An unexpected role of neutrophils in clearing apoptotic hepatocytes in vivo.

Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here, we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver, and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.

Every day, the immune cells clears the remains of billions of old and damaged cells that have undergone a controlled form of death. Removing them quickly helps to prevent inflammation or the development of autoimmune diseases. While immune cells called neutrophils are generally tasked with removing invading bacteria, macrophages are thought to be responsible for clearing dead cells. However, in healthy tissue, the process occurs so efficiently that it can be difficult to confirm which cells are responsible. To take a closer look, Cao et al. focused on the liver by staining human samples to identify both immune and dead cells. Unexpectedly, there were large numbers of neutrophils visible inside dead liver cells. Further experiments in mice revealed that after entering the dead cells, neutrophils engulfed the contents and digested the dead cell from the inside out. This was a surprising finding because not only are neutrophils not usually associated with dead cells, but immune cells usually engulf cells and bacteria from the outside rather than burrowing inside them. The importance of this neutrophil behaviour was shown when Cao et al. studied samples from patients with an autoimmune disease where immune cells attack the liver. In this case, very few dead liver cells contained neutrophils, and the neutrophils themselves did not seem capable of removing the dead cells, leading to inflammation. This suggests that defective neutrophil function could be a key contributor to this autoimmune disease. The findings identify a new role for neutrophils in maintaining healthy functioning of the liver and reveal a new target in the treatment of autoimmune diseases. In the future, Cao et al. plan to explore whether compounds that enhance clearance of dead cells by neutrophils can be used to treat autoimmune liver disease in mouse models of the disease.

Trained immunity of alveolar macrophages enhances injury resolution via KLF4-MERTK-mediated efferocytosis.

Recent studies suggest that training of innate immune cells such as tissue-resident macrophages by repeated noxious stimuli can heighten host defense responses. However, it remains unclear whether trained immunity of tissue-resident macrophages also enhances injury resolution to counterbalance the heightened inflammatory responses. Here, we studied lung-resident alveolar macrophages (AMs) prechallenged with either the bacterial endotoxin or with Pseudomonas aeruginosa and observed that these trained AMs showed greater resilience to pathogen-induced cell death. Transcriptomic analysis and functional assays showed greater capacity of trained AMs for efferocytosis of cellular debris and injury resolution. Single-cell high-dimensional mass cytometry analysis and lineage tracing demonstrated that training induces an expansion of a MERTKhiMarcohiCD163+F4/80low lung-resident AM subset with a proresolving phenotype. Reprogrammed AMs upregulated expression of the efferocytosis receptor MERTK mediated by the transcription factor KLF4. Adoptive transfer of these trained AMs restricted inflammatory lung injury in recipient mice exposed to lethal P. aeruginosa. Thus, our study has identified a subset of tissue-resident trained macrophages that prevent hyperinflammation and restore tissue homeostasis following repeated pathogen challenges.

Endosomal trafficking of two-pore K+ efflux channel TWIK2 to plasmalemma mediates NLRP3 inflammasome activation and inflammatory injury.

Potassium efflux via the two-pore K+ channel TWIK2 is a requisite step for the activation of NLRP3 inflammasome, however, it remains unclear how K+ efflux is activated in response to select cues. Here, we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in the extrusion of K+. We showed that ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP-ligated purinergic receptor P2X7 each prevented endosomal fusion with the plasmalemma and K+ efflux as well as NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-depleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. We conclude that Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 localization and activity at the cell surface and the downstream activation of the NLRP3 inflammasome. Results show that endosomal trafficking of TWIK2 to the plasmalemma is a potential therapeutic target in acute or chronic inflammatory states.

An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) neutralize infection and are efficacious for the treatment of COVID-19. However, SARS-CoV-2 variants, notably sublineages of B.1.1.529/omicron, have emerged that escape antibodies in clinical use. As an alternative, soluble decoy receptors based on the host entry receptor ACE2 broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE22 .v2.4-IgG1 was previously shown to be effective against SARS-CoV-2 variants when administered intravenously. Here, inhalation of aerosolized sACE22 .v2.4-IgG1 increased survival and ameliorated lung injury in K18-hACE2 mice inoculated with P.1/gamma virus. Loss of catalytic activity reduced the decoy's therapeutic efficacy, which was further confirmed by intravenous administration, supporting dual mechanisms of action: direct blocking of S and turnover of ACE2 substrates associated with lung injury and inflammation. Furthermore, sACE22 .v2.4-IgG1 tightly binds and neutralizes BA.1, BA.2, and BA.4/BA.5 omicron and protects K18-hACE2 mice inoculated with a high dose of BA.1 omicron virus. Overall, the therapeutic potential of sACE22 .v2.4-IgG1 is demonstrated by the inhalation route and broad neutralization potency persists against highly divergent SARS-CoV-2 variants.

Single-cell transcriptomic profiling of lung endothelial cells identifies dynamic inflammatory and regenerative subpopulations.

Studies have demonstrated the phenotypic heterogeneity of vascular endothelial cells (ECs) within a vascular bed; however, little is known about how distinct endothelial subpopulations in a particular organ respond to an inflammatory stimulus. We performed single-cell RNA-Seq of 35,973 lung ECs obtained during baseline as well as postinjury time points after inflammatory lung injury induced by LPS. Seurat clustering and gene expression pathway analysis identified 2 major subpopulations in the lung microvascular endothelium, a subpopulation enriched for expression of immune response genes such as MHC genes (immuneEC) and another defined by increased expression of vascular development genes such as Sox17 (devEC). The presence of immuneEC and devEC subpopulations was also observed in nonhuman primate lungs infected with SARS-CoV-2 and murine lungs infected with H1N1 influenza virus. After the peak of inflammatory injury, we observed the emergence of a proliferative lung EC subpopulation. Overexpression of Sox17 prevented inflammatory activation in ECs. Thus, there appeared to be a "division of labor" within the lung microvascular endothelium in which some ECs showed propensity for inflammatory signaling and others for endothelial regeneration. These results provide underpinnings for the development of targeted therapies to limit inflammatory lung injury and promote regeneration.

Nanoparticle targeting of de novo profibrotic macrophages mitigates lung fibrosis.

The pathogenesis of lung fibrosis involves hyperactivation of innate and adaptive immune pathways that release inflammatory cytokines and growth factors such as tumor growth factor (TGF)ß1 and induce aberrant extracellular matrix protein production. During the genesis of pulmonary fibrosis, resident alveolar macrophages are replaced by a population of newly arrived monocyte-derived interstitial macrophages that subsequently transition into alveolar macrophages (Mo-AMs). These transitioning cells initiate fibrosis by releasing profibrotic cytokines and remodeling the matrix. Here, we describe a strategy for leveraging the up-regulation of the mannose receptor CD206 in interstitial macrophages and Mo-AM to treat lung fibrosis. We engineered mannosylated albumin nanoparticles, which were found to be internalized by fibrogenic CD206+ monocyte derived macrophages (Mo-Macs). Mannosylated albumin nanoparticles incorporating TGFß1 small-interfering RNA (siRNA) targeted the profibrotic subpopulation of CD206+ macrophages and prevented lung fibrosis. The findings point to the potential utility of mannosylated albumin nanoparticles in delivering TGFß-siRNA into CD206+ profibrotic macrophages as an antilung fibrosis strategy.

Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants.

Vaccine hesitancy and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) escaping vaccine-induced immune responses highlight the urgency for new COVID-19 therapeutics. Engineered angiotensin-converting enzyme 2 (ACE2) proteins with augmented binding affinities for SARS-CoV-2 spike (S) protein may prove to be especially efficacious against multiple variants. Using molecular dynamics simulations and functional assays, we show that three amino acid substitutions in an engineered soluble ACE2 protein markedly augmented the affinity for the S protein of the SARS-CoV-2 WA-1/2020 isolate and multiple VOCs: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). In humanized K18-hACE2 mice infected with the SARS-CoV-2 WA-1/2020 or P.1 variant, prophylactic and therapeutic injections of soluble ACE22.v2.4-IgG1 prevented lung vascular injury and edema formation, essential features of CoV-2-induced SARS, and above all improved survival. These studies demonstrate broad efficacy in vivo of an engineered ACE2 decoy against SARS-CoV-2 variants in mice and point to its therapeutic potential.

Mechanisms of Lung Injury Induced by SARS-CoV-2 Infection.

The lung is the major target organ of SARS-CoV-2 infection, which causes COVID-19. Here, we outline the multistep mechanisms of lung epithelial and endothelial injury induced by SARS-CoV-2: direct viral infection, chemokine/cytokine-mediated damage, and immune cell-mediated lung injury. Finally, we discuss the recent progress in terms of antiviral therapeutics as well as the development of anti-inflammatory or immunomodulatory therapeutic approaches. This review also provides a systematic overview of the models for studying SARS-CoV-2 infection and discusses how an understanding of mechanisms of lung injury will help identify potential targets for future drug development to mitigate lung injury.

CD38-mediated Inhibition of Bruton's Tyrosine Kinase in Macrophages Prevents Endotoxemic Lung Injury.

TLR4 signaling via endotoxemia in macrophages promotes macrophage transition to the inflammatory phenotype through NLRP3 inflammasome activation. This transition event has the potential to trigger acute lung injury (ALI). However, relatively little is known about the regulation of NLRP3 and its role in the pathogenesis of ALI. Here we interrogated the signaling pathway activated by CD38, an ectoenzyme expressed in macrophages, in preventing ALI through suppressing NLRP3 activation. Wild-type and Cd38-knockout (Cd38-/-) mice were used to assess inflammatory lung injury, and isolated macrophages were used to delineate underlying TLR4 signaling pathway. We showed that CD38 suppressed TLR4 signaling in macrophages by inhibiting Bruton's tyrosine kinase (Btk) through the recruitment of Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) and resulting in the dephosphorylation of activated Btk. Cd38-/- mice show enhanced lung polymorphonuclear leukocyte extravasation and severe lung injury. LPS- or polymicrobial sepsis-induced mortality in Cd38-/- mice were markedly augmented compared with wild types. CD38 in macrophages functioned by inhibiting Btk activation through activation of SHP2 and resulting dephosphorylation of Btk, and thereby preventing activation of downstream targets NF-κB and NLRP3. Cd38-/- macrophages displayed markedly increased activation of Btk, NF-κB, and NLRP3, whereas in vivo administration of the Btk inhibitor ibrutinib (a Food and Drug Administration-approved drug) prevented augmented TLR4-induced inflammatory lung injury seen in Cd38-/- mice. Our findings together show upregulation of CD38 activity and inhibition of Btk activation downstream of TLR4 activation as potential strategies to prevent endotoxemic ALI.

KIF13B-mediated VEGFR2 trafficking is essential for vascular leakage and metastasis in vivo.

VEGF-A induces vascular leakage and angiogenesis via activating the cell surface localized receptor VEGF receptor 2 (VEGFR2). The amount of available VEGFR2 at the cell surface is however tightly regulated by trafficking of VEGFR2 by kinesin family 13 B (KIF13B), a plus-end kinesin motor, to the plasma membrane of endothelial cells (ECs). Competitive inhibition of interaction between VEGFR2 and KIF13B by a peptide kinesin-derived angiogenesis inhibitor (KAI) prevented pathological angiogenesis in models of cancer and eye disease associated with defective angiogenesis. Here, we show the protective effects of KAI in VEGF-A-induced vascular leakage and cancer metastasis. Using an EC-specific KIF13B knockout (Kif13b iECKO ) mouse model, we demonstrated the function of EC expressed KIF13B in mediating VEGF-A-induced vascular leakage, angiogenesis, tumor growth, and cancer metastasis. Thus, KIF13B-mediated trafficking of VEGFR2 to the endothelial surface has an essential role in pathological angiogenesis induced by VEGF-A, and is therefore a potential therapeutic target.

VEGFR2 Trafficking by KIF13B Is a Novel Therapeutic Target for Wet Age-Related Macular Degeneration.

Purpose: Vascular endothelial growth factor (VEGF) and its receptor VEGFR2 are promising therapeutic targets for wet age-related macular degeneration (AMD). As a topically applicable option, we developed the peptide KAI to selectively interfere with VEGFR2 trafficking to the cell surface where it receives VEGF. This study sought to determine the efficacy of KAI in the mouse model of choroidal neovascularization (CNV). Methods: The specificity of KAI was tested by surface plasmon resonance. The drug delivery was analyzed by cryosection and the ELISA after treatment of KAI eyedrop to the mouse eyes. For the laser-induced CNV model, mice with laser-induced ruptures in Bruch's membrane received daily treatment of KAI eyedrop or control peptide. The other groups of mice received intravitreal injection of anti-VEGF or IgG control. After two weeks, CNV was quantified and compared. Results: First, we showed the specificity and high affinity of KAI to VEGFR2. Next, biodistribution revealed successful delivery of KAI eyedrop to the back of the mouse eyes. KAI significantly reduced the disease progression in laser-induced CNV. The comparison with current therapy suggests that KAI eyedrop is as effective as current therapy to prevent CNV in wet AMD. Moreover, the genetic deletion of a kinesin KIF13B, which mediates VEGFR2 trafficking to the cell surface, confirmed the pivotal role of KIF13B in disease progression of wet AMD and neovascularization from choroidal vessels. Conclusions: Taken together, pharmacologic inhibition and genetic deletion complementarily suggest the therapeutic possibility of targeting VEGFR2 trafficking to inhibit pathological angiogenesis in wet AMD.

The angiocrine Rspondin3 instructs interstitial macrophage transition via metabolic-epigenetic reprogramming and resolves inflammatory injury.

Macrophages demonstrate remarkable plasticity that is essential for host defense and tissue repair. The tissue niche imprints macrophage identity, phenotype and function. The role of vascular endothelial signals in tailoring the phenotype and function of tissue macrophages remains unknown. The lung is a highly vascularized organ and replete with a large population of resident macrophages. We found that, in response to inflammatory injury, lung endothelial cells release the Wnt signaling modulator Rspondin3, which activates ß-catenin signaling in lung interstitial macrophages and increases mitochondrial respiration by glutaminolysis. The generated tricarboxylic acid cycle intermediate α-ketoglutarate, in turn, serves as the cofactor for the epigenetic regulator TET2 to catalyze DNA hydroxymethylation. Notably, endothelial-specific deletion of Rspondin3 prevented the formation of anti-inflammatory interstitial macrophages in endotoxemic mice and induced unchecked severe inflammatory injury. Thus, the angiocrine-metabolic-epigenetic signaling axis specified by the endothelium is essential for reprogramming interstitial macrophages and dampening inflammatory injury.

High-loading Gα13-binding EXE peptide nanoparticles prevent thrombosis and protect mice from cardiac ischemia/reperfusion injury.

Inefficient delivery is a major obstacle to the development of peptide-based drugs targeting the intracellular compartment. We recently showed that selectively inhibiting integrin outside-in signaling using a peptide (mP6) derived from the Gα13-binding ExE motif within the integrin ß3 cytoplasmic domain had antithrombotic effects. Here, we engineered lipid-stabilized, high-loading peptide nanoparticles (HLPN), in which a redesigned ExE peptide (M3mP6) constituted up to 70% of the total nanoparticle molarity, allowing efficient in vivo delivery. We observed that M3mP6 HLPN inhibited occlusive thrombosis more potently than a clopidogrel/aspirin combination without adverse effects on hemostasis in rodents. Furthermore, M3mP6 HLPN synergized with P2Y12 receptor inhibitors or the clopidogrel/aspirin combination in preventing thrombosis, without exacerbating hemorrhage. M3mP6 HLPN also inhibited intravascular coagulation more potently than the P2Y12 inhibitor cangrelor. Postischemia injection of M3mP6 HLPN protected the heart from myocardial ischemia-reperfusion injury in a mouse model. This study demonstrates an efficient in vivo peptide delivery strategy for a therapeutic that not only efficaciously prevented thrombosis with minimal bleeding risk but also protected from myocardial ischemia-reperfusion injury in mice.

Angiocrine Sphingosine-1-Phosphate Activation of S1PR2-YAP Signaling Axis in Alveolar Type II Cells Is Essential for Lung Repair.

Lung alveolar epithelium is composed of alveolar type I (AT1) and type II (AT2) cells. AT1 cells mediate gas exchange, whereas AT2 cells act as progenitor cells to repair injured alveoli. Lung microvascular endothelial cells (LMVECs) play a crucial but still poorly understood role in regulating alveolar repair. Here, we studied the role of the LMVEC-derived bioactive lipid sphingosine-1-phosphate (S1P) in promoting alveolar repair using mice with endothelial-specific deletion of sphingosine kinase 1 (Sphk1), the key enzyme promoting S1P generation. These mutant lungs developed airspace-enlargement lesions and exhibited a reduced number of AT1 cells after Pseudomonas-aeruginosa-induced lung injury. We demonstrated that S1P released by LMVECs acted via its receptor, S1PR2, on AT2 cells and induced nuclear translocation of yes-associated protein (YAP), a regulator of AT2 to AT1 transition. Thus, angiocrine S1P released after injury acts via the S1PR2-YAP signaling axis on AT2 cells to promote AT2 to AT1 differentiation required for alveolar repair.

PV1 in Caveolae Controls Lung Endothelial Permeability.

Caveolae are prominent plasmalemmal invaginations in endothelial cells, especially in the lung vasculature, which comprises a vast surface area. PV1 (plasmalemmal vesicle-associated protein-1), a 60-kD glycoprotein expressed in endothelial cells, is essential for generating spoke-like diaphragmatic structures that span the neck region of endothelial caveolae. However, their role in caveolae-mediated uptake and endothelial-barrier function is unknown. Here, we generated mice with endothelial cell-specific deletion of PV1 through tamoxifen-induced Cdh5.Cre.ERT2 (endothelial-specific vascular cadherin.Cre.estrogen receptor 2)-mediated excision of the floxed PV1 allele. We observed that loss of PV1 specifically in endothelial cells increased lung vascular permeability of fluid and protein, indicating that PV1 is required for maintenance of lung vascular-barrier integrity. Endothelial-specific PV1 deletion also increased caveolae-mediated uptake of tracer albumin compared with controls, promoted Au-albumin accumulation in the bulb of caveolae, and induced caveolar swelling. In addition, we observed the progressive loss of plasma proteins from the circulation and reduced arterial pressure resulting from transudation of water and protein as well as edema formation in multiple tissues, including lungs. These changes seen after endothelial-specific PV1 deletion occurred in the absence of disruption of endothelial junctions. We demonstrated that exposure of wild-type mice to endotoxin, which is known to cause acute lung injury and increase protein permeability, also significantly reduced PV1 protein expression. We conclude that the key function of PV1 is to regulate lung endothelial permeability through its ability to restrict the entry of plasma proteins such as albumin into caveolae and their transport through the endothelial barrier.

IL-1ß suppression of VE-cadherin transcription underlies sepsis-induced inflammatory lung injury.

Unchecked inflammation is a hallmark of inflammatory tissue injury in diseases such as acute respiratory distress syndrome (ARDS). Yet the mechanisms of inflammatory lung injury remain largely unknown. Here we showed that bacterial endotoxin lipopolysaccharide (LPS) and cecal ligation and puncture-induced (CLP-induced) polymicrobial sepsis decreased the expression of transcription factor cAMP response element binding (CREB) in lung endothelial cells. We demonstrated that endothelial CREB was crucial for VE-cadherin transcription and the formation of the normal restrictive endothelial adherens junctions. The inflammatory cytokine IL-1ß reduced cAMP generation and CREB-mediated transcription of VE-cadherin. Furthermore, endothelial cell-specific deletion of CREB induced lung vascular injury whereas ectopic expression of CREB in the endothelium prevented the injury. We also observed that rolipram, which inhibits type 4 cyclic nucleotide phosphodiesterase-mediated (PDE4-mediated) hydrolysis of cAMP, prevented endotoxemia-induced lung vascular injury since it preserved CREB-mediated VE-cadherin expression. These data demonstrate the fundamental role of the endothelial cAMP-CREB axis in promoting lung vascular integrity and suppressing inflammatory injury. Therefore, strategies aimed at enhancing endothelial CREB-mediated VE-cadherin transcription are potentially useful in preventing sepsis-induced lung vascular injury in ARDS.

mtDNA Activates cGAS Signaling and Suppresses the YAP-Mediated Endothelial Cell Proliferation Program to Promote Inflammatory Injury.

Cytosolic DNA acts as a universal danger-associated molecular pattern (DAMP) signal; however, the mechanisms of self-DNA release into the cytosol and its role in inflammatory tissue injury are not well understood. We found that the internalized bacterial endotoxin lipopolysaccharide (LPS) activated the pore-forming protein Gasdermin D, which formed mitochondrial pores and induced mitochondrial DNA (mtDNA) release into the cytosol of endothelial cells. mtDNA was recognized by the DNA sensor cGAS and generated the second messenger cGAMP, which suppressed endothelial cell proliferation by downregulating YAP1 signaling. This indicated that the surviving endothelial cells in the penumbrium of the inflammatory injury were compromised in their regenerative capacity. In an experimental model of inflammatory lung injury, deletion of cGas in mice restored endothelial regeneration. The results suggest that targeting the endothelial Gasdermin D activated cGAS-YAP signaling pathway could serve as a potential strategy for restoring endothelial function after inflammatory injury.

Alveolar Stretch Activation of Endothelial Piezo1 Protects Adherens Junctions and Lung Vascular Barrier.

Disruption of alveolar-capillary barriers is a major complication of high-volume mechanical ventilation referred to as "ventilator-induced lung injury." The stretching force in alveoli is transmitted to endothelial cells, increasing the tension on underlying endothelial plasma membrane. The mechanosensor Piezo1, a plasma membrane cation channel, was inducibly deleted in endothelial cells of mice (Piezo1iEC-/-), which allowed us to study its role in regulating the endothelial barrier response to alveolar stretch. We observed significant increase in lung vascular permeability in Piezo1iEC-/- mice as compared with control Piezo1fl/fl mice in response to high-volume mechanical ventilation. We also observed that human lung endothelial monolayers depleted of Piezo1 and exposed to cyclic stretch had increased permeability. We identified the calcium-dependent cysteine protease calpain as a downstream target of Piezo1. Furthermore, we showed that calpain maintained stability of the endothelial barrier in response to mechanical stretch by cleaving Src kinase, which was responsible for disassembling endothelial adherens junctions. Pharmacological activation of calpain caused Src cleavage and thereby its inactivation, and it restored the disrupted lung endothelial barrier seen in Piezo1iEC-/- mice undergoing high-volume mechanical ventilation. Our data demonstrate that downregulation of Piezo1 signaling in endothelium is a critical factor in the pathogenesis of ventilator-induced lung injury, and thus augmenting Piezo1 expression or pharmacologically activating Piezo1 signaling may be an effective therapeutic strategy.

Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling.

The vascular endothelial growth factor-A (VEGF-A)-VEGFR2 pathway drives tumor vascularization by activating proangiogenic signaling in endothelial cells (ECs). Here, we show that EC-sphingosine-1-phosphate receptor 1 (S1PR1) amplifies VEGFR2-mediated angiogenic signaling to enhance tumor growth. We show that cancer cells induce S1PR1 activity in ECs, and thereby, conditional deletion of S1PR1 in ECs (EC-S1pr1-/- mice) impairs tumor vascularization and growth. Mechanistically, we show that S1PR1 engages the heterotrimeric G-protein Gi, which amplifies VEGF-VEGFR2 signaling due to an increase in the activity of the tyrosine kinase c-Abl1. c-Abl1, by phosphorylating VEGFR2 at tyrosine-951, prolongs VEGFR2 retention on the plasmalemma to sustain Rac1 activity and EC migration. Thus, S1PR1 or VEGFR2 antagonists, alone or in combination, reverse the tumor growth in control mice to the level seen in EC-S1pr1-/- mice. Our findings suggest that blocking S1PR1 activity in ECs has the potential to suppress tumor growth by preventing amplification of VEGF-VEGFR2 signaling.