Arginine therapy of transgenic-knockout sickle mice improves microvascular function by reducing non-nitric oxide vasodilators, hemolysis, and oxidative stress

Abstract

In sickle cell disease, nitric oxide (NO) depletion by cell-free plasma hemoglobin and/or oxygen radicals is associated with arginine deficiency, impaired NO bioavailability, and chronic oxidative stress. In transgenic-knockout sickle (BERK) mice that express exclusively human alpha- and beta(S)-globins, reduced NO bioavailability is associated with induction of non-NO vasodilator enzyme, cyclooxygenase (COX)-2, and impaired NO-mediated vascular reactivity. We hypothesized that enhanced NO bioavailability in sickle mice will abate activity of non-NO vasodilators, improve vascular reactivity, decrease hemolysis, and reduce oxidative stress. Arginine treatment of BERK mice (5% arginine in mouse chow for 15 days) significantly reduced expression of non-NO vasodilators COX-2 and heme oxygenase-1. The decreased COX-2 expression resulted in reduced prostaglandin E(2) (PGE(2)) levels. The reduced expression of non-NO vasodilators was associated with significantly decreased arteriolar dilation and markedly improved NO-mediated vascular reactivity. Arginine markedly decreased hemolysis and oxidative stress and enhanced NO bioavailability. Importantly, arteriolar diameter response to a NO donor (sodium nitroprusside) was strongly correlated with hemolytic rate (and nitrotyrosine formation), suggesting that the improved microvascular function was a response to reduced hemolysis. These results provide a strong rationale for therapeutic use of arginine in sickle cell disease and other hemolytic diseases.

Fig. 1.

Effect of arginine on endothelial nitric oxide synthase (eNOS), COX-2, COX-1, and heme oxygenase-1 (HO-1) expression. A: arginine had caused 52 and 31% decreases in eNOS expression, respectively, in BERK-hemizygous (Hemi) and BERK mice. B: arginine resulted in distinct decreases in COX-2 expression in BERK-Hemi and BERK mice. C: arginine had no appreciable effect on COX-2 expression in C57BL mice. D: COX-1 expression showed no changes after arginine treatment. E: arginine markedly decreased in HO-1 expression in BERK mice.

Fig. 2.

Densitometric analysis of COX-2 expression and plasma prostaglandin E₂ (PGE₂) levels. A: COX-2 expression was elevated in untreated groups of BERK-Hemi and BERK mice (*P < 0.05 and **P < 0.01, respectively), with BERK mice showing the maximal increase. Arginine caused a markedly reduced expression of COX-2 in BERK-Hemi and BERK mice (52 and 43% decreases, respectively, +P < 0.01 and ++P < 0.038). B: untreated BERK-Hemi and BERK mice showed greater PGE₂ levels compared with C57BL mice (*P < 0.05 and **P < 0.0001, respectively). Arginine caused marked 56% decrease in BERK mice (+P < 0.004 vs. untreated BERK).

Fig. 3.

Effect of arginine on the arteriolar diameter (branching orders: A2, A3, and A4) and flow parameters in A2 arterioles in the cremaster microcirculation. A: in C57BL and BERK-Hemi mice, arginine caused diameter increases in all arteriolar orders, whereas, in BERK mice, arginine caused either a decrease (A2 arterioles) or had no effect on the diameter. The postarginine values in each group were strikingly similar, as indicated by horizontal lines. B: arginine resulted in increased wall shear rates in BERK mice. C: the reduced A2 diameter in arginine-treated BERK mice was accompanied by reduced volumetric flow (Q), which was in contrast to the increase in Q in controls. *P < 0.05–0.001 vs. prearginine (untreated) values. +P < 0.01 vs. untreated C57BL mice.

Fig. 4.

Effect of arginine on vascular responses. A and B: attenuated response of arterioles in BERK mice to ACh and SNP. Arginine-treated BERK mice showed markedly improved responses to acetylcholine (ACh) and sodium nitroprusside (SNP) (*P < 0.00001 vs. C57BL and BERK-Hemi mice, +P < 0.00001 vs. untreated BERK mice). C: baseline mean arterial pressure (MAP) in untreated BERK-Hemi and BERK mice was significantly lower than in C57BL mice (*P < 0.005 and **P < 0.0001, respectively). In contrast to significant increases in untreated C57BL and BERK-Hemi mice (+P < 0.01 and ++P < 0.05, respectively), N^G-nitro-l-arginine methyl ester (l-NAME) caused attenuated response in untreated BERK mice, but resulted in 43% increase in MAP in arginine (Arg)-treated BERK mice (#P < 0.001).

Fig. 5.

Effect of arginine on nitrotyrosine formation. A: BERK mice showed maximal tyrosine nitration of both ∼60- and 26-kDa proteins. Arginine caused marked decreases in tyrosine nitration of 60- and 26-kDa proteins in BERK-Hemi and BERK mice. B and C: BERK mice showed maximal tyrosine nitration of both 66- and 26-kDa nitrated proteins, i.e., 4.6- and 5.6-fold increase over C57BL mice. Arginine supplementation caused significant decreases in the nitrated proteins in both BERK-Hemi and BERK mice compared with corresponding untreated groups. *P < 0.05–0.01 vs. C57BL mice. +P < 0.05 vs. untreated BERK-Hemi mice. ++P < 0.004 vs. untreated BERK mice.

Fig. 6.

A: relationship between arteriolar diameter response to SNP and plasma heme levels. Percent arteriolar diameter increase after topical SNP (10⁻⁶ M) was strongly correlated with plasma hemoglobin levels. B: relationship between arteriolar diameter response to SNP and nitrotyrosine formation showed a similar strong correlation between the two parameters. Data from BERK+γ mice (*) are added from Kaul et al. (19) to the above regression plots to demonstrate the dependence of arteriolar diameter response to SNP on hemolytic rate and nitrotyrosine formation.