PULMONARY HYPERTENSION IN HIGH ALTITUDE NEWBORNS

ANÍBAL LLANOS M.D.

Laboratorio de Fisiología y Fisiopatología del DesarrolloPrograma de Fisiopatología, ICBM, Facultad de Medicina e INCAS,

UNIVERSIDAD DE CHILE.

ALFA-HAPPOM, DECEMBER 3, 2005-LYON, FRANCE

ATMOSPHERIC O2 LEVEL VS TIME

PAP in HIGH ALTITUDE

Moore y cols, 1998

Abman, 1999

PPHT IN THE NEWBORN

PULMONARY CIRCULATIONTRANSITION FROM FETUS TO NEONATE

Modificado de Abman, 1999

Hem

NITRIC OXIDE (NO)

Derived from L-arginina and catalyzed by NOS

Three NOS isoforms : nNOS (I), iNOS(II) y eNOS(III)

eNOS in the blood vessels → vasodilation

Travali y Patole, 2003

NO is an important pulmonary vasodilator

20 mmHg

22 mmHg

18 17 17 18 mmHg

Ross y cols, 2005

HYPOTHESIS

Chronic fetal hypoxia determines pulmonary vascular hypereactivity in the neonatal period. This hypereactivity will be associated with a lesser expression/activity of the NO pathway and can be assessed as pulmonary hypertension

p<0.05 : a, vs RNTB; b, vs basal; *, vs todo (dentro del grupo).

NEWBORN SHEEPPULMONARY CIRCULATION DURING ACUTE HYPOXIA-

0

25

50

PAP

(mm

Hg)

0

150

300

450

B B+I H+I R

GC

(ml.m

in-1

.kg-1

)

*a *

0

0,04

0,08

0,12

B B+I H+I RR

VP (m

mH

g.m

l-1.m

in.k

g)

*a aab

*

HIPOXIA

RNTB

RNTA

0 60 120 180Tiempo (min)

0

150

300

450

B B+I H+I R

GC

(ml.m

in-1

.kg-

1)

L-NAME

**

a

0,0

0,1

0,2

0,3

B B+I H+I R

RVP

(mm

Hg.

ml-1

.min

.kg)

L-NAME

*

*

a

a

b b

0

20

40

60

B B+I H+I R

PAP

(mm

Hg)

*

*

bb

a

a

a

b

L-NAME

P<0,05: a vs RNTB, b vs basal, * vs todos.

NEWBORN SHEEPPULMONARY CIRCULATION DURING ACUTE HYPOXIA + L-NAME

0

20

40

60

B B+I H+I R

PAP

(mm

Hg)

*a a

a

ab

*

RNTB

RNTANaCl O.9%

SUMMARY I

THE HIGH ALTITUDE NEWBORN SHEEP PRESENTS:

HIGH PULMONARY ARTERIAL PRESSURE AND RESISTANCE

NITRERGIC TONE IS IMPORTANT IN THE PULMONARY CIRCULATION.

NEVERTHELESS THERE IS PULMONARY HYPERTENSION

CONTRACTILITY OF THE PULMONARY ARTERIES?

PULMONARY ARTERIES

RESPONSES TO KCl

0 20 40 60 80 100 120

0

1

2

3

0,0

1,0

2,0

pD2

0,0

1,0

2,0

3,0

N/m

Tens

ión,

N/m

KCl, mM

p<0.05 a vs RNTB

RNTB RNTA

a

RESPONSES TO NPS

p<0.05 † vs LLNB

-10 -9 -8 -7 -6 -5 -4 -3

0

50

100

0,0

2,5

5,0

7,5

pD2

Tens

ión,

% K

MA

X

Log [SNP], M RNTB RNTA

a

p<0.05 a vs RNTB

PULMONARY ARTERIES

SUMMARY II

THE HIGH ALTITUDE NEWBORN SHEEP PRESENTS:

HIGHER CONTRACTILE CAPACITY

HIGHER SENSITIVITY TO NPS, CONSISTENT WITH A HIGHER NITRERGIC ACTIVITY

eNOS EXPRESSION & NOS ACTIVITY IN THE PULMONARY ARTERIES & LUNG?

0

15

30

45

60

RNTB RNTA

L-ci

trul

ine

form

atio

n (p

mol

. μg

prot

-1.3

0 m

in-1

)

0

0,25

0,5

0,75

1

RNTB RNTA

eNO

S/β

-act

in

(arb

itrar

y un

its)

0

0,25

0,5

0,75

1

RNTB RNTA

mR

NA

eN

OS/ β

-act

in

(arb

itrar

y un

its)

0

0,25

0,5

RNTB RNTA

sGC

/ β-a

ctin

(a

rbitr

ary

units

)

Significant differences p<0.05 : a, low-altitude vs high-altitude.

a

a

a

mRNA eNOS prot eNOS

actividad eNOS prot sGC

NOS-NO PATHWAY

SUMMARY III

THE HIGH ALTITUDE NEWBORN SHEEP PRESENTS:

DECREASED eNOS PROTEIN EXPRESSION, HOWEVER ITS ACTIVITY IS AUGMENTED

DECREASED sGC PROTEIN EXPRESSION

CONCLUSIONS NOS-NO

HIGH ALTITUDE & PULMONARY NO- PAP & PVR ↑ (with ↑ nitrergic tone)- eNOS mRNA =- eNOS protein ↓- eNOS activity ↑- sGC protein ↓

PARTIAL COMPENSATION OF THEHIPOXIC VASOCONSTRICTION

Remodelation? Vasodil/vasoconstriction?

0

10

20

30

40

PAP

(mm

Hg)

RNTARNTB

0 60 120 180TIEMPO (min)

HIPOXEMIA

0

10

20

30

40

PAP

(mm

Hg)

HIPOXEMIA

0 60 120 180TIEMPO (min)

NEONATAL LLAMA & SHEEPPulmonary circulation during acute hypoxia

LLAMA OVEJA

Herrera EA et alNEWBORN SHEEP NEWBORN LLAMA

0

10

20

30

40

B B + LNAME

PAP

(mm

Hg)

LASHHASH

0

10

20

30

40

B B + LNAME

PAP

(mm

Hg)

LALLHALL

0

0,1

0,2

0,3

B B + LNAME

PVR

(mm

Hg.

mL-1

.min

.Kg)

0

0,1

0,2

0,3

B B + LNAME

PVR

(mm

Hg.

mL-1

.min

.Kg)

ab

a

bc

b

a

ab

a

bc

aa

c

A. PAP in NB sheep with and without L-NAME, B. PAP in NB llama with and without L-NAME, C. PVR in NB sheep with and without L-NAME, D. PVR in NB llama with and without L-NAME

A. B.

C. D.

Significant differences p<0.05 : a, vs basal; b, low-altitude vs high-altitude; c, sheep vs llama (at same altitude).

Herrera EA et al

0

15

30

45

60

LASH HASH LALL HALL

L-ci

trul

ine

form

atio

n (p

mol

. μg

prot

-1.3

0 m

in-1

)

0

0,25

0,5

0,75

1

LASH HASH LALL HALL

eNO

S/β

-act

in

(arb

itrar

y un

its)

0

0,25

0,5

0,75

1

LASH HASH LALL HALL

sGC

/ β-a

ctin

(a

rbitr

ary

units

)A. B.

C.

A. Western blot of eNOS, B. NOS total activity, C. Western blot of sGCSignificant differences p<0.05 : b, low-altitude vs high-altitude ; c, sheep vs llama (at same altitude).

b

b

b

Herrera EA et al

0

0,25

0,5

0,75

1

LASH HASH LALL LASH

HO

-1/ β

-act

in

(arb

itrar

y un

its)

0

0,5

1

1,5

LASH HASH LALL HASH

HO

-2/ β

-act

in

(arb

itrar

y un

its)

A. B.

C.

A. Western blot of HO-1, B. Western blot of HO-2, C. CO lung production. Significant differences p<0.05 : b, low-altitude vs high-altitude; c, sheep vs llama (at same altitude).

0

1

2

3

4

5

LASH HASH LALL HALL

CO

pro

duct

ion

(mm

oles

CO

.min

-1.m

l-1)

b

b,c

b

bb

0

0.2

0.4

0.6

0.8

1

1.2

OVTB OVTA0

0.2

0.4

0.6

0.8

1

1.2

LLTB LLTA

OVEJA LLAMA

SHEEP & LLAMA NEWBORNPDEV mRNA EXPRESSION IN PULMONARY TISSUE

Uni

dade

s re

latia

vs(P

DEV

/ 18

S)

Uni

dade

s re

latia

vs(P

DEV

/ 18

S)

OVTB: recién nacido de oveja de tierras bajas (n=5), OVTA: recién nacido de oveja de tierras altas (n=5), LLTB: recién nacido de llama de tierras bajas (n=5), LLTA: recién nacido de llama de tierras altas (n=5), p<0.05 (test de Student).

*

VASODILATADOR NO-CO-cGMP MECHANISMS

Modificado de Carvajal et al. J. Cell. Physiol. , 2000.

K+

(+)

SUMMARY IV

IN THE HIGH ALTITUDE NEWBORN LLAMA:

HEM OXYGENASE-CO, APPEARS TO BE CENTRAL IN THE REGULATION OF THE PULMONARY CIRCULATION

Enero 2004

HIGHLANDERS 2005RAQUEL RIQUELMEEMILIA SANHUEZAEMILIO HERRERA ROBERTO REYES

GERMAN EBENSPERGERBERNARDO KRAUSE

PAULA GÁLVEZANIBAL LLANOS

CARLOS BRITOMARIO MORALESGABINO LLUSCO

RENATO EBENSPERGERNATALIA MENDEZ

IRMA VALENZUELADANIEL PEÑA

EVELYN CRUZ RODRIGO ROJAS

JULIAN PARERENRIQUE VALDEZ

MARK HANSONCARLOS BLANCO

DINO GIUSSANI

FFDD

FINANCIAMIENTOFONDECYT 1010636 & 1050479-CHILE

THE WELLCOME TRUST- CRIG 072256-UK

0 60 120 1800

10

20

30

40

50

Hipoxemia

45

Indometacina

Tpo (min)

PAP

(mm

Hg)

0 60 120 1800

10

20

30

40

50

H solaH indo

45

Indometacina

Hipoxemia

Tpo (min)

PAP RNOV Stgo (580m)PAP RNOV Putre (3600m)

PULMONARY ARTERIAL PRESSURE (PAP) IN NEWBORN SHEEPH ALONE VS H+INDOMETHACIN

n=5 n=6

0

0,01

0,02

0,03

0,04

0,05

N H 20 40 80 100

CO (ppm)

PVR

(mm

Hg·

ml-1

·min

·kg-1

)INHALATORY CO & PVR

HANB LANB

CHRONIC FETAL HYPOXIA FETAL NORMOXIA

PUTRE 3,600 m SANTIAGO 580 m

PDE5

PDE5

NOSsGCNO

NOSsGCNO

O2n

PayachatasPayachatas 6330 mts6240 mts

0

50

100

150

SAP

(mm

Hg)

0

100

200

300

400

HR (m

in-1

)

0 60 120 180 Time (min)

0,00

0,20

0,40

Basal Hypoxemia Recovery

SVR

(mm

Hg.

ml-1

.min

.Kg)

†* *

SYSTEMIC CIRCULATION - NEWBORN SHEEP

p<0.05, † vs basal, * vs RNTB.

SLSH

HLSH

MECANISMO VASODILATADOR NO-CO

Hampl y Herget, 2000

SYSTEM PHYSIOLOGYNEWBORN SHEEPCHRONIC INSTRUMENTATIONSUPERIMPOSED ACUTEHYPOXIA

L-NAME (20 mg.kg-1 + 0.5 mg.kg-1.min-1)

MEASUREMENT OF CARDIOVASCULAR VARIABLESPAP, PcP, CARDIAC OUTPUT, SAP, HR

- 1h BASAL- 1h HYPOXIA- 1h RECOVERY

PAP & NO in High Altitude

Pulmonary NO in the chronic hypoxic neonate?

NO is a key mediator in thetransition from fetus ti

neonate

0

10

20

30

40

PAP

(mm

Hg)

RNTARNTB

0 60 120 180TIEMPO (min)

HIPOXEMIA

0

10

20

30

40

PAP

(mm

Hg)

HIPOXEMIA

0 60 120 180TIEMPO (min)

NEONATAL LLAMA & SHEEPPulmonary Circulation during acute hypoxia

LLAMA OVEJA

HOW THE OXYGEN IS SENSED?

• THE OXYGEN IS SENSED AS PO2

• CHEMORECEPTORS (e.g. carotid body, by inhibition of a potassium channel, generating an action potential traveling by the sinus nerve)

•In the PULMONARY ARTERY, in the ENDOTHELIUM and/or SMOOTH MUSCLE CELLS by inhibition of a potassium channel

• ADRENAL MEDULLA by inhibition of a potassium channel

• In all cells by means of the PROLYL HYDROXILASES and HIF-1

EX VIVO STUDIES

ARTERIES 150-400 umMOUNTED ON A WIRE MYOGRAPH Optimal Diameter (KCl, 65 mM K+)

Maximal Contraction (KCl, 4,75-125 mM K+)PHARMACOLOGIC RESPONSE

SNP (10-10-10-3M)

HIPOTHESIS

THE SMALL RESISTANCE ARTERIES OF THE PULMONARY CIRCULATION IN THE CHRONICALLY HYPOXIC NEWBORN SHEEP AT HIGH ALTITUDE HAVE ENHANCED CONTRACTILE RESPONSES THAN LOWLAND NEWBORN LAMBS

RESPONSES ex vivoPOTASSIUM CHLORIDESNP

AIM

TO DETERMINE THE SMALL RESISTENCE ARTERIES RESPONSES OF THE PULMONARY CIRCULATION IN HA AND LA NB SHEEP

HYPOTHESIS

THE EXPRESSION & ACTIVITY OF THE NITRERGIC PATHWAY IS INCREASED IN THE LUNG OF HA-NB LAMBS, AS A COMPENSATORY MECHANISM TO COPE WITH THE PULMONARY HYPERTENSION IN HIGHLANDS

AIM

TO DETERMINE THE mRNA & PROTEIN EXPRESSION OF eNOS, sGC, AND NOS ACTIVITY

BIOCHEMISTRY & MOLECULAR BIOLOGY TISSUE OBTENTION- mRNA EXPRESSION (RT-PCR)

eNOS, PDEV vs β-actin, - PROTEIN EXPRESSION (WB)

eNOS, sGCα vs β-actin- ACTIVITY

NOS