pubs.acs.org/Langmuir © 2009 American Chemical Society
Erythrocyte-Endothelium Adhesion Can Be Induced by Dextran Yang Yang, Huiling Eng, and Bj€orn Neu* Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, Singapore 639798 Received August 11, 2009. Revised Manuscript Received September 24, 2009 Plasma proteins have been identified to play a key role in the increased adhesiveness of red blood cells (RBC) to endothelial cells (EC) in various diseases associated with vascular complications. However, the underlying mechanisms on how plasma proteins facilitate adhesion remain unclear. In this study, we investigated if macromolecular depletion is able to induce adhesion of RBC to EC. RBC were suspended in solutions containing the neutral polyglucose dextran to mimic the effects of nonadsorbing macromolecules and the dynamics of RBC-EC adhesion were recorded using a parallel plate flow chamber system. Cell adhesion was markedly increased in the presence of dextran with a molecular mass larger than 70 kDa, with this increase reflected by both the number of cells adhering and the strength of the adhesion. This increased adhesiveness is attributed to reduced surface concentrations of the large polymers and hence attractive forces due to depletion interaction. Our results thus provide a rational explanation on how nonadsorbing plasma proteins could play a significant role in abnormal RBC-EC adhesion in vivo.
1. Introduction Red blood cell (RBC) adhesion to endothelial cells (EC) is usually insignificant but abnormal RBC adhesion to EC has been observed in various diseases such as sickle cell anemia or diabetes mellitus.1-4 Although several cell surface alterations have been linked to the increased RBC adhesiveness in disease states, such as the expression of receptors like VCAM-1, ICAM-1 or CD36þ,5,6 the detailed underlying mechanisms often remain unclear.7 In addition various plasma factors have been identified to enhance adhesion of pathological RBC to EC.8-10 For example fibrinogen has been identified as a promoter of RBC-EC adhesion,11,12 which is consistent with the observation that the onset of vasoocclusive crisis in sickle cell disease is always accompanied by a temporal elevated level of this protein.11,13 As a likely mechanism, it has been suggested that the various plasma factors act as ligands, cross-linking receptors on adjacent cells, and thus inducing or promoting RBC adhesion to EC. On the other hand, various nonspecific forces such as van der Waals interaction, electrostatic repulsion, sterical interaction, and membrane undulations are also known to play an important role
in cell adhesion. Another nonspecific force, which has received less attention in the area of cell-cell interaction, is induced by macromolecular depletion. Depletion interaction is a result of a lower localized protein or polymer concentration near the cell surface as compared to the suspending medium.14 This exclusion of macromolecules near the cell surface leads to an osmotic gradient and as two cells approach, solvent is displaced from the depletion zone into the bulk phase leading to an attractive force. More recently, it has been demonstrated that depletion interaction is most likely the driving force behind the reversible aggregation of RBC and that it can induce RBC adhesion to glass surfaces.15,16 However, if macromolecular depletion might also influence or control other cell-cell interactions and the role of depletion interaction in the stabilization and destabilization of blood flow in vivo remains to be clarified. In this study, we investigated if macromolecular depletion could be an alternative mechanism for the adhesion promoting effect of plasma proteins in RBC-EC adhesion. For this purpose, RBC-EC adhesion was studied in the presence of dextran, a neutral poly glucose, which is known to be depleted from the RBC surface.17,18
*Corresponding author. E-mail:
[email protected]. Telephone: (65) 6790 6951. Fax: (65) 6791 1761. (1) Hebbel, R. P.; Boogaerts, M. A.; Eaton, J. W.; Steinberg, M. H. N. Engl. J. Med. 1980, 302, 992-995. (2) Hebbel, R. P.; Yamada, O.; Moldow, C. F.; Jacob, H. S.; White, J. G.; Eaton, J. W. J. Clin. Invest. 1980, 65, (1), 154-160. (3) Wautier, J. L.; Wautier, M. P.; Schmidt, A. M.; Anderson, G. M.; Hori, O.; Zoukourian, C.; Capron, L.; Chappey, O.; Yan, S. D.; Brett, J.; Guillausseau, P. J.; Stern, D. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7742-7746. (4) Yedgar, S.; Koshkaryev, A.; Barshtein, G. Pathophysiology Haemostasis Thrombosis 2002, 32, (5-6), 263-268. (5) Gee, B. E.; Platt, O. S. Blood 1995, 85, 268-274. (6) Swerlick, R. A.; Eckman, J. R.; Kumar, A.; Jeitler, M.; Wick, T. M. Blood 1993, 82, 1891-1899. (7) Hebbel, R. P. Transfusion Clin. Biol. 2008, 15, (1-2), 14-18. (8) Wautier, J. L.; Wautier, M. P.; Pintigny, D.; Galacteros, F.; Courillon, A.; Passa, P.; Caen, J. P. Blood Cells 1983, 9, (2), 221-234. (9) Shiu, Y. T.; McIntire, L. V. Ann. Biomed. Eng. 2003, 31, 1299-1313. (10) Mohandas, N.; Evans, E. Blood 1984, 64, 282-287. (11) Hebbel, R. P.; Moldow, C. F.; Steinberg, M. H. Blood 1981, 58, 947-952. (12) Wautier, J. L.; Pintigny, D.; Wautier, M. P.; Paton, R. C.; Galacteros, F.; Passa, P.; Caen, J. P. J. Lab. Clin. Med. 1983, 101, 911-920. (13) Richardson, S. G.; Matthews, K. B.; Stuart, J.; Geddes, A. M.; Wilcox, R. M. Br. J. Hamaetol. 1979, 41, (1), 95-103.
2680 DOI: 10.1021/la902977y
2. Materials and Methods Red Blood cells. Blood was drawn from the antecubital vein of healthy adult volunteers into EDTA (1.5 mg/mL). Red blood cells were separated from whole blood by gentle centrifugation (1,000 g, 10 min), then washed thrice with phosphate buffered saline (PBS, 10 mM phosphate, 285 mOsm/kg, pH = 7.4) containing 0.2% BSA. RBC were resuspended in either dextranfree PBS or in dextran-PBS solutions: dextran 70 kDa, dextran 150 kDa, dextran 500 kDa, and dextran 2 MDa (Sigma-Aldrich, Singapore) were dissolved in PBS at the desired final concentrations. (14) Feign, R. I.; Napper, D. H. J. Colloid Interface Sci. 1980, 75, 525-541. (15) Zhang, Z.; Neu, B. Biophys. J. 2009, 97, 1031-1037. (16) Neu, B.; Meiselman, H. J. Biophys. J. 2002, 83, 2482-2490. (17) B€aumler, H.; Donath, E.; Krabi, A.; Knippel, W.; Budde, A.; Kiesewetter, H. Biorheology 1996, 33, (4-5), 333-351. (18) Rad, S.; Gao, J.; Baskurt, O. K.; Meiselman, H. J.; Neu, B. Electrophoresis 2009, 30, 450-456.
Published on Web 10/07/2009
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Endothelial Cells. Human umbilical vein endothelial cells (HUVEC) were obtained from Lonza, UK. The culture medium consisted of 90% basal medium (Ham’s F-12K with 2 mM L-glutamine (Sigma), 1.5 g/L sodium bicarbonate (Sigma), 0.1 mg/mL heparin (Sigma), 0.2 vol % bovine brain extract (BBE; Hammond Cell Tech), 120 U/mL penicillin/streptomycin (Sigma) and 10% fetal bovine serum (FBS; PAC)). HUVEC were placed in tissue culture flasks precoated with gelatin and cultured at 37 °C in a CO2 (5%) incubator. Once the cells reached 80% confluence, the cells were subcultured in 35 mm Petri dishes (Greiner) precoated with collagen from fish skin (Sigma) and grown to confluence. Parallel Plate Flow Chamber System. The flow system consists of an acrylic flow deck and a silicone rubber gasket (Glycotech, USA) with the cutout area of the gasket forming the flow channel. Both the gasket and flow deck were placed into 35 mm Petri-dishes coated with confluent layers of HUVEC. This flow chamber was then placed on an inverted microscope (IX71, Olympus). The inlet of the chamber was connected by silicone tubing to a miniature low displacement electronic valve that allowed switching between reservoirs containing either RBC suspensions or rinse solution. The outlet of the chamber was connected to a variable speed syringe pump (Harvard Apparatus Co., Millis, MA) that drew either RBC suspension or rinse solution through the flow chamber at a selected volumetric flow rates Q. The microscope, valve, inflow tubing and the reservoirs for the two fluids were maintained at 37 °C via a thermostated enclosure. The wall shear stress τ was calculated via τ = 6μQ/a2b, where μ is the dynamic viscosity of the solution, a the channel height (0.254 mm) and b is the channel width (2.5 mm). The density of the solutions was measured by a density meter (Anton Paar DMA35) and the dynamic viscosities were measured by an automated micro capillary viscometer (Anton Paar AMVn). Experimental Protocol. The chamber was initially filled with RBC suspension and the RBC were allowed settling without flow to the bottom of the chamber for a period of 8 min. The chamber was then rinsed with stepwise increasing flow rates corresponding to wall shear stresses between 0.01 and 0.1 Pa. At the end of each rinse, captures at 20 random locations were taken at 20 magnification. The number of adherent RBC was counted in each picture. The adherent RBC value for each shear stress was calculated as the mean plus/minus the standard deviation (SD) of 20 random captures and converted to adherent RBC per mm2. Unless otherwise stated, RBC concentration was fixed at 5 106/mL by hemacytometer counting. Between two investigated solutions Student’s t test was performed on 20 unpaired adherence values at the same shear stress, while the Wilcoxon-MannWhitney U test, a nonparametric method for two unpaired samples, was performed on the serial mean of adherence values against shear stress.
3. Results In Figure 1, RBC were suspended in polymer-free suspensions and in solutions containing dextran 500 kDa at concentrations of 0.5 and 1.0 g/dL. After allowing RBC to settle onto EC, a stepwise increase of the shear stress was applied as described above and the number of adherent cells was determined. In polymer-free suspensions only a few cells adhered to EC and were quickly removed at shear stresses larger than 0.02 Pa. The addition of dextran 500 kDa led to a significant increase in the number of adherent cells. At 0.01 Pa a 4-fold increase was observed with 0.5 g/dL of dextran 500 kDa and a 10-fold increase with 1.0 g/dL. The strength of the adhesion did also increase significantly, with the adherent RBC withstanding shear stresses of up to 0.05 Pa. Further increasing the shear stress to 0.1 Pa eventually removed all the cells. In Figure 2, a similar experiment shows the adhesion of RBC to EC in solutions containing dextran 2 MDa at 0.5 and 1.0 g/dL and Langmuir 2010, 26(4), 2680–2683
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Figure 1. Number of RBC adherent to EC as a function of the applied shear stress. Cells were suspended in dextran-free buffer or 0.5 g/dL and 1.0 g/dL of dextran 500 kDa and allowed to settle for 8 min. Mann-Whitney U test was performed on the serial mean of adherence values against shear stress between control and dextran 500 kDa at 0.5 g/dL (P = 0.03) and 1.0 g/dL (P = 0.02).
Figure 2. Number of RBC adherent to EC as a function of the applied shear stress. Cells were suspended in dextran-free buffer or 0.5 and 1.0 g/dL of dextran 2 MDa and allowed to settle for 8 min. Mann-Whitney U test was performed on the serial mean of adherence values against shear stress between control and dextran 2 MDa at 1.0 g/dL (P = 0.008).
in polymer-free solution. In 1.0 g/dL of dextran 2 MDa, a significant increase in the number of adherent cells can be found (P = 0.08), which also withstand higher shear stresses of up to 0.05 Pa. At the smaller dextran concentration of 0.5 g/dL, only a small increase in the number of adherent cells (∼40%) can be observed and the cells were washed away quickly with the applied shear stress exceeding 0.03 Pa. In Figure 3, dextrans with lower molecular mass were tested for their ability to induce RBC-EC adhesion. Dextran 70 kDa at 1.5 and 2.0 g/dL was found unable to induce adhesion strong enough to increase the number of adherent cells, even after rinsing with the lowest applied shear stress of 0.01 Pa. Above 0.02 Pa, all cells were quickly washed away, without a significant difference between polymer containing and polymer-free solutions. In solutions containing dextran 150 kDa, a 4-fold increase in the number of adherent cells can be observed at 0.01 Pa and increasing the shear stress to 0.05 Pa removes most of the cells. In order to test if the adhesion promoted by dextran requires the presence of dextran after the adhesion has been induced, the effects of rinsing with either polymer containing or polymer-free solution were compared. RBC suspensions containing 1.0 g/dL of dextran 500 kDa were first injected into the flow chamber. DOI: 10.1021/la902977y
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Figure 3. Number of RBC adherent to EC as a function of the applied shear stress. Cells were suspended in dextran-free buffer, dextran 70 kDa (1.5 and 2.0 g/dL) or dextran 150 kDa (1.0 and 2.0 g/dL). RBC were allowed to settle for 8 min. Mann-Whitney U test was performed on the serial mean of adherence values against shear stress between control and dextran 150 kDa at 1.0 g/dL (P = 0.167) and 2.0 g/dL (P = 0.008).
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Figure 5. The impact of RBC concentrations on number of RBC adherent to EC as a function of the applied shear stress. Cells were suspended in 1 g/dL of dextran 500 kDa and allowed to settle for 8 min. Mann-Whitney U test was performed on the serial mean of adherence values against shear stress: P = 0.159 for the adherence at 10 106/mL compared to 5.0 106/mL, P = 0.055 for the adherence of 50 106/mL compared to 5.0 106/mL.
25 cells/mm2 at cell concentrations of 5 106/mL and 10 106/mL, respectively.
4. Discussion
Figure 4. Adherence of RBC to EC settled in solutions containing 1 g/dL of dextran 500 kDa followed by rinsing with either the same solution or with polymer-free PBS. Student’s t test was performed on the 20 captures of random locations at 0.02 and 0.03 Pa.
After the cells were allowed to settle onto the EC monolayer, the chamber was rinsed either with the same dextran solution, i.e. 1.0 g/dL dextran 500 kDa, or with polymer-free buffer. As shown in Figure 4, rinsing with polymer-free solution resulted in a significantly reduced number of adherent cells as compared to rinsing with dextran solution. This reduction of adherence clearly indicates that the presence of dextran is required to maintain the adhesive interaction. Lastly, the impact of RBC concentration on RBC-EC adhesion was investigated. RBC were suspended at cell concentrations ranging from 5 106/mL to 50 106/mL in solutions containing 1.0 g/dL of dextran 500 kDa. After a settling time of 8 min, a stepwise increasing shear profile was applied and the number of adherent cells was determined. RBC aggregation was observed in all three samples but the aggregates were much less at smaller cell concentrations (i.e., 5 106/mL and 10 106/mL). Both RBC aggregates and individual cells were found to adhere to EC, with the former being washed away quickly at lower shear stress (e.g., 0.01 Pa) while the latter remaining adherent at higher shear stress. Figure 5 demonstrates that increasing the cell concentration from 5 106/mL to 10 106/mL led to a significant increase of the adhesion efficiency (P = 0.159): at 0.02 Pa the number of cells adherent increased by more than 50%. Further increasing the RBC concentration to 50 106/mL reduced the total number of adherent cells (P = 0.055): after applying a shear stress of 0.2 Pa about 11 cells/mm2 were adherent as compared to 16 and 2682 DOI: 10.1021/la902977y
The presented results clearly illustrate that large dextran molecules can induce RBC adhesion to EC under low shear stress. Taking into consideration that dextran is a neutral, uncharged polymer without the ability to develop attractive electrostatic interactions and that it has been shown repeatedly to be depleted from RBC surfaces,17-19 these findings suggest that macromolecular depletion can induce an attractive interaction between RBC and EC. This is also supported by the overall dependence of the adhesion rates and strength on the polymer concentrations and molecular mass.20 Depletion interaction is mainly determined by two factors, the osmotic pressure gradient between the depletion layer and the bulk phase, and the depletion layer thickness. However, evaluating depletion interaction between RBC and EC also requires to consider that both cells have a glycocalyx, i.e., a layer of attached macromolecules, that can be penetrated in part or entirely by the free polymers in solution.21 For two adjacent cell surfaces, the depletion energy can be estimated as15 ωD ¼ -Πð2Δ -dþδ1 -p1 þδ2 -p2 Þ
ð1Þ
where Π is the osmotic pressure difference, d is the separation distance, Δ the depletion layer thickness and p and δ represent the penetration depth and thickness of the two glycocalyces. For small polymer concentrations, the osmotic pressure is proportional to the polymer concentration: Π ¼
RT b c M
ð2Þ
where R, T, and M are the gas constant, absolute temperature, and molecular mass of the polymer and cb represents the bulk polymer concentration. The depletion layer thickness Δ usually depends on the polymer concentrations but since we are looking at small concentrations it (19) B€aumler, H.; Donath, E. Studia Biophys. 1987, 120, (2), 113-122. (20) Neu, B.; Meisleman, H. J. Red blood cell aggregation. In Handbook of Hemorheology and Hemodynamics; Baskurt, O. K., Hardeman, M. R., Meiselman, H. J., Rampling, M. W., Eds.; IOS Press: Amsterdam, 2007; Vol. 69, pp 114-136. (21) Vincent, B.; Edwards, J.; Emmett, S.; Jones, A. Colloids Surf. 1986, 18, 261-281.
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Figure 6. Theoretical dependence of depletion energy on polymer molecular mass for a suspending phase polymer concentrations of 1.0 g/dL and total penetration of 0, 10, 20, and 30 nm, respectively.
can be approximated by 1.4Rg.22 Rg is the polymer’s radius of gyration and can be calculated via Rg = AecMw0.5 with Aec = 0.88 nm 3 mol0.5 3 kg-0.5.23 The depletion energy for two cells at close proximity (i.e., d ≈ δ1 þ δ2) then becomes: wD ¼ -
pffiffiffiffiffi 2RT b c ð1:4Aec M -pÞ M
ð3Þ
with p being the sum of the penetration into both adjacent surfaces. Figure 6 presents calculated depletion energies at a bulk polymer concentration of 1.0 g/dL employing eq 3. For a hard surface (p = 0), the depletion energy depends linearly on cb/M0.5 and thus decreases with increasing molecular mass. On the basis of the reported wide range of EC glycocalyx thickness (20500 nm) and the much thinner one of RBC (
