Sandra Isabel Cunha Silveira

outubro de 2013

Redox changes in aged placental bed: consequences for cell signalling

UM

inho

|201

3Sa

ndra

Isab

el C

unha

Silv

eira

Re

do

x ch

an

ge

s in

ag

ed

pla

cen

tal b

ed

: co

nse

qu

en

ces

for

cell

sig

na

llin

g

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.