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-1 (TGF-Beta1), 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-Beta1 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-Beta1 mRNA was determined by quantitative reverse transcription-polymerase chain reaction and TGF-Beta1 protein was localized by immunohistochemical analysis. Serum and peritoneal fluid TGF-Beta1 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-Beta1 mRNA expression at days 2, 4, and 7. Immunostaining intensity of TGF-Beta1 in adhesions from the peritonitis group also steadily rose through day 7. In peritoneal fluid, the ratio of active:total TGF-Beta1 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-Beta1, a mechanism that may exacerbate adhesion formation.

Cardiovascular disease in the diabetic woman.

The incidence of cardiovascular disease (CVD) with age is increasing in the United States, and elderly women constitute a disproportional component of the aging population. Elderly women also have a relatively high incidence of diabetes, which contributes to this relatively high CVD risk. Although CVD is less common in premenopausal women than in men, this difference begins to disappear after the onset of menopause, presumably related to decreased levels of female sex hormones (estrogen and/or progesterone). Diabetes mellitus removes the normal premenopausal gender-related differences in the prevalence of CVD by mechanisms that are not clearly defined, including metabolic and hemodynamic factors associated with diabetes. Dyslipidemia in diabetes mellitus consists of low high density lipoprotein cholesterol, elevated triglyceride levels, and a small, dense, more atherogenic low density lipoprotein particle (i.e. oxidized). Dyslipidemia interacts with associated hemodynamic (i.e. hypertension) and metabolic abnormalities (i.e. increased platelet aggregation and plasminogen activator inhibitor-1 levels) to promote CVD risks in diabetic women. Recent controlled trials underscore the critical importance of aggressively treating CVD risk factors, especially dyslipidemia, in women with diabetes.

Effect of CGS 20267 on ovarian aromatase and gonadotropin levels in the rat.

Aromatase catalyzes the rate limiting step that converts androgens to estrogens. Postmenopausal women with hormone dependent breast cancer respond to first generation aromatase inhibitors such as aminoglutethimide with a marked suppression of circulating estradiol levels. In contrast, premenopausal women appear to be resistant to first generation aromatase inhibitors. The inability to block ovarian aromatase results from the low affinity of first generation inhibitors for the active site of the enzyme. Under these circumstances, the high substrate levels in the premenopausal ovary compete effectively with these inhibitors and do not allow binding of inhibitor to the active site of the enzyme. Second and third generation aromatase inhibitors with higher affinity for aromatase have now been developed and potentially could block ovarian aromatase. To test this possibility, we administered CGS 20267 (letrozole), a highly potent aromatase inhibitor, to cycling female rats. A dose dependent inhibition of uterine weight occurred with maximum effects produced by the 5 mg/kg/day dosage. During a period of 4 weeks, uterine weight was reduced to levels induced by ovariectomy. Ovarian tissue estradiol levels were inhibited by approximately 80%. As a reflection of inhibition of ovarian aromatase activity, the levels of androstenedione in the ovary increased by an order of magnitude. Both LH and FSH plasma levels increased but not to those observed after ovariectomy. The rise in gonadotropin levels induced a statistically significant but relatively small increase in ovarian weights. These results demonstrate the ability to persistently block ovarian aromatase activity in cycling rats with a potent aromatase inhibitor. This study provides a rationale for clinical trials of potent aromatase inhibitors in pre-menopausal women with breast cancer.

Estrogen production via the aromatase enzyme in breast carcinoma: which cell type is responsible?

Studies of breast tumor homogenates from women with breast cancer have demonstrated the synthesis of estrogens in situ through the enzyme aromatase. The present series of investigations sought to determine which cell type within the tumor is responsible for local estrogen biosynthesis, and whether or not the amount produced is biologically important. Accordingly, we utilized an indirect immunohistochemical scoring method (H-score) to determine the relative amount of enzyme present in tumor epithelial and stromal cells. This revealed a value of 13 for tumor stromal cells and 4.8 for the epithelial component. Contributing to this difference is the fact that a greater percentage of cells in the tumor were stromal (45%) than epithelial (37%). To obtain direct evidence that tumor stromal cells could synthesize estrogens, we isolated and grew these cells in tissue culture. Stromal cells originating from within the tumor could be stimulated by known enhancers of transcription to produce nearly as much aromatase as is found in placental microsomes. Stromal cells isolated from benign tissue distal to the tumor exhibited properties similar to those of the tumor stroma. Epithelial cells, in contrast, did not respond to these enhancers and had low levels of aromatase basally. To obtain proof of the principle that local estrogen synthesis can be biologically meaningful, we measured tumor tissue estradiol levels and growth rates in aromatase-transfected MCF-7 cells implanted into nude mice. Local synthesis resulted in tumor levels ranging from 300 to 800 pg/g and growth rates substantially higher than in non-aromatase-containing tumors. These data suggest that tumor stromal cells contribute the major portion of estrogen synthesized in tumors, and that this local synthesis can increase tumor estradiol levels and growth rates.

Aromatase activity and expression in breast cancer and benign breast tissue stromal cells.

In situ estrogen synthesis by hormone-dependent breast cancers could potentially regulate cellular proliferation through autocrine or paracrine mechanisms. Several biochemical studies have demonstrated activity of the enzyme aromatase, the rate-limiting step for estrogen synthesis, in breast tumor homogenates. Prior immunohistochemical studies in breast neoplasms demonstrated aromatase antibody binding to both stroma and parenchyma, but biochemically measured enzyme activity significantly correlated only with the level of staining in the stromal component. The present study sought to provide more direct evidence of the predominant role for stromal cell aromatase in breast tumor tissue. Accordingly, breast tumor stromal and epithelial cells were examined for aromatase enzyme activity and messenger ribonucleic acid (mRNA) expression. Stromal and epithelial cells from benign tissue served as a means of comparing activity and regulation in benign and tumor tissue. Enzyme activity in stromal cells from breast tumor tissue was low basally, but increased by 30- to 1200-fold when induced by dexamethasone. Combining dexamethasone with phorbol esters and cAMP produced an additional 1.2- to 4.1-fold stimulation. Analyses of exons III/V and exons IX/X demonstrated that aromatase mRNA expression was also substantially increased by these treatments. Increases in enzyme activity and mRNA expression in cells from benign breast stroma paralleled those observed in tumor stroma, although the increases in enzyme activity were generally lower. In contrast to the responses observed in stromal cells, epithelial cells from breast tumor or nonmalignant breast tissue were unresponsive to dexamethasone, either added alone or in combination with phorbol esters and cAMP. This study provides direct biochemical evidence that aromatase is present in stroma within breast tumors, as in surrounding tissues, and suggests that estrogen synthesis within the tumor may modulate tumor growth via a paracrine mechanism.