Examining atherosclerotic lesions in three dimensions at the nanometer scale with cryo-FIB-SEM.

We employed in a correlative manner an unconventional combination of methods, comprising cathodoluminescence, cryo-scanning electron microscopy (SEM), and cryo-focused ion beam (FIB)-SEM, to examine the volumes of thousands of cubed micrometers from rabbit atherosclerotic tissues, maintained in close-to-native conditions, with a resolution of tens of nanometers. Data from three different intralesional regions, at the media-lesion interface, in the core, and toward the lumen, were analyzed following segmentation and volume or surface representation. The media-lesion interface region is rich in cells and lipid droplets, whereas the core region is markedly richer in crystals and has lower cell density. In the three regions, thin crystals appear to be associated with intracellular or extracellular lipid droplets and multilamellar bodies. Large crystals are independently positioned in the tissue, not associated with specific cellular components. This extensive evidence strongly supports the idea that the lipid droplet surfaces and the outer membranes of multilamellar bodies play a role in cholesterol crystal nucleation and growth and that crystal formation occurs, in part, inside cells. The correlative combination of methods that allowed the direct examination of cholesterol crystals and lipid deposits in the atherosclerotic lesions may be similarly used for high-resolution examination of other tissues containing pathological or physiological cholesterol deposits.

Apolipoprotein CIII Deficiency Protects Against Atherosclerosis in Knockout Rabbits.

OBJECTIVE: Apo (apolipoprotein) CIII mediates the metabolism of triglyceride (TG)-rich lipoproteins. High levels of plasma apoCIII are positively correlated with the plasma TG levels and increase the cardiovascular risk. However, whether apoCIII is directly involved in the development of atherosclerosis has not been fully elucidated. Approach and Results: To examine the possible roles of apoCIII in lipoprotein metabolism and atherosclerosis, we generated apoCIII KO (knockout) rabbits using ZFN (zinc finger nuclease) technique. On a normal standard diet, apoCIII KO rabbits exhibited significantly lower plasma levels of TG than those of WT (wild type) rabbits while total cholesterol and HDL (high-density lipoprotein) cholesterol levels were unchanged. Analysis of lipoproteins isolated by sequential ultracentrifugation revealed that reduced plasma TG levels in KO rabbits were accompanied by prominent reduction of VLDLs (very-low-density lipoproteins) and IDLs (intermediate-density lipoproteins). In addition, KO rabbits showed faster TG clearance rate after intravenous fat load than WT rabbits. On a cholesterol-rich diet, KO rabbits exhibited constantly and significantly lower levels of plasma total cholesterol and TG than WT rabbits, which was caused by a remarkable reduction of ß-VLDLs-the major atherogenic lipoproteins. ß-VLDLs of KO rabbits showed higher uptake by cultured hepatocytes and were cleared faster from the circulation than ß-VLDLs isolated from WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. CONCLUSIONS: These results indicate that apoCIII deficiency facilitates TG-rich lipoprotein catabolism, and therapeutic inhibition of apoCIII expression may become a novel means not only for the treatment of hyperlipidemia but also for atherosclerosis.

Sex hormones affect endothelial lipase-mediated lipid metabolism and atherosclerosis.

BACKGROUND: Endothelial lipase (EL) plays an important role in lipoprotein metabolism and atherosclerosis. To study the functional roles of EL, we recently generated transgenic (Tg) rabbits and reported that increased hepatic expression of EL in male Tg rabbits significantly reduced diet-induced hypercholesterolemia compared with non-Tg controls. This gender difference suggests that sex hormones may mediate EL functions thereby influencing lipoprotein metabolism. To examine this hypothesis, we compared the effects of orchiectomy and ovariectomy on plasma lipids and diet-induced atherosclerosis in both Tg and non-Tg rabbits. METHODS: Male rabbits were under orchiectomy whereas female rabbits were under ovariectomy. We compared plasma lipids, lipoproteins, and apolipoproteins of rabbits before and after surgery in each group fed either a chow diet or cholesterol-rich diet. RESULTS: On a chow diet, both male and female Tg rabbits showed lower plasma lipids than non-Tg counterparts and this lipid-lowering effect of EL was not affected by either orchiectomy in male or ovariectomy in female Tg rabbits. On a cholesterol diet; however, male Tg rabbits but not female Tg rabbits showed significant resistance to diet-induced hypercholesterolemia and atherosclerosis. The EL-mediated atheroprotective effect was eliminated after orchiectomy in male Tg rabbits. Female Tg rabbits showed similar levels of total cholesterol and lesion size of atherosclerosis compared with non-Tg rabbits and ovariectomy did not affect diet-induced hypercholesterolemia or atherosclerosis. CONCLUSION: These results suggest that increased EL protects against diet-induced hypercholesterolemia and atherosclerosis. The beneficial effect of EL was dependent upon the presence of androgenic hormones.

Detection of potential new biomarkers of atherosclerosis by probe electrospray ionization mass spectrometry.

INTRODUCTION: Atherosclerotic diseases are the leading cause of death worldwide. Biomarkers of atherosclerosis are required to monitor and prevent disease progression. While mass spectrometry is a promising technique to search for such biomarkers, its clinical application is hampered by the laborious processes for sample preparation and analysis. METHODS: We developed a rapid method to detect plasma metabolites by probe electrospray ionization mass spectrometry (PESI-MS), which employs an ambient ionization technique enabling atmospheric pressure rapid mass spectrometry. To create an automatic diagnosis system of atherosclerotic disorders, we applied machine learning techniques to the obtained spectra. RESULTS: Using our system, we successfully discriminated between rabbits with and without dyslipidemia. The causes of dyslipidemia (genetic lipoprotein receptor deficiency or dietary cholesterol overload) were also distinguishable by this method. Furthermore, after induction of atherosclerosis in rabbits with a cholesterol-rich diet, we were able to detect dynamic changes in plasma metabolites. The major metabolites detected by PESI-MS included cholesterol sulfate and a phospholipid (PE18:0/20:4), which are promising new biomarkers of atherosclerosis. CONCLUSION: We developed a remarkably fast and easy method to detect potential new biomarkers of atherosclerosis in plasma using PESI-MS.

Increased Hepatic Expression of Endothelial Lipase Inhibits Cholesterol Diet-Induced Hypercholesterolemia and Atherosclerosis in Transgenic Rabbits.

OBJECTIVE: Endothelial lipase (EL) is a key determinant in plasma high-density lipoprotein-cholesterol. However, functional roles of EL on the development of atherosclerosis have not been clarified. We investigated whether hepatic expression of EL affects plasma lipoprotein metabolism and cholesterol diet-induced atherosclerosis. APPROACH AND RESULTS: We generated transgenic (Tg) rabbits expressing the human EL gene in the liver and then examined the effects of EL expression on plasma lipids and lipoproteins and compared the susceptibility of Tg rabbits with cholesterol diet-induced atherosclerosis with non-Tg littermates. On a chow diet, hepatic expression of human EL in Tg rabbits led to remarkable reductions in plasma levels of total cholesterol, phospholipids, and high-density lipoprotein-cholesterol compared with non-Tg controls. On a cholesterol-rich diet for 16 weeks, Tg rabbits exhibited significantly lower hypercholesterolemia and less atherosclerosis than non-Tg littermates. In Tg rabbits, gross lesion area of aortic atherosclerosis was reduced by 52%, and the lesions were characterized by fewer macrophages and smooth muscle cells compared with non-Tg littermates. CONCLUSIONS: Increased hepatic expression of EL attenuates cholesterol diet-induced hypercholesterolemia and protects against atherosclerosis.

Deficiency of Cholesteryl Ester Transfer Protein Protects Against Atherosclerosis in Rabbits.

OBJECTIVE: CETP (cholesteryl ester transfer protein) plays an important role in lipoprotein metabolism; however, whether inhibition of CETP activity can prevent cardiovascular disease remains controversial. APPROACH AND RESULTS: We generated CETP knockout (KO) rabbits by zinc finger nuclease gene editing and compared their susceptibility to cholesterol diet-induced atherosclerosis to that of wild-type (WT) rabbits. On a chow diet, KO rabbits showed higher plasma levels of high-density lipoprotein (HDL) cholesterol than WT controls, and HDL particles of KO rabbits were essentially rich in apolipoprotein AI and apolipoprotein E contents. When challenged with a cholesterol-rich diet for 18 weeks, KO rabbits not only had higher HDL cholesterol levels but also lower total cholesterol levels than WT rabbits. Analysis of plasma lipoproteins revealed that reduced plasma total cholesterol in KO rabbits was attributable to decreased apolipoprotein B-containing particles, while HDLs remained higher than that in WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. Apolipoprotein B-depleted plasma isolated from CETP KO rabbits showed significantly higher capacity for cholesterol efflux from macrophages than that from WT rabbits. Furthermore, HDLs isolated from CETP KO rabbits suppressed tumor necrosis factor-α-induced vascular cell adhesion molecule 1 and E-selectin expression in cultured endothelial cells. CONCLUSIONS: These results provide evidence that genetic ablation of CETP activity protects against cholesterol diet-induced atherosclerosis in rabbits.

Angiotensin II Destabilizes Coronary Plaques in Watanabe Heritable Hyperlipidemic Rabbits.

OBJECTIVE: Increased plasma concentrations of angiotensin II (Ang II) have been implicated in many cardiovascular diseases, such as atherosclerosis, aortic aneurysms, and myocardial infarction, in humans. However, it is not known whether high levels of plasma Ang II affect coronary plaque stability and subsequent myocardial infarction. This study was designed to examine whether elevated plasma Ang II can directly induce coronary events, such as acute coronary syndrome. APPROACH AND RESULTS: To examine the above hypothesis, we infused Ang II (100 ng/min per kg [low group] and 200 ng/min per kg [high group]) or saline vehicle via osmotic minipumps into Watanabe heritable hyperlipidemic rabbits, a model of human familial hypercholesterolemia and atherosclerosis. Infusion of Ang II resulted in mortality rates of 50% and 92% in the low- and high-Ang II groups, respectively, whereas there were no deaths in the vehicle group. Pathological analysis revealed that Ang II-infused Watanabe heritable hyperlipidemic rabbits that died showed myocardial infarction. Furthermore, Ang II-infused Watanabe heritable hyperlipidemic rabbits exhibited coronary plaque erosion and rupture that were associated with thrombosis. CONCLUSIONS: These findings suggest that increased blood levels of Ang II can destabilize coronary plaques and trigger the thrombosis, which possibly induces myocardial infarction. The model described in this study provides a novel means for the study of human acute coronary syndrome.

Suramin inhibits antibody binding to cell surface antigens and disrupts complement-mediated mesangial cell lysis.

Suramin inhibits immune responses and protects cells against inflammatory cell injury. However, little is known about its mechanisms. Using an in vitro model of glomerular mesangial cell (MC) lysis induced by antibodies plus complement, we investigated the potential protective effects and mechanisms of suramin on immunologic cell injury. Exposure of rat MCs to anti-Thy-1 antibody plus complement or anti-MC rabbit serum caused complement-dependent cell lysis, which was blocked by suramin and its structural analogue NF023 and NF049, but not by PPADS, an antagonist of purinergic receptors. Addition of exogenous ATP also failed to affect MC lysis. Further analysis revealed that suramin interfered with antibody binding to cell membrane antigens and suppressed antibody-induced phosphorylation of several proteins, including p38. Inhibition of p38 with chemical inhibitor significantly attenuated cell injury. Collectively, our results indicate that suramin protects cells against antibody-initiated and complement-dependent cell injury through inhibition of antibody binding to cell surface antigens and suppression of p38 activation. Our study thus provides novel mechanistic insights into the actions of suramin and suggests that suramin might be used to treat certain immune diseases.

Dietary cholesterol atherogenic changes in juvenile rabbits.

Atherosclerotic lesion formation starts during fetal development and progresses with age after adolescence. However, atherogenesis during the juvenile period has not been studied thoroughly. In this study, we examined the atherogenic susceptibility of juvenile rabbits to cholesterol feeding. Male New Zealand White rabbits aged 8 (younger group) and 12 (older group) weeks were fed a 0.5% cholesterol-containing diet for 8 weeks, and then their aortic atherosclerotic lesion areas were evaluated. Plasma concentrations of total cholesterol, triglycerides, and phospholipids did not differ between the two groups; however, plasma concentrations of high-density lipoprotein cholesterol were 23% lower in the younger than in the older group. Atherosclerotic lesion areas were significantly larger in the younger group (32±21%). However, only moderate changes were observed in these areas in the older group (3.3±0.3%). Histological examination showed marked intimal thickening and macrophage accumulation in the aortic lesions of rabbits in the younger group. To the best of our knowledge, this is the first study to show that dietary cholesterol-induced atherogenic changes markedly occur during a short period in juvenile rabbits.

Bisphenol A exposure induces metabolic disorders and enhances atherosclerosis in hyperlipidemic rabbits.

Bisphenol A (BPA) is an artificial environmental endocrine disrupter. Excess exposure to BPA may induce many disorders in the metabolism and cardiovascular system. However, the underlying toxicological mechanisms remain largely unknown. In this study, we administered genetically hyperlipidemic Watanabe heritable hyperlipidemic (WHHL-MI) rabbits (male, 14 week old), which have more common features with humans than the mouse and rat especially in the metabolism and cardiovascular system, with BPA at 40 mg kg(-1) day(-1) for 8 weeks by gavage and compared their plasma lipids, glucose and insulin response with those of the vehicle group. All of the rabbits were sacrificed, and their pancreas, liver, adipose tissue, heart and aorta were analyzed using histological and morphometric methods. Furthermore, we treated human hepatoma HepG2 cells and human umbilical cord vein endothelial cells (HUVECs), with different doses of BPA based on the serum BPA levels in the WHHL rabbits for 6 h to investigate the possible molecular mechanisms. Our results showed that BPA-treated rabbits showed insulin resistance, prominent adipose accumulation and hepatic steatosis. Additionally, BPA exposure also caused myocardial injury and enhanced the development of atherosclerosis in the aortic arch with increased macrophage number (86%) and advanced lesion areas (69%). Increased expression of inflammatory genes found in the liver of BPA-treated rabbits along with the up-regulation of ER stress, lipid and glucose homeostasis and inflammatory genes in the cultured HepG2 cells and HUVECs suggest that BPA may induce metabolic disorders and enhance atherosclerosis through regulating above molecular pathways in the liver and endothelium.

Human apolipoprotein A-II protects against diet-induced atherosclerosis in transgenic rabbits.

OBJECTIVE: Apolipoprotein (apo) A-II is the second major apo of high-density lipoproteins, yet its pathophysiological roles in the development of atherosclerosis remain unknown. We aimed to examine whether apo A-II plays any role in atherogenesis and, if so, to elucidate the mechanism involved. METHODS AND RESULTS: We compared the susceptibility of human apo A-II transgenic (Tg) rabbits to cholesterol diet-induced atherosclerosis with non-Tg littermate rabbits. Tg rabbits developed significantly less aortic and coronary atherosclerosis than their non-Tg littermates, while total plasma cholesterol levels were similar. Atherosclerotic lesions of Tg rabbits were characterized by reduced macrophages and smooth muscle cells, and apo A-II immunoreactive proteins were frequently detected in the lesions. Tg rabbits exhibited low levels of plasma C-reactive protein and blood leukocytes compared with non-Tg rabbits, and high-density lipoproteins of Tg rabbit plasma exerted stronger cholesterol efflux activity and inhibitory effects on the inflammatory cytokine expression by macrophages in vitro than high-density lipoproteins isolated from non-Tg rabbits. In addition, ß-very-low-density lipoproteins of Tg rabbits were less sensitive to copper-induced oxidation than ß-very-low-density lipoproteins of non-Tg rabbits. CONCLUSIONS: These results suggest that enrichment of apo A-II in high-density lipoprotein particles has atheroprotective effects and apo A-II may become a target for the treatment of atherosclerosis.

Gut bacteria exacerbates TNBS-induced colitis and kidney injury through oxidative stress.

Gut microbiota has been implicated in the initiation and progression of various diseases; however, the underlying mechanisms remain elusive and effective therapeutic strategies are scarce. In this study, we investigated the role and mechanisms of gut microbiota in TNBS-induced colitis and its associated kidney injury while evaluating the potential of dietary protein as a therapeutic intervention. The intrarectal administration of TNBS induced colitis in mice, concurrently with kidney damage. Interestingly, this effect was absent when TNBS was administered intraperitoneally, indicating a potential role of gut microbiota. Depletion of gut bacteria with antibiotics significantly attenuated the severity of TNBS-induced inflammation, oxidative damage, and tissue injury in the colon and kidneys. Mechanistic investigations using cultured colon epithelial cells and bone-marrow macrophages unveiled that TNBS induced cell oxidation, inflammation and injury, which was amplified by the bacterial component LPS and mitigated by thiol antioxidants. Importantly, in vivo administration of thiol-rich whey protein entirely prevented TNBS-induced colonic and kidney injury. Our findings suggest that gut bacteria significantly contribute to the initiation and progression of colitis and associated kidney injury, potentially through mechanisms involving LPS-induced exaggeration of oxidative cellular damage. Furthermore, our research highlights the potential of dietary thiol antioxidants as preventive and therapeutic interventions.

Probucol inhibits the initiation of atherosclerosis in cholesterol-fed rabbits.

BACKGROUND: Probucol and statin are often prescribed for treating atherosclerosis. These two drugs exhibit different mechanisms but it is unknown whether they have the same anti-atherogenic properties. In the current study, we examined whether these two drugs at optimal doses could inhibit the initiation of atherosclerosis in cholesterol-fed rabbits in the same way. METHODS: New Zealand White rabbits were fed a cholesterol-rich diet for 5 weeks to produce the early-stage lesions of atherosclerosis. Drug-treated rabbits were administered either probucol or atorvastatin and serum lipids and aortic atherosclerotic lesions were compared with those in a control group. RESULTS: Atorvastatin treatment significantly reduced serum total cholesterol levels while probucol treatment led to significant reduction of high-density lipoprotein cholesterol levels without changing total cholesterol levels compared with those in the control group. Compared with the control, probucol treatment led to 65% (p < 0.01) reduction while atorvastatin treatment led to 23% (p = 0.426) reduction of the aortic lesion area. Histological and immunohistochemical analyses revealed that the lesions of the probucol-treated group were characterized by remarkable reduction of monocyte adherence to endothelial cells and macrophage accumulation in the intima compared with those of both atorvastatin and control groups. Furthermore, low-density lipoprotein (LDL) isolated from the probucol group exhibited prominent anti-oxidative reaction, which was not present in LDL isolated from either the atorvastatin-treated or the control group. CONCLUSIONS: This study suggests that probucol inhibits the initiation of atherosclerosis by reducing monocyte adherence and infiltration into the subintima. Anti-oxidization of LDL by probucol protects more effectively against early-stage lesion formation than statin-mediated lipid-lowering effects.

A Simple Telemetry Sensor System for Monitoring Body Temperature in Rabbits-A Brief Report.

Continuous body temperature measurement is an important means of studying inflammation and metabolic changes using experimental animals. Although expensive telemetry equipment for collecting multiple parameters is available for small animals, readily used devices for mediate- or large-sized animals are rather limited. In this study, we developed a new telemetry sensor system that can continuously monitor rabbit body temperature. The telemetry sensor was easily implanted subcutaneously in rabbits housed in the animal facility while temperature changes were continuously recorded by a personal computer. Temperature data obtained by the telemetry was consistent with the rectal temperature measured by a digital device. Analysis of body temperature changes of unstrained rabbits, either under the normal condition or fever induced by endotoxin confirms the reliability and usefulness of this system.

The Combined Administration of Vitamin C and Copper Induces a Systemic Oxidative Stress and Kidney Injury.

Vitamin C (ascorbic acid; AA) and copper (Cu2+) are well used supplements with many health-promoting actions. However, when they are used in combination, the Fenton reaction occurs, leading to the formation of highly reactive hydroxyl radicals. Given that kidney is vulnerable to many toxicants including free radicals, we speculated that the in vivo administration of AA plus Cu2+ may cause oxidative kidney injury. The purpose of this study was to address this possibility. Mice were administered with AA and Cu2+, alone or in combination, via oral gavage once a day for various periods. Changes in the systemic oxidative status, as well renal structure and functions, were examined. The administration of AA plus Cu2+ elevated protein oxidation in serum, intestine, bladder, and kidney, as evidenced by the increased sulfenic acid formation and decreased level of free sulfhydryl groups (-SH). The systemic oxidative stress induced by AA plus Cu2+ was associated with a significant loss of renal function and structure, as indicated by the increased blood urea nitrogen (BUN), creatinine and urinary proteins, as well as glomerular and tubular cell injury. These effects of AA and Cu2+ were only observed when used in combination, and could be entirely prevented by thiol antioxidant NAC. Further analysis using cultured renal tubular epithelial cells revealed that AA plus Cu2+ caused cellular protein oxidation and cell death, which could be abolished by NAC and catalase. Moreover, coincubation of AA and Cu2+ led to H2O2 production. Collectively, our study revealed that a combined administration of AA and Cu2+ resulted in systemic oxidative stress and renal cell injury. As health-promoting supplements, AA and Cu2+ should not be used together.

Development of Dietary Thiol Antioxidant via Reductive Modification of Whey Protein and Its Application in the Treatment of Ischemic Kidney Injury.

Thiol antioxidants play important roles in cell and body defense against oxidative stress. In body fluid, albumin is the richest source of thiol antioxidants. One recent study showed that the reductive modification of thiol residues in albumin potentiated its antioxidative activity. Given that whey protein (WP) contains albumin and other thiol-active proteins, this property of WP could be exploited to develop novel thiol antioxidants. The aim of this study was to address this possibility. WP was reductively modified with dithiothreitol (DTT). The modified protein exhibited significantly elevated free sulfhydryl groups (-SH) and thiol antioxidative activity. It detoxified H2O2 and prevented H2O2-initiated protein oxidation and cell death in a -SH group-dependent way in vitro. In addition, it reacted with GSH/GSSG and altered the GSH/GSSG ratio via thiol-disulfide exchange. In vivo, oral administration of the reductively modified WP prevented oxidative stress and renal damage in a mouse model of renal injury caused by ischemia reperfusion. It significantly improved renal function, oxidation, inflammation, and cell injury. These protective effects were not observed in the WP control and were lost after blocking the -SH groups with maleimide. Furthermore, albumin, one of the ingredients of WP, also exhibited similar protective effects when reductively modified. In conclusion, the reductive modification of thiol residues in WP transformed it into a potent thiol antioxidant that protected kidneys from ischemia reperfusion injury. Given that oxidative stress underlies many life-threatening diseases, the reductively modified dietary protein could be used for the prevention and treatment of many oxidative-stress-related conditions, such as cardiovascular diseases, cancer, and aging.

Use of Rabbit Models to Study Atherosclerosis.

Rabbits are a useful animal model for examining human hyperlipidemia and atherosclerosis because they have unique features of lipoprotein metabolism that are similar to those in humans. Feeding rabbits a cholesterol-rich diet is a simple means to induce experimental atherosclerosis. Indeed, cholesterol-fed rabbits were first applied to address the relationship between dietary cholesterol and atherosclerosis more than 100 years ago. However, the methods for investigating atherosclerosis using cholesterol-fed rabbits have not been well formulated. In this chapter, we attempt to provide readers with the essential methods to use cholesterol-fed rabbits in the examination of atherosclerosis from cholesterol diet preparation to lesion analysis. These protocols are compiled for both experienced and young researchers who intend to use rabbits for the investigation of lipoprotein metabolism and atherosclerosis.

Pathological Investigations of Intracranial Atherosclerosis Using Multiple Hypercholesterolemic Rabbit Models.

Background: Intracranial atherosclerosis (ICAS) is one of the most common causes of ischemic stroke, but there are few animal models that can recapitulate its pathological features. In this study, we examined ICAS pathological features and anatomic distributions using three types of hyperlipidemic rabbit models. We also investigated the effect of different lipoprotein profiles and hypertension on ICAS. Materials and Methods: We examined Watanabe heritable hyperlipidemic (WHHL) rabbits, apoE knockout (KO) rabbits and wild-type rabbits (WT) fed a cholesterol diet, in addition to WT rabbits fed a standard diet as a control. The whole brain was dissected and embedded in paraffin. Serial sections were stained with either hematoxylin/eosin or elastica van Gieson, or immunohistochemically stained with monoclonal antibodies against macrophages and smooth muscle cells. We investigated (1) the presence of cerebral atherosclerosis; (2) the lesion locations in the cerebral arteries; (3) the degree of lumen stenosis; (4) pathological features and cellular components of the lesions in these rabbits; and (5) whether hypertension affects ICAS. Results: ICAS was detected in apoE and WHHL rabbits, but not in WT rabbits. Compared with apoE KO rabbits, WHHL rabbits had greater ICAS. The lesions of cerebral atherosclerosis were mainly distributed at the bifurcations of the posterior cerebral artery, basilar artery and vertebral artery, and they were basically characterized by smooth muscle cells and extracellular matrix with few macrophages. The extent of the ICAS in WHHL rabbits was significantly increased by hypertension. Conclusions: ICAS was detected in WHHL and apoE KO rabbits, and occurred in specific locations in the cerebral arteries. Hypertension promotes the development of ICAS in the setting of hypercholesterolemia.

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation.

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and atherosclerosis. Although there are several methods for analyzing plasma lipoproteins, ultracentrifugation is still one of the most popular and reliable methods. Because of its intact separation procedure, the lipoprotein fractions isolated by this method can be used for analysis of lipoproteins, apolipoproteins, proteomes, and functional study of lipoproteins with cultured cells in vitro. Here, we provide a detailed protocol to isolate seven lipoprotein fractions including VLDL (d<1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL) from rabbit plasma using sequential floating ultracentrifugation. In addition, we introduce the readers how to analyze apolipoproteins such as apoA-I, apoB, and apoE by SDS-PAGE and Western blotting and show representative results of lipoprotein and apolipoprotein profiles using hyperlipidemic rabbit models. This method can become a standard protocol for both clinicians and basic scientists to analyze lipoprotein functions.

Tanshinone IIA Stimulates Cystathionine γ-Lyase Expression and Protects Endothelial Cells from Oxidative Injury.

Tanshinone IIA (Tan IIA), an active ingredient of Danshen, is a well-used drug to treat cardiovascular diseases. Currently, the mechanisms involved remain poorly understood. Given that many actions of Tan IIA could be similarly achieved by hydrogen sulfide (H2S), we speculated that Tan IIA might work through the induction of endogenous H2S. This study was to test this hypothesis. Exposure to endothelial cells to Tan IIA elevated H2S-synthesizing enzyme cystathionine γ-Lyase (CSE), associated with an increased level of endogenous H2S and free thiol activity. Further analysis revealed that this effect of Tan IIA was mediated by an estrogen receptor (ER) and cAMP signaling pathway. It stimulated VASP and CREB phosphorylation. Inhibition of ER or PKA abolished the CSE-elevating effect, whereas activation of ER or PKA mimicked the effect of Tan IIA. In an oxidative endothelial cell injury model, Tan IIA potently attenuated oxidative stress and inhibited cell death. In support of a role of endogenous H2S, inhibition of CSE aggerated oxidative cell injury. On the contrary, supplement of H2S attenuated cell injury. Collectively, our study characterized endogenous H2S as a novel mediator underlying the pharmacological actions of Tan IIA. Given the multifaceted functions of H2S, the H2S-stimulating property of Tan IIA could be exploited for treating many diseases.