Familial Hypercholesterolemic Research Articles

Effects of lipid-modifying pharmacological agents on circulating PCSK9 in severe and familial hypercholesterolemic (FH) patients in argentina

Background and Aims : PCSK9 is a key regulator of cholesterol levels by its action on LDL receptors and would promotes inflammation and atherogenesis. PCSK9 represents a novel pharmacological target for hypercholesterolemia Frequently prescribed lipid-lowering therapy (LLT), particularly statins, has been reported to increase circulating PCSK9, decreasing the effect of statins on lowering LDL-C. Little is known -and with contradictory results- about the effect of statin+ezetimibe (S+E) combination on PCSK9 levels.

Insulin‐like growth factor I reduces atherosclerosis in Rapacz pigs

Cardiovascular disease is the leading cause of mortality in the Western world and its manifestations result in large part from atherosclerotic lesion development and destabilization. We have reported previously that administration of insulin-like growth factor I (IGF-1) reduced atherosclerosis in the murine model. To test the effect of IGF-1 on human-like atherosclerosis we injected 50 ug/kg recombinant human IGF-1 or saline (control) (n=9/group) into high-fat diet fed Rapacz familial hypercholesterolemic (FH) swine for 6 months. We quantified vessel volume, atherosclerotic burden (% of volume of atherosclerotic plaque + media per vessel volume), lumen volume and assessed plaque phenotype in coronary arteries using intravascular ultrasound (IVUS) at time 0 and after 3 and 6 months on the diet. IGF-1-injected pigs had on average 2.6-fold increase in total plasma IGF-1 (ELISA, p<0.001 vs. control pigs). Pigs in the IGF-1 and control group gradually gained weight over the time of experiment, however pigs in the IGF-1 group had a larger increase in BW compared to controls at 6 months (IGF-1, 60.6±7.4% increase vs. saline, 44.3±4.0% increase, p<0.05) consistent with growth-stimulating effects of IGF-1. Pigs in both groups had a gradual increase in vessel volume in the right coronary artery (RCA), left anterior descending artery (LAD) and left circumflex artery (LCX) with time as quantified by serial IVUS. However, IGF-1-injected pigs had a larger increase in vessel volume after 3 and 6 months compared to controls suggesting vascular hypertrophy. The atherosclerotic burden was increased in RCA, LAD and LCX in both groups after 3 and 6 months of injections compared to the pre-injection time point. We found a significant reduction in atherosclerotic burden in RCA and LAD in IGF-1 group compared to control at 6 months’ time-point (RCA: IGF-1, 203±13%, vs. control, 244±8; LAD: IGF-1, 200±6%, vs. control, 228±11%). The lumen volume of coronaries was decreased in both groups, however, coronaries in IGF-1-injected pigs had on average 24±3% larger lumen volume compared to control pigs at 6 months’ time-point. We used virtual histology (VH) post-IVUS image processing to assess plaque composition and to classify plaque sub-types. Pigs in both groups had a progressive increase in plaque calcification, necrotic core area, fibrous plaque and fibro-fatty plaque area. IGF-1 did not significantly change plaque composition. In summary, we report that IGF-1 increased pig BW, and total volume of coronaries suggesting hypertrophy and these data are consistent with growth-stimulating effects of IGF-1. We found a significant reduction in atherosclerotic burden and increase in lumen volume in coronaries in the IGF-1 group after 6 months of injections consistent with an anti-atherosclerotic effect of IGF-1. Our data support the feasibility of using IGF-1 as an anti-atherogenic therapy.

Clinically used selective oestrogen receptor modulators increase LDL receptor activity in primary human lymphocytes.

Treatment with selective oestrogen receptor modulators (SERMs) reduces low-density lipoprotein (LDL) cholesterol levels. We assessed the effect of tamoxifen, raloxifene and toremifene and their combinations with lovastatin on LDL receptor activity in lymphocytes from normolipidaemic and familial hypercholesterolaemic (FH) subjects, and human HepG2 hepatocytes and MOLT-4 lymphoblasts. Lymphocytes were isolated from peripheral blood, treated with different compounds, and 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (DiI)-labelled LDL uptake was analysed by flow cytometry. Tamoxifen, toremifene and raloxifene, in this order, stimulated DiI-LDL uptake by lymphocytes by inhibiting LDL-derived cholesterol trafficking and subsequent down-regulation of LDL receptor expression. Differently to what occurred in HepG2 and MOLT-4 cells, only tamoxifen consistently displayed a potentiating effect with lovastatin in primary lymphocytes. The SERM-mediated increase in LDL receptor activity was not altered by the anti-oestrogen ICI 182,780 nor was it reproduced by 17β-oestradiol. However, the tamoxifen-active metabolite endoxifen was equally effective as tamoxifen. The SERMs produced similar effects on LDL receptor activity in heterozygous FH lymphocytes as in normal lymphocytes, although none of them had a potentiating effect with lovastatin in heterozygous FH lymphocytes. The SERMs had no effect in homozygous FH lymphocytes. Clinically used SERMs up-regulate LDL receptors in primary human lymphocytes. There is a mild enhancement between SERMs and lovastatin of lymphocyte LDLR activity, the potentiation being greater in HepG2 and MOLT-4 cells. The effect of SERMs is independent of oestrogen receptors but is preserved in the tamoxifen-active metabolite endoxifen. This mechanism may contribute to the cholesterol-lowering action of SERMs.

Carotid Endothelial VCAM-1 Is an Early Marker of Carotid Atherosclerosis and Predicts Coronary Artery Disease in Swine.

ObjectiveThe aim was to determine if endothelial VCAM-1 (eVCAM-1) expression in the common carotid artery (CCA) would correlate with predictive markers of atherosclerotic disease, would precede reduction of markers of endothelial cell function and would predict coronary artery disease (CAD).Methods and resultsCarotid arterial segments (bifurcation, proximal and distal CCA) were harvested from 14 and 24 month-old male castrated familial hypercholesterolemic (FH) swine, a model of spontaneous atherosclerosis. Quantification of local expression of eVCAM-1, intimal macrophage accumulation, oxidative stress, intima-media (I/M) ratio, intima-media thickness (IMT), endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) in selected regions of the carotids revealed a relationship between local inflammation and atheroscle-rotic plaque progression. Importantly, inflammation was not uniform throughout the CCA. Endo-thelial VCAM-1 expression was the greatest at the bifurcation and increased with age. Finally, eV-CAM-1 best estimated the severity of CAD compared to blood levels of glucose, hypercholestero-lemia, carotid IMT, and p-eNOS.ConclusionOur data suggested that eVCAM-1 was closely associated with atherosclerotic plaque progression and preceded impairment of EDD. Thus, this study supported the use of carotid VCAM-1 targeting agents to estimate the severity of CAD.

Gene Expression Profiling of Valvular Interstitial Cells in Rapacz Familial Hypercholesterolemic Swine.

Rapacz familial hypercholesterolemic (RFH) swine is a well-established model of human FH, a highly prevalent hereditary disease associated with increased risk of coronary artery disease and calcific aortic valve disease (CAVD). However, while these animals have been used extensively for the study of atherosclerosis, the heart valves from RFH swine have not previously been examined. We report the analysis of valvular interstitial cell gene expression in adult (two year old) and juvenile (three months old) RFH and WT swine by microarray analysis via the Affymetrix Porcine Genome Array (GEO #: GSE53997). Principal component and hierarchical clustering analysis revealed grouping and almost no variability between the RFH juvenile and WT juvenile groups. Additionally, only 21 genes were found differentially expressed between these two experimental groups whereas over 900 genes were differentially expressed when comparing either RFH or WT juvenile swine to RFH adults.

Measurements of wall shear stress and aortic pulse wave velocity in swine with familial hypercholesterolemia

To assess measurements of pulse wave velocity (PWV) and wall shear stress (WSS) in a swine model of atherosclerosis. Nine familial hypercholesterolemic (FH) swine with angioplasty balloon catheter-induced atherosclerotic lesions to the abdominal aorta (injured group) and 10 uninjured FH swine were evaluated with a 4D phase contrast (PC) magnetic resonance imaging (MRI) acquisition, as well as with radial and Cartesian 2D PC acquisitions, on a 3T MR scanner. PWV values were computed from the 2D and 4D PC techniques, compared between the injured and uninjured swine, and validated against reference standard pressure probe-based PWV measurements. WSS values were also computed from the 4D PC MRI technique and compared between injured and uninjured groups. PWV values were significantly greater in the injured than in the uninjured groups with the 4D PC MRI technique (P = 0.03) and pressure probes (P = 0.02). No significant differences were found in PWV between groups using the 2D PC techniques (P = 0.75-0.83). No significant differences were found for WSS values between the injured and uninjured groups. The 4D PC MRI technique provides a promising means of evaluating PWV and WSS in a swine model of atherosclerosis, providing a potential platform for developing the technique for the early detection of atherosclerosis.

The influence of apoE-deficiency and LDL-receptor-deficiency on the HDL subpopulation profile in mice and in humans

ObjectiveAs apoE−/− and LDL-Receptor−/− mice are commonly used in atherosclerosis research; our objective was to point out the differences in HDL metabolism between mice and humans regarding the roles of apoE and LDLR. MethodsWe examined HDL particles obtained from wild type (WT), LDLR−/−, and apoE−/− mice, as well as from normal, homozygous familial hypercholesterolemic (FH), and apoE-deficient human subjects by 2-dimensional non-denaturing PAGE followed by immunoblot and image analysis. ResultsIn WT mice, the majority of apoA-I was in large (9.0–12.0 nm), α-mobility HDL with trace amounts of apoA-I in small, preβ-1 HDL. In LDL−/− mice, both apoA-I- and apoE-containing HDL looked normal. About one-third of apoE was associated with large apoA-I-containing HDL (LpA-I:E) and two-thirds formed large HDL without apoA-I (LpE). In apoE−/− mice, apoA-I was detected in multiple, β-preβ-mobility, tightly-packed bands (7.0–13.0 nm) indicating that apoA-I in these animals was present only in poorly-lipidated, discoidal particles. Neither FH nor apoE-deficient humans showed significant alterations in apoA-I-containing HDL particles as compared to non-carriers. ConclusionsOur data indicate that apoE is necessary for the formation of spherical, lipidated HDL particles in mice, but not in humans, probably because mice lack CETP. Based on our data, we hypothesize that apoE−/− mice have little or no functional HDL, therefore results from apoE−/− mice cannot be extrapolated to humans without taking this significant difference into consideration.

Familial hypercholesterolemia impairs exercise-induced systemic vasodilation due to reduced NO bioavailability

Hypercholesterolemia impairs endothelial function [e.g., the nitric oxide (NO)-cyclic GMP-phosphodiesterase 5 (PDE5) pathway], limits shear stress-induced vasodilation, and is therefore expected to reduce exercise-induced vasodilation. To assess the actual effects of hypercholesterolemia on endothelial function and exercise-induced vasodilation, we compared the effects of endothelial NO synthase (eNOS) and PDE5 inhibition in chronically instrumented Yucatan (Control) and Rapacz familial hypercholesterolemic (FH) swine, at rest and during treadmill exercise. The increases in systemic vascular conductance produced by ATP (relative to nitroprusside) and exercise were blunted in FH compared with Control swine. The vasoconstrictor response to eNOS inhibition, with nitro-l-arginine (NLA), was attenuated in FH compared with Control swine, both at rest and during exercise. Furthermore, whereas the vasodilator response to nitroprusside was enhanced slightly, the vasodilator response to PDE5 inhibition, with EMD360527, was reduced in FH compared with Control swine. Finally, in the pulmonary circulation, FH resulted in attenuated vasodilator responses to ATP, while maintaining the responses to both NLA and EMD360527. In conclusion, hypercholesterolemia reduces exercise-induced vasodilation in the systemic but not the pulmonary circulation. This reduction appears to be the principal result of a decrease in NO bioavailability, which is mitigated by a lower PDE5 activity.

Exercise training does not increase muscle FNDC5 protein or mRNA expression in pigs

BackgroundExercise training elevates circulating irisin and induces the expression of the FNDC5 gene in skeletal muscles of mice. Our objective was to determine whether exercise training also increases FNDC5 protein or mRNA expression in the skeletal muscles of pigs as well as plasma irisin. MethodsCastrated male pigs of the Rapacz familial hypercholesterolemic (FHM) strain and normal (Yucatan miniature) pigs were sacrificed after 16–20weeks of exercise training. Samples of cardiac muscle, deltoid and triceps brachii muscle, subcutaneous and epicardial fat were obtained and FNDC5 mRNA, along with that of 6 other genes, was measured in all tissues of FHM pigs by reverse transcription polymerase chain reaction. FNDC protein in deltoid and triceps brachii was determined by Western blotting in both FHM and normal pigs. Citrate synthase activity was measured in the muscle samples of all pigs as an index of exercise training. Irisin was measured by an ELISA assay. ResultsThere was no statistically significant effect of exercise training on FNDC5 gene expression in epicardial or subcutaneous fat, deltoid muscle, triceps brachii muscle or heart muscle. Exercise-training elevated circulating levels of irisin in the FHM pigs and citrate synthase activity in deltoid and triceps brachii muscle. A similar increase in citrate synthase activity was seen in muscle extracts of exercise-trained normal pigs but there was no alteration in circulating irisin. ConclusionExercise training in pigs does not increase FNDC5 mRNA or protein in the deltoid or triceps brachii of FHM or normal pigs while increasing circulating irisin only in the FHM pigs. These data indicate that the response to exercise training in normal pigs is not comparable to that seen in mice.

Gene expression differences during the heterogeneous progression of peripheral atherosclerosis in familial hypercholesterolemic swine

BackgroundThe heterogeneous progression of atherosclerotic disease in the peripheral arteries is currently not well understood. In humans, artery specific disease progression is partly attributed to the local hemodynamic environments. However, despite similar hemodynamic environments, porcine brachial arteries are protected while femoral arteries are highly susceptible to advanced lesion formation. The aim of this investigation was to determine whether artery specific gene expression patterns contribute to the uneven distribution of peripheral arterial disease (PAD) in Rapacz Familial-Hypercholesterolemic (FHC) swine.ResultsHistological results confirmed rapid atherosclerotic disease progression in femoral but not brachial arteries. A total of 18,922 probe sets had sufficient signal abundance. A main effect for age and artery was observed for 1784 and 1256 probe sets, respectively. A significant age x artery interaction was found for 184 probe sets. Furthermore, comparison between arteries found a decrease from 714 to 370 differentially expressed transcripts from nine months to two years of age. Gene ontology analysis of the 56 genes with a main effect for artery and an age x artery interaction identified vascular smooth muscle contraction as enhanced biological signaling pathway.ConclusionThis is the first investigation to report that the total number of differential genes decreases with diverging atherosclerotic disease pattern between porcine brachial and femoral arteries.

Carotid Inflammation Is Unaltered by Exercise in Hypercholesterolemic Swine

Reduction of vascular inflammation might contribute to the beneficial effects of exercise. We hypothesized that 1) exercise would reduce carotid endothelial vascular cell adhesion molecule-1 (VCAM-1) and that 2) in vivo detection of carotid inflammation can be achieved in a large animal model using contrast-enhanced ultrasound (CEU) with VCAM-1-targeted microbubbles (MBs). Familial hypercholesterolemic (FH) swine were divided into sedentary (Sed) and exercise-trained (Ex) groups. Ex pigs underwent 16-20 wk of treadmill aerobic exercise. At the end of the study, in vivo CEU with VCAM-1-targeted MBs and assessment of endothelial-dependent dilation (EDD) were performed in carotid arteries. VCAM-1 mRNA and protein expression were compared with markers of atherosclerotic disease and health, and in vitro EDD was assessed in carotid arteries. Exercise training neither reduced inflammation nor improved EDD in carotid arteries of FH swine. Markers of atherosclerosis including VCAM-1 were prominent in the bifurcation compared with the proximal or distal common carotid artery and inversely associated with phosphorylated and total endothelial nitric oxide synthase. Signal intensity from VCAM-1-to-control MBs positively correlated with carotid VCAM-1 protein expression, validating our technique. These results first demonstrate that aerobic exercise has no effect on carotid endothelial inflammatory markers and EDD in FH swine. Second, our findings indicate that CEU using VCAM-1-targeted MBs can detect inflammation in vivo, providing strong foundations for longitudinal studies examining the effect of therapeutic interventions on the inflammatory status of the endothelium.

Reduced NO Bioavailability Hampers Exercise Induced Vasodilation in Familial Hypercholesterolemic Swine

Hypercholesterolemia impairs endothelial function (e.g. the NO‐cGMP‐ phosphodiesterase 5 (PDE5) pathway), limits shear stress induced vasodilation, and is thereby expected to reduce exercise induced vasodilation. To assess the actual effects of hypercholesterolemia on endothelial function and exercise induced vasodilation, we compared the effects of eNOS and PDE5 inhibition in chronically instrumented Yorkshire (Y) and Rapacz Familial Hypercholesterolemic (FH) swine, at rest and during exercise.Exercise induced systemic vasodilation, i.e. the increase in systemic vascular conductance index (SVCi = SVC per Kg bodyweight), was markedly reduced in FH swine (27±5%) compared to Y swine (119±4%). As a result, during exercise mean arterial pressure remained constant in Y swine while it significantly increased in FH swine (from 91±6 to 110±8 mmHg). The vasoconstrictor effect of eNOS inhibition, with Nitro‐L‐Arginine, was attenuated in FH compared to Y swine, both at rest and during exercise. Furthermore, the vasodilator effect of PDE5 inhibition, with EMD360527, was also markedly reduced in FH compared to Y swine.In conclusion, hypercholesterolemia hampers exercise‐induced vasodilation via reduced NO bioavailability. The reduced NO bioavailability results in a decreased activation of the NO‐cGMP pathway, as evidenced by a blunted vasodilator response to PDE5 inhibition.

Exercise alone limits reductions in intimal smooth muscle K+ current in a swine model of coronary artery disease

We previously demonstrated in Rapacz familial hypercholesterolemic (FH) swine with coronary artery disease that exercise (EX) combined with a low fat diet attenuated reductions in intimal coronary smooth muscle (iCSM) whole cell K+ current. The goal of the present study was to determine if EX alone is sufficient in attenuating these reductions and to identify the K+ channels responsible. FH swine were placed on a high fat diet at 14 months of age and then were treadmill trained (EX; n=5) or remained sedentary (SED; n=6) from 20 to 24 months of age. iCSM whole cell K+ current was measured in the presence and absence of blockers for voltage‐gated (Kv; 4‐AP, 2mM), large‐conductance (BK; Iberiotoxin, IBTX, 100nM) and intermediate‐conductance Ca2+ activated K+ (IK; TRAM‐34, 100nM) channels from −70 to +100 mV. Compared to weight matched non‐FH control swine (84.3 ± 9.3 pA/pF, n=20), FH SED had reduced peak (+100 mV) whole cell K+ current (14.3 ± 2.1, n=33), which was attenuated by EX (32.3 ± 4.6, n=42; p&lt;0.001). SED (0.10 ± 0.29, n=30) had reduced Kv current compared to EX (1.72 ± 0.74, n=20; +30 mV; p=0.02). BK current was not different between groups [SED (6.0 ± 1.6, n=24) vs. EX (12.5 ± 5.4, n=17); p=0.19]. Neither SED nor EX had measurable IK current. In conclusion, EX alone attenuated reductions in iCSM K+ current in FH swine, in part via the maintenance of Kv current. NIH HL52490

Blunted exercise‐induced vasodilation in familial hypercholesterolemic swine does not involve enhanced ET‐1‐mediated vasoconstriction

Hypercholesterolemia causes endothelial dysfunction and activation of the vascular endothelin‐1 (ET‐1) system. The procontractile effect of ET‐1 action on resistance vessels would therefore be expected to limit exercise‐induced vasodilation. To assess the role of hypercholesterolemia on ET‐1 activation and exercise‐induced vasodilation, we compared the effects of ET receptor blockade in instrumented Yucatan (Y) and familial hypercholesterolemic (FH) swine, at rest and during exercise. Exercise induced systemic vasodilation, measured as systemic vascular conductance index (SVCi = SVC/kg bodyweight), that was reduced in FH swine (36%) compared to Y swine (91%). Unexpectedly, the ETA/ETB receptor blocker tezosentan did not enhance exercise‐induced vasodilation in FH swine and the vasodilator effect of tezosentan at rest was reduced in FH swine (16%) versus Y swine (48%). Isolated skeletal muscle 2A arterioles from FH swine demonstrated enhanced sensitivity to ET‐1; however, plasma ET‐1 levels tended to be lower in conscious FH swine compared to Y swine (p=0.08). Thus, our data suggest that although the skeletal muscle vasculature of FH swine exhibits enhanced sensitivity to ET‐1, an ET‐1‐mediated enhancement of systemic vascular tone does not account for the reduced exercise‐induced dilation in FH swine, possibly as a result of reduced production/release of ET‐1 in FH swine. Supported by NIH‐HL52490.

Abstract P237: Exercise Training Prevents Hypercholesterolemia-Induced Cardiac Mitochondrial Dysfunction

Hypercholesterolemia has been suggested to have direct negative effects on myocardial function due to increased reactive oxygen species (ROS) generation and increased myocyte death. Mitochondrial permeability transition (MPT) is a significant mediator of cell death, which is enhanced by ROS generation and attenuated by exercise training. The purpose of this study was to investigate the effect of hypercholesterolemia on the MPT response of cardiac mitochondria. We hypothesized that familial hypercholesterolemic (FH) pigs would have an enhanced MPT response, and that exercise training could reverse this phenotype. FH pigs were obtained from the University of Wisconsin. Control, normolipidemic farm pigs were maintained on standard pig chow. After 4 months on a high-fat diet, the FH pigs were switched to the standard pig chow, and randomized to sedentary or exercise groups. The exercise group underwent a progressive treadmill-based training program for 4 months. At the end of the training protocol the animals were sacrificed and the heart removed. MPT was assessed by mitochondrial swelling in response to Ca2+. Protein nitrotyrosylation, GSH levels, and antioxidant enzyme expression were also examined. FH pigs did show an increased MPT response despite no change in the expression of putative MPT pore components adenine nucleotide translocase (ANT), mitochondrial phosphate carrier (PiC), and cyclophilin-D (CypD). FH also caused increased oxidative stress, depicted by increased protein nitrotyrosylation and decreased GSH levels. This was associated with concomitant decreases in the expression of mitochondrial antioxidant enzymes manganese superoxide dismutase (MnSOD) and thioredoxin-2 (Trx2). However, chronic exercise training was able to normalize the MPT response in FH pigs, reduce oxidative stress, and increase MnSOD expression. We conclude that hypercholesterolemia causes increased oxidative stress and enhances the MPT response in the porcine myocardium, and that exercise training can correct for both the increased oxidative stress and MPT alterations observed with hypercholesterolemia.

Hypercholesterolemia increases mitochondrial oxidative stress and enhances the MPT response in the porcine myocardium: beneficial effects of chronic exercise

Hypercholesterolemia has been suggested to have direct negative effects on myocardial function due to increased reactive oxygen species (ROS) generation and increased myocyte death. Mitochondrial permeability transition (MPT) is a significant mediator of cell death, which is enhanced by ROS generation and attenuated by exercise training. The purpose of this study was to investigate the effect of hypercholesterolemia on the MPT response of cardiac mitochondria. We tested the hypothesis that familial hypercholesterolemic (FH) pigs would have an enhanced MPT response and that exercise training could reverse this phenotype. MPT was assessed by mitochondrial swelling in response to 10-100 μM Ca(2+). FH pigs did show an increased MPT response to Ca(2+) that was associated with decreases in the expression of the putative MPT pore components mitochondrial phosphate carrier (PiC) and cyclophilin-D (CypD). FH also caused increased oxidative stress, depicted by increased protein nitrotyrosylation, as well as decreased levels of reduced GSH in cardiac mitochondria. Expression of the mitochondrial antioxidant enzymes manganese superoxide dismutase (MnSOD), thioredoxin-2 (Trx2), and peroxiredoxin-3 (Prx3) was greatly reduced in the FH pigs. In contrast, cytosolic catalase expression and activity were increased. However, chronic exercise training was able to normalize the MPT response in FH pigs, reduce mitochondrial oxidative stress, and return MnSOD, Trx2, Prx3, and catalase expression/activities to normal. We conclude that FH reduces mitochondrial antioxidants, increases mitochondrial oxidative stress, and enhances the MPT response in the porcine myocardium, and that exercise training can reverse these detrimental alterations.

A novel type of familial hypercholesterolemia: Double heterozygous mutations in LDL receptor and LDL receptor adaptor protein 1 gene

BackgroundAutosomal recessive hypercholesterolemia (ARH) is an extremely rare inherited hypercholesterolemia, the cause of which is mutations in low-density lipoprotein (LDL) receptor adaptor protein 1 (LDLRAP1) gene. MethodsA total of 146 heterozygous familial hypercholesterolemic (FH) patients with a mutation in LDLR gene were screened for genes encoding proprotein convertase subtilisin/kexin type 9 (PCSK9) and LDLRAP1. ResultsAmong the 146 subjects, we identified a 79-year-old Japanese female with double mutations in LDLR gene (c.2431A>T) and LDLRAP1 gene (c.606dup). Two other relatives with double mutations in those genes in her family were also identified. Although the proband exhibited massive Achilles tendon xanthoma and coronary and aortic valvular disease, serum LDL-C level of subjects with double mutations was similar with that of subjects with single LDLR mutation (284.0±43.5 versus 265.1±57.4mg/dl). ConclusionAdditional mutation in LDLRAP1 may account for severer phenotype in terms of xanthoma and atherosclerotic cardiovascular disease in FH patients.

Relationship between plasma coenzyme Q10, asymmetric dimethylarginine and arterial stiffness in patients with phenotypic or genotypic familial hypercholesterolemia on long-term statin therapy

ObjectiveWe investigated whether statin-treated heterozygous familial hypercholesterolemic (FH) patients have lower plasma coenzyme Q10 (CoQ10) levels than low-density lipoprotein receptor (LDLR) mutation negative FH patients on equivalent statin doses, and whether lower CoQ10 concentrations are associated with increased arterial stiffness. MethodsThirty LDLR mutation negative patients with clinical FH and 30 mutation positive FH patients matched for gender, statin duration and dose, and a further 30 controls were studied. Plasma CoQ10 and asymmetric dimethylarginine (ADMA) levels were measured by HPLC and the augmentation index by pulse wave analysis. ResultsPlasma CoQ10 levels, and the ratios of CoQ10 to total cholesterol and LDL-cholesterol were similar in treated FH patients with identified LDLR mutations to mutation negative patients on equivalent doses of statin therapy (p>0.05). CoQ10 and lipid levels were also comparable to controls not using any lipid modifying treatment. Arterial stiffness was higher in mutation negative patients (p=0.04) and there was a trend for an increase in mutation positive patients (p=0.09). ADMA was higher in the mutation positive group (p<0.01). The augmentation index corrected for age, blood pressure, and heart rate, was negatively correlated with plasma CoQ10 within FH patients (p<0.05). ConclusionLong-term, high-dose statin therapy does not lead to subnormal CoQ10 concentrations in patients with phenotypic or genotypic FH. Arterial stiffness is elevated in FH patients compared to untreated controls, and low CoQ10 levels are associated with increased arterial stiffness. CoQ10 supplementation trials are warranted in FH patients.

Gene expression differences in healthy brachial and femoral arteries of Rapacz familial hypercholesterolemic swine

The mechanisms underlying the unequal distribution of atherosclerotic disease in the peripheral arteries are currently unclear. Gene expression differences in healthy arteries may influence the heterogeneous distribution of atherosclerosis. Therefore, this investigation compares gene expression in healthy atheroprotected brachial and atherosusceptible femoral arteries of young and disease free Rapacz familial hypercholesterolemic (FHC) swine. We hypothesized that transcripts related to atherosusceptibility would be differentially expressed between these arteries prior to the onset of disease. Femoral and brachial arteries were harvested from four 13-day-old Rapacz FHC swine. No atherosclerotic disease was detected using Sudan IV, Verhoeff-van Gieson, and hematoxylin-eosin staining. Gene expression was quantified using Affymetrix GeneChip Porcine Genome Arrays. An average of 15,552 probe sets had detectable transcripts, while 430 probe sets showed a significant differential expression between arteries (false discovery rate < 0.05). The human orthologs of 63 probe sets with differential expression and a 1.5-fold or greater transcript abundance between arteries are associated with Wnt/β-catenin, lysophospholipid, and Ca-signaling, as well as apoptosis. This is the first investigation reporting that differences in relative abundance of gene expression exist between brachial and femoral arteries in young Rapacz FHC swine prior to the development of atherosclerotic lesions.

KCa3.1 upregulated in familial hypercholesterolemic (FH) coronary smooth muscle

Familial hypercholesterolemia (FH) is a key risk factor for development of atherosclerosis. Vascular smooth muscle (VSMC) cell phenotypic modulation plays a key role in the development and stability of atherosclerotic plaques. We have previously shown upregulation of KCa3.1 channels mediates phenotypic modulation of coronary smooth muscle (CSM) cells and inhibition of these channels reduces restenosis following angioplasty. It remains unclear whether KCa3.1 channels are altered in atherosclerotic CSM of individuals with FH. Our objective was to determine whether KCa3.1 channels are upregulated in CSM isolated from FH swine. RCA sections from 2 year old FH swine showed a ~15 fold increase in artery stenosis, accompanied by elevated triglyceride and cholesterol values. In the media of FH coronaries, there was a trend for increased KCa3.1mRNA expression and KCa3.1 protein expression was elevated ~20%. KCa3.1 channel activity was also increased ~2‐fold in CSMCs isolated from FH swine. Our findings demonstrate that KCa3.1 is upregulated in CSM of FH swine and support previous research indicating KCa3.1 plays a key role in the development and progression of atherosclerosis.