Limits...
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.

Show MeSH
Significant retention of FGF-2 occurs under flow.(A) FGF-2 (5.0+/−0.4 ng) was injected into the inlet reservoir, pumped through the cartridge at 0.65+/−0.01 mL/min (1.3 mm/sec), and measured in the output stream samples from three independent runs on three separate cartridges. The average retention of FGF-2 within the cell-lined cartridge was 40+/−0.5% FGF-2 (mean +/− standard deviation of the three runs shown) with a specific binding of 9+/−2.5% (B) FGF-2 ((•) 0.92 ng , (○) 6.9 ng, (X) 12 ng, and (▪) 18 ng FGF-2) was injected into the initial reservoir, run through the system at 0.64 mL/min, and the FGF-2 in the output stream measured using ELISA. (C) FGF-2 (ng) retained within the cell-lined cartridge versus the FGF-2 (ng) injected into the system is shown. The flow rate for this study varied between 0.60 and 0.67 mL/min (1.2 and 1.34 mm/sec respectively). Results are from individual runs with 9 independent cartridges.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2965741&req=5

pcbi-1000971-g003: Significant retention of FGF-2 occurs under flow.(A) FGF-2 (5.0+/−0.4 ng) was injected into the inlet reservoir, pumped through the cartridge at 0.65+/−0.01 mL/min (1.3 mm/sec), and measured in the output stream samples from three independent runs on three separate cartridges. The average retention of FGF-2 within the cell-lined cartridge was 40+/−0.5% FGF-2 (mean +/− standard deviation of the three runs shown) with a specific binding of 9+/−2.5% (B) FGF-2 ((•) 0.92 ng , (○) 6.9 ng, (X) 12 ng, and (▪) 18 ng FGF-2) was injected into the initial reservoir, run through the system at 0.64 mL/min, and the FGF-2 in the output stream measured using ELISA. (C) FGF-2 (ng) retained within the cell-lined cartridge versus the FGF-2 (ng) injected into the system is shown. The flow rate for this study varied between 0.60 and 0.67 mL/min (1.2 and 1.34 mm/sec respectively). Results are from individual runs with 9 independent cartridges.

Mentions: The average fluid velocity in human capillaries is <1 mm/sec [4]. We hypothesized that capture of regulatory growth factors from solution would be significant at these flow rates thereby facilitating growth factor activity. Using the lowest velocity setting with the standard pulsatile pump included with the Cellmax system (∼1.3 mm/sec, ∼0.65 mL/min), FGF-2 (5.0±0.4 ng) was injected into the cartridge inlet reservoir and flow was commenced. As shown in Figure 3, there is a delay in FGF-2 appearance in the outflow corresponding to the time for FGF-2 to travel through the cartridge and exit the system. The majority of FGF-2 added exited the cartridge as a large peak approximately 1 mL (or 1.5 min at this flow rate) after flow was initiated. Non-specific binding within the injection cartridge reservoir was measured directly (31+/−2.5%). Specific binding within the cell-lined hollow fibers accounted for 9+/−2.5% of total FGF-2 added to the cartridge at this concentration and ∼13% of the FGF-2 entering the cell-lined fibers, after taking into account non-specific binding (Figure 3). The results shown in Figure 3A are from three independent experiments conducted using three different cartridges illustrating the reproducibility of the system. Repeat runs conducted using the same cartridge as well as runs using radiolabeled FGF-2 instead of unlabeled FGF-2 both produced similar results (data not shown). The peak appearance time or volume in the outflow from the cartridge was insensitive to FGF-2 injection concentration in the range studied (data not shown). However, the size of the FGF-2 peak correlated with the injection concentration with the highest peak corresponding to the highest concentration of FGF-2 added (Figure 3B).


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)

Significant retention of FGF-2 occurs under flow.(A) FGF-2 (5.0+/−0.4 ng) was injected into the inlet reservoir, pumped through the cartridge at 0.65+/−0.01 mL/min (1.3 mm/sec), and measured in the output stream samples from three independent runs on three separate cartridges. The average retention of FGF-2 within the cell-lined cartridge was 40+/−0.5% FGF-2 (mean +/− standard deviation of the three runs shown) with a specific binding of 9+/−2.5% (B) FGF-2 ((•) 0.92 ng , (○) 6.9 ng, (X) 12 ng, and (▪) 18 ng FGF-2) was injected into the initial reservoir, run through the system at 0.64 mL/min, and the FGF-2 in the output stream measured using ELISA. (C) FGF-2 (ng) retained within the cell-lined cartridge versus the FGF-2 (ng) injected into the system is shown. The flow rate for this study varied between 0.60 and 0.67 mL/min (1.2 and 1.34 mm/sec respectively). Results are from individual runs with 9 independent cartridges.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1000971-g003: Significant retention of FGF-2 occurs under flow.(A) FGF-2 (5.0+/−0.4 ng) was injected into the inlet reservoir, pumped through the cartridge at 0.65+/−0.01 mL/min (1.3 mm/sec), and measured in the output stream samples from three independent runs on three separate cartridges. The average retention of FGF-2 within the cell-lined cartridge was 40+/−0.5% FGF-2 (mean +/− standard deviation of the three runs shown) with a specific binding of 9+/−2.5% (B) FGF-2 ((•) 0.92 ng , (○) 6.9 ng, (X) 12 ng, and (▪) 18 ng FGF-2) was injected into the initial reservoir, run through the system at 0.64 mL/min, and the FGF-2 in the output stream measured using ELISA. (C) FGF-2 (ng) retained within the cell-lined cartridge versus the FGF-2 (ng) injected into the system is shown. The flow rate for this study varied between 0.60 and 0.67 mL/min (1.2 and 1.34 mm/sec respectively). Results are from individual runs with 9 independent cartridges.
Mentions: The average fluid velocity in human capillaries is <1 mm/sec [4]. We hypothesized that capture of regulatory growth factors from solution would be significant at these flow rates thereby facilitating growth factor activity. Using the lowest velocity setting with the standard pulsatile pump included with the Cellmax system (∼1.3 mm/sec, ∼0.65 mL/min), FGF-2 (5.0±0.4 ng) was injected into the cartridge inlet reservoir and flow was commenced. As shown in Figure 3, there is a delay in FGF-2 appearance in the outflow corresponding to the time for FGF-2 to travel through the cartridge and exit the system. The majority of FGF-2 added exited the cartridge as a large peak approximately 1 mL (or 1.5 min at this flow rate) after flow was initiated. Non-specific binding within the injection cartridge reservoir was measured directly (31+/−2.5%). Specific binding within the cell-lined hollow fibers accounted for 9+/−2.5% of total FGF-2 added to the cartridge at this concentration and ∼13% of the FGF-2 entering the cell-lined fibers, after taking into account non-specific binding (Figure 3). The results shown in Figure 3A are from three independent experiments conducted using three different cartridges illustrating the reproducibility of the system. Repeat runs conducted using the same cartridge as well as runs using radiolabeled FGF-2 instead of unlabeled FGF-2 both produced similar results (data not shown). The peak appearance time or volume in the outflow from the cartridge was insensitive to FGF-2 injection concentration in the range studied (data not shown). However, the size of the FGF-2 peak correlated with the injection concentration with the highest peak corresponding to the highest concentration of FGF-2 added (Figure 3B).

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