| BackgroundAtherosclerosis-associated CVD remains as the leading cause of mortality in the Western society and in most other parts of the world. Despite tremendous efforts devoted to CVD research, little progress has been made during the last decades for effective prevention and treatment. Several recent studies have shown that colder ambient temperature has been associated with an increased incidence of myocardial infarction (MI) and other atherosclerosis-associated vascular disorders such as stroke. Accumulating evidence from epidemiological studies have provided compelling evidence linking cardiovascular disorders to the cold seasons or low ambient temperature. In a recent short-term study, daily reduction of1℃of average temperature could cumulatively increase2.2%risk of MI during a month period, demonstrating the causal role of low ambient temperature in promoting CVD. Despite this long-known linkage between cold and high CVD risks, the mechanisms underlying cold-induced CVD remain unknown although snow-removal during the winter season has been claimed to be associated to the increased incidence.Recent research showed that the level of LDL-C, as an independent risk factor for CVD, shared similar seasonal variation pattern as cardiovascular events. In the62th annual scientific conference of the newest ACC, Dr. Filipe of Brazil Campinas University reported an interesting large population study. They collected blood samples from227,359individuals among2008-2010, found that plasma LD-C>130mg/dl was8%more prevalent during winter than summer, HDL-C<40mg/dl and TG>150mg/dl were respectively9%and5%more prevalent than summer. This showed that there was significant connection between temperature and human blood lipid level, which provides an novel possible mechanism for further understanding the cold induced high prevelance of cardiovascular events.Many research showed that metabolism of adipose tissue is closely related to temperature, the metabolic state of adipose tissue changed a lot under cold environment. Adipose tissue is an important metabolic organ of the human, mainly consists of two different adipose tissue, white adipose tissue (WAT) and brown adipose tissue (BAT). WAT is an important tissue for energy store, regulator of metabolism and the secretion of insulin and cytokines, while BAT maintain the body temperature by heat production from breakdown of TG into CO2, water and heat is important for basal and stimulated energy production. Under cold environment the heat production of the adipose tissue is accomplished by BAT.In rodents, it is known that cold exposure can sufficiently activate BAT, additionally, low ambient temperature can sufficiently stimulate phenotypic and functional conversion of WAT into BAT-like tissue (BRITE), which undergoes high rates of metabolism in an UCP1-dependent manner. Several recent studies show that a substantial amount of BAT exists in adult humans and cold augments BAT activation. Based on those articles and research, we proposed the following hypothesizes:1. Cold exposure could influence the development of atherosclerosis;2. Cold could significantly activate the BAT, then influence the AS progress;3. The WAT could be converted into BRITE under cold temperature, therefore influence the development of atherosclerosis.In order to verify the hypothesis, we used the cold exposure model, choosed the classic AS mouse model, apoE-/-mice to study and use C57wild type mice as a control, to see if the activation of BAT and conversion of WAT into BRITE could influence the metabolic state of the mice, further change the adipose tissue metabolism and therefore promote the development of atherosclerosis. We focused on studying the following fat depots:sWAT, eWAT and iBAT.Aims(1) To see if cold exposure could activate BAT.(2) To see if cold exposure could promote the WAT-BRITE conversion.(3) To see if cold exposure could promote the development of AS.Experiment MaterialsAnimalsMale ApoE-/-mice and wild type mice in C57BL/6background at the age of8-wk were purchased from Beijing Wei Tong Li Hua Experimental Animal Technology Co. LTD (Beijing, China). Mice were sacrificed by anesthetizing with intraperitoneal (i.p.) injection of0.8%pentobarbital sodium (60mg/kg), followed by cervical dislocation. All animal studies were complied with the Animal Management Rules of the Chinese Ministry of Health and approved by the Ethical and Use Committee of the Qilu Hospital of the Shandong University.Cold AcclimationEight-wk-old male ApoE-/-and C57BL/6mice caged in22℃were fed with a high-fat diet. Each of the strain of mice was randomly divided into different groups. While one group of mice was exposed to30℃directly, the other groups were first adapted at18℃for1week, followed by exposure to4℃for the following wks.Tissue Sample Collection For collection of blood samples, animals exposed under different conditions were fasted overnight and anesthetized with0.8%pentobarbital sodium (i.p,60mg/kg). Blood was intracardially collected using a syringe. After sacrificing animals, various adipose depots, heart and arteries were dissected. For analysis of mRNA expression, adipose tissue samples were immediately placed in liquid nitrogen. The samples were kept in-80℃until use. For histological analysis, samples were fixed with4%paraformaldehyde overnight, followed by washing with lx PBS. A portion of the samples was paraffin-embedded and the rest were used for cryo-sections.Blood Lipid AnalysisAfter collection of blood samples, plasma from each mouse was prepared and stored at-80℃until use. Free fatty acids were measured using a commercially available ELISA kit (Roche, Shanghai, China). Total plasma levels of CHO, TG, LDL-C, and high-density lipoprotein cholesterol (HDL-C) were measured using commercially available agents (Zhejiang Dongou Biotechnology, Wenzhou, China).Measurement of cAMP and GlycerolTissue homogenates from eWAT exposed to different temperatures were used to measure the levels of cAMP using a commercially available kit (Cayman. Florida). For detection of glycerol, adipocytes from eWAT were isolated using collagenase Ⅱ digestion (0.25mg/ml) at37℃for1h. The single cell suspension was cultured at37℃for1h in the presence or absence of isoproterenol in an AIS buffer containing125mM NaCl,5mM KC1,1mM CaC12,2.5mM MgC12,1mM KH2PO4,2%BSA,4mM glucose, and25mM Tris (pH7.4). The released glycerol was measured using a commercial kit (Sigma, Shanghai, China).Metabolic RateOxygen consumption was measured using an open-circuit system (Sable, USA) as previously described. Prior to measurement, mice were anesthetized by i.p. injection with pentobarbital at the dose of90mg/kg. Animals were immediately transferred to a33℃metabolic chamber, where02consumption and release of CO2were uninterruptedly recorded for30min. By the end of30min, a single subcutaneous injection of NE was administrated.,02consumption and release of CO2were further recorded for successive90min.Histological Staining and ImmunohistochemistrySome tissue samples were stained with HE and Oil-Red O using a standard protocol. Tissues prepared from cryosections or paraffin-embedded samples (5μm thickness) were immunohistologically stained with various primary antibodies (see above), followed by further staining with secondary antibodies as previously described. For staining of blood vessels in the adipose tissue, the whole-mount technology was used and the positive signals were detected using a confocal microscope. AP instability index was calculated according to the standard formula:(Oil Red O+signal plus MOMA-2+signal)/(SMA+signal plus collagen I+signal).Statistical AnalysisAll statistical analyses were performed using a standard two-tail student t-test. Mean determinants were presented as+SEM.ResultsCold Exposure Activates BAT and Induces BRITEHistological analysis of sWAT and eWAT showed that the average sizes of the adipocytes were markedly smaller in the cold-exposed group as compared with those exposed to the termoneutral temperature, leading to reduced total weights of sWAT and eWAT. Cellular contents of4℃-sWAT and4℃-eWAT were markedly increased and contained high density of HE-stained structures. Immunohistochemical analysis demonstrated that high expression levels of UCP1, a specific mitochondrial protein for BAT, were present in4℃-sWAT and4℃-eWAT whereas UCP1levels were completely undetectable in30℃-sWAT and30℃-eWAT. These findings demonstrate that exposure of ApoE-/-Amice to cold led to sufficient conversion of various WAT into BRITE. Cold exposure also augmented the activation of interscapular BAT (iBAT) by increasing UCP1expression. In addition to alterations of adipocytes, the micro vessel density in4℃-WATs and4℃-iBAT was significantly increased.Activation of Lipolysis and Metabolism by Cold ExposureExposure of ApoE-/-and wild type mice to cold resulted in significant decrease of the body weight and body mass index (BMI) as compared to the30℃group, supporting the fact that cold increases metabolism, which leads to a lean phenotype. Exposure of ApoE-/-mice to cold led to marked elevation of the metabolic rate as measured by O2consumption and CO2production. The level of plasma triglyceride (TG) was significantly decreased, conversely, the levels of CHO, LDL-C and glycerol were increased2-3folds, whereas the level of free fatty acid (FFA) remained unchanged.Elevated Expression Levels of Lipolysis-Associated Gene ProductsTo study alteration of expression levels of metabolically associated genes, a genome-wide microarray analysis was performed in four groups i.e.,4℃-1wk,30℃-1wk, using the inguinal WAT (iWAT), which exhibited an obvious BRITE phenotype. Gene cluster and principle component analyses demonstrated that gene expression patterns were highly reproducible. As expected, a number of lipolysis-related genes were highly expressed in4℃-1-wk-iWAT. Noticeably, many of these cold highly-induced lipolysis-associated genes are crucial enzymes for oxidative and lipid degradation. Consistent with gene profile alterations, high expression levels of several key lipolysis genes were further validated by quantitative real-time PCR (qPCR) analysis.Interestingly, the cAMP level in the4℃-eWAT group was significantly increased relative to the30℃-eWAT group. Furthermore, adipocytes from4℃-eWAT and30℃-eWAT were isolated and incubated in the presence or absence of isoproterenol (ISO). As expected, stimulation of4℃-eWAT adipocytes with isoproterenol significantly increased the release of glycerol, indicating that the high rate of lipolysis occurred in these cells.Cold Exposure Stimulates AP Growth in ApoE-/-MiceSurprisingly, gross examination by general Oil Red O staining revealed that arteries including the aorta arch, thoracic artery (Tho.A), abdominal artery (Abd.A) and common iliac artery (C.I.A) contained wide-spread APs throughout the artery stem after exposure to cold. Cross-section analysis followed by HE staining showed that significantly larger APs were present the aortic root after exposure to cold as compared to the30℃group. The existence of large APs in the4℃group was further validated by Oil Red O staining. These findings demonstrate that cold significantly augments AP growth in main arteries of ApoE-/-mice.Conclusion1. Cold exposure noticeably activated the BAT, sufficiently converted several WATs into BRITE.2. Cold exposure induced high level of LDL-C through high level of lipolysis caused by the activated of BAT/BRITE.3. Cold exposure significantly promoted the development of atherosclerosis. BackgroundAccording to the newest WHO publication, AS related cardiovascular diseases such as acute myocardial infarction, angina, cardiac arrest, ischemic stroke and heart failure etc, have become the number1mortality cause of the world, with a20000thousand or so annual death. It is estimated that by2030, the CVD will still be the first mortality cause of the world. There was one person died of CVD every11.6seconds,3times of the death rate of United States, which makes CVD the first killer of China. Therefore, further studying about the mechanisms and therapeutic targets of AS are urgently needed.The major danger of AS is its related acute cardiovascular events. Traditionally, acute cardiovascular events were induced by luminal stenosis and ischemia suffered from the sudden intrusion of atherosclerotic plaque. Recent research showed that the formation of thrombosis caused by plaque rupture and erosion is the major mechanism of cardiovascular event, therefore this type of plaque was called vulnerable plaque. The atherosclerotic plaque of stable angina with thicker fibrous cap, more extracelluar matrix, less lipid and inflammatory cells such as macrophages belonged to stable plaque.The deposition of cholesterol in the blood vessels followed through the whole process of atherosclerosis, also played an import role in the rupture of atherosclerotic plaque. Because LDL-C could promote the apoptosis of endothelium and smooth muscle cells, activate the matrix metalloproteinases, weaken fibrous cap and acclerate the inflammatory reaction which causing the plaque rupture and formation of thrombosis. Atherosclerosis is an chronic inflammatory reaction, sudden conversion from chronic inflammation into acute inflammation is the key factor to the plaaque rupture. A lot of research showed that intraplaque hemorrhage siginificantly increased the instability of atherosclerotic plaque, and its main resource was the immature neovessels within the plaque.In order to rule out the cold effect on atherosclerosis only happened in apoE-/-mice, this special mouse model, we choose another classic AS mouse model, LDLR-/-mice to further study the influence and mechanism of cold on atherosclerosis. According to those reference and previous research, we put forward the following hypothesises:1. Cold exposure could significantly activate BAT and promote the conversion from WAT into BRITE, which could increase the lipolysis and hepatic cholesterol synthesis, finally influence the metabolic balance of blood lipid and promote the development and instablity of atherosclerotic plaque;2.Cold exposure significantly increased the circulating and local inflammation and promoted intraplaque angiogenesis, which significantly increased the instablity of atherosclerotic plaque;3. Cold exposure significantly promoted the progress and instablity of atherosclerotic plaque in LDLR-/-mice. Based on these hypothesis, we chosed apoE-/-mice and LDLR-/-mice to further study the impact and exact mechanism of cold exposure on atherosclerotic lesions.Aims(1) To see if cold exposure could promote the development of AS in LDLR-/mice.(2) To see if cold exposure could activate BAT and promote the WAT-BRITE conversion in LDLR-/-mice. (3) To see if cold exposure could promote the instability of atherosclerotic plaque in apoE-/-and LDLR-/-mice.Experimental proceduresAnimalsMale ApoE-/-mice at the age of8-wk were purchased from Beijing Wei Tong Li Hua Experimental Animal Technology Co. LTD (Beijing, China). Male LDLR-/-mice in C57BL/6background at the age of8-wk were purchased from the Animal Model Institute, Nanjing University (Nanjing, China). Mice were sacrificed by anesthetizing with intraperitoneal (i.p.) injection of0.8%pentobarbital sodium (60mg/kg), followed by cervical dislocation. All animal studies were complied with the Animal Management Rules of the Chinese Ministry of Health and approved by the Ethical and Use Committee of the Qilu Hospital of the Shandong University.Cold AcclimationEight-wk-old male ApoE-/-, LDLR-/-mice caged in22℃were fed with a high-fat diet for8wks. Each of the strain of mice was randomly divided into two groups (n=20/group) and switched to pair-fed feeding with regular diet. While one group of mice was exposed to30℃for4or8wks, the other group were first adapted at18℃for1week, followed by exposure to4℃for another7wks.Tissue Sample CollectionFor collection of blood samples, animals exposed under different conditions were fasted overnight and anesthetized with0.8%pentobarbital sodium (i.p,60mg/kg). Blood was intracardially collected using a syringe. After sacrificing animals, various adipose depots, heart, arteries and liver tissue were dissected. For analysis of mRNA expression, tissue samples were immediately placed in liquid nitrogen. The samples were kept in-80℃until use. For histological analysis, samples were fixed with4% paraformaldehyde overnight, followed by washing with1x PBS. A portion of the samples was paraffin-embedded and the rest were used for cryo-sections.FPLCFPLC chromatography separation technique was used for detailed analysis of various cholesterol components and TG (n=6/group). Briefly, a aliquot of100μl of plasma was chromatographed on a Superose6HR10/300GL column (GE Healthcare, Sweden) and eluted with lx PBS (PH7.2) at a speed of0.5ml/min. A total number of60fractions (0.5ml/fraction) were collected, and each fraction was analyzed for cholesterol and TG concentrations using standard ELISA Kits (Cayman, Florida). Values from fractions5-10were used for calculation of VLDL/IDL, fractions11-19were LDL, and fractions20-30were HDL.In vivo Cholesterol SynthesisVarious groups of ApoE-/-mice (n=6/group) were injected (i.p.) with20mCi of [3H]-water (PerkinElmer, USA) in0.1ml of phosphate saline. After1h of injection, mice were anesthetized and blood samples were immediately collected and used for measurement of plasma [H]. Liver tissues were homogenized and200-300mg of homogenate portions was saponified, and the digitoninprecipitable sterols were isolated as previously described. The rates of hepatic cholesterol synthesis were calculated as the mmol of [jH]-water incorporated into digitonin-precipitable sterols per hour per gram of tissues.Indirect CalorimetryOxygen consumption was measured using an open-circuit system (Sable, USA) as previously described. Prior to measurement, mice were anesthetized by i.p. injection with pentobarbital at the dose of90mg/kg. Animals were immediately transferred to a33℃metabolic chamber, where02consumption and release of CO2were uninterruptedly recorded for30min. By the end of30min, a single subcutaneous injection of NE at the dose of20μg/20g-mouse was administrated.O2consumption and release of CO2were further recorded for successive90min.Histological StainingSome tissue samples were stained with H&E using a standard protocol. Oil red O (Sigma, Shanghai, China) at the final concentration of0.5%was used to stain tissue sections (5μm thickness) for10min. The stained samples were washed with37℃water for a few seconds. The samples were counter-stained with hematoxylin. Picrosirus red (Sigma, Shanghai, China) at the final concentration of0.5%was used for staining collagens.ImmunohistochemistryTissues prepared from cryosections or paraffin-embedded samples (5μm thickness) were immunohistologically stained with various primary antibodies (see above), followed by further staining with secondary antibodies as previously described. For staining of blood vessels in the adipose tissue, the whole-mount technology was used and the positive signals were detected using a confocal microscope. AP instability index was calculated according to the standard formula:(Oil Red O+signal plus MOMA-2+signal)/(SMA+signal plus collagen I+signal).Statistical AnalysisAll statistical analyses were performed using a standard two-tail student t-test. Mean determinants were presented as+SEM.Results Prolonged Cold Exposure Activates BAT and Induces BRITENecropathic gross examination of subcutaneous WAT (sWAT) and epididymal WAT (eWAT) at wk8revealed an obvious brownish phenotype of both depots in the4℃group relative to the30℃group. Histological analysis of sWAT and eWAT showed that the average sizes of the adipocytes were markedly smaller in the cold-exposed group as compared with those exposed to the termoneutral temperature, leading to reduced total weights of sWAT and eWAT. Owing to smaller adipocyte sizes, the number of adipocytes per field was relatively increased.Cellular contents of4℃-sWAT and4℃-eWAT were markedly increased and contained high density of H&E-stained structures. Immunohistochemical analysis demonstrated that high expression levels of UCP1, a specific mitochondrial protein for BAT, were present in4℃-sWAT and4℃-eWAT whereas UCP1levels were completely undetectable in30℃-sWAT and30℃-eWAT. Kinetic studies demonstrated that these alterations in WATs were readily detected after4-wk-exposure to cold. These findings demonstrate that exposure of ApoE-’-mice to cold led to sufficient conversion of various WAT into BRITE. Cold exposure also augmented the activation of interscapular BAT (iBAT) by increasing UCP1expression. In addition to alterations of adipocytes, the microvessel density in4℃-WATs and4℃-iBAT was significantly increased; validating the fact that cold exposure could sufficiently switch on an angiogenic phenotype in the adipose tissue.Activation of Lipolysis and Metabolism by Cold ExposureAs conversion from WAT into BRITE and activation of BAT are instrumental for NST and lipolysis, we next analyzed alterations of blood lipid profiles, metabolism and expression levels of crucial genes involved in lipolysis. Interestingly, exposure of pair-fed ApoE-/-mice to cold resulted in significant decrease of the body weight and body mass index (BMI) as compared to the30℃group, supporting the fact that cold increases metabolism, which leads to a lean phenotype. NST-related metabolism can be detected by measuring the metabolic rate in response to norepinephrine (NE). As expected, exposure of ApoE-/-mice to cold for4or8wks led to marked elevation of the metabolic rate as measured by O2consumption and CO2production. These findings are consistent with the decrease of body weight and BMI owing to a high metabolic rate during cold acclimation.Cold Exposure Increase Blood LipidFast protein liquid chromatography (FPLC) analysis of plasma cholesterol under cold-exposure showed that the proportion of VLDL was particularly increased. Similarly, intermediate-density lipoprotein (IDL) and the proportion of LDL were also markedly increased by cold exposure. Conversely, plasma levels of the high-density lipoprotein (HDL) cholesterol component remained virtually unchanged. We next studied cholesterol synthesis by intraperitoneal injection with [H]-water. Intriguingly, the hepatic cholesterol synthesis rate was markedly increased in cold-exposed ApoE-/-mice. Consistent with the increased level of cholesterol synthesis, hydroxymethylglutaryl-coenzyme A reductase (HMG-CoA reductase) and transcriptional factors essential for cholesterol synthesis, including Sterol Regulatory Element-Binding Proteins (SREBPs) transcription factor and its partner protein SREBP-cleavage-activating protein (SCAP) were also markedly increased. Inversely, plasma levels of TG were markedly decreased in cold-exposed ApoE-/-mice relative to those of the thermoneutral temperature-exposed group.Cold Exposure Stimulates AP Growth in ApoE-/-MiceThe average size of plaque areas was substantially increased after prolonged8-wk-exposure to cold although the plaques in the30℃control group showed a tendency to grow at a slow rate. Prolonged exposure of ApoE-/-mice to cold for8wks led to markedly accelerated AP growth whereas the average plaque size in the30℃remained unchanged. These findings demonstrate that cold significantly augments AP growth in main arteries of ApoE-/-mice. Cold Acclimation Augments AP Instability in ApoE-/-MiceDeposition of lipid contents, infiltration of inflammatory cells, mobilization of smooth muscle cells, and decrease of collagen I are considered as crucial elements for AP instability. By the end of wk8, an approximate4-fold increase of IMs was found in4℃-APs relative to the30℃control group. Consistent with high numbers of inflammatory cells, the circulating level of IL-6was increased by approximately2-fold in the8-wk-4℃group relative to the30℃control group. Intriguingly, levels of IL-6and other inflammatory cytokines including MCP-1and MMP-2within4℃-APs were also markedly increased as compared with30℃-APs. Additionally, high levels of IL-6and MCP-1were also detected in4℃-iWAT, suggesting that the adipocytes are the primary source of high production of inflammatory cytokines.Further, a-smooth muscle actin (a-SMA) positive structures in APs were significantly decreased after4-wk and8-wk cold acclimation. Additionally, Sirus-Red+collagen I structures were significantly decreased in4℃groups as compared with30℃controls. In the light of these changes, the plaque instability indexes in8-wk-4℃groups were increased in several magnitudes relative to the30℃control groups. Interestingly, microvessels were often present in the adventitial area of the aorta stem, occasionally penetrating into APs, in cold-exposed ApoE-/-mice relative to30℃-exposed mice. Penetration of microvessels in the adventitial region and within APs might further increase instability as previously reported. Taken together, these findings provide compelling evidence that cold acclimation significantly increases the plaque instability in ApoE-/-mice.Cold Acclimation Induces AP Growth and Instability in LDLR-/-MiceTo study if the cold-augmented AP growth and instability were not only restricted to ApoE-/-mice and generally occurred in other mouse models, we used the LDLR-/-mouse strain to validate our findings. Similar to ApoE-/-mice, cold acclimation for8wks led to the marked transition from eWAT into a BRITE phenotype by significantly increasing UCPl expression. Additionally, iBAT became highly activated during cold exposure by expressing a high level of UCP1relative to the30℃control group. Under the pair-fed condition, cold acclimation significantly reduced body weight and BMI, suggesting active lipolysis occurred in these mice. Indeed, measurement of the NST-related metabolic rate showed an increased level of metabolism in LDLR-/-mice under cold acclimation.Similar to ApoE-/-mice, the key lipolysis-associated signaling component cAMP was noticeably increased in4℃-eWAT relative to30℃-eWAT, demonstrating active lipolysis occurring in WAT. Consistent with the increased level of lipolysis, the level of glycerol as a lipolysis product was also increased in4℃-eWAT. Reconciling with active lipolysis, plasma levels of cholesterol and LDL-cholesterol were significantly increased to levels similar to those of4℃-exposed Apo E-/-mice. Plasma levels of the VLDL and small LDL remnants were markedly increased in the cold-exposed Ldlr-/-mice relative to those exposed to30℃. Again, plasma levels of HDL remained similar in both groups. Plasma TG levels in the4℃-exposed group was considerably decreased in all fractions.In general, development of atherosclerotic plaques in the Ldlr-/-mouse strain under30℃was delayed and less prominent as compared with that in ApoE-/-mice. Nevertheless, histological examination of the main arteries revealed a striking increase of atherosclerotic plaque sizes in the4℃-exposed group (n=20/group). The plaque instability-related parameters including infiltration of the number of inflammatory macrophages and lipid deposition were markedly increased in4℃-plaques as compared with those in30℃-plaques. Additionally, significant decreases of α-SMA+and collagen I+structures were also observed in4℃-plaques. Calculation of the plaque instability index demonstrated that an approximate8-fold-increase of the atherosclerotic plaque instability index was found in the4℃group after8-wk-exposure to cold. Similar to ApoE-/-mice, increased necrotic core areas and decreased thickness of fibrous caps in association with plaque instability were observed in the4℃group as compared to30℃group. Taken together, these findings demonstrate that cold acclimation induces atherosclerotic plaque growth and instability in Ldlr-/-mice and thus our findings may be reasonably generalized to other mouse strains.Conclusions1. Cold-induced high level of hepatic cholesterol sythesis rate significantly increased the plasma levels of VLDL-C and LDL-C.2. Cold significantly increased the circulating and local inflammation, intraplaque angiogenesis, which promotes the instability of apoE"’"mice.3. Cold exposure significantly increased the growth and instability of AS in apoE-/-mice and LDLR-/-mice. BackgroundTremendous research showed that mortality rate suffering from CVD significantly increased during winter. Despite this long-known linkage between cold and high CVD risks, the mechanisms underlying cold-induced CVD remain unknown although snow-removal during the winter season has been claimed to be associated to the increased incidence. In our previous study, we verified that cold exposure could significantly promoted the growth and instablity of atherosclerotic plaque, and increased the plasma level of LDL-C. Cold exposure fullfilled those effects through the axis of activation of SNS-NE-BAT/BRITE-UCP1-lipolysis. In order to verify that mechanistic axis, we focused on two targets, NE and UCP1.As we all know that, cold exposure could immediately acitvate SNS, release a lot of NE. To clarify the role of β3-adrenoceptor in physiological function of NE, researchers usually compare the difference between NE stimulus and β3-adrenoceptor agonists. Among all the others agonists, CL-316243was mostly used. While in the in vivo studies, if you want to exclude the effect of β3-adrenoceptor, the antiagonist SR59230A was often used. According to our previous study, we speculated the mechanistic axis of cold accelerating AS progress was NE acitivate β3-adrenoceptor, activate BAT and promote the conversion from WAT into BRITE, increase lipolysis, change lipid metabolism, accelerate synthesis of cholesterol, then the hypercholesterolemia promote the progress of atherosclerotic lesions. The relation between adipokine, especially adiponectin, and atherosclerosis attracted a lot of attention. The adiponectin transgeneic mice verified that adiponectin could improve insulin resistance and diabetes; while adiponectin knockout mice showed moderate indulin resistance, glucose intolerence and some other metabolic syndrom related sympotoms, including dyslipidemia and hypertension. Study showed that low adiponectin level is related to a lot of symptoms of human insulin resistance, such as dyslipidemia, CVD and hypertension. Given the close connection among adipose tissue and adiponectin, adiponectin and AS, we speculated that cold exposure could significantly change the metabolic state of adipose tissue, then influence the plasma level of adiponectin, while injection of adiponectin will reversely influence the metabolism of adipose tissue.In order to verify the role of (33-adrenoceptor in the cold induced development of atherosclerosis, we used (33-adrenoceptor agonist (CL316243) and antiagonist (SR59230A) to study the exact mechanism. In order to verify the role of UCP1in the cold induced development of atherosclerosis, we created apoE-/-UPC1-/-double knockout (AUDK) mice by crossbreeding apoE-/-mice with UCP1-/-mice. All8weeks old male AUDK mice were randomly divided into30℃group and4℃group. This experiment focused on studying if the deletion of UCP1gene could impact the cold effects on the metabolic state of adipose tissue, the atherosclerosis development and plaque instability, to furether clarify the exact molecular mechanism of cold exposure accelerating athersosclerosis.Aims(1) To see if CL316243has similar effect on the apoE-/-mice as cold stimulus.(2) To see if SR59230A could block the effect of cold on the apoE-/-mice.(3) To see if the cold exposure influence plasma level of adiponectin and the relation between adiponectin and UCPl.(4) To study the exact role of UCP1in cold accelerating AS progress. Experiment MaterialsAnimalsMale ApoE-/-mice and wild type mice in C57BL/6background at the age of8-wk were purchased from Beijing Wei Tong Li Hua Experimental Animal Technology Co. LTD. Crossbreed apoE-/-mice with UCP1-/-mice to generate apoE-/-UCP-/-double knockout mice (AUDK). Mice were sacrificed by anesthetizing with intraperitoneal (i.p.) injection of0.8%pentobarbital sodium (60mg/kg), followed by cervical dislocatio... |
PDF Full Text Request