Cytosol Of Endothelial Cells Research Articles

MtDNA Activates cGAS Signaling and Suppresses the YAP-Mediated Endothelial Cell Proliferation Program to Promote Inflammatory Injury

Cytosolic DNA acts as a universal danger-associated molecular pattern (DAMP) signal; however, the mechanisms of self-DNA release into the cytosol and its role in inflammatory tissue injury are not well understood. We found that the internalized bacterial endotoxin lipopolysaccharide (LPS) activated the pore-forming protein Gasdermin D, which formed mitochondrial pores and induced mitochondrial DNA (mtDNA) release into the cytosol of endothelial cells. mtDNA was recognized by the DNA sensor cGAS and generated the second messenger cGAMP, which suppressed endothelial cell proliferation by downregulating YAP1 signaling. This indicated that the surviving endothelial cells in the penumbrium of the inflammatory injury were compromised in their regenerative capacity. In an experimental model of inflammatory lung injury, deletion of cGas in mice restored endothelial regeneration. The results suggest that targeting the endothelial Gasdermin D activated cGAS-YAP signaling pathway could serve as a potential strategy for restoring endothelial function after inflammatory injury.

Abstract P316: Cysteine-Independent Interactions of Thioredoxin-1 and Apex1: Implications for Endothelial Cell Apoptosis and Aging?

Aging processes are associated with increased intracellular formation of reactive oxygen species. One redox regulator is the ubiquitously expressed oxidoreductase Thioredoxin-1 (Trx). It contains two active site cysteines and has anti-oxidative and anti-apoptotic properties. Trx levels are reduced in senescent endothelial cells (EC), leading to increased apoptosis sensitivity. Several functions of Trx are enhanced by its interaction with AP-endonuclease 1 (APEX1). APEX1 is a multifunctional protein and in concert with Trx-1 a reductive activator of e.g. the transcription factor AP-1, suggesting at least an interaction of Trx and APEX1 in vivo. Recent evidence suggests that also APEX1 levels are decreased in senescent cells. Therefore, the aim of this study is to understand the function of APEX1 in concert with Trx in EC. We first investigated whether APEX1, similar to Trx has anti-apoptotic properties in EC. Indeed, overexpression of APEX1 inhibited apoptosis. To analyze where the Trx/APEX1 interaction occurs, we performed fluorescence microscopy demonstrating a colocalization throughout the whole cell. To study the interaction in more detail, we carried out coimmunoprecipitations from nuclear and cytosolic extracts and demonstrated their association in both fractions. To determine whether disulfide bridges are involved in the Trx/APEX1 association, we mutated APEX1 cysteines 65, 93 and 310 previously shown to interact with cysteines in other proteins, to serines. This mutant bound Trx in the nucleus and cytosol of EC with the same efficiency as APEX1 wildtype. Moreover, a Trx mutant with serine mutations of active site cysteines (Trx(C32S/C35S)) was still able to bind to endogenous APEX1. These data demonstrate that the Trx/APEX-1 interaction is independent of disulfide bridge formation. We are currently analyzing a set of APEX1 deletion mutants for their interaction with Trx-1 and a potential loss of anti-apoptotic properties Exact delineation of the Trx/APEX1 interaction surface will in the future allow the development of small peptides containing the interaction domains of Trx or APEX1 respectively, in order to enhance transcription factor activity and bypass the loss of antioxidative capacity during aging processes.

P. aeruginosa ExoY Disrupts Microtubules and Induces Endothelial Cell Gap Formation

Microtubule architecture controls endothelial cell barrier function, as acute microtubule disruption leads to inter‐endothelial gap formation. The P. aeruginosa toxin, ExoY, is injected through a type III secretion system (TTSS) into the cytosol of endothelial cells, where it associates with microtubules, and produces a cytosolic cAMP pool that disrupts the endothelial barrier. However, it is unclear whether cytosolic cAMP reorganizes microtubules as a necessary prerequisite. To test this idea, confluent pulmonary microvascular endothelial cell monolayers were infected with two strains of P. aeruginosa that possess a: (1) functional TTSS, and only injects ExoY (ExoY+), and (2) functional TTSS, but injects a catalytically inactive ExoY (ExoYK81M) due to a single point mutation at the ATP binding site. After 4 hours of incubation, cells were stained for microtubule organization. Although microtubules were disrupted and inter‐endothelial gaps were formed in ExoY+‐treated cells, neither microtubules nor inter‐endothelial gaps were formed in ExoYK81M or vehicle control‐treated cells. However, stabilizing microtubules prior to infection prevented ExoY+‐induced microtubule disruption and inter‐endothelial gap formation. Collectively, these data suggest that ExoY generates a cytosolic cAMP pool that reorganizes microtubules and causes endothelial barrier disruption. [HL‐60024 & HL‐66299]

Endothelial calcium signaling in rabbit arteries and its local alterations in early-stage atherosclerosis

This study is to examine whether endothelial calcium signaling is different between atherosclerosis-prone thoracic aortas (TA) and atherosclerosis-resistant carotid arteries (CA) in normal rabbits and how it changes in early-stage atherosclerosis. Local endothelial calcium signaling was examined in arterial segments obtained from rabbits fed with normal or high-cholesterol diet for 1-4 weeks. Contrasting to normal CA, normal TA showed lower endothelial calcium signaling with more concentrated NF-kappaB in the endothelial nuclei. In the same hypercholesterolemic animal, fatty streak formation was much more prominent in TA than in CA. TA endothelial calcium signaling became augmented in the second week of hypercholesterolemia, being most pronounced in smooth regions adjacent to miniature fatty streaks. It was sporadically elevated even in regions away from any detectable TA fatty streak. When the entire TA was covered with fatty streaks in the fourth week of hypercholesteremia, endothelial calcium signaling returned to the original level. In comparison, CA endothelial calcium signaling was reduced around scattered fatty streaks. Reduced calcium signaling happened where CA fatty streaks were 150 microm long (covering 15-30 cells); and it extended to areas adjacent to larger fatty streaks. Moreover, NF-kappaB remained in the cytosol of endothelial cells covering CA fatty streaks. Our results indicate that inter-vascular differences in endothelial calcium signaling may provide partial explanation in their differential susceptibility in atherosclerosis.

Unsaturated fatty acids isolated from human lipoproteins activate protein phosphatase type 2Cβ and induce apoptosis in endothelial cells

Activity of serine/threonine protein phosphatase type 2C is known to be stimulated by certain unsaturated fatty acids and this enzyme dephosphorylates Bad, thus acting on apoptosis. This prompted us to investigate endothelial cell death. Here, we present evidence for the presence of protein phosphatase type 2Cβ (PP2Cβ) in human umbilical vein endothelial cells (HUVECs) and report on colocalization of PP2Cβ and Bad in the cytosol of endothelial cells. Lipophilic compounds that stimulated PP2Cβ activity in vitro were found to induce cell death of HUVECs. Lipoproteins did neither influence PP2Cβ activity nor affect cell behaviour. Lipoproteins treated with the lipoprotein lipase, however, stimulated the activity of PP2Cβ at least 10-fold concomitantly triggering cell death. Analytical methods revealed that both effects – stimulation of PP2Cβ and apoptosis – were caused by free fatty acids liberated from VLDL, LDL and HDL with oleic acid and linoleic acid as major constituents. The results provide novel insights in endothelial apoptosis and suggest that PP2Cβ participates in the development and progress of atherosclerosis.

Modulation of ICAM-1 Expression in ECV304 Cells by Macrophage-Released Cytokines

ABSTRACTTransendothelial leukocyte trafficking during inflammation requires the expression of adhesion molecules such as human intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is constitutively expressed on the surface of endothelial cells and its levels increase in response to a variety of inflammatory mediators, including cytokines. Monocyte/macrophage cells play a crucial role in this context because, upon stimulation, they release proinflammatory cytokines which are responsible for the upregulation of adhesion molecules in endothelial cells. In the present study we investigated whether the modulation of macrophage activation and cytokine release is able to modulate ICAM-1 expression in endothelial cells. Dexamethasone was selectively delivered to macrophages by means of a red blood cell-mediated delivery system. Subsequent stimulation of macrophages by lipopolysaccharide (LPS) was found to inhibit NF-kB activation and tumor necrosis factor-α (TNF-α) release [R. Crinelli, A. Antonelli, M. Bianchi, L. Gentilini, S. Scaramucci, and M. Magnani (2000) Blood Cells Mol. Dis. 26, 211–222]. Incubation with conditioned medium derived from LPS-stimulated macrophages receiving dexamethasone resulted in a 45% inhibition of ICAM-1 mRNA expression in ECV304 cells. In the same experimental system this reduced ICAM-1 expression was paralleled by a reduced NF-kB DNA binding activity and a twofold higher level of IkBα in the cytosol of endothelial cells. Activation of ICAM-1 expression in ECV304 cells by macrophage-conditioned medium is not due to IFN-γ stimulation since STAT-1 DNA binding remained unchanged. Furthermore, treatment of the macrophage-conditioned medium with a TNF-α-inactivating antibody resulted in the complete abrogation of induced ICAM-1 expression. These results suggest that TNF-α is the main cytokine released by LPS-stimulated macrophages able to promote ICAM-1 gene expression in endothelial cells. Modulation of the NF-kB activation pathway in macrophages by targeted delivery of dexamethasone could potentially be used as a therapeutic strategy with which to inhibit the expression of ICAM-1 in endothelial cells.

Incorporation of the elderberry anthocyanins by endothelial cells increases protection against oxidative stress

The objective of this study was to investigate the ability of endothelial cells (EC) to incorporate anthocyanins and to examine their potential benefits against various oxidative stressors. Endothelial dysfunction has been proposed to play an important role in the initiation and development of vascular disease, with studies having shown that administration of antioxidants improves endothelial function. Elderberry extract contains 4 anthocyanins, which where incorporated into the plasma membrane and cytosol of EC following 4 h incubation at 1 mg.ml −1. However, incorporation within the cytosol was considerably less than that in the membrane. Uptake within both regions appeared to be structure dependent, with monoglycoside concentrations higher than that of the diglucosides in both compartments. The enrichment of EC with elderberry anthocyanins conferred significant protective effects in EC against the following oxidative stressors: hydrogen peroxide (H 2O 2); 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH); and FeSO 4/ascorbic acid (AA). These results show for the first time that vascular EC can incorporate anthocyanins into the membrane and cytosol, conferring significant protective effects against oxidative insult. These findings may have important implications on preserving EC function and preventing the initiation of EC changes associated with vascular diseases.

Regulation of Leukotriene A4 Hydrolase Activity in Endothelial Cells by Phosphorylation

Endothelial cells contain leukotriene (LT) A4 hydrolase (LTA-H) as detected by Northern and Western blotting, but several studies have been unable to detect the activity of this enzyme. Since LTA-H could play a key role in determining what biologically active lipids are generated by activated endothelium during the inflammatory process, we studied possible mechanisms by which this enzyme may be regulated. We find that LTA-H is phosphorylated under basal conditions in human endothelial cells and in this state does not exhibit epoxide hydrolase activity (i.e. conversion of LTA4 to LTB4). LTA-H purified from endothelial cells is efficiently dephosphorylated by incubation with protein phosphatase-1 in the presence of an LTA-H peptide substrate and not at all in the absence of substrate. Under conditions that lead to dephosphorylation, protein phosphatase-1 activates the epoxide hydrolase activity of LTA-H. Using peptide mapping and site-directed mutagenesis, we have identified serine 415 as the site of phosphorylation of LTA-H by a kinase found in endothelial cell cytosol. In parallel, we have studied a human lung carcinoma cell line that expresses active LTA-H. Although these cells have cytosolic kinases that phosphorylate recombinant LTA-H, they do not target serine 415 and thus do not inhibit LTA-H activity. We believe that LTA-H is regulated in intact cells by a kinase/phosphatase cycle and further that the kinase in endothelial cells specifically recognizes and phosphorylates a regulatory site in the LTA-H.

Colocalization of Tissue Transglutaminase and Stress Fibers in Human Vascular Smooth Muscle Cells and Human Umbilical Vein Endothelial Cells

The subcellular distribution of tissue transglutaminase in human umbilical vein endothelial cells and human arterial and venous smooth muscle cells was examined. Double-immunofluorescence staining of smooth muscle cells and endothelial cells with anti-transglutaminase antisera and rhodamine-tagged phalloidin revealed codistribution of transglutaminase with the stress fibers, with endothelial cells also containing a cytoplasmic pool. This pattern of distribution was confirmed by confocal microscopy. Immunoprecipitation experiments demonstrated that transglutaminase co-immunoprecipitated with myosin in high-molecular-weight complexes, but not with actin, suggesting that the association of transglutaminase with the stress fibers was due to its cross-linking to myosin. About 97% of endothelial cell transglutaminase activity was present in the cytosolic fraction and 3% in the particulate fraction. The detergent-insoluble fraction was practically devoid of activity as measured by the putrescine assay, but was active as evidenced by the covalent cross-linking of 125I-fibronectin. Western blotting with a polyclonal rabbit antiserum raised against human erythrocyte transglutaminase detected high levels of enzyme in endothelial cell cytosol and both detergent-soluble and detergent-insoluble membrane fractions. In contrast, smooth muscle cells contained much less cytosolic transglutaminase, as determined either functionally or antigenically. Furthermore, within the particulate fraction of the smooth muscle cells, most of the enzyme was located in the detergent-insoluble fraction, as assessed by Western blot analysis. Retinoic acid increased the levels of enzyme in the cytosol of all cell types and the increases were correlated with increases in mRNA. Thus, tissue transglutaminase is present in various particulate fractions of vascular smooth muscle cells and endothelial cells and may be present in this cellular fraction by virtue of autocross-linking of the enzyme itself to stress fiber-associated myosin.