Fibroblast growth factors (FGF) in human myometrium and uterine leiomyomas in various stages of tumour growth

We previously described that the growth of human uterine leiomyomas was associated with a significant remodelling of the extracellular matrix of these tumours. Significant weight-related increase of collagen and heparan sulphate contents was detected. The latter was known as a component, which bound some peptide growth factors, mainly FGFs, therefore it was decided to evaluate the amounts of acidic FGF (aFGF) and basic FGF (bFGF) in human myometrium and in leiomyomas of various weight and FGF-binding to tissue components. It was found that myometrium and uterine leiomyomas contain picogram amount of aFGF and nanogram amounts of bFGF. No free aFGF was found. Slight amounts of free bFGF were detected both in myometrium and in the tumours. The aFGF and most of bFGF existed in a form of complex with a high molecular component(s). These complexes were very stable and they did not dissociate in denaturation conditions. In comparison to myometrium the tumours contained several times more FGFs and their amounts distinctly increased during the tumour growth. The expression of FGF-receptor I (FGF RI) in the tumours was more distinct in comparison to myometrium. The extracts from myometrium did not bind exogenous 125I-bFGF. In contrast to that the tumours of different weights contained at least two high molecular weight FGF-binding components. One of them (150 kDa) corresponded to FGF-receptor. The other one (190-200 kDa) might be a heparan sulphate-proteoglycan. It seems that aFGF and bFGF play an important role in transformation of normal myometrium into leiomyoma and further growth of this tumour. The action of FGFs on tumour cells enhances biosynthesis of collagen and sulphated glycosaminoglycans, especially heparan sulphate which binds FGFs in the vicinity of cells and facilitates their interaction with membrane receptors. The effect of these processes may be further stimulation of tumour growth and remodelling of tumour extracellular matrix.     

Altered hormonal responsiveness of proliferation and apoptosis during myometrial maturation and the development of uterine leiomyomas in the rat

Uterine leiomyomas are responsive to the ovarian steroids, estrogen and progesterone; however, a mechanistic understanding of the role of these hormones in the development of this common gynecologic lesion remains to be elucidated. We have used the Eker rat uterine leiomyoma model to investigate how ovarian hormones regulate or promote the growth of these tumors. Proliferative and apoptotic rates were quantitated in normal uterine tissues and leiomyomas in response to endogenous ovarian steroids. In 2- to 4-mo-old animals, cell proliferation in the normal uterus corresponded with high serum levels of steroid hormones during the estrous cycle, and apoptosis occurred in the rat uterus in all cell types following sharp, cyclical declines in serum hormone levels. It is interesting that the responsiveness of uterine mesenchymal cells changed between 4 and 6 mo of age, with significant decreases in both proliferative and apoptotic rates observed in myometrial and stromal cells of cycling animals. Leiomyomas displayed much higher levels of proliferation than did age-matched myometrium; however, their apoptotic index was significantly decreased in comparison with normal myometrium. This disregulation between proliferative and apoptotic responses, which were tightly regulated during ovarian cycling in the normal myometrium, may contribute to the disruption of tissue homeostasis and underlie neoplastic growth of these tumors.     

Expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in human uterine leiomyomas and myometrium during the menstrual cycle and after menopause

To investigate the expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak proteins in human uterine leiomyomas and homologous myometrium during the menstrual cycle and after menopause.The expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in leiomyomas (n=24) and myometrial samples (n=22) from women with leiomyomas was measured by immunohistochemistry and Western blot. Measured by immunohistochemistry, a significant difference between leiomyomas and myometrium was observed only for the Bax protein, in tissues obtained from women in the secretory phase of the menstrual cycle. The Bcl-2 staining was more abundant in leiomyomas than in myometrium only in tissues obtained in the proliferative phase of the cycle. Bcl-2 was more abundant in leiomyomas from women of fertile age than in leiomyomas from menopausal women. No significant differences were observed for the Bcl-x or Bak proteins, whereas the Mcl-1 protein was significantly less abundant in secretory phase leiomyomas than in leiomyomas from menopausal women. Western blot analysis based on pools of tissue extracts from the different groups essentially confirmed the data obtained by immunohistochemistry. Bcl-2 family proteins are expressed in leiomyomas and myometrium in different phases related to and influenced by gonadal steroids. These proteins are suggested to interact with each other in the regulation of programmed cell death, apoptosis, but their specific role in growth control of uterine leiomyomas remains to be investigated.     

Tissue-specific expression of messenger ribonucleic acids for insulin-like growth factors and insulin-like growth factor-binding proteins during perinatal development of the rat uterus

Insulin-like growth factor (IGF)-I and IGF-II play a number of important roles in growth and differentiation, and IGF-binding proteins (IGFBPs) modulate IGF biological activity. IGF-I has been shown previously to be essential for normal uterine development. Therefore, we used in situ hybridization assays to characterize the unique tissue- and developmental stage-specific pattern of expression for each IGF and IGFBP gene in the rat uterus during perinatal development (gestational day [GD]-20 to postnatal day [PND]-24). IGF-I and IGFBP-1 mRNAs were expressed in all uterine tissues throughout this period. IGFBP-3 mRNA was not detectable at GD-20 but became detectable beginning at PND-5, and the signal intensity appeared to increase during stromal and muscle development. IGFBP-4 mRNA was abundant throughout perinatal development in the myometrium and in the stroma, particularly near the luminal epithelium. IGFBP-5 mRNA was abundantly expressed in myometrium throughout perinatal development. IGFBP-6 mRNA was detected throughout perinatal development in both the stroma and myometrium in a diffuse expression pattern. IGF-II and IGFBP-2 mRNAs were not detected in perinatal uteri. Our results suggest that coordinated temporal and spatial expression of IGF-I and its binding proteins (IGFBP-1,-3,-4,-5, and -6) could play important roles in perinatal rodent uterine development.     

Identification of differentially expressed genes in human uterine leiomyomas using differential display

INTRODUCTIONUterine leiomyomas (ULs) have been consideredto be of uniceIIular origin[l1. It is one of the mostcommon benign tumors, occurring in 20% to 30% ofwomen[2], accounting for significant morbidity andusually need major surgery[3] which might causesome side effects afterwards[4]. Therefore, to de-velop certain drug treatments instead has been thehope of these patients for a long time. Using alter-native approaches fOr studying patients sufferingfrom leiomyoma in various ethnic gr…     

Effects of progesterone on growth factor expression in human uterine leiomyoma

It is now evident that the use of levonorgestrel-releasing intrauterine system (LNg-IUS) is effective for long-term management of menorrhagic women with uterine myomas because of a striking reduction in menorrhagia. This prompted us to characterize the effects of progesterone (P4) on the growth and apoptosis of uterine leiomyoma cells. On the other hand, we have recently noted that epidermal growth factor (EGF) and IGF-I play a crucial role in prompting uterine leiomyoma growth through stimulating the proliferative potential and inhibiting apoptosis of cultured human leiomyoma cells. In the present review, attention was paid to evaluate the effects of P4 on the expression of growth factors (EGF, IGF-I) and apoptosis-related factors (TNFalpha, Bcl-2 protein) in cultured uterine leiomyoma cells. Treatment with P4 augmented EGF and Bcl-2 protein expression, but inhibited IGF-I and TNFalpha expression in cultured leiomyoma cells. It is known that TNFalpha induces apoptosis in a variety of cell types and Bcl-2 protein is an apoptosis-inhibiting gene product. Thus, the results obtained suggest that P4 has dual actions on uterine leiomyoma growth: one is to stimulate leiomyoma cell growth and survival through up-regulating EGF and Bcl-2 protein expression as well as down-regulating TNFalpha expression in those cells, and the other is to inhibit leiomyoma cell growth through down-regulating IGF-I expression in those cells. This may explain why the size of uterine myomas during use of LNg-IUS increases in some but decreases in other instances. This may also explain why the size of uterine myomas during pregnancy does not increase despite the overwhelming increase in circulating concentrations of sex steroid hormones.     

Pathophysiology of uterine leiomyomas.

Uterine leiomyomas is the most common benign neoplasia in women, one of the most frequent causes of infertility in reproductive years, and the leading cause for hysterectomy. The pathophysiology of uterine leiomyomas is uncertain. Therefore, therapeutic approaches have been primarily empirical. It is now well documented that growth factors control the functional and possibly the histological integrity of several tissues. Recently the presence of growth substances in uterine tissues suggested that the role of sex steroid hormones in the pathophysiology of leiomyomas may be mediated by substances influencing the proliferation of smooth muscle cells and fibroblasts. This report summarizes the data related to the pathophysiology of leiomyomas, which indicate a possible role of growth factors in uterine leiomyomas.     

Elastin distribution in the normal uterus, uterine leiomyomas, adenomyosis and adenomyomas: a comparison

OBJECTIVE: To describe the histologic distribution of elastin in the nonpregnant human uterus, uterine leiomyomas, adenomyosis and adenomyomas. STUDY DESIGN: Uteri were obtained from women undergoing hysterectomy for benign conditions, including 26 cases of uterine leiomyomas, 24 cases of adenomyosis, 18 adenomyomas and 6 cases of autopsy specimens. Specific histochemical staining techniques were employed in order to demonstrate the distribution of elastin. RESULTS: The distribution of elastin components in the uterus was markedly uneven and showed a decreasing gradient from outer to inner myometrium. No elastin was present within leiomyomas, adenomyomas or adenomyosis. CONCLUSION: The distribution of elastin may help explain the normal function of the myometrium in labor. It implies that the uneven distribution of elastin components and absence of elastin within leiomyomas, adenomyomas and adenomyosis could be of some clinical significance. The altered elastin distribution in disease states may help explain such symptoms as dysmenorrhea in uterine endometriosis.     

Estrogen regulation of aquaporins in the mouse uterus: potential roles in uterine water movement

Estrogen stimulates water imbibition in the uterine endometrium. This water then crosses the epithelial cells into the lumen, leading to a decrease in viscosity of uterine luminal fluid. To gain insight into the mechanisms underlying this estrogen-stimulated water transport, we have explored the expression profile and functionality of water channels termed aquaporins (AQPs) in the ovariectomized mouse uterus treated with ovarian steroid hormones. Using immunocytochemical analysis and immunoprecipitation techniques, we have found that AQP-1, -3, and -8 were constitutively expressed. AQP-1 expression was restricted to the myometrium and may be slightly regulated by ovarian steroid hormones. AQP-3 was expressed at low levels in the epithelial cells and myometrium, whereas AQP-8 was found in both the stromal cells and myometrium. AQP-2 was absent in vehicle controls but strongly up-regulated by estrogen in the epithelial cells and myometrium of the uterus. This localization implicates all four isotypes in movement of water during uterine imbibition and, based on their localization to the luminal epithelial cells, AQP-2 and -3 in facilitating water movement into the lumen of the uterus. The analysis of the plasma membrane permeability of luminal epithelial cells by two separate cell swelling assays confirmed a highly increased water permeability of these cells in response to estrogen treatment. This finding suggests that estrogen decreases the luminal fluid viscosity, in part, by enhancing the water permeability of the epithelial layer, most likely by increasing the expression of AQP-2 and/or the availability of AQP-3. Together these results provide novel information concerning the mechanism by which estrogen controls water imbibition and luminal fluid viscosity in the mouse uterus.     

Expression of insulin-like growth factor binding protein-4, insulin-like growth factor-I receptor, and insulin-like growth factor-I in the mouse uterus throughout the estrous cycle

Recent evidence suggests that a regulated insulin-like growth factor (IGF) system mediates the effects of estrogen, promoting the proliferation and differentiation of specific uterine cell types throughout the estrous cycle and during gestation in the rodent. Previous studies have shown that IGFs are differentially expressed in the mouse uterus during the periimplantation period. In the current study, we examined the expression of IGF binding protein-4 (IGFBP-4), IGF-I receptor (IGF-IR), and IGF-I in the mouse uterus throughout the estrous cycle. Ligand blot analysis was conducted on uterine homogenates using [125I]IGF-I. IGFBP-4 was detected in all uterine homogenates, varying in intensity throughout the estrous cycle. In situ hybridization studies at metestrus and diestrus demonstrated an intense IGFBP-4 mRNA signal in antimesometrial stromal cells between the luminal epithelium and the myometrium, but at proestrus and estrus, no IGFBP-4 signal was detected. No IGF-I mRNA was detected at any stage of the estrous cycle by in situ hybridization. However, by RT-PCR analysis, IGF-I mRNA was detected at all stages of the estrous cycle. RT-PCR analysis also showed IGF-IR mRNA throughout the estrous cycle. Using immunohistochemistry, IGF-IR immunostaining was detected throughout the estrous cycle and on days 2-7 of gestation, but was restricted to the glandular epithelium. These results suggest that uterine IGFBP-4 expression may not be dependent on uterine IGF-I expression. They also suggest that IGFBP-4 may play a role in uterine physiology independent of the inhibition of IGF-I action, and that IGF-IR is constitutively expressed in the mouse uterus.     

Analysis of liquid extracts from tree and grass pollens by capillary electromigration methods

Capillary electromigration methods, zone electrophoresis (CZE), micellar electrokinetic chromatography (CMEKC) and isotachophoresis (CITP), have been used for analysis of water and water-buffer extracts from tree-common birch (Betula verrucosa) and grass-orchardgrass (Dactylis glomerata) pollen samples. Water extracts were analyzed by CZE using acetic acid as background electrolyte (BGE), by CMEKC in tris-phosphate BGE with anionic detergent sodium dodecyl sulfate (SDS) micellar pseudophase (TP-SDS) and by CITP in cationic mode with leading/terminating cations K+/BALA+ (beta-alanine (BALA)) and in anionic mode with leading/terminating anions Cl-/MES- (2-(N-morpholino)ethanesulphonic acid (MES)). Moreover, acetic acid extracts were analyzed by CZE using acetic acid as BGE, and alkaline water-SDS-buffer extracts were analyzed by CMEKC using TP-SDS as BGE. Extracted amounts of pollen allergens and other UV-absorbing compounds and the number of resolved components were evaluated from CZE, CMEKC and CITP analyses of the liquid extracts. Larger amounts of UV-absorbing material were found in the water-buffer pollen extracts than in the water extracts. More UV-absorbing material was found in all extracts from D. glomerata pollen than in relevant extracts from B. verrucosa pollen. It was found by CITP that the extracted amounts of anionic components and their number were much higher than those of cationic components. Concentrations of some inorganic ions (e.g. Cl-, K+, Na+, Ca2+) in pollen samples were also determined by CITP.     

Insulin-like growth factor I-like immunoreactivity in serum and tissues of the tilapia, Oreochromis mossambicus

Tilapia serum was acidified with 0.5 M HCl and then chromatographed on an octadecasily-silica column. After washing with 4% acetic acid, the column was eluted with methanol. The eluate was evaporated to dryness. The sample cross-reacted in a human insulin-like growth factor I (IGF-I) radioimmunoassay, suggesting immunochemical similarity to human IGF-I. IGF-I-like immunoreactivity was present at high levels in tilapia liver. Other tissues containing IGF-I-like immunoreactivity included the gonad, kidney, heart, spleen, brain and muscle. The serum IGF-I-like immunoreactivity was attributed to substances with a molecular weight of 9,000 and 45,000 respectively, and it was elevated after treatment with bovine growth hormone and carp pituitary extract.     

Ovarian expression of human insulin-like growth factor-I in transgenic mice results in cyst formation

Insulin-like growth factor-I (IGF-I) has been implicated in a wide variety of physiological processes including ovarian function. To better understand the ovarian role of IGF-I, transgenic mice harbouring a human IGF-I cDNA (hIGF-I) under the control of the mouse LH receptor promoter were generated. Expression of the hIGF-I, determined by Northern blot, was found to occur in the gonad tissues of these transgenic mice. The hIGF-I protein was also detectable by radioimmunoassay in ovarian extracts as well as in the plasma. The fertility of mating transgenic females, as estimated by the number of implantation sites post-coitum, did not appear to be affected. However, transgenic females who failed to mate and produce offspring were found to possess polycystic ovaries. Evaluation of testosterone, estradiol, and LH levels revealed that transgenic animals had significantly elevated circulating levels of testosterone compared to their non-transgenic littermates, while LH levels in transgenic females were significantly lower. Yet, estradiol appeared to be unaffected. These results support the contention that the IGF system plays an important role in ovarian function and that an imbalance in this system may result in ovarian pathology.     

Glycoproteins from the cuticle of the Atlantic shore crab Carcinus maenas: I. Electrophoresis and Western-blot analysis by use of lectins

The protein and glycoprotein content of four different neutral or acidic solvent extracts (0.5 M KCl, 10% EDTA, 0.1 N HCl, or 2% acetic acid) from the mineralized exoskeleton of a decapod crustacean, the Atlantic shore crab Carcinus maenas, were characterized by quantitative analysis of proteins, SDS-PAGE analysis, and probing with lectins on blots. The lectins used were Conconavalin A, Jacalin, soybean agglutinin, Maackia amurensis agglutinin II, and Sambucus nigra agglutinin. The results show that many proteins can be obtained from the crab cuticle without strong denaturants in the extraction medium. Many of the extracted cuticle proteins appeared to be glycosylated, bearing O-linked oligosaccharides and N-linked mannose-rich glycans. N-acetyl-galactosamine and N-acetylneuraminic acids were revealed, for the first time, as terminal residues on N-linked mannose-rich structures of crab cuticle glycoproteins. Sialylated glycoproteins might thus be involved in organic-mineral interactions in the calcified crab exoskeleton. The amount and variety of glycoproteins extracted with the acidic solvents are obviously different from those extracted with neutral solvents. HCl proved to be the best of the tested extraction solvents and a valuable alternative to EDTA.     

The endothelin axis in uterine leiomyomas: new insights

The endothelin axis, comprising endothelin-1 (ET-1) and its receptors (ETA and ETB), is involved in the pathophysiology of different human tumors. Here we review conventional approaches and gene expression profiling indicating the association of ET-1 and its cognate receptors with human and rat leiomyomas, the most common benign tumors of myometrium. Specifically, ET-1/ETA interactions affect human and rat leiomyoma cell proliferation through protein kinase C and mitogen-activated protein kinase-dependent signaling pathways. Recent experiments demonstrate that the ET-1 axis exerts a potent antiapoptotic effect involving sphingolipid metabolism and prostaglandin-endoperoxide synthase 2/prostaglandin system in the rat Eker leiomyoma tumor-derived ELT3 cell line. Evidence supports that steroid hormones, growth factors, and extracellular matrix are key regulators of the leiomyoma growth. Interestingly, the ET-1 axis is under steroid hormones and can cooperate with these growth factors. Therefore, ET-1 alone or in association with these factors could contribute to the complex regulation of uterine tumor growth, such as proliferation, survival, and extracellular matrix production. This review summarizes current knowledge and emerging data on ET-1 in uterine leiomyoma pathology.