Vascular Hyperpermeability, Angiogenesis, and Stroma...

Vascular Hyperpermeability, Angiogenesis,and Stroma Generation

Janice A. Nagy, Ann M. Dvorak, and Harold F. Dvorak

Center for Vascular Biology Research and the Department of Pathology, Beth Israel DeaconessMedical Center and Harvard Medical School, Boston, Massachusetts 02115

Correspondence: [email protected]

It has been known for more than half a century that the tumor microvasculature ishyperpermeable to plasma proteins. However, the identity of the leaky vessels and the con-sequences of vascular hyperpermeability have received little attention. This article placestumor vascular hyperpermeability in a broader context, relating it to (1) the low-level“basal” permeabilityof the normal vasculature; (2) the “acute,” short-term hyperpermeabilityinduced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A)and other vascular permeabilizing agents; and (3) the “chronic” hyperpermeability associ-ated with longer-term exposure to agents such as VPF/VEGF-A that accompanies manytypes of pathological angiogenesis. Leakage of plasma protein-rich fluids is importantbecause it activates the clotting system, depositing an extravascular fibrin gel provisionalmatrix that serves as the first step in stroma generation.

All cells require a continuing supply ofnutrients and a means of clearing waste

products. Vertebrates have solved this problemby developing a vascular system that projectsinto all organs and most tissues. The vertebratevasculature is often described as “closed,” but itis sufficiently “open” (i.e., “permeable”) toallow the ready exchange of small moleculenutrients and waste products between the bloodand tissues. Plasma proteins also need to crossthe vascular barrier, at least in small amounts.Albumin, for example, transports fatty acidsand some vitamins and immunoglobulins thatare required for host defense. Vascular perme-ability, then, is essential for the health of normaltissues and is also an important feature of many

disease states (e.g., cancer, in which it is greatlyincreased).

WHAT IS VASCULAR PERMEABILITYAND HOW SHOULD IT BE MEASURED?

Surprisingly, there is considerable disagreementas to the meaning of the term vascular perme-ability and the methods by which it shouldbe measured (Bates and Harper 2003). Perme-ability is a complicated process that, howeverdefined, is affected by many different variables.These include the intrinsic properties of thedifferent types of microvessels involved (i.e.,capillaries, venules, “mother” vessels); the size,shape, and charge of extravasating molecules

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(water and solutes); the anatomic pathways(trans- vs. intercellular [paracellular]) throughwhich molecules cross the endothelial cell bar-rier; the time course over which permeabilityis measured; and the vascular beds that arebeing investigated. A related issue, that of thepassage of inflammatory cells across the micro-vasculature, is reviewed elsewhere (Feng et al.1998; Kamei and Carman 2010).

The Physiologists’ View of VascularPermeability

Over the last half century, eminent physiologistshave investigated the mechanisms by whichplasma components cross the vascular barrier(Pappenheimer 1953; Rippe and Haraldsson1994; Michel and Curry 1999; Guyton andHall 2000; Bates and Harper 2003; Curry2005). They recognized that capillaries werethe vascular segment involved in molecularexchange in normal tissues and that water,gases, and other small molecules crossed capil-lary endothelial cells freely, whereas the passageof larger molecules such as plasma proteinswas tightly restricted. Physiologists have oftenregarded capillary endothelium as a passive bar-rier, likening it to a thin, cellophane-like mem-brane that is punctuated by large numbers ofsmall pores and smaller numbers of large pores.They postulated that the numerous small poresallowed the ready passage of small moleculesand that the smaller number of large poresallowed limited extravasation of plasma pro-teins. With these assumptions in mind theydeveloped elegant methods for investigatingthe flux of water and of plasma solutes acrossindividual cannulated microvessels and for cal-culating the important parameters that governpermeability: (1) vascular surface area availablefor molecular exchange; (2) thickness of the ves-sel wall; (3) hydraulic conductivity, a measure ofcapillary permeability to water; (4) reflectioncoefficient, a measure of solvent drag in relationto that of water; (5) diffusion; and (6) transvas-cular pressure gradients. For a given capillary,diffusion is the most important of these forthe exchange of small molecules and dependson differences in solute concentration across

vascular endothelium. Diffusion is importantlyaffected by the molecular properties of solute;for example, despite a substantial concentrationgradient, diffusion of a large molecule such asalbumin is �1000-fold less than that of water(Pappenheimer 1953; Guyton and Hall 2000).Therefore, for large molecules such as plasmaproteins, flux is largely determined by trans-vascular hydrostatic and osmotic pressure dif-ferences, by hydraulic conductivity, and by thereflection coefficient characteristic of a givensolute.

Permeability as Understood byVascular Biologists

Vascular biologists have used the term vascularpermeability in a different sense. Rather thanbeing concerned with the filtration rate acrossa single cannulated microvessel, they havesought to measure the net amount of a solute,often a macromolecule such as plasma albumin,that has crossed a vascular bed and accumulatedin the interstitium in response to a vascularpermeabilizing agent or at a site of pathologicalangiogenesis. Generally, the vessels presentare a mixture of several different types, andthe measurements made combine together allof the factors that regulate extravasation,including both intrinsic properties of the bloodvessels as well as extrinsic properties such asblood flow and the molecular properties of thesolute.

To obtain this type of information, we intro-duced the Miles assay (Miles and Miles 1952)into tumor biology many years ago (Dvoraket al. 1979b). Typically, a dye such as Evansblue that binds noncovalently to albumin isinjected intravenously and its accumulation ismeasured in a skin test site, a tumor, etc. Per-meability is then defined as the amount ofalbumin-dye complex that is present at sometime, for example, 30 min, after Evans blueinjection. The intensity of local bluing observedvisually provides sufficient information forsome purposes. For example, we originallyidentified VPF/VEGF-A as a vascular permea-bilizing agent using this assay in guinea pigskin (Dvorak et al. 1979b) and used local bluing

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to evaluate column fractions in the originalpurification of VPF/VEGF-A (Senger et al.1983). Quantitative measurements can also bemade by extracting the dye from tissues andmeasuring it spectrophotometrically (Nagyet al. 2008b). A limitation of the Miles assay isthat it does not distinguish between dye thathas extravasated from that which is presentwithin the vasculature. However, serious errordoes not result if (1) comparisons of permeabil-ity are made at similar test sites in the same ani-mal and (2) intravascular volumes are smallrelative to the amounts of dye that have leaked.Another limitation is that the Miles assay meas-ures net accumulation of dye-albumin complexover a period of time, and return of extravasatedmolecules to the circulation, either by way ofcapillaries or lymphatics, is not considered.Despite these limitations the Miles assay iswidely used and has provided much usefulinformation.

However, in tumors and in other examplesof pathological angiogenesis, the vasculatureundergoes dramatic structural and functionalchanges and is not comparable to that of adja-cent normal control tissues. In such instancesit is desirable to measure both the content ofmacromolecular tracer within the vasculatureand the dye that has extravasated. Flushingthe vasculature to remove intravascular dyehas been used but can result in error asperfusion may be incomplete and/or mayextract extravasated dye. A better approach isto use a double tracer method (e.g., combining131I- and 125I-albumin or Evans blue dye and125I-albumin [Nagy et al. 2008b]). The firsttracer is used to measure total albumin accumu-lation in a tumor over 30 min (i.e., albuminboth leaked and intravascular). The secondtracer, injected 5 min before tumor harvest,measures only albumin that is present withinthe blood vasculature, because extravascularleakage is negligible over that short time period.By subtracting values of the second tracer fromthat of the first, one obtains quantitative meas-ures of (1) intravascular plasma volume, anexcellent, unbiased measure of the extent ofnew blood vessel formation and (2) the volumeof leaked plasma.

IN VITRO PERMEABILITY ASSAYS

The assays described above measure permeabil-ity in living animals. However, many investiga-tors have used in vitro assays to measuremolecular flux across lawns of confluent endo-thelial cells cultured on membrane filters intranswell chambers. These assays are appealingin that they are easy to perform and avoid thecomplexities of experiments in living animals.However, they suffer from some important lim-itations. Confluent cultured endothelial cellmonolayers, whether isolated from large orsmall vessels, are generally leakier than normalblood vessels in vivo for several reasons: (1)absence of normal basement membrane; (2)lack of pericytes or smooth muscle cells; and(3) relative loss of cytoplasmic vesicles andvacuoles, structures that are numerous inendothelial cells in vivo and that provide animportant pathway for macromolecular extra-vasation (Palade 1988; Feng et al. 1996, 1999b).Cultured endothelial cell monolayers, therefore,most closely resemble “mother” vessels, the firstnew type of blood vessel to develop in tumors,wounds, etc.; therefore, they perhaps best modelthe vascular hyperpermeability associated withpathological angiogenesis. Finally, the kineticsof leakage in response to agents such as VEGF-Aoften differs markedly from that in vivo.In vivo, leakage in response to a single exposureto VEGF-A begins within a minute and islargely complete by �20–30 min. In contrast,increased permeability often develops moreslowly in cultured endothelium and may con-tinue over a period of hours, suggesting thatthe permeability observed results from a longer-term loosening of intercellular connections.

VASCULAR PERMEABILITY OCCURSIN THREE DIFFERENT SETTINGS

Low levels of vascular permeability to plasmaproteins are essential for the health of normaltissues and these levels may vary considerablyin different organs and tissues and in responseto different physiological stimuli (e.g., localtemperature, exercise). However, it is importantto distinguish between the basal permeability

Permeability, Angiogenesis, and Stroma Generation

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levels of normal tissues and the greatly increasedlevels of plasma protein extravasation that occurin pathology. These hyperpermeable states maybe acute or chronic and differ from each otherand from basal levels of permeability withrespect to the vessels that leak, the compositionof the extravasate, and the anatomic pathwaysthat solutes follow in crossing vascular endothe-lium. Each of the three types of permeability isnow discussed in turn.

Basal Vascular Permeability

Molecular exchange in normal tissues takesplace primarily in capillaries. The moleculesexchanged consist largely of water, gases, andsmall solutes such as salts and sugars. Exchangeis driven largely by diffusion and only smallamounts of plasma proteins extravasate. Basalvascular permeability (BVP) varies consider-ably in different normal tissues and is modu-lated by changes in blood flow, opening ofpreviously closed vessels, etc.

How do plasma and solutes of different sizetraverse capillaries? No “pores” of the type thathad been postulated by physiologists have beenfound in normal capillary endothelium. None-theless, it was not difficult to explain the trans-port pathways followed by small molecules.Water, lipophilic solutes (e.g., gases such as O2

and CO2), and other small solutes are able topass between endothelial cells or throughthem by receptor-mediated transcytosis, andalso cross through the endothelial fenestraefound in some capillaries. Small proteins suchas horseradish peroxidase (MW 44 kDa) canpass through interendothelial cell junctions,but do so at rates that are much slower than theiroverall entry into tissues (Karnovsky 1967;Dvorak 2007a).

Plasma proteins such as albumin (MW 69kDa) are too large to pass between intact endo-thelial cells. A solution to their extravasationwas offered by George Palade who observedthat capillary endothelium contained largenumbers of small (50–100 nm diameter),uncoated vesicles, now commonly referred toas caveolae (Palade 1988). The majority ofcaveolae are found connected to the luminal

and abluminal plasma membranes by stomatathat are generally closed by thin diaphragms(Fig. 1). Little is known about the compositionof these diaphragms other than that they con-tain a unique protein, PV-1, and likely sulfatedproteoglycans (Stan 2007). Palade postulatedthat caveolae shuttled across capillary endo-thelium carrying cargoes of plasma fluid andproteins and this was subsequently demon-strated experimentally with tracers (reviewedin Dvorak 2007a). Thus, it seemed that the largepores postulated by physiologists were notpores at all but shuttling caveolae, and that trans-port of large molecules across capillaries was any-thing but passive. This concept has recently beenchallenged by the finding that caveolin-12/2

mice, which lack caveolae, actually clear plasmaalbumin more rapidly than their wild-type coun-terparts (Schubert et al. 2002; Rosengren et al.2006). Further work will be required to explain

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Figure 1. Basal vascular permeability (BVP). (Upper)Electron micrograph illustrating a typical capillaryendothelial cell with numerous caveolae. Many ofthese are connected to the luminal or abluminalplasma membranes (arrows). Scale bar, 100 nm.(Lower) Schematic diagram illustrating pathways bywhich molecules can cross the capillary barrier.(1) Interendothelial cell cleft, (2) caveolae that shuttleacross the capillary endothelium or form a chainof vesicles connecting the lumen and albumen.(BL) Basal lamina, (EC) endothelial cell, (BVP) basalvascular permeability. (Figure adapted from Nagyet al. 2008a; reproduced, with permission, fromSpringer # 2008.)

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this conundrum, but it should be noted thatcapillary endothelium in caveolin-12/2 miceretain 10%–20% of the uncoated caveolae-sizedvesicles present in wild-type mice, though thesevesicles do not stain for caveolin-1 (Chang et al.2009a).

Acute Vascular Hyperpermeability

A rapid increase in vascular permeability occurswhen the microvasculature is exposed acutelyto vascular permeabilizing factors such asVPF/VEGF-A, histamine, serotonin, PAF (pla-telet activating factor), etc. Some of these(e.g., histamine, serotonin, VPF/VEGF-A) arestored in tissue mast cells (Boesiger et al. 1998;Galli 2000) and so may be released by agentsthat cause mast cell degranulation (e.g., expo-sure to allergens, insect bites). Single exposureto any of these permeability factors results ina rapid but self-limited (complete by 20–30 min) influx of plasma into the tissues. Boththe quantity of extravasated fluid and its com-position differ strikingly from that in BVP.The fluid passing from the circulation into nor-mal tissues under basal conditions is a plasmafiltrate (i.e., a fluid consisting largely of waterand small solutes but containing only smallamounts of plasma proteins). However, thefluid that extravasates in acute vascular hyper-permeability (AVH) is rich in plasma proteinsapproaching the levels found in plasma (i.e.,an exudate). The consequences of plasma fibri-nogen leakage and resulting extravascularclotting are discussed below under chronicvascular hyperpermeability (CVH).

AVH also differs from BVP in that vascularleakage takes place not from capillaries butfrom postcapillary venules (Majno et al. 1961,1969). Whereas capillaries have a flattenedendothelium, venules are lined by taller, cuboi-dal endothelium. Majno proposed that hista-mine and other vascular permeabilizing agentsinduced endothelial cells to contract and pullapart to form intercellular (paracellular) gapsof sufficient size to permit plasma proteinextravasation.

More recently, a structure was discoveredin venular endothelium, the vesiculo–vacuolar

organelle (VVO), which offers an alternative,transendothelial cell route for plasma extravasa-tion (Kohn et al. 1992; Dvorak et al. 1996; Fenget al. 1996, 1997, 2000). VVOs are grape-likeclusters comprised of hundreds of uncoated,cytoplasmic vesicles and vacuoles that togetherform an organelle that traverses venular endo-thelial cytoplasm from lumen to ablumen(Figs. 2A,B and 3A). VVOs also open to theinterendothelial cell cleft, either below or abovesites of specific junctional (adherens or tightjunctions) attachment (Fig. 3A). The vesiclesand vacuoles comprising VVOs vary in sizefrom caveolae-sized vesicles to vacuoles withvolumes as much as 10-fold larger (Feng et al.1999a). These vesicles and vacuoles are linkedto each other and to the plasma membrane bystomata that are normally closed by thin dia-phragms that appear similar to those found incaveolae. We conjectured some years ago thatVVOs formed from the linking together of indi-vidual caveolae, and that larger vesicles andvacuoles resulted from the fusion of two ormore caveolae-sized vesicles (Feng et al.1999a). Evidence for this was that the smallestVVO vesicles were indistinguishable structurallyfrom caveolae; further, larger VVO vesicles andvacuoles have volumes that do not fall on a con-tinuum but have a modal distribution (i.e.,occur as multiples of the volume of caveolae,the unit vesicle, up to 10 mers). However,more recent data render this conclusion lesslikely. As in capillary endothelium, caveolin-1staining, 50–100 nm diameter, uncoatedvesicles were greatly reduced in the venularendothelium of caveolin-12/2 mice, butVVOs were not diminished in either numberor composition (Chang et al. 2009a). It wouldseem, therefore, that VVOs are comprised of apopulation of uncoated vesicles and vacuolesthat is distinct from caveolae, though inwild-type mice many stain with anti-caveolin-1antibodies. Whether VVOs somehow take theplace of caveolae in caveolin-12/2 mice andthereby contribute to the increased perme-ability observed in these animals needs to beinvestigated. A further conundrum is thatalthough VVOs appear structurally normal incaveolin-12/2 mice, AVH is greatly reduced



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Figure 2. Transmission electron micrographs of normal venules (A,B) and hyperpermeable microvessels (C–G).(A) Typical normal venules lined by cuboidal endothelium. The cytoplasm contains prominent VVOs and isenveloped by a complete coating of pericytes (P). (R) Red blood cell. (B) Ultrathin section from a set of 36 con-secutive serial sections demonstrates three sets of VVO vesicles (x,y,z) that could be traced from the vascularlumen to albumen. (C) Mother vessels (MVs) are characterized by greatly enlarged size, extensive endothelialcell thinning, striking reduction in VVOs and other cytoplasmic vesicles, prominent nuclei that project intothe vascular lumen, mitotic figures (arrows), and pericyte (P) detachment. MV lumens are packed with redblood cells, indicating extensive plasma extravasation. (Inset) The normal venule depicted in A is reproducedin C at the same magnification as the MVs to illustrate relative size differences. (D–G) Leakage of ferritin tracer(black dots, some encircled) through MV VVOs (D,E) and fenestrae (F, arrows), and through VVOs of a normalvenule in response to VPF/VEGF-A (G). Scale bars, 1 mm (A); 50 nm (B,D,G); 5 mm (C); 200 nm (E,F). (A, C,and E adapted from Nagy et al. 2006; reproduced, with permission, from Nature Publishing # 2006. D adaptedfrom Kohn et al. 1992; reproduced, with permission, from Nature Publishing # 1992. B and G adapted fromFeng et al. 1996; reproduced, with permission, from The Rockefeller University Press # 1996. F adaptedfrom Feng et al. 1999a; reproduced, with permission, from John Wiley and Sons # 1999.)

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(Chang et al. 2009a). This raises the possibilitythat caveolin-1, although not necessary forVVO formation, is necessary for their properfunction. Further evidence to this effect is thatangiogenesis and CVH are also greatly reducedin caveolin-12/2 mice (see below).

Although very little is known about themechanisms of VVO function, it is clear that,with exposure to histamine, VPF/VEGF-A,etc., large macromolecular tracers such as ferri-tin pass through a sequence of interconnectedVVO vesicles and vacuoles from the vascularlumen to the ablumen (Figs. 2D,E and 3)

(Feng et al. 1996, 1997). It seems that vascularpermeability-inducing agents cause the dia-phragms interconnecting vesicles and vacuolesto open, thereby providing a transcellular path-way for plasma and plasma protein extravasa-tion. The underlying mechanism could bemechanical, as was Majno’s endothelial cell con-traction model (Majno et al. 1969). If so, endo-thelial cell–cell junctions would serve as intact“anchors” and the actin–myosin contractionsinduced by permeability factors would pullapart the diaphragms linking adjacent VVOvesicles and vacuoles, resulting in a transcellular

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Figure 3. Pathways of acute and chronic vascular hyperpermeability. (A) Schematic diagram of a normal venulecomprised of cuboidal endothelium with prominent VVOs and closed interendothelial cell junctions. Note thatsome VVO vesicles attach to the intercellular cleft below the tight and adherens junctions. 1 and 2 indicate poten-tial pathways for transcellular (VVO) and intercellular (paracellular) plasma extravasation, respectively. Basallamina (BL) is intact and the endothelium is covered by pericytes. (B) AVH. Acute exposure to VEGF-A causesVVOs to open, allowing transcellular passage of plasma contents, possibly by mechanical pulling apart of sto-matal diaphragms. Others have suggested that fluid extravasation takes place through an opening of intercellularjunctions. (C) CVH. Prolonged VEGF-A stimulation causes venules to transform into MVs. Plasma may extrav-asate either through residual VVO vesicles (4) or through fenestrae (5). (Figure adapted from Nagy et al. 2008a;reproduced, with permission, from Springer # 2008.)



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rather than a paracellular route of plasmaextravasation (Fig. 3B). Determining whethersolutes cross venular endothelium by inter- ortranscellular (by VVOs) pathways is difficultbecause of the tortuosity of the interendothelialcell interface and the proximity of VVOs to thatinterface (Fig. 3A,B). Electron microscopicthree-dimensional reconstructions have dem-onstrated that many of the openings inducedin venular endothelium by permeability fac-tors are in fact transendothelial cell pores(Fig. 4) (Neal and Michel 1995; Feng et al.1997, 1999a). To demonstrate for sure that apore is paracellular it would be necessary tolocalize specialized junction proteins such asVE-cadherin in the plasma membrane border-ing the pore (e.g., by electron microscopicimmunocytochemistry), an enormously diffi-cult feat.

Chronic Vascular Hyperpermeability

Whereas acute exposure to VEGF-A causes animmediate but self-limited hyperpermeabilityof normal venules, chronic exposure inducesprofound changes in venular structure andfunction. Together, these result in the chronichyperpermeability of pathological angiogenesisas found in tumors (Fig. 5A–C), healingwounds, and chronic inflammatory diseasessuch as rheumatoid arthritis, psoriasis, cellularimmunity, etc. (Nagy et al. 1995; Dvorak 2003,2007b). As in AVH, the fluid that extravasatesis an exudate whose protein content approachesthat of plasma.

In contrast to the capillaries and venules in-volved in BVP and AVH, the blood vessels thatleak in CVH do not correspond to any typeof normal blood vessel. Instead, they consist

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Figure 4. Computer-generated three-dimensional reconstruction of serial electron microscope sections of acolloidal carbon-filled pore induced in rat cremaster venule by VPF/VEGF-A. Panels represent successiverotations toward the viewer around a horizontal axis at the angles indicated. 08 (not shown) corresponds to avascular cross section taken at right angles to the direction of blood flow and 908 corresponds to a view lookingdown on the luminal surface; views �2408 illustrate the abluminal surface. Endothelial cells are labeled 1(yellow) and 2 (orange-brown). Tracer colloidal carbon (black) fills the pores in the left panels. In the middlepanels, carbon was deleted from the reconstruction to facilitate visualization into the pore interior, which isseen to pass transcellularly through the cytoplasm of both endothelial cells, revealing the underlying turquoisebackground. A small portion of the large pore, which passes through cell 1 (yellow), does not traverse cell 2, asevidenced by the underlying orange-brown background (middle panel, 308 and 908). Also, a second smallerpore, as well as small portions of the larger pore, penetrate cell 2 without passing through cell 1 (recognizedby yellow instead of turquoise background in panels �2408). Right panels depict endothelial cells 1 and 2separately, again with carbon deleted. (L) Lumen. (Figure adapted from Feng et al. 1997; reproduced, with per-mission, from Wiley-Blackwell # 1997.)

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largely of “mother” vessels (MVs), a well-defined subset of the several types of new andhighly abnormal blood vessels that form intumors and other examples of pathologicalangiogenesis (Dvorak 2003; Nagy et al. 2007,2010). MVs begin to develop within hours afterinjection of tumor cells into mice, and similarMVs form in healing wounds and in nude

mouse tissues injected with an adenovirusexpressing VEGF-A164 (Ad-VEGF-A164).

MVs are greatly enlarged sinusoids that arisefrom preexisting normal venules by a processthat initially involves vascular basementmembrane (VBM) degradation, followed bypericyte detachment, extensive endothelial cellthinning, and a substantial increase in lumen

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Figure 5. Chronic vascular hyperpermeability and tumor stroma generation. (A–C) Blood vessels (arrows) sup-plying line 10 guinea pig tumors are hyperpermeable to circulating macromolecular fluoresceinated dextran, out-lining tumor nodules. (D,E) Line 10 tumor cells 48 h after transplant into the subcutaneous space of syngeneicstrain 2 guinea pigs. Fibrin forms a water-trapping gel (F) that serves as a provisional stroma that separates tumorcells into discrete islands and that provides a favorable matrix for fibroblast (white arrows) and endothelial cellmigration. (F,G) Immunohistochemical demonstration of fibrin (brown staining) in guinea pig line 1 and humancolorectal adenocarcinoma, respectively. (H ) Fibroblasts and blood vessels (black arrows) invade line 1 tumorfibrin gel, replacing it with fibrous connective tissue. (I) Fibroblasts (arrows) migrate through fibrin gel (F) inculture. (J ) Implanted fibrin gel (F) in subcutaneous space is replaced by ingrowing fibroblasts and new bloodvessels, creating granulation-like vascular connective tissue. Scale bars, 25 mm (B,I), 50 mm (A,C,D,H ), and100 mm (E,F,J ). (A–F and H–J adapted from Dvorak 2003; reproduced, with permission, from the author. Gadapted from Dvorak et al. 1988; reproduced, with permission, from the American Journal of Pathology# 1988.)



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size (Figs. 2C and 3C). VBMs are noncompliant(nonelastic) structures that limit microvesselexpansion to �30% (Swayne et al. 1989).Therefore, to acquire a cross-sectional areathat is four- to fivefold greater than that of thevenules from which they arise, VBM must bedegraded. Recent data have shown that twoimportant VBM components, collagen IV andlaminin b1 chain, are degraded in developingMVs in both tumors and at Ad-VEGF-A164

injection sites (Chang et al. 2009b). Surpris-ingly, matrix metalloproteases did not seem tobe involved. Instead, VBM degradation resultedfrom an altered balance between cathepsins(B . S . L) and their endogenous, high-affinity competitive inhibitors, a family of small(11–13 kDa) proteins called cysteine proteaseinhibitors (CPIs). Pericytes of venules thatwere evolving into MVs were identified as thesource of increased cathepsin expression andactivity (Chang et al. 2009b). In parallel, expres-sion of several CPIs, including stefin A (cystatinA) and cystatins B and C, was strikingly reducedin developing MVs. Taken together, these dataindicate that MV formation results from anupsetting of the normal cathepsin/CPI balancethat regulates vascular size. Increased pericytecathepsins, relieved of CPI inhibition, degradeVBM, causing pericytes to detach, resulting inMVs that are lined only by endothelial cells.

Vascular enlargement, the final step in MVformation, is likely driven by centripetal vascu-lar pressure on endothelial cells that have lostthe constraints normally imposed by VBMand pericytes. To accommodate this increasein luminal size, MV endothelial cells thin andexpand to cover a greatly enlarged surface area,a process that requires substantial increases inplasma membrane. Although some membranemay derive from new synthesis, a significantamount comes from the transfer to the cell sur-face of membrane that is normally stored in thevenular endothelial cell cytoplasm in the formof VVOs. Thus, in addition to providing a path-way for transcellular extravasation of plasmafrom normal venules in response to acute expo-sure to vascular permeabilizing factors (Dvoraket al. 1996; Feng et al. 1996, 1997, 2000), VVOshave a second function, that of contributing

their membrane to the cell surface to providethe additional plasma membrane required tocover an enlarged surface area (Nagy et al.2006, 2007). As noted above, CVH and MV for-mation are strikingly deficient in caveolin-12/2

mice (Chang et al. 2009a).MVs are transient structures that do not

persist long before evolving into several differ-ent types of “daughter” vessels. Only one ofthese, glomeruloid microvascular proliferations(GMPs), is hyperpermeable. GMPs are poorlyorganized vascular structures that are so namedbecause of their macroscopic resemblance torenal glomeruli (Pettersson et al. 2000; Sund-berg et al. 2001). They are present in a varietyof human tumors, particularly those such asglioblastoma multiforme that make unusuallylarge amounts of VPF/VEGF-A; however, theyare also found in breast and other carcinomaswhere their presence has been associated withan unfavorable prognosis (Straume et al. 2002;Goffin et al. 2003). The mechanisms by whichGMPs form from MVs have been described else-where (Sundberg et al. 2001).

Pathways of Macromolecular Extravasationfrom MVs and GMPs

There continues to be considerable debate as tothe pathways by which macromolecules crosshyperpermeable tumor blood vessels (Robertsand Palade 1995; McDonald et al. 1999; Fenget al. 2000). Our data indicate that macro-molecules such as ferritin or peroxidase extra-vasate from MVs and GMPs largely by atranscellular route (Figs. 2D and 3C) (Nagyet al. 2006, 2008a). MVs and GMPs containmany fewer and less complex VVOs than nor-mal venular endothelium. However, the pathlength for molecular extravasation is also greatlyshortened, such that tracers need to passthrough only a few, often only one or two,vesicles or vacuoles to reach the ablumen. Mac-romolecules also extravasate through fenestraethat develop in both MVs and GMPs (Figs. 2Fand 3C). Pores of the type that have beendescribed in AVH (Fig. 4) have also been foundin the endothelial cells of blood vessels supply-ing tumors (Roberts and Palade 1995), and as in

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AVH, there is debate as to whether these poresare trans- or paracellular.

VASCULAR HYPERPERMEABILITYAND STROMA FORMATION

Vascular hyperpermeability results not only inthe leakage of albumin and immunoglobulins,but also of fibrinogen and other clotting pro-teins such as factors V, VII, X, XIII, and pro-thrombin. These interact with tissue factor, aprotein that is expressed by many tumor andnormal mesenchymal cells, to activate theclotting system with two major consequences:conversion of extravasated plasma to serumand extravascular deposition of fibrin (Dvoraket al. 1981; VanDeWater et al. 1985). Both ofthese events have important sequelae. Normally,interstitial cells are bathed in a plasma pro-tein-poor filtrate. However, vascular hyperper-meability changes the composition of theinterstitial fluid dramatically. Not only is thefluid enriched in proteins, but with clotting ithas also changed from plasma to serum. Serumhas been shown to cause dramatic changes inthe gene expression patterns of cultured mesen-chymal cells (Chang et al. 2004), and it is likelythat similar changes occur in vivo, thoughexperiments to test this possibility have not asyet been performed.

Insertion of fibrin into tissues also hasimportant consequences. Fibrin takes the formof a gel that traps water and other solutes,thereby inhibiting their clearance by lymphaticvessels or capillaries and resulting in tissueedema (Fig. 5D,E). Fibrin also serves as a “pro-visional” stroma, providing a matrix that ini-tially organizes tumors into parenchymal andstromal compartments, before being replacedby mature stroma (Fig. 5D–H) (Dvorak et al.1979a). The amount of fibrin present at anyone time represents a balance between fibrindeposition and degradation. Degradation isthought to result primarily from plasmin, theprotease generated when another leaked plasmaprotein, plasminogen, is activated by tumor-secreted plasminogen activator.

With RGD and other sequences that arerecognized by avb3 and other integrins, fibrin

provides a favorable matrix for the migrationof tumor, mesenchymal, and inflammatory cells(Senger 1996; Hynes 2009). The capacity offibrin to support cell migration can be readilydemonstrated in vitro (Fig. 5I). When adherentfibroblasts are overlaid with appropriately con-stituted fibrin gels, they migrate upward intoand through these gels even in the absence ofchemotactic factors (Brown et al. 1993). Otherplasma proteins that extravasate from leakyblood vessels include fibronectin, vitronectin,and osteopontin. These likely also contributeto cell migration by virtue of sequences thatfavor cell attachment and detachment (Joshiet al. 1993; Hauptmann et al. 1995; Senger1996). Fibronectin may also be integrated intofibrin as the result of factor XIII–mediatedcrosslinking.

Fibrin has additional properties that con-tribute to angiogenesis and mature stroma for-mation. Some of these are clearly understood.For example, fibrin binds to and thereby seques-ters growth factors, protecting them fromdegradation. It also induces the expression ofproangiogenic molecules such as IL-8 and tissuefactor. Fragment E, a fibrin breakdown product,is directly proangiogenic (Dvorak and Rickles2006; Dvorak 2007b). Less clearly understoodare other mechanisms by which fibrin providesan environment in which endothelial cellsorganize to form blood vessels and in whichfibroblasts synthesize and secrete the matrixproteins and proteoglycans characteristic ofmature stroma. Together with macrophages,fibroblasts degrade the provisional fibrin stromaand eventually replace it by synthesizing maturestromal proteins. The vessel-promoting proper-ties of fibrin have been demonstrated in vitro.Endothelial cells cultured as a monolayer onfibrin gels rapidly organized into vascular struc-tures when they were overlaid with a secondlayer of fibrin (Chalupowicz et al. 1995). Thestroma-promoting properties of fibrin havealso been demonstrated in vivo by experimentsin which fibrin gels were planted into the sub-cutaneous space of guinea pigs (Dvorak et al.1987). Fibroblasts and new blood vessels soonentered these gels and proceeded to synthesizecollagen and replace fibrin with granulation



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tissue similar to that deposited in tumors andhealing wounds (Fig. 5J). Some of the proteinsthat comprise granulation tissue, such as typesI and III collagen, are also abundant in normaladult tissues. However, granulation tissue alsoincludes tenascin, alternatively spliced formsof fibronectin, and proteoglycans that are notnormally expressed in most normal adult tis-sues (Yeo et al. 1991; Hauptmann et al. 1995;Hashimoto-Uoshimka et al. 1997; Sage 1997).

CONCLUDING REMARKS

In summary, vascular permeability occurs inthree contexts, that of the low-level basal perme-ability of normal tissues and the greatlyincreased permeability of acute and chronicpathologic states, which are often associatedwith acute release or chronic synthesis andrelease of VPF/VEGF-A. Different types ofblood microvessels participate in each of thethree contexts. Also, the composition of theextravasate differs: a low protein transudate inthe case of BVP, and high plasma protein exu-dates in the case of AVH and CVH. Althoughmuch remains to be learned, plasma and plasmaproteins apparently traverse endothelial cells byboth transcellular and paracellular pathways inall three contexts.

CVH and its consequences of fibrin deposi-tion, angiogenesis, and stroma generation havelong been known to be important features ofhealing wounds, whether in the form of cutsor bruises in skin or healing myocardial infarctsand strokes (Haddow 1974; Dvorak 1986,2003). It is now clear that tumors have co-optedthis response by secreting VPF/VEGF inamounts similar to those found in healingwounds. Whether in wounds or tumors, secre-tion of VPF/VEGF leads to deposition of afibrin gel provisional stroma that changes theextracellular matrix of normal tissues fromanti- to proangiogenic and stromagenic. Thisnew matrix favors and supports inward migra-tion of mesenchymal and inflammatory cells,new blood vessel formation, and the layingdown of granulation tissue and subsequentmature matrix that resembles but is nonethelessdistinct from the connective tissue found in

normal adults. The amount of mature stromathat results depends on many factors, such asthe extent of leakage, activation of clottingand fibrinolysis, etc. The end result is scarformation in the case of healed wounds anddesmoplasia in the case of tumors. The processof tumor angiogenesis thus differs strikinglyfrom that of the physiological angiogenesisof development in which newly formingvessels are structurally normal and not hy-perpermeable.

ACKNOWLEDGMENTS

This work was supported by National Insti-tutes of Health grants P01 CA92644 andR01CA142262 and by a contract from theNational Foundation for Cancer Research.

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