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HSP70-mediated control of endothelial cell apoptosis during pre-eclampsia
European Journal of Obstetrics & Gynecology and Reproductive Biology 156 (2011) 158–164
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
HSP70-mediated control of endothelial cell apoptosis during pre-eclampsia Padmini Ekambaram*, Lavanya Srinivasan Postgraduate Department of Biochemistry, Bharathi Women’s College, Affiliated to University of Madras, Chennai 600108, Tamil Nadu, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 June 2010 Received in revised form 22 December 2010 Accepted 25 January 2011
Objective: Pre-eclampsia is a hypertensive disorder characterized by maternal vascular endothelial dysfunction. It is likely that this enhanced rate of endothelial cell stress is associated with the pre- and post-partum complications of both mother and fetus. Deciphering the expression pattern of factors involved in altering placental endothelial cell viability in pre-eclampsia aids in identifying components that may protect the fetus from the consequences of placental dysfunction and oxidative stress. Study design: Expression of thioredoxin (Trx), an antioxidant protein; heat shock protein (HSP) 70, a cytoprotective protein; heat shock factor (HSF)1, a transcriptional factor of HSPs; and apoptosis signalregulating kinase 1 (ASK1), a pro-apoptotic protein, was elucidated in endothelial cells from human term placentas of normotensive and pre-eclamptic subjects (n = 35). Results: A significant increase in HSP70 (p < 0.05), HSF1 (p < 0.05), Trx (p < 0.05) and an insignificant increase in ASK1 were noted in pre-eclamptic endothelial cells. Conclusion: This analysis supports the role of HSP70 expression in promoting cell survival by regulating ASK expression in pre-eclampsia. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Pre-eclampsia Endothelial cell Apoptosis HSP70 ASK1 Thioredoxin
1. Introduction Pre-eclampsia, a pregnancy-specific disorder, is characterized by placental abnormalities and maternal vascular endothelial dysfunction [1]. The significance of pre-eclampsia is substantial when considering the requirement for regular antenatal monitoring of symptoms, preservation of maternal health and care of the premature or low birth weight fetus whose incidence of acute morbidity is increasing. This condition may be life-threatening to mother and fetus if it is not properly managed, but it usually ends when the baby and placenta are delivered [2]. A longer-term burden also exists, as pre-eclamptic women are two and half times more likely to develop ischaemic heart disease later in life [3]. The infants born to pre-eclamptic woman are at a higher risk of developing respiratory diseases and long-term neurological morbidity [4]. The increasing association between oxidative and nitrative stress with pre-eclampsia indicates that hypoxia is one of its common complications. The reactive oxygen species (ROS) formed contribute to the imbalance in antioxidant status and oxidative
Abbreviations: HSP, heat shock protein; Trx, thioredoxin; HSF, heat shock factor; ASK1, apoptosis signal-regulating kinase 1; ROS, reactive oxygen species; MAPKKK, mitogen activated protein kinase kinase kinase; BMI, body mass index; PROM, premature rupture of membrane; IUGR, intrauterine growth retardation. * Corresponding author. Tel.: +91 044 26213748; fax: +91 044 25280473. E-mail addresses: [email protected], [email protected] (E. Padmini). 0301-2115/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2011.01.026
stress as seen in pre-eclampsia. This imbalance is known to induce cellular damage by altering cell integrity [5]. The present study focuses on the changes in the expression of the various signalling molecules involved in control of oxidative stress mediated via heat shock proteins (HSPs). HSPs are highly conserved, found in all cell types and expressed as a result of stressful environmental, pathological or physiological stimuli [6]. Apart from protein transport and degradation during unstressed conditions, they are involved in preventing stress-mediated accumulation of misfolded or damaged proteins [7]. HSP acts as an antioxidant in maintaining cellular redox homeostasis. HSPs are found to inhibit intracellular ROS sand increase the glutathione level [8]. There is evidence that HSPs are produced by the placental tissues and that they have a physiological role in stress management during pre-eclampsia [9,10]. HSP70 induction may involve different mechanisms under different circumstances. The regulation of heat shock gene expression in eukaryotes is mediated at the transcriptional level by transcriptional activators, heat shock factor (HSF) [11]. HSF1 is the ubiquitous stressresponsive transcriptional activator essential for the inducible transcription of genes encoding HSPs [12], by binding to regulatory heat shock elements present in the promoter region of all heat shock genes [13]. Thioredoxin (Trx) is a family of small proteins that contains a conserved redox active center [14]. It scavenges ROS such as H2O2 and free radicals through its direct reducing activity [15]. It has been reported that thioredoxin works as an antioxidant, stabilizing redox balance, and is expressed in the placenta [16].
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Table 1 Clinical characteristics of normotensive pregnant women and pre-eclamptic patients. Criteria
Normotensive subjects (n = 35)
Preeclamptic subjects (n = 35)
Maternal age (years) Gestational age (weeks) Pregnancy weight at the time of admission (kg) Pre-pregnancy blood pressure (mmHg) Systolic Diastolic Pregnancy blood pressure at the time of delivery (mmHg) Systolic Diastolic Proteinuria (mg/dL) Xanthine oxidase (U/mg protein) Infant birth weight
27 5.2 38.2 0.4 57.5 7.8
30 4.8NS 30.8 2.5* 72.1 6.3*
114.2 5.1 76.2 5.7
117.2 5.4NS 78.5 4.9NS
122.6 7.6 81.4 7.2 Nil 1.72 0.84 3.42 0.54
167.5 7.9* 112.3 7.4* >300** 2.83 0.96* 2.19 0.54*
NS, non-significant. * p < 0.05 when compared to normotensive subjects. ** p < 0.01 when compared to normotensive subjects.
ASK1 (apoptosis signal regulating kinase 1) is the member of the mitogen-activated protein kinase kinase kinase (MAPKKK) that was shown to be an important signalling kinase in apoptotic cell death [17,18]. Over-expression of ASK1 induces apoptosis through multiple cell death pathway (intrinsic and extrinsic, caspasedependent and independent) induced by various pro-apoptotic stimuli such as lipopolysaccharide, reactive oxygen species (ROS), ischaemic insult and genotoxic stress [19,20]. This paper aims to identify factors that may protect the fetus from the consequences of placental dysfunction and oxidative stress. It is investigating the extent to which the expression of certain cytoprotective and apoptotic proteins could be involved in altering placental endothelial cell viability, thus promoting fetal survival. 2. Materials and methods 2.1. Selection of subjects Patients registered in the Department of Obstetrics & Gynecology of a public sector hospital at Chennai in India were enrolled in the study. The study was carried out for a period of 1 year. The sample consisted of 35 mild pre-eclamptic patients and 35 normotensive subjects of the age 18–40 years. Patients with mild pre-eclampsia were defined on the basis of the following clinical and laboratory criteria: systolic blood pressure in the range of 140–160 mmHg and diastolic blood pressure in the range of 90–110 mmHg noted on at least two occasions; proteinuria concentrations >300 mg/dL measured on at least two random specimens and xanthine oxidase activity of approximately 2.6 U/mg protein [21]. Patients with severe pre-eclampsia were excluded from the study as most of them were advised immediate medication to avoid further complications. Healthy volunteers who were normotensive, of similar race, body mass index (BMI) and without maternal and fetal complications during pregnancy were selected as control subjects. Clearance was obtained from the Hospital Ethical Committee prior to the commencement of the study and informed consent was obtained from all subjects. Pregnant women with other complications such as PROM, IUGR, gestational diabetes, chorioamnionitis, other clinical infections and those undergoing medication were excluded. The clinical characteristics of the pre-eclamptic patients were tabulated and compared with the normotensive pregnant subjects and the data are presented in Table 1.
1 million cells per culture flask (125 mm2) in M199 medium containing 20% fetal calf serum in a 5% CO2 atmosphere at 37 8C. Non-adherent cells and debris were removed by washing three times with PBS the following day. Viability of the endothelial cells was assessed using trypan blue exclusion test [23]. 2.3. Scanning electron microscopy (SEM) analysis of endothelial cells Endothelial cells were fixed for scanning electron microscopy with 4% glutaraldehyde overnight at 4 8C followed by centrifugation at 100 g for 5 min. The supernatant was discarded and the pellet was resuspended in 2% osmium tetroxide for 2 h at room temperature. The centrifugation process was repeated and the samples were dehydrated with an ascending ethanol series (10– 100%). The absolute ethanol was finally displaced by liquid carbon dioxide which served as the transitional fluid for critical point drying. Dried samples were mounted on aluminium stubs and sputter coated with gold (JEC-1100). Electron accelerators for SEM were operated at 15 kV and the samples were viewed with an AMR100 scanning electron microscope (Jeol JSM-6360, Jeol Ltd., Tokyo, Japan). 2.4. Co-immunofluorescence of HSP70 and HSF1 Indirect immunofluorescence technique was performed with the placental sections taken from the subjects of the study according to the method of Pringle et al. [24] with some modifications. The
[()TD$FIG]
2.2. Isolation of endothelial cells Placental endothelial cells were prepared by the method previously mentioned [22]. The cells were cultured overnight at
Fig. 1. Cell viability of endothelial cells isolated from placentas of normotensive and pre-eclamptic subjects.
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primary antibodies used were HSP70 (mouse monoclonal) (SPA810) and HSF1 (Rabbit polyclonal) (SPA 901) antibody, Stressgen, Canada. The primary antibody of HSF1 was detected using an antirabbit fluorescein isothiocyanate (FITC) conjugated secondary antibody (1:3000 dilution) and the primary antibody of HSP70 was detected with anti-mouse IgG Alexa fluor 594. Then the fluorescence of HSP70 and HSF1 was observed using confocal microscope (Leica TCS Sp-2 XL) in 40 magnification that could be [()TD$FIG]visualized as red fluor and green fluor, or as a combined orange fluor.
2.5. Immunohistochemical expression of ASK1 Immunohistochemical expression of ASK1 was achieved using an immunohistochemical staining kit with specific anti-ASK1 antibodies. Placental sections 7-mm thick were mounted onto poly-L-lysine coated slides and stored under dry conditions until histologic analysis. To reduce non-specific binding, slides were incubated in 10% normal goat serum for 10 min at room temperature before 1 h incubation with
Fig. 2. Scanning electron microscopy of endothelial cells isolated from placentas of normotensive women (panels A, C, and E) and pre-eclamptic women (panels B, D, and F). SEM analysis of the endothelial cells isolated from normotensive placenta showed characteristic arrangement of densely packed appearance (panels A, C, and E). While the SEM of endothelial cells isolated from pre-eclamptic placenta (panels B, D, and F) showed changes in the surface morphology. The membrane structures were dispersed with significant alteration in the shape of the cells which contributes to the endothelial cells dysfunction during pre-eclampsia.
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way ANOVA test. The differences were considered significant at p 0.05, 0.01.
primary anti ASK1 (1:2500) antibody in a humidified chamber at 4 8C. The immunohistochemical images were acquired with Zeiss microscopes (Carl Zeiss Microimaging Inc, Thornwood, USA).
3. Results
2.6. Immunoblot analysis of HSP70, ASK1, HSF1 and Trx
3.1. Viability of endothelial cells
The placental endothelial cell protein aliquots containing 50 mg proteins were run on two 10% SDS-polyacrylamide gels that were placed together in the Dual gel electrophoretic system. Both the gels were then blotted onto PVDF membranes (BioTrace PVDF 0.4 (m, Pall Corporation, Germany) according to the method of Towbin et al. [25]. The antibodies used were HSP70 (SPA 810), ASK1 (AAP 480), HSF1 (SPA 901), Trx (MSA150), and b-actin (CSA 400); followed by goat anti-mouse IgG secondary antibody treatment, and colour development was done using BCIP-NBT substrate system. The band intensities were scanned with the HP Scan Imager and quantified using the TotalLab Software (GELS, USA). The results were confirmed by individually performing the blotting studies of HSP70, ASK1, HSF1 and Trx.
Viability of endothelial cells assessed using trypan blue exclusion test showed a significant decrease (p < 0.05) in the viability during pre-eclampsia (78%) compared to the normotensive subjects (89%) immediately after isolation (Fig. 1). The endothelial cells from both the groups were also grown for a period of 5 days in medium M199: the cell viability was >75% in all the groups of cultured cells.
2.7. Statistical analysis All results were expressed as mean standard deviation. Each experiment was performed thrice and data were analyzed by two-
[()TD$FIG]
3.2. Scanning electron microscopy analysis of endothelial cells, coimmunofluorescence of HSP70 and HSF1 and immunohistochemical expression of ASK1 in whole placenta The increased cell disruption and damage to the endothelial cells in pre-eclamptic placenta are clearly demonstrated through the SEM analysis of the endothelial cells isolated from the subjects of the study (Fig. 2). The immunoblot of HSP70 shows that the protein is significantly increased during pre-eclampsia in the placenta compared to normotensive subjects (Fig. 3). The histochemistry slides of the placenta of pre-eclamptic subjects
Fig. 3. Co-immunoblot of HSP70 and HSF1 in the placental sections of normotensive women (panels A–C) and pre-eclamptic women (panels D–F). Co-immunofluorescence images of sections stained for HSF1 with Alexa fluor 594 conjugated goat anti-rabbit IgG secondary antibody (panels A and D) and HSP70 with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG secondary antibody (B and E). Overlay image of HSF1 and HSP70 expressions is shown in panels (C) and (F). HSF1 and HSP70 show positive fluorescent staining in the tissue sections of both normotensive and pre-eclamptic placenta. Intense fluorescent staining for HSP70 and enhanced localization of HSF1 was noted in the pre-eclamptic condition (panels (D) and (E) for HSF1 and HSP70, respectively). In contrast, normotensive placental section demonstrates only a diffuse distribution of HSF1 accompanied with a moderate immunostaining for HSP70 (panels (A) and (B) for HSF1 and HSP70, respectively). Scale bar: 75 mm.
[()TD$FIG]
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Fig. 4. Immunohistochemical staining of ASK1 in the placenta from normotensive women and pre-eclamptic women. Placental sections of both normotensive and preeclamptic subjects show only a diffuse distribution of ASK1. Scale bar: 20 mm.
depict that there is only insignificant increase in the expression of ASK1 in the pre-eclamptic condition compared to normotensive subjects (Fig. 4).
HSF1 (p < 0.05) and Trx (p < 0.05) in pre-eclamptic subject compared to normotensive subjects (Fig. 5). It was observed that there was only an insignificant increase in ASK1 expression which was confirmed by comparison with two-way ANOVA (Fig. 6).
3.3. Immunoblot analysis of HSP70, ASK1, HSF1 and Trx 4. Comments Analysis of the signalling changes was performed using Western blotting. The experiment was performed in triplicate to confirm the results and exclude handling errors. The co-immunoblot analysis revealed a significant increase in HSP70 (p < 0.05),
[()TD$FIG]
Fig. 5. (A) Western blot analysis of normotensive and pre-eclamptic endothelial cells for HSP70 with b-actin as a loading control. (B) Western blot analysis of normotensive and pre-eclamptic endothelial cells for HSF1 with b-actin as a loading control. (C) Western blot analysis of normotensive and pre-eclamptic endothelial cells for ASK1 with b-actin as a loading control. (D) Western blot analysis of normotensive and pre-eclamptic endothelial cells with b-actin as a loading control.
Diminished or poor placental function in pre-eclampsia increases the chances of apoptosis [26]. Most of the clinical findings in pre-eclampsia can be attributed to placental cell damage and abnormalities in endothelial cell function, which are evident from the SEM analysis of the placenta and clinical characteristics exhibited by the pre-eclamptic patients observed in the present study [27]. Endothelial cells were isolated and used freshly in order to annul the influence of cultivation process. All the cases taken for analysis were associated with live fetal delivery in the present study. This suggests the presence of a protective adaptive mechanism in pre-eclampsia that functions to fight the existing condition. Studies in our laboratory have demonstrated an increase in ROS accumulation in placental endothelial cell mitochondria under the condition of pre-eclampsia [22]. The various enzymatic and nonenzymatic antioxidants fail to combat the excessive ROS generated during such complications which stimulate the over-expression of HSPs. The HSP70 quantified using ELISA showed an increase in the expression of the protein in pre-eclamptic endothelial cells (p < 0.05) (unpublished data). HSP forms a powerful system in many intra- and extra-cellular processes including protection against oxidative stress, anti-apoptotic functions and cell proliferation. HSP70 is a potent anti-apoptotic protein that can influence the apoptotic cascade at multiple sites [28]. This study suggests that pre-eclampsia stimulates a multi-faceted response in endothelial cell by inducing HSP70 that acts at different levels to suppress cell death. The oxidative stress noted during pre-eclampsia results in the activation of secondary antioxidant defense system. This might be the prime reason for the increased expression of Trx and HSP70 in these conditions as noted in our study. Trx is a widely distributed intracellular redox protein which constitutes an endogenous antioxidant system in addition to glutathione and superoxide dismutase systems by suppressing free radical formation. It controls the redox state of proteins with its active site by contributing to the reduction of disulfide bonds [29]. Trx plays a role in many cellular processes and its active participation in cell
[()TD$FIG]
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Fig. 6. Comparison of the ASK, Trx, HSF and HSP70 between the two groups of analysis using two way ANOVA.
growth stimulation, chemotaxis and activation of transcription factors in addition to the antioxidant function is prominent [30]. HSP70 has an effect in increasing the active Trx concentration in the local environment. The mechanism by which HSP70 increases free Trx level is not yet understood. The mechanism is thought to be due to the anti-apoptotic function of HSP70 through its chaperoning effect on Trx activating proteins like Trx reductase. Trx is implicated in apoptosis regulation through two different mechanisms: the first is by scavenging reactive oxygen species (ROS) to protect against oxidative stress [30] and the second, by its direct binding and inhibiting the activity of the pro-apoptotic protein apoptosis signal-regulating kinase 1 (ASK1) [31], regulating the activities of transcription factors, such as NF-kB. In mammalian cells, ASK1 participates in the JNK and p38 MAPK signalling cascades by phosphorylating MKK4/MKK7 and MKK3/ MKK6, respectively [32]. ASK1 is activated in response to various extracellular and intracellular stimuli, such as lipopolysaccharide, and ROS [33]. Over-expression of ASK1 induces apoptosis through induction of the mitochondrial pathway [34]. The Trx–ASK1 complex subjects to the ubiquitination and proteasomic attack of the bound ASK1 are achieved [35]. The free Trx protein also has a potential role in stimulating the activation of HSF1 [36], a transcriptional regulator of HSPs. In a native state HSF1 remains as a complex with HSP; in stressed conditions the damaged proteins are thought to compete with HSF1 for binding to HSP, thus leading to the appearance of unbound HSF1 monomer that is free to trimerize, translocate into the nucleus, undergo phosphorylation and activate HSP expression [37]. Disruption of HSF1 causes decreased constitutive expression of several HSPs, which is intimately associated with impaired redox homeostasis and mitochondrial damage [38]. Here we analyze the role of HSP70, a major stress-inducible protein in promoting endothelial cell survival under conditions of preeclampsia by its antioxidant property. The entire process is therefore a systematic cycle where each and every element has a control on the other. Thus, the natural adaptive response is programmed in order to maintain the level of HSP70 in such a way to restore viability of the cell. HSP70 and Trx function to control generated oxidative stress. ASK1 is one of the transcription factors involved in signal transduction that are sensitive to regulation by reactive species. It was found to be expressed with minimal changes from the normotensive samples in spite of the existing complication. This might be due to the control established by the cytoprotective
proteins like HSP70 and Trx. Direct interactions between proapoptotic and anti-apoptotic proteins lead to cell death, in the absence of trophic factors. Binding of trophic factors like HSP70 can trigger changes in these interactions, resulting in cell survival [39]. HSP70 can directly bind and antagonize the function of proapoptotic proteins like ASK1 [40,41]. In the present study, expression of ASK1 was monitored in the samples as it has a direct control over apoptosis [42]. The HSP70–ASK1 complex promotes the ubiquitin-mediated degradation of bound ASK1. HSP70 can indirectly control apoptosis using Trx. Activation of Trx regulates intracellular redox-dependent processes, including transcriptional activity, proliferation and apoptosis [43]. Trx– ASK1 complex pre-exists in the normal unstressed cells and appears to be the target of many extracellular stimuli such as ROS that activate ASK1 by disrupting the Trx–ASK1 complex [44]. The Trx-red (reduced Trx) controls apoptosis by interacting with ASK1 [45]. This modulation of ASK1 through HSP70 up-regulation might be one of the reasons for live birth noted in the present study. As noted in the current study, the delicate balance in the expression of HSP70 promotes the survival of the fetus and aids in live birth in spite of the existing complications. Therefore, the control in the expression of HSP70 plays a crucial role in maintenance of cell viability, which paves way for its usage as a potential therapeutic target for treatment of such conditions as pre-eclampsia. Acknowledgement The authors thank National Tea Research Foundation, Tea Board of India, for providing funds for conducting this work. Project referral number 115/07. References [1] Roberts JM, Cooper DW. Pathogenesis and genetics of pre-eclampsia. Lancet 2001;357:53–6. [2] Higgins JR, de Swiet M. Blood pressure measurement and classification in pregnancy. Lancet 2001;357:131–5. [3] Jonsdottir LS, Arngrimsson R, Geirsson RT, et al. Death rates from ischemic heart disease in women with a history of hypertension in pregnancy. Acta Obstet Gynecol Scand 1995;74:772–6. [4] Van Wijk MJ, Kublickiene K, Boer K, et al. Vascular function in pre-eclampsia. Cardiovasc Res 2000;47:38–48. [5] Muschel RJ, Bernhard EJ, Garza L, et al. Induction of apoptosis at different oxygen tensions: evidence that oxygen radicals do not mediate apoptotic signaling. Cancer Res 1995;55:995–8. [6] Young RA. Stress proteins and immunology. Annu Rev Immunol 1990;8:401–20.
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HSP70-mediated control of endothelial cell apoptosis during pre-eclampsia Padmini Ekambaram*, Lavanya Srinivasan Postgraduate Department of Biochemistry, Bharathi Women’s College, Affiliated to University of Madras, Chennai 600108, Tamil Nadu, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 June 2010 Received in revised form 22 December 2010 Accepted 25 January 2011
Objective: Pre-eclampsia is a hypertensive disorder characterized by maternal vascular endothelial dysfunction. It is likely that this enhanced rate of endothelial cell stress is associated with the pre- and post-partum complications of both mother and fetus. Deciphering the expression pattern of factors involved in altering placental endothelial cell viability in pre-eclampsia aids in identifying components that may protect the fetus from the consequences of placental dysfunction and oxidative stress. Study design: Expression of thioredoxin (Trx), an antioxidant protein; heat shock protein (HSP) 70, a cytoprotective protein; heat shock factor (HSF)1, a transcriptional factor of HSPs; and apoptosis signalregulating kinase 1 (ASK1), a pro-apoptotic protein, was elucidated in endothelial cells from human term placentas of normotensive and pre-eclamptic subjects (n = 35). Results: A significant increase in HSP70 (p < 0.05), HSF1 (p < 0.05), Trx (p < 0.05) and an insignificant increase in ASK1 were noted in pre-eclamptic endothelial cells. Conclusion: This analysis supports the role of HSP70 expression in promoting cell survival by regulating ASK expression in pre-eclampsia. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Pre-eclampsia Endothelial cell Apoptosis HSP70 ASK1 Thioredoxin
1. Introduction Pre-eclampsia, a pregnancy-specific disorder, is characterized by placental abnormalities and maternal vascular endothelial dysfunction [1]. The significance of pre-eclampsia is substantial when considering the requirement for regular antenatal monitoring of symptoms, preservation of maternal health and care of the premature or low birth weight fetus whose incidence of acute morbidity is increasing. This condition may be life-threatening to mother and fetus if it is not properly managed, but it usually ends when the baby and placenta are delivered [2]. A longer-term burden also exists, as pre-eclamptic women are two and half times more likely to develop ischaemic heart disease later in life [3]. The infants born to pre-eclamptic woman are at a higher risk of developing respiratory diseases and long-term neurological morbidity [4]. The increasing association between oxidative and nitrative stress with pre-eclampsia indicates that hypoxia is one of its common complications. The reactive oxygen species (ROS) formed contribute to the imbalance in antioxidant status and oxidative
Abbreviations: HSP, heat shock protein; Trx, thioredoxin; HSF, heat shock factor; ASK1, apoptosis signal-regulating kinase 1; ROS, reactive oxygen species; MAPKKK, mitogen activated protein kinase kinase kinase; BMI, body mass index; PROM, premature rupture of membrane; IUGR, intrauterine growth retardation. * Corresponding author. Tel.: +91 044 26213748; fax: +91 044 25280473. E-mail addresses: [email protected], [email protected] (E. Padmini). 0301-2115/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2011.01.026
stress as seen in pre-eclampsia. This imbalance is known to induce cellular damage by altering cell integrity [5]. The present study focuses on the changes in the expression of the various signalling molecules involved in control of oxidative stress mediated via heat shock proteins (HSPs). HSPs are highly conserved, found in all cell types and expressed as a result of stressful environmental, pathological or physiological stimuli [6]. Apart from protein transport and degradation during unstressed conditions, they are involved in preventing stress-mediated accumulation of misfolded or damaged proteins [7]. HSP acts as an antioxidant in maintaining cellular redox homeostasis. HSPs are found to inhibit intracellular ROS sand increase the glutathione level [8]. There is evidence that HSPs are produced by the placental tissues and that they have a physiological role in stress management during pre-eclampsia [9,10]. HSP70 induction may involve different mechanisms under different circumstances. The regulation of heat shock gene expression in eukaryotes is mediated at the transcriptional level by transcriptional activators, heat shock factor (HSF) [11]. HSF1 is the ubiquitous stressresponsive transcriptional activator essential for the inducible transcription of genes encoding HSPs [12], by binding to regulatory heat shock elements present in the promoter region of all heat shock genes [13]. Thioredoxin (Trx) is a family of small proteins that contains a conserved redox active center [14]. It scavenges ROS such as H2O2 and free radicals through its direct reducing activity [15]. It has been reported that thioredoxin works as an antioxidant, stabilizing redox balance, and is expressed in the placenta [16].
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Table 1 Clinical characteristics of normotensive pregnant women and pre-eclamptic patients. Criteria
Normotensive subjects (n = 35)
Preeclamptic subjects (n = 35)
Maternal age (years) Gestational age (weeks) Pregnancy weight at the time of admission (kg) Pre-pregnancy blood pressure (mmHg) Systolic Diastolic Pregnancy blood pressure at the time of delivery (mmHg) Systolic Diastolic Proteinuria (mg/dL) Xanthine oxidase (U/mg protein) Infant birth weight
27 5.2 38.2 0.4 57.5 7.8
30 4.8NS 30.8 2.5* 72.1 6.3*
114.2 5.1 76.2 5.7
117.2 5.4NS 78.5 4.9NS
122.6 7.6 81.4 7.2 Nil 1.72 0.84 3.42 0.54
167.5 7.9* 112.3 7.4* >300** 2.83 0.96* 2.19 0.54*
NS, non-significant. * p < 0.05 when compared to normotensive subjects. ** p < 0.01 when compared to normotensive subjects.
ASK1 (apoptosis signal regulating kinase 1) is the member of the mitogen-activated protein kinase kinase kinase (MAPKKK) that was shown to be an important signalling kinase in apoptotic cell death [17,18]. Over-expression of ASK1 induces apoptosis through multiple cell death pathway (intrinsic and extrinsic, caspasedependent and independent) induced by various pro-apoptotic stimuli such as lipopolysaccharide, reactive oxygen species (ROS), ischaemic insult and genotoxic stress [19,20]. This paper aims to identify factors that may protect the fetus from the consequences of placental dysfunction and oxidative stress. It is investigating the extent to which the expression of certain cytoprotective and apoptotic proteins could be involved in altering placental endothelial cell viability, thus promoting fetal survival. 2. Materials and methods 2.1. Selection of subjects Patients registered in the Department of Obstetrics & Gynecology of a public sector hospital at Chennai in India were enrolled in the study. The study was carried out for a period of 1 year. The sample consisted of 35 mild pre-eclamptic patients and 35 normotensive subjects of the age 18–40 years. Patients with mild pre-eclampsia were defined on the basis of the following clinical and laboratory criteria: systolic blood pressure in the range of 140–160 mmHg and diastolic blood pressure in the range of 90–110 mmHg noted on at least two occasions; proteinuria concentrations >300 mg/dL measured on at least two random specimens and xanthine oxidase activity of approximately 2.6 U/mg protein [21]. Patients with severe pre-eclampsia were excluded from the study as most of them were advised immediate medication to avoid further complications. Healthy volunteers who were normotensive, of similar race, body mass index (BMI) and without maternal and fetal complications during pregnancy were selected as control subjects. Clearance was obtained from the Hospital Ethical Committee prior to the commencement of the study and informed consent was obtained from all subjects. Pregnant women with other complications such as PROM, IUGR, gestational diabetes, chorioamnionitis, other clinical infections and those undergoing medication were excluded. The clinical characteristics of the pre-eclamptic patients were tabulated and compared with the normotensive pregnant subjects and the data are presented in Table 1.
1 million cells per culture flask (125 mm2) in M199 medium containing 20% fetal calf serum in a 5% CO2 atmosphere at 37 8C. Non-adherent cells and debris were removed by washing three times with PBS the following day. Viability of the endothelial cells was assessed using trypan blue exclusion test [23]. 2.3. Scanning electron microscopy (SEM) analysis of endothelial cells Endothelial cells were fixed for scanning electron microscopy with 4% glutaraldehyde overnight at 4 8C followed by centrifugation at 100 g for 5 min. The supernatant was discarded and the pellet was resuspended in 2% osmium tetroxide for 2 h at room temperature. The centrifugation process was repeated and the samples were dehydrated with an ascending ethanol series (10– 100%). The absolute ethanol was finally displaced by liquid carbon dioxide which served as the transitional fluid for critical point drying. Dried samples were mounted on aluminium stubs and sputter coated with gold (JEC-1100). Electron accelerators for SEM were operated at 15 kV and the samples were viewed with an AMR100 scanning electron microscope (Jeol JSM-6360, Jeol Ltd., Tokyo, Japan). 2.4. Co-immunofluorescence of HSP70 and HSF1 Indirect immunofluorescence technique was performed with the placental sections taken from the subjects of the study according to the method of Pringle et al. [24] with some modifications. The
[()TD$FIG]
2.2. Isolation of endothelial cells Placental endothelial cells were prepared by the method previously mentioned [22]. The cells were cultured overnight at
Fig. 1. Cell viability of endothelial cells isolated from placentas of normotensive and pre-eclamptic subjects.
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primary antibodies used were HSP70 (mouse monoclonal) (SPA810) and HSF1 (Rabbit polyclonal) (SPA 901) antibody, Stressgen, Canada. The primary antibody of HSF1 was detected using an antirabbit fluorescein isothiocyanate (FITC) conjugated secondary antibody (1:3000 dilution) and the primary antibody of HSP70 was detected with anti-mouse IgG Alexa fluor 594. Then the fluorescence of HSP70 and HSF1 was observed using confocal microscope (Leica TCS Sp-2 XL) in 40 magnification that could be [()TD$FIG]visualized as red fluor and green fluor, or as a combined orange fluor.
2.5. Immunohistochemical expression of ASK1 Immunohistochemical expression of ASK1 was achieved using an immunohistochemical staining kit with specific anti-ASK1 antibodies. Placental sections 7-mm thick were mounted onto poly-L-lysine coated slides and stored under dry conditions until histologic analysis. To reduce non-specific binding, slides were incubated in 10% normal goat serum for 10 min at room temperature before 1 h incubation with
Fig. 2. Scanning electron microscopy of endothelial cells isolated from placentas of normotensive women (panels A, C, and E) and pre-eclamptic women (panels B, D, and F). SEM analysis of the endothelial cells isolated from normotensive placenta showed characteristic arrangement of densely packed appearance (panels A, C, and E). While the SEM of endothelial cells isolated from pre-eclamptic placenta (panels B, D, and F) showed changes in the surface morphology. The membrane structures were dispersed with significant alteration in the shape of the cells which contributes to the endothelial cells dysfunction during pre-eclampsia.
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way ANOVA test. The differences were considered significant at p 0.05, 0.01.
primary anti ASK1 (1:2500) antibody in a humidified chamber at 4 8C. The immunohistochemical images were acquired with Zeiss microscopes (Carl Zeiss Microimaging Inc, Thornwood, USA).
3. Results
2.6. Immunoblot analysis of HSP70, ASK1, HSF1 and Trx
3.1. Viability of endothelial cells
The placental endothelial cell protein aliquots containing 50 mg proteins were run on two 10% SDS-polyacrylamide gels that were placed together in the Dual gel electrophoretic system. Both the gels were then blotted onto PVDF membranes (BioTrace PVDF 0.4 (m, Pall Corporation, Germany) according to the method of Towbin et al. [25]. The antibodies used were HSP70 (SPA 810), ASK1 (AAP 480), HSF1 (SPA 901), Trx (MSA150), and b-actin (CSA 400); followed by goat anti-mouse IgG secondary antibody treatment, and colour development was done using BCIP-NBT substrate system. The band intensities were scanned with the HP Scan Imager and quantified using the TotalLab Software (GELS, USA). The results were confirmed by individually performing the blotting studies of HSP70, ASK1, HSF1 and Trx.
Viability of endothelial cells assessed using trypan blue exclusion test showed a significant decrease (p < 0.05) in the viability during pre-eclampsia (78%) compared to the normotensive subjects (89%) immediately after isolation (Fig. 1). The endothelial cells from both the groups were also grown for a period of 5 days in medium M199: the cell viability was >75% in all the groups of cultured cells.
2.7. Statistical analysis All results were expressed as mean standard deviation. Each experiment was performed thrice and data were analyzed by two-
[()TD$FIG]
3.2. Scanning electron microscopy analysis of endothelial cells, coimmunofluorescence of HSP70 and HSF1 and immunohistochemical expression of ASK1 in whole placenta The increased cell disruption and damage to the endothelial cells in pre-eclamptic placenta are clearly demonstrated through the SEM analysis of the endothelial cells isolated from the subjects of the study (Fig. 2). The immunoblot of HSP70 shows that the protein is significantly increased during pre-eclampsia in the placenta compared to normotensive subjects (Fig. 3). The histochemistry slides of the placenta of pre-eclamptic subjects
Fig. 3. Co-immunoblot of HSP70 and HSF1 in the placental sections of normotensive women (panels A–C) and pre-eclamptic women (panels D–F). Co-immunofluorescence images of sections stained for HSF1 with Alexa fluor 594 conjugated goat anti-rabbit IgG secondary antibody (panels A and D) and HSP70 with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG secondary antibody (B and E). Overlay image of HSF1 and HSP70 expressions is shown in panels (C) and (F). HSF1 and HSP70 show positive fluorescent staining in the tissue sections of both normotensive and pre-eclamptic placenta. Intense fluorescent staining for HSP70 and enhanced localization of HSF1 was noted in the pre-eclamptic condition (panels (D) and (E) for HSF1 and HSP70, respectively). In contrast, normotensive placental section demonstrates only a diffuse distribution of HSF1 accompanied with a moderate immunostaining for HSP70 (panels (A) and (B) for HSF1 and HSP70, respectively). Scale bar: 75 mm.
[()TD$FIG]
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Fig. 4. Immunohistochemical staining of ASK1 in the placenta from normotensive women and pre-eclamptic women. Placental sections of both normotensive and preeclamptic subjects show only a diffuse distribution of ASK1. Scale bar: 20 mm.
depict that there is only insignificant increase in the expression of ASK1 in the pre-eclamptic condition compared to normotensive subjects (Fig. 4).
HSF1 (p < 0.05) and Trx (p < 0.05) in pre-eclamptic subject compared to normotensive subjects (Fig. 5). It was observed that there was only an insignificant increase in ASK1 expression which was confirmed by comparison with two-way ANOVA (Fig. 6).
3.3. Immunoblot analysis of HSP70, ASK1, HSF1 and Trx 4. Comments Analysis of the signalling changes was performed using Western blotting. The experiment was performed in triplicate to confirm the results and exclude handling errors. The co-immunoblot analysis revealed a significant increase in HSP70 (p < 0.05),
[()TD$FIG]
Fig. 5. (A) Western blot analysis of normotensive and pre-eclamptic endothelial cells for HSP70 with b-actin as a loading control. (B) Western blot analysis of normotensive and pre-eclamptic endothelial cells for HSF1 with b-actin as a loading control. (C) Western blot analysis of normotensive and pre-eclamptic endothelial cells for ASK1 with b-actin as a loading control. (D) Western blot analysis of normotensive and pre-eclamptic endothelial cells with b-actin as a loading control.
Diminished or poor placental function in pre-eclampsia increases the chances of apoptosis [26]. Most of the clinical findings in pre-eclampsia can be attributed to placental cell damage and abnormalities in endothelial cell function, which are evident from the SEM analysis of the placenta and clinical characteristics exhibited by the pre-eclamptic patients observed in the present study [27]. Endothelial cells were isolated and used freshly in order to annul the influence of cultivation process. All the cases taken for analysis were associated with live fetal delivery in the present study. This suggests the presence of a protective adaptive mechanism in pre-eclampsia that functions to fight the existing condition. Studies in our laboratory have demonstrated an increase in ROS accumulation in placental endothelial cell mitochondria under the condition of pre-eclampsia [22]. The various enzymatic and nonenzymatic antioxidants fail to combat the excessive ROS generated during such complications which stimulate the over-expression of HSPs. The HSP70 quantified using ELISA showed an increase in the expression of the protein in pre-eclamptic endothelial cells (p < 0.05) (unpublished data). HSP forms a powerful system in many intra- and extra-cellular processes including protection against oxidative stress, anti-apoptotic functions and cell proliferation. HSP70 is a potent anti-apoptotic protein that can influence the apoptotic cascade at multiple sites [28]. This study suggests that pre-eclampsia stimulates a multi-faceted response in endothelial cell by inducing HSP70 that acts at different levels to suppress cell death. The oxidative stress noted during pre-eclampsia results in the activation of secondary antioxidant defense system. This might be the prime reason for the increased expression of Trx and HSP70 in these conditions as noted in our study. Trx is a widely distributed intracellular redox protein which constitutes an endogenous antioxidant system in addition to glutathione and superoxide dismutase systems by suppressing free radical formation. It controls the redox state of proteins with its active site by contributing to the reduction of disulfide bonds [29]. Trx plays a role in many cellular processes and its active participation in cell
[()TD$FIG]
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Fig. 6. Comparison of the ASK, Trx, HSF and HSP70 between the two groups of analysis using two way ANOVA.
growth stimulation, chemotaxis and activation of transcription factors in addition to the antioxidant function is prominent [30]. HSP70 has an effect in increasing the active Trx concentration in the local environment. The mechanism by which HSP70 increases free Trx level is not yet understood. The mechanism is thought to be due to the anti-apoptotic function of HSP70 through its chaperoning effect on Trx activating proteins like Trx reductase. Trx is implicated in apoptosis regulation through two different mechanisms: the first is by scavenging reactive oxygen species (ROS) to protect against oxidative stress [30] and the second, by its direct binding and inhibiting the activity of the pro-apoptotic protein apoptosis signal-regulating kinase 1 (ASK1) [31], regulating the activities of transcription factors, such as NF-kB. In mammalian cells, ASK1 participates in the JNK and p38 MAPK signalling cascades by phosphorylating MKK4/MKK7 and MKK3/ MKK6, respectively [32]. ASK1 is activated in response to various extracellular and intracellular stimuli, such as lipopolysaccharide, and ROS [33]. Over-expression of ASK1 induces apoptosis through induction of the mitochondrial pathway [34]. The Trx–ASK1 complex subjects to the ubiquitination and proteasomic attack of the bound ASK1 are achieved [35]. The free Trx protein also has a potential role in stimulating the activation of HSF1 [36], a transcriptional regulator of HSPs. In a native state HSF1 remains as a complex with HSP; in stressed conditions the damaged proteins are thought to compete with HSF1 for binding to HSP, thus leading to the appearance of unbound HSF1 monomer that is free to trimerize, translocate into the nucleus, undergo phosphorylation and activate HSP expression [37]. Disruption of HSF1 causes decreased constitutive expression of several HSPs, which is intimately associated with impaired redox homeostasis and mitochondrial damage [38]. Here we analyze the role of HSP70, a major stress-inducible protein in promoting endothelial cell survival under conditions of preeclampsia by its antioxidant property. The entire process is therefore a systematic cycle where each and every element has a control on the other. Thus, the natural adaptive response is programmed in order to maintain the level of HSP70 in such a way to restore viability of the cell. HSP70 and Trx function to control generated oxidative stress. ASK1 is one of the transcription factors involved in signal transduction that are sensitive to regulation by reactive species. It was found to be expressed with minimal changes from the normotensive samples in spite of the existing complication. This might be due to the control established by the cytoprotective
proteins like HSP70 and Trx. Direct interactions between proapoptotic and anti-apoptotic proteins lead to cell death, in the absence of trophic factors. Binding of trophic factors like HSP70 can trigger changes in these interactions, resulting in cell survival [39]. HSP70 can directly bind and antagonize the function of proapoptotic proteins like ASK1 [40,41]. In the present study, expression of ASK1 was monitored in the samples as it has a direct control over apoptosis [42]. The HSP70–ASK1 complex promotes the ubiquitin-mediated degradation of bound ASK1. HSP70 can indirectly control apoptosis using Trx. Activation of Trx regulates intracellular redox-dependent processes, including transcriptional activity, proliferation and apoptosis [43]. Trx– ASK1 complex pre-exists in the normal unstressed cells and appears to be the target of many extracellular stimuli such as ROS that activate ASK1 by disrupting the Trx–ASK1 complex [44]. The Trx-red (reduced Trx) controls apoptosis by interacting with ASK1 [45]. This modulation of ASK1 through HSP70 up-regulation might be one of the reasons for live birth noted in the present study. As noted in the current study, the delicate balance in the expression of HSP70 promotes the survival of the fetus and aids in live birth in spite of the existing complications. Therefore, the control in the expression of HSP70 plays a crucial role in maintenance of cell viability, which paves way for its usage as a potential therapeutic target for treatment of such conditions as pre-eclampsia. Acknowledgement The authors thank National Tea Research Foundation, Tea Board of India, for providing funds for conducting this work. Project referral number 115/07. References [1] Roberts JM, Cooper DW. Pathogenesis and genetics of pre-eclampsia. Lancet 2001;357:53–6. [2] Higgins JR, de Swiet M. Blood pressure measurement and classification in pregnancy. Lancet 2001;357:131–5. [3] Jonsdottir LS, Arngrimsson R, Geirsson RT, et al. Death rates from ischemic heart disease in women with a history of hypertension in pregnancy. Acta Obstet Gynecol Scand 1995;74:772–6. [4] Van Wijk MJ, Kublickiene K, Boer K, et al. Vascular function in pre-eclampsia. Cardiovasc Res 2000;47:38–48. [5] Muschel RJ, Bernhard EJ, Garza L, et al. Induction of apoptosis at different oxygen tensions: evidence that oxygen radicals do not mediate apoptotic signaling. Cancer Res 1995;55:995–8. [6] Young RA. Stress proteins and immunology. Annu Rev Immunol 1990;8:401–20.
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