Seth Thacker

HDL-transferred microRNA-223 regulates ICAM-1 expression in endothelial cells.

High-density lipoproteins (HDL) have many biological functions, including reducing endothelial activation and adhesion molecule expression. We recently reported that HDL transport and deliver functional microRNAs (miRNA). Here we show that HDL suppresses expression of intercellular adhesion molecule 1 (ICAM-1) through the transfer of miR-223 to endothelial cells. After incubation of endothelial cells with HDL, mature miR-223 levels are significantly increased in endothelial cells and decreased on HDL. However, miR-223 is not transcribed in endothelial cells and is not increased in cells treated with HDL from miR-223(-/-) mice. HDL inhibit ICAM-1 protein levels, but not in cells pretreated with miR-223 inhibitors. ICAM-1 is a direct target of HDL-transferred miR-223 and this is the first example of an extracellular miRNA regulating gene expression in cells where it is not transcribed. Collectively, we demonstrate that HDLs anti-inflammatory properties are conferred, in part, through HDL-miR-223 delivery and translational repression of ICAM-1 in endothelial cells.

Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(−/−)] mice, which have a secondary defect in cholesterol esterification. Scarab(−/−)×LCAT-null [Lcat(−/−)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(−/−)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(−/−)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(−/−)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(−/−) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.

Scavenger Receptor BI and High-Density Lipoprotein Regulate Thymocyte Apoptosis in Sepsis

Objective—Thymocyte apoptosis is a major event in sepsis; however, how this process is regulated remains poorly understood. Approach and Results—Septic stress induces glucocorticoids production which triggers thymocyte apoptosis. Here, we used scavenger receptor BI (SR-BI)–null mice, which are completely deficient in inducible glucocorticoids in sepsis, to investigate the regulation of thymocyte apoptosis in sepsis. Cecal ligation and puncture induced profound thymocyte apoptosis in SR-BI+/+ mice, but no thymocyte apoptosis in SR-BI−/− mice because of lack of inducible glucocorticoids. Unexpectedly, supplementation of glucocorticoids only partly restored thymocyte apoptosis in SR-BI−/− mice. We demonstrated that high-density lipoprotein (HDL) is a critical modulator for thymocyte apoptosis. SR-BI+/+ HDL significantly enhanced glucocorticoid-induced thymocyte apoptosis, but SR-BI−/− HDL had no such activity. Further study revealed that SR-BI+/+ HDL modulates glucocorticoid-induced thymocyte apoptosis via promoting glucocorticoid receptor translocation, but SR-BI−/− HDL loses such regulatory activity. To understand why SR-BI−/− HDL loses its regulatory activity, we analyzed HDL cholesterol contents. There was 3-fold enrichment of unesterified cholesterol in SR-BI−/− HDL compared with SR-BI+/+ HDL. Normalization of unesterified cholesterol in SR-BI−/− HDL by probucol administration or lecithin cholesteryl acyltransferase expression restored glucocorticoid-induced thymocyte apoptosis, and incorporating unesterified cholesterol into SR-BI+/+ HDL rendered SR-BI+/+ HDL dysfunctional. Using lckCre-GRfl/fl mice in which thymocytes lack cecal ligation and puncture–induced thymocyte apoptosis, we showed that lckCre-GRfl/fl mice were significantly more susceptible to cecal ligation and puncture–induced septic death than GRfl/fl control mice, suggesting that glucocorticoid-induced thymocyte apoptosis is required for protection against sepsis. Conclusions—The findings in this study reveal a novel regulatory mechanism of thymocyte apoptosis in sepsis by SR-BI and HDL.

Lipoprotein X Causes Renal Disease in LCAT Deficiency

Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat-/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.

High Density Lipoprotein reduces inflammation from cholesterol crystals by inhibiting inflammasome activation

Interleukin‐1β (IL‐1β), a potent pro‐inflammatory cytokine, has been implicated in many diseases, including atherosclerosis. Activation of IL‐1β is controlled by a multi‐protein complex, the inflammasome. The exact initiating event in atherosclerosis is unknown, but recent work has demonstrated that cholesterol crystals (CC) may promote atherosclerosis development by activation of the inflammasome. High‐density lipoprotein (HDL) has consistently been shown to be anti‐atherogenic and to have anti‐inflammatory effects, but its mechanism of action is unclear. We demonstrate here that HDL is able to suppress IL‐1β secretion in response to cholesterol crystals in THP‐1 cells and in human‐monocyte‐derived macrophages. HDL is able to blunt inflammatory monocyte cell recruitment in vivo following intraperitoneal CC injection in mice. HDL appears to modulate inflammasome activation in several ways. It reduces the loss of lysosomal membrane integrity following the phagocytosis of CC, but the major mechanism for the suppression of inflammasome activation by HDL is decreased expression of pro‐IL‐1β and NLRP3, and reducing caspase‐1 activation. In summary, we have described a novel anti‐inflammatory effect of HDL, namely its ability to suppress inflammasome activation by CC by modulating the expression of several key components of the inflammasome.

Addition of aspirin to a fish oil-rich diet decreases inflammation and atherosclerosis in ApoE-null mice.

Aspirin (ASA) is known to alter the production of potent inflammatory lipid mediators, but whether it interacts with omega-3 fatty acids (FAs) from fish oil to affect atherosclerosis has not been determined. The goal was to investigate the impact of a fish oil-enriched diet alone and in combination with ASA on the production of lipid mediators and atherosclerosis. ApoE(-/-) female mice were fed for 13weeks one of the four following diets: omega-3 FA deficient (OD), omega-3 FA rich (OR) (1.8g omega-3 FAs/kg·diet per day), omega-3 FA rich plus ASA (ORA) (0.1g ASA/kg·diet per day) or an omega-3 FA deficient plus ASA (ODA) with supplement levels equivalent to human doses. Plasma lipids, atherosclerosis, markers of inflammation, hepatic gene expression and aortic lipid mediators were determined. Hepatic omega-3 FAs were markedly higher in OR (9.9-fold) and ORA (7-fold) groups. Mice in both OR and ORA groups had 40% less plasma cholesterol in very low-density lipoprotein-cholesterol and low-density lipoprotein fractions, but aortic plaque area formation was only significantly lower in the ORA group (5.5%) compared to the OD group (2.5%). Plasma PCSK9 protein levels were approximately 70% lower in the OR and ORA groups. Proinflammatory aortic lipid mediators were 50%-70% lower in the ODA group than in the OD group and more than 50% lower in the ORA group. In summary, less aortic plaque lesions and aortic proinflammatory lipid mediators were observed in mice on the fish oil diet plus ASA vs. just the fish oil diet.

Synthesis of Cholesterol Analogues Bearing BODIPY Fluorophores by Suzuki or Liebeskind–Srogl Cross-Coupling and Evaluation of Their Potential for Visualization of Cholesterol Pools

We report a synthetic route to BODIPY–cholesterol conjugates in which the key steps were Suzuki or Liebeskind–Srogl cross‐coupling of cholesterol phenyl moieties with structurally diverse BODIPY scaffolds. All conjugates feature single‐bonded and hydrophobic linkages between the fluorophore and sterol that are devoid of heteroatoms. Using HeLa cells, we show that these BODIPY–cholesterol analogues can be used simultaneously with the parent BODIPY–cholesterol for cell imaging and flow cytometry. The BODIPY–cholesterol analogues exhibit similar cellular localization in HeLa cells and show similar cholesterol efflux properties from THP‐1 cells to HDL acceptors. These results demonstrate that the red‐shifted BODIPY–cholesterol analogues behave in a manner similar to unlabeled cholesterol and are useful probes for simultaneous visualization of intracellular cholesterol pools and for monitoring cholesterol efflux from cells to extracellular acceptors.

Dietary α‐cyclodextrin reduces atherosclerosis and modifies gut flora in apolipoprotein E‐deficient mice

SCOPE α-Cyclodextrin (α-CD), a cyclic polymer of glucose, has been shown to lower plasma cholesterol in animals and humans; however, its effect on atherosclerosis has not been previously described. METHODS AND RESULTS apoE-knockout mice were fed either low-fat diet (LFD; 5.2% fat, w/w), or Western high fat diet (21.2% fat) containing either no additions (WD), 1.5% α-CD (WDA); 1.5% β-CD (WDB); or 1.5% oligofructose-enriched inulin (WDI). Although plasma lipids were similar after 11 weeks on the WD vs. WDA diets, aortic atherosclerotic lesions were 65% less in mice on WDA compared to WD (P < 0.05), and similar to mice fed the LFD. No effect on atherosclerosis was observed for the other WD supplemented diets. By RNA-seq analysis of 16S rRNA, addition of α-CD to the WD resulted in significantly decreased cecal bacterial counts in genera Clostridium and Turicibacterium, and significantly increased Dehalobacteriaceae. At family level, Comamonadaceae significantly increased and Peptostreptococcaceae showed a negative trend. Several of these bacterial count changes correlated negatively with % atherosclerotic lesion and were associated with increased cecum weight and decreased plasma cholesterol levels. CONCLUSION Addition of α-CD to the diet of apoE-knockout mice decreases atherosclerosis and is associated with changes in the gut flora.

Scavenger receptor BI and HDL regulate thymocyte apoptosis in sepsis

Objective—Thymocyte apoptosis is a major event in sepsis; however, how this process is regulated remains poorly understood. Approach and Results—Septic stress induces glucocorticoids production which triggers thymocyte apoptosis. Here, we used scavenger receptor BI (SR-BI)–null mice, which are completely deficient in inducible glucocorticoids in sepsis, to investigate the regulation of thymocyte apoptosis in sepsis. Cecal ligation and puncture induced profound thymocyte apoptosis in SR-BI+/+ mice, but no thymocyte apoptosis in SR-BI−/− mice because of lack of inducible glucocorticoids. Unexpectedly, supplementation of glucocorticoids only partly restored thymocyte apoptosis in SR-BI−/− mice. We demonstrated that high-density lipoprotein (HDL) is a critical modulator for thymocyte apoptosis. SR-BI+/+ HDL significantly enhanced glucocorticoid-induced thymocyte apoptosis, but SR-BI−/− HDL had no such activity. Further study revealed that SR-BI+/+ HDL modulates glucocorticoid-induced thymocyte apoptosis via promoting glucocorticoid receptor translocation, but SR-BI−/− HDL loses such regulatory activity. To understand why SR-BI−/− HDL loses its regulatory activity, we analyzed HDL cholesterol contents. There was 3-fold enrichment of unesterified cholesterol in SR-BI−/− HDL compared with SR-BI+/+ HDL. Normalization of unesterified cholesterol in SR-BI−/− HDL by probucol administration or lecithin cholesteryl acyltransferase expression restored glucocorticoid-induced thymocyte apoptosis, and incorporating unesterified cholesterol into SR-BI+/+ HDL rendered SR-BI+/+ HDL dysfunctional. Using lckCre-GRfl/fl mice in which thymocytes lack cecal ligation and puncture–induced thymocyte apoptosis, we showed that lckCre-GRfl/fl mice were significantly more susceptible to cecal ligation and puncture–induced septic death than GRfl/fl control mice, suggesting that glucocorticoid-induced thymocyte apoptosis is required for protection against sepsis. Conclusions—The findings in this study reveal a novel regulatory mechanism of thymocyte apoptosis in sepsis by SR-BI and HDL.