Expression of surfactant protein C (SP-C), which is restricted to alveolar type II epithelial cells of the adult lung, is critically dependent on thyroid transcription <em>factor</em> 1 (TTF-1). In the present study we have demonstrated that Erm, a member of the Ets family of transcription <em>factors</em>, is expressed in the distal lung epithelium during development and is also restricted to alveolar type II cells in the adult. Erm was up-regulated by <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) in culture, and blocking FGF signaling inhibited Erm expression both in vivo and in vitro. The SP-C minimal promoter was found to contain two potential Ets binding sites, and electrophoretic mobility shift assays showed that two <em>20</em>-bp wild-type oligonucleotides containing the 5'-GGA(A/T)-3' Ets consensus binding motif were shifted by nuclear extracts from MLE15 cells. Co-transfection assays showed that Erm by itself had little effect on SP-C promoter activity but that Erm significantly enhanced TTF-1-mediated SP-C transcription. Mutation of one of the Ets binding sites reduced SP-C transcription to background levels, whereas mutation of the other site resulted in increased SP-C transcription. Protein-protein interactions between Erm and TTF-1 were demonstrated by mammalian two-hybrid assays and by co-immunoprecipitation assays. Mapping studies showed that the Ets domain of Erm and the combined N terminus and homeodomain of TTF-1 were critical for this interaction. Treatment of primary cultures of adult alveolar type II cells with siRNA targeting Erm diminished expression of both Erm and SP-C but had no effect on beta-actin or GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Taken together, these results demonstrate that Erm is involved in SP-C regulation, which results from an interaction with TTF-1.

Following cataract surgery, many patients suffer secondary loss of vision because of posterior capsule opacification (PCO), which arises when residual lens epithelial cells become aberrant and migrate into the light path. Transforming <em>growth</em> <em>factor</em>-beta (TGFbeta)-induced transdifferentiation of lens cells appears to play a key role in this process. <em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) may also play a role by promoting the survival of TGFbeta-affected cells and influencing their subsequent behaviour. In the present study, the effects of two different TGFbeta and FGF treatment regimes were compared in rat lens epithelial explants with either low or high initial cell coverage. Explants treated with 50 pg ml(-1) TGFbeta2 and <em>20</em> ng ml(-1) FGF-2 sequentially (day 0, day 1) or simultaneously (day 0), then cultured for up to 30 days with FGF, were assessed by light microscopy and immunolocalisation of markers for transdifferentiation (alpha-smooth muscle actin (alphaSMA) and type I collagen) or lens epithelial phenotype (Pax6) and fibre differentiation (beta-crystallin). By day 4, most cells had lost Pax6 reactivity, alphaSMA reactivity was evident, and there were differences between <em>growth</em> <em>factor</em> treatment groups, low and high initial cell coverage explants, and peripheral and central regions of explants. On day 30 of culture, all explants were well populated with cells, irrespective of treatment and initial cell coverage, and exhibited diverse PCO-like morphological changes, with expression of transdifferentiation markers and beta-crystallin in virtually all cells. Such overall resilience to variations in conditions may contribute to the insidious nature of PCO, while <em>factors</em> related to observed early differences between groups may contribute to PCO pleiomorphism.

Bovine corneal endothelial (BCE) cells seeded and grown on plastic dishes were labeled with 35S-sulfate or 3H-glucosamine for 48 h at various phases of <em>growth</em> of the cultures. Newly synthesized proteoglycans were isolated from the culture medium and from the extracellular matrix (ECM) produced by the BCE cells, and the glycosaminoglycan (GAG) component of the proteoglycans was analyzed. Cells actively proliferating on plastic surfaces secreted an ECM that contained heparan sulfate as the major 35S-labeled GAG (86%) and dermatan sulfate as a minor component (13%). Upon reaching confluence, the BCE cells incorporated 35S-labeled chondroitin sulfate (<em>20</em>%), as well as heparan sulfate (66%) and dermatan sulfate (14%), into the EC. Seven-day postconfluent cells incorporated newly synthesized heparan sulfate and dermatan sulfate into the matrix in approximately equal proportions. Dermatan sulfate was the main 35S-labeled GAG (60-65%) in the medium of both confluent and postconfluent cultures. 35S-Labeled chondroitin sulfate (<em>20</em>-25%) and heparan sulfate (15%) were also secreted into the culture medium. The type of GAG incorporated into newly synthesized ECM was affected when BCE cells were seeded onto ECM-coated dishes instead of plastic. BCE cells actively proliferating on ECM-coated dishes incorporated newly synthesized heparan sulfate and dermatan sulfate into the ECM in a ratio that was very similar to the ratio of these GAGs in the underlying ECM. Addition of mitogens such as <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) to the culture medium altered the type of GAG synthesized and incorporated into the ECM by BCE cells seeded onto ECM-coated dishes if the cells were actively <em>growing</em>, but had no effect on postconfluent cultures.

Rat resident peritoneal macrophages in primary culture were found to elaborate a mitogenic <em>factor</em> (or <em>factors</em>) for rat osteoblast-like cells and chondrocytes but not for skin <em>fibroblasts</em>. <em>Growth</em>-promoting activity appeared in the incubation medium within the first <em>20</em> hr of macrophage culture and was released in amounts that paralleled the number of macrophages per culture. After their proliferative response, as judged by increases in DNA synthesis and cell number, the osteoblast-like cells became enriched in alkaline phosphatase, an index of osteoblast specialization. The macrophage-derived activity was nondialyzable and heat-stable, and it was eliminated by exposure to trypsin. Inhibition of prostaglandin cyclooxygenase failed to modify its generation. Partial purification (Amicon filter concentration, gel filtration) disclosed principal peaks of activity corresponding to Mr of 43,000 and 10,000. The crude conditioned medium and the Mr 43,000-peak, but not the low-molecular-weight peak, exhibited interleukin 1 activity, as judged by the ability to stimulate the proliferation of mouse thymic lymphocytes. The macrophage-derived <em>growth</em> <em>factor</em> described herein may participate in bone remodeling and repair and in primary bone and cartilage <em>growth</em>.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was shown to enhance rat stromal bone marrow cells in culture to produce mineralized bone-like tissue in response to dexamethasone (Dex) treatment (Pitaru et al., J Bone Miner Res 8:919; 1993). The purpose of this study was to explore the effect of bFGF on Dex-treated human stromal bone marrow cells (hSBMC) in culture. Human SBMC from 6 patients were cultured for 14 days (P0) and then subcultured and grown for 28 days in the presence of Dex (10(-8) mol/L). The effect of bFGF on cell proliferation at P0 and protein content, DNA content, alkaline phosphatase activity (ALP), osteocalcin secretion, and formation of mineralized bone-like tissue (MBT) at P1 was analyzed. bFGF treatment resulted in a 2.4-fold increase in cell number at P0 and a concentration-dependent increase in [3H]-thymidine incorporation at P1, reaching a maximum increase of 3.7-fold at a concentration of 0.3 ng/mL. Furthermore, bFGF significantly increased both DNA content (two- to threefold), protein content (five- to sixfold), and the amount of MBT (up to <em>20</em>-fold) at P1 cultures. Morphological evaluation of the MBT at the electron microscope level revealed a mineralization process along collagen fibrils similar to the natural process. The osteogenic nature of the bFGF-treated cultures was further shown by their ALP activity, as well as osteocalcin secretion in response to 1,25-dihydroxyvitamin D3. In conclusion, bFGF demonstrated a stimulatory effect on the proliferation of Dex-treated hSBMC-derived osteoprogenitors while maintaining their capacity to fully differentiate and form bone-like tissue in culture.

A fluorescence assay of in vivo endosome fusion was developed and applied to define the kinetics of endosome fusion in baby hamster kidney (BHK) <em>fibroblasts</em>. The assay is based on an approximately 10-fold enhancement of the green fluorescence of BODIPY-avidin upon biotin binding. The BODIPY-avidin fluorescence enhancement occurred in < 25 ms, was pH-independent, and involved a BODIPY-tryptophan interaction. For endocytosis in vivo, BHK <em>fibroblasts</em> were pulse-labeled with BODIPY-avidin together with a red (rhodamine) fluorescent fusion-independent chromophore (TMR). After specified chase times in a nonfluorescent medium, a second cohort of endosomes was pulse-labeled with biotin-conjugated albumin, dextran, or transferrin. Fusion of biotin-containing endosomes with avidin-containing endosomes was quantified by ratio imaging of BODIPY-to-TMR fluorescence in individual endosomes, using imaging methods developed for endosome pH studies. Analysis of BODIPY-to-TMR ratio distributions in avidin-labeled endosomes exposed to zero and maximum biotin indicated>> 90% sensitivity for detection of endosome fusion. In avidin pulse (10 min) -chase-biotin albumin pulse (10 min) studies, both fused and unfused endosomes were identified; the fractions of avidin-labeled endosomes that fused with biotin-labeled endosomes were 0.48, 0.21, 0.16, and 0.07 for 0-, 5-, 10-, and <em>20</em>-min chase times. Fitting of fusion data to a mathematical model of in vivo endosome fusion required the existence of an intermediate fusion compartment. Pulse-chase studies performed with biotin-transferrin to label the early/recycling endosomes indicated that after a 10-min chase, avidin-labeled endosomes reached a compartment that was inaccessible to biotin-transferrin. The assay was also applied to determine whether endosome fusion was influenced by temperature, pH (bafilomycin A1), second messengers (cAMP agonists, phorbol 12-myristate 13-acetate, staurosporine), and <em>growth</em>-related <em>factors</em> (platelet-derived <em>growth</em> <em>factor</em>, genistein). The results establish a sensitive fluorescence assay to quantify the fusion of vesicular compartments in living cells.

A large-scale purification procedure for brain-derived <em>growth</em> <em>factors</em>, without chromatography in 0.1% trifluoracetic acid, has been developed. Two related brain-derived <em>growth</em> <em>factors</em> have been purified to homogeneity from bovine brain using this procedure involving ammonium sulfate fractionation, followed by chromatography on CM-Sephadex C-50, sulfated Sephadex G-50, and heparin-Sepharose 4B. Brain-derived <em>growth</em> <em>factor</em> A (BDGF-A, molecular weight approximately 16,000) and brain-derived <em>growth</em> <em>factor</em> B (BDGF-B, molecular weight approximately 17,000) were eluted from heparin-Sepharose 4B at 1.2 and 1.6 M NaCl, respectively. Both BDGF-A and BDGF-B have a pI value of 5.7, have the same specific mitogenic activity, and react with mouse anti-BDGF-A antiserum. Both <em>growth</em> <em>factors</em> have a broad spectrum of mitogenic activity (vascular and aorta endothelial cells, chondrocytes, osteoblasts, epithelial cells, glial cells, <em>fibroblasts</em>, and smooth muscle cells), with a half-maximum effect at 10-<em>20</em> pM. The binding of BDGF to bovine aorta endothelial cells and Swiss mouse 3T3 cells has been characterized. Binding of 125I-BDGF-A to these cells reached equilibrium in less than 15 min. Scatchard plot analysis of the binding of 125I-BDGF-A to endothelial cells and 3T3 cells showed a single class of high-affinity receptor with Kd values of <em>20</em> +/- 5 and 13 +/- 3 pM and receptor numbers/cell of 7,000 +/- 1,000 and <em>20</em>,000 +/- 3,000, respectively. BDGF-B competed for 125I-BDGF-A binding in the same manner as unlabeled BDGF-A, suggesting that BDGF-A and BDGF-B bind to the same receptor. Basic pituitary <em>fibroblast</em> <em>growth</em> <em>factor</em> appeared to be a weak inhibitor, whereas platelet-derived <em>growth</em> <em>factor</em> and epidermal <em>growth</em> <em>factor</em> had no effect on 125I-BDGF-A binding. Protamine, histone, polylysine, and polyarginine are potent inhibitors of the mitogenic activity of BDGF-A and BDGF-B and of binding of 125I-BDGF-A to responsive cells. The half-time (t1/2) of internalization and degradation of cell surface-bound 125I-BDGF is 3 h.

An inorganic phosphate (P(i))-restricted diet is important for patients with chronic kidney disease and patients on hemodialysis. Phosphate binders are essential for preventing hyperphosphatemia and ectopic calcification. The sodium-dependent P(i) (Na/P(i)) transport system is involved in intestinal P(i) absorption and is regulated by several <em>factors</em>. The type II sodium-dependent P(i) transporter Npt2b is expressed in the brush-border membrane in intestinal epithelial cells and transports P(i). In the present study, we analyzed the phenotype of Npt2b(-/-) and hetero(+/-) mice. Npt2b(-/-) mice died in utero soon after implantation, indicating that Npt2b is essential for early embryonic development. At 4 wk of age, Npt2b(+/-) mice showed hypophosphatemia and low urinary P(i) excretion. Plasma <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 levels were significantly decreased and 1,25(OH)(2)D(3) levels were significantly increased in Npt2b(+/-) mice compared with Npt2b(+/+) mice. Npt2b mRNA levels were reduced to 50% that in Npt2b(+/+) mice. In contrast, renal Npt2a and Npt2c transporter protein levels were significantly increased in Npt2b(+/-) mice. At <em>20</em> wk of age, Npt2b(+/-) mice showed hypophosphaturia and reduced Na/P(i) cotransport activity in the distal intestine. Npt2b(+/+) mice with adenine-induced renal failure had hyperphosphatemia and high plasma creatinine levels. Npt2b(+/-) mice treated with adenine had significantly reduced plasma P(i) levels compared with Npt2b(+/+) mice. Intestinal Npt2b protein and Na(+)/P(i) transport activity levels were significantly lower in Npt2b(+/-) mice than in the Npt2b(+/+) mice. The findings of the present studies suggest that Npt2b is an important target for the prevention of hyperphosphatemia.

Connective tissue <em>growth</em> <em>factor</em> (CTGF) is a 38 Kda cysteine-rich, heparin-binding peptide that has been implicated in several normal and abnormal physiological processes. CTGF has been shown to be induced by transforming <em>growth</em> <em>factor</em>-beta. Previous studies in our pig model of skin wound healing showed a coordinate expression of transforming <em>growth</em> <em>factor</em>-beta and CTGF during the healing process. To better understand the function of CTGF during wound healing, normal porcine <em>fibroblasts</em> were isolated from skin samples from SPF Yorkshire pigs. At fourth passage the cells were cultured in Dulbecco's modified Eagle's medium supplemented with fetal calf serum and at 80% confluence the medium was replaced with supplemented serum-free medium. After a further 24 hours, cells were treated with 0, 10, 25, 50, 100, and 500 ng/ml of 38 Kda or 16-<em>20</em> Kda (C-terminal truncated form) recombinant expressed human CTGF for 24 hours or treated with 100 ng/ml for 0, 12, 24, and 48 hours. Subsequently, CTGF effects on cell DNA synthesis and mRNA levels for a subset of relevant molecules were assessed. The results showed that in cells treated with 38 Kda rhCTGF, mRNA levels for types I and III collagen, fibromodulin, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> were significantly up-regulated, but mRNA levels for HSP47, decorin, biglycan, and versican were not significantly altered. mRNA levels for CTGF were also significantly increased, indicating autoregulation of expression. However, mRNA levels for transforming <em>growth</em> <em>factor</em>-beta, inteleukins 1 and 6, tumor necrosis <em>factor</em>-alpha, and nerve <em>growth</em> <em>factor</em> did not change. Interestingly, mRNA levels for the tissue inhibitors of metalloproteinase-1, -2, -3 and -4 were observed to significantly increase, but in contrast, mRNA levels for matrix metalloproteinases-1, -2, -9 were not significantly altered by exposure of the cells to the 38 Kda form of CTGF. In addition, DNA synthesis was augmented in the presence of 38 Kda rhCTGF. However, the truncated 16-<em>20</em> Kda form of rhCTGF appeared to have none of these effects on porcine <em>fibroblasts</em>. These results indicate that in order to induce changes in porcine <em>fibroblasts</em> a molecule with an intact C-terminal domain is required, and that CTGF regulates porcine <em>fibroblast</em> extracellular matrix molecule, <em>growth</em> <em>factor</em>, and proteinase inhibitor gene expression without apparently affecting matrix metalloproteinase mRNA levels. These findings suggest that CTGF contributes to the anabolic environment during skin wound healing via selective modulation of <em>fibroblast</em> proliferation and changes to gene expression.

Phorbol ester binding to intact GH4C1 cells, a continuous strain of rat pituitary cells, was measured using [3H]phorbol 12,13-dibutyrate. The binding was saturable; Scatchard analysis indicated one class of high-affinity binding sites (Kd, 11 nM) with 6.4 pmol [3H]phorbol 12,13-dibutyrate bound per mg cell protein at saturation. The relative binding affinities for other phorbol esters and analogs were similar to those demonstrated for binding to homogenates of chick embryo <em>fibroblasts</em> and mouse skin, as well as for tumor promotion in vivo. A close correlation was shown to exist between the binding affinities of these derivatives and their potencies for inducing biological responses in GH4C1 cells, such as decreases in binding of epidermal <em>growth</em> <em>factor</em> and thyrotropin-releasing hormone. A distinctive new finding is the down modulation of phorbol ester binding sites on GH4C1 cells by both homologous and heterologous ligands. Prolonged exposure to phorbol esters or thyrotropin-releasing hormone produced a loss of available [3H]phorbol 12,13-dibutyrate binding sites with a maximal decrease to about <em>20</em>% of control after 24 hr of treatment. Scatchard analysis indicated that the decrease in binding was due to a loss of receptors with no change in affinity. The biological significance of phorbol ester receptor down modulation is not yet known; however, it may represent a mechanism for attenuating cellular responsiveness to phorbol esters in their continued presence.

OBJECTIVE

The purpose of this study was to assess the effects of gadolinium (Gd3+), provided as gadolinium chloride, on fibroblast function.

METHODS

Human dermal fibroblasts in monolayer culture and intact skin in organ culture were exposed to the lanthanide metal (1-20 μM).

RESULTS

Increased proliferation was observed, in association with upregulation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1, without an apparent increase in production of type I procollagen. A platelet-derived growth factor (PDGF) receptor-blocking antibody inhibited fibroblast proliferation in response to Gd3+ as did inhibitors of signaling pathways--that is, mitogen-activated protein kinase and phosphatidylinositol-3 kinase pathways--that are activated by PDGF.

CONCLUSIONS

The responses to gadolinium chloride are similar to responses previously seen with chelated Gd3+ in clinically used magnetic resonance imaging contrast agents. Fibroblast responses appear to reflect Gd3+-induced PDGF receptor activation and downstream signaling. Increased dermal fibroblast proliferation in conjunction with effects on matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1 could contribute to the fibroplastic/fibrotic changes seen in the lesional skin of individuals with nephrogenic systemic fibrosis.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a drug synthesized in this laboratory that halts tumor blood flow and induces tumor hemorrhagic necrosis in transplantable murine tumors, is known to induce the synthesis of antiangiogenic cytokines in vitro. We have measured the induction of mRNA for modulators of angiogenesis in vivo and investigated whether DMXAA may also have an additional antiangiogenic action through the production of these cytokines. The genes for IFN-alpha and for interferon-inducible protein 10 (IP-10) were strongly induced in both spleen and Colon 38 tumor tissue after DMXAA treatment, whereas that for IFN-gamma was induced in spleen but not in tumor. Expression of mRNA for IFN-beta and for the p35 or the p40 subunits of interleukin 12 was not observed in either tissue. Splenic IP-10 mRNA induction was not a result of IFN-gamma production induced with DMXAA because spleen tissue from DMXAA-treated mice that lacked functional IFN-gamma receptors expressed similar amounts of IP-10 mRNA as those from wild-type mice. A single i.p. injection of DMXAA (<em>20</em> mg/kg) was sufficient to reduce <em>fibroblast</em> <em>growth</em> <em>factor</em>-induced endothelial cell invasion of Matrigel implants in athymic nude mice by nearly 100%. The inactive analogue 8-methylxanthenone-4-acetic acid did not up-regulate the genes for IP-10 or IFNs and did not inhibit endothelial cell invasion. Antibodies to IP-10 reversed the inhibition of DMXAA of endothelial cell invasion by 58%; antibodies to tumor necrosis <em>factor</em>-alpha, IFN-gamma, and IFN-alpha reversed inhibition by 7%, 5%, and 0%, respectively. The data support the hypothesis that DMXAA, in addition to antivascular effects mediated by tumor necrosis <em>factor</em>-alpha, may have an antiangiogenic effect mediated largely by the induction of IP-10.

BACKGROUND

Luminal, estrogen receptor-positive breast cancers represent more than 70% of cases. Despite initial good prognoses one third of Luminal cancers eventually recur locally or at distant sites and exhibit hormone resistance. Here we demonstrate that factors elaborated by malignant stromal cells can induce Luminal tumor cells proliferation and promote angiogenesis and hormone independence. We recently isolated a malignant mouse mammary gland stromal cell line named BJ3Z that increases proliferation and angiogenesis in estrogen-free xenografted Luminal MCF-7 breast cancer cells.

METHODS

BJ3Z and Normal mouse mammary Fibroblasts (NMFs) were expression profiled using microarray assays. Messenger RNA levels were confirmed by RT-PCR and by immunohistochemistry (IHC). Breast cancer MCF-7, BT-474, BT-20 and MDA-MB-231cell lines and stromal BJ3Z and NMFs were grown for in vitro assays: breast cancer cell lines were treated with stromal cells conditioned media, for three-dimensional (3D) mono and co-cultures in Matrigel, proliferation was measured by Bromo-deoxyuridine (BrdU) incorporation using IHC. Tubule formation in vitro, a proxy for angiogenesis, was assessed using 3D cultured Human Umbilical cord Vascular Endothelial Cells (HUVEC).

RESULTS

We show that under estrogen-free conditions, BJ3Z cells but not NMFs increase proliferation of co-cultured Luminal but not basal-like human breast cancer cells in 2D or as 3D Matrigel colonies. Gene expression profiling, RT-PCR analysis and IHC of colony-derived BJ3Z cells and NMFs shows that Platelet Derived Growth Factor ligands (PDGF-A and -B) are elaborated by BJ3Z cells but not NMFs; while PDGF receptors are present on NMFs but not BJ3Z cells. As a result, in colony co-culture assays, BJ3Z cells but not NMFs increase MCF-7 cell proliferation. This can be mimicked by direct addition of PDGF-BB, and blocked by the PDGF receptor inhibitor Imatinib Mesylate. Both normal and malignant stromal cells enhance angiogenesis in an in vitro model. This effect is also due to PDGF and is suppressed by Imatinib.

CONCLUSIONS

We provide evidence that Luminal breast cancer cells can be targeted by the PDGF signaling pathway leading to estrogen-independent proliferation and angiogenesis. We speculate that stroma-directed therapies, including anti-PDGFR agents like Imatinib, may be useful in combination with other therapies for treatment of luminal cancers.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has been shown to be involved in epiretinal membrane formation in proliferative vitreoretinal disorders. However, up to now, little knowledge exists; as to the actual cellular source of this potent mitogen. We examined <em>20</em> epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) (n = 12) and proliferative vitreoretinopathy (PVR) (n = 8) for the presence of bFGF peptide, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-1 (FGFR-1) and bFGF messenger ribonucleic acid (mRNA). Using a specific antibody, we detected bFGF peptide in most (8/10) examined PDR membranes and in all (8/8) PVR membranes. Moreover, we found positive staining for the corresponding receptor. Local production of bFGF in epiretinal membranes was confirmed by nonisotopic in situ hybridisation for bFGF mRNA in some (4/7) examined PDR membranes and some (3/4) examined PVR membranes. All membranes which contained bFGF mRNA were also positive for bFGF peptide. In conclusion, bFGF is produced and stored in epiretinal membranes. Together with the corresponding receptor, bFGF may play a role in the auto- and paracrine control of the proliferative processes at the vitreoretinal interface.

<em>Fibroblasts</em> from ovine skin, and from the perirenal and subcutaneous adipose tissues of sheep were grown at clonal densities in medium MCDB <em>20</em>2 supplemented with 1 microgram/ml bovine insulin, 1 microM dexamethasone, 100 ng/ml <em>fibroblast</em> <em>growth</em> <em>factor</em> and <em>20</em> micrograms/ml of the lipid preparation described by Bettger, W. J., Boyce, S. T., Walthall, B. J. and Ham, R. G. [(1981) Proc. Natl Acad. Sci, USA, 78, 5588-5592]. When maintained as a confluent monolayer in this medium, the <em>fibroblasts</em> from the adipose tissues spontaneously underwent an adipose differentiation. This was accelerated by substituting medium F12 for medium MCDB <em>20</em>2, and by raising the CO2 tension from 2% to 7.5% in air over the cultures. The differentiation was inhibited by deleting FGF from the <em>growth</em> medium, or by coating the culture surface with fibronectin or poly-D-lysine. Differentiation also failed to occur when the defined supplements were replaced with fetal bovine serum. The synthesis of triacylglycerol by the cells, as seen by the increased specific activity of [14C]acetate incorporated into this lipid class, was accompanied by an increase in the specific activity of glycerol-3-phosphate dehydrogenase.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) is one of the mitogens that facilitate epithelial proliferation and angiogenesis. We analysed the expression of FGF-2 and type I <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR1) in <em>20</em> selected cases of human pancreatic carcinoma (PC) in connection with proliferation of tumour cells and intratumour endothelial cells (ECs), using immunohistochemistry and in situ hybridization (ISH). By FGF-2 immunostaining, tumour cells were strongly positive in 10 cases (50%). By FGFR1 immunostaining, stromal <em>fibroblasts</em> and ECs occasionally showed positive staining. Tumour cells in 12 cases (60%) were strongly positive. Expression of FGF-2 mRNA, as examined by ISH, was detected in 12 cases (60%) of PC, and its distribution pattern was similar to that of FGF-2 immunostaining. We divided these cases into two groups according to the result of FGF-2 immunostaining, and examined the Ki67 labelling indices of tumour cells and ECs between these two groups. These two proliferative indices were significantly higher in FGF-2-positive than in FGF-2-negative cases (P<0.05, P<0.05, respectively). These findings suggest that the expression of FGF-2 in PC is strongly associated with the proliferation of tumour cells and ECs and its increased expression may give tumour a <em>growth</em> advantage.

Induced pluripotent stem cells (iPSCs) can be artificially reprogrammed from somatic cells by overexpression of exogenous transcription <em>factors</em>. The pig has increasingly become an important large animal model for preclinical tests and studies of human diseases; thus, the generation of porcine iPSCs will facilitate research into the efficacy and safety of stem cell therapy. A current major problem facing the generation of porcine iPSCs is the failure to silence exogenous transgenes. We hypothesized that this problem can be resolved by reducing the number of transcriptional <em>factors</em> used for porcine iPSCs induction. Here, we report the successful generation of porcine iPSCs using the porcine <em>factors</em> Oct4 and Klf4 in combination with specific small molecules. In comparison with high oxygen conditions (<em>20</em>%), the efficiency of porcine iPSC generation was higher under low oxygen conditions (5%). Porcine iPSCs exhibited a normal karyotype and morphology, like mouse embryonic stem cells (ESCs), and could proliferate in the absence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and in the presence of human leukemia inhibitory <em>factor</em> (hLIF) and mouse embryonic <em>fibroblast</em> feeder cells. These iPSCs also expressed ESC-like markers (Oct4, Nanog, Klf4, c-Myc, Bmp4, bFgf). Importantly, the porcine iPSCs showed pluripotency, as evidenced by differentiation into three germ layers in vitro following embryoid body formation, as well as by efficiently forming teratomas containing three germ layers in immunodeficient mice. Thus, pluripotent porcine iPSCs can be generated from somatic stem cells by using only two porcine transcription <em>factors</em> in combination with small molecules. These attempts represent the first step toward generating truly pluripotent porcine iPSCs with fewer exogenous genes and less integration.

BACKGROUND

Bronchiolitis obliterans syndrome (BOS), chronic lung allograft rejection, remains an impediment for the function of the transplanted organ. In this study, we defined the role of the microRNA (miRNA) miR-144 in fibroproliferation leading to BOS.

METHODS

Biopsy specimens were obtained from <em>20</em> lung transplant recipients with BOS((+)) and 19 without BOS((-)). Expression of miR-144 and its target, transforming <em>growth</em> <em>factor</em>-β (TGF-β)-induced <em>factor</em> homeobox 1(TGIF1), were analyzed by real-time polymerase chain reaction and Western blot. Overexpression of miR-144 and luciferase reporter genes were performed to elucidate miRNA-target interactions. The function of miR-144 was evaluated by transfecting <em>fibroblasts</em> and determining the response to TGF-β by analyzing Sma- and Mad-related family (Smads), <em>fibroblast</em> <em>growth</em> <em>factor</em>, TGF-β, and vascular endothelial <em>growth</em> <em>factor</em>. Smooth muscle actin-α-positive stress fibers and F-actin filaments in lung <em>fibroblasts</em> were analyzed by immunofluorescence.

RESULTS

Analysis of miR-144 in the biopsy specimens demonstrated 4.1 ± 0.8-fold increases in BOS(+) compared with BOS(-) patients, with a significant reduction in TGIF1 (3.6 ± 1.2-fold), a corepressor of Smads. In vitro transfection confirmed that over-expression of miR-144 results in a reduction in TGIF1 and an increase in SMAD2, SMAD4, fibroblast growth factor-6, TGF-β, and vascular endothelial growth factor. Increasing miR-144 by transfecting, increased smooth muscle actin-α and fibronectin, and knockdown of miR-144 diminished fibrogenesis in MRC-5 fibroblasts.

CONCLUSIONS

miR-144 is a critical regulator of the TGF-β signaling cascade and is over-expressed in lungs with BOS. Therefore, miR-144 is a potential target toward preventing fibrosis leading to BOS after lung transplant.

Thyroid cancer is the most common type of endocrine neoplasia. Differentiated thyroid carcinoma (DTC) represents 94% of all thyroid cancer types. Approximately <em>20</em>% experience local recurrence and 10% distant metastasis. The recurrent DTC often becomes less differentiated, loses the iodine uptake capability and consequently loses the radioactive iodine treatment option. Under these circumstances survivability drops below 10% at 10 years. The treatment options for dedifferentiated thyroid cancers are extremely limited. This category sometimes referred to as poorly differentiated thyroid cancer (PDTC), is characterised by a missing response to radioiodine treatment and a remarkably reduced survivability. Therefore, new drugs have been developed to fill this gap in treatment.

The goal of this work is to review the effects and roles of the multikinase inhibitors sorafenib, sunitinb and lenatinib in thyroid cancer.

The new tyrosine kinase inhibitors (TKIs) aimed to inhibit tumour angiogenesis. Current clinical trials with novel drugs have shown promising results. A phase III trial (DECISION) of sorafenib in radioiodine (RAI)-refractory thyroid cancer showed a median progression-free survival (PFS) of 10.8 months in the sorafenib group, compared to 5.8 months in the placebo group. Sunitinib, another TKI, exhibited significant antitumour effects in patients with advanced DTC. Nevertheless, treatment with TKIs can lead to the development of resistance against these anti-angiogenic treatments, partly due to compensatory mechanisms. Lenvatinib, the recently approved drug for RAI-refractory thyroid cancer, blocks a different receptor than the currently available drugs. Lenvatinib inhibits fibroblast growth factor receptor (FGFR), as well as other receptors. FGFR plays a key role in the development of resistance against anti-angiogenic drugs. In a phase III trial (SELECT) on RAI-refractory DTC, the lenvatinib group showed a PFS of 18.3 months, compared to 3.6 months in the placebo group. This led to the approval of lenvatinib, the first drug capable of reversing anti-angiogenic mechanisms.

The frequently adverse effects seen in TKI treatment require further investigation. A well-adjusted balance between efficacy and adverse effects is desirable. No effects on overall survival were reported. Therefore, further studies are required.

Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix. hMVECs stimulated with <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) or vascular endothelial <em>growth</em> <em>factor</em> (VEGF) together with tumor necrosis <em>factor</em>-alpha (TNF-alpha) formed 2- to 3-fold more tubular structures under hypoxic conditions than in normoxic (<em>20</em>% oxygen) conditions. In both conditions the in-<em>growth</em> of capillary-like tubular structures into fibrin required cell-bound urokinase-type plasminogen activator (uPA) and plasmin activities. The hypoxia-induced increase in tube formation was accompanied by a decrease in uPA accumulation in the conditioned medium. This decrease in uPA level was completely abolished by uPA receptor-blocking antibodies. During hypoxic culturing uPA receptor activity and messenger RNA (mRNA) were indeed increased. This increase and, as a consequence, an increase in plasmin formation contribute to the hypoxia-induced stimulation of tube formation. A possible contribution of VEGF-A to the increased formation under hypoxic conditions is unlikely because there was no increased VEGF-A expression detected under hypoxic conditions, and the hypoxia-induced tube formation by FGF-2 and TNF-alpha was not inhibited by soluble VEGFR-1 (sVEGFR-1), or by antibodies blocking VEGFR-2. Furthermore, although the alpha(v)-integrin subunit was enhanced by hypoxia, blocking antibodies against alpha(v)beta(3)- and alpha(v)beta(5)-integrins had no effect on hypoxia-induced tube formation. Hypoxia increases uPA association and the angiogenic response of human endothelial cells in a fibrin matrix; the increase in the uPA receptor is an important determinant in this process. (Blood. <em>20</em>00;96:2775-2783)

Lung cancer remains a leading cause of death globally, with the most frequent type, nonsmall cell lung cancer (NSCLC), having a 5-year survival rate of less than <em>20</em>%. While platinum-based doublet chemotherapy is currently first-line therapy for advanced disease, it is associated with only modest clinical benefits at the cost of significant toxicities. In an effort to overcome these limitations, recent research has focused on targeted therapies, with recently approved agents targeting the epidermal <em>growth</em> <em>factor</em> receptor and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) signaling pathways. However, these agents (gefitinib, erlotinib, and bevacizumab) provide antitumor activity for only a small proportion of patients, and patients whose tumors respond inevitably develop resistance to treatment. As angiogenesis is a crucial step in tumor <em>growth</em> and metastasis, antiangiogenic treatments might be expected to have antitumor activity. Important targets for the development of novel antiangiogenic therapies include VEGF, <em>fibroblast</em> <em>growth</em> <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>, and their receptors. It is hypothesized that targeting multiple angiogenic pathways may not only improve antitumor activity but also reduce the risk of resistance. Several novel agents, such as BIBF 11<em>20</em>, sorafenib, sunitinib, and cediranib have shown promising preliminary activity and tolerability in Phase II studies, and results of ongoing Phase III randomized studies will be necessary to establish the potential place of these new therapies in the management of individual patients with NSCLC.

This study evaluated gene expression changes in gastrocnemius slow-twitch myosin heavy chain I (MHC I) and fast-twitch (MHC IIa) muscle fibers of collegiate cross-country runners (n = 6, <em>20</em>±1 y, VO₂max = 70±1 ml•kg-1•min-1) during two distinct training phases. In a controlled environment, runners performed identical 8 kilometer runs (30:18±0:30 min:s, 89±1% HRmax) while in heavy training (∼72 km/wk) and following a 3 wk taper. Training volume during the taper leading into peak competition was reduced ∼50% which resulted in improved race times and greater cross-section and improved function of MHC IIa fibers. Single muscle fibers were isolated from pre and 4 hour post run biopsies in heavily trained and tapered states to examine the dynamic acute exercise response of the <em>growth</em>-related genes <em>Fibroblast</em> <em>growth</em> <em>factor</em>-inducible 14 (FN14), Myostatin (MSTN), Heat shock protein 72 (HSP72), Muscle ring-finger protein-1 (MURF1), Myogenic <em>factor</em> 6 (MRF4), and Insulin-like <em>growth</em> <em>factor</em> 1 (IGF1) via qPCR. FN14 increased 4.3-fold in MHC IIa fibers with exercise in the tapered state (P<0.05). MSTN was suppressed with exercise in both fiber types and training states (P<0.05) while MURF1 and HSP72 responded to running in MHC IIa and I fibers, respectively, regardless of training state (P<0.05). Robust induction of FN14 (previously shown to strongly correlate with hypertrophy) and greater overall transcriptional flexibility with exercise in the tapered state provides an initial molecular basis for fast-twitch muscle fiber performance gains previously observed after taper in competitive endurance athletes.

OBJECTIVE

To elucidate molecular aspects of the mechanisms of expansion of chronic subdural haematomas (CSH), we examined the expression of two representative angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in CSH.

METHODS

We quantified VEGF and bFGF in haematoma fluid and serum of 20 patients with CSH using an enzyme-linked immunosorbent assay. Mean concentrations of VEGF in the haematoma fluid (10277 pg/ml) and in serum, (355 pg/ml) were much greater than those of bFGF (haematoma, 3.04 pg/ml; serum, 4.74 pg/ml). Surgical specimens, including dura and the outer membrane of the CSH were analysed by in situ hybridisation to detect VEGF mRNA. Macrophages and vascular endothelial cells in the outer membrane over expressed VEGF mRNA.

CONCLUSIONS

Enhanced production of VEGF by macrophages and vascular endothelial cells in the outer membrane is thought to be pathogenetically important in CSH.

OBJECTIVE

Serum biomarkers may be useful in the evaluation of suspected head and neck squamous cell cancer (HNSCC) and as indicators of treatment success or failure in adjuvant and chemopreventive clinical trials.

OBJECTIVE

To determine serum cytokine and chemokine concentrations altered in patients with HNSCC compared with healthy volunteers to identify potential biomarkers.

METHODS

A retrospective experimental laboratory study at Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport. Serum samples from 50 patients with stages II, III, and IV HNSCC and <em>20</em> healthy volunteers were available for study. Primary tumor sites represented in the patient group included the nasal cavity, oral cavity, oropharynx, hypopharynx, and larynx.

METHODS

Following institutional review approval and written informed consent, blood samples were drawn from patients. No intervention, to include any kind of diagnostic workup or treatment, was provided to patients during the course of this study.

METHODS

The main outcome measures were the quantification of cytokine and chemokine concentrations in serum samples. Luminex multiplex panel technology was used for simultaneous measurement of 18 analytes, including fibroblast growth factor 2, granulocyte-macrophage colony-stimulating factor, growth-related oncogene, interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, inducible protein (IP)-10, soluble CD40 ligand, tumor necrosis factor, and vascular endothelial growth factor.

RESULTS

The serum samples of patients with HNSCC contained lower levels of IFN-γ (mean patient serum level, 6.08 pg/mL, compared with the mean control level, 26.<em>20</em> pg/mL; P = .004), IL-13 (mean patient serum level, 2.85 pg/mL, compared with the control mean level, 7.23 pg/mL; P = .02), and macrophage inflammatory protein-1β (MIP-1β) (mean patient serum level, 14.91 pg/mL, compared with the mean control level, 28.98 pg/mL; P = .004), and elevated levels of IP-10 (mean patient serum level, 359.24 pg/mL, compared with mean control level, 216.40 pg/mL; P = .04). All other markers tested were not significantly different between patients with cancer and controls.

CONCLUSIONS

This pilot study demonstrated a significant decrease in serum IFN-γ, IL-13, and MIP-1β levels and a significant elevation of serum IP-10 concentration in patients with HNSCC, irrespective of primary tumor site. If validated in larger, independent studies, these serum biomarkers may be useful in the diagnosis and treatment of HNSCC. In the future, a defined, multianalyte screening panel could facilitate early diagnosis of HNSCC, allowing for earlier treatment and thereby reducing patient mortality.