Vascular endothelial growth factor increases vascular ... · PDF fileVascular endothelial...

22
Revista de Medicină şi Farmacie Orvosi és Gyógyszerészeti Egyetem Szemle, 2009, 55: 227-230 Vascular endothelial growth factor increases vascular permeability in acute respiratory distress syndrome Raluca Solomon 1 , L. Azamfirei 2 , Simona Gurzu 3 , Ruxandra Copotoiu 2 , Sanda Copotoiu 2 , I. Jung 3 , J. Szederjesi 2 , Judit Kovacs 2 Objective: investigation of the VEGF expression in the lung tissue of ARDS patients and also the VEGFS plasmatic levels of these patients. Material and method: there were examined immunohistochemical lung specimens from 10 ARDS patiens which were compared to a control group. The VEGF seric levels was determined using ELISA method, at the same time with the tissue sampling from ARDS patients and during the hospital stay in case of the control group of non ARDS patients. Results: patients who died because of ARDS had VEGF pulmonary expression significantly decreased comparing to non-ARDS patients (p<0.001). The seric VEGF levels of ARDS patients were raised comparing to non-ARDS patients (p<0.001). Conclusions: A decreased alveolar type II cells, was noticed in ARDS evolution, therefore reducing the VEGF production in the alveolar space and also contributing to the decrease in lung perfusion, but also to the consecutive increase of VEGF plasmatic level. Keywords: ARDS, Vascular endothelial growth factor Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury (ALI), remains a devastating condition characterised by difuse injury of the alveolo-capillary wall and alveolar and interstitial oedema consecutive to increased pulmonary vascular permeability. It concerns 1,5-8,3 cases in every 100 000 patients, still having a signifiant mortality of 30-50% despite improvements in the management of sepsis and lung protective ventilation strategy. 2 There is a heterogeneous group of conditions, both direct or indirect insults, such as, sepsis, trauma, apiration, massive blood transfusion or burns which predispose to ARDS. The pathogenetic basis of ARDS and factors governing susceptibility are incompletely understood. Markers of both epithelial and endothelial injury have been correlated with outcome 8 , wheras the severity of ARDS depends significantly on the balance between alveolar epithelial and/or vascular endothelial injuries and their repair rnechanisms. 13 Vascular endothelial cell growth factor (VEGF) was identified by its properties to increase permeability and acts as a cellular growth factor, hence its potential 1 Anesthesiology and Intensive Care Clinic, Mureş County Emergency Hospital 2 Anesthesiology and Intensive care Department, Faculty of Medicine, University of medicine and Pharmacy Tg.Mureş 3 Department of pathology, Faculty of Medicine, University of Medicine and Pharmacy Tg. Mureş for a key role in the pathogenesis of ALI/ARDS. The VEGF family has several members, and each acts through specific receptors. The human VEGF gene family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E., and placenta growth factor (PlGF), all with multiple and diverse biological functions. The genes for VEGF family members also rely on alternative exon splicing to confer various isoforms for biological and functional specificity. 9 The most studied molecule of the VEGF family is VEGF-A. lts double effect, both permogen and mitogen 3 induces vascular endothelial cell proliferation and promotes survival by induction of anti-apoptotic proteins bcl-2 and Al. 5 VEG . F increases microvascular permeability, but also it also exerts bioactivity outside the vascular endotheliurn: macrophages, type II pneumocytes, and monocytes for which it may be chernotactic. 4 The biological activity of VEGF is dependent on interaction with specific receptors (VEGF-R1, 2, and 3), which are expressed not only by endothelial cells, but also by activated macrophages and alveolar type II epithelial cells. 1 VEGF acts also on a family of coreceptors, the neuropilins. 10 The objective of this study was to investigate the VEGF expression in the lung tissue of ARDS patients and also the VEGF plasmatic levels of these patients compared to a control group.

Transcript of Vascular endothelial growth factor increases vascular ... · PDF fileVascular endothelial...

Revista de Medicină şi Farmacie – Orvosi és Gyógyszerészeti Egyetem Szemle, 2009, 55: 227-230

Vascular endothelial growth factor increases vascular permeability in acute respiratory distress syndrome

Raluca Solomon1, L. Azamfirei

2, Simona Gurzu

3, Ruxandra Copotoiu

2, Sanda

Copotoiu2, I. Jung

3, J. Szederjesi

2, Judit Kovacs

2

Objective: investigation of the VEGF expression in the lung tissue of ARDS patients and also the VEGFS

plasmatic levels of these patients.

Material and method: there were examined immunohistochemical lung specimens from 10 ARDS patiens which were compared to a control group. The VEGF seric levels was determined using ELISA method, at the

same time with the tissue sampling from ARDS patients and during the hospital stay in case of the control

group of non ARDS patients.

Results: patients who died because of ARDS had VEGF pulmonary expression significantly decreased comparing to

non-ARDS patients (p<0.001). The seric VEGF levels of ARDS patients were raised comparing to non-ARDS patients

(p<0.001).

Conclusions: A decreased alveolar type II cells, was noticed in ARDS evolution, therefore reducing the VEGF

production in the alveolar space and also contributing to the decrease in lung perfusion, but also to the consecutive

increase of VEGF plasmatic level. Keywords: ARDS, Vascular endothelial growth factor

Acute respiratory distress syndrome (ARDS), the

most severe form of acute lung injury (ALI), remains a devastating condition characterised by difuse injury of the alveolo-capillary wall and alveolar and interstitial oedema consecutive to increased pulmonary vascular permeability. It concerns 1,5-8,3 cases in every 100 000 patients, still having a signifiant mortality of 30-50% despite improvements in the management of sepsis and lung protective ventilation strategy.

2 There is a heterogeneous group

of conditions, both direct or indirect insults, such as, sepsis, trauma, apiration, massive blood transfusion or burns which predispose to ARDS. The pathogenetic basis of ARDS and factors governing susceptibility are incompletely understood. Markers of both epithelial and endothelial injury have been correlated with outcome

8, wheras the severity of ARDS depends

significantly on the balance between alveolar epithelial and/or vascular endothelial injuries and their repair rnechanisms.

13

Vascular endothelial cell growth factor (VEGF) was identified by its properties to increase permeability and acts as a cellular growth factor, hence its potential 1Anesthesiology and Intensive Care Clinic, Mureş County

Emergency Hospital 2Anesthesiology and Intensive care Department, Faculty of

Medicine, University of medicine and Pharmacy Tg.Mureş 3Department of pathology, Faculty of Medicine, University of

Medicine and Pharmacy Tg. Mureş

for a key role in the pathogenesis of ALI/ARDS. The VEGF family has several members, and each acts through specific receptors. The human VEGF gene family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E., and placenta growth factor (PlGF), all with multiple and diverse biological functions. The genes for VEGF family members also rely on alternative exon splicing to confer various isoforms for biological and functional specificity.

9

The most studied molecule of the VEGF family is VEGF-A. lts double effect, both permogen and mitogen

3 induces vascular endothelial cell

proliferation and promotes survival by induction of anti-apoptotic proteins bcl-2 and Al.

5 VEG

.F

increases microvascular permeability, but also it also exerts bioactivity outside the vascular endotheliurn: macrophages, type II pneumocytes, and monocytes for which it may be chernotactic.

4 The biological

activity of VEGF is dependent on interaction with specific receptors (VEGF-R1, 2, and 3), which are expressed not only by endothelial cells, but also by activated macrophages and alveolar type II epithelial cells.

1 VEGF acts also on a family of coreceptors, the

neuropilins.10

The objective of this study was to investigate the VEGF expression in the lung tissue of ARDS patients and also the VEGF plasmatic levels of these

patients compared to a control group.

userr
Line
userr
Textbox
1. Solomon2009
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
Administrator
Typewriter
Digitized

228 Raluca Solomon, L. Azamfirei, Simona Gurzu, Ruxandra Copotoiu, Sanda Copotoiu, I. Jung, J. Szederjesi, Judit Kovacs

MATERIAL AND METHOD

We realized a prospective study including 10 patients

diagnosed with ARDS. These patients were evaluated

daily during their stay in the ICU and those included in

this study accomplished the ARDS diagnosis criteria as

recommended by the international American-European

consensus conference. The parameters needed to assess

the SOFA score were determined daily and were refered

to the score obtained on the fist day of setting the ARDS

diagnostic.

Lung specimens from ARDS patients were obtained by

bronchoscopy or by autopsy in case of the deceased

patients. The controls were 10 patients deceased from

other causes than ARDS from whom necroptic pulmonary

tissue samples were obtained and processed as for the

ARDS patients.

All lung samples were standard stained using

Haematoxylin Eosine and were examined in order to

assess the histopathological diagnosis of ARDS: presence

of hyaline membranes, type II pneumocyte proliferation

associated with myofibroblast proliferation.

The determination of VEGF expression in the

pulmonary tissue was performed using specific

monoclonal antibodies VEGF, clones VG1 and JH 121

produced by LabVision firm. A charge-coupled video

camera coupled with an optical microscope, was used to

view the sections and to digitize images to a PC host

computer using a program of assisted quantification. At

least 10 fields per section were analysed and the results

were given as the ratio (%) of stained surface area over

the total field surface area.

The VEGF seric level was determined using the ELISA method, at the same time with the tissue sampling of the ARDS patients and during the hospital stay in case of the control group of non ARDS patients. Plasma probes were aquired for VEGF measurement using the Human VEGF ELISA kit with crossed reactivity for VEGF 165, VEGF 121 and PGDE (BioLife Group). The statistical analysis of data. was made using media and standard deviation through soft SPSS, version 17.0. RESULTS The ARDS etiology of the studied patients was mainly extrapulmonary (7 cases out of 10) (Figure 1) and the demografical and clinical data of the ARDS patients appear in Table I.

Figure 1. Causes of ARDS

Table I. Demographic and clinical data of ARDS patients

Parameters Value

Age 52 (31-71)

Male/Female 6/4

PaO2/FiO2 (average, extremes) 128(61-191)

Period of mechanical ventilation

(average days, extremes) 11.37 (4-26)

ICU stay (average days, extremes) 18.37 (4-39)

SOFA score (the ARDS diagnostic day)

(average extreme values) 6 (4-9)

Mortality 40% (4/10)

The mortality of ARDS patients was of 40 % (4 out of 10 patients). Pa02/ Fi02 ratio was lower than 200 for all the ARDS patients according to one of diagnostic criteria, but this ratio will be subsequent modified for the survivors (assessed in the day of extubation) comparing to the non-survivors (assessed in the day of death), with a mean of 282 for survivors versus 128 for the non-survivors (p<0.05) Figure 2).

Figure 2. Dynamic of PaO2/FiO2 ratio in ARDS patients

The mean period of ICU stay was 18 days (with a maximum of 39 days) and the mean ventilation period was 11 days (with a maximum of 26 days). The mean SOFA score aquired in the first day of ICU stay was 6, with similar values for survivors and for the non survivors. Patients who died because of ARDS had a VEGF pulmonary expression significantly decreased compared to non-ARDS patients: 8,5 (4,1-9,9) versus 28,7 (9,5-48,6) (p<0,001) (Figure 3).

Figure 3. Distribution of VEGF expression in ARDS vs non ARDS patients

userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
Administrator
Typewriter
Digitized

Vascular endothelial growth factor increases vascular permeability in acute respiratory distress syndrome 229

On the other hand, the seric VEGF levels of ARDS patients were raised (230 pg/ml) compared to non- ARDS patients (131 pg/ml) (p<0,001) (Figure 4).

Figure 4. Distribution of serum VEGF level in ARDS vs non ARDS patients

Alveolar macrophages were immunopositive in both groups. No significant statistical differences were noted between the two groups with regard to age, gender, period of ARDS conditioned, number of ICU days. DISCUSSIONS

In the normal lung tissue, VEGF is highly

compartmentalised, with levels within the alveolar

space 500 times than over the plasma figures8, despite

VEGF production being closely associated with

hypoxia response element. In vitro studies have

demonstrated an abundance of VEGF especially in the

alveolar epithelium (including the A549 cell line and

primary human cultured type II pneumocytes) which

suggests that the alveolar epithelium is the

predominant source.1,12

The expression of VEGF in

ARDS varies, depending of epithelial and endothelial

damage. In the early stages of ARDS plasma VEGF

levels rise and intrapulmonary levels fall, with

normalisation of both during recovery.11

A possible

explication of this variation is that the alveolar injury

is the primary modulator of the alveolar level of VEGF

in ARDS. This variation is probably the consequence

of VEGF degradation by proteases released from

infiltrated neutrophils and other inflammatory cells in

the alveolar space.

VEGF release takes place in 3 phases paralleling

the development of ARDS. The initial injury of the

lung and the proinflammatory cytokines stimulate the

production and the release of VEGF from type II

pneumocytes, alveolar macrophages and marginal neutrophils. Subsequently, the endothelial-alveolar barrier is exposed to high concentration of VEGF, which alters the adherence jonction complexes (AJC) leading to microvascular injury and interstitial oedema. As the ARDS lesions develop, there is a distruction of type I

and II alveolar epithelium, with a release of protease from neutrophils which leads to the decrease of VEGF concentration in the alveolar compartment. This pulmonary compartment deprivation of VEGF besides the release of VEGF from other organs increases the VEGF seric concentration, as ARDS represents only the pulmonary manifestation of a widespread endothelial injury in multiple organs. During the recovery period of ALI (when this appears!) type I and II alveolar epithelial cells begin to reapear, the VEGF production rises, wich may participate in the angiogenesis, an important component of lung repair. The over-expression of pulmonary VEGF, leads to increased vascular pulmonary permeability and consequently pulmonary oedema.

6 Nevertheless, the

VEGF expression in human alveolar epithelial cells also facilitates neovascularization, contributing to endothelial injury repair.

9

Low VEGF pulmonary levels were associated with the severity of ARDS, whereas high VEGF levels were associated with the recovery from ARDS, signalling the role of VEGF in the pulmonary injury repairing process.

11

CONCLUSIONS

A decreased alveolar type II cells, induced by

apoptosis was noticed in ARDS evolution, therefore

reducing the VEGF production in the alveolar space

and also contributing to the decrease in lung

perfusion, but also to the consecutive increase of

VEGF plasmatic level. REFERENCES

1. 1. Abadie Y, Bregeon F, Papazian L et al. Decreased

VEGF concentration in lung tissue and vascular injury

during ARDS. Eur Respir J 2005; 25: 139-146 2. Chiumello D, Valente Barbas CS, Pelosi P, Patho-physiology of ARDS. Springer Milan, 2008; 101-117 3. Dvorak HE Angiogenesis: update 2005, J Thromb Haemost,. 2005; 3:1835-42. 4. Fehrenbach II. Development of the pulmonary

surfactant system. Pneumologie 2007; 61:488

5. Gerber HP, Wu X, Yu L, Wiesmann C. Mice

expressing a humanized form of VEGILA may provide

insights into the safety and efficacy of anti-VEGF

antibodies.Proc Natl Acad Sci U.S.A. 2007; 104: 3478-

83.

6. Kaner RJ, Ladetto JV, Singh R et al. Lung

overexpression of the vascular endothelial growth

factor gene induces pulmonary edema. Am J Respir

Cell Mol Biol 2000; 22:657-64

7. Medford ARL, Keen LJ, Bidwell LJ et al. Vascular

endothelial growth factor gene polymorphism and acute

respiratory distress syndrome. Thorax 2005;60;244-248

8. Medford ARL, Millar AB. Vascular endothelial

growth factor (VEGI) in acute lung injury (ALI) and

acute respiratory distress syndrome (ARDS): paradox or

paradigm? Thorax 2006; 61:621

9. Mura M, dos Santos CC, Stewart D et al. Vascular

endothelial growth factor and related molecules in

acute lung injury. J Appl Physiol 2004; 97:1605-1617

10. Neufeld G, Kessler 0, Herzog Y. The interaction of

userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
Administrator
Typewriter
Digitized

230 Raluca Solomon, L. Azamfirei, Simona Gurzu, Ruxandra Copotoiu, Sanda Copotoiu, I. Jung, J. Szederjesi, Judit Kovacs

neuropilin-1 and neuropilin-2 with tyrosine-kinase

receptors for VEGF. Adv Exp Med Bio1 2002;515: 81-90

11. Thickiett DR, Armstrong A., Christie SJ et al.

Vascular endothelial growth factor may contribute to

increased vascular permeability in acute respiratory

distress syndrome, Am 1 Rcspir Crit Care Med 2001;

164:1601-1605

12. Van 'der FM, van Lceuwen Fij, van Kessel KP et al.

Plasma vascular endothelial growth factor in severe

sepsis. Shock 23: 35-38, 2005.

13. Zhai R, Gong MN, Zhou W, Thompson TB et al,

Genotypes and halotypes of the VEGF gene are

associated with higher mortality and lower VEGF

plasma levels in patients with ARDS, Thorax, 2007; 62:

718-722.

userr
Highlight
Administrator
Typewriter
Digitized

Vol. 76 - No. 8 MINERVA ANESTESIOLOGICA 609

O R I G I N A L A R T I C L E

Vascular endothelial growth factor: a possiblemediator of endothelial activation in acute

respiratory distress syndrome

L. AZAMFIREI 1, S. GURZU 2, R. SOLOMON 3, R. COPOTOIU 1, S. COPOTOIU 1, I. JUNG 2,M. TILINCA 4, K. BRANZANIUC 5, D. CORNECI 6, J. SZEDERJESI 1, J. KOVACS 1

1Department of Intensive Care, University of Medicine and Pharmacy, Targu-Mures, Romania; 2Department of Pathology,University of Medicine and Pharmacy, Targu-Mures, Romania;3Department of Intensive Care, University Hospital, Targu-Mures, Romania; 4Department of Cell Biology, University of Medicine and Pharmacy, Targu-Mures, Romania; 5Departmentof Anatomy and Embryology, University of Medicine and Pharmacy, Targu-Mures, Romania; 6Department of Intensive Care,University of Medicine and Pharmacy Carol Davila Bucharest, Romania

Acute respiratory distress syndrome (ARDS) isthe most severe form of acute lung injury

(ALI) that remains as a devastating condition. It ischaracterized by the diffuse injury of the alveolo-capillary wall and alveolar and interstitial edemaassociated with increased pulmonary vascular per-meability. The incidence is 1.5-8.3 cases in every100 000 patients, and there is a significant mortal-ity of 30% to 50% despite improvements in the

management of sepsis and lung-protective venti-lation strategy.1 Admission to intensive care unitsis usually planned for these patients at high riskof mortality and morbidity.2 Moreover, survivalanalyses have shown that the group with delayedextubation (ARDS patients usually have a pro-longed ventilation) had an eight-fold higher mor-tality rate after 180 days compared with patientswith early extubation.3 There is a heterogeneous

A B S T R A C T

Aim. Vascular endothelial growth factor (VEGF) is a potent angiogenic and endothelial factor, which is abundantlyfound in the normal lung tissue. The objective of the study was to assess the VEGF levels in lung tissue and plasma inacute respiratory distress syndrome (ARDS) patients compared with controls who died from non-ARDS causes.Methods. Plasma and tissue samples were prospectively collected from 20 patients with ARDS within 6 hours afterintubation (VEGF in plasma and tissue samples) and on the day of extubation (plasma VEGF) or postmortem (lungtissue). We used an ELISA to measure the VEGF level in plasma. Lung specimens were obtained by bronchoscopic biop-sy or by open biopsy during autopsy. All lung samples were stained for standard histopathological analysis and forimmunohistochemical methods. Biomarker levels were compared between survivors (N=12), non-survivors (N=8)and controls (N=10).Results. Compared with the levels in controls, in the early stages of ARDS, plasma VEGF levels rose and intrapulmonarylevels fell, but during recovery, these levels went back to normal levels. Conclusion. The initial phase of ARDS is associated with a decrease in VEGF in the lung and an increase in the plas-ma. This down-regulation may represent a protective mechanism aimed at limiting endothelial permeability and mayparticipate in the decrease in the capillary number that is observed during early ARDS. A persistent elevation of plas-ma VEGF over time predicts poor outcome. (Minerva Anestesiol 2010;76:609-16)

Key words: ARDS - Vascular Endothelial Growth Factor - Lung - Endothelium.

MINERVA MEDICA COPYRIGHT®

Thi

s do

cum

ent i

s pr

otec

ted

by in

tern

atio

nal c

opyr

ight

law

s.N

o ad

ditio

nal r

epro

duct

ion

is a

utho

rized

.It i

s pe

rmitt

ed fo

r pe

rson

al u

se to

dow

nloa

d an

d sa

ve o

nly

one

file

and

prin

t onl

y on

e co

py o

f thi

s A

rtic

le.I

t is

not p

erm

itted

to m

ake

addi

tiona

l cop

ies

(eith

ersp

orad

ical

ly o

r sy

stem

atic

ally

, eith

er p

rinte

d or

ele

ctro

nic)

of t

he A

rtic

le fo

r an

y pu

rpos

e.It

is n

ot p

erm

itted

to d

istr

ibut

e th

e el

ectr

onic

cop

y of

the

artic

le th

roug

h on

line

inte

rnet

and

/or

intr

anet

file

sha

ring

syst

ems,

ele

ctro

nic

mai

ling

or a

ny o

ther

mea

ns w

hich

may

allo

w a

cces

s to

the

Art

icle

.The

use

of a

ll or

any

par

t of t

he A

rtic

le fo

r an

y C

omm

erci

al U

se is

not

per

mitt

ed.T

he c

reat

ion

of d

eriv

ativ

e w

orks

from

the

Art

icle

is n

ot p

erm

itted

.The

pro

duct

ion

of r

eprin

ts fo

r pe

rson

al o

r co

mm

erci

al u

se is

not

per

mitt

ed.I

t is

not p

erm

itted

to r

emov

e, c

over

, ove

rlay,

obs

cure

, blo

ck, o

r ch

ange

any

cop

yrig

ht n

otic

es o

r te

rms

of u

se w

hich

the

Pub

lishe

r m

ay p

ost o

n th

e A

rtic

le.I

t is

not p

erm

itted

to fr

ame

or u

se fr

amin

g te

chni

ques

to e

nclo

se a

ny tr

adem

ark,

logo

, or

othe

r pr

oprie

tary

tion

of th

e P

ublis

her.

userr
Textbox
2. Azamfirei2010

AZAMFIRE VASCULAR ENDOTHELIAL GROWTH FACTOR

610 MINERVA ANESTESIOLOGICA August 2010

group of conditions, both direct or indirect insults,such as sepsis, trauma, aspiration, massive bloodtransfusion or burns which predispose to ARDS.The pathogenic basis of ARDS and factors gov-erning susceptibility are incompletely understood.Great attention has been recently paid on thepotential of determining the possible genetic deter-minants of more susceptible endothelium, andthe onset, severity and outcome of septic syndromewith ALI/ARDS.4

Markers of both epithelial and endothelialinjury have been correlated with outcome, where-as the severity of ARDS depends significantlyon the balance between alveolar epithelial and/orvascular endothelial injuries and their repairmechanisms.5, 6

Vascular endothelial cell growth factor (VEGF)increase endothelial permeability and acts as a cel-lular growth factor, hence its potential for a keyrole in the pathogenesis of ALI/ARDS. The VEGFfamily has several members, and each acts throughspecific receptors. The human VEGF gene fami-ly includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta growth factor (PlGF),all with multiple and diverse biological functions.The VEGF family members’ genes also rely onalternative exon splicing to confer various isoformsfor biological and functional specificity.7,8 Themost studied molecule of the VEGF family isVEGF-A. Its dual effects as both permogen andmitogen induce vascular endothelial cell prolifer-ation and promote survival by induction of anti-apoptotic proteins bcl-2 and A1.9, 10 VEGF increas-es microvascular permeability, but it also exertsbioactivity outside the vascular endothelium:macrophages, type II pneumocytes, and mono-cytes, for which it may be chemotactic.11 The bio-logical activity of VEGF is dependent on its inter-action with specific receptors (VEGF-R1, 2, and3), which are expressed not only by endothelialcells, but also by activated macrophages and alve-olar type II epithelial cells.12 VEGF acts also on afamily of co-receptors, the neuropilins.13

The objective of this study was to assess theVEGF levels in lung tissue and plasma from acuterespiratory distress syndrome (ARDS) patientscompared with controls who died from non-ARDScauses.

Materials and methods

We evaluated prospectively 20 patients accord-ing to the international American-European con-sensus conference criteria for ARDS.14 The studyhas been approved by the ethics committee, andinformed consent was obtained.

The SOFA score was obtained daily for all thepatients and was compared with the score obtainedon the first day of the ARDS diagnosis.

Plasma and tissue samples were prospectivelycollected from 20 patients with ARDS withoutany previous lung diseases before admittance inICU, within 6 hours after intubation (plasmaVEGF and tissue samples) and on the day of extu-bation (plasma VEGF) or postmortem (lung tis-sue). Lung specimens from ARDS patients wereobtained by bronchoscopy or by autopsy in con-trols. The control group was comprised of 10patients who died from causes other than ARDS.Pulmonary tissue samples were retrieved at autop-sy and processed similarly to the samples from theARDS patients.

All lung samples were stained in a standard man-ner using hematoxylin-eosin and were examinedto assess the histopathological diagnosis of ARDS:presence of hyaline membranes and type II pneu-mocyte proliferation associated with myofibrob-last proliferation.

The determination of VEGF expression in thepulmonary tissue was performed using specificmonoclonal antibodies VEGF, clones VG1 andJH 121 produced by the LabVision company. Acharge-coupled video camera connected to an opti-cal microscope was used to view the sections andto digitize images on a PC host computer, using aprogram of assisted quantification. For angiogen-esis quantification, we chose the most relevantregions, and we realized the digital photo capturesat 400x high power fields. The pictures were tak-en with the optical microscope Nikon 800E thatwas coupled to a color video camera. The countwas made using NIH’s ImageJ software for imageprocessing. We batch-measured the stained sur-face versus total tissue area ratio. We eliminatedthe ulcerated regions and also the regions rich inlymphocytes. At least 10 fields per section wereanalyzed, and the results were given as the ratio(%) of the stained surface area over the total fieldsurface area.

MINERVA MEDICA COPYRIGHT®

Thi

s do

cum

ent i

s pr

otec

ted

by in

tern

atio

nal c

opyr

ight

law

s.N

o ad

ditio

nal r

epro

duct

ion

is a

utho

rized

.It i

s pe

rmitt

ed fo

r pe

rson

al u

se to

dow

nloa

d an

d sa

ve o

nly

one

file

and

prin

t onl

y on

e co

py o

f thi

s A

rtic

le.I

t is

not p

erm

itted

to m

ake

addi

tiona

l cop

ies

(eith

ersp

orad

ical

ly o

r sy

stem

atic

ally

, eith

er p

rinte

d or

ele

ctro

nic)

of t

he A

rtic

le fo

r an

y pu

rpos

e.It

is n

ot p

erm

itted

to d

istr

ibut

e th

e el

ectr

onic

cop

y of

the

artic

le th

roug

h on

line

inte

rnet

and

/or

intr

anet

file

sha

ring

syst

ems,

ele

ctro

nic

mai

ling

or a

ny o

ther

mea

ns w

hich

may

allo

w a

cces

s to

the

Art

icle

.The

use

of a

ll or

any

par

t of t

he A

rtic

le fo

r an

y C

omm

erci

al U

se is

not

per

mitt

ed.T

he c

reat

ion

of d

eriv

ativ

e w

orks

from

the

Art

icle

is n

ot p

erm

itted

.The

pro

duct

ion

of r

eprin

ts fo

r pe

rson

al o

r co

mm

erci

al u

se is

not

per

mitt

ed.I

t is

not p

erm

itted

to r

emov

e, c

over

, ove

rlay,

obs

cure

, blo

ck, o

r ch

ange

any

cop

yrig

ht n

otic

es o

r te

rms

of u

se w

hich

the

Pub

lishe

r m

ay p

ost o

n th

e A

rtic

le.I

t is

not p

erm

itted

to fr

ame

or u

se fr

amin

g te

chni

ques

to e

nclo

se a

ny tr

adem

ark,

logo

, or

othe

r pr

oprie

tary

tion

of th

e P

ublis

her.

userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight

Vol. 76 - No. 8 MINERVA ANESTESIOLOGICA 611

VASCULAR ENDOTHELIAL GROWTH FACTOR AZAMFIRE

The plasma VEGF level was determined usingthe ELISA method, at the same as the tissue sam-pling of the ARDS patients, and during the hos-pital stay in the case of the control group of non-ARDS patients. Plasma samples were acquired forVEGF measurement using the Human VEGFELISA kit with crossed reactivity for VEGF 165(BioLife Group), according to the manufacturer’sinstructions. Biomarker levels were comparedbetween survivors, non-survivors and controlgroup.

Statistical analysis

The statistical analysis of the data was conduct-ed using media and the standard deviation throughsoft GraphPadPrism, version 5. The results arepresented as medians (interquartile range) in thetext. The data are presented as box-plots indicat-

ing the median and the 10th, 25th, 75th and 90th

percentiles. The differences between groups wereestimated using the Kruskal-Wallis test with posthoc Mann-Whitney analysis. P-values <0.05 wereconsidered statistically significant.

Results

The causes of ARDS are shown in Table I. Thedemographic and clinical data of the ARDSpatients are shown in Table II.

The mortality of ARDS patients was 8 out of 20patients.

The mean period of ICU stay was 18.3 days(with a maximum of 39.5 days), and the meanventilation period was 11.4 days (with a maxi-mum of 26.3 days). The mean SOFA scoreacquired on the first day of ICU stay was 6.4 withsimilar values for survivors and for the non-sur-vivors.

The PaO2/FiO2 ratio was lower than 200 forall the ARDS patients according to one of the diag-nostic criteria, but this ratio would be subsequent-ly modified for the survivors (assessed in the dayof extubation) compared with the non-survivors(assessed in the day of death), with a mean of 247.2(167-342) for survivors versus 115.8 (67-156) thenon-survivors (P<0.05)

Patients in the early phase of ARDS (day ofintubation) had a VEGF pulmonary expressionsignificantly decreased compared with non-ARDSpatients: 8.5 (4.1-10.7) (Figure 1, column A) ver-sus 26.7 (9.5-38.6) (P<0.05) (Figure 1, columnC). The lower VEGF expression was observedmainly in patients who died of ARDS: 8.05 (5.4-11.2) (Figure 1, column B). The plasma VEGF

MINERVA MEDICA COPYRIGHT®

Thi

s do

cum

ent i

s pr

otec

ted

by in

tern

atio

nal c

opyr

ight

law

s.N

o ad

ditio

nal r

epro

duct

ion

is a

utho

rized

.It i

s pe

rmitt

ed fo

r pe

rson

al u

se to

dow

nloa

d an

d sa

ve o

nly

one

file

and

prin

t onl

y on

e co

py o

f thi

s A

rtic

le.I

t is

not p

erm

itted

to m

ake

addi

tiona

l cop

ies

(eith

ersp

orad

ical

ly o

r sy

stem

atic

ally

, eith

er p

rinte

d or

ele

ctro

nic)

of t

he A

rtic

le fo

r an

y pu

rpos

e.It

is n

ot p

erm

itted

to d

istr

ibut

e th

e el

ectr

onic

cop

y of

the

artic

le th

roug

h on

line

inte

rnet

and

/or

intr

anet

file

sha

ring

syst

ems,

ele

ctro

nic

mai

ling

or a

ny o

ther

mea

ns w

hich

may

allo

w a

cces

s to

the

Art

icle

.The

use

of a

ll or

any

par

t of t

he A

rtic

le fo

r an

y C

omm

erci

al U

se is

not

per

mitt

ed.T

he c

reat

ion

of d

eriv

ativ

e w

orks

from

the

Art

icle

is n

ot p

erm

itted

.The

pro

duct

ion

of r

eprin

ts fo

r pe

rson

al o

r co

mm

erci

al u

se is

not

per

mitt

ed.I

t is

not p

erm

itted

to r

emov

e, c

over

, ove

rlay,

obs

cure

, blo

ck, o

r ch

ange

any

cop

yrig

ht n

otic

es o

r te

rms

of u

se w

hich

the

Pub

lishe

r m

ay p

ost o

n th

e A

rtic

le.I

t is

not p

erm

itted

to fr

ame

or u

se fr

amin

g te

chni

ques

to e

nclo

se a

ny tr

adem

ark,

logo

, or

othe

r pr

oprie

tary

tion

of th

e P

ublis

her.

TABLE I.—Causes of ARDS.

Cause Number of patients

Pulmonary 6— Chest trauma 4— Bronchopneumonia 2

Extrapulmonary 14— Sepsis 6— Liver dysfunction 4— Massive transfusion 2— Acute pancreatitis 2

TABLE II.—Demographic and clinical data of ARDS patients.

Parameters Value

Age (yr)range 52.6 (31.1-71.3)

Male/Female (number) 12/8PaO2/FiO2 134.6 (56 -198)Period of mechanical ventilation

(days) 11.4 (4.8-26.3)ICU stay (days) 18.3 (4.1-39.5)SOFA score (day of the ARDS diagnosis) 6.4 (4.7-9.3)Mortality (%) 40% (8/20)

Data are expressed as median (interquartile range).

50

40

30

20

10

0VE

GF

pulm

onar

y ex

pres

sion

(%

)

A B C

*

**

Figure 1.—The lung tissue expression of VEGF in ARDSpatients.

userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Rectangle
userr
Callout
Red highlighted values coincide with corresponding values from Table II in Solomon2010-AMM

AZAMFIRE VASCULAR ENDOTHELIAL GROWTH FACTOR

612 MINERVA ANESTESIOLOGICA August 2010

levels of ARDS patients were 234.4 pg/mL (140-323 pg/mL) (Figure 2, column A) in the earlyphase of ARDS (day of intubation) compared withnon-ARDS patients: 141 pg/mL (76-209 pg/mL)(Figure 2, column B) (P<0.05). The level remainedhigh until the day of death (mean: 262.8 pg/mL),(Figure 2, column C) but this level decreased atthe day of extubation (mean: 138.5 pg/mL) (Figure2, column D) for the patients with a favorableevolution, compared with the patients from thecontrol group (P>0.05).

Alveolar macrophages were immunopositive inboth groups.

No significant statistical differences were notedbetween the two groups regarding age, gender,period of ARDS condition, and number of ICUdays.

Discussion

In the normal lung tissue, VEGF is highly com-partmentalized, with levels within the alveolarspace being 500 times more than those in plasma,even though VEGF production is closely associat-ed with the hypoxia response element.4 In vitrostudies have demonstrated an abundance of VEGFespecially in the alveolar epithelium (includingthe A549 cell line and primary human culturedtype II pneumocytes), which suggests that the alve-olar epithelium is the predominant source.12, 15-17

The expression of VEGF in ARDS varies, depend-ing on epithelial and endothelial damage.18, 19 Ourresults showed that in the early stages of ARDS,plasma VEGF levels rose and intrapulmonary lev-els fell, and during recovery, both levels showednormalization. A possible explanation for this vari-

ation is that the alveolar injury is the primary mod-ulator of the alveolar level of VEGF in ARDS.This variation is probably the consequence ofVEGF degradation by proteases released from theinfiltrated neutrophils and other inflammatorycells in the alveolar space.20, 21

VEGF release takes place in 3 phases parallelingthe development of ARDS. The initial injury of thelung and the pro-inflammatory cytokines stimu-late the production and the release of VEGF fromtype II pneumocytes, alveolar macrophages andmarginal neutrophils.22, 23

Subsequently, the endothelial-alveolar barrier isexposed to a high concentration of VEGF, whichalters the adherence junction complexes (AJC)leading to microvascular injury and interstitial ede-ma.24 As the ARDS lesions develop, there is adestruction of type I and II alveolar epithelium,with a release of proteases from neutrophils thatleads to the decrease of VEGF concentration inthe alveolar compartment. This pulmonary com-partment deprivation of VEGF, independent fromthe release of VEGF from other organs, increasesthe VEGF plasma concentration, as ARDS repre-sents only the pulmonary manifestation of a wide-spread endothelial injury in multiple organs.25, 26

During the early stage of ARDS, plasma cross-es the capillary and alveolar walls, subsequentlyflooding the alveoli. VEGF is present in higherconcentrations than normal in plasma. It is aproven fact that in early stages of ARDS, the lev-el of VEGF decreases.18

VEGF signaling is required for the maintenanceof adult lung alveolar structures, 27 and the with-drawal of VEGF leads to endothelial cell apopto-sis. The increased septal cell death in the lungs isassociated with reduced lung expression of VEGFand VEGFR-2.28 This is consistent with dimin-ished lung perfusion, suggesting a protective mech-anism for endothelial cell survival. This was provenby some authors in experimental models whoobserved that endothelial cell survival was increasedthrough increases in oxidative stress, alveolarenlargement and alveolar cell apoptosis followingthe blocking of VEGF.29 In neonatal and pretermmice with respiratory distress syndrome generat-ed by the hypoxia inducible factor (HIF) knock-out, the administration of VEGF increased sur-factant biosynthesis and improved survival rates.30

MINERVA MEDICA COPYRIGHT®

Thi

s do

cum

ent i

s pr

otec

ted

by in

tern

atio

nal c

opyr

ight

law

s.N

o ad

ditio

nal r

epro

duct

ion

is a

utho

rized

.It i

s pe

rmitt

ed fo

r pe

rson

al u

se to

dow

nloa

d an

d sa

ve o

nly

one

file

and

prin

t onl

y on

e co

py o

f thi

s A

rtic

le.I

t is

not p

erm

itted

to m

ake

addi

tiona

l cop

ies

(eith

ersp

orad

ical

ly o

r sy

stem

atic

ally

, eith

er p

rinte

d or

ele

ctro

nic)

of t

he A

rtic

le fo

r an

y pu

rpos

e.It

is n

ot p

erm

itted

to d

istr

ibut

e th

e el

ectr

onic

cop

y of

the

artic

le th

roug

h on

line

inte

rnet

and

/or

intr

anet

file

sha

ring

syst

ems,

ele

ctro

nic

mai

ling

or a

ny o

ther

mea

ns w

hich

may

allo

w a

cces

s to

the

Art

icle

.The

use

of a

ll or

any

par

t of t

he A

rtic

le fo

r an

y C

omm

erci

al U

se is

not

per

mitt

ed.T

he c

reat

ion

of d

eriv

ativ

e w

orks

from

the

Art

icle

is n

ot p

erm

itted

.The

pro

duct

ion

of r

eprin

ts fo

r pe

rson

al o

r co

mm

erci

al u

se is

not

per

mitt

ed.I

t is

not p

erm

itted

to r

emov

e, c

over

, ove

rlay,

obs

cure

, blo

ck, o

r ch

ange

any

cop

yrig

ht n

otic

es o

r te

rms

of u

se w

hich

the

Pub

lishe

r m

ay p

ost o

n th

e A

rtic

le.I

t is

not p

erm

itted

to fr

ame

or u

se fr

amin

g te

chni

ques

to e

nclo

se a

ny tr

adem

ark,

logo

, or

othe

r pr

oprie

tary

tion

of th

e P

ublis

her.

400

300

200

100

0

Plas

ma

VE

GF

(pg/

mL

)

A B C

*

***

D

**

Figure 2.—The plasma VEGF level in ARDS patients.

userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight

Vol. 76 - No. 8 MINERVA ANESTESIOLOGICA 613

VASCULAR ENDOTHELIAL GROWTH FACTOR AZAMFIRE

Likewise, the over-expression of pulmonary IL-13 protected mice from hyperoxic lung injurythrough a VEGF-dependent pathway, and aden-oviral-delivered VEGF has been reported toincrease survival from hypoxic pulmonary hyper-tension.31, 32 VEGF protects endothelial cells fromapoptotic cell death through the coordinate sig-naling of phosphatidyl 3-kinase/Akt and inhibitionof p38 MAPK.33, 34 Furthermore, insufficientmaintenance of VEGF levels has been reported toinhibit capillary formation in neonatal mice andto lead to regression of newly formed vessels inthe retina, heart, and liver.35-37

In our study, the lung tissue expression of VEGFin ARDS patients was low on the day of intubationas well after death, and these results point to thepotential importance of this factor. The presenceof VEGF has a pro-survival and anti-apoptoticrole in endothelial cells. The failure of pulmonaryendothelial cell survival induced by VEGF recep-tor blockade results in lung alveolar septal cellapoptosis and may contribute to the genesis ofvascular injury. A vicious circle might be estab-lished, because cells undergoing apoptosisdisplayincreased oxidative stress, which further contributesto the apoptosis.38 Our result supports the ideathat the protective effect of VEGF depends on theexisting reserves of VEGF on lung tissue.

At the same time, the decrease in VEGF mayprotect lungs from alveolar flooding. In a previ-ous study using an adenovirus-mediatedgene trans-fer, Kaner et al. has shown that VEGF over-expres-sion induces pulmonary edema.39 Because VEGFincreases the permeability, the decreased VEGFproduction does not exclude its involvement inthe early increase of endothelial permeability thatmay occur concomitantly with extravascular neu-trophil migration at the onset of ARDS.40

Potential explanations for the reduction in intra-pulmonary VEGF levels in early ARDS are man-ifold and not mutually exclusive. They includeincreased membrane-bound rather than solubleisoforms, changes in isoform expression and dam-age to the alveolar-capillary membrane with con-sequent leakage of intrapulmonary VEGF intothe vascular bed.41 The intrapulmonary VEGFlevels are known to be lower also in non-survivorswith ARDS.29

In patients in the early ARDS stage, intubation

induces the secretion of hypoxic inducing factorslike HIF-1-α or HIF-2-α, and NO. They all con-tribute to the release of VEGF and to the genesisof endothelial cells as a consequence. The role ofhypoxia in releasing VEGF and angiogenesis ini-tiation was observed in tumors, especially in breastand colorectal carcinomas in which the anti-angio-genic treatment was approved by the US Food andDrug Administration (FDA) in 2004 and 2008,respectively.42, 43 So, after intubation (includingthe day of death), the level of VEGF could be slow-ly increased compared with the level before intu-bation, although it was still not enough for a pro-tective effect. However, VEGF tissue expressionis lower compared with the normal lung.

The ARDS patients who passed on died ofMSOF induced by the initial morbidity, thus, theydid not live enough to reach the late ARDS stage.

During the recovery period of ALI (when itdoes appear), type I and II alveolar epithelial cellsbegin to reappear, and the VEGF production ris-es, which may lead to VEGF stimulating angiogen-esis, an important component of lung repair.44

The over-expression of pulmonary VEGF leadsto increased vascular pulmonary permeability andsubsequent pulmonary edema.39 Nevertheless, theVEGF expression in human alveolar epithelialcells also facilitates neovascularization, contribut-ing to endothelial injury repair.7

The three receptors of VEGF-A (VEGF-R1, -R2 and –R3) are present in different types of cells,such as tumor cells, endothelial cells, activatedmacrophages or alveolar type II cells.12, 45 In nor-mal lung tissue, VEGF-R1 and -R2 are expressedby alveolar cells.26 VEGF-R2 mediates endothelialsurvival in relationship with KDR (Kinase insertdomain-containing receptor) and in this way,VEGF plays an important role in the regulation ofalveolar-capillary permeability.46 Although themechanism remains unclear, in vitro studies haveshown that VEGF can increase the rate of alveo-lar cell proliferation after acidic injury and at thesame time, can stimulate surfactant productionfrom alveolocytes.30, 47

In our study, the low VEGF pulmonary levelswere associated with the severity of ARDS, where-as high VEGF levels were associated with the recov-ery from ARDS, signaling the role of VEGF inthe pulmonary injury repairing process.

MINERVA MEDICA COPYRIGHT®

Thi

s do

cum

ent i

s pr

otec

ted

by in

tern

atio

nal c

opyr

ight

law

s.N

o ad

ditio

nal r

epro

duct

ion

is a

utho

rized

.It i

s pe

rmitt

ed fo

r pe

rson

al u

se to

dow

nloa

d an

d sa

ve o

nly

one

file

and

prin

t onl

y on

e co

py o

f thi

s A

rtic

le.I

t is

not p

erm

itted

to m

ake

addi

tiona

l cop

ies

(eith

ersp

orad

ical

ly o

r sy

stem

atic

ally

, eith

er p

rinte

d or

ele

ctro

nic)

of t

he A

rtic

le fo

r an

y pu

rpos

e.It

is n

ot p

erm

itted

to d

istr

ibut

e th

e el

ectr

onic

cop

y of

the

artic

le th

roug

h on

line

inte

rnet

and

/or

intr

anet

file

sha

ring

syst

ems,

ele

ctro

nic

mai

ling

or a

ny o

ther

mea

ns w

hich

may

allo

w a

cces

s to

the

Art

icle

.The

use

of a

ll or

any

par

t of t

he A

rtic

le fo

r an

y C

omm

erci

al U

se is

not

per

mitt

ed.T

he c

reat

ion

of d

eriv

ativ

e w

orks

from

the

Art

icle

is n

ot p

erm

itted

.The

pro

duct

ion

of r

eprin

ts fo

r pe

rson

al o

r co

mm

erci

al u

se is

not

per

mitt

ed.I

t is

not p

erm

itted

to r

emov

e, c

over

, ove

rlay,

obs

cure

, blo

ck, o

r ch

ange

any

cop

yrig

ht n

otic

es o

r te

rms

of u

se w

hich

the

Pub

lishe

r m

ay p

ost o

n th

e A

rtic

le.I

t is

not p

erm

itted

to fr

ame

or u

se fr

amin

g te

chni

ques

to e

nclo

se a

ny tr

adem

ark,

logo

, or

othe

r pr

oprie

tary

tion

of th

e P

ublis

her.

AZAMFIRE VASCULAR ENDOTHELIAL GROWTH FACTOR

614 MINERVA ANESTESIOLOGICA August 2010

Elevated plasma VEGF levels in ARDS probablyreflect multiple cellular sources. VEGF has beendemonstrated to induce fenestrations in previous-ly non-fenestrated capillaries. These actions aredependent on intracellular third messengerproduc-tion and the release of nitric oxide.48 Elevated VEGFlevels in ARDS plasma would, therefore, be expect-ed to contribute to abnormal capillary permeabili-ty, which is a characteristic feature of ARDS. Theelevated VEGF levels in hypotensive patients withARDS would also support a role for tissue hypox-ia in determining plasma levels.20

This study has demonstrated that VEGF levelsare significantly greater in non-survivors withARDS than non-ARDS. Furthermore, changes inVEGF appear to be associated with either a goodoutcome if VEGF levels fall, or a bad outcome ifVEGF levels rise. These findings, therefore, needfurther confirmation before they can be consid-ered useful as a clinical marker of pooroutcome inpatients with ARDS.

The soluble form of VEGF-R1 is present in thebronchial lavage fluid, and it could be an antago-nist for the soluble form of VEGF. Therefore,VEGF165 decreases in plasma in the early stagesof ARDS, and there are concurrent VEGF121 andVEGF-R1 increases in bronchial fluid andbronchial epithelium. It is possible that one ofthese forms of VEGF can stimulate VEGF-R2 topromote endothelial activation.49

Endothelial activation refers to a specific changein endothelial phenotype, characterized mostnotably by an increase in endothelial-leukocyteinteractions and permeability, which is pivotal toinflammatory responses in both physiologic andpathologic settings.50 VEGF has been well char-acterized as an endothelial survival factor, and itprevents microvascular apoptotic cell loss.51 VEGFsurvival signals in endothelial cells (ECs) are medi-ated by VEGFR-2 through the phosphatidylinos-itol 3-kinase/Akt signal transduction pathway. Aconstitutively active Akt is sufficient to promotesurvival of serum-starved endothelial cells in tran-sient transfection experiments. In contrast, theover-expression of a dominant-negative form ofAkt blocks the survival effect of VEGF. Thesefindings identify the Flk-1/KDR receptor and thePI3-kinase/Akt signal transduction pathway asimportant elements in the processes leading to

endothelial activation and cell survival inducedby VEGF.52

VEGF also promotes the expression of anti-apoptotic proteins Bcl-2 and A1 in vascular ECsand can specifically blockextrinsic signal-inducedapoptosis of ECs, mediated by TNF receptors andFas. Pro-survival signaling by VEGF may, therefore,alter the threshold level of ECs susceptibility tointrinsic and extrinsic inducers of apoptosis.51

On the other hand, VEGF recruits lactosylce-ramide (a member of the glycosphingolipid fam-ily, which has been implicated in the inflamma-tory process and in the vascular lesion formation)to induce endothelial cellular adhesion molecules(CAMs) expression.53 CAMs play an essential rolein tethering circulating leukocytes to the vascularendothelium activation at sites of inflammation.They are also instrumental in enabling leukocytesto transmigrate from blood vessels into adjacentinflamed tissues. In the absence of signals to stim-ulate expression of CAMs, the adhesive forcesbetween leukocytes and the vascular endotheliumare below the threshold level required to tetherleukocytes. Several cytokines, including tumornecrosis factor alpha (TNFα) and interleukin-1β(IL-1β), potently increase the expression of manyCAMs and, thus, increase the adhesiveness betweenleukocytes and the endothelium. The CAM-induc-ing activity of these cytokines is crucial to the reg-ulation of the inflammatory processes.54

Conclusions

The initial phase of ARDS is associated with adecrease in VEGF in the lung and with an increasein the plasma, suggesting that VEGF in the alve-olar space may reflect the development of, and therecovery from, ARDS in a manner opposite tothat in plasma. This down-regulation may repre-sent a protective mechanism aimed at limitingendothelial permeability and may participate atthe decrease in capillary number that is observedduring early ARDS. Persistent elevation of plas-ma VEGF over time predicts a poor outcome, andincreased production of VEGF may contribute tothe resolution of inflammation in the lung.

References1. Chiumello D, Valente Barbas CS, Pelosi P. Pathophysiology

of ARDS. In: Lucangelo U, Pelosi P, Zin WA, Aliverti A, edi-

MINERVA MEDICA COPYRIGHT®

Thi

s do

cum

ent i

s pr

otec

ted

by in

tern

atio

nal c

opyr

ight

law

s.N

o ad

ditio

nal r

epro

duct

ion

is a

utho

rized

.It i

s pe

rmitt

ed fo

r pe

rson

al u

se to

dow

nloa

d an

d sa

ve o

nly

one

file

and

prin

t onl

y on

e co

py o

f thi

s A

rtic

le.I

t is

not p

erm

itted

to m

ake

addi

tiona

l cop

ies

(eith

ersp

orad

ical

ly o

r sy

stem

atic

ally

, eith

er p

rinte

d or

ele

ctro

nic)

of t

he A

rtic

le fo

r an

y pu

rpos

e.It

is n

ot p

erm

itted

to d

istr

ibut

e th

e el

ectr

onic

cop

y of

the

artic

le th

roug

h on

line

inte

rnet

and

/or

intr

anet

file

sha

ring

syst

ems,

ele

ctro

nic

mai

ling

or a

ny o

ther

mea

ns w

hich

may

allo

w a

cces

s to

the

Art

icle

.The

use

of a

ll or

any

par

t of t

he A

rtic

le fo

r an

y C

omm

erci

al U

se is

not

per

mitt

ed.T

he c

reat

ion

of d

eriv

ativ

e w

orks

from

the

Art

icle

is n

ot p

erm

itted

.The

pro

duct

ion

of r

eprin

ts fo

r pe

rson

al o

r co

mm

erci

al u

se is

not

per

mitt

ed.I

t is

not p

erm

itted

to r

emov

e, c

over

, ove

rlay,

obs

cure

, blo

ck, o

r ch

ange

any

cop

yrig

ht n

otic

es o

r te

rms

of u

se w

hich

the

Pub

lishe

r m

ay p

ost o

n th

e A

rtic

le.I

t is

not p

erm

itted

to fr

ame

or u

se fr

amin

g te

chni

ques

to e

nclo

se a

ny tr

adem

ark,

logo

, or

othe

r pr

oprie

tary

tion

of th

e P

ublis

her.

userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Underline
userr
Underline

Vol. 76 - No. 8 MINERVA ANESTESIOLOGICA 615

VASCULAR ENDOTHELIAL GROWTH FACTOR AZAMFIRE

tors. Respiratory system and artificial ventilation. Milan:Springer; 2008. p. 101-17

2. Cavaliere F, Mascia L, Terragni P. Year in Review in MinervaAnestesiologica, 2008. Minerva Anestesiol 2009;75:163-7.

3. Chiumello D, Caironi P, Colombo R, Grasso S. Year in reviewin Minerva Anestesiologica, 2007. I Critical Care.Experimental and clinical studies. Minerva Anestesiol2008;74:35-40.

4. Chiumello D, Caironi P, Colombo R, Grasso S, GattinoniL. A year in review in Minerva Anestesiologica, 2008. I CriticalCare. Experimental and clinical studies. Minerva Anestesiol2009;75:47-57.

5. Medford AR, Millar AB. Vascular endothelial growth factor(VEGF) in acute lung injury (ALI) and acute respiratory dis-tress syndrome (ARDS): paradox or paradigm? Thorax2006;61:621-6.

6. Zhai R, Gong MN, Zhou W, Thompson TB, Kraft P, Su L etal. Genotypes and haplotypes of the VEGF gene are associ-ated with higher mortality and lower VEGF plasma levels inpatients with ARDS. Thorax 2007;62:718-22.

7. Mura M, dos Santos CC, Stewart D, Liu M. Vascular endothe-lial growth factor and related molecules in acute lung injury.J Appl Physiol 2004;97:1605-17.

8. Medford AR, Keen LJ, Bidwell LJ, Millar AB. Vascularendothelial growth factor gene polymorphism and acute res-piratory distress syndrome. Thorax 2005;60;244-8.

9. Dvorak HF. Angiogenesis: update 2005. J Thromb Haemost2005;3:1835-42.

10. Gerber HP, Wu X, Yu L, Wiesmann C. Mice expressing ahumanized form of VEGF-A may provide insights into thesafety and efficacy of anti-VEGF antibodies. Proc Natl AcadSci USA 2007;104:3478-83.

11. Fehrenbach H. Development of the pulmonary surfactantsystem. Pneumologie 2007;61:488-9.

12. Abadie Y, Bregeon F, Papazian L, Lange F, Chailley-Heu B,Thomas P et al. Decreased VEGF concentration in lung tis-sue and vascular injury during ARDS. Eur Respir J2005;25:139-46.

13. Neufeld G, Kessler O, Herzog Y. The interaction of neu-ropilin-1 and neuropilin-2 with tyrosine-kinase receptors forVEGF. Adv Exp Med Biol 2002;515:81-90.

14. Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K,Hudson L et al. Report of the American-European consensusconference on ARDS: definitions, mechanisms, relevant out-comes and clinical trial coordination. The ConsensusCommittee. Intensive Care Med 1994;20:225-32.

15. Kaner RJ, Crystal RG. Compartmentalization of vascularendothelial growth factor to the epitelial surface of the humanlung. Mol Med 2001;7:240-6.

16. Shibuya M. Differential roles of vascular endothelial growthfactor receptor-1 and receptor-2 in angiogenesis. J BiochemMol Biol 2006;39:469-78.

17. Van der Flier M, van Leeuwen HJ, van Kessel KP, KimpenJL, Hoepelman AI, Geelen SP. Plasma vascular endothelialgrowth factor in severe sepsis. Shock 2005;23:35–8.

18. Strieter RM, Gomperts BN, Keane MP. The role of CXCchemokines in pulmonary fibrosis J Clin Invest 2007;117:549-56.

19. Dobyns EL, Eells PL, Griebel JL, Abman SH. Elevated plas-ma endothelin-1 and cytokine levels in children with severeacute respiratory distress syndrome. J Pediatr 1999;135:246-9.

20. Thickett DR, Armstrong L, Christie SJ, Millar AB. Vascularendothelial growth factor may contribute to increased vas-cular permeability in acute respiratory distress syndrome. AmJ Respir Crit Care Med 2001;164:1601-5.

21. Keane MP, Donnelly SC, Belperio JA, Goodman RB, Dy M,Burdick MD et al. Imbalance in the expression of CXCchemokines correlates with bronchoalveolar lavage fluid angio-genic activity and procollagen levels in acute respiratory dis-tress syndrome. J Immun 2002;169:6515-21.

22. Meduri GU, Eltorky M, Winer-Muram HT. The fibroprolif-erative phase of late adult respiratory distress syndrome. SeminRespir Infect 1995;10:154-75.

23. Su G, Hodnett M, Wu N, Atakilit A, Kosinski C, Godzich Met al. Integrin regulates lung vascular permeability and pul-monary endothelial barrier function. Am J Respir Cell MolBiol 2007;36:377-86.

24. Van der Heijden M, Van Nieuw Amerongen GP, KoolwijkP, Van Hinsbergh VWM, Groeneveld J. Angiopoietin-2, per-meability oedema, occurrence and severity of ALI/ARDS inseptic and non-septic critically ill patients. Thorax 2008;63:903-9.

25. Papaioannou AI. Kostikas K, Kollia P, Gourgoulianis. Clinicalimplications for vascular endothelial growth factor in thelung: friend or foe? Resp Res 2006;7:128-41.

26. Perkins GD, Roberts J, McAuley DF, Armstrong L, MillarA, Gao F et al. Regulation of vascular endothelial growth fac-tor bioactivity in patients with acute lung injury. Thorax2005;60:153-8.

27. Kasahara Y, Tuder RM, Cool CD, Lynch DA, Flores SC,Voelkel NF. Endothelial cell death and decreased expressionof vascular endothelial growth factor and its receptor KDR/Flk-1 in smoking-induced emphysema. Am J Respir Crit CareMed 2001;163:737-44

28. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, Le Cras TD,Abman S, Hirth PK et al. Inhibition of VEGF receptors caus-es lung cell apoptosis and emphysema. J Clin Invest 2000;106:1311-9.

29. Tuder RM, Zhen L, Cho CY, Taraseviciene-Stewart L,Kasahara Y, Salvemini D et al. Oxidative stress and apopto-sis interact and cause emphysema due to vascular endothe-lial growth factor receptor blockade. Am J Respir Cell Mol Biol2003;29:88-97.

30. Compernolle V, Brusselmans K, Acker T, Hoet P, Tjwa M,Beck H et al. Loss of HIF-2 and inhibition of VEGF impairfetal lung maturation, whereas treatment with VEGF pre-vents fatal respiratory distress in premature mice. Nat Med2002;8:702-10.

31. Corne J, Chupp G, Lee CG, Homer RJ, Zhu Z, Chen Q etal. IL-13 stimulates vascular endothelial cell growth factorand protects against hyperoxic acute lung injury. J Clin Invest200;106:783-91.

32. Partovian C, Adnot S, Raffestin B, Louzier V, Levame M,Mavier IM et al. Adenovirus-mediated lung vascular endothe-lial growth factor overexpression protects against hypoxic pul-monary hypertension in rats. Am J Respir Cell Mol Biol2000;23:762-71.

33. Gerber HP, McMurtrey A, Kowalski J, Yan M, Keyt BA, DixitV et al. Vascular endothelial growth factor regulates endothe-lial cell survival through the phosphatidylinositol 3'-kinase/Aktsignal transduction pathway. Requirement for Flk-1/KDRactivation. J Biol Chem 1998;273:30336-43.

34. Gratton JP, Morales-Ruiz M, Kureishi Y, Fulton D, Walsh K,Sessa WC. Akt down-regulation of p38 signaling provides anovel mechanism of vascular endothelial growth factor-medi-ated cytoprotection in endothelial cells. J Biol Chem2001;276:30359-65.

35. Benjamin LE, Hemo I, Keshet E. A plasticity window forblood vessel remodelling is defined by pericyte coverage ofthe preformed endothelial network and is regulated by PDGF-B and VEGF. Development 1998;125:1591-8.

36. Dor Y, Djonov V, Abramovitch R, Itin A, Fishman GI,Carmeliet P et al. Conditional switching of VEGF providesnew insights into adult neovascularization and pro-angio-genic therapy. EMBO J 2002;21:1939-47.

37. Gerber HP, Hillan KJ, Ryan AM, Kowalski J, Keller GA,Rangell L et al. VEGF is required for growth and survival inneonatal mice. Development 1999;126:1149-59.

38. Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL,

MINERVA MEDICA COPYRIGHT®

Thi

s do

cum

ent i

s pr

otec

ted

by in

tern

atio

nal c

opyr

ight

law

s.N

o ad

ditio

nal r

epro

duct

ion

is a

utho

rized

.It i

s pe

rmitt

ed fo

r pe

rson

al u

se to

dow

nloa

d an

d sa

ve o

nly

one

file

and

prin

t onl

y on

e co

py o

f thi

s A

rtic

le.I

t is

not p

erm

itted

to m

ake

addi

tiona

l cop

ies

(eith

ersp

orad

ical

ly o

r sy

stem

atic

ally

, eith

er p

rinte

d or

ele

ctro

nic)

of t

he A

rtic

le fo

r an

y pu

rpos

e.It

is n

ot p

erm

itted

to d

istr

ibut

e th

e el

ectr

onic

cop

y of

the

artic

le th

roug

h on

line

inte

rnet

and

/or

intr

anet

file

sha

ring

syst

ems,

ele

ctro

nic

mai

ling

or a

ny o

ther

mea

ns w

hich

may

allo

w a

cces

s to

the

Art

icle

.The

use

of a

ll or

any

par

t of t

he A

rtic

le fo

r an

y C

omm

erci

al U

se is

not

per

mitt

ed.T

he c

reat

ion

of d

eriv

ativ

e w

orks

from

the

Art

icle

is n

ot p

erm

itted

.The

pro

duct

ion

of r

eprin

ts fo

r pe

rson

al o

r co

mm

erci

al u

se is

not

per

mitt

ed.I

t is

not p

erm

itted

to r

emov

e, c

over

, ove

rlay,

obs

cure

, blo

ck, o

r ch

ange

any

cop

yrig

ht n

otic

es o

r te

rms

of u

se w

hich

the

Pub

lishe

r m

ay p

ost o

n th

e A

rtic

le.I

t is

not p

erm

itted

to fr

ame

or u

se fr

amin

g te

chni

ques

to e

nclo

se a

ny tr

adem

ark,

logo

, or

othe

r pr

oprie

tary

tion

of th

e P

ublis

her.

userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight

AZAMFIRE VASCULAR ENDOTHELIAL GROWTH FACTOR

616 MINERVA ANESTESIOLOGICA August 2010

Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway toprevent apoptosis. Cell 1993;75:241-51

39. Kaner RJ, Ladetto JV, Singh R, Fukuda N, Matthay MA,Crystal RG. Lung overexpression of the vascular endothelialgrowth factor gene induces pulmonary edema. Am J RespirCell Mol Biol 2000;22:657-64.

40. Hoidal JR. Reactive oxygen species and cell signaling. Am JRespir Cell Mol Biol 2001;25:661-3.

41. Maitre B, Biussat S, Jean D, Gouge L, Brochard L, HoussetB et al. Vascular endothelial growth factor synthesis in theacute phase of experimental and clinical lung injury. EurRespir J 2001;18:100-6.

42. Saad RS, Liu YL, Nathan G, Celebrezze J, Medich D,Silverman JF. Endoglin (CD105) and vascular endothelialgrowth factor as a prognostic markers in colorectal cancer.Mod Pathol 2004;17:197-203.

43. Cebe-Suarez S, Zehnder-Fjallman A, Ballmer-Hofer K. Therole of VEGF receptors in angiogenesis; complex partner-ships. Cell Mol Life Sci 2006;63:601-15.

44. Barleon B, Reusch P, Totzke F. Soluble VEGFR-1 secreted byendothelial cells and monocytes is present in human serum andplasma from healthy donors. Angiogenesis 2001;4:143-54.

45. Akslen LA, Jacobsen GK, Vainio O. Angiogenesis – recentdevelopments in molecular pathogenesis, morphologicalmarkers and therapeutic applications. APMIS 2004;112:399-401.

46. Zachary I, Gliki G. Signaling transduction mechanisms medi-

ating biological actions of the vascular endothelial growthfactor family. Cardiovasc Res 2001;49:568-81.

47. Ohwada A, Yashioka Y, Iwabuki K, Nagaoka I, Dambara T,Fukuchi Y. VEGF regulates the proliferation of acid-exposedalveolar lining epithelial cells. Thorax 2003;58:328-32.

48. Roberts WG, Palade GE. Increased microvascular permeabil-ity and endothelial fenestration induced by vascular endothe-lial growth factor. J Cell Sci 1995;108:2369-79

49. Robinson CJ, Stringer SE. The splice variants of vascularendothelial growth factor (VEGF) and their receptors. J CellSci 2001;114:853-65.

50. Alom-Ruiz SP, Anilkumar N, Shah AM. Antioxidants &Redox Signaling 2008;10:1089-100.

51. Alavi A, Hood JD, Frausto R, Stupack DG, Cheresh DA.Role of Raf in vascular protection from distinct apoptoticstimuli. Science 2003;301:94-6.

52. Gerber HP, Dixit V, Ferrara N. Vascular endothelial growthfactor induces expression of the antiapoptotic proteins Bcl-2and A1 in vascular endothelial cells. J Biol Chem1998;273:13313-6.

53. Kolmakova A, Rajesh M, Zang D, Pili R, Chatterjee S. VEGFrecruits lactosylceramide to induce endothelial cell adhesionmolecule expression and angiogenesis in vitro and in vivo.Glycoconj J 2009;26:547-58.

54. Meager A. Cytokine regulation of cellular adhesion moleculeexpression in inflammation. Cytokine Growth Factor Rev1999;10:27-39.

MINERVA MEDICA COPYRIGHT®

Thi

s do

cum

ent i

s pr

otec

ted

by in

tern

atio

nal c

opyr

ight

law

s.N

o ad

ditio

nal r

epro

duct

ion

is a

utho

rized

.It i

s pe

rmitt

ed fo

r pe

rson

al u

se to

dow

nloa

d an

d sa

ve o

nly

one

file

and

prin

t onl

y on

e co

py o

f thi

s A

rtic

le.I

t is

not p

erm

itted

to m

ake

addi

tiona

l cop

ies

(eith

ersp

orad

ical

ly o

r sy

stem

atic

ally

, eith

er p

rinte

d or

ele

ctro

nic)

of t

he A

rtic

le fo

r an

y pu

rpos

e.It

is n

ot p

erm

itted

to d

istr

ibut

e th

e el

ectr

onic

cop

y of

the

artic

le th

roug

h on

line

inte

rnet

and

/or

intr

anet

file

sha

ring

syst

ems,

ele

ctro

nic

mai

ling

or a

ny o

ther

mea

ns w

hich

may

allo

w a

cces

s to

the

Art

icle

.The

use

of a

ll or

any

par

t of t

he A

rtic

le fo

r an

y C

omm

erci

al U

se is

not

per

mitt

ed.T

he c

reat

ion

of d

eriv

ativ

e w

orks

from

the

Art

icle

is n

ot p

erm

itted

.The

pro

duct

ion

of r

eprin

ts fo

r pe

rson

al o

r co

mm

erci

al u

se is

not

per

mitt

ed.I

t is

not p

erm

itted

to r

emov

e, c

over

, ove

rlay,

obs

cure

, blo

ck, o

r ch

ange

any

cop

yrig

ht n

otic

es o

r te

rms

of u

se w

hich

the

Pub

lishe

r m

ay p

ost o

n th

e A

rtic

le.I

t is

not p

erm

itted

to fr

ame

or u

se fr

amin

g te

chni

ques

to e

nclo

se a

ny tr

adem

ark,

logo

, or

othe

r pr

oprie

tary

tion

of th

e P

ublis

her.

Fundings.—This work was supported by CNCSIS-UEFISCSU, project number PNII - IDEI code 136/2008.Received on September 25, 2009. - Accepted for publication on March 14, 2010.Corresponding author: Prof. Dr. L. Azamfirei, Aleea Izvorului 2, Livezeni, 547365, Mures, Romania. E-mail: [email protected]

userr
Highlight

Invited Review

Vascular endothelial growth factor and related molecules in acute lung injury

Marco Mura,1 Claudia C. dos Santos,1,2 Duncan Stewart,3,4 and Mingyao Liu1,4

1Thoracic Surgery Research Laboratories, Toronto General Research Institute, University Health Network,Toronto M5G 2C4; Departments of 2Critical Care Medicine and 3Cardiology and Division of Molecularand Cell Biology Research, St. Michael’s Hospital, Toronto M5B 1W8; and 4Institute of Medical Science,Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8

Mura, Marco, Claudia C. dos Santos, Duncan Stewart, and Mingyao Liu.Vascular endothelial growth factor and related molecules in acute lung injury.J Appl Physiol 97: 1605–1617, 2004. doi:10.1152/japplphysiol.00202.2004.—VEGFsand their receptors have been implicated in the regulation of vascular permeabilityin many organ systems, including the lung. Increased permeability and interstitialand pulmonary edema are prominent features of acute lung injury (ALI)/acuterespiratory distress syndrome (ARDS). Extrapolating data from other organ sys-tems and animal experiments have suggested that overexpression of VEGF func-tions primarily as proinjurious molecules in the lung. Recent data, from animalmodels as well as from patients with ARDS, have shown decreased levels of VEGFin the lung. The role of VEGF and related molecules in ALI/ARDS is, therefore,controversial: what has become clear is that there are many unique features in theregulation of pulmonary vascular permeability and in VEGF expression in the lung.In this review, we explore a growing body of literature looking at the expressionand function of VEGF and related molecules in different models of ALI and inpatients with ALI/ARDS. Novel evidence points to a potential role of VEGF inpromoting repair of the alveolar-capillary membrane during recovery from ALI/ARDS. Understanding the role of VEGF in this disease process is crucial fordeveloping new therapeutic strategies for ALI/ARDS.

acute respiratory distress syndrome; pulmonary edema; angiopoietins; hypoxia;hyperoxia

ACUTE LUNG INJURY (ALI ) AND its most severe manifestation,acute respiratory distress syndrome (ARDS), are clinicallydefined as severe dysfunction of gas exchange and chestradiographic abnormalities following a predisposing injury, inthe absence of heart failure (14). ARDS and ALI may occurfollowing various inciting events, including serious illness,such as sepsis, trauma, or organ transplantation. Overwhelmingintrapulmonary inflammation, endothelial and epithelial injury,and consequent reparative responses are key components of theevolving ALI and progression to ARDS (14, 91).

The hallmarks of ALI are increased capillary permeability,interstitial and alveolar edema, influx of circulating inflamma-tory cells, and formation of hyaline membranes. Increasedpermeability leads to pulmonary edema, a life-threateningcondition resulting from an imbalance between forces drivingfluid into the air spaces and biological mechanisms for itsremoval. The severity and outcome of ALI depend on thebalance between alveolar epithelial and/or vascular endothelialinjuries and their repair mechanisms. The importance of endo-thelial injury and increased vascular permeability to the for-mation of pulmonary edema in this disorder is well established(14, 91).

VEGF plays an important role in several organs by directlyregulating vascular permeability to water and proteins. Forexample, in the brain, VEGF is responsible for hypoxia-

induced vascular leakage and edema formation; inhibition ofVEGF activity by a neutralizing antibody can block thehypoxia-induced increase in vascular permeability (117).The role of VEGF in the control of pulmonary permeabilityis, however, controversial. Systemic expression of VEGFhas been shown to cause widespread multiorgan capillaryleakage in an animal model, suggesting that the overexpres-sion of VEGF plays a pivotal role in the development ofpulmonary edema (67). However, recent animal studies andclinical data support a protective role for VEGF in ALI andARDS patients (30, 133). Understanding the relationshipbetween VEGF and pulmonary permeability in ALI/ARDSmay lead to the development of novel therapeutic interven-tions for this syndrome.

In the lung, the regulation of pulmonary permeability andthe expression of VEGF and VEGF-related molecules havemany unique features. Consequently, it is not appropriate tosimply extrapolate knowledge from other organ systems andapply them to the lung. Therefore, we have undertaken asystematic review of the literature, focusing on features per-taining to VEGF regulation and function in the lung and inparticular its potential role in the pathophysiology of ALI/ARDS. In addition to reviewing the current state of knowledge,the objective of this paper is to further discuss controversialobservations related to the role of VEGF, its related factors,

Address for reprint requests and other correspondence: M. Liu, Professor ofSurgery, Toronto General Hospital, Room: MBRC5R422, 200 Elizabeth St.,Toronto, Ontario, Canada M5G 2C4 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Appl Physiol 97: 1605–1617, 2004;doi:10.1152/japplphysiol.00202.2004.

8750-7587/04 $5.00 Copyright© 2004 the American Physiological Societyhttp://www. jap.org 1605

on January 30, 2012jap.physiology.org

Dow

nloaded from

userr
Textbox
Mura2004 (truncated)

and their receptors in ALI/ARDS, offering alternative perspec-tives on this intricate system.

BIOLOGY OF VEGF AND RELATED MOLECULES

The VEGF family has several members, and each actsthrough specific receptors. The biology of VEGFs has recentlybeen the focus of many excellent reviews (9, 10, 43). Interac-tions between VEGFs and angiopoietins (Ang) is very impor-tant in the regulation of angiogenesis and vascular permeability(83). For the purpose of this review, we will focus on the roleof VEGF and related molecules that have been implicated toplay in the lung. The characteristics and properties of VEGFsand Ang and the potential interplay between VEGF and relatedmolecules and their receptors in the lung are summarized inTable 1 and illustrated in Fig. 1.

VEGFs

The human VEGF gene family includes VEGF-A, VEGF-B,VEGF-C, VEGF-D, VEGF-E, and placenta growth factor(PlGF), all with multiple and diverse biological functions (43).The genes for VEGF family members also rely on alternativeexon splicing to confer various isoforms for biological andfunctional specificity (109, 113).

The most studied molecule of the VEGF family is VEGF-A.The gene encoding human VEGF-A is organized into eightexons. Multiple protein isoforms are generated through alter-native splicing of the pre-mRNA (136). Human VEGF-A iso-forms include 121, 145, 165, 183, 189, and 206 amino acids(VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, and

VEGF206, respectively). VEGF121 is a soluble isoform, whereasVEGF189, VEGF206, and 60–70% of VEGF165 are found inassociation with cells or sequestered in the extracellular matrix(43). Most VEGF-A isoforms contain a heparin-binding domain.VEGF189 and VEGF206 bind to heparin with high affinity (60).The absence of heparin-binding domain in VEGF121 results in aloss of mitogenic activity (73), as demonstrated by the observationthat transgenic mice expressing exclusively VEGF120 (mouseVEGF is one amino acid shorter) die shortly after delivery, due tosevere angiogenic defects (22). VEGF145 and VEGF183 are lessfrequent splicing variants (109, 113). Due to its bioavailability andbiological potency, VEGF165 is the predominant isoform ofVEGF-A, and from hereon the abbreviation VEGF refers toVEGF165, except if otherwise specified.

The major site for VEGF-B expression is the heart (103).VEGF-B forms heterodimers with VEGF and may, therefore,modulate its signaling (103). A study on VEGF-B knockoutmice suggested that VEGF-B may have a role in the develop-ment of chronic hypoxic pulmonary hypertension in mice bycontributing to pulmonary vascular remodeling (142).

VEGF-C and VEGF-D induce growth of the lymphaticvasculature in vivo (63). They can also induce capillary endo-thelial cell (EC) migration and proliferation in culture (19, 95)and act as vascular permeability factors at higher concentra-tions (68, 115). VEGF-D shares 61% identity with VEGF-C(111). VEGF-C and VEGF-D mRNA are most abundant in theheart, lung, skeletal muscle, colon, and small intestine (95,104). Furthermore, VEGF-C is highly expressed by activatedmacrophages (124).

Table 1. Characteristics and properties of VEGF and related molecules in the lung and theirpotential role in acute lung injury.

Factor Sources Receptor Stimulating Factor Potential Functions

VEGF-A Alveolar type II cellsAirway epithelial cellsMesenchymal cellsMacrophagesNeutrophils

VEGFR-1VEGFR-2NRP-1

HypoxiaMechanical stretchROSGlucose deprivationTGF-�1IL-6IFN-�IL-13Endothelin

EC proliferation1 Vascular permeabilityAngiogenesisVasculogenesisAntiapototic for ECsMigration of monocytes

VEGF-B (Heart) VEGFR-1NRP-1

? Pulmonary vascular remodeling

VEGF-C/D Lung ?Macrophages

VEGFR-3VEGFR-2

Proinflammatorycytokines

LymphangiogenesisEC migration and proliferation

VEGF-E ? VEGFR-2 ? EC proliferation1 Vascular permeability

PIGF Alveolar type II cells VEGFR-1NRP-1

? Angiogenesis1 Vascular permeabilityApoptotic for type II cellsMigration of ECs and monocytes

Ang-1 Airway epithelial cellsInterstitial cells

Tie-2 HypoxiaVEGF-A

2 Vascular permeabilityVasculogenesisAntiapototic for ECs

Ang-2 Airway epithelial cellsECs (at sites of active vascular remodeling)

Tie-2 HypoxiaVEGFHIF-1�

1 Vascular permeabilityAntiapototic for ECsPostnatal vascular remodeling

Ang-3/4 Airway epithelial cellsInterstitial cells

Tie-2 ? 2 Vascular permeabilityVasculogenesisAntiapototic for ECs

PlGF, placenta growth factor; Ang, angiopoietin; EC, endothelial cells; NRP, neuropilin; ROS, reactive oxygen species; TGF, transform growth factor; HIF,hypoxia-induced factor; 1, increase; 2, decrease.

Invited Review

1606 VEGF AND LUNG INJURY

J Appl Physiol • VOL 97 • NOVEMBER 2004 • www.jap.org

on January 30, 2012jap.physiology.org

Dow

nloaded from

userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight

Decreased VEGF concentration in lung

tissue and vascular injury during ARDSY. Abadie*,#, F. Bregeon", L. Papazian+, F. Lange1, B. Chailley-Heu*,, P. Thomase,P. Duvaldestin#, S. Adnot*,**, B. Maitre*,## and C. Delclaux*,**

ABSTRACT: Endothelial injury is an important prognostic factor in acute respiratory distress

syndrome (ARDS). Decreased production of vascular endothelial growth factor (VEGF) in ARDS

may favour vascular lesions, since VEGF promotes endothelial survival by inhibiting apoptosis.

This study sought to document low VEGF levels in lung tissue from ARDS patients, to determine

whether the cause was injury to alveolar type II cells (the main pulmonary source of VEGF) and to

evaluate the vascular consequences. Lung specimens were obtained by open biopsy or autopsy

from 29 patients with severe ARDS (two survivors) and five controls.

As compared with controls, homogenates of lung tissue from ARDS patients contained less

VEGF (median (interquartile range) ARDS 8.2 (4.7–12.2) versus controls 28.4 (9.9–47.1) ng?g-1

protein). Increased immunostaining with surfactant protein B was seen in ARDS lungs. Extensive

cellular apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate

nick-end labelling staining), including endothelial and alveolar type II cells, was demonstrated,

and vascular bed density (CD31 immunostaining) decreased in ARDS lungs as compared with

controls. VEGF levels were negatively correlated to apoptotic endothelial cell counts.

In conclusion, decreased vascular endothelial growth factor levels in lung tissue may

participate in the decrease in lung perfusion in acute respiratory distress syndrome.

KEYWORDS: Alveolar type II cells, endothelial apoptosis, lung tissue

The detailed morphological descriptionof acute respiratory distress syndrome(ARDS) by BACHOFEN and WEIBEL [1]

includes a decrease in lung capillary density.Conceivably, the resulting decrease in lungperfusion may lead to an increase in alveolardead space, which has been recently shown to bea major prognostic factor during ARDS [2]. Thepathophysiology of vascular lesions in ARDS is achallenging issue that has received little attention[3]. Recently, HAMACHER et al. [4] have demon-strated that bronchoalveolar lavage fluids ofARDS patients exhibit an ex vivo pro-apopoticactivity against pulmonary microvascularendothelial cells [4]. Conversely, a decrease insurvival factors (anti-apoptotic) of the endothe-lium may be an alternative hypothesis. Lowlevels of vascular endothelial growth factor(VEGF) have been found in the lungs of patientswith early ARDS [5, 6]. This VEGF decreasemay contribute to the genesis of vascular injury,inasmuch as VEGF has been demonstrated to be amajor survival factor for endothelium [7]. VEGF(or VEGF-A) is a highly conserved, dimeric,heparin-binding glycoprotein (molecular weight

46 kDa). At least four different VEGF transcriptsresulting from alternate splicing of a single genehave been identified in human cells. VEGF121and VEGF165 are secreted in a soluble form,whereas VEGF189 and VEGF206 remain cell-surface associated or are primarily depositedin the extracellular matrix. VEGF seems tospecifically affect endothelial cell growth,survival and permeability. In the lung, VEGFis expressed primarily by epithelial cells andmacrophages. VEGF is also produced by andstored in both human platelets and polymorpho-nuclear neutrophils. The biological activity ofVEGF is dependent on interaction with specificreceptors (VEGF-R1, 2, and 3), which areexpressed not only by endothelial cells, but alsoby activated macrophages and alveolar type IIepithelial cells. Endothelial survival is mediatedvia VEGF-R2/kinase insert domain-containingreceptor (KDR) [8]. The aim of this study wastwo-fold: to determine whether the decrease inlung VEGF is related to epithelial injury, andwhether it may be associated with increasedendothelial apoptosis and decreased capillarydensity.

AFFILIATIONS

*INSERM Unit 492, Paris XII

University, and

Depts of 1Pathology,#Anaesthesiology,##Pulmonary Medicine Unit, and

**Physiology, Hopital Henri Mondor,

Creteil,"Dept of Respiratory Physiology,

Hopital Nord, andeDept of Thoracic Surgery, and+Medical Intensive Care Unit, Hopital

Sainte-Marguerite, Marseille, France.

CORRESPONDENCE

C. Delclaux

Service de Physiologie –

Explorations Fonctionnelles

Hopital Henri Mondor

51 avenue du Marechal de Lattre de

Tassigny

94 010 Creteil

France

Fax: 33 148981777

E-mail: christophe.delclaux@

creteil.inserm.fr

Received:

June 04 2004

Accepted after revision:

September 20 2004

European Respiratory Journal

Print ISSN 0903-1936

Online ISSN 1399-3003

EUROPEAN RESPIRATORY JOURNAL VOLUME 25 NUMBER 1 139

Eur Respir J 2005; 25: 139–146

DOI: 10.1183/09031936.04.00065504

Copyright�ERS Journals Ltd 2005

c

userr
Textbox
Abadie2005 (truncated)

Kit (Qbiogen Inc., Carlsbad, CA, USA), according to themanufacturer’s instructions. Nuclei of apoptotic cells appearedbrown and granular. To quantify the extent of microvascularendothelial apoptosis, co-staining with an anti-human mousemonoclonal antibody to CD31 (WM-59; PharMingen, SanDiego, CA, USA) was used, which recognises the endothelialsurface marker platelet/endothelial cell adhesion molecule 1.TUNEL sections were incubated with 2% normal goat serum(in PBS with 0.05% Tween 20), and, subsequently, with theprimary anti-CD31 antibody (1:100,000 dilution, in PBS with1% BSA) overnight at 4 C. Mouse nonimmune IgG1 (Sigma)was used as the negative control. Biotinylated goat anti-mouseantibodies (Sigma) were applied for 30 min at room tempera-ture, followed by avidin-biotin peroxidase (Sigma) complexesfor 30 min (room temperature). True Blue Peroxidase substratewas used as the final chromogen (KPL Laboratories,Gaithersburg, MD, USA). No counterstaining was used, toavoid possible interference with the specific dark-blue immu-nostaining of the endothelial cell surface. Double stainingwas considered positive when specific cells displayed abrown nucleus with a surrounding blue-to-black membrane-immunoreactive pattern. Positive cells were counted in 10randomly selected areas (final magnification 4006).

Image analysisA charge-coupled device Iris camera (CDD Iris; Sony France,Paris, France), coupled with an optical microscope (LeitzLaborlux, Wetzlar, Germany), was used to view the sectionsand to digitise colour images to a PC host computer. CD31 andTUNEL staining were estimated with Perfect Image software(ClaraVision, Orsay, France), which differentiates coloursbased on their red–green–blue proportions with a ¡10%variation. At least 10 fields per section were analysed, withapproximately the same number of alveoli. Results are given asthe ratio (%) of the blue- (CD31) or brown- (TUNEL) stainedsurface area over the total field surface area (CD31 or TUNELstaining relative to total field surface).

Statistical analysisResults are presented as medians (interquartile range) in thetext. Data are presented as box-plots indicating the median andthe 10th, 25th, 75th and 90th percentiles. Differences betweengroups were estimated using the Kruskal-Wallis test with posthoc Mann-Whitney analysis. To assess the possible influence ofVEGF on pulmonary microcirculation, the relationships link-ing VEGF levels to endothelial quantification and apoptosiswere assessed by Spearman’s rank correlation. p-Values ,0.05were considered statistically significant.

RESULTSPatient characteristicsA total of 29 ARDS patients were studied (20 males and ninefemales; median age 64 yrs (50–73)). Their median acutephysiology and chronic health evaluation II score at admissionwas 16.9 (13.6–23.6). Open lung biopsy was performed as adiagnostic procedure in 15 patients and immediately afterdeath in 14 patients.

At the time of lung tissue sampling, the arterial oxygentension/inspiratory oxygen fraction ratio was ,200 mmHg in22 patients and 200–300 mmHg in seven patients. The cause ofARDS was direct lung injury in 17 patients (related to infection

in 11 and to inhalation in six) and indirect lung injury in 12patients (nonpulmonary sepsis in seven, nonseptic shock infour, and pancreatitis in one). The six patients who died within5 days after ARDS onset were classified as having acute ARDS,whereas the remaining 23 patients were classified as havinglate ARDS; histological findings confirmed the classificationin every case. Survival was only 7% (two out of 29) in thisselected population.

The control group was composed of five patients (all males;median age 65 years (54–70)).

Vascular endothelial growth factor expression in lungs fromacute respiratory distress syndrome and control patientsVEGF levels in the lung homogenates were lower in the ARDSpatients than in the controls: 8.2 (4.7–12.2) versus 28.4 (9.9–47.1)ng?g-1 protein, respectively (p50.03). There was no differencebetween the early and late ARDS subgroups (fig. 1). Resultswere similar when VEGF concentrations were normalised pergram of lung weight. In the controls, immunostaining forVEGF (fig. 2) labelled the bronchiolar cells and alveolarmacrophages and strongly labelled some alveolar type IIepithelial cells. Alveolar type I epithelial cells were negative. Inthe ARDS patients, staining was highly heterogeneous.Bronchiolar and alveolar epithelial cells were negative in someareas and positive in others, even within a tissue section. Inearly ARDS tissue samples, inflammatory cells were alsopositive.

Vascular endothelial growth factor receptor 2 expression inlungs from acute respiratory distress syndrome and controlpatientsThere was no significant difference between VEGF-R2 con-centrations measured by ELISA in lung homogenates fromARDS and control patients (116 (78–157) versus 156 (131–191) ng?g-1 protein). No difference was found between earlyand late ARDS patients.

Immunostaining for VEGF-R2 was noted in endothelial cells inthe control group; however, not all endothelial cells were

����������� �������������

��

��

��

��

��

����������� ��

������

FIGURE 1. Vascular endothelial growth factor (VEGF) concentrations in the

lung homogenates of patients with early-stage (,5 days; n56) or late-stage (o5

days; n523) acute respiratory distress syndrome (ARDS) and of control patients

(n55). VEGF was measured by immunoassay. The box-plot shows the median and

the 10th, 25th, 75th and 90th percentiles. *: p,0.05 compared with control patients.

Y. ABADIE ET AL. VASCULAR ENDOTHELIAL GROWTH FACTOR AND ARDS

cEUROPEAN RESPIRATORY JOURNAL VOLUME 25 NUMBER 1 141

userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight

OCCASIONAL REVIEW

Vascular endothelial growth factor (VEGF) in acute lunginjury (ALI) and acute respiratory distress syndrome (ARDS):paradox or paradigm?

A R L Medford, A B Millar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Thorax 2006;61:621–626. doi: 10.1136/thx.2005.040204

Acute respiratory distress syndrome (ARDS), the mostsevere form of acute lung injury (ALI), remains adevastating condition with a high mortality. It ischaracterised by alveolar injury and increased pulmonaryvascular permeability. Vascular endothelial cell growthfactor (VEGF) was identified by its properties to increasepermeability and act as a cellular growth factor, hence itspotential for a key role in the pathogenesis of ALI/ARDS.This review describes the basic biology of VEGF and itsreceptors as an essential prerequisite to discussing theavailable and sometimes paradoxical published data,before considering a paradigm for the role of VEGF in thehuman lung.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

See end of article forauthors’ affiliations. . . . . . . . . . . . . . . . . . . . . . .

Correspondence to:Dr A B Millar, LungResearch Group,Department of ClinicalScience at North Bristol,University of Bristol LifelineCentre, SouthmeadHospital, Westbury-on-Trym, Bristol BS10 5NB,UK; [email protected]

Received 20 May 2005Accepted20 December 2005. . . . . . . . . . . . . . . . . . . . . . .

Ahealthy alveolar capillary membrane isessential for the gas exchange function ofthe human lung. Injury and loss of this

tissue contributes to the pathology of manyforms of lung disease of which the archetypalexample would be the most extreme form ofacute lung injury (ALI)—namely, acute respira-tory distress syndrome (ARDS).1 An understand-ing of the mechanisms involved in the injury andrepair of this tissue would have significantimpact on the clinical management and treat-ment of this and many other lung conditions.Vascular endothelial growth factor (VEGF) wasoriginally identified by its properties as both apermogen and a mitogen, key elements in thefunction of the alveolar capillary membrane,leading to interest in its role in many forms oflung disease, particularly ARDS.2 3 Intriguingly,in healthy human subjects VEGF protein levelsare highly compartmentalised, with the directlyoxygenated alveolar levels 500 times higher(2 nM) than plasma levels, despite VEGF pro-duction being closely associated with a hypoxiaresponse element.4 5 These levels in normalalveoli are significant, twice the concentrationpreviously shown to induce permeability andmitogenesis (particularly angiogenesis) in vivo.3

However, in healthy lung these processes areextremely restricted. These data suggest animportant persistent or additional function ofVEGF within the human lung that has not yetbeen characterised, which is normally tightlyregulated and which goes awry in ALI/ARDS.Current in vitro work, animal models, andclinical studies are somewhat conflicting as to

the role of VEGF in ALI/ARDS. We attempt toresolve these apparent conflicts in the availabledata by proposing a unifying hypothesis for therole of VEGF in injured lung pertinent to ALI/ARDS—namely, that VEGF protects the alveolarepithelium with a role in repair following lunginjury, but causes fluid flux across the exposedendothelium if the alveolar capillary membraneis functionally breached.

ACUTE RESPIRATORY DISTRESSSYNDROME (ARDS)ARDS, the most extreme form of ALI, was firstdescribed in 1967.1 It is more common than isperhaps appreciated with an estimated incidenceof 75 per 100 000 in some studies.6 It is estimatedto account for nearly 16 500 deaths annually inthe USA, roughly equal to the number of deathsdue to HIV and emphysema, increasing to 74 500if ALI is considered overall.7 ARDS continues tohave a significant mortality of more than 35%despite recent improvements in ventilator stra-tegies and in sepsis management.8 A host ofconditions, including sepsis, trauma, aspiration,massive blood transfusion and burns, both directand indirect insults, predispose to ARDS.However, exposure to a given ‘‘insult’’ does notguarantee that ARDS will follow; for example,there is a 40–60% risk of ARDS following Gramnegative sepsis.9 Although the underlyingmechanisms and factors governing susceptibilityremain unclear, ARDS is characterised by alveo-lar epithelial injury and increased vascularpermeability.6 Markers of both epithelial andendothelial injury have been correlated withoutcome.6 10–12 An additional factor is the poten-tial to induce damage by mechanical ventilationitself.13 14 Survival from ARDS requires resolutionof these features and renewed integrity of thealveolar capillary membrane.

BIOLOGY OF VASCULAR ENDOTHELIALGROWTH FACTOR (VEGF)To appraise and understand the publishedevidence in this area, it is essential to have someunderstanding of the basic biology of VEGF.

Abbreviations: AE, alveolar epithelial; ALI, acute lunginjury; AP, activator protein; ARDS, acute respiratorydistress syndrome; FLT, fms-like tyrosine kinase; HUVEC,human umbilical venous endothelial cell; LPS,lipopolysaccharide; NRP, neuropilin; VEGF, vascularendothelial growth factor; VEGF-R1, VEGF-R2, vascularendothelial growth factor receptor 1 and 2

621

www.thoraxjnl.com

VEGFThe superfamily of VEGF proteins consists of at least sixmembers that are structurally and functionally related butwith predominantly differing key roles.15 This review isconfined to the importance of VEGF-A, termed VEGFthroughout the text. These properties have led to investiga-tion of this molecule in cancer, vascular diseases, chronicinflammatory disorders, and ALI as well as many other lungdiseases including asthma, emphysema, pulmonary fibrosis,lung cancer, and pulmonary hypertension. VEGF is a 34–46 kDa glycoprotein that was first isolated from tumour cellsbut other cellular sources include macrophages, smooth cells,and epithelial cells.2 16 It is a potent angiogenic factor andcritically regulates vasculogenesis such that embryos lackinga single VEGF allele have a lethal phenotype due to abnormalvascular development including that of the lung.17 It bothinduces vascular endothelial cell proliferation and promotessurvival by induction of anti-apoptotic proteins bcl-2 andA1.18–21 VEGF increases microvascular permeability 20 000times more potently than histamine.2 3 Targets for VEGFbioactivity outside the vascular endothelium include macro-phages, type II pneumocytes, and monocytes for which itmay be chemotactic.22–26 It also has a vasodilatory function.27

VEGF isoformsAlternate splicing of the VEGF gene (6p21.3) transcript leadsto the generation of several splice variants (isoforms) ofdiffering sizes, the subscript relating to the number of aminoacids present (VEGF121, VEGF145, VEGF148, VEGF165,VEGF183, VEGF189 and VEGF206).28–30 VEGF165 is the pre-dominant isoform and most biologically active in thephysiological state.31 The longer isoforms are cell associated(exons 6 and 7 have heparin binding activity allowingbinding to the extracellular matrix) compared with theshorter diffusible isoforms.29 Plasmin, the acute phaseprotein, can also cleave the isoforms to form PL-VEGF110

and a recently identified isoform, VEGF165b, is inhibitory infunction and may not be detected in standard VEGF assaysnecessitating reappraisal of current VEGF data.32 Themechanisms by which splicing occurs and is regulated arepoorly understood.

VEGF-R1 and VEGF-R2All VEGF isoforms bind to the tyrosine kinase receptors,VEGF receptor 1 (VEGF-R1) and VEGF receptor 2 (VEGF-R2).15 31 33 They were initially thought to be largely confinedto vascular endothelium but have subsequently been detectedelsewhere including activated macrophages, AE2 cells, andneutrophils.22–25 34–36 Hence, VEGF is capable of having itseffect on both sides of the alveolar capillary membrane onboth the epithelial and endothelial surfaces. There is evidencethat the signal transduction cascades for VEGF-R1 andVEGF-R2 are different and, although VEGF-R1 has greateraffinity for VEGF, VEGF-R2 is tyrosine phosphorylated muchmore efficiently upon ligand binding.22 31 VEGF-R2 isregarded as the main signalling receptor for VEGF bioactivity(angiogenesis, proliferation and permeability) and can causeproliferation in cells lacking VEGF-R1.37 38 VEGF-R2 knock-out mice fail to develop blood islands or organised bloodvessels resulting in early death.39 VEGF-R2 also has a pro-survival function with anti-apoptotic effects on humanumbilical venous endothelial cells (HUVECs).40 In contrast,VEGF-R1 rarely induces cellular proliferation in cells lackingVEGF-R2.26 26 41–44 This has led to the suggestion that VEGF-R1 may function mainly as a decoy receptor, although this isstill contentious. Nevertheless, VEGF-R12/2 mice diebetween days 8.5 and 9.5 in utero from excessive prolifera-tion of angioblasts, supporting a negative regulatory role onVEGF by VEGF-R1 at least during early development.45 46 Inaddition, targeted deletion of the tyrosine kinase domain but

not the VEGF binding domain on VEGF-R1 does not causedeath or obvious vascular defects, although it is required forsome other functions such as monocyte chemotaxis.26 42

Alternate splicing leads to a soluble form of VEGF-R1 (sflt)which can act as an inhibitor of VEGF activity.47

Neuropilins (NRP-1, 2)By contrast, the neuropilins (NRP-1, NRP-2) are isoform-specific VEGF binding sites of different size and affinity toVEGF-R1 and VEGF-R2.48 49 They are expressed by endothe-lial cells in many adult tissues but lack the intracellularcomponent containing tyrosine kinase activity and areregarded as VEGF co-receptors, being unable to signalthemselves without the involvement of VEGF-R2. NRP-1 isisoform-specific, recognising exon 7 of VEGF (bindingVEGF165 but not VEGF121), and increases the effect ofVEGF165 by enhancing its binding to VEGF-R2.50 This mayalso partially account for the greater mitogenic potency ofVEGF165 compared with the VEGF121 isoform. Studies alsosupport a role for NRP-1 in vasculogenesis and angiogenesis.NRP-1 knockout and over expressing mice both die prema-turely from vascular defects.51 52 In contrast, NRP-22/2 areviable but do have absent or reduced lymphatic vessels andcapillaries.53 54

VEGF polymorphismSeveral functional human VEGF polymorphisms have beendescribed.55–57 Significant interindividual variations in plasmaVEGF levels and gene expression related to the presence ofpolymorphism have been reported.58–60 In one study a CTsubstitution at position 936 distal to the start of translation inthe 39-untranslated region of the VEGF gene on chromosome6 was associated with a 75% reduction in plasma levels inboth heterozygotes and homozygotes in a Caucasian popula-tion.55 No such changes in plasma levels were detected inanother genetic association study, but this may have beendue to different racial populations.61 This polymorphismresults in altered binding of the transcription factor activatorprotein 4 (AP-4), although whether the abolition of the AP-4binding site is of specific relevance to the reduction in VEGFprotein expression remains unclear.55 The effect of the CTgenotype on intrapulmonary levels remains unknown at thecurrent time.

VEGF AND THE ALVEOLAR SPACEStudies of ARDS/ALI need to consider both sides of thealveolar capillary membrane (fig 1A). Isolated cellular studiesof epithelial or microvascular endothelial cells give additionalinsight to animal models and clinical studies, as discussedbelow. In vitro studies have demonstrated an abundance ofVEGF in lung tissue, especially in alveolar epithelium,including the A549 cell line and primary human culturedtype II pneumocytes.62–65 Indeed, the highest levels of VEGFmRNA are found in animal and human lung, which suggeststhat the alveolar epithelium is the predominant source.62 66

Although the embryonic role of VEGF is undoubted, in allspecies studied to date adult lungs contain higher amounts ofVEGF mRNA transcript than the developing lung. Changes inrelative isoforms have also been observed with maturity,suggesting an ongoing role.67 VEGF-R1, NRP-1, and NRP-2are all expressed in normal lung.25 68 Primary human type 2alveolar epithelial (AE2) cells are known to express VEGF-R2,the main functioning VEGF receptor, which would facilitatean autocrine role in the air space for VEGF in addition to itswell known paracrine effects on the vascular bed.15 24

Studies suggesting a pathological role for VEGF in thealveolar spaceThe properties of VEGF described previously have led manyworkers to the hypothesis that VEGF would be solely

622 Medford, Millar

www.thoraxjnl.com

userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Underline
userr
Underline
userr
Highlight

1059PERIOPERATIVE TISSUE OXYGENATION(STO2) IS RELATED TO LONG TERMSURGICAL OUTCOME

M.A. Hamilton1, M. Cecconi1, N. Al-Subaie1, D. Dawson1, M. Canete1, A. Rhodes1,R.M. Grounds1

1St. George’s Hospital Intensive Care Unit, London, UK

INTRODUCTION. There is increasing evidence to suggest perioperative complications arepredictive of long term survival and that reducing them may improve survival rates1. Goaldirected therapy has been shown to reduce mortality and morbidity perioperatively, with thoseunable to increase oxygen delivery perioperatively having demonstrably worse outcomes. Theadvent of non invasive tissue oxygenation monitors using near infrared spectroscopy hasallowed further study of oxygen flux during goal directed therapy.

OBJECTIVES. To observe changes in tissue oxygenation during an 8 h oxygen deliverytargeted post surgical optimisation program and provide long term mortality followup of asurgical cohort of high risk patients.

METHODS. 40 patients undergoing high risk surgery and postoperative optimisation (tar-geting of oxygen delivery index of [600 ml/min/m2) on the tensive care unit at a Londonteaching hospital were enrolled. Each patient underwent a protocolised haemodynamicoptimisation protocol as per our standard unit policy for 8 h with consecutive recordings oftissue oxygenation at the thenar eminence using an inspectra 325 monitor. Additional vari-ables relating to global and tissue perfusion were measured concurrently. Patients werefollowed up for survival status at 3.5 years using routinely available information held withinour hospital records.

RESULTS. In hospital mortality was 17.5% (N = 7), whilst at 3.5 years this had increased to50% (N = 20). There was no significant difference between APII scores 11 (4) versus 9.5 (4),Age 61.95 ± 16.23 versus 65.8 ± 16.35 or operation type for survivors and non-survivors at3.5 years respectively. Significant differences between groups were found however foradmission and mid optimisation protocol (4 h) HR and STO2 (see Table 1), But not for DO2i,Lactae, or Base excess. results are shown as mean and sd or median and IQR where notnormally distributed.

STO2 DIFFERENCES FOR SURVIVORS AND NON-SURVIVORSOptimisation time point Ovear all Survivors Non-Survivors Significance (Mann–Whitney)

Admission 72.5 (29) 79.5 (29) 66.5 (29) p = 0.0094 h 69 (24) 75.5 (15) 58.5 (20) p = 0.007End of optimisation 79 (13) 79.5 (10) 77.5 (25) p = 0.098(ns)

There were no significant differences in measured variables for 30 day mortality.

CONCLUSIONS. There appears to be a statistical and clinical difference in HR and tissueoxygenation between the long term survivors of high risk surgery who undergo monitoredpostoperative goal directed optimisation.

REFERENCE(S). 1. Rhodes A, Cecconi M, Hamilton M et al: Goal-directed therapy in high-risk surgical patients: a 15-year follow-up study. Intensive Care Med 2010

GRANT ACKNOWLEDGMENT. Inspectra 325 monitor and probes were loaned byHutchinson technology.

1060CORRELATION OF THORACIC FLUID CONTENT DERIVED BY BIOREAC-TANCE TECHNOLOGY WITH PULMONARY ARTERY PRESSURE

P. Squara1, P. Estagnasie1, A. Brusset1

1Clinique Ambroise Pare, ICU, Neuilly-sur-Seine, France

INTRODUCTION. Bioreactance, a new noninvasive technology for cardiac output moni-toring, has shown to be accurate, precise and time responsive enough for clinical use.1 It alsoallows assessing changes of thoracic fluid content(TFC).2 However, the practical interest ofthe absolute value of TFC is actually limited by the absence of proven relationship withtraditional hemodynamic variables.

OBJECTIVES. To optimize TFC absolute value for clinical use.

METHODS. We used the bioreactance validation database1 to determine first the relationshipbetween TFC and other hemodynamic variables coming from a pulmonary artery cather-ization (PAC). Then, we modeled PAC-related hemodynamic variables from TFC,demographic data (Age, Height, Weight, BSA, BMI) and bioreactance variables (TFC, HeartRate, chest impedance, first time derivative of reactance, ventricle ejection time) using amultiple regression. Finally, we tested the best results on a second new data base.

RESULTS. Our data base included 32231 hemodynamic point in 119 post cardiac surgerypatients. Although weak, the best relationship was found between TFC and systolic pul-monary artery pressure (PAPs: R = 0.15, p \ 0.001). A multiple regression includingdemographic data and bioreactance variables allows to model an estimated PAPS that wasacceptably related with PAPs: R = 0.57, p \ 0.0001). When reported on the second data base(70 patients, 12254 points), the relationship between estimated PAPs and PAPs was stillacceptably good: R = 0.40 p \ 0.0001)

CONCLUSIONS. It seems possible to derive a standardized TFC that roughly correlates withPAPs. Further researches are needed to find better models and to determine if suitable forclinical use.

REFERENCE(S). 1. Squara et al, Intensive Care Med 2007;33:1191–11942. Kossarti et al, Hemodial Int 2009;13:512–517

GRANT ACKNOWLEDGMENT. Cheetah medical

Acute lung injury: Experimental studies: 1061–10741061SIGNIFICANCE OF VEGF RECEPTORS IN THE ACUTE RESPIRATORYDISTRESS SYNDROME

L. Azamfirei1, S. Gurzu2, R. Copotoiu1, R. Solomon3, S.M. Copotoiu1, I. Jung2, J. Szederjesi1,

G. Grigorescu4

1University of Medicine and Pharmacy Targu-Mures, Anesthesiology and Intensive Care,Targu-Mures, Romania, 2University of Medicine and Pharmacy Targu-Mures, Pathology,Targu-Mures, Romania, 3University Hospital Targu-Mures, Anesthesiology and IntensiveCare, Targu-Mures, Romania, 4University Hospital Targu-Mures, Pneumology, Targu-Mures,Romania

INTRODUCTION. Vascular endothelial growth factor (VEGF) is produced by many type ofcells, including the alveolar ones. Its activity depends on the interaction with kinase receptorsVEGF-R1 and -R2. Many studies showed that in normal lung tissue both VEGF and itsreceptors were immunohistochemically expressed by the alveolocytes, macrophages, epi-thelial and endothelial cells. Their exact role in pathogenesis of Acute Respiratory DistressSyndrome (ARDS) is not yet known.

OBJECTIVE. To investigate the expression of VEGF-A, VEGF-R1 and -R2 in the lung ofARDS patients.

METHODS. Lung specimens were obtained by bronchoscopic biopsy from 11 living ARDSpatients in the early phase or by open biopsy in the proliferative phase during autopsy (50patients). Patients with fibrosis were excluded. For the immunohistochemical study we usedthe following antibodies, provided by LabVision: VEGF-A—clone VG1 and polyclonalantibodies Flt-1/VEGFR1 and Flk-1/KDR/VEGFR2. A charge-coupled video camera con-nected to an optical microscope, was used to view the sections and to digitize images on a PChost computer, using a program of assisted quantification

RESULTS. For the specimens obtained from living ARDS patients, the early ARDS phasewas characterized by both VEGF and VEGFR1 endothelial and alveolar expression: 9.5(5.3–11.8) and 26.7 (9.5–38.6). Moving along the time frame, VEGF expression decreased inthe lung tissue: 6.05 (4.4–9.2) (p \ 0.05), while alveolocytes were still marked by VEGFR1,but to a lesser extent: 23.5 (15.7–36.2) (p \ 0.05). For the specimens obtained from autopsy,in the proliferative phase the hyaline membranes strongly expressed VEGF, being negativefor VECFR1. However, the alveolocytes in lung areas without hyaline membranes werepositive for VEGFR1 but the number of alveolar cells positive for VEGFR1 was smaller.VEGFR2 marked only bronchial epithelium in both early and proliferative phase with noexpression on alveolocytes or hyaline membranes.

CONCLUSIONS. Three steps in the pathogenesis of ARDS could be defined: decrease ofVEGF expression in alveolocytes, damage of surfactant and hyaline membranes constitutionand subsequent apoptosis of the alveolar cells in those areas. Persistence of VEGFR1 im-munostain in alveolar cells outside hyaline membranes areas proved that only a part of thesecells were destroyed during ARDS. The number of non-damaged alveolar cells seems to bedecisive for survival.

GRANT ACKNOWLEDGMENT. Research Grant no 136/IDEI/National Authority ofScientific Research, Romania.

1062EVALUATION OF NONBRONCHOSCOPIC LAVAGE BY AIRWAY EXCHANGECATHETER AS A DIAGNOSTIC INSTRUMENT FOR MONITORING CHANGES INTHE ALVEOLAR MILIEU

N. Larsson1, S. Lehtipalo1, J. Pourazar2, A.F. Behndig2, J. Claesson1

1Umea University Hospital, Dept of Surgical and Perioperative Sciences, Anesthesiology andIntensive Care, Umea, Sweden, 2Umea University Hospital, Department of RespiratoryMedicine and Allergy, Umea, Sweden

INTRODUCTION. Bronchoscopic bronchoalveolar lavage (B-BAL) is today the goldstandard for sampling of inflammatory markers in the distal airways. Nonbronchoscopicbronchoalveolar lavage (N-BAL) by ordinary suction catheter has been investigated as a moreeasily accessible method for alveolar sampling in the setting of acute respiratory distresssyndrome (ARDS). The results, however, were disappointing, probably due to more proximalsampling by the N-BAL.

OBJECTIVES. To investigate wether N-BAL by a catheter with physical properties similarto those of the bronchoscope is comparable to B-BAL.

METHODS. B-BAL and N-BAL by Cook’s airway exchange catheter was performed with3 9 30 ml normal saline on opposite sides 15 min apart at nine different occasions on 5anesthetized and intubated pigs. The volume of the recovered lavage was noted, after whichthe fluid was analyzed for albumin, total cell count, viability and differential cell count.Statistical analysis was performed using Wilcoxon’s rank-sum test.

RESULTS. N-BAL yielded significantly higher albumin content than B-BAL (20.1 ± 8.7 vs.11.7 ± 3.4 mg/L, p = 0.027). In all other measurements there were no significant differencesbetween N-BAL and B-BAL (recovered volume 52.1 ± 19.2 vs. 67.9 ± 9.4 ml, total cellcount 40.0 ± 28.4 vs. 40.7 ± 17.3 9 106 cells, viability 49.6 ± 20.4 vs. 62.6 ± 13.1% andpercent macrophages 70.5 ± 15.5 vs. 77.1 ± 6.6%.

CONCLUSIONS. N-BAL by airway exchange catheter does not differ significantly inrecovered volume and cell count when compared to B-BAL. N-BAL for alveolar samplingdeserves further investigation.

REFERENCE(S). Perkins GD, Chatterjie S, McAuley DF, Gao F, Thickett DR. Role ofnonbronchoscopic lavage for investigating alveolar inflammation and permeability in acuterespiratory distress syndrome. Crit Care Med. 2006 Jan;34(1):57–64.

S352 23rd ESICM Annual Congress – Barcelona, Spain – 9–13 October 2010

userr
Rectangle
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Underline
userr
Underline
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Underline
userr
Highlight
userr
Highlight
userr
Highlight
userr
Underline
userr
Highlight
userr
Highlight
userr
Highlight
userr
Underline
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Underline
userr
Highlight
userr
Highlight
userr
Underline
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Underline
userr
Underline
userr
Rectangle
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Polyline
userr
Rectangle
userr
Callout
Ideas reported in Solomon2010 (the same study under different Title with several authors)
userr
Callout
Coincidental values with those reported in Solomon2010
userr
Callout
Identical conclusions with those reported in Solomon2010 (the same study under different Title with several authors)

IntroductionEmphysema is estimated to affect 1.65 million peoplein the US (1). Emphysema is defined as abnormal per-manent enlargement of the airspaces distal to termi-nal bronchioles. The pathogenesis of pulmonaryemphysema, which is characterized by the disappear-ance of alveolar septa, remains enigmatic, althoughthe protease-antiprotease imbalance hypothesis ofinflammation is widely supported (2). Briefly, the con-cept is that activated alveolar macrophages releaseelastase, which destroys the lung tissue, overwhelminglocal antiprotease activities (3). However, becausemany cigarette smokers and patients with severeinflammatory lung parenchyma diseases (like pneu-monia and adult respiratory distress syndrome) do notdevelop significant emphysema, this hypothesis maynot fully explain the loss of lung tissue in cigarettesmoking–induced emphysema.

Preceding the discovery of the association betweenpanacinar emphysema and a hereditary deficiency ofα-1-antitrypsin (4), A.A. Liebow (5), based on histo-logical examination of emphysema lungs, pointed outthat the alveolar septa in centrilobular emphysemaappear to be remarkably thin and almost avascular. He

also considered that a reduction in the blood supply ofthe small precapillary blood vessels might induce thedisappearance of alveolar septa. We revisit this earlyvascular hypothesis of emphysema development andpropose that the disappearance of lung tissue inemphysema may involve the progressive loss of capil-lary endothelial and epithelial cells through theprocess of programmed cell death, apoptosis (6). Oneknown survival factor for endothelial cells is VEGF,initially characterized as a factor that increases en-dothelial permeability (7) and induces endothelial cellgrowth (8). It is not only essential for the normal devel-opment of blood vessels in the embryo (9, 10), but it isalso required for survival of endothelial cells. With-drawal of VEGF leads to endothelial cell apoptosis invitro (11, 12) and in vivo (13). VEGF binds to two tyro-sine kinase receptors present on endothelial cells:VEGF receptor 1 (VEGFR-1; Flt-1) and VEGFR-2(KDR/Flk-1). Although VEGF is highly abundant inthe lung, its normal biological activity in the lung isnot well understood. We postulate that VEGF signal-ing may be required for the maintenance of adult lungalveolar structures. We have recently reported in-creased septal cell death in human emphysematous

The Journal of Clinical Investigation | December 2000 | Volume 106 | Number 11 1311

Inhibition of VEGF receptors causes lung cell apoptosis and emphysema

Yasunori Kasahara,1 Rubin M. Tuder,1,2 Laimute Taraseviciene-Stewart,2

Timothy D. Le Cras,3 Steven Abman,3 Peter K. Hirth,4 Johannes Waltenberger,5

and Norbert F. Voelkel1

1Division of Pulmonary Sciences and Critical Care Medicine, Pulmonary Hypertension Center, Department of Medicine,2Department of Pathology, and3Pediatric Heart Lung Center, Division of Pediatric Pulmonary Medicine, Department of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado, USA

4SUGEN Inc., South San Francisco, California, USA5Department of Internal Medicine II, Division of Cardiology, Ulm University Medical Center, Ulm, Germany

Address correspondence to: Norbert F. Voelkel, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Box C 272, 4200 E. Ninth Avenue, Denver, Colorado 80262, USA. Phone: (303) 315-7047; Fax: (303) 315-5632; E-mail: [email protected].

Received for publication May 8, 2000, and accepted in revised form August 30, 2000.

Pulmonary emphysema, a significant global health problem, is characterized by a loss of alveolarstructures. Because VEGF is a trophic factor required for the survival of endothelial cells and is abun-dantly expressed in the lung, we hypothesized that chronic blockade of VEGF receptors could inducealveolar cell apoptosis and emphysema. Chronic treatment of rats with the VEGF receptor blockerSU5416 led to enlargement of the air spaces, indicative of emphysema. The VEGF receptor inhibitorSU5416 induced alveolar septal cell apoptosis but did not inhibit lung cell proliferation. Viewed byangiography, SU5416-treated rat lungs showed a pruning of the pulmonary arterial tree, althoughwe observed no lung infiltration by inflammatory cells or fibrosis. SU5416 treatment led to a decreasein lung expression of VEGF receptor 2 (VEGFR-2), phosphorylated VEGFR-2, and Akt-1 in the com-plex with VEGFR-2. Treatment with the caspase inhibitor Z-Asp-CH2-DCB prevented SU5416-inducedseptal cell apoptosis and emphysema development. These findings suggest that VEGF receptor sig-naling is required for maintenance of the alveolar structures and, further, that alveolar septal cellapoptosis contributes to the pathogenesis of emphysema.

J. Clin. Invest. 106:1311–1319 (2000).

userr
Textbox
Kasahara2000 (truncated)
userr
Highlight
userr
Highlight

lungs, which was associated with reduced lung expres-sion of VEGF and VEGFR-2 (KDR/Flk-1) (6). Here wereport that chronic treatment of rats with the VEGFreceptor blocker SU5416 causes alveolar cell apopto-sis–dependent emphysema.

MethodsAnimals. The animal protocol was approved by the Ani-mal Care and Use Committee of the University of Col-orado Health Sciences Center. Male Sprague-Dawleyrats were purchased from a commercial vendor and keptfor 7 days in the Animal Care Facility of the Universityof Colorado Health Sciences Center. In the first set ofexperiments, rats then were divided into three groups:(a) control group (n = 6), (b) SU5416 {3-[(2,4-di-methylpyrrol-5-yl)methylidenyl]-indolin 2-one} group(n = 6), and (c) SU5416+caspase inhibitor group (n = 6).SU5416 (provided by SUGEN Inc.) and SU5416+cas-pase inhibitor groups were injected subcutaneouslythree times per week for 3 weeks. SU5416 (20 mg/kg)was suspended in diluent (0.5% carboxymethylcellulosesodium, 0.9% sodium chloride, 0.4% polysorbate 80,0.9% benzyl alcohol in deionized water). The controlgroup received only the diluent. SU5416+caspaseinhibitor group was injected intraperitoneally with 1 mgof Z-Asp-CH2-DCB (Alexis Corp., San Diego, California,USA), dissolved in 50 µl of PBS solution containing 20%DMSO, every day for 3 weeks. Control and SU5416-alone groups were injected only with 50 µl of PBS solu-tion containing 20% DMSO. To study the time courseof the effect of SU5416, rats (n = 6) were treated for 3, 7,and 21 days.

Tissue processing. After completion of the treatmentperiod, the chest was opened and the cardiopul-monary block was quickly isolated and excised, andthe ratio of right ventricular weight/left ventricle plusseptum weight was obtained. The right main bronchuswas cross-clamped and the left lung was filled with0.5% low melting agarose in 10% formalin at a con-stant pressure of 25 cm H2O, allowing for homoge-nous expansion of lung parenchyma (14). After that,the lungs were fixed in 10% formalin for 48 hours. Theparaffin-embedded tissues were sectioned and pre-pared for histological analysis. Lung sections weretaken from the same lung regions in both the treatedand control groups, representative of upper and lowerlobes of the left lung.

Morphological assessment. After fixation, 5-µm sectionswere stained with hematoxylin and eosin. The meanlinear intercept, as a measure of interalveolar wall dis-tance, was determined by light microscopy at a totalmagnification of ×100. The mean linear intercept wasobtained by dividing the total length of a line drawnacross the lung section by the total number of inter-cepts encountered in 72 lines per each rat lung, asdescribed by Thurlbeck (15).

Angiograms. Arteriograms were performed in lungsthat were infused with barium sulfate (16). Immedi-ately after sacrifice, PBS was infused through a main

pulmonary artery catheter to flush the pulmonary cir-culation free of blood. A barium sulfate–gelatin mix-ture was heated to 65°C, and infused into the mainpulmonary artery at a perfusion pressure of 74 mmHg.This pressure was maintained for at least 5 minutes toensure penetration of the barium mixture. Lung tissuewas subsequently fixed for angiograms. The quantita-tion of the peripheral pulmonary arteries filled withbarium was performed as described recently (17).

Immunohistochemistry. Anti–caspase 3 polyclonal anti-body CM1 (1:250 dilution; kindly provided by AnuSrinivasan, Idun Pharmaceuticals, La Jolla, California,USA) (18), proliferating cell nuclear antigen (PCNA;1:50 dilution; sc-56 from Santa Cruz BiotechnologyInc., Santa Cruz, California, USA), and anti-mac-rophage antibody (1:50 dilution for immunohisto-chemistry; Chemicon International, Temecula, Cali-fornia, USA) were used. Immunolocalization wasperformed on paraffin-embedded, formalin-fixed ratlungs. Briefly, after paraffin removal in xylene, the sec-tions were rehydrated and submitted to microwavetreatment (800 W/15 min) in 10 mM citric acid mono-hydrate solution. After quenching of endogenous per-oxidase with 3% H2O2 for 20 minutes, the sections wereexposed to CM1 polyclonal antibody, which recog-nizes active caspase-3, or to PCNA monoclonal anti-body for 30 minutes. After incubation with the pri-mary antibody, immunodetection was performedusing biotinylated anti-rabbit or anti-mouse IgG. Per-oxidase-conjugated streptavidin (Vector Laboratories,Burlingame, California, USA), with diaminobenzidine(DAB) as the substrate, completed the immunostain-ing. Negative controls for nonspecific binding includ-ed normal rabbit or mouse serum.

TUNEL. Terminal deoxynucleotidyl transferase–medi-ated dUTP nick end-labeling (TUNEL) was performedwith TACS 2 TdT DAB kit (Trevigen, Gaithersburg,Maryland, USA), following the manufacturer’s instruc-tions. Briefly, after deparaffinization and rehydration,sections were digested with proteinase K at a concen-tration of 20 µg/ml for 15 minutes. Endogenous perox-idase activity was quenched with 2% H2O2 for 5 minutes.The slides were immersed in terminal deoxynucleotidyltransferase (TdT) buffer. TdT, 1 mM Mn2+, and biotiny-lated dNTP in TdT buffer were then added to cover thesections and incubated in a humid atmosphere at 37°Cfor 60 minutes. The slides were washed with PBS andincubated with streptavidin–horseradish peroxidase for10 minutes. After rinsing with PBS, the slides wereimmersed in DAB solution. The slides were counter-stained for 3 minutes with 1% methyl green.

In situ ligation of labeled DNA fragments. A 200-bp dou-ble-stranded DNA fragment was prepared by poly-merase chain reaction with Taq polymerase usingprimers 5′-CACTAGAGACAGTTTGCCATG-3′ and 5′-GTC-CCACACCCTTTAGAGAAG-3′ complementary to humanadenocarcinoma DNA. Deparaffinized 5-µm sectionswere subjected to Taq polymerase–based DNA in situligation assay using the DNA fragments labeled with

1312 The Journal of Clinical Investigation | December 2000 | Volume 106 | Number 11

userr
Highlight
userr
Highlight
userr
Rectangle

Chest, October 2009, Vol 136, No. 4, Meeting Abstracts

RELATIONSHIP BETWEEN LUNG TISSUE EXPRESSION AND PLASMATIC LEVEL

OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN PATIENTS WITH ACUTE

RESPIRATORY DISTRESS SYNDROME

Leonard Azamfirei, PhD; Ruxandra Copotoiu, MD*; Simona Gurzu, PhD; Sanda-Maria Copotoiu, PhD;

Ioan Jung, PhD; Klara Branzaniuc, PhD; Raluca Solomon, MD; Janos Szederjesi, MD

Abstract

PURPOSE: To investigate the vascular endothelial growth factor (VEGF) expression in the lung tissue of acute respiratory distress syndrome (ARDS) patients and also the VEGF plasmatic levels of these patients.

METHODS: We realized a prospective study including 10 patients diagnosed with ARDS. Lung specimens from ARDS patients were obtained by bronchoscopy or by autopsy in case of the deceased patients. The controls were 10 patients deceased from other causes than ARDS from whom necroptic pulmonary tissue samples. The determination of VEGF expression in the pulmonary tissue was performed using specific monoclonal antibodies VEGF, clones VG1 and JH 121. The controls were 10 patients deceased from other causes than ARDS from whom necroptic pulmonary tissue samples.

RESULTS: The ARDS etiology of the studied patients was mainly extrapulmonary (7 cases out of 10). Patients who died because of ARDS had a VEGF pulmonary expression significantly decreased compared to non-ARDS patients: 8.5 (4.1–9.9) versus 28.7 (9.5–48.6) (p < 0.001). (Fig. 1). The seric VEGF levels of ARDS patients were raised (230 pg/ml) compared to non-ARDS patients (131 pg/ml) (p < 0.001). Alveolar macrophages were immunopositive in both groups. No significant statistical differences were noted between the two groups with regard to age, gender, period of ARDS condition, number of ICU days.

CONCLUSION: A decreased alveolar type II cells, induced by apoptosis was noticed in ARDS

evolution, therefore reducing the VEGF production in the alveolar space and also contributing to the decrease in lung perfusion, but also to the consecutive increase of VEGF plasmatic level.

CLINICAL IMPLICATIONS: The over-expression of pulmonary VEGF, leads to increased vascular pulmonary permeability and consequently pulmonary oedema. Nevertheless, the VEGF expression in human alveolar epithelial cells also facilitates neovascularization, contributing to endothelial injury repair.Low VEGF pulmonary levels were associated with the severity of ARDS, whereas high VEGF levels were associated with the recovery from ARDS, signalling the role of VEGF in the pulmonary injury repairing process.

DISCLOSURE: Ruxandra Copotoiu, Grant monies (from sources other than industry) The study was

supported by National University Research Council (CNCSIS) Grant, Romania, IDEI no 136/2008; No Product/Research Disclosure Information

Wednesday, November 4, 2009

userr
Highlight
userr
Highlight
userr
Highlight
userr
Highlight
userr
Textbox
Azamfirei2009-Chest