Enriched cultures of human oligodendrocytes were obtained from fetal brain specimens between <em>16</em> and 21 gestational weeks. Brain cells were separated over a Percoll density gradient and collected as two fractions with initial relative densities of approximately 1.035 g/ml and 1.102 g/ml, for fractions 1 and 2, respectively. After separation, 58.3 and 67.7% of the cells in fractions 1 and 2, respectively, were labeled by the antibody O4 that recognizes immature oligodendrocytes. A total of 15.5 and 29.4% of the cells in fractions 1 and 2, respectively, were positive for tubulin-beta(III), a marker for neurons but none of the freshly isolated cells were positive for glial fibrillary acidic protein (GFAP), a protein associated with astrocytes in the central nervous system. When the fractionated cells were cultured on poly-ornithine coated coverslips for 3 days and processed for immunocytochemistry, the percentage of O4+ oligodendrocytes decreased to less than 4% whereas GFAP+ cells increased to 1.8 and 12.4% for fractions 1 and 2 respectively. The percentage of tubulin-betaIII+ cells increased to 46 and 61% in cultures from the two Percoll fractions. This increase is probably due to the decrease in the number of oligodendrocytes. To avoid the loss of oligodendrocytes, cells were cultured as free-floating aggregates in the presence of 20 ng/ml of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 for 2 weeks. The resultant cultures became enriched for oligodendrocytes as demonstrated by cellular morphology and by positive O4 labeling. The method described here provides a means of obtaining enriched cultures of immature human oligodendrocytes for developmental and transplantation studies.

In the brain, 5'-deiodinase (5'-D) is responsible for the metabolic activation of thyroxine (T4) into 3,5,3'-triiodothyronine (T3) and 5-deiodinase (5-D) deiodinates T4 and T3 into inactive metabolites. This study examines the effects of <em>factors</em> known to induce astroglial 5'-D activity on the 5-D activity in cultured rat astroglial cells. The potencies of these <em>factors</em> were compared after 8 h of incubation, when stimulations by these <em>factors</em> near their maximal effects. 12-O-Tetradecanoylphorbol 13-acetate (TPA) at 10(-7) M was a potent inducer of 5-D activity, producing a 30- to 80-fold increase after 8 h. The maximal effect of TPA was observed after about 14 h. The TPA stimulation of 5-D activity was not dependent on glucocorticoids, unlike 5'-D activity. In comparison with TPA, 8-bromo-cyclic AMP (10(-3) M) was a poor inducer of 5-D activity whereas it is an excellent inducer of 5'-D activity. It produced a 2- to 20-fold increase in 5-D activity after 8 h. Natural acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (20 ng/ml) produced a degree of stimulation similar to that of TPA after 8 h. The maximal effect of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> was observed after about <em>16</em> h (until a 120-fold increase). Recombinant acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> also induced 5-D activity. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> was less potent than acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> for increasing 5-D activity (maximal increase by 40- to 50-fold after 8 h).(ABSTRACT TRUNCATED AT 250 WORDS)

It has been proposed that bile acid suppression of CYP7A1 gene expression is mediated through a gut-liver signaling pathway <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)15/19-<em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 which is initiated by activation of farnesoid X receptor in the ileum but not in the liver. This study evaluated whether FGF15/19 protein levels in the portal blood reflected changes in FGF15/19 mRNA in the ileum. Studies were conducted in Sprague Dawley rats and New Zealand white rabbits fed regular chow (controls), supplemented with cholesterol (Ch) or cholic acid (CA). After feeding CA, ileal FGF15 mRNA increased 8.5-fold in rats and FGF19 rose <em>16</em>-fold in rabbits associated with 62 and 75% reduction of CYP7A1 mRNA, respectively. Neither FGF15 nor FGF19 protein levels changed in the portal blood to correspond with the marked increase of FGF15/19 mRNA levels in the ileum or inhibited CYP7A1 expression in the liver. Further, in Ch-fed rats, CYP7A1 mRNA increased 1.9-fold (P < 0.001) although FGF15 mRNA levels in the ileum and portal blood FGF15 protein levels were not decreased. In Ch-fed rabbits, although FGF19 mRNA levels in the ileum and liver did not increase significantly, CYP7A1 mRNA declined 49% (P < 0.05). We were unable to find corresponding changes of FGF15/19 protein levels in the portal blood in rats and rabbits where the mRNA levels of FGF15/19 in the ileum and CYP7A1 in the liver change significantly.

A number of cytokines and <em>growth</em> <em>factors</em> are known to modulate proliferation and differentiation of human endometrium. In this study, the expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and VEGF receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing region (KDR), and bFGF receptor 1 (Flg) were examined in the endometrium of rhesus monkey on Day 5, 10, <em>16</em>, 20, 25 of menstrual cycle and on Day 19 of early pregnancy. Western blot analysis showed the specificity of the anti-human antibodies with the monkey tissue. The expression of mRNA and protein of VEGF was correlated with that of its receptor KDR, which was detected in epithelial, vascular, and myometrial cells. The localization of bFGF and its receptor Flg was similar to that of VEGF, except that the Flg was absent in the endothelial cells. Strong expression of VEGF and bFGF in the glandular epithelial cells was observed in the proliferative phase, declined in the secretory phase during the cycle. Stronger staining of these <em>factors</em> was also observed in the decidual cells of the pregnant uterus, as compared with the stromal cells of cycling uterus. No expression of Flt1 was detected in the tissue examined in this study. These data suggest that VEGF, bFGF, and their receptors play important roles in epithelial and stromal development, angiogenesis, and blood vessel function in the endometrium during the menstrual cycle and early pregnancy of the rhesus monkey.

<em>Fibroblast</em> <em>Growth</em> <em>Factor</em> Receptor 3 (FGFR3) related skeletal dysplasias are caused by mutations in the FGFR3 gene that result in increased activation of the receptors causing alterations in the process of endochondral ossification in all long bones, and include achondroplasia, hypochondroplasia, thanatophoric dysplasia, and SADDAN. Reports of prenatal diagnosis of FGFR3 related skeletal dysplasias are not rare; however, the correlation between 2nd trimester ultrasonographic findings and underlying molecular defect in these cases is relatively poor. There is a need for specific ultrasound (U/S) predictors than can distinguish lethal from non-lethal cases and aid an earlier prenatal diagnosis. Here we present one familial and <em>16</em> sporadic cases with FGFR3 related skeletal dysplasia, and we evaluate biometric parameters and U/S findings consistent with the diagnosis of skeletal dysplasia. U/S scan performed even at the 18th week of gestation can indicate a decreased rate of development of the femora (femur length (FL) <5th centile), while the mean gestational age at diagnosis is still around the 26th week. The utility of other biometric parameters and ratios is discussed (foot length, BPD, HC, FL/foot, and FL/AC). Prenatal cytogenetic and molecular genetic analyses were performed. A final diagnosis was reached by molecular analysis. In two cases of discontinued pregnancy, fetal autopsy led to a phenotypic diagnosis and confirmed the prenatal prediction of lethality. We conclude that the combination of U/S and molecular genetic approach is helpful for establishing an accurate diagnosis of FGFR3-related skeletal dysplasias in utero and subsequently for appropriate genetic counselling and perinatal management.

Noncovalent protein delivery into cells via peptide carriers is an emerging concept. Only a handful of such peptides are known. To address various limitations associated with protein delivery for therapeutic purposes, a greater number of different delivery peptides would be required. No general method exists for creating such peptides. By combining a sequence of <em>16</em> lysine residues (K<em>16</em>) with the signal peptide (SP) sequence of Kaposi's <em>fibroblast</em> <em>growth</em> <em>factor</em> (K-FGF), we have synthesized a peptide (K<em>16</em>SP) that efficiently and noncovalently delivers functionally intact proteins (immunoglobulin G molecules, beta-galactosidase, and green fluorescent protein) into mammalian cells. The peptides K<em>16</em> and SP each alone did not show any noncovalent protein-carrying capacity. K<em>16</em>SP appears to be nontoxic to cells and three to four times more efficient than a commercially available peptide reagent. Our approach offers proof-of-concept of a general strategy for creating a diverse array of peptide carriers for eventual therapeutic applications.

Autostimulatory <em>growth</em> <em>factors</em> may contribute to the ability of malignant cells to escape normal <em>growth</em> controls. We have previously shown that Hs0294 human malignant melanoma cells release into culture medium an acid-soluble, heat-stable, trypsin-sensitive, autostimulatory monolayer mitogen which can be purified from acetic acid extracts of conditioned medium by gel filtration, reverse-phase high-performance liquid chromatography, and preparative electrophoresis. The majority of this melanoma <em>growth</em>-stimulatory activity (MGSA) resides in a <em>16</em>-Kd moiety, though bioactivity is also associated with 24-26 and less than 14-Kd forms of MGSA (Richmond and Thomas: J Cell Physiol 129:375, 1986). In order to further characterize this <em>growth</em> <em>factor</em>, monoclonal antibodies were prepared against a partially purified preparation of the autostimulatory melanoma mitogen. Monoclonal antibody clones were selected based on supernate inhibition of 3H-thymidine incorporation in serum-free Hs0294 melanoma cultures. One of these, termed FB2AH7, slows, but does not completely block, the <em>growth</em> of Hs0294 cells in serum-free medium in a dose-dependent manner. This antibody does not slow the <em>growth</em> of normal rat kidney <em>fibroblasts</em>, which neither produce nor require this mitogen, in either serum-free medium or medium containing 0.8% calf serum. This monoclonal antibody also blocks the mitogenic effects of partially purified preparations of this melanoma <em>growth</em> stimulatory activity (MGSA) on both Hs0294 cells and normal rat kidney <em>fibroblasts</em>. The FB2AH7 antibody has been demonstrated to bind MGSA by Western blot and by immunoprecipitation procedures. Western blot analysis of reverse-phase high-performance liquid chromatography purified <em>growth</em> <em>factor</em> demonstrated that FB2AH7 antibody binds to the <em>16</em>-Kd and approximately 13-14-Kd forms of MGSA. FB2AH7 antibody can be used in immunoprecipitation experiments to bind the approximately 13-<em>16</em>-Kd forms of MGSA. The specificity of the binding of FB2AH7 antibody for MGSA but not other <em>growth</em> <em>factors</em> has been demonstrated in a modified dot blot assay. These data thus support the hypothesis that MGSA is an autostimulatory melanoma mitogen distinct from other <em>growth</em> <em>factors</em>.

OBJECTIVE

We have developed a technique for biologic coronary artery bypass grafting, which is a revival of a classic concept with modern biotechnology.

METHODS

Acute myocardial infarction was created by ligating the major branch of the circumflex artery in rabbits. Animals were divided into four groups: a nontreated group (group N), a group in which omentum was used to wrap the infarcted area (group G), a group in which a gelatin hydrogel sheet incorporating 100 microg basic fibroblast growth factor was placed over the infarcted area (group F), and a group in which the infarcted area was similarly treated with basic fibroblast growth factor followed by omental wrapping (group FG). Cardiac function was subsequently assessed by echocardiography. Postmortem angiography through the gastroepiploic artery was done in groups G and FG. Infarct size and arteriolar density were evaluated.

RESULTS

Group FG showed a better fractional area change than did the other groups (group N P <.001, group G P =.002, group F P <.001). Angiography revealed that communication from the gastroepiploic artery to the coronary artery was created through a rich bed of neovascularization in all 7 animals of group FG, whereas poor collaterals were recognized in only 2 of 7 animals in group G. Infarct size was reduced to a greater extent in group FG than in groups F, G, and N (10% +/- 3%, 16% +/- 5%, 19% +/- 7%, 23% +/- 2%, respectively, group F P =.04, groups G and N P <.01). The number of arterioles 20 to 100 microm in diameter was increased to a greater extent in group FG than in groups F, G, and N (23 +/- 5 arterioles/mm(2), 14 +/- 3 arterioles/mm(2), 10 +/- 1 arterioles/mm(2), 4 +/- 2 arterioles/mm(2), respectively), with the differences being significant.

CONCLUSIONS

These results show that bypass from the gastroepiploic artery to coronary arteries can be achieved without surgical anastomosis through slow release of basic fibroblast growth factor in this rabbit acute myocardial infarction model. This new revascularization concept, biologic coronary artery bypass grafting, could be applicable for revascularizing many tiny coronary vessels in patients who are difficult to treat with conventional surgery or catheter intervention.

BACKGROUND

Vascular growth factors are upregulated in stroke patients, but it remains unknown if they correlate with carotid atherosclerosis.

RESULTS

A case-control study was conducted to determine: (1) possible association between biomarkers of angiogenesis or inflammation and carotid stenosis; and (2) the impact of revascularization on the same biomarkers. Circulating vascular endothelial growth factor (VEGF), basic fibroblast GF (bFGF), tissue kallikrein (tK), and high-sensitivity C-reactive protein (hs-CRP) were measured in 89 patients with carotid obstruction and 45 age-matched controls. Patients were stratified as <50% carotid stenosis (CAS; n=16); 50% to 69% CAS (n=12); 70% to 99% CAS (n=43); and carotid occlusion (CAO; n=18). No association was found between VEGF, bFGF, or hs-CRP and obstruction grading. TK augmented from 360+/-30 in <50% CAS (P=NS versus controls) to 509+/-72 in moderate CAS (P<0.05), 1159+/-178 in high-grade CAS (P<0.02), and 1616+/-403 pg/mL in CAO (P<0.01). A threshold of 508 pg/mL provided the maximized predictive value of high-grade obstruction. After revascularization, tK decreased from 1410+/-352 to 782+/-86 pg/mL (P<0.01), whereas no change was detected in nonoperated cases. Hs-CRP was unaffected by revascularization.

CONCLUSIONS

Angiogenic factors are heterogeneously expressed in patients with carotid atherosclerosis. The tK measurement may be useful for the diagnosis and monitoring of atherosclerotic disease.

In NIH 3T3 <em>fibroblasts</em> insulin is a much less potent mitogen than platelet-derived <em>growth</em> <em>factor</em> or <em>fibroblast</em> <em>growth</em> <em>factor</em>. Here we demonstrate that addition of choline phosphate (1 mM) or ethanolamine (1 mM) to these <em>fibroblasts</em> for a prolonged (<em>16</em> h) period specifically and greatly enhanced, particularly when added in combination, the stimulatory effects of both insulin and insulin-like <em>growth</em> <em>factor</em> I on DNA synthesis. The results suggest that increased production of choline phosphate and ethanolamine, often observed in tumors and carcinogen-treated cells, may promote the mitogenic activity of insulin and insulin-like <em>growth</em> <em>factor</em> I.

Transforming <em>growth</em> <em>factor</em> (TGF)-beta is a family of multifunctional cytokines controlling cell <em>growth</em>, differentiation, and extracellular matrix deposition in the lung. The biological effects of TGF-beta are mediated by type I (TbetaR-I) and II (TbetaR-II) receptors. Our previous studies show that the expression of TbetaR-II is highly regulated in a spatial and temporal fashion during lung development. In the present studies, we investigated the temporal-spatial pattern and cellular expression of TbetaR-I during lung development. The expression level of TbetaR-I mRNA in rat lung at different embryonic and postnatal stages was analyzed by Northern blotting. TbetaR-I mRNA was expressed in fetal rat lungs in early development and then decreased as development proceeded. The localization of TbetaR-I in fetal and postnatal rat lung tissues was investigated by using in situ hybridization performed with an antisense RNA probe. TbetaR-I mRNA was present in the mesenchyme and epithelium of gestational day 14 rat lungs. An intense TbetaR-I signal was observed in the epithelial lining of the developing bronchi. In gestational day <em>16</em> lungs, the expression of TbetaR-I mRNA was increased in the mesenchymal tissue. The epithelium in both the distal and proximal bronchioles showed a similar level of TbetaR-I expression. In postnatal lungs, TbetaR-I mRNA was detected in parenchymal tissues and blood vessels. We further studied the expression of TbetaR-I in cultured rat lung cells. TbetaR-I was expressed by cultured rat lung <em>fibroblasts</em>, microvascular endothelial cells, and alveolar epithelial cells. These studies demonstrate a differential regulation and localization of TbetaR-I that is different from that of TbetaR-II during lung development. TbetaR-I, TbetaR-II, and TGF-beta isoforms exhibit distinct but overlapping patterns of expression during lung development. This implies a distinct role for TbetaR-I in mediating TGF-beta signal transduction during lung development.

To begin to understand the molecular mechanisms by which basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) stimulates proliferation of coronary venular endothelial cells (CVEC), we have characterized the kinetic interactions of bFGF with various binding sites on CVEC and determined the kinetics of nuclear translocation of bFGF. We report that bFGF rapidly binds to its receptor and is immediately internalized at 37 degrees C with a half-time for receptor binding of 0.9 min. After internalization bFGF is processed by two kinetically and biochemically distinguishable pathways. Up to 40-50% of total internalized bFGF is translocated to the nuclei of serum-starved, quiescent cells at early time points (0-2 h). This proportion declines to less than or equal to 20% by 24 h. Cytoplasmic accumulation continued to increase for up to 24 h. Nuclear-bound 125I-labeled bFGF consisted primarily of the intact 18-kDa species with small amounts of a <em>16</em>-kDa degradation fragment. Nuclear-bound 125I-labeled bFGF showed little evidence of degradation even after 24 h, whereas cytoplasmic 125I-labeled bFGF showed increased degradation to smaller fragments with time. Nuclear-binding of bFGF reached equilibrium by 8 h, just before initiation of DNA synthesis, which began 9-12 h after <em>growth</em> <em>factor</em> addition. These results suggest that nuclear-bound bFGF may function in triggering division (proliferation) of CVEC subsequent to binding of the <em>growth</em> <em>factor</em> to cell surface receptors.

Elastic cartilage responds mitogenically in vitro to transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (basic FGF). We studied the effects of these <em>growth</em> <em>factors</em> separately or in a combination on porcine auricular chondrocytes in vitro and on the autologous elastic cartilage produced. Cells were harvested from the elastic auricular cartilage of <em>16</em>- to 18-kg Yorkshire swine. Viability and quantification of the cells was determined. Cells were plated at equal concentration and studied in vitro in one of four identical media environments except for the <em>growth</em> <em>factors</em>: Group I contained Ham's F-12 with supplements but no <em>growth</em> <em>factors</em>, Group II also contained basic-FGF, Group III also contained TGF-beta, and Group IV also contained a combination of both <em>growth</em> <em>factors</em>. After 3 weeks in vitro, the cells were chemically dissociated with 0.25% trypsin. Cell suspensions composed of 3 x 10(7) cells/cc in 30% Pluronic F-127/Ham's F-12 were injected subcutaneously. Implants were harvested at 6, 8, 10, and 12 weeks of in vivo culture and then were examined with histologic stains. After 3 weeks of in vitro culture the total number of cells was as follows: Group I, 1.8 x 10(8); Group II, 3.5 x 10(8); Group III, 1.3 x 10(8); Group IV, 2.5 x 10(8). After 8 weeks of in vivo autologous implantation, the average weight (g) and volume (cm3) of each group was as follows: Group I, 0.7 g/0.15 cm3; Group II, 1.5 g/0.8 cm3; Group III, 0.6 g/0.1 cm3; Group IV, 1.2 g/0.3 cm3. Histologically, Groups I, II, and IV generated cartilage similar to native elastic cartilage, but Group III specimens demonstrated fibrous tissue in<em>growth</em>. Basic FGF produced the most positive enhancement on the quantity and quality of autologous tissue engineered elastic cartilage produced in this porcine model both in vitro and in vivo.

The distributions of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) and basic FGF (bFGF) in extracts of various cultured mammalian cells were determined from their elution profiles on heparin-affinity chromatography, and assay of activity as ability to stimulate DNA synthesis in BALB/c3T3 cells. Only aFGF was found in extracts of mouse melanoma B <em>16</em> cell and rat Morris hepatoma cell (MH1C1) lines. Other tumor cell lines established from solid tumors and some normal cells contained bFGF as a main component, but blood tumor cell lines contained no aFGF or bFGF. The FGFs in extracts of solid tumor tissues derived by transplantations of these cultured tumor cells and various normal tissues of mice were also examined. Tumors formed by all cell lines, regardless of whether they produced aFGF, bFGF, or neither, contained bFGF that was probably derived from host cells including capillary endothelial cells, in addition to the tumor-derived aFGF or bFGF, if produced. The content of bFGF, possibly derived from the host, in these tumor tissues was comparable to those of various mouse organs other than thymus, lung, spleen, and testis, which have higher bFGF contents. Tumor tissues derived from cultured cells producing bFGF had relatively higher bFGF contents. Like bFGF, aFGF was distributed almost ubiquitously in normal mouse tissues.

The purpose of this study was to examine the hypoxic and inflammatory conditions after immobilization in the joint capsule of rat knees. The unilateral knee joints of adult male rats were immobilized with an internal fixator (Im group) for 1 day, 3 days, and 1, 2, 4, 8, and <em>16</em> weeks. Sham-operated animals had holes drilled in the femur and tibia and screws inserted without a plate (control group). The number of cells and blood vessels in the capsule were histologically examined. The hypoxic condition in the capsule was histologically examined with a Hypoxyprobe™-1. The gene expressions related to the hypoxic (hypoxia inducible <em>factor</em>-1α, vascular endothelial <em>growth</em> <em>factor</em>, and <em>fibroblast</em> <em>growth</em> <em>factor</em> 2) and inflammatory conditions [interleukin-6 (IL-6), IL-1α, IL-1β, tumor necrosis <em>factor</em>-α, and tumor necrosis <em>factor</em>-β] were evaluated by quantitative reverse transcription polymerase chain reaction. The number of cells was unchanged at 1 day in the two groups; however, the number significantly increased at 3 days in the Im group. The number of blood vessels in the Im group gradually decreased. Strong immunostaining of Hypoxyprobe™-1 around the blood vessels was observed in the Im group. The gene expressions of hypoxia inducible <em>factor</em>-1α and <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 were significantly higher in the Im group compared with those in the control group. The gene expressions of IL-6, IL-1α, IL-1β, and tumor necrosis <em>factor</em>-β were significantly higher in the Im group compared with those in the control group. These data indicated that joint immobilization induced hypoxic and inflammatory conditions in the joint capsule, which might be an initiating <em>factor</em> for joint contracture.

In the rat, there is a marked but transient increase in hypothalamic aromatase activity during the last week of fetal life. The present study was undertaken to gain insight into the regulation of this developmental pattern. Hypothalamic fragments comprising the medial basal hypothalamus and the suprachiasmatic region (henceforth referred to as preoptic area) were explanted and cultured in serum-free medium for 2 to 5 days. Aromatase activity was measured by the formation of (3) H(2) O, utilizing either [1ß-(3) H]androstene-dione or [1ß-(3) H]testosterone as substrate. Maximal rates of activity were obtained at a saturating concentration of 0.3 μM [1ß-(3) H]testosterone. Confirmation of the identity of the [(3) H]estradiol formed was demonstrated by recrystallization of the derivatized estradiol to constant specific activity following incubation with [1,2,6,7-(3) H]testosterone. In agreement with previous reports, in vivo hypothalamic aromatase activity was negligible before gestational day (GD) <em>16</em>, increased strikingly by GD19 (>5.0 pmol/h/mg protein) and decreased, thereafter, to low levels at GD22 (∼1.0 pmol/h/mg protein). Medial basal hypothalamus-preoptic area fragments explanted before GD17 failed to develop aromatase activity in vitro. If the tissue was explanted on GD17 or 18 (i.e. when the in vivo rate of activity was increasing), the enzyme activity did not continue to increase, but it was rather maintained for 2 days before decreasing in a manner that closely mimicked the decline observed in vivo. A similar, butimmediate decline was observed when the tissue was explanted on GD19 (i.e. at the time when theactivity peaks in vivo). Exposure of explants to either <em>growth</em> <em>factors</em> (insulin-like <em>growth</em> <em>factor</em> II, epidermal <em>growth</em> <em>factor</em>, and basic or acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>), or steroids (estradiol-17ß, progesterone, testosterone, dihydrotestosterone and corticosterone) failed to either increase aromatase activity before the peak at GD19 or ameliorate its perinatal decline. Increase of Ca(2+) fluxes with the ionophore A23187 or activation of the cyclic AMP, cyclic GMP, or protein kinase C pathways were similarly ineffective, as was angiotensin II, a recently proposed stimulator of neural aromatase. In contrast, aromatase activity was suppressed 2- to 4-fold by activation of the cyclic AMP pathway (with either forskolin or 8-bromo-cyclic AMP) or by the androgens, testosterone and dihydrotestosterone. These results suggest that: 1) the appearance of aromatase activity in the rat hypothalamus before GD17 requires the unfolding of extrahypothalamic events, 2) the increase in aromatase activity that occurs before GD19 also requires extrahypothalamic inputs and does not involve any of the known intracellular signal transduction pathways, and 3) the decline in activity observed after GD19 is regulated within the hypothalamus, and appears to be determined, at least in part, by the activation of cyclic AMP formation. A potential role for androgens is discussed.

Bovine embryos at the 8- or <em>16</em>-cell stage were cultured singly, or in groups (10-12 embryos), in the presence or absence of bovine oviduct epithelial cells (BOEC) in a defined medium which was used as a basic culture medium. A higher (P < 0.05) proportion of 8-cell embryos (48.3-50.8%) cultured singly developed beyond the 8-cell stage after the addition of platelet-derived <em>growth</em> <em>factor</em> (PDGF)-AB (1 ng mL-1) only, or with PDGF-AB + basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF; 1 ng ml-1) + transforming <em>growth</em> <em>factor</em> (TGF)-beta 1 beta 2 (1 ng mL-1) than in basic medium alone (30.3%). In contrast, a significantly (P < 0.02) higher percentage (62.6-65.8%) of <em>16</em>-cell embryos developed to the morula stage after the addition of TGF-beta 1 beta 2 only, or the addition of TGF-beta 1 beta 2 + bFGF + PDGF-AB than in basic medium alone (30.2%). These proportions were not significantly (P>> 0.05) different from the proportions obtained when embryos were cultured in groups, but were significantly (P < 0.005) lower than the proportions obtained when embryos were cultured in groups on BOEC monolayers. Arachidonic acid (50 ng mL-1), beta-mercaptoethanol (10 microM) and glutathione (10-1000 microM) stimulated the development of 8-cell embryos in the presence of PDGF and TGF-beta 1 beta 2; blastocyst formation was observed for the first time in 8-cell embryos cultured singly in the presence of these embryotrophic substances (2.2-6.2%).

Platelet-derived <em>growth</em> <em>factor</em> (PDGF), epidermal <em>growth</em> <em>factor</em> (EGF), insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1), and insulin protect density-inhibited murine Balb/c-3T3 <em>fibroblasts</em> against death by distinctive mechanisms. Determination of the cell survival-enhancing activity of <em>growth</em> <em>factors</em> by cell enumeration and neutral red uptake measurement gives equivalent results. PDGF displays a steep dose-response relationship in the 1-5 ng/ml range. The other <em>factors</em> display shallow log-linear relationships in the following ranges: EGF: 0.2-5 ng/ml; IGF-1: 2-80 ng/ml; and insulin: 57-4,500 ng/ml. Agonists that lead to the activation of protein kinase A, including forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate (Br-cAMP) and N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (db-cAMP), markedly increase both short-term (5-h) and long-term (20-h) survival of cells. 2-Isobutyl-1-methylxanthine (IBMX) markedly enhances short-term survival, but its effect decays with time. The protein kinase C agonist 12-O-tetradecanoyl phorbol-13-acetate (TPA) has a moderate protective effect at concentrations of <em>16</em>-32 nM, and 64 nM TPA is highly effective. The synthetic diaclglycerols 1,2-dioctanoylglycerol (DiC8) and 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore ionomycin show low activity. Supplementation of EGF with a protein kinase A or C agonist results in a varying additive increase in short-term (5-h) cell survival and supplementation of EGF + insulin or PDGF + EGF + insulin increases further the already high level of protection given by the <em>growth</em> <em>factor</em> combinations. Combining a protein kinase A and a protein kinase C agonist in the absence of <em>growth</em> <em>factors</em> gives an approximately additive increase in cell survival. Results obtained with kinase, RNA, and protein synthesis inhibitors suggest that: 1) activated protein kinase C catalyzes one or more phosphorylation events in quiescent Balb/c-3T3 cells that lead to gene expression with the protein product(s) mediating protection of quiescent cells against death, and 2) phosphorylation events catalyzed by protein kinase A largely serve to protect cells by a mechanism not requiring de novo RNA and protein biosynthesis.

The role of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in regulating the functional state of neuropeptide Y (NPY) neurons in the brain was investigated, using aggregate cultures, derived from 17-day-old fetal rat cortex maintained for <em>16</em> days in serum-free medium, as a model. The criterion for the functional state was NPY production in response to a 24-h exposure to forskolin + phorbol 12-myristate 13-acetate (For + PMA). bFGF (0.1 nM) induced a approximately 2-fold increase in NPY production under basal conditions as well as after For + PMA (p < 0.001 vs control). To address the possibility that bFGF may interact with other <em>growth</em> <em>factors</em>, we assessed the effect of bFGF in the presence of long R3-insulin-like <em>growth</em> <em>factor</em>-I (l-IGF-I; 1 nM) and found that NPY production in response to For + PMA was even greater than with bFGF alone (2-fold; p < 0.001); even though l-IGF-I by itself was ineffective; suggesting that bFGF is the driving force of this amplification. To assess the selectivity of this process, we evaluated SRIF production in response to For + PMA and found that it was not amplified by bFGF, l-IGF-I, or bFGF + l-IGF-I. These results are consistent with bFGF selectively amplifying the functional state of the cAMP and protein kinase C (PKC) pathways leading to increased NPY-production, with cooperative interaction(s) between bFGF and IGF-I, and with a role for bFGF and IGF-I in the developmental expression/survival of the NPY neurons.

Studies were conducted to evaluate the expression and regulation of cytochrome P450 17 alpha-hydroxylase (P450 17 alpha-OH) mRNA levels and androstenedione production in 6-8-mm-diameter follicle granulosa cells from the domestic hen. Although P450 17 alpha-OH mRNA was detected within granulosa cells from follicles at all stages of development (3-12-mm and preovulatory follicles), 8-12-fold higher levels were found in small, less developed follicles (3-12 mm in diameter) compared to the three largest follicles within the preovulatory hierarchy (F3 to F1 follicles). By comparison, P450 17 alpha-OH mRNA levels were 25-89-fold higher in theca tissue compared to the granulosa layer at comparable stages of development. Despite detection of P450 17 alpha-OH mRNA in granulosa cells from 6-8-mm follicles, androstenedione production was low to nondetectable when cells were cultured in the presence of exogenous progesterone (10 ng/ml). Treatment with FSH increased levels of P450 17 alpha-OH mRNA (by 6-fold after <em>16</em> h of treatment) and induced androstenedione production in cultured granulosa cells; these actions were mimicked by the cAMP analog, 8-bromo-cAMP. By contrast, addition of the <em>growth</em> <em>factors</em> transforming <em>growth</em> <em>factor</em> alpha (TGF alpha), epidermal <em>growth</em> <em>factor</em> (EGF), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) completely suppressed the stimulatory effects of FSH on both mRNA levels and androstenedione production, while insulin-like <em>growth</em> <em>factor</em> I (IGF-I) inhibited only androstenedione production. We conclude that in the granulosa layer of developing (< or = 12 mm) hen follicles the expression of P450 17 alpha-OH mRNA does not directly reflect P450 17 alpha-OH enzyme activity. Moreover, P450 17 alpha-OH mRNA levels dramatically decrease in granulosa cells from preovulatory (compared to developing) follicles, and in the theca layer from the F1 preovulatory follicle (compared to remaining follicles). It is proposed that FSH initiates P450 17 alpha-OH enzyme activity (and perhaps to a lesser extent regulates mRNA levels) at the time a follicle is recruited into the preovulatory hierarchy, and that this action is mediated, at least in part, by the adenylyl cyclase/cAMP second messenger pathway. On the other hand, <em>growth</em> <em>factors</em> (including TGF alpha, EGF, bFGF, and possibly IGF-I) may act to prevent premature expression of P450 17 alpha-OH activity in the granulosa layer of follicles that have yet to enter the rapid <em>growth</em> phase of follicle development.

Incubation of <em>16</em>-kDa 125I-labeled heparin binding (acidic <em>fibroblast</em>) <em>growth</em> <em>factor</em> type one (HBGF-1) with human hepatoma cells and normal rat hepatocytes resulted in the appearance of a stable 125I-labeled complex with an apparent molecular mass of 40 kDa. The complex could be isolated with specific antibodies against HBGF-1. In contrast to membrane receptor-bound 125I-HBGF-1, the complex was resistant to dissociation by detergents, acid, heat, and reducing or denaturing agents. Formation of a stable complex did not require treatment with cross-linking agents. Appearance of the 40-kDa complex was dependent on time, temperature, and enriched culture medium. Conditions that enhanced or reduced display of specific HBGF-1 membrane receptor sites enhanced or reduced the appearance of the 40-kDa complex. Dansylcadaverine, chloroquine, and staurosporine blocked the appearance of the 40-kDa complex concurrent with the blockage of internalization of the receptor-bound HBGF-1. Two-dimensional gel electrophoretic analysis, metabolic labeling with L-[35S]cysteine, and recovery of <em>16</em>-kDa HBGF-1 from the 40-kDa complex after base treatment suggest involvement of a 24-kDa cellular protein in the complex formation. These results suggest a potentially novel receptor-dependent pathway for metabolism of HBGF-1.

Studies were undertaken to characterize the cytokines and cytokine-cytokine interactions that stimulate human lung <em>fibroblast</em> leukemia inhibitory <em>factor</em> (LIF) production and the mechanisms of these regulatory effects were investigated. Unstimulated <em>fibroblasts</em> did not produce significant amounts of LIF, whereas recombinant interleukin-1 alpha (rIL-1 alpha), transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), and recombinant tumor necrosis <em>factor</em> (rTNF) were dose-dependent stimulators of LIF production. TGF-beta and rIL-1 alpha also interacted in a synergistic fashion to further increase LIF elaboration. Under all conditions alterations in LIF production were associated with comparable alterations in LIF mRNA accumulation. The kinetics of mRNA induction, however, differed with rIL-1-induced LIF mRNA being readily detected after 2 h, TGF-beta 1 induction peaking after <em>16</em>-24 h, and the induction caused by rIL-1 alpha plus TGF-beta 1 being most prominent after 2-4 h and decreasing with additional incubation. Protein synthesis was not required for LIF induction. In addition, even though A23187 was an effective stimulator of LIF production, the calmodulin antagonists W-7 and trifluoperazone dichoride (TFP) did not significantly alter the LIF-stimulatory effects of IL-1 and TGF-beta. PKC did appear to play an important role in this induction, however, since LIF was induced by PMA and cytokine induction of LIF production was markedly diminished by chronic phorbol ester preincubation, staurosporine, and H-7, but not by HA1004. These studies demonstrate that 1) rIL-1, TGF-beta, TNF, agents that increase intracellular calcium and agents that activate PKC, stimulate lung <em>fibroblast</em> LIF production; 2) rIL-1 and TGF-beta interact in a synergistic fashion to further increase <em>fibroblast</em> LIF production; and 3) rIL-1 and TGF-beta stimulate lung <em>fibroblast</em> LIF production via a pretranslational activation pathway that is largely PKC-dependent and protein synthesis-, cyclic nucleotide-, and calmodulin-independent. Cytokine-stimulated LIF production may play an important role in homeostasis and repair in the human lung.

Rat embryo <em>fibroblasts</em> transformed with the HPV-<em>16</em> E7 gene and the activated c-H-ras gene fall into two distinct phenotypic classes. At high cell density, clones of one class form colonies in methylcellulose supplemented with low serum; at low cell density, these cells display responsiveness to mitogenic <em>factors</em> present in serum-free conditioned medium from rat embryo <em>fibroblasts</em>. In contrast, clones of the second class exhibit an absolute dependency on <em>growth</em> <em>factors</em> present in serum at all cell densities in the methylcellulose colony assay and fail to respond to conditioned medium. We find that the status of the endogenous p53 gene is tightly correlated with these two classes of clones. Clones of the first class contain missense mutations in the p53 gene and have lost the wild-type allele. Clones of the second class express wild-type p53 protein. The importance of mutant p53 expression in reducing the <em>growth</em> <em>factor</em> dependency of transformed clones was confirmed in a separate series of experiments in which rat embryo <em>fibroblasts</em> were transformed with three genes, E7 + ras + mutant p53. The <em>growth</em> behaviour of these triply transfected clones was similar to that of the E7 + ras clones expressing endogenous mutant p53. We demonstrate that the enhanced proliferation of E7 + ras clones expressing mutant p53 protein involves an autocrine mechanism.

OBJECTIVE

This study investigated the metabolism and excretion of dovitinib (TKI258), a tyrosine kinase inhibitor that inhibits fibroblast, vascular endothelial, and platelet-derived growth factor receptors, in patients with advanced solid tumors.

METHODS

Four patients (cohort 1) received a single 500 mg oral dose of (14)C-dovitinib, followed by the collection of blood, urine, and feces for ≤10 days. Radioactivity concentrations were measured by liquid scintillation counting and plasma concentrations of dovitinib by liquid chromatography-tandem mass spectrometry. Both techniques were applied for metabolite profiling and identification. A continuous-dosing extension phase (nonlabeled dovitinib 400 mg daily) was conducted with the 3 patients from cohort 1 and 9 additional patients from cohort 2.

RESULTS

The majority of radioactivity was recovered in feces (mean 61 %; range 52-69 %), as compared with urine (mean 16 %; range 13-21 %). Only 6-19 % of the radioactivity was recovered in feces as unchanged dovitinib, suggesting high oral absorption. (14)C-dovitinib was eliminated predominantly via oxidative metabolism, with prominent primary biotransformations including hydroxylation on the fluorobenzyl ring and N-oxidation and carbon oxidation on the methylpiperazine moiety. Dovitinib was the most prominent radioactive component in plasma. The high apparent volume of distribution (2,160 L) may indicate that dovitinib distributes extensively to tissues. Adverse events were predominantly mild to moderate, and most common events included nausea, vomiting, constipation, diarrhea, and fatigue.

CONCLUSIONS

Dovitinib was well absorbed, extensively distributed, and eliminated mainly by oxidative metabolism, followed by excretion, predominantly in feces. The adverse events were as expected for this class of drug.