INTRODUCTION
Three-hydroxy-3methylglutaryl-CoA reductase inhibitors (statins) significantly decrease cardiovascular morbidity and mortality in hypercholesterolemic patients.1 2 3 4 5 6 7,8
Statins block mevalonate synthesis and are potent inhibitors of cholesterol biosynthesis. Statins also inhibit the synthesis of isoprenoid intermediates in the cholesterol biosynthesis pathway, such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). These intermediates serve as lipid attachments that are implicated in the activation of the small GTP-binding proteins, Ras and Rho.9
The expression of the VEGF gene is regulated by a transcription factor, hypoxia-inducible factor-1 (HIF-1).10 11
In this study, we investigated the effects of simvastatin on VEGF expression in a human endothelial cell line, EA hy.926 cells.12
MATERIALS AND METHODS
Materials
Mevalonate, FPP, and GGPP were purchased from Sigma. Simvastatin was obtained from Merck.
Cell Culture
EA hy.926 cells, a gift from Dr. Cora-Jean S. Edgell (University of North Carolina at Chapel Hill, Chapel Hill, NC), were maintained at 37°C under atomosphere of 5% CO2 in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/mL streptomysin.
Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from cells with Isogen (Nippon gene, Japan) following the manufacturer's protocol. cDNA synthesis was performed using 2 μg of total RNA and a RETROscript (Ambion, Austin, TX) following the manufacture's protocol. The specific primer sets for the target genes were as follows; VEGF 5′-CCA TGA ACT TTC TGC TGT CTT-3′, 5′-TCG ATC GTT CTG TAT CAG TCT-3′ (GeneBank Accession No. AF022375), HIF-1α 5′-GTC GGA CAG CCT CAC CAA ACA GAG C-3′, 5′-GTT AAC TTG ATC CAA AGC TCT GAG-3′ (GeneBank Accession No. BT009776), β-actin was amplified using Quantum RNA β-actin Internal Standards from Ambion.
After an initial denaturation step at 94°C for 5 minutes, the reactions underwent 28 cycles at 94°C (1 minutes), 65°C (2 minutes), and 72°C (2 minutes). PCR products were electrophoresed on 2% agarose gels before ethidium bromide staining. The mRNA expression levels were normalized by β-actin expression and presented as the relative expression level compared to that of control cells.
VEGF Enzyme-linked Immunosorbent Assay (ELISA)
Conditioned media were collected from serum-starved cells treated with control vehicle or simvastatin for 24 hours. Commercially available ELISA kit to measure human VEGF (Biosourse International) was used following the manufacture's protocol.
Recombinant Adenovirus Transfection
After cells had attained confluence, they were infected with recombinant adenovirus expressing LacZ, RhoA T19N13 14
Preparation of Cytosolic and Nuclear Fractions and Immunoblot Analysis
Proteins from the cytosolic and nuclear fractions of cells were isolated using Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology) following the manufacturer's protocol. Immunoblotting was performed as described previously.13
HIF-1α Depletion Using siRNA
HIF-1α was depleted using Silencer validated siRNA (Genbank accession No. NM-001530 and NM-181054, purchased from Ambion) following the manufacturer's protocol. Transfection of siRNA was performed at a concentration of 100 nM using lipofectamin 2000 (Invitrogen).
Statistical Analyses
Data from 5 independent experiments are shown as means ± SEM. Statistical significance, calculated using Mann-Whitney U test and Wilcoxson signed-rank test, was taken as P < 0.05.
RESULTS
Increased Expression of VEGF mRNA and Protein by Simvastatin
We examined the effects of simvastatin on VEGF mRNA expression in EA hy.926 cells by RT-PCR. As shown in Figure 1A , mRNA expression of VEGF121 and VEGF165 was increased by 0.1 and 1 μmol/L of simvastatin. Next, we studied the effects of simvastatin on the secretion of VEGF protein into conditioned medium. VEGF protein in conditioned medium measured by ELISA was increased by 1 μmol/L of simvastatin in EA hy.926 cells (Figure 1B ). These results suggest that simvastatin increases the expression of mRNA and protein of VEGF in EA hy.926 cells.
FIGURE 1:
Simvastatin induces vascular endothelial growth factor (VEGF) expression in EA hy.926 cells. (A) The mRNA expression of VEGF121 and VEGF165 was examined by reverse transcriptase polymerase chain reaction (RT-PCR) using total RNA extracted from cells treated with control vehicle and simvastatin at the indicated concentration for 8 hours. A representative gel image is shown (upper panel). The mRNA expression level of VEGF121 and VEGF165 normalized relative to that of β-actin was expressed compared to control (lower panel). Black and white bars indicate VEGF121 and VEGF165 , respectively. * and # indicate P < 0.05 versus VEGF121 and VEGF165 of vehicle-treated control cells, respectively. N = 5 in each group. (B) VEGF concentration in conditioned medium measured by ELISA. Secretion of VEGF was increased in culture medium in EA hy.926 cells treated with 1 μmol/L of simvastatin for 24 hours. *P < 0.05 versus control. N = 5 in each group.
Reversal of Simvastatin-Induced VEGF mRNA Expression by GGPP and Mevalonate but not FPP
Simvastatin inhibits cholesterol biosynthesis by decreased synthesis of mevalonate and isoprenoid intermediates such as FPP and GGPP. We examined the involvement of downstream isoprenoid intermediates in simvastatin-induced mRNA expression of VEGF121 and VEGF165 . EA hy.926 cells were treated with simvastatin in the presence or absence of FPP, GGPP, or mevalonate. As shown in Figure 2 , treatment with GGPP or mevalonate reversed the effects of simvastatin-induced mRNA expression of VEGF121 and VEGF165 , whereas treatment with FPP had no significant effect. These data indicate that simvastatin induces mRNA expression of VEGF121 and VEGF165 by inhibiting production of mevalonate and GGPP.
FIGURE 2:
Geranylgeranylpyrophosphate (GGPP) and mevalonate reverse simvastatin-induced mRNA expression of VEGF121 and VEGF165 . Serum-starved cells were treated with 1 μmol/L simvastatin for 8 hours in the presence or absence of farnesylpyrophosphate (FPP) (10 μmol/L), GGPP (20 μmol/L), or mevalonate (200 μmol/L). Vascular endothelial growth factor (VEGF) and β-actin mRNA expression was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). A representative gel image is shown (upper panel). The mRNA expression level of VEGF121 and VEGF165 normalized relative to that of β-actin was expressed compared to control (lower panel). Black and white bars indicate VEGF121 and VEGF165 , respectively. * and # indicate P < 0.05 versus VEGF121 and VEGF165 of simvastatin-treated cells, respectively. N = 5 in each group.
Inhibition of RhoA and Rho-kinase Increases mRNA Expression of VEGF121 and VEGF165 .
It has been shown that GGPP is required for small G proteins such as Rho, Rab, and Raf to anchor to cell membranes during their activation through geranylgeranylation. We studied the involvement of RhoA and Rho-kinase in VEGF expression. As shown in Figures 3A and 3B , the mRNA expression of VEGF121 and VEGF165 was dose-dependently increased by adenovirus-mediated expression of dominant negative mutants of RhoA and Rho-kinase. As a control, adenovirus-mediated expression of LacZ showed no obvious effect on the mRNA expression of VEGF121 and VEGF165 . Also, the concentration of VEGF protein secreted into conditioned medium was increased by adenovirus-mediated expression of the mutants (Figures 3C and 3D ). These results suggest that RhoA and Rho-kinase negatively regulate VEGF expression in EA hy.926 cells.
FIGURE 3:
Overexpression of dominant-negative mutants of RhoA and Rho-kinase increases the expression of mRNA and protein of vascular endothelial growth factor (VEGF). Total RNA extracted from cells overexpressing RhoA T19N (A) and ROCK DN (B) with adenovirus was subjected to reverse transcriptase polymerase chain reaction (RT-PCR) analysis for VEGF121 and VEGF165 . A representative gel image is shown (upper panel). The mRNA expression level of VEGF121 and VEGF165 normalized relative to that of β-actin was expressed compared to control (lower panel). Black and white bars indicate VEGF121 and VEGF165 , respectively. * and # indicate P < 0.05 versus VEGF121 and VEGF165 of control cells, respectively. N = 5 in each group. VEGF was measured by ELISA in conditioned medium prepared from cells overexpressing RhoA T19N (C) and ROCK DN (D) with adenovirus. *P < 0.05 versus control. N = 5 in each group.
Increased HIF-1α Protein Levels by Simvastatin or the Inhibition of Rho and Rho-kinase
It has been demonstrated that HIF-1α increases VEGF mRNA expression.11
It has been demonstrated that translocation of HIF-1α to the nucleus plays an important role in its activation. We investigated the changes of HIF-1α protein levels in both cytosolic and nuclear fractions by simvastatin or by the inhibition of RhoA and Rho-kinase by overexpressing their dominant negative mutants in EA hy.926 cells. As shown in Figure 4A , HIF-1α protein levels increased in whole cell lysates by simvastatin, and also tended to increase in cytosolic and nuclear fractions by simvastatin. HIF-1β protein levels in whole cell lysates, and cytosolic and nuclear fractions were not changed by treatment with simvastatin (Figure 4A ). By inhibition of RhoA and Rho-kinase by adenovirus-mediated expression of RhoA T19N and ROCK DN, HIF-1α protein was increased in whole cell lysates and nuclear fractions, but it was decreased in cytosolic fractions (Figure 4B ). HIF-1β protein levels in whole cell lysates and cytosolic and nuclear fractions were not changed by adenovirus-mediated expression of RhoA T19N and ROCK DN (Figure 4B ). These data suggest that simvastatin and the inhibition of RhoA and Rho-kinase increase HIF-1α protein levels in whole cell and nuclear fractions without changing HIF-1α mRNA expression in EA hy.926 cells. It is also indicated that HIF-1β protein levels are not changed either by simvastatin or by inhibition of RhoA and Rho-kinase in EA hy.926 cells.
FIGURE 4:
(A) Protein level of HIF-1α was increased by simvastatin in EA hy.926 cells. Serum-depleted cells were treated with vehicle or simvastatin (1 μmol/L) for 8 hours. Whole cell lysates, cytosolic fractions, and nuclear fractions (40 μg of protein each) were prepared from the cells. The expression of HIF-1α and HIF-1β was examined by immunoblotting. Representative immunoblots are shown (HIF-1α: upper panel, HIF-1β: lower panel). The bar graphs indicate quantitative band intensity of HIF-1α normalized to each control. *P < 0.05 versus control. N = 5 in each group. (B) HIF-1α protein level was increased by overexpression of dominant negative mutants of RhoA and Rho-kinase in whole cell lysates and nuclear fractions from EA hy.926 cells. Whole cell lysates, cytosolic fractions, and nuclear fractions (40 μg of protein each) were prepared from noninfected control cells and the cells overexpressing RhoA T19N and ROCK DN with adenovirus. The expression of HIF-1α and HIF-1β was examined by immunoblotting. Representative immunoblots are shown (HIF-1α: upper panel, HIF-1β: lower panel). The bar graphs indicate quantitative band intensity of HIF-1α normalized to each control. *P < 0.05 versus control. N = 5 in each group.
Attenuation of Simvastatin-Induced VEGF mRNA Expression by HIF-1α Depletion
We examined the involvement of HIF-1α in simvastatin-induced VEGF mRNA expression by HIF-1α depletion using siRNA in EA hy.926 cells. As shown in Figure 5 , HIF-1α siRNA transfection reduced HIF-1α mRNA expression to 38% of control, whereas control transfection had no obvious effect on HIF-1α mRNA expression. HIF-1α siRNA transfection reduced HIF-1α protein level to 26% of control, whereas control transfection had no obvious effect on HIF-1α protein levels. Basal mRNA expression of VEGF121 and VEGF165 was decreased by HIF-1α depletion using RNA interference. Simvastatin-induced mRNA expression of VEGF121 and VEGF165 was attenuated by HIF-1α depletion using RNA interference. These results suggest that HIF-1α is implicated in simvastatin-mediated VEGF mRNA expression in EA hy.926 cells.
FIGURE 5:
The mRNA expression of VEGF121 and VEGF165 is decreased by HIF-1α depletion using RNA interference in EA hy.926 cells. After transfection with siRNA against HIF-1α for 24 hours, the cells were treated with vehicle or simvastatin (1 μmol/L) for 8 hours. Total RNA was extracted from the cells, and HIF-1α mRNA expression was examined by reverse transcriptase polymerase chain reaction (RT-PCR). A representative gel image is shown (upper panel). Total protein was extracted from the cells and HIF-1α protein expression was examined by immunoblotting. A representative blot is shown (the second panel). Vascular endothelial growth factor (VEGF) mRNA expression was examined by RT-PCR. A representative gel image is shown (the third panel). The mRNA expression level of VEGF121 and VEGF165 normalized relative to that of β-actin was expressed compared to control (the bottom panel). Black and white bars indicate VEGF121 and VEGF165 , respectively. * and # indicate P < 0.05 versus VEGF121 and VEGF165 of control transfection in the absence of simvastatin. ** and ## indicate P < 0.05 versus VEGF121 and VEGF165 of control transfection in the presence of simvastatin. & and && indicate P < 0.05 versus VEGF121 and VEGF165 of HIF-1α RNAi transfection in the absence of simvastatin. N = 5 in each group.
DISCUSSION
In our study, we first evaluated the effects of simvastatin on VEGF expression in cultured endothelial cells. It has been reported that 1 μmol/L pitavastatin inhibited angiogenesis, whereas 0.3 μmol/L increased angiogenesis in vitro.15 6 16 Figure 1 ). We believe that our data support the results of previous studies.7,8
We also examined the involvement of isoprenoid intermediates in simvastatin-induced VEGF expression. Our results suggest that simvastatin-induced VEGF expression was mediated by the inhibition of mevalonate and GGPP. Although geranylgeranylation of Rho is important for its activation, the involvement of RhoA in VEGF expression has not been well documented.9 Figure 4A ). Inhibition of RhoA had a similar effect (Figure 4B ). In addition, inhibition of RhoA and Rho-kinase increased VEGF expression in endothelial cells (Figure 3 ). These results provide novel evidence that inhibition of RhoA and Rho-kinase upregulates HIF-1α that plays a pivotal role in VEGF expression in endothelial cells. Previous reports demonstrated that RhoA activation upregulates HIF-1α expression levels in hypoxia in certain type of cells.17,18
In this study, simvastatin increased HIF-1α protein in both cytosolic and nuclear fractions in EA hy.926 cells. Under normoxic conditions, HIF-1α is hydroxylated and subsequently degraded by proteasome. In contrast, under hypoxic conditions, HIF-1α is stabilized by inhibiting its hydroxylation. Increased HIF-1α is translocated to the nucleus and stimulates target gene transcription after dimerization with HIF-1β.19 20 21
It has been reported that vascular smooth muscle cells express 2- to 3-fold higher amounts of VEGF protein than endothelial cells under basal conditions7 22,23 24,25 26
CONCLUSION
This study demonstrates that simvastatin stimulates VEGF production by RhoA downregulation and HIF-1α upregulation in endothelial cells. These data indicate a novel role for RhoA as a negative regulator of HIF-1α. Also, these effects of simvastatin may contribute to vascular protection in addition to cholesterol lowering.
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