Erika S. Wittchen

The tension mounts: Stress fibers as force-generating mechanotransducers

Stress fibers (SFs) are often the most prominent cytoskeletal structures in cells growing in tissue culture. Composed of actin filaments, myosin II, and many other proteins, SFs are force-generating and tension-bearing structures that respond to the surrounding physical environment. New work is shedding light on the mechanosensitive properties of SFs, including that these structures can respond to mechanical tension by rapid reinforcement and that there are mechanisms to repair strain-induced damage. Although SFs are superficially similar in organization to the sarcomeres of striated muscle, there are intriguing differences in their organization and behavior, indicating that much still needs to be learned about these structures.

Direct activation of RhoA by reactive oxygen species requires a redox-sensitive motif.

Background Rho family GTPases are critical regulators of the cytoskeleton and affect cell migration, cell-cell adhesion, and cell-matrix adhesion. As with all GTPases, their activity is determined by their guanine nucleotide-bound state. Understanding how Rho proteins are activated and inactivated has largely focused on regulatory proteins such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, recent in vitro studies have indicated that GTPases may also be directly regulated by redox agents. We hypothesized that this redox-based mechanism occurs in cells and affects cytoskeletal dynamics, and in this report we conclude this is indeed a novel mechanism of regulating the GTPase RhoA. Methodology/Principal Findings In this report, we show that RhoA can be directly activated by reactive oxygen species (ROS) in cells, and that this requires two critical cysteine residues located in a unique redox-sensitive motif within the phosphoryl binding loop. First, we show that ROS can reversibly activate RhoA and induce stress fiber formation, a well characterized readout of RhoA activity. To determine the role of cysteine residues in this mechanism of regulation, we generated cysteine to alanine RhoA mutants. Mutation of these cysteines abolishes ROS-mediated activation and stress fiber formation, indicating that these residues are critical for redox-regulation of RhoA. Importantly, these mutants maintain the ability to be activated by GEFs. Conclusions/Significance Our findings identify a novel mechanism for the regulation of RhoA in cells by ROS, which is independent of classical regulatory proteins. This mechanism of regulation may be particularly relevant in pathological conditions where ROS are generated and the cellular redox-balance altered, such as in asthma and ischemia-reperfusion injury.

Endothelial cell junctions and the regulation of vascular permeability and leukocyte transmigration

Summary.  The endothelial lining of the vasculature forms the physical barrier between the blood and underlying tissues. Junctions between adjacent endothelial cells are dynamically modulated to sustain vascular homeostasis and to support the transendothelial migration of leukocytes during inflammation. A variety of factors initiate intracellular signaling pathways that regulate the opening and resealing of junctional complexes. This review focuses on three primary signaling pathways initiated within endothelial cells by the binding of vasoactive factors and leukocyte adhesion: Rho GTPases, reactive oxygen species, and tyrosine phosphorylation of junctional proteins. These pathways converge to regulate junctional permeability, either by affecting the stability of junctional proteins or by modulating their interactions. Although much progress has been made in understanding the relationships of these pathways, many questions remain to be answered. A full understanding of the signaling cascades that affect endothelial junctions should identify novel therapeutic targets for diseases that involve excessive permeability or inappropriate leukocyte infiltration into tissues.

Trading spaces: Rap, Rac, and Rho as architects of transendothelial migration

Purpose of reviewThis review focuses on recent developments in understanding regulation of leukocyte transendothelial migration by small GTPase signaling. Recent findingsNew studies are refining the model for GTPase regulation of leukocyte-endothelial cell interactions that occur during leukocyte transmigration. An emerging theme is that the endothelial cell is an active participant in this process; an example of this is the identification of a novel leukocyte docking structure. The role of second messengers such as reactive oxygen species downstream and the involvement of kinases such as Pyk2 and Tec kinases upstream of GTPase activation is becoming appreciated. In the leukocyte, finer distinctions between closely related GTPases like Rac1 and Rac2 are being made, and a new role for RhoH has been characterized. Finally, the focus on Rap1 as a key regulator of leukocyte integrin-dependent adhesion is expanding to include roles in endothelial cell-cell adhesion and junctional regulation during transmigration. SummaryUnderstanding the complex series of events involved in cell-cell interactions during leukocyte transendothelial migration is a prerequisite for designing novel therapies to treat clinical conditions in which an inappropriate inflammatory response leads to disease. A discussion is provided of recent developments in the molecular regulation of leukocyte recruitment.

Isoform-specific differences between Rap1A and Rap1B GTPases in the formation of endothelial cell junctions.

Rap1 is a Ras-like GTPase that has been studied with respect to its role in cadherin-based cell adhesion. Rap1 exists as two separate isoforms, Rap1A and Rap1B, which are 95% identical and yet the phenotype of the isoform-specific knockout mice is different. We and others have previously identified a role for Rap1 in regulating endothelial adhesion, junctional integrity, and barrier function; however, these early studies did not distinguish a relative role for each isoform. To dissect the individual contribution of each isoform in regulating the endothelial barrier, we utilized an engineered microRNA-based approach to silence Rap1A, Rap1B, or both, then analyzed barrier properties of the endothelium. Electrical impedance sensing experiments show that Rap1A is the predominant isoform involved in endothelial cell junction formation. Quantification of monolayer integrity by VE-cadherin staining revealed that knockdown of Rap1A, but not Rap1B, increased the number of gaps in the confluent monolayer. This loss of monolayer integrity could be rescued by re-expression of exogenous Rap1A protein. Expression of GFP-tagged Rap1A or 1B revealed quantifiable differences in localization of each isoform, with the junctional pool of Rap1A being greater. The junctional protein AF-6 also co-immunoprecipitates more strongly with expressed GFP-Rap1A. Our results show that Rap1A is the more critical isoform in the context of endothelial barrier function, indicating that some cellular processes differentially utilize Rap1A and 1B isoforms. Studying how Rap1 isoforms differentially regulate EC junctions may thus reveal new targets for developing therapeutic strategies during pathological situations where endothelial barrier disruption leads to disease.

Upregulation of CCR3 by age-related stresses promotes choroidal endothelial cell migration via VEGF-dependent and -independent signaling.

PURPOSE To explore the molecular mechanisms by which the C-C chemokine receptor type 3 (CCR3) and chemokine (C-C motif) ligand 11 (CCL11) regulate choroidal endothelial cell (CEC) migration and the interactions with the vascular endothelial growth factor (VEGF) signaling pathway. METHODS Human retinal sections from young and aged donor normal eyes were immunolabeled. By real-time PCR, CCR3 mRNA was measured in retinal pigmented epithelium (RPE)/choroids obtained from young and aged human donor eyes and in cultured CECs exposed to hydrogen peroxide. CCR3 ligand and CCL11- or VEGF-stimulated CEC migration was also measured in the presence of the CCR3 inhibitor or control using fluorescence microscopy. Activation of Rac1, phosphorylated Akt as a readout for phosphoinositol 3-kinase signaling, and VEGFR2 activation were measured in CECs incubated with CCL11, VEGF, or combined CCL11/VEGF. RESULTS CCR3 was expressed to a greater level in older compared with that in younger human retinas or RPE/choroids. Ligand-activated CCR3 increased CEC migration, which was inhibited by the CCR3 inhibitor. Rac1 activity, p-Akt, and p-VEGFR2 were significantly increased in CECs incubated with CCL11. The CCR3 inhibitor prevented VEGF-induced CEC migration and Rac1 activation in CECs. Rac1 activity was additively increased in CECs treated with CCL11 and VEGF compared with that in cells with CCL11 or VEGF treatment alone. Ligand-activated CCR3 caused VEGFR2 phosphorylation and coimmunoprecipitation of VEGFR2 and CCR3. CONCLUSIONS Activated CCR3 promotes CEC migration and Rac1 activation and causes an association with and activation of VEGFR2. Cross-talk between CCR3 and VEGF signaling exists and may be important in choroidal neovascularization in human age-related macular degeneration.

The Role of RPE Cell-Associated VEGF189 in Choroidal Endothelial Cell Transmigration across the RPE

PURPOSE To determine the role of vascular endothelial growth factor 189 (VEGF₁₈₉) in choroidal endothelial cell (CEC) migration across the retinal pigment epithelium (RPE) and to explore the molecular mechanisms involved. METHODS Using real-time PCR, the expression of VEGF splice variants VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉ was determined in human RPE from donor eyes, cultured human RPE in contact with CECs exposed to hydrogen peroxide (H₂O₂) or hypoxia, and RPE/choroid specimens from mice treated with laser to induce choroidal neovascularization (CNV). Activation of VEGF receptors (VEGFRs), phosphoinositol 3-kinase (PI-3K) or Rac1 was measured in CECs cocultured in contact with RPE exposed to peroxide or silenced for VEGF₁₈₉ expression. Migration of CECs across the RPE was determined using fluorescence microscopy. RESULTS VEGF₁₈₉ expression was increased in human RPE from aged compared with young donor eyes and from mouse RPE/choroids after laser to induce CNV. VEGF₁₈₉ was also upregulated in human RPE challenged with peroxide, hypoxia, or cultured in contact with CECs. CEC migration across RPE was greater after RPE exposure to peroxide to induce VEGF₁₈₉; VEGFR2 and Rac1 activities were also increased in these CECs. When CECs were cocultured with RPE silenced for VEGF₁₈₉, VEGFR2 and Rac1 activities in CECs were significantly reduced, as was CEC migration across the RPE. Inhibition of Rac1 activity significantly inhibited CEC transmigration without affecting PI-3K activity. CONCLUSIONS RPE-derived cell-associated VEGF₁₈₉ facilitates CEC transmigration by Rac1 activation independently of PI-3K signaling and may have importance in the development of neovascular AMD.

The Small GTPase Rap1 Is a Novel Regulator of RPE Cell Barrier Function

PURPOSE To determine whether the small GTPase Rap1 regulates the formation and maintenance of the retinal pigment epithelial (RPE) cell junctional barrier. METHODS An in vitro model was used to study RPE barrier properties. To dissect the role of Rap1, two techniques were used to inhibit Rap1 function: overexpression of RapGAP, which acts as a negative regulator of endogenous Rap1 activity, and treatment with engineered, adenovirally-transduced microRNAs to knockdown Rap1 protein expression. Transepithelial electrical resistance (TER) and real-time cellular analysis (RTCA) of impedance were used as readouts for barrier properties. Immunofluorescence microscopy was used to visualize localization of cadherins under steady state conditions and also during junctional reassembly after calcium switch. Finally, choroidal endothelial cell (CEC) migration across RPE monolayers was quantified under conditions of Rap1 inhibition in RPE. RESULTS Knockdown of Rap1 or inhibition of its activity in RPE reduces TER and electrical impedance of the RPE monolayers. The loss of barrier function is also reflected by the mislocalization of cadherins and formation of gaps within the monolayer. TER measurement and immunofluorescent staining of cadherins after a calcium switch indicate that junctional reassembly kinetics are also impaired. Furthermore, CEC transmigration is significantly higher in Rap1-knockdown RPE monolayers compared with control. CONCLUSIONS Rap1 GTPase is an important regulator of RPE cell junctions, and is required for maintenance of barrier function. This observation that RPE monolayers lacking Rap1 allow greater transmigration of CECs suggests a possible role for potentiating choroidal neovascularization during the pathology of neovascular age-related macular degeneration.

Rap1 GTPase Activation and Barrier Enhancement in RPE Inhibits Choroidal Neovascularization In Vivo

Loss of barrier integrity precedes the development of pathologies such as metastasis, inflammatory disorders, and blood-retinal barrier breakdown present in neovascular age-related macular degeneration. Rap1 GTPase is involved in regulating both endothelial and epithelial cell junctions; the specific role of Rap1A vs. Rap1B isoforms is less clear. Compromise of retinal pigment epithelium barrier function is a contributing factor to the development of AMD. We utilized shRNA of Rap1 isoforms in cultured human retinal pigment epithelial cells, along with knockout mouse models to test the role of Rap1 on promoting RPE barrier properties, with emphasis on the dynamic junctional regulation that is triggered when the adhesion between cells is challenged. In vitro, Rap1A shRNA reduced steady-state barrier integrity, whereas Rap1B shRNA affected dynamic junctional responses. In a laser-induced choroidal neovascularization (CNV) model of macular degeneration, Rap1b−/− mice exhibited larger CNV volumes compared to wild-type or Rap1a−/−. In vivo, intravitreal injection of a cAMP analog (8CPT-2′-O-Me-cAMP) that is a known Rap1 activator significantly reduced laser-induced CNV volume, which correlated with the inhibition of CEC transmigration across 8CPT-2′O-Me-cAMP-treated RPE monolayers in vitro. Rap1 activation by 8CPT-2′-O-Me-cAMP treatment increased recruitment of junctional proteins and F-actin to cell-cell contacts, increasing both the linearity of junctions in vitro and in cells surrounding laser-induced lesions in vivo. We conclude that in vitro, Rap1A may be important for steady state barrier integrity, while Rap1B is involved more in dynamic junctional responses such as resistance to junctional disassembly induced by EGTA and reassembly of cell junctions following disruption. Furthermore, activation of Rap1 in vivo inhibited development of choroidal neovascular lesions in a laser-injury model. Our data suggest that targeting Rap1 isoforms in vivo with 8CPT-2′-O-Me-cAMP may be a viable pharmacological means to strengthen the RPE barrier against the pathological choroidal endothelial cell invasion that occurs in macular degeneration.

Analysis of low molecular weight GTPase activity in endothelial cell cultures.

GTPases control a myriad of cellular processes, including cell migration, proliferation, polarity, and cell adhesion, which includes cell-cell junction regulation. Angiogenesis requires many of these diverse cellular events to occur appropriately. In this chapter, we describe the techniques for in vitro assessment of GTPase activity in endothelial cells grown in monolayer culture as a model system for studying their role during angiogenesis.