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
EGF and VEGF are retained under flow.(A) EGF (1.49 ng) was injected into the input reservoir, pumped through the system at 0.61 mL/min (1.22 mm/sec), and EGF quantified in the output flow by ELISA. Data shown are from the same cartridge either untreated (○) or enzyme-treated (•). FGF-2 (1.01ng - x) is shown for comparison. (B) VEGF was injected into the input reservoir of untreated (0.95ng - ○) or heparinase-treated (0.98ng -•) cartridges, run through the system at 0.66 mL/min (1.32 mm/sec), and VEGF quantified in the output flow by ELISA. Data are representative of at least three runs quantified in Table 4.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2965741&req=5

pcbi-1000971-g004: EGF and VEGF are retained under flow.(A) EGF (1.49 ng) was injected into the input reservoir, pumped through the system at 0.61 mL/min (1.22 mm/sec), and EGF quantified in the output flow by ELISA. Data shown are from the same cartridge either untreated (○) or enzyme-treated (•). FGF-2 (1.01ng - x) is shown for comparison. (B) VEGF was injected into the input reservoir of untreated (0.95ng - ○) or heparinase-treated (0.98ng -•) cartridges, run through the system at 0.66 mL/min (1.32 mm/sec), and VEGF quantified in the output flow by ELISA. Data are representative of at least three runs quantified in Table 4.

Mentions: VEGF, a heparin binding protein, and EGF, which does not bind heparin, were next tested in this system. Both the initial appearance time and outflow volume for the protein as well as the general shape of the outflow peak for both VEGF and EGF were similar to FGF-2 (Figure 4). To ensure that the measured effects seen with heparinase-treatment on FGF-2 retention were due to specific responses of the growth factor to the removal of heparan sulfate and not a general response by all proteins, flow studies were done with VEGF and EGF following enzymatic treatment. EGF retention and outflow were unaffected by treatment with a cocktail of heparinase, chondroitinase, and keratanase (Table 4). Treatment with heparinase without chondroitinase or keratanase also had no effect on EGF retention or outflow (data not shown). In contrast, VEGF showed a significant decrease in specific retention between control and heparinase treated cartridges (16+/−5.8% versus −2.5+/−6.1% VEGF retained) indicating the critical role HSPG can have in heparin-binding growth factor capture under flow. The lack of a change in EGF binding or outflow profile under heparinase treatment is supportive that there are no gross changes in the cell glycocalyx that might impact the shear stress in the system.


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)

EGF and VEGF are retained under flow.(A) EGF (1.49 ng) was injected into the input reservoir, pumped through the system at 0.61 mL/min (1.22 mm/sec), and EGF quantified in the output flow by ELISA. Data shown are from the same cartridge either untreated (○) or enzyme-treated (•). FGF-2 (1.01ng - x) is shown for comparison. (B) VEGF was injected into the input reservoir of untreated (0.95ng - ○) or heparinase-treated (0.98ng -•) cartridges, run through the system at 0.66 mL/min (1.32 mm/sec), and VEGF quantified in the output flow by ELISA. Data are representative of at least three runs quantified in Table 4.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1000971-g004: EGF and VEGF are retained under flow.(A) EGF (1.49 ng) was injected into the input reservoir, pumped through the system at 0.61 mL/min (1.22 mm/sec), and EGF quantified in the output flow by ELISA. Data shown are from the same cartridge either untreated (○) or enzyme-treated (•). FGF-2 (1.01ng - x) is shown for comparison. (B) VEGF was injected into the input reservoir of untreated (0.95ng - ○) or heparinase-treated (0.98ng -•) cartridges, run through the system at 0.66 mL/min (1.32 mm/sec), and VEGF quantified in the output flow by ELISA. Data are representative of at least three runs quantified in Table 4.
Mentions: VEGF, a heparin binding protein, and EGF, which does not bind heparin, were next tested in this system. Both the initial appearance time and outflow volume for the protein as well as the general shape of the outflow peak for both VEGF and EGF were similar to FGF-2 (Figure 4). To ensure that the measured effects seen with heparinase-treatment on FGF-2 retention were due to specific responses of the growth factor to the removal of heparan sulfate and not a general response by all proteins, flow studies were done with VEGF and EGF following enzymatic treatment. EGF retention and outflow were unaffected by treatment with a cocktail of heparinase, chondroitinase, and keratanase (Table 4). Treatment with heparinase without chondroitinase or keratanase also had no effect on EGF retention or outflow (data not shown). In contrast, VEGF showed a significant decrease in specific retention between control and heparinase treated cartridges (16+/−5.8% versus −2.5+/−6.1% VEGF retained) indicating the critical role HSPG can have in heparin-binding growth factor capture under flow. The lack of a change in EGF binding or outflow profile under heparinase treatment is supportive that there are no gross changes in the cell glycocalyx that might impact the shear stress in the system.

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