Regulation of mesangial cell growth by polypeptide mitogens. Inhibitory role of transforming growth factor beta.

Proliferation of mesangial cells is a common histologic abnormality in glomerular diseases. In vivo studies suggest a role for platelets and monocytes-macrophages in mediating glomerular hypercellularity. The authors recently reported that several peptide growth factors stimulate DNA synthesis and growth of human mesangial cells. This article reports that transforming growth factor beta (TGF-beta), a peptide released by inflammatory cells and platelets, inhibits DNA synthesis and growth of human mesangial cells. The stimulatory and inhibitory effects of these mitogens on DNA synthesis and growth was confirmed by autoradiography and cell counting. The inhibitory effect of TGF-beta is not mediated at the receptor level because TGF-beta did not inhibit the binding of epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) to mesangial cells. Because peptide growth factors that stimulate DNA synthesis in mesangial cells induce expression of PDGF mRNAs, the effect of TGF-beta on PDGF mRNAs expression induced by peptide growth factors was studied. TGF-beta did not lower the increased levels of PDGF mRNAs caused by EGF or PDGF. These data show that TGF-beta is a potent inhibitor of DNA synthesis and growth of mesangial cells. The mechanism of the inhibitory effect of TGF-beta remains to be determined.     

Comparative effects of heparin-binding epidermal growth factor-like growth factor on the growth of cultured human uterine leiomyoma cells and myometrial cells

BACKGROUND: The objective of this study was to investigate the comparative effects of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on the growth of cultured human leiomyoma cells and myometrial cells. METHODS: Isolated 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 24 and 48 h in the presence or absence of graded concentrations of HB-EGF (0.1, 1, 10 and 100 ng/ml). These cells were used for immunocytochemical analysis for Ki67, western blot analysis for proliferating cell nuclear antigen (PCNA) and human EGF receptor (HER1), and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay. RESULTS: Treatment with HB-EGF at concentrations >1 ng/ml significantly increased the Ki67-positive rate of cultured leiomyoma cells and myometrial cells. Treatment with HB-EGF also resulted in a dose-dependent increase in PCNA expression in both cells compared with untreated control cultures. A significant increase in PCNA expression in cultured myometrial cells was noted following treatment with HB-EGF at concentrations >1 ng/ml, whereas an increase in PCNA expression in cultured leiomyoma cells was noted following treatment with HB-EGF at concentrations >10 ng/ml. HER1 expression was significantly higher in untreated myometrial cells than in untreated leiomyoma cells. A significant increase in HER1 expression in myometrial cells was observed when treated with HB-EGF at concentrations >10 ng/ml, whereas a significant increase in HER1 expression in leiomyoma cells was noted only by the treatment with HB-EGF at concentrations >100 ng/ml. Treatment with HB-EGF decreased the TUNEL-positive rate of those cells with no significant differences between the two cell types. CONCLUSIONS: The results obtained suggest that HB-EGF plays a role in stimulating the proliferation of leiomyoma cells and myometrial cells and in inhibiting apoptosis of those cells through augmentation of HER1 expression. Since the proliferative potential of myometrial cells responded better to HB-EGF than that of leiomyoma cells, HB-EGF may play a more vital role in myometrial growth than leiomyoma growth.     

A novel selective progesterone receptor modulator asoprisnil (J867) down-regulates the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured uterine leiomyoma cells

BACKGROUND: This study was conducted to evaluate the effects of a novel selective progesterone receptor modulator (SPRM) asoprisnil on the expression of growth factors and their receptors and on growth factor-induced proliferation of cultured uterine leiomyoma and matching myometrial cells. METHODS: The expression of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and transforming growth factor (TGFbeta3) was assessed by immunocytochemistry and semi-quantitative RT-PCR. The expression of phosphorylated EGF receptor (p-EGFR), IGF-I receptor alpha subunit (IGF-IRalpha) and phosphorylated TGFbeta receptor type II (p-TGFbeta RII) was assessed by Western blot analysis. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. RESULTS: Treatment with 10(-7) M asoprisnil decreased EGF, IGF-I and TGFbeta3 mRNA and protein expression as well as p-EGFR, IGF-IRalpha and p-TGFbeta RII protein expression in leiomyoma cells cultured for 72 h. EGF (100 ng/ml), IGF-I (100 ng/ml) and TGFbeta3 (10 ng/ml) increased the number of viable leiomyoma cells cultured for 72 h, whereas the concomitant treatment with 10(-7) M asoprisnil antagonized the growth factor-induced increase in leiomyoma cell proliferation. In cultured myometrial cells, however, asoprisnil affected neither the growth factor and their receptor expression nor the cell proliferation. CONCLUSION: Asoprisnil inhibits the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured leiomyoma cells without affecting their expressions in myometrial cells.     

Early signals in the mitogenic response of Swiss 3T3 cells: a comparative study of purified PDGF homodimers

Platelet-derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB. Two distinct receptor subunits, alpha and beta, have been identified which bind either all three isoforms of PDGF (alpha) or PDGF-BB only (beta). Here, we have compared the effect of purified PDGF homodimers on the early intracellular signaling events and mitogenesis in Swiss 3T3 cells, which possess equivalent numbers of the alpha and beta subunits. Both PDGF-AA and PDGF-BB stimulated receptor phosphorylation, inositol phosphate formation, activation of protein kinase C, calcium mobilization, EGF receptor transmodulation, sodium uptake, arachidonic acid release, cyclic AMP accumulation, and c-fos induction in a comparable, dose-dependent manner (half-maximal values for all these response were in the 2-10 ng/ml range for both homodimers). At high concentrations of PDGF (greater than 10 ng/ml), the BB homodimer effect on early membrane and cytosolic signals was 20-30% greater than PDGF-AA, reflecting the greater number of available binding sites for PDGF-BB. DNA synthesis studies indicated that PDGF-AA and PDGF-BB were potent mitogens for Swiss 3T3 cells, displaying identical dose-response effects. Moreover, the mitogenic activities of both homodimers were equally potentiated in the presence of insulin. These results indicate that both PDGF-AA and PDGF-BB stimulate the full complement of molecular responses required for the synergistic interactions mediating long-term mitogenesis. We conclude that alpha and beta receptor subunits do not differ in their ability to transduce PDGF-mediated signals leading to DNA synthesis in Swiss 3T3 cells.     

Insulinlike growth factor-1 is a progression factor for human mesangial cells.

Mesangial cell hyperplasia is a feature common to several human glomerular diseases. The cause of this increased cell number is unknown. The authors assessed human mesangial cells in vitro and found that they possessed an insulinlike growth factor-1 (IGF-1) receptor consisting of alpha and beta units (Mr, 130 k and 90 k respectively). Fifty percent inhibition of IGF-1 specific binding to the receptor required 1 X 10(-9) M IGF-1, greater than 1 X 10(-6) M insulin and 1 X 10(-7) M multiplication stimulating activity (MSA). Analysis of binding by the method of Scatchard revealed one type of IGF-1 receptor with a Kd of 1.35 X 10(-9) M, and a number per cell of 1.04 X 10(5). Binding studies on whole glomeruli had similar specificity and there were 7.17 X 10(7) receptors per glomerulus (Kd, 1.12 X 10(-9) M). Examination of the effect of IGF-1 on the cell cycle revealed that exposure of cells to both IGF-1 and platelet-derived growth factor (PDGF) led to a significant increase in 3H-thymidine incorporation into cell layers. Antibody to PDGF abolished only that response due to PDGF. Similarly, the labeling index of cells pretreated with PDGF, washed, and then exposed to IGF-1 was increased, whereas if the order of ligand exposure was reversed, there was no such additive effect. Finally, PDGF increased RNA and protein synthesis, and this response was not enhanced by IGF-1. In summary, human mesangial cells and whole glomeruli possess IGF-1-specific receptors and IGF-1 was found to act as a progression factor in the cell cycle.     

Early Signals in the Mitogenic Response of Swiss 3T3 Cells: A Comparative Study of Purified PDGF Homodimers

Abstract

Platelet-derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB. Two distinct receptor subunits, α and β, have been identified which bind either all three isoforms of PDGF (α) or PDGF-BB only (β). Here, we have compared the effect of purified PDGF homodimers on the early intracellular signaling events and mitogenesis in Swiss 3T3 cells, which possess equivalent numbers of the α and β subunits. Both PDGF-AA and PDGF-BB stimulated receptor phosphorylation, inositol phosphate formation, activation of protein kinase C, calcium mobilization, EGF receptor transmodulation, sodium uptake, arachidonic acid release, cyclic AMP accumulation, and c-fos induction in a comparable, dose-dependent manner (half-maximal values for all these responses were in the 2–10 ng/ml range for both homodimers). At high concentrations of PDGF (> 10 ng/ml), the BB homo-dimer effect on early membrane and cytosolic signals was 20–30% greater than PDGF-AA, reflecting the greater number of available binding sites for PDGF-BB. DNA synthesis studies indicated that PDGF-AA and PDGF-BB were potent mitogens for Swiss 3T3 cells, displaying identical dose-response effects. Moreover, the mitogenic activities of both homodimers were equally potentiated in the presence of insulin. These results indicate that both PDGF-AA and PDGF-BB stimulate the full complement of molecular responses required for the synergistic interactions mediating long-term mitogenesis. We conclude that α and β receptor subunits do not differ in their ability to transduce PDGF-mediated signals leading to DNA synthesis in Swiss 3T3 cells.     

Role of endothelin-1 in regulating proliferation of cultured human uterine smooth muscle cells

Endothelin-1 (ET-1) exhibits vasoconstricting and growth-promoting properties in vascular smooth muscle. Whether ET-1 has mitogenic properties in uterine smooth muscle cells, and which ET receptor subtype mediates this response, is unknown. The present study was undertaken to examine the proliferative potential of the ET family on human myometrial cells in culture. ET-1 stimulated DNA synthesis and proliferation of myometrial cells. The absence of a stimulating effect of endothelin-3 (ET-3) or the ETB agonist sarafotoxin 6c (S6c) was observed. The proliferative effect of 100nM ET-1 was blocked by the two ETA antagonists (BQ 123 and FR 139317), whereas the ETB antagonist IRL 1038 was ineffective. These data indicated that ET-1-induced DNA synthesis was mediated only by the ETA receptor subtype. Pertussis toxin (PTX) pretreatment completely abolished this effect, indicating that this pathway was coupled to the ETA receptor via the Gi protein family. PTX treatment partially decreased serum-induced DNA synthesis. This suggests that some factors from serum may operate via the G- protein in initiation of mitogenesis. Insulin-like growth factors (IGFs), epidermal growth factor (EGF) and insulin were found to be mitogens in the absence of serum, and they had no potentiating effect on ET-1-induced DNA synthesis. In the presence of 0.5% serum, EGF alone caused a weak increase in DNA synthesis, while all the growth factors were able to reduce the proliferative effect of ET-1. These findings on human myometrial cells in culture raise the possibility that, under certain conditions, ET-1 may function as a positive or as a negative modulator of smooth muscle proliferation.      

Epidermal growth factor as a candidate for ex vivo expansion of bone marrow-derived mesenchymal stem cells

Bone marrow mesenchymal stem cells (BMMSCs) are pluripotent cells capable of differentiating into several cell types and are thus an attractive cell source for connective tissue engineering. A challenge in such a use is expansion and directed seeding in vitro, requiring proliferation and survival, and directed migration, respectively, prior to functional differentiation. The epidermal growth factor (EGF) receptor (EGFR) is the prototypal growth factor receptor and elicits these responses from a wide variety of stromal, epithelial, and endothelial cells. Ligands for this receptor are appealing for use in tissue engineering because they are relatively resistant to biological extremes and amenable to high-volume production. Therefore, we determined whether an EGFR ligand, EGF, could be used for ex vivo expansion of BMMSCs. EGF stimulated motility in rat and immortalized human BMMSCs. EGF-induced proliferation was observed in immortalized human BMMSCs but was not apparent in rat BMMSCs under our experimental conditions. EGF did not, however, rescue either type of BMMSC from apoptosis due to lack of serum. During our examination of key signaling intermediaries, EGF caused robust phosphorylation of extracellular signal-regulated protein kinase (ERK) and protein kinase B/akt (AKT) but only minimal phosphorylation of EGFR and phospholipase C-gamma in rat BMMSCs, whereas in the human BMMSCs these intermediaries were all strongly activated. EGF also induced robust ERK activation in primary porcine mesenchymal stem cells. EGF pretreatment or cotreatment did not interfere with secondarily induced differentiation of either type of BMMSC into adipogenic or osteogenic lineages. Platelet-derived growth factor (PDGF) effects were similar to but not additive with those elicited by EGF, with some quantitative differences; however, PDGF did interfere with the differentiation of these BMMSCs. These findings suggest that EGFR ligands could be used for ex vivo expansion and direction of BMMSCs.     

Spontaneously transformed NRK cells lose their mitogenic response to epidermal growth factor

To understand the relationship between growth factor-induced mitogenesis and spontaneous cell transformation, a clonal isolate of epidermal growth factor (EGF)-responsive NRK cells was passed in vitro until morphologically transformed variants arose. Subclones of EGF responsive (Cl-3) and EGF nonresponsive (Cl-10) NRK cells were isolated. Cl-3 cells grew as flat, contact-inhibited monolayers, while Cl-10 cells grew as rounded or spindle-shaped cells that formed dense foci. Cl-10 cells formed colonies in soft agar more efficiently (p less than 0.01) and formed larger tumors in nude mice (p less than 0.05) than Cl-3 cells. Cl-3 cells exhibited a sixfold increase in DNA synthesis in response to 1.0 nM EGF. Cl-10 cells did not increase DNA synthesis on exposure to 100 nM EGF. These different responses to EGF occurred despite similar numbers of receptors and similar receptor.binding affinities for EGF (Cl-3: 7000 receptors, Kd = 0.67 nM; Cl-10: 8000 receptors, Kd = 0.72 nM). No evidence of transforming growth factor-alpha was detected in either of these cell lines using Northern blots, Western blots, or biologic assays. We conclude that NRK cells which undergo spontaneous morphologic transformation and exhibit enhanced anchorage-independent growth lose their mitogenic response to EGF.     

胰岛素样生长因子1及血小板源性生长因子对人退变髓核细胞生物学活性的影响

背景：有研究表明胰岛素样生长因子1和血小板源性生长因子可抑制人椎间盘细胞凋亡。 目的：观察在体外培养条件下胰岛素样生长因子1、血小板源性生长因子对人退变髓核细胞生物学活性的影响。 方法：体外单层培养人退变髓核细胞,通过免疫组织化学鉴定细胞。对传3代人退变髓核细胞采用分别不同生长因子干预,实验分4组：胰岛素样生长因子1组,血小板源性生长因子组,胰岛素样生长因子1+血小板源性生长因子组及对照组。 结果与结论：胰岛素样生长因子1、血小板源性生长因子均可促进细胞增殖,促进细胞合成Ⅱ型胶原和聚集蛋白聚糖,其中胰岛素样生长因子1促Ⅱ型胶原合成作用强于血小板源性生长因子(P < 0.05),血小板源性生长因子促蛋白聚糖合成作用强于胰岛素样生长因子1(P < 0.05)。胰岛素样生长因子1促进细胞合成Ⅰ型胶原,血小板源性生长因子抑制细胞合成Ⅰ型胶原。结果证实,胰岛素样生长因子1、血小板源性生长因子均可通过促进细胞增殖、促进细胞合成Ⅱ型胶原和聚集蛋白聚糖,从而提高人退变髓核细胞的生物学活性。     

Ideal concentration of growth factors in rabbit's flexor tendon culture

Growth factors have the ability to stimulate matrix synthesis and cell proliferation in rabbit flexor tendon. Maximal stimulation effects of growth factors have a wide variation. It depends upon the different anatomic sites of the tendon segment, the kinds of growth factor, the concentration of growth factors, and the time sequence. Since proliferation was an early component of intrinsic tendon healing, we investigated the short-term dose response to four different growth factors on in vitro rabbit's tendon culture. We evaluated the effects according to the various concentrations of recombinant human insulin-like growth factor 1 (IGF), recombinant human epidermal growth factor (EGF), fibroblast growth factor (FGF), and recombinant human platelet-derived growth factor-BB (PDGF). Fetal calf serum was the most potent stimulator of cell proliferation and protein synthesis in in vitro rabbit's tendon culture. Matrix synthesis and cell proliferation were stimulated dose-dependently by IGF between the doses of 50 and 150 ng/ml. The maximum mitogenic effect of EGF was observed at the concentration of 100 ng/ml (1.3 times more than the media-only control culture). The rabbit's tendon responded significantly dose-dependently to PDGF, whereas there was no significant response to FGF.     

Progesterone receptor modulator CDB-2914 down-regulates vascular endothelial growth factor, adrenomedullin and their receptors and modulates progesterone receptor content in cultured human uterine leiomyoma cells

BACKGROUND: This study was conducted to evaluate the effects of graded concentrations (10(-8), 10(-7) and 10(-6) M) of progesterone receptor (PR) modulator CDB-2914 on the protein contents of PR, of vascular endothelial growth factor (VEGF), adrenomedullin (ADM) and their receptors in cultured human uterine leiomyoma and matching myometrial cells. METHODS: PR-A, PR-B, VEGF-A, VEGF-B, VEGF receptor (VEGFR)-1, VEGFR-2, ADM and ADM receptor (ADMR) contents were assessed by Western blot analysis. RESULTS: Treatment with 100 ng/ml progesterone increased VEGF-A, VEGF-B and ADM contents in cultured leiomyoma cells and normal myometrial cells. The concomitant treatment with 10(-6) M CDB-2914 significantly decreased the progesterone-induced VEGF-A, VEGF-B and ADM contents in cultured leiomyoma cells but not in normal myometrial cells. CDB-2914 treatment alone decreased VEGFR-1, VEGFR-2 and ADMR contents in cultured leiomyoma cells but not in normal myometrial cells. CDB-2914 treatment increased PR-A and decreased PR-B contents in cultured leiomyoma cells in a dose-dependent manner compared with untreated cultures, whereas no significant changes in PR isoform contents were observed in normal myometrial cells. CONCLUSIONS: These results suggest that CDB-2914 down-regulates VEGF, ADM and their receptor contents and modulates PR isoform contents in cultured leiomyoma cells in a cell-type-specific manner.     

Influence of platelet-derived growth factor on lens epithelial cell proliferation and differentiation

The expression pattern of platelet-derived growth factor (PDGF) and its receptor suggest a role in lens cell proliferation. PDGF is strongly expressed in the iris and ciliary body, situated opposite the proliferative cells of the lens epithelium which express the PDGF-alpha receptor. In this study, using lens epithelial explant cultures, we report that PDGF can induce a dose and time dependent increase in lens cell DNA synthesis. Culturing lens explants with both PDGF and FGF (a mitogen and differentiation factor for lens cells) resulted in responses greater than those induced by either growth factor alone. PDGF did not induce any changes typical of fibre differentiation; however, in combination with FGF it potentiated the fibre differentiating activity of FGF. Results obtained in this study support previous indications that PDGF has an important role in regulating lens cell proliferation. In addition, PDGF may have a role in potentiating FGF-induced lens fibre differentiation in vivo.     

Epidermal growth factor-mediated cell maturation and apoptosis in RGM-1 rat gastric mucosal cells.

Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) exert their activities through the same cell surface receptor. In the present study, the role of EGF in cell kinetics of rat gastric epithelial cell line, RGM-1, in which HB-EGF is known to function in an autocrine fashion, was investigated. EGF stimulated the mucus glycoprotein synthesis which is characterized with cell maturation, but competed with HB-EGF for cell proliferation. By flow cytometric analysis, EGF inhibited G2/M transition by 48 h after stimulation and induced apoptosis manifested by hypodiploid DNA by 72 h after stimulation. Consistent with this, DNA ladder formation together with the histone-associated DNA fragments was found in EGF-treated cells. These results indicate that EGF and HB-EGF possess different functions in RGM-1 cells and that EGF acts as a mediator of both cell maturation and apoptosis in these cells.     

Fetal mouse kidney maturation in vitro: Coordinated influences of epidermal growth factor, transferrin and hydrocortisone

Summary We have investigated the individual and combined actions of epidermal growth factor (EGF), transferrin and hydrocortisone on the maturation of whole fetal mouse metanephrol maintained in serum-free conditions for up to 5 days. The presence of EGF (100 ng/ml) resulted in elevated levels of [³H]-thymidine incorporation when compared to controls; autoradiograms showed that the proliferation of mesenchymal cells in the nephrogenic zone is particularly enhanced as verified by cell counting. Brush border hydrolase activities (alkaline phosphatase and -glutamyltransferase), on the other hand, were significantly diminished. Transferrin (5 g/ml) slightly stimulated DNA synthesis and potentiated EGF mitogenic action. The activation of DNA replication by the growth factor seems to be mediated through the protein kinase C pathway. When added alone, hydrocortisone (10^–6 M) strongly inhibited DNA synthesis, stimulated hydrolase activities and exerted a positive effect on brush border differentiation. When combined with EGF or to EGF + transferrin, hydrocortisone counteracted the effects of these latter peptides on DNA synthesis and enzyme activities. Considering the earlier observation of a reciprocal relation between proliferation and differentiation during the neotubulogenic phase of kidney development, the results described in the current study suggest that synergistic and synarchic actions of these heterologous factors are involved in the regulation of tubulogenesis.This investigation was supported by the Kidney Foundation of Canada     

Vascular endothelial growth factor, platelet-derived growth factor, and insulin-like growth factor-1 promote rat aortic angiogenesis in vitro.

The purpose of this study was to evaluate the vasoformative response of isolated vascular explants to a variety of growth factors that have been shown to stimulate angiogenesis. Rings of rat aorta were cultured in collagen gels under serum-free conditions in the presence or absence of vascular endothelial growth factor (VEGF), natural platelet-derived growth factor (PDGF), PDGF-AA, PDGF-BB, insulin-like growth factor-1 (IGF-1), transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta 1 (TGF-beta 1), epidermal growth factor (EGF), interleukin-1 alpha (IL-1 alpha), or hepatocyte growth factor (HGF). The angiogenic response of the rat aorta was stimulated by VEGF, PDGF, PDGF-AA, PDGF-BB, and IGF-1. Maximum stimulatory effects were obtained with VEGF and PDGF-BB. By contrast, TGF-beta 1 and IL-1 alpha had inhibitory activity. No significant effects were observed with TGF-alpha, EGF, or HGF. The vascular outgrowth of VEGF-stimulated cultures was primarily composed of microvessels, whereas that of PDGF- and IGF-1-stimulated cultures contained an increased number of fibroblast-like cells. The inability of TGF-alpha, TGF-beta 1, IL-1 alpha, EGF, and HGF to stimulate rat aortic angiogenesis in serum-free culture suggests that either these factors require the mediatory activity of accessory cells that are not present in the rat aorta model or that blood vessels are heterogeneous in their capacity to respond to different angiogenic factors.     

Expression of PDGF alpha- and beta-receptors in rat arterial smooth muscle cells is phenotype and growth state dependent

Adult rat arterial smooth muscle cells were shown to express mRNA for the platelet-derived growth factor (PDGF) alpha- and beta-receptors and to bind radioiodinated PDGF-AA and PDGF-BB in a phenotype-dependent and growth state-dependent manner. PDGF alpha-receptor mRNA was not detected in the intact aortic media, but appeared as the cells converted from a contractile to a synthetic phenotype during serum-free primary culture. PDGF beta-receptor mRNA was expressed already in vivo, and increased further as the cells were isolated and cultured in vitro. Exposure of the cells to human platelet PDGF resulted in increased PDGF alpha-receptor mRNA levels, decreased PDGF beta-receptor mRNA levels, and decreased binding of both PDGF-AA and PDGF-BB. Following removal of the exogenous mitogen, the content of PDGF alpha- and beta-receptor mRNA increased, as did the binding of PDGF-AA and PDGF-BB. Subsequently, the content of PDGF A-chain mRNA started to rise, and the cells retained a high rate of DNA synthesis in a serum-free medium. As a result of this autocrine stimulation, the PDGF receptors were down-regulated. Although smooth muscle cells in serum-free primary cultures bound the different PDGF isoforms to a varying extent (AA less than AB less than BB), the replicative response was of a similar magnitude. Subcultured cells bound the different PDGF isoforms in similar proportions as the primary cells. Contrary to the situation in primary cells, there was a direct correlation between the binding level and the DNA synthetic response. Moreover, the subcultured cells did not replicate in a serum-free medium. These observations support the idea that the phenotypic modulation of arterial smooth muscle cells in primary culture prepares the cells to activate autocrine growth mechanisms. When stimulated with an exogenous mitogen, they enter the cell cycle and are thereafter able to stimulate their own growth in an autocrine manner by production of PDGF-AA or a closely related molecule.     

Cell proliferation and apoptosis in human uterine leiomyomas and myometria

To determine the role of cell proliferation and apoptosis in uterine leiomyoma growth, we studied protein expression of two major regulatory proteins of apoptosis -- Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) -- and two endogenous markers of cell replication - proliferating cell nuclear antigen (PCNA) and Ki-67 - in tumors and matched myometrium from premenopausal women. Conventional mitotic indices also were determined, and all proliferation data were correlated to tumor size. In situ end-labeling of fragmented DNA and routine histology were used to assess apoptosis. Our results showed that the apoptosis-regulating proteins (Bcl-2 and Bax) were expressed in the cytoplasm of the leiomyoma and myometrial smooth muscle cells throughout the menstrual cycle. Bax expression differed from Bcl-2 in that it also was found in the cytoplasm of vascular smooth muscle cells of the myometria and tumors. Both tumors and myometrial samples expressed 26-kDa and 21-kDa proteins that reacted with antibodies directed towards Bcl-2 and Bax, respectively. Apoptosis was not a prominent feature of uterine leiomyomas or myometrium. PCNA- and Ki-67-labeling and mitotic counts were significantly ( P<0.05) higher in leiomyomas than in matched myometrial samples. Proliferative activity was variable for individual tumors of the same patient and independent of tumor size. Our results suggest that altered apoptosis by overexpression of Bcl-2 or by decreased expression of Bax does not appear to be a major factor in uterine leiomyoma growth. We conclude that increased cell proliferation is the most significant contributor to growth and that the proliferative state is autonomous for each tumor in a given patient and is independent of tumor size.