CLIC4 Regulates Endothelial Barrier Control by Mediating PAR1 Signaling via RhoA.

BACKGROUND: Endothelial CLICs (chloride intracellular channel proteins) CLIC1 and CLIC4 are required for the GPCRs (G-protein-coupled receptors) S1PR1 (sphingosine-1-phosphate receptor 1) and S1PR3 to activate the small GTPases Rac1 (Ras-related C3 botulinum toxin substrate 1) and RhoA (Ras homolog family member A). To determine whether CLIC1 and CLIC4 function in additional endothelial GPCR pathways, we evaluated CLIC function in thrombin signaling via the thrombin-regulated PAR1 (protease-activated receptor 1) and downstream effector RhoA. METHODS: We assessed the ability of CLIC1 and CLIC4 to relocalize to cell membranes in response to thrombin in human umbilical vein endothelial cells (HUVEC). We examined CLIC1 and CLIC4 function in HUVEC by knocking down expression of each CLIC protein and compared thrombin-mediated RhoA or Rac1 activation, ERM (ezrin/radixin/moesin) phosphorylation, and endothelial barrier modulation in control and CLIC knockdown HUVEC. We generated a conditional murine allele of Clic4 and examined PAR1-mediated lung microvascular permeability and retinal angiogenesis in mice with endothelial-specific loss of Clic4. RESULTS: Thrombin promoted relocalization of CLIC4, but not CLIC1, to HUVEC membranes. Knockdown of CLIC4 in HUVEC reduced thrombin-mediated RhoA activation, ERM phosphorylation, and endothelial barrier disruption. Knockdown of CLIC1 did not reduce thrombin-mediated RhoA activity but prolonged the RhoA and endothelial barrier response to thrombin. Endothelial-specific deletion of Clic4 in mice reduced lung edema and microvascular permeability induced by PAR1 activating peptide. CONCLUSIONS: CLIC4 is a critical effector of endothelial PAR1 signaling and is required to regulate RhoA-mediated endothelial barrier disruption in cultured endothelial cells and murine lung endothelium. CLIC1 was not critical for thrombin-mediated barrier disruption but contributed to the barrier recovery phase after thrombin treatment.

Nanofibrous Cathode Catalysts with MoC Nanoparticles Embedded in N-Rich Carbon Shells for Low-Overpotential Li-CO2 Batteries.

Li-CO2 batteries with high theoretical energy densities are recognized as next-generation energy storage devices for addressing the range anxiety and environmental issues encountered in the field of electric transportation. However, cathode catalysts with unsatisfactory activity toward CO2 absorption and reduction/evolution reactions hinder the development of Li-CO2 batteries with desired specific capacities and sufficient cycle numbers. In this work, a multifunctional nanofibrous cathode catalyst that integrates N-rich carbon shells embedded with molybdenum carbide nanoparticles and multiwalled carbon nanotube cores was designed and prepared. The N-rich carbon shell could strengthen the absorption capacity of CO2 and Li2CO3. The molybdenum carbide nanoparticles would improve the catalytic activity of both CO2 reduction and evolution reactions. The carbon nanotube cores would provide an efficient network for electron transportation. The synergistic effect of the cathode catalysts enhances the electrochemical performance of Li-CO2 batteries. A high cycling stability of more than 150 cycles at a current density of 250 mA g-1 with a cutoff capacity of 1000 mAh g-1 and a charge/discharge overpotential of less than 1.5 V is achieved. This work provides a feasible strategy for the design of a high-performance cathode catalyst for lithium-air batteries.

CLIC1 and CLIC4 mediate endothelial S1P receptor signaling to facilitate Rac1 and RhoA activity and function.

Chloride intracellular channels 1 (CLIC1) and 4 (CLIC4) are expressed in endothelial cells and regulate angiogenic behaviors in vitro, and the expression of Clic4 is important for vascular development and function in mice. Here, we found that CLIC1 and CLIC4 in endothelial cells regulate critical G protein-coupled receptor (GPCR) pathways associated with vascular development and disease. In cultured endothelial cells, we found that CLIC1 and CLIC4 transiently translocated to the plasma membrane in response to sphingosine 1-phosphate (S1P). Both CLIC1 and CLIC4 were essential for mediating S1P-induced activation of the small guanosine triphosphatase (GTPase) Rac1 downstream of S1P receptor 1 (S1PR1). In contrast, only CLIC1 was essential for S1P-induced activation of the small GTPase RhoA downstream of S1PR2 and S1PR3. Neither were required for other S1P-S1PR signaling outputs. Rescue experiments revealed that CLIC1 and CLIC4 were not functionally interchangeable, suggesting distinct and specific functions for CLICs in transducing GPCR signaling. These CLIC-mediated mechanisms were critical for S1P-induced stimulation of the barrier function in endothelial cell monolayers. Our results define CLICs as previously unknown players in the pathways linking GPCRs to small GTPases and vascular endothelial function.

Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling.

The vascular endothelial growth factor-A (VEGF-A)-VEGFR2 pathway drives tumor vascularization by activating proangiogenic signaling in endothelial cells (ECs). Here, we show that EC-sphingosine-1-phosphate receptor 1 (S1PR1) amplifies VEGFR2-mediated angiogenic signaling to enhance tumor growth. We show that cancer cells induce S1PR1 activity in ECs, and thereby, conditional deletion of S1PR1 in ECs (EC-S1pr1-/- mice) impairs tumor vascularization and growth. Mechanistically, we show that S1PR1 engages the heterotrimeric G-protein Gi, which amplifies VEGF-VEGFR2 signaling due to an increase in the activity of the tyrosine kinase c-Abl1. c-Abl1, by phosphorylating VEGFR2 at tyrosine-951, prolongs VEGFR2 retention on the plasmalemma to sustain Rac1 activity and EC migration. Thus, S1PR1 or VEGFR2 antagonists, alone or in combination, reverse the tumor growth in control mice to the level seen in EC-S1pr1-/- mice. Our findings suggest that blocking S1PR1 activity in ECs has the potential to suppress tumor growth by preventing amplification of VEGF-VEGFR2 signaling.

Omega-3 fatty acids suppress Fusobacterium nucleatum-induced placental inflammation originating from maternal endothelial cells.

Fusobacterium nucleatum is an oral anaerobe prevalent in intrauterine infection associated with a wide spectrum of adverse pregnancy outcomes. We demonstrate here that F. nucleatum triggers placental inflammation through maternal, rather than paternal, TLR4-mediated signaling. Elimination of TLR4 from maternal endothelial cells alleviated placental inflammation and reduced fetal and neonatal death, while elimination of TLR4 in the hematopoietic cells had no effect. The placental inflammatory response followed a spatiotemporal pattern, with NF-κB activation observed first in the maternal endothelial cells and then in the decidual cells surrounding the endothelium, followed by induction of inflammatory cytokines and chemokines. Supplementation of pregnant mice with fish oil as a source of omega-3 fatty acids suppressed placental inflammation, reduced F. nucleatum proliferation in the placenta, and increased fetal and neonatal survival. In vitro analysis illustrates that omega-3 fatty acids inhibit bacterial-induced inflammatory responses from human umbilical cord endothelial cells. Our study therefore reveals a mechanism by which microbial infections affect pregnancy and identifies a prophylactic therapy to protect against intrauterine infections.