Nrf2 Activation With CDDO-Methyl Promotes Beneficial and Deleterious Clinical Effects in Transgenic Mice With Sickle Cell Anemia

Abstract

Activation of Nrf2, a major transcription factor that drives the antioxidant defense system, is an emerging therapeutic strategy in Sickle Cell Disease (SCD). In this study, transgenic Sickle Cell Anemia mice (SS mice) treated with CDDO-Methyl (CDDO-Me), a potent Nrf2 activator, showed reduced progression of hemolytic anemia with aging, but surprisingly also showed reduced endothelial function. Pulmonary vessels isolated from SS mice treated for 4 months with CDDO-Me displayed a diminished response to nitric oxide (NO)-induced vasodilation compared to littermates given vehicle. It is unclear what molecular mechanism underly the vascular impairment, however, our in vitro assays revealed that CDDO-Me induced the expression of the endothelin receptor (ET_A and ET_B) in vascular smooth muscle cells. Endothelin signaling is associated with increased vascular tone and vasoconstriction. This study underscores the importance of pre-clinical benefit-risk investigations of Nrf2 activating compounds which may be used to treat patients with SCD.

Keywords: CDDO-methyl (CDDO-Me); dimethyl fumarate (DMF); endothelin receptor (ET); nuclear factor erythroid 2-like 2 (Nrf2); sickle cell anemia.

FIGURE 1

CDDO-Me is a potent activator of endothelial and erythroid NRF2 in vitro and improves hemolytic anemia with aging in SS mice. (A,B) Immunoblots were used to assess drug specific temporal differences in Nrf2 nuclear translocation in HMVEC and K562 cells. Quantification was done using NIH Image J processing with aribitrary units shown for the y-axis. Cells were cultured and treated with (A) CDDO-Me (100 nM) or vehicle, and (B) DMF (20 μM) or vehicle for 6, 12, and 24 h. Subsequently, nuclear extracts were isolated and probed for NRF2 and the loading control mutS homolog 2 (MSH2). (C–F) SS mice were randomly chosen at 4–6 weeks of age to be treated for 16 weeks with CDDO-Me (20 μmol/kg) or vehicle (DMSO) by oral gavage. (C) Schematic illustrating the experimental dosing regimen in SS mice. To assess drug induced changes in hematological chemistry with aging, mice were phlebotomized with retro-orbital bleeds to compare baseline (Bl) values to values from the same mouse after 16 weeks (16 weeks). For total hemoglobin quantification, we used a portable Co-Oximeter. Pairwise changes in (D) absolute total hemoglobin values and (E) total hemoglobin percent change are shown (n = 6 CDDO-Me group, n = 10 vehicle group, ^∗ p < 0.05, Student’s t-test). (F) For reticulocyte quantification we used a Heska HemaTrue analyzer to compare baseline and 16-week values for SS mice treated with CDDO-Me (20 mmol/kg) or vehicle (DMSO) by oral gavage. Pairwise changes in absolute reticulocyte percentage values are shown (n = 6 CDDO-Me group, n = 10 vehicle group, ^∗ p < 0.05, Student’s t-test).

FIGURE 2

CDDO-Me worsens the vasodilatory response of pulmonary artery vessels from SS mice. CDDO-Me and DMF induced the genetic expression of the endothelin receptor A (ET_A) and B (ET_B) in rat smooth muscle cells. SS mice were randomly chosen at 4–6 weeks of age to be treated for 16 weeks with CDDO-Me (20 μmol/kg) or vehicle (DMSO) by oral gavage as described in Figure 1. (A) For nitric oxide (NO) response analysis, we used wire myography which quantified changes in vessel vasodilation and vasoconstriction. Lungs of SS mice treated for 16 weeks with CDDO-Me or vehicle were contracted with a continuous dose response of Endothelin-1 (100 pM-10 nM). After reaching plateau (max constriction), endothelial function was examined with a continuous dose response curve of acetylcholine (10 nM–100 mM) to produce relaxation (n = 5 for each group, ^∗∗∗ p < 0.001, 2-way ANOVA).(B) We used ELISA assays for soluble vCAM to assess the effect CDDO-Me had on inflammation. Plasma samples were isolated from whole blood collected at baseline and after 16 weeks of treatment (vehicle n = 8, CDDO-Me n = 8; ^* p < 0.05, ^∗∗ p < 0.01, ns, not significant, Student’s t-test). Real-time PCR was used to analyze and compare the drug-induced temporal expression of the endothelin receptor A (ET_A) and B (ET_B). (C,D) We cultured rat smooth muscle cells and treated them with CDDO-Me (50,100, and 200 nM), DMF (10, 20, and 40 μM) or vehicle for 6–24 h. RNA was isolated for cDNA synthesis and analyzed using real-time PCR (n = 6 biological replicates; ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, NS, not significant).