Gene expression of connective tissue growth factor in adult mouse

The connective tissue growth factor (CTGF) is a well-known fibroblast mitogen and angiogenic factor that plays an important role in bone formation during embryogenesis. In the adult, CTGF is involved in wound healing as well as fibrotic and vascular disease. However, little is known about its physiological functions under non-pathological conditions in the adult organism. Here, we describe the cellular site of the CTGF mRNA expression in adult male and female mice as revealed by in situ hybridization histochemistry. Strong and persistent CTGF gene expression was particularly prominent in the mesenchyme of the cardiovascular system (aorta, auricular tissue, renal glomeruli), the mesenchyme surrounding the ovarian follicles or the testicular tubes in the gonadal tissue, and the subcapsular mesenchyme bordering densely innervated parts of whisker hair vibrissae. CTGF hybridization signals were not observed in the mesenchyme of many other organs including gut, muscle, liver or most parts of the lymphatic tissue. Strong expression was also present in the primary (early) ovarian follicles, the epithelium of the deep uterine glands and on myenteric ganglia neurons. These data suggest a selective and continuous mesenchymal function in the gonads and those tissues attracting very strong vascular supply or peripheral innervation. CTGF may also be involved in the cyclical proliferation of the uterine gland epithelium and in the early stages of follicular maturation, as well as in the neuropeptide regulation in the gut, cardiovascular and renal systems.     

Follicle stimulating hormone receptor protein is expressed in ovine uterus during the estrous cycle and utero-placenta during early pregnancy: An immunohistochemical study

Follicle stimulating hormone (FSH) is a well characterized gonadotropin that controls primarily development and functions of ovarian follicles in mammalian species. FSH binds to a specific G protein-coupled receptor (FSHR) belonging to the glycoprotein hormone receptor family that plays an essential role in reproduction. Although the primary location of FSHR is in the gonads (mainly in ovarian follicles), FSHR protein and/or mRNA have also been detected in extragonadal female reproductive tissues including embryo, placenta, endometrium, cervix, ovarian cancer tissues, and/or endometriotic lesions in several species. To determine the pattern of FSHR expression in the uterus and placenta, uterine tissues were collected at the early, mid- and/or late luteal phases of the estrous cycle from non-treated or FSH-treated ewes, and utero-placental tissues were collected during early pregnancy followed by immunohistochemistry and image generation. FSHR was immunolocalized to several uterine and utero-placental compartments including luminal epithelium, endometrial glands and surrounding stroma, myometrium, and endothelium and vascular smooth muscle cells in endometrium, myometrium and mesometrium. Intensity of staining and distribution of FSHR in selected compartments differed and seems to depend on the stage of the estrous cycle or pregnancy, and FSH-treatment. These novel data demonstrate differential expression of FSHR protein indicating that FSH plays a specific role in regulation of uterine and utero-placenta functions in sheep.     

Connective tissue growth factor: a novel marker of layer VII neurons in the rat cerebral cortex

Connective tissue growth factor (CTGF) belongs to a family of secreted, extracellular matrix-associated proteins that are involved in the regulation of cellular functions such as adhesion, migration, mitogenesis, differentiation and survival. Recent studies have also suggested the up-regulation of CTGF in response to trauma, scar formation and excitotoxicity in the CNS. To further elucidate the localization and regulation of this molecule in the rat brain we performed in situ hybridization experiments and found a very strong and selective expression of CTGF messenger ribonucleic acid (mRNA) on the band of layer VII neurons throughout the adult cerebral cortex. Similarly strong neuronal expression was also present in the dorsal endopiriform nucleus, extending rostrally from the ventrocaudal cortical layer VII, and in the deep layers of the olfactory glomeruli and the accessory olfactory nucleus. Double in situ hybridization confirmed selective CTGF mRNA expression on a subpopulation (approximately 35%) of microtubule-associated protein 2 mRNA-positive neurons in the cortical layer VII and the dorsal endopiriform nucleus. The nucleus of lateral olfactory tract showed moderate signal intensity; other parts of the forebrain, mesencephalon and brain stem only revealed a very weak level of CTGF mRNA expression. Non-neuronal expression was rare, considerably weaker than on cortical layer VII neurons, and normally associated with blood vessels. Developmental analysis of CTGF mRNA expression in embryonic and postnatal mouse also showed a moderately late onset at embryonic day 16-18, and confirmed the presence of CTGF mRNA in cortical layer VII in a second rodent species. Interestingly, injury experiments using direct cerebral trauma or injection of excitotoxic kainic acid into rat brain failed to up-regulate CTGF mRNA after injury and during the ensuing period of neuronal cell death, gliosis and neural scar tissue formation. Altogether, the current data suggest a constitutive role of CTGF, particularly in the adult cerebral cortex. In view of the strong ascending projections of subplate neurons into cortical layer 1, this molecule may be involved in the modulation of synaptic input to apical dendrites of pyramidal neurons.     

Tissue interactions mediate early events in pulmonary vasculogenesis.

Extensive study has provided considerable insight into the mechanisms governing branching morphogenesis and developmental maturation of the pulmonary epithelium. The process by which the vascular tree arises in the mesodermal mesenchyme of the developing lung, however, is not known. Because normal epithelial branching and differentiation have been shown to be dependent on interactions with the lung mesenchyme, we hypothesized that the developing pulmonary vasculature is dependent on a reciprocal interaction with pulmonary epithelium. In this study we have defined the temporal and spatial expression of flk-1 mRNA, which encodes an endothelial cell-specific vascular endothelial growth factor (VEGF) receptor, in fetal and neonatal rat lung. Flk-1-positive cells were observed in the lung at every prenatal stage from fetal day 11 through birth, demonstrating that vascularization has been initiated as soon as the lung evaginates from the foregut epithelium. The spatial distribution of vascular precursors was distinct and consistent in early lung (fetal days 11-16): clusters of flk-1-positive cells were localized in the mesenchyme closely apposed to the developing epithelium. This spatial relationship between vascular precursors and the developing epithelium suggested that vascular development in the lung may be dependent on interactions between the two tissue types. To investigate this possibility, day-13 distal lung mesenchyme was cultured in the presence and absence of lung epithelium. Lung mesenchyme cultured in the absence of epithelium degenerated significantly, and few flk-1-positive cells were maintained. In contrast, lung mesenchyme recombined with lung epithelium contained abundant flk-1-positive cells, and their spatial distribution mimicked that observed in vivo. These studies provide the first detailed information regarding the temporal and spatial pattern of pulmonary vascularization in early development and suggest that tissue interactions play an important role in growth and maintenance of the developing lung vasculature.     

Farnesoid X receptor immunolocalization in reproductive tissues of adult female rabbits

Farnesoid X receptor (FXR) has been involved in lipid metabolism, cell proliferation, apoptosis, and aromatase expression, as well as in the steroid synthesis and signaling. Considering that these events occur in reproductive tissues in females, the aim of the present study was to determine the immunolocalization of FXR in the ovary, oviduct, uterus, and vagina of rabbits. Rabbits were sacrificed and their reproductive tissues were excised and histologically processed. Immunohistochemistry for FXR was done and reproductive tissues were photographed. FXR immunoreactivity was found in all types of ovarian follicles, ovarian stroma, and corpus luteum of virgin and pregnant rabbits. Also, oviductal and vaginal epithelium of virgins, as well as the oviductal smooth muscle, showed anti-FXR immunoreactivity. The uterine epithelium and musculature of virgins had scarce anti-FXR immunoreactivity. Although the role of FXR in female reproductive tissues is still not known, it is possible to consider various functions related to the reproductive tissue.     

Hh pathway expression in human gut tissues and in inflammatory gut diseases

Sonic hedgehog (Shh) directs early gut patterning via epithelial-mesenchymal signaling and remains expressed in endoderm-derived tissues into the adult period. In human adult gut epithelium SHH/SHH expression is strongest in basal layers, which suggests that SHH may function in the maintenance of gut epithelial stem or progenitor cells. Recent publications suggest a role for aberrant SHH/SHH expression in gut epithelial neoplasias. We hypothesized that the regenerating gut epithelium in inflammatory gut disorders would show an upregulation of SHH/SHH signaling and this abnormal signal may explain the increased incidence of neoplasia in these diseases. Archived healthy gut and inflammatory gut diseased tissues were analyzed by RNA in situ hybridization and immunohistochemistry to describe location and levels of SHH signaling. We show that SHH/SHH and its receptor PTCH1/PTCH1 expression is restricted to the glandular epithelium of the gut, in an antiluminal pattern (strongest in basal layers and weak to absent in luminal epithelium). Inflammatory diseases of the gut show dramatic increases in epithelial SHH signaling. Expression increases in inflamed glandular epithelium (including metaplastic glandular epithelium), losing its radial (crypt-villous) polarity, and expression appears upregulated and present in all epithelial cells. We also describe strong SHH/SHH and PTCH1/PTCH1 expression in intraepithelial and mucosal inflammatory cells. We suggest that SHH signaling in inflammatory diseases of the gut acts to ensure stem cell restitution of damaged mucosal epithelium. However, such signaling may also present a risk for neoplastic transformation.     

ALK-5 mediates endogenous and TGF-beta1-induced expression of connective tissue growth factor in embryonic lung

Transforming growth factor-beta1 (TGF-beta1) has been implicated as a major negative regulator of lung branching morphogenesis. Since connective tissue growth factor (CTGF) is a downstream mediator of TGF-beta1 effects on mesenchymal cells, we hypothesized that TGF-beta1 induces CTGF expression in mouse embryonic lung explants and that CTGF mediates TGF-beta1 inhibition of branching morphogenesis. We show that addition of TGF-beta1 to the serum-free medium of embryonic day (E)12.5 lung explant cultures inhibited branching morphogenesis and induced CTGF mRNA expression in time- and dose-dependent manners. In contrast to basal endogenous CTGF protein, which was exclusively localized in the distal airway epithelium, TGF-beta1-induced CTGF protein was localized in both the epithelium and the mesenchyme. Addition of exogenous CTGF to culture medium directly inhibited branching morphogenesis. To identify the signal transduction pathway through which TGF-beta1 induces CTGF, we used SB431542, a specific inhibitor for TGF-beta type I receptor (TbetaRI)/ALK-5 to block TGF-beta1-induced Smad2/3 phosphorylation. Consequently, SB431542 stimulated normal branching morphogenesis and blocked TGF-beta1 inhibition of branching. Furthermore, SB-431542 blocked both endogenous and TGF-beta1-induced expression of CTGF mRNA and protein. These results demonstrate for the first time that TGF-beta1 induces CTGF expression in mouse embryonic lung explants, that CTGF inhibits branching morphogenesis, and that both endogenous and TGF-beta1-induced CTGF expression are mediated by the TbetaRI/ALK-5-dependent Smad2 signaling pathway.     

Ontogeny of the Kidney and Renal Developmental Markers in the Rhesus Monkey (Macaca Mulatta)

Nonhuman primates share many developmental similarities with humans, thus they provide an important preclinical model for understanding the ontogeny of biomarkers of kidney development and assessing new cell‐based therapies to treat human disease. To identify morphological and developmental changes in protein and RNA expression patterns during nephrogenesis, immunohistochemistry and quantitative real‐time PCR were used to assess temporal and spatial expression of WT1, Pax2, Nestin, Synaptopodin, alpha‐smooth muscle actin (α‐SMA), CD31, vascular endothelial growth factor (VEGF), and Gremlin. Pax2 was expressed in the condensed mesenchyme surrounding the ureteric bud and in the early renal vesicle. WT1 and Nestin were diffusely expressed in the metanephric mesenchyme, and expression increased as the Pax2‐positive condensed mesenchyme differentiated. The inner cleft of the tail of the S‐shaped body contained the podocyte progenitors (visceral epithelium) that were shown to express Pax2, Nestin, and WT1 in the early second trimester. With maturation of the kidney, Pax2 expression diminished in these structures, but was retained in cells of the parietal epithelium, and as WT1 expression was upregulated. Mature podocytes expressing WT1, Nestin, and Synaptopodin were observed from the mid‐third trimester through adulthood. The developing glomerulus was positive for α‐SMA (vascular smooth muscle) and Gremlin (mesangial cells), CD31 (glomerular endothelium), and VEGF (endothelium), and showed loss of expression of these markers as glomerular maturation was completed. These data form the basis for understanding nephrogenesis in the rhesus monkey and will be useful in translational studies that focus on embryonic stem and other progenitor cell populations for renal tissue engineering and repair. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

The transient disappearance of cytokeratin in human fetal and adult ovaries

Cells from the inner and outer granulosa cell layers of the ovarian follicles differ in function, probably because of their different origins from the surface epithelium and from the rete. This suggestion has not so far been thoroughly investigated in the human ovary. We examined fetal ovaries from the early, middle and late gestational periods, ovaries from fertile women, and preovulatory follicular cells obtained from patients under in vitro fertilization therapy (IVF). Indirect immunohistology and immunocytology were used to detect the presence of cytokeratin (CK)-positive epithelial cells. In fetal ovaries from the early gestational period, prominent rete tubules (sometimes with oocytes) appeared to be fused with the sex cords and primordial follicles. Both showed CK-positively, detected with the pan-CK antibody Lu-5. Cytokeratin 19 was clearly expressed in the fusion area. In the fetal and adult ovaries, CK-positive follicular or granulosa cells were noted in the primordial and primary follicles as well as the preovulatory follicles. Cytokeratin was not detected in the granulosa cells of growing follicles, CK-positive and -negative luteal cells were identified in the developing corpus luteum. We conclude for the human ovary: (1) the heterogeneous morphology of granulosa cells may be explained by their twofold origin from the surface epithelium and the rete, (2) the rete tubules appear to be involved in folliculogenesis, (3) the transient absence of CK expression in growing follicles compared to resting and mature follicles or to the developing corpus luteum indicates a particular role of CK-positive cells at the periovulatory period. Accepted: 20 September 1999     

Temporal and spatial expression of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta type 1 (TGF-beta1) at the utero-placental interface during early pregnancy in the pig.

AIMS: To determine the localisation and distribution of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta type 1 (TGF-beta1) in uterine tissues from cycling and early pregnant pigs. METHODS: In situ hybridisation and immunohistochemistry were used to localise CCN2 (CTGF) or TGF-beta1 in uteri obtained from gilts on days 0, 5, 10, 12, 15, and 18 of the oestrous cycle or days 10, 12, 14, 16, 17, and 21 of gestation. RESULTS: In cycling animals, CCN2 (CTGF) mRNA and protein were abundant in luminal epithelial cells (LECs) and glandular epithelial cells (GECs), with lesser amounts in stromal fibroblasts and little or none in endothelial cells. A similar pattern of staining was seen up to day 10 of pregnancy, except that overall staining intensities for CCN2 (CTGF) mRNA or protein were higher and that stromal and endothelial cells were CCN2 (CTGF) positive. However, on days 12-17 there was a striking decrease in the amount of CCN2 (CTGF) in LECs at the utero-conceptus interface, which was associated with maternal stromal matrix reorganisation and the onset of subepithelial neovascularisation. This differential distribution of CCN2 (CTGF) was localised to those LECs that were in close proximity to or in apposition with trophoblast cells. This decrease in CCN2 (CTGF) staining was transient in nature and high amounts of CCN2 (CTGF) were again apparent in LECs on days 17-21, when endometrial neovascularisation and matrix remodelling were complete. The expression of uterine TGF-beta1 was comparable to that of CCN2 (CTGF) at most stages of the oestrous cycle or early pregnancy. Pre-elongation blastocysts recovered on day 10 were positive for both CCN2 (CTGF) and TGF-beta1 in the extra-embryonic trophectoderm, endoderm, and inner cell mass. On day 12, trophectoderm expressed low amounts of TGF-beta1 mRNA and non-detectable amounts of TGF-beta1 protein or CCN2 (CTGF) mRNA or protein. By days 17-21, the expression of both growth factors in the extra-embyronic/placental membranes increased and frequently exceeded that seen in LECs. CONCLUSIONS: The pattern of CCN2 (CTGF) production during the initial attachment phase supports a role for this factor in stromal remodelling and neovascularisation, although alternative functions at later stages such as epithelial-epithelial interactions are also possible. In most major cell types in the uterus or utero-placental unit, CCN2 (CTGF) expression was highly correlated with that of TGF-beta(1), indicating that CCN2 (CTGF) may mediate some of the functions of TGF-beta in the reproductive tract during the oestrous cycle and pregnancy. The data further highlight epithelium as an important source of CCN2 (CTGF) in the regulation of uterine function.     

Epithelial and connective tissue cell CTGF/CCN2 expression in gingival fibrosis

Gingival overgrowth is a side effect of certain medications and occurs in non-drug-induced forms either as inherited (human gingival fibromatosis) or idiopathic gingival overgrowth. The most fibrotic drug-induced lesions develop in response to therapy with phenytoin; the least fibrotic lesions are caused by cyclosporin A; and intermediate fibrosis occurs in nifedipine-induced gingival overgrowth. Connective tissue growth factor (CTGF/CCN2) expression is positively related to the degree of fibrosis in these tissues. The present study has investigated the hypothesis that CTGF/CCN2 is expressed in human gingival fibromatosis tissues and contributes to this form of non-drug-induced gingival overgrowth. Histopathology/immunohistochemistry studies showed that human gingival fibromatosis lesions are highly fibrotic, similar to phenytoin-induced lesions. Connective tissue CTGF/CCN2 levels were equivalent to the expression in phenytoin-induced gingival overgrowth. The additional novel observation was made that CTGF/CCN2 is highly expressed in the epithelium of fibrotic gingival tissues. This finding was confirmed by in situ hybridization. Real-time polymerase chain reaction (PCR) analyses of RNA extracted from drug-induced gingival overgrowth tissues for CTGF/CCN2 were fully consistent with these findings. Finally, normal primary gingival epithelial cell cultures were analysed for basal and transforming growth factor beta1 (TGF-beta1) or lysophosphatidic acid-stimulated CTGF/CCN2 expression at protein and RNA levels. These data indicate that fibrotic human gingival tissues express CTGF/CCN2 in both the epithelium and connective tissues; that cultured gingival epithelial cells express CTGF/CCN2; and that lysophosphatidic acid further stimulates CTGF/CCN2 expression. These findings suggest that interactions between epithelial and connective tissues could contribute to gingival fibrosis.     

Gonadotropin-releasing hormone receptor mRNA expression in the ovaries of neonatal and adult rats

Recent studies have shown that gonadotropin-releasing hormone (GnRH) can exert various effects on the rat ovary by acting through its specific receptors. To determine the cell types responsive to the action of GnRH under physiological conditions in the ovary, distribution of the GnRH receptor mRNA was studied histologically by in situ hybridization in neonatal and adult rats. Expression of the luteinizing hormone receptor mRNA was also examined to judge the growing state of follicles and the corpora lutea. In neonatal rat ovaries, no significant GnRH receptor mRNA signal was detected until 5 days after birth. The expression was first observed at 10 days in the interstitial cells. At 15 days of age, the receptor mRNA was expressed in the granulosa cells of most preantral and early antral follicles, while no hybridization signal was detected in oocyte and theca cells. In adult cycling rats, GnRH receptor mRNA was detected mainly in the granulosa cells of most follicles and luteal cells. The granulosa cells of atretic follicles showed a very high level of the mRNA expression throughout their degenerating process. A strong hybridization signal was also detected in the mural granulosa cells of mature follicles. Newly formed (developing) corpora lutea exhibited signals with moderate intensity in the luteal cells, and the older ones showed weaker signals. The finding that the initial expression of GnRH receptor mRNA was seen in the interstitial cells of neonatal ovaries implies an unknown function of the ovarian GnRH receptor in ovarian development. The high level expression of GnRH receptor mRNA in atretic and mature follicles supports the putative roles of GnRH in the induction of follicular atresia and ovulation in rat ovaries.     

结缔组织生长因子在肾间质纤维化中的表达及其调控

CTGF, a member of the CCN family of immediate early genes, is a recently discovered profibrotic growth factor, which is involved in many pathophysiologic procedures. CTGF acts as a downstreame ffector of TGF-β acting on interstitial ceils to enhance the progression of fibrotic renal diseases. It has been shown that CTGF gene expression can be induced or blocked by some kinds of cytokine and drugs. It is an interesting candidate target for future intervention strategies of renal interstitial fibrosis.     

Comprehensive analysis of HE4 expression in normal and malignant human tissues.

The HE4 (WFDC2) gene encodes a WAP-type four disulphide core domain-containing protein with a presumptive role in natural immunity. Multiple studies have consistently identified upregulation of HE4 gene expression in carcinomas of the ovary; however, the expression in normal and malignant adult tissues has not been examined in detail. Here, we examined the expression of the HE4 gene and protein in a large series of normal and malignant adult tissues by oligonucleotide microarray and tissue microarray, respectively. HE4 gene expression was highest in normal human trachea and salivary gland, and to a lesser extent, lung, prostate, pituitary gland, thyroid, and kidney. In a series of 175 human adult tumors, gene expression was highest in ovarian serous carcinomas. However, adenocarcinomas of the lung, and occasional breast, transitional cell and pancreatic carcinomas had moderate or high levels of HE4 expression. Using tissue microarrays and full tissue sections of normal and 448 neoplastic tissues, HE4 immunoreactivity was found in normal glandular epithelium of the female genital tract and breast, the epididymis and vas deferens, respiratory epithelium, distal renal tubules, colonic mucosa, and salivary glands, consistent with HE4 gene expression. In addition to consistent positivity in ovarian carcinoma, some pulmonary, endometrial, and breast adenocarcinomas, mesotheliomas, and less often, gastrointestinal, renal and transitional cell carcinomas were also positive. Knowledge of the expression patterns of HE4 in our survey is useful for application in histopathologic diagnosis, and should be taken into consideration in future studies that examine the role of HE4 as a serological tumor biomarker or as a target for gene-based therapy.