| Methods: CMECs were isolated from myocardial tissue of adult male SD rats andcharacterized by the immunocytochemistry staining with Factor VIII and the capacity oftube formation on Matrigel. The mRNAs and protein expressions of ghrelin and itsreceptor (growth hormone secretagogue receptor, GHSR) of CMECs were determinedby RT-PCR, immunoflurescence, ELISA and Western-blot. Proliferation, migration andtube formation of CMECs were tested in the presence of10-9M～10-6M ghrelin.Results: Purity of CMECs characterized by immunocytochemistry staining withFactor VIII was about95%, and the cells showed a high ability to form tube formationon Matrigel. Ghrelin and GHSR were constitutively expressed in CMECs.10-8M～10-7M ghrelin significantly stimulated CMECs proliferation, migration, and tubeformation on Matrigel. However, ghrelin at the higher concentration of10-6Msignifiantly suppressed proliferation, migration and tube formation on Matrigel inCMECs.Conclusions:Ghrelin and its receptor are expressed in CMECs and ghrelin at theconcentration of10-8M～10-7M could significantly stimulate proliferation, migration and tube formation on Matrigel in CMECs. Objective:The aim of our study was to investigate the mechanisms of ghrelin onproliferation, migration, and tube formation in rat cardiac microvascular endothelialcells (CMECs).Methods: CMECs were incubated with10-9M～10-6M ghrelin for15min for assayof ERK, Akt and eNOS phosphorylation. CMECs were incubated with10-7M ghrelinfor0～60min for assay of ERK, Akt and eNOS phosphorylation. CMECs werepretreated with PD98059, LY294002, L-NAME or [D-Lys3]-GHRP-6for30min andthen further incubated with ghrelin for15min for assay of ERK, Akt and eNOSphosphorylation, levels of phosphorylated and total ERK and Akt were determined byWestern blot analysis, respectively. Proliferation, migration, tube formation on Matrigeland NO production were tested in the presence of ghrelin with or without pretreatmentwith each inhibitor.Results: Western blot analyses revealed that treatment of CMECs with ghrelinincreased phosphorylation of ERK, Akt, and eNOS in a time-and dose-dependentmanner. Ghrelin-induced angiogenesis was accompanied by phosphorylation of ERK,Akt, and eNOS, as well as an increase in NO production. These biochemical events andangiogenesis were completely inhibited by GHSR1a blocker [D-Lys3]-GHRP-6.Ghrelin-induced ERK activation and angiogenic events were significantly inhibited bythe MEK inhibitor PD98059, without affecting Akt and eNOS phosphorylation. ThePI3K inhibitor LY294002suppressed ghrelin-mediated angiogenesis, Akt and eNOS activation, and NO production without affecting ERK phosphorylation. The eNOSinhibitor L-NAME suppressed ghrelin-mediated angiogenesis, eNOS activation, andNO production, without affecting ERK and Akt phosphorylation.Conclusions:Our results demonstrated that ghrelin stimulated angiogenesis viaGHSR1a-dependent MEK/ERK and PI3K/AKT/eNOS/NO signal pathways in CMECs,indicating that two pathways are required for full angiogenic activity of ghrelin. Thisstudy suggests that ghrelin may play an important role in myocardial angiogenesis. Objective:To investigate the state of serum ghrelin concentration and ghrelin gene(C408A,G346A) polymorphisms in patients with coronary heart disease (CHD).Methods: The artery blood of316patients were collected by using coronaryangiography. The levels of serum ghrelin were assayed by RIA, and all subjects weregenotyped for these two single nucleotide polymorphisms of ghrelin by polymerasechain reaction-restriction fragment length polymorphism (PCR-RFLP) assay.Results:The levels of serum ghrelin in CHD group were lower than those incontrol group significantly(P＜0.05). Correlation analysis between serum ghrelinconcentration and other variances revealed that it was negatively associated with BMIand WHR, stepwise multiple regression analysis demonstrated that BMI and WHR wereindependently correlated with the serum ghrelin level in CHD group. The allelefrequencies and genotype distribution of the C408A was not different between controland CHD groups. serum ghrelin concentrations were not associated with the polymorphism(P＞0.05). In two groups,HDL-C levels in subjects carrying CCgenotype was higher than that of subjects without CC genotype(P＜0.001). There wasno G346A variants existed in our study subject.Conclusion:Serum ghrelin concentration decreased in patients with CHD. Thelevels of serum ghrelin were not associated with C408A polymorphism; C408A variantsis related with HDL-C. G346A polymorphism was not found in control and CHDgroups. |
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