Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.     

Redox regulation of 4-hydroxy-2-nonenal-mediated endothelial barrier dysfunction by focal adhesion, adherens, and tight junction proteins

4-Hydroxy-2-nonenal (4-HNE), one of the major biologically active aldehydes formed during inflammation and oxidative stress, has been implicated in a number of cardiovascular and pulmonary disorders. 4-HNE has been shown to increase vascular endothelial permeability; however, the underlying mechanisms are unclear. Hence, in the current study, we tested our hypothesis that 4-HNE-induced changes in cellular thiol redox status may contribute to modulation of cell signaling pathways that lead to endothelial barrier dysfunction. Exposure of bovine lung microvascular endothelial cells (BLMVECs) to 4-HNE induced reactive oxygen species generation, depleted intracellular glutathione, and altered cell-cell adhesion as measured by transendothelial electrical resistance. Pretreatment of BLM-VECs with thiol protectants, N-acetylcysteine and mercaptopropionyl glycine, attenuated 4-HNE-induced decrease in transendothelial electrical resistance, reactive oxygen species generation, Michael protein adduct formation, protein tyrosine phosphorylation, activation of ERK, JNK, and p38 MAPK, and actin cytoskeletal rearrangement. Treatment of BLMVECs with 4-HNE resulted in the redistribution of FAK, paxillin, VE-cadherin, beta-catenin, and ZO-1, and intercellular gap formation. Western blot analyses confirmed the formation of 4-HNE-derived Michael adducts with the focal adhesion and adherens junction proteins. Also, 4-HNE decreased tyrosine phosphorylation of FAK without affecting total cellular FAK contents, suggesting the modification of integrins, which are natural FAK receptors. 4-HNE caused a decrease in the surface integrin in a time-dependent manner without altering total alpha5 and beta3 integrins. These results, for the first time, revealed that 4-HNE in redox-dependent fashion affected endothelial cell permeability by modulating cell-cell adhesion through focal adhesion, adherens, and tight junction proteins as well as integrin signal transduction that may lead dramatic alteration in endothelial cell barrier dysfunction during heart infarction, brain stroke, and lung diseases.     

Bone marrow‐derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion‐induced lung injury

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow‐derived mesenchymal stem cells (BM‐MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM‐MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion‐induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM‐MSCs. Seventy mice were pre‐treated with BM‐MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro‐vascular endothelial cells (HPMVECs) were pre‐conditioned with BM‐MSCs by oxygen‐glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3‐kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI‐treated mice, administration of BM‐MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD‐treated HPMVECs, co‐culture with BM‐MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM‐MSCs decreased the level of PI3K class I and p‐Akt while the expression of PI3K class III was increased. Finally, BM‐MSCs‐induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM‐MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM‐MSCs and will help to develop new cell‐based therapeutic strategies in lung injury.     

VEGF controls endothelial-cell permeability by promoting the beta-arrestin-dependent endocytosis of VE-cadherin

How vascular endothelial growth factor (VEGF) induces vascular permeability, its first described function, remains poorly understood. Here, we provide evidence of a novel signalling pathway by which VEGF stimulation promotes the rapid endocytosis of a key endothelial cell adhesion molecule, VE-cadherin, thereby disrupting the endothelial barrier function. This process is initiated by the activation of the small GTPase Rac by VEGFR-2 through the Src-dependent phosphorylation of Vav2, a guanine nucleotide-exchange factor. Rac activation, in turn, promotes the p21-activated kinase (PAK)-mediated phosphorylation of a highly conserved motif within the intracellular tail of VE-cadherin. Surprisingly, this results in the recruitment of beta-arrestin2 to serine-phosphorylated VE-cadherin, thereby promoting its internalization into clathrin-coated vesicles and the consequent disassembly of intercellular junctions. Ultimately, this novel biochemical route by which VEGF promotes endothelial permeability through the beta-arrestin2-dependent endocytosis of VE-cadherin may help identify new therapeutic targets for the treatment of many human diseases that are characterized by vascular leakage.     

Actin-binding protein,IQGAP1, regulates LPS-induced RPMVECs hyperpermeability and ICAM-1 upregulation via Rap1/Src signalling pathway

Pulmonary microvascular barrier dysfunction is a hallmark feature of acute lung injury (ALI). IQGAP1 is a ubiquitously expressed scaffolding protein known to regulate cancer metastasis, angiogenesis, and barrier stability. However, the function of IQGAP1 in lipopolysaccharide (LPS)-induced microvascular endothelial hyperpermeability remains poorly understood. In the present study, we demonstrated that IQGAP1 was markedly upregulated in LPS-induced ALI models and rat pulmonary microvascular endothelial cells (RPMVECs). Lentivirus-mediated knockdown of IQGAP1 significantly attenuated the formation of actin stress fibers, phosphorylation of myosin light chain (MLC), and disruption of VE-cadherin, thereby protecting the RPMVECs barrier failure from LPS damage. In addition, IQGAP1 depletion reduced the reactive oxygen species (ROS)-mediated increase in intracellular adhesion molecule-1 (ICAM-1) in RPMVECs stimulated with LPS. Mechanistically, we found that the upregulation of IQGAP1 affected the activity of Rap1 and the downstream phosphorylation of Src. In conclusion, these findings reveal an essential mechanism by which increased IQGAP1 in LPS-treated RPMVECs promotes barrier dysfunction and ICAM-1 upregulation, at least in part by regulating Rap1/Src signalling, indicating that IQGAP1 may be a potential therapeutic target to prevent endothelial hyperpermeability and inflammation in ALI.     

Urokinase-type plasminogen activator (uPA) induces pulmonary microvascular endothelial permeability through low density lipoprotein receptor-related protein (LRP)-dependent activation of endothelial nitric-oxide synthase

Urokinase plasminogen activator (uPA) and PA inhibitor type 1 (PAI-1) are elevated in acute lung injury, which is characterized by a loss of endothelial barrier function and the development of pulmonary edema. Two-chain uPA and uPA-PAI-1 complexes (1-20 nM) increased the permeability of monolayers of human pulmonary microvascular endothelial cells (PMVECs) in vitro and lung permeability in vivo. The effects of uPA-PAI-1 were abrogated by the nitric-oxide synthase (NOS) inhibitor L-NAME (N(D)-nitro-L-arginine methyl ester). Two-chain uPA (1-20 nM) and uPA-PAI-1 induced phosphorylation of endothelial NOS-Ser(1177) in PMVECs, which was followed by generation of NO and the nitrosylation and dissociation of β-catenin from VE-cadherin. uPA-induced phosphorylation of eNOS was decreased by anti-low density lipoprotein receptor-related protein-1 (LRP) antibody and an LRP antagonist, receptor-associated protein (RAP), and when binding to the uPA receptor was blocked by the isolated growth factor-like domain of uPA. uPA-induced phosphorylation of eNOS was also inhibited by the protein kinase A (PKA) inhibitor, myristoylated PKI, but was not dependent on PI3K-Akt signaling. LRP blockade and inhibition of PKA prevented uPA- and uPA-PAI-1-induced permeability of PMVEC monolayers in vitro and uPA-induced lung permeability in vivo. These studies identify a novel pathway involved in regulating PMVEC permeability and suggest the utility of uPA-based approaches that attenuate untoward permeability following acute lung injury while preserving its salutary effects on fibrinolysis and airway remodeling.     

Heterozygous Null Bone Morphogenetic Protein Receptor Type 2 Mutations Promote SRC Kinase-dependent Caveolar Trafficking Defects and Endothelial Dysfunction in Pulmonary Arterial Hypertension

Hereditary pulmonary arterial hypertension (HPAH) is a rare, fatal disease of the pulmonary vasculature. The majority of HPAH patients inherit mutations in the bone morphogenetic protein type 2 receptor gene (BMPR2), but how these promote pulmonary vascular disease is unclear. HPAH patients have features of pulmonary endothelial cell (PEC) dysfunction including increased vascular permeability and perivascular inflammation associated with decreased PEC barrier function. Recently, frameshift mutations in the caveolar structural protein gene Caveolin-1 (CAV-1) were identified in two patients with non-BMPR2-associated HPAH. Because caveolae regulate endothelial function and vascular permeability, we hypothesized that defects in caveolar function might be a common mechanism by which BMPR2 mutations promote pulmonary vascular disease. To explore this, we isolated PECs from mice carrying heterozygous null Bmpr2 mutations (Bmpr2^+/−) similar to those found in the majority of HPAH patients. We show that Bmpr2^+/− PECs have increased numbers and intracellular localization of caveolae and caveolar structural proteins CAV-1 and Cavin-1 and that these defects are reversed after blocking endocytosis with dynasore. SRC kinase is also constitutively activated in Bmpr2^+/− PECs, and localization of CAV-1 to the plasma membrane is restored after treating Bmpr2^+/− PECs with the SRC kinase inhibitor 3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2). Late outgrowth endothelial progenitor cells isolated from HPAH patients show similar increased activation of SRC kinase. Moreover, Bmpr2^+/− PECs have impaired endothelial barrier function, and barrier function is restored after treatment with PP2. These data suggest that heterozygous null BMPR2 mutations promote SRC-dependent caveolar trafficking defects in PECs and that this may contribute to pulmonary endothelial barrier dysfunction in HPAH patients.     

ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration

Leukocyte transendothelial migration (TEM) has been modeled as a multistep process beginning with rolling adhesion, followed by firm adhesion, and ending with either transcellular or paracellular passage of the leukocyte across the endothelial monolayer. In the case of paracellular TEM, endothelial cell (EC) junctions are transiently disassembled to allow passage of leukocytes. Numerous lines of evidence demonstrate that tyrosine phosphorylation of adherens junction proteins, such as vascular endothelial cadherin (VE-cadherin) and beta-catenin, correlates with the disassembly of junctions. However, the role of tyrosine phosphorylation in the regulation of junctions during leukocyte TEM is not completely understood. Using human leukocytes and EC, we show that ICAM-1 engagement leads to activation of two tyrosine kinases, Src and Pyk2. Using phospho-specific Abs, we show that engagement of ICAM-1 induces phosphorylation of VE-cadherin on tyrosines 658 and 731, which correspond to the p120-catenin and beta-catenin binding sites, respectively. These phosphorylation events require the activity of both Src and Pyk2. We find that inhibition of endothelial Src with PP2 or SU6656 blocks neutrophil transmigration (71.1 +/- 3.8% and 48.6 +/- 3.8% reduction, respectively), whereas inhibition of endothelial Pyk2 also results in decreased neutrophil transmigration (25.5 +/- 6.0% reduction). Moreover, overexpression of the nonphosphorylatable Y658F or Y731F mutants of VE-cadherin impairs transmigration of neutrophils compared with overexpression of wild-type VE-cadherin (32.7 +/- 7.1% and 38.8 +/- 6.5% reduction, respectively). Our results demonstrate that engagement of ICAM-1 by leukocytes results in tyrosine phosphorylation of VE-cadherin, which is required for efficient neutrophil TEM.     

Reversibility of increased microvessel permeability in response to VE-cadherin disassembly

We determined the role of vascular endothelial (VE)-cadherin complex in regulating the permeability of pulmonary microvessels. Studies were made in mouse lungs perfused with albumin-Krebs containing EDTA, a Ca(2+) chelator, added to study the VE-cadherin junctional disassembly. We then repleted the perfusate with Ca(2+) to restore VE-cadherin integrity. Confocal microscopy showed a disappearance of VE-cadherin immunostaining in a time- and dose-dependent manner after Ca(2+) chelation and reassembly of the VE-cadherin complex within 5 min after Ca(2+) repletion. We determined the (125)I-labeled albumin permeability-surface area product and capillary filtration coefficient (K(fc)) to quantify alterations in the pulmonary microvessel barrier. The addition of EDTA increased (125)I-albumin permeability-surface area product and K(fc) in a concentration-dependent manner within 5 min. The permeability response was reversed within 5 min after repletion of Ca(2+). An anti-VE-cadherin monoclonal antibody against epitopes responsible for homotypic adhesion augmented the increase in K(fc) induced by Ca(2+) chelation and prevented reversal of the response. We conclude that the disassembled VE-cadherins in endothelial cells are mobilized at the junctional plasmalemmal membrane such that VE-cadherins can rapidly form adhesive contact and restore microvessel permeability by reannealing the adherens junctions.     

Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions

Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE- cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE- cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen- reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE-cadherin distribution was affected by PMN adhesion to the vessel wall in vivo too. This work suggests that PMN adhesion could trigger intracellular signals in EC that possibly regulate VE-cadherin /catenin complex disorganization. This effect could increase EC permeability and facilitate PMN transmigration during the acute inflammatory reaction.     

Angiopoietin-1 prevents VEGF-induced endothelial permeability by sequestering Src through mDia

Vascular endothelial growth factor (VEGF) and Angiopoietin 1 (Ang1) are both potent proangiogenic factors, but, whereas VEGF causes vascular permeability, Ang1 stabilizes blood vessels and protects them from VEGF-induced plasma leakage. The antivascular permeability mechanisms deployed by Ang1 are still undefined. Here, we demonstrate that Ang1 halts the ability of VEGF to induce the phosphorylation-dependent redistribution of the adhesion molecule VE-cadherin, thereby rescuing the endothelial barrier function. Ang1 inhibits the activation of Src by VEGF, the most upstream component of the pathway linking VEGF receptors to VE-cadherin internalization. Indeed, Ang1 promotes the activation of mDia through RhoA, resulting in the association of mDia with Src. This ultimately deprives VEGF receptors of an essential molecule required for promoting the disruption of endothelial cell-cell contacts and paracellular permeability.     

Mesenchymal stem cells protect cigarette smoke‐damaged lung and pulmonary function partly via VEGF–VEGF receptors

Progressive pulmonary inflammation and emphysema have been implicated in the progression of chronic obstructive pulmonary disease (COPD), while current pharmacological treatments are not effective. Transplantation of bone marrow mesenchymal stem cells (MSCs) has been identified as one such possible strategy for treatment of lung diseases including acute lung injury (ALI) and pulmonary fibrosis. However, their role in COPD still requires further investigation. The aim of this study is to test the effect of administration of rat MSCs (rMSCs) on emphysema and pulmonary function. To accomplish this study, the rats were exposed to cigarette smoke (CS) for 11 weeks, followed by administration of rMSCs into the lungs. Here we show that rMSCs infusion mediates a down‐regulation of pro‐inflammatory mediators (TNF‐α, IL‐1β, MCP‐1, and IL‐6) and proteases (MMP9 and MMP12) in lung, an up‐regulation of vascular endothelial growth factor (VEGF), VEGF receptor 2, and transforming growth factor (TGFβ‐1), while reducing pulmonary cell apoptosis. More importantly, rMSCs administration improves emphysema and destructive pulmonary function induced by CS exposure. In vitro co‐culture system study of human umbilical endothelial vein cells (EA.hy926) and human MSCs (hMSCs) provides the evidence that hMSCs mediates an anti‐apoptosis effect, which partly depends on an up‐regulation of VEGF. These findings suggest that MSCs have a therapeutic potential in emphysematous rats by suppressing the inflammatory response, excessive protease expression, and cell apoptosis, as well as up‐regulating VEGF, VEGF receptor 2, and TGFβ‐1. J. Cell. Biochem. 114: 323–335, 2013. © 2012 Wiley Periodicals, Inc.     

Opposing effects of the angiopoietins on the thrombin-induced permeability of human pulmonary microvascular endothelial cells

Background

Angiopoietin-2 (Ang-2) is associated with lung injury in ALI/ARDS. As endothelial activation by thrombin plays a role in the permeability of acute lung injury and Ang-2 may modulate the kinetics of thrombin-induced permeability by impairing the organization of vascular endothelial (VE-)cadherin, and affecting small Rho GTPases in human pulmonary microvascular endothelial cells (HPMVECs), we hypothesized that Ang-2 acts as a sensitizer of thrombin-induced hyperpermeability of HPMVECs, opposed by Ang-1.

Methodology/Principal Findings

Permeability was assessed by measuring macromolecule passage and transendothelial electrical resistance (TEER). Angiopoietins did not affect basal permeability. Nevertheless, they had opposing effects on the thrombin-induced permeability, in particular in the initial phase. Ang-2 enhanced the initial permeability increase (passage, P = 0.010; TEER, P = 0.021) in parallel with impairment of VE-cadherin organization without affecting VE-cadherin Tyr685 phosphorylation or increasing RhoA activity. Ang-2 also increased intercellular gap formation. Ang-1 preincubation increased Rac1 activity, enforced the VE-cadherin organization, reduced the initial thrombin-induced permeability (TEER, P = 0.027), while Rac1 activity simultaneously normalized, and reduced RhoA activity at 15 min thrombin exposure (P = 0.039), but not at earlier time points. The simultaneous presence of Ang-2 largely prevented the effect of Ang-1 on TEER and macromolecule passage.

Conclusions/Significance

Ang-1 attenuated thrombin-induced permeability, which involved initial Rac1 activation-enforced cell-cell junctions, and later RhoA inhibition. In addition to antagonizing Ang-1, Ang-2 had also a direct effect itself. Ang-2 sensitized the initial thrombin-induced permeability accompanied by destabilization of VE-cadherin junctions and increased gap formation, in the absence of increased RhoA activity.     

Endothelial signaling in leukocyte transmigration

Leukocyte transendothelial migration is a multistep process coordinated by chemokine receptors, integrins and cell adhesion molecules. The interaction between leukocytes and endothelial cells is accompanied by bidirectional signaling in both cell types, which is initiated following formation of specialized, "docking" structures. In recent years, it has become clear that signaling in the endothelial cells importantly contributes to the transmigration process. This signaling induces a focal and transient loss of endothelial cell-cell adhesion, which is dependent on vascular-endothelial cadherin. Recent work from several groups has implicated Rho-like GTPases, reactive oxygen species, changes in the actin cytoskeleton and protein tyrosine phosphorylation in the control of VE-cadherin and associated proteins. This review discusses what is currently known about control of VE-cadherin function via intracellular signaling, and its induction by endothelial adhesion molecules of the immunoglobulin superfamily.     

Novel mechanism regulating endothelial permeability via T-cadherin-dependent VE-cadherin phosphorylation and clathrin-mediated endocytosis

T-cadherin is a unique member of the cadherin superfamily of adhesion molecules. In contrast to “classical” cadherins, T-cadherin lacks transmembrane and cytoplasmic domains and is anchored to the cell membrane via a glycosilphosphoinositol moiety. T-cadherin is predominantly expressed in cardiovascular system. Clinical and biochemical studies evidence that expression of T-cadherin increases in post-angioplasty restenosis and atherosclerotic lesions—conditions associated with endothelial dysfunction and pathological expression of adhesion molecules. Here, we provide data suggesting a new signaling mechanism by which T-cadherin regulates endothelial permeability. T-cadherin overexpression leads to VE-cadherin phosphorylation on Y731 (β-catenin-binding site), VE-cadherin clathrin-dependent endocytosis and its degradation in lysosomes. Moreover, T-cadherin overexpression results in activation of Rho GTPases signaling and actin stress fiber formation. Thus, T-cadherin up-regulation is involved in degradation of a key endothelial adhesion molecule, VE-cadherin, resulting in the disruption of endothelial barrier function. Our results point to the role of T-cadherin in regulation of endothelial permeability and its possible engagement in endothelial dysfunction.     

Assessing the permeability of engineered capillary networks in a 3D culture

Many pathologies are characterized by poor blood vessel growth and reduced nutrient delivery to the surrounding tissue, introducing a need for tissue engineered blood vessels. Our lab has developed a 3D co-culture method to grow interconnected networks of pericyte-invested capillaries, which can anastamose with host vasculature following implantation to restore blood flow to ischemic tissues. However, if the engineered vessels contain endothelial cells (ECs) that are misaligned or contain wide junctional gaps, they may function improperly and behave more like the pathologic vessels that nourish tumors. The purpose of this study was to test the resistance to permeability of these networks in vitro, grown with different stromal cell types, as a metric of vessel functionality. A fluorescent dextran tracer was used to visualize transport across the endothelium and the pixel intensity was quantified using a customized MATLAB algorithm. In fibroblast-EC co-cultures, the dextran tracer easily penetrated through the vessel wall and permeability was high through the first 5 days of culture, indicative of vessel immaturity. Beyond day 5, dextran accumulated at the periphery of the vessel, with very little transported across the endothelium. Quantitatively, permeability dropped from initial levels of 61% to 39% after 7 days, and to 7% after 2 weeks. When ECs were co-cultured with bone marrow-derived mesenchymal stem cells (MSCs) or adipose-derived stem cells (AdSCs), much tighter control of permeability was achieved. Relative to the EC-fibroblast co-cultures, permeabilities were reduced 41% for the EC-MSC co-cultures and 50% for the EC-AdSC co-cultures after 3 days of culture. By day 14, these permeabilities decreased by 68% and 77% over the EC-fibroblast cultures. Co-cultures containing stem cells exhibit elevated VE-cadherin levels and more prominent EC-EC junctional complexes when compared to cultures containing fibroblasts. These data suggest the stromal cell identity influences the functionality and physiologic relevance of engineered capillary networks.     

Distinct roles for IκB kinases alpha and beta in regulating pulmonary endothelial angiogenic function during late lung development

Pulmonary angiogenesis is essential for alveolarization, the final stage of lung development that markedly increases gas exchange surface area. We recently demonstrated that activation of the nuclear factor kappa‐B (NFκB) pathway promotes pulmonary angiogenesis during alveolarization. However, the mechanisms activating NFκB in the pulmonary endothelium, and its downstream targets are not known. In this study, we sought to delineate the specific roles for the NFκB activating kinases, IKKα and IKKβ, in promoting developmental pulmonary angiogenesis. Microarray analysis of primary pulmonary endothelial cells (PECs) after silencing IKKα or IKKβ demonstrated that the 2 kinases regulate unique panels of genes, with few shared targets. Although silencing IKKα induced mild impairments in angiogenic function, silencing IKKβ induced more severe angiogenic defects and decreased vascular cell adhesion molecule expression, an IKKβ regulated target essential for both PEC adhesion and migration. Taken together, these data show that IKKα and IKKβ regulate unique genes in PEC, resulting in differential effects on angiogenesis upon inhibition, and identify IKKβ as the predominant regulator of pulmonary angiogenesis during alveolarization. These data suggest that therapeutic strategies to specifically enhance IKKβ activity in the pulmonary endothelium may hold promise to enhance lung growth in diseases marked by altered alveolarization.     

VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts

VE-cadherin is the essential adhesion molecule in endothelial adherens junctions, and the regulation of protein tyrosine phosphorylation is thought to be important for the control of adherens junction integrity. We show here that VE-PTP (vascular endothelial protein tyrosine phosphatase), an endothelial receptor-type phosphatase, co-precipitates with VE-cadherin, but not with beta-catenin, from cell lysates of transfected COS-7 cells and of endothelial cells. Co-precipitation of VE-cadherin and VE-PTP required the most membrane-proximal extracellular domains of each protein. Expression of VE-PTP in triple-transfected COS-7 cells and in CHO cells reversed the tyrosine phosphorylation of VE-cadherin elicited by vascular endothelial growth factor receptor 2 (VEGFR-2). Expression of VE-PTP under an inducible promotor in CHO cells transfected with VE-cadherin and VEGFR-2 increased the VE-cadherin-mediated barrier integrity of a cellular monolayer. Surprisingly, a catalytically inactive mutant form of VE-PTP had the same effect on VE-cadherin phosphorylation and cell layer permeability. Thus, VE-PTP is a transmembrane binding partner of VE-cadherin that associates through an extracellular domain and reduces the tyrosine phosphorylation of VE-cadherin and cell layer permeability independently of its enzymatic activity.     

Human mesenchymal stromal cells small extracellular vesicles attenuate sepsis-induced acute lung injury in a mouse model: the role of oxidative stress and the mitogen-activated protein kinase/nuclear factor kappa B pathway

Background aimsAcute lung injury (ALI) secondary to sepsis is a complex disease associated with high morbidity and mortality. Mesenchymal stem cells (MSCs) and their conditioned medium have been demonstrated to reduce alveolar inflammation, improve lung endothelial barrier permeability and modulate oxidative stress in vivo and in vitro. Recently, MSCs have been found to release small extracellular vesicles (sEVs) that can deliver functionally active biomolecules into recipient cells. The authors’ study was designed to determine whether sEVs released by MSCs would be effective in sepsis-induced ALI mice and to identify the potential mechanisms.MethodsA total of 6 h after cercal ligation and puncture, the mice received saline, sEV-depleted conditioned medium (sEVD-CM) or MSC sEVs via the tail vein.ResultsThe administration of MSC sEVs improved pulmonary microvascular permeability and inhibited both histopathological changes and the infiltration of polymorphonuclear neutrophils into lung tissues. In addition, the activities of antioxidant enzymes were significantly increased in the group treated with sEVs compared with the saline and sEVD-CM groups, whereas lipid peroxidation was significantly decreased. Furthermore, sEVs were found to possibly inhibit phosphorylation of the mitogen-activated protein kinase/nuclear factor kappa B (MAPK/NF-κB) pathway and degradation of IκB but increase the activities of nuclear factor erythroid 2-related factor 2 and heme oxygenase 1.ConclusionsThese findings suggest that one of the effective therapeutic mechanisms of sEVs against sepsis-induced ALI may be associated with upregulation of anti-oxidative enzymes and inhibition of MAPK/NF-κB activation.