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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 indicate coupling is critical for FGF-2 retention.(A) FGF-2 bound on cell surfaces plus internalized FGF-2 as a function of time for kc values of 0 (○), 0.0001(•), 0.001(□), and 0.1(▪) (#/cell)−1 min−1; (B,C) FGF-2 bound (▪), internalized (•), bound plus internalized (○) and exited (□) under flow with kc = 0.0024 (B) or 0 (C) (#/cell)−1min−1 following addition of FGF-2 (1ng) at 0.63 mL/min (1.26 mm/sec) pulsatile flow(30% non-specific loss). Capillaries were simulated to include 1×104 FGFR/cell and 2.5×105 HSPG/cell on the cell-lined hollow fibers. 300s corresponds to the time when essentially all of the FGF-2 has entered the hollow fiber from the reservoir.
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pcbi-1000971-g012: Simulations indicate coupling is critical for FGF-2 retention.(A) FGF-2 bound on cell surfaces plus internalized FGF-2 as a function of time for kc values of 0 (○), 0.0001(•), 0.001(□), and 0.1(▪) (#/cell)−1 min−1; (B,C) FGF-2 bound (▪), internalized (•), bound plus internalized (○) and exited (□) under flow with kc = 0.0024 (B) or 0 (C) (#/cell)−1min−1 following addition of FGF-2 (1ng) at 0.63 mL/min (1.26 mm/sec) pulsatile flow(30% non-specific loss). Capillaries were simulated to include 1×104 FGFR/cell and 2.5×105 HSPG/cell on the cell-lined hollow fibers. 300s corresponds to the time when essentially all of the FGF-2 has entered the hollow fiber from the reservoir.

Mentions: The results with the FGF-2-HSPG affinity simulations and the density studies indicated the importance of coupling in facilitating effective FGF-2-FGFR interactions. We next looked at how varying the coupling rate constant impacted binding and internalization using simulations (Figure 12). In the absence of HSPG-FGFR coupling (kc = 0), there is a reduction in peak binding of FGF-2 and the majority of FGF-2 bound is not internalized but dissociates and exits from the system in the outflow. Even with a low level of coupling, the FGF-2 binding and internalization is dramatically increased until a peak effect is seen with kc = 0. 01 (#/cell)−1 min−1. If we looked at later times in the simulation (Figure 12B), we would find that a large fraction of the FGF-2 injected is bound during the initial pass and that this bound FGF-2 is largely internalized with little exiting the system. If coupling between HSPG and FGFR is eliminated (Figure 12C), this is not the case. In this scenario, the cells bind a smaller but still significant level of FGF-2 during the initial pass but this FGF-2 is not retained and nearly all of the FGF-2 captured ultimately exits the system in the outflow.


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 indicate coupling is critical for FGF-2 retention.(A) FGF-2 bound on cell surfaces plus internalized FGF-2 as a function of time for kc values of 0 (○), 0.0001(•), 0.001(□), and 0.1(▪) (#/cell)−1 min−1; (B,C) FGF-2 bound (▪), internalized (•), bound plus internalized (○) and exited (□) under flow with kc = 0.0024 (B) or 0 (C) (#/cell)−1min−1 following addition of FGF-2 (1ng) at 0.63 mL/min (1.26 mm/sec) pulsatile flow(30% non-specific loss). Capillaries were simulated to include 1×104 FGFR/cell and 2.5×105 HSPG/cell on the cell-lined hollow fibers. 300s corresponds to the time when essentially all of the FGF-2 has entered the hollow fiber from the reservoir.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965741&req=5

pcbi-1000971-g012: Simulations indicate coupling is critical for FGF-2 retention.(A) FGF-2 bound on cell surfaces plus internalized FGF-2 as a function of time for kc values of 0 (○), 0.0001(•), 0.001(□), and 0.1(▪) (#/cell)−1 min−1; (B,C) FGF-2 bound (▪), internalized (•), bound plus internalized (○) and exited (□) under flow with kc = 0.0024 (B) or 0 (C) (#/cell)−1min−1 following addition of FGF-2 (1ng) at 0.63 mL/min (1.26 mm/sec) pulsatile flow(30% non-specific loss). Capillaries were simulated to include 1×104 FGFR/cell and 2.5×105 HSPG/cell on the cell-lined hollow fibers. 300s corresponds to the time when essentially all of the FGF-2 has entered the hollow fiber from the reservoir.
Mentions: The results with the FGF-2-HSPG affinity simulations and the density studies indicated the importance of coupling in facilitating effective FGF-2-FGFR interactions. We next looked at how varying the coupling rate constant impacted binding and internalization using simulations (Figure 12). In the absence of HSPG-FGFR coupling (kc = 0), there is a reduction in peak binding of FGF-2 and the majority of FGF-2 bound is not internalized but dissociates and exits from the system in the outflow. Even with a low level of coupling, the FGF-2 binding and internalization is dramatically increased until a peak effect is seen with kc = 0. 01 (#/cell)−1 min−1. If we looked at later times in the simulation (Figure 12B), we would find that a large fraction of the FGF-2 injected is bound during the initial pass and that this bound FGF-2 is largely internalized with little exiting the system. If coupling between HSPG and FGFR is eliminated (Figure 12C), this is not the case. In this scenario, the cells bind a smaller but still significant level of FGF-2 during the initial pass but this FGF-2 is not retained and nearly all of the FGF-2 captured ultimately exits the system in the outflow.

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