Cyclic mechanical stretch augments both interleukin-8 and monocyte chemotactic protein-3 production in the cultured human uterine cervical fibroblast cells

Intensive local leukocyte infiltration in the uterine cervix is a characteristic feature in the process of cervical ripening. The infiltrated leukocytes include neutrophils, macrophages and monocytes, which are believed to play important roles in cervical ripening by secreting elastase, matrix metalloproteinase and interleukin-1 (IL-1). Interleukin-8 (IL-8) and monocyte chemotactic protein-3 (MCP-3) belong to the CXC and CC chemokine families, and mediate the chemotaxis of neutrophils and monocytes/macrophages respectively. The aim of the present study was to investigate the possible involvement of IL-8 and MCP-3 in leukocyte chemotaxis in cervical ripening. Immunohistochemistry and RT-PCR detected both IL-8 and MCP-3 expression in human pregnant uterine cervices. Labour-like cyclic mechanical stretch for 48 h significantly elevated both IL-8 (555%) and MCP-3 (360%) secretion from cultured human uterine cervical fibroblast (CxF) cells (P<0.05 for both). Cyclic mechanical stretch for 24, 36 and 48 h significantly increased both IL-8 and MCP-3 mRNA expression in CxF cells (P<0.05 for all). The stretch-induced augmentation of both IL-8 and MCP-3 expression was significantly suppressed by an activator protein-1 (AP-1) inhibitor, curcumin. These data suggest that cyclic mechanical stretch of the uterine cervix by the presenting part of the fetus during labour may augment both IL-8 and MCP-3 production in the uterine cervix via AP-1 activation.     

Prostanoid receptors in human uterine myocytes: the effect of reproductive state and stretch

In the human, prostanoids are known to be important mediators of uterine relaxation and contraction during pregnancy and parturition. We have previously shown that stretch of uterine smooth muscle cells increased prostaglandin H synthase 2 (PGHS-2) mRNA expression, PGHS-2 peptide synthesis and activity, however, the net effect on uterine contractility of this increase in prostaglandin synthesis would be determined by the expression of the different prostanoid receptors. Therefore, the aims of this study were to establish the expression of prostanoid receptor mRNA in uterine myocytes obtained from women in different reproductive states and to test the hypothesis that stretch of uterine myocytes alters prostanoid receptor mRNA expression to promote uterine contractility. Myocytes were isolated from women undergoing hysterectomy (NP) and pregnant women undergoing LSCS either before (NL) or after the onset of labour (L) and were subjected to 11% stretch for 1 h (n = 6 in all cases). Copy numbers of the individual receptors varied widely with reproductive state but followed the pattern: FP > IP = DP = EP-4 > TP = EP-3 = EP-2 > EP-1. FP mRNA expression was significantly lower in the NL group compared to the NP group and EP-3, EP-4 and TP mRNA expression was significantly lower in both NL and L groups compared to NP group levels. The level of mRNA expression of EP-1, EP-2, DP and IP did not differ between NP, NL and L groups. Stretch of cells derived from the NP group resulted in a significant decrease in EP-4 mRNA expression alone and of the NL group a significant decrease in EP-2 mRNA expression alone. Stretch had no effect on cells derived from the L group. These data show that pregnancy is associated with a significant reduction in 3 of 4 pro-contraction associated prostanoid receptor mRNA expression and 1 of 4 pro-relaxant. Stretch elicited no consistent change in prostanoid receptor mRNA expression.     

Mechanical stretch of human uterine smooth muscle cells increases IL-8 mRNA expression and peptide synthesis

Labour is associated with increased synthesis of interleukin-8 (IL-8) by the fetal membranes and myometrium, which leads to an inflammatory infiltrate. Stretch has been shown to increase the expression of contraction-associated proteins in animal models of labour and in human myocytes in vitro. In this study, we tested the hypothesis that mechanical stretch of human myometrial cells increases IL-8 messenger ribonucleic acid (mRNA) expression. We isolated myocytes from non-pregnant women undergoing hysterectomy and pregnant women undergoing Caesarean section before and after the onset of labour. Myocytes in culture were subjected to stretch of varying intensity (6-16%) and duration (1 or 6 h) using the Flexercell system. IL-8 mRNA expression was lowest in myocytes from pregnant women not in labour, intermediate in those from non-pregnant women and greatest in those from pregnant women in labour. Stretch increased IL-8 mRNA expression independent of reproductive state. The stretch-induced increase in IL-8 mRNA expression was associated with higher IL-8 levels in the culture supernatant and enhanced promoter activity. These data suggest that stretch contributes to the increase in myometrial IL-8 synthesis associated with the onset of labour in humans.     

Prostaglandin F(2alpha), cytokines and cyclic mechanical stretch augment matrix metalloproteinase-1 secretion from cultured human uterine cervical fibroblast cells

Human uterine cervical tissue is composed mainly of fibroblast cells and the extracellular matrix in which collagen types I and III predominate. It is hypothesized that these collagens are degraded by matrix metalloproteinases (MMPs) in the initial step of uterine cervical ripening during parturition. Among the MMPs, MMP-1, -8 and -13 have substrate selectivity for collagen types I and III. In the present study, we examined the regulation of MMP-1 secretion from the human uterine cervix. Immunohistochemistry detected strong staining of MMP-1, but not of MMP-8 or -13, in stromal cells of the pregnant uterine cervix. The MMP-1 expression in the pregnant uterine cervix was further confirmed by Western blot analysis and RT-PCR. To clarify the regulation of MMP-1 production, we subsequently investigated the effects of prostaglandins, inflammatory cytokines and cyclic mechanical stretch on the secretion of MMP-1 from cultured human uterine cervical fibroblast cells. Treatment with prostaglandin (PG)F(2alpha) (10(-7) to 10(-5) mol/l) or interleukin (IL)-1alpha (0.01-1.0 ng/ml) or stimulation with cyclic mechanical stretch increased MMP-1 secretion from cultured human uterine cervical fibroblast cells, with maximal increases of 3.4-, 4.5- and 1.9-fold respectively (24 h of treatment, P < 0.05 for all comparisons). These data suggest that MMP-1 may play a significant role in the degradation of extracellular collagen types I and III in the pregnant uterine cervix during the process of cervical ripening, in response to various stimulations such as PGF(2alpha), IL-1alpha and mechanical stretch.     

Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta

Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.     

Progesterone down-regulates insulin-like growth factor-I expression in cultured human uterine leiomyoma cells

BACKGROUND: Insulin-like growth factor-I (IGF-I) plays crucial roles in uterine leiomyoma cell growth through stimulating proliferation and inhibiting apoptosis. The present study was conducted to elucidate whether progesterone affects IGF-I and its receptor expression in cultured leiomyoma cells. METHODS: Isolated leiomyoma cells were subcultured in Phenol Red-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions for an additional 48 h in the presence or absence of 17beta-estradiol (E(2)) (10 ng/ml) or progesterone (100 ng/ml). IGF-I and its receptor mRNA and immunoreactive IGF-I in the cultured cells were assessed by quantitative RT-PCR with Southern blot analysis and by radioimmunoassay with Seppak C18 chromatography, respectively. The presence of estrogen receptor (ER) and progesterone receptor (PR) in cultured leiomyoma cells was immunocytochemically examined. RESULTS: Both treatment with progesterone alone and treatment with E(2) and progesterone combined significantly decreased IGF-I mRNA and protein expression in cultured leiomyoma cells compared with that in untreated cultures, but treatment with E(2) alone did not. IGF-I receptor mRNA expression in those cells was not affected by treatment with either E(2) or progesterone. Immunocytochemical analysis revealed that PR protein expression in cultured leiomyoma cells maintained in a serum-free condition for 48 h whereas ER protein expression in the cells remarkably decreased after 24 h culture under the serum-free condition. CONCLUSIONS: The present study provided evidence for the first time that progesterone down-regulates IGF-I mRNA and protein expression in cultured leiomyoma cells without affecting IGF-I receptor mRNA expression.     

Physiology: Autonomic nervous modulation and effects of a prostaglandin synthase inhibitor on human cervical secretion

Modulation of cervical secretion at ovulation time was studiedin 10 women with regular menstruations. In an in-vivo modelwith repeated collection of mucus samples during three 90-minperiods, the amounts of mucus in a control cycle and in threeexperimental cycles were compared. Drugs interacting with theautonomic nervous system and a prostaglandin synthase inhibitorwere administered at time of ovulation. The cholinomimetic drugcarbacholine significantly increased cervical secretion, whilethe anticholinergic drug butylscopolamine markedly inhibitedthis secretion. A long-lasting decrease in secretion was seenafter administration of the prostaglandin synthase inhibitordiclofenac. Beside regulation of cervical secretion by the ovarianhormones, these results suggest an autonomic nervous modulationof cervical secretion, and in addition an impact on cervicalsecretion by a prostaglandin synthase inhibitor. The effectson fertility regulation in the female are discussed.     

Apoptosis in woman uterine cervix in pathologies associated with human papillomavirus

The level of apoptosis in uterine cervical tissue was evaluated in healthy women and in patients with various pathologies. No signs of apoptosis were found in unchanged stratified epithelium, condyloma latum, and condyloma acuminatum. The level of apoptosis decreased with progression of neoplastic epithelial transformations, usually no apoptosis was observed in samples of stage III cervical intraepithelial neoplasms. The development of preinvasive carcinoma was accompanied by activation of apoptotic processes most pronounced in the upper third of the epithelium. In some stage I and stage I-II cervical intraepithelial neoplasms, apoptosis and elimination of the basal layer cells caused rejection of the epithelium which can explain regression of this pathology at the initial stages. The prevalence of human papillomavirus infection directly correlated with neoplastic changes in the cervical epithelium.     

The importance of fibroblasts in remodelling of the human uterine cervix during pregnancy and parturition

It is well established that fibroblasts play a crucial role in pathophysiological extracellular matrix remodelling. The aim of this project is to elucidate their role in normal physiological remodelling. Specifically, the remodelling of the human cervix during pregnancy, resulting in an enabled passage of the child, is used as the model system. Fibroblast cultures were established from cervices of non-pregnant women, women after 36 weeks of pregnancy and women directly after partus. The cells were immunostained and quantified by western blots for differentiation markers. The cultures were screened for cytokine and metalloproteinase production and characterized by global proteome analysis. The cell cultures established from partal donors differ significantly from those from non-pregnant donors, which is in accordance with in vivo findings. A decrease in alpha-smooth actin and prolyl-4-hydroxylase and an increase in interleukin (IL)-6, IL-8 and matrix metalloproteinases (MMP)-1 and MMP-3 were observed in cultures from partal donors. 2D-gel electrophoresis followed by mass spectrometry showed that the expression of 59 proteins was changed significantly in cultures of partal donors. The regulated proteins are involved in protein kinase C signalling, Ca2+ binding, cytoskeletal organization, angiogenesis and degradation. Our data suggest that remodelling of the human cervix is orchestrated by fibroblasts, which are activated or recruited by the inflammatory processes occurring during the ripening cascade.     

Physiology: The production of placental protein 14 by human uterine tubal epithelial cells in culture

Cells prepared from the mucosal layer obtained from the fimbrial,proximal ampullary and distal ampullary regions of the humanuterine (Fallopian) tube have been grown in monolayer culture.Immunocytochemistry with anti-cytokeratin, anti-vimentin oranti-CD 45 antibodies indicated that the overwhelming numberof cells were epithelial in nature and were free of fibroblastsand leukocytes. Basal and steroid-stimulated placental protein14 (PP14) production was investigated in tissue obtained fromnine patients undergoing hysterectomy, by addition of oestradioland/or progesterone to confluent cultures. Basal PP14 productionvaried considerably between experiments, probably due to differencesbetween individuals from whom the tissue had been obtained.However, there was no difference in basal PP14 production bycells prepared from the fimbrial, proximal ampullary and distalampullary parts of the tube obtained from the same patient.When total PP14 production by cells obtained from an individualuterine tube was pooled both progesterone and oestradiol significantly(P < 0.05) stimulated the production of PP14 but the effectof progesterone either alone or in the presence of oestradiolwas numerically greater than that of oestradiol alone. ConsideringPP14 production by cells prepared from the different regionsof the tube showed that cells from the fimbrial region weremore responsive to steroid stimulation than cells prepared fromeither the proximal or the distal ampullary regions. All combinationsof hormonal supplementation stimulated PP14 production by cellsfrom the fimbrial region on all days measured (P < 0.05 -P < 0.001). In contrast oestradiol alone had no effect onPP14 production by cells prepared from the proximal and distalampullary parts of the tube on any day measured. Maximum oestradiol-stimulatedPP14 values, expressed as percentages of control, were (mean± SEM) 146 ± 11, 113 ± 7.5 and 108 ±8.5 for cells prepared from the fimbrial, proximal ampullaryand distal ampullary portions of the tube respectively. Maximumprogesterone or progesterone plus oestradiol-stimulated valueswere very similar; those for progesterone plus oestradiol were(mean ± SEM) 151 ± 8.6, 130 ± 6.2 and 118± 10 for cells from the fimbrial, proximal ampullaryand distal ampullary parts of the tube. The results show thata cell culture system has been established which allows theinvestigation of the function of human uterine tubal cells inculture. Using this system we have shown that PP14 productionby these cells is stimulated by steroids and that cells preparedfrom the fimbrial region of the uterine tube appear more responsiveto steroid stimulation than those prepared from other regionsof the tube.     

Secretory products of breast cancer cells upregulate hyaluronan production in a human osteoblast cell line

Hyaluronan (HA) has been implicated in breast cancer progression and metastasis to lymph nodes. Although breast cancer has a strong propensity for metastasis to the bone, the role of HA in the development of bone metastasis in breast cancer is not well delineated. In order to determine the role of HA in breast cancer induced osteolysis, we examined the effect of secretory products in the conditioned medium of breast cancer cell lines on the expression of hyaluronan synthases (HAS), accumulation of HA in pericellular matrix, secretion of HA in the culture media, and on expression of surface HA receptors, in a human immature osteoblast cell line (Hfob). Our results show that conditioned medium derived from breast cancer cells upregulate the expression of hyaluronan synthases, HAS1 and HAS2, followed by significant increase in pericellular and secreted HA in Hfob cells. Our results further demonstrate that both CD44 and receptor for hyaluronan-mediated motility (RHAMM) are involved in binding cell surface associated HA on Hfob cells. Analysis of the growth factors in the conditioned medium implicates TGF-β1 in the modulation of HAS1 and HAS2, as well as in the increase in pericellular and secreted HA. This report is the first to show that soluble factors produced by breast cancer cells mediate increase in HA production in osteoblasts.     

牵张应力对体外骨髓间充质干细胞形态、排列及迁移的影响

目的观察牵张应力对体外骨髓间充质干细胞(BMSC)的细胞形态、排列及迁移生物学行为的影响。方法利用应力装置对体外培养的BMSC施以周期性牵张应力刺激,在相差显微镜下观察细胞形态、排列及迁移的变化,借助计算机图像处理和分析系统(ImageJ软件)进行定量分析。结果 BMSC受力后,形态明显变长,轮廓鲜明。强度为12%应力干预BMSC12h后,细胞长度增长1.56倍;干预24h后,细胞长度增长1.66倍。BMSC受力刺激后,细胞排列规则,沿受力环切线方向分布。受力12h及24h后,BMSC与切线夹角统计值分别为9.48°±2.83°、8.30°±6.03°,与切线夹角<10°的细胞比例分别为88.52%±6.43%,92.33%±4.98%,明显较对照组规律,并且细胞的迁移速度明显快于对照组。结论 BMSC加载应力刺激后,其形态学及细胞迁移速度均发生明显变化。     

Lymphotoxin production by regional lymph node lymphocytes in patients with uterine cervical cancer

The cytotoxin production by regional lymph node cells was examined in 25 patients with uterine cervical cancer and 10 patients with uterine myoma. The patients in stage I had significantly increased spontaneous release of cytotoxins compared with that in stages II, III, and IV. The spontaneous release in stages III and IV was markedly reduced. There was no difference in the release of cytotoxins from peripheral blood lymphocytes between cancer patients and patients with myoma or healthy controls. The cytotoxin production by lymph node cells was increased in stage III by stimulating with formalin-fixed QG-K cells derived from uterine cervical cancer, but not in stages I and II. Almost all of the cytotoxic activity of cytotoxin was abrogated by antilymphotoxin antibody. However, the cytotoxin activity was partially inhibited by anti-tumor necrosis factor antibody. These results suggest that cytotoxins released from the regional lymph node cells of uterine cancer patients are derived from, most of all, lymphotoxin.     

Mechanical stretch activates type 2 cyclooxygenase via activator protein-1 transcription factor in human myometrial cells

The uterus is subject to stretch throughout pregnancy, which, in the presence of progesterone, is a potent stimulus for uterine growth. However, in the absence of progesterone or when stretch is excessive, as in multiple pregnancy, it may provoke the onset of labour. We have investigated the effect of stretch on prostaglandin synthesis in primary human uterine myocytes [non-pregnant (NP), pregnant not in labour (NL) and pregnant in labour (L)]. The cells were grown on flexible bottom culture plates and subjected to 1 or 6 h static stretch. Expression of type 2 cyclooxygenase (COX-2) mRNA was similar in samples obtained from NP and L groups and both were significantly greater than those found in the NL group. Stretch of cells from all groups resulted in increased COX-2 mRNA expression. In further studies carried out on cells taken from the NL group, 6 h of stretch resulted in increased COX-2 protein levels and, in the media, increases in prostaglandin (PG) I(2) metabolite and PGE(2) concentrations and a reduction in the concentration of PGF(2)alpha metabolites. After stretch, EMSA studies showed increased activator protein-1 (AP-1) nuclear protein DNA binding activity but not of nuclear factor kappaB. These data demonstrate that stretch of human myocytes results in increased COX-2 activity and suggest that this may occur through activation of the AP-1 system.     

8-硝基白杨素对人宫颈癌Hela细胞增殖和凋亡的影响

目的：观察8-硝基白杨素(8-NOChR)抑制体外培养人宫颈癌Hela细胞增殖和诱导凋亡作用。方法：体外培养人宫颈癌Hela细胞系细胞。MTT比色法测定Hela细胞增殖活性。软琼脂培养克隆形成法检测Hela细胞集落形成能力。AO/EB染色荧光显微镜观察Hela细胞凋亡形态学改变。DNA凝胶电泳观察梯形DNA条带。结果：MTT比色测定显示,8-NOChR抑制Hela细胞增殖,呈剂量依赖性。软琼脂培养克隆形成法检测表明,8-NOChR显著抑制Hela细胞集落形成,呈剂量依赖性。AO/EB染色荧光显微镜观察发现,8-NOChR诱导Hela细胞呈现典型凋亡细胞形态特征。8-NOChR（30μmol/L）处理Hela细胞72h,琼脂糖凝胶电泳出现“梯形”DNA条带。结论：8-NOChR具有抑制人宫颈癌Hela细胞增殖和诱导细胞凋亡作用     

Seminal plasma differentially regulates inflammatory cytokine gene expression in human cervical and vaginal epithelial cells

Exposure to semen elicits an inflammatory response in the female reproductive tract of rodents and other animals. The nature and regulation of any similar response in humans is poorly understood. This study investigated seminal plasma induction of inflammatory cytokine and chemokine gene regulation in human cervical and vaginal epithelial cells in vitro. Affymetrix microarray gene profiling revealed that inflammatory cytokine genes were prevalent among 317 known genes differentially expressed in immortalized ectocervical epithelial (Ect1) cells after incubation with pooled human seminal plasma. A dose- and time-dependent induction by seminal plasma of IL8, IL6, CSF2 and CCL2 mRNA expression in Ect1 cells was verified by quantitative RT-PCR. This was accompanied by increases in Ect1 secretion of immunoactive gene products IL-8, IL-6, GM-CSF and MCP-1. Similar cytokine responses were elicited in primary ectocervical epithelial cells. Endocervical epithelial (End1) and vaginal epithelial (Vk2) cells were less responsive to seminal fluid, with induction of IL-8 and MCP-1, but not GM-CSF or IL-6. In a panel of 10 seminal plasma samples, considerable variation in inflammatory cytokine-inducing activity was evident. These experiments show that seminal plasma can elicit expression of a range of inflammatory cytokines and chemokines in reproductive tract epithelia, and implicate the ectocervix as the primary site of responsiveness, with gene-specific differences in the kinetics and site-restrictedness of the response. Seminal factor regulation of inflammatory cytokines in the cervical epithelium is implicated in controlling the immune response to seminal antigens, and defence against infectious agents introduced at intercourse.