J Clin Endocrin Metab. First published ahead of print November 27, 2013 as doi:10.1210/jc.2013-2103
Vitamin D protects Human Endothelial Cells from oxidative stress through the autophagic and survival pathways Uberti F.1, Lattuada D.1, Morsanuto V.1,3, Nava U.1, Bolis G.2, Vacca G.3, Squarzanti D.F.3, Cisari C.4, Molinari C.3 1.Department of Obstetrics and Gynecology, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy; 2. Dipartimento di Scienze Cliniche e di Comunità, Università degli Studi di Milano, Milan, Italy 3.Dipartimento di Medicina Traslazionale, Università degli Studi del Piemonte Orientale A. Avogadro, Novara, Italy; 4.Dipartimento di Scienze della Salute, Università degli Studi del Piemonte Orientale A. Avogadro, Novara, Italy
Context. Recently, vitamin D (VitD) has known an increasing importance in many cellular functions of several tissues and organs other than bone. In particular, VitD showed important beneficial effects in cardiovascular system. Although the relationship among VitD, endothelium and cardiovascular disease is well established, little is known about the antioxidant effect of VitD. Objective. This research was carried on in order to study the intracellular pathways activated by VitD in cultured human umbilical vein endothelial cells undergoing to oxidative stress. Design. Nitric oxide production, cell viability, reactive oxygen species, mitochondrial permeability transition pore, membrane potential and caspase-3 activity were measured during oxidative stress induced by administration of 200M hydrogen peroxide for 20 minutes. Experiments were repeated in presence of specific VDR ligand ZK191784. Results. Pre-treatment with VitD alone or in combination with ZK191784 is able to reduce the apoptosis-related gene expression, involving both intrinsic and extrinsic pathways. At the same time it has been shown the activation of pro-autophagic Beclin 1 and the phosphorylation of ERK1/2 and Akt, indicating a modulation between apoptosis and autophagy. Moreover VitD alone or in combination with ZK191784 is able to prevent the loss of mitochondrial potential and the consequent cytochrome C release and caspase activation. Conclusions. The present study shows that VitD may prevent endothelial cell death through modulation of interplay between apoptosis and autophagy. This effect is obtained by inhibiting superoxide anion generation, maintaining mitochondria function and cell viability, activating survival kinases and inducing NO production.
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he hormonally active form of vitamin D, 1␣25(OH)2D3 (VitD), influences the expression of various genes, whose products not only are involved in the control of calcium and phosphate homeostasis (1) but are able to interact with a wide range of nonclassic organs and target tissues, including the heart and the vasculature as well (2). Recent clinical studies have demonstrated that VitD
may cause regression of cardiac hypertrophy, and reduces cardiovascular morbidity and mortality in patients who frequently suffer from atherosclerosis (3, 4), and it may improve the cardiac structure and function (5). VitD exerts its physiological effects principally through the binding with the nuclear vitamin D receptor (VDR), regulating the expression of genes responsible for cellular proliferation, differentiation, apoptosis, and angiogenesis in local
ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2013 by The Endocrine Society Received May 2, 2013. Accepted November 11, 2013.
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doi: 10.1210/jc.2013-2103
J Clin Endocrinol Metab
Copyright (C) 2013 by The Endocrine Society
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tissue (6, 7, 8). In particular many vascular effects, such as increased expression of Ca-ATPase, induction of contractile protein synthesis, and hence, increased vascular resistance were reported. VitD is a modulator of vascular wall growth as well (9), and induces a decrease in the expression and/or secretion of proinflammatory and proatherosclerotic factors in the endothelium (2). VitD exerts its effects on many target tissues through genomic and nongenomic mechanisms. The non genomic mechanism induces rapid responses, from seconds to minutes (10), and activates signal transduction pathways through a putative membrane receptor including activation of adenylyl cyclase-cAMP-protein kinase A and phospholipase C-diacylglicerol-inositol (1, 4, 5)-trisphosphate-protein kinase C signal transduction pathways (11). In particular between the second messengers RAF/MAPK play an important role because they may engage in cross-talk with the nucleus to modulate gene expression (10). The role of endothelium as a target of VitD is demonstrated by the study published by Zehnder et al (12), in which the expression of mRNA and protein for l␣-hydroxylase in human endothelium was shown for the first time. Altogether these findings demonstrated the direct effects of VitD on the endothelial function, whose alteration plays an important role in the development of atherosclerosis. Xiang et al (13) showed the ability of VitD to stimulate endothelial cell proliferation and to inhibit apoptosis by increasing endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production. NO plays a key role in cardiovascular physiology (14) and its production in the heart by eNOS phosphorylation represents an important regulator of both myocardial perfusion after ischemia and myocardial contractility, and has important effects on cardiac cell functions such as oxygen consumption, hypertrophic remodeling, apoptosis and myocardial regeneration (15–17). In particular, our previous study demonstrated that VitD can induce a concentration-dependent increase in endothelial NO production through the eNOS activation consequential to the phosphorylation of p38, AKT and ERK (18). Moreover, VitD is able to prevent the myocardial ischemia, as well reported in a case-control study in 1990 on 179 patients (19) in which the odds of having a myocardial infarction (MI) increased along with decreasing of serum VitD concentration. Another study by Giovannucci et al (20) demonstrates that men with VitD deficiency had higher risk of MI compared to those with normal VitD concentration. Recent investigations on cardiac myocytes show that reactive oxygen species (ROS) produced by mitochondrial and oxidative stress can cause multiple changes in cell structure and function that are associated with the failing heart (21) and apoptosis (22) or autophagy (23). On the other hand,
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ROS, along with NO, show antiapoptotic effects involving several signaling pathways: for example the increase in cytosolic-free Ca2⫹ concentration not only activates proapoptotic signals (23) but also potently induces autophagy by activating calmodulin-dependent kinase (24). Autophagy can be induced either by starvation, or hypoxia, or biological and chemical agents. The functional role of autophagy during ischemia/reperfusion is complex, because its pathophysiological functions depends on the severity and duration of ischemia (hypoxia) and the consequent tissue damage during reperfusion in the heart. The level of autophagy may determine whether autophagy itself is protective or detrimental in response to ischemia/ reperfusion injury in the heart (25). VitD is able to induce autophagy in various human cell types. The signaling pathways regulated by VitD include for example Bc1–2, Beclin 1, and mammalian target of rapamycin (mTOR) (26). During autophagy, intracellular contents are enveloped in double-layered membrane vesicles that fuse with lysosomes for what could be called “recycling process” (27). The interplay between autophagic and apoptotic pathways is a crucial point to determine the initiation of programmed cell death and whether the members of the Beclin l and Bc1–2 family were involved. Beclin l directly interacts not only with Bc1–2 but also with other antiapoptotic Bc1–2 family proteins such as Bcl-xl, and Bc1–2 was able to inhibit the Beclin l-dependent autophagy (28). In this context the aim of the study was to analyze the intracellular pathways activated by VitD under oxidative stress in human endothelial cells.
Materials and Methods Endothelial cell culture HUVEC were obtained from 25 human umbilical vein cord preparations as previously described (18, 29) in accordance with the procedures approved by the local institutional ethics committee. Only the cells positive for vWF/Factor VIII, CD31 and Dil-Ac-LDL were selected. The cells were cultured in 0.1% gelatin-coated flask with endothelial growth medium 2 (EGM-2) as previously described (18). The cells used for all the experiments were at passage 3 to 6. When the cells were at 70% confluence, they were divided for the experiment: in the first group of experiments 1 ⫻ 105 cells were plated in gelatin-coated 24 well plates in complete medium and after the adhesion the cells were incubated for 4 – 6 in DMEM without red phenol and FBS and supplemented with 2 mM glutamine and 1% penicillin-streptomycin (starvation medium; Sigma-Aldrich, Milan, Italy) in incubator to study the cell viability. The nitric oxide production, the reactive oxygen species production (ROS), the mitochondrial permeability transition pore (MPTP) opening and the potential membrane were analyzed in 5 ⫻ 105 cells plated on gelatincoated 6 well plates and maintained in the same conditions used to 24-well plates. To analyze the protein expression, HUVEC were plated on 0.1% gelatin-coated flask with EGM-2 complete
doi: 10.1210/jc.2013-2103
medium, and after the attachment were incubated overnight in starvation medium in incubator. To confirm the protein expression and cell death through TUNEL assay, 3.5 ⫻ 103 were plated on chamber slide (BD Biosciences, Milan, Italy) with EGM-2 complete medium to perform immunohistochemistry and immunofluorescence analysis. All the experiments were conducted in the oxidative stress condition, which was generated using 200M hydrogen peroxide (H2O2) for 20 minutes in DMEM without FBS and phenol red. In some experiments the cells were pretreated with 1 nM VitD for 15 minutes alone or in combination with 1 nM ZK191784 (Bayer Pharma AG, Berlin, Germany) for 15 minutes and in other experiments VitD or the combination VitD⫹ZK191784 was added after H2O2.
Griess assay In 5 ⫻ 105 cells plated as described before (18), the NO production was measured in culture supernatants using Griess reagents (Promega, Madison, WI, USA) and the results are expressed as percentage (%).
Cell viability (MTT assay) In Vitro Toxicology Assay Kit, MTT based (Sigma-Aldrich) was used to determine cell viability. Details on the method are reported on Supplemental Data. Cell viability was calculated by comparing results to control cells (100% viable).
ROS production The rate of superoxide anion release was used to examine the effects of VitD against the oxidative stress. The superoxide anion production was measured as superoxide dismutase-inhibitable reduction of Cytochrome C. In all samples (stimulated and untreated) 100l of Cytochrome C was added and in another one 100l of superoxide dismutase was also added for 30 minutes in incubator (all substances from Sigma-Aldrich). The absorbance changes in the supernatants of the sample was measured at 550nm in a Wallac Victor mod. 1421 spectrometer (PerkinElmer). The O2. was expressed as nanomoles per reduced Cytochrome C for microgram of protein, using an extinction coefficient of 21000 ml/cm, after the interference absorbance subtraction (30).
Measurement of mitochondrial permeability transition pore (MPTP) After each stimulation, conducted in the same condition and with the same agents used before, and in the other sample pretreated with lM cyclosporin A for 30 minutes (31), the medium was removed and the sarcolemmal membrane was permeabilized using 10 M digitonin (32) per 60 seconds in an intracellular solution buffer (135 mM KC1, 10 mM NaCL, 20 mM HEPES, 5 mM pyruvate, 2 mM glutamate, 2 mM malate, 0.5 mM KH2PO4, 0.5 mM MgC12, 15 mM 2,3-butanedione monoxime, 5 mM ethylene glycol tetraacetic acid, 1.86 mM CaC12; SigmaAldrich) and then loaded with 5 M calcein/AM for 40 minutes at 37°C. After this time the cells were washed with Tyrode solution for 10 minutes and then the fluorescence was measured by a Wallac Victor mod. 1421 at fluorescence excitation and emission of 488nm and 510nm respectively.
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Membrane potential assay After each stimulation as seen before, the medium was removed and the cells were incubated with 5,5⬘,6,6⬘-tetrachloro1,1⬘,3,3⬘-tetraethylbenzimidazolylcarbocyanine iodide following the manufacturer’s instruction (APO-LOGIX JC-1, Bachem, San Carlos, CA, USA). The rate of apoptosis cells was obtained by determining the ratio of red to green fluorescence (33). Details are available in Supplemental Data.
Western blot analysis 40 g of protein from each lysate were resolved on 8% or 15% SDS-PAGE gels and after the transfer of proteins to polyvinylidene fluoride membranes (PVDF) were incubated overnight at 4°C with specific antibodies. Details are available in Supplemental Data.
Caspase-3 activity The same samples used to western blot analysis were used to study the Caspase-3 activity following manifacturer’s instructions (Assay Designs Temaricerca, Bologna, Italy). The results are expressed as percentage. Details are available in Supplemental Data.
Immunohistochemistry The immunostaining procedure was performed on HUVEC plated on chamber slide, using Annexin V (13 g/ml; SigmaAldrich) and Beclin 1 antibody (4 g/ml; Santa-Cruz). At the end the cells were visualized using light microscopic (Leica, Germany). Details are available in Supplemental Data.
Immunofluorescence The immunofluorescence protocol was performed on HUVEC plated on chamber slide to study Caspase-3 specific antibody (13 g/ml; Santa-Cruz). At the end, the cells were visualized using fluorescence microscope (Leica, Germany). Details are available in Supplemental Data.
TUNEL assay For the detection of the endonucleolytic cleavage of chromatin, characteristic of apoptosis, HUVEC cells plated on chamber slide, were analyzed through TUNEL assay kit following the manifacturer’s instruction (ApopTag Plus Peroxidase Apoptosis Detection Kit, Merck Millipore, Milan, Italy). Details are available in Supplemental Data.
Statistical analysis Results are expressed as means ⫾SD of five independent experiments. One-way ANOVA followed by Bonferroni post hoc test were used for statistical analysis. The percentage values were compared through Mann-Whitney U test. The value of P ⬍ .05 was considered as statistically significant.
Results Effects of VitD on NO production during oxidative stress (H2O2) To analyze the VitD effects on NO production induced by oxidative stress, HUVEC were treated with H2O2, VitD
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and/or agonist of VitD receptor (ZK191784) alone or combined. The results obtained by Griess method demonstrated that 1 nM VitD alone was able to cause NO release (P ⬍ .05) of about 70.33 ⫾ 4.73 compared to controls and this effect was amplified (P ⬍ .05) in the sample treated in combination with 1 nM ZK191784 (123.3 ⫾ 6.51%; Figure 1). These data confirmed which was observed in a previously study on NO production caused by VitD in endothelial cells (18). During the oxidative stress induced by 200 M H2O2, we observed a reduction on NO release of about 10 ⫾ 1.52% (P ⬍ .05) compared to controls, which was a significantly (P ⬍ .05) reverse in the samples pretreated with 1 nM VitD (26.33 ⫾ 4.16%) alone and amplified in combined pretreatment with 1 nM ZK191784 (53.33 ⫾ 7.64%). In the samples treated before with H2O2, the stimulations with VitD alone or in combination with ZK191784 were not able to counteract the negative effects on NO production caused by H2O2 (Figure 1). These results suggest that VitD is able to counteract the negative effects caused by oxidative stress and confirme the ability of VitD to induce NO release. Effects of VitD on cell viability during oxidative stress (H2O2) In the same experimental conditions using MIT test, it has been studied if VitD was able to prevent a reduction of cell viability during the oxidative stress (Figure 2). After the stimulation with 1 nM VitD alone the cell viability showed a significant increase of about 18.57 ⫾ 2.13% (P ⬍ .05) and in combination with 1 nM ZK191784 this effect was amplified of about 39.5% (25.91 ⫾ 1.02%, P ⬍ .05). These data indicate the ability of VitD to increase the
Figure 1. Effects on NO production determined by means of Griess method, induced by VitD (1 nM) alone or in combination with ZK191784 (1 nM) and/or H2O2 (200 M) in HUVEC cultures. C ⫽ control; Z19 ⫽ ZK191784; Z19⫹VitD ⫽ combination; H ⫽ 11202; VitD⫹H ⫽ combination; Z19⫹VitD⫹H ⫽ combination; H⫹VitD ⫽ combination; H⫹Z19⫹VitD ⫽ combination. * P ⬍ .05 vs. control; arrows indicate: H⫹VitD P ⬍ .05 vs. VitD⫹H; H⫹Z19⫹VitD P ⬍ .05 vs. Z19⫹VitD⫹H. Reported data are means (%) ⫾ SD of 5 independent experiments.
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cell viability in physiological condition. The pretreatment with 200 M H2O2 for 20 minutes caused a reduction of cell viability (-9.77 ⫾ 0.91%, P ⬍ .05) that was prevented only by the pretreatment with 1 nM VitD alone (13.16 ⫾ 1.37% compared to controls) or in combination with 1 nM ZK191784 (16.95 ⫾ 1.66% compared to controls; Figure 2). This effect is similar to what observed on NO production. For this reason we hypothesize that VitD is able to prevent the negative effects of H2O2 only if was used before the oxidative stress. Effects of VitD on reactive oxygen species production In HUVEC treated with 1 nM VitD alone or in combination with 1 nM ZK191784 it has not been observed significant changes in superoxide anion production compared to controls (P ⬎ .05), but in the sample treated with 200 M H2O2, the superoxide anion production was increased to 23.08 ⫾ 1.17 nmol per reduced Cytochrome C per g of protein (P ⬍ .05) from control values of 4.7 ⫾ 1.096 nmol per reduced Cytochrome C per g of protein (Figure 3). The pretreatment with 1 nM VitD alone was able to reduce superoxide anion production (P ⬍ .05) and this effect was amplified of 19.7% (P ⬍ .05) in presence of 1 nM ZK191784 (15.17 ⫾ 0.38, 12.19 ⫾ 1.6 nmol per reduced Cytochrome C per g of protein respectively). The stimulation with 1 nM VitD alone or in combination with 1 nM ZK191784 after the treatment with 200 M H2O2, did not induced an important superoxide anion reduction compared to the effect of H2O2 alone (P ⬎ .05). These data confirmed previous findings on NO production and MTT test, and they demonstrate the ability of VitD to prevent the negative effects of the oxidative stress only if it used before the oxidative event.
Figure 2. Effects on cell viability determined by means of MTT test, induced by VitD (1 nM) alone or in costimulation with ZK191784 (1 nM) and/or H2O2 (200 M) in HUVEC cultures. The abbreviations are the same used in Figure 1. * P ⬍ .05 vs. control; arrows indicate: H⫹VitD P ⬍ .05 vs. VitD⫹H; H⫹Z19⫹VitD P ⬍ .05 vs. Z19⫹VitD⫹H. Reported data are means (%) ⫾ SD of 5 independent experiments.
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VitD counteracts apoptosis caused by H2O2 through activation of autophagy and survival signaling To investigate the mechanisms activated by VitD during the oxidative stress to counteract the negative effects of H2O2, HUVEC were treated with the same experimental protocol previously used. As reported in Figure 4 and 5, the effects induced by the stimulation with VitD alone were able to induce significant changes (P ⬍ .05) in expression of apoptosis- and autophagy-associated proteins. The only VitD administration reduced Bax, Caspase-3, Caspase-8, Caspase-9 and Cytochrome C expression compared to control (P ⬍ .05). Similar data were also obtained by immunohistochemistry and immunofluorescence analysis on Annexin V, TUNEL assay and Caspase-3 fluorescence analysis, confirming a protective role of VitD on cell death. In addition VitD was able to increase Beclin 1, ERK and Akt compare to control (P ⬍ .05). Graded and amplified changes in apoptosis and autophagy expression were found also in presence of combination with VitD and VDR agonist ZK191784; a reduction of Bax, Caspase-3, Caspase-8, Caspase-9, Annexin V, TUNEL and Cytochrome C expression and an increase of Beclin 1, ERK and Akt were observed as well (P ⬍ .05) (Figure 4, 5 and 7). Samples treated with H2O2 alone showed the opposite effects: the markers of apoptosis (Bax, Cytochrome C, Caspases-8,3,9, TUNEL and Annexin V) were increased compared to controls (P ⬍ .05) and the signaling of survival was decreased. Similar data were obtained in Caspase-3 activity test (Figure 5F), in which was observed that VitD alone caused a reduction of Caspase-3 activity
Figure 3. Effects on reactive oxygen species production (ROS) induced by VitD (1 nM) alone or in combination with ZK191784 (1 nM) and/or H2O2 (200 M) in HUVEC cultures undergoing to oxidative stress. The abbreviations are the same used in previous Figures. * P ⬍ .05 vs. control; arrows indicate: H⫹VitD P ⬍ .05 vs. VitD⫹H; H⫹Z19⫹VitD P ⬍ .05 vs. Z19⫹VitD⫹H. Reported data are means ⫾ SD of 5 independent experiments.
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(-4.48 ⫾ 0.78%; P ⬍ .05) and in combination with ZK191784 this effect was amplified (-5.67 ⫾ 2.08%; P ⬍ .05). The stimulation with H2O2 alone induced a significant increase in Caspase-3 activity respect to control values (41.46 ⫾ 1.46%; P ⬍ .05). The pretreatment with VitD alone or in combination with ZK191784 was able to prevent the activity of Caspase-3 during the oxidative stress (3.5 ⫾ 1.5% and 2.6 ⫾ 0.6%, respectively), but the same stimulation after the treatment with H2O2 not reverse the effect (50.26 ⫾ 1.78% and 50.67 ⫾ 4.16%, respectively). These results were also confirmed by immunofluorescence analysis (Figure 7D). As shown in Figure 5, pretreatment with H2O2 and the successive stimulation with VitD alone or in costimulation with ZK191784 showed that VitD and its agonist were able to reduce the activation of apoptosis and to increase expression of autophagic and survival signaling compared to treatment with H2O2 alone.
Figure 4. Effects of VitD (1 nM) alone or in combination with ZK191784 (1 nM) and/or H2O2 (200 M) on the activation of Beclin-1 (A), Box (B), and the phosphorylation of ERK 1/2 (C). In the panel are reported of immunoblots of activation and phosphorylation relative to specific proteins. The abbreviations are the same as used above. The results represented an example of 5 independent experiments.
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Effects of VitD during oxidative stress on mitochondrial membrane potential and MPTP opening As shown in Figure 6A, 1 nM VitD was able to prevent (45.33 ⫾ 8.39%, P ⬍ .05) the loss of mitochondrial membrane potential induced by 200 M H2O2 (-11 ⫾ 4.58%) and this effect was amplified in presence of 1 nM ZK191784 (71 ⫾ 3.61%, P ⬍ .05). These results demonstrated that VitD could prevent the loss of mitochondrial membrane potential through the modulation of apoptosis/ autophagy pathways. Moreover, additional experiments were performed to examine the modulation of MPTP opening. As reported in Figure 6B, the stimulation with 200 M H2O2 caused a significant (P ⬍ .05) reduction of mitochondria-trapped calcein intensity (-34 ⫾ 5.92%; P ⬍ .05), which was prevented by the treatment with 1 nM VitD and in presence of 1 nM ZK191784 this effect was amplified (P ⬍ .05). In the sample pretreated with 1 M cyclosporine A, agent that interacts with cyclophilin D to
Figure 5. Densitometric analysys of the effects of VitD (1 nM) alone or in combination with ZK191784 (1 nM) and/or H2O2 (200 M) on various kinases. In A the phosphorylation of Akt, in B the activation of cytochrome C, in C the activation of caspase-8, in D the activation of caspase-9, in E and F the activation of caspase-3 and relative protein activity. The abbreviations are the same used in previous. * P ⬍ .05 vs. control; arrows indicate: H⫹VitD P ⬍ .05 vs. VitD⫹H; H⫹Z19⫹VitD P ⬍ .05 vs. Z19⫹VitD⫹H. Reported data are means (%) ⫾ SD of 5 independent experiments.
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delay MPTP opening, was observed a significant increase of calcein intensity in the sample stimulated with H2O2 alone (28 ⫾ 6.03%; P ⬍ .05), and when VitD alone or in combination with ZK191784 was added these effects were amplified (34.03 ⫾ 4% and 92.33 ⫾ 5.86% compared to controls), respectively. In conclusion the results demonstrated that VitD was able to prevent MPTP opening during the oxidative stress.
Discussion The present research explains the mechanism activated by VitD to counteract the negative effects induced by oxidative stress in endothelial cells. As reported in literature, VitD is able to reduce the damage following H2O2-mediated stress in a dose- and time-dependent manner through the decrease of anion superoxide generation and apoptotic cells (34). In the present study it has been clearly demon-
Figure 6. Study of mitochondrial membrane potential (A) and MPTP opening (B) in HUVEC treated with VitD (1 nM) alone or in combination with ZK191784 (1 nM) and/or H2O2 (200 M). In A the abbreviations are the same used in previous Figures. * P ⬍ .05 vs. control; arrows indicate: Z19⫹VitD⫹HP P ⬍ .05 vs. VitD⫹H. In B CsA ⫽ cyclosporin A 1 M, the other abbreviation are the same used above, * P ⬍ .05 vs. control; arrows indicate: Z19⫹VitD⫹H P ⬍ .05 vs. VitD⫹H; CsA⫹VitD⫹H P ⬍ .05 vs. V⫹H; CsA⫹Z19⫹VitD⫹H P ⬍ .05 vs. Z19⫹VitD⫹H; CsA⫹Z19⫹VitD⫹H P ⬍ .05 vs. CsA⫹VitD⫹H. Reported data are means (%) ⫾ SD of 5 independent experiments.
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strated that the administration of VitD to endothelial cells before the induction of an oxidative stress can improve cell viability. The mechanisms involved include the prevention of free oxygen radical release and the modulation of the interplay between apoptosis and autophagy. These effects were also accompanied by NO production and preservation of mitochondrial function. In these experiments HUVEC cultures received an oxidative stress by means of H2O2. This method is widely used in order to reproduce a cellular damage similar to what occurs in myocardial ischemia/reperfusion injury. In the light of these data, a cardioprotective role of VitD against the ischemic injury can be hypothesized. As observed in NO production and MTT tests, the effect induced by VDR agonist ZK191784 is greater than the one observed after VitD alone. Moreover, the combined administration of the two VDR agonists (VitD and ZK191784) induced an amplified effect. The reason why VitD and ZK191784 combined administration induces a more potent effect on HUVEC could be an interesting issue for successive research. These beneficial effects were observed when VDR agonists have been administered before the induction of oxidative stress. This fact supports the hypothesis that VitD is able to counteract the negative effects of the oxidant event on endothelial cells increasing the cell viability. In addition, NO release induced by VitD during oxidative stress is able to protect cells from death. This finding is demonstrated by the observation that rate of NO production observed in this study was below to 2 M/s. This threshold prevents the opening of mitochondrial transition pore and the release of cytochrome C and avoids the mitochondrial collapse leading to cell death (35). Oxidative stress plays an important role in the pathogenesis of atherosclerosis (36, 37). In last years several studies have indicated that antioxidant vitamins such as vitamin C or E may restore endothelial function and may have anti-inflammatory and antithrombotic properties (38). Moreover an overwhelming body of evidence demonstrated that NO plays a fundamental biological role in protecting the heart against ischemia/reperfusion injury. Recent investigations on cardiac myocytes show that ROS produced by mitochondrial and oxidative stress can cause multiple changes in cell structure and function that are associated with the failing heart (21) and apoptosis (22) or autophagy (23). The antiapoptotic effects of NO and ROS involved several signaling pathways and the interplay between autophagic and apoptotic pathways is a crucial point to determining the initiation of programmed cell death. In the present study, the pretreatment with VitD, alone or in combination with ZK191784, is able to reduce the apoptosis-related gene expression (Bax, Caspase-3, Caspase-9, Caspase-8, cytochrome C), involving both in-
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trinsic and extrinsic pathways. These findings were confirmed by immunohistochemistry analysis of Annexin V and TUNEL assay in which we observed a signal reduction. At the same time it has been shown the activation of proautophagic Beclin 1 and the phosphorylation of ERK1/2 and Akt, member of reperfusion injury salvage kinase (RISK) pathway, indicating a modulation between apoptosis and autophagy. Moreover VitD, alone or in combination with ZK191784, administered before the oxidative stress, is able to prevent the loss of mitochondrial potential and the consequent cytochrome C release and caspase activation. In addition, VitD alone or in combination with ZK191784 was able to prevent the MPTP opening caused by H2O2. These findings depend on changes of mitochondria-trapped calcein intensity and on effects of cyclosporine A, which inhibits MPTP opening, modulating the cyclophilin D activity (38). This work adds new information to the debate on the benefits of VitD supplementation. In fact the present study shows for the first time that VitD may prevent endothelial cell death through modulation of interplay between apoptosis and autophagy. This effect is obtained by inhibiting superoxide anion generation, maintaining mitochondria function and cell viability, activating survival kinases (ERK and Akt) and inducing NO production.
Acknowledgments This work was supported by SISDIM (Società Italiana per lo studio della Sarcopenia e delle Disabilità Muscoloscheletriche). The authors thank Bayer Pharma AG (Berlin, Germany) for donating the VDR agonist ZK191784. Thanks to Dr. Mariangela Fortunato for her help. Address all correspondence and requests for reprints to: Corresponding author (to whom reprint requests should be addressed): Francesca Uberti, [email protected]. Laboratorio di ricerca molecolare per lo studio e la cura delle patologie riproduttive, U.O. Ostetricia e Ginecologia 2, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, via Manfredo Fanti, 6 20122 Milano. Disclosure Summary: The authors have nothing to disclose. This work was supported by .
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hypertrophy in hemodialysis patients with secondary hyperparathyroidism. Am J Kidney Dis. 1999;33:73– 81. Shoji T, Shinohara K, Kimoto E, Emoto M, Tahara H, Koyama H, Inaba M, Fukumoto S, Ishimura E, Miki T, Tabata T, Nishizawa Y. Lower risk for cardiovascular mortality in oral lalphahydroxy vitamin D3 users in a haemodialysis population. Nephrol Dial Transplant. 2004;19:179 –184. Talmor Y, Golan E, Benchetrit S, Berheim J, Klein O, Green J. Calcitriol blunts the deleterious impact of advanced glycation end products on endothelial cells. Am J Physiol Renal Physiol. 2008; 294:F1059- Fl 064. Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Thompson PD, Selznick SH, Dominguez CE, Jurutka PW. The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res. 1998;13:325–349. Wu-Wong JR, Kawai M, Chen Y, Nakane M. VS-105: a novel vitamin D receptor modulator with the cardiovascular protective effects. Br J Pharmacol. 2011;164:551–560. Thompson L, Wang A, Tawfik O, Templeton K, Tancabelic J, Pinson D, Anderson HC, Keighley J, Garimella R. Effects of 25-hydroxyvitamin D3 and 1 a, 25 dihydroxyvitamin D3 on differentiation and apoptosis of human osteosarcoma cell lines. J Orthop Res. 2012;30(5):831– 844. Somjen D, Kohen F, Amir-Zaltsman, Knoll E, Stern N. Vitamin D analogs modulate the action of gonadal steroids in human vascular cells in vitro. Am J Hypertens. 2000;13(4 pt 1):396 – 403. Norman AW. Minireview: Vitamin D receptor: new assignments for an already busy receptor. Endocrinology. 2006;147(12):5542– 5548. Boland R, Buitrago C, Russo De Boland A. Modulation of tyrosine phosphorylation signalling pathways by l␣,25 (011)2- vitamin D3. Trends Endocrinol Metab. 2005;16(6):280 –287. Zehnder D, Bland R, Chana RS, Wheeler DC, Howie AJ, Williams MC, Stewart PM, Hewison M. Synthesis of 1,25-dihydroxyvitamin D3 by human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion. J Am Soc Nephrol. 2002;13(3):621– 629. Xiang W, He XJ, Ma YL, Yi ZW, Cao Y, Zhao SP, Yang JF, Ma ZC, Wu M, Fu SM, Ma JL, Wang J, Zheng W, Kang H. 1, 25 (OH)(2)D(3) influences endothelial cell proliferation, apoptosis and endothelial nitric oxide synthase expression of aorta in apoliprotein E-deficient mice. Zhonghua Er Ke Za Zhi. 2011;49(11):829 – 833. Kanno Y, Into T, Lowenstein CJ, Matsushita K. Nitric oxide regulates vascular calcification by interfering with TGF- signaling. Cardiovasc Res. 2008;77(1):221–230. Massion PB, Pelat M, Belge C, Balligand JL. Regulation of the mammalian heart function by nitric oxide. Comp Biochem Physiol, Part A Mol Integr Physiol. 2005;142(2):144 –150. Mount PF, Kemp BE, Power DA. Regulation of endothelial and myocardial NO synthesis by multi-site eNOS phosphorylation. J Mol Cell Cardio. 2007;142(2):271–279. Smith RS, Agata J, Xia CF, Chao L, Chao J. Human endothelial nitric oxide synthase gene delivery protects against cardiac remodeling and reduces oxidative stress after myocardial infarction. Life Sci. 2005;76(21):2457–2571. Molinari C, Uberti F, Grossini E, Vacca G, Carda S, Invernizzi M, Cisari C. l␣, 25- dihydroxycholecalciferol induces nitric oxide production in coltured endothelial cells. Cell Physiol Biochem. 2011; 27(6):661– 668. Scragg R, Jackson R, Holdaway IM, Lim T, Beaglehole R. Myocardial infarction is inversely associated with plasma 25-hydroxyvitamin D3 levels: a community-based study. Int J Epidemiol. 1990; 19(3):559 –563. Giovannucci E, Liu Y, Hollis BW, Rimm EB. 25-hydroxyvitamin D
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Vitamin D protects Human Endothelial Cells from oxidative stress through the autophagic and survival pathways Uberti F.1, Lattuada D.1, Morsanuto V.1,3, Nava U.1, Bolis G.2, Vacca G.3, Squarzanti D.F.3, Cisari C.4, Molinari C.3 1.Department of Obstetrics and Gynecology, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy; 2. Dipartimento di Scienze Cliniche e di Comunità, Università degli Studi di Milano, Milan, Italy 3.Dipartimento di Medicina Traslazionale, Università degli Studi del Piemonte Orientale A. Avogadro, Novara, Italy; 4.Dipartimento di Scienze della Salute, Università degli Studi del Piemonte Orientale A. Avogadro, Novara, Italy
Context. Recently, vitamin D (VitD) has known an increasing importance in many cellular functions of several tissues and organs other than bone. In particular, VitD showed important beneficial effects in cardiovascular system. Although the relationship among VitD, endothelium and cardiovascular disease is well established, little is known about the antioxidant effect of VitD. Objective. This research was carried on in order to study the intracellular pathways activated by VitD in cultured human umbilical vein endothelial cells undergoing to oxidative stress. Design. Nitric oxide production, cell viability, reactive oxygen species, mitochondrial permeability transition pore, membrane potential and caspase-3 activity were measured during oxidative stress induced by administration of 200M hydrogen peroxide for 20 minutes. Experiments were repeated in presence of specific VDR ligand ZK191784. Results. Pre-treatment with VitD alone or in combination with ZK191784 is able to reduce the apoptosis-related gene expression, involving both intrinsic and extrinsic pathways. At the same time it has been shown the activation of pro-autophagic Beclin 1 and the phosphorylation of ERK1/2 and Akt, indicating a modulation between apoptosis and autophagy. Moreover VitD alone or in combination with ZK191784 is able to prevent the loss of mitochondrial potential and the consequent cytochrome C release and caspase activation. Conclusions. The present study shows that VitD may prevent endothelial cell death through modulation of interplay between apoptosis and autophagy. This effect is obtained by inhibiting superoxide anion generation, maintaining mitochondria function and cell viability, activating survival kinases and inducing NO production.
T
he hormonally active form of vitamin D, 1␣25(OH)2D3 (VitD), influences the expression of various genes, whose products not only are involved in the control of calcium and phosphate homeostasis (1) but are able to interact with a wide range of nonclassic organs and target tissues, including the heart and the vasculature as well (2). Recent clinical studies have demonstrated that VitD
may cause regression of cardiac hypertrophy, and reduces cardiovascular morbidity and mortality in patients who frequently suffer from atherosclerosis (3, 4), and it may improve the cardiac structure and function (5). VitD exerts its physiological effects principally through the binding with the nuclear vitamin D receptor (VDR), regulating the expression of genes responsible for cellular proliferation, differentiation, apoptosis, and angiogenesis in local
ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2013 by The Endocrine Society Received May 2, 2013. Accepted November 11, 2013.
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J Clin Endocrinol Metab
Copyright (C) 2013 by The Endocrine Society
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tissue (6, 7, 8). In particular many vascular effects, such as increased expression of Ca-ATPase, induction of contractile protein synthesis, and hence, increased vascular resistance were reported. VitD is a modulator of vascular wall growth as well (9), and induces a decrease in the expression and/or secretion of proinflammatory and proatherosclerotic factors in the endothelium (2). VitD exerts its effects on many target tissues through genomic and nongenomic mechanisms. The non genomic mechanism induces rapid responses, from seconds to minutes (10), and activates signal transduction pathways through a putative membrane receptor including activation of adenylyl cyclase-cAMP-protein kinase A and phospholipase C-diacylglicerol-inositol (1, 4, 5)-trisphosphate-protein kinase C signal transduction pathways (11). In particular between the second messengers RAF/MAPK play an important role because they may engage in cross-talk with the nucleus to modulate gene expression (10). The role of endothelium as a target of VitD is demonstrated by the study published by Zehnder et al (12), in which the expression of mRNA and protein for l␣-hydroxylase in human endothelium was shown for the first time. Altogether these findings demonstrated the direct effects of VitD on the endothelial function, whose alteration plays an important role in the development of atherosclerosis. Xiang et al (13) showed the ability of VitD to stimulate endothelial cell proliferation and to inhibit apoptosis by increasing endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production. NO plays a key role in cardiovascular physiology (14) and its production in the heart by eNOS phosphorylation represents an important regulator of both myocardial perfusion after ischemia and myocardial contractility, and has important effects on cardiac cell functions such as oxygen consumption, hypertrophic remodeling, apoptosis and myocardial regeneration (15–17). In particular, our previous study demonstrated that VitD can induce a concentration-dependent increase in endothelial NO production through the eNOS activation consequential to the phosphorylation of p38, AKT and ERK (18). Moreover, VitD is able to prevent the myocardial ischemia, as well reported in a case-control study in 1990 on 179 patients (19) in which the odds of having a myocardial infarction (MI) increased along with decreasing of serum VitD concentration. Another study by Giovannucci et al (20) demonstrates that men with VitD deficiency had higher risk of MI compared to those with normal VitD concentration. Recent investigations on cardiac myocytes show that reactive oxygen species (ROS) produced by mitochondrial and oxidative stress can cause multiple changes in cell structure and function that are associated with the failing heart (21) and apoptosis (22) or autophagy (23). On the other hand,
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ROS, along with NO, show antiapoptotic effects involving several signaling pathways: for example the increase in cytosolic-free Ca2⫹ concentration not only activates proapoptotic signals (23) but also potently induces autophagy by activating calmodulin-dependent kinase (24). Autophagy can be induced either by starvation, or hypoxia, or biological and chemical agents. The functional role of autophagy during ischemia/reperfusion is complex, because its pathophysiological functions depends on the severity and duration of ischemia (hypoxia) and the consequent tissue damage during reperfusion in the heart. The level of autophagy may determine whether autophagy itself is protective or detrimental in response to ischemia/ reperfusion injury in the heart (25). VitD is able to induce autophagy in various human cell types. The signaling pathways regulated by VitD include for example Bc1–2, Beclin 1, and mammalian target of rapamycin (mTOR) (26). During autophagy, intracellular contents are enveloped in double-layered membrane vesicles that fuse with lysosomes for what could be called “recycling process” (27). The interplay between autophagic and apoptotic pathways is a crucial point to determine the initiation of programmed cell death and whether the members of the Beclin l and Bc1–2 family were involved. Beclin l directly interacts not only with Bc1–2 but also with other antiapoptotic Bc1–2 family proteins such as Bcl-xl, and Bc1–2 was able to inhibit the Beclin l-dependent autophagy (28). In this context the aim of the study was to analyze the intracellular pathways activated by VitD under oxidative stress in human endothelial cells.
Materials and Methods Endothelial cell culture HUVEC were obtained from 25 human umbilical vein cord preparations as previously described (18, 29) in accordance with the procedures approved by the local institutional ethics committee. Only the cells positive for vWF/Factor VIII, CD31 and Dil-Ac-LDL were selected. The cells were cultured in 0.1% gelatin-coated flask with endothelial growth medium 2 (EGM-2) as previously described (18). The cells used for all the experiments were at passage 3 to 6. When the cells were at 70% confluence, they were divided for the experiment: in the first group of experiments 1 ⫻ 105 cells were plated in gelatin-coated 24 well plates in complete medium and after the adhesion the cells were incubated for 4 – 6 in DMEM without red phenol and FBS and supplemented with 2 mM glutamine and 1% penicillin-streptomycin (starvation medium; Sigma-Aldrich, Milan, Italy) in incubator to study the cell viability. The nitric oxide production, the reactive oxygen species production (ROS), the mitochondrial permeability transition pore (MPTP) opening and the potential membrane were analyzed in 5 ⫻ 105 cells plated on gelatincoated 6 well plates and maintained in the same conditions used to 24-well plates. To analyze the protein expression, HUVEC were plated on 0.1% gelatin-coated flask with EGM-2 complete
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medium, and after the attachment were incubated overnight in starvation medium in incubator. To confirm the protein expression and cell death through TUNEL assay, 3.5 ⫻ 103 were plated on chamber slide (BD Biosciences, Milan, Italy) with EGM-2 complete medium to perform immunohistochemistry and immunofluorescence analysis. All the experiments were conducted in the oxidative stress condition, which was generated using 200M hydrogen peroxide (H2O2) for 20 minutes in DMEM without FBS and phenol red. In some experiments the cells were pretreated with 1 nM VitD for 15 minutes alone or in combination with 1 nM ZK191784 (Bayer Pharma AG, Berlin, Germany) for 15 minutes and in other experiments VitD or the combination VitD⫹ZK191784 was added after H2O2.
Griess assay In 5 ⫻ 105 cells plated as described before (18), the NO production was measured in culture supernatants using Griess reagents (Promega, Madison, WI, USA) and the results are expressed as percentage (%).
Cell viability (MTT assay) In Vitro Toxicology Assay Kit, MTT based (Sigma-Aldrich) was used to determine cell viability. Details on the method are reported on Supplemental Data. Cell viability was calculated by comparing results to control cells (100% viable).
ROS production The rate of superoxide anion release was used to examine the effects of VitD against the oxidative stress. The superoxide anion production was measured as superoxide dismutase-inhibitable reduction of Cytochrome C. In all samples (stimulated and untreated) 100l of Cytochrome C was added and in another one 100l of superoxide dismutase was also added for 30 minutes in incubator (all substances from Sigma-Aldrich). The absorbance changes in the supernatants of the sample was measured at 550nm in a Wallac Victor mod. 1421 spectrometer (PerkinElmer). The O2. was expressed as nanomoles per reduced Cytochrome C for microgram of protein, using an extinction coefficient of 21000 ml/cm, after the interference absorbance subtraction (30).
Measurement of mitochondrial permeability transition pore (MPTP) After each stimulation, conducted in the same condition and with the same agents used before, and in the other sample pretreated with lM cyclosporin A for 30 minutes (31), the medium was removed and the sarcolemmal membrane was permeabilized using 10 M digitonin (32) per 60 seconds in an intracellular solution buffer (135 mM KC1, 10 mM NaCL, 20 mM HEPES, 5 mM pyruvate, 2 mM glutamate, 2 mM malate, 0.5 mM KH2PO4, 0.5 mM MgC12, 15 mM 2,3-butanedione monoxime, 5 mM ethylene glycol tetraacetic acid, 1.86 mM CaC12; SigmaAldrich) and then loaded with 5 M calcein/AM for 40 minutes at 37°C. After this time the cells were washed with Tyrode solution for 10 minutes and then the fluorescence was measured by a Wallac Victor mod. 1421 at fluorescence excitation and emission of 488nm and 510nm respectively.
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Membrane potential assay After each stimulation as seen before, the medium was removed and the cells were incubated with 5,5⬘,6,6⬘-tetrachloro1,1⬘,3,3⬘-tetraethylbenzimidazolylcarbocyanine iodide following the manufacturer’s instruction (APO-LOGIX JC-1, Bachem, San Carlos, CA, USA). The rate of apoptosis cells was obtained by determining the ratio of red to green fluorescence (33). Details are available in Supplemental Data.
Western blot analysis 40 g of protein from each lysate were resolved on 8% or 15% SDS-PAGE gels and after the transfer of proteins to polyvinylidene fluoride membranes (PVDF) were incubated overnight at 4°C with specific antibodies. Details are available in Supplemental Data.
Caspase-3 activity The same samples used to western blot analysis were used to study the Caspase-3 activity following manifacturer’s instructions (Assay Designs Temaricerca, Bologna, Italy). The results are expressed as percentage. Details are available in Supplemental Data.
Immunohistochemistry The immunostaining procedure was performed on HUVEC plated on chamber slide, using Annexin V (13 g/ml; SigmaAldrich) and Beclin 1 antibody (4 g/ml; Santa-Cruz). At the end the cells were visualized using light microscopic (Leica, Germany). Details are available in Supplemental Data.
Immunofluorescence The immunofluorescence protocol was performed on HUVEC plated on chamber slide to study Caspase-3 specific antibody (13 g/ml; Santa-Cruz). At the end, the cells were visualized using fluorescence microscope (Leica, Germany). Details are available in Supplemental Data.
TUNEL assay For the detection of the endonucleolytic cleavage of chromatin, characteristic of apoptosis, HUVEC cells plated on chamber slide, were analyzed through TUNEL assay kit following the manifacturer’s instruction (ApopTag Plus Peroxidase Apoptosis Detection Kit, Merck Millipore, Milan, Italy). Details are available in Supplemental Data.
Statistical analysis Results are expressed as means ⫾SD of five independent experiments. One-way ANOVA followed by Bonferroni post hoc test were used for statistical analysis. The percentage values were compared through Mann-Whitney U test. The value of P ⬍ .05 was considered as statistically significant.
Results Effects of VitD on NO production during oxidative stress (H2O2) To analyze the VitD effects on NO production induced by oxidative stress, HUVEC were treated with H2O2, VitD
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and/or agonist of VitD receptor (ZK191784) alone or combined. The results obtained by Griess method demonstrated that 1 nM VitD alone was able to cause NO release (P ⬍ .05) of about 70.33 ⫾ 4.73 compared to controls and this effect was amplified (P ⬍ .05) in the sample treated in combination with 1 nM ZK191784 (123.3 ⫾ 6.51%; Figure 1). These data confirmed which was observed in a previously study on NO production caused by VitD in endothelial cells (18). During the oxidative stress induced by 200 M H2O2, we observed a reduction on NO release of about 10 ⫾ 1.52% (P ⬍ .05) compared to controls, which was a significantly (P ⬍ .05) reverse in the samples pretreated with 1 nM VitD (26.33 ⫾ 4.16%) alone and amplified in combined pretreatment with 1 nM ZK191784 (53.33 ⫾ 7.64%). In the samples treated before with H2O2, the stimulations with VitD alone or in combination with ZK191784 were not able to counteract the negative effects on NO production caused by H2O2 (Figure 1). These results suggest that VitD is able to counteract the negative effects caused by oxidative stress and confirme the ability of VitD to induce NO release. Effects of VitD on cell viability during oxidative stress (H2O2) In the same experimental conditions using MIT test, it has been studied if VitD was able to prevent a reduction of cell viability during the oxidative stress (Figure 2). After the stimulation with 1 nM VitD alone the cell viability showed a significant increase of about 18.57 ⫾ 2.13% (P ⬍ .05) and in combination with 1 nM ZK191784 this effect was amplified of about 39.5% (25.91 ⫾ 1.02%, P ⬍ .05). These data indicate the ability of VitD to increase the
Figure 1. Effects on NO production determined by means of Griess method, induced by VitD (1 nM) alone or in combination with ZK191784 (1 nM) and/or H2O2 (200 M) in HUVEC cultures. C ⫽ control; Z19 ⫽ ZK191784; Z19⫹VitD ⫽ combination; H ⫽ 11202; VitD⫹H ⫽ combination; Z19⫹VitD⫹H ⫽ combination; H⫹VitD ⫽ combination; H⫹Z19⫹VitD ⫽ combination. * P ⬍ .05 vs. control; arrows indicate: H⫹VitD P ⬍ .05 vs. VitD⫹H; H⫹Z19⫹VitD P ⬍ .05 vs. Z19⫹VitD⫹H. Reported data are means (%) ⫾ SD of 5 independent experiments.
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cell viability in physiological condition. The pretreatment with 200 M H2O2 for 20 minutes caused a reduction of cell viability (-9.77 ⫾ 0.91%, P ⬍ .05) that was prevented only by the pretreatment with 1 nM VitD alone (13.16 ⫾ 1.37% compared to controls) or in combination with 1 nM ZK191784 (16.95 ⫾ 1.66% compared to controls; Figure 2). This effect is similar to what observed on NO production. For this reason we hypothesize that VitD is able to prevent the negative effects of H2O2 only if was used before the oxidative stress. Effects of VitD on reactive oxygen species production In HUVEC treated with 1 nM VitD alone or in combination with 1 nM ZK191784 it has not been observed significant changes in superoxide anion production compared to controls (P ⬎ .05), but in the sample treated with 200 M H2O2, the superoxide anion production was increased to 23.08 ⫾ 1.17 nmol per reduced Cytochrome C per g of protein (P ⬍ .05) from control values of 4.7 ⫾ 1.096 nmol per reduced Cytochrome C per g of protein (Figure 3). The pretreatment with 1 nM VitD alone was able to reduce superoxide anion production (P ⬍ .05) and this effect was amplified of 19.7% (P ⬍ .05) in presence of 1 nM ZK191784 (15.17 ⫾ 0.38, 12.19 ⫾ 1.6 nmol per reduced Cytochrome C per g of protein respectively). The stimulation with 1 nM VitD alone or in combination with 1 nM ZK191784 after the treatment with 200 M H2O2, did not induced an important superoxide anion reduction compared to the effect of H2O2 alone (P ⬎ .05). These data confirmed previous findings on NO production and MTT test, and they demonstrate the ability of VitD to prevent the negative effects of the oxidative stress only if it used before the oxidative event.
Figure 2. Effects on cell viability determined by means of MTT test, induced by VitD (1 nM) alone or in costimulation with ZK191784 (1 nM) and/or H2O2 (200 M) in HUVEC cultures. The abbreviations are the same used in Figure 1. * P ⬍ .05 vs. control; arrows indicate: H⫹VitD P ⬍ .05 vs. VitD⫹H; H⫹Z19⫹VitD P ⬍ .05 vs. Z19⫹VitD⫹H. Reported data are means (%) ⫾ SD of 5 independent experiments.
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VitD counteracts apoptosis caused by H2O2 through activation of autophagy and survival signaling To investigate the mechanisms activated by VitD during the oxidative stress to counteract the negative effects of H2O2, HUVEC were treated with the same experimental protocol previously used. As reported in Figure 4 and 5, the effects induced by the stimulation with VitD alone were able to induce significant changes (P ⬍ .05) in expression of apoptosis- and autophagy-associated proteins. The only VitD administration reduced Bax, Caspase-3, Caspase-8, Caspase-9 and Cytochrome C expression compared to control (P ⬍ .05). Similar data were also obtained by immunohistochemistry and immunofluorescence analysis on Annexin V, TUNEL assay and Caspase-3 fluorescence analysis, confirming a protective role of VitD on cell death. In addition VitD was able to increase Beclin 1, ERK and Akt compare to control (P ⬍ .05). Graded and amplified changes in apoptosis and autophagy expression were found also in presence of combination with VitD and VDR agonist ZK191784; a reduction of Bax, Caspase-3, Caspase-8, Caspase-9, Annexin V, TUNEL and Cytochrome C expression and an increase of Beclin 1, ERK and Akt were observed as well (P ⬍ .05) (Figure 4, 5 and 7). Samples treated with H2O2 alone showed the opposite effects: the markers of apoptosis (Bax, Cytochrome C, Caspases-8,3,9, TUNEL and Annexin V) were increased compared to controls (P ⬍ .05) and the signaling of survival was decreased. Similar data were obtained in Caspase-3 activity test (Figure 5F), in which was observed that VitD alone caused a reduction of Caspase-3 activity
Figure 3. Effects on reactive oxygen species production (ROS) induced by VitD (1 nM) alone or in combination with ZK191784 (1 nM) and/or H2O2 (200 M) in HUVEC cultures undergoing to oxidative stress. The abbreviations are the same used in previous Figures. * P ⬍ .05 vs. control; arrows indicate: H⫹VitD P ⬍ .05 vs. VitD⫹H; H⫹Z19⫹VitD P ⬍ .05 vs. Z19⫹VitD⫹H. Reported data are means ⫾ SD of 5 independent experiments.
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(-4.48 ⫾ 0.78%; P ⬍ .05) and in combination with ZK191784 this effect was amplified (-5.67 ⫾ 2.08%; P ⬍ .05). The stimulation with H2O2 alone induced a significant increase in Caspase-3 activity respect to control values (41.46 ⫾ 1.46%; P ⬍ .05). The pretreatment with VitD alone or in combination with ZK191784 was able to prevent the activity of Caspase-3 during the oxidative stress (3.5 ⫾ 1.5% and 2.6 ⫾ 0.6%, respectively), but the same stimulation after the treatment with H2O2 not reverse the effect (50.26 ⫾ 1.78% and 50.67 ⫾ 4.16%, respectively). These results were also confirmed by immunofluorescence analysis (Figure 7D). As shown in Figure 5, pretreatment with H2O2 and the successive stimulation with VitD alone or in costimulation with ZK191784 showed that VitD and its agonist were able to reduce the activation of apoptosis and to increase expression of autophagic and survival signaling compared to treatment with H2O2 alone.
Figure 4. Effects of VitD (1 nM) alone or in combination with ZK191784 (1 nM) and/or H2O2 (200 M) on the activation of Beclin-1 (A), Box (B), and the phosphorylation of ERK 1/2 (C). In the panel are reported of immunoblots of activation and phosphorylation relative to specific proteins. The abbreviations are the same as used above. The results represented an example of 5 independent experiments.
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Vitamin D and endothelial oxidative stress
Effects of VitD during oxidative stress on mitochondrial membrane potential and MPTP opening As shown in Figure 6A, 1 nM VitD was able to prevent (45.33 ⫾ 8.39%, P ⬍ .05) the loss of mitochondrial membrane potential induced by 200 M H2O2 (-11 ⫾ 4.58%) and this effect was amplified in presence of 1 nM ZK191784 (71 ⫾ 3.61%, P ⬍ .05). These results demonstrated that VitD could prevent the loss of mitochondrial membrane potential through the modulation of apoptosis/ autophagy pathways. Moreover, additional experiments were performed to examine the modulation of MPTP opening. As reported in Figure 6B, the stimulation with 200 M H2O2 caused a significant (P ⬍ .05) reduction of mitochondria-trapped calcein intensity (-34 ⫾ 5.92%; P ⬍ .05), which was prevented by the treatment with 1 nM VitD and in presence of 1 nM ZK191784 this effect was amplified (P ⬍ .05). In the sample pretreated with 1 M cyclosporine A, agent that interacts with cyclophilin D to
Figure 5. Densitometric analysys of the effects of VitD (1 nM) alone or in combination with ZK191784 (1 nM) and/or H2O2 (200 M) on various kinases. In A the phosphorylation of Akt, in B the activation of cytochrome C, in C the activation of caspase-8, in D the activation of caspase-9, in E and F the activation of caspase-3 and relative protein activity. The abbreviations are the same used in previous. * P ⬍ .05 vs. control; arrows indicate: H⫹VitD P ⬍ .05 vs. VitD⫹H; H⫹Z19⫹VitD P ⬍ .05 vs. Z19⫹VitD⫹H. Reported data are means (%) ⫾ SD of 5 independent experiments.
J Clin Endocrinol Metab
delay MPTP opening, was observed a significant increase of calcein intensity in the sample stimulated with H2O2 alone (28 ⫾ 6.03%; P ⬍ .05), and when VitD alone or in combination with ZK191784 was added these effects were amplified (34.03 ⫾ 4% and 92.33 ⫾ 5.86% compared to controls), respectively. In conclusion the results demonstrated that VitD was able to prevent MPTP opening during the oxidative stress.
Discussion The present research explains the mechanism activated by VitD to counteract the negative effects induced by oxidative stress in endothelial cells. As reported in literature, VitD is able to reduce the damage following H2O2-mediated stress in a dose- and time-dependent manner through the decrease of anion superoxide generation and apoptotic cells (34). In the present study it has been clearly demon-
Figure 6. Study of mitochondrial membrane potential (A) and MPTP opening (B) in HUVEC treated with VitD (1 nM) alone or in combination with ZK191784 (1 nM) and/or H2O2 (200 M). In A the abbreviations are the same used in previous Figures. * P ⬍ .05 vs. control; arrows indicate: Z19⫹VitD⫹HP P ⬍ .05 vs. VitD⫹H. In B CsA ⫽ cyclosporin A 1 M, the other abbreviation are the same used above, * P ⬍ .05 vs. control; arrows indicate: Z19⫹VitD⫹H P ⬍ .05 vs. VitD⫹H; CsA⫹VitD⫹H P ⬍ .05 vs. V⫹H; CsA⫹Z19⫹VitD⫹H P ⬍ .05 vs. Z19⫹VitD⫹H; CsA⫹Z19⫹VitD⫹H P ⬍ .05 vs. CsA⫹VitD⫹H. Reported data are means (%) ⫾ SD of 5 independent experiments.
doi: 10.1210/jc.2013-2103
strated that the administration of VitD to endothelial cells before the induction of an oxidative stress can improve cell viability. The mechanisms involved include the prevention of free oxygen radical release and the modulation of the interplay between apoptosis and autophagy. These effects were also accompanied by NO production and preservation of mitochondrial function. In these experiments HUVEC cultures received an oxidative stress by means of H2O2. This method is widely used in order to reproduce a cellular damage similar to what occurs in myocardial ischemia/reperfusion injury. In the light of these data, a cardioprotective role of VitD against the ischemic injury can be hypothesized. As observed in NO production and MTT tests, the effect induced by VDR agonist ZK191784 is greater than the one observed after VitD alone. Moreover, the combined administration of the two VDR agonists (VitD and ZK191784) induced an amplified effect. The reason why VitD and ZK191784 combined administration induces a more potent effect on HUVEC could be an interesting issue for successive research. These beneficial effects were observed when VDR agonists have been administered before the induction of oxidative stress. This fact supports the hypothesis that VitD is able to counteract the negative effects of the oxidant event on endothelial cells increasing the cell viability. In addition, NO release induced by VitD during oxidative stress is able to protect cells from death. This finding is demonstrated by the observation that rate of NO production observed in this study was below to 2 M/s. This threshold prevents the opening of mitochondrial transition pore and the release of cytochrome C and avoids the mitochondrial collapse leading to cell death (35). Oxidative stress plays an important role in the pathogenesis of atherosclerosis (36, 37). In last years several studies have indicated that antioxidant vitamins such as vitamin C or E may restore endothelial function and may have anti-inflammatory and antithrombotic properties (38). Moreover an overwhelming body of evidence demonstrated that NO plays a fundamental biological role in protecting the heart against ischemia/reperfusion injury. Recent investigations on cardiac myocytes show that ROS produced by mitochondrial and oxidative stress can cause multiple changes in cell structure and function that are associated with the failing heart (21) and apoptosis (22) or autophagy (23). The antiapoptotic effects of NO and ROS involved several signaling pathways and the interplay between autophagic and apoptotic pathways is a crucial point to determining the initiation of programmed cell death. In the present study, the pretreatment with VitD, alone or in combination with ZK191784, is able to reduce the apoptosis-related gene expression (Bax, Caspase-3, Caspase-9, Caspase-8, cytochrome C), involving both in-
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trinsic and extrinsic pathways. These findings were confirmed by immunohistochemistry analysis of Annexin V and TUNEL assay in which we observed a signal reduction. At the same time it has been shown the activation of proautophagic Beclin 1 and the phosphorylation of ERK1/2 and Akt, member of reperfusion injury salvage kinase (RISK) pathway, indicating a modulation between apoptosis and autophagy. Moreover VitD, alone or in combination with ZK191784, administered before the oxidative stress, is able to prevent the loss of mitochondrial potential and the consequent cytochrome C release and caspase activation. In addition, VitD alone or in combination with ZK191784 was able to prevent the MPTP opening caused by H2O2. These findings depend on changes of mitochondria-trapped calcein intensity and on effects of cyclosporine A, which inhibits MPTP opening, modulating the cyclophilin D activity (38). This work adds new information to the debate on the benefits of VitD supplementation. In fact the present study shows for the first time that VitD may prevent endothelial cell death through modulation of interplay between apoptosis and autophagy. This effect is obtained by inhibiting superoxide anion generation, maintaining mitochondria function and cell viability, activating survival kinases (ERK and Akt) and inducing NO production.
Acknowledgments This work was supported by SISDIM (Società Italiana per lo studio della Sarcopenia e delle Disabilità Muscoloscheletriche). The authors thank Bayer Pharma AG (Berlin, Germany) for donating the VDR agonist ZK191784. Thanks to Dr. Mariangela Fortunato for her help. Address all correspondence and requests for reprints to: Corresponding author (to whom reprint requests should be addressed): Francesca Uberti, [email protected]. Laboratorio di ricerca molecolare per lo studio e la cura delle patologie riproduttive, U.O. Ostetricia e Ginecologia 2, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, via Manfredo Fanti, 6 20122 Milano. Disclosure Summary: The authors have nothing to disclose. This work was supported by .
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