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i
The Role of Vascular Matrix Metalloproteinase-2 and Heme Oxygenase-2 in Mediating the Response to
Hypoxia
by
Jeff ZiJian He
A thesis submitted in conformity with the requirements for the degree of doctor of philosophy
Department of Laboratory Medicine and Pathobiology University of Toronto
© Copyright by Jeff ZiJian He, 2009
ii
The Role of Vascular Matrix Metalloproteinase-2 and Heme
Oxygenase-2 in Mediating the Response to Hypoxia
Jeff ZiJian He
Doctor of Philosophy
Department of Laboratory Medicine and Pathobiology University of Toronto
2009
ABSTRACT Systemic hypoxia frequently occurs in patients with cardiopulmonary diseases.
Maintenance of vascular reactivity and endothelial viability is essential to preserving
oxygen delivery in these patients. The role of matrix metalloproteinase-2 (MMP-2) and
heme oxygenase-2 (HO-2) in the vascular response to hypoxia were investigated. In the
first part of the thesis, the role of MMP-2 in regulating systemic arterial contraction after
prolonged hypoxia was investigated. MMP-2 inhibition with cyclic peptide
CTTHWGFTLC (CTT) reduced phenylephrine (PE)-induced contraction in aortae and
mesenteric arteries harvested from rats exposed to hypoxia for 7 d. Responses to PE
were reduced in MMP-2-/- mice exposed to hypoxia for 7 d compared to wild-type
controls. CTT reduced contraction induced by big endothelin-1 (big ET-1) in aortae
harvested from rats exposed to hypoxia. Increased contraction to big ET-1 after hypoxia
was observed in wild-type controls, but not MMP-2-/- mice. Rat aortic MMP-2 and
MT1-MMP protein levels and MMP activity were increased after 7 d of hypoxia. Rat
aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular
iii
smooth muscle. These results suggest that hypoxic induction of MMP-2 activity
potentiates contraction in systemic conduit and resistance arteries through proteolytic
activation of big ET-1.
The second part of the thesis investigated oxygen regulation of HO-2 protein and
whether it plays a role in preserving endothelial cell viability during hypoxia. HO-2, but
not HO-1, protein level was maintained during hypoxia in human endothelial cells
through enhanced translation of HO-2 transcripts. Inhibition of HO-2 expression
increased the production of reactive oxygen species, decreased mitochondrial membrane
potential, and enhanced apoptotic cell death and activated caspases during hypoxia, but
not during normoxia. These data indicate that HO-2 is translationally regulated and
important in maintaining endothelial viability and function during hypoxia.
In summary, the thesis demonstrates the importance of MMP-2 and HO-2 in
preserving vascular function during prolonged systemic hypoxia. These enzymatic
pathways may, therefore, represent novel therapeutic targets that may be exploited to
ameliorate the effects of hypoxia in patients with cardiopulmonary disease.
iv
ACKNOWLEDGMENTS
I would like to thank all of the people who have helped and inspired me during my
doctoral study.
I especially want to thank my supervisor, Dr. Michael Ward, for his guidance during my
research. He has provided me with encouragement, sound advice, good teaching, and
lots of good ideas. I would have been lost without him. I want to thank my co-
supervisor, Dr. Philip Marsden, for constantly challenging me to look beyond the
obvious and ensuring that I am knowledgeable in the field. I also want to thank my
advisory members for their constructive criticism, advice, and continuous support.
My deepest gratitude goes to my parents, Ying Wong and Wing Ming Ho, for their
unflagging love and support throughout my life; this dissertation is simply impossible
without them. I am thankful everyday that they are part of my life.
I am indebted to my many student colleagues for providing a stimulating and fun
environment in which to learn and grow. I am especially grateful to Ben Lai, Shirley
Mei, Diana Wong, Julie Basu-Ray, Orisha Yacyshyn, Lakshmi Kugathasan for all the
emotional support, entertainment, and caring they provided. I look forward to their
company in the next phase of my life.
v
TABLE OF CONTENTS
ACKNOWLEDGMENTS iv
TABLE OF CONTENTS v
LIST OF TABLES viii
LIST OF FIGURES ix
ABBREVIATIONS xii
PREFACE xiv
CHAPTER 1 Introduction
1.1 Oxygen Delivery 2
1.2 Mechanisms of Oxygen Sensing 5
1.3 Regulation of Gene Expression by Hypoxia 6
1.4 Physiological Responses to Hypoxia 11
1.5 The Endothelium 14
1.6 Matrix Metalloproteinase-2 17
1.6.1 Matrix Metalloproteinase-2 Protein Structure 17
1.6.2 Regulation of Matrix Metalloproteinase-2 Activity 18
1.7 Endothelin 20
1.7.1 Activation of Endothelin-1 21
1.7.2 Endothelin Receptor Signalling 22
1.8 Heme Oxygenase 23
1.8.1 Functions of Heme Oxygenase-2 24
1.8.2 Structure and Expression of Heme Oxygenase-2 27
1.8.3 Regulation of Heme Oxygenase Protein Expression 29
1.9 Thesis Objectives 31
vi
CHAPTER 2 Induction of matrix metalloproteinase-2 enhances systemic arterial
contraction after hypoxia
2.1 Introduction 34
2.2 Materials and Methods
2.2.1 Exposure to Hypoxia 36
2.2.2 Chemicals/Antibodies 36
2.2.3 Aortic and Mesenteric Contractile Responses 37
2.2.4 Immunohistochemistry 38
2.2.5 Western Blots 38
2.2.6 Gelatin Zymography 39
2.2.7 MMP and ECE Activity 40
2.2.8 Aortic MMP-2, MT1-MMP, TIMPs 1 to 4 mRNA Levels 40
2.2.9 Contractile Responses in Aorta of MMP-2-/- and
MMP-2+/+ Mice 41
2.3 Results 43
2.4 Discussion 65
CHAPTER 3 Enhanced translation of HO-2 transcripts preserves human
endothelial cell viablility during prolonged hypoxia
3.1 Introduction 72
3.2 Materials and Methods
3.2.1 Chemicals and Reagents 73
3.2.2 Cell Culture Studies 73
3.2.3 Quantitative Real Time PCR 74
3.2.4 Western Blotting 75
3.2.5 3H-uridine and 3H-leucine Incorporation 75
3.2.6 35S-methionine Incorporation 76
3.2.7 Polysome Profiling 77
3.2.8 Measurement of Intracellular Reactive Oxygen Species 77
vii
3.2.9 Mitochondrial Membrane Depolarization 77
3.2.10 Annexin V/Propidium Iodide Labeling 78
3.2.11 Total Caspase Activation 78
3.3 Results 79
3.4 Discussion 99
CHAPTER 4 Perspective 106
REFERENCES 112
viii
LIST OF TABLES
Table 1. Effect of MMP inhibition on maximum contraction and EC50 values
during PE and big ET-1-induced rat aortic contraction 46
Table 2. Maximum contraction and EC50 values during PE- induced contraction and response to big ET-1 in MMP-2-/- and MMP-2+/+ mice 64
ix
LIST OF FIGURES
CHAPTER 1
Figure 1.1 Regional distribution of pO2 from the airways to the cytosol 3
Figure 1.2 Oxygen dissociation curve from adult haemoglobin 3
Figure 1.3 Oxygen regulation of HIF activity 7
Figure 1.4 Control of translation initiation during hypoxia 9
Figure 1.5 Domain structure of matrix metalloproteinase-2 18
Figure 1.6 Generation of endothelin-1 22
Figure 1.7 Metabolism of heme by heme oxygenase 24
Figure 1.8 Structural organization of the human HO-1 and HO-2 gene 28
CHAPTER 2
Figure 2.1 Concentration-response relationships for phenylephrine in aortic rings
and mesenteric arteries and concentration response relationships for
big ET-1 in aortic rings from normoxic rats and rats exposed to
hypoxia for 7 d 44
Figure 2.2 Immunohistochemistry for MMP-2 on aortic sections from normoxic
rats and rats exposed to hypoxia for 7 d 47
Figure 2.3 Aortic MMP-2 protein levels in normoxic rats and rats exposed to
hypoxia for 16 h, 48 h, and 7 d 49
Figure 2.4 MMP and ECE activity in aortae from normoxic rats and rats exposed
to hypoxia for 7 d 51
Figure 2.5 Levels of MT1-MMP, TIMP-1, -2, -3, and -4 proteins in aortae
from normoxic rats and rats exposed to hypoxia for 16 and 48 h
and 7 d 52
Figure 2.6 Levels of MMP-2, MT1-MMP, TIMP-1, -2, -3, -4 mRNAs in
aortae from normoxic rats and rats exposed to hypoxia for 16 h, 48 h,
and 7 d 56
x
Figure 2.7 MMP-2 and MT1-MMP mRNA levels in endothelial, sub-endothelial
VSMC, and deep medial VSMC from aortae of normoxic rats and from
rats exposed to hypoxia for 7 d 60
Figure 2.8 Concentration-response relationship for phenylephrine and contractile
response to big ET-1 in in aortic rings from MMP-2+/+ and MMP-2-/-
mice exposed to normoxia or hypoxia for 7 d 63
CHAPTER 3
Figure 3.1 HO-1 and HO-2 mRNA and protein levels in HUVEC exposed to
normoxia or 1% oxygen for either 16 or 48 h 81
Figure 3.2 HO-1 and HO-2 protein levels in HAEC and EPC exposed to normoxia
or 1% oxygen for 16 h 83
Figure 3.3 Rate of 3H-uridine (A) and 3H-leucine (B) incorporation into RNA and
protein of HUVEC exposed to normoxia or hypoxia for 16 or 48 h. (C)
Rate of 35S-methionine incorporation into HO-1 and HO-2 protein of
HUVECs exposed to normoxia or hypoxia for 16 h. (D) Quantification
of the abundance of HO-2 mRNA in various polysome fractions from
HUVEC exposed to normoxia or hypoxia for 6 or 24 h 85
Figure 3.4 ROS levels in HUVEC transfected with scrambled or HO-2 siRNA
exposed to normoxia or hypoxia for 48 h, or exposed to normoxia or
hypoxia for 16 h treated with TNF-α or H2O2 88
Figure 3.5 Mitochondrial membrane potential in HUVEC transfected with
scrambled or HO-2 siRNA exposed to normoxia or hypoxia for 48 h,
or exposed to normoxia or hypoxia for 16 h treated with TNF-α or
H2O2 91
Figure 3.6 Cell death in HUVEC transfected with scrambled or HO-2 siRNA
exposed to normoxia or hypoxia for 48 h, or exposed to normoxia or
hypoxia for 16 h treated with TNF-α or H2O2 93
xi
Figure 3.7 Total activated caspase level in HUVEC transfected with scrambled or
HO-2 siRNA exposed to normoxia or hypoxia for 48 h, or exposed to
normoxia or hypoxia for 16 h treated with TNF-α or H2O2 95
Figure 3.8 Cell death and total activated caspase level in HAEC transfected with
scrambled or HO-2 siRNA exposed to normoxic or hypoxia for 48 h or
or exposed to normoxia or hypoxia for 16 h treated with TNF-α or
H2O2 97
xii
ABBREVIATIONS
ANOVA analysis of variance ARD1 arrest defective 1 ATF activating transcription factor ATP adenosine triphosphate bHLH basic helix-loop-helix CGRP calcitonin gene related peptide CRC concentration response curve CO carbon monoxide CTT CTTHWGFTLC DCF 2’,7’-dichlorofluorescein ECE endothelin converting enzyme eIF eukaryotic initiation factor EPC endothelial progenitor cell ET-1 endothelin-1 ETAR endothelin type A receptor ETBR endothelin type B receptor ETC electron transfer chain FIH factor-inhibiting HIF H2O2 hydrogen peroxide HAEC human aortic endothelial cell HIF hypoxic inducible factor HO heme oxygenase HRM heme regulatory motif HUVEC human umbilical vein endothelial cell HVR hypoxic ventilatory response IRES internal ribosomal entry site JC-1 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide MAPK mitogen-activated protein kinase MARE Maf recognition element miRNA microRNA MMP-2 matrix metalloproteinase-2 MT1-MMP membrane type-1 matrix metalloproteinase mTOR mammalian target of rapamycin NO nitric oxide ODD oxygen dependent degradation domain PE phenylephrine PERK PKR-like endoplasmic reticulum kinase PHD prolyl hydroxylase PI propidium iodide pO2 partial pressure of oxygen ROS reactive oxygen species TIMP tissue inhibitors of matrix metalloproteinase
xiii
TNF-α tumor necrosis factor α UTR untranslated region uORF upstream open reading frame VSMC vascular smooth muscle cell
xiv
PREFACE
The work presented in Chapter 2 has been published in Am J Physiol Heart Circ Physiol
292:H684-H693, 2007. Jeff Z. He, Adrian Quan, Yi Xu, Hwee Teoh, Guilin Wang,
Jason E. Fish, Brent M. Steer, Shigeyoshi Itohara, Philip A. Marsden, Sandra T. Davidge
and Michael E. Ward. Induction of matrix metalloproteinase-2 enhances systemic
arterial contraction after hypoxia. Permission has been obtained from the American
Physiological Society and all of the authors for inclusion of the paper in the thesis and
for the National Library to make use of the thesis.
As the first author of the publication, I contributed to study design, figure making
and manuscript writing. I performed all of the experiments and data analysis except
i) Figure 2.1 B: Adrian Quan measured the contractile response from
mesenteric arteries
ii) Figure 2.3: Experiment was done by Yi Xu from Dr. Davidge’s lab
iii) Figure 2.6 and 2.7: mRNA extraction and measurements were done by
Jason Fish and Brent Steer from Dr. Marsden’s lab
The work presented in Chapter 3 has been written into a manuscript and is expected to
be submitted for publication before December 2008. As the first author of this
manuscript, I contributed to study design, figure making and manuscript writing. I
performed all of the experiments and data analysis except
Figure 3.2: Blood outgrowth endothelial cells were a gift from Dr. Courtman
Figure 3.3 D: Experiment was done by members of Dr. Marsden’s Lab
Chapter 1
- 1 -
CHAPTER 1
Introduction
Chapter 1
- 2 -
In mammalian cells, oxygen serves as the terminal electron acceptor in the
mitochondria during generation of adenosine triphosphate (ATP) and as substrate for
numerous enzymes, such as heme oxygenases and prolyl hydroxylases.[1, 2] Depending
on the metabolic activity and the distance away from vessels, cells experience a variety
of oxygen concentrations under physiological conditions. Hypoxia is thus a relative
term, and is most usefully described as a condition in which normal tissue function is
inhibited due to insufficient oxygen supply. Reductions in systemic oxygen delivery
occur commonly in patients suffering from cardiopulmonary diseases, hemorrhagic
shock, or sepsis and in normal individuals during ascent to high altitudes.[3-5] The
molecular mechanisms that underlie the physiologic adaptations to hypoxia and its
pathophysiological consequences are complex and incompletely understood.
Investigation into these mechanisms will lead to the development of strategies to
mitigate the effects of hypoxia in these conditions. The role of vascular matrix
metalloproteinase-2 (MMP-2) and heme oxygenase-2 (HO-2) in the adaptive response to
hypoxia were investigated in the current study.
1.1 Oxygen Delivery
The delivery of oxygen to tissues is one of the main functions performed by the
cardiovascular system (Figure 1.1). The partial pressure of oxygen (pO2) in inspired air
at sea level is 21 kPa (~150 mmHg).[6] Oxygen extraction at the terminal airways and
CO2 accumulation in the alveolar gas decreases the alveolar pO2 to 14 kPa (~100
mmHg). At the alveoli, oxygen diffuses down its pressure gradient into the pulmonary
capillaries where it binds to haemoglobins in red blood cells. The median pO2 in
Chapter 1
- 3 -
systemic arteries is 13 kPa (~92 mmHg), falling to 7 kPa (~50 mmHg) in arterioles and
~3-4 kPa (~25 mmHg) in precapillary arterioles and capillaries.[6, 7]
Oxygen is off-loaded from haemoglobin in red blood cells to tissues as it travels
through the cardiovascular system according to the oxygen-haemoglobin dissociation
curve (Figure 1.2). A substantial amount of oxygen diffuses out from the lumen of the
arterioles.[7, 8] The rate of diffusion increases from the larger to the smaller arterioles,
Figure 1.1 Regional distribution of pO2 from the airways to the cytosol. Source: Ward J (2007) Oxygen sensors in context.
Figure 1.2 Oxygen dissociation curve from adult haemoglobin. Source: http://www.anaesthesiamcq.com/downloads/odc.pdf
Chapter 1
- 4 -
consistent with the lower velocity of blood in the smaller arterioles and their larger
surface area-to-volume ratio.[7, 9] Because the pO2 gradient between capillaries and the
tissue interstitium is small and pO2 actually increases in the venular circulation as blood
moves from the collecting venules to the larger veins, the capillary bed contributes
relatively little oxygen to tissues under resting conditions. The capillary circulation’s
function at rest appears predominantly to extract from the tissue byproducts of
metabolism and participate in oxygen exchange primarily when the tissue is active.
The amount of oxygen delivered to tissues is determined mainly by bulk blood
flow, haemoglobin saturation and the sympathetic nervous system. The cardiovascular
system constantly matches tissue oxygen supply to tissue oxygen demand by adjusting
these parameters. Oxygen supply to tissues could be enhanced by increasing vessel
diameter through the release of vasodilators (nitric oxide) and/or inhibition of
vasoconstrictors (endothelin-1). Increased capillary perfusion augments oxygen delivery
during exercise.[7] Oxygen delivery to tissues could also be increased by decreasing the
oxygen affinity for haemoglobin, as occurs during increases in body temperature or
carbon dioxide concentration. Other factors that are known to alter the affinity of
oxygen binding to haemoglobin include pH, carbon monoxide, and 2,3-
disphosphoglycerate. Lastly, activation of the adrenergic nervous system increases
oxygen delivery by optimizing of blood flow among different vascular beds to increase
oxygen extraction.[10, 11] The role of vascular MMP-2 in the contractile response to
adrenoceptor stimulation after exposure to hypoxia was investigated in the current study.
Chapter 1
- 5 -
1.2 Mechanisms of oxygen sensing
Cells generally respond to hypoxia, especially if it is prolonged, by reducing
energy use and upregulating energetically efficient ATP producing pathways.[12] The
existence of these homeostatic processes in every cell implies that all cells have the
ability to sense changes in oxygen concentration. Changes in oxygen concentration are
detected by O2 sensors, which in turn regulate the activity of the effectors that determine
the modifications of specific cellular functions.[13] Despite recent progress in the
characterization of the cellular effectors of hypoxia, understanding of this process
remains incomplete.
Mitochondria have long been considered as a potential O2 sensing site since they
are the site of oxidative phosphorylation and electron transport and their activity is
altered by changes in oxygen concentration.[1, 14] Single electrons are lost to molecular
O2 to form ROS at various points in the electron transfer chain (ETC). The tendency for
the ETC to generate ROS increases during hypoxia because the Vmax of the cytochorome
oxidase is reversibly decreased due to reduced O2 availability.[1, 14] The increase in
ROS levels provides a favourable environment for iron to be in the Fe3+ state which,
along with decreased available oxygen, results in rapid inhibition of prolyl hydroxylase
activity leading to HIF-α stabilization and transactivation of hypoxia-inducible gene
expression. In addition to the mitochondria, plasma membrane-associated NAD(P)H
regulates intracellular ROS levels and function as an oxygen sensor in some cells.[15]
Other potential oxygen sensors that do not regulate intracellular ROS levels include
heme containing enzymes such as heme oxygenase and enzymes that utilize oxygen as
Chapter 1
- 6 -
substrate, such as proly hydroxylase.[6, 16] It is now recognised that a number of
mechanisms with different sensitivities may effectively act as O2 sensors for different
cellular processes in different cell types.
1.3 Regulation of gene expression by hypoxia
Hypoxia activates a variety of complex pathways with the ultimate aim of
reinstating oxygen homeostasis. The identification of the hypoxia inducible factors
(HIFs) has greatly advanced our understanding of gene regulation during hypoxia.[17]
The HIFs are a family of transcription factors which are heterodimers containing of α
and β subunits.[18] They are characterized by the presence of basic helix-loop-helix
(bHLH) and Per/ARNT/Sim (PAS) domains. HIF-α, but not HIF-β, subunits possess an
oxygen-dependent degradation domain (ODD), rendering these proteins labile in the
presence of oxygen.[19]
HIF mediates a large number of critical responses that restore tissue oxygenation
and limit tissue injury.[14, 20] HIF activity is primarily controlled via the stability of
the alpha subunit (Figure 1.3). Under physiological conditions, prolyl hydroxylases
(PHD) catalyse hydroxylation of the HIF-α subunit on proline residues 402 and/or 564
within the ODD.[21] Upon hydroxylation, HIF-α binds to the VHL protein, which is
the recognition component of an E3 ubiquitin-protein ligase. Binding of VHL targets
HIF-α for ubiquitination and subsequent degradation by the 26S proteasome.[22]
Binding of HIF-α to VHL is enhanced by acetylation of HIF-α at lysine 532 in the ODD
by arrest defective 1 (ARD1).[23] In addition to PHDs and ARD1, HIF-α activity is
Chapter 1
- 7 -
Figure 1.3 Oxygen regulation of HIF activity. Source: Schmid et. al. (2006) Lights on for Low Oxygen: A Noninvasive Mouse Model Useful for Sensing Oxygen Deficiency
regulated by factor-inhibiting HIF (FIH), which hydrolyzes asparagine 803 located in
the C-transactivation domains.[24, 25] Hydroxylation of asparagine 803 prevents
binding of p300/cAMP-response element-binding protein and reduces the transcriptional
activity of HIF-1 during normoxic conditions. During hypoxia, activities of both PHD
and FIH are inhibited.[16] Inhibition of HIF-α hydroxylation prevents HIF-α binding to
VHL, leading to stabilization and accumulation of HIF-α in the cytoplasm. The
stabilized HIF-α enters the nucleus to dimerize with HIF-β and activates transcription of
genes that contain hypoxic response element (5’-RCGTG-3’) in their promoter or
enhancer regions.[17, 26] Apart from the HIF family, hypoxia activates a number of
other important transcription factors, including nuclear factor κB, activator protien-1,
and p53.[27]
Normoxia
Hypoxia
HIF-1 accumulation + transcriptional activity
HIF-1 inactivation+ degradation
Chapter 1
- 8 -
Whereas HIFs enable long-term cellular survival, a variety of HIF-independent
pathways promote ATP conservation by limiting energy-consuming processes.[14, 28]
Protein synthesis is the second costliest cellular process in terms of ATP demand and is
tightly linked to cellular oxygen availability.[29-33] In human cells, protein synthesis
drops to less than 50% of that in normoxic cells during hypoxic incubation (<1% O2)
due to inhibition of translation and later through effects on transcription.[34-37]
Translation of mRNA transcripts occurs in three stages: initiation, elongation, and
termination. Initiation is the most complex step and is tightly controlled by a number of
eukaryotic initiation factors (eIF).[38] The two central mechanisms for regulating
translation initiation are the assembly of eIF4F (comprised of eIF4A, eIF4E, and eIF4G
subunits) and the phosphorylation status of the eIF2α subunit.[39] Binding of the eIF4F
complex to the mRNA 5’cap structure is essential for initiating cap dependent
translation.[40, 41] Phosphorylation of the eIF2α subunit regulates the availability of
the ternary eIF2-GTP-Met-tRNA complex upon which 60S ribosomal subunit
recruitment depends.[12]
During acute hypoxia, global protein translation initiation is inhibited by
phosphorylation of eIF2α, preventing the exchange of GDP for GTP and sequestering
eIF2B in an inactive complex (Figure 1.4).[35, 42] Phosphorylation of eIF2α can be
achieved by several different kinases: interferon-inducible double-stranded RNA-
activated kinase, heme-regulated inhibitor of translation, kinase activated by nutrient
starvation, and PKR-like endoplasmic reticulum kinases (PERK).[43, 44] PERK is the
main kinase responsible for eIF2α phosphorylation following acute hypoxia.[36]
Chapter 1
- 9 -
During prolonged hypoxia, cap-dependent protein translation initiation is
inhibited by preventing eIF4F complex formation (Figure 1.4).[35] A family of eIF4E-
binding proteins (4E-BPs) competes with eIF4G for binding to limited amounts of
eIF4E. Binding affinity of 4E-BPs to eIF4E is regulated by mammalian target of
rapamycin (mTOR). mTOR-mediated phosphorylation of 4E-BPs reduces the affinitiy
of 4E-BP binding to eIF4E.[45] Hypoxia inhibits mTOR activity, leading to
hypophosphorylation of 4E-BP and its binding to eIF4E.[38, 46, 47]
Logic dictates that mechanisms must exist to ensure that proteins which comprise
of the phenotypic modulation on which hypoxic adaptation depends are synthesized in
the face of global suppression of cap-dependent mRNA translation. Polysome profile
comparisons of cells cultured under normoxia and hypoxia have identified mRNA
Figure 1.4 Control of translation initiation during hypoxia. Source: Van Den Beuken et al. (2006) Translational Control of Gene Expression During hypoxia
Chapter 1
- 10 -
transcripts that remain actively translated during hypoxia.[48-50] Translationally
regulated transcripts show an increased association with polysomes during hypoxia
compared with normoxia. These genes generally have long and GC rich nucleotide
sequences in their 5’ untranslated region (UTR) and significant secondary structures that
impede ribosomal scanning for the initiation codon.[12] One of the genes regulated at
the translational level during hypoxia is activating transcription factor 4 (ATF4).[48, 51]
The 5’-UTR of ATF4 contains two conserved upstream open reading frames (uORF).
Under normoxia, ATF4 protein synthesis is repressed because most ribosomes that
translate the first uORF reinitiate translation at the second uORF rather than at the ATF4
ORF. During hypoxia, ribosome reintiation is delayed by low ternary complex
availability. The delay increases the proportion of ribosomes that scan through the
second uORF and reinitiate, instead, at the ATF4 ORF. The presence of uORF,
therefore, represents an RNA element that can stimulate translation during hypoxia.
Other genes containing uORFs and are selectively translated when eIF2α is
phosphorylated include CHOP and GADD34.[35, 52]
In an environment of low eIF4F availability, a competitive advantage over other
mRNAs for ribosome binding could be conferred through activation of transcripts
containing a 5’-UTR lacking secondary structure that obviate the need to engage the
helicase activity of the eIF4F complex. This was identified as the operative mechanism
regulating expression of the hypoxia inducible nNOS variant.[27] Another gene with
extensive variation in the 5’-UTR that influences its translational efficiency is Dicer.[53]
Enhanced protein translation during hypoxia could also occur for transcripts containing
Chapter 1
- 11 -
internal ribosomal entry sites (IRES) within the untranslated regions of the mRNA.[28]
IRES increases translation by facilitating direct ribosome binding independent of
formation of eIF4F at the cap, which is inhibited during hypoxia. Translation of the
angiogenic factor Tie2 transcript is enhanced through its IRES activity at the 5’-UTR
during hypoxia in human umbilical vein endothelial cells (HUVECs).[54] MicroRNAs
(miRNA) are small noncoding RNAs that regulate gene expression post transcriptionally
by preventing initiation or elongation.[55] Selective relief from micro-RNA mediated
suppression could be another mechanism that selectively enhances translation during
hypoxia. For example, the cationic amino acid transporter 1 translation under stress is
enhanced due to relief of micro-RNA mediated suppression by miR-122 in human
hepatic cell lines.[56] In the current study, the effect of hypoxia on HO-2 translation in
human endothelial cells was investigated.
1.4 Physiological responses to hypoxia
Oxygen supply to essential organs during acute systemic hypoxia is initially
maintained through three mechanisms. First, ventilation is increased through increases
in respiratory rate and tidal volume.[57] This response, termed the hypoxic ventilatory
response (HVR), in humans is almost solely due to depolarization of glomus cells in the
carotid body.[16] Second, alveolar oxygen uptake is enhanced by improved ventilation-
perfusion matching. This response is mediated by hypoxia-induced modulation of
pulmonary arterial and bronchial smooth muscle tone.[58] Pulmonary arterial
vasoconstriction redirects blood to better oxygenated regions of the lung while changes
in bronchial and bronchiolar tone optimize the distribution of gas flow within the lung.
Chapter 1
- 12 -
Third, oxygen delivery to essential organs is maintained by activation of the sympathetic
system and release of local vasodilators.
Activation of the sympathetic system increases oxygen extraction by increasing
the heart rate and diverting unnecessary blood flow away from organs such as the
kidneys and splanchnic viscera toward the heart and brain. Schlichtig et al.
demonstrated that redistribution of blood flow improves oxygen delivery during
progressive hemorrhage.[11] Cain demonstrated that vigourous vasoconstrictor
sympathetic tone during hypoxia increased survival time by promoting greater O2
extraction.[10] Vessels in essential organs accommodate the increased blood flow
through both a sympathetically-mediated increase in arteriolar tone and the release of
vasodilators in areas of imbalance between metabolic demand and oxygen supply.
Hypoxic vasodilation is particularly well manifested in coronary and cerebral vessels
where it ensures sufficient oxygen to support the activity of the working myocardium
and the neuronal activity of the brain.[3]
Conduit vessels are also subject to dual regulatory mechanisms. Sympathetic
responses regulate blood flow among organs while local paracrine responses modulate
tone in response to regional environmental conditions.[59] Contractile response to
adrenoceptor stimulation is impaired in conduit and resistance vessels from animals
exposed to prolonged systemic hypoxia in vivo.[60-64] Hu et al. demonstrated that
chronic hypoxia attenuates coupling efficiency of α1-adrenoceptors to inositol 1,4,5-
trisphosphate synthesis in the uterine artery.[65] Others have shown impaired smooth
muscle activation in aorta of rats after prolonged exposure to hypoxia.[62, 63] These
Chapter 1
- 13 -
results suggest that as the duration of systemic hypoxia is prolonged, as occurs in
patients suffering from cardiovascular and pulmonary diseases, shock, or sepsis, the
sympathetically mediated responses to hypoxia are impaired and lead to reduced oxygen
extraction. In this setting, the endothelium plays an increasingly important role in
preserving the capacity to regulate the systemic circulation by releasing vasoconstrictor
substances which maintain vasoreactivity.[61, 64] In the current work, the role of
matrix metalloproteinase-2 in preserving vascular reactivity and the role of heme
oxygenase-2 in maintaining endothelial viability during prolonged exposure to hypoxia
are, therefore, investigated.
Chapter 1
- 14 -
1.5 The endothelium
The endothelium consists of a single layer of cells lining the luminal side of all
blood vessels. Under physiological conditions, the endothelium works in concert with
other cells in the vessel wall to maintain vascular homeostasis by 1) maintaining a non-
thrombogenic blood-tissue interface; (2) regulating leukocyte adhesion/migration; 3)
participating in the regulation of vascular tone; and 4) producing cytokines and other
paracrine signalling molecules in response to external stimuli.[66]
In addition to its homeostatic functions, the endothelium constitutes an active
gate for the passage of oxygen into the interstitium.[67, 68] The endothelium consumes
oxygen at a rate one to two orders of magnitudes higher than is observed in most tissues
(about 0.1 ml O2/min/cm3 for arterioles) and alterations in its metabolic activity can
affect the gradient in oxygen concentration that determines tissue oxygen delivery.[7,
68] As a result, induction of vasoconstriction lowers tissue pO2 and increases vessel
wall oxygen consumption, whereas vasodilation is associated with a decrease in the
arteriolar vessel wall oxygen concentration gradient with a concomitant increase in
tissue pO2. [7] This effect compounds the detrimental effect of excessive
vasoconstriction on tissue perfusion and contributes to the impairment of viability and
function during hypoxia by further reducing tissue oxygen delivery.
Because of their location, endothelial cells are directly and frequently subjected
to changes in oxygen tension. In comparison to other cell types, endothelial cells are
relatively resistant to hypoxia-induced cell death.[69, 70] However, hypoxia triggers
profound changes in endothelial phenotype leading to disruption of the homeostatic
balance in coagulation, vascular permeability and vascular tone. These changes, if
Chapter 1
- 15 -
uncontrolled, result in failure of microcirculatory regulation of oxygen extraction, local
inflammation, thrombosis and eventually organ failure and death. Endothelial injury and
dysfunction occur in many disease processes, including diabetes, atherosclerosis,
systemic and pulmonary hypertension, and inflammatory syndromes.[71]
Regulation of blood flow is one of the essential functions of the endothelium,
especially during hypoxia. Under normal physiological conditions, the vasoconstricting
and vasodilating substances produced by endothelium are in dynamic balance and
influence each other through multiple feedback mechanisms.[72] Vasodilating
substances released by the endothelium include CO, NO, prostacylin, and endothelium-
derived hyperpolarizing factor. Vasoconstricting factors released by the endothelium
include prostagladins (Thromboxane A2 and Prostaglandin F2α) and endothelins. Upon
exposure to hypoxia, the endothelium initiates a rapid but transient vasoconstriction
followed by relaxation. As the duration of hypoxic exposure is prolonged, smooth
muscle contractility is reduced and endothelial function is dramatically altered in both
conduit and resistance vessels.[60-62, 64, 73-75] Specifically, the inhibitory effect of the
endothelium on basal myogenic tone is lost and endothelium-dependent relaxation is
impaired. Furthermore, the contractile responses to α-agonist, KCl, and increased
transmural pressure are enhanced in endothelium-intact compared to denuded vessels,
indicating an excess endothelial vasoconstrictor over vasodilator release. Superoxide
ion, endothelin-1 and vasoconstrictor prostanoids are some of the factors implicated in
endothelium-dependent vasoconstriction during hypoxia.[69]
Chapter 1
- 16 -
Changes in the endothelial function during hypoxia have both adaptive and
pathophysiological consequences. On one hand, since systemic vasoreactivity to
adrenoceptor stimulation is impaired following hypoxia,[60, 64] and since the reflexes
that redistribute blood flow and preserve vital organ oxygen supply are sympathetically
mediated,[76-78] they represent an important compensatory response that preserves the
capacity to regulate the circulation in the early phases of hypoxic stress. On the other
hand, impairment of the endothelium dependent mechanisms that optimize tissue
perfusion and adjust arteriolar tone in response to hypoxia[79-81] will compromise the
capacity to match blood flow to metabolic demand in vital vascular beds. Hence,
understanding pathways that regulate vascular reactivity during prolonged hypoxia is
important in understanding the pathophysiology of diseases associated with tissue
hypoxia and the development of effective treatment strategies. In the studies described
in this thesis, the role of two important pathways of endothelium-dependent regulation
of vascular function, matrix metalloproteinase-2 and heme oxygenase-2, are
investigated.
Chapter 1
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1.6 Matrix metalloproteinase-2
Matrix Metalloproeinase-2 (MMP-2) is produced constitutively by a wide range
of cell types, including endothelial cells and vascular smooth muscle cells.[82] Its
expression and activity increases during hypoxia.[83] MMP-2 metabolizes a wide
variety of matrix proteins, including gelatin, type I, IV and V collagens, elastin, and
vitronectin.[84] Based on its ability to degrade extracellular matrix proteins, MMP-2 is
thought to play a major role in vascular remodelling and angiogenesis during
hypoxia.[84-91] A broader biological role for MMP-2 has become apparent as novel
MMP-2 substrates are identified.[84, 92] For example, MMP-2 catalyzed degradation of
contractile proteins contributes to contractile dysfunction after ischemia/reperfusion
injury and dampens inflammatory responses due to the effects of its activity on
monocyte chemotactic protein-3.[83, 93-95] MMP-2 also regulates vascular tone
through modification of the activity of vasoactive molecules. MMP-2 catalyzed
inactivation of the potent vasodilatory neuropeptide calcitonin gene-related peptide
(CGRP) and activation of big endothelin-1 (ET-1) may both contribute to altered
vasoreactivity during hypoxic exposure [96, 97]. Therefore, hypoxic regulation of
vascular MMP-2 expression and activity and the role it plays in altered contractile
responses observed in systemic conduit and resistance arteries after prolonged hypoxia
in vivo was investigated.
1.6.1 Matrix Metalloproteinase-2 protein structure
Matrix metalloproteinases are a family of 28 related zinc-containing
endopeptidases that share structural similarities, but differ from each other in their
Chapter 1
- 18 -
expression profiles and substrates.[82] MMPs are classified on the basis of substrate
specificity, sequence similarity , and domain organization into collagenases,
stromelysins, gelatinases, and membrane type metalloproteinases.[82] The two
gelatinases in the MMP family are MMP-2 and MMP-9. The activated MMP-2 (64
kDa) protein contains a hemopexin/vitronectin-like domain that is connected to the
catalytic domain by a hinge region (Figure 1.5). The hemopexin domain contains four
repeats with the first and last repeat linked by a disulfide bond. It influences substrate
specificity and binding of tissue inhibitors of matrix metalloproteinase (TIMP) to MMP-
2.[82] The catalytic domain contains three head-to-tail cysteine-rich repeats resembling
the collagen-binding type II repeats of fibronectin. The cysteine-rich repeats are
required for MMP-2 to bind and cleave collagen and elastin.[82]
1.6.2 Regulation of matrix metalloproteinase-2 activity
Like all MMPs, MMP-2 is secreted as ab inactive proenzyme.[86] Although
MMP-2 is constitutively released, its activity is tightly controlled posttranslationally
through a unique mechanism of proenzyme activation and interaction with its
endogenous inhibitors.[98] Unlike other MMPs, MMP-2 is refractory to activation by
Figure 1.5 Domain structure of matrix metalloproteinase-2. Source: Schulz R. (2007) Intracellular targets of matrix metalloproteinase-2 in cardiac disease: rationale and therapeutic approaches
Chapter 1
- 19 -
serine proteinases and is instead, activated at the cell surface by membrane type-matrix
metalloproteinases (MT-MMP).[82] The activity of activated MMP-2 is inhibited by
TIMPs and the broad spectrum proteinase inhibitor α2-macroglobulin. Therefore, the
overall activity of MMP-2 depends not only on its abundance but also on the relative
bioactivity of its activators and inhibitors.
MMP-2 is produced and secreted as a 72 kDa proenzyme called proMMP-2.[85]
Activation of proMMP-2 occurs through a multistep pathway involving MT1-MMP and
TIMP-2.[98, 99] Initially, a cell surface MT1-MMP binds to the N-terminal domain of
TIMP-2. The C-terminal domain of the bound TIMP-2 then acts as a receptor for the
hemopexin domain of proMMP-2. Once proMMP-2 is tethered to TIMP-2, an adjacent
MT1-MMP that is free of TIMP-2 cleaves portions of the MMP-2 prodomain.
Following the initial cleavage by MT1-MMP, the residual portion of the MMP-2
propeptide is removed by another MMP-2 molecule to yield the fully active form of
MMP-2 that is 64 kDa in size.[98, 99]
In addition to proteolytic activation, reactive oxygen and nitrogen species are
known to activate MMP-2 without proteolytic removal of the autoinhibitory propeptide
domain.[84] The cysteine residues of the propeptide domain are highly sensitive to
changes in the redox environment and may exist in one of several oxidation states
depending on the type and level of oxidative challenge. Human recombinant 72 kDa
MMP-2 has been show to be activated by very low concentrations of peroxynitrite and
this occurred without evidence for the formation of the lower-molecular-weight 64 kDa
enzyme.[84]
Chapter 1
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1.7 Endothelin
The endothelins are a family of three small peptides, endothelin-1 (ET-1),
endothelin-2, and endothelin-3, that are central to regulating vascular function.[100]
ET-1, ET-2, and ET-3 are encoded by distinct genes located on chromosomes 6, 1, and
20, respectively which encode their respective propeptides (big ET-1, -2 and -3) which
require proteolytic activation to generate the vasoactive peptides.[101] ET-1 is
recognized as the major isoform of relevance in human cardiovascular physiology and
pathophysiology and regulation of vascular tone is one of the major functions of ET-
1.[100, 102-105] Altered expression/activity of ET-1 is involved in the development of
diseases such as hypertension, atherosclerosis, and vasospasm.[100]
Previous results suggest that ET-1 plays a central role in the adaptation to
hypoxia. Hypoxia increases circulating ET-1 concentrations, as well as its production
by cultured endothelial cells and rat arteries in vitro.[106-110] ET-1 has been
demonstrated to potentiate vascular responses that enhance pulmonary gas exchange and
tissue oxygen extraction,[61, 111, 112] however, the predominant mechanism of big
ET-1 activation in the systemic vasculature during hypoxia is unknown. Accordingly,
the current study was undertaken to investigate the contribution of ECE and MMP-2 in
the activation of big ET-1 in systemic arteries of rats exposed to prolonged hypoxia.
ET-1 acts primarily as a local hormone in an autocrine and paracrine
fashion.[113] It has been shown to be secreted primarily into the basolateral
compartment and not into the apical compartment.[114] Thus, plasma concentrations of
ET-1 are typically very low, ranging between 1 and 5 pM and may not be representative
of locally released ET-1 at its site of action.[115, 116] The effects of endothelins are
Chapter 1
- 21 -
mediated primarily by two receptor subtypes ETA and ETB. In the systemic and
pulmonary vessels under physiological condidtions, endothelin type A receptor (ETAR)
are located primarily on vascular smooth muscle cells while endothelin type B receptor
(ETBR) are expressed on both vascular endothelial and smooth muscle cells. Both
receptors expressed on vascular smooth muscle cells mediate vasoconstriction while
endothelial ETBR activate the release of vasodilating factors, such as prostacyclin or
nitric oxide.[117] The net effect produced by ET-1, whether vasoconstriction or
vasodilation, is determined by the balance between ETAR and ETBR expression and
localisation.
1.7.1 Activation of big endothelin-1
Human endothelin-1 protein is encoded by the preproendothelin-1 mRNA.[118]
Removal of the signal peptide from preproET-1 in the rough endoplasmic reticulum
yields the ~200 amino acid proET-1 (Figure 1.6).[119] ProET-1 is, in turn, cleaved by a
furin-like convertase to release the precursor peptide big ET-1 which has minimal
biological activity and requires further proteolytic activation. The primary pathway for
big ET-1 activation is cleavage by the type II integral membrane zinc metalloproteinases
endothelin converting enzymes (ECE) 1 & 2.[120] These enzymes catalyse hydrolysis
of the Trp21-Val22/Ile22 bond of big ET-1 to release the 21 amino acid active peptide
termed ET-1[1-21]. Other enzymes also cleave big ET-1, including chymase, and MMP-
2. MMP-2 cleavage of big ET-1 at Gly32-Leu33 generates the isopeptide ET-1[1-32] with
enhanced potency compared to ET-1[1-21].[96, 121]
Chapter 1
- 22 -
1.7.2 Endothelin receptor signalling
Endothelin-1 exerts its effects by activating cell-surface ETAR or ETBR.[122]
ETAR and ETBR are members of the G-protein-coupled receptor superfamily containing
seven hydrophobic transmembrane domains, an intracytoplasmatic C terminus and an
extracellular N terminus. Binding of ET-1 to ETAR or ETBR stimulates phospholipase C
activity through a pertussis toxin-insensitive G protein that is coupled to the ET receptor
intracellular domain. Phospholipase C hydrolyzes phosphatidyl inositol bisphosphate
into diacylglycerol and inositol triphosphate, leading to increase intracellular Ca2+ levels
and signalling through the RAF/MEK/MAPK pathway to activate genes that promote
cell growth and mitogenesis.[123-127] Increases in intracellular Ca2+ leads to SMC
contraction and activation of eNOS in endothelial cells.
Figure 1.6 Generation of endothelin-1.
preproET-1 proET-1furin like endopeptidase
Chapter 1
- 23 -
1.8 Heme Oxygenase
Given the multifaceted actions of ET-1 and consequences of excessive ET-1
production, numerous mechanisms have evolved to modulate the local bioavailability
and potency of ET-1. Another enzyme in the vasculature that modulates ET-1
bioavailability and potency is heme oxygenase.[74, 128-130] Heme oxygenases (HO)
are the only enzymes that catalyze the oxidative breakdown of heme into biliverdin IXα,
carbon monoxide (CO), and ferrous iron (Fe2+).[131, 132] Biliverdin IXα is
subsequently reduced to bilirubin IXα by biliverdin IXα reductase (Figure 1.7).[133]
Biliverdin/bilirubin possesses the capacity to suppress intracellular concentrations of the
reactive oxygen species (ROS) that regulate ET-1 precursor mRNA expression through
its antioxidant properties.[134] CO mimics many NO functions including cGMP-
dependent and –independent inhibition of agonist-induced vascular smooth muscle
contraction.[135] HO activity decreases ET-1-mediated potentiation of contraction to α-
adrenoceptor stimulation in aorta of rats exposed to hypoxia.[74] Taken together, these
findings suggest that HO-2 is an important enzyme in regulating vascular function in
systemic vessels during hypoxia.
The HO enzymatic activity requires three moles of molecular oxygen per heme
molecule oxidized, and the reducing equivalents from NAD(P)H.[136] Bilirubin
produced in the process is excreted from cells and passes through the blood in
association with serum proteins such as albumin to the liver where it is excreted in bile.
CO binds to haemoglobin to form carboxyhemoglobin, which is transported to the lung
Chapter 1
- 24 -
and is excreted in exhaled air. Iron is mainly transported to the bone marrow where it is
reused for heme biosynthesis and erythropoiesis.
1.7.1 Functions of heme oxygenase-2
By virtue of its catalytic action, heme oxygenase 2 regulates intracellular
concentrations of heme, CO, biliverdin/bilirubin, and ferrous iron, each of which has
important cellular functions.[135] Investigations into the function of heme oxygenases
have revealed that heme oxygenase activity possesses antioxidant, vasodilatory, and
antiapoptotic properties. These findings suggest that HO activity may ameliorate the
deleterious effects of hypoxia on endothelial function. Therefore, the role of HO-2 in
preserving endothelial cell viability during prolonged hypoxic exposure was investigated
during my studies.
The antioxidant function of HO-2 is supported by the findings that HO-2-/- mice
are more susceptible to hyperoxic lung damage, streptozotocin-induced renal
dysfunction, and intracerebral hemorrhage than wild-type mice.[137-139] Furthermore,
Figure 1.7 Metabolism of heme by heme oxygenase. Source: Ryter et al. (2005) Heme Oxygenase-1/Carbon Monoxide: From Basic Science to Therapeutic Applications.
Chapter 1
- 25 -
overexpression of the catalytically inactive HO-2 protein protects against H2O2-induced
oxidative stress.[140] Heme oxygenase exerts its antioxidative effects by reducing
levels of the oxidative heme molecules and by increasing antioxidant bilirubin and
ferritin levels. Unsequestered or free heme is a potent oxidant.[141, 142] Together with
iron, heme catalyzes free radical reactions to create ROS that damage DNA and proteins.
Heme oxygenases are the only known enzymes that can degrade heme and thus play a
critical role in heme homeostasis. In addition to metabolising heme, HO-2 can also
reduce intracellular heme levels by sequestering heme at its heme binding motifs, which
is not present in HO-1.[143] Bilirubin exerts its antioxidant effect through a repeated
process by which it is oxidized to biliverdin and then recycled back to bilirubin by
biliverdin reductase.[144] At micromolar concentrations in cell culture media, bilirubin
protects against cytotoxicity induced by H2O2 and/or enzymatically generated ROS in
endothelial and vascular smooth muscle cells.[145, 146] The antioxidant protection of
HO with respect to ferrous iron is facilitated by ferritin. The enhancement of ferritin
synthesis induced by ferrous iron increases the iron storage capacity of the cell as well as
protecting cells from oxidative stress.[147, 148]
The vasodilatory role of HO-2 has been demonstrated in numerous studies.
Govindaraju et al. has shown that HO-2 contributes to impaired contractile responses in
systemic arteries of rats exposed to hypoxia.[74] HO-2 has also been shown to regulate
blood flow in the uteroplancental vascular system.[149, 150] The vasodilatory effects of
heme oxygenase-2 are mainly mediated through the effects of CO, although bilirubin
could play an indirect role.[151-154] CO is a stable non-radical that binds the heme iron
moiety in a number of hemoproteins and metalloenzymes. It exerts its vasodilatory
Chapter 1
- 26 -
properties through several mechanisms. Binding of CO to the heme iron of soluble
guanlylate cyclase stimulates its enzymatic activity and increases cGMP
production.[153] Other mechanisms of CO-mediated vasodilation include activation of
calcium-dependent potassium channels and/or blocking expression of endothelial-
derived vasoconstrictors, such as ET-1.[128, 129, 155, 156] Bilirubin may regulate
vascular tone by suppressing intracellular concentrations of the ROS that serve as
second messengers in signalling the response to a number of contractile agonists,
including ET-1. During hypoxia, increases in ET-1 and HO-2 immunoreactivity are
localized to the endothelium [74] and HO inhibition increases the contractile response to
phenylephrine and ET-1 in endothelium-intact, but not –denuded aortic rings from
hypoxia-exposed rats.[74]
An anti-apoptotic role for HO-2 was demonstrated in studies in which inhibition
of HO-2 protein levels was shown to increase apoptosis induced by TNF-α, glutamate,
or hydrogen peroxide.[140, 157-159] The anti-apoptotic properties of CO are mediated
mainly through activation of mitogen-activated protein kinases (MAPK).[160, 161]
MAPK are a family of Ser/Thr protein kinases that are activated in response to a variety
of stimuli.[162] The three major MAPK signalling pathways identified in mammalian
cells include extracellular signal-regulated protein kinase, p38 MAPK, and c-Jun NH2-
terminal protein kinase.[162] The inhibitory effect of CO on TNF-α induced apoptosis
was abolished with a p38 MAPK dominant negative mutant in endothelial cells,
implying a critical role for the p38 MAPK pathway.[163] In addition to directly
Chapter 1
- 27 -
activating anti-apoptotic pathways, HO activity could be cytoprotective through its anti-
oxidant and anti-inflammatory effects.
1.7.2 Structure and expression heme oxygenase-2
Heme oxygenases consist of two structurally related isozymes representing
products of distinct genes. The human HO-1 and HO-2 genes are localized to
chromosome 22q12 and 16q13.3, respectively (Figure 1.8).[164] Rat HO-1 and HO-2
gene share similar organization into five exons and four introns, but only share 43% in
amino acid homology.[165, 166] A highly conserved sequence of 24 amino acid
residues has been identified in common to both HO-1 and HO-2.[167] Both HO-1 and
HO-2 also share similar hydrophobic regions at the extreme COOH terminus that serve
to anchor the protein in cellular membranes. Although HO-1 lacks Cys residues, HO-2
contain three Cys-Pro sequences in regions that have been proposed to contain heme
regulatory (or responsive) motifs (HRM) centered at Cys127, Cys265, and Cys282.[143]
Interactions between heme and the HRM have been proposed to control the activity or
stability of several regulatory proteins, including the transcriptional repressor Bach1 and
eukaryotic initiation factor-2α kinase. Therefore, these motifs could regulate HO-2
activity and provide additional heme binding sites that function to maintain intracellular
free heme level and act as a sink for CO.
Chapter 1
- 28 -
HO-1 is the inducible form of heme oxygenase with a high level of expression in
the spleen and other tissues that degrade senescent red blood cells, including specialized
reticuloendothelial cells of the liver and bone marrow.[135] In most other tissue not
directly involved in erythrocyte or haemoglobin metabolism, HO-1 typically occurs at
low to undetectable levels under basal conditions but responds to rapid transcriptional
activation by diverse chemical and phycial stimuli. HO-2 is constitutively expressed.
HO-2 is expressed in abundance in the testes, but the protein is also found ubiquitously
in other tissues including central nervous system, vasculature, and the gut.[2] Both HO-
1 and HO-2 catalyze the same biochemical reaction with similar substrate specificity and
co-factor requirements. However, in a comparative analysis of rat HO-1 and HO-2,
differential properties with respect to enzyme kinetics and substrate Km values have
been reported, as well as differences in thermostability.[168, 169]
Figure 1.8 Structural organization of the human HO-1 (top) and HO-2 (bottom) gene. Source: Shibahara et al. (2007) Hypoxia and heme oxygenases: oxygen sensing and regulation of expression.
Chapter 1
- 29 -
1.7.3 Regulation of heme oxygenase protein expression
Protein expression of HO-1 and HO-2 is differentially regulated. Human HO-1
is mainly regulated at the level of transcription. Its promoter contains one copy of the
functional Maf recognition element (MARE) immediately downstream from the
cadmium-responsive element (Figure 1.8).[170] HO-1 expression is regulated by
members of the small Maf family, which are basic region leucine zipper proteins that
can function as transcriptional activators or repressors. NF-E2-related factor-2 functions
as a transcriptional activator of HO-1 by forming a heterodimer with a member of the
Maf family, whereas Bach1 heterodimerizes with MafK to inhibit HO-1
expression.[171, 172]
Analysis of the genomic sequences 5’ to the rat and human HO-2 genes reveals
no regulatory elements corresponding to transcription factors known to participate in the
hypoxic or other cellular stress responses (Figure 1.8).[165, 173-175] The two
noticeable features that exist in the organization of the human HO-2 gene are the
presence of a potential bidirectional promoter and a large intron 1 of ~30 kb.[176] The
HO-2 gene and the gene encoding HSCARG of unknown function appear to share a
common promoter region. Similar to other bidirectional promoters, the HO-2 gene
promoter lacks the TATA box and contains GC-rich sequences.[177] The exon 1 of the
HO-2 gene encodes the 5’untranslated region of the HO-2 mRNA. Diversity of HO-2 5’
UTR sequences has been documented previously in the rodent, suggesting the use of
alternate transcription initiation sites (and promoters).[174, 178]
Despite the lack of evidence for transcriptional control, HO-2 protein expression
is not entirely constitutive. Development stage-specific changes in HO-2 protein level
Chapter 1
- 30 -
have been reported and cigarette smoke increases the number of lung cells expressing
HO-2 protein.[179-181] HO-2 protein increases 1.3 fold in the heart of mice exposed to
hypoxia for 28 days compared with age-matched normoxic controls.[182] Spatial and
temporal dissociation between HO-2 protein and mRNA expression have been noted in
rodent brain and testis and changes in HO-2 protein levels have been detected without
changes in HO-2 mRNA during hypoxia.[165, 173-175] These findings strongly
suggest that HO-2 protein expression is regulated mainly at the posttranscriptional level.
Therefore, posttranscriptional regulation of HO-2 protein expression during prolonged
hypoxic exposure was investigated in the studies described in the current work.
Chapter 1
- 31 -
1.8 Thesis Objectives
Aim 1:
• To determine the role that altered matrix metalloproteinase-2 expression and
activity play in the changes in contractile responses observed in systemic conduit
and resistance arteries after prolonged hypoxia in vivo
Hypothesis 1:
• Increased matrix metalloproteinase-2 activity potentiates contraction in systemic
conduit and resistance arteries after hypoxia in vivo by proteolytic activation of
big endothelin-1
Rationale:
• Endothelin-1 protein is increased in aorta of rats exposed to hypoxia without a
concomitant increase in preproET-1 mRNA expression.
• Increases in rat aortic ECE-1 expression, the activator of endothelin-1, could not
be detected
• Matrix metalloproteinase-2 and its activator, MT1-MMP have been shown to be
increased during hypoxic exposure.
Chapter 1
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Aim 2:
• To determine if hypoxia alters HO-2 expression through effects on protein
translation and whether HO-2 preserves endothelial cell viability during
concurrent hypoxic and oxidative stress in human endothelial cells.
Hypothesis 2:
• Heme oxygenase-2 translation is enhanced during hypoxia in human endothelial
cells.
Rationale:
• Enhanced HO-2 protein expression has been detected in rat aortic endothelium
without a concomitant increase in HO-2 mRNA expression.
• Discordance between HO-2 protein level and mRNA expression has previously
been identified.
Hypothesis 3:
• Endothelial HO-2 expression during hypoxia preserves endothelial viability.
Rationale
• Products of the HO-catalyzed reaction have properties that favours enhanced
endothelial cell survival.
• HO-2 is the predominate HO isoform expressed in the endothelium.
• HO-1 protein and mRNA expression are decreased by hypoxia in endothelial
cells.
Chapter 2
- 33 -
CHAPTER 2
Induction of Matrix Metalloproteinase-2 Enhances Systemic Arterial
Contraction After Hypoxia
Chapter 2
- 34 -
2.1 Introduction
Hypoxia is frequently observed in patients with cardiopulmonary diseases and in
normal subjects at high altitude. Studies done to date have primarily focused on the
short term systemic circulatory responses which redistribute blood flow [183] and
enhance the capacity for oxygen extraction [184] in these conditions. As the duration of
hypoxia increases, however, systemic vascular smooth muscle and endothelial function
are impaired, limiting the efficacy of the acute responses [61, 62, 64, 185] while
concurrent structural remodelling plays an increasing role in maintaining the balance
between oxygen delivery and metabolic demand [183]. Although the clinical and
physiological relevance of these longer term effects on vascular function are being
increasingly recognized [186], the mechanisms that mediate them remain unknown.
Matrix metalloproteinase-2 (MMP-2) is a zinc-dependent proteinase secreted by
both endothelial and smooth muscle cells, and its expression is increased in regions of
matrix turnover and remodelling [187]. Recently, it was discovered that MMP-2 can
mediate the posttranslational modification of several vasoactive peptides [96, 97, 188],
suggesting that it has a vasoregulatory role as well. MMP-2 protein and mRNA levels
are increased after hypoxic incubation in endothelial cells [83]. Its activating protease,
membrane type 1-matrix metalloproteinase (MT1-MMP), and its endogenous inhibitors,
tissue inhibitors of matrix metalloproteinase (TIMPs), are also oxygen-regulated in some
cell types [189-191]. If hypoxia induces a functionally significant increase in MMP-2
expression in systemic arteries, modulation of MMP-2 activity may, in addition to
mediating structural adaptations, contribute to the changes in vascular tone that occur
during prolonged reductions in oxygen delivery. This study was, therefore, carried out
Chapter 2
- 35 -
to determine the role that altered MMP-2 expression and activity play in the changes in
contractile responses observed in systemic conduit and resistance arteries after
prolonged hypoxia in vivo.
Chapter 2
- 36 -
2.2 Materials and Methods
Exposure to Hypoxia: All protocols were in compliance with standards set by the
Canadian Council on Animal Care and were approved by the institutional animal care
committee. Male Sprague-Dawley rats (200–250 g) and C56/B16J mice (20-25 g) were
placed in a Plexiglas chamber into which the flow of air and nitrogen was controlled
independently. In preliminary experiments, the arterial PO2 averaged 38 Torr (range
35–42 Torr) in rats breathing a gas mixture containing 10% O2 [64] and 38.1 Torr
(range 35-40 Torr) in mice breathing 8% O2,
Rats and mice exposed to hypoxia breathed gas mixtures containing 10% or 8%
oxygen, respectively for 16 h, 48 h, or 7 d. Normoxic control animals breathed room air
under otherwise identical conditions. At the end of the exposure period, rats were
decapitated and mice scarified by cervical dislocation. Thoracic aortae were excised, cut
into 4 mm segments and mounted in tissue bath myographs (Radnoti Glass Technology
Inc.), frozen in liquid nitrogen, or fixed in 10% paraformaldehyde. Rat mesenteric
arteries (100-200 µm internal diameter) were either mounted in wire myographs (Living
Systems) or frozen in liquid nitrogen for later protein extraction.
Chemicals/Antibodies: The cyclic peptide MMP-2/9 antagonist, CTTHWGFTLC
(CTT) was purchased from Calbiochem. Polyclonal MMP-2- and TIMP-1 to 3-specific
antibodies and monoclonal MT1-MMP-specific antibody were purchased from
Chemicon. Polyclonal TIMP-4-specific antibody was obtained from Biomol Research
Laboratories. Mca-RPPGFSAFK(Dnp)-OH and Mca-PLGL-Dpa-AR-NH were from R
& D systems. ECE-1-specific polyclonal antibody was from Zymed Laboratories.
Histostain and PicoPureTM RNA Isolation Kit were purchased from Arcturus Bioscience
Chapter 2
- 37 -
Inc. SYBR green universal master mix was from Applied Biosystems. Primers and
Superscript II were from Invitrogen. All other reagents were from Sigma.
Rat Studies
Aortic and Mesenteric Artery Contractile Responses: Rat aortic segments were
mounted in tissue bath myographs and mesenteric arterial segments in wire myographs
containing Krebs-Henseleit solution (KHS) composed of (in mmol/l) 120 NaCl, 25
NaHCO3, 11.1 glucose, 4.76 KCl, 1.18 MgSO4, 1.18 KH2PO4, 2.5 CaCl2 aerated with
95% O2-5% CO2 at 37°C. Thoracic aortae and mesenteric arteries were equilibrated in
warmed aerated KHS for 1 hour under a resting tension of 2 g or 500 mg, respectively,
before drug-induced changes in tension were monitored. When necessary, the
endothelium was removed by gentle abrasion of the luminal surface. The failure of
acetylcholine (1 µmol/l) to elicit relaxation after contraction with phenylephrine (1
µmol/l) was taken as evidence of functional endothelial ablation [185].
Redistribution of blood flow during hypoxia is mediated by neurohumoral
stimulation of α-adrenoceptors [76]. Cumulative concentration-response curves (CRCs)
for the α1-adrenoceptor agonist phenylephrine (PE, 1 nmol/l to 10 µmol/l) were,
therefore, generated in endothelium-intact aortic rings. MMP-2 cleaves big ET-1 to
release the potent vasoconstrictor ET-1[1-32] [96]. Since big ET-1 itself has minimal
biological activity prior to proteolytic activation and since ECE-1 protein levels and
ECE activity are unchanged after hypoxia (see below), the contractile response to big
ET-1 was used as a bioassay for changes in vascular MMP-2 activity. To eliminate the
confounding influence of endogenous endothelium-derived ET-1 [96], CRCs for rat big
Chapter 2
- 38 -
ET-1 (1 nmol/l to 300 nmol/l) were generated in endothelium-denuded aortic rings from
normoxic rats and rats exposed to hypoxia for 7 d after 45 min incubation with, and in
the continuous presence of CTT (30 µmol/l) or vehicle.
To assess the effect of hypoxia on contractile response of resistance vessels,
CRCs for PE (10 nmol/l to 100 µmol/l) were generated in mesenteric arteries from rats
exposed to normoxia or hypoxia for 7 d after 30 min incubation with and in the
continuous presence of CTT (10 µmol/l) or vehicle.
Immunohistochemistry: Paraffin embedded sections (5 µm) of rat aortae from
normoxic rats and rats exposed to hypoxia for 7 d were analyzed using MMP-2 specific
polyclonal antibodies as described [185]. Slides processed in an identical manner,
except incubated with non-specific rabbit IgG instead of primary antibody, served as
negative controls.
Western Blots: Thoracic aortic proteins from rats exposed to normoxia or to
hypoxia for 16 h, 48 h, and 7 d, and mesenteric arteries proteins from rats exposed to
normoxia or to hypoxia for 7 d were extracted in 1% SDS, 0.001 mol/l sodium
orthovanadate, and 0.01 mol/l Tris (pH 7.4). After protein concentrations in aortic and
mesenteric arterial extracts were determined by the Lowry method, total proteins (60 µg
for MMP-2 and MT1-MMP, 40µg for TIMPs 1-4, 100 µg for ECE-1) were resolved by
4-12% SDS-PAGE (Helixx Technologies Inc) and transferred to nitrocellulose. MT1-
MMP membranes were blocked in 3% milk-0.1% tween tris-buffered saline (TTBS).
All other membranes were blocked in 5% milk-TTBS. Membranes were then incubated
for 3 h at room temperature with goat polyclonal anti-MMP-2 (1:100), 1 hour at room
Chapter 2
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temperature with rabbit polyclonal anti-TIMP-1 (1:2500) or anti-TIMP-4 (1:10000), or
overnight at 4°C with rabbit polyclonal anti-ECE-1 (1:400), anti-TIMP-2 (1:2500), anti-
TIMP-3 (1:2500), or mouse monoclonal anti-MT1-MMP (1:400). Immunoblots were
probed with horseradish peroxidase (HRP)-donkey anti-goat IgG (1:4000 for MMP-2) or
HRP-anti-rabbit IgG (1:4000 for ECE-1, TIMP-1, TIMP-2, TIMP-3, TIMP-4) and
visualized by enhanced chemiluminescence (Amersham Biosciences). HRP-goat anti-
mouse IgG (1:1000) was used to probe for MT1-MMP and the resulting bands were
visualized by chemiluminescence (Sigma). Bands were quantified by densitometry.
Samples from normoxic and hypoxic groups were paired on each gel to control for inter-
experimental variation. Protein loading and transfer efficiency were corroborated
following transfer, using full-lane densitometry of the Ponceau red-stained membranes.
Gelatin Zymography: Aortae from normoxic rats and rats exposed to hypoxia for
16 h, 48 h, and 7 d were extracted with 10 mmol/l Tris-HCl (pH 7.5) extraction buffer.
Zymography was performed using 7.5% SDS-PAGE with co-polymerized gelatin (2
mg/ml) as substrate. At the end of each run, gels were washed with 2.5% Triton X-100
and incubated for 48 h in an enzyme assay buffer (50 mmol/l Tris, pH 7.0, 5 mmol/l
CaCl2, 0.15 mol/l NaCl, 0.05% Na3N) to allow for the development of enzyme activity
bands. Gels were stained with 0.05% Coomassie brilliant blue G-250 in a mixture of
methanol: acetic acid: water (2.5:1:6.5) and de-stained in 4% methanol with 8% acetic
acid. The gelatinolytic activities were detected as transparent bands against the
background of Coomassie brilliant blue-stained gelatin. Gels were scanned using Fluor-
S Multi-Imager (Bio-Rad) and analyzed for pro- and activated MMP-2 (72 and 64 kDa
bands, respectively).
Chapter 2
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MMP and ECE Activity: To further ensure that the change in the response to big
ET-1 was not attributable to a change in endothelin converting enzyme (ECE) activity,
total MMP and ECE activities were measured in aortae from normoxic rats and rats
exposed to hypoxia for 7 d. Thoracic aortic proteins (50 µg) from normoxic rats and rats
exposed to hypoxia for 7 d were incubated for 1 hour at 37° C with either 20 µmol/l of
fluorogenic MMP substrate Mca-PLGL-Dpa-AR-NH [192] in 100 µl of reaction
mixture (pH 7.5) composed of (in mmol/l): 50 Tris-HCl, 150 NaCl, and 1 CaCl2 or with
20 µmol/l of fluorogenic ECE substrate Mca-RPPGFSAFK(Dnp)-OH [193] in 100 µl of
reaction mixture (pH 6.0) composed of (in mmol/l): 100 MES and 200 NaCl. Blanks
containing the substrate dissolved in assay buffer were analyzed in parallel. Increases in
fluorescence as a result of substrate cleavage were continuously measured using a
fluorescence plate reader (Thermo Labsystems). Samples were run in triplicate and final
values were derived by subtracting the blank reading from the raw data.
Aortic MMP-2, MT1-MMP, TIMPs 1 to 4 mRNA levels: Total aortic RNA was
isolated as previously described [185]. In addition, pure populations of aortic
endothelial cells were isolated from aortae of rats exposed to normoxia or hypoxia for 7
d using the Hautchen technique [194]. Pure cell populations of vascular smooth muscle
cells from immediately below the endothelial cell layer (subendothelial VSMC) or from
deep within the media of the vessel (deep medial VSMC, Figure 2.7) were obtained
using the PixCell IITM Laser Capture Microdissection System according to the
manufacturer’s instructions and RNA was extracted using the PicoPureTM RNA Isolation
Kit. Aortic levels of specific mRNAs were measured by quantitative real-time RT-PCR
Chapter 2
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(ABI PRISM 7900 HT, Applied Biosystems and the SYBR Green detection system
[185]) using the following primers: MMP-2 (sense 5’-ACA CTG GGA CCT GTC ACT
CC-3’, antisense 5’-ACA CGG CAT CAA TCT TTT CC-3’); MT1-MMP (sense 5’-
TCC TGC TCC CCC TGC TCA CG, antisense 5’-GTG ACT GGG GTG AGC GTT
GTG T-3’); TIMP-1 (sense 5’-GGA TAT GTC CAC AAG TCC CAG AAC C-3’,
antisense 5’-TTA TGC CAG GGA ACC AGG AAG C-3’); TIMP-2 (sense 5’-GGC
CAA AGC AGT GAG CGA GAA -3’, antisense 5’-GGA GGG GGC CGT GTA GAT
AAA T-3’); TIMP-3 (sense 5’-CCC TTT GGC ACT CTG GTC TAC ACT A-3’,
antisense 5’- AGG CCA CAG AGA CTT TCA GAG GCT-3’); and TIMP-4 (sense 5’-
TAC ACG CCA TTT GAC TCT TCT CTC TG-3’, antisense 5’-CCT CCC AGG GCT
CAA TGT AGT TG-3’). 18S (sense 5’-GAC GAT CAG ATA CCG TCG TAG TTC-
3’, antisense 5’-GTT TCA GCT TTG CAA CCA TAC TCC-3’) and TATA binding
protein (sense 5’-CCC CTA TCA CTC CTG CCA CAC C-3’, antisense 5’-CGC AGT
TGT TCG TGG CTC TCT T-3’) transcripts were used as control genes for
normalization and the average change in the target gene with respect to 18S and TATA
binding protein was determined.
Studies in MMP-2-/- and MMP-2+/+ mice
Animals: To corroborate the results of the pharmacological studies described
above, aortic contraction was assessed in mice deficient in MMP-2. The MMP-2+/- mice
on the C57/Bl6J background previously described [195] were interbred to generate
MMP-2 knockout (MMP-2-/-) and littermate control (MMP-2+/+) groups. Mouse
genotypes were assessed by polymerase chain reaction of genomic DNA. Primers for
wild-type alleles were located in exon-1 (5’-CAA CGA TGG AGG CAC GAG TG-3’
Chapter 2
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and 5’-GCC GGG GAA CTT GAT CAT GG-3’), and primers for the mutant allele were
located in the neo cassette (5’-CTT GGG TGG AGA GGC TAT TC-3’ and 5’-AGG
TGA GAT GAC AGG AGA TC-3’).
Aortic Contractile Responses: Mouse aortic segments were equilibrated in
warmed KHS aerated with 95% O2-5% CO2 at 37°C for 1 hour under a resting tension of
1g before drug-induced changes in tension were monitored. As in rat aortic segments the
endothelium was removed by lumenal abrasion and the success of endothelial ablation
assessed by acetylcholine-induced relaxation of phenylephrine-induced contraction
[185]. CRCs were generated for PE (1 nmol/l to 10 µmol/l) in endothelium-intact
thoracic aortic rings, and the contractile response to human big ET-1 (100 nmol/L) was
determined in endothelium-denuded aortic rings from MMP-2+/+ and MMP-2-/- mice
exposed to normoxia or hypoxia for 7d.
Data Analysis
Results are presented as mean ± S.E.M. for n number of animals with P<0.05
representing statistical significance. Paired means were compared by two-tailed
Student’s t-test. Differences among multiple means were evaluated by analysis of
variance (ANOVA) corrected for multiple measures where appropriate and, when
overall differences were detected, individual means were compared post-hoc using
Dunnet's test.
Chapter 2
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2.3 Results
Rat Studies
Aortic and Mesenteric Artery Contractile Responses: Figure 2.1A shows the
concentration-response relationship for PE in endothelium-intact aortic rings from
normoxic rats and rats exposed to hypoxia for 7 d in the presence or absence of CTT (30
µmol/l). Inhibition of MMP-2 decreased the maximum tension generated during PE-
induced contraction in aortic rings from rats exposed to hypoxia (Table 1), but had no
effect in rings from normoxic rats. Figure 2.1B illustrates the response of endothelium-
intact rat mesenteric artery segments to PE in the presence or absence of CTT. As in
aortic rings, CTT had no effect on the response of mesenteric arteries from normoxic
rats but reduced contraction in those from hypoxia-exposed animals (Table 1).
The contractile responses to big ET-1 in endothelium-denuded aortic rings from
rats exposed to normoxia or hypoxia for 7 d in the presence or absence of CTT (30
µmol/l) are illustrated in Figure 2.1C. Maximal contractions achieved in rings from
hypoxic animals were higher than those in the normoxic controls (Table 1). This
hypoxia-dependent augmentation of big ET-1 mediated contraction was abolished by
CTT (Table 1). At this concentration, CTT had no effect on ET-1-induced contraction
in aortic rings from either normoxic or hypoxia-exposed rats (data not shown),
indicating that its effect is mediated by inhibition of big ET-1 conversion.
Chapter 2
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Figure 2.1 (A) Concentration-response relationships for phenylephrine (PE) in the
presence and absence of the MMP-2/9 inhibitor CTTHWGFTLC (CTT, 30 µmol/l) in
endothelium-intact aortic rings from normoxic rats and rats exposed to hypoxia for 7 d
(n = 8 per group). (B) Concentration-response relationships for PE in the presence and
absence of CTT (10 µmol/l) in endothelium-intact mesenteric arteries from normoxic
rats and rats exposed to hypoxia for 7 d (n = 5-6 per group).
A Endothelium-Intact Rat Aorta
B Endothelium-Intact Rat Mesenteric Arteriole
Chapter 2
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Figure 2.1 (C) Concentration response relationships for big ET-1 in the presence and
absence of CTT (30 µmol/l) in endothelium-denuded aortic rings from normoxic rats and
rats exposed to hypoxia for 7 d (n = 10 per group). *P<0.05 for differences between
CTT-treated and -untreated group. †P<0.05 for difference from corresponding
normoxic control group.
C Endothelium-Denuded Rat Aorta
Chapter 2
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Table 1: Effect of MMP inhibition on maximum contraction and EC50 values
during PE and big ET-1 big ET-1-induced rat aortic contraction.
Values are means ± SEM; *P<0.05 for differences between CTT-treated and -untreated
group. †P<0.05 for difference from corresponding normoxic control group.
Maximum Tension (g/mg dry weight)
Rat Vessels Treatment Nor. Hyp. (7 d)
PE KHS 2.89 ± 0.10 3.41 ± 0.21† Aortae
CTT (30 µmol/L) 3.11 ± 0.17 2.73 ± 0.19*
KHS 678.15 ± 52.20 911.93 ± 76.51† Mesenteric Arteries CTT (30 µmol/L) 748.95 ± 47.11 715.32 ± 101.21*
Big ET-1 KHS 1.35 ± 0.08 1.77 ± 0.10† Aorta
CTT (30 µmol/L) 1.31 ± 0.18 1.28 ± 0.12*
-log EC50, mol/L Rat
Vessels Treatment Nor. Hyp. (7 d) PE
KHS 7.412 ± 0.05 7.63 ± 0.09 Aortae CTT (30 µmol/L) 7.32 ± 0.09 7.43 ± 0.10
KHS 5.81 ± 0.07 5.79 ± 0.09 Mesenteric
Arteries CTT (30 µmol/L) 5.73 ± 0.08 5.64 ± 0.02 Big ET-1
KHS 7.25 ± 0.05 7.25 ± 0.03 Aorta CTT (30 µmol/L) 7.21 ± 0.08 6.92 ± 0.17
Chapter 2
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Immunohistochemistry: Figure 2.2 depicts representative immunohistochemical
staining for MMP-2 in aortic sections from normoxic rats (Figure 2.2A) and rats
exposed to hypoxia for 7 d (Figure 2.2C), along with the respective negative controls
(Figures 2.2B and 2.2D). Although MMP-2 protein was detected in both the intima and
media of the thoracic aorta from normoxic and hypoxic animals, staining was more
intense in the hypoxic group with no apparent inhomogeneity in its distribution across
the aortic wall.
Figure 2.2 Immunohistochemistry for MMP-2 on aortic sections from normoxic rats
and rats exposed to hypoxia for 7 d. Immunoreactivity (brown diaminobenzidine
staining) is apparent in the aortic endothelium and media layer of both the normoxic (A)
and hypoxic (C) groups, but not in the normoxic (B) or hypoxic (D) negative controls
(40X objective).
Chapter 2
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MMP-2 and ECE Protein and Activity Levels: Western analysis shown in Figure
2.3A indicates that rat aortic proMMP-2 (72 kDa) protein levels increased after
prolonged hypoxia. These differences reached statistical significance after 48 h and 7 d
of hypoxia. Activated MMP-2 (64 kDa) protein levels were also found to be elevated
with increasing duration of hypoxia, achieving statistical significance after 7 d. Aortic
MMP-2 activity, as determined by gelatin zymography, was also significantly greater
after 7 d of hypoxia compared to the normoxic control group (Figure 2.3B). No bands
corresponding to the expected molecular weight of MMP-9 were detected in these
samples, suggesting that MMP-2 is the predominant source of gelatinase activity.
Figure 2.3C illustrates that proMMP-2 protein is also increased in mesenteric arteries
from rats exposed to hypoxia for 7 d compared to the normoxic animals. Protein
concentrations obtained from these small vessels was insufficient to quantify levels of
the cleaved (activated) form. Aortic ECE-1 protein levels did not differ between
normoxic and hypoxia-exposed rats (data not shown).
The results of fluorometric assays of total MMP and ECE activities are presented in
figure 2.4. MMP activity was higher in aortae from hypoxia-exposed rats compared to
the normoxic group (Figure 2.4A), whereas ECE activity was unchanged (Figure 2.4B).
Chapter 2
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Figure 2.3 (A) Aortic MMP-2 protein levels in normoxic rats and rats exposed to
hypoxia for 16 h, 48 h, and 7 d (n = 9 per group). (B) Gelatin zymography showing the
levels of activated MMP-2 in aortae of normoxic rats and from rats exposed to hypoxia
for 16 h, 48 h, and 7 d (n = 6 per group). *P<0.05 for differences from corresponding
normoxic group.
Chapter 2
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Figure 2.3 (C) MMP-2 protein levels in mesenteric arteries from normoxic rats and
rats exposed to hypoxia for 7 d (n = 8 per group). *P<0.05 for differences from
corresponding normoxic group.
Chapter 2
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Figure 2.4 MMP (A; n = 10) and ECE (B; n=9) activity in aortae from normoxic rats
and rats exposed to hypoxia for 7 d. *P<0.05 for differences from corresponding
normoxic group.
Chapter 2
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MT1-MMP and TIMPs Protein Levels: MMP-2 activity is regulated by its activator
protease MT1-MMP, and its tissue inhibitors TIMPs. Western analysis demonstrated
that rat aortic MT1-MMP protein levels increased progressively with increasing duration
of hypoxic exposure, reaching statistical significance after 7 d (Figure 2.5A). Although
aortic levels of TIMPs 1 to 4 exhibited an upward trend, these changes did not reach
statistical significance during the same period of hypoxic exposure (Figures 2.5B-2.5E).
Figure 2.5 Levels of MT1-MMP (A) proteins in aortae from normoxic rats and rats
exposed to hypoxia for 16 h, 48 h, and 7 d (n = 6 per group). *P<0.05 for differences
from corresponding normoxic group.
Chapter 2
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Figure 2.5 Levels of TIMP-1 (B) and 2 (C) proteins in aortae from normoxic rats
and rats exposed to hypoxia for 16 h, 48 h, and 7 d (n = 6 per group). *P<0.05 for
differences from corresponding normoxic group.
Chapter 2
- 54 -
Figure 2.5 Levels of TIMP-3 (D) and 4 (E) proteins in aortae from normoxic rats
and rats exposed to hypoxia for 16 h, 48 h, and 7 d (n = 6 per group). *P<0.05 for
differences from corresponding normoxic group.
Chapter 2
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MMP-2, MT1-MMP and TIMPs mRNA Levels: Figure 2.6 illustrates MMP-2, MT1-
MMP and TIMPs 1 to 4 mRNA levels in aortae from normoxic rats and rats exposed to
hypoxia for 16 h, 48 h, or 7 d. After exposure to hypoxia for 7 d, MMP-2 and MT1-
MMP mRNA levels are increased compared to the normoxic control group. An increase
in levels of TIMPs -1 to -3 mRNA was observed after 7 d of hypoxia while TIMP-4
mRNA expression was upregulated at the earlier time points (16 h and 48 h) as well.
Chapter 2
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Figure 2.6 Levels of MMP-2 (A) and MT1-MMP (B) mRNAs in aortae from
normoxic rats and rats exposed to hypoxia for 16 h, 48 h, and 7 d (n = 7 per group).
*P<0.05 for differences from corresponding normoxic group.
Chapter 2
- 57 -
Figure 2.6 Levels of TIMP-1 (C) and TIMP-2 (D) mRNAs in aortae from normoxic
rats and rats exposed to hypoxia for 16 h, 48 h, and 7 d (n = 7 per group). *P<0.05 for
differences from corresponding normoxic group.
Chapter 2
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Figure 2.6 Levels of TIMP-3 (E) and TIMP-4 (F) mRNAs in aortae from normoxic
rats and rats exposed to hypoxia for 16 h, 48 h, and 7 d (n = 7 per group). *P<0.05 for
differences from corresponding normoxic group.
Chapter 2
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MMP-2 and MT1-MMP mRNA Levels: To identify the cell type responsible for the
observed increase in MMP-2 and MT1-MMP expression, MMP-2 and MT1-MMP
mRNAs were quantified using RNA extracted from pure populations of aortic
endothelial cells, sub-endothelial vascular smooth muscle cells (VSMC) and deep
medial VSMCs (Figure 2.7A-C). MMP-2 (Figure 2.7D) and MT1-MMP mRNA levels
(Figure 2.7E) are increased in deep medial aortic VSMCs from rats exposed to hypoxia
for 7 d, whereas, no change was observed in endothelial or subintimal smooth muscle
cells.
Chapter 2
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Figure 2.7 (A-C) Illustration of aortic regions where cells were collected for mRNA
extraction.
Chapter 2
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Figure 2.7 MMP-2 (D) and MT1-MMP (E) mRNA levels in endothelial, sub-
endothelial VSMC, and deep medial VSMC from aortae of normoxic rats and from rats
exposed to hypoxia for 7 d (n = 6 per group). *P<0.05 for differences from
corresponding normoxic group.
Chapter 2
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Studies in MMP-2-/- and MMP-2+/+ Mice
Aortic Contractile Responses: Figure 2.8A presents the concentration-response
relationships for PE in endothelium-intact aortic rings from mice exposed to normoxia
or hypoxia for 7 d. In contrast to rats, hypoxia did not enhance the response to PE in
MMP-2+/+ mice possibly reflecting differences in the adaptive response to hypoxia
between the two species. Nevertheless, after hypoxic exposure, the effect of MMP-2
deletion in mice mimics the effect of MMP inhibition in rats in that, after hypoxia, the
maximum tensions generated during PE-induced contraction are reduced in MMP-2-/-
compared to their MMP-2+/+ littermate controls (Table 2). The responses of
endothelium-denuded aortic rings from MMP-2-/- and MMP-2+/+ mice to big ET-1 (100
nmol/l) are illustrated in figure 2.8B. Similar to the results obtained in rat aortae,
contraction to big ET-1 is greater in aortic segments from MMP-2+/+ mice exposed to
hypoxia than in segments from the corresponding normoxic control group (Table 2). In
contrast, the aortic response to big ET-1 in normoxic and hypoxia-exposed MMP-2-/-
mice do not differ.
Chapter 2
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Figure 2.8 (A) Concentration-response relationship for phenylephrine in
endothelium intact aortic rings from MMP-2 deficient (MMP-2-/-) and littermate control
(MMP-2+/+) mice exposed to normoxia or hypoxia for 7 d (n = 8-9 per group). *P<0.05
for differences between MMP-2+/+ and MMP-2-/- groups. (B) Contractile response to big
ET-1 (100 nmol/l) in endothelium-denuded aortic rings from MMP-2+/+ and MMP-2-/-
mice exposed to normoxia or hypoxia for 7 d (n = 9 for each group). *P<0.05 for
difference from corresponding normoxic group.
Chapter 2
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Table 2: Maximum contraction and EC50 values during PE- induced contraction
and response to big ET-1 (100 nmol/l) in MMP-2-/- and MMP-2+/+ mice.
Values are means ± SEM; *P<0.05 for differences between MMP-2+/+ and MMP-2-/- groups. †P<0.05 for difference from corresponding normoxic control group.
Maximum Tension (mg) Mouse Vessels Genotype Nor. Hyp. (7 d) PE
+/+ 462.11 ± 73.77 429.95 ± 51.27 Aortae -/- 444.42 ± 61.66 328.84 ± 31.17*
Big ET-1 +/+ 67.49 ± 11.32 172.62 ± 25.33† Aortae -/- 98.50 ± 10.30 125.18 ± 12.57
-log EC50, mol/L Mouse Vessels Genotype Nor. Hyp. (7 d) PE
+/+ 7.85 ± 0.17 8.03 ± 0.15 Aortae -/- 7.50 ± 0.17 8.04 ± 0.24
Big ET-1 +/+ n/a n/a Aortae -/- n/a n/a
Chapter 2
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2.4 Discussion
The results of this study show that after prolonged hypoxia: 1) MMP-2 inhibition
(rat) or deletion (mouse) reduces aortic and mesenteric arterial contraction to
phenylephrine; 2) the aortic contractile response to big ET-1 is enhanced in rats and
mice through an MMP-2-dependent mechanism; 3) MMP-2 protein levels in rat aortae
and mesenteric arteries, and MMP activity in rat aortae are increased; 4) aortic MT1-
MMP protein levels are increased; 5) aortic MMP-2, MT-1 MMP and TIMPs 1 to 4
mRNA levels are increased; and 6) the increase in rat aortic MMP-2 and MT1-MMP
mRNA expression is localized to the deep medial vascular smooth muscle.
The 23 MMPs identified to date are divided, based on substrate preference, into
collagenases, gelatinases, stromelysins, matrilysins and membrane-type MMPs. MMP-2
and MMP-9 are the gelatinases which efficiently degrade collagen type IV [82] and,
hence, are involved in the restructuring of vascular basement membranes. A broader
biological role for MMP-2 has become apparent with the recognition that its substrates
also include a number of vasoregulatory peptides [96, 188, 196-199]. Inactivation of
vasodilators (calcitonin gene-related peptide [97] and adrenomedullin [188]) and
activation or release of vasoconstrictors (big endothelin-1 (ET-1) [96], heparin binding
epidermal growth factors (HB-EGF) [197, 199], and integrin binding RGD peptides
[200]) all contribute to its vasoactive effects. Although the relative importance of each
of these pathways and the possible existence of others remains to be explored, our
present results emphasize that the net effect of increased MMP-2 activity in the systemic
circulation, as occurs after hypoxia, is to potentiate vascular smooth muscle contraction.
Chapter 2
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In regions affected by arterial insufficiency, MMP-2-mediated enhancement of
vascular contraction may be maladaptive since it will exacerbate ischemic injury.
During global hypoxia, however, the primary defensive vascular response depends on
the capacity of the adrenergic nervous system to regulate the regional distribution of
blood flow and oxygen extraction [76]. In previous studies in rats, systemic arterial
smooth muscle contraction to adrenoceptor stimulation is impaired after 48 hours of
hypoxia due to induction of heme oxygenase and nitric oxide synthase expression and
inhibition of myosin phosphorylation [61-63, 74, 185]. Hence, the ability to target
oxygen delivery to areas of greatest metabolic demand [76, 78, 183] is impaired. Our
present results indicate that after 7 days of hypoxia, rat aortic and mesenteric arterial
contraction are increased and that this is concomitant with and dependent on enhanced
MMP-2 activity. In this setting, therefore, upregulation of vascular MMP-2 provides a
mechanism to reinforce adrenergic regulation in the period during which maintenance of
oxygen delivery to vital organs is mediated by changes in vascular tone prior to the
structural change on which the redistribution of blood flow will ultimately depend.
In the rat aorta, MMP-2 and endothelin converting enzyme-1 (ECE-1) are the
major enzymes that convert big ET-1, the inactive prohormone, into the active
vasoconstrictor ET-1. Activation of big ET-1 by ECE-1 releases ET-1 [1-21] whereas
cleavage at Gly32-Leu33 by MMP-2 generates an isopeptide ET-1 [1-32] with enhanced
potency at the smooth muscle ETA receptor [96]. In the current study, the maximum
contraction that could be elicited by big ET-1 was increased after hypoxia. Since this
was reversed by MMP inhibition or MMP-2 deletion, MMP-2-mediated formation of the
Chapter 2
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more potent isopeptide ET-1 [1-32] appears to be a prominent pathway for big ET-1
conversion during prolonged hypoxia.
MMP-2 is secreted as a zymogen whose activity is regulated by its activating
protease MT1-MMP [187] and the endogenous tissue inhibitors of MMPs (TIMPs) [82].
An increase in MT1-MMP relative to the TIMPs is, therefore, requisite to any significant
enhancement of bioactivity. The possibility that MT1-MMP activity may be regulated
by an oxygen-sensitive mechanism has been suggested previously. MT1-MMP protein
is increased after hypoxic incubation in HepG2 cells and in the myocardium after
ischemia-reperfusion injury [201, 202] and the intracellular proprotein convertase furin,
responsible for its activation, is transcriptionally regulated by Hypoxia Inducible Factor-
1 [201]. Our current results confirm the functional relevance of these findings in the
systemic circulation and demonstrate that upregulation of MT1-MMP in the aorta
parallels the expression of its substrate, MMP-2. We also considered the possibility that
TIMPs may be oxygen regulated in order to provide an additional level of control. In
cultured cells, TIMP-1 and -2 have been observed to increase [189, 203, 204], decrease
[83, 205, 206], or remain unchanged [207] after hypoxic incubation. Our results confirm
that aortic levels of TIMPs mRNAs are sensitive to oxygen tension in vivo. However, in
contrast to MT1-MMP and MMP-2, changes in TIMP protein levels did not reach
statistical significance. Since MMP-2 activity was increased, this suggests that elevated
TIMP expression is insufficient to offset the increase in MT1-MMP and MMP-2.
Nevertheless, the fact that the expression of these endogenous inhibitors is hypoxia
inducible suggests that they may provide an important negative feedback mechanism in
some conditions.
Chapter 2
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The genes encoding MMP-2 and MT1-MMP contain consensus binding elements
for a number of hypoxia inducible transcription factors [191, 208], and their
transcriptional regulation by oxygen tension would be anticipated. Nevertheless, the
results of previous studies in cultured cells, are in conflict on this point [83, 203, 209-
211]. Our present results provide both pharmacological and biochemical evidence that,
in vivo, the expression of MMP-2 and MT1-MMP is upregulated in the systemic
circulation after hypoxia as a result of increases in their steady state mRNA levels and
that this change in systemic vascular cell phenotype is functionally relevant. Vascular
cells experience a broad range of oxygen tensions under physiological conditions. In the
aorta, oxygen concentrations from 11.2% (90 mm Hg) at the luminal surface to 2.2% (20
mmHg) at a depth of 150µm [212] are reported and longitudinal gradients of similar
magnitude occur in the microcirculation [213]. Since the severity of the hypoxic
stimulus varies significantly across the aortic wall, production of MMP-2 and MT1-
MMP would also be expected to demonstrate regional heterogeneity.
Immunohistochemical analysis may be confounded because MMP-2 is secreted and,
hence, distributed in the intracellular space across the aortic wall (see Figure 2.2).
Accordingly, we evaluated the regional expression of MMP-2 and MT1-MMP mRNA
and found that these transcripts are selectively enriched in VSMC located deep within
the aortic media, the most hypoxic region of the tissue [214]. This supports our
hypothesis that the expression of MMP-2 correlates with the severity of the hypoxic
stimulus and suggests that proteolytic activation of MMP-2 proenzyme occurs as it is
produced in the deep medial layer.
Chapter 2
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It is well recognized that changes in vascular tone precede the structural
alterations that occur when changes in blood flow persist chronically [200, 215, 216].
Such remodelling of the circulation is important in adapting the mature circulation to
chronic changes in tissue perfusion as well as arterial growth to meet the changing blood
flow demands of developing peripheral tissues. A role for MMP-2 in this process is
supported by observations that MMP-2-/- mice demonstrate impaired angiogenesis [90]
and that inhibitors of MMP reduce the pathological structural remodelling that
accompanies monocrotaline-induced pulmonary arterial hypertension [217]. Hypoxia is
a potent stimulus for both changes in vascular tone and structural remodelling in the
systemic circulation [79, 218]. The results of the current study indicate that vascular
MMP-2 levels and activity are tightly regulated by oxygen tension and, hence, represent
a pivotal regulatory pathway by which the acute vascular responses associated with
hypoxia may be integrated with the longer term structural changes in both conduit and
resistance arteries. Further investigation to determine the specific roles of MMP-2 and
each of its newly identified substrates will advance our understanding of the
pathobiology of this process in cardiopulmonary diseases and offer new therapeutic
targets in their management.
Chapter 3
- 70 -
During exposure to prolonged hypoxia, maintenance of vascular reactivity is
essential to ensuring adequate oxygen supply to vital organs.[10, 76, 184, 219] In the
previous chapter, increased vascular MMP-2 was demonstrated to induce
vasoconstriction in systemic conduit and resistance arteries of rats exposed to hypoxia
for 7 days. This is mediated through cleavage of big ET-1 by vascular MMP-2 to
release the vasoconstrictor ET-1[1-32].
Given the multifaceted actions of ET-1 and consequences of excessive ET-1
production, numerous mechanisms have evolved to modulate the local bioavailability
and potency of ET-1. Another enzyme in the vasculature that modulates ET-1
bioavailability and potency is HO-2. HO-2 catalyzes the degradation of heme to release
CO, biliverdin/bilirubin, and ferrous iron. Biliverdin/bilirubin possesses the capacity to
suppress intracellular concentrations of the reactive oxygen species (ROS) that regulate
ET-1 precursor mRNA expression through its antioxidant properties. CO mimics many
NO functions including cGMP-dependent and –independent inhibition of agonist-
induced vascular smooth muscle contraction. Govindaraju et al. demonstrated that
enhanced endothelial HO-2 protein expression reduces aortic reactivity in rats exposed
to hypoxia. Taken together, these findings suggest that HO-2 is another important
enzyme in regulating vascular function in systemic vessels during hypoxia.
Accordingly, hypoxic regulation of HO-2 protein expression and the physiological
effects of HO-2 activity during exposure to prolonged hypoxia are investigated.
Chapter 3
- 71 -
CHAPTER 3
Enhanced translation of HO-2 transcripts preserves human endothelial
cell viablility during prolonged hypoxia
Chapter 3
- 72 -
3.1 Introduction
Hypoxia is frequently observed in patients with shock, cardiopulmonary diseases
and in normal subjects at high altitudes. The compensatory mechanisms that preserve
blood flow to vital organs under these conditions are, in part, dependent on the release of
endothelium derived vasoregulatory factors.[64, 79, 185] During prolonged exposure to
hypoxia, endothelial function is impaired due to changes in endothelial phenotype and
cell death, and the adaptive responses that they mediate are compromised.[61, 64, 79]
Investigation of mechanisms that preserve endothelial cell survival and function in this
setting is, therefore, needed in order to develop therapeutic strategies to mitigate the
effects of hypoxia in patients with disorders associated reduced oxygen delivery.
Heme oxygenases (HO) are the rate limiting enzymes in the heme catabolic
pathway that cleaves heme to release carbon monoxide (CO), biliverdin, and ferrous
iron.[135] These products possess anti-apoptotic, anti-oxidant, and anti-inflammatory
properties, and so, may ameliorate the deleterious effects of hypoxia on endothelial
function. HO-1 and HO-2 are the heme oxygenase isoforms identified in the
endothelium. HO-1 expression is suppressed during hypoxia in human endothelial cells
and this is mediated by increased expression of the transcription repressor Bach1.[172]
Although a protective role for HO-2 has been suggested in other conditions associated
with impaired endothelium-dependent vasoregulation (diabetes and ischemia), the effect
of hypoxia on endothelial HO-2 expression and its functional role are unknown.[74, 137,
157] The current study was, therefore, carried out to test the hypothesis that HO-2
expression is oxygen regulated in human endothelial cells and to determine whether it
plays a role in preserving endothelial cell viability during hypoxic stress.
Chapter 3
- 73 -
3.2 Materials and Methods
Chemicals and Reagents: The Protein and RNA isolation system (PARIS) kit and
SYBR green universal master mix was purchased from Applied Biosystems (Foster City,
CA). The cDNA Synthesis Kit and DC Protein Quantifiation kit were from BioRad
laboratories (Hercules, CA). Anti-HO-1 and HO-2 antibodies were from Assay
Designs (Ann Arbor, MI). HO-2 siRNA, scrambled siRNA, and siRNA transfection
reagents were from Santa Cruz Biotechnology Inc (Santa Cruz, CA). 3H-uridine, 3H-
leucine and 35S-methionine were purchased from Perkin Elmer Life Science (Waltham,
MA). Carboxy-5-(6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (carboxy-
H2DCFDA), MitoProbe JC-1 Assay Kit, and custom primers were from Invitrogen
(Carlsbad, CA). Annexin V–FLUOS staining kit was purchased from Roche and the
CaspACE FITC-VAD-FMK in situ marker were from Promega (Madison, WI). All
other chemicals were from Sigma (St. Louis, MO).
Cell Culture Studies: Pooled human umbilical vein endothelial cells (HUVEC) and
human aortic endothelial cells (HAEC) were purchased from Lonza (Basel, Switzerland)
and cultured in EGM-2 medium according to manufacture’s instructions. Human blood
outgrowth endothelial cells were derived from peripheral blood as described.[220]
Healthy volunteers underwent a mononuclear cell collection (100 ml, 3-8% hematocrit)
procedure on a Cobe Spectra (Gambro, BCT, Denver CO). Samples were cultured in
EGM-2MV medium containing 20% human serum in tissue culture flasks pre-coated
with fibronectin (10µg/mL) for 7 to 10 days to obtain pure population of blood
outgrowth EC. After which, these cells were cultured for an additional 3 passages
Chapter 3
- 74 -
before being used in experiments. HUVEC at passages 3-5 and HAEC at passages 6-7
were used.
For siRNA transfections, HUVEC (105 cells/cm2) were seeded in antibiotic free
EGM-2 media in 60 mm dishes for 16-24 hours and transfected with human HO-2
siRNA (sc-35556) or non-specific control siRNA (sc-37007) using siRNA Transfection
Reagent following the manufacturer’s instructions. Transfected cells were replated in
either 60 mm dishes or 12 well plates after 24 h. Cells exposed to hypoxia were grown
to 70% confluence and transferred, after changing the medium, to a humidified Plexiglas
chamber maintained at 37°C and continuously flushed with gas composed of 1% O2/5%
CO2/balanced N2. Normoxic control cells were exposed to air/5% CO2/balanced N2
under otherwise identical conditions.
Quantitative Real Time PCR: Total RNA was extracted from HUVEC exposed to
either normoxia or hypoxia for 16 or 48 h using the PARIS kit and reverse transcribed
(1µg) with the cDNA synthesis kit containing random primers. All quantitative RT-
PCR analyses of were performed in triplicate using the ABI PRISM 7900 HT sequence
detection system (Applied Biosystems, Foster City, CA) with SYBR® green technology.
Levels of heme oxygenase 1 and 2 cDNA were detected using the following primers:
HO-1 (sense 5’- GTC CGC AAC CCG ACA G -3’, antisense 5’- ACC AGC TTG AAG
CCG TCT C -3’, exon 1/2); HO-2 (sense 5’- CCC TGG ACC TGA ACA TGA A -3’,
antisense 5’- ACC CAT CCT CCA AGG TCT C -3’, exon 4/5). The exponential
portion of the amplification curve for 1000 copies of target amplicon passed through the
cycle threshold (CT1000) at 24.34 ± 0.63 cycles for HO-1 and 24.35 ± 0.30 cycles for
Chapter 3
- 75 -
HO-2. 28S (sense 5’- TTG AAA ATC CGG GGG AGA G -3’, antisense 5’- ACA TTG
TTC CAA CAT GCC AG -3’) transcripts were used as controls for normalization.
Results obtained under each experimental condition were compared with their own
corresponding normoxic control values.
Western Blotting: Total protein was extracted from HUVEC, HAEC, and human
blood outgrowth endothelial cells exposed to either normoxia or hypoxia for 16 h or 48 h
using the PARIS kit according to the manufacturer’s instructions. After protein
concentrations were determined by the Lowry method, total proteins (20µg/lane) were
resolved by 8-16% SDS-PAGE and transferred to nitrocellulose. To detect HO-1 and
HO-2 protein, membranes were blocked in 5% milk-TTBS and incubated with rabbit
polyclonal anti-HO-1 (1:2000) or anti-HO-2 (1:2000) antibody overnight follow by
peroxidase conjugated anti-rabbit IgG (1:2500). HO-1 and HO-2 protein was
normalized to β-actin detected by reprobing the membranes with anti-β-actin
monoclonal antibodies (1:40,000, Sigma). The immunocomplexes were visualized with
the ECL plus kit purchased from GE HealthCare (Uppsala, Sweden) and quantified by
digital densitometry using the Quantity One software provided by BioRad laboratories.
Results obtained under each experimental condition were compared with their own
corresponding normoxic controls.
3H-uridine and 3H-leucine Incorporation: 3H-uridine and 3H-leucine incorporation
was performed as previously described.[221] HUVEC plated in 12-well plates were
incubated under either normoxia or hypoxia for 16 h or 48 h. 3H-uridine or 3H-leucine
were added to media (10 µCi/well) for the last 15, 30, 45, or 60 minutes of normoxic or
Chapter 3
- 76 -
hypoxic exposure. Cells were washed with cold PBS, incubated in 10% trichloroacetic
acid for 20 minutes, washed 3 times with 100% ethanol, and dried in oven at 45°C. The
residues were dissolved in 0.3 N NaOH for 20 min and neutralized with 0.3 N HCl. The
resultant mixture (400 µl) from each well was added to 5ml of scintillation fluid and
radioactivity in each sample was counted in a liquid scintillation counter. The rate of 3H-
leucine or 3H-uridine incorporation is represented by the slope of the radioactivity-
incubation time relationship.
35S-methionine Incorporation: HUVEC were exposed to either normoxia or
hypoxia for 16 h and incubated in methionine-free RPMI 1640 medium supplemented
with 10% fetal calf serum and 35S-methionine (10 µCi/ml) for 2 additional hours. Total
proteins were extracted in RIPA buffer (50 mM Tris, 150 mM NaCl, 50 mM NaF, 1 mM
Na Orthovanadate, 5 mM Benzamidine, 1 mM EDTA, 1% Igepal CA630, 0.5% Sodium
Deoxycholate, and 0.1% SDS) and quantified by the Lowry method. Cell lysates (500
µg), precleared with protein G-Sepharose, are then immunoprecipitated using rabbit
polyclonal anti-HO-1 (1:50) or HO-2 antibodies (1:50) overnight. The resulting
immunoprecipitates were separated by 10% SDS-PAGE and bands were visualized by
autoradiography.
Polysome Profiling: As decribed previously,[222] HUVEC were exposed to either
normoxic or hypoxia for 6 or 24 h. At the end of exposure periods, HUVEC were
washed with PBS containing 100µg/ml cycloheximide and lysed using 200µl of lysis
buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 7.4, 100 mg/ml cycloheximide,
and 1000 units/ml RNAseOUT). After centrifugation to remove cell debris,
Chapter 3
- 77 -
supernatants were layered onto a sucrose gradient (15% - 45%) and centrifuged for 2
hours at 35,000 rpm. A programmable density gradient fraction collector was used to
divide the gradient into 15 fractions so that HO-2 mRNA from each fraction could be
measured using quantitative real-time PCR.
Measurement of Intracellular Reactive Oxygen Species (ROS): Intracellular ROS
levels were measured in intact HUVEC using carboxy-H2DCFDA. This method is
based on the oxidation of non-fluorescent carboxy-H2DCFDA resulting in the formation
of the fluorescent compound 2’,7’-dichlorofluorescein (DCF). The fluorescence
generated by DCF is proportional to the rate of carboxy-H2DCFDA oxidation, which is
in turn indicative of the cellular oxidizing activity and intracellular ROS levels.
HUVEC grown to 70% confluence in 12-well plates were incubated with water, TNF-
α (10 ng/ml), or H2O2 (100 µM), prior to exposure to normoxia or hypoxia for 16 or 48
h. Cells were washed twice in HBSS and then incubated in HBSS containing of
carboxy-H2DCFDA (15 µM) for 30 min in the dark at 37 degrees. After rinsing with
HBSS once to remove free probe, fluorescence (Ex484/Em518 nm) from each well was
measured using the Fluroskan Ascent & FL fluorescent plate reader (Thermo Fisher,
Pittsburgh, PA). Cell number in each well was counted using the Z2 Coulter particle
count and size analyzer (Beckman Coulter, Fullerton, CA) so that fluorescence could be
normalized to cell number.
Mitochondrial Membrane Depolarization: Depolarization of the mitochondrial
membrane was detected using the cationic dye, 5,5’,6,6’-tetrachloro-1,1’,3,3’-
tetraethylbenzimidazolcarbocyanine Iodide (JC-1). JC-1 localizes to and aggregates
Chapter 3
- 78 -
within the mitochondria in proportion to mitochondrial membrane potential, emitting red
fluorescence. When the mitochondrial membrane depolarizes, JC-1 leaks into the
cytoplasm and forms monomers that emit green fluorescence. The ratio of red to green
fluorescence is an index of mitochondrial membrane depolarization. HUVEC grown to
70% confluence in 12-well plates were incubated with water, TNF-α (10 ng/ml), or
H2O2 (100 µM), prior to exposure to normoxia or hypoxia for 16 or 48 h. At the end of
the exposure period, HUVEC were incubated with PBS containing JC-1 (2 µM) for 15
min in the dark. Cells were then washed once with PBS and fluorescence
(Ex485/Em518nm (green) and Ex544/Em590nm (red)) was measured using the
Fluroskan Ascent & FL fluorescent plate reader (Thermo Fisher, Pittsburgh, PA).
Annexin V/Propidium Iodide labeling: The Roche Annexin V–FLUOS staining kit
was used to detect phosphatidylserine externalization (a marker of apoptosis) in HUVEC
and HAEC exposed to normoxia or hypoxia for 16 or 48 h. HUVEC and HAEC treated
with TNF-α or H2O2 were exposed to normoxia or hypoxia for 16 h. Cells in the media
were included in the sample. After trypsinization, cells were washed once with PBS
before addition of 100 µl of labeling solution that contains 2µl Annexin V-Fluos
labeling reagent and 2µl Propidium Iodide (PI) solution. Labeled cells were analyzed
using the cytomicsTM FC 500 flow cytometer (Beckman Coulter, Fullerton, CA).
Total Caspase Activation: Total caspase activation was measured in HUVEC and
HAEC exposed to normoxia or hypoxia for 16 or 48 h. HUVEC and HAEC treated with
TNF-α or H2O2 were exposed to normoxia or hypoxia. CaspACE FITC-VAD-FMK is a
FITC conjugate of the cell permeable inhibitor of caspases. This structure allows
Chapter 3
- 79 -
delivery of the inhibitor into the cell where binding to activated caspase serves as an in
situ marker for apoptosis. After trypsinization, cells were suspended in PBS containing
FITC-VAD-FMK (1 µM) at room temperature in the dark for 20 min. Cells were then
washed, resuspended in PBS, and analyzed using the cytomicsTM FC 500 flow cytometer
(Beckman Coulter, Fullerton, CA).
Data Analysis: Results are presented as mean ± S.E.M. for n number of
independent experiments with P<0.05 representing statistical significance. The
significance of differences between individual means was determined by two-tailed
Student’s t test. Differences among multiple means were evaluated by analysis of
variance corrected for multiple measures where appropriate and, when overall
differences were detected, differences between individual means were evaluated post-
hoc using the Student Neuman - Keuls procedure.
Chapter 3
- 80 -
3.3 Results
Effect of hypoxia on HO-1 and HO-2 mRNA and protein expression in systemic
vascular endothelial cells.
The effects of hypoxia on the expression of HO-1 and HO-2 mRNA and protein
were compared in HUVEC incubated under normoxic or hypoxic conditions for 16 and
48 h. After 16 and 48 h of hypoxic exposure, HO-1 mRNA is reduced to 27.82 ± 1.8 %
and 29.14 ± 10% of the corresponding normoxic control values, respectively (Figure
3.1A). HO-2 mRNA expression was decreased (42.93 ± 7.52 % of normoxic control,
Figure 3.1B) after 16 h of hypoxia and returned to the normoxic control level after 48 h.
HO-1 protein (Figure 3.1C) is reduced after 16 h and 48 h of hypoxia (79.57 ± 2.11%
and 64.93 ± 5.75% of normoxic control values, respectively) whereas HO-2 protein
levels were unaltered (Figure 3.1D). To corroborate this finding in other systemic
vascular endothelial cells, HO-1 and HO-2 proteins were also measured in HAEC and
human blood outgrowth EC exposed to normoxia or hypoxia for 16 h. Blood outgrowth
ECs were characterized by the expression of cell-surface markers. These cells stained
positive for CD34, KDR, VEGFR2, CD146, and CD31 and negative for CD14 and
CD45. As shown in Figure 3.2, hypoxia decreased HO-1 protein levels in both HAEC
and human blood orgin EC (38.50 ± 8.47% and 59.1 ± 8.85% of normoxic control
values, respectively) but, as in HUVEC, HO-2 was unaltered.
Chapter 3
- 81 -
Figure 3.1 HO-1 and HO-2 mRNA (A and B) in HUVEC exposed to 1% oxygen for
either 16 or 48 h. mRNA data are normalized to 28S rRNA. Bars represent means ±
S.E.M. n = 6 independent experiments, *P<0.05 for differences from corresponding
normoxic control.
A
16hrs 48hrs0
25
50
75
100
125 21% O21% O2
HO
-1 m
RNA
/ 28
S rR
NA(%
of N
orm
oxic
Con
trol
)
* *
B
16hrs 48hrs0
25
50
75
100
125
*
1% O2
21% O2
HO
-2 m
RNA
/ 28
S rR
NA(%
of N
orm
oxic
Con
trol
)
*
Chapter 3
- 82 -
16hrs 48hrs0
25
50
75
100
125
150
Duration of Hypoxia (1% O2)
*
HO-2
β-actin
1% O2
21% O2
HO
-2 /
β-a
ctin
Pro
tein
Lev
els
(% o
f Nor
mox
ic C
ontr
ol)
16hrs 48hrs0
25
50
75
100
125
150 21% O21% O2
Duration of Hypoxia (1% O2)
*
HO-1
β-actin
HO
-1 /
β-a
ctin
Pro
tein
Lev
els
(% o
f Nor
mox
ic C
ontr
ol)
* *
Figure 3.1 HO-1 and HO-2 mRNA protein levels (C and D) in HUVEC exposed to
1% oxygen for either 16 or 48 h. Protein data are normalized to β-actin. Bars represent
means ± S.E.M. n = 6 independent experiments, *P<0.05 for differences from
corresponding normoxic control.
C
D
Chapter 3
- 83 -
30405060708090
100110
21% O21% O2
HO-1β-actin
*
*
HO
-1 /
β-ac
tin P
rote
in L
evel
s(%
of N
orm
oxic
Con
trol
)
HAEC Blood Outgrowth EC
30405060708090
100110
21% O21% O2
HO-2
β-actin
HO
-2 /
β-ac
tin P
rote
in L
evel
s(%
of N
orm
oxic
Con
trol
)
HAEC Blood Outgrowth EC
Figure 3.2 (A) Representative blots of HO-1 and HO-2 protein in HAEC and human
blood outgrowth EC exposed to normoxia or hypoxia for 16 h. (B) Quantification of
HO-1 and HO-2 protein in HAEC and human blood outgrowth EC exposed to normoxia
or hypoxia for 16 h. Bars represent means ± S.E.M. n = 4 independent experiments,
*P<0.05 for differences from corresponding normoxic control.
A
B
Chapter 3
- 84 -
Effect of hypoxia on the translation of HO-2 transcripts in systemic vascular
endothelial cells.
In order to compare the effects of hypoxia on HO-1 and HO-2 protein synthesis
with its non-selective effects on total cellular mRNA and protein synthesis, 3H-uridine
and 3H-leucine incorporation were assessed in HUVEC after 16 and 48 h of hypoxic
incubation and 35S-methionine incorporation into HO-1 and HO-2 protein was measured
in HUVEC after exposure to hypoxia for 16 h (Fig. 3). Figure 3.3A illustrates that RNA
synthesis is decreased to 37.93 ± 3.71% and 28.78 ± 4.88% of normoxic control values,
respectively, after 16 and 48 h of hypoxic exposure. Protein synthesis is reduced to 56.6
± 2.77% at 16 h and 34.80 ± 2.97% at 48 h of the normoxic control value (Figure 3.3B).
Synthesis of HO-2 protein is less affected by hypoxia than HO-1 protein synthesis
(74.80 ± 7.80% vs. 47.01 ± 6.55% of normoxic control values, respectively). The
relative preservation of HO-2 protein synthesis, despite the 57% reduction in steady state
mRNA level and 43% reduction in total protein synthesis, suggests that HO-2 protein
expression is preserved during hypoxia, possibly through enhanced translation and/or
reduced protein degradation. Hypoxic incubation for 16 h with cycloheximide, an
inhibitor of RNA translation, reduced HO-2 protein levels. This suggests that translation
of HO-2 is important in maintaining HO-2 protein levels during hypoxia (data not
shown). To demonstrate the effect of hypoxia on HO-2 translation, ribosmal association
of HO-2 mRNA was assessed by polysome profiling. As shown in Figure 3.3D, after 6
h of hypoxia, HO-2 mRNA transcripts are located in higher polysome fractions which,
together with the results of the metabolic labelling and immunoprecipitation studies
supports enhanced translation of HO-2 transcripts during hypoxia.
Chapter 3
- 85 -
Normoxia 16 h 48 h0
25
50
75
100
Hypoxia (1% O2)
3 H-u
ridin
e In
corp
orat
ion
(% N
orm
oxic
Con
trol
)
**
Normoxia 16 h 48 h0
25
50
75
100
Hypoxia (1% O2)
**
3 H-le
ucin
e In
corp
orat
ion
(% N
orm
oxic
Con
trol
)
Figure 3.3 Rate of 3H-uridine (A) and 3H-leucine (B) incorporation into RNA and
protein of HUVEC exposed to normoxia or hypoxia for 16 or 48 h Bars represent means
± S.E.M. n = 4 independent experiments, *P<0.05 for differences from corresponding
normoxic control.
A
B
Chapter 3
- 86 -
HO-1 HO-20
25
50
75
100
125
*
21% O2, 16 h1% O2, 16 h
*35
S-m
ethi
onin
e In
corp
orat
ion
(% o
f Nor
mox
ic C
ontr
ol)
3 4 5 6 7 8 9 10 11 12 13 14 150.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 21% O2,6 h
1% O2, 6 h
Fraction Number
Rela
tive
Copi
es o
f HO
-2 T
rans
crip
t(p
er c
opy
in n
orm
oxic
frac
tion)
Figure 3.3 (C) Rate of 35S-methionine incorporation into HO-1 and HO-2 protein of
HUVECs exposed to normoxia or hypoxia for 16 h. Bars represent means ± S.E.M. n =
4 independent experiments, *P<0.05 for differences from corresponding normoxic
control. (D) Quantification of the abundance of HO-2 mRNA in various polysome
fractions from HUVEC exposed to normoxia or hypoxia for 6 h.
C
D
Chapter 3
- 87 -
Effect of decreased HO-2 protein level on ROS production in human endothelial
cells during hypoxia with or without treatment with TNF-α or H2O2
HO-2 protects cells from oxidative stress by reducing intracellular concentrations
of heme (a pro-oxidant) and by increasing levels of bilirubin and ferritin, which are
potent anti-oxidants. To determine the importance of HO-2 in protecting cells from
oxidative stress during hypoxia, we compared reactive oxygen specie (ROS) levels in
HUVEC transfected with scrambled or HO-2 siRNA exposed to normoxia or hypoxia
for 16 or 48 h, and also in HUVEC treated with TNF-α or H2O2 exposed to normoxia or
hypoxia for 16 h. Inhibition of HO-2 protein expression had no significant effects on
HO-1 protein expression in any of the conditions tested (data not shown). As illustrated
in figure 3.4B, exposure to hypoxia for 16 or 48 h increases ROS levels in HUVEC.
Compared to the scrambled siRNA control, inhibition of HO-2 expression increases
ROS levels in HUVEC exposed to hypoxia for 48h, but had no effect in cells exposed to
hypoxia for 16 h or in cells incubated under normoxic conditions. In HUVEC treated
with either TNF-α or H2O2, inhibition of HO-2 expression increases ROS levels in cells
exposed to hypoxia for 16 h (figure 3.4C).
Chapter 3
- 88 -
Scrambled siRNA HO-2 siRNA0
25
50
75
100H
O-2
/β
-act
in P
rote
in L
evel
s(%
of N
orm
oxic
Con
trol
)
*
HO-2
β-actin
21% O2 1% O2 21% O2 1% O20
50
100
150
200
250Scrambled siRNAHO-2 siRNA
16 h 48 h
†*†
DCF
Flu
ores
cenc
e/10
6 cel
ls(%
of N
orm
oxic
Con
trol
)
††
Figure 3.4 (A) Representative blots and quantification of HO-2 protein in HUVEC
transfected with scrambled or HO-2 siRNA. (B) ROS levels in HUVEC transfected
with scrambled or HO-2 siRNA exposed to normoxia or hypoxia for 16 or 48 h. Bars
represent means ± S.E.M. n = 6 independent experiments, *P<0.05 for differences
between with or without inhibition of HO-2 protein. †P<0.05 for differences between
hypoxia and corresponding normoxic control.
A
B
Chapter 3
- 89 -
21% O2 1% O2 21% O2 1% O20
50
100
150
200
250Scrambled siRNAHO-2 siRNA
H2O2 (100µM)TNF-α (10ng/ml)
**
DCF
Fluo
resc
ence
/106
cells
(% o
f Nor
mox
ic C
ontr
ol)
††
Figure 3.4 (C) ROS levels in HUVEC transfected with scrambled or HO-2 siRNA
exposed to normoxia or hypoxia for 16 h treated with TNF-α or H2O2. Bars represent
means ± S.E.M. n = 6 independent experiments, *P<0.05 for differences between with
or without inhibition of HO-2 protein. †P<0.05 for differences between hypoxia and
corresponding normoxic control.
C
Chapter 3
- 90 -
Inhibition of HO-2 protein expression decreases mitochondrial membrane potential
in human endothelial cell during hypoxia in the presence and absence of TNF-α or
H2O2
Both hypoxia and increased intracellular ROS levels may trigger programmed
cell death (apoptosis) or necrosis depending on their severity/magnitude. To determine
the functional significance of preservation of HO-2 levels during hypoxia, the effect of
HO-2 expression inhibition using HO-2 siRNA on HUVEC viability during hypoxia in
the presence or absence of TNF-α or H2O2 was assessed. Mitochondrial membrane
depolarization is an early event in the intrinsic apoptotic pathway activated by hypoxia.
The ratio of JC-1 aggregates/JC-1 monomer, which is a measure of mitochondrial
membrane potential (13), is decreased during hypoxic exposure (Fig. 5). Inhibition of
HO-2 expression further reduced mitochondrial membrane potential after exposure to
hypoxia for 48 h (Fig. 5A), suggesting that HO-2 protein or its activity increases the
capacity to maintain mitochondrial membrane potential during hypoxia. In HUVEC
treated with TNF-α or H2O2, inhibition of HO-2 enhanced mitochondrial membrane
depolarization in both normoxic and hypoxic cells (Figure 3.5B).
Chapter 3
- 91 -
21% O2 1% O2 21% O2 1% O20
25
50
75
100
16 h 48 h
Scrambled siRNAHO-2 siRNA
*
† †
JC-1
Agg
rega
tes/
JC-1
Mon
omer
s(%
of N
orm
oxic
Con
trol
)
21% O2 1% O2 21% O2 1% O20
25
50
75
100
TNF-α H2O2
Scrambled siRNAHO-2 siRNA
*
**
† †
JC-1
Agg
rega
tes/
JC-1
Mon
omer
s(%
of N
orm
oxic
Con
trol
)
Figure 3.5 (A) Mitochondrial membrane potential in HUVEC transfected with
scrambled or HO-2 siRNA exposed to normoxia or hypoxia for 16 or 48 h. (B)
Mitochondrial membrane potential in HUVEC transfected with scrambled or HO-2
siRNA exposed to normoxia or hypoxia for 16 and treated with TNF-α, or H2O2. Bars
represent means ± S.E.M. n = 6 independent experiments, *P<0.05 for differences
between with or without inhibition of HO-2 protein. †P<0.05 for differences between
hypoxia and corresponding normoxic control.
A
B
Chapter 3
- 92 -
HO-2 preserves human endothelial cell viability during hypoxia in the presence and
absence of TNF-α or H2O2
Annexin V/PI double staining was used to detect externalization of
phosphatidylserine, an early event in apoptosis, and cell membrane permeabilization, an
indicator of cell death. Representative plots of annexin V/PI staining are shown in Fig
6A. Non-viable cells are cells that are stained by annexin V and/or PI (Figure 3.6A).
Figure 3.6B demonstrates that exposure of HUVEC to hypoxia for 48 h increases cell
death and that cell death is further increased by inhibition of HO-2 expression. In
HUVEC treated with TNF-α or H2O2, HO-2 expression inhibition had no effect on
normoxic cells and increases cell death after hypoxic exposure for 16 h (Figure 3.6C).
To confirm that the effect of decreased HO-2 activity on cell viability is mediated by
inhibition of apoptosis, caspase activation was assessed in HUVEC exposed to normoxia
or hypoxia for 16 or 48 h and in HUVEC treated with TNF-α or H2O2 exposed to
normoxia or hypoxia for 16 h (Figure 3.7). In HUVEC where HO-2 protein expression
is inhibited, exposure to hypoxia for 48 h increased activated caspase by 1.5 fold
compare to scrambled siRNA control (Figure 3.7A). Total activated caspase is also
increased in HUVEC treated with TNF-α or H2O2 and exposed to hypoxia for 16 h
(Figure 3.7B).
Chapter 3
- 93 -
Annexin V
Scrambled siRNA, 21% O2, 48 h HO-2 siRNA 21% O2, 48 h
Scrambled siRNA, 1% O2, 48 h HO-2 siRNA 1% O2, 48 h
Pro
pidi
um Io
dide
Figure 3.6 (A) Representative annexin V/PI staining plots of HUVEC transfected
with scrambled or HO-2 siRNA exposed to normoxia or hypoxia for 48 h.
A
Chapter 3
- 94 -
21% O2 1% O2 21% O2 1% O20
255075
100125150175200
16 h 48 h
Scrambled siRNAHO-2 siRNA *
†
Cell
Deat
h(%
of N
orm
oxic
Con
trol
) †
21% O2 1% O2 21% O2 1% O20
255075
100125150175200
Scrambled siRNAHO-2 siRNA
H2O2 (100µM)TNF-α (10ng/ml)
**
Cell
Deat
h(%
of N
orm
oxic
Con
trol
)
Figure 3.6 The amount of cell death in HUVEC transfected with scrambled or HO-2
siRNA exposed to normoxia or hypoxia for 16 or 48 h (B) or exposed to normoxia or
hypoxia for 16 h in the presence of TNF-α, or H2O2 (C). Bars represent means ± S.E.M.
n = 6 independent experiments, *P<0.05 for differences between with or without
inhibition of HO-2 protein. †P<0.05 for differences between hypoxia and corresponding
normoxic control.
C
B
Chapter 3
- 95 -
21% O2 1% O2 21% O2 1% O20
50
100
150
200Scrambled siRNAHO-2 siRNA
H2O2 (100µM)TNF-α (10ng/ml)
**
Tota
l Act
ivat
ed C
aspa
se(%
of N
orm
oxic
Con
trol
)
†
Figure 3.7 Total activated caspase level in HUVEC transfected with scrambled or
HO-2 siRNA exposed to normoxia or hypoxia for 16 or 48 h or exposed to normoxia (A)
or hypoxia for 16 h and treated with TNF-α or H2O2 (B). Bars represent means ±
S.E.M. n = 5 independent experiments, *P<0.05 for differences between with or without
inhibition of HO-2 protein. †P<0.05 for differences between hypoxia and corresponding
normoxic control.
21% O2 1% O2 21% O2 1% O20
50
100
150
200
Scrambled siRNAHO-2 siRNA
16 h 48 h
*†
Tota
l Act
ivat
ed C
aspa
se(%
of N
orm
oxic
Con
trol
) †
A
B
Chapter 3
- 96 -
HO-2 preserves human endothelial cell viability during hypoxia in the presence and
absence of TNF-α or H2O2
To confirm the cytoprotective effect of HO-2 protein during hypoxia is not
specific to HUVEC, annexin V/PI staining and total caspase activation were assessed in
HAEC exposed to normoxic or hypoxia for 48 h and in HAEC treated with TNF-α or
H2O2 exposed to normoxia or hypoxia for 16 h. As observed in HUVEC, inhibition of
HO-2 increased cell death in HAEC exposed to hypoxia for 48 h (Figure 3.8A) and in
HAEC exposed to hypoxia for 16 h treated with TNF-α or H2O2 (Figure 3.8C). When
HO-2 expression was suppressed, total activated caspase increased in HAEC exposed to
hypoxia for 48 h and in HAEC treated with TNF-α or H2O2 and exposed to hypoxia for
16 h (Figure 3.8B and Figure 3.8D).
Chapter 3
- 97 -
21% O2 1% O20
50
100
150
200
250
300 Scrambled siRNA, 48 h
HO-2 siRNA, 48 h **
Cell
Deat
h(%
of N
orm
oxic
Con
trol
)
21% O2 1% O20
50
100
150
200
250
300 Scrambled siRNA, 48 hHO-2 siRNA, 48 h
**
Tota
l Act
ivat
ed C
aspa
se(%
of N
orm
oxic
Con
trol
)
Figure 3.8 Cell death (A) and total activated caspase level (B) in HAEC transfected
with scrambled or HO-2 siRNA exposed to normoxic or hypoxia for 48 h. Bars
represent means ± S.E.M. n = 5 independent experiments, *P<0.05 for differences
between with or without inhibition of HO-2 protein.
B
A
Chapter 3
- 98 -
21% O2 1% O2 21% O2 1% O20
255075
100125150175200
H2O2 (100µM)TNF-α (10ng/ml)
Scrambled siRNA, 16 hHO-2 siRNA, 16 h
**
**
Cell
Deat
h(%
of N
orm
oxic
Con
trol
)
21% O2 1% O2 21% O2 1% O20
50
100
150
200
250
H2O2 (100µM)TNF-α (10ng/ml)
Scrambled siRNA, 16 hHO-2 siRNA, 16 h
**
*
*
Tota
l Act
ivat
ed C
aspa
se(%
of N
orm
oxic
Con
trol
)
Figure 3.8 Cell death (C) and total activated caspase level (D) in HAEC transfected
with scrambled or HO-2 siRNA exposed to normoxia or hypoxia for 16 h and treated
with TNF−α or H2O2. Bars represent means ± S.E.M. n = 5 independent experiments,
*P<0.05 for differences between with or without inhibition of HO-2 protein.
C
D
Chapter 3
- 99 -
DISCUSSION
The results of this study show that in human endothelial cells incubated under
hypoxic conditions: 1) HO-1 mRNA and protein levels are decreased; 2) HO-2 protein
level is unaltered despite a 40% reduction in HO-2 mRNA expression and 50%
reduction in total protein synthesis; 3) HO-2 protein level is maintained through
enhanced translation of HO-2 transcripts; and 4) inhibition of HO-2 expression increases
production of reactive oxygen species, decreases mitochondrial membrane potential and
enhances apoptotic cell death.
Previous studies indicate cell type- and inter-species differences in the regulation
of HO expression by hypoxia. Aortic HO-2 protein is increased in rats exposed to
hypoxia,[74] remains unchanged in cultured rat aortic smooth muscle cells and human
cytotrophoblast[223, 224] and is decreased in Jurkat, K562, and YN-1 cells after
hypoxic incubation.[176] HO-1 mRNA and protein levels decrease in HUVEC, human
astrocytes, and human coronary artery endothelial cells[172, 225] but increase in bovine
aortic and rat pulmonary artery endothelial cells and in human fibroblasts and smooth
muscle cells after hypoxic incubation.[226-229] In HUVEC, decreased expression of
HO-1 after hypoxia is mediated by induction of the transcription repressor Bach1.[172]
The current study, the first to directly compare the effects of hypoxia on HO-1 and HO-2
expression in human endothelial cells, demonstrates that although both HO-1 and HO-2
mRNA levels are decreased, HO-2, but not HO-1 protein level remains unchanged. HO-
1 and HO-2 protein levels are, therefore, differentially regulated by oxygen tension and
HO-2 is the predominant isoform present under these conditions.
Chapter 3
- 100 -
HO-1 is primarily regulated transcriptionally and the genomic sequences 5’ to its
coding region contain cis-acting response elements that bind transcription factors
including HIF-1α, AP1, SP1, as well as the heme response element GC-
NNNGTCA.[135] In contrast, the 5’-flanking region of the HO-2 gene contains no
regulatory elements corresponding to transcription factors known to participate in the
response to hypoxia.[165, 173-175] Not surprisingly, therefore, hypoxic incubation did
not result in increased HO-2 mRNA levels in the current study, or in any of the cell
culture systems in which it has previously been evaluated.[223, 230] Nevertheless, HO-
2 expression is not entirely constitutive. Development stage-specific changes in HO-2
protein levels have been reported and HO-2 protein is increased in the aortic
endothelium of rats exposed to hypoxia without a corresponding increase in HO-2
mRNA.[74, 181, 231] Similarly, spatial and temporal dissociation between HO-2
protein and mRNA expression have been noted in the rodent brain and testis.[165, 174,
181] Our current results, therefore, reconcile these observations by demonstrating that
HO-2 expression is regulated at the post transcriptional level.
Hypoxia results in decreased cap-dependent translation due to increased
formation of the eIF4E/4E-BP1 inhibitory complex and increased phosphorylation of
eIF2F-α.[35] When hypoxia is severe, or prolonged, transcription is also inhibited and
mRNA levels decrease, as observed in the present study. Accordingly, maintenance of
protein levels under these conditions requires enhanced translation of existing mRNA
transcripts and/or reduced degradation of protein. Our current observation that HO-2
transcripts are localized to larger polysome fractions after hypoxic incubation (Figure
Chapter 3
- 101 -
3D) supports enhanced translation as an important mechanism by which HO-2 protein
levels are preserved. Consistent with this conclusion, we observed that the reduction in
HO-2 protein synthesis after exposure to hypoxia for 16 h is small relative to the
decreases in HO-2 mRNA levels and the rate of total protein synthesis. Furthermore,
HO-2 protein levels were decreased after hypoxic incubation in HUVEC treated with the
protein translation inhibitor cycloheximide, but not with the RNA synthesis inhibitor
actinomycin D or the proteasome inhibitor epoximycin. In other transcripts for which
this has been described, structural features that enhance cap dependent (nNOS) and cap-
independent (Tie-2) ribosomal association have been identified in their 5’ untranslated
regions.[54, 185] Using the BD Marathon-Ready human testis cDNA library, Zhang et
al. has demonstrated that HO-2 transcription is initiated from multiple sites.[176] Thus,
translation of HO-2 could be enhanced through selective transcriptional activation of a
promoter that produces more efficiently translated mRNA species during hypoxia, a
mechanism we have previously shown to mediate hypoxic enhancement of nNOS
expression in vascular smooth muscle.[185] In view of the current findings, therefore,
further examination of HO-2 mRNA structure and its functional relevance in the
regulation of HO-2 protein expression during hypoxia are now warranted.
Oxidant injury occurs when there is an imbalance between the formation of ROS
and the antioxidant capacity of the cell and is implicated in the pathogenesis of organ
dysfunction in diseases associated with reduced oxygen delivery.[232, 233] Previous
studies demonstrate that HO-2 is a component of the endogenous cell defence against
oxidative stress; HO-2 gene deletion increases hemin-induced injury in astrocytes and
sensitizes cerebral vascular endothelial cells to glutamate and TNF-α induced
Chapter 3
- 102 -
apoptosis.[157, 158, 234] The results of the current study confirm the essential role that
HO-2 plays in oxidant stress defence in human endothelial cells exposed to hypoxia
since inhibition of its expression increases intracellular ROS levels after 48 hours of
hypoxic incubation (Figure 4B). Compensation of HO activity by increased HO-1
expression was not observed. To corroborate the conclusion that HO-2 is important in
modulating oxidant stress, the effect of inhibition of HO-2 expression on the response to
other oxidative stimuli (TNF-α or H2O2) was also evaluated. In cells deficient in HO-2,
significant increases in ROS levels were observed only after hypoxic incubation. Its role,
relative to other defence mechanisms, therefore, is specifically enhanced during hypoxia.
HO activity is required for catabolism the prooxidant heme and for production of
bilirubin, a scavenger of superoxide and peroxyl radicals.[135] HO-2 also plays a
specific role in regulating intracellular free iron which increases the generation of
reactive hydroxyl radicals through the Fenton reaction.[138, 235] Since HO-1
expression is inhibited by hypoxia, HO-2 becomes the predominant isoform under these
conditions and a significant mechanism of defence against oxidant stress and hypoxic
injury.
Apoptosis may be triggered in response to stimuli extrinsic or intrinsic to the
affected cell. Hypoxia-induced apoptosis occurs mainly through the intrinsic
pathway.[236, 237] The lack of oxygen limits ATP synthesis required for maintenance
of the mitochondrial membrane potential. Depolarization of the mitochondrial
membrane potential elevates cytoplasmic ROS levels and further inhibits oxidative ATP
synthesis because the electromotive force for electron transport is reduced. This, in turn,
Chapter 3
- 103 -
activates Bax or Bak and leads to cytochrome C release into the cytosol, caspase
activation and chromatin fragmentation.[238] In the current study, we demonstrate that
mitochondrial membrane potential is decreased after exposure to hypoxia for 16 h,
whereas increase in cell death (annexin V/PI staining) and total activated caspase
activity was detected only after 48 h. These results support involvement of the intrinsic
pathway since mitochondrial membrane depolarization precedes caspase activation.
HO-2 protects against apoptotic cell death induced by TNF-α and glutamate in
cerebrovascular endothelial cells and by hydrogen peroxide in HEK cells.[140, 157-159]
In the present study, inhibition of HO-2 expression exacerbated mitochondrial
membrane depolarization and increased cell death and activated caspase levels in
hypoxic, but not normoxic human endothelial cells. When cells where concomitantly
treated with TNF-α or H2O2, the anti-apoptotic effect of HO-2 was detected after
exposure to hypoxia for 16 h, instead of 48 h. These results indicate that HO-2 also
protects against hypoxia-induced apoptosis in human endothelial cells, and plays an
even greater role in preserving cell viability during concomitant oxidative stress induced
by TNF-α or H2O2, perhaps because generalized inhibition of protein synthesis by
hypoxia suppresses the expression of components of conventional cytoprotective
pathways.
Consistent with previous studies, our present results support a central role for
reactive oxygen species in triggering apopototic endothelial death induced by hypoxia,
TNF-α and H2O2. Elevated ROS levels induce apoptosis through activating the JNK-
cJUN pathway and/or damaging mitochondrial membrane integrity resulting in reduced
Chapter 3
- 104 -
mitochondrial membrane potential.[239] These may be abrogated by the antioxidative
effects of HO-2, however, several other mechanisms have been invoked. For example,
CO inhibits TNF-α-induced apoptosis by activation of p38 MAPK pathway.[161] In
addition HO-2 protects cell viability through mechanism(s) separate from its role in
heme degradation since transfection of HEK cells with a catalytically inactive HO-2
mutant protects against oxidative injury, although the mechanism of protection is
unknown.[140]
Although HO-2 and HO-1 catalyze the same reaction, the differences between
these enzymes could provide insight into the advantages of maintaining HO-2, but not
HO-1 during prolonged hypoxia. In HEK cells transfected with plasmids containing
either HO-1 or HO-2 and treated with hydrogen peroxide, HO-2 was found to colocalize
with its cofactor NAPH-cytochrome P450 reductase in the microsomal fraction, whereas
HO-1 was more widely dispersed.[159] Accordingly, HO-2 may provide a more
efficient pathway for heme degradation, hence greater cytoprotective capacity, due to its
subcellular localization in association with this co-factor. Additionally, HO-2 contains
Cys-Pro repeats, termed heme regulatory domains, not present in HO-1 that provide
heme binding sites distinct from the heme catalytic domain.[135] During hypoxia or
ischemia injury, large amounts of prooxidant heme are release by cells undergoing
necrosis or apoptosis. HO-2, but not HO-1, could sequester this excess free heme.
Finally, differential regulation of the expression of these isoforms enables fine control of
the antioxidant capacity of the endothelium; by maintaining HO-2 and downregulating
Chapter 3
- 105 -
HO-1 during hypoxia, endothelial cells reserve the capacity to increase HO activity in
response to additional stress.
Previously, we identified a role for HO-2 in preserving endothelium-dependent
modulation of vasoconstrictor responses to endothelin-1 and phenylephrine in rats
exposed to prolonged hypoxia.[74] HO-2 knockout mice exhibit hypoxemia and
myocardial hypertrophy while breathing room air, indicating that HO-2 contributes to
pulmonary ventilation-perfusion matching.[240] Our current results highlight the
importance of HO-2 in modulating endothelial cell apoptosis which is a prominent
feature in a variety of diseases including atherosclerosis, ischemia/reperfusion injury,
and transplantation. Accumulating evidence, therefore, supports a central role for HO-2
in the cardiopulmonary adaptation to hypoxia and in the pathophysiology of disorders in
which endothelial injury contributes to vascular dysfunction. Accordingly it represents a
potential novel target for therapeutic intervention.
Chapter 4
- 106 -
CHAPTER 4
Perspective
Chapter 4
- 107 -
Hypoxia occurs in a variety of cardiopulmonary diseases and in normal
individuals during ascent to high altitude. In response to acute hypoxia, oxygen delivery
to vital organs is maintained through adrenergically mediated sympathetic responses and
endothelial release of vasoactive peptides.[77] As hypoxic exposure is prolonged,
sympathetic regulation of vascular tone is impaired due to reduced contractile response
to adrenergic stimulation.[62, 64] Studies in rat aorta indicate that enhanced targeting of
type 1 phosphatase activity to the contractile myofilaments and increased expression of
the inhibitory thin-filament proteins caldesmon and calponin may contribute.[62, 63]
The impairment of vascular smooth muscle contractility is partially compensated by
alteration in the function of the endothelium, in that it becomes an agency of
vasoconstrictors as opposed to its normal role as a source of vasorelaxing factors.[60,
61]
Endothelin-1 is a potent vasoconstrictor released by the endothelium and
previous studies have shown that ET-1 plays a central role in the adaptation to
hypoxia.[61, 76, 111, 112, 241] The increased vascular ET-1 production during hypoxia
potentiates vascular reactivity and enhances oxygen extraction. However, the
mechanism of its activation in the vasculature has not been fully investigated.
Classically, ET-1 is produced by the cleavage of big ET-1 by ECE-1.[242] Recently,
vascular MMP-2 mediated cleavage of big ET-1 was found to release a vasoconstrictive
ET-1 isopeptide (ET-1[1-32]).[96] MMP-2 also regulates vascular tone by inactivating the
vasodilators CGRP and adrenomudulin.[97, 188] Given that hypoxia increases MMP-2
production and activation,[83] MMP-2 has the potential to play a significant role in
regulating vascular reactivity during hypoxia.
Chapter 4
- 108 -
In the studies presented in chapter 2, the vasoregulatory role of vascular MMP-2 was
investigated, along with the effect of prolonged exposure to hypoxia in vivo on vascular
MMP-2 production and activity. The novel findings of this study are:
1) Vascular MMP-2 mediates vasoconstriction in systemic conduit and resistance
vessels of rats exposed to hypoxia for 7 days.
2) Vascular MMP-2 mediated activation of Big ET-1 is a prominent mechanism of
regulation of vascular reactivity during hypoxia
3) Hypoxia increases vascular MMP-2 and MT1-MMP protein levels without
altering TIMPs 1-4 protein levels
4) Hypoxia induces MMP-2 and MT1-MMP mRNA expression in the deep medial
vascular smooth muscle
It is well recognized that changes in vascular tone precede the structural alterations
that occur when changes in blood flow persist chronically, as occurs during prolonged
hypoxia.[215, 216] Such remodeling of the circulation is important in adapting the
mature circulation to chronic changes in tissue perfusion. Given vascular MMP-2’s
vasoregulatory role and its role in basement membrane degradation, hypoxic activation
of vascular MMP-2 represents a pivotal pathway by which the acute vascular responses
to hypoxia may be integrated with the longer-term structural changes in both conduit and
resistance arteries. MMP-2 activation alters the activity of a number of vasoregulatory
peptides in addition to big ET-1.[96, 198, 199, 243] Although the present study
demonstrated that activation of big ET-1 plays a role, the relative importance of other
pathways remains to be explored. Given that ET-1 also promotes proliferation and
inhibits apoptosis of endothelial and smooth muscle cells,[100] investigation into other
Chapter 4
- 109 -
physiological roles of MMP-2 mediated release of ET-1[1-32] during hypoxia is
warranted. Expression of MMP-2 and big ET-1 is also be observed during conditions of
tissue injury, inflammation and cancer.[244-246] It is also important, therefore, to
elucidate the effects of MMP-2 mediated activation of big ET-1, and whether it is
influenced by tissue oxygenation, in these pathophysiological settings. Selective
inhibition of MMP-2 may represent a new pharmacological strategy for regulating
vascular reactivity and remodeling in pathological conditions.
Heme oxygenase-2 is another vasoregualtory enzyme with the potential to
regulate the production and potency of ET-1.[74] HO-2 increases in the endothelium
and alters aortic reactivity after exposure to hypoxia in rats. Based on the properties of
its products (CO and biliverdin) endothelial HO-2 may play additional roles, potentially
acting to increase endothelial cell viability and reduce inflammatory responses.[135]
Hypoxia reduces HO-1 mRNA and protein levels in human endothelial cells,[172, 225]
and it is likely that HO-2 is the dominate HO enzyme in these cells during hypoxia.
Accordingly we proposed that it may play a meaningful role in preserving endothelial
function in conditions associated with reduced oxygen delivery. In the absence of active
transcriptional regulation others have suggested that HO-2 protein expression may be
regulated posttranscriptionally.[173, 179, 181] In the studies presented in chapter 3,
therefore, oxygen regulation of HO-2 in human endothelial cells and its role in
preserving endothelial cell viability during hypoxic stress was investigated. The novel
findings are:
1) HO-2 protein level is unaltered despite a 40% reduction in HO-2 mRNA
expression and 50% reduction in total protein synthesis
Chapter 4
- 110 -
2) Hypoxia enhances translation of HO-2 transcripts
3) Inhibition of HO-2 protein expression increases production of reactive oxygen
species, decreases mitochondrial membrane potential and enhances apoptotic cell
death.
These results demonstrate that HO-2, but not HO-1, protein level is selectively
maintained in human endothelial cells during hypoxia through enhanced translation of
HO-2 transcripts. In some cases, a competitive advantage over other mRNAs for
ribosome binding is conferred through activation of an alternate promoter that drives
expression of an mRNA containing a 5’ UTR lacking secondary structure. This
mechanism regulates the expression of the hypoxia inducible nNOS variant.[185] In
other cases, the presence of an internal ribsosomal entry site in the 5’ UTR that enables
cap-independent translation increases translation in situations where cap-dependent
translation is inhibited, such as during hypoxia.[37, 247] In view of these findings,
therefore, further examination of HO-2 mRNA structure and its functional relevance in
the regulation of HO-2 protein expression during hypoxia are now warranted.
In the studies described in chapter 3, HO-2 protein was found to be anti-
oxidative and anti-apoptotic in human endothelial cells, suggesting that HO-2 is
essential in maintaining endothelial integrity in conditions associated with hypoxia.
Further study to assess the pathophysiological relevance of this effect in vivo is now
warranted. HO-2 could maintain endothelial cell viability through the effects of
bilirubin, carbon monoxide, ferritin, or other unidentified intracellular signaling
pathways. Therefore, the molecular mechanisms mediating the effects on HO-2 in the
current setting remain to be identified. Lastly, given that HO-2 activity reduces ROS
Chapter 4
- 111 -
levels in human endothelial cells during hypoxic exposure, and ROS increase both
MMP-2 and ET-1 mRNA expression, future studies will investigate the link between
HO-2 and oxygen regulation of MMP-2 expression and ET-1 bioavailability.
In conclusion, my work has revealed that vascular MMP-2 and HO-2 play
important roles in the adaptive response to hypoxia. Further investigation into signaling
pathways altered by these enzymes could lead to the development of novel therapeutic
strategies to mitigate the effects of hypoxia in patients with disorders associated reduced
oxygen delivery.
- 112 -
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