Endothelial barrier dysfunction and inflammation
Mariya Y. Radeva and Alexander Garcia Ponce (group leaders)
Ibrahim Hamad (MD student)
Marlene Delivuk (technician)
Under allergic and inflammatory conditions, the functional integrity of the endothelium lining the inner surface of the blood vessels is compromised primarily by intercellular gap formation. This effect is associated with reduced endothelial barrier function and increased paracellular permeability.
Modulation of endothelial barrier function can be achieved by a number of molecules such as the second messenger cyclic adenosine monophosphate (cAMP), members of the Rho family of small GTPases, i.e RhoA and Rac1, as well as actin-binding proteins (ABPs).
Our previous studies demonstrated that cAMP-mediated activation of the small GTPase Rac1 is an essential signaling step required for stabilization of endothelial barrier function. In this respect, it was shown that Rac1 is activated by both the exchange protein directly activated by cAMP (Epac1)/ Ras-related protein 1 (Rap1) and protein kinase A (PKA)-dependent signaling pathways (Figure 1).
Figure 1: Mechanisms required for barrier maintenance and stabilization (from Radeva MY, Waschke J. Acta Physiol (Oxf). 2018 Jan;222(1))
On another side, tight regulation of PKA function can be achieved by discrete compartmentalization of the enzyme via physical interaction with A-kinase anchoring proteins (AKAPs). Apparently, this signaling pathway is impaired in inflammation and sepsis, since the levels of cAMP become lower and Rac1 is inactivated, contributing to endothelial barrier breakdown (Figure 2). Therefore, our group worked on discovering the functional relevance of AKAP molecules for maintenance of endothelial barrier integrity and cytoskeleton reorganization. So far we found AKAP220 and AKAP12 as new candidates relevant for maintenance of endothelial barrier integrity (Figure 1).
Figure 2: Mechanisms leading to barrier disruption in inflammation (from Radeva MY, Waschke J. Acta Physiol (Oxf). 2018 Jan;222(1))
In addition, we studied the role of Epac1-dependent pathways in the maintenance of endothelial barrier. These investigations were designed as follow-up research in Epac1−/− (Epac1-knockout) mice that increased the basal permeability to high and low molecular weight tracers in different microvascular beds such as in the skin, small and large intestine, and adipose tissue.
Epac1 is a central player working through multiple mechanisms to promote cAMP-mediated endothelial barrier stabilization, including modulation of junctional integrity and rearrangement of cortical actin as well as by cross-talk between GTPases. In this respect, our recent study emphasize that part of the Epac1 effect might be mediated by regulation of not only Rap1, but also by simultaneous involvement of Rac1 and RhoA molecules.
Furthermore, the control of actin dynamics is crucial for endothelial barrier stability in vivo, since strengthening of the cortical actin cytoskeleton, partially regulated by actin-binding proteins (ABPs), controls the dynamics of junctional proteins and thus endothelial barrier integrity. In line with this, a growing number of ABPs such as vasodilator-stimulated phosphoprotein (VASP), cortactin (CTTN) and adducin (ADD) were shown to play a significant role in modulation of endothelial barrier (Figure 1).
The interaction of ABPs with the actin cytoskeleton is diverse. In this respect, it was shown that CTTN contributes to branching of pre-existent actin filaments necessary for different cellular functions. On the other hand, ADD is a key component for the assembly of actin-spectrin networks important for supporting plasma membrane. Although, the ABPs associations with the actin cytoskeleton have been extensively studied, their relation with intercellular contacts and thus, endothelial barrier homeostasis is still not completely understood. For this reason, our group is currently interested in elucidating the role of CTTN and ADD in the maintenance of cAMP-mediated endothelial barrier integrity.
Another topic our group is focused on is the role of γ-catenin or more commonly known as plakoglobin (PG) in endothelial barrier integrity. PG is a structural and functional homologue of ß-catenin and therefore, in a similar fashion to the latter, exerts a dual function of being structural/ cell adhesion related and signaling molecule, capable of translocating to the nucleus and modulating cell transcription or protein stability in both a ß-catenin- dependent and – independent manner (Figure 1). Recent studies have shown the important role of PG in the regulation of endothelia barrier integrity. However, the relationship between PG and cAMP- mediated endothelial barrier stabilization is not clear yet, and therefore our current studies are focused on this topic.
As part of our experimental procedures, we employ in vitro cellular models and measure Transendothelial Electrical Resistance (TER) from different cell monolayers. These analyses are combined with structural, pharmacological and biochemical investigations. Moreover, atomic force microscopy and optical laser tweezer are readily available to study junctional proteins binding forces.
Waschke J, Baumgartner W, Adamson RH, Zeng M, Aktories K, Barth H, Wilde C, Curry FE, Drenckhahn D (2004) Requirement of Rac activity for maintenance of capillary endothelial barrier properties. Am. J. Physiol. Heart Circ. Physiol. 286: H394-401
Waschke J, Drenckhahn D, Adamson RH, Curry FE (2004) Role of adhesion and contraction in Rac 1-regulated endothelial barrier function in vivo and in vitro. Am. J. Physiol. Heart Circ. Physiol. 287: H704-711
Waschke J, Drenckhahn D, Adamson RH, Barth H, Curry FE (2004) cAMP protects endothelial barrier functions by preventing Rac-1 inhibition. Am. J. Physiol. Heart Circ. Physiol. 287: H2427-433
Waschke J, Adamson RH, Curry FE, Drenckhahn D (2005) Regulation of actin dynamics is critical for endothelial barrier functions. Am. J. Physiol. Heart Circ. Physiol. 288: H1296-305
Waschke J, Burger S, Curry FE, Drenckhahn D, Adamson RH (2006) Activation of Rac-1 and Cdc42 stabilizes the microvascular endothelial barrier. Histochem. Cell Biol. 125(4): 397-406
Waschke J, Golenhofen N, Kurzchalia, TV, Drenckhahn D (2006) Protein kinase C-mediated endothelial barrier regulation is caveolin 1-dependent. Histochem. Cell Biol.126 (1): 17-26
Förster C, Waschke J, Burek M, Leers J, Drenckhahn D (2006) Glucocorticoid effects on microvascular endothelial barrier permeability are brain specific. J. Physiol. 573(Pt2): 413-425
Chtcheglova L, Waschke J, Wildling L, Drenckhahn D, Hinterdorfer P (2007) Nano-Scale dynamic recognition imaging on vascular endothelial cells. Biophys. J., 15; 93: L11-13
Müller-Marschhausen K, Waschke J, Drenckhahn, D (2008) Physiological hydrostatic pressure protects endothelial monolayer integrity in vitro. Am. J. Physiol. Cell Physiol., 294(1):C324-332
Baumer Y, Burger S, Curry FE, Golenhofen N, Drenckhahn D, Waschke J (2008) Different role of Rho GTPases in endothelial barrier regulation dependent on endothelial cell origin. Histochem. Cell Biol., 129(2): 179-191
Schlegel N, Burger S, Golenhofen N, Walter U, Drenckhahn D, Waschke J (2008) The role of VASP in the regulation of cAMP- and Rac 1-mediated endothelial barrier stabilization. Am. J. Physiol. Cell Physiol., 294(1): c178-188
Baumer Y, Drenckhahn D, Waschke J (2008) cAMP induced Rac 1-mediated cytoskeletal reorganization in microvascular endothelium. Histochem. Cell Biol., 129(6): 765-778
Samarin J, Rehm M, Krueger B, Waschke J, Goppelt-Struebe M (2009) Up-regulation of connective tissue growth factor in endothelial cells by the microtubule-destabilizing agent combretastatin A-4. Mol. Cancer Res, 7(2): 180-188
Schlegel N, Waschke J (2009) VASP is involved in cAMP-mediated Rac 1 activation in microvascular endothelial cells. AJP Cell Physiology, 296(3): C453-462
Schlegel N, Baumer Y, Drenckhahn D, Waschke J (2009) LPS-induced endothelial barrier breakdown is cAMP-dependent in vivo and in vitro. Crit. Care Med., 37(5): 1735-43
Schlegel N, Waschke J (2009) Impaired integrin-mediated adhesion contributes to reduced barrier properties in VASP-deficient microvascular endothelium. J. Cell. Physiol, 220:357-366
Baumer Y, Spindler V, Werthmann R, Buenemann M, Waschke J (2009) Role of Rac 1 and cAMP in endothelial barrier stabilization and thrombin-induced barrier breakdown, J. Cell Physiol., 220: 716-726
Schlegel N, Waschke J (2009) Impaired cAMP and Rac 1 signalling contribute to TNF-α-induced endothelial barrier breakdown in microvascular endothelium. Microcirculation, 16: 521-533
Schick MA, Isbary TJ, Schlegel N, Brugger J, Waschke J, Muellenbach R, Roewer N, Wunder C (2010) The impact of crystalloid infusion on the kidney in rodent sepsis. Intensive Care Med., 36(3). 541-548
Benz PM, Blume C, Seifert S, Wilhelm S, Waschke J, Schuh K, Gertler F, Münzel T, Renné T (2010) Differential VASP phosphorylation controls remodeling of the actin cytoskeleton. J. Cell Sci., 122:3954-3965
Chtcheglova L, Wildling L, Waschke J, Drenckhahn D, Hinterdorfer P (2010) AFM functional imaging on vascular endothelial cells.. J.Mol.Recognit., 23(6): 598-596
Spindler V, Waschke J (2011) Beta-adrenergic stimulation contributes to maintenance of endothelial functions under baseline conditions. Microcirculation, 18(2): 118-127
Chen W, Gaßner B, Börner S, Nikolaev VO, Schlegel N, Waschke J, Steinbronn N, Strasser R, Kuhn M (2012) Atrial natriuretic peptide enhances microvascular albumin permeability by the caveolae-mediated transcellular pathway. Cardiovasc Res., 93(1): 141-151
Spindler V, Peter D, Harms GS, Asan E, Waschke J (2011). Ultrastructural analysis reveals cAMP-dependent enhancement of microvascular endothelial barrier functions via Rac1-mediated reorganization of intercellular junctions. Am J Pathol., 178(5):2424-2436
Schick MA, Wunder C, Wollborn J, Roewer N, Waschke J, Germer CT, Schlegel N (2012) Phosphodiesterase-4 inhibition as a therapeutic approach to treat capillary leakage in systemic inflammation J. Physiol., 590(Pt 11):2693-2708
Schlegel N, Leweke R, Meir M., Germer CT, Waschke J (2012) Role of NF-κB activation in LPS-induced endothelial barrier breakdown. Histochem. Cell Biol., 138(4):627-641
Radeva MY, Kugelmann D, Spindler V, Waschke J (2014) PKA compartmentalization via AKAP220 and AKAP12 contributes to endothelial barrier regulation. PLoS One.; 9(9)
Adamson RH, Clark JF, Radeva M, Kheirolomoom A, Ferrara KW, Curry FE. (2014) Albumin modulates S1P delivery from red blood cells in perfused microvessels: mechanism of the protein effect. Am J Physiol Heart Circ Physiol. 2014 Apr 1;306(7):H1011-7. doi: 10.1152/ajpheart.00829.2013. Epub 2014 Feb 14. Erratum in: Am J Physiol Heart Circ Physiol. 2014 Jul 1;307(1):H120
Flemming S, Burkard N, Renschler M, Vielmuth F, Meir M, Schick MA, Wunder C, Germer CT, Spindler V, Waschke J, Schlegel N (2015) Soluble VE-cadherin is involved in endothelial barrier breakdown in systemic inflammation and sepsis. Cardiovasc Res., 107(1):32-44
Kugelmann D, Waschke J, Radeva MY (2015) Adducin is involved in endothelial barrier stabilization. PLoS One;10(5)
Kugelmann D, Rotkopf LT, Radeva MY, Garcia-Ponce A, Walter E, Waschke J. (2018) Histamine causes endothelial barrier disruption via Ca2+-mediated RhoA activation and tension at adherens junctions. Sci Rep. 2018 Sep 5;8(1):13229. doi: 10.1038/s41598-018-31408-3
Epac1 Is Crucial for Maintenance of Endothelial Barrier Function through A Mechanism Partly Independent of Rac1. García-Ponce A, Schuster K, Døskeland SO, Reed RK, Curry FE, Waschke J, Radeva MY. Cells. 2020 Sep 25; 9(10):E2170. doi: 10.3390/cells9102170.PMID: 32992982
Spindler V, Schlegel N, Waschke J (2010) Role of GTPases in control of microvascular permeability. Cardiovasc.Res., 87(2):243-253
Schlegel N, Waschke J (2010) Vasodilator-stimulated phosphoprotein (VASP): Crucial for activation of Rac1 in endothelial barrier maintenance. Cardiovasc.Res., 87(1):1-3
Schlegel N, Waschke J (2014) cAMP with other signaling cues converges on Rac1 to stabilize the endothelial barrier- a signaling pathway compromised in inflammation. Cell Tissue Res. 355(3):587-96
Radeva M, Waschke J (2017) Mind the gap- mechanisms regulating the endothelial barrier. Acta Physiol., 222(1)