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Endothelial cell NADPH oxidase mediates the cerebral microvascular dysfunction in sickle cell transgenic mice

Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USA; and
^* Vascular Biology Center and Division of Hematology-Oncology-Transplantation, University of Minnesota Medical School, Minneapolis, Minnesota, USA

¹Correspondence: Department of Molecular and Cellular Physiology, LSU Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130-3932, USA. E-mail: dgrang{at}lsuhsc.edu

SPECIFIC AIMS

Although blood cell-endothelial cell adhesion and oxidative stress have been implicated in the pathogenesis of sickle cell disease (SCD), the nature of the linkage between these vascular responses in SCD remains unclear. The objectives of this study were to determine whether reactive oxygen species (ROS) contribute to enhanced leukocyte and platelet adhesion responses in the microvasculature during SCD and to identify potential enzymatic and cellular sources of these ROS.

PRINCIPAL FINDINGS

1. Reduced leukocyte and platelet adhesion in ß^S/SOD-1TgN chimeric mice, with normal SOD expression by circulating ß^S blood cells but overexpression of SOD in the vessel wall, strongly implicates endothelial cell-associated superoxide in these adhesion responses
One objective of this study was to determine whether genetic overexpression of cytosolic (CuZn) superoxide dismutase (SOD) in vascular endothelial cells alters the elevated blood cell adhesion responses noted in a mouse model of SCD. The role of endothelial cell-associated superoxide in mediating the leukocyte and platelet adhesion responses to 2 h of whole body hypoxia (10% O₂, 0.05% CO₂, balance nitrogen), followed by 4 h reoxygenation (room air), in ß^S mice was addressed in bone marrow chimeric mice (Fig. 1 ). Transfer of marrow from ß^S mice into WT mice (ß^S/WT) yielded significantly higher hypoxia-reoxygenation (H/R) -induced leukocyte and platelet adhesion responses (observed using intravital fluorescence microscopy) than in WT mice receiving bone marrow from WT mice (WT/WT). However, the transfer of bone marrow from ß^S mice into SOD1-TgN mice (ß^S/SOD-1TgN) yielded leukocyte and platelet adhesion responses to H/R that were significantly reduced (to WT/WT levels) compared with leukocyte and platelet adhesion responses observed in ß^S/WT chimeras.

View larger version (29K): [in this window] [in a new window]   Figure 1. Leukocyte and platelet adhesion responses in wild-type (WT) mice receiving bone marrow from WT (WT/WT) or ßS (ßS/WT) donor mice and in copper-zinc superoxide dismutase (SOD-1) transgenic (TgN) mice receiving bone marrow from ßS mice (ßS/SOD1-TgN). *P <0.05 compared with WT/WT, #P <0.05 relative to ßS/WT mice.

2. Attenuated leukocyte and platelet adhesion in ß^S/gp91phox^–/– chimeras, with functional NADPH oxidase expression by circulating ß^S blood cells and inactive NADPH oxidase in the vessel wall, implicates endothelial cell-associated NADPH oxidase as a source of the superoxide that elicits these cell-cell interactions
A second objective was to define the contribution of endothelial cell NADPH oxidase to the superoxide-dependent recruitment of platelets and leukocytes in venules of sickle cell transgenic mice. The involvement of endothelial cell NADPH oxidase (Fig. 2 ) was addressed in gp91phox^–/– mice that were transplanted with bone marrow derived from ß^S mice (ß^S/gp91phox^–/–). These chimeras exhibited platelet and leukocyte adhesion responses to H/R that were significantly reduced (to WT/WT levels) compared with platelet and leukocyte adhesion responses observed in ß^S/WT chimeras.

View larger version (30K): [in this window] [in a new window]   Figure 2. Leukocyte and platelet adhesion responses in wild-type (WT) mice receiving bone marrow from either WT (WT/WT) or ßS (ßS/WT) donor mice and in gp91phox–/– mice receiving bone marrow from ßS mice (ßS/ gp91phox–/–). *P <0.05 compared with WT/WT, #P <0.05 relative to ßS/WT mice.

3. Unchanged leukocyte and platelet adhesion in cerebral venules of ß^S/WT mice treated with allopurinol indicates that xanthine oxidase is not a major source of the superoxide that mediates SCD-associated blood cell adhesion
The contribution of xanthine oxidase to H/R-stimulated leukocyte and platelet adhesion responses in ß^S mice was addressed in ß^S/WT mice treated with the xanthine oxidase inhibitor allopurinol (Fig. 3 ). Regardless of the route of administration, oral or intraperitoneal (50 mg/kg for 2 days and 1 day, respectively), allopurinol pretreatment did not significantly reduce leukocyte and platelet adhesion responses in ß^S/WT chimeras.

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic diagram summarizing the relative importance of NADPH-oxidase-derived superoxide and iron-dependent radicals in the production of SCD-associated adhesion of leukocytes and platelets in the cerebral microcirculation.

4. Reduced leukocyte and platelet adhesion in cerebral venules of ß^S/WT mice treated with the iron chelator desferroxamine implicates catalytically active iron in these adhesion responses
The role of catalytically active iron in mediating leukocyte and platelet adhesion responses to H/R in ß^S mice was addressed in ß^S/WT chimeras treated with desferroxamine (DFO; 300 mg/kg i.p. for 3 days). Intraperitoneal DFO pretreatment of ß^S/WT mice yielded platelet and leukocyte adhesion responses to H/R that were significantly reduced (to WT/WT levels) compared with platelet and leukocyte adhesion responses in untreated ß^S/WT chimeras.

CONCLUSIONS AND SIGNIFICANCE

Novel findings of the present study include 1) the first evidence for vascular endothelial cells as the cellular source of the superoxide that mediates the cerebral blood cell-vessel wall interactions elicited by SCD, 2) a demonstration that endothelial cell-associated NADPH oxidase is the major enzymatic source of the superoxide that mediates SCD-induced blood cell adhesion, and 3) evidence invoking a role for catalytically active iron in the proinflammatory and prothrombogenic response associated with SCD.

The potential involvement of superoxide, which has been implicated in the defective arteriolar and venular responses of SCD mice, was studied in our model using SOD1-TgN mutants transplanted with bone marrow from ß^S mice to achieve overexpression of this cytosolic form (CuZn) of SOD. Overexpression of intracellular CuZnSOD in these chimeric mice resulted in dramatically reduced leukocyte and platelet adhesion responses, strongly implicating superoxide in the SCD-associated inflammatory process and suggesting that the source of superoxide lies at or near the endothelial cell surface.

Xanthine oxidase, auto-oxidation of sickle hemoglobin, and NADPH oxidase are potential sources of the superoxide generated in postcapillary venules of ß^S mice. Studies have implicated xanthine oxidase as a major source of ROS produced in arterioles and venules of SCD mice. Treatment of ß^S/WT mice with allopurinol had no effect on the proinflammatory and prothrombogenic responses seen in the cerebral venules of these mice. These findings suggest that xanthine oxidase is not an important source of the superoxide generated in the cerebral microvasculature of SCD mice.

Another enzymatic source of superoxide implicated in several disease processes is NADPH oxidase. Vascular endothelial cells are one of several cell types where this enzyme has been localized. Our findings, based on mice unable to produce superoxide through NADPH oxidase due to a genetic deficiency in the gp91phox subunit of this enzyme, indicate that NADPH oxidase inactivation is as effective as SOD overexpression in abolishing the proinflammatory and prothrombogenic adhesion responses normally observed in SCD mice.

SCD-associated hemolytic anemia is a potential contributor to increased plasma levels of redox active free iron and heme. Treatment of ß^S/WT chimeras with DFO produced leukocyte and platelet adhesion responses in cerebral venules that were reduced to normal WT/WT levels. Considered in the context of our findings with SOD1-TgN overexpression and gp91phox (NADPH oxidase) deficiency, the results of this study suggest that NADPH oxidase-derived superoxide ultimately results in the formation of iron-dependent radicals that mediate the microvascular responses observed in ß^S mice.

Leukocytes, platelets, and endothelial cells are all capable of producing NADPH-oxidase-derived superoxide. Bone marrow chimeras, wherein ß^S marrow was transferred to gp91phox^–/– or SOD1-TgN recipients to create chimeric mice with typical ß^S levels of gp91phox and CuZnSOD in all circulating blood cells and vascular endothelial cells with deficiency in gp91phox or overexpression of CuZnSOD, demonstrated leukocyte and platelet adhesion responses that were reduced to normal WT/WT levels. These reduced adhesion responses suggest that the vessel wall is the major source of NADPH oxidase-derived superoxide rather than leukocytes or platelets. Results obtained with the allopurinol and DFO treatment groups, although derived from less cell-specific approaches, nonetheless agree with the possibility that the vessel wall is the dominant source of ROS in the brain of ß^S mice.

The experimental strategies used in this study reveal a strong dependence of the blood cell interactions in SCD to NADPH oxidase-derived superoxide and catalytically active iron (Fig. 3) . Furthermore, our findings point to endothelial cells as a potentially important source of the ROS that contribute to the microvascular dysfunction elicited by SCD.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3218fje; doi: 10.1096/fj.04-3218fje

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This Article Full Text (PDF) All Versions of this Article: 19/8/989 04-3218fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wood, K. C. Articles by Granger, D. N. Search for Related Content PubMed PubMed Citation Articles by Wood, K. C. Articles by Granger, D. N.