Fibrin deposits drives thromboinflammation and neutrophil extracellular trap formation (NETosis) in various pathophysiological situations but the underlying mechanisms remain unclear. Similarly, it is unknown how, irrespective of diabetes, hyperglycemia exacerbates thromboinflammation. Using a microfluidic model of thromboinflammation based on the perfusion of partially recalcified citrated human blood at venous flow rate over immobilized fibrin, we investigated these mechanisms. Blood was stained to detect P-selectin, phosphatidylserine, polyphosphates, fibrin, neutrophils, and NETs. The roles of glycoprotein VI (GPVI), integrin α2bβ3, polyphosphates, factor XIIa (FXIIa), tissue factor, thrombin, fibrin polymerization, glucose, and glucose uptake were explored using specific inhibitors, and by spiking blood with glucose. Perfusion of partially recalcified blood over a fibrin monolayer consistently triggered a platelet-dependent thromboinflammatory cascade. Pharmacological inhibition and analysis of activation at the individual platelet level revealed that this cascade was initiated by a subpopulation of procoagulant platelets, which activated second-responder platelets via GPVI-, FXIIa-, and polyphosphate-dependent thrombin generation, leading to fibrin polymerization, and neutrophil recruitment. This sequence was maintained but exacerbated in hyperglycemia, drastically increasing intra-thrombus neutrophil recruitment and NETosis, the latter prevented by inhibition of glucose transporters. Our results highlight the importance of a GPVI-FXIIa-polyphosphate axis in mediating thromboinflammation and NETosis when human flowing blood encounters immobilized fibrin, and the interest of targeting this axis and glucose transporters to prevent thromboinflammation and its exacerbation by hyperglycemia.

G protein-coupled receptors (GPCRs) are key regulators of cardiovascular function that provide targets for the treatment of cardiovascular disease. Sphingosine 1-phosphate (S1P) is an erythrocyte- and platelet-derived lipid mediator with cognate GPCRs on endothelial cells (EC), vascular smooth muscle cells (VSMC) and cardiomyocytes. S1P circulates in plasma bound to apolipoprotein M (ApoM)-containing high-density lipoproteins (HDL) and to albumin. Circulating S1P levels correlate positively with systolic blood pressure in hypertension and negatively with severity in septic shock and with left ventricular (LV) function in coronary heart disease. In mice, impaired S1P binding to HDL or signaling to EC both trigger hypertension, supporting an essential role for HDL-S1P in supporting endothelial function. The roles of albumin-S1P and myocyte S1PRs in cardiovascular homeostasis remain incompletely defined. Contrasting isolated HDL-S1P deficiency, we report that non-selective depletion of circulating S1P pools in mice impairs LV contractile function and induces hypotension and resistance to the spontaneous increase in blood pressure with age. Cardiac output was preserved in naive S1P deficient mice by compensatory LV dilation, but cardiac reserve reduced in a dobutamine stress test. These phenotypes tracked with hematopoietic cell S1P production and were partially or fully reversed by erythrocyte transfusion. Hypotension was accompanied by reduced peripheral resistance, and S1P infusion dose-dependently increased vascular resistance in isolated perfused kidneys from wild-type mice but not mice with compound deficiency in S1PR2&3. Epistatic analysis supported a critical role for S1PR3 in S1P-dependent blood pressure regulation and pointed to a distinct origin of the cardiac phenotype. Although circulating S1P is elevated in hypertensive mice and humans, increasing circulating S1P was not sufficient to induce hypertension in naive mice. These observations suggests that albumin-S1P crosses the endothelium in resistance arteries to gain access to contractile VSMC S1P receptors, and that myocyte S1PR signaling is essential for vascular resistance and blood pressure maintenance in mice. They also highlight the role for plasma chaperones in specifying vascular responses to S1P and the relevance of S1P as a biomarker and potential therapeutic target for blood pressure regulation and heart failure. ### Competing Interest Statement The authors have declared no competing interest.

Efficient lymph flow is ensured by lymphatic valves (LVs). The mechanisms that regulate LV development are incompletely understood. Here, we show that the deletion of the GPCR sphingosine 1-phosphate receptor-1 (S1PR1) from lymphatic endothelial cells (LECs) results in fewer LVs. Interestingly, LVs that remained in the terminal ileum-draining lymphatic vessels were specifically dysfunctional. Furthermore, tertiary lymphoid organs (TLOs) formed in the terminal ileum of the mutant mice. TLOs in this location are associated with ileitis in humans and mice. However, mice lacking S1PR1 did not develop obvious characteristics of ileitis. Mechanistically, S1PR1 regulates shear stress signaling and the expression of the valve-regulatory molecules FOXC2 and connexin-37. Importantly, Foxc2+/- mice, a model for lymphedema-distichiasis syndrome, also develop TLOs in the terminal ileum. Thus, we have discovered S1PR1 as a previously unknown regulator of LV and TLO development. We also suggest that TLOs are a sign of subclinical inflammation that can form due to lymphatic disorders in the absence of ileitis.

Background Primary aldosteronism is the most common form of secondary arterial hypertension, due to autonomous aldosterone production from the adrenal cortex. Genome wide association studies discovered genetic risk loci associated with the disease, which may affect adrenal cortex renewal, differentiation and lineage conversion. Genetic susceptibility may be modulated by environmental challenges. Method Here we investigate how environmental cues affecting mineralocorticoid output modulate adrenal cortex homeostasis, cell lineage conversion and zone-specific transcriptional landscape. We have used a newly developed Cyp11b2Cre mouse model and characterised its adaptation to a high or low salt diet (HSD, LSD ) , as well as dexamethasone (DEX) treatment. To explore underlying mechanisms, deep functional and morphological phenotyping, lineage tracing and spatial transcriptomics of the adrenal cortex were performed. Results Cyp11b2 expression was detected in Cyp11b2Cre - m T m G mice as early as day P1 in different areas of the ZG, associated with high plasma aldosterone levels, with lineage conversion of zona glomerulosa (ZG) into zona fasciculata (ZF) cells progressing between 2 and 9 weeks of age and a progressive reduction of ZG size and evolution of cell components of the adrenal cortex over time. Transdifferentiation progressed into the X-zone (ZX) in females, revealing a previously unrecognized connection between ZF and ZX cells in adult mice. A sexually dimorphic, reciprocal interaction between the ZG and the ZF in adapting to salt diets or DEX treatment was observed, involving changes in cell composition and transcriptional reprogramming of the three zones. Conclusion HSD, LSD and DEX induce a sexually dimorphic cellular and transcriptional response, involving all layers of the adrenal cortex, and the reciprocal contribution of ZG and ZF cells, indicating functional interaction between adrenocortical zones in adapting to external cues. ### Competing Interest Statement The authors have declared no competing interest.

Data from epidemiological and genetic studies as well as animal experiments indicate that high-density lipoproteins (HDL) play a role in the pathogenesis of central nervous system (CNS) diseases. Apolipoprotein A-I, the major protein of HDL, has been immunolocalized in the brain although it is produced exclusively by the liver and intestine. We therefore investigated how HDL cross the blood-brain barrier (BBB), using both in vitro and in vivo approaches. In vitro , we found that HDL bind to, are internalized by, and are transported through human brain endothelial cells via mechanisms involving the scavenger receptor BI (SR-BI) and the low-density lipoprotein receptor (LDLR). Notably, we discovered that LDLR facilitates only the transport of HDL particles containing apolipoprotein E (apoE). In vivo, HDL injected into the bloodstream enter into the brain through brain endothelial cells, and accumulated in medulla, cerebellum, olfactory bulb, hippocampus and cortex. Further investigation in Ldlr-/- mice revealed region-specific changes in HDL accumulation with reduced levels in the medulla and mixture of midbrain/cortex/hippocampus, no change in the olfactory bulb, and increased levels in the cerebellum. Together, these findings provide new insight on the interaction of the lipoprotein metabolism between the periphery and CNS. For the first time, we show that brain endothelial receptors and HDL composition jointly dictate HDL’s crossing through the BBB and their localization within the brain. ### Competing Interest Statement Jerome Robert consults for Cellerys AG outside the topic of this work, BrightFocus Foundation, https://ror.org/03cvfxv40, A2021037S, A2021045S Synapsis Foundation, 2022-PI04 Swiss Heart Foundation, https://ror.org/01k789405, FF22028 Swiss National Science Foundation, https://ror.org/00yjd3n13, 185109

BACKGROUND:Antiplatelet drugs represent potential candidates for protecting the penumbral microcirculation during cerebral ischemia and improving the benefits of arterial recanalization in ischemic stroke. Yet while the efficacy of such adjuvant strategies has been shown to be highly time dependent, antiplatelet therapy at the acute phase of ischemic stroke cannot be envisioned until the diagnosis of stroke and its ischemic nature have been confirmed because of the presumed risk of worsening bleeding in case of intracranial hemorrhage (ICH). Here, we investigated this risk for 2 antiplatelet drugs currently being tested in clinical trials for ischemic stroke, glenzocimab and eptifibatide, in 2 mouse models of ICH. METHODS AND RESULTS:The severity of ICH was assessed in mice humanized for glycoprotein VI treated or not with glenzocimab or eptifibatide at effective dose, in a model of primary ICH caused by unilateral striatal injection of collagenase type VII, and in a model of hyperglycemia-induced hemorrhagic transformation of cerebral ischemia-reperfusion injury. Glenzocimab had no impact on bleeding severity in either model of ICH. Conversely, eptifibatide caused a significant increase in intracranial bleeding in both models, and a drastic increase in death after hyperglycemia-induced hemorrhagic transformation of cerebral ischemia-reperfusion injury. CONCLUSIONS:Unlike eptifibatide, glenzocimab is safe in the setting of ICH. These results suggest that glenzocimab could be administered upon suspicion of ischemic stroke, before assessment of its ischemic nature, thus opening the way to hastening of treatment initiation.

Lymphatic vessels function throughout the body to drain interstitial fluids. Efficient lymph flow is ensured by lymphatic valves (LVs). However, the mechanisms that regulate LV development are incompletely understood. Here, we show that the deletion of the GPCR sphingosine 1-phosphate receptor-1 (S1PR1) from lymphatic endothelial cells (LECs) results in fewer LVs. Interestingly, LVs that remained in the terminal-ileum draining lymphatic vessels were specifically dysfunctional, and tertiary lymphoid organs (TLOs) formed in this location. TLOs in the terminal ileum are associated with ileitis in humans and mice. However, mice lacking S1PR1 did not develop obvious characteristics of ileitis. Sphingosine kinases 1 and 2 (SPHK1/2) are required for the synthesis of S1P, the ligand of S1PR1. Mice that lack Sphk1/2 in LECs recapitulate the LV and TLO phenotypes of mice that lack S1PR1. Mechanistically, S1PR1 regulates shear stress signaling and the expression of the valve-regulatory molecules FOXC2 and connexin-37. Importantly, Foxc2+/- mice, a model for lymphedema-distichiasis syndrome, also develop TLOs in the terminal ileum. Thus, we have discovered S1PR1 as a previously unknown regulator of LV and TLO development. We also suggest that TLOs are a sign of subclinical inflammation that can form due to lymphatic disorders in the absence of ileitis. ### Competing Interest Statement The authors have declared no competing interest.

Objective: Lineage tracing studies have shown that advanced atherosclerosis causes vascular smooth muscle cells (VSMCs) to become macrophage-like (MLCs), which increases inflammation. We investigated whether the RNA binding protein human antigen R (HuR) and the inflammatory receptor protease activated receptor 2 (PAR2) accelerate VSMC-MLC in a mouse model of atherosclerosis. Methods and Results: Downregulation of VSMC migration and cytokine release in Par2 -/- mice reduces atherosclerotic lesion area compared to proficient controls. To examine the role of PAR2 in the VSMC-MLC dedifferentiation, Par2 +/+ and Par2 -/- VSMCs were treated with 10μg/mL methyl-β-cyclodextrin cholesterol for 72 hours. While Par2 +/+ VSMCs demonstrated a loss of VSMCs markers and upregulated macrophage markers, Par2 -/- VSMCs remained stable. When comparing 101 mice strains with induced atherosclerosis, PAR2 was significantly correlated with Krüppel-like factor 4 (KLF4), a gene that regulates VSMC dedifferentiation. Also, Par2-/- VSMCs had lower basal KLF4 expression. Previous studies have demonstrated PAR2 mRNA is bound and stabilized by the RNA binding protein HuR. Our studies confirm these findings where Par2 -/- VSMCs have less HuR mRNA expression, suggesting a role of HuR in PAR2 mRNA stability. Our studies have also demonstrated a dampening of HuR activation in Par2 -/- VSMCs, suggesting a potential feedforward mechanism between HuRs stabilizing capabilities of PAR2 mRNA and PAR2s presence necessary for HuR activation. To delineate the role of HuR in VSMC dedifferentiation and atherosclerosis, a HuR flox mouse line has been bred on to a SM22 Cre as well as a Myh11 Cre lineage tracing reporter line. To date, we have determined male and female Ldlr -/- HuR flox/flox SM22 Cre+ mice have attenuated atherosclerosis compared to their littermate Cre - controls at both 12 and 24 weeks. Conclusions: These results suggest that VSMC PAR2 activation mediates the dedifferentiation of VSMCs via KLF4 and subsequent binding by HuR, which stabilizes PAR2 mRNA and upregulates PAR2. We also have established a definitive role of VSMC HuR in the development of atherosclerosis. Future studies will continue to elucidate the specific role of HuR in VSMC dedifferentiation.

Aims Circulating levels of sphingosine 1-phosphate (S1P), an HDL-associated ligand for the endothelial cell (EC) protective S1P receptor-1 (S1PR1), are reduced in disease states associated with endothelial dysfunction. Yet, as S1PR1 has high affinity for S1P and can be activated by ligand-independent mechanisms and EC autonomous S1P production, it is unclear if relative reductions in circulating S1P can cause endothelial dysfunction. It is also unclear how EC S1PR1 insufficiency, whether induced by deficiency in circulating ligand or by S1PR1-directed immunosuppressive therapy, affects different vascular subsets.Methods and results We here fine map the zonation of S1PR1 signalling in the murine blood and lymphatic vasculature, superimpose cell-type-specific and relative deficiencies in S1P production to define ligand source and dose dependence, and correlate receptor engagement to essential functions. In na & iuml;ve blood vessels, despite broad expression, EC S1PR1 engagement was restricted to resistance-size arteries, lung capillaries, and a subset of high-endothelial venules (HEVs). Similar zonation was observed for albumin extravasation in EC S1PR1-deficient mice, and brain extravasation was reproduced with arterial EC-selective S1pr1 deletion. In lymphatic ECs, S1PR1 engagement was high in collecting vessels and lymph nodes and low in blind-ended capillaries that drain tissue fluids. While EC S1P production sustained S1PR1 signalling in lymphatics and HEV, haematopoietic cells provided similar to 90% of plasma S1P and sustained signalling in resistance arteries and lung capillaries. S1PR1 signalling and endothelial function were both surprisingly sensitive to reductions in plasma S1P with apparent saturation around 50% of normal levels. S1PR1 engagement did not depend on sex or age but modestly increased in arteries in hypertension and diabetes. Sphingosine kinase (Sphk)-2 deficiency also increased S1PR1 engagement selectively in arteries, which could be attributed to Sphk1-dependent S1P release from perivascular macrophages.Conclusion This study highlights vessel subtype-specific S1PR1 functions and mechanisms of engagement and supports the relevance of S1P as circulating biomarker for endothelial function. Graphical Abstract

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.

Objective: Recent lineage tracing studies have shown vascular smooth muscle cells (VSMCs) develop macrophage-like characteristics (MLCs) in late-stage atherosclerosis resulting in accelerated inflammation and disease severity. Our objective was to determine if the inflammatory receptor, protease activated receptor 2 (PAR2), accelerates VSMC-MLC in a mouse model of atherosclerosis. Methods and Results: Our lab demonstrated Par2 -/- mice have decreased atherosclerotic lesion area versus proficient controls via downregulation of VSMC migration and cytokine release. To examine the role of PAR2 in VSMC dedifferentiation, Par2 +/+ and Par2 -/- VSMCs were treated with 10 ug/mL methyl- β-cyclodextrin cholesterol for 72 hours. While Par2 +/+ VSMCs demonstrated a loss of VSMCs markers (α - actin and myosin heavy chain) and upregulated macrophage markers (CD68 and Mac-2), Par2 -/- VSMCs remained stable. Looking at macrophage characteristics, Par2 -/- VSMCs phagocytized significantly less latex beads than Par2 +/+ VSMCs. Analysis of a hybrid mouse diversity panel (HMDP), conducted on 101 strains of mice with induced atherosclerosis, determined PAR2 was significantly correlated with the MLC super-gene Krüppel-like factor 4 (KLF4), which was found to have lower genetic expression in Par2 deficient VSMCs. Previous studies have demonstrated Par2 mRNA is bound and stabilized by the RNA binding protein human antigen R (HuR). Our studies confirm these findings where Par2 -/- VSMCs have less HuR mRNA expression and activity, suggesting a role of HuR in PAR2 mRNA stability. Par2 flox/flox mice bred with transgelin (SM22-alpha) Cre and low-density lipoprotein receptor deficient mice l have been establishing in our lab to investigate the effects of cell-specific deletions of PAR2 in atherosclerosis. VSMC-specific Par2 deficiency blunts the formation of atherosclerosis versus the Cre + littermates. Conclusions: These results suggest that VSMC-specific PAR2 activation mediates the dedifferentiation of VSMCs via upregulation of KLF4 and subsequent binding by HuR, which stabilizes PAR2 mRNA and upregulates PAR2. Future studies will continue to study VSMC dedifferentiation utilizing tagged lineage-tracing mouse models and various in vitro techniques.

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that is secreted by several cell types. We recently showed that Mfsd2b is an S1P transporter from hematopoietic cells that contributes approximately 50% plasma S1P. Here we report the characterization of compound deletion of Mfsd2b and Spns2, another S1P transporter active primarily in endothelial cells. Global deletion of Mfsd2b and Spns2 (global double knockout [gDKO]) results in embryonic lethality beyond embryonic day 14.5 (E14.5), with severe hemorrhage accompa-nied by defects of tight junction proteins, indicating that Mfsd2b and Spns2 provide S1P for signaling, which is essential for blood vessel integrity. Compound postnatal deletion of Mfsd2b and Spns2 using Mx1Cre (ctDKO-Mx1Cre) results in maximal 80% reduction of plasma S1P. ctDKO-Mx1Cre mice exhibit severe susceptibility to anaphylaxis, indicating that S1P from Mfsd2b and Spns2 is indispensable for vascular homeostasis. Our re-sults show that S1P export from Mfsd2b and Spns2 is essential for developing and mature vasculature.

HomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 42, No. 7Distinct GEFs Couple S1PR1 to Rac for Endothelial Barrier Enhancement and Lymphocyte Trafficking Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBDistinct GEFs Couple S1PR1 to Rac for Endothelial Barrier Enhancement and Lymphocyte Trafficking Ilaria Del Gaudio and Eric Camerer Ilaria Del GaudioIlaria Del Gaudio From the Université Paris Cité, Inserm, PARCC, F-75015 Paris, France. and Eric CamererEric Camerer Correspondence to: Eric Camerer, PhD, Université Paris Cité, Inserm, PARCC, F-75015 Paris, France. Email E-mail Address: [email protected] https://orcid.org/0000-0002-6271-7125 From the Université Paris Cité, Inserm, PARCC, F-75015 Paris, France. Originally published26 May 2022https://doi.org/10.1161/ATVBAHA.122.317794Arteriosclerosis, Thrombosis, and Vascular Biology. 2022;42:903–905This article is a commentary on the followingDOCK4 Regulation of Rho GTPases Mediates Pulmonary Vascular Barrier FunctionOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: May 26, 2022: Ahead of Print See accompanying article on page 886The vascular endothelium provides a barrier between circulating fluids and the tissue parenchyma.1,2 Edema caused by loss of endothelial barrier function can impair gas exchange, increase interstitial pressure, and reduce tissue perfusion. In the lungs, endothelial barrier dysfunction may contribute to acute respiratory distress syndrome and pulmonary hypertension.3,4 In the brain, it can extend tissue damage after an ischemic stroke and may contribute to cognitive and functional decline in Alzheimer disease and other dementias.2,5Endothelial barrier function is secured by transmembrane adhesive proteins that anchor to the actin cytoskeleton through adaptor proteins and seal the borders between adjacent endothelial cells by homophilic interactions.1,2,4 They assemble into tight junctions, which form stable barriers to paracellular passage, and adherens junctions, which form dynamic barriers that facilitate vascular remodeling and the extravasation of immune cells. The actin cytoskeleton is regulated in a tug-of-war between Rho family GTPases Rho (Ras homolog), Rac (Ras-related C3 botulinum toxin), and CDC42 (cell division control protein 42 homolog). Excess RhoA activity, which is counteracted by Rac1, induces stress fiber formation and opening of adherens junctions. Mechanical stress, integrin clustering, and ligation of membrane receptors can regulate the actin cytoskeleton by engaging GEFs (guanine nucleotide exchange factors) to drive the transition of Rho family GTPases from their inactive GDP-bound state to their active GTP-bound state. GEFs are thus important intermediates in the regulation of cytoskeletal dynamics and endothelial barrier function in response to extracellular cues.In this issue of ATVB, Yazbeck et al6 identify DOCK4 as a critical GEF for Rac1 and a mediator of S1PR1 (sphingosine-1-phosphate receptor-1) signaling in endothelial cells. They report that similar to a deficiency in plasma S1P (sphingosine-1-phosphate) or endothelial S1PR1,7–9 a deficiency in DOCK4 in mice results in the extravasation of small and medium sized molecular tracers to the lung and of small tracers to the brain.6 And similar to a deficiency in S1P export or inhibition of S1PR1,8,10 knockdown of DOCK4 substantially reduces basal resistance in monolayers of cultured endothelial cells.6 Knockdown of DOCK4 also results in spontaneous stress fiber formation due to loss of Rac1-dependent RhoA inhibition and impairs the ability of exogenous S1P to activate Rac1 and acutely increase barrier function in cultured endothelial cells.6 Together, this positions DOCK4 as a critical intermediate between S1PR1 and Rac1 in the maintenance of endothelial barrier function and vascular integrity (Figure).Download figureDownload PowerPointFigure. Cell type–specific mechanisms for S1PR1 (sphingosine-1-phosphate receptor-1) coupling to Rac1 (Ras-related C3 botulinum toxin substrate 1). The lipid mediator S1P (sphingosine-1-phosphate) circulates in plasma in complex with HDL (high-density lipoprotein)-associated ApoM (apolipoprotein M) and albumin. Upon HDL binding to SR-B1 (scavenger receptor class B type 1) on endothelial cells (left), ApoM delivers S1P to S1PR1 to promote barrier function, NO production, and transendothelial transport of HDL. In this issue of ATVB, Yazbeck et al demonstrate that the guanine nucleotide exchange factor DOCK4 is required for S1P-induced Rac1 activation and barrier enhancement in cultured endothelial cells and that DOCK4 deficiency in mice induces spontaneous extravasation of tracers to lung and brain similar to what has been reported for S1PR1 deficiency.6 In vitro, stress fiber formation induced by DOCK4 deficiency was reversed by ROCK (Rho-associated protein kinase) inhibition, suggesting that DOCK4-mediated Rac1 activation is continuously required to counterbalance RhoA (Ras homolog family member A) activity. Intriguingly, SR-BI has also been suggested to couple directly to DOCK4 to mediate LDL (low-density lipoprotein) transcytosis in atherosclerosis-prone regions of the aorta. Unlike endothelial S1PR1, lymphocyte S1PR1 (right) uses DOCK2 to couple to Rac1/2 for egress from secondary lymphoid organs.12,18 AJ indicates adherens junctions; and TJ, tight junctions. This figure was created with BioRender (BioRender.com).Although this study points to evident parallels between deficiencies in DOCK4 and S1PR1, there are also notable differences. While endothelial-specific deletion of S1pr1 results in embryonic failure in late gestation,11 global Dock4 deletion did not induce embryonic lethality in this study.6 This may suggest either that other Rac1 GEFs are active downstream of S1PR1 in the embryo or that Rac1 plays a less important role downstream of S1PR1 in angiogenesis than in endothelial barrier stabilization. On the contrary, global Dock4 deletion had more severe consequences than endothelial S1pr1 deletion for vascular integrity of the mature lung,8 with defective vascular smooth muscle cell coverage and bleeding in addition to protein and fluid leak.6 This may suggest additional upstream modulators of DOCK4 activity in the developing lung, or potential compound effects of DOCK4 targeting in endothelial cells and other cell types, as Yazbeck et al6 did not take a tissue-specific approach to Dock4 deletion.DOCK4 is a member of a family of 11 DOCK (dedicator of cytokinesis) proteins, a subfamily of Rho GEFs that lack Dbl homology domains.12,13 It is a GEF for Rac and Rap1 that is enriched in endothelial cells and expressed primarily in the lung and the brain (https://www.proteinatlas.org).13 An essential role for DOCK4 downstream of S1PR1 in endothelial barrier enhancement raises the question of whether it is also implicated in other endothelial functions regulated by S1PR1 signaling, such as NO synthase activation, suppression of inflammation, and a recently identified role in HDL (high-density lipoprotein) transcytosis.14–16 The bulk of S1P in the blood circulation associates with HDL through high-affinity binding to ApoM (apolipoprotein M).15 The binding of HDL to SR-B1 (scavenger receptor class B type 1) may present S1P to S1PR1 (Figure),15 and S1PR1 activation was recently shown to promote the translocation of SR-B1 to the plasma membrane and the selective transendothelial transport of HDL.16 Intriguingly, DOCK4 was recently attributed a critical role in the selective endothelial transcytosis of LDL (low-density lipoprotein) in atherosclerosis-prone vessels by coupling SR-B1 to Rac1.17 How SR-B1, S1PR1, and DOCK4 coordinate to specify HDL- versus LDL-selective transendothelial transport remains to be clarified. Regardless, as DOCK4 is likely to mediate not only endothelial barrier promoting6 but also antiatherogenic actions of endothelial S1PR1,15 DOCK4 inhibition may not be a strategy of choice for preventing LDL uptake in atherosclerosis-prone vessels.16S1PR1 also plays an essential role in lymphocyte trafficking that is dependent on Rac GTPases.18,19 Clinical targeting of S1PR1 to block the egress of autoreactive T cells in multiple sclerosis patients induces side effects by activation and desensitization of S1PR1 on cardiomyocytes and endothelial cells, respectively.20 While DOCK4 deficiency mimics endothelial S1PR1 deficiency and impairs endothelial responses to S1P,6 deficiency in DOCK2, which is expressed primarily in hematopoietic cells, partially mimics lymphocyte S1PR1 deficiency and impairs lymphocyte migration to S1P and other chemokines.12 Cell type specificity in the use of GEFs and other downstream mediators could open for cell type selective targeting of S1PR1 signaling (Figure).In conclusion, Yazbeck et al identify DOCK4 as a critical mediator of barrier protective signaling in the endothelium of lung and brain. This raises the possibility that reduced DOCK4 expression or function could predispose to disease conditions that involve loss of vascular integrity in these organs.Article InformationSources of FundingThis work was supported by the French Foundation for Medical Research (DCP20171138945-EC) and the French National Research Agency (ANR-19-CE14-0028-01 and ANR-21-CE17-0023-01).Disclosures None.FootnotesFor Sources of Funding and Disclosures, see page 905.The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to: Eric Camerer, PhD, Université Paris Cité, Inserm, PARCC, F-75015 Paris, France. Email eric.[email protected]frReferences1. Vandenbroucke E, Mehta D, Minshall R, Malik AB. Regulation of endothelial junctional permeability.Ann NY Acad Sci. 2008; 1123:134–145. doi: 10.1196/annals.1420.016CrossrefMedlineGoogle Scholar2. Dejana E, Tournier-Lasserve E, Weinstein BM. 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Background Innate immune responses to influenza A virus (IAV) infection are initiated in part by toll-like receptor 3 (TLR3). TLR3-dependent signaling induces an antiviral immune response and an NFκB-dependent inflammatory response. Protease-activated receptor 2 (PAR2) inhibits the antiviral response and enhances the inflammatory response. PAR2 deficiency protected mice during IAV infection. However, the PAR2 expressing cell-types contributing to IAV pathology in mice and the mechanism by which PAR2 contributes to IAV infection is unknown. Methods IAV infection was analyzed in global (Par2-/- ), myeloid (Par2 fl/fl;LysMCre+) and lung epithelial cell (EpC) Par2 deficient (Par2fl/fl ;SPCCre+) mice and their respective controls (Par2 +/+ and Par2 fl/fl). In addition, the effect of PAR2 activation on polyinosinic-polycytidylic acid (poly I:C) activation of TLR3 was analyzed in bone marrow-derived macrophages (BMDM). Lastly, we determined the effect of PAR2 inhibition in wild-type (WT) mice. Results After IAV infection, Par2-/- and mice with myeloid Par2 deficiency exhibited increased survival compared to infected controls. The improved survival was associated with reduced proinflammatory mediators and reduced cellular infiltration in bronchoalveolar lavage fluid (BALF) of Par2-/- and Par2 fl/fl;LysMCre+ 3 days post infection (dpi) compared to infected control mice. Interestingly, Par2 fl/fl;SPCCre+ mice showed no survival benefit compared to Par2fl/fl . In vitro studies showed that Par2-/- BMDM produced less IL6 and IL12p40 than Par2 +/+ BMDM after poly I:C stimulation. In addition, activation of PAR2 on Par2 +/+ BMDM increased poly I:C induction of IL6 and IL12p40 compared to poly I:C stimulation alone. Importantly, PAR2 inhibition prior to IAV infection protect WT mice. Conclusion Global Par2 or myeloid cell but not lung EpC Par2 deficiency was associated with reduced BALF inflammatory markers and reduced IAV-induced mortality. Our study suggests that PAR2 may be a therapeutic target to reduce IAV pathology.

Aims: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis. Methods: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times. Results: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα. Conclusions: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology.

There is now a consensus that a profound reduction in platelet numbers will not only impair classical hemostasis, but also the barrier function of the vascular wall.1 Recent evidence suggests that platelets not only seal vascular breaches caused by infiltrating neutrophils,2,3 but also coordinate the tightening of the circumferential endothelial belt around transmigrating leukocytes,4 thus preventing both bleeding and the extravasation of plasma during diapedesis. Most experimental studies and clinical observations indicate that thrombocytopenia is only associated with bleeding when insults‐such as traumatic injury or leukocyte infiltration and activation during inflammation and immune responses‐cause damage to the endothelial basement membrane.

RATIONALE:Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. OBJECTIVE:To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. METHODS AND RESULTS:Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. CONCLUSIONS:This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.