Endothelial permeability is controlled by spatially defined cytoskeletal mechanics: Atomic force microscopy force mapping of pulmonary endothelial monolayer

107

148

Immune cell trafficking requires the frequent breaching of the endothelial barrier either directly through individual cells ('transcellular' route) or through the inter-endothelial junctions ('paracellular' route). What determines the loci or route of breaching events is an open question with important implications for overall barrier regulation. We hypothesized that basic biomechanical properties of the endothelium might serve as crucial determinants of this process. By altering junctional integrity, cytoskeletal morphology and, consequently, local endothelial cell stiffness of different vascular beds, we could modify the preferred route of diapedesis. In particular, high barrier function was associated with predominantly transcellular migration, whereas negative modulation of junctional integrity resulted in a switch to paracellular diapedesis. Furthermore, we showed that lymphocytes dynamically probe the underlying endothelium by extending invadosome-like protrusions (ILPs) into its surface that deform the nuclear lamina, distort actin filaments and ultimately breach the barrier. Fluorescence imaging and pharmacologic depletion of F-actin demonstrated that lymphocyte barrier breaching efficiency was inversely correlated with local endothelial F-actin density and stiffness. Taken together, these data support the hypothesis that lymphocytes are guided by the mechanical 'path of least resistance' as they transverse the endothelium, a process we term 'tenertaxis'.

Section: Ilps Might Function In Local Sampling Of Endothelial Stiffnesssupporting

confidence: 91%

Probing the biomechanical contribution of the endothelium to lymphocyte migration: diapedesis by the path of least resistance

Martinelli

Zeiger

Whitfield

et al. 2014

107

148

“…Atomic force microscopy (AFM) studies revealed that a single cell in a monolayer of HUVECs or bovine aortic ECs, either inflamed or non-inflamed, is stiffer at the cell periphery compared to the cell center and the nucleus (Sato et al, 2000;Schaefer et al, 2014;Stroka and Aranda-Espinoza, 2011a). However, it has also been shown that non-inflamed HUVECs have a softer cell periphery compared to their nucleus and that non-inflamed ECs from human pulmonary aortas show no stiffness gradient (Birukova et al, 2009;Mathur et al, 2001). These discrepancies might be due to the differences in cytokine stimulation, the cell type used, and experimental conditions, such as the size and geometry of the AFM probe.…”

Section: Mechanotransduction In Ecs Substrate Stiffness Controls Endomentioning

confidence: 99%

Cell-stiffness-induced mechanosignaling – a key driver of leukocyte transendothelial migration

Schaefer

Hordijk

2015

104

The breaching of cellular and structural barriers by migrating cells is a driving factor in development, inflammation and tumor cell metastasis. One of the most extensively studied examples is the extravasation of activated leukocytes across the vascular endothelium, the inner lining of blood vessels. Each step of this leukocyte transendothelial migration (TEM) process is regulated by distinct endothelial adhesion receptors such as the intercellular adhesion molecule 1 (ICAM1). Adherent leukocytes exert force on these receptors, which sense mechanical cues and transform them into localized mechanosignaling in endothelial cells. In turn, the function of the mechanoreceptors is controlled by the stiffness of the endothelial cells and of the underlying substrate representing a positive-feedback loop. In this Commentary, we focus on the mechanotransduction in leukocytes and endothelial cells, which is induced in response to variations in substrate stiffness. Recent studies have described the first key proteins involved in these mechanosensitive events, allowing us to identify common regulatory mechanisms in both cell types. Finally, we discuss how endothelial cell stiffness controls the individual steps in the leukocyte TEM process. We identify endothelial cell stiffness as an important component, in addition to locally presented chemokines and adhesion receptors, which guides leukocytes to sites that permit TEM.

“…Birukova et al analysed nonTNFa-treated human pulmonary arterial endothelial cells using a 30-nm tip as compared to a 10-mm bead, which is similar to the size of a neutrophil, as used in our assays (Birukova et al, 2009). They detected, in contrast, hardly any differences in elastic module between the periphery and the centre of the resting cells.…”

Section: Actin-binding Proteins Differentially Regulate Endothelial Cmentioning

confidence: 99%

Actin-binding proteins differentially regulate endothelial cell stiffness, ICAM-1 function and neutrophil transmigration

Schaefer

Riet

Ritz

et al. 2014

Chronic vascular inflammation is driven by interactions between activated leukocytes and the endothelium. Leukocyte b2-integrins bind to endothelial intercellular adhesion molecule 1 (ICAM-1), which allows leukocyte spreading, crawling and transendothelial migration. Leukocytes scan the vascular endothelium for permissive sites to transmigrate, which suggests that there is apical membrane heterogeneity within the endothelium. However, the molecular basis for this heterogeneity is unknown. Leukocyte adhesion induces ICAM-1 clustering, which promotes its association to the actinbinding proteins filamin B, a-actinin-4 and cortactin. We show that these endothelial proteins differentially control adhesion, spreading and transmigration of neutrophils. Loss of filamin B, a-actinin-4 and cortactin revealed adaptor-specific effects on a nuclear-toperipheral gradient of endothelial cell stiffness. By contrast, increasing endothelial cell stiffness stimulates ICAM-1 function. We identify endothelial a-actinin-4 as a key regulator of endothelial cell stiffness and of ICAM-1-mediated neutrophil transmigration. Finally, we found that the endothelial lining of human and murine atherosclerotic plaques shows elevated levels of a-actinin-4. These results identify endothelial cell stiffness as an important regulator of endothelial surface heterogeneity and of ICAM-1 function, which in turn controls the adhesion and transmigration of neutrophils.