A Phase 1b Trial of Prexasertib in Combination with Standard-of-Care Agents in Advanced or Metastatic Cancer.

BACKGROUND: The checkpoint kinase 1 (CHK1) inhibitor prexasertib exhibited modest monotherapy antitumor activity in prior trials, suggesting that combination with chemotherapy or other targeted agents may be needed to maximize efficacy. OBJECTIVES: The aim of this study was to determine the recommended phase II dose and schedule of prexasertib in combination with either cisplatin, cetuximab, pemetrexed, or 5-fluorouracil in patients with advanced and/or metastatic cancer, and to summarize preliminary antitumor activity of these combinations. PATIENTS AND METHODS: This phase Ib, nonrandomized, open-label study comprised dose-escalation phase(s) with multiple sub-arms evaluating different prexasertib-drug combinations: Part A, prexasertib + cisplatin (n = 63); Part B, prexasertib + cetuximab (n = 41); Part C, prexasertib + pemetrexed (n = 3); Part D, prexasertib + 5-fluorouracil (n =8). Alternate dose schedules/regimens intended to mitigate toxicity and maximize dose exposure and efficacy were also explored in sub-parts. RESULTS: In Part A, the maximum tolerated dose (MTD) of prexasertib in combination with cisplatin (75 mg/m2) was declared at 80 mg/m2, with cisplatin administered on Day 1 and prexasertib on Day 2 of a 21-day cycle. The overall objective response rate (ORR) in Part A was 12.7%, and 28 of 55 evaluable patients (50.9%) had a decrease in target lesions from baseline. The most frequent treatment-related adverse events (AEs) in Part A were hematologic, with the most common being white blood cell count decreased/neutrophil count decreased, experienced by 73.0% (any grade) and 66.7% (grade 3 or higher) of patients. In Part B, an MTD of 70 mg/m2 was established for prexasertib administered in combination with cetuximab (500 mg /m2), both administered on Day 1 of a 14-day cycle. The overall ORR in Part B was 4.9%, and 7 of 31 evaluable patients (22.6%) had decreased target lesions compared with baseline. White blood cell count decreased/neutrophil count decreased was also the most common treatment-related AE (56.1% any grade; 53.7% grade 3 or higher). In Parts A and B, hematologic toxicities, even with the addition of prophylactic granulocyte colony-stimulating factor, resulted in frequent dose adjustments (> 60% of patients). In Part C, evaluation of prexasertib + pemetrexed was halted due to dose-limiting toxicities in two of the first three patients; MTD was not established. In Part D, the MTD of prexasertib in combination with 5-fluorouracil (label dose) was declared at 40 mg /m2, both administered on Day 1 of a 14-day cycle. In Part D, overall ORR was 12.5%. CONCLUSIONS: This study demonstrated the proof-of-concept that prexasertib can be combined with cisplatin, cetuximab, and 5-fluorouracil. Schedule was a key determinant of the tolerability and feasibility of combining prexasertib with these standard-of-care agents. Reversible hematologic toxicity was the most frequent AE and was dose-limiting. Insights gleaned from this study will inform future combination strategies for the development of prexasertib and next-generation CHK1 inhibitors. CLINICALTRIALS. GOV IDENTIFIER: NCT02124148 (date of registration 28 April 2014).

ARHGEF26 enhances Salmonella invasion and inflammation in cells and mice.

Salmonella hijack host machinery in order to invade cells and establish infection. While considerable work has described the role of host proteins in invasion, much less is known regarding how natural variation in these invasion-associated host proteins affects Salmonella pathogenesis. Here we leveraged a candidate cellular GWAS screen to identify natural genetic variation in the ARHGEF26 (Rho Guanine Nucleotide Exchange Factor 26) gene that renders lymphoblastoid cells susceptible to Salmonella Typhi and Typhimurium invasion. Experimental follow-up redefined ARHGEF26's role in Salmonella epithelial cell infection. Specifically, we identified complex serovar-by-host interactions whereby ARHGEF26 stimulation of S. Typhi and S. Typhimurium invasion into host cells varied in magnitude and effector-dependence based on host cell type. While ARHGEF26 regulated SopB- and SopE-mediated S. Typhi (but not S. Typhimurium) infection of HeLa cells, the largest effect of ARHGEF26 was observed with S. Typhimurium in polarized MDCK cells through a SopB- and SopE2-independent mechanism. In both cell types, knockdown of the ARHGEF26-associated protein DLG1 resulted in a similar phenotype and serovar specificity. Importantly, we show that ARHGEF26 plays a critical role in S. Typhimurium pathogenesis by contributing to bacterial burden in the enteric fever murine model, as well as inflammation in the colitis infection model. In the enteric fever model, SopB and SopE2 are required for the effects of Arhgef26 deletion on bacterial burden, and the impact of sopB and sopE2 deletion in turn required ARHGEF26. In contrast, SopB and SopE2 were not required for the impacts of Arhgef26 deletion on colitis. A role for ARHGEF26 on inflammation was also seen in cells, as knockdown reduced IL-8 production in HeLa cells. Together, these data reveal pleiotropic roles for ARHGEF26 during infection and highlight that many of the interactions that occur during infection that are thought to be well understood likely have underappreciated complexity.

Decreased inspired oxygen stimulates de novo formation of coronary collaterals in adult heart.

RATIONALE: Collateral vessels lessen myocardial ischemia when acute or chronic coronary obstruction occurs. It has long been assumed that although native (pre-existing) collaterals enlarge in obstructive disease, new collaterals do not form in the adult. However, the latter was recently shown to occur after coronary artery ligation. Understanding the signals that drive this process is challenged by the difficulty in studying collateral vessels directly and the complex milieu of signaling pathways, including cell death, induced by ligation. Herein we show that hypoxemia alone is capable of inducing collateral vessels to form and that the novel gene Rabep2 is required. OBJECTIVE: Hypoxia stimulates angiogenesis during embryonic development and in pathological states. We hypothesized that hypoxia also stimulates collateral formation in adult heart by a process that involves RABEP2, a recently identified protein required for formation of collateral vessels during development. METHODS AND RESULTS: Exposure of mice to reduced FiO2 induced collateral formation that resulted in smaller infarctions following LAD ligation and that reversed on return to normoxia. Deletion of Rabep2 or knockdown of Vegfa inhibited formation. Hypoxia upregulated Rabep2, Vegfa and Vegfr2 in heart and brain microvascular endothelial cells (HBMVECs). Knockdown of Rabep2 impaired migration of HBMVECs. In contrast to systemic hypoxia, deletion of Rabep2 did not affect collateral formation induced by ischemic injury caused by LAD ligation. CONCLUSIONS: Hypoxia induced formation of coronary collaterals by a process that required VEGFA and RABEP2, proteins also required for collateral formation during development. Knockdown of Rabep2 impaired cell migration, providing one potential mechanism for RABEP2's role in collateral formation. This appears specific to hypoxia, since formation after acute ischemic injury was unaffected in Rabep2-/- mice. These findings provide a novel model for studying coronary collateral formation, and demonstrate that hypoxia alone can induce new collaterals to form in adult heart.

The role of endothelial MERTK during the inflammatory response in lungs.

As a key homeostasis regulator in mammals, the MERTK receptor tyrosine kinase is crucial for efferocytosis, a process that requires remodeling of the cell membrane and adjacent actin cytoskeleton. Membrane and cytoskeletal reorganization also occur in endothelial cells during inflammation, particularly during neutrophil transendothelial migration (TEM) and during changes in permeability. However, MERTK's function in endothelial cells remains unclear. This study evaluated the contribution of endothelial MERTK to neutrophil TEM and endothelial barrier function. In vitro experiments using primary human pulmonary microvascular endothelial cells found that neutrophil TEM across the endothelial monolayers was enhanced when MERTK expression in endothelial cells was reduced by siRNA knockdown. Examination of endothelial barrier function revealed increased passage of dextran across the MERTK-depleted monolayers, suggesting that MERTK helps maintain endothelial barrier function. MERTK knockdown also altered adherens junction structure, decreased junction protein levels, and reduced basal Rac1 activity in endothelial cells, providing potential mechanisms of how MERTK regulates endothelial barrier function. To study MERTK's function in vivo, inflammation in the lungs of global Mertk-/- mice was examined during acute pneumonia. In response to P. aeruginosa, more neutrophils were recruited to the lungs of Mertk-/- than wildtype mice. Vascular leakage of Evans blue dye into the lung tissue was also greater in Mertk-/- mice. To analyze endothelial MERTK's involvement in these processes, we generated inducible endothelial cell-specific (iEC) Mertk-/- mice. When similarly challenged with P. aeruginosa, iEC Mertk-/- mice demonstrated no difference in neutrophil TEM into the inflamed lungs or in vascular permeability compared to control mice. These results suggest that deletion of MERTK in human pulmonary microvascular endothelial cells in vitro and in all cells in vivo aggravates the inflammatory response. However, selective MERTK deletion in endothelial cells in vivo failed to replicate this response.

Software for lattice light-sheet imaging of FRET biosensors, illustrated with a new Rap1 biosensor.

Lattice light-sheet microscopy (LLSM) is valuable for its combination of reduced photobleaching and outstanding spatiotemporal resolution in 3D. Using LLSM to image biosensors in living cells could provide unprecedented visualization of rapid, localized changes in protein conformation or posttranslational modification. However, computational manipulations required for biosensor imaging with LLSM are challenging for many software packages. The calculations require processing large amounts of data even for simple changes such as reorientation of cell renderings or testing the effects of user-selectable settings, and lattice imaging poses unique challenges in thresholding and ratio imaging. We describe here a new software package, named ImageTank, that is specifically designed for practical imaging of biosensors using LLSM. To demonstrate its capabilities, we use a new biosensor to study the rapid 3D dynamics of the small GTPase Rap1 in vesicles and cell protrusions.

Hepatic Glucagon Signaling Regulates PCSK9 and Low-Density Lipoprotein Cholesterol.

RATIONALE: Glucagon is a key hormone that regulates the adaptive metabolic responses to fasting. In addition to maintaining glucose homeostasis, glucagon participates in the regulation of cholesterol metabolism; however, the molecular pathways underlying this effect are incompletely understood. OBJECTIVE: We sought to determine the role of hepatic Gcgr (glucagon receptor) signaling in plasma cholesterol regulation and identify its underlying molecular mechanisms. METHODS AND RESULTS: We show that Gcgr signaling plays an essential role in LDL-C (low-density lipoprotein cholesterol) homeostasis through regulating the PCSK9 (proprotein convertase subtilisin/kexin type 9) levels. Silencing of hepatic Gcgr or inhibition of glucagon action increased hepatic and plasma PCSK9 and resulted in lower LDLR (LDL receptor) protein and increased plasma LDL-C. Conversely, treatment of wild-type (WT) mice with glucagon lowered LDL-C levels, whereas this response was abrogated in Pcsk9-/- and Ldlr-/- mice. Our gain- and loss-of-function studies identified Epac2 (exchange protein activated by cAMP-2) and Rap1 (Ras-related protein-1) as the downstream mediators of glucagon's action on PCSK9 homeostasis. Moreover, mechanistic studies revealed that glucagon affected the half-life of PCSK9 protein without changing the level of its mRNA, indicating that Gcgr signaling regulates PCSK9 degradation. CONCLUSIONS: These findings provide novel insights into the molecular interplay between hepatic glucagon signaling and lipid metabolism and describe a new posttranscriptional mechanism of PCSK9 regulation.

Small GTPase Rap1A/B Is Required for Lymphatic Development and Adrenomedullin-Induced Stabilization of Lymphatic Endothelial Junctions.

Objective- Maintenance of lymphatic permeability is essential for normal lymphatic function during adulthood, but the precise signaling pathways that control lymphatic junctions during development are not fully elucidated. The Gs-coupled AM (adrenomedullin) signaling pathway is required for embryonic lymphangiogenesis and the maintenance of lymphatic junctions during adulthood. Thus, we sought to elucidate the downstream effectors mediating junctional stabilization in lymphatic endothelial cells. Approach and Results- We knocked-down both Rap1A and Rap1B isoforms in human neonatal dermal lymphatic cells (human lymphatic endothelial cells) and genetically deleted the mRap1 gene in lymphatic endothelial cells by producing 2 independent, conditional Rap1a/b knockout mouse lines. Rap1A/B knockdown caused disrupted junctional formation with hyperpermeability and impaired AM-induced lymphatic junctional tightening, as well as rescue of histamine-induced junctional disruption. Less than 60% of lymphatic- Rap1a/b knockout embryos survived to E13.5 exhibiting interstitial edema, blood-filled lymphatics, disrupted lymphovenous valves, and defective lymphangiogenesis. Consistently, inducible lymphatic- Rap1a/b deletion in adult animals prevented AM-rescue of histamine-induced lymphatic leakage and dilation. Conclusions- Rap1 (Ras-related protein) serves as the dominant effector downstream of AM to stabilize lymphatic junctions. Rap1 is required for maintaining lymphatic permeability and driving normal lymphatic development.

A Rnd3/p190RhoGAP pathway regulates RhoA activity in idiopathic pulmonary fibrosis fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is an incurable disease of the lung that is characterized by excessive deposition of extracellular matrix (ECM), resulting in disruption of normal lung function. The signals regulating fibrosis include both transforming growth factor beta (TGF-ß) and tissue rigidity and a major signaling pathway implicated in fibrosis involves activation of the GTPase RhoA. During studies exploring how elevated RhoA activity is sustained in IPF, we discovered that not only is RhoA activated by profibrotic stimuli but also that the expression of Rnd3, a major antagonist of RhoA activity, and the activity of p190RhoGAP (p190), a Rnd3 effector, are both suppressed in IPF fibroblasts. Restoration of Rnd3 levels in IPF fibroblasts results in an increase in p190 activity, a decrease in RhoA activity and a decrease in the overall fibrotic phenotype. We also find that treatment with IPF drugs nintedanib and pirfenidone decreases the fibrotic phenotype and RhoA activity through up-regulation of Rnd3 expression and p190 activity. These data provide evidence for a pathway in IPF where fibroblasts down-regulate Rnd3 levels and p190 activity to enhance RhoA activity and drive the fibrotic phenotype.

Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization.

To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 10(8) viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/ß-catenin complexes, increased transepithelial electrical resistance through an interaction of ß-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes.

TNF-α mediates choroidal neovascularization by upregulating VEGF expression in RPE through ROS-dependent ß-catenin activation.

PURPOSE: Inflammation, oxidative stress, and angiogenesis have been proposed to interact in age-related macular degeneration. It has been postulated that external stimuli that cause oxidative stress can increase production of vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells. In this study, we tested the hypothesis that the inflammatory cytokine, tumor necrosis factor alpha (TNF-α), contributed to choroidal neovascularization (CNV) by upregulating VEGF in RPE through intracellular reactive oxygen species (ROS)-dependent signaling and sought to understand the mechanisms involved. METHODS: In a murine laser-induced CNV model, 7 days after laser treatment and intravitreal neutralizing mouse TNF-α antibody or isotype immunoglobulin G (IgG) control, the following measurements were made: 1) TNF-α protein and VEGF protein in RPE/choroids with western blot, 2) CNV volume in RPE/choroidal flatmounts, and 3) semiquantification of oxidized phospholipids stained with E06 antibody within CNV with immunohistochemistry (IHC). In cultured human RPE cells treated with TNF-α or PBS control, 1) ROS generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence assay, and 2) NOX4 protein and VEGF protein or mRNA were measured with western blot or quantitative real-time PCR in cells pretreated with apocynin or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, VAS 2870, or transfected with p22phox siRNA, and each was compared to its appropriate control. Western blots of phosphorylated p65 (p-p65), total p65 and ß-actin, and quantitative real-time PCR of VEGF mRNA were measured in human RPE cells treated with TNF-α and pretreatment with the nuclear factor kappa B inhibitor, Bay 11-7082 or control. Western blots of ß-catenin, VEGF, and p22phox and coimmunoprecipitation of ß-catenin and T-cell transcriptional factor were performed in human RPE cells treated with TNF-α following pretreatment with ß-catenin transcriptional inhibitors, XAV939 or JW67, or transfection with p22phox siRNA and compared to appropriate controls. RESULTS: Compared to the non-lasered control, TNF-α and VEGF protein were increased in the RPE/choroids in a murine laser-induced CNV model (p<0.05). An intravitreal neutralizing antibody to mouse TNF-α reduced CNV volume, and VEGF protein in the RPE/choroids (p<0.01) and oxidized phospholipids within CNV compared to IgG control (p<0.05). In cultured RPE cells and compared to controls, TNF-α induced ROS generation and increased activation of NOX4, an isoform of NADPH oxidase; both were prevented by pretreatment with the apocynin or VAS2870 or knockdown of p22phox, a subunit of NADPH oxidase. TNF-α treatment increased VEGF expression (p<0.001) and the formation of a transcriptional complex of ß-catenin and T-cell transcriptional factor; both were prevented by pretreatment with apocynin or knockdown of p22phox. Inhibition of ß-catenin by XAV939, but not the nuclear factor kappa B inhibitor, Bay 11-7082, prevented TNF-α-induced VEGF upregulation. CONCLUSIONS: Our results support the thinking that TNF-α contributes to CNV by upregulating VEGF production in RPE cells through ROS-dependent activation of ß-catenin signaling. These results provide mechanisms of crosstalk between inflammatory mediator, TNF-α, and ROS in RPE cells.

Rap1 GTPase Inhibits Tumor Necrosis Factor-α-Induced Choroidal Endothelial Migration via NADPH Oxidase- and NF-κB-Dependent Activation of Rac1.

Macrophage-derived tumor necrosis factor (TNF)-α has been found in choroidal neovascularization (CNV) surgically removed from patients with age-related macular degeneration. However, the role of TNF-α in CNV development remains unclear. In a murine laser-induced CNV model, compared with un-lasered controls, TNF-α mRNA was increased in retinal pigment epithelial and choroidal tissue, and TNF-α colocalized with lectin-stained migrating choroidal endothelial cells (CECs). Inhibition of TNF-α with a neutralizing antibody reduced CNV volume and reactive oxygen species (ROS) level around CNV. In CECs, pretreatment with the antioxidant apocynin or knockdown of p22phox, a subunit of NADPH oxidase, inhibited TNF-α-induced ROS generation. Apocynin reduced TNF-α-induced NF-κB and Rac1 activation, and inhibited TNF-α-induced CEC migration. TNF-α-induced Rac1 activation and CEC migration were inhibited by NF-κB inhibitor Bay11-7082. Overexpression of Rap1a prevented TNF-α-induced ROS generation and reduced NF-κB and Rac1 activation. Activation of Rap1 by 8-(4-chlorophenylthio)adenosine-2'-O-Me-cAMP prevented TNF-α-induced CEC migration and reduced laser-induced CNV volume, ROS generation, and activation of NF-κB and Rac1. These findings provide evidence that active Rap1a inhibits TNF-α-induced CEC migration by inhibiting NADPH oxidase-dependent NF-κB and Rac1 activation and suggests that Rap1a de-escalates CNV development by interfering with ROS-dependent signaling in several steps of the pathogenic process.

Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization.

Activation of Rap1 GTPase can improve the integrity of the barrier of the retina pigment epithelium (RPE) and reduce choroidal neovascularization (CNV). Inhibition of NADPH oxidase activation also reduces CNV. We hypothesize that Rap1 inhibits NADPH oxidase-generated ROS and thereby reduces CNV formation. Using a murine model of laser-induced CNV, we determined that reduced Rap1 activity in RPE/choroid occurred with CNV formation and that activation of Rap1 by 2'-O-Me-cAMP (8CPT)-reduced laser-induced CNV via inhibiting NADPH oxidase-generated ROS. In RPE, inhibition of Rap1 by Rap1 GTPase-activating protein (Rap1GAP) increased ROS generation, whereas activation of Rap1 by 8CPT reduced ROS by interfering with the assembly of NADPH oxidase membrane subunit p22phox with NOX4 or cytoplasmic subunit p47phox. Activation of NADPH oxidase with Rap1GAP reduced RPE barrier integrity via cadherin phosphorylation and facilitated choroidal EC migration across the RPE monolayer. Rap1GAP-induced ROS generation was inhibited by active Rap1a, but not Rap1b, and activation of Rap1a by 8CPT in Rap1b(-/-) mice reduced laser-induced CNV, in correlation with decreased ROS generation in RPE/choroid. These findings provide evidence that active Rap1 reduces CNV by interfering with the assembly of NADPH oxidase subunits and increasing the integrity of the RPE barrier.

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.

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.

Unique structural and nucleotide exchange features of the Rho1 GTPase of Entamoeba histolytica.

The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.

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.

In vitro analyses of endothelial cell permeability.

Endothelial cells lining the vessels of the vasculature and the cell-cell junctions, which join them, -provide the primary barrier to the passage of fluids, immune cells, and macromolecules between the bloodstream and the tissues. Appropriate and dynamic regulation of this barrier is required during normal -physiological processes; however, if not tightly controlled, increased permeability of the endothelium can also contribute to many pathological situations, including chronic inflammatory diseases and edema. The development of in vitro methods to study endothelial barrier function has been key in the identification of molecular mechanisms underlying many of these disease states. In this chapter, we describe three complementary approaches to measure endothelial monolayer permeability and barrier function in vitro.

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.

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.