Activating mutations of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-3 (FGFR3) have been described in approximately 75% of low-grade papillary bladder tumors. In muscle-invasive disease, FGFR3 mutations are found in <em>20</em>% of tumors, but overexpression of FGFR3 is observed in about half of cases. Therefore, FGFR3 is a particularly promising target for therapy in bladder cancer. Up to now, most drugs tested for inhibition of FGFR3 have been small molecule, multityrosine kinase inhibitors. More recently, a specific inhibitory monoclonal antibody targeting FGFR3 (R3Mab) has been described and tested preclinically. In this study, we have evaluated mutation and expression status of FGFR3 in 19 urothelial cancer cell lines and a cohort of 170 American patients with bladder cancer. We have shown inhibitory activity of R3Mab on tumor <em>growth</em> and corresponding cell signaling in three different orthotopic xenografts of bladder cancer. Our results provide the preclinical proof of principle necessary to translate FGFR3 inhibition with R3Mab into clinical trials in patients with bladder cancer.

OBJECTIVE

This study objectively characterized the microenvironment of indolent, chronic wounds by developing a method by which minute quantities of cytokines could be extracted from chronic wounds and separately identified.

BACKGROUND

Recombinant DNA technology and the ability to clone compounds such as cytokines allow new management schemes for the treatment of acute and chronic wounds. Before treatment with an exogenous cytokine is started, it would be helpful to know the endogenous level of that cytokine in the wound. Although various methods of extracting cytokines from acute wounds have been reported, no techniques have existed to reliably measure endogenous levels of cytokines in chronic wounds.

METHODS

Porous, inert hydrophilic dextranomer beads were tested for their ability to absorb or adsorb protein and cytokines in vitro with either albumin or albumin laced with various known amounts of cytokines, and then from chronic human pressure ulcers. The Bradford protein assay was used to determine protein levels. Enzyme-linked immunosorbent assay (ELISA) techniques were used to determine levels of platelet-derived growth factor (PDGF)-AB, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and transforming growth factor-Beta (TGF-beta) extracted by the beads.

RESULTS

Between 88% and 98% of known amounts of albumin could be recovered. Similarly, more than 90% of the laced cytokines could be recovered. In 20 grade III/IV pressure ulcers, although protein concentrations were remarkably similar, endogenous levels of cytokine growth factors varied tremendously. Platelet-derived growth factor-AB ranged from 49 to 867 pg/mL; bFGF from 47 to 697 pg/mL; and EGF from nondetectable to 247.5 pg/mL. TGF-B was not detected in 17 of the 20 pressure ulcers.

CONCLUSIONS

This new technique appears useful for measuring endogenous levels of cytokines. Levels of cytokines found in these chronic wounds are much lower than those reported from acute wounds. The marked variation found among the 20 wounds may help to explain the differences reported in recent wound healing trials with exogenous cytokines.

Chronic allograft nephropathy is characterized by chronic inflammation and fibrosis. Because retinoids exhibit anti-proliferative, anti-inflammatory, and anti-fibrotic functions, the effects of low and high doses of 13-cis-retinoic acid (13cRA) were studied in a chronic Fisher344->>Lewis transplantation model. In 13cRA animals, independent of dose (2 or <em>20</em> mg/kg body weight/day) and start (0 or 14 days after transplantation) of 13cRA administration, serum creatinine was significantly lower and chronic rejection damage was dramatically reduced, including subendothelial fibrosis of preglomerular vessels and chronic tubulointerstitial damage. The number of infiltrating mononuclear cells and their proliferative activity were significantly diminished. The mRNA expression of chemokines (MCP-1/CCL2, MIP-1alpha/CCL3, IP-10/CXCL10, RANTES/CCL5) and proteins associated with fibrosis (plasminogen activator inhibitor-1, transforming <em>growth</em> <em>factor</em>-beta1, and collagens I and III) were strikingly lower in treated allografts. In vitro, activated peritoneal macrophages of 13cRA-treated rats showed a pronounced decrease in protein secretion of inflammatory cytokines (eg, tumor necrosis <em>factor</em>-alpha, interleukin-6). The suppression of the proinflammatory chemokine RANTES/CCL5 x 13cRA in <em>fibroblasts</em> could be mapped to a promoter module comprising IRF-1 and nuclear <em>factor</em>-kappaB binding elements, but direct binding of retinoid receptors to promoter elements could be excluded. In summary, 13cRA acted as a potent immunosuppressive and anti-fibrotic agent able to prevent and inhibit progression of chronic allograft nephropathy.

<em>Fibroblast</em> <em>Growth</em> <em>Factors</em> (FGFs) have been implicated in malignant transformation, tumor mitogenesis, angiogenesis and chemoresistance. The aim of this study was to determine which FGFs and FGFRs play functional roles in epithelial ovarian cancer. Restriction enzyme analysis of mRNA revealed that transformation was associated with a switch in FGFR2 and FGFR3, from the IIIc to the IIIb isoform. There was widespread expression of FGFs, including FGF7, in all tissues but, FGF3 and FGF19 were expressed by malignant cell lines and cancer tissue but were not present in normal tissue. Using FGFR-specific shRNAi we demonstrated that reductions in FGFR2 inhibited proliferation of ovarian cancer cell lines in vitro (>50%, p < 0.006) and reduced cisplatin IC(50) (>60%, p < 0.0001). Cell cycle analysis revealed increased cisplatin sensitivity was associated with increased G(2)/M arrest and increased apoptosis. FGFR2 shRNAi reduced <em>growth</em> rates of ovarian tumor xenografts by <em>20</em>% (p < 0.006) and when combined with cisplatin caused a 40% reduction in proliferation rates (p < 0.007). In contrast, RNAi-induced reductions in FGFR1 increased SKOV3 cell numbers, with associated changes in cell cycle but had no effect on ES2 cells. However, the cisplatin IC(50) was reduced (>50%, p < 0.0001) by FGFR1 shRNAi in both cell lines and there was increased apoptosis (46-50%) compared with control cells (35%) (p < 0.004). Together our data suggest that combining FGFR2 inhibitors with platinum-containing cytotoxic agents for the treatment of epithelial ovarian cancer may yield increased antitumor activity. However, data on the inhibition of FGFR1 suggest that broad spectrum FGFR inhibitors may have unexpected effects on proliferation.

The goal of this study was to compare the <em>growth</em> and differentiation potential of fetal and adult rhesus monkey (Macaca mulatta) mesenchymal stem cells (rhMSCs). rhMSCs were obtained from healthy early third-trimester fetal (n = 3) and adult (n = 3) rhesus monkey bone marrow. Fetal rhMSCs were plated at 10, 50, 100, or 1,000 cells/cm(2) in medium containing 10% or <em>20</em>% infant monkey serum (IMS) or fetal bovine serum (FBS). Fetal rhMSCs grown at 1,000 cells/cm(2) in <em>20</em>% FBS showed faster <em>growth</em> rates and differentiation toward adipogenic, chondrogenic, and osteogenic lineages when compared to other culture conditions and to adult cells (p < 0.05). Fetal rhMSC showed higher population doubling times (11.3 +/- 0.5) when compared to adult cells (7.3 +/- 0.8) during the first three passages. Adult rhMSC did not grow beyond the third passage under all culture conditions, including those supplemented with insulin-like <em>growth</em> <em>factor</em> (IGF)-I, IGF-II, platelet-derived <em>growth</em> <em>factor</em> (PDGF), and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2). After the third passage, adult rhMSC cultures were observed with large syncytia and with evidence of apoptosis. Cells obtained from these cultures tested positive for simian foamy virus (SFV) by PCR, RT-PCR, and immunofluorescent assay. Adult rhMSCs cultured with 10 microM tenofovir, an antiviral agent, showed normal <em>growth</em> and differentiation for over <em>20</em> population doublings. These findings suggest that: (1) fetal rhMSCs possess greater self-renewal and differentiation potential when compared to adult cells; and (2) SFV can inhibit proliferation of adult rhMSCs in culture, whereas the addition of tenofovir can successfully suppress SFV replication in vitro and result in resumed <em>growth</em>.

While much has been learned about how endothelial cells transform to mesenchyme during cardiac cushion formation, there remain fundamental questions about the developmental fate of cushions. In the present work, we focus on the <em>growth</em> and development of cushion mesenchyme. We hypothesize that proliferative expansion and distal elongation of cushion mesenchyme mediated by <em>growth</em> <em>factors</em> are the basis of early valve leaflet formation. As a first step to test this hypothesis, we have localized <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-4 protein in cushion mesenchymal cells at the onset of prevalve leaflet formation in chick embryos (Hamburger and Hamilton stage <em>20</em>-25). Ligand distribution was correlated with FGF receptor (FGFR) expression. In situ hybridization data indicated that FGFR3 mRNA was confined to the endocardial rim of the atrioventricular (AV) cushion pads, whereas FGFR2 was expressed exclusively in cushion mesenchymal cells. FGFR1 expression was detected in both endocardium and cushion mesenchyme as well as in myocardium. To determine whether the FGF pathways play regulatory roles in cushion mesenchymal cell proliferation and elongation into prevalvular structure, FGF-4 protein was added to the cushion mesenchymal cells explanted from stage 24-25 chick embryos. A significant increase in proliferative ability was strongly suggested in FGF-4-treated mesenchymal cells as judged by the incorporation of 5'-bromodeoxyuridine (BrdU). To determine whether cushion cells responded similarly in vivo, a replication-defective retrovirus encoding FGF-4 with the reporter, bacterial beta-galactosidase was microinjected into stage 18 chick cardiac cushion mesenchyme along the inner curvature where AV and outflow cushions converge. As compared with vector controls, overexpression of FGF-4 clearly induced expansion of cushion mesenchyme toward the lumen. To further test the proliferative effect of FGF-4 in cardiac cushion expansion in vivo (ovo), FGF-4 protein was microinjected into stage 18 chick inner curvature. An assay for BrdU incorporation indicated a significant increase in proliferative ability in FGF-4 microinjected cardiac cushion mesenchyme as compared with BSA-microinjected controls. Together, these results suggest a role of FGF-4 for cardiac valve leaflet formation through proliferative expansion of cushion mesenchyme.

PTH stimulates bone formation and increases hematopoietic stem cells through mechanisms as yet uncertain. The purpose of this study was to identify mechanisms by which PTH links actions on cells of hematopoietic origin with osteoblast-mediated bone formation. C57B6 mice (10 d) were nonlethally irradiated and then administered PTH for 5-<em>20</em> d. Irradiation reduced bone marrow cellularity with retention of cells lining trabeculae. PTH anabolic activity was greater in irradiated vs. nonirradiated mice, which could not be accounted for by altered osteoblasts directly or osteoclasts but instead via an altered bone marrow microenvironment. Irradiation increased <em>fibroblast</em> <em>growth</em> <em>factor</em> 2, TGFβ, and IL-6 mRNA levels in the bone marrow in vivo. Irradiation decreased B2<em>20</em> cell numbers, whereas the percent of Lin(-)Sca-1(+)c-kit(+) (LSK), CD11b(+), CD68(+), CD41(+), Lin(-)CD29(+)Sca-1(+) cells, and proliferating CD45(-)Nestin(+) cells was increased. Megakaryocyte numbers were reduced with irradiation and located more closely to trabecular surfaces with irradiation and PTH. Bone marrow TGFβ was increased in irradiated PTH-treated mice, and inhibition of TGFβ blocked the PTH augmentation of bone in irradiated mice. In conclusion, irradiation created a permissive environment for anabolic actions of PTH that was TGFβ dependent but osteoclast independent and suggests that a nonosteoclast source of TGFβ drives mesenchymal stem cell recruitment to support PTH anabolic actions.

Oncostatin M (OSM) is a multifunctional cytokine, a member of the interleukin-6/leukemia inhibitory <em>factor</em> (IL-6/LIF) family, that can regulate a number of connective-tissue cell types in vitro including cartilage and synovial tissue-derived <em>fibroblasts</em>, however its role in joint inflammation in vivo is not clear. We have analyzed murine OSM (muOSM) activity in vitro and in vivo in mouse joint tissue, to determine the potential role of this cytokine in local joint inflammation and pathology. The effects of muOSM and other IL-6/LIF cytokines on mouse synovial <em>fibroblast</em> cultures were assessed in vitro and showed induction of monocyte chemotactic protein-1, interleukin-6, and tissue inhibitor metalloproteinase-1, as well as enhancement of colony <em>growth</em> in soft agarose culture. Other IL-6/LIF cytokines including IL-6, LIF, or cardiotrophin-1, did not have such effects when tested at relatively high concentrations (<em>20</em> ng/ml). To assess effects of muOSM in articular joints in vivo, we used recombinant adenovirus expressing muOSM cDNA (AdmuOSM) and injected purified recombinant virus (10(6) to 10(8) pfu) intra-articularly into the knees of various mouse strains. Histological analysis revealed dramatic alterations in the synovium but not in synovium of knees treated with the control virus Ad-dl70 or knees treated with Adm-IL-6 encoding biologically active murine IL-6. AdmuOSM effects were characterized by increases in the synovial cell proliferation, infiltration of mononuclear cells, and increases in extracellular matrix deposition that were evident at day 4, but much more marked at days 7, 14, and 21 after administration. The synovium took on characteristics similar to pannus and appeared to contact and invade cartilage. Collectively, these results provide good evidence that OSM regulates synovial <em>fibroblast</em> function differently than other IL-6-type cytokines, and can induce a proliferative invasive phenotype of synovium in vivo in mice on overexpression. We suggest that OSM may contribute to pathology in arthritis.

Hepatocyte <em>growth</em> <em>factor</em> (HGF), a humoral mediator for regeneration of liver and kidney, possesses multiple biological activities. To investigate target cell specificity and to examine whether multiple actions of HGF are related to properties of the HGF receptor on target cells, we examined the effects of HGF on cell <em>growth</em> and motility and analyzed the HGF receptor in various species of cells. HGF stimulated <em>growth</em> and DNA synthesis of PAM212 (naturally immortalized mouse keratinocytes), Mv1Lu (mink lung epithelia), and A431 (human epidermoid carcinoma) cells, as well as mature hepatocytes, but inhibited those of IM-9 (human B-lymphoblasts). Conversely, HGF had a marked stimulatory effect on cell motility of MDCK (Mardin-Darby canine kidney epithelia) cells, but not on their <em>growth</em>. Also, HGF enhanced the motility of various species of cells, including A431, PAM212, HepG2 (human hepatoma), KB (human epidermoid carcinoma), and J-111 (human monocytes) cells. Scatchard analysis of 125I-HGF binding to hepatocytes indicated that the cells expressed both high- and low-affinity binding sites for HGF with Kd values of 23 and 260 pM, respectively. High-affinity HGF receptor with Kd values of <em>20</em>-25 pM was detected at 40-7<em>20</em> sites/cell in MDCK, A431, PAM212, Lu99, and IM-9 cells, but not in <em>fibroblasts</em> and hematopoietic cells. In contrast, low-affinity binding sites were detected in all cell lines examined, even in those not responsive to HGF. Northern blots revealed that cells possessing a high-affinity HGF receptor expressed c-MET/HGF receptor mRNA. Therefore, HGF probably regulates both cell <em>growth</em> and motility of various types of epithelial cells and some types of mesenchymal cells. The multiple biological activities of HGF may be exerted through a high-affinity HGF receptor linked to multiple distinct intracellular signaling pathways.

BACKGROUND

The mechanism of chronic progressive conjunctival cicatrization in mucous membrane pemphigoid is not well understood, and current therapy is often of limited use. Rapid progression of cicatrization follows exacerbations of clinical inflammation, and the investigation of immune mechanisms related to disease activity may provide a clue for more effective therapeutic strategies.

METHODS

The authors undertook an immunohistochemical study, using monoclonal and polyclonal antibodies in glycol methacrylate-embedded tissues, of epibulbar conjunctival biopsy specimens obtained from <em>20</em> patients with ocular cicatricial pemphigoid and from 12 matched healthy controls. The study patients were classified according to the ocular disease activity as acute ulcerative (n = 4), subacute (n = 8), and chronic (n = 8).

RESULTS

The composition of the subepithelial cellular infiltrate varied with disease activity. Acute disease was characterized by an abundance of macrophages and neutrophils. The number of T lymphocytes was significantly raised in all the disease groups, but were most marked in subacute disease. Of the T-cell subsets, there were more CD8- than CD4-positive cells observed, except in acute disease where there were equal numbers. Only approximately 5% of the T cells in all disease groups were activated as demonstrated by expression of interleukin-2 receptor. There was increased expression of major histocompatibility complex class II (MHC II) molecules on macrophages, fibroblasts, and other cells in all the groups. The number of B cells and natural killer cells was not increased. Staining for the fibrogenic cytokines, transforming growth factor-beta (TGF-beta), platelet-derived growth factor, and basic fibroblast growth factor was found in both pemphigoid patients and control persons, but the intensity of TGF-beta staining was significantly greater in acute disease.

CONCLUSIONS

The composition of the cellular infiltrate in the bulbar conjunctiva depends on clinical disease activity. The numbers of neutrophils and macrophages seem to reflect clinical disease activity. Fibrogenic cytokines, especially TGF-beta, may play an important role in the formation of conjunctival scar tissue.

BACKGROUND

Tumor angiogenesis, or new blood vessel formation, is regulated by a balance between pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and anti-angiogenic factors such as endostatin.

METHODS

To investigate this angiogenic balance in soft tissue sarcomas (STS), blood samples were collected from 76 STS patients and 15 healthy controls, and analyzed for VEGF, bFGF and endostatin using quantitative enzyme-linked immunosorbent assays (ELISA).

RESULTS

Forty-one patients (54%) had primary tumors, 20 (26%) had local recurrences and 15 (20%) had metastatic disease with or without local disease. Levels of all three angiogenic factors were highly variable in STS patients. Mean levels of VEGF and bFGF were 12 and 14 times higher, respectively, in patients compared with controls (P<0.0001). VEGF levels correlated with size of tumor, with the highest levels found in tumors >10 cm in size. Patients with metastases had endostatin levels 45% lower than patients without metastases (P=0.047). In 54 patients who underwent resection of primary disease or local recurrence, low pre-operative bFGF level was associated with a higher risk of subsequent recurrence (P=0.044).

CONCLUSIONS

STS secrete widely variable levels of angiogenic factors, and levels of specific factors may correlate with extent of disease, predict risk of recurrence and possibly guide the use of anti-angiogenic agents.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a secreted multifunctional cytokine and a potent stimulator of angiogenesis. We measured bFGF concentrations from serum samples taken from 103 patients with small cell lung cancer at the time of diagnosis. Serum concentration of bFGF (S-bFGF) ranged from undetectable to 54 pg/ml (median, 6 pg/ml). S-bFGF was not associated with age, sex, performance status, or stage. A high pretreatment S-bFGF was associated with poor overall survival. The 1- and 2-year survival rates of the patients within the highest quartile of S-bFGF >>or=17 pg/ml) were only 26% and 11%, respectively, in contrast to the 49% and <em>20</em>% 1- and 2-year survival rates of those patients with S-bFGF < 17 pg/ml (P = 0.013). The 1- and 2-year survival rates of the patients with extensive-stage disease were 33% and 10%, respectively (P = 0.0091). Interestingly, S-bFGF provided additional prognostic information to the stage because the 1- and 2-year survival rates of patients with extensive-stage disease and a high S-bFGF >>or=17 pg/ml) were as low as 16% and 5%, respectively (P = 0.0026). Similarly, in the multivariate model of survival analysis, patients with both extensive-stage disease and a high S-bFGF >>or=17 pg/ml) were found to have a particularly poor prognosis (relative risk of death, 2.1; 95% confidence interval, 1.2-3.6; P = 0.0057). We conclude that a high S-bFGF at diagnosis is associated with poor outcome in small cell lung cancer, possibly reflecting active angiogenesis and rapid tumor <em>growth</em>, and may complement prognostic information obtained by staging.

We have examined the effect of atrial natriuretic peptide (ANP) and its guanylyl cyclase/natriuretic peptide receptor-A (NPRA) on mitogen-activated protein kinase/extracellular signal-regulated kinase 2 (MAPK/ERK2) activity in rat mesangial cells overexpressing NPRA. Agonist hormones such as platelet-derived <em>growth</em> <em>factor</em> (PDGF), <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), angiotensin II (ANG II), and endothelin-1 (ET-1) stimulated 2.5- to 3.5-fold immunoreactive MAPK/ERK2 activity in these cells. ANP inhibited agonist-stimulated activity of MAPK/ERK2 by 65-75% in cells overexpressing NPRA, whereas in vector-transfected cells, its inhibitory effect was only 18-<em>20</em>%. NPRA antagonist A71915 and KT5823, a specific inhibitor of cGMP-dependent protein kinase (PKG) completely reversed the inhibitory effect of ANP on MAPK/ERK2 activity. ANP also inhibited the PDGF-stimulated [(3)H]thymidine uptake by almost 70% in cells overexpressing NPRA, as compared with only <em>20</em>-25% inhibition in vector-transfected cells. These results demonstrate that ANP/NPRA system negatively regulates MAPK/ERK2 activity and proliferation of mesangial cells in a PKG-dependent manner.

Limited proteolysis of insulin-like <em>growth</em> <em>factor</em> binding protein-3 (IGFBP-3) is increasingly becoming recognized as an essential mechanism in the regulation of insulin-like <em>growth</em> <em>factor</em> (IGF) bioavailability, both in the bloodstream and at cellular level. Plasmin generated on contact with various cell types provokes proteolytic cleavages that are similar to those induced in vivo by (as yet unidentified) IGFBP-3 proteases. Experimental conditions were determined to achieve plasmin-induced limited proteolysis of recombinant human nonglycosylated IGFBP-3. Two major fragments of 22/25 kilodaltons (kDa) and one of 16 kDa were identified by Western immunoblotting and isolated by reverse-phase chromatography. The 22/25-kDa fragments correspond to the major approximately 30-kDa glycosylated fragment of IGFBP-3 in serum and the 16-kDa fragment, to one of the same size, that is nonglycosylated. Western ligand blot analysis, affinity cross-linking, and competitive binding experiments using radiolabeled IGF and unlabeled IGF-I or -II showed that in the high performance liquid chromatography eluate containing the 16-kDa fragment, all affinity for IGFs had been lost, whereas the affinity of the 22/25-kDa fragments was considerably reduced. Scatchard analysis of the data indicated a <em>20</em>-fold loss of affinity for IGF-II and an 50-fold loss for IGF-I compared with that of recombinant human IGFBP-3. In a chick embryo <em>fibroblast</em> assay in which DNA synthesis was stimulated both by IGF-I and by insulin (at 100-fold concentrations, so that interaction with the Type 1 IGF receptor would occur), IGFBP-3 was found to inhibit IGF-I-induced stimulation almost totally. It had no effect on stimulation by insulin, which has no affinity for the IGFBPs. With the 22/25-kDa fragments, barely 50% inhibition of IGF-I stimulation was achieved and no inhibition of insulin stimulation. Unexpectedly, with the fraction containing the 16-kDa fragment (despite the total lack of affinity for IGF-I), IGF-I-induced stimulation was inhibited to nearly the same extent as with intact IGFBP-3. In addition, insulin-induced stimulation was inhibited with similar potency. IGFBP-3 proteolysis therefore generates two types of fragment with different activities. One has weak affinity for IGF-I and is only a weak antagonist of IGF action. The other lacks affinity for the IGFs, but nevertheless inhibits IGF-stimulated mitogenesis, thus acting by a mechanism that is independent of the IGFs.

The aim of this paper was to determine if <em>growth</em> <em>factors</em>, known to be upregulated in proliferative diabetic retinopathy, exerted combined effects on retinal endothelial cells. The authors explored the individual and collective actions of insulin-like <em>growth</em> <em>factor</em> I (IGF-I), vascular endothelial <em>growth</em> <em>factor</em> (VEGF), platelet-derived <em>growth</em> <em>factor</em>-BB (PDGF-BB), <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) and placenta <em>growth</em> <em>factor</em> (PlGF) on several parameters that reflect the angiogenic potential of endothelial cells. The effect of <em>growth</em> <em>factors</em> on cell migration and survival/proliferation was examined using primary cultures of bovine retinal endothelial cells (BREC). The authors also determined the <em>growth</em> <em>factor</em> action on capillary-like tube formation on a reconstituted basement membrane matrix and on the newly described phenomenon of secondary sprouting, in which endothelial cell colonies spontaneously survive, proliferate, migrate and invade the matrix after the original capillary-like tubes have collapsed. Sprouting cells were positive for von Willebrand <em>factor</em> and could aggregate into larger tubes with lumens. Incubation with VEGF+IGF-I or PlGF+FGF-2 enhanced tube stability by 40-50%, more than each <em>growth</em> <em>factor</em> alone or other combinations (5-<em>20</em>%). The concurrent addition of four <em>growth</em> <em>factors</em> did not improve the response seen with <em>growth</em> <em>factor</em> pairs. Surprisingly, PDGF-BB induced tube collapse. IGF-I and FGF-2 mildly enhanced BREC proliferation/survival (5-15%). However, VEGF+IGF-I or PlGF+FGF-2 increased BREC proliferation/survival by 25% under low serum conditions, whereas combinations of all four <em>growth</em> <em>factors</em> exerted a clearly synergistic effect (250% increase). PDGF-BB or FGF-2 stimulated secondary sprouting and were the only <em>factors</em> capable of exerting this effect alone. Even though VEGF, IGF-I or PlGF were not effective, if administered in pairs, they demonstrated increased responses. PDGF-BB was also able to enhance the effect of FGF-2+IGF-I+VEGF on BREC secondary sprouting, but not of any of them individually. No other <em>growth</em> <em>factor</em> tested was able to significantly improve the action of combinations of three other <em>growth</em> <em>factors</em>. VEGF increased cell migration in a wounded monolayer assay two-fold and PDGF-BB, 2.5 times, but other individual <em>growth</em> <em>factors</em> were ineffective. PlGF+FGF-2 enhanced cell migration more than each <em>factor</em> alone. VEGF+IGF-I+PlGF+FGF-2, however, increased cell migration four-fold. In summary, this study indicates that <em>growth</em> <em>factors</em>, overexpressed in diabetic retinopathy eyes, enhance the angiogenic characteristics of cultured cells (tube formation, proliferation, secondary sprouting and migration). Their effects, however, can be greatly augmented by other <em>growth</em> <em>factors</em> that alone exert little or no action. Therefore, diabetic retinal neovascularization may result from the additive or synergistic action of several <em>growth</em> <em>factors</em>.

Antibodies raised against the 51C/SHIP2 inositol polyphosphate 5'-phosphatase were used to examine the effects of <em>growth</em> <em>factors</em> and insulin on the metabolism of this protein. Immunoblot analysis revealed that the 51C/SHIP2 protein was widely expressed in <em>fibroblast</em> and nonhematopoietic tumor cell lines, unlike the SHIP protein, which was found only in cell lines of hematopoietic origin. The 51C/SHIP2 antiserum precipitated a protein of approximately 145 kDa along with an activity which hydrolyzed phosphatidylinositol 3,4, 5-trisphosphate to phosphatidylinositol 3,4-bisphosphate. Tyrosine phosphorylation of the 51C/SHIP2 protein occurred in response to treatment of cells with epidermal <em>growth</em> (EGF), platelet-derived <em>growth</em> <em>factor</em> (PDGF), nerve <em>growth</em> <em>factor</em> (NGF), insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1), or insulin. EGF and PDGF induced transient tyrosine phosphorylation of 51C/SHIP2, with maximal tyrosine phosphorylation occurring at 5-10 min following treatment and returning to near basal levels within <em>20</em> min. In contrast, treatment of cells with NGF, IGF-1, or insulin resulted in prolonged tyrosine phosphorylation of 51C/SHIP2 protein, with 40-80% maximal phosphorylation sustained for up to 2 h following agonist treatment. The kinetics of activation of the Akt/PKB protein kinase by the various <em>factors</em> correlated well with the kinetics of tyrosine phosphorylation of 51C/SHIP2. EGF, NGF, and PDGF stimulated the association of 51C/SHIP2 protein with the Shc adapter protein; however, no Shc could be detected in 51C/SHIP2-immune precipitates from cells treated with IGF-1 or insulin. The data suggest that 51C/SHIP2 may play a significant role in regulation of phosphatidylinositol 3'-kinase signaling by <em>growth</em> <em>factors</em> and insulin.

BACKGROUND

Low vitamin D concentrations are prevalent in patients with chronic kidney disease (CKD). We investigated the relationship between plasma 25-hydroxyvitamin D (25[OH]D) or 1,25-dihydroxyvitamin D (1,25[OH](2)D) concentrations with death, cardiovascular events, and dialysis therapy initiation in patients with advanced CKD.

METHODS

The HOST (Homocysteinemia in Kidney and End Stage Renal Disease) Study was a randomized double-blind trial evaluating the effects of high doses of folic acid on death and long-term dialysis therapy initiation in patients with advanced CKD (stages 4 and 5 not yet on dialysis therapy). 25(OH)D and 1,25(OH)(2)D were measured in stored plasma samples obtained 3 months after trial initiation and evaluated at clinically defined cutoffs (<10, 10-30, and >30 ng/mL) and tertiles (<15, 15-22, and >22 pg/mL), respectively. Cox proportional hazard models were used to examine the association between vitamin D concentrations and clinical outcomes.

METHODS

1,099 patients with advanced CKD from 36 Veteran Affairs Medical Centers.

METHODS

25(OH)D and 1,25(OH)(2)D concentrations.

RESULTS

Death, cardiovascular events, and time to initiation of long-term dialysis therapy.

RESULTS

After a median follow-up of 2.9 years, 41% (n = 453) died, whereas 56% (n = 615) initiated dialysis therapy. Mean 25(OH)D and 1,25(OH)(2)D concentrations were 21 ± 10 ng/mL and <em>20</em> ± 11 pg/mL, respectively. After adjustment for potential confounders, the lowest tertile of 1,25(OH)(2)D was associated with death (HR, 1.33; 95% CI, 1.01-1.74) and initiation of long-term dialysis therapy (HR, 1.78; 95% CI, 1.40-2.26) compared with the highest tertile. The association with death and initiation of dialysis therapy was moderately attenuated after adjustment for plasma <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF-23) concentrations (HRs of lower tertiles of 1.<em>20</em> [95% CI, 0.91-1.58] and 1.56 [95% CI, 1.23-1.99], respectively, compared with highest tertile). There was no association between 25(OH)D concentrations and outcomes.

CONCLUSIONS

Participants were mostly men.

CONCLUSIONS

Low plasma 1,25(OH)(2)D concentrations are associated with death and initiation of long-term dialysis therapy in patients with advanced CKD. FGF-23 level may attentuate this relationship.

Inappropriate <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signaling is involved in most tissue-specific pathologies including cancer. Previously we showed that inappropriate expression and chronic activity of FGF receptor (FGFR) 1 in hepatocytes accelerated diethylnitrosamine (DEN)-initiated hepatocarcinogenesis. Here we showed that although widely expressed FGF1 and FGF2 are frequently upregulated in hepatocellular carcinoma (HCC), germline deletion of both FGF1 and FGF2 had no effect on DEN-initiated hepatocarcinogenesis. Thus overexpression of FGF1 or FGF2 may be a consequence rather than contributor to hepatoma progression. FGF21 is the first of 22 homologues whose expression has been reported to be preferentially in the liver. We showed that similar to FGF1 and FGF2, FGF21 mRNA was upregulated in neoplastic and regenerating liver after partial hepatectomy (PH) and CCl4 administration. In situ hybridization analysis confirmed that in contrast to FGF1 and FGF2, expression of FGF21 mRNA was limited to hepatocytes. Forced overexpression of FGF21 in hepatocytes by gene targeting had no apparent impact on normal liver development and compensatory response to injury. Surprisingly, overexpression of FGF21 delayed the appearance of DEN-induced liver tumors. At 8 and 10 mo, only 10% and 30% of transgenic mice, respectively, developed adenomas compared to 50% (all adenomas) and 80% (60% adenoma/<em>20</em>% HCC) in the wild-type (WT) mice. However, the incidence and burden of HCC at 10 mo and later was equal in the FGF21 transgenic and WT mice. We propose that FGF21 may delay development of adenomas through activation of resident hepatocyte FGFR4 at early times, but counteracts the delay by acceleration of progression to HCC through interaction with ectopic FGFR1 once it appears in hepatoma cells. This indicates a dual function of FGF21 that may reflect changes in FGFR isotype during progression of differentiated hepatoma cells.

BACKGROUND

Iron deficiency anemia and serum phosphate levels>> 4.0mg/dL are relatively common in chronic kidney disease stages 3 to 5 and are associated with higher risks of progressive loss of kidney function, cardiovascular events, and mortality.

METHODS

Double-blind, placebo-controlled, randomized trial.

METHODS

149 patients with estimated glomerular filtration rates < 60 mL/min/1.73 m(2), iron deficiency anemia (hemoglobin, 9.0-12.0 g/dL; transferrin saturation [TSAT]≤ 30%, serum ferritin ≤ 300 ng/mL), and serum phosphate levels ≥ 4.0 to 6.0mg/dL. Use of intravenous iron or erythropoiesis-stimulating agents was prohibited.

METHODS

Randomization to treatment for 12 weeks with ferric citrate coordination complex (ferric citrate) or placebo.

METHODS

Coprimary end points were change in TSAT and serum phosphate level from baseline to end of study. Secondary outcomes included change from baseline to end of treatment in values for ferritin, hemoglobin, intact fibroblast growth factor 23 (FGF-23), urinary phosphate excretion, and estimated glomerular filtration rate.

RESULTS

Ferric citrate treatment increased mean TSAT from 22% ± 7% (SD) to 32% ± 14% and reduced serum phosphate levels from 4.5 ± 0.6 to 3.9 ± 0.6 mg/dL, while placebo exerted no effect on TSAT (21% ± 8% to 20% ± 8%) and less effect on serum phosphate level (4.7 ± 0.6 to 4.4 ± 0.8 mg/dL; between-group P<0.001 for each). Ferric citrate increased hemoglobin levels (from 10.5 ± 0.8 to 11.0 ± 1.0 g/dL; P<0.001 vs placebo), reduced urinary phosphate excretion 39% (P<0.001 vs placebo), and reduced serum intact FGF-23 levels from a median of 159 (IQR, 102-289) to 105 (IQR, 65-187) pg/mL (P=0.02 vs placebo). The incidence and severity of adverse effects were similar between treatment arms.

CONCLUSIONS

The study is limited by relatively small sample size and short duration and by having biochemical rather than clinical outcomes.

CONCLUSIONS

Short-term use of ferric citrate repletes iron stores, increases hemoglobin levels, and reduces levels of serum phosphate, urinary phosphate excretion, and FGF-23 in patients with chronic kidney disease stages 3 to 5.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signaling has been shown to play critical roles in vertebrate segmentation and elongation of the embryonic axis. Neither the exact roles of FGF signaling, nor the identity of the FGF ligands involved in these processes, has been conclusively determined. Fgf8 is required for cell migration away from the primitive streak when gastrulation initiates, but previous studies have shown that drastically reducing the level of FGF8 later in gastrulation has no apparent effect on somitogenesis or elongation of the embryo. In this study, we demonstrate that loss of both Fgf8 and Fgf4 expression during late gastrulation resulted in a dramatic skeletal phenotype. Thoracic vertebrae and ribs had abnormal morphology, lumbar and sacral vertebrae were malformed or completely absent, and no tail vertebrae were present. The expression of Wnt3a in the tail and the amount of nascent mesoderm expressing Brachyury were both severely reduced. Expression of genes in the NOTCH signaling pathway involved in segmentation was significantly affected, and somite formation ceased after the production of about 15-<em>20</em> somites. Defects seen in the mutants appear to result from a failure to produce sufficient paraxial mesoderm, rather than a failure of mesoderm precursors to migrate away from the primitive streak. Although the epiblast prematurely decreases in size, we did not detect evidence of a change in the proliferation rate of cells in the tail region or excessive apoptosis of epiblast or mesoderm cells. We propose that FGF4 and FGF8 are required to maintain a population of progenitor cells in the epiblast that generates mesoderm and contributes to the stem cell population that is incorporated in the tailbud and required for axial elongation of the mouse embryo after gastrulation.

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K(D) values varying between 38 nM (FGF-18) and 6<em>20</em> nM (FGF-9) and association rate constants spanning over <em>20</em>-fold (FGF-1, 2,900,000 M(-1) s(-1) and FGF-9, 130,000 M(-1) s(-1)). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.

The so-called triple-negative breast cancer, as defined by tumors that lack estrogen receptor, progesterone receptor and human epidermal <em>growth</em> <em>factor</em> receptor 2 (HER2) overexpression, has generated <em>growing</em> interest in recent years despite representing less than <em>20</em>% of all breast cancers. These tumors constitute an important clinical challenge, as they do not respond to endocrine treatment and other targeted therapies. As a group they harbor an aggressive clinical phenotype with early development of visceral metastases and a poor long-term prognosis. While chemotherapy remains effective in triple-negative disease, research continues to further identify potential new targets based on phenotypical and molecular characteristics of these tumors. In this respect, the presence of a higher expression of different biomarkers including epidermal <em>growth</em> <em>factor</em> receptor, vascular endothelial <em>growth</em> <em>factor</em> receptor, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor and Akt activation has led to a proliferation of clinical trials assessing the role of inhibitors to these pathways in triple-negative tumors. Moreover, the described overlap between triple-negative and basal-like tumors, and the similarities with tumors arising in the BRCA1 mutation carriers has offered potential therapeutic avenues for patients with these cancers including poly (ADP-ribose) polymerase inhibitors and a focus on a higher sensitivity to alkylating chemotherapy agents. Results from these trials have shown some benefit in small subgroups of patients, even in single-agent therapy, which reflects the heterogeneity of triple-negative breast cancer and highlights the need for a further subclassification of these types of tumors for better prognosis identification and treatment individualization.

OBJECTIVE

To investigate the effect(s) of cataract surgery on the expression of pro-inflammatory genes and proteins in the retina using an experimental rodent model.

METHODS

An extracapsular lens extraction was performed in one eye of C57BL/6 mice (n = 24); the contralateral unoperated eyes (n = 24) as well as eyes from unoperated animals (n = 9) served as controls. The neurosensory retina and retinal pigment epithelium (RPE)/choroid were collected postoperatively. Expression of genes involved in the acute inflammatory/injury response, including IL-1β, fibroblast growth factor, transforming growth factor β, chemokine CCL2, SDF-1, and complements C3, C4, and factor B (CFB), were examined by real-time PCR and, selectively, by immunohistochemistry.

RESULTS

The expression of IL-1β and CCL2 genes was markedly upregulated (>20-fold, P < 0.01) in the neurosensory retina 30 minutes postoperatively and maintained for the 2-week postoperative period of observation; IL-1β expression was also upregulated in RPE/choroid. The expression of complement C3 (>5-fold) and CFB (>30-fold) genes in the neurosensory retina was also significantly upregulated (P < 0.01 in both cases). Increased IL-1b, CCL2, and CFB as well as enhanced C5b-9 immunostaining were observed by confocal microscopy.

CONCLUSIONS

In rodents, lens extraction elicited an acute pro-inflammatory gene and protein response in the posterior segment of the eye, indicating induction of the inflammasome as well as complement activation, as occurs in the "danger" response. A similar response in humans might explain the pathogenesis of cataract surgery-associated retinal complications such as cystoid macular edema.

Numerous studies of the proliferative effects of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in culture, including neonatal and adult hippocampal precursors, suggest that the <em>factor</em> plays a ubiquitous and life-long role in neurogenesis. In contrast, in vivo, bFGF is devoid of effects on neurons in mature hippocampus, raising the possibility that bFGF exhibits developmental stage-specific activity in the complex animal environment. To define neurogenetic effects in the newborn, a single subcutaneous injection of bFGF (<em>20</em> ng/gm) was administered to postnatal day 1 (P1) rats, and hippocampal DNA content was quantified: bFGF elicited an increase in total DNA throughout adulthood, by 48% at P4, 25% at P22, and 17% at P180, suggesting that bFGF increases hippocampal cell number. To define mechanisms, bromodeoxyuridine (BrdU) was injected at P1 and mitotically labelled cells were assessed at P22: there was a twofold increase in BrdU-positive cells in the dentate granule cell layer (GCL), indicating that bFGF enhanced the generation of neurons, or neuronogenesis, from a cohort of precursors. Moreover, enhanced mitosis and survival led to a 33% increase in absolute GCL neuron number, suggesting that neuron production depends on environmental levels of bFGF. To evaluate this possibility, bFGF-knockout mice were analyzed: hippocampal DNA content was decreased at all ages examined (P3, -42%; P21, -28%; P360, -18%), and total GCL neuron and glial fibrillary acidic protein (GFAP)-positive cell number were decreased by 30%, indicating that bFGF is necessary for normal hippocampal neurogenesis. We conclude that environmental levels of bFGF regulate neonatal hippocampal neurogenesis. As adult hippocampal neuronogenesis was unresponsive to bFGF manipulation in our previous study [Wagner, J.P., Black, I.B. & DiCicco-Bloom, E. (1999) J. Neurosci., 19, 6006], these observations suggest distinct, stage-specific roles of bFGF in the dentate gyrus granule cell lineage.