Metformin synergizes with PD-1 blockade to promote normalization of tumor vessels via CD8T cells and IFNγ.

Tumor blood vessels are highly leaky in structure and have poor blood perfusion, which hampers infiltration and function of CD8T cells within tumor. Normalizing tumor vessels is thus thought to be important in promoting the flux of immune T cells and enhancing ant-tumor immunity. However, how tumor vasculature is normalized is poorly understood. Metformin (Met) combined with ant-PD-1 therapy is known to stimulate proliferation of and to produce large amounts of IFNγ from tumor-infiltrating CD8T lymphocytes (CD8TILs). We found that the combination therapy promotes the pericyte coverage of tumor vascular endothelial cells (ECs) to improve blood perfusion and that it suppresses the hyperpermeability through the increase of VE-cadherin. Peripheral node addressin(PNAd) and vascular cell adhesion molecule (VCAM)-1, both implicated to promote tumor infiltration of CD8T cells, were also increased. Importantly, tumor vessel normalization, characterized as the reduced 70-kDa dextran leakage and the enhancement of VE-cadherin and VCAM-1, were canceled by anti-CD8 Ab or anti-IFNγ Ab injection to mice. The increased CD8TILs were also abrogated by anti-IFNγ Ab injection. In vascular ECs, flow cytometry analysis revealed that pSTAT1 expression was found to be associated with VE-cadherin expression. Moreover, in vitro treatment with Met and IFNγ enhanced VE-cadherin and VCAM-1 on human umbilical vein endothelial cells (HUVECs). The Kaplan-Meier method revealed a correlation of VE-cadherin or VCAM-1 levels with overall survival in patients treated with immune checkpoint inhibitors. These data indicate that IFNγ-mediated cross talk of CD8TILs with tumor vessels is important for creating a better tumor microenvironment and maintaining sustained antitumor immunity.

TMEM16F scramblase regulates angiogenesis via endothelial intracellular signaling.

TMEM16F (also known as ANO6), a Ca2+-activated lipid scramblase (CaPLSase) that dynamically disrupts lipid asymmetry, plays a crucial role in various physiological and pathological processes, such as blood coagulation, neurodegeneration, cell-cell fusion and viral infection. However, the mechanisms through which it regulates these processes remain largely elusive. Using endothelial cell-mediated angiogenesis as a model, here we report a previously unknown intracellular signaling function of TMEM16F. We demonstrate that TMEM16F deficiency impairs developmental retinal angiogenesis in mice and disrupts angiogenic processes in vitro. Biochemical analyses indicate that the absence of TMEM16F enhances the plasma membrane association of activated Src kinase. This in turn increases VE-cadherin phosphorylation and downregulation, accompanied by suppressed angiogenesis. Our findings not only highlight the role of intracellular signaling by TMEM16F in endothelial cells but also open new avenues for exploring the regulatory mechanisms for membrane lipid asymmetry and their implications in disease pathogenesis.

Ubiquitination of VE-cadherin regulates inflammation-induced vascular permeability in vivo.

VE-cadherin is a major component of the cell adhesion machinery which provides integrity and plasticity of the barrier function of endothelial junctions. Here, we analyze whether ubiquitination of VE-cadherin is involved in the regulation of the endothelial barrier in inflammation in vivo. We show that histamine and thrombin stimulate ubiquitination of VE-cadherin in HUVEC, which is completely blocked if the two lysine residues K626 and K633 are replaced by arginine. Similarly, these mutations block histamine-induced endocytosis of VE-cadherin. We describe two knock-in mouse lines with endogenous VE-cadherin being replaced by either a VE-cadherin K626/633R or a VE-cadherin KallR mutant, where all seven lysine residues are mutated. Mutant mice are viable, healthy and fertile with normal expression levels of junctional VE-cadherin. Histamine- or LPS-induced vascular permeability in the skin or lung of both of these mutant mice are clearly and similarly reduced in comparison to WT mice. Additionally, we detect a role of K626/633 for lysosomal targeting. Collectively, our findings identify ubiquitination of VE-cadherin as important for the induction of vascular permeability in the inflamed skin and lung.

PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin.

Leukocyte extravasation is an essential step during the immune response and requires the destabilization of endothelial junctions. We have shown previously that this process depends in vivo on the dephosphorylation of VE-cadherin-Y731. Here, we reveal the underlying mechanism. Leukocyte-induced stimulation of PECAM-1 triggers dissociation of the phosphatase SHP2 which then directly targets VE-cadherin-Y731. The binding site of PECAM-1 for SHP2 is needed for VE-cadherin dephosphorylation and subsequent endocytosis. Importantly, the contribution of PECAM-1 to leukocyte diapedesis in vitro and in vivo was strictly dependent on the presence of Y731 of VE-cadherin. In addition to SHP2, dephosphorylation of Y731 required Ca2+ -signaling, non-muscle myosin II activation, and endothelial cell tension. Since we found that ß-catenin/plakoglobin mask VE-cadherin-Y731 and leukocyte docking to endothelial cells exert force on the VE-cadherin-catenin complex, we propose that leukocytes destabilize junctions by PECAM-1-SHP2-triggered dephosphorylation of VE-cadherin-Y731 which becomes accessible by actomyosin-mediated mechanical force exerted on the VE-cadherin-catenin complex.

ARAP3 protects from excessive formylated peptide-induced microvascular leakage by acting on endothelial cells and neutrophils.

Vascular permeability is temporarily heightened during inflammation, but excessive inflammation-associated microvascular leakage can be detrimental, as evidenced in the inflamed lung. Formylated peptides regulate vascular leakage indirectly via formylated peptide receptor-1 (FPR1)-mediated recruitment and activation of neutrophils. Here we identify how the GTPase-activating protein ARAP3 protects against formylated peptide-induced microvascular permeability via endothelial cells and neutrophils. In vitro, Arap3-/- endothelial monolayers were characterised by enhanced formylated peptide-induced permeability due to upregulated endothelial FPR1 and enhanced vascular endothelial cadherin internalisation. In vivo, enhanced inflammation-associated microvascular leakage was observed in Arap3-/- mice. Leakage of plasma protein into the lungs of Arap3-/- mice increased within hours of formylated peptide administration. Adoptive transfer experiments indicated this was dependent upon ARAP3 deficiency in both immune and non-immune cells. Bronchoalveolar lavages of formylated peptide-challenged Arap3-/- mice contained neutrophil extracellular traps (NETs). Pharmacological inhibition of NET formation abrogated excessive microvascular leakage, indicating a critical function of NETs in this context. The observation that Arap3-/- mice developed more severe influenza suggests these findings are pertinent to pathological situations characterised by abundant formylated peptides. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Regulation of aortic morphogenesis and VE-cadherin dynamics by VEGF.

In amniote vertebrates, the definitive dorsal aorta is formed by the fusion of two primordial aortic endothelial tubes. Formation of the definitive dorsal aorta requires extensive cellular migrations and rearrangements of the primordial tubes in order to generate a single vessel located at the embryonic ventral midline. This study examines the role of VEGF signaling in the generation of the definitive dorsal aorta. Through gain- and loss-of-function studies in vivo in the chick embryo, we document a requirement for VEGF signaling in growth and remodeling of the paired primordia. We find that regions of the aorta are differentially sensitive to levels of VEGF signaling, and present evidence that areas of low blood flow are more sensitive to the loss of VEGF signaling. We also find that VEGF signaling regulates the intracellular distribution between membrane and cytoplasm of the cell-cell adhesion molecule VE-cadherin in aortic endothelial cells in vivo. Together, these finding identify mechanisms that likely contribute to the dynamic behavior of endothelial cells during aorta morphogenesis.

Spleen Tyrosine Kinase phosphorylates VE-cadherin to cause endothelial barrier disruption in acute lung injury.

Increased endothelial cell (EC) permeability is a cardinal feature of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Tyrosine phosphorylation of VE-cadherin is a key determinant of EC barrier disruption. However, the identity and role of tyrosine kinases in this context are incompletely understood. Here we report that Spleen Tyrosine Kinase (Syk) is a key mediator of EC barrier disruption and lung vascular leak in sepsis. Inhibition of Syk by pharmacological or genetic approaches, each reduced thrombin-induced EC permeability. Mechanistically, Syk associates with and phosphorylates VE-cadherin to cause EC permeability. To study the causal role of endothelial Syk in sepsis-induced ALI, we used a remarkably efficient and cost-effective approach based on gene transfer to generate EC-ablated Syk mice. These mice were protected against sepsis-induced loss of VE-cadherin and inflammatory lung injury. Notably, the administration of Syk inhibitor R788 (fostamatinib); currently in phase II clinical trial for the treatment of COVID-19, mitigated lung injury and mortality in mice with sepsis. These data identify Syk as a novel kinase for VE-cadherin and a druggable target against ALI in sepsis.

Control of dynamic cell behaviors during angiogenesis and anastomosis by Rasip1.

Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveals distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tricellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics the junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function in junction formation and stabilization during sprouting angiogenesis.

Electronic vape fluid activates the pulmonary endothelium and disrupts vascular integrity in vitro through an ARF6-dependent pathway.

The use of e-cigarettes or vapes is increasingly popular amongst a range of different demographics however the research in this area is surprisingly sparse. Clinical reports of e-cigarette- or vaping use-associated lung injury (EVALI) and vascular disruption, in both nicotine-containing and nicotine-free e-cigarette smokers, prompts the need for further research with a focus on the pulmonary endothelium. Using a common brand of e-cigarette (eVape) and an in vitro model of the human lung microvasculature, we investigated the effect of nicotine-free eVape fluid on pulmonary endothelial barrier integrity, oxidative stress and inflammation profile. Findings demonstrate reactive oxygen species-dependent breakdown of the pulmonary endothelium and release of inflammatory cytokines. These phenotypic changes, following exposure to nicotine-free eVape fluid, were accompanied by dysregulation of a number of adheren junctions-related genes of which ARF6 was most abundantly overexpressed. Further investigation of ARF6 identified it as a key regulator in eVape-induced barrier disruption and ROS accumulation. This study demonstrates, for the first time, the barrier disruptive effect of nicotine-free e-cigarette fluid on the pulmonary microvasculature and the ARF6 and ROS-dependent molecular mechanisms underlying this damage. Whilst these studies focus on a human in vitro model of the pulmonary microvasculature, the results support clinical case studies on EVALI and demonstrate a need for further investigation of the impact of nicotine-free e-cigarettes on the lung.

Umbilical cord mesenchymal stem cells from gestational diabetes show impaired ability to up-regulate paracellular permeability from sub-endothelial niche.

In vitro studies have shown that Wharton's jelly mesenchymal stem cells (WJ-MSCs) can cross umbilical and uterine endothelial barriers and up-regulate endothelial junctional integrity from sub-endothelial niches. This pericytic behaviour may be lost in pregnancies complicated by gestational diabetes (GDM), where increased vascular permeability and junctional disruption are reported. The aim of the present study was to investigate whether WJ-MSCs isolated from GDM pregnancies displayed any changes in morphology, proliferation, VEGF-A secretion, and their ability to influence paracellular junctional composition and permeability. WJ-MSCs were isolated from human umbilical cords from normal pregnancies (nWJ-MSCs, n=13) and those complicated by GDM (gWJ-MSCs), either diet-controlled (d-GDM, n=13) or metformin-treated (m-GDM, n=9). We recorded that 4-fold more WJ-MSCs migrated from m-GDM, and 2.5-fold from d-GDM cord samples compared with the normal pregnancy. gWJ-MSCs showed a less predominance of spindle-shaped morphology and secreted 3.8-fold more VEGF-A compared with nWJ-MSCs. The number of cells expressing CD105 (Endoglin) was higher in gWJ-MSCs compared with nWJ-MSCs (17%) at P-2. The tracer leakage after 24 h across the HUVEC + gWJ-MSCs bilayer was 22.13% and 11.2% higher in the m-GDM and d-GDM, respectively, HUVEC + nWJ-MSCs. Transfection studies with siRNAs that target Endoglin were performed in n-WJ-MSCs; transfected cells were co-cultured with HUVEC followed by permeability studies and VE-cadherin analyses. Loss of Endoglin also led to increased VEGF-A secretion, increased permeability and affected endothelial stabilization. These results reinforce the pericytic role of nWJ-MSCs to promote vascular repair and the deficient ability of gWJ-MSCs to maintain endothelial barrier integrity.

CD93 maintains endothelial barrier function by limiting the phosphorylation and turnover of VE-cadherin.

Regulation of vascular permeability to plasma is essential for tissue and organ homeostasis and is mediated by endothelial cell-to-cell junctions that tightly regulate the trafficking of molecules between blood and tissue. The single-pass transmembrane glycoprotein CD93 is upregulated in endothelial cells during angiogenesis and controls cytoskeletal dynamics. However, its role in maintaining homeostasis by regulating endothelial barrier function has not been elucidated yet. Here, we demonstrate that CD93 interacts with vascular endothelial (VE)-cadherin and limits its phosphorylation and turnover. CD93 deficiency in vitro and in vivo induces phosphorylation of VE-cadherin under basal conditions, displacing it from endothelial cell-cell contacts. Consistent with this, endothelial junctions are defective in CD93-/- mice, and the blood-brain barrier permeability is enhanced. Mechanistically, CD93 regulates VE-cadherin phosphorylation and turnover at endothelial junctions through the Rho/Rho kinase-dependent pathway. In conclusion, our results identify CD93 as a key regulator of VE-cadherin stability at endothelial junctions, opening up possibilities for therapeutic strategies directed to control vascular permeability.

Atorvastatin-induced intracerebral hemorrhage is inhibited by berberine in zebrafish.

Intracerebral hemorrhage (ICH), for which there are currently no effective preventive or treatment methods, has a very high fatality rate. Statins, such as atorvastatin (ATV), are the first-line drugs for regulating blood lipids and treating hyperlipidemia-related cardiovascular diseases. However, ATV-associated ICH has been reported, although its incidence is rare. In this study, we aimed to investigate the protective action and mechanisms of berberine (BBR) against ATV-induced brain hemorrhage. We established an ICH model in zebrafish induced by ATV (2 µM) and demonstrated the effects of BBR (10, 50, and 100 µM) on ICH via protecting the vascular network using hemocyte staining and three transgenic zebrafish. BBR was found to reduce brain inflammation and locomotion injury in ICH-zebrafish. Mechanism research showed that ATV increased the levels of VE-cadherin and occludin proteins but disturbed their localization at the cell membrane by abnormal phosphorylation, which decreased the number of intercellular junctions between vascular endothelial cells (VECs), disrupting the integrity of vascular walls. BBR reversed the effects of ATV by promoting autophagic degradation of phosphorylated VE-cadherin and occludin in ATV-induced VECs examined by co-immunoprecipitation (co-IP). These findings provide crucial insights into understanding the BBR mechanisms involved in the maintenance of vascular integrity and in mitigating adverse reactions to ATV.

Molecular Mechanisms Regulating Vascular Endothelial Permeability.

Vascular endothelial cells form a monolayer in the vascular lumen and act as a selective barrier to control the permeability between blood and tissues. To maintain homeostasis, the endothelial barrier function must be strictly integrated. During acute inflammation, vascular permeability temporarily increases, allowing intravascular fluid, cells, and other components to permeate tissues. Moreover, it has been suggested that the dysregulation of endothelial cell permeability may cause several diseases, including edema, cancer, and atherosclerosis. Here, we reviewed the molecular mechanisms by which endothelial cells regulate the barrier function and physiological permeability.

The Protein Kinase A Inhibitor KT5720 Prevents Endothelial Dysfunctions Induced by High-Dose Irradiation.

High-dose irradiation can trigger numerous endothelial dysfunctions, including apoptosis, the overexpression of adhesion molecules, and alteration of adherens junctions. Altogether, these endothelial dysfunctions contribute to the development of tissue inflammation and organ damage. The development of endothelial dysfunctions may depend on protein phosphorylation by various protein kinases, but the possible role of protein kinase A (PKA) has not been investigated so far, and efficient compounds able to protect the endothelium from irradiation effects are needed. Here we report the beneficial effects of the PKA inhibitor KT5720 on a panel of irradiation-induced endothelial dysfunctions in human pulmonary microvascular endothelial cells (HPMECs). High-dose X-irradiation (15 Gy) triggered the late apoptosis of HPMECs independent of the ceramide/P38 MAP kinase pathway or p53. In contrast, the treatment of HPMECs with KT5720 completely prevented irradiation-induced apoptosis, whether applied before or after cell irradiation. Immunostainings of irradiated monolayers revealed that KT5720 treatment preserved the overall integrity of endothelial monolayers and adherens junctions linking endothelial cells. Real-time impedance measurements performed in HPMEC monolayers confirmed the overall protective role of KT5720 against irradiation. Treatment with KT5720 before or after irradiation also reduced irradiation-induced ICAM-1 overexpression. Finally, the possible role for PKA in the development of endothelial dysfunctions is discussed, but the potency of KT5720 to inhibit the development of a panel of irradiation-induced endothelial dysfunctions, whether applied before or after irradiation, suggests that this compound could be of great interest for both the prevention and treatment of vascular damages in the event of exposure to a high dose of radiation.

Lipopolysaccharide, VE-cadherin, HMGB1, and HIF-1α levels are elevated in the systemic circulation in chronic migraine patients with medication overuse headache: evidence of leaky gut and inflammation.

OBJECTIVE: Medication overuse headache (MOH) was recently shown to be associated with leaky gut in rodents. We aimed to investigate whether chronic migraine (CM) patients with MOH have elevated lipopolysaccharide levels and inflammatory molecules in blood circulation. MATERIALS AND METHODS: The study included women participants (40 CM patients with NSAID overuse headache, 35 episodic migraine (EM) patients, and 20 healthy non-headache sufferers). Migraine duration, monthly migraine headache days, MigSCog, HADS-D, HADS-A, and HIT-6 scores were recorded. Serum samples were collected to measure circulating LPS, LPS binding protein (LBP), tight junction protein occludin, adherens junction protein vascular endothelial cadherin (VE-cadherin), CGRP, HMGB1, HIF-1α, IL-6, and IL-17 levels. RESULTS: Serum LPS, VE-Cadherin, CGRP, HIF-1α, and IL-6 levels were significantly higher in the CM + MOH group compared to the EM group and healthy controls while serum LBP and HMGB1 were higher in the CM + MOH group compared to healthy controls. IL-17 and occludin levels were comparable between the three groups. Serum HMGB1 levels in EM patients were higher compared to the control group. Mig-SCog and HIT-6 scores were higher in the CM + MOH group compared to EM patients. HADS-A and HADS-D scores were significantly higher in the CM + MOH group compared to EM patients and healthy controls, and they were also higher in EM patients compared to healthy subjects. LPS levels were correlated with VE-cadherin and occludin levels. The number of monthly migraine headache days was positively correlated with serum LPS, HIF-1α, VE-cadherin, and IL-6 levels, HADS-A, HADS-D, HIT-6, and MigSCog scores. CONCLUSION: We have evidence for the first time that CM + MOH is associated with elevated serum LPS and LBP levels suggestive of LPS leak into the systemic circulation. Higher levels of nociceptive and/or pro-inflammatory molecules such as HMGB1, HIF-1α, IL-6, and CGRP may play a role in trigeminal sensitization and neurobiology of MOH. Intestinal hyperpermeability and consequent inflammatory response should be considered as a potential contributory factor in patients with MOH.

Reactive astrocytes associated with prion disease impair the blood brain barrier.

BACKGROUND: Impairment of the blood-brain barrier (BBB) is considered to be a common feature among neurodegenerative diseases, including Alzheimer's, Parkinson's and prion diseases. In prion disease, increased BBB permeability was reported 40 years ago, yet the mechanisms behind the loss of BBB integrity have never been explored. Recently, we showed that reactive astrocytes associated with prion diseases are neurotoxic. The current work examines the potential link between astrocyte reactivity and BBB breakdown. RESULTS: In prion-infected mice, the loss of BBB integrity and aberrant localization of aquaporin 4 (AQP4), a sign of retraction of astrocytic endfeet from blood vessels, were noticeable prior to disease onset. Gaps in cell-to-cell junctions along blood vessels, together with downregulation of Occludin, Claudin-5 and VE-cadherin, which constitute tight and adherens junctions, suggested that loss of BBB integrity is linked with degeneration of vascular endothelial cells. In contrast to cells isolated from non-infected adult mice, endothelial cells originating from prion-infected mice displayed disease-associated changes, including lower levels of Occludin, Claudin-5 and VE-cadherin expression, impaired tight and adherens junctions, and reduced trans-endothelial electrical resistance (TEER). Endothelial cells isolated from non-infected mice, when co-cultured with reactive astrocytes isolated from prion-infected animals or treated with media conditioned by the reactive astrocytes, developed the disease-associated phenotype observed in the endothelial cells from prion-infected mice. Reactive astrocytes were found to produce high levels of secreted IL-6, and treatment of endothelial monolayers originating from non-infected animals with recombinant IL-6 alone reduced their TEER. Remarkably, treatment with extracellular vesicles produced by normal astrocytes partially reversed the disease phenotype of endothelial cells isolated from prion-infected animals. CONCLUSIONS: To our knowledge, the current work is the first to illustrate early BBB breakdown in prion disease and to document that reactive astrocytes associated with prion disease are detrimental to BBB integrity. Moreover, our findings suggest that the harmful effects are linked to proinflammatory factors secreted by reactive astrocytes.

AMP-activated protein kinase is a key regulator of acute neurovascular permeability.

Many neuronal and retinal disorders are associated with pathological hyperpermeability of the microvasculature. We have used explants of rodent retinae to study acute neurovascular permeability, signal transduction and the role of AMP-activated protein kinase (AMPK). Following stimulation with either vascular endothelial growth factor (VEGF-A) or bradykinin (BK), AMPK was rapidly and strongly phosphorylated and acted as a key mediator of permeability downstream of Ca2+. Accordingly, AMPK agonists potently induced acute retinal vascular leakage. AMPK activation led to phosphorylation of endothelial nitric oxide synthase (eNOS, also known as NOS3), which in turn increased VE-cadherin (CDH5) phosphorylation on Y685. In parallel, AMPK also mediated phosphorylation of p38 MAP kinases (hereafter p38) and HSP27 (HSPB1), indicating that it regulated paracellular junctions and cellular contractility, both previously associated with endothelial permeability. Endothelial AMPK provided a missing link in neurovascular permeability, connecting Ca2+ transients to the activation of eNOS and p38, irrespective of the permeability-inducing factor used. Collectively, we find that, due to its compatibility with small molecule antagonists and agonists, as well as siRNA, the ex vivo retina model constitutes a reliable tool to identify and study regulators and mechanisms of acute neurovascular permeability.

Role of the blood-spinal cord barrier: An adheren junction regulation mechanism that promotes chronic postsurgical pain.

Chronic postsurgical pain (CPSP) is a serious postoperative complication with high incidence, and its pathogenesis involves neuroimmune interactions and the breakdown of the blood-spinal cord barrier (BSCB), the decreased level of adheren junction (AJ)-related proteins is an important cause of BSCB injury. Vascular endothelial-cadherin (VE-cadherin) and p120 catenin (p120) constitute the endothelial barrier adheren junction. The Src/p120/VE-cadherin pathway is involved in the regulation of the endothelial barrier function. However, the role of the BSCB-AJ regulatory mechanism in CPSP has not been reported. In this study, we established a skin/muscle incision and retraction (SMIR) model and evaluated the paw withdrawal threshold (PWT), the effects of an Src inhibitor and p120 knockdown on p-Src, p120 and VE-cadherin expression, as well as BSCB-AJ function in rat spinal cord were observed to explore the regulation of BSCB-AJ function by the p-Src/p120/VE-cadherin pathway in promoting SMIR-induced CPSP. The levels of p-Src, p120 and VE-cadherin in the spinal cord were detected by Western blot. Meanwhile, BSCB permeability test was used to detect the changes in BCSB function. Finally, the spatial and temporal localization of p120 in spinal cord was detected by immunofluorescence. Our findings indicated that p-Src/p120/VE-cadherin could induce BSCB-AJ dysfunction and promote the development of CPSP.

Hematopoietic Stem Cell-Independent Differentiation of Mast Cells From Mouse Intraembryonic VE-Cadherin+ Cells.

Hematopoietic stem cell (HSC)-independent hematopoiesis from hemogenic endothelial cells (HECs) in the mouse embryo has been recognized as a source of tissue-resident hematopoietic cells in adult mice. Connective tissue mast cells (MCs) have been reported to originate from VE-cadherin (VE-cad)-expressing HECs in the yolk sac and embryo proper (EP) by a VE-cad-Cre-mediated lineage-tracing analysis. However, it remains unclear whether MCs are generated via a conventional HSC-dependent hematopoietic differentiation pathway, or whether through a fast-track pathway bypassing the emergence of HSCs. Here, we investigated whether EP-derived VE-cad+ cells differentiate into MCs independently of HSCs. VE-cad+ cells isolated from the embryonic day (E) 9.5-10.5 EP robustly formed connective tissue-type MCs in a newly established co-culture system using PA6 stromal cells. In contrast, bone marrow (BM) reconstitution assays of cultured cells indicated that E9.5 VE-cad+ cells did not differentiate into transplantable HSCs in this culture condition. Lymphoid-biased HSCs with a limited self-renewal capacity were occasionally detected in some cultures of E10.5 VE-cad+ cells, while MC growth was constantly observed in all cultures examined. HSCs purified from adult BM required a more extended culture period to form MCs in the PA6 co-culture than the embryonic VE-cad+ cells. Furthermore, E9.5-E10.5 VE-cad+ cells contributed to tissue-resident MCs in postnatal mice when transplanted into the peritoneal cavity of newborn mice. These results suggest that EP-derived VE-cad+ cells generate MCs independently of HSC development in vitro and possess the potential of generating connective tissue MCs in vivo, although the exact differentiation program remains unsolved.

Inhibition of fatty acid synthase protects obese mice from acute lung injury via ameliorating lung endothelial dysfunction.

BACKGROUND: Obesity has been identified as a risk factor for acute lung injury/acute respiratory distress syndrome (ALI/ARDS). However, the underlying mechanisms remain elusive. This study aimed to investigate the role of fatty acid synthase (FASN) in lipopolysaccharide (LPS)-induced ALI under obesity. METHODS: A high-fat diet-induced obese (DIO) mouse model was established and lean mice fed with regular chow diet were served as controls. LPS was intratracheally instilled to reproduce ALI in mice. In vitro, primary mouse lung endothelial cells (MLECs), treated by palmitic acid (PA) or co-cultured with 3T3-L1 adipocytes, were exposed to LPS. Chemical inhibitor C75 or shRNA targeting FASN was used for in vivo and in vitro loss-of-function studies for FASN. RESULTS: After LPS instillation, the protein levels of FASN in freshly isolated lung endothelial cells from DIO mice were significantly higher than those from lean mice. MLECs undergoing metabolic stress exhibited increased levels of FASN, decreased levels of VE-cadherin with increased p38 MAPK phosphorylation and NLRP3 expression, mitochondrial dysfunction, and impaired endothelial barrier compared with the control MLECs when exposed to LPS. However, these effects were attenuated by FASN inhibition with C75 or corresponding shRNA. In vivo, LPS-induced ALI, C75 pretreatment remarkably alleviated LPS-induced overproduction of lung inflammatory cytokines TNF-α, IL-6, and IL-1ß, and lung vascular hyperpermeability in DIO mice as evidenced by increased VE-cadherin expression in lung endothelial cells and decreased lung vascular leakage. CONCLUSIONS: Taken together, FASN inhibition alleviated the exacerbation of LPS-induced lung injury under obesity via rescuing lung endothelial dysfunction. Therefore, targeting FASN may be a potential therapeutic target for ameliorating LPS-induced ALI in obese individuals.