G. P. van Nieuw Amerongen

Driving Rho GTPase activity in endothelial cells regulates barrier integrity

In the past decade understanding of the role of the Rho GTPases RhoA, Rac1 and Cdc42 has been developed from regulatory proteins that regulate specific actin cytoskeletal structures - stress fibers, lamellipodia and filopodia - to complex integrators of cytoskeletal structures that can exert multiple functions depending on the cellular context. Fundamental to these functions are three-dimensional complexes between the individual Rho GTPases, their specific activators (GEFs) and inhibitors (GDIs and GAPs), which greatly outnumber the Rho GTPases themselves, and additional regulatory proteins. By this complexity of regulation different vasoactive mediators can induce various cytoskeletal structures that enable the endothelial cell (EC) to respond adequately. In this review we have focused on this complexity and the consequences of Rho GTPase regulation for endothelial barrier function. The permeability inducers thrombin and VEGF are presented as examples of G-protein coupled receptor- and tyrosine kinase receptor-mediated Rho GTPase activation, respectively. These mediators induce complex but markedly different networks of activators, inhibitors and effectors of Rho GTPases, which alter the endothelial barrier function. An interesting feature in this regulation is that Rho GTPases often have both barrier-protecting and barrier-disturbing functions. While Rac1 enforces the endothelial junctions, it becomes part of a barrier-disturbing mechanism as activator of reactive oxygen species generating NADPH oxidase. Similarly RhoA is protective under basal conditions, but becomes involved in barrier dysfunction after activation of ECs by thrombin. The challenge and promise lies in unfolding this complex regulation, as this will provide leads for new therapeutic opportunities.

Angiopoietin-2, permeability oedema, occurrence and severity of ALI/ARDS in septic and non-septic critically ill patients

Background: Angiopoietin-2 and vascular endothelial growth factor (VEGF) may impair vascular barrier function while angiopoietin-1 may protect it. It was hypothesised that circulating angiopoietin-2 is associated with pulmonary permeability oedema and severity of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) during septic or non-septic critical illness. Methods: Plasma levels of angiopoietin-1 and angiopoietin-2 were measured in mechanically ventilated patients (24 with sepsis, 88 without sepsis), together with the pulmonary leak index (PLI) for 67-gallium-labelled transferrin and extravascular lung water (EVLW) by transpulmonary thermal-dye dilution as measures of pulmonary permeability and oedema, respectively. ALI/ARDS was characterised by consensus criteria and the lung injury score (LIS). Plasma VEGF and von Willebrand factor (VWF) levels were assayed. Results: Angiopoietin-2, VWF, PLI, EVLW and LIS were higher in patients with sepsis than in those without sepsis and higher in patients with ALI/ARDS (n = 10/12 in sepsis, n = 19/8 in non-sepsis) than in those without. VEGF was also higher in patients with sepsis than in those without. Patients with high PLI, regardless of EVLW, had higher angiopoietin-2 levels than patients with normal PLI and EVLW. Angiopoietin-2 correlated with the PLI, LIS and VWF levels (minimum r = 0.34, p<0.001) but not with EVLW. Angiopoietin-2 and VWF were predictive for ARDS in receiver operating characteristic curves (minimum area under the curve = 0.69, p = 0.006). Angiopoietin-1 and VEGF did not relate to the permeability oedema of ALI/ARDS. Conclusion: Circulating angiopoietin-2 is associated with pulmonary permeability oedema, occurrence and severity of ALI/ARDS in patients with and without sepsis. The correlation of angiopoietin-2 with VWF suggests activated endothelium as a common source.

Involvement of Rho Kinase in Endothelial Barrier Maintenance

Objective—Rho kinase mediates vascular leakage caused by many vasoactive agents including thrombin. Enhanced Rho kinase activity induces endothelial barrier dysfunction by a contractile mechanism via inactivation of Myosin Phosphatase (MP). Here, we investigated the contribution of basal Rho kinase activity to the regulation of endothelial barrier integrity. Methods and Results—Using a phospho-specific antibody against the myosin phosphatase targeting subunit (Thr696−MYPT1) as a marker for Rho kinase activity, basal endothelial Rho kinase activity was observed at cell-cell contact sites, in vitro and in situ. Thrombin enhanced MYPT phosphorylation at F-actin stress fibers. Inhibition of basal Rho kinase activity for 24 hours or depletion of Rho kinase (ROCK-I and -II) by siRNA disrupted endothelial barrier integrity, opposite to the previously observed protection from the thrombin-enhanced endothelial permeability. This barrier dysfunction could not be explained by changes in RhoA, Rac1, eNOS, or apoptosis. Remarkably, basal Rho kinase activity was essential for proper expression of the adhesion molecule VE-cadherin. Conclusions—Rho kinase has opposing activities in regulation of endothelial barrier function: (1) an intrinsic barrier-protective activity at the cell margins, and (2) an induced barrier-disruptive activity at contractile F-actin stress fibers. These findings may have implications for long-term antivascular leak therapy.

Fasudil reduces monocrotaline-induced pulmonary arterial hypertension: comparison with bosentan and sildenafil

Pulmonary arterial hypertension (PAH) still cannot be cured, warranting the search for novel treatments. Fasudil (a Rho kinase inhibitor) was compared with bosentan (an endothelin receptor blocker) and sildenafil (a phosphodiesterase 5 inhibitor), with emphasis on right ventricular (RV) function, in a reversal rat model of monocrotaline (MCT)-induced PAH. In addition, the effects of combining bosentan or sildenafil with fasudil were studied. MCT (40 mg·kg body weight−1) induced clear PAH in male Wistar rats (n = 9). After 28 days, echocardiography, RV catheterisation and histochemistry showed that cardiac frequency, stroke volume and RV contractility had deteriorated, accompanied by RV dilatation and hypertrophy, and marked pulmonary arterial wall thickening. Mean pulmonary arterial pressure and pulmonary vascular resistance increased significantly compared to healthy rats (n = 9). After 14 days, MCT-treated rats received a 14-day oral treatment with bosentan, sildenafil, fasudil or a combination of fasudil with either bosentan or sildenafil (all n = 9). All treatments preserved cardiac frequency, stroke volume and RV contractility, and reduced pulmonary vascular resistance and RV dilatation. Fasudil lowered RV systolic pressure and mean pulmonary arterial pressure significantly, by reducing pulmonary arterial remodelling, which reduced RV hypertrophy. Combining bosentan or sildenafil with fasudil had no synergistic effect. Fasudil significantly improved PAH, to a greater degree than did bosentan and sildenafil.

Thrombin-induced endothelial barrier disruption in intact microvessels: role of RhoA/Rho kinase-myosin phosphatase axis

Endothelial hyperpermeability is regulated by a myosin light chain-2 (MLC2) phosphorylation-dependent contractile mechanism. Thrombin is a potent inducer of hyperpermeability of cultured monolayers of endothelial cells (ECs) via Rho kinase-mediated MLC2-phosphorylation. The aim of the present study was to investigate the effects of thrombin on in situ endothelial morphology and barrier integrity. Cytoskeletal dynamics, regions of paracellular flux, and MLC2-phosphorylation of ECs were visualized by digital three-dimensional imaging microscopy of pressurized rat kidney arterioles. Myosin phosphatase targeting subunit (MYPT1)-phosphorylation was used as a surrogate marker for Rho kinase activity. Thrombin induced the formation of F-actin filaments in ECs in situ and rounding of the ECs in the absence of obvious formation of gaps between ECs. These changes were accompanied by an increase in MLC2 phosphorylation and a decrease in barrier integrity. In vitro analysis revealed that Rho kinase activity on F-actin filaments was associated with a contractile response that enhanced opening of the barrier. Rho kinase activity was not detectable on F-actin filaments induced by histamine, an inducer of a more transient hyperpermeability response. Inhibition of the myosin phosphatase mimicked the effects of thrombin on barrier function. The thrombin-induced changes in in situ MLC2 phosphorylation and barrier function were Rho kinase dependent. These data demonstrate a direct effect of thrombin on EC morphology and barrier integrity in intact microvessels. Furthermore, they establish an important contribution of enhanced Rho kinase activity to the development of prolonged but not transient types of endothelial barrier dysfunction.

Possible role of imatinib in clinical pulmonary veno-occlusive disease

The platelet-derived growth factor receptor inhibitor imatinib has demonstrated clinical and haemodynamical improvement in both animal models of pulmonary hypertension (PH) and patients with PH. It has been suggested that anti-proliferative effects on pulmonary vascular smooth muscle cells are responsible for these beneficial effects. The current study describes a patient with pulmonary arterial hypertension associated with a suspected pulmonary veno-occlusive disease. Treatment with imatinib resulted in rapid clinical improvement and decrease of ground-glass opacities and lobular septal thickening on high-resolution computed tomography. Based on these findings and on in vitro effects of imatinib on permeability of the endothelium, the authors hypothesise that the rapid clinical outcome is partly due to effects of imatinib on vascular integrity.

Effect of prior statin therapy on capillary permeability in the lungs after cardiac or vascular surgery

Cholesterol-lowering statins can ameliorate severe forms of vascular hyperpermeability in experimental studies, and may thereby ameliorate acute lung injury and sepsis. It is unknown whether this also applies to humans. This study aimed to define whether or not prior statin therapy reduces mild post-operative increases in pulmonary capillary protein permeability associated with acute lung injury after cardiac or major vascular surgery. A prospective observational study was performed in an intensive care unit of a university hospital on 64 patients, 37 after elective cardiac and 27 after major vascular surgery, of whom 68 and 44%, respectively, had received prior statin therapy. A mobile probe system was used to measure the pulmonary leak index (PLI), i.e. the transvascular transport rate of gallium-67-radiolabelled transferrin. For all of the patients together, the mean PLI did not differ between the statin and control groups (22.9 versus 24.4×10−3 min−1). The prevalence of an elevated PLI was 57% in the statin and 59% in the control group. Subgroup analysis did not reveal significant differences caused by statins in the PLI of these patients. Prior statin therapy neither has an adverse effect on mildly increased pulmonary capillary permeability in patients after cardiac or major vascular surgery nor does it ameliorate this increased capillary permeability.

Endothelial hyperpermeability after cardiac surgery with cardiopulmonary bypass as assessed using an in vitro bioassay for endothelial barrier function

BACKGROUND The mechanisms causing increased endothelial permeability after cardiopulmonary bypass (CPB) have not been elucidated. Using a bioassay for endothelial barrier function, we investigated whether endothelial hyperpermeability is associated with alterations in plasma endothelial activation and adhesion markers and can be attenuated by the use of pulsatile flow during CPB. METHODS Patients undergoing cardiac surgery were randomized to non-pulsatile (n=20) or pulsatile flow CPB (n=20). Plasma samples were obtained before (pre-CPB) and after CPB (post-CPB), and upon intensive care unit (ICU) arrival. Changes in plasma endothelial activation and adhesion markers were determined by enzyme-linked immunosorbent assay. Using electric cell-substrate impedance sensing of human umbilical vein endothelial monolayers, the effects of plasma exposure on endothelial barrier function were assessed and expressed as resistance. RESULTS Cardiopulmonary bypass was associated with increased P-selectin, vascular cell adhesion molecule-1, and von Willebrand factor plasma concentrations and an increase in the angiopoietin-2 to angiopoietin-1 ratio, irrespective of the flow profile. Plasma samples obtained after CPB induced loss of endothelial resistance of 21 and 23% in non-pulsatile and pulsatile flow groups, respectively. The negative effect on endothelial cell barrier function was still present with exposure to plasma obtained upon ICU admission. The reduction in endothelial resistance after exposure to post-CPB plasma could not be explained by CPB-induced haemodilution. CONCLUSION The change in the plasma fingerprint during CPB is associated with impairment of in vitro endothelial barrier function, which occurs irrespective of the application of a protective pulsatile flow profile during CPB. CLINICAL TRIAL REGISTRATION NTR2940.

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.