Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) are both recognized as stimulators of migration and angiogenesis during the progression of melanoma. However, the timepoints during tumour progression at which the expression of these angiogenic <em>factors</em> is most essential is still controversial. Using immunohistochemical analyses, melanoma cells were found to express bFGF in 18 out of 19 primary tumours and in 13 out of 20 metastases. Eleven of the 19 primary tumours and <em>15</em> of the 20 metastases were found to contain VEGF-positive melanoma cells; five of the 19 patients showed no VEGF-expressing melanoma cells at all. This indicates that VEGF expression may be a later event in the progression of melanoma than bFGF expression. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of the melanoma cell lines showed that all cell lines were positive for both bFGF and VEGF mRNA. CD31-positive endothelial cells were primarily seen in the metastases (17 out of 20). Only four of the primary tumours contained CD31-positive cells, but these tumours expressed bFGF as well as VEGF, indicating that both angiogenic <em>factors</em> may be important for the formation of vessels in tumours.

Quiescent 3T3 <em>fibroblasts</em> grown on microcarrier beads and loaded with the [Ca2+] indicator quin2 had a cytosolic free Ca2+ concentration ( [Ca2+]i) of <em>15</em>4 +/- 11 nM (SE; n = 32). Stimulation with the mitogens vasopressin, epidermal <em>growth</em> <em>factor</em> (EGF) or prostaglandin F2 alpha (PGF2 alpha) caused a very rapid increase in [Ca2+]i to a maximum of 200-500 nM after 60-90 s. [Ca2+]i declined thereafter to a level above that in quiescent cells which was maintained for at least <em>15</em> min. In contrast no immediate effects on [Ca2+]i were detected after the addition of the mitogens insulin or 12-O-tetradecanoylphorbol 13-acetate (TPA). These studies indicate that early changes in [Ca2+]i may be involved in the action on <em>fibroblasts</em> of some, but not all, mitogens.

The expression of 1B6, a <em>growth</em>-regulated sequence isolated from a Syrian hamster <em>fibroblast</em> cDNA library, was studied in BALB/c 3T3 cells. The level of cytoplasmic 1B6 mRNA (1600 bases) was low in quiescent cells and plateaued in mid/late G1 after the cells were stimulated with <em>15</em>% fetal calf serum (FCS). Protein synthesis was not required for the induction of 1B6 mRNA; therefore, the expression of 1B6 is a primary response to serum stimulation. The induction of 1B6 mRNA was also observed after stimulation with insulin, epidermal <em>growth</em> <em>factor</em>, and <em>fibroblast</em> <em>growth</em> <em>factor</em> but not with platelet-derived <em>growth</em> <em>factor</em>. When quiescent cells were serum-stimulated, the percentage of cells that became committed to enter DNA synthesis was proportional to the length of their incubation with serum. To determine if 1B6 expression was also correlated with the time of exposure to serum, quiescent cells were stimulated with a pulse of <em>15</em>% FCS and the abundance level of 1B6 induced by that pulse was determined. The amount of 1B6 mRNA increased with increasing time of exposure to serum and paralleled the increase in the percentage of nuclei that were induced into DNA synthesis by the serum pulse. Comparison of the nucleotide sequence of the p1B6 cDNA to the GenBank database revealed a striking identity of 1B6 to the 3' end of p36, the heavy chain of calpactin I. The previous characterization of p36 as a substrate for tyrosine kinases suggests a possible role for 1B6/p36 in cell proliferation.

We recently proposed a hypothesis for the molecular mechanism of the osteogenic action of fluoride in which it stimulates osteoblast proliferation via the inhibition of an osteoblastic acid phosphatase-like phosphotyrosyl protein phosphatase activity. To test this hypothesis, we investigated whether orthovanadate, a known phosphotyrosyl protein phosphatase inhibitor, would mimic fluoride in the stimulation of bone cell proliferation and bone collagen synthesis in vitro. Orthovanadate inhibited the osteoblastic acid phosphatase activity and stimulated bone cell proliferation at the same low concentrations (i.e. 5-<em>15</em> microM). At the mitogenic doses, orthovanadate also showed a dose-dependent increase in alkaline phosphatase (a marker of mature osteoblasts) in cultured calvarial cells and stimulated bone collagen synthesis, as measured by the incorporation of [3H]proline and the conversion into [3H] hydroxyproline in organ calvaria cultures. Therefore, orthovanadate stimulated bone formation by increasing the number of mature osteoblasts mediated via stimulation of cell proliferation and differentiation. Orthovanadate was dependent on the presence of a mitogen in cell medium for its mitogenic action in vitro and synergistically potentiated the mitogenic actions on osteoblasts of those <em>growth</em> <em>factors</em>, i.e. insulin, epidermal <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em> I, and skeletal <em>growth</em> <em>factor</em>, whose mitogenic action involved tyrosyl protein phosphorylation. However, the interaction between orthovanadate and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, a <em>growth</em> <em>factor</em> that does not appear to involve tyrosyl protein phosphorylation, on bone cell proliferation was additive. In summary, these data are consistent with the hypothesis that inhibition of the osteoblastic phosphotyrosyl protein phosphatases can prolong and/or potentiate the mitogenic actions of <em>growth</em> <em>factors</em>, and thereby stimulates cell proliferation.

BACKGROUND

P450 oxidoreductase (POR) deficiency is a disorder of steroidogenesis affecting the microsomal P450 enzymes that use POR as an electron donor. The clinical presentation is variable; patients can be asymptomatic or can present with genital anomalies and the Antley-Bixler syndrome, characterized by craniosynostosis and other bony anomalies. Obligately heterozygous parents are normal. Combined POR and 21-hydroxylase deficiencies have not been reported.

OBJECTIVE

The aim was to explore the manifestations of combined deficiencies of 21-hydroxylase and POR and to search for lesions in apparent manifesting POR heterozygotes.

METHODS

A newborn female had craniosynostosis, severe salt wasting, minimal virilization, grossly elevated 17OH-progesterone, and minimally elevated androgens. DNA encoding 21-hydroxylase, POR, and fibroblast growth factor receptor 2 was sequenced. For POR, the first untranslated exon (exon 1U), 5' flanking DNA, and most introns were sequenced in five apparent manifesting POR heterozygotes.

RESULTS

CYP21B mutations were found on both alleles, proving classical 21-hydroxylase deficiency. Fibroblast growth factor receptor 2 exons 8 and 10 were normal. A POR mutation, A287P, was found only on the maternal allele. Five previously reported patients had POR mutations found on only one allele, but their clinical characteristics were indistinguishable from patients with mutations on both alleles. Sequencing of exon 1U, 274 bp of POR 5' flanking DNA, and 12 of the 15 POR introns did not identify additional mutations affecting gene expression or splicing.

CONCLUSIONS

Manifesting heterozygosity is a possible feature of POR deficiency and may ameliorate the findings in coexisting 21-hydroxylase deficiency.

Rheumatoid arthritis is characterized by chronic inflammation and proliferation of a number of important elements within the joint including the synovial <em>fibroblasts</em>. Elevated levels of a number of cytokines such as Il-1, IL-2, IL-6, interferon-gamma (IFN-gamma), transforming <em>growth</em> <em>factor</em>-beta and tumour necrosis <em>factor</em>-alpha (TNF-alpha) have been detected in the synovial fluid of patients with rheumatoid arthritis and other inflammatory arthritides. It seems likely that local release of such mediators may be responsible for the proliferation and over<em>growth</em> of connective tissue elements in these disorders. In order to ascertain whether there was evidence to suggest local production or release of <em>fibroblast</em> <em>growth</em> <em>factors</em> in the joint in inflammatory arthritis, and to determine their identity, cells were obtained from the synovial fluid of <em>15</em> patients with chronic inflammatory arthritides. All subjects' synovial fluid cells spontaneously released <em>growth</em> <em>factor</em> activity for <em>fibroblasts</em>. This was present in large amounts, being detectable in culture supernatants diluted to a titre of at least 1/625. By a series of depletion experiments using solid-phase bound antibodies to cytokines, it was possible to demonstrate that this activity was due to TNF-alpha and platelet-derived <em>growth</em> <em>factor</em> (PDGF). Thus, this study showed for the first time that functionally active PDGF was released from synovial fluid cells. Both PDGF and TNF-alpha appeared to contribute in approximately equal amounts to this <em>fibroblast</em> <em>growth</em> <em>factor</em> activity, and were synergistic in effect. Thus this study provides evidence for the local production and release of these two cytokines and suggests that together they are the dominant <em>factors</em> in <em>fibroblast</em> proliferation within the synovial cavity.

We have shown previously that thromboxane A2 stimulates hypertrophy of cultured rat aortic smooth muscle cells defined as protooncogene expression and protein synthesis without DNA synthesis or cellular proliferation (Dorn, G.W., II, Becker, M.W., Davis, M.G. (1992) J. Biol. Chem. 267, 24897-24905). Since endogenous <em>growth</em> modulators could possibly regulate vascular smooth muscle <em>growth</em> to this vasoconstrictor, we tested the hypothesis that thromboxane-stimulated vascular smooth muscle hypertrophy was due to increased expression of endogenously produced basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The thromboxane mimetic (<em>15S</em>)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) (1 microM) increased cultured rat aorta derived smooth muscle cell immunoreactive bFGF content by 331 +/- 40% over untreated controls after 24 h. Co-incubation of vascular smooth muscle cells with a specific antisense oligodeoxynucleotide (AS) against codon 60 of bFGF coding sequence reduced thromboxane-stimulated bFGF expression by 72 +/- 5% and prevented thromboxane-stimulated hypertrophy (nonsense oligonucleotide had no effects). Addition of exogenous bFGF (20 ng/ml) restored <em>growth</em> to AS-treated/thromboxane-stimulated vascular smooth muscle cells. Furthermore, addition to the culture medium of neutralizing antibody against bFGF inhibited U46619-stimulated vascular smooth muscle hypertrophy by 69 +/- 17%, whereas nonimmune IgG had no effect. Since protein tyrosine phosphorylation is a cell signal associated with <em>growth</em>, thromboxane-stimulated tyrosine phosphorylation was also examined. Exposure to 1 microM U46619 for 10 min increased vascular smooth muscle immunoreactive phosphotyrosine content of 130-144-, 86-, 80-, 75-, and 58-kDa proteins. The tyrosine kinase inhibitor herbimycin A (5 microM) prevented thromboxane-stimulated tyrosine phosphorylation, but not thromboxane-stimulated hypertrophy, suggesting that tyrosine phosphorylation was not required for thromboxane-stimulated vascular smooth muscle <em>growth</em>. These results indicate that increased expression and release of endogenous bFGF, but not direct tyrosine phosphorylation, mediates the hypertrophic vascular smooth muscle response to thromboxane.

BACKGROUND

Microvascular proliferation, a prominent feature of tumors of the central nervous system, is a prime target for anti-cancer therapy.

METHODS

Because basic fibroblast growth factor (bFGF) plays a key role in the regulation of angiogenesis, surgical specimens from 52 human brain tumors were examined by immunocytochemical studies with a murine monoclonal antibody to bFGF. Sections from these tumors also were incubated with Ki-67 monoclonal antibody to measure the growth fraction.

RESULTS

Immunostaining for bFGF was observed in 45 of 52 (87%) neoplasms, reacting with 97% of the malignant brain tumors and 67% of benign tumors (P < 0.01). The nonreactive tumors were a medulloblastoma and 7 of 21 (33%) benign, noninvasive, slow-growing neoplasms (1 acoustic schwannoma, 3 meningiomas, 2 pituitary adenomas, and 1 cholesteatoma). The indices of proliferation (Ki-67 labeling) were lower for the 21 benign tumors (1.2 +/- 1.1%) than the 31 malignant tumors (10.3 +/- 10.5%; P < 0.001). The bFGF was immunolocalized in the tumor cell nuclei in 23 of 52 tumors (44%) and in the cytoplasm of 8 of 52 (15%) tumors. Immunostaining to bFGF was prominent in the microvascular endothelial compartment in 84% of the malignant tumors and only 52% of benign tumors (P < 0.01). Immunostaining was not present after preabsorption of the antibody with pure human recombinant bFGF.

CONCLUSIONS

The presence of bFGF predominantly within the tumor microvasculature indicates a cellular depot for this potent growth factor that mediates angiogenesis and tumorigenesis. These data support a role for bFGF in the transition from the benign to the malignant phenotype.

High circulating levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) have been demonstrated in kidney failure, but mechanisms of this are not well understood. Here we examined the impact of the kidney on the early regulation of intact FGF23 in acute uremia as induced by bilateral or unilateral nephrectomy (BNX and UNX, respectively) in the rat. BNX induced a significant increase in plasma intact FGF23 levels from 112 to 267 pg/ml within <em>15</em> min, which remained stable thereafter. UNX generated intact FGF23 levels between that seen in BNX and sham-operated rats. The intact to C-terminal FGF23 ratio was significantly increased in BNX rats. The rapid rise in FGF23 after BNX was independent of parathyroid hormone or FGF receptor signaling. No evidence of early stimulation of FGF23 gene expression in the bone was found. Furthermore, acute severe hyperphosphatemia or hypercalcemia had no impact on intact FGF23 levels in normal and BNX rats. The half-life of exogenous recombinant human FGF23 was significantly prolonged from 4.4 to 11.8 min in BNX rats. Measurements of plasma FGF23 in the renal artery and renal vein demonstrated a significant renal extraction. Thus the kidney is important in FGF23 homeostasis by regulation of its plasma level and metabolism.

Elastase/anti-elastase imbalance is a hallmark of emphysema, a chronic obstructive pulmonary disease associated with the rupture and inefficient repair of interstitial elastin. We report that neutrophil elastase (NE) at low physiologic concentrations, ranging from 35 nm to 1 microm, invokes transient, peaking at <em>15</em> min, activation of extracellular signal-regulated kinases 1 and 2 (ERK) in elastogenic lung <em>fibroblasts</em>. ERK activation is preceded by the release of soluble 25-26-kDa forms of epidermal <em>growth</em> <em>factor</em> (EGF) and transactivation of EGF receptor (EGFR) in NE-exposed cells. The stimulatory effect of NE on ERK is abrogated in the presence of anti-EGF-neutralizing antibodies, EGFR tyrosine kinase inhibitor (AG1478), and ERK kinase inhibitor (PD98059), as well as abolished in both EGFR-desensitized and endocytosis-arrested <em>fibroblasts</em>. Nuclear accumulation of activated ERK is associated with transient, peaking at 30 min, induction of c-Fos and sustained, observed at 24-48 h, decrease of tropoelastin mRNA levels in NE-challenged cells. Pretreatment of <em>fibroblasts</em> with AG1478 or PD98059 abrogates the NE-initiated tropoelastin mRNA suppression. We conclude that proteolytically released EGF signals directly via EGFR and ERK to down-regulate tropoelastin mRNA in NE-challenged lung <em>fibroblasts</em>.

BACKGROUND

Decreased glomerular filtration rate (GFR) leads to reduced production of 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3 (25[OH]D3). Effects of low GFR on vitamin D catabolism are less well understood. We tested associations of estimated GFR (eGFR) with the circulating concentration of 24,25-dihydroxyvitamin D3 (24,25[OH]2D3), the most abundant product of 25(OH)D3 catabolism, across populations with a wide range of GFRs.

METHODS

Cross-sectional study.

METHODS

9,596 participants in 5 cohort studies and clinical trials: the Diabetes Control and Complications Trial (N=1,193), Multi-Ethnic Study of Atherosclerosis (N=6,470), Cardiovascular Health Study (N=932), Seattle Kidney Study (N=289), and Hemodialysis Study (N=712).

METHODS

eGFR.

RESULTS

Circulating 24,25(OH)2D3 concentration.

METHODS

GFR was estimated from serum creatinine using the Chronic Kidney Disease Epidemiology Collaboration equation. Vitamin D metabolites were measured by mass spectrometry.

RESULTS

Circulating 24,25(OH)2D3 concentration was correlated with circulating 25(OH)D3 concentration (Pearson r range, 0.64-0.88). This correlation was weaker with lower eGFRs. Moreover, the increment in 24,25(OH)2D3 concentration associated with higher 25(OH)D3 concentration (slope) was lower with lower eGFRs: 2.06 (95% CI, 2.01-2.10), 1.77 (95% CI, 1.74-1.81), 1.55 (95% CI, 1.48-1.62), 1.17 (95% CI, 1.05-1.29), 0.92 (95% CI, 0.74-1.10), 0.61 (95% CI, 0.22-1.00), and 0.37 (95% CI, 0.35-0.39) ng/mL of 24,25(OH)2D3 per 10 ng/mL of 25(OH)D3 for eGFRs≥90, 60-89, 45-59, 30-44, <em>15</em>-29, and (<em>15</em> mL/min/1.73 m2 and end-stage renal disease treated with hemodialysis, respectively. As a result, at a 25(OH)D3 concentration of 20 ng/mL, mean 24,25(OH)2D3 concentrations were 2.92 (95% CI, 2.87-2.96), 2.68 (95% CI, 2.64-2.72), 2.35 (95% CI, 2.26-2.45), 1.92 (95% CI, 1.74-2.10), 1.69 (95% CI, 1.43-1.95), 1.14 (95% CI, 0.62-1.66), and 1.04 (95% CI,1.02-1.07) ng/mL for each category, respectively. This interaction was independent of other relevant clinical characteristics. Race, diabetes, urine albumin excretion, and circulating parathyroid hormone and <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 concentrations more modestly modified the association of 24,25(OH)2D3 with 25(OH)D3.

CONCLUSIONS

Lack of direct pharmacokinetic measurements of vitamin D catabolism.

CONCLUSIONS

Lower eGFR is associated strongly with reduced vitamin D catabolism, as measured by circulating 24,25(OH)2D3 concentration.

The differentiation and function of cumulus cells depend upon oocyte-derived paracrine <em>factors</em>, but studies on the estrogen receptor knockout mice suggested that estrogen also participates in these processes. This study investigates the possible coordination of estrogen and oocytes in the development and function of cumulus cells using cumulus expansion and the expression of transcripts required for expansion as functional endpoints. Preantral granulosa cell-oocyte complexes developed in vitro with 17β-estradiol (E2) exhibited increased levels of cumulus expansion and Has2 transcripts, encoding hyaluronan synthase 2, compared with those developed without E2. Moreover, cumulus cell-oocyte complexes (COCs) isolated from antral follicles and maintained in culture without E2 exhibited reduced cumulus expansion and Has2 mRNA levels compared with freshly isolated COCs. Exogenous E2, provided during the maintenance culture, alleviated these deficiencies. However, when oocytes were removed from COCs, E2 supplementation did not maintain competence to undergo expansion; the presence in culture of either fully grown oocytes or recombinant <em>growth</em> differentiation <em>factor</em> 9 (GDF9) was required. Recombinant bone morphogenetic protein <em>15</em>, but not <em>fibroblast</em> <em>growth</em> <em>factor</em> 8, augmented the GDF9 effect. Oocytes or GDF9 suppressed cumulus cell levels of Nrip1 transcripts encoding nuclear receptor-interacting protein 1, a potential inhibitor of estrogen receptor signals. Therefore, E2 and oocyte-derived paracrine <em>factors</em> GDF9 and bone morphogenetic protein <em>15</em> coordinate to promote the development of cumulus cells and maintain their competence to undergo expansion. Furthermore, suppression of Nrip1 expression in cumulus cells by oocyte may be one mechanism mediating cross talk between oocyte and E2 signals that promotes follicular development.

ADAM<em>15</em> (a disintegrin and metalloproteinase <em>15</em>) is a membrane-anchored metalloproteinase, which is overexpressed in several human cancers and has been implicated in pathological neovascularization and prostate cancer metastasis. Yet, little is known about the catalytic properties of ADAM<em>15</em>. Here, we purified soluble recombinant ADAM<em>15</em> to test for its ability to cleave a library of peptide substrates. However, we found no processing of any of the peptide substrates tested here, and therefore turned to cell-based assays to characterize the catalytic properties of ADAM<em>15</em>. Overexpression of full-length membrane-anchored ADAM<em>15</em> or the catalytically inactive ADAM<em>15</em>E->>A together with various membrane proteins resulted in increased release of the extracellular domain of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2iiib (FGFR2iiib) by ADAM<em>15</em>, but not ADAM<em>15</em>E->>A. This provided a robust assay for a characterization of the catalytic properties of ADAM<em>15</em> in intact cells. We found that increased expression of ADAM<em>15</em> resulted in increased FGFR2iiib shedding, but that ADAM<em>15</em> was not stimulated by phorbol esters or calcium ionophores, two commonly used activators of ectodomain shedding. Moreover, ADAM<em>15</em>-dependent processing of FGFR2iiib was inhibited by the hydroxamate-based metalloproteinase inhibitors marimastat, TAPI-2 and GM6001, and by 50 nM TIMP-3 (tissue inhibitor of metalloproteinases 3), but not by 100 nM TIMP-1, and only weakly by 100 nM TIMP-2. These results define key catalytic properties of ADAM<em>15</em> in cells and its response to stimulators and inhibitors of ectodomain shedding. A cell-based assay for the catalytic activity of ADAM<em>15</em> could aid in identifying compounds, which could be used to block the function of ADAM<em>15</em> in pathological neovascularization and cancer.

OBJECTIVE

Transforming <em>growth</em> <em>factor</em> β superfamily members are involved in the pathogenesis of systemic sclerosis (SSc). <em>Growth</em> differentiation <em>factor</em> <em>15</em> (GDF-<em>15</em>) is a distant member of this family. We undertook this study to evaluate the role of GDF-<em>15</em> in SSc pathogenesis.

METHODS

A longitudinal prospective cohort of SSc patients was screened for serum GDF-<em>15</em> levels using enzyme-linked immunosorbent assay, and associations with clinical data were analyzed. In vitro stimulation experiments were performed on lung fibroblasts. The role of GDF-<em>15</em> in fibrosis development in vivo was evaluated in the bleomycin lung fibrosis model in GDF-<em>15</em>-deficient mice.

RESULTS

GDF-<em>15</em> was measured at baseline in serum samples from 119 SSc patients. An increase in GDF-<em>15</em> levels was observed in patients classified as having no skin involvement, those with limited cutaneous SSc, and those with diffuse cutaneous SSc. Moreover, baseline serum GDF-<em>15</em> levels correlated strongly with disease activity and extent of organ involvement, particularly clinical symptoms of lung fibrosis, including impact on lung function at prospective followup. This was mimicked in the bleomycin model of SSc, in which animals exposed to bleomycin had elevated expression levels of GDF-<em>15</em> in lung tissue. Lung fibroblasts isolated from GDF-<em>15</em>-deficient mice showed reduced induction of interleukin-6 and CCL2 upon bleomycin stimulation. Surprisingly, no differences in fibrosis development were observed between wild-type and GDF-<em>15</em>-deficient animals.

CONCLUSIONS

An intriguing profile of serum GDF-<em>15</em> levels was found in SSc patients. GDF-<em>15</em> expression is induced during fibrosis development and markedly correlates with lung function impairment in this disease. The protein may participate in fibrosis initiation, but is not indispensable in the course of fibrosis development in vivo.

OBJECTIVE

Paracrine/autocrine interactions between orbital fibroblasts (OF) and infiltrating lymphocytes/macrophages are thought to play a central role in the evolution of Graves' ophthalmopathy (GO). Compounds capable of stimulating the proliferation and synthetic capacities of OF may be of particular importance to these processes, because fibroblasts are known to both produce and respond to certain paracrine factors.

METHODS

The effects of interleukin-1 alpha, interleukin-2, interleukin-4, interleukin-6, insulin-like growth factor I, transforming growth factor beta, and platelet-derived growth factor on OF monolayers derived from orbital fatty connective tissue and extraocular muscle endomysium of patients with severe GO undergoing orbital decompression (n = 3), and from connective tissue of normal persons (n = 3) were investigated. Stimulation of proliferation in growth-arrested OF was determined using immunocytochemical staining for the cell-proliferation-related nuclear antigen recognized by a monoclonal anti-Ki 67 antibody. In addition, the effects of OF coincubation with one of the aforementioned compounds and hydrocortisone (10(-7) M), the selective glucocorticoid receptor agonist RU 28362 (10(-7) M), or the glucocorticoid receptor antagonist RU 38486 (10(-7) M) were assessed.

RESULTS

Under baseline conditions (0.1% fetal bovine serum), the proportion of proliferating cells was significantly higher in GO-OF compared with normal OF (p < 0.001). Significant stimulation of GO-OF proliferation was observed with interleukin-1 alpha (10 U/ml), interleukin-4 (1 ng/ml), insulin-like growth factor I (10 ng/ml), transforming growth factor beta (10 ng/ml), platelet-derived growth factor (1 ng/ml), and 1% or 15% fetal bovine serum (all P < 0.01), but not with interleukin-2 (10 U/ml) and interleukin-6 (100 U/ml). Compared with GO-OF, proliferation of normal OF was stimulated by fetal bovine serum to a similar degree, by interleukin-4, insulin-like growth factor I, transforming growth factor beta, and platelet-derived growth factor to a significantly lesser degree (all P < 0.01), and was unaffected by interleukin-1 alpha, interleukin-2, and interleukin-6. Compared with normal OF, either glucocorticoid receptor agonists, but not testosterone or progesterone, specifically inhibited the cytokine-stimulated proliferation of GO-OF to a significantly greater degree (P < 0.01).

CONCLUSIONS

The enhanced proliferative capacity of GO-OF at baseline and in response to certain cytokines could play a role in the evolution of the clinical manifestations in GO. Inhibition of cytokine-activated cellular functions may be one mechanism by which glucocorticosteroids exert clinically useful effects in GO.

Pregnancy invokes a doubling of intestinal calcium absorption whereas lactation programs skeletal resorption to provide calcium to milk. Postweaning bone formation restores the skeleton's bone mineral content (BMC), but the <em>factors</em> that regulate this are not established. We used Pth-null mice to test whether parathyroid hormone (PTH) is required for postweaning skeletal recovery. On a normal 1% calcium diet, wild-type (WT) and Pth-null mice each gained BMC during pregnancy, declined <em>15</em>% to 18% below baseline during lactation, and restored the skeleton above baseline BMC within 14 days postweaning. A 2% calcium diet reduced the lactational decline in BMC without altering the gains achieved during pregnancy and postweaning. The hypocalcemia and hyperphosphatemia of Pth-null mice normalized during lactation and serum calcium remained normal during postweaning. Osteocalcin and propeptide of type 1 collagen (P1NP) each rose significantly after lactation to similar values in WT and Pth-null. Serum calcitriol increased fivefold during pregnancy in both genotypes whereas vitamin D binding protein levels were unchanged. Absence of PTH blocked a normal rise in <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF23) during pregnancy despite high calcitriol. A 30-fold higher expression of Cyp27b1 in maternal kidneys versus placenta suggests that the pregnancy-related increase in calcitriol comes from the kidneys. Conversely, substantial placental expression of Cyp24a1 may contribute significantly to the metabolism of calcitriol. In conclusion, PTH is not required to upregulate renal expression of Cyp27b1 during pregnancy or to stimulate recovery from loss of BMC caused by lactation. A calcium-rich diet in rodents suppresses skeletal losses during lactation, unlike clinical trials that showed no effect of supplemental calcium on lactational decline in BMC.

Mesenchymal stem cells (MSCs) are promising tools for studying the mechanisms of development and for the regeneration of injured tissues. Correct selection of the MSCs source is crucial in order to obtain a more efficient treatment and, in this respect Periosteum-Derived Cells (PDPCs) may represent an interesting alternative to bone marrow MSCs for osteochondral tissue regeneration. In the present study we have isolated and characterized a MSCs population from the periosteum of human adult donors. PDPCs were expanded under specific culture conditions that prevent <em>fibroblast</em> contamination and support the maintenance of their undifferentiated phenotype. We show, for the first time, that PDPCs expresses VEGF receptor (Flt1 and KDR/Flk1) proteins and that they were similar to bone marrow Multipotent Adult Progenitor Cells (MAPCs). Since the latter are able to differentiate into endothelial cells, we tested the possible PDPCs commitment toward an endothelial phenotype in view of bone tissue engineering approaches that takes into account not only bone formation but also vascularization. PDPCs were treated with two different VEGF concentrations for 7 and <em>15</em> days and, alternatively, with the supernatant of human primary osteoblasts. Differently from MAPCs our PDPCs were unable to differentiate into endothelial cells after their in vitro VEGF treatment. On the contrary, <em>growth</em> <em>factor</em> stimulation induces PDPCs differentiation toward osteoblasts. We concluded that in PDPCs the presence of VEGF receptors is related to different cross-talk between osteogenesis and angiogenesis that could involve in situ PDPCs recruitment.

The selenoenzyme glutathione peroxidase 4 (GPx4) has been described to control specific cyclooxygenases (COXs) and lipoxygenases (LOXs) that exert substantiated functions in tumor <em>growth</em> and angiogenesis. Therefore, we hypothesized a putative regulatory role of GPx4 during tumor progression and created transformed murine embryonic <em>fibroblasts</em> with inducible disruption of GPx4. GPx4 inactivation caused rapid cell death in vitro, which could be prevented either by lipophilic antioxidants or by 12/<em>15</em>-LOX-specific inhibitors, but not by inhibitors targeting other LOX isoforms or COX. Surprisingly, transformed GPx4(+/-) cells did not die when grown in Matrigel but gave rise to tumor spheroids. Subcutaneous implantation of tumor cells into mice resulted in knockout tumors that were indistinguishable in volume and mass in comparison to wild-type tumors. However, further analysis revealed a strong vascular phenotype. We observed an increase in microvessel density as well as a reduction in the number of large diameter vessels covered by smooth muscle cells. This phenotype could be linked to increased 12/<em>15</em>-LOX activity that was accompanied by an up-regulation of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and down-regulation of vascular endothelial <em>growth</em> <em>factor</em> A protein expression. Indeed, pharmacological inhibition of 12/<em>15</em>-LOX successfully reversed the tumor phenotype and led to "normalized" vessel morphology. Thus, we conclude that GPx4, through controlling 12/<em>15</em>-LOX activity, is an important regulator of tumor angiogenesis as well as vessel maturation.

In asthma, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) plays an important (patho)physiological role. This study examines the effects of FGF-2 on the transforming <em>growth</em> <em>factor</em>-β (TGF-β)-stimulated differentiation of airway smooth muscle (ASM) cells in vitro. The differentiation of human ASM cells after incubation with TGF-β (100 pM) and/or FGF-2 (300 pM) for 48 hours was assessed by increases in contractile protein expression, actin-cytoskeleton reorganization, enhancements in cell stiffness, and collagen remodeling. FGF-2 inhibited TGF-β-stimulated increases in transgelin (SM22) and calponin gene expression (n = <em>15</em>, P < 0.01) in an extracellular signal-regulated kinase 1/2 (ERK1/2) signal transduction-dependent manner. The abundance of ordered α-smooth muscle actin (α-SMA) filaments formed in the presence of TGF-β were also reduced by FGF-2, as was the ratio of F-actin to G-actin (n = 8, P < 0.01). Furthermore, FGF-2 attenuated TGF-β-stimulated increases in ASM cell stiffness and the ASM-mediated contraction of lattices, composed of collagen fibrils (n = 5, P < 0.01). However, the TGF-β-stimulated production of IL-6 was not influenced by FGF-2 (n = 4, P>> 0.05), suggesting that FGF-2 antagonism is selective for the regulation of ASM cell contractile protein expression, organization, and function. Another mitogen, thrombin (0.3 U ml(-1)), exerted no effect on TGF-β-regulated contractile protein expression (n = 8, P>> 0.05), α-SMA organization, or the ratio of F-actin to G-actin (n = 4, P>> 0.05), suggesting that the inhibitory effect of FGF-2 is dissociated from its mitogenic actions. The addition of FGF-2, 24 hours after TGF-β treatment, still reduced contractile protein expression, even when the TGF-β-receptor kinase inhibitor, SB43<em>15</em>42 (10 μM), was added 1 hour before FGF-2. We conclude that the ASM cell differentiation promoted by TGF-β is antagonized by FGF-2. A better understanding of the mechanism of action for FGF-2 is necessary to develop a strategy for therapeutic exploitation in the treatment of asthma.

Vitamin D insufficiency has been increasingly recognized in the general population worldwide and has been associated with several lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and respiratory tract infections. <em>Fibroblasts</em> play a critical role in tissue repair and remodeling, which is a key feature of COPD and asthma. <em>Fibroblasts</em> modulate tissue repair by producing and modifying extracellular matrix components and by releasing mediators that act as autocrine or paracrine modulators of tissue remodeling. The current study was designed to investigate if vitamin D alters <em>fibroblast</em> release of key autocrine/paracrine repair <em>factors</em>. First, we demonstrated that human fetal lung (HFL)-1 cells express the vitamin D receptor (VDR) and that vitamin D, 25-hydroxyvitamin D [25(OH)D], or 1,25-dihydroxyvitamin D [1,25(OH)2D] induce VDR nuclear translocation and increase VDR-DNA binding activity. We next demonstrated that vitamin D, 25(OH)D, and 1,25(OH)2D significantly reduced prostaglandin (PG)E2 production by human lung <em>fibroblasts</em> (HFL-1) but had no effect on transforming <em>growth</em> <em>factor</em> β1, vascular endothelial <em>growth</em> <em>factor</em>, or fibronectin production. Vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibited IL-1β-induced microsomal PGE synthase (mPGES)-1 expression; in contrast, all three forms of vitamin D stimulated <em>15</em>-hydroxy PG dehydrogenase, an enzyme that degrades PGE2. Cyclooxygenase-1 and -2 and the other two PGE2 synthases (mPGES-2 and cytosolic PGE synthase) were not altered by vitamin D, 25(OH)D, or 1,25(OH)2D. Finally, the effect of PGE2 inhibition by 25(OH)D was observed in adult lung <em>fibroblasts</em>. These findings suggest that vitamin D can regulate PGE2 synthesis and degradation and by this mechanism can modulate <em>fibroblast</em>-mediated tissue repair function.

Different <em>growth</em> <em>factors</em> have been shown to influence the development of form-deprivation myopia and lens-induced ametropias. However, <em>growth</em> <em>factors</em> have relatively little effect on the <em>growth</em> of eyes with unrestricted vision. We investigate whether the combination of insulin-like <em>growth</em> <em>factor</em> 1 (IGF1) and <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) influence ocular <em>growth</em> in eyes with unrestricted vision. Different doses of IGF1 and FGF2 were injected into the vitreous chamber of postnatal chicks. Measurements of ocular dimensions and intraocular pressure (IOP) were made during and at the completion of different treatment paradigms. Histological and immunocytochemical analyses were performed to assess cell death, cellular proliferation and integrity of ocular tissues. Treated eyes had significant increases in equatorial diameter and vitreous chamber depth. With significant variability between individuals, IGF1/FGF2-treatment caused hypertrophy of lens and ciliary epithelia, lens thickness was increased, and anterior chamber depth was decreased. Treated eyes developed myopia, in excess of <em>15</em> diopters of refractive error. Shortly after treatment, eyes had increased intraocular pressure (IOP), which was increased in a dose-dependent manner. Seven days after treatment with IGF1 and FGF2 changes to anterior chamber depth, lens thickness and elevated IOP were reduced, whereas increases in the vitreous chamber were persistent. Some damage to ganglion cells was detected in peripheral regions of the retina at 7 days after treatment. We conclude that the extreme myopia in IGF1/FGF2-treated eyes results from increased vitreous chamber depth, decreased anterior chamber depth, and changes in the lens. We propose that <em>factor</em>-induced ocular enlargement and myopia result from changes to the sclera, lens and anterior chamber depth.

Ursodeoxycholic acid (UDCA) is no longer recommended for management of adult patients with primary sclerosing cholangitis (PSC). We undertook a prospective evaluation of UDCA withdrawal in a group of consecutive patients with PSC. Twenty six patients, all treated with UDCA (dose range: 10-<em>15</em> mg/kg/day) were included. Paired blood samples for liver biochemistry, bile acids, and <em>fibroblast</em> <em>growth</em> <em>factor</em> 19 (FGF19) were collected before UDCA withdrawal and 3 months later. Liquid chromatography/tandem mass spectrometry was used for quantification of 29 plasma bile acid metabolites. Pruritus and health-related quality of life (HRQoL) were assessed with a 10-point numeric rating scale, the Medical Outcomes Study Short Form-36 (SF-36), and PBC-40 questionnaires. UDCA withdrawal resulted in a significant deterioration in liver biochemistry (increase of alkaline phosphatase of 75.6%; P<0.0001; gamma-glutamyl transpeptidase of 117.9%, P<0.0001; bilirubin of 50.0%, P<0.001; alanine aminotransferase of 63.9%, P<0.005; and aspartate aminotransferase of 45.0%, P<0.005) and increase of Mayo Risk Score for PSC (change from baseline of +0.5 point; P<0.003). Bile acid analysis revealed a significant decrease in lithocholic acid and its derivatives after UDCA withdrawal, but no effect on concentrations of primary bile acids aside from an increased accumulation of their taurine conjugates. After UDCA removal cholestatic parameters, taurine species of cholic acid and chenodeoxycholic acid correlated with serum FGF19 levels. No significant effect on HRQoL after UDCA withdrawal was observed; however, 42% of patients reported a deterioration in their pruritus.

CONCLUSIONS

At 3 months, discontinuation of UDCA in patients with PSC causes significant deterioration in liver biochemistry and influences concentrations of bile acid metabolites. A proportion of patients report increased pruritus, but other short-term markers of quality of life are unaffected.

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory <em>factor</em> (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-<em>15</em> days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

Neural stem cells (NSCs) are a small population of resident cells that can grow, migrate and differentiate into neuro-glial cells in the central nervous system (CNS). Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription <em>factor</em> that regulates cell <em>growth</em> and differentiation. In this study we analyzed the influence of PPARγ agonists on neural stem cell <em>growth</em> and differentiation in culture. We found that in vitro culture of mouse NSCs in neurobasal medium with B27 in the presence of epidermal <em>growth</em> <em>factor</em> (EGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) induced their <em>growth</em> and expansion as neurospheres. Addition of all-trans retinoic acid (ATRA) and PPARγ agonist ciglitazone or <em>15</em>-Deoxy-Δ(12,14)-Prostaglandin J(2) (<em>15</em>d-PGJ2) resulted in a dose-dependent inhibition of cell viability and proliferation of NSCs in culture. Interestingly, NSCs cultured with PPARγ agonists, but not ATRA, showed significant increase in oligodendrocyte precursor-specific O4 and NG2 reactivity with a reduction in NSC marker nestin, in 3-7 days. In vitro treatment with PPARγ agonists and ATRA also induced modest increase in the expression of neuronal β-III tubulin and astrocyte-specific GFAP in NSCs in 3-7 days. Further analyses showed that PPARγ agonists and ATRA induced significant alterations in the expression of many stemness and differentiation genes associated with neuro-glial differentiation in NSCs. These findings highlight the influence of PPARγ agonists in promoting neuro-glial differentiation of NSCs and its significance in the treatment of neurodegenerative diseases.