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. Author manuscript; available in PMC: 2011 Mar 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2009 Dec 30;30(3):554–560. doi: 10.1161/ATVBAHA.109.201087

BMPER is upregulated by statins and modulates endothelial inflammation by ICAM-1

Thomas Helbing 1, René Rothweiler 1, Jennifer Heinke 1,3, Lena Goetz 1, Philipp Diehl 1, Andreas Zirlik 1, Cam Patterson 2, Christoph Bode 1, Martin Moser 1
PMCID: PMC2853692  NIHMSID: NIHMS172812  PMID: 20042706

Abstract

Objective

Besides cholesterol lowering statins exert pleiotropic effects on endothelial cells. Bone morphogenetic proteins (BMPs) have recently been implicated in vascular inflammation and disease. We set out to investigate the effect of statins on BMPER, a novel member of the BMP pathway.

Methods and Results

Mevastatin enhanced BMPER expression in cultured endothelial cells in a time and concentration dependent manner as determined by immunocytochemistry, RT-PCR, and western blotting. Similar effects were observed in vitro and in vivo using simvastatin.

Actinomycin D chase analysis and BMPER promoter reporter assays revealed that this is mostly a posttranscriptional event resulting in prolonged BMPER RNA half-life. We confirmed that the RhoA/Rock/actin pathway is involved using the specific pathway activator CNFY that prevented upregulation of BMPER expression by mevastatin and pathway inhibitors (C3-toxin, RhoAN19 mutant, fasudil, cytochalasin D) that enhanced BMPER expression.

Increasing concentrations of BMPER exert antiinflammatory features in endothelial cells as reflected by ICAM-1 downregulation. Accordingly, silencing of BMPER enhances ICAM-1 expression. Furthermore mevastatin reduced the expression of proinflammatory BMP4, a well known direct interaction partner of BMPER.

Conclusion

Mevastatin modulates the BMP pathway by enhancing BMPER via the RhoA/Rock/actin pathway as well as by reducing BMP4 expression. BMP4 down- and BMPER upregulation contribute to the antiinflammatory pleiotropic effects of statins.

Keywords: Angiogenesis, Vascular biology

Introduction

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily. BMPs are important regulators in blood vessel formation and vascular disease 1. BMP2 and BMP4 are upregulated in athero-prone regions in blood vessels, induce 2, 3 a proinflammatory endothelial phenotype and may contribute to the development of atherosclerotic plaques and vascular calcification 4, 5. Infusion of BMP4 in vivo leads to endothelial dysfunction and arterial hypertension 6, 7. Important insights also came from the discovery of mutations of the BMP receptors in patients with familial pulmonary artery hypertension or teleangiectasia 8.

BMP endothelial cell precursor-derived regulator (BMPER) is a secreted glycoprotein that binds directly to BMPs and modulates their function in a dose dependent fashion. In gain of function assays BMPER behaves as a BMP-antagonist 9, 10, whereas in loss of function models BMPER may also exert pro-BMP functions 11–14. BMPER was originally identified in a screen for differentially expressed proteins in embryonic endothelial precursor cells 9. In mouse and zebrafish, it is expressed at sites and at the time of vasculogenesis consistent with a regulatory role for BMPER in vascular events. When BMPER is inactivated in zebrafish embryos intersomitic angiogenesis is severely perturbed 11. Consistent with this vascular phenotype BMPER may confer proangiogenic activity in endothelial cells in a dose-dependent fashion 15. Taken together, BMPER acts as a context dependent BMP modulator and is essential for BMP4 function in endothelial cells 15.

It has been shown that BMP4 exerts its proinflammatory effects by increased NF-kB activation and induction of ICAM-1 16, 17. ICAM-1 is an adhesion molecule that is expressed on the endothelium and leukocytes and is upregulated in inflammation by proinflammatory cytokines like TNF-α, IL-1β, IFN-γ 18. Increased expression of ICAM-1 was identified in all subtypes of atherosclerotic lesions and is involved in the recruitment of monocytes to the lesion, as suggested by its role in the entry of leukocytes into foci of inflammation. Along the same lines, ICAM-1 enhanced monocyte recruitment is a potential mechanism for the growth of an atherosclerotic plaque 19. Therefore it is important to understand the regulation of ICAM-1 on the endothelial surface and to identify regulators of ICAM-1 expression because of their potential in the treatment of vascular inflammation.

In addition to their ability to lower plasma cholesterol level statins have been shown to decrease ICAM-1 expression in endothelial cells 20, 21. They possess anti-atherogenic properties by improving endothelial function, stabilizing atherosclerotic plaques, reducing oxidative stress as well as endothelial inflammation and thrombogeneity 22. Therefore statins are used in the primary and secondary prevention of cardiovascular disease. By inhibition of the 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase statins block the conversion of HMG-CoA to mevalonate and cause a depletion of isoprenoids such as mevalonate, farnesylpyrophosphate (FPP), and geranylgeranylpyrophosphate (GGPP). These isoprenoids serve as important lipid anchors for the posttranscriptional modification of small GTPases such as Ras, Rho, Rac and Rap by isoprenylation. Small GTPases are involved in cell signalling and perturbed isoprenylation of small GTPases by statins mediates anti-inflammatory effects partially by downregulation of proinflammatory BMP2 6.

In this manuscript we identify the extracellular BMP modulator BMPER as a new mediator of antiinflammatory effects of statins in endothelial cells.

Methods

Reagents, antibodies, cell culture, immunocytochemistry, transfection of promoter constructs, luciferase assays, RT-PCR, quantitative Real-Time PCR, siRNA transfection, western blotting and animal procedures are described in the online data supplement.

Statistical analysis and quantification

Statistical analyses were performed using GraphPad Prism 4.0. Data are presented as mean±SD, and comparisons were calculated by Student’s t-test (2-sided, unpaired). Results were considered statistically significant when p<0.05. Densitometric analysis of Western blots was performed using Quantity One 1-DAnalysis Software (version 4.4, Bio-Rad) and levels of significance were calculated by one-sample t-test.

Results

Mevastatin upregulates BMPER expression in endothelial cells

In order to test the hypothesis that statins may exert pleiotropic effects by regulating BMPER we treated HUVECs with mevastatin. Indeed, as visualized by immunocytochemistry and quantified by western blotting, mevastatin increased BMPER protein expression (Figure 1A&B). Treatment with 10μM mevastatin for 24h resulted in a four fold upregulation of BMPER protein. Similar results were obtained for BMPER RNA levels in a concentration and time dependent manner (Figure 1C&D). To investigate if BMPER regulation was confined to mevastatin or if other statins have similar properties we tested simvastatin and pravastatin. Indeed, these compounds also increased BMPER RNA levels in vitro suggesting a class effect of statins on BMPER regulation (Figure 1E). These data were confirmed in vivo by treating C57/BL6 mice with subcutaneous injection of simvastatin for 14 days. In simvastatin treated animals BMPER RNA levels were upregulated in the lungs compared to control. These data clearly demonstrate that statins increase BMPER expression in vitro and in vivo.

Figure 1. Inhibition of endothelial HMG-CoA reductase upregulates BMPER expression in endothelial cells.

Figure 1

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(A) After 24 hours mevastatin (10μM) treatment, BMPER expression is increased in HUVECs showed by immuncytochemistry (right panel) compared to negative control with corresponding serum (left panel). Nuclei were stained with DAPI. (B) After 24h mevastatin (10μM) HUVECs were lysed and used for western blot with the indicated antibody. β-tubulin was used as a loading control. Expression was quantified by densitometric analysis of three independent experiments. (C&D) BMPER RNA expression depends on concentration (C) and duration (D) of mevastatin treatment shown by RT-PCR (gel) and by RT-qPCR (bar graph) of three independent experiments. RNA expression was analyzed by specific primers for human BMPER and human RNA polymerase II (hRPII). (E) Simvastatin increased BMPER RNA level in HUVECs in vitro. (F) C57/BL5 mice were treated with activated simvastatin (n=5) s.c. or PBS (n=5) as control for 14 days. Lungs were isolated and RNA was prepared and used for qPCR (*= p<0.05 vs. control).

Mevastatin increases BMPER expression by posttranscriptional modification

To analyze the mechanism how statins regulate BMPER expression we pursued two separate approaches. First, we tested the effect of mevastatin on two BMPER promoter constructs of different size that contain luciferase as a reporter of BMPER promoter activity. Krüpple-like factor (KLF) 15, a known activator of the BMPER promoter was used as positive control 23. As demonstrated in Figure 2A both BMPER promoter constructs did not respond to mevastatin suggesting a regulatory mechanism that is independent from the promoter. In the second set of experiments we pretreated HUVECs with actinomycin D, an inhibitor of de novo transcription (Figure 2B). When these cells, in which RNA content depends completely on RNA degradation, were exposed to mevastatin we found that BMPER RNA half-life was prolonged compared to cells that were not exposed to mevastatin. This indicates that mevastatin stabilized BMPER RNA. Taken together these data cannot completely exclude transcriptional regulation but strongly suggest a posttranscriptional mechanism of regulation.

Figure 2. Mevastatin increases BMPER levels by RNA half life prolongation.

Figure 2

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(A) Mevastatin does not induce BMPER promoter activity in endothelial cells. Cells were transfected with the respective promoter construct and luciferase activity was quantified after 24h mevastatin (10μM) treatment. KLF15 was used as a positive control for BMPER promoter activation. Values represent the mean +/− SD of three independent experiments normalized to β-galactosidase. (B) After 24h preincubation with or without mevastatin HUVECs were treated with de novo transcription inhibitor Actinomycin D (ActD) for indicated times. RNA levels were normalized to the RNA level at 0h of the same group. Each experiment was performed at least three times with similar results (*= p<0.05 vs. ActD control).

Mevastatin mediated induction of BMPER expression is dependent on isoprenoid intermediates

Statins inhibit the HMG-CoA reductase and cause a depletion of downstream isoprenoids such as mevalonate, geranylpyrophosphate (GPP), farnesylpyrophosphate (FPP) or geranylgeranylpyrophosphate (GGPP) in the cells 24. To determine which downstream isoprenoid in the cholesterol biosynthetic pathway regulates BMPER expression HUVECs were exposed to mevastatin alone or in combination with individual downstream isoprenoids. As shown in Figure 3A supplementation of mevastatin treated cells with mevalonate completely reversed the mevastatin dependent induction of BMPER confirming the specificity of the mevastatin effect. Similarly the mevastatin effect could be reversed using GPP, FPP and GGPP, whereas the respective isoprenoid alone had no effect (Figure 3B&C). These data underline that the statin mediated BMPER upregulation is dependent on the cholesterin synthesis pathway and demonstrate that all tested isoprenoids are able to reverse the mevastatin effect on BMPER.

Figure 3. Mevastatin induced BMPER expression is reversed by isoprenoids in endothelial cells.

Figure 3

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HUVECs were treated with mevastatin alone or in combination with mevalonate (A), FPP (A), GPP (B) and GGPP (C) for 24h. Total RNA was harvested and assessed for BMPER expression by RT-PCR. hRPII was used as loading control. One representative gel out of three independent experiments with similar results is shown. Additionally, corresponding quantitative PCRs were performed (C) and results are shown as bar graphs. (*= p<0.05 vs. control or #= p<0.05 vs. mevastatin).

RhoA is involved in BMPER regulation in endothelial cells

The isoprenoids FPP and GGPP have regulatory roles in a number of signaling cascades such as the Ras or the Rho pathway. Since Rho is a major target of geranylgeranylation, inhibition of Rho and its downstream target, Rho-kinase, mediates some of the pleiotropic effects of statins on the vascular wall 24, 25. As a consequence we hypothesized that activation of RhoA reverses mevastatin induced BMPER expression. Therefore we tested if activation of RhoA using a direct and highly specific RhoA activator (cytotoxic necrotizing factor of yersinia pseudotuberculosis [CNFY]) may reverse the statin effect 26. As expected mevastatin increased BMPER RNA level to 241% compared to basal level whereas CNFY alone reduced BMPER RNA level to 71%. Co-treatment of cells with mevastatin and CNFY completely reversed the upregulation of BMPER RNA by mevastatin (Figure 4A). These findings indicate that the statin-mediated inhibition of RhoA contributes to the increased BMPER expression.

Figure 4. Mevastatin mediated upregulation of BMPER depends on RhoA and Rho-kinase activity.

Figure 4

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(A) Specific activation of the Rho pathway by CNFY prevents mevastatin induced BMPER upregulation. HUVECs were treated with CNFY (400ng/ml) and Mevastatin (15μM) alone or in combination. RT-qPCR was performed to quantify BMPER RNA levels. hRPII was used as loading control. (*= p<0.05 vs. control or #= p<0.05 vs. mevastatin). (B–E) Inhibition of the Rho pathway enhances BMPER expression. (B) The Rho inhibitor C3 toxin augments BMPER RNA levels. HUVECs were treated with C3 toxin (250ng/ml) for 24h. (C) Overexpression of the dominant negative RhoA (RhoN19) mutant in HUVECs increases BMPER RNA levels compared to control (empty vector). (D&E) Rock inhibitor fasudil upregulates BMPER RNA and protein expression in endothelial cells. HUVECs were treated with fasudil (50μM) for 24h before cells were harvested for RNA or protein analysis. (F) Disruption of actin cytoskeleton increases BMPER RNA level in endothelial cells. HUVECs were incubated with the actin cytoskeleton disruptor cytochalasin D (5μM) for 8h (*= p<0.05 vs. control).

To confirm these results we tested the effect of RhoA inhibition on BMPER. Therefore HUVECs were co-incubated with clostridium botulinum C3 transferase, an exotoxin that inactivates Rho by ADP-ribosylation 27. Indeed as shown in Figure 4B treatment of HUVECs with the C3 toxin for 24 hours augmented BMPER RNA level to 140%. To increase the specificity we overexpressed a dominant negative RhoA mutant (RhoA19N) in HUVECs (Figure 4C). Specific inhibition of RhoA resulted in upregulation of BMPER RNA. These findings demonstrate that RhoA inhibition increases BMPER expression to a similar extent as inhibition of Rho GTPases by mevastatin. Taken together, these findings indicate that RhoA is an important regulator of BMPER RNA levels in endothelial cells and that the statin-mediated inhibition of geranylgeranylation of RhoA is responsible for the increased BMPER expression.

Inhibition of Rock increases BMPER RNA in endothelial cells

Rho-associated coiled-coil containing protein kinase Rho-kinase (Rock) is an important downstream target of RhoA activity 24, 28. To determine the involvement of Rock in BMPER expression, endothelial cells were incubated with the specific Rock- inhibitor fasudil for 24h. Confirming our hypothesis, fasudil upregulates BMPER RNA levels (246%) and protein levels (134%) compared to control (Figure 4D&E). These findings support the notion that the RhoA/Rock pathway is involved in the regulation of BMPER RNA levels.

Disruption of the endothelial actin cytoskeleton increases BMPER expression

Rock phosphorylates various targets and mediates a range of cellular responses that involve the assembly of the actin cytoskeleton. The amount of actin stress fibers and the reorganisation of the cytoskeleton are mediated in part by Rocks. To address the question if this downstream step of RhoA/Rock signaling is also involved in BMPER regulation, we treated endothelial cells with cytochalasin D, a well characterized disruptor of the actin cytoskeleton, for 8 hours (Figure 4F). Cytochalasin D causes a strong increase in BMPER RNA levels to 338% compared to control suggesting that the actin cytoskeleton is indeed involved in the regulation of BMPER expression.

Mevastatin downregulates proinflammatory BMP4 in endothelial cells

Having demonstrated that mevastatin upregulates BMPER we asked if other BMP pathway members are also regulated by statins. We decided to focus on BMP4 because we have shown earlier that BMPER interacts directly with BMP4 and because BMP4 is a known inducer of vascular inflammation 17. In contrast to BMPER, BMP4 was markedly downregulated by mevastatin (Figure 5A&B). This finding is consistent with the notion that downregulation of BMP4 contributes to the antiinflammatory effect of statins.

Figure 5. Mevastatin downregulates BMP4.

Figure 5

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(A&B) HUVECs were incubated with mevastatin. BMP4 RNA (A) and BMP4 protein (B) were quantified at 24h and 48h respectively. These experiments were performed three times with comparable results (*= p<0.05 vs. control). BMPER regulates ICAM-1 expression. (C) Serum-starved HUVECs were treated with or without BMPER with the indicated concentrations for 24h before RNA was harvested, reverse transcribed and used for RT-qPCR to quantify ICAM-1. (D) HUVECs were stimulated with TNFα (2ng/ml) and increasing concentration of BMPER. After 8 hours cells were harvested and used for western blotting (*= p<0.05 vs. control). (E&F) Specific silencing of BMPER in endothelial cells by two different siRNAs results in increased ICAM-1 expression. (E) Two different siRNAs for BMPER or control siRNA were transfected in HUVECs. After 48 hours protein and RNA was prepared. Sufficient BMPER knock-down at the protein level is shown by western blotting compared to tubulin as the loading control (top panels). ICAM-1 expression is quantified by RT-qPCR as shown in the bar graph. Three independent experiments were quantified (*= p<0.05 vs. control). (F) After silencing of BMPER expression HUVECs were treated with mevastatin (10μM) for 24 h and RNA was used for quantitative Real-Time PCR to analyze ICAM-1 RNA expression (*= p<0.05 vs. control). (G) After silencing of BMPER in HUVECs ICAM-1 protein expression (right panel) is increased compared to control (left panel) shown by immuncytochemistry. DAPI is used for staining of nuclei.

BMPER modulates ICAM-1 expression

Next we asked if regulation of BMPER by statins is also involved in vascular inflammation. To quantify vascular inflammation we chose ICAM-1 expression as a surrogate marker. When we added BMPER to HUVECs ICAM-1 RNA was downregulated (Figure 5C). To investigate if BMPER was capable to antagonize proinflammatory stimuli, we coincubated HUVECs with tumor necrosis factor alpha (TNF-α), a strong inducer of endothelial inflammation, and with increasing concentrations of BMPER. Indeed, BMPER inhibited TNF-α-induced ICAM-1 expression in a concentration dependent manner (Figure 5D). To confirm these findings we silenced BMPER in HUVECs. Two different siRNAs designed to target BMPER were used in these experiments. Consistent with an inhibitory role of BMPER on ICAM-1 expression we found that ICAM-1 RNA and protein is increased in BMPER silenced endothelial cells (Figure 5E&G). In our hands siBMPERII consistently reached higher knockdown efficiencies than siBMPERI and consequently more pronounced effects on ICAM1 were observed using siBMPERII. To analyze if BMPER is a mediator of ICAM-1 regulation in the presence of statins HUVECs were transfected with siBMPER and treated with mevastatin for 24h. As expected silencing of BMPER increased ICAM-1 expression in mevastatin treated cells (Figure 5F).

Taken together, these data suggest that statins reduce vascular inflammation by interfering with the BMP pathway at two ends. First they downregulate proinflammatory BMP4 and second they increase BMPER for which we suggest an antiinflammatory role by its suppressing activity on ICAM-1 expression.

Discussion

In this study, we characterize the BMP modulator BMPER as a novel pleiotropic target of statins in endothelial cells and present novel findings with regard to the regulation of BMPER and the BMP pathway: First, HMG-CoA reductase inhibitors upregulate BMPER expression in vitro and in vivo. Second, upregulation of BMPER expression by mevastatin is a post-transcriptional event. Third, the mevastatin effect on BMPER involves inhibition of the RhoA/Rock/actin pathway. Forth, mevastatin differentially regulates BMPER and BMP4 and thereby inhibits vascular inflammation as reflected by ICAM-1.

BMPER is an extracellular BMP modulator that is expressed by endothelial cells. Data from drosophila, xenopus, zebrafish, chicken and mouse reveal that BMPER is necessary to sharpen BMP gradients and that its activity is sensitive to dose changes 9, 12–14, 29–32. At low doses BMPER is needed to enhance BMP4 activity, but at higher doses BMPER increasingly inhibits BMP4 activity 9, 15. Therefore detailed understanding of BMPER regulation is crucial to control BMP effects.

Here we present data demonstrating that mevastatin increases BMPER expression in endothelial cells (Figure 1). Until now, this is the first drug that has been shown to increase BMPER expression. Pleiotropic effects of statins are frequently controlled by posttranscriptional events rather than by control of gene promoter activity 25, 33. This is also the case for BMPER. Although our data cannot completely rule out transcriptional modification of BMPER expression by statins they strongly suggest a posttranscriptional effect; resulting in prolongation of BMPER RNA half-life (Figure 2). The isoprenoids downstream of HMG-CoA reductase are important modulators of small GTPases. E.g., inhibition of Rho geranylgeranylation and membrane translocation of Rho by mevastatin leads to a greater accumulation of inactive Rho in the cytoplasm 25. Since supplementation of GGPP could reverse the statin effect on BMPER we hypothesized that the Rho/Rock/actin pathway was involved in BMPER regulation (Figure 3). Indeed, specific activation of this pathway by CNFY reduced BMPER expression, whereas pathway inhibition by either a specific RhoA inhibitor (C3-transferase toxin), a dominant negative RhoA mutant (RhoA19N) or a Rock inhibitor (fasudil) results in up-regulation of BMPER (Figure 4). Similarly, inhibition of downstream actin cytoskeleton assembly increases BMPER RNA. Taken together this is compelling evidence that the RhoA/Rock/actin pathway plays a pivotal role in mevastatin mediated BMPER expression. Remarkably, this is a very similar mechanism to the regulation of eNOS by statins 25, 34, 35. The mechanistic data dissecting the RhoA/Rock/actin pathway of BMPER activation is of great value as recently specific inhibitors of Rock have emerged as novel therapeutic strategies to treat vascular dysfunction and its longterm consequences such as atherosclerosis or pulmonary artery hypertension 36, 37. Our data demonstrate that Rock inhibition in addition to the well described consequences for eNOS also modifies the BMP pathway.

Modification of the BMP pathway has a major impact on vascular inflammation. BMP4 exerts prooxidant, proinflammatory and prohypertensive effects on endothelial cells and is involved in vascular calcification. By downregulation of BMP4 expression statins decrease BMP activity which mediates antiinflammatory, antiatherogenic and vasculoprotective effects 17, 38. Another new target of statins within the BMP pathway is BMPER. Here we show that high levels of BMPER modulates expression of adhesion molecules on endothelial cells. This is demonstrated by downregulation of ICAM-1, a marker of endothelial cell activation and inflammation (Figure 5). Thus statins modulate the BMP pathway at different levels: they downregulate BMP4 and at the same time they upregulate the dose dependent BMP4 modulator BMPER together resulting in a strong antiinflammatory activity.

In conclusion, we demonstrate that BMPER is upregulated by mevastatin via posttranscriptional modification involving the RhoA/Rock/actin pathway. At the same time BMP4 is downregulated by mevastatin. This dual modification of the BMP pathway results in decreased vascular inflammation and thus represents a so far unknown antiinflammatory effect of statins.

Supplementary Material

Supp1

Acknowledgments

Funding

This work was supported by Deutsche Forschungsgemeinschaft SFB-TR23 (A1) to M.M.

We are indebted to Bianca Engert for her outstanding technical assistance. We thank Klaus Aktories (Freiburg) for kindly providing CNFY and C3 transferase toxin and Keith Burridge (Chapel Hill) for sharing the dominant negative RhoN19 mutant.

Footnotes

Conflict of interest

None declared

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