Acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-1), a prototype member of the heparin-binding <em>growth</em> <em>factor</em> family, influences proliferation, differentiation, and protein synthesis in different cell types. However, its possible role on lung extracellular matrix (ECM) metabolism has not been evaluated. In this study we examined the effects of FGF-1 and FGF-1 plus heparin on type I collagen, collagen-binding stress protein HSP47, interstitial collagenase (matrix metalloproteinase [MMP]-1), gelatinase A, and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 expression by normal human lung <em>fibroblasts</em>. Heparin was used because it enhances the biologic activities of FGF-1. <em>Fibroblasts</em> were exposed either to <em>20</em> ng/ml FGF-1 plus 100 micrograms/ml heparin for 48 h or to FGF-1 or heparin alone. Messenger RNA (mRNA) expression was analyzed by Northern blot. Collagen synthesis was evaluated by digestion of [3H]collagen with bacterial collagenase, MMP-1 by Western blot, and gelatinolytic activities by zymography. Our results show that FGF-1 induced collagenase mRNA expression, which was strongly enhanced when FGF-1 was used with heparin. Likewise, both FGF-1 and FGF-1 plus heparin reduced by 70 to 80% the expression of type I collagen transcript, in part through effect on pro-alpha1(I) collagen mRNA stability. A downregulation of HSP47 gene expression was also observed. Synthesis of collagen and collagenase proteins paralleled gene expression results. FGF-1 activities were abolished with genistein, a tyrosine kinase inhibitor. Neither FGF-1 nor FGF-1 plus heparin affected the expression of TIMP-1, TIMP-2, and gelatinase A. These findings demonstrate that FGF-1, mostly in the presence of heparin, upregulates collagenase and downregulates type I collagen expression that might have a protective role in avoiding collagen accumulation during lung ECM remodeling.

Exposure of quiescent density arrested BALB/c-3T3 cells (clone A31) to platelet-derived <em>growth</em> <em>factor</em> (PDGF; 6-12 ng/ml) results in a rapid, reversible, time- and dose-dependent removal of vinculin from adhesion plaques (Herman and Pledger, 1985). Potential cellular mechanisms involved in PDGF-induced removal of vinculin from adhesion plaques were examined. Removal of vinculin from adhesion plaques following exposure of cells to PDGF was temperature dependent, occurred in many <em>fibroblast</em> cell lines, and could be mimicked by 12-tetradecanoyl phorbol-13-acetate (TPA; 5-125 nM) or melittin (0.35 microM). Unlike the effect of PDGF, TPA- or melittin-induced vinculin disruption was not reversible. The removal of vinculin from adhesion plaques was inhibited by trifluoroperazine (TFP; 2.5 microM). 8-(N,N-diethylamino) octyl-3,4,5-trimethoxy benzoate (TMB-8; 1.0 microM), mepacrine (2<em>20</em> microM), n-alpha-p-tosyl-L-lysine chloromethylketone (TLCK; 100 microM), phenylmethoxysulphonylfluoride (PMSF; 500 microM), and epsilon-aminocaproic acid (epsilon-ACA; 100 microM); however, amiloride (100 microM), A23187 (<em>20</em> microM), and chloroquine (1 mM) were unable to inhibit this effect. Melittin disruption of vinculin was inhibited by (in order of decreasing effectiveness) mepacrine greater than TMB-8 greater than TFP greater than leupeptin greater than PMSF, whereas A23187 and amiloride had no effect. The return of vinculin to adhesion plaques following PDGF treatment required de novo mRNA transcription and protein synthesis and was associated with PDGF-stimulated synthesis of vinculin. The observation that both PDGF- and melittin-induced removal of vinculin from adhesion plaques is inhibited by mepacrine suggests that phospholipase activation may be an early and important step in PDGF-induced disruption of vinculin from adhesion plaques. In addition, TFP, TMB-8 and protease inhibitor inhibition of both the PDGF and melittin effects on vinculin distribution, coupled with the finding that TPA can mimic the PDGF or melittin response, suggests that Ca2+, calmodulin, protein kinase C, and/or proteolysis may play an important role(s) in the removal of vinculin from adhesion plaques following PDGF addition. The lack of effect of A23187 addition on vinculin distribution suggests that alterations in cellular Ca2+ is necessary but not sufficient for vinculin removal from adhesion plaques.

In preparation for studies on the <em>growth</em> <em>factor</em> requirements of normal and transformed human <em>fibroblasts</em>, we have developed a serum-free medium that supports vigorous long-term serial subculture of diploid human <em>fibroblasts</em> and allows them to form large-sized colonies with high efficiency (40 to 60%) when plated at cloning density (2 to 5 cells/cm2). This medium, which is a modification of Ham's MCDB 110 base medium with its serum replacement supplements, is relatively easy to prepare and the cost of the serum replacements is approximately the same as that of fetal bovine serum supplied at 10%. The ingredients of "Supplement B" of MCDB 110 medium were added in an ethanol solution, rather than in the form of liposomes, and were combined with bovine serum albumin (0.5%), a lipid carrier. Gelatin and fetuin were included as attachment <em>factors</em> instead of polylysine. Bioassays indicated that none of the ingredients in the medium were contaminated with either epidermal <em>growth</em> <em>factor</em> or platelet-derived <em>growth</em> <em>factor</em>. In this modified serum-free medium, which we have designated McM+SR1, diploid human <em>fibroblasts</em> grew for 21 days at the same rate as in the base medium, McM, supplemented with 10% FBS (i.e., 21 population doublings). During the next <em>20</em> days, they underwent 15 population doublings which was 75% of the rate of cells <em>growing</em> in the medium containing serum.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>20</em> (FGF-<em>20</em>) has been shown to protect dopaminergic neurons against a range of toxic insults in vitro, through activation of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1). This study set out to examine whether FGF-<em>20</em> also displayed protective efficacy in the unilateral, 6-hydroxydopamine (6-OHDA) lesion rat model of Parkinson's disease. Initial studies demonstrated that, in embryonic ventral mesencephalic (VM) cultures, FGFR1 was expressed on tyrosine hydroxylase (TH)-positive neurons and that, in line with previous data, FGF-<em>20</em> (100 and 500 ng/ml) almost completely protected these TH-positive neurons against 6-OHDA-induced toxicity. Co-localisation of FGFR1 and TH staining was also demonstrated in the substantia nigra pars compacta (SNpc) of naïve adult rat brain. In animals subject to 6-OHDA lesion of the nigrostriatal tract, supra-nigral infusion of FGF-<em>20</em> (2.5 μg/day) for 6 days post-lesion gave significant protection (∼40%) against the loss of TH-positive cells in the SNpc and the loss of striatal TH immunoreactivity. This protection of the nigrostriatal tract was accompanied by a significant preservation of gross locomotion and fine motor movements and reversal of apomorphine-induced contraversive rotations, although forelimb akinesia, assessed using cylinder test reaching, was not improved. These results support a role for FGF-<em>20</em> in preserving dopamine neuron integrity and some aspects of motor function in a rodent model of Parkinson's disease (PD) and imply a potential neuroprotective role for FGF-<em>20</em> in this disease.

BACKGROUND

Although the systemic administration of deferoxamine (DFO) is protective in experimental models of normal ischemic flap and diabetic wound, its effect on diabetic flap ischemia using a local injection remains unknown.

OBJECTIVE

To explore the feasibility of local injection of DFO to improve the survival of ischemic random skin flaps in streptozotocin (STZ)-induced diabetic mice.

METHODS

Ischemic random skin flaps were made in 125 mice. Animals were divided into the DFO-treated (n = <em>20</em>), PBS-treated (n = 16) and untreated (n = 16) groups. Surviving area, vessel density, and expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and hypoxia-inducible <em>factor</em>-1α (HIF-1α) were evaluated on the seventh day after local injection.

RESULTS

The viability of DFO-treated flap was significantly enhanced, with increased regional blood perfusion and capillary density compared with those in the two control groups. Fluorescence-activated cell sorting (FACS) analysis demonstrated a marked increase in systemic Flk-1+/CD11b- endothelial progenitor cells (EPCs) in DFO-treated mice. Furthermore, the expression of VEGF and HIF-1α was increased not only in diabetic flap tissue, but also in dermal fibroblasts cultured under hyperglycemic and hypoxic conditions.

CONCLUSIONS

Local injection of DFO could exert preventive effects against skin flap necrosis in STZ-induced diabetic mice by elevating the expression of HIF-1α and VEGF, increased EPC mobilization, which all contributed to promote ischemic diabetic flap survival.

OBJECTIVE

Myofibroblasts and transforming growth factor-beta(1) (TGF-beta(1)) are key elements of cardiac tissue fibrosis development. The aim of this study was to determine whether the ability of TGF-beta(1) to affect the contractile activity of cardiac fibroblasts depends on their differentiation into myofibroblasts.

METHODS

Cardiac fibroblasts (from male adult Wistar rats) from passage two were cultured to confluency and incubated on a hydrated collagen gel with and without TGF-beta(1) (0, 20, 40, 100, 200, 400 or 600 pmol/L) for one, two and three days in a Dulbecco's Modified Eagle's Medium without foetal bovine serum.

RESULTS

TGF-beta(1) dose-dependently increased the contraction of collagen gel mediated by cardiac fibroblasts, reaching a maximal effect at 100 pmol/L TGF-beta(1). TGF-beta(1) also stimulated 3(H)-thymidine incorporation and total protein content in cardiac fibroblasts in the collagen gel lattice. TGF-beta(1) dose-dependently induced an increase in beta-smooth muscle actin, a marker of myofibroblasts. The TGF-beta(1)-induced reduction of area of the collagen gel was negatively correlated to the TGF-beta(1)-evoked appearance of a-smooth muscle actin in the collagen gel matrix.

CONCLUSIONS

Our data demonstrate that TGF-beta(1) increased the contractile activity of adult rat cardiac fibroblasts and their ability to differentiate into myofibroblasts. Because contractile activity was correlated with differentiation, the influence of TGF-beta(1) on cardiac fibroblast-induced collagen gel contraction might depend on the promotion of myofibroblast differentiation.

Exposure of human lung <em>fibroblasts</em> to human cytomegalovirus (HCMV) stimulated a rapid increase in the release of [3H] from cells prelabelled with radiolabelled arachidonic acid ([3H]AA). Maximum stimulation of [3H] release was observed at <em>20</em> min postinfection and was quantitatively similar to that induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA: 10 nM) or fetal calf serum (5%). The level of [3H] release was dependent on the multiplicity of infection, and appeared to be mediated by a component(s) of the virion, since the findings from three series of experiments suggested that neither infectious virus, nor HCMV-specific macromolecular synthesis was required for stimulation of [3H] release. (1) Inactivation of HCMV infectivity with ultra-violet (UV) light (approximately 254 nm, 4.80 x 10(4) ergs/mm2) did not diminish the stimulation of [3H] release. (2) Significant reduction in the level of [3H] release was not observed when infected cells were maintained in the presence of a protein synthesis inhibitor, cycloheximide (50 micrograms/ml), or an inhibitor of mRNA synthesis, 3'-deoxyadenosine (cordycepin, 50 micrograms/ml). (3) No correlation was established between the expression of HCMV immediate early (IE) antigens and the induction of [3H] release, since there was little, if any, synthesis of HCMV IE antigen detectable by anticomplement immunofluorescence through the first 30 min postinfection. These findings suggesting that the HCMV particle rapidly stimulates AA metabolism are consistent with the view that the interaction of a HCMV virion component(s) with the cell surface may initiate membrane-associated events similar to those induced by <em>growth</em> <em>factors</em>.

The addition of non-anticoagulant heparin [periodate-oxidized (IO4) heparin] and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 to a viscous water-soluble chitosan (CH-LA) aqueous solution produces an injectable FGF-2/CH-LA/IO4-heparin hydrogel. The purpose of this study was to examine the ability of the injected FGF-2/CH-LA/IO4-heparin hydrogel to induce vascularization and fibrous tissue formation. FGF-2/CH-LA/IO4-heparin hydrogels (100 microL of hydrogel consisting of <em>20</em> mg/mL of CH-LA, 2 mg/mL of IO4-heparin, and 50 microg/mL of FGF-2) were subcutaneously injected into the backs of wound healing-impaired diabetic (db/db) mice. Furthermore, the effect of percutaneous injection of FGF-2/CH-LA/IO4-heparin hydrogel at eight sites (25 microL/site) into ischemic left lower limbs of rats was examined from day 4 to at least day 28 postinjection. The injection of FGF-2/CH-LA/IO4-heparin hydrogels into the backs of db/db mice resulted in significant increases in blood vessel formation, significant vascularization, and fibrous tissue formation near the injection site. Injection of FGF-2/CH-LA/IO4-heparin hydrogel into ischemic left lower limbs of rats also significantly recovered and increased blood flow and blood oxygen in the calf and thigh. These results indicate that the controlled release of biologically active FGF-2 molecules from FGF-2/CH-LA/IO4-heparin induces angiogenesis and possibly collateral circulation in db/db mice and the ischemic limbs of rats.

We recently reported that the subcutaneous (s.c.) administration of a low-molecular-weight heparin (LMWH) fraction significantly inhibited de novo angiogenesis in the mesentery induced by the intraperitoneal (i.p.) injection of saline to adult rats compared with unfractionated heparin and high-molecular-weight heparin (HMWH) fractions. The present study assesses the effect on basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF)-mediated de novo angiogenesis in the mesentery of the systemic administration of a LMWH fraction (2.6 kD) and a series of four HMWH fractions (about <em>20</em> kD) with varying degrees of polydispersity, charge density and anticoagulant activity. bFGF, a prototypic heparin-binding angiogenic <em>growth</em> <em>factor</em>, was injected i.p. at 2<em>20</em> pM on days 0-4. The heparins were given s.c. on days 0-13 or 0-14 at doses which were approximately within the range used clinically. Angiogenesis was assessed by microscopic morphometry and image analysis in groups of animals killed on days 14 and 15. Compared with the saline control, the LMWH and three of the HMWHs significantly inhibited angiogenesis in terms of microvascular length (MVL), a measure of microvascular density. Interestingly, the vascularized area (VA), a measure of microvascular spatial extension, and the total microvascular length (VA x MVL) were significantly lower in the LMWH-treated animals than in the animals treated with one of the HMWHs. The total microvascular length was, moreover, significantly reduced in the LMWH-treated animals compared with the combined data of all the HMWH-treated animals. No significant effects were related to the degree of charge density and anticoagulant activity of the heparins. In view of the putative significant angiogenic role of bFGF in human angiogenesis diseases, the present findings may have implications for the choice of anticoagulant treatment modality for patients suffering from cancer and other angiogenesis diseases.

The mammalian kidney develops from the ureteric bud and the metanephric mesenchyme. In mice, the ureteric bud invades the metanephric mesenchyme at day E10.5 and begins to branch. The tips of the ureteric bud induce the metanephric mesenchyme to condense and form the cap mesenchyme. Some cells of this cap mesenchyme undergo a mesenchymal-to-epithelial transition and differentiate into renal vesicles, which further develop into nephrons. The developing kidney expresses <em>Fibroblast</em> <em>growth</em> <em>factor</em> (Fgf)1, 7, 8, 9, 10, 12 and <em>20</em> and Fgf receptors Fgfr1 and Fgfr2. Fgf7 and Fgf10, mainly secreted by the metanephric mesenchyme, bind to Fgfr2b of the ureteric bud and induce branching. Fgfr1 and Fgfr2c are required for formation of the metanephric mesenchyme, however the two receptors can substitute for one another. Fgf8, secreted by renal vesicles, binds to Fgfr1 and supports survival of cells in the nascent nephrons. Fgf9 and Fgf<em>20</em>, expressed in the metanephric mesenchyme, are necessary to maintain survival of progenitor cells in the cortical region of the kidney. FgfrL1 is a novel member of the Fgfr family that lacks the intracellular tyrosine kinase domain. It is expressed in the ureteric bud and all nephrogenic structures. Targeted deletion of FgfrL1 leads to severe kidney dysgenesis due to the lack of renal vesicles. FgfrL1 is known to interact mainly with Fgf8. It is therefore conceivable that FgfrL1 restricts signaling of Fgf8 to the precise location of the nascent nephrons. It might also promote tight adhesion of cells in the condensed metanephric mesenchyme as required for the mesenchymal-to-epithelial transition.

The aim of this study was to define prognostic <em>factors</em> that might be predictive for response to thalidomide (Thal) in progressive multiple myeloma (n = 54). We examined the concentration of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), two potent heparin-binding mediators of angiogenesis in peripheral blood (PB; PB-VEGF and PB-bFGF) and bone marrow (BM; BM-VEGF and BM-bFGF), in combination with well-characterized predictors for response and survival to chemotherapy. After a median follow-up time of 15 months (range, 0.3-<em>20</em>), 29 patients (pts.) showed at least a minimal response to Thal therapy, whereas 25 pts. were nonresponsive. As shown by univariate analysis, responsive pts. had statistically significant higher concentrations of PB-bFGF (P = 0.009) and beta2-microglobulin (P = 0.03) before therapy, as well as lower hemoglobin (P = 0.008) and albumin (P = 0.02) levels, whereas no statistically significant difference was found for PB-VEGF (P = 0.93). When a multiple logistic regression analysis was performed, PB-bFGF was the only statistically significant predictor for response to therapy (P = 0.01). None of these variables was associated with a prolonged progression-free survival. In conclusion, our findings indicate that high pretreatment plasma bFGF levels in pts. with progressive multiple myeloma are associated with unfavorable parameters of response and survival but nevertheless predict for response to Thal therapy.

BACKGROUND

High levels of circulating fibroblast growth factor 23 (FGF23) are associated with chronic kidney disease (CKD) progression and high mortality. In the Phosphate Reduction Evaluation of FGF23 in Early CKD Treatment (PREFECT) study, we assessed the effect of reducing intestinal phosphate absorption using lanthanum carbonate on FGF23 levels in normophosphatemic patients with CKD stage 3.

METHODS

Thirty-five individuals were randomized to lanthanum carbonate 3000 mg/day (n=23) or placebo (n=12) for 12 weeks. Levels of intact FGF23 (iFGF23), C-terminal FGF23, serum and urinary phosphate and calcium, intact parathyroid hormone and 1,25-dihydroxyvitamin D were assessed.

RESULTS

The median age was 65 years in the lanthanum group and 73 years in the placebo group; 58.8% and 41.7% were men, respectively. No significant difference was seen in mean iFGF23 between groups at week 12. There was, however, a transient reduction from baseline in iFGF23 in the lanthanum group at week 1, from 70.5 pg/ml to 51.9 pg/ml, which was not seen in the placebo group; this between-group difference in percentage change from baseline was significant in post hoc analyses (p=0.0102). Urinary phosphate decreased after 1 week of lanthanum treatment and remained low at week 12.

CONCLUSIONS

Reducing intestinal phosphate absorption with lanthanum carbonate did not lead to sustained reductions in iFGF23 in patients with CKD stage 3, although phosphaturia decreased. This suggests that factors other than phosphate burden may be responsible for driving increases in circulating FGF23 in patients with CKD.

BACKGROUND

ClinicalTrials.gov NCT01128179, 20 May 2010.

The survival of isolated neurons from chick embryo ciliary, sympathetic, and dorsal root ganglia is greatly enhanced by concentrations of extracellular potassium that significantly depolarize the neurons (ED50 = <em>20</em>-25 mM). The survival-promoting effect of elevated potassium on each of these 3 types of neurons appears to be the result of the opening of voltage-gated calcium channels. The dihydropyridine, Bay K 8644, which increases calcium influx through L-type voltage-gated calcium channels in neurons, strongly potentiated the survival-promoting action of elevated potassium (ED50 = 10.8 +/- 7.0 nM). In contrast, chemically closely related dihydropyridines, PN<em>20</em>0-110 (ED50 = 0.33 +/- 0.15 nM) and nitrendipine (ED50 = 1.3 +/- 0.3 nM), which block calcium influx through the same voltage-gated channels, completely inhibited potassium-mediated neuronal survival. Chemically different agents that also block calcium influx through voltage-gated channels also inhibited potassium-mediated neuronal survival: the phenylalkylamine verapamil (ED50 = 0.78 +/- 0.38 microM), the benzothiazepine diltiazem (ED50 = 1.7 microM), and the inorganic ion cadmium (ED50 = 5.8 microM). These calcium-channel blockers are not simply toxic to neurons, since they did not inhibit neuronal survival mediated by the neurotrophic proteins, nerve <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, or ciliary neurotrophic <em>factor</em>, also suggesting that voltage-gated calcium channels are not involved in the action of these <em>factors</em>. These results suggest that neuronal survival in elevated potassium in ciliary, sympathetic, and dorsal root ganglion neurons is the result of calcium influx through dihydropyridine-sensitive, L-type voltage-gated calcium channels. These findings are discussed in relation to the neuronal toxicity of excitatory amino acids which is also thought to occur through increased calcium influx.

OBJECTIVE

Liver X receptors (LXRs) transcriptionally regulate inflammation, metabolism, and immunity. Synthetic LXR agonists have been evaluated for their efficacy in the cardiovascular system; however, they elicit prolipogenic side effects which substantially limit their therapeutic use. AZ876 is a novel high-affinity LXR agonist. Herein, we aimed to determine the cardioprotective potential of LXR activation with AZ876.

RESULTS

Cardiac hypertrophy was induced in C57Bl6/J mice via transverse aortic constriction (TAC) for 6 weeks. During this period, mice received chow supplemented or not with AZ876 (<em>20</em> µmol/kg/day). In murine hearts, LXRα protein expression was up-regulated ∼7-fold in response to TAC. LXR activation with AZ876 attenuated this increase, and significantly reduced TAC-induced increases in heart weight, myocardial fibrosis, and cardiac dysfunction without affecting blood pressure. At the molecular level, AZ876 suppressed up-regulation of hypertrophy- and fibrosis-related genes, and further inhibited prohypertrophic and profibrotic transforming <em>growth</em> <em>factor</em> β (TGFβ)-Smad2/3 signalling. In isolated cardiac myocytes and <em>fibroblasts</em>, immunocytochemistry confirmed nuclear expression of LXRα in both these cell types. In cardiomyocytes, phenylephrine-stimulated cellular hypertrophy was significantly decreased in AZ876-treated cells. In cardiac <em>fibroblasts</em>, AZ876 prevented TGFβ- and angiotensin II-induced <em>fibroblast</em> collagen synthesis, and inhibited up-regulation of the myo<em>fibroblast</em>ic marker, α-smooth muscle actin. Plasma triglycerides and liver weight were unaltered following AZ876 treatment.

CONCLUSIONS

AZ876 activation of LXR protects from adverse cardiac remodelling in pathological pressure overload, independently of blood pressure. LXR may thus represent a putative molecular target for antihypertrophic and antifibrotic therapies in heart failure prevention.

Clear cell renal cell carcinoma (ccRCC) is the most common renal cell carcinoma subtype, and metastatic ccRCC is associated with 5-year survival rates of 10% to <em>20</em>%. Genetically, ccRCC originates from sequential losses of multiple tumor suppressor genes. Remarkably, chromosome 3p loss occurs in more than 90% of sporadic ccRCCs. This results in concurrent one-copy loss of four tumor suppressor genes that are also mutated individually at high frequency in ccRCC (ie, VHL, 80%; PBRM1, 29% to 46%; BAP1, 6% to 19%; and SETD2, 8% to 30%). Pathogenically, 3p loss probably represents the first genetic event that occurs in sporadic ccRCC and the second genetic event in VHL-mutated hereditary ccRCC. VHL constitutes the substrate recognition module of the VCB-Cul2 E3 ligase that degrades HIF1/2α, whereas PBRM1, BAP1, and SETD2 are epigenetic modulators that regulate gene transcription. Because 3p loss and VHL inactivation are nearly universal truncal events in ccRCC, the resulting HIF1/2 signaling overdrive and accompanied tumor hypervascularization probably underlie the therapeutic benefits observed with vascular endothelial <em>growth</em> <em>factor</em> receptor inhibitors, including sorafenib, sunitinib, pazopanib, axitinib, bevacizumab, cabozantinib, and lenvatinib. Furthermore, recent marked advances in ccRCC genomics, transcriptomics, proteomics, metabolomics, molecular mechanisms, mouse models, prognostic and predictive biomarkers, and clinical trials have rendered invaluable translational insights concerning precision kidney cancer therapeutics. With an armamentarium encompassing 13 drugs that exploit seven unique therapeutic mechanisms (ie, cytokines, vascular endothelial <em>growth</em> <em>factor</em> receptor, mTORC1, cMET/AXL, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor, programmed cell death-1 and programmed death-ligand 1, and cytotoxic T-cell lymphocyte associated-4) to treat metastatic renal cell carcinoma, one of the imminent clinical questions concerning care of patients with metastatic ccRCC is how a personalized treatment strategy, through rationally combining and sequencing different therapeutic modalities, can be formulated to offer the best clinical outcome for individual patients. Here, we attempt to integrate recent discoveries of immediate translational impacts and discuss future translational challenges and opportunities.

This study was carried out to investigate the expression of various <em>growth</em> <em>factors</em> (GFs) involved in mucosal healing and thereby to clarify whether there are potential differences in the expression of GFs between normal colonic mucosa and the uninvolved mucosa of ulcerative colitis (UC). GF mRNA was investigated by reverse transcription polymerase chain reaction in colorectal biopsies from <em>20</em> normal controls and 15 UC patients. The positive rates (%) for mRNA expression for normal/UC were: epidermal <em>growth</em> <em>factor</em> (EGF) 65/53, transforming <em>growth</em> <em>factor</em> (TGF)-alpha 100/87, TGF-beta 1 60/33, insulin like <em>growth</em> <em>factor</em>-I 45/33, platelet-derived <em>growth</em> <em>factor</em>-A 55/67, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> 0/0, hepatocyte <em>growth</em> <em>factor</em> (HGF) 50/53, EGF receptor <em>20</em>/27, erb-B2 75/73, and HGF receptor (c-MET) 55/67. Semiquantitation of mRNA showed significantly lower expression of TGF-beta 1 (P < 0.05) in UC. Differences in expression and mRNA levels were not statistically significant for any other GFs. Our results indicate that mucosa in chronic persistent UC has a low basal expression of TGF-beta 1 mRNA, and, since TGF-beta 1 is a multifunctional GF that plays important roles in regulating repair and regeneration following tissue injury, this low expression may be partially responsible for the intractability of the disease.

Objectives. Human breast milk contains a heterogeneous population of cells that have the potential to provide a noninvasive source of cells for cell therapy in many neurodegenerative diseases without any ethical concern. The objectives of this study were to differentiate the breast milk-derived stem cells (BMDSC) toward neural stem cells and then into the neurons and neuroglia. Materials and Methods. To do this, the BMDSC were isolated from human breast milk and cultured in Dulbecco's modified Eagle medium/F12 (DMEM/F12) containing <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The cells were then characterized by evaluation of the embryonic and stem cell markers. Then, the cells were exposed to culture medium containing 1% B27 and 2% N2 for 7-10 days followed by medium supplemented with B27, N2, bFGF 10 µg/mL, and endothelial <em>growth</em> <em>factor</em> (EGF) <em>20</em> µg/mL. Then, the sphere-forming assay was performed. The spheres were then differentiated into three neural lineages by withdrawing <em>growth</em> <em>factor</em> in the presence of 5% FBS (fetal bovine serum). The immunofluorescence was done for β-tubulin III, O4, and GFAP (glial fibrillary acidic protein). Results. The results indicated that the cells expressed both embryonic and mesenchymal stem cell (MSC) markers. They also showed neurospheres formation that was nestin-positive. The cells were also differentiated into all three neural lineages. Conclusion. The BMDSC can behave in the same way with neural stem cells. They were differentiated into oligodendrocytes, and astrocytes as well as neurons.

BACKGROUND

Hyperphosphatemic Familial Tumoral Calcinosis (HFTC) and Hyperphosphatemic Hyperostosis Syndrome (HHS) are associated with autosomal recessive mutations in three different genes, FGF23, GALNT3 and KL, leading to reduced levels of fibroblast growth factor 23 (FGF23) and subsequent clinical effects.

RESULTS

We describe a consanguineous family with two affected siblings with HFTC and HHS caused by a novel homozygous G-to T substitution in exon 3 of GALNT3 (c.767 G>> T; p.Gly256Val), demonstrating great phenotypic variation and long asymptomatic intervals. Calcific tumors appeared at 14 years of age in the male, and the female displayed episodic diaphysitis from age 9 years. Symptoms of eye involvement were present in both from childhood, and progressed into band keratopathy in the female. Abnormal dental roots and tooth loss, as well as myalgia were present in both from their mid-twenties, while the female also had calcifications in the placenta, the iliac vessels and thyroid cartilage. New calcific tumors appeared more than 20 years after the initial episodes, delaying diagnosis and treatment until the ages of 37 and 50 years, respectively. Both siblings had elevated serum phosphate levels, inappropriately elevated tubular maximum phosphate reabsorption per unit glomerular filtration rate (TmP/GFR), reduced levels of intact FGF23 and increased levels of c-terminal FGF23. Review of all 54 previously published cases of GALNT3, FGF23, and KL associated HFTC and HHS demonstrated that more subjects than previously recognized have a combined phenotype.

CONCLUSIONS

We have described HFTC and HHS in a consanguineous Caucasian family with a novel GALNT3 mutation, demonstrating new phenotypic features and significant variability in the natural course of the disease. A review of the literature, show that more subjects than previously recognized have a combined phenotype of HFTC and HHS. HHS and HFTC are two distinct phenotypes in a spectrum of GALNT3 mutation related calcification disorders, where the additional factors determining the phenotypic expression, are yet to be clarified.

Pediatric burn wounds can be problematic because an accurate evaluation is difficult as the result of anatomically immature vasculature or immobilization failure, especially in patients with second-degree burns, and because the burn surface areas and the burn depth tend to worsen over the course of time. Delayed wound healing results in unsightly scarring, such as hypertrophic scars, which are problematic both esthetically and functionally. Among cytokines and <em>growth</em> <em>factors</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is clinically proven, having demonstrated accelerated acute and chronic wound healing. Accelerated wound healing may lead to improved scarring. To elucidate the effects of bFGF on second-degree pediatric burn wounds, a comparative study was performed. A total of <em>20</em> pediatric patients ranging from 8 month to 3 years (average 1 year, 3 months +/- 6 months) who suffered from the burns by various causes were divided into two groups, conventional (n = 10) and treatment with bFGF (n = 10). A moisture meter, used to objectively measure the stratum corneum and epithelial-mesenchymal functions, was used to assess scars at least 1 year after wound healing. Clinical evaluation of pigmentation, pliability, height, and vascularity demonstrated significant differences between conventional and bFGF-treated scars (1.7 +/- 0.55 vs 0.7 +/- 0.58, 2.4 +/- 0.82 vs 1.1 +/- 0.69, 1.8 +/- 0.66 vs 0.5 +/- 0.57, 1.9 +/- 0.63 vs 0.8 +/- 0.68; conventional vs bFGF-treated, pigmentation, pliability, height, and vascularity, respectively, P < .01). The effective contact coefficient was significantly greater in conventional wounds than bFGF-treated wounds (14.6 +/- 1.68 % vs 8.7 +/- 2.82 %; conventional vs bFGF, P < .01) and bFGF-treated wounds demonstrated significantly less transepidermal water loss values than conventional treatment (8.3 +/- 1.90 g/m/h vs 5.7 +/- 1.85 g/m/hr; conventional vs bFGF, P < .01). Pediatric burn patients treated with bFGF showed less damaging function of the stratum corneum after healing both in clinical assessment and moisture meter analysis.

BACKGROUND

Triple-negative breast cancer (TNBC) makes up about 10 - <em>20</em>% of all breast cancers and the lack of hormone receptors and human epidermal <em>growth</em> <em>factor</em> receptor-2/Neu expression is responsible for poor prognosis, no targeted therapies and trouble in the clinical management. Tumor heterogeneity, also within the same tumor, is a major cause for this difficulty. Based on the introduction of new biological drugs against different kinds of tumor, many efforts have been made for classification of genetic alterations present in TNBC, leading to the identification of several oncogenes and tumor suppressor genes involved in breast cancer carcinogenesis.

METHODS

In this review we investigated the molecular alteration present in TNBC which could lead to the creation of new targeted therapies in the future, with the aim to counteract this disease in the most effective way.

CONCLUSIONS

In this context some hormone receptors like G-protein-coupled receptor 30 and androgen receptors may be a fascinating area to investigate; also, angiogenesis, represented not only by the classical VEGF/VEGFR relationship, but also by other molecules, like semaphorins, fibroblast growth factor and heparin-binding-EGF-like, is a mechanism in which new developments are expected. In this perspective, one technique that may show promise is the gene therapy; in particular the gene transfer could correct abnormal genetic function in cancer cells.

OBJECTIVE

To determine the protective effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo.

METHODS

In Experiment I, cultured spiral ganglion neurons (SGNs) prepared from postnatal Day 3 mice were exposed to 20 mM glutamate for 2 h before the culture medium was replaced with fresh medium containing 0, 25, 50 or 100 ng/ml bFGF. Fourteen days later, all cultures were fixed with 4% paraformaldehyde and stained with 1% toluidine blue. The number of surviving SGNs was counted and the length of the neurites of the SGNs was measured. In Experiment II, in vivo studies were carried out with guinea pigs in which bFGF or normal saline was injected intramuscularly to assess possible protective effects of bFGF on cochlear hair cells and to accelerate the recovery of the auditory brainstem response (ABR). The ABRs were measured before, immediately after and 2 and 4 weeks after exposure to noise.

RESULTS

Exposure to 20 mM glutamate for 2 h resulted in an inhibition of neurite outgrowth of SGNs and an increase in cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and an increase in the number of surviving SGNs. In Experiment II, significant (p < 0.05) differences in ABR thresholds were observed between the groups injected with bFGF and saline (t = 2.689) at 4 weeks after noise exposure. Cochleae were removed and hair cell loss analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 240 and 2160 inner hair cells in the groups injected with bFGF and saline, respectively. Similarly, more outer hair cells were lost in the normal saline injection group (99,291) than in the group treated with bFGF (70,377).

CONCLUSIONS

Our results demonstrate that bFGF protects SGNs against glutamate neurotoxicity in vitro. In addition, treatment with bFGF protects hair cells from acoustic trauma.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2) and its main receptor FGFR1 have been shown to promote hepatic stellate cell (HSC) activation and proliferation. However, scant information is available on the anti-fibrogenic activity of FGFR1 inhibitors. The aim of this study was to assess the impact of a selective FGFR1 tyrosine kinase inhibitor NP603 on HSC proliferation and hepatic fibrosis. We demonstrated that rat primary HSCs secreted significant amounts of FGF-2, and its tyrosine phosphorylation of FGFR1 was attenuated by NP603. NP603 inhibited HSC activaton by measuring the expression of α-smooth muscle actin (α-SMA) and the production of type I collagen using ELISA. Furthermore, NP603 (25 μM) in vitro strongly suppressed HSC <em>growth</em> induced by FGF-2 (10 ng/ml) and FCS. This effect correlated with the suppression of extracellular-regulated kinase (ERK) activity and its downstream targets cyclin D1 and p21. In addition, PO NP603 (<em>20</em> mg·kg(-1)·day(-1)) administration significantly decreased hepatic collagen deposition and α-SMA expression in CCl(4)-treated rats. Collectively, these studies suggest that selective blocking of the FGFR1-mediated pathway could be a promising therapeutic approach for the treatment of hepatic fibrosis.

1. Currently, there is no satis<em>factor</em>y treatment for pulmonary fibrosis. Emodin, a component in Chinese herbs, has been shown to have an antifibrotic effect on pancreatic fibrosis and liver fibrosis. In the present study, we tested the hypothesis that emodin may attenuate the development of pulmonary fibrosis. 2. Mice were randomly divided into five groups (n = 16 in each). One group was a control group; the remaining four groups were treated with intratracheal instillation of 3 mg/kg bleomycin (BLM). The following day, emodin (5, 10 or <em>20</em> mg/kg per day, p.o.) treatment was started for three of the BLM-treated groups and was continued for 21 days. The fourth BLM-treated group (and the control group) received daily 0.5% sodium carboxymethyl cellulose (placebo) by gavage over the same period. 3. Bleomycin challenge provoked severe pulmonary fibrosis, with marked increases in fibrosis fraction, hydroxyproline content and myeloperoxidase activity in lung tissue. Emodin treatment (10 and <em>20</em> mg/kg per day, p.o.) attenuated all these biochemical indices, as well as histopathological alterations induced by BLM. Furthermore, in mice injected with BLM, elevated levels of transforming <em>growth</em> <em>factor</em>-beta1, interleukin (IL)-4 and IL-13 were found in bronchoalveolar lavage fluid. These increases were significantly inhibited by 10 and <em>20</em> mg/kg per day emodin. 4. In cell culture, exposure of cells to 6.25, 12.5, 25 or 50 micromol/L emodin for 24 h decreased <em>fibroblast</em> proliferation. Treatment of cells with the same concentrations of emodin for 72 h decreased collagen production by <em>fibroblasts</em>. In addition, emodin (6.25, 12.5, 25 or 50 micromol/L) inhibited the steady state expression of alpha1 (I) procollagen and alpha2 (I) procollagen mRNA in a dose-dependent manner. 5. The results of the present study suggest that emodin may be effective in the treatment of pulmonary fibrosis.

Expression of early <em>growth</em> response protein (Egr)-1, a protein of the Egr family of zinc finger transcription <em>factors</em>, is stimulated in glucose-treated pancreatic β-cells and insulinoma cells. The purpose of this study was to elucidate the role of Egr transcription <em>factors</em> in pancreatic β-cells in vivo. To overcome the problem associated with redundancy of functions between Egr proteins, conditional transgenic mice were generated expressing a dominant-negative mutant of Egr-1 in pancreatic β-cells. The Egr-1 mutant interferes with DNA binding of all Egr proteins and thus impairs the biological functions of the entire Egr family. Expression of the Egr-1 mutant reduced expression of TGFβ and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, known target genes of Egr-1, whereas the expression of Egr-1, Egr-3, Ets-like gene-1 (Elk-1), and specificity protein-3 was not changed in the presence of the Egr-1 mutant. Expression of the homeobox protein pancreas duodenum homeobox-1, a major regulator of insulin biosynthesis, was reduced in islets expressing the Egr-1 mutant. Accordingly, insulin mRNA and protein levels were reduced by 75 or 25%, respectively, whereas expression of glucagon and somatostatin was not altered after expression of the Egr-1 mutant in β-cells. Glucose tolerance tests revealed that transgenic mice expressing the Egr-1 mutant in pancreatic β-cells displayed impaired glucose tolerance. In addition, increased caspase-3/7 activity was detected as a result of transgene expression, leading to a <em>20</em>% decrease of the size of the islets. These results show that Egr proteins play an important role in controlling insulin biosynthesis, glucose homeostasis, and islet size of pancreatic β-cells in vivo.