Statins differentially regulate vascular endothelial growth factor synthesis in endothelial and vascular smooth muscle cells

Atherosclerosis 170 (2003) 229–236

Statins differentially regulate vascular endothelial growth factor synthesis in endothelial and vascular smooth muscle cells Matthias Frick a,1 , Jozef Dulak a,b,∗,1 , Jarosław Cisowski b , Alicja Józkowicz b,c , Ralf Zwick a , Hannes Alber a , Wolfgang Dichtl a , Severin P. Schwarzacher a , Otmar Pachinger a , Franz Weidinger a b

a Division of Cardiology, Department of Medicine, Innsbruck University, Innsbruck, Austria Department of Cell Biochemistry, Faculty of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland c Department of Vascular Surgery, University of Vienna, Vienna, Austria

Received 5 February 2003; received in revised form 7 July 2003; accepted 14 July 2003

Abstract Objectives: HMG-CoA reductase inhibitors (statins) can modulate the formation of new blood vessels, but the reports on their contribution to angiogenesis are contradictory. Therefore, we investigated whether the effect of statins is dependent either on the concentration of the drug or on the cell type. Methods and results: Under basal conditions human vascular smooth muscle cells (HVSMC) and microvascular endothelial cells (HMEC-1) constitutively generate and release vascular endothelial growth factor (VEGF). In contrast, primary macrovascular endothelial cells (HUVEC) produce minute amounts of VEGF. Different statins (atorvastatin, simvastatin and lovastatin, 1–10 ␮mol/l) significantly reduced basal and cytokine-, nitric oxide- or lysophosphatidylcholine (LPC)-induced VEGF synthesis in HMEC-1 and HVSMC. Interestingly, at the same concentrations statins upregulated VEGF generation in HUVEC. Furthermore, statins exerted dual, concentration-dependent influence on angiogenic activities of HUVEC as determined by tube formation assay. At low concentrations (0.03–1 ␮mol/l) the pro-angiogenic activity of statins is prevalent, whereas at higher concentrations statins inhibit angiogenesis, despite increasing VEGF synthesis. Conclusion: Our data show that statins exert concentration- and cell type-dependent effects on angiogenic activity of endothelial cells and on VEGF synthesis. The data are of relevance for elucidating the differential activity of statins on angiogenesis in cardiovascular diseases and cancer. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Statins; VEGF; Angiogenesis; HMG-CoA reductase; Nitric oxide

1. Introduction Statins are potent inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase via blocking the substrate accessibility to the enzyme and thereby effectively subverting cholesterol metabolism. Several large trials have revealed an impressive clinical benefit of statins which has led to a widespread use of these drugs in the primary and secondary prevention of coronary artery disease (for review see [1]). In the last 5 years compelling evidence has accumulated suggesting a beneficial effect of statins beyond their inhibition of cholesterol synthesis (for review see [2–4]). Indeed, for ∗ Corresponding author. Tel.: +48-12-252-63-75; fax: +48-12-252-69-02. E-mail address: [email protected] (J. Dulak). 1 These authors contributed equally to this study.

example, simvastatin and lovastatin have been reported to increase the half-life of the mRNA for eNOS. Atorvastatin, pravastatin and cerivastatin were able to scavenge oxygen derived free radicals and simvastatin and atorvastatin have been shown to decrease the precursor for endothelin-1 [2–4]. Thus, statins may exert directly vasculoprotective, perhaps cholesterol-independent effects. Vascular endothelial growth factor (VEGF) has been proven to be an important growth factor critical for blood vessel formation [5]. Recently, it has been suggested that statins may also modulate VEGF synthesis and consequently angiogenesis. Indeed, lovastatin at micromolar concentrations inhibited the production of VEGF in transformed fibroblasts [6] and cytokine-induced VEGF synthesis in rat vascular smooth muscle cells (VSMC) [7], while mevastatin suppressed the VEGF synthesis in rat primary aortic endothelial cells [8]. In contrast, statins at

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nanomolar concentrations have been recently demonstrated to enhance angiogenesis via activation of Akt kinase in human umbilical vein endothelial cells [9] or endothelial progenitor cells [10,11]. However, it is not known so far whether the decreasing effect is dependent on the dose of statins or associated to its efficacy on different cell types. Therefore, the aim of this study was to investigate whether the effect of statins on endothelial cell angiogenic activity and on VEGF expression is dependent on the concentration of the drug and/or on the cell type.

phatidylcholine, LPC) (3 ␮g/ml) [14]. VEGF synthesis was determined by ELISA in culture supernatant (for HVSMC and HMEC-1) or in the cell lysate (HUVEC) at 24 h after stimulation, while VEGF mRNA expression was measured by quantitative ELISA mRNA hybridization assay at 12 h after stimulation according to vendor’s protocol. Finally, we determined the effect of statins on angiogenic activity by measuring endothelial tube formation in a Matrigel matrix [9]. HUVEC seeded on Matrigel were treated for 12 h with VEGF (20 ng/ml) or statins or mevalonic acid and the number of fully formed polygons has been counted under microscope by observers blinded to the treatment.

2. Material and methods

2.4. Reverse transcription–polymerase chain reaction (RT–PCR)

2.1. Reagents Lovastatin was purchased from Calbiochem (Vienna, Austria), atorvastatin and simvastatin were gifts from Pfizer and MSD, respectively (Vienna, Austria). SNAP (S-nitroso-N-acetyl-d,l-penicillamine) was obtained from Alexis Biochemicals (Laufelfingen, Switzerland). Total RNA isolation kit, AMV reverse transcriptase and PCR core kit were purchased from Promega (Madison, USA). All other chemicals were obtained from Sigma (St. Louis, USA). Concentration of VEGF in the cell culture media or in cell lysates were measured using an ELISA kit for human VEGF. The quantitative measurements of human VEGF mRNA was done by ELISA mRNA quantitative assay and the amount of eNOS protein in endothelial cell lysate was determined by eNOS ELISA (all three ELISA kits were from R&D, Abingdon, UK). Lovastatin, simvastatin and mevalonic acid were activated by alkaline hydrolysis. 2.2. Cell culture Human coronary vascular smooth muscle cells (HVSMC) and human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics CellSystems (St. Katharinen, Germany). HVSMC were cultured in SmGM-2 medium supplemented with 5% FCS. HUVEC were cultured in EGM-2 medium containing 2.5% FCS and used between passages 2 and 4. Human microvascular endothelial cells (HMEC-1, Center for Disease Control and Prevention, Atlanta, USA) were cultured like HUVEC. 2.3. Experimental protocols Cells were cultured to confluence and starved for 12 h (HUVEC) or 24 h (HVSMC, HMEC-1) in medium containing 0.5% serum before treatment with different concentrations (0.01–10 ␮mol/l) of lovastatin, simvastatin or atorvastatin. HVSMC and HMEC-1 were additionally treated with several known inducers of VEGF, such as IL-1␤ (10 ng/ml) [12], SNAP (100 ␮mol/l) [13] or by lysophos-

Total RNA was isolated from the cells by acid guanidinum thiocyanate-phenol-chloroform extraction. Synthesis of cDNA was performed on 2 ␮g of total RNA with oligo-dT primers for 1 h at 42 ◦ C using AMV reverse transcriptase, according to vendor’s instruction. Then PCR with Taq polymerase was performed on cDNA for 35 cycles using the following protocol: 95 ◦ C–40 s, 58 ◦ C–40 s and 72 ◦ C–50 s. The primers recognizing VEGF (5 -CAC CGC CTC GGC TTG TCA CAT and 5 -CTG CTG TCT TGG GTG CAT TGG) and ␤-actin (5 -AGC GGG AAA TCG TGC GTG and 5 -CAG GGT ACA TGG TGG TGC) were used. PCR products were analyzed by electrophoresis in 2% agarose gel. The product length for the VEGF121 was 431 bp, for VEGF165 : 563 bp and for ␤-actin: 310 bp. 2.5. Statistical analysis All data are expressed as mean ± S.E.M. Statistical analysis were performed using ANOVA followed by post-hoc LSD test. Differences at P < 0.05 were considered as statistically significant. All experiments were repeated at least twice for each statin. All analyses were performed with the use of statistical software (SPSS for Windows, version 7.5.2G). 3. Results 3.1. Baseline synthesis of VEGF is cell-dependent (Fig. 1) Untreated HVSMC, cultured in 24-well plates (∼105 cells), generate during 24 h 100–200 pg of VEGF per ml of culture supernatant (Fig. 1). Under the same conditions, HMEC-1 produce only about 20 pg/ml, while no detectable VEGF was found in HUVEC conditioned media (Fig. 1). However, up to a few pg of VEGF could be detected in the cell lysates of unstimulated HUVEC (Fig. 1). RT–PCR demonstrated that VEGF mRNA expression is also cell-dependent (Fig. 1A) and related to the amount of VEGF protein detected by ELISA (Fig. 1B).

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Fig. 1. Production of VEGF by different vascular cell types. (A) The cell-dependent differences of unstimulated VEGF mRNA expression using RT–PCR. (B) An overview on VEGF synthesis measured in the supernatant of untreated HVSMC, HMEC-1 and HUVEC as well as in the HUVEC lysates. VEGF: vascular endothelial growth factor; HVSMC: human vascular smooth muscle cells; HMEC-1: human microvascular endothelial cell line; HUVEC: human umbilical vein endothelial cells.

3.2. Dual effects of statins on VEGF production and differentiation of endothelial cells Simvastatin at 1–3 ␮M enhanced VEGF synthesis, whereas very low concentrations (0.01 and 0.1 ␮mol/l)

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did not significantly influence the VEGF production by HUVEC (Fig. 2A). The induction of VEGF synthesis by statins was accompanied by an increase in eNOS expression, as shown for simvastatin (Fig. 2B). Then data were also confirmed by the quantitative mRNA ELISA

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Fig. 2. Dual effect of statins on VEGF synthesis in HUVEC. (A) The effect of different concentrations of simvastatin (Simv) on VEGF production determined in the HUVEC lysates. (B) The increase in VEGF synthesis is paralleled by an increase in eNOS expression. (C) VEGF mRNA increase in relation to the house-keeping gene expression (GAPDH) after 12 h of simvastatin (1 ␮mol/l) or atorvastatin (10 ␮mol/l) treatment (P = 0.08 vs. control). (D) The effect of atorvastatin (Atorv) on VEGF production in HUVEC lysate: ∗ P < 0.05 vs. control; # P < 0.01 vs. control.

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Fig. 3. Effect of statins on differentiation of endothelial cells. (A) Simvastatin at Acid) reverses this effect. (B) Four examples of the HUVEC-tube formation assay in cultures treated with high concentrations of simvastatin: # P < 0.001 vs. control;

0.1 ␮mol/l enhances tube formation, whereas mevalonic acid (Mev on Matrigel matrix. The arrows indicate the pycnotic cells observed § P < 0.001 vs. VEGF; † P < 0.05 vs. VEGF.

Fig. 4. Inhibitory effect of statins on basal and induced VEGF synthesis in HVSMC. (A and B) Decreased release of VEGF after lovastatin (Lov) and simvastatin (Simv) treatment in HVSMC. (C and D) Atorvastatin (Atorv) reduced SNAP (S-nitroso-N-acetyl-d,l-penicillamine) or IL-1␤-induced VEGF release in HVSMC: # P < 0.001 vs. control; ∗ P < 0.05 vs. control.

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Fig. 5. Statins decrease basal and induced VEGF synthesis in HMEC-1. (A and B) Diminished synthesis of VEGF after atorvastatin (Atorv) and lovastatin (Lov) treatment in HMEC-1. (C) Atorvastatin (Atorv) reduces SNAP induced VEGF synthesis. (D) Simvastatin (Simv) inhibits the lysophosphatidylcholine (LPC) induced VEGF expression: # P < 0.001 vs. control; ∗ P < 0.05 vs. control.

demonstrating increased amounts of VEGF mRNA after simvastatin and atorvastatin treatment (Fig. 2C). The stimulatory effect of simvastatin was the strongest at 1 ␮M (Fig. 2A), while atorvastatin potently augmented VEGF synthesis at a concentration of 3–10 ␮mol/l (Fig. 2D). The observed effect of statins on VEGF synthesis by HUVEC was not, however, paralleled by their influence on angiogenic activity of endothelial cells. HUVEC seeded on Matrigel did not form well organized tubes without stimulation (Fig. 3B, control). In turn, simvastatin enhanced tube formation at low concentrations (0.1 ␮mol/l) to a level similar as the induction by VEGF (Fig. 3A and B). This effect, however, was lost at higher concentrations (1–10 ␮mol/l), which was likely due to the toxicity of these high doses of simvastatin, as identified by the appearance of pycnotic cells (Fig. 3B). Mevalonic acid (100 ␮mol/l) reversed this stimulatory effect of simvastatin (0.1 ␮mol/l) on tube formation (Fig. 3A). Experiments with atorvastatin and lovastatin yielded similar results (data not shown).

3.3. Effect of statins on basal and stimulated VEGF synthesis in HVSMC and HMEC-1 Interestingly, in contrast to the stimulatory effect of statins on VEGF generation in HUVEC (Fig. 2), treatment with statins at concentrations 1–10 ␮mol/l decreased the basal release of VEGF in HVSMC (Fig. 4A and B). More importantly, statins reversed the IL-1␤ and SNAP-induced VEGF synthesis (Fig. 4B and D) without influencing HVSMC and HMEC-1 viability as detected by LDH measurement (data not shown). Also in HMEC-1 statins decreased the basal production of VEGF (Fig. 5A and B). In addition, all three statins reversed SNAP or LPC-induced VEGF synthesis, as shown for simvastatin and atorvastatin (Fig. 5C and D).

4. Discussion The crucial finding of this study is the demonstration of differential effects of statins on VEGF synthesis, which

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appear to be cell type dependent. At micromolar concentrations, statins inhibited the generation of VEGF in HVSMC and HMEC-1 which constitutively produce this growth factor. In contrast, HUVEC under basal conditions do not release VEGF and produce only very small amounts of intracellular stored VEGF. Interestingly, statins at micromolar concentration can enhance the formation of VEGF in HUVEC. This effect is, however, not paralleled by angiogenic activity of HUVEC. Regarding the latter activity of statins our data are in line with earlier observations [15,16] demonstrating a dual effect of statins, being pro-angiogenic at lower, nanomolar and inhibitory at higher, micromolar concentrations. A modulation of VEGF synthesis by statins has been shown recently in different angiogenic in-vitro models [6,8]. Indeed, very high concentrations of lovastatin (12 and 24 ␮mol/l) were reported to reverse the Ha-ras oncogene induced up-regulation of VEGF in NIH 3T3 cells [6]. Additionally, similar doses of mevastatin (25 ␮mol/l) have been shown to completely block bFGF-induced VEGF expression in cultured rat primary aortic endothelial cells [8]. Also, it has been observed that cerivastatin (0.5 ␮mol/l) diminished the VEGF synthesis in primary human dermal microvascular endothelial cells [15]. Our data show that basal and induced synthesis of VEGF in HVSMC and HMEC-1 were significantly decreased by statins, especially at higher concentrations of the drug. Recent data from our laboratories suggest that the inhibitory effect of statins on VEGF expression in HMEC-1 and VSMC may be due to the downregulation of transcription factors such as AP-1, HIF-1␣ and NF-␬B [17]. Those transcription factors may play a role in induced VEGF synthesis, hence their inhibition, and in consequence the attenuation of VEGF production, may be relevant in conditions of inflammation-induced angiogenesis. The in vitro inhibitory effect of statins on VEGF synthesis is strongly confirmed by recent in vivo data. Blann et al. have demonstrated that fluvastatin decreased VEGF in hypercholesterolemic patients [18], while we have shown that atorvastatin decreased VEGF plasma levels in CAD patients [19]. The observed anti-VEGF effect of statins may be of relevance for the therapy of coronary artery disease patients. Indeed, it has been demonstrated that angiogenesis is implicated in the pathogenesis of atherosclerosis [20]. Also, VEGF expression is potently induced by inflammatory factors including the modified lipids which have an important role in the development of atherosclerosis [14,21,22]. Several studies have demonstrated that VEGF expression is increased in atherosclerotic lesions in human coronary arteries [23] and recent data strongly indicate that at least in a hypercholesterolemic setting VEGF may be pro-atherogenic [24,25]. Consequently, one could speculate that VEGF production may be enhanced or dysregulated in atherosclerosis, which can be efficiently blocked by statin

therapy. This may result in attenuation of plaque growth and/or stabilisation of plaque. Interestingly, the experiments performed on HUVEC revealed a different effect of statins on VEGF synthesis. Lower micromolar concentrations (1–3 ␮mol/l) of simvastatin significantly increased intracellular VEGF in HUVEC, whereas a stimulatory effect of atorvastatin appeared at higher concentrations (3–10 ␮mol/l; Fig. 2A and D). The observed upregulation of VEGF synthesis in HUVEC was associated with an increase in eNOS expression. As nitric oxide induces VEGF expression in VSMC [12,13,26], the enhancement of VEGF synthesis in HUVEC could be at least partially due to a NO-dependent pathway (Dulak et al., unpublished data). The stimulatory effect of statins on VEGF expression in HUVEC, however, did not parallel their recently reported pro-angiogenic activity [10,11,15,16]. In our study simvastatin was pro-angiogenic at low concentrations (0.1 ␮mol/l) as assessed in the Matrigel assay (Fig. 3A), but VEGF production was not enhanced at this concentration of the drug (Fig. 2A). Interestingly, higher concentrations (1–10 ␮mol/l) of statins induced VEGF synthesis (Fig. 2A and D) but they did not promote tube formation in the Matrigel assay (Fig. 3). It might be speculated that the stimulatory influence of statins on VEGF synthesis in HUVEC reflects a kind of unspecific protective activity induced under stress conditions. A similar effect has been observed when acidosis inhibited proliferation and migration of cultured endothelial cells, but simultaneously increased expression of VEGF and bFGF [27]. It can be suggested that under stressful conditions VEGF may at least partially protect endothelial cells from apoptosis. Our data on angiogenic activity of statins in HUVEC are in agreement with two recent papers demonstrating dual (biphasic) effects of those drugs on angiogenesis in HUVEC and HMEC-1 [15,16]. First, Weis et al. [15] showed that cerivastatin and atorvastatin enhanced proliferation, migration and differentiation of HMEC at low concentrations (0.005–0.01 ␮mol/l) but inhibited those events at higher concentrations (0.05–1 ␮mol/l). Additionally, they demonstrated that the inhibitory effect at higher concentrations of statins was associated with increased apoptosis and was reversed by geranylgeranyl pyrophosphate suggesting an involvement of isoprenoids in the inhibiton of angiogenesis [15]. In the second relevant study, Urbich et al. [16] demonstrated that atorvastatin or mevastatin up to 0.1 ␮mol/l promoted migration of mature macrovascular endothelial cells (HUVEC) and endothelial progenitor cells as well as tube formation. Higher concentrations, however, inhibited angiogenesis and migration by inducing endothelial cell apoptosis. The pro-angiogenic effect of statins was also attributed to the stimulation of PI3/Akt kinase pathway [9] followed by eNOS activation [28,29]. Both PI3/Akt [30] and eNOS activity [12,13] can augment VEGF synthesis. Therefore, one can speculate that the different effects of statins on VEGF

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synthesis in VSMC and HMEC-1 in comparison to HUVEC might be due to different effects on Akt kinase in different cell types. Indeed, activation of Akt by statins has been observed in macrovascular endothelial cells but inhibition occurred in SMC [31,32]. Among endothelial cells, statins have been shown to enhance Akt in macrovascular while they inhibited its phosphorylation in microvascular cells [28]. Atorvastatin has been shown to enhance eNOS phosphorylation in HUVEC but not in microvascular endothelial cells [28]. Consequently, atorvastatin promoted tube formation in macro- but not microvascular endothelial cells [28]. Thus, according to endothelial cell type, the extent of Akt/eNOS interaction may vary dramatically and the effect of statins on eNOS activators do likely differ among endothelial beds [28]. It remains to be established whether those findings are related to the influence of statins on VEGF synthesis, being stimulatory in HUVEC and inhibitory in HMEC-1. The extrapolation of the pro-angiogenic effect of statins to potential clinical applications should be tempered, however, by the still uncertain role of angiogenesis in the development and progression of atherosclerosis. Further studies in different models of atherosclerosis should give more insight into the effects of VEGF on plaque stability or progression before this growth factor can be recommended for therapeutic use in patients.

Acknowledgements We are grateful to Prof. Aleksander Koj for useful comments. J. Dulak was a recipient of a fellowship from the Austrian Society of Cardiology (1999–2001). The study was in part supported by the Grant 3 PO4 049 22 awarded by the Polish State Committee for Scientific Research and by the Polish–Austrian Collaborative Grant (17/2002).

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