A brain-specific angiogenic mechanism enabled by tip cell specialization.

Vertebrate organs require locally adapted blood vessels1,2. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals3-5. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-ß-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.

Molecular mechanism of the interaction between Megalocytivirus-induced virus-mock basement membrane (VMBM) and lymphatic endothelial cells.

IMPORTANCE: Viruses are able to mimic the physiological or pathological mechanism of the host to favor their infection and replication. Virus-mock basement membrane (VMBM) is a Megalocytivirus-induced extracellular structure formed on the surface of infected cells and structurally and functionally mimics the basement membrane of the host. VMBM provides specific support for lymphatic endothelial cells (LECs) rather than blood endothelial cells to adhere to the surface of infected cells, which constitutes a unique phenomenon of Megalocytivirus infection. Here, the structure of VMBM and the interactions between VMBM components and LECs have been analyzed at the molecular level. The regulatory effect of VMBM components on the proliferation and migration of LECs has also been explored. This study helps to understand the mechanism of LEC-specific attachment to VMBM and to address the issue of where the LECs come from in the context of Megalocytivirus infection.

SARS-CoV-2 spike protein induces lung endothelial cell dysfunction and thrombo-inflammation depending on the C3a/C3a receptor signalling.

The spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can interact with endothelial cells. However, no studies demonstrated the direct effect of the spike protein subunit 1 (S1) in inducing lung vascular damage and the potential mechanisms contributing to lung injury. Here, we found that S1 injection in mice transgenic for human angiotensin converting enzyme 2 (ACE2) induced early loss of lung endothelial thromboresistance at 3 days, as revealed by thrombomodulin loss and von Willebrand factor (vWF) increase. In parallel, vascular and epithelial C3 deposits and enhanced C3a receptor (C3aR) expression were observed. These changes preceded diffuse alveolar damage and lung vascular fibrin(ogen)/platelets aggregates at 7 days, as well as inflammatory cell recruitment and fibrosis. Treatment with C3aR antagonist (C3aRa) inhibited lung C3 accumulation and C3a/C3aR activation, limiting vascular thrombo-inflammation and fibrosis. Our study demonstrates that S1 triggers vascular dysfunction and activates complement system, instrumental to lung thrombo-inflammatory injury. By extension, our data indicate C3aRa as a valuable therapeutic strategy to limit S1-dependent lung pathology.

Sphingosine-1-phosphate and its receptors in vascular endothelial and lymphatic barrier function.

The vascular and lymphatic systems both comprise a series of structurally distinct vessels lined with an inner layer of endothelial cells that function to provide a semipermeable barrier to blood and lymph. Regulation of the endothelial barrier is critical for maintaining vascular and lymphatic barrier homeostasis. One of the regulators of endothelial barrier function and integrity is sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite secreted into the blood by erythrocytes, platelets, and endothelial cells and into the lymph by lymph endothelial cells. Binding of S1P to its G protein-coupled receptors, known as S1PR1-5, regulates its pleiotropic functions. This review outlines the structural and functional differences between vascular and lymphatic endothelium and describes current understanding of the importance of S1P/S1PR signaling in regulation of barrier functions. Most studies thus far have been primarily focused on the role of the S1P/S1PR1 axis in vasculature and have been summarized in several excellent reviews, and thus, we will only discuss new perspectives on the molecular mechanisms of action of S1P and its receptors. Much less is known about the responses of the lymphatic endothelium to S1P and the functions of S1PRs in lymph endothelial cells, and this is the major focus of this review. We also discuss current knowledge related to signaling pathways and factors regulated by the S1P/S1PR axis that control lymphatic endothelial cell junctional integrity. Gaps and limitations in current knowledge are highlighted together with the need to further understand the role of S1P receptors in the lymphatic system.

Direct Reprogramming of Resident Non-Myocyte Cells and Its Potential for In Vivo Cardiac Regeneration.

Cardiac diseases are the foremost cause of morbidity and mortality worldwide. The heart has limited regenerative potential; therefore, lost cardiac tissue cannot be replenished after cardiac injury. Conventional therapies are unable to restore functional cardiac tissue. In recent decades, much attention has been paid to regenerative medicine to overcome this issue. Direct reprogramming is a promising therapeutic approach in regenerative cardiac medicine that has the potential to provide in situ cardiac regeneration. It consists of direct cell fate conversion of one cell type into another, avoiding transition through an intermediary pluripotent state. In injured cardiac tissue, this strategy directs transdifferentiation of resident non-myocyte cells (NMCs) into mature functional cardiac cells that help to restore the native tissue. Over the years, developments in reprogramming methods have suggested that regulation of several intrinsic factors in NMCs can help to achieve in situ direct cardiac reprogramming. Among NMCs, endogenous cardiac fibroblasts have been studied for their potential to be directly reprogrammed into both induced cardiomyocytes and induced cardiac progenitor cells, while pericytes can transdifferentiate towards endothelial cells and smooth muscle cells. This strategy has been indicated to improve heart function and reduce fibrosis after cardiac injury in preclinical models. This review summarizes the recent updates and progress in direct cardiac reprogramming of resident NMCs for in situ cardiac regeneration.

BMAL1 involved in autophagy and injury of thoracic aortic endothelial cells of rats induced by intermittent heat stress through the AMPK/mTOR/ULK1 pathway.

Physiological activities of the body exhibit an obvious biological rhythm. At the core of the circadian rhythm, BMAL1 is the only clock gene whose deletion leads to abnormal physiological functions. However, whether intermittent heat stress influences cardiovascular function by altering the circadian rhythm of clock genes has not been reported. This study aimed to investigate whether intermittent heat stress induces autophagy and apoptosis, and the effects of BMAL1 on thoracic aortic autophagy and apoptosis. An intermittent heat stress model was established in vitro, and western blotting and immunofluorescence were used to detect the expression of autophagy, apoptosis, the AMPK/mTOR/ULK1 pathway, and BMAL1. After BMAL1 silencing, RT-qPCR was performed to detect the expression levels of autophagy and apoptosis-related genes. Our results suggest that heat stress induces autophagy and apoptosis in RTAECs. In addition, intermittent heat stress increased the phosphorylation of AMPK and ULK1, but reduced the phosphorylation of mTOR, AMPK inhibitor Compound C reversed the phosphorylation of AMPK, mTOR, and ULK1, and Beclin1 and LC3-II/LC3-I were downregulated. Furthermore, BMAL1 expression was elevated in vitro and shBMAL1 decreased autophagy and apoptosis. We revealed that intermittent heat stress induces autophagy and apoptosis, and that BMAL1 may be involved in the occurrence of autophagy and apoptosis.

Ultraviolet B irradiation induces senescence of human corneal endothelial cells in vitro by DNA damage response and oxidative stress.

The human corneal endothelial cells (HCEnCs) play a vital role in the maintenance of corneal transparency and visual acuity. In our daily life, HCEnCs are inevitably exposed to ultraviolet B (UVB) radiation leading to decreases of visual acuity and corneal transparency resulting in visual loss eventually. Therefore, understanding the UVB-induced cytotoxicity in HCEnCs is of importance for making efficient strategies to protect our vision from UVB-damage. However, in-depth knowledge about UVB-induced cytotoxicity in HCEnCs is missing. Herein, we pulse-irradiated the HCEnCs in vitro with 150 mJ/cm2 UVB (the environmental dose) at each subculture for 4 passages to explore the insights into UVB-induced phototoxicity. The results showed that the UVB-treated HCEnCs exhibit typical senescent characteristics, including significantly enlarged relative cell area, increased senescence-associated ß-galactosidase positive staining, and upregulated p16INK4A and senescence associated secretory phenotypes (SASPs) such as CCL-27, IL-1α/6/8/10, TGF-ß1 and TNF-α, as well as decreased cell proliferation and Lamin B1 expression, and translocation of Lamin B1. Furthermore, we explored the causative mechanisms of senescence and found that 150 mJ/cm2 UVB pulse-irradiation impairs DNA to activate DNA damage response (DDR) pathway of ATM-p53-p21WAF1/CIP1 with downregulated DNA repair enzyme PARP1, leading to cell cycle arrest resulting in DDR-mediated senescence. Meanwhile, UVB pulse-irradiation also elicits a consistent increase of ROS production to aggravate DNA damage and impose oxidative stress on energy metabolism leading to metabolic disturbance resulting in metabolic disturbance-mediated senescence. Altogether, the repeated pulse-irradiation of 150 mJ/cm2 UVB induces HCEnC senescence via both DDR pathway and energy metabolism disturbance.

Extracellular vesicles secreted by human uterine stromal cells regulate decidualization, angiogenesis, and trophoblast differentiation.

In humans, the uterus undergoes a dramatic transformation to form an endometrial stroma-derived secretory tissue, termed decidua, during early pregnancy. The decidua secretes various factors that act in an autocrine/paracrine manner to promote stromal differentiation, facilitate maternal angiogenesis, and influence trophoblast differentiation and development, which are critical for the formation of a functional placenta. Here, we investigated the mechanisms by which decidual cells communicate with each other and with other cell types within the uterine milieu. We discovered that primary human endometrial stromal cells (HESCs) secrete extracellular vesicles (EVs) during decidualization and that this process is controlled by a conserved HIF2α-RAB27B pathway. Mass spectrometry revealed that the decidual EVs harbor a variety of protein cargo, including cell signaling molecules, growth modulators, metabolic regulators, and factors controlling endothelial cell expansion and remodeling. We tested the hypothesis that EVs secreted by the decidual cells mediate functional communications between various cell types within the uterus. We demonstrated that the internalization of EVs, specifically those carrying the glucose transporter 1 (GLUT1), promotes glucose uptake in recipient HESCs, supporting and advancing the decidualization program. Additionally, delivery of HESC-derived EVs into human endothelial cells stimulated their proliferation and led to enhanced vascular network formation. Strikingly, stromal EVs also promoted the differentiation of trophoblast stem cells into the extravillous trophoblast lineage. Collectively, these findings provide a deeper understanding of the pleiotropic roles played by EVs secreted by the decidual cells to ensure coordination of endometrial differentiation and angiogenesis with trophoblast function during the progressive phases of decidualization and placentation.

Effects of intermittent hypoxia with thrombin in an in vitro model of human brain endothelial cells and their impact on PAR-1/PAR-3 cleavage.

Patients with obstructive sleep apnea/hypopnea (OSA) are at high risk of cerebrovascular diseases leading to cognitive impairment. The oxidative stress generated by intermittent hypoxia (IH) could lead to an increase in blood-brain barrier (BBB) permeability, an essential interface for the protection of the brain. Moreover, in patients with OSA, blood coagulation could be increased leading to cardiovascular complications. Thrombin is a factor found increased in these populations that exerts various cellular effects through activation of protease activated receptors (PARs). Thus, we have evaluated in an in vitro BBB model the association of IH with thrombin at two concentrations. We measured the apparent BBB permeability, expression of tight junctions, ROS production, HIF-1α expression, and cleavage of PAR-1/PAR-3. Pre-treatment with dabigatran was performed. IH and higher thrombin concentrations altered BBB permeability: high levels of HIF-1α expression, ROS and PAR-1 activation compared to PAR-3 in such conditions. Conversely, lower concentration of thrombin associated with IH appear to have a protective effect on BBB with a significant cleavage of PAR-3. Dabigatran reversed the deleterious effect of thrombin at high concentrations but also suppressed the beneficial effect of low dose thrombin. Therefore, thrombin and PARs represent novel attractive targets to prevent BBB opening in OSA.

Carbonic anhydrase IX proteoglycan-like and intracellular domains mediate pulmonary microvascular endothelial cell repair and angiogenesis.

The lungs of patients with acute respiratory distress syndrome (ARDS) have hyperpermeable capillaries that must undergo repair in an acidic microenvironment. Pulmonary microvascular endothelial cells (PMVECs) have an acid-resistant phenotype, in part due to carbonic anhydrase IX (CA IX). CA IX also facilitates PMVEC repair by promoting aerobic glycolysis, migration, and network formation. Molecular mechanisms of how CA IX performs such a wide range of functions are unknown. CA IX is composed of four domains known as the proteoglycan-like (PG), catalytic (CA), transmembrane (TM), and intracellular (IC) domains. We hypothesized that the PG and CA domains mediate PMVEC pH homeostasis and repair, and the IC domain regulates aerobic glycolysis and PI3k/Akt signaling. The functions of each CA IX domain were investigated using PMVEC cell lines that express either a full-length CA IX protein or a CA IX protein harboring a domain deletion. We found that the PG domain promotes intracellular pH homeostasis, migration, and network formation. The CA and IC domains mediate Akt activation but negatively regulate aerobic glycolysis. The IC domain also supports migration while inhibiting network formation. Finally, we show that exposure to acidosis suppresses aerobic glycolysis and migration, even though intracellular pH is maintained in PMVECs. Thus, we report that 1) the PG and IC domains mediate PMVEC migration and network formation, 2) the CA and IC domains support PI3K/Akt signaling, and 3) acidosis impairs PMVEC metabolism and migration independent of intracellular pH homeostasis.

Dose-dependent dominance: How cell densities design stromal cell functions during soft tissue healing.

Regular soft tissue healing relies on the well-organized interaction of different stromal cell types with endothelial cells. However, spatiotemporal conditions might provoke high densities of one special stromal cell type, potentially leading to impaired healing. Detailed knowledge of the functions of rivaling stromal cell types aiming for tissue contraction and stabilization as well as vascular support is mandatory. By the application of an in vitro approach comprising the evaluation of cell proliferation, cell morphology, myofibroblastoid differentiation, and cytokine release, we verified a density-dependent modulation of these functions among juvenile and adult fibroblasts, pericytes, and adipose-derived stem cells during their interaction with microvascular endothelial cells in cocultures. Results indicate that juvenile fibroblasts rather support angiogenesis via paracrine regulation at the early stage of healing, a role potentially compromised in adult fibroblasts. In contrast, pericytes showed a more versatile character aiming at angiogenesis, vessel stabilization, and tissue contraction. Such a universal character was even more pronounced among adipose-derived stem cells. The explicit knowledge of the characteristic functions of stromal cell types is a prerequisite for the development of new analytical and therapeutic approaches for impaired soft tissue healing. The present study delivers new considerations concerning the roles of rivaling stromal cell types within a granulation tissue, pointing to extraordinary properties of pericytes and adipose-derived stem cells.

Flow goes forward and cells step backward: endothelial migration.

Systemic and pulmonary circulations constitute a complex organ that serves multiple important biological functions. Consequently, any pathological processing affecting the vasculature can have profound systemic ramifications. Endothelial and smooth muscle are the two principal cell types composing blood vessels. Critically, endothelial proliferation and migration are central to the formation and expansion of the vasculature both during embryonic development and in adult tissues. Endothelial populations are quite heterogeneous and are both vasculature type- and organ-specific. There are profound molecular, functional, and phenotypic differences between arterial, venular and capillary endothelial cells and endothelial cells in different organs. Given this endothelial cell population diversity, it has been challenging to determine the origin of endothelial cells responsible for the angiogenic expansion of the vasculature. Recent technical advances, such as precise cell fate mapping, time-lapse imaging, genome editing, and single-cell RNA sequencing, have shed new light on the role of venous endothelial cells in angiogenesis under both normal and pathological conditions. Emerging data indicate that venous endothelial cells are unique in their ability to serve as the primary source of endothelial cellular mass during both developmental and pathological angiogenesis. Here, we review recent studies that have improved our understanding of angiogenesis and suggest an updated model of this process.

Immunomodulation of Acellular Dermal Matrix Through Interleukin 4 Enhances Vascular Infiltration.

BACKGROUND: Acellular dermal matrix (ADM) supported implant-based reconstruction remains the most commonly performed mode of reconstruction after breast cancer. Acellular dermal matrix clinical usage has reported benefits but requires rapid and efficient vascular and cellular incorporation into the recipient to have the best outcomes. Orderly transition from M1 to M2 macrophage phenotypic profile, coordinated in part by interleukin 4 (IL-4), is an important component of vascular stabilization and remodeling. Using the ADM substrate as a delivery device for immunomodulation of macrophage phenotype holds the potential to improve integration. METHODS: Interleukin 4 was adsorbed onto ADM samples and drug elution curves were measured. Next, experimental groups of 8 C57BL/6 mice had 5-mm ADM discs surgically placed in a dorsal window chamber with a vascularized skin flap on one side and a plastic cover slip on the other in a model of implant-based breast reconstruction. Group 1 consisted of IL-4 (5 µg) adsorbed into the ADM preoperatively and group 2 consisted of an untreated ADM control. Serial gross examinations were performed with histology at day 21 for markers of vascularization, mesenchymal cell infiltration, and macrophage lineage. RESULTS: Drug elution curves showed sustained IL-4 release for 10 days after adsorption. Serial gross examination showed similar rates of superficial vascular investment of the ADM beginning at the periphery by day 14 and increasing through day 21. Interleukin-4 treatment led to significantly increased CD31 staining of vascular endothelial cells within the ADM over the control group (P < 0.05) at 21 days. Although vimentin staining did not indicate a significant increase in fibroblasts overall, IL-4 did result in a significant increase in expression of α-smooth muscle actin. The expression of macrophage phenotype markers Arginase1 and iNOS present within the ADM were not significantly affected by IL-4 treatment at the day 21 time point. CONCLUSIONS: Acellular dermal matrix has the potential to be used for immunomodulatory cytokine delivery during the timeframe of healing. Using implanted ADM as a delivery vehicle to drive IL-4 mediated angiogenesis and vascular remodeling significantly enhanced vascularity within the ADM substrate.

Generation of specialized blood vessels via lymphatic transdifferentiation.

The lineage and developmental trajectory of a cell are key determinants of cellular identity. In the vascular system, endothelial cells (ECs) of blood and lymphatic vessels differentiate and specialize to cater to the unique physiological demands of each organ1,2. Although lymphatic vessels were shown to derive from multiple cellular origins, lymphatic ECs (LECs) are not known to generate other cell types3,4. Here we use recurrent imaging and lineage-tracing of ECs in zebrafish anal fins, from early development to adulthood, to uncover a mechanism of specialized blood vessel formation through the transdifferentiation of LECs. Moreover, we demonstrate that deriving anal-fin vessels from lymphatic versus blood ECs results in functional differences in the adult organism, uncovering a link between cell ontogeny and functionality. We further use single-cell RNA-sequencing analysis to characterize the different cellular populations and transition states involved in the transdifferentiation process. Finally, we show that, similar to normal development, the vasculature is rederived from lymphatics during anal-fin regeneration, demonstrating that LECs in adult fish retain both potency and plasticity for generating blood ECs. Overall, our research highlights an innate mechanism of blood vessel formation through LEC transdifferentiation, and provides in vivo evidence for a link between cell ontogeny and functionality in ECs.

Cell-Crossing Functional Network Driven by microRNA-125a Regulates Endothelial Permeability and Monocyte Trafficking in Acute Inflammation.

Opening of the endothelial barrier and targeted infiltration of leukocytes into the affected tissue are hallmarks of the inflammatory response. The molecular mechanisms regulating these processes are still widely elusive. In this study, we elucidate a novel regulatory network, in which miR-125a acts as a central hub that regulates and synchronizes both endothelial barrier permeability and monocyte migration. We found that inflammatory stimulation of endothelial cells induces miR-125a expression, which consecutively inhibits a regulatory network consisting of the two adhesion molecules VE-Cadherin (CDH5) and Claudin-5 (CLDN5), two regulatory tyrosine phosphatases (PTPN1, PPP1CA) and the transcription factor ETS1 eventually leading to the opening of the endothelial barrier. Moreover, under the influence of miR-125a, endothelial expression of the chemokine CCL2, the most predominant ligand for the monocytic chemokine receptor CCR2, was strongly enhanced. In monocytes, on the other hand, we detected markedly repressed expression levels of miR-125a upon inflammatory stimulation. This induced a forced expression of its direct target gene CCR2, entailing a strongly enhanced monocyte chemotaxis. Collectively, cell-type-specific differential expression of miR-125a forms a synergistic functional network controlling monocyte trafficking across the endothelial barrier towards the site of inflammation. In addition to the known mechanism of miRNAs being shuttled between cells via extracellular vesicles, our study uncovers a novel dimension of miRNA function: One miRNA, although disparately regulated in the cells involved, directs a biologic process in a synergistic and mutually reinforcing manner. These findings provide important new insights into the regulation of the inflammatory cascade and may be of great use for future clinical applications.

c-Maf: The magic wand that turns on LSEC fate.

In this issue of Cell Stem Cell, Gómez-Salinero et al. (2022) identify c-Maf as a driver for murine liver sinusoidal endothelial cell (LSEC) fate and function during liver development, homeostasis, and repair. Similarly, c-Maf defines human LSECs, and its overexpression specializes generic HUVECs into functional induced-LSECs, potentiating regenerative therapeutics.

Dynamics of Endothelial Engagement and Filopodia Formation in Complex 3D Microscaffolds.

The understanding of endothelium-extracellular matrix interactions during the initiation of new blood vessels is of great medical importance; however, the mechanobiological principles governing endothelial protrusive behaviours in 3D microtopographies remain imperfectly understood. In blood capillaries submitted to angiogenic factors (such as vascular endothelial growth factor, VEGF), endothelial cells can transiently transdifferentiate in filopodia-rich cells, named tip cells, from which angiogenesis processes are locally initiated. This protrusive state based on filopodia dynamics contrasts with the lamellipodia-based endothelial cell migration on 2D substrates. Using two-photon polymerization, we generated 3D microstructures triggering endothelial phenotypes evocative of tip cell behaviour. Hexagonal lattices on pillars ("open"), but not "closed" hexagonal lattices, induced engagement from the endothelial monolayer with the generation of numerous filopodia. The development of image analysis tools for filopodia tracking allowed to probe the influence of the microtopography (pore size, regular vs. elongated structures, role of the pillars) on orientations, engagement and filopodia dynamics, and to identify MLCK (myosin light-chain kinase) as a key player for filopodia-based protrusive mode. Importantly, these events occurred independently of VEGF treatment, suggesting that the observed phenotype was induced through microtopography. These microstructures are proposed as a model research tool for understanding endothelial cell behaviour in 3D fibrillary networks.

Temporal analyses of postnatal liver development and maturation by single-cell transcriptomics.

The postnatal development and maturation of the liver, the major metabolic organ, are inadequately understood. We have analyzed 52,834 single-cell transcriptomes and identified 31 cell types or states in mouse livers at postnatal days 1, 3, 7, 21, and 56. We observe unexpectedly high levels of hepatocyte heterogeneity in the developing liver and the progressive construction of the zonated metabolic functions from pericentral to periportal hepatocytes, which is orchestrated with the development of sinusoid endothelial, stellate, and Kupffer cells. Trajectory and gene regulatory analyses capture 36 transcription factors, including a circadian regulator, Bhlhe40, in programming liver development. Remarkably, we identified a special group of macrophages enriched at day 7 with a hybrid phenotype of macrophages and endothelial cells, which may regulate sinusoidal construction and Treg-cell function. This study provides a comprehensive atlas that covers all hepatic cell types and is instrumental for further dissection of liver development, metabolism, and disease.

IL-6-induced FOXO1 activity determines the dynamics of metabolism in CD8 T cells cross-primed by liver sinusoidal endothelial cells.

Liver sinusoidal endothelial cells (LSECs) are liver-resident antigen (cross)-presenting cells that generate memory CD8 T cells, but metabolic properties of LSECs and LSEC-primed CD8 T cells remain understudied. Here, we report that high-level mitochondrial respiration and constitutive low-level glycolysis support LSEC scavenger and sentinel functions. LSECs fail to increase glycolysis and co-stimulation after TLR4 activation, indicating absence of metabolic and functional maturation compared with immunogenic dendritic cells. LSEC-primed CD8 T cells show a transient burst of oxidative phosphorylation and glycolysis. Mechanistically, co-stimulatory IL-6 signaling ensures high FOXO1 expression in LSEC-primed CD8 T cells, curtails metabolic activity associated with T cell activation, and is indispensable for T cell functionality after re-activation. Thus, distinct immunometabolic features characterize non-immunogenic LSECs compared with immunogenic dendritic cells and LSEC-primed CD8 T cells with memory features compared with effector CD8 T cells. This reveals local features of metabolism and function of T cells in the liver.

A human pluripotent stem cell-based model of SARS-CoV-2 infection reveals an ACE2-independent inflammatory activation of vascular endothelial cells through TLR4.

To date, the direct causative mechanism of SARS-CoV-2-induced endotheliitis remains unclear. Here, we report that human ECs barely express surface ACE2, and ECs express less intracellular ACE2 than non-ECs of the lungs. We ectopically expressed ACE2 in hESC-ECs to model SARS-CoV-2 infection. ACE2-deficient ECs are resistant to the infection but are more activated than ACE2-expressing ones. The virus directly induces endothelial activation by increasing monocyte adhesion, NO production, and enhanced phosphorylation of p38 mitogen-associated protein kinase (MAPK), NF-κB, and eNOS in ACE2-expressing and -deficient ECs. ACE2-deficient ECs respond to SARS-CoV-2 through TLR4 as treatment with its antagonist inhibits p38 MAPK/NF-κB/ interleukin-1ß (IL-1ß) activation after viral exposure. Genome-wide, single-cell RNA-seq analyses further confirm activation of the TLR4/MAPK14/RELA/IL-1ß axis in circulating ECs of mild and severe COVID-19 patients. Circulating ECs could serve as biomarkers for indicating patients with endotheliitis. Together, our findings support a direct role for SARS-CoV-2 in mediating endothelial inflammation in an ACE2-dependent or -independent manner.