Manjunath B. Joshi

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.

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)

T-cadherin is present on endothelial microparticles and is elevated in plasma in early atherosclerosis

AIMS The presence of endothelial cell (EC)-derived surface molecules in the circulation is among hallmarks of endothelial activation and damage in vivo. Previous investigations suggest that upregulation of T-cadherin (T-cad) on the surface of ECs may be a characteristic marker of EC activation and stress. We investigated whether T-cad might also be shed from ECs and in amounts reflecting the extent of activation or damage. METHODS AND RESULTS Immunoblotting showed the presence of T-cad protein in the culture medium from normal proliferating ECs and higher levels in the medium from stressed/apoptotic ECs. Release of T-cad into the circulation occurs in vivo and in association with endothelial dysfunction. Sandwich ELISA revealed negligible T-cad protein in the plasma of healthy volunteers (0.90 ± 0.90 ng/mL, n = 30), and increased levels in the plasma from patients with non-significant atherosclerosis (9.23 ± 2.61 ng/mL, n = 63) and patients with chronic coronary artery disease (6.93 ± 1.31 ng/mL, n = 162). In both patient groups there was a significant (P = 0.043) dependency of T-cad and degree of endothelial dysfunction as measured by reactive hyperaemia peripheral tonometry. Flow cytometry analysis showed that the major fraction of T-cad was released into the EC culture medium and the plasma as a surface component of EC-derived annexin V- and CD144/CD31-positive microparticles (MPs). Gain-of-function and loss-of-function studies demonstrate that MP-bound T-cad induced Akt phosphorylation and activated angiogenic behaviour in target ECs via homophilic-based interactions. CONCLUSION Our findings reveal a novel mechanism of T-cad-dependent signalling in the vascular endothelium. We identify T-cad as an endothelial MP antigen in vivo and demonstrate that its level in plasma is increased in early atherosclerosis and correlates with endothelial dysfunction.

T-cadherin attenuates insulin-dependent signalling, eNOS activation, and angiogenesis in vascular endothelial cells

AIMS T-cadherin (T-cad) is a glycosylphosphatidylinositol-anchored cadherin family member. Experimental, clinical, and genomic studies suggest a role for T-cad in vascular disorders such as atherosclerosis and hypertension, which are associated with endothelial dysfunction and insulin resistance (InsRes). In endothelial cells (EC), T-cad and insulin activate similar signalling pathways [e.g. PI3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)] and processes (e.g. angiogenesis). We hypothesize that T-cad is a regulatory component of insulin signalling in EC and therefore a determinant of the development of endothelial InsRes. METHODS AND RESULTS We investigated T-cad-dependent effects on insulin sensitivity using human EC stably transduced with respect to T-cad overexpression or T-cad silencing. Responsiveness to insulin was examined at the level of effectors of the insulin signalling cascade, EC nitric oxide synthase (eNOS) activation, and angiogenic behaviour. Overexpression and ligation of T-cad on EC attenuates insulin-dependent activation of the PI3K/Akt/mTOR signalling axis, eNOS, EC migration, and angiogenesis. Conversely, T-cad silencing enhances these actions of insulin. Attenuation of EC responsiveness to insulin results from T-cad-mediated chronic activation of the Akt/mTOR-dependent negative feedback loop of the insulin cascade and enhanced degradation of the insulin receptor (IR) substrate. Co-immunoprecipitation experiments revealed an association between T-cad and IR. Filipin abrogated inhibitory effects of T-cad on insulin signalling, demonstrating localization of T-cad-insulin cross-talk to lipid raft plasma membrane domains. Hyperinsulinaemia up-regulates T-cad mRNA and protein levels in EC. CONCLUSION T-cad expression modulates signalling and functional responses of EC to insulin. We have identified a novel signalling mechanism regulating insulin function in the endothelium and attribute a role for T-cad up-regulation in the pathogenesis of endothelial InsRes.

T-cadherin attenuates the PERK branch of the unfolded protein response and protects vascular endothelial cells from endoplasmic reticulum stress-induced apoptosis

Endoplasmic reticulum (ER) stress activated by perturbations in ER homeostasis induces the unfolded protein response (UPR) with chaperon Grp78 as the key activator of UPR signalling. The aim of UPR is to restore normal ER function; however prolonged or severe ER stress triggers apoptosis of damaged cells to ensure protection of the whole organism. Recent findings support an association of ER stress-induced apoptosis of vascular cells with cardiovascular pathologies. T-cadherin (T-cad), an atypical glycosylphosphatidylinositol-anchored member of the cadherin superfamily is upregulated in atherosclerotic lesions. Here we investigate the ability of T-cad to influence UPR signalling and endothelial cell (EC) survival during ER stress. EC were treated with a variety of ER stress-inducing compounds (thapsigargin, dithiothereitol, brefeldin A, tunicamycin, A23187 or homocysteine) and induction of ER stress validated by increases in levels of UPR signalling molecules Grp78 (glucose-regulated protein of 78kDa), phospho-eIF2alpha (phosphorylated eukaryotic initiation factor 2alpha) and CHOP (C/EBP homologous protein). All compounds also increased T-cad mRNA and protein levels. Overexpression or silencing of T-cad in EC respectively attenuated or amplified the ER stress-induced increase in phospho-eIF2alpha, Grp78, CHOP and active caspases. Effects of T-cad-overexpression or T-cad-silencing on ER stress responses in EC were not affected by inclusion of either N-acetylcysteine (reactive oxygen species scavenger), LY294002 (phosphatidylinositol-3-kinase inhibitor) or SP6000125 (Jun N-terminal kinase inhibitor). The data suggest that upregulation of T-cad on EC during ER stress attenuates the activation of the proapoptotic PERK (PKR (double-stranded RNA-activated protein kinase)-like ER kinase) branch of the UPR cascade and thereby protects EC from ER stress-induced apoptosis.

BRN2 is a transcriptional repressor of CDH13 (T-cadherin) in melanoma cells

T-cadherin (cadherin 13, H-cadherin, gene name CDH13) has been proposed to act as a tumor-suppressor gene as its expression is significantly diminished in several types of carcinomas, including melanomas. Allelic loss and promoter hypermethylation have been proposed as mechanisms for silencing of CDH13. However, they do not account for loss of T-cadherin expression in all carcinomas, and other genetic or epigenetic alterations can be presumed. The present study investigated transcriptional regulation of CDH13 in melanoma. Bioinformatical analysis pointed to the presence of known BRN2 (also known as POU3F2 and N-Oct-3)-binding motifs in the CDH13 promoter sequence. We found an inverse correlation between BRN2 and T-cadherin protein and transcript expression. Reporter gene analysis and electrophoretic mobility shift assays in melanoma cells demonstrated that CDH13 is a direct target of BRN2 and that BRN2 is a functional transcriptional repressor of CDH13 promoter activity. The regulatory binding element of BRN2 was located −219 bp of the CDH13 promoter proximal to the start codon and was identified as 5′-CATGCAAAA-3′. Ectopic expression of BRN2 in BRN2-negative/T-cadherin-positive melanoma cells resulted in suppression of CDH13 promoter activity, whereas BRN2 knockdown in BRN2-positive/T-cadherin-negative melanoma cells resulted in re-expression of T-cadherin transcripts and protein. Transcriptional repression of CDH13 by BRN2 may participate in malignant transformation of melanoma by increasing invasion and migration potentials of melanoma cells. The study has identified CDH13 as a novel direct BRN2 transcriptional target gene and has advanced knowledge of mechanisms underlying loss of T-cadherin expression in melanoma.

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.

Extracellular cadherin repeat domains EC1 and EC5 of T-cadherin are essential for its ability to stimulate angiogenic behavior of endothelial cells

T‐cadherin (T‐cad) promotes survival, proliferation, and migration of endothelial cells and induces angiogenesis. We aimed to identify domains of T‐cad functionally relevant to its effects on endothelial cell behavior. To specifically target the functional properties of the 5 cadherin repeat domains (EC1–EC5) of T‐cad, endothelial cells were transduced with lentivectors containing specific T‐cad‐domain‐deletion mutant constructs (ΔΙ, ΔΠ, ΔΠΙ, ΔIV, ΔV). Empty (E) lentivector‐transduced cells served as control. Similarly to overexpression of native T‐cad, cells expressing ΔΙΙ, ΔΙΙΙ, or ΔΙV displayed elevated levels of p‐Akt and p‐GSK3β and increased proliferation rates (for ΔΙΙ, ΔΙΙΙ) vs. E. ΔΙ‐ and ΔV‐transduced cells exhibited reduced levels of p‐Akt and p‐GSK3β and retarded growth rates vs. E. Stimulatory effects of native T‐cad overexpression on Akt and GSK3β phosphorylation were dose dependently inhibited by coexpression of ΔΙ or ΔV. Subsequent functional analyses compared only ΔΙ‐, ΔΙΙ‐, and ΔV‐mutant constructs with E as a negative control. Unlike ΔΙΙ cells, ΔΙ and ΔV cells failed to exhibit homophilic ligation and deadhesion responses on a substratum of T‐cad protein. In the wound assay, migration was increased for ΔΙΙ cells but impaired for ΔΙ and ΔV cells. m endothelial cell‐spheroid assay, angiogenic sprouting was augmented for ΔΙΙ cells but inhibited for ΔΙ and ΔV cells. We conclude that EC1 and EC5 domains of T‐cad are essential for its proan‐giogenic effects. ΔΙ and ΔV constructs may serve as dominant‐negative mutants and as potential tools targeting excessive angiogenesis.—Joshi, M. B., Kyriakakis, E., Pfaff, D., Rupp, K., Philippova, M., Erne, P., Resink, T. J. Extracellular cadherin repeat domains EC1 and EC5 of T‐cadherin are essential for its ability to stimulate angiogenic behavior of endothelial cells. FASEBJ. 23, 4011–4021 (2009). www.fasebj.org