Macrophages of genetically characterized familial hypercholesterolaemia patients show up‐regulation of LDL‐receptor‐related proteins

Abstract

Familial hypercholesterolaemia (FH) is a major risk for premature coronary heart disease due to severe long‐life exposure to high LDL levels. Accumulation of LDL in the vascular wall triggers atherosclerosis with activation of the innate immunity system. Here, we have investigated (i) gene expression of LDLR and LRPs in peripheral blood cells (PBLs) and in differentiated macrophages of young FH‐patients; and (ii) whether macrophage from FH patients have a differential response when exposed to high levels of atherogenic LDL. PBLs in young heterozygous genetically characterized FH patients have higher expression of LRP5 and LRP6 than age‐matched healthy controls or patients with secondary hypercholesterolaemia. LRP1 levels were similar among groups. In monocyte‐derived macrophages (MACs), LRP5 and LRP1 transcript levels did not differ between FHs and controls in resting conditions, but when exposed to agLDL, FH‐MAC showed a highly significant up‐regulation of LRP5, while LRP1 was unaffected. PBL and MAC cells from FH patients had significantly lower LDLR expression than control cells, independently of the lipid‐lowering therapy. Furthermore, exposure of FH‐MAC to agLDL resulted in a reduced expression of CD163, scavenger receptor with anti‐inflammatory and atheroprotective properties. In summary, our results show for first time that LRPs, active lipid‐internalizing receptors, are up‐regulated in innate immunity cells of young FH patients that have functional LDLR mutations. Additionally, their reduced CD163 expression indicates less atheroprotection. Both mechanisms may play a synergic effect on the onset of premature atherosclerosis in FH patients.

miR‐505‐3p controls chemokine receptor up‐regulation in macrophages: role in familial hypercholesterolemia

“…In a previous study, we demonstrated that FH‐MAC have lower anti‐inflammatory activity and less resistance to LDL‐mediated oxidative stress, but that they retain their capacity to internalize LDL via receptors of the LDL receptor family, such as LRP5 and LRP6 (15). Here, by using quantitative real‐time PCR in FH‐MACs and non‐FH control participants (co‐MACs) of the SAFEHEART cohort, we found that miR‐505–3p expression is decreased in FH‐MACs independently of agLDL loading (FH‐MAC vs. co‐MAC: baseline, 1.33‐fold decrease, P = 0.005; lipid‐loaded cells: 1.38‐fold decrease, P = 0.002; Fig .…”

“…PBMNs—obtained by using the Vacutainer CPT System (samples from the SAFEHEART cohort) or isolated from leukocyte‐rich buffy coats from heathy donors (Barcelona Blood Bank, Barcelona, Spain) by centrifugation on a Ficoll‐Hypaque gradient (GE Healthcare, Waukesha, WI, USA) (15)—were suspended in RPMI 1640 GlutaMax culture medium that was supplemented with 100 U/ml penicillin/streptomycin, 2 mM l ‐glutamine, and 10 mM Hepes buffer (Thermo Fisher Scientific, Waltham, MA, USA) and containing 10% (v/v) human serum (HS; blood group AB; Lonza, Basel, Switzerland). On the basis of the cell counts of each individual sample, PBMNs were platted on 6‐well polystyrene plates (Becton Dickinson) to obtain a cellular density of 2 × 10 6 monocytes/ml, as previously described.…”

“…On the basis of the cell counts of each individual sample, PBMNs were platted on 6‐well polystyrene plates (Becton Dickinson) to obtain a cellular density of 2 × 10 6 monocytes/ml, as previously described. After incubation for 24 h, nonadherent cells were removed by gently washing and adherent monocytes were allowed to differentiate into MACs for 7 d (5, 15). This procedure generates homogenous cell cultures, with low variability between cell preparations.…”

“…Cellular localization of RUNX1 was proven by using a rabbit polyclonal anti‐RUNX1 Ab (1:50 dilution; Abcam, Cambridge, United Kingdom) in MACs that were fixed with 4% paraformaldehyde (15). Analysis was performed in an inverted confocal microscope (Leica TCS SP2‐AOBS; Leica, Wetzlar, Germany) with the HCX PL APO × 63/1.2W Corr/0.17CS objective.…”

“…Studies that have used fluorodeoxyglucose positron emission tomography imaging have provided evidence that patients with FH have noticeably increased arterial wall inflammation compared with healthy controls (14). Furthermore, we have recently demonstrated that MACs derived from mononuclear cells that were obtained from patients with FH (FH-MACs), although presenting with low or null classic LDL receptor expression, retain the LDL internalization function via receptors of the LDL receptor-related protein family and can evolve into lipid-loaded cells (15). In addition, FH monocytes display a greater response to atherogenic stimulus than do innate immunity cells that originate from non-FH individuals (15).…”