Tangential flow filtration facilitated fractionation and PEGylation of low and high-molecular weight polymerized hemoglobins and their biophysical properties

Various types of hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as red blood cell substitutes for treating blood loss when blood is not available. Among those HBOCs, glutaraldehyde polymerized Hbs have attracted significant attention due to their facile synthetic route, and ability to expand the blood volume and deliver oxygen. Hemopure®, Oxyglobin®, and PolyHeme® are the most well-known commercially developed glutaraldehyde polymerized Hbs. Unfortunately, only Oxyglobin® was approved by the FDA for veterinary use in the United States, while Hemopure® and PolyHeme® failed phase III clinical trials due to their ability to extravasate from the blood volume into the tissue space which facilitated nitric oxide scavenging and tissue deposition of iron, which elicited vasoconstriction, hypertension and oxidative tissue injury. Fortunately, conjugation of poly (ethylene glycol) (PEG) on the surface of Hb is capable of reducing the vasoactivity of Hb by creating a hydration layer surrounding the Hb molecule, which increases its hydrodynamic diameter and reduces tissue extravasation. Several commercial PEGylated Hbs (MP4®, Sanguinate®, Euro-PEG-Hb) have been developed for clinical use with a longer circulatory half-life and improved safety compared to Hb. However, all of these commercial products exhibited relatively high oxygen affinity compared to Hb, which limited their clinical use. To dually address the limitations of prior generations of polymerized and PEGylated Hbs, this current study describes the PEGylation of polymerized bovine Hb (PEG-PolybHb) in both the tense (T) and relaxed (R) quaternary state via thiol-maleimide chemistry to produce an HBOC with low or high oxygen affinity. The biophysical properties of PEG-PolybHb were measured and compared with those of commercial polymerized and PEGylated HBOCs. T-state PEG-PolybHb possessed higher hydrodynamic volume and P₅₀ than previous generations of commercial PEGylated Hbs. Both T- and R-state PEG-PolybHb exhibited significantly lower haptoglobin binding rates than the precursor PolybHb, indicating potentially reduced clearance by CD163 + monocytes and macrophages. Thus, T-state PEG-PolybHb is expected to function as a promising HBOC due to its low oxygen affinity and enhanced stealth properties afforded by the PEG hydration shell.     

Polynitroxyl hemoglobin: a pharmacokinetic study of covalently bound nitroxides to hemoglobin platforms

Adding antioxidant activities to hemoglobin-based oxygen carriers (HBOCs) represents a means of reducing cell-free hemoglobin-mediated oxidative cascades. We have covalently bound nitroxides, a class of antioxidant enzyme mimetics, to HBOCs. The objectives of this study were (1) to evaluate the pharmacokinetic (PK) effects of administering nitroxide covalently bound to HBOCs compared to those of free nitroxide coadministered with HBOCs and (2) to elucidate the effects of differing molecular weight HBOCs on the PK of bound nitroxide in a conscious guinea pig model of 25% blood exchange transfusion. Two HBOC platforms were used, intramolecular cross-linked hemoglobin (XLHb) and dextran polymerized/conjugated XLHb (PolyHb). Polynitroxylation was achieved by reacting 4-(2-bromoacetamido)-2,2,6,6,-tetramethylpiperidine-1-oxyl with XLHb or PolyHb to form polynitroxylated XLHb and polynitroxylated PolyHb, respectively, whereas a physical mixture of XLHb or PolyHb with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl was prepared to reflect a molar equivalence to HBOC-bound nitroxide. Plasma concentrations of two redox states, nitroxide and hydroxylamine, were determined by electron paramagnetic resonance spectroscopy. Results are presented to illustrate the influence of covalent labeling and HBOC molecular weight on nitroxide PK. The therapeutic potential of polynitroxylation of HBOCs as it relates to observations from the current and previously reported studies is discussed.     

A recombinant polymeric hemoglobin with conformational,functional, and physiological characteristics of an in vivo O2 transporter

With the objective of developing a recombinant oxygen carrier suitable for therapeutic applications, we have employed an Escherichia coli expression system to synthesize in high-yield hemoglobin (Hb) Minotaur, containing alpha-human and beta-bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position beta9 and substituting the naturally occurring Cys. This homogeneous polymer, Hb Polytaur, has a molecular mass of approximately 500 kDa and was resistant toward reducing agents present in blood. In mice, the circulating half-time (3 h) was fivefold greater than adult human Hb (HbA). The half-time of autooxidation measured in blood (46 h) exceeded the circulating retention time. Hypervolemic exchange transfusion resulted in increased arterial blood pressure similar to that with albumin. The increase in pressure was less than that obtained by transfusion of cross-linked tetrameric Hb known to undergo renovascular extravasation. The nitric oxide reactivity of Hb Polytaur was similar to HbA, suggesting that the diminished pressor response to Hb Polytaur was probably related to diminished extravasation. Transfusion of 3% Hb Polytaur during focal cerebral ischemia reduced infarct volume by 22%. Therefore, site-specific Cys insertion on the Hb surface results in uniform size polymers that do not produce the large pressor response seen with tetrameric Hb. Polymerization maintains physiologically relevant oxygen and heme affinity, stability toward denaturation and oxidation, and effective oxygen delivery as indicated by reduced cerebral ischemic damage.     

High O2 affinity hemoglobin-based oxygen carriers synthesized via polymerization of hemoglobin with ring-opened 2-chloroethyl-beta-D-fructopyranoside and 1-o-octyl-beta-D-glucopyranoside

Second generation hemoglobin-based O(2) carriers (HBOCs) are being developed with high O(2) affinity (low P(50)) in order to suppress vasoconstriction elicited by over-oxygenating tissues, a problem associated with low O(2) affinity first generation HBOCs. Our group has previously investigated the polymerization of hemoglobin (Hb) with dialdehydes as a strategy for engineering high O(2) affinity HBOCs. In this study, two novel reactive dialdehydes were synthesized by ring-opening 2-chloroethyl-beta-D-fructopyranoside (2-CEFP) and 1-o-octyl-beta-D-glucopyranoside (1-OGP) at the 1,2-diol position, respectively, to yield novel Hb polymerizing reagents. High-affinity polymerized HBOCs were synthesized by reacting R-state bovine hemoglobin (bHb) with ring-opened 2-CEFP and 1-OGP at cross-linker to bHb molar ratios ranging from 10:1 to 30:1. The resulting polymerized bovine HBOCs (bHBOCs) displayed P(50)s ranging from 7 to 18 mmHg, cooperativities ranging from 0.8 to 1.4, and methemoglobin (metHb) levels ranging from 3% to 10%. The cross-linking reaction also stabilized the third stepwise Adair coefficient for bHbs reacted with ring-opened 1-OGP at cross-linker to bHb molar ratios of 20:1 and 30:1 and for bHbs reacted with ring-opened 2-CEFP at molar ratios of 30:1. Additionally, the number-averaged molecular weight, M(n), of each polymerized bHBOC was larger compared to bHb. Molecular weight distributions leaning towards larger molecular weight bHBOCs were obtained by increasing the cross-linker to bHb molar ratio. Taken together, the results of this study have identified novel Hb polymerization reagents that are easy to synthesize, and that are capable of yielding bHBOCs with higher O(2) affinities and weight-averaged molecular weights compared to bHb.     

Effects of calcium on transendothelial albumin transfer and electrical resistance

Nicolaysen, and more recently Kern and Malik, reported that chelation of calcium increased microvascular hydraulic conductivity and albumin permeability in isolated perfused lungs. To begin to understand how calcium affects endothelial function we examined the effect of calcium chelation on an in vitro endothelium. Chelation of calcium with ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid increased the rate of transendothelial albumin transfer by 125%. Reincubation of the endothelium in calcium-repleted medium restored the rate of transfer to its original value. Chelation of extracellular calcium abolished transendothelial electrical resistance. The transendothelial electrical resistance was also restored to normal by reincubation of the endothelium in calcium-repleted medium. Chelation of extracellular calcium caused adjacent endothelial cells to retract from one another, and normal apposition of adjacent cells was restored after reincubation in calcium-repleted medium. Chelation of extracellular calcium produced a centripetal retraction of the peripheral band of actin in individual endothelial cells, and the actin band resumed its normal location after reincubation in calcium-repleted medium. Calcium is an important determinant of endothelial integrity and alterations in calcium produce dynamic changes in endothelial barrier properties and in endothelial-cell shape.     

Molecular dimensions of Hb-based O(2) carriers determine constriction of resistance arteries and hypertension

The effect of molecular dimension of hemoglobin (Hb)-based O(2) carriers on the diameter of resistance arteries (A(0), 158 +/- 21 microm) and arterial blood pressure were studied in the conscious hamster dorsal skinfold model. Cross-linked Hb (XLHb), polyethylene glycol (PEG)-conjugated Hb, hydroxyethylstarch-conjugated XLHb, polymerized XLHb, and PEG-modified Hb vesicles (PEG-HbV) were synthesized. Their molecular diameters were 7, 22, 47, 68, and 224 nm, respectively. The bolus infusion of 7 ml/kg of XLHb (5 g/dl) caused an immediate hypertension (+34 +/- 13 mmHg at 3 h) with a simultaneous decrease in A(0) diameter (79 +/- 8% of basal value) and a blood flow decrease throughout the microvascular network. The diameter of smaller arterioles did not change significantly. Infusion of larger O(2) carriers resulted in lesser vasoconstriction and hypertension, with PEG-HbV showing the smallest changes. Constriction of resistance arteries was found to be correlated with the level of hypertension, and the responses were proportional to the molecular dimensions of the O(2) carriers. The underlying mechanism is not evident from these experiments; however, it is likely that the effects are related to the diffusion properties of the different Hb molecules.     

Involvement of ROCK-mediated endothelial tension development in neutrophil-stimulated microvascular leakage

Neutrophil-induced coronary microvascular barrier dysfunction is an important pathophysiological event in heart disease. Currently, the precise cellular and molecular mechanisms of neutrophil-induced microvascular leakage are not clear. The aim of this study was to test the hypothesis that rho kinase (ROCK) increases coronary venular permeability in association with elevated endothelial tension. We assessed permeability to albumin (P(a)) in isolated porcine coronary venules and in coronary venular endothelial cell (CVEC) monolayers. Endothelial barrier function was also evaluated by measuring transendothelial electrical resistance (TER) of CVEC monolayers. In parallel, we measured isometric tension of CVECs grown on collagen gels. Transference of constitutively active (ca)-ROCK protein into isolated coronary venules or CVEC monolayers caused a significant increase in P(a) and decreased TER in CVECs. The ROCK inhibitor Y-27632 blocked the ca-ROCK-induced changes. C5a-activated neutrophils (10(6)/ml) also significantly elevated venular P(a), which was dose-dependently inhibited by Y-27632 and a structurally distinct ROCK inhibitor, H-1152. In CVEC monolayers, activated neutrophils increased permeability with a concomitant elevation in isometric tension, both of which were inhibited by Y-27632 or H-1152. Treatment with ca-ROCK also significantly increased CVEC monolayer permeability and isometric tension, coupled with actin polymerization and elevated phosphorylation of myosin regulatory light chain on Thr18/Ser19. The data suggest that during neutrophil activation, ROCK promotes microvascular leakage in association with actin-myosin-mediated tension development in endothelial cells.     

Model of nitric oxide diffusion in an arteriole: impact of hemoglobin-based blood substitutes

Administration of hemoglobin-based oxygen carriers (HBOCs) frequently results in vasoconstriction that is primarily attributed to the scavenging of endothelium-derived nitric oxide (NO) by cell-free hemoglobin. The ensuing pressor response could be caused by the high NO reactivity of HBOC in the vascular lumen and/or the extravasation of hemoglobin molecules. There is a need for quantitative understanding of the NO interaction with HBOC in the blood vessels. We developed a detailed mathematical model of NO diffusion and reaction in the presence of an HBOC for an arteriolar-size vessel. The HBOC reactivity with NO and degree of extravasation was studied in the range of 2-58 x 10(6) M(-1) x s(-1) and 0-100%, respectively. The model predictions showed that the addition of HBOC reduced the smooth muscle (SM) NO concentration in the activation range (12-28 nM) for soluble guanylate cyclase, a major determinant of SM contraction. The SM NO concentration was significantly reduced when the extravasation of HBOC molecules was considered. The myoglobin present in the parenchymal cells scavenges NO, which reduces the SM NO concentration.     

Rate of NO scavenging alters effects of recombinant hemoglobin solutions on pulmonary vasoreactivity.

Many hemoglobin-based oxygen carriers (HBOCs) produce systemic and pulmonary hypertension and may increase microvascular permeability as a consequence of nitric oxide (NO) scavenging. In this study, we examined the effects of two recombinant human hemoglobin solutions, rHb1.1 and rHb2.0 for injection (rHb2.0), with different rates of NO scavenging on vasoconstrictor reactivity and vascular permeability in isolated, saline-perfused rat lungs. We hypothesized that rHb1.1, a first-generation HBOC with an NO scavenging rate similar to that of native human hemoglobin, would exacerbate pulmonary vasoconstriction and permeability and that rHb2.0, a second-generation HBOC with an NO scavenging rate approximately 20- to 30-fold lower than that of rHb1.1, would minimally influence these responses. Consistent with this hypothesis, rHb1.1 enhanced pulmonary vasoconstrictor reactivity to both hypoxia and thromboxane mimetic U-46619 in a dose-dependent fashion. In contrast, rHb2.0 produced little or no change in reactivity to these stimuli. Furthermore, whereas rHb1.1 abrogated pulmonary vasodilation to the NO-donor S-nitroso-N-acetyl-penicillamine (SNAP), dose-dependent responses to SNAP were preserved, albeit attenuated, in lungs treated with rHb2.0. Finally, the capillary filtration coefficient was unaltered by either rHb1.1 or rHb2.0. We conclude that pulmonary hemodynamic responses to rHb2.0 are greatly reduced compared with those observed with rHb1.1, consistent with rHb2.0 having a diminished capacity to scavenge NO. In addition, neither hemoglobin solution measurably altered microvascular permeability in this preparation.     

A review of experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms

Experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms are reviewed. Effective diffusive permeabilities, the parameter appropriate to the analysis of reaction-diffusion interactions, depend on solute type and biofilm density. Three categories of solute physical chemistry with distinct diffusive properties were distinguished by the present analysis. In order of descending mean relative effective diffusive permeability (De/Daq) these were inorganic anions or cations (0.56), nonpolar solutes with molecular weights of 44 or less (0.43), and organic solutes of molecular weight greater than 44 (0.29). Effective diffusive permeabilities decrease sharply with increasing biomass volume fraction suggesting a serial resistance model of diffusion in biofilms as proposed by Hinson and Kocher (1996). A conceptual model of biofilm structure is proposed in which each cell is surrounded by a restricted permeability envelope. Effective diffusion coefficients, which are appropriate to the analysis of transient penetration of nonreactive solutes, are generally similar to effective diffusive permeabilities in biofilms of similar composition. In three studies that examine diffusion of very large molecular weight solutes (>5000) in biofilms, the average ratio of the relative effective diffusion coefficient of the large solute to the relative effective diffusion coefficient of either sucrose or fluorescein was 0.64, 0.61, and 0.36. It is proposed that large solutes are effectively excluded from microbial cells, that small solutes partition into and diffuse within cells, and that ionic solutes are excluded from cells but exhibit increased diffusive permeability (but decreased effective diffusion coefficients) due to sorption to the biofilm matrix.     

Hemoglobin-based O2 carrier O2 affinity and capillary inlet pO2 are important factors that influence O2 transport in a capillary

Hemopure (Biopure; Cambridge, MA) and PolyHeme (Northfield Laboratories; Evanston, IL) are two acellular hemoglobin-based O2 carriers (HBOCs) currently in phase III clinical trials for use as red blood cell substitutes. The most common adverse side effect that these HBOCs exhibit is increased vasoconstriction. Autoregulatory theory has been presented as a possible explanation for this physiological effect, where it is hypothesized that low-affinity HBOCs over-deliver O2 to tissues surrounding arterioles, thereby eliciting vasoconstriction. In this paper, we wanted to investigate HBOC oxygenation of tissue surrounding a capillary, which is the smallest element of the circulatory system. An a priori model has been developed in which the performance of mixtures of acellular HBOCs (synthesized by our group and others) and human red blood cells (hRBCs) has been simulated using a Krogh tissue cylinder model (KTCM) comprising a capillary surrounded by a capillary membrane and skeletal muscle tissue in cylindrical coordinates with specified tissue O2 consumption rates and Michaelis-Menten kinetics. In this study, the total hemoglobin (hRBCs and HBOCs) concentration was kept constant. The HBOCs studied possessed O2 affinities that were higher and lower compared to hRBCs (P50's spanned 5-55 mmHg), and the equilibrium binding/release of oxygen to/from the HBOCs was modeled using the Adair equation. At normoxic inlet pO2's, there was no correlation between O2 flux out of the capillary and the O2 affinity of the HBOC. However, a correlation was found between the average pO2 tension in the capillary and the O2 affinity of the HBOC. Additionally, we studied the change in the O2 equilibrium curve of HBOCs with different O2 affinities over a wide range of inlet pO2's and found that changing the inlet pO2 greatly affected which HBOC, having a unique O2 affinity, best delivered O2 to the surrounding tissue. The analysis of oxygen transport presented could lead to a better prediction of which acellular HBOC is best suited for a specific transfusion application that many times depends on the capillary inlet pO2 tension.     

Permeability characteristics of cultured endothelial cell monolayers

The purpose of this study was to characterize the permeability characteristics of an in vitro endothelial cell monolayer system and relate this information to available in vivo data. We cultured bovine fetal aortic endothelial cells on fibronectin-coated polycarbonate filters and confirmed that our system was similar to others in the literature with regard to morphological appearance, transendothelial electrical resistance, and the permeability coefficient for albumin. We then compared our system with in vivo endothelium by studying the movement of neutral and negatively charged radiolabeled dextran tracers across the monolayer and by using electron microscopy to follow the pathways taken by native ferritin. There were a number of differences. The permeability of our monolayer was 10-100 times greater than seen in intact endothelium, there was no evidence of "restricted" diffusion or charge selectivity, and ferritin was able to move freely into the subendothelial space. The reason for these differences appeared to be small (0.5-2.0 micron) gaps between 5 and 10% of the endothelial cells. Although the current use of cultured endothelial cells on porous supports may provide useful information about the interaction of macromolecules with the endothelium, there appear to be differences in the transendothelial permeability characteristics of these models and in vivo blood vessels.     

Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules

We have developed a method for studying the permeability properties of human endothelia in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured on a substrate of human amnion. Confluent monolayers of these cells demonstrated 6-12 delta.cm2 of electrical resistance (a measure of their permeability to ions) and restricted the transendothelial passage of albumin from their apical to their basal surface. To determine whether leukocyte emigration alters endothelial permeability in this model, we examined the effects of migrating human polymorphonuclear leukocytes (PMN) on these two parameters. Few PMN migrated across the HUVEC monolayers in the absence of chemoattractants. In response to chemoattractants, PMN migration through HUVEC monolayers was virtually complete within 10 minutes and occurred at random locations throughout the monolayer. PMN migrated across the monolayer via the paracellular pathway. Although one PMN migrated across the monolayer for each HUVEC, PMN migration induced no change in electrical resistance or albumin permeability of these monolayers. At this PMN:HUVEC ratio, these permeability findings were correlated morphologically to measurements that HUVEC paracellular pathway size increases by less than 0.22% with PMN migration. This increase is insufficient to effect a measurable change in the electrical resistance of the endothelial cell monolayer. These findings demonstrate that increased permeability of cultured endothelial cell monolayers is not a necessary consequence of PMN emigration.     

Erythrocytic ATP release in the presence of modified cell-free hemoglobin

The red blood cell (RBC) has been proposed as an O₂ sensor through a direct link between the desaturation of intracellular hemoglobin (Hb) and ATP release, leading to vasodilation. We hypothesized that the addition of cell-free Hb to the extracellular space provides a supplementary O₂ source that reduces RBC desaturation and, consequently, ATP release. In this study, the saturation of RBC suspensions was lowered by additions of deoxygenated hemoglobin-based oxygen carrier (HBOC) and then assayed for extracellular ATP. When an acellular human Hb intramolecularly cross-linked between α subunits (ααHb, p50 = 33 mmHg) was added to the red cell suspension, ATP production was significantly less than that in the presence of a lower p50 HBOC (Hb cross-linked between β subunits, ββHb, p50 = 8 mmHg). These results provide a potential mechanism for the O₂ affinity of HBOCs to interfere with a vasodilatory signal.     

Adrenomedullin Improves the Blood–Brain Barrier Function Through the Expression of Claudin-5

Summary 1. Aims: Brain vascular endothelial cells secret Adrenomedullin (AM) has multifunctional biological properties. AM affects cerebral blood flow and blood–brain barrier (BBB) function. We studied the role of AM on the permeability and tight junction proteins of brain microvascular endothelial cells (BMEC).2. Methods: BMEC were isolated from rats and a BBB in vitro model was generated. The barrier functions were studied by measuring the transendothelial electrical resistance (TEER) and the permeability of sodium fluorescein and Evans’ blue albumin. The expressions of tight junction proteins were analyzed using immunocytochemistry and immunoblotting.3. Results: AM increased TEER of BMEC monolayer dose-dependently. Immunocytochemistry revealed that AM enhanced the claudin-5 expression at a cell–cell contact site in a dose-dependent manner. Immunoblotting also showed an overexpression of claudin-5 in AM exposure.4.Conclusions: AM therefore inhibits the paracellular transport in a BBB in vitro model through claudin-5 overexpression.     

Numerical simulation of oxygen delivery to muscle tissue in the presence of hemoglobin-based oxygen carriers

This work represents a culmination of research on oxygen transport to muscle tissue, which takes into account oxygen transport due to convection, diffusion, and the kinetics of simultaneous reactions between oxygen and hemoglobin and myoglobin. The effect of adding hemoglobin-based oxygen carriers (HBOCs) to the plasma layer of blood in a single capillary surrounded by muscle tissue based on the geometry of the Krogh tissue cylinder is examined for a range of HBOC oxygen affinity, HBOC concentration, capillary inlet oxygen tension (pO(2)), and hematocrit. The full capillary length of the hamster retractor muscle was modeled under resting (V(max) = 1.57 x 10(-4) mLO(2) mL(-1) s(-1), cell velocity (v(c)) = 0.015 cm/s) and working (V(max) = 1.57 x 10(-3) mLO(2) mL(-1) s(-1), v(c) = 0.075 cm/s) conditions. Two spacings between the red blood cell (RBC) and the capillary wall were examined, corresponding to a capillary with and without an endothelial surface layer. Simulations led to the following conclusions, which lend physiological insight into oxygen transport to muscle tissue in the presence of HBOCs: (1) The reaction kinetics between oxygen and myoglobin in the tissue region, oxygen and HBOCs in the plasma, and oxygen and RBCs in the capillary lumen should not be neglected. (2) Simulation results yielded new insight into possible mechanisms of oxygen transport in the presence of HBOCs. (3) HBOCs may act as a source or sink for oxygen in the capillary and may compete with RBCs for oxygen. (4) HBOCs return oxygen delivery to muscle tissue to normal for varying degrees of hypoxia (inlet capillary pO(2) < 30 mmHg) and anemia (hematocrit < 46%) for the hamster model.     

Lysophosphatidylcholine increases endothelial permeability: role of PKCalpha and RhoA cross talk

Lysophosphatidylcholine (LPC) is a bioactive proinflammatory lipid that can be generated by pathological activities. We investigated the hypothesis that LPC signals increase in endothelial permeability. Stimulation of human dermal microvascular endothelial cells and bovine pulmonary microvascular endothelial cells with LPC (10-50 microM) induced decreases (within minutes) in transendothelial electrical resistance and increase of endothelial permeability. LPC activated (within 5 min) membrane-associated PKC phosphotransferase activity in the absence of translocation. Affinity-binding analysis indicated that LPC induced increases (also by 5 min) of GTP-bound RhoA, but not Rac1 or Cdc42. By 60 min, both signaling pathways decreased toward baseline. Inhibition of RhoA with C3 transferase inhibited approximately 50% of LPC-induced resistance decrease. Pretreatment with PKC inhibitor G?-6983 (concentrations selective for classic PKC), PMA-induced depletion of PKCalpha, and transfection of antisense PKCalpha oligonucleotide each prevented 40-50% of the LPC-induced resistance decrease. Furthermore, these three PKC inhibition strategies inhibited 60-80% of the LPC-induced GTP-bound RhoA. These results show that LPC directly impairs the endothelial barrier function that was dependent, at least in part, on cross talk of PKCalpha and RhoA signals. The evidence indicates that elevated LPC levels can contribute to the activation of a proinflammatory endothelial phenotype.     

Synthesis and characterization of a novel DTPA polymerized hemoglobin based oxygen carrier

OBJECTIVE: The purpose of this study was to prepare a novel polymerized hemoglobin (Hb) based oxygen carrier (HBOC) designed to minimize Hb induced hypertension, while employing a simple and inexpensive method of preparation. Cyclic-diethylenetriaminepentaacetic acid (DTPA) was used to polymerize stroma free Hb (SF-Hb). METHODS: SF-Hb was isolated from red blood cells and reacted with DTPA at a constant concentration, pH, and duration. Low molar mass fractions (<100 kDa) were removed using ultrafiltration. Reactions and subsequent ultrafiltration steps were determined to be reproducible by analyzing molar mass, colloid osmotic pressure and oxygen affinity. Finally, a model of 50% exchange transfusion (ET) in rats was used to evaluate the blood pressure response to DTPA polymerized SF-Hb (Poly-DTPA-Hb). RESULTS: Poly-DTPA-Hb demonstrated a number averaged molar mass of 128.7 kDa and a weighted average of 223.0 kDa. Oxygen binding equilibrium indicated high oxygen affinity (P50 = 5.1+/-0.01 mm Hg) and little cooperativity (n = 1.4). Poly-DTPA-Hb and a control DTPA polymerized human serum albumin (Poly-DTPA-HSA) unexpectedly caused acute hypotension during the period of ET in rats (mean arterial pressure approximately 45% less than baseline). Hypotension occurring over the period of ET was determined to be mediated by calcium binding to protein associated DTPA. This effect was attenuated by the addition of calcium chloride (CaCl2) to the Poly-DTPA protein preparations. CONCLUSIONS: Cyclic DTPA anhydride can be used to create cross-linked and polymerized hemoglobin, using a simple and inexpensive process. However, the addition of CaCl2 to the preparation appears to be required to prevent calcium chelation and subsequent hypotension during infusion.     

Role of mitogen-activated protein kinases in 4-hydroxy-2-nonenal-induced actin remodeling and barrier function in endothelial cells

In vivo and in vitro studies indicate that 4-hydroxy-2-nonenal (4-HNE), generated by cellular lipid peroxidation or after oxidative stress, affects endothelial permeability and vascular tone. However, the mechanism(s) of 4-HNE-induced endothelial barrier function is not well defined. Here we provide evidence for the first time on the involvement of mitogen-activated protein kinases (MAPKs) in 4-HNE-mediated actin stress fiber formation and barrier function in lung endothelial cells. Treatment of bovine lung microvascular endothelial cells with hydrogen peroxide (H(2)O(2)), as a model oxidant, resulted in accumulation of 4-HNE as evidenced by the formation of 4-HNE-Michael protein adducts. Exposure of cells to 4-HNE, in a dose- and time-dependent manner, decreased endothelial cell permeability measured as transendothelial electrical resistance. The 4-HNE-induced permeability changes were not because of cytotoxicity or endothelial cell apoptosis, which occurred after prolonged treatment and at higher concentrations of 4-HNE. 4-HNE-induced changes in transendothelial electrical resistance were calcium independent, as 4-HNE did not alter intracellular free calcium levels as compared with H(2)O(2) or diperoxovanadate. Stimulation of quiescent cells with 4-HNE (1-100 microm) resulted in phosphorylation of ERK1/2, JNK, and p38 MAPKs, and actin cytoskeleton remodeling. Furthermore, pretreatment of bovine lung microvascular endothelial cells with PD 98059 (25 microm), an inhibitor of MEK1/2, or SP 600125 (25 microm), an inhibitor of JNK, or SB 202190 (25 microm), an inhibitor of p38 MAPK, partially attenuated 4-HNE-mediated barrier function and cytoskeletal remodeling. These results suggest that the activation of ERK, JNK, and p38 MAP kinases is involved in 4-HNE-mediated actin remodeling and endothelial barrier function.     

Influenza Infects Lung Microvascular Endothelium Leading to Microvascular Leak: Role of Apoptosis and Claudin-5

Severe influenza infections are complicated by acute lung injury, a syndrome of pulmonary microvascular leak. The pathogenesis of this complication is unclear. We hypothesized that human influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed by spectrofluorimetry and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. We found that both clinical and laboratory strains of human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced degradation of the tight junction protein claudin-5; the adherens junction protein VE-cadherin and the actin cytoskeleton were unaffected. Over-expression of claudin-5 was sufficient to prevent replication-deficient virus-induced permeability. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dose-dependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Thus, we describe two distinct mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza.