Altered Purinergic Signalling in Endothelial Cells Represents a Novel
Mechanism of Pulmonary Vascular Disease
University of Helsinki
Department of Biosciences
Physiology and Neuroscience
Tiedekunta – Fakultet – Faculty
Laitos – Institution– Department
Faculty of Biological and Environmental Sciences
Department of Biosciences
Tekijä – Författare – Author
Anne Johanna Komulainen
Työn nimi – Arbetets titel – Title
Altered Purinergic Signalling in Endothelial Cells Represents a Novel Mechanism of Pulmonary Vascular Disease
Oppiaine – Läroämne – Subject
Työn laji – Arbetets art – Level
Tiivistelmä – Referat – Abstract
Aika – Datum – Month and year
Sivumäärä – Sidoantal – Number of pages
Pulmonary arterial hypertension (PAH) is a progressive and devastating disease with poorly
understood pathogenesis. It is characterized by abnormal remodelling of pulmonary vasculature
due to uncontrolled apoptosis and proliferation of endothelial (ECs) and smooth muscle cells
(SMCs) in vascular wall. In severe PAH pulmonary ECs exhibit hyperproliferative and apoptosis
resistant phenotype contributing to the formation of neointima and development of plexiformic
lesions. Structural changes promote occlusion of vascular lumen, and thus, increase in
pulmonary vascular resistance. To date we lack efficient therapy to prevent vascular
remodelling and restore normal vascular function in PAH.
Purinergic signalling is potential modulator of pulmonary vascular homeostasis. It comprises of
extracellular nucleotides, such as ATP, which signal through their receptors on cell membrane.
Ectoenzymes with nucleotide hydrolyzing activity have an essential part in controlling
homeostasis and physiologic concentration of extracellular nucleotides. Ectoenzyme CD39
plays a crucial role in dephosphorylating ATP, which is a known mediator of inflammation,
angiogenesis, thrombosis and vasoconstriction according to previous research.
Aims of this project were to study the role of extracellular ATP in pulmonary endothelial
dysfunction during PAH pathogenesis. The goal was to evaluate the significance of ATPases,
such as CD39, in the disease process and to identify significant ATP receptors on pulmonary
ECs. We utilized a previously unused strategy to monitor ATPase activity in vivo in pulmonary
endothelium of rats with PAH. With this strategy we could identify changes in a time-line
manner. Our results indicate that ATPase activity is significantly attenuated in ECs during
disease process. Similar finding was also observed in human pulmonary EC isolated from PAH
patients suggesting that loss of ATPase activity mediated increase of extracellular ATP could
play a role in disease pathogenesis. Our in vitro experiments reveal that loss-of CD39 in human
pulmonary ECs leads to an apoptosis resistant and hyperproliferative phenotype. We also
identify that purinergic receptor P2Y11 is a critical mediator of ATP responses in these ECs.
Suppression of ATP mediated P2Y11 response in apoptosis resistant PAH patient ECs restores
normal EC phenotype and thus, suggests a novel therapeutic strategy for pulmonary occlusive
Avainsanat – Nyckelord – Keywords
IPAH - vascular remodelling - endothelial cells - purinergic signalling - ATP- CD39 - P2Y11
Ohjaaja tai ohjaajat – Handledare – Supervisor or supervisors
Dr. Tero-Pekka Alastalo
Säilytyspaikka – Förvaringställe – Where deposited
Department of Physiology and Neuroscience
Muita tietoja – Övriga uppgifter – Additional information
Tiedekunta – Fakultet – Faculty
Laitos – Institution– Department
Bio- ja ympäristötieteellinen tiedekunta
Tekijä – Författare – Author
Anne Johanna Komulainen
Työn nimi – Arbetets titel – Title
Altered Purinergic Signalling in Endothelial Cells Represents a Novel Mechanism of Pulmonary Vascular Disease
Oppiaine – Läroämne – Subject
Työn laji – Arbetets art – Level
Tiivistelmä – Referat – Abstract
Aika – Datum – Month and year
Sivumäärä – Sidoantal – Number of pages
Keuhkovaltimoiden verenpainetauti on kohtalokas ja etenevä keuhkoverisuonten sairaus, jonka
syntymekanismeja ja kehitykseen vaikuttavia tekijöitä tunnetaan puutteellisesti. Taudinkuvalle on
ominaista keuhkoverisuonten seinämän endoteeli- ja sileälihassolujen häiriintynyt apoptoosi- ja
proliferaatioherkkyys, joka johtaa suonenseinämän poikkeavaan paksuuntumiseen ja toiminnan
häiriöön. Pitkälle edenneessä taudissa erityisesti pienet valtimot ovat alttiita tukkeumille, koska
endoteelisolut ovat muuttuneet apoptoosille vastustuskykyisiksi ja hyperproliferatiivisiksi
muodostaen komplekseja pleksiformisia leesioita suonenseinämään. Muutokset valtimon
seinämärakenteessa johtavat verisuonen ontelon kaventumiseen ja keuhkoverenpaineen
kohoamiseen. Keuhkovaltimoiden verenpainetaudille ei ole tehokasta lääkitystä, joka pysäyttäisi
rakenteellisten muutosten kehittymisen verisuonissa.
Puriinisignalointi säätelee verisuonten toimintaa. Yksinkertaistettuna se tarkoittaa endoteelisoluista
vapautettuja nukleotideja, kuten ATP:ta, jotka toimivat solun ulkopuolisina signaalimolekyyleinä
välittäen soluvasteen solukalvon puriinireseptorien kautta. Solunulkopuoliset ektoentsyymit
säätelevät nukleotidien konsentraatiota ja verisuonten homeostasiaa pilkkomalla nukleotideja.
Tärkeä merkitys on ATP:ta pilkkovalla ektoentsyymi CD39:llä, sillä solun ulkopuolisen ATP:n
tiedetään aiheuttavan suonistossa mm. tulehdusta, angiogeneesia, tromboosien muodostumista
Projektimme tarkoituksena oli tutkia CD39-aktiivisuuden ja keuhkovaltimoiden verenpainetaudin
yhteyttä toisiinsa. Lisäksi halusimme tutkia kohonneen solunulkopuolisen ATP-pitoisuuden
vaikutusta keuhkoendoteelisolujen fenotyyppiin. Halusimme myös määrittää reseptorin, joka välittää
ATP-signaalin soluun. Tutkimme endoteelisolujen CD39-aktiivisuuden muutoksia aikajanalla
uudella in vivo-menetelmällä rottamallissa, jossa keuhkoverenpainetauti oli aiheutettu
monokrotaliini-injektiolla. Tutkimme myös tämän entsyymin aktiivisuutta ja ekspressiota
terminaalivaiheen potilaiden keuhkoendoteeleissa. Sen lisäksi mallinsimme CD39-vaimennettujen
solujen toimintaa siRNA-menetelmällä ja elatusliuokseen lisätyn ATP:n avulla, koska halusimme
selvittää alentuneen CD39-aktiivisuuden vaikutusta endoteelisolujen apoptoosiin ja
proliferaatioherkkyyteen. Lopuksi tutkimme keuhkoendoteelisolujen P2Y11-reseptorin osallisuutta
ATP-signaloinnissa siRNA-menetelmää hyödyntäen.
Havaitsimme CD39-aktiivisuuden alenemisen ja siitä johtuvan solunulkopuolisen ATP-pitoisuuden
lisääntymisen olevan yhteydessä keuhkovaltimoiden verenpainetaudin patofysiologiaan. ATP lisää
keuhkoendoteelien apoptoosiresistenssiä ja proliferaatioherkkyyttä, mikä on myös tyypillistä
endoteelisoluille pitkälle kehittyneessä keuhkovaltimoiden verenpainetaudissa. ATP-vaste välittyy
endoteelisoluihin P2Y11-reseptorin kautta. Tutkimuksessani esitetty purinergisen signaloinnin
epätasapaino antaa mahdollisuuden jatkotutkimuksille jotka tähtäävät purinergiseen signalointiin
kohdennettujen PAH-terapioiden kehittämisen.
Avainsanat – Nyckelord – Keywords
Keuhkovaltimoverenpainetauti - verisuonten rakenteen muuttuminen - endoteelisolut - puriinisignalointi - ATP- CD39 - P2Y11
Ohjaaja tai ohjaajat – Handledare – Supervisor or supervisors
Dr. Tero-Pekka Alastalo
Säilytyspaikka – Förvaringställe – Where deposited
Fysiologian ja neurotieteen osasto
Muita tietoja – Övriga uppgifter – Additional information
Table of contents
Abbreviations .................................................................................................................... 5
1. Introduction ................................................................................................................... 6
1.1 Pulmonary arterial hypertension ............................................................................. 6
1.2 Purinergic signalling ............................................................................................. 11
1.3 Ectoenzymes ......................................................................................................... 13
1.4 Purinergic reseptors .............................................................................................. 14
1.5 Study objectives .................................................................................................... 17
2. Material and methods .................................................................................................. 18
2.1 Isolation of rat pulmonary vascular endothelial cells ........................................... 18
2.2 Enzyme activity assay ........................................................................................... 19
2.3 Enzyme histochemistry ......................................................................................... 21
2.5 Basal ATP excretion and hydrolysis measurement .............................................. 23
2.6 mRNA expression ................................................................................................. 24
2.7 Western immunoblotting ...................................................................................... 25
2.8 Immunohistochemistry ......................................................................................... 26
2.9 Gene transfection .................................................................................................. 26
2.10 Apoptosis experiment ......................................................................................... 27
2.11 Proliferation assay ............................................................................................... 28
2.12 Statistical analysis ............................................................................................... 28
3. Results ......................................................................................................................... 29
3.1 Suppression of CD39 activity in pulmonary endothelium in a rat PAH model ... 29
3.2 ATPase activity is attenuated in ECs isolated from end-stage IPAH patients ...... 31
3.3 Down-regulation of CD39 leads to a phenotypic change in pulmonary ECs ....... 34
3.4 P2Y11 mediates cell survival signalling in ECs ................................................... 36
4. Discussion ................................................................................................................... 39
Acknowledgements ......................................................................................................... 45
References ....................................................................................................................... 46
Alk-1 Activin-like kinase type 1
ADP Adenosine diphosphate
AMP Adenosine monophosphate
ATP Adenosine 5’-triphosphate
BMPR2 Bone morphogenetic receptor 2
BSA Albumin from bovine serum
BSS Balanced salt solution
cAMP Cyclic adenosine monophosphate
cDNA comlementary DNA
DMEM Dulbecco´s modified eagle medium
E-NPP Ectonucleotide pyrophosphatase/phosphoriesterases
EBM2 Endothelial basal cell medium 2
EC Endothelial cell
ER Endoplasmic reticulum
ERK1/2 Extracellular-signal regulated kinases
HCL Hydrochloric acid
hMVEC Human microvascular endothelial cell
hUVEC Human umbilical vein endothelial cell
IPAH Idiopathic pulmonary arterial hypertension
LDS Lithium dodecyl sulfate
MAPK Mitogen activated protein kinase
MCL-1 Myeloid leukemia cell differentiation protein-1
mRNA messenger RNA
NO Nitric oxide
NOGO B Neurite outgrowth inhibitorB
NTPDase Nucleoside triphosphate diphosphorylase
PBS Phosphate buffered saline
PKA Protein kinase A
PMSF Phenylmethane sulfonylfluoride
qPCR Quantitative real time polymerase chain reaction
SDS Sodium dodecyl sulfate
SMC Smooth muscle cell
STAT3 Signal transducer and activator of transcription 3
TBS Tris buffered saline
TGFβ Transforming growth factor β
TLC-plate Thin-layer chromatography plate
VEGFR2 Vascular endothelial growth factor receptor 2
1.1 Pulmonary arterial hypertension
Pulmonary arterial hypertension (PAH) is a fatal disease of the pulmonary vasculature
and defined by mean pulmonary arterial pressure greater than 25 mm Hg at rest.
Chronic elevation of pulmonary arterial pressure and resistance result in restricted blood
flow in pulmonary circulation, increasing right ventricle work load. Eventually this may
lead to heart failure and premature death (Hoeper, 2009; ESC, ERS, ISHLT, Galie et al,
2009). World health organisation (WHO) classifies PAH with different subgroups based
on the aetiology of disease; idiopathic PAH (IPAH), heritable PAH, drug- and toxin
induced PAH, connective tissue disease-, HIV infection-, congenital heart disease-,
schistosomiasis-, chronic haemolytic anaemia- and portal hypertension associated PAH,
and persistent pulmonary hypertension of newborns (Simonneau et al, 2009). As PAH
is associated with number of different conditions it is estimated that over 100 million
people world-wide suffers from elevated pulmonary resistance. Unfortunately for many
patients it is a progressive disease with no effective cure, when despite the advanced
medical therapies, three-year survival from diagnosis is still less than 60% in severe
types of PAH (Humbert et al, 2010). IPAH is considered as multifactorial disease with
unknown specific origin, and it represents often aggressively progressing form of PAH.
According to The Registry to Evaluate Early and Long-term pulmonary arterial
hypertension disease management (REVEAL Registry), IPAH diagnosis is most
commonly given to patients at the age of 45-54, and females are more susceptible for
this disease as the female to male ratio is approximately 4:1 (Badesch et al, 2010).
Pathophysiology of IPAH is poorly understood, but similarly to other PAH subtypes, it
is characterized with critical morphological changes in pulmonary vasculature due to
imbalance between cell proliferation and apoptosis (ESC, ERS, ISHLT, Galie et al,
2009). Healthy arteries comprise of inner monolayer of endothelial cells (ECs) in intima,
muscular media of smooth muscle cells (SMCs) and external tunica adventitia of
connective tissue (Figure 1). Prominent feature in early phase of IPAH is media
thickening and muscularization of small peripheral arteries, which normally lack the
media, due to SMC proliferation and migration (Heath and Edwards, 1958). Other
associated features are increased EC injury and apoptosis, which leads to loss of precapillary
arteries and thus leads to critical changes in pulmonary hemodynamics (Jurasz
et al, 2010). During the course of PAH, endothelial cells with hyper-proliferative and
apoptosis-resistant phenotype appear and contribute to intima hyperplasia and occlusion
of arteries (Masri et al, 2007; Kawano, 1994; Magee et al, 1988). Hallmarks for endstage
severe PAH is plexogenic arteriopathy characterized by tumor-like cell mass with
capillary-like channels (Figure 2). Pathobiology of these lesions is still a matter of
debate and the origin of involved cells is also obscure (Yi et al, 2000; Abe et al, 2010;
Toshner et al, 2009).
Figure 1. Vascular layers of healthy muscular artery. Hypertrophy of intima and media
layers are hallmarks of IPAH. (240113, http://bme.ccny.cuny.edu)
Figure 2. Simplified model of vascular pathophysiology of PAH. After initiation,
PAH manifests on a timeline involving early vasoconstriction (1), medial hyperplasia
caused by smooth muscle cell (SMC) proliferation (2), intimal hyperplasia due to
endothelial cell (EC) proliferation and abnormal apoptosis sensitivity (3) leading to total
occlusion and formation of plexogenic lesions (4). Hallmark of severe PAH is a poorly
understood phenotypic switch of ECs.
Endothelial dysfunction is considered as a critical step in PAH development (Friedman
et al, 2012; Wolff et al, 2007). Endothelium is responsible for vascular homeostasis by
regulation of vascular tone, inflammatory responses, permeability, and anti-thrombotic
action (Furchgott and Zawadzki, 1980; Drexler, 1998; Vane et al, 1990). Characteristic
for activated, dysfunctional endothelium is loss of its vascular modulating functions,
such as lost of endothelium-dependent vasodilation due to imbalanced nitric oxide (NO),
endothelin-1 and prostacyclin production. Prevailing vasoconstriction elevates blood
pressure in pulmonary circulation resulting in increased shear stress, which further
injures endothelium and promotes vascular pathology. In addition, dysfunctional
endothelium is pro-inflammatory as there is infiltration of macrophages and
lymphocytes into the vascular wall. Inflammation is also tightly associated with
pathophysiology of PAH (Budhiraja and Hassoun, 2004; Lockette et al, 1986; Feletou
and Vanhoutte, 2006). Dysfunctional and apoptotic endothelium promotes vascular
SMC proliferation due to release of growth factors and suppressed NO secretion, which
is known to have a suppressive effect on proliferation (Yang et al, 2011; Scott-Burden
and Vanhoutte, 1994). Thus, dysfunctional and apoptotic ECs contribute to media
hypertrophy and initial stage of PAH.
During the course of PAH pathogenesis there is a switch in EC phenotype. Appearance
of apoptosis resistant hyperproliferative ECs, especially in areas of intimal hyperplasia
is characteristic for the human disease. Several studies have shown that these aberrant
cells are monoclonal in origin (Sakao et al, 2005). It is likely that the aberrant ECs
contribute to the occlusive vasculopathy in PAH. One potential mediator in this process
is signal transducer and activator of transcription 3 (STAT3), which promotes apoptosis
resistant and hyperproliferative phenotype, and abnormal and persist activation of
STAT3 has been observed in diseased ECs. (Masri et al, 2007). Furthermore, ECs from
plexiform lesions over-express vascular endothelial growth factor receptor 2 (VEGFR2),
which is associated with regulation of EC proliferation and apoptosis and, thus, could
explain certain features of plexogenic arteriopathy (Tuder et al, 2001).
Characteristic for aberrant ECs of severe IPAH is altered energy metabolism and other
mitochondrial modifications. Similarly to cancer cells, these abnormal ECs have
suppressed mitochondrial glucose oxidation and increased cytoplasmic glycolysis
instead of more efficient cellular respiration (Xu et al, 2007). Aerobic glycolysis
associates with uncontrolled neoplastic proliferation, as aerobic glycolysis induces upregulated
DNA synthesis (Wang et al, 1976). The primary cause of metabolic switch in
IPAH and cancer are unknown. However, NO is known modulator of mitochondrial
function and expression, and generally suppressed NO production of IPAH patients may
mediate bioenergetics alteration in EC metabolism and proliferation tendency (Xu et al,
2007). Among the altered energy metabolism, defective mitochondrial function in IPAH
might be associated with abnormal mitochondrial apoptosis. In vascular SMCs,
endoplasmic reticulum (ER) stress often triggered by known PAH risk factors,
suppresses mitochondria induced apoptosis. Neurite outgrowth inhibitor B (Nogo B)
activation impairs connection between ER and mitochondria, which is needed for
mitochondrial dependent apoptosis. Nogo B is expressed in pulmonary vascular SMCs
and ECs of IPAH patients, and thus, Nogo B activation may be associated with
vasculature remodelling due to abnormal apoptosis sensitivity (Sutendra et al, 2011).
Somatic mutations and genomic instability, which are also characteristic for
development of cancer, have been identified in abnormal ECs from IPAH patients.
Somatic abnormalities in genes regulating cell survival may be related with aberrant
phenotype of ECs of IPAH patients. For instance, microsatellite instability of DNA
epair associated mutS homolog 2 (MSH2) gene, microsatellite site mutations in
transforming growth factor beta receptor 2 (TGFBR2), and in proapoptotic BAX genes
have been identified in pulmonary ECs of IPAH patients (Yeager et al, 2001). In
addition, severe somatic chromosomal mutations, such as total loss of active X
chromosome in female patients, have been shown in pulmonary ECs isolated from
IPAH patients (Aldred et al, 2010). The role of genomic abnormality in IPAH
pathogenesis is poorly understood and the mechanisms behind these changes in ECs are
unknown. It is also unclear when these genetic instabilities develop and whether they
are associated with impaired signalling pathways of IPAH, such as bone morphogenetic
receptor 2 (BMPR2) pathways.
Mutations in BMPR2 have been found in patients with heritable or idiopathic forms of
PAH. Interestingly, suppressed expression of BMPR2 in pulmonary vasculature has
been detected in other forms of PAH as well as in animal models of PAH. Extensive
research during past years has revealed that BMPR2 plays a critical role in pulmonary
endothelial homeostasis (Yang et al, 2011; Atkinson et al, 2002; Teichert-Kuliszewska
et al, 2006). Dysfunctional BMPR2-signalling due to mutations or suppressed
expression, leads to decreased survival and attenuated regeneration capacity of
pulmonary ECs (Teichert-Kuliszewska et al, 2006; de Jesus Perez et al, 2009).
Interestingly, BMPR2-signalling has an opposite function in SMCs where dysfunction
leads to increased proliferation and apoptosis resistance (Zhang et al, 2003). BMPR2 is
a member of TGFβ family, which seems to be crucial for normal vascular development
and function (Dijke and Hill, 2004). Interestingly, several other member of this family
are linked to the pathogenesis of PAH, as disease causing mutations have also been
found in activin like kinase 1 (ALK1) (Harrison et al, 2003), endoglin (ENG) (Chaouat
et al, 2004), activin like kinase 5 (ALK5) (Thomas et al, 2009) and SMAD9 (SMAD9)
(Shintani et al, 2009). However, despite the significance of abnormalities in BMPR2,
80% of people carrying BMPR2 mutation do not develop PAH suggesting low
penetrance of disease phenotype (Hamid et al, 2009). BMPR2 mutation frequencies of
60-80% in heritable PAH and 10-20% in IPAH suggest that other factors are involved
in the pathogenesis of these PAH types (Rabinovitch, 2008).
There is no cure for PAH. Current medications including calcium channel antagonists,
endothelin inhibitors, phosphodiesterase inhibitors and prostanoids are potent
vasodilators of pulmonary vasculature but have no effects on vascular remodelling.
These medications mainly relieve symptoms but have poor affects on mortality
(Badesch et al, 2007). As the disease progresses lung transplantation is eventually the
only rescue for many patients. New therapeutic approaches with capabilities to suppress
vascular remodelling and restore normal vascular function and structure are warranted.
1.2 Purinergic signalling
Adenosine 5’-triphosphate (ATP) is an evolutionary conserved molecule. It is found in
all living organisms and generally known as a fundamental intracellular energy
molecule. Because of its ancient origin, it has also a unique role as a cellular signalling
molecule. ATP is released in stressed condition and extracellular ATP activates cell
signalling pathways (Burnstock and Verkhratsky, 2009). As ATP has an ancient history
as a signalling molecule, it is not surprising that it is a potent mediator of essential
cellular actions in many cell types. The concentration of extracellular ATP is
approximately 10nM while intracellular concentration is 1000 000 fold higher (10mM).
Thus, extracellular release of ATP does not compromise intracellular energy
metabolism as the amount of released ATP is very low and still can initiate maximum
responses in cell surface purinergic receptors. There is no consensus on the mechanism
how ATP is released, although, multiple theories have been suggested such as ATP
releasing channels, ATP transporters and vesicle-mediated release. Probably ATP
secretion is specific for cell type and there are multiple overlapping systems
(Schwiebert and Zsembery, 2003). To date purinergic signalling is known to comprise
extracellular nucleotides modulating tissue functions including development, blood flow,
secretion, inflammation and immune responses through purinergic receptors on cell
surfaces. Roles of ATP and its breakdown product, adenosine are the most prominent
extracellular signalling molecules in purinergic system. ATP is released from various
different cell types such as neurons, lymphocytes, platelets and endothelial cells and
functions as a paracrine- or autocrine signalling molecule (Burnstock, 1972), (Chen et
al, 2006; Kirby et al, 2012; Burnstock, 1989).
In the vasculature extracellular ATP and other nucleotides regulate vascular tone and
remodelling. Both ATP and adenosine, trigger vasoactive substrate secretion from ECs,
which can modify SMC contractility and functions (Burnstock, 2009). For example,
ATP secretion from ECs is up-regulated during hypoxia and thus, could play a role in
hypoxia-mediated vascular responses and remodelling (Bodin et al, 1992). ATP induces
vascular SMC proliferation through the mobilization of cytosolic calcium (Zhang et al,
2004; Lee et al, ; Erlinge, 1998). Calcium is essential for proliferation response as it
switches on mitotic cell cycle (Kahl and Means, 2003). ATP is also involved in
inflammation as it promotes leukocyte recruitment, which is associated with
degradation of elastic lamina and induced SMC proliferation (Bours et al, 2011).
Opposite to ATP, adenosine has usually balancing effect on vascular function, and it has
been shown to induce EC mediated vasodilatation (Duza and Sarelius, 2003).
Adenosine receptor activation limits inflammation and tissue injury, and is thus known
as an anti-inflammatory compound (Ohta and Sitkovsky, 2001). As noted, inflammation
is associated with cell proliferation, so adenosine also prevents hyperproliferation
(Bours et al, 2011). According to Jackson and co-workers, adenosine can suppress
aortic and coronary SMC proliferation (Jackson et al, 2011).
Less is known about ATP-mediated remodelling in ECs, but still there is some evidence
of the association of ATP with altered endothelial phenotype. For example, in vasa
vasorum ECs, extracellular ATP induces proliferation and angiogenesis. However, in
the same study, Gerasimoskaya and co-workers did not see the same response in
pulmonary or aortic ECs (Gerasimovskaya et al, 2008). ATP-dependent cardiac EC
proliferation has also been reported (Rumjahn et al, 2009). Further support for ATP
mediated EC remodelling came from recent research on ATP-mediated suppressed
apoptosis sensitivity of human umbilical vein ECs (hUVECs) in ischemic condition
(Urban et al, 2012).
Despite of the well known role of ATP in vascular modulation, its association with
pathophysiology of PAH is undefined. ATP mediates pulmonary vascular SMCs
proliferation (Zhang et al, 2004), but the role of ATP in remodelling of pulmonary ECs
is poorly understood.
Ectoenzymes are essential part of purinergic signalling, because they control
extracellular nucleotide concentrations and their optimal balance in vascular
homeostasis. As we discussed above, excess ATP may be harmful for vascular
phenotype whereas adenosine promotes vascular homeostasis. Under normal
physiological condition, released ATP is rapidly hydrolysed to ADP and further AMP
and adenosine by membrane-bound ectoenzymes.
There are four major families of extracellular nucleotide-hydrolyzing enzymes:
nucleoside triphosphate diphosphorylases (NTPDases) with ATP and ADP hydrolyzing
activity, ectonucleotide pyrophosphatase/phosphoriesterases (E-NPPs,) hydrolyzing also
ATP and ADP, alkaline phosphatases with adenosine nucleotide hydrolyzing activity
and ecto-5’-nucleotidases (CD73), which plays a significant role in hydrolyzing AMP to
adenosine (Schetinger et al, 2007). NTPDase family consist of eight members of
nucleoside tri- and diphosphate hydrolyzing enzymes with separate localization and
other features. Four of them are expressed on the external cell membrane, two of them
are soluble and can be secreted to extracellular space and the rest two are intracellulary
located. NTPDases need millimolar concentration of Ca 2+ or Mg 2+ for their hydrolyzing
activity and moreover, they are not inhibited by most prevalent inhibitors of ATPases.
Especially NTPDase1 is not inhibited by natural ATPase inhibitors (Schetinger et al,
2007; Pearson et al, 1980).
NTPDase1, also named as CD39, is highly expressed on endothelial cells and plays a
role in modulating extracellular ATP concentration in the vasculature. For instance,
study of Goepfert co-workers shows that up regulated CD39 activity suppressed
extracellular ATP concentration on hUVECs. CD39 dephosphorylates ATP to ADP and
further AMP releasing hardly any free ADP during the reaction cascade (Marcus et al,
2003; Imai et al, 1999; Goepfert et al, 2000). It is an integral membrane protein with
two transmembrane domains and a large extracellular region (Wang et al, 1998). In
addition to endothelial cells, CD39 is also expressed in different lymphocytes and
SMCs. It has been suggested to play a role in controlling inflammation and immune
response, blood fluidity and cell proliferation (Dwyer et al, 2007; Kauffenstein et al,
2010). Suppressed CD39 activity induces EC activation and thus, their pro-
inflammatory reactions (Goepfert et al, 2000). Moreover, endothelial CD39 regulates
blood fluidity and thrombosis due to its ADP hydrolysing activity. ADP is a potent
prothrombotic factor inducing platelet aggregation (Robson et al, 2005b). CD39 is the
major modulator of extracellular nucleotide concentration on vascular SMCs
(Kauffenstein et al, 2010). Overexpression of CD39 in injured rat aortas has been
reported to suppress vascular SMCs proliferation (Koziak et al, 2008).
Down-regulation of CD39 activity is linked with many diseases. There are studies
showing association of suppressed vascular CD39 expression with ischemic injury
(Koehler, 2007) and transition of coronary atherosclerotic plaque from stable to
unstable (Hatakeyama et al, 2005). The role of CD39 in pulmonary vascular
remodelling during PAH pathogenesis has not been elucidated.
1.4 Purinergic reseptors
Effects of extracellular nucleotides are mediated through purinergic receptors on cell
surface. There are two known main families of receptors, P1 and P2, and both of them
express several receptor subtypes. Positive action of adenosine is mediated via P1
receptors, whereas P2 receptors are sensitive for nucleotides such as ATP and ADP
(Kukulski et al, 2011).
Four different adenosine-reactive P1-receptors (A1, A2a, A2b and A3) have been
characterized to date, and they are G-protein coupled receptors with adenylate cyclase
modulating activity. (Fredholm et al, 2001). Especially A2a and A2b are expressed in
vasculature and mediate vasodilation and inflammatory control. P1-receptors are also
considered as protectors against ischemic injury (Fredholm et al, 2001; Collis and
Brown, 1983; Jordan et al, 1997; Lankford et al, 2006).
As mentioned before, P2-receptors are more diverse group with different cellular
actions. Activation of P2X-type receptor opens ion channels on cell membrane, while
P2Y-type receptors act as G-protein coupled receptors. Multiple receptor subtypes and
their complexes are expressed on cell membrane, but as usual, expression of different
eceptors is highly cell type specific, and neither their interactions nor effects on cellular
function are yet fully understood (Burnstock, 2007).
Seven subtypes of ligand-gated P2X-receptors have been defined, and they are
unspecific cation selective ion channels. Conformational changes and opening of ion
channels are regulated by ATP-binding on active site (Coddou et al, 2011). P2X4 is the
dominant P2X-receptor on vascular ECs with Ca 2+ influx increasing action. Elevation of
free cytosolic calcium concentration induce NO and prostacyclin production inducing
relaxation of vascular SMCs (Yamamoto et al, 2000; Wang et al, 2002). Among
vascular endothelium, P2X-receptors are also expressed on SMC where P2X1 is the
prevailing P2X-type receptor. P2X4 and P2X7 are expressed in SMCs in low levels
(Wang et al, 2002). Activation of P2X-receptors on SMCs, is associated with
vasoconstriction (Erlinge et al, 1998). In addition, P2X7 has been shown to induce a
large ion pore opening on cell membrane resulting cytotoxin secretion and
inflammatory condition. Activation of P2X7 may lead to apoptosis in some cell types
(Volonte et al, 2012).
Eight members of P2Y-receptors have been identified to date, and they are G-protein
coupled activating different signal transduction routes. Activation of some of these
receptors increases intracellular calcium concentration, which is known to induce cell
proliferation as well as NO production (Zheng et al, 1991). P2Y-receptors are also often
linked with mitogen activated protein kinases (MAPKs) activation, especially
extracellular-signal regulated kinases (ERK 1/2), which modulate cell survival related
functions (Burnstock, 2007; Zarubin and Han, 2005). P2Y2 and P2Y6 are the most
dominating P2Y-type receptors on vascular SMCs, and they may modulate proliferation.
In ECs, several subtypes of P2Y-receptors are expressed, and they induce vasodilation
due to increase in NO production. According to analysis of P2-receptor expression by
Wang and co-workers, P2Y11 is the dominating P2-receptor on hUVECs (Wang et al,
P2Y11 is a unique P2-receptor with special affinity to activate two separate cellular
pathways, adenylyl cyclase and phospholipase C. Phospholipase C activation promotes
intracellular calcium mobilization (White et al, 2003) contributing to NO production
and proliferation (Devader et al, 2007; Erlinge, 1998). A unique feature of P2Y11 is its
ability to activate adenylyl cyclase, which mediates cyclic AMP-dependent (cAMP)
signalling (Communi et al, 1997a). Activation of this pathway is not a feature of other
P2-receptors. cAMP is responsible for protein kinase A (PKA) activation, which
mediates several cellular functions depending on cell type. For instance, in neutrophils
PKA activation impairs apoptosis inducing expression of myeloid leukemia cell
differentiation protein-1 (MCL-1) in form, which prevents activation of apoptotic
pathway (Kato et al, 2006). P2Y11 receptor is characterized by considerably larger
second and third extracellular loops than the other described P2Y-receptors. Genomic
sequence of P2Y11 differs significantly from others, while P2Y11 exhibits only 33%
amino acid identity with the P2Y1 receptor, its closest homolog (Communi et al, 1997b).
This far only human, canine and Xenopus P2Y11 sequences have been defined
(Devader et al, 2007). According to Amisten and co-workers, mutation in P2Y11 risks
for acute myocardial infarction, suggesting its prominent role in cardiovascular system
(Amisten et al, 2007). Interestingly, a microarray analysis of BMPR2 down stream
targets shows that expression of P2Y11 receptor was significantly suppressed in
BMPR2-deficient pulmonary ECs (Alastalo et al, 2011). This unpublished observation
links for the first time PAH associated BMPR2 pathway and purinergic system.
In addition to the down-regulation of P2Y11 in BMPR2-deficient cells, there are some
studies showing association of purinergic receptors with PAH. Genome wide expression
analysis from pulmonary samples from IPAH patients shows up regulated expression of
purinergic P2Y1-receptors (Rajkumar et al, 2010). Moreover, adenosine A2A-receptor
deficient mice exhibit increased pulmonary artery media hypertrophy (Xu et al, 2011).
In short, the role of purinergic receptors in PAH is currently an unexplored area.
Figure 3. Overview of purinergig signalling system. ATP is released from cells, and
then extracellular nucleotides act as signalling molecules. ATP activates P2X- or P2Yreceptors,
or are further hydrolyzed by ectoenzymes located on cell membrane.
NTPDase1 (CD39) hydrolyze ATP and ADP to AMP, witch is hydrolyzed by e5’NT
(CD73) to adenosine. P1-receptor mediates adenosine effect on cell. (300113, unileipzig.de)
1.5 Study objectives
Pulmonary arterial hypertension is a devastating disease with relatively unknown origin
and inefficient therapy. The main goal of this study was to evaluate the role of
purinergic signalling in PAH pathogenesis and evaluate potential for new therapeutic
strategies. Based on our observation on down-regulated P2Y11 in BMPR2-deficient
ECs, we hypothesized that unbalanced ATP-mediated signalling could play a role in
PAH pathogenesis. First, as ectoenzyme CD39 is the dominant modulator of
extracellular ATP concentration, we were interested in studying the activity and
expression of CD39 in PAH. We utilized a novel in vivo assay to study CD39 activity
during PAH development in rats. Results were then tested in human IPAH patientderived
pulmonary ECs and lung tissue. Then mechanistical studies were initiated with
healthy human pulmonary ECs. With these strategies our goals was to identify the role
of extracellular ATP in pulmonary vascular remodelling and to determine the role of
P2Y11 in this process. Our main goal was to identify novel targets for PAH therapy that
would not only suppress disease process but could restore normal pulmonary vascular
function and structure.
2. Material and methods
2.1 Isolation of rat pulmonary vascular endothelial cells
Monocrotaline (MCT) model is widely used for modelling the pathophysiology of PAH.
It is derived from the seeds of Crotalaria spectabilis, and the effects of MCT on
pulmonary vasculature have been known since 1960s (Kay et al, 1967). Oxidation
products of MCT formed by the liver cause inflammatory reaction, vascular lesions
formation and hyperproliferation of SMCs in pulmonary arteries. Remodelling of
pulmonary vasculature is associated with increase in vascular resistance and formation
of obstructions in small arteries (Lame et al, 2000). It is the only animal model of PAH
with lethal end-point caused by severe pulmonary vascular disease and right ventricle
With our EC profiling in vivo-method, we measured the enzyme activity in MCT rat
model straight after cell isolation with magnetic beads avoiding the risk of phenotype
change related to cell culture. There is significant evidence that the phenotype of ECs
changes dramatically upon in vitro conditions (Durr et al, 2004). Animal procedures
were performed in compliance with Finnish National Animal Experiment Board.
Young Sprague-Dawley male rats were treated with subcutaneous 60mg/kg MCT
(Sigma, Crotaline) or saline injection. 5, 10, 15 or 19 days after the injection, rats were
sacrificed and whole lung samples were collected in RPMI medium (Gibco) containing
100 u/ml penicillin and 0,1 mg/ml streptomycin (Lonza). A sample of pulmonary tissue
was stored in -70 ºC for later enzyme histochemistry staining, and the rest of the tissue
were chopped and then incubated for 40 minutes at 37ºC in 7ml of digestion solution
(Hanks' balanced salt solution (Gibco), 0,5mg/ml Collagenase IV from Clostrium
histolyticum (Sigma) and 1μM Hepes Buffer (Lonza)), and afterwards collagenase was
inactivated with RPMI supplemented with 10% Fetal calf serum (Lonza). Suspension
was properly mixed and filtered through 70µm cell strainers (BD Falcon) and then
centrifuged and washed twice to separate the cell solution from tissue debris. Finally,
the formed cell pellet was solved in 1,5ml buffer-1 solution (100 μM EDTA (Ambion),
1 mg/ml BSA (Sigma)), and purified Mouse Anti-Rat CD31 antibody (BD Pharmingen)
coated magnetic beads (Dynabeads goat anti-mouse, Invitrogen) was added on cell
solution. Mixture was gently shaken for ten minutes to ensure effective adhesion of
antibodies on the endothelial cells. Eventually endothelial cells attached to magnetic
beads were washed and isolated from the solution with magnet, and then resuspended in
200 μl RPMI medium.
2.2 Enzyme activity assay
Because CD39 is known to be the most dominant ectoenzyme with ATP hydrolyzing
affinity on ECs (Kaczmarek et al, 1996; Robson et al, 2005a), we hypothesized that the
ATP hydrolyzing efficiency in samples is especially associated with the activity of
CD39. In short, we studied CD39 enzyme activity by incubating samples with
radiolabelled ATP, and then quantifying the amount of its breakdown products based on
To measure CD39 activity on ECs, samples were incubated for 30 min at 37ºC warm
bath with 450 μM ATP-substrate solution (Sigma) including tritium labelled [2,8- 3 ]
ATP (ATP-tetrasodiumsalt, spec.act.19,0 Ci/mmol, American Radiolabelled Chemicals,
Inc.). Substrate solution contained also 4 mM β-glycerophosphate to prevent undesired
hydrolysis of radiolabelled nucleotides by unspecific phosphatases. Suitable
concentration of ATP and incubation time was set so, that amount of radiolabelled
metabolites did not exceed 10-15% of initial substrate concentration.
After the incubation, 8μl aliquots of samples were applied on silica thin-layer
chromatography plates (TLC-plate, Alugram® Sil G/UV254), prior treated with
nucleotide standard solution (containing 0,5-1mM ATP, ADP, AMP, adenosine, inosine
and hypoxanthine, Sigma) for separating and visualizing the breakdown products of
ATP. Fractions of metabolites were separated with two hours incubation in glass
chamber containing 50ml running buffer (1-butanol, isoamyl alcohol, diethylene glycol
monoethylether, ammonia solution and milli-Q-aqua (9:6:18:9:15), Sigma).
Separation of metabolites was visualized with UV-light from dry TLC-plate and bands
containing metabolites of ATP were scratched from the TLC-plate into 4ml plastic vials
(PerkinElmer) including 0,5ml 0,2M HCL. After extracting nucleotides from silica with
hour incubation and vortexing tubes, 2,5ml scintillation mixture (Optiphase Hisafe 2,
PerkinElmer) was added into tubes to quantify radiation in the samples with microbeta²
Enzyme activity of CD39 in samples was calculated (Formula 1) from the counts (dpm,
disintegrations per minute) measured by microbeta² Lumijet, and the results were
reported as μmoles of radiolabelled ATP metabolized per hour by sample or 10 000
Enzyme activity = ( Smpl-Bl) × 60 × [ATP] × AV ×1000
Total × t × s
Smpl = counts from the sample (dpm)
Bl = counts from well without any cells (dpm)
[ATP] = ATP concentration (μmol/ml)
Total = total counts of used substrate solution (dpm)
t = incubation time (min)
s = mixture aliquot added to TLC-plate (μl)
AV = total reaction volume (μl)
10 μl of endothelial cell solution from MCT rat model was applied in 4 ml FACS tubes
(BD Falcon) for enzyme activity assay, whereas with cell cultures, assay was performed
with 10000 cells per well seeded on 96-well plate.
To confirm the comparable validity of results between different samples, protein
concentrations in them were measured. This was highly significant in analysis of rat
samples, while the amount of cells in experiment was impossible to determine. So the
results from enzyme activity assay are proportioned to the protein concentration in rat
samples. To quantify the protein concentration, cells were firstly lysated with
mammalian cell lysis solution (ATP Detection Assay System, PerkinElmer) and the
protein concentration was defined with commercial analysis kit (DC Protein Assay,
BioRad). Absorbance of cell solutions was measured at 750 nm (Original Multiskan EX,
Thermo Labsystems) from clear 96-well plates and finally, the protein concentration
(μg/ml) was calculated from BSA standard curve (Figure 4, Albumin from bovine
We applied approximately 70% of harvested rat ECs to protein lysate for concentration
measurement. Instead, concentration in cell culture samples were determined from cell
lysate from single well of 96-plate well plate, in which was seeded 10 000 cells day
before experiments. Protein concentration was mainly used as a loading control of
cultured cells, but as noted above, in the rat study, protein concentration played a
Absorbance 750 nm
R 2 = 0.9923
0.0 20.0 40.0 60.0 80.0 100.0 120.0
BSA concentration (µg/ml)
Figure 4. BSA-calibration curve. Protein concentration of standard BSA samples
associated with absorption level.
2.3 Enzyme histochemistry
To study ATPase activity in situ in MCT rat model, we sliced 8 μm sections from
frozen pulmonary tissue samples with cryotom (HM 500 O, Microm) and incubated
sections for three minutes in acetone (Sigma) to fix them on the microscope slides.
Before staining, slides were pre-treated first in Trizma-Maleate sucrose buffer (40mM
Trizma-Maleate, 8% sucrose, Sigma, pH 7,4) and then in TMSB including 5mM
levamisol (Sigma) and 3 μM oubain (Sigma) to prevent the action of alkaline
phosphatases and Na/K/ATP-ases. Slides were incubated 25 minutes at 37ºC with 100
μM ATP and 2mM lead nitrate (Pb(NO 3 ) 2 , Sigma), and then washed with MQ. Finally,
short incubation in 0,5% ammonium sulfide solution (Sigma) caused colour reaction.
Staining is based on precipitation of lead phosphate, which was formed because of
presence of inorganic phosphate released by ATPase activity. So by adding yellow
ammonium sulphide, the lead phosphate is converted to lead sulphide, which is the
brownish reaction product (Zaprianova et al, 1983). Before inspecting the results with
microscope (DM RXA, Leica), slides were mounted (Aquamount, Gurr). Pulmonary
arteries from sections were analyzed and photographed with DP70 (Olympus).
2.4 Cell culturing
All the human cells were cultured on 10cm cell culture dishes (BD Falcon) coated with
0,2% gelatin from bovine skin (Sigma) to ensure better attachment of cells, and
endothelial basal cell medium 2 (EBM2, Lonza) was applied as a culturing media.
Dishes were cultured in a fully humidified atmosphere at 37ºC and 5% CO 2 in CO 2
incubator (Sanyo) in the sterile cell culturing room, and cells were always treated in the
sterile fume hood with aseptic manners.
To seed cells for experiments or divide culture for further use, they were first washed
with PBS (from HUSLAB) and dissociated with short Tryple express-treatment (Gibco).
Trypsin was inactivated with RPMI medium (Lonza) containing 10% fetal calf serum.
Centrifuged cell pellet was resuspended in 1ml culturing media, before division for new
cultures or cell counting. Tryptan blue stained (Sigma) cells were counted under light
microscope using hemazytometer, and then needed amount of cells were seeded on 24-
or 96-well plates for the next day experiments. Cell passages 4-10 were applied in our
Commercial human microvascular endothelial cells from lungs (hMVECs, Lonza) were
used as healthy control cells. Dr Vinicio de Jesus Perez from Stanford University kindly
sent us pulmonary vascular ECs from end-stage patients suffering idiopathic pulmonary
hypertension. Cells were isolated with magnetic beads and then further cultured.
2.5 Basal ATP excretion and hydrolysis measurement
On 0,2% gelatine (Sigma) coated 24-plate were seeded 50 000 cells per well in 1ml of
EBM2, and on the next day cell were washed with balanced salt solution (BSS)
followed by incubation with 2μM ATP (Sigma) in BSS. After an hour incubation ATP
concentration was measured and set against the ATP concentration of the well without
any cells. Result of the experiment was expressed as percentage of hydrolysed ATP.
ATP concentration was measured with commercial ATP Detection Assay System
(PerkinElmer). Aliquot of medium from each sample was added on the well of white
walled 96-well plate, and two parallel measurements were done. Luminescence was
measured with Victor² 1420 multilabelcounter (Wallac) after increasing luciferase into
wells, which products detectable light signal in presence of ATP trough the enzymatic
reaction. ATP content of samples was analyzed with ATP calibration curve generated
from 2μM ATP aliquots (Figure 5).
R 2 = 0.9992
0 100 200 300
Figure 5. ATP concentration was clearly proportional to emitted light intensity.
2.6 mRNA expression
Messenger RNA expression was measured with real time polymerase chain reaction
based on amplification and simultaneous quantification of target mRNA determined
with wanted primers. Specific heat resistance polymerase enzyme doubles the amount
of target mRNA in every thermal cycle, and the increase is detected as more intense
fluorescence signal due to the fluorescent of dyeing substrate bound to the synthesized
First, RNA was isolated from confluent cell culture with commercial Nucleo Spin RNA
II kit (Machery-Nagel) according the procedure of manufacturer. Before measuring
1000ng of RNA for synthesis of complementary DNA, RNA concentration of samples
was measured with Spectrophotometer ND-1000 (Nanodrop). SuperScript® VILO
cDNA Synthesis Kit (Invitrogen) containing reverse transcriptase, DNA-polymerase
and random primers was applied according to instruction of manufacturer, and cDNA
synthesis was performed with recommended programming of MJ Research PTC-200
Peltimet Thermal Cycler (Biorad) to generate optimal thermal conditions for enzymes
synthesising reverse transcript.
Mesa Green qPCR MasterMix Plus for SYBR assay (Eurogentec) was applied
according to instructions to run quantitative real time polymerase chain reaction with
799HT Sequence Detection System (Applied Biosystem) to analyse CD39 mRNA
expression in the samples. Mastermix contained Meteor Taq- polymerase for mRNA
synthesis and SYBR green fluorescent for mRNA detection and quantification. 20ng of
cDNA was added in every sample wells, and three parallel measurements were
performed. Standard calibration curve was generated with maximum of 40ng containing
equal concentration of cDNA from every sample. β-actin acted as a loading control, and
CD39 mRNA expression level was normalized to β-actin mRNA expression. Genes
under interest were detected with suitable primers; CD39 forward: 5'- GAG GAG CCT
CAG CAA CTA CC -3', CD39 reverse: 5'- TGA ATT TGC CCA GCA GAT AG-3' β-
actin forward: 5'- CAC TCT TCC AGC CTT CCT TC-3', β-actin reverse: 5'- GGA TGT
CCA CGT CAC ACT TC-3' (Oligomer).
Efficiency of PCR was analysed with the slope of standard curve. Ideal, 100%
efficiency means, that in every cycle amount of mRNA doubled, and for efficiency of
100% slope is -3,2 (Efficiency= 10^(-1/slope) -1) x 100). Slopes of our curves situated
between -2,8 and -3,0, so the efficiencies were 115-127%. General explanation for the
efficiency greater than 110% is the impaired action of polymerase in the higher
concentrations of standard curve due to ethanol waste or other contaminant.
2.7 Western immunoblotting
To analyse protein expression in cultured cell, confluent dishes were first washed with
PBS, and 100μl boiling protein lysis buffer (10mM Tris-HCL, 1% SDS and 0.2mM
PMSF) supplemented with phenymethanesulfonyl fluoride solution (1:1000), protease
inhibitor solution, phosphate inhibitor cocktails 2 and 3 (1:100, all from Sigma), was
added on the top of the cell monolayer. Protein lysates were collected in tubes and
boiled 10 minutes before centrifuged. Before protein concentration determination with
DC Protein Assay (Biorad, see above), supernatant was gently shifted in new tube.
Samples were stored at -20ºC prior use.
For electrophoresis, we applied 30μg proteins diluted in protein lysis buffer, and
samples were completed with LDS sample buffer (1:4) and reducing agent (1:10) to
denature and reduce protein disulfide bonds in samples. After 10 minutes boiling of
solutions, 35μl samples were loaded together with standard marker (See Blue Plus2
prestained Standards) on Bolt 4-12% Bis-tris Plus polyacrylamide gel in a tank
containing Mes SDS running buffer. During the electrophoresis (150W), reduced,
negatively charged proteins migrated across the gel toward positive anode, and the rate
of protein migration was size-depended. Protein bands from the gel were then
transferred on nitrocellulose membrane with iBlot dryBlotting System according the
instructions. (All the materials and reagents used in electrophoresis from Bolt,
Invitrogen Life technologies.)
Membranes were incubated over night at 4°C in blocking solution (TBS + 0,2% Tween
20 (Sigma), 3% BSA (Sigma)) with primary antibody; CD39 (H-85) rabbit polyclonal
IgG, 200 μg/ml (1:500), P2Y11 (W-17) goat polyclonal IgG, 200 μg/ml (1:500) or
Actin (C-11) goat polyclonal IgG, 200 μg/ml (1:2000). Actin expression was used for
normalization of CD39 and P2Y11 protein expression. On next day membranes were
washed three times with TBST before hour incubation with appropriate horseradish
peroxidase conjugated secondary antibody (1:5000) (All antibodies from Santa Cruz
Biotechnologies). Finally, protein bands were visualized with enhanced
chemiluminescence (Amersham ECL Prime Western Blotting Detection Reagent, GE
Healthcare or Western Lightning- ECL Enhanced chemiluminescence Substrat,
PerkinElmer), which utilized the detectable signal produced by oxidation reaction of
horseradish peroxidase and the substrate. Photographs of membranes were developed
with CURIX 60 (Agfa) on BioMax MR Films (Kodak) for later analysis of protein band
Sections from folmaldehyde-fixed and paraffin-embedded lung tissue was
deparaffinized and rehydrated. Epitope retrieval was performed by boiling the sections
in citrate buffer. Sections were reacted with hydrogen peroxide to block endogenous
peroxidase, washed and blocked with 5% goat serum. The sections were then incubated
with the primary CD39 rabbit anti-human antibody (1:500, Centre de Recherche en
Rhumatologie et Immunologie CHUQ research centre) overnight at 4°C. After
streptoavidin-biotin amplification (LSAB2+ kit DAKO), the slides were incubated with
3, 3'-diaminobenzidine. Van Gieson stainings were done at the pathology department of
University Hospital of Turku.
2.9 Gene transfection
Transfection is a method to silence specific gene expression in cells with small
interfering RNAs. Negatively charged nucleic acids are carried through the negative cell
membrane in positive liposome avoiding the rejection of same charges, (Dalby et al,
2004; Zhao et al, 2008).
9,3 μl of liposome forming Lipofectamine RNAiMAX reagent (Invitrigen) was solved
in 750 μl Opti-MEM medium (Gibco) and after five minutes incubation it combined
with 750 μl Opti-MEM containing human CD39-, P2Y11- or control NT- siRNA (On-
Target plus SMART pool, Thermo Scientific Dharmacon). After 20 minutes, 1490 μl of
this siRNA (100nM) solution was added on 60-80% confluent cell cultures, which were
just before washed with PBS. Following four hours incubation at 37ºC, 5 ml EBM2
medium was added on dishes, and cells were ready for experiments after 48 hours of
transfection. Validity of transfection was verified with qPCR analysis (Figure 6) or with
Western immunoblotting (Figure 7).
Figure 6. CD39-mRNA expression was significantly suppressed in CD39-deficient
cells (n=3). p
0,1% fetal calf serum, Lonza) or in starvation medium containing 2μM ATP for four
hours at 37˚C. Starvation medium with 10μl synthetic P2Y11 antagonist NF340
(Bioscience) was also applied. To measure intracellular caspase level, which indicates
the apoptotic state of cell, 100μl of Caspase-Glo 3/7- reagent (Caspase-Glo 3/7,
Promega) was added on each well. Substrates of reagent were cleaved by intracellular
caspase 3/7 proteases, generating a measurable bioluminescent signal (Scabini et al,
2011). Samples were protected from light and incubated for one hour at room
temperature, and eventually caspase 3/7 level was determined by luminescence
measurement with Victor² 1420 multilabelcounter (Wallac).
2.11 Proliferation assay
To quantify cell proliferation efficiency, 4000 cells per well were seeded on clear, flatbottomed
96-well plate in EBM2 medium day before begin of the experiment. On next
day cells were washed with RPMI (Gibco) and incubated for six hours in 100 μl of
starvation media (RPMI, 0,1% fetal calf serum) at 37˚C. Cells were incubated overnight
either in full EBM2 medium, RPMI with 2% serum or RPMI with 2% serum and 2 μM
ATP. On third day, 10 μl of XTT- reagent (XTT cell proliferation assay kit, Cayman
chemical) were added in every well and cells were incubated for three hours at 37˚C.
Quantification is based on mitochondrial enzyme activity, which reduces XTT-substrate
to orange-colored reaction product (Meshulam et al, 1995). Absorbance and thus,
amount of viable cells was determined with 450 nm wavelength of Original Multiskan
EX (Thermo Labsystems).
2.12 Statistical analysis
Statistical analysis of the results was performed with Graph Pad Prisma 6.0. N shows an
independent value of cell sample from at least three separate experiments. Significance
of the difference between two groups was analyzed with Mann–Whitney U test (also
called Wilcoxon rank-sum test), nonparametric counterpart of unpaired t-test, because
number of samples was often limited, and thus, groups unlikely followed normal
distribution. Although all experiments and results were highly reproducible and in line
with other produced results, more samples are needed in some experiments for
statistical significance in multiple variation analysis such as ANOVA. Difference was
considered to statistically significant when p
Figure 8. CD39 ATPase activity of PAH rat model. a) CD39 ATPase
activity of isolated pulmonary vascular endothelial cells of PAH rat model at
different time points. ATPase activity was significantly suppressed on day
five and on day ten after MCT injection, but it reached the activity of control
level by day 15 and 19. Analysis of every time point included five parallel
samples from five different rats, except day 19 comprising samples from
four different animals. p
3.2 ATPase activity is attenuated in ECs isolated from endstage
Our studies on the rat PAH model suggest that CD39 activity is suppressed in diseased
pulmonary endothelium and could contribute to the pathologic vascular remodelling of
these vessels. To test weather this phenomenon could take place also in human patients,
we obtained pulmonary microvascular ECs isolated from end-stage IPAH patient who
underwent lung transplantation. We found a significant down-regulation of CD39
enzyme activity in these patient ECs compared to our human control cells from healthy
donors (hMVEC). As shown in figure 9a, CD39 ATPase activity of IPAH cells was
59% lower than in control cells. Due to the ATP concentration (500μM) used in the
experiment, which highly exceeded the physiological range of extracellular ATP level,
we measured the basal ATP concentration and hydrolysis also with 2μM ATP. As
expected, control cells hydrolyzed 50% of increased ATP, whereas IPAH cells
hydrolyzed 10% of it (Figure 9b), suggesting that CD39 ATPase activity of the patient
ECs were suppressed regardless of extracellular ATP concentration.
Figure 9. CD39 ATPase activity and dephosphorylation efficiency in patient
pulmonary ECs. a) CD39 ATPase activity of patient endothelial cells. The ability of
hydrolyzing extracellular ATP was suppressed in IPAH cells when they were incubated
with 500 μM ATP (n=6). b) ATP-hydrolyzing efficiency of IPAH cells after one hour
incubation in cell culturing media containing 2 μM ATP. Control cells hydrolyzed about
50% of increased ATP meanwhile IPAH cells hydrolyzed approximately 10% of ATP
The attenuation of CD39 activity is partly mediated through significant down-regulation
of CD39 expression. Results from qPCR showed 48% difference between control and
IPAH cells in mRNA expression (Figure 10a) and results from Western blotting
confirmed the down-regulation of CD39 in IPAH ECs (Figures 10b). Furthermore,
immunohistochemistry staining from end-stage IPAH patient lung tissue samples
showed attenuated CD39 protein expression in the endothelium of diseased and
remodelled pulmonary arteries in comparison to arteries with normal structure (Figure
11). This observation together with our rat studies strongly suggests correlation between
pulmonary vascular remodelling and suppressed CD39 expression and activity.
Figure 10. CD39 mRNA and protein expression level in IPAH cells. a) CD39-
mRNA expression in IPAH cells. Analysis from qPCR results showed down regulation
of the CD39-mRNA expression in IPAH cells (n=3). b) Western blot-picture from the
protein lysates of patient ECs. Protein expression of CD39 (kD 68) was decreased in
IPAH cells in contrast to control cells (n=3). p
Figure 11. Immunohistochemistry staining from end-stage IPAH patient pulmonary
arteries. a) In normal pulmonary artery sections, CD39 expression indicated by brown
colour is clearly visualized in vascular endothelium. b) Expression of CD39 is
suppressed in diseased vascular ECs in comparison to healthy arteries. c and d) CD34
acts as a marker protein of ECs and it is shown in brown in these staining. e and f) Van
Gieson staining for differentiating collagen and SMCs. Arrows indicate endothelium, *
intima and # media hyperplasia.
3.3 Down-regulation of CD39 leads to a phenotypic change in
After discovering the suppressed CD39 activity and expression level in pulmonary
endothelium in PAH, we wanted to model the consequences of attenuated ATPase
activity in our healthy control pulmonary microvascular ECs. Interestingly, downregulation
of CD39 by siRNA strategy decreased the level of apoptosis by 25% under
serum-starvation induced stress (Figure 12a). This could suggest that increased
extracellular ATP has antiapoptotic properties in these ECs. This was confirmed in
experiments, where 2 μM ATP treatments decreased the level of apoptosis by 40% in
control cells (Figure 12b). Suppression of CD39 activity leads to apoptosis resistant
phenotype, which could also partly explain the apoptosis resistant phenotype of IPAH
cells, which was shown in the experiment described in figure 12c. In contrast to control
cells, the pro-survival affects of 2 μM ATP is totally abolished in IPAH cells. This may
be caused by saturation of all critical ATP binding receptors via increased extracellular
Among the effect of ATP on cell survival, it also influenced the proliferation capacity of
ECs. According to literature, apoptosis resistance and cell proliferation are often linked
together, while both associated with cell survival signalling (Zarubin and Han, 2005).
As illustrated in figure 13a, 2μM ATP induced a significant proliferation response.
Proliferation results from CD39-knock down experiment (Figure 13b) were also in line
with other results, as silenced cells were 45% more proliferative compared to control
Figure 12. ATP effect on apoptosis sensitivity. Caspase 3/7 signal indicates the cell
death in the samples. a) In CD39-knock down cells sensitivity for apoptosis was
suppressed. Because of CD39-gene silencing there was a greater ATP niche on the cells
(n=20). b) Control cells performed suppressed apoptosis when treated with 2 μM ATP
(n=20). c) Between control and IPAH cells there was a significant difference in
responsiveness to 2 μM ATP. In control cells ATP affected negatively apoptosis
sensitivity, but IPAH cells were basically more resistant to apoptosis (Con: n= 20, IPAH:
ATP - +
Con CD39 -/-
Figure 13. ATP effect on proliferation. a) Control cells became more proliferative
after 2 μM ATP exposure (n=6). b) CD39-knock down showed a greater affinity to
proliferate in comparison to control cells (n=6). p
Furthermore, P2Y11-deficient ECs showed a significant attenuation in their
proliferation capacity and were not responsive to ATP mediated proliferation (Figure
14c). So according to these results, P2Y11 drastically regulates the ATP-mediated
survival and proliferation of pulmonary ECs.
Figure 14. P2Y11-silencing regulated apoptosis sensitivity and proliferation.
a) Blocking of P2Y11 receptor with 10 μM synthetic antagonist (NF340), rose the
apoptosis sensitivity of control cells (n=6). b) 2 μM ATP increased the apoptotic
sensitivity of P2Y11-knock down cells, which is just the opposite to the control cell
performing (n=14). c) P2Y11-knock down cells showed attenuated proliferation affinity
in comparison to control cells. ATP did not contribute any effect on proliferation (Con:
n=6, P2Y11: n=11). p
Our experiments suggest that P2Y11 is a critical mediator of ATP responses in
pulmonary ECs. This would mean that P2Y11 could have an important role in
mediating the aberrant phenotypes of IPAH ECs. To test this we transfected the IPAH
ECs with P2Y11 siRNA and interestingly, these pathologic cells became significantly
more sensitive to apoptosis (Figure 15a) and less proliferative after loss-of P2Y11
Figure 15. Influence of P2Y11-gene silencing on apoptosis and proliferation in
IPAH cells. a) Stressed P2Y11-knock down IPAH cells (P2Y11-/-) were more
apoptosis sensitive than stressed IPAH cells without P2Y11-gene silencing . Stress was
induced with 0,1% starvation media and full media incubation was used as a baseline
condition (n=8). b) Proliferation was suppressed in P2Y11-deficient IPAH cells in
comparison to unmanipulated IPAH cell (n=8). p
Our study on purinergic signalling in PAH pathogenesis reveals, for the first time,
significant suppression of CD39 ATPase activity in diseased pulmonary endothelium.
This phenomenon was detected both in a rat model of PAH and in pulmonary
endothelium of end-stage human IPAH patients. Loss-of CD39 activity leads to
significant increase in extracellular ATP and ATP mediated responses in vascular wall.
Modelling loss-of CD39 activity with CD39 siRNA in healthy human pulmonary ECs
reveals that loss-of CD39 leads to apoptosis resistant and hyperproliferative phenotype.
This phenotypic switch can be reproduced also by stimulating these cells with extra
ATP. Finally, we show that P2Y11 receptor is a critical purinergic receptor for
mediating the ATP responses in pulmonary ECs and thus a crucial receptor in regulating
EC survival and proliferation responses (Figure 16). Furthermore, we suggest that this
imbalanced purinergic homeostasis could be a novel therapeutic target for future PAH
therapy as suppression of P2Y11 in aberrant pulmonary ECs from IPAH patients
restores, at least partly, normal EC phenotype.
Majority of studies on EC biology are based on in vitro work or studies on whole tissue.
There is significant evidence that the phenotype of ECs changes dramatically upon in
vitro conditions (Durr et al, 2004). To avoid these problems we developed a strategy to
monitor pulmonary EC biology as close to in vivo as possible. By utilizing CD31-
antibody coated magnetic beads we could pull down pulmonary ECs from digested
tissue and experiments were done straight from these beads bound to ECs. Previously
this approach has been used in our laboratory for global gene-expression profiling and
other expression studies. We have previously validated the method and also confirmed
the purity of these magnet bead-bound cells (Li et al, submitted). This strategy can be a
powerful asset in our goal to understand biological events that really take part in the
endothelium during disease process.
Figure 16. Influence of CD39 ATPase activity on ECs. A) In a healthy pulmonary
vasculature extracellular ATP is rapidly hydrolyzed to AMP suppressing ATP mediated
effects on ECs and SMCs. B) In PAH ECs, CD39 activity is impaired and contributes to
increase in extracellular ATP concentration. Excess ATP induces apoptosis resistance
and hyperproliferative phenotype of ECs through P2Y11 activation and could promote
intimal hyperplasia. Overload of ATP may also promote media hypertrophy by induced
proliferation of SMCs
Our unique In vivo EC profiling strategy was now used for the first time to monitor
CD39 ATPase enzyme activity in a MCT rat model. This study showed significant
suppression of ATPase activity at early stages of disease. Seen down-regulation of
ATPase activity enables accumulation of ATP and heightened ATP responses.
Interestingly, the suppression of ATPase is seen at 5 and 10 days after MCT injection,
which present stages where vascular remodelling is initiated and takes place. This could
suggest that excessive ATP would play a significant role in PAH pathogenesis. The
signalling cascade could be further potentiated through ATP release from severely
injured or stressed ECs (Bodin et al, 1992; Burnstock and Verkhratsky, 2009). It would
be likely event in our rat MCT model as EC stress and injury are hallmarks of the model.
In our experiments ATPase enzyme activity normalized during the later stages of the
disease. Toxic metabolites of MCT, present in the very early stages, could contribute to
our observations on ATPase activity. Perhaps the degradation of these metabolites
reflects our observation on normalization of ATPase activity. This phenomenon has
been suggested in SMCs as their phenotype becomes more sensitive for vasoconstrictors
in the early stages of disease but loose this sensitivity at later stages (Altiere et al, 1986).
A likely explanation is also massive EC death at later stages of the disease (Jurasz et al,
2010), which could affect our analysis by causing an experimental bias. ATPase activity
in our assay is normalized at stages where the animals become severely symptomatic.
There is no intimal hyperplasia in rat MCT model, which is a significant difference to
human disease and could explain why the attenuated ATPase activity is sustained in
human disease model.
MCT-induced vascular remodelling is a widely used model of PAH and the only rodent
model with 100% mortality. Metabolites of MCT cause endothelial injury resulting in
media hypertrophy and leakage of vasculature (Ghodsi and Will, 1981; Bauer et al,
2007). Muscularization of peripheral arteries is detectable since day three after MCT
injection. At later stages inflammation and media hypertrophy of larger arteries are also
identified (Meyrick and Reid, 1979). ECs are likely responsible for the vasoconstriction
and elevated resistance soon after MCT injection (Ito et al, 1988; Ghodsi and Will,
1981). Disadvantage of this model is the lack of neointima or plexiform lesion
formation, which are characteristic for severe human PAH (Bauer et al, 2007).
Metabolites of MCT affect DNA synthesis of ECs and impair cell-cycle progression
(Lappin et al, 1998). Although similar EC defects can be observed in the early stages of
human disease where ECs have lost their regeneration capacity and are highly
susceptible to apoptosis, MCT model is not the ideal method to model severe PAH as
there is no phenotypic switch in ECs that leads to the intimal disease. Thus, there are
limitations on making conclusions on EC dysfunction and PAH by using this model
only. Further experiments would be interesting with animal models that show features
of intimal disease. These include combination of hypoxia and MCT, and MCT together
with pneumonectomy (Morimatsu et al, 2012; White et al, 2007). Another interesting
PAH model with plexiform lesions is induced with VEGFR antagonist and hypoxia
exposure (Abe et al, 2010).
As mentioned above, PAH rodent models are not fully comparable with human disease.
From an unknown reason rodent models differ dramatically in symptoms and structural
modifications from the human disease (Heath, 1992). As our study from MCT model
suggests the contribution of suppressed endothelial ATPase activity to pathologic
vascular remodelling, we wanted to confirm the phenomenon in human IPAH patient
ECs. In line with our rat model, a significant suppression of ATPase activity was
observed in IPAH ECs in vitro, and CD39 expression was significantly down-regulated
in pulmonary arteries with severe vascular remodelling. Our results suggest that at least
partly this suppression of enzyme activity may be mediated through attenuation of
CD39 expression in the diseased cells. This is intriguing as the pathways responsible for
this down-regulation are unknown. It could be possible that the suppression of enzyme
activity is also mediated at the protein level by so far unknown mechanism. These
mechanisms could involve protein conformation changes, variations in interacting
proteins or changes in protein distribution. Pathways behind the CD39 down-regulation
needs to be studied in the future.
In a recent study Visovatti and co-workers studied the level of CD39 in circulating
microparticles in IPAH patients. Circulating microparticles are vesicles derived mainly
from platelets, but also from ECs in response to activation or apoptosis. Interestingly,
they showed increased CD39 activity in these microparticles (Visovatti et al, 2012).
This could reflect a compensatory response to the attenuated activity detected by us in
vascular wall. On the other hand we do not know the exact origin of these particles and
thus cannot be sure how they link to the pulmonary vascular pathology. There is also a
possibility that the microparticles are reflecting changes in systemic vasculature as there
is evidence of systemic endothelial dysfunction and EC activation in IPAH patients
(Peled et al, 2009). In conclusion, circulating microparticles seem not to be an indicator
of CD39 activity in pulmonary vasculature.
Our study reveals that suppression of CD39 in pulmonary ECs leads to an apoptosis
resistant phenotype, at least partly, through increased extracellular ATP levels. Similar
conclusion was found in a study by Urban and co-workers on hUVECs and ischemic
conditions. In this study hUVECs treated with excessive extracellular ATP or
impairment of ATPaes activity increased significantly survival under stress (Urban et al,
2012). Thus, ATP may generally act as a pro-survival factor on ECs. Interestingly,
pulmonary ECs from end-stage IPAH patients show an apoptosis resistant phenotype
and treatment of these cells with ATP did not further increase survival when compared
to healthy ECs. We speculate that this loss-of ATP response is caused by saturation of
ATP binding receptors through suppressed CD39 mediated excess of naturally
occurring extracellular ATP. In addition to the anti-apoptotic actions of ATP, we
demonstrate that ATP also promotes pulmonary EC proliferation. Similar ATP effects
on cell proliferation have also been detected in human cardiac ECs and pulmonary
artery vasa vasorum ECs (Gerasimovskaya et al, 2008; Rumjahn et al, 2009). In our
studies the pro-proliferative effects are established through suppression of ATPase
activity. One potential mechanism underlying the proliferative response is ATP induced
increase in intracellular free Ca 2+ concentration, which can promote proliferation
(Erlinge, 1998). As increased EC proliferation and apoptosis resistance are a critical
component of intimal hyperplasia in PAH, it is tempting to speculate that suppression of
CD39 activity in IPAH ECs plays a fundamental role in the process of severe occlusive
To identify purinergic receptors in pulmonary ECs that could mediate the ATP
responses observed in our studies we analyzed a previously published microarray
analysis of BMPR2-deficient human pulmonary ECs (Alastalo et al, 2011). BMPR2-
deficient ECs become highly susceptible to apoptosis and loose their proliferation
capacity and thus, could be a model to search for alterations in purinergic signalling.
Interestingly, purinergic receptor P2Y11 was one of the most down-regulated genes
after acute loss-of BMPR2 by siRNA strategy. The role and function of P2Y11 in ECs
is largely unknown. In our further studies we confirmed that P2Y11 plays a significant
role in ATP-mediated survival and proliferation. It is intriguing that P2Y11 deficient
ECs show abolished ATP mediated survival and proliferation response suggesting that
this receptor is the major ATP binding receptor in pulmonary ECs. Interestingly,
P2Y11-mediated apoptosis resistance has been described in human neutrophils
(Vaughan et al, 2007). The effects of P2Y11 both on apoptosis and proliferation can be
mediated either through calcium mobilization or through cAMP pathways or both. As
remembered, capability to activate both cAMP-mediated- and phospholipase C-
pathways is a unique feature of P2Y11 in comparison to other purin receptors
(Communi et al, 1997a). Although, we strongly highlight the possible role for P2Y11 in
the apoptosis resistant phenotype of aberrant pulmonary ECs, it could have an important
role in normal pulmonary endothelium in situations of vascular injury and stress. Based
on our studies, the P2Y11 pathway is only partly active in normal ECs as increasing
extracellular ATP leading to dramatic responses, which are abolished in P2Y11
deficient ECs. Furthermore, IPAH patient ECs show complete resistance to extra ATP
and loose their apoptosis-resistant and hyperproliferative characteristics after
suppression of P2Y11-mediated signalling.
In contradiction to our results Xiao and co-workers have shown that P2Y11 does not
have effects on apoptosis, and activation of P2Y11 actually inhibits proliferation of
hUVECs (120 Xiao et al., 2011). One possible explanation for these differences
between results may arise from ATP concentrations applied in the experiments. Xiao
and co-workers reached their finding with 50-100μM ATP, which highly exceeds
physiological extracellular ATP content (normally around 10nM) (Schwiebert and
Zsembery, 2003). In our experiments we found the P2Y11 actions with 2μM ATP.
Other explanation might be the difference of cell types as pulmonary microvascular ECs
can vary dramatically form ECs isolated umbilical vein. Further support for our finding
come from experiments on apoptosis resistant and hyperproliferative IPAH ECs, as
normal EC phenotype was restored by silenced P2Y11.
Our study suggests, for the first time, possibility of therapeutic strategies that restore
CD39 activity and normalize extracellular ATP. We are currently searching for
strategies to increase CD39 ATPase activity in rodent pulmonary endothelium to test
the applicability of our findings for PAH therapy. Furthermore, a similar strategy would
be to inhibit ATP mediated effects by suppressing P2 receptors such as P2Y11 mediated
signalling. In this case modulation of imbalanced purinergic system could enable
restoration of both intimal and media pathology. Synthetic P2Y11 antagonist may
present a plausible option for future therapy for severe PAH. Further research is needed
to define the efficiency and side effects of these antagonists. The major challenge in
these studies is that P2Y11 is not expressed in rodents. For example in mouse genome,
there is no homologous gene for human P2Y11. P2Y11 has only been cloned in human,
canine and Xenopus (Devader et al, 2007). So applying rodent models in these
experiments would be inappropriate, at least without over-expression of human P2Y11
in a transgenic mouse model. In the light of P2Y11 missing in rodents, the significant
role of P2Y11 in controlling the fate of human pulmonary ECs is highly interesting. It is
still totally obscure why human PAH is so different from rodent models of PAH.
However, one of the hallmark differences between humans and rodents is the behaviour
of endothelium. Identifying genes and pathways which significantly differ between
humans and rodents, such as P2Y11 may reveal important insight to the pathogenesis
and treatment of human PAH.
I would like to thank the personnel of Pediatric Research Laboratory at Biomedicum
Helsinki for kindness and help they showed me during my research periode. Special
thanks to my supervisor Dr. Tero-Pekka Alastalo and PhD student Mikko Helenius for
guidance and support in completing my master’s thesis project.
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