Cell Biology

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
JASN December 2004, 15 (12) 3026-3034; DOI: https://doi.org/10.1097/01.ASN.0000146425.58046.6A

Abstract

Oxidized LDL (OxLDL) induces proliferation in human umbilical vein endothelial cells (HUVEC). The influence of OxLDL on the cyclin-dependent kinase inhibitor p27Kip1, on the activity of the small GTPase RhoA as a known regulator of p27Kip1, and on resulting cell proliferation and hypertrophy was studied. HUVEC were stimulated with OxLDL (1 to 50 μg/ml). Proliferation was quantified by 3H-thymidine incorporation, colorimetric 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2h-tetrazolium bromide assay, and cell count and was compared with proliferation of HUVEC that were transfected with dominant negative RhoA or treated with the Rho-kinase inhibitor Y27632. Hypertrophy was quantified by 3H-leucine incorporation and by planimetry. p27Kip1 expression was determined by Western blot analysis. p27Kip1 was downregulated by transient transfection with antisense oligonucleotides. Low concentrations of OxLDL induced proliferation of HUVEC, paralleled by a persistent decrease of p27Kip1 expression. With the use of antisense oligonucleotides, further downregulation of p27Kip1 expression enhanced the OxLDL-induced proliferative response. High concentrations of OxLDL resulted in cellular hypertrophy and caused a delayed increase in p27Kip1 expression after initial downregulation. Concomitant, OxLDL caused a significant activation of the small GTPase RhoA. In cells that were transfected with dominant negative RhoA, the effect of OxLDL on p27Kip1 expression and on cellular proliferation was abolished. HUVEC that were preincubated with the Rho-kinase inhibitor Y27632 also showed a significantly decreased proliferative response to OxLDL stimulation. In summary, OxLDL has a dual effect on cell-cycle progression via regulation of p27Kip1 expression, resulting in cellular proliferation and hypertrophy, involving activation of RhoA. OxLDL may importantly contribute to vascular hyperplasia in atherosclerosis and other diseases associated with increased levels of OxLDL.

Cardiovascular disease, resulting from atherosclerotic remodeling of the vessel system, reflects the main cause of death in patients with ESRD (1). The progression of atherosclerosis and the stability of an atherosclerotic plaque determine cardiovascular disease and depend on a fragile balance between proinflammatory stimuli on one side and anti-inflammatory and antioxidative defense mechanisms on the other side (2,3⇓). A typical feature of atherosclerotic plaques is increased cellular turnover (4,5⇓), with the parallel existence of cell proliferation and cell death, the latter being either of apoptotic or of necrotic nature (6,7⇓). Vascular proliferation and inflammation are linked (8), and excessive proliferation of vascular cells plays an important role in the pathobiology of vascular occlusive disease (e.g., transplant vasculopathy, in-stent restenosis, vessel bypass graft failure). Enhanced oxidative stress is considered to play a causal role in this setting (3), contributing to the inflammatory cascade in the vessel wall (9) and influencing cell-cycle decisions (10). Another typical feature of atherosclerotic plaques is the accumulation of oxidatively modified LDL (OxLDL) (11,12⇓), and these lipoproteins are 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 stress via induction of NAD(P)H oxidase (16,17⇓) and that OxLDL-induced O2− formation leads to either cell proliferation or apoptotic cell death of endothelial and other cells, depending on the OxLDL concentration and its exposure time (16,18,19⇓⇓). Thus, OxLDL-induced O2− formation has a dual effect on the cell cycle, inducing both proliferation and cell death (20). These data are well in line with other recent studies showing that NAD(P)H oxidase-derived O2− stimulates vascular cell proliferation that can be prevented by NAD(P)H oxidase inhibition (21).

Proliferation and apoptosis are well known end points of cell-cycle progression, whereas hypertrophy (increase in cell size) occurs when a cell is arrested in the G1 phase of the cell cycle (22). These end points are closely connected to the activity of certain cell-cycle proteins, the cyclins and their partners, the cyclin-dependent kinases (CDK) (23). An important representative of these cell-cycle regulators is CDK-2 (24–28⇓⇓⇓⇓). Activation of CDK-2 is considered to be essential for the progress of cells from the G1 to the G2 phase in the cell cycle (23), and its activity is controlled by a CDK inhibitor called p27Kip1 (29,30⇓). It is generally believed that p27Kip1 levels in a cell have to be decreased to pass through the cell cycle. The results of lowered p27Kip1 levels can be that a cell finally enters mitosis, or, in case of, e.g., lack of growth-relevant substrate, enters the apoptotic cascade (22).

One important pathway leading to decreased p27Kip1 activity involves the small GTPase RhoA. It was demonstrated that RhoA brings about changes in DNA synthesis in vascular smooth muscle cells through reduced expression of p27Kip1 (31). It is interesting that we identified OxLDL as a potent stimulator of RhoA in smooth muscle cells, leading to enhanced vascular responsiveness (32). In view of the abovementioned dual effect of OxLDL on cell-cycle decisions in human umbilical vein endothelial cells (HUVEC), we asked whether OxLDL influences p27Kip1 levels in these cells and, if so, whether this involves the RhoA pathway. For this purpose, we studied RhoA activation in HUVEC after stimulation with OxLDL, detected its impact on p27Kip1 expression, and analyzed the effects of p27Kip1 inhibition and of inhibition of the RhoA pathway on cell proliferation.

Materials and Methods

Reagents

CuSO4, trypsin, PBS, Tris-buffered saline (TBS), hydrocortisone, gentamicin, amphotericin B, proteinase K, lauryl sulfate sodium salt (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 from Merck (Darmstadt, Germany). EGF, Lipofectin, and FCS were from Life Technologies (Gaithersburg, MD). Genporter II was from Gene Therapy Systems (San Diego, CA). 3-(4,5-Dimethyl-2-thiazyl)-2,5-diphenyl-2h-tetrazolium bromide (MTT) assay, Phenol, and protease inhibitor (complete mini) were obtained from Roche Diagnostics (Mannheim, Germany). PVDF membranes, ECL plus kit, 3H-thymidine, and 3H-leucine were obtained 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 the local blood bank and isolated by ultracentrifugation. LDL (0.3 mg protein/ml) was oxidized with CuSO4 (5 μM) in PBS for 30 h at 23°C as described previously (33). Protein content of OxLDL was measured, and the degree of oxidation was quantified by the increase in relative mobility on agarose gel (lipidophor electrophoresis kits; IMMUNO, Heidelberg, Germany), as described previously (33). The index for lipoprotein oxidation thereby was typically 2.5- to 3.0-fold increased compared with native LDL. Lipoproteins were stored at 4°C in the dark and freshly prepared every 2 wk.

Culture of HUVEC and Incubation with OxLDL

HUVEC were purchased from Clonetics (Walkersville, MD) and were cultured in endothelial basal medium (Clonetics), supplemented with hydrocortisone (1 μg/ml), bovine brain extract (12 μg/ml; Clonetics), gentamicin (50 μg/ml), amphotericin B (50 ng/ml), EGF (10 ng/ml), and 10% FCS. Cells in the fifth passage were starved in basal medium supplemented with 0.1% FCS for 15 h before they were incubated with various concentrations of 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 with 3H-thymidine, (2) a nonradioactive proliferation assay using MTT, and (3) cell count using flow cytometry (FACScan; Becton Dickinson, Franklin Lakes, NJ). For the radioactive assay, cells (1 × 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 with phenol/chloroform and resuspended in water. The amount of DNA was determined photometrically, and the content of 3H-thymidine as indicator for proliferation was measured in a β-counter (Canberra-Packard GmbH, Dreieich, Germany). For the nonradioactive MTT proliferation assays, cells (1 × 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.

Assessment of Hypertrophy in HUVEC

De novo protein synthesis of HUVEC was measured by 3H-leucine incorporation as described previously (35). In brief, cells (1.4 × 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, and precipitated twice with 10% TCA on ice. Proteins were redissolved in 0.5 M NaOH that contained 0.1% Triton X-100. The amount of protein was determined with the Lowry reaction, and the content of 3H-leucine as indicator for protein synthesis was measured in a β-counter. For discriminating between cellular proliferation and hypertrophy, data are presented as a relative ratio of 3H-leucine to 3H-thymidine incorporation. In addition, cellular hypertrophy was determined by measuring HUVEC surface area by planimetry with phase-contrast microscopy and a digital image analyzer (SigmaScan Pro 5.0). Therefore, cells were stimulated for 22 h in the absence (control) or the presence of OxLDL (1 to 50 μg/ml).

Detection of p27Kip1 Protein Expression in HUVEC

Cells (6 × 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 with the Lowry reaction. Ten micrograms of protein from each fraction was loaded per lane, transferred to a PVDF membrane, and blocked with 3% nonfat dried milk. Primary antibodies were mouse monoclonal anti-p27Kip1 antibody (PharMingen, San Diego, CA) diluted 1:500 in Tris-buffered saline/Tween 20 (TBS-T), and monoclonal mouse anti-β-actin (Sigma) was diluted 1:20000 in TBS-T. Secondary antibody was horseradish peroxidase-conjugated goat anti-mouse (Dako, Hamburg, Germany) diluted 1:2000. For detection, ECL Plus was used. When indicated, cells were transiently transfected with dominant inhibitory RhoA N19 or vector alone as control on the day before OxLDL stimulation (OxLDL 50 μg/ml for 4 h).

Downregulation of p27Kip1 by Transient Transfection with Antisense Oligonucleotides

We used antisense oligonucleotides for transient downregulation of p27Kip1 as described previously (36). The oligonucleotides were labeled with fluorescein to determine the efficiency of transfection by flow cytometry. For transfection, HUVEC (1 × 105) 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 either OxLDL (5 μg/ml) or its respective buffer as control, and proliferation was measured by MTT proliferation assays.

Analysis of RhoA Stimulation by Immunohistochemistry

RhoA stimulation was detected using the same method as described previously for smooth muscle cells (32). HUVEC were seeded on coverslips and cultured in serum-free media for 4 h. After stimulation with OxLDL for various times, cells were fixed in 4% paraformaldehyde at 4°C for 10 min, washed, and incubated in 50 mM NH4Cl for 10 min. After washing and incubation in 0.1% Triton X-100 in PBS for 10 min, cells were blocked with 10% goat serum in PBS that contained 0.1% Triton X-100 for 30 min. Immunostaining was 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–conjugated goat anti-mouse (Dianova, Hamburg, Germany) diluted 1:2000 in 10% goat serum in PBS that contained 0.1% Triton X-100 was used as a secondary antibody. Specimens were embedded with Mowiol (Calbiochem, La Jolla, CA) and examined using an argon/krypton laser confocal microscope (Leica, Solms, Germany) with an excitation wavelength of 514 nm.

Statistical Analyses

Data are presented as means ± SEM and analyzed by ANOVA or t test. P < 0.05 was considered significant.

Results

Effect of OxLDL on Cellular Proliferation in HUVEC

Confirming a recent publication of our laboratory (16), incubation of 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 proliferation assay (data not shown). This proliferative response of endothelial cells to OxLDL showed a maximal effect at 5 μg/ml (236 ± 35%), whereas higher concentrations of OxLDL (10 to 50 μg/ml) exhibited a lower or abolished proliferative response compared with controls. Cell number counting by FACS analysis confirmed that increased DNA synthesis indeed reflected proliferation (Figure 1B).

Figure1

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 novo synthesized proteins in endothelial cells in response to incubation with OxLDL (1 to 50 μg/ml). 3H-leucine incorporation is increased not only during cellular hypertrophy but also during cellular proliferation. Therefore, the 3H-leucine/3H-thymidine ratio was calculated to discriminate between hypertrophy and proliferation, as shown in Figure 2A. When viewed together with Figure 1A, Figure 2A indicates that low concentrations of OxLDL (1 to 20 μg/ml) induce 3H-leucine incorporation as a result of cellular proliferation, whereas high concentrations of OxLDL induce 3H-leucine incorporation as a result of cellular hypertrophy.

Figure2

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 as an exclusive measurement of cellular hypertrophy. OxLDL significantly increased the cell surface area of HUVEC (127 ± 8%) at a concentration of 50 μg/ml (Figure 2B), indicating that hypertrophy was induced at high OxLDL concentration and thus confirming the interpretation derived from the 3H-leucine/3H-thymidine ratio calculation. We then asked whether OxLDL influences cellular proliferation and hypertrophy by regulation of the CDK inhibitor p27Kip1 and therefore studied the effect of OxLDL on p27Kip1 expression.

Impact of Oxidized LDL on p27Kip1 Protein Expression in HUVEC

Expression of the CDK inhibitor p27Kip1 in response to incubation of endothelial cells with OxLDL is dependent on the concentration and incubation period of OxLDL (Figure 3). As an early effect (4 h of incubation), OxLDL (1 to 50 μg/ml) significantly decreased p27Kip1 expression in endothelial cells by ∼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).

Figure3

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. β-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), inducing the maximal proliferative response of HUVEC, showed a persistent decrease of p27Kip1 expression, whereas high concentrations of OxLDL (50 μg/ml), resulting in cellular hypertrophy, caused a delayed increase in p27Kip1 expression, after initial downregulation. To demonstrate further a direct effect of OxLDL on cellular proliferation via regulation of the p27Kip1 expression, we used antisense oligonucleotides to suppress p27Kip1 expression before 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′-untranslated region of the p27Kip1 mRNA including the AUG translational start site, was used for the downregulation of the p27Kip1 protein expression in HUVEC. Transfection efficacy of the cells was >80%, as seen by FACS analysis of cells that were transfected with the fluorescence-labeled oligonucleotides, and antisense transfection 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 increased proliferative response (32 ± 5%) in comparison with sense-transfected controls, resulting from incubation with OxLDL (Figure 4). Because p27Kip1 is known to be regulated by RhoA and because we recently identified OxLDL as a potent stimulator of RhoA in smooth muscle cells (32), we next aimed to investigate whether OxLDL also activates RhoA in endothelial cells.

Figure4

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 translocation from the cytosolic to the particulate fraction, an effect that is associated with RhoA activation. To visualize RhoA translocation directly and to study the time course of OxLDL-induced RhoA translocation, we performed immunocytochemistry with a RhoA-specific antibody in endothelial cells using confocal laser scan microscopy. Under control conditions, RhoA remained distributed homogeneously in the cellular cytosol (Figure 5). Translocation of RhoA to the cell membrane was visible already after 1 min of OxLDL stimulation and peaked after 5 min of stimulation. After 30 min, RhoA enrichment in the cell membrane decreased but was still clearly above control levels. We next investigated the influence of RhoA activation on OxLDL-induced downregulation of p27Kip1 expression.

Figure5

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 transfection with the dominant inhibitory RhoA N19 mutant before stimulation with OxLDL. Under control conditions with HUVEC that were transfected with the pcDNA3 vector alone, OxLDL stimulation (4 h) induced significant downregulation of p27Kip1 expression (66 ± 7%; see Figure 6), thus confirming results as described above. In contrast, cells that were transfected with dominant inhibitory RhoA 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 was significantly inhibited in RhoA N19–transfected endothelial cells. To demonstrate further a direct effect of RhoA activation on OxLDL-induced cellular proliferation, we next transfected HUVEC with dominant inhibitory RhoA N19 before analyzing cellular proliferation in response to OxLDL stimulation.

Figure6

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%; Embedded Image, 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. β-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 completely abolished proliferative response of these cells to OxLDL, compared with cells that were transfected with the pcDNA3 vector alone as control (Figure 7). The maximal OxLDL-induced proliferative response in control cells that were transfected with pcDNA3 alone was 206 ± 13%, whereas the maximal proliferative response in RhoA N19–transfected cells was only 113 ± 6%. Because Rho-kinase is an effector of RhoA signaling, we next investigated the influence of the Rho-kinase inhibitor Y27632 on OxLDL-mediated cellular proliferation.

Figure7

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%; Embedded Image, 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 endothelial cell proliferation, we incubated HUVEC with various concentrations of OxLDL in the presence of the Rho-kinase inhibitor Y27632 (Figure 8). Nonradioactive MTT proliferation assays showed a significantly decreased OxLDL-induced proliferation rate in Y27632-treated cells, compared with PBS-treated controls (maximum proliferation, 200 ± 21%, versus 166 ± 19%).

Figure8

Figure 8. Rho-kinase inhibition reduces the proliferative effect of OxLDL in HUVEC. Proliferation of HUVEC was measured by MTT assays (n = 6). Embedded Image, 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.

Discussion

We investigated the impact of OxLDL on cell-cycle regulation in endothelial cells, with particular emphasis on the CDK-2 inhibitor 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 proliferation and hypertrophy, resulting from stimulation of endothelial cells with OxLDL. We found that low concentrations of OxLDL (1 to 10 μg/ml) caused a strong proliferative response in endothelial cells, 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 increased the 3H-leucine/3H-thymidine ratio (20 to 50 μg/ml) as well as the cell surface area (50 μg/ml). Hence, we conclude that low concentrations of OxLDL induce endothelial cell proliferation, whereas high concentrations of OxLDL induce endothelial cell hypertrophy. Of note, these concentrations of OxLDL were still lower compared with concentrations necessary for significant induction of apoptosis (used in the same cell line, under comparable experimental conditions) (37).

It has been shown that not only proliferation but also cellular hypertrophy and apoptosis are regulated by CDK-2 (36,38⇓). The CDK inhibitor p27Kip1 plays different roles in cell-cycle progression. Besides inhibiting CDK-2 activity, it facilitates the formation of the active cyclin D/CDK complex that triggers the transition from G1 to S phase of the cell cycle (39–41⇓⇓). Therefore, the CDK-2 inhibitor p27Kip1 acts as an important regulator for cellular signals controlling cellular growth, proliferation, and death. Our results provide several lines of evidence that OxLDL regulates endothelial cell proliferation and hypertrophy via regulation of p27Kip1 expression. First, within 4 h of incubation, all concentrations of OxLDL (1 to 50 μg/ml) induced cell-cycle progression in endothelial cells, reflected by downregulation of the p27Kip1 expression, because downregulation of p27Kip1 is known to result in activation of CDK-2, enabling cell-cycle progression to enter mitosis (40). Second, transient downregulation of p27Kip1 in human endothelial cells with p27Kip1 antisense oligonucleotides further increased cellular proliferation after stimulation with OxLDL compared with sense-transfected controls, as measured by nonradioactive MTT proliferation assays. Therefore, the proliferative response of endothelial cells to OxLDL presumably results from downregulation of p27Kip1 after OxLDL stimulation. Third, ongoing stimulation (24 h) of endothelial cells with high concentrations of OxLDL caused a delayed increase of p27Kip1 expression. High expression of p27Kip1 arrests cell-cycle progression by inhibition of CDK-2 and therefore could be responsible for cellular hypertrophy seen in endothelial cells stimulated with higher concentrations of OxLDL. Thus, we postulate a dual effect of OxLDL on p27Kip1 expression and on cell-cycle progression in HUVEC: Low concentrations of OxLDL cause a persistent downregulation of p27Kip1 expression, resulting in proliferation of HUVEC, whereas higher concentrations of OxLDL cause a late increase of p27Kip1 (after initial downregulation), resulting in cellular hypertrophy.

We hypothesized that OxLDL regulates p27Kip1 expression through activation of RhoA. An increased RhoA activity downregulates p27Kip1 after overexpression of GTPase-deficient activated L63RhoA in rat aortic smooth muscle cells (31). Furthermore, decreased p27Kip1 expression as a result of activation of RhoA is believed to be responsible for cellular proliferation in thrombin-stimulated aortic smooth muscle cells (31). RhoA function depends on its conversion from the cytosolic GDP- to the membrane-associated GTP-bound state. Increases in membrane-associated RhoA are commonly accepted as an indicator of active RhoA (31,42⇓). We showed previously that OxLDL stimulates RhoA by demonstrating its translocation from the cytosol to the plasma membrane in smooth muscle cells (32). Our present results provide several lines of evidence that OxLDL induces endothelial cell proliferation via activation of the RhoA pathway. First, we found a fast and transient activation of RhoA as a result of OxLDL stimulation in endothelial cells, as shown by its translocation from the cytosol to the plasma membrane. Second, we found that activation of RhoA is an essential process for the signaling of endothelial cell proliferation in response to OxLDL stimulation, because inhibition of RhoA activation by transient transfection with dominant inhibitory RhoA N19 prevented downregulation of p27Kip1 and almost completely abolished the proliferative response of HUVEC to OxLDL stimulation. Third, we confirmed the role of the RhoA/Rho-kinase signaling pathway in OxLDL-induced endothelial cell proliferation by demonstrating a reduced proliferative response of OxLDL in endothelial cells that were treated with the Rho-kinase inhibitor Y27632. Rho kinases are downstream effectors of RhoA (43–45⇓⇓), known to play an important role in cell-cycle regulation by influencing cytokinesis, centrosome positioning, and G1- to S-phase progression, depending on the cell type (46,47⇓). The observation that Y27632 does not completely inhibit OxLDL-induced endothelial cell proliferation might be explained by studies that provide evidence for an additional role of phosphatidylinositol-3 kinase in RhoA-mediated decreases in p27Kip1 expression and cell-cycle progression (31). To clarify further to what extent OxLDL-induced RhoA stimulation was responsible for the proliferative response of the cells, we also attempted specific inhibition of RhoA, using a fusion toxin with botulinus toxin C3, as previously reported (32). However, C3 treatment was too toxic for HUVEC, making it impossible to study proliferation in C3-treated cells (data not shown).

In conclusion, OxLDL has a dual effect on cell-cycle progression via regulation of p27Kip1 expression, involving stimulation of the small GTPase RhoA. We suggest that OxLDL may importantly contribute to vascular hyperplasia in atherosclerosis and other diseases associated with increased levels of oxidized lipoproteins. Because renal diseases and renal insufficiency are frequently associated with lipid disorders that can give rise to the formation of OxLDL, we speculate that our findings are particularly relevant for 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 Institute at the University of Würzburg for help with the confocal laser microscopy.

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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, Jan Galle
JASN Dec 2004, 15 (12) 3026-3034; DOI: 10.1097/01.ASN.0000146425.58046.6A
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