hypoxia-inducible factors in physiology and medicine2. physiology: even modest gain or loss of...

Gregg L. Semenza, M.D., Ph.D.Vascular Program, Institute for Cell Engineering;

Departments of Genetic Medicine, Pediatrics, Oncology, Medicine, Radiation Oncology, and Biological Chemistry;

Johns Hopkins University School of MedicineBaltimore, Maryland USA

Hypoxia-Inducible Factors in Physiology and Medicine

Oxygen Homeostasis: A Balancing Act

Hypoxia-Inducible Factors

Blood

Heart

Blood Vessels

Lungs

CNS

O2 Supply

O2 Demand

Control of Red Blood Cell Production

Cardiovascular Disease

Cancer

Erythropoietin (EPO)

Bonemarrow

O2 deliveryto every cellIn the body

Erythropoietin Controls Red Blood Cell Production


Bonemarrow


Erythropoietin Controls Red Blood Cell Production

ChronicKidney Disease


Bonemarrow


?

Erythropoietin Controls Red Blood Cell ProductionWhat Controls Erythropoietin Production?

ChronicKidney Disease

G. L. Semenza and G. L. Wang, Mol. Cell. Biol. 12: 5447, 1992G. L. Wang and G. L. Semenza, J. Biol. Chem. 270: 1230, 1995

G. L. Wang et al. Proc. Natl. Acad. Sci. USA 92: 5510, 1995

Hypoxia-Inducible Factor 1 (HIF-1) Binds to the EPO Gene and Activates Transcription

HypoxiaResponseElement

HATs

HIF-1α is Regulated by Oxygen-dependent Hydroxylation

CO2 + succinate

O2 + α-ketoglutarate

PHDs = Prolyl Hydroxylase Domain proteins target HIF-1α for destruction when O2 is available.

HIF-1α Protein Accumulates in Response to Hypoxia Leading to Increased Transcription of HIF-1 Target Genes

% O2

B.-H. Jiang et al.Am. J. Physiol. 1996;271:C1172

Arterial PO2~100 mm Hg

Venous PO2~40 mm Hg

HIF-1 Mediates Homeostatic Responses to Reduced O2 Levels

> 4,000 Target Genes

HIF-1α is Required for Development of the Circulatory System

+/+ -/-

Heart

Blood

+/+ -/-

Blood

Yolk sac

Embryo

Blood Vessels

N. V. Iyer et al., Genes Dev. 12: 149, 1998 D. Yoon et al., J. Biol. Chem. 281:25703, 2006

HIF-1α, HIF-2α and HIF-3α Heterodimerize with HIF-1β and Activate Gene Transcription

HIF2α

HIF1β

All nucleatedcell types of

~all metazoanspecies

Certain cell types of

vertebratespecies

HIF1α

HIF1β

HIF3α

HIF1β

Certain cell types of

vertebratespecies

HIF-2HIF-1 HIF-3

HIF-1αHIF-2α

HIF-αPro-OH

HIF-αPro-OHLys-Ubin

HIF degradation

PHD2

VHL

O2

EPO

EPOR

Type 4HIF2α

Gain of Function

Red blood cell production

Congenital Polycythemia (Too Many Red Cells) is Caused by Mutations in the HIF Pathway

Type 3PHD2

Loss of function

Type 2VHL

Loss of function

Type 1EPOR

Gain of Function



Cancer

Cardiovascular Disease is the Leading Cause of DeathIn the Industrialized World

CORONARY ARTERY CHEST PAINHEART ATTACK

PERIPHERAL ARTERY LEG PAINAMPUTATION

stenosis

Cardiovascular Disease is the Leading Cause of DeathIn the Industrialized World

CORONARY ARTERY CHEST PAINHEART ATTACK

PERIPHERAL ARTERY LEG PAINAMPUTATION

Critical Limb Ischemia:End-Stage Peripheral Arterial Disease

Critical Limb Ischemia

Perfusion is not sufficient to maintain tissue viability, leading to:

Ischemic pain at rest

Ischemic ulcers

Gangrene

Limb amputation

stenosis

collateral

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Pre-Op

Post-Op

3 7 14 21 28 35

Time (days)

Isch

emic

:Non

-isch

emic

Lim

b Pe

rfusi

on R

atio

WTHET 2 mon old

WTHET 8 mon old

WTHET 20 mon old

Analysis of Vascularization after Femoral Artery Ligation in Wild-type (WT) and Heterozygous HIF-1α−Null (HET) Mice

Laser Doppler Perfusion Imaging

M. Bosch-Marcé et al.Circ. Res. 2007;101:1310

0

20

40

60

80

100

120

WT

(n = 10)

HET

(n = 9)

WT

(n = 10)

HET

(n = 15)

WT

(n = 7)

HET

(n = 8)

% o

f Ani

mal

s in

Gro

up

Limb salvage

BAA/necrosis

AAA

Additive Effects of Aging and Hif1a Genotype on Limb Salvage

2 mon old 8 mon old 20 mon old

M. Bosch-Marcé et al., Circ. Res. 2007;101:1310

WT HET WT HET WT HET

NIS ISC NIS ISC NIS ISC NIS ISC NIS ISC NIS ISC

2-month-old 8-month-old 20-month-old

HIF-1α

β-actin

Effects of Aging and Hif1a Genotype on Ischemia-induced HIF-1α Protein Levels

M. Bosch-Marcé et al., Circ. Res. 2007;101:1310

***

0.0

0.2

0.4

0.6

0.8

1.0

1.2Li

mb

Perfu

sion

Rat

io

AdLacZAdCA5

Pre Post 3 7 14 21Time (days)

Improved Recovery of Perfusion in Mice by HIF-1α Gene Therapy

2-month-old C57BL/6J miceAd: 6x108 pfu


***

0.0

0.2

0.4

0.6

0.8

1.0

1.2Li

mb

Perfu

sion

Rat

io

AdLacZAdCA5

**

0.0

0.2

0.4

0.6

0.8

1.0

1.2AdLacZAdCA5



Lim

b Pe

rfusi

on R

atio





***

0.0

0.2

0.4

0.6

0.8

1.0

1.2Li

mb

Perfu

sion

Rat

io

AdLacZAdCA5

**

0.0

0.2

0.4

0.6

0.8

1.0

1.2AdLacZAdCA5



Lim

b Pe

rfusi

on R

atio





AdCA5 gene therapy corrects age-relatedimpairment of vascularization

HIF-1 Regulates the Expression of Angiogenic Growth Factors

EPC Recruitment,EC Proliferation/Survival,

Activation

Modulation ofEC-SMC

Interactions

VEGF-R2 TIE2 PDGF-R

Increased Tissue Vascularization

VEGF-R1

Hypoxia/Ischemia

CXCR4

Mobilization and Recruitment of

MSCs and BMDACs

EPOR

SDF1 PLGF VEGF EPO ANGPT1 ANGPT2 PDGFBSCF

C-KIT

Increased HIF-1 Activity



Activation

Modulation ofEC-SMC

Interactions

VEGF-R2 TIE2 PDGF-R


VEGF-R1

Hypoxia/Ischemia

CXCR4


MSCs and BMDACs

EPOR


C-KIT

Increased HIF-1 ActivityAging



Activation

Modulation ofEC-SMC

Interactions

VEGF-R2 TIE2 PDGF-R


VEGF-R1

Hypoxia/Ischemia

CXCR4


MSCs and BMDACs

EPOR


C-KIT

Increased HIF-1 ActivityAging AdCA5



Cancer

Advanced Human Cancers Commonly ContainRegions of Intratumoral Hypoxia

Direct measurements of O2 concentration in human tumors have demonstrated that

PO2 < 10 mm Hg is associated with a significantly increased risk of invasion,

metastasis, and patient mortality.

HIF-1αexpression by hypoxic cancer cells

HIF-1α Overexpression is Associated with Patient Mortality

K. Lee et al.Proc. Natl. Acad. Sci. USA

2009;106:2353

HIF Inhibitor Acriflavine Inhibits Tumor Growth and Vascularization in a Mouse Model of Prostate Cancer

Acriflavine (ACF) inhibitsdimerization of HIF-αand HIF-1β subunits

HIF Inhibitor Digoxin Decreases Primary Tumor Growth and Metastasis in a Mouse Model of Breast Cancer

H. Zhang et al. Oncogene 2012;31:1757L. Schito et al. Proc. Natl. Acad. Sci. USA 2012;109:E2707

C.C. Wong et al. J. Mol. Med. 2012;90:803

Tum

or c

ell a

rea

in a

xilla

ry L

N

Lung metastasis

Lymph nodemetastasis Primary tumor growth

Saline Digoxin

Treatment with Gemcitabine + HIF Inhibitor DigoxinCauses Tumor Eradication

No treatment

Gemcitabine

Gemcitabine+ Digoxin

Tum

or v

olum

e

Days after injection of tumor cells

D. Samanta et al. Proc. Natl. Acad. Sci. USA 2014;111:E5429

Migration/InvasionMicrovesicle Formation

RHOA, ROCK1P4HA1, P4HA2, PLOD2

RAB22A

Immune EvasionCD47, CD73, PDL1

Margination/Extravasation

L1CAM, ANGPTL4

MSC/Mφ CooptationCXCR3, CCR5

CXCL16, PGF, CSF1

Premetastatic Niche Formation

LOX, LOXL2, LOXL4

Cancer Stem Cell Specification

WWTR1, SIAH1IL6, IL8, MDR1, CD47

SLC7A11, GCLMALKBH5, ZNF217,

PHGDH, GSTO1

HIF-1β

HIF-1α

Deconvoluting the Role of HIFs in Breast Cancer Metastasis

D Samanta et al. PNAS 2018;115:E1239H Lu et al. Cell Rep 2017;18:1946

D Samanta et al. Cancer Res 2016;76:4430C Zhang et al. PNAS 2016;113:E2047

C Zhang et al. Oncotarget 2016;7:64527H Zhang et al. PNAS 2015;112:E6215

H Lu et al. PNAS 2015;112:E4600D Samanta et al. PNAS 2014;111:E5429L Xiang et al. Oncotarget 2014;5:12509T Wang et al. PNAS 2014;111:E3234DM Gilkes et al. PNAS 2014;111:E384L Xiang et al. J Mol Med 2014;92:151

P Chaturvedi et al. PNAS 2014;111:E2120DM Gilkes et al. Cancer Res 2013;73:3285

DM Gilkes et al. Mol Cancer Res 2013;11:456P Chaturvedi et al. J Clin Invest 2013;123:189

L Schito et al. PNAS 2012;109:E2707CC Wong et al. J Mol Med 2012;90: 803 H Zhang et al. Oncogene 2012;31:1757 CC Wong et al. PNAS 2011;108:16369

SLC7A11

GCLM

ERK

FOXO3

NANOG

GSH

ALKBH5

ZNF217

GSTO1

RYR1

PYK2

SRC

STAT3

IL6

JAK2

Ca2+ Cu

MEK

NANOG mRNAtranscription

NANOG mRNAstabilitym6A

Breast cancer stem cellspecification and self-renewal

DUSP9 REST

DUSP16

p38MAPK

MK2ZFP36L1

CD73

A2BR

Ado

PKCδ

HIFs MediateBreast Cancer

Stem CellSpecification

In Response toHypoxia or

Chemotherapy

H. Lu, PNAS 2015;112:E4600H. Lu, Cancer Res 2018;78:4191J. Lan, PNAS 2018;115:E9640

C. Zhang, PNAS 2016;113:E2047C. Zhang, Oncotarget 2016;7:64527

H. Lu, Cell Rep 2017;18:1946

AMP Ado

CD73

HIFs

A2A

Anergy/death

PDL1

PD-1

CD47

CRT

Breast cancer cell

Adaptive immunity

Innate immunity

C

H

E

M

O

T lymphocyte

SIRPα

LRP

Phagocytosis

Macrophage Chemotherapy-Induced & HIF-Dependent Evasion of Innate and Adaptive Immunity

HIF Pathway in Biology and Medicine

1. Development: HIF-1α, HIF-1β, HIF-2α, PHD2, and VHL are all required for normal mammalian embryonic development.

2. Physiology: Even modest gain or loss of function of pathway components interferes with normal postnatal physiological responses to hypoxia (congenital polycythemia).

3. Medicine: HIFs play key roles in cancer and cardiovascular disease, the major causes of mortality in the U.S. population. Clinical trials are planned or underway to evaluate novel therapies that target HIFs for inhibition (cancer, retinopathy) or activation (anemia, cardiovascular disease).

4. Evolution: HIF-1α, HIF-1β, PHD, and VHL homologs are found in all metazoan species. Genetic evidence implicates the HIF pathway as the critical genetic target for successful adaptation to high altitude in Tibetan and Andean populations.

Acknowledgments

Rose Nelson

Haig Kazazian Stylianos Antonarakis Victor McKusick

Guang Wang

Larissa Shimoda

Josef Prchal Mike ArmstrongJohn Gearhart Thomas Kelly

Akrit Sodhi

Chi Dang

Nanduri Prabhakar

Acknowledgments

Guang WangRaquel DurezaBing-Hua JiangNarayan IyerLori KotchAimee YuFaton AganiErik LaughnerSandra LeungBrian KellyKiichi HirotaHiroaki OkuyamaAshley RowanSharon Berg-DixonRyo FukudaBalaji KrishnamacharyConnor MahonJane OhDom ManaloYifu ZhouKaren Rainey Jake WesleyHideko NagasawaHideo KimuraZheqing CaiHua ZhongJin BaekYe LiuMarta Bosch-Marce

Xiaofei WeiShaoping ChenRigu GuptaDavid QianHuafeng ZhangKangAe LeeYee Sun TanKakali SarkarSergio ReyWeibo LuoHong WeiMaimon HubbiHongxia HuRyan ChangJasper ChenStephen PitcairnTing WangPallavi ChaturvediNaoharu TakanoDaniele GilkesSun Joo LeeJohn BullenYoungrok ParkIvan ChenLisha XiangHuimin ZhangChuanzhao ZhangDebangshu SamantaJie Lan

Stelios AntonarakisHaig KazazianJohn GearhartTom KellyAugustine ChoiMike KastanJonathan SimonsLarissa ShimodaCharles WienerJim SylvesterRaj RatanPeter CampochiaroAtul BediJay ZweierAngelo DeMarzoSkip GarciaJon ResarRusty HofmannSeva PolotskyDavid HusoBob ColeChi DangJohn HarmonSteve GeorasRoberto PiliJun LiuCharles SteenbergenAkhilesh PandeyAndre Levchenko

Josef PrchalChristian BauerAgata GiallongoRoger JohnsLowell DavisStella KourembanasKurt StenmarkDavid ZagzagFrank SharpElsken van der WallDaniel AebersoldRubin TuderJeffrey IsnerNanduri PrabhakarMikhail SitkovskyLee EllisShinae Kizaka-KondohKiichi HirotaHua YuMichal HorowitzMichael HadjiargyrouMichiko WatanabeBob KerbelTheresa LaValleYe SunFrank GonzalezCharles GrahamNajie JingGerd Schmitz

Tina HuangWendy XieHaiquan LuCaroline VissersLinh TranWalter JacksonShaima SalmanYongkang YangYufeng WangYajing LyuRu WangYiwei AiYayun ZhuChelsey ChenRima ShahSophia LeeNaveena MuruganKazuyo Yamaji-KeeganDavid KassKathy GabrielsonJenny van EykDenis WirtzJordan GreenEd GabrielsonSol SnyderFan PanCindy ZahnowAkrit SodhiDavid Meyers

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hypoxia-inducible factors in physiology and medicine2. physiology: even modest gain or loss of...

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