kekeijiiji narusenaruse, m.d. & ph.d., m.d. & ph.d....cardiovascular physiology okayama...

Cardiovascular Physiology Okayama University Graduate School of MedicineCardiovascular Physiology Okayama University Graduate School of Medicine

MechanobiologyMechanobiology and its Clinical Applicationand its Clinical Application

KeijiKeiji NaruseNaruse, M.D. & Ph.D., M.D. & Ph.D.Professor and ChairProfessor and Chair

Cardiovascular Physiology, Okayama Cardiovascular Physiology, Okayama UnivUniv Grad Grad SchSch MedMedCSO, STREX Inc.CSO, STREX Inc.

OVERVIEWOVERVIEW


MECHANOBIOLOGYMECHANOBIOLOGY

MECHANOTRANSDUCTIONMECHANOTRANSDUCTION

REPRODUCTIVE MEDICINEREPRODUCTIVE MEDICINE

Modified from”ICURUS”by HENRY MATISSE

HEARINGHEARING


TOUCHTOUCH

JOINTSJOINTS

CARDIOVASCULARCARDIOVASCULAR

REPRODUCTIONREPRODUCTION

MECHANOBIOLOGYMECHANOBIOLOGY

1xG1xG

We are continually exposed to mechanical stimuliWe are continually exposed to mechanical stimuli

TEETHTEETHperiodontal ligament cells Bone

Endothelial cellsEndothelial cells

NO, ET1 etc.NO, ET1 etc.Gene expressionGene expressionMorphological changesMorphological changes


Hemodynamic forcesHemodynamic forces

pressurepressurestretchstretch

shear stressshear stress

STRETCHSTRETCH--ACTIVATED ACTIVATED ION CHANNELION CHANNEL


Biomedical EngineeringBiomedical Engineering

MEMSMEMSBiomedicalBiomedical

SOFT LITHOGRAPHYSOFT LITHOGRAPHY•• MEMS(MEMS(micro electro mechanical systems))

MechanobiologyMechanobiology

•• PDMS PDMS ((polydimethylsiloxanepolydimethylsiloxane))•• nmnm--mmmm•• MeritsMerits

–– TransparentTransparent–– StableStable–– FlexibleFlexible–– High throughput High throughput –– InexpensiveInexpensive

millingmillingphotolithographyphotolithography

Light, EBLight, EBphotomaskphotomask

registregist

Silicon waferSilicon wafer

curecure

PDMSPDMS

porepore

lift offlift off

Mechanotransduction


Soft LithographySoft Lithography

Stretch chamberStretch chamber

mouldmould

PDMSPDMS

Mechanotransduction


Stretch ChamberStretch Chamber

Mechanotransduction


Stretch SystemStretch System

1 Hz 20 %Cardiovascular Physiology Okayama University Graduate School of MedicineCardiovascular Physiology Okayama University Graduate School of Medicine

StretchStretch--induced Morphological changeinduced Morphological change

1 Hz20 %

0 min0 min

30 min30 min

60 min60 min

120 min120 min

1 Hz, 20 %Cardiovascular Physiology Okayama University Graduate School of MedicineCardiovascular Physiology Okayama University Graduate School of Medicine

0 30 60 90 120 150 18005

101520

%

0 30 60 90 120 150 18005

101520

%

0 30 60 90 120 150 18005

101520

%

0 30 60 90 120 150 18005

101520

%

StretchStretch--induced morphological changeinduced morphological change

1 Hz, 20 % 60 min1 Hz, 20 % 60 min

CaCa2+2+ freefree

GdGd3+3+ GdGd3+3+ CaCa2+2+ freefree


0 min0 min

30 min30 min

60 min60 min

120 min120 min

0 30 60 90 120 150 18005

101520

%

0 30 60 90 120 150 18005

101520

%0 30 60 90 120 150 1800

5101520

%

0 30 60 90 120 150 18005

101520

%

0 30 60 90 120 150 18005

101520

%

0 30 60 90 120 150 18005

101520

%

0 30 60 90 120 150 18005

101520

%

0 30 60 90 120 150 18005

101520

%

StretchStretch--induced morphological changeinduced morphological change

StretchStretch--activated channel dependentactivated channel dependent

Plasma membranePlasma membrane

SA channelSA channel

CaCa2+2+influxinflux

Fura2Fura2

FluorsecenceFluorsecence MicroscopyMicroscopy

Gd blocks SA channel


StretchStretch--induced Ca responseinduced Ca response

(10%)(5%) (15%) (20%) (25%)

Br J Pharmacol, 2002

200 400 600 800Time (sec)

00.4

0.6

0.8

1.0

1.2

1.4

1.6

Ratio

(340

/380

)

Stretch for 3 sec



0 100 200 300 400 500 6000.4

Time (sec)

Gd3+

(10%)(5%) (15%) (20%) (25%) (10%)(5%) (15%) (20%) (25%)

Br J Pharmacol, 2002

00.4

0.6

0.8

1.0

1.2

1.4R

atio

(340

/380

)

Stretch for 3 sec



StretchStretch--activated channel dependentactivated channel dependent

•• MscLMscL, , MscSMscS (Non(Non--selective)selective)

••SAKCA (K SAKCA (K ++ selective)selective)

••MidMid--1 (1 (NonNon--selective)selective)••TREK/TRAAK TREK/TRAAK ((KK++ selective)selective)

••TRPs (TRPs (cationcation selective)selective)



Science, 285:882Science, 285:882--885 1999885 1999

Molecular Identification of a Eukaryotic, StretchMolecular Identification of a Eukaryotic, Stretch--Activated Nonselective Activated Nonselective CationCation ChannelChannel ：：Mid1Mid1


SA channelSA channel Mid1Mid1

MDALIIPVTMEVPCDSRGQRMWWAFLASSMVTFFGGLFIILLWRTLKYLW 50MDALIIPVTMEVPCDSRGQRMWWAFLASSMVTFFGGLFIILLWRTLKYLW 50TVCCHCGVKNKEAQKINGGGDTQADGACKPTDEKEENVAAEVGWMTSVKD 100TVCCHCGVKNKEAQKINGGGDTQADGACKPTDEKEENVAAEVGWMTSVKD 100WAGVMISAQTLTGRVLVVLVFALSIGALVIYFIDSSNPIESCQNFYKDFT 150WAGVMISAQTLTGRVLVVLVFALSIGALVIYFIDSSNPIESCQNFYKDFT 150LQIDMAFNVFFLLYFGLRFIAANDKLWFWLEVNSVVDFFTVPPVFVSVYL 200LQIDMAFNVFFLLYFGLRFIAANDKLWFWLEVNSVVDFFTVPPVFVSVYL 200NRSWLGLRFLRALRLIQFSEILQFLNILKTSNSIKLVNLCSIFISTWLTA 250NRSWLGLRFLRALRLIQFSEILQFLNILKTSNSIKLVNLCSIFISTWLTA 250AGFIHLVENSGDPWENFQNNQQLTYWECVYLLMVTMSTVGYGDVYAKTTL 300AGFIHLVENSGDPWENFQNNQQLTYWECVYLLMVTMSTVGYGDVYAKTTL 300GRLFMVFFILGGLAMFASYVPEIIELIGNRKKYGGSYSAVSGRKHIVVCG 350GRLFMVFFILGGLAMFASYVPEIIELIGNRKKYGGSYSAVSGRKHIVVCG 350HITLESVSNFLKDFLHKDRDDVNVEIVFLHNISPNPELEALFKRHFTQVE 400HITLESVSNFLKDFLHKDRDDVNVEIVFLHNISPNPELEALFKRHFTQVE 400FYQGSVLNPHDLARVKIESADACLILANKYCADPDAEDASNIMRVISIKN 450FYQGSVLNPHDLARVKIESADACLILANKYCADPDAEDASNIMRVISIKN 450YHPKIRIITQMLQYHNKAHLLNIPSWNWKEGDDAICLAELKLGFIAQSCL 500YHPKIRIITQMLQYHNKAHLLNIPSWNWKEGDDAICLAELKLGFIAQSCL 500APGLSTMLANLFSMRSFIKIEEDTWQKYYLEGVANEMYTEYLSSAFVGLS 550APGLSTMLANLFSMRSFIKIEEDTWQKYYLEGVANEMYTEYLSSAFVGLS 550FPAVCELVFAKLKLLMIAIEYKSEKRESSILINPGNHVKIQEGTLGFFIA 600FPAVCELVFAKLKLLMIAIEYKSEKRESSILINPGNHVKIQEGTLGFFIA 600SDAKEVKRAFFYCKACHDDITDPKRIKKCGCKRSDAKEVKRAFFYCKACHDDITDPKRIKKCGCKRPKMSIYKRMKLACCFDCPKMSIYKRMKLACCFDC 650650GRSERDCSCMSGSVHSNMDTLERAFPLSSVSVNDCSTSLRAFGRSERDCSCMSGSVHSNMDTLERAFPLSSVSVNDCSTSLRAFEDEQPSTL 700EDEQPSTL 700SPKKKQRNGGMRNSPNSSPKLMRHDPLLIPGNEQIDNMDANVKKYDSTGM 750SPKKKQRNGGMRNSPNSSPKLMRHDPLLIPGNEQIDNMDANVKKYDSTGM 750FHWCPAKDIEKVILTRSEAAMTVLSGHVVVCIFGDVKSALIGLRNLVMPL 800FHWCPAKDIEKVILTRSEAAMTVLSGHVVVCIFGDVKSALIGLRNLVMPL 800RASNFHYHELKHIVFVGSLEYLRREWETLHNFPKVSILPGTPLSRADLRA 850RASNFHYHELKHIVFVGSLEYLRREWETLHNFPKVSILPGTPLSRADLRA 850VNINLCDMCVILSANQNNIDDASLQDKECILASLNIKSMQFDDSIGVLQA 900VNINLCDMCVILSANQNNIDDASLQDKECILASLNIKSMQFDDSIGVLQA 900NSQGFTPPGMDRSSPDNSPVHGLLRQPSITTGANIPIITELVNDSNVQFL 950NSQGFTPPGMDRSSPDNSPVHGLLRQPSITTGANIPIITELVNDSNVQFL 950DQDDDDDPDTELYLTQPFACGTAFAVSVLDSLMSATYFNDNILTLIRTLV 1000DQDDDDDPDTELYLTQPFACGTAFAVSVLDSLMSATYFNDNILTLIRTLV 1000TGGATPELEALIAEENALRGGYSTPQTLANRDRCRVAQLALYDGPFADLG 1050TGGATPELEALIAEENALRGGYSTPQTLANRDRCRVAQLALYDGPFADLG 1050DGGCYGDLFCKALKTYNMFCFGIYRLRDAHLSTPSQCTKRYVITNPPYEF 1100DGGCYGDLFCKALKTYNMFCFGIYRLRDAHLSTPSQCTKRYVITNPPYEF 1100ELVPTDLIFCLMQFDHNAGQSRASLSHSSHSSYSSSKKSSSVHSIPSTAN 1150ELVPTDLIFCLMQFDHNAGQSRASLSHSSHSSYSSSKKSSSVHSIPSTAN 1150RPNRTKTRDSREKQKYVQEDRL 1172RPNRTKTRDSREKQKYVQEDRL 1172

SAKCA

STREX: STREX: StStress Axis ress Axis RReguratedegurated EXEXonon

`

STREX

S0S1S2S3S4S5 S6

COOH

NH2



0 10 20 30 4005

1015202530

pressure(-mmHg) P

o (%

)50

`STREX

S0S1S2S3S4S5S6

`S0S1S2S3S4S5S6

TRPM

TRPP

TRPML

TRPC

TRPV

TRPATRPN

Mechanosensitivechannel



Transient Receptor Potential channels・ Drosophila Retina・ non-selective cation channel・ 6 putative transmembrane helices ・ ankyrin repeats (N-terminal)・ variety of sensory system・ TRPV1→Thermosensor・ Mechanosensor?・ TRPV2→SA channel?

COS7（Control）10%

20% 40%

10 20 40

COS7（hTRPV2-His） 10%

20% 40% 10 20 40

Transient Rceptor Potential channels


COS7（hTRPV2-His）

SA channelSA channel TRPV2TRPV2

1

0 100000 200000

0.3

0.4

0.5

0.6

0.7

0.8

Rat

io (

340nm

/380nm

)

Time (msec)

4

5

6

23

1

2

34

56

ratio380 nm TRPV2-siRNA

Effect of TRPV2-siRNA on stretch-induced [Ca2+]i response

HUVEC



心筋症・心不全におけるTRPV2の役割

NEW YORK, ManhattanCardiovascular Physiology Okayama University Graduate School of MedicineCardiovascular Physiology Okayama University Graduate School of Medicine


TRPV2 TG, KO, KI mouseTRPV2 TG, KO, KI mouse

Inducible, Tissue specific, Human KIDouble KO

Wild V2 TG



Ito et al., ARCMB, 2008

“Nano-crack”

Fabrication of reconfigurable protein matrices by crackingNature Materials 4（5）：403‐406，2005



Conventional cantileverFast Scanning cantilever

10μ10μｍｍ

200μ200μｍｍ

Olympus


Fast Scanning Atomic Force MicroscopyFast Scanning Atomic Force Microscopy

plasmid (800 x 600 nm)

Fibronectin(７４0 x 560 nm)

1 nm10 nm

MscL / WT MscL / G22NOpen mutant

5 nm

Mold & cure

moldcure

Ink

Print

PDMS stretch-chamber

Fibronectin

PDMS

Fibronectin

(A)

(B) (C)

(D) (E)

(F) (G)

(H)


Microcontact Printing

StretchStretch--inducedinduced Responses Responses in Patterned in Patterned HUVECHUVEC

Bef

ore

stre

tch

Aft

er s

tret

ch(1

5 m

in)

Aft

er s

tret

ch(6

0 m

in)

Anti- PY Phalloidine

20 um


Microcontact Printing on Stretch chamber

StretchStretch--inducedinduced Responses Responses in Patterned in Patterned HUVECHUVEC

00.10.20.30.40.50.60.7

Ratio (A

340/

A38

0)

stretch

35μm×35μm25μm×25μm20μm×20μm10μm×10μm

1min

Z

X-Y

Fura 2

Tyrosine phosphorylation Stretch-induced [Ca2+]i

J Biotech 2008

Coverslip

Cells in microchannel

W=200μm

H=50μm①High shear②Laminar flow



CONTROL

SHEAR-STRESS

Nuclear Factor kB

Cell response in microchannel

0.4

0.5

0.6

0.7

0.8

0.9

1

500 600 700 8 00 9 00 10 00

ti me (se c)

rati

o (F

340/

F380

)

0.25μM ATP

9 dyn/ cm2

18 dyn/cm2

[Ca2+]i in response to shear stress

0.4

0.5

0.6

0.7

0.8

0.9

1

500 600 700 8 00 9 00 10 00

ti me (se c)

rati

o (F

340/

F380

)

0.25μM ATP

9 dyn/ cm2

18 dyn/cm2



Principle of laminar flow

～500μm in width～ 50μm in hight

W=500μm

H=50μm




W=200μm

H=50μm

Principle of laminar flow

PARTCEL(pattented)

“Subcellular positioning of small molecules”

Nature, 2001Cardiovascular Physiology Okayama University Graduate School of MedicineCardiovascular Physiology Okayama University Graduate School of Medicine

A

B

C

HHuman uman AAssisted ssisted

RReproductive eproductive TTherapyherapy


INFERTILITYINFERTILITY

MechanobiologyMechanobiology in Reproductive Medicinein Reproductive Medicine

••Decreasing in PopulationDecreasing in Population••Death(Death(1,077,0001,077,000)>Birth()>Birth(1,067,0001,067,000))

••Decreasing in BirthrateDecreasing in Birthrate••Total fertility rate : ~1.3Total fertility rate : ~1.3••Various causeVarious cause

••InfertilityInfertility••One in 5One in 5--10 couples10 couples

Male factor=Female factorMale factor=Female factor

••22--3 % : IVF3 % : IVF平成18年6月2日日経新聞


INFERTILITY in JAPANINFERTILITY in JAPAN

MechanobiologyMechanobiology in Reproductive Medicinein Reproductive Medicine


INFERTILIGY TREATMENTINFERTILIGY TREATMENT

•• Timing treatment (Ovulation)Timing treatment (Ovulation)

•• AIH (Artificial Insemination by Husband)AIH (Artificial Insemination by Husband)

•• IVF (In Vitro Fertilization)IVF (In Vitro Fertilization)–– IVFIVF‐‐conventionalconventional

–– IVFIVF‐‐ICSIICSI（（IntracytoplasmicIntracytoplasmic sperm injectionsperm injection））

Sperm

Artificial Insemination Conventional IVF

ICSI


Conventional Conventional HHuman uman AAssisted ssisted RReproductive eproductive TTreatment (HART)reatment (HART)

Density gradient

Swim‐up

centrifuge400×g, 20min

Sperm preparationSperm preparation

Culture dishMimeral oil

Culture mediumEmbryo

STATIC CULTURE

Egg cultureEgg culture


NonNon--physiologicalphysiological

1. Microfluidic Sperm Sorter System2. Mechanically Active Embryo Culture System

STREX www.strex.jpwww.strex.jpCardiovascular Physiology Okayama University Graduate School of MedicineCardiovascular Physiology Okayama University Graduate School of Medicine


STREX www.strex.jpwww.strex.jp

W=500μm, H=50μm

Sperm Sorter /microfluidics



Takayama S, Analytical Chemistry, 75:1671-1675, 2003STREX www.strex.co.jpwww.strex.co.jpInitialInitial SortedSorted

00

2020

4040

6060

8080

100100S

perm

Mot

ility

(%

)S

perm

Mot

ility

(%

)

n=10n=10

n=10n=10

aa

bba,b; P<0.05a,b; P<0.05

Sperm Sorter /microfluidics



400 x ｇ

Conventional protocol

Motile sperm

SWIM UP !!SWIM UP !!

MFSS & Dilution

+semen medium Dilution

> X 3MFSS

No centrifugation

＜岡山大学医学部＞

＜名古屋大学医学部＞Cardiovascular Physiology Okayama University Graduate School of MedicineCardiovascular Physiology Okayama University Graduate School of Medicine


No CentrifugationNo Centrifugation

conv

MFSS separation

min ０３０６０９０１２０

semen prep

centrifugation density grad Swim up

＜名古屋大学医学部＞Cardiovascular Physiology Okayama University Graduate School of MedicineCardiovascular Physiology Okayama University Graduate School of Medicine


Short processing timeShort processing time

010203040506070

%

59.6%59.6%59/9959/99 46.8%46.8%

51/10951/109

Sperm SorterSperm Sorter conventionalconventional



Fertilization rate ↑Fertilization rate ↑

•IRB: Passed 13/5/2005•40 couples with informed consent•> 4 eggs ⇒ ICSI•Embryo transplantation

＜プラスチック製＞

0.45 mm

0.08 mm

1 JPY

Quartz

Plastic

0.49 mm

0.18 mm

a

b

c

d



http://www2.edu.ipa.go.jp/gz/ Cardiovascular Physiology Okayama University Graduate School of MedicineCardiovascular Physiology Okayama University Graduate School of Medicine


http://www2.edu.ipa.go.jp/gz/ Cardiovascular Physiology Okayama University Graduate School of MedicineCardiovascular Physiology Okayama University Graduate School of Medicine


RAT FALLOPIAN TUBERAT FALLOPIAN TUBE Mouse eggs in fallopian tubeMouse eggs in fallopian tube


Shear StressCompressionStretch TurbulenceFriction

Mechanical stresses applied to embryo in oviductMechanical stresses applied to embryo in oviduct



TECSTECS

Tilting Embryo Culture System (TECS) Tilting Embryo Culture System (TECS)

Shear stress(dynes/cm2)

Velocity(mm/sec) 0.003 0.0018

*p=0.035

Blastocysts/total %

Control 63/136 46

TECS 77/130 59

Tilting culture unitControl unit

STREX Inc.

20deg tilting for 10 min, 4-6 mouse eggs/drop

Roll

• Nagoya Univ– M. Sokabe– T. Yamada– X. Sai– K. Wang– H. Inoh– H. Takeda

• Nagoya UnivHospital– H. Ando– D. Shibata

• Harvard Univ– D. Ingber– G.Whitesides

• Michigan Univ– S. Takayama– G. Smith

• ICORP/JST– X. Tang– F. Lopez

• OKAYAMA Univ– S. Mohri– Y. Katanosaka– G. Iribe– A. Yamada– H. Kishio– H. Bao– T. Suemori– T. Komatsu– M. Kajiya

Acknowledgement


kekeijiiji narusenaruse, m.d. & ph.d., m.d. & ph.d....cardiovascular physiology okayama...

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