Vascular remodeling in response to altered blood flow is mediated by fibroblast growth factor-2

Vascular structures adapt to changes in blood flow by adjusting their diameter accordingly. The factors mediating this process are only beginning to be identified. We have recently established a mouse model of arterial remodeling in which flow in the common carotid artery is interrupted by ligation of the vessel near the carotid bifurcation, resulting in a dramatic reduction in vessel diameter as a consequence of inward remodeling and intimal lesion formation. In the present study, we used this model to determine the role of fibroblast growth factor-2 (FGF-2) in the remodeling response by maintaining neutralizing serum levels of a mouse monoclonal antibody against FGF-2 for 4 weeks. Morphometric analysis revealed that intimal lesion formation was not affected by the antibody. However, lumen narrowing was significantly inhibited, resulting in a greater than 3-fold increase in lumen area in anti-FGF-2-treated animals compared with controls. Treatment with anti-FGF-2 antibody significantly inhibited the reduction in vessel diameter (inward remodeling) and shortening of the internal elastic lamina in the ligated vessel. In addition, anti-FGF-2 treatment also caused outward remodeling of the contralateral carotid artery. These findings identify FGF-2 as an important factor in vascular remodeling, and its effects are likely to be mediated by increasing vascular tone. The results are consistent with the recent observation of reduced vascular tone in the FGF-2-deficient mouse.     

Duration-dependent cytoprotective versus inflammatory effects of lung epithelial fibroblast growth factor-7 expression

Fibroblast growth factor-7 (FGF7) is a lung epithelial cell mitogen that is cytoprotective during injury. Transgenic mice that conditionally expressed FGF7 were used to dissect the mechanisms of FGF7 protection during lung injury. FGF7 improved survival when induced 3 days prior to acute lung injury. In contrast, FGF7 caused pulmonary inflammation and lung injury after 7 days or longer. Gene expression analysis of mouse lung mRNA identified mRNAs that contribute to the protective effects of FGF7. FGF7 improved survival during acute lung injury in adult mouse lung after short-term expression, but paradoxically induced inflammation and injury after persistent expression.     

Basic fibroblast growth factor: a potential mediator of stromal growth in the human prostate.

Studies were undertaken, using isolated prostatic epithelial and stromal cells, to evaluate the role of basic fibroblast growth factor (bFGF) in the regulation of benign prostatic growth. bFGF was detected in lysates, but not the conditioned media, of cultured prostatic epithelial and stromal cells by Western immunoblotting and immunoprecipitation of metabolically labeled proteins. Immunofluorescence analysis of benign human prostate localized the majority of bFGF to the prostatic stroma. In addition, bFGF was a potent stimulator of stromal cell proliferation in vitro, but was not mitogenic to cultured epithelial cells. Further studies demonstrated bFGF receptors (Kd = 258 pM; 61,400 receptors/cell) on stromal cells, but not epithelial cells. Epithelial cell-conditioned medium was mitogenic for stromal cells, suggesting the presence of paracrine interactions. However, bFGF does not appear to be the mediator of this interaction, since the mitogenic effect of epithelial cell-conditioned medium on stromal cells was not significantly reduced by the addition of anti-bFGF. Additional studies showed that concentrated stromal cell-conditioned medium was not mitogenic to cultured stromal cells under serum-free defined conditions, indicating the lack of an external autocine mechanism. These studies demonstrate that bFGF is actively synthesized by isolated prostatic epithelial and stromal cells, but is largely not secreted. Prostatic stroma, but not epithelia, are responsive to the mitogenic effect of bFGF in vitro. However, because of the limited secretion of bFGF by prostatic cells, the mechanism(s) of bFGF-mediated regulation of stromal growth remains unclear.     

Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells

This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (P<0.01). A 50% increase in FGF-2 content versus control (P<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm(2) versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.     

Functional insulin-like growth factor-1/insulin-like growth factor-1 receptor-mediated circuit in human and murine thymic epithelial cells

Interactions between thymocytes and thymic epithelial cell (TEC) can be modulated by growth hormone via insulin-like growth factor-1 (IGF-1). In this study, we showed IGF-1 and IGF-1 receptor mRNA expression by human and murine TEC and thymocytes. Functionally, IGF-1 stimulates extracellular matrix production by human TEC. Moreover, pretreatment of murine TEC with IGF-1 increases their adhesion to thymocytes. Interestingly, we observed an increase in the frequency of CD4-CD8-CD90+ T cells which adhered to pretreated TEC, supporting the concept that IGF-1 may also act indirectly on intrathymic T cell differentiation and migration through the thymic epithelium.     

Regulation of endothelial cell branching morphogenesis by endogenous chemokine stromal-derived factor-1

The chemokine stromal-derived factor-1 (SDF-1) and its unique receptor, CXCR4, are required for normal cardiovascular development, but a critical role for SDF-1 in postnatal vascular remodeling and the mechanisms underlying SDF-1/CXCR-4 vasculogenesis are unclear. Here we show that SDF-1 is expressed by the vascular endothelium from selected healthy and tumor tissues. In vitro, primary endothelial cells constitutively express SDF-1 that is detected in the cytoplasm, on the cell surface, and in the culture supernatant. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) increase SDF-1 expression in endothelial cells. In functional studies, pertussis toxin and antibodies to SDF-1 or CXCR-4 disrupt extracellular matrix-dependent endothelial cell tube formation in vitro. This morphogenic process is associated with time-dependent modulation of surface CXCR-4 expression that changes from being diffuse to being polarized and subsequently lost. In vivo, pertussis toxin and neutralizing antibodies directed at SDF-1 inhibit growth factor-dependent neovascularization. These results indicate that SDF-1/CXCR-4 identifies VEGF- and bFGF-regulated autocrine signaling systems that are essential regulators of endothelial cell morphogenesis and angiogenesis.     

Tumor suppression and apoptosis of human prostate carcinoma mediated by a genetic locus within human chromosome 10pter-q11.

Prostate cancer is the second leading cause of male cancer deaths in the United States. Yet, despite a large international effort, little is known about the molecular mechanisms that underlie this devastating disease. Prostate secretory epithelial cells and androgen-dependent prostate carcinomas undergo apoptosis in response to androgen deprivation and, furthermore, most prostate carcinomas become androgen independent and refractory to further therapeutic manipulations during disease progression. Definition of the genetic events that trigger apoptosis in the prostate could provide important insights into critical pathways in normal development as well as elucidate the perturbations of those key pathways in neoplastic transformation. We report the functional definition of a novel genetic locus within human chromosome 10pter-q11 that mediates both in vivo tumor suppression and in vitro apoptosis of prostatic adenocarcinoma cells. A defined fragment of human chromosome 10 was transferred via microcell fusion into a prostate adenocarcinoma cell line. Microcell hybrids containing only the region 10pter-q11 were suppressed for tumorigenicity following injection of microcell hybrids into nude mice. Furthermore, the complemented hybrids undergo programmed cell death in vitro via a mechanism that does not require nuclear localization of p53. These data functionally define a novel genetic locus, designated PAC1, for prostate adenocarcinoma 1, involved in tumor suppression of human prostate carcinoma and furthermore strongly suggest that the cell death pathway can be functionally restored in prostatic adenocarcinoma.     

Heparinase treatment of bovine smooth muscle cells inhibits fibroblast growth factor-2 binding to fibroblast growth factor receptor but not FGF-2 mediated cellular proliferation

On the surface of smooth muscle cells there are two types of receptors for the mitogenic and angiogenic growth factor fibroblast growth factor-2 (FGF-2); a high affinity tyrosine kinase FGF receptor (FGFR1) and low affinity heparin./heparan-like glycosaminoglycan (HLGAG) component of surface expressed proteoglycans. It is believed that all three components; FGFR1, FGF-2, and the HLGAG chains, must form a ternary complex for maximal cellular stimulation. To carefully examine the role surface HLGAGs play in FGF-2-mediated proliferation of SMCs we have utilized HLGAG degrading enzymes heparinase I, II and III. We report that heparinase treatment of bovine smooth muscle cells inhibits the binding of ¹²⁵I-FGF-2 to FGFR1, but does not inhibit FGF-2 induced cellular proliferation. Through the use of both sodium chlorate and FGF-2 mutants with deficient HLGAG-binding capabilities, we show the FGF-2-HLGAG interaction is important for FGF-2's ability to induce SMC proliferation. Finally, we report conditioned media from heparinase treated SMCs is capable of supporting FGF-2 induced proliferation in an HLGAG-free lymphoid F32 cells, suggesting that the heparinase generated fragments are responsible for the proliferative response. The data presented here suggest FGF-2 is capable of stimulating smooth muscle cell proliferation through an FGFR independent, HLGAG dependent mechanism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Acceleration of fracture healing in nonhuman primates by fibroblast growth factor-2

One of the greatest needs in the clinical bone field is a bioactive agent to stimulate bone formation. We previously reported that fibroblast growth factor-2 (FGF-2) exhibited strong anabolic actions on bone formation in models of rodents and dogs. Aiming at a clinical application, this study was undertaken to clarify the effect of a single local application of recombinant human FGF-2 on fracture healing in nonhuman primates. After a fracture was created at the midshaft of the right ulna of animals and stabilized with an intramedullary nail, gelatin hydrogel alone (n = 10) or gelatin hydrogel containing 200 microg FGF-2 (n = 10) was injected into the fracture site. Although 4 of 10 animals treated with the vehicle alone remained in a nonunion state even after 10 weeks, bone union was complete at 6 weeks in all 10 animals treated with FGF-2. Significant differences in bone mineral content and density at the fracture site between the vehicle and FGF-2 groups were seen at 6 weeks and thereafter. FGF-2 also increased the mechanical property of the fracture site. We conclude that FGF-2 accelerates fracture healing and prevents nonunion in primates, and therefore propose that it is a potent bone anabolic agent for clinical use.     

Role of stromal-derived factor-1 in the hematopoietic-supporting activity of human mesenchymal stem cells

Mesenchymal stem cells (MSC) have the ability to support and maintain hematopoiesis in vitro. However, mechanisms implicated in this support are not fully characterized. In the present study, the role of stromal-derived factor-1 (SDF-1)/CXCR4 axis in the interactions between MSC and hematopoietic stem/progenitor cells (HSPC) was studied. Human bone marrow MSC were plated as feeder layers in Dexter-type long-term cultures (LTC) with human cord blood CD34(+) HSPC. Cultures were supplemented weekly with neutralizing antibodies against CXCR4 or SDF-1 for 5 wk. LTC-initiating cell (IC) activity was strongly dependent on the SDF-1/CXCR4 axis, as both antibodies significantly decreased secondary colony-forming cell production. To assess the effect of SDF-1/CXCR4 axis on progenitor cell proliferation, LTC-IC killing assays were carried out: in LTC of CD34(+) cells in contact with MSC, treatment with anti-CXCR4 antibody significantly reduced the number of cycling progenitors. These results indicate that the SDF-1/CXCR4 axis promotes HSPC proliferation in contact with MSC. Interestingly, when HSPC were separated from MSC by a semipermeable membrane, LTC-IC activity became CXCR4 independent. Multiplex analysis of MSC-conditioned medium revealed that in addition to SDF-1, MSC produced stimulatory and inhibitory factors, such as interleukin (IL)-6, IL-11, granulocyte macrophage-colony stimulating factor as well as monocyte-chemoattractant protein-1. Altogether, human MSC support hematopoiesis in Dexter-type cultures through the activation of the SDF-1/CXCR4 axis. Our data further suggest that SDF-1 stimulates retention of HSPC in MSC niches which expose them to stimulatory and inhibitory factors in a paracrine manner.     

Injectable fibroblast growth factor-2 coacervate for persistent angiogenesis

Enhancing the maturity of the newly formed blood vessels is critical for the success of therapeutic angiogenesis. The maturation of vasculature relies on active participation of mural cells to stabilize endothelium and a basal level of relevant growth factors. We set out to design and successfully achieved robust angiogenesis using an injectable polyvalent coacervate of a polycation, heparin, and fibroblast growth factor-2 (FGF2). FGF2 was loaded into the coacervate at nearly 100% efficiency. In vitro assays demonstrated that the matrix protected FGF2 from proteolytic degradations. FGF2 released from the coacervate was more effective in the differentiation of endothelial cells and chemotaxis of pericytes than free FGF2. One injection of 500 ng of FGF2 in the coacervate elicited comprehensive angiogenesis in vivo. The number of endothelial and mural cells increased significantly, and the local tissue contained more and larger blood vessels with increased circulation. Mural cells actively participated during the whole angiogenic process: Within 7 d of the injection, pericytes were recruited to close proximity of the endothelial cells. Mature vasculature stabilized by vascular smooth muscle cells persisted till at least 4 wk. On the other hand, bolus injection of an identical amount of free FGF2 induced weak angiogenic responses. These results demonstrate the potential of polyvalent coacervate as a new controlled delivery platform.     

A subpopulation of fibroblast growth factor-2-binding heparan sulfate is lost in human papillary thyroid carcinomas.

We hypothesized that a change in composition of proteoglycans can regulate the bioactivity of fibroblast growth factor (FGF)-2 in the thyroid. In order to test this hypothesis, we established a simple and sensitive method for detecting FGF-2-binding heparan sulfates and characterized them in papillary thyroid carcinomas and normal thyroids. The thyroid extracts were applied to a Q-Sepharose anion exchange column. After the column was washed with 10 mM of phosphate buffer, 1 microgram of human recombinant FGF-2 was added onto the column. The column was eluted with a gradient of NaCl (0.3-1.5 M). Each fraction was blotted onto nitrocellulose membrane. Immunoreactivity of heparan sulfate and FGF-2 was revealed by the incubation of membranes with the specific antibodies, and quantitatively estimated by measuring the density of the color product. In normal thyroids, immunoreactivity of heparan sulfate was detected as two peaks at 0.7 and 0.9 M of NaCl. Heparan sulfate-containing fractions also showed FGF-2 immunoreactivity, indicating the complex formation of FGF-2 and heparan sulfate. In papillary thyroid carcinomas, immunoreactivity of heparan sulfate showed various elution profiles on Q-Sepharose chromatography, including single peak at 0.7 M of NaCl and the one similar to that of the normal thyroids. However, FGF-2 immunoreactivity was detected only in the fractions eluting at 0.7 M of NaCl. This loss of a subpopulation of FGF-2-binding heparan sulfate in human papillary thyroid carcinomas may lead to the increase of free FGF-2 bioavailable in extracellular matrix.     

The metabolic state of diabetic monkeys is regulated by fibroblast growth factor-21

Fibroblast growth factor (FGF)-21 has been recently characterized as a potent metabolic regulator. Systemic administration of FGF-21 reduced plasma glucose and triglycerides to near normal levels in genetically compromised diabetic rodents. Importantly, these effects were durable and did not come at the expense of weight gain, hypoglycemia, or mitogenicity. To explore the therapeutic properties of FGF-21 in a nongenetically modified primate species, and thus demonstrate the potential for efficacy in humans, we evaluated its bioactivity in diabetic nonhuman primates. When administered daily for 6 wk to diabetic rhesus monkeys, FGF-21 caused a dramatic decline in fasting plasma glucose, fructosamine, triglycerides, insulin, and glucagon. Of significant importance in regard to safety, hypoglycemia was not observed at any point during the study. FGF-21 administration also led to significant improvements in lipoprotein profiles, including lowering of low-density lipoprotein cholesterol and raising of high-density lipoprotein cholesterol, beneficial changes in the circulating levels of several cardiovascular risk markers/factors, and the induction of a small but significant weight loss. These data support the development of FGF-21 for the treatment of diabetes and other metabolic diseases.     

Transgenic mice expressing human fibroblast growth factor-19 display increased metabolic rate and decreased adiposity

The fibroblast growth factors (FGFs), and the corresponding receptors, are implicated in more than just the regulation of epithelial cell proliferation and differentiation. Specifically, FGF23 is a regulator of serum inorganic phosphate levels, and mice deficient in FGF receptor-4 have altered cholesterol metabolism. The recently described FGF19 is unusual in that it is nonmitogenic and appears to interact only with FGF receptor-4. Here, we report that FGF19 transgenic mice had a significant and specific reduction in fat mass that resulted from an increase in energy expenditure. Further, the FGF19 transgenic mice did not become obese or diabetic on a high fat diet. The FGF19 transgenic mice had increased brown adipose tissue mass and decreased liver expression of acetyl coenzyme A carboxylase 2, providing two mechanisms by which FGF19 may increase energy expenditure. Consistent with the reduction in expression of acetyl CoA carboxylase 2, liver triglyceride levels were reduced.     

Plasmin-mediated fibroblast growth factor-2 mobilisation supports smooth muscle cell proliferation in human saphenous vein

The focus of this study was identification of the contribution of the plasminogen activator-plasmin system to smooth muscle cell proliferation and migration in human saphenous vein. Segments of human saphenous vein were grown in organ culture for up to 14 days. Smooth muscle cell proliferation and migration were measured by incubating vein segments in bromodeoxyuridine, and smooth muscle cell death was detected by in situ end-labelling. Tissue-type (tPA) and urokinase-type (uPA) plasminogen activator enzymic activities were detectable in cultured saphenous vein segments, and were concentrated in focal zones. Inhibition of plasmin activity with alpha-N-acetyl-L-lysine methyl ester (NALME) or of uPA activity with a neutralising antibody caused significant decreases in smooth muscle cell proliferation in the media and the intima, but no significant changes in smooth muscle cell migration. Intimal thickness was also significantly decreased. Incubation with plasminogen or plasmin caused fibroblast growth factor-2 (FGF2) to be released into the medium. Addition of FGF2 to segments cultured with NALME reversed the inhibition of smooth muscle cell proliferation, and blocking the activity of FGF2 with a neutralising antibody caused a significant decrease in medial smooth muscle cell proliferation. These data suggest that plasmin mobilises FGF2 bound to the extracellular matrix of human saphenous vein, so that it can support smooth muscle cell proliferation and intimal thickening.     

Mechanisms of fibroblast growth factor-2 modulation of vascular endothelial growth factor expression by osteoblastic cells

Normal bone growth and repair is dependent on angiogenesis. Fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGFbeta) have all been implicated in the related processes of angiogenesis, growth, development, and repair. The purpose of this study was to investigate the relationships between FGF-2 and both VEGF and TGFbeta in nonimmortalized and clonal osteoblastic cells. Northern blot analysis revealed 6-fold peak increases in VEGF mRNA at 6 h in fetal rat calvarial cells and MC3T3-E1 osteoblastic cells after stimulation with FGF-2. Actinomycin D inhibited these increases in VEGF mRNA, whereas cycloheximide did not. The stability ofVEGF mRNA was not increased after FGF-2 treatment. Furthermore, FGF-2 induced dose-dependent increases in VEGF protein levels (P < 0.01). Although in MC3T3-E1 cells, TGFbeta1 stimulates a 6-fold peak increase in VEGF mRNA after 3 h of stimulation, we found that both TGFbeta2 and TGFbeta3 yielded 2- to 3-fold peak increases in VEGF mRNA levels noted after 6 h of stimulation. Similarly, both TGFbeta2 and TGFbeta3 dose dependently increased VEGF protein production. To determine whether FGF-2-induced increases in VEGF mRNA may have occurred independently of TGFbeta, we disrupted TGFbeta signal transduction (using adenovirus encoding a truncated form of TGFbeta receptor II), which attenuated TGFbeta1 induction of VEGF mRNA, but did not impede FGF-2 induction ofVEGF mRNA. In summary, FGF-2-induced VEGF expression by osteoblastic cells is a dose-dependent event that may be independent of concomitant FGF-2-induced modulation of TGFbeta activity.