IntroductionApoptosis is essential to maintain homeostasis in multicellular organisms and apoptotic cells are rapidly and effectively cleared by professional phagocytes before they undergo secondary necrosis and release their noxious cytoplasmic content into the environment. 1 Ineffective clearance of apoptotic cells contributes to disease pathogenesis. 2,3 This may be true especially for autoimmune diseases such as systemic lupus erythematosus (SLE) because apoptotic cells are thought to be a potential source of autoantigens and disturbed clearance of apoptotic corpses may initiate and drive autoimmunity. [4][5][6] This concept of acquired autoimmunity has also been confirmed in several animal models that showed disturbed engulfment of apoptotic cells. [7][8][9][10] Vascular endothelial cells (ECs) serve as a crucial barrier between tissues and the circulation. They secrete a variety of substances, regulate coagulation, and participate in the immune response. 11 Under physiologic conditions circulating ECs (CECs) are almost not traceable, whereas in a variety of vascular diseases such as myocardial infarction, small-vessel vasculitis, transplantation, or cancer high numbers of CECs are detectable in the peripheral circulation. [12][13][14][15][16][17] Consequentially, accumulation of CECs may affect the homeostasis of the vessel wall by interfering with the EC layer both in the vicinity of endothelial lesions or even distant from the site of injury.Clearance of dying cells or cellular debris has been mostly ascribed to professional phagocytes, for example, antigenpresenting macrophages, neutrophils, or dendritic cells, but it is conceivable that other cell types such as epithelial cells or ECs may also partake in this process. 18,19 ECs are not professional phagocytes although specific subpopulations, such as liver ECs or cells from high endothelial venules, are able to phagocytose apoptotic cells. 20,21 Phagocytosis of circulating endothelial debris by healthy endothelium thus appears to be an intriguing concept.The role of endothelial-or platelet-derived microparticles in the circulation is the subject of several recently published studies but few, if any, data, shed light on the impact of apoptotic endothelial corpses on the adjacent endothelium. [22][23][24][25][26][27][28][29][30][31] The aim of the present study was to establish an in vitro model to study the interaction of apoptotic or necrotic ECs with a healthy EC layer. We here demonstrate that microvascular ECs, when exposed to apoptotic cells, react with the release of proinflammatory chemotactic cytokines and that this response triggers enhanced adhesion of primary neutrophils and macrophages. Patients, materials, and methodsThe study was approved by the Hannover Medical School Ethics Committee and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients included in this study. EC cultureHuman umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords by exposure of the vein...

BackgroundEndothelial tight and adherens junctions control a variety of physiological processes like adhesion, paracellular transport of solutes or trafficking of activated leukocytes. Formation and maintenance of endothelial junctions largely depend on the microenvironment of the specific vascular bed and on interactions of the endothelium with adjacent cell types. Consequently, primary cultures of endothelial cells often lose their specific junctional pattern and fail to establish tight monolayer in vitro. This is also true for endothelial cells isolated from the vein of human umbilical cords (HUVEC) which are widely used as model for endothelial cell-related studies.ResultsWe here compared the effect of cyclic 3'-5'-adenosine monophosphate (cAMP) and its derivates on formation and stabilization of tight junctions and on alterations in paracellular permeability in HUVEC. We demonstrated by light and confocal laser microscopy that for shorter time periods the sodium salt of 8-bromoadenosine-cAMP (8-Br-cAMP/Na) and for longer incubation periods 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) exerted the greatest effects of all compounds tested here on formation of continuous tight junction strands in HUVEC. We further demonstrated that although all compounds induced protein kinase A-dependent expression of the tight junction proteins claudin-5 and occludin only pCPT-cAMP slightly enhanced paracellular barrier functions. Moreover, we showed that pCPT-cAMP and 8-Br-cAMP/Na induced expression and membrane translocation of tricellulin.ConclusionspCPT-cAMP and, to a lesser extend, 8-Br-cAMP/Na improved formation of continuous tight junction strands and decreased paracellular permeability in primary HUVEC. We concluded that under these conditions HUVEC represent a feasible in vitro model to study formation and disassembly of endothelial tight junctions and to characterize tight junction-associated proteins

Abstract-Aldosterone (Aldo) is involved in vascular remodeling and inflammation; however, the mechanisms are imperfectly defined. We hypothesized that Aldo alters endothelial integrity and modifies paracellular permeability. Human umbilical vein endothelial cells were exposed to Aldo (10 -9 mol/L) and alterations in paracellular permeability, assembly of tight and adherens junctions and activation of intracellular signaling pathways were determined. Aldo increased endothelial permeability for molecules ≤70 kDa within 60 minutes. A transient loss of cortical actin with formation of actin stress fibers and disruption of continuous adherens and tight junction strands accompanied these changes. Mineralocorticoid receptor blockade, inhibition of RhoA, or disruption of extracellular-regulated protein kinase1/2 signaling pathways attenuated the Aldo-related effects. Moreover, Aldo-induced cytoskeletal rearrangement led to rapid dephosphorylation of protein kinase B and subsequent deactivation of endothelial nitric oxide synthase. Ex vivo tracer flux experiments with Evans blue-conjugated albumin demonstrated a concordant response to Aldo in freshly isolated umbilical arteries. Furthermore, low-dose cortisol (3×10 -10 to 3×10 -9 mol/L) mimics the effect of Aldo on endothelial integrity, and Aldo, by upregulating11β-hydroxysteroid dehydrogenase type 2, might even aggravate this deleterious effect of low-dose cortisol. We suggest that these mechanisms may contribute to the vasculopathy induced by inappropriate mineralocorticoid receptor activation. Results Aldo Modulates Paracellular Permeability In Vitro and Ex VivoWe measured Aldo-induced alterations in vascular permeability toward Evans blue-conjugated albumin in human umbilical arteries. Preliminary experiments revealed that Aldo exerts its most prominent effect on paracellular permeability after 60 minutes at concentrations of 10 -9 to 10 -10 mol/L ( Figure S1, available in the online-only Data Supplement). Therefore, the following experiments were performed with an Aldo concentration of 10 -9 mol/L. After 60 minutes, albumin flux significantly increased compared with Ringer solution alone (242±108 µg of BSA/mg of dry tissue versus 120±63 µg of BSA/mg of dry tissue; P<0.001). Elevated albumin extravasation could be prevented by pretreating the arteries with EPL for 30 minutes (111±51 µg of BSA/mg of dry tissue; P<0.001; Figure 1A).We then determined the effects of Aldo on paracellular permeability in cAMP-treated human umbilical vein endothelial cells (HUVECs). 14 To test, whether the density of endothelial monolayer influenced expression of the MR, transcript amounts were analyzed by real-time quantitative PCR. MR mRNA level decreased with increasing density of the HUVEC monolayer, and stimulation with cAMP even further reduced the amount of MR transcript ( Figure 1B). However, intracellular cAMP levels were not regulated by Aldo itself (10 -6 to 10 -11 mol/L; data not shown). Tracer flux assays revealed that Aldo induced a clear, time-dependent transient increas...

Rapid apoptotic cell engulfment is crucial for prevention of inflammation and autoimmune diseases and is conducted by special immunocompetent cells like macrophages or immature dendritic cells. We recently demonstrated that endothelial cells (ECs) also participate in apoptotic cell clearance. However, in contrast to conventional phagocytes they respond with an inflammatory phenotype. To further confirm these pro-inflammatory responses human ECs were exposed to apoptotic murine ECs and changes in thrombospondin-1 (TSP-1) expression and in activation of intracellular signalling cascades were determined by real-time qPCR, immunoblotting and immunocytochemistry. Human primary macrophages or monocytic lymphoma cells (U937) were incubated with conditioned supernatant of human ECs exposed to apoptotic cells and changes in activation, migration and phagocytosis were monitored. Finally, plasma levels of TSP-1 in patients with anti-neutrophil cytoplasmic antibody(ANCA)-associated vasculitis (AAV) were determined by ELISA. We provided evidence that apoptotic cells induce enhanced expression of TSP-1 in human ECs and that this increase in TSP-1 is mediated by the mitogen-activated protein kinases (MAPK) ERK1 and 2 and their upstream regulators MEK and B-Raf. We also showed that plasma TSP-1 levels are increased in patients with AAV. Finally, we showed that conditioned supernatant of ECs exposed to apoptotic cells induces pro-inflammatory responses in monocytes or U937 cells and demonstrated that increased TSP-1 expression enhances migration and facilitates engulfment of apoptotic cells by monocyte-derived macrophages or U937 cells. These findings suggest that under pathological conditions with high numbers of uncleared dying cells in the circulation endothelial-derived elevated TSP-1 level may serve as an attraction signal for phagocytes promoting enhanced recognition and clearance of apoptotic cells.