Developmental differences in focal adhesion kinase expression modulate pulmonary endothelial barrier function in response to inflammation

Compromised pulmonary endothelial cell (PEC) barrier function characterizes acute respiratory distress syndrome (ARDS), a cause of substantial morbidity and mortality. Survival from ARDS is greater in children compared with adults. Whether developmental differences intrinsic to PEC barrier function contribute to this survival advantage remains unknown. To test the hypothesis that PEC barrier function is more well-preserved in neonatal lungs compared with adult lungs in response to inflammation, we induced lung injury in neonatal and adult mice with systemic lipopolysaccharide (LPS). We assessed PEC barrier function in vivo and in vitro, evaluated changes in the expression of focal adhesion kinase 1 (FAK1) and phosphorylation in response to LPS, and determined the effect of FAK silencing and overexpression on PEC barrier function. We found that LPS induced a greater increase in lung permeability and PEC barrier disruption in the adult mice, despite similar degrees of inflammation and apoptosis. Although baseline expression was similar, LPS increased FAK1 expression in neonatal PEC but increased FAK1 phosphorylation and decreased FAK1 expression in adult PEC. Pharmacologic inhibition of FAK1 accentuated LPS-induced barrier disruption most in adult PEC. Finally, in response to LPS, FAK silencing markedly impaired neonatal PEC barrier function, whereas FAK overexpression preserved adult PEC barrier function. Thus, developmental differences in FAK expression during inflammatory injury serve to preserve neonatal pulmonary endothelial barrier function compared with that of adults and suggest that intrinsic differences in the immature versus pulmonary endothelium, especially relative to FAK1 phosphorylation, may contribute to the improved outcomes of children with ARDS.

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Abstract 73: Bone Morphogenetic Protein Activity Modulates Endothelial Barrier Function

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a73 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Thomas Helbing ◽

Elena Ketterer ◽

Bianca Engert ◽

Jennifer Heinke ◽

Sebastian Grundmann ◽

...

Keyword(s):

Acute Lung Injury ◽

Lung Injury ◽

Barrier Function ◽

Endothelial Permeability ◽

Bmp Signaling ◽

Endothelial Barrier ◽

Endothelial Barrier Function

Introduction: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, are associated with high morbidity and mortality in patients. During the progression of ALI, the endothelial cell barrier of the pulmonary vasculature becomes compromised, leading to pulmonary edema, a characteristic feature of ALI. It is well-established that EC barrier dysfunction is initiated by cytoskeletal remodeling, which leads to disruption of cell-cell contacts and formation of paracellular gaps, allowing penetration of protein-rich fluid and inflammatory cells. Bone morphogenetic proteins (BMPs) are important players in endothelial dysfunction and inflammation but their effects on endothelial permeability in ALI have not been investigated until now. Methods and Results: As a first approach to assess the role of BMPs in acute lung injury we analysed BMP4 and BMPER expression in an infectious (LPS) and a non-infectious (bleomycin) mouse models of acute lung injury. In both models BMP4 and BMPER protein expression levels were reduced demonstrated by western blots, suggesting that BMPs are involved in progression ALI. To assess the role of BMPs on vascular leakage, a key feature of ALI, BMP activity in mice was inhibited by i.p. administration of LDN193189, a small molecule that blocks BMP signalling. After 3 days Evans blue dye (EVB) was administered i.v. and dye extravasation into the lungs was quantified as a marker for vascular leakage. Interestingly, LDN193189 significantly increased endothelial permeability compared to control lungs, indicating that BMP signaling is involved in maintenance of endothelial barrier function. To quantify effects of BMP inhibition on endothelial barrier function in vitro, HUVECs were seeded onto transwell filters and were exposed to LDN193189. After 3 days FITC-dextrane was added and passage into the lower chamber was quantified as a marker for endothelial barrier function. Thrombin served as a positive control. As expected from our in vivo experiments inhibition of BMP signaling by LDN193189 enhanced FITC-dextrane passage. To study specific effects of BMPs on endothelial barrier function, two protagonist of the BMP family, BMP2 and BMP4, or BMP modulator BMPER were tested in the transwell assay in vitro. Interestingly BMP4 and BMPER, but not BMP2, reduced FITC-dextrane passage demonstrating that BMP4 and BMPER improved endothelial barrier function. Vice versa, specific knock down of BMP4 or BMPER increased leakage in transwell assays. Im immuncytochemistry silencing of BMPER or BMP4 induced hyperpermeability as a consequence of a pro-inflammatory endothelial phenotype characterised by reduced cell-cell contacts and increased actin stress fiber formation. Additionally, the pro-inflammatory endothelial phenotype was confirmed by real-time revealing increased expression of adhesion molecules ICAM-1 or proinflammatory cytokines such as IL-6 and IL-8 in endothelial cells after BMPER or BMP4 knock down. Confirming these in vitro results BMPER +/- mice exhibit increased extravasation of EVB into the lungs, indicating that partial loss of BMPER impairs endothelial barrier function in vitro and in vivo. Conclusion: We identify BMPER and BMP4 as local regulators of vascular permeability. Both are protective for endothelial barrier function and may open new therapeutic avenues in the treatment of acute lung injury.

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Effect of p18 on Endothelial Barrier Function by Mediating Vascular Endothelial Rab11a-VE-cadherin Recycling

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab172 ◽

2021 ◽

Author(s):

Bo-Wen Xu ◽

Zhi-Qiang Cheng ◽

Xu-Ting Zhi ◽

Xiao-Mei Yang ◽

Zhi-Bo Yan

Keyword(s):

Barrier Function ◽

Adherens Junctions ◽

Cecal Ligation ◽

Underlying Mechanism ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Barrier Dysfunction ◽

Recycling Endosome

Abstract Endothelial barrier integrity requires recycling of VE-cadherin to adherens junctions. Both p18 and Rab11a play significant roles in VE-cadherin recycling. However, the underlying mechanism and the role of p18 in activating Rab11a have yet to be elucidated. Performing in vitro and in vivo experiments, we showed that p18 protein bound to VE-cadherin before Rab11a through its VE-cadherin-binding domain (aa 1–39). Transendothelial resistance showed that overexpression of p18 promoted the circulation of VE-cadherin to adherens junctions and the recovery of the endothelial barrier. Silencing of p18 caused endothelial barrier dysfunction and prevented Rab11a-positive recycling endosome accumulation in the perinuclear recycling compartments. Furthermore, p18 knockdown in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide and cecal ligation puncture. This study showed that p18 regulated the pulmonary endothelial barrier function in vitro and in vivo by regulating the binding of Rab11a to VE-cadherin and the activation of Rab11a.

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A Potential Role of Progestin-Induced Laminin-5/α6-Integrin Signaling in the Formation of Side Branches in the Mammary Gland

Endocrinology ◽

10.1210/en.2012-1518 ◽

2012 ◽

Vol 153 (10) ◽

pp. 4990-5001 ◽

Cited By ~ 7

Author(s):

Gabriele Meyer ◽

Jeffrey Leipprandt ◽

Jianwei Xie ◽

Mark D. Aupperlee ◽

Sandra Z. Haslam

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Nuclear Factor Κb ◽

Inhibitory Effect ◽

Collagen Gels ◽

Paracrine Factor ◽

Laminin 5 ◽

Adult Mice

Abstract Mammary organoids from adult mice produce tubules, analogous to mammary ducts in vivo, in response to hepatocyte growth factor (HGF) when cultured in collagen gels. The combination of HGF plus progestin (R5020) causes reduced tubule number and length. We hypothesized that the inhibitory effect on tubulogenesis was due to progestin-mediated alteration of HGF/c-Met signaling. Using molecular inhibitors and short hairpin RNA, it was determined that HGF activation of Ras-related C3 botulinum toxin substrate (Rac1) was required for the formation of cytoplasmic extensions, the first step of tubulogenesis, and that Rac1 activity was Src kinase (Src) and focal adhesion kinase (FAK) dependent. The highly novel finding was that R5020 reduced tubulogenesis by up-regulating and increasing extracellular laminin and α6-integrin ligation to reduce activation of the Src, focal adhesion kinase, and Rac1 pathway. Receptor activator of nuclear factor-κB ligand, another progesterone-induced paracrine factor, did not replicate this effect of R5020. The inhibitory effect of R5020 on tubulogenesis was likely mediated through progesterone receptor (PR) isoform A (PRA), because PRA is the predominant PR isoform expressed in the organoids, and the progestin-induced effect was prevented by the PR antagonist RU486. These results provide a plausible mechanism that explains progestin/PRA-mediated blunting of HGF-induced tubulogenesis in vitro and is proposed to be relevant to progesterone/PRA-induced side-branching in vivo during pregnancy.

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Modulation of Rac1 Activity by ADMA/DDAH Regulates Pulmonary Endothelial Barrier Function

Molecular Biology of the Cell ◽

10.1091/mbc.e08-04-0395 ◽

2009 ◽

Vol 20 (1) ◽

pp. 33-42 ◽

Cited By ~ 46

Author(s):

Beata Wojciak-Stothard ◽

Belen Torondel ◽

Lan Zhao ◽

Thomas Renné ◽

James M. Leiper

Keyword(s):

Nitric Oxide ◽

Actin Cytoskeleton ◽

Barrier Function ◽

Endothelial Permeability ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Nitric Oxide Synthase Inhibitor ◽

Rac1 Activity

Endogenously produced nitric oxide synthase inhibitor, asymmetric methylarginine (ADMA) is associated with vascular dysfunction and endothelial leakage. We studied the role of ADMA, and the enzymes metabolizing it, dimethylarginine dimethylaminohydrolases (DDAH) in the regulation of endothelial barrier function in pulmonary macrovascular and microvascular cells in vitro and in lungs of genetically modified heterozygous DDAHI knockout mice in vivo. We show that ADMA increases pulmonary endothelial permeability in vitro and in in vivo and that this effect is mediated by nitric oxide (NO) acting via protein kinase G (PKG) and independent of reactive oxygen species formation. ADMA-induced remodeling of actin cytoskeleton and intercellular adherens junctions results from a decrease in PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and a subsequent down-regulation of Rac1 activity. The effects of ADMA on endothelial permeability, Rac1 activation and VASP phosphorylation are prevented by overexpression of active DDAHI and DDAHII, whereas inactive DDAH mutants have no effect. These findings demonstrate for the first time that ADMA metabolism critically determines pulmonary endothelial barrier function by modulating Rac1-mediated remodeling of the actin cytoskeleton and intercellular junctions.

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Adenosine protected against pulmonary edema through transporter- and receptor A2-mediated endothelial barrier enhancement

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00330.2009 ◽

2010 ◽

Vol 298 (6) ◽

pp. L755-L767 ◽

Cited By ~ 32

Author(s):

Qing Lu ◽

Elizabeth O. Harrington ◽

Julie Newton ◽

Brian Casserly ◽

Gregory Radin ◽

...

Keyword(s):

Pulmonary Edema ◽

Adenosine Deaminase ◽

Focal Adhesion ◽

Adenosine Receptor ◽

Barrier Function ◽

Endothelial Barrier ◽

Gtpase Activity ◽

Endothelial Barrier Function ◽

Rac1 Gtpase

We have previously demonstrated that adenosine plus homocysteine enhanced endothelial basal barrier function and protected against agonist-induced barrier dysfunction in vitro through attenuation of RhoA activation by inhibition of isoprenylcysteine-O-carboxyl methyltransferase. In the current study, we tested the effect of elevated adenosine on pulmonary endothelial barrier function in vitro and in vivo. We noted that adenosine alone dose dependently enhanced endothelial barrier function. While adenosine receptor A1 or A3 antagonists were ineffective, an adenosine transporter inhibitor, NBTI, or a combination of DPMX and MRS1754, antagonists for adenosine receptors A2A and A2B, respectively, partially attenuated the barrier-enhancing effect of adenosine. Similarly, inhibition of both A2A and A2B receptors with siRNA also blunted the effect of adenosine on barrier function. Interestingly, inhibition of both transporters and A2A/A2B receptors completely abolished adenosine-induced endothelial barrier enhancement. The adenosine receptor A2A and A2B agonist, NECA, also significantly enhanced endothelial barrier function. These data suggest that both adenosine transporters and A2A and A2B receptors are necessary for exerting maximal effect of adenosine on barrier enhancement. We also found that adenosine enhanced Rac1 GTPase activity and overexpression of dominant negative Rac1 attenuated adenosine-induced increases in focal adhesion complexes. We further demonstrated that elevation of cellular adenosine by inhibition of adenosine deaminase with Pentostatin significantly enhanced endothelial basal barrier function, an effect that was also associated with enhanced Rac1 GTPase activity and with increased focal adhesion complexes and adherens junctions. Finally, using a non-inflammatory acute lung injury (ALI) model induced by α-naphthylthiourea, we found that administration of Pentostatin, which elevated lung adenosine level by 10-fold, not only attenuated the development of edema before ALI but also partially reversed edema after ALI. The data suggest that adenosine deaminase inhibition may be useful in treatment of pulmonary edema in settings of ALI.

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Focal adhesion kinase regulation of endothelial barrier function, apoptosis, and neovascularization

Microvascular Research ◽

10.1016/j.mvr.2011.10.001 ◽

2012 ◽

Vol 83 (1) ◽

pp. 1-2 ◽

Cited By ~ 6

Author(s):

Dolly Mehta

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Barrier Function ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Kinase Regulation

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Adrenomedullin and endothelial barrier function

Thrombosis and Haemostasis ◽

10.1160/th07-02-0128 ◽

2007 ◽

Vol 98 (11) ◽

pp. 944-951 ◽

Cited By ~ 58

Author(s):

Bettina Temmesfeld-Wollbrück ◽

Andreas Hocke ◽

Norbert Suttorp ◽

Stefan Hippenstiel

Keyword(s):

Septic Shock ◽

Barrier Function ◽

Severe Infection ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Oedema Formation ◽

In Vivo Models ◽

Sepsis And Septic Shock

SummaryAlthough loss of endothelial barrier function is a hallmark of every acute inflammation and contributes to fatal loss of organ function during severe infections, there is no sufficient therapy for stabilization of endothelial barrier function. Endogenous peptide adrenomedullin (AM) serum levels were shown to be increased during severe infection including sepsis and septic shock. In different in-vitro and in-vivo models AM acted as a potent therapeutic endothelial barrier function-stabilizing agent. Activation of specific receptors by AM results in elevation of second messenger cAMP. AM inhibits actin-myosin based endothelial cell contraction and junctional disassembly, thereby preventing paracellular permeability and oedema formation. The peptide furthermore possesses several protective cardiovascular qualities, including protection of the microcirculation during inflammation, and was proven as an efficient counter-regulatory molecule in various models of sepsis and septic shock. Overall, AM may be an attractive molecule to combat against cardiovascular malfunction during severe infection.

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