Nerve <em>growth</em> <em>factor</em>-like substance(s) were identified in both conditioned media of a human prostatic tumor epithelial cell line (TSU-pr1) and a human prostatic stromal cell line (HPS) by Western blot analysis and bioassay of neurite out<em>growth</em> of PC12 cells. Nerve <em>growth</em> <em>factor</em>-beta (NGF) immunofluorescence was also localized to secretory vesicles in the cytoplasm of both the TSU-pr1 and HPS cells. Western blot of the TSU-pr1 and HPS cell-secreted protein identified an Mr 65,000 major protein which immunoreacted with murine NGF antibody. NGF Western blot of HPS cell-secreted protein also identified an Mr 42,000 minor band under reduced and nonreduced conditions and an Mr 61,000 minor band under reduced conditions. The secreted protein from the TSU-pr1 cells (50 micrograms/ml) and HPS (50 micrograms/ml), as well as murine NGF (50 ng/ml) or human recombinant NGF (50 ng/ml), stimulated neurite out<em>growth</em> from PC12 cells. This neurite out<em>growth</em> activity was partially inhibited by treatment with NGF antibody. Neither the serum containing <em>growth</em> medium nor bovine serum albumin (50 micrograms/ml) stimulated neurite out<em>growth</em>. The NGF-like secretory protein appeared to play a role in the paracrine regulation of prostatic <em>growth</em> between TSU-pr1 cells and HPS cells. The relative <em>growth</em> of TSU-pr1 cells, as indicated by [3H]thymidine incorporation, in response to HPS secretory protein was stimulated 2.8-fold in a dose-dependent manner. In the converse interaction, the relative <em>growth</em> of HPS cells in response to TSU-pr1 secretory protein was stimulated 1.8-fold in a dose-dependent manner. Immunoneutralization of TSU-pr1 and HPS secretory protein was performed with antibody against NGF, acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Removal of the NGF-like protein from the maximal stimulatory dose of TSU-pr1 secretory protein (100 micrograms/ml) with NGF antibody reduced HPS proliferation to 52% of maximal levels, and immunoneutralization of the NGF-like protein in the maximal stimulatory dose of HPS secretory protein (<em>20</em> micrograms/ml) also reduced TSU-pr1 proliferation to 16% of maximal levels. Addition of normal rabbit serum or prior immunoprecipitation of either TSU-pr1 or HPS secretory protein with antibody against acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> did not inhibit the proliferation of either cell type. These results suggest that TSU-pr1 tumor cells and HPS cells secrete NGF-like protein(s) which modulate their paracrine interactive <em>growth</em> in vitro.

Basic and acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF and aFGF, respectively) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) exert angiogenic actions and have a role in wound healing, inflammation, and tumor <em>growth</em>. Monocytes and endothelial cells are involved in these processes, but the effect of FGF and VEGF on monocyte-endothelial cell interactions has not been defined. We observed that monocyte adhesion to resting or cytokine (tumor necrosis <em>factor</em>-alpha or interleukin-1 alpha)-stimulated human umbilical vein endothelial cells (HUVECs) was markedly inhibited (40 to 65%) by culture (1 to 6 days) of HUVECs with aFGF or bFGF. Monocyte transendothelial migration induced by C5a and chemokines (MCP-1, SDF-1 alpha, RANTES, MIP-1 alpha) was also suppressed (by 50 to 75%) on bFGF-stimulated HUVECs. VEGF did not have these effects at the concentrations used (10 to <em>20</em> ng/ml), although like bFGF, it promoted HUVEC proliferation. Culture of HUVECs with bFGF and aFGF significantly down-regulated intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expression on resting or tumor necrosis <em>factor</em>-alpha-stimulated HUVECs, but had no influence on platelet endothelial cell adhesion molecule (PECAM)-1 and VE-cadherin expression. bFGF also inhibited MCP-1 production by HUVECs. The inhibitory effects of bFGF on monocyte transendothelial migration and adhesion molecule expression were reversed by SU6668, an anti-angiogenic agent and bFGF receptor tyrosine kinase inhibitor. Our results suggest that bFGF and aFGF may suppress endothelial-dependent monocyte recruitment and thus have an anti-inflammatory action during angiogenesis in chronic inflammation but inhibit the immunoinflammatory tumor defense mechanism. However, SU6668 is an effective agent to prevent this down-regulatory action of bFGF on monocyte-endothelial cell interactions.

OBJECTIVE

The role of myofibroblasts in vocal fold scarring has not been extensively studied, partly because of the lack of a robust in vitro model. The objective of this investigation was to develop and characterize a myofibroblast in vitro model that could be utilized to investigate the molecular mechanism of myofibroblast differentiation and function in injured vocal fold tissue.

METHODS

Differentiation of human primary vocal fold <em>fibroblasts</em> (hVFFs) to myo<em>fibroblasts</em> was stimulated with 5, 10, or <em>20</em> ng/mL of recombinant transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1). Cultures were analyzed by immunofluorescence and Western blotting, with an alpha-smooth muscle actin (alpha-SMA) antibody used as a myofibroblast marker. Normal rabbit vocal folds were treated with 10 ng/mL of TGF-beta1 for 7 days for in vivo corroboration. The effects of interleukin-6 (IL-6) and hepatocyte <em>growth</em> <em>factor</em> (HGF) on myofibroblast differentiation were studied with Western blots.

RESULTS

The hVFFs demonstrated positive alpha-SMA labeling in cells stimulated by 10 and <em>20</em> ng/mL TGF-beta1, indicating that hVFFs were capable of differentiation to myo<em>fibroblasts</em>. Transforming <em>growth</em> <em>factor</em>-beta1 induced the largest increase in alpha-SMA at 10 ng/mL on day 5 of treatment. Both HGF and IL-6 suppressed the expression of TGF-beta1-induced alpha-SMA.

CONCLUSIONS

Our work characterizes a useful in vitro model of TGF-beta1-mediated vocal fold fibroblast-myofibroblast differentiation. The extent of differentiation appears to be attenuated by HGF, suggesting a potential mechanism to support prior work indicating that HGF plays a protective role in reducing scar formation in vocal fold injuries. Paradoxically, IL-6, which has been shown to play a profibrotic role in dermal studies, also attenuated the TGF-beta1 response.

Advanced hypertension (HT), associated with left ventricular hypertrophy (LVH), impairs myocardial microvascular function and structure and leads to increased myocardial hypoxia and <em>growth</em> <em>factor</em> activation. However, the effect of HT on microvascular architecture and its relation to microvascular function, before the development of LVH (early HT), remains unclear. By way of method, pigs were studied after 12 wk of renovascular HT (n = 7) or control (n = 7) animals. Myocardial microvascular function (blood volume and blood flow at baseline and in response to adenosine) was assessed by using electron beam computed tomography (CT). Microvascular architecture was subsequently studied ex vivo using micro-CT, and microvessels (diameter, <500 microm) were counted in situ in three-dimensional images (40-microm on-a-side cubic voxels). Myocardial expression of vascular endothelial <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and hypoxia-inducible <em>factor</em>-1alpha were also measured. By way of results, left ventricular muscle mass was similar between the groups. The blood volume response to intravenous adenosine was attenuated in HT animals compared with normal animals (+7.4 +/- 17.0 vs. +46.2 +/- 12.3% compared with baseline, P = 0.48 and P = 0.01, respectively). Microvascular spatial density in HT animals was significantly elevated compared with normal animals (246 +/- 26 vs. 125 +/- <em>20</em> vessels/cm2, P < 0.05) and correlated inversely with the blood volume response to adenosine. <em>Growth</em> <em>factors</em> expression was increased in HT animals compared with control animals. In conclusion, early HT elicits changes in myocardial microvascular architecture, which are associated with microvascular dysfunction and precede changes in muscle mass. These observations underscore the direct and early effects of HT on the myocardial vasculature.

Myo<em>fibroblasts</em> are important but transient mediators of normal wound contraction and are characterized phenotypically by their high levels of alpha-smooth-muscle actin (SMA). During wound maturation, these cells disappear. We have examined the mechanisms that lead to myo<em>fibroblast</em> deletion in a <em>fibroblast</em> culture model. Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) was used to increase SMA content in gingival <em>fibroblasts</em> (three- to sixfold). After replating TGF-beta-induced cells at low density with serum, there was a fivefold decrease in SMA protein content, SMA protein synthesis, and SMA mRNA as cells proliferated. These reductions were due to reduced SMA mRNA stability. For TGF-beta-induced cells plated at high density without serum (ie, quiescent conditions), protein content was reduced by only <em>20</em>% over 12 days. TGF-beta protected SMA-positive cells against apoptosis in serum-free cultures. Those cells that were protected against apoptosis exhibited well-developed stress fibers enriched in SMA. We conclude that, in quiescent myo<em>fibroblasts</em>, SMA protein turnover is slow, and cells are long-lived. In proliferative conditions SMA protein and mRNA turn over quickly, and the myo<em>fibroblast</em> phenotype dissipates. The reduced apoptosis of myo<em>fibroblasts</em> in quiescent conditions is due in part to the organization of SMA into stress fibers.

NSILA-s (nonsuppressible insulin-like activity, soluble in acid ethanol) is a serum peptide that has insulin-like and <em>growth</em>-promoting activities. We have demonstrated previously that liver plasma membranes possess separate receptors for NSILA-s and insulin and have characterized the insulin receptor in detail. In the present study we have characterized the properties and specificity of the NSILA-s receptor and compared them to those of the insulin receptor in the same tissue. Both 125I-NSILA-s and 125I-insulin bind rapidly and reversibly to their receptors in liver membranes; maximal NSILA-s binding occurs at <em>20</em> degrees while maximal insulin binding is seen at 1-4 degrees. The pH optimum for NSILA-s binding is broad (6.0 to 8.0), in contrast to the very sharp pH optimum (7.5 to 8.0) for insulin binding. Both receptors exhibit a high degree of specificity. With the insulin receptor, NSILA-s and insulin analogues compete for binding in proportion to their insulin-like potency: insulin greater than proinsulin greater than NSILA-s. With the NSILA-s receptor, NSILA-s is most potent and the order is reversed: NSILA-s greater than proinsulin greater than insulin. Furthermore, six preparations of NSILA-s which varied 70-fold in biological activity competed for 125I-NSILA-s binding in order of their potencies. NSILA-s which had been inactivated biologically by reduction and aminoethylation and <em>growth</em> hormone were less than 1/100,000 as potent as the most purified NSILA-s preparation. Purified preparations of <em>fibroblast</em> <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>, nerve <em>growth</em> <em>factor</em>, and somatomedins B and C were less than 1% as effective as NSILA-s in competing for the 125I-NSILA-s suggesting that these <em>factors</em> act through other receptors. In contrast, somatomedin A was 10% as active as NSILA-s and multiplication-stimulating activity was fully as active as NSILA-s in competing for the NSILA-s receptor. Analysis of the data suggests that there are approximately 50 times more insulin receptors than NSILA-s receptors per liver cell, while the apparent affinity of NSILA-s receptors is somewhat higher than that of the insulin receptor.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family of signaling molecules has been implicated in normal developmental and physiological processes, as well as in human malignancy. Using a homology-based genomic DNA mining process, we identified a human gene encoding a novel member of the FGF family, that we designate FGF-<em>20</em>. The FGF-<em>20</em> cDNA was isolated, and its sequence confirmed the gene prediction. FGF-<em>20</em> is expressed in normal brain, particularly the cerebellum, and in some cancer cell lines. Recombinant FGF-<em>20</em> protein induces DNA synthesis in a variety of cell types and is recognized by multiple FGF receptors. Ectopic expression of FGF-<em>20</em> in NIH 3T3 cells renders the cells transformed in vitro and tumorigenic in nude mice. These results underscore the utility of mining genomic DNA databases and reveal FGF-<em>20</em> to be a novel oncogene that may play a role in human cancer.

The D-type cyclins (D1, D2, and D3) are involved in progression through the G1 phase of the cell cycle and are induced as part of the delayed early response to <em>growth</em> <em>factor</em> stimulation. To better understand the role of D-type cyclins in pituitary cell function and the regulatory role of <em>growth</em> <em>factors</em> in the cell cycle, we analyzed the expression and regulation of D-type cyclins in normal and neoplastic rat pituitary cells. Immunocytochemical and RT-PCR analyses showed expression of all three D-type cyclins in the normal pituitary, with higher percentages of positive cells by immunocytochemistry in the nuclei of normal pituitaries (D1, <em>20</em>-30%; D2, 50-60%; D3, 70-80%), compared with GH3 cells. In the normal pituitary, there were significantly higher levels of cyclins D2 and D3 in PRL, GH, LH, and TSH cells, compared with ACTH cells. Cyclin D1 protein was not detected in GH3 cells, while D2 was present in less than 1 percent and D3 in 10-15 percent of GH3 cells. There were low levels of cyclin D1 and D2 messenger RNA expression in GH3 cells, by RT-PCR. When dissociated rat pituitary cells were cultured in the presence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (5.6 nM) for 3 days, cyclin D2 was up-regulated 2-fold in normal PRL cells (control, 33 +/- 1%; treated, 68 +/- 2%). Similarly, bFGF treatment stimulated cyclin D3 expression 3-fold in GH3 cells (control, 15 +/- 1%; treated, 44 +/- 1%). Treatment of GH3 cells with 5-aza-2'-deoxycytidine, which induces gene demethylation, produced marked increases in cyclin D2 and D3 expression. Transfection of mouse cyclin D1 complementary DNA, driven by a cytomegalovirus promoter into GH3 cells, led to ectopic cyclin D1 expression; and there was a slight stimulation of cell proliferation and increased apoptosis in GH3 cells. These results indicate that there is a differential expression of various D-type cyclins in different types of normal pituitary cells and between normal pituitary and GH3 cells. <em>Growth</em> <em>factors</em>, such as bFGF and demethylation increased D-type cyclin expression, whereas ectopic overexpression of cyclin D1 stimulates cell proliferation and increases apoptosis in GH3 pituitary tumor cells.

Postnatal rat retinal ganglion cells (RGCs) were identified with specific fluorescent labels and placed in culture. Under these conditions, the out<em>growth</em> of processes by RGCs was found to be promoted to a far greater degree by acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) than by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The effect of aFGF and bFGF on process extension by solitary RGCs was quantified after 24 hr in culture, a time when neither a FGF nor bFGF enhanced RGC survival. The action of aFGF on process out<em>growth</em> was markedly potentiated by the addition of heparin (10 micrograms/ml) to the medium, but heparin alone had no effect. In the presence of heparin, half-maximal process out<em>growth</em> occurred at an aFGF concentration of less than <em>20</em> pg/ml (1 pM). Since all of the centrally projecting processes have already been formed in the living animal prior to use (at 7-12 days of age), at least a portion of the process out<em>growth</em> in culture appears to represent a regenerative phenomenon. Statistical analysis of the increase in process <em>growth</em> revealed that aFGF with heparin contributed to both neurite initiation and elongation. The mean number of glial cells, identified with polyclonal antiserum against glial fibrillary acidic protein, was slightly increased in cultures receiving aFGF plus heparin, but this effect was variable, and these glial cells were not in contact with the solitary RGCs that were scored for regeneration of processes. Thus, glial cells probably did not exert a direct physical influence on the degree of process out<em>growth</em> observed in the solitary RGCs, although a humoral effect cannot be totally excluded. These results suggest that aFGF has a potent influence on the out<em>growth</em> of processes by a neuron in the mammalian central nervous system. The potentiation of this effect by heparin leads us to speculate that the interaction of aFGF with a heparin-like molecule located in the extracellular matrix (such as heparan sulfate proteoglycan) may produce physiological effects in vivo. Furthermore, the lack of a substantial effect of bFGF in this system under these conditions shows that a specific population of mammalian central neurons may be differentially influenced by these two closely related peptide <em>growth</em> <em>factors</em>.

Strong epidemiological and pathological evidence supports a role for Chlamydia pneumoniae infection in atherosclerosis and human coronary heart disease. Animal models have shown that C. pneumoniae disseminates hematogenously in infected monocytes and macrophages, while in vitro data suggest that infected macrophages can transmit C. pneumoniae infection directly to endothelial cells. Endothelial cells may be key in vivo targets for C. pneumoniae infection; given that these cells are important in regulating the dynamics of the vessel wall, we used cDNA microarrays to study the transcriptional response of endothelial cells to infection with C. pneumoniae. cDNA arrays were used to characterize the mRNA expression profiles for 268 human genes following infection with C. pneumoniae, which were compared to mRNA profiles of uninfected cells. Selected genes of interest were further investigated by reverse transcription-PCR throughout a 24-h period of infection. C. pneumoniae infection upregulated mRNA expression for approximately <em>20</em> (8%) of the genes studied. Genes coding for cytokines (interleukin-1), chemokines (monocyte chemotactic protein 1 and interleukin-8), and cellular <em>growth</em> <em>factors</em> (heparin-binding epidermal-like <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and platelet-derived <em>growth</em> <em>factor</em> B chain) were the most prominently upregulated. In addition to these families of genes, increases in mRNA levels for intracellular kinases and cell surface receptors with signal transduction activities were observed. Time course experiments showed that mRNA levels were upregulated within 2 h following infection. These results expand our knowledge of the response of endothelial cells to C. pneumoniae by further defining the repertoire of C. pneumoniae-inducible genes and provide new insight into potential mechanisms of atherogenesis. In addition, the use of cDNA microarrays may prove useful for the study of host cell responses to C. pneumoniae infection during latent and replicative stages of infection and related pathology.

Myo<em>fibroblasts</em> and transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1) are key elements of cardiac tissue fibrosis development. The aim of this study was to determine whether the ability of TGF-beta 1 to affect the contractile activity of cardiac <em>fibroblasts</em> depends on their differentiation into myo<em>fibroblasts</em>. Cardiac <em>fibroblasts</em> (from male adult Wistar rats) from passage 2 were therefore cultured to confluency and incubated on a hydrated collagen gel, both with and without TGF-beta 1 (0, <em>20</em>, 40, 100, <em>20</em>0, 400 or 600 pmol/l), for 1, 2 and 3 days in a Dulbecco's Modified Eagle's Medium (DMEM) without fetal bovine serum (FBS). <em>Growing</em> cultures of cardiac <em>fibroblasts</em> were obtained by incubating second-passage <em>fibroblasts</em> in DMEM with 10% FBS with or without TGF-beta 1 (0 to 600 pmol/l) for 6 days. These <em>fibroblasts</em> were then further incubated on the collagen gel for 1, 2 and 3 days in DMEM without FBS. TGF-beta 1 dose-dependently increased the contraction of collagen gel mediated by cardiac <em>fibroblasts</em>, either added directly to the gel or after <em>growing</em> of the cardiac <em>fibroblasts</em> in the presence of TGF-beta 1 for 6 days, reaching a maximal effect at 100 pmol/l TGF-beta 1. In both culturing conditions, TGF-beta 1 also stimulated the [3H]-thymidine incorporation and the total protein content in the cardiac <em>fibroblasts</em> in the collagen gel lattice. TGF-beta 1 dose-dependently induced an increase in alpha-smooth muscle actin, a marker of myo<em>fibroblasts</em>, in both culturing conditions. The TGF-beta 1-induced reduction of area of the collagen gel was negatively correlated to the TGF-beta 1-evoked appearance of alpha-smooth muscle actin in the collagen gel matrix. TGF-beta 1 increased the contractile activity of adult rat cardiac <em>fibroblasts</em> and their ability to differentiate into myo<em>fibroblasts</em>. Because contractile activity was correlated with differentiation, the influence of TGF-beta 1 on cardiac <em>fibroblast</em>-induced collagen gel contraction may depend on the promotion of myo<em>fibroblast</em> differentiation.

Approximately 60% of all breast tumors are estrogen-responsive and chemicals that show estrogenic or anti-estrogenic properties are able to interact with breast tumor <em>growth</em>. In a breast tumor, adipose stromal cells (<em>fibroblasts</em>) surrounding the epithelial tumor contain the aromatase enzyme, which converts androgens into estrogens. Exposure to aromatase inducers can therefore lead to increased estrogen levels and possibly to accelerated breast tumor <em>growth</em>. Subsequently, breast tumor cells synthesize and secrete elevated levels of <em>factors</em> such as prostaglandin E2 (PGE2), interleukin-6 (IL-6), and IL-6 soluble receptor (IL-6sR), which in turn have the ability to stimulate aromatase gene transcription in <em>fibroblasts</em>, establishing a positive feedback loop. In this study, a technique that allows for culturing MCF-7 epithelial breast tumor cells and healthy primary human mammary <em>fibroblasts</em> together in one compartment was developed. To establish the positive feedback loop, the co-culture was exposed to estrogenic compounds. RNA was isolated and reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on the aromatase and pS2 genes. Exposure of the co-culture to estradiol (E2), diethylstilbestrol (DES), and bisphenol-A (BPA), resulted in a three- to seven-fold increase of pS2 transcription levels. Furthermore, pS2 transcription levels increased even more when the aromatase substrate testosterone (<em>20</em> nM) was present in the co-culture medium. Exposure of the co-culture to the aromatase inducer dexamethasone (DEX) resulted in increased pS2 transcription levels, as well as increased aromatase transcription levels. Simultaneous exposure to DEX and the synthetic anti-estrogen ICI 182,780 almost completely blocked the pS2 response. The aromatase induction response was not altered by ICI 182,780 treatment. Simultaneous exposure to DEX and the non-steroidal aromatase inhibitor fadrozole, abolished the effect of the presence of testosterone in the co-culture medium, but did not result in pS2 gene transcription levels as low as seen after exposure to ICI 182,780. These observations indicate the presence of a positive feedback loop in our co-culture system. This co-culture provides a more sophisticated and sensitive system to detect direct and indirect estrogenic effects of compounds and their possible effects on breast tumor promotion.

Vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and transforming <em>growth</em> <em>factor</em>-beta(1) (TGF-beta(1)) mRNA increase in rat skeletal muscle in response to a single acute exercise bout. Nitric oxide (NO) is released locally by muscle vascular endothelium and muscle fibers during exercise, contributes to the blood flow response to exercise, and regulates mitochondrial respiration. We hypothesized that a reduction in NO production, via NO synthase inhibition, would demonstrate a link between NO and the VEGF, bFGF, and TGF-beta(1) gene responses to exercise. To investigate this hypothesis, 9-wk-old female Wistar rats were divided into eight treatment groups (n = 6 each): 1) saline + rest, 2) saline + exercise, 3) 30 mg/kg N(omega)-nitro-L-arginine methyl ester (L-NAME, a known NOS inhibitor) + rest, 4) 30 mg/kg L-NAME + exercise, 5) 300 mg/kg L-NAME + rest, 6) 300 mg/kg L-NAME + exercise, 7) 300 mg/kg N(omega)-nitro-D-arginine methyl ester (D-NAME, inactive enantiomer of L-NAME) + rest, and 8) 300 mg/kg D-NAME + exercise. Exercise consisted of 1 h of running at <em>20</em> m/min on a 10 degrees incline. VEGF, TGF-beta(1), and bFGF mRNA from left gastrocnemius were analyzed by quantitative Northern blot. Submaximal exercise for 1 h increased VEGF mRNA 4.2-fold and TGF-beta(1) mRNA 1.5-fold in untreated rats but did not increase bFGF mRNA. The exercise-induced increase in VEGF mRNA was attenuated approximately 50% by 30 and 300 mg/kg L-NAME; the TGF-beta(1) mRNA increase was unaffected by 300 mg/kg L-NAME. In addition, 300 mg/kg D-NAME had no effect on the exercise-induced increase in VEGF mRNA. Administration of 300 mg/kg L-NAME had no effect on bFGF mRNA. These findings suggest that NO is important in the regulation of the VEGF gene response to exercise through increases in VEGF transcription or by increases in the VEGF mRNA half-life.

Serotonin [5-hydroxytryptamine (5-HT)] has a dual effect on bovine pulmonary artery smooth muscle cells (SMC) in culture (S.-L. Lee, W. W. Wang, B. J. Moore, and B. L. Fanburg. Circ. Res. 68: 1362-1368, 1991.). Cellular internalization of 5-HT stimulates DNA synthesis and cellular proliferation, whereas the action of 5-HT on a cell surface receptor elevates adenosine 3',5'-cyclic monophosphate and inhibits proliferation. The present study shows that 5-HT causes proliferation of both pulmonary artery and aortic SMC but not of endothelial cells or <em>fibroblasts</em>. Furthermore, c-myc and alpha- and beta-actin gene expressions of pulmonary artery SMC were elevated after 2-h incubation with 5-HT, before stimulation of [3H]thymidine incorporation. Actinomycin D (0.05 micrograms/ml) but not cycloheximide (1 microgram/ml) inhibited the gene expression produced by 5-HT. <em>Growth</em>-arrested SMC progressed from a G0 quiescent state through a normal cell cycle when subjected to 5-HT, 5-HT plus 25 ng/ml insulin-like <em>growth</em> <em>factor</em>, or 5-HT plus 0.5 ng/ml <em>fibroblast</em> <em>growth</em> <em>factor</em>. Cell number increased by <em>20</em>-40% at 40 h and 50-140% at 7 days. Protein content of cells treated with 5-HT was elevated by <em>20</em>-40% at 7 days, whereas the rate of protein synthesis, measured by [35S]methionine incorporation, increased by 50-70% at 24 h. In the presence of 1 microM 5-HT, cells enlarged by 70 and 100-<em>20</em>0% at 40 h and 7 days, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

To characterize mitogens that might contribute to Schwann cell proliferation during development or in tumors, we tested the ability of hepatocyte <em>growth</em> <em>factor</em> (HGF) to stimulate Schwann cell division in vitro. HGF is a potent mitogen for purified rat Schwann cells; DNA synthesis in rat Schwann cells was stimulated <em>20</em>-40-fold by 3-10 ng/ml HGF. Rat Schwann cells express c-met mRNA, encoding the HGF receptor, but not HGF mRNA, implying that HGF might act as a paracrine Schwann cell <em>growth</em> <em>factor</em>. HGF-stimulated Schwann cell proliferation differs from that of previously described Schwann cell mitogens in that its activity is abolished by forskolin and is not inhibited or potentiated by addition of transforming <em>growth</em> <em>factor</em> beta (TGF beta) or <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). HGF is probably not a component of the axonal signal thought to cause Schwann cell division during development, as anti-HGF neutralizing antibodies failed to block neuron-stimulated Schwann cell proliferation. In contrast, mitogenic activity present in normal human adult nerves and in neurofibromas from patients with type 1 neurofibromatosis analyzed in the absence of forskolin is largely inhibitable by anti-HGF. Thus, HGF is a novel mitogen for Schwann cells in vitro and it is present in Schwann cell tumors, suggesting a potential role for HGF after wounding of peripheral nerves or in tumor <em>growth</em>.

Overexpression of the cyclin D1/PRAD1 oncogene has been observed in a number of tumorigenic cell lines, suggesting that regulation of D1 expression may represent an important step in the control of cellular proliferation. We have examined the mRNA expression of cyclin D1, as well as two related D-type cyclins, D2 and D3, in response to defined <em>growth</em> <em>factors</em> that control the <em>growth</em> of Balb/c-3T3 <em>fibroblasts</em>. Transcripts for all three D-type cyclins were expressed during the G1 phase of the Balb cell cycle, however only D1 and D3 exhibited periodic induction. Although redundantly expressed, message levels of cyclin D1 and D3 were differentially regulated in regard to kinetics of induction; a modest increase in D3 mRNA was detected near the G1/S boundary, 12 h after serum stimulation of quiescent cells, while abundance of D1 transcript increased <em>20</em> to 30-fold, peaking 6 h after addition of serum. <em>Factors</em> such as platelet-derived <em>growth</em> <em>factor</em> (PDGF) that induce competence formation in Balb cells, increased D1 message and protein levels to the same extent as serum but did not affect expression of cyclin D3 and did not stimulate entry into S phase. Progression <em>factors</em> contained within platelet-poor plasma stimulated D1 expression only weakly but acted synergistically with low concentrations of PDGF to increase D1 mRNA to maximum levels. Depletion of protein kinase C severely reduced the ability of PDGF and serum to induce D1 mRNA. PDGF- and serum-mediated elevation of steady-state D1 message levels was in part because of a transcriptional activation of the D1 gene that was independent of protein synthesis. However, protein synthesis was required 3-4 h after serum stimulation for the shut down of D1 transcription leading to the normal decline in message levels after peak induction. Our results indicate that overexpression of cyclin D1 message may result from a disruption of negative regulatory events that repress D1 transcription.

Diploid cells derived from the portio surface of the cervix uteri of adult women can be grown in culture, from single cells. Ultrastructurally the colonies of <em>growing</em> cells show the features of stratified squamous epithelium and exhibit some of the differentiated characteristics of this epithelium in vivo. The successful serial cultivation of cervical epithelial cells, HCE, depends upon the presence of a non-dividing <em>fibroblast</em> feeder layer which provides both attachment and <em>growth</em> <em>factors</em> for epithelial cells. Lethally irradiated Wi-38, cervical <em>fibroblasts</em> or mouse <em>fibroblast</em> lines are all effective in promoting HCE colony formation and <em>growth</em>. Hydrocortisone and epidermal <em>growth</em> <em>factor</em> increase HCE colony size in the presence of a feeder cell layer. Culture lifetime was studied in five strains and ranged from <em>20</em>-38 population doublings, with cells from older patients exhibiting a shorter culture life-time.

It was recently reported that transplantation of clonally derived murine neurosphere cells into sublethally irradiated allogeneic hosts leads to a donor-derived hematopoietic reconstitution. The confirmation of the existence of a common neurohematopoietic stem cell in the human brain will have a significant effect on stem cell research and on clinical transplantation. Here, it is demonstrated that the human fetal brain contains separate but overlapping epidermal <em>growth</em> <em>factor</em> (EGF)-responsive and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2)-responsive neural stem cells. The majority >> 85%) of cells within these EGF- and/or FGF-2-generated neurospheres express characteristic neural stem/progenitor cell markers including nestin, EGF receptor, and FGF-2 receptor. These neural stem cells can be continuously passaged in vitro, and demonstrate a constant <em>20</em>-fold expansion in every passage for up to the fifth passage (the longest period that has been carried out in the authors' laboratory). These neural stem cells are multipotential for neurons, astrocytes, and oligodendrocytes. After transplantation into SCID-hu mice, all neural stem cells, regardless of passages, culture conditions, and donors, are able to establish long-term hematopoietic reconstitution in the presence of an intact human bone marrow microenvironment.

OBJECTIVE

Fibroblast growth factors (FGFs) and their receptors (FGFRs) have been identified in a variety of carcinomas, but there are few studies concerning their presence in oral cancers. The objective of this study was to determine whether FGF-1, FGF-2, and high affinity receptors FGFR2 and FGFR3 are present in the pathogenesis of oral epithelial dysplasias and oral squamous cell carcinoma.

METHODS

Sections from formalin-fixed, paraffin-embedded samples of oral normal mucosa (n = 14), epithelial dysplasia (n = 20), carcinoma in situ (n = 10), and squamous cell carcinoma (n = 12) were tested for cytoplasmic staining by standard in situ immunohistochemistry with antibodies for FGF-1, FGF-2, FGFR2, and FGFR3.

RESULTS

Staining for FGF-1 is decreased or lost in the development of epithelial dysplasia and carcinoma. Staining for FGF-2 showed increased intensity (although not statistically significant) in oral epithelial dysplasias and squamous cell carcinomas and showed a significant increased expression in the upper layers of dysplasias and stratum spinosum-like cells in squamous cell carcinomas. Staining for FGFR2 showed a statistically significant increase in intensity in all layers of epithelial dysplasias and squamous cell carcinomas. Staining for FGFR3 was found in the upper stratum spinosum cells of normal and dysplastic epithelium and well-differentiated squamous cells in squamous cell carcinomas, with a statistically significant increase in staining intensity in dysplastic and carcinomatous tissues.

CONCLUSIONS

The loss of FGF-1 is consistent with loss of differentiation in dysplasias and some squamous cell carcinomas. Changes in the localization of FGF-2 and FGFR2 into upper epithelial layers with increasing dysplasia suggest increased mitotic potential of high level cells. The co-localization of FGF-2 and its high affinity receptors in neoplastic tissues suggests an autocrine mechanism of influence on carcinogenesis.

We have analysed DNA from 183 primary breast cancers for amplification or rearrangement of a number of cellular proto-oncogenes, focusing primarily on a cluster of markers on the long arm of chromosome 11. Two of these oncogenes, INT2 and HST1, both of which encode members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family, are implicated in the generation of virally induced mammary tumours in mice. Here we confirm earlier reports that the q13 region of chromosome 11, in which these genes are tandemly linked, is modestly amplified in approximately 15% of primary human breast cancers. This amplification is confined, with one exception, to cases in which the oestrogen receptor (ER) levels are in excess of <em>20</em> fmol/mg protein (P = 0.001). However, DNA amplification does not usually result in detectable expression of either the INT2 or HST1 gene. The data imply that some other gene in the vicinity must contribute to the development of a subset of ER-positive tumours and that assessing the amplification of this region of DNA may be of value in defining a separate category of ER-positive tumour.

The implication of protein phosphorylation in the mitogenic action of high density lipoproteins (HDL) on bovine vascular endothelial cells was investigated by incubating endothelial cell cultures in the presence of 32P-labeled phosphoric acid. The incorporation of 32P into proteins was measured after fractionation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and autoradiography of the gel. In endothelial cells seeded at low density and made quiescent by serum starvation, HDL markedly and consistently enhanced the degree of phosphorylation of a Mr 27,000 protein in a time- and dose-dependent manner. Using 500 micrograms/ml HDL, 32P labeling of the 27-kDa protein was already measurable after 10 min of incubation and reached a maximum at <em>20</em>-30 min. Minimal effective dose of HDL during a 30-min incubation period was in the range of 5-10 micrograms/ml. While the apolipoprotein moiety of HDL was able to mimic the effect of total HDL, the lipid part of HDL was not. Furthermore, <em>fibroblast</em> <em>growth</em> <em>factor</em> appeared to potentiate the effect of HDL on 27-kDa protein phosphorylation, in agreement with the synergism observed between <em>fibroblast</em> <em>growth</em> <em>factor</em> and HDL on endothelial cell proliferation. Two activators of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetylglycerol also induced the phosphorylation of the 27-kDa protein. These results suggest that the 27-kDa protein may be a physiological substrate for protein kinase C and that HDL could exert their mitogenic effect on endothelial cells through activation of protein kinase C and subsequent protein phosphorylation.

We prepared a 6-O-desulfated (DS-) heparin (Hep) hydrogel as an excellent carrier for the controlled release of Hep-binding <em>growth</em> <em>factors</em>, such as <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2. This material, which is partially derived from photoreactive groups, such as cinnamate, is easily crosslinked upon ultraviolet light (UV)-irradiation, resulting in a water-insoluble, viscous, and injectable hydrogel. In the present study, we examined the capacity of 6-O-DS-Hep hydrogel to immobilize FGF-2, as well as the controlled release of FGF-2 molecules from this hydrogel in vitro and in vivo. Only 10% of FGF-2 was gradually released from the FGF-2-containing 6-O-DS-Hep hydrogel (photocrosslinked 6-O-DS-Hep (4%; w/w) hydrogel containing 50 microg/mL FGF-2) into PBS (phosphate-buffered saline) within first 7 days. The 6-O-DS-Hep hydrogel in vitro maintained the original form through 1 weeks incubation in PBS, but it was gradually fragmented and could not maintain the original form by 2-3 week-washing. When the FGF-2-containing 6-O-DS-Hep hydrogel was subcutaneously injected into the back of rats, significant neovascularization and fibrous tissue formation were induced near the injected site from day 3 after the injection. And, the hydrogel had been biodegraded and completely disappeared from the injected sites in vivo within about 15-<em>20</em> days after the injection. These findings indicate a controlled release of biologically active FGF-2 molecules together with fragmentation and biodegradation of 6-O-DS-Hep hydrogel and the subsequent induction of neovascularization in vivo.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) has a complex spatial and temporal pattern of expression and is essential for the normal development of a diverse set of tissues. Recently, mutations have been identified in FGFR3 that result in constitutive tyrosine kinase activity and cause a number of different human skeletal disorders. To examine the regulatory mechanisms governing FGFR3 expression, the promoter for the FGFR3 gene was identified and characterized. It resides in a CpG island, which encompasses the 5' end of the FGFR3 gene and lacks classical cis-regulatory motifs. As little as 100 base pairs of sequence 5' to the initiation site can confer a <em>20</em>-40-fold increase in transcriptional activity upon a promoter-less vector. The transcriptional activity of these cis-regulatory sequences is further stimulated by elements found within the first intron. Mapping of the enhancer activity found within intron I identified two purine-rich sequence motifs between +340 and +395. Electrophoretic mobility shift assays demonstrated that sequences within this region bind members of the Sp1 family of transcription <em>factors</em>. In a background lacking Sp1-like activity, we demonstrate that Sp1 can enhance transcription of the minimal promoter (which contains five classical Sp1 sites), whereas both Sp1 and Sp3 can enhance transcription through the elements found in intron I. Although these transcription <em>factors</em> are ubiquitously expressed, we demonstrate that the sequences between -2<em>20</em> and +609 of the FGFR3 gene are sufficient to promote the tissue-specific expression of a reporter gene in transgenic mice.

The tyrosine kinase Src has been implicated in the process of osteoclast-mediated bone resorption. Here, we describe a novel class of Src inhibitors, substituted 5,7-diphenyl-pyrrolo[2,3-d]pyrimidines, and characterize one of them, CGP77675, in vitro and in models of bone resorption in vivo. In vitro, CGP77675 inhibited phosphorylation of peptide substrates and autophosphorylation of purified Src (concentration producing half-maximal inhibition [IC50] values 5-<em>20</em> and 40 nmol/L, respectively). The compound was selective toward other protein kinases: the Src IC50 value was lower than those for Cdc2 (>500-fold), epidermal <em>growth</em> <em>factor</em> (EGF) receptor (7.5-fold), and vascular endothelial <em>growth</em> <em>factor</em> receptor (>50-fold), and for v-Abl (15-fold) and focal adhesion kinase (Fak) (>25-fold). The Src kinase family members Lck and Yes were inhibited with IC50 values <em>20</em>-fold higher than or equal to Src. To measure the inhibition of cellular Src activity, we identified the major tyrosine-phosphorylated proteins in an Src-overexpressing cell line IC8.1 as Src, Fak, and paxillin. CGP77675 potently inhibited tyrosine phosphorylation of the Src substrates Fak and paxillin, but had much less effect on Src (IC50 values 0.3, 0.5, and 5.7 micromol/L). The phosphorylation of Src in IC8.1 cells reflected phosphorylation of the negative regulatory tyrosine 527 (Y527); thus, the inhibitor was selective against the Y527 C-terminal Src kinase Csk. In osteoblastic MC3T3-E1 cells, CGP77675 inhibited signaling induced by PDGF at the receptor level, but not signaling by EGF, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>-1, and phorbol 12-myristate 13-acetate. The effect of CGP77675 on bone resorption was evaluated in vitro and in vivo. The parathyroid hormone-induced bone resorption in rat fetal long bone cultures was inhibited with an IC50 of 0.8 micromol/L. CGP77675 dose-dependently reduced the hypercalcemia induced in mice by interleukin-1beta and partly prevented bone loss and microarchitectural changes in young ovariectomized rats, showing that the protective effect on bone was exerted via the inhibition of bone resorption. Thus, specific Src family kinase inhibitors may be useful for the treatment of diseases associated with elevated bone loss.