Mexenone protects mice from LPS-induced sepsis by EC barrier stabilization.

Blood vessels permit the selective passage of molecules and immune cells between tissues and circulation. Uncontrolled inflammatory responses from an infection can increase vascular permeability and edema, which can occasionally lead to fatal organ failure. We identified mexenone as a vascular permeability blocker by testing 2,910 compounds in the Clinically Applied Compound Library using the lipopolysaccharide (LPS)-induced vascular permeability assay. Mexenone suppressed the LPS-induced downregulation of junctional proteins and phosphorylation of VE-cadherin in Bovine Aortic Endothelial Cells (BAECs). The injection of mexenone 1 hr before LPS administration completely blocked LPS-induced lung vascular permeability and acute lung injury in mice after 18hr. Our results suggest that mexenone-induced endothelial cell (EC) barrier stabilization could be effective in treating sepsis patients.

Vascular Inflammatory Diseases and Endothelial Phenotypes.

The physiological functions of endothelial cells control vascular tone, permeability, inflammation, and angiogenesis, which significantly help to maintain a healthy vascular system. Several cardiovascular diseases are characterized by endothelial cell activation or dysfunction triggered by external stimuli such as disturbed flow, hypoxia, growth factors, and cytokines in response to high levels of low-density lipoprotein and cholesterol, hypertension, diabetes, aging, drugs, and smoking. Increasing evidence suggests that uncontrolled proinflammatory signaling and further alteration in endothelial cell phenotypes such as barrier disruption, increased permeability, endothelial to mesenchymal transition (EndMT), and metabolic reprogramming further induce vascular diseases, and multiple studies are focusing on finding the pathways and mechanisms involved in it. This review highlights the main proinflammatory stimuli and their effects on endothelial cell function. In order to provide a rational direction for future research, we also compiled the most recent data regarding the impact of endothelial cell dysfunction on vascular diseases and potential targets that impede the pathogenic process.

DJ-1 promotes cell migration by interacting with Mena, the mammalian homolog of Drosophila enabled.

DJ-1 has gained extensive attention after being identified in 2003 as a protein implicated in the pathogenesis of early-onset Parkinson's disease. Since then, efforts have revealed versatile DJ-1 functions in reactive oxygen species (ROS) control, transcriptional regulation, chaperone function, fertility, and cell transformation. Herein, we report a novel function of DJ-1 in actin cytoskeletal rearrangements. DJ-1 was identified as a new binding partner of Mena, a protein of the Enah/VASP family, and it promoted cancer cell migration by Mena-dependent actin polymerization and filopodia formation. These results suggest a novel molecular mechanism for DJ-1-dependent cancer cell invasion and metastasis.

Shinjulactone A Blocks Vascular Inflammation and the Endothelial-Mesenchymal Transition.

Objective: The endothelial inflammatory response plays an important role in atherogenesis by inducing nuclear factor (NF)κB-dependent cell adhesion molecule expression and monocyte recruitment. Here, we screened for natural ligands and investigated the ability of shinjulactone A to inhibit interleukin-1ß (IL-1ß)-induced endothelial inflammatory signaling. Methods: The natural compound library included 880 single compounds isolated from medicinal plants by the Korean Medicinal Material Bank. Primary endothelial cells were pretreated with single compounds before stimulation with IL-1ß to induce endothelial inflammation. Endothelial inflammation was measured by assaying NFκB activation and monocyte adhesion. The endothelial-mesenchymal transition (EndMT) was evaluated using cell type-specific marker protein expression and morphology. Results: Shinjulactone A was identified as an efficient blocker of IL-1ß -induced NFκB activation, with a half-maximal inhibitory concentration of approximately 1 µM, and monocyte recruitment in endothelial cells. However, it did not affect lipopolysaccharide-induced NFκB activation in macrophages. Compared to Bay 11-782, a well-known NFκB inhibitor that shows considerable cytotoxicity during long-term treatment, shinjulactone A did not affect endothelial cell viability. Furthermore, it also significantly inhibited the EndMT, which is known to promote atherosclerosis and plaque instability. Conclusion: We suggest that shinjulactone A may be an effective and safe drug candidate for atherosclerosis because it targets and inhibits both endothelial inflammation and the EndMT, without impairing NFκB-dependent innate immunity in macrophages.

Fibronectin-Integrin α5 Signaling in Vascular Complications of Type 1 Diabetes.

Vascular complications are a major cause of illness and death in patients with type 1 diabetes (T1D). Diabetic vascular basement membranes are enriched in fibronectin (FN), an extracellular matrix protein that amplifies inflammatory signaling in endothelial cells through its main receptor, integrin α5ß1. Binding of the integrin α5 cytoplasmic domain to phosphodiesterase 4D5 (PDE4D5), which increases phosphodiesterase catalytic activity and inhibits antiinflammatory cAMP signaling, was found to mediate these effects. Here, we examined mice in which the integrin α5 cytoplasmic domain is replaced by that of α2 (integrin α5/2) or the integrin α5 binding site in PDE4D is mutated (PDE4Dmut). T1D was induced via injection of streptozotocin and hyperlipidemia induced via injection of PCSK9 virus and provision of a high-fat diet. We found that in T1D and hyperlipidemia, the integrin α5/2 mutation reduced atherosclerosis plaque size by ∼50%, with reduced inflammatory cell invasion and metalloproteinase expression. Integrin α5/2 T1D mice also had improved blood-flow recovery from hindlimb ischemia and improved biomechanical properties of the carotid artery. By contrast, the PDE4Dmut had no beneficial effects in T1D. FN signaling through integrin α5 is thus a major contributor to diabetic vascular disease but not through its interaction with PDE4D.

Defective Flow-Migration Coupling Causes Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia.

BACKGROUND: Activin receptor-like kinase 1 (ALK1) is an endothelial transmembrane serine threonine kinase receptor for BMP family ligands that plays a critical role in cardiovascular development and pathology. Loss-of-function mutations in the ALK1 gene cause type 2 hereditary hemorrhagic telangiectasia, a devastating disorder that leads to arteriovenous malformations. Here, we show that ALK1 controls endothelial cell polarization against the direction of blood flow and flow-induced endothelial migration from veins through capillaries into arterioles. METHODS: Using Cre lines that recombine in different subsets of arterial, capillary-venous, or endothelial tip cells, we show that capillary-venous Alk1 deletion was sufficient to induce arteriovenous malformation formation in the postnatal retina. RESULTS: ALK1 deletion impaired capillary-venous endothelial cell polarization against the direction of blood flow in vivo and in vitro. Mechanistically, ALK1-deficient cells exhibited increased integrin signaling interaction with vascular endothelial growth factor receptor 2, which enhanced downstream YAP/TAZ nuclear translocation. Pharmacologic inhibition of integrin or YAP/TAZ signaling rescued flow migration coupling and prevented vascular malformations in Alk1-deficient mice. CONCLUSIONS: Our study reveals ALK1 as an essential driver of flow-induced endothelial cell migration and identifies loss of flow-migration coupling as a driver of arteriovenous malformation formation in hereditary hemorrhagic telangiectasia disease. Integrin-YAP/TAZ signaling blockers are new potential targets to prevent vascular malformations in patients with hereditary hemorrhagic telangiectasia.

Author Correction: Mechanosensation of cyclical force by PIEZO1 is essential for innate immunity.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mechanosensation of cyclical force by PIEZO1 is essential for innate immunity.

Direct recognition of invading pathogens by innate immune cells is a critical driver of the inflammatory response. However, cells of the innate immune system can also sense their local microenvironment and respond to physiological fluctuations in temperature, pH, oxygen and nutrient availability, which are altered during inflammation. Although cells of the immune system experience force and pressure throughout their life cycle, little is known about how these mechanical processes regulate the immune response. Here we show that cyclical hydrostatic pressure, similar to that experienced by immune cells in the lung, initiates an inflammatory response via the mechanically activated ion channel PIEZO1. Mice lacking PIEZO1 in innate immune cells showed ablated pulmonary inflammation in the context of bacterial infection or fibrotic autoinflammation. Our results reveal an environmental sensory axis that stimulates innate immune cells to mount an inflammatory response, and demonstrate a physiological role for PIEZO1 and mechanosensation in immunity.

Integrin α5ß1 regulates PP2A complex assembly through PDE4D in atherosclerosis.

Fibronectin in the vascular wall promotes inflammatory activation of the endothelium during vascular remodeling and atherosclerosis. These effects are mediated in part by fibronectin binding to integrin α5, which recruits and activates phosphodiesterase 4D5 (PDE4D5) by inducing its dephosphorylation on an inhibitory site Ser651. Active PDE then hydrolyzes anti-inflammatory cAMP to facilitate inflammatory signaling. To test this model in vivo, we mutated the integrin binding site in PDE4D5 in mice. This mutation reduced endothelial inflammatory activation in athero-prone regions of arteries, and, in a hyperlipidemia model, reduced atherosclerotic plaque size while increasing markers of plaque stability. We then investigated the mechanism of PDE4D5 activation. Proteomics identified the PP2A regulatory subunit B55α as the factor recruiting PP2A to PDE4D5. The B55α-PP2A complex localized to adhesions and directly dephosphorylated PDE4D5. This interaction also unexpectedly stabilized the PP2A-B55α complex. The integrin-regulated, pro-atherosclerotic transcription factor Yap is also dephosphorylated and activated through this pathway. PDE4D5 therefore mediates matrix-specific regulation of EC phenotype via an unconventional adapter role, assembling and anchoring a multifunctional PP2A complex with other targets. These results are likely to have widespread consequences for control of cell function by integrins.

Inhibiting Integrin α5 Cytoplasmic Domain Signaling Reduces Atherosclerosis and Promotes Arteriogenesis.

BACKGROUND: Fibronectin in endothelial basement membranes promotes endothelial inflammatory activation and atherosclerosis but also promotes plaque stability and vascular remodeling. The fibronectin receptor α5 subunit is proinflammatory through binding to and activating phosphodiesterase 4D5, which inhibits anti-inflammatory cyclic adenosine monophosphate and protein kinase A. Replacing the α5 cytoplasmic domain with that of α2 resulted in smaller atherosclerotic plaques. Here, we further assessed plaque phenotype and compensatory vascular remodeling in this model. METHODS AND RESULTS: α5/2 mice in the hyperlipidemic apolipoprotein E null background had smaller plaques in the aortic root, with reduced endothelial NF-κB activation and inflammatory gene expression, reduced leukocyte content, and much lower metalloproteinase expression. However, smooth muscle cell content, fibrous cap thickness, and fibrillar collagen were unchanged, indicating no shift toward vulnerability. In vivo knockdown of phosphodiesterase 4D5 also decreased endothelial inflammatory activation and atherosclerotic plaque size. α5/2 mice showed improved recovery from hindlimb ischemia after femoral artery ligation. CONCLUSIONS: Blocking the fibronectin-Integrin α5 pathway reduces atherosclerotic plaque size, maintains plaque stability, and improves compensatory remodeling. This pathway is therefore a potential therapeutic target for treatment of atherosclerosis.

Inhibitory effect of tartrate against phosphate-induced DJ-1 aggregation.

The DJ-1 protein engages in diverse cellular and pathological processes, including tumorigenesis, apoptosis, sperm fertilization, and the progression of Parkinson's disease (PD). The functional dimeric form of DJ-1 transforms into non-functional filamentous aggregates in an inorganic phosphate (Pi)-dependent manner in vitro. Here, we demonstrated that Pi and reactive oxygen species (ROS) induce DJ-1 aggregation in Neuro2A and SH-SY5Y cells. Remarkably, tartrate treatment significantly reduced Pi- and ROS-induced DJ-1 aggregation and restored Pi- and ROS-provoked cell death using quantitative data as mean±standard deviation, and statistics. Mechanistically, tartrate prevented DJ-1 aggregation via occupying the Pi-binding site. These findings revealed an unexpected physiological role of tartrate in the maintenance of DJ-1 function, and thus, a potential use as an inhibitor of DJ-1 aggregation.

Interaction between integrin α5 and PDE4D regulates endothelial inflammatory signalling.

Atherosclerosis is primarily a disease of lipid metabolism and inflammation; however, it is also closely associated with endothelial extracellular matrix (ECM) remodelling, with fibronectin accumulating in the laminin-collagen basement membrane. To investigate how fibronectin modulates inflammation in arteries, we replaced the cytoplasmic tail of the fibronectin receptor integrin α5 with that of the collagen/laminin receptor integrin α2. This chimaera suppressed inflammatory signalling in endothelial cells on fibronectin and in knock-in mice. Fibronectin promoted inflammation by suppressing anti-inflammatory cAMP. cAMP was activated through endothelial prostacyclin secretion; however, this was ECM-independent. Instead, cells on fibronectin suppressed cAMP via enhanced phosphodiesterase (PDE) activity, through direct binding of integrin α5 to phosphodiesterase-4D5 (PDE4D5), which induced PP2A-dependent dephosphorylation of PDE4D5 on the inhibitory site Ser651. In vivo knockdown of PDE4D5 inhibited inflammation at athero-prone sites. These data elucidate a molecular mechanism linking ECM remodelling and inflammation, thereby identifying a new class of therapeutic targets.

Endothelial fluid shear stress sensing in vascular health and disease.

Endothelial cells transduce the frictional force from blood flow (fluid shear stress) into biochemical signals that regulate gene expression and cell behavior via specialized mechanisms and pathways. These pathways shape the vascular system during development and during postnatal and adult life to optimize flow to tissues. The same pathways also contribute to atherosclerosis and vascular malformations. This Review covers recent advances in basic mechanisms of flow signaling and the involvement of these mechanisms in vascular physiology, remodeling, and these diseases. We propose that flow sensing pathways that govern normal morphogenesis can contribute to disease under pathological conditions or can be altered to induce disease. Viewing atherosclerosis and vascular malformations as instances of pathological morphogenesis provides a unifying perspective that may aid in developing new therapies.

Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex.

Endothelial responses to fluid shear stress are essential for vascular development and physiology, and determine the formation of atherosclerotic plaques at regions of disturbed flow. Previous work identified VE-cadherin as an essential component, along with PECAM-1 and VEGFR2, of a complex that mediates flow signaling. However, VE-cadherin's precise role is poorly understood. We now show that the transmembrane domain of VE-cadherin mediates an essential adapter function by binding directly to the transmembrane domain of VEGFR2, as well as VEGFR3, which we now identify as another component of the junctional mechanosensory complex. VEGFR2 and VEGFR3 signal redundantly downstream of VE-cadherin. Furthermore, VEGFR3 expression is observed in the aortic endothelium, where it contributes to flow responses in vivo. In summary, this study identifies a novel adapter function for VE-cadherin mediated by transmembrane domain association with VEGFRs.

Integrins in mechanotransduction.

Forces acting on cells govern many important regulatory events during development, normal physiology, and disease processes. Integrin-mediated adhesions, which transmit forces between the extracellular matrix and the actin cytoskeleton, play a central role in transducing effects of forces to regulate cell functions. Recent work has led to major insights into the molecular mechanisms by which these adhesions respond to forces to control cellular signaling pathways. We briefly summarize effects of forces on organs, tissues, and cells; and then discuss recent advances toward understanding molecular mechanisms.

DJ-1 promotes angiogenesis and osteogenesis by activating FGF receptor-1 signaling.

Communication between osteoblasts and endothelial cells is essential for bone fracture repair, but the molecular identities of such communicating factors are not well defined. Here we identify DJ-1 as a novel mediator of the cross-talk between osteoblasts and endothelial cells through an unbiased screening of molecules secreted from human mesenchymal stem cells during osteogenesis. We show that DJ-1 stimulates the differentiation of human mesenchymal stem cells to osteoblasts and that DJ-1 induces angiogenesis in endothelial cells through activation of fibroblast growth factor receptor-1 signalling. In a rodent model of bone fracture repair, extracellular application of DJ-1 enhances bone regeneration in vivo by stimulating the formation of blood vessels and new bones. Both these effects are blocked by antagonizing fibroblast growth factor receptor-1 signalling. These findings uncover previously undefined extracellular roles of DJ-1 to promote angiogenesis and osteogenesis, suggesting DJ-1 may have therapeutic potential to stimulate bone regeneration.

Crystal structure of filamentous aggregates of human DJ-1 formed in an inorganic phosphate-dependent manner.

Mutations in the DJ-1 gene have been implicated in the autosomal recessive early onset parkinsonism. DJ-1 is a soluble dimeric protein with critical roles in response to oxidative stress and in neuronal maintenance. However, several lines of evidence suggest the existence of a nonfunctional aggregated form of DJ-1 in the brain of patients with some neurodegenerative diseases. Here, we show that inorganic phosphate, an important anion that exhibits elevated levels in patients with Parkinson disease, transforms DJ-1 into filamentous aggregates. According to the 2.4-A crystal structure, DJ-1 dimers are linearly stacked through P(i)-mediated interactions to form protofilaments, which are then bundled into a filamentous assembly.

Multiple roles of phosphoinositide-specific phospholipase C isozymes.

Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-beta, -gamma, -delta, -epsilon, -zeta and -eta. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.

Siah proteins induce the epidermal growth factor-dependent degradation of phospholipase Cepsilon.

Phospholipase Cepsilon (PLCepsilon) is activated by various growth factors or G-protein-coupled receptor ligands via different activation mechanisms. The Ras association (RA) domain of PLCepsilon is known to be important for its ability to bind with Ras-family GTPase upon growth factor stimulation. In the present study, we identified Siah1 and Siah2 as novel binding partners of the PLCepsilon RA domain. Both Siah1 and Siah2 interacted with the RA2 domain of PLCepsilon, and the mutation of Lys-2186 of the PLCepsilon RA2 domain abolished this association. Moreover, Siah induced the ubiquitination and degradation of PLCepsilon upon epidermal growth factor (EGF) stimulation, and Siah proteins were phosphorylated on multiple tyrosine residues via an Src-dependent pathway upon EGF treatment. The Src inhibitor abolished the EGF-dependent ubiquitination of PLCepsilon, and the Siah1 phosphorylation-deficient mutant could not increase the EGF-dependent ubiquitination and degradation of PLCepsilon. The EGF-dependent degradation of PLCepsilon was blocked in mouse embryonic fibroblast (MEF) cells derived from Siah1a/Siah2 double knockout mice, and the extrinsic expression of wild-type Siah1 restored the degradation of PLCepsilon, whereas the phosphorylation-deficient mutant did not. Siah1 expression abolished PLCepsilon-dependent potentiation of EGF-dependent cell growth. In addition, the expression of wild-type Siah1 in Siah1a/Siah2-double knockout MEF cells inhibited EGF-dependent cell growth, and this inhibition was abolished by PLCepsilon knockdown. Our results suggest that the Siah-dependent degradation of PLCepsilon plays a role in the regulation of growth factor-dependent cell growth.