, and support structures [3]
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The authors found two groups of perivascular fibroblasts marked by DCN+ and APOD+ (Fig.
1 HI) and no other markers of brain fibroblasts, confirming the presence of perivascular fibroblasts in the adult brain
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The authors identified cell populations based on low expression of contractile proteins (TAGLN and ACTA2) and high expression of fibroblast (DCN and LUM) and macrophage (LGALS3) genes in peripheral arteries such as aorta and internal carotid arteries For "fibrous muscle cells" [15-17]
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Differential gene expression identified IGFBP5, KCNT2 and CCL19 as more specific for fibromuscular cells, validating the presence of KCNT2+ and CCL19+ fibromuscular cells in the cortex (Fig.
2 AC), a feature not found in previous mouse cell maps [3,9-10], indicating the existence of fibromuscular cells in human cerebral blood vessels
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Retinoic acid synthase and receptor gene expression was enriched in brain fibromyal cell clusters and perivascular fibroblasts (Fig.
1I), spatially confirming that ALDH1A1 and RARA are in DCN+ perivascular fibroblasts and CCL19+ fibroblasts, respectively expression in cells (Fig.
2 BC)
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Therefore, fibromyocytes and perivascular fibroblasts may be endogenous sources of retinoic acid in the adult brain
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Figure 2 Spatial RNA analysis (Source: Ethan A.
Winkler, et al.
, Science, 2022) Next, in order to verify the usefulness of the data, the authors performed angiography from 5 patients with arteriovenous malformation (AVM) Brain tissue was harvested to generate high-quality whole-cell transcriptome data from 106,853 cells, and 11 major cell populations were identified (Figure 3 BC)
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And spatially verified CLDN5+ endothelial cells, TAGLN+ SMCs, CCL19+ fibromyocytes and COL1A2+ perivascular fibroblasts in AVMs (Fig.
3D)
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To determine molecular changes in endothelial and perivascular cells in AVMs, the authors identified differentially expressed genes in control and AVMs (Fig.
3 EF), identifying aberrant gene expression and cell-specific patterns in AVMs
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AVMs are caused by pathological molecular alterations of endothelial cells [19-20], which induce direct connections between arteries and veins and lead to tortuous, malformed vascular tangles called “focals” [18]
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Combined analysis of the control and AVM datasets revealed that in AVM, endothelial cell subsets were more abundant in arteries and veins (Fig.
expression differences) (Fig.
3H)
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Through gene set enrichment analysis (GSEA), Nd2 enrichment of endothelial cells in AVMs may lead to pathogenic cascades such as angiogenesis, inflammation, and epithelial-mesenchymal transition (Fig.
3J) [21] -22]
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Capillary endothelial cells in the control group highly expressed blood-brain barrier nutrient transporters, including MFSD2A, SLC16A1 and SLC38A5 (Fig.
3K), while AVM Nd2 endothelial cells suppressed the expression of nutrient transporters and up-regulated pro-inflammatory genes (CCL14), Angiogenic genes (PGF and STC1) and permeability-promoting genes (PLVAP and ANGPT2) (Fig.
3 KL)
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In addition, the localization of Nd2 endothelial cells was also verified in AVM lymph nodes (Fig.
3M)
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 Figure 3 Cell abnormalities in malformed cerebral blood vessels (Source: Ethan A.
Winkler, et al.
, Science, 2022) Inflammation may play an important role in the formation of AVMs[18,23]
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Iterative analysis of immune cell populations associated with cerebral blood vessels revealed 17 immune cell populations (Fig.
4 AB), 9 of which consisted of myeloid cells, including vascular-associated microglia, conventional dendritic cells (conventional dendritic cells, cDCs), 3 are perivascular macrophages (perivascular macrophage, pvMφ) subsets, 3 are monocytes (monocyte, Mo) subsets
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In addition, isolated myeloid cells (conventional dendritic cells, ExV) with evidence of in vitro activation [24] found that 8 lymphocyte populations were composed of CD4+ T cells, CD8+ T cell subsets, regulatory T cells (Treg), B cells, natural killer cells (NK), plasmacytoid dendritic cells (pDCs), and dividing lymphocytes (Div) composed of Treg cells (Fig.
4 AB)
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pvMφs were the most abundant immune cell population, accounting for 31.
2% and 28.
3% of immune cells in control and AVMs, respectively (Fig.
4C)
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Greater than 90% of circulating immune cells, such as CD8+ T cells, are confined within the quiescent cerebral vasculature, but in AVMs they infiltrate the perivascular space or adjacent areas, and a greater abundance of bone marrow immune cells also suggests suggested its possible activation in AVMS (Fig.
4DG)
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Vascular-associated CD11c+ antigen-presenting cells are effective activators of in situ responses of brain CD4+ T cells [25-26]
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Vascular-associated CD11c+ cells were observed in the present results to be composed of myeloid cells, including cDCs, pvMφs, and some microglia, and a heterogeneous spatial distribution of vascular-associated antigen-presenting myeloid cells was found in AVM lesions (Fig.
4F).
, discrete domains have more IBA1+ P2RY12- macrophages or IBA1+ P2RY12+ microglia
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A marked perivascular myeloid cell response was observed in AVMs, and IBA1+P2RY12− macrophages were present farther away from the blood vessels, consistent with the infiltration of AVMs (Fig.
4G)
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Thus, there are multiple cellular and spatially heterogeneous cerebrovascular inflammatory responses in AVMs
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Figure 4 Cerebrovascular inflammation and malformation (Source: Ethan A.
Winkler, et al.
, Science, 2022) The authors next sought to identify the deleterious cellular state associated with AVM rupture
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Using Bulk RNA sequencing, scMappR and other calculations, 39 unbroken AVMs and 26 ruptured AVMs were isolated (Fig.
5A) [27]
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We found 871 differential gene expression associated with AVM rupture, which were enriched in vascular development pathways (such as vascular development and morphogenesis) and inflammatory processes (such as cell recruitment)
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AIF1+ (encoding IBA1), a subset of P2RY12− monocytes, and GPNMB+ Mo3 monocytes were abundantly present in ruptured AVMs (Fig.
5B,D)
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It suggested that the infiltration state of immune cells was significantly enhanced after the rupture of AVM
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Inflammation leads to the loss of vascular integrity, which leads to intracerebral hemorrhage after SMCs depletion, and the abundance of GPNMB+ monocytes and SMCs in ruptured AVMs is negatively correlated [23,28]
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Therefore, the authors next investigated whether GPNMB+ monocytes were involved in SMC death
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Co-culture of GPNMB+ monocytes isolated from ruptured AVMs with primary cerebral vascular SMCs (VSMCs) increased apoptotic cleaved caspase-3+ VSMCs (Fig.
5E)
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SPP1 (encoding osteopontin (OPN)) was the most dysregulated export signaling pathway in GPNMB+ monocytes in AVMs (Fig.
5F)
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The ligand OPN was shown to interact with CD44 and integrin receptors on SMCs [29]
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Soluble OPN induced a 2.
7-fold increase in apoptosis in VSMC cells, while neutralizing CD44 antibody and integrin inhibitor ameliorated VSMC apoptosis (Fig.
5G)
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These results suggest that GPNMB+ monocytes contribute to SMC depletion and are associated with AVM rupture and intracerebral hemorrhage
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 Figure 5.
The cellular state of ruptured brain arteriovenous malformations (Source: Ethan A.
Winkler, et al.
, Science, 2022) Conclusions and discussions, inspiration and prospects This paper provides the basic cellular functions and insights of the transcriptome of the human adult cerebral vasculature.
Interaction maps identified endothelial molecular compartments that are critical for altered arteriovenous phenotypes and expanded vascular cellular diversity around brain cells, including fibromuscular cells not previously identified in the cerebral vasculature [3,9-10]
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The atlas has important implications for neuroscience and clinical medicine, and informs future studies in other brain regions or cerebrovascular diseases to accelerate mechanistic understanding and therapeutic targeting of the human cerebrovascular system
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However, this map is only the first step in a comprehensive census of human cerebral vessels.
Due to the limitations of unanticipated biases such as cell capture, isolation and random sampling, the relative cell ratio may change.
Identify differences between cell types and cell states, such as transient or metabolic changes
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Link to the original article: https://doi.
org/10.
1093/cercor/bhac017 Selected articles from previous issues [1] J Neurosci︱ Guo Weixiang's research group reveals the pathological mechanism of neurometabolic disorders in type II hyperlysinemia [2] Cereb Cortex︱Cui Fang/Liu Jie/Gu Ruolei's research group collaborated to reveal the neural mechanism of resource shortage inhibiting prosocial behavior [3] Neuron︱Cao Gang research group revealed a new mechanism by which the nervous system senses pathogenic infection and fine-tunes immune response [4] J Neuroinflammation︱Ge Jinfang/Xia Qingrong's research group reveals the partial therapeutic mechanism of bone marrow mesenchymal stem cell exosomes for Alzheimer's disease【5】Sci Transl Med︱GABAB receptor may rescue visual processing abnormalities in autistic patients【5】 6] Sci Adv︱ Xu Yong/Xu Wenping/He Yanlin collaborated to discover the neural circuit mechanism of estrogen receptor neurons regulating body temperature and movement [7] PNAS︱ Han Chun’s research group revealed a new mechanism of neuronal degeneration caused by external phagocytosis [8] ] Nat Neurosci︱VTA dopaminergic neurons are involved in encoding social prediction error and social reinforcement learning [9]Nature︱New discovery! Inflammatory lymphocytes or new targets mediating CNS inflammation? 【10】Neurosci Bull︱Hu Bo’s research group revealed that neurons in the deep cerebellar nucleus projecting to the ventromedial thalamus are specifically involved in the regulation of combined sensory-motor learning behavior.
Recommended for excellent scientific research training courses 【1】Introduction to multimodal magnetic resonance brain network analysis Class (Online: 2022.
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6~4.
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