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Background: High cholesterol levels in pancreatic β-cells cause oxidative stress and decrease insulin secretion. β-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves β-cell insulin secretion by reducing oxidative stress., Methods: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-β-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by β-cells was monitored by flow cytometry. The effects of apoA-I internalization on β-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the β-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in β-cells and isolated islets with MitoSOX and confocal microscopy., Results: An F 1 -ATPase β-subunit on the β-cell surface was identified as the main apoA-I-binding partner. β-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F 1 -ATPase β-subunit-dependent. β-cells with internalized apoA-I (apoA-I + cells) had higher cholesterol and cell surface F 1 -ATPase β-subunit levels than β-cells without internalized apoA-I (apoA-I - cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF 1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E β-cells and isolated mouse islets. Differentially expressed genes in apoA-I + and apoA-I - Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function., Conclusions: These results establish that β-cells are functionally heterogeneous, and apoA-I restores insulin secretion in β-cells with elevated cholesterol levels by improving mitochondrial redox balance., Competing Interests: Disclosures None.

Background: Thrombin (via PAR [protease-activated receptor]-1 and PAR-4) and ADP (via P2Y 12 receptors) are potent endogenous platelet activators implicated in the development of cardiovascular disease. We aimed to assess whether platelet pathways alter with aging., Methods: We characterized platelet activity in community-dwelling volunteers (n=174) in the following age groups: (1) 20 to 30 (young); (2) 40 to 55 (middle-aged); (3) ≥70 years (elderly). Platelet activity was assessed by aggregometry; flow cytometry (surface markers [P-selectin: alpha granule release, CD63: dense granule release, PAC-1: measure of conformationally active GPIIb/IIIa at the fibrinogen binding site]) measured under basal conditions and after agonist stimulation [ADP, thrombin, PAR-1 agonist or PAR-4 agonist]); receptor cleavage and quantification; fluorometry; calcium flux; ELISA., Results: The elderly had higher basal platelet activation than the young, evidenced by increased expression of P-selectin, CD63, and PAC-1, which correlated with increasing inflammation (IL [interleukin]-1β/IL-6). The elderly demonstrated higher P2Y 12 receptor density, with greater ADP-induced platelet aggregation ( P <0.05). However, elderly subjects were resistant to thrombin, achieving less activation in response to thrombin (higher EC 50 ) and to selective stimulation of both PAR-1 and PAR-4, with higher basal PAR-1/PAR-4 cleavage and less inducible PAR-1/PAR-4 cleavage (all P <0.05). Thrombin resistance was attributable to a combination of reduced thrombin orienting receptor GPIbα (glycoprotein Ibα), reduced secondary ADP contribution to thrombin-mediated activation, and blunted calcium flux. D-Dimer, a marker of in situ thrombin generation, correlated with platelet activation in the circulation, ex vivo thrombin resistance, and circulating inflammatory mediators (TNF [tumor necrosis factor]-α/IL-6)., Conclusions: Aging is associated with a distinctive platelet phenotype of increased basal activation, ADP hyperreactivity, and thrombin resistance. In situ thrombin generation associated with systemic inflammation may be novel target to prevent cardiovascular disease in the elderly.

Objective: Atherosclerotic coronary artery disease is well recognised as an inflammatory disorder that is also influenced by oxidative stress. β2-GPI (β-2-glycoprotein-I) is a circulating plasma protein that undergoes post-translational modification and exists in free thiol as well as oxidized forms. The aim of this study was to assess the association between these 2 post-translational redox forms of β2-GPI and atherosclerotic coronary artery disease. Approach and Results: Stable patients presenting for elective coronary angiography or CT coronary angiography were prospectively recruited. A separate group of patients after reperfused ST-segment-elevation myocardial infarction formed an acute coronary syndrome subgroup. All patients had collection of fasting serum and plasma for quantification of total and free thiol β2-GPI. Coronary artery disease extent was quantified by the Syntax and Gensini scores. A total of 552 patients with stable disease and 44 with acute coronary syndrome were recruited. While total β2-GPI was not associated with stable coronary artery disease, a higher free thiol β2-GPI was associated with its presence and extent. This finding remained significant after correcting for confounding variables, and free thiol β2-GPI was a better predictor of stable coronary artery disease than hs-CRP (high-sensitivity C-reactive protein). Paradoxically, there were lower levels of free thiol β2-GPI after ST-segment-elevation myocardial infarction., Conclusions: Free thiol β2-GPI is a predictor of coronary artery disease presence and extent in stable patients. Free thiol β2-GPI was a better predictor than high-sensitivity C-reactive protein.

Objective: Patients with schizophrenia have increased long-term mortality attributable to cardiovascular disease and commonly demonstrate features of mixed dyslipidemia with low HDL-C (high-density lipoprotein cholesterol). The removal of cholesterol from cells by HDL via specific ATP-binding cholesterol transporters is a major functional property of HDL, and its measurement as cholesterol efflux capacity (CEC) can predict cardiovascular risk. Whether HDL function is impaired in patients with schizophrenia is unknown. Approach and Results: We measured basal and ABCA1 (ATP-binding cassette transporter A1)- and ABCG1 (ATP-binding cassette transporter G1)-dependent CEC, comparing patients with schizophrenia with age- and sex-matched healthy controls, and related our findings to nuclear magnetic resonance analysis of lipoprotein subclasses. Total plasma cholesterol and LDL-C (low-density lipoprotein cholesterol) were comparable between healthy controls (n=51) and patients (n=120), but patients with schizophrenia had increased total plasma triglyceride, low HDL-C and apo (apolipoprotein) A-I concentrations. Nuclear magnetic resonance analysis indicated a marked (15-fold) increase in large triglyceride-rich lipoprotein particle concentration, increased small dense LDL particles, and fewer large HDL particles. Despite lower HDL-C concentration, basal CEC was 13.7±1.6% higher, ABCA1-specific efflux was 35.9±1.6% higher, and ABCG1 efflux not different, in patients versus controls. In patients with schizophrenia, ABCA1-specific efflux correlated with the abundance of small 7.8 nm HDL particles but not with serum plasminogen or triglyceride levels., Conclusions: Patients with schizophrenia have increased concentrations of atherogenic apoB-containing lipoproteins, decreased concentrations of large HDL particles, but enhanced ABCA1-mediated CEC. In this population, preventative strategies should focus on reducing atherogenic lipoproteins rather than increasing CEC.

Background: Multiple crossing techniques in chronic total occlusion (CTO) percutaneous coronary intervention have been developed. This study compared recovery of quantitative myocardial blood flow (MBF) after different CTO percutaneous coronary intervention techniques., Methods: Consecutive patients with [ 15 O]H 2 O positron emission tomography perfusion imaging before and 3 months after successful CTO percutaneous coronary intervention between 2013 and 2018 were included. Changes in hyperemic MBF, coronary flow reserve, and perfusion defect size were compared between antegrade wire escalation, retrograde wire escalation, antegrade dissection and reentry (ADR), and retrograde dissection and reentry., Results: One hundred ninety-three patients were treated with antegrade wire escalation (N=90), retrograde wire escalation (N=24), ADR (N=35), and retrograde dissection and reentry (N=44). Increase in hyperemic MBF (1.19±0.77, 0.94±0.65, 1.09±0.63, and 1.02±0.75 mL·min -1 ·g -1 , respectively; P =0.40) and coronary flow reserve (1.34±1.08, 1.14±1.09, 1.31±0.96, and 1.24±0.99, respectively; P =0.84) and decrease in defect size (3.2±2.1, 3.0±2.2, 2.7±2.1, and 2.9±1.9 segments, respectively; P =0.77) were comparable between the 4 approaches. In addition, recovery of hyperemic MBF was less pronounced after subintimal crossing with knuckle-wire-technique compared with CrossBoss in controlled ADR and retrograde dissection and reentry (0.93±0.69 versus 1.54±0.65 mL·min -1 ·g -1 , P =0.02), and less after reentry using subintimal tracking and reentry in ADR compared with controlled ADR (Stingray) or limited antegrade subintimal tracking (0.60±0.53 versus 1.18±0.54 [ P =0.04] and versus 1.49±0.57 mL·min -1 ·g -1 , [ P <0.01])., Conclusions: Recovery of hyperemic MBF, coronary flow reserve, and perfusion defect size after CTO percutaneous coronary intervention was comparable between different approaches. Although sometimes necessary to cross a complex CTO lesion, subintimal knuckle wiring and subintimal tracking and reentry resulted in less hyperemic MBF improvement compared with other subintimal crossing and reentry techniques.

Objective: Cyclosporin A (CsA) is an immunosuppressant commonly used to prevent organ rejection but is associated with hyperlipidemia and an increased risk of cardiovascular disease. Although studies suggest that CsA-induced hyperlipidemia is mediated by inhibition of low-density lipoprotein receptor (LDLr)-mediated lipoprotein clearance, the data supporting this are inconclusive. We therefore sought to investigate the role of the LDLr in CsA-induced hyperlipidemia by using Ldlr-knockout mice (Ldlr(-/-))., Approach and Results: Ldlr(-/-) and wild-type (wt) C57Bl/6 mice were treated with 20 mg/kg per d CsA for 4 weeks. On a chow diet, CsA caused marked dyslipidemia in Ldlr(-/-) but not in wt mice. Hyperlipidemia was characterized by a prominent increase in plasma very low-density lipoprotein and intermediate-density lipoprotein/LDL with unchanged plasma high-density lipoprotein levels, thus mimicking the dyslipidemic profile observed in humans. Analysis of specific lipid species by liquid chromatography-tandem mass spectrometry suggested a predominant effect of CsA on increased very low-density lipoprotein-IDL/LDL lipoprotein number rather than composition. Mechanistic studies indicated that CsA did not alter hepatic lipoprotein production but did inhibit plasma clearance and hepatic uptake of [(14)C]cholesteryl oleate and glycerol tri[(3)H]oleate-double-labeled very low-density lipoprotein-like particles. Further studies showed that CsA inhibited plasma lipoprotein lipase activity and increased levels of apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9., Conclusions: We demonstrate that CsA does not cause hyperlipidemia via direct effects on the LDLr. Rather, LDLr deficiency plays an important permissive role for CsA-induced hyperlipidemia, which is associated with abnormal lipoprotein clearance, decreased lipoprotein lipase activity, and increased levels of apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9. Enhancing LDLr and lipoprotein lipase activity and decreasing apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9 levels may therefore provide attractive treatment targets for patients with hyperlipidemia receiving CsA., (© 2016 American Heart Association, Inc.)

Rationale: High-density lipoprotein (HDL) is a heterogeneous population of particles. Differences in the capacities of HDL subfractions to remove cellular cholesterol may explain variable correlations between HDL-cholesterol and cardiovascular risk and inform future targets for HDL-related therapies. The ATP binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux to lipid-free apolipoprotein A-I, but the majority of apolipoprotein A-I in the circulation is transported in a lipidated state and ABCA1-dependent efflux to individual HDL subfractions has not been systematically studied., Objective: Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process., Methods and Results: We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions., Conclusions: Small, dense HDL subfractions are the most efficient mediators of cholesterol efflux, and ABCA1 mediates cholesterol efflux to small dense HDL and to lipid-free apolipoprotein A-I. HDL-directed therapies should target increasing the concentrations or the cholesterol efflux capacity of small, dense HDL species in vivo., (© 2015 American Heart Association, Inc.)

Apolipoprotein E has critical roles in the protection against atherosclerosis and is understood to follow the classical constitutive secretion pathway. Recent studies have indicated that the secretion of apoE from macrophages is a regulated process of unexpected complexity. Cholesterol acceptors such as apolipoprotein A-I, high density lipoprotein, and phospholipid vesicles can stimulate apoE secretion. The ATP binding cassette transporter ABCA1 is involved in basal apoE secretion and in lipidating apoE-containing particles secreted by macrophages. However, the stimulation of apoE secretion by apoA-I is ABCA1-independent, indicating the existence of both ABCA1-dependent and -independent pathways of apoE secretion. The release of apoE under basal conditions is also regulated, requiring intact protein kinase A activity, intracellular calcium, and an intact microtubular network. Mathematical modeling of apoE turnover indicates that whereas some pools of apoE are committed to either secretion or degradation, other pools can be diverted from degradation toward secretion. Targeted inhibition or stimulation of specific apoE trafficking pathways will provide unique opportunities to regulate the biology of this important molecule.

Macrophage-specific expression of apolipoprotein (apo)E protects against atherosclerosis; however, the signaling and trafficking pathways regulating secretion of apoE are unknown. We investigated the roles of the actin skeleton, microtubules, protein kinase A (PKA) and calcium (Ca2+) in regulating apoE secretion from macrophages. Disrupting microtubules with vinblastine or colchicine inhibited basal secretion of apoE substantially, whereas disruption of the actin skeleton had no effect. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI(14-22)) all decreased basal secretion of apoE by between 50% to 80% (P<0.01). Pulse-chase experiments demonstrated that inhibition of PKA reduced the rate of apoE secretion without affecting its degradation. Confocal microscopy and live cell imaging of apoE-green fluorescent protein-transfected RAW macrophages identified apoE-green fluorescent protein in vesicles colocalized with the microtubular network, and inhibition of PKA markedly inhibited vesicular movement. Chelation of intracellular calcium ([Ca2+]i) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester (BAPTA-AM) inhibited apoE secretion by 77.2% (P<0.01). Injection of c57Bl6 apoE+/+ bone marrow-derived macrophages into the peritoneum of apoE-/- C57Bl6 mice resulted in time-dependent secretion of apoE into plasma, which was significantly inhibited by transient exposure of macrophages to BAPTA-AM and colchicine and less effectively inhibited by H89. We conclude that macrophage secretion of apoE occurs via a PKA- and calcium-dependent pathway along the microtubule network.

Objective: To study the acceptor specificity for human ABCG1 (hABCG1)-mediated cholesterol efflux., Methods and Results: Cells overexpressing hABCG1 were created in Chinese Hamster Ovary (CHO-K1) cells and characterized in terms of lipid composition. hABCG1 expressed in these cells formed homodimers and was mostly present intracellularly. Cholesterol efflux from hABCG1 cells to HDL2 and HDL3 was increased but not to lipid-free apolipoproteins. A range of phospholipid containing acceptors apart from high-density lipoprotein (HDL) subclasses were also efficient in mediating ABCG1-dependent export of cholesterol. Importantly, a buoyant phospholipid-containing fraction generated from incubation of lipid-free apoA-I with macrophages was nearly as efficient as HDL2. The capacity of acceptors to induce ABCG1-mediated efflux was strongly correlated with their total phospholipid content, suggesting that acceptor phospholipids drive ABCG1-mediated efflux. Most importantly, acceptors for ABCG1-mediated cholesterol export could be generated from incubation of cells with lipid-free apoA-I through the action of ABCA1 alone., Conclusions: These results indicate a synergistic relationship between ABCA1 and ABCG1 in peripheral tissues, where ABCA1 lipidates any lipid-poor/free apoA-I to generate nascent or pre-beta-HDL. These particles in turn may serve as substrates for ABCG1-mediated cholesterol export.

Objective: Vein grafts fail because of the development of intimal hyperplasia and accelerated atherosclerosis. Placement of an external stent around vein grafts resulted in an inhibition of intimal hyperplasia in several animal studies. Here, we assess the effects of external stenting on accelerated atherosclerosis in early vein grafts in carotid arteries in hypercholesterolemic apolipoprotein E*3-Leiden transgenic mice., Methods and Results: Venous interposition grafting was performed in apolipoprotein E*3-Leiden mice fed standard chow or a highly cholesterol-rich diet for 4 weeks. After engraftment, external stents with different inner diameters (0.4 or 0.8 mm) were placed. In unstented vein grafts in hypercholesterolemic mice, thickening up to 50 times the original thickness, with foam cell-rich lesions, calcification, and necrosis, was observed within 28 days. The atherosclerotic lesions observed show high morphological resemblance to atherosclerotic lesions observed in human vein grafts. In stented vein grafts in hypercholesterolemic mice, no foam cell accumulation or accelerated atherosclerosis was observed. Compared with unstented vein grafts, stenting of vein grafts in a hypercholesterolemic environment resulted in a 94% reduction of vessel wall thickening. These effects were independent of stent size., Conclusions: Extravascular stent placement results in strong inhibition of accelerated vein graft atherosclerosis in hypercholesterolemic transgenic mice and thereby provides a perspective for therapeutic intervention in vein graft diseases.

An increasing body of evidence from both animal models and human specimens suggests that apoptosis or programmed cell death is a major event in the pathophysiology of atherosclerosis. Although the significance of apoptosis in atherosclerosis remains unclear, it has been proposed that apoptotic cell death contributes to plaque instability, rupture and thrombus formation. Biochemical and genetic analyses of apoptosis provide an increasingly detailed picture of the intracellular signaling pathways involved. Nevertheless, it remains to be determined whether apoptosis can become a clinically important approach to modulate plaque progression. In this review, we have outlined some of the most recent results concerning apoptosis in atherosclerosis with a special focus on oxidized lipids, inflammation and therapeutic regulation of the apoptotic cell death process.

Cell proliferation and cell death (either necrosis or apoptosis) are key processes in the progression of atherosclerosis. The tumor suppressor gene p53 is an essential gene in cell proliferation and cell death and is upregulated in human atherosclerotic plaques, both in smooth muscle cells and in macrophages. In the present study, we investigated the importance of macrophage p53 in the progression of atherosclerosis using bone marrow transplantation in APOE*3-Leiden transgenic mice, an animal model for human-like atherosclerosis. APOE*3-Leiden mice were lethally irradiated and reconstituted with bone marrow derived from either p53-deficient (p53(-/-)) or control (p53(+/+)) donor mice. Reconstitution of mice with p53(-/-) bone marrow did not result in any hemopoietic abnormalities as compared with p53(+/+) transplanted mice. After 12 weeks on an atherogenic diet, APOE*3-Leiden mice reconstituted with p53(-/-) bone marrow showed a significant (P=0.006) 2.3-fold increase in total atherosclerotic lesion area as compared with mice reconstituted with p53(+/+) bone marrow. Although likely a secondary effect of the increased lesion area, p53(-/-) transplanted mice also showed significantly more lesion necrosis (necrotic index, 1.1+/-1.3 versus 0.2+/-0.7; P=0.04) and lesion macrophages (macrophage area, 79.9+/-40.0 versus 39.7+/-27.3x10(3) micrometer(2) per section; P=0.02). These observations coincided with a tendency toward decreased apoptosis (terminal deoxynucleotidyl transferase end-labeling [TUNEL]-positive nuclei going from 0.42+/-0.39 to 0.14+/-0.15%, P=0.071), whereas the number of proliferating cells (5'-bromo-2'-deoxyuridine-positive nuclei) was not affected (3.75+/-0.98 versus 4.77+/-2.30%; P=0.59). These studies indicate that macrophage p53 is important in suppressing the progression of atherosclerosis and identify a novel therapeutic target for regulating plaque stability.

Increased oxidative stress is a major characteristic of hypercholesterolemia-induced atherosclerosis. The oxidative environment is mainly created by the production of reactive oxygen species, which are assumed to mediate vascular tissue injury. Oxidative DNA damage resulting from free radical attack remains, however, a poorly examined field in atherosclerosis. Male New Zealand White rabbits were fed a cholesterol-rich diet (0.3%) for 24 weeks. The induced atherosclerotic plaques showed elevated levels of the DNA damage marker 7,8-dihydro-8-oxoguanine (8-oxoG) as demonstrated by immunohistochemistry. 8-oxoG immunoreactivity was found predominantly in the superficial layer of the plaque containing numerous macrophage-derived foam cells but not in the media or in arteries of age-matched control animals. Alkaline single-cell gel electrophoresis revealed that the number of DNA strand breaks was significantly higher in the plaque as compared with control samples of normolipemic animals. These changes were associated with the upregulation of DNA repair enzymes (poly[ADP-ribose] polymerase-1, p53, phospho-p53 [phosphorylated at Ser392], and XRCC1 [x-ray repair cross-complementing 1]). DNA strand breaks normalized after 4 weeks of dietary lipid lowering. However, a significant reduction of 8-oxoG immunoreactivity was only observed after a prolonged period of lipid lowering (12 to 24 weeks). Repair pathways started to decline progressively when cholesterol-fed animals were placed on a normal diet. In conclusion, oxidative DNA damage and increased levels of DNA repair, both associated with diet-induced hypercholesterolemia, are strongly reduced during dietary lipid lowering. These findings may provide a better insight into the benefits of lipid-lowering therapy on plaque stabilization.

Positioning a silicone collar around the rabbit carotid artery induces a smooth muscle cell-rich intimal thickening. We investigated the localization of inducible nitric oxide synthase (iNOS) during thickening of the intima, the effect of iNOS inhibition on intimal thickness, and the effect of oxidized LDL (ox-LDL) on iNOS expression in the vessel wall. Collars were positioned for 18 hours or for 3, 7, or 14 days. Arterial cross sections were immunostained for iNOS, including naïve, sham-operated, and carotid arteries in which ox-LDL had been infused locally for 14 days. Furthermore, collars were connected to osmotic minipumps for local delivery (5 microL. h(-1), 14 days, n=12) of saline or the iNOS inhibitor L-N(6)-(1-iminoethyl)-lysine-HCl (L-NIL, 10 mmol/L). In the adventitia and the periadventitial granulation tissue of collared arteries, iNOS-positive macrophages and T lymphocytes were present from 18 hours onward. The media and intima were negative for iNOS. Reverse transcription-polymerase chain reaction revealed iNOS mRNA in collared but not in sham-operated arteries. Local inhibition of iNOS doubled the intimal thickness and decreased nitrotyrosine staining. Ox-LDL-treated arteries, which had a thicker intima, showed a pronounced influx of macrophages and T lymphocytes in all layers of the vessel wall, accompanied by iNOS expression in a subpopulation of these cells. Our study indicates that iNOS was not induced in intimal thickenings predominantly consisting of smooth muscle cells. However, iNOS was expressed in (peri)adventitial tissue and counteracted the progression of intimal thickening. Ox-LDL treatment was accompanied by an abundant influx of iNOS-positive macrophages and T lymphocytes in the vessel, but this could not prevent the progression of intimal thickening.

During the formation of intimal thickening in normocholesterolemic rabbits, von Willebrand factor (vWF) is increased in the endothelial cells (ECs) and deposited in the intima. We investigated whether this also occurs during cholesterol-induced plaque formation, whether the synthesis of vWF increases, and whether this influences platelet adhesion. Rabbits were fed a cholesterol-rich (0.3%) diet for 26 weeks. Thereafter, half of the animals received a normal diet for another 26 weeks (cholesterol withdrawal). To induce intimal thickening in normocholesterolemic rabbits, collars were positioned around the carotid artery. Arterial segments were studied using immunohistochemistry, reverse transcription-polymerase chain reaction, electron microscopy, and platelet adhesion tests. Cholesterol treatment induced plaque formation in the aorta. The ECs had a cuboidal aspect, showed a dense immunoreactivity for vWF, a pronounced rough endoplasmic reticulum, and numerous Weibel-Palade bodies. There were subendothelial vWF deposits in the plaques and vWF mRNA was significantly increased as compared with controls. Similar changes were seen after collar-induced intimal thickening. After cholesterol withdrawal, both vWF mRNA and the ultrastructural morphology of the ECs normalized, and the vWF deposits disappeared from the plaque. Perfusion studies with anticoagulated rabbit blood over cross-sections of atherosclerotic aortas revealed increased vWF-mediated platelet adhesion in the subendothelial plaque region. Whereas rabbit platelets perfused through the lumen adhered to the same extent to de-endothelialized aortas of normocholesterolemic and atherosclerotic rabbits, vWF mediated platelet adhesion to endothelium was observed in atherosclerotic but not in normal aortas. Our results show an increased synthesis and (sub)endothelial presence of vWF after vascular injury, with functional consequences for platelet deposition on the vessel wall.

The aim of this study was to evaluate whether vascular remodeling and neointimal thickening occur after balloon dilatation of the nonatherosclerotic rabbit carotid artery, and whether both processes are influenced by continuous perivascular delivery of L-arginine or the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). In the first experiment, histological and morphometric evaluation of arteries was performed at different time points after balloon dilatation: 10 minutes (n=7), and 1 (n=7), 2 (n=9), 3 (n=20), or 10 (n=5) weeks. Neointimal thickening progressively contributed to luminal narrowing for at least 10 weeks after angioplasty. During the first 2 weeks after dilatation, a significant decrease of the total vessel area was measured. Ten weeks after dilatation, both the neointimal and total vessel area were increased without further changing of the luminal area. In the second experiment, endothelial injured rabbits were randomly assigned to receive 2 weeks of continuous local perivascular physiological salt solution (n=6), L-arginine (n=8), or L-NAME (n=7), starting immediately after balloon dilatation (ie, local drug delivery during the first phase of the biphasic vascular remodeling process). Perivascular L-arginine delivery significantly reduced the neointimal area, despite an increased number of neointimal Ki-67-positive smooth muscle cells. Both the luminal area and total vessel area were significantly increased. Serum L-arginine levels remained unchanged. L-NAME administration had no effect on the neointimal area, nor on the luminal and total vessel area. Neointimal formation and biphasic vascular remodeling occur after experimental balloon dilatation of the nonatherosclerotic rabbit carotid artery, and can be influenced by continuous local perivascular delivery of L-arginine.

Background: This study documents (1) the progression of atherosclerosis along the entire arterial tree in APOE*3-Leiden mice after 1, 4, 6, 9, and 12 months of a high-fat/high-cholesterol (HFC) diet and (2) the amount and phenotype of DNA-synthesizing and apoptotic cells in different lesion types after 6 months of HFC diet., Methods and Results: Diet duration was correlated with a craniocaudal progression of lesion development and with an increase in severity of the lesion. Typically, the lesions contained smooth muscle cells, macrophages, and T lymphocytes and were covered by an intact endothelium. Whereas DNA synthesis (BrdU uptake) was usually elevated in type II lesions (8.6+/-0.8% versus 1.0+/-0.2% in the nondiseased arterial wall; P<0.05), apoptosis was found primarily in advanced lesions (type IV, 1.3+/-0.1% and type V, 1.2+/-0.2% versus 0.04+/-0.04% in the nondiseased arterial wall [P<0.05]). Cell phenotyping revealed that the majority of DNA synthesis and apoptosis was confined to the macrophage-derived foam cell (68.6+/-3. 0% and 82.2+/-4.6%, respectively)., Conclusions: This study shows that in APOE*3-Leiden mice, duration of an HFC diet is associated with (1) a craniocaudal progression of lesion development and (2) an increased complexity of atherosclerotic lesions. Furthermore, DNA synthesis is predominant in early lesions, whereas apoptosis is present mainly in more advanced lesions. Both parameters of cell turnover are confined primarily to the macrophage-derived foam cell.

Several laboratories have demonstrated the presence of apoptotic cell death in atherosclerotic plaques. Apoptosis occurs in at least 2 stages. The final "execution" phase, which includes DNA fragmentation, is brief ( approximately 6 hours) and irreversible and can be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique. The TUNEL technique is only selective (rather than specific) for apoptotic nuclei, because these contain a far greater degree of DNA fragmentation than do nonapoptotic nuclei. Nonapoptotic cell nuclei that show high levels of RNA synthesis and splicing can also be labeled. This could explain the large variation in the reported percentages of TUNEL-positive nuclei in the plaques. Therefore, the TUNEL technique should be combined with additional techniques, such as markers of transcription and morphological criteria. Recent studies indicate that human fatty streaks differ from adaptive intimal thickenings by the presence of cells containing pro-apoptotic proteins. However, apoptotic cell death is present only in advanced atherosclerotic plaques that show a dense macrophage infiltration. This indicates that although both smooth muscle cells and macrophages within the human fatty streaks become susceptible to apoptosis, additional factors (mainly macrophage- and lipid-derived factors) are necessary to terminate the cell death pathway.

Unstable human atherosclerotic plaques are characterized by a thin fibrous cap that contains few smooth muscle cells (SMCs) and numerous foam cells of macrophagic origin. Apoptosis of SMCs in the fibrous cap could destabilize the plaque and promote plaque rupture. In an experimental approach, we have studied apoptotic cell death and related proteins in atherosclerotic plaques of cholesterol-fed rabbits and examined the effects of cholesterol withdrawal. The induced atherosclerotic plaques at the thoracic aorta were composed of both fibromuscular tissue and foam cells. The presence of SMCs overlying macrophage accumulation was reminiscent of the structure of human atherosclerotic plaques. The plaques showed signs of cell replication and apoptotic cell death (1.8+/-0.5% terminal deoxynucleotidyl transferase end-labeling [TUNEL]-positive nuclei). Cell replication was confined mostly to the macrophages, whereas 34% of the TUNEL-labeled cells were SMCs. Both the macrophages and SMCs in the plaques expressed BAX, a proapoptotic protein of the BCL-2 family. After 6 months of cholesterol withdrawal, the thickness of the plaques in all localizations of the aorta was unchanged, but apoptosis was nearly absent (<0.1% of nuclei). Moreover, macrophages disappeared from the plaques, whereas the SMCs that remained present lost their lipid accumulation and strongly reduced their BAX expression. These changes were associated with a reduction of cell replication and increased deposition of fibrillar collagen fibers in the plaques, which pointed to plaque stabilization. In conclusion, the cell composition but not the thickness of atherosclerotic plaques was profoundly altered after a 6-month cholesterol withdrawal period. These changes were associated with a strong reduction of cell replication and apoptotic cell death. Moreover, the expression of the proapoptotic factor, BAX, was reduced in the remaining cells, which were mainly SMCs. These findings could help to explain the benefit of lipid-lowering therapy on plaque stabilization.

Background: The transition of a fatty streak into an atherosclerotic plaque is characterized by the appearance of focal and diffuse regions of cell death. We have investigated the distribution of apoptotic cell death and apoptosis-related proteins in early and advanced atherosclerotic lesions., Methods and Results: Human atherosclerotic plaques were studied by whole-mount carotid endarterectomy specimens (n=18). This approach allowed comparison of adaptive intimal thickenings, fatty streaks, and advanced atherosclerotic plaques of the same patient. The fatty streaks differed from adaptive intimal thickenings by the presence of BAX (P<0.01), a proapoptotic protein of the BCL-2 family. Both regions were composed mainly of smooth muscle cells (SMCs), and macrophage infiltration was low and not different. Apoptosis, as detected by DNA in situ end labeling (terminal deoxynucleotidyl transferase end labeling [TUNEL] and in situ nick translation) was not present in these regions. Apoptosis of SMCs and macrophages, however, was present in advanced atherosclerotic plaques that were present mainly in the carotid sinus. A dense infiltration of macrophages (5.8+/-3% surface area) was present in these advanced atherosclerotic plaques. Cytoplasmic remnants of apoptotic SMCs, enclosed by a cage of thickened basal lamina, were TUNEL negative and remained present in the plaques as matrix vesicles., Conclusions: We conclude that SMCs within human fatty streaks express BAX, which increases the susceptibility of these cells to undergo apoptosis. The localization of these susceptible SMCs in the deep layer of the fatty streaks could be important in our understanding of the transition of fatty streaks into atherosclerotic plaques, which are characterized by regions of cell death. Matrix vesicles are BAX-immunoreactive cytoplasmic remnants of fragmented SMCs that can calcify and may be considered the graves of SMCs that have died in the plaques.

In this study, the involvement of polymorphonuclear leukocytes (PMNs) in the development of intimal thickening was investigated. A fibromuscular intima was induced by placing a silicone collar around the rabbit carotid artery for 3 days or 2 weeks; the contralateral artery was sham operated. Rabbits received placebo treatments (groups 1 and 3), granulocyte-colony stimulating factor (group 2; G-CSF, 20 microg x kg(-1) x d(-1), delivered by subcutaneous osmotic pumps), or an anti-CD18 monoclonal antibody (group 4; 1.5 mg/kg i.v.). The G-CSF treatment raised the peripheral PMN count 5- to 12-fold but had no effect on intimal thickening on day 3, 12, or 14. A single injection of anti-CD18 prevented PMN extravasation 6 hours after collar implantation without influencing intimal hyperplasia on day 14. Repeated daily administration of anti-CD18 strongly bound to CD18 on peripheral PMNs and inhibited both PMN-dependent plasma extravasation in the skin and accumulation of CD14-immunoreactive leukocytes in the intima and media. However, anti-CD18 did not suppress early intimal thickening or accumulation of alpha-smooth muscle actin-immunoreactive cells by day 3. It thus appears that the PMN influx in the intima and media evoked by the perivascular collar is of little functional relevance to the subsequent smooth muscle cell migration and intimal thickening in this model.

Oxidized LDL (oxLDL) has been implicated in atherogenesis on the basis of in vitro studies and is present in atherosclerotic lesions. The aim of this study was to investigate the effects of LDL and oxLDL on intimal thickening in vivo. Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of rabbits for 2 weeks. The collars were connected to osmotic minipumps containing LDL (7 micrograms h-1, n = 16 arteries), oxLDL (Cu2+ oxidized, 7 micrograms h-1, n = 16), or phosphate-buffered saline (5 microL h-1, n = 16). Segments proximal to the collars served as controls. Collar placement without lipoprotein application resulted in the appearance of alpha-SMC actin-immunoreactive cells in the intima, thereby increasing the intimal thickness from 5 +/- 1 to 26 +/- 5 microns. The perivascular infusion of LDL or oxLDL within the collar significantly enhanced the development of the intima ninefold and sevenfold, respectively. The large intimas resulting from lipoprotein exposure were infiltrated by macrophages and T lymphocytes, and the intimal collagen area was increased from 5 +/- 2% in the discrete collar-induced intima to approximately 20% in the lipoprotein-evoked lesions. In conclusion, the local vascular application of LDL, oxidized in vitro or possibly in vivo, elicited an inflammatory-fibroproliferative response characteristic of arteriosclerotic lesions, thereby demonstrating an active role for this class of lipoproteins in the disease process.

The positioning of a soft silicone collar around the rabbit carotid artery induces intimal thickening. We investigated to which extent occlusion of the vasa vasorum, damage of the perivascular nerve network, and/or changes in blood flow velocity contribute to intimal thickening. To this end, collars with different bores (diameter of inlet and outlet) were positioned around the carotid artery of male rabbits for 14 days. In another experiment, 75% of the wall of fitting collars was removed (open collar). In the midcollar region, the cross-sectional area of the intima reached a maximum (72 +/- 14 mm2/1000) when the endings of the collar fitted the artery closely. Removal of the side wall of these fitting collars reduced intimal thickening by 90%. Examination of unoperated carotid arteries never showed penetration of the adventitia or the media by vasa vasorum. The perivascular neuronal network in the region surrounded by a closed or an open collar was almost completely lost as compared with the zones outside the collar. Both the closed and open collar slightly bent the artery and increased the peak systolic velocity, measured with pulsed color Doppler after 6 hours, to a similar extent as compared with the proximal zone outside the collar. After 2 weeks, the peak systolic velocity within both the closed and open collar was partly normalized and was statistically not different from the proximal zone outside the collar. In conclusion, the geometry of the collar influenced the extent of intimal thickening, whereby more intimal thickening was obtained with a collar whose endings fit the carotid artery, rather than with a loose collar. Moreover, a closed structure was essential. The results obtained with the open collar exclude occlusion of vasa vasorum, damage of the perivascular neuronal network, kinking of the artery, and changes in blood flow velocity as major factors in the collar-induced intimal thickening. Our findings are consistent with the possibility that intimal thickening is the consequence of the combination of both vascular injury and hindrance of transmural flow by the collar. The obstruction of transmural fluid transport may then lead to retention of toxic metabolites, and/or cytokines within the segment enclosed by the collar.

Differentiation of vascular smooth muscle cells (SMCs) is characterized by several molecular transitions. As differentiation proceeds, proteins of the cytoskeletal and contractile apparatus, such as alpha-smooth muscle actin, smooth muscle myosin, calponin, and heavy caldesmon, and the expression of the membrane-related protein smooth muscle phosphoglucomutase-related protein increase, whereas the expression of other proteins, such as fibronectin splice variants with extradomains A (EDA) and B (EDB), decreases. In this study, we investigated the differentiation of the SMCs of the ductus arteriosus during the development of intimal thickening. Ascending and descending aortas of the same age were used for comparison because these vessels lack intimal thickening. In the fetal ductus arteriosus, a relatively early differentiation of the contractile apparatus was observed compared with the ascending and descending aortas. EDA and EDB expression was already low, being similar in the ductus and descending aorta and even lower in the ascending aorta. In the neonatal ductus, SMCs of the media and outer intima were well differentiated and comparable with SMCs of the ascending aorta. Dedifferentiated SMCs, with a low expression of cytoskeletal and contractile proteins and a high expression of EDA and EDB, were found in regions in the inner intima that show features of progression of intimal thickening and in areas of cytolytic necrosis in the media. With a technique using in situ end labeling of DNA fragments, we found extensive apoptosis in the area of cytolytic necrosis and to a lesser extent in these areas of the inner intima. In conclusion, SMCs of the fetal ductus arteriosus have an advanced differentiation of the contractile apparatus compared with the adjacent aorta. Reexpression of fetal characteristics is seen in a number of cells in inner intima and media of the neonatal ductus arteriosus. The finding of apoptosis in these areas suggests that dedifferentiation and apoptosis are associated processes that may play a role in vascular remodeling.

The aim of the study was to assess the contribution of thrombus incorporation into neointimal thickening in the rabbit carotid artery after deep vascular injury induced by balloon angioplasty compared with superficial vascular injury induced by a perivascular collar. Besides CD 31 (PECAM 1), vimentin, alpha-smooth muscle actin, rabbit anti-macrophage monoclonal antibody and proliferating cell nuclear antigen, fibrin(ogen) and von Willebrand factor (vWF) deposition was assessed immunohistochemically. Angioplasty was performed in 47 rabbits and evaluated immediately (n = 7), after 6 hours (n = 4), and after 1 (n = 7), 2 (n = 9), or 3 (n = 20) weeks. A collar was placed in 29 rabbits and evaluated immediately (n = 5), after 6 hours (n = 5), and after 1 (n = 7), 2 (n = 10), or 3 (n = 2) weeks. After dilatation, the arteries were extensively denuded of endothelium, the internal elastic membrane was ruptured and blood-filled clefts were present in the media, pointing to deep vascular (type III) injury. Six hours later, mural fibrin(ogen) thrombi were formed, specially at sites with severe damage. This fibrin(ogen) matrix became infiltrated by phagocytes and smooth muscle cells. A luminal cap covered by regenerating endothelium was formed, demonstrating increased immunoreactivity to vWF. vWF was deposited in the extracellular neointimal spaces. Fibrin(ogen) thrombus deposition and incorporation appeared to be protracted phenomena for at least 2 weeks. After collar placement, minimal endothelial denudation was documented, pointing to focal superficial (type I) vascular injury. In subsequent weeks, neointimal thickening was associated with vWF deposition but not with fibrin(ogen) thrombus incorporation. In conclusion, mural fibrin(ogen) thrombus formation and incorporation contribute to neointima formation after deep vascular injury and seem to occur for several weeks after the initial insult.

Fibrates are widely used drugs in hyperlipidemic disorders. In addition to lowering serum triglyceride levels, fibrates have also been shown to reduce elevated plasma plasminogen activator inhibitor-1 (PAI-1) levels in vivo. We demonstrate that fibrates suppress PAI-1 synthesis in cultured cynomolgus monkey hepatocytes in a concentration-dependent way (0.1 to 1.0 mmol/L) and independent of their lipid-lowering effect. Different fibrates showed different potency in suppressing PAI-1 production: gemfibrozil and clofibric acid, at a concentration of 1 mmol/L, reduced PAI-1 synthesis over 24 hours to 52 +/- 20% and 60 +/- 5%, while clofibrate and bezafibrate lowered PAI-1 synthesis to only 86 +/- 17% and 84 +/- 15% of control values, respectively. These changes in PAI-1 production by fibrates correlated with changes in PAI-1 mRNA levels and were also visible at the level of gene transcription. Fibrates did not lower basal PAI-1 synthesis but attenuated an acceleration of PAI-1 production during culture. The suppressing effect of fibrates on PAI-1 synthesis could not be mimicked with activators or inhibitors of protein kinase C (PKC). Furthermore, fibrates did not inhibit the increase in PAI-1 synthesis induced by epidermal growth factor or transforming growth factor-beta. These results make mechanisms involving PKC modulation or growth factor receptor inactivation as a mode of action of fibrates unlikely. The suppressing effect of fibrates on PAI-1 synthesis could involve the nuclear receptor peroxisome proliferator-activated receptor (PPAR) and its heterodimeric partner, the retinoid X receptor (RXR). The alpha forms of PPAR and RXR were both found to be expressed in cynomolgus monkey hepatocytes. the ligand for RXR alpha, 9-cis retinoic acid, suppressed PAI-1 synthesis to the same extent as gemfibrozil, while a combination of gemfibrozil and 9-cis retinoic acid had no more effect on PAI-1 synthesis than any of these compounds alone at optimal concentrations. In conclusion, fibrates downregulate an induced PAI-1 production in cynomolgus monkey hepatocytes independent of a decrease in triglyceride levels. A possible involvement of PPAR alpha/RXR alpha in this down-regulation is discussed.

Background: Occlusion of saphenous vein grafts is a major problem after coronary artery bypass graft surgery. Diffuse intimal thickening develops in all implanted aortocoronary saphenous vein grafts within 6 months to 1 year. In some regions of the thickened intima, foam cells accumulate along the luminal margin. This particular morphology resembles the morphology of unstable atherosclerotic plaques as they occur in coronary arteries. In the present study, we focused on the possible topographic relation between luminal foam cell accumulation and cell death of smooth muscle cells (SMCs) within the adjacent thickened intima., Methods and Results: Segments of occluded and suboccluded implanted human aortocoronary saphenous vein grafts were obtained during reintervention coronary artery bypass graft surgery in 30 patients. In the regions of the vein grafts with luminal foam cell accumulation, the percentage of SMC alpha-actin immunoreactive area of the superficial intimal thickening was 6 +/- 1.4%, which was different from the 17.6 +/- 2.3% of the deep intimal thickening. A strong negative correlation between the number of foam cell nuclei and the percentage of SMC alpha-actin immunoreactive area in the adjacent superficial intimal thickening was present (r = -.77, P < .001). Within the superficial intimal thickening, cytoplasmic and DNA fragmentation could be detected, which points to apoptotic cell death. A fraction of the cytoplasmic fragments fitted the ultrastructural characteristics of matrix vesicles and showed pronounced calcium and phosphorus accumulation as demonstrated with the use of x-ray microanalysis., Conclusions: The close spatial relation among foam cell accumulation, pronounced intimal SMC loss, and cell death suggests the presence of a foam cell-derived factor that can induce cell death in the SMC population of the intimal thickening. The depletion of the intimal SMC population could promote plaque rupture and thrombotic complications in the grafts.

The positioning of a nonocclusive silicone collar around the rabbit carotid artery results in the formation of a neointima under a morphologically continuous endothelium. We wished to determine whether oral treatment with angiotensin-converting enzyme (ACE) inhibitors prevents or retards intimal thickening and whether this is related to the blood pressure (BP) lowering effects of such drugs. Silicone collars were placed around the left carotid artery of 104 male New Zealand white rabbits for 14 days. The contralateral carotid artery was sham-operated. Three ACE inhibitors were administered from 7 days before collar placement until the end of the experiment: zabicipril (0, 0.03, 0.10, or 0.30 mg/kg/day), moexipril (0, 0.3, 1, or 3 mg/kg twice daily, b.i.d.), and enalapril (0 or 3 mg/kg/day). Each group consisted of 6-12 animals. BP and plasma ACE activity were measured in the nonanesthetized rabbits after 3-week treatment. To evaluate intimal thickening, we measured the cross-sectional area of intima and media. The positioning of the collar led to significant intimal thickening after 14 days. Although the ACE inhibitors decreased BP (zabicipril, 9, 16, 16%; moexipril, 10, 22, 31%; enalapril, 15%) and plasma ACE activity (zabicipril, 87, 88, 92%; moexipril, 79, 92, 93%; enalapril, 88%) significantly and dose dependently, they did not reduce intimal thickening or the cross-sectional area of the media. Angiotensin II does not play a dominant role in collar-induced intimal thickening in rabbits. Furthermore, reducing the BP of normotensive rabbits does not alter neointima formation in this model.

Intimal thickening in arteries is considered a site of predilection for atherosclerosis. We investigated whether oral application of the nitric oxide (NO) donors SPM-5185 [N-nitratopivaloyl-S-(N'-acetylalanyl)-cysteine ethylester, 10 mg/kg body weight twice daily (b.i.d.)] and molsidomine (10 mg/kg body weight/day) can retard neointima formation and changes in vascular reactivity induced by a nonocclusive, soft silicone collar positioned around the left carotid artery of rabbits. The contralateral carotid artery was sham operated and served as a control. Drug and placebo (diet without drug) treatments were initiated 7 days before placement of the collar. At the end of the experiments, two segments were cut from each collared and sham-treated artery, one for measurement of the cross-sectional area of intima and media and the other for isometric tension recording. Sham treatment did not result in intimal thickening in either group. In contrast, the intima/media (I/M) ratio was considerably increased after 14 days of collar treatment as a result of neointima formation. Intimal thickening was significantly inhibited by SPM-5185 (I/M ratio 0.05 +/- 0.01 vs. 0.11 +/- 0.02, p < 0.05), but not by molsidomine (0.06 +/- 0.02 vs. 0.08 +/- 0.02, p = 0.49), which is a donor of both NO and superoxide anions. Neither collar nor NO donor treatment altered the area of the media. SPM-5185 did not alter the percentage of replicating smooth muscle cells (SMC) in the media after collar treatment, as demonstrated by their immunoreactivity for proliferating cell nuclear antigen (PCNA).(ABSTRACT TRUNCATED AT 250 WORDS)