INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

PRECLINICAL DATA on various chemically or genetically modified hemoglobins developed as hemoglobin-based oxygen carriers (HBOC) have demonstrated their efficacy in ensuring oxygen transport and maintenance of the circulating volume (21, 22). However, these blood substitutes cause side effects among which increased blood pressure (10-50 mmHg) was reported in both preclinical and clinical studies (5, 6, 15). This pressure effect explained by systemic vasoconstriction is thought to be due essentially to scavenging of the vasodilating substance nitric oxide (NO) by free hemoglobin in the vascular lumen and/or by penetration into the vascular wall. Both the ferrous and ferric iron of the heme and the thiols of the -93 cysteine residues are likely to react with NO, thus causing a decrease in the steady-state level of biologically active endothelium-derived NO in the blood (7, 19). However, the NO-scavenging action of free hemoglobin cannot fully account for the vasoconstriction elicited by HBOC, because other elements of the regulation of the vascular tone have been demonstrated to be influenced by free hemoglobin (16). Although the mechanisms are not known, peripheral vascular -adrenergic receptors, endothelin-1 production/release, and increased O₂-carrying capacity of blood have been proposed to be involved in the HBOC pressor effect (2, 6, 18, 20). Few authors reported that free hemoglobin could cross the endothelial barrier by getting between the endothelial cells and could cause vasoconstriction by interacting with NO before it could stimulate its target hemoprotein, guanylate cyclase, in smooth muscle cells (13). However, this hypothesis does not take into account the interaction of hemoglobin with other regulatory elements such as endothelin-1. To induce vasoconstriction via these mechanisms, hemoglobin should indeed penetrate into the endothelial cell by mechanisms such as endocytosis.

Therefore, we investigated this penetration by immunohistochemical techniques in guinea pig arteries perfused in vivo with a HBOC. In addition, to evaluate whether the Hb-NO interaction is a prerequisite for this potential penetration, the effect of pretreatment with N^G-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of the NO synthesis pathway, has been studied.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Experimental models. The experiments were conducted in male Hartley VAF guinea pigs (300-350 g, Charles River, St. Aubain les Elbeuf, France), an animal model for which the principal hemostatic characteristics (platelet function, responsiveness to ADP, arachidonic cascade) appeared to most closely resemble to those of humans (9, 17). With the guinea pigs under general anesthesia with halothane (Belamont, France, 1% in 95% O₂-5% CO₂), we inserted a polyethylene tube into the left femoral artery and tunneled it subcutaneously to emerge at the top of the back. The animals were treated with penicillin (200,000 U/kg im) and heparin (150 U/kg iv). An isovolemic exchange transfusion of 50% of the estimated blood volume was performed via the femoral artery tube without inducing significant hemodynamic disturbances as described by Faivre et al. (3). The animals were randomly allocated to one of the experimental groups consisting of hemodilution with a HBOC (described below) (groups I and II; n = 5 and n = 3, respectively), with the same HBOC 2 min after pretreatment with L-NAME (20 mg/kg body wt, Fluka Chemica, Switzerland) (group III, n = 3), and with a control solution of human albumin (group IV and V, n = 5 and n = 3, respectively).

In groups I and IV, the right carotid artery was cannulated and connected to a pressure transducer (Viggo-Spectramed, France) to measure the pulsatile arterial pressure (in mmHg) during the first hour before exchange transfusion (baseline) and for 3 h after exchange. This transducer was connected to a computer for on-line data acquisition at a rate of 75 Hz (Acqknowledge hardware and software, Biopac Systems).

Groups II, III, and V were used for immunohistochemical experiments. One hour after the beginning of the exchange transfusion (~30 min after the end of exchange) when arterial pressure was maximal (cf. Fig. 1), a fixing aqueous solution (1.94 × 10² M picric acid, acetic acid 6.7%, formaldehyde 10.7%, absolute ethylic alcohol 52.8%) was perfused for 30 min in situ from the right carotid artery to the left femoral artery. The following technique was described by Zarins et al. (23), but the perfusion way was modified to wash and rapidly fix the abdominal aorta with the bloodstream. This technique makes it possible to keep the structural integrity of the vascular wall and to limit the detachment and the alteration of the endothelial barrier. Rings of abdominal aorta were collected, kept in the fixing solution, and then included into paraffin. The rings were cut into 5- and 10-µm sections and stained according to the immunohistochemical protocol described in Immunohistochemical staining.

Solutions. A human HBOC prepared as described by Prouchayret et al. (14) by chemical modification of human stroma-free hemoglobin [dextran 10-benzene-tetracarboxylate (Dex-BTC-Hb), 8 ± 0.5 g/dl, pH 7.4, supplied by Pasteur-Mérieux, sérums et vaccins, Marcy l'Etoile, France] was used. Briefly, Dex-BTC-Hb has a mean molecular mass of 300 kDa (none at 32 kDa, 64 kDa < 5%, 64-500 kDa > 90%, more than 500 kDa < 5% determined on HPLC columns TSKG3000SW and TSKG4000SW, Varian LC 5020), physiological O₂ affinity [P₅₀ = 21.7 ± 0.4 mmHg, n = 1, 7 ± 0.05, Hemox Analyzer (TCS Medical Product)] in a bis-Tris buffer (pH 7.4 at 37°C), pyrogen-free, endotoxin-free (<0.5 EU/ml), and a half-life of 9.5 ± 0.5 h in guinea pigs. Neither vascular leakage nor hemoglobinuria was found in guinea pigs (10). The study of the dissociation state of plasma Dex-BTC-Hb in vivo, determined with HPLC (column TSKG3000SW on Varian LC 5020) in phosphate buffer pH 7.2 (flow rate 1 ml/min, 403 nm), demonstrated a very slow dissociation because >70% of circulating Dex-BTC-Hb had a molecular mass > 64 kDa during the first 24 h after the exchange transfusion (4). Purified human albumin was used as a control solution (5 g/dl, pH 7.6) and was supplied by Pasteur-Mérieux sérums et vaccins. Both solutions were dissolved in Tyrode medium (in mM: 6.7 glucose, 141.0 Na⁺, 5.0 K⁺, 2.5 Ca²⁺, 1.1 Mg²⁺, 115.8 Cl, 0.8 phosphates, and 30.0 carbonates).

Immunohistochemical staining. The presence of HBOC inside guinea pig aortic endothelial cells was assessed using a three-step indirect immunoassay protocol, which, using avidin-biotin complex, is a powerful detection system. Primary antibodies were obtained from New Zealand White rabbits immunized with Dex-BTC-Hb according to Bleeker's protocol (1). With the use of ELISA methods, primary antibodies against HBOC have been demonstrated to be specific to human hemoglobin, and no cross-reactions have been found with guinea pig hemoglobin. Before addition of the primary antibody on hydrate deparaffinized sections, 5% normal goat serum was added to minimize nonspecific protein bindings. After the addition of the primary antibody, rabbit anti-human modified hemoglobin, at a dilution of 1:200, sections were incubated 30 min at room temperature. Control sections were exposed to normal dilute rabbit serum. After we rinsed the sections with phosphate-buffered saline (PBS), sections were incubated 30 min at room temperature with the secondary antibody (goat anti-rabbit IgG covalently linked to biotin, Microm, France). After being rinsed with PBS, sections were incubated 30 min at room temperature with avidin conjugated to alkaline phosphatase and were then rinsed with PBS. The alkaline phosphatase was visualized 15 min at room temperature using a medium containing p-nitrophenylphosphate as the substrate. Sections were counterstained with hematoxylin to enhance nuclear detail.

Data analysis. Mean arterial pressure (MAP) was calculated as one-third (systolic pressure  diastolic pressure) + diastolic pressure and expressed as means ± SE. Statistical comparisons were made before (baseline) and after exchange transfusion (t = 15, 30, 60, 120, and 180 min) for groups I and III using analysis of variance for repeated measures. Comparisons between groups I and III were made for each time, using analysis of variance for repeated measures with Bonferroni-Dunn correction. A value of P < 0.05 was considered significant.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Figure 1 shows the variations of MAP with time in groups I and IV before exchange transfusion (baseline) and 15, 30, 60, 120, and 180 min after the end of the exchange transfusion. The exchange transfusion was achieved in 25 ± 5 min. With HBOC, MAP increased maximally and significantly (P < 0.05) immediately after the end of exchange transfusion. The increase lasted 60 min, and then MAP progressively decreased and was not significantly different from baseline values after this time. With albumin, MAP was unchanged during the first hour following the end of exchange transfusion and a moderate fall in MAP appeared after 120 min.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Variation of mean arterial pressure (MAP) with time expressed as means ± SE after hemodilution with albumin (n = 5) or hemoglobin-based O2 carriers (HBOC) dextran 10-benzene-tetracarboxylate (Dex-BTC-Hb) (n = 5). * P < 0.05 time vs. baseline. § P < 0.05 HBOC vs. albumin.

Considering these results, we collected rings of abdominal aorta when HBOC-induced pressor effect was maximal, 30 min after the end of exchange transfusion. At the same time, MAP was unchanged with albumin. Figure 2 illustrates the immunohistochemical staining in the vascular wall and particularly in the endothelium of abdominal aorta perfused with HBOC (Fig. 2A), with HBOC after pretreatment with L-NAME (Fig. 2B), and with albumin (Fig. 2C). Figure 2, A and B, reveals positive immunohistochemical staining into the endothelial cells and in the adventitia. The endothelial cells' cytoplasm was strongly stained in HBOC groups. The pretreatment with L-NAME did not seem to limit the endothelial uptake of hemoglobin. Moreover, we observed systematically large amounts of hemoglobin pasted onto the endothelial membrane that were not removed by the fixation technique. In the adventitia, the staining was due to HBOC staying in vasa vasorum. In the media, no staining was observed. As expected, no staining was revealed in the aortic vascular wall of albumin-hemodiluted animals.

View larger version (131K): [in this window] [in a new window]   Fig. 2.   Immunohistochemical staining for human hemoglobin in aortic vascular wall of guinea pig perfused with HBOC: without NG-nitro-L-arginine methyl ester (L-NAME) pretreatment (A), with L-NAME pretreatment (B); or with albumin (C). Red staining represents human hemoglobin molecule. Nuclei counterstained with hematoxylin appear blue. A, B, and C: ×1,000. I, intima; M, media; A, adventitia; EI, elastica interna; EC, endothelial cell; SMC, smooth muscular cell.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The present study is, to our knowledge, the first to report the presence of free hemoglobin in endothelial cells in the early time following administration of a HBOC.

The penetration of hemoglobin in the subendothelial space and between the smooth muscle cells has been proposed to explain in vitro and in vivo the vasoconstriction elicited by HBOC (11). As a result, hemoglobin could interact with some of the mechanisms involved in the regulation of the vascular tone. In this study, we confirm that HBOC induce an immediate increase in MAP, and we propose that it would be due to the rapid penetration of hemoglobin into the endothelial cells, because hemoglobin was detected there 30 min after the end of the exchange transfusion. At this time, the pressor effect of hemoglobin was maximal (Fig. 1), and the plasma concentration of human hemoglobin was high, ~3 g/dl (10). The progressive decrease in MAP after 120 min could be due to experimental conditions, such as anesthesia influence, rather than to the disappearance of the pressor effect of HBOC, because a decrease in MAP was also observed with albumin after this time. Therefore, the rings of abdominal aorta were collected at t = 30 min to be sure that the pressor effect of HBOC was maximal. In abdominal aorta, immunohistochemical staining was aimed to detect human hemoglobin contained in HBOC in the vascular wall. The advantage of fixing arteries in situ by perfusion of a fixing solution is to remove blood entirely from the lumen of large arteries. However, with this technique blood was not removed from capillaries and vasa vasorum of large arteries adventitia. The absence of staining in the vascular wall of aorta perfused with albumin (Fig. 2C, group V) indicates that no cross-reaction occurred between primary antibodies anti-human hemoglobin and guinea pig blood in the vasa vasorum of large arteries. Therefore, we can assume that the staining obtained in the adventitia (Fig 2, A and B) with the HBOC was due to human hemoglobin from HBOC in the numerous vasa vasorum. In addition, because HBOC contained in circulating blood was removed from the lumen during the fixation, Fig. 2A clearly shows that human hemoglobin was present in the intima and more specifically into the endothelial cells. Besides, Fig. 2B shows that human hemoglobin seems to be able to be fixed on the abluminal endothelial membrane.

The presence of human hemoglobin in these cells will have to be discussed. It is known that HBOC generates an increase of the endothelial heme oxygenase activity and a higher endothelial heme content than with unmodified hemoglobin (HbA₀) in cultured aortic endothelial cells (11, 12). These results involved the oxidation and the catabolism of hemoglobin before the endothelial heme uptake. In our study, we demonstrated that at least the antigenic globin moiety of hemoglobin was present early in endothelial cells. Thus heme and globin can penetrate in endothelial cells. Because catabolism of hemoglobin and release of heme are relatively slow mechanisms, the presence of the globin moiety into endothelial cells in the early times following HBOC administration should demonstrate the presence of the whole molecule of hemoglobin and the ability of endothelial cells to take up modified hemoglobin. The pretreatment with L-NAME at a dose for which the NO synthesis was fully inhibited did not limit the uptake of hemoglobin by endothelial cells. This result brings about the hypothesis that the Hb-NO interaction is not a prerequisite for endothelial uptake of hemoglobin. Besides, in the presence of L-NAME, the removal of hemoglobin was impaired, indicating that hemoglobin seems to be fixed at the abluminal side of the endothelial membrane. So when the NO synthesis is blocked, hemoglobin could be fixed more easily on the endothelial membrane.

In all cases, two uptake mechanisms are possible. First, hemoglobin could penetrate into the cytoplasm of the endothelial cells by endocytosis; in this case the presence of a specific receptor to hemoglobin on the membrane is required. Such a hemoglobin-binding molecule (94.5 kDa) has been found on the membrane of cultured bovine endothelial cells (8), and the presence of such a receptor seemed to be confirmed by our results. Second, hemoglobin could be trapped by a vesicular system of transendothelial transport, which is known to be involved in the transport of light lipoprotein. Because our results did not permit us to reject this hypothesis, both mechanisms have to be considered.

In perspective, considering our results, it appears that despite the chemical modifications applied to hemoglobin that were aimed to increase the molecular weight, thus prevent the subunit dissociation, and thereby the extravascular leakage, hemoglobin was detected in endothelial cells. This suggests that the studies describing the pressor effect of HBOC, which hypothesized that the interactions of hemoglobin with the mechanisms of regulation of the vascular tone are confined in the subendothelial space or media, have to be newly considered. Thus hemoglobin could scavenge NO directly at the site of its synthesis (endothelial cells) and would prevent it from diffusing to vascular smooth muscle cells where it acts. In addition, hemoglobin could interfere with endothelin-1 synthesis as has been previously described (2, 6, 18). However, numerous questions have to be resolved: How does hemoglobin penetrate into endothelial cells? How long does hemoglobin stay in endothelial cells? Is the duration of the pressor effect of hemoglobin dependent on the presence of hemoglobin into endothelial cells? Do endothelial cells release hemoglobin in the media? Because the pressor effect is immediate, do endothelial cells instantaneously take up hemoglobin from the lumen?