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Promotion of autophagy and inhibition of apoptosis by low concentrations of cadmium in vascular endothelial cells
Toxicology in Vitro 23 (2009) 105–110
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Promotion of autophagy and inhibition of apoptosis by low concentrations of cadmium in vascular endothelial cells Zhiwu Dong a,b, Li Wang a,b, Jiping Xu c,*, Yulian Li c, Yun Zhang b, Shangli Zhang a,b, Junying Miao a,b,* a
Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University, Qilu Hospital, Jinan 250012, China c The Department of Neural Medicine, The Second Hospital of Shandong University, Jinan 250033, China b
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Article history: Received 10 July 2008 Accepted 10 November 2008 Available online 20 November 2008 Keywords: Cadmium Autophagy Integrin b4 Phosphatidylcholine-specific phospholipase C (PC-PLC) Caveolin-1
a b s t r a c t Recent reports, highlighting the relationships of cadmium exposure and vascular diseases, indicated that vascular endothelial cell was the target of cadmium (Cd) toxicity. However, the underlying mechanisms have not been fully elucidated. In this study, we evaluated the internalization of Cd(2+) into human umbilical vein endothelial cells (HUVECs) by a novel Cd(2+)-selective sensor suitable for living cells. Then, we detected apoptosis in the treated cells. Our results showed that Cd(2+) at low concentrations (<10 lmol/l) inhibited apoptosis induced by deprivation of serum and basic fibroblast growth factor (bFGF). To investigate the corresponding molecular mechanisms, we employed acridine orange staining and Western blotting of MAP1 LC3 to detect autophagy, and analyzed the levels of integrin b4, caveolin-1 and activity of PC-PLC. Our results showed that low concentrations of Cd(2+) promoted autophagy and depressed the levels of integrin b4, caveolin-1 and PC-PLC activity. The data suggested that autophagy played a key role in Cd(2+) induced endothelial dysfunction; integrin b4, caveolin-1 and PC-PLC might be the targets of Cd(2+) in vascular endothelial cells. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Cadmium (Cd) is an important industrial and environmental pollutant which produces a variety of adverse effects in humans and animals. Although the concentration of Cd(2+) is relative low in natural environment, its wide distribution and high half period make it cumulative in living cultures (Sugita and Tsuchiya, 1995). Chronic inhalation of Cd(2+) leads to various pathological manifestations, such as inflammation and fibrosis, and eventually causes organ dysfunction and injury. Moreover, Cd(2+) is considered carcinogenic and Cd(2+) exposure has already been linked to cancers of prostate, liver and lung. Recent publications revealed that Cd(2+) is involved in cardiovascular diseases including hypertension, atherosclerosis and myocardial infarction. Cd(2+)-induced cardiovascular impairment may be considerably due to endothelial dysfunction (Kolluru et al., 2006; Wolf and Baynes, 2007; Majumder et al., 2008). Nevertheless its molecular mechanisms are not fully elucidated. Deprivation of serum and growth factor during ischemia induces apoptosis in several cell types, including endothelial cell.
* Corresponding author. Address: Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China. Tel.: +86 531 88364929; fax: +86 531 88565610. E-mail address: [email protected] (J. Miao). 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.11.003
Our previous studies showed that deprivation of serum and bFGF induced apoptosis through elevating membrane protein integrin b4 (Miao et al., 1997b) and phosphatidylcholine-specific phospholipase C (PC-PLC) in vascular endothelial cells (Miao et al., 1997a; Miao et al., 1997c; Du et al., 2003; Zhao et al., 2004, 2005). D609, a specific inhibitor of PC-PLC, promoted cell survival of VECs (Liu et al., 2006). Bae et al. (2003) reported that in immortal human epidermal (RHEK-1) keratinocytes treated with a mixture of arsenic (As), cadmium (Cd), chromium (Cr), and lead (Pb), gene expression level of integrin b4 was altered in a chemical-specific manner. Increasing data showed that PC-PLC was involved in inflammation and cytotoxicity. D609 could effectively rescue cell death induced by Methylmercury in MDCK cells (Kang et al., 2006). However the effect of cadmium on integrin b4 and PC-PLC in normal VECs is still not known. It was reported that autophagy was implicated in the response of human hematopoietic stem cells to toxic concentrations of Cd(2+) (Di et al., 2008). But whether low concentrations of Cd(2+) induce autophagy in VECs is not clear. Recent report indicated that Cd(2+) induced apoptotic cell death in mouse brain microvascular endothelial cells (Jung et al., 2008). Interestingly, when we studied the effect of Cd(2+) on vascular endothelial cells, we found that low concentrations of Cd(2+) inhibit apoptosis induced by serum and bFGF deprivation in vascular endothelial cells. This phenomenon attracted us to further investigate the underlying mechanisms.
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2. Materials and methods
2.5. Immunofluorescence staining
2.1. Cell culture and Cd(2+) exposure
To detect early signal events in the process, we exposed HUVECs of subconfluence to M199 medium without serum and bFGF (control) or different concentrations of Cd(2+) for 4 h. Then cells were washed with 0.1 M PBS three times, fixed with 4% paraformaldehyde and blocked with 3% normal goat serum at room temperature. Specific polyclonal IgG antibody (1:200) served as primary Ab and cells were incubated at 4 °C over night. After rinsing three times with 0.1 M PBS, a corresponding FITC-conjugated secondary Ab was applied for 1 h at 37 °C in a humid chamber. Uncombined secondary Ab was eliminated by washing with 0.1 M PBS three times. Specimens were visualized with confocal laser scanning microscope. The fluorescent levels in HUVECs were quantified with the software of Leica microscope. Random areas were applied to fluorescent assessment and photographs are representatives of three independent experiments.
Human umbilical vein endothelial cells (HUVECs) were obtained as described previously (Jaffe et al., 1973). Cells were normally cultured in M199 medium supplemented with 10% fetal bovine serum (FBS) and 2 ng/ml basic fibroblast growth factor (bFGF) (both deprived in control group) in 5% CO2 and humidified atmosphere. Culture plates were precoated with 0.1% gelatin. Cadmium nitrate was dissolved in distilled water to the concentration of 0.05 mol/l as stock solution, and further diluted in basic M199 medium to different concentrations as indicated for Cd(2+) exposure. 2.2. Cd(2+) probing with novel sensor A novel Cd(2+)-selective sensor suitable for living cells was recently synthesized. It can distinguish Cd(2+) from Zn(2+) and many other metal ions with both emission shift and fluorescent intensity (Peng et al., 2007). We employed this sensor to detect different concentrations of Cd(2+). The changes of fluorescent intensity were observed in HUVECs. The assay was performed as described preciously (Peng et al., 2007). Cells were incubated with the sensor for 30 min, washed with 0.1 mol/l PBS three times, and further incubated with Cd(2+) for 30 min. Before detecting, we rinsed cells thrice to eliminate free Cd(2+) in the medium. The fluorescent photographs of HUVECs were obtained with confocal laser scanning microscope (Leica, Germany) with the excitation and emission wavelength of 543 nm and 597 nm, respectively. Fluorescence quantification was processed with Leica Confocal Software (LCS Lite). The fluorescence per cell of random area was calculated as follows:
Fluorescence per cell ¼ ðTotal fluorescence Background fluorescenceÞ=Cell number:
2.3. Autophagy detection with acridine orange (AO) staining and Western blot assay
2.6. Determination of PC-PLC activity PC-PLC activity assay was performed as described before (Wu et al., 1997) with a slight modification. After 4 h Cd(2+) exposure, HUVECs were washed with 1 PBS, trypsinized and centrifuged at 1000g for 10 min. Cell sedimentum was re-suspended in 1 ml homogenate solution (20 mmol/l Tris–HCl pH 7.0, 10 mmol/l EGTA, 1 mmol/l EDTA, 1 mmol/l DTT, 1 mmol/l PMSF, 0.34 mol/l sucrose) and sonicated in ice–water mixture for 5 min. After centrifugation at 1000g, 4 °C for 10 min, supernatant was utilized as enzyme sample for PC-PLC activity. The activities were evaluated as described previously (Zhao et al., 2005). 2.7. Statistics analysis Data are shown in mean ± SEM. Statistical analysis was processed in paired t-test by SPSS 11.5 (SPSS Inc., Chicago, IL). A value of P < 0.05 was considered statistically significant. All data are from three independent experiments. 3. Results 3.1. Cd(2+) internalization into HUVECs
The volume of the cellular acidic compartment, as a marker of autophagy, was visualized by lysosomotropic agent acridine orange (Paglin et al., 2001; Arthur et al., 2007). Acridine orange moves freely across biological membrane and accumulates in acidic compartment, where it is seen as fluorescence bright red. Cells were treated for 4 h and stained with 5 lg/ml acridine orange at room temperature for 1 min. Then cells were washed with PBS and visualized under a fluorescent microscope. Cell lysates were extracted in lysis buffer (25 mM Tris-HCl (PH 6.8), 2% SDS, 6% glycerol, 1% 2-mercaptoethanol, 2 mM PMSF, 0.2‰ bromphenol blue and a protease inhibitor cocktail (Sigma, St. Louis, MO)) and prepared for Western blot analysis. Fifty micrograms proteins were separated by 12% SDS–PAGE and transferred onto PVDF membrane. b-actin was used as a loading control and the relative quantities of the proteins were evaluated by quantity one software (BioRad, Hercules, CA, USA). 2.4. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay
To detect the transportation of Cd(2+) ions into cytoplasm, we used a novel Cd(2+)-selective fluorescent chemosensor to image Cd(2+) in living HUVECs. After cells were exposed to 1, 5 and 10 lmol/l Cd(2+) for 30 min, enhanced fluorescence intensity was markedly visualized compared with sensor group in which HUVECs were treated with sensor alone. Although certain amount of fluorescence presented in sensor group, the variation of fluorescence intensity exhibits in a dose dependent manner (Fig. 1). 3.2. Cd(2+) inhibited endothelium apoptosis TUNEL assay is a reliable test for detecting apoptosis and we observed that Cd(2+) at low concentrations could inhibit apoptosis induced by deprivation of serum and bFGF (Fig. 2A,B, *p < 0.05 vs. control, n = 3). Phase contrast graphs showed that low concentrations of Cd(2+) reduced cell detachment which was obviously visualized in control group (Fig. 2C). 3.3. Cd(2+) promoted autophagy in endothelium
HUVECs were treated with Cd(2+) for 24 h as mentioned above. DeadEndTM Fluorometric TUNEL System (Promega, USA) was used to detect the nuclear fragmentation according to the manufacturer’s protocol. Photographs were taken under confocal laser scanning microscope.
Acridine orange staining was performed to detect autophagy. Autophagy is morphologically characterized by an accumulation of autophagosomes (also called autophagic vacuoles). Autophagosomes subsequently fuse with endosomes and eventually with
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Fig. 1. Cd(2+) internalization into HUVECs. A novel sensor imaged the internalization of cadmium into VECs. (A) HUVECs were treated with cadmium at concentrations of 1, 5 and 10 lM. Enhanced fluorescent intensities were visualized compared with control group. (B) bar graph indicated the changes of Fluorescent intensities (**p < 0.01 vs. control, n = 3).
Fig. 2. Inhibiton of apoptosis by Cd(2+) in vascular endothelial cells. Cd(2+) inhibited apoptosis induced by serum and bFGF deprivation. (A) TUNEL assay showed that Cd(2+) at concentrations of 1, 5 and 10 lM inhibited endothelium apoptosis compared with control group. (B) bar graph showed the percentage of positive cells in TUNEL assay (*p < 0.05 vs. control, n = 3). (C) Cd(2+) at concentrations of 1, 5 and 10 lM inhibited the detachment induced by serum and bFGF deprivation in control group.
lysosomes, then forming autophagolysosomes or autolysosomes. The cells in control group after AO staining displayed green fluorescence in cytoplasm and nucleolus but displayed considerable red
fluorescent dots in cytoplasm of Cd(2+)-treated cells, as suggested the formation of acidic autophagolysosomal vacuoles (Fig. 3A). This is consistent with autophagic changes.
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blotting showed that LC3 processing, namely increased ratio of LC3-II/LC3-I, was markedly enhanced after Cd(2+) treatment (Fig. 3B). The results indicated that low concentrations of Cd(2+) induced autophagy which might be one of the causes of apoptosis inhibition in vascular endothelial cells. 3.4. Effect of Cd(2+) on integrin b4 level
Fig. 3. Promotion of autophagy by Cd(2+) in vascular endothelial cells. Low concentrations of cadmium induced autophagy in vascular endothelial cells. (A) AO staining indicated that Cd(2+) at concentrations of 1, 5 and 10 lM induced autophagic vacuolation compared with control group. (B) conversion of LC3-I to autophagic LC3-II was detected by Western blot assay. The ratio of LC3-II/LC3-I (indicated under each lane) was increased after Cd(2+) challenge. The photograph is a representative of three independent experiments.
Microtubule-associated protein 1 light chain 3 (MAP1 LC3-I) localizes in cytoplasm and phosphatidylethanolamine (PE)-conjugated LC3-II on the membrane of autophagic vacuoles. Conversion of soluble 18 kD LC3-I to membrane-bound, 16 kD LC3-II, assessed by Western blot assay, presents hallmark of autophagy. Western
To test whether integrin b4 was involved in Cd(2+)-induced endothelial dysfunction and VEC apoptosis, HUVECs were treated with indicated concentrations of Cd(2+) for 4 h. Then integrin b4, which localizes in glycosphingolipid (GSL)-enriched domains, was imaged by the method of immunofluorescence staining. Our data indicated level of integrin b4 was markedly decreased in HUVECs in a perfect dose dependent manner. The relative levels of integrin b4 were 72.9%, 62.7% and 49.0% in 1, 5 and 10 lM Cd(2+) groups, respectively (presumed as 100% in the control group, Fig. 4). 3.5. Effect of Cd(2+) on caveolin-1 and caveolae Caveolin-1 is marker protein of caveolae. To elucidate the involvement of caveolae after Cd(2+) exposure, we visualized caveolin-1 with immunohistochemical method. Different concentrations of Cd(2+) depressed caveolin-1 level. Caveolin-1 showed a minimum value of 37.2% (*p < 0.05 vs. control, n = 3) in 5 lM Cd(2+)-treated group and it was 55.7% (*p < 0.05 vs. control, n = 3) in 10 lM Cd(2+) group (Fig. 5).
Fig. 4. Cd(2+) depressed the level of integrin b4 in vascular endothelial cells. (A) Fluorescent photographs indicated that Cd(2+) at concentrations of 1, 5 and 10 lM decreased the level of integrin b4 compared with control group. (B) Bar graph showed the relative level of integrin b4 in vascular endothelial cells (*p < 0.05 vs. control, n = 3).
Fig. 5. Cd(2+) depressed the level of caveolin-1 in vascular endothelial cells. (A) fluorescent photographs indicated that Cd(2+) at concentrations of 1, 5 and 10 lM attenuated the level of caveolin-1 compared with control group. (B) bar graph showed the relative level of caveolin-1 in vascular endothelial cells (*p < 0.05 vs. control, n = 3).
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Fig. 6. Cd(2+) decreased the activity of PC-PLC in vascular endothelial cells. PC-PLC activities were depressed by cadmium at concentrations of 1, 5 and 10 lM compared with control group (*p < 0.05, **p < 0.01 vs. control, n = 3).
3.6. Effect of Cd(2+) on PC-PLC activity Previous study showed that PC-PLC was involved in metal induced cytotoxicity (Kang et al., 2006) and VEC apoptosis. We therefore hypothesized that PC-PLC might participate in Cd(2+) induced endothelial dysfunction. After 4 h of Cd(2+) treatment, HUVECs were harvested and cell lysates were employed for PC-PLC assay. PC-PLC activities, which appeared in similar variation tendency to caveolin-1 level, were sharply attenuated after cadmium exposure (*p < 0.05, **p < 0.01 vs. control, n = 3, Fig. 6). In 5 lM group PC-PLC activity was 53.8% of that in control group and it was 64.7% in 10 lM Cd(2+)-treated group. 4. Discussion Recent studies indicated that cadmium had various effects on cardiovascular system, mostly on endothelial cells. Several molecules, such as endothelial nitric oxide synthase (eNOS), cyclooxygenase 2 (COX-2), heat shock protein (HSP), mammalian metallothionein (MT), have been reported to illustrate its pathways in endothelial cells, nevertheless the molecular mechanisms underlying Cd(2+)-induced endothelial dysfunction are not fully elucidated. Our study showed that Cd(2+) at low concentrations promoted autophagy and depressed the levels of integrin b4, caveolin-1 and PC-PLC activity in VEC. The results provided new evidence for understanding the mechanism of Cd(2+) action in VECs. Apoptosis is mediated by caspases and factors released from mitochondria. In contrast, autophagy is characterized by the formation of acidic vesicular organelle (AVO) in cytoplasm. During autophagy macromolecules and organelles sequestered within double-membrane vesicles are delivered to the lysosome/vacuole for degradation and recycling of nutrients. It is crucial for cell survival under environmental stress (Cuervo, 2004; Edinger and Thompson, 2004). In addition, autophagy may promote a type of programmed cell death termed type II programmed cell death. Apoptosis, necrosis and autophagy all contribute to cell death and which is reigning depends on the cell type and triggering factor (Golstein and Kroemer, 2007; Abedin et al., 2007). As we know, in conditions of nutrient depletion inhibition of autophagy by siRNA specific for Atg genes or by the autophagy inhibitor 3-methyladenine can induce cell death. In this study, when challenged with Cd(2+) HUVECs did not undergo apoptosis in 1, 5 and 10 lM groups, whereas autophagy was detected. Although low concentrations of cadmium could inhibit apoptosis and promote autophagy, apoptotic cell death was induced by higher concentrations of Cd(2+) (approximately >20 lM, data not shown). Taken together, our findings support the notion that moderate level of autophagy may prevent apoptosis, but excessive autophagic vacuolization
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also lead to cellular stress, even cell death (Tasdemir et al., 2008). The homeostasis of apoptosis and autophagy in VECs is an intriguing issue. To demonstrate their relationships further studies are in progress in our laboratory. Membrane protein integrin b4, together with its partner integrin a6, functions as receptor of Laminin-5. It plays a pivotal role in hemidesmosome assembly and signal transduction. Recently, increased level of integrin b4 mRNA was observed in larger tumors and in higher grade tumors (Diaz et al., 2005). Although considerable progress has been made to elucidate roles of integrin b4, most researches focused on tumorigenesis and angiogenesis in keratinocytes and cancer cells. Our previous study showed that integrin b4 was involved in apoptotic signal transduction in vascular endothelial cells (Miao et al., 1997b). Elevating or depressing its level by small chemical molecules could promote or inhibit apoptosis, respectively (Zhao et al., 2005, 2008; Wang et al., 2007; Zhang et al., 2008), suggesting that integrin b4 plays a pivotal role in VEC apoptosis. PC-PLC is a key upstream signal molecule. It hydrolyzes phosphatidylcholine (PC) to produce phosphocholine (PCho) and diacylglycerol (DAG). The latter could educe prolonged activation of protein kinase C (PKC). Our previous studies showed that PC-PLC played a significant role during VEC senescence and apoptosis. VEC impairment of senescence and apoptosis leads to enhanced vessel wall permeability to cytokines, growth factors, lipids and immune cells, increases coagulatory activity of VECs and induces atherosclerotic plaque rupture (Choy et al., 2001). Consistent with our previous reports, the data of this study suggested that depressing integrin b4 and PC-PLC inhibited apoptosis in vascular endothelial cells challenged with cadmium. But it is necessary to test whether downregulation of integrin b4 and/or PC-PLC could promote autophagy in VECs. Emerging evidence focused on the alteration of endothelial caveolae in cardiovascular diseases and endothelial dysfunction. A previous study showed that Cd(2+), as a blocker of voltage-dependent Ca(2+) channel, reduced transient K(Ca) current frequency through caveolin-1 (Cheng and Jaggar, 2006). Imaging caveolae by the marker caveolin-1 provided indirect clue that caveolae might be one of primal targets of cadmium. In this study, we found that low concentrations of Cd(2+) depressed the level of caveolin-1 obviously, indicating that cadmium exposure could lead to severe interference of caveolae in VECs. Because some signal molecules exert their function through localization to and interaction with other lipid raft components, we supposed that cadmium might affect the natural characteristics of signal transduction within lipid rafts. Furthermore, recent reports suggested that integrin b4 and PC-PLC might be involved in caveolae maintenance and distribution. The findings encouraged us to further investigate their relationships in autophagy. In summary, our results firstly highlighted the important relationships of apoptosis and autophagy in VECs during cadmium exposure. It also might provide underlying mechanisms of cadmium induced endothelial dysfunction, as well as potential strategy for the prevention of cadmium induced endothelial impairment. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements Here, we thank Dr. Peng (State Key Laboratory of Fine Chemicals, Dalian University of Technology) for kindly providing the Cd(2+) selective sensor. This study was supported by the National 973 Research Project (No. 2006CB503803) and by National Natural Science Foundation of China (No. 90813022).
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Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Promotion of autophagy and inhibition of apoptosis by low concentrations of cadmium in vascular endothelial cells Zhiwu Dong a,b, Li Wang a,b, Jiping Xu c,*, Yulian Li c, Yun Zhang b, Shangli Zhang a,b, Junying Miao a,b,* a
Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University, Qilu Hospital, Jinan 250012, China c The Department of Neural Medicine, The Second Hospital of Shandong University, Jinan 250033, China b
a r t i c l e
i n f o
Article history: Received 10 July 2008 Accepted 10 November 2008 Available online 20 November 2008 Keywords: Cadmium Autophagy Integrin b4 Phosphatidylcholine-specific phospholipase C (PC-PLC) Caveolin-1
a b s t r a c t Recent reports, highlighting the relationships of cadmium exposure and vascular diseases, indicated that vascular endothelial cell was the target of cadmium (Cd) toxicity. However, the underlying mechanisms have not been fully elucidated. In this study, we evaluated the internalization of Cd(2+) into human umbilical vein endothelial cells (HUVECs) by a novel Cd(2+)-selective sensor suitable for living cells. Then, we detected apoptosis in the treated cells. Our results showed that Cd(2+) at low concentrations (<10 lmol/l) inhibited apoptosis induced by deprivation of serum and basic fibroblast growth factor (bFGF). To investigate the corresponding molecular mechanisms, we employed acridine orange staining and Western blotting of MAP1 LC3 to detect autophagy, and analyzed the levels of integrin b4, caveolin-1 and activity of PC-PLC. Our results showed that low concentrations of Cd(2+) promoted autophagy and depressed the levels of integrin b4, caveolin-1 and PC-PLC activity. The data suggested that autophagy played a key role in Cd(2+) induced endothelial dysfunction; integrin b4, caveolin-1 and PC-PLC might be the targets of Cd(2+) in vascular endothelial cells. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Cadmium (Cd) is an important industrial and environmental pollutant which produces a variety of adverse effects in humans and animals. Although the concentration of Cd(2+) is relative low in natural environment, its wide distribution and high half period make it cumulative in living cultures (Sugita and Tsuchiya, 1995). Chronic inhalation of Cd(2+) leads to various pathological manifestations, such as inflammation and fibrosis, and eventually causes organ dysfunction and injury. Moreover, Cd(2+) is considered carcinogenic and Cd(2+) exposure has already been linked to cancers of prostate, liver and lung. Recent publications revealed that Cd(2+) is involved in cardiovascular diseases including hypertension, atherosclerosis and myocardial infarction. Cd(2+)-induced cardiovascular impairment may be considerably due to endothelial dysfunction (Kolluru et al., 2006; Wolf and Baynes, 2007; Majumder et al., 2008). Nevertheless its molecular mechanisms are not fully elucidated. Deprivation of serum and growth factor during ischemia induces apoptosis in several cell types, including endothelial cell.
* Corresponding author. Address: Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China. Tel.: +86 531 88364929; fax: +86 531 88565610. E-mail address: [email protected] (J. Miao). 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.11.003
Our previous studies showed that deprivation of serum and bFGF induced apoptosis through elevating membrane protein integrin b4 (Miao et al., 1997b) and phosphatidylcholine-specific phospholipase C (PC-PLC) in vascular endothelial cells (Miao et al., 1997a; Miao et al., 1997c; Du et al., 2003; Zhao et al., 2004, 2005). D609, a specific inhibitor of PC-PLC, promoted cell survival of VECs (Liu et al., 2006). Bae et al. (2003) reported that in immortal human epidermal (RHEK-1) keratinocytes treated with a mixture of arsenic (As), cadmium (Cd), chromium (Cr), and lead (Pb), gene expression level of integrin b4 was altered in a chemical-specific manner. Increasing data showed that PC-PLC was involved in inflammation and cytotoxicity. D609 could effectively rescue cell death induced by Methylmercury in MDCK cells (Kang et al., 2006). However the effect of cadmium on integrin b4 and PC-PLC in normal VECs is still not known. It was reported that autophagy was implicated in the response of human hematopoietic stem cells to toxic concentrations of Cd(2+) (Di et al., 2008). But whether low concentrations of Cd(2+) induce autophagy in VECs is not clear. Recent report indicated that Cd(2+) induced apoptotic cell death in mouse brain microvascular endothelial cells (Jung et al., 2008). Interestingly, when we studied the effect of Cd(2+) on vascular endothelial cells, we found that low concentrations of Cd(2+) inhibit apoptosis induced by serum and bFGF deprivation in vascular endothelial cells. This phenomenon attracted us to further investigate the underlying mechanisms.
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2. Materials and methods
2.5. Immunofluorescence staining
2.1. Cell culture and Cd(2+) exposure
To detect early signal events in the process, we exposed HUVECs of subconfluence to M199 medium without serum and bFGF (control) or different concentrations of Cd(2+) for 4 h. Then cells were washed with 0.1 M PBS three times, fixed with 4% paraformaldehyde and blocked with 3% normal goat serum at room temperature. Specific polyclonal IgG antibody (1:200) served as primary Ab and cells were incubated at 4 °C over night. After rinsing three times with 0.1 M PBS, a corresponding FITC-conjugated secondary Ab was applied for 1 h at 37 °C in a humid chamber. Uncombined secondary Ab was eliminated by washing with 0.1 M PBS three times. Specimens were visualized with confocal laser scanning microscope. The fluorescent levels in HUVECs were quantified with the software of Leica microscope. Random areas were applied to fluorescent assessment and photographs are representatives of three independent experiments.
Human umbilical vein endothelial cells (HUVECs) were obtained as described previously (Jaffe et al., 1973). Cells were normally cultured in M199 medium supplemented with 10% fetal bovine serum (FBS) and 2 ng/ml basic fibroblast growth factor (bFGF) (both deprived in control group) in 5% CO2 and humidified atmosphere. Culture plates were precoated with 0.1% gelatin. Cadmium nitrate was dissolved in distilled water to the concentration of 0.05 mol/l as stock solution, and further diluted in basic M199 medium to different concentrations as indicated for Cd(2+) exposure. 2.2. Cd(2+) probing with novel sensor A novel Cd(2+)-selective sensor suitable for living cells was recently synthesized. It can distinguish Cd(2+) from Zn(2+) and many other metal ions with both emission shift and fluorescent intensity (Peng et al., 2007). We employed this sensor to detect different concentrations of Cd(2+). The changes of fluorescent intensity were observed in HUVECs. The assay was performed as described preciously (Peng et al., 2007). Cells were incubated with the sensor for 30 min, washed with 0.1 mol/l PBS three times, and further incubated with Cd(2+) for 30 min. Before detecting, we rinsed cells thrice to eliminate free Cd(2+) in the medium. The fluorescent photographs of HUVECs were obtained with confocal laser scanning microscope (Leica, Germany) with the excitation and emission wavelength of 543 nm and 597 nm, respectively. Fluorescence quantification was processed with Leica Confocal Software (LCS Lite). The fluorescence per cell of random area was calculated as follows:
Fluorescence per cell ¼ ðTotal fluorescence Background fluorescenceÞ=Cell number:
2.3. Autophagy detection with acridine orange (AO) staining and Western blot assay
2.6. Determination of PC-PLC activity PC-PLC activity assay was performed as described before (Wu et al., 1997) with a slight modification. After 4 h Cd(2+) exposure, HUVECs were washed with 1 PBS, trypsinized and centrifuged at 1000g for 10 min. Cell sedimentum was re-suspended in 1 ml homogenate solution (20 mmol/l Tris–HCl pH 7.0, 10 mmol/l EGTA, 1 mmol/l EDTA, 1 mmol/l DTT, 1 mmol/l PMSF, 0.34 mol/l sucrose) and sonicated in ice–water mixture for 5 min. After centrifugation at 1000g, 4 °C for 10 min, supernatant was utilized as enzyme sample for PC-PLC activity. The activities were evaluated as described previously (Zhao et al., 2005). 2.7. Statistics analysis Data are shown in mean ± SEM. Statistical analysis was processed in paired t-test by SPSS 11.5 (SPSS Inc., Chicago, IL). A value of P < 0.05 was considered statistically significant. All data are from three independent experiments. 3. Results 3.1. Cd(2+) internalization into HUVECs
The volume of the cellular acidic compartment, as a marker of autophagy, was visualized by lysosomotropic agent acridine orange (Paglin et al., 2001; Arthur et al., 2007). Acridine orange moves freely across biological membrane and accumulates in acidic compartment, where it is seen as fluorescence bright red. Cells were treated for 4 h and stained with 5 lg/ml acridine orange at room temperature for 1 min. Then cells were washed with PBS and visualized under a fluorescent microscope. Cell lysates were extracted in lysis buffer (25 mM Tris-HCl (PH 6.8), 2% SDS, 6% glycerol, 1% 2-mercaptoethanol, 2 mM PMSF, 0.2‰ bromphenol blue and a protease inhibitor cocktail (Sigma, St. Louis, MO)) and prepared for Western blot analysis. Fifty micrograms proteins were separated by 12% SDS–PAGE and transferred onto PVDF membrane. b-actin was used as a loading control and the relative quantities of the proteins were evaluated by quantity one software (BioRad, Hercules, CA, USA). 2.4. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay
To detect the transportation of Cd(2+) ions into cytoplasm, we used a novel Cd(2+)-selective fluorescent chemosensor to image Cd(2+) in living HUVECs. After cells were exposed to 1, 5 and 10 lmol/l Cd(2+) for 30 min, enhanced fluorescence intensity was markedly visualized compared with sensor group in which HUVECs were treated with sensor alone. Although certain amount of fluorescence presented in sensor group, the variation of fluorescence intensity exhibits in a dose dependent manner (Fig. 1). 3.2. Cd(2+) inhibited endothelium apoptosis TUNEL assay is a reliable test for detecting apoptosis and we observed that Cd(2+) at low concentrations could inhibit apoptosis induced by deprivation of serum and bFGF (Fig. 2A,B, *p < 0.05 vs. control, n = 3). Phase contrast graphs showed that low concentrations of Cd(2+) reduced cell detachment which was obviously visualized in control group (Fig. 2C). 3.3. Cd(2+) promoted autophagy in endothelium
HUVECs were treated with Cd(2+) for 24 h as mentioned above. DeadEndTM Fluorometric TUNEL System (Promega, USA) was used to detect the nuclear fragmentation according to the manufacturer’s protocol. Photographs were taken under confocal laser scanning microscope.
Acridine orange staining was performed to detect autophagy. Autophagy is morphologically characterized by an accumulation of autophagosomes (also called autophagic vacuoles). Autophagosomes subsequently fuse with endosomes and eventually with
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Fig. 1. Cd(2+) internalization into HUVECs. A novel sensor imaged the internalization of cadmium into VECs. (A) HUVECs were treated with cadmium at concentrations of 1, 5 and 10 lM. Enhanced fluorescent intensities were visualized compared with control group. (B) bar graph indicated the changes of Fluorescent intensities (**p < 0.01 vs. control, n = 3).
Fig. 2. Inhibiton of apoptosis by Cd(2+) in vascular endothelial cells. Cd(2+) inhibited apoptosis induced by serum and bFGF deprivation. (A) TUNEL assay showed that Cd(2+) at concentrations of 1, 5 and 10 lM inhibited endothelium apoptosis compared with control group. (B) bar graph showed the percentage of positive cells in TUNEL assay (*p < 0.05 vs. control, n = 3). (C) Cd(2+) at concentrations of 1, 5 and 10 lM inhibited the detachment induced by serum and bFGF deprivation in control group.
lysosomes, then forming autophagolysosomes or autolysosomes. The cells in control group after AO staining displayed green fluorescence in cytoplasm and nucleolus but displayed considerable red
fluorescent dots in cytoplasm of Cd(2+)-treated cells, as suggested the formation of acidic autophagolysosomal vacuoles (Fig. 3A). This is consistent with autophagic changes.
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blotting showed that LC3 processing, namely increased ratio of LC3-II/LC3-I, was markedly enhanced after Cd(2+) treatment (Fig. 3B). The results indicated that low concentrations of Cd(2+) induced autophagy which might be one of the causes of apoptosis inhibition in vascular endothelial cells. 3.4. Effect of Cd(2+) on integrin b4 level
Fig. 3. Promotion of autophagy by Cd(2+) in vascular endothelial cells. Low concentrations of cadmium induced autophagy in vascular endothelial cells. (A) AO staining indicated that Cd(2+) at concentrations of 1, 5 and 10 lM induced autophagic vacuolation compared with control group. (B) conversion of LC3-I to autophagic LC3-II was detected by Western blot assay. The ratio of LC3-II/LC3-I (indicated under each lane) was increased after Cd(2+) challenge. The photograph is a representative of three independent experiments.
Microtubule-associated protein 1 light chain 3 (MAP1 LC3-I) localizes in cytoplasm and phosphatidylethanolamine (PE)-conjugated LC3-II on the membrane of autophagic vacuoles. Conversion of soluble 18 kD LC3-I to membrane-bound, 16 kD LC3-II, assessed by Western blot assay, presents hallmark of autophagy. Western
To test whether integrin b4 was involved in Cd(2+)-induced endothelial dysfunction and VEC apoptosis, HUVECs were treated with indicated concentrations of Cd(2+) for 4 h. Then integrin b4, which localizes in glycosphingolipid (GSL)-enriched domains, was imaged by the method of immunofluorescence staining. Our data indicated level of integrin b4 was markedly decreased in HUVECs in a perfect dose dependent manner. The relative levels of integrin b4 were 72.9%, 62.7% and 49.0% in 1, 5 and 10 lM Cd(2+) groups, respectively (presumed as 100% in the control group, Fig. 4). 3.5. Effect of Cd(2+) on caveolin-1 and caveolae Caveolin-1 is marker protein of caveolae. To elucidate the involvement of caveolae after Cd(2+) exposure, we visualized caveolin-1 with immunohistochemical method. Different concentrations of Cd(2+) depressed caveolin-1 level. Caveolin-1 showed a minimum value of 37.2% (*p < 0.05 vs. control, n = 3) in 5 lM Cd(2+)-treated group and it was 55.7% (*p < 0.05 vs. control, n = 3) in 10 lM Cd(2+) group (Fig. 5).
Fig. 4. Cd(2+) depressed the level of integrin b4 in vascular endothelial cells. (A) Fluorescent photographs indicated that Cd(2+) at concentrations of 1, 5 and 10 lM decreased the level of integrin b4 compared with control group. (B) Bar graph showed the relative level of integrin b4 in vascular endothelial cells (*p < 0.05 vs. control, n = 3).
Fig. 5. Cd(2+) depressed the level of caveolin-1 in vascular endothelial cells. (A) fluorescent photographs indicated that Cd(2+) at concentrations of 1, 5 and 10 lM attenuated the level of caveolin-1 compared with control group. (B) bar graph showed the relative level of caveolin-1 in vascular endothelial cells (*p < 0.05 vs. control, n = 3).
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Fig. 6. Cd(2+) decreased the activity of PC-PLC in vascular endothelial cells. PC-PLC activities were depressed by cadmium at concentrations of 1, 5 and 10 lM compared with control group (*p < 0.05, **p < 0.01 vs. control, n = 3).
3.6. Effect of Cd(2+) on PC-PLC activity Previous study showed that PC-PLC was involved in metal induced cytotoxicity (Kang et al., 2006) and VEC apoptosis. We therefore hypothesized that PC-PLC might participate in Cd(2+) induced endothelial dysfunction. After 4 h of Cd(2+) treatment, HUVECs were harvested and cell lysates were employed for PC-PLC assay. PC-PLC activities, which appeared in similar variation tendency to caveolin-1 level, were sharply attenuated after cadmium exposure (*p < 0.05, **p < 0.01 vs. control, n = 3, Fig. 6). In 5 lM group PC-PLC activity was 53.8% of that in control group and it was 64.7% in 10 lM Cd(2+)-treated group. 4. Discussion Recent studies indicated that cadmium had various effects on cardiovascular system, mostly on endothelial cells. Several molecules, such as endothelial nitric oxide synthase (eNOS), cyclooxygenase 2 (COX-2), heat shock protein (HSP), mammalian metallothionein (MT), have been reported to illustrate its pathways in endothelial cells, nevertheless the molecular mechanisms underlying Cd(2+)-induced endothelial dysfunction are not fully elucidated. Our study showed that Cd(2+) at low concentrations promoted autophagy and depressed the levels of integrin b4, caveolin-1 and PC-PLC activity in VEC. The results provided new evidence for understanding the mechanism of Cd(2+) action in VECs. Apoptosis is mediated by caspases and factors released from mitochondria. In contrast, autophagy is characterized by the formation of acidic vesicular organelle (AVO) in cytoplasm. During autophagy macromolecules and organelles sequestered within double-membrane vesicles are delivered to the lysosome/vacuole for degradation and recycling of nutrients. It is crucial for cell survival under environmental stress (Cuervo, 2004; Edinger and Thompson, 2004). In addition, autophagy may promote a type of programmed cell death termed type II programmed cell death. Apoptosis, necrosis and autophagy all contribute to cell death and which is reigning depends on the cell type and triggering factor (Golstein and Kroemer, 2007; Abedin et al., 2007). As we know, in conditions of nutrient depletion inhibition of autophagy by siRNA specific for Atg genes or by the autophagy inhibitor 3-methyladenine can induce cell death. In this study, when challenged with Cd(2+) HUVECs did not undergo apoptosis in 1, 5 and 10 lM groups, whereas autophagy was detected. Although low concentrations of cadmium could inhibit apoptosis and promote autophagy, apoptotic cell death was induced by higher concentrations of Cd(2+) (approximately >20 lM, data not shown). Taken together, our findings support the notion that moderate level of autophagy may prevent apoptosis, but excessive autophagic vacuolization
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also lead to cellular stress, even cell death (Tasdemir et al., 2008). The homeostasis of apoptosis and autophagy in VECs is an intriguing issue. To demonstrate their relationships further studies are in progress in our laboratory. Membrane protein integrin b4, together with its partner integrin a6, functions as receptor of Laminin-5. It plays a pivotal role in hemidesmosome assembly and signal transduction. Recently, increased level of integrin b4 mRNA was observed in larger tumors and in higher grade tumors (Diaz et al., 2005). Although considerable progress has been made to elucidate roles of integrin b4, most researches focused on tumorigenesis and angiogenesis in keratinocytes and cancer cells. Our previous study showed that integrin b4 was involved in apoptotic signal transduction in vascular endothelial cells (Miao et al., 1997b). Elevating or depressing its level by small chemical molecules could promote or inhibit apoptosis, respectively (Zhao et al., 2005, 2008; Wang et al., 2007; Zhang et al., 2008), suggesting that integrin b4 plays a pivotal role in VEC apoptosis. PC-PLC is a key upstream signal molecule. It hydrolyzes phosphatidylcholine (PC) to produce phosphocholine (PCho) and diacylglycerol (DAG). The latter could educe prolonged activation of protein kinase C (PKC). Our previous studies showed that PC-PLC played a significant role during VEC senescence and apoptosis. VEC impairment of senescence and apoptosis leads to enhanced vessel wall permeability to cytokines, growth factors, lipids and immune cells, increases coagulatory activity of VECs and induces atherosclerotic plaque rupture (Choy et al., 2001). Consistent with our previous reports, the data of this study suggested that depressing integrin b4 and PC-PLC inhibited apoptosis in vascular endothelial cells challenged with cadmium. But it is necessary to test whether downregulation of integrin b4 and/or PC-PLC could promote autophagy in VECs. Emerging evidence focused on the alteration of endothelial caveolae in cardiovascular diseases and endothelial dysfunction. A previous study showed that Cd(2+), as a blocker of voltage-dependent Ca(2+) channel, reduced transient K(Ca) current frequency through caveolin-1 (Cheng and Jaggar, 2006). Imaging caveolae by the marker caveolin-1 provided indirect clue that caveolae might be one of primal targets of cadmium. In this study, we found that low concentrations of Cd(2+) depressed the level of caveolin-1 obviously, indicating that cadmium exposure could lead to severe interference of caveolae in VECs. Because some signal molecules exert their function through localization to and interaction with other lipid raft components, we supposed that cadmium might affect the natural characteristics of signal transduction within lipid rafts. Furthermore, recent reports suggested that integrin b4 and PC-PLC might be involved in caveolae maintenance and distribution. The findings encouraged us to further investigate their relationships in autophagy. In summary, our results firstly highlighted the important relationships of apoptosis and autophagy in VECs during cadmium exposure. It also might provide underlying mechanisms of cadmium induced endothelial dysfunction, as well as potential strategy for the prevention of cadmium induced endothelial impairment. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements Here, we thank Dr. Peng (State Key Laboratory of Fine Chemicals, Dalian University of Technology) for kindly providing the Cd(2+) selective sensor. This study was supported by the National 973 Research Project (No. 2006CB503803) and by National Natural Science Foundation of China (No. 90813022).
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