Epimorphin expression in interstitial pneumonia

Epimorphin modulates epithelial morphogenesis in embryonic mouse organs. We previously suggested that epimorphin contributes to repair of bleomycin-induced pulmonary fibrosis in mice via epithelium-mesenchyme interactions. To clarify the role of epimorphin in human lungs, we evaluated epimorphin expression and localization in normal lungs, lungs with nonspecific interstitial pneumonia (NSIP), and lungs with usual interstitial pneumonia (UIP); we also studied the effect of recombinant epimorphin on cultured human alveolar epithelial cells in vitro. Northern and Western blotting analyses revealed that epimorphin expression in NSIP samples were significantly higher than those in control lungs and lungs with UIP. Immunohistochemistry showed strong epimorphin expression in mesenchymal cells of early fibrotic lesions and localization of epimorphin protein on mesenchymal cells and extracellular matrix of early fibrotic lesions in the nonspecific interstitial pneumonia group. Double-labeled fluorescent images revealed expression of matrix metalloproteinase 2 in re-epithelialized cells overlying epimorphin-positive early fibrotic lesions. Immunohistochemistry and metalloproteinase activity assay demonstrated augmented expression of metalloproteinase induced by recombinant epimorphin in human alveolar epithelial cells. These findings suggest that epimorphin contributes to repair of pulmonary fibrosis in nonspecific interstitial pneumonia, perhaps partly by inducing expression of matrix metalloproteinase 2, which is an important proteolytic factor in lung remodeling.     

Autocrine stimulation of human pancreatic duct-like development by soluble isoforms of epimorphin in vitro

Epimorphin was recently described as a mesenchymal factor modulating morphogenesis of murine mammary ducts, skin, liver, and lung in vitro. In this study epimorphin was analyzed in a human, pancreatic adenocarcinoma cell line (A818-6) which develops single layer epithelial hollow spheres resembling normal pancreatic ductal structures in vitro. Soluble 34- and 31-kD isoforms of epimorphin were found in the culture supernatant of A818-6 cells. In lysates of A818-6 cells we detected the 34-and 31-kD isoforms and the dimers, and in lysates of fibroblasts the 150-kD tetramers of epimorphin additionally. A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells. Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5-6-d-old hollow spheres. A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1. In this novel system for human duct-like differentiation of pancreatic epithelial cells, we provide evidence for an autocrine and paracrine function of epimorphin as a major mediator for morphogenesis.     

The development of the connective tissue in the human orbit.

The development of the mesenchymal structures of the human orbit was studied using 10 mu or 60 mu serial sections of orbits of foetuses of 35 to 320 mm stages (C.R. length; 2-9 months). The ontogenesis of the orbital walls, the eye muscles, the blood vessels and the connective tissue was compared. This comparison revealed that the eye muscles and their fasciae together with the adventitial layers of the larger blood vessels and nerves develop first, closely linked in time to the progress of ossification and growth of most of the orbital bones. Only the (endochondral) ossification of the ethmoid starts much later. The orbital connective tissue septa development commences later, i.e. from the third month onwards, concurrently with the development of special mesenchymal condensations, a particular capillary system and adipose tissue. About five months later the adult configuration is attained.     

Haymaker gene expression in malignant and normal gynecologic tissues.

We previously reported that cell lines established from human carcinomas and leukemias/lymphomas expressed high levels of an intracellular membrane-bound protein, Haymaker, whereas cell lines derived from non-malignant connective tissue cells and lymphoid cells expressed low levels of this gene product. To determine whether these findings reflect neoplastic transformation or, alternatively, tissue specificity, we examined by immunohistochemical and molecular methods the expression of Haymaker in gynecologic organs with and without tumor. A highly specific, affinity-purified rabbit polyclonal antibody against a 25-mer Haymaker peptide was used for immunohistochemical staining and morphometric analysis of 85 tissue specimens. Immunohistochemical studies demonstrate, for the first time, that Haymaker protein is highly expressed in epithelial cells of the endometrium of the normal uterus and to a somewhat lesser extent in the mucosa of the normal vagina and cervix, but is poorly expressed or absent in cells of the connective tissue and smooth muscle strata of these organs (p < 0.005). Significant differences in Haymaker expression, as assessed by immunohistochemistry, between malignant and normal gynecologic tissues were not observed (p = 0.27). The expression of Haymaker protein does not appear, therefore, to be a marker of malignant transformation of the epithelium of gynecologic organs but rather distinguishes both normal and malignant epithelial cells from normal connective tissue and smooth muscle cells.     

Connective tissue influences on patterns of epithelial architecture and keratinization in skin and oral mucosa of the adult mouse

Summary Epithelial-mesenchymal interactions play an important role during embryogenesis but it is uncertain whether such interactions influence the maintenance of epithelial structure in the adult. To examine this problem, separated epithelial and connective tissue components of skin and mucosae from various regions of adult mice were homoor heterotypically recombined and transplanted to histocompatible hosts. The patterns of tissue architecture and keratinization of the resultant epithelia were examined for changes indicative of mesenchymal influences on the epithelial phenotype. Each type of epithelium, in some recombinations, fully conserved its normal pattern of phenotypic expression indicating that subepithelial connective tissue from all regions is permissive and that regionally-specific connective tissue influences are not necessary for conservation of epithelial specificity. In other recombinations, however, the epithelium acquired features of tissue architecture or keratinization typical of the epithelium normally associated with the connective tissue component, indicating directive influ ences from the connective tissue. The patterns of epithelial response observed suggest that there may be separate connective tissue influences on epithelial architecture and cytodifferentiation and that there is a regionally-related variation in the competence of epithelia to respond to these influences.Supported by NIH NIDR 2 R01 DE05190     

Localization of NTPDase1/CD39 in normal and transformed human pancreas.

Elevated levels of extracellular ATP have been observed in many tumors. We have localized NTPDase1/CD39, one of the principal extracellular nucleotide-hydrolyzing enzymes, in normal and cancerous human pancreas. NTPDase/E-ATPDase activity was demonstrated with an enzyme histochemical technique on cryosections of human pancreas. Acinar and duct epithelial cells were devoid of E-ATPDase activity in both normal and transformed tissue. Endothelial cells and smooth muscle around blood vessels and larger ducts showed strong activity. Nerves, connective tissue, and the beta-cells of the islets were also stained. In cancerous tissue this activity was diminished in the smooth muscle around the ducts and was absent from newly formed connective tissue. Immunostaining for CD39 supported these results but revealed the presence of inactive CD39 in the duct epithelial cells. We hypothesize that the significantly diminished activity of NTPDase1 in the tissues surrounding the ducts may be associated with the processes that lead to tumor formation in human pancreas.     

WWOX protein expression in normal human tissues

WWOX is a putative tumor suppressor gene that spans approximately a 1 Mb genomic region and is the site for the second most common chromosomal fragile site, FRA16D at 16q23. Various studies have focused on the expression of WWOX in human cancer mostly at the RNA level, but little is known about the normal pattern of WWOX protein expression in non-neoplastic tissues. In this study, a comprehensive analysis of WWOX protein expression in normal tissues was performed by means of immunohistochemistry utilizing a very specific anti-WWOX polyclonal antibody. We analyzed tissue cores of human samples representing more than 30 organs, using various tissue microarray (TMA) slides. Due to the potential role of WWOX in sex-steroid metabolism, whole sections from hormonally regulated organs like breast, ovaries, testes and prostate were also analyzed. The results from our study indicate that WWOX is preferentially highly expressed in secretory epithelial cells of reproductive, endocrine and exocrine organs, as well as in ductal epithelial cells from specific segments of the urinary system. Interestingly, we also observed significant WWOX protein expression in various cell types of neural origin including neurons, ependymal cells and astrocytes. No expression of WWOX was detected in adipose, connective, and lymphoid tissues, myelinized structures and blood vessels. By better defining the topographic distribution of WWOX in normal tissues this study provides some insight on the potential physiological role of this novel protein.     

Clock gene Per2 modulates epidermal tissue repair in vivo

Wound healing can be influenced by genes that control the circadian cycle, including Per2 and BMAL1, which coordinate the functions of several organs, including the skin. The aim of the study was to evaluate the role of PER2 during experimental skin wound healing. Two groups (control and Per2-KO), consisting of 14 male mice each, were anesthetized by inhalation, and two 6 mm wounds were created on their dorsal skin using a punch biopsy. A silicone ring was sutured around the wound perimeter to restrict contraction. The wound healing process was clinically measured daily (closure index) until complete wound repair. On Day 6, histomorphometric analysis was performed using the length and thickness of the epithelial migration tongue, in addition to counting vessels underlying the lesion by immunofluorescence assay and maturation of collagen fibers through picrosirius staining. Bromodeoxyuridine (BrdU) incorporation and quantification were performed using the subcutaneous injection technique 2 h before euthanasia and through immunohistochemical analysis of the proliferative index. In addition, the qualitative analysis of myofibroblasts and periostin distribution in connective tissue was performed by immunofluorescence. Statistically significant differences were observed in the healing time between the experimental groups (means: 15.5 days for control mice and 13.5 days for Per2-KO; p = 0.001). The accelerated healing observed in the Per2-KO group (p < 0.05) was accompanied by statistical differences in wound diameter and length of the migrating epithelial tongue (p = 0.01) compared to the control group. Regarding BrdU immunoreactivity, higher expression was observed in the intact epithelium of Per2-KO animals (p = 0.01), and this difference compared to control was also present, to a lesser extent, at the wound site (p = 0.03). Immunofluorescence in the connective tissue underlying the wound showed a higher angiogenic potential in the Per2-KO group in the intact tissue area and the wound region (p < 0.01), where increased expression of myofibroblasts was also observed. Qualitative analysis revealed the distribution of periostin protein and collagen fibers in the connective tissue underlying the wound, with greater organization and maturation during the analyzed period. Our research showed that the absence of the Per2 gene positively impacts the healing time of the skin in vivo. This acceleration depends on the increase of epithelial proliferative and angiogenic capacity of cells carrying the Per2 deletion.     

Changes in Expression of Fibroblast Surface Antigen (SFA) during Cytodifferentiation and Heterokaryon Formation

Fibroblast surface antigen (SF antigen, SFA) is a major glycoprotein antigen detected in connective tissue cells (primitive mesenchymal cells, fibroblasts, and astroglial cells). In this study the expression of SFA was followed during differentiation of the mesenchymal cells of the mouse metanephros and during heterokaryon formation produced by Sendai-virus induced fusion of human fibroblasts and chick red blood cells. It was demonstrated by immunofluorescence that SFA was lost from the kidney mesenchymal cells when they differentiate into epithelial cells of the secretory tubuli. During this process SFA became detectable in the basement membrane formed around the tubuli. In cell fusion experiments human SFA which was present as fibrillar network on the surface of cultured fibroblasts, was gradually lost from the heterokaryons when the incorporated chick nuclei became activated. These two sets of experiments indicate that SFA can be used as a phenotypic marker of Cytodifferentiation.     

Delivering the message: epimorphin and mammary epithelial morphogenesis

The mammary gland consists of a highly branched tubular epithelium surrounded by a complex mesenchymal stroma. Epimorphin is an extracellular protein that is expressed by mammary mesenchymal cells that directs epithelial morphogenesis. Depending upon the context of presentation--polar versus apolar--epimorphin can selectively direct two key processes of tubulogenesis: branching morphogenesis (processes involved in tubule initiation and extension) and luminal morphogenesis (required for enlargement of tubule caliber). Here, we outline the fundamentals of mammary gland development and describe the function of epimorphin in these processes. We conclude with a review of recent studies that suggest similar morphogenic roles for epimorphin in other glandular organs.     

Effects of subepithelial fibroblasts on epithelial differentiation in human skin and oral mucosa: heterotypically recombined organotypic culture model

The stratified squamous epithelia differ regionally in their patterns of morphogenesis and differentiation. Although some reports suggested that the adult epithelial phenotype is an intrinsic property of the epithelium, there is increasing evidence that subepithelial connective tissue can modify the phenotypic expression of the epithelium. The aim of this study was to elucidate whether the differentiation of cutaneous and oral epithelia is influenced by underlying mesenchymal tissues. Three normal skin samples and three normal buccal mucosa samples were used for the experiments. Skin equivalents were constructed in four ways, depending on the combinations of keratinocytes (cutaneous or mucosal keratinocytes) and fibroblasts (dermal or mucosal fibroblasts), and the effects of subepithelial fibroblasts on the differentiation of oral and cutaneous keratinocytes were studied with histological examinations and immunohistochemical analyses with anti-cytokeratin (keratins 10 and 13) antibodies. For each experiment, three paired skin equivalents were constructed by using single parent keratinocyte and fibroblast sources for each group; consequently, nine (3 x 3) organotypic cultures per group were constructed and studied. The oral and cutaneous epithelial cells maintained their intrinsic keratin expression. The keratin expression patterns in oral and cutaneous epithelia of skin equivalents were generally similar to their original patterns but were partly modified exogenously by the topologically different fibroblasts. The mucosal keratinocytes were more differentiated and expressed keratin 10 when cocultured with dermal fibroblasts, and the expression patterns of keratin 13 in cutaneous keratinocytes cocultured with mucosal fibroblasts were different from those in keratinocytes cocultured with cutaneous fibroblasts. The results suggested that the epithelial phenotype and keratin expression could be extrinsically modified by mesenchymal fibroblasts. In epithelial differentiation, however, the intrinsic control by epithelial cells may still be stronger than extrinsic regulation by mesenchymal fibroblasts.     

Contribution of Subcutaneous Connective Tissues to the Epithelialization and Cyst Formation by the Skin Transplanted Subcutaneously

Skins and hollow organs have been shown to form epithelialized cysts when transplanted into subcutaneous tissue of a recipient animal, expanding their surface areas. This system seems to offer a good potential for regenerating organs. We investigated the functional and structural contribution of epithelia and connective tissue compartments in this regeneration system with two experimental systems.Key Words: skin, epithelialization, transplantation, GFP, epidermis, cystDispase-separated epidermis often forms epithelialized cysts when combined with dermal connective tissue whereas dispase-separated epidermis alone does not form cysts or epithelialize, indicating the functional importance of the dermal connective tissue in the regeneration process.When GFP rats were used as donors for the skin, the donor-derived tissue was composed of whole epidermis and parts of the connective tissue cells and blood vessels under the newly epithelialized portion of the cyst wall. Small capillaries of granulation tissues were shown to be of recipient origin, but some large vessels were of donor origin. These results showed the significant functional and structural contribution of dermal connective tissue in the regeneration of the skin in subdermal transplant.     

Contribution of Subcutaneous Connective Tissues to the Epithelialization and Cyst Formation by the Skin Transplanted Subcutaneously

Skins and hollow organs have been shown to form epithelialized cysts when transplanted into subcutaneous tissue of a recipient animal, expanding their surface areas. This system seems to offer a good potential for regenerating organs. We investigated the functional and structural contribution of epithelia and connective tissue compartments in this regeneration system with two experimental systems.

Dispase-separated epidermis often forms epithelialized cysts when combined with dermal connective tissue whereas dispase-separated epidermis alone does not form cysts or epithelialize, indicating the functional importance of the dermal connective tissue in the regeneration process.

When GFP rats were used as donors for the skin, the donor-derived tissue was composed of whole epidermis and parts of the connective tissue cells and blood vessels under the newly epithelialized portion of the cyst wall. Small capillaries of granulation tissues were shown to be of recipient origin, but some large vessels were of donor origin. These results showed the significant functional and structural contribution of dermal connective tissue in the regeneration of the skin in subdermal transplant.     

Changes in the distribution of the 34-kdalton tyrosine kinase substrate during differentiation and maturation of chicken tissues

We examined the distribution of the 34-kilodalton (34-kD) tyrosine kinase substrate in tissues of adult and embryonic chicken using both a mouse monoclonal antibody and a rabbit polyclonal antibody raised against the affinity purified 34 kD protein. We analyzed the localization by immunoblotting of tissue extracts, by immunofluorescence staining of frozen tissue sections, and by staining sections of paraffin-embedded organs by the peroxidase antiperoxidase method. The 34-kD protein was present in a variety of cells, including epithelial cells of the skin, gastrointestinal, and respiratory tracts, as well as in fibroblasts and chondrocytes of connective tissue and mature cartilage, and endothelial cells of blood vessels. The 34-kD protein was also found in subpopulations of cells in thymus, spleen, bone marrow, and bursa. The protein was not detected in cardiac, skeletal, or smooth muscle cells, nor in epithelial cells of liver, kidney, pancreas, and several other glands. Although most neuronal cells did not contain the 34-kD protein, some localized brain regions did contain detectable amounts of this protein. The 34-kD protein was not detected in actively dividing cells of a number of tissues. Changes in the distribution of the 34-kD protein were observed during the differentiation or maturation of cells in several tissues including epithelial cells of the skin and gastrointestinal tract, fibroblasts of connective tissue, and chondroblasts.     

gC1qR expression in normal and pathologic human tissues: differential expression in tissues of epithelial and mesenchymal origin

The gC1qR (i.e., gC1q receptor, gC1q binding protein, p32, p33) is a multifunctional cellular protein that interacts with components of the complement, kinin, and coagulation cascades and select microbial pathogens. Enhanced gC1qR expression has been reported in adenocarcinomas arising in a variety of organs. The present study compared gC1qR expression in normal, inflammatory, dysplastic, and malignant tissue of epithelial and mesenchymal origin. gC1qR expression was visualized in tissue sections by immunohistochemistry using the 60.11 monoclonal antibody (i.e., IgG(1) mouse monoclonal antibody directed against gC1qR) and the UltraVision LP Detection System. Sections were counterstained with hematoxylin and examined by light microscopy. Strongest gC1qR expression was noted in epithelial tumors of breast, prostate, liver, lung, and colon, as well as in squamous and basal cell carcinoma of the skin. However, increased gC1qR staining was appreciated also in inflammatory and proliferative lesions of the same cell types, as well as in normal continuously dividing cells. In contrast, tumors of mesenchymal origin generally stained weakly, with the exception of osteoblasts, which stained in both benign and malignant tissues. The data suggest that increased gC1qR expression may be a marker of benign and pathologic cell proliferation, particularly in cells of epithelial origin, with potential diagnostic and therapeutic applications.     

Mesenchymal epimorphin is important for pancreatic duct morphogenesis

Epithelial-mesenchymal interactions are crucial for the proper development of many organs, including the pancreas. Within the pancreas, the ducts are thought to harbor stem/progenitor cells, and possibly to give rise to pancreatic ductal carcinoma. Little is known about the mechanism of formation of pancreatic ducts in the embryo. Pancreatic mesenchyme contains numerous soluble factors which help to sustain the growth and differentiation of exocrine and endocrine structures. Here, we report that one such morphoregulatory mesenchymal protein, epimorphin, plays an important role during pancreatic ductal proliferation and differentiation. We found that epimorphin is expressed in pancreatic mesenchyme during early stages of development, and at mesenchymal-epithelial interfaces surrounding the ducts at later stages. Strong upregulation of epimorphin expression was seen during in vitro pancreatic duct differentiation. Similarly, in vitro pancreatic duct formation was inhibited by a neutralizing antibody against epimorphin, whereas addition of recombinant epimorphin partially rescued duct formation. Together, our study demonstrates the role of epimorphin in pancreatic ductal morphogenesis.     

Morphogenic protein epimorphin protects intestinal epithelial cells from oxidative stress by the activation of EGF receptor and MEK/ERK, PI3 kinase/Akt signals

Epimorphin is a mesenchymal protein that regulates morphogenesis of epithelial cells. Our preliminary study suggested a novel function of epimorphin in enhancing survival of intestinal epithelial cells (IEC). Oxidative stress leads to cell injury and death and is suggested to be a key contributor to pathogenesis of inflammatory bowel disease. This study was conducted to determine whether epimorphin protects IEC from oxidative stress. Rat intestinal epithelial cell line IEC-6 was cultured with epimorphin (10 and 20 mug/ml), and the life span of IEC was assessed. The mean life span of IEC-6 cells was prolonged 1.9-fold (P < 0.0006) by treatment with epimorphin. We then examined the epimorphin signaling pathways. Epimorphin phosphorylated epidermal growth factor (EGF) receptor, activated the MEK/extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase and phosphatidylinositol 3 (PI3) kinase/Akt pathways, phosphorylated Bad, and induced Bcl-X(L) and survivin. Hydrogen peroxide (1 mM) induced cell death in 92% of IEC-6 cells, but epimorphin dramatically diminished (88.7%) cell death induced by hydrogen peroxide (P < 0.0001). This protective effect of epimorphin was significantly attenuated by inhibitors of MEK and PI3 kinase (P < 0.0001) or EGF receptor-neutralizing antibody (P = 0.0007). In wound assays, the number of migrated cells in the wound area decreased (72.5%) by treatment with 30 muM hydrogen peroxide, but epimorphin increased the number of migrated cells 3.18-fold (P < 0.0001). These results support a novel function of epimorphin in protecting IEC from oxidative stress. This anti-oxidative function of epimorphin is dramatic and is likely mediated by the activation of EGF receptors and the MEK/extracellular signal-regulated kinase and PI3 kinase/Akt signaling pathways and through the induction of anti-apoptotic factors.     

A loss of telocytes accompanies fibrosis of multiple organs in systemic sclerosis

Systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of the skin and various internal organs. In SSc, telocytes, a peculiar type of stromal (interstitial) cells, display severe ultrastructural damages and are progressively lost from the clinically affected skin. The aim of the present work was to investigate the presence and distribution of telocytes in the internal organs of SSc patients. Archival paraffin‐embedded samples of gastric wall, myocardium and lung from SSc patients and controls were collected. Tissue sections were stained with Masson's trichrome to detect fibrosis. Telocytes were studied on tissue sections subjected to CD34 immunostaining. CD34/CD31 double immunofluorescence was performed to unequivocally differentiate telocytes (CD34‐positive/CD31‐negative) from vascular endothelial cells (CD34‐positive/CD31‐positive). Few telocytes entrapped in the fibrotic extracellular matrix were found in the muscularis mucosae and submucosa of SSc gastric wall. In the muscle layers and myenteric plexus, the network of telocytes was discontinuous or even completely absent around smooth muscle cells and ganglia. Telocytes were almost completely absent in fibrotic areas of SSc myocardium. In SSc fibrotic lung, few or no telocytes were observed in the thickened alveolar septa, around blood vessels and in the interstitial space surrounding terminal and respiratory bronchioles. In SSc, the loss of telocytes is not restricted to the skin, but it is a widespread process affecting multiple organs targeted by the fibrotic process. As telocytes are believed to be key players in the regulation of tissue/organ homoeostasis, our data suggest that telocyte loss might have important pathophysiological implications in SSc.