Protective role of a novel erythrocyte-derived depressing factor on blood vessels of renovascular hypertensive rats.
1. We have isolated a novel human erythrocyte-derived depressing factor (EDDF) that has a significant antihypertensive effect in various rat models of hypertension. The aim of the present study was to examine the mechanisms of action of EDDF on vascular function in two-kidney, one-clip (2K1C) renovascular hypertensive rats. 2. The EDDF was prepared from human erythrocytes. Experiments were performed in 18 male Wistar rats. The vascular ring perfusion assay and a two-photon laser scanning fluorescence microscope (TMP) were used to evaluate the vascular contractile response. The effects of EDDF on phenylephrine (PE)- and noradrenaline (NA)-induced vascular contraction were evaluated in 2K1C hypertensive rats. The proliferation and DNA synthesis in vascular smooth muscle cells (VSMC) were determined using the [3H]-TdR (thymidine) incorporation and 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. Flow cytometry, reverse transcription-polymerase chain reaction and western blots were used to measure cell cycle and apoptotic profiles, platelet-derived growth factor (PDGF)-A expression and the activity of extracellular signal-regulated kinase (ERK)-1/2, as well as the expression of cyclin D1 and cyclin-dependent kinase (CDK) 4. 3. At 10(-5) g/mL, EDDF significantly decreased the PE- and NA-induced hypertensive vascular contraction. In addition, EDDF inhibited DNA synthesis in primary VSMC from 2K1C rats. The mRNA expression of PDGF-A in VSMC was twofold higher in 2K1C rats compared with control rats, whereas EDDF significantly inhibited the increment in PDGF-A mRNA expression. In addition, EDDF inhibited the phosphorylation of ERK1/2 and decreased the expression of cyclin D1 and CDK4; p21 (Cip1) levels were increased after treatment with EDDF. 4. In conclusion, EDDF inhibits VSMC proliferation in 2K1C rats through G0/G1 cell cycle arrest. The effects may be mediated, in part, by enhanced expression of p21 (Cip1) and the inhibition of ERK1/2 phosphorylation and the expression of cyclin D1/CDK4 and PDGF-A.
Pang
Huan
H
Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing, China.
Wen
Yun-yi
YY
Ma
Ning
N
Wang
Yu-tang
YT
Shi
Lei
L
D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects* / Q000503:physiopathology; D001685:Biological Factors / Q000737:chemistry / Q000494:pharmacology*; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000737:chemistry*; D006801:Humans; D006978:Hypertension, Renovascular / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control*; D008297:Male; D008856:Microscopy, Fluorescence; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D009638:Norepinephrine / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014661:Vasoconstriction / Q000187:drug effects
D001685:Biological Factors; D050759:Cyclin-Dependent Kinase Inhibitor p21; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; C075278:platelet-derived growth factor A; D019938:Cyclin D1; D010656:Phenylephrine; D051358:Cyclin-Dependent Kinase 4; D048052:Mitogen-Activated Protein Kinase 3; D009638:Norepinephrine