Matthew A. Nugent

Fibroblast growth factor-2.

Fibroblast growth factor-2 (FGF-2) is a member of a large family of proteins that bind heparin and heparan sulfate and modulate the function of a wide range of cell types. FGF-2 stimulates the growth and development of new blood vessels (angiogenesis) that contribute to the pathogenesis of several diseases (i.e. cancer, atherosclerosis), normal wound healing and tissue development. FGF-2 contains a number of basic residues (pI 9.6) and consists of 12 anti-parallel beta-sheets organized into a trigonal pyrimidal structure. FGF-2 binds to four cell surface receptors expressed as a number of splice variants. Many of the biological activities of FGF-2 have been found to depend on its receptors intrinsic tyrosine kinase activity and second messengers such as the mitogen activated protein kinases. However, considerable evidence suggest that intracellular FGF-2 might have a direct biological role particularly within the nucleus. In addition, heparan sulfate proteoglycans have been demonstrated to enhance and inhibit FGF-2 activity. The possibility that FGF-2 activity can be manipulated through alterations in heparan sulfate-binding is currently being exploited in the development of clinical applications aimed at modulating either endogenous or administered FGF-2 activity.

Basic fibroblast growth factor enhances the coupling of intimal hyperplasia and proliferation of vasa vasorum in injured rat arteries.

Basic fibroblast growth factor (bFGF) is mitogenic for smooth muscle cells (SMC) and angiogenic. We examined the in vivo effects of bFGF in balloon denuded carotid arteries of laboratory rats. bFGF was administered continuously from polymer-based devices at 34 ng/d into the periadventitial space of rat carotid arteries for 2 wk. Intimal hyperplasia was not observed in the absence of injury or with lipopolysaccharide induced endothelial dysfunction. Different degrees of vascular injury produced proportionally more intimal hyperplasia. bFGF increased the intimal hyperplastic response 1.3-fold with severe vascular injury, and 2.4-fold with more mild injury. Increased cell proliferation, not extracellular matrix production, accounted for these effects. Cell density was unchanged for the control and bFGF-treated groups, and the number of proliferating intimal cells at 2 wk rose to an amount equivalent to the increase in mass; 1.9- and 4.0-fold for severe and lesser injury, respectively. The relative ability of heparin to reduce SMC proliferation was not altered by the presence of bFGF.bFGF also induced profound angiogenesis within and surrounding the polymeric releasing device, and in the vasa vasorum immediately around the injured arteries. bFGFs effect on vasa was linearly related to the amount of SMC proliferation within the blood vessel. Thus, the in vivo mitogenic and angiogenic potential of bFGF are coupled, and may be similarly modulated by the products of local injury and/or factors in the vessel wall.

Mechanisms of Venous Leakage: A Prospective Clinicopathological Correlation of Corporeal Function and Structure

PURPOSE We investigated the pathophysiology of structurally based corporeal veno-occlusive dysfunction. MATERIALS AND METHODS We prospectively evaluated 24 impotent patients (mean age plus or minus standard error 46 +/- 3 years) who had exposure to vascular risk factors and/or disorders inducing diffuse trabecular structure alterations and who underwent penile prosthesis insertion. Preoperative indexes of veno-occlusive function (flow to maintain, venous outflow resistance and pressure decay measurements using repeat dosing pharmacocavernosometry) were correlated with postoperative erectile tissue computer assisted color histomorphometry (percent trabecular smooth muscle to total erectile tissue area). To develop further study findings and correlate histomorphometric findings with molecular biological properties molecular biological studies (ribonuclease protection analysis, reverse transcription-polymerase chain reaction assay for expression of transforming growth factor-beta 1 messenger [m] ribonucleic acid [RNA] and protein affinity labeling techniques for specific transforming growth factor-beta receptors) were performed in representative patients with high (39 to 43%), intermediate (30 to 37%) and low (13 to 29%) trabecular smooth muscle content (normal 42 to 50%). RESULTS Flow to maintain, venous outflow resistance and pressure decay values significantly correlated with trabecular smooth muscle cell content (r = -0.89, 0.82 and -0.85, respectively). In the high, intermediate and low smooth muscle content subgroups flow to maintain, venous outflow resistance and pressure decay values were 1 to 5, 9 to 30 and 50 to 120 ml. per minute, 17 to 84, 3 to 9 and 1 to 2 mm. Hg/ml. per minute, and 40 to 60, 48 to 80 and 110 to 120 mm. Hg decrease in 30 seconds from 150 mm. Hg, respectively. There were no significant differences in patient age or prevalence of risk factors among the 3 subgroups. Patients representative of all 3 subgroups had transforming growth factor-beta 1 mRNA, auto-induction of transforming growth factor-beta 1 mRNA and induction and/or increased availability of all 3 types of transforming growth factor-beta receptors. CONCLUSIONS The pathophysiology of structurally based corporeal veno-occlusive dysfunction is related to elevated corporeal connective tissue content. Based on our data and those in the literature corporeal fibrosis is hypothesized to develop secondary to abnormalities in the regulation of normal collagen synthesis and degradation, most likely associated with adverse influences of chronic ischemia.

Antisense targeting of perlecan blocks tumor growth and angiogenesis in vivo.

Perlecan, a ubiquitous heparan sulfate proteoglycan, possesses angiogenic and growth-promoting attributes primarily by acting as a coreceptor for basic fibroblast growth factor (FGF-2). In this report we blocked perlecan expression by using either constitutive CMV-driven or doxycycline- inducible antisense constructs. Growth of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression and these effects correlated with reduced responsiveness to and affinity for mitogenic keratinocyte growth factor (FGF-7). Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan sulfate-independent manner. In both tumor xenografts induced by human colon carcinoma cells and tumor allografts induced by highly invasive mouse melanoma cells, perlecan suppression caused substantial inhibition of tumor growth and neovascularization. Thus, perlecan is a potent inducer of tumor growth and angiogenesis in vivo and therapeutic interventions targeting this key modulator of tumor progression may improve cancer treatment.

Sustained Down-regulation of the Epidermal Growth Factor Receptor by Decorin A MECHANISM FOR CONTROLLING TUMOR GROWTH IN VIVO

The small leucine-rich proteoglycan decorin interacts with the epidermal growth factor receptor (EGFR) and triggers a signaling cascade that leads to elevation of endogenous p21 and growth suppression. We demonstrate that decorin causes a sustained down-regulation of the EGFR. Upon stable expression of decorin, the EGFR number is reduced by ∼40%, without changes in EGFR expression. However, EGFR phosphorylation is nearly completely abolished. Concurrently, decorin attenuates the EGFR-mediated mobilization of intracellular calcium and blocks the growth of tumor xenografts by down-regulating the EGFR kinase in vivo. Thus, decorin acts as an autocrine and paracrine regulator of tumor growth and could be utilized as an effective anti-cancer agent.

Basic Fibroblast Growth Factor Binds Its Receptors, Is Internalized, and Stimulates DNA Synthesis in Balb/c3T3 Cells in the Absence of Heparan Sulfate

We have investigated the interaction of basic fibroblast growth factor (bFGF) with its receptors and heparan sulfate proteoglycans (HSPG). It has been suggested that in the absence of HSPG, cells are not able to bind bFGF or respond to treatment with bFGF. In our studies, Balb/c3T3 fibroblasts were treated with 50 mM sodium chlorate to completely inhibit (99%) sulfation of proteoglycans. We found that bFGF was able to bind, be internalized, and stimulate DNA synthesis in the absence of HSPG in a dose-dependent manner. bFGF bound to its receptors on chlorate-treated cells with a lower apparent affinity and no change in receptor number. To determine if this decreased affinity bFGF-receptor interaction is functional, we quantitatively analyzed bFGF internalization and stimulation of DNA synthesis in control and chlorate-treated cells. Endocytotic rate constants (ke) for chlorate-treated and control cells were ke = 0.078 ± 0.022 min−1 and ke = 0.043 ± 0.012 min−1, respectively, suggesting that the process of bFGF internalization is not dramatically altered by HSPG. bFGF stimulated DNA synthesis to the same maximal level under both conditions, but chlorate-treated cells were significantly less responsive at low bFGF doses (∼10-fold increase in ED50). The differences observed for control and chlorate-treated cells in the dose-response curves for stimulation of DNA synthesis and receptor binding correlated directly, suggesting that receptors are equally capable of eliciting a mitogenic signal under both conditions. It is unlikely that these results are due to residual HSPG since heparinase (I and III) digestion of chlorate-treated cells had little effect. Although the presence of HSPG on the cell surface increases the affinity of bFGF for its receptors, our observations suggest that HSPG are not “absolutely” required for binding, internalization, or stimulation of mitogenic activity.

Heparan sulfate-FGF10 interactions during lung morphogenesis

Signaling by fibroblast growth factor 10 (FGF10) through FGFR2b is essential for lung development. Heparan sulfates (HS) are major modulators of growth factor binding and signaling present on cell surfaces and extracellular matrices of all tissues. Although recent studies provide evidence that HS are required for FGF-directed tracheal morphogenesis in Drosophila, little is known about the HS role in FGF10-mediated bud formation in the vertebrate lung. Here, we mapped HS expression in the early lung and we investigated how HS interactions with FGF10-FGFR2b influence lung morphogenesis. Our data show that a specific set of HS low in O-sulfates is dynamically expressed in the lung mesenchyme at the sites of prospective budding near Fgf10-expressing areas. In turn, highly sulfated HS are present in basement membranes of branching epithelial tubules. We show that disrupting endogenous gradients of HS or altering HS sulfation in embryonic lung culture systems prevents FGF10 from inducing local responses and markedly alters lung pattern formation and gene expression. Experiments with selectively sulfated heparins indicate that O-sulfated groups in HS are critical for FGF10 signaling activation in the epithelium during lung bud formation, and that the effect of FGF10 in pattern is in part determined by regional distribution of O-sulfated HS. Moreover, we describe expression of a HS 6-O-sulfotransferase preferentially at the tips of branching tubules. Our data suggest that the ability of FGF10 to induce local budding is critically influenced by developmentally regulated regional patterns of HS sulfation.

Vascular cell-derived heparan sulfate shows coupled inhibition of basic fibroblast growth factor binding and mitogenesis in vascular smooth muscle cells.

Basic fibroblast growth factor (bFGF) has been previously shown to be mitogenic for vascular smooth muscle cells (VSMCs) in vivo, but only after vascular injury. We show in the present study that the regulation of bFGF-stimulated VSMC proliferation, by vascular cell-secreted heparin-like compounds, correlates with inhibition of bFGF binding to cell-associated heparin sulfate proteoglycans. The stimulation of cultured VSMC proliferation by bFGF was markedly reduced when these cells were cocultured with confluent endothelial cells or confluent VSMCs (100.8 +/- 8.4% and 55.6 +/- 2.3% inhibition, respectively) or with conditioned media from these two cell types. Balb/c3T3 fibroblasts had no statistically significant effect on bFGF-stimulated VSMC proliferation. Vascular cell-conditioned media also inhibited bFGF binding to heparan sulfate proteoglycans on VSMCs, and the inhibition of binding correlated linearly with the inhibition of proliferation after a critical amount of binding was inhibited (44%) (r = .952, P < .0001). Heparinase or heparitinase treatment of conditioned media removed the bFGF-inhibitory effects, presumably by degrading heparin-like compounds. Indeed, heparin itself mimicked the inhibitory effects of conditioned media on bFGF-mediated proliferation and binding to heparin sulfate proteoglycans. These results suggest a bFGF regulatory role for vascular cell-produced heparin-like compounds, linking the mitogenic effects with binding to heparan sulfate proteoglycans for this heparin-binding growth factor.

Endothelial proteoglycans inhibit bFGF binding and mitogenesis.

Basic fibroblast growth factor (bFGF) is a known mitogen for vascular smooth muscle cells and has been implicated as having a role in a number of proliferative vascular disorders. Binding of bFGF to heparin or heparan sulfate has been demonstrated to both stimulate and inhibit growth factor activity. The activity, towards bFGF, of heparan sulfate proteoglycans present within the vascular system is likely related to the chemical characteristics of the glycosaminoglycan as well as the structure and pericellular location of the intact proteoglycans. We have previously shown that endothelial conditioned medium inhibits both bFGF binding to vascular smooth muscle cells and bFGF stimulated cell proliferation in vitro. In the present study, we have isolated proteoglycans from endothelial cell conditioned medium and demonstrated that they are responsible for the bFGF inhibitory activity. We further separated endothelial secreted proteoglycans into two fractions, PG‐A and PG‐B. The larger sized fraction (PG‐A) had greater inhibitory activity than did PG‐B for both bFGF binding and bFGF stimulation of vascular smooth muscle cell proliferation. The increased relative activity of PG‐A was attributed, in part, to larger heparan sulfate chains which were more potent inhibitors of bFGF binding than the smaller heparan sulfate chains on PG‐B. Both proteoglycan fractions contained perlecan‐like core proteins; however, PG‐A contained an additional core protein (approximately 190 kDa) that was not observed in PG‐B. Both proteoglycan fractions bound bFGF directly, and PG‐A bound a significantly greater relative amount of bFGF than did PG‐B. Thus the ability of endothelial heparan sulfate proteoglycans to bind bFGF and prevent its association with vascular smooth muscle cells appears essential for inhibition of bFGF‐induced mitogenesis. The production of potent bFGF inhibitory heparan sulfate proteoglycans by endothelial cells might contribute to the maintenance of vascular homeostasis. J. Cell. Physiol. 172:209–220, 1997.

Heparan Sulfate Proteoglycans Function as Receptors for Fibroblast Growth Factor-2 Activation of Extracellular Signal–Regulated Kinases 1 and 2

Abstract— Fibroblast growth factor-2 (FGF2) activates the extracellular signal–regulated kinases 1 and 2 (ERK1/2) through its specific receptors. Interaction of FGF2 with cell-surface heparan sulfate proteoglycans has also been suggested to induce intracellular signals. Thus, we investigated whether FGF2 can stimulate ERK1/2 activation through heparan sulfate proteoglycans using mechanisms that do not depend on receptor activation in vascular smooth muscle cells. The activation of FGF receptors was inhibited by treating cells with 5′-deoxy-5′methyl-thioadenosine and by expressing truncated dominant-negative FGF receptors. In both cases, FGF2 was able to stimulate the phosphorylation of ERK1/2 despite the absence of detectable FGF receptor tyrosine kinase activity. The FGF2 activation of ERK1/2 in the absence of receptor activity was completely dependent on heparan sulfate, because this activity was abolished by heparinase III digestion of the cells. In contrast, heparinase III treatment of control cells, with functional FGF receptors, showed only slight changes in FGF2-mediated ERK1/2 activation kinetics. Thus, in addition to serving as coreceptors for FGF receptor activation, heparan sulfate proteoglycans might also function directly as receptors for FGF2-induced ERK1/2 activation. Activation of ERK1/2 via cell-surface proteoglycans could have significant biological consequences, potentially directing cell response toward growth, migration, or differentiation.