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Physiology American Journal of Physiology - Heart and Circulatory about Additional material and information www.the-aps/publications/ajpheart This information is current as of January 31, 2013.
cardiovascular function at all levels of organization ranging from the intact animal to the cellular, subcellular, and physiology of the heart, blood vesls, and lymphatics, including experimental and theoretical studies of
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Vasoactive factors and growth factors alter vascular smooth muscle cell EC-SOD expression PONTUS STRÅLIN AND STEFAN L.MARKLUND
Department of Medical Biosciences and Department of Clinical Chemistry,
UmeåUniversity Hospital,SE-90185Umeå,Sweden
Received5November1999;accepted infinal form24May2001
Strålin,Pontus,and Stefan L.Marklund.Vasoactive
factors and growth factors alter vascular smooth muscle cell
EC-SOD expression.Am J Physiol Heart Circ Physiol281:
H1621–H1629,2001.—Oxygen free radicals have been sug-
gested to play important roles in atherogenesis and other
pathological process in the blood vesl wall.The vascular
wall contains large amounts of extracellular superoxide dis-
muta(EC-SOD),which is produced and creted to the
extracellular space by smooth muscle cells.In this study,we
investigated the influence of factors regulating tension and
proliferation of vascular smooth muscle cells and of some
interstitial matrix components on EC-SOD expression.The
expression and cretion of EC-SOD were upregulated by
histamine,vasopressin,oxytocin,endothelin-1,angiotensin
个体户纳税标准>母乳和奶粉的区别II,rotonin,heparin,and heparan sulfate and were down-
regulated by platelet-derived growth factors-AA and-BB,
acidic and basicfibroblast growth factors,and epidermal
growth factor.The respons were slow and developed over
veral days.Thefindings suggest that various physiological
and pathological conditions might markedly influence EC-
SOD expression,significantly altering the susceptibility of
the vascular wall to effects of the superoxide radical.
oxygen radicals;atherosclerosis;vascular smooth muscle
cells;glycosaminoglycans;heparin;extracellular superoxide
dismuta
with either 1%BSA or 15%FCS containing the indicated concentrations of factors or medium with only 1%BSA or 15%FCS (controls).Every 24h,media were collected and replaced with fresh media containing active factors.At the end of the experiments,after 4days,media were collected and the wells were washed three times with 0.15mol/l NaCl.To collect and homogenize the cells,0.5ml of ice-cold 50mmol/l sodium phosphate (pH 7.4)containing 0.3mol/l KBr,10mmol/l diethylene triamine pentaacetic acid,0.5mmol/l phenylmethylsulfonyl fluoride,and 107IU/l aprotinin (the latter three additions to inhibit proteas)was added to the wells.After sonication in the wells,with the plate bathing in ice water,the homogenates were centrifuged (20,000g for 10min),and the supernatants were collected for analysis.All samples were kept at Ϫ80°C until assay.A heparin wash step was introduced in experiments related to heparin and other glycosaminoglycans.In the experiments,the SMCs were incubated for 10min at 37°C with culture media containing 105IU/l heparin followed by three washes with 0.15mol/l NaCl and homogenization as described above.
Analysis of EC-SOD.EC-SOD protein in culture media and cell homogenates was determined with an ELISA as previously described (15).Turnover of EC-SOD in cell cultures.To asss the rate of uptake of EC-SOD by cultured cells,conditioned media from SMCs containing EC-SOD were incubated with human cell lines not producing EC-SOD.Thus Waymouth medium sup-plemented with 1%BSA was incubated with confluent SMCs for 4days,after which it was diluted with unconditioned medium to ϳ3g/l EC-SOD,a typical level in the SMC experiments (c.f.Fig.1).Diluted medium (0.5ml)was then added to triplicate wells with different human cell lines in 12-well culture plates.The cell lines were MG-251,a glioma cell line;Hep-G2,a hepatoma cell line;and PL-3and DU-145,prostate cancer cell lines.The concentrations of EC-SOD in the media were determined at intervals over 24h.
Protein and DNA analysis.For protein analysis,Coomas-sie brilliant blue G-250was employed (1),standardized with human rum albumin.The DNA concentration was deter-mined with fluorimetry as a complex with bisbenzimidazol (Hoechst 33258)(20)using calf thymus DNA as a standard.Incorporation of [35S]methionine into protein.SMCs were cultured in 12-well culture plates (3.8cm 2)for 4days as described above.At the end of the experiment,the cells were incubated with [35S]methionine for 1h followed by homoge-nization,protein precipitation with 10%trichloroacetic
acid,
Fig.1.Effects of some factors on extracellular superoxide dismuta (EC-SOD)expression in the Au-1smooth muscle cell line.The cells were cultured in 3.8-cm 2wells,and culture media (0.5ml)containing factors at indicated concentrations were exchanged daily for 4days.At the end of the experiment,the cells were homogenized,and analys were made on culture media (left )and cell homogenates (right ).EC-SOD content was determined by ELISA as described in MATERIALS AND METHODS .The data prented are the means of results from 2wells.A and D :effects of platelet-derived growth factor (PDGF)-BB (A )and heparin (D )on cells with media supplemented with 15%fetal calf rum.B and C :effects of vasopressin (B )and A-23187(C )on cells with media supplemented with 1%BSA.
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and analysis of [35S]methionine incorporation into protein as previously described (25).Aliquots of the cell homogenates were also analyzed for EC-SOD,protein,and DNA as de-scribed above.
RNA extraction and Northern blot analysis.Total cellular RNA was isolated from SMCs using the TRIzol reagent (GIBCO-BRL Life Sciences;Gaithersburg,MD).RNA (10g/lane)was electrophored in formaldehyde-containing 1.2%agaro gels,transferred to nylon filters (Hybond N,Amersham),and immobilized by ultraviolet linkage.The filters were then prehybridized for 15min and hybridized for 1h with Quickhyb solution (Stratagene;La Jolla,CA)at 65°C in a hybridization oven.For EC-SOD detection,a DNA probe was ud that corresponded to nucleotides 1,018–1,211in the cDNA quence (11).For glyceraldehyde-3-phosphate dehydrogena (GAPDH)detection,a 1,100-bp cDNA -quence (Clontech;Palo Alto,CA)was ud.32P-labeling was achieved by random priming (Megaprime DNA-labeling sys-tems,Amersham).To allow rehybridization,boiling 0.1%SDS was poured twice onto the filters.Radiolabeled filters were expod to imaging screens for 3–4days and analyzed in a Molecular Imager (Bio-Rad Life Technologies;Hercules,CA).
Statistics.Experiments were carried out on two to six wells for each data point.All major findings were replicated at least once on at least two cell lines.For analysis of signifi-cance levels,the individual results from each well were normalized to the mean of the controls of that experiment.Student’s t -test was then applied to the t of relativized values from all the experiments on that data point (Table 1).Materials.Heparin was obtained from Lo ¨vens La ¨kemedel (Malmo ¨,Sweden).Fragmin wa
s purchad from Pharmacia Upjohn (London,UK).Chondroitin sulfate A,isolated from bovine nasal pta (containing 1.0sulfate residues/disaccha-ride unit),was kindly donated by Dr.Å.Wasteson (Linko ¨p-ing,Sweden).Heparan sulfate,derived from pig intestinal mucosa,was kindly supplied by Dr.U.Lindahl (Uppsala,Sweden).All other factors were obtained from Sigma (St.Louis,MO).Waymouth cell culture medium and FCS were purchad from Flow (Irvine,UK)and GIBCO-BRL Life Technologies.
RESULTS
General obrvations.The effects of the analyzed factors on EC-SOD expression and total cell protein were found to naturally group into three patterns of respons (Table 1).The first group,hereafter called the “growth factor”group,included acidic (aFGF)and basic fibroblast growth factors (bFGF),platelet-derived growth factors (PDGF)-AA and -BB,and epidermal growth factor (EGF).The cond group,hereafter called the “vasoactive factor”group,included hista-mine,vasopressin,oxytocin,angiotensin II (ANG II),endothelin-1(ET-1),and rotonin.The third group,hereafter called the “heparan sulfate”group,included heparin and some other glycosaminoglycans.Charac-teristic for all factors was a slow development of effects on EC-SOD in culture media over the 4-day experi-ments and a correspondence between results for EC-SOD in m
edia and in cell homogenates.The four cell lines tested generally responded in similar ways to factors tested.All active respons found were repro-duced veral times with two of the SMC lines,mostly Au-1and Au-2.Additional confirmatory two-well do-respon experiments (c.f.Fig.1)were carried out with two additional cell lines.
Respons to growth factors.All factors in this group decread the levels of EC-SOD creted to the me-dium and in the cell homogenates in cultures supple-mented with 15%FCS.The cell protein and DNA content in the cultures were in some cas to a moder-ate extent influenced.The levels of EC-SOD protein in the 4-h day media corrected for cell protein and relative to controls were between 0.3and 0.8in cell cultures expod to the factors (Fig.1A and Table 1).
In the abnce of rum,the respons varied.PDGF-AA and -BB tended to stimulate overall protein synthesis and downregulate EC-SOD,whereas aFGF,bFGF,and EGF influenced the cells less.The cell respon was strongest to PDGF-BB,with approxi-mately a doubling in the protein level after 4days of exposure concomitant with a downregulation of EC-SOD (Table 1).The increas in DNA content of the cultures were overall less pronounced.
The respons of EC-SOD mRNA levels were mea-sured by Northern analysis for bFGF,PDGF-BB,a
nd EGF.The respons on EC-SOD mRNA levels relative to GAPDH mRNA were similar to the respons en in EC-SOD protein expression (Fig.2B )
Respons to vasoactive factors.Respons to this group were discrete in cell cultures supplemented with 15%FCS (Table 1).In cell cultures supplemented with 1%BSA,increas in the amount of EC-SOD protein in both culture media and cell homogenates were ob-rved (Fig.1B and Table 1).The factors often also caud an increa in general protein synthesis as measured by the Bradford technique and by [35S]me-thionine incorporation (Table 1;data on [35S]methi-onine incorporation not shown),but the increas in EC-SOD protein relative to total protein were most often significant.There was also often a minor prolif-erative effect,as indicated by increas in the DNA content of the cultures.
For ANG II,the respon was found to differ between the Au-1and Au-2cell lines.In repeated experiments on the Au-1cell line,there was no increa in the amount of EC-SOD in the medium and in cell homog-enates relative to protein level,whereas repeated ex-periments displayed increas in the Au-2cell line (Table 1).
Northern blot experiments showed increas in the levels of EC-SOD mRNA relative to GAPDH mRNA for all factors tested in this group (Fig.2B ).
For histamine and rotonin,experiments were per-formed to determine which receptor subtypes mediated the respons.The addition of 10mol/l diphenhy-dramin,a lective H1receptor antagonist,markedly diminished the respons to histamine in the Au-2cell line,whereas addition of 10mol/l cimetidin,a lec-tive H2receptor antagonist,did not affect the hista-mine respon,indicating a H1receptor-mediated his-tamine respon.Also,the cells did not respond to 10mol/l dimaprit,a lective H2receptor agonist (Table 2).
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Table 1.Collection of data for factors influencing EC-SOD expression
Cell Line
No.of Experiments
蛋糕学习No.of Wells
EC-SOD
DNA
恒心教
Protein
In media
In cells
Media supplemented with 1%BSAL
ANG II (10mol/l)Au-1270.82Ϯ0.16* 1.27Ϯ0.48 1.17Ϯ0.05‡ 1.91Ϯ0.27‡Au-228 2.00Ϯ0.13† 2.56Ϯ0.43‡ 1.15Ϯ0.06‡ 1.31Ϯ0.07‡Au-412 2.1;2.2 2.0;2.0 1.2;1.2 1.4;1.9Au-5
12 1.5;1.8 1.4;1.6 1.4;1.5 1.2;1.6Serotonin (10mol/l)Au-1516 1.66Ϯ0.70† 1.42Ϯ0.30‡ 1.08Ϯ0.08† 1.32Ϯ0.17‡Au-2413 1.40Ϯ0.27‡ 1.63Ϯ0.42‡ 1.06Ϯ0.09* 1.13Ϯ0.16†Au-412 1.0;1.10.93;1.2 1.1;1.1 1.3;1.4Au-5
12 1.3;1.4 1.0;1.0 1.2;1.4 1.3;1.5Histamine (10mol/l)Au-1617 2.26Ϯ1.62† 1.92Ϯ0.52‡ 1.17Ϯ0.09‡ 1.55Ϯ0.29‡Au-2413 1.52Ϯ0.32‡ 2.33Ϯ1.30† 1.06Ϯ0.08* 1.22Ϯ0.16‡Au-412 1.4;2.2 1.5;2.5 1.2;1.2 1.2;2.0Au-5
12 2.0;2.3 1.4;1.5 1.4;1.4 1.5;1.7Vasopressin (100nmol/l)Au-136 2.75Ϯ1.32† 2.43Ϯ0.64‡ 1.14Ϯ0.05‡ 1.61Ϯ0.30†Au-236 2.02Ϯ0.10‡ 2.31Ϯ0.62‡ 1.13Ϯ0.12* 1.57Ϯ0.27‡Au-412 3.9;4.8 2.3;2.8 1.1;1.2 1.1;1.4Au-5
12 1.7;1.9 1.4;1.7 1.0;1.1 1.0;1.2Oxytocin (1nmol/l)Au-137 1.40Ϯ0.40* 1.56Ϯ0.26‡ 1.12Ϯ0.03‡ 1.72Ϯ0.34‡Au-237 1.52Ϯ0.78 1.74Ϯ1.13 1.02Ϯ0.04 1.25Ϯ0.17†Au-412 2.3;2.3 1.7;1.7 1.1;1.2 1.1;1.2Au-5
12 1.5;1.8 1.3;1.40.93;1.0 1.0;1.1Endothelin-1(1mol/l)Au-1413 1.71Ϯ0.70† 1.51Ϯ0.56† 1.13Ϯ0.10‡ 1.67Ϯ0.76†Au-2311 1.13Ϯ0.26 2.30Ϯ1.28† 1.06Ϯ0.07* 1.22Ϯ0.28*Au-412 2.2;2.5 2.5;2.6 1.2;1.2 1.2;1.6Au-5
12 1.6;1.7 1.6;1.6 1.3;1.3 1.4;1.4PDGF-AA (50g/l)Au-1120.57;0.880.81;1.1 1.1;1.1 1.4;1.6Au-2
120.68;0.83 1.0;1.3 1.2;1.2 1.4;1.8PDGF-BB (50g/l)Au-1250.66Ϯ0.380.78Ϯ0.44 1.39Ϯ0.15‡ 2.02Ϯ0.27‡Au-2
心成语接龙
250.44Ϯ0.05‡0.52Ϯ0.15‡ 1.36Ϯ0.08‡ 2.25Ϯ0.24‡aFGF (50g/l)Au-1120.43;0.450.63;0.69 1.1;1.2 1.5;1.7Au-2
120.75;0.81 1.0;1.2 1.0;1.1 1.1;1.1bFGF (100g/l)Au-113 1.36Ϯ0.11† 1.28Ϯ0.210.99Ϯ0.120.95Ϯ0.13Au-2
120.84;0.950.95;1.0 1.0;1.1 1.1;1.2EGF (10g/l)Au-125 1.09Ϯ0.22 1.11Ϯ0.25 1.07Ϯ0.05 1.13Ϯ0.15Au-2
250.90Ϯ0.220.91Ϯ0.15 1.13Ϯ0.03‡ 1.11Ϯ0.19Heparin (105IU/l)Au-137 1.12Ϯ0.260.56Ϯ0.05‡ 1.03Ϯ0.05 1.05Ϯ0.16Au-23
7
1.40Ϯ0.27†
1.15Ϯ0.11†
0.95Ϯ0.06
0.94Ϯ0.10
Media supplemented with 15%FCS
ANG II (500nmol/l)Au-1120.92;1.00.90;1.0 1.0;1.0 1.1;1.1Au-2
12 1.0;1.1 1.2;1.30.76;0.790.78;0.83Serotonin (10mol/l)Au-1120.72;0.940.81;1.0 1.0;1.0 1.1;1.2Au-2
120.94;1.00.93;1.1 1.1;1.1 1.0;1.0Histamine (10mol/l)Au-112 1.0;1.0 1.1;1.20.92;0.950.93;1.0Au-2
120.83;1.0 1.1;1.3 1.0;1.1 1.1;1.2Vasopressin (100nmol/l)Au-112 1.0;1.0 1.0;1.0 1.0;1.0 1.0;1.1Au-2
12 1.2;1.4 1.1;1.30.95;1.00.87;0.95Oxytocin (1nmol/l)Au-1120.82;1.0 1.0;1.10.93;1.0 1.0;1.1Au-2
1
2
1.2;1.4
四个人
1.4;1.8
0.93;1.0
0.92;0.93Continued
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