Oxidized LDL Induces Proliferation and Hypertrophy in Human Umbilical Vein Endothelial Cells via Regulation of p27Kip1 Expression: Role of RhoA
Stefan Seibold*,
Dorothea Schürle*,
Alexandra Heinloth*,
Gunter Wolf,
Martin Wagner* and
Jan Galle*
*Department of Medicine, Division of Nephrology, University Hospital, Würzburg, Germany; and Department of Medicine, Division of Nephrology, University Hospital, Hamburg, Germany
Correspondence to Prof. Dr. Jan Galle, Department of Medicine, University Hospital Würzburg, Joseph-Schneider-Strasse 2, D-97080 Würzburg, Germany. Phone: +49-931-201-36194; Fax: +49-931-201-36502; E-mail: J-C.Galle{at}mail.uni-wuerzburg.de
Oxidized LDL (OxLDL) induces proliferation in human umbilicalvein endothelial cells (HUVEC). The influence of OxLDL on thecyclin-dependent kinase inhibitor p27Kip1, on the activity ofthe small GTPase RhoA as a known regulator of p27Kip1, and onresulting cell proliferation and hypertrophy was studied. HUVECwere stimulated with OxLDL (1 to 50 µg/ml). Proliferationwas quantified by 3H-thymidine incorporation, colorimetric 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2h-tetrazoliumbromide assay, and cell count and was compared with proliferationof HUVEC that were transfected with dominant negative RhoA ortreated with the Rho-kinase inhibitor Y27632. Hypertrophy wasquantified by 3H-leucine incorporation and by planimetry. p27Kip1expression was determined by Western blot analysis. p27Kip1was downregulated by transient transfection with antisense oligonucleotides.Low concentrations of OxLDL induced proliferation of HUVEC,paralleled by a persistent decrease of p27Kip1 expression. Withthe use of antisense oligonucleotides, further downregulationof p27Kip1 expression enhanced the OxLDL-induced proliferativeresponse. High concentrations of OxLDL resulted in cellularhypertrophy and caused a delayed increase in p27Kip1 expressionafter initial downregulation. Concomitant, OxLDL caused a significantactivation of the small GTPase RhoA. In cells that were transfectedwith dominant negative RhoA, the effect of OxLDL on p27Kip1expression and on cellular proliferation was abolished. HUVECthat were preincubated with the Rho-kinase inhibitor Y27632also showed a significantly decreased proliferative responseto OxLDL stimulation. In summary, OxLDL has a dual effect oncell-cycle progression via regulation of p27Kip1 expression,resulting in cellular proliferation and hypertrophy, involvingactivation of RhoA. OxLDL may importantly contribute to vascularhyperplasia in atherosclerosis and other diseases associatedwith increased levels of OxLDL.
Cardiovascular disease, resulting from atherosclerotic remodelingof the vessel system, reflects the main cause of death in patientswith ESRD (1). The progression of atherosclerosis and the stabilityof an atherosclerotic plaque determine cardiovascular diseaseand depend on a fragile balance between proinflammatory stimulion one side and anti-inflammatory and antioxidative defensemechanisms on the other side (2,3). A typical feature of atheroscleroticplaques is increased cellular turnover (4,5), with the parallelexistence of cell proliferation and cell death, the latter beingeither of apoptotic or of necrotic nature (6,7). Vascular proliferationand inflammation are linked (8), and excessive proliferationof vascular cells plays an important role in the pathobiologyof vascular occlusive disease (e.g., transplant vasculopathy,in-stent restenosis, vessel bypass graft failure). Enhancedoxidative stress is considered to play a causal role in thissetting (3), contributing to the inflammatory cascade in thevessel wall (9) and influencing cell-cycle decisions (10). Anothertypical feature of atherosclerotic plaques is the accumulationof oxidatively modified LDL (OxLDL) (11,12), and these lipoproteinsare considered to contribute to the inflammatory state of atherosclerosis(13) and to play a key role in its pathogenesis (14,15).
We demonstrated that OxLDL potently stimulates oxidative stressvia induction of NAD(P)H oxidase (16,17) and that OxLDL-inducedO2– formation leads to either cell proliferation or apoptoticcell death of endothelial and other cells, depending on theOxLDL concentration and its exposure time (16,18,19). Thus,OxLDL-induced O2– formation has a dual effect on the cellcycle, inducing both proliferation and cell death (20). Thesedata are well in line with other recent studies showing thatNAD(P)H oxidase-derived O2– stimulates vascular cell proliferationthat can be prevented by NAD(P)H oxidase inhibition (21).
Proliferation and apoptosis are well known end points of cell-cycleprogression, whereas hypertrophy (increase in cell size) occurswhen a cell is arrested in the G1 phase of the cell cycle (22).These end points are closely connected to the activity of certaincell-cycle proteins, the cyclins and their partners, the cyclin-dependentkinases (CDK) (23). An important representative of these cell-cycleregulators is CDK-2 (24–28). Activation of CDK-2 is consideredto be essential for the progress of cells from the G1 to theG2 phase in the cell cycle (23), and its activity is controlledby a CDK inhibitor called p27Kip1 (29,30). It is generally believedthat p27Kip1 levels in a cell have to be decreased to pass throughthe cell cycle. The results of lowered p27Kip1 levels can bethat a cell finally enters mitosis, or, in case of, e.g., lackof growth-relevant substrate, enters the apoptotic cascade (22).
One important pathway leading to decreased p27Kip1 activityinvolves the small GTPase RhoA. It was demonstrated that RhoAbrings about changes in DNA synthesis in vascular smooth musclecells through reduced expression of p27Kip1 (31). It is interestingthat we identified OxLDL as a potent stimulator of RhoA in smoothmuscle cells, leading to enhanced vascular responsiveness (32).In view of the abovementioned dual effect of OxLDL on cell-cycledecisions in human umbilical vein endothelial cells (HUVEC),we asked whether OxLDL influences p27Kip1 levels in these cellsand, if so, whether this involves the RhoA pathway. For thispurpose, we studied RhoA activation in HUVEC after stimulationwith OxLDL, detected its impact on p27Kip1 expression, and analyzedthe effects of p27Kip1 inhibition and of inhibition of the RhoApathway on cell proliferation.
Reagents
CuSO4, trypsin, PBS, Tris-buffered saline (TBS), hydrocortisone,gentamicin, amphotericin B, proteinase K, lauryl sulfate sodiumsalt (SDS), ammonium chloride, paraformaldehyde, Tris/HCl, NaCl,TCA, Triton X-100, Tween-20, Igepal Ca-630, Lowry kit, acrylamide,TEMED, ammoniumpersulfate, and EDTA were obtained from Sigma(Munich, Germany). Chloroform, NaOH, and isopropanol were fromMerck (Darmstadt, Germany). EGF, Lipofectin, and FCS were fromLife Technologies (Gaithersburg, MD). Genporter II was fromGene Therapy Systems (San Diego, CA). 3-(4,5-Dimethyl-2-thiazyl)-2,5-diphenyl-2h-tetrazoliumbromide (MTT) assay, Phenol, and protease inhibitor (completemini) were obtained from Roche Diagnostics (Mannheim, Germany).PVDF membranes, ECL plus kit, 3H-thymidine, and 3H-leucine wereobtained from Amersham (Braunschweig, Germany). Y27632 [((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl))]was purchased from Calbiochem (San Diego, CA).
Isolation and Oxidation of LDL
Pooled human LDL was obtained from healthy subjects from thelocal blood bank and isolated by ultracentrifugation. LDL (0.3mg protein/ml) was oxidized with CuSO4 (5 µM) in PBS for30 h at 23°C as described previously (33). Protein contentof OxLDL was measured, and the degree of oxidation was quantifiedby the increase in relative mobility on agarose gel (lipidophorelectrophoresis kits; IMMUNO, Heidelberg, Germany), as describedpreviously (33). The index for lipoprotein oxidation therebywas typically 2.5- to 3.0-fold increased compared with nativeLDL. Lipoproteins were stored at 4°C in the dark and freshlyprepared every 2 wk.
Culture of HUVEC and Incubation with OxLDL
HUVEC were purchased from Clonetics (Walkersville, MD) and werecultured in endothelial basal medium (Clonetics), supplementedwith hydrocortisone (1 µg/ml), bovine brain extract (12µg/ml; Clonetics), gentamicin (50 µg/ml), amphotericinB (50 ng/ml), EGF (10 ng/ml), and 10% FCS. Cells in the fifthpassage were starved in basal medium supplemented with 0.1%FCS for 15 h before they were incubated with various concentrationsof OxLDL or its buffer as control.
Detection of Proliferation in HUVEC
Proliferation of HUVEC was detected by three different methods:(1) quantification of the DNA synthesis after incubation with3H-thymidine, (2) a nonradioactive proliferation assay usingMTT, and (3) cell count using flow cytometry (FACScan; BectonDickinson, Franklin Lakes, NJ). For the radioactive assay, cells(1 x 105) were stimulated with various concentrations of OxLDL(1 to 50 µg/ml) for a total of 22 h, and 3H-thymidine(0.37 MBq) was added after 15 h for the last 7 h of OxLDL stimulation.Cells were subsequently lysed, and the DNA was extracted withphenol/chloroform and resuspended in water. The amount of DNAwas determined photometrically, and the content of 3H-thymidineas indicator for proliferation was measured in a -counter (Canberra-PackardGmbH, Dreieich, Germany). For the nonradioactive MTT proliferationassays, cells (1 x 104) were seeded on 96-well plates and incubatedwith OxLDL for 24 h. Absorbance was measured photometricallyat 570 nm (reference wavelength 700 nm) after adding MTT solutionand solubilization solution as recommended by the manufacturer.When indicated, cells were transiently transfected with dominantinhibitory RhoA N19 or vector alone as control before OxLDL-stimulation,using Geneporter II transfection reagent as recommended by themanufacturer. The dominant inhibitory RhoA N19 construct wasderived from pCEV29 RhoA N19 (34), and cloned into the pcDNA3vector (Invitrogen, Karlsruhe, Germany), using the BamHI andEcoRI restriction sites. The correct sequence of RhoA N19 pcDNA3was confirmed by sequencing analysis. Transient transfectionwas carried out 24 h before stimulation with OxLDL. When indicated,the Rho-kinase inhibitor Y27632 (10 µM in PBS) or itsbuffer as control was added 1 h before addition of OxLDL.
Assessment of Hypertrophy in HUVEC De novo protein synthesis of HUVEC was measured by 3H-leucineincorporation as described previously (35). In brief, cells(1.4 x 104) were stimulated with OxLDL (1 to 50 µg/ml)or its buffer as control for a total of 22 h, and 3H-leucine(0.37 MBq) was added after 10 h for the last 12 h of OxLDL stimulation.Afterward, cells were washed in ice-cold PBS, harvested, andprecipitated twice with 10% TCA on ice. Proteins were redissolvedin 0.5 M NaOH that contained 0.1% Triton X-100. The amount ofprotein was determined with the Lowry reaction, and the contentof 3H-leucine as indicator for protein synthesis was measuredin a -counter. For discriminating between cellular proliferationand hypertrophy, data are presented as a relative ratio of 3H-leucineto 3H-thymidine incorporation. In addition, cellular hypertrophywas determined by measuring HUVEC surface area by planimetrywith phase-contrast microscopy and a digital image analyzer(SigmaScan Pro 5.0). Therefore, cells were stimulated for 22h in the absence (control) or the presence of OxLDL (1 to 50µg/ml).
Detection of p27Kip1 Protein Expression in HUVEC
Cells (6 x 105) were stimulated with OxLDL (1 to 50 µg/ml)and harvested after 4, 12, and 24 h of incubation, respectively.Cells were lysed, and protein concentration was determined withthe Lowry reaction. Ten micrograms of protein from each fractionwas loaded per lane, transferred to a PVDF membrane, and blockedwith 3% nonfat dried milk. Primary antibodies were mouse monoclonalanti-p27Kip1 antibody (PharMingen, San Diego, CA) diluted 1:500in Tris-buffered saline/Tween 20 (TBS-T), and monoclonal mouseanti--actin (Sigma) was diluted 1:20000 in TBS-T. Secondaryantibody was horseradish peroxidase-conjugated goat anti-mouse(Dako, Hamburg, Germany) diluted 1:2000. For detection, ECLPlus was used. When indicated, cells were transiently transfectedwith dominant inhibitory RhoA N19 or vector alone as controlon the day before OxLDL stimulation (OxLDL 50 µg/ml for4 h).
Downregulation of p27Kip1 by Transient Transfection with Antisense Oligonucleotides
We used antisense oligonucleotides for transient downregulationof p27Kip1 as described previously (36). The oligonucleotideswere labeled with fluorescein to determine the efficiency oftransfection by flow cytometry. For transfection, HUVEC (1 x105) were incubated with 0.1 µM oligonucleotide and Lipofectin(20 µl/ml medium) for 4 h. Twenty-four hours after transfection,cells were washed with PBS and incubated for 24 h with eitherOxLDL (5 µg/ml) or its respective buffer as control, andproliferation was measured by MTT proliferation assays.
Analysis of RhoA Stimulation by Immunohistochemistry
RhoA stimulation was detected using the same method as describedpreviously for smooth muscle cells (32). HUVEC were seeded oncoverslips and cultured in serum-free media for 4 h. After stimulationwith OxLDL for various times, cells were fixed in 4% paraformaldehydeat 4°C for 10 min, washed, and incubated in 50 mM NH4Clfor 10 min. After washing and incubation in 0.1% Triton X-100in PBS for 10 min, cells were blocked with 10% goat serum inPBS that contained 0.1% Triton X-100 for 30 min. Immunostainingwas carried out with monoclonal mouse anti-RhoA (Santa Cruz,CA) diluted 1:200 in 10% goat serum in PBS that contained 0.1%Triton X-100 for 60 min at 37°C. After washing, Cy 3–conjugatedgoat anti-mouse (Dianova, Hamburg, Germany) diluted 1:2000 in10% goat serum in PBS that contained 0.1% Triton X-100 was usedas a secondary antibody. Specimens were embedded with Mowiol(Calbiochem, La Jolla, CA) and examined using an argon/kryptonlaser confocal microscope (Leica, Solms, Germany) with an excitationwavelength of 514 nm.
Statistical Analyses
Data are presented as means ± SEM and analyzed by ANOVAor t test. P < 0.05 was considered significant.
Effect of OxLDL on Cellular Proliferation in HUVEC
Confirming a recent publication of our laboratory (16), incubationof HUVEC with various concentrations of OxLDL (1 to 20 µg/ml)resulted in a significant increase in cellular proliferation,as measured by radioactive 3H-thymidine incorporation (Figure 1A),and identically with the nonradioactive MTT proliferationassay (data not shown). This proliferative response of endothelialcells to OxLDL showed a maximal effect at 5 µg/ml (236± 35%), whereas higher concentrations of OxLDL (10 to50 µg/ml) exhibited a lower or abolished proliferativeresponse compared with controls. Cell number counting by FACSanalysis confirmed that increased DNA synthesis indeed reflectedproliferation (Figure 1B).
Figure 1. Oxidized LDL (OxLDL) induces proliferation in human umbilical vein endothelial cells (HUVEC), as measured by 3H-thymidine incorporation (n = 6; A) and flow cytometry, counting cell number (n = 3; B). Control cells without OxLDL stimulation (buffer alone) were defined as 100%. Data are means ± SEM. *P < 0.05; **P < 0.01.
Effect of OxLDL on Hypertrophy in HUVEC
We used incorporation of 3H-leucine as a measurement of de novosynthesized proteins in endothelial cells in response to incubationwith OxLDL (1 to 50 µg/ml). 3H-leucine incorporation isincreased not only during cellular hypertrophy but also duringcellular proliferation. Therefore, the 3H-leucine/3H-thymidineratio was calculated to discriminate between hypertrophy andproliferation, as shown in Figure 2A. When viewed together withFigure 1A, Figure 2A indicates that low concentrations of OxLDL(1 to 20 µg/ml) induce 3H-leucine incorporation as a resultof cellular proliferation, whereas high concentrations of OxLDLinduce 3H-leucine incorporation as a result of cellular hypertrophy.
Figure 2. OxLDL induces hypertrophy in HUVEC. (A) 3H-leucine/3H-thymidine ratio. 3H-leucine incorporation was measured in HUVEC that were stimulated with OxLDL (1 to 50 µg/ml) for 22 h. For ratio calculation, data of 3H-thymidine incorporation were taken from Figure 1A. Control cells without OxLDL stimulation (buffer alone) were defined as a ratio of 1.0. (B) Planimetry of cell surface area of HUVEC with phase-contrast microscopy, evaluated by a digital image analyzer (SigmaScan Pro 5.0). Control cells without OxLDL stimulation (buffer alone) were defined as 100%. Data are means ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01.
In addition, we determined cell surface area by planimetry asan exclusive measurement of cellular hypertrophy. OxLDL significantlyincreased the cell surface area of HUVEC (127 ± 8%) ata concentration of 50 µg/ml (Figure 2B), indicating thathypertrophy was induced at high OxLDL concentration and thusconfirming the interpretation derived from the 3H-leucine/3H-thymidineratio calculation. We then asked whether OxLDL influences cellularproliferation and hypertrophy by regulation of the CDK inhibitorp27Kip1 and therefore studied the effect of OxLDL on p27Kip1expression.
Impact of Oxidized LDL on p27Kip1 Protein Expression in HUVEC
Expression of the CDK inhibitor p27Kip1 in response to incubationof endothelial cells with OxLDL is dependent on the concentrationand incubation period of OxLDL (Figure 3). As an early effect(4 h of incubation), OxLDL (1 to 50 µg/ml) significantlydecreased p27Kip1 expression in endothelial cells by 50% (Figure 3A).After 12 h of incubation, OxLDL significantly decreasedp27Kip1 expression in HUVEC only at a concentration of 5 µg/ml(69 ± 7%), whereas all other OxLDL concentrations testeddid not significantly alter p27Kip1 expression (Figure 3B).After an incubation period of 24 h, p27Kip1 expression in HUVECremained significantly decreased to 69 ± 13% of the baselevel after incubation with 5 µg/ml OxLDL. However, incontrast to the earlier time points, stimulation of HUVEC with50 µg/ml OxLDL resulted in significantly increased p27Kip1expression, as compared with 5 µg/ml of OxLDL (Figure 3C).
Figure 3. OxLDL regulates p27Kip1 expression in HUVEC, as measured by Western blot analysis using a mouse monoclonal anti-p27Kip1 antibody. (A) Treatment with various concentrations of OxLDL (1 to 50 µg/ml) for 4 h. (B) Treatment with various concentrations of OxLDL (1 to 50 µg/ml) for 12 h. (C) Treatment with various concentrations of OxLDL (1 to 50 µg/ml) for 24 h. The blots shown are representative of at least three independent experiments. -Actin staining was used as a control to ensure equal loading of protein. Control cells without OxLDL stimulation (buffer alone) were defined as 100%. Data are means ± SEM. *P < 0.05; **P < 0.01.
In summary, low concentrations of OxLDL (5 µg/ml), inducingthe maximal proliferative response of HUVEC, showed a persistentdecrease of p27Kip1 expression, whereas high concentrationsof OxLDL (50 µg/ml), resulting in cellular hypertrophy,caused a delayed increase in p27Kip1 expression, after initialdownregulation. To demonstrate further a direct effect of OxLDLon cellular proliferation via regulation of the p27Kip1 expression,we used antisense oligonucleotides to suppress p27Kip1 expressionbefore incubation with OxLDL.
Effects of Oxidized LDL on Proliferation of Endothelial Cells with Downregulated p27Kip1 Expression Using an Antisense Strategy
An antisense oligonucleotide, complementary to the 5'-untranslatedregion of the p27Kip1 mRNA including the AUG translational startsite, was used for the downregulation of the p27Kip1 proteinexpression in HUVEC. Transfection efficacy of the cells was>80%, as seen by FACS analysis of cells that were transfectedwith the fluorescence-labeled oligonucleotides, and antisensetransfection resulted in a >60% decrease in the p27Kip1 expression,compared with the sense-transfected control (data not shown).p27Kip1 antisense-transfected endothelial cells showed an increasedproliferative response (32 ± 5%) in comparison with sense-transfectedcontrols, resulting from incubation with OxLDL (Figure 4). Becausep27Kip1 is known to be regulated by RhoA and because we recentlyidentified OxLDL as a potent stimulator of RhoA in smooth musclecells (32), we next aimed to investigate whether OxLDL alsoactivates RhoA in endothelial cells.
Figure 4. Downregulation of p27Kip1 using p27Kip1 antisense oligonucleotides enhances the proliferative response of OxLDL in HUVEC. Transfected cells were stimulated with OxLDL (5 µg/ml) for 24 h. 3-(4,5-Dimethyl-2-thiazyl)-2,5-diphenyl-2h-tetrazolium bromide (MTT) proliferation assay of HUVEC that were transfected with p27Kip1 antisense oligonucleotides as compared with the sense-transfected control (n = 11). Control cells that were transfected with sense oligonucleotides were defined as 100%. Data are means ± SEM. *P < 0.05.
Effects of Oxidized LDL on RhoA Activation in HUVEC
RhoA stimulation was assessed by means of its translocationfrom the cytosolic to the particulate fraction, an effect thatis associated with RhoA activation. To visualize RhoA translocationdirectly and to study the time course of OxLDL-induced RhoAtranslocation, we performed immunocytochemistry with a RhoA-specificantibody in endothelial cells using confocal laser scan microscopy.Under control conditions, RhoA remained distributed homogeneouslyin the cellular cytosol (Figure 5). Translocation of RhoA tothe cell membrane was visible already after 1 min of OxLDL stimulationand peaked after 5 min of stimulation. After 30 min, RhoA enrichmentin the cell membrane decreased but was still clearly above controllevels. We next investigated the influence of RhoA activationon OxLDL-induced downregulation of p27Kip1 expression.
Figure 5. OxLDL induces RhoA activation in HUVEC. (A through D) Immunocytochemistry with confocal laser scan microscopy using a RhoA-specific antibody for direct visualization of RhoA translocation as a marker for RhoA activation. OxLDL 5 µg/ml (or its buffer as control) was added to HUVEC for 1, 5, or 30 min.
Effects of RhoA Inhibition on OxLDL-Induced p27Kip1 Expression in HUVEC
RhoA activity was inhibited in HUVEC by transient transfectionwith the dominant inhibitory RhoA N19 mutant before stimulationwith OxLDL. Under control conditions with HUVEC that were transfectedwith the pcDNA3 vector alone, OxLDL stimulation (4 h) inducedsignificant downregulation of p27Kip1 expression (66 ±7%; see Figure 6), thus confirming results as described above.In contrast, cells that were transfected with dominant inhibitoryRhoA N19 mutant showed no downregulation of p27Kip1 expression(117 ± 21%) after 4 h of OxLDL stimulation (Figure 6).Therefore, OxLDL-induced suppression of p27Kip1 expression wassignificantly inhibited in RhoA N19–transfected endothelialcells. To demonstrate further a direct effect of RhoA activationon OxLDL-induced cellular proliferation, we next transfectedHUVEC with dominant inhibitory RhoA N19 before analyzing cellularproliferation in response to OxLDL stimulation.
Figure 6. RhoA inhibition abolishes the OxLDL-induced downregulation of p27Kip1 expression in HUVEC, as measured by Western blot analysis using a mouse monoclonal anti-p27Kip1 antibody. Cells were transfected with either pcDNA3 vector or dominant negative RhoA N19 mutant and were treated with OxLDL or its buffer as control. , Control cells (transfected with either dominant negative RhoA N19 or pcDNA3 vector) stimulated with buffer alone for 4 h, defined as 100%; , HUVEC (transfected with either dominant negative RhoA N19 or pcDNA3 vector) stimulated with OxLDL (50 µg/ml for 4 h). The blots shown are representative of six independent experiments. -Actin staining was used as a control to ensure equal loading of protein. Data are means ± SEM. *P < 0.05; **P < 0.01.
Effects of RhoA Inhibition on OxLDL-Induced Proliferation of HUVEC
Nonradioactive MTT proliferation assays showed an almost completelyabolished proliferative response of these cells to OxLDL, comparedwith cells that were transfected with the pcDNA3 vector aloneas control (Figure 7). The maximal OxLDL-induced proliferativeresponse in control cells that were transfected with pcDNA3alone was 206 ± 13%, whereas the maximal proliferativeresponse in RhoA N19–transfected cells was only 113 ±6%. Because Rho-kinase is an effector of RhoA signaling, wenext investigated the influence of the Rho-kinase inhibitorY27632 on OxLDL-mediated cellular proliferation.
Figure 7. RhoA inhibition abolishes the proliferative effect of OxLDL in HUVEC. Proliferation of HUVEC was measured by MTT assays (n = 4). , HUVEC transfected with pcDNA3 vector alone as control; control cells without OxLDL stimulation (buffer alone) were defined as 100%; , HUVEC that were transfected with the dominant inhibitory RhoA N19 mutant. Data are means ± SEM. *P < 0.05.
Effects of Rho Kinase Inhibition on OxLDL-Induced Proliferation of HUVEC
To investigate further RhoA involvement in OxLDL-induced endothelialcell proliferation, we incubated HUVEC with various concentrationsof OxLDL in the presence of the Rho-kinase inhibitor Y27632(Figure 8). Nonradioactive MTT proliferation assays showed asignificantly decreased OxLDL-induced proliferation rate inY27632-treated cells, compared with PBS-treated controls (maximumproliferation, 200 ± 21%, versus 166 ± 19%).
Figure 8. Rho-kinase inhibition reduces the proliferative effect of OxLDL in HUVEC. Proliferation of HUVEC was measured by MTT assays (n = 6). , HUVEC that were treated with the Rho kinase inhibitor Y27632 (10 µM); , control cells that were treated with buffer alone. Control cells without OxLDL stimulation were defined as 100%. Data are means ± SEM. *P < 0.05.
We investigated the impact of OxLDL on cell-cycle regulationin endothelial cells, with particular emphasis on the CDK-2inhibitor p27Kip1 and its regulator, the small GTPase RhoA.We previously demonstrated that OxLDL, depending on its concentration,influences cell-cycle regulation by inducing cellular proliferation(16) as well as apoptotic and necrotic cell death in HUVEC (37).In this study, we focused on the induction of cellular proliferationand hypertrophy, resulting from stimulation of endothelial cellswith OxLDL. We found that low concentrations of OxLDL (1 to10 µg/ml) caused a strong proliferative response in endothelialcells, whereas higher concentrations (20 to 50 µg/ml)resulted in a diminished or abolished proliferative response,besides an ongoing relative increase in de novo protein synthesis.Instead, high concentrations of OxLDL significantly increasedthe 3H-leucine/3H-thymidine ratio (20 to 50 µg/ml) aswell as the cell surface area (50 µg/ml). Hence, we concludethat low concentrations of OxLDL induce endothelial cell proliferation,whereas high concentrations of OxLDL induce endothelial cellhypertrophy. Of note, these concentrations of OxLDL were stilllower compared with concentrations necessary for significantinduction of apoptosis (used in the same cell line, under comparableexperimental conditions) (37).
It has been shown that not only proliferation but also cellularhypertrophy and apoptosis are regulated by CDK-2 (36,38). TheCDK inhibitor p27Kip1 plays different roles in cell-cycle progression.Besides inhibiting CDK-2 activity, it facilitates the formationof the active cyclin D/CDK complex that triggers the transitionfrom G1 to S phase of the cell cycle (39–41). Therefore,the CDK-2 inhibitor p27Kip1 acts as an important regulator forcellular signals controlling cellular growth, proliferation,and death. Our results provide several lines of evidence thatOxLDL regulates endothelial cell proliferation and hypertrophyvia regulation of p27Kip1 expression. First, within 4 h of incubation,all concentrations of OxLDL (1 to 50 µg/ml) induced cell-cycleprogression in endothelial cells, reflected by downregulationof the p27Kip1 expression, because downregulation of p27Kip1is known to result in activation of CDK-2, enabling cell-cycleprogression to enter mitosis (40). Second, transient downregulationof p27Kip1 in human endothelial cells with p27Kip1 antisenseoligonucleotides further increased cellular proliferation afterstimulation with OxLDL compared with sense-transfected controls,as measured by nonradioactive MTT proliferation assays. Therefore,the proliferative response of endothelial cells to OxLDL presumablyresults from downregulation of p27Kip1 after OxLDL stimulation.Third, ongoing stimulation (24 h) of endothelial cells withhigh concentrations of OxLDL caused a delayed increase of p27Kip1expression. High expression of p27Kip1 arrests cell-cycle progressionby inhibition of CDK-2 and therefore could be responsible forcellular hypertrophy seen in endothelial cells stimulated withhigher concentrations of OxLDL. Thus, we postulate a dual effectof OxLDL on p27Kip1 expression and on cell-cycle progressionin HUVEC: Low concentrations of OxLDL cause a persistent downregulationof p27Kip1 expression, resulting in proliferation of HUVEC,whereas higher concentrations of OxLDL cause a late increaseof p27Kip1 (after initial downregulation), resulting in cellularhypertrophy.
We hypothesized that OxLDL regulates p27Kip1 expression throughactivation of RhoA. An increased RhoA activity downregulatesp27Kip1 after overexpression of GTPase-deficient activated L63RhoAin rat aortic smooth muscle cells (31). Furthermore, decreasedp27Kip1 expression as a result of activation of RhoA is believedto be responsible for cellular proliferation in thrombin-stimulatedaortic smooth muscle cells (31). RhoA function depends on itsconversion from the cytosolic GDP- to the membrane-associatedGTP-bound state. Increases in membrane-associated RhoA are commonlyaccepted as an indicator of active RhoA (31,42). We showed previouslythat OxLDL stimulates RhoA by demonstrating its translocationfrom the cytosol to the plasma membrane in smooth muscle cells(32). Our present results provide several lines of evidencethat OxLDL induces endothelial cell proliferation via activationof the RhoA pathway. First, we found a fast and transient activationof RhoA as a result of OxLDL stimulation in endothelial cells,as shown by its translocation from the cytosol to the plasmamembrane. Second, we found that activation of RhoA is an essentialprocess for the signaling of endothelial cell proliferationin response to OxLDL stimulation, because inhibition of RhoAactivation by transient transfection with dominant inhibitoryRhoA N19 prevented downregulation of p27Kip1 and almost completelyabolished the proliferative response of HUVEC to OxLDL stimulation.Third, we confirmed the role of the RhoA/Rho-kinase signalingpathway in OxLDL-induced endothelial cell proliferation by demonstratinga reduced proliferative response of OxLDL in endothelial cellsthat were treated with the Rho-kinase inhibitor Y27632. Rhokinases are downstream effectors of RhoA (43–45), knownto play an important role in cell-cycle regulation by influencingcytokinesis, centrosome positioning, and G1- to S-phase progression,depending on the cell type (46,47). The observation that Y27632does not completely inhibit OxLDL-induced endothelial cell proliferationmight be explained by studies that provide evidence for an additionalrole of phosphatidylinositol-3 kinase in RhoA-mediated decreasesin p27Kip1 expression and cell-cycle progression (31). To clarifyfurther to what extent OxLDL-induced RhoA stimulation was responsiblefor the proliferative response of the cells, we also attemptedspecific inhibition of RhoA, using a fusion toxin with botulinustoxin C3, as previously reported (32). However, C3 treatmentwas too toxic for HUVEC, making it impossible to study proliferationin C3-treated cells (data not shown).
In conclusion, OxLDL has a dual effect on cell-cycle progressionvia regulation of p27Kip1 expression, involving stimulationof the small GTPase RhoA. We suggest that OxLDL may importantlycontribute to vascular hyperplasia in atherosclerosis and otherdiseases associated with increased levels of oxidized lipoproteins.Because renal diseases and renal insufficiency are frequentlyassociated with lipid disorders that can give rise to the formationof OxLDL, we speculate that our findings are particularly relevantfor patients who have renal diseases.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft(grants Ga431/5-3 and SFB 355, TP B11).
The skillful technical assistance of Traudel Baier, Elke Baumeister,Carmen Bauer, and Margarete Röder is gratefully acknowledged.We thank Dr. Cornelius Krasel from the Pharmacologic Instituteat the University of Würzburg for help with the confocallaser microscopy.
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Received for publication August 8, 2003.
Accepted for publication September 6, 2004.
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Top
Abstract
Introduction
-counter (Canberra-Packard GmbH, Dreieich, Germany). For the nonradioactive MTT proliferation assays, cells (1 x 104) were seeded on 96-well plates and incubated with OxLDL for 24 h. Absorbance was measured photometrically at 570 nm (reference wavelength 700 nm) after adding MTT solution and solubilization solution as recommended by the manufacturer. When indicated, cells were transiently transfected with dominant inhibitory RhoA N19 or vector alone as control before OxLDL-stimulation, using Geneporter II transfection reagent as recommended by the manufacturer. The dominant inhibitory RhoA N19 construct was derived from pCEV29 RhoA N19 (34), and cloned into the pcDNA3 vector (Invitrogen, Karlsruhe, Germany), using the BamHI and EcoRI restriction sites. The correct sequence of RhoA N19 pcDNA3 was confirmed by sequencing analysis. Transient transfection was carried out 24 h before stimulation with OxLDL. When indicated, the Rho-kinase inhibitor Y27632 (10 µM in PBS) or its buffer as control was added 1 h before addition of OxLDL. 
50% (Figure 3A). After 12 h of incubation, OxLDL significantly decreased p27Kip1 expression in HUVEC only at a concentration of 5 µg/ml (69 ± 7%), whereas all other OxLDL concentrations tested did not significantly alter p27Kip1 expression (Figure 3B). After an incubation period of 24 h, p27Kip1 expression in HUVEC remained significantly decreased to 69 ± 13% of the base level after incubation with 5 µg/ml OxLDL. However, in contrast to the earlier time points, stimulation of HUVEC with 50 µg/ml OxLDL resulted in significantly increased p27Kip1 expression, as compared with 5 µg/ml of OxLDL (Figure 3C). 
B and overcomes protection from apoptosis in activated endothelial cells. J Am Soc Nephrol 12: 456–463, 2001
, Control cells (transfected with either dominant negative RhoA N19 or pcDNA3 vector) stimulated with buffer alone for 4 h, defined as 100%; 
, HUVEC (transfected with either dominant negative RhoA N19 or pcDNA3 vector) stimulated with OxLDL (50 µg/ml for 4 h). The blots shown are representative of six independent experiments. 
