Abstract
Background: Vasoregulation is impaired in human and murine sickle cell disease (SCD). Chronic inflammation and oxidative stress impair vasodilation. High-density lipoprotein (HDL) plays an important role in attenuating inflammatory responses. Previously we showed 4F, an apoA-I mimetic designed to improve HDL function, dramatically restores vasodilation in SCD mice. Here, we examine mechanisms by which D-4F restores vasodilation in SCD mice and in mice made to develop SCD via fetal liver hematopoietic stem cell transplantation (HSCT). Effects of proinflammatory lipids and D-4F were determined in HSCT-SCD- LDL receptor null (Ldlr−/−) mice fed either chow or western diet (WD). The role of HDL was examined in HSCT-SCD-apoA-I null (apoA-I−/−) mice. Finally, the role of eNOS was examined in HSCT-SCD-eNOS deficient (eNOS−/−) mice. Mice were treated with or without D-4F (1mg/kg/d for 6–8 wks).
Results: Total cholesterol concentrations in HSCT-SCD-Ldlr−/− mice fed lab chow were slightly increased compared to transgenic SCD mice (40–60 vs. 90–130 mg/dL, p<0.05) with no change in HDL. Acetylcholine-mediated vasodilation (Ach, 10-7 to 10-4M) in HSCT-SCD-Ldlr−/− mice was impaired compared to untreated non-SCD Ldlr−/− mice (10 vs 43%, p<0.05). D-4F restored eNOS-dependent vasodilation in HSCT-SCD-Ldlr−/− mice to the level in non-SCD Ldlr−/− mice. D-4F did not alter total cholesterol or HDL in HSCT-SCD-Ldlr−/− mice but did decrease proinflammatory HDL (580 vs 380, p<0.05), an index of oxidizability. In contrast to HSCT-SCD-Ldlr−/− mice fed chow diet, HSCT-SCD-Ldlr−/− mice fed WD had little to no ACh vasodilation (0–3%). D-4F increased vasodilation slightly in HSCT-SCD-Ldlr−/− fed WD (~12%). Total cholesterol and HDL increased in response to WD in HSCT-SCD-Ldlr−/− mice (p<0.01). D-4F induced minimal changes in total cholesterol, HDL or proinflammatory HDL in these mice. To examine the role of HDL, we found that vasodilation in HSCT-SCD-apoA-I−/− mice was reduced to ~25% compared to 65% in C57BL/6 mice (p<0.01). D-4F nearly doubled vasodilation to ~43% in HSCT-SCD-apoA-I−/− mice (p<0.05). L-NAME (100μM) blocked vasodilation in all HSCT-SCD-apoA-I−/− mice, indicating vasodilation was mediated exclusively by eNOS. In contrast, when we examined the effect of eNOS deficiency, ACh induced minimal increases in vasodilation (~22%). Dissection of cellular mechanisms mediating vasodilation revealed that a small portion HSCT-SCD-eNOS−/− mice was inhibited by L-NAME (i.e., NOS, ~12%), with none mediated by COX-prostacyclin (0%) and a small portion mediated by cytochrome P450 (~10%). Inhibitor studies revealed D-4F restored vasodilation in HSCT-SCD-eNOS−/− mice to ~52% (p<0.05) by predominately a L-NAME-inhibitable mechanism (NOS = 40%; COX-prostacylcin = 0% and cytochrome P450 = 11%).
Conclusions: D-4F improves eNOS-dependent vasodilation even when hypercholesterolemia is superimposed on SCD. Measurements of proinflammatory HDL reveal D-4F restores vasodilation by protecting HDL against oxidation. Interestingly, D-4F protects vasodilation even in mice that have low levels of apoA-I-deficient HDL. Taken together, these data indicate proinflammatory HDL plays a critical role in mechanisms by which SCD impairs eNOS-dependent vasodilation and D-4F increases vasodilation, at least in part, by decreasing proinflammatory HDL in SCD.
Critical role of C5a in sickle cell disease
