RESUMEN
Pericytes are mural cells that play an important role in regulation of angiogenesis and endothelial function. Cadherins are a superfamily of adhesion molecules mediating Ca2+-dependent homophilic cell-cell interactions that control morphogenesis and tissue remodeling. To date, classical N-cadherin is the only cadherin described on pericytes. Here, we demonstrate that pericytes also express T-cadherin (H-cadherin, CDH13), an atypical glycosyl-phosphatidylinositol (GPI)-anchored member of the superfamily that has previously been implicated in regulation of neurite guidance, endothelial angiogenic behavior, and smooth muscle cell differentiation and progression of cardiovascular disease. The aim of the study was to investigate T-cadherin function in pericytes. Expression of T-cadherin in pericytes from different tissues was performed by immunofluorescence analysis. Using lentivirus-mediated gain-of-function and loss-of-function in cultured human pericytes, we demonstrate that T-cadherin regulates pericyte proliferation, migration, invasion, and interactions with endothelial cells during angiogenesis in vitro and in vivo. T-cadherin effects are associated with the reorganization of the cytoskeleton, modulation of cyclin D1, α-smooth muscle actin (αSMA), integrin ß3, metalloprotease MMP1, and collagen expression levels, and involve Akt/GSK3ß and ROCK intracellular signaling pathways. We also report the development of a novel multiwell 3-D microchannel slide for easy analysis of sprouting angiogenesis from a bioengineered microvessel in vitro. In conclusion, our data identify T-cadherin as a novel regulator of pericyte function and support that it is required for pericyte proliferation and invasion during active phase of angiogenesis, while T-cadherin loss shifts pericytes toward the myofibroblast state rendering them unable to control endothelial angiogenic behavior.
Asunto(s)
Células Endoteliales , Pericitos , Humanos , Pericitos/metabolismo , Células Endoteliales/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Morfogénesis , Neovascularización FisiológicaRESUMEN
Modeling chronic cortical demyelination allows the study of long-lasting pathological changes observed in multiple sclerosis such as failure of remyelination, chronically disturbed functions of oligodendrocytes, neurons and astrocytes, brain atrophy and cognitive impairments. We aimed at generating an animal model for studying the consequences of chronic cortical demyelination and meningeal inflammation. To induce long-lasting cortical demyelination and chronic meningeal inflammation, we immunized female Lewis rats against myelin oligodendrocyte glycoprotein (MOG) and injected lentiviruses for continuing overexpression of the cytokines TNFα and IFNγ in the cortical brain parenchyma. Immunization with MOG and overexpression of TNFα and IFNγ led to widespread subpial demyelination and meningeal inflammation that were stable for at least 10 weeks. We demonstrate here that immunization with MOG is necessary for acute as well as chronic cortical demyelination. In addition, long-lasting overexpression of TNFα and IFNγ in the brain parenchyma is sufficient to induce chronic meningeal inflammation. Our model simulates key features of chronic cortical demyelination and inflammation, reminiscent of human multiple sclerosis pathology. This will allow molecular, cellular and functional investigations for a better understanding of the adaptation mechanisms of the cerebral cortex in multiple sclerosis.
Asunto(s)
Esclerosis Múltiple , Factor de Necrosis Tumoral alfa , Ratas , Animales , Humanos , Femenino , Ratas Endogámicas Lew , Factor de Necrosis Tumoral alfa/genética , Modelos Animales , Glicoproteína Mielina-Oligodendrócito , Corteza Cerebral , InflamaciónRESUMEN
Bone morphogenetic protein (BMP) signalling plays a significant role during embryonic cartilage development and has been associated with osteoarthritis (OA) pathogenesis, being in both cases involved in triggering hypertrophy. Inspired by recent findings that BMP inhibition counteracts hypertrophic differentiation of human mesenchymal progenitors, we hypothesized that selective inhibition of BMP signalling would mitigate hypertrophic features in OA cartilage. First, a 3D in vitro OA micro-cartilage model was established using minimally expanded OA chondrocytes that was reproducibly able to capture OA-like hypertrophic features. BMP signalling was then restricted by means of two BMP receptor type I inhibitors, resulting in reduction of OA hypertrophic traits while maintaining synthesis of cartilage extracellular matrix. Our findings open potential pharmacological strategies for counteracting cartilage hypertrophy in OA and support the broader perspective that key signalling pathways known from developmental processes can guide the understanding, and possibly the mitigation, of adult pathological features.
Asunto(s)
Cartílago Articular , Osteoartritis , Adulto , Proteína Morfogenética Ósea 2 , Condrocitos , Humanos , Hipertrofia , Osteoartritis/tratamiento farmacológico , Osteoartritis/genéticaRESUMEN
It is generally accepted that adult human bone marrow-derived mesenchymal stromal cells (hMSCs) are default committed toward osteogenesis. Even when induced to chondrogenesis, hMSCs typically form hypertrophic cartilage that undergoes endochondral ossification. Because embryonic mesenchyme is obviously competent to generate phenotypically stable cartilage, it is questioned whether there is a correspondence between mesenchymal progenitor compartments during development and in adulthood. Here we tested whether forcing specific early events of articular cartilage development can program hMSC fate toward stable chondrogenesis. Inspired by recent findings that spatial restriction of bone morphogenetic protein (BMP) signaling guides embryonic progenitors toward articular cartilage formation, we hypothesized that selective inhibition of BMP drives the phenotypic stability of hMSC-derived chondrocytes. Two BMP type I receptor-biased kinase inhibitors were screened in a microfluidic platform for their time- and dose-dependent effect on hMSC chondrogenesis. The different receptor selectivity profile of tested compounds allowed demonstration that transient blockade of both ALK2 and ALK3 receptors, while permissive to hMSC cartilage formation, is necessary and sufficient to maintain a stable chondrocyte phenotype. Remarkably, even upon compound removal, hMSCs were no longer competent to undergo hypertrophy in vitro and endochondral ossification in vivo, indicating the onset of a constitutive change. Our findings demonstrate that adult hMSCs effectively share properties of embryonic mesenchyme in the formation of transient but also of stable cartilage. This opens potential pharmacological strategies to articular cartilage regeneration and more broadly indicates the relevance of developmentally inspired protocols to control the fate of adult progenitor cell systems.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Ingeniería de Tejidos/métodos , Receptores de Activinas Tipo I/metabolismo , Adulto , Animales , Células de la Médula Ósea/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/metabolismo , Cartílago Articular/metabolismo , Células Cultivadas , Condrocitos/metabolismo , Condrogénesis/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Osteogénesis/efectos de los fármacos , Cultivo Primario de Células , Transducción de Señal/efectos de los fármacosRESUMEN
Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best-understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine-tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF-R2 activation by VEGF nor its internalization, but it modulates VEGF-R2 downstream signaling through phospho-ERK1/2. In vivo inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF-induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential.
Asunto(s)
Efrina-B2/metabolismo , Mioblastos/metabolismo , Neovascularización Patológica/metabolismo , Receptor EphB4/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Células Endoteliales/metabolismo , Femenino , Humanos , Intususcepción , Isquemia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Músculo Esquelético/patología , Neovascularización Patológica/patología , Fosforilación , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Introduction: An in vitro model that appropriately recapitulates the degenerative disc disease (DDD) microenvironment is needed to explore clinically relevant cell-based therapeutic strategies for early-stage degenerative disc disease. We developed an advanced 3D nucleus pulposus (NP) microtissues (µT) model generated with cells isolated from human degenerating NP tissue (Pfirrmann grade: 2-3), which were exposed to hypoxia, low glucose, acidity and low-grade inflammation. This model was then used to test the performance of nasal chondrocytes (NC) suspension or spheroids (NCS) after pre-conditioning with drugs known to exert anti-inflammatory or anabolic activities. Methods: NPµTs were formed by i) spheroids generated with NP cells (NPS) alone or in combination with ii) NCS or iii) NC suspension and cultured in healthy or degenerative disc disease condition. Anti-inflammatory and anabolic drugs (amiloride, celecoxib, metformin, IL-1Ra, GDF-5) were used for pre-conditioning of NC/NCS. The effects of pre-conditioning were tested in 2D, 3D, and degenerative NPµT model. Histological, biochemical, and gene expression analysis were performed to assess matrix content (glycosaminoglycans, type I and II collagen), production and release of inflammatory/catabolic factors (IL-6, IL-8, MMP-3, MMP-13) and cell viability (cleaved caspase 3). Results: The degenerative NPµT contained less glycosaminoglycans, collagens, and released higher levels of IL-8 compared to the healthy NPµT. In the degenerative NPµT, NCS performed superior compared to NC cell suspension but still showed lower viability. Among the different compounds tested, only IL-1Ra pre-conditioning inhibited the expression of inflammatory/catabolic mediators and promoted glycosaminoglycan accumulation in NC/NCS in DDD microenvironment. In degenerative NPµT model, preconditioning of NCS with IL-1Ra also provided superior anti-inflammatory/catabolic activity compared to non-preconditioned NCS. Conclusion: The degenerative NPµT model is suitable to study the responses of therapeutic cells to microenvironment mimicking early-stage degenerative disc disease. In particular, we showed that NC in spheroidal organization as compared to NC cell suspension exhibited superior regenerative performance and that IL-1Ra pre-conditioning of NCS could further improve their ability to counteract inflammation/catabolism and support new matrix production within harsh degenerative disc disease microenvironment. Studies in an orthotopic in vivo model are necessary to assess the clinical relevance of our findings in the context of IVD repair.
RESUMEN
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.
Asunto(s)
Células Endoteliales , Osteogénesis , Adiponectina/metabolismo , Tejido Adiposo , Cadherinas , Diferenciación Celular , Células Cultivadas , Humanos , Células del Estroma/metabolismo , Fracción Vascular Estromal , Linfocitos T , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
By permeabilizing the cell membrane with ultrasound and facilitating the uptake of iron oxide nanoparticles, the magneto-sonoporation (MSP) technique can be used to instantaneously label transplantable cells (like stem cells) to be visualized via magnetic resonance imaging in vivo. However, the effects of MSP on cells are still largely unexplored. Here, we applied MSP to the widely applicable adipose-derived stem cells (ASCs) for the first time and investigated its effects on the biology of those cells. Upon optimization, MSP allowed us to achieve a consistent nanoparticle uptake (in the range of 10 pg/cell) and a complete membrane resealing in few minutes. Surprisingly, this treatment altered the metabolic activity of cells and induced their differentiation towards an osteoblastic profile, as demonstrated by an increased expression of osteogenic genes and morphological changes. Histological evidence of osteogenic tissue development was collected also in 3D hydrogel constructs. These results point to a novel role of MSP in remote biophysical stimulation of cells with focus application in bone tissue repair.
RESUMEN
Cell-based strategies for nucleus pulposus (NP) regeneration that adequately support the engraftment and functionality of therapeutic cells are still lacking. This study explores a scaffold-free approach for NP repair, which is based on spheroids derived from human nasal chondrocytes (NC), a resilient cell type with robust cartilage-regenerative capacity. We generated NC spheroids (NCS) in two types of medium (growth or chondrogenic) and analyzed their applicability for NP repair with regard to injectability, biomechanical and biochemical attributes, and integration potential in conditions simulating degenerative disc disease (DDD). NCS engineered in both media were compatible with a typical spinal needle in terms of size (lower than 600µm), shape (roundness greater than 0.8), and injectability (no changes in morphology and catabolic gene expression after passing through the needle). While growth medium ensured stable elastic modulus (E) at 5 kPa, chondrogenic medium time-dependently increased E of NCS, in correlation with gene/protein expression of collagen. Notably, DDD-mimicking conditions did not impair NCS viability nor NCS fusion with NP spheroids simulating degenerated NP in vitro. To assess the feasibility of this approach, NCS were injected into an ex vivo-cultured bovine intervertebral disc (IVD) without damage using a spinal needle. In conclusion, our data indicated that NC cultured as spheroids can be compatible with strategies for minimally invasive NP repair in terms of injectability, tuneability, biomechanical features, and resilience. Future studies will address the capacity of NCS to integrate within degenerated NP under long-term loading conditions. STATEMENT OF SIGNIFICANCE: Current regenerative strategies still do not sufficiently support the engraftment of therapeutic cells in the nucleus pulposus (NP). We present an injectable approach based on spheroids derived from nasal chondrocytes (NC), a resilient cell type with robust cartilage-regenerative capacity. NC spheroids (NCS) generated with their own matrix and demonstrated injectability, tuneability of biomechanical/biochemical attributes, and integration potential in conditions simulating degenerative disc disease. To our knowledge, this is the first study that explored an injectable spheroid-based scaffold-free approach, which showed potential to support the adhesion and viability of therapeutic cells in degenerated NP. The provided information can be of substantial interest to a wide audience, including biomaterial scientists, biomedical engineers, biologists and medical researchers.
Asunto(s)
Degeneración del Disco Intervertebral , Disco Intervertebral , Núcleo Pulposo , Animales , Bovinos , Condrocitos , Condrogénesis , Colágeno , Humanos , Degeneración del Disco Intervertebral/terapiaRESUMEN
Design criteria for tissue-engineered materials in regenerative medicine include robust biological effectiveness, off-the-shelf availability, and scalable manufacturing under standardized conditions. For bone repair, existing strategies rely on primary autologous cells, associated with unpredictable performance, limited availability and complex logistic. Here, a conceptual shift based on the manufacturing of devitalized human hypertrophic cartilage (HyC), as cell-free material inducing bone formation by recapitulating the developmental process of endochondral ossification, is reported. The strategy relies on a customized human mesenchymal line expressing bone morphogenetic protein-2 (BMP-2), critically required for robust chondrogenesis and concomitant extracellular matrix (ECM) enrichment. Following apoptosis-driven devitalization, lyophilization, and storage, the resulting off-the-shelf cartilage tissue exhibits unprecedented osteoinductive properties, unmatched by synthetic delivery of BMP-2 or by living engineered grafts. Scalability and pre-clinical efficacy are demonstrated by bioreactor-based production and subsequent orthotopic assessment. The findings exemplify the broader paradigm of programming human cell lines as biological factory units to engineer customized ECMs, designed to activate specific regenerative processes.
Asunto(s)
OsteogénesisRESUMEN
Exposure of cells to externally applied magnetic fields or to scaffolding materials with intrinsic magnetic properties (magnetic actuation) can regulate several biological responses. Here, we generated novel magnetized nanocomposite hydrogels by incorporation of magnetic nanoparticles (MNPs) into polyethylene glycol (PEG)-based hydrogels containing cells from the stromal vascular fraction (SVF) of human adipose tissue. We then investigated the effects of an external Static Magnetic Field (SMF) on the stimulation of osteoblastic and vasculogenic properties of the constructs, with MNPs or SMF alone used as controls. MNPs migrated freely through and out of the material following the magnetic gradient. Magnetically actuated cells displayed increased metabolic activity. After 1 week, the enzymatic activity of Alkaline Phosphatase (ALP), the expression of osteogenic markers (Runx2, Collagen I, Osterix), and the mineralized matrix deposition were all augmented as compared to controls. With magnetic actuation, strong activation of endothelial, pericytic and perivascular genes paralleled increased levels of VEGF and an enrichment in the CD31+ cells population. The stimulation of signaling pathways involved in the mechanotransduction, like MAPK8 or Erk, at gene and protein levels suggested an effect mediated through the mechanical stimulation. Upon subcutaneous implantation in mice, magnetically actuated constructs exhibited denser, more mineralized and faster vascularized tissues, as revealed by histological and micro-computed tomographic analyses. The present study suggests that magnetic actuation can stimulate both the osteoblastic and vasculogenic potentials of engineered bone tissue grafts, likely at least partially by mechanically stimulating the function of progenitor cells.
Asunto(s)
Tejido Adiposo/citología , Hidrogeles/química , Nanopartículas de Magnetita/química , Nanocompuestos/química , Osteoblastos/citología , Tejido Adiposo/patología , Animales , Regeneración Ósea , Proliferación Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Campos Magnéticos , Imagen por Resonancia Magnética , Ratones Desnudos , Microscopía Electrónica de Rastreo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Transducción de Señal , Células Madre/citología , Ingeniería de Tejidos/métodos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Microtomografía por Rayos XRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Cadherinas/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Amidas/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Humanos , Integrinas/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Piridinas/farmacologíaRESUMEN
Modulators of cofactor recruitment by nuclear receptors are expected to play an important role in the coordination of hormone-induced transactivation processes. To identify such factors interacting with the N-terminal domain (NTD) of the progesterone receptor (PR), we used this domain as bait in the yeast Sos-Ras two-hybrid system. cDNAs encoding the C-terminal MYST (MOZ-Ybf2/Sas3-Sas2-Tip60 acetyltransferases) domain of HBO1 [histone acetyltransferase binding to the origin recognition complex (ORC) 1 subunit], a member of the MYST acetylase family, were thus selected from a human testis cDNA library. In transiently transfected CV1 cells, the wild-type HBO1 [611 amino acids (aa)] enhanced transcription mediated by steroid receptors, notably PR, mineralocorticoid receptor, and glucocorticoid receptor, and strongly induced PR and estrogen receptor coactivation by steroid receptor coactivator 1a (SRC-1a). As assessed by two-hybrid and glutathione-S-transferase pull-down assays, the HBO1 MYST acetylase domain (aa 340-611) interacts mainly with the NTD, and also contacts the DNA-binding domain and the hinge domains of hormone-bound PR. The HBO1 N-terminal region (aa 1-340) associates additionally with PR ligand-binding domain (LBD). HBO1 was found also to interact through its NTD with SRC-1a in the absence of steroid receptor. The latter coassociation enhanced specifically activation function 2 activation function encompassed in the LBD. Conversely, the MYST acetylase domain specifically enhanced SRC-1 coupling with PR NTD, through a hormone-dependent mechanism. In human embryonic kidney 293 cells expressing human PRA or PRB, HBO1 raised selectively an SRC-1-dependent response of PRB but failed to regulate PRA activity. We show that HBO1 acts through modification of an LBD-controlled structure present in the N terminus of PRB leading to the modulation of SRC-1 functional coupling with activation function 3-mediated transcription. Importantly, real-time RT-PCR analysis also revealed that HBO1 enhanced SRC-1 coactivation of PR-dependent transcription of human endogenous genes such as alpha-6 integrin and 11beta-hydroxydehydrogenase 2 but not that of amphiregulin. Immunofluorescence and confocal microscopy of human embryonic kidney-PRB cells demonstrated that the hormone induces the colocalization of HBO1 with PR-SRC-1 complex into nuclear speckles characteristic of PR-mediated chromatin remodeling. Our results suggest that HBO1 might play an important physiological role in human PR signaling.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Receptores de Progesterona/metabolismo , Transcripción Genética/genética , Activación Transcripcional/genética , Animales , Línea Celular , Chlorocebus aethiops , Hormonas/metabolismo , Humanos , Ligandos , Coactivador 1 de Receptor Nuclear , Complejo de Reconocimiento del Origen/genética , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores de Progesterona/genética , Receptores de Esteroides/metabolismo , Transducción de Señal , Factores de TranscripciónRESUMEN
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.
RESUMEN
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.
Asunto(s)
Autofagia/genética , Cadherinas/genética , Estrés del Retículo Endoplásmico/genética , Músculo Liso Vascular/metabolismo , Animales , Aorta/citología , Aorta/metabolismo , Apoptosis/genética , Flavonoides/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 2/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Activación Transcripcional/genéticaRESUMEN
Participation of the cadherin superfamily of adhesion molecules in smooth muscle cell (SMC) phenotype modulation is poorly understood. Immunohistochemical analyses of arterial lesions indirectly suggest upregulated expression of atypical glycosylphosphatidylinositol-anchored T-cadherin on vascular SMCs as a molecular indicator of the dedifferentiated/proliferative phenotype. This study investigated the role of T-cadherin in SMC phenotypic modulation. Morphological, molecular and functional SMC-signature characteristics of rat, porcine and human arterial SMCs stably transduced with respect to T-cadherin upregulation (Tcad+) or T-cadherin-deficiency (shTcad) were compared with their respective control transductants (E-SMCs or shC-SMCs). Tcad+-SMCs displayed several characteristics of the dedifferentiated phenotype including loss of spindle morphology, reduced/disorganized stress fiber formation, decay of SMC-differentiation markers (smooth muscle α-actin, smooth muscle myosin heavy chain, h-caldesmon), gain of SMC-dedifferentiation marker calmodulin, reduced levels of myocardin, nuclear-to-cytoplasmic redistribution of the myocardin related transcription factors MRTFA/B and increased proliferative and migratory capacities. T-cadherin depletion enforced features of the differentiated SMC phenotype. PI3K/Akt is a major signal pathway utilized by T-cadherin in SMCs and we investigated mTORC1/S6K1 and GSK3ß axes as mediators of T-cadherin-induced dedifferentiation. Inhibition of mTORC1/S6K1 signalling by rapamycin suppressed proliferation in both E-SMCs and Tcad+-SMCs but failed to restore expression of contractile protein markers in Tcad+-SMCs. Ectopic adenoviral-mediated co-expression of constitutively active GSK3ß mutant S9A in Tcad+-SMCs restored the morphological and molecular marker characteristics of differentiated SMCs and normalized rate of proliferation to that in control SMCs. In conclusion our study demonstrates that T-cadherin promotes acquisition of the dedifferentiated phenotype via a mechanism that is dependent on GSK3ß inactivation.
Asunto(s)
Cadherinas/fisiología , Desdiferenciación Celular , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Transducción de Señal , Animales , Movimiento Celular , Proliferación Celular , Células Cultivadas , Citoesqueleto/ultraestructura , Humanos , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/ultraestructura , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/ultraestructura , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Endogámicas WKY , PorcinosRESUMEN
Close relationships exist between presence of adiponectin (APN) within vascular tissue and expression of T-cadherin (T-cad) on vascular cells. APN and T-cad are also present in the circulation but here their relationships are unknown. This study investigates associations between circulating levels of high molecular weight APN (HMW-APN) and T-cad in a population comprising 66 women and 181 men with angiographically proven stable coronary artery disease (CAD). Plasma HMW-APN and T-cad were measured by ELISA and analysed for associations with baseline clinical characteristics and with each other. In multivariable analysis BMI and HDL were independently associated with HMW-APN in both genders, while diabetes and extent of coronary stenosis were independently associated with T-cad in males only. Regression analysis showed no significant association between HMW-APN and T-cad in the overall study population. However, there was a negative association between HMW-APN and T-cad (P=0.037) in a subgroup of young men (age <60 years, had no diabetes and no or 1-vessel CAD) which persisted after multivariable analysis with adjustment for all potentially influential variables (P=0.021). In the corresponding subgroup of women there was a positive association between HMW-APN and T-cad (P=0.013) which disappeared after adjustment for HDL. After exclusion of the young men, a positive association (P=0.008) between HMW-APN and T-cad was found for the remaining participants of the overall population which disappeared after adjustment for HDL and BMI. The existence of opposing correlations between circulating HMW-APN and T-cad in male and female patient populations underscores the necessity to consider gender as a confounding variable when evaluating biomarker potentials of APN and T-cad.
Asunto(s)
Adiponectina/sangre , Biomarcadores/sangre , Cadherinas/sangre , Enfermedad de la Arteria Coronaria/sangre , Angiografía Coronaria , Enfermedad de la Arteria Coronaria/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Peso Molecular , Pronóstico , Estudios Prospectivos , Factores SexualesRESUMEN
AIMS: This study evaluated associations between plasma T-cadherin levels and severity of atherosclerotic disease. METHODS AND RESULTS: Three hundred and ninety patients undergoing coronary angiography were divided into three groups based on clinical and angiographic presentation: a group (n=40) with normal coronary arteries, a group (n=250) with chronic coronary artery disease and a group (n=100) with acute coronary syndrome. Plasma T-cadherin levels were measured by double sandwich ELISA. Intravascular ultrasound data of the left-anterior descending artery were acquired in a subgroup of 284 patients. T-cadherin levels were lower in patients with acute coronary syndrome than in normal patients (p=0.007) and patients with chronic coronary artery disease (p=0.002). Levels were lower in males (p=0.002), in patients with hypertension (p=0.002) and inpatients with diabetes (p=0.008), and negatively correlated with systolic blood pressure (p=0.014), body mass index (p=0.001) and total number of risk factors (p=0.001). T-cadherin negatively associated with angiographic severity of disease (p=0.001) and with quantitative intravascular ultrasound measures of lesion severity (p<0.001 for plaque, necrotic core and dense calcium volumes). Significant associations between T-cadherin and intravascular ultrasound measurements persisted even if the regression model was adjusted for the presence of acute coronary syndrome. Multivariate analysis identified a strong (p=0.002) negative association of T-cadherin with acute coronary syndrome, and lower T-cadherin levels significantly (p=0.002) associated with a higher risk of acute coronary syndrome independently of age, gender and cardiovascular risk factors. CONCLUSIONS: A reduction in plasma T-cadherin levels is associated with increasing severity of coronary artery disease and a higher risk for acute coronary syndrome.
Asunto(s)
Síndrome Coronario Agudo/sangre , Cadherinas/sangre , Enfermedad de la Arteria Coronaria/sangre , Placa Aterosclerótica/diagnóstico por imagen , Síndrome Coronario Agudo/diagnóstico , Síndrome Coronario Agudo/diagnóstico por imagen , Adulto , Anciano , Angiografía Coronaria , Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Vasos Coronarios/diagnóstico por imagen , Vasos Coronarios/patología , Diabetes Mellitus/sangre , Femenino , Humanos , Hipertensión/sangre , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de Riesgo , Índice de Severidad de la Enfermedad , Ultrasonografía Intervencional/métodosRESUMEN
A study comparing five different cAMP detection technologies in terms of sensitivity, robustness, and feasibility for HTS is presented. In this report, the following methods are described: a nonhomogeneous DELFIA, and the homogeneous methods based on time-resolved fluorescence (HTRF), luminescent singlet oxygen channeling or ALPHAScreen, FP, and high-affinity enzyme complementation. DELFIA had the highest sensitivity, whereas ALPHAScreen and HTRF shared several advantages, including high sensitivity, broad dynamic range, and minimal reagent addition steps. For G(s)-coupled antagonist screens, we found HTRF and ALPHAScreen the more sensitive and HTS-compatible techniques.