Hypoxia-Inducible Factor-1 in Physiological and Pathophysiological

Review ArticleHypoxia-Inducible Factor-1 in Physiological andPathophysiological Angiogenesis Applications and Therapies

Agnieszka Zimna and Maciej Kurpisz

Institute of Human Genetics Polish Academy of Sciences Strzeszynska 32 60-479 Poznan Poland

Correspondence should be addressed to Maciej Kurpisz kurpimacmanpoznanpl

Received 19 December 2014 Revised 10 April 2015 Accepted 17 April 2015

Academic Editor Alessio DrsquoAlessio

Copyright copy 2015 A Zimna and M Kurpisz This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The cardiovascular system ensures the delivery of oxygen and nutrients to all cells tissues and organs Under extended exposureto reduced oxygen levels cells are able to survive through the transcriptional activation of a series of genes that participate inangiogenesis glucose metabolism and cell proliferation The oxygen-sensitive transcriptional activator HIF-1 (hypoxia-induciblefactor-1) is a key transcriptional mediator of the response to hypoxic conditions The HIF-1 pathway was found to be a masterregulator of angiogenesisWhether the process is physiological or pathological HIF-1 seems to participate in vasculature formationby synergistic correlations with other proangiogenic factors such as VEGF (vascular endothelial growth factor) PlGF (placentalgrowth factor) or angiopoietins Considering the important contributions of HIF-1 in angiogenesis and vasculogenesis it shouldbe considered a promising target for treating ischaemic diseases or cancer In this review we discuss the roles of HIF-1 in bothphysiologicalpathophysiological angiogenesis and potential strategies for clinical therapy

1 Angiogenesis

The circulatory system is the first biological system that isestablished during mammalian development [1] Vessel for-mation occurs via only two basicmechanisms vasculogenesisand angiogenesis [2] During embryonic development theprimary vascular plexus is formed by vasculogenesis Thisphenomenon involves de novo blood vessel formation fromprecursor cells called angioblasts (precursors of endothelialcells) whereas angiogenesis is the process by which bloodvessels are formed from preexisting vessels This processinvolves the remodelling of blood vessels into the large andsmall vessels that are typical for networks containing arteriescapillaries and veins [3 4] Angiogenesis occurs in adultorganisms and in embryos during development [3]

Angiogenesis consists of several basic steps Briefly bio-logical signals such as hypoxia ischaemia andor bloodvessel damage upregulate the expression of proangiogenicgrowth factors that activate their receptors [5 6] Vascularpermeability increases in response to VEGF thereby allowingthe extravasation of plasma proteins that form a primitive

scaffold for migrating endothelial cells [7] Angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2) exert antagonisticfunctions during vessel development Ang-1 which is aknown natural inhibitor of vascular permeability protectsagainst plasma leakage whereas Ang-2 is involved in vesseldestabilisation via the detachment of smooth muscle cellsand the promotion of permeabilisation [8 9] Subsequentlymatrix metalloproteinases (MMPs) enhance angiogenesisthrough the degradation of matrix components [10] Pro-liferating endothelial cells migrate to distant sites and thenassemble as a solid cord that subsequently forms a lumen[11] Integrins 120572120573 promote endothelial cell adhesion andmigration whereas VE-cadherin increases cell survival andpromotes endothelial cell adhesion [12ndash14] Once the vesselsare formed pericytes and smooth muscle cells surround thenewly created capillaries to stabilise the walls and to preventleakage Other factors such as Ang-1 PDGF-BB (platelet-derived growth factor BB) and PDGFR (platelet-derivedgrowth factor receptor) participate in thematuration of bloodvessels [8 15] Further expansion of the lumen diameter iscalled arteriogenesis [11]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 549412 13 pageshttpdxdoiorg1011552015549412

2 BioMed Research International

Angiogenesis occurs in physiological states such asembryonic development wound healing or vessel penetra-tion into avascular regions and in pathological states suchas solid tumours formation eye diseases or chronic inflam-matory disorders such as rheumatoid arthritis psoriasisand periodontitis [16 17] Pathophysiological angiogenesisexhibits differences in molecular pathways in comparisonto physiological angiogenesis Mutations in oncogenes andtumour suppressor genes and disruptions in growth factoractivity play crucial roles during tumour angiogenesis [17]The activation of the most prominent proangiogenic factorVEGF might be due to physiological stimuli such as hypoxiaor inflammation or due to oncogene activation and tumoursuppression function loss [18 19] Additionally physiologicalangiogenesis such as that which occurs during embryonicdevelopment or wound healing seems to be dependent onVEGF signalling whereas tumour angiogenesis adopts theability to shift its dependence from VEGF to other proan-giogenic pathways for example through the recruitment ofmyeloid cells and the upregulation of alternative vasculargrowth factors (PlGF and FGF fibroblast growth factor) [17]Moreover tumour vessels are distinct from normal vascula-ture because they are disorganised and tortuous Many mor-phological and functional differences exist between normaland tumour vasculature For instance tumour vessels areleakier than normal vessels and endothelial cells growingwithin tumours carry genetic abnormalities [20 21] Elucidat-ing the molecular mechanism of pathological angiogenesismight lead to the identification of potential therapeutictargets

Angiogenesis is a multistep process that requires theinvolvement ofmany biological signals and stimuli regardlessof whether it is a physiological or pathological action Proan-giogenic factors are activated in response to some physicalsignals Blood vessel damage infarction and blood flowreduction lead to decreases in O

2supply [11] This state is

called hypoxia which is a potent angiogenic trigger thatstimulates proangiogenic factor activity

2 Hypoxia

21 O2 Homeostasis Constant oxygen supply is essential forproper tissue function development and homeostasis Thusthe vasculature network plays a crucial role in delivering oxy-gen particles (O

2) nutrients and other molecules within the

entire human body [22] Significantly the first physiologicalsystem that becomes functional during mammalian embry-onic development is the circulatory system Governing O

2

homeostasis in tissues by supplying an oxygen concentrationadequate for the demand generated by the metabolic outputsof the tissue is essential Oxygenic balance can be upset byrapid cellular division during embryonic development bytumour growth or by vasculature dysfunction due to vesselocclusion or rupture [23] Notably normal physiological O

2

concentrations vary greatly from normal oxygen tension inthe air depending on the type of tissue For instance arterialblood has a normal pO

2of 14 myocardium 10 and

skeletalmuscle 5 In contrast the natural pO2levels of bone

marrow thymus and cartilage are at or below 1 [24 25]The

state when the O2level decreases relative to physiological lev-

els (characteristic for particular tissues) is called hypoxia [1]

22 Hypoxia-Inducible Factor-1 Adaptation to low oxygentension (hypoxia) in cells and tissues requires the acti-vation of several genes that participate in angiogenesiscell proliferationsurvival glucose and iron metabolism Ineukaryotic cells hypoxia-inducible factor-1 (HIF-1) is a pri-mary transcriptional mediator of the hypoxic response andmaster regulator of O

2homeostasis [26] Hypoxia-inducible

factor-1 was first discovered as a transcription factor thatregulates erythropoietin (EPO) expression in response to lowoxygen levels in the blood [27 28] HIF-1 consists of twodifferent subunits 120572 and 120573 (also known as aryl hydrocarbonnuclear receptor translocator (ARNT)) both subunits aremembers of the basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor family [29 30] PAS and bHLHmotifs are required for heterodimerisation between the HIF-1120572 and HIF-1120573 subunits [31] Additionally the bHLH domainof the HIF-1120572ARNT dimer is essential for DNA bindingon hypoxia response elements (HREs) with the consensussequence (GACGTG) in the promoters or enhancers oftarget genes [32] Transcriptional activation and interactionswith coactivators (such as CBPp300) of HIF-1120572 aremediatedby two domains that is C-TAD andN-TAD which are on theC-terminus of the protein [33ndash35] HIF-1120573 is a constitutivelyexpressed subunit whereas HIF-1120572 is translated continuouslyand degraded subsequently through ubiquitination undernormoxic conditions [36 37]Thus far three isoforms ofHIF-120572 have been discovered (Figure 1(a)) that is the previouslydiscussed subunit HIF-1120572 and two other subunits HIF-2120572(also called endothelial PAS protein (EPAS)) and HIF-3120572(IPAS) The second isoform is expressed constitutively in theendothelium lung and cartilage and shares 48 amino acidsequence identity with HIF-1120572 The third protein HIF-3120572also called inhibitory PAS (IPAS) is a negative regulator ofHIF-1 that dimerises with the HIF-1120572 subunit and preventsits DNA-binding activity The entire set of HIF-120572 subunitsdimerises with ARNT and binds to HREs [38ndash40]

Under extended exposure to hypoxic conditions HIF-1120572 is expressed as long as a balance between O

2supply

and usage in tissues cannot be reached [36 37] Whenoxygen tension is sufficient de novo synthesised cytoplasmicHIF-1120572 is hydroxylated by a family of prolyl hydroxylaseenzymes (PHDs) on proline 402 and 564 residues locatedwithin ODDD (O

2-dependent degradation domain) [41 42]

The hydroxylation of the abovementioned prolines is thekey mechanism of negative regulation of HIF-1120572 activityand results in the binding of the von Hippel-Lindau (VHL)E3 ligase complex which ubiquitinates HIF-1120572 targetingit to proteasomal degradation [43ndash45] The second majormechanism that modulates HIF-1120572 activity is the hydrox-ylation of asparagine residue 803 (Asn803) in the C-TADdomain In this case asparagine is hydroxylated undernormoxic conditions by factor inhibiting HIF-1 (FIH-1)which prevents the interaction of HIF-1120572 within CBPp300(CREB-binding proteinE1A binding protein p300) [46ndash49]Hydroxylases (PHDs and FIH-1) are strictly Fe(II)- and2-oxoglutarate-dependent dioxygenases that are activated

BioMed Research International 3

bHLH PAS ODDD N-TAD

bHLH PAS ODDD N-TAD C-TAD

bHLH PAS ODDD

NLS NLSPro 402 Pro 564

N-TAD C-TADHIF-1120572 826aa

HIF-2120572-EPAS 870aa

HIF-3120572-IPAS 557aa

(a)

CBP

bHLH PAS TAD

NLS

Normoxia

Hypoxia

bHLH PAS ODDD N-TAD

N-TAD

C-TAD

Pro 402 Pro 564OH OH

Asn 803OH

CBPp300

bHLH PAS ODDDAsn 803Pro 564Pro 402

pVHL

Proteasomaldegradation

HIF-1120572 826aa

N-TAD C-TAD

HIF-1120573 789aa

Dimerization with HIF-1120573

pVHL

p300

(b)

Figure 1 Schematic representation of HIF-120572 gene structures andDNA binding (a) HIF-1120572 andHIF-2120572 contain the following domains a nuclearlocalisation domain (NLS) DNA binding and dimerisation domains (bHLHPAS) oxygen-dependent degradation domain (ODDD) andcofactor interaction and transcriptional activity domains (N-TADC-TAD) HIF-3120572 lacks a C-TAD domain NLS nuclear localisation signalbHLH basic helix-loop-helix domain PAS Per-ARNT-Sim motif ODDD oxygen-dependent degradation domain N-TAD N-terminaltransactivation domain C-TAD C-terminal transactivation domain (b) Dimerisation of HIF-1120572 with HIF-1120573 under hypoxic conditionsresults in the formation of the HIF-1 transcription factor which binds to hypoxia response elements (HREs) and activates the transcriptionof O2-dependent genes (according to [134])

only in the presence of molecular oxygen Under hypoxicconditions substrates and coactivators of hydroxylation suchas O2 Fe(II) and 2-oxoglutarate become limited which leads

to the attenuation of HIF-1120572 hydroxylation [50 51] HIF-1120572accumulates in the cytosol and is subsequently translocatedinto the nucleus where it dimerises with the HIF-1120573 subunitThe HIF-1120572120573 dimer binds to HREs that are located withinO2-regulated genes (Figure 2) [34 35] HIF-1 target gene

members include a stress response gene family that mediatesthe adaptation of cellstissues to chronic or acute hypoxia thisfamily includes glucose transporters glycolytic enzymes andangiogenic and haematopoietic growth factors [52]

23 HIF-1 Activity and Target Genes Recently HIF-1 hasbeen shown to regulate more than 2 of genes in vascular

endothelial cells either directly or indirectly [53] The mod-ulation of cell responses is followed by the transcriptionalactivation of target genes by theHIF-1120572120573 dimer (Figure 1(b))[54 55] For example hypoxia upregulates the expression oferythropoietin (EPO) which is required for red blood cellproductionThe generation of new erythrocytes increases thedelivery of oxygen to tissues to reach O

2homeostasis [56]

Additionally low oxygen levels influence glucosemetabolismin cells Under hypoxic conditions cells generate only 2ATP molecules by oxygen-independent glycolysis instead of38 ATP molecules by the oxygen-dependent tricarboxylicacid cycle (TCA) under normoxic conditions [57 58] Tomaintain the energetic balance under hypoxic conditionscells increase their ability to produce ATP by increasingglucose uptake via the enhancement of glycolytic enzyme and


CBP

CBPp300CBPp300

p300

Transcr

iption of ta

rget g

ene

Normoxia Hypoxia

HIF-1120572

HIF-1120572

HIF-1120572

HIF-1120572HIF-1120572

HIF-1120572HIF-1120572

HIF-1120572

HIF

-1120572

HIF-1120572

CBPp300

HIF

-1120573

Cytosol Nucleus HRE elements

PHD PHD


pVHL

Ubiquitin

OH-Pro 402

OH-Asp 803

OH-Asp 803

564

CBPp300 cannot bind tohydroxylated Asp 803

HIF-1 degradation

FIHFIH

Pro 402 564

Asp 803

2-Oxoglutarate+ O2 + Fe+2


Figure 2 HIF-1120572 under normoxic and hypoxic conditions In the presence of molecular oxygen 2-oxoglutarate and Fe2+ HIF-1120572 ishydroxylated on proline 402 and 564 residues located within ODDD (O

2-dependent degradation domain) by prolyl hydroxylase enzymes

(PHDs) Hydroxylation results in the binding of the von Hippel-Lindau (VHL) E3 ligase complex which ubiquitinates HIF-1120572 targeting itto proteasomal degradation The hydroxylation of the asparagine residue prevents CBPp300 binding to HIF-1120572 Under hypoxic conditionssubstrates and coactivators of hydroxylation such as O

2 Fe(II) and 2-oxoglutarate become limited which leads to the attenuation of HIF-

1120572 hydroxylation HIF-1120572 accumulates in the cytosol and is subsequently translocated into the nucleus where it dimerises with the HIF-1120573subunit The HIF-1120572120573 dimer binds to HREs and regulates target gene expression

transporter expression [59ndash61] Furthermore cell prolifera-tion and survivalmay be enhanced under hypoxic conditionsFactors such as IGF-2 (insulin growth factor-2) and TGF120572(transforming growth factor) are upregulated through HIF-1 activity [62 63] Additionally myoblasts cultured underhypoxic conditions show increased proliferation comparedto cells maintained under normoxic conditions Myogenicgene expression analysis revealed that the genes MyoD andMyf5 (both involved in myogenic cell proliferation anddifferentiation) were upregulated in hypoxic cells whereasno significant influence of myogenic gene expression wasobserved in normoxic cells [64]

3 Hypoxia-Induced Angiogenesis

31 Physiological Vasculogenesis and Angiogenesis under Hyp-oxic Conditions Vasculogenesis is a characteristic embryonicprocess that involves de novo blood vessel formation and thatleads to establishing a primary vascular plexus O

2tension

plays a crucial role in organogenesis and vasculogenesisduring embryonic development [1] During the first stageof embryogenesis before the circulatory system developsthe oxygen tension is relatively low and does not exceed3 [65 66] The developing embryo requires an increase inthe oxygen level which leads to the formation of primaryvessels from angioblasts Because the uterine environment ishypoxic it is thereby obvious that hypoxiamay be the primarystimulus of vessel formation [3 4] Hypoxia also stimulatesEC (endothelial cell) behaviour Under in vitro conditionsHIF-1120572 promotes the arterial differentiation of endothe-lial progenitor cells (EPCs) over venous differentiation byregulating the expression of genes that inhibit the venousspecification factor Coup-TFII (Hey2 and delta-like ligand 4(DII4)) [67] In contrast HIF-1120573 enhances the differentiationof hemangioblasts frommesodermal progenitors [68]Mousemodel studies have demonstrated the participation of bothHIF-1 dimer subunits in vasculogenesis [69 70] For instanceHIF-1120572- orHIF-1120573 (ARNT) deficientmouse embryos showed


aberrant placental architecture with fewer foetal blood vessels[71ndash73] Additionally a lack of the HIF-1120573 gene results indefective vascular development in the yolk sac branchialarches cranium and somites [74 75]These defects are lethalfor embryos at day 105 HIF-1 subunit deficiencies markedlydecrease VEGF mRNA and protein expression leading todefects in blood vessel formation and neural fold termination[70 74 75]

Current studies have indicated that hypoxia and HIF-1 expression in adult organisms may contribute to angio-genesis in the following ways by transcriptionally activatingseveral angiogenic genes and their receptors (VEGF PlGFPDGFB ANGPT1 and ANGPT2) [76 77] by regulatingproangiogenic chemokines and receptors (SDF-1120572 stromalcell derived factor 1120572 and S1P sphingosine-1-phosphate andreceptors CXCR4 C-X-C chemokine receptor type 4 andS1PRs sphingosine-1-phosphate receptors) thus facilitatingthe recruitment of endothelial progenitor cells to the siteof hypoxia [78] and by enhancing EC proliferation anddivision (regulating genes involved in the cell cycle and DNAreplication) [53] Summarising these findings we concludethat HIF-1 can orchestrate the process of angiogenesis

HIF-1 participates in every step of angiogenesis Crossactivity between HIF-1 and proangiogenic factors is a basicrelationship during capillary formation under hypoxia Everystep of the vessel formation cascade is supported by HIF-1Notably vascular endothelial growth factor isoforms (VEGF-A VEGF-B VEGF-C and VEGF-D) are the primary factorsthat participate in angiogenesis [79] and angiogenesis ini-tiated by hypoxia and by HIF-1 is often VEGF-dependentprimarily because HIF-1 is a master stimulator of vascularendothelial growth factor The sprouting of new vessels isa complex process that involves a wide range of proangio-genic factors and their receptors Under hypoxic conditionsHIF-1 accumulation upregulates the principal proangiogenicfactor VEGF directly [80 81] VEGF activity induces theexpression of Flt-1 (fms-related tyrosine kinase) and KDR(kinase insert domain receptor) receptors [82 83] As longas the oxygen balance is disrupted VEGF will bind itsreceptors and stimulate capillary outgrowth AdditionallyVEGF is known as a factor that enhances the expressionof other proangiogenic factors such as PlGF and FGF Wecan assume that when the blood flow is sufficient to supplyoxygen to cells and tissues HIF-1120572 will be degraded therebyinhibiting VEGF gene and protein expression and stoppingthe entire cascade of proangiogenic factors In conclusionHIF-1 may regulate the expression of proangiogenic factorseither directly (by binding to HREs) or indirectly (cascadeeffect) [84] Aside from VEGFs other factors participatein angiogenesis including placental growth factor (PlGF)platelet-derived growth factor (PDGF) angiopoietins 1 and2 (ANGPT1 and ANGPT2) andmetalloproteinases (MMPs)Receptors such as Flt-1 KDR Tie 1 and Tie 2 that transducethese signals and thereby maintain the cascade of new vesselformation are also important Analyses of proangiogenicgenes revealed the presence of HREs within the promotersof some genes [85 86]

As was mentioned above angiogenesis is a multistepprocess [87] During the first step hypoxia and HIF-1

stimulate VEGF and their receptors directly and the cascadeof new vessel creation begins Second the extracellularmatrix must be degraded by metalloproteinases to allow themigrating endothelial cells to form tubes Metalloproteinase2 (MMP-2) expression has been shown to be enhancedby HIF-1120572 [88] Next integrins 120572120573 (induced by HIF) [89]stimulate endothelial cell proliferation and adhesion andHIF-1 controls EC behaviour The Manalo group examinedthe influence of hypoxia and HIF-1 on the endothelial celltranscriptome Microarray analysis demonstrated the upreg-ulation of several genes that are responsible for the cell cycleand DNA replication Additionally it was proven that ECscultured under low density and hypoxic conditions exhibitincreased proliferation In conclusion HIF-1 can stimulatecell proliferation and division under hypoxia-induced angio-genesis by transcriptionally regulating the genes involvedin the basic biological functions of cells [53] Finally thelast step of angiogenesis is vessel maturation which involvesthe recruitment of vascular supporting cells (pericytes andsmooth muscle cells) and the formation of the basementmembrane In this case HIF-2120572 is a primary enhancer ofgenes that determine blood vessel stabilisation For examplefibronectin is a component of the basement membraneand HIF-2120572 is involved in the upregulation of this gene[90] Considering these findings we can assume that HIF-1regulates almost every step of capillary formation

Increasing knowledge regarding the influence of HIF-1 on angiogenesis has been applied to in vitro studies thatattempt to develop treatments for ischaemic diseases Forexample the adenoviral transfer of HIF-1120572 and HIF-2120572 intorabbit ischaemic limbs resulted in an increase in blood flowfollowed by induction of vessel sprouting Additionally thesecapillaries were highly enlarged compared to those of rabbitstransduced with VEGF-A As another consequence of VEGFgene transfer the newly formed vessels were leaky and thearea of transfer was surrounded by oedema compared toanimals treated with AdHIF-1120572 and AdHIF-2120572 [91] In turnin vivo studies demonstrated that the deletion of HIF-2120572 inmurine ECs caused VEGF-induced acute vessel permeabilityFurthermore immortalised HIF-2120572-deficient ECs exhibiteddecreased adhesion to extracellular matrix proteins anddiminished expression of fibronectin integrins endothelinB receptor angiopoietin-2 and delta-like ligand 4 (Dll4)[90] Artificial hypoxia induced by CoCl

2(05mM) stimu-

lates angiogenic responses in SCAPs (stem cells from apicalpapilla) cocultured with HUVECs (human umbilical veinendothelial cells) Hypoxic conditions induced upregulatedHIF-1120572 and VEGF expression in HUVECs whereas ephrin-B2 gene was enhanced in SCAPs Notably this gene playsa central role in heart morphogenesis and angiogenesis byregulating cell adhesion and cell migration Additionallysynergistic effects between HIF-1 VEGF and ephrin-B2 ledto an increase in the endothelial tubule number vessel lengthand branching points [92]

In conclusion hypoxia-induced angiogenesis is a com-plex process that has been tested in both in vitro and in vivostudies Moreover the application of HIF-1 therapy in phase Iclinical studies of critical limb ischaemia resulted in completerecovery of the ischaemic region [93] Increased knowledge


of the role of HIF-1 in angiogenesis may provide promisingtreatment methods for ischaemic diseases as partly describedin Section 41 of this paper

32 The Effect of Hypoxia on Pathophysiological Angiogenesisand the Role of HIF-1 in Tumour Angiogenesis Low oxygentension has been linked with many pathophysiological disor-ders and human diseases Hypoxia is a component of tumourdevelopment and metastasis while angiogenesis is principalfor tumour growth and progression [94 95] Current studieshave suggested that in terms of malignant transformation theactivation of HIF-dependent angiogenesis in cancer occursin two basic ways by hypoxic conditions prevailing in thetumour cell mass or by genetic alterations caused by tumourtransformation genetic disorders or molecular interactionsthat stimulate HIF activity irrespective of oxygen tensionUndeniably HIF plays a critical role in stimulating angio-genesis However notably proangiogenic activity is directlylinked with HIF-dependent VEGF activation which resultsin an ldquoangiogenic switchrdquo in growing tumour masses [96]

Hypoxia is best characterised as an HIF activator Whenintensively proliferating cells form a solid tumour the balancebetween oxygen supply and demand is impaired thereforehypoxic environments (intratumoural hypoxia) prevail ingrowing cell masses This prevalence is the reason whynewly activated HIF-1120572 is not ubiquitinated and targeted toproteasomal degradation Accumulating HIF-1 upregulatesthe expression of a number of proangiogenic genes includingVEGF and their receptors Flt-1 Flk-1 Ang-1 Ang-2 and Tie-2receptor and all of these genes are essential for sprouting newvessels Among these factors VEGF is considered a primaryeffector of tumour angiogenesis Moreover the expressionof VEGF can enhance the expression of other proangio-genic factors and their receptors thus vessel outgrowth isstimulated by multiple factors [95 97] This phenomenonwhich is called ldquoangiogenic switchingrdquo allows tumour cells toinduce angiogenesis thereby stimulating tumour progressionby supplying oxygen and nutrients through the newly createdcapillaries [96]

The above-presented data suggest that hypoxia directlyenhances angiogenesis by promoting VEGF expression Incontrast HIF-1-dependent angiogenesis can be activatedby factors other than hypoxia HIF-1120572 accumulation doesnot always occur under low O

2tension Thus HIF-1 can

also be regulated by oxygen-independent mechanisms Themalignant transformation of cells may cause a whole rangeof genetic alterations that block the ubiquitination andproteasomal degradation of HIF-1120572 [84 98] Carcinogenesisis linked with aberrations in tumour suppressor genes alsoknown as antioncogene genes The protein products of thesegenes may control cell division the cell cycle or apoptosisWhen cells exhibit DNA damage these proteins are respon-sible for repressing the cell cycle or cell division or for pro-moting apoptosis The most important tumour suppressorsare pRb p53 p21 and PTEN Some evidence has indicatedthat altered antioncogenes can enhance angiogenesis viaHIF-dependent VEGF stimulation For instance deleting the p53tumour suppressor gene in a human cancer cell line promotesthe neovascularisation and growth of tumours in mice This

vascularisation was followed by increased HIF-1120572 levels andaugmented HIF-1-dependent transcriptional activation ofvascular endothelial growth factor (VEGF) [99] Addition-ally mutation in the tumour suppressor gene PTEN led tohypoxia-independent HIF-1120572 accumulation and to activatedHIF-1-mediated proangiogenic gene expression [100] Inbreast cancer HER2 (receptor tyrosine kinase) signallinginduces HIF-1120572 protein synthesis rather than inhibiting itsdegradation thus manifesting a novel mechanism of HIF-1-dependent VEGF expression regulation [101] These findingssuggest that not only the hypoxic environment can leadto angiogenesis in the case of malignant transformationAdditionally the genetic alteration of single gene may bea cause of hypoxia-independent stimulation of HIF Someevidence has indicated that genetic diseases are involvedin the activation of HIF and thereby the stimulation ofangiogenesis von Hippel-Lindau is a genetic disease thatpredisposes individuals to benign and malignant tumours[102] This condition is defined by a mutation in the VHLgene thus pVHL is not translated This protein is a ligasethat ubiquitinates HIF-1120572 and causes its degradation byproteasomes thus maintaining the balance of HIF-1 undernormoxia is crucial A loss of pVHL allows HIF-1120572 todimerise with HIF-1120573 and to activate the transcription of anumber of proangiogenic genes including VEGF Patientssuffering from this disease have frequent malignancies in thecentral nervous system andor retinal hemangioblastomasor clear cell renal carcinomas [103ndash106] However despitehomology between HIF-1120572 and HIF-2120572 there are evidencesthat in VHL-defective renal cell carcinoma HIF isoformsexhibit different or opposite effect on gene expression andproliferation of tumour cells [107] Genetic predisposition isnot the only factor that can stimulate oxygen-independentHIF angiogenesis involving VEGF gene activation Caseswhere some other molecules can stimulate HIF-dependentangiogenesis have been demonstrated As an example we canrecall a phenomenon based on the feedback loop of HIF-1 and a product of the anaerobic metabolic pathway Theabovementioned mechanism that activates HIF-1-dependentangiogenesis in cancer cells is the phenomenon called theWarburg effect Because anaerobic conditions prevail intumour cell masses cancer cells produce energy primarily byglycolysis As a side effect cells also produce high levels oflactates and pyruvates These molecules have been reportedto increase HIF-1120572 accumulation and to regulate hypoxia-inducible gene expression thus promoting VEGF transcrip-tion and translation and resulting in tumour angiogenesis[108]

Strong interactions between HIF-1120572 and VEGF lead tothe rapid activation of the vessel formation cascade howeverthese vessels exhibit several dysfunctions The process ofintratumoural vessel formation is a consequence of imbal-anced activity of angiogenic activators and inhibitors Thesevessels are not fully functional and exhibit structural abnor-malities A loss of stabilisation with pericytes and smoothmuscle cells and a lack of an arterial and venous phenotypelead to leakage poor blood flow and perfusion [109 110]However these vessels are able to deliver nutrients andoxygen to growing tumour cell masses Thus we can also


conclude that tumours with vasculature are more sensitive tovarious therapies Due to vascularisation chemotherapeuticsand inhibitors may be better distributed across tumour cellsthus leading to the inhibition of malignant cell growth

4 Hypoxia as a Potential Therapeutic Tool

Hypoxia-inducible factor-1 has been considered a potentialtherapeutic target in many diseases Predominantly HIF-1therapies focus on diseases common to developing countriessuch as ischaemic disorders (including cardiovascular diseaseand limb ischaemia) and cancer Nonetheless HIF-1 is alsoa potential therapeutic agent for treating endometriosis andblindness Depending on the type of disease and the expectedtherapeutic effect HIF-1 therapies have different approachesThe function of HIF-1 in therapies can be classified into twodifferent strategies HIF-1 upregulation (ischaemia) and HIF-1 inhibition (cancer and endometriosis) (Figure 3)

41 Activation of HIF-1-Dependent Angiogenesis in IschaemicDiseases The neovascularisation of ischaemic regions is afundamental assumption of therapeutic angiogenesis Theinduction of capillary outgrowth for therapeutic purposesis stimulated by the administration of angiogenic growthfactors or sequences that encode these proteins Thus farmultiple angiogenic factors such as VEGF PlGF FGF andPDGF have been applied in in vitro studies and in preclinicaland clinical trials [111ndash114] However therapies using only oneproangiogenic agent to initiate angiogenesis were shown tobe insufficient thus these therapies may require supplemen-tation with other factors that can stabilise new capillariesTherefore hypoxia-induced angiogenesis may be a successfulstrategy [115]HIF-1 regulates cell-type specific proangiogenicfactors and cytokines either directly or indirectly PromotingHIF-1 activity is essential in ischaemic diseases Thus farHIF-1 therapies have been based on two approaches theadministration of HIF-11205722120572 or the induction of HIF-1expression by the modificationadministrationinhibition ofmolecules associated with HIF activity

Basic strategies of therapeutic angiogenesis involve theadministration of proangiogenic factors using vectors com-bined therapies with stem cells or fusionrecombinant pro-teins Hypoxia-inducible gene transfer led to the enhance-ment of angiogenesis in both myocardium and ischaemicskeletal muscles Additionally AdHIF-1120572 and AdHIF-2120572injection did not induce tissue oedema in contrast to regionstreated with AdVEGF In conclusion HIF application inthis case resulted in the formation of stable and maturevessels compared to VEGF treatment where the newlyformed vessels were leaky [91] To prevent oxygen-dependentdegradation of HIF-1120572 attempts to modify this subunit weremade An alternative approach of applying AdCA5 aden-ovirus encoding a constitutively active form of the HIF-1120572subunit due to a deletion and a point mutation in the regionresponsible for O

2-dependent degradation was proposed

In a model of endovascular limb ischaemia intramuscularinjection of AdCA5 improved the recovery of blood flowby stimulating both angiogenesis and arteriogenesis [116]

Furthermore therapy with AdCA5 induced the upregulationof several proangiogenic genesproteins that are targets fortherapeutic angiogenesis in hindlimb and cardiac ischaemiamodels including FGF-2 hepatocyte growth factor MCP-1PDGF-B PlGF SDF-1 and VEGF Moreover AdCA5 injec-tion intomouse eyes enhanced neovascularisation inmultiplecapillary beds including those not responsive to VEGF aloneDue to the upregulation of PlGF and VEGF expressionafter AdCA5 treatment both genes acted synergisticallywhich led to neovascularisation of the retina [117] Combinedtherapy using AdCA5 gene therapy and prolyl-4-hydroxylaseinhibitor dimethyloxalylglycine- (DMOG-) treated BMDACs(bone marrow-derived angiogenic cells) acted synergisticallyto increase the recovery of blood flow after femoral arteryligation thereby preventing tissue necrosis This synergisticeffect is due to AdCA5 enhancement of BMDAC homingwhereas DMOG treatment increases the retention of graftedcells in the ischaemic tissues Another combined therapywith modified stem cells was applied in cerebral ischaemiaRat bone marrow-derived mesenchymal stem cells (BMSCs)were infected with adenoviral particles containing consti-tutively expressed HIF-1120572 due to mutations in proline 564and asparagine 803 sites Genetically modified cells weretransplanted in a rat middle cerebral artery occlusion model(MCAO) At 7 days after intervention improved motorfunction reduced cerebral infarction and increased VEGFprotein expression that led to revascularisationwere observed[118] The stabilisation and administration of HIF-1120572 may beachieved by the construction of a fusion protein For instanceDNA-binding and dimerisation domains of HIF-1120572 werefusedwith the transactivation domain of herpes simplex virus(VP16) A plasmid vector encoding this structure was usedin a rabbit hindlimb ischaemia model The administrationof HIF-1120572VP16 resulted in the improvement of blood flowin the ischaemic region as determined by an increase in thenumber of blood vessels [119] Additionally using promotersspecific for particular cells or tissues angiogenesis couldhave occurred in the designated site The construction of avector encoding O

2-independent HIF-1120572 under the keratin

14 promoter (K14-HIF-1ΔODD) resulted in the upregulationof HIF-1120572 in a skin The activation of HIF-1120572 increasedskin capillary density Moreover the new vessels were notleaky and enhanced skin vascularity was not associated withoedema compared to the capillaries that formed after K14-VEGF therapy [120]

Another approach leading to HIF-1-dependent angio-genesis is associated with the modificationadministrationof molecules that regulate HIF-1120572 activity The stabilisationof HIF-1120572 under normoxic conditions is focused on theinhibition of prolyl hydroxylase activity Asmentioned abovePHDs require O

2 Fe(II) and 2-oxoglutarate (2-OG) for their

enzymatic activity [50 51] The delivery of small moleculessuch as an iron chelator (DFO) or 2-OG analogue (N-oxalylglycine) may inhibit the enzyme activity [121 122]For instance HIF-1 can be enhanced by the suppressionof prolyl hydroxylase activity using dimethyloxalylglycine(DMOG)The systemic administration of DMOG in amousemodel of hindlimb ischaemia results in the elevation of HIF-1120572 protein expression which leads to neovascularisation in


Administration of

Ischaemic diseases

Inhibition of

dimerization

Inhibition of

transcription and transcriptional activity

protein translation

Cancer

Angiogenesis

Angiogenesis

(i) Acriflavine

(i) Bortezomib

(ii) Amphotericin B

(i) Silibinin

(i) Topotecan

(i) SAHA(ii) FK228

(i) DMOG(ii) DFO (iii) PR39

Modificationadministrationinhibition of molecules

associated with HIFs activity

HIF-11205722120572(i) AdHIF-1120572AdHIF-2120572(ii) AdCA5(iii) BMDAC AdCA5 + DMOG(iv) BMSC + constitutively

expressed HIF-1120572(v) HIF-1120572VP16(vi) K14 HIF-1ΔODDD

HIF-1120572 and HIF-1120573

HIF-1120572 DNA-binding activity

Inhibition of HIF-1120572

Attenuation of HIF-1120572

HIF-1120572 protein degradation

HIF-1120572

1120572Upregulation of HIF-

Downregulation of HIF-1120572

Figure 3 Different strategies in HIF-1120572 therapies Depending on the type of disease and the expected therapeutic effect different approachesare used in HIF-1 therapies HIF-1 upregulation (ischaemia) to induce angiogenesis or HIF-1 inhibition to attenuate angiogenesis (cancer)DMOG dimethyloxalylglycine DFO 2-OG analogue (N-oxalylglycine) PR39 macrophage-derived peptide AdHIF-1120572 and AdHIF-2120572adenoviral vector AdCA5 adenovirus encoding a constitutively active form of the HIF-1120572 BMDAC bone marrow-derived angiogenic cellsHIF-1120572VP16 DNA-binding and dimerisation domains of HIF-1120572 fused with the transactivation domain of herpes simplex virus K14-HIF-1ΔODD vector encoding O

2-independent HIF-1120572 under the keratin 14 promoter Silibinin nontoxic flavonoid topotecan chemotherapeutic

agent that is a topoisomerase inhibitor SAHA and FK228 histone deacetylase inhibitors acriflavine bortezomib amphotericin B anti-HIFdrugs

the infarcted region [121] Then capillary growth is followedby HIF-induced VEGF and Flk-1 upregulation A novelstrategy involves the preconditioning of rat bone marrow-derived mesenchymal stem cells (BMSCs) using DMOG toenhance their survival and therapeutic efficacy after trans-plantation into infarcted rat hearts After DMOG treatment

these cells exhibited enhanced expression of survival andproangiogenic factors such as HIF-1120572 vascular endothelialgrowth factor and glucose transporter 1Thus the transplan-tation of DMOG-treated BMSCs reduced heart infarctionsize and promoted angiogenesis in the ischaemic region [123]An alternative to the use of PHD inhibitor molecules is


the administration of a molecule that stabilises HIF-1120572 undernormoxic conditions PR39 which is a macrophage-derivedpeptide achieved this effect by inhibitingHIF-1120572 degradationthrough the ubiquitin-proteasome system The introductionof the PR39 peptide resulted in a significant increase inHIF-1120572 protein levels irrespective of ischaemia or hypoxiaTherefore angiogenesis can be induced in the postinfarctedmyocardium of transgenic mice [124]

42 Inhibition of HIF-1-Dependent Angiogenesis in CancerTherapies Cancer therapies are based on the targeted inhibi-tion of proangiogenic factors Current treatments are focusedon the inhibition of VEGF activity Because of studies regard-ing VEGF antiangiogenic therapy patients were treatedwith bevacizumab (VEGF inhibitor) or sunitinib (VEGFR2inhibitor) [125] Since the discovery that the HIF-1 pathwayregulates the activation of many proangiogenic factors intumours and may promote metastasis the HIF-1120572 subunithas been considered an attractive target for new cancertherapeuticsThe inhibition ofHIF-1-dependent angiogenesisinvolves the regulation of HIF-1120572 activity by molecules thatmodulate HIF-1120572 transcription and transcriptional activityHIF-1120572 and HIF-1120573 dimerisation HIF-1120572 protein translationHIF-1120572 DNA binding and HIF-1120572 protein degradation [24126]

Thus far the Food andDrugAdministration has approvedseveral anti-HIF drugs For instance bortezomib and ampho-tericin B functionally inhibit HIF-1120572 thereby preventingp300 recruitment by enhancing the interaction betweenFIH-1 and the HIF-1120572 C-terminal transactivation domain(C-TAD) [127 128] In contrast silibinin is a nontoxicflavonoid that is able to inhibit hypoxia-dependent HIF-1120572accumulation and to inhibit HIF-1 transcriptional activityin HeLa and hepatoma cells [129] Silibinin is also a potentinhibitor of cell proliferation Two other drugs SAHA andFK228 are histone deacetylase inhibitors that were found topromote HIF-1120572 degradation by upregulating p53 and VHLThese drugs have been successfully used in the United StatesHIF-1 activity might also be attenuated by previously usedchemotherapeutics such as anthracycline and doxorubicinwhich act by inhibiting HIF-1120572 DNA-binding activity andacriflavine which prevents HIF subunit dimerisation [130131]

Clinical trials are presently focused on other drugsthat may attenuate HIF-1-dependent angiogenesis Currentlyflavopiridol (Alvocidib) is in phase III clinical trials Thissynthetic flavonoid which is derived from an alkaloid Indianplant was found to be a potent inhibitor of the transcriptionof HIF-1120572 and of many other genes involved in cell cyclearrest [132 133] Other clinical studies (phases I and II) arebeing conducted with many other drugs that involve HIF-1degradation downregulation or inactivation

Conflict of Interests

The authors declare that no conflict of interests exists regard-ing the publication of this paper

Acknowledgments

This study was supported by the National Science Cen-tre Grants nos 201207NNZ301687 and 201413BNZ304646 and by the National Centre for Research and Devel-opment Grant no STRATEGMED12336245NCBR2014

References

[1] G L Semenza ldquoHypoxia-inducible factor 1 and cardiovasculardiseaserdquo Annual Review of Physiology vol 76 pp 39ndash56 2014

[2] P Carmeliet ldquoMechanisms of angiogenesis and arteriogenesisrdquoNature Medicine vol 6 no 4 pp 389ndash395 2000

[3] W Risau ldquoMechanisms of angiogenesisrdquo Nature vol 386 no6626 pp 671ndash674 1997

[4] W Risau and I Flamme ldquoVasculogenesisrdquo Annual Review ofCell and Developmental Biology vol 11 pp 73ndash91 1995

[5] P Carmeliet ldquoAngiogenesis in health and diseaserdquo NatureMedicine vol 9 no 6 pp 653ndash660 2003

[6] G D Yancopoulos S Davis N W Gale J S Rudge S JWiegand and J Holash ldquoVascular-specific growth factors andblood vessel formationrdquoNature vol 407 no 6801 pp 242ndash2482000

[7] B P Eliceiri R Paul P L Schwartzberg J D Hood J Lengand D A Cheresh ldquoSelective requirement for Src kinasesduringVEGF-induced angiogenesis and vascular permeabilityrdquoMolecular Cell vol 4 no 6 pp 915ndash924 1999

[8] G Thurston J S Rudge E Ioffe et al ldquoAngiopoietin-1 protectsthe adult vasculature against plasma leakagerdquo Nature Medicinevol 6 no 4 pp 460ndash463 2000

[9] P C Maisonpierre C Suri P F Jones et al ldquoAngiopoietin-2 anatural antagonist for Tie2 that disrupts in vivo angiogenesisrdquoScience vol 277 no 5322 pp 55ndash60 1997

[10] A R Nelson B Fingleton M L Rothenberg and L MMatrisian ldquoMatrix metalloproteinases biologic activity andclinical implicationsrdquo Journal of Clinical Oncology vol 18 no5 pp 1135ndash1149 2000

[11] E M Conway D Collen and P Carmeliet ldquoMolecular mecha-nisms of blood vessel growthrdquo Cardiovascular Research vol 49no 3 pp 507ndash521 2001

[12] B P Eliceiri and D A Cheresh ldquoThe role of 120572v integrins duringangiogenesis insights into potential mechanisms of action andclinical developmentrdquo Journal of Clinical Investigation vol 103no 9 pp 1227ndash1230 1999

[13] M Corada M Mariotti G Thurston et al ldquoVascularendothelial-cadherin is an important determinant of microvas-cular integrity in vivordquo Proceedings of the National Academy ofSciences of the United States of America vol 96 no 17 pp 9815ndash9820 1999

[14] K A Knudsen C Frankowski K R Johnson and M JWheelock ldquoA role for cadherins in cellular signaling anddifferentiationrdquo Journal of Cellular Biochemistry vol 30-31 pp168ndash176 1998

[15] P Lindahl M Hellstrom M Kalen and C BetsholtzldquoEndothelial-perivascular cell signaling in vascular develop-ment lessons from knockout micerdquo Current Opinion in Lipidol-ogy vol 9 no 5 pp 407ndash411 1998

[16] P J Polverini ldquoThe pathophysiology of angiogenesisrdquo CriticalReviews in Oral Biology and Medicine vol 6 no 3 pp 230ndash2471995


[17] A S Chung J Lee and N Ferrara ldquoTargeting the tumourvasculature insights from physiological angiogenesisrdquo NatureReviews Cancer vol 10 no 7 pp 505ndash514 2010

[18] R S Kerbel ldquoTumor angiogenesisrdquoTheNew England Journal ofMedicine vol 358 no 19 pp 2039ndash2049 2008

[19] N Ferrara and R S Kerbel ldquoAngiogenesis as a therapeutictargetrdquo Nature vol 438 no 7070 pp 967ndash974 2005

[20] R K Jain ldquoMolecular regulation of vessel maturationrdquo NatureMedicine vol 9 no 6 pp 685ndash693 2003

[21] J A Nagy S-H Chang A M Dvorak and H F Dvorak ldquoWhyare tumour blood vessels abnormal and why is it important toknowrdquo British Journal of Cancer vol 100 no 6 pp 865ndash8692009

[22] G L Semenza ldquoLife with oxygenrdquo Science vol 318 no 5847 pp62ndash64 2007

[23] J A Bertout S A Patel and M C Simon ldquoThe impact of O2

availability on human cancerrdquoNature Reviews Cancer vol 8 no12 pp 967ndash975 2008

[24] B L Krock N Skuli and M C Simon ldquoHypoxia-inducedangiogenesis good and evilrdquo Genes and Cancer vol 2 no 12pp 1117ndash1133 2011

[25] Z Yun Q Lin and A J Giaccia ldquoAdaptive myogenesis underhypoxiardquo Molecular and Cellular Biology vol 25 no 8 pp3040ndash3055 2005

[26] J I Bardos and M Ashcroft ldquoNegative and positive regulationof HIF-1 a complex networkrdquo Biochimica et Biophysica ActamdashReviews on Cancer vol 1755 no 2 pp 107ndash120 2005

[27] G L Semenza and G L Wang ldquoA nuclear factor induced byhypoxia via de novo protein synthesis binds to the human ery-thropoietin gene enhancer at a site required for transcriptionalactivationrdquo Molecular and Cellular Biology vol 12 no 12 pp5447ndash5454 1992

[28] G L Wang and G L Semenza ldquoCharacterization of hypoxia-inducible factor 1 and regulation of DNA binding activity byhypoxiardquo The Journal of Biological Chemistry vol 268 no 29pp 21513ndash21518 1993

[29] R J Kewley M L Whitelaw and A Chapman-Smith ldquoThemammalian basic helix-loop-helixPAS family of transcrip-tional regulatorsrdquo International Journal of Biochemistry and CellBiology vol 36 no 2 pp 189ndash204 2004

[30] G L Wang B-H Jiang E A Rue and G L SemenzaldquoHypoxia-inducible factor 1 is a basic-helix-loop-helix-PASheterodimer regulated by cellular O

2tensionrdquo Proceedings of the

National Academy of Sciences of theUnited States of America vol92 no 12 pp 5510ndash5514 1995

[31] S T Crews ldquoControl of cell lineage-specific development andtranscription by bHLH-PAS proteinsrdquo Genes and Developmentvol 12 no 5 pp 607ndash620 1998

[32] R H Wenger D P Stiehl and G Camenisch ldquoIntegration ofoxygen signaling at the consensus HRErdquo Sciencersquos STKE vol2005 no 306 article re12 2005

[33] J L Ruas L Poellinger and T Pereira ldquoFunctional analysisof hypoxia-inducible factor-1 alpha mediated transactivationIdentification of amino acid residues critical for transcriptionalactivation andor interaction with CREB-binding proteinrdquoTheJournal of Biological Chemistry vol 277 no 41 pp 38723ndash387302002

[34] B-H Jiang J Z Zheng SW Leung R Roe andG L SemenzaldquoTransactivation and inhibitory domains of hypoxia-induciblefactor 1120572 modulation of transcriptional activity by oxygentensionrdquoThe Journal of Biological Chemistry vol 272 no 31 pp19253ndash19260 1997

[35] C W Pugh J F OrsquoRourke M Nagao J M Gleadle and P JRatcliffe ldquoActivation of hypoxia-inducible factor-1 definitionof regulatory domains within the 120572 subunitrdquo The Journal ofBiological Chemistry vol 272 no 17 pp 11205ndash11214 1997

[36] B-H Jiang G L Semenza C Bauer and H H MartildquoHypoxia-inducible factor 1 levels vary exponentially over aphysiologically relevant range of O

2tensionrdquo The American

Journal of PhysiologymdashCell Physiology vol 271 no 4 pp C1172ndashC1180 1996

[37] S Salceda and J Caro ldquoHypoxia-inducible factor 1120572 (HIF-1120572) protein is rapidly degraded by the ubiquitin-proteasomesystem under normoxic conditions Its stabilization by hypoxiadepends on redox-induced changesrdquo Journal of BiologicalChemistry vol 272 no 36 pp 22642ndash22647 1997

[38] M Ema S Taya N Yokotani K Sogawa Y Matsuda and YFujii-Kuriyama ldquoA novel bHLH-PAS factor with close sequencesimilarity to hypoxia-inducible factor 1120572 regulates the VEGFexpression and is potentially involved in lung and vasculardevelopmentrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 9 pp 4273ndash42781997

[39] Y-Z Gu S M Moran J B Hogenesch L Wartman and CA Bradfield ldquoMolecular characterization and chromosomallocalization of a third 120572-class hypoxia inducible factor subunitHIF3120572rdquo Gene Expression vol 7 no 3 pp 205ndash213 1998

[40] Y Makino R Cao K Svensson et al ldquoInhibitory PAS domainprotein is a negative regulator of hypoxia-inducible gene expres-sionrdquo Nature vol 414 no 6863 pp 550ndash554 2001

[41] W G Kaelin Jr ldquoProline hydroxylation and gene expressionrdquoAnnual Review of Biochemistry vol 74 pp 115ndash128 2005

[42] V Srinivas L-P Zhang X-H Zhu and J Caro ldquoCharacteri-zation of an oxygenredox-dependent degradation domain ofhypoxia-inducible factor 120572 (HIF-120572) proteinsrdquo Biochemical andBiophysical Research Communications vol 260 no 2 pp 557ndash561 1999

[43] N Masson and P J Ratcliffe ldquoHIF prolyl and asparaginylhydroxylases in the biological response to intracellular O

2

levelsrdquo Journal of Cell Science vol 116 no 15 pp 3041ndash30492003

[44] N Masson C Willam P H Maxwell C W Pugh and P JRatcliffe ldquoIndependent function of two destruction domains inhypoxia-inducible factor-120572 chains activated by prolyl hydroxy-lationrdquo EMBO Journal vol 20 no 18 pp 5197ndash5206 2001

[45] L A McNeill K S Hewitson J M Gleadle et al ldquoThe useof dioxygen by HIF prolyl hydroxylase (PHD1)rdquo Bioorganic ampMedicinal Chemistry Letters vol 12 no 12 pp 1547ndash1550 2002

[46] K S Hewitson L A McNeill M V Riordan et al ldquoHypoxia-inducible factor (HIF) asparagine hydroxylase is identical tofactor inhibitingHIF (FIH) and is related to the cupin structuralfamilyrdquoThe Journal of Biological Chemistry vol 277 no 29 pp26351ndash26355 2002

[47] D Lando D J Peet D A Whelan J J Gorman and M LWhitelaw ldquoAsparagine hydroxylation of theHIF transactivationdomain a hypoxic switchrdquo Science vol 295 no 5556 pp 858ndash861 2002

[48] D Lando D J Peet J J Gorman D AWhelanM LWhitelawand R K Bruick ldquoFIH-1 is an asparaginyl hydroxylase enzymethat regulates the transcriptional activity of hypoxia-induciblefactorrdquo Genes and Development vol 16 no 12 pp 1466ndash14712002

[49] P C Mahon K Hirota and G L Semenza ldquoFIH-1 a novel pro-tein that interacts with HIF-1120572 and VHL to mediate repression


of HIF-1 transcriptional activityrdquo Genes and Development vol15 no 20 pp 2675ndash2686 2001

[50] W G Kaelin Jr and P J Ratcliffe ldquoOxygen sensing bymetazoans the central role of the HIF hydroxylase pathwayrdquoMolecular Cell vol 30 no 4 pp 393ndash402 2008

[51] C J Schofield and Z Zhang ldquoStructural and mechanisticstudies on 2-oxoglutarate-dependent oxygenases and relatedenzymesrdquo Current Opinion in Structural Biology vol 9 no 6pp 722ndash731 1999

[52] Q Ke and M Costa ldquoHypoxia-inducible factor-1 (HIF-1)rdquoMolecular Pharmacology vol 70 no 5 pp 1469ndash1480 2006

[53] D J Manalo A Rowan T Lavoie et al ldquoTranscriptionalregulation of vascular endothelial cell responses to hypoxia byHIF-1rdquo Blood vol 105 no 2 pp 659ndash669 2005

[54] P J Kallio I Pongratz K Gradin J McGuire and L PoellingerldquoActivation of hypoxia-inducible factor 1120572 posttranscriptionalregulation and conformational change by recruitment of theArnt transcription factorrdquo Proceedings of the National Academyof Sciences of the United States of America vol 94 no 11 pp5667ndash5672 1997

[55] P J Kallio K Okamoto S OrsquoBrien et al ldquoSignal transduction inhypoxic cells inducible nuclear translocation and recruitmentof the CBPp300 coactivator by the hypoxia-inducible factor-1120572rdquoThe EMBO Journal vol 17 no 22 pp 6573ndash6586 1998

[56] G L Semenza M K Nejfelt S M Chi and S E AntonarakisldquoHypoxia-inducible nuclear factors bind to an enhancer ele-ment located 31015840 to the human erythropoietin generdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 88 no 13 pp 5680ndash5684 1991

[57] T N Seagroves H E Ryan H Lu et al ldquoTranscriptionfactor HIF-1 is a necessary mediator of the pasteur effect inmammalian cellsrdquo Molecular and Cellular Biology vol 21 no10 pp 3436ndash3444 2001

[58] C V Dang and G L Semenza ldquoOncogenic alterations ofmetabolismrdquo Trends in Biochemical Sciences vol 24 no 2 pp68ndash72 1999

[59] G L Semenza P H Roth H-M Fang and G L Wang ldquoTran-scriptional regulation of genes encoding glycolytic enzymes byhypoxia-inducible factor 1rdquoThe Journal of Biological Chemistryvol 269 no 38 pp 23757ndash23763 1994

[60] R H Wenger ldquoCellular adaptation to hypoxia O2-sensing

protein hydroxylases hypoxia-inducible transcription factorsand O

2-regulated gene expressionrdquoThe FASEB Journal vol 16

no 10 pp 1151ndash1162 2002[61] G L Semenza ldquoRegulation of metabolism by hypoxia-

inducible factor 1rdquoCold SpringHarbor Symposia onQuantitativeBiology vol 76 pp 347ndash353 2011

[62] D Feldser F Agani N V Iyer B Pak G Ferreira and G LSemenza ldquoReciprocal positive regulation of hypoxia-induciblefactor 1alpha and insulin-like growth factor 2rdquo Cancer Researchvol 59 no 16 pp 3915ndash3918 1999

[63] B Krishnamachary S Berg-Dixon B Kelly et al ldquoRegulationof colon carcinoma cell invasion by hypoxia-inducible factor 1rdquoCancer Research vol 63 no 5 pp 1138ndash1143 2003

[64] M Koning P M N Werker M J A van Luyn and M CHarmsen ldquoHypoxia promotes proliferation of humanmyogenicsatellite cells a potential benefactor in tissue engineering ofskeletalmusclerdquoTissue Engineering A vol 17 no 13-14 pp 1747ndash1758 2011

[65] J A Mitchell and J M Yochim ldquoIntrauterine oxygen tensionduring the estrous cycle in the rat its relation to uterine

respiration and vascular activityrdquo Endocrinology vol 83 no 4pp 701ndash705 1968

[66] F Rodesch P Simon C Donner and E Jauniaux ldquoOxygenmeasurements in endometrial and trophoblastic tissues duringearly pregnancyrdquo Obstetrics and Gynecology vol 80 no 2 pp283ndash285 1992

[67] H Diez A Fischer A Winkler et al ldquoHypoxia-mediatedactivation of Dll4-Notch-Hey2 signaling in endothelial progen-itor cells and adoption of arterial cell faterdquo Experimental CellResearch vol 313 no 1 pp 1ndash9 2007

[68] D M Adelman E Maltepe and M C Simon ldquoMultilineageembryonic hematopoiesis requires hypoxic ARNT activityrdquoGenes and Development vol 13 no 19 pp 2478ndash2483 1999

[69] N V Iyer L E Kotch F Agani et al ldquoCellular and develop-mental control of O

2homeostasis by hypoxia-inducible factor

1120572rdquo Genes amp Development vol 12 no 2 pp 149ndash162 1998[70] K R Kozak B Abbott and O Hankinson ldquoARNT-deficient

mice and placental differentiationrdquo Developmental Biology vol191 no 2 pp 297ndash305 1997

[71] D M Adelman M Gertsenstein A Nagy M C Simon andE Maltepe ldquoPlacental cell fates are regulated in vivo by HIF-mediated hypoxia responsesrdquo Genes and Development vol 14no 24 pp 3191ndash3203 2000

[72] K D Cowden Dahl B H Fryer F A Mack et al ldquoHypoxia-inducible factors 1120572 and 2120572 regulate trophoblast differentiationrdquoMolecular and Cellular Biology vol 25 no 23 pp 10479ndash104912005

[73] K Takeda V C Ho H Takeda L J Duan A Nagy and G-H Fong ldquoPlacental but not heart defects are associated withelevated hypoxia-inducible factor 120572 levels inmice lacking prolylhydroxylase domain protein 2rdquoMolecular and Cellular Biologyvol 26 no 22 pp 8336ndash8346 2006

[74] EMaltepe J V Schmidt D Baunoch C A Bradfield andMCSimon ldquoAbnormal angiogenesis and responses to glucose andoxygen deprivation in mice lacking the protein ARNTrdquo Naturevol 386 no 6623 pp 403ndash407 1997

[75] D L Ramırez-Bergeron A Runge D M Adelman M Gohiland M C Simon ldquoHIF-dependent hematopoietic factors reg-ulate the development of the embryonic vasculaturerdquo Develop-mental Cell vol 11 no 1 pp 81ndash92 2006

[76] A E Greijer P van der Groep D Kemming et al ldquoUp-regualtion of gene expression by hypoxia is mediated pre-dominantly by hypoxia-inducible factor I (HIF-I)rdquo Journal ofPathology vol 206 no 3 pp 291ndash304 2005

[77] G L Semenza ldquoAngiogenesis in ischemic and neoplasticdisordersrdquo Annual Review of Medicine vol 54 pp 17ndash28 2003

[78] D J Ceradini A R Kulkarni M J Callaghan et al ldquoProgenitorcell trafficking is regulated by hypoxic gradients through HIF-1induction of SDF-1rdquoNatureMedicine vol 10 no 8 pp 858ndash8642004

[79] J E Park G-A Keller andN Ferrara ldquoThe vascular endothelialgrowth factor (VEGF) isoforms differential deposition into thesubepithelial extracellular matrix and bioactivity of extracellu-lar matrix-bound VEGFrdquo Molecular Biology of the Cell vol 4no 12 pp 1317ndash1326 1993

[80] Y Liu S R Cox T Morita and S Kourembanas ldquoHypoxiaregulates vascular endothelial growth factor gene expression inendothelial cells Identification of a 51015840 enhancerrdquo CirculationResearch vol 77 no 3 pp 638ndash643 1995


[81] J A Forsythe B-H Jiang N V Iyer et al ldquoActivation of vas-cular endothelial growth factor gene transcription by hypoxia-inducible factor 1rdquoMolecular and Cellular Biology vol 16 no 9pp 4604ndash4613 1996

[82] H-P Gerber F Condorelli J Park and N Ferrara ldquoDifferen-tial transcriptional regulation of the two vascular endothelialgrowth factor receptor genes Flt-1 but not Flk-1KDR is up-regulated by hypoxiardquo The Journal of Biological Chemistry vol272 no 38 pp 23659ndash23667 1997

[83] JWaltenberger UMayr S Pentz andVHombach ldquoFunctionalupregulation of the vascular endothelial growth factor receptorKDR by hypoxiardquoCirculation vol 94 no 7 pp 1647ndash1654 1996

[84] C W Pugh and P J Ratcliffe ldquoRegulation of angiogenesis byhypoxia role of the HIF systemrdquo Nature Medicine vol 9 no 6pp 677ndash684 2003

[85] D R Mole C Blancher R R Copley et al ldquoGenome-wideassociation of hypoxia-inducible factor (HIF)-1120572 and HIF-2120572DNA binding with expression profiling of hypoxia-inducibletranscriptsrdquoThe Journal of Biological Chemistry vol 284 no 25pp 16767ndash16775 2009

[86] J Schodel S Oikonomopoulos J Ragoussis C W Pugh PJ Ratcliffe and D R Mole ldquoHigh-resolution genome-widemapping of HIF-binding sites by ChIP-seqrdquo Blood vol 117 no23 pp e207ndashe217 2011

[87] P Carmeliet and R K Jain ldquoMolecularmechanisms and clinicalapplications of angiogenesisrdquoNature vol 473 no 7347 pp 298ndash307 2011

[88] Y Ben-Yosef A Miller S Shapiro and N Lahat ldquoHypoxiaof endothelial cells leads to MMP-2-dependent survival anddeathrdquo The American Journal of PhysiologymdashCell Physiologyvol 289 no 5 pp C1321ndashC1331 2005

[89] S Keely L E Glover C FMacManus et al ldquoSelective inductionof integrin 1205731 by hypoxia-inducible factor implications forwound healingrdquo The FASEB Journal vol 23 no 5 pp 1338ndash1346 2009

[90] N Skuli L Liu A Runge et al ldquoEndothelial deletion ofhypoxia-inducible factor-2120572 (HIF-2120572) alters vascular functionand tumor angiogenesisrdquo Blood vol 114 no 2 pp 469ndash4772009

[91] H Niemi K Honkonen P Korpisalo et al ldquoHIF-1120572 and HIF-2120572 induce angiogenesis and improve muscle energy recoveryrdquoEuropean Journal of Clinical Investigation vol 44 no 10 pp989ndash999 2014

[92] C Yuan P Wang L Zhu et al ldquoCoculture of stem cells fromapical papilla and human umbilical vein endothelial cell underhypoxia increases the formation of three-dimensional vessel-like structures in vitrordquo Tissue Engineering Part A vol 21 no5-6 2015

[93] S Rajagopalan J Olin S Deitcher et al ldquoUse of a constitutivelyactive hypoxia-inducible factor-1120572 transgene as a therapeuticstrategy in no-option critical limb ischemia patients phase Idose-escalation experiencerdquo Circulation vol 115 no 10 pp1234ndash1243 2007

[94] L Holmgren M S OrsquoReilly and J Folkman ldquoDormancy ofmicrometastases balanced proliferation and apoptosis in thepresence of angiogenesis suppressionrdquo Nature Medicine vol 1no 2 pp 149ndash153 1995

[95] J Folkman ldquoTumour angiogenesisrdquo in Cancer Medicine J FHolland R C Bast Jr D L Morton E Frei III D W Kufeand R RWeichselbaum Eds pp 181ndash204WilliamsampWilkinsBaltimore Md USA 1997

[96] E B Rankin and A J Giaccia ldquoThe role of hypoxia-induciblefactors in tumorigenesisrdquo Cell Death and Differentiation vol 15no 4 pp 678ndash685 2008

[97] J M Brown and A J Giaccia ldquoThe unique physiology of solidtumours opportunities (and problems) for cancer therapyrdquoCancer Research vol 58 no 7 pp 1408ndash1416 1998

[98] G L Semenza ldquoHypoxia clonal selection and the role of HIF-1 in tumor progressionrdquo Critical Reviews in Biochemistry andMolecular Biology vol 35 no 2 pp 71ndash103 2000

[99] R Ravi B Mookerjee Z M Bhujwalla et al ldquoRegulation oftumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1120572rdquo Genes and Development vol 14 no 1 pp34ndash44 2000

[100] W Zundel C Schindler D Haas-Kogan et al ldquoLoss of PTENfacilitatesHIF-1-mediated gene expressionrdquoGenes andDevelop-ment vol 14 no 4 pp 391ndash396 2000

[101] E Laughner P Taghavi K Chiles P C Mahon and G LSemenza ldquoHER2 (neu) signaling increases the rate of hypoxia-inducible factor 1120572 (HIF-1120572) synthesis novel mechanism forHIF-1-mediated vascular endothelial growth factor expressionrdquoMolecular and Cellular Biology vol 21 no 12 pp 3995ndash40042001

[102] W G Kaelin Jr ldquoMolecular basis of the VHL hereditary cancersyndromerdquo Nature Reviews Cancer vol 2 no 9 pp 673ndash6822002

[103] P H Maxwell M S Wlesener G W Chang et al ldquoThe tumoursuppressor protein VHL targets hypoxia-inducible factors foroxygen-dependent proteolysisrdquo Nature vol 399 no 6733 pp271ndash275 1999

[104] O Iliopoulos A Kibel S Gray and W G Kaelin Jr ldquoTumoursuppression by the human von Hippel-Lindau gene productrdquoNature Medicine vol 1 no 8 pp 822ndash826 1995

[105] K Kondo J Klco E Nakamura M Lechpammer and W GKaelin Jr ldquoInhibition of HIF is necessary for tumor suppressionby the von Hippel-Lindau proteinrdquo Cancer Cell vol 1 no 3 pp237ndash246 2002

[106] J K Maranchie J R Vasselli J Riss J S Bonifacino WM Linehan and R D Klausner ldquoThe contribution of VHLsubstrate binding and HIF1-120572 to the phenotype of VHL lossin renal cell carcinomardquo Cancer Cell vol 1 no 3 pp 247ndash2552002

[107] R R Raval K W Lau M G B Tran et al ldquoContrastingproperties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 invon Hippel-Lindau-associated renal cell carcinomardquoMolecularand Cellular Biology vol 25 no 13 pp 5675ndash5686 2005

[108] H Lu R A Forbes and A Verma ldquoHypoxia-inducible factor1 activation by aerobic glycolysis implicates the Warburg effectin carcinogenesisrdquoThe Journal of Biological Chemistry vol 277no 26 pp 23111ndash23115 2002

[109] Z Wang C Dabrosin X Yin et al ldquoBroad targeting ofangiogenesis for cancer prevention and therapyrdquo Seminars inCancer Biology 2015

[110] P N Span and J Bussink ldquoBiology of hypoxiardquo Seminars inNuclear Medicine vol 45 no 2 pp 101ndash109 2015

[111] A Zimna A Janeczek N Rozwadowska et al ldquoBiologicalproperties of human skeletal myoblasts genetically modified tosimultaneously overexpress the pro-angiogenic factors vascularendothelial growth factor-A and fibroblast growth factor-4rdquoJournal of Physiology and Pharmacology vol 65 no 2 pp 193ndash207 2014


[112] S Kolakowski Jr M F Berry P Atluri et al ldquoPlacental growthfactor provides a novel local angiogenic therapy for ischemiccardiomyopathyrdquo Journal of Cardiac Surgery vol 21 no 6 pp559ndash564 2006

[113] S Rajagopalan E RMohler III R J Lederman et al ldquoRegionalangiogenesis with vascular endothelial growth factor in periph-eral arterial disease a phase II randomized double-blindcontrolled study of adenoviral delivery of vascular endothelialgrowth factor 121 in patients with disabling intermittent claudi-cationrdquo Circulation vol 108 no 16 pp 1933ndash1938 2003

[114] R J Lederman F O Mendelsohn R D Anderson et alldquoTRAFFIC InvestigatorsTherapeutic angiogenesiswith recom-binant fibroblast growth factor-2 for intermittent claudication(the TRAFFIC study) a randomised trialrdquoThe Lancet vol 359no 9323 pp 2953ndash2958 2002

[115] K Hirota and G L Semenza ldquoRegulation of angiogenesisby hypoxia-inducible factor 1rdquo Critical Reviews in Oncol-ogyHematology vol 59 no 1 pp 15ndash26 2006

[116] T H Patel H Kimura C R Weiss G L Semenza and LV Hofmann ldquoConstitutively active HIF-1120572 improves perfusionand arterial remodeling in an endovascular model of limbischemiardquo Cardiovascular Research vol 68 no 1 pp 144ndash1542005

[117] B D Kelly S F Hackett K Hirota et al ldquoCell type-specificregulation of angiogenic growth factor gene expression andinduction of angiogenesis in nonischemic tissue by a consti-tutively active form of hypoxia-inducible factor 1rdquo CirculationResearch vol 93 no 11 pp 1074ndash1081 2003

[118] C Yang H Liu and D Liu ldquoMutant hypoxia-inducible factor1120572 modified bone marrow mesenchymal stem cells amelioratecerebral ischemiardquo International Journal of Molecular Medicinevol 34 no 6 pp 1622ndash1628 2014

[119] K-G Shyu M-T Wang B-W Wang et al ldquoIntramyocardialinjection of naked DNA encoding HIF-1120572VP16 hybrid toenhance angiogenesis in an acute myocardial infarction modelin the ratrdquo Cardiovascular Research vol 54 no 3 pp 576ndash5832002

[120] D A Elson G Thurston L E Huang et al ldquoInduction ofhypervascularity without leakage or inflammation in transgenicmice overexpressing hypoxia-inducible factor-1120572rdquo Genes andDevelopment vol 15 no 19 pp 2520ndash2532 2001

[121] M Milkiewicz C W Pugh and S Egginton ldquoInhibitionof endogenous HIF inactivation induces angiogenesis inischaemic skeletal muscles of micerdquo Journal of Physiology vol560 no 1 pp 21ndash26 2004

[122] G L Wang and G L Semenza ldquoDesferrioxamine induceserythropoietin gene expression and hypoxia-inducible factor1 DNA-binding activity Implications for models of hypoxiasignal transductionrdquo Blood vol 82 no 12 pp 3610ndash3615 1993

[123] X Liu J Wang X Ji S Yu and L Wei ldquoPreconditioning ofbone marrow mesenchymal stem cells by prolyl hydroxylaseinhibition enhances cell survival and angiogenesis in vitro andafter transplantation into the ischemic heart of ratsrdquo Stem CellResearch ampTherapy vol 5 no 5 p 111 2014

[124] J Li M Post R Volk et al ldquoPR39 a peptide regulator ofangiogenesisrdquo Nature Medicine vol 61 pp 49ndash55 2000

[125] K Mittal H Koon P Elson et al ldquoDual VEGFVEGFRinhibition in advanced solid malignanciesrdquo Cancer Biology ampTherapy vol 15 no 8 pp 975ndash981 2014

[126] Y Hu J Liu andH Huang ldquoRecent agents targeting HIF-1120572 forcancer therapyrdquo Journal of Cellular Biochemistry vol 114 no 3pp 498ndash509 2013

[127] D H Shin Y-S Chun D S Lee L E Huang and J-WPark ldquoBortezomib inhibits tumor adaptation to hypoxia bystimulating the FIH-mediated repression of hypoxia-induciblefactor-1rdquo Blood vol 111 no 6 pp 3131ndash3136 2008

[128] E J Yeo Y S Chun Y S Cho et al ldquoYC-1 a potentialanticancer drug targeting hypoxia-inducible factor 1rdquo Journal ofthe National Cancer Institute vol 95 no 7 pp 516ndash525 2003

[129] P Garcıa-Maceira and J Mateo ldquoSilibinin inhibits hypoxia-inducible factor-1120572 andmTORp70S6K4E-BP1 signalling path-way in human cervical and hepatoma cancer cells implicationsfor anticancer therapyrdquo Oncogene vol 28 no 3 pp 313ndash3242009

[130] Y M Lee S-H Kim H-S Kim et al ldquoInhibition of hypoxia-induced angiogenesis by FK228 a specific histone deacetylaseinhibitor via suppression of HIF-1120572 activityrdquo Biochemical andBiophysical Research Communications vol 300 no 1 pp 241ndash246 2003

[131] S Shankar R Davis K P Singh R Kurzrock D DRoss and R K Srivastava ldquoSuberoylanilide hydroxamic acid(Zolinzavorinostat) sensitizes TRAIL-resistant breast cancercells orthotopically implanted in BALBc nudemicerdquoMolecularCancer Therapeutics vol 8 no 6 pp 1596ndash1605 2009

[132] E W Newcomb M A Ali T Schnee et al ldquoFlavopiridoldownregulates hypoxia-mediated hypoxia-inducible factor-1120572expression in human glioma cells by a proteasome-independentpathway implications for in vivo therapyrdquoNeuro-Oncology vol7 no 3 pp 225ndash235 2005

[133] M V Blagosklonny ldquoFlavopiridol an inhibitor of transcriptionimplications problems and solutionsrdquo Cell Cycle vol 3 no 12pp 1537ndash1542 2004

[134] C J Yeom L Zeng Y Zhu M Hiraoka and H HaradaldquoStrategies to assess hypoxicHIF-1-active cancer cells for thedevelopment of innovative radiation therapyrdquo Cancers vol 3no 3 pp 3610ndash3631 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Angiogenesis occurs in physiological states such asembryonic development wound healing or vessel penetra-tion into avascular regions and in pathological states suchas solid tumours formation eye diseases or chronic inflam-matory disorders such as rheumatoid arthritis psoriasisand periodontitis [16 17] Pathophysiological angiogenesisexhibits differences in molecular pathways in comparisonto physiological angiogenesis Mutations in oncogenes andtumour suppressor genes and disruptions in growth factoractivity play crucial roles during tumour angiogenesis [17]The activation of the most prominent proangiogenic factorVEGF might be due to physiological stimuli such as hypoxiaor inflammation or due to oncogene activation and tumoursuppression function loss [18 19] Additionally physiologicalangiogenesis such as that which occurs during embryonicdevelopment or wound healing seems to be dependent onVEGF signalling whereas tumour angiogenesis adopts theability to shift its dependence from VEGF to other proan-giogenic pathways for example through the recruitment ofmyeloid cells and the upregulation of alternative vasculargrowth factors (PlGF and FGF fibroblast growth factor) [17]Moreover tumour vessels are distinct from normal vascula-ture because they are disorganised and tortuous Many mor-phological and functional differences exist between normaland tumour vasculature For instance tumour vessels areleakier than normal vessels and endothelial cells growingwithin tumours carry genetic abnormalities [20 21] Elucidat-ing the molecular mechanism of pathological angiogenesismight lead to the identification of potential therapeutictargets

Angiogenesis is a multistep process that requires theinvolvement ofmany biological signals and stimuli regardlessof whether it is a physiological or pathological action Proan-giogenic factors are activated in response to some physicalsignals Blood vessel damage infarction and blood flowreduction lead to decreases in O

2supply [11] This state is

called hypoxia which is a potent angiogenic trigger thatstimulates proangiogenic factor activity

2 Hypoxia

21 O2 Homeostasis Constant oxygen supply is essential forproper tissue function development and homeostasis Thusthe vasculature network plays a crucial role in delivering oxy-gen particles (O

2) nutrients and other molecules within the

entire human body [22] Significantly the first physiologicalsystem that becomes functional during mammalian embry-onic development is the circulatory system Governing O

2

homeostasis in tissues by supplying an oxygen concentrationadequate for the demand generated by the metabolic outputsof the tissue is essential Oxygenic balance can be upset byrapid cellular division during embryonic development bytumour growth or by vasculature dysfunction due to vesselocclusion or rupture [23] Notably normal physiological O

2

concentrations vary greatly from normal oxygen tension inthe air depending on the type of tissue For instance arterialblood has a normal pO

2of 14 myocardium 10 and

skeletalmuscle 5 In contrast the natural pO2levels of bone

marrow thymus and cartilage are at or below 1 [24 25]The

state when the O2level decreases relative to physiological lev-

els (characteristic for particular tissues) is called hypoxia [1]

22 Hypoxia-Inducible Factor-1 Adaptation to low oxygentension (hypoxia) in cells and tissues requires the acti-vation of several genes that participate in angiogenesiscell proliferationsurvival glucose and iron metabolism Ineukaryotic cells hypoxia-inducible factor-1 (HIF-1) is a pri-mary transcriptional mediator of the hypoxic response andmaster regulator of O

2homeostasis [26] Hypoxia-inducible

factor-1 was first discovered as a transcription factor thatregulates erythropoietin (EPO) expression in response to lowoxygen levels in the blood [27 28] HIF-1 consists of twodifferent subunits 120572 and 120573 (also known as aryl hydrocarbonnuclear receptor translocator (ARNT)) both subunits aremembers of the basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor family [29 30] PAS and bHLHmotifs are required for heterodimerisation between the HIF-1120572 and HIF-1120573 subunits [31] Additionally the bHLH domainof the HIF-1120572ARNT dimer is essential for DNA bindingon hypoxia response elements (HREs) with the consensussequence (GACGTG) in the promoters or enhancers oftarget genes [32] Transcriptional activation and interactionswith coactivators (such as CBPp300) of HIF-1120572 aremediatedby two domains that is C-TAD andN-TAD which are on theC-terminus of the protein [33ndash35] HIF-1120573 is a constitutivelyexpressed subunit whereas HIF-1120572 is translated continuouslyand degraded subsequently through ubiquitination undernormoxic conditions [36 37]Thus far three isoforms ofHIF-120572 have been discovered (Figure 1(a)) that is the previouslydiscussed subunit HIF-1120572 and two other subunits HIF-2120572(also called endothelial PAS protein (EPAS)) and HIF-3120572(IPAS) The second isoform is expressed constitutively in theendothelium lung and cartilage and shares 48 amino acidsequence identity with HIF-1120572 The third protein HIF-3120572also called inhibitory PAS (IPAS) is a negative regulator ofHIF-1 that dimerises with the HIF-1120572 subunit and preventsits DNA-binding activity The entire set of HIF-120572 subunitsdimerises with ARNT and binds to HREs [38ndash40]

Under extended exposure to hypoxic conditions HIF-1120572 is expressed as long as a balance between O

2supply

and usage in tissues cannot be reached [36 37] Whenoxygen tension is sufficient de novo synthesised cytoplasmicHIF-1120572 is hydroxylated by a family of prolyl hydroxylaseenzymes (PHDs) on proline 402 and 564 residues locatedwithin ODDD (O

2-dependent degradation domain) [41 42]

The hydroxylation of the abovementioned prolines is thekey mechanism of negative regulation of HIF-1120572 activityand results in the binding of the von Hippel-Lindau (VHL)E3 ligase complex which ubiquitinates HIF-1120572 targetingit to proteasomal degradation [43ndash45] The second majormechanism that modulates HIF-1120572 activity is the hydrox-ylation of asparagine residue 803 (Asn803) in the C-TADdomain In this case asparagine is hydroxylated undernormoxic conditions by factor inhibiting HIF-1 (FIH-1)which prevents the interaction of HIF-1120572 within CBPp300(CREB-binding proteinE1A binding protein p300) [46ndash49]Hydroxylases (PHDs and FIH-1) are strictly Fe(II)- and2-oxoglutarate-dependent dioxygenases that are activated


bHLH PAS ODDD N-TAD


bHLH PAS ODDD





(a)

CBP

bHLH PAS TAD

NLS

Normoxia

Hypoxia

bHLH PAS ODDD N-TAD

N-TAD

C-TAD


Asn 803OH

CBPp300


pVHL


HIF-1120572 826aa

N-TAD C-TAD

HIF-1120573 789aa


pVHL

p300

(b)







2homeostasis [56]



CBP

CBPp300CBPp300

p300

Transcr

iption of ta

rget g

ene

Normoxia Hypoxia

HIF-1120572

HIF-1120572

HIF-1120572

HIF-1120572HIF-1120572

HIF-1120572HIF-1120572

HIF-1120572

HIF

-1120572

HIF-1120572

CBPp300

HIF

-1120573


PHD PHD


pVHL

Ubiquitin

OH-Pro 402

OH-Asp 803

OH-Asp 803

564


HIF-1 degradation

FIHFIH

Pro 402 564

Asp 803











2tension









2(05mM) stimu-



























Administration of

Ischaemic diseases

Inhibition of

dimerization

Inhibition of


protein translation

Cancer

Angiogenesis

Angiogenesis

(i) Acriflavine

(i) Bortezomib

(ii) Amphotericin B

(i) Silibinin

(i) Topotecan

(i) SAHA(ii) FK228






HIF-1120572 and HIF-1120573





HIF-1120572















Acknowledgments


References


















































2














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


bHLH PAS ODDD N-TAD


bHLH PAS ODDD





(a)

CBP

bHLH PAS TAD

NLS

Normoxia

Hypoxia

bHLH PAS ODDD N-TAD

N-TAD

C-TAD


Asn 803OH

CBPp300


pVHL


HIF-1120572 826aa

N-TAD C-TAD

HIF-1120573 789aa


pVHL

p300

(b)







2homeostasis [56]



CBP

CBPp300CBPp300

p300

Transcr

iption of ta

rget g

ene

Normoxia Hypoxia

HIF-1120572

HIF-1120572

HIF-1120572

HIF-1120572HIF-1120572

HIF-1120572HIF-1120572

HIF-1120572

HIF

-1120572

HIF-1120572

CBPp300

HIF

-1120573


PHD PHD


pVHL

Ubiquitin

OH-Pro 402

OH-Asp 803

OH-Asp 803

564


HIF-1 degradation

FIHFIH

Pro 402 564

Asp 803











2tension









2(05mM) stimu-



























Administration of

Ischaemic diseases

Inhibition of

dimerization

Inhibition of


protein translation

Cancer

Angiogenesis

Angiogenesis

(i) Acriflavine

(i) Bortezomib

(ii) Amphotericin B

(i) Silibinin

(i) Topotecan

(i) SAHA(ii) FK228






HIF-1120572 and HIF-1120573





HIF-1120572















Acknowledgments


References


















































2














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


CBP

CBPp300CBPp300

p300

Transcr

iption of ta

rget g

ene

Normoxia Hypoxia

HIF-1120572

HIF-1120572

HIF-1120572

HIF-1120572HIF-1120572

HIF-1120572HIF-1120572

HIF-1120572

HIF

-1120572

HIF-1120572

CBPp300

HIF

-1120573


PHD PHD


pVHL

Ubiquitin

OH-Pro 402

OH-Asp 803

OH-Asp 803

564


HIF-1 degradation

FIHFIH

Pro 402 564

Asp 803











2tension









2(05mM) stimu-



























Administration of

Ischaemic diseases

Inhibition of

dimerization

Inhibition of


protein translation

Cancer

Angiogenesis

Angiogenesis

(i) Acriflavine

(i) Bortezomib

(ii) Amphotericin B

(i) Silibinin

(i) Topotecan

(i) SAHA(ii) FK228






HIF-1120572 and HIF-1120573





HIF-1120572















Acknowledgments


References


















































2














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








2(05mM) stimu-



























Administration of

Ischaemic diseases

Inhibition of

dimerization

Inhibition of


protein translation

Cancer

Angiogenesis

Angiogenesis

(i) Acriflavine

(i) Bortezomib

(ii) Amphotericin B

(i) Silibinin

(i) Topotecan

(i) SAHA(ii) FK228






HIF-1120572 and HIF-1120573





HIF-1120572















Acknowledgments


References


















































2














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























Administration of

Ischaemic diseases

Inhibition of

dimerization

Inhibition of


protein translation

Cancer

Angiogenesis

Angiogenesis

(i) Acriflavine

(i) Bortezomib

(ii) Amphotericin B

(i) Silibinin

(i) Topotecan

(i) SAHA(ii) FK228






HIF-1120572 and HIF-1120573





HIF-1120572















Acknowledgments


References


















































2














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Administration of

Ischaemic diseases

Inhibition of

dimerization

Inhibition of


protein translation

Cancer

Angiogenesis

Angiogenesis

(i) Acriflavine

(i) Bortezomib

(ii) Amphotericin B

(i) Silibinin

(i) Topotecan

(i) SAHA(ii) FK228






HIF-1120572 and HIF-1120573





HIF-1120572















Acknowledgments


References


















































2














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Administration of

Ischaemic diseases

Inhibition of

dimerization

Inhibition of


protein translation

Cancer

Angiogenesis

Angiogenesis

(i) Acriflavine

(i) Bortezomib

(ii) Amphotericin B

(i) Silibinin

(i) Topotecan

(i) SAHA(ii) FK228






HIF-1120572 and HIF-1120573





HIF-1120572















Acknowledgments


References


















































2














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Acknowledgments


References


















































2














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


































2














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Hypoxia-Inducible Factor-1 in Physiological and Pathophysiological