Cadmium overkill: autophagy, apoptosis and necrosis signalling in endothelial cells exposed to cadmium
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Apoptosis, necrosis, or autophagy—it is the mode of cell demise that defines the response of surrounding cells and organs. In case of one of the most toxic substances known to date, cadmium (Cd), and despite a large number of studies, the mode of cell death induced is still unclear. As there exists conflicting data as to which cell death mode is induced by Cd both across various cell types and within a single one, we chose to analyse Cd-induced cell death in primary human endothelial cells by investigating all possibilities that a cell faces in undergoing cell death. Our results indicate that Cd-induced death signalling starts with the causation of DNA damage and a cytosolic calcium flux. These two events lead to an apoptosis signalling-related mitochondrial membrane depolarisation and a classical DNA damage response. Simultaneously, autophagy signalling such as ER stress and phagosome formation is initiated. Importantly, we also observed lysosomal membrane permeabilization. It is the integration of all signals that results in DNA degradation and a disruption of the plasma membrane. Our data thus suggest that Cd causes the activation of multiple death signals in parallel. The genotype (for example, p53 positive or negative) as well as other factors may determine the initiation and rate of individual death signals. Differences in the signal mix and speed may explain the differing results recorded as to the Cd-induced mode of cell death thus far. In human endothelial cells it is the sum of most if not all of these signals that determine the mode of Cd-induced cell death: programmed necrosis.
KeywordsApoptosis Necrosis Autophagy p53 Lysosome Calcium
Human umbilical vein cells
Intracellular adhesion molecule-1
Vascular cell adhesion molecule-1
Reactive oxygen radicals
Ethylene glycol tetra-acetic acid
Lysosomal membrane permeabilization
Cadmium (Cd) is a toxic heavy metal and pollutant which is ubiquitously distributed in our environment. Human uptake occurs mainly by diet and exposure to cigarette smoke [1, 2]. The primary target organs in the human body are the kidneys, liver, testes, bones [1, 2, 3, 4, 5] and those of the cardiovascular system . Furthermore, Cd is also known to be carcinogenic within different organs and tissues, which has led the International Agency for Research on Cancer to classify Cd as a Group I human carcinogen [7, 8].
Based on the variety of target organs of Cd-induced toxicity, a mass of in vitro data exists investigating the effect of Cd on different cell types. Further, Cd triggers different pathways within different cell types, rendering the landscape even more complex. In this respect one of the most useful examples is in Cd-induced cell death. In general and based upon knowledge gained thus far cell death may be classified into apoptosis, necrosis and autophagy, whereby, above all, detailed molecular and biochemical analyses are indicative of an alternative classification. In this regard, the morphological system of classification is being replaced by a functional system based on the demonstration of detailed signalling pathways and involved molecules . Further adding to the complexity of signalling pathways in response to Cd toxicity, outcome is also dependent on Cd concentration; this may serve to explain some of the contradictory reports presently existing as to Cd-induced cell death. Interestingly, a review of literature revealed that even within one cell type different pathway outcomes are described. Important examples of contradictory findings regarding the Cd-induced cell death signals and final outcome are found in experiments performed upon kidney cells. Different in vitro studies have revealed highly diverse signalling pathways, ranging from caspase-independent  to caspase-dependent [11, 12, 13, 14] apoptosis, although nearly all apoptotic cell death signals have in common an attendant impairment of the mitochondria. Additionally, several studies performed upon kidney cells have demonstrated autophagic signals , as well as signals for apoptosis and necrosis [16, 17, 18] or apoptosis and autophagy [19, 20] within the same cell type. Similarly, Lemaire et al. has reported caspase independent Cd-induced cell death in liver cells , whereas within the same cells, Oh et al. and Lasfer et al. provided evidence of caspase-dependent apoptotic signals [22, 23].
In addition to accumulating in known tissues such as the kidney, liver and bone, increased Cd concentrations were detected in blood serum and aortic walls (up to 20 µM)  of young smokers . Beyond that, we and others have demonstrated that Cd is a risk factor for the development of atherosclerosis leading to fatal cardiovascular outcomes [24, 25, 26, 27, 28, 29]. Thus, in addition to activating the cell death machinery [30, 31, 32] Cd exerts further effects on endothelial cells. These effects include increased expression of the adhesion molecules ICAM-1 [33, 34] and VCAM-1  which possibly facilitates the adhesion and trans-endothelial migration of leucocytes leading to vascular inflammation. Furthermore, Cd treatment is known to attenuate the production of the essential vascular signalling molecule nitric oxide (NO) [36, 37, 38] thereby reducing the responsiveness of the vascular wall to essential signals. In 2000, Liu et al. demonstrated the genotoxic effect of Cd in endothelial cells as a result of the production of reactive oxygen radicals (ROR).  Cd-caused endothelial dysfunction is triggered by defective migratory ability of endothelial cells and inhibited angiogenesis. [40, 41] Ultimately, exposure of endothelial cells to Cd once again initiates cell death signals with inconsistent findings regarding the final fatal outcome. Kim et al. and Jung et al. have postulated a caspase-dependent apoptotic signalling pathway [30, 31] whereas Wolf et al. detected evidence of cell membrane damage and therefore necrosis . Recently, a study conducted by Dong et al. revealed a novel pathway, whereby Cd-induced apoptotic signals are inhibited and rather autophagy is induced . The process of Cd-induced endothelial cell death becomes further complicated still, as we have now shown that Cd triggers a programmed form of necrotic cell death accomplished by the rupture of lysosomes .
Taken together, the results concerning Cd-induced cell death signalling in endothelial cells are inconsistent. Proceeding from the results of a previous study by our group  in which we analysed the effect of Cd as a new risk factor for atherosclerosis development on endothelial cells, the present study aims to provide a highly detailed description of cell death signals within Cd-exposed endothelial cells, from Cd uptake over initiated signalling pathways and involved organelles right up to cell execution.
Materials and methods
A detailed description of the isolation of endothelial cells (HUVECs) of umbilical cords is described elsewhere . The isolation and analyses of the used cells is approved by the Ethics Committee of the Medical University of Vienna (EK Nr. 1183/2012). Endothelial cells were cultured on gelatine-coated (Sigma Aldrich, Austria) polysterene culture flasks (TPP, Switzerland) in a specialised endothelial cell culture medium (EGM-2, Lonza). Prior to each experiment, the medium was replaced by fresh medium containing the indicated Cd concentrations. The following inhibitors were used: 50 µM calpain-inhibitor III (MDL 28170, Sigma Aldrich, Germany), 2 mM 3-Methyladenine (3MA; M9281, Sigma Aldrich, Germany) and 50 µM 2-Aminoethyl diphenylborinate (2APB, D9754, Sigma Aldrich, Germany), 100 µM Dantrolene (550-072-M050, Eubio) and 10 µM BAPTA (BML-CA411-0025, Enzo Lifesciences).
To detect DNA damage in endothelial cells after Cd treatment, a Comet-assay was performed according to the manufacturer’s instructions (Trevigen, Cat.No.: #4250-050-K).
Generation of stable BCL-XL OE cells as well as p53 KD HUVECs
Generation of stable BCL-XL OE cells and knock-down of p53 in endothelial cells was performed as previously described . Monitoring of stable p53 KD and BCL-XL OE in endothelial cells is shown in the Supplemental Material, Figure S10, S11.
Quantification of intracellular Ca2+ concentration
After the treatment, cells were incubated with 1 µM FLUO 3/AM (Calbiochem, # 343242), a cell-permeable Ca2+ indicator that exhibits an increase in fluorescence upon Ca2+ binding. Increase in fluorescence intensity after Cd treatment was detected using flow cytometry analysis (FACS Canto II, BD Biosciences) and quantified as change in the intensity as compared to the control level. We used BAPTA-AM (BML-CA411-0025, Enzo Life Sciences) at a concentration of 10 µM as a sensitive intracellular Ca2+ chelator.
Detection of intracellular Cd
After treatment the cells were handled as indicated by the manufacturer’s instructions (Leadmium™ Green AM Dye for intracellular Detection of Lead and Cadmium; Life Technologies, Cat.No.: 10024) and analysed by flow cytometry (FACS Canto II, BD Biosciences, Germany).
Detection of changes in the mitochondrial membrane potential and staining of mitochondria
After the treatment, cells were enzymatically detached, washed with PBS and incubated with 5 µM JC-1 according to the manufacturer’s instructions (Molecular Probes, Austria). Analysis and quantification of changes in the fluorescence intensity, indicating depolarised mitochondria, was performed using a FACS Canto II (BD Biosciences, Germany). Tracking of the cellular distribution of mitochondria in Cd-treated cells as compared to the control cells was performed using Mitotracker® Red FM and according to the manufacturer’s instructions (Life Technologies, Molecular Probes).
Tracking of lysosome stability
Labelling of lysosomes was performed as previously described in Messner et al. . Endothelial cells with or without an inhibitor and p53 KD cells were incubated with 15 µM Cd and 30 µM Cd, respectively for the indicated times.
Western blot analyses
Whole protein extracts from Cd-treated and control cells were obtained by incubation of detached cells in a triple detergent lysis buffer (50 mM Tris-Chloride, 150 mM Sodium Chloride, 0.02 % Sodium Azide, 0.1 % SDS, 1 % Nonident P-40, 0.5 % Sodium Deoxycholate, 5 µg/ml Aprotinin, 1 µg/ml Leupeptin, 1 µg/ml Pepstatin and 1 mM ABESF). Cells were lysed by repeated freezing and thawing and subsequent sonication. After determination of protein concentration, equal amounts of proteins were separated (20 µg) on SDS–polyacrylamide gels and subsequently transferred onto a nitrocellulose membrane (Schleicher and Schuell, Germany). The primary antibody used was anti-phospho S51 eIF2 alpha (ab47769; Abcam, UK; dilution: 1:750). The secondary antibody used was goat anti-rabbit IgG HRP-conjugate (#31460, Thermo Scientific, Austria). To control loading of equal protein amounts, membranes were stained with Ponceau S and the GAPDH expression (ab9485; Abcam, UK, dilution 1:2500) was analysed using Western blotting (secondary antibody: goat anti-rabbit IgG HRP-conjugate #31460, Thermo Scientific, Austria). Quantification of bands was performed using ImageJ software (National Institute of Health) and the ratios of the band intensities (GAPDH to specific eIF2 alpha S51) were calculated.
Quantification of cell death
For the analysis and quantification of Cd-induced cell death, different methods were used. Annexin V/PI staining served for the discrimination of apoptotic and necrotic cell death. The staining of the cells was performed as described in Bernhard et al. . And the quantification was performed using a flow cytometry device (FACS Canto II, BD Biosciences, Germany). Annexin V and PI positive cells were summarised and quantified compared to corresponding controls to display all dead cells. Furthermore, the effect of Cd on the cell membrane integrity of endothelial cells was analysed by the quantification of lactate dehydrogenase (LDH) release using the LDH cytotoxicity kit II (Biovision) according to the manufacturer’s instructions. Released LDH was quantified as compared to the LDH content of the entire cell.
Detection and quantification of cellular DNA content and PI staining
After permeabilization of cell membranes with 1 mg/ml of saponin, the DNA was stained with PI and the DNA content was then quantified by flow cytometry (FACS Canto II, BD Biosciences, Germany). Moreover, cells were cultivated in Lab Teks (BD Biosciences) and treated with 15 and 30 µM Cd, respectively for 48 h. After fixation with 4.5 % paraformaldehyde, cells were permeabilized with 0.2 % Triton X-100 and cell nuclei were stained with PI (1 mg/ml).
Scanning electron microscopic images of Cd-treated endothelial cells
Fixation of treated cells was performed using 2.5 % glutaraldehyde. Thereafter, cells were dehydrated in a graded ethanol series (70, 90, 100, 100, 100 %, and acetone), desiccated by critical point drying (Emitech K850; Quorum Technologies LTD, West Sussex, UK), mounted, sputtered with gold–palladium (Polaron CA 508; Fisons Instruments, Mainz-Kastel, Germany) and examined with a JEOL JSM—5400 scanning electron microscope (Eching, Germany).
Primary data were tested for a Gaussian distribution and equality of variances. Equal distributed data were subjected either to student’s t Test or to one-sided ANOVA. Statistical analyses were performed using IBM SPSS Statistics 20.0 (SPSS Inc. USA).
Chelation of Cd by EGTA prevents toxicity and Cd treatment induces DNA strand breaks in endothelial cells
Cd treatment provokes an increase in intracellular Ca2+ concentration
Involvement of BCL-XL in Cd-induced Ca2+ flux was analysed using endothelial cells overexpressing this anti-apoptotic protein, and Fig. 2c shows that this overexpression is highly efficient in inhibiting Ca2+ flux. To test the hypothesis that Cd treatment induces a substantial release of Ca2+ from the endoplasmatic reticulum (ER), 2APB (2-Aminoethoxydiphenyl borate) was used to inhibit InsP3 (Inositol 1,4,5-triphosphate)-mediated Ca2+ release. Flow cytometry-based analyses uncovered that 2APB incubation is not able to inhibit Cd-induced Ca2+ flux entirely, but does so significantly as observed 24 h after treatment with 30 µM (Fig. 2d). Referring to the controversially discussed specificity of 2APB as restricted to ER Ca2+ pumps, we analysed the effect of this inhibitor upon the cellular Cd uptake. Leadmium-dye-based intracellular Cd quantification indicates that 2APB significantly inhibits Cd uptake (Fig. 2e).
Cd treatment induces mitochondrial membrane depolarisation and changes in the mitochondrial distribution
Lysosomal membrane permeabilization and induction of autophagy signals are characteristic of death signalling in Cd-treated endothelial cells
Classical AnnexinV/PI based flow cytometry analyses reveal apoptosis as the prevailing mode of Cd-induced cell death
DNA degradation is characteristic of end-stage processes of Cd-induced endothelial cell death
Cd induces the permeabilization of the plasma membrane and release of LDH
The chelating agents EGTA and EDTA (ethylenediamineteteaacetic acid) which are clinically applied for the treatment of metal intoxications clearly reduce Cd-induced DNA degradation in a concentration-dependent manner by forming extracellular complexes with the metal ion, thereby hindering the cellular uptake of Cd.  Once taken up by cells, Cd is known to cause DNA damage as already proven in different cell types such as liver cells [46, 47], fibroblasts , lung cells , and epithelial cells . Similarly, endothelial cells are also sensitive to Cd-induced DNA damage as demonstrated by our previous data showing an increase in the expression of the DNA damage response protein p53  and the latest data showing a pronounced amount of Comet-positive cells after 12 h of Cd treatment. Interestingly, the extent of damage is comparable between 15 and 30 µM Cd, although the number of dead cells is significantly higher after treatment with 30 µM Cd. Furthermore, the involvement of DNA damage and p53 in Cd-induced cell death signalling is shown by the inhibition of cell death (and the preservation of proliferation after Cd treatment, Supplemental Material, Figure S3 A, B) after the knock-down of p53. This demonstrates that Cd causes DNA damage in endothelial cells even at lower concentrations leading to cell death with the involvement of p53.
In addition to the induction of DNA damage, Cd treatment of endothelial cells induces a rapid and substantial increase in intracellular Ca2+ concentration within 6 h. Ca2+, as a universal intracellular messenger, influences a wide range of cellular processes (such as cell death signalling) when the concentration is elevated in the cytosolic compartment [51, 52]. Cytosolic Ca2+ increase is accomplished by either uptake from the extracellular space or release from intracellular Ca2+ stores such as the ER and the mitochondria. 2APB, a critically discussed blocker of Ca2+ release from the ER, reduces Cd-induced cytosolic Ca2+ concentration but simultaneously inhibits both cellular Cd uptake and Cd-induced cell death significantly. Moreover, chelation of extracellular Ca2+ by EGTA protects endothelial cells from Cd-induced cell death. Ca2+ concentration showed no link to p53-dependent DNA damage signalling. Another indication of the release of Ca2+ from the ER through Cd treatment is the fact that the over-expression of BCL-XL—an anti-apoptotic BCL-2 family member located in the ER membrane—is able to inhibit the increase of cytosolic Ca2+ concentration in endothelial cells. However, BCL-XL OE did not effectively prevent endothelial cell death following Cd incubation. Withe and Li et al. confirmed this hypothesis by providing evidence of a regulation of the inositol trisphosphate receptor (InsP3R) by BCL-XL through direct interaction to increase apoptosis resistance . Transmission of the cell death signal from Ca2+ is mediated by a family of Ca2+-dependent non-lysosomal cysteine proteases known as calpains . Interestingly, inhibition of calpain activity reduces intracellular Ca2+ concentration significantly at low Cd concentrations. Although an involvement of autophagy in Cd-induced endothelial cell death is already proven , no interplay between increased cytosolic Ca2+ and autophagy could be detected.
The complex signalling pathway is extended by an involvement of the mitochondria and a variety of factors influencing mitochondrial function . Depolarisation of mitochondria as well as the simultaneous loss of mitochondrial mass certainly occurs late in the Cd-induced cell death process (after 48 h). Similarly to p53-mediated cell death in neuronal cells , Cd toxicity leads to mitochondrial depolarisation through p53, which was reduced by the p53 KD. Since outer membrane permeabilization is weakly inhibited by BCL-XL OE, Bax activation is arguably involved in mitochondrial depolarisation. Additionally, Cd-induced mitochondrial depolarisation is also indirectly induced by calpain activation (as already shown in endothelial cells ) as well as directly through elevated Ca2+ levels as indicated by the limited inhibitory effect of intracellular Ca2+ chelation. Lastly, the close proximity between ER cisternae and the mitochondria allows for local Ca2+ oscillation into mitochondria . All these factors ultimately lead to a central element in apoptosis, which is signal transduction through caspases, known as apoptosis executors. In the case of Cd-treated endothelial cells we have previously shown that caspase-3 is cleaved, but Cd inhibits its activity . Therefore, the apoptotic signalling is halted at this point, underpinned by unchanged intracellular ATP concentration (Supplemental Material, Figure S2).
Ca2+ imbalance and calpain activation not only lead to mitochondrial signalling but also result in the initiation of the UPR and induce ER stress, indicated by the phosphorylation of eIF2 alpha. Phosphorylation of eIF2 alpha disables initiation of translation and thereby inhibits the production of novel unfolded proteins to reduce ER stress. If this protective feedback loop fails, apoptotic signalling is induced—likely, once again, via the mitochondria . Importantly, the PERK-eIF2 alpha complex is responsible for the up-regulation of Atg12 and thereby induces autophagosome formation. The induction of autophagy signals by Cd treatment, shown by an increased LC3 II/LC3 I ratio  as well as cell death protection through 3MA incubation, might therefore be triggered by UPR and eIF2 alpha phosphorylation. However, autophagy is inhibited since Cd treatment of endothelial cells provokes lysosomal membrane permeabilization (LMP) .
Lysosomes are known as “suicide bags” indicating their role in both cell death mainly through necrosis but also in survival mechanisms in the case of autophagy . As previously described, Cd-induced LMP provokes the release of nucleases triggering complete DNA degradation, as well as proteases and lipases causing permeabilization of the plasma membrane . Previously, BCL-XL OE demonstrated a stabilizing effect on LMP which suggests the involvement of the mitochondria . An explanation of this effect could be the fact that BCL-XL attenuates BAX activity, a pro-apoptotic protein which is known to induce LMP. Moreover, as BCL-XL is also present in the ER-membrane, an involvement of this organelle in LMP cannot be excluded. Lastly, we were able to provide a connection between a DNA damage-induced increased expression of p53 and LMP, as the knock-down of p53 rescues cells from Cd-induced LMP, which is already shown in myeloid leukaemic cells . The signalling cascade from increased p53 protein levels to LMP is further confirmed by the protection of DNA degradation and the inhibition of plasma membrane rupture by p53 KD. In contrast to data from neuronal cells by Villalpando Rodriguez et al. , in endothelial cells no connection between calpain activation, LMP induction and DNA degradation could be observed. However, an inhibition of calpain activity inhibits cell membrane permeabilization and preserves proliferation (Supplemental Material, Figure S4 A, B), possibly through an as yet unknown mechanism. Comparable to the inhibition of calpain activity and the knock-down of p53, the disruption of autophagy signals also inhibits plasma membrane permeabilization. Remarkably, an inhibition of apoptotic signalling (by p53 KD and inhibition of calpain activity) combined with autophagy (by using 3MA) was able to completely prevent necrotic cell death induced by 15 µM Cd, and to significantly reduce the number of necrotic cell deaths induced by 30 µM Cd.
The present study is based on several hypotheses, which limit the extensibility of the observed results to the situation in humans. A central limitation is that all observations made are based on an in vitro cell culture model including the chosen Cd concentrations, which are not necessarily applicable to the situation in humans. Cd-concentrations chosen are based on studies by Abu-Hayyeh et al.  who reported on the occurrence of up to 20 µM of Cd in the aortic wall of chronic smokers, and a study by Bergström et al. reporting up to 50 fold increased Cd concentrations in the vascular intima compared to the blood stream. .
The authors would like to thank Anneliese Steinacher-Nigisch, Birgitta Winter, and Eva Eichmair for their excellent technical assistance and Jackson Shaw Kern for careful proof-reading of the manuscript.
Compliance with ethical standards
This project was supported by the Austrian National Bank: Anniversary Fund, Project # 14590 to B.M. and Anniversary Fund, Project # 14745 to D.B.
Conflict of interest
The authors declare no conflict of interest.
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