Michael Fannon

PPARα agonist fenofibrate suppresses tumor growth through direct and indirect angiogenesis inhibition

Angiogenesis and inflammation are central processes through which the tumor microenvironment influences tumor growth. We have demonstrated recently that peroxisome proliferator-activated receptor (PPAR)α deficiency in the host leads to overt inflammation that suppresses angiogenesis via excess production of thrombospondin (TSP)-1 and prevents tumor growth. Hence, we speculated that pharmacologic activation of PPARα would promote tumor growth. Surprisingly, the PPARα agonist fenofibrate potently suppressed primary tumor growth in mice. This effect was not mediated by cancer-cell-autonomous antiproliferative mechanisms but by the inhibition of angiogenesis and inflammation in the host tissue. Although PPARα-deficient tumors were still susceptible to fenofibrate, absence of PPARα in the host animal abrogated the potent antitumor effect of fenofibrate. In addition, fenofibrate suppressed endothelial cell proliferation and VEGF production, increased TSP-1 and endostatin, and inhibited corneal neovascularization. Thus, both genetic abrogation of PPARα as well as its activation by ligands cause tumor suppression via overlapping antiangiogenic pathways. These findings reveal the potential utility of the well tolerated PPARα agonists beyond their use as lipid-lowering drugs in anticancer therapy. Our results provide a mechanistic rationale for evaluating the clinical benefits of PPARα agonists in cancer treatment, alone and in combination with other therapies.

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.

Control of Growth Factor Networks by Heparan Sulfate Proteoglycans

Growth factor binding to transmembrane protein receptors is generally understood to initiate cell signaling. Receptor binding of heparin-binding growth factors (HB-GFs), such as fibroblast growth factor-2 (FGF-2), is regulated by interactions with heparan sulfate proteoglycans. While there is some specificity for binding to heparan sulfate, overlap in sites for different growth factors may allow for cross regulation. Here we demonstrate, using experiments and computer simulations, that the HB-GFs FGF-2 and heparin-binding EGF-like growth factor (HB-EGF) can cross regulate receptor binding of the other despite having unique receptors. The ability of HSPG to stabilize HB-GF receptor binding is critical for competing growth factors to modulate receptor binding with both enhanced and reduced binding possible depending on this stabilization process. HSPG density and affinity for HB-GF are also critical factors for HB-GF cross regulation. Simulations further reveal that HB-GF can regulate receptor binding of non-HB-GFs such as EGF even when the two proteins share no binding sites when other HB-GF are present within the network. Proliferation studies demonstrate potentiation of HB-EGF-induced growth by FGF-2 indicating that competition networks can alter biological response. Exogenous manipulation of cellular responses to growth factors in complex living systems will require understanding the HSPG-controlled network.

Binding inhibition of angiogenic factors by heparan sulfate proteoglycans in aqueous humor: potential mechanism for maintenance of an avascular environment

Aqueous humor is a clear fluid, primarily a blood filtrate, which circulates through the anterior chamber of the eye and bathes the cornea. We explored the possibility that components in the aqueous humor play a direct part in maintaining the avascular environment of the cornea. We report here that heparan sulfate proteoglycan (HSPG) was found in bovine aqueous humor and that it directly inhibits binding of basic fibroblast growth factor and vascular endothelial growth factor to cell‐surface heparan sulfate. We demonstrate that this holds true for all heparin binding proteins tested but not for epidermal growth factor, which does not bind heparin. Furthermore, we show, with mathematical modeling, that the concentration of HSPG in aqueous humor (~4 µg/ml), when combined with the clearance of aqueous humor from the eye due to circulation, is sufficient to block the binding of heparin binding growth factors to corneal endothelium. This mechanism suggests a physiological process to control bioavailability of angiogenic growth factors in the cornea.

Vitamin D Binding Protein-Macrophage Activating Factor Directly Inhibits Proliferation, Migration, and uPAR Expression of Prostate Cancer Cells

Background Vitamin D binding protein-macrophage activating factor (DBP-maf) is a potent inhibitor of tumor growth. Its activity, however, has been attributed to indirect mechanisms such as boosting the immune response by activating macrophages and inhibiting the blood vessel growth necessary for the growth of tumors. Methods and Findings In this study we show for the first time that DBP-maf exhibits a direct and potent effect on prostate tumor cells in the absence of macrophages. DBP-maf demonstrated inhibitory activity in proliferation studies of both LNCaP and PC3 prostate cancer cell lines as well as metastatic clones of these cells. Flow cytometry studies with annexin V and propidium iodide showed that this inhibitory activity is not due to apoptosis or cell death. DBP-maf also had the ability to inhibit migration of prostate cancer cells in vitro. Finally, DBP-maf was shown to cause a reduction in urokinase plasminogen activator receptor (uPAR) expression in prostate tumor cells. There is evidence that activation of this receptor correlates with tumor metastasis. Conclusions These studies show strong inhibitory activity of DBP-maf on prostate tumor cells independent of its macrophage activation.

A Computational Model of FGF-2 Binding and HSPG Regulation Under Flow

A novel convection--diffusion--reaction model is developed to simulate fibroblast growth factor (FGF-2) binding to cell surface receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) under flow conditions within a cylindrical-shaped vessel or capillary. The model consists of a set of coupled nonlinear partial differential equations (PDEs) and a set of coupled nonlinear ordinary differential equations (ODEs). The time-dependent PDE system is discretized and solved by a second-order implicit Euler scheme using the finite volume method. The ODE system is solved by a stiff ODE solver VODE using backward differencing formulation (BDF). The transient solution of FGF-2, FGFR, HSPG, and their bound complexes for three different flow rates are computed and presented. Simulation results indicate that the model can predict growth factor transport and binding to receptors with/without the presence of heparan sulfate, as well as the effect of flow rate on growth factor-receptor binding. Our computational model may provide a useful means to investigate the impact of fluid flow on growth factor dynamics, and ultimately, signaling within the circulation.

Sucrose octasulfate regulates fibroblast growth factor-2 binding, transport, and activity: Potential for regulation of tumor growth

The antithrombotic activity of heparin has largely been credited with the success found in some cancer treatment by heparin. There are, however, many potent growth factors involved in tumor and blood vessel growth that bind to heparin with high affinity and their regulation by heparin may play a role in heparins efficacy. We therefore chose to study the activity of a heparin analog, sucrose octasulfate (SOS), which has been similarly shown to interact with heparin‐binding growth factors. Using mouse melanoma and lung carcinoma models, we demonstrate in vivo inhibition of tumor growth by SOS. SOS, however, showed little effect in coagulation assays indicating that this activity was not a primary mechanism of action for this molecule. Studies were then performed to assess the effect of SOS on basic fibroblast growth factor (FGF‐2) activity, a growth factor which promotes tumor and blood vessel growth and is produced by B16 melanoma cells. SOS potently inhibited FGF‐2 binding to endothelial cells and stripped pre‐bound FGF‐2 from cells. SOS also regulated FGF‐2 stimulated proliferation. Further, SOS facilitated FGF‐2 diffusion through Descemets membrane, a heparan sulfate‐rich basement membrane from the cornea, suggesting a possible role in FGF‐2 clearance. Our results suggest that molecules such as SOS have the potential to remove growth factors from tumor microenvironments and the approach offers an attractive area for further study. J. Cell. Physiol. 215: 434–441, 2008.

Endothelial Cell Capture of Heparin-Binding Growth Factors under Flow

Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecules fate, are difficult to interpret using traditional approaches. In this work, we analyzed and predicted growth factor capture under flow using computer modeling and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process was desired. The experimental module consisted of a bioreactor with synthetic endothelial-lined hollow fibers under flow. The physical design of the system was incorporated into the model parameters. The heparin-binding growth factor fibroblast growth factor-2 (FGF-2) was used for both the experiments and simulations. Our computational model was composed of three parts: (1) media flow equations, (2) mass transport equations and (3) cell surface reaction equations. The model is based on the flow and reactions within a single hollow fiber and was scaled linearly by the total number of fibers for comparison with experimental results. Our model predicted, and experiments confirmed, that removal of heparan sulfate (HS) from the system would result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicted a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that should prove advantageous for biologicals or drug delivery investigations.

Facilitated diffusion of VEGF165 through descemet's membrane with sucrose octasulfate

Vascular endothelial growth factor A (VEGF‐A) is a promoter of neovascularization and thus a popular therapeutic target for diseases involving excessive growth of blood vessels. In this study, we explored the potential of the disaccharide sucrose octasulfate (SOS) to alter VEGF165 diffusion through Descemets membrane. Descemets membranes were isolated from bovine eyes and used as a barrier between two chambers of a diffusion apparatus to measure VEGF transport. Diffusion studies revealed a dramatic increase in VEGF165 transport in the presence of SOS, with little diffusion of VEGF165 across the membrane over a 10‐h time course in the absence of SOS. Diffusion studies with VEGF121, a non‐heparin binding variant of VEGF, showed robust diffusion with or without SOS. To determine a possible mechanism, we measured the ability of SOS to inhibit VEGF interactions with extracellular matrix (ECM), using cell‐free and cell surface binding assays. Binding studies showed SOS had no effect on VEGF165 binding to either heparin‐coated plates or endothelial cell surfaces at less than mg/ml concentrations. In contrast, we show that SOS inhibited VEGF165 binding to fibronectin in a dose dependent manner and dramatically accelerated the rate of release of VEGF165 from fibronectin. SOS also inhibited the binding of VEGF165 to fibronectin‐rich ECM deposited by vascular smooth muscle cells. These results suggest that fibronectin‐rich extracellular matrices serve as barriers to VEGF165 diffusion by providing a network of binding sites that can trap and sequester the protein. Since the content of Descemets membrane is typical of many basement membranes it is possible that they serve throughout the body as formidable barriers to VEGF165 diffusion and tightly regulate its bioavailability and distribution within tissues. J. Cell. Physiol. 227: 3693–3700, 2012.

A Numerical Study of Pulsatile Flow Through a Hollow Fiber Cartridge: Growth Factor-Receptor Binding and Dissociation Analysis

This paper presents a numerical solution to describe growth factor-receptor binding under flow through hollow fibers of a bioreactor. The multi-physics of fluid flow, the kinetics of fibroblast growth factor (FGF-2) binding to its receptor (FGFR) and heparan sulfate proteoglycan (HSPG) and FGF-2 mass transport is modeled by a set of coupled nonlinear partial differential equations (PDEs) and coupled nonlinear ordinary differential equations (ODEs). A finite volume method is used to discretize the PDEs. The ODEs are solved by a stiff ODE solver CVODE. Overall, second order accuracy in time and space is achieved with the second order implicit Euler scheme. In order to obtain a reasonable accuracy of the binding and dissociation from cells, a uniform mesh is used. To handle pulsatile flow, several assumptions are made including neglecting any entrance effects and an analytical solution for axial velocity within the fibers is obtained. Qualitative and quantitative analysis are presented. Computational results and experimental measurements are compared and observed to agree quite well, indicating that the simulation model and methods could be used as a complementary and even predictable tool for the study of biochemical reactions in a similar flow environment.