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CONTRIBUTORS
O.Baretella
StateKeyLaboratoryofPharmaceuticalBiotechnology,TheUniversityofHongKong,
HongKongS.A.R.,China
M.Barton
MolecularInternalMedicine,UniversityofZu€rich,Zu€rich,Switzerland
K.Bian
SchoolofMedicine,GeorgeWashingtonUniversity,Washington,DC,UnitedStates
S.L.K.Bowers
DaltonCardiovascularResearchCenter,UniversityofMissouriSchoolofMedicine,
Columbia,MO,UnitedStates
G.E.Davis
DaltonCardiovascularResearchCenter,UniversityofMissouriSchoolofMedicine,
Columbia,MO,UnitedStates
C.deWit
Institutfu€rPhysiologie,Universit€atzuLu€beck;DeutschesZentrumfu€rHerz-Kreislauf-
Forschung(DZHK)e.V.(GermanCenterforCardiovascularResearch),PartnerSite
Hamburg/Kiel/Lu€beck,Lu€beck,Germany
L.Desbiens
Universit(cid:2)edeSherbrooke,Sherbrooke,QC,Canada
P.D’Orl(cid:2)eans-Juste
Universit(cid:2)edeSherbrooke,Sherbrooke,QC,Canada
D.J.R.Fulton
MedicalCollegeofGeorgiaatAugustaUniversity,Augusta,GA,UnitedStates
M.S.Goligorsky
NewYorkMedicalCollegeatTouroUniversity,Valhalla,NY;YaleUniversity,
NewHaven,CT,UnitedStates
K.Hirschi
NewYorkMedicalCollegeatTouroUniversity,Valhalla,NY;YaleUniversity,
NewHaven,CT,UnitedStates
M.Houde
Universit(cid:2)edeSherbrooke,Sherbrooke,QC,Canada
J.D.Imig
MedicalCollegeofWisconsin,Milwaukee,WI,UnitedStates
R.A.Khalil
VascularSurgeryResearchLaboratories,BrighamandWomen’sHospital,HarvardMedical
School,Boston,MA,UnitedStates
xi
xii Contributors
R.Ko€hler
AragonInstituteofHealthSciences&IISAragon;AragonAgencyforResearchand
Development(ARAID),Zaragoza,Spain
M.R.Meyer
TriemliCityHospital,Zu€rich,Switzerland
F.Z.Mo´nica
SchoolofMedicine,GeorgeWashingtonUniversity,Washington,DC,UnitedStates;State
UniversityofCampinas(UNICAMP),Campinas,Brazil
F.Murad
SchoolofMedicine,GeorgeWashingtonUniversity,Washington,DC,UnitedStates
P.R.Norden
DaltonCardiovascularResearchCenter,UniversityofMissouriSchoolofMedicine,
Columbia,MO,UnitedStates
A.Oliva´n-Viguera
AragonInstituteofHealthSciences&IISAragon;AragonAgencyforResearchand
Development(ARAID),Zaragoza,Spain
J.S.Possomato-Vieira
VascularSurgeryResearchLaboratories,BrighamandWomen’sHospital,HarvardMedical
School,Boston,MA,UnitedStates
K.Schmidt
Institutfu€rPhysiologie,Universit€atzuLu€beck;DeutschesZentrumfu€rHerz-Kreislauf-
Forschung(DZHK)e.V.(GermanCenterforCardiovascularResearch),PartnerSite
Hamburg/Kiel/Lu€beck,Lu€beck,Germany
P.M.Vanhoutte
StateKeyLaboratoryofPharmaceuticalBiotechnology,TheUniversityofHongKong,
HongKongS.A.R.,China
R.Windler
Institutfu€rPhysiologie,Universit€atzuLu€beck;DeutschesZentrumfu€rHerz-Kreislauf-
Forschung(DZHK)e.V.(GermanCenterforCardiovascularResearch),PartnerSite
Hamburg/Kiel/Lu€beck,Lu€beck,Germany
H.Wulff
UniversityofCalifornia,Davis,CA,UnitedStates
PREFACE
Thevascularsystemplaysanimportantroleinthedistributionofbloodand
nutrientsupplytodifferenttissuesandorgans.Itcomprisesavastandintricate
networkoflargeandsmall,superficialanddeeparteriesandveins.Structur-
ally,abloodvesseliscomposedofthreelayers:theintimaandendothelium,
themediaandvascularsmooth muscle,andtheadventitia andextracellular
matrix.Eachoftheselayershasaspecificroleinordertomaintaintheblood
vessel diameter and to handle the changes in blood pressure. Interestingly,
the blood vessel wall is lined with only one layer of endothelial cells.
However, this single layer of cells is capable of releasing several relaxing
factors including nitric oxide, prostacyclin, and hyperpolarizing factor that
could promote vasodilation and decrease vascular resistance and blood
pressure. Endothelial cells also release powerful contracting factors such as
endothelin and thromboxane A2 with multiple targets in the blood vessels
and other tissues, an important role in the control of the circulation, the
hemodynamics, and cardiovascular and renal function. An imbalance
betweenendothelium-derivedvasodilatorsandvasoconstrictorscouldcause
pathological changes that lead to major disorders such as hypertension,
coronary heart disease, and diabetes.
ThisvolumeofAdvancesinPharmacologyfocusesonthevascularendothe-
lium and its role under physiological and pathological conditions. The
volume will highlight the current knowledge regarding the various
endothelium-derived mediators and how they may change with gender,
duringvasculardevelopmentandsenescence,andinhypertensivedisorders.
Renownedscientistsandresearchershaveagreedtosharetheirexpertiseand
advancedknowledgeontheendothelium.Thefirsthalfofthevolumewill
coverimportanttopicsregardingthebasicfunctionoftheendotheliumand
therolesofnitricoxide,gapjunctions,potassiumchannels,andendothelin.
Thesecondhalfwillincludetopicsthatdiscusstheroleoftheendothelium
in vascular development and the changes in endothelial and vascular func-
tionwithgenderandadvancedageandincertainhypertensivestatessuchas
hypertension in pregnancy and preeclampsia. These important review
articles were written by research investigators and clinician–scientists from
different parts of the world, thus promoting different viewpoints and
highlighting differentapproachesinthemanagementofcardiovasculardis-
ease.Thankstothegoodworkofthecontributingauthors,andthecareful
xiii
xiv Preface
reviewofourdedicatedreviewersandEditors,theAdvancesinPharmacology
was able to put together these important topics, and present them to our
readers in a clear, concise, and informative fashion. I encourage every
researcher, clinician, medical, graduate, and undergraduate student with
aspirationtoworkinthevascularfieldtoreadthisstate-of-the-artsynopsis
on the endothelium.
Iwouldliketotakethisopportunitytoexpressmydeepestgratitudeto
Dr.S.J.Enna,theSeriesEditorofAdvancesinPharmacology,whogavemethe
opportunitytobetheEditorofthisspecialandtimelyvolume.Ialsowould
liketothankouroutstandingManagingEditor,Ms.LynnLeCount,andour
hard-workingEditorialStaffwhosparednoefforttoensurethehighestqual-
ityofthearticles.Ialsowouldliketoacknowledgeourcontributingauthors
notonlyfortheirexcellentarticlesbutalsoforsharingsomeofthereviewers’
duties, and for being very generous with their time and effort in providing
otherauthorswithtimelyreviewofthearticlesaswellashelpfulcomments
andconstructivecriticism.Iparticularlywishtothankourreadersfortheir
interest in vascular pharmacology and the endothelium. I encourage all of
youtoprovidefeedbackandcontactmedirectlyifyouhaveanyquestions,
comments, suggestions, criticism, or ideas that could further enhance our
knowledgeandhelpusachieveourgoalsandmeetthehighestexpectations
of our readers.
RAOUF A. KHALIL
Harvard Medical School, Brigham and Women’s Hospital,
Boston, MA, United States
CHAPTER ONE
The Endothelium-Dependent
–
Nitric Oxide cGMP Pathway
F.Z. Mónica*,†, K. Bian*,1, F. Murad*,1
*SchoolofMedicine,GeorgeWashingtonUniversity,Washington,DC,UnitedStates
†StateUniversityofCampinas(UNICAMP),Campinas,Brazil
1Correspondingauthors:e-mailaddress:[email protected];[email protected]
Contents
1. Introduction 3
2. PartI:NO–sGCSignaling 4
2.1 EndothelialDysfunctionandNODeficiency 4
2.2 eNOS(NOS-3)Uncoupling 4
2.3 EndogenousInhibitorsofeNOS(NOS-3) 7
2.4 NOReceptor:sGC 7
2.5 Phosphodiesterases 8
3. PartII:NO–sGCPathwayasaTherapeuticTargetforCardiovascularDiseases 9
3.1 TherapeuticApproachforPulmonaryHypertensionTherapy 9
3.2 TherapeuticApproachforHeartDisease 13
4. PartIII:EpigeneticsRegulationofNO–sGCPathwayComponentsinthe
CardiovascularSystem 14
4.1 OutlineofEpigeneticRegulation 14
4.2 EpigeneticRegulationofeNOS(NOS-3) 15
4.3 EpigeneticRegulationofiNOS 17
4.4 EpigeneticRegulationofnNOS 17
4.5 EpigeneticRegulationofsGC 18
4.6 NitricOxideModulatingEpigeneticsPathways 19
5. Conclusion 20
ConflictofInterest 20
Acknowledgments 20
References 21
Abstract
Nitric oxide (NO)–cyclic 30-50 guanosine monophosphate (cGMP) signaling plays
a critical role on smooth muscle tone, platelet activity, cardiac contractility, renal
functionandfluidbalance,andcellgrowth.Studiesofthe1990sestablishedendothe-
lium dysfunction as one of the major causes of cardiovascular diseases. Therapeutic
strategies that benefit NO bioavailability have been applied in clinical medicine
AdvancesinPharmacology,Volume77 #2016ElsevierInc. 1
ISSN1054-3589 Allrightsreserved.
http://dx.doi.org/10.1016/bs.apha.2016.05.001
2 F.Z.Mónicaetal.
extensively.BasicandclinicalstudiesofcGMPregulationthroughactivationofsoluble
guanylyl cyclase (sGC) or inhibition of cyclic nucleotide phosphodiesterase type 5
(PDE5)haveresultedineffectivetherapiesforpulmonaryhypertension,erectiledysfunc-
tion,andmorerecentlybenignprostatichyperplasia.Thissectionreviews(1)howendo-
thelialdysfunctionandNOdeficiencyleadtocardiovasculardiseases,(2)howsoluble
cGMPregulationleadstobeneficialeffectsondisordersofthecirculationsystem,and
(3) the epigenetic regulation of NO–sGC pathway components in the cardiovascular
system. In conclusion, the discovery of the NO–cGMP pathway revolutionized the
comprehension of pathophysiological mechanisms involved in cardiovascular and
other diseases. However, considering the expression “from bench to bedside” the
therapeutic alternatives targeting NO–cGMP did not immediately follow the marked
biochemicalandpathophysiologicalrevolution.Sometherapeuticoptionshavebeen
effective and released on the market for pulmonary hypertension and erectile dys-
functionsuchasinhaledNO,PDE5inhibitors,andrecentlysGCstimulators.Thethera-
peuticarmamentariumformanyotherdisordersisexpectedinthenearfuture.There
arecurrentlynumerousactivebasicandclinicalresearchprogramsinuniversitiesand
industries attempting to develop novel therapies for many diseases and medical
applications.
ABBREVIATIONS
ADMA asymmetricdimethylarginine
BH4 tetrahydrobiopterin
cGMP cyclic30-50 guanosinemonophosphate
CHF congestiveheartfailure
COPD chronicobstructivepulmonarydisease
CTEPH chronicthromboembolicpulmonaryhypertension
EDCF endothelium-derivedcontractingfactor
EDRF endothelium-derivedrelaxingfactor
FAD flavinadeninenucleotide
FMN flavinmononucleotide
GTP guanosinetriphosphate
HAT histoneacetyltransferase
HDAC histonedeacetylase
L-NMMA NG-monomethtyl-L-arginine
NO nitricoxide
NOS nitricoxidesynthase
O (cid:2)(cid:3) superoxideanion
2
ONOO2 peroxynitrite
PAH pulmonaryarterialhypertension
PDE phosphodiesterases
PH pulmonaryhypertension
sGC solubleguanylylcyclase
Endothelium-DependentNO–cGMPPathway 3
1. INTRODUCTION
In Oct. 1998 the Nobel Prize in Physiology and Medicine was
awardedtoRobertFurchgott,LouisIgnarro,andFeridMuradfortheirdis-
coveriesofnitricoxide(NO)asakeymoleculeinthecardiovascularsystem.
In the 1970s, Ferid Murad’s group showed that nitroglycerin and other
nitrovasodilators acted by forming nitric oxide and increasing soluble gua-
0 0
nylyl cyclase (sGC) activity and cyclic 3-5 guanosine monophosphate
(cGMP) formation, thus leading to smooth muscle relaxation (Arnold,
Mittal, Katsuki, & Murad, 1977; Katsuki, Arnold, & Murad, 1977;
Murad, Mittal, Arnold, Katsuki, & Kimura, 1978). In 1980, Furchgott’s
group demonstrated that the presence of endothelial cells was essential for
the relaxation induced by acetylcholine, bradykinin, adenosine triphos-
phate, and histamine, and these substances released a relaxing factor from
endothelial cells, which they named at that time as endothelium-derived
relaxingfactor(EDRF)(Furchgott&Zawadzki,1980).In1986Muradpro-
posed that EDRF is an “endogenous nitrovasodilator” and that NO
explainedtheeffectsofEDRFoncyclicGMPformationandvascularrelax-
ation. Later in 1997 Ignarro, Furchgott, and Moncada concluded that
EDRF is nitric oxide (Ignarro, Buga, Wood, Byrns, & Chaudhuri, 1987;
Palmer, Ferrige, & Moncada, 1987).
Nitricoxideisanimportantgasandfreeradicalthatisubiquitouslysyn-
thesized in various cells by three different types of nitric oxide synthases
(NOSs), namely endothelial (eNOS, NOS-3), neuronal (nNOS, NOS-1),
andinducible(iNOS,NOS-2)(Nakane,Schmidt,&Murad,1991).Nitric
oxide stimulates its intracellular receptor sGC, which converts guanosine
triphosphate (GTP) into cGMP. The physiological effects of cGMP are
exerted through the activation of cGMP-dependent protein kinases,cyclic
nucleotide-gated ion channels, and the activation and/or inhibition of
PDEs.In thecardiovascularsystem, NO–sGCsignaling playsan important
role for antithrombotic and antiatherogenic agents by inhibiting platelet
aggregation (Marcondes et al., 2006), leukocyte adhesion and chemotaxis
(Conran et al., 2001), and vascular smooth muscle relaxation (Murad,
1986;Rapoport&Murad,1983).Alterationson(1)formationandNObio-
availabilitymainlyduetobylowerproductionbyNOSand/orgreaterinac-
tivationandclearanceand(2)cGMPlevelsaresomeofthemajoralterations
seen in cardiovascular- and vascular-related diseases, such as hypertension,
4 F.Z.Mónicaetal.
pulmonaryhypertension(PH),congestiveheartfailure(CHF),diabetes,and
erectile dysfunction.
Thisreviewwillbedividedintothreesections:(1)howendothelialdys-
function and NO deficiency lead to cardiovascular diseases; (2) how sGC
regulation leads to beneficial effects on disorders of the circulation system,
and (3) the epigenetic regulation of NO–sGC pathway components in the
cardiovascular system.
2. PART I: NO–sGC SIGNALING
2.1 Endothelial Dysfunction and NO Deficiency
Theendothelium,thethininnerliningofvessels,canbeactivatedbychemical
and physical stimuli that lead to the formation and release of EDRF prosta-
cyclin, bradykinin, and contracting (EDCF) factors such as endothelin-1
and angiotensin II (Versari, Daghini, Virdis, Ghiadoni, & Taddei, 2009).
Endothelialcellsproduceawiderangeoffactorsthatcontrolcellularadhesion,
smooth muscle reactivity, proliferation, and vessel wall inflammation and
atherogenesis. The imbalance between vasodilating and vasoconstricting
substances leads to endothelium dysfunction, which contributes to several
cardiovascular diseases including hypertension, heart failure (HF), PH, and
vascular-related diseases such as diabetes and erectile dysfunction (Bian,
Doursout, & Murad, 2008; Mo´nica, Murad, & Bian, 2014; Murad, 2006).
Nitric oxide plays a pivotal role in vascular hemostasis since a defect in
NO production or activity has been proposed as a major mechanism that
leadstoendothelialdysfunction.Besidesitsvasorelaxingactionthatopposes
the effects of EDCF, NO also inhibits platelet adherence and aggregation
(Marcondes et al., 2006), leukocyte adhesion/infiltration (Benjamim
et al., 2002), and proliferation of vascular smooth muscle cells (VSMCs)
(Wolf,Rasmussen,Sherman,Bundens,&Hye,1995)andpreventstheoxi-
dation of low-density lipoprotein cholesterol (Mehta, Bryant, & Mehta,
1995).LowerNOproductionandincreasedNOinactivationaremajorfac-
torsthatcontributetotheinitiation,progression,andclinicalphenotypeof
atherosclerosis (Fig. 1).
2.2 eNOS (NOS-3) Uncoupling
NOSsconvertL-arginineintoL-citrullineandNOinareactiondependent
on oxygen, heme, tetrahydrobiopterin (BH ), nicotinamide adenine dinu-
4
cleotide phosphate (NADPH), calmodulin, flavin adenine nucleotide
Endothelium-DependentNO–cGMPPathway 5
Smooth muscle
Platelet contraction
aggregation
Smooth muscle
proliferation
Monocyte and NO
platelet adhesion
Expression
adhesion
molecules
Oxidation LDL
Fig.1 Nitricoxide(NO)playsakeyroleintheinitiationofendothelialdysfunctionsince
theloweritslevel,thehigherwillbeplateletandmonocyteaggregationandadhesion,
smoothmusclecontractility,expressionofadhesionmolecules,andoxidationoflow-
densityprotein.Theseeventscontributetotheformationofatheroscleroticplaque.
(FAD), and flavin mononucleotide (FMN) (Schmidt, Smith, Nakane, &
Murad,1992).eNOS(NOS-3)isthemajorisoformexpressedintheendo-
theliallayerandisthecriticalenzymeinthemaintenanceofvascularhomeo-
stasis by NO production.
ThefunctionaleNOS(NOS-3)isadimerthatconsistsofaC-reductase
domain(bindsNADPH,FAD,andFMN),whichislinkedtotheN-terminal
oxygenase domain (binds the prosthetic group, BH , molecular oxygen,
4
and the substrate L-arginine). Heme group is essential for the linkage
between the reductase and oxygenase domains and allows the NOS
dimerization. BH , an essential cofactor of eNOS (NOS-3), is necessary
4
for optimal since it facilitates the NADPH electron transferring from
the eNOS reductase to the oxygenase domain. There is evidence that
eNOS uncoupling is the underlying cause of endothelial dysfunction seen
in atherosclerotic lesions (Herranz et al., 2012; Margaritis et al., 2013),
hypercholesterolemia (Warnholtz et al., 1999), hypertension (Li et al.,
2006), and diabetes (Cassuto et al., 2014). In these situations, NAPDH
oxidases are upregulated and the levelsof reactive oxygen species as super-
oxideðO (cid:2)(cid:3)Þ,whichreactswithNOtoproduceperoxynitrite(ONOO(cid:3)),
2
(cid:3)
are high. The cofactor BH is sensitive to oxidation by ONOO and
4
(cid:2)(cid:3)
lowerlevels ofBH promotesuperoxide O rather thanNO production
4 2
by eNOS in a process referred as eNOS uncoupling (Fo€rstermann &
Mu€nzel, 2006). L-Arginine deficiency or the presence of NOS inhibitors
may lead to eNOS uncoupling (Sydow & Mu€nzel, 2003) (Fig. 2).
