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Maspin, an inhibitor of cell migration and a stimulator of adhesion of cells to the ECM, is synthesized and released by corneal keratocytes into the extracellular matrix. When the cornea is wounded, the quiescent stromal keratocytes underlying the wound undergo apoptosis and cells adjacent to this apoptotic area convert to fibroblasts or myofibroblasts. This study explores the effect of extracellular maspin on the plasminogen-plasminogen activator system of corneal stromal cells following wounding. Treatment of corneal fibroblasts and myofibroblasts with r-maspin increased extracellular but not cell-associated tissue-type plasminogen activator (tPA), urinary-type plasminogen activator (uPA) or plasminogen activator inhibitor-1 (PAI-1). Despite the extracellular increase in PAI-1, the net effect of maspin treatment was an increase in plasminogen activation. At physiological levels, maspin did not alter uPA or tPA mRNA levels, in these cells. The increase in pro and active uPA was due to decreased clearance in the presence of maspin for myofibroblasts but not for fibroblasts. The clearance of pro and active tPA was normal in fibroblasts indicating different mechanisms for the increase of these homologous enzymes in the two cell types. Increased generation of plasmin by maspin treated corneal stromal fibroblasts and myofibroblasts led to conversion of plasminogen to active plasmin degradation products and angiostatin-like molecules. This study suggests that extracellular maspin increased pro and active uPA and tPA released by corneal fibroblasts and myofibroblasts on the short time scale of 1-4 h, but by 24 h there was no increase over the levels produced without maspin. This augmentation of plasminogen activator activity increases plasmin activation and angiostatin generation. It further indicates that the effect of maspin on uPA and tPA levels is cell type dependent., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down-regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation is involved in the mechanism of down-regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-beta1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for Western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-beta1 were hypermethylated. Down-regulation of maspin synthesis was also associated with histone H3 dimethylation at lysine 9. Both maspin mRNA and protein were re-expressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down-regulation of maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation.

Background: Recently we reported that bacterial lipopolysaccharide (LPS) stimulates release of tumor necrosis factor alpha (TNF-alpha) from porcine coronary arteries and smooth muscle cells cultured from those vessels. It has also been reported that plasma levels of TNF-alpha are elevated after myocardial infarction. Since it is known that the production of reactive oxygen intermediates (ROI) occurs during ischemia and ROI are suggested activators of the nuclear regulatory factor kappaB (NF-kappaB), we tested the hypothesis that release of TNF-alpha from smooth muscle cells could also be stimulated with a ROI-generating system., Materials and Methods: Smooth muscle cells were isolated from porcine coronary arteries. Confluent cells in 48-well culture dishes were treated for 30 min with 0.003 units/ml xanthine oxidase (XO) and 2 mM hypoxanthine (HX) added to the culture medium. The medium was then removed and the cells were washed three times and fresh medium without HX-XO was added. Then, at 1, 3, and 6 h the medium was removed and analyzed for biologically active TNF-alpha. In other experiments, smooth muscle cells were treated with 20 micrograms/ml LPS for 6 h and aliquots of medium analyzed for TNF-alpha. Untreated cells served as controls. Data were analyzed by two-way ANOVA with repeated measures. Extracts of total cell protein were prepared and activation of NF-kappaB was determined by electrophoretic mobility shift assay., Results: Treatment of cells with HX-XO stimulated release of TNF-alpha, which rose to a maximum of 17.5 +/- 1.7 units/mg cell protein at 6 h. This was significantly higher (P < 0. 05) than release stimulated by LPS (10.2 +/- 1.0 units/mg at 6 h) or TNF-alpha detected in the culture medium from untreated control cells (4.2 +/- 0.9 units/mg protein at 6 h). Both HX/XO and LPS activated NF-kappaB., Conclusions: These results support the conclusion that coronary smooth muscle cells are a potential source of TNF-alpha during events that are associated with formation of ROI such as myocardial ischemia., (Copyright 1999 Academic Press.)

Background: Evidence suggests that tumor necrosis factor-alpha (TNF-alpha) is involved in heart diseases such as atherosclerosis. We used porcine coronary arteries and smooth muscle cells cultured from these vessels to study the regulation of production of TNF-alpha. The aims were to determine if bacterial lipopolysaccharide (LPS) could stimulate production; if activation of the nuclear regulatory factor, NF-kappaB, was associated with production; and if intracellular cAMP regulates TNF-alpha in coronary vasculature through a mechanism involving NF-kappaB., Material and Methods: LPS was used to stimulate TNF-alpha production. Forskolin (FSK) and 8-Br-cAMP were added to tissue and cells in order to elevate intracellular cAMP. TNF-alpha release into the bathing medium was measured by the L929 cell cytotoxicity assay. Intracellular cAMP was determined by radioimmunoassay. NF-kappaB activation was determined in whole cell extracts by electrophoretic mobility shift assay., Results: In segments of coronary arteries, LPS stimulated TNF-alpha release which increased with time to a maximum at 6 h (485 +/- 19 units/g tissue) and remained elevated at this level for 24 h. In contrast, the level of TNF-alpha measured at 24 h in medium from coronary tissue not exposed to LPS was 11.1 +/- 4.1 units/g tissue. In the presence of LPS, both FSK and 8-Br-cAMP significantly reduced TNF-alpha release. For instance at 6 h in the presence of LPS and FSK or 8-Br-cAMP, TNF-alpha was 126 +/- 24 and 71.6 +/- 22 units/g tissue, respectively (P < 0.05 vs LPS alone). Tissue levels of cAMP were significantly elevated in the presence of FSK. Similar results were obtained with smooth muscle cells cultured from the coronary arteries; i.e., LPS stimulated TNF-alpha release which was inhibited in a concentration-dependent manner by a rise in intracellular cAMP induced by FSK. In cultured cells release of TNF-alpha stimulated by LPS was associated with activation of NF-kappaB. Neither FSK nor 8-Br cAMP inhibited activation of NF-kappaB by LPS., Conclusions: Porcine coronary arteries produce TNF-alpha from a smooth muscle cell source. Production stimulated by LPS was inhibited by elevated intracellular cAMP and was associated with activation of NF-kappaB. However, activation of NF-kappaB was not inhibited by elevated cAMP, suggesting that the regulatory action of this cyclic nucleotide could lie downstream from activation of the TNF-alpha gene. These results support the view that coronary vessels can be a source of TNF-alpha possibly involved in heart disease., (Copyright 1998 Academic Press.)

Transforming growth factor-beta (TGF-beta) is an important factor in regulating the inflammatory response and the production of extracellular matrix by fibroblasts. These two processes are linked in the formation of fibrous adhesions after abdominal surgery. When the mesothelium is injured a fibrin strand is produced which is populated first by inflammatory cells then by fibroblasts which secrete extracellular matrix forming a permanent adhesion. TGF-beta promotes both chemotaxis of monocytes and the production of extracellular matrix by fibroblasts. We have used a model of abdominal adhesions in rats in which a circle of peritoneum is dissected and then sutured into place again. After 2 weeks the rats are euthanized and the adhesions are scored. Six groups of 10 rats each underwent this surgery. Group I served as the operative control. Group II was treated with saline which was injected immediately after surgery and on Days 1 and 2 after surgery (vehicle control). Using the same protocol with saline as vehicle, the other four groups of rats were treated with nonspecific IgG (150 microgram per day), anti-TGF-beta (panspecific, 167 microgram per day), anti-TGF-beta1 (67 microgram per day), or anti-TGF-beta2 (50 microgram per day). The rats injected with anti-TGF-beta1 had significantly lower adhesion scores (P < 0.05) than the controls. Rats injected with anti-TGF-beta2 or anti-TGF-beta (panspecific) did not differ significantly from the control saline-injected rats. The results indicate that specifically reducing levels of TGF-beta1 alone can be effective in preventing abdominal adhesions.

One of the common and most serious side effects of abdominal surgery is the formation of adhesions within the peritoneal cavity during healing. Efforts to prevent adhesion formation have concentrated on inhibiting the inflammatory response, inhibiting the formation or encouraging the lysis of fibrin, and protection of the damaged serosal surface. We are interested in regenerating the serosal surface by providing a source of mesothelial progenitor cells. Rats were divided into groups of 10 each. Abdominal adhesions were created by removing a circle of peritoneum and suturing it back into place. Two weeks later the rats were euthanized and the adhesions scored on a scale of 0-5. A population of mesenchymal stem cells (MSCs) isolated from the skeletal muscle of neonatal rats was tested. The cells were grown in primary culture to expand the population and then trypsinized and frozen at -80 degrees C. They are then thawed and grown in secondary culture before use. The control group were injected with saline i.p. immediately after surgery. The experimental groups received (1) 1.4 X 10(6) MSCs, (2) 5 X 10(6) MSCs, (3) 7.5 X 10(6) dead MSCs, (4) 5 X 10(6) rat smooth muscle cells immediately post-op, and (5) 5 X 10(6) MSCs 4-6 hours after surgery. Only live MSCs given immediately after surgery by i.p. injection significantly decreased the adhesion scores of the rats (mean score of 3.5 vs 0.9). MSCs injected i.p. 4-6 hours after surgery actually increased the adhesion scores (3.5 vs 4.7), and rat smooth muscle cells injected i.p. immediately after surgery had no effect on adhesions. The exact mechanism of action of the MSCs is unknown at this time. However, we postulate that the MSCs have the capacity to differentiate into mesothelial cells capable of repopulating the injured mesothelium.

A population of stem cells has been isolated from embryonic avian and neonatal rat skeletal muscle. These cells differentiate into several mesodermal phenotypes in culture upon treatment with dexamethasone. This study reports the isolation of a similar population of stem cells from another mesodermal tissue, the heart. Hearts were excised from 3- to 5-day- old rats, minced, and treated with a collagenase-dispase solution. Single cells were collected by centrifugation, washed, and plated in dishes. The cells were grown to confluence, trypsinized, and frozen at -80 degrees C in 7.5% dimethylsulfoxide. After at least 24 hr, the cells were thawed and plated in 24-well plates and treated with media containing dexamethasone at concentrations of 10(-6)-10(-10) M for 4 weeks. Control cultures contained mononucleated cells with a stellate morphology. Treatment with dexamethasone resulted in the appearance of several mesodermal phenotypes. Bone and cartilage nodules were identified with von Kossa and Alcian blue staining respectively. Adipocytes were identified using Sudan black B stain. Smooth muscle cells were identified by an anti-smooth muscle alpha-actin antibody, and skeletal myotubes were stained with anti-myosin antibody. Large binuclear cells with obvious fibers were noted and stained with anti-desmin. These binuclear cells appeared in both the control and the dexamethasone-treated cultures and were tentatively identified as cardiomyocytes. These data strongly suggest the existence of a population of mesenchymal stem cells in neonatal rat heart.