<em>Fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) promotes phosphaturia and suppresses 1,25-dihydroxyvitamin D [1,25(OH)(2)D] production. PTH also promotes phosphaturia, but, in contrast, stimulates 1,25(OH)(2)D production. The relationship between FGF23 and PTH is unclear, and the acute effect of pharmacologically dosed PTH on FGF23 secretion is unknown. Twenty healthy men were infused with human PTH(1-34) [hPTH(1-34)] at 44 ng/kg/h for 24 h. Compared with baseline, FGF23, 1,25(OH)(2)D, ionized calcium (iCa), and serum N-telopeptide (NTX) increased significantly over the 18-h hPTH(1-34) infusion (p < 0.0001), whereas serum phosphate (PO(4)) transiently increased and then returned to baseline. FGF23 increased from 35 +/- 10 pg/ml at baseline to 53 +/- <em>20</em> pg/ml at 18 h (p = 0.0002); 1,25(OH)(2)D increased from 36 +/- 16 pg/ml at baseline to 80 +/- 33 pg/ml at 18 h (p < 0.0001); iCa increased from 1.23 +/- 0.03 mM at baseline to 1.46 +/- 0.05 mM at hour 18 (p < 0.0001); and NTX increased from 17 +/- 4 nM BCE at baseline to 28 +/- 8 nM BCE at peak (p < 0.0001). PO(4) was 3.3 +/- 0.6 mg/dl at baseline, transiently rose to 3.7 +/- 0.4 mg/dl at hour 6 (p = 0.016), and then returned to 3.4 +/- 0.5 mg/dl at hour 12 (p = 0.651). hPTH(1-34) infusion increases endogenous 1,25(OH)(2)D and FGF23 within 18 h in healthy men. Whereas it is possible that the rise in PO(4) contributed to the observed increase in FGF23, the increase in 1,25(OH)(2)D was more substantial and longer sustained than the change in serum phosphate. Given prior data that suggest that neither PTH nor calcium stimulate FGF23 secretion, these data support the assertion that 1,25(OH)(2)D is a potent physiologic stimulator of FGF23 secretion.

Stem cells from the adult forebrain of mice were stimulated to form clones in vitro using <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2). At concentrations above 10 ng/ml of FGF-2, very few clones gave rise to neurons; however, if FGF-2 was removed after 5 days, <em>20</em>-30% of clones subsequently gave rise to neurons. The number of neuron-containing clones and the number of neurons per clone was significantly enhanced, if insulin-like <em>growth</em> <em>factor</em> (IGF)-1 or heparin were added subsequent to FGF-2 removal. The spontaneous production of neurons after FGF-2 removal was shown to be due to endogenous IGF-1, since antibodies to IGF-1 and an IGF-1 binding protein totally inhibited neuronal production. Similarly, these reagents also abrogated the neuron-promoting effects of heparin. Thus, it appears that endogenous IGF-1 may be a major regulator of stem cell differentiation into neurons. Furthermore, it was found that high levels of IGF-1 or insulin promoted the maturation and affected the neurotransmitter phenotype of the neurons generated.

OBJECTIVE

The aim of this study was to test the hypothesis that angiotensin (Ang)-converting enzyme-2 (ACE2) overexpression may inhibit myocardial collagen accumulation and improve left ventricular (LV) remodeling and function in diabetic cardiomyopathy.

BACKGROUND

Hyperglycemia activates the renin-Ang system, which promotes the accumulation of extracellular matrix and progression of cardiac remodeling and dysfunction.

METHODS

Ninety male Wistar rats were divided randomly into treatment (n = 80) and control (n = 10) groups. Diabetes was induced in the treatment group by a single intraperitoneal injection of streptozotocin. Twelve weeks after streptozotocin injection, rats in the treatment group were further divided into adenovirus-ACE2, adenovirus-enhanced green fluorescent protein, losartan, and mock groups (n = <em>20</em> each). LV volume; LV systolic and diastolic function; extent of myocardial fibrosis; protein expression levels of ACE2, Ang-converting enzyme, and Ang-(1-7); and matrix metalloproteinase-2 activity were evaluated. Cardiac myocyte and <em>fibroblast</em> culture was performed to assess Ang-II and collagen protein expression before and after ACE2 gene transfection.

RESULTS

Four weeks after ACE2 gene transfer, the adenovirus-ACE2 group showed increased ACE2 expression, matrix metalloproteinase-2 activity, and LV ejection fractions and decreased LV volumes, myocardial fibrosis, and ACE, Ang-II, and collagen expression in comparison with the adenovirus-enhanced green fluorescent protein and control groups. ACE2 was superior to losartan in improving LV remodeling and function and reducing collagen expression. The putative mechanisms may involve a shift in balance toward an inhibited fibroblast-myocyte cross-talk for collagen and transforming growth factor-beta production and enhanced collagen degradation by matrix metalloproteinase-2.

CONCLUSIONS

ACE2 inhibits myocardial collagen accumulation and improves LV remodeling and function in a rat model of diabetic cardiomyopathy. Thus, ACE2 provides a promising approach to the treatment of patients with diabetic cardiomyopathy.

Recent studies have revealed that 1,2,3,4,6-penta-O-galloyl-beta-d-glucose (PGG) has anti-tumorigenic activity in vitro. In the present work, we evaluated the in vitro and in vivo antiangiogenic and antitumor activities of PGG and examined its molecular mechanisms. PGG significantly inhibited the proliferation and tube formation in basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF)-treated human umbilical vein endothelial cells (HUVECs) at non-cytotoxic concentrations. PGG effectively disrupted the bFGF-induced neo-vascularization in chick chorioallantoic membrane (CAM) and in Matrigel plugs in the mice. When mice were intraperitoneally injected, PGG also significantly inhibited tumor angiogenesis induced by Lewis lung carcinoma (LLC) and the <em>growth</em> of LLC by 57 and 91% of control tumor weight at 4 and <em>20</em> mg/kg, respectively. Immunohistochemical analysis revealed decreased microvessel density, decreased expression of cyclooxygenase-2 (COX-2) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF), reduced tumor cell proliferation and increased tumor cell apoptosis. Similarly, PGG significantly attenuated the expression of COX-2 and VEGF and reduced the secretion of VEGF and prostaglandin E2 in bFGF-treated HUVECs. Furthermore, the COX-2 inhibitor NS398 significantly inhibited tube formation and neo-vascularization in CAM, supporting the role of COX-2 in PGG inhibition of angiogenesis. PGG diminished the phosphorylation of extracellular signal regulated kinase 1/2, Jun NH2-terminal kinase and activated phospho-p38 mitogen-activated protein kinase (MAPK) in a dose-dependent manner in bFGF-treated HUVECs. In addition, p38 inhibitor SB<em>20</em>3580 abolished the downregulation of COX-2, VEGF and the antiproliferative activity by PGG. Taken together, our data demonstrate that PGG exerts antitumor activity primarily via inhibition of angiogenesis through COX-2 and MAPK- dependent pathways.

OBJECTIVE

Low-level laser irradiation at certain fluences and wavelengths can enhance the release of growth factors from fibroblasts and stimulate cell proliferation in vitro. We evaluated whether low-level laser irradiation can improve wound healing in diabetes mellitus.

METHODS

Genetically diabetic mice (C57BL/Ksj/db/db) were used as the animal model for this wound healing study. The experimental animals were divided among four groups: negative control, positive control (topical basic fibroblast growth factor [bFGF] on wound), laser therapy group; and a combination group of laser therapy and topical bFGF. An argon dye laser (Lexel Auora Model 600) at a wavelength of 630 nm and an output of 20 m W/cm2 was used as the light source. The speed of wound closure and histological evaluation were used to analyze the experimental results.

RESULTS

Laser irradiation enhanced the percentage of wound closure over time as compared to the negative control group (58.4 +/- 2.6 vs. 40.8 +/- 3.4 at day 10 and 95.7 +/- 2 vs. 82.3 +/- 3.6 at day 20, P < .01). Histological evaluation showed that laser irradiation improved wound epithelialization, cellular content, granulation tissue formation, and collagen deposition in laser-treated wounds as compared to the negative control group (6.4 +/- 0.16 vs. 3.8 +/- 0.13 at day 10 and 12 +/- 0.21 vs. 8.2 +/- 0.31, P < .01).

CONCLUSIONS

This study of laser biostimulation on wound healing in diabetic mice suggests that such therapy may be of great benefit in the treatment of chronic wounds that occur as a complication of diabetes mellitus.

Loss of the tumor suppressor gene NF1 in neurofibromatosis type 1 (NF1) contributes to the development of a variety of tumors, including malignant peripheral nerve sheath tumors (MPNST) and benign neurofibromas. Of the different cell types found in neurofibromas, Schwann cells usually provide between 40 and 80%, and are thought to be critical for tumor <em>growth</em>. Here we describe the identification of <em>growth</em> <em>factors</em> that are upregulated in NF1-/- mouse Schwann cells and are potential regulators of angiogenesis and cell <em>growth</em>. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2), platelet-derived <em>growth</em> <em>factor</em> (PDGF) and midkine (MK) were found to be induced by loss of neurofibromin and MK was further characterized. MK was induced in human neurofibromas, schwannomas, and various nervous system tumors associated with NF1 or NF2; midkine showed an expression pattern overlapping but distinct from its homolog pleiotrophin (PTN). Immunohistochemistry revealed expression of MK in S-100 positive Schwann cells of dermal and plexiform neurofibromas, and in endothelial cells of tumor blood vessels, but not in normal blood vessels. Furthermore, MK demonstrated potent mitogenic activity for human systemic and brain endothelial cells in vitro and stimulated proliferation and soft agar colony formation of human MPNST derived S100 positive cells and <em>fibroblast</em>oid cells derived from an NF1 neurofibroma. The data support a possible central role for MK as a mediator of angiogenesis and neurofibroma <em>growth</em> in NF1. Oncogene (<em>20</em>01) <em>20</em>, 97 - 105.

OBJECTIVE

To characterize the effects of small applied electric fields (EFs) (100 to 250 mV/ mm) on cultured bovine corneal epithelial cell (CEC) sheets and to determine how EFs interact with other environmental cues in directing CEC sheet migration.

METHODS

Primary cultures of bovine CECs were exposed to EFs in medium with or without serum, epithelial growth factor, basic fibroblast growth factor, or transforming growth factor-beta 1. Cell sheet migration was traced using an image analyzer.

RESULTS

Cell sheets migrated toward the cathode (negative pole). The directional migration was voltage dependent, and, at low field strength (up to 200 mV/mm), it required serum in the medium. Sheets showed no migration responses up to 200 mV/mm in serum-free medium, whereas those in medium with serum showed evident migration toward the cathode, at an average rate of approximately 15 microns/h (n = 15 approximately 20) at 150 mV/mm. When serum was present, the threshold was below 100 mV/mm, very close to the measured wound field strength (approximately 42 mV/mm). After supplementing serum-free medium with individual growth factors or with combinations of epithelial growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1, significant restoration of cathode-directed migration occurred at 150 mV/ mm. Lamellipodia were abundant at the leading edges of migrating sheets, extending the area of sheets covered. The extension of cell membranes toward the cathode was more prominent in cell sheets than in single cells.

CONCLUSIONS

The endogenous EFs generated by wounded cornea could play an important role by interacting with other environmental factors to promote changes in shape and in directed migration of CEC sheets.

Matrix accumulation in the renal tubulointerstitium is predictive of a progressive decline in renal function. Transforming <em>growth</em> <em>factor</em>-beta(1) (TGF-beta(1)) and, more recently, connective tissue <em>growth</em> <em>factor</em> (CTGF) are recognized to play key roles in mediating the fibrogenic response, independently of the primary renal insult. Further definition of the independent and interrelated effects of CTGF and TGF-beta(1) is critical for the development of effective antifibrotic strategies. CTGF (<em>20</em> ng/ml) induced fibronectin and collagen IV secretion in primary cultures of human proximal tubule cells (PTC) and cortical <em>fibroblasts</em> (CF) compared with control values (P < 0.005 in all cases). This effect was inhibited by neutralizing antibodies to either TGF-beta or to the TGF-beta type II receptor (TbetaRII). TGF-beta(1) induced a greater increase in fibronectin and collagen IV secretion in both PTC (P < 0.01) and CF (P < 0.01) compared with that observed with CTGF alone. The combination of TGF-beta(1) and CTGF was additive in their effects on both PTC and CF fibronectin and collagen IV secretion. TGF-beta(1) (2 ng/ml) stimulated CTGF mRNA expression within 30 min, which was sustained for up to 24 h, with a consequent increase in CTGF protein (P < 0.05), whereas CTGF had no effect on TGF-beta(1) mRNA or protein expression. TGF-beta(1) (2 ng/ml) induced phosphorylated (p)Smad-2 within 15 min, which was sustained for up to 24 h. CTGF had a delayed effect on increasing pSmad-2 expression, which was evident at 24 h. In conclusion, this study has demonstrated the key dependence of the fibrogenic actions of CTGF on TGF-beta. It has further uniquely demonstrated that CTGF requires TGF-beta, signaling through the TbetaRII in both PTCs and CFs, to exert its fibrogenic response in this in vitro model.

Endostatin (<em>20</em> kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor <em>growth</em>. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (K(D) approximately 2 microm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg(47) and Arg(66), participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl(2) to endostatin enhanced its binding to heparan sulfate by approximately 40% as well as its antiproliferative effect on endothelial cells stimulated by <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial <em>growth</em> <em>factor</em> was observed upon the addition of zinc.

We have used aequorin as an indicator for the intracellular free calcium ion concentration [( Ca++]i) of Swiss 3T3 <em>fibroblasts</em>. Estimated [Ca++]i of serum-deprived, subconfluent <em>fibroblasts</em> was 89 (+/-<em>20</em>) nM, almost twofold higher than that of subconfluent cells <em>growing</em> in serum, whose [Ca++]i was 50 (+/-19) nM. Serum, partially purified platelet-derived <em>growth</em> <em>factor</em> (PDGF), and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) stimulated DNA synthesis by the serum-deprived cells, whereas epidermal <em>growth</em> <em>factor</em> (EGF) did not. Serum immediately and transiently elevated the [Ca++]i of serum-deprived cells, which reached a maximal value of 5.3 microM at 18 s poststimulation but returned to near prestimulatory levels within 3 min. Moreover, no further changes in [Ca++]i were observed during 12 subsequent h of continuous recording. PDGF produced a peak rise in [Ca++]i to approximately 1.4 microM at 115 s after stimulation, and FGF to approximately 1.2 microM at 135 s after stimulation. EGF caused no change in [Ca++]i. The primary source of calcium for these transients was intracellular, since the magnitude of the serum-induced rise in [Ca++]i was reduced by only 30% in the absence of exogenous calcium. Phorbol 12-myristate 13-acetate (PMA) had no effect on resting [Ca++]i. When, however, quiescent cells were treated for 30 min with 100 nM PMA, serum-induced rises in [Ca++]i were reduced by sevenfold. PMA did not inhibit <em>growth</em> <em>factor</em>-induced DNA synthesis and was by itself partially mitogenic. We suggest that if calcium is involved as a cytoplasmic signal for mitogenic activation of quiescent <em>fibroblasts</em>, its action is early, transient, and can be partially substituted for by PMA. Activated protein kinase C may regulate <em>growth</em> <em>factor</em>-induced increases in [Ca++]i.

Ciliary neurotrophic <em>factor</em> (CNTF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) were tested for effects on sprouting by motor neurons innervating the adult mouse gluteus muscle. <em>Factors</em> were delivered by subcutaneous injection directly over the surface of the superior gluteus muscle once daily for 7 d and then end plates and axons were visualized by combined silver and cholinesterase staining. CNTF (500 ng daily) induced sprouting both from end plates and from the subset of nodes of Ranvier that are closest to the end plate. The effect of CNTF was potentiated twofold by coadministration of bFGF at doses of 2-<em>20</em> ng daily, whereas treatment with bFGF alone failed to induce sprouting from either end plates or nodes of Ranvier. The sprouting stimulus delivered by the <em>factors</em> showed limited penetrance into the muscle and restricted lateral spread from the injection site.

Endothelial cell seeding, a promising method to improve the performance of small-diameter vascular grafts, requires a suitable substrate, e.g. crosslinked collagen. In addition, the <em>growth</em> of seeded endothelial cells can be improved by local release of a heparin-binding protein, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). In this study, the influence of immobilization of heparin to collagen, crosslinked using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), on the binding and release of bFGF was determined. Heparin was immobilized also using EDC and NHS. Furthermore, the effects of the release of bFGF from (heparinized) EDC/NHS-crosslinked collagen on the proliferation of seeded endothelial cells was studied in vitro. Immobilization of increasing amounts of heparin to EDC/NHS-crosslinked collagen (containing 14 free epsilon-amino groups per 1000 amino acid residues, E/N14C) resulted in binding of increasing amounts of bFGF to the material. Maximal bFGF binding was observed for E/N14C containing <em>20</em>-30 mg heparin immobilized per gram of collagen which was obtained using a molar ratio of EDC to heparin-carboxylic acid groups of 0.4 for heparin immobilization (E/N14C-H(0.4)). Up to concentrations of 3<em>20</em> ng bFGF/ml, 10% of the added bFGF bound to E/N14C, while binding of bFGF to E/N14C-H(0.4) was 22%. The initial release rate of bFGF bound to E/N14C was much higher compared to bFGF bound to E/N14C-H(0.4): respectively, 30 vs. 2% in the first 6 h. After 10 days, the bFGF release from E/N14C and E/N14C-H(0.4) amounted to 83 vs. 42%, respectively. Binding of increasing amounts of bFGF resulted in increased <em>growth</em> of human umbilical vein endothelial cells (HUVECs) seeded on both E/N14C and E/N14C-H(0.4). Nevertheless, after 6 and 10 days of proliferation cell numbers on E/N14C-H(0.4) where higher than cell numbers on E/N14C, irrespective of the bFGF concentration used for loading of the matrix. It is concluded that heparinized, EDC/NHS-crosslinked collagen is a good synthetic vascular graft coating for in vivo endothelial cell seeding.

The specific expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>20</em> (FGF-<em>20</em>) in the adult substantia nigra and the association between FGF-<em>20</em> mutations and Parkinson's disease provoked exploration of the function of this <em>growth</em> <em>factor</em>. We show by gain- and loss-of-function in vitro experiments that FGF-<em>20</em> promotes survival and stimulates dopamine (DA) release in a calbindin-negative subset of cells that are preferentially lost in Parkinson's disease. FGF-<em>20</em> selectively activates tyrosine hydroxylase in calbindin-negative neurons. In the adult substantia nigra, calbindin-negative neurons specifically express high levels of FGFR1 (FGF receptor 1). These data show that FGF signals to elevate DA levels and protect the specific midbrain neuron type at most risk in Parkinson's patients.

Rough titanium (Ti) surface microarchitecture and high surface energy have been shown to increase osteoblast differentiation, and this response occurs through signaling via the alpha(2)beta(1) integrin. However, clinical success of implanted materials is dependent not only upon osseointegration but also on neovascularization in the peri-implant bone. Here we tested the hypothesis that Ti surface microtopography and energy interact via alpha(2)beta(1) signaling to regulate the expression of angiogenic <em>growth</em> <em>factors</em>. Primary human osteoblasts (HOB), MG63 cells and MG63 cells silenced for alpha(2) integrin were cultured on Ti disks with different surface microtopographies and energies. Secreted levels of vascular endothelial <em>growth</em> <em>factor</em>-A (VEGF-A), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2), epidermal <em>growth</em> <em>factor</em> (EGF), and angiopoietin-1 (Ang-1) were measured. VEGF-A increased 170% and 250% in MG63 cultures, and 178% and 435% in HOB cultures on SLA and modSLA substrates, respectively. In MG63 cultures, FGF-2 levels increased <em>20</em> and 40-fold while EGF increased 4 and 6-fold on SLA and modSLA surfaces. These <em>factors</em> were undetectable in HOB cultures. Ang-1 levels were unchanged on all surfaces.Media from modSLA MG63 cultures induced more rapid differentiation of endothelial cells and this effect was inhibited by anti-VEGF-A antibodies. Treatment of MG63 cells with 1 alpha,25(OH)(2)D3 enhanced levels of VEGF-A on SLA and modSLA.Silencing the alpha(2) integrin subunit increased VEGF-A levels and decreased FGF-2 levels. These results show that Ti surface microtopography and energy modulate secretion of angiogenic <em>growth</em> <em>factors</em> by osteoblasts and that this regulation is mediated at least partially via alpha(2)beta(1) integrin signaling.

In hair follicle development, a placode-derived signal is believed to induce formation of the dermal condensation, an essential component of ectodermal organs. However, the identity of this signal is unknown. Furthermore, although induction and patterning of hair follicles are intimately linked, it is not known whether the mesenchymal condensation is necessary for inducing the initial epithelial pattern. Here, we show that <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>20</em> (Fgf<em>20</em>) is expressed in hair placodes and is induced by and functions downstream from epithelial ectodysplasin (Eda)/Edar and Wnt/β-Catenin signaling to initiate formation of the underlying dermal condensation. Fgf<em>20</em> governs formation of primary and secondary dermal condensations in developing hair follicles and subsequent formation of guard, awl, and auchene hairs. Although primary dermal condensations are absent in Fgf<em>20</em> mutant mice, a regular array of hair placodes is formed, demonstrating that the epithelial patterning process is independent of known histological and molecular markers of underlying mesenchymal patterns during the initial stages of hair follicle development.

Through the use of a keratinocyte/<em>fibroblast</em> co-culture system, we have recently identified a potent keratinocyte-derived anti-fibrogenic <em>factor</em> (KDAF) for dermal <em>fibroblasts</em>. A sequential chromatography of the active fractions of keratinocyte-conditioned medium (KCM) and peptide mapping of the candidate proteins identified KDAF as being the keratinocyte-releasable 14-3-3 sigma (14-3-3sigma) protein, which is also known as stratifin. In this study, we hypothesize that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3sigma in conditioned medium. To address this hypothesis, in a longitudinal study, keratinocyte differentiation was induced by <em>growing</em> these cells in a medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% Dulbecco's modified eagle's medium without any additives for up to <em>20</em> d. When KCM was collected every other day and added to <em>fibroblasts</em>, the level of matrix metalloproteinase (MMP)-1 mRNA expression was markedly increased in <em>fibroblasts</em> receiving KCM and this increase was even greater in cells receiving conditioned media collected at later time points relative to that of controls. The results of a western blot analysis further showed a marked increase in the expression of 14-3-3sigma protein in keratinocytes grown in test medium from day 4 to day 10. This finding was consistent with the levels of 14-3-3sigma mRNA expression in differentiated keratinocytes. In contrast to a very high level of 14-3-3sigma mRNA expression seen in keratinocytes, <em>fibroblasts</em> that are highly responsive to14-3-3sigma were unable to express this <em>factor</em>. Interestingly, the level of 14-3-3sigma mRNA expression was markedly higher in keratinocytes co-cultured with <em>fibroblasts</em> relative to that of mono-cultured keratinocytes. In conclusion, this study provides evidence that keratinocytes express a high level of 14-3-3sigma at the levels of mRNA and protein. But the releasable form of 14-3-3sigma protein was only found in conditioned medium derived from differentiated keratinocytes. Further, our recently purified recombinant 14-3-3sigma protein mimics the collagenase stimulatory effect of KCM in dermal <em>fibroblasts</em>.

One of the earliest events in vertebrate eye development is the establishment of the pigmented epithelium and neural retina. These fundamentally different tissues derive from the invaginated optic vesicle, or optic cup. Even after achieving a fairly advanced state of differentiation, the pigmented epithelium exhibits the same potential as the optic cup in that it can "transdifferentiate" into neural retina. C. M. Park and M. J. Hollenberg (Dev. Biol. 134, <em>20</em>1-<em>20</em>5, 1989) discovered that administration of basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, coupled with retinal removal, could trigger this transformation in vivo. We have developed a quantitative in vitro assay to study the role(s) of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family in this phenomenon and more generally in early retinal development. We found that several aspects of the process, including inhibition of pigmented epithelium differentiation, proliferation, and conversion to a retinal fate, were not strictly correlated. Both acidic and basic FGFs were found to potentiate all aspects of the process, with acidic FGF being 4 to <em>20</em> times more potent than basic FGF for inhibition of pigmentation and induction of retinal antigens. Depending upon its concentration, acidic FGF induced from 40% to 80% of the cells in the explants to produce antigens normally expressed by retinal ganglion cells, the first cell type to be generated in retinal development. Expression of such a ganglion cell marker could be directly stimulated in non-dividing cells as well as in dividing cells, indicating that conversion from the pigmented epithelial to retinal fate did not require cell division. These data suggest that acidic FGF, or a related molecule, may function in establishment of retinal fate from the optic cup. This effect may be directly or indirectly mediated by induction of retinal ganglion cell fate among multipotent progenitor cells.

2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol-17beta and the oral contraceptive agent 17-ethylestradiol. 2-ME was recently reported to inhibit endothelial cell proliferation. The current study was undertaken to explore the mechanism of 2-ME effects on endothelial cells, especially whether 2-ME induces apoptosis, a prime mechanism in tissue remodeling and angiogenesis. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to 2-ME showed morphological (including ultrastructural) features characteristic of apoptosis: cell shrinkage, cytoplasmic and nuclear condensation, and cell blebbing. 2-ME-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 0.45 +/- 0.09 microM, n = 8). Nucleosomal DNA fragmentation in BPAEC treated with 2-ME was identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end labeling. Under the same experimental conditions, estradiol-17beta and two of its other metabolites, estriol and 2-methoxyestriol (< or =10 microM), did not have an apoptotic effect on BPAEC. 2-ME activated stress-activated protein kinase (SAPK)/c-Jun amino-terminal protein kinase in BPAEC in a concentration-dependent manner. The activity of SAPK was increased by 170 +/- 27% and 314 +/- 22% over the basal level in the presence of 0.4 and 2 microM 2-ME (n = 3-6), respectively. The activation of SAPK was detected at 10 min, peaked at <em>20</em> min, and returned to basal levels at 60 min after exposure to 2-ME. Inhibition of SAPK/c-Jun amino-terminal protein kinase activation by basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>, or forskolin reduced 2-ME-induced apoptosis. Immunohistochemical analysis of BPAEC indicated that 2-ME up-regulated expression of both Fas and Bcl-2. In addition, 2-ME inhibited BPAEC migration (IC50 = 0.71 +/- 0.11 microM, n = 4) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-induced angiogenesis in the chick chorioallantoic membrane model. Taken together, these results suggest that promotion of endothelial cell apoptosis, thereby inhibiting endothelial cell proliferation and migration, may be a major mechanism by which 2-ME inhibits angiogenesis.

The effects of radiation on the release of mitogenic <em>factors</em> into the media of cultured endothelial cells of bovine, porcine, and human origins were studied. Although unirradiated controls revealed a significant background activity, single doses of irradiation (<em>20</em>-60 Gy) resulted in a dose-related increased release of <em>growth</em> <em>factor</em> activity, measured by the mitogenic effects of the conditioned media on both 3T3 mouse <em>fibroblasts</em> and unirradiated endothelial cells serving as target cells. Receptor binding competition assays for the platelet-derived <em>growth</em> <em>factor</em> receptor revealed that 12-28% of the total mitogenic activity was due to platelet-derived <em>growth</em> <em>factor</em>-like mitogens. Mitogenic assays using endothelial cells and specific antibody mediated inhibition assays suggested that another component of the mitogenic activity was due to a <em>fibroblast</em> <em>growth</em> <em>factor</em>-like <em>factor</em>. Although radiation resulted in a significant increase in cell death, the enhanced <em>growth</em> <em>factor</em> activities did not appear to result from cell lysis-related leakage of intracellular stores of <em>growth</em> <em>factor</em>. Instead, our data suggest that the <em>growth</em> <em>factors</em> were synthesized de novo and secreted at elevated levels by the cells which maintained postradiation a high level of metabolic activity. Time course studies demonstrated that the <em>growth</em> <em>factors</em> accumulation in the conditioned media started within the first 24 h after radiation and reached a plateau within 72 h after treatment. Radiation-induced release of endothelial cell-derived <em>growth</em> <em>factors</em> may be involved in the pathogenesis of both early vascular damage and the late fibrosis which represents a prominent feature of late radiation damage in normal tissues.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) supports the survival of neurons from many regions of the E18 fetal rat brain. Survival was significantly increased for neurons derived from the hippocampus, entorhinal cortex (EC), frontal cortex, parietal cortex (PC), occipital cortex, striatum, septum, and thalamus, but not from the subiculum (Sb). The proportion of neurons rescued by bFGF varied among brain regions, suggesting the existence of subpopulations of responsive neurons. Like hippocampal neurons, neurons from the EC and PC required about 1 pM bFGF (10-<em>20</em> pg/ml) for half-maximal response; striatal neurons, in contrast, required about 3 pM bFGF. Neurite out<em>growth</em> after 24 hr exposure was significantly increased for neurons from the hippocampus, EC, and PC, while striatal neurons had only a marginal response. Although bFGF stimulated some astrocytic proliferation in the cultures, glial contamination was maintained at 2% or less. Acidic FGF (aFGF) supported smaller numbers of neurons from each region, although it significantly increased survival of neurons from hippocampus, EC, PC, striatum, and Sb. The concentration required for half-maximal survival was around 100-300 pM (2-5 ng/ml). It appears that bFGF and aFGF are potent trophic <em>factors</em> for many populations of CNS neurons and could potentially play a significant role in nervous system development.

Microtubules (MTs) contribute to the directional locomotion of many cell types through an unknown mechanism. Previously, we showed that low concentrations ((<em>20</em>0 nM) of nocodazole or taxol reduced the rate of locomotion of NRK <em>fibroblasts</em> over 60% without altering MT polymer level [Liao et al., 1995: J. Cell Sci. 108:3473-3483]. In this paper, we directly measured the dynamics of MTs in migrating NRK cells injected with rhodamine tubulin and treated with low concentrations of nocodazole or taxol. Both drug treatments caused statistically significant reductions (approx. twofold) in <em>growth</em> and shortening rates and less dramatic effects on rescue and catastrophe transition frequencies. The percent time MTs were inactive (i.e., paused) increased greater than twofold in nocodazole- and taxol-treated cells, while the percent time <em>growing</em> was substantially reduced. Three parameters of MT dynamics were linearly related to the rates of locomotion determined previously: rate of shortening, percent time pausing and percent time <em>growing</em>. The number of MTs that came within 1 microm of the leading edge was reduced in drug-treated cells, suggesting that reduced MT dynamics may affect actin arrays necessary for cell locomotion. We examined two such structures, lamellipodium and adhesion plaques, and found that lamellipodia area was coordinately reduced with MT dynamics. No effect was detected on adhesion plaque density or distribution. In time-lapse recordings, MTs did not penetrate into the lamellipodium of untreated cells, suggesting that MTs affect lamellipodia either through their interaction with <em>factors</em> at the base of the lamellipodium or by releasing <em>factors</em> that diffuse into the lamellipodia. In support of the latter hypothesis, when all MTs were rapidly depolymerized by <em>20</em> microM nocodazole, we detected the rapid formation of exaggerated protrusions from the leading edge of the cell. Our results show for the first time a linear relationship between MT dynamics and the formation of the lamellipodium and support the idea that MT dynamics may contribute to cell locomotion by regulating the size of the lamellipodium, perhaps through diffusable <em>factors</em>.

Basic or acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), alone, was found to be as potent as alpha-thrombin to reinitiate DNA synthesis in G0-arrested Chinese hamster lung <em>fibroblasts</em> (CCL39). Basic FGF at 50 ng/ml or thrombin at 1 unit/ml rapidly initiated early events such as cytoplasmic alkalinization (0.2-0.3 pH units), rise in cytoplasmic Ca2+, phosphorylation of ribosomal protein S6 and increased c-myc expression, followed by a 30-40-fold increase in labeled nuclei. Whereas thrombin is a potent activator of phospholipase C as judged by the rapid release of inositol trisphosphate, inositol bisphosphate and by the massive accumulation of total inositol phosphate (IP) in the presence of <em>20</em> mM Li+, FGF failed to induce the breakdown of polyphosphoinositides in quiescent CCL39 cells. Indeed, no inositol trisphosphate nor inositol bisphosphate could be detected in response to FGF; in presence of Li+ the total IP release never exceeded 8% of the IP released by the action of thrombin. Two additional findings indicated that FGF and thrombin activate different signaling pathways. First, we found that, in contrast to thrombin, the FGF-induced rise in the cytoplasmic free Ca2+ concentration measured by quin-2 fluorescence, is strictly dependent upon the presence of Ca2+ in the external medium. Second, we found that FGF failed to activate protein kinase C as judged by the epidermal <em>growth</em> <em>factor</em>-receptor binding assay. Treatment of the cells with either thrombin or phorbol esters, rapidly inhibited 125I-labeled epidermal <em>growth</em> <em>factor</em> binding (50-60%). Basic or acidic FGF had no effect. We conclude that: the FGF-receptor signaling pathway is not coupled to phospholipase C activation, and early mitogenic events and reinitiation of DNA synthesis can be initiated independently of inositol lipid breakdown and protein kinase C activation.

Abnormal stiffness and altered cardiac function arising from abnormal collagen deposition occur in hypertrophy and heart failure. ANG II has been shown to play a role in this process. To evaluate the mechanism, we developed an in vitro model by subjecting <em>fibroblasts</em> to ANG II treatment in the presence or absence of myocytes in coculture (25). Employing this model, we demonstrated that ANG II-induced collagen gene transcription in cardiac <em>fibroblasts</em> was potentiated by myocyte-derived <em>factors</em>. In attempting to identify mechanisms of collagen upregulation and to define the role of myocytes, we found that interleukin (IL)-6, tumor necrosis <em>factor</em> (TNF)-alpha, and the transforming <em>growth</em> <em>factor</em> (TGF)-beta superfamily were also involved in collagen upregulation. Collagen transcripts were increased after <em>fibroblasts</em> were treated with IL-6 (<em>20</em>-50 ng/ml) and TNF-alpha (0.1-0.5 ng/ml). In this study, we show that cardiomyocytes induce secretion of active TGF-beta in the presence of ANG II and that a paracrine action of TGF-beta subsequently induces different cytokines (IL-6) in <em>fibroblasts</em>, thereby promoting collagen synthesis. The cross-talk between myocytes and <em>fibroblasts</em> and involvement of these cytokines in the upregulation of collagen transcript levels are novel findings that may explain their possible roles in the upregulation of collagen.

Fat-storing cells (FSCs), perisinusoidal cells which normally participate in metabolism of vitamin A, have been suggested to participate in collagen synthesis in fibrotic liver. However, key mediators which regulate collagen metabolism in FSCs are yet to be elucidated. In <em>fibroblasts</em>, Interleukin-1 (IL-1), Tumor Necrosis <em>Factor</em> alpha (TNF alpha), and Transforming <em>Growth</em> <em>Factor</em> beta (TGF beta) have been shown to induce diverse modulations of collagen metabolism and cell proliferation. In the present study, these cytokines were tested for their abilities to regulate collagen formation and proliferation by cultured rat FSCs. FSCs primary culture was established and incubated in the absence or presence of various concentrations of IL-1 alpha, TNF alpha, and TGF beta 1. Tritiated proline and thymidine were used to examine collagen formation and cell proliferation. IL-1 alpha (2.5-10 U/ml) had a concentration-dependent stimulatory effect on FSC proliferation with a maximal response of 160% compared to that of untreated FSCs. This mitogenic effect resulted in slight but significant increases (15-<em>20</em>%) in the net collagen formation. However, when this parameter was standardized relative to DNA content, significant inhibition of both collagen and noncollagen protein formation by IL-1 alpha was demonstrated. TNF alpha also exhibited a similar mitogenic effect but induced a more selective inhibition of collagen formation. In contrast, TGF beta 1 (0.01-1 ng/ml) specifically enhanced collagen formation by 60-80%, as also evidenced by significant increases in the ratio of [3H]hydroxyproline to [3H]proline incorporated in newly formed proteins.(ABSTRACT TRUNCATED AT 250 WORDS)