Danila Ivanov

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.

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.

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.

RhoA and Rac mediate endothelial cell polarization and detachment induced by T-cadherin

T‐cadherin (T‐cad) is an atypical GPI‐anchored member of the cadherin superfamily. Ligation of T‐cad receptors on endothelial cells prevents cell spreading, promotes elongation and polarization, decreases adhesion to the matrix, and facilitates migration. This study investigates involvement of Rho GTPases in T‐cad signaling. Human umbilical vein endothelial cells were infected with adenoviral vectors expressing dominant‐negative and/or constitutively active mutants of RhoA (N19RhoA/RhoA63), ROCK (RB/PH(TT)/CAT), and Rac1 (N17RAC). Mutant‐infected and empty vector‐infected cells were compared with respect to their ability to detach and polarize when plated on substratum containing recombinant T‐cad protein used as a ligand mimicking homophilic T‐cad interactions. ROCK involvement was also studied using specific inhibitor Y‐27632. Adhesion assays, analysis of cell phenotype, and actin cytoskeleton organization using TRITC‐labeled phalloidin demonstrated that T‐cad‐induced cell polarization includes two complementary components: RhoA/ROCK pathway is necessary for cell contraction, stress fiber assembly, and inhibition of spreading, whereas Rac is required for formation of actin‐rich lamellipodia at the leading edges of polarized cells. Individual repression of either pathway only partially prevented cell polarization and detachment, while simultaneous repression of RhoA and Rac pathways fully eliminated responses to homophilic T‐cad ligation. In conclusion, these data suggest that T‐cad induces cell deadhesion and polarization via RhoA‐ROCK‐ and Rac‐dependent mechanisms.

Polarisation of T-cadherin to the leading edge of migrating vascular cells in vitro: a function in vascular cell motility?

Both histological and in vitro studies indicate a relationship between T-cadherin levels and acquisition of a modulated, migratory phenotype by vascular cells. This study further examines a role for T-cadherin in relation to cell migration and adhesion. Fluorescence microscopic examination of T-cadherin localisation in confluent cultures of human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells and the human carcinoma cell line ECV-304 revealed global distribution over the entire cell body, and with only slight enrichment at cell borders. This contrasts with restricted cell–cell junction localisation of classical cadherin (for example, VE-cadherin in HUVEC). In wounded cultures, T-cadherin polarised to the leading edge of cells migrating into the wound area, again contrasting with classical VE-cadherin, which was undetectable in this region. Confocal microscopy demonstrated that potential signalling functions of T-cadherin at the leading edge are unrelated to physical interactions with caveolin. Adherence of HUVEC onto a monolayer of T-cadherin-transfected L929 cells is significantly reduced compared with adhesion onto control (T-cadherin-negative) L929. Thus T-cadherin is not required for maintenance of intercellular adhesion, but may rather function as a signalling molecule involved in cell–cell recognition and sensing of the environment in processes where cell detachment occurs.

LDL induces intracellular signalling and cell migration via atypical LDL-binding protein T-cadherin.

Cadherins are a superfamily of adhesion molecules that mediate Ca2+-dependent cell–cell adhesion. T-cadherin (T-cad), a unique glycosylphosphatidylinositol-anchored member of the cadherin superfamily, was initially identified by immunoblotting of vascular cell membranes as an atypical low affinity low density lipoprotein (LDL)-binding protein. It is not known whether this heterophilic interaction is physiologically relevant. Expression of T-cadherin is upregulated in vascular cells during atherosclerosis, restenosis and tumour angiogenesis, conditions characterized by enhanced cell migration and growth. Elevated levels of serum low density lipoproteins (LDL), which result in cholesterol accumulation in vascular wall, is a widely accepted risk factor in atherosclerosis development. Additionally to its metabolic effects, LDL can produce hormone-like effects in a number of cell types. This study has utilized HEK293 cells and L929 cells stably transfected with T-cadherin cDNA to investigate T-cad-dependent responses to LDL. Stable expression of T-cad in both HEK293 and L929 cells results in significantly (p < 0.05) elevated specific surface binding of [I125]-LDL. Compared with mock-transfectants, cells expressing T-cad exhibit significantly (p < 0.01) enhanced LDL-induced mobilization of intracellular Ca2+-stores and a significantly (p < 0.01) increased migration toward an LDL gradient (0.1% BSA + 60 μg/ml LDL) in Boyden chamber migration assay. Thus LDL-binding to T-cad is capable of activating physiologically relevant intracellular signaling and functional responses.

A requirement for thioredoxin in redox-sensitive modulation of T-cadherin expression in endothelial cells

T-cad (T-cadherin), a glycosylphosphatidylinositol-anchored cadherin superfamily member, is expressed widely in the brain and cardiovascular system, and absent, decreased, or even increased, in cancers. Mechanisms controlling T-cad expression are poorly understood. The present study investigated transcriptional regulation of T-cad in ECs (endothelial cells). Conditions of oxidative stress (serum-deprivation or presence of H(2)O(2)) elevate T-cad mRNA and protein levels in ECs. Reporter gene analysis, using serially deleted T-cad promoter stretches ranging from -99 to -2304 bp, located the minimal promoter region of T-cad within -285 bp from the translation start site. Reporter activity in ECs transfected with the -285 bp construct increased under conditions of oxidative stress, and this was normalized by antioxidant N-acetylcysteine. An electrophoretic-mobility-shift assay revealed a specific nucleoprotein complex unique to -156 to -203 bp, which increased when nuclear extracts from oxidatively stressed ECs were used, suggesting the presence of redox-sensitive binding element(s). MS analysis of the nucleoprotein complex unique to -156 to -203 bp after streptavidin-agarose pull-down detected the presence of the redox-active protein thioredoxin. The presence of thioredoxin-1 in a nuclear extract from oxidatively stressed ECs was demonstrated after immunoprecipitation and immunoblotting. Transfection of ECs with thioredoxin-1 small interfering RNA abrogated oxidative-stress-induced up-regulation of T-cad transcripts and protein. We conclude that thioredoxin-1 is an important determinant of redox-sensitive transcriptional up-regulation of T-cad in ECs.