Hemoglobin (Hb)-based O2 carriers (HBOC) are undergoing extensive development as potential “blood substitutes.” A major problem associated with these molecules is an increase in microvascular permeability and peripheral vascular resistance. In this paper, we utilized bovine lung microvascular endothelial cell monolayers and simultaneously measured Hb-induced changes in transendothelial electrical resistance, diffusive albumin permeability, and diffusive Hb permeability (PDH) for three forms of Hb: natural tetrameric human Hb-A and two polymerized recombinant HBOCs containing α-human and β-bovine chains designated Hb-Polytaur (molecular mass: 500 kDa) and Hb-(Polytaur)n (molecular mass: ∼1,000,000 Da), respectively. Hb-Polytaur and Hb-(Polytaur)n are being evaluated for clinical use as HBOCs. All three Hb molecules induce a rapid decline of transendothelial electrical resistance to 30% of baseline. Diffusive albumin permeabiltiy increases, on average, approximately ninefold (2.78 × 10−7 vs. 2.47 × 10−6 cm/s) in response to Hb exposure. All three Hb molecules induce an increase in their own permeability, a process that we have called Hb-induced Hb permeability. The magnitude of change of PDH is also related to Hb size. When PDH is corrected for the diffusive coefficient for each Hb species, no evidence of restricted diffusion is found. Immunofluorescent images demonstrate Hb-induced actin stress fiber formation and large intercellular gaps. These data provide the first quantitative assessment of the effect of polymerized HBOC on their own diffusion rates over time. We discuss the importance of these findings in terms of Hb extravasation rates, molecular sieving, and clinical consequences of HBOC use.
- blood substitutes
- restricted diffusion
hemoglobin (hb)-based o2 carriers (HBOC) have received considerable attention as blood substitutes, and interest in developing these therapeutic macromolecules remains high, given the shortage and limited shelf-life of banked red blood cells, infection concerns, and, at times, unacceptable delays in cross-matching banked blood (1, 24, 26, 33). Unfortunately, a number of unexpected problems have been associated with the use of HBOC, including extravasation, vasospasm, renal toxicity, and patient death (1, 24, 33). The ultimate utility of HBOC is to provide adequate oxygen delivery to patients requiring immediate resuscitation, as may occur following major trauma or surgical procedures associated with large-volume blood losses. Two untoward consequences of HBOC are extravasation of the HBOC itself, which is no longer able to participate in oxygen delivery, and the associated vasoconstriction due, at least in part, to local nitric oxide (NO) scavenging. The decrease in plasma Hb and localized vasospasms in the setting of anemia may create regional hypoxia. Overcoming these issues will be paramount in the development of clinically useful HBOC.
Winslow and colleagues (32, 34) have investigated HBOC-induced vasoactivity and have demonstrated that HBOCs with similar NO reactivity induce notably different degrees of vasoconstriction. They concluded that 1) vasoconstriction is mediated by arteriole autoregulation by local oxygen tension, and 2) differences in the extravasation rates of HBOC may affect the magnitude of NO scavenging and, therefore, the degree of vasoconstriction. In support of this notion, Matheson et al. (25) and Sakai et al. (31) demonstrated that the hypertensive response was inversely related to the size of the HBOC, suggesting that extravasation rates are clinically important. Our data extend these observations by demonstrating that HBOCs increase endothelial permeability; therefore, HBOC extravasation rates may actually increase over time, thus altering the magnitude of NO scavenging. We refer to this process as Hb-induced Hb permeability. Therefore, three distinct but interdependent mechanisms may play a role in HBOC-induced vasoactivity.
A number of published studies have modeled the complex interrelationship of HBOCs on O2 delivery (6, 18, 21), wall shear stress (22), NO scavenging (23), and altered plasma rheology (7). It is clear from these studies that a precise quantitative assessment of Hb extravasation and HBOC-induced changes in vascular permeability is fundamentally important toward understanding their intravascular behavior and oxygen delivery potential. Predicting the microcirculatory mass transport of oxygen, drugs, and other therapeutic agents in the setting of HBOC utilization should prove to be crucially important in the development of HBOCs.
The study of transendothelial transport of macromolecules, like HBOC, in situ can be hampered by the inability to discern the effects of other cell types (mast cells, neutrophils) on the measured endothelial transport parameters. For example, inflammatory agonists frequently act through multiple mechanisms, including neutrophil and mast cell activation (3, 4, 11); therefore, the direct effects of the test agonist on the endothelium are confounded by the release of secondary mediators. We have exploited the ability to control all aspects of the microenvironment by utilizing endothelial monolayers grown on porous substrates to measure both water (8, 12, 13) and solute transport (12, 20) during precisely defined conditions and controlled Starling forces. Using these techniques, we have demonstrated that cultured monolayers have hydraulic conductivity coefficients and albumin permeability coefficients (PDA) within the physiological range.
Here, we report the simultaneous measurement of both PDA and PDH, evaluate these data as a function of the diffusivity coefficient (P/D), and examine both the change in these indexes over time as well as their interrelationship. To date, only one other study has examined the transport of HBOC across endothelial monolayers (27). In that study, Nakai and colleagues (27) examined the permeability of bovine aortic endothelial cells to a variety of low molecular mass (MM) HBOCs under control and LPS-stimulated conditions. A major drawback to their data was that the permeability coefficient was obtained at a single time point only, and they did not consider the effect of Hb-induced changes on endothelial permeability. We extend their observations by quantifying the permeability of microvascular endothelial cell monolayers to a new class of large, polymerized, recombinant HBOC, and we examine the phenomenon of Hb-induced Hb permeability.
Cell Culture and Reagents
Bovine lung microvascular endothelial cells (BLMVEC) were obtained from American Type Culture Collection (Rockville, MD) and Vec Technologies (Rensselaer, NY) and cultured in DMEM and MCDB-131, respectively, supplemented with 10% fetal calf serum. Cells were used from passages 6–10. All reagents were from Sigma (St. Louis, MO), unless otherwise stated. Texas Red-labeled (TR)-BSA and TR-phalloidin were purchased from Molecular Probes (Portland, OR).
Preparation of Hb
Human Hb (Hb-A).
Stroma-free human Hb was prepared from outdated blood samples. The red cells were washed three times with 0.09% NaCl and hemolyzed in 0.005 M phosphate buffer at pH 7.0. Red blood cell stroma were eliminated by treatment of the solution with 5% chloroform followed by centrifugation at 5,000 g. The supernatant was collected, dialyzed, and stored at −80°C.
Hb-Polytaur and (Hb-Polytaur)n.
Hb-Polytaur and (Hb-Polytaur)n were obtained by the polymerization of Hb-Minotaur (αHβBv), a hybrid Hb containing α-human and β-bovine subunits (6). Polymerization of Hb-Minotaur occurred spontaneously via the oxidation of thiol groups and formation of S-S bonds between Cys residues introduced at position β9, which is external and exposed to the solvent. Two polymerized forms are obtained. A homogeneous polymer (MM = 500 kDa), designated as Hb-Polytaur (αHC104SβBvA9C+C93A), is obtained when the naturally occurring α104 and β93 cysteine are replaced with serine and alanine, respectively. This polymer probably assembles into a globular form, and its biochemical properties have been extensively investigated (6, 15). The oxygen affinity is similar to that of Hb-A [O2 half-saturation pressure (P50) = 16.0 Torr, cooperativity is maintained, n = 1.7]. Polymerization increases the affinity of the protein for heme, a positive effect, but also increases the autooxidation rate, a negative effect. When the naturally occurring cysteines are not substituted, polymerization is faster but heterogenous with the prevalent species, having an MM of 1,000 kDa or higher. This polymer, designated as Hb-(Polytaur)n, has modified functionality; namely, high oxygen affinity (P50 = 2.0 Torr) and absent cooperativity (n = 1) (15).
The polymerized Hb was dialyzed against Ringer solution and filtered through a 0.45-μm filter. Endotoxins were removed by gentle mixing for 18 h with Detoxy-gel (Pierce, Rockford, IL), following the manufacturer's instructions, which reduces the endotoxin content to <2 endotoxin units/ml. All Hb were stored in sterile glass vials at −80°C.
Transendothelial electrical resistance.
BLMVEC were grown to confluence in polycarbonate wells containing evaporated gold microelectrodes (surface area 10−3 cm2) in series with a gold counter electrode (surface area 1.0 cm2) connected to a phase-sensitive, locked-in amplifier (16). Measurements of transmonolayer electrical resistance (TER) were performed by using an electrical cell-substrate impedance-sensing system (Applied BioPhysics, Troy, NY). TER was monitored for at least 30 min to establish baseline resistance (∼6–10 × 103 Ω) before the addition of Hb solutions.
Diffusive albumin permeability.
BLMVECs were cultured on Costar Transwell polycarbonate filters (12-mm diameter, 0.4-μm pore size; Corning Costar, Acton, MA) that were pretreated with 0.2% gelatin (Sigma) for 1 h followed by 30-μg bovine fibronectin for 1 h. When cells reached confluence, typically at 5 days, monolayers were washed three times with protein-free medium and covered with 475 μl of medium containing 1% BSA + 25 mM HEPES. The Transwell chambers were placed into custom-fabricated Sylgard beakers (lower chamber; Dow Corning, Midland, MI) containing 3 ml of 1% BSA + 25 mM HEPES medium, and the media levels in each chamber were set at the same height to negate any hydrostatic gradient. The beakers, containing the Transwell chambers, were then placed on a nine-position stir plate to allow for constant stirring of the medium in the lower chamber. The stir plates were housed in a convection-heated incubator to maintain a constant temperature of 37°C. The monolayers were allowed to equilibrate for 1 h before the experimental protocol was begun. To begin the experiment, 25 μl of 1% TR-BSA were added to the Transwell chamber. Baseline permeability of each monolayer was monitored for 2 h by sampling 30 μl/h from the lower chamber. An equal volume of media containing 1% BSA + 25 mM HEPES was added to each chamber, following sample removal, to maintain constant volume. After the 2-h control sample was taken, the Hb were added to the Transwell chamber to yield a final concentration of 1%. The flux of the TR-BSA through the monolayer was converted into a diffusive permeability coefficient (PDA) by: (1) where ΔC is the change in concentration of the TR-BSA in the abluminal (lower) chamber, Abvol is the volume (3 ml) of the abluminal chamber, A is the area of the monolayer (1 cm2), t is time (s) between samples, and Lconcn is the concentration of TR-BSA in the luminal chamber. PDA has units of cm/s.
Hb concentration was determined from 30-μl samples removed from the lower chamber every hour. Samples were diluted with 20 mM phosphate buffer at pH 7.2 that contained 2 mg/ml sodium dithionite after equilibration with carbon monoxide. The sodium dithionite reduced any methemoglobin present, and the total Hb was transformed into the carbonmonoxy form. Spectra were recorded at wavelengths between 460 and 400 nm and Hb concentration (Hbconcn) was determined at 419 nm by using: (2) where OD is the optical density at 419 nm, MMsubunit = 16.500 is the MM of a single Hb subunit, and Mheme is the molar extinction coefficient (190,000 M−1/cm−1) of the carbonmonoxyheme. Changes in Hb concentration were converted to a diffusive permeability coefficient (PDH) by using Eq. 1 as described above for albumin permeability.
Confluent monolayers of BLMVECs were cultured on glass coverslips and treated with Hb-A for 30 min. Untreated monolayers served as controls. Cells were washed with MCDB-131 and fixed with 3.7% paraformaldehyde, permeabilized with 0.25% Triton X-100, and incubated with TR-phalloidin (Molecular Probes, Portland, OR) to visualize F-actin. Cell monolayers were then examined by fluorescence microscopy (Nikon Eclipse TE-200 inverted microscope).
For the variables PDA, PDH, and P/D, the effect of the three Hb treatment species and changes over time were analyzed by developing a Linear Mixed Effect Model using S-Plus statistical software (version 6.1.2 release 2; Insightful, Seattle, WA). The Linear Mixed Effect Model tested for differences in the slope of each variable per time. Treatment effects, time effects, and time-treatment interactions were also modeled. For albumin permeability, both raw values and delta values (raw minus baseline, where baseline values are from the control period, i.e., 1- and 2-h data) were modeled separately; for the raw values of albumin permeability, the model included the baseline value as a covariate; for PDH and P/D, only raw values were modeled. Variance-covariance structures and variance functions were modeled, and estimation was by maximum likelihood. Models were evaluated and compared by t statistics on parameter estimates and likelihood ratio χ2 statistics between models. Parameter estimates were presented with means ± SE. For clarity, significant differences between groups and over time are given in Fig. 1 and 2 legends. Sample was n = 4–6 per group. Null hypotheses were rejected at an α < 0.05.
Hb Reduce TER
To screen the three Hb molecules for an effect on endothelial permeability, we examined the change in electrical resistance following Hb exposure. BLMVEC had a baseline TER of 6–10 × 103 Ω in medium containing 1% albumin, which is consistent with previous use of this cell type by our laboratory (10). Addition of Hb-A, Hb-Polytaur, or Hb-(Polytaur)n to a final concentration of 1% (vol/vol) caused a rapid (within 30 min) decrease in TER to ∼60% below baseline (data not shown). All Hb tested caused a similar decrease in TER. There was no recovery of TER during the duration of exposure of the endothelial cells to the Hb solution.
Hb Increase PDA
Baseline permeability of every monolayer was measured, and, therefore, each group has an average control PDA for samples taken over the 2-h control period (Fig. 1). BLMVEC demonstrated an average baseline PDA during the 2-h control period of 2.78 × 10−7 cm/s. The three Hb species [Hb-A, Hb-Polytaur, Hb-(Polytaur)n] were added to the endothelial monolayers just after the 2-h control sample was obtained. As can be seen in Fig. 1, Hb exposure increased PDA by approximately five- to ninefold among the three groups. During the first hour after exposure, Hb-A increased PDA to 1.4 × 10−6 cm/s, Hb-Polytaur increased PDA to 1.5 10−6 cm/s, and Hb-(Polytaur)n increased PDA to 2.47 × 10−6 cm/s. The PDA values for all groups at 3–6 h were significantly different than the control period (P < 0.001), but no intra- or intergroup differences were noted during the 3- to 6-h time interval. The P/D for albumin was calculated by dividing PDA by the diffusional coefficient, D = 0.067 × 10−5 (cm2/s) for albumin (17). Peak P/D for albumin during exposure to each Hb species is presented in Table 1.
Extravasation of Hb represents a major drawback to the clinical utility of HBOC as it is associated with local NO scavenging, increased vascular reactivity, and, often, vasospasm. With the exception of a single study by Nakai et al. (27), we were not aware of any quantitative measures of the rate of Hb extravasation or quantification of Hb-induced Hb permeability. Therefore, we directly measured the changes in Hb concentration per time across microvascular endothelial monolayers and converted this change into a PDH. To examine sieving properties of the monolayers, we calculated P/D by dividing each PDH value by D, the diffusivity coefficient, for each Hb molecule. Figure 2A demonstrates the ability of the microvascular endothelial monolayer to sieve the three Hb species, and, as expected, an inverse correlation exists between the size of the Hb species and the change in abluminal Hb concentration over time. Figure 2B presents the change in PDH over time for each Hb species tested. Hb-A increased its own permeability by approximately twofold during the 4-h exposure period, reaching a peak PDH of 5.88 × 10−6 cm/s. Hb-Polytaur demonstrated an 11-fold increase in PDH over the 4-h exposure, reaching a value of 5.32 × 10−6 cm/s. Hb-(Polytaur)n had the lowest rate of change in PDA during the 1- through 3-h time interval but demonstrated a large rate of change from hour 3 to hour 4. Hb-(Polytaur)n reached a peak PDH of 2.6 × 10−6 cm/s, which represents a 9.5-fold increase from the 1-h value. The increases in PDH over the 4-h test period were significant (P < 0.001) for all three groups. No statistical differences were noted between PDH for Hb-A and Hb-Polytaur (P = 0.268), whereas Hb-(Polytaur)n was statistically different from both Hb-A and Hb-Polytaur (P = 0.0167).
MM and hydrodynamic radius are important determinants of the D for macromolecules. To evaluate the sieving characteristics of the endothelial monolayer during exposure to the Hb solutions, we calculated the P/D for each Hb species over the 4-h experiment. Hb-A has an MM of 64 kDa and is a globular molecule with a radius of 31.9 Å and a D = 0.068 × 10−5 cm2/s (14). Hb-Polytaur has an MM of nearly 500 kDa and is also likely globular (7, 15) with a radius of 62.1 Å. We have estimated its D, based on MM and radius, to be ∼0.05 × 10−5 cm2/s (14). Little is known regarding the overall structure of Hb-(Polytaur)n, although it likely includes globular domains; based on its size, we estimated its D to be ∼0.02 × 10−5 cm2/s. These data for each Hb species are presented in Fig. 2C and Table 1. When PDH is corrected for D, the P/D (cm−1) for the three Hb molecules are not statistically different (P = 0.1974), and we conclude that there is not restricted diffusion of the Hb molecules across the endothelial monolayer. Because the calculation of P/D required estimation of D for both Hb-Polytaur and Hb-(Polytaur)n, we wanted to validate these estimations. As D is dependent on both MM and molecular radius (Å), we plotted P/D vs. MM and P/D vs. radius (Fig. 3, A and B, respectively) for PDH at both 1 h (bottom curve) and 4 h (top curve). The shapes of the curves in each plot are virtually identical, suggesting that the relationship between MM and radius used to estimate D was accurate.
Hb-A Induces Actin Stress Fiber Formation
To validate that the lack of restricted diffusion of HBOC across the endothelial monolayers following Hb exposure correlates with structural changes in cytoskeletal organization (9), we examined actin distribution before and after Hb-A exposure. P/D were similar for all three Hb molecules, suggesting that similar alterations in the endothelial barrier occurred. Thus we provide representative images of endothelial actin for only Hb-A-treated monolayers. Control monolayers (Fig. 4A) display a prominent band of cortical actin that is typically found in nonactivated endothelium. We do not serum-starve our cells before staining for actin, as serum starvation results in an artificially reduced amount of stress fibers. Therefore, a few centrally located stress fibers are present. After exposure to 1% Hb-A, actin undergoes a marked reorganization from a cortical distribution to centrally located stress fibers (Fig. 4B). Note the presence of large intercellular gaps (arrows) induced by Hb-A exposure.
In this study, we simultaneously quantified the diffusion rates of two new HBOC (polymerized recombinant Hb) and albumin diffusion across confluent monolayers of capillary endothelial cells in response to Hb exposure. We compared tetrameric human Hb-A and two polymerized recombinant human/bovine Hb, Hb-Polytaur and Hb-(Polytaur)n, with respect to their capacity to directly induce endothelial barrier dysfunction. The changes in Hb and albumin fluxes were converted into diffusive permeability coefficients to allow direct comparison of our results to other published data. Evidence of restricted diffusion was evaluated by calculating P/D for each Hb molecule. We found that 1) monolayers of BLMVEC have significantly lower PDA compared with other commonly cultured endothelial cells, 2) all Hb tested induced endothelial barrier dysfunction, 3) Hb exposure resulted in complete loss of restricted diffusion and, 4) Hb exposure was associated with formation of actin stress fiber and large intercellular gaps. We believe that this is the first detailed quantitative assessment of PDH in the literature.
The rationale for these studies was twofold: 1) Baldwin et al. (2–4) reported increased venular permeability, in the rat, to fluorescently labeled albumin in response to polymerized Hb administration, and 2) Sakai et al. (31), Matheson et al. (25), and Winslow et al. (28, 32, 34) reported that Hb extravasation rates may account for differences in HBOC-induced vascular reactivity independent of NO reactivity. Taken in order, Baldwin et al. (2, 3) concluded that Hb extravasation and subsequent mast-cell degranulation were, in part, responsible for the changes in microvascular permeability. Follow-up studies (4) demonstrated that heme oxidation (35) and the formation of reactive oxygen species play a role in Hb-induced changes in microvascular permeability in addition to mast cell degranulation. We sought to quantify the direct effects of recombinant Hb on Hb-induced albumin permeability and to provide a quantitative assessment of Hb-induced Hb permeability. Using an established in vitro approach for the quantification of macromolecular permeability allowed us to quantitatively examine the direct effects of Hb-A and two forms of polymerized recombinant human/bovine Hb on endothelial barrier function.
Winslow and colleagues (28, 32, 34) have examined the relationship between NO-binding properties and P50 on the vasopressor activity of a variety of polymerized HBOC. They demonstrated that HBOC with similar NO binding have significantly different effects on peripheral vascular resistance. These observations lead to the important insight that HBOC P50 and subsequent oxygen unloading at the arteriolar level may lead to marked vasoconstriction. Thus the arteriole oxygen content plays an important role in an autoregulatory process that modulates blood flow and, hence, oxygen delivery into the microcirculation. Importantly, however, they also concluded that differences in Hb extravasation rates are likely to further influence the process of vasoactivity via NO scavenging. The idea that extravasation rates have important clinical consequences had been tested previously by Sakai et al. (31), who showed that hypertension and arteriole constriction inversely correlate to the molecular dimensions of the HBOC. Here, we provide a quantitative analysis of the extravasation rates of three different Hb molecules and demonstrate that, in addition to altering endothelial permeability to albumin, they induce a profound barrier dysfunction that alters their own extravasation rate. In fact, by 4 h, all three Hb resulted in large intracellular gap formation and the complete loss of restricted diffusion. We believe that this is the first detailed assessment of HBOC diffusion rates, and our analysis takes into account the influence of the HBOC on its own permeability coefficient over time.
The baseline PDA rates for BLMVEC monolayers reported here are among the lowest values to date for in vitro models. We measured control PDA to be 2.78 × 10−7 cm/s. Our laboratory has previously published PDA data for bovine aortic endothelial cell monolayers in the range of 4.5 × 10−6 cm/s (12) to 5.09 × 10−6 cm/s (20) and for human umbilical vein endothelial cell as 1.93 × 10−6 cm/s (8). All of these studies were done with the use of the same Transwell culture system, identical filter pretreatment, and similar culture duration. We conclude that BLMVEC monolayers form a more restrictive barrier to albumin diffusion than endothelial cells from larger vessels.
One notable difference between those studies and the present study was the fluorescent tag used to label BSA. In previous studies, we utilized FITC as our tracer, and, in the present study, we used TR-BSA. Our decision to change tracers was based on recent reports that some fluorescent dyes may both adversely affect the endothelial cell directly and alter the physiochemical properties of albumin in solution. For example, Rumbaut and colleagues (29) reported that the albumin leakage response to NO inhibition varied, depending on the fluorescent dye used, whereas Harris et al. (19) reported similar differences in a model of ischemia-reperfusion injury. The phototoxicity of various dyes was evaluated by Rumbaut and Sial (30), and the modification of labeled proteins by fluorescent dyes has been examined by Bingaman et al. (5). TR-BSA had minimal alterations in MM, charge, and isoelectric point compared with other dyes. In the studies cited above, the difference in measured albumin leakage that could be attributed to the specific fluorescent dye varied by severalfold (range 2–5 times), but much less than the 16-fold difference that we observed between bovine aortic endothelial cells and BLMVEC (4.5 × 10−6 vs. 2.78 × 10−7 cm/s).
Exposure of BLMVEC monolayers to Hb-A, Hb-Polytaur, or Hb-(Polytaur)n at a clinically relevant concentration of 1% caused a five- to ninefold increase in PDA within the first 1 h. There was no further statistical increase in PDA over the ensuing 4 h of exposure, and there was no difference in the magnitude of PDA increase between groups (Fig. 1). When control PDA (before Hb exposure) is normalized for the D for albumin, the resulting ratio P/D has a value of 4.15 × 10−6. Peak P/D (at 4 h) values for albumin following exposure to Hb-A, Hb-Polytaur, and Hb-(Polytaur)n were 3.45, 3.46, and 2.39 × 10−5 cm/s, respectively (Table 1). No differences in P/D were noted among the three Hb groups.
In addition to measuring changes in albumin permeability, which has been the gold standard for evaluating endothelial macromolecular permeability, we simultaneously measured the flux of each Hb species across BLMVEC monolayers. We found that BLMVEC barrier function was directly altered by exposure to Hb solutions at what may be considered clinically relevant concentrations. Figure 2A shows the apparent ability of the capillary endothelial monolayer to effectively restrict (sieve) the diffusion of each Hb form as assessed by changes in abluminal Hb concentrations. As expected, the Hb flux was directly related to the size of each Hb molecule. The change in Hb concentration was then converted into a diffusive permeability coefficient (PDH, cm/s), presented in Fig. 2B. Several important observations can be made: 1) each Hb increased its own permeability coefficient over time, and 2) the percent change in PDH over 4 h was larger for the two polymerized forms compared with Hb-A. That is, PDH for Hb-A increased twofold, whereas the polymerized forms, Hb-Polytaur and Hb-(Polytaur)n, increased 11- and 9.5-fold, respectively, over the 4-h experiment. This complex relationship suggests that the Hb polymers induced more endothelial injury compared with Hb-A. However, the absolute increase in measured permeability is limited by the inherent diffusivity of the HBOC, as explained below. Notice also that the largest increase in PDH for each Hb occurred over the time interval from 3 to 4 h and suggests the loss of common compensatory mechanism(s) of the endothelial monolayer.
We conclude from these data that the polymerized Hb induced a greater degree of endothelial barrier dysfunction compared with Hb-A, possibly due to the increased autoxidation rate of the Hb polymer. The in vitro half-time (T1/2) for autoxidation of Hb-Polytaur is 3.3 h, 10 times faster than for Hb-A (T1/2 = 33 h) (6). Therefore, autoxidation may be relevant over the time course of the experiments that we have done (4 h). However, the process of autoxidation in vivo is dramatically different, where the T1/2 = 46 and 146 h for Hb-Polytaur and Hb-A, respectively. Given that the circulation retention time T1/2 for polymerized Hb in humans is ∼24 h (19a, 19b), the autoxidation process in vivo may not be so relevant.
To further analyze the relationship between Hb size and permeability and to determine whether the monolayers offered restricted diffusion, we calculated P/D for each Hb species over the 4-h exposure period. P/D normalize the permeability coefficient for inherent differences in macromolecular diffusion rates (D), which are related to mass and radius. In Table 1, we provide MM, radius (Å), D, and mean P/D. It was necessary to estimate radius and D for Hb-(Polytaur)n, as its three dimensional structure is unknown (although it is likely to possess globular domains); therefore, our estimates of radius and D are based on proteins with similar mass and shape. Notice that, although the absolute values of PDH differ among the three HBOC (Fig. 2B), all of these differences can be accounted for the D of each Hb molecule. That is, when PDH is corrected for D, the P/D are statistically similar (Fig. 2C). This means that, over time, the endothelial monolayer is altered such that a Hb polymer of MM of ∼1,000,000 Da crosses as easily as Hb-A (MM 60 kDa). Unrestricted diffusion can only be explained by the development of large intercellular gaps in response to the HBOC.
The intent of these studies was solely to quantify the simultaneous changes in albumin and Hb permeability in response to polymerized recombinant HBOC. While we make no attempt to identify the mechanism(s) responsible for these changes in permeability, we did confirm that predictable changes in cell structure occurred. For example, alterations in the actin cytoskeleton are a common phenotypical change indicative of altered paracellular permeability (9). During endothelial activation, actin undergoes a reorganization from a cortical distribution to centrally located stress fibers. As can be seen in Fig. 4A, control BLMVEC display cortical actin and, following exposure to Hb-A, demonstrate a marked conversion to stress fibers (Fig. 4B). As anticipated, the appearance of stress fibers is associated with intercellular gaps. We believe that these intercellular gaps represent the dominant pathway for both albumin and Hb transport across the monolayer.
In summary, we describe changes in microvascular endothelial barrier function following exposure to Hb-A and two new polymerized recombinant Hb-based blood substitutes [Hb-Polytaur and Hb-(Polytaur)n] and present the first quantitative measurement of Hb-induced permeability (PDH, P/D). We find that these Hb molecules directly increase microvascular endothelial albumin permeability, and Hb-induced Hb permeability is, in part, related to the size of the Hb polymers. The methods and results presented here should be of benefit in the design and testing of new HBOC.
This work was funded by National Heart, Lung, and Blood Institute Grant K08 HL-068063 (to R. O. Dull) and by funding from the Eugene and Mary B. Meyer Center for Advanced Transfusion Practice and Blood Research, Johns Hopkins School of Medicine (to C. Fronticelli).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2004 the American Physiological Society