Although the first <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) was discovered as a mitogen on 3T3 <em>fibroblasts</em> [Gospodarowicz D: Localization of a <em>fibroblast</em> <em>growth</em> <em>factor</em> and its effect alone and with hydrocortisone on 3T3 cell <em>growth</em>. Nature 1974, 249:123-127], this name is functionally misleading. This group of secreted proteins consisting now of <em>22</em> members was composed based on common structural characteristics rather than on functional similarity. Thus, only a few members of the human FGF family promote <em>growth</em> and strictly act on <em>fibroblasts</em>. While the research in the last century firmly established FGFs as key players in development, morphogenesis, angiogenesis, hematopoiesis, and survival, this decade provided clues on FGF roles in metabolism. In particular, 'hormone-like' FGF19, FGF21, and FGF23, were shown to be involved in glucose, lipid, bile acid, phosphate, and vitamin D metabolism but the mechanisms underlying their functions as metabolic regulators are still being defined.

Fibulin-4 is a member of the fibulin family, a group of extracellular matrix proteins prominently expressed in medial layers of large veins and arteries. Involvement of the FBLN4 gene in cardiovascular pathology was shown in a murine model and in three patients affected with cutis laxa in association with systemic involvement. To elucidate the contribution of FBLN4 in human disease, we investigated two cohorts of patients. Direct sequencing of 17 patients with cutis laxa revealed no FBLN4 mutations. In a second group of <em>22</em> patients presenting with arterial tortuosity, stenosis and aneurysms, FBLN4 mutations were identified in three patients, two homozygous missense mutations (p.Glu126Lys and p.Ala397Thr) and compound heterozygosity for missense mutation p.Glu126Val and frameshift mutation c.577delC. Immunoblotting analysis showed a decreased amount of fibulin-4 protein in the <em>fibroblast</em> culture media of two patients, a finding sustained by diminished fibulin-4 in the extracellular matrix of the aortic wall on immunohistochemistry. pSmad2 and CTGF immunostaining of aortic and lung tissue revealed an increase in transforming <em>growth</em> <em>factor</em> (TGF)beta signaling. This was confirmed by pSmad2 immunoblotting of <em>fibroblast</em> cultures. In conclusion, patients with recessive FBLN4 mutations are predominantly characterized by aortic aneurysms, arterial tortuosity and stenosis. This confirms the important role of fibulin-4 in vascular elastic fiber assembly. Furthermore, we provide the first evidence for the involvement of altered TGFbeta signaling in the pathogenesis of FBLN4 mutations in humans.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family consists of <em>22</em> members and regulates a broad spectrum of biological activities by activating diverse isotypes of FGF receptor tyrosine kinases (FGFRs). Among the FGFs, FGF7 and FGF10 have been implicated in the regulation of prostate development and prostate tissue homeostasis by signaling through the FGFR2 isoform. Using conditional gene ablation with the Cre-LoxP system in mice, we demonstrate a tissue-specific requirement for FGFR2 in urogenital epithelial cells--the precursors of prostatic epithelial cells--for prostatic branching morphogenesis and prostatic <em>growth</em>. Most Fgfr2 conditional null (Fgfr2(cn)) embryos developed only two dorsal prostatic (dp) and two lateral prostatic (lp) lobes. This contrasts to wild-type prostate, which has two anterior prostatic (ap), two dp, two lp and two ventral prostatic (vp) lobes. Unlike wild-type prostates, which are composed of well developed epithelial ductal networks, the Fgfr2(cn) prostates, despite retaining a compartmented tissue structure, exhibited a primitive epithelial architecture. Moreover, although Fgfr2(cn) prostates continued to produce secretory proteins in an androgen-dependent manner, they responded poorly to androgen with respect to tissue homeostasis. The results demonstrate that FGFR2 is important for prostate organogenesis and for the prostate to develop into a strictly androgen-dependent organ with respect to tissue homeostasis but not to the secretory function, implying that androgens may regulate tissue homeostasis and tissue function differently. Therefore, Fgfr2(cn) prostates provide a useful animal model for scrutinizing molecular mechanisms by which androgens regulate prostate <em>growth</em>, homeostasis and function, and may yield clues as to how advanced-tumor prostate cells escape strict androgen regulations.

Purified type beta transforming <em>growth</em> <em>factor</em> from human platelets (TGF beta) radioiodinated with 125I-labeled Bolton and Hunter reagent was found to bind to a variety of cultured cells of both epithelial and mesenchymal origin, including normal human <em>fibroblasts</em> and keratinocytes. TGF beta binding sites have also been found on three mouse embryo-derived <em>fibroblast</em>-like cell lines with lower levels of TGF beta binding on the chemically transformed derivatives of these cell lines. A variety of human tumor cell lines was shown to have an inverse correlation between their level of TGF beta binding and their ability to form colonies in soft agar. The mouse embryo-derived AKR-2B (clone 84A) cells reached maximal binding of 125I-labeled TGF beta after 2 hr at <em>22</em> degrees C. Scatchard analysis of the equilibrium binding of TGF beta to AKR-2B (clone 84A) cells gives a Kd of 33 pM with approximately equal to 10,500 binding sites per cell. This Kd for TGF beta binding to AKR-2B (clone 84A) cells agreed well with the ED50 of 40 pM for stimulation of colony formation of these cells by TGF beta. The TGF beta binding sites on the AKR-2B cells were shown to be specific for TGF beta with no significant competition with epidermal <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>, or insulin and only a small level of competition with high concentrations of platelet-derived <em>growth</em> <em>factor</em>. Partially purified preparations with TGF beta-like activity from mouse embryos and medium conditioned by mouse embryo-derived cells competed effectively for binding to the TGF beta receptor.

OBJECTIVE

Interaction between neoplastic and stromal cells plays an important role in tumour progression. It was recently found that WNT2 was frequently overexpressed in fibroblasts isolated from tumour tissue tumour fibroblasts (TF) compared with fibroblasts from non-tumour tissue normal fibroblasts in oesophageal squamous cell carcinoma (OSCC). This study aimed to investigate the effect of TF-secreted Wnt2 in OSCC development via the tumour-stroma interaction.

METHODS

Quantitative PCR, western blotting, immunohistochemistry and immunofluorescence were used to study the expression pattern of Wnt2 and its effect on the Wnt/β-catenin pathway. A Wnt2-secreting system was established in Chinese hamster ovary cells and its conditioned medium was used to study the role of Wnt2 in cell proliferation and invasion.

RESULTS

Expression of Wnt2 could only be detected in TF but not in OSCC cancer cell lines. In OSCC tissues, Wnt2(+) cells were mainly detected in the boundary between stroma and tumour tissue or scattered within tumour tissue. In this study, Wnt2-positive OSCC was defined when five or more Wnt2(+) cells were observed in 200× microscopy field. Interestingly, Wnt2-positive OSCC (22/51 cases) was significantly associated with lymph node metastases (p=0.001), advanced TNM stage (p=0.001) and disease-specific survival (p<0.0001). Functional study demonstrated that secreted Wnt2 could promote oesophageal cancer cell growth by activating the Wnt/β-catenin signalling pathway and subsequently upregulated cyclin D1 and c-myc expression. Further study found that Wnt2 could enhance cell motility and invasiveness by inducing epithelial-mesenchymal transition.

CONCLUSIONS

TF-secreted Wnt2 acts as a growth and invasion-promoting factor through activating the canonical Wnt/β-catenin signalling pathway in oesophageal cancer cells.

This review describes the dose-dependent health benefits of resveratrol, a polyphenolic antioxidant that is found in a variety of foods, especially grape skin and red wine. Resveratrol provides diverse health benefits including cardioprotection, inhibition of low-density lipoprotein, activation of nitric oxide (NO) production, hindering of platelet aggregation [32] A.A.E. Bertelli, D.E. Giovannini, R.L. Caterina, W. Bernini, M. Migliori and M. Fregoni et al., Antiplatelet activity of cis-resveratrol, Drugs Exp Clin Res <em>22</em> (1996), pp. 61-63. View Record in Scopus | Cited By in Scopus (111) and promotion of anti-inflammatory effects. Studies have shown that at a lower dose, resveratrol acts as an anti-apoptotic agent, providing cardioprotection as evidenced by increased expression in cell survival proteins, improved postischemic ventricular recovery and reduction of myocardial infarct size and cardiomyocyte apoptosis and maintains a stable redox environment compared to control. At higher dose, resveratrol acts as a pro-apoptotic compound, inducing apoptosis in cancer cells by exerting a death signal. At higher doses, resveratrol depresses cardiac function, elevates levels of apoptotic protein expressions, results in an unstable redox environment, increases myocardial infarct size and number of apoptotic cells. At high dose, resveratrol not only hinders tumor <em>growth</em> but also inhibits the synthesis of RNA, DNA and protein, causes structural chromosome aberrations, chromatin breaks, chromatin exchanges, weak aneuploidy, higher S-phase arrest, blocks cell proliferation, decreases wound healing, endothelial cell <em>growth</em> by <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) and vascular endothelial <em>growth</em> <em>factor</em>, and angiogenesis in healthy tissue cells leading to cell death. Thus, at lower dose, resveratrol can be very useful in maintaining the human health whereas at higher dose, resveratrol has pro-apoptotic actions on healthy cells, but can kill tumor cells.

The regulation of creatine phosphokinase (CPK) expression by polypeptide <em>growth</em> <em>factors</em> has been examined in the clonal mouse muscle BC3H1 cell line. After arrest of cell <em>growth</em> by exposure to low concentrations of serum, BC3H1 cells accumulate high levels of muscle-specific proteins including CPK. The induction of this enzyme is reversible in the presence of high concentrations of fetal calf serum, which cause quiescent, differentiated cells to reenter the cell cycle. Under these conditions, the rate of CPK synthesis is drastically reduced. We show in the present communication that either pituitary-derived <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) or brain-derived FGF are as effective as serum in repressing the synthesis of CPK when added to quiescent, differentiated cells. The decrease in the rate of synthesis of CPK occurs within <em>22</em> h after the addition of pituitary FGF to the cells. Pituitary FGF had very little effect, if any, on the rate CPK degradation. The overall rate of protein synthesis and the pattern of synthesis of the major polypeptides made by these cells was not altered by the addition of FGF. Although pituitary FGF was mitogenic for BC3H1 cells, the rate of cell <em>growth</em> was not absolutely correlated with the extent of repression of CPK. Brain-derived FGF fully repressed CPK induction under conditions where it showed no significant mitogenic activity. These results show that the expression of a muscle-specific protein, CPK, can be controlled by a single defined polypeptide <em>growth</em> <em>factor</em> in fully differentiated cultures, and that initiation of cell division is not required for their regulation to take place.

Semaphorin-3B (sema3B) and semaphorin-3F (sema3F) are secreted tumor suppressors of lung cancer. Sema3F functions as an antiangiogenic <em>factor</em> that repels endothelial cells and compromises their proliferation/survival. However, tumor cells expressing either endogenous or recombinant sema3B fail to repel endothelial cells efficiently. Sema3B found in the conditioned medium of such cells is almost completely cleaved by furin-like pro-protein convertases, generating inactive 61- and <em>22</em>-kDa fragments. We have generated a sema3B variant that was point mutated at the cleavage site (sema3B-m), thereby conferring partial resistance to cleavage. Conditioned medium from HEK293 cells expressing sema3b-m and conditioned medium of HEK293 cells expressing sema3B contained similar concentrations of semaphorin but sema3B-m was cleaved much less than sema3B. In contrast to HEK293 cells expressing native sema3B, cells expressing sema3b-m strongly repel endothelial cells. Conditioned medium from sema3B-m-expressing cells rapidly caused disassembly of focal adhesions and a collapse of the actin cytoskeleton of endothelial cells, inhibited vascular endothelial <em>growth</em> <em>factor</em>-induced phosphorylation of extracellular signal-regulated kinase 1/2, induced apoptosis of endothelial cells, and inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay more potently than conditioned medium from cells expressing sema3B. Furthermore, HEK293 cells expressing sema3B-m inhibited basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-induced angiogenesis in vivo much more potently than cells expressing sema3B. Repulsion of human umbilical vascular endothelial cells by sema3B-m was mediated primarily by the neuropilin-1 (np1) receptor but sema3B-m was also able to transduce signals via neuropilin-2 (np2). These results suggest that up-regulation of furin-like pro-protein convertases in malignant cells may enable tumors to evade the antiangiogenic effects of sema3B.

The novel hematopoietic <em>growth</em> <em>factor</em> FLT3 ligand (FL) is the cognate ligand for the FLT3, tyrosine kinase receptor (R), also referred to as FLK-2 and STK-1. The FLT3R belongs to a family of receptor tyrosine kinases involved in hematopoiesis that also includes KIT, the receptor for SCF (stem cell <em>factor</em>), and FMS. the receptor for M-CSF (macrophage colony- stimulating <em>factor</em>). Restricted FLT3R expression was seen on human and murine hematopoietic progenitor cells. In functional assays recombinant FL stimulated the proliferation and colony formation of human hematopoietic progenitor cells, i.e. CD34+ cord and peripheral blood, bone marrow and fetal liver cells. Synergy was reported for co-stimulation with G-CSF (granulocyte-CSF). GM-CSF (granulocyte-macrophage CSF), M-CSF, interleukin-3 (IL-3), PIXY-321 (an IL-3/GM-CSF fusion protein) and SCF. In the mouse, FL potently enhanced <em>growth</em> of various types of progenitor/precursor cells in synergy with G-CSF, GM-CSF, M-CSF, IL-3, IL-6, IL-7, IL-11, IL-12 and SCF. The well-documented involvement of this ligand-receptor pair in physiological hematopoiesis brought forth the question whether FLT3R and FL might also have a role in the pathobiology of leukemia. At the mRNA level FLT3R was expressed by most (80-100%) cases of AML (acute myeloid leukemia) throughout the different morphological subtypes (MO-M7), of ALL(acute lymphoblastic leukemia) of the immunological subtypes T-ALL and BCP-ALL (B cell precursor ALL including pre-pre B-ALL, cALL and pre B-ALL), of AMLL (acute mixed-lineage leukemia), and of CML (chronic myeloid leukemia) in lymphoid or mixed blast crisis. Analysis of cell surface expression of FLT3R by flow cytometry confirmed these observations for AML (66% positivity when the data from all studies are combined), BCP-ALL (64%) and CML lymphoid blast crisis (86%) whereas less than 30% of T-ALL were FLT3R+. The myeloid, monocytic and pre B cell type categories also contained the highest proportions of FLT3R+ leukemia cell lines . In contrast to the selective expression of the receptor, FL expression was detected in 90-100% of the various cell types of leukemia cell lines from all hematopoietic cell lineages. The potential of FL to induce proliferation of leukemia cells in vitro was also examined in primary and continuously cultured leukemia cells. The data on FL-stimulated leukemia cell <em>growth</em> underline the extensive heterogeneity of primary AML and ALL samples in terms of cytokine-inducible DNA synthesis that has been seen with other effective cytokines. While the majority of T-ALL (0-33% of the cases responded proliferatively; mean 11%) and BCP-ALL (0-30%; mean 20%) failed to proliferate in the presence of FL despite strong expression of surface FLT3R, FL caused a proliferative response in a significantly higher percentage of AML cases (<em>22</em>-90%; mean 53%). In the panel of leukemia cell lines examined only myeloid and monocytic <em>growth</em> <em>factor</em>- dependent cell lines increased their proliferation upon incubation with FL, whereas all <em>growth</em> <em>factor</em>-independent cell lines were refractory to stimulation. Combinations of FL with G-CSF, GM-CSF, M-CSF, IL-3, PIXY- 321 or SCF and FL with IL-3 or IL-7 had synergistic or additive mitogenic effects on primary AML and ALL cells, respectively. The potent stimulation of the myelomonocytic cell lines was further augmented by addition of bFGF (basic <em>fibroblast</em> <em>growth</em> <em>factor</em>), GM-CSF, IL-3 or SCF. The inhibitory effects of TGF-beta 1 (transforming <em>growth</em> <em>factor</em>-beta 1) on FL- supported proliferation were abrogated by bFGF. Taken together, these results demonstrate the expression of functional FLT3R capable of mediating FL- dependent mitogenic signaling in a subset of AML and ALL cases further underline the heterogeneity of AML and ALL samples in their proliferative response to cytokine.

Type I and type III procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced procollagen synthesis. In the present study, we investigated type I procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I procollagen gene expression by in situ hybridization and for type I procollagen protein by immunostaining. Both mRNA and protein were reduced ( approximately 65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I procollagen production was because of inherently reduced capacity of skin <em>fibroblasts</em> in severely photodamaged forearm skin to synthesize procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I procollagen synthesis. For these studies, <em>fibroblasts</em> from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of <em>fibroblasts</em> were isolated from the two skin sites. <em>Fibroblasts</em> from the two sites had similar <em>growth</em> capacities and produced virtually identical amounts of type I procollagen protein. These findings indicate that the lack of type I procollagen synthesis in sun-damaged skin is not because of irreversible damage to <em>fibroblast</em> collagen-synthetic capacity. It follows, therefore, that <em>factors</em> within the severely photodamaged skin may act in some manner to inhibit procollagen production by cells that are inherently capable of doing so. Interactions between <em>fibroblasts</em> and the collagenous extracellular matrix regulate type I procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. <em>Fibroblasts</em> are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is approximately 3.6-fold greater in photodamaged skin than in sun-protected skin, and some <em>fibroblasts</em> are surrounded by debris. To model this situation, skin <em>fibroblasts</em> were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal <em>fibroblasts</em>, whereas intact collagen did not. <em>Fibroblasts</em> cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (<em>22</em> and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) <em>fibroblasts</em> from photoaged and sun-protected skin are similar in their capacities for <em>growth</em> and type I procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I procollagen synthesis.

Synapses form after <em>growing</em> axons recognize their appropriate targets. The subsequent assembly of aligned pre and postsynaptic specializations is critical for synaptic function. This highly precise apposition of presynaptic elements (i.e. active zones) to postsynaptic specializations (i.e. neurotransmitter receptor clusters) strongly suggests that communication between the axon and target is required for synaptic differentiation. What trans-synaptic <em>factors</em> drive such differentiation at vertebrate synapses? First insights into the answers to this question came from studies at the neuromuscular junction (NMJ), where axon-derived agrin and muscle-derived laminin beta2 induce post and presynaptic differentiation, respectively. Recent work has suggested that axon- and target-derived <em>factors</em> similarly drive synaptic differentiation at central synapses. Specifically, WNT-7a, neuroligin, synaptic cell adhesion molecule (SynCAM) and <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>22</em> (FGF-<em>22</em>) have all been identified as target-derived presynaptic organizers, whereas axon-derived neuronal activity regulated pentraxin (Narp), ephrinB and neurexin reciprocally co-ordinate postsynaptic differentiation. In addition to these axon- and target-derived inducers of synaptic differentiation, <em>factors</em> released from glial cells have also been implicated in regulating synapse assembly. Together, these recent findings have profoundly advanced our understanding of how precise appositions are established during vertebrate nervous system development.

Fibrillin-1 (FBN1) contains 47 epidermal <em>growth</em> <em>factor</em> (EGF)-like domains characterized by six conserved cysteine residues. Cysteine substitutions that disrupt one of the three disulfide bonds are frequent causes of Marfan syndrome (MFS). We identified 19 new substitutions involving cysteine residues in each of the six positions of EGF-like domains. Allele-specific mRNA assays revealed equal abundance of mutant and normal FBN1 transcripts in all 10 individuals studied. Quantitative pulse-chase analysis of fibrillin protein was performed on 25 mutant <em>fibroblast</em> strains with substitutions of <em>22</em> different cysteine residues in 18 different EGF-like domains spanning the entire gene. Normal synthesis and stability of mutant fibrillin molecules was seen in 20/25 individuals, 11 of whom showed delayed intracellular processing and/or secretion. In the remaining five cases, the mutant protein was apparently unstable. In four of these five cases, the second or third disulfide bond of EGF-like domains immediately preceding an 8-cysteine or hybrid domain was affected. All but two mutations caused severe reduction of matrix deposition, which was attributed to a dominant-negative effect of mutant molecules. For genotype/phenotype comparisons, clinical data on 25 probands and 19 mutation-positive family members were analyzed. Ocular manifestations were among the most consistent features (ectopia lentis in 86%, myopia in 80%). Nine mutations encoded by exons 26-32 resulted in early-onset classic MFS and, in one case, neonatal-lethal MFS. Mutations outside this region were associated with variable clinical phenotypes, including individuals with fibrillinopathies not meeting diagnostic criteria for MFS.

<em>Growth</em> <em>factors</em> secreted by platelets and macrophages may play roles in atherogenesis and in wound repair. The multiple biologic effects of these <em>factors</em> are being studied extensively in vitro, but their roles in vivo are relatively unexplored. The cellular responses to platelet-derived <em>growth</em> <em>factor</em> (PDGF), transforming <em>growth</em> <em>factor</em> beta (TGF beta), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and epidermal <em>growth</em> <em>factor</em> (EGF) were examined in a wound chamber model in rats. <em>Growth</em> <em>factors</em> were emulsified in bovine dermal collagen suspensions, placed in 1 X 30-mm porous polytetrafluoroethylene tubes, inserted subcutaneously, and removed after 10 days. The presence of PDGF (400 ng), TGF beta (200 ng), or bFGF (100 ng) increased the DNA content of the chambers two- to sixfold, compared with controls. Regardless of dose, EGF (100-800 ng) did not affect the DNA content. The increases in DNA observed for PDGF, TGF beta, or bFGF resulted from accumulations of varying numbers of <em>fibroblasts</em>, capillaries, macrophages, and leukocytes in 10-day chambers. The addition of 250 micrograms/ml heparin to the collagen suspension potentiated the response to PDGF and bFGF, but not to TGF beta or EGF. The clearance of 125I-labeled <em>growth</em> <em>factors</em> from the chambers was biphasic. After an initial rapid phase, the remaining <em>growth</em> <em>factor</em> was slowly cleared. The half-life of the initial phase was rapid for PDGF (12 hours) and bFGF (9 hours) and somewhat slower for TGF beta (<em>22</em> hours). There was no difference in the rate of clearance between collagen and collagen/heparin matrices for any of the <em>growth</em> <em>factors</em> examined. These studies demonstrate that PDGF, bFGF, and TGF beta can induce granulation tissue development in normal animals. The similarity in cellular responses to three peptides with differing in vitro actions suggests that the responses observed at 10 days reflect a secondary process, possibly mediated by effector cells such as macrophages, lymphocytes, or granulocytes that are attracted into the chamber by each <em>growth</em> <em>factor</em>, rather than a direct effect of the <em>factors</em> themselves.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) comprise a family of <em>22</em> distinct proteins with pleiotropic signaling functions in development and homeostasis. These functions are mediated principally by four <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs), members of the receptor tyrosine kinase family, with heparin glycosaminoglycan as an important co<em>factor</em>. Developmental studies in chick and mouse highlight the critical role of FGF-receptor signaling in multiple phases of limb development, including the positioning of the limb buds, the maintenance of limb bud out<em>growth</em>, the detailed patterning of the limb elements, and the <em>growth</em> of the long bones. Corroborating these important roles, mutations of two members of the FGFR family (FGFR1 and FGFR2) are associated with human disorders of limb patterning; in addition, mutations of FGFR3 and FGF23 affect <em>growth</em> of the limb bones. Analysis of FGFR2 mutations in particular reveals a complex pattern of genotype/phenotype correlation, which will be reviewed in detail. Circumstantial evidence suggests that the more severe patterning abnormalities are mediated by illegitimate paracrine signaling in the mesoderm, mediated by FGF10 or by a related FGF, and this is beginning to gain some experimental support. A further test of this hypothesis is provided by a unique family segregating two FGFR2 mutations in cis (S252L; A315S), in which severe syndactyly occurs in the absence of the craniosynostosis that typically accompanies FGFR2 mutations.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGF) are a family of ligands that bind to four different types of cell surface receptor entitled, FGFR1, FGFR2, FGFR3 and FGFR4. These receptors differ in their ligand binding affinity and tissue distribution. The prototypical receptor structure is that of an extracellular region comprising three immunoglobulin (Ig)-like domains, a hydrophobic transmembrane segment and a split intracellular tyrosine kinase domain. Alternative gene splicing affecting the extracellular third Ig loop also creates different receptor isoforms entitled FGFRIIIb and FGFRIIIc. Somatic <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) mutations are implicated in different types of cancer and germline FGFR mutations occur in developmental syndromes particularly those in which craniosynostosis is a feature. The mutations found in both conditions are often identical. Many somatic FGFR mutations in cancer are gain-of-function mutations of established preclinical oncogenic potential. Gene amplification can also occur with 19-<em>22</em>% of squamous cell lung cancers for example having amplification of FGFR1. Ontologic comparators can be informative such as aberrant spermatogenesis being implicated in both spermatocytic seminomas and Apert syndrome. The former arises from somatic FGFR3 mutations and Apert syndrome arises from germline FGFR2 mutations. Finally, therapeutics directed at inhibiting the FGF/FGFR interaction are a promising subject for clinical trials.

Lipocortin I is a high affinity substrate for the epidermal <em>growth</em> <em>factor</em> receptor/kinase that can be purified by reversible Ca2+-dependent association with cellular particulate fractions. Purified human lipocortin I was shown by ultraviolet spectroscopy to undergo conformational changes in response to high concentrations of Ca2+ (30-700 microM). Equilibrium dialysis of lipocortin I alone against 45Ca2+ showed barely detectable binding at 5-60 microM Ca2+. However, in the presence of phosphatidylserine, binding was significantly enhanced, and Scatchard analysis of the binding data showed that lipocortin I contained four Ca2+-binding sites with an apparent Kd of 75 microM. Lipocortin I associated with phosphatidylserine vesicles and F-actin in a Ca2+-dependent manner with half-maximal association occurring at <em>22</em> and 180 microM Ca2+, respectively. Neither phosphatidylcholine vesicles nor intact cultured <em>fibroblasts</em> bound detectable amounts of lipocortin I. Phosphorylation of lipocortin I by the epidermal <em>growth</em> <em>factor</em>-stimulated kinase in A431 membranes required Ca2+ (half-maximum = 28 microM) in the presence of Mg2+, but phosphorylation was not Ca2+-dependent in the presence of Mn2+. The difference in Ca2+ requirement for phosphorylation probably is a reflection of the fact that Mn2+, but not Mg2+, promoted the association of lipocortin I with phospholipid. Although the physiological role of lipocortin I phosphorylation has not yet been determined, it was observed that phosphorylation of Tyr-21 reduced by 5-fold the amount of Ca2+ required for half-maximal association of the protein with phosphatidylserine vesicles.

Congenital cutis laxa, a rare syndrome with marked skin laxity and pulmonary and cardiovascular compromise, is due to defective elastic fiber formation. In several cases, skin <em>fibroblast</em> tropoelastin production is markedly reduced yet reversed in vitro by transforming <em>growth</em> <em>factor</em>-beta treatment. We previously showed that this reversal was due to elastin mRNA stabilization in one cell strain, and here this behavior was confirmed in skin <em>fibroblasts</em> from two generations of a second family. cDNA sequencing and heteroduplex analysis of elastin gene transcripts from three <em>fibroblast</em> strains in two kindreds now identify two frameshift mutations (2012DeltaG and 2039DeltaC) in elastin gene exon 30, thus leading to missense C termini. No other mutations were present in the ELN cDNA sequences of all three affected individuals. Transcripts from both alleles in each kindred were unstable and responsive to transforming <em>growth</em> <em>factor</em>-beta. Exons <em>22</em>, 23, 26A, and 32 were always absent. Since exon 30 underwent alternative splicing in <em>fibroblasts</em>, we speculate that a differential splicing pattern could conceivably lead to phenotypic rescue. These two dominant-acting, apparently de novo mutations in the elastin gene appear to be responsible for qualitative and quantitative defects in elastin, resulting in the cutis laxa phenotype.

OBJECTIVE

Fibroblast growth factor receptor 3 (FGFR3) mutations have been associated with achondroplastic syndromes and urinary bladder carcinomas. Here we describe changes in FGFR3 mRNA and protein expression in transitional carcinomas and determine the effect of monoclonal antibodies against FGFR3 in RT-112 cell line proliferation.

METHODS

We used microarray tools to evaluate FGFR3 mRNA expression in 22 urinary bladder carcinomas at different stages (noninvasive pTa, lamina propria invasive pT1, and muscular invasive pT2) and 7 nonneoplastic tissue controls. FGFR3 protein expression was evaluated by Western blotting in 15 different carcinomas and 3 nonneoplastic controls. Two hundred thirty-seven urinary bladder and renal pelvis carcinomas and 21 negative controls were tested on tissue microarrays by immunohistochemistry. The effect on cell proliferation in the RT-112 bladder cancer cell line of monoclonal antibodies against FGFR3 was also evaluated.

RESULTS

Overexpression of FGFR3 mRNA was found in pTa and pT1 stage carcinomas (fold change >8) and in pT2 carcinomas (fold change >4). Nonneoplastic urinary bladder samples do not express FGFR3 protein. However, 83% of pTa, 100% of pT1, and 50% of pT2 carcinomas expressed FGFR3 as determined by Western blotting. By immunohistochemistry, FGFR3 was positive in 71.4% of pTa, 72% of pT1, and 49.2% of pT2 cases as well as 61.5% of upper urinary tract carcinomas. Proliferation of the RT-112 cell line was inhibited with monoclonal antibodies against FGFR3.

CONCLUSIONS

FGFR3 seems to play an important role in transitional cell carcinoma development. Our results suggest that FGFR3 antagonists could be developed as possible therapeutics for treatment of urinary tract carcinoma.

The heparan sulfate (HS) chains of proteoglycans are a key regulatory component of the extracellular matrices of animal cells, including the pericellular matrix around the plasma membrane. In these matrices they regulate transport, gradient formation, and effector functions of over 400 proteins central to cell communication. HS from different matrices differs in its selectivity for its protein partners. However, there has been no direct test of how HS in the matrix regulates the transport of its partner proteins. We address this issue by single molecule imaging and tracking in <em>fibroblast</em> pericellular matrix of <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2), stoichiometrically labelled with small gold nanoparticles. Transmission electron microscopy and photothermal heterodyne imaging (PHI) show that the spatial distribution of the HS-binding sites for FGF2 in the pericellular matrix is heterogeneous over length scales ranging from <em>22</em> nm to several µm. Tracking of individual FGF2 by PHI in the pericellular matrix of living cells demonstrates that they undergo five distinct types of motion. They spend much of their time in confined motion (∼110 nm diameter), but they are not trapped and can escape by simple diffusion, which may be slow, fast, or directed. These substantial translocations (µm) cover distances far greater than the length of a single HS chain. Similar molecular motion persists in fixed cells, where the movement of membrane PGs is impeded. We conclude that FGF2 moves within the pericellular matrix by translocating from one HS-binding site to another. The binding sites on HS chains form non-random, heterogeneous networks. These promote FGF2 confinement or substantial translocation depending on their spatial organisation. We propose that this spatial organisation, coupled to the relative selectivity and the availability of HS-binding sites, determines the transport of FGF2 in matrices. Similar mechanisms are likely to underpin the movement of many other HS-binding effectors.

BACKGROUND

Lenvatinib is an oral inhibitor of vascular endothelial growth factor receptor 1-3, fibroblast growth factor receptor 1-4, platelet-derived growth factor receptor alpha, RET, and KIT. This phase 2, single-arm, open-label multicenter study evaluated lenvatinib in advanced hepatocellular carcinoma (HCC).

METHODS

Patients with histologically/clinically confirmed advanced HCC who did not qualify for surgical resection or local therapies received lenvatinib at a dosage of 12 mg once daily (QD) in 28-day cycles. The primary efficacy endpoint was time to progression (TTP) per modified Response Evaluation Criteria in Solid Tumors v1.1; secondary efficacy endpoints included objective response rate (ORR), disease control rate (DCR), and overall survival (OS).

RESULTS

Between July 2010 and June 2011, 46 patients received lenvatinib at sites across Japan and Korea. The median TTP, as determined by independent radiological review, was 7.4 months [95 % confidence interval (CI): 5.5-9.4]. Seventeen patients (37 %) had partial response and 19 patients (41 %) had stable disease (ORR: 37 %; DCR: 78 %). Median OS was 18.7 months (95 % CI: 12.7-25.1). The most common any-grade adverse events (AEs) were hypertension (76 %), palmar-plantar erythrodysesthesia syndrome (65 %), decreased appetite (61 %), and proteinuria (61 %). Dose reductions and discontinuations due to AEs occurred in 34 (74 %) and 10 patients (22 %), respectively. Median body weight was lower in patients with an early (<30 days) dose withdrawal or reduction than in those without.

CONCLUSIONS

Lenvatinib 12-mg QD showed clinical activity and acceptable toxicity profiles in patients with advanced HCC, but early dose modification was necessary in patients with lower body weight. Further development of lenvatinib in HCC should consider dose modification by body weight. TRIAL REGISTRATION ID: www.ClinicalTrials.gov NCT00946153.

Catecholamine stimulation of alpha1-adrenoceptors exerts <em>growth</em> <em>factor</em>-like activity, mediated by generation of reactive oxygen species, on arterial smooth muscle cells and adventitial <em>fibroblasts</em> and contributes to hypertrophy and hyperplasia in models of vascular injury and disease. Adrenergic trophic activity also contributes to flow-mediated positive arterial remodeling by augmenting proliferation and leukocyte accumulation. To further examine this concept, we studied whether catecholamines contribute to collateral <em>growth</em> and angiogenesis in hindlimb insufficiency. Support for this hypothesis includes the above-mentioned studies, evidence that ischemia augments norepinephrine release from sympathetic nerves, and proposed involvement of reactive oxygen species in angiogenesis and collateral <em>growth</em>. Mice deficient in catecholamine synthesis [by gene deletion of dopamine beta-hydroxylase (DBH-/-)] were studied. At 3 wk after femoral artery ligation, increases in adductor muscle perfusion were similar in DBH-/- and wild-type mice, whereas recovery of plantar perfusion and calf microsphere flow were attenuated, although not significantly. Preexisting collaterals in adductor of wild-type mice showed increases in lumen diameter (60%) and medial and adventitial thickness (57 and 119%, P < 0.05 here and below). Lumen diameter increased similarly in DBH-/- mice (52%); however, increases in medial and adventitial thicknesses were reduced (30 and 65%). Leukocyte accumulation in the adventitia/periadventitia of collaterals was 39% less in DBH-/- mice. Increased density of alpha-smooth muscle actin-positive vessels in wild-type adductor (45%) was inhibited in DBH-/- mice (2%). Although both groups experienced similar atrophy in the gastrocnemius (approximately <em>22</em>%), the increase in capillary-to-muscle fiber ratio in wild-type mice (21%) was inhibited in DBH-/- mice (7%). These data suggest that catecholamines may contribute to collateral <em>growth</em> and angiogenesis in tissue ischemia.

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25-D(3)) has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, its calcemic effect in vivo limits its therapeutic applications. Here, we report the efficacy of <em>22</em>-oxa-1alpha,25-dihydroxyvitamin D(3) (<em>22</em>-oxa-1alpha,25-D(3)), a low calcemic analog of vitamin D, against the development of metastatic lung carcinoma after an intravenous injection of green fluorescent protein-transfected Lewis lung carcinoma (LLC-GFP) cells in C57BL/6 mice. The mice injected with tumor cells were implanted simultaneously with osmotic minipumps containing either 1alpha,25-D(3), <em>22</em>-oxa-1alpha,25-D(3) or vehicle. The 1alpha,25-D(3) treatment group had been hypercalcemic, but the <em>22</em>-oxa-1alpha,25-D(3) and vehicle treatment groups remained normocalcemic for the duration of the experiment. The total number of lung metastases, lung weight and the expression of GFP mRNA in the lung were markedly decreased in 1alpha,25-D(3) and <em>22</em>-oxa-1alpha,25-D(3)-treated mice. In the in vitro experiment, 1alpha,25-D(3) and <em>22</em>-oxa-1alpha,25-D(3) reduced the expression of matrix metalloproteinase (MMP)-2, MMP-9, vascular endothelial <em>growth</em> <em>factor</em> and parathyroid hormone-related protein in LLC-GFP cells. Furthermore, in the angiogenesis assay, the number of tumor cells or basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-induced angiogenesis was reduced in 1alpha,25-D(3) and <em>22</em>-oxa-1alpha,25-D(3)-treated mice. Moreover, using a new experimental model of vitamin D receptor (VDR) null mutant (VDR(-/-)) mice with corrected hypocalcemia and hypervitaminosis D, we examine the anti-cancer effect of <em>22</em>-oxa-1alpha,25-D(3) without other functions induced by <em>22</em>-oxa-1alpha,25-D(3) in the host. In the VDR(-/-) mice, <em>22</em>-oxa-1alpha,25-D(3) directly inhibited the metastatic activity of LLC-GFP cells in a dose-dependent manner without exerting a direct influence on the calcemic activity or other actions regulated by <em>22</em>-oxa-1alpha,25-D(3) in the host. These results indicate that the inhibition of metastasis and angiogenesis-inducing activity in cancer cells seemed to be a major mechanism responsible for the anti-cancer effects of <em>22</em>-oxa-1alpha,25-D(3). Our findings show that <em>22</em>-oxa-1alpha,25-D(3) is beneficial for the prevention of metastasis in lung carcinoma.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2), also known as basic FGF, is a multi-functional <em>growth</em> <em>factor</em>. One of the <em>22</em>-member FGF family, it signals through receptor tyrosine kinases encoding FGFR1-4. FGF2 activates FGFRs in cooperation with heparin or heparin sulfate proteoglycan to induce its pleiotropic effects in different tissues and organs, which include potent angiogenic effects and important roles in the differentiation and function of the central nervous system (CNS). FGF2 is crucial to development of the CNS, which explains its importance in adult neurogenesis. During development, high levels of FGF2 are detected from neurulation onwards. Moreover, developmental expression of FGF2 and its receptors is temporally and spatially regulated, concurring with development of specific brain regions including the hippocampus and substantia nigra pars compacta. In adult neurogenesis, FGF2 has been implicated based on its expression and regulation of neural stem and progenitor cells in the neurogenic niches, the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus. FGFR1 signaling also modulates inflammatory signaling through the surface glycoprotein CD200, which regulates microglial activation. Because of its importance in adult neurogenesis and neuroinflammation, manipulation of FGF2/FGFR1 signaling has been a focus of therapeutic development for neurodegenerative disorders, such as Alzheimer's disease, multiple sclerosis, Parkinson's disease and traumatic brain injury. Novel strategies include intranasal administration of FGF2, administration of an NCAM-derived FGFR1 agonist, and chitosan-based nanoparticles for the delivery of FGF2 in pre-clinical animal models. In this review, we highlight current research towards therapeutic interventions targeting FGF2/FGFR1 in neurodegenerative disorders.

Angiogenesis, the process of new blood vessel formation from pre-existing ones, plays a key role in various physiological and pathological conditions, including embryonic development, wound repair, inflammation, and tumor <em>growth</em>. The 1980s saw for the first time the identification, purification, and sequencing of the two prototypic heparin-binding angiogenic <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF) 1 and 2. Since then, <em>22</em> structurally-related members of the FGF family and different classes of FGF receptors have been identified. Several experimental evidences point to a role for various FGFs in the neovascularization process that takes place in inflammation, angioproliferative diseases, and tumor <em>growth</em>. Thus, the FGF/FGF receptor system represents a target for the development of anti-angiogenic therapies. Purpose of this review is to summarize the different modalities that have been approached to impair the pro-angiogenic activity of the FGF/FGF receptor system and discuss their possible therapeutic implications.