PLoS Biol v83 2010 Mar PMC2830450 PLoS Biol 2010 Mar 83 e1000321 Published online 2010 Mar 2 doi 101371journalpbio1000321 PMCID PMC2830450 Studying Function and Behavior in the Fossil Record Michael J Benton Author information Copyright and License information See the article Predation upon Hatchling Dinosaurs by a New Snake from the Late Cretaceous of India e1000322 This article has been cited by other articles in PMC Inferring the behavior and function of ancient organisms is hard Some paleontologists would say that it can not be done because such hypotheses can never be testable whereas others would say that this is surely a prime task for paleontologyto seek to bring ancient organisms back to life These issues have long troubled paleontologists The founder of comparative anatomy Georges Cuvier 17691832 insisted on the common pattern of the skeleton of living and fossil vertebrates and that anatomy could be reconstructed with confidence from incomplete fossil remains Further he argued that the skeleton of a living or extinct animal held unequivocal clues about function and behavior Cuvier saw his mission to establish rules for comparative anatomy that would allow paleontologists to make certain statement with clarity and confidence 1 a key principle today what one might call evidencebased reconstruction for example sharp teeth indicate a diet of meat rather than plants or mammalian characters in the teeth indicate that the unknown animal was endothermic and nourished its young from mammary glands as opposed to speculation this dinosaur was purple because I guess it was Form Function and Behavior It can be assumed that biological structures are adapted in some way and that they have evolved to be reasonably efficient at doing something So an elephants trunk has evolved to act as a grasping and sucking organ to allow the huge animal to reach the ground and to gather food and drink Cuvier realized that form reflected function even though he interpreted such exquisite adaptations as evidence for design rather than evolution But we must be cautious of overinterpretation perhaps assuming that everything is an adaptation and that adaptations are all perfect 2 Fossils can provide a great deal of evidence about function For example the hard skeleton of a fossil arthropod reveals the number and shape of the limbs the nature of each joint in each limb perhaps also the mouthparts and other structures relating to locomotion and feeding Exceptionally preserved fossils may reveal additional structures such as the outline of the tentacles of a belemnite or ammonite hair and feathers and muscle tissue or sensory organs In vertebrates there may be muscle scars on the surface of the bone and particular knobs and ridges processes that show where the muscles attached 3 There are three approaches to inferring function and behavior from fossilsempirical evidence comparison with modern analogs and biomechanical modeling Empirical Evidence Paleontologists are inquisitive by nature and they gather evidence of all kinds to test their hypotheses Evidence about the lifestyle of an ancient plant or animal may come from the enclosing rocks associated fossil remains associated trace fossils and particular features of the body fossils themselves The rocks can give clear evidence about ancient climates and associated fossils indicate possible prey and predator relationships Trace fossils such as tracks and burrows can sometimes be linked with their makers and then used to look at modes of locomotion and whether animals burrowed or not 4 5 Tracks also give surer evidence about some aspects of locomotion than the bones themselves For example manipulating bones can allow a paleontologist to work out whether a tetrapod stood upright like a mammal or held its arms and legs sideways in a sprawling posture But its not always easy Footprints show precisely whether the feet fell in a single line or were far apart and a study across the famous endPermian mass extinction 252 million years ago shows a dramatic shift from sprawling to upright posture among virtually all tetrapods at the same time 6 Fossil dung or stomach contents can often be associated with the producer and paleobiologists who are so inclined can tease apart fossil dung under the microscope and determine the key constituents of diet A famous 44cmlong coprolite dropped by T rex contains pulverized bones of ornithischian dinosaurs that had been corroded to some extent by stomach acids but not entirely destroyed 7 This suggests a relatively rapid transit of food material through the gut The teeth of ancient animals can indicate diet and detailed study of teeth of fossil mammals can even indicate the kinds of plants they were eating based on fine scratches and grooves seen under the microscope 8 Sometimes one organism is preserved in flagrante delicto as it were feeding upon anotherfor example small leafeating insects within fossil plant stems 9 or a fish that died choking on a fish it was trying to swallow 10 Jeff Wilson and colleagues report another such remarkable specimen in this issue of PLoS Biology a snake preserved complete and wrapped around a crushed dinosaur egg in a nest of otherwise unbroken eggs from the Late Cretaceous of India 11 It seems most likely as the authors argue that this 35meterlong snake was waiting and snatching juveniles as they hatched see Figure 1 Of course we can not be entirely sure unless further specimens come to light showing the bones of juvenile dinosaurs in the stomach region of the snake In this case and others the specimens are key and the care taken by their collectors and investigators to extract every fine detail Figure 1 A Cretaceous snake feeding on hatchling sauropod dinosaurs In all cases these are not mere observations or descriptions of rather obvious data but rather the statements are hypotheses like any other subject to refutation at any time The Cuvierian example noted earlier that a jaw that bears mammalian teeth can tell the paleontologist that it came from say a marsupial mole of a particular family and the needlelike teeth indicate that it fed on insects is based entirely within the hypotheticodeductive model Just as the classic assertion that all swans are white was refuted by the discovery of the black Tasmanian swan each of the assertionsclaims made by Comparison with Modern Analogs It is probable that function and behavior of a fossil bat should be inferred from comparisons with living bats But should a dinosaur be compared with living relatives eg birds or crocodiles or with living animals with apparently similar function eg elephants or rhinos Phylogeny might be thought to trump general similarity but does it Perhaps it would be pointless to compare a Diplodocus with a sparrowtheir body size morphology and presumed modes of life are wildly different But something informative does come from phylogeny At one level parsimony allows paleobiologists to infer the presence of softtissue characters and behaviors A development of the parsimony principle is the extant phylogenetic bracket EPB 12 According to this principle osteological correlates of unpreserved features are identified and these allow inferences about the presence of unpreserved features At a simple level we could say that Tyrannosaurus rex presumably had an eyeball with certain properties because its bracketing living relativesbirds and crocodilesshare many common characters in their eyes A further example perhaps a little more impressive is the prediction that fossil eggs will some day be found in the Carboniferous The reasoning is that all living amniotes ie reptiles birds and mammals lay hardshelled eggs even though egg laying has been replaced by live birth in most mammals and some snakes and lizards Thus the first amniote in the Carboniferous over 300 million years ago presumably laid a hardshelled egg even though the oldest fossil eggs are known 13 Parsimony and the EPB are now widely used in discourse about the remarkable feathered birds and dinosaurs from the Jehol Group of China Early Cretaceous 131120 Ma When specimens of the small theropod Sinosauropteryx were announced 14 with simple filamentlike feathers paleontologists looked at the phylogenetic trees and realized that this took the origin of feathers back to the base of the Middle Jurassic some 175 million years ago This is because Sinosauropteryx is a basal coelurosaur and the first coelurosaurs are known from the base of the Middle Jurassic the most parsimonious assumption is that all coelurosaurs possessed some kind of feathers from the start Note that the Sinosauropteryx filaments are debated and some 15 argue they are not feathers but connective tissue but close study suggests otherwise 16 In any case feathers have been reported from nearly every other coelurosaur lineage and so their origin deep within the phylogeny of theropod dinosaurs appears assured Knowing the arrangement of feathers and perhaps their colors and patterns 16 18 may allow paleobiologists soon to speculate rationally and calmly of course about whether certain dinosaurs used their brightly colored and patterned feathers for camouflage warning sexual display establishing pecking order or other behaviors and functions Biomechanical Modeling Biomechanical models combined with considerations of modern analogs provide powerful insight into certain aspects of the moving parts and skeletons of ancient organisms Opportunities have been hugely expanded by the relative ease with which 3D structures such as shells bones and skeletons may be scanned and imaged These images may then be tested using standard engineering software to determine how the structure was shaped by stresses and strains of walking running feeding or head butting A useful modeling approach is finite element analysis FEA 19 20 a wellestablished method used by engineers to assess the strength of bridges and buildings before they are built and now applied to dinosaur skulls see Figure 2 among other fossil problems Figure 2 Finite element analysis of the skull of T rex A number of attempts have been made to understand how dinosaurs walked and ran and of course everyone focuses on T rex It is reasonable to assume that the laws of physics and the principles of biomechanics were the same in the past as they are now For example the starting point in studying the locomotion of any animal especially a biped is to establish the center of masswhatever happens the animal must not fall over The center of mass for a living or extinct animal can be determined either from solid models or from calculations of the distribution of tissues and air spaces through slices of the 3D restored body 21 22 In T rex the center of mass lay just in front of the hips and the tail balanced the body over the hips that acted as a fulcrum giving a most natural stance with the backbone held almost horizontal This is a major improvement over the oldstyle kangaroo poses that people used to use for dinosaurs where the animals body was more vertical than horizontal and the tail rested on the ground This is only the beginning however and T rex could be imagined walking and running in a variety of poses 23 24 But how fast could T rex run Here many estimates have been made ranging from a speedy 20 meters per second 72 kmh 42 mph to a more sedate 5 meters per second 18 kmh 11 mph the speed of a human longdistance runner Many approaches were used and these illustrate the ingenuity of paleobiologists For example fossil trackways can indicate speeds there is a constant relationship between the spacing of footprints stride length leg length and speed 25 Others made calculations based on relative lengths of the leg bones or on assumptions about the risk of injury if the animal fell or by using calculations of stress and strain the faster you run the greater the impact as the foot hits the ground Most recently the question has been resolved by simple calculations based on estimated leg muscle volume the major leg muscles that power the stride are proportional to body mass and speed 26 At speeds faster than 5 meters per second the 6tonne T rex would have needed leg muscles that were proportional to those of a chicken see Figure 3 and at 20 meters per second the highest speed previously assumed the legpowering muscles would have made up to 86 of total body mass In a further set of calculations Pontzer and collegues 27 show that the biomechanics of running and metabolic rate are intimately linked and based on evidence from extant tetrapods they can identify that the larger dinosaurs at least exceeded the maximum aerobic capabilities of modern ectotherms This means they were functionally endothermic although this may well have arisen through inertial homeothermy because of their large size Figure 3 T rex trotting along beside a T rex sized chicken Paleobiologists then need not make wild guesses in reconstruction of behavior and function in the past Ingenious interpretations and spectacular discoveries such as the snake on the dinosaur nest 11 can sometimes give us remarkable insights into the longlost life of the past Abbreviations extant phylogenetic bracket EPB finite element analysis FEA Footnotes The author has declared that no competing interests exist No specific funding was received for this article References 1 Rudwick M Chicago Chicago University Press 2005 Bursting the limits of time732 2 Gould S Lewontin R The spandrels of San Marco and the Panglossian paradigm a critique of the adaptationist programme Proceedings of the Royal Society Series B 1979 205 281288 PubMed 3 Unwin D Pterosaurs back to the traditional model Trends in Evolution and Ecology 1999 14 263268 PubMed 4 Hasiotis S Wellner R Martin A Demko T Vertebrate burrows from Triassic and Jurassic continental deposits of North America and Antarctica their paleoenvironmental and paleoecological significance Ichnos 2004 11 103124 5 Varricchio D Martin A Katsura Y First trace and body fossil evidence of a burrowing denning dinosaur Proceedings of the Royal Society Series B 2007 274 13611368 PMC free article PubMed 6 Kubo T Benton M Tetrapod postural shift estimated from Permian and Triassic trackways Palaeontology 2009 52 10291037 7 Chin K Tokaryk T Erickson G Calk L A kingsized theropod coprolite Nature 1998 393 680682 8 Williams V Barrett P Purnell M Quantitative analysis of dental microwear in hadrosaurid dinosaurs and the implications for hypotheses of jaw mechanics and feeding Proceedings of the National Academy of Sciences USA 2009 106 1119411199 PMC free article PubMed 9 Wilf P Insectdamaged fossil leaves record food web response to ancient climate change and extinction New Phytologist 2008 178 486502 PubMed 10 Kriwet J Witzmann F Klug S Heidtke U First direct evidence of a vertebrate threelevel trophic chain in the fossil record Proceedings of the Royal Society Series B 2008 275 181186 PMC free article PubMed 11 Wilson J Mohabey D Peters S Head J Predation upon hatchling dinosaurs by a new snake from the Late Cretaceous of India PLoS Biology 2010 83 e1000322 doi 101371journalpbio1000322 PMC free article PubMed 12 Witmer L The Extant Phylogenetic Bracket and the importance of reconstructing soft tissues in fossils In Thomason J editor Functional Morphology in Vertebrate Paleontology New York Cambridge University Pres 1995 pp 1933 13 Benton M Oxford WileyBlackwell 2005 Vertebrate palaeontology455 14 Chen P Dong Z Zhen S An exceptionally wellpreserved theropod dinosaur from the Yixian Formation of China Nature 1998 391 147152 15 LinghamSoliar T Feduccia A Wang X A new Chinese specimen indicates that protofeathers in the Early Cretaceous theropod dinosaur Sinosauropteryx are degraded collagen fibres Proceedings of the Royal Society Series B 2007 274 18231829 PMC free article PubMed 16 Zhang F Kearns S Orr P Benton M Zhou Z et al Fossilized melanosomes and the colour of Cretaceous dinosaurs and birds Nature Epub ahead of print 2010 doi nature087403d PubMed 17 Vinther J Briggs D Prum R Saranathan V The colour of fossil feathers Biology Letters 2008 4 522525 PMC free article PubMed 18 Vinther J Briggs D Clarke J Mayr G Prum R Structural coloration in a fossil feather Biology Letters Epub ahead of print 2009 doi 101098rsbl20090524 PMC free article PubMed 19 Rayfield E Cranial mechanics and feeding in Tyrannosaurus rex Proceedings of the Royal Society Series B 2004 271 14511459 PMC free article PubMed 20 Rayfield E Finite element analysis in vertebrate morphology Annual Reviews in Earth and Planetary Sciences 2007 35 541576 21 Henderson D Estimating the masses and centers of mass of extinct animals by 3D mathematical slicing Paleobiology 1999 25 88106 22 Henderson D Burly gaits centers of mass stability and the trackways of sauropod dinosaurs Journal of Vertebrate Paleontology 2006 26 907921 23 Hutchinson J Gatesy S Dinosaur locomotion beyond the bones Nature 2006 440 292294 PubMed 24 Gatesy S Baeker M Hutchinson J Constraintbased exclusion of limb poses for reconstructing theropod dinosaur locomotion Journal of Vertebrate Paleontology 2009 29 535544 25 Alexander R Estimates of speeds of dinosaurs Nature 1976 261 129130 26 Hutchinson J Garcia M Tyrannosaurus was not a fast runner Nature 2002 415 10181021 PubMed 27 Pontzer H Allen V Hutchinson J Biomechanics of running indicates endothermy in bipedal dinosaurs PLoS One 2009 411 e7783 doi 101371journalpone0007783 PMC free article PubMed Articles from PLoS Biology are provided here courtesy of Public Library of Science

O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

fossil record
paleontology
georges cuvier


Cardiovasc

Res

Jul

Published

online

Apr

doi

PMCID

EMSID

Vascular

endothelial

growth

factors

and

vascular

permeability

David

O

Bates

Author

information

Article

notes

Copyright

and

License

information

This

article

has

been

cited

by

other

articles

in

PMC

Go

to

Abstract

Vascular

endothelial

growth

factors

VEGFs

are

key

regulators

of

permeability

The

principal

evidence

behind

how

they

increase

vascular

permeability

in

vivo

and

in

vitro

and

the

consequences

of

that

increase

are

addressed

here

Detailed

analysis

of

the

published

literature

has

shown

that

in

vivo

and

in

vitro

VEGFmediated

permeability

differs

in

its

time

course

but

has

common

involvement

of

many

specific

signalling

pathways

in

particular

VEGF

activation

calcium

influx

through

transient

receptor

potential

channels

activation

of

phospholipase

C

gamma

and

downstream

activation

of

nitric

oxide

synthase

Pathways

downstream

of

endothelial

nitric

oxide

synthase

appear

to

involve

the

guanylyl

cyclasemediated

activation

of

the

RhoRac

pathway

and

subsequent

involvement

of

junctional

signalling

proteins

such

as

vascular

endothelial

cadherin

and

the

tight

junctional

proteins

zona

occludens

and

occludin

linked

to

the

actin

cytoskeleton

The

signalling

appears

to

be

coordinated

through

spatial

organization

of

the

cascade

into

a

signalplex

and

Keywords

VEGF

Vascular

permeability

Calcium

Capillary

Endothelium

Go

to

Importance

of

vascular

endothelial

growth

factormediated

permeability

in

health

and

disease

The

discovery

of

vascular

endothelial

growth

factor

VEGF

in

the

reflects

to

some

degree

the

continuing

revision

of

our

understanding

of

what

this

critical

molecule

does

in

humans

its

mechanisms

of

action

and

its

cellular

targets

For

while

VEGF

was

originally

purified

as

a

tumoursecreted

factor

it

was

done

so

using

an

assay

designed

not

for

vessel

growth

but

increased

extravasation

of

dye

Thus

it

was

termed

vascular

permeability

factor

by

Dvorak

and

Senger

when

they

partially

purified

it

although

neither

investigator

measured

its

effect

on

vascular

permeability

per

se

It

was

subsequently

also

identified

in

to

be

the

long

sought

after

tumour

angiogenesis

factor

separately

by

two

endocrinologists

who

had

recently

worked

in

the

same

laboratory

Ferrara

and

the

late

Plouet

who

both

worked

with

Denis

Gospadorowicz

at

UCSF

in

and

was

termed

vasculotropin

by

Plouet

et

al

and

VEGF

by

Ferrara

and

coworkers

However

VEGF

acts

on

many

cell

types

including

epithelial

nerve

and

tumour

cells

and

can

act

as

either

a

stimulator

or

inhibitor

of

endothelial

cell

growth

Our

understanding

has

therefore

undergone

such

a

radical

revision

since

that

the

term

VEGF

now

applies

to

a

molecule

that

may

or

may

not

be

either

vascular

endothelial

or

growth

specific

Subsequent

investigations

on

how

VEGF

acts

to

increase

vascular

permeability

are

also

complex

confusing

and

in

many

cases

contradictory

This

review

will

attempt

to

pull

together

much

of

the

work

on

how

VEGF

acts

to

increase

vascular

permeability

and

what

its

role

is

in

health

and

disease

What

is

VEGF

The

VEGF

family

of

proteins

consists

in

humans

of

five

separate

gene

products

termed

VEGFA

or

VEGF

VEGFB

VEGFC

VEGFD

and

placental

growth

factor

PlGF

They

are

members

of

the

superfamily

of

platelet

derived

growth

factors

cysteine

knot

proteins

that

are

characterized

by

eight

cysteines

that

form

stable

dimeric

bonds

resulting

in

dimers

in

a

head

to

tail

conformation

VEGFA

can

be

generated

by

almost

all

cells

under

hypoxic

or

other

stress

conditions

including

endothelial

cells

and

are

highly

expressed

in

tissues

undergoing

growth

or

remodelling

including

cancers

atherosclerosis

and

during

remodelling

of

the

female

reproductive

cycle

All

five

human

VEGFs

are

generally

alternatively

spliced

to

form

multiple

isoforms

with

widely

varying

properties

How

do

VEGFs

work

VEGFs

act

through

ligand

binding

to

their

cognate

VEGF

receptors

in

particular

in

the

mouse

KDR

in

the

human

and

All

three

receptors

can

be

used

by

various

isoforms

of

the

various

genes

to

signal

to

increase

vascular

permeability

angiogenesis

and

other

actions

of

VEGFs

All

the

receptors

act

by

homo

and

heterodimerization

and

activation

of

an

intracellular

tyrosine

kinase

activity

recruitment

of

domain

binding

proteins

and

subsequent

downstream

signalling

through

multiple

common

signalling

pathways

The

link

between

these

signalling

pathways

and

vascular

permeability

regulation

is

however

not

so

clear

What

is

their

effect

The

effects

of

VEGF

on

endothelial

cells

are

profound

complex

and

not

simple

to

interpret

It

is

clearly

a

potent

though

not

very

powerful

endothelial

growth

factor

It

is

a

potent

and

powerful

vascular

permeabilizing

agent

and

a

potent

vasodilator

It

increases

migration

of

endothelial

cells

new

vessel

formation

and

has

effects

on

the

pericytes

that

surround

endothelial

cells

and

on

the

matrix

metalloproteinases

secreted

by

endothelial

cells

The

net

result

of

treatment

with

VEGF

is

hypotension

when

given

centrally

leakage

of

fluid

and

protein

from

the

vasculature

to

the

tissue

when

secreted

locally

and

chaotically

increased

vascular

growth

if

given

alone

However

to

understand

the

published

evidence

on

how

VEGF

acts

to

increase

vascular

permeability

it

is

necessary

to

define

what

we

mean

by

vascular

permeability

Go

to

What

is

permeability

Permeability

coefficients

The

permeability

of

the

vasculature

is

a

property

of

the

capillary

wall

to

impede

movement

of

fluid

or

solutes

driven

by

a

physical

force

It

is

a

primary

contributor

to

the

regulation

of

exchange

of

nutrients

and

water

between

tissues

and

blood

The

other

primary

contributors

which

are

not

dependent

on

the

barrier

include

size

of

the

physical

forces

pressure

for

convective

flux

of

fluid

and

concentration

gradient

for

diffusion

of

solutes

and

surface

area

of

the

available

barrier

Thus

it

is

critical

to

identify

whether

parameters

being

measured

during

experiments

to

identify

mechanisms

through

which

VEGF

acts

are

ones

There

are

three

principal

permeability

coefficients

of

the

capillary

wall

that

can

be

used

to

indicate

the

quality

of

the

barrier

to

impede

flow

of

fluid

or

solutes

Lphydraulic

conductivity

The

fluid

flow

per

unit

pressure

gradient

driving

fluid

across

the

barrier

is

the

hydraulic

conductivity

This

parameter

can

be

measured

relatively

straightforwardly

in

vitro

and

in

vivo

by

determining

the

flow

rate

at

specific

hydrostatic

or

osmotic

pressures

across

the

vascular

wall

In

continuous

capillaries

the

pathway

is

thought

to

reside

in

the

intercellular

clefts

of

the

vascular

wall

although

a

significant

proportion

up

to

may

be

across

the

plasma

membrane

and

through

the

cell

In

fenestrated

capillaries

glomerulus

synovium

salivary

gland

the

pathway

will

be

dominated

by

the

fenestra

which

have

a

high

hydraulic

conductivity

and

surface

area

relative

to

the

intercellular

clefts

In

endothelial

cells

in

culture

the

hydraulic

conductivity

will

depend

on

confluence

and

the

basement

membrane

may

come

into

play

when

measurements

are

made

at

higher

pressures

as

the

cells

compress

the

basement

membrane

a

sealing

effect

may

be

seen

Pssolute

permeability

Solute

permeability

is

the

solute

flux

by

diffusion

per

unit

concentration

gradient

per

unit

area

The

caveat

by

diffusion

is

critical

here

as

solutes

may

also

be

carried

across

the

vessel

wall

by

convection

and

therefore

if

the

convective

flux

is

not

accounted

for

the

permeability

calculated

is

overestimated

apparent

permeability

Ps

can

be

measured

in

vivo

and

in

vitro

often

by

extrapolating

measurements

of

apparent

permeability

back

to

the

point

at

which

flow

would

cease

Oncotic

reflection

coefficient

σ

A

mixture

of

large

and

small

molecular

weight

molecules

will

exert

an

osmotic

pressure

across

a

semipermeable

membrane

proportional

to

the

size

of

the

channels

through

which

molecules

may

cross

Thus

if

the

membrane

is

permeable

to

water

but

not

solutes

then

the

solution

will

exert

its

full

osmotic

pressure

and

hence

σ

If

the

membrane

is

freely

permeable

to

all

solutes

then

no

osmotic

pressure

can

be

exerted

and

σ

The

inverse

of

σ

is

the

sieving

coefficient

Measurement

of

the

effect

of

VEGF

on

permeability

The

effect

of

VEGF

on

permeability

has

been

investigated

using

both

in

vitro

and

in

vivo

models

In

vitro

The

permeability

of

endothelial

monolayers

has

been

extensively

studied

but

using

three

principal

methodologiesmeasurement

of

hydraulic

conductivity

Lp

transendothelial

electrical

resistance

TEER

and

albumin

transport

P

alb

Lp

is

measured

as

described

above

fluid

flow

per

unit

pressure

per

unit

area

of

membrane

TEER

is

the

current

per

unit

voltage

applied

and

is

inversely

proportional

to

the

permeability

to

the

principal

charged

ions

and

Cl

P

alb

is

usually

measured

by

adding

labelled

albumin

often

fluorescent

to

one

side

of

a

monolayer

and

measuring

the

amount

on

the

well

on

the

other

side

of

the

layer

Using

these

three

assays

the

effect

of

VEGF

has

been

shown

to

increase

permeability

in

all

three

models

and

there

are

a

number

of

biochemical

studies

that

have

been

carried

out

to

investigate

the

mechanisms

described

below

In

most

but

not

all

studies

of

cultured

endothelial

cells

treatment

with

VEGF

results

in

increased

L

p

or

Ps

to

either

or

albumin

that

develops

over

a

period

of

h

peaks

and

then

a

second

wave

occurs

between

and

h

Figure

This

has

been

shown

for

venous

arterial

and

capillary

endothelial

cells

for

cells

from

the

brain

glomerulus

aorta

retina

skin

umbilical

cord

and

many

other

endothelial

cell

types

One

caveat

however

is

that

in

almost

all

in

vitro

models

permeability

is

usually

significantly

higher

than

in

in

vivo

equivalents

This

needs

to

be

borne

in

mind

when

interpreting

data

from

monolayers

in

culture

Figure

Time

course

of

VEGFinduced

permeability

Alterations

in

hydraulic

conductivity

Lp

solute

permeability

to

albumin

P

alb

or

small

solutes

such

as

fluorescein

P

NaF

ions

TEER

or

sucrose

or

sieving

coefficient

are

shown

in

vivo

bold

or

in

In

vivo

In

vivo

measurement

of

the

effects

of

VEGF

on

barrier

function

per

se

have

been

done

by

the

measurement

of

solute

permeability

to

albumin

by

hydraulic

conductivity

and

by

oncotic

reflection

coefficient

However

the

vast

majority

of

studies

that

have

attempted

to

investigate

the

effect

of

VEGF

on

permeability

have

used

indirect

measures

and

in

particular

the

accumulation

of

albumin

associated

dyes

or

labelled

albumin

into

the

tissue

These

latter

studies

can

not

be

ignored

but

must

be

interpreted

with

caution

The

most

widely

used

assay

was

that

originally

used

by

Senger

and

Dvorak

to

identify

VEGF

which

is

to

inject

Evans

blue

incubated

with

albumin

into

an

animal

and

then

treat

the

animal

usually

by

subcutaneous

injection

with

VEGF

or

inhibitors

The

tissue

is

then

photographed

and

often

excised

and

the

amount

of

Evans

blue

measured

after

extraction

There

are

a

number

of

assumptions

that

need

to

be

borne

in

mind

when

considering

these

studies

The

delivery

of

a

labelled

solute

to

a

tissue

depends

on

the

surface

area

concentration

difference

and

driving

forces

for

flux

across

the

vessel

wall

as

well

as

the

permeability

Thus

as

VEGF

is

a

vasodilator

it

results

in

perfusion

of

more

capillaries

and

therefore

the

mechanisms

through

which

it

acts

may

be

due

to

its

action

as

a

vasodilator

rather

than

its

barrier

effects

The

flux

of

a

large

molecular

weight

molecule

is

primarily

convective

under

lowpermeability

conditions

and

therefore

increased

hydrostatic

pressure

will

increase

solute

flux

and

as

again

VEGF

is

a

vasodilator

it

will

increase

hydrostatic

pressure

In

addition

any

agent

investigated

that

acts

to

increase

venous

pressure

will

increase

postcapillary

resistance

and

therefore

increase

solute

flux

Evans

blue

forms

an

equilibrium

binding

with

albumin

It

can

be

bound

to

albumin

but

its

free

dye

concentration

is

highly

dependent

on

preparation

and

both

dye

and

albumin

concentration

For

instance

at

mM

Evans

blue

there

is

free

dye

in

albumin

As

Evans

blue

is

smaller

than

albumin

it

would

be

expected

to

have

a

permeability

of

at

least

that

of

albumin

Moreover

while

it

is

in

equilibrium

in

the

plasma

and

predominantly

bound

as

soon

as

it

crosses

the

vessel

wall

it

will

again

bind

albumin

and

therefore

the

concentration

gradient

of

Evans

blue

will

remain

high

as

the

interstitial

concentration

is

effectively

zero

However

if

plasma

flows

slowly

through

a

capillary

as

it

reaches

the

end

of

the

capillary

the

plasma

concentration

will

fall

as

molecules

have

diffused

into

the

tissue

If

flow

rate

is

increased

this

concentration

will

not

fall

so

quickly

so

maintaining

a

higher

concentration

difference

across

the

vessel

wall

and

so

delivering

more

Evans

blue

this

can

explain

a

large

proportion

of

Evans

blue

extravasation

Moreover

as

permeability

increases

albumin

flux

now

also

becomes

flow

limited

so

combining

increased

flow

with

increased

permeability

will

amplify

the

solute

flux

Use

of

a

single

label

such

as

Evans

blue

does

not

distinguish

between

extravasated

dye

and

dye

bound

to

the

surface

layer

of

the

endothelium

or

still

in

the

blood

and

hence

increased

blood

flow

will

again

contribute

Go

to

How

does

VEGF

increase

permeability

Time

course

Whereas

in

vitro

VEGF

results

in

a

slow

increase

in

permeability

that

peaks

at

h

and

then

a

second

continuing

peak

occurs

that

lasts

for

between

and

days

in

vivo

there

is

a

great

discrepancy

in

published

data

Application

of

a

single

bolus

of

VEGFA

or

VEGFC

and

measurement

of

hydraulic

conductivity

and

solute

permeability

to

albumin

in

single

capillariespostcapillary

venules

results

in

a

very

rapid

and

transient

permeability

increase

that

resolves

within

min

This

is

then

followed

by

a

longer

and

more

sustained

increase

that

occurs

at

some

point

after

h

and

lasts

for

up

to

a

few

days

In

contrast

application

of

VEGF

and

measurement

of

solute

flux

by

Evans

blue

or

labelled

albumin

extravasation

results

in

a

slow

accumulation

over

the

first

hour

similar

to

that

seen

in

vitro

There

is

thus

a

biphasic

response

to

VEGFan

initial

transient

increase

lasting

just

a

few

minutes

and

a

sustained

increase

that

lasts

many

hours

The

problem

is

deciphering

the

contribution

of

the

vasodilatory

effects

of

VEGF

towards

this

sustained

effect

the

acute

effect

is

measured

in

vessels

where

flow

and

driving

forces

are

controlled

and

there

are

very

few

studies

measuring

the

chronic

response

in

the

same

way

The

following

is

therefore

an

account

of

the

signalling

pathways

where

acute

and

chronic

response

and

in

vitro

vs

in

vivo

data

are

compared

Signalling

pathways

VEGFR

activation

VEGFA

has

been

shown

to

bind

to

and

and

VEGFC

binds

to

and

The

splice

variants

of

VEGFA

that

are

proangiogenic

eg

VEGF

act

to

increase

Lp

in

vivo

acutely

Lp

in

vitro

and

albumin

extravasation

through

activation

PlGF

which

only

binds

appears

not

to

increase

Lp

acutely

VEGFC

increases

Lp

acutely

in

vivo

by

acting

on

It

therefore

appears

that

is

the

principal

signalling

pathway

for

VEGFA

and

VEGFCmediated

increased

permeability

However

the

antiangiogenic

isoforms

of

VEGF

such

as

VEGF

b

can

also

increase

Lp

acutely

and

appear

to

do

so

through

the

activation

of

not

In

endothelial

cells

in

culture

and

in

vivo

is

phosphorylated

inside

min

after

the

addition

of

VEGFA

and

this

phosphorylation

is

required

for

the

acute

and

chronic

phases

of

the

increase

in

permeability

phosphorylation

results

in

initiation

of

a

series

of

signalling

pathways

including

increased

intracellular

calcium

src

activation

and

stimulation

of

the

pathway

and

the

kinase

pathway

Increased

intracellular

Ca

A

series

of

detailed

experiments

examining

how

calcium

was

involved

in

the

VEGF

response

have

indicated

that

activation

by

VEGFA

stimulates

phospholipase

C

activation

and

production

of

DAG

and

However

it

appears

to

be

the

DAG

production

that

is

responsible

for

the

acutely

increased

calcium

and

Lp

and

this

acts

independently

of

calcium

store

release

but

through

activation

of

the

storeindependent

channels

This

has

now

been

shown

to

be

the

case

in

vitro

for

human

microvascular

endothelial

cells

HMVEC

a

microvascular

endothelial

cell

type

but

the

calcium

response

is

much

slower

than

in

vivo

and

there

is

evidence

that

in

HUVECs

the

response

to

VEGF

is

dependent

on

calcium

storemediated

channels

indicating

that

there

is

a

significant

difference

between

endothelial

cell

types

It

has

now

been

shown

that

in

HMVECs

inhibition

of

by

a

dominant

negative

overexpression

inhibits

the

calcium

response

but

also

endothelial

cell

migration

and

tube

formation

induced

by

VEGF

Src

activation

VEGF

also

activates

src

kinase

in

endothelial

cells

in

culture

and

inhibition

of

src

kinase

inhibits

Evans

blue

extravasation

induced

by

VEGF

suggesting

that

src

may

also

be

required

for

the

increase

in

permeability

However

there

is

no

clear

separation

yet

between

srcdependent

VEGFmediated

extravasation

and

VEGFmediated

permeability

and

this

remains

to

be

seen

Subcellular

localization

VEGFR

can

be

localized

to

caveolae

in

endothelial

cells

and

this

appears

to

form

part

of

a

signalling

complex

or

signalplex

that

regulates

the

downstream

signalling

There

is

a

close

association

between

the

structural

protein

caveolin

and

the

Cterminal

of

including

PLCγ

and

hence

DAG

eNOS

and

the

and

downstream

signalling

pathways

such

as

protein

kinase

Cα

MAPK

kinase

and

Moreover

it

has

recently

been

shown

that

the

lipid

raft

associated

proteins

such

as

the

storeoperated

calcium

channel

Orai

and

the

scaffolding

protein

Stim

can

also

interact

with

receptoroperated

calcium

channels

such

as

to

couple

both

receptoroperated

and

storeoperated

calcium

entry

in

microdomains

within

the

cell

thus

providing

a

molecular

mechanism

through

which

VEGF

signalling

could

depend

upon

the

components

of

the

signalplex

for

its

mechanisms

of

cation

entry

Thus

if

VEGFRs

are

activated

outside

the

caveolae

then

the

associated

activation

would

lead

to

a

Stimdependent

recruitment

to

the

lipid

raftcaveolar

domain

to

initiate

a

signalling

pathway

that

may

be

different

from

that

when

is

dissociated

from

the

caveolae

The

concept

that

a

coordinated

regulation

of

caveolar

signalling

is

central

to

the

mechanisms

and

subsequent

downstream

signalling

regulating

permeability

is

becoming

clearer

but

their

link

to

increased

vascular

permeability

has

still

not

shown

The

in

vivo

particularly

compared

with

in

vitro

signalling

suggests

that

the

components

involved

must

be

very

closely

associated

and

it

is

perhaps

no

coincidence

that

the

density

of

caveolae

in

microvessels

in

vivo

is

substantially

higher

than

in

cultured

endothelial

cells

Ultrastructural

changes

VEGF

activation

of

endothelial

cells

in

vivo

and

in

vitro

results

in

marked

ultrastructural

changes

that

follow

the

appropriate

time

course

for

the

permeability

responses

Vesicular

vacuolar

organelles

to

gaps

One

of

the

first

ultrastructural

changes

to

be

described

for

VEGF

was

the

formation

of

clusters

of

linked

vesicles

through

endothelial

cells

thought

to

link

the

luminal

and

abluminal

surfaces

These

vesicular

vacuolar

organelles

VVOs

occur

very

rapidly

after

activation

with

VEGF

within

a

few

seconds

in

vivo

The

VVO

formation

is

seen

by

electron

microscopy

and

has

been

shown

to

consist

of

fenestrated

linked

caveolar

vesicles

Perfusion

of

electron

dense

molecules

eg

ferritin

has

shown

that

VVOs

can

contain

macromolecules

and

thus

they

are

thought

to

form

pathways

for

fluid

and

solute

movement

across

the

vessel

wall

during

VEGFmediated

permeability

increase

Perfusion

of

single

vessels

in

frogs

and

rats

with

VEGF

has

also

shown

the

presence

of

transcellular

gaps

running

close

to

but

not

at

the

endothelial

cell

junction

as

well

as

intercellular

gaps

at

the

cell

junction

themselves

It

is

not

known

whether

these

processes

are

part

of

the

same

mechanism

and

that

VVOs

are

precursors

of

transcellular

gaps

or

whether

the

processes

are

entirely

separate

but

what

is

clear

is

that

large

holes

can

appear

in

endothelial

cell

barrier

in

response

to

VEGF

and

that

this

can

occur

within

a

few

seconds

after

administration

of

VEGF

Junctional

changes

As

discussed

above

addition

of

VEGF

in

endothelial

cells

in

vitro

results

in

differing

responses

from

endothelial

cells

in

vitro

and

this

also

applies

to

the

effect

on

endothelial

junctions

In

vitro

within

min

a

reduction

in

staining

of

junctional

proteins

such

as

occludin

VEcadherin

at

the

endothelial

junctions

coincident

with

the

onset

of

increased

vascular

permeability

and

prevention

of

endothelial

junctional

changes

inhibit

the

VEGF

permeability

response

These

changes

are

not

apparent

at

min

after

the

activation

of

VEGF

but

VEcadherin

is

phosphorylated

at

min

after

the

addition

of

VEGF

to

endothelial

cells

in

culture

earlier

time

points

have

not

been

investigated

However

in

vivo

changes

in

immunofluorescence

staining

of

junctional

proteins

have

been

less

well

described

making

it

difficult

to

associate

the

timedependent

changes

in

vivo

with

those

in

vitro

Ultrastructural

studies

show

that

there

is

a

loss

of

junctional

integrity

with

VEGF

activation

very

rapidly

and

there

is

loss

of

tight

and

adherens

junctions

widening

of

the

cleft

and

in

many

cases

micronsized

gaps

and

VVOs

through

endothelial

cells

and

mathematical

modelling

of

the

VEGF

response

predicts

an

opening

of

the

tight

junctional

strands

Fenestrae

VEGF

in

vivo

and

in

vitro

induces

the

formation

of

fenestrations

again

many

minutes

after

the

initial

transient

increase

in

permeability

but

consistent

with

the

development

of

a

chronic

increase

This

is

also

seen

in

endothelial

cells

in

culture

where

fenestrations

have

been

described

in

response

to

VEGF

treatment

over

h

The

link

from

the

signalplex

to

the

structural

changes

eNOS

One

of

the

earliest

demonstrated

downstream

signal

targets

of

VEGF

activation

was

endothelial

nitric

oxide

synthase

eNOS

has

been

shown

to

be

required

for

VEGFmediated

increased

solute

permeability

In

isolated

perfused

rat

coronary

venules

eNOS

inhibition

resulted

in

a

significant

decrease

in

the

transient

increase

in

solute

permeability

to

albumin

induced

by

VEGF

confirming

its

role

as

a

downstream

target

involved

in

the

permeability

pathways

Subsequent

work

has

identified

soluble

guanylyl

cyclase

as

the

NOactivated

target

involved

in

this

transient

increase

in

solute

permeability

and

extravasation

of

dye

The

sGC

activation

results

in

the

conversion

of

GTP

to

cGMP

which

activates

protein

kinase

G

PKG

which

also

appears

to

be

required

for

the

permeability

increase

The

downstream

targets

for

protein

kinase

G

PKG

have

not

yet

been

identified

Members

of

the

Ras

superfamily

of

small

GTPases

have

been

implicated

in

the

regulation

of

permeability

by

many

agents

These

are

prime

candidates

for

the

connection

between

VEGFR

signalling

and

the

ultrastructural

changes

that

occur

in

response

to

VEGF

VEGFR

as

part

of

its

caveolin

complex

is

associated

with

which

also

regulates

activity

in

membrane

ruffling

and

actin

cytoskeleton

dynamics

The

Rho

kinase

ROCK

is

also

activated

by

VEGF

and

appears

to

be

required

for

the

transient

solute

permeability

response

Activation

of

ROCK

results

in

phosphorylation

of

RhoGTPases

including

Rho

Rac

and

These

have

been

clearly

identified

as

regulators

of

cytoskeletal

element

within

the

cell

and

classically

Rac

is

required

for

lamellipodia

protrusion

Rho

promoting

assembly

of

actin

stress

fibres

and

contributing

to

filopodia

and

membrane

ruffles

In

endothelial

cells

in

culture

the

different

members

of

the

family

have

different

functions

with

Rho

resulting

in

stress

fibre

formation

Rac

and

forming

actin

bundles

and

lamellipodia

It

is

not

yet

clear

how

any

of

these

processes

may

be

involved

in

the

regulation

of

permeability

but

it

has

been

suggested

from

in

vitro

endothelial

experiments

that

the

polymerization

of

actin

can

result

in

cell

retraction

which

could

lead

to

an

increase

in

tension

on

the

intercellular

junctions

and

hence

a

force

for

disassociation

of

the

adherens

junctions

as

described

below

or

for

generation

of

actindependent

contraction

of

areas

of

endothelial

cells

to

form

transcellular

gaps

Rac

in

particular

appears

to

be

linked

to

VEGFinduced

fenestration

formation

While

these

associations

have

been

made

there

is

as

yet

no

clear

evidence

for

how

cell

cytoskeletal

changes

are

linked

to

barrier

changes

in

endothelial

cells

in

vivo

or

how

these

link

to

VEGF

VEcadherin

VEGF

activation

of

endothelial

cells

results

in

phosphorylation

and

disassembly

of

VEcadherin

one

of

the

principal

signalling

and

structural

proteins

associated

with

the

adherens

junction

that

forms

part

of

the

intercellular

cleft

VEcadherin

phosphorylation

is

initiated

within

min

but

peaks

at

min

after

activation

by

VEGF

in

endothelial

cells

in

culture

It

is

thus

able

to

be

responsible

at

least

in

part

for

the

chronic

increase

in

permeability

but

is

too

slow

to

account

for

the

acute

increase

seen

in

response

to

VEGF

The

contribution

of

VEcadherin

to

vascular

permeability

in

vivo

is

not

as

clearly

established

as

in

the

in

vitro

models

There

is

considerable

evidence

that

the

adherens

junction

itself

is

regulated

by

VEGF

during

alterations

in

vascular

permeability

but

whether

it

structurally

forms

a

barrier

to

fluid

or

protein

flux

is

a

component

of

the

extracellular

matrix

or

whether

it

is

primarily

a

signalling

function

is

still

not

clearly

demonstrated

in

vivo

For

instance

it

has

recently

been

shown

that

the

tight

junction

can

be

regulated

by

VEcadherin

signalling

Additional

data

show

that

VEGF

mediates

a

dephosphorylation

of

VEcadherin

which

disassociates

from

src

and

Csk

a

negative

regulator

of

src

thus

enabling

src

to

be

activated

This

would

fit

with

a

dual

mechanism

of

VEGFinduced

permeabilitya

transient

initial

calcium

dependence

followed

by

the

activation

of

src

FAK

and

VEcadherin

followed

by

a

cadherindependent

signalling

pathway

through

the

tight

junction

that

involves

src

activation

for

the

chronic

effect

Zona

and

occludin

The

tight

junction

barrier

has

been

closely

linked

with

contributing

to

maintenance

of

permeability

through

a

number

of

studies

linking

tight

junctional

barrier

presence

to

baseline

permeability

in

different

organs

However

recent

work

indicates

that

the

role

of

the

tight

junction

may

be

to

provide

a

narrowing

of

the

fluid

exit

pathway

regulating

junctional

fluid

velocity

and

hence

providing

a

restriction

to

both

protein

and

fluid

flux

by

maintaining

an

oncotic

reflection

coefficient

as

well

as

its

role

as

a

regulator

of

fluid

and

solute

flux

There

is

good

evidence

that

in

vitro

VEGF

activation

stimulates

phosphorylation

of

the

tight

junctional

regulator

Zona

and

it

has

been

suggested

that

this

mediates

the

VEGFmediated

permeability

increase

in

vitro

Again

however

the

phosphorylation

of

the

takes

min

in

vitro

too

long

to

account

for

the

acute

increase

in

permeability

There

are

no

data

on

the

time

course

of

VEGFmediated

phosphorylation

of

in

vivo

VEGF

induces

fenestration

in

endothelial

cells

in

vitro

within

min

of

activation

and

in

fenestrated

tissues

the

inhibition

of

VEGF

mediated

activation

of

either

by

overexpression

of

the

antiangiogenic

isoform

VEGF

b

or

by

administration

of

VEGFR

tyrosine

kinase

inhibitors

or

VEGF

blocking

antibodies

results

in

a

reduction

in

the

fenestral

density

and

reduction

in

the

hydraulic

conductivity

and

dye

extravasation

Fenestrae

in

many

vascular

beds

are

spanned

by

a

diaphragm

formed

from

the

protein

has

been

shown

to

be

both

induced

and

repressed

by

VEGF

indicating

that

this

may

also

contribute

to

the

chronic

increase

in

permeability

brought

about

by

VEGF

Again

this

is

too

slow

to

account

for

the

acute

increase

but

may

be

responsible

for

the

high

hydraulic

conductivity

maintained

in

many

fenestrated

vascular

beds

in

which

VEGF

is

constitutively

produced

eg

salivary

gland

adrenal

gland

ovary

pituitary

etc

There

is

therefore

a

dichotomy

between

in

vivo

and

in

vitro

experiments

and

a

biphasic

response

to

VEGF

However

VEGF

expression

can

occur

either

as

a

sudden

onset

of

VEGF

or

as

a

gradual

response

as

the

VEGF

concentration

increases

or

decreases

in

response

to

physiological

or

pathophysiological

stimuli

Interestingly

there

is

yet

to

be

a

clear

and

concise

assessment

of

how

such

effects

of

VEGF

on

permeability

investigated

through

multiple

arbitrary

systemsfrom

bolus

administration

of

VEGF

to

isolated

microvessels

in

vivo

injection

into

skin

or

application

to

endothelial

cells

in

cultureapply

to

the

VEGF

seen

by

blood

vessels

during

pathological

and

physiological

states

Go

to

Consequences

of

the

effects

of

VEGF

on

permeability

Physiological

effects

Reproduction

VEGF

is

highly

expressed

during

the

menstrual

cycle

in

a

manner

that

facilitates

follicle

maturation

from

primary

to

secondary

antral

follicles

and

during

corpus

luteal

development

following

ovulation

Moreover

the

growing

endometrium

induces

a

strong

VEGF

driven

vascularization

in

readiness

for

implantation

of

the

fertilized

embryo

As

the

mammary

glands

remodel

the

mammary

alveolar

epithelium

massive

angiogenesis

occurs

which

provides

sufficient

vasculature

to

generate

milk

The

permeability

of

the

capillaries

is

extremely

variable

Endometrial

and

corpus

luteal

vessels

are

relatively

tight

with

permeabilities

approaching

normal

continuous

vessels

ovarian

theca

interna

vessels

are

highly

fenestrated

and

are

thought

to

contribute

to

follicular

fluid

formation

The

mammary

gland

vasculature

is

variable

with

alveolar

epithelium

fenestrated

but

ductal

epithelium

tight

despite

both

vascular

beds

seeing

continuous

high

concentrations

of

VEGF

Wound

healing

VEGF

is

also

highly

expressed

during

wound

healing

and

the

permeability

of

vessels

growing

into

the

wound

is

raised

compared

with

normal

is

fenestrated

and

contains

large

transcellular

gaps

but

recent

studies

on

patients

undergoing

treatment

with

antiVEGF

antibodies

do

not

report

wound

healing

side

effects

Glomerular

function

In

the

glomerulus

on

the

other

hand

there

is

a

high

vascular

permeability

in

particular

the

hydraulic

conductivity

of

the

endothelium

in

response

to

substantial

VEGF

secretion

and

production

by

the

periendothelial

podocytes

VEGF

inhibition

can

lead

to

breakdown

of

glomerular

function

and

this

may

be

due

to

the

effects

of

VEGF

on

endothelial

fenestrations

combined

with

its

survival

effects

on

podocytes

themselves

A

similar

story

can

be

painted

for

other

tissues

with

highVEGF

expression

including

salivary

gland

adrenal

cortex

and

pituitary

glandhigh

secretion

rates

but

no

angiogenesis

It

therefore

appears

that

in

the

physiological

system

the

two

pathways

are

kept

separate

In

disease

In

contrast

to

VEGFdriven

physiological

angiogenesis

where

excess

fluid

formation

is

rarely

seen

or

VEGFinduced

physiologically

increased

permeability

where

angiogenesis

is

rarely

seen

in

disease

states

VEGF

overexpression

often

leads

to

excess

of

both

actions

Cancer

ascites

cerebral

oedema

and

so

on

VEGF

expression

driven

by

hypoxia

is

considered

to

be

a

principal

inducer

of

increased

vascular

supply

to

tumours

VEGF

expression

in

tumours

is

as

high

or

higher

than

seen

in

most

normal

states

where

VEGF

expression

is

substantial

eg

renal

glomerulus

or

corpus

luteum

The

VEGF

produced

drives

not

only

angiogenesis

but

also

increased

vascular

permeability

and

resulting

oedema

Inhibition

of

VEGF

in

brain

tumours

ovarian

carcinoma

as

well

as

in

benign

conditions

such

as

vestibular

Schwannomas

reduces

the

fluid

efflux

around

the

tumours

reducing

morbidity

and

in

the

case

of

hearing

loss

associated

with

vestibular

Schwannomas

restoring

normal

function

In

fact

the

contribution

of

reduced

vascular

permeability

to

the

effect

of

antiVEGF

agents

has

not

yet

been

clearly

discriminated

from

their

antiangiogenic

actions

and

as

will

be

seen

below

may

actually

be

a

more

important

effect

Blindness

diabetic

macular

edema

DME

wet

agerelated

macular

degeneration

VEGF

is

also

overexpressed

in

two

key

causes

of

blindness

in

the

western

world

neovascular

or

wet

agerelated

macular

degeneration

wAMD

and

diabetic

retinopathy

These

two

conditions

have

very

different

fundamental

causes

wAMD

results

from

a

buildup

of

lipid

deposits

in

the

retinal

pigmented

epithelial

cells

of

the

retina

and

progressive

breakdown

of

the

underlying

Bruchs

membrane

Increased

VEGF

expression

results

in

a

neovascularization

from

the

underlying

choroidal

vasculature

into

the

retina

Whereas

retinal

blood

vessels

are

part

of

the

blood

brain

barrier

and

have

extremely

low

hydraulic

conductivity

the

choroidal

vasculature

has

a

very

high

permeability

partly

due

to

the

presence

of

fenestrations

in

the

endothelial

cells

which

may

be

maintained

by

constitutive

VEGF

expression

from

the

basolateral

part

of

the

retinal

pigmented

epithelial

cells

When

these

choroidal

vessels

invade

into

the

retina

the

increased

vascular

permeability

results

in

subretinal

oedema

which

results

in

a

rapid

loss

of

sight

over

a

few

weeks

to

months

Intraocular

injection

with

either

bevacizumab

or

the

variable

fragment

of

a

similar

VEGF

antibody

termed

ranibizumab

can

initially

reverse

the

vision

loss

and

then

maintain

vision

This

is

associated

with

a

resolution

of

the

subretinal

oedema

indicating

that

it

is

the

antipermeability

properties

of

VEGF

therapy

that

is

effective

at

least

initially

in

this

disease

The

second

leading

cause

of

blindness

in

the

developed

world

is

diabetic

retinopathy

This

has

quite

a

different

aetiology

from

AMD

but

is

also

VEGF

dependent

The

endothelial

dysfunction

associated

with

diabetes

occurs

systemically

resulting

in

loss

of

endothelial

cells

from

the

retina

and

localized

areas

of

ischaemia

This

results

in

hypoxic

upregulation

of

VEGF

which

acts

on

the

vasculature

that

is

still

perfused

in

the

periphery

of

the

ischaemic

area

to

produce

a

proliferation

and

increased

leakage

of

vessels

resulting

in

haemorrhage

oedema

and

neovascularization

intraretinally

causing

disrupted

vision

AntiVEGF

therapies

are

being

tried

in

diabetic

retinopathy

and

particularly

in

the

form

of

retinopathy

that

results

in

demonstrable

fluid

formation

in

the

retinadiabetic

macular

oedema

and

phase

II

trials

have

been

promising

for

ranibizumab

Proliferative

diabetic

retinopathy

is

a

more

complicated

initial

target

because

VEGF

inhibition

results

in

endothelial

cell

regression

and

vascular

loss

in

animal

models

which

is

a

potential

hazard

for

diabetic

patients

where

endothelial

loss

is

an

underlying

cause

of

the

disease

Other

conditions

VEGFmediated

permeability

has

also

been

implicated

in

renal

failure

arthritis

psoriasis

and

coronary

disease

but

their

consideration

is

beyond

the

scope

of

this

review

Go

to

Conclusion

Importance

of

VEGF

antagonists

in

clinical

situations

The

increasingly

widespread

use

of

VEGF

antagonists

in

clinical

conditions

in

particular

in

cancer

and

eye

disease

has

focused

attention

on

how

these

antagonists

exert

their

effects

It

has

been

presumed

that

as

VEGF

is

a

potent

angiogenic

agent

the

effects

are

mediated

through

this

mechanism

However

it

is

increasingly

becoming

the

case

that

antiVEGF

agents

are

acting

on

the

symptoms

of

the

disease

by

blocking

the

VEGFmediated

increase

in

permeability

rather

than

any

direct

effect

on

angiogenesis

In

AMD

DME

gliomas

ovarian

carcinoma

and

lung

cancer

this

is

probably

the

principal

mechanism

of

action

and

it

is

still

unknown

Synthesis

of

current

knowledge

to

summarize

best

case

VEGFmediated

permeability

experimentally

appears

to

occur

in

a

biphasic

manner

both

in

vitro

and

in

vivo

although

these

two

time

courses

do

not

compare

The

molecular

mechanisms

underlying

this

increase

in

permeability

are

still

not

clear

not

least

due

to

the

difficulty

in

reconciling

different

methodologies

of

permeability

measurement

with

the

respective

changes

in

molecular

interactions

We

still

do

not

know

if

src

activation

for

instance

in

vivo

occurs

within

a

time

frame

of

the

acute

increase

in

permeability

or

is

only

required

for

the

chronic

response

We

do

not

know

whether

the

ultrastructural

features

seen

initially

in

vivo

VVOs

gaps

are

consistent

with

altered

junctional

properties

measured

in

the

long

term

It

therefore

appears

to

be

critical

for

the

effect

of

VEGF

on

permeability

to

be

examined

with

care

to

the

timing

of

the

response

when

investigating

the

molecular

mechanisms

and

not

to

overinterpret

in

vitro

results

to

the

in

vivo

situation

Equally

there

is

a

danger

in

assuming

that

abrupt

VEGF

application

to

a

tissue

represents

a

physiological

or

even

pathological

state

and

that

VEGF

expression

in

vivo

occurs

in

a

more

diffuse

and

gradual

time

scale

which

needs

to

be

reproduced

experimentally

to

investigate

the

mechanisms

underlying

the

permeability

changes

that

are

clearly

important

clinically

Future

directions

The

synthesis

of

existing

molecular

changes

is

becoming

more

and

more

difficult

to

reconcile

with

the

generation

of

results

that

are

not

comparable

across

systems

Endothelial

cells

from

different

vascular

beds

are

not

the

same

and

cells

in

identical

vascular

beds

can

respond

differently

according

to

their

environmentoxygen

tension

shear

stress

circulating

leucocyte

interactions

etc

Finally

the

transport

of

molecules

across

the

endothelial

wall

is

dependent

on

multiple

factors

in

addition

to

the

barrier

properties

of

that

wall

It

will

therefore

be

critical

in

the

future

to

be

able

to

build

a

molecular

picture

of

VEGFmediated

permeability

that

is

specific

responses

additive

or

synergistic

Perhaps

the

key

area

required

for

investigation

is

the

dissection

of

the

signalplex

involving

VEGF

receptors

ion

channels

and

scaffolding

proteins

that

regulate

the

signalling

pathways

downstream

for

while

this

is

being

resolved

for

angiogenesis

it

is

some

way

off

for

permeability

Conflict

of

interest

declared

Go

to

Funding

This

was

supported

by

the

British

Heart

Foundation

Funding

to

pay

the

open

access

publication

charge

was

provided

by

the

British

Heart

Foundation

Go

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