Blockade of vascular smooth muscle cell proliferation and intimal thickening after balloon injury by the sulfated oligosaccharide PI-88: Phosphomannopentaose sulfate directly binds FGF-2, blocks cellular signaling, and inhibits proliferation

Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (K-i 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.

FGF-23 as a Predictor of Renal Outcome in Diabetic Nephropathy

Background and objectives Fibroblast growth factor 23 (FGF-23) has emerged as a new factor in mineral metabolism in chronic kidney disease (CKD). An important regulator of phosphorus homeostasis, FGF-23 has been shown to independently predict CKD progression in nondiabetic renal disease. We analyzed the relation between FGF-23 and renal outcome in diabetic nephropathy (DN). Design, setting, participants, & measurements DN patients participating in a clinical trial (enalapril+placebo versus enalapril+losartan) had baseline data collected and were followed until June 2009 or until the primary outcome was reached. Four patients were lost to follow-up. The composite primary outcome was defined as death, doubling of serum creatinine, and/or dialysis need. Results At baseline, serum FGF-23 showed a significant association with serum creatinine, intact parathyroid hormone, proteirturia, urinary fractional excretion of phosphate, male sex, and race. Interestingly, FGF-23 was not related to calcium, phosphorus, 25OH-vitamin D, or 24-hour urinary phosphorus. Mean follow-up time was 30.7 +/- 10 months. Cox regression showed that FGF-23 was an independent predictor of the primary outcome, even after adjustment for creatinine clearance and intact parathyroid hormone (10 pg/ml FGF-23 increase = hazard ratio, 1.09; 95% CI, 1.01 to 1.16, P = 0.02). Finally, Kaplan-Meier analysis showed a significantly higher risk of the primary outcome in patients with FGF-23 values of >70 pg/ml. Conclusions FGF-23 is a significant independent predictor of renal outcome in patients with macroalbuminuric DN. Further studies should clarify whether this relation is causal and whether FGF-23 should be a new therapeutic target for CKD prevention. Clin J Am Soc Nephrol 6: 241-247, 2011. doi: 10.2215/CJN.04250510

Dilated cardiomyopathy and impaired cardiac hypertrophic response to angiotensin II in mice lacking FGF-2.

FGF-2 has been implicated in the cardiac response to hypertrophic stimuli. Angiotensin II (Ang II) contributes to maintain elevated blood pressure in hypertensive individuals and exerts direct trophic effects on cardiac cells. However, the role of FGF-2 in Ang II-induced cardiac hypertrophy has not been established. Therefore, mice deficient in FGF-2 expression were studied using a model of Ang II-dependent hypertension and cardiac hypertrophy. Echocardiographic measurements show the presence of dilated cardiomyopathy in normotensive mice lacking FGF-2. Moreover, hypertensive mice without FGF-2 developed no compensatory cardiac hypertrophy. In wild-type mice, hypertrophy was associated with a stimulation of the c-Jun N-terminal kinase, the extracellular signal regulated kinase, and the p38 kinase pathways. In contrast, mitogen-activated protein kinase (MAPK) activation was markedly attenuated in FGF-2-deficient mice. In vitro, FGF-2 of fibroblast origin was demonstrated to be essential in the paracrine stimulation of MAPK activation in cardiomyocytes. Indeed, fibroblasts lacking FGF-2 expression have a defective capacity for releasing growth factors to induce hypertrophic responses in cardiomyocytes. Therefore, these results identify the cardiac fibroblast population as a primary integrator of hypertrophic stimuli in the heart, and suggest that FGF-2 is a crucial mediator of cardiac hypertrophy via autocrine/paracrine actions on cardiac cells.

Role of fibroblast growth factor (FGF) signaling in the neuroendocrine control of human reproduction.

Fibroblast growth factor (FGF) signaling is critical for a broad range of developmental processes. In 2003, Fibroblast growth factor receptor 1 (FGFR1) was discovered as a novel locus causing both forms of isolate GnRH Deficiency, Kallmann syndrome [KS with anosmia] and normosmic idiopathic hypogonadotropic hypogonadism [nIHH] eventually accounting for approximately 10% of gonadotropin-releasing hormone (GnRH) deficiency cases. Such cases are characterized by a broad spectrum of reproductive phenotypes from severe congenital forms of GnRH deficiency to reversal of HH. Additionally, the variable expressivity of both reproductive and non-reproductive phenotypes among patients and family members harboring the identical FGFR1 mutations has pointed to a more complex, oligogenic model for GnRH deficiency. Further, reversal of HH in patients carrying FGFR1 mutations suggests potential gene-environment interactions in human GnRH deficiency disorders.

FGF receptors control vitamin D and phosphate homeostasis by mediating renal FGF-23 signaling and regulating FGF-23 expression in bone.

The functional interaction between fibroblast growth factor 23 (FGF-23) and Klotho in the control of vitamin D and phosphate homeostasis is manifested by the largely overlapping phenotypes of Fgf23- and Klotho-deficient mouse models. However, to date, targeted inactivation of FGF receptors (FGFRs) has not provided clear evidence for an analogous function of FGFRs in this process. Here, by means of pharmacologic inhibition of FGFRs, we demonstrate their involvement in renal FGF-23/Klotho signaling and elicit their role in the control of phosphate and vitamin D homeostasis. Specifically, FGFR loss of function counteracts renal FGF-23/Klotho signaling, leading to deregulation of Cyp27b1 and Cyp24a1 and the induction of hypervitaminosis D and hyperphosphatemia. In turn, this initiates a feedback response leading to high serum levels of FGF-23. Further, we show that FGFR inhibition blocks Fgf23 transcription in bone and that this is dominant over vitamin D-induced Fgf23 expression, ultimately impinging on systemic FGF-23 protein levels. Additionally, we identify Fgf23 as a specific target gene of FGF signaling in vitro. Thus, in line with Fgf23- and Klotho-deficient mouse models, our study illustrates the essential function of FGFRs in the regulation of vitamin D and phosphate levels. Further, we reveal FGFR signaling as a novel in vivo control mechanism for Fgf23 expression in bone, suggesting a dual function of FGFRs in the FGF-23/Klotho pathway leading to vitamin D and phosphate homeostasis.

Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome.

Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, which is characterized by cleft palate and severe defects of the skin, is an autosomal dominant disorder caused by mutations in the gene encoding transcription factor p63. Here, we report the generation of a knock-in mouse model for AEC syndrome (p63(+/L514F) ) that recapitulates the human disorder. The AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting progenitor cell expansion during ectodermal development leading to a defective epidermal stem cell compartment. These phenotypes are associated with impairment of fibroblast growth factor (FGF) signalling resulting from reduced expression of Fgfr2 and Fgfr3, direct p63 target genes. In parallel, a defective stem cell compartment is observed in humans affected by AEC syndrome and in Fgfr2b(-/-) mice. Restoring Fgfr2b expression in p63(+/L514F) epithelial cells by treatment with FGF7 reactivates downstream mitogen-activated protein kinase signalling and cell proliferation. These findings establish a functional link between FGF signalling and p63 in the expansion of epithelial progenitor cells and provide mechanistic insights into the pathogenesis of AEC syndrome.

FGF signaling controls caudal hindbrain specification through Ras-ERK1/2 pathway

Background: During early steps of embryonic development the hindbrain undergoes a regionalization process along the anterior-posterior (AP) axis that leads to a metameric organization in a series of rhombomeres (r). Refinement of the AP identities within the hindbrain requires the establishment of local signaling centers, which emit signals that pattern territories in their vicinity. Previous results demonstrated that the transcription factor vHnf1 confers caudal identity to the hindbrain inducing Krox20 in r5 and MafB/Kreisler in r5 and r6, through FGF signaling [1].Results: We show that in the chick hindbrain, Fgf3 is transcriptionally activated as early as 30 min after mvHnf1 electroporation, suggesting that it is a direct target of this transcription factor. We also analyzed the expression profiles of FGF activity readouts, such as MKP3 and Pea3, and showed that both are expressed within the hindbrain at early stages of embryonic development. In addition, MKP3 is induced upon overexpression of mFgf3 or mvHnf1 in the hindbrain, confirming vHnf1 is upstream FGF signaling. Finally, we addressed the question of which of the FGF-responding intracellular pathways were active and involved in the regulation of Krox20 and MafB in the hindbrain. While Ras-ERK1/2 activity is necessary for MKP3, Krox20 and MafB induction, PI3K-Akt is not involved in that process.Conclusion: Based on these observations we propose that vHnf1 acts directly through FGF3, and promotes caudal hindbrain identity by activating MafB and Krox20 via the Ras-ERK1/2 intracellular pathway.

FGF-2 controls the differentiation of resident cardiac precursors into functional cardiomyocytes.

Recent evidence suggests that the heart possesses a greater regeneration capacity than previously thought. In the present study, we isolated undifferentiated precursors from the cardiac nonmyocyte cell population of neonatal hearts, expanded them in culture, and induced them to differentiate into functional cardiomyocytes. These cardiac precursors appear to express stem cell antigen-1 and demonstrate characteristics of multipotent precursors of mesodermal origin. Following infusion into normal recipients, these cells home to the heart and participate in physiological and pathophysiological cardiac remodeling. Cardiogenic differentiation in vitro and in vivo depends on FGF-2. Interestingly, this factor does not control the number of precursors but regulates the differentiation process. These findings suggest that, besides its angiogenic actions, FGF-2 could be used in vivo to facilitate the mobilization and differentiation of resident cardiac precursors in the treatment of cardiac diseases.

Pharmacological inhibition of fibroblast growth factor (FGF) receptor signaling ameliorates FGF23-mediated hypophosphatemic rickets.

Fibroblast growth factor 23 (FGF23) is a circulating factor secreted by osteocytes that is essential for phosphate homeostasis. In kidney proximal tubular cells FGF23 inhibits phosphate reabsorption and leads to decreased synthesis and enhanced catabolism of 1,25-dihydroxyvitamin D3 (1,25[OH]2 D3 ). Excess levels of FGF23 cause renal phosphate wasting and suppression of circulating 1,25(OH)2 D3 levels and are associated with several hereditary hypophosphatemic disorders with skeletal abnormalities, including X-linked hypophosphatemic rickets (XLH) and autosomal recessive hypophosphatemic rickets (ARHR). Currently, therapeutic approaches to these diseases are limited to treatment with activated vitamin D analogues and phosphate supplementation, often merely resulting in partial correction of the skeletal aberrations. In this study, we evaluate the use of FGFR inhibitors for the treatment of FGF23-mediated hypophosphatemic disorders using NVP-BGJ398, a novel selective, pan-specific FGFR inhibitor currently in Phase I clinical trials for cancer therapy. In two different hypophosphatemic mouse models, Hyp and Dmp1-null mice, resembling the human diseases XLH and ARHR, we find that pharmacological inhibition of FGFRs efficiently abrogates aberrant FGF23 signaling and normalizes the hypophosphatemic and hypocalcemic conditions of these mice. Correspondingly, long-term FGFR inhibition in Hyp mice leads to enhanced bone growth, increased mineralization, and reorganization of the disturbed growth plate structure. We therefore propose NVP-BGJ398 treatment as a novel approach for the therapy of FGF23-mediated hypophosphatemic diseases.

Role of FGF and Wnt pathways in endoderm patterning and pancreas organogenesis

Summary Between gastrulation and gut tube formation, the endoderm becomes regionally specified along the anterior-posterior axis. An early sign of patterning is the expression of organ-specific genes in restricted endoderm domains. We studied the role of the fibroblast growth factor (FGF) and Wnt pathways in the establishment of the antero-posterior (A-P) axis domains. Here we report the first evidence that graded FGF4-mediated signaling establishes gut tube domains along the A-P axis in vivo from gastrulation to somitogenesis. At gastrulation, FGF4 may act cooperatively with Wnts, since both of them affect the gut tube patterning by promoting posterior and inhibiting anterior endoderm cell fate. The activity of the Wnt pathway is however time restricted, since. it does not affect patterning at somitogenesis. Our experiments point to a global mechanism that coordinates the A-P patterning of all three primary germ layers. Soon after regionalization of the gut tube, morphogenetic evidences of organogenesis appear. We focused our attention on one of these organs, the pancreas. We report a comprehensive investigation of the activity and the role of the Wnt pathway in pancreas organogenesis. We have used two mouse reporter lines to monitor canonical Wnt-pathway activity during development and after birth and demonstrate activity in early pancreatic bud, endocrine cells and in the mesenchyme. We have specifically deleted the ß-catenin .gene, a key component of the Wnt pathway, in the epithelium of the pancreas and duodenum using Pdxl -Cre mice. In agreement with Wnt pathway activity in pancreatic endocrine cells, we find a reduction in endocrine islet numbers. Our study reveals that ß-catenin deletion also affects cells in which Wnt pathway activity is not detected. Indeed, ß-catenin mutant cells have a competitive disadvantage during development that also' affects the exocrine compartment. Moreover, the conditional KO mice develop acute edematous pancreatitis perinatally due to the disruption of the epithelial structure of acini. These effects are likely to be due to the function of ß-catenin at the membrane. Résumé Entre la gastrulation et la formation du tube digestif, l'endoderme est progressivement régionalisé le long de l'axe antéropostérieur (A-P). Un des premiers signes de cette régionalisation est l'expression de gènes spécifiques à certains organes dans une région restreinte. Nous avons étudié l'implication des voies de signalisation FGF et Wnt dans l'établissement de la régionalisation A-P. Nous rapportons les premières preuves que FGF4 établit la ségrégation des domaines de l'endoderme le long de l'axe A-P in vivo de la gastrulation à la somitogenèse. Cette activité peut être menée en collaboration avec les Wnts, puisque ceux-ci influencent aussi l'endoderme en inhibant le destin antérieur et en induisant le destin postérieur des cellules. Cette activité des Wnts est perdue à la somitogenèse. Nos expériences démontrent une régionalisation coordonnée des trois feuillets germinaux le long de l'axe A-P. Peu après la régionalisation, les premiers signes morphologiques de l'organogenèse apparaissent. Nous nous sommes intéressés au rôle des Wnts dans un des dérivés de l'endoderme : le pancréas. Nous avons utilisés deux lignés de souris rapportrices de l'activité de la voie canonique des Wnts, qui montrent une activité dans le bourgeon précoce du pancréas avant la différentiation, puis plus tard dans les cellules endocrines et le mésenchyme. Nous avons utilisé la souris transgénique Pdxl -Cre pour inactiver spécifiquement le gène de la ß-caténine, un intermédiaire de la voie des Wnts, dans la région pancréatique. En accord avec l'activité de la voie de signalisation Wnt, la perte de la ßcaténine conduit à une réduction du nombre de cellules endocrines. De plus certaines cellules qui ne montrent aucune activité de la voie Wnt sont aussi affectées. En effet, les cellules ayant perdu la ß-caténine ont un désavantage compétitif face aux cellules sauvages dans un environnement mosaïque. Cette compétition résulte en l'absence de cellules déplétées en ßcaténine chez l'adulte. De plus, vers la naissance, les animaux déficients pour la ß-caténine développent une pancréatite aiguë due à la destruction de l'architecture des acini. Ceci est probablement aux fonctions d'adhésion de la ß-caténine à la membrane.

The role of fibroblast growth factor (FGF) and type beta transforming growth factot beta (TGF beta1, beta2 and beta3) during rat craniofacial development

Growth factors seem to be part of a complex cellular signalling language, in which individual growth factors are the equivalents of the letters that compose words. According to this analogy, informational content lies, not in an individual growth factor, but in the entire set of growth factors and others signals to which a cell is exposed. The ways in which growth factors exert their combinatorial effects are becoming clearer as the molecular mechanisms of growth factors actions are being investigated. A number of related extracellular signalling molecules that play widespread roles in regulating development in both invertebrates and vertebrates constitute the Fibroblast Growth Factor (FGF) and type beta Transforming Growth Factor ((TGF beta). The latest research literature about the role and fate of these Growth factors and their influence in the craniofacial bone growth ad development is reviewed

The role of fibroblast growth factor (FGF) and type beta transforming growth factot beta (TGF beta1, beta2 and beta3) during rat craniofacial development

Growth factors seem to be part of a complex cellular signalling language, in which individual growth factors are the equivalents of the letters that compose words. According to this analogy, informational content lies, not in an individual growth factor, but in the entire set of growth factors and others signals to which a cell is exposed. The ways in which growth factors exert their combinatorial effects are becoming clearer as the molecular mechanisms of growth factors actions are being investigated. A number of related extracellular signalling molecules that play widespread roles in regulating development in both invertebrates and vertebrates constitute the Fibroblast Growth Factor (FGF) and type beta Transforming Growth Factor ((TGF beta). The latest research literature about the role and fate of these Growth factors and their influence in the craniofacial bone growth ad development is reviewed

Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion.

Carcinoma-associated fibroblasts were reported to promote colorectal cancer (CRC) invasion by secreting motility factors and extracellular matrix processing enzymes. Less is known whether fibroblasts may induce CRC cancer cell motility by contact-dependent mechanisms. To address this question we characterized the interaction between fibroblasts and SW620 and HT29 colorectal cancer cells in 2D and 3D co-culture models in vitro. Here we show that fibroblasts induce contact-dependent cancer cell elongation, motility and invasiveness independently of deposited matrix or secreted factors. These effects depend on fibroblast cell surface-associated fibroblast growth factor (FGF) -2. Inhibition of FGF-2 or FGF receptors (FGFRs) signaling abolishes these effects. FGFRs activate SRC in cancer cells and inhibition or silencing of SRC in cancer cells, but not in fibroblasts, prevents fibroblasts-mediated effects. Using an RGD-based integrin antagonist and function-blocking antibodies we demonstrate that cancer cell adhesion to fibroblasts requires integrin αvβ5. Taken together, these results demonstrate that fibroblasts induce cell-contact-dependent colorectal cancer cell migration and invasion under 2D and 3D conditions in vitro through fibroblast cell surface-associated FGF-2, FGF receptor-mediated SRC activation and αvβ5 integrin-dependent cancer cell adhesion to fibroblasts. The FGF-2-FGFRs-SRC-αvβ5 integrin loop might be explored as candidate therapeutic target to block colorectal cancer invasion.

Fibroblast growth factor-23 (FGF-23) levels differ across populations by degree of industrialization.

CONTEXT: Compensatory increases in FGF23 with increasing phosphate intake may adversely impact health. However, population and clinical studies examining the link between phosphate intake and FGF23 levels have focused mainly on populations living in highly industrialized societies in which phosphate exposure may be homogenous. OBJECTIVE: Contrast dietary phosphate intake, urinary measures of phosphate excretion and FGF23 levels across populations that differ by level of industrialization. DESIGN: Cross-sectional analysis of three populations Setting: Maywood, IL, U.S., Mah|fe Island, Seychelles, and Kumasi, Ghana Participants: Adults with African ancestry aged 25-45 years Main Outcome: Fibroblast growth factor 23 (FGF23) levels Results: The mean age was 35.1 (6.3) years and 47.9% were male. Mean phosphate intake and fractional excretion of phosphate were significantly higher in the U.S. vs. Ghana while no significant difference in phosphate intake or fractional excretion of phosphate was noted between U.S. and Seychelles for men or women. Overall, median FGF23 values were 57.41 RU/ml (IQR 43.42, 75.09) in U.S., 42.49 RU/ml (IQR 33.06, 55.39) in Seychelles and 33.32 RU/ml (IQR 24.83, 47.36) in Ghana. In the pooled sample, FGF23 levels were significantly and positively correlated with dietary phosphate intake (r=0.11; P < 0.001), and the fractional excretion of phosphate (r=0.13; P < 0.001) but not with plasma phosphate levels (-0.001; P = 0.8). Dietary phosphate intake was significantly and positively associated with the fractional excretion of phosphate (r=0.23; P < 0.001). CONCLUSION: The distribution of FGF23 levels in a given population may be influenced by the level of industrialization, likely due to differences in access to foods preserved with phosphate additives.

Fibroblast Growth Factor 8 and its Receptors in The Growth and Angiogenesis of Experimental Breast Cancer . With Special Reference to Thrombospondin-1 as a Novel Target Gene for FGF-8

The growth of breast cancer is regulated by hormones and growth factors. Recently, aberrant fibroblast growth factor (FGF) signalling has been strongly implicated in promoting the progression of breast cancer and is thought to have a role in the development of endocrine resistant disease. FGFs mediate their auto- and paracrine signals through binding to FGF receptors 1-4 (FGFR1-4) and their isoforms. Specific targets of FGFs in breast cancer cells and the differential role of FGFRs, however, are poorly described. FGF-8 is expressed at elevated levels in breast cancer, and it has been shown to act as an angiogenic, growth promoting factor in experimental models of breast cancer. Furthermore, it plays an important role in mediating androgen effects in prostate cancer and in some breast cancer cell lines. We aimed to study testosterone (Te) and FGF-8 regulated genes in Shionogi 115 (S115) breast cancer cells, characterise FGF-8 activated intracellular signalling pathways and clarify the role of FGFR1, -2 and -3 in these cells. Thrombospondin-1 (TSP-1), an endogenous inhibitor of angiogenesis, was recognised as a Te and FGF-8 regulated gene. Te repression of TSP-1 was androgen receptor (AR)-dependent. It required de novo protein synthesis, but it was independent of FGF-8 expression. FGF-8, in turn, downregulated TSP-1 transcription by activating the ERK and PI3K pathways, and the effect could be reversed by specific kinase inhibitors. Differential FGFR1-3 action was studied by silencing each receptor by shRNA expression in S115 cells. FGFR1 expression was a prerequisite for the growth of S115 tumours, whereas FGFR2 expression alone was not able to promote tumour growth. High FGFR1 expression led to a growth advantage that was associated with strong ERK activation, increased angiogenesis and reduced apoptosis, and all of these effects could be reversed by an FGFR inhibitor. Taken together, the results of this thesis show that FGF-8 and FGFRs contribute strongly to the regulation of the growth and angiogenesis of experimental breast cancer and support the evidence for FGF-FGFR signalling as one of the major players in breast cancers.