Maria Philippova

Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death

Neutrophil interaction with activated endothelial cells (EC) is required for transmigration. We examined consequences of this interaction on NETosis. Co‐culture of activated EC with neutrophils induced neutrophil extracellular trap (NET) formation, which was partially dependent on production of IL‐8 by activated EC. Extended neutophil/EC co‐culture resulted in EC damage, which could be abrogated by inclusion of either diphenyleneiodonium to inhibit the NAPDH oxidase pathway required for NETosis, or DNAse to disrupt NETs. These findings offer new insight into mechanisms whereby NETs trigger damage to the endothelium in sepsis, small vessel vasculitis and possibly the villous trophoblast in preeclampsia.

Expression of cell adhesion molecule T-cadherin in the human vasculature.

Abstract. Alterations in expression of surface adhesion molecules on resident vascular and blood-derived cells play a fundamental role in the pathogenesis of cardiovascular disease. Smooth muscle cells (SMCs) have been shown to express T-cadherin (T-cad), an unusual GPI-anchored member of the cadherin family of adhesion molecules. Particular relevance for T-cad in cardiovascular tissues is indicated by our present screen (immunoblotting) of human tissues and organs whereby highest expression of T-cad was found in aorta, carotid, iliac and renal arteries and heart. To explore the (patho)physiological role for T-cad in the vasculature we performed an immunohistochemical analysis of T-cad expression in normal human aorta and atherosclerotic lesions of varying severity. T-cad was present both in the intima and media and was expressed in endothelial cells (ECs), SMCs and pericytes, but not in monocytes/macrophages, foam cells and lymphocytes. In the adventitia T-cad was present in the wall of vasa vasorum and was expressed in ECs, SMCs and pericytes. T-cad was differentially expressed in SMCs from distinct vascular layers of normal aorta (for example, high in the subendothelial (proteoglycan) layer of the intima, low in the musculoelastic intimal layer and in the media), as well as at different stages of lesion progression. In SMCs there was an apparent inverse relationship between the intensities of T-cad and smooth muscle α-actin expression, this being most prominent in lesions. The findings suggest a phenotype-associated expression of T-cad which may be relevant to control of the normal vascular architecture and its remodelling during atherogenesis.

Structure and Functions of Classical Cadherins

Cadherins are a family of membrane receptors that mediate calcium-dependent homophilic cell–cell adhesion. Cadherins play a key role in the regulation of organ and tissue development during embryogenesis. In adult organisms, these proteins are responsible for formation of stable cell–cell junctions and maintenance of normal tissue structure. Disruption in expression or function of cadherins may cause uncontrolled cell migration and proliferation during tumor development. This review focuses on the structure and physiological functions of classical cadherins.

Oxidized Phospholipids Stimulate Angiogenesis Via Autocrine Mechanisms, Implicating a Novel Role for Lipid Oxidation in the Evolution of Atherosclerotic Lesions

Angiogenesis is a common feature observed in advanced atherosclerotic lesions. We hypothesized that oxidized phospholipids (OxPLs), which accumulate in atherosclerotic vessels can stimulate angiogenesis. We found that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) stimulated the formation of sprouts from endothelial cell spheroids and promoted growth of capillaries into Matrigel plugs in mice. OxPLs stimulated expression of vascular endothelial growth factor (VEGF) in vivo and in several normal and tumor cell types in vitro. In addition, OxPAPC upregulated cyclooxygenase (COX)-2 and interleukin (IL)-8. COX-2 inhibitors, as well as blocking antibodies to IL-8 suppressed activation of sprouting by OxPAPC. We conclude that OxPAPC stimulates angiogenesis via autocrine mechanisms involving VEGF, IL-8, and COX-2–generated prostanoids. Our data suggest that accumulation of OxPLs may contribute to increased growth of blood capillaries in advanced lesions, thus leading to progression and destabilization of atherosclerotic plaques.

Identification of Proteins Associating with Glycosylphosphatidylinositol- Anchored T-Cadherin on the Surface of Vascular Endothelial Cells: Role for Grp78/BiP in T-Cadherin-Dependent Cell Survival†

ABSTRACT There is scant knowledge regarding how cell surface lipid-anchored T-cadherin (T-cad) transmits signals through the plasma membrane to its intracellular targets. This study aimed to identify membrane proteins colocalizing with atypical glycosylphosphatidylinositol (GPI)-anchored T-cad on the surface of endothelial cells and to evaluate their role as signaling adaptors for T-cad. Application of coimmunoprecipitation from endothelial cells expressing c-myc-tagged T-cad and high-performance liquid chromatography revealed putative association of T-cad with the following proteins: glucose-related protein GRP78, GABA-A receptor α1 subunit, integrin β3, and two hypothetical proteins, LOC124245 and FLJ32070. Association of Grp78 and integrin β3 with T-cad on the cell surface was confirmed by surface biotinylation and reciprocal immunoprecipitation and by confocal microscopy. Use of anti-Grp78 blocking antibodies, Grp78 small interfering RNA, and coexpression of constitutively active Akt demonstrated an essential role for surface Grp78 in T-cad-dependent survival signal transduction via Akt in endothelial cells. The findings herein are relevant in the context of both the identification of transmembrane signaling partners for GPI-anchored T-cad as well as the demonstration of a novel mechanism whereby Grp78 can influence endothelial cell survival as a cell surface signaling receptor rather than an intracellular chaperone.

Oxidized Phospholipids Regulate Expression of ATF4 and VEGF in Endothelial Cells via NRF2-Dependent Mechanism: Novel Point of Convergence Between Electrophilic and Unfolded Protein Stress Pathways

Objective—The ATF4 arm of the unfolded protein response is increasingly recognized for its relevance to pathology, and in particular to angiogenic reactions. Oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, were shown to upregulate vascular endothelial growth factor (VEGF) and induce angiogenesis via an ATF4-dependent mechanism. In this study, we analyzed the mechanism of ATF4 upregulation by OxPLs and more specifically the involvement of NRF2, the major transcriptional mediator of electrophilic stress response. Methods and Results—Using reverse transcription/real-time polymerase chain reaction and Western blotting, we found that OxPLs induced upregulation of ATF4 mRNA and protein in several types of endothelial cells and that these effects were suppressed by short interfering RNA (siRNA) against NRF2. Electrophilic (iso)prostaglandins and oxidized low-density lipoprotein, similarly to OxPLs, elevated ATF4 mRNA levels in an NRF2-dependent mode. Chromatin immunoprecipitation revealed OxPL-dependent binding of NRF2 to a putative antioxidant response element site in the ATF4 gene promoter. Knockdown of NRF2 inhibited OxPL-induced elevation of VEGF mRNA and endothelial cell sprout formation. Conclusion—Our data characterize NRF2 as a positive regulator of ATF4 and identify a novel cross-talk between electrophilic and unfolded protein responses, which may play a role in stress-induced angiogenesis.

T-cadherin and signal-transducing molecules co-localize in caveolin-rich membrane domains of vascular smooth muscle cells

Cadherins are a family of cellular adhesion proteins mediating homotypic cell‐cell binding. In contrast to classical cadherins, T‐cadherin does not possess the transmembrane and cytosolic domains known to be essential for tight mechanical coupling of cells, and is instead attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. This study explores the hypothesis that T‐cadherin might function as a signal‐transducing protein. Membranes from human and rat vascular smooth muscle cells were fractionated using Triton X‐100 solubilization and density gradient centrifugation techniques. We demonstrate that T‐cadherin is enriched in a minor detergent‐insoluble low‐density membrane domain and co‐distributes with caveolin, a marker of caveolae. This domain was enriched in other GPI‐anchored proteins (CD‐59, uPA receptor) and signal‐transducing molecules (Gαs protein and Src‐family kinases), but completely excluded cell‐cell and cell‐matrix adhesion molecules (N‐cadherin and β1‐integrin). Coupling of T‐cadherin with signalling molecules within caveolae might enable cellular signal transduction.

T-cadherin protects endothelial cells from oxidative stress-induced apoptosis

In vascular tissue, T‐cadherin (T‐cad) is up‐regulated in vivo under disease conditions associated with oxidative stress and concomitant cell migration, proliferation and apoptosis/survival. Using cultures of human umbilical vein endothelial cells (HUVEC), we examined whether there is a functional relationship between oxidative stress, T‐cad expression, and cell survival status. Culture of HUVEC under conditions of oxidative stress (e.g., serum deprivation, inclusion of 2H2O2) resulted in increased T‐cad expression. Oxidative stress‐induced increases in T‐cad were inhibited by the free radical‐scavenging antioxidant, N‐acetylcysteine, and the flavin‐containing oxidase inhibitor, diphenyleneiodonium. Thus reactive oxygen species (ROS) contribute to stress‐induced elevation of T‐cad in HUVEC. Compared with control cells, HUVEC overexpressing T‐cad (T‐cad+‐HUVEC) had higher phosphorylation levels for phosphatidylinositol 3‐kinase (PI3K) target Akt and mTOR target p70S6K (survival pathway regulators), but lower levels for p38MAPK (death pathway regulator). T‐cad+‐HUVEC exposed to stress (serum‐deprivation, TNF‐α, actinomycin D, staurosporine) exhibited reduced caspase activation together with increased cell survival. Protection against stress‐induced apoptosis in T‐cad+‐HUVEC was abrogated by either PI3K‐inhibitor wortmannin or mTOR‐inhibitor rapamycin. We conclude that T‐cad overexpression in HUVEC protects against stress‐induced apoptosis through activation of the PI3K/Akt/mTOR survival signal pathway and concomitant suppression of the p38 MAPK proapoptotic pathway. ROS‐induced changes in T‐cad expression may play an important role in controlling tissue cellularity during vascular remodeling.

Integrin-linked kinase is an essential mediator for T-cadherin-dependent signaling via Akt and GSK3β in endothelial cells

Glycosylphosphatidylinositol‐anchored T‐cadherin (T‐cad) influences several parameters of angio‐genesis including endothelial cell (EC) differentiation, migration, proliferation, and survival. This presupposes signal transduction networking via mediatory regulators and molecular adaptors since T‐cad lacks transmembrane and cytosolic domains. Here, using pharmacological inhibition of PI3K, adenoviral‐mediated T‐cad‐overexpression, siRNA‐mediated T‐cad‐depletion, and agonistic antibody‐mediated ligation, we demonstrate signaling by T‐cad through PI3K‐Akt‐GSK3β pathways in EC. T‐cad‐overexpressing EC exhibited increased levels and nuclear accumulation of active β‐catenin, which was transcriptionally active as shown by increased Lef/Tcf reporter activity and cyclin D1 levels. Cotransduction of EC with constitutively active GSK3β (S9A‐GSK3β) abrogated the stimulatory effects of T‐cad on active β‐catenin accumulation, proliferation, and survival. Integrin‐linked kinase (ILK), a membrane proximal upstream regulator of Akt and GSK3β, was considered a candidate signaling mediator for T‐cad. T‐cad was present in anti‐ILK immunopre‐cipitates, and confocal microscopy revealed colocal‐ization of T‐cad and ILK within lamellipodia of migrating cells. ILK‐siRNA abolished T‐cad‐dependent effects on Ser‐473Akt/Ser‐9GSK3β phosphorylation, active β‐catenin accumulation, and survival. We conclude ILK is an essential mediator for T‐cad signaling via Akt and GSK3β in EC. This is the first demonstration that ILK can regulate inward signaling by GPI‐anchored proteins. Furthermore, ILK‐GSK3β‐dependent modulation of active β ‐catenin levels by GPI‐anchored T‐cad represents a novel mechanism for controlling cellular β‐catenin activity.—Joshi, M. B., Ivanov, D., Philippova, M., Erne, P., Resink, T. J. Integrin‐linked kinase is an essential mediator for T‐cadherin‐dependent signaling via Akt and GSK3β in endothelial cells. FASEB J. 21, 3083–3095 (2007)

Atypical GPI-Anchored T-Cadherin Stimulates Angiogenesis In Vitro and In Vivo

Objective—T-cadherin (T-cad) is an atypical GPI-anchored member of the cadherin superfamily. In vascular tissue, T-cad expression is increased during atherosclerosis, restenosis, and tumor neovascularization. In vitro, overexpression and/or homophilic ligation of T-cad on endothelial cells (ECs) facilitates migration, proliferation, and survival. This study investigated T-cad effects on angiogenesis. Methods and Results—In vitro, T-cad homophilic ligation induced arrangement of ECs into a capillary-like network in a 2-dimensional model of EC differentiation and stimulated in-gel endothelial sprout outgrowth in an EC spheroid model and a modified Nicosia tissue assay. Sprouting from spheroids composed of adenoviral-infected T-cad overexpressing ECs or T-cad siRNA transfected ECs were significantly increased or reduced, respectively. In vivo, T-cad potentiated VEGF effects on neovascularization in a model of myoblast-mediated gene transfer to mouse skeletal muscle; vessel caliber after co-delivery of T-cad and VEGF was significantly greater than after delivery of VEGF alone. Conclusions—We unequivocally identify T-cad as a novel modulator of angiogenesis and suggest that this molecule can be exploited as a target for modulation of therapeutic angiogenesis, as well as for prevention of pathological conditions associated with abnormal neovascularization.