Hydroxyurea with AKT2 inhibition decreases vaso-occlusive events in sickle cell disease mice

Abstract

Heterotypic cell-cell adhesion and aggregation mediate vaso-occlusive events in patients with sickle cell disease (SCD). Although hydroxyurea (HU), an inducer of fetal hemoglobin, is the main therapy for treatment of SCD, it is unclear whether it has immediate benefits in acute vaso-occlusive events in SCD patients. Using real-time fluorescence intravital microscopy, we demonstrated that short-term coadministration of HU and Akti XII, an AKT2 inhibitor, efficiently reduced neutrophil adhesion and platelet-neutrophil aggregation in venules of Berkeley (SCD) mice challenged with tumor necrosis factor α (TNF-α) or hypoxia/reoxygenation. Importantly, compared with HU or Akti XII treatment alone, short-term treatment with both agents significantly improved survival in those mice. We found that the level of plasma nitric oxide species was elevated by HU but not Akti XII, AKT2 phosphorylation levels in activated neutrophils and platelets were reduced by Akti XII but not HU, and the expression of endothelial E-selectin and intercellular adhesion molecule 1 was decreased by either agent. Our results suggest that short-term coadministration of HU and Akti XII has immediate benefits for acute vaso-occlusive events and survival in SCD mice exceeding those seen for single therapy.

Figure 1

Coadministration of HU and Akti XII efficiently inhibits neutrophil adhesion and platelet-neutrophil aggregation in venules, improves survival, and impairs neutrophil transmigration into alveoli in TNF-α-challenged Berkeley mice. TNF-α was intraperitoneally injected into Berkeley mice to induce severe inflammatory conditions. Intravital microscopy was performed as described in supplemental Methods. Neutrophils and platelets were labeled by infusion of Alexa Fluor 647-conjugated anti-Gr-1 and Dylight 488-conjugated anti-CD42c antibodies. (A) Timeline for the treatment and surgery (jugular cannulation and cremaster muscle exposure) in Berkeley mice. White arrows show direction of blood flow. (B) Representative images of intravital captures at various time points. Time 0 was set as the image capture was initiated at each vessel. (C-D) Number of rolling and adherent neutrophils. (E) The integrated median fluorescence intensities of anti-CD42c antibodies (F platelets) were normalized to the number of adherent neutrophils and plotted as a function of time. Data were obtained from 45-57 venules in 6-8 mice per group. (F) Survival curves of Berkeley mice during or after intravital microscopy. (G-H) Representative images from histochemistry of lung sections. The number of transmigrated neutrophils (arrow heads) was quantified in the field of view (110 mm²). Data represent the mean ± standard deviation (n = 25-30 sections in 6-8 mice per group). Bar = 20 μm. The survival rate was assessed with Mantel-Cox log-rank test. *P < .05, **P < .01, and ***P < .001 vs vehicle control; analysis of variance (ANOVA) and Dunnett’s test. #P < .05, ##P < .01, and ###P < .001 between two groups; Student t test.

Figure 2

The effects of HU and Akti XII on the expression of E-selectin and ICAM-1, plasma NO_x levels, and AKT2 phosphorylation in TNF-α-challenged Berkeley mice. (A-D) Following intravital microscopy, the cremaster muscle was excised, fixed, and embedded in paraffin for immunohistochemistry. Sections of the muscle were labeled with rat anti-mouse E-selectin or ICAM-1 and then Dylight 488-labeled anti-rat immunoglobulin G antibodies, followed by incubation with phycoerythrin-labeled anti-PECAM-1 antibodies and a mounting reagent containing 4,6 diamidino-2-phenylindole. (A,C) Representative images of E-selectin or ICAM-1 and PECAM-1 staining. (B,D) The geometric mean fluorescence intensities (MFI) of E-selectin or ICAM-1 expression in venules of Berkeley mice. Data represent the mean ± standard deviation (SD) (n = 45-57 venules in 6-8 mice per group). (E) Following intravital microscopy, plasma NO_x levels were measured as described in supplemental Methods. Data represent the mean ± SD (n = 6-8 mice per group). (F-I) Berkeley mice were treated with saline, HU, Akti XII, or both HU and Akti XII as described in supplemental Methods. Neutrophils and platelets were isolated and stimulated with fMLF and thrombin, respectively. Immunoblotting was performed by using equal amounts of protein (50 μg) followed by densitometry using Image J software. Representative blots (F,H) and quantitation of AKT2 phosphorylation after normalization to total AKT expression in neutrophils (G) and platelets (I). Data represent the mean ± standard error of the mean (n = 6 mice per group). *P < .05, **P < .01, and ***P < .001 vs vehicle control; ANOVA and Dunnett’s test. #P < .05 and ##P < .01 between two groups; Student t test.