Simvastatin, a Novel Stimulator of Eryptosis, the Suicidal Erythrocyte Death

Simvastatin, a 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase inhibitor decreasing cholesterol synthesis [1], is widely used for the treatment of dyslipidemia with hypercholesterolemia [2-15]. Further putative therapeutic effects include decrease of hippocampal amyloid-beta abundance in Alzheimer’s disease [16], anticoagulation [10], inhibition of inflammatory disease, such as asthma [1, 17-19], protection against contrast-induced nephropathy [20], protection of cardiac allografts against ischemia/reperfusion injury [21], as well as counteraction of osteoporosis and support of bone fracture healing [22, 23]. Side effects of simvastatin include rhabdomyolysis [6, 8, 24-29] and thrombocytopenia [30].

Simvastatin has been shown to stimulate apoptosis of neurons [31] and tumor cells [32-39]. In view of its effect in tumor cells simvastatin was considered as a potential therapeutic option in malignancy [32, 35-37]. On the other hand, simvastatin may inhibit apoptosis of osteoblasts [22, 40].

Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis [41], the suicidal death of erythrocytes characerized by cell shrinkage [42] and by cell membrane scrambling with phosphatidylserine translocation to the outer face of the erythrocyte [41, 43] and release of exosomes [44]. Signaling stimulating eryptosis includes increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) [41, 45], ceramide [46], caspases [41, 47, 48], G-protein Galphai2 [49], casein kinase 1α [41, 50], Janus-activated kinase JAK3 [41, 51], protein kinase C [41, 52], and p38 kinase [41, 53]. Eryptosis is suppressed by AMP activated kinase AMPK [41, 54], cGMP-dependent protein kinase [41, 55], mitogen and stress activated kinase MSK1/2 [56], PAK2 kinase [41, 57] and sorafenib/sunitinib sensitive kinases [41, 58, 59]. Eryptosis could be triggered by a wide variety of cell stressors including hyperosmotic shock [41], oxidative stress [41], energy depletion [41], and a myriad of xenobiotics [41, 60-117]. Enhanced eryptosis is observed in multiple clinical conditions including iron deficiency [41], dehydration [118], hyperphosphatemia [119], vitamin D excess [70], chronic kidney disease (CKD) [120-124], hemolytic-uremic syndrome [125], diabetes mellitus [126], hepatic failure [94, 127], malignancy [128, 129], arteritis [130], sepsis [131], sickle-cell disease [41], beta-thalassemia [41], Hb-C and G6PD-deficiency [41], and Wilsons disease [132]. Eryptosis is further enhanced in elderly individuals [133], sensitive to erythrocyte age [107], and increased following extended storage of erythrocytes for transfusion [134].

To possibly uncover an effect of simvastatin on eryptosis, human erythrocytes from healthy volunteers were exposed to simvastatin and phosphatidylserine surface abundance, cell volume, [Ca²⁺]_i, reactive oxygen species (ROS) and ceramide abundance determined by flow cytometry.

Fresh Li-Heparin-anticoagulated blood samples were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003 V). The blood was centrifuged at 120 g for 20 min at 21 °C and the supernatant with platelets as well as leukocytes was disposed. Erythrocytes were incubated in vitro at a packed cell volume of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO₄, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; pH 7.4), 5 glucose, 1 CaCl₂,at 37°C for 48 h. Where indicated, erythrocytes were exposed for 48 h to simvastatin (Sigma Aldrich, Hamburg, Germany). In order to estimate the impact of Ca²⁺ entry on simvastatin induced eryptosis, erythrocytes were exposed to simvastatin in the absence of extracellular Ca²⁺. To test for an involvement of p38 kinase, erythrocytes were exposed for 48 hours to a combination of simvastatin and p38 kinase inhibitor SB203580 (Tocris bioscience, Bristol, UK).

After incubation under the respective experimental condition, a 150 µl cell suspension was washed in Ringer solution containing 5 mM CaCl₂ and then stained with Annexin-V-FITC (1: 200 dilution; ImmunoTools, Friesoythe, Germany) in this solution at 37°C for 15 min under protection from light. The annexin-V-abundance at the erythrocyte surface was subsequently determined on a FACS Calibur (BD, Heidelberg, Germany). Annexin-V-binding was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. A marker (M1) was placed to set an arbitrary threshold between annexin-V-binding cells and control cells. The same threshold was used for untreated and simvastatin treated erythrocytes. The threshold of forward scatter was set at the default value of “52”.

After incubation, erythrocytes were washed in Ringer solution and loaded with Fluo-3/AM (Biotium, Hayward, USA) in Ringer solution containing 5 mM CaCl₂ and 5 µM Fluo-3/AM. The cells were incubated at 37°C for 30 min. Ca²⁺-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur. Afterwards, the geomean of the Ca²⁺ dependent fluorescence was determined.

Oxidative stress was determined utilizing 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA). After incubation, a 150 µl suspension of erythrocytes was washed in Ringer solution and stained with DCFDA (Sigma, Schnelldorf, Germany) in Ringer solution containing DCFDA at a final concentration of 10 µM. Erythrocytes were incubated at 37°C for 30 min in the dark and washed two times in Ringer solution. The DCFDA-loaded erythrocytes were resuspended in 200 µl Ringer solution and ROS-dependent fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD). Subsequently, the geomean of the DCFDA dependent fluorescence was determined.

For the determination of ceramide, a monoclonal antibody-based assay was used. To this end, cells were stained for 1 h at 37°C with 1 µg/ml anti ceramide antibody (clone MID 15B4, Enzo Life Science GmbH, Lörrach, Germany) in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1: 10. The samples were washed twice with PBS-BSA. The cells were stained for 30 min with polyclonal fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG and IgM specific antibody (Pharmingen, Hamburg, Germany) diluted 1: 50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. The samples were analyzed by flow cytometric analysis with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. As a control, secondary antibody alone was used.

For the determination of hemolysis, the samples were centrifuged (3 min at 1600 rpm in room temperature) after incubation, and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis.

Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as post-test and t test as appropriate. n denotes the number of different erythrocyte specimens studied. Since different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.

In order to test, whether simvastatin modifies eryptosis, erythrocytes drawn from healthy individuals were exposed for 48 h to Ringer solution without or with simvastatin (0.5 – 2 µg/ml). Eryptotic erythrocytes were identified from cell shrinkage and cell membrane scrambling.

Cell volume was estimated from forward scatter in flow cytometry. As illustrated in Fig. 1A,B, a 48 h exposure to simvastatin decreased the forward scatter, an effect reaching statistical significance at 1 µg/ml simvastatin concentration. Thus, simvastatin triggered cell shrinkage. Along those lines, at all simvastatin concentrations tested, simvastatin significantly increased the percentage of shrunken erythrocytes (Fig. 1C) and significantly decreased the percentage of erythrocytes with large volumes (Fig. 1D).

Cell membrane scrambling with phosphatidylserine translocation to the cell surface was evidenced from annexin-V-binding to phosphatidylserine, as determined by flow cytometry. As illustrated by Fig. 2A,B, a 48 h exposure to simvastatin increased the percentage of phosphatidylserine exposing erythrocytes, an effect reaching statistical significance at 1 µg/ ml simvastatin.

Eryptosis could be triggered by increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i). Fluo3 fluorescence was thus employed in order to quantify [Ca²⁺]_i. As illustrated in Fig. 3A,B, a 48 h exposure to simvastatin increased the Fluo3 fluorescence, an effect reaching statistical significance at 2 µg/ml simvastatin.

In order to test, whether the increase of [Ca²⁺]_i required entry of extracellular Ca²⁺, erythrocytes were incubated for 48 h in the absence or presence of 2 µg/ml simvastatin in the presence or nominal absence of extracellular Ca²⁺. As illustrated in Fig. 4, removal of extracellular Ca²⁺ tended to blunt the effct of simvastatin on [Ca²⁺]_i, an effect, however, not reaching statistical significance. Exposure of erythrocytes for 48 h to simvastatin (2 µg/ml) significantly increased the Fluo3 fluorescence both, in the presence and nominal absence of extracellular Ca²⁺.

As illustrated in Fig. 5, a 48 h treatment of erythrocytes with 2 µg/ml of simvastatin deceased the forward scatter significantly in both, the presence and absence of extracellular Ca²⁺. The effect tended to be smaller in the nominal absence than in the presence of extracellular Ca²⁺, a diffference, however, not reaching statistical significance.

In order to test whether simvastatin-induced cell membrane scrambling required entry of extracellular Ca²⁺, erythrocytes were incubated for 48 h in the absence or presence of 2 µg/ml simvastatin in the presence or nominal absence of extracellular Ca²⁺. As illustrated in Fig. 6A-C, removal of extracellular Ca²⁺ significantly blunted the effect of simvastatin on annexin-V-binding. However, even in the nominal absence of extracellular Ca²⁺, simvastatin significantly increased the percentage of annexin-V-binding erythrocytes. Thus, the simvastatin-induced cell membrane scrambling was in part but not completely due to entry of extracellular Ca²⁺.

Eryptosis is further stimulated by oxidative stress. The abundance of reactive oxygen species (ROS) was thus quantified utilizing 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA). As illustrated in Fig. 7A,B, a 48 h exposure to simvastatin (2 µg/ml) increased the average DCFDA fluorescence of erythrocytes from 15.6 ± 0.4 a.u. (n = 22) to 20.8 ± 1.3 a.u. (n = 22). Moreover, simvastatin significantly increased the percentage of erythrocytes with high DCFDA fluorescence (Fig. 7C). The observations suggest that simvastatin did induce oxidative stress.

Again, oxidative stress was measured following incubation for 48 h in the absence or presence of 2 µg/ml simvastatin in the presence or nominal absence of extracellular Ca²⁺. As illustrated in Fig. 8, DCFDA fluorescence of erythrocytes significantly increased both, in the presence and absence of extracellular calcium. The average DCFDA fluorescence reached significantly higher values in the absence than in the presence of extracellular Ca²⁺.

A further stimulator of eryptosis is ceramide. Ceramide abundance at the erythrocyte surface was thus quantified utilizing specific antibodies. As a result, the ceramide abundance tended to be higher following exposure to 2 µg/ml simvastatin (8.72 ± 0.34 a.u., n = 15) than following incubation in the absence of simvastatin (7.77 ± 0.32 a.u., n = 15), a difference, however, not reaching statistical significance.

To explore, whether the effects of simvastatin involved p38 kinase activity, the influence of simvastatin on annexin-V-binding was tested in the absence and presence of p38 kinase inhibitor SB203580. As illustrated in Fig. 9, SB203580 (2 µM) significantly blunted the effect of simvastatin (2 µg/ml) on annexin-V-binding. However, even in the presence of SB203580, simvastatin significantly increased the percentage of annexin-V-binding erythrocytes.

A final series of experiments explored the influence of simvastatin on hemolysis. The percentage of hemolytic cells was quantified from the hemoglobin concentration in the supernatant. As illustrated in Fig. 10, a 48 h exposure to simvastatin increased the percentage of hemolytic erythrocytes, an effect reaching statistical significance at 1 µg/ml simvastatin.

Again the role of extracellular Ca²⁺ was tested by incubation for 48 h in the absence or presence of 2 µg/ml simvastatin in the presence or nominal absence of extracellular Ca²⁺. As illustrated Fig. 11, the percentage of hemolytic erythrocytes increased significantly to virtually the same extent in the presence and absence of extracellular Ca²⁺.

The present observations reveal that simvastatin triggers erythrocyte shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Accordingly, simvastatin does not only trigger apoptosis, the suicidal death of nucleated cells [31-39], but by the same token triggers eryptosis, the suicidal erythrocyte death. The simvastatin concentrations required for the stimulation of eryptosis were in the range of concentrations (0.9 µg/ml) measured in the plasma of patients [39].

The effect of simvastatin on cell volume and cell membrane scrambling was paralleled by an increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i). Moreover, the effect of simvastatin on cell membrane scrambling was partially blunted by removal of extracellular Ca²⁺, an observation pointing to a role of Ca²⁺ entry in the triggering of eryptosis. However, simvastatin triggered cell membrane scrambling even in the nominal absence of extracellular Ca²⁺, an observation suggesting the participation of additional mechanisms in simvastatin induced cell membrane scrambling. It must be kept in mind that removal of Ca²⁺ from and addition of EDTA to the Ringer solution (nominal absence of Ca²⁺) does not completely eliminate extracellular Ca²⁺. We cannot rule out that residual extracellular Ca²⁺ enters the cell and elicits the observed effects.

Cells could be sensitized for the scrambling effect of Ca²⁺ by ceramide [41]. Simvastatin treatment tended to increase the ceramide abundance. The effect did, however, not reach statistical significance.

Simvastatin significantly enhanced the abundance of reactive oxygen species, a well known trigger of eryptosis [41] . Simvastatin is therefore partially effective by inducing oxidative stress. Similar to the triggring effect of simvastatin on apoptosis of osteosarcoma cells [135], the stimulation of eryptosis by simvastatin on cell membrane scrambling requires apparently activity of p38 kinase, which is known to participate in the orchestration of eryptosis [53]. Accordingly, the effect of simvastatin on annexin-V-binding is significantly blunted in the presence of p38 kinase inhibitor SB203580.

The simvastatin-induced erythrocyte shrinkage was not significantly blunted in the nominal absence of extracellular Ca²⁺. This observation does not rule out the involvement of Ca²⁺ entry. An increase of [Ca²⁺]_i activates Ca²⁺-sensitive K⁺ channels followed by K⁺ exit, cell membrane hyperpolarization, Cl^- exit and thus cellular loss of KCl with water [42]. Whether additional factors contribute to simvastatin induced erythrocyte shrinkage, remained elusive The present experiments cannot rule out the participation of further mechanisms in the stimulation of cell membrane scrambling and cell shrinkage following simvastatin treatment of erythrocytes.

Simvastatin further triggered hemolysis, pointing to disruption of the cell membrane. The function of eryptosis is the removal of defective erythrocytes from circulating blood prior to hemolysis [41]. Without timely removal of defective erythrocytes hemoglobin released following hemolysis passes the renal glomerular filter, precipitates in the acidic lumen of renal tubules, occludes nephrons and may thus lead to renal failure [136].

The clearance of phosphatidylserine exposing erythrocytes may result in the development of anemia. A combined therapy with simvastatin and cetuximab frequently leads to anemia [137]. However, we did not find reports of anemia following treatment with simvastatin alone. Anemia develops only, when the loss of erythrocytes surpasses the formation of new erythrocytes by erythropoiesis [41]. It should further be kept in mind that simvastatin decreases plasma concentrations of C-reactive protein (CRP) [138], a stimluator of eryptosis [139]. Thus the direct stimulation of eryptosis may be attenuated by the indirect decrease of CRP-induced eryptosis.

Besides its potential effect on anemia, eryptosis may interfere with microcirculation [46, 140-144], as phosphatidylserine exposing erythrocytes adhere to other phosphatidylserine exposing erythrocytes [145] and the vascular wall [146], stimulate blood clotting and trigger thrombosis [140, 147, 148]. The present observations may thus not only be relevant for development of anemia but may suggest to carefully consider the risk of thrombosis following simvastatin treatment. This may be particularly important in patients suffering from clinical disorders associated with enhanced eryptosis. However, the present data cannot be taken as evidence that simvastatin treatment triggers thrombosis.

In conclusion, simvastatin stimulates Ca²⁺ entry into erythrocytes, triggers oxidative stress and thus triggers erythrocyte shrinkage and erythrocyte cell membrane scrambling, the hallmarks of eryptosis, the suicidal death of erythrocytes.

The authors acknowledge the meticulous preparation of the manuscript by Lejla Subasic. The study was supported by the Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of Tuebingen University.

None.