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Introduction: Lecithin cholesterol acyltransferase (LCAT) plays a crucial role in acyl-esterifying cholesterol in plasma, which is essential for reverse cholesterol transport (RCT). Previous studies indicated that its activity on both α and β lipoproteins interpret its effects on lipoproteins for many controversial investigations of atherosclerosis. Objectives: To better understand the relationship between LCAT, diet-induced dyslipidemia and atherosclerosis, we developed a double knockout (LCAT−/−&LDLR−/−, DKO) hamster model to evaluate the specific role of LCAT independent of LDL clearance effects. Methods: Plasma triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), and free cholesterol (FC) levels were measured using biochemical reagent kits. FPLC was performed to analyze the components of lipoproteins. Apolipoprotein content was assessed using western blotting (WB). The hamsters were fed a high cholesterol/high fat diet (HCHFD) to induce atherosclerosis. Oil Red O staining was employed to detect plaque formation. Peritoneal macrophages were studied to investigate the effects of LCAT on cholesterol uptake and efflux. Results: On HCHFD, DKO hamsters exhibited significantly elevated levels of TG and FC, while HDL-C was nearly undetectable without affecting TC levels, as compared to low-density lipoprotein receptor (LDLR)-deficient (LDLR−/−, LKO) hamsters. Lipoprotein profiling revealed a marked increase in plasma chylomicron/very low-density lipoprotein (CM/VLDL) fractions, along with an unexpected reduction in LDL fraction in DKO hamsters. Furthermore, DKO hamsters displayed aggravated atherosclerotic lesions in the aorta, aortic root, and coronary artery relative to LKO hamsters, attributed to a pro-atherogenic lipoprotein profile and impaired cholesterol efflux in macrophages. Conclusions: Our study demonstrates the beneficial role of LCAT in inhibiting atherosclerotic development and highlights the distinctive lipid metabolism characteristics in hamsters with familial hypercholesterolemia.

IntroductionApolipoprotein E (apoE) acts as a binding molecule for both the low-density lipoprotein receptor and the lipoprotein receptor-related protein and this function is essential for facilitating the hepatocyte uptake of lipoproteins containing apoB. The absence of apoE leads to increased atherogenicity in both humans and mice, although the precise molecular mechanisms remain incompletely understood.ObjectivesThis study aimed to investigate the susceptibility of apoE knockout (KO) rabbits, in comparison with wild-type (WT) rabbits, to diet-induced hyperlipidemia and atherosclerosis.MethodsApoE KO rabbits and WT rabbits were fed a diet containing 0.3% cholesterol for 16 weeks. Plasma lipid levels, lipoproteins, and apolipoproteins were analyzed. Atherosclerosis was evaluated at the endpoint of experiments. In addition, we evaluated the oxidizability of those lipoproteins containing apoB to investigate the possible mechanisms of atherosclerosis.ResultsMale apoE KO rabbits showed significantly elevated levels of total cholesterol and triglycerides compared to WT rabbits, while female apoE KO rabbits displayed similar high total cholesterol levels, albeit with significantly higher triglycerides levels than WT controls. Notably, both male (2.1-fold increase) and female (1.6-fold increase) apoE KO rabbits exhibited a significantly augmented aortic lesion area compared to WT controls. Pathological examination showed that the increased intimal lesions in apoE KO rabbits were featured by heightened infiltration of macrophages (2.7-fold increase) and smooth muscle cells (2.5-fold increase). Furthermore, coronary atherosclerotic lesions were also increased by 1.3-fold in apoE KO rabbits. Lipoprotein analysis revealed that apoB48-rich beta-very-low-density lipoproteins were notably abundant in apoE KO rabbits, suggesting that these remnant lipoproteins of intestinal origin serve as the primary atherogenic lipoproteins. Moreover, apoB48-rich remnant lipoproteins isolated from apoE KO rabbits exhibited heightened susceptibility to copper-induced oxidation.ConclusionsThe findings indicate that apoB48-rich remnant lipoproteins, resulting from apoE deficiency, possess greater atherogenic potential than apoB100-rich remnant lipoproteins, regardless of plasma TC levels.

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.

Abstract Early pressure injury (PI) progression is associated with multi-circulatory disorders and they interplay with each other, resulting in a lack of a satisfactory diagnostic method. We generated early PI and blanchable erythema hairless rat models. Transparent disc method and capillary refilling time test (CRTT) results were recorded with ultraviolet camera to capture the dynamics changes, and the blanching index and refilling index were set for comprehensive analysis. The deteriorated areas of early PI showed non-blanchable erythema (NBE) and an increase in erythema at 0.5 and 6 h with the transparent disc method. CRTT showed a marked refilling delay at 12 h. The comprehensive analysis of blanching index and refilling index showed a significant change in erythema from NBE at 0.5 h and ischemia progressing to hemorrhage at 18 h. There was also a marked difference in the deteriorating and improving areas within the same erythema. Pathological analysis showed inflammatory cell infiltration, with marked edema accompanied by increased hemorrhage and tissue necrosis. Furthermore, small arteries and veins with thrombosis and microthrombi were observed. Consistent ischemia after decompression and subsequent hemorrhage are important indicators, and comprehensive analysis can help increase the positive diagnosis rate over that for other circulatory disorders alone.

Abstract As a multifunctional fluorescent nanomaterial, carbon dots (CDs) not only have small size, stable chemical properties, excellent photoluminescence characteristics, but also exhibit good biocompatibility and low toxicity. It has attracted considerable attention in the field of nanotechnology and biological science. CDs contain abundant functional groups on the surface, which not only retain part of the properties of raw materials, but also may have new photoelectric, catalytic, biomedical, and other functions. In this review, we systematically summarize the synthesis methods, modifications, optical properties, and main biological functions of CDs in recent years. The application of functionalized modified CDs in biological detection, biological imaging, photodynamic therapy, photothermal therapy, targeted therapy, drug delivery, gene delivery, protein delivery, and other biomedical fields is introduced. The latest progress of CDs with its own biomedical function in antioxidant, anti‐pathogen, and disease treatment is summarized. Finally, we discuss some problems in the practical application of CDs and look forward to the future development trend of self‐functional CDs combined with surface modification to achieve multimodal treatment of diseases.

Base on the precipitation forecast of ECWMF model from 2016 to 2019 and the observation data, five schemes are designed to carry out revision experiment on numerical model clear-rainy forecast, including Optimal Percentile (OP), the Optimal Threat Score (OTS), the Probability Matching (PM), the sub-district OTS and the sub-district PM-OTS fusion scheme. The results show that: (1) The clear-rainy revised thresholds of OP and PM are static, and that of OTS is dynamic. The thresholds of the 5 schemes are all applicable to Area A(basin, northern Aba Prefecture and northern Garze Prefecture), among which the thresholds of PM and sub-district PM-OTS are more applicable to Area B(southern Ganzi Prefecture and panxi area) with obvious wet deviation of the numerical model. (2) Each scheme has obvious revised ability for ECWMF model clear-rainy forecast. The revised effect of 24 h is the best, and that of Area B is superior to Area B, autumn and winter is better than spring and summer. (3) Revised scheme scores after subdivision are better than before, and the sub-district PM-OTS is the best. Individual cases and batch tests show that the revised effects of the schemes in Area A are comparable, the rainfall distribution in Area B are more close to the observed precipitation after revised of PM, sub-district OTS and PM-OTS fusion schemes, among which the sub-district PM-OTS is the best.

Abdominal aortic aneurysm (AAA) is pathologically characterized by intimal atherosclerosis, disruption and attenuation of the elastic media, and adventitial inflammatory infiltrates. Although all these pathological events are possibly involved in the pathogenesis of AAA, the functional roles contributed by adventitial inflammatory macrophages have not been fully documented. Recent studies have revealed that increased expression of matrix metalloproteinase-12 (MMP-12) derived from macrophages may be particularly important in the pathogenesis of both atherosclerosis and AAA. In the current study, we developed a carrageenan-induced abdominal aortic adventitial inflammatory model in hypercholesterolemic rabbits and evaluated the effect of adventitial macrophage accumulation on the aortic remodeling with special reference to the influence of increased expression of MMP-12. To accomplish this, we compared the carrageenan-induced aortic lesions of transgenic (Tg) rabbits that expressed high levels of MMP-12 in the macrophage lineage to those of non-Tg rabbits. We found that the aortic medial and adventitial lesions of Tg rabbits were greater in degree than those of non-Tg rabbits, with the increased infiltration of macrophages and prominent destruction of elastic lamellae accompanied by the frequent appearance of dilated lesions, while the intimal lesions were slightly increased. Enhanced aortic lesions in Tg rabbits were focally associated with increased dilation of the aortic lumens. RT-PCR and Western blotting revealed high levels of MMP-12 in the lesions of Tg rabbits that were accompanied by elevated levels of MMP-2 and -3, which was caused by increased number of macrophages. Our results suggest that adventitial inflammation constitutes a major stimulus to aortic remodeling and increased expression of MMP-12 secreted from adventitial macrophages plays an important role in the pathogenesis of vascular diseases such as AAA.

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.

BackgroundIntracranial 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 MethodsWe 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.ResultsICAS 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.ConclusionsICAS 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.

Abstract Background Marmots are large Holarctic rodents with unique biological features, making them potential animal models in various research fields. Due to the rapid accumulation of the genetic data in marmots, a highly integrative database is urgent needed. Description iMarmot is freely available on the web at http://www.marmotdb.org/ and currently contains the biological information of 14 marmots, genomic sequence of 6 marmots, syntenic relationship and orthologs among 3 marmots, and expression profiles of several hibernators and plague hosts. To assist with the genomic and transcriptomic analysis, we also integrated a set of analysis and visualization tools, such as KEGG or GO enrichment analysis, PCA, Blast, Muscle, GeneWise, Lastz, and JBrowse. Particularly, one DEGs (differentially expressed genes) module has been implemented in this database to visualize the gene expression changes in hibernators and plague hosts. Conclusion This database will provide comprehensive information and analysis platform for researchers interested in understanding the biological features of marmots.

By using the precipitation observations (including the encrypted automatic stations) and the ECMWF forecasts for Sichuan during June to August from 2016-2018, based on the probability forecast of short-time heavy rain every 3h derived by the "ingredients method", we calculate the times of various transition probability threshold at each grid, and then a method to improve forecast accuracy of heavy rainfall of 24h cumulative precipitation forecast in Sichuan was developed. We then applied it to daily test of ECMWF model precipitation forecast for 24-72 h on the precipitation concentration period from June to August of 2018. The results show that:1) According to the composite experiment indicators of precipitation above 25 and 50mm, the hit rate, missing rate and TS score of each period validity have been significantly improved after the calibration, and with the extension of the period validity, the value of each index has been further increased greatly. Although the false alarm rate increases a bit of 0-24 h and 24-48 h forecast, the increase of false alarm rate is far less than the decrease of missing rate. 2) According to the experiment of individual cases, the precipitation value increased significantly, the effect of precipitation area forecast above 50mm is greatly improved, which means that the distribution of precipitation area after the calibration is basically consistent with the actual situation, especially for the 0-24h cumulative precipitation forecast.

C1q/tumor necrosis factor-related protein 9 (CTRP9) is a newly discovered adipokine that is the closest paralog of adiponectin. Proteolytic cleavage of CTRP9 leads to the release of the globular domain (gCTRP9), which serves as the major circulating subtype. After binding with adiponectin receptor 1 (AdipoR1) and N-cadherin, CTRP9 activates various signaling pathways to regulate glucose and lipid metabolism, vasodilation and cell differentiation. Throughout human development and adult life, CTRP9 controls many biological phenomena. simultaneously, abnormal gene or protein expression of CTRP9 is accompanied by a wide range of human pathological phenomena. In this review, we briefly introduce CTRP9 and its associated signaling pathways and physiological functions, which may be helpful in the understanding of the occurrence of diseases. Moreover, we summarize the broader research prospects of CTRP9 and advances in therapeutic intervention. In recent years, CTRP9 has attracted extensive attention due to its role in the pathogenesis of various diseases, providing further avenues for its exploitation as a potential biomarker or therapeutic target.

Elevated triglycerides are associated with an increased risk of cardiovascular disease (CVD). Therefore, it is very important to understand the metabolism of triglyceride-rich lipoproteins (TRLs) and their atherogenic role in animal models. Using low-density lipoprotein receptor knockout (LDLR−/−) Syrian golden hamsters, this study showed that unlike LDLR−/− mice, when LDLR−/− hamsters were fed a high cholesterol high-fat diet (HFD), they had very high plasma levels of triglycerides and cholesterol. We found that LDLR−/− hamsters exhibited increased serum TRLs and the ApoB100 and 48 in these particles after being fed with HFD. Treatment with ezetimibe for 2 weeks decreased these large particles but not the LDL. In addition, ezetimibe simultaneously reduced ApoB48 and ApoE in plasma and TRLs. The expression of LRP1 did not change in the liver. These findings suggested that the significantly reduced large particles were mainly chylomicron remnants, and further, the remnants were mainly cleared by the LDL receptor in hamsters. After 40 days on an HFD, LDLR−/− hamsters had accelerated aortic atherosclerosis, accompanied by severe fatty liver, and ezetimibe treatment reduced the consequences of hyperlipidemia. Compared with the serum from LDLR−/− hamsters, that from ezetimibe-treated LDLR−/− hamsters decreased the expression of vascular adhesion factors in vascular endothelial cells and lipid uptake by macrophages. Our results suggested that in the LDLR−/− hamster model, intestinally-derived lipoprotein remnants are highly atherogenic and the inflammatory response of the endothelium and foam cells from macrophages triggered atherosclerosis. The LDL receptor might be very important for chylomicrons remnant clearance in the Syrian golden hamster, and this may not be compensated by another pathway. We suggest that the LDLR−/− hamster is a good model for the study of TRLs-related diseases as it mimics more complex hyperlipidemia.

Abstract Although liver‐humanized animals are desirable tools for drug development and expansion of human hepatocytes in large quantities, their development is restricted to mice. In animals larger than mice, a precondition for efficient liver humanization remains preliminary because of different xeno‐repopulation kinetics in livers of larger sizes. Since rats are ten times larger than mice and widely used in pharmacological studies, liver‐humanized rats are more preferable. Here, Fah–/–Rag2–/–IL2rg–/– (FRG) rats are generated by CRISPR/Cas9, showing accelerated liver failure and lagged liver xeno‐repopulation compared to FRG mice. A survival‐assured liver injury preconditioning (SALIC) protocol, which consists of retrorsine pretreatment and cycling 2‐(2‐nitro‐4‐trifluoromethylbenzoyl)‐1,3‐cyclohexanedione (NTBC) administration by defined concentrations and time intervals, is developed to reduce the mortality of FRG rats and induce a regenerative microenvironment for xeno‐repopulation. Human hepatocyte repopulation is boosted to 31 ± 4% in rat livers at 7 months after transplantation, equivalent to approximately a 1200‐fold expansion. Human liver features of transcriptome and zonation are reproduced in humanized rats. Remarkably, they provide sufficient samples for the pharmacokinetic profiling of human‐specific metabolites. This model is thus preferred for pharmacological studies and human hepatocyte production. SALIC may also be informative to hepatocyte transplantation in other large‐sized species.

Abstract 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.

Abstract Background In recent years, an increasing number of studies have proved that circulating miRNAs could be used for the early diagnosis of cardiovascular diseases and even play vital roles in the evaluation of therapeutic effects or prognosis. This study was conducted to examine the correlation between serum microRNAs and hyperlipidemia to provide a theoretical basis for the early screening and intervention of atherosclerotic cardiovascular diseases (ASCVD). Methods The serum samples and clinical data of 122 patients with hyperlipidemia and 168 healthy subjects were collected. Related clinical information was statistically analyzed for the two groups. Expression of circulating miRNAs was detected by miRNA microarray analysis and further verified by reverse transcription-quantitative PCR (RT-qPCR). Results Statistical analysis of clinical information revealed a significant difference in the incidence of ASCVD between the two groups. The MiRNA microarray analysis (n = 10) showed 22 miRNAs with significantly different expression, among which 12 showed upregulation, and the others showed downregulation. Those possessing obvious differences and stable expression in the miRNA microarray, including miRNA-191-3p, miRNA-933, and miRNA-425-3p, were chosen for further investigation using RT-qPCR. The results demonstrated that several miRNAs were related to lipid metabolism disorders, especially miRNA-933. The area under the curve (AUC) of miRNA-933 in distinguishing the hyperlipidemia and ASCVD patients was 0.739 (95% CI, 0.682–0.795; P

Abstract Objective To generate novel rabbit models with a large-fragment deletion of either LDL receptor (LDLR) and/or apolipoprotein (apoE) genes for the study of hyperlipidemic and atherosclerosis. Methods CRISPR/Cas9 system directed by a multiple sgRNAs system was used in rabbit embryos to edit their LDLR and apoE genes. The LDLR and apoE genes of founder rabbits were sequenced, and their plasma lipids and lipoprotein profiles on a normal chow diet were analyzed, western blotting was also performed to evaluate the expression of apolipoprotein. Sudan IV and HE staining of aortic were performed to confirm the formation of atherosclerosis. Results Six knockout (KO) rabbits by injection of both LDLR and apoE sgRNAs were obtained, including four LDLR KO rabbits and two LDLR/apoE double- KO rabbits. Sequence analysis of these KO rabbits revealed that they contained multiple mutations including indels, deletions, and substitutions, as well as two rabbit lines containing biallelic large fragment deletion in the LDLR region. Analysis of their plasma lipids and lipoprotein profiles of these rabbits fed on a normal chow diet revealed that all of these KO rabbits exhibited remarkable hyperlipidemia with total cholesterol levels increased by up to 10-fold over those of wild-type rabbits. Pathological examinations of two founder rabbits showed that KO rabbits developed prominent aortic and coronary atherosclerosis. Conclusion Large fragment deletions can be achieved in rabbits using Cas9 mRNA and multiple sgRNAs. LDLR KO along with LDLR/apoE double KO rabbits should provide a novel means for translational investigations of human hyperlipidemia and atherosclerosis.

Summary: The Himalayan marmot (Marmota himalayana) is a hibernating mammal that inhabits the high-elevation regions of the Himalayan mountains. Here we present a draft genome of the Himalayan marmot, with a total assembly length of 2.47 Gb. Phylogenetic analyses showed that the Himalayan marmot diverged from the Mongolian marmot approximately 1.98 million years ago. Transcriptional changes during hibernation included genes responsible for fatty acid metabolism in liver and genes involved in complement and coagulation cascades and stem cell pluripotency pathways in brain. Two selective sweep genes, Slc25a14 and ψAamp, showed apparent genotyping differences between low- and high-altitude populations. As a processed pseudogene, ψAamp may be biologically active to influence the stability of Aamp through competitive microRNA binding. These findings shed light on the molecular and genetic basis underlying adaptation to extreme environments in the Himalayan marmot. : Physiology; Genetics; Evolutionary Biology; Bioinformatics; Omics Subject Areas: Physiology, Genetics, Evolutionary Biology, Bioinformatics, Omics

Background: Pressure injuries (PIs) generally result from prolonged ischemia through localized skin compression, and ischemia persists and exacerbates damage even post-decompression. The mechanisms of ischemia post-decompression are still unclear, and appropriate methods for detection are lacking. Methods: We used blanchable erythema (BE) and early PI rat models. We assessed the perfusion using Evans Blue (EB) and thrombus formation under a light microscope. Furthermore, we performed a capillary refill time test (CRTT) to detect ischemia after depression coupled with the transparent disk method using a spectrophotometer. Results: Compared with the BE group, the early PI group showed significantly slow and insufficient perfusion, as determined by EB staining (p < 0.001). Histological observations revealed that ischemia during post-decompression of early PI was caused by a greater amount of thrombi. The CRTT results showed that although both groups exhibited varying degrees of insufficient refilling volume, the early PI group had significantly slower refilling than the BE group (p < 0.001), which persisted during the deterioration or disappearance of erythema. Conclusions: Our results showed that persistent ischemia caused by thrombi is an important cause of early PI deterioration post-decompression. Therefore, the performance of CRTT coupled with the transparent disc method may become a promising method for detecting ischemia post-decompression.

Albumin (Alb) is the most abundant plasma protein with multiple biological functions, including antioxidative property through its thiol activity. Given that inflammatory bowel disease is associated with a decreased level of Alb and an increased level of Alb oxidation, we asked whether Alb could have a therapeutic effect on colitis. Here we tested this possibility. Bovine serum albumin (BSA) was reductively modified with dithiothreitol (DTT) and administrated via gavage or intraperitoneal injection. Dextran sulfate sodium (DSS)-induced mice colitis was associated with massive oxidative stress, as indicated by the elevated sulfenic acid formation in blood, colon tissues, and feces. Treatment of mice with the reductively modified albumin (r-Alb) attenuated the oxidative stress and reduced local inflammation and tissue injury. These effects of r-Alb were only partially achieved by unmodified Alb and wholly lost after blocking the –SH groups with maleimide. In cultured colon epithelial cells, r-Alb prevented DSS- and H2O2-induced ROS elevation and barrier dysfunction, preceded by inhibition of sulfenic acid formation and P38 activation. Further analysis revealed that Alb was susceptible to H2O2-induced oxidation, and it detoxified H2O2 in a –SH group-dependent way. Moreover, Alb reacted with GSH/GSSG via thiol-disulfide exchange and reciprocally regulated the availability of –SH groups. Collectively, our study shows that r-Alb effectively attenuates DSS colitis via –SH group-mediated antioxidative action. Given that the oxidative stress underlies many life-threatening diseases, r-Alb, functioning as a potent antioxidant, could have a wide range of applications.

Rabbits are one of the most used experimental animals for investigating the mechanisms of human cardiovascular disease and lipid metabolism because they are phylogenetically closer to human than rodents (mice and rats). Cholesterol-fed wild-type rabbits were first used to study human atherosclerosis more than 100 years ago and are still playing an important role in cardiovascular research. Furthermore, transgenic rabbits generated by pronuclear microinjection provided another means to investigate many gene functions associated with human disease. Because of the lack of both rabbit embryonic stem cells and the genome information, for a long time, it has been a dream for scientists to obtain knockout rabbits generated by homologous recombination-based genomic manipulation as in mice. This obstacle has greatly hampered using genetically modified rabbits to disclose the molecular mechanisms of many human diseases. The advent of genome editing technologies has dramatically extended the applications of experimental animals including rabbits. In this review, we will update genetically modified rabbits, including transgenic, knock-out, and knock-in rabbits during the past decades regarding their use in cardiovascular research and point out the perspectives in future.

Background: Non-blanchable erythema is used as a diagnostic indicator for stage 1 pressure injury (early PI); it is distinguished from blanchable erythema (BE) by the application of “light pressing”. Considering the low of the accuracy of the degree of pressure applied, it is difficult to use this method in clinical settings. Methods: We constructed models of BE and early PI in order to determine the most appropriate pressure values using the transparent disc method. We observed erythema by using a Dermo-camera to quantify the gray and a* values of the wound area along with a spectrophotometer. Results: BE started to fade at 50 mmHg, while the gray values became statistically significant when the pressure was increased to 100 mmHg (p < 0.05). However, erythema remained even when the pressure was increased to 150 mmHg soon after decompression. By contrast, the early PI was showed to be non-blanchable for the longest time under a pressure of 150 mmHg, but by 18 h it had decreased and the erythema faded more obviously after applying pressure. Conclusions: We proposed that a pressure of 50–100 mmHg was more appropriate for light pressure, but this may vary when different instruments are used. Variations may occur in either BE or early PI, therefore, careful attention should be paid during observations.

Background/Aims: The proliferation and migration of vascular smooth muscle cells (VSMCs) are key steps in the progression of atherosclerosis. The aim of the present study was to investigate the potential roles of salusin-α in the functions of VSMCs during the development of atherosclerosis. Methods: In vivo, the effects of salusin-α on atherogenesis were examined in rabbits fed a cholesterol diet. The aortas were en face stained with Sudan IV to evaluate the gross atherosclerotic lesion size. The cellular components of atherosclerotic plaques were analyzed by immunohistochemical methods. In vitro, Cell Counting Kit-8 and wound-healing assays were used to assess the effects of salusin-α on VSMC proliferation and migration. In addition, western blotting was used to evaluate the total and phosphorylated levels of Akt (also known as protein kinase B) and mammalian target of rapamycin (mTOR) in VSMCs. Results: Salusin-α infusion significantly reduced the aortic lesion areas of atherosclerosis, with a 39% reduction in the aortic arch, a 71% reduction in the thoracic aorta, and a 71% reduction in the abdominal aorta; plasma lipid levels were unaffected. Immunohistochemical staining showed that salusin-α decreased both macrophage- and VSMC-positively stained areas in atherosclerotic lesions by 54% and 69%, cell proliferative activity in the intima and media of arteriosclerotic lesions, and matrix metalloproteinase 2 (MMP-2) and MMP-9 expression in plaques. Studies using cultured VSMCs showed that salusin-α decreased VSMC migration and proliferation via reduced phosphorylation of Akt and mTOR. Conclusion: Our data indicate that salusin-α suppresses the development of atherosclerosis by inhibiting VSMC proliferation and migration through the Akt/mTOR pathway.

Rabbits (Oryctolagus cuniculus) have been the very frequently used as animal models in the study of human lipid metabolism and atherosclerosis, because they have similar lipoprotein metabolism to humans. Most of hyperlipidemia and atherosclerosis rabbit models are produced by feeding rabbits a high-cholesterol diet. Gene editing or knockout (KO) offered another means of producing rabbit models for study of the metabolism of lipids and lipoproteins. Even so, apolipoprotein (Apo)E KO rabbits must be fed a high-cholesterol diet to induce hyperlipidemia.In this study, we used the CRISPR/Cas9 system anchored exon 7 of low-density lipoprotein receptor (LDLR) in an attempt to generate KO rabbits. We designed two sgRNA sequences located in E7:g.7055–7074 and E7:g.7102–7124 of rabbit LDLR gene, respectively. Seven LDLR-KO founder rabbits were generated, and all of them contained biallelic modifications. Various mutational LDLR amino acid sequences of the 7 founder rabbits were subjected to tertiary structure modeling with SWISS-MODEL, and results showed that the structure of EGF-A domain of each protein differs from the wild-type. All the founder rabbits spontaneously developed hypercholesterolemia and atherosclerosis on a normal chow (NC) diet. Analysis of their plasma lipids and lipoproteins at the age of 12 weeks revealed that all these KO rabbits exhibited markedly increased levels of plasma TC (the highest of which was 1013.15 mg/dl, 20-fold higher than wild-type rabbits), LDL-C (the highest of which was 730.00 mg/dl, 35-fold higher than wild-type rabbits) and TG accompanied by reduced HDL-C levels. Pathological examinations of a founder rabbit showed prominent aortic atherosclerosis lesions and coronary artery atherosclerosis.In conclusion, we have reported the generation LDLR-KO rabbit model for the study of spontaneous hypercholesterolemia and atherosclerosis on a NC diet. The LDLR-KO rabbits should be a useful rabbit model of human familial hypercholesterolemia (FH) for the simulations of human primary hypercholesterolemia and such models would allow more exact research into cardio-cerebrovascular disease. Keywords: LDLR, Hypercholesterolemia, Atherosclerosis, Rabbits, Cas9, Knockout

Abstract Background Gut microbiota plays an important role in many metabolic diseases such as diabetes and atherosclerosis. Apolipoprotein E (apoE) knock-out (KO) mice are frequently used for the study of hyperlipidemia and atherosclerosis. However, it is unknown whether apoE KO mice have altered gut microbiota when challenged with a Western diet. Methods In the current study, we assessed the gut microbiota profiling of apoE KO mice and compared with wild-type mice fed either a normal chow or Western diet for 12 weeks using 16S pyrosequencing. Results On a western diet, the gut microbiota diversity was significantly decreased in apoE KO mice compared with wild type (WT) mice. Firmicutes and Erysipelotrichaceae were significantly increased in WT mice but Erysipelotrichaceae was unchanged in apoE KO mice on a Western diet. The weighted UniFrac principal coordinate analysis exhibited clear separation between WT and apoE KO mice on the first vector (58.6%) with significant changes of two dominant phyla (Bacteroidetes and Firmicutes) and seven dominant families (Porphyromonadaceae, Lachnospiraceae, Ruminococcaceae, Desulfovibrionaceae, Helicobacteraceae, Erysipelotrichaceae and Veillonellaceae). Lachnospiraceae was significantly enriched in apoE KO mice on a Western diet. In addition, Lachnospiraceae and Ruminococcaceae were positively correlated with relative atherosclerosis lesion size in apoE KO. Conclusions Collectively, our study showed that there are marked changes in the gut microbiota of apoE KO mice, particularly challenged with a Western diet and these alterations may be possibly associated with atherosclerosis.

Background/Aims: Secreted frizzled-related protein 4 (SFRP4) is a member of the SFRP family that acts as soluble modulators of Wnt signaling. Given the substantial rise in obesity, depot-specific fat accumulation and its associated diseases like diabetes, it is important to understand the molecular basis of depot-specific adipocyte differentiation. In the current study, we investigated the expression of SFRP4 in both subcutaneous and visceral adipose tissue in terms of their differentiation. Methods: White preadipocytes were isolated from the inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) from C57BL/6J mice (age: 8-week-old, male). SFRP4 expression in iWAT and eWAT preadipocytes was silenced by siRNA transfection and harvested cells for gene and protein expression analysis was performed during the differentiation. Furthermore, iWAT and eWAT preadipocytes treated with or without IL-1β were harvested for gene and protein expression analysis. Results: SFRP4 expression levels were gradually increased and proportionally associated with eWAT adipocyte differentiation toward maturation at 14 days, while iWAT adipocyte just showed an opposite tendency. Moreover, genetic (adiponectin, C/EBPα, C/EBPβ, FABP4, GLUT4 and PPARγ) analysis demonstrated that depot-specific adipogenesis in response to SFRP4 silencing in eWAT and iWAT preadipocytes. Upon IL-1β treatment, SFRP4 mRNA expression decreased significantly in iWAT adipocyte, but the expression was no significant difference in eWAT adipocyte. Conclusion: These results suggest that SFRP4 expression differentially mediates adipocyte differentiation and may play an important role in adipogenesis.

Antioxidant glutathione (GSH) plays an important role in the regulation of immunity. However, little is known about its effects on humoral immunity, especially its action on effector molecules like antibody and complement. Given that these molecules contain abundant disulfide bonds, we speculated that GSH might influence the action of these proteins via its thiol function. Using a model of a glomerular mesangial cell (MC) lysis induced by antibodies plus complement, we addressed this hypothesis. Exposure of rat MCs to anti-Thy-1 antibody plus complement or anti-MC rabbit serum caused a complement-dependent cell lysis, which was completely blocked by GSH. Moreover, GSH potently prevented the antibody-mediated agglutination of red blood cells and aggregation of antibody-sensitized microspheres. Further analysis revealed that GSH inhibited antibody binding to antigens and promoted the conversion of the antibodies to its reduced forms. GSH also potently inhibited the formation and deposition of C5b-9 in MCs and suppressed both the classic and alternative complement activation pathway. Lastly, GSH attenuated P38 activation, an oxidative sensitive kinase that partially mediated the antibody- and complement-dependent MC lysis. Depletion of GSH via inhibiting gamma-glutamylcysteine synthetase or xCT transporter augmented P38 activation and sensitized MCs to the cell lysis. Collectively, our results indicate that GSH protects cells from immunological cell damage via mechanisms involving inhibition of antibody binding to the antigens, suppression of complement activation and augmentation of cellular defense mechanism. Our study provides novel mechanistic insights into the actions of GSH in the regulation of immune responses and suggests that GSH might be used to treat certain immune disorders. Keywords: Glutathione, Antibody, Complement, Cell lysis, p38, Mesangial cells

The rabbit is a valuable animal for both the economy and biomedical sciences. Sperm cryopreservation is one of the most efficient ways to preserve rabbit strains because it is easy to collect ejaculate repeatedly from a single male and inseminate artificially into multiple females. During the cooling, freezing and thawing process of sperms, the plasma membrane, cytoplasm and genome structures could be damaged by osmotic stress, cold shock, intracellular ice crystal formation, and excessive production of reactive oxygen species. In this review, we will discuss the progress made during the past years regarding efforts to minimize the cell damage in rabbit sperms, including freezing extender, cryoprotectants, supplements, and procedures.

Hyperlipidemia or dyslipidemia is a major risk factor for atherosclerotic diseases. Experimental animals play an important role in elucidating the molecular mechanisms of the pathophysiology of hyperlipidemia as well as in drug development. Rabbits are one of the most suitable models to study human hyperlipidemia because many features of the lipoprotein metabolism of rabbits are similar to those of humans such as LDL-rich lipoproteins in plasma, apolipoprotein B mRNA editing, and cholesteryl ester transfer protein. Currently, three types of rabbit models are commonly used for studying hyperlipidemia: (1) diet-induced hyperlipidemic rabbits, (2) spontaneous hyperlipidemic rabbits, and (3) gene-manipulated rabbits (transgenic and knockout rabbits). In this review, we give an overview of the features of hyperlipidemic rabbits and discuss the usefulness of rabbits for the development of anti-atherogenic drugs.

Transgenic rabbits have contributed to the progress of biomedical science as human disease models because of their unique features, such as the lipid metabolism system similar to humans and medium body size that facilitates handling and experimental manipulation. In fact, many useful transgenic rabbits have been generated and used in research fields such as lipid metabolism and atherosclerosis, cardiac failure, immunology, and oncogenesis. However, there have been long-term problems, namely that the transgenic efficiency when using pronuclear microinjection is low compared with transgenic mice and production of knockout rabbits is impossible owing to the lack of embryonic stem cells for gene targeting in rabbits. Despite these limitations, the emergence of novel genome editing technology has changed the production of genetically modified animals including the rabbit. We are finally able to produce both transgenic and knockout rabbit models to analyze gain- and loss-of-functions of specific genes. It is expected that the use of genetically modified rabbits will extend to various research fields. In this review, we describe the unique features of rabbits as laboratory animals, the current status of their development and use, and future perspectives of transgenic rabbit models for human diseases.

Cholesteryl ester transfer protein (CETP) is a plasma protein that mediates bidirectional transfers of cholesteryl esters and triglycerides between low-density lipoproteins and high-density lipoproteins (HDL). Because low levels of plasma CETP are associated with increased plasma HDL-cholesterol, therapeutic inhibition of CETP activity is considered an attractive strategy for elevating plasma HDL-cholesterol, thereby hoping to reduce the risk of cardiovascular disease. Interestingly, only a few laboratory animals, such as rabbits, guinea pigs, and hamsters, have plasma CETP activity, whereas mice and rats do not. It is not known whether all CETPs in these laboratory animals are functionally similar to human CETP. In the current study, we compared plasma CETP activity and characterized the plasma lipoprotein profiles of these animals. Furthermore, we studied the three CETP molecular structures, physicochemical characteristics, and binding properties with known CETP inhibitors in silico. Our results showed that rabbits exhibited higher CETP activity than guinea pigs and hamsters, while these animals had different lipoprotein profiles. CETP inhibitors can inhibit rabbit and hamster CETP activity in a similar manner to human CETP. Analysis of CETP molecules in silico revealed that rabbit and hamster CETP showed many features that are similar to human CETP. These results provide novel insights into understanding CETP functions and molecular properties.

High levels of plasma high-density lipoprotein-cholesterol (HDL-C) are inversely associated with the risk of atherosclerosis and other cardiovascular diseases; thus, pharmacological inhibition of cholesteryl ester transfer protein (CETP) is considered to be a therapeutic method of raising HDL-C levels. However, many CETP inhibitors have failed to achieve a clinical benefit despite raising HDL-C. In the study, we generated transgenic (Tg) rabbits that overexpressed the human CETP gene to examine the influence of CETP on the development of atherosclerosis. Both Tg rabbits and their non-Tg littermates were fed a high cholesterol diet for 16 weeks. Plasma lipids and body weight were measured every 4 weeks. Gross lesion areas of the aortic atherosclerosis along with lesional cellular components were quantitatively analyzed. Overexpression of human CETP did not significantly alter the gross atherosclerotic lesion area, but the number of macrophages in lesions was significantly increased. Overexpression of human CETP did not change the plasma levels of total cholesterol or low-density lipoprotein cholesterol but lowered plasma HDL-C and increased triglycerides. These data revealed that human CETP may play an important role in the development of atherosclerosis mainly by decreasing HDL-C levels and increasing the accumulation of macrophage-derived foam cells.

N-glycans play important roles in various pathophysiological processes and can be used as clinical diagnosis markers. However, plasma N-glycans change and their pathophysiological significance in the setting of hypercholesterolemia, a major risk factor for atherosclerosis, is unknown. Here, we collected plasma from both hypercholesterolemic patients and cholesterol-fed hypercholesterolemic rabbits, and determined the changes in the whole-plasma N-glycan profile by electrospray ionization mass spectrometry. We found that both the hypercholesterolemic patients and rabbits showed a dramatic change in their plasma glycan profile. Compared with healthy subjects, the hypercholesterolemic patients exhibited higher plasma levels of a cluster of high-mannose and complex/hybrid N-glycans (mainly including undecorated or sialylated glycans), whereas only a few fucosylated or fucosylated and sialylated N-glycans were increased. Additionally, cholesterol-fed hypercholesterolemic rabbits also displayed increased plasma levels of high-mannose in addition to high complex/hybrid N-glycan levels. The whole-plasma glycan profiles revealed that the plasma N-glycan levels were correlated with the plasma cholesterol levels, implying that N-glycans may be a target for treatment of hypercholesterolemia.

This study focused on the unique properties of both the Ldlr knockout defect (closely mimicking the human situation) and the BALB/c (C) inbred mouse strain (Th-2 slanted immune response). We generated two immunodeficient strains with severe combined B- and T-cell immunodeficiency with or without a complete lack of natural killer cells to revisit the role of adaptive immune responses on atherogenesis. C-Ldlr-/- Rag1-/- mice, which show severe combined B- and T-cell immunodeficiency and C-Ldlr-/- Rag1-/- Il2rg-/- mice, which combine the T- and B-cell defect with a complete lack of natural killer cells and inactivation of multiple cytokine signalling pathways were fed an atherogenic Western type diet (WTD). Both B6-Ldlr-/- and C-Ldlr-/- immunocompetent mice were used as controls. Body weights and serum cholesterol levels of both immunodeficient strains were significantly increased compared to C-Ldlr-/- controls, except for cholesterol levels of C-Ldlr-/- Rag1-/- double mutants after 12 weeks on the WTD. Quantification of the aortic sinus plaque area revealed that both strains of immunodeficient mice developed significantly more atherosclerosis compared to C-Ldlr-/- controls after 24 weeks on the WTD. Increased atherosclerotic lesion development in C-Ldlr-/- Rag1-/- Il2rg-/- triple mutants was associated with significantly increased numbers of macrophages and significantly decreased numbers of smooth muscle cells compared to both C-Ldlr-/- wild type and C-Ldlr-/- Rag1-/- double mutants pointing to a plaque destabilizing effect of NK cell loss. Collectively, the present study reveals a previously unappreciated complexity with regard to the impact of lymphocytes on lipoprotein metabolism and the role of lymphocyte subsets in plaque composition.

Cocoa powder is rich in flavonoids, which have many beneficial effects on human health, including antioxidative and anti-inflammatory effects. The aim of our study was to investigate whether the intake of cocoa powder has any influence on hyperlipidemia and atherosclerosis and examine the underlying molecular mechanisms. We fed apoE knockout mice a Western diet supplemented with either 0.2% (low group) or 2% (high group) cocoa powder for 12 weeks. The groups fed dietary cocoa powder showed a significant reduction in both plasma cholesterol levels and aortic atherosclerosis compared to the control group. Analysis of mRNA profiling of aortic atherosclerotic lesions revealed that the expression of several genes related to apoptosis, lipid metabolism, and inflammation was significantly reduced, while the antiapoptotic gene Bcl2 was significantly increased in the cocoa powder group compared to the control. RT-PCR analysis along with Western blotting revealed that a diet containing cocoa powder inhibited the expression of hepatic endoplasmic reticulum stress. These data suggest that cocoa powder intake improves hyperlipidemia and atherosclerosis, and such beneficial effects are possibly mediated through the suppression of hepatic endoplasmic reticulum stress.

In the present study, we examined the effect of primary cooling rates on the motility and fertility of frozen-thawed rabbit spermatozoa. Rabbit semen diluted with an egg-yolk acetamide extender was cooled from room temperature to 5°C at four different rates (-0.1, -0.2, -0.4, -0.8°C/min) as a primary cooling step, then semen was frozen in liquid nitrogen vapour. After thawing, sperm cooled at -0.1°C/min showed the highest motility (40.7 ± 7.3%); there were no significant differences between the motilities of the -0.1, -0.2, and -0.4°C/min groups. The motility of frozen-thawed sperm cooled at -0.8°C/min (29.2 ± 6.8%) was significantly lower than that of sperm cooled at -0.1 and -0.2°C/min. The viability (-0.1°C/min, 38.1 ± 4.0%; -0.8°C/min, 24.3 ± 7.3%) of frozen-thawed sperm was closely related to its motility (-0.1°C/min, 36.7 ± 7.2%; -0.8°C/min, 22.3 ± 4.7%). Quality of post-thaw motile sperm cooled at different rates was estimated by comparing the fertilisation ability of the -0.1 and -0.8°C/min groups following artificial insemination. There were no significant differences in pregnancy rates and mean litter sizes. These data suggest that cooling rabbit semen at rates ranging from -0.1 to -0.8°C/min affects the viability but not the fertilisation capacity of motile spermatozoa after thawing.

High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

Hot spring or hot spa bathing (Onsen) is a traditional therapy for the treatment of certain ailments. There is a common belief that hot spring bathing has therapeutic effects for wound healing, yet the underlying molecular mechanisms remain unclear. To examine this hypothesis, we investigated the effects of Nagano hot spring water (rich in carbonate ion, 42°C) on the healing process of the skin using a nude rat skin wound model. We found that hot spring bathing led to an enhanced healing speed compared to both the unbathed and hot-water (42°C) control groups. Histologically, the hot spring water group showed increased vessel density and reduced inflammatory cells in the granulation tissue of the wound area. Real-time RT-PCR analysis along with zymography revealed that the wound area of the hot spring water group exhibited a higher expression of matrix metalloproteinases-2 and -9 compared to the two other control groups. Furthermore, we found that the enhanced wound healing process induced by the carbonate ion-enriched hot spring water was mediated by thermal insulation and moisture maintenance. Our results provide the evidence that carbonate ion-enriched hot spring water is beneficial for the treatment of skin wounds.

ABSTRACT Herpes simplex virus type 1 (HSV-1) establishes latency in neurons and can cause severe disseminated infection with neurological impairment and high mortality. This neurodegeneration is thought to be tightly associated with virus-induced cytoskeleton disruption. Currently, the regulation pattern of the actin cytoskeleton and the involved molecular mechanisms during HSV-1 entry into neurons remain unclear. Here, we demonstrate that the entry of HSV-1 into neuronal cells induces biphasic remodeling of the actin cytoskeleton and an initial inactivation followed by the subsequent activation of cofilin, a member of the actin depolymerizing factor family that is critical for actin reorganization. The disruption of F-actin dynamics or the modulation of cofilin activity by mutation, knockdown, or overexpression affects HSV-1 entry efficacy and virus-mediated cell ruffle formation. Binding of the HSV-1 envelope initiates the epidermal growth factor receptor (EGFR)-phosphatidylinositide 3-kinase (PI3K) signaling pathway, which leads to virus-induced early cofilin phosphorylation and F-actin polymerization. Moreover, the extracellular signal-regulated kinase (ERK) kinase and Rho-associated, coiled-coil-containing protein kinase 1 (ROCK) are recruited as downstream mediators of the HSV-1-induced cofilin inactivation pathway. Inhibitors specific for those kinases significantly reduce the virus infectivity without affecting virus binding to the target cells. Additionally, lipid rafts are clustered to promote EGFR-associated signaling cascade transduction. We propose that HSV-1 hijacks cofilin to initiate infection. These results could promote a better understanding of the pathogenesis of HSV-1-induced neurological diseases. IMPORTANCE The actin cytoskeleton is involved in many crucial cellular processes and acts as an obstacle to pathogen entry into host cells. Because HSV-1 establishes lifelong latency in neurons and because neuronal cytoskeletal disruption is thought to be the main cause of HSV-1-induced neurodegeneration, understanding the F-actin remodeling pattern by HSV-1 infection and the molecular interactions that facilitate HSV-1 entry into neurons is important. In this study, we showed that HSV-1 infection induces the rearrangement of the cytoskeleton as well as the initial inactivation and subsequent activation of cofilin. Then, we determined that activation of the EGFR-PI3K-Erk1/2 signaling pathway inactivates cofilin and promotes F-actin polymerization. We postulate that by regulating actin cytoskeleton dynamics, cofilin biphasic activation could represent the specific cellular machinery usurped by pathogen infection, and these results will greatly contribute to the understanding of HSV-1-induced early and complex changes in host cells that are closely linked to HSV-1 pathogenesis.

Herpes simplex virus type 1 (HSV-1), a member of the herpesviridae, causes a variety of human viral diseases globally. Although a series of antiviral drugs are available for the treatment of infection and suppression of dissemination, HSV-1 remains highly prevalent worldwide. Therefore, the development of novel antiviral agents with different mechanisms of action is a matter of extreme urgency. During the proliferation of HSV-1, capsid assembly is essential for viral growth, and it is highly conserved in all HSV-1 strains. In this study, small interfering RNAs (siRNAs) against the HSV-1 capsid protein were screened to explore the influence of silencing capsid expression on the replication of HSV-1. We designed and chemically synthesized siRNAs for the capsid gene and assessed their inhibitory effects on the expression of target mRNA and the total intracellular viral genome loads by quantitative real-time PCR, as well as on the replication of HSV-1 via plaque reduction assays and electron microscopy. Our results showed that siRNA was an effective approach to inhibit the expression of capsid protein encoding genes including UL18, UL19, UL26, UL26.5, UL35 and UL38 in vitro. Interference of capsid proteins VP23 (UL18) and VP5 (UL19) individually or jointly greatly affected the replication of clinically isolated acyclovir-resistant HSV-1 as well as HSV-1/F and HSV-2/333. Plaque numbers and intracellular virions were significantly reduced by simultaneous knockdown of UL18 and UL19. The total intracellular viral genome loads were also significantly decreased in the UL18 and UL19 knockdown groups compared with the viral control. In conclusion, interfering with UL18 and UL19 gene expression could inhibit HSV-1 replication efficiently in vitro. Our research offers new targets for an RNA interference-based therapeutic strategy against HSV-1.

Urotensin II (UII) is a vasoactive peptide composed of 11 amino acids that has been implicated to contribute to the development of cardiovascular disease. The purpose of this study was to investigate whether UII affects the development of atherosclerosis in cholesterol-fed rabbits. UII was infused for 16 weeks through an osmotic mini-pump into male Japanese White rabbits fed on a high-cholesterol diet. Plasma lipids and body weight were measured every 4 weeks. Aortic atherosclerotic lesions along with cellular components, collagen fibers, matrix metalloproteinase-1 and -9 were examined. Moreover, vulnerability index of atherosclerotic plaques was evaluated. UII infusion significantly increased atherosclerotic lesions within the entire aorta by 21% over the control (P = 0.013). Atherosclerotic lesions were increased by 24% in the aortic arch (P = 0.005), 11% in the thoracic aorta (P = 0.054) and 18% in the abdominal aorta (P = 0.035). These increases occurred without changes in plasma levels of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides or body weight. Immunohistochemical staining revealed that macrophages and matrix metalloproteinase-9 were significantly enhanced by 2.2-fold and 1.6-fold in UII group. In vitro studies demonstrated that UII up-regulated the expression of vascular cell adhesion protein-1 and intercellular adhesion molecule-1 in human umbilical vein endothelial cells, which was inhibited by the UII receptor antagonist urantide. In conclusion, our results showed that UII promotes the development of atherosclerotic lesions and destabilizes atherosclerotic plaques in cholesterol-fed rabbits.

Increased plasma levels of C-reactive protein (CRP) are closely associated with cardiovascular diseases, but whether CRP is directly involved in the pathogenesis of atherosclerosis is still under debate. Many controversial and contradictory results using transgenic mice and rabbits have been published but it is also unclear whether CRP lowering can be used for the treatment of atherosclerosis. In the current study, we examined the effects of the rabbit CRP antisense oligonucleotides (ASO) on the development of atherosclerosis in WHHL rabbits. CRP ASO treatment led to a significant reduction of plasma CRP levels; however, both aortic and coronary atherosclerotic lesions were not significantly changed compared to those of control WHHL rabbits. These results suggest that inhibition of plasma CRP does not affect the development of atherosclerosis in WHHL rabbits.

Bisphenol A (BPA) is an environmental endocrine disrupter. Excess exposure to BPA may increase susceptibility to many metabolic disorders, but it is unclear whether BPA exposure has any adverse effects on the development of atherosclerosis. To determine whether there are such effects, we investigated the response of Watanabe heritable hyperlipidemic (WHHL) rabbits to 400-µg/kg BPA per day, administered orally by gavage, over the course of 12 weeks and compared aortic and coronary atherosclerosis in these rabbits to the vehicle group using histological and morphometric methods. In addition, serum BPA, cytokines levels and plasma lipids as well as pathologic changes in liver, adipose and heart were analyzed. Moreover, we treated human umbilical cord vein endothelial cells (HUVECs) and rabbit aortic smooth muscle cells (SMCs) with different doses of BPA to investigate the underlying molecular mechanisms involved in BPA action(s). BPA treatment did not change the plasma lipids and body weights of the WHHL rabbits; however, the gross atherosclerotic lesion area in the aortic arch was increased by 57% compared to the vehicle group. Histological and immunohistochemical analyses revealed marked increases in advanced lesions (37%) accompanied by smooth muscle cells (60%) but no significant changes in the numbers of macrophages. With regard to coronary atherosclerosis, incidents of coronary stenosis increased by 11% and smooth muscle cells increased by 73% compared to the vehicle group. Furthermore, BPA-treated WHHL rabbits showed increased adipose accumulation and hepatic and myocardial injuries accompanied by up-regulation of endoplasmic reticulum (ER) stress and inflammatory and lipid metabolism markers in livers. Treatment with BPA also induced the expression of ER stress and inflammation related genes in cultured HUVECs. These results demonstrate for the first time that BPA exposure may increase susceptibility to atherosclerosis in WHHL rabbits.

In this study, we created LDL receptor (LDLr) defective (WHHL) transgenic rabbits expressing human apo[a] to examine whether LDLr mediates the Lp[a] clearance from the plasma. By crossbreeding WHHL rabbits with human apo[a] transgenic rabbits, we obtained two groups of human apo[a] transgenic rabbits with defective LDLr functions: apo[a]+/0 WHHL heterozygous (LDLr+/−) and apo[a]+/0 WHHL homozygous (LDLr−/−) rabbits. The lipid and lipoprotein levels of human apo[a] WHHL rabbits were compared to those of human apo[a] transgenic rabbits with normal LDLr functions (LDLr+/+). The apo[a] production rate was evaluated by analyzing apo[a] mRNA expression in the liver, the major site for apo[a] synthesis in transgenic rabbits. We found that pre-β lipoproteins were markedly increased accompanied by a 2-fold increase in the plasma Lp[a] in apo[a]+/0/LDLr+/− rabbits and a 4.2-fold increase in apo[a]+/0/LDLr−/− rabbits compared with that in apo[a]+/0 rabbits with normal LDLr function. In apo[a]+/0/LDLr−/− rabbits, there was a marked increase in plasma total cholesterol and triglycerides, as was found in their counterpart non-transgenic WHHL rabbits. Northern blot analysis revealed that hepatic apo[a] expression in WHHL transgenic rabbits was similar to that in LDLr+/+ transgenic rabbits, suggesting the accumulation of plasma Lp[a] in WHHL transgenic rabbits was not due to increased apo[a] synthesis.In conclusion, absence of a functional LDLr leads to a marked accumulation of plasma Lp[a] in human apo[a] transgenic WHHL rabbits and LDLr may participate in the catabolism of Lp[a] in rabbits.

The triglyceride lipase gene subfamily plays a central role in lipid and lipoprotein metabolism. There are three members of this subfamily: lipoprotein lipase, hepatic lipase, and endothelial lipase. Although these lipases are implicated in the pathophysiology of hyperlipidemia and atherosclerosis, their structures have not been fully solved. In the current study, we established homology models of these three lipases, and carried out analysis of their activity sites. In addition, we investigated the kinetic characteristics for the catalytic residues using a molecular dynamics simulation strategy. To elucidate the molecular interactions and determine potential key residues involved in the binding to lipase inhibitors, we analyzed the binding pockets and binding poses of known inhibitors of the three lipases. We identified the spatial consensus catalytic triad "Ser-Asp-His", a characteristic motif in all three lipases. Furthermore, we found that the spatial characteristics of the binding pockets of the lipase molecules play a key role in ligand recognition, binding poses, and affinities. To the best of our knowledge, this is the first report that systematically builds homology models of all the triglyceride lipase gene subfamily members. Our data provide novel insights into the molecular structures of lipases and their structure-function relationship, and thus provides groundwork for functional probe design towards lipase-based therapeutic inhibitors for the treatment of hyperlipidemia and atherosclerosis.