The role of endothelial-mesenchymal transition in development and pathological process

Epithelial-mesenchymal transition is an important developmental process, participates in tumor's formation, invasion, and metastasis and has been extensively studied. Recently, endothelial-mesenchymal transition (EndMT), a newly recognized type of cellular transdifferentiation, has been demonstrated to participate in a number of diseases by causing morphology changes and pathological processes. Previous studies showed that EndMT was a critical process of embryonic cardiac development. Not only that recent advances also suggested that EndMT occurred postnatally in cancer and cardiac fibrosis and emerged as a possible source of cancer-associated fibroblasts (CAFs). CAFs were found to acquire properties that promoted tumor development and metastasis formation. Resident endothelial cells undergoing EndMT lose their endothelial markers, acquire a mesenchymal or myofibroblastic phenotype, express mesenchymal cell products such as α-smooth muscle actin and type I collagen and develop invasive and migratory abilities. EndMT-derived cells are believed to function as fibroblasts in damaged tissue and may therefore have an important role in pathological process. However, little is known about the signaling mechanisms that cause endothelial cells to transform into mesenchymal cells. Transforming growth factor-β, Notch, or other signaling pathways could direct or interact to mediate EndMT. Therefore, to explore the signaling mechanisms of EndMT may provide novel therapeutic strategies for treating cancer. ? 2012 IUBMB IUBMB Life, 64(9): 717-723, 2012.     

Tie1 deficiency induces endothelial-mesenchymal transition

Endothelial-mesenchymal transition (EndMT) has a significant role in embryonic heart formation and in various pathologies. However, the molecular mechanisms that regulate EndMT induction remain to be elucidated. We show that suppression of receptor tyrosine kinase Tie1 but not Tie2 induces human endothelial cells to undergo EndMT and that Slug deficiency reverts this process. We find that Erk1/2, Erk5 and Akt cascades control Slug promoter activity induced by Tie1 deficiency. Interestingly, EndMT is present in human pancreatic tumour. We propose that EndMT associated with Tie1 downregulation participates in the pathological development of stroma observed in tumours.     

Potential role for insulin-like growth factor II and vitronectin in the endothelial-mesenchymal transition process

Endothelial-to-mesenchymal transition (EndoMT) is a process through which certain subsets of endothelial cells lose endothelial characteristics and transform into mesenchymal or smooth muscle-like cells. Emerging evidence suggests that this process plays an important role during vascular development and in many vascular pathologies. As in epithelial-mesenchymal transition, EndoMT seems to progress through a series of important steps whose interdependence and order are not clear, and that some of them are regulated by soluble growth factors. Insulin-like growth factor II (IGFII), apart from being considered important in cancer, angiogenesis, and atherosclerotic lesions, is also considered as essential to embryonic development. Here, we report that addition of IGFII promoted the EndoMT process in the presence of very low amounts of chicken serum to arrested primary embryonic aortic chicken endothelial cells attached to fibronectin (FN), gelatin, or native type I collagen. This was demonstrated by cell spreading, loss of cell-cell contacts, detachment, migration, and transformation. These cellular events also occurred when IGFII was added to medium containing vitronectin (VN). Additionally, we demonstrated that these proteins were present in the spontaneous intimal thickenings that are observed at day 11-13 of chicken embryo development. We also show that alterations in the distribution of VE-cadherin and beta-catenin occur after IGFII and serum or VN stimulation, and propose that the via VN IGFII effects may be facilitated by interaction of the mannose-6-phosphate/IGFII receptor (M6P/IGFIIR) with the urokinase-type plasminogen activator receptor (uPAR) and its ligand (uPA). Collectively, these findings provide the first evidence for a potential role of the IGFII-VN complex during the EndoMT process. From our observations and previous studies, we postulate a working hypothesis supporting a fundamental role for these molecules during EndoMT.     

Engagement of circular RNA HECW2 in the nonautophagic role of ATG5 implicated in the endothelial-mesenchymal transition

Endothelial-mesenchymal transition (EndoMT) is associated with damage to blood-brain barrier (BBB) integrity. Circular RNAs (circRNAs) are highly expressed in the brain and are involved in brain diseases; however, whether circRNAs regulate the EndoMT in the brain remains unknown. Our study demonstrated that circHECW2 regulated the EndoMT by directly binding to MIR30D, a significantly downregulated miRNA from miRNA profiling, which subsequently caused an increased expression of ATG5. These findings shed new light on the understanding of the noncanonical role of ATG5 in the EndoMT induced by methamphetamine (Meth) or lipopolysaccharide (LPS). The in vivo relevance was confirmed as microinjection of circHecw2 siRNA lentivirus into the mouse hippocampus suppressed the EndoMT induced by LPS. These findings provide novel insights regarding the contribution of circHECW2 to the nonautophagic role of ATG5 in the EndoMT process in the context of drug abuse and the broad range of neuroinflammatory disorders.     

Upregulation of the high mobility group AT-hook 2 gene in acute aortic dissection is potentially associated with endothelial-mesenchymal transition

The high mobility group AT-hook 2 (HMGA2) gene is proposed to regulate the genes involved in the epithelial-mesenchymal transition (EMT). One form of EMT is endothelial-mesenchymal transition (EndMT). We analyzed the expression profile of the HMGA2 gene in different human aortic diseases. Aortic specimens were collected from 51 patients, including 19 with acute aortic dissection, 26 with aortic aneurysm, two with Marfan syndrome and four aortic valves. Quantitative real-time polymerase chain reaction was carried out for HMGA2 and immunohistochemical analyses were performed for HMGA2, SNAI1, Vimentin, CD34, MKI-67 and TGFB1. The expression of let-7d microRNA, which is assumed to play a role in the regulation of HMGA2, was also quantified. The level of HMGA2 gene expression was significantly higher in acute aortic dissection compared with all the other samples (193.1 vs. 8.1 fold normalized to calibrator, P<0.001). The immunohistochemical investigation showed that HMGA2, SNAI1, and Vimentin proteins were mainly detected in the endothelial cells of the vasa vasorum. The HMGA2 gene is upregulated in acute aortic dissection. This is the first report describing a link between HMGA2 and acute aortic dissection. The HMGA2, SNAI1 and Vimentin proteins were mainly detected in the endothelium of the vasa vasorum. It seems that HMGA2 overexpression in acute aortic dissection occurs in a let-7d-independent manner and is associated with EndMT of the vasa vasorum.     

Thrombin and its protease-activated receptor-1 (PAR1) participate in the endothelial-mesenchymal transdifferentiation process

The serine protease thrombin, independently of its participation in hemostasis and thrombosis, has been involved in tissue repair and remodeling, embryogenesis, angiogenesis, and development and progression of atherosclerosis. Many of these functions appear to be mediated by specific thrombin receptors, particularly the protease-activated receptor-1 (PAR1). In this study, we investigated whether both thrombin and PAR1 were present in the aortic wall of chicken embryos at days 11 and 12 of development. We found that PAR1 was limited to some cells of the intimal thickening and the inner media, whereas thrombin appeared distributed across the aortic wall. We also investigated whether PAR1 was present during endothelial-mesenchymal transdifferentiation (EMT) in vitro. A moderate immunoreactivity was detected in the monolayer of endothelial cells. In contrast, a strong cytoplasmic immunoreactivity was observed in the detaching and migrating cells and those that had acquired mesenchymal characteristics. This PAR1 expression was confirmed by flow cytometry. In this study, the addition of thrombin to arrested endothelial cell cultures was assessed. We found that thrombin stimulated endothelial cell spreading and migration, as no migrating cells were observed in serum-free medium (SFM) condition. Immunolocalization of PAR1 in the thrombin-treated cultures showed strong cytoplasmic immunoreactivity in the monolayers and in spreading and migrating cells, whereas in the SFM condition undetectable PAR1 immunoreactivity was observed. Flow cytometry of these cultures revealed an elevated expression of PAR1 in the presence of thrombin, in contrast to that detected in SFM and complete medium. These data indicate that both thrombin and PAR1 are involved in the remodeling of the aortic wall and intimal thickening formation, and in the endothelial-mesenchymal transdifferentiation process.     

Immunolocalization of Periostin-like factor and Periostin during embryogenesis.

Periostin-like factor (PLF) and Periostin are alternatively spliced mRNAs. Our findings are the first to show similarities and differences between PLF and Periostin location using isoform-specific antibodies. The differences in when and where they are present during mouse embryogenesis suggest that they may have different functions. Using immunostaining techniques, we observed that PLF was highly expressed at 12.5 days postconception (dpc) in the intermediate and outer zones of most brain regions, spinal cord, cranial and spinal nerves, and chondrocytes in developing bone and in the heart wall. By 16.5 dpc, PLF was also present in ameloblasts and odontoblasts in developing teeth, and by 19.5 dpc, PLF was present at low levels only in vagal nerve bundles, discrete white matter bundles in the brain, and chondrocytes of developing ribs. Periostin, on the other hand, was absent at 12.5 dpc from dorsal spinal cord and from cranial and spinal nerves. By 16.5 dpc, Periostin was present in many spinal nerves, but absent thereafter, and at 19.5 dpc, Periostin was present in chondrocytes in developing bone but not in neural tissues. The different spatial and temporal location of PLF and Periostin in cartilage and bone cells suggests different roles for these proteins in endochondral bone formation. The early expression of PLF in brain differentiation zones and in developing axon bundles and nerves suggests that it may facilitate axon growth.     

Expression and distribution of endocan in human tissues

Abstract

Endocan is a novel human endothelial cell specific molecule. Its expression is regulated by cytokines and vascular endothelial growth factor (VEGF). The distribution of endocan in normal human tissues, however, remains unclear. We examined the expression of endocan in normal human tissue using immunohistochemical stains. Endocan was expressed in actively proliferative or neogeneic tissues and cells such as glandular tissues, endothelium of neovasculature, bronchial epithelium, germinal centers of lymph nodes etc. Endocan was not present in silent or resting tissues or cells such as endothelium of great arteries and spleen etc. Our findings suggest that endocan may act as a marker for angiogenesis or oncogenesis and could be regarded as a candidate gene for inflammatory tissue, neoplasia, tumor development and metastasis. The expression level of endocan may assist early diagnosis and prognosis of some tumors.     

Immunolocalization of a thiol-protease induced during the senescence of unpollinated pea ovaries

A thiol-endoprotease induced during the senescence of unpollinated ovaries of Pisum sativum L. cv. Alaska has been localized at both cellular and subcellular levels using purified antibodies. Immunoblot analysis showed a single band of 30 kDa in extracts from senescent ovaries 3 and 4 days post-anthesis. Immunolocalization showed the accumulation of the protease within the exocarp and in the outer cell layers of the mesocarp of the senescent ovaries, although with an asymmetric distribution as illustrated in transverse sections. Ultrastructural localization indicates that the protease is associated with the tonoplast and with electron dense materials within the vacuole, where lysis of cell components occurs in senescent ovaries.     

Immunolocalization of basic fibroblast growth factor during chicken cardiac development

Basic fibroblast growth factor (bFGF) has been identified in cultured cardiac myocytes as well as in myocardial tissue of both embryonic and adult organisms; bFGF has also been demonstrated to regulate proliferation and differentiation of these cells in culture. Such studies suggest a possible role for bFGF in cardiac myogenesis. In vitro studies using cultured endothelial and neuronal cells also suggest that myocyte-derived bFGF may be involved in the regulation of vascularization and/or innervation of the developing heart. We have generated a spatial and temporal map for bFGF in the developing chick heart using immunohistochemical techniques and our monospecific polyclonal rabbit antihuman bFGF IgG. A progressive decrease in bFGF expression was seen in the highly trabeculated region of the ventricular myocardium, relative to the myocardium directly underlying the epicardial tissue, with increasing developmental age. bFGF expression was limited to the cytoplasm of cardiac myocytes; neither vascular endothelium nor smooth muscle contained anti-bFGF immunoreactive material. A correlation between the temporal and spatial pattern of bFGF expression seen here, with the pattern of myocyte proliferation and differentiation reported by others, suggests a role for bFGF in the autocrine regulation of myocyte proliferation and differentiation.     

Immunolocalization of matricial components during the early stages of chick embryonic liver development

In the chick embryo, the first liver primordium is observed at the end of the second day of incubation. At 3 and 4 days, ultrastructural analysis of the primitive vascular spaces showed that the endothelial limiting plate was constituted by one or several cell layers. At the vascular pole of the hepatoblasts, mesenchymal cells and connective matrix, present as fibrillar and non fibrillar components, were closely associated. At 5 days, some vascular spaces were limited by a simple endothelial layer. The limiting plate was fenestrated and the connective matrix was reduced to rare collagen fibrils and fibers. Collagen types I, III, IV, procollagen type III, fibronectin and laminin were visualized in the perivascular spaces using immunoperoxidase labeling methods. These components were also detected in the endoplasmic reticulum of hepatoblastic, endothelial and mesenchymal cells. All these appeared to be involved in connective matrix synthesis. Comparing 4 and 5 days, we demonstrated that the number of cells showing intracellular labelling of matricial components dropped dramatically at 5 days, indicating a possible decrease of connective matrix synthesis. Quantification of parenchymal and vascular surfaces was carried out using a semi-automatic image analyzer on consecutive parasagittal sections chosen in the axial part of the embryonic liver. These measurements were performed in order to quantitate the vascular distribution pattern during early development of the liver. These combined immunomorphological studies and morphometrical analyses suggest that during embryogenesis of the liver the synthesis of connective matrix precedes and possibly initiates the vascular differentiation.     

Immunolocalization of laminin during estrogen-induced differentiation of quail oviduct epithelial cells

During estrogen-induced development of the quail oviduct, tubular glands are formed by evagination of epithelial cells into the stroma. The distribution of laminin was studied during the early stages by means of immunofluorescence and immunoperoxidase techniques. Ultrastructural changes in the basal lamina were studied by electron microscopy. Basement membranes at all stages of development were delineated with 3 polyclonal antilaminin antisera. However, in ovariectomized birds, laminin could not be detected by one of the polyclonal antilaminin antisera. Subsequently, this antibody detected laminin as epithelial cell evaginations were induced by estradiol benzoate. The heavy and light chains of Engelbreth Holm sarcoma (EHS) laminin were revealed in immunoblotting by all antibodies. By electron microscopy after the immunoperoxidase technique with antilaminin antisera laminin appears to be accumulated mainly in the lamina densa. Furthermore, the thickness of the basal lamina increases during oviduct development. These data indicate that basal lamina organization is modified during oviduct cell differentiation and that immunoreactivity of epithelial basement membrane laminin changes during development.     

Immunolocalization of the 100 kDa nucleolar protein during the mitotic cycle in CHO cells

The localization of a major nucleolar protein with a molecular weight of 100,000 has been followed during mitosis in Chinese hamster ovary CHO cells using specific antibodies to this protein and immunocytochemical techniques. The 100 kDa protein was visualized at discrete sites on metaphase chromosomes, corresponding to nucleolus organizer regions, and in large, immunostained nucleolar remnants that are discarded in the cytoplasm after nucleolar disintegration. After mitosis, the 100 kDa protein was shown to play an early role in nucleolar reformation. It was first detected in small deposits around the anaphase chromosomes. In telophase, the protein accumulated simultaneously in prenucleolar bodies and in the reforming nucleoli. The early presence of the 100 kDa protein in the telophase nucleus suggests that it is essential for the reestablishment of nucleolar function after mitosis. Thus this protein is present throughout the CHO cell cycle, an observation which supports the hypothesis that it plays a fundamental role in cell organization.     

Immunolocalization patterns of cytokeratins during salivary acinar cell development in mice

Embryonic development of the mouse salivary glands begins with epithelial thickening and continues with sequential changes from the pre-bud to terminal bud stages. After birth, morphogenesis proceeds, and the glands develop into a highly branched epithelial structure that terminates with saliva-producing acinar cells at the adult stage. Acinar cells derived from the epithelium are differentiated into serous, mucous, and seromucous types. During differentiation, cytokeratins, intermediate filaments found in most epithelial cells, play vital roles. Although the localization patterns and developmental roles of cytokeratins in different epithelial organs, including the mammary glands, circumvallate papilla, and sweat glands, have been well studied, their stage-specific localization and morphogenetic roles during salivary gland development have yet to be elucidated. Therefore, the aim of this study was to determine the stage and acinar cell type-specific localization pattern of cytokeratins 4, 5, 7, 8, 13, 14, 18, and 19 in the major salivary glands (submandibular, sublingual, and parotid glands) of the mouse at the E15.5, PN0, PN10, and adult stages. In addition, cell physiology, including cell proliferation, was examined during development via immunostaining for Ki67 to understand the cellular mechanisms that govern acinar cell differentiation during salivary gland morphogenesis. The distinct localization patterns of cytokeratins in conjunction with cell physiology will reveal the roles of epithelial cells in salivary gland formation during the differentiation of serous, mucous or seromucous salivary glands.     

Immunolocalization of acidic and basic fibroblast growth factors during mouse odontogenesis.

Acidic and basic fibroblast growth factors (aFGF and bFGF), are both known to bind to extracellular matrix components, particularly proteoheparin sulfates, and to regulate in vitro proliferation, differentiation and morphology of cells of neuroectodermal and mesodermal origins. Their patterns of distribution were studied during mouse odontogenesis by means of indirect immunofluorescence and immunoperoxidase histochemistry on frozen fixed sections and after Bouin's fixative and paraffin embedding. Localization of aFGF on frozen fixed sections was observed in the oral epithelium, dental lamina and oral mesenchyme (day-12 of gestation), the stellate reticulum and oral epithelium (day-14), the stratum intermedium and at the basal and apical poles of preameloblasts at bell stage. After birth aFGF epitopes were localized within the predentin-dentin area, the stratum intermedium and at the secretory pole of ameloblasts. There was no staining with anti-aFGF antibodies after Bouin's fixative and paraffin embedding. In contrast, using this protocol, intense stainings were found with anti-bFGF antibodies predominantly within dental and peridental basement membranes and mesenchyme: staining of the dental basement membranes was transient (bud and cap stage) and discontinuous; a preferential concentration of bFGF epitopes in the condensed dental mesenchyme of incisors (cap stage) and the dental papillae mesenchymal cells of molars (bell stage) was observed in the posterior and the cervical part of tooth germs. An intense immunostaining of the stellate reticulum with anti-bFGF antibodies was also found on paraffin sections from bud to bell stage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Computer simulation of the folding-unfolding transition of island-model proteins--folding pathway, transition process, and fluctuations

Monte Carlo computer simulations were performed to elucidate the dynamic aspects of the folding and unfolding transitions of island-model protein. Five different types of model proteins were designed, according to characteristics of backbone structure. The computer simulations clearly show that the unfolding and folding transitions are all-or-none processes between the N-and U-states. They are typical Poisson processes. From the Arrhenius plots of rate constants, the activation enthalpies of folding and unfolding were determined. In addition, the folding pathways were determined along the reaction coordinate. Formations of several local structures along a polypeptide chain are almost simultaneous, but the most probable time sequence of events exists at the moment of transition. That is the most probable folding pathway. The unfolding pathway was found to be just the reverse process of the most probable folding pathway. The relationship between the fluctuations in each equilibrium state and the transition process was considered. In contrast to the theory of absolute reaction rate, the transient states are widely distributed along the reaction coordinate. From analysis of the “transient process,” we tried to determine the critical states from which the transient process starts. As a result, we found that the unfolding transition occurs at the stage near the N-state. During the U-state, large joined blocks rarely appear, but they appear in the transient process towards the N-state. However, the “branch point” between the N- and U-states lies near the N-state, and joined blocks tend to unfold prior to passing over the branch point. We concluded that the stability of later folding intermediates is important for selection of the folding pathway, while preferential selection of an early folding intermediate is important in acceleration of the folding rate. The effects of intrachain cross-linking and peptide fragment binding on the rate constants were examined by using computer simulations of model proteins. In general, a small-sized loop formed by cross-linking accelerates the folding rate and a large-sized loop contributes much to the stabilization of the native conformation. We also found that peptide fragment binding contributes little to the acceleration of the folding rate of the residual protein.