Robert M. Badeau

Depletion of FOXP3+ regulatory T cells promotes hypercholesterolemia and atherosclerosis

Atherosclerosis is a chronic inflammatory disease promoted by hyperlipidemia. Several studies support FOXP3-positive regulatory T cells (Tregs) as inhibitors of atherosclerosis; however, the mechanism underlying this protection remains elusive. To define the role of FOXP3-expressing Tregs in atherosclerosis, we used the DEREG mouse, which expresses the diphtheria toxin (DT) receptor under control of the Treg-specific Foxp3 promoter, allowing for specific ablation of FOXP3+ Tregs. Lethally irradiated, atherosclerosis-prone, low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice received DEREG bone marrow and were injected with DT to eliminate FOXP3(+) Tregs. Depletion of Tregs caused a 2.1-fold increase in atherosclerosis without a concomitant increase in vascular inflammation. These mice also exhibited a 1.7-fold increase in plasma cholesterol and an atherogenic lipoprotein profile with increased levels of VLDL. Clearance of VLDL and chylomicron remnants was hampered, leading to accumulation of cholesterol-rich particles in the circulation. Functional and protein analyses complemented by gene expression array identified reduced protein expression of sortilin-1 in liver and increased plasma enzyme activity of lipoprotein lipase, hepatic lipase, and phospholipid transfer protein as mediators of the altered lipid phenotype. These results demonstrate that FOXP3(+) Tregs inhibit atherosclerosis by modulating lipoprotein metabolism.

The impact of gender and serum estradiol levels on HDL-mediated reverse cholesterol transport

Premenopausal women have a lower incidence of cardiovascular disease compared to men of the same age. Endogenous oestrogens, especially estradiol, presumably protect against atherosclerosis by a variety of mechanisms. Reverse cholesterol transport (RCT) mechanisms also provide protection against this disease. RCT is defined as the removal of cholesterol from peripheral macrophage foam cells, via high‐density lipoproteins (HDL), and cholesterol transportation to the liver for excretion. We have previously shown in a preliminary study that HDL, isolated from premenopausal women, enhanced macrophage cholesterol efflux compared to HDL derived from age‐matched male subjects.

Investigating the membrane orientation and transversal distribution of 17β-estradiol in lipid membranes by solid-state NMR

17beta-Estradiol (E(2)) is a potent estrogen, which modulates many important cellular functions by binding to specific estrogen receptors located in the cell nucleus and also on the plasma membrane. We have studied the membrane interaction of E(2) using a combination of solid-state NMR methods. (2)H NMR results indicate that E(2) does not cause a condensation effect of the surrounding phospholipids, which is contrary to the effects of cholesterol, and only very modest E(2) induced alterations of the membrane structure were detected. (1)H magic-angle spinning NMR showed well resolved signals from E(2) as well as of POPC in the membrane-lipid layer. Two-dimensional NOESY spectra revealed intense cross-peaks between E(2) and the membrane lipids indicating that E(2) is stably inserted into the membrane. The determination of intermolecular cross-relaxation rates revealed that E(2) is broadly distributed in the membrane with a maximum of the E(2) distribution function in the upper chain region of the membrane. We conclude that E(2) is highly dynamic in lipid membranes and may undergo rotations as it exhibits two polar hydroxyl groups on either side of the molecule.

Systemic metabolic markers and myocardial glucose uptake in type 2 diabetic and coronary artery disease patients treated for 16 weeks with rosiglitazone, a PPARγ agonist

Abstract Introduction. Treatment with rosiglitazone, a peroxisome proliferator-activated receptor-γ agonist, in type 2 diabetic mellitus (T2DM) patients is under scrutiny because it affects adversely cardiovascular outcomes. In T2DM patients, with existing coronary heart disease, short-term treatment with rosiglitazone increases myocardial glucose uptake (MGU). Serum metabolic and lipoprotein subclass changes, which may be associated with this rosiglitazone-induced improvement, are unknown. Methods. Patients with both T2DM and coronary heart disease were separated into placebo (n = 26) and treatment (rosiglitazone 4–8 mg; n = 25) groups. After 16 weeks of treatment, serum NMR metabolomics was used to measure circulating low-molecular-weight metabolites and lipoprotein subclasses and lipids that are associated with T2DM before and after the treatment. Significant metabolic measure changes after rosiglitazone treatment were correlated to MGU values assessed with [18F]fluorodeoxyglucose positron emission tomography. Results. Compared to placebo, the treatment significantly increased circulating glutamine and decreased lactate concentrations. Circulating lactate concentrations showed a significant inverse association with MGU after rosiglitazone treatment. Conclusion. In T2DM patients with existing coronary heart disease, short-term rosiglitazone treatment caused minor improvements in metabolism: serum lactate and glutamine concentrations changed, reflecting improvements in insulin sensitivity, and circulating lactate concentrations inversely correlated to increases in myocardial glucose uptake.

Macrophage cholesterol efflux to plasma and HDL in subjects with low and high homocysteine levels: A FIELD substudy

OBJECTIVES Increases of homocysteine (Hcy) by fenofibrate correlated inversely to changes in HDL-C and apoA-I in the FIELD study. This finding raised the question whether high Hcy may influence HDL function and counteract benefits of fenofibrate on cardiovascular outcomes. In a subset of the FIELD study we investigated whether fenofibrate therapy or high Hcy, separately or in concert, modulate: (1) ability of plasma or HDL to facilitate cholesterol efflux from THP-1 foam cells; (2) plasma potential to generate preβ-HDL; (3) plasma phospholipid transfer protein (PLTP) activity, serum PON-1 mass and activity, HDL particle size and distribution. METHODS We selected 33 subjects in the FIELD fenofibrate arm according to quartiles of Hcy at 5th year: 17 subjects were in the lowest (Low Hcy group) and 16 subjects were in the highest quartile (High Hcy group). In addition, 14 subjects allocated to placebo were matched by close-out Hcy levels to Low Hcy group. This design allowed us to examine the effects of both fenofibrate (comparison between placebo vs Low Hcy groups) and Hcy (comparison between close-out Low and High Hcy groups) on plasma and HDL ability to facilitate cellular cholesterol removal in the efflux assay in vitro using THP-1 foam cells. RESULTS Hcy levels were 13.3±0.7 μmol/L (placebo), 13.2±2 μmol/L (Low Hcy) and 27.4±6.5 μmol/L (High Hcy). Cholesterol efflux values to HDL and plasma, percentage of plasma preβ-HDL, PLTP activity, serum PON-1 mass and HDL particle size and distribution were similar in both fenofibrate groups and comparable to those of the placebo group. CONCLUSIONS In the present study cohort fenofibrate and high Hcy levels did not modulate HDL and plasma functions in the first step of reverse cholesterol transport, cholesterol efflux from foam cells.

Vertebral bone marrow glucose uptake is inversely associated with bone marrow fat in diabetic and healthy pigs: [18F]FDG-PET and MRI study

OBJECTIVES Diabetes induces osteoporosis and during osteoporosis, vertebral bone marrow (VBM) adipose tissue amount increases. The association between this adiposity and bone marrow metabolism is unclear. Here, we compared VBM glucose metabolism and fat content in healthy and diabetic pigs, in vivo, using positron emission tomography (PET), in-phase and out-of-phase magnetic resonance imaging and magnetic resonance proton spectroscopy ((1)H MR spectroscopy). MATERIALS/METHODS Eleven pigs (n=11) were used. The intervention group had five diabetic and the control group had six healthy pigs. To measure metabolism, PET-imaging with [(18)F]fluoro-deoxy-glucose ([(18)F]FDG) intravenous tracer was used. 1.5-T MRI with (1)H spectroscopy, in-phase and out-of-phase imaging and chemical TAG analysis of the VBM were performed. RESULTS We found a significant inverse correlation between VBM glucose uptake (GU) and VBM fat content (R=-0.800, p<0.01) and TAG concentration assay (R=-0.846, p<0.05). There was a trend, although non-significant, of a linear correlation between VBM (1)H MR spectroscopy and TAG concentration (R=0.661) and (1)H MR spectroscopy and in-phase and out-of-phase MR imaging (R=0.635). CONCLUSIONS VBM glucose metabolism coupled with VBM fat content may impact diabetic induced osteoporosis.

Effect of isolated isoflavone supplementation on ABCA1-dependent cholesterol efflux potential in postmenopausal women.

Objective: Isoflavones may display beneficial health effects in postmenopausal women. We studied in a clinical trial whether isolated isoflavone treatment in postmenopausal women could affect reverse cholesterol transport as evaluated by adenosine triphosphate-binding cassette A1- (ABCA1), dependent cholesterol efflux from macrophages. In addition, various serum lipid and lipoprotein parameters were investigated. Furthermore, we separately assessed equol-producing and non-equol-producing women. Design: Postmenopausal women (n = 56) were treated with either isoflavone or placebo tablets for 3 months in a crossover design, separated by a 2-month washout period. Fifteen women were classified as equol producers, and 15 women were classified as non-equol producers. Serum samples were collected before and after each treatment period. [3H]-Cholesterol-labeled J774 macrophage cells, with and without ABCA1 up-regulation, were incubated with the samples, and ABCA1-dependent cholesterol efflux and serum lipid and lipoprotein levels were assessed. Results: Serum promoted 3.1% ± 1.1% and 3.2% ± 1.1% cholesterol efflux from macrophages after isoflavone and placebo treatment, respectively. Thus, isoflavone supplementation did not affect ABCA1-dependent cholesterol efflux to serum. However, as a novel finding, isoflavone treatment increased a subclass of high-density lipoprotein, the pre-&bgr; high-density lipoprotein levels by 18% without affecting any other serum lipid concentrations. ABCA1-facilitated cholesterol efflux and lipid parameters did not differ between equol-producing and non-equol-producing women. Conclusion: In postmenopausal women, isolated isoflavone treatment does not affect ABCA1-dependent cholesterol efflux potential from macrophages but increases circulating pre-&bgr; high-density lipoprotein level, which could provide beneficial vascular effects.

The orientation and dynamics of estradiol and estradiol oleate in lipid membranes and HDL disc models.

Estradiol (E2) and E2 oleate associate with high-density lipoproteins (HDLs). Their orientation in HDLs is unknown. We studied the orientation of E2 and E2 oleate in membranes and reconstituted HDLs, finding that E2 and E2 oleate are membrane-associated and highly mobile. Our combination of NMR measurements, molecular dynamics simulation, and analytic theory identifies three major conformations where the long axis of E2 assumes a parallel, perpendicular, or antiparallel orientation relative to the membranes z-direction. The perpendicular orientation is preferred, and furthermore, in this orientation, E2 strongly favors a particular roll angle, facing the membrane with carbons 6, 7, 15, and 16, whereas carbons 1, 2, 11, and 12 point toward the aqueous phase. In contrast, the long axis of E2 oleate is almost exclusively oriented at an angle of ∼60° to the z-direction. In such an orientation, the oleoyl chain is firmly inserted into the membrane. Thus, both E2 and E2 oleate have a preference for interface localization in the membrane. These orientations were also found in HDL discs, suggesting that only lipid-E2 interactions determine the localization of the molecule. The structural mapping of E2 and E2 oleate may provide a design platform for specific E2-HDL-targeted pharmacological therapies.

Steroid Hormone–Fatty Acid Esters and Bone

The recent paper by Brodeur et al. on the metabolism of low(LDL) and high-density lipoproteins (HDL) by osteoblastic cells showed for the first time that these cells express scavenger receptors of class B (SR-B). This important study presented a novel mechanism, which explains how cholesterol and estradiol carried by LDL and HDL3 can enter the osteoblast. The authors refer to the previously reported correlation between atherosclerosis and the development of osteoporosis, as well as the well-defined importance of estrogen in bone metabolism. The results of their study suggested that lipoproteins have a significant role in the routing of cholesterol and estradiol to bone cells, to be taken up selectively by a SR-B–mediated process. Interestingly, Gong et al. showed SR-B type I–dependent uptake of HDL-associated estradiol into vascular endothelial cells, which implies the possibility of a similar mechanism in these cell types. According to our newly published data, HDL-associated, esterified estradiol is also taken up by liver-derived cells by both SR-B type I and LDL receptors (LDLRs) and becomes hydrolyzed in the cells to free estradiol. As Brodeur et al. mention in their paper, estradiol is known to associate with LDL and HDL in fatty acid ester form, a mechanism shared with also other steroid hormones. Steroid fatty acid esters are water-insoluble molecules, which are transported in lipoprotein particles in the circulation. Unesterified steroids are incorporated in lipoproteins only to a small extent. Brodeur et al. did not discuss the possibility that the lipoprotein bound estradiol in their study could be, in fact, esterified with fatty acids. The fatty acid esterification of estradiol would be important to specify, because the esterification has been shown to enhance the potential of estradiol to protect lipoproteins from oxidation. The antioxidant protection of LDL by bound estradiol esters even after entering the arterial intima could hinder the progress of atherosclerosis, and a similar mechanism could have a role also in bone health, assuming that oxidized LDL participates in the pathogenesis of osteoporosis. It is known that estradiol becomes esterified in plasma on HDL3 particles by lecithin:cholesterol acyltransferase (LCAT) and is transferred from HDL to LDL in a process at least partly mediated by cholesteryl ester transfer protein (CETP). In our in vitro incubations with plasma and also isolated total plasma HDL and HDL3 subfraction, under conditions similar to the ones used by Brodeur et al., estradiol associates with lipoprotein particles in mostly esterified form. In HDL particles after plasma incubation, 83% of bound hormone was fatty acid ester and only 17% was free, unesterified E2. In isolated HDL and HDL3 subfractions, without any purification, there is still enough lipoprotein associated LCAT left to support the fatty acid esterification of estradiol. In serum samples from pregnant women, estradiol accumulates in lipoproteins in esterified form, and free estradiol is mostly undetectable by time-resolved fluoroimmunoassay (TR-FIA) in isolated very LDL (VLDL), LDL, and HDL fractions. We have also quantitated estradiol fatty acid esters in blood and adipose tissue samples from postmenopausal women, as well as premenopausal and pregnant women. The results confirmed the abundance of these lipophilic estrogen derivatives in adipose tissue and showed that the proportion of esterified estradiol compared with free hormone is enhanced during low estrogen status. The serum estradiol ester/free ratio was lowest in pregnancy (0.5%) and increased to 15% in premenopausal women and to 110% in postmenopausal women. In postmenopausal women, the overwhelming majority of estradiol was present in adipose tissue, most of it in fatty acid ester form. Estradiol fatty acid esterification and hydrolysis have been described in humans and various tissues from mammals and invertebrates, showing that these mechanisms are evolutionally conserved and probably of importance. Although the function of estradiol esters has not been clarified, they are known to have long half-lives and to display prolonged hormonal activity, which suggests a function as a reservoir for the release of active estradiol, probably by hormone sensitive lipase (HSL) present in the cytosol. The release of estradiol from its fatty acid ester form would also be a faster way to obtain active hormone compared with biosynthesis from cholesterol. The recently published results imply that the fatty acid esterification of estradiol and subsequent transportation of these esters in plasma lipoprotein particles may have an important role in the selective routing of the active hormone to target tissues, such as bone, vascular endothelium, or liver cells. It is intriguing to note that other steroid hormones, such as dehydroepiandrosterone (DHEA), pregnenolone, and 5androstenediol, are esterified by LCAT, after which they become associated with HDL and are taken up by animal cells, resulting in hydrolysis of the esters and subsequent metabolic conversion of the hormones. With pregnenolone, there is also evidence of lipoprotein receptor– mediated uptake mechanism. Future studies must be designed to increase our understanding of the lipoprotein associated steroid hormone esters and their uptake into tissues, as well as to put this knowledge into use in designing therapies targeted to postmenopausal osteoporosis and atherosclerosis in particular.

Widespread vascular inflammation in a patient with antineutrophil cytoplasmic antibody-associated vasculitis as detected by positron emission tomography

A 47-year-old man had fever, fatigue, abdominal pain, and claudication due to antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Whole-body [18 F] FDG PET/CT scan was performed as a diagnostic procedure, and it revealed abnormal [18 F] FDG accumulation in smalland medium-sized vessels of the upper extremities and in the lower extremities appearing as a tree-root-like [18 F] FDG uptake pattern (Panel A). Increased [18 F] FDG uptake in vertebral bone marrow and the spleen was observed due to hematopoietic stimulation. For comparison, two different [18 F] FDG PET/CT scans are presented. A 76-year-old woman had an atypical [18 F] FDG uptake pattern in the larger arteries, which indicated giant cell vasculitis (Panel B). A 39-year-old man had Staphylococcus aureus septicaemia. He had no [18 F] FDG metabolic activity in vessel walls, but the activation of axillary lymph nodes and metastatic infectious foci in the left leg and spleen was observed (Panel C). [F] FDG PET/CT is a useful tool for evaluating the distribution of inflammation and infection, as in our case. It can detect the affected organs and also show unexpected localizations of ANCA-associated vasculitis [1].