Table 1.
Lipid profiles of the plasma from 32 sickle cell disease (SCD) patients and 28 healthy normal donors.
Table 2.
Levels of inflammatory markers in plasma in 32 sickle cell disease (SCD) patients and 28 healthy normal donors.
Table 3.
Levels of hemoglobin (Hb), haptoglobin (Hp) and hemopexin (Hx) associated with HDL in 32 sickle cell disease (SCD) patients and 28 healthy normal donors (Control).
Fig 1.
Plasma hemoglobin (Hb), haptoglobin (Hp) and hemopexin (Hx) in HDL are linearly correlated with lipid hydroperoxide (LOOH) and HDL inflammatory index (HII). “[ApoA-1]” means “in ApoA-1 particles”.
Results in SCD patients (n = 32): ApoA-1, Hb, Hp and Hx levels in plasma (A1–-A4, respectively) or ApoA-1 particles, Hb, Hp, and Hx associated with ApoA-1 (B1–B4, respectively) vs. LOOH content in ApoA-1 particles. ApoA-1, Hb, Hp and Hx levels in plasma (C1–C4) or those associated with ApoA-1 (D1–D4) vs. HII measured by monocyte chemotaxis activity (MCA). Linear regression was performed individually and p values and R2 values are shown in the figures. (NS = not significant).
Fig 2.
Hemoglobin (Hb) concentrations associated with HDL were positively correlated with heme, reactive oxygen species (ROS) level and IL-6/IL-10.
Relative Hb levels in the plasma (A1–A4 and C1–C4). Relative Hb [ApoA-1] = Hb associated with ApoA-1 particles from HDL (B1–B4 and D1–D4). Fig A1-A4 and B1-B4 present the data from the SCD group (n = 32) and Fig C1-C4 and D1-D4 are those from healthy donors (n = 28). Linear regression was performed individually and and p values and R2 values are shown in the Figs. (NS = not significant).
Fig 3.
Hemoglobin (Hb), haptoglobin (Hp) and hemopexin (Hx) content is increased in ApoA-1 particles of HDL in SCD mice vs. wild-type (WT) mice.
(A,B) ApoA-1 and Hb levels, respectively, in the plasma of SCD mice (n = 8) and WT mice (n = 8). (C,D) ApoA-1 and Hb levels, respectively, in HDL measured by sandwich ELISA. (E) Immunoprecipitation was used to obtain the ApoA-1 particles. We measured the level of these particles by ELISA and found fewer ApoA-1 particles in the HDL of SCD mice compared to WT. (F) Hb, Hp and Hx levels associated with ApoA-1 of HDL in the two groups. The association of ApoA-1 with Hb/Hp/Hx was enhanced because ApoA-1 was reduced in the HDL of the SCD sample.
Fig 4.
The association of Hb/Hp/Hx complexes with HDL in SCD mice.
(A) Cell-free hemoglobin (cf-Hb) or associated Hb was elevated in the HDL or plasma of SCD mice compared to wild-type (WT). (B) The curve in the Octet system shows HDL had its association for Hp (upper) and Hx (lower) on the basis of HDL/ApoA-1 inside HDL. Hemoglobin/Haptoglobin/ Hemopexin = loading biotin-labelled Antibody of Hemoglobin/Haptoglobin/Hemopexin. It suggests that Hb/Hp/Hx association has a strong relationship with ApoA-1 particles in HDL. (C) The result of immunoprecipitation and immunoblot analysis show that Hb, Hp and Hx associated with ApoA-1 was increased under conditions in which ApoA-1 was at the same level in both groups. * p<0.05; *** p<0.001; **** p<0.0001.
Fig 5.
Hemopexin (Hp) is the protein necessary to promote the association of hemoglobin (Hb) with HDL.
(A) HDL cholesterol (A1) and associated ApoA-1 (A2) concentrations in C57BL/6J wild-type (WT), Hp−/− or Hx−/− mice with or without SCD (n = 8 each). (B,C) Hb, Hp and hemopexin (Hx) in plasma (B1–B3, respectively), determined by direct ELISA, and in ApoA-1 (C1–C3, respectively), determined by sandwich ELISA. ANOVA were performed for statistical analysis. * = p<0.05, ** = p<0.01; # = p<0.05, ## = p<0.01. The symbol “#” shown statistical difference between SCD and control in same genotype.
Table 4.
Plasma lipoprotein levels (mg/dL) in wild-type (WT), Hp−/− and Hx−/− mice with and without sickle cell disease (SCD; n = 8 each group).
Fig 6.
Hemopexin (Hp) is required to increase levels of p-HDL in SCD.
The lipoprotein fractions were isolated by HPLC assay to detect p-HDL (accumulation of ROS) in wild-type (WT), Hp−/− and Hx−/− mice. A significantly lower level of p-HDL is seen in SCD mice with Hp knock-out, compared with other SCD groups, confirming that Hp is the protein necessary for the proinflammatory properties of HDL. (Other = other fractions of lipoprotein; RFU, relative fluorescence units).
Fig 7.
Hb/Hp/Hx complexes play a role in the proinflammatory functions of HDL.
HDL was separated from wild-type (WT), Hp−/− and Hx−/− mice either with or without SCD (n = 8 each). (A) The HDL inflammatory index (HII) was measured by monocyte chemotaxis activity (MCA). (B) Proinflammatory HDL (p-HDL) level was measured by plate assay after HDL was isolated by HPLC as previously described. HII<1 and lower p-HDL level were only found in the Hp knock-out SCD mice. Comparisons were performed by ANOVA (* = p<0.05; # = p<0.01).
Fig 8.
D-4F inhibits the proinflammatory properties of HDL and increases the Hb isolated from HDL.
Three groups of mice (n = 8 each) were compared: control, mice with SCD and mice with SCD that were treated with D-4F (10μg/day for 4 weeks) (A) D-4F, an ApoA-1 mimetic peptide, increased the anti-inflammatory properties of HDL (HII<1). (B) Results of analysis with the Octet system to detect interactions among proteins. Biotin-labeled HDL from SCD mice treated with D-4F or placebo was loaded and detected with anti-ApoA-1 and anti-hemoglobin antibodies. D-4F decreased the Hb level in HDL by making Hb dissociate from HDL. (C) Levels of hemoglobin (Hb), haptoglobin (Hp) and hemopexin (Hx) associated with HDL were reduced by the administration of D-4F (* = p<0.005).