(Schub-)Spannendes aus der Biotechnologie – Blutstrom als Fitness-Training für die Gefäßwand

Mechanische Beanspruchungen verändern bei nahezu jeder Zelle ihre Funktion und ihre Form. Wir interessieren uns besonders für die durch mechanische Beanspruchungen hervorgerufene Effekte im Blutgefäßsystem, dessen Innenseite von den sogenannten Endothelzellen ausgekleidet ist, die eine Permeabilitätsbarriere zwischen Blut und Gewebe darstellen. Durch den Blutstrom sind diese Zellen permanent einer erheblichen mechanischen Beanspruchung ausgesetzt, die nicht nur ihre Form, sondern auch ihre Funktionen wesentlich verändert. Wir haben in unserem Labor einen experimentellen Aufbau entwickelt, mit dem wir erstmalig zeigen konnten, dass laminare Strömungen zu einer Verstärkung der endothelialen Barrierefunktion führen und so vermutlich der Entwicklung der Gefäßverkalkung entgegenwirken. Neben diesen Experimenten wird das neue System auch zur dynamischen Untersuchung der Zellhaftung auf Biomaterialien verwendet.Mechanical loads change the function and morphology of nearly every cell. We are particularly interested in the effects of mechanical loads on the endothelial cells which line the inner surface of blood vessels and control the exchange of water and solutes between blood and tissue (barrier function). These cells are exposed permanently to mechanical forces from the blood stream, which induces changes not only in cell morphology but also in function. We have developed an experimental setup which allows the endothelial barrier function to be measured under defined flow conditions. We have demonstrated for the first time that laminar shear stress enhances the endothelial barrier function, and thus a possible explanation for the anti-arteriosclerotic effect. Importantly, our setup can also be used to dynamically test the adhesion of cells on biomaterials

Caveolin-1 opens endothelial cell junctions by targeting catenins

Aims A fundamental phenomenon in inflammation is the loss of endothelial barrier function, in which the opening of endothelial cell junctions plays a central role. However, the molecular mechanisms that ultimately open the cell junctions are largely unknown. Methods and results Impedance spectroscopy, biochemistry, and morphology were used to investigate the role of caveolin-1 in the regulation of thrombin-induced opening of cell junctions in cultured human and mouse endothelial cells. Here, we demonstrate that the vascular endothelial (VE) cadherin/catenin complex targets caveolin-1 to endothelial cell junctions. Association of caveolin-1 with VE-cadherin/catenin complexes is essential for the barrier function decrease in response to the pro-inflammatory mediator thrombin, which causes a reorganization of the complex in a rope ladder-like pattern accompanied by a loss of junction-associated actin filaments. Mechanistically, we show that in response to thrombin stimulation the protease-activated receptor 1 (PAR-1) causes phosphorylation of caveolin-1, which increasingly associates with β- and γ-catenin. Consequently, the association of β- and γ-catenin with VE-cadherin is weakened, thus allowing junction reorganization and a decrease in barrier function. Thrombin-induced opening of cell junctions is lost in caveolin-1-knockout endothelial cells and after expression of a Y/F-caveolin-1 mutant but is completely reconstituted after expression of wild-type caveolin-1. Conclusion Our results highlight the pivotal role of caveolin-1 in VE-cadherin-mediated cell adhesion via catenins and, in turn, in barrier function regulatio

Polarized actin and VE-Cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis

MAGI1 mediates eNOS activation and NO production in endothelial cells in response to fluid shear stress

Fluid shear stress stimulates endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) production through multiple kinases, including protein kinase A (PKA), AMP-activated protein kinase (AMPK), AKT and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Membrane-associated guanylate kinase (MAGUK) with inverted domain structure-1 (MAGI1) is an adaptor protein that stabilizes epithelial and endothelial cell-cell contacts. The aim of this study was to assess the unknown role of endothelial cell MAGI1 in response to fluid shear stress. We show constitutive expression and co-localization of MAGI1 with vascular endothelial cadherin (VE- cadherin) in endothelial cells at cellular junctions under static and laminar flow conditions. Fluid shear stress increases MAGI1 expression. MAGI1 silencing perturbed flow-dependent responses, specifically, Krüppel-like factor 4 (KLF4) expression, endothelial cell alignment, eNOS phosphorylation and NO production. MAGI1 overexpression had opposite effects and induced phosphorylation of PKA, AMPK, and CAMKII. Pharmacological inhibition of PKA and AMPK prevented MAGI1- mediated eNOS phosphorylation. Consistently, MAGI1 silencing and PKA inhibition suppressed the flow-induced NO production. Endothelial cell-specific transgenic expression of MAGI1 induced PKA and eNOS phosphorylation in vivo and increased NO production ex vivo in isolated endothelial cells. In conclusion, we have identified endothelial cell MAGI1 as a previously unrecognized mediator of fluid shear stress- induced and PKA/AMPK dependent eNOS activation and NO productio

(Schub-)Spannendes aus der Biotechnologie – Blutstrom als Fitness-Training für die Gefäßwand

Mechanische Beanspruchungen verändern bei nahezu jeder Zelle ihre Funktion und ihre Form. Wir interessieren uns besonders für die durch mechanische Beanspruchungen hervorgerufene Effekte im Blutgefäßsystem, dessen Innenseite von den sogenannten Endothelzellen ausgekleidet ist, die eine Permeabilitätsbarriere zwischen Blut und Gewebe darstellen. Durch den Blutstrom sind diese Zellen permanent einer erheblichen mechanischen Beanspruchung ausgesetzt, die nicht nur ihre Form, sondern auch ihre Funktionen wesentlich verändert. Wir haben in unserem Labor einen experimentellen Aufbau entwickelt, mit dem wir erstmalig zeigen konnten, dass laminare Strömungen zu einer Verstärkung der endothelialen Barrierefunktion führen und so vermutlich der Entwicklung der Gefäßverkalkung entgegenwirken. Neben diesen Experimenten wird das neue System auch zur dynamischen Untersuchung der Zellhaftung auf Biomaterialien verwendet.Mechanical loads change the function and morphology of nearly every cell. We are particularly interested in the effects of mechanical loads on the endothelial cells which line the inner surface of blood vessels and control the exchange of water and solutes between blood and tissue (barrier function). These cells are exposed permanently to mechanical forces from the blood stream, which induces changes not only in cell morphology but also in function. We have developed an experimental setup which allows the endothelial barrier function to be measured under defined flow conditions. We have demonstrated for the first time that laminar shear stress enhances the endothelial barrier function, and thus a possible explanation for the anti-arteriosclerotic effect. Importantly, our setup can also be used to dynamically test the adhesion of cells on biomaterials

(Schub-)Spannendes aus der Biotechnologie – Blutstrom als Fitness-Training für die Gefäßwand

Mechanische Beanspruchungen verändern bei nahezu jeder Zelle ihre Funktion und ihre Form. Wir interessieren uns besonders für die durch mechanische Beanspruchungen hervorgerufene Effekte im Blutgefäßsystem, dessen Innenseite von den sogenannten Endothelzellen ausgekleidet ist, die eine Permeabilitätsbarriere zwischen Blut und Gewebe darstellen. Durch den Blutstrom sind diese Zellen permanent einer erheblichen mechanischen Beanspruchung ausgesetzt, die nicht nur ihre Form, sondern auch ihre Funktionen wesentlich verändert. Wir haben in unserem Labor einen experimentellen Aufbau entwickelt, mit dem wir erstmalig zeigen konnten, dass laminare Strömungen zu einer Verstärkung der endothelialen Barrierefunktion führen und so vermutlich der Entwicklung der Gefäßverkalkung entgegenwirken. Neben diesen Experimenten wird das neue System auch zur dynamischen Untersuchung der Zellhaftung auf Biomaterialien verwendet.Mechanical loads change the function and morphology of nearly every cell. We are particularly interested in the effects of mechanical loads on the endothelial cells which line the inner surface of blood vessels and control the exchange of water and solutes between blood and tissue (barrier function). These cells are exposed permanently to mechanical forces from the blood stream, which induces changes not only in cell morphology but also in function. We have developed an experimental setup which allows the endothelial barrier function to be measured under defined flow conditions. We have demonstrated for the first time that laminar shear stress enhances the endothelial barrier function, and thus a possible explanation for the anti-arteriosclerotic effect. Importantly, our setup can also be used to dynamically test the adhesion of cells on biomaterials

Imbalance of SMC1 and SMC3 Cohesins Causes Specific and Distinct Effects

<div><p>SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.</p></div

Two-species system: knockdown of human SMC3 by siRNA and rescue by stably expressed EGFP-msSMC3.

<p>(<b>A</b>) Cells stably transfected with EGFP-msSMC3 were analyzed by IF microscopy. (<b>B</b>) Total cell (RIPA), cytoplasmic and nuclear extracts from cells stably expressing EGFP-msSMC3 collected 72 h after treatment with siSMC1 or siSMC3 were analyzed by IB using anti-EGFP, -SMC3 and -SMC1 antibodies. RIPA buffer was used for the final extraction step after the nuclear extraction with 250 mM ammonium sulfate. Topo II and Karyopherin ß1 were used as a loading control. Relative protein levels are shown at the bottom (representative of 3 experiments).</p