Invasion Of Stromal Cells Research Articles

Increased matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 secretion but unaffected invasiveness of endometrial stromal cells in adenomyosis

To investigate cell invasiveness and the production of matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in endometrial stromal cells (ESCs) of adenomyosis. Prospective study. Tertiary university hospital. Twenty-one women with adenomyosis (study group) and 25 women without adenomyosis (control group). Endometrial stromal cells were purified from eutopic endometrium and were cultured in vitro. Matrigel invasion assay and measurement of MMP-2, MMP-9, TIMP-1, and TIMP-2 concentrations. Endometrial stromal cell invasiveness of adenomyosis was not different from that of the control group (0.95 vs. 1, using the latter as the bench mark) in ESCs cultured alone. After the ESCs were cultured with IL-6, anti-IL-6, or GM6001 for 24 hours, the indices of cell invasion were not different between the two groups. MMP-2 (median 7.3 vs. 3.5 ng/mL) and TIMP-1 (median 12.1 vs. 3.9 ng/mL), but not MMP-9 and TIMP-2, secreted by ESCs in women with adenomyosis were much higher than those in women without adenomyosis. The formation of adenomyosis does not result from altered invasiveness of ESCs. The concomitant elevation of MMP-2 and TIMP-1 may partially justify why invasiveness was not increased, but other enzymes should also be considered.

Modeling the early endometriotic lesion: mesothelium-endometrial cell co-culture increases endometrial invasion and alters mesothelial and endometrial gene transcription

To determine the role of peritoneal mesothelial cells (PMCs) in the process of endometrial invasion into the peritoneum and to evaluate gene expression after endometrial-PMC co-culture. In vitro study. University laboratory. Reproductive-age women without endometriosis. None. The rate of endometrial invasion through modeled peritoneum in the presence and absence of PMCs was evaluated. The influence of endometrial-PMC attachment on the expression of target genes, implicated in the pathogenesis of endometriosis, was examined by using reverse transcription polymerase chain reaction. Endometrial stromal cell (ESC) invasion through invasion chambers coated with Matrigel (MTGL) and with growth factor-reduced Matrigel (GFR-MTGL) was increased 10-fold when a PMC monolayer was present. Endometrial epithelioid cell (EM42) invasion increased greater than threefold through the MTGL and GFR-MTGL-coated membranes when a PMC monolayer was present. Endometrial stromal cell, EM42, and PMC transcription of extracellular signal-related kinase, colony stimulating factor-1, c-fms, and c-Met was increased after endometrial-PMC attachment. Similar changes were not seen when endometrial cells were exposed to PMC-conditioned media and when PMCs were exposed to endometrial cell conditioned media. Peritoneal mesothelial cells increased invasion of ESCs and EM42s through modeled peritoneum. Endometrial-PMC co-culture led to alterations in gene transcription by endometrial cells and PMCs. This study suggests that PMCs contribute to the process of endometrial invasion into the peritoneum.

Cyclooxygenase-2 Regulates Survival, Migration, and Invasion of Human Endometriotic Cells through Multiple Mechanisms

Endometriosis is a debilitating disease characterized by the presence of functional endometrial glandular epithelium and stroma outside the uterine cavity that affects up to 20% of women of child-bearing age. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of prostaglandin E(2) (PGE(2)), is highly expressed in endometriotic tissues and results in increased concentrations of peritoneal PGE(2) in women. In this study, we determined the expression of COX-2 protein in ectopic and eutopic endometria in humans and the role of COX-2 in endometriotic cell survival, migration, and invasion in humans. Our results indicate that COX-2 protein is abundantly expressed in ectopic endometria compared with eutopic endometria. Comparatively, expression of COX-2 protein is higher in eutopic endometria from women with endometriosis compared with women without endometriosis. Inhibition of COX-2 decreases survival, migration, and invasion of endometriotic cells that are associated with decreased production of PGE(2). Cell growth inhibitory effects of COX-2 inhibition/silencing are mediated through nuclear poly (ADP-ribose) polymerase-mediated apoptosis. Cell motility and invasion inhibitory effects of COX-2 inhibition/silencing are mediated through matrix metalloproteinase-2 and -9 activities. Interestingly, effects of COX-2 inhibition is more profound in endometriotic epithelial than in stromal cells. Furthermore, inhibition of COX-2 affects invasion rather than migration of endometriotic epithelial and stromal cells. It is the first evidence showing that inhibition of COX-2 decreases endometriotic epithelial and stromal cell survival, migration, and invasion in humans. Our results support the emerging concept that COX-2/PGE(2) promotes the pathophysiology and pathogenesis of endometriosis in humans.

PROSTAGLANDIN E2 SIGNALING IN PATHOPHYSIOLOGY OF ENDOMETRIOSIS IN HUMAN

Endometriosis is a debilitating disease affects up to 20% of reproductive age women characterized by the presence of functional endometrial glandular epithelium and stroma out side the uterine cavity. The pathophysiology of endometriosis remains as enigma in reproductive medicine. Accumulating evidence indicates that steroids, growth factors, and cytokines, and prostaglandins may promote establishment and maintenance of endometriosis. Treatment options include oral contraceptives, aromatase inhibitors, androgenic agents, and gonadotrophin releasing hormone analogues and nonsteroidal anti-inflammatory drugs and surgical removal. But the recurrence rate is up to 70% after cessation of therapy. Prostaglandin E2 (PGE2) promotes cell proliferation, migration, invasion, angiogenesis, anti-apoptosis, pain and immunomodulation. PGE2 mediated effects are primarily mediated through G-protein coupled membrane receptors designated EP that includes EP1, EP2, EP3 and EP4. EP2 and EP4 receptors are coupled to adenyl cyclase generating cAMP that in turn activates the protein kinase A signaling pathway. EP1 receptor is coupled to phospholipase C generating two second messengers, inositol triphosphate (IP3) involved in the liberation of intracellular calcium (Ca2+), and diacyl glycerol an activator of protein kinase C. There are several EP3 isoforms exhibiting a wide range of actions from inhibition of cAMP production to increases in Ca2+ and IP3. In this study, we used immortalized human endometriotic epithelial cells (11-Z, 12-Z, 49-Z, and 108-Z) and stromal cells (22-B) as in vitro models to unravel PGE2 signaling in pathophysiology of endometriosis in human. Normal endometrial epithelial and stromal cells were used as control. We investigated: (1) PGE2 production; (2) expression profiles of EP1, EP2, EP3, and EP4 receptors and (3) role of EP receptors in endometriotic cell proliferation, migration and invasion, and unraveled the underlined mechanisms. We used RT-PCR, western blot, zymography, matrigel migration and invasion assays, and ELISA. Endometriotic cells produced 8–10 fold more PGE2 than normal endometrial cells at basal condition. EP2 and EP4 receptor mRNAs and proteins were abundantly expressed whereas EP1 and EP3 receptor mRNAs and proteins are barely detectable in endometriotic epithelial and stromal cells. Inhibition of either EP2 or EP4 decreased (60%) the endometriotic epithelial and stromal cell proliferation, migration and invasion. Combined inhibition of EP2 and EP4 synergistically decreased (80–85%) endometriotic epithelial and stromal cell proliferation, migration and invasion. Inhibition of EP1 receptor did not have any effect. Inhibition of EP2 and/or EP4 decreased the expression and phosphorylation of Bcl2 protein. Inhibition of EP2 and/or EP4 decreased MMP2 and MMP9 activities. These results suggest that EP2 and/or EP4 mediated cell proliferation and invasion might be mediated respectively through Bcl2 and MMP2 and MMP9 pathways in endometriotic epithelial and stromal cells. Although endometriosis is not a malignancy its pathophysiology seems to be analogous to the mechanisms of cancer metastasis. In conclusion, these endometriotic epithelial and stromal cells can be used as ideal model to study the cellcell communications and interactions in the establishment and maintenance of endometriosis in human. Targeting PGE2 receptors and downstream signaling might be a novel therapy for endometriosis in human. (poster)

TRANSCRIPTIONAL REGULATION OF INFLAMMATORY MEDIATORS IN ENDOMETRIOSIS

Endometriosis affects more than 7 million women in North America and accounts for ∼$1 billion in medical and surgical health expenses annually. A variety of theories has been promulgated to explain the derivation of extrauterine implants of endometrial tissue that define this syndrome, but the precise pathogenic mechanisms responsible for pelvic pain and infertility associated with endometriosis remain unknown. Investigations from our laboratory have supported the hypothesis that recruitment of immune cells is a critical etiological step in disease establishment. The implantation of endometriosis lesions on ectopic peritoneal surfaces requires attachment and invasion of stromal and epithelial cells into the extracellular matrix of the host tissue. These proliferating cells attract immune cell infiltrates, particularly macrophages, via the selective production of chemokines. Recruitment of macrophages exacerbates implant growth and fibrosis via paracrine secretion of mitogens, cytokines, oxygen free radicals and prostaglandins. In addition, environmental endocrine disruptors have been invoked as putative etiologic agents. We observed that peritoneal concentrations of RANTES (Regulated on Activation Normal T cell Expressed and Secreted) was highly correlated with the presence of endometriosis in women and this protein accounts for ∼70% of monocyte chemotaxis in Boyden chamber assays containing pelvic fluid from endometriosis patients. Endometriosis stromal cells are a potent source of RANTES secretion and RANTES mRNA expression. Proinflammatory cytokines, particularly IL-1beta and TNF-alpha, activate RANTES gene transcription in these cells through an NF-kappaB-dependent mechanism. Estradiol enhances this effect at transcriptional and post-transcriptional levels. An environmental toxicant, 2,3,7,8-TCDD (dioxin), also appears to activate RANTES gene expression directly in endometriosis stromal cells. RANTES mRNA production also can be negatively regulated in these cells. PPAR-gamma ligands inhibit RANTES expression. Prolonged exposure to medroxyprogesterone acetate or treatment with the non-steroidal anti-inflammatory drug, sulindac, represses RANTES transcription via interference with NF-kappaB. It is hoped that identification and characterization of the latter pathways will provide opportunities for new drug discovery and refinement of the treatment of endometriosis. Supported by NIH grant HD37321.

Effect of a statin on an in vitro model of endometriosis

To determine the inhibitory effect of a statin on angiogenesis in a three-dimensional (3-D) culture of human endometrial fragments in vitro. Angiogenesis has been proposed as an important mechanism in the pathogenesis of endometriosis, and statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) have been shown to have anti-inflammatory and anti-angiogenic activity. Experimental in vitro study of human endometrial biopsies and 3-D culture in fibrin matrix. Research laboratory at a university-affiliated infertility center. Forty-six normal ovulating women undergoing infertility treatment. Endometrial samples obtained from the fundus of the uterine cavity were minced, and the fragments were placed in a three-dimensional fibrin matrix culture system. Presence or absence of proliferation of stromal cells and invasion of the fibrin matrix, presence or absence of vessel sprouting, and immunohistochemical characterization of cellular components. During the 1st week of culture, invasion of stromal cells into the fibrin matrix occurred in the control group and in some wells outgrowths were observed. After 2 weeks, endometrial glands were observed in the outgrowths at a distance from the main tissue and were growing in conjunction with new vessel formation until the end of culture period. A concentration-dependent effect of lovostatin was seen on cell growth and angiogenesis in the experimental groups. In the presence of 5 and 10 microM of statin, angiogenesis was abolished, and cell proliferation was inhibited. In the presence of 1 microM of lovastatin, angiogenesis was reduced, but cell proliferation was not affected. The statins were shown to be effective in inhibiting the mechanisms of cell proliferation and angiogenesis in an experimental model for the development of endometriosis-like tissue.

Effect of a Statin on an In Vitro Model of Endometriosis

Effect of a Statin on an In Vitro Model of Endometriosis

Integrin Function and Signaling as Pharmacological Targets in Cardiovascular Diseases and in Cancer

The microenvironment is now considered as an important source of potential therapeutic targets in diverse pathologies. In cardiovascular diseases and in cancer, common processes involving stromal remodeling, cell invasion, and angiogenesis can promote progression of the pathology. At each step of the pathogenesis, cell adhesion needs to be modulated to allow adaptation of cell survival/motility/proliferation functions to the microenvironment. Among adhesion receptors, integrins, responsible for cell/matrix or cell/cell interactions, play a key role in the cellular responses. Moreover, their engagement conditions the sensitivity to apoptosis induced by therapeutic drugs. Targeting of the extracellular side of integrins in order to modulate their adhesive functions is under development and has reached clinical indications. However, improvement of oral availability and of cell signaling control is required in the future. Targeting of the extracellular or the intracellular key proteins involved in integrin-dependent signaling pathway seems promising. Yet, although some common key enzyme inhibitors are under development, a better knowledge of the specificity of integrin activation and interaction with partners upon pathogenesis is of major importance in envisaging the antagonism of integrin-linked signals as a therapeutic tool alone or in association with other therapies.

Light microscopic study of periapical lesions associated with asymptomatic apical periodontitis

The purpose of the study has been to evaluate the degree of chronic inflammation in tissues surrounding the apex of the tooth root in patients with apical periodontitis in the remission phase. The material included 37 apical granulomas and radicular cysts obtained as a result of apiectomy, and 20 teeth which were removed together with the focus of the periapical inflammation. Routine histological techniques, as well as the immunofluorescent and immuno-chemical methods were used to examine the material. Despite the absence of clinical symptoms in 23 of 57 cases, the morphological signs of chronic inflammation were observed in the apical area of the tooth root. Morphological signs of viral invasion of epithelial and stromal cells in the radicular cyst wall were revealed in six cases. The presence of the virus of Herpes simplex I in epithelial cells (five cases) and adenoviral invasion (one case) was confirmed by immuno-fluorescent and immuno-chemical methods. Histological examination often reveals morphological signs of an active inflammatory process in the periapical tissues of patients treated during clinical remission. In our opinion, the presence of viruses in the epithelial cells of the radicular cyst may contribute to the persistence of the active stage of the inflammatory process.

Critical role of N-cadherin in myofibroblast invasion and migration in vitro stimulated by colon-cancer-cell-derived TGF-beta or wounding.

Invasion of stromal host cells, such as myofibroblasts, into the epithelial cancer compartment may precede epithelial cancer invasion into the stroma. We investigated how colon cancer-derived myofibroblasts invade extracellular matrices in vitro in the presence of colon cancer cells. Myofibroblast spheroids invade collagen type I in a stellate pattern to form a dendritic network of extensions upon co-culture with HCT-8/E11 colon cancer cells. Single myofibroblasts also invade Matrigel trade mark when stimulated by HCT-8/E11 colon cancer cells. The confrontation of cancer cells with extracellular matrices and myofibroblasts, showed that cancer-cell-derived transforming growth factor-beta (TGF-beta) is required and sufficient for invasion of myofibroblasts. In myofibroblasts, N-cadherin expressed at the tips of filopodia is upregulated by TGF-beta. Functional N-cadherin activity is implicated in TGF-beta stimulated invasion as evidenced by the neutralizing anti-N-cadherin monoclonal antibody (GC-4 mAb), and specific N-cadherin knock-down by short interference RNA (siRNA). TGF-beta1 stimulates Jun N-terminal kinase (also known as stress-activated protein kinase) (JNK) activity in myofibroblasts. Pharmacological inhibition of JNK alleviates TGF-beta stimulated invasion, N-cadherin expression and wound healing migration. Neutralization of N-cadherin activity by the GC-4 or by a 10-mer N-cadherin peptide or by siRNA reduces directional migration, filopodia formation, polarization and Golgi-complex reorientation during wound healing. Taken together, our study identifies a new mechanism in which cancer cells contribute to the coordination of invasion of stromal myofibroblasts.

Regulation of endometrial stromal cell matrix metalloproteinase activity and invasiveness by interleukin-8

Objective To evaluate the effect of interleukin-8 (IL-8) on endometrial stromal cell metalloproteinase (MMP) activity and invasiveness. Design Experimental laboratory study. Setting University medical center. Patient(s) Reproductive age women without endometriosis. Intervention(s) Endometrial stromal cells were grown in culture and treated with recombinant IL-8 (0.0001–10 ng/mL) for 24 to 48 hours. Supernatants were collected for soluble assay of collagenase activity and gelatin zymography. Endometrial stromal cells were plated on 8-μm pore membranes coated with Matrigel or human simple matrix and treated with IL-8 for 48 hours. Main outcome measure(s) Collagenase activity and MMP2 and 9 activity of control vs. IL-8 treated endometrial cells, and number of endometrial cells that invade through Matrigel or human simple matrix. Result(s) Collagenase activity in cells treated with IL-8 (1–10 ng/mL) was higher than in control cells. On gelatin zymograms, MMP2 and MMP9 activity of endometrial stromal cells was stimulated by IL-8 treatment (1–10 ng/mL). The number of cells that invaded the Matrigel or human simple matrix was 1.7-fold higher in the group treated with 10 ng/mL of IL-8 compared with the control group. Conclusion(s) IL-8 increases MMP activity and invasive capability of endometrial stromal cells in culture; IL-8 may be involved in the pathogenesis of endometriosis and menstrual physiology.

Hepatocyte growth factor/Met system promotes endometrial and endometriotic stromal cell invasion via autocrine and paracrine pathways.

Endometrial stromal cells reportedly have a role in the initial invasion of endometrial tissue into the peritoneum. Hepatocyte growth factor (HGF), which is a ligand for the c-met protooncogene product (Met), stimulates proliferation and invasion of a large number of cells. In this study we investigated the role of the HGF/Met system in the pathogenesis of endometriosis. HGF concentrations in the peritoneal fluid of patients with endometriosis were significantly higher than in those without endometriosis and correlated positively with revised American Society of Reproductive Medicine scores. We showed that the peritoneum and endometriotic stromal cells may be major sources of HGF in peritoneal fluid. Endometrial and endometriotic stromal cells expressed the Met receptor, which was activated by endogenous and exogenous HGF. HGF enhanced stromal cell proliferation and invasion. We also demonstrated that the HGF-stimulated stromal cell invasion was due in part to the induction of urokinase-type plasminogen activator, a member of the extracellular proteolysis system. In conclusion, the HGF/Met system is involved in the pathogenesis of endometriosis by promoting stromal cell proliferation and invasion of shed endometria and endometrial lesions via autocrine and paracrine pathways.

4P-1122 Catechin inhibits directly matrix metalloproteinases activity

Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. We recently reported that inhibition of COX-2 decreased migration as well as invasion of human endometriotic epithelial and stromal cells. Results of the present study indicates that selective inhibition of PGE2 receptors EP2 and EP4 suppresses expression and/or activity of MMP1, MMP2, MMP3, MMP7 and MMP9 proteins and increases expression of TIMP1, TIMP2, TIMP3, and TIMP4 proteins and thereby decreases migration and invasion of human immortalized endometriotic epithelial and stromal cells into matrigel. The interactions between EP2/EP4 and MMPs are mediated through Src and β-arrestin 1 protein complex involving MT1-MMP and EMMPRIN in human endometriotic cells. These novel findings provide an important molecular and cellular framework for further evaluation of selective inhibition of EP2 and EP4 as potential nonsteroidal therapy for endometriosis in childbearing-age women.

An in vitro model to study the pathogenesis of the early endometriosis lesion.

To determine if whole fragments of endometrium can adhere to peritoneum with intact mesothelium. Tissue culture and immunohistochemical study. University Medical Center. Reproductive-age women undergoing surgery for benign conditions. Whole explants of human peritoneum from the anterior abdominal wall and the posterior surface of the uterus were cultured with whole fragments of mechanically dispersed endometrium. Adhesion of endometrial fragments to the surface of the peritoneum was evaluated. Adherent fragments of endometrium were identified using the dissecting microscope and by performing serial sections of the peritoneum explants for light and confocal laser-scanning microscopy. Immunohistochemical staining of the mesothelium with antibodies to cytokeratin and vimentin was used to ensure an intact layer of mesothelium beneath the endometrial implants. Transmission electron microscopy was also used to evaluate the adhesion of endometrium to the mesothelium. Endometrium was identified attached to the surface of the peritoneum. After 18-24 hours of culture, the majority of implants did not have identifiable mesothelium beneath them, but most had intact mesothelium running up to the point of attachment. Approximately 10% of the endometrial implants had intact mesothelium at the site of attachment. After 1 hour of culture, both endometrial stromal and epithelial cells were attached to intact mesothelium in nearly all cases. Early transmesothelial invasion involves endometrial stromal cells. Endometrial stromal and epithelial cells can attach to the intact mesothelial surface of the peritoneum. Endometrial stromal cell invasion through the mesothelium occurs in less than 18-24 hours.

Increased invasion activity of endometrial stromal cells and elevated expression of matrix metalloproteinase messenger RNA in the uterine tissues of mice with experimentally induced adenomyosis

Objective: This study was undertaken to determine whether changes in stromal cell invasiveness and matrix metalloproteinase activity in mouse uterine tissues were involved in the development of adenomyosis that was induced by ectopic pituitary grafting. Study Design: The invasion activity of endometrial stromal cells into various kinds of extracellular matrix components was examined in experimentally induced adenomyotic and control uteri in vitro. The effect of an inhibitor of matrix metalloproteinase on the invasion activity was also examined. Differences between the expression of matrix metalloproteinases in adenomyotic and control uteri were examined with the use of degenerate primer-driven reverse transcription-polymerase chain reaction, dot-blot hybridization, and gelatin zymography. Results: Stromal cells that were obtained from adenomyotic uteri markedly invaded the reconstituted basement membrane matrix and gelatin, and the matrix metalloproteinase inhibitor suppressed the invasion. Expression of membrane-type matrix metalloproteinase –14 messenger RNA and gelatinase activity was elevated in accordance with the development of adenomyosis. Conclusion: An elevation of the invasion activity of stromal cells into the extracellular matrix is related to the development of adenomyosis; matrix metalloproteinase –14 may play an important role in the invasion of endometrial tissues into the myometrium. (Am J Obstet Gynecol 2001;185: 1374-80.)

Development of endometriosis-like lesions after transplantation of human endometrial fragments onto the chick embryo chorioallantoic membrane.

The chick embryo chorioallantoic membrane (CAM) bioassay was used to investigate the early pathogenesis of endometriosis. Endometrial fragments were explanted onto the CAM. The grafts including the surrounding CAM were excised at 24, 48 or 72 h after explantation, fixed and embedded in paraffin. Immunohistochemical analysis was used to distinguish endometrial cells. To identify cells of human origin, in-situ hybridization was performed using a probe specific for human chromosome 1. After 24 h, direct contact between endometrial stromal as well as epithelial cells and the mesenchymal layer of the CAM was observed. Invasion of both stromal cells and intact endometrial glands into the mesenchymal layer was observed after 48 h. At 72 h, endometriosis-like lesions were observed in the mesenchymal layer. Positive staining with antibodies to vimentin and pan-cytokeratin was observed in the invading cells as well as in the lesions. In the lesions these positively stained cells showed in-situ hybridization signals for human chromosome 1, confirming their human origin. In conclusion, after 3 days of incubation, endometriosis-like lesions consisting of human endometrial glands and stromal cells were found in the mesenchymal layer of the CAM. These lesions apparently resulted from the invasion of intact human epithelial structures and stromal cells.

Defects of basement membrane and hemidesmosome structure correlate with malignant phenotype and stromal interactions in HaCaT-Ras xenografts

Benign and malignant HaCaT-ras clones, derived from immortalized HaCaT cells were grown as nude mouse surface transplants rendering a human tumor progression model. Searching for malignancy-related alterations, the deposition, localization and mRNA of basement membrane and hemidesmosome components were analysed by immunofluorescence, in situ hybridization and electron microscopy. Initially, at 1 week epithelia of benign and malignant cells revealed a similarly low polarity and an enlarged ‘activated basal’ compartment, reflected by partial dislocation and extended pericellular staining of the hemidesmosome constituent integrin α6β4 seen by immunofluorescence. Whereas benign grafts eventually normalized, closely resembling grafts of HaCaT cells, malignant growth was correlated with a persisting epithelial activation state and continuing higher expression of α6 (by immunoflourescence and in situ hybridization). The basement membrane components bullous pemphigoid antigen 1, laminin-5 and collagen IV exhibited a largely linear distribution at 1 week. However, in the malignant cell transplants initially minor basement membrane discontinuities became more severe at around 2 weeks, associated with close stromal cell contacts, angiogenesis and invasion. Most striking were basement membrane alterations seen by electron microscopy. At 1 week stretches of basement membrane had developed in malignant transplants, though to a much lesser extent than in benign specimens. With invasion these basement membrane structures mostly disappeared despite persistent although variable immunofluorescence, suggesting high turnover without ultrastructural assembly. The hemidesmosome structures were defective throughout, completely lacking anchoring plaques with keratin filaments, whereas they were still associated with basement membrane deposits. Thus, malignant HaCaT-ras transplants, while initially resembling regenerating wounds, revealed an increasing loss of tissue polarity and basement membrane structures, which seemed to be accelerated upon stromal cell contacts.

Defects of basement membrane and hemidesmosome structure correlate with malignant phenotype and stromal interactions in HaCaT-Ras xenografts

Benign and malignant HaCaT-ras clones, derived from immortalized HaCaT cells were grown as nude mouse surface transplants rendering a human tumor progression model. Searching for malignancy-related alterations, the deposition, localization and mRNA of basement membrane and hemidesmosome components were analysed by immunofluorescence, in situ hybridization and electron microscopy. Initially, at 1 week epithelia of benign and malignant cells revealed a similarly low polarity and an enlarged ‘activated basal’ compartment, reflected by partial dislocation and extended pericellular staining of the hemidesmosome constituent integrin α6β4 seen by immunofluorescence. Whereas benign grafts eventually normalized, closely resembling grafts of HaCaT cells, malignant growth was correlated with a persisting epithelial activation state and continuing higher expression of α6 (by immunoflourescence and in situ hybridization). The basement membrane components bullous pemphigoid antigen 1, laminin-5 and collagen IV exhibited a largely linear distribution at 1 week. However, in the malignant cell transplants initially minor basement membrane discontinuities became more severe at around 2 weeks, associated with close stromal cell contacts, angiogenesis and invasion. Most striking were basement membrane alterations seen by electron microscopy. At 1 week stretches of basement membrane had developed in malignant transplants, though to a much lesser extent than in benign specimens. With invasion these basement membrane structures mostly disappeared despite persistent although variable immunofluorescence, suggesting high turnover without ultrastructural assembly. The hemidesmosome structures were defective throughout, completely lacking anchoring plaques with keratin filaments, whereas they were still associated with basement membrane deposits. Thus, malignant HaCaT-ras transplants, while initially resembling regenerating wounds, revealed an increasing loss of tissue polarity and basement membrane structures, which seemed to be accelerated upon stromal cell contacts.

Collagen type IX and developmentally regulated swelling of the avian primary corneal stroma.

A critical event in avian corneal development occurs when the acellular primary stroma swells and becomes populated by mesenchymal cells that migrate from the periphery. These cells then deposit the mature stromal matrix that exhibits the unique features necessary for corneal function. Our previous work correlated the disappearance of collagen type IX immunoreactivity at stage 27 (5 1/2-6 days) with matrix swelling and invasion. To investigate further the mechanism of this disappearance, we employed immunohistochemistry after tissue fixation with Histochoice, a non-crosslinking fixative, immunoblot analysis of protein extracts, and gel substrate chromatography (zymography) to detect endogenous proteolytic activity. We found that corneas fixed in Histochoice retain immunoreactivity for type IX collagen for 1-2 days after corneal swelling. This immunoreactivity, however, becomes extractable from tissue sections of unfixed corneas at the time of initiation of stromal swelling and mesenchymal cell invasion. Immunoblot analysis confirmed that, following swelling, immunoreactivity for collagen IX decreased substantially in corneas, but not in the vitreous body, which served as a comparison. Analysis of ammonium sulfate (AS) fractions of such extracts indicated that, at the time of swelling, much of the immunoreactivity for type IX collagen in cornea shifted from the AS precipitate (containing high molecular weight molecules) to the AS supernatant (containing smaller fragments). In contrast, collagen IX immunoreactivity from the vitreous was precipitated by ammonium sulfate throughout the period of study. Collagen type II, a major fibrillar collagen in both the corneal stroma and vitreous, remained in the high molecular weight fraction at all times examined. Zymography detected the presence of the latent (proenzyme) form of gelatinase A (MMP-2) before corneal swelling and invasion (4 days), and both the latent and active forms of the enzyme after corneal swelling. This suggests tissue-specific, developmentally regulated proteolysis of collagen IX as a trigger for corneal matrix swelling.

Collagen type IX and developmentally regulated swelling of the avian primary corneal stroma

A critical event in avian corneal development occurs when the acellular primary stroma swells and becomes populated by mesenchymal cells that migrate from the periphery. These cells then deposit the mature stromal matrix that exhibits the unique features necessary for corneal function. Our previous work correlated the disappearance of collagen type IX immunoreactivity at stage 27 (5½–6 days) with matrix swelling and invasion. To investigate further the mechanism of this disappearance, we employed immunohistochemistry after tissue fixation with Histochoice, a non-crosslinking fixative, immunoblot analysis of protein extracts, and gel substrate chromatography (zymography) to detect endogenous proteolytic activity. We found that corneas fixed in Histochoice retain immunoreactivity for type IX collagen for 1–2 days after corneal swelling. This immunoreactivity, however, becomes extractable from tissue sections of unfixed corneas at the time of initiation of stromal swelling and mesenchymal cell invasion. Immunoblot analysis confirmed that, following swelling, immunoreactivity for collagen IX decreased substantially in corneas, but not in the vitreous body, which served as a comparison. Analysis of ammonium sulfate (AS) fractions of such extracts indicated that, at the time of swelling, much of the immunoreactivity for type IX collagen in cornea shifted from the AS precipitate (containing high molecular weight molecules) to the AS supernatant (containing smaller fragments). In contrast, collagen IX immunoreactivity from the vitreous was precipitated by ammonium sulfate throughout the period of study. Collagen type II, a major fibrillar collagen in both the corneal stroma and vitreous, remained in the high molecular weight fraction at all times examined. Zymography detected the presence of the latent (proenzyme) form of gelatinase A (MMP-2) before corneal swelling and invasion (4 days), and both the latent and active forms of the enzyme after corneal swelling. This suggests tissue-specific, developmentally regulated proteolysis of collagen IX as a trigger for corneal matrix swelling. Dev. Dyn. 1998;212:27–37. © 1998 Wiley-Liss, Inc.