Cholesterol efflux capacity of large, small and total HDL particles is unaltered by atorvastatin in patients with type 2 diabetes.

BACKGROUND AND AIMS: Research on the biologic activities of HDL, such as cholesterol efflux capacity and HDL composition, has allowed the understanding of the effect of interventions directed to improve cardiovascular risk. Previously, statin therapy has shown conflicting results about its effects on cholesterol efflux capacity of HDL; the underlying mechanisms are unclear but studies with positive effects are associated with an increase of HDL-cholesterol levels. We investigated if 10 weeks of atorvastatin therapy changes HDL efflux capacity and the chemical composition of its subpopulations. METHODS: In a before-after design basis, HDL-cholesterol levels, chemical composition and cholesterol efflux capacity from HDL subpopulations isolated by isophynic ultracentrifugation were assessed in plasma samples from 60 patients with type 2 diabetes mellito (T2DM) at baseline and after 10 weeks of treatment with 20 mg atorvastatin. Cholesterol efflux was measured from human THP-1 cells using large, light HDL2b and small, dense 3c subpopulations as well as total HDL as acceptors. Changes of cholesterol efflux and chemical composition of HDL after treatment were analyzed. Correlations among variables potentially involved in cholesterol efflux were evaluated. RESULTS: A significant decrease of 4% in HDL-cholesterol levels was observed from 47 (42-54) to 45 (39-56) mg/dL, p = 0.02. Cholesterol efflux from total-HDL and HDL2b and 3c subfractions was maintained unchanged after treatment. The total mass of HDL remained unaffected, except for the HDL3a subpopulation accounted for by a significant increase in total protein content. No significant correlations for variables previously known to be associated with cholesterol efflux were found in our study. CONCLUSIONS: Short therapy of 10 weeks with 20 mg of atorvastatin does not modify the cholesterol efflux capacity neither the total mass of HDL2b, HDL3c and total HDL. The discrepancy with previous reports may be due to the selective effects among different classes of statins or differences in the approaches to measure cellular cholesterol efflux.

Small dense HDLs display potent vasorelaxing activity, reflecting their elevated content of sphingosine-1-phosphate.

The functional heterogeneity of HDL is attributed to its diverse bioactive components. We evaluated whether the vasodilatory effects of HDL differed across HDL subpopulations, reflecting their distinct molecular composition. The capacity of five major HDL subfractions to counteract the inhibitory effects of oxidized LDL on acetylcholine-induced vasodilation was tested in a rabbit aortic rings model. NO production, an essential pathway in endothelium-dependent vasorelaxation, was studied in simian vacuolating virus 40-transformed murine endothelial cells (SVECs). Small dense HDL3 subfractions displayed potent vasorelaxing activity (up to +31% vs. baseline, P < 0.05); in contrast, large light HDL2 did not induce aortic-ring relaxation when compared on a total protein basis. HDL3 particles were enriched with sphingosine-1-phosphate (S1P) (up to 3-fold vs. HDL2), with the highest content in HDL3b and -3c that concomitantly revealed the strongest vasorelaxing properties. NO generation was enhanced by HDL3c in SVECs (1.5-fold, P < 0.01), a phenomenon that was blocked by the S1P receptor antagonist, VPC 23019. S1P-enriched reconstituted HDL (rHDL) was a 1.8-fold (P < 0.01) more potent vasorelaxant than control rHDL in aortic rings. Small dense HDL3 particles displayed potent protective effects against oxidative stress-associated endothelium dysfunction, potentially reflecting their elevated content of S1P that might facilitate interaction with S1P receptors and ensuing NO generation.

Paradoxical coronary artery disease in humans with hyperalphalipoproteinemia is associated with distinct differences in the high-density lipoprotein phosphosphingolipidome.

BACKGROUND: Plasma high-density lipoprotein cholesterol (HDL-C) levels are inversely associated with risk of coronary artery disease (CAD) in epidemiologic studies. Despite this, the directionality of this relationship and the underlying biology behind it remain to be firmly established, especially at the extremes of HDL-C levels. OBJECTIVE: We investigated differences in the HDL phosphosphingolipidome in a rare population of subjects with premature CAD despite high HDL-C levels to gain insight into the association between the HDL lipidome and CAD disease status in this unusual phenotype. We sought to assess differences in HDL composition that are associated with CAD in subjects with HDL-C >90th percentile. We predicted that quantitative lipidomic analysis of HDL particles would reveal novel differences between CAD patients and healthy subjects with matched HDL-C levels. METHODS: We collected plasma samples from 25 subjects with HDL-C >90th percentile and clinically manifest CAD and healthy controls with HDL-C >90th percentile and without self-reported CAD. More than 140 individual HDL phospholipid and sphingolipid species were analyzed by LC/MS/MS. RESULTS: Significant reductions in HDL phosphatidylcholine (-2.41%, Q value = 0.025) and phosphatidylinositol (-10.7%, Q value = 0.047) content, as well as elevated sphingomyelin (+10.0%, Q value = 0.025) content, and sphingomyelin/phosphatidylcholine ratio (+12.8%, P value = .005) were associated with CAD status in subjects with high HDL-C. CONCLUSIONS: These differences may lay the groundwork for further analysis of the relationship between the HDL lipidome and disease states, as well as for the development of biomarkers of CAD status and HDL function.

Oxidative stress, HDL functionality and effects of intravenous iron administration in women with iron deficiency anemia.

BACKGROUND AND AIMS: Iron deficiency anemia (IDA) affects around 20-30% of adults worldwide. An association between IDA and cardiovascular disease (CVD) has been reported. Oxidative stress, inflammation and low concentration of high-density lipoproteins (HDL) were implicated on endothelial dysfunction and CVD in IDA. We studied the effects of iron deficiency and of an intravenous iron administration on oxidative stress and HDL characteristics in IDA women. METHODS: Two studies in IDA women are presented: a case-control study, including 18 patients and 18 age-matched healthy women, and a follow-up study 72hr after the administration of intravenous iron (n = 16). Lipids, malondialdehyde, cholesteryl ester transfer protein (CETP), paraoxonase-1 (PON-1) and HDL chemical composition and functionality (cholesterol efflux and antioxidative activity) were measured. Cell cholesterol efflux from iron-deficient macrophages to a reference HDL was also evaluated. RESULTS: IDA patients showed higher triglycerides and CETP activity and lower HDL-C than controls (all p < 0.001). HDL particles from IDA patients showed higher triglyceride content (+30%,p < 0.05) and lower antioxidative capacity (-23%,p < 0.05). Although HDL-mediated cholesterol efflux was similar between the patients and controls, iron deficiency provoked a significant reduction in macrophage cholesterol efflux (-25%,p < 0.05). Arylesterase activity of PON-1 was significantly lower in IDA patients than controls (-16%,p < 0.05). The intravenous administration of iron was associated with a decrease in malondialdehyde levels and an increase in arylesterase activity of PON-1 (-22% and +18%, respectively, p < 0.05). CONCLUSION: IDA is associated with oxidative stress and functionally deficient HDL particles. It remains to be determined if such alterations suffice to impair endothelial function in IDA.

D25V apolipoprotein C-III variant causes dominant hereditary systemic amyloidosis and confers cardiovascular protective lipoprotein profile.

Apolipoprotein C-III deficiency provides cardiovascular protection, but apolipoprotein C-III is not known to be associated with human amyloidosis. Here we report a form of amyloidosis characterized by renal insufficiency caused by a new apolipoprotein C-III variant, D25V. Despite their uremic state, the D25V-carriers exhibit low triglyceride (TG) and apolipoprotein C-III levels, and low very-low-density lipoprotein (VLDL)/high high-density lipoprotein (HDL) profile. Amyloid fibrils comprise the D25V-variant only, showing that wild-type apolipoprotein C-III does not contribute to amyloid deposition in vivo. The mutation profoundly impacts helical structure stability of D25V-variant, which is remarkably fibrillogenic under physiological conditions in vitro producing typical amyloid fibrils in its lipid-free form. D25V apolipoprotein C-III is a new human amyloidogenic protein and the first conferring cardioprotection even in the unfavourable context of renal failure, extending the evidence for an important cardiovascular protective role of apolipoprotein C-III deficiency. Thus, fibrate therapy, which reduces hepatic APOC3 transcription, may delay amyloid deposition in affected patients.

Defective functionality of small, dense HDL3 subpopulations in ST segment elevation myocardial infarction: Relevance of enrichment in lysophosphatidylcholine, phosphatidic acid and serum amyloid A.

BACKGROUND: Low plasma levels of high-density lipoprotein-cholesterol (HDL-C) are typical of acute myocardial infarction (MI) and predict risk of recurrent cardiovascular events. The potential relationships between modifications in the molecular composition and the functionality of HDL subpopulations in acute MI however remain indeterminate. METHODS AND RESULTS: ST segment elevation MI (STEMI) patients were recruited within 24h after diagnosis (n=16) and featured low HDL-C (-31%, p<0.05) and acute-phase inflammation (determined as marked elevations in C-reactive protein, serum amyloid A (SAA) and interleukin-6) as compared to age- and sex-matched controls (n=10). STEMI plasma HDL and its subpopulations (HDL2b, 2a, 3a, 3b, 3c) displayed attenuated cholesterol efflux capacity from THP-1 cells (up to -32%, p<0.01, on a unit phospholipid mass basis) vs. CONTROLS: Plasma HDL and small, dense HDL3b and 3c subpopulations from STEMI patients exhibited reduced anti-oxidative activity (up to -68%, p<0.05, on a unit HDL mass basis). HDL subpopulations in STEMI were enriched in two proinflammatory bioactive lipids, lysophosphatidylcholine (up to 3.0-fold, p<0.05) and phosphatidic acid (up to 8.4-fold, p<0.05), depleted in apolipoprotein A-I (up to -23%, p<0.05) and enriched in SAA (up to +10.2-fold, p<0.05); such changes were most marked in the HDL3b subfraction. In vitro HDL enrichment in both lysophosphatidylcholine and phosphatidic acid exerted deleterious effects on HDL functionality. CONCLUSIONS: In the early phase of STEMI, HDL particle subpopulations display marked, concomitant alterations in both lipidome and proteome which are implicated in impaired HDL functionality. Such modifications may act synergistically to confer novel deleterious biological activities to STEMI HDL. SIGNIFICANCE: Our present data highlight complex changes in the molecular composition and functionality of HDL particle subpopulations in the acute phase of STEMI, and for the first time, reveal that concomitant modifications in both the lipidome and proteome contribute to functional deficiencies in cholesterol efflux and antioxidative activities of HDL particles. These findings may provide new biomarkers and new insights in therapeutic strategy to reduce cardiovascular risk in this clinical setting where such net deficiency in HDL function, multiplied by low circulating HDL concentrations, can be expected to contribute to accelerated atherogenesis.

Metabolic alterations, HFE gene mutations and atherogenic lipoprotein modifications in patients with primary iron overload.

Iron overload (IO) has been associated with glucose metabolism alterations and increased risk of cardiovascular disease (CVD). Primary IO is associated with mutations in the HFE gene. To which extent HFE gene mutations and metabolic alterations contribute to the presence of atherogenic lipoprotein modifications in primary IO remains undetermined. The present study aimed to assess small, dense low-density lipoprotein (LDL) levels, chemical composition of LDL and high-density lipoprotein (HDL) particles, and HDL functionality in IO patients. Eighteen male patients with primary IO and 16 sex- and age-matched controls were recruited. HFE mutations (C282Y, H63D and S65C), measures of insulin sensitivity and secretion (calculated from the oral glucose tolerance test), chemical composition and distribution profile of LDL and HDL subfractions (isolated by gradient density ultracentrifugation) and HDL functionality (as cholesterol efflux and antioxidative activity) were studied. IO patients compared with controls exhibited insulin resistance (HOMA-IR (homoeostasis model assessment-estimated insulin resistance): +93%, P< 0.001). Metabolic profiles differed across HFE genotypes. C282Y homozygotes (n=7) presented a reduced ß-cell function and insulin secretion compared with non-C282Y patients (n=11) (-58% and -73%, respectively, P< 0.05). In addition, C282Y homozygotes featured a predominance of large, buoyant LDL particles (C282Y: 43±5; non-C282Y: 25±8; controls: 32±7%; P< 0.001), whereas non-C282Y patients presented higher amounts of small, dense LDL (C282Y: 23±5; non-C282Y: 39±10; controls: 26±4%; P< 0.01). HDL particles were altered in C282Y homozygotes. However, HDL functionality was conserved. In conclusion, metabolic alterations and HFE gene mutations are involved in the presence of atherogenic lipoprotein modifications in primary IO. To what extent such alterations could account for an increase in CVD risk remains to be determined.

Altered lipidome and antioxidative activity of small, dense HDL in normolipidemic rheumatoid arthritis: relevance of inflammation.

OBJECTIVE: High-density lipoprotein (HDL) particles exert potent antiatherogenic activities, including antioxidative actions, which are relevant to attenuation of atherosclerosis progression. Such activities are enriched in small, dense HDL and can be compromised under conditions of chronic inflammation like rheumatoid arthritis (RA). However, structure-function relationships of HDL largely remain indeterminate. METHODS: The relationships between HDL structure and function were evaluated in normolipidemic patients with active RA (DAS28 > 3.2; n = 12) and in normolipidemic age-matched controls (n = 10). Small, dense HDL3b and 3c particles were isolated from plasma or serum by density gradient ultracentrifugation and their physicochemical characteristics, lipidome (by LC/MS/MS) and antioxidative function (as protection of normolipidemic LDL from free radical-induced oxidation) were evaluated. RESULTS: As expected, active RA patients featured significantly elevated plasma levels of high-sensitivity C-reactive protein (hsCRP; p < 0.001) and serum amyloid A (SAA; p < 0.01) relative to controls. Antioxidative activity and weight % chemical composition of small, dense HDL did not differ between RA patients and controls (p > 0.05), whereas HDL phosphosphingolipidome was significantly altered in RA. Subgroup analyses revealed that RA patients featuring high levels of inflammation (hsCRP>10 mg/l) possessed small, dense HDL with reduced antioxidative activities (p < 0.01). Furthermore, antioxidative activity of HDL was inversely correlated with plasma hsCRP (p < 0.01). CONCLUSIONS: These data revealed that (i) despite normolipidemic state, the lipidome of small, dense HDL was altered in RA and (ii) high levels of inflammation can be responsible for the functional deficiency of small, dense HDL in RA.

Defective functionality of HDL particles in familial apoA-I deficiency: relevance of alterations in HDL lipidome and proteome.

To evaluate functional and compositional properties of HDL in subjects from a kindred of genetic apoA-I deficiency, two homozygotes and six heterozygotes, with a nonsense mutation at APOA1 codon -2, Q[-2]X, were recruited together with age- and sex-matched healthy controls (n = 11). Homozygotes displayed undetectable plasma levels of apoA-I and reduced levels of HDL-cholesterol (HDL-C) and apoC-III (5.4% and 42.6% of controls, respectively). Heterozygotes displayed low HDL-C (21 ± 9 mg/dl), low apoA-I (79 ± 24 mg/dl), normal LDL-cholesterol (132 ± 25 mg/dl), and elevated TG (130 ± 45 mg/dl) levels. Cholesterol efflux capacity of ultracentrifugally isolated HDL subpopulations was reduced (up to -25%, P < 0.01, on a glycerophospholipid [GP] basis) in heterozygotes versus controls. Small, dense HDL3 and total HDL from heterozygotes exhibited diminished antioxidative activity (up to -48%, P < 0.001 on a total mass basis) versus controls. HDL subpopulations from both homozygotes and heterozygotes displayed altered chemical composition, with depletion in apoA-I, GP, and cholesteryl ester; enrichment in apoA-II, free cholesterol, and TG; and altered phosphosphingolipidome. The defective atheroprotective activities of HDL were correlated with altered lipid and apo composition. These data reveal that atheroprotective activities of HDL particles are impaired in homozygous and heterozygous apoA-I deficiency and are intimately related to marked alterations in protein and lipid composition.