Iris A. Montoya-Rodriguez

Short-term culture of peritoneum explants confirms attachment of endometrium to intact peritoneal mesothelium.

OBJECTIVE To evaluate the initial adhesion of endometrium to the peritoneum. DESIGN Descriptive study using light and confocal laser-scanning microscopy, immunohistochemistry, and transmission electron microscopy. SETTING University-based laboratory. PATIENT(S) Women without endometriosis undergoing surgery for benign conditions. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Explants of peritoneum (n = 20), prepared from four patients, were cultured for 1 hour with mechanically dispersed proliferative or secretory endometrium. Peritoneum was cultured with endometrium from the same patient. Specimens were fixed and serially sectioned for hematoxylin and eosin stain, immunohistochemistry using an anti-cytokeratin monoclonal antibody, and transmission electron microscopy. RESULT(S) In 17 of 20 explants, endometrium was adherent to intact mesothelium. There was no evidence of transmesothelial invasion at any sites of attachment. Although in most cases endometrium was adherent to mesothelium via endometrial stroma, there were many sites of endometrial epithelium-mesothelium attachment. Confocal laser scanning microscopy demonstrated an intact monolayer of cytokeratin-positive cells below the sites of endometrial implantation. Transmission electron microscopy demonstrated intact, viable, mesothelial cells below sites of attachment. CONCLUSION(S) This study demonstrates that endometrium rapidly adheres to intact peritoneal mesothelium. In addition, this study demonstrates that endometrial epithelial cells, as well as stroma, can attach to mesothelium. Further studies are needed that characterize the mechanism of endometrial-mesothelial cell adhesion.

Time Series Analysis of Transmesothelial Invasion by Endometrial Stromal and Epithelial Cells Using Three-Dimensional Confocal Microscopy

OBJECTIVE To evaluate endometrial adhesion and invasion of peritoneal mesothelium. DESIGN Descriptive study using confocal laser-scanning microscopy. SETTING University-based laboratory. PATIENT(S) Women undergoing surgery for benign conditions. INTERVENTION(S) Fluorescence-labeled peritoneal mesothelial cells (PMCs) were grown on coverslips. Fluorescence-labeled endometrial stromal cells (ESCs) and epithelial cells (EECs) and myometrial cells (Myos) were plated on the PMCs. Cultures were examined at 1, 6, 12, and 24-27 hours with differential interference contrast and confocal laser-scanning microscopy. MAIN OUTCOME MEASURE(S) Demonstration of adherence and invasion of endometrial cells through peritoneal mesothelium. RESULT(S) At 1 hour, there was adherence of the ESCs, EECs, and Myos on the perimeter of PMCs. There was no invasion by the Myos. By 6 hours, ESCs and EECs spread over the surface of the PMCs and extended cell processes through PMC junctions. Extension of pseudopodia under the PMCs followed. By 12 hours, there was vacuolization and lifting of PMCs that had been undermined by endometrial cells. CONCLUSION(S) This is the first time-phase study to demonstrate adherence and the process of invasion of endometrial cells through the mesothelium. The application of three-dimensional confocal laser-scanning microscopy is a novel technique that can be used to further examine mechanisms involved in the pathogenesis of the early endometriotic lesion.

Composition of the extracellular matrix of the peritoneum.

OBJECTIVE To localize the extracellular matrix proteins collagen I, collagen IV, fibronectin, and laminin in the peritoneal membrane. STUDY DESIGN Peritoneal biopsies (n = 13) from the anterior abdominal wall and the uterine serosa (n = 3) were incubated with antibodies to collagen IV, laminin, collagen I, and fibronectin. Specimens were examined using light and confocal laser scanning microscopy. RESULTS All of the extracellular matrix (ECM) proteins were present immediately under the mesothelium. Collagen (Col) IV and laminin (LM) were seen in the smooth muscle of microvascular structures, in the subendothelial basement membrane, and were present in a fascicular pattern in the peritoneal stroma. Collagen I was distributed diffusely in the peritoneal stroma. Fibronectin was also present in the subendothelial basement membrane. CONCLUSIONS The resolution of the confocal microscope allowed for localization of extracellular matrix proteins in relation to the mesothelium. The presence of collagen IV, laminin, collagen I, and fibronectin under the mesothelium suggests that cells invading the peritoneum must have the ability to degrade and remodel this matrix.

Mesothelial cell–associated hyaluronic acid promotes adhesion of endometrial cells to mesothelium

OBJECTIVE To evaluate the role of hyaluronic acid in the attachment of endometrial cells to mesothelium. DESIGN In vitro study of adhesion of endometrial stromal and epithelial cells to mesothelial cells. SETTING University medical center. PATIENT(S) Reproductive-age women without endometriosis undergoing surgery for benign conditions. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The effect of hyaluronidase treatment of mesothelial cells or endometrial cells on adhesion of (51)Cr labeled endometrial stromal and epithelial cells to monolayers of mesothelium was evaluated. The expression of CD44, the hyaluronate receptor, was evaluated by western blot. RESULT(S) Hyaluronidase pretreatment of mesothelial cells decreased the binding of endometrial stromal and epithelial cells to mesothelium by 39% (P< .02) and 31% (P< .03), respectively. There was no effect on endometrial cell binding to mesothelial cells or to collagen IV when the endometrial cells were pretreated with hyaluronidase. CD44 expression by endometrial stromal and epithelial cells was demonstrated by western blot. CONCLUSIONS This study demonstrates that mesothelial cell-associated hyaluronic acid is involved in attachment of endometrial stromal and endometrial epithelial cells to the mesothelium. We hypothesize that binding of hyaluronic acid by endometrial cells is involved in the pathogenesis of the early endometriotic lesion.

Mesothelium expression of integrins in vivo and in vitro.

To characterize the expression of α subunits of integrin adhesion molecules in peritoneal tissue in vivo and in vitro. Peritoneum from the anterior abdominal wall (n = 22) and the serosa of the posterior uterus (n = 11) was obtained from women of reproductive age without endometriosis who were undergoing surgery for benign conditions. Immunohistochemical studies were performed on serial of peritoneum from the anterior abdominal wall, the uterine serosa, mesothelial monolayer cultures, and peritoneum explants from the abdominal wall using monoclonal antibodies to α subunits of integrin adhesion molecules. Electron microscopy was performed to localize these adhesion molecules in the mesothelium. The mesothelial expression of α integrin subunits was identical in the anterior peritoneum and uterine serosa. In vivo the mesothelium strongly expressed α2 and α3 and variably expressed α6. In the monolayer cultures there was moderate/strong staining for α2, α3, and α5; and αv; α6 was variably expressed. The ultrastructure of the mesothelium was unique in the anterior peritoneum, uterine serosa, and the nonolayer cultures. The integrin subunits were distributed throughout the cytoplasm, were expressed in the plasma membrane, and were present on the surface (ie, towards the peritoneal cavity) of the mesothelium. Integrins are expressed by the mesothelium of the peritoneum. The mesothelium expression of integrins in vivo differs from that of the mesothelium integrin expression in monolayer culture and explant culture.

Expression of the α2β1 and α3β1 integrins at the surface of mesothelial cells: a potential attachment site of endometrial cells

Abstract Objective: To localize α2β1 and α3β1 integrins in the cell membrane of peritoneal mesothelium in vivo and in vitro. Design: Descriptive study using confocal and two-photon laser-scanning microscopy. Setting: University-based laboratory. Patient(s): Women without endometriosis undergoing surgery for benign conditions. Intervention(s): None. Main Outcome Measure(s): Peritoneal biopsies (n = 9) and mesothelial monolayer cultures (n = 4) were incubated with antibodies to the α2 and α3 subunits and to the intact α2β1 and α3β1 integrins. Specimens were examined with laser-scanning microscopy. Result(s): The α2 and α3 subunits and the intact α2β1 and α3β1 integrins were identified at the base of the mesothelial cells (i.e., toward the basement membrane). There was also expression of the α2 and α3 subunits and the intact α2β1 and α3β1 integrins at the cell surface (i.e., toward the peritoneal cavity). Conclusion(s): The resolution of the confocal and two-photon laser-scanning microscope enabled localization of integrins in mesothelial cells. The presence of α2β1 (collagen-laminin receptor) and α3β1 integrins (collagen-laminin-fibronectin receptor) at the base of mesothelial cells suggests a role for these molecules in adhesion to the basement membrane. The presence of these molecules at the cell surface suggests a potential locus for cell adhesion in such processes as endometriosis and cancer metastasis.

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

Abstract: Objective—To determine if whole fragments of endometrium can adhere to peritoneum with intact mesothelium. Design—Tissue culture and immunohistochemical study. Setting—University Medical Center. Patients—Reproductive‐age women undergoing surgery for benign conditions. Interventions—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. Main Outcome Measures—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. Results—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. Conclusions—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.

The α2β1 and α3β1 integrins do not mediate attachment of endometrial cells to peritoneal mesothelium

Abstract Objective: To evaluate the possible role of mesothelial α 2 β 1 and α 3 β 1 integrins in the attachment of endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs). Design: In vitro study. Setting: University medical center. Patient(s): Women of reproductive age (n = 26). Main Outcome Measure(s): Mesothelial cells were grown on collagen IV. Endometrial stromal cells and EECs were plated on mesothelial cells for 1 hour. Before plating, mesothelial cells or endometrial cells were incubated with antibodies to α2, α3, and β1 integrin subunits. The effect of these antibodies on ESC and EEC binding to collagen IV and collagen I was also examined. The expression of collagen I, collagen IV, fibronectin, and laminin by cultured ESCs and EECs was examined. Result(s): The anti-integrin antibodies had no effect on endometrial binding to mesothelium. The β1 integrin antibody decreased binding of ESCs and EECs to the collagen matrices. In culture, ESCs and EECs expressed collagen I, collagen IV, fibronectin, and laminin to varying degrees. Conclusion(s): The initial adhesion of ESCs and EECs to mesothelium is not mediated by β1 integrins. In contrast, ESC and EEC attachment to collagen IV and collagen I, which are present in the submesothelial extracellular matrix, is mediated by β1 integrins.