| 1. BackgroudThe rapture of atherosclerotic plaque is responsible for the clinical acute events of vascular, such as stroke and myocardial infarction, which is the most common cause of mortality and morbidity in developed countries. To stabilize atherosclerotic plaques is an essential strategy to reduce the clinical acute events of vascular.Collagen is one kind important composition in atherosclerotic plaque. Type I and type III collagen are the predominant collagen in atherosclerotic plaques and arterial wall. The vulnerable plaque is characterized by less collagen and more lipids. Otherwise, the stable one with more collagen and less lipids.Prolyl-4-hydroxylase （P4H） is a key intracellular enzyme involved in the synthesis of collagens. It catalyzes the proline residues repeating X-Pro-Gly triplets located in the procollagens to format hydroxyproline. It is essential for collagen posttranslational modification, and this modification facilitate folding the polypeptide procollagen chains into stable triple-helical molecules.P4H is composed of a and P subunits; The a subunit is a rate-limiting enzyme.The active enzyme is a tetramer composed of two pairs of non-identical subunits （a2p2）. P4Hal is the most important form of all kinds of P4Hs. It is responsible for the posttranslational modifications of procollagens. Studies demonstrated that less P4Hal in plaques is closely related to destability of plaques. So to increase the expression of P4Hal in plaques is a good strategy to avoid the plaque rupture. In theory, overexpression of P4Hal can make over production of collagen under the condition of sufficient procollagen. But whether or not overexpression of P4Ha1influences the collagens and other components in atherosclerotic plaques, and whether or not overexpression of P4Hal influences the vulnerable index are unknown.Collagen metabolism is composed of collagen synthesis and collagen degradation modulated by extracellular matrix metallopeptidase （MMPs）, along with their endogenous tissue inhibitors （TIMPs） constituting an important system for regulating extracellular matrix （ECM）, especially, collagen turnover. But whether or not overexpression of P4Hal influences the MMPs and TIMPs is also unknown.Guided by above theories, We put forward such scientific hypothesis as:（1） The expession of P4Hal is developmental decrease in the pathological course of atherosclerotic plaques in ApoE-/-mice.（2） Overexpression of P4Ha1can increase the product of Type I and type III collagen both in evolving plaques and in established ones.（3） Overexpression of P4Hal can increase the stability of evolving plaques and established ones. But there may be some different influence in evolving plaques from established ones.Objectives（1） To investigate the developmental expession of P4Hal in the pathological course of atherosclerotic plaques of ApoE-/-mice.（2） To investigate effects of overexpression of P4H a1on the collagen metabolism both in evolving plaques and in established ones of ApoE-/-mice.（3） To elucidate the effect of overexpression of P4Hal on the stability of evolving plaques and established ones of ApoE-/-mice.（4） Clarify the different influence of overexpression of P4Hal on evolving plaques from established ones and explore the mechanism under the appearance.2. Material and Methods（1） Plasmids and Lentivirus-P4Ha1Construction The Lentivirus-P4Haland empty Lentivirus （control） were constructed by Inventrogen, Shang hai.（2） Animal ModelEthics StatementThe animal experimental protocol complied with the Animal Management Rules of the Chinese Ministry of Health （document No55,2001） and was approved by Animal Care Committee of Shandong University.Spontaneous plaque of aortic rootMale apolipoprotein E-deficient mice （n=80,8weeks old, weighing25-30g） were obtained from Peking University （Beijing, China）, and were divided into randomly4groups:10weeks old group;12weeks old group;14weeks old group and16weeks old group （n=20）. All of them had high-fat diet. At the end of10,12,14,16week, they were killed kindly. Histopathology-Immunohistochemistry and Western, Blot, were carried on for detecting the expression of P4Hal in Spontaneous plaque of aortic root.Carotid Collar Placement and Transgenic therapyMale apolipoprotein E-deficient mice （n=120,8weeks old, weighing25-30g） m were obtained from Peking University （Beijing, China）. Atherosclerotic lesions were induced by placing a perivascular collar on the right common carotid artery of mice. The Lenti-EGFP groups were used as controls.120mice were divided randomly into two groups:established plaques and early ones.①Evolving Plaques:60mice （male,8weeks old） were induced carotid atherosclerotic lesions by placement of a restraint perivascular silica collar as above. After surgery2weeks,60were divided into randomly3groups:saline group; Lenti-EGFP group and Lenti-P4H a1group. Three group mice were given intravenously saline, Lentivirus-P4Ha1and empty Lentivirus transgenic therapy.2weeks later, all mice were given euthanasia for research.②Established plaques:60mice （male,8weeks old） were induced carotid atherosclerotic lesions by placement of a restraint perivascular silica collar, and fed with high-fat diet combined stimulation of mental to create a vulnerable plaque model. After surgery10weeks,60were divided into randomly3groups:saline group; Lenti-EGFP group and Lenti-P4Hal group. Three group mice were also given intravenously saline, Lentivirus-P4Hal and empty Lentivirus transgenic therapy.2weeks later, all mice were given euthanasia for research.（3） Micro-ultrasonographyVevo770system was used to measure the baseline ultrasonography parameters of plaques（4） Blood samples and Biological MeasurementsBlood samples were taken to detect:total cholesterol? high-density lipoprotein cholesterol,triglycerides,low-density lipoprotein cholesterol, and hydroxyproline.Weight was also detected before surgery and euthanasia.（5） Histopathology and ImmunohistochemistryCryosections were stained with hematoxylin and eosin （H&E）, oil O and picosirius red as previous.For immunohistochemistry, Sections were incubated with different primary antibodies and second antibodies for immunohistochemistry.（6） Western BlotProteins were extracted from carotid artery specimens, separated on14%SDS-PAGE and transferred to nitrocellulose membranes. After incubation with primary antibodies and second antibodies and exposure, protein expression was determined.（7） RT-PCRCarotid artery specimens were pooled for RNA isolation with TriZol to detect the mRNA of P4Ha1, collagen I （procollagen I） and collagen III （procollagen III）.（8） Gelatin zymography assayMMP-9and MMP-2activity was detected using the gelatin zymography assay. The tissue proteins were extracted using lysis buffer. The protein concentration was determined. The sample was electrophoresed on SDS-polyacrylamide gels copolymerized with1%gelatin. After electrophoresis, the gels were washed twice in2.5%Triton X-100and incubated in1%Triton X-100,5mM CaC12and5jiM ZnC12 （pH7.5） at37℃for36h. The gels were stained with0.1%Coomassie blue R250, and destained in10%isopropanol and10%acetic acid in H2O. MMP-9/MMP-2was detected as transparent bands on the blue background of a Coomassie blue-stained gel.4. ResultsOne mouse died of liver fibrosis, and others finished this study.4.1Blood samples and Biological MeasurementsThere was no significant difference in serum levels of TC, TG, LDL-C and HDL-C among the3groups of ApoE-/-mice both in established plaque group and in evolving plaque group. But the hydroxyproline serum levels was significant higher in lenti-P4Hα1group than that of mock group and lenti-EGFP group both in established plaque group and in evolving plaque group （P<0.05）.4.2Identification of PlaqueMicro-ultrasonography was used to evaluate the stage of the plaques. Small atherosclerotic plaques could be seen in the right common carotid artery in4weeks after collar surgery. And there were obvious plaques in10weeks and12weeks after collar surgery, otherwise, no plaque was detected in the left common carotid artery of animals.4.3Transfection Efficiency AssaysGFP expression（about70%） was found in atherogenic plaque of carotid arteries in Lenti-P4Hα1and Lenti-EGFP groups. The mRNA of P4Hα1increased about five fold than Lenti-EGFP group and PBS group. All demonstrated efficient transfection.4.4Effect of Lenti-P4Hα1transgenic therapy on P4Hal protein expression①Evolving Plaque group:Western blot and Immunohistochemistry showed that the expression of P4Hα1protein in lenti-P4Hα1group was significant higher than saline and Lenti-EGFP groups （P<0.05）.②Established plaque group:consistent with the result of established plaque group （P<0.05）.4.5Effect of overexpression of P4Hal on procollagen Ⅰ, collagen Ⅰ and procollagen Ⅲ, collagen Ⅲ in plaques①Evolving Plaque group:Western blot and Immunohistochemistry showed that there was no significant difference in the expression of procollagen Ⅰ and procollagen Ⅲ in3groups, but the collagen Ⅰ and collagen Ⅲ protien expression in Lenti-P4Hal was significant higher than saline and Lenti-EGFP groups （P<0.05）.②Established plaque group:consistent with the result of evolving plaque group （P<0.05）.4.6Effect of overexpression of P4Hal on carotid plaque composition and stability③Evolving Plaque group:Histological and immunohistochemical staining of carotid plaque in3groups were significantly different. Collagen content was increased significantly in the Lenti-P4H a1group; And VSMC content in the atherosclerotic lesion area, detected by a-actin staining, was also significantly increased （P<0.05）. But the content of macrophages was significantly decreased in Lenti-P4Hal group compared with saline group and lenti-EGFP control groups （P<0.05）. And there was no significant difference in lipid deposition among3groups.②) Established plaque group:consistent with the result of evolving plaque group （P<0.05）.4.7Effect of overexpression of P4Hal on the morphology changes of the carotid plaque①Evolving plaque group:The fibrous cap area, fibrous cap thickness and collagen content in fibrous cap were higher significantly in lenti-P4Hal group than control groups （P<0.05）. And the vulnerability index was lower significantly than control groups. The plaque area was significant lager and the total plaque burden was significant higher in lenti-P4Ha1group, compared with control groups （P<0.05）.②Established plaque group:It was inconsistent with the result of evolving plaque group. The plaque area and total plaque burden in lenti-P4Ha1group had no significant difference from control groups （P<0.05）, although in lenti-P4Hal group, the fibrous cap area, fibrous cap thickness and collagen content in fibrous cap were higher significantly than control groups （P<0.05）. And the vulnerability index was also lower significantly than control groups. This was consistent with the result of evolving plaque group group,4.8Effect of overexpression of P4Hal on inflammatory markers and on MMPs and TIMPs in established plaque groupThe protein expression levels of IL-6, TNF-a, MCP-l,MMP-2, MMP-9in the carotid plaque of in lenti-P4Hal group were lower significantly compared with control groups （P<0.05）, based on quantitative analysis of immunohistochemical staining, immunohistochemical staining. And the relative activity of MMP9, MMP2also decreased significantly in lenti-P4Hal group.4.9Effect of overexpression of P4Hal on PTEN expression〦volving Plaque group:Western blot and Immunohistochemistry showed that PTEN expression in lenti-P4Ha1group decreased significantly, compared with control groups （P<0.05）.②Established plaque group:It was different from established plaque group: there was no significant difference on PTEN expression among three groups （P>0.05）.5. ConclusionP4Hal overexpression of can increase collagen Ⅰ and Ⅲ significantly in both evolving plaques and in established ones. It can increases the stability of established plaques but accelerates intimal hyperplasia and the total lesion area （%） in evolving plaques of Apolipoprotein E-knockout mice. 1. BackgroundmiRNAs are short ribonucleic acid （RNA） molecules, approximately18-24nucleotide RNAs that modulate gene expression by base pairing with the3’-untranslated regions （3’-UTRs） of miRNAs to repress protein expression by inhibiting translation or promoting degradation of miRNAs. Untill now, several hundreds of miRNAs have been identified. They involved in cell proliferation, apoptosis, differentiation, development and metabolism. Studies on miRNAs show that about60%of mammalian genes are targeted by special miRNAs. One miRNA can target one or more genes.P4H a1is the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Studies show P4Hal are losely related to the vulnerable atherosclerosis plaque. Our previous studies demonstrate that inflammatory cytokines, interleukin6, can downregulate P4H a1via a mitogen-activated protein kinase and c-Jun pathway to destabilize atherosclerotic plaques. And TNFa can also downregulate P4Hal via affecting the histone （H）4lysine12acetylation. Otherwise, Adiponectin can upregulate prolyl-4-hydroxylase al expression human aortic smooth muscle cells by regulating ERK1/2and Spl. But how miRNAs regulate P4Hal in atherosclerotic plaque is unknown till now. miR-124is a brain-enriched microRNA that has been shown to be down-regulated in glioma and medulloblastoma and plays a crucial role in brain tumor progression. Keiwa Kin observed that miR-124was up-regulated in the tissue of atherosclerotic abdominal aortic aneurysm. It suggests that miR-124was related to the collagen metabolism of aortic remolding. But whether or not miR-124involves the progression of atherosclerotic plaque is unclear.P4Hal is miR-124’s target gene by the famous softwears, such as:MIRBASE, STARBASE, TARBASE, TARGETSCAN. But whether or not miR-124can bind to the3’-untranslated region （3’-UTR） of P4Hal gene mRNA to regulate the translation of P4Hal is unknown in fact. Guided by above theories, We put forward such scientific hypothesis as:（1） The expression of miR-124is different in stressed plaques from unstressed plaques. And miR-124is up-regulated probably in stressed plaques.（2） miR-124can bind to the wild3’-untranslated region （3’-UTR） of P4Hal gene mRNA but can not bind to the mutation one.（3） P4Hal is miR-124’s target gene. In human aortic smooth muscle cells, miR-124mimics can inhibit P4Hal expression and down-regulate "collagen I, collagen Ⅲ. Otherwise, miR-124inhibitors can up regulate P4Hal and increase the production of collagen Ⅰ, collagen Ⅲ.Aims:（1） To identify miR-124alteration in atherosclerotic plaques and the different expession of miR-124between stressed plaques and unstressed ones.（2）To clarify the relationship between miR-124and P4Hal.（3）To clarify the mechanism on how miR-124regulate P4Hal and to identify miR-124’s special binding site on3’-UTR of P4Hal.（4） To clarify the relationship between miR-124and collagen Ⅰ and collagen3.2. Materials and methods（1） AnimalsMale apolipoprotein E-deficient mice （n=60,8weeks old, weighing25-30g） were obtained from Peking University （Beijing, China）. 60mice were divided randomly into2groups:unstressed group （n=30） and stressed group （n=30）. Carotid collar placement and mental stress protocol is consistent with paper1.（2） Carotid artery isolationAfter12weeks, the carotid artery of E-deficient mice, restricted by collar placement, were isolated carefully to store at-80℃refrigerator for RT-PCR and Western blot detected.（3） Computational targeted gene predictionsmiRanda, TargetScan, RNAhybrid and PicTar etal..（4） Plasmids, mutagenesis, transfection and luciferase reporter assaysThe3’-UTR of the mouseP4Hal gene was amplified. All constructs were verified by sequencing. The wild and mutated3’-UTR of theP4Hal gene were obtained from Jinan Biolabs. The luciferase reporter vector pGL3-basic vector [luc2] was also from Jinan Biolabs.（5） miRNA mimics, miRNA inhibitorsmiRNA mimics or miRNA inhibitors were transfected using Lipofectamine2000（Invitrogen）. Luciferase activity was detecked by using Dual-lo Luciferase Reporter Assay Kit （Promega） on a FLUOstar Omega luminometer （BMG Labtech, Cary, NC）, after48hours of transfections.（6） HASMCs cultureHASMCs were obtained from ScienCell. They were cultured with5%CO2at37℃in smooth muscle cell medium （ScienCell, USA）. HASMCs （passages4to8） were used.（7） RNA isolation and qRT-PCRTotal RNA was isolated from carotid artery or HASMCs using TRIzol, and cDNA was synthesized according to manufacturer’s instructions （Invitrogen, San Diego, CA）.（8） Western blot analysisWestern blots were performe d using anti-P4HAl, anti-COLlA1, anti-Collagen3, and anti-β-actin antibodies （Santa Cruz Biotechnology） following our published method[10].（9） ImmunofluorescenceHASMCs were grown on chamber slides and rmiR-124transfection was carried out. Forty-eight hours following the transfection, HASMCs were fixed in4%paraformaldehyde for15min at room temperature and was rised with PBS three times.5%rabbit serum was used to block the cells, following by incubation with anti-P4H ol antibody overnight at4℃.3. Results3.1miR-124is upregulated in stressed plaques and in TNFa treated HVSMCs（1） The mRNA was isolated from unstressed carotid plaques and stressed carotid plaques in vivol. The expression of miR-124was determined by real-time; PCR. The relative levels of miR-124expression in stressed plaques were significantly increased （P<0.05）.（2） The mRNA was isolated from TNFa treated HVSMCs and from control HVSMCs in vitrol. The expression of miR-124was also determined by real-time PCR. The relative levels of miR-124expression in TNFa treated HVSMCs were significantly higher （P<0.05）, compared with control HVSMCs.3.2miR-124is predicted to target P4Hal genemiRanda, TargetScan, RNAhybrid and PicTar et al., the famous computational approaches for the identification of mammalian microRNA targets, identified that Homo sapiens prolyl4-hydroxylase, alpha polypeptide I （P4Hal）, transcript variant1, mRNA. NM000917is miR-124’s targe gene and miR-124is P4Hal’s target mirRNA.3.3miR-124targetsP4HalThe protein expression ofP4Hal was significantly reduced after miR-124mimics transfection （P<0.05）. Otherwise, miR-124inhibitor can make the reduction of P4Hal induced by TNFa significantly abrogated （P<0.05）.3.4miR-124downregulates the expression of P4Hal via targeting the 3’-UTR of P4Hal mRNATransient co-transfection of miR-124mimics with luciferase reporter plasmids resulted in a significant repression of luciferase reporter gene expression in HVSMCs, whereas co-transfection of HVSMCs with negative control miRNA or mutations did not have any effect on the expression of luciferase.3.5Inhibition of miR-124increases collagen maturation.To addresse whether miR-124overexpression had an inhibitory effect on collagen protein expression. After48hours transfection of miR-124mimics in HVSMCs, collagen Ⅰ and Ⅲ were all significantly decreased （P<0.05） as expected. Whereas, miR-124inhibitor can make the reduction of collagen Ⅰ and Ⅲ induced by TNFa significantly abrogated （P<0.05）.4. ConclusionsmiR-124involved in and play important roles in atherosclerotic plaques. It is upregulated in stressed plaques and TNFa induced HVSMCs. miR-124can regulate collagen maturation via targeting its target gene, the P4Ha1mRNA. |
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