Generation of induced Pluripotent Stem Cells as disease modelling of NLSDM.

Neutral Lipid Storage Disease with Myopathy (NLSDM) is a rare defect of triacylglycerol metabolism, characterized by the abnormal storage of neutral lipid in organelles known as lipid droplets (LDs). The main clinical features are progressive myopathy and cardiomyopathy. The onset of NLSDM is caused by autosomal recessive mutations in the PNPLA2 gene, which encodes adipose triglyceride lipase (ATGL). Despite its name, this enzyme is present in a wide variety of cell types and catalyzes the first step in triacylglycerol lipolysis and the release of fatty acids. Here, we report the derivation of NLSDM-induced pluripotent stem cells (NLSDM-iPSCs) from fibroblasts of two patients carrying different PNPLA2 mutations. The first patient was homozygous for the c.541delAC, while the second was homozygous for the c.662G>C mutation in the PNPLA2 gene. We verified that the two types of NLSDM-iPSCs possessed properties of embryonic-like stem cells and could differentiate into the three germ layers in vitro. Immunofluorescence analysis revealed that iPSCs had an abnormal accumulation of triglycerides in LDs, the hallmark of NLSDM. Furthermore, NLSDM-iPSCs were deficient in long chain fatty acid lipolysis, when subjected to a pulse chase experiment with oleic acid. Collectively, these results demonstrate that NLSDM-iPSCs are a promising in vitro model to investigate disease mechanisms and screen drug compounds for NLSDM, a rare disease with few therapeutic options.

Lower endothelial progenitor cell number, family history of cardiovascular disease and reduced HDL-cholesterol levels are associated with shorter leukocyte telomere length in healthy young adults.

BACKGROUND AND AIMS: Leukocyte telomere length (LTL) is a novel marker of cardiovascular (CV) risk. The aim of the study was to investigate the major determinants of LTL in a healthy young population at very low CV risk. METHODS AND RESULTS: LTL was determined in 82 healthy subjects (49M/33F; age37 ± 9yrs), normotensive and not taking any medication with different family history of cardiovascular disease (CVD) (24yes/58no). Fasting blood samples were drawn in all subjects for the determination of lipid profile, high sensitive C-reactive protein, uric acid, Plasminogen Activator Inhibitor-1 (PAI-1), LTL and Endothelial Progenitor Cell (EPC) number. LTL was assessed with a specific real-time PCR reaction in leukocyte DNA samples. LTL resulted inversely correlated with family history of CVD (t = 2.70; p = 0.009), age (r = -0.238; p = 0.032), waist circumference (r = -0.256; p = 0.02), triglycerides (r = -0.218; p = 0.049), PAI-1 (r = -0.288; p = 0.009) and directly correlated with HDL-cholesterol (r = 0.316; p = 0.004) and EPC number (r = 0.358; p = 0.002). At a multivariate analysis, family history of CVD (p = 0.013), EPC count (p = 0.003), and HDL-cholesterol (p = 0.017) were independently associated with LTL (r = 0.62). CONCLUSION: LTL is independently associated to CV risk factors also in healthy young adults.

Parkinson's disease patient-specific neuronal networks carrying the LRRK2 G2019S mutation unveil early functional alterations that predate neurodegeneration.

A deeper understanding of early disease mechanisms occurring in Parkinson's disease (PD) is needed to reveal restorative targets. Here we report that human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons (DAn) obtained from healthy individuals or patients harboring LRRK2 PD-causing mutation can create highly complex networks with evident signs of functional maturation over time. Compared to control neuronal networks, LRRK2 PD patients' networks displayed an elevated bursting behavior, in the absence of neurodegeneration. By combining functional calcium imaging, biophysical modeling, and DAn-lineage tracing, we found a decrease in DAn neurite density that triggered overall functional alterations in PD neuronal networks. Our data implicate early dysfunction as a prime focus that may contribute to the initiation of downstream degenerative pathways preceding DAn loss in PD, highlighting a potential window of opportunity for pre-symptomatic assessment of chronic degenerative diseases.

Different tyrosine autophosphorylation requirements in fibroblast growth factor receptor-1 mediate urokinase-type plasminogen activator induction and mitogenesis.

Among the seven tyrosine autophosphorylation sites identified in the intracellular domain of tyrosine kinase fibroblast growth factor receptor-1 (FGFR1), five of them are dispensable for FGFR1-mediated mitogenic signaling. The possibility of dissociating the mitogenic activity of basic FGF (FGF2) from its urokinase-type plasminogen activator (uPA)-inducing capacity both at pharmacological and structural levels prompted us to evaluate the role of these autophosphorylation sites in transducing FGF2-mediated uPA upregulation. To this purpose, L6 myoblasts transfected with either wild-type (wt) or various FGFR1 mutants were evaluated for the capacity to upregulate uPA production by FGF2. uPA was induced in cells transfected with wt-FGFR1, FGFR1-Y463F, -Y585F, -Y730F, -Y766F, or -Y583/585F mutants. In contrast, uPA upregulation was prevented in L6 cells transfected with FGFR1-Y463/583/585/730F mutant (FGFR1-4F) or with FGFR1-Y463/583/585/730/766F mutant (FGFR1-5F) that retained instead a full mitogenic response to FGF2; however, preservation of residue Y730 in FGFR1-Y463/583/585F mutant (FGFR1-3F) and FGFR1-Y463/583/585/766F mutant (FGFR1-4Fbis) allows the receptor to transduce uPA upregulation. Wild-type FGFR1, FGFR1-3F, and FGFR1-4F similarly bind to a 90-kDa tyrosine-phosphorylated protein and activate Shc, extracellular signal-regulated kinase (ERK)2, and JunD after stimulation with FGF2. These data, together with the capacity of the ERK kinase inhibitor PD 098059 to prevent ERK2 activation and uPA upregulation in wt-FGFR1 cells, suggest that signaling through the Ras/Raf-1/ERK kinase/ERK/JunD pathway is necessary but not sufficient for uPA induction in L6 transfectants. Accordingly, FGF2 was able to stimulate ERK1/2 phosphorylation and cell proliferation, but not uPA upregulation, in L6 cells transfected with the FGFR1-Y463/730F mutant, whereas the FGFR1-Y583/585/730F mutant was fully active. We conclude that different tyrosine autophosphorylation requirements in FGFR1 mediate cell proliferation and uPA upregulation induced by FGF2 in L6 cells. In particular, phosphorylation of either Y463 or Y730, dispensable for mitogenic signaling, represents an absolute requirement for FGF2-mediated uPA induction.

alphavbeta3 integrin mediates the cell-adhesive capacity and biological activity of basic fibroblast growth factor (FGF-2) in cultured endothelial cells.

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38-61 and 82-101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-alphavbeta3 antibodies prevent cell adhesion to FGF-2. Also, purified human alphavbeta3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-alphavbeta3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with alphavbeta3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.

A distinct basic fibroblast growth factor (FGF-2)/FGF receptor interaction distinguishes urokinase-type plasminogen activator induction from mitogenicity in endothelial cells.

Basic fibroblast growth factor (FGF-2) induces cell proliferation and urokinase-type plasminogen activator (uPA) production in fetal bovine aortic endothelial GM 7373 cells. In the present paper we investigated the role of the interaction of FGF-2 with tyrosine-kinase (TK) FGF receptors (FGFRs) in mediating uPA up-regulation in these cells. The results show that FGF-2 antagonists suramin, protamine, heparin, the synthetic peptide FGF-2(112-155), and a soluble form of FGFR-1 do not inhibit FGF-2-mediated uPA up-regulation at concentrations that affect growth factor binding to cell surface receptors and mitogenic activity. In contrast, tyrosine phosphorylation inhibitors and overexpression of a dominant negative TK- mutant of FGFR-1 abolish the uPA-inducing activity of FGF-2, indicating that FGFR and its TK activity are essential in mediating uPA induction. Accordingly, FGF-2 induces uPA up-regulation in Chinese hamster ovary cells transfected with wild-type FGFR-1, -2, -3, or -4 but not with TK- FGFR-1 mutant. Small unilamellar phosphatidyl choline:cholesterol vesicles loaded with FGF-2 increased uPA production in GM 7373 cells in the absence of a mitogenic response. Liposome-encapsulated FGF-2 showed a limited but significant capacity, relative to free FGF-2, to interact with FGFR both at 4 degrees C and 37 degrees C and to be internalized within the cell. uPA up-regulation by liposome-encapsulated FGF-2 was quenched by neutralizing anti-FGF-2 antibodies, indicating that the activity of liposome-delivered FGF-2 is mediated by an extracellular action of the growth factor. Taken together, the data indicate that a distinct interaction of FGF-2 with FGFR, quantitatively and/or qualitatively different from the one that leads to mitogenicity, is responsible for the uPA-inducing activity of the growth factor.

Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): comparative analysis of Y373C, K650E and the novel G384D mutations.

The t(4;14)(p16.3;q32) chromosomal translocation occurs in approximately 20% of multiple myelomas (MM) and leads to the apparent deregulation of two genes located on 4p16.3: the fibroblast growth factor receptor 3 (FGFR3) and the putative transcription factor WHSC1/MMSET. Interestingly, FGFR3 mutations known to be associated with autosomal dominant human skeletal disorders have also been found in some MM cell lines with t(4;14) but their pathogenetic role in MM is still controversial. Since cell lines may represent useful models for investigating the effects of deregulated FGFR3 mutants in MM, we analysed the expression, activation, signaling pathways and oncogenic potential of three mutants identified so far: the Y373C and K650E in the KMS-11 and OPM-2 cell lines respectively, and the novel G384D mutation here identified in the KMS-18 cell line. All of the cell lines present a heterozygous FGFR3 gene mutation and transcribe the mutated allele; unlike KMS-11 and OPM-2 (which express the IIIc isoform), the KMS-18 cell line expresses prevalently the isoform IIIb. We demonstrated that, under serum-starved conditions, KMS-11 and OPM-2 cells express appreciable levels of phosphorylated FGFR3 mutants indicating a constitutive activation of the Y373C and K650E receptors; the addition of the aFGF ligand further increased the level of receptor phosphorylation. Conversely, the FGFR3 mutant in KMS-18 does not seem to be constitutively activated since it was phosphorylated only in the presence of the ligand. In all three MM cell lines, ligand-stimulated FGFR3 mutants activated the MAP kinase signaling pathway but did not apparently involve either the STAT1 or STAT3 cascades. However, when transfected in 293T cells, G384D, like Y373C and K650E, was capable of activating MAPK, STAT1 and STAT3 under serum-starved condition. Finally, a focus formation assay of NIH3T3 cells transfected with FGFR3-expressing plasmid vectors showed that Y373C and K650E (albeit at different levels) but not G384D or the wild-type receptor, can induce transformed foci. Overall, our results support the idea that FGFR3 mutations are graded in terms of their activation capability, thus suggesting that they may play a critical role in the tumor progression of MM patients with t(4;14).

Paracrine and autocrine effects of fibroblast growth factor-4 in endothelial cells.

Recombinant Fibroblast Growth Factor-4 (FGF4) and FGF2 induce extracellular signal-regulated kinase-1/2 activation and DNA synthesis in murine aortic endothelial (MAE) cells. These cells co-express the IIIc/Ig-3 loops and the novel glycosaminoglycan-modified IIIc/Ig-2 loops isoforms of FGF receptor-2 (FGFR2). The affinity of FGF4/FGFR2 interaction is 20-30 times lower than that of FGF2 and is enhanced by heparin. Overexpression of FGF2 or FGF4 cDNA in MAE cells results in a transformed phenotype and increased proliferative capacity, more evident for FGF2 than FGF4 transfectants. Both transfectants induce angiogenesis when applied on the top of the chick embryo chorioallantoic membrane. However, in contrast with FGF2-transfected cells, FGF4 transfectants show a limited capacity to growth under anchorage-independent conditions and lack the ability to invade 3D fibrin gel and to undergo morphogenesis in vitro. Also, they fail to induce hemangiomas when injected into the allantoic sac of the chick embryo. In conclusion, although exogenous FGF2 and FGF4 exert a similar response in MAE cells, significant differences are observed in the biological behavior of FGF4 versus FGF2 transfectants, indicating that the expression of the various members of the FGF family can differently affect the behavior of endothelial cells and, possibly, of other cell types, including tumor cells.

Differential expression of fibroblast growth factor receptors by human neurones, astrocytes and microglia.

Expression of fibroblast-growth factor receptors (FGFRs) was studied in human fetal neurones, astrocytes and microglia in culture. Northern blot analysis showed that neurones and microglia expressed the mRNAs for FGFR-1, FGFR-2, FGFR-3, FGFR-4 at different levels, whereas astrocytes expressed only FGFR-1 and FGFR-4 mRNAs. Immunocytochemical localization of FGFR-1 revealed that this receptor was predominantly localized on the axon hillock membrane in neurones, and was associated with the plasma membrane of ameboid, activated microglia and of glial-fibrillar acidic protein positive astrocytes. The expression of various members of the FGFR family in all the cell types investigated implicates FGFs in human brain development and functions.

Expression of basic fibroblast growth factor and its receptors in human fetal microglia cells.

The presence of basic fibroblast growth factor (bFGF) and FGF receptors was investigated in microglia cells derived from human fetal brain long-term cultures. Production of bFGF was suggested through the capability of microglial extracts to stimulate plasminogen activator (PA) synthesis in endothelial cells. The identity of PA-stimulating activity with bFGF was confirmed by its high affinity for heparin and its cross-reactivity with polyclonal antibodies to human recombinant bFGF. These antibodies recognized a cell-associated M(r) 18,000 protein as well as trace amounts of the M(r) 24,000 bFGF isoform in Western blot. All microglial cells showed bFGF immunoreactivity in the cytoplasm and, sometimes, in the nucleus. Scatchard plot analysis of 125I-bFGF binding data revealed the presence of low affinity heparansulphate proteoglycans (380,000 +/- 60,000 sites/cell; Kd = 730 +/- 200 nM) and of high affinity tyrosine-kinase receptors (10,300 + 2500 sites/cell; Kd = 30 +/- 9 pM). Immunocytochemistry confirmed the presence of FGF receptor (1/flg) on the cell surface of some, but not all microglial cells, with prevalent association to ameboid microglia. Transcripts for FGF receptors 1, 2, 3 and 4 were found in microglia by Northern blot analysis. Co-expression of bFGF and its receptors in human fetal microglia suggests an autocrine role of bFGF in these cells.

Examining new models for the study of autocrine and paracrine mechanisms of angiogenesis through FGF2-transfected endothelial and tumour cells.

Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumour neovascularization. Several growth factors and cytokines have been shown to stimulate endothelial cell proliferation in vitro and in vivo and among them FGF2 was one of the first to be characterised. FGF2 is a Mr 18,000 heparin-binding cationic polypeptide that induces proliferation, migration, and protease production in endothelial cells in culture and neovascularization in vivo. FGF2 interacts with endothelial cells through two distinct classes of receptors, the high affinity tyrosine-kinase receptors (FGFRs) and low affinity heparan sulfate proteoglycans (HSPGs) present on the cell surface and in the extracellular matrix. Besides experimental evidence for paracrine mode of action for FGF2, some observations raise the hypothesis that FGF2 may also play an autocrine role in endothelial cells. FGF2 may therefore represent a target for anti-angiogenic therapies. In order to assess the angiostatic potential of different classes of compounds, novel experimental models have been developed based on the autocrine and/or the paracrine capacity of FGF2.

Nuclear localization of endogenous basic fibroblast growth factor in cultured endothelial cells.

Indirect immunofluorescence using anti-human placental bFGF antibodies demonstrates the presence of bFGF-like reactivity in the cytoplasm and in the nucleus of adult bovine aortic endothelial cells and of normal and transformed fetal bovine aortic endothelial AG 7680 and GM 7372 cells. Biologically active immunoreactive Mr 18,000 bFGF can be isolated by heparin-Sepharose affinity chromatography from the extract of GM 7372 cell nuclei. Quantitation of bFGF content by biological and immunological methods indicates that 100,000 bFGF molecules per nucleus are present in GM 7372 cells, with nuclear bFGF corresponding to 25-30% of total cellular bFGF. Immunoprecipitation experiments demonstrate that the nuclear localization of newly synthesized bFGF occurs when GM 7372 cells are biosynthetically labeled both in the absence and in the presence of suramin, a molecule that inhibits the binding of bFGF to its plasma membrane receptor. Thus the data indicate that endogenous bFGF undergoes an intracellular sorting to the nucleus of the endothelial cell.

Basic fibroblast growth factor requires a long-lasting activation of protein kinase C to induce cell proliferation in transformed fetal bovine aortic endothelial cells.

Basic fibroblast growth factor (bFGF) induces a protein kinase C (PKC)-dependent mitogenic response in transformed fetal bovine aortic endothelial GM 7373 cells. A long-lasting interaction of bFGF with the cell is required to induce cell proliferation. bFGF-treated cells are in fact committed to proliferate only after they have entered the phase S of the cell cycle, 12-14 h after the beginning of bFGF treatment. Before that time, the mitogenic response to bFGF is abolished by 1) removal of extracellular bFGF by suramin, 2) addition of neutralizing anti-bFGF antibodies to the culture medium, 3) inhibition of PKC activity by the protein kinase inhibitor H-7, and 4) down-regulation of PKC by cotreatment with phorbol ester. Thus the requirement for a prolonged interaction of bFGF with the cell reflects the requirement for a prolonged activation of PKC. Similar conclusions can be drawn for the PKC activators 12-O-tetradecanoyl phorbol 13-acetate and 1,2-dioctanoyl-sn-glycerol. The two molecules require 16 and 6 h, respectively, of activation of PKC to induce 50% of maximal cell proliferation. The requirement for a long-lasting activation of PKC appears to be a mechanism for the control of cell proliferation capable of discriminating among transient nonmitogenic stimuli and long-lasting mitogenic stimuli.

Characterization of the murine BEK fibroblast growth factor (FGF) receptor: activation by three members of the FGF family and requirement for heparin.

The bek gene encodes a member of the high-affinity fibroblast growth factor receptor family. The BEK/FGFR-2 receptor is a membrane-spanning tyrosine kinase with the typical features of FGF receptors. We have cloned a murine bek cDNA and expressed it in receptor-negative Chinese hamster ovary cells and in 32D myeloid cells. The BEK receptor expressed in Chinese hamster ovary cells binds acidic FGF, basic FGF, and Kaposi FGF equally well but does not bind keratinocyte growth factor or FGF-5 appreciably. Upon treatment with basic FGF or Kaposi FGF, the BEK receptor is phosphorylated and a mitogenic response is achieved. Heparan sulfate proteoglycans have been shown to play an obligate role in basic FGF binding to the high-affinity FLG receptor. Unlike the BEK-expressing Chinese hamster ovary cells, 32D cells expressing the BEK receptor require the addition of exogenous heparin in order to grow in the presence of basic FGF or Kaposi FGF. We show that the addition of heparin greatly enhances the binding of radio-labeled basic FGF to the receptor. Thus the BEK receptor, like FLG, also requires an interaction with heparan sulfate proteoglycans to facilitate binding to its ligands.

Developmentally regulated expression and localization of fibroblast growth factor receptors in the human muscle.

Fibroblast growth factors (FGFs) are believed to play a key role in tissue differentiation and maturation. Thus, the expression of the four members of the high-affinity tyrosine kinase FGF receptor family (FGFRs) and of the low-affinity heparan sulphate proteoglycan binding sites, syndecan-1 and perlecan, was studied in the human skeletal muscle during development. Northern blot analysis demonstrated a developmentally regulated expression of the mRNAs for FGFR-1, FGFR-3, FGFR-4, whereas only traces of FGFR-2 mRNA were found. Each receptor type had a different developmental pattern, suggesting an independent regulation. Signal for FGFR-3 was retained only in the adult muscle. Among the low-affinity FGF binding sites, perlecan was absent, whereas RNA transcript for syndecan-1 peaked at week 13 of gestation, after which a significant decrease was observed. Immunohistochemistry for FGFRs revealed that their localization changed with muscle maturation. At early embryonic stages, FGFR-3 and FGFR-4 had a scattered distribution in the tissue, and FGFR-1 was found on myotube and myofiber plasma membranes. At later stages, FGFR-1 positivity decreased and was found in a few areas of the muscle, FGFR-3 was concentrated in the nuclei of some, but not all, muscle fibers, and FGFR-4 maintained an association with plasma membrane. In adult tissue, weak positivity for FGFR-3 and FGFR-4 was observed in the connective tissue only. When immunocytochemistry was performed on human fetal myoblasts in culture, confocal microscope analysis revealed a nonhomogeneous cell membrane distribution of FGFRs. Taken together, the data strongly suggest that developmentally regulated expression and cell distribution of FGFRs play a role during muscle maturation.

Chemical structure and genotoxic activity of the hepatocarcinogenic beta-blocker DL-ZAMI 1305.

A single administration of the sex-dependent hepatocarcinogenic beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)3-tert-butylamino-propan-2-ol (DL-ZAMI 1305) induces liver DNA damage, as evaluated by alkaline sucrose gradient analysis, in female but not in male Fisher 344 rats. The shift of the methyl-group from the 3-position of the aromatic ring of DL-ZAMI 1305 to the 4- and 5-position causes the progressive decrease of the genotoxic activity of the molecule. No effect on the DNA-damaging capacity of DL-ZAMI 1305 is instead observed when the NO2 group of the aromatic ring is reduced to an NH2 group. Bis-demethylation of the side chain of DL-ZAMI 1305, or its modification to an alpha-hydroxicarboxylic acid or glycol, increases the DNA-damaging capacity of the molecule, which becomes genotoxic also for the liver of the male rat. Thus, modifications of the chemical structure of the aromatic ring or of the side chain of DL-ZAMI 1305 affect the genotoxic activity of the molecule both in male and female rat liver. The modifications of the side chain of DL-ZAMI 1305 investigated in the present work are likely to occur in vivo as a consequence of the hepatic metabolism of the molecule. Sex-dependent differences in the activity of the liver drug-metabolizing system might explain the different genotoxic and oncogenic activity of DL-ZAMI 1305 in the two sexes.

Characterization of a Mr 25,000 basic fibroblast growth factor form in adult, regenerating, and fetal rat liver.

A heparin-binding Mr 25,000 immunoreactive bFGF-like protein (ir-bFGF) is recognized in adult rat liver extract by affinity-purified polyclonal anti-human placental bFGF antibodies. Hepatic levels of this protein increase 4-fold in regenerating rat liver during the first 48 h after partial hepatectomy. Also, they appear to be higher in embryonic than in newborn or in adult rat liver. Mr 25,000 ir-bFGF from regenerating rat liver, partially purified by heparin-affinity chromatography, induces plasminogen activator activity and cell proliferation in transformed fetal bovine aortic endothelial GM 7373 cells and competes with Mr 18,000 [125I]bFGF for the binding to high affinity bFGF receptors. The data indicate the presence in rat liver of a high molecular weight form of bFGF whose expression is modulated during embryonic development and liver regeneration.

Distinct role of 2-O-, N-, and 6-O-sulfate groups of heparin in the formation of the ternary complex with basic fibroblast growth factor and soluble FGF receptor-1.

Interaction of basic fibroblast growth factor (bFGF) with heparan sulfate proteoglycans (HSPGs) plays an important role in the binding of bFGF to its tyrosine kinase receptor (FGFR). The molecular bases of this interaction were investigated by evaluating the capacity of conventional and selectively desulfated heparins i) to affect the binding of bFGF to FGFR and HSPGs of NIH 3T3 cells transfected with FGFR-1/flg cDNA, ii) to facilitate the interaction of bFGF with a recombinant soluble form of the extracellular domain of FGFR-1/flg (xcFGFR-1), and iii) to protect xcFGFR-1 from tryptic cleavage. 6-O-desulfated (6-O-DS) heparin, but not 2-O-desulfated (2-O-DS) and N-desulfated/N-acetylated (N-DS/N-Ac) heparins, retains the capacity to bind bFGF, as assessed by its ability to inhibit bFGF-binding to cell-associated FGFR-1 and HSPGs. On the other hand, at variance with conventional heparin, 2-O-DS, N-DS/N-Ac, and 6-O-DS heparins are all ineffective in potentiating the binding of bFGF to xcFGFR-1 and protecting xcFGFR-1 from tryptic cleavage. The data indicate that 6-O-sulfate groups are not essential for the interaction of heparin with bFGF but are involved in the interaction with xcFGFR-1. Our findings support the hypothesis that HSPGs modulate the binding of bFGF to FGFR through the formation of a ternary complex in which the glycosaminoglycan chains interact with bFGF via 2-O- and N-sulfate groups and with FGFR also via 6-O-sulfate groups.

Nitric oxide promotes proliferation and plasminogen activator production by coronary venular endothelium through endogenous bFGF.

We reported previously that NO is responsible for the angiogenesis produced by endothelium-dependent vasodilating peptides. To investigate the mechanisms by which NO controls angiogenesis, NO was assessed for the ability to affect cell proliferation and upregulation of urokinase-type plasminogen activator (uPA) induced by basic fibroblast growth factor (bFGF) when added exogenously to or when produced endogenously by coronary venular endothelial cells (CVECs). The treatment of the cells with the NO donor sodium nitroprusside (NaNp) induced uPA upregulation and cell proliferation, which were prevented by anti-bFGF antibodies. Similarly, the NO-dependent mitogenic activity of the vasodilating peptide substance P (SP) was blocked by anti-bFGF antibodies, thus implicating endogenous bFGF in the NO-induced response. NaNp and SP induced bFGF expression as measured by Western blot analysis of CVEC extracts and by differential reverse transcriptase-polymerase chain reaction of bFGF mRNA. SP-induced upregulation of bFGF was prevented by the NO synthase inhibitor N omega-monomethyl-L-arginine. We conclude that NO promotes cell proliferation and uPA upregulation in CVECs by inducing endogenous bFGF and that this pathway mediates the angiogenetic response to the vasoactive neuropeptide SP. This signaling paradigm may provide an important link between shear rate, NO, bFGF, and coronary angiogenesis.

Urokinase-type plasminogen activator overexpression enhances the invasive capacity of endothelial cells.

Fetal bovine aortic endothelial GM 7373 cells were transfected with a viral expression vector harboring the human urokinase-type plasminogen activator (uPA) gene. The stable transfectant clone uPA-R5 overexpressed and secreted human uPA as shown by Northern blot analysis, immunoprecipitation of metabolically labeled proteins, plasmin chromogenic assays, and SDS-PAGE zymography of cell extracts and conditioned media. The uPA-R5 cells were analyzed for their invasive capacity in vitro in the Matrigel chemoinvasion assay in the presence of serine- or metalloprotease inhibitors. uPA overexpression enhanced the invasive capacity of GM 7373 cells through a mechanism which differs from that mediated by metalloproteases. Endothelial cell uPA transfectants may represent an useful experimental model to investigate the role of uPA in angiogenesis and angioproliferative diseases.