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Endothelial cell capture of heparin-binding growth factors under flow.

Zhao B, Zhang C, Forsten-Williams K, Zhang J, Fannon M - PLoS Comput. Biol. (2010)

Bottom Line: 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.Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule's 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.

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Simulations agree well with FGF-2 outflow measurements.(A) FGF-2 (0.92 ng) was injected into the cartridge reservoir and then flowed through the cell-lined hollow fibers at 0.63 mL/min (1.26 mm/sec), pulsatile flow. FGF-2 collected from the exit fluid (•) is shown. Simulation results based on cells expressing 1×104 FGFR/cell and 2.5×105 HSPG/cell with 32% loss in the entrance reservoir having the same FGF-2 amount injected at the same flow rate (○) are also shown. (B) Similar outflow FGF-2 measurements are shown following FGF-2 (0.92 ng) addition for heparinase-treated (experimental - •) and simulation results with out HSPG (simulations - ○). Simulations were run with cells expressing 1×104 FGFR/cell and 30% loss in the entrance reservoir.
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pcbi-1000971-g005: Simulations agree well with FGF-2 outflow measurements.(A) FGF-2 (0.92 ng) was injected into the cartridge reservoir and then flowed through the cell-lined hollow fibers at 0.63 mL/min (1.26 mm/sec), pulsatile flow. FGF-2 collected from the exit fluid (•) is shown. Simulation results based on cells expressing 1×104 FGFR/cell and 2.5×105 HSPG/cell with 32% loss in the entrance reservoir having the same FGF-2 amount injected at the same flow rate (○) are also shown. (B) Similar outflow FGF-2 measurements are shown following FGF-2 (0.92 ng) addition for heparinase-treated (experimental - •) and simulation results with out HSPG (simulations - ○). Simulations were run with cells expressing 1×104 FGFR/cell and 30% loss in the entrance reservoir.

Mentions: Capture of FGF-2 by endothelial cells within the vasculature is a critical step in growth factor activity and our bioreactor is an excellent tool for investigating the capture process. However, it has limitations with regard to quantification of cellular binding behavior. The cartridges are expensive for short-term experiments and culture time and preparation can be relatively lengthy. Visualization of individual cell behavior within the culture is not feasible. In addition, the ability to predict the capture of molecules by cells under flow has value across a wide range of areas and the development of a flow-based tool for the design and testing of mechanisms related to retention is desireable. Our computer model was designed based on media flow equations and mass transport equations [35] with cell surface reaction equations to reflect the cell-growth factor interactions (see Materials and Methods-Model development). To validate the model, simulations were performed using the variables (ie FGF input concentration and flow rate) specific for an experimental series and a comparison was made. Experimental trials were run in which FGF-2 (0.92 ng) was added to the reservoir, pumped through the cartridge, and outflow collected and analyzed for FGF-2. FGF-2 in the outflow showed a characteristic peak outflow approximately 100s after flow was initiated at 0.63 mL/min (1.26 mm/sec) and 17±6.3% of the input FGF-2 was retained within the cartridge after non-specific binding was subtracted (Figure 5). Simulations performed using the same input FGF-2 value and flow rate were run and comparison was made between the simulations and experimental outflow from control (Figure 5A) or heparinase-treated (Figure 5B) cartridges. We defined good agreement based on two criteria; the amount of FGF-2 recovered and the curve similarity. Criteria one requires the relative difference in FGF-2 outlow from the experimental and simulation studies to be less than 1% while the second criteria compares the actual amounts of FGF-2 exiting from the experimental and the simulation system (see Materials and Methods). We did note that FGF-2 retention with the simulations was very dependent on the level of HSPGs with higher densities resulting in too much retention via HSPG-FGF-2 binding and subsequent FGFR coupling while lower HSPG densities resulted in too little retention (data not shown). Comparison of simulation results with our heparinase-treated data showed fine agreement with regard to our criteria when non-specific loss in the reservoir was subtracted.


Endothelial cell capture of heparin-binding growth factors under flow.

Zhao B, Zhang C, Forsten-Williams K, Zhang J, Fannon M - PLoS Comput. Biol. (2010)

Simulations agree well with FGF-2 outflow measurements.(A) FGF-2 (0.92 ng) was injected into the cartridge reservoir and then flowed through the cell-lined hollow fibers at 0.63 mL/min (1.26 mm/sec), pulsatile flow. FGF-2 collected from the exit fluid (•) is shown. Simulation results based on cells expressing 1×104 FGFR/cell and 2.5×105 HSPG/cell with 32% loss in the entrance reservoir having the same FGF-2 amount injected at the same flow rate (○) are also shown. (B) Similar outflow FGF-2 measurements are shown following FGF-2 (0.92 ng) addition for heparinase-treated (experimental - •) and simulation results with out HSPG (simulations - ○). Simulations were run with cells expressing 1×104 FGFR/cell and 30% loss in the entrance reservoir.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965741&req=5

pcbi-1000971-g005: Simulations agree well with FGF-2 outflow measurements.(A) FGF-2 (0.92 ng) was injected into the cartridge reservoir and then flowed through the cell-lined hollow fibers at 0.63 mL/min (1.26 mm/sec), pulsatile flow. FGF-2 collected from the exit fluid (•) is shown. Simulation results based on cells expressing 1×104 FGFR/cell and 2.5×105 HSPG/cell with 32% loss in the entrance reservoir having the same FGF-2 amount injected at the same flow rate (○) are also shown. (B) Similar outflow FGF-2 measurements are shown following FGF-2 (0.92 ng) addition for heparinase-treated (experimental - •) and simulation results with out HSPG (simulations - ○). Simulations were run with cells expressing 1×104 FGFR/cell and 30% loss in the entrance reservoir.
Mentions: Capture of FGF-2 by endothelial cells within the vasculature is a critical step in growth factor activity and our bioreactor is an excellent tool for investigating the capture process. However, it has limitations with regard to quantification of cellular binding behavior. The cartridges are expensive for short-term experiments and culture time and preparation can be relatively lengthy. Visualization of individual cell behavior within the culture is not feasible. In addition, the ability to predict the capture of molecules by cells under flow has value across a wide range of areas and the development of a flow-based tool for the design and testing of mechanisms related to retention is desireable. Our computer model was designed based on media flow equations and mass transport equations [35] with cell surface reaction equations to reflect the cell-growth factor interactions (see Materials and Methods-Model development). To validate the model, simulations were performed using the variables (ie FGF input concentration and flow rate) specific for an experimental series and a comparison was made. Experimental trials were run in which FGF-2 (0.92 ng) was added to the reservoir, pumped through the cartridge, and outflow collected and analyzed for FGF-2. FGF-2 in the outflow showed a characteristic peak outflow approximately 100s after flow was initiated at 0.63 mL/min (1.26 mm/sec) and 17±6.3% of the input FGF-2 was retained within the cartridge after non-specific binding was subtracted (Figure 5). Simulations performed using the same input FGF-2 value and flow rate were run and comparison was made between the simulations and experimental outflow from control (Figure 5A) or heparinase-treated (Figure 5B) cartridges. We defined good agreement based on two criteria; the amount of FGF-2 recovered and the curve similarity. Criteria one requires the relative difference in FGF-2 outlow from the experimental and simulation studies to be less than 1% while the second criteria compares the actual amounts of FGF-2 exiting from the experimental and the simulation system (see Materials and Methods). We did note that FGF-2 retention with the simulations was very dependent on the level of HSPGs with higher densities resulting in too much retention via HSPG-FGF-2 binding and subsequent FGFR coupling while lower HSPG densities resulted in too little retention (data not shown). Comparison of simulation results with our heparinase-treated data showed fine agreement with regard to our criteria when non-specific loss in the reservoir was subtracted.

Bottom Line: 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.Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule's 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.

Show MeSH