EGFR and IGF-1R in regulation of prostate cancer cell phenotype and polarity: opposing functions and modulation by T-cadherin.

T-cadherin is an atypical glycosylphosphatidylinsoitol-anchored member of the cadherin superfamily of adhesion molecules. We found that T-cadherin overexpression in malignant (DU145) and benign (BPH-1) prostatic epithelial cell lines or silencing in the BPH-1 cell line, respectively, promoted or inhibited migration and spheroid invasion in collagen I gel and Matrigel. T-cadherin-dependent effects were associated with changes in cell phenotype: overexpression caused cell dissemination and loss of polarity evaluated by relative positioning of the Golgi/nuclei in cell groups, whereas silencing caused formation of compact polarized epithelial-like clusters. Epidermal growth factor receptor (EGFR) and IGF factor-1 receptor (IGF-1R) were identified as mediators of T-cadherin effects. These receptors per se had opposing influences on cell phenotype. EGFR activation with EGF or IGF-1R inhibition with NVP-AEW541 promoted dissemination, invasion, and polarity loss. Conversely, inhibition of EGFR with gefitinib or activation of IGF-1R with IGF-1 rescued epithelial morphology and decreased invasion. T-cadherin silencing enhanced both EGFR and IGF-1R phosphorylation, yet converted cells to the morphology typical for activated IGF-1R. T-cadherin effects were sensitive to modulation of EGFR or IGF-1R activity, suggesting direct involvement of both receptors. We conclude that T-cadherin regulates prostate cancer cell behavior by tuning the balance in EGFR/IGF-1R activity and enhancing the impact of IGF-1R.

Paradoxical effects of T-cadherin on squamous cell carcinoma: up- and down-regulation increase xenograft growth by distinct mechanisms.

Mechanisms underlying cutaneous squamous cell carcinoma (SCC) tumour growth and invasion are incompletely understood. Our previous pathological and in vitro studies suggest that cell surface glycoprotein T-cadherin (T-cad) might be a controlling determinant of the behaviour of SCC. Here we used a murine xenograft model to determine whether T-cad modulates SCC tumour progression in vivo. Silencing or up-regulation of T-cad in A431 (shTcad or Tcad(+) , respectively) both resulted in increased tumour expansion in vivo. To explain this unanticipated outcome, we focused on proliferation, apoptosis and angiogenesis/lymphangiogenesis, which are important determinants of the progression of solid tumours in vivo. shTcad exhibited enhanced proliferation potential in vitro and in vivo, and their signalling response to EGF was characterized by a higher Erk1/2:p38MAPK activity ratio, which has been correlated with more aggressive tumour growth. T-cad over-expression did not affect proliferation but staining for cleaved caspase 3 revealed a minimal occurrence of extensive apoptosis in Tcad(+) tumours. Immunofluoresence staining of xenograft sections revealed increased intra-tumoural total microvessel (CD31(+)) and lymphatic vessel (LYVE-1(+)) densities in Tcad(+) tumours. shTcad tumours exhibited decreased microvessel and lymphatic densities. Tcad(+) expressed higher levels of transcripts for VEGF-A, VEGF-C and VEGF-D in vitro and in vivo. Culture supernatants collected from Tcad(+) enhanced sprout outgrowth from spheroids composed of either microvascular or lymphatic endothelial cells, and these in vitro angiogenic and lymphangiogenic responses were abrogated by inclusion of neutralizing VEGF antibodies. We conclude that T-cad can exert pleiotropic effects on SCC progression; up- or down-regulation of T-cad can promote SCC tumour expansion in vivo but through distinct mechanisms, namely enhancement of angio/lymphangiogenic potential or enhancement of proliferation capacity.

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 Expressing Cells in the Stromal Vascular Fraction of Human Adipose Tissue: Role in Osteogenesis and Angiogenesis.

Cells of the stromal vascular fraction (SVF) of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, cultured adipose-derived stromal cells (ASCs), even after minimal monolayer expansion, lose osteogenic capacity in vivo. Communication between endothelial and stromal/mesenchymal cell lineages has been suggested to improve bone formation and vascularization by engineered tissues. Here, we investigated the specific role of a subpopulation of SVF cells positive for T-cadherin (T-cad), a putative endothelial marker. We found that maintenance during monolayer expansion of a T-cad-positive cell population, composed of endothelial lineage cells (ECs), is mandatory to preserve the osteogenic capacity of SVF cells in vivo and strongly supports their vasculogenic properties. Depletion of T-cad-positive cells from the SVF totally impaired bone formation in vivo and strongly reduced vascularization by SVF cells in association with decreased VEGF and Adiponectin expression. The osteogenic potential of T-cad-depleted SVF cells was fully rescued by co-culture with ECs from a human umbilical vein (HUVECs), constitutively expressing T-cad. Ectopic expression of T-cad in ASCs stimulated mineralization in vitro but failed to rescue osteogenic potential in vivo, indicating that the endothelial nature of the T-cad-positive cells is the key factor for induction of osteogenesis in engineered grafts based on SVF cells. This study demonstrates that crosstalk between stromal and T-cad expressing endothelial cells within adipose tissue critically regulates osteogenesis, with VEGF and adiponectin as associated molecular mediators.

Invariant natural killer T cells: linking inflammation and neovascularization in human atherosclerosis.

Atherosclerosis, a chronic inflammatory lipid storage disease of large arteries, is complicated by cardiovascular events usually precipitated by plaque rupture or erosion. Inflammation participates in lesion progression and plaque rupture. Identification of leukocyte populations involved in plaque destabilization is important for effective prevention of cardiovascular events. This study investigates CD1d-expressing cells and invariant NKT cells (iNKT) in human arterial tissue, their correlation with disease severity and symptoms, and potential mechanisms for their involvement in plaque formation and/or destabilization. CD1d-expressing cells were present in advanced plaques in patients who suffered from cardiovascular events in the past and were most abundant in plaques with ectopic neovascularization. Confocal microscopy detected iNKT cells in plaques, and plaque-derived iNKT cell lines promptly produced proinflammatory cytokines when stimulated by CD1d-expressing APC-presenting α-galactosylceramide lipid antigen. Furthermore, iNKT cells were diminished in the circulating blood of patients with symptomatic atherosclerosis. Activated iNKT cell-derived culture supernatants showed angiogenic activity in a human microvascular endothelial cell line HMEC-1-spheroid model of in vitro angiogenesis and strongly activated human microvascular endothelial cell line HMEC-1 migration. This functional activity was ascribed to IL-8 released by iNKT cells upon lipid recognition. These findings introduce iNKT cells as novel cellular candidates promoting plaque neovascularization and destabilization in human atherosclerosis.

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.

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 (DeltaI, DeltaII, DeltaIII, DeltaIV, DeltaV). Empty (E) lentivector-transduced cells served as control. Similarly to overexpression of native T-cad, cells expressing DeltaII, DeltaIII, or DeltaIV displayed elevated levels of p-Akt and p-GSK3beta and increased proliferation rates (for DeltaII, DeltaIII) vs. E. DeltaI- and DeltaV-transduced cells exhibited reduced levels of p-Akt and p-GSK3beta and retarded growth rates vs. E. Stimulatory effects of native T-cad overexpression on Akt and GSK3beta phosphorylation were dose dependently inhibited by coexpression of DeltaI or DeltaV. Subsequent functional analyses compared only DeltaI-, DeltaII-, and DeltaV-mutant constructs with E as a negative control. Unlike DeltaII cells, DeltaI and DeltaV cells failed to exhibit homophilic ligation and deadhesion responses on a substratum of T-cad protein. In the wound assay, migration was increased for DeltaII cells but impaired for DeltaI and DeltaV cells. In endothelial cell-spheroid assay, angiogenic sprouting was augmented for DeltaII cells but inhibited for DeltaI and DeltaV cells. We conclude that EC1 and EC5 domains of T-cad are essential for its proangiogenic effects. DeltaI and DeltaV constructs may serve as dominant-negative mutants and as potential tools targeting excessive angiogenesis.

Cadherins and cardiovascular disease.

Cardiovascular diseases encompass an enormous range of conditions arising through an equally diverse aetiology. The cadherin superfamily of cell surface adhesion molecules have long been recognised for their crucial roles in morphogenesis and controlled growth and turnover in adult tissues. Thus, their involvement in the development of cardiovascular diseases characterised by tissue remodelling can be predicted. However, given the diversity of cadherins expressed on resident cells in cardiac and vascular tissue and their assorted and frequently overlapping functions that extend beyond mere mediation of adhesive interactions, definition of specific roles in the progression of cardiovascular diseases can be confounding. Compared with the fields of embryogenesis and oncology, investigations targeted specifically toward delineation of the participation of cadherins in cardiovascular disease are remarkably scant. In this article we offer the reader a brief introduction to members of the cadherin superfamily, and review the involvement of cadherins in cardiac diseases (dilated and dysplastic cardiomyopathies) and vascular diseases (atherosclerosis and restenosis) in which prominent alterations in tissue architecture occur and ultimately cause the clinical manifestations and complications of the diseases. Putative functions of the different cadherins expressed in cardiomyocytes, smooth muscle cells and endothelial cells are discussed.

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.

Analysis of fragmented oxidized phosphatidylcholines in human plasma using mass spectrometry: Comparison with immune assays.

Circulating oxidized phospholipids are increasingly recognized as biomarkers of atherosclerosis. Clinical association studies have been mainly performed using an immune assay based on monoclonal antibody E06, which recognizes a variety of molecular species of oxidized phosphatidylcholine (OxPC) in lipoproteins, cell membranes or covalently bound to plasma proteins. Accumulating evidence shows that individual molecular species of OxPC demonstrate different biological activities and have different half-life times. Therefore, it is likely that certain molecular species can be associated with pathology more strongly than others. This hypothesis can only be tested using LC-MS/MS allowing quantification of individual molecular species of OxPCs. In order to ensure that laborious LC-MS/MS methods do not simply replicate the results of a technically simpler E06-OxPCs assay, we have performed relative quantification of 8 truncated molecular species of OxPCs in plasma of 132 probands and compared the data with the results of the E06-OxPCs and OxLDL assays. We have found a strong correlation between individual molecular species of OxPCs but only a weak correlation of LC-MS/MS-OxPCs data with the E06-OxPCs assay and no correlation with the OxLDL assay. Furthermore, in contrast to the results of E06-OxPCs or OxLDL assays, 7 out of 8 OxPC species were associated with hypertension. The data suggest that the results of the LC-MS/MS-OxPCs assay do not replicate the results of two ELISA-based lipid oxidation tests and therefore may produce additional diagnostic information. These findings necessitate development of simplified mass spectrometric procedures for high-throughput and affordable analysis of selected molecular species of OxPCs.

Integrin-linked kinase is an essential mediator for T-cadherin-dependent signaling via Akt and GSK3beta in endothelial cells.

Glycosylphosphatidylinositol-anchored T-cadherin (T-cad) influences several parameters of angiogenesis 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-GSK3beta pathways in EC. T-cad-overexpressing EC exhibited increased levels and nuclear accumulation of active beta-catenin, which was transcriptionally active as shown by increased Lef/Tcf reporter activity and cyclin D1 levels. Cotransduction of EC with constitutively active GSK3beta (S9A-GSK3beta) abrogated the stimulatory effects of T-cad on active beta-catenin accumulation, proliferation, and survival. Integrin-linked kinase (ILK), a membrane proximal upstream regulator of Akt and GSK3beta, was considered a candidate signaling mediator for T-cad. T-cad was present in anti-ILK immunoprecipitates, and confocal microscopy revealed colocalization of T-cad and ILK within lamellipodia of migrating cells. ILK-siRNA abolished T-cad-dependent effects on (Ser-473)Akt/(Ser-9)GSK3beta phosphorylation, active beta-catenin accumulation, and survival. We conclude ILK is an essential mediator for T-cad signaling via Akt and GSK3beta in EC. This is the first demonstration that ILK can regulate inward signaling by GPI-anchored proteins. Furthermore, ILK-GSK3beta-dependent modulation of active beta-catenin levels by GPI-anchored T-cad represents a novel mechanism for controlling cellular beta-catenin activity.

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.

Smooth muscle cell-driven vascular diseases and molecular mechanisms of VSMC plasticity.

Vascular smooth muscle cells (VSMCs) are the major cell type in blood vessels. Unlike many other mature cell types in the adult body, VSMC do not terminally differentiate but retain a remarkable plasticity. Fully differentiated medial VSMCs of mature vessels maintain quiescence and express a range of genes and proteins important for contraction/dilation, which allows them to control systemic and local pressure through the regulation of vascular tone. In response to vascular injury or alterations in local environmental cues, differentiated/contractile VSMCs are capable of switching to a dedifferentiated phenotype characterized by increased proliferation, migration and extracellular matrix synthesis in concert with decreased expression of contractile markers. Imbalanced VSMC plasticity results in maladaptive phenotype alterations that ultimately lead to progression of a variety of VSMC-driven vascular diseases. The nature, extent and consequences of dysregulated VSMC phenotype alterations are diverse, reflecting the numerous environmental cues (e.g. biochemical factors, extracellular matrix components, physical) that prompt VSMC phenotype switching. In spite of decades of efforts to understand cues and processes that normally control VSMC differentiation and their disruption in VSMC-driven disease states, the crucial molecular mechanisms and signalling pathways that shape the VSMC phenotype programme have still not yet been precisely elucidated. In this article we introduce the physiological functions of vascular smooth muscle/VSMCs, outline VSMC-driven cardiovascular diseases and the concept of VSMC phenotype switching, and review molecular mechanisms that play crucial roles in the regulation of VSMC phenotypic plasticity.

Cadherins in vascular smooth muscle cell (patho)biology: Quid nos scimus?

Vascular smooth muscle cells (SMCs) phenotypes span a reversible continuum from quiescent/contractile (differentiated) to proliferative/synthetic (dedifferentiated) enabling them to perform a diversity of functions that are context-dependent and important for vascular tone-diameter homeostasis, vasculogenesis, angiogenesis or vessel reparation after injury. Dysregulated phenotype modulation and failure to maintain/regain the mature differentiated and contractile phenotypic state is pivotal in the development of vascular diseases such as atherosclerosis and restenosis after angioplasty and coronary bypass grafting. Many functions of SMCs such as adhesion, migration, proliferation, contraction, differentiation and apoptosis are regulated by a broad spectrum of cell-cell and cell-matrix adhesion molecules. Cadherins represent a superfamily of cell surface homophilic adhesion molecules with fundamental roles in morphogenetic and differentiation processes during development and in the maintenance of tissue integrity and homeostasis in adults. The cadherins have major inputs on signalling pathways and cytoskeletal assemblies that participate in regulating processes such as cell polarity, migration, proliferation, survival, phenotype and differentiation. Abnormalities in these processes have long been recognized to underlie pathological SMC-driven reparation, but knowledge on the involvement of cadherins is remarkably limited. This article presents a comprehensive review of cadherin family members currently identified on vascular SMCs in relation to their functions, molecular mechanisms of action and relevance for vascular pathology.

Actin cytoskeleton regulates functional anchorage-migration switch during T-cadherin-induced phenotype modulation of vascular smooth muscle cells.

Vascular smooth muscle cell (SMC) switching between differentiated and dedifferentiated phenotypes is reversible and accompanied by morphological and functional alterations that require reconfiguration of cell-cell and cell-matrix adhesion networks. Studies attempting to explore changes in overall composition of the adhesion nexus during SMC phenotype transition are lacking. We have previously demonstrated that T-cadherin knockdown enforces SMC differentiation, whereas T-cadherin upregulation promotes SMC dedifferentiation. This study used human aortic SMCs ectopically modified with respect to T-cadherin expression to characterize phenotype-associated cell-matrix adhesion molecule expression, focal adhesions configuration and migration modes. Compared with dedifferentiated/migratory SMCs (expressing T-cadherin), the differentiated/contractile SMCs (T-cadherin-deficient) exhibited increased adhesion to several extracellular matrix substrata, decreased expression of several integrins, matrix metalloproteinases and collagens, and also distinct focal adhesion, adherens junction and intracellular tension network configurations. Differentiated and dedifferentiated phenotypes displayed distinct migrational velocity and directional persistence. The restricted migration efficiency of the differentiated phenotype was fully overcome by reducing actin polymerization with ROCK inhibitor Y-27632 whereas myosin II inhibitor blebbistatin was less effective. Migration efficiency of the dedifferentiated phenotype was diminished by promoting actin polymerization with lysophosphatidic acid. These findings held true in both 2D-monolayer and 3D-spheroid migration models. Thus, our data suggest that despite global differences in the cell adhesion nexus of the differentiated and dedifferentiated phenotypes, structural actin cytoskeleton characteristics per se play a crucial role in permissive regulation of cell-matrix adhesive interactions and cell migration behavior during T-cadherin-induced SMC phenotype transition.

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.

Pleiotropic effects of oxidized phospholipids.

Oxidized phospholipids (OxPLs) are increasingly recognized to play a role in a variety of normal and pathological states. OxPLs were implicated in regulation of inflammation, thrombosis, angiogenesis, endothelial barrier function, immune tolerance and other important processes. Rapidly accumulating evidence suggests that OxPLs are biomarkers of atherosclerosis and other pathologies. In addition, successful application of experimental drugs based on structural scaffold of OxPLs in animal models of inflammation was recently reported. This review briefly summarizes current knowledge on generation, methods of quantification and biological activities of OxPLs. Furthermore, receptor and cellular mechanisms of these effects are discussed. The goal of the review is to give a broad overview of this class of lipid mediators inducing pleiotropic biological effects.

T-cadherin in prostate cancer: relationship with cancer progression, differentiation and drug resistance.

Prostate cancer represents the second leading cause of cancer-related death in men. T-cadherin (CDH13) is an atypical GPI-anchored member of the cadherin family of adhesion molecules. Its gene was reported to be downregulated in a small series of prostate tumours. T-cadherin protein expression/localisation in prostate tissue has never been investigated. The purpose of our study was to analyse CDH13 gene and protein levels in large sets of healthy and cancer prostate tissue specimens and evaluate CDH13 effects on the sensitivity of prostate cancer cells to chemotherapy. Analysis of CDH13 gene expression in the TCGA RNAseq dataset for prostate adenocarcinoma (N = 550) and in tissue samples (N = 101) by qPCR revealed weak positive correlation with the Gleason score in cancer and no difference between benign and malignant specimens. Immunohistochemical analysis of tissue sections (N = 12) and microarrays (N = 128 specimens) demonstrated the presence of CDH13 on the apical surface and at intercellular contacts of cytokeratin 8-positive luminal cells and cells double-positive for cytokeratin 8 and basal marker p63. T-cadherin protein expression was markedly upregulated in cancer as compared to benign prostate hyperplasia, the increase being more prominent in organ-confined than in advanced hormone-resistant tumours, and correlated negatively with the Gleason pattern. T-cadherin protein level correlated strongly with cytokeratin 8 and with an abnormal diffuse/membrane localisation pattern of p63. Ectopic expression of CDH13 in metastatic prostate cancer cell line DU145 reduced cell growth in the presence of doxorubicin. We conclude that CDH13 protein, but not its gene expression, is strongly upregulated in early prostate cancer, correlates with changes in luminal/basal differentiation and p63 localisation, and promotes sensitivity of cancer cells to doxorubicin. These data identify CDH13 as a novel molecule relevant for prostate cancer progression and response to therapy.

T-cadherin promotes autophagy and survival in vascular smooth muscle cells through MEK1/2/Erk1/2 axis activation.

Autophagy is an evolutionary conserved intracellular catabolic process of vital importance to cell and tissue homeostasis. Autophagy is implicated in the pathogenesis of atherosclerosis but participating cells, molecular mechanisms and functional outcomes have not been fully elucidated. T-cadherin, an atypical glycosylphosphatidylinositol-anchored member of the cadherin superfamily of adhesion molecules, is upregulated on smooth muscle cells (SMCs)1 in atherosclerotic lesions. Here, using rat and murine aortic SMCs as experimental models, we surveyed the ability of T-cadherin to regulate autophagy in SMCs during serum-starvation stress. Ectopic upregulation of T-cadherin in SMCs resulted in augmented autophagy characterized by increased autophagic flux, LC3-II abundance and autophagosome formation. Analysis of signal transduction pathway effectors and use of specific pharmacological inhibitors demonstrated that T-cadherin-associated enhancement of the autophagic response to serum-deprivation was dependent on MEK1/2/Erk1/2 activation and independent of PI3K/Akt/mTORC1, reactive oxygen species or endoplasmic reticulum stress. T-cadherin upregulation on SMCs conferred a survival advantage during prolonged serum-starvation which was sensitive to inhibition of MEK1/2/Erk1/2 by PD98059 or UO126 and to blockade of autophagy by chloroquine. Loss of T-cadherin expression in SMCs diminished autophagy responsiveness and compromised survival under conditions of serum-starvation. Overall our findings have identified T-cadherin as a novel positive regulator of autophagy and survival in SMCs.

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.