Post on 10-Nov-2018
Sandra Isabel Cunha Silveira
outubro de 2013
Redox changes in aged placental bed: consequences for cell signalling
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Universidade do Minho
Escola de Ciências
Sandra Isabel Cunha Silveira
outubro de 2013
Dissertação de Mestrado Mestrado em Genética Molecular
Redox changes in aged placental bed: consequences for cell signalling
Universidade do Minho
Escola de Ciências
Trabalho realizado sob a orientação daDoutora Elisabete Silvae doProfessor Doutor Henrique Almeidae daProfessora Doutora Cristina Pereira-Wilson
III
Acknowledgements
Em primeiro lugar, gostaria de agradecer à minha orientadora, Doutora Elisabete
Silva, por me integrar no seu próprio projecto de investigação, acompanhar as suas
experiências e participar na sua execução, pela sua orientação, aconselhamento, apoio e
paciência.
Gostaria ainda de agradecer ao Professor Henrique Almeida por me aceitar no
grupo Ageing and Stress e assumir a co-orientação.
Gostaria de agradecer à Sra. Professora Deolinda Lima, directora do
Departamento de Biologia Experimental da Faculdade de Medicina da Universidade do
Porto, onde o trabalho se realizou, e por extensão, a todos os quantos que aí exercem,
em especial os meus colegas de laboratório, Ana Isabel Soares e Anabela Silvestre,
pelas ajudas ao longo do ano.
Um agradecimento especial ao Dr. Luis Guedes-Martins do Centro Hospitalar do
Porto, Unidade Maternidade Júlio Dinis, por nos facultar os tecidos que permitiram a
realização desta dissertação de mestrado.
Gostaria, igualmente, de agradecer aos meus pais e irmã pelo apoio.
Por fim, gostaria de agradecer a Brian Baron por todo o apoio, ajuda, por
acreditar em mim e por estar sempre presente quando preciso.
Redox changes in aged placental bed: consequences for cell signalling
V
Abstract
The occurrence of pregnancy-related disorders such as preeclampsia, IUGR or
foetal death is associated with increased maternal age. These pregnancy complications
are thought to be a consequence of a local imbalance in redox homeostasis, leading to
an abnormal placentation, placental development and function. In placental bed, the
uterine place where placenta adheres, ROS can react with biomolecules and alter cell
function, though modification of cell signalling pathways involved in placentation or
induction of cellular damage.
In this project it was hypothesized that at older reproductive age loss of redox
homeostasis at the placental bed contributes to disruption of foetal/placental interactions
and enhances the development of pregnancy complications. Therefore, to address this
hypothesis, evaluation of oxidative stress markers and activation of cell signalling
pathways was performed in samples of uterine placental bed of women at different ages.
Employing a human cell stress-related protein expression array kit, it was observed that
in reproductively older women the protein expression of cytochrome c, FABP-1 and
ADAMTS1 were significantly increased. The array was validated by western blot, using
antibodies for SODII, HSP70, Sirt2 and phospho-p38α. Western blot, also demonstrated
a significant age-related increase SODII expression. Results show a local redox
imbalance in placental bed of reproductively older women that could lead to increased
cellular oxidative stress (OS). As marker of cellular OS, a protein carbonylation assay
was used. Although no significant changes were observed in total protein carbonylation
content, quantification of specific carbonylated proteins showed a positive correlation
with maternal age. As mild increases in OS can alter signalling pathways, an additional
human phospho-kinase array kit was employed to study the same tissue. STAT3 protein
phosphorylation had over 100% increase in expression. OS status was also evaluated in
pregnancy-related disorders. SODI, SODII and Sirt2 protein expression was evaluated
by western blot. SODI and Sirt2 expression was increased in pregnancies of
hypertensive women with or without pre-eclampsia. SODII expression had no
significant changes in all groups. Once again, only a specific protein was significantly
increased in the group of women with gestational hypertension and IUGR of the foetus.
This project gathered information on local uterine redox status and cell signal in
reproductively older women and demonstrated that alterations in tissue redox balance
are present in a maternal tissue, even in pregnancies without associated complications
Alterações do balanço redox no local de placentação envelhecido: consequências
para a sinalização celular
VII
Resumo
A ocorrência de complicações relacionadas com a gravidez como a pré-
eclâmpsia, restrição ao crescimento intra-uterino ou morte fetal, está associada com o
aumento da idade materna. Elas podem ser consequência de um desequilíbrio local no
balanço redox, que estará na origem de uma placentação anormal. No local de
placentação, o local uterino onde a placenta adere, as espécies reactivas de oxigénio
podem reagir com biomoléculas e alterar funções celulares, quer por modificação de
vias de sinalização, quer por indução de danos celulares.
Neste projeto, colocou-se a hipótese de em idade reprodutiva avançada ocorrer
um desequilíbrio do balanço redox no local de placentação, que contribui para a
instabilidade da interacção feto/placenta e o aumento da probabilidade de ocorrerem
complicações. Para testar esta hipótese foram avaliados marcadores de stress oxidativo e
indicadores de activação de vias de sinalização celular em amostras de local de
placentação de mulheres com diferentes idades. Recorrendo a um array de expressão de
proteínas relacionadas com o stress celular, observou-se que em mulheres com idade
reprodutiva avançada a expressão do citocromo c, FABP-1 e ADAMTS1 estava
aumentada significativamente. A validação do array foi realizada por Western blotting
utilizando anticorpos para SODII, HSP70, Sirtuin2 e fosfo-p38α. Os resultados do
Western blot demonstraram aumento significativo da expressão da SOD II com a idade
materna. Nesta primeira parte do trabalho, observou-se um desequilíbrio redox no local
de placentação em mulheres com idade reprodutiva avançada, que poderá originar um
aumento do stress oxidativo. Quando se empregou a carbonilação de proteínas como
marcador de stress oxidativo, não se observaram alterações significativas no conteúdo
total de carbonilos, mas a quantificação de proteínas específicas mostrou uma
correlação positiva entre a carbonilação e a idade materna. Além disso, atendendo a que
um ligeiro aumento do stress oxidativo pode alterar vias de sinalização, empregou-se
um array de cínases de fosfatases para o seu estudo. A proteína STAT3 fosforilada teve
um aumento da expressão superior a 100%.
Na segunda parte do trabalho, em gravidezes com complicações associadas, a
expressão proteica das enzimas antioxidantes SODI, SODII e da Sirtuin2, avaliadas por
Western Blotting, mostrou que em mulheres hipertensas com ou sem pré-eclampsia, a
SODI e Sirt2 estavam aumentadas, enquanto a expressão da SODII não apresentou
alterações significativas. O estudo da carbonilação proteica demonstrou que apenas uma
Alterações do balanço redox no local de placentação envelhecido: consequências
para a sinalização celular
VIII
proteína específica se encontra significativamente aumentada no grupo de mulheres com
hipertensão gestacional e restrição ao crescimento intra-uterino.
Este projecto reuniu informação sobre o estado redox e sinalização celular no
local de placentação em mulheres com idade reprodutiva avançada e demonstrou a
existência de alterações no equilíbrio redox uterino, mesmo em gravidezes sem
complicações associadas.
IX
Table of contents
Abbreviations ................................................................................................................ XI
Figures ......................................................................................................................... XIII
Tables .......................................................................................................................... XIV
1. Introduction ............................................................................................................. 3
1.1. Placentation and placental bed ....................................................................... 3
1.2. Oxidative stress ................................................................................................ 5
1.3. Oxidative stress biomarkers ........................................................................... 7
1.3.1. Protein carbonylation ............................................................................... 7
1.3.2. DNA Damage ............................................................................................ 8
1.3.3. Lipid Peroxidation .................................................................................... 8
1.4. Cellular signalling ............................................................................................ 8
1.4.1. Extracellular signal-regulated protein kinase 1 and 2 pathway ........ 10
1.4.2. p38 Mitogen-activated kinase pathway ................................................ 10
1.4.3. C-jun amino terminal kinase pathway ................................................. 11
1.5. Pregnancy-associated disorders .................................................................... 11
1.5.1. Hypertension ........................................................................................... 11
1.5.1.1. Pre-eclampsia ................................................................................... 11
1.5.1.2. Intrauterine Growth Restriction.................................................... 13
1.6. Aims ................................................................................................................. 14
2. Methods .................................................................................................................. 17
2.1. Tissue collection ............................................................................................. 17
2.2. Tissue lysation ................................................................................................ 17
2.3. Protein quantification – Lowry method ....................................................... 17
2.4. Western Blotting ............................................................................................ 18
2.5. Protein carbonylation .................................................................................... 19
2.6. Human Cell Stress Array .............................................................................. 20
2.7. Protein quantification – Bradford method .................................................. 21
2.8. Human Phospho-kinase Array kit ................................................................ 22
2.9. Statistical analysis .......................................................................................... 23
3. Results .................................................................................................................... 27
X
3.1. Reproductive ageing and redox balance in placental bed. ......................... 27
3.1.1. Protein array. .......................................................................................... 27
3.1.2. Western blotting – Protein array validation. ....................................... 28
3.1.3. Oxidative stress marker - protein carbonylation. ............................... 30
3.2. Reproductive ageing and cell signalling. ...................................................... 31
3.2.1. Protein array. .......................................................................................... 31
3.2.2. Western blotting – Protein array validation. ....................................... 33
3.3. Redox balance in placental bed and pregnancy-associated disorders. ..... 33
3.3.1. Western blotting – cell stress related proteins. .................................... 33
3.3.2. Oxidative stress marker – protein carbonylation. ............................... 36
4. Discussion ............................................................................................................... 41
5. Conclusions and future perspectives ................................................................... 49
6. Final remarks......................................................................................................... 53
7. Bibliography .......................................................................................................... 57
XI
Abbreviations
ADAMTS1 – A desintegrin and metalloproteinase with thrombospondin motifs 1
BSA – Bovine serum albumin
CO• – Carbonyl group
CuSO4 – Copper (II) sulphate
DNP – 2,4 –Dinitrofenol
DNPH – 2,4-Dinitrophenylhydrazine
DTT – Dithiothreitol
ECM – Extracellular matrix
EDTA – Ethylenediaminetetraacetic acid
ERK – Extracellular signal-regulated kinase
FABP-1 – Fatty acid-binding protein 1
HCl – Hydrochloric acid
HRP – Horseradish peroxidase
HSP70 – Heat shock protein 70
HT – Hypertension
H2O2 – Hydrogen peroxide
IUGR – Intrauterine growth restriction
JNK/SAPK – c-Jun N-terminal kinase/ Stress-activated protein kinase
KNaC4H4O6 – Potassium sodium tartrate
MAPK – Mitogen-activated protein kinase
MAPKK – Mitogen – activated protein kinase kinase
XII
MAPKKK – Mitogen-activated protein kinase kinase kinase
NaCl – Sodium chloride
NaCO3 – Sodium carbonate
NADPH – Nicotinamide adenine dinucleotide phosphate
NaOH – Sodium hydroxide
NO• – Nitric oxide
OH• – Hydroxyl ion
OS – Oxidative stress
O2•- – Superoxide anion
PE – Pre-eclampsia
RNS – Reactive nitrogen species
ROS – Reactive oxygen species
SDS – Sodium dodecyl sulphate
Sirt 2 – Sirtuin 2
SOD – Superoxide dismutase
STAT3 – Signal transducer and activator of transcription 3
TBS – Tris-buffer saline
TBS-T – Tris-buffer saline with Tween 20
TFA – Trifluoroacetic acid
UV – Ultraviolet
XIII
Figures
Figure 1 – Scheme of human Placentation.
Figure 2 – Production of reactive oxygen species and nitrogen reactive species and its
neutralization.
Figure 3 – Protein scaffold of MAPK pathways involved in oxidative stress.
Figure 4 – Scheme of trophoblast invasion and spiral remodelling in normal pregnancy
and in pregnancy-associated disorders.
Figure 5 – Human cell stress array profile in human placental bed.
Figure 6 – Effect of age on cell stress-related proteins expression.
Figure 7 – Effect of age on DNP expression.
Figure 8 – Effect of age on DNP expression in different proteins.
Figure 9 – Human phospho-kinase array profile in human placental bed.
Figure 10 – Effect of age on phospho-proteins expression.
Figure 11 – Effect of pregnancy-related disorders on SOD I expression.
Figure 12 – Effect of pregnancy-related disorders on SOD II expression.
Figure 13 – Effect of pregnancy-related disorders on Sirtuin 2 expression.
Figure 14 – Effect of pregnancy-related disorders on DNP expression.
Figure 15 – Effect of pregnancy-related disorders on DNP expression in different
proteins.
XIV
Tables
Table I – BSA standard curve for protein quantification by Lowry method.
Table II – Primary antibodies used.
Table III – Capture antibodies in human cell stress array.
Table IV – BSA standard curve for protein quantification by Bradford method.
Table V – Capture antibodies in human phospho-kinase array.
Table VI – Identification of cell stress-related proteins expressed in human placental
bed tissues.
Table VII – Identification of phospho-kinases proteins expressed in human placental
bed tissues.
Introduction
Introduction
3
1. Introduction
Nowadays, in modern societies, women are postponing childbearing beyond the
age of 35 years old (Balasch and Gratacós 2011). Higher education, pursuit of a career,
economic independence, effective contraception, longer life expectation and the
promise of advanced reproductive technologies are reasons for the decision of having
their first child in later age (van Katwijk and Peeters 1998, Istance and Theisens 2008).
As women’s fertility declines with age, being more notorious after 35 years-old, it leads
to an increased risk of developing pregnancy-related complications like hypertension,
pre-eclampsia and intrauterine growth restriction (IUGR), that can affect both mother
and foetus during pregnancy and labour (van Katwijk and Peeters 1998).
Various observations favour the view that some pregnancy-related complications
are due to an abnormal placental bed, the uterine environment where the placenta
adheres, which in turn may result, from altered redox status, consequence to an
imbalance between the activity of pro-oxidant and anti-oxidant molecules. This
promotes reactive oxygen species (ROS) effects and the establishment of oxidative
stress (OS). This condition can induce alterations in signalling pathways and damage to
biomolecules such as proteins, DNA and lipids (Myatt and Cui 2004).
1.1. Placentation and placental bed
Placentation is the process of formation and development of the placenta and the
associated anatomical modifications in maternal tissue. In humans, it involves the
development of three regions (Figure 1).
The first region is called foetal placenta and contains both foetal and maternal
blood and is the place where physiological exchange of nutrients and waste products
occurs (Georgiades, Ferguson-Smith et al. 2002). The second region is termed basal
plate and is a region that borders the maternal surface next to the foetal placenta
although it does not contain any foetal blood and is transversed by maternal vessels.
This region can also be called implantation site. The third region is known as placental
bed and is constituted by maternal uterine tissue that contains the decidua basalis, which
is the term given to the uterine endometrium specially modified to enable implantation.
Introduction
4
Figure 1. Human Placentation. Placentation involves the development of three regions: foetal placenta,
which protects the foetus and serve as a gas and hormone exchange; basal plate which borders the
maternal surface and the foetal placenta; and placental bed where trophoblast invasion and the
physiological change occurs. Extracted from Georgiades, Ferguson-Smith et al. 2002
Placental bed contains 100-150 maternal spiral arteries (Georgiades, Ferguson-
Smith et al. 2002, Lyall 2002). Maternal spiral arteries supply nutrients and oxygen to
the placenta (Lyall 2005). But, for the success of a healthy pregnancy it is necessary that
maternal spiral arteries are transformed into flaccid vessels in a physiological process
where endovascular invasion of the arterial lumen by trophoblasts is a crucial process
(Redline 2008). Trophoblast invasion through the uterine stroma is also fundamental for
anchoring the placenta into the uterine wall. There is no doubt that placental bed plays a
role is supporting placenta function and that failure in the setting up of the physiological
changes may lead to pre-eclampsia, foetal growth restriction or even spontaneous
miscarriage (Lyall 2005).
An imbalance in placental bed redox status is thought to contribute to the
development of the above-mentioned pregnancy complications (Myatt and Cui 2004).
Introduction
5
1.2. Oxidative stress
OS is described as an imbalance between the production of ROS/reactive
nitrogen species (RNS) and the levels of antioxidant molecules, able to scavenge them
(Myatt and Cui 2004). It is the basis of one of the most widely accepted theories of
ageing (Harman 1956).
Pregnancy is known as a state of OS arising from an increased metabolic activity
of mitochondria and increased ROS and RNS production in the placenta and the
reduction of antioxidant defence mechanisms (Myatt and Cui 2004, Agarwal, Aponte-
Mellado et al. 2012). A balance between ROS and RNS production and antioxidant
defence is necessary to maintain cellular homeostasis (Lázár 2012), since ROS and RNS
are needed for a correct cell function (Agarwal, Aponte-Mellado et al. 2012). However,
when the production of pro-oxidant molecules increases or when the antioxidant
defences decrease (Gupta, Malhotra et al. 2009) this originates an imbalance in the
redox equilibrium and leads to a disturbance in cellular metabolism and regulatory
pathways (Lázár 2012).
ROS play an important signalling role in female fertility and pregnancy. Indeed
they are needed for physiological processes involved in oocyte maturation, fertilization,
implantation and embryo development (Burton and Jauniaux 2011). ROS can alter
trophoblasts proliferation and differentiation and placental vascular reactivity, thus
being important regulators of placental function (Myatt and Cui 2004, Webster, Roberts
et al. 2008). As such, an imbalance in cellular levels of ROS, leading to OS, may be
implied in placental dysfunction and the development of pregnancy complications.
Most ROS are free radicals containing one or more unpaired electrons but some
ROS are not free radicals. Nevertheless, both have high reactivity (Burton and Jauniaux
2011) and can induce cellular damage (Myatt and Cui 2004). One of the most reactive
ROS is superoxide anion (O2-) (Myatt and Cui 2004, Gupta, Malhotra et al. 2009,
Agarwal, Aponte-Mellado et al. 2012, Lázár 2012). The major source of O2- is the
mitochondria, through the mitochondrial respiratory chain (Agarwal, Aponte-Mellado et
al. 2012) but other sources of O2- include the short electron chain in the endoplasmic
reticulum, xanthine oxidase, nitric oxide synthase (Myatt and Cui 2004), cytochrome
P450 and the enzyme adenine dinucleotide phosphate (NADPH) oxidase (Figure 2).
NADPH oxidase produces substantial quantities of O2- during early pregnancy
(Burton and Jauniaux 2011) that it is believed to be implicated in cell signalling events,
Introduction
6
which leads to up-regulation of transcription factors, antioxidant production,
angiogenesis, proliferation and matrix remodelling (Myatt and Cui 2004, Webster,
Roberts et al. 2008, Gupta, Malhotra et al. 2009). O2- can be neutralized enzymatically
by superoxide dismutase (SOD) and converted into hydrogen peroxide (H2O2), which,
although less reactive is more stable and membrane permeable. In the cells H2O2 can be
convert into water by catalase and glutathione peroxidase (Burton and Jauniaux 2011).
SOD has three isoforms: copper/zinc SOD (Cu/Zn SOD) or SODI that is located in the
cytosol, Manganese SOD (Mn SOD) or SODII, located in the mitochondria and SODIII,
an extracellular isoform that is structurally similar to Cu/Zn SOD. SODI and SODII
isoforms have cell specific distribution in the placenta. SODI is intensely expressed in
villous stroma whereas SODII is expressed strongly in the villous endothelium and
faintly expressed in syncytiotrophoblast (Myatt and Cui 2004).
ROS can be neutralized by antioxidants. Antioxidants are molecules that protect
the cell against ROS by dramatically delaying or preventing oxidation. They can be
enzymatic or non-enzymatic: SOD, thioredoxin, thioredoxin reductase, catalase,
glutathione peroxidase are endogenous enzymatic antioxidants (Agarwal, Aponte-
Mellado et al. 2012), while vitamins C and E and glutathione are non-enzymatic
antioxidants (Gupta, Malhotra et al. 2009).
Figure 2. Production of reactive oxygen and nitrogen species and its neutralization. Superoxide
anion is produced by the mitochondria, endoplasmic reticulum, NADPH oxidase, cytochrome P450 and
xanthine oxidase and is scavenged by superoxide dismutase that converts it into hydrogen peroxide.
Catalase and glutathione peroxidase may breakdown hydrogen peroxide into water. The action of
antioxidants is necessary because an imbalance between superoxide and hydrogen peroxide leads to the
formation of hydroxyl ion. In the other side, superoxide can interact with nitric oxide and form a powerful
pro-oxidant, peroxynitrite. Extracted from Burton and Janiaux 2011.
Introduction
7
Nitrative stress is also a biological process that alters the placental function. The
most important RNS are nitric oxide (NO). It has a role, likely a signalling one, in the
trophoblast invasion and in the maternal-foetal circulation. When NO and O2- interact
with each other, they generate a potent pro-oxidant, peroxynitrite that affects placental
physiology (Webster, Roberts et al. 2008).
It is believed that local cellular redox imbalance may lead to alterations in
cellular signalling pathways and also induce damage to biomolecules such as proteins,
lipids or DNA. These events originate cellular dysfunction and may contribute to the
development and maintenance of pregnancy-related complications (Myatt and Cui
2004).
1.3. Oxidative stress biomarkers
1.3.1. Protein carbonylation
Protein carbonylation is an irreversible protein oxidation process promoted by
ROS (Dalle-Donne, Aldini et al. 2006, Suzuki, Carini et al. 2010), which includes the
formation of stable carbonyl groups like aldehydes and ketones on protein side chains
when they are oxidized. The amino acids that are more susceptible to be oxidized in this
way are proline, arginine, lysine and threonine (Dalle-Donne, Rossi et al. 2003, Dalle-
Donne, Aldini et al. 2006, Wong, Cheema et al. 2008).
Protein oxidation results in alteration of protein conformation, exposure of
hydrophobic domain and loss of catalytic function and structural properties (Levine
2002, Wong, Cheema et al. 2008). This phenomena is often mentioned as a process
needed in the cells because it may act as a signal mechanism to ensure that defective
proteins enter the degradation pathway and are degraded by the proteasome (Nystrom
2005).
Accumulation of protein carbonyls has been observed in various human age-
related diseases (Dalle-Donne, Rossi et al. 2003) and during the ageing process (Levine
2002). Since protein carbonyls are very stable alterations they have been used as
oxidative stress biomarker. (Dalle-Donne, Aldini et al. 2006, Wong, Cheema et al.
2008).
Introduction
8
1.3.2. DNA Damage
DNA damage is described as an alteration in the structure of the DNA caused by
oxidative stress, UV radiation, alkylating agents or hydrolytic deamination. These
alterations comprise strand breaks and modifications in purines and pyrimidines
(Kumari, Rastogi et al. 2008). Free radicals like OH often attack DNA, leading to
guanine modification and producing of 8-hydroxy-2’-deoxyguanosine (Evans,
Dizdaroglu et al. 2004, Burton and Jauniaux 2011) that can be measured biochemically
or detected immunohistochemically (Burton and Jauniaux 2011). This type of
modification was found increased in placental tissues of pregnancy-associated disorders
when compared with normal pregnancies (Wiktor, Kankofer et al. 2004).
1.3.3. Lipid Peroxidation
Lipid peroxidation has been defined as the oxidative degradation of lipids that
contain a number of carbon-carbon double bonds (Devasagayam, Boloor et al. 2003).
Lipid peroxides are formed when ROS attack the phospholipids presents in cell
membranes and react with polyunsaturated fatty acids leading to cellular damage
contribute to the formation of lipid peroxides, lipid hydroperoxides and a secondary
product of lipid peroxidation, malonaldehyde (Lázár 2012). In fact, it was demonstrated
that in pregnancy related disorders, an accumulation of lipid peroxides occurs mainly in
placenta (Agarwal, Gupta et al. 2005).
1.4. Cellular signalling
Environmental signals can regulate cellular activity by binding to cellular
receptors and activating specific intracellular pathways (Aouadi, Binetruy et al. 2006,
Berridge 2012). Many of these extracellular signals are transduced by highly conserved
eukaryotic signalling mechanisms called mitogen-activated protein kinases (MAPKs)
(Aouadi, Binetruy et al. 2006), members of important signalling pathways that regulate
physiological processes like cell growth, differentiation and cell death (Junttila, Li et al.
2008).
When a stimulus is generated, a phosphorylation cascade is initiated originating
a protein scaffold where a specific mitogen-activated protein kinase kinase kinase is
Introduction
9
phosphorylated. It in turn phosphorylates a specific mitogen-activated protein kinase
kinase that phosphorylates a specific mitogen-activated protein kinase (MAPK) such as
the extracellular signal-regulated kinase (ERK) 1 and 2, the p38 mitogen-activated
protein kinase (p38 MAPK) and JNK. In turn, these MAPKs can activate several
transcription factors and/or cytoplasmatic and nuclear kinases (figure 3) (Aouadi,
Binetruy et al. 2006, Junttila, Li et al. 2008).
So far, in mammals, four distinct MAPK signalling pathways were described:
ERK 1 and ERK 2, that are responsible for cell survival and development, c-jun amino
terminal kinase or stress-activated protein kinase (JNK/SAPK), p38 MAPK which are
described as stress activated proteins (SAPK) and play a role in host defence
mechanisms (Coulthard, White et al. 2009), and ERK5 (Ono and Han 2000, Aouadi,
Binetruy et al. 2006, Cuenda and Rousseau 2007, Berridge 2012).
When a redox imbalance occurs, ROS can induce the activation of MAPK
signalling pathways. Modification of essential amino acids residues by ROS that
consequently alters the protein structure and function is one of the plausible
mechanisms of MAPKs activation (Son, Cheong et al. 2011). In addition, MAPKs
activation may occur directly by interaction with membrane receptors, such as
Figure 3. Protein scaffold of MAPK pathways involved in oxidative stress. When an intracellular or an
extracellular stimulus occurs, the phosphorylation of MAPKKK (MAP3K) is initiated. MAP3K in turn,
phosphorylates MAPKK (MAP2K), which in succession phosphorylates MAPKs. The activated MAPKs
consequently phosphorylate several substrates that are important for cellular activities such as development,
differentiation, growth and apoptosis. Extracted from Son, Cheong et al 2011.
Introduction
10
endothelial growth factor receptor and platelet-derived growth factor receptor
(McCubrey, LaHair et al. 2006) or indirectly due to oxidation of MAPKs related
proteins, such as protein tyrosine phosphatases (Finkel 2003, Son, Cheong et al. 2011).
1.4.1. Extracellular signal-regulated protein kinase 1 and 2 pathway
ERK 1/2 is a member of one of the most important pathways of MAPK
signalling (Berridge 2012) once it participates in essential cellular functions as control
of cell proliferation, differentiation, cell survival and the synaptic plasticity responsible
for learning and memory (Junttila, Li et al. 2008, Berridge 2012). The ERK 1/2 pathway
can be activated by mitogens, phorbol esters, growth factors and oxidative stress
(Daoud, Amyot et al. 2005, Junttila, Li et al. 2008).
In pregnancy events, ERK 1/2 is also necessary because it is implicated in
placental development (Aouadi, Binetruy et al. 2006), trophoblast differentiation and
decidual invasion (Daoud, Amyot et al. 2005, Moon, Park et al. 2008). In fact, it has
been shown that inhibition of ERK 1/2 alone reduces trophoblast migration (Moon, Park
et al. 2008) and when in parallel with p38 MAPK decreases trophoblast differentiation
(Daoud, Amyot et al. 2005).
1.4.2. p38 Mitogen-activated kinase pathway
The p38 MAPK pathway is also an important MAPK signalling pathway
(Berridge 2012), that is activated by cytokines (Coulthard, White et al. 2009) and a wide
range of environmental stress agents, as osmotic redox and radiation stress (Berridge
2012). This pathway is important in the control of apoptosis, inflammation, cell cycle
regulation, senescence and oncogenesis (Ono and Han 2000, Coulthard, White et al.
2009).
The p38 MAPK has four isoforms: p38α, p38β, p38γ and p38δ (Cuenda and
Rousseau 2007). The p38α isoform is the most well characterized and is described to
play a vital role in placental embryonic development and placental angiogenesis
(Mudgett, Ding et al. 2000). In a mouse model, it was demonstrated that disruption of
p38α results in defective placental angiogenesis, leading to abnormal placental
development or even to embryo death (Mudgett, Ding et al. 2000).
Introduction
11
1.4.3. C-jun amino terminal kinase pathway
It is believed that JNK pathway is involved in the control several cellular
processes such as cellular proliferation, apoptosis and embryonic development
(Berridge 2012). It was also demonstrated that JNK pathway plays a role on promoting
cell survival during pre-eclampsia (Padmini, Uthra et al. 2012).
1.5. Pregnancy-associated disorders
1.5.1. Hypertension
Hypertension is the most common disorder during pregnancy. It occurs
approximately 15 % of pregnancies and may cause maternal and foetal morbidity and
mortality (James and Nelson-Piercy 2004).
Hypertension can be divided into 3 groups (James and Nelson-Piercy 2004):
chronic hypertension, which presently complicates 3% to 5% of pregnancies;
gestational hypertension complicates 6–7% of pregnancies and resolves post-partum;
and pre-eclampsia, which complicates 3%-14% of pregnancies (Agarwal, Aponte-
Mellado et al. 2012). Pre-existing hypertension is a risk factor for the development of
pre-eclampsia.
1.5.1.1. Pre-eclampsia
Pre-eclampsia is known as a complex pregnancy multisystem disorder that
affects both the mother and foetus (Hubel 1999, Gupta, Agarwal et al. 2005). It has an
incidence between 3%-14% (Agarwal, Aponte-Mellado et al. 2012) and is the leading
cause of maternal and foetal morbidity and mortality (Hubel 1999, Agarwal, Aponte-
Mellado et al. 2012). Pre-eclampsia is characterized by hypertension, edema and urinary
albumin loss (Serdar, Gür et al. 2002), generalized vasospasm (Lázár 2012) that may
lead to renal, liver and brain damage in the setting of eclampsia. In addition, it can lead
to placental abruption, preterm delivery, IUGR and foetal death (Sibai, Dekker et al.
2005, Steegers, von Dadelszen et al. 2010). In this pregnancy disorder, trophoblast
invasion is limited into the decidua basalis and insufficient spiral arteries remodelling
occurs (figure 4) (Hubel 1999, Jauniaux, Poston et al. 2006, Redman and Sargent 2009).
Introduction
12
Figure 4. Trophoblast invasion and spiral arteries remodelling. Trophoblasts invade the decidua
basalis and the adjacent myometrium leading to a remodelling of spiral arteries that become flaccid
vessels. In pregnancy disorders like preeclampsia and IUGR the trophoblasts invade only the decidua
basalis and the remodeling of spiral arteries is limited to the same tissue. Extracted from Bell 2004.
In a normal pregnancy, trophoblasts invade both decidua basalis and the adjacent
myometrium leading to a remodelling of the spiral arteries (Lyall 2002). However, in
the case of pre-eclampsia, trophoblast invasion and spiral arteries remodelling into
flaccid vessels is restricted to the decidua basalis (Hubel 1999, Roberts and Escudero
2012). Deficient placentation will alter blood flow and result in an intermittent velocity
of the utero-placental arterial blood flow with phenomena of hypoxia/reperfusion,
which in turn leads to enhanced ROS production and OS establishment (Redman and
Sargent 2009). It is believed that OS is an important factor in the development of this
pathology (Hubel 1999, Gupta, Agarwal et al. 2005).
In placentae from pre-eclamptic women it was demonstrated that both the
expression and activity of total SOD were decreased (Hubel 1999). Phospho-p38α and
ERK1/2 were also significantly reduced (Webster, Brockman et al. 2006). Protein
carbonylation, lipid peroxidation and DNA damage were also reported to be higher in
the placenta and in decidua basalis from pregnancies complicated with preeclampsia
Introduction
13
when compared with normal pregnancies (Serdar, Gür et al. 2002, Myatt and Cui 2004,
Fujimaki, Watanabe et al. 2011).
1.5.1.2. Intrauterine Growth Restriction
IUGR is described as a pregnancy complication in which there is a failure of the
foetus from achieving its genetic growth potential (Scifres and Nelson 2009, Lázár
2012). It may lead to foetal death, perinatal morbidity and in child it may lead to
lifelong disorders such as hypertension, renal and cardiovascular diseases (Scifres and
Nelson 2009). Aneuploidies, malformations, infections, metabolic factors, placental
disorders and OS are recognized as causes for IUGR (Scifres and Nelson 2009, Lázár
2012).
In IUGR, as in pre-eclampsia, a deficient trophoblast invasion and insufficient
spiral arteries remodelling, confined to the decidua basalis, is also observed (Jauniaux,
Poston et al. 2006, Redman and Sargent 2009, Scifres and Nelson 2009). This leads to a
hypoxia/reperfusion phenomena resulting from reduced velocity of the utero-placental
arterial flow which, once again, leads to local OS (Redman and Sargent 2009) that is
also thought to antedate the development of this pathology (Hubel 1999, Gupta,
Agarwal et al. 2005).
Introduction
14
1.6. Aims
Information about the age-related role of oxidative and nitrative stress in
signalling mechanisms of placenta and placental bed remain rather vague. It is thus
important to improve our knowledge on the damage caused by redox imbalance in these
tissues. Although no single theory purposes an inclusive explanation for the
mechanisms underlying ageing, a better understanding of the downstream effects of
imbalanced redox status in cells from the reproductive tract may enhance our
understanding of female reproductive ageing. In the future, it may lead to the
development of preventive or therapeutic strategies answering to improve placenta
function and reduce morbidity and mortality associated to pregnancy in aged women.
Based on the published knowledge about OS in the placental bed and its
consequence on pregnancies, it is hypothesized that at older reproductive age, the uterus
is more prone to redox homeostasis imbalance, which, as consequence, alters key
signalling pathways at the uterine placental site that contribute to disruption of
foetal/placental interactions and increases the likelihood of developing pregnancy
complications. Therefore, to address this hypothesis three major aims were defined:
1) Determine oxidative stress status in uterine samples from placental bed of young
and aged pregnant women;
2) Evaluate, the effect of women’s age in the local modulation of cell signalling;
3) Determine oxidative stress status in uterine samples from placental bed of young
and aged pregnant women with chronic hypertension, chronic hypertension and
pre-eclampsia and gestational hypertension and intrauterine growth restriction of
the foetus.
Methods
Methods
17
2. Methods
2.1. Tissue collection
Dr. Luis Guedes-Martins, MD, PhD Student, from Centro Hospitalar do Porto,
EPE, collected all human samples. The protocol was approved by ethical committee of
“Centro Materno-Infantil do Porto”. Volunteers, in accordance with the Institution’s
guidelines, gave written consent to be included in this study. All human samples were
collected at delivery by elective caesarean section. Uterine pieces were taken from the
placental site, processed for structural studies and a stock was stored at – 80 ºC for
molecular study.
Women were separated into four groups. Group one had no pathologies; group
two had gestational hypertension plus IUGR; group three had chronic hypertension;
group four had chronic hypertension plus pregnancy-associated pre-eclampsia.
2.2. Tissue lysation
A small unthawed portion of tissue was cut from samples frozen at -80 ºC and
cut again to smaller portions. Tissue was homogenized, using a glass homogenizer
(Heidolph), in 500 µL of ice-cold lysis buffer (10 mM NaCl, 5 mM EDTA, 50 mM
Tris-HCl pH 7.5 and 1% SDS) containing proteases inhibitors (P8340, Sigma-Aldrich)
and phosphatases inhibitors (P0044, P5726, Sigma-Aldrich). Afterwards, the lysates
were sonicated and centrifuged at 1500 rpm for 5 minutes. The pellet was rejected and
supernatants were stored at -80 ºC for future experiments.
2.3. Protein quantification – Lowry method
The protein content of tissue lysates was determined by Lowry method using
Folin-Ciocalteau reagent (F-9252, Sigma-Aldrich) and BSA (A9647-1006, Sigma-
Aldrich) as standard. The albumin standard curve was prepared as shown in table I
departing from an albumin stock solution of 2 µg/µL.
Methods
18
Table I - BSA standard curve.
Samples Albumin solution (µL) Protein mass (µg) Sample buffer (µL)
Blank 0 0 200
P1 40 0.4 160
P2 80 0.8 120
P3 120 1.2 80
P4 160 1.6 40
P5 200 2 0
Tissue lysate samples (50 µL) were pre-incubated with the reactive mixture (250
µL) for 10 minutes, at room temperature, which was prepared by mixing 2.5 µl of 1%
CuSO4 with 2.5 µl of 2% KNaC4H4O6 followed by the addition of 245 µl of 2% NaCO3
in 0.1 N NaOH. After pre-incubation Folin-Ciocalteau reagent (25 µl diluted with water
in the proportion of 1:1) was added to the mixture and in the dark for 30 minutes, at
room temperature for colour development. The mixture was transferred to a 96 well
plate (Sarstedt) and protein content was measured by spectrophotometry (Tecan ®), at
750 nm.
2.4. Western Blotting
Equal amounts of protein were mixed with 4x sample buffer (1,875 M Tris-HCl
pH 8.8, 8% SDS, 40% glycerol, 0.5 M EDTA, 1 M DTT and 0.2% Bromophenol blue),
heated at 65 ºC for 15 minutes, then at 95 ºC for 5 minutes and centrifuged at 15 000 for
5 minutes. The samples were resolved in Laemmli electrophoresis running buffer (125
mM Tris Base, 250 mM Glycine, 0,1% SDS), in a SDS-Page System set (BioRad) for 1
hour, at 25 mA/gel unit, and electrotransferred to a nitrocellulose membrane in Tris-
glycine transfer buffer (25 mM Tris Base, 390 mM Glycine and 0.37% SDS) containing
20% methanol for 2 hours, at 25 volts. Afterwards, membranes were washed in distilled
water, stained with Ponceau S (0.2% Ponceau, 3% trichloroacetic acid and 3%
sulfosalicylic acid) and photographed in a ChemidocTM
MP imaging system (BioRad).
Then, they were washed with TBS-T (20 mM Tris, 137 mM NaCl, 0.05 mM tween ®
20), blocked for 1 hour in 5% non-fat dry milk in TBS-T and incubated with the
appropriated primary antibody overnight at 4 ºC, with agitation. In the following day,
membranes were washed with TBS-T for 30 minutes (TBS-T was changed every 10
Methods
19
minutes) and probed with donkey secondary antibody [1/15000 anti-rabbit HRP (711-
035-152, Jackson ImmunoResearch)] in 5% non-fat dry milk in TBS- T for 1 hour at
room temperature, also with agitation. Blots were then washed with TBS-T for 30
minutes (TBS-T was changed every 10 minutes) and detected with a chemiluminescent
agent (170-5061, BioRad) for 5 minutes, at room temperature. The reaction was
visualized in a ChemidocTM
MP imaging system (BioRad).
The Ponceau S staining was used for protein normalization and quantification
was performed using Image version 4.0.1 (BioRad).
Table II – Primary antibodies used
Primary antibodies Dilution Reference, Company
SOD I 1:2000 SOD-100 Stressgen Biotechnologies
SOD II 1:2000 SOD-110, Stressgen Biotechnologies
Sirtuin 2 1:1000 S8447, Sigma-Aldrich
HSP70 1:1000 #4873, Cell Signal
Phospho-p38α 1:500 #9215, Cell Signal
Phospho-JNK 1:1000 Sc-6254, Santa Cruz Biotechnologies
2.5. Protein carbonylation
Tissue lysation, protein quantification, electrophoresis, electrotransference and
Ponceau S staining were performed as described in Western Blotting. Membranes were
then equilibrated in 20% methanol (32213, Sigma-Aldrich) in TBS for 5 minutes and
washed in 10% TFA (20751-261, Merck) for 5 minutes. Afterwards, membranes were
incubated in 10 mM DNPH (42210-256-F, Sigma-Aldrich) in 10% TFA for 10 minutes
in the dark, at room temperature, with agitation. Membranes were washed first in 10%
TFA for 25 minutes (TFA was changed every 5 minutes) and then in 50% methanol for
5 minutes. Next, membranes were blocked in 5% BSA in TBS-T for 1 hour and
incubated with appropriated rabbit primary antibody (1/10000 anti-DNP) (D9656,
Sigma-Aldrich) overnight, at 4 ºC. In the next day, membranes were washed with TBS-
T for 30 minutes (TBS-T was changed every 10 minutes) and probed with fluorescent
secondary antibody (1/15000 goat anti-rabbit; 926-32213, Li-Cor Biosciences) in 5%
BSA in TBS-T for 1 hour in the dark, at room temperature, with agitation. Blots were
then washed, in the dark, with TBS-T for 20 minutes, (TBS-T was changed every 10
minutes) and washed again with TBS-Triton 0.2% (T9284, Sigma-Aldrich) for 10
Methods
20
minutes. Membranes were visualized in an Odyssey® CLx Infrared Imaging System
(Li-Cor Biosciences).
The Ponceau S staining was used for protein normalization and quantification
was performed using Image Studio Lite version 3.1 (Li-Cor Biosciences).
2.6. Human Cell Stress Array
The antibody array (ARY018, R&D Systems) is composed of 26 cell stress-
related proteins, listed in table III. All the above-described procedures were performed
following the manufacturer’s instructions.
A similar small portion of tissue was cut from samples frozen at -80 ºC (without
thawing) and crushed in a glass homogenizer (Heidolph) with PBS (10 mM dissodium
phosphate, 1,8 mM monopotassium phosphate, 137 mM sodium chloride, 2,7 mM
potassium chloride) containing proteases inhibitors (P8340, Sigma-Aldrich) and
phosphatases inhibitors (P0044, P5726, Sigma-Aldrich). After homogenization, Triton
X-100 (T9284, Sigma-Aldrich) was added to a final concentration of 1% and samples
were frozen at -80 ºC. Tissue lysates were then thawed and centrifuged at 1000 rpm for
5 minutes. Protein quantification was performed by Bradford method (described below).
Nitrocellulose membranes containing the antibodies were blocked with array
buffer 6 for 1 hour, at room temperature, with agitation in an appropriate 4-well multi-
dish plate. While the membranes were blocking, 600 µg of protein (up to 1 ml) was
mixed with 500 µL of array buffer 4 and then with array buffer 6 to a final volume of
1.5 ml. Reconstituted Detection Antibody Cocktail (20 µl) was added to the mixture and
incubated for 1 hour, at room temperature. After aspiration of the array buffer 6 from
the 4-well multi-dish plate sample/antibody mixtures were added to the nitrocellulose
membranes and incubated overnight at 4 ºC, with agitation.
In the following day, membranes were washed with wash buffer for 30 minutes
(wash buffer was changed every 10 minutes) and incubated with diluted streptavidin-
HRP in array buffer 6 for 30 minutes, at room temperature, with agitation. Membranes
were then washed for 30 minutes (wash buffer was changed every 10 minutes) and
incubated with Chemi Reagent Mix for 5 minutes, at room temperature. The reaction
was visualized in a ChemidocTM
MP imaging system (BioRad).
Methods
21
Table III. Capture antibodies in human cell stress array.
ADAMTS1 HIF-2α Phospho-p38α (T180/Y182)
Bcl-2 Phospho-HSP27 (S78/S82) Phospho-p53 (S46)
Carbonic Anhydrase IX HSP60 PON1
Cited-2 HSP70 PON2
COX-2 IDO PON3
Cytochrome c Phospho-JNK Pan (T183/Y185) Thioredoxin
Dkk-4 NFkB1 Sirtuin2
FABP1/ L-FABP p21/CIP1 SOD2
HIF-1α p27/ Kip1
Table abbreviations: ADAMTS1 - A Disintegrin And Metalloproteinase with Thrombospondin Motifs 1, Bcl-2 - B-cell lymphoma
2, Cited-2 - Cbp/p300-interacting transactivator 2, COX-2 - ciclo-oxigenase 2, Dkk-4 - Dickkopf-related protein 4, FABP-1 - Fatty
acid protein binding 1, HIF - Hypoxia-inducible factor , HSP27 – Heat shock protein 27, HSP60 – Heat shock protein 60, Heat
shock protein 70, IDO - Indoleamine-pyrrole 2,3-dioxygenase, JNK - c-Jun N-terminal kinases, NFkB1 - Nuclear factor NF-kappa-
B p105 subunit , p21/CIP1 - Cyclin-dependent kinase inhibitor 1A , p27/ Kip1 - Cyclin-dependent kinase inhibitor 1B, PON -
Serum paraoxonase/arylesterase, SOD2 – Superoxide dismutase 2
2.7. Protein quantification – Bradford method
A commercial Bradford solution 5x (BioRad) was diluted to a bradford solution
1x, in water. The albumin standard curve was prepared as shown in table IV departing
from an albumin stock solution of 1 µg/µL.
Table IV – BSA standard curve.
Samples Albumin solution (µL) Protein mass (µg) Sample buffer (µL)
Blank 0 0 100
P1 20 0.2 80
P2 40 0.4 60
P3 60 0,6 40
P4 80 0,8 20
P5 100 1 0
In a 96 well plate (Sarstedt) 5 µL of each sample or standards were mixed with
95 µL of Bradford solution 1x. Mixture was left reacting 5 minutes at room temperature
and protein content was measured by spectrophotometry (Tecan ®) at 595 nm.
Methods
22
2.8. Human Phospho-kinase Array kit
Tissue lysation and protein quantification were performed as described in human
cell stress array Kit. The human phospho-kinase array kit (Ary 003B, R&D Systems)
comprises a panel of site-specific phosphorylation of 43 kinases and 2 related total
proteins listed in table V. All the above-described procedures were employed following
the manufacturer’s instructions.
For each sample two nitrocellulose membranes (Part A and Part B) containing
the antibodies were blocked with array buffer 1 for 1 hour, at room temperature, with
agitation. While membranes were blocking, 600 µg of protein (up to 334 µL) was mixed
with array buffer 1 to a final volume of 2 ml. After aspiration of blocking buffer, 1 ml
of sample mixture was added to part A and B of the nitrocellulose membrane and
incubated overnight at 4 ºC, with agitation.
In the following day, membranes were washed for a total time of 30 minutes
(with buffer changes every 10 minutes) and each membrane A and B were incubated
with the respective detection antibody cocktail for 2 hours, at room temperature, with
agitation. Then, membranes were washed again for 30 minutes (wash buffer was
changed every 10 minutes) and incubated with diluted streptavidin-HRP for 30 minutes,
at room temperature, with agitation. Finally, membranes were severally washed for 30
minutes (wash buffer was changed every 10 minutes) and incubated with Chemi
Reagent Mix for 5 minute, at room temperature. The reaction was visualized in
ChemidocTM
MP imaging system (BioRad).
Methods
23
Table V. Capture antibodies in human phospho-kinase array kit.
Akt (S473) Hck (Y411) PRAS40 (T246)
Akt (T308) HSP27 (S78/S82) PyK2 (Y402)
AMPKα1 (T174) HSP60 RSK1/2/3 (S380/S386/S377)
AMPKα2 (T172) JNK pan (T183/Y185, T221/Y223) Src (Y419)
Β-catenin Lck (Y394) STAT2 (Y689)
Chk-2 (T68) Lyn (Y397) STAT3 (S727)
c-jun (S63) MSK 1/2 (S376/S360) STAT3 (Y705)
CREB (S133) P27 (T198) STAT5a (Y694)
EGF R (Y1086) P38α (T180/Y182) STAT5a/b (Y694/Y699)
eNOS (S1177) P53 (S1S) STAT5b (Y699)
ERK 1/2 (T202/Y204, T185/Y187) P53 (S392) STAT6 (Y641)
FAK (Y397) p 53 (S46) TOR (S2448)
Fgr (Y412) p70 S6 Kinase (T421/S424) WNK-1 (T60)
Fyn (Y420) PDGF Rβ (Y751) Yes (Y426)
GSK-3α/β (S21/S9) PLCγ-1 (Y783)
Table abbreviations: Akt – Protein kinase B, AMPKα1 - AMP-activated protein kinase alpha 1, AMPKα2 - AMP-activated protein
kinase alpha 2, Chk-2 - Checkpoint kinase 2, CREB - cAMP response element-binding protein, EGF R - Epidermal growth factor
receptor, eNOS – Endothelial nitric oxide synthases, ERK1/2 – Extracellular signal-regulated kinase 1 and 2, FAK - Focal
Adhesion Kinase, Fgr - Feline Gardner-Rasheed sarcoma viral oncogene, GSK-3α/β - Glycogen Synthase Kinase 3 alpha and beta,
Hck - hemopoietic cell kinase, Lck - Lymphocyte-specific protein tyrosine kinase, Lyn - v-yes-1 Yamaguchi sarcoma viral, MSK
1/2- mitogen- and stress-activated kinase 1 and 2, PDGF Rβ - Platelet-derived growth factor receptor beta, PLCγ-1 -
Phosphoinositide-specific phospholipase C gama 1, PyK2 - Pyruvate kinase 2, RSK1/2/3 - Ribosomal s6 kinase 1, 2 and 3, Src- v-
src avian sarcoma (Schmidt-Ruppin A-2) viral oncogene, STAT - Signal transducer and activator of transcription, WNK-1 - WNK
lysine deficient protein kinase 1.
2.9. Statistical analysis
Arithmetic means are given with standard error mean (SEM). Statistical analysis
was carried out with a Student's t-test. All the association between protein expression
and increasing maternal age was calculated using the Pearson correlation coefficient (r).
The respective plots display a fitted linear regression line. A P-value less than 0.05 was
assumed to denote a significant difference.
Results
Results
27
3. Results
3.1. Reproductive ageing and redox balance in placental bed.
3.1.1. Protein array.
It is known that cellular oxidative stress may alter cell function and contribute to
the development of several pregnancy-related pathologies. Knowing that age is also a
risk factor for their development, it was decided to evaluate the expression of several
cell stress related proteins in tissue lysates from placental bed of young (less than 25
years-old) and reproductively older (more than 34 years-old) women. The array was
performed according to manufacturer’s instructions and results are shown in table VI
and figure 5.
Table VI enumerates the 16 cell stress-related proteins that are expressed in
placental bed.
Table VI - Identification of cell stress-related proteins in placental bed tissue.
Cell stress-related proteins expressed
ADAMTS1 NFkB1
Cited-2 P27
Cytochrome c Phospho-p38α
FABP-1 PON1
HSP60 PON2
HSP70 Thioredoxin-1
IDO SIRT2
Phospho-JNK Pan SOD2
For each protein expressed the average signal of protein duplicate spots was
quantified and a ratio between reproductive older versus young women was determined.
A ratio superior to 1 indicates increased protein expression in reproductively aged
women. It was considered that a protein increase over 20% might denote biological
significance. Cytochrome c, FABP-1, sirtuin 2, SODII, thioredoxin, phospho-p38α and
ADAMST-1 all had a mean increase in protein expression superior to 20%. However, in
the human cell stress array statistical significance was only obtained for cytochrome c,
FABP-1 and ADAMTS1 (figure 5).
Results
28
A B
Figure 5. Human cell stress array profile in placental bed. Relative expression of 26 cell stress related
proteins was determined in placental bed lysates from young (less than 25) and reproductively older
(more than 34) women. (A) Representative arrays. (B) Ratio reproductive older/young of the
quantification of the average signal of protein duplicates. Columns represent the mean of 3 experiments
per group; vertical lines show error bars. (*) Significantly different from control values using the student t
test (P < 0.05).
3.1.2. Western blotting – Protein array validation.
To validate the results obtained in the cell stress-related protein array the effect
of maternal age in the expression of sirtuin 2, SODII, HSP70 and phospho-p38α was
evaluated by western blotting.
In the cell stress-related protein array, sirtuin 2 expression in placental bed had a
mean increase over 20%. Western blot results for sirtuin 2 denote an increase in protein
expression in women with advanced reproductive age (Figure 6 A and B). A Pearson
correlation value of 0.52 suggests a moderate positive correlation, but it did not reach
statistical significance. Western blot for SODII was also performed and results are
shown in figure 6 C and D. SODII expression increased significantly along maternal
age. As for sirtuin 2, a Pearson correlation value of 0.51 suggests a moderate positive
correlation between SODII expression and maternal age. HSP70 western blot results
confirm the expression pattern obtained in the cell stress array. No correlation was
observed between maternal age and HSP 70 expression in placental bed (Figure 6 E and
F). No correlation was also observed between maternal age and protein expression
regarding phospho-p38α protein expression (Figure 6 G and H). The great dispersion
observed in the plotted values confirms the major error bar obtained in the array.
CTS Cit
FABPSirt
2
SOD2
Thiore
d
HSP70 p38
pon1
ADAM
TS1
0
1
2
3
4
Pro
tein
exp
ress
ion
ra
tio(a
.u.)
* * *
Results
29
The results obtained in western blot studies are consistent with the results from
the array and thus validate the obtained data.
A C
B D
E G
F H
Figure 6. Effect of age on cell stress-related protein expression. Protein expression was determined in
placental bed tissue obtained from women between 22 and 41 years old. (A, C, E, G) Representative
western blots for Sirtuin 2, SODII, HSP70 and phospho- p38, respectively. Quantification of sirtuin 2 (B),
SODII (D), HSP70 (F) and phospho p38 (H) protein expression. In each experiment the Ponceau S
staining was used for protein normalization. Data are expressed as relative densitometric units and are
plotted according to maternal age (r Pearson coefficient).
20 25 30 35 40 450.6
0.8
1.0
1.2
1.4
Age (years)
Sirt2
(re
lativ
e e
xp
ressio
n)
r = 0.5163p= 0.0709
20 25 30 35 40 450.0
0.5
1.0
1.5
2.0
2.5
r=0,5050p=0.0387
Age (years)
SO
D I
I exp
ressio
n
(rela
tive
units)
20 25 30 35 40 450.0
0.5
1.0
1.5
Age (years)
HS
P7
0 e
xp
ressio
n(r
ela
tive
un
its)
r = 0.1292p= 0.6832
20 25 30 35 40 450.0
0.5
1.0
1.5
2.0
Age (years)
ph
osp
ho
p3
8 e
xp
ressio
n(r
ela
tive
un
its)
r = 0.1668p= 0.6043
Results
30
3.1.3. Oxidative stress marker - protein carbonylation.
Oxidative stress may modify biological molecules. In the case of proteins, ROS
may lead to the oxidation of amino acid residue side chains, to the formation of protein-
protein cross-linkages and to oxidation of protein backbone, which result in loss of
function and protein fragmentation (Berlett and Stadtman 1997). These oxidative
modifications lead to the formation of protein carbonyl derivatives, which are
commonly seen as a biomarker of oxidative stress. Western blotting using an anti-DNP
antibody was the method used to detect and quantify protein carbonylation in placental
bed samples.
A B
Figure 7. Effect of age on DNP expression. DNP expression was determined in placental bed tissue
obtained from women between 22 and 41 years old. (A) Representative western blots. (B) Quantification
of protein expression. In each experiment the Ponceau S staining was used for protein normalization. Data
are expressed as relative densitometric units and are plotted according to maternal age (r Pearson
coefficient).
After quantification of total protein carbonylation in the analysed samples, no
correlation was observed between these two variables. However, when performing
quantification of protein carbonylation by western blot it is possible to measure specific
carbonylation bands. As such, proteins highly carbonylated were given random letters
from A to D and quantified separately. Figure 8 shows that a moderate positive
correlation, with statistical significance, between age and specific carbonylation of
protein A is occurring.
20 25 30 35 40 450.0
0.5
1.0
1.5
2.0
2.5
Age (years)
Pro
tein
ca
bo
nyla
tion
(re
lativ
e u
nits)
r = 0.3132p= 0.2755
Results
31
A B
C D
Figure 8. Effect of age on DNP expression in different proteins . DNP expression was determined in
placental bed tissue obtained from women between 22 and 41 years. (A, B, C, D) Quantification of
protein A, protein B, protein C and protein D carbonylation, respectively. In each experiment the Ponceau
S staining was used for protein normalization. Data are expressed as relative densitometric units and are
plotted according to maternal age (r Pearson coefficient).
3.2. Reproductive ageing and cell signalling.
3.2.1. Protein array.
As oxidative stress can alter cell signalling and thus affect important cell
functions it was evaluated a panel of site-specific phosphorylation of 43 kinases using
the human phospho-kinase array kit (Ary 003). The array was performed according to
manufacturer’s instructions and results are shown in figure 9 and table VII.
20 25 30 35 40 450.0
0.5
1.0
1.5
2.0
2.5
r = 0.5272p= 0.0434
Age (years)
Pro
tein
A c
ab
on
yla
tio
n(r
ela
tive
un
its)
20 25 30 35 40 450.0
0.5
1.0
1.5
2.0
2.5
r = 0.0145p= 0.9608
Age (years)
Pro
tein
B c
ab
on
yla
tio
n(r
ela
tive
un
its)
20 25 30 35 40 450
1
2
3
4
r = 0.2133p= 0.0455
Age (years)
Pro
tein
C c
ab
on
yla
tion
(re
lativ
e u
nits
)
20 25 30 35 40 450.0
0.5
1.0
1.5
2.0
2.5
r=-0,0885p=0,7635
Age (Years)
pro
tein
D e
xpre
ssio
n
(rela
tive u
nits)
Results
32
Table VII - Identification of phospho-kinase proteins expressed in placental bed tissue.
Phospho-kinase proteins expressed
p38 α RSK 1/2/3 PDGF Rβ Lck
Erk 1/2 eNOS STAT5a/b STAT 2
JNK Pan Fyn WNK 1 STAT 5a
GSK-3α/β Yes PYK2 p70 S6 kinase
P53 Fgr PRAS40 PLC-γ1
EGF R STAT 6 HSP60 Hck
MSK1/2 STAT 5b c-Jun Chk-2
AMPKα1 STAT3 (S727) Src FAK
Akt STAT3 (Y705) Lyn CREB
TOR p27 β-catenin HSP27
AMPKα2
After quantification of the average signal of phosphorylated protein duplicate
spots, a ratio between reproductive older versus young women was determined. A ratio
superior to 1 indicates increased protein expression in reproductively aged women. The
criteria used in the analysis of the present array were the same as for the human cell
stress array. It was considered that an increase/decrease over 20%, in the specific
phosphorylation site analysed, might denote biological significance. It was observed
that the protein with a ratio superior to 1 was phospho-Stat3. Stat 3 had an increase in
protein phosphorylation of 139% in S727 and 22% in Y705. Proteins with a decrease
over 20% were JNK pan (T183/Y185, T221/Y223), MSK1/2 (S376/S360), Akt (S473),
c-jun (S63), p53 (S392), Gsk3-α/β (S21,S9), STAT5b (Y699), LCK (Y394) and Fgr
(Y412) (figure 9).
Figure 9. Human phospho-kinase array profile in placental bed. Relative expression of 43 kinase and
2 related proteins was determined in placental bed lysates from young (less than 25) and reproductively
older (more than 34) women. (A) Representative arrays. (B) Ratio reproductive older/young of the
quantification of the average signal of protein duplicates obtained in placental bed tissue lysates.
A B
Ct
RSK
p70
Stat3
Stat3p5
3
c-Ju
n
GSK
3Akt
Stat5JN
KSta
t6
PDG
F
MSK1/
2LC
KFgr
CREB
Src
0
1
2
3
Pro
tein
exp
ressio
n ra
tio
(a.u
.)
Results
33
For array confirmation, phospho-JNK was evaluated by western blot.
3.2.2. Western blotting – Protein array validation.
To validate the results obtained in the human phospho-kinase array kit, phospho-
JNK1/2 expression was determined in lysates from placental bed tissue. Figures 10
represents the relative blots of phospho-JNK1 and phosho-JNK2, respectively.
After normalization with Ponceau S, no significant changes were observed for
both phospho-JNK1 and phospho-JNK2.
A C
B D
Figure 10. Effect of age on phospho-kinase protein expression. Protein expression was determined in
placental bed tissue obtained from women between 22 and 41 years old. (A, C) Representative western
blots for phospho-JNK 1 and phospho-JNK2, respectively. Quantification of phospho-JNK1 (B),
phospho-JNK2 (D). In each experiment the Ponceau S staining was used for normalization. Data are
expressed as relative densitometric units and are plotted according to maternal age (r Pearson coefficient).
3.3. Redox balance in placental bed and pregnancy-associated disorders.
3.3.1. Western blotting – cell stress related proteins.
Previous studies reported differences in placental antioxidant levels in normal
and pregnancies associated disorders such as hypertension, preeclampsia and IUGR.
20 25 30 35 40 450.0
0.5
1.0
1.5
2.0
Age (years)
ph
osp
ho
JN
K1
exp
ressio
n(r
ela
tive
un
its)
r = 0.2495p= 0.4110
20 25 30 35 40 450.0
0.5
1.0
1.5
Age (years)
ph
osp
ho
JN
K2
exp
ressio
n(r
ela
tive
un
its)
r = 0.1190p= 0.6985
A
Results
34
Results for the expression of SODI, SODII and sirtuin 2 in placental bed of normal
pregnancies and pregnancies with associated-disorders are depicted in figures 11-13.
Figure 11 shows the expression of SODI in normal pregnancies and in pregnancies with
associated disorders. Following normalization with Ponceau S staining no changes were
observed in SODI, in pregnancies with gestational hypertension plus IUGR. In the
group of pregnancies complicated with chronic hypertension and chronic hypertension
plus pre-eclampsia SODI expression is increased (Figure 11).
A C
B D
Figure 11. Effect of pregnancy related disorders on superoxide dismutase I expression. Protein
expression was determined in placental bed tissue. (A,C) Representative western blots for SOD I in
gestational HT plus IUGR, in chronic hypertension and chronic hypertension plus preeclampsia,
respectively. Quantification of SOD I protein expression in gestational HT plus IUGR (B), in chronic
hypertension and in chronic hypertension plus pre-eclampsia (D). In each experiment Ponceau S staining
was used for normalization. Data are expressed as relative densitometric units (r Pearson coefficient).
No changes in the expression levels of SODII isoform were observed in
placental bed from women with the studied pregnancy-associated disorders (Figure 12).
The expression of sirtuin 2 was also evaluated in pregnancy-related disorders.
Sirtuin 2 expression is only significantly increased in placental bed from women with
pregnancy-associated chronic hypertension and pre-eclampsia (Figure 13).
Normal gHT+ IUGR0.0
0.5
1.0
1.5
2.0
SO
DI
expre
ssio
n
(rela
tive u
nits)
Normal cHT cHT+PE0.0
0.5
1.0
1.5
2.0 *
SO
DI
exp
ressio
n
(rela
tive
units)
Results
35
A C
B D
Figure 12. Effect of pregnancy related disorders on superoxide dismutase II expression. Protein
expression was determined in placental bed tissue. (A, C) Representative western blots for SOD II in
gestational HT plus IUGR, in chronic hypertension and chronic hypertension plus preeclampsia,
respectively. Quantification of SOD II protein expression in gestational HT plus IUGR (B), in chronic
hypertension and in chronic hypertension plus pre-eclampsia (D). In each experiment the Ponceau S
staining was used for protein normalization. Data are expressed as relative densitometric units.
A C
B D
Figure 13. Effect of pregnancy related disorders on Sirtuin 2 expression. Protein expression was
determined in placental bed tissue. (A,C) Representative western blots for Sirtuin2 in gestational HT plus
IUGR, in chronic hypertension and chronic hypertension plus preeclampsia, respectively. Quantification
of Sirtuin2 protein expression in gestational HT plus IUGR (B), in chronic hypertension and in chronic
hypertension plus pre-eclampsia (D). In each experiment Ponceau S staining was used for normalization.
Data are expressed as relative densitometric units.
Normal gHT+IUGR0.0
0.5
1.0
1.5
SO
DII
expre
ssio
n
(rela
tive u
nits)
Normal cHT cHT+PE0.0
0.5
1.0
1.5
2.0
2.5
SO
DII
exp
ressio
n
(rela
tive
units)
Normal gHT+IUGR0.0
0.5
1.0
1.5
Sir
t2 e
xpre
ssio
n
(rela
tive u
nits)
Normal cHT cHT+PE0.0
0.5
1.0
1.5 *
Sir
t2 e
xpre
ssio
n
(rela
tive
units)
Results
36
3.3.2. Oxidative stress marker – protein carbonylation.
It is evidenced that protein carbonylation is increased several pathologies. Figure
14 shows protein carbonylation in placental bed tissues of the studied pregnancy-
associated disorders. Total protein carbonylation did not show any significant changes.
A C
B D
Figure 14. Effect of pregnancy related disorders on DNP expression. Protein
expression was determined in placental bed tissue. (A,C) Representative western blots for DNP in
gestational HT plus IUGR, in chronic hypertension and in chronic hypertension plus preeclampsia,
respectively. Quantification of DNP protein expression in gestational HT plus IUGR (B), in chronic
hypertension and in chronic hypertension plus pre-eclampsia (D). In each experiment the Ponceau S
staining was used for protein normalization. Data are expressed as relative densitometric units.
Specific carbonylation of proteins was performed as in 3.1.3 section. Proteins
highly carbonylated were given random letters taking into consideration that the letters
were the same for proteins already quantified in 3.1.3 section and between groups.
Figure 15 shows that a positive correlation in protein F in gestational HT plus IUGR
occurs with statistical significance.
Normal gHT+IUGR0.0
0.5
1.0
1.5
2.0
2.5
Pro
tein
cabonyla
tion
(rela
tive u
nits)
Normal cHT cHT+PE0.0
0.5
1.0
1.5
2.0
2.5
Pro
tein
cabonyla
tion
(rela
tive
units)
Results
37
A B
C D
E F
Figure 15. Effect of gestational hypertension with IUGR on DNP expression in different proteins.
Protein expression was determined in placental bed tissue. Quantification of protein A expression (A),
protein C expression (B), protein E expression (C), protein F expression (D), protein G expression (E)
and protein H expression (F) in gestational HT plus IUGR. In each experiment Ponceau S staining was
used for normalization. Data are expressed as relative densitometric units.
Normal gHT+ IUGR0.0
0.5
1.0
1.5
2.0
2.5pro
tein
A e
xpre
ssio
n
(rela
tive u
nits)
Normal gHT+IUGR0.0
0.5
1.0
1.5
2.0
2.5
Pro
tein
C e
xpre
ssio
n
(rela
tive u
nits)
Normal gHT+IUGR0.0
0.5
1.0
1.5
2.0
2.5
Pro
tein
E e
xpre
ssio
n
(rela
tive u
nits)
Normal gHT+IUGR0.0
0.5
1.0
1.5
2.0
2.5 *
Pro
tein
F e
xpre
ssio
n
(rela
tive u
nits)
Normal gHT+IUGR0.0
0.5
1.0
1.5
2.0
2.5
Pro
tein
G e
xpre
ssio
n
(rela
tive u
nits)
Normal gHT+IUGR0.0
0.5
1.0
1.5
2.0
2.5
Pro
tein
H e
xpre
ssio
n
(rela
tive u
nits)
Results
38
No changes were observed in placental bed protein carbonylation levels for
chronic hypertension and chronic hypertention plus pre-eclampsia.
A B
C D
Figure 16. Effect of chronic hypertension with or without pre-eclampsia on DNP expression in
different proteins. Protein expression was determined in placental bed. Quantification of protein A
expression (A), protein C expression (B), protein F expression (C), protein G expression (D) in chronic
hypertension with or without PE. In each experiment the Ponceau S staining was used for protein
normalization. Data are expressed as relative densitometric units.
Normal cHT cHT+PE0.0
0.5
1.0
1.5
2.0
2.5
Pro
tein
A e
xpre
ssio
n
(rela
tive
units)
Normal cHT cHT+PE0.0
0.5
1.0
1.5
2.0
2.5
Pro
tein
C e
xpre
ssio
n
(rela
tive u
nits)
Normal cHT cHT+PE0
1
2
3
4
5
Pro
tein
F e
xpre
ssio
n
(rela
tive u
nits)
Normal cHT cHT+PE0.0
0.5
1.0
1.5
2.0
2.5P
rote
in G
exp
ressio
n
(rela
tive
units)
Discussion
Discussion
41
4. Discussion
An age-related decline in female fertility is a well known fact in several species
(Ito, Miyado et al. 2010). It is considered that there are a number of reasons for this
decline, being the loss of quality of the eggs released by the ovary one of the most well
studied (Wu, Tang et al. 2007, Yung, Maman et al. 2010). Eventually, when women
become pregnant, there is also an age-related increase in the risk of developing
pregnancy-related disorders that may lead several serious complications that include
abortion, foetal death, preterm delivery, pre-eclampsia, IUGR and abruption placenta.
Although during the last decades many studies addressed the placenta of these
pregnancies, currently it is believed that most of these disorders arise from
abnormalities in the transformation of the uterine placental bed. There is evidence that
redox process intervenients, including ROS and anti-oxidant enzymes are important
players in placentation and an imbalance in this system may lead/contribute to the loss
of placenta function. It was based on these findings that the hypothesis of the present
work was developed and the major aims of this project were defined.
A human cell-stress array kit was used to evaluate oxidative stress status in
placental bed samples from young and reproductively aged women. The use of the cell
stress array kit Ary 018 from R&D Systems was chosen because it is a sensitive tool
that enables the simultaneous detection of 26 cell stress-related proteins in a single
tissue sample and the detection of changes between samples. As one of the limitations
of the arrays is the number of samples used, it was necessary to validate the results
obtained using western blotting. Validation was performed with 4 antibodies against
proteins that had substantially different expression in the array and similar results were
obtained. Of the proteins studied in the array, cytochrome c, FABP-1 and ADAMTS1
were significantly increased. The western blot results also demonstrated that there was a
positive correlation between maternal age and SODII expression.
Cytochrome c is an electron-transport protein located in the inner membrane of
the mitochondria (Velayutham, Hemann et al. 2011). About 15% of cytochrome c is
tightly bound to the membrane, mediates electron transfer and upon interaction with
cardiolopin may have a peroxidase activity. The remaining cytochrome c is loosely
attached and besides participating in electron transport mediates superoxide removal
and prevents OS. Thus, on different contexts cytochrome c can act as a pro or an anti-
oxidant protein (Hüttemann, Pecina et al. 2011, Velayutham, Hemann et al. 2011).
Discussion
42
Moreover, cytochrome c can also be an apoptotic factor (Hüttemann, Pecina et al.
2011). Upon apoptotic stimulation it is released from the mitochondria, activates pro-
caspase 9 and may initiate apoptosis. Further studies would be necessary to elucidate
the meaning of the age-related increase observed in placental site tissue.
Regarding FABP-1, it is mainly a cytoplasmatic protein, most extensively
studied in the liver. It promotes cellular uptake and intracellular utilization of fatty
acids. In vitro studies using human hepatoma cell lines demonstrated that hat FABP-1
has an anti-oxidant function (Wang, Gong et al. 2005). In the placenta, elevated levels
of this protein were found in pregnancies complicated by diabetes and IUGR
(Magnusson, Waterman et al. 2004). Its expression in placental site tissue has not been
previously described, nor its possible function in this tissue.
ADAMTS are known as a desintegrin and metalloproteinase with
thrombospodin motifs that belongs to the metalloproteinase family ADAMS (a
desintegrin and metalloproteinase) (Kaushal and Sahah 2000) and are often incorporated
into the extracellular matrix (Shindo, Kurihara et al. 2000). ADAMTS1 appears to have
a role in fertility, organ morphology and function and normal growth (Shindo, Kurihara
et al. 2000). Knock out mice for ADAMTS1 had uterine abnormalities (Kaushal and
Shah 2000, Shindo, Kurihara et al. 2000). As ADAMST1 is a metalloproteinase present
in the extracellular matrix (ECM), it may influence ECM remodelling and modify cell
adhesion (Kaushal and Shah 2000). This remodelling is important for many
differentiative processes such as trophoblast invasion, uterine and mammary changes
during menstrual cycle and pregnancy (Streuli 1999). Moreover, in vitro studies
demonstrated that ADAMTS1 has a potent anti-angiogenic function (Hohberg, Knöchel
et al. 2011). Having in mind that both uterine remodelling and angiogenesis are
important for a successful pregnancy and increased expression of ADAMTS1 may
increase the risk for developing pregnancy-related complications.
Besides these proteins that had a significantly increased expression in
reproductively aged women, SODII expression also correlated positively with
reproductive ageing. Although studies performed in other tissues demonstrated that
SODII expression decreases with age (Matos, Stevenson et al. 2009, Priyanka, Sharma
et al. 2013) , it is possible that at 35-40 years this increase reflects a compensation
facing an increased production of ROS. Evaluation of ROS production in placental site
could validate this hypothesis. It would be also interesting to measure catalase activity,
since a decrease in catalase activity along maternal age could cause a reduction of the
Discussion
43
catalase/SODII ratio indicating a reduction of the placental site tissue scavenging
efficiency along ageing (Carbone, Tatone et al. 2003).
All together these results point to an age-related alteration of placental bed redox
balance.
It is believed that OS can damage biomolecules like proteins, DNA and lipids.
As proteins are believed to be the main biomolecule of cells and tissues, they are
potentially the major targets for oxidative damage (Bozaykut, Sozen et al. 2013).
Moreover, protein carbonylation is an irreversible protein oxidation that leads to the
formation of stable carbonyl groups on the protein side chain (Dalle-Donne, Aldini et al.
2006, Suzuki, Carini et al. 2010). For the above-mentioned reasons, placental bed OS
status during maternal ageing was evaluated by quantification of protein carbonylation.
No significant correlation was found between total protein carbonylation and maternal
age. However, the method used (determination of protein carbonyl groups by western
blot) allowed the quantification of specific carbonylated proteins (Suzuki, Carini et al.
2010). As such, specific proteins were assigned letters from A to D. Following this
approach it was demonstrated that carbonylation of protein A was significantly affected
by maternal ageing. Knowing that proteins especially rich in arginine, proline, lysine
and threonine are more prone to protein oxidation these results suggest that in
reproductively aged women there are mild increases in OS that might be affecting, at
this point, only specific proteins. Although it is believed that the general function of the
cells is not compromised this does not rule out the possibility that, at some extent, mild
increases in OS may induce alterations in cell signalling, even in pregnancies without
complications.
The human phospho-kinase array performed in placental site samples from
young and reproductively aged women demonstrated that there was a slight age-related
decrease in several kinases, but the phosphorylation level of signal transducer and
activator of transcription (STAT) 3 was increased more than 100%. STATs are a family
of proteins involved in cell growth, apoptosis, organogenesis, innate and adaptive
immunity, cancer and cancer metastasis (Horvath 2000) and embryogenesis (Horvath
2000, Levy and Lee 2002). It is suggested that STAT3 may be directly activated by
oxidative stress (Carballo, Conde et al. 1999), which could explain the present finding.
It has been suggested to exist a connection between STAT3 activity and trophoblast
invasiveness (Poehlmann, Fitzgerald et al. 2006). The meaning of the observed increase
of STAT3 expression in placental site needs further research. However, the results
Discussion
44
obtained from the human phospho-kinase array demonstrate that there is an age-related
alteration in cell signalling, in placental site tissues obtained from healthy women who
had pregnancies without complications.
Oxidative stress has been suggested to play an important role in the
pathophysiology of several pregnancy-related complications (Many, Hubel et al. 2000,
Scifres and Nelson 2009) as well as in cardiovascular diseases (Elahi, Kong et al. 2009),
cancer (Sosa, Moliné et al. 2013) and in neurodegenerative diseases (Reynolds, Laurie
et al. 2007). As such, in the second part of this work it was evaluated the expression of
two main anti-oxidant enzymes (SOD I and SOD II) and sirtuin 2 (attending to the fact
that it showed a moderate positive correlation with increased reproductive age) in
women with pregnancy-related disorders. Two groups of study were defined. Group one
with women who had gestational hypertension and IUGR of the foetus and group two
with women that have chronic hypertension with or without pre-eclampsia. SOD I is an
antioxidant enzyme located in the cytosol while SOD II is located in the mitochondria.
Both these antioxidant enzymes scavenge superoxide anion into hydrogen peroxide and
have an important role in the regulation of redox-balance (Burton and Jauniaux 2011).
Sirtuin 2 is a protein mainly located in the cytoplasm. Sirtuin 2 deacetylates α-tubulin
and has been implicated in cytoskeleton regulation and progression of cell mitosis
(Wang, Nguyen et al. 2007, Dan, Klimenkova et al. 2012, Machado de Oliveira,
Sarkander et al. 2012).
No significant changes were observed in both SODI and sirtuin 2 expressions in
women with gestational hypertension and IUGR of the foetus while these enzymes were
increased in women with chronic HT and chronic hypertension and PE. In the case of
SODII no changes were observed in placental bed of all the pregnancy disorders
studied. After western blot analysis it was demonstrated that, although as mentioned in
the introduction both in IUGR and in PE a shallow trophoblast invasion of placental bed
occurs, the cellular mechanisms altered in placental bed of these pathologies differ.
Favouring this statement are the results obtained by the evaluation of protein carbonyl
contents in the same groups. Protein assigned with the letter F is significantly increased
only in pregnancies complicated by gestational hypertension and IUGR of the foetus.
The fact that no significant changes were observed in protein carbonylation in
pregnancies complicated by PE and only one of the 6 proteins analysed in gestational
HT and IUGR of the foetus is significantly increased are in agreement with recent
Discussion
45
findings, that have been well reviewed pointing to an important role of protein nitration
in the development of pregnancy-related pathologies (Webster, Roberts et al. 2008,
White, Capobianco et al. 2010).
Conclusions and
future perspectives
Conclusions and future perspectives
49
5. Conclusions and future perspectives
This work focused in the evaluation of local redox balance and altered cell
signalling in placental bed tissues from young and reproductively older women.
Additionally, redox imbalance in placental bed of pregnancies with associated
pathologies was also addressed.
The results of the present thesis indicate an age- and pathology-related redox
imbalance in placental bed that is accompanied by alterations in cell signalling.
Furthermore, specific protein modifications, expression and carbonylation, were found
altered in placental bed. This observation further supports the hypothesis that local
uterine redox imbalance contributes to the development of placental bed dysfunction.
Future studies should focus on elucidating the role of these altered proteins in the
regulation of uterine tissue remodelling.
The present thesis also emphasises that redox status varies among distinct
pregnancy-associated disorders. This supports the need to develop specific antioxidants,
since it is believed that prevention of severe cellular OS during pregnancy can avoid the
development of pregnancy-associated complications and currently there are no such
specific molecules available.
Final Remarks
Final remarks
53
6. Final remarks
1) Part of this work was presented in the following meetings:
Summary in an international congress
Soares A, Silveira S, Almeida H, Silva E. “Anti-oxidant supplementation and
reproductive outcome in aged female mice”. Livro de resumos do 7th Young
European Scientist Meeting (YES Meeting). 2012: 91.
Communication in an international congress
Soares A, Silveira S, Almeida H, Silva E. “Anti-oxidant supplementation and
reproductive outcome in aged female mice”. 7th Young European Scientist
Meeting (YES Meeting). Porto. 2012.
Summary in a national meeting
Soares A, Silveira S, Almeida H, Silva E. “Aged female diet anti-oxidant
supplementation improves reproductive outcome”. 3rd
I3S Scienticic Retreat.
Póvoa de Varzim. 2012.
2) Part of this work was funded by “Prémio Crioestaminal 2011” (Federação da
Sociedade Portuguesa da Ginecologia e Obstetrícia), awarded to Henrique
Almeida, Luis Guedes-Martins, Elisabete Silva and Liliana Matos.
Bibliography
Bibliography
57
7. Bibliography
Agarwal, A., et al. (2012). "The effects of oxidative stress on female
reproduction: a review." Reproductive Biology and Endocrinology 10(1): 49.
Agarwal, A., et al. (2005). "Role of oxidative stress in female reproduction."
Reproductive Biology and Endocrinology 3(1): 1-21.
Aouadi, M., et al. (2006). "Role of MAPKs in development and differentiation:
lessons from knockout mice." Biochimie 88(9): 1091-1098.
Balasch, J. and E. Gratacós (2011). "Delayed Childbearing: Effects on Fertility
and the Outcome of Pregnancy." Fetal Diagnosis and Therapy 29(4): 263-273.
Bell, E. (2004). "A bad combination." Nat Rev Immunol 4(12): 927-927.
Berlett, B. S. and E. R. Stadtman (1997). "Protein Oxidation in Aging, Disease,
and Oxidative Stress." Journal of Biological Chemistry 272(33): 20313-20316.
Berridge, M. J. (2012). "Cell signalling Pathways." Cell signalling Biology 2.
Bozaykut, P., et al. (2013). "The role of heat stress on the age related protein
carbonylation." Journal of Proteomics 89(0): 238-254.
Burton, G. J. and E. Jauniaux (2011). "Oxidative stress." Best practice &
research. Clinical obstetrics & gynaecology 25(3): 287-299.
Carballo, M., et al. (1999). "Oxidative Stress Triggers STAT3 Tyrosine
Phosphorylation and Nuclear Translocation in Human Lymphocytes." Journal of
Biological Chemistry 274(25): 17580-17586.
Carbone, M. C., et al. (2003). "Antioxidant enzymatic defences in human
follicular fluid: characterization and age‐dependent changes." Molecular Human
Reproduction 9(11): 639-643.
Coulthard, L. R., et al. (2009). "p38MAPK: stress responses from molecular
mechanisms to therapeutics." Trends in Molecular Medicine 15(8): 369-379.
Bibliography
58
Cuenda, A. and S. Rousseau (2007). "p38 MAP-Kinases pathway regulation,
function and role in human diseases." Biochimica et Biophysica Acta (BBA) -
Molecular Cell Research 1773(8): 1358-1375.
Dalle-Donne, I., et al. (2006). "Protein carbonylation, cellular dysfunction, and
disease progression." Journal of Cellular and Molecular Medicine 10(2): 389-
406.
Dalle-Donne, I., et al. (2003). "Protein carbonyl groups as biomarkers of
oxidative stress." Clinica Chimica Acta 329(1–2): 23-38.
Dan, L., et al. (2012). "The role of sirtuin 2 activation by nicotinamide
phosphoribosyltransferase in the aberrant proliferation and survival of myeloid
leukemia cells." Haematologica 97(4): 551-559.
Daoud, G., et al. (2005). "ERK1/2 and p38 regulate trophoblasts differentiation
in human term placenta." The Journal of Physiology 566(2): 409-423.
Devasagayam, T., et al. (2003). "Methods for estimating lipid peroxidation: an
analysis of merits and demerits." Indian Journal of biochemistry & Biophysics
40: 300-308.
Elahi, M. M., et al. (2009). "Oxidative Stress as a Mediator of Cardiovascular
Disease." Oxidative Medicine and Cellular Longevity 2(5): 259-269.
Evans, M. D., et al. (2004). "Oxidative DNA damage and disease: induction,
repair and significance." Mutation Research/Reviews in Mutation Research
567(1): 1-61.
Finkel, T. (2003). "Oxidant signals and oxidative stress." Current Opinion in
Cell Biology 15(2): 247-254.
Fujimaki, A., et al. (2011). "Placental oxidative DNA damage and its repair in
preeclamptic women with fetal growth restriction." Placenta 32(5): 367-372.
Georgiades, P., et al. (2002). "Comparative Developmental Anatomy of the
Murine and Human Definitive Placentae." Placenta 23(1): 3-19.
Bibliography
59
Gupta, S., et al. (2005). "The role of placental oxidative stress and lipid
peroxidation in preeclampsia." Obstetrical and Gynecological survey 60(12).
Gupta, S., et al. (2009). "Oxidative Stress and its Role in Female Infertility and
Assisted Reproduction: Clinical Implications." International Journal of Fertility
and Sterility 2(4): 147-164.
Harman, D. (1956). "Aging: a theory based on free radical and radiation
chemistry." Gerontol 11(3): 298-300.
Hohberg, M., et al. (2011). "Expression of ADAMTS1 in endothelial cells is
induced by shear stress and suppressed in sprouting capillaries." Journal of
Cellular Physiology 226(2): 350-361.
Horvath, C. M. (2000). "STAT proteins and transcriptional responses to
extracellular signals." Trends in biochemical sciences 25(10): 496-502.
Hubel, C. A. (1999). "Oxidative Stress in the Pathogenesis of Preeclampsia."
Proceedings of the Society for Experimental Biology and Medicine. Society for
Experimental Biology and Medicine (New York, N.Y.) 222(3): 222-235.
Hüttemann, M., et al. (2011). "The multiple functions of cytochrome c and their
regulation in life and death decisions of the mammalian cell: From respiration to
apoptosis." Mitochondrion 11(3): 369-381.
Istance, D. and H. Theisens (2008). "Ageing OECD Societies in Trends shaping
education 2008. Centre for Educational Research and Innovation." Organization
for Economic Co-operation and Development, OECD: 13-20.
Ito, M., et al. (2010). "Age-associated changes in the subcellular localization of
phosphorylated p38 MAPK in human granulosa cells." Molecular Human
Reproduction 16(12): 928-937.
James, P. R. and C. Nelson-Piercy (2004). "Management of hypertension before,
during, and after pregnancy." Heart 90(12): 1499-1504.
Bibliography
60
Jauniaux, E., et al. (2006). "Placental-related diseases of pregnancy:
involvement of oxidative stress and implications in human evolution." Human
Reproduction Update 12(6): 747-755.
Junttila, M. R., et al. (2008). "Phosphatase-mediated crosstalk between MAPK
signaling pathways in the regulation of cell survival." The FASEB Journal
22(4): 954-965.
Kaushal, G. P. and S. V. Shah (2000). "The new kids on the block: ADAMTSs,
potentially multifunctional metalloproteinases of the ADAM family." The
Journal of Clinical Investigation 105(10): 1335-1337.
Kumari, S., et al. (2008). "DNA damage: detection strategies." EXCLI Journal
7: 44-62
Lázár, L. (2012). "The Role of Oxidative stress in Female Reproduction and
Pregnancy." Oxidative Stress and Diseases 14.
Levine, R. L. (2002). "Carbonyl modified proteins in cellular regulation, aging,
and disease." Free Radical Biology and Medicine 32(9): 790-796.
Levy, D. E. and C.-k. Lee (2002). "What does Stat3 do?" The Journal of Clinical
Investigation 109(9): 1143-1148.
Lyall, F. (2002). "The Human Placental Bed Revisited." Placenta 23(8): 555-
562.
Lyall, F. (2005). "Priming and remodelling of human placental bed spiral
arteries during pregnancy – A Review." Placenta 26, Supplement(0): S31-S36.
Machado de Oliveira, R., et al. (2012). "SIRT2 as a therapeutic target for age-
related disorders." Frontiers in Pharmacology 3.
Magnusson, A. L., et al. (2004). "Triglyceride Hydrolase Activities and
Expression of Fatty Acid Binding Proteins in the Human Placenta in
Pregnancies Complicated by Intrauterine Growth Restriction and Diabetes."
Journal of Clinical Endocrinology & Metabolism 89(9): 4607-4614.
Bibliography
61
Many, A., et al. (2000). "Invasive Cytotrophoblasts Manifest Evidence of
Oxidative Stress in Preeclampsia." The American journal of pathology 156(1):
321-331.
Matos, L., et al. (2009). "Superoxide dismutase expression in human cumulus
oophorus cells." Molecular Human Reproduction 15(7): 411-419.
McCubrey, J. A., et al. (2006). "Reactive oxygen species-induced activation of
the MAP Kinase pathways." Antioxidants & Redox Signaling 8(9-10).
Moon, K. C., et al. (2008). "Expression of Extracellular Signal-Regulated
Kinase1/2 and p38 Mitogen-Activated Protein Kinase in the Invasive
Trophoblasts at the Human Placental Bed." Placenta 29(5): 391-395.
Mudgett, J. S., et al. (2000). "Essential role for p38α mitogen-activated protein
kinase in placental angiogenesis." Proceedings of the National Academy of
Sciences 97(19): 10454-10459.
Myatt, L. and X. Cui (2004). "Oxidative stress in the placenta." Histochemistry
and Cell Biology 122(4): 369-382.
Nystrom, T. (2005). "Role of oxidative carbonylation in protein quality control
and senescence." EMBO J 24(7): 1311-1317.
Ono, K. and J. Han (2000). "The p38 signal transduction pathway Activation
and function." Cellular Signalling 12(1): 1-13.
Padmini, E., et al. (2012). "Effect of HSP70 and 90 in Modulation of JNK, ERK
Expression in Preeclamptic Placental Endothelial Cell." Cell Biochemistry and
Biophysics 64(3): 187-195.
Poehlmann, T. G., et al. (2006). "Trophoblast invasion: tuning through LIF,
signalling via Stat3." Placenta 26.
Priyanka, H. P., et al. (2013). "Menstrual cycle and reproductive aging alters
immune reactivity, NGF expression, antioxidant enzyme activities, and
intracellular signaling pathways in the peripheral blood mononuclear cells of
healthy women." Brain, Behavior, and Immunity 32(0): 131-143.
Bibliography
62
Redline, R. W. (2008). "Placental Pathology: A Systematic Approach with
Clinical Correlations." Placenta 29: 86-91.
Redman, C. W. G. and I. L. Sargent (2009). "Placental Stress and Pre-eclampsia:
A Revised View." Placenta 30: 38-42.
Reynolds, A., et al. (2007). Oxidative Stress and the Pathogenesis of
Neurodegenerative Disorders. International Review of Neurobiology. M. T. C.
Giacinto Bagetta and A. L. Stuart, Academic Press. Volume 82: 297-325.
Roberts, J. and C. Escudero (2012). "The placenta in preeclampsia." Pregnancy
hypertension: An International Journal of Women´s Cardiovascular Health 2:
72-83.
Scifres, C. M. and D. M. Nelson (2009). "Intrauterine growth restriction, human
placental development and trophoblast cell death." The Journal of Physiology
587(14): 3453-3458.
Serdar, Z., et al. (2002). "Placental and decidual lipid peroxidation and
antioxidant defenses in preeclampsia: Lipid peroxidation in preeclampsia."
Pathophysiology 9(1): 21-25.
Shindo, T., et al. (2000). "ADAMTS-1: a metalloproteinase-disintegrin essential
for normal growth, fertility, and organ morphology and function." The Journal
of Clinical Investigation 105(10): 1345-1352.
Sibai, B., et al. (2005). "Pre-eclampsia." The Lancet 365(9461): 785-799.
Son, Y., et al. (2011). "Mitogen-Activated Protein Kinases and Reactive Oxygen
Species: How can ROS activate MAPK pathways." Journal of Signal
Transduction.
Sosa, V., et al. (2013). "Oxidative stress and cancer: An overview." Ageing
Research Reviews 12(1): 376-390.
Steegers, E. A. P., et al. (2010). "Pre-eclampsia." The Lancet 376(9741): 631-
644.
Bibliography
63
Streuli, C. (1999). "Extracellular matrix remodelling and cellular
differentiation." Current Opinion in Cell Biology 11(5): 634-640.
Suzuki, Y. J., et al. (2010). "Protein Carbonylation." Antioxidants and Redox
Signalling 13(3).
van Katwijk, C. and L. Peeters (1998). "Clinical aspects of pregnancy after the
age of 35 years: a review of the literature." Human Reproduction Update 4(2):
185-194.
Velayutham, M., et al. (2011). "Removal of H2O2 and generation of superoxide
radical: Role of cytochrome c and NADH." Free Radical Biology and Medicine
51(1): 160-170.
Wang, F., et al. (2007). "SIRT2 deacetylates FOXO3a in response to oxidative
stress and caloric restriction." Aging Cell 6(4): 505-514.
Wang, G., et al. (2005). "Antioxidative function of L-FABP in L-FABP stably
transfected Chang liver cells." Hepatology 42(4): 871-879.
Webster, R. P., et al. (2006). "Nitration of p38 MAPK in the placenta:
association of nitration with reduced catalytic activity of p38 MAPK in pre-
eclampsia." Molecular Human Reproduction 12(11): 677-685.
Webster, R. P., et al. (2008). "Protein Nitration in Placenta – Functional
Significance." Placenta 29(12): 985-994.
White, V., et al. (2010). "Increased nitration and diminished activity of
copper/zinc superoxide dismutase in placentas from diabetic rats." Free Radical
Research 44(12): 1407-1415.
Wiktor, H., et al. (2004). "Oxidative DNA damage in placentas from normal and
pre-eclamptic pregnancies." Virchows Archiv 445(1): 74-78.
Wong, C. M., et al. (2008). "Protein Carbonylation as a Novel Mechanism in
Redox Signaling." Circulation Research 102(3): 310-318.
Bibliography
64
Wu, Y.-T., et al. (2007). "High bone morphogenetic protein-15 level in follicular
fluid is associated with high quality oocyte and subsequent embryonic
development." Human Reproduction 22(6): 1526-1531.
Yung, Y., et al. (2010). "ADAMTS-1: A new human ovulatory gene and a
cumulus marker for fertilization capacity." Molecular and Cellular
Endocrinology 328(1–2): 104-108.