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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 show FGF-2 binding and internalization under flow.For the simulations, FGF-2 (1 ng) was introduced into the reservoir (30% nonspecific loss) and sent into the cell-lined hollow fibers under pulsatile flow (0.63 mL/min, 1.26 mm/sec). (A) The sum of all cell surface bound FGF-2 (•) and FGF-2 internalized (○) within the cell-lined hollow fiber are shown. (B, C) Plot of % FGFR bound to FGF-2 versus time at the entrance (•), middle (▴) and at the exit (▪) cell when the diffusion coefficient is 1.67×10−10 (B) or 1.67×10−9 m2/s (C). The fluid entering the system is essentially free of FGF-2 by 150s after flow is initiated.
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pcbi-1000971-g007: Simulations show FGF-2 binding and internalization under flow.For the simulations, FGF-2 (1 ng) was introduced into the reservoir (30% nonspecific loss) and sent into the cell-lined hollow fibers under pulsatile flow (0.63 mL/min, 1.26 mm/sec). (A) The sum of all cell surface bound FGF-2 (•) and FGF-2 internalized (○) within the cell-lined hollow fiber are shown. (B, C) Plot of % FGFR bound to FGF-2 versus time at the entrance (•), middle (▴) and at the exit (▪) cell when the diffusion coefficient is 1.67×10−10 (B) or 1.67×10−9 m2/s (C). The fluid entering the system is essentially free of FGF-2 by 150s after flow is initiated.

Mentions: Our experimental system does not allow easy separation between internalized FGF-2 and that bound to the cell surface or visualization of FGF-2 distribution within the cell-lined hollow fiber. Using our computer model we examined how FGF-2 would be distributed with respect to time after flow was initiated (Figure 7). At a relatively low flow rate (0.63 mL/min, 1.26 mm/sec), the FGF-2 in the reservoir had essentially all entered the hollow fibers by 150s and the peak outflow of FGF-2 was evident ∼200s after flow was initiated corresponding to the time when the bulk FGF-2 had exited the hollow fibers. Later times showed cell-bound FGF-2 either internalized or dissociated from the cell surface with little chance to reassociate. The vast majority of binding is predicted to occur near the entrance to the cell-lined hollow fibers as opposed to the middle or end of the fibers (Figure 7B). The impact of time was more pronounced in the front section also as fluid entering the hollow fiber after ∼150s was devoid of FGF-2 (<0.1% of initial FGF-2). Increasing the diffusion rate for FGF-2 in solution by increasing the diffusion coefficient by an order of magnitude is predicted to have a negligible impact on FGF-2 capture in the front of the capillary but increased significantly the FGF-2 bound down the length of the cell-lined hollow fiber. This was due to changes in the depletion zone near the cell-lined walls (Figure 8). After 44s, an FGF-2 depletion zone near the surface was evident which was reduced when the diffusive transport of FGF-2 was increased. The replenishment of FGF-2 near the wall promoted greater FGF-2 binding as complex formation is a second-order process and illustrates the importance of surface depletion in growth factor capture.


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 show FGF-2 binding and internalization under flow.For the simulations, FGF-2 (1 ng) was introduced into the reservoir (30% nonspecific loss) and sent into the cell-lined hollow fibers under pulsatile flow (0.63 mL/min, 1.26 mm/sec). (A) The sum of all cell surface bound FGF-2 (•) and FGF-2 internalized (○) within the cell-lined hollow fiber are shown. (B, C) Plot of % FGFR bound to FGF-2 versus time at the entrance (•), middle (▴) and at the exit (▪) cell when the diffusion coefficient is 1.67×10−10 (B) or 1.67×10−9 m2/s (C). The fluid entering the system is essentially free of FGF-2 by 150s after flow is initiated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2965741&req=5

pcbi-1000971-g007: Simulations show FGF-2 binding and internalization under flow.For the simulations, FGF-2 (1 ng) was introduced into the reservoir (30% nonspecific loss) and sent into the cell-lined hollow fibers under pulsatile flow (0.63 mL/min, 1.26 mm/sec). (A) The sum of all cell surface bound FGF-2 (•) and FGF-2 internalized (○) within the cell-lined hollow fiber are shown. (B, C) Plot of % FGFR bound to FGF-2 versus time at the entrance (•), middle (▴) and at the exit (▪) cell when the diffusion coefficient is 1.67×10−10 (B) or 1.67×10−9 m2/s (C). The fluid entering the system is essentially free of FGF-2 by 150s after flow is initiated.
Mentions: Our experimental system does not allow easy separation between internalized FGF-2 and that bound to the cell surface or visualization of FGF-2 distribution within the cell-lined hollow fiber. Using our computer model we examined how FGF-2 would be distributed with respect to time after flow was initiated (Figure 7). At a relatively low flow rate (0.63 mL/min, 1.26 mm/sec), the FGF-2 in the reservoir had essentially all entered the hollow fibers by 150s and the peak outflow of FGF-2 was evident ∼200s after flow was initiated corresponding to the time when the bulk FGF-2 had exited the hollow fibers. Later times showed cell-bound FGF-2 either internalized or dissociated from the cell surface with little chance to reassociate. The vast majority of binding is predicted to occur near the entrance to the cell-lined hollow fibers as opposed to the middle or end of the fibers (Figure 7B). The impact of time was more pronounced in the front section also as fluid entering the hollow fiber after ∼150s was devoid of FGF-2 (<0.1% of initial FGF-2). Increasing the diffusion rate for FGF-2 in solution by increasing the diffusion coefficient by an order of magnitude is predicted to have a negligible impact on FGF-2 capture in the front of the capillary but increased significantly the FGF-2 bound down the length of the cell-lined hollow fiber. This was due to changes in the depletion zone near the cell-lined walls (Figure 8). After 44s, an FGF-2 depletion zone near the surface was evident which was reduced when the diffusive transport of FGF-2 was increased. The replenishment of FGF-2 near the wall promoted greater FGF-2 binding as complex formation is a second-order process and illustrates the importance of surface depletion in growth factor capture.

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