title: Effects and mechanisms of ghrelin on cardiac microvascular endothelial cells in rats.
abstract: Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague-Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil-ac-LDL (1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine-labelled acetylated low-density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non-treated group; three ghrelin dosage groups (1×10-9, 1×10-8, 1×10-7 mol/l) and one ghrelin+PI3K inhibitor group (1×10-7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10-8 mol/l (0.271±0.041 compared with 0.199±0.021, P = 0.03) and 10-7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin-mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P = 0.15). At a concentration between 10-8 and 10-7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P = 0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P = 0.32). Ghrelin treatment significantly enhanced NO production in a dose-dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P = 0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin-stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P = 0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway.
authors.affiliation: *Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi 710032, Peoples Republic of China.
authors.forename: Yabin
authors.initials: Y
authors.lastname: Wang
authors.forename: Kazim
authors.initials: K
authors.lastname: Narsinh
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Dongdong
authors.initials: D
authors.lastname: Sun
authors.forename: Dongjuan
authors.initials: D
authors.lastname: Wang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhang
authors.forename: Zhongchan
authors.initials: Z
authors.lastname: Sun
authors.forename: Rongqing
authors.initials: R
authors.lastname: Zhang
authors.forename: Haichang
authors.initials: H
authors.lastname: Wang
authors.forename: Feng
authors.initials: F
authors.lastname: Cao
mesh_terms: D000818:Animals; D002465:Cell Movement; D049109:Cell Proliferation; D002867:Chromones / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology*; D054439:Ghrelin / Q000494:pharmacology*; D006352:Heart Ventricles / Q000166:cytology; D008297:Male; D055806:Microvessels / Q000166:cytology; D009025:Morpholines / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction
chemical_list: D002867:Chromones; D054439:Ghrelin; D009025:Morpholines; D009569:Nitric Oxide; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D051057:Proto-Oncogene Proteins c-akt; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one