Targeting β1-integrin inhibits vascular leakage in endotoxemia

Hakanpää, Laura; Kiss, Elina A.; Jacquemet, Guillaume; Miinalainen, Ilkka; Lerche, Martina; Guzmán, Camilo; Mervaala, Eero; Eklund, Lauri; Ivaska, Johanna; Saharinen, Pipsa

doi:10.1073/pnas.1722317115

Cited by 77 publications

(101 citation statements)

References 34 publications

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“…Prior to FBSR, our confocal-based TFM analyses have yielded between 0.2 to 0.5 trackable beads per square micrometre (Jacquemet et al, 2019;Närvä et al, 2017;Georgiadou et al, 2017;Hakanpaa et al, 2018) (Figure 1f), in agreement with values reported by others (Colin-York et al, 2016). Here, by implementing FBSR, and conservative reconstruction parameters, we were able to substantially increase the number of trackable beads to 1.2 beads per square micrometre ( Figure 1f).…”

Section: Increasing the Bead Density Of Tfm Gels Using Fluctuationbassupporting

confidence: 86%

“…Next, we sought to demonstrate that our improved TFM pipeline could be used to answer biologically relevant questions. In particular, TFM is very commonly used to assess how a protein or a drug treatment influences the ability of cells to exert forces on their environment (Jacquemet et al, 2019;Hakanpaa et al, 2018;Närvä et al, 2017). For this purpose, we aimed to cause a mild perturbation to simulate a plausible biological response and treated cells with either DMSO or the myosin II inhibitor blebbistatin for 15 minutes (Figure 4a and 4b).…”

Section: Biologically Relevant Applications Of Fluctuation-based Tfmmentioning

confidence: 99%

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Fluctuation-Based Super-Resolution Traction Force Microscopy

Stubb

Laine

Guzmán

et al. 2019

Preprint

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Cellular mechanics play a crucial role in tissue morphogenesis and homeostasis and are often misregulated in disease. Traction force microscopy (TFM) is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, the power of TFM is limited by poor resolution and low throughput. Here, we propose a simplified protocol and imaging strategy, relying on super-resolution microscopy enabled by fluorophore fluctuation analysis, to enhance the output of TFM, by increasing both bead density as well as the accuracy of bead tracking in TFM gels. Our analysis pipeline can be used on either camera-based confocal or widefield microscopes and is fully compatible with available TFM analysis software. In addition, we demonstrate that our workflow can be used to gain biologically relevant information and is suitable for longterm live measurement of traction forces even in light-sensitive cells. Finally, we propose that our strategy could be used to considerably simplify the implementation of TFM screens. Our streamlined protocol can be performed with minimal hardware and software investment, and has the potential to standardize high-resolution TFM. Fluctuation-based super-resolution microscopy | SACD, LiveSRRF | Traction force microscopy | Live imaging | Mechanobiology Correspondence: guillaume.jacquemet@abo.fi or johanna.ivaska@utu.fi Stubb et al. | bioRχiv |

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Section: Increasing the Bead Density Of Tfm Gels Using Fluctuationbassupporting

confidence: 86%

Section: Biologically Relevant Applications Of Fluctuation-based Tfmmentioning

confidence: 99%

Fluctuation-Based Super-Resolution Traction Force Microscopy

Stubb

Laine

Guzmán

et al. 2019

Preprint

Self Cite

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“…Intriguingly, work by Hakanpaa et al showed that the deletion of a single β1 integrin allele in ECs of established blood vessels did not alter basal permeability but rather protected β1 EC heterozygous mice from LPS-induced hyperpermeability compared to wild type littermates. 67 Furthermore, pharmacological inhibition of β1 integrin also mitigated LPS-induced tracheal permeability. Prior to LPS treatment, wildtype mice were prophylactically administered either a β1 blocking antibody (HMβ1-1) or an isotype nonimmune IgG control with the former group showing reduced levels of FITC-dextran extravasation relative to control.…”

Section: Role Of β1 Integrins In Ec Barrier Functionmentioning

confidence: 98%

“…Prior to LPS treatment, wildtype mice were prophylactically administered either a β1 blocking antibody (HMβ1-1) or an isotype nonimmune IgG control with the former group showing reduced levels of FITC-dextran extravasation relative to control. 67 It was therefore proposed that in quiescent endothelium β1 integrin predominantly localizes to FAs whereas inflammatory molecules acting in an angiopoietin-2-dependent manner induce β1 integrin association with tensin at fibrillar adhesions. β1 integrin-positive fibrillar adhesions in turn promote cytoskeletal tension that alters cell-cell junctions and increases vascular permeability.…”

Section: Role Of β1 Integrins In Ec Barrier Functionmentioning

confidence: 99%

Integrin-dependent regulation of the endothelial barrier

Pulous

Petrich

2019

Tissue Barriers

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The endothelium physically separates blood from surrounding tissue and yet allows for the regulated passage of nutrients, waste, and leukocytes into and out of the circulation. Trans-endothelium flux occurs across endothelial cells (transcellular) and between endothelial cells (paracellular). Paracellular endothelial barrier function depends on the regulation of cell-cell junctions. Interestingly, a functional relationship between cell-cell junctions and cell-matrix adhesions has long been appreciated but the molecular mechanisms underpinning this relationship are not fully understood. Here we review the evidence that supports the notion that cell-matrix interactions contribute to the regulation of cell-cell junctions, focusing primarily on the important adherens junction protein VE-cadherin. In particular, we will discuss recent insights gained into how integrin signaling impacts VE-cadherin stability in adherens junctions and endothelial barrier function. ARTICLE HISTORY

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“…Integrins are involved in a vast spectrum of biological processes, including cell adhesion, gene expression, proliferation, and cell migration 40 . Deletion of fibronectin or fibronectin-binding integrins (α5β1, αvβ3) limits endothelial NF-κB activation and proinflammatory gene expression in atherosclerosis models 9,11 , whereas β1 integrin inhibition reduces vascular permeability in response to proinflammatory mediators (LPS, IL-1β) 41 . To dissect the role of integrin signaling in endothelial cell phenotype, several groups have used variety of integrin inhibitors and blocking antibodies to implicate integrin activation and new integrin-matrix interactions in endothelial proinflammatory responses, particularly in response to flow and oxLDL 8,12 .…”

Section: Discussionmentioning

confidence: 99%

Integrin Affinity Modulation Critically Regulates Atherogenic Endothelial Activationin vitroandin vivo

Al‐Yafeai

Peretik

Pearson

et al. 2020

Preprint

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While vital to platelet and leukocyte adhesion, integrin activation's role in adherent cells remains controversial. In endothelial cells, atheroprone hemodynamics and oxidized lipoproteins promote endothelial integrin activation in vitro, and integrin inhibitors reduce endothelial activation to these stimuli in vitro and in vivo. However, integrin activation's importance to endothelial phenotype remains unknown. We now show that integrin activation-deficient endothelial cells (talin1 L325R) adhere and spread, suggesting that integrin activation is dispensable for endothelial cell adhesion. However, talin1 L325R endothelial cells fail to support integrin activation, fibronectin deposition, and proinflammatory responses to atheroprone hemodynamics and oxidized lipoproteins. Rescuing integrin activation in talin1 L325R cells partially restores fibronectin deposition, whereas NF-B activation and maximal fibronectin deposition require both integrin activation and other integrin-independent signaling.Similarly, atheroprone hemodynamics fail to promote inflammation and macrophage recruitment in endothelial-specific talin1 L325R mice. These studies demonstrate a vital role for integrin activation in regulating endothelial phenotype. Materials and MethodsCell culture-Mouse lung endothelial cells were isolated from Talin1 fl/L325R mice (gift of Brian Petrich, Emory, Atlanta, GA) as previously described 21 ). Briefly, lung tissue was harvested, minced, and pushed through a 16G needle and subjected to enzymatic digestion to obtain a single cell suspension. After sorting with magnetic beads coupled to ICAM2 antibodies (eBiosource), cells were transformed using a retrovirus expressing temperature-sensitive large T-antigen. The floxed allele of talin1 was deleted using adenovirus harboring either GFP (green fluorescence protein)-Cre or GFP alone and then sorted for GFP positivity. Lung endothelial cells were grown in DMEM with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% glutamax. For endothelial shear stress treatments, cells were plated on 38 x 75 mm glass slides (Corning) at confluency, and slides were assembled into a parallel plate flow chamber as previously described 24 . For acute shear stress, cells were exposed to 12 dynes/cm2 flow for up to 1 hour. For chronic oscillatory flow (model of disturbed flow), cells were exposed to ±5 dynes/cm2 with 1 dyne/cm2 forward flow for media exchange. Human LDL (Intracell) was oxidized by dialysis in PBS containing 13.8 M Cu2SO4 followed by 50 M EDTA as previously described 25 . Computationally designed transmembrane α-helical peptides (CHAMP) peptides were a gift of Dr. William DeGrado (University of California -San Francisco) 26 .Western blot-Cell lysis and western blot was done as previously described 25 . Briefly, cells were lysed in 2X laemmli buffer, separated by SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA). Consequently, membranes were blocked in 5% dry milk in TBST for an hour, then incubated with pr...

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Targeting β1-integrin inhibits vascular leakage in endotoxemia

Cited by 77 publications

References 34 publications

Fluctuation-Based Super-Resolution Traction Force Microscopy

Fluctuation-Based Super-Resolution Traction Force Microscopy

Integrin-dependent regulation of the endothelial barrier

Integrin Affinity Modulation Critically Regulates Atherogenic Endothelial Activationin vitroandin vivo

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