Chunfeng Ma

Expression of matrix metalloproteinases and tissue inhibitor of matrix metalloproteinases in mesothelial cells and their regulation by transforming growth factor-beta1.

Tissue injury and pelvic inflammation often results in peritoneal scar tissue formation. The objective of this study was to determine whether mesothelial cells which line the peritoneal cavity express matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), and if their expression is regulated by transforming growth factor‐β1, a key regulator of tissue fibrosis. For this purpose we used Met‐5A cells, a cell line derived from human normal mesothelial cells, and for comparative analysis we used U‐937 cells, a human monocytic/macrophage cell line. The cells were treated with transforming growth factor‐β1 (1 ng/ml) for various time periods and the levels of MMP and TIMP mRNA and protein expression were determined using quantitative reverse transcription‐polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. The results indicate that the mesothelial cells and macrophages express MMP‐1 (collagenase‐1), MMP‐3 (stromelysin‐1), TIMP‐1 and TIMP‐2 mRNA and protein at various levels, with significantly higher TIMPs than MMPs, and higher MMP‐1 than MMP‐3 (p < 0.001). The mesothelial cells express significantly less MMP‐1, higher MMP‐ 3 and similar levels of TIMP mRNA compared to macrophages. In a time‐dependent manner, treatment of the mesothelial cells with transforming growth factor‐β1 resulted in a significant decrease in the expression of MMP‐1, while increasing the expression of TIMP‐1 mRNA (p = 0.05). In contrast, MMP‐3 and TIMP‐2 expression was unaffected in mesothelial cells and in macrophages, compared to untreated controls. There was a significant increase in secreted MMP‐1 and TIMP‐2 by mesothelial cells following transforming growth factor‐β1 treatments in a time‐dependent manner (p = 0.05 and p = 0.01), without affecting the secretion of these proteins by macrophages. A major portion of MMP‐1 in the culture conditioned media of both cell types was found in complex with TIMP‐1. The ratios of MMP‐1/TIMPs production were significantly higher than MMP‐3/TIMPs in mesothelial cells and macrophages, and progressively decreased following transforming growth factor‐β1 treatments (p < 0.05). In conclusion, these results indicate that mesothelial cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by transforming growth factor‐β1, a mechanism that in part may influence the outcome of peritoneal tissue repair and adhesion formation.

Differential expression of interleukins (IL) IL-13 and IL-15 throughout the menstrual cycle in endometrium of normal fertile women and women with recurrent spontaneous abortion

Interleukins (IL)-13 and IL-15 are novel cytokines and key regulators of immune/inflammatory related responses that are critical in the outcome of various normal biological and associated abnormalities of the endometrium. The present study determined the temporal and spatial expression of IL-13 and IL-15 mRNA and protein in endometrium of normal fertile women throughout the menstrual cycle, and examined whether profiles of their expression differ from endometrium of women with unexplained recurrent spontaneous abortion (RSA). Using quantitative RT-PCR, ELISA and immunohistochemistry we found that IL-13 and IL-15 mRNA and protein are expressed in control endometrium throughout the menstrual cycle and in RSA (cycle days 21-23) with peak expression detected immediately after and prior to onset of menses, and two distinct periods corresponding to late proliferative and the early-mid secretory phases, respectively. The ratio of IL-13:IL-15 expression revealed a predominance in IL-13 expression during late proliferative/early secretory phase and IL-15 during the mid secretory phase. Compared to control endometrium, endometrium of women with RSA expresses elevated levels of IL-13 and IL-15, with IL-13:IL-15 ratio favoring IL-13. The immunoreactive IL-13 and IL-15 were localized primarily in endometrial luminal epithelial cells with an increased intensity in glandular epithelial and stromal cells in RSA. In conclusion, the results indicate that endometrium of normal fertile women expresses IL-13 and IL-15, with a distinct profile during the menstrual cycle and elevated expression in women with RSA. Although the biological significance of IL-13 and IL-15 in human endometrium and their elevated expression in RSA await investigation, these cytokines with distinct biological functions may regulate endometrial inflammatory/immune responses, tissue repair and receptivity for embryo implantation.

Role of transforming growth factor beta-1 in peritonitis-induced adhesions.

Peritonitis is a major cause of intra-abdominal adhesion formation. The overexpression of transforming growth factor beta-l (TGF-Pl), a potent mitogen, chemoattractant, and stimulant for collagen synthesis by fibroblasts, has been linked to tissue fibrosis at various sites throughout the body including peritoneal adhesion formation. Hence we hypothesized that the mechanism(s) involved in peritonitis-induced adhesion formation may be mediated through the upregulation of TGF-β expression. Peritonitis was induced in rats by cecal ligation and puncture, while a control group underwent sham operation. Adhesions were scored and harvested from both groups at 0,6 and 12 hours and at 1,2,4, 7, and 28 days. Tissue expression of TGF-PI mRNA was determined by quantitative reverse transcription-polymerase chain reaction and TGF-β protein was localized by immunohistochemical analysis. Serum and peritoneal fluid TGF-β concentrations were quantified by enzyme-linked immunosorbent assay. Compared with sham operation, peritonitis was associated with a significantly greater incidence of abdominal adhesions and a significant increase in the levels of TGF-β 1 mRNA expression at days 2,4, and 7. Immunostaining intensity of TGF-β in adhesions from the peritonitis group also steadily rose through day 7. In peritoneal fluid, the ratio of active:total TGF-βl was significantly increased in the peritonitis group on days 1, 2, and 4 compared with the sham group. These results suggest that peritonitis is associated with the upregulation of TGF-βl, a mechanism that may exacerbate adhesion formation.

Expression of matrix metalloproteinase (MMP-1) and tissue inhibitor of MMP in serosal tissue of intraperitoneal organs and adhesions

OBJECTIVE To compare expression of matrix metalloproteinase (MMP-1) and tissue inhibitor of MMP (TIMP-1) in serosal tissue of intraperitoneal organs and adhesions. DESIGN Prospective and cross-sectional study. SETTING Academic research centers. PATIENT(S) Patients undergoing abdominal or pelvic surgery. INTERVENTION(S) MMP-1 and TIMP-1 expression. MAIN OUTCOME MEASURE(S) Expression of messenger ribonucleic acid (mRNA) and protein was measured by using quantitative reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. RESULT(S) Serosal tissue of intraperitoneal organs and adhesions express MMP-1 and TIMP-1 mRNA and protein at levels that are consistently varied with 10- to 10,000-fold and 2- to 10-fold higher TIMP, mRNA and protein, respectively. Parietal peritoneum, fallopian tubes and ovaries express higher MMP-1 mRNA levels compared with uterus and adhesions; the lowest expression is found in small and large bowels, subcutaneous tissue. and omentum. Expression of TIMP-1 mRNA was less variable; the highest level was found in the uterus and the lowest in subcutaneous tissue and small bowels. There was less variability in MMP-1 and TIMP-1 protein content than mRNA expression; ovaries and adhesions contained the highest MMP-1 and TIMP-1 levels, respectively, and peritoneum contained the lowest. The MMP-1 and TIMP-1 content and ratios further indicate limited MMP-1 proteolytic activity. Although tissues from premenopausal women express more MMP-1 and TIMP-1, expression did not differ by sex or age. CONCLUSION(S) Because MMP-1 and TIMP-1 expression varies consistently among the serosal tissues of peritoneal organs and adhesions, and because tissue injury alters their expression, site-specific variations in expression of these substances may predispose a particular organ to develop more adhesions.

Differential expression of matrix metalloproteinase and tissue inhibitor of MMP in serosal tissue of intraperitoneal organs and adhesions

Objective To comparatively analyse the expression of matrix metalloproteinase (MMP‐3) and tissue inhibitor of MMP (TIMP‐2) in serosal tissue of intraperitoneal organs and adhesions, as well as peritoneal fluid and serum of subjects with and without adhesions.

Differential Expression of Transforming Growth Factor-β1 and Transforming Growth Factor-β Receptors in Myometrium of Women With Failed Induction of Labor, No Labor, and Preterm Labor

Objective: Comparative analysis of transforming growth factor-β1 (TGF-β1) and TGF-β receptor type I and type II messenger RNA (mRNA) and protein expression in myometrium of women who had unsuccessful labor induction, with those without labor or in preterm labor complicated by chorioamnionitis. Methods: Small segments of myometrium were collected from women who were undergoing cesarean delivery for unsuccessful labor induction (n = 5), elective cesarean without labor (n = 5), or cesarean delivery for complications related to preterm labor and chorioamnionitis (n = 5). Total RNA was isolated from these tissues and subjected to competitive quantitative reverse-transcription-polymerase chain reaction (Q-RT-PCR) to determine the level of TGF-β1, and TGF-β type I and type II receptor mRNA expression. Tissue sections were prepared from paraffin-embedded specimens and immunostained for TGF-β1 and receptor proteins using specific polycolonal antibodies. The data were analyzed by unpaired Student t test and Kruskal-Wallis analysis of variance. Results: Myometrium from women who had unsuccessful labor induction expressed higher levels of TGF-β1 mRNA (2.21 ± 0.28 × 106 copies/μg of total cellular RNA) than those with preterm labor (4.53 ± 0.2 × 105 copies), or without labor [3.13 ± 2.6 × 104 copies (P < .05)]. The level of TGF-β type I receptor mRNA expression did not vary; however, type II receptor expression was significantly lower in myometrium from preterm labor (1.36 ± 0.36 × 105 copies) compared with those from unsuccessful labor induction (3.42 ± × 106 copies) or without labor (9.65 ± 3.2 × 105 copies). Immunoreactive TGF-β1 and TGF-β receptor proteins were present in all myometrial tissues, and their intensity reflected that of the mRNA expression in these tissues. Conclusion: TGF-β1 and TFG-β type II receptors are expressed differently in myometrium of women who had unsuccessful labor induction compared with those without labor or with preterm labor complicated by chorioamnionitis. Because TGF-β is a key regulator of tissue remodeling which is central to initiation of normal labor, alterations in TGF-β and/or TGF-β receptor expression may lead to changes in the outcome of labor, at least at the myometrial level.

Increased expression of interferon-inducible protein-10 during surgically induced peritoneal injury

Interferon‐inducible protein 10 (IP‐10) is a key regulator of neutrophils, monocytes, and lymphocytes, cells infiltration whose secretory products play a key role in peritoneal wound healing. The objective of the present study was to determine whether IP‐10 is expressed by parietal peritoneum and whether its temporal and spatial expression is altered following surgically induced peritoneal injury during healing. Peritoneal sidewall injuries were induced in mice (N = 60), and the severity of adhesions were graded at 12 hours and 1, 2, 4, and 7 days postsurgery. After collection of peritoneal washes, the injured peritoneum with associated adhesion and intact parietal peritoneum were collected to determine IP‐10 mRNA and protein expression using quantitative reverse transcription‐polymerase chain reaction, enzyme‐linked immunosorbent assay, and immunohistochemistry. Peritoneal injury resulted in adhesion formation with increased severity by day 7 postsurgery. The intact parietal peritoneum expressed IP‐10 mRNA, whose level of expression significantly increased following peritoneal injury and reached a maximum at day 4 (p = 0.001), declining to the uninjured control levels by day 7 post‐injury. The level of IP‐10 in peritoneal washes also increased as a result of peritoneal injury. Immunohistochemically, IP‐10 was localized to various inflammatory and immune cells, adhesion fibroblasts, and mesothelial cells, and its intensity increased during the course of wound healing. In conclusion, we showed that parietal peritoneum expresses IP‐10 and peritoneal tissue injury results in an elevated level of its expression throughout the early phase of wound healing. The results suggest that IP‐10 and its elevated expression may play a role in peritoneal cellular activities that influence the early phases of tissue repair and, possibly, the development of peritoneal adhesions. (WOUND REP REG 2003;11:120–126)

Differential expression of integrin αv and β3 in serosal tissue of human intraperitoneal organs and adhesion

Abstract Objective: To assess the expression of integrin αv and β3 in the serosal tissue of intraperitoneal organs and adhesions in persons with and without adhesions. Design: Prospective study. Setting: Academic research centers. Patient(s): Fifty-seven patients undergoing abdominal or pelvic surgery. Main Outcome Measure(s): Integrin αv and β3 messenger RNA (mRNA) expression was evaluated by quantitative reverse transcription polymerase chain reaction. Result(s): The serosal tissue of the parietal peritoneum, uterus, fallopian tubes, ovary, and the large and small bowel, as well as peritoneal adhesions, skin, fascia, subcutaneous tissue, and omentum, expresses integrin αv and β3 mRNA. The level of αv and β3 mRNA expression varied among these tissues; expression of the former substance was highest in uterine serosa and lowest in the skin and small bowel, and expression of the latter substance was highest in the fallopian tubes and skin and lowest in the uterine serosa. Parietal peritoneum and adhesions express equal levels of integrin αv; however, integrin β3 expression was >100-fold lower in adhesions than in peritoneum. The level of integrin β3 expression in omentum, small and large bowels, and subcutaneous tissue was 100-fold to 10,000-fold lower than in other tissues. Conclusion(s): Serosal tissue of peritoneal organs and adhesions express variable levels of integrin αv and β3 mRNA. On the basis of such variation and the knowledge that tissue injury alters local integrin expression, integrins may play a key role in adhesion development, particularly in tissue with higher integrin expression.