Relationship Between Adiponectin And Atherosclerotic Plaque And The Mechanism Of Prolyl-4-Hydroxylase Expression

IntroductionAtherosclerosis, which causes ischemic cardiopathy and stroke, is the most common cause of mortality and morbidity in developed countries. In the event of cap rupture, thrombosis occurs and causes acute coronary syndrome. Finding the mechanism of the stability of preexisting plaques is a hot study recently years.In recent years, theories about the role of extracellular matrix (ECM) in the pathogenesis of atherosclerosis have been developed. ECM components, especially collagen—the main constituent of the Fibrouscapin atheroma—is thought to be important in the progression of atherosclerosis and plaque rupture. Functional tissue structure, such as the distensibility of an artery, is maintained by a balanced ECM metabolism of production and degradation. At present, many groups have studied the degradation of collagen, but it is not very clear about the relationship between collagen biosynthesis and stability of plaques. Collagen biosynthesis involves a number of posttranslational modifications of procollagens and proteolytic conversion to collagens. The synthesis of all known types of collagen depends on prolyl 4-hydroxylase (P4H), one of the key intracellular enzymes. P4H is composed of a andβsubunits; the a subunit is rate limiting and essential for collagen maturation and secretion. The subunit folds the procollagen polypeptide chains into stable triple helical molecules. Inhibition of P4H produces unstable collagen associated with decreased collagen level. Cytokines, including various interleukins, TNF-αand transforming growth factorβ, may participate in ECM metabolism by decreasing the activity of the typeⅠisoenzyme of P4H. Adiponectin is an adipocyte-specific plasma protein that directly contributes to obesity-linked metabolic and vascular diseases. Adiponectin has been found to play an important role in preventing atherosclerosis in which it regulates inflammation, oxidative stress and ECM metabolism. Previous investigation found that plasma adiponectin level was positively correlated with the level of typeⅠcollagenⅠ.But it is not very clear about the relationship between adiponectin and prolyl 4-hydroxylase.In the present study, we aim to find the effection of adiponectin on the stability of preexisting plaques and the interaction between adiponectin and prolyl 4-hydroxylase in the plaque.Objectives 1. Elucidate the effect of adiponectin on the stability of preexisting plaques and thecorresponding mechanisms.2. Elucidate the interaction between adiponectin and P4Hal in the plaque and the corresponding mechanisms.Materials and Methods1 Animals Male apolipoprotein E-deficient mice (n=120),12 weeks old, weighing 25-30 grams, were given a high-fat Western-type diet containing 0.25% cholesterol.2 Carotid Collar Placement and Transgene ExpressionAtherosclerotic lesions were induced by perivascular collar placement on the left common carotid artery of mice. Six weeks after surgery,120 mice were divided into phosphate-buffered saline (PBS) (n=40), empty adenovirus (Ad.Empty) (n=40) and adiponectin adenovirus (Ad.Adipo) groups (n=40). The mice of the PBS group were intravenously injected with 0.1 ml PBS, and the mice of the Ad.Adipo and Ad.Empty groups were intravenously injected with an adenovirus suspension (2×108 plaque-forming units [pfu]) carrying murine adiponectin and an adenovirus suspension (2×108 pfu) carrying an empty transgene, respectively.3 Micro-ultrasonographyWe used the Vevo770 system to measure the baseline ultrasonography parameters of the carotid artery before transfection.4 Blood samples and Biological MeasuermentsBlood samples were taken before transfection and euthanization to monitor the levels of glucose, insulin, total cholesterol, high-density lipoprotein cholesterol, triglycerides, low-density lipoprotein cholesterol and plasma adiponectin.5 Tissue Harvesting and Preparation for Histological AnalysisSerial cryosections were stained with hematoxylin and eosin; picosirius red staining, Oil-red O staining, Perl's staining. Corresponding sections were stained immunohistochemically with antibodies against mouse metallophilic macrophages, a-smooth muscle (SM)-actin, P4Hal, and typesⅠandⅢcollagen. Plaque area, cap area, core area, cap thickness and intima-media thickness (IMT) were measured by use of an automated image analysis system (Image-Pro Plus 5.0, Media Cybernetics, USA).6 RT-PCRThe mRNA levels of P4Hal were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology.7 Western BlotThe protein levels of P4Hα1, typesⅠandⅢcollagen were quantified by Western Blot.Results1 Blood samples and Biological MeasuermentsThe values for body weight or plasma concentration of glucose, insulin, total cholesterol, high-density lipoprotein cholesterol, triglycerides and low-density lipoprotein cholesterol did not differ among the groups before transfection and euthanization or between before and after transfection.2 Identification of Advanced Plaque and Transfection Efficiency Assays Micro-ultrasonography was used to evaluate the stage of the plaques. Six weeks later, atherosclerotic plaques could be seen in the left common carotid artery of animals and no plaque was detected in the right common carotid artery of animals. Histological analysis for plaque morphology and composition also suggested that plaques had developed.Plasma adiponectin concentration in mice after intravenous administration of Ad.Adipo was increased about threefold that before transfer (12.7±0.6 vs.4.4±0.8μg/mL, P<0.05). The plasma adiponectin concentration in Ad.Adipo-transfected mice was about two fold higher that of endogenous adiponectin in Ad.Empty mice (12.7±0.6 vs.6.3±0.8μg/mL; P<0.05), with no significant difference in both control groups(4.3±0.5 vs.6.3±0.8μg/mL; P>0.05). Three days after transfection, analysis of GFP expression (about 60%) in atherogenic plaque of carotid arteries of 1 mouse demonstrated efficient transfection.3 Effect of Adiponectin on Plaque Stability and CompositionAd.Adipo-transfected mice showed a lower prevalence of unstable plaques than did Ad.Empty-transfected mice (15% vs.70%; P<0.05), with no significant difference in both control groups (65% vs.70%; P>0.05). Adverse events (class 5), in this case, healed rupture, were not detected in Ad.Adipo or Ad.Empty plaques but, rather, in 5% (n=1) of PBS plaques; plaque rupture (an established sign of plaque vulnerability) occurred not in Ad.Adipo plaques but in 20%(n=4) of Ad.Empty plaques and 10% (n=2) of PBS plaques; no intralesional bleeding was detected in Ad.Adipo plaques or Ad.Empty plaques but, rather, in 5%(n=1) of PBS plaques.Macrophage(0.07±0.01 vs.0.17±0.01, P<0.05) and lipid content (0.11±0.02 vs. 0.35±0.02, P<0.05)were lower on transfection with Ad.Adipo than with Ad.Empty, SMCS (0.43±0.11 vs.0.26±0.03, P<0.05)and collagen content (0.39±0.08 vs. 0.22±0.03, P<0.05) were higher on transfection with Ad.Adipo than with Ad.Empty. However, both control groups showed no difference in Macrophage, lipid, SMCs and collagen content(P>0.05).The vulnerable index for the Ad.Adipo group was 0.22±0.15, significantly lower than that for both control groups (1.08±0.5 for with Ad.Empty group, P<0.05; 0.96±0.42 for with PBS group, P<0.05), there was no difference in both control groups (0.96±0.42 vs.1.08±0.5, P>0.05).Mean cap thickness was higher, by 41%, on transfection with Ad.Adipo than with Ad.Empty (16.5±1.1 vs.8.7±2.6μm; P<0.05), and cap area was higher (7680±460 vs.4210±530μm2; P<0.05), as was cap-to-core ratio (0.16±0.02 vs. 0.09±0.01; P<0.05;). However, both control groups showed no difference in mean cap thickness (8.9±2.3 vs 8.7±2.6μm; P>0.05), cap area (4280±490 vs.4210±530μm2; P>0.05) or cap-to-core ratio (0.10±0.02 vs.0.09±0.01; P>0.05). Ad.Adipo and Ad.Empty groups did not differ in plaque area (0.17±0.02 vs.0.19±0.02 mm2, P>0.05) or MT (31±7 vs.34±9μm, P>0.05), nor did control groups differ in plaque area (0.18±0.03 vs.0.19±0.02 mm2, P>0.05) or IMT (33±6 vs.34±9μm, P>0.05). Furthermore, plasma adiponectin level was positively correlated with intimal collagen content (R=0.45, P<0.05).4 Effect of Adiponectin Overexpression on P4Hal and Collagen ExpressionAdiponectin overexpression increased the expression of P4Hal in plaque as compared with Ad.Empty transfection. Ad.Adipo transfection significantly increased P4Hal mRNA and protein levels as compared with Ad.Empty transfection (P<0.05), with no changes in both control groups (P>0.05), In addition, plasma adiponectin level was positively correlated with intimal P4Hal content (R=0.32, P<0.05).Ad.Adipo transfection also significantly increased type I and type III collagen levels as compared with Ad.Empty transfection, as shown by histological analysis and western blot analysis(P<0.05), with no changes in both control groups (P>0.05).ConclusionAdiponectin increases collagen production by inducing the expression of prolyl 4-hydroxylase, which may play a major role in the development of a thick fibrous cap in advanced atherosclerotic plaque. BackgroundAtherosclerosis, which causes ischemic cardiopathy and stroke, is the most common cause of mortality and morbidity in developed countries. In the event of cap rupture, thrombosis occurs and causes acute coronary syndrome. ECM components, especially collagen—the main constituent of the fibrouscapin atheroma—is thought to be important in the progression of atherosclerosis and plaque rupture. Finding the relationship of the ECM metabolism and stability of plaques is a hot study recently years.In recent years, theories about the role of extracellular matrix (ECM) in the pathogenesis of atherosclerosis have been developed. Functional tissue structure, such as the distensibility of an artery, is maintained by a balanced ECM metabolism of production and degradation. At present, many groups have studied the degradation of collagen, but it is not very clear about the relationship between collagen biosynthesis and stability of plaques. Collagen biosynthesis involves a number of posttranslational modifications of procollagens and proteolytic conversion to collagens. The synthesis of all known types of collagen depends on prolyl 4-hydroxylase (P4H), one of the key intracellular enzymes. Inhibition of P4H produces unstable collagen associated with decreased collagen level. Cytokines, including various interleukins, TNF-a and transforming growth factorβ, may participate in ECM metabolism by decreasing the activity of the type I isoenzyme of P4H. Among them, IL-6 is one of the most potent cytokines involved in cardiovascular pathogenesis and actively regulates ECM metabolism. In our study, we found that the expression of P4Hα1 was mostly induced at the level of 20ng/ml IL-6 and the time point of 24 hr. Adiponectin is an adipocyte-specific plasma protein that directly contributes to obesity-linked metabolic and vascular diseases. Adiponectin has been found to play an important role in preventing atherosclerosis in which it regulates inflammation, oxidative stress and ECM metabolism. All of these data suggest that adiponectin has anti-inflammatory properties and might regulate ECM metabolism.Our previous study showed that adiponectin increases collagen production by inducing the expression of prolyl 4-hydroxylase. But the regulation of signal transduction is known little.Intracellular signaling transduction pathways activated by cytokines are mitogen-activated protein kinase (MAPK) pathways, of which there are 3 distinct groups:extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Among these 3 kinase, ERK is most correlated with the biological effect of inflammation, adiponectin or ECM metabolism. Some studies demonstrated that inflammation and adiponectin are involved in ERK1/2-MAPK pathway; Other studies demonstrated that ECM metabolism are also involved in ERK1/2-MAPK pathway. Spl, as a vital downstream molecule in the MAPK pathway, which can be activated by ERK and induces the Spl DNA-binding activity. We used the web-based motif program (http://motif.genome.jp/) and found that the promoter of P4Hα1 possesses Spl site. So we think that ERK1/2-MAPK pathway can regulate the effect of adiponectin up-regulate P4Hα1 expression in IL-6-mediated HASMCs and most correlated with transcription factor Sp1.To sum up, we hypothesize that adiponectin attenuates the IL-6-inhibited P4Hα1 synthesis in HASMCs. This enhancement of adiponectin is accomplished through ERK1/2-dependent pathways; it is achieved by inhibition of Sp1 activation.Objectives1. To identify whether adiponectin up-regulate P4Hα1 expression at the level of mRNA and protein in IL-6-induced HASMCs.2. To identify whether Spl is involved in the effect of adiponectin up-regulate P4Hα1 expression in IL-6-induced HASMCs.3. To identify whether ERK1/2 is involved in the effect of adiponectin up-regulate P4Hα1 expression in IL-6-induced HASMCs.Methods1. Cell CultureHuman aortic smooth muscle cells (HASMCs) were obtained from ScienCell-and cultured in smooth-muscle-cell culture medium containing 10% fetal bovine serum. Cells were cultured up to passage 4 before the experiments were conducted. In all experiments, HASMCs were incubated in a humidified incubator at 37℃in a 95% air-5% CO2 atmosphere until cells reached 80-90% confluence and rendered quiescent by serum free starvation for at least 24h.2. Cell Pretreatment2.1 Different Dose and Time Point of of IL-6 to P4Hα1 ExpressionHASMCs were treated with 0,10,20,50, and 100 ng/mL recombinant human IL-6 for 0,6,12,24,48h before being harvested for measurement of target gene mRNA and protein levels.2.2 Different Dose and Time Point of Adiponectin to P4Hal ExpressionThe IL-6-induced HASMCs were treated with 5 MOI,10 MOI,20 MOI,50 MOI,100MOI of adiponectin adenovirus for 4h,8h,12h,24h before being harvested for measurement of target gene mRNA and protein levels.2.3 Spl Regulates the Effect of Adiponectin Up-regulate P4Hα1 Expression in IL-6-Mediated HASMCs1) control group:no any intervention;2) WP631 group:Exposing HASMCs to 0.1μM WP631 for 1h;3) Adiponectin group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h;4) WP631+Adiponectin group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h, following 1 h pretreatment with 0.1μM WP631;5) IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 0 min, 15min,30min,1h,2h,24h, repectively;6) Adiponectin+IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 1h,24h, repectively; following 8 h pretreatment with 10 MOI adiponectin adenovirus. 7) WP631+IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 1h,24h, repectively, following 1 h pretreatment with 0.1μM WP631.8) WP631+Adiponectin+IL-6 group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h, following 1 h pretreatment with 0.1μM WP631, and then following 1 h treatment with 20ng/ml IL-6.2.4 ERK1/2 Leads to Adiponectin Up-regulate P4Hal Expression in IL-6-Mediated HASMCs in a Spl-dependent Manner in HASMCs1) control group:no any intervention;2) PD98059 group:Exposing HASMCs to 20μM PD98059 for 1h;3) Adiponectin group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h;4) PD98059+Adiponectin group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h, following 1h pretreatment with 20μM PD98059;5) IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 0 min,15min,30min,lh, 2h,24h, repectively;6) Adiponectin+IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for 1h,24h, repectively; following 8 h pretreatment with 10 MOI adiponectin adenovirus;7) PD98059+IL-6 group:Exposing HASMCs to 20ng/ml IL-6 for lh,24h, repectively, following 1h pretreatment with 20μM PD98059;8) PD98059+Adiponectin+IL-6 group:Exposing HASMCs to 10 MOI adiponectin adenovirus for 8h, following 1h pretreatment with 20μM PD98059, and then following 1h treatment with 20ng/ml IL-6.3. RT-PCR AnalysisThe mRNA levels of P4Hal were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology.4. Western Blot AnalysisThe protein levels of p-ERK and P4Hal were quantified by Western Blot.5. Nuclear extracts and electrophoretic mobility shift assay(EMSA)Activation of specific protein 1 (Sp1) was determined using Nuclear extracts and electrophoretic mobility shift assay according to the manufacturer's instructions. 6. ImmunofluorescenceSubcellular localization of specific protein 1 (Sp1) was determined using immunofluorescence.Results1. Different Dose and time point of of adiponectin to P4Hα1 ExpressionHASMCs were treated with 0,10,20,50, and 100 ng/mL recombinant human IL-6 for 0,6h,12h,24h,48h before being harvested for measurement of target gene mRNA and protein levels. And find the expression of P4Hal was mostly induced at the level of 20ng/ml IL-6 and at time point of 24h.2. Different Dose and time point of adiponectin to P4Hα1 ExpressionThe IL-6-induced HASMCs were treated with 0MOI,5MOI,10MOI,20MOI,50MOI,100MOI adiponectin adenovirus for 4h,8h,12h,24h before being harvested for measurement of target gene mRNA and protein levels. And find the expression of P4Hal was mostly induced at the level of 10MOI adiponectin and at time point of 8h.3. Spl regulates effect of adiponectin up-regulate P4Hal expression in IL-6-mediated HASMCs.3.1 Effect of Adiponectin on Sp1 Activation Induced by IL-6 in HASMCsWe examined the effect of adiponectin on the binding activity of Sp1 in HASMCs induced by IL-6. Our results shows that IL-6 significantly up-regulate the binding activity of Sp1 in HASMCs compared to the control cells (P<0.05) Adiponectin alone had no effect on the binding activity of the Sp1 in HASMCs, but it inhibited the IL-6 induced binding activity of the Sp1 by &60%(P<0.05), by treatment with WP631, an inhibitor of Sp1, it inhibited the IL-6 induced binding activity of the Sp1 by 95%(P<0.05), the combination of WP631 and adiponectin abrogate the Spl binding activity induced by IL-6 by &90%(P<0.05), while adiponectin or WP631 alone didn,t abrogate the Sp1 binding activity.3.2 Effect of Adiponectin on Subcellular Localization and Expression of Sp1 Induced by IL-6 in HASMCs The great majority of unstimulated cells under normal growth conditions had diffuse staining in the cytoplasm, as well as weak staining in the nucleus.1h of treatment with IL-6, staining for Spl in the nucleus raise 8 times as compared with the control cells (P<0.05). Eight hours after the addition of adiponectin to the cells, the level of nuclear staining was abrogated by 70%(P<0.05). After preincubation of WP631 for 1h, the level of nuclear staining was weak (P<0.05). The combination of WP631 and adiponectin also had weak staining in the nuclear (P<0.05). However, staining for Spl in the nucleus is not influenced by adiponectin or WP631 alone (P> 0.05).3.3 Effect of Adiponectin on the expression of P4Hα1 Induced by IL-6 in HASMCsOur results show that IL-6 significantly down-regulate P4Hal mRNA by 65% (P<0.05) and P4Hα1 protein by 70%(P<0.05) compared to the control cells. Eight hours after the addition of adiponectin to the cells, P4Hal mRNA raise 5.8 times (P<0.05) and P4Hα1 protein raise 6 times (P<0.05). After preincubation of WP631 for 1h or the combination of WP631 and adiponectin, P4Hα1 mRNA and P4Hα1 protein return to the level of the control cells. However, the expression of P4Hal are not influenced by adiponectin or WP631 alone (P>0.05)4. ERK1/2 leads to adiponectin up-regulate P4Hα1 expression in IL-6-mediated HASMCs in a Spl-dependent manner in HASMCs.4.1 Effect of adiponectin on phosphorylation of ERK induced by IL-6 in HASMCsOur results show that IL-6 induce phosphorylation of ERK compared to the control cells (P<0.05), Eight hours after the addition of adiponectin to the cells, the activity of phosphorylation of ERK induced by IL-6 was abrogated by 70%(P<0.05). After preincubation of PD98059 for 1h, the activity of phosphorylation of ERK induced by IL-6 was abrogated by 95%(P<0.05). After preincubation of the combination of PD98059 and adiponectin, the activity of phosphorylation of ERK induced by IL-6 was abrogated by 92%(P<0.05). However, the activity of phosphorylation of ERK is not influenced by adiponectin or PD98059 alone (P> 0.05).4.2 Effect of ERK on Spl Activation Induced by IL-6 in HASMCsOur results show that IL-6 significantly up-regulate the binding activity of Sp1 in HASMCs compared to the control cells (P<0.05). Eight hours after the addition of adiponectin to the cells, the binding activity of Sp1 induced by IL-6 was abrogated by 80%(P<0.05). After preincubation of PD98059 for lh, the binding activity of Spl induced by IL-6 was abrogated by95%(P<0.05).The combination of PD98059 and adiponectin abrogate the binding activity of Spl induced by IL-6 by 93%(P<0.05). However, the binding activity of Spl is not influenced by adiponectin or PD98059 alone (P>0.05)4.3 Effect of ERK on Subcellular Localization and Expression of Spl Induced by IL-6 in HASMCsThe great majority of unstimulated cells under normal growth conditions had diffuse staining in the cytoplasm, as well as weak staining in the nucleus, 1h of treatment with IL-6, staining for Spl in the nucleus raise 7 times as compared with the control cells (P<0.05). Eight hours after the addition of adiponectin to the cells, the level of nuclear staining was abrogated by 70%(P<0.05). After preincubation of PD98059 for 1h, the level of nuclear staining was weak (P<0.05).The combination of PD98059 and adiponectin also had weak staining in the nuclear (P<0.05). However, staining for Sp1 in the nucleus is not influenced by adiponectin or PD98059 alone (P >0.05).4.4 Effect of ERK on the expression of P4Hα1 Induced by IL-6 in HASMCs Our results show that IL-6 significantly down-regulate P4Hα1 mRNA by 65% (P<0.05) and P4Hal protein by 70%(P<0.05) compared to the control cells. Eight hours after the addition of adiponectin to the cells, P4Hα1 mRNA raise 6.3 times (P<0.05) and P4Hal protein raise 7 times (P<0.05). After preincubation of PD98059 for lh or the combination of PD98059 and adiponectin, P4Hα1 mRNA and P4Hal protein return to the level of the control cells. However, the expression of P4Hal is not influenced by adiponectin or PD98059 alone (P>0.05).Conclusions1. Adiponectin can up-regulate P4Hα1 expression at the level of mRNA and protein in IL-6-induced HASMCs.2. Sp1 was involved in the effect of adiponectin up-regulate P4Hal expression in IL-6-induced HASMCs.3. ERK1/2 was involved in the effect of adiponectin up-regulate P4Hα1 expression in IL-6-induced HASMCs.