A transgenic mouse model of sickle cell disorder

We investigated a transgenic mouse model of sickle cell disease, homozygous for deletion of mouse β-globin and containing transgenes for human βSand βS-antillesglobins linked to the transgene for human α-globin. In these mice, basal cGMP production in aortic rings is increased, whereas relaxation to an endothelium-dependent vasodilator, A-23187, is impaired. In contrast, aortic expression of endothelial nitric oxide synthase (NOS) is unaltered in sickle mice, whereas expression of inducible NOS is not detected in either group; plasma nitrate/nitrite concentrations and NOS activity are similar in both groups. Increased cGMP may reflect the stimulatory effect of peroxides (an activator of guanylate cyclase), because lipid peroxidation is increased in aortae and in plasma in sickle mice. Despite increased vascular cGMP levels in sickle mice, conscious systolic blood pressure is comparable to that of aged-matched controls; sickle mice, however, evince a greater rise in systolic blood pressure in response to nitro-l-arginine methyl ester, an inhibitor of NOS. Systemic concentrations of the vasoconstrictive oxidative product 8-isoprostane are increased in sickle mice. We conclude that vascular responses are altered in this transgenic sickle mouse and are accompanied by increased lipid peroxidation and production of cGMP; we suggest that oxidant-inducible vasoconstrictor systems such as isoprostanes may oppose nitric oxide-dependent and nitric oxide-independent mechanisms of vasodilatation in this transgenic sickle mouse. Destabilization of the vasoactive balance in the sickle vasculature by clinically relevant states may predispose to vasoocclusive disease.

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Systematic Enhancement of Polymerization of Recombinant Sickle Hemoglobin Mutants: Implications for Transgenic Mouse Model for Sickle Cell Anemia

Blood ◽

10.1182/blood.v90.11.4620 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4620-4627 ◽

Cited By ~ 6

Author(s):

Xianfeng Li ◽

Urooj A. Mirza ◽

Brian T. Chait ◽

James M. Manning

Keyword(s):

Mouse Model ◽

Transgenic Mouse ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

High Performance ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Quantitative Information ◽

Transgenic Mouse Model ◽

Sickle Hemoglobin

Abstract To provide quantitative information on the sites that promote polymerization of sickle hemoglobin (HbS) after formation of the initial hydrophobic bond involving Val-6(β) [E6V(β)] and also to provide hemoglobins with an enhanced polymerization that could be used in a mouse model for sickle cell anemia, we have expressed recombinant double, triple, and quadruple HbS mutants with substitutions on both the α- and β-chains, E6V(β)/E121R(β), D75Y(α)/E6V(β)/E121R(β) and D6A(α)/D75Y(α)/E6V(β)/E121R(β). These recombinant hemoglobins were extensively characterized by high-performance liquid chromatography analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, amino acid analysis, and mass spectroscopy. They retained the functional properties of the Hb tetramer and polymerized in a linear manner at progressively lower Hb concentration as a function of the degree of substitution, suggesting that these remote sites (αD6A, αD75Y, and βE121R) on the α- and β-chains exhibit additive, enhanced polymerization properties. The quadruple mutant has a polymerization concentration close to that of the purified SAD hemoglobin from transgenic mouse red blood cells consisting of HbS, Hb Antilles, and Hb D-Punjab. Normal mouse Hb increases the polymerization concentration of each mutant. Thus, the general approach of using recombinant Hbs as described here should prove useful in elucidating the quantitative aspects of the mechanism of HbS polymerization and in identifying the contribution of individual sites to the overall process. The strategy described here demonstrates the feasibility of a systematic approach to achieve future recombinant HbS mutants that could provide a new generation of the transgenic mouse model for sickle cell anemia.

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