Molecular Human Reproduction, Vol. 5, No. 2, 139-145,
February 1999
© 1999 European Society of Human Reproduction and Embryology
Heparin inhibits proliferation of myometrial and leiomyomal smooth muscle cells through the induction of 
-smooth muscle actin, calponin h1 and p27
1 Department of Obstetrics and Gynecology, Shinshu University School of Medicine, 3–1–1 Asahi, Matsumoto 390–8621, and 2 Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606–8397, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Mast cells are widely distributed in human tissues, including the human uterus. However, the function of mast cells in uterine smooth muscle has not been clearly established. Mast cells possess secretory granules containing such substances as heparin, serotonin, histamine and many cytokines. To help establish the role of mast cells in the human myometrium, the action of heparin was investigated using smooth muscle cells (SMC) from normal myometrium and from leiomyoma. The proliferation of cultured myometrial and leiomyomal SMC was inhibited by heparin treatment. Flow cytometric analysis showed that the population in the G1 phase of the cell cycle increased under heparin treatment. Western blotting analysis showed that markers of SMC differentiation such as
-smooth muscle actin (-SMA), calponin h1 and cyclin-dependent kinase inhibitor p27 were induced by heparin, whereas cell-cycle-related gene products from the G1 phase of the cell cycle, such as cyclin E and cdk2, were not changed. Taken together, these results indicate that heparin inhibits the proliferation of myometrial and leiomyomal SMC through the induction of -SMA, calponin h1 and p27. We suggest that heparin from mast cells may induce differentiation in uterine SMC and may influence tissue remodelling and reconstruction during physiological and pathophysiological events.
-smooth muscle actin/heparin/mast cells/myometrium and leiomyoma/p27
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Mast cells (MC) are widely distributed in most human tissues and neoplasms, including those of the uterus (Crow et al., 1988
; Mori et al., 1997a). There are many reports suggesting that MC play a wide variety of roles in the female reproductive tract. For example, it has been suggested that degranulation of myometrial MC, thus releasing mediators such as histamine and serotonin, might promote contractions at parturition (Rudolph et al., 1993). Furthermore, there is a high incidence of uterine inversions in mutant strains of mice that lack MC, and this suggests that MC may be associated with the process of implantation and the formation of a decidual cell response, or with relaxation of the myometrium and cervix (Yokoi et al., 1994). However, in humans, the role played by uterine MC is not clearly understood.
Heparin is released from MC (Lydyard and Grossi, 1993) and is a well-known anticoagulant. Moreover, it has also been shown to decrease vascular smooth muscle cell (SMC) mitotic activity both in vivo and in vitro (Clowes and Karnovsky, 1977; Guyton et al., 1980; Clowes et al., 1988), to prevent the phenotypic modulation of SMC and to induce -smooth muscle actin (-SMA) (Desmouliere et al., 1992). These functions have also been found in airway and intestinal smooth muscle (Cochran et al., 1987; Johnson et al., 1995). It has been reported that heparin binds directly to the SMC surface and that it inhibits SMC proliferation (Reilly et al., 1986). In addition, in a study using heparin–sepharose chromatography, it has been found that heparin retained full activity in a medium containing serum depleted of all heparin-binding proteins (Orlandi et al., 1994). In contrast, it has been suggested that heparin may inhibit cellular proliferation by binding endogenous growth factors and cytokines, or by displacing growth factors from their binding sites (Reilly et al., 1988; Bono et al., 1997). Its ability to inhibit proliferation seems to be related to the negative charge on the heparin molecule, as the most highly charged fragments are the most potent inhibitors (Wright et al., 1989). Heparin probably acts in the G1 phase of the cell cycle since it must be present before cells enter the S phase if it is to produce its maximum inhibitory effect on SMC proliferation (Reilly et al., 1989). Furthermore, heparin may suppress the expression of proto-oncogenes such as c-fos and c-myc (Pukac et al., 1992) and it interferes directly with the protein kinase C pathway (Herbert et al., 1996). However, the effect of heparin on human myometrial and leiomyomal SMC remains unknown.
In the present study, in an attempt to gain a better understanding of the role of MC in the human uterus, we analysed the responses of both normal uterine myometrial tissue and leiomyomal tumours to heparin treatment. Analysis of the proliferation of cultured cells was performed by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. In addition, cell cycle analysis was carried out using flow cytometry and Western blotting using antibodies against -SMA, calponin h1 and the G1 cell-cycle-related gene products, cyclin E, cyclin-dependent kinase 2 (cdk2) and the cdk inhibitor, p27.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cell culture
Tissue for cell culture was obtained from seven premenopausal women with symptomatic uterine leiomyoma who were undergoing elective hysterectomy. None were receiving any type of hormonal or drug therapy. The tissues were used after obtaining written consent from the patients. Endometrial dating of the experimental uteri was carried out by the method of Noyes et al. (1950). Of the seven patients, four were in the proliferative phase and three in the secretory phase of the menstrual cycle.
Briefly, tissue was collected in Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical Co, Tokyo, Japan) with 10% fetal bovine serum (Biological Industries, Grand Island, NY, USA) and 1% antibiotic–antimycotic solution (Gibco Laboratories, Grand Island, NY, USA), then transported directly to the laboratory at room temperature. The tissue was rinsed in medium and minced into fine pieces. Samples were aspirated and placed into tubes containing DMEM and 0.4% collagenase (Wako, Osaka, Japan) in which they were kept for 4 h at 37°C with continuous mixing until a suspension was evident. The cell suspension was separated from the undigested fragments, diluted in an equal volume of calcium-, and magnesium-free Dulbecco's phosphate-buffered saline [PBS(–); Nissui], then centrifuged at 100 g for 10 min. The supernatant was discarded and the cell pellet was suspended in DMEM with 10% fetal bovine serum and 1% antibiotic–antimycotic solution. A cell solution containing 4x104 cells/ml was placed in a 75 ml flask (Becton Dickinson Laboratories, Lincoln Park, NJ, USA). Incubation was carried out at 37°C in 5% CO2 in air. Cultures were determined to be pure SMC cultures (>98%) by immunostaining for -SMA (Dako, Glostrup, Denmark), which are markers for SMC (Nowak et al., 1993). Cells used in the experiments were from passages 1 or 2.
Analysis of cell proliferation
Cell proliferation in cultures of myometrial or leiomyomal tissue was analysed by the MTT colorimetric method, which is a convenient way to detect enzymes associated with DNA synthesis in dividing cells (Mosmann, 1983). Secondary cultured cells from myometrial or leiomyomal cultures were plated at a density of 4x103 cells into 96-well flat-bottomed tissue-culture plates (Corning Laboratory Sciences Co., Corning, NY, USA). The cells were incubated at 37°C in DMEM with 10% fetal bovine serum under 5% CO2 in air. The following day, heparin (Wako, Osaka, Japan) was added at a concentration of 10, 50 or 100 IU/ml, the concentrations being selected on the basis of previous reports about heparin inhibition of vascular and airway SMC (see Introduction). After 3 days incubation, an MTT assay was performed using each of the above concentrations. Other MTT assays were performed each day using only the 100 IU/ml concentration. MTT reagent [3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; Research Organics Inc, Cleveland, OH, USA] was diluted to 5 mg/ml in PBS, and this solution was filter-sterilized and stored at 4°C. At designated intervals during the incubation of the secondary cultured cells in the tissue-culture plates, 20 µl of the MTT reagent was added to each well, and the plates were then re-incubated under the prescribed conditions for an additional 4 h. Next, 100 µl of extraction buffer was dispensed per well, and incubation was continued until all the blue crystals had been solubilized. The absorbance of the wells was measured using an enzyme-linked immunosorbent assay (ELISA) microplate reader (Molecular Devices, Menlo Park, CA, USA) at a wavelength of 570 nm.
Cell cycle analysis by flow cytometry
Cultures of secondary cells from normal myometrium or leiomyoma were plated at a density of 1x105 cells in a 75 ml flask. The following day, heparin was added at a concentration of 100 IU/ml. The cells were incubated in culture flasks in DMEM with 10% fetal bovine serum with or without heparin to allow an analysis of the cell cycle. After 3 days incubation, each group of cells was collected and washed with PBS(–) three times. The cells were fixed in 70% ethanol and stored at –20°C. To reduce the effects of contact inhibition, control cells were adjusted to reach 70% confluency at the time of Fluorescence Activated Cell Sorter (FACS) analysis. Then, the cells were resuspended in a DNA stain solution containing propidium iodide (20 µg/ml; Calbiochem, CA, USA) and RNAase (1.8 IU/ml; Sigma, St Louis, MO, USA). The cells were analysed with a FACScan flow cytometer equipped with an argon laser (488 nm; Becton Dickinson Immunocytometry System, Mountain View, CA, USA). The experiments were repeated three times for each of six cultures. When assessed by the Mann–Whitney U-test, the differences were considered to be significant when P < 0.05.
Western blotting analysis
Cultures of secondary cells from normal myometrium or leiomyoma were plated at a density of 1x105 cells in a 75 ml flask in DMEM with 10% fetal bovine serum and 1% antibiotic–antimycotic solution. The following day, heparin was added at a concentration of 100 IU/ml. After 3 days incubation, the cultured cells were analysed by Western blotting for -SMA, calponin h1 and G1 cell-cycle-related gene products. Cultured cells from normal myometrium or leiomyoma were lysed in a lysis buffer, consisting of 50 mM Tris–HCl, pH 8.0, 0.25 M NaCl, 0.5% NP-40, 1 mM phenylmethylsulphonyl fluoride (PMSF; Sigma), 1 µg/ml aprotinin (Boehringer Mannheim, Mannheim, Germany), 1 µg/ml leupeptin (Boehringer Mannheim), and 20 µg/ml N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK; Boehringer Mannheim). The lysates were centrifuged at 13 000 g for 20 min at 4°C and the supernatants were stored at –80°C. Extracts equivalent to 30µg of total protein were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (10% acrylamide) and transferred onto nitrocellulose membranes (HybondTM-C super; Amersham, High Wycome, UK). The membranes were blocked in TBST (0.2 mM NaCl, 10 mM Tris, pH 7.4, 0.2% Tween-20) containing 5% non-fat dry milk and 0.02% NaN3 for 1 h. This was followed by incubation first with specific antibodies against -SMA (Dako), calponin h1 (Sigma) and the cell-cycle-related gene products, cyclin E, cdk2 and p27 (Santa Cruz Bio Inc, Santa Cruz, CA, USA)a diluted 1:500 in TBST containing 5% milk, and second with 1:1000 dilutions of anti-mouse immunoglobulin (Ig) (-SMA, calponin h1 and cyclin E) or anti-rabbit Ig (cdk2 and p27) (Amersham) in TBST containing 2% milk. Membranes were then incubated with sheep anti-mouse Ig (Amersham) in TBST containing 2% non-fat dry milk. Bound antibody was detected with the aid of an enhanced chemiluminescence (ECL) system (Amersham). The relative expression of -SMA, calponin h1 and cell-cycle-related gene products on the immunoblotting filters was analysed by densitometric analysis using a Quantity One Scan System (ATTO Co, Tokyo, Japan). The intensity generated under heparin treatment is expressed as a percentage of the corresponding untreated control value.
Statistical analysis
The data are presented as the mean ± SE. The significance of differences was assessed by the Kruskal–Wallis test or by the Mann–Whitney U-test. Differences were considered to be significant when P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Analysis of cell proliferation
After 3 days of heparin treatment at a concentration of 10, 50 or 100 IU/ml, the MTT assay showed that the number of cultured cells per 96-well plate was reduced by heparin in a dose-dependent manner (Figure 1
). The cell numbers indicated significant inhibition of cell proliferation in myometrial cells at the two highest concentrations: to 70% of control with 50 IU/ml heparin and to 57% of control with 100 IU/ml heparin (P < 0.05). The cell numbers also indicated significant inhibition in leiomyomal cells at the highest concentration: to 70% of control with 100 IU/ml heparin (P < 0.05). There was no significant difference between myometrial cells and leiomyomal cells in terms of the inhibition of cell proliferation. In addition, no significant differences in response to heparin were noted between cells obtained at different phases of the menstrual cycle. Growth curves showed that a significant inhibition of myometrial cell proliferation was first obtained on day 2 (P < 0.01; Figure 2A), whereas a significant inhibition of leiomyomal cell proliferation was not obtained until day 3 (P < 0.05; Figure 2B). The proliferative activity of myometrial SMC tended to be higher than that of leiomyomal SMC under control conditions. However, the growth curves showed no significant difference between myometrial cells and leiomyomal cells with respect to the inhibition of cell proliferation.
|
|
Cell cycle analysis
Representative results of the cell cycle analysis performed on cells from myometrial and leiomyomal smooth muscle are shown in Figure 3
. A significant increase in the fraction of cells in the G1 phase of the cell cycle (81.7 ± 1.3%; P < 0.05) together with significant decreases in the S and G2/M phases (5.2 ± 3.5 and 12.9 ± 3.9%; P < 0.05) occurred after heparin treatment (by comparison with the corresponding values for cultured myometrial SMC without heparin, which were: G1, 64.8 ± 3.9%; S, 14.1 ± 1.3%; G2/M, 20.4 ± 2.4%). Similar results were obtained for leiomyomal SMC. The values obtained were: in the G1 phase, 81.0 ± 0.9% versus 68.1 ± 2.9% (P < 0.05); in the S phase, 7.5 ± 2.3% versus 13.1 ± 3.1% (P < 0.05); and in the G2/M phase, 11.5 ± 2.2% versus 18.7 ± 2.0% (P < 0.05). The results suggest that heparin produces a block of the G1–S transition or G1 arrest in both myometrial and leiomyomal SMC.
|
Western blotting analysis
Protein extracts from cells treated for 3 days with 100 IU/ml of heparin were analysed by Western blotting to detect the expression of
-SMA and calponin h1, which are markers of the differentiation of SMC. The results are shown in Figure 4A. The 42 kDa band of -SMA and the 34 kDa band of calponin h1 were both increased by treatment with heparin in myometrial SMC and in leiomyomal SMC. For myometrial SMC, the ratio between the values obtained for the expression of -SMA with or without heparin treatment was 1.59 (P < 0.01). For leiomyomal SMC, the values obtained for this ratio were 1.48 (P < 0.01) (Figure 4B). The corresponding ratios for the expression of calponin h1 were: 1.17 in myometrial SMC (P < 0.05), 1.17 in leiomyomal SMC (P < 0.05) (Figure 4B).
|
To confirm that heparin induced a block of the G1–S transition or G1 arrest in these SMC, we analysed the expression of the G1-phase-related gene products cyclin E, cdk2 and p27 (Figure 5A
). Neither the 50 kDa band of cyclin E nor the 33 kDa band of cdk2 was changed by heparin treatment in myometrial or leiomyomal SMC (Figure 5B). In contrast, the 27 kDa band of p27, which is a cdk inhibitor, was increased by treatment with heparin in both myometrial and leiomyomal SMC (Figure 5A). The ratio between the values obtained for the expression of p27 in myometrial SMC with or without heparin treatment was 1.67 (P < 0.05). The values obtained for this ratio in leiomyomal SMC was 1.71 (P < 0.05) (Figure 5B). In contrast, suppression of proliferating cell nuclear antigen (PCNA), which is a cofactor of DNA polymerase 
, was observed after heparin treatment, the ratios being 0.59 for myometrial SMC (P < 0.05) and 0.51 for leiomyomal SMC (P < 0.05) (Figure 5B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We have demonstrated that heparin inhibits the proliferation of SMC from the human myometrium and from leiomyoma, both effects occurring in dose-dependent manner. With respect to this inhibitory action, there was no significant difference between myometrial and leiomyomal SMC. However, leiomyomal SMC inhibition began later than that in the normal myometrium. We postulate this lag occurred because the proliferative rate of myometrial SMC tended to be higher than that of leiomyomal SMC under control conditions, or because leiomyomal SMC were in some way more resistant to heparin treatment than myometrial SMC. The results also revealed a marked increase in the fraction of cells in the G1 phase of the cell cycle after heparin treatment. This suggests that the anti-proliferative effect of heparin on SMC from human myometrium and leiomyoma is related either to a block of the G1–S transition or to a G1 arrest.
The transition from G1 to S is controlled by the concerted actions of protein kinases, the activities of which are modulated by families of regulatory proteins in both a positive (cyclins) and a negative [cyclin-dependent kinase inhibitors (CDI)] manner. A family of CDI plays a major role in the cell cycle machinery (Sherr and Roberts, 1995). In this study, our analysis of Western blotting showed: (i) a suppression of PCNA after heparin treatment that was consistent with the results obtained using the MTT method; and (ii) that heparin increased the expressions of -SMA, calponin h1 and p27. In contrast, no marked change in the expressions of cyclin E and cdk2 was observed in cells treated with heparin after 3 days incubation. It is possible that the other G1 cell-cycle-related molecules or an increased activity of cyclin-associated kinase may have participated in the block of the G1–S transition or G1 arrest induced by heparin treatment. Since -SMA and calponin h1 are expressed in differentiated SMC, our data suggest that the anti-proliferative effect of heparin is closely associated with an up-regulation of p27 and an induced differentiation of SMC.
With respect to the effect of heparin on myometrial and leiomyomal SMC, our results suggest that: (i) heparin inhibits SMC proliferation; (ii) heparin increases the expression of -SMA; and (iii) heparin most likely acts in the G1 phase of the cell cycle. This is consistent with results previously obtained from vascular SMC. Although there are many reports showing heparin inhibition of vascular SMC proliferation (see Introduction), this is the first report showing heparin inhibition of myometrial and leiomyomal SMC. Our results show that the effect of heparin is associated with an up-regulation of p27 and an induced differentiation of SMC. On these grounds, we postulate that heparin may be of therapeutic or preventative value in cases of leiomyoma.
It is becoming increasingly clear that in many instances the cellular phenotypic features of vascular SMC are modulated locally by cytokines or growth factors and by extracellular matrix (ECM) components, rather than being controlled by genetically irreversible steps, as was believed for a long time (Ross, 1993). However, the control of SMC proliferation and cellular phenotypic features in the uterus have not been clearly explained. It has been reported that a variety of hormones, growth factors and mitogens can affect the proliferation of uterine SMC (Fayed et al., 1989; Koutsilieris et al., 1992; van der Ven et al., 1994). Recently, heparin-binding growth factors, which include platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), have been isolated from human leiomyomas and normal myometrium, and it has suggested that the enhanced growth of leiomyomas may be due, in part, to the presence of the large quantities of bFGF that are stored in the ECM of leiomyomas (Mangrulkar et al., 1995). Since heparin may inhibit cellular proliferation by binding endogenous growth factors or by displacing growth factors from their binding sites (Reilly et al., 1988; Bono et al., 1997), heparin may operate by binding heparin-binding bFGF and so inhibit the proliferation of normal myometrial and leiomyomal SMC.
In addition, many of the heparin-binding group of growth factors-including bFGF, vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor (HBEGF), PDGF and transforming growth factor-ß (TGF-ß) regulate the process of angiogenesis or have other effects on vascular structures. These growth factors or their receptors are differentially regulated in leiomyomas and the endometrium of patients with leiomyoma-related complications (Stewart and Nowak, 1996). This being so, heparin may potentially be of use to regulate angiogenesis in women with leiomyoma or abnormal bleeding.
We have reported that there are many MC among the myometrial SMC (Mori et al., 1997a) and that the SMC of the myometrium produce stem cell factor (SCF) (Mori et al., 1997b), which is a major regulator of human MC (Valent et al., 1992; Sperr et al., 1993) through the receptor for SCF (c-kit) (Zsebo et al., 1990). Our results and those obtained by others, suggest that there is an interaction between uterine SMC and MC. In addition, it has been shown that the number of MC in the rodent uterus decreases around the time of implantation (Hore and Mehrotra, 1988) and that a factor secreted by the human embryo stimulates cytokine release of endometorial MC in the uterus (Cocchiara et al., 1996). These findings suggest that there is a physiologically relevant interaction between MC and the adjacent endometrium. However, we have reported that the endometrium contains a significantly smaller number of MC than the myometrium (Mori et al., 1997a). Comparison of the interaction between the granulated lymphocytes that exist in the functional layer of the endometrium and the endometrial stromal cells during the menstrual cycle (Bhartiya et al., 1996), indicates that the interaction between MC and the endometrial stromal cells in the functional layer may be negligible. However, while the proliferative activity of myometrium and leiomyoma increases in the secretary phase of the menstrual cycle and during pregnancy (Kawaguchi et al., 1989; 1991), the number of MC does not change during the menstrual cycle (Mori et al., 1997a,b; Orii et al., 1998). Thus, the physiological interactions between MC and the adjacent endometrium, and between MCs and SMC proliferation cannot be explained simply as a heparin effect.
In a recent study, the number of MC was found to be lower in usual leiomyomas than in the normal myometrium (Orii et al., 1998). Since differences in cellularity and in the collagen matrix were found to correlate with the number of MC in usual leiomyomas (Orii et al., 1998), changes in the levels of MC products such as heparin may possibly be involved in the maintenance of a differentiated state as well as in phenotypic modulations of pathological phenomena such as inflammation and tumour formation (Singh et al., 1992). These results suggest that an interaction between SMC and MC may influence tissue remodelling and reconstruction during both physiological and pathophysiological events. However, the functional roles of uterine MC and the details of their relationship with the myometrium and uterine smooth muscle tumours have not been fully characterized. Clearly, further research will be needed to establish the functional importance of the effects of heparin on leiomyoma.
In conclusion, we have demonstrated an inhibitory effect of heparin on the proliferation of myometrial and leiomyomal SMC. A marked increase in the fraction of cells in the G1 phase of the cell cycle was observed in both tissues after heparin treatment. This study also showed that heparin induced cytoskeletal muscle proteins, such as -SMA, calponin h1 and cell cycle inhibitor p27, suggesting that heparin inhibits the proliferation of uterine SMC and may induce differentiation of SMC, and that the interaction between SMC and MC products such as heparin may influence tissue remodelling and reconstruction during pathophysiological events.
|
Acknowledgments |
|---|
This work was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture (No.08457438, 09877318, 09671671), Japan.
|
Notes |
|---|
3> To whom correspondence should be addressed
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bhartiya, D., Chowdhury, S.R. and Bajpai, V.K. (1996) Stromal cell interaction and relevance to predecidual events and menstruation. Hum. Reprod., 11, 850–856.
Bono, F., Rigon, P., Lamarche, I. et al. (1997) Heparin inhibits the binding of basic fibroblast growth factor to cultured human aortic smooth-muscle cells. Biochem. J., 326, 661–668.
Clowes, A.W. and Karnovsky, M.J. (1977) Suppression by heparin of smooth muscle cell proliferation in injured arteries. Nature, 265, 625–626.[Medline]
Clowes, A.W., Clowes, M.M., Kocher, O. et al. (1988) Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin. J. Cell Biol., 107, 1939–1945.
Cocchiara, R., Lampiasi, N., Albeggiani, G. et al. (1996) A factor secreted by human embryo stimulates cytokine release by uterine mast cells. Mol. Hum. Reprod., 2, 781–791.
Cochran, D.L., Perr, H.A., Graham, M.F. et al. (1987) Heparin induces specific protein release from human intestinal smooth muscle cells. Biochem. Biophys. Res. Commun., 142, 542–551.[Web of Science][Medline]
Crow, J., More, L. and Howe, S. (1988) The mast cell of the human uterus. APMIS, 96, 921–926.[Web of Science][Medline]
Desmouliere, A., Rubbia-Brandt, L., Grau, G. et al. (1992) Heparin induces alpha-smooth muscle actin expression in cultured fibroblasts and in granulation tissue myofibroblasts. Lab. Invest., 67, 716–726.[Web of Science][Medline]
Fayed, Y.M., Tsibris, J.C., Langenberg, P.W. et al. (1989) Human uterine leiomyoma cells: binding and growth responses to epidermal growth factor, platelet-derived growth factor, and insulin. Lab. Invest., 60, 30–37.[Web of Science][Medline]
Guyton, J.R., Rosenberg, R.D., Clowes, A.W. et al. (1980) Inhibition of rat arterial smooth muscle cell proliferation by heparin. In vivo studies with anticoagulant and nonanticoagulant heparin. Circ. Res., 46, 625–634.
Herbert, J.M., Clowes, M., Lea, H.J. et al. (1996) Protein kinase C alpha expression is required for heparin inhibition of rat smooth muscle cell proliferation in vitro and in vivo. J. Biol. Chem., 271, 25928–25935.
Hore, A. and Mehrotra, P.N. (1988) Presence of a blastocyst and mast cell depletion of the mouse uterus. Acta Anat., 132, 6–8.[Web of Science][Medline]
Johnson, P.R., Armour, C.L., Carey, D. et al. (1995) Heparin and PGE2 inhibit DNA synthesis in human airway smooth muscle cells in culture. Am. J. Physiol. (Lung Cell Mol Physiol.), 269, L514–519.
Kawaguchi, K., Fujii, S., Konishi, I. et al. (1989) Mitotic activity in uterine leiomyomas during the menstrual cycle. Am. J. Obstet. Gynecol., 160, 637–641.[Web of Science][Medline]
Kawaguchi, K., Fujii, S., Konishi, I. et al. (1991) Immunohistochemical analysis of oestrogen receptors, progesterone receptors and Ki-67 in leiomyoma and myometrium during the menstrual cycle and pregnancy. Virchows Archiv A Pathol. Anat., 419, 309–315.
Koutsilieris, M., Elmeliani, D., Frenette, G. et al. (1992) Leiomyoma-derived growth factors for smooth muscle cells. In vivo, 6, 579–585.[Medline]
Lydyard, P. and Grossi, C. (1993) Cells involved in immune responses. In Roitt, I., Brostoff, J. and Male, D. (eds), Immunology. 3rd edn. Mosby, London, UK, pp. 2.1–2.20
Mangrulkar, R.S., Ono, M., Ishikawa, M. et al. (1995) Isolation and characterization of heparin-binding growth factors in human leiomyomas and normal myometrium. Biol. Reprod., 53, 636–646.[Abstract]
Mori, A., Zhai, Y.L., Toki, T. et al. (1997a) Distribution and heterogeneity of masts cell in the human uterus. Hum. Reprod., 12, 368–372.
Mori, A., Nakayama, K., Suzuki, J. et al. (1997b) Analysis of stem cell factor for mast cell proliferation in the human myometrium. Mol. Hum. Reprod., 3, 411–418.
Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytoxicity assays. J. Immunol. Meth., 65, 55–63.[Web of Science][Medline]
Nowak, R.A., Rein, M.S., Heffner, L.J. et al. (1993) Production of prolactin by smooth muscle cells cultured from human uterine fibroid tumors. J. Clin. Endocrinol. Metab., 76, 1308–1313.[Abstract]
Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3–25.
Orii, A., Mori, A., Zhai, Y.L. et al. (1998) Number of mast cells present in smooth muscle tumors of the uterus depends on histological subtype. Int. J. Gynecol. Pathol., in press.
Orlandi, A., Ropraz, P. and Gabbiani, G. (1994) Proliferative activity and alpha-smooth muscle actin expression in cultured rat aortic smooth muscle cells are differently modulated by transforming growth factor-beta 1 and heparin. Exp. Cell Res., 214, 528–536.[Web of Science][Medline]
Pukac, L.A., Ottlinger, M.E. and Karnovsky, M.J. (1992) Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells. J. Biol. Chem., 267, 3707–3711.
Reilly, C.F., Fritze, L.M. and Rosenberg, R.D. (1986) Heparin inhibition of smooth muscle cell proliferation: a cellular site of action. J. Cell. Physiol., 129, 11–19.[Web of Science][Medline]
Reilly, C.F., Fritze, L.M. and Rosenberg, R.D. (1988) Heparin-like molecules regulate the number of epidermal growth factor receptors on vascular smooth muscle cells. J. Cell. Physiol., 136, 23–32.[Web of Science][Medline]
Reilly, C.F., Kindy, M.S., Brown, K.E. et al. (1989) Heparin prevents vascular smooth muscle cell progression through the G1 phase of cell cycle. J. Biol. Chem., 264, 6990–6995.
Ross, R. (1993) The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature, 362, 801–809.[Medline]
Rudolph, M.I., Reinicke, K., Cruz, M.A., et al. (1993) Distribution of mast cells and the effect of their mediators on contractility in human myometrium. Br. J. Obstet. Gynaecol., 100, 1125–1130.[Web of Science][Medline]
Sherr, C.J. and Roberts, J.M. (1995) Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev., 9, 1149–1163.
Singh, S., Ross, S.R., Acena, M. et al. (1992) Stroma is critical for preventing or permitting immunological destruction of antigenetic cancer cells. J. Exp. Med., 175, 139–146.
Sperr, W.R., Czerwenka, K., Mundigler, G. et al. (1993) Specific activation of human mast cells by the ligand for c-kit: comparison between lung, uterus and heart mast cells. Int. Arch. Allergy Imunol., 102, 170–175.
Stewart, E.A. and Nowak, R.A. (1996) Leiomyoma-related bleeding: a classic hypothesis updated for the molecular era. Hum. Reprod. Update, 2, 295–306.
Valent, P., Spanblochl, E., Sperr, W.R. et al. (1992) Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/ kit-ligand in long-term culture. Blood, 80, 2237–2245.
van der Ven, L.T., Gloudemans, T., Roholl, P.J. et al. (1994) Growth advantage of human leiomyoma cells compared to normal smooth muscle cells due to enhanced sensitivity toward insulin-like growth factor I. Int. J. Cancer, 59, 427–434.[Web of Science][Medline]
Wright,T.C. Jr., Castellot, JJ. Jr., Petitou, M. et al. (1989) Structural determinants of heparin's growth inhibitory activity. Interdependence of oligosaccharide size and charge. J. Biol. Chem., 264, 1534–1542.
Yokoi, H., Nakayama, H., Horie, K. et al. (1994) High incidence of uterine inversion in mast cell-deficient osteopetrotic mutant mice of mi/mi genotype. Biol. Reprod., 50, 1034–1039.[Abstract]
Zsebo, K.M., Wypych, J., McNiece, I.K. et al. (1990) Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver-conditioned medium. Cell, 63, 195–201.[Web of Science][Medline]
Submitted on July 20, 1998; accepted on October 16, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Zaitseva, B. J. Vollenhoven, and P. A.W. Rogers Retinoids regulate genes involved in retinoic acid synthesis and transport in human myometrial and fibroid smooth muscle cells Hum. Reprod., May 1, 2008; 23(5): 1076 - 1086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. V. Rodriguez, Z. Alfonso, R. Zhang, J. Leung, B. Wu, and L. J. Ignarro Clonogenic multipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells PNAS, August 8, 2006; 103(32): 12167 - 12172. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Yu, D. A. Quinn, H. G. Garg, and C. A. Hales Cyclin-Dependent Kinase Inhibitor p27Kip1, But Not p21WAF1/Cip1, Is Required for Inhibition of Hypoxia-Induced Pulmonary Hypertension and Remodeling by Heparin in Mice Circ. Res., October 28, 2005; 97(9): 937 - 945. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Fasciano, R. C. Patel, I. Handy, and C. V. Patel Regulation of Vascular Smooth Muscle Proliferation by Heparin: INHIBITION OF CYCLIN-DEPENDENT KINASE 2 ACTIVITY BY p27kip1 J. Biol. Chem., April 22, 2005; 280(16): 15682 - 15689. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. R. Mason, A. C. Lake, J. E. Wubben, R. A. Nowak, and J. J. Castellot Jr The growth arrest-specific gene CCN5 is deficient in human leiomyomas and inhibits the proliferation and motility of cultured human uterine smooth muscle cells Mol. Hum. Reprod., March 1, 2004; 10(3): 181 - 187. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. R. Mason, R. A. Nowak, C. C. Morton, and J. J. Castellot Jr. Heparin Inhibits the Motility and Proliferation of Human Myometrial and Leiomyoma Smooth Muscle Cells Am. J. Pathol., June 1, 2003; 162(6): 1895 - 1904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Chegini, J. Verala, X. Luo, J. Xu, and R. S. Williams Gene Expression Profile of Leiomyoma and Myometrium and the Effect of Gonadotropin Releasing Hormone Analogue Therapy Reproductive Sciences, April 1, 2003; 10(3): 161 - 171. [Abstract] [PDF]
|
|
|
|
|
|
A. Horiuchi, T. Nikaido, T. Yoshizawa, K. Itoh, Y. Kobayashi, T. Toki, I. Konishi, and S. Fujii HCG promotes proliferation of uterine leiomyomal cells more strongly than that of myometrial smooth muscle cells in vitro Mol. Hum. Reprod., June 1, 2000; 6(6): 523 - 528. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Zhao, J. Letterman, and B. M. Schreiber beta -Migrating Very Low Density Lipoprotein (beta VLDL) Activates Smooth Muscle Cell Mitogen-activated Protein (MAP) Kinase via G Protein-coupled Receptor-mediated Transactivation of the Epidermal Growth Factor (EGF) Receptor. EFFECT OF MAP KINASE ACTIVATION ON beta VLDL PLUS EGF-INDUCED CELL PROLIFERATION J. Biol. Chem., August 10, 2001; 276(33): 30579 - 30588. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||