BACKGROUND

First full term pregnancy (FFTP) completed at a young age has been linked to low long term breast cancer risk, whereas late FFTP pregnancy age confers high long term risk, compared to nulliparity. Our hypothesis was that proteins linked to breast cancer would be differentially expressed in human milk collected at three time points during lactation based on age at FFTP.

METHODS

We analyzed breast milk from 72 lactating women. Samples were collected within 10 days of the onset of lactation (baseline-BL), two months after lactation started and during breast weaning (W). We measured <em>16</em> proteins (11 kallikreins (KLKs), basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, YKL-40, neutrophil gelatinase-associated lipocalin and transforming <em>growth</em> <em>factor</em> (TGF) β-1 and -2) associated with breast cancer, most known to be secreted into milk.

RESULTS

During lactation there was a significant change in the expression of 14 proteins in women < 26 years old and 9 proteins in women>> = 26 at FFTP. The most significant (p < .001) changes from BL to W in women divided by FFTP age (< 26 vs.>> = 26) were in KLK3,6, 8, and TGFβ2 in women < 26; and KLK6, 8, and TGFβ2 in women>> = 26. There was a significant increase (p = .022) in KLK8 expression from BL to W depending on FFTP age. Examination of DNA methylation in the promoter region of KLK6 revealed high levels of methylation that did not explain the observed changes in protein levels. On the other hand, KLK6 and TGFβ1 expression were significantly associated (r2 = .43, p = .0050).

CONCLUSIONS

The expression profile of milk proteins linked to breast cancer is influenced by age at FFTP. These proteins may play a role in future cancer risk.

In heart disease, transforming <em>growth</em> <em>factor</em>-β1 (TGF-β1) converts <em>fibroblasts</em> into myo<em>fibroblasts</em>, which synthesize and secrete fibrillar type I and III collagens. The purpose of the present study was to investigate how hydrogen sulfide (H2S) suppresses TGF-β1-induced differentiation of human cardiac <em>fibroblasts</em> to myo<em>fibroblasts</em>. Human cardiac <em>fibroblasts</em> were serum-starved in <em>fibroblast</em> medium for <em>16</em> h before exposure to TGF-β1 (10 ng mL(-1)) for 24 h with or without sodium hydrosulfide (NaHS, 100 µmol L(-1), 30 min pretreatment) treatment. NaHS, an exogenous H2S donor, potently inhibited the proliferation and migration of TGF-β1-induced human cardiac <em>fibroblasts</em> and regulated their cell cycle progression. Furthermore, NaHS treatment led to suppression of <em>fibroblast</em> differentiation into myo<em>fibroblasts</em>, and reduced the levels of collagen, TGF-β1, and activated Smad3 in TGF-β1-induced human cardiac <em>fibroblasts</em> in vitro. We therefore conclude that H2S suppresses TGF-β1-stimulated conversion of <em>fibroblasts</em> to myo<em>fibroblasts</em> by inhibiting the TGF-β1/Smad3 signaling pathway, as well as by inhibiting the proliferation, migration, and cell cycle progression of human cardiac myo<em>fibroblasts</em>. These effects of H2S may play significant roles in cardiac remodeling associated with heart failure.

BACKGROUND

Fibroblast growth factors (FGFs) are multifunctional proteins that play important roles in cell communication, proliferation and differentiation. However, many aspects of their activities are not well defined. LET-756, one of the two C. elegans FGFs, is expressed throughout development and is essential for worm development. It is both expressed in the nucleus and secreted.

RESULTS

To identify nuclear factors associated with LET-756, we used three approaches. First, we screened a two-hybrid cDNA library derived from mixed stages worms and from a normalized library, using LET-756 as bait. This direct approach allowed the identification of several binding partners that play various roles in the nucleus/nucleolus, such as PAL-1, a transcription regulator, or RPS-16, a component of the small ribosomal subunit. The interactions were validated by co-immunoprecipitation and determination of their site of occurrence in mammalian cells. Second, because patterns of protein interactions may be conserved throughout species, we searched for orthologs of known mammalian interactors and measured binary interaction with these predicted candidates. We found KIN-3 and KIN-10, the orthologs of CK2alpha and CK2beta, as new partners of LET-756. Third, following the assumption that recognition motifs mediating protein interaction may be conserved between species, we screened a two-hybrid cDNA human library using LET-756 as bait. Among the few FGF partners detected was 14-3-3beta. In support of this interaction we showed that the two 14-3-3beta orthologous proteins, FTT-1 and FTT-2/PAR-5, interacted with LET-756.

CONCLUSIONS

We have conducted the first extensive search for LET-756 interactors using a multi-directional approach and established the first interaction map of LET-756/FGF with other FGF binding proteins from other species. The interactors identified play various roles in developmental process or basic biochemical events such as ribosome biogenesis.

Purinergic signaling mediates many cellular processes, including embryonic development and regulation of endocrine signaling. The ADP P2Y13 receptor is known to regulate bone and stem cells activities, although relatively little is known about its role in bone development. In this study we demonstrate, using contemporary techniques, that deletion of the P2Y13 receptor results in an age-dependent skeletal phenotype that is governed by changes in phosphate metabolism and hormone levels. Neonatal and postnatal (2 wk) P2Y13 receptor-knockout (KO) mice were indistinguishable from their wild-type (WT) littermate controls. A clear bone phenotype was observed in young (4-wk-old) KO mice compared WT controls, with 14% more trabecular bone, 35% more osteoblasts, 73% fewer osteoclasts, and a 17% thicker <em>growth</em> plate. Mature (>10 wk of age) KO mice showed the opposite bone phenotype, with 14% less trabecular bone, 22% fewer osteoblasts, and 10% thinner <em>growth</em> plate. This age-dependent phenotype correlated with serum <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF-23) and phosphorus levels that were 65 and <em>16</em>% higher, respectively, in young KO mice but remained unchanged in mature mice. These findings provide novel insights for the role of the P2Y13 receptor in skeletal development via coordination with hormonal regulators of phosphate homeostasis.

Opioid peptides are known to play a role in the function of the mammalian cardiovascular system in the newborn. To learn about mechanisms underlying the morphogenesis of the developing vasculature and the principles contributing to the organization and maintenance of adult blood vessels, we have investigated the expression of the opioid <em>growth</em> <em>factor</em> (OGF), [Met5]-enkephalin, and its receptor, zeta. Moreover, gene expression for preproenkephalin mRNA, which encodes OGF, was studied to determine the source(s) of this inhibitory <em>growth</em> <em>factor</em>. By using immunocytochemistry, both OGF and the zeta opioid receptor were detected at embryonic day (E) <em>16</em> in the mesenchymal cells of the aortic wall. Staining appeared to be abundant in endothelial cells, smooth muscle cells, and <em>fibroblasts</em> at E20 and in the neonate. Immunoreactivity was noted to decrease progressively from day 5 to 10, but by weaning (day 21) and continuing into adulthood intense staining for both the peptide and receptor were observed. Preproenkephalin mRNA was detected throughout the aortic wall at E<em>16</em>, and the number of silver grains increased up today. Message was progressively reduced at days 5, 10, and 21, but signal in the adult aorta was comparable to that observed at day 5. These results indicate that components related to an endogenous opioid system regulating <em>growth</em> are present in the embryo, display a distinct spatial and temporal pattern of ontogeny, and persist into adulthood. In addition, these data indicate that OGF is an autocrine produced <em>growth</em> <em>factor</em> that is related to the emergence of vascular architecture and the maintenance of homeostasis in blood vessels.

BACKGROUND

Wound-healing disorders frequently present a clinical problem in patients with squamous epithelial carcinomas of the head and neck region after surgical interventions such as grafts of free flaps in preirradiated graft bed tissues. Here, inflammatory changes and the expression of cytokines can lead to delayed healing and to the induction of fibrosis. The isoforms of the transforming growth factor beta (TGF-beta(1-3)) play a central role in this process. While it was possible to experimentally show a fibrosis-inducing activity for TGF-beta(1) and TGF-beta(2), a fibrosis-reducing and radioprotective effect has been described for TGF-beta(3) on epithelial cells and fibroblasts. The influence of irradiation and tissue grafting on the TGF-beta(3) expression, however, remains uncertain. The objective of the in vivo study was therefore to analyze the expression profile of TGF-beta(3) in the graft bed and in the transition area between the graft and the graft bed after irradiation and/or after surgery.

METHODS

A total of 48 Wistar rats (male, weight 300-500 g) were used in the study. A free myocutaneous gracilis flap was transplanted in 30 rats: group 1 (n = 18 rats) no transplantation, only irradiation (3 x 10 Gy); group 2 (n = 14 rats) transplantation without preoperative irradiation; group 3 (n = 16 rats) transplantation following preoperative irradiation (3 x 10 Gy). The interval between radiotherapy and grafting was 4 weeks. Tissue samples were taken perioperatively and postoperatively after 3, 4, 7, 11, 14, 28, and 30 days from the transition area between the graft and the graft bed and from the graft bed itself. The TGF-beta(3) expression was analyzed immunohistochemically (labeling index) both qualitatively and quantitatively and checked for statistical differences between the groups (p < 0.05).

RESULTS

The success rate for graft healing was 75% in the group irradiated with 30 Gy, and 86% in the nonirradiated group. The rats that were irradiated but not operated on showed significantly (p = 0.04) reduced TGF-beta(3) expression in the graft bed immediately after the end of the irradiation. Whereas only minor differences in TGF-beta(3) expression were observed postoperatively in the graft bed and in the transition area between the graft and the graft bed in the group where a graft was carried out without preirradiation, the group that was preirradiated with subsequent grafting showed a significantly reduced expression in the bed (p = 0.018).

CONCLUSIONS

After irradiation, it was possible to measure reduced TGF-beta(3) expression in the irradiated graft bed. The exogenous application of TGF-beta(3) could therefore present a new therapeutic approach for improving wound healing through radioprotection of nontransformed epithelial cells and fibroblasts after preoperative radiotherapy and surgery.

The cellular distribution of insulin-like <em>growth</em> <em>factors</em> I and II (IGF-I and -II) was examined in bovine corpora lutea at different stages of the luteal phase using specific antibodies and the avidin-biotin immunoperoxidase technique. At the cellular level, intense immunostaining for IGF-I was exclusively observed in large and small luteal cells and in a limited number of endothelial cells. Positive IGF-I immunoreactivity in luteal cells was thereby distributed in a distinct topographical, lobule-specific manner. Immunoreactivity in central areas of luteal lobules was most abundant in large luteal cells, whereas in peripheral zones significantly (P < 0.05) more small luteal cells exhibited IGF-I immunoreactivity. This distribution pattern was evident from day 4 of the cycle onwards and occurred at all the stages investigated. The percentage of positive small (SLC) and large (LLC) luteal cells revealed by semiquantitative analysis depended on the stage of the cycle as follows: days 4-7: 34% LLC versus 21.3% SLC in central areas and 25.1% LLC versus 32.7% SLC in peripheral zones; days 8-12: 42.9% LLC versus 19.9% SLC in central areas and 23.5% LLC versus 35.2% SLC in peripheral zones; days 13-<em>16</em>: 47.7% LLC versus 19.4% SLC in central areas and 19.2% LLC versus 41.4% SLC in peripheral zones. In contrast to IGF-I, no expression of IGF-II immunoreactivity was seen in large or small luteal cells. Positive immunoreactivity was restricted to the perivascular <em>fibroblasts</em> of large blood vessels and to the pericytes of capillaries.(ABSTRACT TRUNCATED AT 250 WORDS)

Keratoacanthomas are rapidly <em>growing</em> hyperproliferative skin tumors that may clinically or histologically be difficult to distinguish from well-differentiated squamous cell cancers (SCCs). UV light, trauma, and immune suppression represent their etiological <em>factors</em>. As matrix metalloproteinases (MMPs) are implicated at all stages of tumorigenesis, we investigated the expression profile of several cancer-related MMPs to find markers that would differentiate keratoacanthomas from SCCs and shed light to the pathobiology of keratoacanthoma. Samples from 31 keratoacanthomas and 15 grade I SCCs were studied using immunohistochemistry for MMP-2, -7, -8, -9, -10, -13, and -19 and p<em>16</em> and laminin-5gamma2 chain. In situ hybridization for MMP-7, -10, and -13 was performed in a subset of tumors. Keratinocytes with atypia, presence of neovascularization, and composition of the inflammatory infiltrate were graded from hematoxylin-eosin stainings. MMP-7 was present in the epithelium of 4/31 keratoacanthomas and 9/15 SCCs, MMP-8 in 3/30 keratoacanthomas and 0/15 SCCs, but MMP-13 in <em>16</em>/31 keratoacanthomas and 10/15 SCCs, and MMP-10 in 28/31 keratoacanthomas and all cancers. MMP-9 was detected in the epithelium in 5/31 keratoacanthomas and 8/15 SCCs, whereas MMP-2 was only present in <em>fibroblasts</em> in both tumors. MMP-19 was upregulated in proliferating epithelium of keratoacanthomas as was p<em>16</em>. Cytoplasmic laminin-5gamma2 was particularly abundant in keratinocytes at the pushing border of MMP-13-positive keratoacanthomas. We conclude that although some MMPs (MMP-10 and -13) are abundantly expressed in keratoacanthomas, the presence of MMP-7 and -9 in their epithelial pushing border is rare and should raise suspicion of SCC. Further, the loss of MMP-19 and p<em>16</em> could aid in making the differential diagnosis between well-differentiated SCC and keratoacanthoma. Frequent expression of the transformation-specific MMP-13 in keratoacanthomas suggests that they are not benign tumors but incomplete SCCs.

Acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF-1 and FGF-2) are mitogenic polypeptides that may play a role in autocrine and paracrine <em>growth</em> control of malignant tumours. We have examined the expression of FGF-1 and FGF-2 in a series of 41 colorectal tumours (24 adenomas, 17 adenocarcinomas) and 50 gastric adenocarcinomas (23 intestinal, 27 diffuse), using immunohistochemistry. Whereas the FGF-1 distribution was cytoplasmic, FGF-2 was restricted to the nuclei of the epithelial cells. FGF-1 immunoreactivity was detected in all samples (100 per cent), whereas FGF-2 immunoreactivity was seen in 17 adenomas (71 per cent), 13 colorectal carcinomas (76 per cent), and 29 gastric carcinomas (58 per cent). Compared with the normal mucosa, FGF-1 was overexpressed in 42 per cent of colorectal adenomas, 76 per cent of colorectal cancers, and 54 per cent of gastric cancers. Conversely, FGF-2 expression was reduced in <em>16</em> (66 per cent), 8 (47 per cent), and 40 (80 per cent) adenomas and colorectal and gastric samples, respectively. We found a significant correlation only between reduced FGF-2 and gastric tumour grade. These data indicate that FGF-1 overexpression occurs in a large proportion of human colorectal and gastric cancers. This may play a role in the progression of these tumours. The topographic variation in FGF-2 expression between normal (nuclear) and tumour (cytoplasmic) cells implies a corresponding functional change that may in turn facilitate tumour <em>growth</em>.

After clinical heart transplantation, ischemia, acute rejection, and repair mechanisms can trigger the up-regulation of cytokines. To investigate the cytokine profile early after transplantation, we monitored messenger RNA (mRNA) expression levels of tumor necrosis <em>factor</em>-alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1), transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), platelet-derived <em>growth</em> <em>factor</em>-A (PDGF-A), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) by reverse transcriptase-polymerase chain reaction (RT-PCR) in serial endomyocardial biopsies ( n=123) from <em>16</em> cardiac allograft recipients during the first 3 post-operative months. In the first month, mRNA expression levels of MCP-1, TNF-alpha, TGF-beta, and bFGF were significantly higher than in the period thereafter (acute rejection episodes excluded). Acute rejection (International Society for Heart and Lung Transplantation (ISHLT) rejection grade >2) was strongly associated with the level of TNF-alpha mRNA. After acute rejection episodes, rising mRNA expression levels of PDGF-A and bFGF were found. The association between TNF-alpha mRNA and acute rejection reflects the importance of this cytokine in allogeneic responses. Elevated <em>growth</em> <em>factor</em> expression levels indicate repair responses after tissue damage due to either the transplantation procedure (surgery, ischemia, reperfusion) or acute allograft rejection.

Previously, we reported a deficient cloning capacity of the bone marrow (BM) mesenchymal stem cells to give colony-forming unit <em>fibroblast</em> (CFU-F) and an inefficient confluence capacity of BM stromal cells in advanced untreated lung cancer patients (LCP) and breast cancer patients (BCP). Moreover, a decreased level of bFGF at day 7 in the conditioned media from BM CFU-F cultures was found in both cancer groups when compared to the normal range. The current study was specially undertaken, to evaluate the percentage of subconfluent <em>fibroblasts</em> expressing receptors (R) of interleukin-1 (IL-1), platelet-derived <em>growth</em> <em>factor</em> (PDGF), <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), transforming <em>growth</em> <em>factor</em> (TGF-beta), epidermal <em>growth</em> <em>factor</em> (EGF), and the proteins c-Fos and c-Myc in BM primary cultures from untreated LCP and BCP. An immunocytochemical study on subconfluent BM <em>fibroblast</em> cultures from 13 healthy patients, <em>16</em> LCP, and 8 BCP was performed, using as primary antibodies, anti-type I of IL-1 R (IL-1R-1), anti-alpha, beta chains of PDGF R (PDGFR-alpha, PDGFR-beta), anti-type I of FGF R (FGFR-I), anti-type I, II, and III of TGF-beta R (TGF-betaR-I, TGF- betaR-II, and TGF-betaR-III), anti-EGF R, anti-c-Fos, and anti-c-Myc. A diminished percentage of subconfluent <em>fibroblasts</em> expressing PDGFR-alpha, TGFbetaR-I, II, III, EGFR, and FGFR-I was found in LCP and BCP compared to healthy patients. A diminished percentage of subconfluent <em>fibroblasts</em> expressing c-Fos and c-Myc was found in patients when compared to healthy patients. The alterations we describe could help to explain the deficiency regarding the proliferative and confluence capacity of BM stroma cells in cancer patients.

The conformational requirements of suramin for its binding to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and platelet-derived <em>growth</em> <em>factor</em> (PDGF) were examined by molecular modeling and docking simulations using the conformational features of suramin determined by the present proton nuclear magnetic resonance (1H-NMR) studies and the crystal structures of <em>growth</em> <em>factors</em> reported previously. The assignment of resonances of suramin to individual protons was accomplished by the combined analysis of the coupling constants, two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY). The NOESY data obtained for suramin were utilized in a conformational search algorithm with constraints to generate a family of conformers which were further refined by restrained energy minimization. A family of nine conformers generated by restrained modeling falls primarily into one of two categories, either the conformer's two naphthyl rings are far apart, approximately 28-30 A, from one another or the conformer's two naphthyl rings are relatively close, approximately <em>16</em>-18 A. The NMR data provide evidence for the presence of more than one conformer in solution. The modeling and docking simulation studies suggest that suramin binds efficiently to bFGF and PDGF by an induced-fit mechanism, wherein suramin complements bFGF or PDGF by adjusting its conformational freedom around the two pairs of single bonds that link the middle phenyl rings to the secondary amide backbone. The interaction of suramin with bFGF or PDGF primarily involves ion-pair, hydrophobic and hydrogen bonding interactions, in addition to van der Waals' contacts. The results indicate that suramin not only sterically blocks the receptor binding loop of the <em>growth</em> <em>factors</em>, but also competes for the binding sites of agonists such as heparin. The results suggest that suramin's propensity to bind to several polypeptides of varying size and structure is due to its conformational flexibility. Collectively, the data emphasize that conformationally constrained suramin analogs that selectively and competitively target angiogenic <em>growth</em> <em>factors</em> could be designed to reduce non-specific binding and, accordingly, toxicity.

Olfaction in rodents provides an excellent modality for the study of cellular mechanisms of information processing and storage, since a single occurrence of precisely timed stimuli has high survival value. We have followed up preliminary evidence of cytokine and proteinase involvement in normal (as opposed to pathologically-induced) brain plasticity by surveying for the presence of these <em>factors</em> in the ol<em>factor</em>y circuitry of the rat. Genes for 25-30 common cytokines and their receptors, and over 30 cell matrix and adhesion molecules were found to be expressed across the ol<em>factor</em>y bulb, insular cortex, amygdala, and dorsal hippocampus. We then measured by real-time PCR the transcriptional expression of seven of these genes following a one-time exposure to the novel odor of blueberry bars or cornnuts, in contrast to presentation of the familiar odor of lab chow. In the amygdala significant up-regulation of interleukin-1 receptor 1 (IL1r1), interleukin-4 receptor (IL4r), <em>fibroblast</em> <em>growth</em> <em>factor</em> 13 (FGF13), and cathepsin-H (CtsH) was observed in males in response to the odor of cornnuts only. Changes were less consistent and widespread in the hippocampus, but were again sex specific for three genes: cathepsin-L (CtsL), matrix metalloproteinase-14 (MMP-14) and MMP-<em>16</em>. Our results show that transcription for several specific cytokines, <em>growth</em> <em>factors</em>, and proteinases responds to a one-time exposure to a novel odor, in a manner that tends to be region- and sex-specific. This suggests considerable variation in the way that ol<em>factor</em>y information is processed at the cellular level in different brain regions and by the two sexes.

Two forms of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> were isolated from bovine brain by a combination of ammonium sulfate precipitation, cation-exchange chromatography, heparin-Sepharose affinity chromatography, and reverse-phase high-performance liquid chromatography. Amino acid analysis, polyacrylamide gel electrophoresis and amino-terminal sequence analysis showed that one form corresponds to a protein with a molecular mass of <em>16</em> kDa and the amino-terminal sequence Phe-Asn-Leu-Pro-Leu-Gly-Asn-Tyr-Lys-Lys-Pro-Lys-Leu-Leu-Tyr- and thus represents the acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> as previously characterized [Böhlen et al. (1985) EMBO J. 5, 1951-1956]. The second mitogen form has a molecular mass of 15.5 kDa. The amino-terminal sequence was established as Asn-Tyr-Lys-Lys-Pro-Lys-Leu-Leu-Tyr-. This evidence indicates that the latter form represents an amino-terminally truncated acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>, lacking the first six amino acid residues. Both forms of the protein are biologically active and equipotent with respect to stimulation of the proliferation in vitro of mesodermal cells such as vascular endothelial and adrenal cortex cells.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>16</em> (FGF-<em>16</em>) has been reported as the sixteenth member of the heparin sulphate proteoglycan binding <em>growth</em> <em>factor</em> family, which includes acidic and basic FGFs (FGF-1 and FGF-2), based on sequence similarity. The sequences of human (h) and rat (r) FGF-<em>16</em> complimentary DNA (cDNA) sequences are known. Rat FGF-<em>16</em> is expressed in brown adipose tissue during embryonic development but also shows some specificity for the postnatal heart. In spite of the importance of other FGF family members in cardiac physiology, there is scant information about FGF-<em>16</em> function. As a first step towards exploiting mouse genetics in this regard, we have used reverse transcriptase-polymerase chain reaction and primers based on the rFGF-<em>16</em> sequence to clone the adult mouse (m) FGF-<em>16</em> cDNA. An mFGF-<em>16</em> cDNA of 624 base pairs was generated. Based on sequence analysis, mFGF-<em>16</em> and hFGF-<em>16</em> share at least 95.2 and 99% nucleotide and amino acid similarity, respectively. In terms of other family members, FGF-<em>16</em> is most closely related to FGF-9. When used as a radiolabeled probe, the mFGF-<em>16</em> cDNA detected a single 1.8 kilobase transcript in adult mouse heart RNA. The mFGF-<em>16</em> cDNA was also used to generate an amino-terminal poly-histidine tagged FGF-<em>16</em> protein in bacteria. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and taking into account the poly-histidine tag, an FGF-<em>16</em> protein of 26.3 kDa was detected. The generation of cardiac mFGF-<em>16</em> cDNA and a purified FGF-<em>16</em> protein preparation are seen as important tools in the further characterization of FGF-<em>16</em> expression and function in the mammalian heart.

Methods for identification and in vitro expansion of ventral mesencephalic dopaminergic precursor cells are of interest in the search for transplantable neurons for cell therapy in Parkinson's disease (PD). We investigated the potential use of <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) and <em>fibroblast</em> <em>growth</em> <em>factor</em> 8 (FGF8) for expansion of such dopaminergic precursor cells, and fetal antigen-1 (FA1), a secreted neuronal protein of unknown function, as a non-invasive dopaminergic marker. Tissue from embryonic day (ED) 12 rat ventral mesencephalon was dissociated mechanically and cultured for 4 days in the presence of FGF2, FGF8, or without mitogens (control). After mitogen withdrawal and addition of 0.5% bovine serum, cells were differentiated for 6 days. Before differentiation, significantly more cells incorporated BrdU in cultures exposed to FGF2 (19-fold; P < 0.001) and FGF8 (3-fold; P < 0.05) compared to controls. After differentiation, biochemical analyses showed significantly more dopamine and FA1 in conditioned medium from both FGF2 and FGF8 expanded cultures than in controls. Correspondingly, numbers of tyrosine hydroxylase (TH)- and FA1-immunoreactive cells had increased <em>16</em>-fold (P < 0.001) and 2.1-fold (P < 0.001), respectively in the FGF2 group and 10-fold (P < 0.001) and 1.8-fold (P < 0.05), respectively in the FGF8 group. In conclusion, the present procedure allows efficient expansion and differentiation of dopaminergic precursor cells and provides novel evidence of FGF8 as a mitogen for these cells. Furthermore, FA1 was identified as a potential supplementary non-invasive marker of cultured dopaminergic neurons.

SH-SY5Y cells differentiate into neuronal-like cells and express marker proteins like <em>growth</em>-associated protein (GAP-43) and neuropeptide tyrosine when treated with a low concentration (<em>16</em> nM) of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of <em>growth</em> <em>factors</em> or serum. Both control and differentiated cells expressed protein kinase C-alpha (PKC-alpha), PKC-epsilon, and PKC-zeta as revealed by Western blot analyses, but the subcellular distribution of the three isoforms was not uniform, indicating specific localized functions of the enzymes. In <em>growth</em> cones prepared from differentiating cells PKC-alpha and PKC-epsilon were enriched. In contrast, PKC-zeta was more evenly distributed within the differentiating cell. Cells treated with a high concentration of TPA (1.6 microM) differentiate poorly and continue to proliferate. In those cells, PKC-alpha and PKC-epsilon were found to be down-regulated while PKC-zeta remained present. Thus, down-regulation of PKC-alpha and PKC-epsilon appears to be incompatible with neuronal differentiation of SH-SY5Y cells. These cells also differentiate when treated with a combination of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and insulin-like <em>growth</em> <em>factor</em> I. <em>Growth</em> cones isolated from such cells are also enriched in PKC-alpha and PKC-epsilon, but not in PKC-zeta. Based on the subcellular distribution of PKC-alpha and epsilon, and that PKC substrates like GAP-43 and pp60c-src are enriched in SH-SY5Y <em>growth</em> cones, a role during neurite <em>growth</em> is suggested.

We describe the derivation and characterization of three novel human embryonic stem (hES) cell lines (YT1, YT2, YT3). One hES line (YT1) was obtained from six discarded blastocysts in a culture medium supplemented with 12 ng/ml basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and two lines (YT2,YT3)were obtained from three discarded blastocysts in the same medium but supplemented with <em>16</em> ng/ml bFGF. These cell lines were derived by partial or whole embryo culture followed by further expansion after manual dissection of the passaged cells. These cells were passaged continuously for more than 6 or 8 months and possessed all of the typical features of pluripotent hES cell lines, such as typical morphological characteristics and the expression of hES-specific markers (TRA-1-60, TRA-1-81, SSEA-4, SSEA-3, alkaline phosphatase, Oct4, Nanog) and pluripotency-related genes (Oct4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex1). The lines maintained normal karyotypes after long-term cultivation. The karyotype of YT1 and YT3 was 46,XX, and that of YT2 was 46, XY. Pluripotency was confirmed by in vitro and in vivo differentiation, and genetic identity was demonstrated by DNA fingerprinting.Our results indicate that higher concentrations of bFGF at the early culture stage support efficient the hES cell derivation.

The stimulation of autocrine and paracrine <em>factors</em> such as basic <em>fibroblast</em>- (bFGF) and platelet-derived (PDGF) <em>growth</em> <em>factors</em> mediates many of the <em>growth</em>-promoting actions of angiotensin II. The aim of this study was to evaluate the effect of chronic AT1-receptor blockade on plasma endothelin-1 (ET-1) and <em>growth</em> <em>factors</em> levels, and on left ventricular mass, in essential hypertension (EH). The study population consisted of <em>16</em> patients with mild-moderate EH, and 25 normotensive controls. In the EH patients under basal conditions, and after 3 and 6 months of chronic therapy with Losartan 50 mg/day, we measured serum levels of ET-1, bFGF and PDGF, and tumor necrosis <em>factor</em> (TNF). At the same time, all patients underwent 24-h ambulatory blood pressure monitoring and an echocardiographic evaluation to measure the thickness of the posterior wall (PWT) of the left ventricle and of the interventricular septum (IVS). The healthy controls underwent the same analyses, under basal conditions, at baseline and after 3 and 6 months of observation. In the EH patients, after 3 months of AT1-receptor blockade bFGF was reduced from 13.6 +/- 0.7 to 10.9 +/- 0.7 pg/mL (P < .004), and both TNF and PDGF were significantly decreased (P < .006 and P < .007, respectively). After 6 months of therapy, ET-1 was significantly diminished in comparison with baseline (6.9 +/- 0.8 v 5.5 +/- 0.1 fmol/mL; P < .05), and the reduction in the levels of <em>growth</em> <em>factors</em> were even more significant than at 3 months of treatment. Both PWT and IVS were significantly changed after 6 months of therapy with losartan after basal evaluation (P < .05, respectively). Systolic and diastolic 24-h blood pressures declined significantly after 3 and 6 months of therapy with losartan (P < .01, respectively). It seems likely that the inhibition of the action of angiotensin II by the specific AT1-receptor blockade, by reducing circulating levels of ET-1 and those of some <em>growth</em> <em>factors</em>, may offer an advantage regarding the effect on hypertensive cardiovascular changes in human hypertension.

Human embryonic stem (hES) cells can self-renew, which enables them to have considerable expansion potential, and are pluripotent. If their differentiation can be controlled, they can offer promise for clinical programs in cell therapies. A novel strategy has been developed to derive early hepatocytic lineage stages from hES cells using four sequential inducing steps lasting <em>16</em> days. First, embryoid bodies (EBs) were generated by <em>growing</em> hES cells in suspension for 2 days; second, EBs were lineage restricted to definitive endoderm with 3 days of treatment with human activin A; third, cells were differentiated further by coculturing for 5 days with human fetal liver stromal cells (hFLSCs) made transgenic to stably release basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF); fourth, treating them for 6 days with soluble signals comprised of hFLSC-derived bFGF, hepatocyte <em>growth</em> <em>factor</em>, oncostatin M, and dexamethasone. Induced cells displayed morphological, immunohistochemical, and biochemical characteristics of hepatocytic committed progenitors and of early lineage stage hepatocytes found in zone 1 of the liver acinus. They expressed alpha-fetoprotein, albumin, cytokeratin 18, glycogen, a fetal P450 isoform, and CYP1B1, and demonstrated indocyanine green uptake and excretion. In conclusion, we have developed a novel method to lineage restrict hES cells into early lineage stages of hepatocytic fates.

BACKGROUND

Vascular endothelial growth factor, a specific endothelial mitogen, plays an important role in myocardial angiogenesis. Previous work has demonstrated increased expression of vascular endothelial growth factor and its receptors in a rat myocardial infarction model, as well as in a pig model of chronic ischemia. The expression of vascular endothelial growth factor and other growth factors after acute myocardial ischemia in patients has not been examined. In this study we examined the expression of vascular endothelial growth factor and its receptors and the responsiveness of human atrial microvessels to vascular endothelial growth factor before and after acute ischemia.

METHODS

Paired specimens of human atrial tissue were harvested before and after atrial devascularization (ligation) in 16 patients undergoing coronary bypass operations.

RESULTS

The messenger RNA (reverse transcriptase-polymerase chain reaction) level of vascular endothelial growth factor and vascular endothelial growth factor receptor 1 were increased by 22.2% +/- 4.2% and 30.7% +/- 7.6%, respectively (P <.05), in the ischemic specimens as compared with the control specimens. Protein expression (Western blotting) of vascular endothelial growth factor and that of vascular endothelial growth factor receptor 1 also were increased significantly by 71.7% +/- 27.8% and 68.2% +/- 27.6%, respectively (P <.05). However, both RNA and protein expressions of another vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2, and fibroblast growth factor and fibroblast growth factor receptor 1 were unchanged. Reactivity of precontracted atrial vessels was examined with video microscopy. Vascular endothelial growth factor-induced (33.9% +/- 2.4% vs 18.3% +/- 2.8% in control and ischemic vessels, respectively; P <.05), fibroblast growth factor-induced (31.6% +/- 3.2% vs 15.8% +/- 4.1%, P <.05), and substance P-induced (84.5% +/- 3.7% vs 54.3% +/- 9.0%, P <.05) microvascular relaxations were decreased in ischemic samples and in the presence of N (G)nitro-L -arginine, whereas responses to sodium nitroprusside were unchanged (90.9% +/- 2.2% vs 91.2% +/- 2.0%).

CONCLUSIONS

This study suggests that acute myocardial ischemia in patients results in increased expression of vascular endothelial growth factor but not fibroblast growth factor and that the functional activity of vascular endothelial growth factor receptors and that of other growth factors may be impaired.

<em>Growth</em> <em>factors</em> have a pivotal role in chondrogenic differentiation of stem cells. The differential effects of known <em>growth</em> <em>factors</em> involved in the maintenance and homeostasis of cartilage tissue have been previously studied in vitro. However, there are few reported researches about the interactional effects of <em>growth</em> <em>factors</em> on chondrogenic differentiation of stem cells. The aim of this study is to examine the combined effects of four key <em>growth</em> <em>factors</em> on chondrogenic differentiation of mesenchymal stem cells (MSCs). Isolated and expanded rabbit bone marrow-derived MSCs underwent chondrogenic differentiation in a micromass cell culture system that used a combination of the following <em>growth</em> <em>factors</em>: transforming <em>growth</em> <em>factor</em> beta 1 (TGF-β1), bone morphogenetic protein 2 (BMP2), parathyroid hormone related protein (PTHrP), and <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) according to a defined program. The chondrogenic differentiation program was analyzed by histochemistry methods, quantitative RT-PCR (qRT-PCR), and measurement of matrix deposition of sulfated glycosaminoglycan (sGAG) and collagen content at days <em>16</em>, 23, and 30. The results showed that the short-term combination of TGF-β1 and BMP-2 increased sGAG and collagen content, Alkaline phosphates (ALP) activity, and type X collagen (COL X) expression. Application of either PTHrP or FGF2 simultaneously decreased TGF-β1/BMP-2 induced hypertrophy and chondrogenic markers (at least for FGF2). However, successive application of PTHrP and FGF2 dramatically maintained the synergistic effects of TGF-β1/BMP-2 on the chondrogenic differentiation potential of MSCs and decreased unwanted hypertrophic markers. This new method can be used effectively in chondrogenic differentiation programs.

OBJECTIVE

We examined the effect of alkali replacement for metabolic acidosis on vascular endothelial function in patients with CKD.

METHODS

We performed a pilot, prospective, open-label 14-week crossover study examining the effect of oral sodium bicarbonate treatment on vascular function in 20 patients with an eGFR of 15-44 ml/min per 1.73 m2 with low serum bicarbonate levels (<em>16</em>-21 mEq/L). Each period was 6 weeks in duration with a 2-week washout period in between. Patients were treated to goal serum bicarbonate of ≥23 mEq/L. The primary end point was change in brachial artery flow-mediated dilation (FMD) between treatment and control conditions. Secondary end points included changes in markers of inflammation, bone turnover, mineral metabolism, and calcification.

RESULTS

Eighteen patients completed the study and were included in the primary efficacy analysis. The mean (SD) age and eGFR were 59 (12) years and 26 (8) ml/min per 1.73 m2, respectively. Serum bicarbonate increased significantly with sodium bicarbonate treatment (+2.7±2.9 mEq/L, P≤0.001), whereas there was no change in bicarbonate levels in the control group. FMD significantly improved after sodium bicarbonate therapy (mean±SD, FMD baseline: 4.1%±4.1%; 6 weeks: 5.2%±2.9%; P=0.04) There was no significant change in FMD in the control group (mean±SD, FMD baseline: 4.6%±3.1%; 6 weeks: 4.1%±3.4%; P=0.20). Compared with control, sodium bicarbonate treatment resulted in a significant increase in FMD (mean, 1.8%; 95% confidence interval, 0.3 to 3.3; P=0.02). There was no significant change in bone markers or serum calcification propensity with treatment. Serum phosphorus and intact fibroblast growth factor 23 increased significantly during treatment.

CONCLUSIONS

Treatment of metabolic acidosis with sodium bicarbonate significantly improved vascular endothelial function in patients with stages 3b and 4 CKD.

Non-alcoholic fatty liver disease (NAFLD), a lipid metabolism disorder characterized by the accumulation of intrahepatic fat, has emerged as a global public health problem. However, its underlying molecular mechanism remains unclear. We previously have found that miR-149 was elevated in NAFLD induced by high-fat diet mice model, whereas decreased by a <em>16</em>-week running programme. Here, we reported that miR-149 was increased in HepG2 cells treated with long-chain fatty acid (FFA). In addition, miR-149 was able to promote lipogenesis in HepG2 cells in the absence of FFA treatment. Moreover, inhibition of miR-149 was capable of inhibiting lipogenesis in HepG2 cells in the presence of FFA treatment. Meanwhile, <em>fibroblast</em> <em>growth</em> <em>factor</em>-21 (FGF-21) was identified as a target gene of miR-149, which was demonstrated by the fact that miR-149 could negatively regulate the protein expression level of FGF-21, and FGF-21 was also responsible for the effect of miR-149 inhibitor in decreasing lipogenesis in HepG2 cells in the presence of FFA treatment. These data implicate that miR-149 might be a novel therapeutic target for NAFLD.