Peter L. Hordijk

Traction force dynamics predict gap formation in activated endothelium.

In many pathological conditions the endothelium becomes activated and dysfunctional, resulting in hyperpermeability and plasma leakage. No specific therapies are available yet to control endothelial barrier function, which is regulated by inter-endothelial junctions and the generation of acto-myosin-based contractile forces in the context of cell-cell and cell-matrix interactions. However, the spatiotemporal distribution and stimulus-induced reorganization of these integral forces remain largely unknown. Traction force microscopy of human endothelial monolayers was used to visualize contractile forces in resting cells and during thrombin-induced hyperpermeability. Simultaneously, information about endothelial monolayer integrity, adherens junctions and cytoskeletal proteins (F-actin) were captured. This revealed a heterogeneous distribution of traction forces, with nuclear areas showing lower and cell-cell junctions higher traction forces than the whole-monolayer average. Moreover, junctional forces were asymmetrically distributed among neighboring cells. Force vector orientation analysis showed a good correlation with the alignment of F-actin and revealed contractile forces in newly formed filopodia and lamellipodia-like protrusions within the monolayer. Finally, unstable areas, showing high force fluctuations within the monolayer were prone to form inter-endothelial gaps upon stimulation with thrombin. To conclude, contractile traction forces are heterogeneously distributed within endothelial monolayers and force instability, rather than force magnitude, predicts the stimulus-induced formation of intercellular gaps.

RhoA, RhoB and RhoC differentially regulate endothelial barrier function

ABSTRACT RhoGTPases are known regulators of intracellular actin dynamics that are important for maintaining endothelial barrier function. RhoA is most extensively studied as a key regulator of endothelial barrier function, however the function of the 2 highly homologous family-members (> 88%) RhoB and RhoC in endothelial barrier function is still poorly understood. This study aimed to determine whether RhoA, RhoB and RhoC have overlapping or distinct roles in barrier function and permeability in resting and activated endothelium. By using primary endothelial cells in combination with siRNA transfection to establish individual, double or triple knockdown of the RhoA/B/C RhoGTPases, we found that RhoB, but not RhoA or RhoC, is in resting endothelium a negative regulator of permeability. Loss of RhoB accounted for an accumulation of VE-cadherin at cell-cell contacts. Thrombin-induced loss of endothelial integrity is mediated primarily by RhoA and RhoB. Combined loss of RhoA/B showed decreased phosphorylation of Myosin Light Chain and increased expression of VE-cadherin at cell-cell contacts after thrombin stimulation. RhoC contributes to the Rac1-dependent restoration of endothelial barrier function. In summary, this study shows that these highly homologous RhoGTPases differentially control the dynamics of endothelial barrier function.

The minor histocompatibility antigen 1 (HMHA1)/ArhGAP45 is a RacGAP and a novel regulator of endothelial integrity

Endothelial cells line the vasculature and act as gatekeepers that control the passage of plasma, macromolecules and cells from the circulation to the interstitial space. Dysfunction of the endothelial barrier can lead to uncontrolled leak or edema. Vascular leakage is a hallmark of a range of diseases and despite its large impact no specialized therapies are available to prevent or reduce it. RhoGTPases are known key regulators of cellular behavior that are directly involved in the regulation of the endothelial barrier. We recently performed a comprehensive analysis of the effect of all RhoGTPases and their regulators on basal endothelial integrity. In addition to novel positive regulators of endothelial barrier function, we also identified novel negative regulators, of which the ArhGAP45 (also known as HMHA1) was the most significant. We now demonstrate that ArhGAP45 acts as a Rac-GAP (GTPase-Activating Protein) in endothelial cells, which explains its negative effect on endothelial barrier function. Silencing ArhGAP45 not only promotes basal endothelial barrier function, but also increases cellular surface area and induces sprout formation in a 3D-fibrin matrix. Our data further shows that loss of ArhGAP45 promotes migration and shear stress adaptation. In conclusion, we identify ArhGAP45 (HMHA1) as a novel regulator, which contributes to the fine-tuning of the regulation of basal endothelial integrity.

A CDC42-centered signaling unit is a dominant positive regulator of endothelial integrity

Endothelial barrier function is carefully controlled to protect tissues from edema and damage inflicted by extravasated leukocytes. RhoGTPases, in conjunction with myriad regulatory proteins, exert both positive and negative effects on the endothelial barrier integrity. Precise knowledge about the relevant mechanisms is currently fragmented and we therefore performed a comprehensive analysis of endothelial barrier regulation by RhoGTPases and their regulators. Combining RNAi with electrical impedance measurements we quantified the relevance of 270 Rho-associated genes for endothelial barrier function. Statistical analysis identified 10 targets of which six promoted- and four reduced endothelial barrier function upon downregulation. We analyzed in more detail two of these which were not previously identified as regulators of endothelial integrity. We found that the Rac1-GEF (Guanine nucleotide Exchange Factor) TIAM2 is a positive regulator and the Cdc42(Rac1)-GAP (GTPase-Activating Protein) SYDE1 is a negative regulator of the endothelial barrier function. Finally, we found that the GAP SYDE1 is part of a Cdc42-centered signaling unit, also comprising the Cdc42-GEF FARP1 and the Cdc42 effector PAK7 which controls the integrity of the endothelial barrier. In conclusion, using a siRNA-based screen, we identified new regulators of barrier function and found that Cdc42 is a dominant positive regulator of endothelial integrity.

Intracellular pH steers vascular wall remodelling

This editorial refers to ‘Na+, HCO3−-cotransporter NBCn1 increases pHi gradients, filopodia, and migration of smooth muscle cells and promotes arterial remodelling’ by E. Boedtkjer et al. , pp. 227–239. Many vascular cell biologists will be well aware of the importance of agonist-induced changes in intracellular Ca2+ levels in vessel wall biology (controlling permeability, contractility, vascular stiffening, etc.). However, the impact of changes in intracellular pH (pHi) or local extracellular pH (pHo) is likely less broadly known. Changes in pHi/pHo drive myriad cellular functions and are regulated by a series of ion transporters, including the Na+/H+ transporters of the NHE family,1 and various types of Na+, HCO3− cotransporters of the Slc4 (Solute carrier 4) family.2 Variations in pHi can regulate protein–protein interactions by changing the protonation of amino acid side chains. In cell adhesion and migration, several examples of such regulation are known, involving actin-binding proteins and integrins. One such protein is talin, a large cytoskeletal protein which is concentrated in focal adhesions, sites of integrin-mediated attachment to the extracellular matrix. Protonation of talin reduces its affinity for F-actin, thus modulating …

Stabilization of cell–cell junctions by active vitamin D ameliorates uraemia-induced loss of human endothelial barrier function

Background Uraemia induces endothelial cell (EC) injury and impaired repair capacity, for which the underlying mechanism remains unclear. Active vitamin D (VD) may promote endothelial repair, however, the mechanism that mediates the effects of VD in chronic kidney disease are poorly understood. Thus, we investigated uraemia-induced endothelial damage and the protection against such damage by active VD. Methods We applied electric cell-substrate impedance sensing (ECIS) to study real-time responses of human ECs exposed to pooled uraemic and non-uraemic plasma with or without the addition of active VD. The effects of indoxyl sulphate and p-cresol were tested in non-uraemic plasma. Structural changes for vascular endothelial (VE)-cadherin and F-actin were assessed by immunostaining and quantified. Results The exposure of ECs to uraemic media significantly decreased endothelial barrier function after 24 h. Cell migration after electrical wounding and recovery of the barrier after thrombin-induced loss of integrity were significantly impaired in uraemic-medium stimulated cells and cells exposed to indoxyl sulphate and p-cresol. This effect on ECIS was dependent on loss of cell-cell interaction. Mechanistically, we found that EC, exposed to uraemic media, displayed disrupted VE-cadherin interactions and F-actin reorganization. VD supplementation rescued both endothelial barrier function and cell-cell interactions in ECs exposed to uraemic media. These events were associated with an increment of VE-cadherin at intercellular junctions. Conclusions Our data demonstrate a potentially clinically relevant mechanism for uraemia-induced endothelial damage. Furthermore, active VD rescued the uraemic medium-induced loss of cell-cell adhesion, revealing a novel role of active VD in preservation of endothelial integrity during uraemia.

Endothelial dysfunction as a long-term effect of late onset hypertensive pregnancy disorders: High BMI is key

OBJECTIVE Hypertensive disorders during pregnancy increase cardiovascular risk later in life by 2 to 9-fold. Endothelial activation is one of the underlying mechanisms of cardiovascular risk. Therefore, we decided to investigate endothelial activation in primiparous women, 2.5 years after a hypertensive pregnancy disorder. STUDY DESIGN Plasma samples were taken from women 2.5 years after gestational hypertension (GH) or late onset preeclampsia (cases) and from women 2.5 years after a normotensive pregnancy (controls). We studied the effects of patient plasma on the endothelial barrier function of primary human umbilical vein endothelial cells (HUVECs) using Electric Cell-Substrate Impedance Sensing (ECIS) and we measured levels of endothelial activation markers soluble intercellular adhesion molecule 1 (sICAM-1) and soluble endothelial selectin (sE-selectin) in the plasma samples of patients. RESULTS Plasma from primiparous women with a history of late onset preeclampsia disrupted the endothelial barrier more than plasma from women with a history of GH. Endothelial resistance was reduced by 22% in samples taken after preeclampsia, 16% after normotensive pregnancy and 3% after GH (p ≤ 0.0001 GH versus preeclampsia and p = 0.0003 versus normotensive pregnancy). We did not find differences in the levels of soluble endothelial activation markers (sICAM-1 p = 0.326 and sE-selectin p = 0.978). However, the BMI ≥25 showed a strong correlation with increased levels of sICAM-1 (p = 0.046) and sE-selectin (p = 0.002). CONCLUSION Our results indicate that GH and late onset preeclampsia are distinct disease entities with a different pathogenic mechanism underlying their cardiovascular risk. Furthermore, this study supports the hypothesis that these two diseases are early manifestations of cardiovascular vulnerability due to an unfavorable risk profile, and that obesity plays a main role. Our results suggest that this high-risk female population would be eligible for preventive care.

The covalently immobilized antimicrobial peptide LL37 acts as a VEGF mimic and stimulates endothelial cell proliferation

The chemical coupling of growth factors to solid substrates are discussed as an alternative to delivery systems. Utilizing entire proteins for this application is hampered by safety and stability considerations. Instead, growth factor mimicking peptides are of great interest for biomedical applications, such as tissue engineering, due to their purity and stability. The human cathelicidin derived antimicrobial peptide LL37, beside its microbicidal activity, was shown to stimulate endothelial cell growth when used in a soluble form. Here, in a novel approach, spacer mediated immobilization, but not direct conjugation of LL37, to a gold substrate was shown to result in a pronounced mitogenic effect on endothelial cells, comparable to that of soluble vascular endothelial growth factor.